9%: 25¢ per day per item RETURNIMS LIBRARY MATERIALS: \ p lace in book return to remove charge from circulation records THE SOLUTION ORDERING OF ALKALI METAL SALTS OF GUANYLIC ACIDS I. THE EFFECT OF PHOSPHATE POSITION ON THE SELF-AGGREGATION OF GUANZLIC ACIDS. II. ETHIDIUM BINDING TO UNSTRUCTURED AND STRUCTURED FORMS OF GUANYLIC ACIDS. III. A NOVEL PROTON EXCHANGE BETWEEN THE AMINO PROTONS AND SOLVENT WATER IN GUANYLIC ACIDS. By Richard Gary Barr A DISSERTATION Submitted to Michigan State University 111 partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department f Chemistry 198]. tux has (0 ABSTRACT THE SOLUTION ORDERING OF ALKALI METAL SALTS OF GUANYLIC ACIDS I. THE EFFECT OF PHOSPHATE POSITION ON THE SELF-AGGREGATION OF GUANYLIC ACIDS. II. ETHIDIUM BINDING TO UNSTRUCTURED AND STRUCTURED FORMS OF GUANYLIC ACIDS. III. A NOVEL PROTON EXCHANGE BETWEEN THE AMINO PROTONS AND SOLVENT WATER IN GUANYLIC ACIDS. By Richard Gary Barr In aqueous solution, the dianion Of guanosine-S-mono- phosphate (S'GMP) forms regular ordered structures that are slow to exchange on the 1H NMR time scale. The most com— pelling evidence for structure formation is the presence of multiple inequivalent H(8) resonances in the limiting spectra of dialkali metal ion salts in D20 solution near 0°C. This self-assembly process is dramatically dependent on the nature of the alkali metal counterion, which is believed to direct structure formation through a size- selective coordination mechanism. Such metal ion dependent ordering is unprecedented in other nucleotide systems. To assess the role of the phosphate group in the solu- tion self-structuring of guanylic acids, guanosine Richard Gary Barr . l A . . aggregation was investigated by H hMR. The addition of Na+, K+, Sr+2, or Ti+2 does not affect the H(8) reson- ance, either in chemical shift or half-width, up to the point of precipitation or gelation. At the gel or pre- cipitation point, all resonances are understandably broad- ened. Although guanosine does not form an NMR-observable ordered structure, selective deuteration experiments were carried out to determine if guanosine could incorporate into the S'GMP structure. Guanosine was found to destabi- lize the Na25'GMP structure, as determined by the loss of H(8) structure resonance intensities as the concentration of d-H(8) guanosine in a Na25'GMP solution increased. The mechanism of destabilization involves formation of a mixed guanosine-S'GMP soluble gel type of structure. That is, S'GMP is incorporated into a guanosine gel structure rather than guanosine incorporated into a S'GMP solution self- structure. Further evidence for the importance of the phosphate position has been provided by detailed studies of 2'GMP + aggregation. The Na+, K+, Rb , and Cs+ salts of 2'GMP have been found to form ordered solution structures as evidenced by multiple H(8) resonances in the 1H NMR and frequency Shifts in the carbonyl region of the IR spectra. The + + e o e 7 + order of stability is K > Na . e Rb+ > Cs The Li+, tetramethylammonium ion (TMA+), and tetrabutylammonium Richard Gary Barr iori (TBA+) salts did not form an NMR-observable self-struc- ture. The stability of the 2'GMP to form self-structures is less than that of the corresponding S'GMP systems. Higher concentrations of (alkali metal)22'GMP and lower temperatures are needed to form the same percent of struc- ture as in the S'GMP case. The number of 2'GMP structure H(8) environments and their chemical shifts are similar to, but not exactly the same as, the corresponding 5'GMP system. An attempt was made to vary the concentration of alkali metal by using TMA+ase1non-inhibiting,rmwvstructure direct- ing ion as in the S'GMP case. TMA+ was found to inhibit the structuring process in 2'GMP. The Na22'GMP self-struc- ture formed by adding NaCl to a TMA22'GMP solution is %5% structured even under conditions where 50% solution self-structure is present in the TMA+ free system. The K+-TMA+ mixed system forms a self-structure, but the extent of structure formation is inhibited by TMA+, It is postulated that ion pairing between the phos- phate and TMA+ in 2'GMP inhibits the formation of tetramers or the stacking of tetramers for steric reasons. This is verified by mixed TMA+-Na+, 2'GMP-5'GMP selective deutera- tion experiments. A destabilization of the S'GMP solution self—structure which is not observed in the pure Na+ mixed rnicleotide system is observed in this system. Nu- clear Overhauser effect (NOE) experiments showed TMA+ Richard Gary Barr was located near the ribose protons and the amino protons. A position near the amino protons would sterically hinder the formation of tetramers. Previous work has shown that TMA+ in the Na25'GMP system is located near H(8) away from the H-bonding positions involved in tetramer formation. The Na+ and K+,3'GMP and guanosine-5'-diphosphate (5'- GDP) systems are similar to those Of guanosine, forming gels in preference to ordered solution structures. The TMA+ inhibition of structure formation is present in both systems, manifested by a destabilization of gel formation. On addition of K+ to a TMA23'GMP solution, new H(8) reson- ances appear. TMA+ is therefore blocking the formation of large aggregates. New resonances in the H(8) NMR spectrum of TMAZE'GDP occur on addition of KC . The H(8) resonance of both 3'GMP and S'GDP is broadened on addition of NaCl but no new resonances occur. Both 3'GMP and S'GMP were found to interact with each other. It 1}; proposed that TMA23'GMP and Na25'GMP form a solution self-structure. Several new resonances occur in the H(8) NMR of S'GDP in mixed TMA2S'GMP-8-deutero-Na25'GMP solutions, indicating mixed solution self-structures of S'GMP and S'GDP can occur. It is clear from the above results that the phosphate group plays a role in the solution ordering of guanylic acids. The structuring ability at pH = 7-8 of the nucleotides is guanosine > 3'GMP > S'GDP > S'GMP > 2'GMP, Richard Gary Barr with guanosine, 3'GMP and S'GDP preferably forming gels and S'GMP and 2'GMP forming solution self-structures. Analyses of NMR spectra taken in lH2O have led to the assignment of a generalized structure for the solution self- structuring of guanylic acids. One Na+ Solution self- structure is composed of two head-to-head stacked planar tetramers with a Na+ located on the outer side of the cavity formed by the carbonyl oxygens. A water molecule may be trapped in the octameric cavity formed by the carbonyl oxygens between the plates, hydrogen bonded to an upper and a lower carbonyl oxygen. A water bridge is formed between a phosphate and an adjacent guanylic acid -NH2. In S'GMP there are hydrogen bonds formed between the 2'OH of the upper plate and the O(l') of the lower plate. A hydrogen bond is also formed between the 3'OH and the 0(5') of the lower plate. A time-averaged NMR value indi- cates two Na+ ions are territorially bound to the outer periphery of the structure. Another structure is similar to that described above except the tetramers are stacked head to tail. A different hydrogen bonding scheme is also present. The other alkali metal solution self—structures are believed to be based on the stacking of these two models with the metal ion located in the octameric cavity. During the investigation of 2'GMP a novel proton ex- change was noted for 2'GMP. The exchange is between the amino protons and water. The most interesting aspect of Richard Gary Barr this exchange is that the integral intensity of the amino protons varies from ml to 2 relative to H(8) in NaQZ'GMP, but is always 2 in TMA22'GMP. The conditions where the integral intensity is <2 are consistent with an ordered form of nucleotide being present. It is proposed that the loss of amino proton relative integral intensity is caused by the formation of a water bridge between the phosphate group of an adjacent nucleotide in a tetramer and the amino proton not involved in tetramer formation. This allows for the rapid exchange of the amino protons with solvent water. This water bridge places the nucleotide in the preferred conformation in S'GMP and 2'GMP. The binding of ethidium was also investigated. Ethi- dium was found to form a 1:1 and a 2:1 ethidium:nucleotide complex with unstructured S'GMP and 2'GMP. The equilibrium constant for the 1:1 complex in both nucleotides is mlOu M'l. For the 1:1 and 1:2 ethidium:2'GMP species the enthalpies were determined to be -l9.8il.0 Kcal/mol and O.li0.3 Kcal/mol, respectively, and the entropies were -53.3i6.6 eu and 3.9:. 1.6 eu, respectively. Ethidium was also found to bind with structured forms of S'GMP and 2'GMP. Formation of a 2:1 and a 1:1 complex occurs. Binding constants could not be determined, but the NMR shifts suggest the binding constants are 103 M'l. The amino proton exchange reaction in the absence of structure was investigated for the TMA+ salts of 2'GMP and 5'GMP afui the Na+ salt of 2'GMP. The process was found to Richard Gary Barr be pseudo first order in amino proton. The exchange re- a action has a AH =+15 to +19 KcalS/mol and a ASE = 44.0 to + 9.0 eu. To My Parents ii ACKNOWLEDGMENTS It is difficult to properly thank everyone who has been of help over the past few years. To those not mentioned, a general thank you is extended. I would like to thank Dr. Harry A. Eick for serving as my second reader. Assistance received from Dr. Tom V. Atkinson on computer Operation and programming, which made much of this work possible, is noted. The encouragement and love of my parents, Frank and Minnie Barr, grandparents and brother, Dennis, has always been a source of guidance for me. Dr. Thomas J. Pinnavaia has always been a continual source of guidance, wisdom and encouragement. Words cannot express the respect and admiration I hold for him as a research director, teacher and friend. I look forward to continued collaboration with him. I would also like to thank Dr. Elene Bouhoutsos-Brown, Dr. Richard Farmer, Dr. Emmanual Schmidt, Mr. Rasik Raythatha, Mr. Walt Cleland, Mr. Steve Christiano, and Dr. Chris Marshall whose collaboration and fruitful dis- cussions were extremely helpful. iii Chapter LIST OF TABLE OF CONTENTS TABLES. LIST OF FIGURES INTRODUCTION. I. General Information on Nucleic Acids. Nomenclature. A B. Structural Information. C Base—pairing and Base Stacking. D Metal Ion Interactions with Nucleic Acids iaggsrtswrw: THEIR? A. General Information on Guanylic Acids. B. Gelation. C. Solution Self-Structuring III. Ethidium Binding to Nucleic Acids A. General Intercalation B. Ethidium Cation General Information . . . IV. Amino Exchange in Mononucleotides V. Statement of Research Aims. EXPERIMENTAL. I. Materials, Techniques, and Preparations. iv . viii xii F4 .2 H U"! 10 10 10 1A 27 27 LO m \n H \J Cow -\] 37 Chapter II. III. RESULTS I. II. Instrumentation A. Nuclear Magnetic Resonance Spectroscopy (NMR). B. Ultraviolet/Visible Absorption Spectroscopy (UV/Vis) . . C. Infrared Spectroscopy (IR). D. Computation Synthesis A. Selective H(8) Deuteration. B. 8-Bromoguanosine. Guanosine A. The Solution Self-Structure of Guanosine . . . . . . . B. Incorporation of Guanosine into the Na25'GMP Solution Self-Structure. The Solution Self-Structuring Of 2'GMP. A. The Li+ Salt of 2'GMP B. The Na+ Salt of 2'GMP c. The K+ Salt of 2'GMP. D. The Rb+ Salt of 2'GMP E. The Cs+ Salt of 2'GMP F. Tetramethylammonium Ion Destabiliza- tion of the 2'GMP Self-Structure. G. Mixed Nucleotide Experiments. H. Stoichiometry of the Na 2'GMP Solution Self-Structure Page Al Al A1 A2 43 AA AA AA A6 A6 A6 A8 55 55 59 71 78 85 85 102 Chapter III. 3'GMP Self-Structuring. A. Alkali Metal Salts. B. Mixed Nucleotide Systems. IV. S'GDP Self-Structuring. A. Alkali Metal Salts. B. Mixed Nucleotide Experiments. V. Ethidium Binding to Guanylic Acids. A. Binding of Ethidium to Unstructured Nucleotide. . . . . . . . . . B. Ethidium Bromide Binding to Structured Forms of Guanylic Acids VI NMR Spectra of Guanylic Acids in lH2O A. NMR Spectra in 1H20 B. Amino Proton Exchange VII. Amino Proton Exchange with Solvent Water DISCUSSION. I. Tetramethylammonium Ion as a Counterion. II Base Stacking Interactions. III. Tetramer Formation. IV. Self-Structuring Tendencies of Guanylic Acids. A. Effect of the Phosphate Position on Self-Structuring. . [13 Experiments on Mixed Nucleotide Solutions vi 127 143 155 155 166 17A 188 189 192 19A 203 203 205 Chapter Page V. The Best Model for Ordered S'GMP. . . . . . 212 A. The Assignment of Resonances. . .7. . . 212 B. Other Models that Have Been Proposed. . . . . . . . . . . . . . . . 21A C. The Model Proposed for the NaZS'GMP Solution SelfeStructure. . . . 217 VI. Ethidium Binding to Unstructured and Structured Forms of 2'GMP and S'GMP . . . . 22A VII. Amino Proton Exchange in the Absence of Solution Self—Structure. . . . . . . . . 228 CONCLUSIONS . . . . . . . . . . . . . . . . . . . . 230 APPENDIX 1 - PROGRAMS USED ON THE ASPECT 2000 ON THE BRUKER WM250 and PROGRAMS USED IN KINFIT. . . . . . . . . . . . . 233 APPENDIX 2 - AMINO PROTON EXCHANGE THEORY AND THE PROGRAM LORENT . . . . . . . . . . 253 APPENDIX 3 - ADDITIONAL DATA AND SPECTRA. . . . . . 252 REFERENCE S. . . . . . . . . . . . . . . . . . . . . 269 vii Table LIST OF TABLES Number Of Isomers and H(8).Lines Expected for Three Exchange Regions. Taken from Reference 97 Results of a Model Building Study of the Octameric Forms of S'GMP Molar Absorptivities Used to Determine Concentration The Effect on the SOlution Self-Structure of 0.32M Na25'GMP on Addition of 8-d Guanosine at 3.5°C The Temperature Dependence for the Absorbances of Various 0.82M K22'GMP IR Bands. Results of a Gated Decoupling Experiment on 0.89M TEA22'GMP at 27°C Results of a Gated Decoupling Experiment in Which the TMA+ Resonance of 0.71M Na22'GMP Solution 1.10M in NaCl and viii *U n) UR (D 20 21 A2 50 80 101 Table m {D m m 1.36M in TMACl was Irradiated at -3°C. . . . . . . . . . . . . . . . .‘. . . 102 8 Deconvolution of the Lowfield Set of H(8) Resonances for Na22'- GMP in the Presence of 3.0M Na+ . . . . . . 112 9 The Best Computer Fits of the Equilibrium Constants for the Binding of Ethidium to TMA22'GMP and TMA25'GMP . . . . . . . . . . . . . . . 136 10 The Best Computer Fit of the H(l') Resonance of 2'GMP to the Binding of Ethidium to TMA22'GMP. in: LA) \{3 11 The Best Computer Pit of the Thermodynamic Parameters of Ethidium Binding to TMA22'GMP. . . . . . . . . . . . 1A2 12 The Best Computer Fit of the Bind- ing Constants for the Binding of Ethidium to Na22'GMP Self- Structures. . . . . . . . . . . . . . . . . 152 13 Percent Decrease in Signal In- tensity on Irradiation of Other Proton Signals of a 0.62M Na25'GMP \fi ‘1 Solution in lH20 at 2°C . . . . . . . . . . 1 1A Integral Intensity of Resonancesin Arbitrary Units Downfield from the ix Table l5 l7 l8 19 20 21 lH20 Resonance of S'GMP Solution in lR20 at 2°C. Integral Intensity of Resonances in Arbitrary Units Downfield from the 1H20 Resonance of 2'GMP Solutions at 1°C. A Comparison of the Integral Intensities Obtained by Proton NMR of the Amide Proton, H(8), 5, and the Amino Protons of Various Nucleotides NMR Lineshape Parameters and First Order Amino Proton Exchange Rate Constants for 0.30M TMA25'GMP NMR Lineshape Parameters and First Order Amino Proton Exchange Rate Constants for 0.61M NaZS'GMP. NMR Lineshape Parameters and First Order Amino Proton Exchange Rate Constants for 0.80M TMA22'GMP NMR Lineshape Parameters and First Order Amino Proton Exchange Rate Constants for 0.15M Na 2' MP. 2 NMR Lineshape Parameters and First Order Amino Proton Exchange Rate EU {1) 09 (D 16A 165 173 180 181 H 0') f0 Table 22 23 2A Constants for 0.91M Na22'GMP. NMR Lineshape Parameters and First Order Amino Proton Exchange Rate Constants for 1.81M Na22'GMP. The Estimated Systematic Un- certainties in the Experimental Input Parameter of LORENT Estimates of the Activation Param- eters for the Amino Proton Exchange in Various Nucleotides. xi '11 In 09 (D 183 18A 187 The purine and pyrimidine.bases, ribose and deoxyribose, and phos- phates of nucleotides. Taken from Reference A9. Newman projections showing the preferred nucleotide conformations and their nomenclature._ Taken from Reference A9 The planar tetramer unit and helix of the guanosine gel structure show- ing hydrogen bonding between N(l) and 0(6) as well as N(2) and N(7). Taken from Reference 2 Schematic representation of the tetrameric arrangement of a 3'GMP gel. Taken from Reference 23. Limiting H(8) resonance lines of the alkali metal salts Of 5'GMP in D20 at high concentration and low tem- peratures. Taken from Reference 9A Figure Schematic diagram of the proposed size selective coordination sites in the structuring of guanylic acids. a) coordination in the center of the tetrameric plate by + Na (r = 0.993). b) Coordination between the plates by K+ and Rb+ (r = 1.51A and 1.60A, respectively). Taken from Reference 97. Schematic diagram of a) normal stack- ing mode and b) the two enantiomeric reversed stacking modes of the octamers of S'GMP. Taken from Reference 97 Browns proposed models for S'GMP solution self-structure. a) The Na+ self-structure; b) the "simple" 4.. K self-structure and c) the "complex" K+ self-structure. (Taken from Reference A9). . Structure proposed by Laszlo for the solution self-structuring of NaQS'GMP. Open circles represent phosphate groups pointing down, while closed circles represent phos- phate groups pointing up. Ja+ xiii 99-106 Page l7 l9 23 Figure 10 ll 13 ions are represented by stripped circles. A is the staircase model proposed by Laszlo. B is an alter- nate model proposed by Laszlo. A. The structure of ethidium. B. A schematic diagram showing the intercalation of ethidium in U-A. Taken from Reference 122 Schematic diagram of binding of ethidium to structured forms of S'GMP. ethidium is represented byC) and the guanylic acid tetramer byCZJ. A is Marshall's (97) model for ethidium binding to the Na25'GMP self-struc- ture. B is Marshall's (97) model for ethidium binding to the K25'GMP self- structure. Exchangeable protons in the G-C pair. The circled protons exchange with each other. Taken from Ref- erence 122 The effect on the H(8) region of the NMR spectrum of 0.32M Na2S'GMP at 3.5°C on the addition of 8-d guano- sine. All solutions were homogeneous and not gels xiv Page 25 28 30 A9 Figure 1A 15 l6 18 19 The H(8) NMR spectrum of a 0.32M NaES'GMP and 0.11M 8-d guanosine solution at 3.5°C. Note the very broad resonance present (shaded) The limiting H(8) resonance lines of alkali metal Salts of 2'GMP. Spectra were recorded in D20 solutions, ex- cept for the spectrum of 1.22M Na22'GMP which was recorded in 1 H2O The H(8) chemical shift of various salts of 2'GMP versus temperature. The H(8) chemical shift of selected salts of 2'GMP versus concentration. The infrared spectra of 0.98M L122'GMP in D O at 50°C and 3°C. 2 1 Neither these spectra nor the H NMR spectra of this solution Shows evidence for structure formation at either temperature The H(8) NMR lines of 0.91M Na22'GMP in D20 at 2°C. The two weak lines at 8.51 ppm and 7.2A ppm are indica- tive of the simple Na22'GMP self- structure. XV 52 57 58 60 61 'gure 20 21 22 23 2A The change in the NMR spectra of H(8) upon addition of NaCl to a 0.69M Na22'GMP solution at 1°C The temperature dependence of the H(8) NMR resonances of a 0.69M Na22'GMP and 1.32M NaCl solution The 1R NMR spectrum of 1.223 Na22'GMP in lH2O at 1°C from 5.00 ppm to 13.00 ppm. The A resonances are attributed to structured N(1)H, the B resonances to H(8), C to the amino protons, the D resonances to H(l') and E to H2O The infrared spectra of 0.30M Na22'GMP in D20 at 3°C and 52°C. Neither of these Spectra nor the 1H NMR spectra of these solutions shows evidence for structure forma- tion at both temperatures. The infrared spectra of 0.95M Na22'GMP in D20 at 2°C and 52°C. Both the H(8) NMR spectrum and the IR spectrum at 52°C Show no evidence of structure formation; while at 2°C the H(8) NMR spectrum is characteristic Page 63 6A 66 68 'TJ igure 25 26 27 28 Of a simple N822'GMP solution struc- ture. The structure resonances account for <5% of the H(8) resonance. The IR spectrum at 2°C shows pertur- bations which have been attributed to solution self—structure. . The infrared spectra of 0.71M Na22'GMP containing 1.1M NaCl in D 0 2 at 2°C and 52°C. Both the H(8) NMR spectrum and the spectrum of this sample at 52°C show no evidence for structure formation. At 2°C the H(8) NMR structure resonances account for W50% of the total H(8) intensity. The IR spectrum at 2°C shows perturba- tion characteristic of structure formation. The concentration dependence of the H(8) NMR spectrum of K 2'GMP at 2 -2°C . . . . . . . The temperature dependence of the H(8) NMR spectrum of 0.82M K22'GMP The infrared spectra of 0.50M K22'GMP in D20 at 51°C and 3°C. The H(8) NMR spectrum and IR xvii '0 to 0Q 69 70 72 7A Figure 29 3O 31 32 33 spectrum at 51°C show no evidence for structure formation; at 3°C the sample is m30% structured as determined by proton NMR. The IR spectrum Show perturbations charac- teristic of structure formation. . . . . . 75 The infrared spectra of 0.82M K 2'GMP in D20 at 50°C and 2°C. At 2 2°C this sample is m80% structured, determined by the H(8) NMR resonance. The IR spectrum shows perturbation characteristic of structure forma- tion. There is no structure present at 50°C determined by proton NMR or by IR . . . . . . . . . . . . . . . . . 76 Comparison of melting profiles of 0.82M K 2'GMP in D20 by IR and 1H 2 NMR. . . . . . . . . . . . . . . . . . . . 77 The concentration dependence of the H(8) NMR spectra of Rb22'GMP at -200 .. . . . .. . . . .. . . . . .. . 79 The temperature dependence of the H(8) NMR spectrum of 0.77M Rb22'GMP. . . . 82 The infrared spectra of 0.30M Rb22'GMP in D20 at 51°C and 3°C. xviii Figure 3A 35 36 Neither these spectra nor the H(8) NMR resonance Shows evidence of structure formation at either tem- perature . . . . . . The infrared spectra of 0.77M Rb22'GMP in D20 at 50°C and 3°C. At 50°C neither the H(8) resonance nor the IR spectrum shows evidence for structure formation, while at 3°C the H(8) structure resonances account for qJA0% of the total H(8) resonance. The IR Spectrum at 3°C shows perturbations characteristic of structure formation . . . . . The temperature dependence of the H(8) NMR spectrum of 0.6AM C322'— GMP in D20 . . . . . . . . . . . The infrared spectra of 0.6AM 0522'GMP in D20 at 51°C and 3°C. Neither the H(8) NMR spectrum nor the IR spectrum at 51°C shows evi- 83 8A 86 dence of structure formation, while at 3°C the sample is %5% structured by proton NMR. The IR spectrum at 3°C shows perturbations characteristic of structure formation xix 38 39 A0 A1 The H(8) region of a 0.68M TMA22'— GMP and 2.2M NaCl solution at 0°C. This solution in absence of TMA+ would be approximately 50% struc- tured. . . . . . . . . . . The effect on the H(8) NMR spectrum on addition of TMACl(s) to a 0.71M Na 2'GMP solution containing 1.1M 2 NaCl at -3°C. B illustrates the H(8) resonance of the sample made 1.36M in TMACI. A illustrates the H(8) resonances of the sample in absence of TMAI. The change in the proton NMR spectra of 0.69M TMA22'GMP upon addition of KCl at 0°C Effect on the H(8) NMR spectrum on making 0.60M K22'GMP 1.33M in TMAC1(S) at -3°C. A is the H(8) NMR spectrum in absence of TMAI. B is the H(8) spectrum after making the solution 1-33M in TMACl(S). The infrared spectra of 0.69M TMA22'GMP at 51°C and 3°C NMR studies of this solution at comparable XX 88 90 91 igure A2 A3 AA A5 *0 m 09 (D temperatures Show no evidence for structure formation. . . . . . . . .5. . . 9A The infrared spectra of a 0.69M TMA22'GMP solution made 1.1M in NaCl at 51°C and 3°C. NMR studies of this solution at comparable tempera— tures Show no evidence for structure formation. . . . . . . . . . . . . . . . . 95 The chemical shift of H(8) versus the concentration of NaCl in 0.69M TMA 2'GMP at 5°C and 0.5°C . . . . . . . . 96 2 The effect on the H(8) NMR spectrum of 0.82M K22'GMP at 0°C on making the solution of 0.08M in TEAP. B is the H(8) region of 0.82M K22'- GMP at 0°C. A is the H(8) region after the solution is made 0.08M. A precipitate formed which was removed by filtration. The concen- tration of 2'GMP was determined to be 0.60M and the concentration of TEAP was approximately 0.01M, determined by NMR.. . . . . . . . . . . . . . . . . . 98 The effect on the H(3'), H(A') and H(5') resonances when the TMA+ xxi Figure A6 A7 A8 A9 resonance is irradiated in a gated decoupling experiment on a solution of 0.71g Na 2'GMP, 1.10g NaCl, and 2 1.36M TMACl at -3°C. . . . . . . . . . . . 103 The effect of 8-d TMA22'GMP on the H(8) NMR spectrum of 0.68M Na25'- GMP at 5°C. The spectra are not drawn to scale. There is no major integral intensity loss. . . . . . . . . . 105 The H(8) spectrum of a 0.69M 8-d Na22'GMP and 0.03M guanosine solu- F“ C) \J tion at 1°C- The H(8) region of 0.18M Na22'GMP and 0.AOM Na22'GMP at 0°C. The[Na+1 for both samples was made 3.0M by the addition of NaCl. . . . . . . . . . 109 The computer deconvolution of the lowfield resonances of the H(8) NMR resonances for 0.38M Na22'GMP made 3-0M in Na+ by the addition of NaCl. The solid line in A is the observed lowfield H(8) spectrum. The x's are the computed values. B shows the three deconvoluted reson- ances. . . . . . . . . . . . . . . . . . . 111 xxii [—1. gure 51 53 \fl U1 The effect on the NMR Spectrum of the H(8) region of 0.A3M TMA23'GMP on addition of NaCl(s) The effect on the NMR spectrum of the H(8) region on addition of KCl to O.A3M TMA23'GMP The temperature dependence of the H(8) region of a 0.A3M TMA23'GMP solution 0.27M in K01- The effect of 8-d TMA23'GMP on the H(8) NMR spectrum of 0.67M Na25'GMP at 7°C - - The H(8) NMR spectra of 0.67M 8-d Na25'GMP and two concentrations of TMA23'GMP at 7°C. A is the H(8) NMR spectrum of a 0.67M 8—d Na25'- GMP and 0.08M TMA23'GMP solution. B is the NMR spectrum of a 0.67M 8-d Na25'GMP and 0.16M TMA23'GMP solution . . . . . ._. The H(8) 1H NMR spectra of 0.11g TMA25'GDP containing KCl. The 1.27M sample contained a precipitate when removed from the NMR spectrometer. xxiii 'U Q) 09 (D 116 118 120 121 123 Figure Page 56 The melting profile of a 0.11M TMA25'GMP solution made 1.27M in K01. . . . . . . . . . . . . . . . . . . . 125 57 The H(8) NMR spectrum of a 0.32M 8-d Na25'GMP solution containing TMA25'GDP at 5°C . . . . . . . . . . . . . 126 58 The effect on the proton NMR spectrum with the addition of TMA22'GMP at 0.020M ethidium bromide at 0°C . . . . . . 128 59 The temperature dependence of the methyl chemical shift of 0.020M ethidium bromide . . . . . . . . . . . . . 130 60 The chemical shift of the ethidium methyl resonance as a function of the mole ratio of TMA22'GMP to ethidium at various temperatures. . . . . . . . . . 131 61 The best computer fit of the ethidium methyl resonance to the binding of ethidium to TMA22'GMP at 0°C. The concentration of ethidium is 0.020M. . . . 137 62 The bestcomputer fit of the ethidium methyl resonance to the binding of ethidium to TMA25'GMP at 5°C. The concentration of ethidium is 0.0205. . . . 138 xxiv Figure 63 6A 65 66 67 The best computer fit of the H(l') resonance of the binding of ethidium‘ to TMA22'GMP at 0°C. The concentra- tion of ethidium is 0.020M . The ethidium methyl chemical shift versus the mole ratio of Na22'GMP to ethidium at various temperatures. The solution was made to have a mole ratio of Na+/2'GMP of 3.28 by the addition of NaCl Plot of the chemical shift of the EtdBr methyl resonance as a function of the structured Na25'GMP/Drug ratio. Extrapolation of the linear portions of the curve included. Taken from Reference 97. The ethidium methyl chemical shift versus the mole fraction of K22'GMP to ethidium at various temperatures. The ethidium concentration was 0.020M. Plot of the chemical shift of the EtdBr methyl resonance as a function of the structured K25'GMP/Drug ratio. Extrapolation of the linear portions of the curve included from Reference 97. XXV 1A0 1AA 1A5 1A6 Figure 68 69 70 71 72 Page The effect on the solution self- structure of 0.72M Na22'GMP upon making the solution 0.020M in ethidium bromide at 0°C. The nucleotide had a ratio of Na+/2'GMP of 3.28 ac- complished by the addition of NaCl. Figure A is the NMR spectrum of ethidium absent system. Figure B is the NMR spectrum on making the solu- tion 0.020M in ethidium. Note the increase in the H(8) and H(l') solution self-structure resonances . . . . 1A8 The best computer fit of the ethidium methyl resonance to the binding of ethidium to Na22'GMP at 0°C. The Na+/2'GMP ratio was made 3.28 by the addition of NaCl . . . . . . . . . . . 15A The proton NMR spectrum of 0.62M Na25'GMP in 1 H20 at 2°C downfield from 5.00 ppm. . . . . . . . . . . . . . . 158 The proton NMR spectrum downfield of 6.00 ppm of a 0.A3M 8-d Na25'GMP solution in lH20 at 2°C. . . . . . . . . . 160 The proton NMR spectra of 1.55M K25'GMP in 1N20 downfield from 5.0 ppm at 1°C and 27°C. . . . . . . . . . . . 167 xxvi Figure 73 7A 75 76 Page The relative integral intensity of the amino resonance and the % solution self-structure determined by H(8) NMR versus the concentration of Na22'GMP at 1°C. . . . . . . . . . . . . . 168 The relative integral intensity of the amino resonance and the % solution self-structure determined by H(8) NMR versus the concentration of Na25'GMP at 2°C. . . . . . . . . . . . . . 169 The relative integral intensity of the amino resonance and the % solution self-structure determined by H(8) NMR for 1.8NflNa22'GMP as a function of temperature . . . . . . . . . . . . . . 171 A typical example of the graphic determination of the amino chemical shift in the absence of exchange and the width at half-height in the ab- sence of exchange. The closed squares represent the observed V1/2 and the open squares the observed chemical Shift of 0.91M Na22'GMP as a function of temperature. The straight lines are the extrapolation from the low xxvii }—Jo gure 77 78 temperature region to give the chemi- cal shift of the amino protons and V1/2 in the absence of exchange. The proposed mechanism for the loss in amino proton NMR intensity for Na22'GMP and Na25'GMP. We expect this mechanism to occur with other alkali metal structure directing ions . . . . . . A segment of the prOposed tetrameric structure for the exchange of the amino proton of Na 2'GMP and Na25'GMP 2 with solvent water. This structure has X for Na25'GMP and Na22'GMP at 60° and 150°, respectively A photograph of a CPK model of the proposed structure for the exchange of the amino proton of Na25'GMP. The arrow points to the water bridge formed between the phosphate group of one nucleotide and the amino proton not involved in the hydrogen bonding of the tetramer unit of an adjacent nucleotide- xxviii Page 175 197 201 81 82 83 8A The proposed structure for a mixed guanosine and Na25'GMP self-structure. The guanosine unit is symbolized as a curved arrow, M+ is a structure directing metal ion, and the solid line extending from a guanosine unit is a phosphate group The proposed model for the destabiliza— tion of Na25'GMP solution self-struc- ture in the presence of TMA22'GMP. The arrow symbolizes a guanosine unit, the solid line extending from a guano- sine unit is a 5' phosphate, an open circle extending from a guano- sine unit is a 2' phosphate, and the solidCiPC1e is a TMA+. The proposed reaction scheme for the structuring of Na25'GMP. Expected upfield shift (in ppm) of a nucleus 3.5 A above or below the aromatic plane due to the ring cur- rent and diamagnetic susceptibility effects. Taken from Reference 97. The prOposed model for the solution self-structure which gives rise to xxix Page 207 209 215 219 Page the a and 5 H(8) resonances in Na25'— GMP. The shaded rectangles represent a tetramer unit. . . . . . . . . . . . . . 220 A photograph of a CPK model of the proposed model for the solution self— structure which gives rise to the a and 6 H(8) resonances in Na25'GMP. . . . 221 A photograph of a CPK model of the proposed model for the solution self- structure which gives rise to the a and 6 H(8) resonances in Na25'GMP. . . . 222 The proposed model for the K+ solu- tion Self-structure of 2'GMP and 5'- GMP. The rectangles represent a tetramer unit and the line extending from the tetramer a ribose-phosphate , . , 225 The 31P NMR spectra of 0.71M Na22'GMP at 0°C and 0.71M Na22'GMP 1.5M in NaCl at 1°C . . . . . . . . . . . . . . . . . . 268 XXX INTRODUCTION I. General Information on Nucleic Acids A. Nomenclature Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are chainlike macromolecules that function in the storage and transfer of genetic information. They are major com- ponents of all cells, together making up from 5 to 15 percent of their dry weight. The monomeric units of the nucleic acids are the nucleotides. DNA and RNA share a number of chemical and physical properties. In both types of polynucleotides the successive units are covalently linked in identical fashion by phosphodiester bridges formed between the 5'hydroxyl group of one nucleotide and the 3' hydroxyl group of the next. The nucleotides and their derivatives occur in the free form in cells in significant amounts. The monomeric units of DNA are called deoxyribonucleo- tides; those of RNA are ribonucleotides. Each nucleotide contains three characteristic components: (1) a nitro- genous heterocyclic base, either a derivative of purine or pyrimidine, (2) a pentose sugar, and (3) a phosphate group (g3. Figure 1). The backbone of both DNA and RNA FJ Pyrimidines 4 P , sN/E: Urines .t .. T 6 IN” 5 'I 5 A ‘ ‘>° 2\ N 4 NH2 3 lgH N/ . _ | cytosine . CAN” . f N A ' N / . \ adenine Q 0 // 2 . . H H" I urocul - > H o H" l \>quonine I CH ' ' \ N A l thymine 1° ~fi DNAomy O 0 ' HO-' -o-i'-?-o-lf»-o- 2 °°_°"y' OH OH OH D-rlbose h—J L monophosphme 1 dipjhosphate ' 1riphosphme D-ribose Figure l. The purine and pyrimidine bases, ribose and deoxyribose , and phosphates of nucleotides . Taken from Reference A9. consists of alternating phosphate and pentose groups, in which phosphodiester bridges provide the covalent con- tinuity. The purine and pyrimidine bases of the nucleo- tide units are not present in the backbone structure but constitute distinctive side chains. Four different deoxyribonucleotides serve as the major components of DNA, differing only in their nitrogenase base components: adenine, guanine, cytosine, and thymine (3:. Figure 1). Similarly, four different ribonucleotides are the major components of RNA: adenine, guanine, cytosine, and uracil (gf. Figure 1). The precise 3-D structures of various pyrimidines and purines have been deduced by X-ray diffraction analysisl-2. Pyrimidines are planar molecules, whereas purines are very nearly planar, with a slight pucker. Both have pronounced aromatic character. Free pyrimidine and purine bases are relatively insoluble in water. They are weakly basic compounds that may exist in two or more tautomeric forms depending on the pH. All the purine and pyrimidine bases of nucleic acids absorb light in the ultra- violet region between 250 and 280 nm. Deoxyribonucleotides contain 2-deoxy-D-ribose as their pentose component whereas ribonucleotides contain D-ribose (3:. Figure 1). Both sugars occur in their furanose forms in the nucleotides. The pentose is joined to the base by a B—N-glycosyl bond between C(1') and N(9) of purine bases or N(l) of pyrimidine bases. The phosphate group of nucleotides in DNA or RNA is in an ester linkage to the C(5') position. Other nucleotides exist in which the phosphate group is in the 2' or 3' position of the sugar. The 5' isomer is the most abundant in cells. When the phosphate group Of a nucleotide is removed by hydrolysis, the structure remaining is called a nucleoside. Thus nucleotides are the phosphates of the corresponding nucleo- sides. The nucleosides are named after the base which they contain; adenosine, cytidine, guanosine, thymidine, and uridine. In cells many polyphosphates also exist. The major polynucleotides are the 5'diphosphates and 5'triphos- phates. Cyclic phosphates can also be found, where a phos- phate group is condensed with two of the ribose hydroxy groups, either in the 2' and 3' position or the 3' and 5' position. Phosphates groups can also be found attached to different hydroxy groups (ELg;, 5'diphosphate 3'diphosphate guanosine). Polyphosphates serve as energy storage com— pounds in cells, whereas cyclic nucleotides and phOSphates attached to several hydroxy groups serve as metabolic regulators. B. Structural Information An almost infinite number of conformations are pos- sible for the nucleotides. The torsion angle between the base and C(1') of the sugar, the ribose conformation, and the exocyclic C(A')—C(5') bond can all vary. A great deal of interest has been expressed in both theoretical and ex- perimental aspects of the stereochemistry of nucleosides and nucleotides3-lo. Two preferred conformations exist about the glycoside bond of purine nucleosides and nucle- tides. Rotation about this bond is defined in terms of X: the angle defined by O(1')-C(1')-N(9)-C(8) as shown in Figure 2. The two common conformations are termed syn (x = 0°i90°) and anti (x = 180°i90°). The different con- formations in the sugar ring have been discussed extensively by Sundaralingamu. The major conformers are shown in Figure 2. In 5'nucleotides, the conformation of the exocyclic C(A')-C(5') bond is specified by the angle T, which is de— fined by the orientation of the C(5')-0(5') bond with respect to the C(A')-O(1') and C(A')-C(3') bonds as shown in Figure 2A. The orientation of the phosphate with respect to C(A') is defined by the angle ¢, and also shown in Figure 2. A number of reviews are available on the stereochemistry of nucleosides and nucleotidesll-l3. C. Base-pairing and Base Stacking Nucleotide bases can associate with each other by two methods. They can hydrogen bond through,>N-H and —OH groups, or they can stack vertically via hydrophobic bonding. The former method is well known to account for base pairing in DNA. In addition to Watson and Crick hydrogen bonding, W a CNN-C(5') , O ' H4. H" H" GAUCHE'GAUCHE TRANS-GAUCHE GAUCHE‘TRANS 99 '9 9' a c¢ c¢ C¢ C (5')'O(5') P ”st So ”at ”5: 9: "Sb p GAUCHE 'GAUCHE TRANS'GAUCHE GAUCHE 'TRANS 90 19 qt X X N(9)-C(l') Ofo‘lc. I ANTI H", 20 5o Figure 2. Newman projections showing the preferred nucleo- tide conformations and their nomenclature. Taken from Reference A9. which allows for semi-conservative replication of cells, other hydrogen bonding schemes have been found which may be of biological importance. Base pairing has_been studied in non-aqueous solutions by infrared spectroscopylu-l7. In both non-aqueous and aqueous solutions, NMR Spectroscopy of nucleotides has shown evidence for hydrogen bond pairing that involves the amino and aromatic ring protonsl8-2l. A review of stacking patterns of the bases and deriva- tives in the crystalline state has been prepared by Bugg22, Base stacking effects have been shown to provide ordered stabilization23. Gupta and Sasisekharan2u have reported theoretical calculations of base-base stacking in the free base systems. Physical methods such as NMR25'29, vapor phase osmometry27-3O, calorimetry3l, and f1uorescence32'3u have been used to study the stacking phenomenon. Base stacking in purine derivatives occurs to a far lesser extent in non-aqueous solventstflnniin water. Hanlon35 and Bugg22 argue that polarizable solvents which possess polar sub- stituents, like dimethyl sulphoxide, interact with the polarizable N-electron systems of the purines and inhibit stacking interactions in these solvents. Purine nucleo- sides have a far greater tendency to form vertical stacks than the corresponding mononucleotides which carry some negative charge on the phosphate moiety26‘28, Although usually deemed responsible for base stacking, hydrophobic effects are not entirely responsible. The stabilization effect of base stacking has been described in terms of 35 36 hydrophobic forces , special Van der Waals interactions , 36, dipole-dipole induced force837, 36 n—electron polarizability and electrostatically stabilized ionic complexes The association is most likely due to a combination of all these forces. D. Metal Ion Interactions With Nucleic Acids A large body of literature related to nucleic acids exists from a wide variety of disciplines due to their multi- faceted involvement in vital life processes. The biologi- cal functions of nucleic acids often involve the participa- tion of metal ions. As pointed out by Makrigiannis et al.38, it is difficult to investigate metal interactions with DNA directly, so that binding must be studied for each base. The binding of pre-transition and post-transition metals to nucleic acids or their constituents has been very im— portant for the understanding of a large number of bio- logical effects. Multiple coordination sites exist on the nucleotides. Interest in metal binding by nucleotides has been stimulated by the finding that Pt(II)-nucleic acid complexes have significant antineoplastic activity39'ul. Several reviews of the literature on metal binding by A2-AA nucleic acids and their constituents have appeared In guanosine the N(7) position, due to its lone pair of electrons, is a favored binding position for many transition metal ionsuz’MS. At neutral pH N(l) is protonated, how- ever, bonding of metal ions to this position can be so A5 favored as to lower the effective pKa Chelation of metal ions by the N(7)-0(6) or N(l)-0(6) positions of guano- sine has been proposed. Shimokawa gt g1.us concluded from 1H NMR work in DMSO that alkaline earth cations formed more stable complexes with guanosine than the transition metal A7 ions. Marzilli and coworker found that alkaline earth 1 nitrates do not cause the same spectral changes in the H NMR spectra found in the chloride salts in guanosine solu- tions. Tetramethylammonium chloride also produced large spectral shifts. Therefore, he concluded that the chloride ion was responsible for the shifts. The order of anions causing shifts, Cl-, Br' > I- > ClOu' > SCN- is the same as the influence of anions on stabilizing DNA. Sohma 22 £1.78 concluded that both cation and anion effects must be con- sidered. A review of X-ray structures of metal ions with guanosine and its derivatives has been prepared by Brownug. Alkali metal-nucleic acid interactions have not been ex- tensively studied for specific coordination binding, even though large amounts of Na+ and K+ are present in cells. The alkali metal ions have been regarded as non-specific electrolytes in balancing the negative charge of DNASO-53. 10 II. Self—Structuring of Guanylic Acids A. General Information on Guanylic Acids Of all the nucleic acid components guanosine has the most complex structure and widest variety of possible hydrogen-bonding interactionssl4 The physio-chemical properties of guanosine—containing regions of nucleic acids are frequently different. Intercalation drugs, such as actiomycinlss-57 and ethidium bromide58'61, and cancer drugs like cis-Pt(NH3)201262 prefer guanosine rich regions of DNA or RNA. The uniqueness of guanosine had been attributed to multiple degrees of freedom about the glycosidic bond63’6u, the different electronic structures of guanosine which re- sults in a completely different orientation of the dipole moment compared to other bases65 5A , and multiple protonation sites B. Gelation The self—association of guanylic acids in aqueous solu- tion may be manifested by the formation of anisotropic gels. As early as 1910 Bang66 reported the formation of gels in concentrated, acidic guanylic acid solutions. This phenomenon has since been found to occur with several other guanosine derivatives (e.g., 3'GMP, 2'GMP, 8-bromo- guanosine, and 2'deoxyguanosine)67“7o. Gel formation has 11 also been observed in methanol and ethanol but the stability of the gels is decreased7l. In order to obtain insight on the nature of gel forma- 6 tion by guanosine, Guschlbauer"7 studied gel formation of different derivatives in which the various functional groups had been substituted or changed under standard conditions (0.02M nucleoside, 0.1M K01). Purine nucleosides which did not contain the guanosine skeleton did not yield gels. These included inosine, xanthosine, adenosine, 2,6-diamino- purine riboside, 2-amino-purine riboside. While the pres- ence of the sugar on the base seems to be necessary, since guanine does not form gels, the nature of the sugar appears to be of little importance. The ribosides, deoxyribosides and arabinosides of guanine form gels. Methylation of N(7) or N(l) inhibited gel formation. Methylation of the amino group is of particular interest, since N2-methy1- guanosine formed a gel, while the doubly methylated com- pound did not. The results indicate that N(7), 6-keto, N(l), and one hydrogen atom of the amino group in position 2 are essential for gel formation. This is in agreement with the stacked planar tetramer model (9:. Figure 3) for the 3'GMP gel structure and the regular helix model (2:. 1.72 Figure 3) for H25'GMP gels reported by Gellert gt based on.X-ray and optical rotation measurements. A seg— ment of the planar tetramer structure is shown in Figure A. Gels of guanylic acids have been studied by IR73’82’83, 12 .m mocmnmmmm Scam coxma .Auvz use Amvz mm flaw: mw.amvo new Aavz consume wcfivcon somehcmn MCH Izonm mhsuosppm flow enamocmzw on» no xaamn new vac: mempumu Landau 059 .m mezwam 13 Figure A. Schematic representation of the tetrameric ar- rangement of a 3'GMP gel. Taken from Reference .23. 1A ORD67368a7u’ CD57-69,71,7A, 70,89,85, 75,86, Raman Calorimetry and X-ray diffraction72’76-81. Among the various conditions necessary for gel formation, the nature of the alkali metal or alkaline earth counterion is of great importanceYl. However, an adequate structural explanation of the cation dependence of gel formation has not been provided. The order of gel stability for guano- + 2+ + ’ Li+ 68,71. sine is Sr > K+ > Na+, Rb > OS The anion dependence of gel formation is much less important7l. Chantot and Guschlbauer have measured the melting tempera- ture, Tm, the temperature at which one-half of the nucleo- tide disordered. They found only a small difference in the Tm of gels formed with various anions68. They attributed the differences in Tm to the "salting out" properties of large anions (e.g., C13CCOO-, C10 ' I"). Their results 3 3 follow the same general trend of the effect of anions on the solubilities and activity coefficients of bases and nucleosides as reported by Robinson and Grant87. 0. Solution Self-Structuring In 1972 Miles and Frazier88 reported IR evidence for a regular, ordered structure formation of Na25'GMP which is not associated with gel formation in neutral aqueous solu- tion. The melting transition of the solution structure is more cooperative than the gel melting transition at pH=5, 15 but the solution structure has a lower melting tempera- ture. Based on IR arguments they postulated that the structure was stacked planar tetramers. Pinnavaia gt al.89 have shown by 1H NMR that the solution self-structure, which is slow to exchange with unstructured nucleotide on the NMR time scale, can be monitored by the appearance of multiple H(8) resonances. The binding constant for com- plexation of alkali metal ions by phosphate groups in poly- phosphatesgo, adenine nucleotidesgl’92 and DNA93 are known to decrease slightly with increasing metal ion radius. In contrast to this electrostatically determined order, the qualitative stability order for self-assembled 5'GMP salts + is K+ > Na , Rb+ > Cs+, Li+, the same as for gel formationgu The 1H NMR spectra of the limiting H(8) lines of the alkali metal salts Of 5'GMP in D20 solutionazmeshown in Figure 5. A size-selective metal complexation mechanism has been A postulated to account for the alkali metal ion dependence9 This order has been observed when a size-selective mechan- 95' 9 ism is Operative although it is unprecedented in other nucleotide systems. The 0(6) positions of the planar tetramer units define the coordination sites in the Na+ self-structure and the 0(6) positions of two stacked tetra- + + mers define an eight-coordinate site for K or Rb . As shown in Figure 6, the cavity of the planar tetramer unit 09A has a diameter of approximately 2.2 A , a value close to 96 that observed for Na-O bonds When two tetramers are 16 (thMP 032! " A 1 l 1 l 1 8.55 0J4 7.88 7.54 7.25 8. mm Figure 5. Limiting H(8) resonance lines of the alkali metal salts of 5'GMP in D O at high concentration and low temperatures. Taken from Reference 9A. Figure 6. 17 Schematic diagram of the proposed size-selec- tive coordination sites in the structuring of guanylic acids. a) coordination in the enter of the tetrameric plate by Na+ (r = 0,99 ). b) Coordinat on between the plates by K+ and Rb+ (r s 1.51 and 1.60A, respectively). Taken from Reference 97. l8 stacked, a central cavity defined by the eight oxygens is formed. The radius of this cavity, approximately 2.8 A assuming a 3.A A spacing between platesgu, can accommodate a K+ or Rb+ ion. The center of a tetramer unit is too large to coordinate a Li+ and too small to coordinate Cs+ properly. The interplatelet distance in an octamer can be increased to accommodate Cs+ (AI6 A)9u, but this reduces the favorable base overlap distance, 3.A A, between plates. The multiple H(8) environments for Na25'GMP have been 97. This model study was 1 based on the results of Zimmerman et al.8* from fiber dif— explained by symmetry arguments fraction studies of Na25'GMP. The plates were assumed to have a twist angle of 30° with respect to each other and the ribose assumed to have an anti configuration and C(3') endo conformation. Due to the directionality of the hydro- gen bonds, stacking can occur two ways in an octamer. Either the direction of hydrogen bonding can be the same in the two tetramers (referred to as head—to-tail or normal stacking) or it can be in Opposite directions (referred to- as head-to-head or reversed stacking). This is shown in Figure 7. The head of an arrow signifies the hydrogen bond donors; the tail the hydrogen bond acceptors, and R the ribose phosphate. The results of this study are shown in Table 1 and Table 2. Models for the solution self-structure of 5'GMP have A9 been proposed by Brown Those for Na25'GMP and K25'GMP Reversed Schematic diagram of a) normal stacking mode and b) the two enantiomeric reversed stacking modes of the octamers of 5'GMP. Taken from Reference 97. Figure 7. “Auoeocosv uosouuou nae: ooosuo>u menu. .ooou.3u.xuo.vo .ooo+.:uo.so.va .obn:.:u.3o.vo A “mocAA .m.= ++ v H »+ v H. ++ v .coAuouamom xuoun amok A “awesomn .ooAu.zu.3uo. a AOOn+.3UU.3U. a AOOM+.3U.3U. U . .ucAunAau amok .ooel.3o.3uovvo 20 ++ v .oom-.3o.3oov a .ooo+.:oo.:o.vo .oomu.:u.:u.vo »+ v ++ v .oom+.3uo.:u. a .oon+.3o.su. o .coAuouomon xooum zoAm v "mocwd .m.= ~ umOCAA .m.= N «mocAA Amy: .0 we coAuonAuosouA n. “nuoeomn . N «muoEOmA A .«uuoEOmH 80A: ocAunA3u anon . . v .ooou so zpuvva ..00n1.3u.3uo.vo v Aooo+.zuu.3o.vn .oo~:.3u.3ovvu . , AOOm+.ZUU.zov a , .00n+.30.30. U .coAuaquon xuoum 30Am .voquuoum >AAasvo a ”moqu .m.= v ”nosed .m.: c .mocAA,.o.= con- n o one oon+ n o o "mHOEOmA . v «muoEOmH N «nuOEOmA :UAz ocAumAzu 30Am Aouoa uchooum vomuo>om. chxuoum Aosuoz ,ucoAuAccou .nm mocmhmmom 50pm :mxme .mcofimmm owcmcoxm tonne pom pouooaxm mocfiq Amvz paw mamEomH ho Lopezz .A oAnma 21 cocoa 3.5.25 .32. 8:9.an me voA o8 oou .oootzooigco meson ..m.o.x.~.=o noon oocoug.m.o . one oAmA com com .oom+szoo.zo.vo mdAxomum cmmuo>oz . ~.o q meson do.A~.=z ucoem opcolx.m.o ~.o coo voH coo bow .Aooc-.=o.zoo. o ~.o apron do.x~.:z groan coco-..nco ~.o one oAaa ooo oo~ .oon-.3o.=oo.vo wfiwxuoum nomum>mm . v.o , . q meson no.x.~.=o noon oped-..mvo o.o one voA com com 1), termed "simple" structures and the other at low ratios of 5'GMP/M+ (<1, >.5), termed "complex" structures. For these structures the metal ion was coordinated to 8 carbonyl oxygens between the tetramer units, the simple structure being an octamer and the complex structure being a hexa- decamer. Marshall97 has determined the structured form of N825'GMP to contain eight nucleotide unitsand four Na+ ions. Thus he modified the model by having only two Na+ ions chelated by interplatelet phosphate oxygens. He also postulates a water molecule in the octameric hole formed A by the two plates to separate the two Na+ ions near the centers of the tetramers. Fisk gt gt.98 have measured the Tl's for the 13C atoms in Na25'GMP and have concluded that the most stable species formed in the self-structure is an octamer, formed by the association of two tetrameric plates. They postulate the existence of at least three other structural species present in solution with octamer, 23 Ls /-—l'i Figure 8. A IVOI No" No" L—[ tetramer [—1 J”-I No“ C Li 1__/ K+' . K+ ’l-J F K+ Ir—q\ \\-—L AJ——J/ K+ re *_-\ Brown'sug proposed models for 5'GMP solution self-structure. a) The Na+ self-structure; b) the "simple" K+ self-structure and c) the "complex" K self-structure. (Taken from Reference A9). 2A probably representing less-stable, stacked complexes. 99-106 Laszlo gt gt. have proposed several models for the Na25'GMP and K25'GMP self-structure. Their first model,based on 23 Na NMR,is a micelle type structure. The structure contained between 12 and 20 monomer units. They then proposed the coordination of Na+ and K+ into the central cavity of the tetramer plate. Based on 1H and 31P NMR evidence they proposed the stacking Of tetramer units as shown in Figure 9. In this staircase model, which is stated to represent the two outer H(8) lines, the metal ion is coordinated by the four oxygens in the tetramer unit and a phosphate group from an adjacent tetramer. The metal ion is located in the center of the tetramer unit. They indi— cate that two of the adjacent phosphates of a tetramer unit point in the Opposite direction of the other two phos- phates. Rapid exchange occurs as shown in Figure 9. They suggest that the non-equivalent H(8) resonances result from different degrees of stacking of this structure. In the same paper they also propose a stacked tetramer. structure where Na+ is found either at the center of two stacked tetramers or in the center of each tetramer unit. In a later paper Laszlo explains the more complex K+ structure by changing the ribose conformation so the number of phosphates pointing "up" and "down" in the octamer staircase unit differ. Brown”9 has determined that the tetramethylammonium 25 Figure 9. Structure prOposed by Laszlogg'106 for the solution self-structuring of Na25'GMP. Open circles represent phosphate groups pointing down, while closed circles represent phosphate groups pointing up. Na+ ions are represented by striped circles, A is the staircase model proposed by Laszlo. B is an alternate model proposed by Laszlo. 26 ion (TMA+) acts as neither a structure directing nor struc- ture inhibiting ion. This has facilitated the study of 5'GMP self-structure at varying ratios of metal to nucleo- tide. She has also found that K+ is the better stabilizing ion and that Na+ is the better structure director in the self-structuring of 5'GMP. This is based on the observa- tion that the addition of K+ to high 5'GMP/Na+ ratios results in the stabilization of the Na+ self-structure and that the addition of Na+ to high ratios of 5'GMP/K+ results in the "simple" K+ self-structure being replaced by a Na+ type of self-structure. 0n the other hand, the addition of K+ to Na25'GMP Causes a decrease in the extent of Na25'GMP self-structure and the formation of the K25'GMP self-structure. 9 Laszlo has investigated the ‘3Na NMR of Na 2'GMP up to 2 0.A5M and found no evidence for solution self-structuring. Brown)49 has reported a brief investigation of 2'GMP, 3'- GMP, 2'3'cGMP, 3'5'CGMP, and 5'dGMP. She found 1H NMR evidence for the self-structuring of 2'GMP; the Na+ salt formed only a very limited structure (<5%), whereas the K+ salt structured to approximately 70% at high concentration and low temperature. 27 III. Ethidium Binding to Nucleic Acids A. General Intercalation The determination of the structures of nucleic acids has been aided in recent years by the use of intercalation drugslo7. With an intercalation drug a portion of the drug molecule, usually an aromatic ring, is inserted between bases of nucleotides. The intercalation mode of binding was first proposed by Lerman108 to explain the binding of the aminoacridines to DNA. There is a wide range of com- pounds in which only a portion of the molecule may inter- calate. The intercalation of aromatic amino acids is probably one of the modes involved in protein-nucleic acid recognitionlog. ‘Purine and pyrimidine bases interact in living things not only with each other but also with pro- teins. They bind to the enzymes of nucleic acid and nucleo- tide metabolism and they serve as a means by which proteins can hold on to many metabolic intermediates and coenzymes. B. Ethidium Cation General Information Ethidium bromide (EtdBr; Figure 10) is biologically active, possessing trypanocidalllo, antivirallll, and anti- bacteriallll vivoll2; and £2 vitro, EtdBr interferes with DNA dependent 113,11A properties. EtdBr inhibits DNA synthesis lg DNA and RNA polymerases of Escherichia Coli EtdBr- DNA complexes exhibit several properties which are 28 IFigure 10. A. The structure of ethidium. B. A schematic diagram showing the intercalation of ethidium in U-A. Taken from Ref. 122. .s. us» 29 indicative Of intercalation. These include a diminished sedimentation coefficientllB, enhanced intrinsic vis- 115 116,117 cosity , and a greatly augmented fluorescence Recently, interest in EtdBr has centered on its special ability to unwind closed circular DNA118'121, Ethidium binding to DNA, RNA, oligonucleotides and di- nucleotides has been studied58-61’122'127, Proton NMR has proven to be a useful tool in studying the interaction of ethidium with short nucleic acid polymers. Krugh gt gl.59’60 and Chan and coworkers128 have used dinucleoside monophos- phates as a model for intercalation of EtdBr into DNA. When the drug is intercalated, the aromatic protons shift up- field due to ring current effects. The side chain methyl group resonance lies upfield of all other resonances and its change in chemical shift as a function Of nucleotide concentration provided stoichiometric information. In all cases it was found that one EtdBr intercalates between two stacked H-bonded base pairs. A preference for binding pyrimidine-purine sequence isomers especially CpG has been determined. To date, no studies have been conducted on the mononucleotides. Studies on the binding to mononucleo- tides may provide information on the mode of binding and sequence preferences in DNA. Marshall97 has investigated the interaction of ethidium cation with the structured forms of Na25'GMP and K25'GMP97. His models are shown in Figure 11- The binding constants of ethidium cation to structured 5'GMP were found to be >103. 30 A 5, GDP r K. r N n... \oN 2 S =2 u. w \ ,4 V , "a I g... 2:! w“ ::::w;::: w r ’ V ' \ AW \w+&4 V.‘—K? ‘- 3;. ‘r ' fig? M’ .fi "hf B HS __.;1_, (22:) F——— 5 ‘/ " ‘ r——I a I Mud \ "' I- : V ' ’m Figure 11. Schematic diagram of binding of ethidium to structured forms of 5'GMP. Ethidium is repre- sented byCDand the guanylic acid tetramer by .EZJ. A is Marshall's (97) model for ethidium binding to the NagS'GMP self-structure. B is Marshall's (97) model for ethidium binding to the K25'GMP self-structure. 31 IV. Amino Exchapgg in Mononucleotides Hydrogen bonding between the monomers of nucleic acids 129-137 Al_ has been the object of numerous investigations though interactions occurring in aqueous solvents are more applicable to biological syStems, little work has been done on hydrogen bonding of nucleotide bases in water until recently. Proton NMR spectrOSCOpy is ideally suited for differentiation between vertical ring stacking and hydrogen bonding. In most cases, upfield shifts of the ring protons are observed upon stacking of aromatic bases, whereas hydrogen bonding results in downfield shifts for the par- ticipating protons. Analysis of 1H NMR linewidths of the amino proton resonances can give detailed data on the rates of proton exchange between the amino groups and the 129,138,139 solvent Spectra taken in lH2O permit the study of slow exchanging NH, NH2, and OH groups. McConnell and Seawell133 have measured the intrinsic hydrogen-exchange of purine amino protons to determine a mechanism for amino exchange in DNA, specifically the nature and position of the structure motility in the se- quence of events that initiate exchange. A space-filling model of the double helix shows one proton of each of the amino pairs is sterically accessible to solvent for exchange in the helical grooves and free from participation in intra- molecular hydrogen bonding. In contrast, the second amino proton and the imino protons of guanine and thymine are 32 much less accessible to solvent molecules because of their location and involvement in hydrogen bonding. Information on the amount of macromolecular structure, Obtained by counting slow-exchanging non-hydrogen bonded amino hydrogens, and the "motility" of the DNA structure derived from exchange rates can be obtained if a quantita- tive knowledge of the hydrogen exchange rates is known in the absence of structure. The latter exchange rates are not well known for nucleic acids. The first-order rates for amino exchange of the purine nucleotides were determined to be several orders of magnitude slower than rates for amino groups of the aliphatic and aromatic compounds, whose rate constants are diffusion controlled. These low intrinsic rates account for the slow exchange of the non-hydrogen bonded amino protons of the helical grooves of DNA. A second observation which explains the distribution of dif- ferent kinetic classes of exchangeable interbase protons in DNA was that the pH dependence is not the same for amino proton exchange of adenine and guanine. It is suggested that proton transfer involving the outside amino protons of DNA base nitrogens might precede structural events such as strand separation. 130 points out that the In another paper McConnell important mechanism of amino exchange at physiological pH appears to involve the ability of the conjugated purine structure to communicate the effects of ring protonation as a regulatory aspect of proton exchange at the exocyclic 33 -NH2. A two-step exchange, in which transfer of the —NH2 proton to solvent water is preceded by protonation of the basic N-l proton of the purine, accounts for properties of hydrogen exchange in A-U double helical structures and polymeric G_ClA0,1Al. This exchange mechanism would provide a means for communication from solvent to the helix interior with consequent effects on Watson-Crick hydrogen—bonding. 136’137 have used the saturation Iwahaski and Kyogoku transfer method of 1H NMR to elucidate the sites and rates of exchangeable nuclei in nonaqueous solvents. An exchange takes place between the imino proton of l-cyclohexyl—S- bromo-uracil (BrU) and the amino proton of 9—ethy1adenine (9EA), and between the amino proton of l-methylcytosine (lMC) and the imino proton of 9-ethylguanine (9E0), while the amino proton of 9EG does not exchange at all with the other protons. Experiments at low temperature Show that, of the two amino protons of cystosine only the proton directly participating in the hydrogen bond exchanges with the imino proton of 9E0 (gt. Figure 12). Activation ener-5 gies of the proton exchange between some l-cyclohexyluracil derivatives and 9EA in chloroform were reported. They vary from 7 to 16 kcal/mole. Although the experiments were carried out on the mono— meric systems, there is no reason that the proton exchange cannot occur in the interior of nucleic acids. The ex- change is possibly due to the presence of the keto-enol tautomerism. 3A Figure 12. Exchangeable protons in the G-C pair. The circled protons exchange with each other. Taken from Reference 122. 35 V. Statement of Research-Aims The phosphate group in Brown's modelug for 5'GMP self- association plays an important role in stabilizing the solu- tion self-structure. The interplatelet phOSphate chelation of Na+ is a major factor in her model in the stability of the structure. Perturbations in the placement of the phos- phate groups should cause an effect on the structure, which may lead to a better understanding of the structure. There- fore the alkali metal dependence of the solution structur- ing of guanosine, 2'GMP, 3'GMP, and 5'GDP were investigated. Mixed nucleotide experiments were carried out to determine if one nucleotide could be incorporated into the structure of another. An ordering of the structure—forming tendencies of the guanylic acid derivatives was to be determined. In preliminary work on 2' MP, a novel proton exchange was noted for unstructured nucleotide. The exchange is between 2-NH2 and solvent water. The most interesting aspect of this exchange is that the integral intensity of NH2 varies from ~1 to 2 relative to H(8) in Na22'GMP, but is always 2 in TMA22'GMP. The conditions where the integral intensity is <2 are consistent with an ordered form, per- haps tetramers, of nucleotide being present. A detailed analysis of the exchange reaction may lead to the determina- tion of thermodynamic parameters for the equilibrium monomers =3 tetramers . A9 The discovery by Brown that TMA+ acts as neither a 36 structure directing nor inhibiting ion has facilitated the study of 5'GMP self-assembly at varying ratios of metal to nucleotide and has allowed the determination of the stoichiometry of the nucleotideixlthe Na25'GMP self-struc- ture. It was found that TMA+ interferes with structure formation in 2'GMP and 3'GMP. Investigations were carried out to determine the mode of this interaction. Marshall97 found that the drug, Ethidium Bromide formed a 1:1 and a 2:1 complex with structured 5'GMP. The binding of EtdBr to unstructured and structured 2'GMP and 5'GMP was investigated. Proton NMR Spectra of nucleotide solu- tions in lH20 were studied to determine if structural in- formation could be obtained from the resonances and in- tensities of the exchangeable protons. EXPERIMENTAL I. Materials, Technicues,_and Preparations Guanosine monohydrate and disodium 5'-guanosine mono- phosphate were purchased from Calbiochem, Inc. Disodium 2'guanosine monophosphate was purchased from P and L Laboratories or Sigma Chemical Co. All other nucleotides were purchased from Sigma Chemical CO. Inosine 5'mono- phosphate was obtained as the free acid, while 5'guano- sine diphosphate and 3'-guanosine phosphate were obtained as the disodium salts. The nucleoside and nucleotides were used without further purification. The desired metal ion forms of free acid nucleotides were obtained by titrating dilute solutions (”0.01M) with the appropriate metal hy- droxide to pH=7.8. Metal ion exchange of the other nucleo- tides was accompliShed using Dowex 50W-X8 (100-200 mesh) cation exchange resin obtained from the Dow Chemical Co. The resin had a cation exchange capacity of 1.7 meq/ml. The resin was prepared by the following washing sequence: (1) 10% HCl, (2) H20 and EDTA, (3) 10% NaOH, (A) H20, (5) 1:1 H2O-CH OH, (6) CH3OH, (7) 1:1 CH3OH-CH2C12, (8) 3 CH2C12, (9) 1:1 CHBOH-CH2C12, (10) CH OH, (11) 1:1 H O- 3 2 CH3OH, (12) 10% HCl, (13) H2O and EDTA, (1A) 1N desired metal hydroxide, and (15) washed to neutrality with H20. 37 38 A dilute solution (<0.1M) of the nucleotide was passed through two columns each containing a twenty-fold excess of resin to ensure complete exchange. The flow rate was approximately 20 ml/hr. The exchanged nucleotide was then lyophilized to dryness. All of the water used in these studies was purified by passing distilled water through an Illinois Water Treatment 00. purification system to remove paramagnetic ion impurities and organic residues. The metal hydroxides were used without further purifica- tion. NaOH and KOH were purchased from Mallinckrodt, RbOH and CsOH from Apache Chemicals, Inc., tetramethylammonium hydroxide pentahydrate and tetrabutylammonium hydroxide (25% in methanol) from Matheson, Coleman and Bell, tetra- ethylammonium hydroxide (20% in water) from Aldrich Chemi- cal Co. and, LiOH and TlOH'H2O from Alfa-Ventron. The metal chlorides were also used without further purification. LiCl, NaCl, and KCl were purchased from Mallinckrodt. RbCl and CsCl were ultrapure quality from Alfa-Ventron. CaCl2 was obtained from Matheson, Coleman and Bell. Tetramethyl; ammonium chloride and tetrabutylammonium iodide was pur- chased from Aldrich Chemical Co. Tetraethylammonium per- chlorate, purchased from Eastman Chemicals, was recrystal- lized twice from water. SrCl2 was purchased from J.T. Baker Chemical Co. TlNO3 was obtained by titrating T10H-H20 with HNO3 to pH=7 and drying. Metal salts were dried by heating and storing in a desiccator. Ethidium 39 bromide, purchased from Sigma Chemical Co., was lyophilized twice from 99.5% D20 to remove ethanol and to replace water of crystallization with D O. 2 NMR samples in D20 were prepared from the nucleoside or desired metal nucleotide lyophilized twice from a 100 f01d weight excess Of 99.5% D20 purchased from Norell Co. or 99.75% D20 obtained from KOR IsOtopes.' The nucleoside was also studied in d-6 DMSO, 99% D, purchased from Strohler Isotope Chemicals, and dried over A A molecular seives. Unless otherwise noted 8 TM sodium or tetramethylammonium 2’2’3’3’-dA-3 trimethylsilylpropionate (TSP), purchased from Dohme Canada Limited, was used as an internal reference. Cation exchange of TSP was accomplished by the same method as the nucleotides. An external reference and lock were used to obtain NMR spectra in lH20. The nucleotide dis- solved in 1H20 was placed in a 5 mm tube. A capillary NMR tube containing 8 EM NaTSP used as an external reference, D20 used as lock, and 1.0M NaCl to prevent freezing at low temperatures was placed in the 5 mm NMR tube containing the sample. To complex paramagnetic impurities 2 EM ethylenediaminetetraacetic acid (EDTA) purchased from Mallinckrodt as the free acid was included in the samples. Some samples were recorded without the addition of EDTA. These samples exhibited the same linewidths and chemical shifts as those containing EDTA. All solutions used for the NMR studies were passed through a 0.65 pm millipore A0 filter obtained from Millipore Corp. Samples were run in 5 mm NMR tubes of good quality. The concentration of the solutions was determined by ultraviolet absorption spectros- copy. The pH of the solutions was determined by an Ingold Electrodes Inc. micro-combination electrode (Wilmad Glass Co.) and a Fisher model 370 pH meter. The p0 of a solution was taken as pH + 0.Alu2. NMR samples used for T1 measure- ments were degassed by bubbling N2 gas through the sample. This was not critical since Tl's were always <<60 see.1“3, Sample height was 10-12 mm to limit diffusion out of the receiver coils. The receiver coils were 10 mm in length. A teflon vortex plug was used to prevent vortexing. Inte- grations were accomplished with a K & E Planimeter. Base- line corrections were accomplished by the 5 parameter base- line correction routine of DISNMR. Zero filling of NMR spectra was used when possible. In the zero filling tech- nique zeros are added to the end of a fully decayed free induction decay (FID). 0n transforming the spectrum, the point to point resolution is increased. This technique allows samples with short Tl's to be accumulated in a shorter time period without loss of point to point resolu- tion. A1 II. Instrumentation A.- Nuclear Magnetic Resonance Spectroscopy (NMR) NMR spectra were obtained on either a Bruker WH—180 interfaced to a Nicolet 1180 computer with 16K of memory or a Bruker WM-250 interfaced to an Aspect 2000 computer with 32K of memory. Both were operated in the Fourier Transform mode. Probe temperature was maintained to i1°C by a Bruker temperature control unit coupled to a probe-mounted thermo- couple. The unit was calibrated by measuring the chemical shift differences between the methanol proton resonances AA and applying the Van Geet equationsl . Nuclear Overhauser Effect (NOE) and T1 experiments were performed on the Bruker WM-250 using a 5 mm lH probe. Calculation of Tl's was ac- complished by the T1 calculation routine of DISNMR version 801015, Bruker Instruments, Inc. (1981). The pulse se- quences used for NOE and T1 data collection are given in Appendix 1. Chemical shifts are accurate to i0.005 ppm. B. Ultraviolet/Visible Absorption §pectroscopyg(UV/Vis) Concentrations of all nucleoside, nucleotide and EtdBr solutions were determined by UV/Vis absorption by dilution of aliquots either on a Beckman DU spectrometer updated with a Gilford photometer 252 or a Cary 219. .Solution concentra- tions were determined using Beer's Law. The molar absorp- tivity (e) at the absorption maximum are listed in Table 3 A2 Table 3. Molar Absorptivities Used to Determine Concentra— tionlus. Compound (M.l cm-l) xmax(nm) 5'GMP 13,700 252 2'GMP 13,300 253 3'GMP 13,300 253 Guanosine 1A,598 253 5'GDP 13,700 253 5'IMP 12,700 2A9 Ethidium 5,A50 A80 for the guanylic acids and EtdBr. It was assumed that the molar absorptivity was independent of metal ion and that the absorbances were additive for mixed solutions. C. Infrared Spectroscopy_(IR) IR spectra were obtained on a Perkin-Elmer 283B infrared spectrophotometer. Matched and sealed CaF2 cells of approximately 15 um pathlength were used. The cells were modified to be contained in a water jacket to enable temperature control. Water jacket temperature was controlled by a Forma Scientific model 2095 bath and circulator. The temperature of the solution was determined using a copper- constantan thermocouple mounted on the cell window. “3 D. Computation i. KINFIT - Non-linear least square fitting was accomplished on a Control Data Corporation, Cyber 170 model 750 computer using the KINFIT program developed by Dyelu6. 1A7 This method uses Wentworth's minimization procedure. Equations used for individual programs are given in Ap- pendix 1. ii. LORENT - Amino exchange rates were esti— mated by LORENT (T. V. Atkinson, Department of Chemistry, Michigan State University) a Fortran program running under RSXllM on a PDP ll/AO. This program calculates theoreti- cal NMR line shapes for the case of general two site ex- change. The equations used are those given by Rogers and WoodbreyluB. iii. R§§gt - Determination of line width in NMR spectra was accomplished with DISNMR (Bruker Instruments, . version, 1981) on the Aspect 2000 for single non-overlapping resonances, and with RESOL (T. V. Atkinson, Department of Chemistry, Michigan State University), a Fortran program running under RSXllM on a PDP ll/AO for overlapping reson- ances. RESOL is a non-linear least squares fitting program which attempts to find the set of a given number of Lorentzian lines which best fits the experimental spectrum. This program uses LMSTRllEO, a subroutine for non—linear AA least squares. The digitized spectra were transferred from the ASPECT 2000 to the PDPll via a multiple step process. The first step involved transfer over an asychronous serial link with the program BEAM (Bruker Instruments, version, 1980) handling the ASPECT end of the link and the program CDC running under RTll handling the PDPll end. The transfer from RTll to RSXllM was accomplished using the utility FILEX. III. Synthesis A. Selective H(8) Deuteration Selective H(8) deuteration of a guanylic acid was accomplished by refluxing a dilute solution of nucleotide (<0.01M) in D20 for 1-2 hrs. and lyophilizing to dryness. If a residual H(8) resonance was observed in the 1H NMR spectra the procedure was repeatedlSO. B. 8-Bromoguanosine 8-Bromoguanosine was synthesized by the method of Holms and RobinslSI. Guanosine (0.5 g) was suspended in 50 ml H20 and saturated bromine water was added dropwise with vigorous stirring. The solution was allowed to become white before adding another drop. When the solution re- mained light yellow no more bromine was added and the A5 solution was allowed to stir 30 minutes. The solution was rotovaporated to 5 m1, filtered cold,-and washed with cold water. The precipitate was recrystallized from water twice. Purity was determined by the loss of H(8) intensity in the NMR spectrum. RESULTS I-Quaaeaina A. The Solution Self-Structure of Guanosine Nuclear magnetic resonance spectroscopy (NMR) is an excellent method for the study of the self-association of guanylic acids. The most compelling evidence for ordered structure formation is the presence of multiple inequivalent H(8) resonances. The resonance in non-structured solutions is a singlet positioned downfield from the other resonances, thus making it facile to monitor. Solution self-structure manifests itself in the formation of new H(8) resonances, showing that the formation of structure is slow on the NMR time scale. Broadening is due to processes fast on the NMR time scale, such as base stacking and possibly tetra- mer formation. Ordered structure in this work will refer to species which are slow to exchange on the NMR time scale and/or show evidence for structure formation in the IR absorption region. Monomeric or "disordered" nucleotide refers to monomeric nucleotide and all weakly aggregated species which are in rapid (NMR time frame) equilibrium with it and exhibit little or no IR frequency perturbation. To access the role of the phOSphate group in the A6 A7 solution self-structuring Of guanylic acids, guanosine aggregation was investigated by 1H NMR. The addition of NaI, KI, SrC12, and TlNO3 to a saturated guanosine_so1ution (0.02 M) at 0°C does not affect the H(8) resonance, neither in chemical shift nor half—width, up to the point of pre- cipitation or gelation. At the gel or precipitation point, all resonances are understandably broadened due to the in- creased viscosity of the medium. To increase the concentra- tion of nucleoside in solution, DMSO-d6 was used as the solvent. The results were similar except that the nucleo- side always precipitated from solution instead of gelling. The addition of small amounts of water (1%) to saturated salt solutions of guanosine in DMSO resulted in instant gelation of the solution. Thus, water is important in guanylic self-structuring. Guschlbauer71 has reported that water is the best solvent for gel formation. Methanol and ethanol can be used but the stability of the gel is greatly reduced. No evidence for solution structuring was found in the 1H NMR of mixed DMSO-d6/D2O salt solutions of- guanosine. To determine the potential importance of the glycosi- dic bond torsion angle in guanosine gelation, 8-bromoguano- sine was investigated. Guanosine is preferentially in the anti conformation68’79, while 8-bromoguanosine is in the syn 68,79 conformation Since H(8) is absent in 8-bromoguanosine, the H(l') region of the NMR spectrum was monitored. The A8 results were the same as with guanosine. The spectra of salt solutions of concentrated 8—bromoguanosine in D20, DMSO-d6/D20 mixtures and DMSO-d6 showed no evidence for solution self-structuring. B. Incorporation of Guanosine into the Na25'GMP Solu- tion Self-Structure Although guanosine does not form an NMR-observable ordered structure, selective deuteration experiments were carried out to determine if guanosine could incorporate into the Na25'GMP structure. Two experiments were per- formed: (1) guanosine was added to an 8-d Na25'GMP (8- deutero-disodium guanosine-5'-monophosphate) solution and (2) 8-d guanosine was added to an Na25'GMP solution. These procedures permit the examination of the H(8) resonance of each guanylic acid individually, because the deuterated nucleotides are not NMR observable. Figure 13 shows the results of the addition of 8-d guanosine to a structured 0.32M Na25'GMP solution. There is a relative intensity loss of the a and 6 resonances and an upfield shift of the B resonance152 (gt. Figure 13), all indicative of a decrease in the amount of structure. The H(l') resonance is sharpened, along with a decrease in the small upfield structure H(l') resonance. There is a linear decrease in structure with addition of 8-d guano- Sine (gt. Table A). The percent structure present in A9 ' r r I [NOZS’GMPI =~ 032M rem p..= 3.5°C 8—d 600 0. 14M O-HM L I 1 8. 5.3 8. 13 7.22 6,me Figure 13. The effect on the H(8) region of the NMR spectrum of 0.32M'Na25'GMP at 3.5°C on the addition of 8-d guanosine. All solutions were homogeneous and not gels. 50 Table A. The Effect on the Solution Self-Structure of 0.32M Na25'GMP on Addition of 8—d Guanosine at 3.5°C. % Structure [8—d Guo] Added ' Absolutea Relativeb 0.1A2m 0.0 0.0 0.107m 10.1 ' 30.0 0.085M 17.8 52.0 0.071m 16.3 A8.0 0.0A7g 2A.3 72.0 0.02AM 26.5 78.0 0.003g 33.2 98.0 0.00M 33.9 100.0 aThe absolute percent structure was determined by integrat- ing the resonances and using the following equation: 3 (Kg-g)/(a+x+6 ). bThe relative percent structure is: [absolute % structure without 8-d guanosine added-absolute % structure with 8-d guanosine added/absolute % structure without 8-d guanosine added]. 51 solution was determined by assuming the 8 resonance was equal in intensity to both the d and 6 resonances. The equivalence of the a, B, and 6 resonances has been con- tinually observed under all conditions in all work done to date on the Na25'GMP self-structureu9’97. Solutions Of 0.32M Na25'GMP containing >0.0AM 8-d guanosine have a broad resonance (01/2 N125 Hz) in the H(8) region, which is not present in an equivalent Na25'GMP solution, as shown in Figure 1A. The broad resonance in- creases in intensity with increasing 8-d guanosine concen- tration. We assign this resonance (shaded resonance in Figure 1A) to a large slow tumbling structure that incor- porates 5'GMP and 8-d guanosine. The broad resonance accounts for 35% of the observable 5'GMP H(8) integral in- tensity in a 0.32M Na25'GMP solution which is 0.11M in 8-d guanosine at 3.5°C. In addition, a decrease in the integral intensity Of H(8) is observed as 8-d guanosine is added to a 0.32M Na25'GMP solution. This loss is attributed to a larger slow—tumbling structure that incorporates 5'GMP and 8-d guanosine, whose half-height is so broad that it is not observable. In a solution containing 0.11M 8-d guanosine and 0.32M Na25'GMP the loss of integral intensity of H(8) accounts for 30% of the total H(8) intensity. It is postulated that large, soluble aggregates are formed that contain 5'GMP and 8—d guanosine. These polymeric structures exist in various sizes. The smaller 52 0.32 M Nazs'GMP + 0.1153 8-d GUO Temp. =3.5°C 8.me Figure 1A. The H(8) NMR spectrum of a 0.32M Naofi'GMP and 0.11M 8-d guanosine solution at 3.5°C. Note the very broad resonance present (shaded). 53 polymeric units give rise to the observable, broad H(8) resonance. The resonance of the larger polymeric structures is broadened into the baseline and is therefore not ob- servable, causing a decrease in H(8) integral intensity. The expected complimentary broad H(8) resonance of guanosine was not observable when guanosine was added to a 0.32m 8-d Na25'GMP solution at 3°C. The guanosine H(8) resonance is assumed to be broadened into the baseline, due to the guanosine being present in large slow-tumbling struc- tures. The Na25'GMP structure was observable in the ribose region of the spectra. No attempt was made to determine the amount of Na25'GMP structure present, but the ribose regions of the Spectra were quantitatively similar to the correspond- ing 8-d guanosine/Na25'GMP solutions. The solubility of guanosine was greatly enhanced in the mixed system. Guanosine is soluble to 0.02M in H20 at 3°C, whereas in a 0.32M Na25'GMP solution its solubility is in- creased to greater than 0.1AM. Increasing the guanosine concentration beyond 0.1AM causes the solution to gel. Guanosine has a greater "structure—forming tendency" than 5'GMP. In all the mixed guanosine and 5'GMP solutions, guanosine was in large aggregates that were NMR unobserv- able. The samples with larger concentrations of guanosine (>0.01M) should have been gels. Gelation was most likely inhibited by phOSphate repulsion of 5'GMP units, which were incorporatedillthe guanosine self-structure. 5A The loss of 5'GMP solution self-structure cannot be attributed to the removal of Na+ of solution by incorpora— tion into the guanosine gel—structure. Assuming there is one Na+ per tetramer unit in the guanosine gel-structure, only a 5% decrease of Na+ concentration would occur in the most concentrated sample. This decrease in [Na+] concentra- tion could not account for the total loss of Na25'GMP self- structure that is found. 55 II. The Solution Self-Structuring of 2'GMP The alkali metal salts of 2'GMP are a better system for study than guanosine. They are more soluble in H20 and do not form gels at neutral pH, regardless of the nature of the alkali metal ion. Figure 15 shows the limiting H(8) resonance lines of the alkali.metal salts of 2'GMP in concentrated D20 solutions, except for the spectrum of 1.22M Na22'GMP which was taken in H2O, near the freezing point of the solvent. It is immediately clear that the Na+, K+, and Rb+ salts form slowly exchanging order struc- tures with multiple H(8) environments, whereas Li+ and Cs+ provide little or no evidence for structure formation. In dilute solution (m0.02M), all Of the salts exhibit a single sharp H(8) resonance at 7.98 ppm at 23°C charac- teristic of disordered 2'GMP. An increase in the nucleo- tide concentration or an increase in temperature causes a downfield shift of the H(8) resonance. Figure 16 and Figure 17 show the effect of temperature and concentra- tion, respectively, on the chemical shift of H(8) of various salts of 2'GMP. A. The L1+ Salt of 2'GMP As seen in Figure 15, a 0.98M Li22'GMP sample at -2°C shows no evidence of structure formation. The H(8) reson- ance is broadened but no new structure resonances are l.22 M Naza'oMP 274° K .68M N022.GMP + l.34MNoCl 274°K 98 MLiz 2'GMP 27PW< Figure 15. T V rT T fi' 1 ‘ d .1 .1 d d A q .64M CSzZ'GMP 27l° K .77 M Rb, 2'GMP 271- K .82 M K22'GMP ififl‘K The limiting H(8) resOnance lines of alkali metals salts of 2'GMP. Spectra were recorded in D 0 solutions, except for the spec rum of l .22 Na22'GMP which was recorded in H20. ___ ‘u I. I: 57 .mpsumhdemu msmpm> mzu.m mo muAmw msoflhm> mo uMAnm AmOAEmso Amvm one A 00 v wmahéwazwh .mA mhswam 0.00 goo 0.0.... 0.9” 0.0N 0.0 _. 0.0 Ofil - u d - d 1 u u - a q d - - d .- d u _ d a q u - 1 di- u. d d u u q _ H 1 Ill 8”.“ . glean: m as o .. . . swans .2. Se 0 . e azeawez a 8.0 o . - m r .. cook 3 . azoawee 2 Rs 4 . A W . genus. a «no 0 . - . m . axing z $5 0 . .. . w . . . . M I . . e 1 Sop d G I .u \ d .. I: 1.. u > . .11. \ n W . l l -\ . a . - n L. n — n b n n — n n n n — - b n n P n n - — — - b p n — F D h n — - A n - 8P.” 58 coflumnucwocoo wsmpm> mso_m no muAmm ocuovom no umfinm AmOAEcno vam 029 75.502 2. ZOFékzuono .00.” pm .2“ themes 0. p 0.0 0.0 5.0 0.0 0.0 To ”.0 N0 v.0 0.0 id! I d .- q u d —)d u 1— d 4 d - A u u — u a .1- di- q — o u A q d u a fi q u a 8h“ . axing: + - - azoammo o . .. sesame. o 1.82 m - sear. o - m - sesame: o - w . S .. genus a .. 82 m .. - u . . .N. - N m A - “a i I 1‘ 0. .ml.) in o 83. w .. . -.P . Lfi .1 .I L - - n — b - n — n n n — b P #1— P p P b n p n _ h h - — P P - — - bf - — - b P 8'.“ 59 evident. The width at half—height for the H(8) line at 2°C increases from 3.7 Hz at 0.05M to 16.0 Hz at 0.98M. No corrections were made for the change in viscosity. An increase in temperature also causes a narrowing of the H(8) resonance. Increasing the temperature of a 0.98M L122'GMP sample from -2°C (vl/2 = 16.0 Hz) to A0°C (v = 6.7 Hz) 1/2 causes a decrease of 9.3 Hz. The carbonyl stretching region of the infrared spec- trum of 0.98M Li22'GMP, Figure 18, does not show any fre- quency shifts or intensity changes over the temperature range 3°-50°C that could be attributed to formation of an ordered structure. A discussion of the expected changes in the infrared spectra on structure formation is pre- sented in the section on the Na+ self-structure of 2'GMP. B. The Na+ Salt of 2'GMP. Two new resonances appear in the H(8) region of a 0.70M Na22'GMP solution at 0°C, one downfield at 8.51 ppm and one upfield at 7.2A ppm of the monomer (7.9A ppm). In a 0.91M solution at 2°C, as shown in Figure 19, the structure resonances account for less than 5% of the total nucleotide concentration. This spectrum is indicative of what will be referred to as the simple structure. As the concentration of Na22'GMP or Na+ is increased, the downfield resonance disappears and a new resonance appears at 7.99 ppm (gt. Figure 15). A 60 1001 00'! %T 70‘ 60" {- Iaoo 16:00 1400 ‘ v 1001 90‘ $7 70‘ ' qu- - 1800 I 600 I400 Figure 18. The infrared spectra of 0.98M L122'GMP in 020 at 50°C and 3°C. Neither thgse spectra nor the 1H NMR spectra of this solution shows evidence for structure formation at either temperature. Figure 19. 61 l L 8.5l 7.97 7.24 8. ppm The H(8) NMR lines of 0.91 M Na52'GMP in D20 at 2°C. The two weak lines at .51 ppm and 7.2A ppm are indicative of the simple Na22'GMP self-structure. . . 62 small shoulder is also present upfield of the monomer. The upfield structure H(8) resonance increases in inten- sity but at a slower rate than the one at 7.99 ppm. As shown in Figure 20, the addition of NaCl to a 0.69M Na2— 2'GMP solution at 1°C, which shows no evidence of struc- turing in absence of added Na+, causes the 7.22 ppm reson- ance, but not the downfield H(8) structure resonance to grow in. The structure line shifts upfield with increasing Na+ concentration. Computer simulation (gt Results IIH), of the spectra reveals the presence of a small broad reson- ance at 7.9A ppm. The structure resonance at 7.99 ppm appears and increases in intensity with increasing Na+ concentration. The Na22'GMP solution at the limiting condi- tion of solubility and low temperature is approximately 60% structured. This form of structure will be referred to as the extended structure. The extent of structure formation is considerably less than in the corresponding Na25'GMP system, which would be m80% structured under the same conditions. The number and chemical shifts of the structure resonances are similar to those of the Na25'- GMP structure, but the intensities are not equivalent. The Na25'GMP structure resonances always appear to be of equal intensity. Figure 21 shows the melting profile of the H(8) region of a 0.69M Na22'GMP solution 1.32M in NaCl. The downfield structure resonance decreases in intensity while shifting upfield and merging with the 9.6m Nozz’om 1°C L111: 1 1 l 7.99 7.91 7.17 tom» Figure 20.- The change in the NMR spectra Of H(8) upon addition of NaCl to a 0.69M Na22'GMP solution at 1°C. 6A I I r T I I If I I I I i I I I U I 1 1 V I I 0.69M No22'GMP H3216 NaCl 1°C 13°C 27°C 7°C 'OC 17°C 4 1 1 1 J 1 1 L 1 I l L A L 1 a 1 l a l l 9.00 8.00 7.00 9.00 8.00 7.00 8 l ppm 8' ppm Figure 21. The temperature dependence of the H(8) NMR resonances of a 0.69M Na22'GMP and 1.32% NaCl solution. 65 monomer resonance. The upfield structure creases in intensity and shifts from 7.17 7.21 ppm at 27°C. Figure 22 shows the spectrum of 1.22M lH20 at 1°C. protons are observable. Under these conditions, the The imide proton to 3 resonances, at ll.Al ppm, 10.98 ppm, resonance at 9.53 ppm. The total area of is less than one proton intensity per total 2'GMP. resonance de- ppm at 1°C to Na22'GMP in amine and imide N(1)H is assigned and a very broad the 3 resonances A non— structured solution at the same temperature does not have the imide proton resonance present, presumably due to rapid exchange with solvent. The imide proton in the ordered structure is presumably involved in intermolecular hydrogen bonding, NMR time scale. than that of the structure H(8) resonances. causing the exchange with solvent to be slow on the The integral intensity of NH(l) is greater The intensity of the resonances is discussed in detail in Results VI. The three lines labelled B are attributed to H(8). f The resonances at 7.75 ppm and 0.95 ppm are solution self— structure resonances. Monomer and structures which ex- change with monomer fast on the NMR time scale give rise to the resonance at 7.A8 ppm. is labelled C. resonance is approximately one. cussed in Results VIB. is a solution self-structure resonance of H(l'). The lines labelled D at The amino proton resonance The integral intensity of the amino proton This discrepancy is dis— 5.77 ppm The other .omz on m can A.Av: on moocmcomOA Q on» .m2090pa ocaew on» Op 0 .Amvm on moocmcomop m can .mAsz consuosnun on nousnAppum one moocmcommn < 0:? .EQQ oo.mA on and oo.m sons ooa no ommH ca nzo.mmoz mmm.A no esneooom mzz mA one .mm enemas Eda .0 9. of. ... cigar: 12.. 67 D resonance at 5.59 ppm is attributed to H(l') of monomeric and fast equilibria structures. The water proton reson- 1-1 ance is labeled n. Perturbations in the carbonyl region of the infrared spectrum similar to those found in the self-structuring of 5'GMP, occur upon ordered structuring of 2'GMP. Figure 23 shows the infrared spectra of disordered 0.30M Na22'GMP at 3°C and 52°C. These spectra are similar to those re- 88 ported by Miles and Frazier and Marshallg7 for disordered Na25'GMP. The absorptions of interest are the carbonyl stretching vibration at 1665 cm-1, the C(A) = C(5) stretch- ing Vibration at 1592 cm‘l, and the shoulder at 1570 cm-1 assigned to a normal mode which involves to a small extent motion of the carbonyl oxygen. The band assignments have 5A,83,88. been discussed extensively in the literature Figures 2A and 25 show the infrared spectra of ordered solutions of 0.95M Na 2'GMP and 0.71M Na22'GMP containing 2 1.1M NaCl. Both solutions exhibit H(8) lines for ordered 1 forms in the H NMR spectra; the 0.95M Na 2'GMP sample 2 having the simple structure (%5% total structure) and the 0.71M Na 2'GMP sample containing 1.1M NaCl the extended 2 structure (m60% total structure). At 2°C, both samples 1 have a new absorption occurring at 15A0 cm- and assigned to a C(8) = N(7) ring vibration and attributed to struc- ture formation. When the sample is heated to 52°C, the 1 15A0 cm- band decreases in intensity and becomes a small ’ IOOT 80' 60‘ %J 40« 204 1000 I 001 001- 60‘ $1 401- 1000 - Figure 23. 68 52°C 1400 3 1690 V cm ' The infrared spectra of 0.30M Na22'GMP in D20 at 3°C and 52°C. Neither of these spectra nor the 1H NMR spectra of these solutions shows evidence for structure formation at both tem- peratures. %T '57 Figure 2A. 1001' .20- 69 V ”l 60‘)‘ 40-17 ell- :- 1800 1400 cm" 1.23. V 100" 60‘- it V «p q)- «r 1300 logo 1400 v cm" The infrared spectracu‘0.95M,Na22'GMP in D20 at 2°C and 52°C. Both the H(8) NMR spectrum and the IR spectrum at 52°C show no evidence of structure formation; while at 2°C the H(8) NMR Spectrum is characteristic of a simple Na22'- GMP solution structure. The structure reson- ances account for <5% of the H(8) resonance. The IR spectrum at 2°C shows perturbations which have been attributed to solution self-structure. 70 1 1001 801- 601)- ‘lo'l’ \ 4o» m up D 1300 logo 14oo v 1001’ 80“ %T 2m. 2 C «4)- GP «r 1300 1690 I400 V cm" Figure 25. The infrared spectra of 0.71M Na22'GMP contain- ing 1.1M NaCl in D20 at 2°C and 52°C. Both the H( ) NMR spectrum and the spectrum of this sample at 52°C show no evidence for structure formation. At 2°C the H(8) NMR structure resonances account for ~50% of the total H(8) intensity. The IR spectrum at 2°C shows perterbation characteristic of structure formation. 71 shoulder on the broad overlapping bands at 158A cm-1 and 1565 cm-1. The intensity of the absorption at 1590 cm—1 is decreased upon structure formation, becoming less intense than the 1ine at 1669 cm-1. The absorbances do not seem to be correlated with the % structure present. The %T of the 15A0 cm.l absorbance is approximately the same in the 0.95M Na22'GMP solution at 2°C, which is %5% structured as determined by 1H NMR of the H(8) resonance, and in 0.71M Na 2'GMP containing 1.1M 2 NaCl at 2°C, which is m50% structured. + C. The K Salt of 2'GMP Figure 26 shows the H(8) NMR spectra of K22'GMP at -2°C as the concentration of K22'GMP is increased. A 0.10M K22'GMP solution at -2°C has a single H(8) resonance at 7.97 ppm with a width at half-height of A.3 Hz. When the concentration of K 2'GMP is increased to 0.50M, several 2 new resonances appear with chemical shifts of 8.29 ppm, 8.06 ppm, 7.93 ppm (monomer), 7.72 ppm, 7.51 ppm and 7.30 ppm. Increasing the concentration to 0.82M causes the ap- pearance of two new resonances, for a total of at least seven solution self-structure resonances. A 0.82M K22'- GMP solution at -2°C is m90% structured. The extent of ordered structure is much greater than in Na22'GMP, but is less than in a comparable K25'CMP solution. There is no 72 [K22’GMP] -2°c 0.50“.“ 0.29 0.60M 0.29 5,... Figure 26. The concentration dependence of the H(8) NMR spectrum of K22'GMP at -2°C. 73 correlation between the number and chemical shifts of the K22'GMP and K25'GMP structure resonances. In K25'GMP, all structure resonances occur upfield of the monomer, whereas in K22'GMP structure resonances occur both upfield and downfield of the monomer. The temperature dependence of the H(8) region of 0.82M K22'GMP is presented in Figure 27. The resonances due to ordered nucleotide do not decrease in intensity at the same rate. The resonances shift toward the monomer resonance while decreasing in intensity. At 50°C, only the monomer resonance is observed. Figures 28 and 29 Show the carbonyl stretching region 2'GMP and 0.82M K 2'GMP. The same perturbations 2 2 are present as in the ordered Na22'GMP system. In the of 0.5014 K 0.50M K22'GMP sample at 3°C (m20% ordered by NMR), the 1587 cm-1 absorption is less intense than the one at 1668 cm-1, and the absorption at 1536 cm-1 is present, both indicative of structure formation. At 51°C these perturba- tions are no longer present. These changes are even more pronounced in 0.82M K22'GMP at 2°C. At 50°C, the spectrum is indicative of a disordered system. The melting pro- 2'GMP by infrared and 1H NMR are compared 1 files of 0.82M K2 in Figure 30 using the change of absorbance at 1568 cm- and the percent structure determined by integration of the H(8) region of the 1H NMR spectra. Both methods show the melting temperature, Tm, defined as the temperature at 7A U'ffIIIFlT IIIIIIIIIT 15° 50° J o 5 30° -2° 25° 1 L L I 1 L L l l L l a L [1 n a l I 1 0.00 7.00 0.00 7.00 5 mm 3 mm Figure 27. The temperature dependence Of the H(8) NMR spectrum of 0.82M K22'GMP. %T %T Figure 28. 75 '°°"\ 30.. \ a... \ I 1 2 f 40" /: 20.. 51°C I. a A i 1800 1690 1400 V cm" 1007‘ 80A 60" 40" 20» 3%: : : a. = 1800 1690 I400 V em" The infrared spectra of 0.50M K22'GMP in D 0 at 51°C and 3°C. The H(8) NMR spectrum and IR spectrum at 51°C show no evidence for struc- ture formation; at 3°C the sample is m30% structured as determined by proton NMR. The IR spectrum show perturbations characteristic of structure formation. 100] 801 60‘ 701’ 20‘ 'F’ .- «r P 1800 1600 I400 1001 80" 60- '57 20‘ «$- 1 moo 1620 moo V cm" .0- ul— Figure 29. The infrared spectra of 0.82fl K22'GMP in D O at 50°C and 2°C. At 2°C this sample is ~88$ structured, determined by the H(8) NMR reson- ance. The IR spectrum shows perturbation characteristic of structure formation. There is no structure present at 50°C determined by proton NMR or by IR. 77 DETERMINATION OF THE MELTING TEMPERATURE BY IR AND NMR FOR 0.82M KZZ'GMP 1w‘o TIIITIT rerlI MO - :1 gm- -‘ a; can - ' ' '- can 3 r- ig coo - c» - can a ' . ' t“ 5&0 - . K - A 'I g; 400 - .A ' 'd 050 r ‘ Em- . ‘00- A o u r- : - I l 1 I LLLJ l I I ‘040 -400 00 100 200 300 400 500 .00 Figure 30.- TEMPERATURE °C Comparison of melting profiles of 0.82% K22'GMP in D20 by IR and 1H NMR. Lu” 899i .lV amuosav 78 which half of the structure remains, to be in the range of 25°-30°C. This is evidence that the same phenomenon is being observed in 1H NMR and infrared spectroscopy. The degree of cooperativity is defined by the temperature range over which the nucleotide goes from structured to unstruc- tured form. The smaller the temperature range, the greater the cooperativity in structure formation. This corresponds to a large positive value of AH upon solution "melting" of structure. The absorbance at wlS30 cm.l does not seem to be correlated with structure formation, although it is always present in structured solutions. Table 5 lists the temperature dependence for the absorbances of 0.82% K22'- GMP IR bands. The absorbance bands at 1590 cm.1 and 1568 cm"1 correlate to the amount of solution self-structure present in solutions. + D. The Rb Salt of 2'GMP The Rb+ salt of 2'GMP at low temperature and high con- centration has four structure resonances, two upfield and two downfield of the monomer over the range of 7.“ to 8.” ppm. Figure 31 shows the effect of concentration on the = 16 Hz) reson- 2 ance is observed in a 0.30% Rb22'GMP at -2°C. When the H(8) resonance of Rb22'GMP. A single (Vl/ concentration is increased to 0.39% Rb22'GMP, the reson- ance broadens to Vl/2 = 32.5 Hz. At 0.77% Rb22'GMP, four structure resonances are present with chemical shifts of 79 I l j I I t I T u Rb22'GMP N -2°c 7. 78 030$ 9.00 8.00 7.00 Figure 31. The concentration dependence of the H(8) NMR spectra of Rb22'GMP at -2°C. 80 Table 5. The Temperature Dependence for the Absorbances of Various 0.82% K22'GMP IR Bands. Abs. at Abs. at ' Abs. at Temp. (°C) 1590 cm-1 lSUO cm-1 1568 cm-1 2.5 0.74 0.37 0.U7 5 0 73 0.36 0 A9 9 0 67 0.35 0 A6 in 0.67 0.35 0.u9 19 0.81 0.33 0.59 2h 0.8M 0.3a 0.62 27 0.91 0.36 0.68 33 0.97 0.38 0.7m 38 1.00 0.32 0.80 an 1.0a 0.31 0.82 81 8.33 ppm, 8.13 ppm (shoulder of monomer), 7.78 ppm (mono- mer), and 7.Ul ppm. The monomer resonance is very broad (01/2 = 33.5 Hz) and is shifted upfield to 7.96 ppm. A 0.77% Rb22'GMP solution at 2°C is approximately U0% struc- tured. The temperature dependence of the H(8) resonance of 0.77% Rb 2'GMP is presented in Figure 32. The small upfield 2 structure resonance decreases in intensity as the tempera- ture is increased and is no longer present at 15°C. The other structure resonance decreases slightly in intensity and merges with the monomer resonance as the temperature is increased. This merging of the lines may be due in part to the structures exchanging in the intermediate range with monomer, while the concentration of the ordered form is decreasing as the temperature is increased. The monomer resonance remains broad even at 35°C (01/2 = 35 Hz). The infrared spectra for 0.30% Rb22'GMP and 0.77% Rb22'GMP are shown in Figures 33 and 34, respectively. 1H NMR data, the spectrum of this sample In agreement with at 3°C shows no evidence of structure formation. The 0.77% Rb 2'GMP spectra at 3°C has the same perturbations 2 as discussed before for structure formation. At 50°C, the spectrum is indicative of an unstructured system, in agreee ment with NMR data. 82 v V V—‘ r V T Y f I 0.771» twp 7 lft wt ‘ Idt 4%: A A A A. L L L L L l 4 g L A L A A A A 000 zoo too 200 5.19M 8.99!" Figure 32. The temperature dependence of the H(8) NMR spectrum of 0.77% Rb22'GMP. 7.7 Figure 33. 100? 83 804. 401 20w . 51%: db- 1 1800 ‘§2° 1400 v «IU- qr dL 1800 1600 1400 The infrared spectra of 0.30% Rb22'GMP in D20 at 51°C and 3°C. Neither these spectra nor the H(8) NMR resonance shows evidence of struc- ture formation at either temperature. '51 7.7 Figure 3H. 8H 10 01- 8°" 6°:- 200 q. 1800 100* 1 4m- 3%: 1 l ' 1:00 ":90 V cm" The infrared spectra at 50°C and 3°C. At resonance nor the IR structure formation, structure resonances total H(8) resonance. 1400 of 0.77%_Rb22'GMP in D20 50°C neither the H(8) spectrum shows evidence for while at 3°C the H(8) account for ~h0% of the The IR spectrum at 3°C shows perturbations characteristic of struc- ture formation. + E. The Cs Salt of 2'GMP The cesium salt of 2'GMP has only a slight tendency for self-association. A 0.6U% Cs22'GMP solution at -2°C has a small H(8) resonance at 7.55 ppm (cf. Figure 15). The structure resonance accounts for m3% of the total nucleo- tide. The H(8) resonance is broad even at high tempera- ture as seen in Figure 35. The infrared spectrum of 0.6M% Cs22'GMP is shown in Figure 36. No evidence for ordered structuring is present. The band at 1587 cm.1 is more intense than the one at 1665 cm.1 and no peak is present at 1536 cm-1. F. Tetramethylammonium Ion Destabilization of the 2'GMP Self-Structure The discovery by Brown“9 that the tetramethylammonium ion (TMA+) acts as neither a structure directing nor in- hibiting ion has facilitated the study of 5'GMP self- structure at varying ratios of metal to nucleotide. Ex- periments were conducted to determine if TMA+ is such a "non-interfering" ion for 2'GMP. Solutions of TMA22'GMP have a single sharp resonance at 8.0M ppm even at high concentration (>l.0%) and low temperature (1°C). The addition of NaCl(S) to 0.68% TMA22'GMP at 0°C causes a broadening of the H(8) resonance and only a small struc- ture resonance at 7.38 ppm at 2.2% NaCl (3:. Figure 37). 86 T I f r I 0.6414 C 522'GM P 40°C L L L l A 8.00 7.00 8,99m Figure 35. The temperature dependence of the H(8) NMR spectrum of 0.6”% 0522'GMP in D20. %T Figure 36. 87 1001 80‘? 60v 20v 51°C, 1 woo 1600 1400 7 3°C 0 20‘ l woo 1620 1400 V cm" qu- .7 up The infrared spectra of 0.6h% Cs 2'GMP in 020 at 51°C and 3°C. Neither the H(8) NMR spec- trum nor the IR spectrum at 51°C shows evidence of structure formation, while at 3°C the sample is ”5% structured by proton NMR. The IR spec- trum at 39C shows perturbations characteristic of structure formations. 88 [ I l I I I l I 0.68M TMA22'GMP +2.2M NaC1 0°C 1 I l 1 1 L 1 I 8.60 8.00 7.40 8,Ppm Figure 37. The H(8) region of a 0.68% TMA 2'GMP and 2.2% NaCl solution at 0°C. This so ution in ab- sence of TMA+ would be approximately 50% structured. 89 This solution in absence of TMA+ is approximately 50% structured. Figure 38 shows the results of a 0.71% Na22'GMP solu- tion made 1.1% in HaCl and 1.36% in TMAClefiz-300. The solution in absence of TMA+ is approximately 60% structured. The resonances in the TMA+-free system are very broad. The structure resonance at 7.99 ppm and the monomer at 7.92 ppm are of the same intensity and overlap making the resonances appear as one. Upon addition of TMACI there is an immediate loss of all structure resonances an the sharpening of the monomer resonance (vl/2 =23 Hz to 01/2 = “.0 Hz). The addition of KCl(S) to 0.69% TNA22'GMP does Show '3 (D 31) (1‘ }_.J Q O. (D I some structure formation but the amount is g creased relative to the TMA+-free system (3:. Figure 39). A 0.60% K22'GMP solution is 80% structured at 0°C while a 0.69% TMA22'GMP solution with 1.08% K01 added is 30% struc- tured at 0°C (32. Figures 39 and 40). Figure M0 shows the *i H w n 1 Lo 0 0 effect of making a 0.60% K22'GMP 1.333 in TMAC There is a drastic change in the spectrum. All structure resonances disappear immediately and the monomer line is sharpened. Similar results were obtained with Rb+ and Cs salts. The addition of TMACl to solutions of Rb22'02? and Cs22'GMP caused the loss of all structure resonances present and a sharpening of n(8). No self—structure reson- ances were found when RbCl or 0301 were added to TMA 2'GIP. Figure 38. A + 1.36& TMACI QJulhhirGMP +IJM NaCl -3°c 8.9m The effect on the H(8) NMR spectrum on addi- tion of TMACl(s) to a 0.71% Na22'GMP solution containing 1.1% NaCl at -3°C. 8 illustrates the H(8) resonance of the sample made 1.36% in TMACl. A illustrates the H(8) resonances of the sample in absence of TMA+. 91 0.6913 IMA22’GMP 0%: 800 8.01 - 7.93 8.02 8.1 7.51 kcfl 8.12 Figure 39. The change in the proton NMR spectra of 0.69% TMA22'GMP upon addition of KCl at 0°C. A +| 1.33M TMACI coon (22'6” -ft 1 l l J l l l l l I l L zoo zoo 8mm Figure “0. Effect on the H(8) NMR spectrum on making 0.60% K22'GMP 1.33% in TMACl(s) at -3°C. A is the H(8) NMR spectrum in absence of TMA+. B is the H(8) spectrum after making the solution 1-33M in TMACl(s). 93 These results of TMA+ with the dialkali metal salts of 2'GMP indicate that TMA+ is not a structure directing ion, but is a structure inhibiting ion. The destabilization of self-structure can be observed in the IR spectra. Figure “1 shows the IR spectra of 0.59% TMA22'GMP at 51°C and 3°C. The strong absorbance at approximately 1500 cm-1 and the broad absorbance at ap- proximately 1970 cm.1 are due to the TMA+ ion. Figure A2 shows the IR spectra of 0.69% TMA22'GMP made 1.1% in NaCl at 51°C and 3°C. There is no evidence of solution self- structure in agreement with proton NMR data. The effect of TMA+ on the chemical shift of the H(8) resonance is shown in Figures 16 and A3. In Figure 16 observed H(8) chemical shift versus temperature for 0.69% TMA22'GMP and 0.86% Na22'GMP are shown. As the temperature is lowered there is an upfield shift of the H(8) resonance which is much more pronounced for the Na22'GMP system, The addition of NaC1(S) to a 0.69% TMA22'GMP solution also. has an effect on the H(8) resonance. A plot of concentra- tion of NaCl versus the chemical shift of H(8) at 5°C and 0.5°C is given in Figure ”3. There is a much greater ef— fect of Na+ ion concentration at low temperature. This suggests that there are at least two forms of nucleotide exchanging rapidly on the NMR time scale. Based on other results in this work, we propose the rapidly exchanging structure is a tetramer unit. 99 1001' 801 60‘ 701’ 404 20' : a e s 1800 1620 1400 V cm" 100 no» 60" 0‘ T 20- 3°C t 11 1* 1 fil 1800 1600 1400 V cm ' Figure “l. The infrared spectra of 0.69% TMAEZ'GMP at 51°C and 3°C NMR studies of this Eolut on at com— parable temperatures show no evidence for struc- ture formation. 701' %1 Figure A2. IooJ 80‘ 95 cos 40‘ 20‘- 58° .L 3 .L 3 1800 logo .1400 V cm" 100'} so? oo~ 4o« 0 201- : t : % moo logo ' moo V cm" The infrared spectra of a 0.69% TMA22'GMP solution made 1.1% in NaCl at 51°C and 3°C. NMR studies of this solution at comparable temperatures show no evidence for structure formation, 96 I .oom.o esm.oom um azo.mmaze - 2mm.o CH Homz ho cofiuwpumooCOO on» mommm> Amvm mo umficm HmOHEono one .m: oszufl A . J '1 .002 no ZO_._.<~:ZMOZOO 9n 9N 9— 0.0 u d J u - J- - q Jq — q u q q 80.“ 1. Own 000d de NI .L-JIHS 'NOMBHO i 009.0 97 In order to determine if the TMA+ destabilization of 2'GMP self-structuring is due to a steric or an ion pair- ing effect, tetraethylammonium (TEA+) and tetrabutylam- monium (TBA+) ions were investigated. The addition of NaCl to 0.89% TEA22'GMF caused a broadening of the H(8) resonance but no structure resonances were present. At 1.10% NaCl a precipitate formed in the solution. Similar results were obtained with the addition of KCl (at 1.17% KCl precipitation occurred). 'Figure AA shows the results of adding TEAP (tetraethyl- ammonium perchlorate) to a 0.82% K 2'GMP solution at 0°C. 2 The solution was made 0.08% in TEAP. The TEAP was not all soluble, so the 1H NMR spectrum was taken of the filtered liquid. The final concentration of 2'GMP was determined to be 0.60%, that of TEA+ was determined by NMR to be m0.01%. The solution containing TEAP has a spectrum similar to a TEAP free system (33. Figure 90), but the extent of structure formation is decreased by the presence of TEA+. Solutions of TBA22'GMP were very Viscous and had a light green color. The H(8) resonance of a 0.1A% TBA22'GMP solu- tion was a very broad singlet. The addition of NaCl or KCl caused a non-miscible oil to form in the solution. Thus, no information could be obtained on the TBA+ system. Nuclear Overhauser experiments were performed to de— termine if the tetraalkylammonium ion was located in a position where it would block the tetramer unit hydrogen Figure MA. The effect on the H(8) NMR spectrum of 0.82% K22'GMP at 0°C on making the solution 0.08% in TEAP. B is the H(8) region of 0.82 M K22'- GMP at 0°C. A is the H(8) region after—the solution is made 0.08%. A precipitate formed which was removed by filtration. The con— centration of 2'GMP was determined to be 0.60% and the concentration of TEAP was ap- proximately 0.01%, determined by NMR. lOO bonding positions. The Nuclear Overhauser effect (NOE) . . . s _ has been used to determine conformations of nucleotidesl/u 156. When a strong radiofrequency field is applied at the frequency of the one resonance, the integral intensity of another resonance of the same nuclei may be reduced, to yield a negative intermolecular Nuclear Overhauser effect. This NOE can originate either from exchange modulation of scalar coupling or it can be the result of dipole-dipole 157. interactions Since the tetraalkylammonium ion protons are not capable of rapid exchange, a dipole-dipole mechan- ism must be invoked. In spin systems which relax primarily through dipole-dipole interactions, such as all proton systems, the magnitude of the Overhauser effect has a 158 l/r6 dependence on the spatial separation of the ob- served and irradiated spins. Because of this, the NOE has found application in studies of molecular conformation and structure. Brown”9 has reported that TMA+ is located near the H(8) of the Na25'GMP self-structure(s) represented by the two outer H(8) lines in the proton NMR spectrum. 1 A 0.80% TMA 2'GMP solution at 25°C in H 0 showed only 2 2 a slight decrease in H(5') when a gated decoupling experi- ment was conducted with the TMA+ resonance irradiated. Small differences in spectra taken in 1H o are hard to 2 quantize due to the dynamic range problems which occur. The results of a gated decoupling experiment on a 0.89% TEA 2'GMP solution at 27°C are given in Table 6. 2 101 Table 6. Results of a Gated Decoupling Experiment on 0.89% TEA22'GMP at 27°C. Decoupler TEA *1 X 100 Power Reson. Applied Irrad. H(8) NH2 H(l') H(3') H(A') H(S') 0.9W -CH3 -20 ll 2 . -18 -2 2 0.1W -CH2— -3O 15 8 -l8 8 3 0.9W -CH2- - 6 22 -1O - 7 2 10 1.1W -CH2- -15 92 U 0 15 3O a1 is the integral intensity with the decoupler power off and Id is the integral intensity with the decoupler on. There is an NOE on the residual NH resonance (solution 2 contained 70% D 0 and 30% 1H20) and the H(5') resonance. 2 The only way TEA+ can be located near both H(5') and -NH2 is for 2'GMP to be in the syn conformation. The NOE is a time averaged effect, so the NOE can be attributed to a TEA+ residing near the NH2 group for at least some time and near H(5') for some time. The TEA+ molecule can there- fore prevent the formation of tetramers by sterically block- ing the -NH group. 2 Table 7 shows the results of irradiating the TMA+ resonance in a gated decoupling experiment on a 0.71% Na22'GMP solution 1.10% in NaCl and 1.36% in TMACl at -3°C. There is a large decrease in H(5') and a smaller r7 Table 7. Results of a Gated Decoupling Experiment in Which the TMA+ Resonance of a 0.71% Na 2'GMP Solution 1.10% in NaCl and 1.36% 15 TMACl was Irradiated at -3°C. Decoupler Io'Ida ——=——— x 100 Power 10 Applied H(8) H(l') H(3') H(A') H(5') 0.1w -5 2 '8 5 8 O.LHJ 10 3 M 6 l3 1.1W 2 l” 23 31 M6 3I is the integral intensity with the decoupler power off and Id is the integral intensity with the decoupler on. decrease in H(l'), H(3') and H(A'). Figure 95 shows the effect on the proton NMR spectrum in the H(3') to H(5) region on irradiation of the TMA+ resonance. TMA+ in this case is located near H(5') as in the other cases. It also spends some time near the other ribose protons, especially H(u'). This is consistent with the other cases. G. Mixed Nucleotide Experiments GMP was added to an 0.68% 8-d Na25'GHP solution and Figure A5. 103 I U T l I I 0.7m N022'GMP —3°-c 1.1M, in NaCl 1.36M TMACI IE: “— _--u- ‘— - 1 1..’ 7"“.5 afll'------- - I 0 o o u 0 o I l l I X~¥ {7 (, H(3’) H(4') H(S') l L L l L L 4.60 4.00 5'me The effect on the H(3'), H(u') and H(S') reson- ances when the TMA+ resonance is irradiated in a gated decoupling experiment on a solution of 0.71M Na22'GMP, 1.10% NaCl, and 1.36% TMACl at -3°C. 10” (2) 8-d TMA22'GMP was added to an 0.68% Na25'GMP solution. Figure 46 shows the effect of adding 8-d TMA 2'GMP to 2 0.68% Na25'GMP solutions at 5°C. There is a relative in- tensity loss of the a and 6 resonances and an upfield shift of the y resonance, both indicative of a decrease in struc- ture. The loss of structure is also seen in the ribose region of the spectra. At 0.67% 84d TMA22'GMP no structure is present. The loss of structure can also be monitored 1 by IR. The absorbance at 1585 cm- increases in intensity and becomes more intense than that at 1665 cm'l. It is interesting to note that the 2'GMP-NH2 resonance shifts downfield on addition of more TMA22'GMP. The addition of TMA 2'GMP to a 0.32% 8-d Na25'GMP solution causes only 2 slight broadening of the H(8) resonance of 2'GMP. Since TMA+ destabilizes the 2'GMP self-structure and high concentrations of Na+ are needed for 2'GMP self-struc- turing, solutions containing Na 2'GMP, Na25'GMP, and NaCl 2 to keep the concentration of Na+ constant, were investi- gated. The addition of large amounts of 8-d Na 2'GMP (up 2 to 0.90%) to 0.37% Na25'GMP at 5°C with the Na+ concentra- tion held constant at 2.5% by addition of NaCl caused a small decrease (<10%) in the amount of Na25'GMP structure present. Thus, Na22'GMP does not inhibit 5'GMP ordering but TMA 2'GMP does inhibit 5'GMP ordering. 2 In the complementary experiment, the addition of Na 2'GMP to 0.90% in 0.37% 8-d Na95'GMP at 3°C, yielded 2 105 T 1 fl 1 0.68 M Nozs’GMP + 8-dTMA22'GMP 5°C [8-d TMA22'GMPI 0.67M. i cam/j w} . 0.001! 1 l I l 8.52 8.23 7.92 7.22 54mm Figure A6.' The effect of 8-d TMA 2'GMP on the H(8) NMR spectrum of 0.68% Na25'GMP at 5°C. The spectra are not drawn to_scale. There is no major integral intensity loss. 106 only one broad (Vl/2 = 23 Hz) 2' MP H(8) resonance. The in- creased broadening may be due to an increase in the vis- cosity or interaction fast on the NMR time scale with 5'GMP. ii. Mixed 2'GMP - Guanosine Systems - Only one H(8) resonance is observed at 8.00 ppm when guanosine is added to 0.69% 8-d TMA 2'GMP at 1°C. This observation implies 2 no long-lived structure is formed. The H(8) resonance is broad (vl/2 = 25 Hz), possibly due to interactions between the nucleoside and nucleotide. One would not expect Solu— tion self-structure to occur in this sample, since no structure directing metal is present. A solution 0.03% in guanosine and 0.69% in 8-d Na22'GMP shows the formation of structure in the NMR spectrum. 'Two new resonances at 8.52 ppm and 7.70 ppm and possibly a shoulder on the down- field side of the monomer (3:. Figure A7) appear. This spectrum is similar to that of Na25'GMP which evidences self-structure. Thus, guanosine and Na22'GMP form a solution self-structure under conditions where neither individually forms a self—structure. H. Stoichiometry of the Na 2'GMP Solution Self-Structure 2 In order to determine the nucleotide stoichiometry for the Na22'GMP self-structure, the concentration of monomer and structure need to be determined at various concentra- tions of nucleotide. Equilibrium (l) is assumed to 107 T T I 0.69M. 8‘d N022'GMP 0.03M, Guanosine 1°C 1 - 1 l ' 8.62 7.96 7.70 6. ppm Figure “7. The H(8) spectrum of a 0.69% 8-d Na22'GMP and 0.03% guanosine solution at 1°C. 108 represent the self-structuring phenomenon. (2n—x)- + = xNa + n2'GMP Z Nax(2'GMP)n (1) + + If [Na ] is constrained such that [Na J >> [Nax(2'GMP)n- _ - + (2n X) 3, then [Na ]x is approximately constant. The equilibrium constant can then be expressed as: (2n-x)- [Na (2'GMP) 1 K' + K[Na+]X = x n- (2) [2'GMP-3n Proton NMR spectra were recorded for solutions of 2'GMP in the concentration range 0.90% to 0.18% while holding the Na+ concentration constant at 3.0% by addition of NaCl(s). The H(8) region of the NMR spectra of 0.U0% and 0.18% Na22'GMP made 3.0% in Na+ by the addition of NaCl(s) at 0°C are given in Figure 98. The H(8) region is composed of four resonances. The monomer resonance at 7.98 ppm, a shoulder at 8.01 ppm which is a structure resonance, and a small resonance(s) at 7.18 ppm which is a structure resonance. The NMR resonance at 7.18 ppm often seems to be composed of two overlapping resonances as seen in Figure U8. It is not known if the peak is composed of l or 2 resonances. The third structure resonance is a small shoulder at 7.9M ppm which is barely detected as a 109 r ' r 1 {Na} total -.- 3'0]! 090 0.1811 N022'GMP 0.40M No22'GMP 8.00 ' 7.20 5. PP'“ Figure A8. The H(8) region of 0.18% Na22'GMP and 0.A0% Na 2'GMP at 0°C. The [Nah for both samples was mage 3.0% by the addition of NaCl. 110 shoulder on the 7.98 ppm resonance. Computer simulation of the spectrum, as described below, shows that the reson— ance at 7.9A ppm is indeed present. A good fit of the H(8) peak shape cannot be obtained if this resonance is not included. The concentrations of 2'GMP and Nax(2'GMP)n- (2n'X)‘]can therefore be determined from the integration of the H(8) proton NMR spectra. Note that on integration of the structure resonances, nENaX(2'GMP)n(2n-x)'] is mea- sured. The program RESOL ( Atkinson ) was used to resolve the overlapped Lorentzian peaks. Digitized spectra were transferred on floppy disks from the ASpect 2000 on the Bruker WM250 to the PDP-ll minicomputer as described in Appendix 2. Given initial estimates of peak height and peak position, RESOL determines the best fit of the reson- ance peak height and peak position using a nonlinear least squares fit and determines the area of each resonance. An example of a computer resolved spectrum is given in Figure A9. The computer fitted results, listed in Table 8 were not accurate enough to attempt a calculation of the stoi- chiometry of the nucleotide. The lowfield set of over- lapping H(8) lines could not be accurately resolved with RESOL due to small changes in relative intensities between spectra. To determine the Na+ stoichiometry, it is necessary to hold the 2'GMP concentration constant while the Na+ lll O .0 .0 Zflfll Figure A9. lllliLLLl‘llLllllLLllllllllll 81H: 1 1850 The computer deconvolution of the lowfield reson- ances of the H(8) NMR resonances for 0.38% Na 2'GMP made 3. 0% in Na+ by the addition of Na 1. The solid line in A is the observed low- field H(8) spectrum. The x' s are the computed values. B shows the three deconvoluted reson- ances. 112 Table 8. Deconvolution of the Lowfield Set of H(8) Reson- ances for Na22'GMP in the Presence of 3.0% Na+. Line 1 [2'GMP] Positiona % Areab vl/2a AmplitudesC 0.38 2003.7 31 15.8 71.1 0.36 2008.1 51 18.8 92.9 0.3A 2008.8 A3 16.3 96.7 0.32 2009.7 58 19.2 107.A 0.30 2006.1 57 18.2 150.3 0.27 2011.1 56 18.A 78.0 0.23 201A.7 55 20.9 113.7 0.21 2023.A A9 1A.9 6A.8 0.20 2022.6 A8 1A.6 6A.9 0.18 2027.1 A0 12.A 71.2 Line 2 (Monomer) 0.38 1977.0 51 8.7 217.7 0.36 1986.5 39 5.5 237.1 0.3A 1987.5 A9 6.1 299.0 0.32 1991.3 35 A.6 271.1 0.30 1990.8 3A A.A 371.7 0.27 1996.3 A2 A.l 260.0 0.23 2002.A A5 5.3 366.A 0.21 2007.1 51 A.A 227.2 0.20 , 2008.6 52 3.8 265.3 0.18 2013.9 60 3.6 370.8 Line 3C1 0.38 1986 2 18 A5.A 1A.2 0.36 1967.7 10 20.0 17.6 0.3A 1959.3 8 19.0 1 .6 0.32 197A 6 7 10.7 21.7 0.30 1966 2 8 20.A 19.7 0.27 1987 6 2 20.A 2.7 0.23 ------ <2% ___- --_ 0.21 ------ <2% ____ _-- 0.20 —————— <2% _-_- -_- 0.18 ------ (2% -_-- -_- 113 Table 8. Continued. ain Hz. bDetermined by integration of deconvoluted spectra. Cln arbitrary units for each sample. dErrors are estimated to be position :2 Hz, % Area i10%, vl/2 i3 Hz, and amplitude i5%. 11A concentration is varied. This was accomplished for Na25'- GMP aggregation by adding TMA25'GMP solutions to Na25'- GMP solutions. This is not possible for 2'GMP since TMA+ was found to interfere with 2'GMP aggregation. The experi- ment might have been accomplished by adding NaCl(s) to a Na22'GMP solution, but this experiment was not attempted. It is unlikely that the solution will gel since 2'GMP does not form gels at neutral pH, but precipitation may be a problem. The stoichiometry of the Na22'GMP solution self-structure therefore could not be determined. III. 3'GMP Self-Structuring A. Alkali Metal Salts Only a single H(8) resonance is observed for solu- tions of Na23'GMP or K23'GMP in D20 at low temperatures. As the concentration is increased the resonance broadens but remains a singlet until the solution gels. At the point of gelation all resonances are understandably broad-I ened. No evidence for solution self-structuring is found in the 1H NMR spectra. An attempt was made to observe a N323'GMP self-structure by adding NaCl to a solution of TMA23'GMP. TMA23'GMP does not gel at high concentrations but the solution becomes very viscous. Only a single H(8) resonance at 8.05 ppm is observed in 0.A3% TMA23'GMP at 1°C. The addition of aliquots of 2.0% NaCl to a 0.A3% 115 TMA23'GMP solution at 1°C caused a broadening of H(8) (3:. Figure 50). At 0.37% TMA23'GMP and 0.29% NaCl the solution gelled. Therefore no evidence of solution self- structuring was obtained. As in 2'GMP, TMA+ is inhibiting the solution self-structure of 3'GMP. If TMA+ did not affect 3'GMP aggregation, the solution should have gelled at a much lower concentration of Na+ Figure 51 shows the spectra of a 0.A3% TMA23'GMP solu- tion on addition of a 2.0% K01 solution. At 0.03% K01 the H(8) resonance is broadened. As the concentration of KCl in the solution is increased to 0.10%, two new reson- ances appear as shoulders of the monomer resonance. The structure resonances grow in intensity as the concentra- tion of K01 is increased. At 0.27% K01, a new intense structure resonance appears at 8.26 ppm. The melting pro- file for this sample is shown in Figure 52. There was no time dependence on the appearance of the resonance at 8.26 ppm. Since gels are known to be less stable in ethanol7l, a concentrated solution of Na23'GMP in l-deutero-ethanol was investigated. If the solution self—structure is a restricted gel structure, then weakening the gel may allow solution self—structure to be stable. The nucleotide in ethanol showed no solution self—structure present. This may have been due to the low solubility (0.02%). A mixed solvent experiment was attempted. A 0.0A% Na23'GMP 116 T . I 1 V r Y Y 1 fl 0.4315, TMA23'GMP 1°C [NaCl] 0.25! 0.16% 0.06M L 1 a l 8.00 7.00 6. PP" Figure 50. The effect on the NMR spectrum of the H(8) region of 0.A3% TMA23'GMP on addition of NaCl(s). 117 0.434 IMAza'oMP ft [‘0] 0.0351 0.005 0.10% J J L L A I L A 8.00 7.60 8.00 7.60 3.09!“ Figure 51. The effect on the NMR Spectrum of the H(8) region on addition of KCl to 0.A3% TMA23'GMP. 118 v v v | I l . I U I ‘ I 0.43;; 1MA23’GMP ‘ +0371; KCI 30°C 20°C 1 . J 15°C :j L. A A L L l I ' A 4 n l L 060 coo ‘ 8.60 0.00 8. ppm Figure 52. The temperature dependence of the H(8) region of a 0.A3% TMA23'GMP solution 0.27% in KCl. 119 solution in 50/50 v/v of DZO/EtOD showed no evidence of solution self-structure. B. Mixed Nucleotide Systems Two experiments were performed to determine if 3'GMP could incorporate into the Na25'GMP solution self-structure; (l) TMA23'GMP was added to an 0.68% 8-d Na25'GMP solution and (2) 8-d TMA23'GMP was added to a 0.68% Na25'GMP solu- tion. Figure 53 shows the effect of adding 8—d TMA23'- GMP to a 0.68% Na25'GMP solution at 7°C. There is a relative intensity loss of the a and 6 resonances and an upfield shift of the B resonance, both indicative of a decrease in structure. The loss of structure is not as drastic as that seen in the 2'GMP case. A shoulder is present on the peak which increases with increasing con- centration of 3'GMP. These samples contain TMA+ as a counterion, which has been shown to destabilize the gel self-structure of 3'GMP. The complimentary experiment, the addition of TMA23'— GMP to 0.67% 8—d Na25'GMP, is shown in Figure 5A. In this experiment the H(8) resonance of 3'GMP is observed. Even at very low concentrations of added 3'GMP a new H(8) resonance which occurs at 7.50 ppm and accounts for 30% of the total H(8) intensity is present. At higher con- centrations all resonances are broadened. The broadening was assumed to be caused by an increase in viscosity. 120 - a T . . .. a F f 0.675: szs’oMP 7°.c. [8—d TMA23'GMP] 0.23M 0.15M 0.08M Figure 53. The effect of 8-d TMA 3'GMP on the H(8) NMR spectrum of 0.67% Na25'GMP at 7°C. 121 0.6 7M 8-d N025'GMP + TMA23'GMP- I 1 7°C BeMfl' 0.IOA_A A. 0.08M L l l 7.91 7.50 8. ppm Figure 5A. The H(8) NMR spectra of 0.67% 8-d NagS'GMP and two concentrations of TMA23'GMP at 7 C. A is the H(8) NMR spectrum of a 0.67% 8-d Na25'GMP and 0.08% TMA23'GMP solution. B is the NMR spectrum of a 0.67% 8-d Na25'GMP and 0.16% TMA23'GMP'solution. 122 Therefore, there is an interaction between 3'GMP and 5'GMP which causes 3'GMP to be in at least two slowly exchanging environments. IV. 5'GDP Self-Structuring A. Alkali Metal Salts Only a single H(8) resonance is observed for solutions of Na25'GDP or K25'GDP in 020 at low temperatures. As the concentration is increased the resonance broadens but remains a singlet until the solution gels. At the point of gelation all resonances are understandably broadened. An attempt was made to observe a Na25'GDP self—structure by adding NaCl to a solution of TMA25'GDP. TMA25'GDP is very viscous and is almost a gel at high concentrations and low temperature. Only a single resonance at 8.12 ppm is observed in 0.11% TMA25'GDP at 0°C. The addition of NaCl(S) to a 0.11% TMA25'GDP solution at 0°C causes line broadening of H(8) but no appearance of new resonances. There is also an upfield shift of the H(8) resonance. At 0.A5% NaCl, the H(8) resonance is present at 8.09 ppm. As in 2'GMP and 3'GMP, TMA+ is affecting the solution self-structure. If TMA+ did not affect structure formation, the solution would have gelled at a much lower concentration of Na+. A 0.1% Na25'GDP at 0°C would be a gel. Figure 55 shows the spectra of a 0.11% TMA25'GDP I l l 1 I I l I l I I I l I l 1 r l I t 1 t [xcnl dug 1 mug 00mg 1 1 n 1 l l 1 1 L l l L l 1 1 l l 1 1 1 l 8.00 7.00 9.00 8 00 7.00 3:09!- Figure 55.- The H(8) 1H NMR spectra of 0.11% TMA25'GDP containing K01. The 1.27% sample contained a piecipitate when removed from the NMR spectrom- e er. 12A solution at 0°C on addition of K01(S). At 0.31% K01, the monomer resonance shifts upfield to 8.0A ppm and a new resonance appears as a Shoulder downfield of the monomer. A small broad resonance is present at 7.A1 ppm. Increasing the concentration of K01 to 0.97% causes the two new resonances to increase in intensity, so the solution is ap- proximately 80% structured. Increasing the concentration to 1.27% K01 causes the formation of a precipitate and a decrease in the extent of solution self—structure. The decrease in the amount of structure can be attributed to the loss of nucleotide in solution caused by the precipitate. Figure 56 Shows the melting profile of a 0.11% TMA25'GDP solution 0.97% in K01. B. Mixed Nucleotide Experiments To determine if 5'GDP could incorporate into the Na25'- GMP solution self-structure, solutions containing 0.32% 8—d Na25'GMP and TMA25'GDP were investigated. The proton. NMR spectra of two solutions at 5°C are shown in Figure 57. Several new resonances are present. TMA25'GDP, as described in the previous section, does not form a solution self-structure in the presence of Na+. Therefore it is believed that 5'GDP has incorporated into the Na25'GMP self-structure. If there were incorporation of 5'GDP into the Na25'GMP solution self-structure, the H(8) region of 5'GMP should be affected. The complimentary experiment, 125 “1°C 50°C 1 j l J 1 l L 1 l 1 l L L l L L 1 1 l 1 9.00 0.00 ' 7.00 9.00 000 7.00 80’” Figure 56. The melting profile of a 0.11% TMAZS'GMP solution made 1.27% in K01. _ 126 r I I l I I 5 ' ‘ 0.3224 8‘0 N025'GMP 5°C ITMA25’GDP] ‘ 0.251 0.50M 1 1 1 l 1 l 1 8.80 8.00 7.20 5.me Figure 57. The H(8) NMR spectrum of a 0.32% 8-d Na85'- GMP solution containing TMAZS'GDP at 5° . 127 8—d TMA25'GDP + Na25'GMP, could not be done because deuteration of H(8) would cause the hydrolysis of 5'GDP to 5'GMP. V. Ethidium Binding to Guanylic Acids A. Bindinggof Ethidium to Unstructured Nucleotide The binding of ethidium (Etd) to 2'GMP and 5'GMP was investigated. Ethidium was found to interact with both unstructured and structured guanylic acids. Figure 58 Shows the change in the NMR spectrum of 0.02% Etd as TMA22'GMP is added to the solution. The lower spectrum represents the Spectrum of the "free" ethidium bromide. The chemical shifts are in agreement with the values re- ported by Kreishman and coworkerslz8. They assigned the resonances as follows (see Figure 10 for numbering se- quence): para- and meta- protons of the phenyl group between 7.80 ppm and 7.70 ppm, H(l) and H(lO) at 7.69 ppm and 7.65 ppm respectively; the orthoprotoncfi‘the phenyl group, H(2), H(9), and H(A) between 7.20 ppm and 7.00 ppm; H(7) at 6.26 ppm; methylene resonance at A.AO ppm; and the methyl resonance at 1.A0 ppm. The splittings of the methyl resonance and methylene resonance are incompletely re- solved at conditions where the drug is stacked, either with itself or with the nucleotide. Kreishman and coworkers128 and Marshall97 have reported 128 T'IfVYV I filtfiilrtitrvvrr . [TMA22'GMP] J U 0.737% JL’ML 1“: J U . (102M LJLIL , m p! 7 011:: 1 11 1411 11114111111111 .1111111111111L1 .800 400 000 L — . Fi ure 58. The effect on the proton NMR spectrum with the g addition of TMA22'GMP at 0.020% ethidium bromide at 0°C. 129 a general upfield movement of the aromatic resonances along with considerable broadening as the drug becomes bound to nucleotide. The H(l) and H(lO) lines of ethidium were observed to shift upfield by m0.u ppm, H(2), H(H) and H(9) shift upfield to 6.60 ppm to 7.20 ppm, and H(7) shifts to a position under the nucleotide's H(l') resonance at m5.90 ppm. The chemical shift of the phenyl group protons exhibits little change on binding to the nucleotide. The shifts in the ethidium methyl resonance are a good indicator of bind- ing to nucleotide. The methyl resonance shows relatively large shifts with stacking interactions, and the resonance is in a region of the 1H NMR spectrum where no other reson- ances are present. The chemical shift of the methyl ethi- dium resonances as a function of temperature for 0.02M ethidium bromide is given in Figure 59. Figure 60 shows the chemical shift of the methyl reson- ance of ethidium as a function of the TMA 2'GMP/Etd 2 ratio at several temperatures. Similar shifts occur in the ethidium methyl resonances when TMA25'GMP is added to solu— tions containing ethidium (of. Figure 62). Under these conditions no IR and NMR evidence exists for an ordered solution self-structure in either nucleotide system. The Etd methyl chemical shift in both the TMA22'GMP and the TMA25'GMP solutions moves upfield as the ratio of nucleo- tide/Etd is increased to 1:1; then the lines move downfield when the ratio exceeds 1:1. This implies the formation of 130 l .ooHEOpn Endoanpo somo.o ho awash HmOHEono Hanuoe on» no mocmocodmo mnzumpoasmp 0:8 .mm opswfim a co . $25.85» 3n 3.. can 98 3: co . q ‘ 1 d - u q d d — q d d d - q q d u - q u q d 8’ p v - II I] go, o x 1 H .. . m . - m r I: r. n lollllo‘ 0 o . 1m: 3a.. m . 1.0!! c 1 u r .. IN. T 1 001p r a fi 330% 229.5 536. Kim .2955 SE: 229.5 _ P P P . — . m b . _ p . a . p P . L f P n p p . p g? 131 .monspmpodsma msoupw> pm Esfiofinuo op mzc.mm..03010 00?, l 09”. 83 .mm opswfim wad NI was woman-Io 139 therefore the most suited for quantitative investigation. The results showed that the nucleotide H(l') chemical shifts and equilibrium constants were coupled so that the equations could not be solved. Setting Kl and K2 equal to the values in Table 9 yields the results in Table 10. Table 10. The Best Computer Fit of the H(l') Resonance of 2'GMP to the Binding of Ethidium to TMA22'GMP. K3 = 1.0:1.o M‘1 M = 6.07110.013 ppm M D 2 5M = 5 687+o 007 ppm MD ' ‘ ' -- 5% = 5.629io.050 ppm The calculated and the experimental chemical shift data are shown in Figure 63. l.Oil.O M , is very close to that 160 The value for K3, reported by Helene and coworker , 2.2:0.2Mfl for purine base stacking in aqueous solution. He used the isodesmic approach to determine a stacking equilibrium constant from 1H NMR data. A better fit may be obtained by using the iso— desmic approach in our fit. In the isodesmic model the stacking constants are equal. The KINFIT program cannot 1110 .momo.o ma assesses no competesmecoe see .000 pm ezc.mm 10. The addition of ethidium to a structured solution of Na22'GMP causes a small increase in the amount of self-structure present (cf. Figure 68). The increase in concentration of solution self-structure for Na25'GMP on the addition of ethidium has been reported by Marsha1197. As seen in Figure 6A and Figure 66, increasing the tempera- ture of both a Na22'GMP solution to which NaCl(S) is added + so a ratio of [Na ]/[2'GMP] = 3.28 is obtained, and a 11111 .Hosz so sofiefipep one an.mm.m so mzo.m\+sz eo oases oHos a 0>mn on 0005 mm: coausaom one .mopsumADQEmp msofihm> no Esacficum OH QEU-waz .HO OHQNH MHOE MSG m3mh0> Qhfifim HGOHE$SO Hhflpmfi SHUHSOfl 05...... .30 wkswfim +0.5 0... n.20..N “.0 00.5. 302 adv . can eds . as. _ 00 d1 d] 1 q - d d d 11 - a 1 d d — d d I - goo T egos -o - .1. o 1 o o v o UOON Q U 11 to. 0 00° 0 11 gw 1 A . L 1 4 1 0 1 ., . 1 H . . 1 m I 1 83 m I r. r l S 1 1 H . . . - a .. a r 1d 4 1a .. H .1 r s 1 u 1- (V w n LI P h p 1— P p n p — n P b n — n P p b 8*0’ lUS 1A -1.J-1.1.1-1.141.1.1-1-i41+4i 7 ‘7' L3 Allnnlllllllllllll Ivvrvvvl'va'V L2 4 1.1 I‘LLIMIIUJJIIIA I'VVVVVWVUV'V'V'IVVV‘IVI 'LO 664}b) AAAJAAA'AI Ivvvvv 03 V'r' 'V' 03 Alllwllllljjlllj [A ’ V r. O 0 OJ € ’1 ‘\ \ .‘r ”\ 0'6 ‘l'l'ltfil'l'lfl'l'l‘l'l'lfil'lfi 0.0 . 2.0 4.0 5.0 0.0 10.0 12.0 «.0 (show- Structured / EtdBr) AIL‘AAAI YVVYVVVVVIVVI VVVV' Figure 65. Plot of the chemical shift of the EtdBr methyl resonance as a function of the structured Na25'- GMP/Drug ratio. Extrapolation of the linear portions of the curve included. Taken from Reference.97. 1146 flomoé 003 coapwnfiaocoo 5:20.230 05. 000300009500 05939,. 00 8:25:00 op 0:0.mms no coduoano «Hoe 0:» m=m00> pofizm HmoH50so Hanums esfiefinuo use .00 opswfim +6.5 0... m20.« no OER 302 93 0.3 08 92 0.0 . ooqo 09w. a0 m 002. M“ S m u 8N. p N 090. r 1 D P n b - b p b b — n n P h b b b P bl 8*. . 1117. 1.4 -1.l.l.l.lll+l.l.Lilal-1Llll. 1.3 ALIJAIILLIAIIAA tuttvlvr‘rvvvyvv 1.2 1.1 1.0 111IAAAIIILJAIAJAAJIIlllllll «41.15) 0.9 llllllllll 0.8 ' I Ilvvv 0.7 lllllLAlllLLlAAlLllllAll ’ A V'T‘Vv" °°5 "1*1'1'1'1'1‘1*1‘1.*1'IrI'PI‘ 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 (5'-GMP' Structurod/ EtdBr) Figure 67. Plot of the chemical shift of the EtdBr methyl resonance as a function of the structured K25'- GMP/Drug ratio. Extrapolation of the linear por- tions of the curve included from Reference 97. Figure 68. 1M8 l L l £00 200 500 $00 3w") The effect on the solution self-structure of 0.72fl Na 2'GMP upon making the solution 0.020fl in e hidium bromide at 0°C. The nucleotide had a ratio of Na+/2'GMP of 3.28 accomplished by the addition of NaCl. Figure A is the NMR spectrum of ethidium absent system. Figure B is the NMR spectrum on making the solution 0.020M in ethidium. Note the increase in the H(8) and H(l') solution self-structure resonances. 1&9 K22'GMP solution causes a decrease in the concentration of self-structure. This decrease in solution self-structure causes a general downfield shift of the ethidium methyl resonance. At temperatures where the solution self-struc- ture is melted out, the ethidium methyl resonance as a func- tion of the ratio of guanylic acid/ethidium is the same as for a non-structured TMA guanylic acid solution. The binding of drug to ordered nucleotide in the pres— ence of disordered nucleotide can be described as D + M I MD K1 (3) MD + M I MZD K2 (u) MD + o : MDo K3 (23) MDo + MD : (MD)20 K“ (2M) 0M: + BNa+ z o<2a+8> KS (25) where O is the structured form of nucleotide, a is the number of monomers and 8 is the number of Na+ ions in the structured unit. This model of drug binding to structured solutions of guanylic acids was chosen because of the large equilibrium constant of the 1:1 unstructured nucleotide drug complex (of. Table 9). Species such as DO and D 0 may exist, but 2 150 they are assumed to be minor species. The concentration of monomer present is always very large compared to that of structured nucleotide. The values for a and 8 were taken as 8 and U, respectively, based on the similarities between the Na22'GMP and Na25'GMP self-structures. Most evidence to date suggests a stoichiometry of NauM8 for the solution self structure97’98. I The total concentration of ethidium can be expressed by. t C = D [D] + [MD] + [M2D] + [MDO] + 2[(MD)2O] (26) Substitution of the equilibrium expressions of Equations (3) + (u) find (23) - (25) yields; t CD = [D] + KlfMJED] + K1K2EMJZED] + Kl K3KSCMJEMJGENa+JBEDJ + 2K12K3KMK5[M]2[M]a[Na+]B[D]2 (27) The total concentration of nucleotide can be expressed by C; = [M] + [MD] + 2EM2D] + (a+1)[MDO] + (a+2)[(MD)Zo] + OLEO] (28) Substitution of the equilibrium expressions of Equations (3) 151 + (D) and (23) - (25) yields; 0; = [M] + Kl [MJED] + 2Kl “[M] [D] + «0+1)K1K 3K5) (CMJEMJG [Na+ ] B[D])+ (0+2)K12K KSEM] 2[D] HEM] [Na+ 38 3 KM + dKSEMJQENa+JB ' (29) Since the reactions are fast on the NMR time scale the observed chemical shift can be expressed as, GB + GB + 5D + D MD XM2D M2D XMDo MDo X(MD)206(MD)2O 6D 5 = D + XMD obs XD (30) The mole fractions of the species containing drug can be expressed through the use of Equations (31) - (35) as; XD = [DJ/cg (31> xMD = KlEMJEDJ/C; (32) XM2D = K1K2EMJ2EDJ/Cg (33) XMDo = K K KSEMJEMJQENa+]B[D]/Cg (3a) X(MD)20 = 2KfK3KuK5£MJ21MJGENafFEDJ2/cg <35) 152 The mass balance equations (27) - (29) are solved for [D] and [M], by using the same procedure as was used in the TMA22'GMP case with ethidium binding. In order to solve for [M]a, it is set equal to [Mestja’ an estimated value of [M] which is updated with each new value of [M] calculated. The [Na+] is arbitrarily set equal to [Na+]total/ 3.0 and updated on each iteration with the correct equation for [Na+], + + [Na 1 = ([Na lT-B([o]+[DMO]+[(MD)2O]))B (36) The values of K K 6D and 0D were assumed e ual to 1’ 2’ MD’ M2D q those calculated for TMA 2'GMP — ethidium binding. 2 The results of the best computer fit are presented in Table 12. Table 12. The Best Computer Fit of the Binding Constants for the Binding of Ethidium to Na22'GMP Self- Structures. K3 = (l.9ltO.lO)xlOu M‘1 K“ - (5.8:0.6)x102 M‘l K5 = o.122:o.oo7 M‘11 D = GMDO 0.93520.006 ppm D = . . 2 5(MD)2O l 200:0 0 0 ppm 153 As described previously these are concentration equilibrium constants. The best computer fit chemical shifts and the experimental chemical shifts are plotted in Figure 69. Other models which were tested for ethidium binding to ordered 2'GMP are given below: M + D 2 MD K M + MD 2 M20 K2 MD + o 2 D0 + M K3 0 + D0 + ODO Ku + O(-20+B) = + 0M + BNa + and MD + M : M2D K2 MD + O I OD + M K3 OD + MD I OD2 + M KM = + _ 0M + BNa Z 0( 2u+B) (3) (H) (37) (38) (39) (3) (u) (40) (Ml) (U2) 15H 0:» an mm.m meme mm: ofipmp azo.m\+wz 0:9 .mo mauocfin 0:» ou 00:0:om0p Hh£p0e +ohu Oh.m2 .H 02 no :oauaccw .Ooo pm mzo.m 02 o» Esfiodnu0 Esfioasp0 0:» no nah n0usasoo 0000 0:9 o.~ no 0:5. 302 can T I d d 1 u (q - d u d d mum-02.57314 L 303005310 1 OQVP l 007' OONé I OQVp ... azohuuz .H P [LP [P n — P h n b _ . n b b h P b h - P g' .mm 0L3wfim thilfl_lflHS'TKMN3HO 155 The values of a and 8 were again taken as 8 and M, respectively. A computer fit could not be obtained for either model. Calculated chemical shift values were diverg- ing from the extrapolated values and the calculated con- centrations of species did not correspond to the integral intensities determined by NMR. A satisfactory computer fit to any model has not been obtained for the binding of ethidium to K22'GMP. Mar- shall's97 model of ethidium binding to solution self- structured Na25'GMP and K25'GMP does not take into account the binding of ethidium to monomeric 5'GMP. The work presented here for the binding of ethidium to TMA25'GMP proves that binding to monomeric 5'GMP is not negligible. No satisfactory fits were obtained for several models of drug binding to structured forms of 5'GMP in the presence of unstructured forms of 5'GMP. VI. NMR Spectra of Guanylic Acids in 1H2Q A. NMR Spectra in 1H2g A wealth of information can be obtained from NMR spectra Of guanylic acids taken in lH20 solutions. Protons which exchange with solvent water can be observed if the exchange is slow. Under conditions of structure formation, the imide protons can be observed. The NH region shows a unique multiline pattern which can be used in conjunction 156 with the H(8) lines to create a picture of the self-struc- ture formed. By comparing the number of hydrogen bonded NH protons with the number of structured H(8) protons, insight into the number of hydrogen bonds per structured nucleotide should be attainable. At about 6.3 ppm in 1 H2O at 0°C, the GMP= monomer is known to exhibit an amino proton signal which shifts to about 6.5 ppm on hydrogen 13H bonding with 5'GMP The hydroxide protons have also been observed in nucleotide solutions in l1120131. Problems occur in distinguishing between NH and OH protons. Chemical shifts of OH protons usually occur less than 6.0 ppm down- field from TSP; they have, however, been reported to have shifts as large as 7.2 ppm in cyclic nucleotide systemsl3l. Iwahaski and Kyogokul36’l37 have used the saturation transfer technique to elucidate the sites and rates of exchangeable nuclei in nonaqueous solvents. In this tech- nique a strong radiofrequency is applied continuously to the frequency of one resonance during the experiment. If this irradiated nuclei exchanges with another, the magni- 1 tization is also transferred, causing a decrease in inte- grated intensity. Saturation transfer experiments were performed on a 0.62M Na25'GMP solution in lH20 at 2°C. The proton NMR spectrum of 0.62M Na25'GMP downfield from 5.0 ppm in 1H20 at 2°C is shown in Figure 70. The results of the saturation transfer experiment are given in Table 13. The resonances are labeled according to Figure 70. 157 .00 00st0 :0 0000000 00 000:0:Om00 msofi00> 0:0 000 050:00 mcfiH020: .OHH 0O 00 O0OOEH0O0 000 000H 00H000020 :H m0c000 0:00 D .co 00Ha3oo00 0:0 :0wz z0fimc00cfi H00m00CH 0:0 0H UH 0:0 000 00Hd§000© 0:0 :00: 0000:000fi H00m0000 0:0 0H H 0 HO HO OO mm Om NO HO HO. HO OOH Om: - O H Om- OH- O- OH O- OH- OH N:2 O - O H- OH ON 0 HO MH- m OHOO: O OH - mm OH O Om- Hm- Om- m OHOV: O- OH- HH- - NO O- NH- O O O >HOO: O O OH OO - OH Om- OH- HH O- OHOO: O OH- O OO- OH - O O O OO OHOOO O- O H OH- 0- O- OH NH - Om Hmvzz mm OH OH- O- O 0 m- OH OO - AHvzz mzz O O > O O HOvzz Hmvzz Hmvzz HHvzz OOOOHOO00H 0000:000m :coflm0m vaz :con0m 0UOEH OH OOH x 1111- O OH-OH .Oom 00 OmmH OH :OHOOHOO 0:0.0mmz am0.0 0 0o 0H0: 1wfim CO0o0m 00:00 00 coa0000000H co m0wmc00cH Hmcwflm :0 0000000o 0000000 .MH 0H009 158 .800 oo.m 2000 OH0H0csoO Oom 00 OmmH :H 0:0.mm02 mm0.0 00 53000000 022 000000 0:0 .OO 00:0H0 Eu...” 3... 8s 8.. , 8.0. 092 8.: 1 1 1 (I — J J 1 d — q q d J u q q q - a — q d a d ‘ q d 1 q - q d d d .— 2902. 3.: 20.00.. 09¢; n v u n 8 . 0 O « rt: 1 Our. 5 sicknoz $3.0 159 Four resonances are present downfield from 9.00 ppm which are not present in samples prepared in D 0. These resonances 2 are attributed to N(l)H protons. Three of the resonances are similar in chemical shift and intensity to the N(l)H protons of Na 2'GMP shown in Figure 22. A new resonance is 2 present in the H(8) region of the spectrum (7.55 ppm,e). This resonance at 7.55 ppm is sometimes present in spectra taken in D20, but the intensity is greatly decreased. Fisk and coworkers98 have attributed the resonance which is seen in D20 to a H(8) proton of a less stable solution self- structure H(8). Since the e resonance is similar in chemical shift to the 2'OH resonances which have been observed hydrogen bonded to H20 in cyclic nucleotideslBl, this resonance at 7.55 ppm may be due to the 2'OH or 3'OH group. Based on the probable difference in relaxation rates between an amino and a hydroxyl proton, a relaxation experiment was done by “9 Brown . Inversion recovery T experiments revealed that 1 very small differences exist between T1 values for all resonances in this region, and thus the results were incon- clusive. As shown in Figure 71, a 0.A3M 8-d Na25'GMP solution in lH20 at 2°C does contain the resonance at 7.55 ppm. The resonance is therefore not exclusively an H(8) resonance. The other four resonances between 7.00 ppm and 9.00 ppm are exclusively H(8) resonances. They have the same chemical shifts and intensities as do solutions run 1 .00m 00 Gama :H coa0saom 02 01m 2m:.o 0 00 San 00.0 no 0H00003o0 53000000 mzz CO0o0d 0:9 .HO 00swfim 160 0%. 220092 O-.. 23.0 161 in D20. The 5'GMP resonance at 6.10 ppm is also present in 2'GMP. For both nucleotides the resonance is absent in D20 solu- tions. This line is attributed to the amino protons. The addition of 1H20 to a 2'GMP solution causes the 6.10 ppm resonance to increase in intensity. After a TMA22'GMP sample is heated to 90°C to allow exchange of H(8) to occur 0 - 1H O solvent, the integral intensity with the mixed D2 2 of the resonance relative to the H(8) line intensities is approximately 2. Repeating the addition of 1H O to the 2 sample and heating yields a relative integral intensity of 2 for the amino proton. The resonance is positioned in the region of the nucleotide -NH2 groups. The resonance is not observed in mixed 1H2O/D2O solutions of Na2'(3') Inosine monOphosphate (IMP; IMP is identical to guanosine except the NH group is absent). The novel characteristics of 2 this resonance in the presence of alkali metal ions are described in detail in the following section. As shown in Table 13, the irradiation of the 1H20 resonance in 0.52% NaZS'GMP in 1H 0 causes a decrease in the 2 intensity of all resonances downfield from 6.00 ppm. Very large intensity decreases occur for the amino protons line at 6.10 ppm, for all of the resonances downfield from 9.00 ppm, and the resonance at 7.55 ppm. All of these lines are not present in D20 solutions. Therefore, the decrease in intensity of these lines upon irradiation of the H20 162 resonance can be attributed to rapid exchange with the sol- vent. The outer H(8) resonances (i.e., the resonance at 8.22 ppm and 7.12 ppm) are also greatly decreased in intensity, when the H20 line is irradiated. These H(8) protons cannot be exchanging with H20 protons at this temperature. There- fore, this decrease in the intensity upon irradiation of the water resonances is attributed to a negative Nuclear Over- hauser effect. Water must therefore be located very close to the H(8) proton in these structures, more so than in the monomer and the other structure which gives rise to the H(8) resonance at 8.10 ppm. As mentioned previously, water is necessary for self-structure formation. Returning to the data in Table 13, we may note that the irradiation of NH(l) causes a U4% decrease in the intensity of the NH(2) resonance, but little change occurs in the intensity of the NH(3) or NH(A) resonances. Irradiation of NH(2) causes a 50% decrease in NH(l) and a small change in, the intensities of NH(3) and NH(A) resonances. This implies the structures giving rise to NH(l) and NH(2) are exchanging faster with each other than they are exchanging with the structures giving rise to NH(3) and NH(A). An alternate explanation is the band width of the decoupler is too broad and is irradiating both resonances to some extent giving rise to the results obtained. (Irradiation of the reson- ance at 7.88 ppm (i.e., H(8)Y) causes a large decrease in 163 the structured H(8) B resonance and a small effect on the other H(8) resonances. Irradiation of H(8)8 also causes a large decrease in the H(8)y intensity with little effect on the other H(8) resonances. This suggests that the struc- ture giving rise to the B resonance exchanges faster with monomer (i.e., the structure giving H(8)y) than to the structure giving rise to the a and 5 resonances. The faster exchange of the structure which gives rise to the B resonance and the monomer is also suggested by the merging of the 8 and y resonances which give rise to the X reson- ance at conditions where structure is present in lower con- centrations. Irradiation of H(8)0 causes a loss of 31% in the intensity of the NH(3) resonance, which suggests that the H(8)5 proton is near the NH(3) proton. This implies they are in the same structure. Table lu and Table 15 give the integral intensities in arbitrary units of the resonances downfield of the H20 resonance for several concentrations of Na25'GMP at 2°C and Na22'GMP at 1°C, respectively. The arbitrary units are not the same for each concentration. For both nucleotides, as the concentration is increased there is an increase in the intensity of H(8) lines assigned to solution self- structures and in the intensities of the amide proton lines. The decrease in the relative intensity of the amino proton line with increasing concentration is discussed in the next section. 160 .:ofi000w00:a 00003000 0o0 00:0:0000 o .ROHM 0: o0 000050000 000 00o00M0 mma 0:0 0: 0000O00H0 000 003 00:0:om00 0:50 .:o0000w00:H.AmV: 5o00 00:050000a0 .000:0:0000 0 0:0 > .0 .0 0:0.uo 00H000:00:H H00M00:H 0:0 no 500 0:90 .000:0:O000 no 00:00H0005o: 0o0 on 00smfim 00mg .0:o00000:00:oo 0:0000000 0o0 0500 0:0 0o: 000 0:0 00000H000 000 000::0 o o.N o.N mm 3N a: III III mm III III IIII IIII III III 80m.o a:0.m~000: 0000: 0:00: 0.0:: AmOaz AmOxz OaHvxz 5:0.Omsz a sz mzz H0008 ho . 0:00 .000 00 OmmH :H OOOHOOHOO 0:0.0 00 managemmm OmzH 0:0 5000 0H000:3oo 0000:: >0000000< :0 000:0:O00m 0o 0000:00:H H00w00cH .OH 0H00B 165 .000000000000 00000000 000 0000:0000 0mm 0 .0000 0: 00 000050000 000 000000 .00000000000 Amy: 5000 0000500000 0 0:0 0: 000000000 000 003 0000:0000 0:90 0 .0000000000 0 000 r .0..0 0:0.00 00000000000 00000000 0:0 00 5:0 0:90 .0000000000 00 000000000500 000 mm 000000 000 D .00000000000000 000000000 000 0500 0:0 00: 000 000000000000 000 000000 0.0 00.0 00.0 0.00 0.00 0.00 -- 0.00 -- -- -- -- 000 200.0 020.00<20 0.0 00.0 00.0 0.00 0.00 0.00 -- 0.00 -- -- -- -- 200.0 0.0 00.0 00.0 0.00 0.00 0.000 -- 0.00 -- -- -- -- 200.0 0.0 00.0 00.0 0.00 0.00 0.00 -- 0.0m -- -- -- -- 200.0 0.0 00.0 00.0 0.00 0.00 0.00 -- 0.00 -- -- -- -- 200.0 00.00 00. -- 0.00 o 0.00 0.0 0.00 . 0.00 0.00 0.00 0.0 200.0 00.00 -- -- 0.000 000 o 0.00 0.000 0.00 ..0.0m 0.0: 0.00 000.0 0.0flW00 mmwwwm MHz: nuwnw 0.00: 0:2 0000: 0:000 00000 00002 00002 000002 mu0ummmu .000 00 020.0 00 000000000 0000 0:0 5000 000000300 0000:: 00000000< 00 000000000m 00 000000000 00000000 .m0 00:09 166 The resonance attributed to the amide proton in both nucleotides should have an integral intensity equal to the integral intensity of the H(8) structure resonances. The NH resonances are only in the slow exchange region when involved in structure formation. Solutions which do not contain self-structure do not have an NH line(s). The results in Table 16 show that the amide resonances are of larger integral intensity for both nucleotides than the structured H(8) resonances. Therefore, some of the in- tensity of the resonances is due to another exchanging proton or to another structure(s) which is different from that giving rise to the H(8) structure lines. The Spectra of 1.55M K22'GMP in lH2O at 1°C and 27°C are shown in Figure 72. The imide resonance(s) are down- field from 8.50 ppm at 1°C, the resonance(s) is very broad. The H(8) resonances are located between 6.70 ppm and 8.MO ppm. At 27°C the spectrum is much more resolved due to a decrease in viscosity. There are several imide resonances present at both temperatures. At 1°C the large imide resonance at 10.7 ppm is most likely composed of several broad resonances. The imide proton resonance is different from that of the sodium system as shown in Figure 70. B. Amino Proton Exchange Figure 73 and Figure 7M show the intensities of the amino proton lines relative to the H(8) line intensities 167 N ..00~.N ocw 00H um and o.m soap efimfioczoo o:fl cw mzo.mme mmm.H mo appomam mzz copopa use .ms magmas .92 .c 80 8.. . . 8.0— . 8N— d I I 1“ I d 1‘ 1 1 1 *d Q a d 4 - d i J 1 qfld‘j .1 1+1~1 4 I 1 168 u.mmm: do cofiumpucoocoo on» .ooa um mzo mzmpo> mzz va: an confiEmemo mgzuospumnuamm cofiosfiom a onu new mocmcomon ocfism on» do apfimcmpcfi Hmpwmucfi o>Humamg one .ms opzmfim azoawoz no zocézmozoo oo.N ooé co; 0*. .. ON... co; and 00.0 09.0 0N6 00.0 Q6 1 q q — d H a - u i q — a u u - d 1 d — d d d ‘ I I I q q u d a d 00° n I q L m 0.2 m. i 3 N" m .H . mm W 93 m. ._. I o; W m 0.0» n. - I. m n 1 Q. m M u m 06* u. l 9 D n 1 t. m E 93 m. ll. IV - I .K n .4 4 l 0% mm m 6.00 ml 4 1 S W .m 1 o; w 065 n... . O T 4. m 98 m. n 3 m n i W n . 7v P 98 W .. «a 008' n n p — b - _ P p n L — - _ — — . p p — u p n b b b — — n n — — n p - — n p *0“ 169 PERCENT STRUCTURE DETERMINED BY NMR usaom x osu 0cm 0.0. 0.0m 0.0m 0.0.. 0.00 0.00 0.05 0.00 0.00 0.00 — 00.0 “.2392 no 20.3.5828 0+.0 00.0 '1 0N0 JOON #6 m50.mmm2 go :ofipmpucmocoo may msmpm> msz Amvz an pocHEpmumo waspoSLumuuamm :oHu monocomop OCHEm can no zpfimcmpcfi Hmpwmpcfi m>aum~mn one 0W0 00.0 0.0 .10.— [*4 l o.— 40.. 9N t.“ .:u opsmfim ZHN- JO ALISNELNI mom: mama 170 versus concentration for Na22'GMP at 1°C and Na25'GMP at 2°C, reSpectively. The amount of solution self-structure for each nucleotide is also plotted in the figures. The relative integral intensity varies-from 2 to approximately 1 in both Na22'GMP and Na25'GMP. Although the amino proton intensity loss parallels structure formation, the onset of amino proton loss occurs at a lower concentration of nucleo- tide thaniflueonset of structure formation. The relative amino proton intensity approaches a minimum of approximately 1, which in the case of Na25'GMP occurs at about 35% solu- tion self-structure. As shown in Figure 75, heating a 1.81fl Na22'GMP solution causes the integral intensity to increase from 1.0 at 0°C to 2.0 at “0°C. The 2'GMP solution self- structure is totally melted out at 27°C as determined by the H(8) resonances. The amino proton relative integral intensity is always found to be 2 in TMA22'GMP or in TMA25'- GMP (3f. Table 14 and Table 15). The amino proton loss can be attributed to the forma- tion of a hydrogen bond, giving rise to a new resonance downfield of the non-hydrogen bonded resonances. Therefore, the proton exchanging with solvent in the H(8) region, H(8)€, occurring at 7.58 ppm in the ordered Na25'GMP solu- tion self—structure or the intensity which cannot be at- tributed to the imide proton can be a resonance of a hydrogen- bonded amino proton. The relative integral intensities of the a resonance and the integral intensity downfield of 171 woLSpMLoQEmu mo cofluocsg a mo mzo.mmmz Saw.a you x22 Amy: an omcasLopmp_opsuozppmumaom :oflpzfiom a on» pcm mocmcommn ocfism ocu no mufimcmucfi ngmeCfi m>fiumamp one .mm mpswfim A oo o manpémazu» 3... 99 con 98 2: ad 92.. 3 u . _ . _ _ . _ . _ . co m as. m. 1 no N m 0|- . m Wm 0.0N w.- l 0.p m w n I ‘ L w 93 ..... a. .. M m i. J m m 0.0.? H. L 9 m 4 1 t. m D 98 V - E .r. I R n ‘ A I 00’ m m 0.00 n. - .. m 92. w .. 3 m m - m mm 060 W .1 on m" m - . 1 H n . Z P. 0.00 n: l NN m l 09' u — u —r n _ p — b — n *0“ 172 9.0 ppm not attributed to an imide proton resonance are given in Table 16. Neither is correlated with the loss of amino proton intensity loss. The hydrogen-bonded amino proton may occur upfield of the amino group. It would not be seen due to the large water resonance. It is unlikely that the hydrogen-bonded amino resonance would shift upfield since hydrogen bonding usually causes a downfield shift. It is likely that the same type of phenomenon is occurring with Na22'GMP. Therefore the hydrogen-bonded amino resonance should occur in both systems. The resonance at 7.58 ppm, H(8)e, does not occur in Na22'GMP. This is added proof that the a resonance is not an amino resonance. The resonance is too far upfield for an imide proton, thus it must be a hydroxyl proton hydrogen-bonded in the Na25'GMP solution self-structure. It does not occur in the Na22'GMP self- structure, because the conformation of the nucleotide is different. This additional hydrogen bond should give the Na25'GMP solution self-structure more stability and should account for its larger percent of self-structure at any given set of conditions. 173 CH moocwcommg mhmvm 00m Hamvm on» mo 53m ocp.no 0:0.m :w mmocmcommn m cum m .5 on» no Sam .uoam on on oomeHumm who whoapmo .cofiuwhwoucfi Amvm thm UoCHELmuoo 0 .mmocmcomop oo030030umcs on» cam mmocmcomoh vanspospum vaz 0:» mo Esme .mzo.m CH 0 .mcoaumpucmocoo.acohommfic.pom mean on» go: who cam hampufinpm ohm muHCDm b o.o u--- oo.~ roots o.o o.o c.o mzo.mmaz mmfl.o o.o n--- ~¢.H co.c o.o o.o o.o mzo.mmmz mmm.o my ..I. Hm.H oo.o o.o o.o o.o mzo.mmmz mo~.o. my u--- mm.H oo.o o.o c.o o.o mzo.-az mom.o m.Hm u--- n--- m~.o ~.om. m.om o.a- mzo.mmmz mmm.H m.=m u--- mp.o ma.c m.m m.m= m.~m use.~mmz mam.fi mv. o.o . ~0.H o.o. o.o o.o o.o mzo.mmaz zmH.o my oo.o mp.” co.o o.o. o.o .o.o mzo.m~mz mofl.o NH mo.c mm.a oH.o- o.>u o.- o.m mzo.m~mz m:~.o pm HH.o so.H Ho.ou H.Ha H.=~ o.m~ azu.mmmz mm~.o mm mo.o mo.H Ho.ou m.on m.a~ >.mm mzo.m~az mam.o a: ofl.o Hp.o_ o=.o 0.0m =.om o.-~ mzc.mmmz m~:.o up mmyo ms.o m.:- =.m~a mmmam mzo.m~az mmo.o E E 3%... 1% ..._M.......l...c.. ”weigh” as? u . «=2 unxz Haaoauv HapmuacH Hayes .moofiuooaosz m30Hhm> no mcouopm ocfiE< on» cam .u .Ame .mcouOLm 00HE< on» no mzz couOAm an UoCHMuno amoapamcmch ngwoucH on» no COmfimeEoov< .oa mHnt 17A VII. Amino Proton Exchange With Solvent Water The unusual properties of the guanylic acid amino pro- tons led us to measure the at of exchange of the amino ’3 (D protons of Na+ and TMA+ salts of 2'GMP and 5'GMP with sol- vent water. The chemical shift and width at half-height, Vl/2’ were measured for the amino proton resonance and the water resonance of several guanylic acid solutions in H2O as a function of temperature. The‘program LORENT, given in Appendix 2, was used to calculate the two—site exchange rate of the amino protons by the method of Rogers and Woodbreylua. This program uses the chemical shift separation between the two exchanging nuclei and the values Of Vl/2 to estimate the rate of exchange. The program requires the chemical shift difference for the proton in sites A and B in absence of exchange, NUAX, and the width at half height in the absence of exchange, NUAX Vl/2abex' and vl/2abex were estimated by extrapolating into the region of fast exchange the chemical shifts and vl/2 of the NH2 protons line in the low temperature region where the amino proton exchange is slow. Figure 76 shows a typical example of this extrapolation. This plot is for the amino protons of 0.91% Na22'GMP, The open squares symbolize the amino proton chemical shift and the closed squares represent the vl/2' The chemical shift is ex- trapolated from the low temperature region to give the igure 76. 175 typical example of the graphic determina- tion of the amino chemical shift in the absence of exchange and the width at half- height in the absence of exchange. The closed squares represent the observed vl/g and the open squares the observed chemical shift of 0.91M N822'GMP as a function of temperature. The straight lines are the extrapolation from the low temperature region to give the chemi- cal shift of the amino protons and 01/2 in the absence of exchange. ,b 7. u l :v «a 0— On 0m ON on mpzmfim “gov 63239.5» Own COM CNN .....q._._J_. _._ .a L -- ....... ....---.. ...... - .............. --..-..Um..i:d/ ‘ . ' \“ .II' 4 .l. . I \\\ - '4 \ 1 fl \sl \ \\. I1 2 -\ A \ \ s \ 1 u s I.‘ \\ L r. \ \ 5 u. . 4 ~ . l S I \\ s u . 5* I \ I Q ~ ‘ s r. .. \ H § 1 All. k . . a A atohuoz 88.0 i _ r _ .Ofd ‘O m '6 006 wdd'g HIHS woman: 177 chemical shift of the amino protons in the absence of ex- change. Similarly, the vl/Q is extrapolated from below 290°K to give u The decrease in 01/2 as the tem- l/2abex' perature is increased from 270°K to 290°K is attributed to a decrease in the viscosity of the solution. The concentration of water is much greater (1 100X) than that of the amino protons, thus the water resonance was assumed not to be affected by the exchange process. Thus the chemical shift in the absence of exchange and V1/2abex were assumed to be equal to the observed chemical shift and the observed half-width, respectively. The popu- lation of the amino protons and water protons was cal- culated with the assumption that aqueous solutions are 55.0% in H20. The equations used in program LORENT are given in Ap- pendix 2. For a given value of T, a mean lifetime param- eter, the program calculates the theoretical NMR lineshape for the case of general two site exchange. Various values of T are tried until the observed NMR spectrum is matched. The mean lifetimes of protons on site A (-NH2) and site B (H20), 1 and 1 respectively, must be equal to the A B’ quantity - TATE TA TB Substituting into (“3) relationship (AM), where PA and PB 178 are the mole fractions of —NH2 and H20, respectively, and realizing that T P = T P‘ (an) PA+PB = 1, one obtains T = PBT (“5) The pseudo first order rate constant for exchange, ka, is given by 1 = .2— k TA (“6) substituting (“5) into (46) gives P k = 7? (A7) The results are tabulated in Tables 17 to 22. The errors generated in ka for estimated systematic uncertainties in the experimental input parameters are given in Table 23. ThUS, the errors in ka propagated by systematic errors in the experimental data are less than 6%. Measuring ka as a function of temperature allows the determination of activation parameters. According to the Arrenhius Theory 179 on Op oopmefipmo ppm mpogpm .oom Ca omcmzoxo Low ucmpmcoo mpmp Loppo umpflm oosomq .00“ on 00 woumefipmo ohm whoppm .N: NH on on poumefiumm mfi Logpm 059 .NE .N2 CH nuoflzamamn mzu mo omeHumm ooumaogmppxo .Nz CH amazm HooHEono oo>pomoo an on on oomefipmo mH LoLLo one .oom :fi Loposmpmq oEHpoMHH :moE .aew 055% m P can mze o 0 new .500 mmo.ow on on ooumepmo who mpopgm .phfizm HonEoco om>pomno one .omcwcoxo mo monomnm ozp CH umficm HmoHEono cowopq ocfiEm onu Lo opmefiumm ooamaoamppxo @590 .500 moo.ow mH pmficm HmoHEoco CH poppo one o .mCOpoLQ Loam; mo cowpompm pace on» ma mm .mCOpOLQ ocfiem mo coauompm oHoE on» ma H.ma H.w> m>>.z mo:.m mam.m mom w.mn mmao.o m.m H.ma m.mz mmw.: mmz.m oa:.m mom >.mw mmao.o m.w H.ma m.o: www.: mm:.w omz.o mom m.>: Hmo.o o.m H.ma o.Hm omm.= m©:.w >m:.w mam s.mm m:o.o m.m H.ma o.mm mom.: moz.m m::.w wmm m.oa H.mH 0.5H moo.m mw:.© mm:.m mmm w.o~ H.ma m.mH m:o.m mw=.m wm:.m mmm s.aa H.WH m.mH mmc.m mm=.m mmz.m mam o.ma H.MH H.ma moa.m moz.w mw:.m owm mg 0 Ommmxa> mzzmxa> m\H> ammo mzz m:z@ x0 oxon< moo nmno oxonme0o .N: Na on o» oopmefipmo ma Logpo one .N: a“ on ow wopmsfipmo ma pogpo ose .EQQ m00.0w mfi umwcm HmOHEoco CH LOLLw one 0%: anew o :90 0:90 0:040 .800 mm0.0fl on Op woumEHpmm opm mLoLLm .o0cmzoxo 0o mocomnm on» CH 00H2m HmoHEoco couOLQ ocflsm ocp mo omeHpmo copmaoamppxo 0:90 .pmflnm HmoHEoco 0o>Lomno 0:9 0 .m000opa Loam: mo :oHpQMLn oHoE on» ma mm amzopoLQ ocfism mo :ofipompm oHoE ozp ma <00 m.m:m 0:00.0 3.5 0.0m 0.0:H 000.: Hmm.0 m>0.0 mmm :.Nmm mm:00.0 m.> 0.0m 0.0ma 020.: m:m.0 sma.w mam m.m>a >m00.0 0.0 0.0m 0.HOH 050.: 0mm.0 m:H.0 mam 0.5ma m000.0 0.0 0.0m >.mm 0m>.: mam.0 00H.0 00m m.mma 2500.0 m.m 0.0m 2.00 mmw.: 00H.0 m0a.0 mom m.w 0.0m m.00 m0>.: mmH.0 HOH.0 now 0.0 0.0m 0.0m 005.: m0H.0 0ma.0 mom m.0 0.0m :.0m mmw.z 00H.0 0:H.0 00m m.m 0.0m ~.m: 300.: :ma.0 :ma.0 mum H.0H 0.0m m.mm H00.: HHH.0 :ma.m 00m 5.0 0.0m 5.0m 0mm.z m00.0 m00.0 mam mx 0 0m:m\fi> mmzmxa> N\H> 0mm© mzzmv mzz go oxmn< 0moo ammo oxo0< 0m000 dame ommm.o n ma oado.o am oumm mmcmsoxm couopm OCHE< Lonpo umpwm 0cm mLoposwme mamcmoCHq mzz .mzo.mmwz 2H0.0 now mandamcoo .mH mant 181 .wfi 0HQ0E Op mmuCCpoom 00m .0000 00:00 Eopm 0:0000H>00 0000a 00 030 Umuomhmp 003 E0000 mHLE0 m.mmm 0.00m 00:000.0 0.0 0.00 000.0 000.0 00m.0 0mm 0.00m 00000.0 0.0 H.0H 0.000 000.0 000.0 000.0 00m 0.000 0000.0 0.00 0.00 m.00 000.0 000.0 000.0 00m m.mHH 0000.0 0.00 0.00 0.00 mH0.: 000.0 000.0 00m 0.00 0000.0 0.00 0.00 0.00 000.: 0m0.0 000.0 00m 0.00 00H0.0 m.HH 0.00 0.0m 000.0 000.0 mm0.0 00m 0.00 0m0.0 0.mH 0.00 0.00 000.0 000.0 000.0 00m 0.00 0.00 0.00 000.0 000.0 000.0 000 0.00 0.0a 0.00 ma0.z 000.0 000.0 000 0.00 0.00 0.00 000.0 000.0 000.0 000 0.00 H.0H 0.00 000.0 000.0 000.0 000 0.00 0.00 0.00 000.0 000.0 000.0 :00 0 0 0 0 0 0 0 :0\H :20\H 0\H 0 2 02 :2 o x p > an<> mno> 0000 xmn<0 00o0 game 0000.0 u 00 m000.0 u <0 .020.0000 00000 00 030 00000000 003 50000 00:90 182 0.000 0000.0 0.0 0.0 0.00 000.0 000.0 000.0 000 00.000 0000.0 0.0 0.0 0.00 000.0 000.0 000.0 . 000 00.000 0000.0 0.0 0.0 0.00 000.0 000.0 000.0 000 0.00 000.0 0.00 0.0 0.00 000.0 000.0 000.0 000 0.00 000.0 0.0 0.0 0.00 000.0 000.0 000.0 000 0.00 0000.0 0.0 0.0 0.00 000.0 000.0 000.0 000 0.00 000.0 0.0 0.0 0.00 000.0 000.0 000.0 000 0.0 0.0 0.0 000.0 000.0 000.0 000 0.0 0.0 0.0 000.0 000.0 000.0 000 0.0 0.0 0.0 000.0 000.0 000.0 000 0.0 0.0 0.0 000.0 000.0 000.0 000 0.0 0.0 0.0 000.0 000.0 000.0 00 0.0 0.0 0.0 000.0 000.0 000.0 00 0 . 0 0 0 0 0 0 0 00\0 020\0 :20\0 0 z 02 :z 00 x 00 > xmn<> 000> 0000 x00<0 0000 0509 00000.0 u 00 00000.0 u 00 .020.0002 200.0 000 000000000 000m 00:0:0xm £00000 0:0E< 00000 00000 000 0000050000 000200200 mzz .00 00009 .NH manmb 0p mmpOCpoom mmm 183 o.HmH mmoo.o m.~ w.m w.wm :mo.= w~:.o mam.o ozm o.mma mmoo.o 9.0 >.m m.~m omo.: mm:.m :5m.m mmm m.NOH mmoo.o m.HH m.m m.~z Hm~.: :m:.m maa.m 0mm 3.05 mmHo.o H.3H :.w . m.om :m».: mom.m mm:.m mmm m.mm mwao.o m.mH m.m c.5m ~w~.: Hflm.o m~:.m 0mm :.mm mmo.o m.mH H.w m.om Hmm.: mam.o oom.o mam mm.mm 320.0 H.Hm o.m m.mH mmw.: wmm.m Nam.m on Hm.zfl 500.0 m.am mw.~ m.mH mmm.: umm.m Hmm.o mom No.0 oaa.o m.mH 5.5 o.HH Hmm.: m:m.m m:m.w com m.~H m.~ w m mmm.: mmm.m mmm.m mam H.mH :.> w m mam.: mom.m mom.m 0mm o.mH m.> H w :mo.m owm.m owm.o mam m.oa H.~ m N mmo.m mwm.o mwm.m 0mm 0. 0.5 a w Nwo.m 5mm.o 5mm.m mbm w.~ 0.5 m.> ooa.m Hmm.m Hmm.© mwm m m m m m m m o :m\H :2m\H :zm\H o m zz :2 x0 x P 9 xmn<> mno> mace xmn<© mace QEmB mam.o u mm mowoo.o n mno> mnomV xmn<© mace .QEmE ompm.o u mm .oamo.o u CH wwcmnoxm :ouopm ocfiE< on» now upoumempmm cofium>fiuo< on» no mmumefiumm .nm magma DISCUSSION Guanylic acids are chemically different from other nucleic acids. For example, enzymatic reactions frequently fail or proceed by a different course when guanosine is in- volved. A number of systems which demonstrate the unusual behavior of guanosine derivatives have been reviewedlBO. The studies presented here on the ordered aggregation of guanylic acids may answer some questions regarding some of these peculiarities. Guanylic acid solutions are a very complex system. The number of possible species in solu- tion is large; monomers, stacked monomers, monomers hy- drogen-bonded to each other, to tetramers, and to stacked tetramers. The requirement of high concentration of nucleotide for solution self-structure formation makes it impossible to use activity coefficients in calculations. Thus, the uncorrected concentrations must be used. Proton. NMR is the best method of study, although the high concentra- tions involved cause problems here also. The viscosity of these solutions becomes a factor in detailed studies which are affected by the relaxation times of the solu- tions. The high concentration and high viscosity do allow the easy accumulation of spectra because of the relatively broad line widths. Even with the unfavorable conditions 188 189 of high concentrations, high ionic strength, and high vis— cosity, proton NMR is still the best method of study of the solution self-structuring. Since the exchange of solu- tion self-structures with monomer is slow on the NMR time scale, non-equivalent environments can be observed for some protons in the NMR spectrum under conditions where struc- ture is present. The study of guanylic acid solution self-structures is also complicated by exchange fast on the NMR time scale between certain types of aggregated units. Base stacking and tetramer formation are reported to occur in guanylic acid solutions with concentrations of 0.02fl71. NMR allows the distinction of two types of aggregates, regular, or— dered self-structures and disordered structures. The ordered solution self-structures exchange slowly and give rise to new lines in the NMR spectrum, while the disordered structures exchange rapidly and are time averaged with the monomer resonance. Other methods such as IR are able to distinguish between ordered and disordered aggregation. I. Tetramethylammonium Ion as a Counterion The effect of TMA+ on nucleic acid structure and stabil- 151 ity has been investigated recently The stability of DNA double helix strands has been found to increase when TMA+ ion pairs to 5'GMP but, the ion is neither a struc- ture directing nor structure inhibiting ion. However, the 190 present work shows that TMA+ is not an "inert" ion in the aggregation of other guanosine nucleotides. Gels are not formed at neutral pH by the tetraalkylammonium salts of 3'GMP, 5'GDP, 5'dGMP, guanosine, and 5'GMP (pH=5) even at high concentration (>O.BOM) and low temperature (0°C). The addition of alkali metal ions to the ditetra- alkylammonium salts of 3'GMP and SiGDP causes gels to form at neutral pH but the concentration of alkali metal ion needed for gel structure formation is increased (of. Re- sults III and Results IV). The aggregation of 2'GMP solu- tion self-structures in the presence of alkali metal ions is greatly decreased in the presence of TMA+ as described in Results II.F. Larger tetraalkylammonium salts also show the same effect (of. Results II.F). Therefore, tetra- alkylammonium salts interact with 2'GMP, 3'GMP, and 5'- GDP in a mode which inhibits the self-aggregation. Since the inhibition of ordered structures by TMA+ does not occur for 5'GMP, the inhibition which occurs for the other nucleotides is most likely a steric effect. The. alkylammonium ions can ion pair to the phosphate group of the guanylic acid and sterically block the positions needed for aggregation. If a steric effect is Operating, TEA+ would have a larger destabilizing effect than TMA+. This assumes that the ion pairing to the phosphates is approxi— mately the same. Solubility problems do not allow the com— parison of TMA+ and TEA+ to be made experimentally. NOE 191 experiments (of. Results II.F) show that some of the TMA+ and TEA+ ions reside near H(S') and possibly near the amino group. This observation is in contrast to those of Brown“9 who found that TMA+ resides near H(8) in structural forms. Obstructing the NH2 group would result in the blocking of tetramer formation. The nucleotide conformation may also cause the tetraalkylammonium ions to block the stacking of tetramers. Figure 16 shows that the H(8) chemical shift of TMA22'- GMP is relatively invarient with temperature while the chemical shifts of Na22'GMP and K22'GMP vary greatly with temperature even under conditions where no solution self- structure is present. The same relationship occurs with chemical shift vs. concentration of nucleotide (3;. Figure 17). TMA+ is therefore interfering with structures which are exchanging fast on the NMR time scale. These results from TMA+-guanylic acid studies are sig— nificant. Guanylic acid substitution and phosphorylation reactions, among others, often have lower yields than the I analogous reactions of other nucleotides. This unusual decrease in reactivity could be due, in part, to the forma- tion of hydrogen-bonded aggregates, such as stacked tetra- mers, in solution. Using a TMA+ exchanged guanylic acid system to replace alkali metals may cause a significant increase in product yield. The use of TMA+ in reaction systems may also affect the product distribution. Another 192 important use of TMA+ as a counterion for guanylic acids is in the elimination of the formation of large ordered aggregates in solution. In some reactions of guanylic acids it may not be desirable to conduct the reaction under conditions complicated by gel formation if an alkali metal ion is present. Replacing the alkali metal ion with TMA+ would eliminate the problem of gel formation. II. Base Stacking Interactions Despite a large body of literature, the base stacking phenomena of nucleic acidsenwanot well understood22’l63‘l65. Interactions involving base-stacking could play an impor- tant role in the selective recognition of individual nucleotides by macromolecules such as proteins and 166,167. enzymes Proton NMR has proven the most informa- tive method of investigation. Most investigations assume hydrogen bonding between nucleic acid bases does not take place in water. This is not reasonable at least for guano- since derivatives. Guanosine derivatives form gels, ex- tensively hydrogen bonded structures, at concentrations as low as 0.01fl7u. Most evidence suggests base stacking proceeds far beyond the dimer stage. Data are usually 160 analyzed by the isodesmic approach which assumes the stacking phenomenon is non-cooperative. The self-stacking tendency decreases within the series adenosine > guanosine > . . . 6" inosine > cytidine % uridinel 3. Sukhorukov and coworkers 193 determined that Na+ > Li 164. + +. .. .. > K in stabiliZing the staCKing phenomenon Neurohr and Manisch163 have reported the isodesmic -1 stacking constant for 5'GMP at 30°C as 1.3M They em— phasize the hydrophobic nature of this phenomenon since base stacking only occurs in water. Classical hydro- phobic interactions are characterized by positive enthalpy 63 and positive entropy changesl . The base stacking self- association of nucleic acids, however, is accomplished by negative enthalpy and entropy changes, as determined by 168-170 calorimetric measurements of several systems and by 163. proton NMR Moreover, the hydrophobic interaction is a rather unspecific phenomenon, which cannot account for the observed high degree of ordered structure formation. Two different geometries have been prOposed for the base stacking of 5'AMP, differing in the placement of the ribosyl substituents relative to each other. Ts'o and 23,27 171 coworkers and Evans and Sarma have proposed a head-to-head arrangement, which places the ribosyl moieties on the same side of the stack. More recent resultslsu’l72’ 173 1 , based on Nuclear Overhauser effect studies and H spin-lattice relaxation measurements, favor a head-to- tail arrangement, where the ribosyl moieties are placed on opposite sides of the stack while retaining the same stack- 163 conclude that the ing pattern. Neurohr and Mantach geometry of interacting adenosine nucleotides can be a combination of both head to head and head to tail stacking. 19“ Individual nucleotide molecules are in rapid motion and base-stacking is a very fast process. III. Tetramer Formation Cation dependent aggregation processes that occur in guanylic acid-alkali metal ion systems cannot be directly observed by the appearance of new H(8) NMR resonances be- cause of fast chemical exchange between the aggregates and the monomeric nucleotide. For example, with 2'GMP, H(8) upfield shifts occur with increasing concentration (of. +, or Rb+. The Figure 17) when the counterion is Na+, K extent of the shift is greatest for K+ (gf, Figure 1?) salt. In contrast the H(8) resonance of the TMA+ salt shows no concentration or temperature dependence (of. Figure 16 and Figure 17). Thus a cation dependent pro- cess similar to that observed for regular ordered struc- ture formation is operating, but the aggregated species which is formed is in fast equilibrium with monomer. We propose that this structure in fast equilibrium with monomers is a tetramer. The metal ion size dependence may be the result of metal binding in the cavity formed by the four carbonyl oxygens of the tetramer unit. The alkali metal ion binding increases the stability of the tetramer unit. Tetramethylammonium ion would bind weakly, if at all, to the carbonyl oxygen cavity. Tetramers are not stable in the absence of alkali metal ions. The increase in 195 half-width of the H(8) resonance (of. Results II and Ref. A9) is also explained by the formation of tetramers. In- creasing the concentration of a dialkali metal 2'GMP solu— tion causes larger line broadening than in a tetraalkyl- ammonium 2'GMP solution even in the absence of solution self-structure. A base stacking interaction is unlikely since the chemical shift of H(8) is alkali metal dependent (of. Figure 16 and Figure 17). Tetramers have been postu- lated previously for alkali metal solutions of guanylic acids. Based on hypochromicity measurements Chantot and 71 have proposed that tetramers are present in Guschlbauer a 0.003M 3'GMP solution at pH=A and at 0°C containing 0.25M CH3COOK. They propose that gel formation occurs by the stacking of tetramers. Other nucleotides were reported to form tetramers at concentrations lower than 0.01%. Lee 17“ have calculated a tetramer formation constant and Chan of 2.5:O.5 x 107 M73 and a tetramer stacking constant of 40:10M'1 in the case of 5'GMP, pD range 3-6. These results were based on the concentration and temperature dependence‘ of the H(8) NMR chemical shift and line width of several guanylic acids at various pD's. The results of Lee and Chan are suspect since EDTA was found to significantly af- fect the H(8) half-width of the sampleslSl. The loss of amino proton relative integral intensity for 2'GMP and 5'GMP (3:. Figure 73 and Figure 7A) may be due to the formation of tetramers. The amino proton 196 intensity loss for both nucleotides occurs under conditions which are favorable for tetramer formation. When the alkali metal ions are replaced by TMA+, as in TMA22'3MP and TMA25'- GMP, the amino proton relative integral intensity is two under all conditions, even at high concentrations and low temperature. Tetramethylammonium ion is not expected to stabilize the formation of tetramers due to its relatively large size and low charge density. The onset of amino proton relative integral intensity loss occurs before the onset of solution self—structure formation, which is con- sistent with the formation of tetramers. A critical con- centration of tetramers must be attained before solution self-structuring (stacking of tetramer units) can occur. In the case of 0.70fl Na22'GMP at 1°C, there is a relative integral intensity loss of approximately 0.2 protons (of. Figure 73) before solution structure-formation occurs. If we assume one amino proton per 2'GMP unit in the tetramer is involved in the intensity loss, the con- centration of tetramer present would be 0.036fl. If the intensity of both amino protons is lost in the formation of tetramers the concentration of tetramers would be 0.018fl. The mechanism we propose for the amino proton intensity loss is shown in Figure 77. Monomers, which undergo slow amino proton exchange with water are in fast equilibrium with tetramers. The tetramers undergo very fast exchange of amino protons with solvent water. Tetramers are in slow 197 Solution Self-Structure Protons exchange between MHZ-solvent may be fast + . M slow / Very fast MHZ-solvent exchange / M fast / Monomer Slow NH2-solvent exchange ’11 09 i ure 77. The proposed mechanism for the loss in amino proton NMR intensity for Na22'GMP and Na25'- GMP. We expect this mechanism to occur with other alkali metal structure directing ions. 198 equilibrium with ordered self-structures, which may also have a very large amino proton exchange rate with water. The very fast amino proton exchange causes the amino proton intensity loss. It is unclear if the relative amino proton integral intensity loss approaches a limit of one proton lost per nucleotide. Solubility limitations do not permit investigation of solutions at concentrations where the integral intensity loss could be significantly less than one proton intensity. If both amino protons of the nucleotide are exchanging in a tetramer unit, the amino proton exchange with solvent water must be much faster than the formation of tetramers by monomer. If only one of the amino protons in a tetramer unit is exchanging, it is unclear which of the two amino protons is exchanging fast. An investigation of N2-methyl- 5'GMP would resolve the question. The amino proton in- volved in the hydrogen bonding of the tetramer would be replaced with a methyl group. If the hydrogen-bonded amino proton is exchanging the amino proton intensity should decrease greatly at high concentration and low temperature. If the bonded amino proton not involved in the hydrogen bonding of the tetramer is exchanging the integral intensity should not decrease on increasing the concentration and decreasing the temperature. Two points must be made clear: (1) the amino proton intensity loss in absence of solution self-structure is 199 not accompanied by the appearance of a new line elsewhere in the spectrum. Thus it must be concluded that fast ex- change is occurring with solvent, (2) exchange is being catalyzed (i.e., exchange is much faster than with TMA+ salts) by the structure directing alkali metal ions. Thus the need for postulating a self-structure which is in fast equilibrium with monomer and for which solvent amino ex— change is very fast. The proposed structure for the exchange of the amino proton with solvent water in a tetramer unit is shown in Figure 78. A water bridge is formed between the amino proton not involvedixiinter-base hydrogen-bonding formation and the phosphates of an adjacent nucleotide in the same tetramer unit. The amino proton is exchanging rapidly with the bridged water molecule and replaced with the hydrogen of another water molecule. Figure 79 is a photograph of a CPK model showing the structure proposed. This model places 5'GMP in an anti conformation with X = 60°. The conformationeflxnu:C(5')-0(5') is gg and about C(A')-C(5') is gg. This is the preferred conformation of 5'GMP in aqueous solutionll’182. The amino proton exchange in this model is extremely dependent on the phosphate position. But because 2'GMP prefers a different conforma- tion, building a model of the tetramer unit in 2'GMP shows that the phosphate is able to form a water bridge with the amino proton. The 2'GMP conformation in the water bridged 200 (DH (DH Figure 78. A segment of the proposed tetrameric struc- ture for the exchange of the amino proton of Na 'GMP and NagS'GMP with solvent water. This structure has x for Na25'GMP and Na22'- GMP at 60° and 150°, respectively. Figure 79. 201 A photograph of a CPK model of the proposed structure for the exchange of the amino pro- ton of NagS'GMP. The arrow points to the water bridge formed between the phosphate group of one nucleotide and the amino proton not involved in the hydrogen bonding of the tetramer unit of an adjacent nucleotide. 202 tetramer is syn with x = 150°. This is the preferred con- formationl75. There is precedence for the occurrence of a water bridge with a phosphate group in a cyclic nucleotide system. Bolton and Kearns have proposed that a water bridge occurs between the phosphate and 2'OH group in cyclic nucleotidesl3l. 176 McConnell has reported evidence that adenylic acid-NH 2 proton exchange at neutral pH occurs almost exclusively by a two step mechanism in which transfer of the -NH2 proton to solvent water is preceded by protonation of the basic endocyclic nitrogen of the purine N(l). This mechanism is 130. In the proposed to occur in the other nucleic acids solution self-structure and in tetramers the N(l) is pro- tonated, with the N(l) involved in a hydrogen bond. Other evidence that a tetramer unit is present in guanylic acid solutions before the onset of solution self- structure is the occurrence of an IR band at 1530-15A0 cm-l. The band is always present when an NMR observable solution self-structure is observed but the band does not behave as if it is associated exclusively with the ordered self- structure, as can be seen in Table 5. A large increase in the extent of ordered solution structure does not cause a large increase in the IR band. This absorbance may arise from the formation of tetramers. The formation of tetra- mers, therefore, occurs before the onset of ordered self- structure. Although one might expect an increase in the 203 absorbance of this band with increasing concentration of monomer, the band intensity in fact remains constant even over the concentration range from 0.95 M Na22YGMP (<5% structure) to 0.71 M Na22'GMP made 1.1 w in NaCl (m50% structure) (cf. Figure 2A and Figure 25). Apparently, a critical concentration of tetramer is retained over the entire concentration range. A small concentration of tetramer would be consistent with a highly cooperative ag- gregation process. IV. Self—Structuring Tendencies of Guanylic Acids A. Effect of the Phosphate Position on Self-Structuring At pH = 7-8 we can separate the guanylic acids into two groups, those that form gels and those that form solu- tion self-structures. The nucleotides that form gels (3'- GMP and 5'GDP) have a greater tendency to self-structure than those that form only solution soluble, ordered self- structures (2'GMP, 5'GMP). The concentration of nucleo- tide needed to cause galtion at constant temperature and metal ion concentrations can be taken as an indication of the tendency for the nucleotide to self-structure. The order guanosine > 3'GMP > 5'GDP > 5'dGMP is obtained67 using this system, when Na+ is the structure directing cation. The amount of solution self-structure formed under a given set of conditions by 5'GMP and 2'GMP indicates that 20“ the former nucleotide has a greater tendency to aggregate. Evidence to date suggests that the solution self-struc- ture is formed by the stacking of tetramer units. Gels have been shown by X-ray fiber diffraction studies to have either a stacked tetramer arrangement or a helical ar- rangement72. The tetrameric arrangement of the bases and the ribose is the same in all of the guanylic acid deriva- tives. The differences in structure forming tendencies for different GMP isomers must therefore be attributed to the position of the phosphate group. The phosphate group can stabilize, destabilize, or not affect the self structuring depending on its position. The phosphate group can stabilize structures by forming hydrogen bonds between tetrameric plates. A destabilizing effect can occur if the build-up of negative charge on the phosphate groups causes a repulsion between plates. This destabilization effect is seen in Na25'GMP. At pH = 5, 72 the phosphate where 5'GMP forms a helical gel structure group is a monoanion. As the pH is raised to 7-8, the phosphate group becomes dianionic, and the gel structure is destroyed and replaced with a solution self-structure. The increased negative charge causes smaller structures to be stable. One can rationalize the structure forming tendencies of the guanylic acids as follows: guanosine, containing no phosphate, has the highest structure forming tendency. 205 The phosphate repulsion must always be greater than its possible stabilizing effect. The differences in ordering tendency for the other nucleotides arise from differences in the phosphate group position. The stability of ordered structures will depend on phosphate-phosphate distances and the ability of the phosphates to form interplatelet hydrogen bonds. B. Experiments on Mixed Nucleotide Solutions It is expected that in systems with two GMP nucleotides tetramers would be formed of compositions [GMP(1)1n[GMP- (2)]U-n where n = 0,1,2,3,A. The bases are the same in all of the guanylic acids. The hydrogen bonding scheme of the mixed nucleotide tetramers should be the same as in the pure systems. The ribose and the phosphate group are not cap- able of sterically blocking the hydrogen-bonding sites. Solution self-structure would then be composed of two or more stacked mixed tetramers. The systems with two nucleo- tides should give rise to more H(8) environments than a homogeneous system. If there is incorporation of one nucleotide unit into the regular ordered structure of another, new lH resonances should appear in the NMR spectrum. Since there is not much difference between the H(8) environments of different guanylic acid self-struc- tures, new peaks may not be resolved, but there should be an increase in the amount of structure because the addition 206 of the second nucleotide will cause an increase in the total nucleotide concentration which should force the equilibrium toward self-structure. In the case of a guanosine-Na25'GMP system, 5'GMP was incorporated into the guanosine self-structure (3:. Re- sults I.B). Guanosine has a greater "structure-forming tendency" than 5'GMP. In all the samples containing guano- sine and 5'GMP, guanosine is in large aggregates that are NMR unobservable. The samples with concentrations of guano- sine >0.01M would have gelled in the absence of 5'GMP. Gelation is most likely inhibited by the 5'GMP which is in- corporated into the guanosine gel structure. The repulsion between adjacent phosphates causes the formation of ag- gregates which are smaller than those needed to form gels. The addition of the negatively charged group into the gel structure, also causes the large increase in the solubility of guanosine. The model proposed for the guanosine-5'GMP structure is given in Figure 80. The guanosine unit is symbolized as a curved arrow, M+ is a structure directing metal ion, and the solid line extending from a guanosine unit is a phosphate group. Thus those guanosine units with a solid line represent 5'GMP units. All of the guanosine present in the guanosine-5'GMP solutions is present in these aggregates. The aggregations are of various sizes. Thus, in the H(8) NMR region a road 5'GMP resonance is present which arises from the Figure 80. 207 The proposed structure for a mixed guanosine and Na95'GMP self-structure. The guanosine unit is symbolized as a curved arrow,M~=+ is a structure directing metal ion, and the solid line extending from a guanosine unit is a phosphate group. 208 smaller structures and an intensity loss occurs in the H(8) line due to incorporation of 5'GMP into guanosine units which are larger and NMR unobservable. The results of a TMA22'GMP-Na25'GMP experiment are dramatically different from those obtained from a Na 2'- 2 GMP-Na25'GMP experiment. In the TMA 2'GMP-Na25'GMP system 2 there is a large decrease in the Na25'GMP solution self- structure (cf. Figure A6) while in the Na22'GMP-Na25'GMP system there is only a small decrease in the NaZS'GMP self- structure (3:. Results II.G.). There is a structure forming interaction between 2'GMP and 5'GMP as evidenced by these results and the fact that TMA+ does not affect the Na25'- GMP solution self-structureug. The results can be ex- plained by the model shown in Figure 81. When mixed, the two nucleotides, 2'GMP and 5'GMP, interact both by stacking interactions and hydrogen bonding. Mixed nucleotide tetra- mers can be formed by 5'GMP and 2'GMP. When TMA+ is present in solution, the TMA+ blocks the formation of mixed nucleo- tide tetramers by inhibiting the 2'GMP from completing a . tetramer unit. This is consistent with the results of TMA+ inhibition of the 2'GMP self-structure. Therefore the decrease in the extent of ordered 5'GMP self-structure which occurs upon addition of TMA22'GMP is due to the inhibition of the formation of mixed nucleotide tetramer units by TMA+. In the TMA+ free 5'GMP-2'GMP system, mixed nucleotide tetramers form but stacking of 2” Figure 81. 209 SOlUTlON SELF-STRUCTURE It 4.: o . F” 2 + 2 x ”k xx The proposed model for the destabilization of Na25'GMP solution self-structure in the pres- ence of TMA22'GMP. The arrow symbolizes a guanosine unit, the solid line extending from a guanosine unit is a 5' phosphate, an open circle extending from a guanosine unit is a 2' phosphate, and the solid curve is a TMA+. 210 mixed tetramers to form solution self—structures is not stable, which accounts for the line broadening of the H(8) resonance in 2'GMP and the decrease in solution self-struc- ture for 5'GMP. The addition of guanosine to an unstructured Na22'GMP solution causes the guanosine H(8) NMR region to be very similar to that evidenced by Na25'0MP self-structure (3:. Figure A7). Thus, the 2'nuc1eotide and the nucleoside, which form no ordered solution self-structures by them- selves under similar conditions, form a solution self- structure when mixed, though guanosine would form an ordered gel structure. A model similar to Figure 77 is proposed. The gel structure of guanosine is destabilized by the forma- tion of mixed nucleotide-nucleoside tetramers. The 2' phosphate group, by the action of its dinegative charge, causes the polymerization of tetramers to be limited to small units. In 3'GMP-Wa25'GMP experiments, TMA+ was shown (gg. Results III) to inhibit structure formation by 3'GMP. There is also a small loss in the Na25'GMP self-structure. We postulate a mechanism similar to that proposed for the TMA22'GMP-Na25'GMP system (of. Figure 81) in which the TMA+ is blocking the formation of tetramers. Since the decrease in the amount of solution self-structure of 5'- GMP is not as dramatic as in the TMA22'GMP-Na25'GMP system, the inhibition must not be as strong in the TMA23'GMP-Na25'GMP 211 system. A new H(8) resonance is observed at 7.50 ppm in the proton NMR spectrum of the TMA23'GMP-8-dNa25'GMP ex- periment (of. Figure 5“). If the same mechanism is oc- curring in the 2'GMP-5'GMP system, a new resonance should also be observed in the 2'GMP H(8) region. No such reson- ance is observed (pf. Results II). This discrepancy is explainable if the 2'GMP units in the 2'GMP-5'GMP aggre- gates exchange rapidly with 2'GMP monomer. In the 3'GMP- 5'GMP system, the 3'GMP units in the mixed aggregates may exchange slowly with 3'GMP monomer, thus giving rise to the 3'GMP H(8) resonance at 8.50 ppm. The 2'GMP-5'GMP mixed aggregate resonance may also be coincidental with the 2'- GMP monomer resonance. As in the 3'GMP-5'GMP experiment, TMA+ was present in the mixed Na25'GMP-TMA25'GDP study. TMA+ has been shown here to inhibit self—structure formation by 5'GDP (of. Results IV). As shown in Figure 57, several new 5'GDP H(8) resonances are present in a Na25'GMP-TMA25'GDP solu- tion. As shown in results IV.A, Na25'GDP does not form an. NMR observable solution self-structure, though it does form a gel structure. Mixed nucleotide tetramers of 5'GMP and 5'GDP must form, then stack to yield solution self- structures which give rise to the new 5'GDP H(8) reson- ances. The phosphate of 5'GDP in the aggregated units may be oriented outward toward the solvent, thereby minimizing the repulsion between phosphates. The larger size of the [‘J [.4 [\J dianion diphosphate allows its negative charge to be dis- persed over more oxygen atoms, and leads to smaller repul- sions relative to 5'GMP. The large number of new H(8) resonances observed for the mixed 5'GDP-5'GMP aggregation suggests that a larger number of structures is formed in this solution than in an ordered 5'GMP system or in any of the other mixed nucleotide-5'GMP systems discussed previously. V. The Best Model for Ordered 5'GMP A. The Assignment of Resonances The study of the exchangeable protons in guanylic acids has provided valuable information on the solution self-structuring of guanylic acids. The amino proton intensity loss with regards to tetramer formation has already been discussed (9:. Discussion III). The e reson- ance (of. Figure 70) of Na25'GMP can be assigned to a hydroxyl proton, though this argument is not unequivocal. If correct this would mean that the 2'OH or the 3'OH of 5'GMP is involved in the stabilization of the Na25'GMP solution self-structure. At least three imide resonances are present in both 2'GMP and 5'GMP, and these suggest the presence of more than one type of structure. The chemical shift differences arise from the magnetic an- isotropy in the tetramer units. Analysis of the symmetry 213 of the stacked tetramer model shows that the possible number of different imide resonances for different twist angles are 1,2, or A. The evidence to date suggests that at least two solution self-structures are present in Na25'- GMPug. The "extra" integral intensity of the imine reson- ance in the structured solutions of 5'GMP may be due to a hydrogen bond between a phosphate group and a hydroxyl (IQ roup (of. Results VI). Alternatively the "extra" inten- sity may be assigned to amino protons which are slow to exchange with solvent (of. Results VI). The decrease in the outer H(8) lines of solution self-structured Na25'GMP (i.e., a and 6 lines) in saturation transfer and NOE experiments where the water resonance is irradiated implies that water must be located very close to the H(8) proton in these structures (of. Result VI), more so than in the monomeric nucleotide and the other structure(s) which gives rise to the H(8) B resonance. It is believed that the outer H(8) lines arise from the same structure. In addition to the NOE results, Pinnavaia89 has shown that the addition of Mn+2 to a 0.60M Na25'GMP solution at 7°C causes the monomer and the B resonance to A 2 paramagnetically broaden until at 9.1 x 10- M Mn+ , the B and 6 resonances are no longer visible. The outer lines are only slightly broadened. The outer H(8) lines 89 also have similar T1 and T2 values , and they grow into the spectrum at equal rates when NaCl is added to 21A TMA25'GMPH9. The results of the saturation transfer experiments (Result VI) suggest the structure giving rise to the 8 resonance exchanges faster with monomer than the struc- ture giving rise to the a and 6 resonances. The tetramers must separate faster in the base stacking pattern which gives rise to the 8 resonance due to differences in the base stacking stability or the hydrogen bonding. These data lead us to suggest the reaction scheme shown in Figure 82 for the structuring of Na25'GMP. B. Other Models That Have Been Proposed 99-106 Laszlo and coworkers have proposed several dif- ferent structures for self—assembled Na25'GMP and K25'GMP, based on 1H, 31F, 23Na, and 39K NMR results. One of his early models is the staircase model shown in Figure 9. There are several discrepancies in the staircase model. These workers propose the same coordination sites and identical arrangements to account for the Na+ structure and the K+ structure. This postulate seems unreasonable since the Na+ and K+ structures behave differently under similar conditionsug. In rationalizing the multi-line H(8) environments in the K+ systems, Laszlo changes the number "up" and "down" in an octamer stair- of phosphates pointing case unit. To achieve these "up" or "down" orientations of the ribose, a conformation other than anti must be 215 Disordered Structures (Stacked Monomers) Very Fast exchange Monomer Very Fast Exchange Tetramers Slow Slow Exchange Exchange Solution Self-Structure Solution Self-Structure giving rise to a and 6 giving rise to the B H(8) NMR Resonances H(8) NMR Resonance exchanges slowly with exchanges slowly with monomer tetramers but the exchange is faster with tetramers than the structure giving rise to the a and 6 reson- ances. Figure 82. The proposed reaction scheme for the structur- ing of Na25'GMP. 216 generated about the glycosidic bond. If Sundarlingham and Westhoflo "rigid" nucleotide concept is correct, there are definite preferred conformations of nucleotides. Las- zlo's model implies the conformation of nucleotide is dependent on the alkali metal counterion. Most importantly, Laszlo's model ignores the precedent for the tetramer stack- ing pattern observed by X-ray fiber diffraction studie822’ 72. The staircase model totally ignores the favorable base overlap of a stacked tetramer model. Models for the solution self-structure of 5'GMP have been proposed by Brownug. Those for Na25'GMP and K25'GMP are shown in Figure 8. The Na25'GMP self-structure consists of 2 stacked tetramers with one Na+ associated with the cavity of each tetramer. There are also four Na+ ions chelated by interplatelet phosphate oxygens. The K+ and Rb+ structures have the metal ion coordinated to 8 carbonyl oxygens between the tetramer units, a simple structure being an octamer and a more complex structure being a hexadecamer. Marshall97 has determined that the structured form of Na25'GMP contains eight nucleotide units and four Na+ ions. Thus he modified Brown's model by having only two Na+ ions chelated by interplatelet phos- phate oxygens. He also postulates a water molecule in the octameric hole formed by the two plates to separate the + 0 two Na ions near the centers of the tetramers. Fisk and coworkers98 have measured the Tl's for the 13C atoms in 217 Na25'GMP and have concluded that the most stable species formed in the self—structure is an octamer, formed by the association of two tetrameric plates. She postulates that at least three other less stable, stacked complexes are pres- ent in solution with octamer. C. The Model Proposed for the Na25'GMP Solution Self- Structure The model we propose for the solution self-structure of Na25'GMP which gives rise to the a and 6 lines is similar to that of Brown“9 and Marsha1197. The structure is composed of two stacked tetramer plates, each having a five coordinate Na+ in the tetramer cavity with a water molecule at the fifth coordination site of each Na+. A time averaged number of two Na+ are territorially bound (solvent separated ion pairing) to the outer sides of the structure. A water molecule may be present in the octa- meric cavity between the two tetrameric plates, hydrogen bonded to the carbonyl oxygens. The two tetrameric plates are in a head to head stacking arrangement with a twist angle of 30°. The ribose has a gg conformation about both C(5')- O(5') and C(A')-C(5') which corresponds to C-2'-endo. The glycosidic bond is anti with x = 60°, the preferred con- 11,182 formation for 5'GMP This conformation allows a water bridge between a phosphate group and the amino group 218 of an adjacent nucleotide in the same plate, which cata- lysis the amino proton exchange with solvent accounting for the intensity loss discussed earlier (3: Discussion III). Intramolecular hydrogen bonds can be formed between the 2'OH and 0(1') of adjacent plates and between the 3'OH and the phosphate oxygen of adjacent plates or between 2'OH and 0(1') of adjacent plates and between the 3'OH and a phosphate oxygen which is bonded to the C(5') of an ad- jacent plate. The imide proton in this conformation is in two non-equivalent sites, which from ring current maps of guanylic acids (of Figure 83) should be m0.2 ppm different in chemical shift. We therefore assign NH(l) and NH(2) to these imide protons. A schematic representation of this structure is illustrated in Figure 8A. Pictures of a CPK model of this structure are shown in Figures 85 and Figure 86. Other evidence which supports this structure is the NOE experiments of Brownug. Placing a TMA+ near a phos- phate group in this model also places it near an H(8), thus when the TMA+ resonance is irradiated a negative Nuclear Overhauser effect results in an intensity loss in the H(8) resonance as seen by Brown. Another observation by Brown“9 was the unequal loss of intensity of the outer H(8) lines on irradiation of the HBO resonance. This model places a water molecule, that involved in the phosphate-amino water bridge, close to H(8). The H(8)'s of the upper and lower 219 Figure 83. Expe ted upfield shift (in ppm) of a nucleus 3.5 above or below the aromatic plane due to the ring.current and diamagnetic susceptibility effects. Taken from Reference 97. 220 Figure 8A. The prOposed model for the solution self- structure which gives rise to the a and 6 H(8) resonances in Na 5'GMP. The shaded rec- tangles represent a t tramer unit. Figure 85. 221 A photograph of a CPK model of the proposed model for the solution self-structure which gives rise to the a and 6 H(8) resonances in Na25'GMP Figure 86. 222 A photograph of a CPK model of the proposed model for the solution self-structure which gives rise to the a and 6 H(8) resonances in Na25'GMP. 223 plates are not equivalent. The H(8)'s of the lower plate are located near the 2'OH of the upper plate, a location which would lead to a larger negative Nuclear Overhauser effect when the HDO resonance is irradiated, since the 2'0H and H20 are most likely in rapid equilibrium. The structure which gives rise to the H(8) 3 reson- ance is proposed to be a head to tail stacked set of tetramers. This structure does not allow the same hydrogen bonding scheme as the head to head stack. Keeping the water bridge between the amino proton and the phosphate, there are two head to tail stacking patterns, one with the ribose phosphates of each plate pointing away from the center of the plates (I) and the other with the ribose phosphates of each plate pointing toward the center of the plates (II). Structure I does not allow for favorable hydrogen bonds to be formed. The plates can rotate freely because there is no steric blocking by the ribose-phosphate. Structure (II) is unfavorable due to steric interactions (e.g., phosphate-phosphate repulsion) if a plate to plate distance of 3.A A is to be maintained. Thus, the H(8) B resonance may arise from a head to tail stacking pattern I. Since the plates are free to rotate only a time averaged signal will be observed for the H(8) resonance. Some of the structures in the more complicated K+ solution self-structure are proposed to have a similar hydrogen bonding scheme as the head to head and head to 22A tail models proposed for Na25'GMP. A K+ is believed to be in the octameric hole between two tetrameric plates. Other structures are believed to arise from stacking of this structure unit as shown in Figure 87. The other nucleotides are believed to have solution self-structures similar to that proposed for 5'GMP. The phosphate and hydroxyl groups are no longer in the same position but assume a conformation which gives the best hydrogen-bonding scheme. VI. Ethidium Binding to Unstructured and Structured Forms of 2'GMP and 5'GMP Ethidium bromide was found to bind to both structured and unstructured guanylic acids (3:. Results V). The inter- actions of ethidium with guanylic acidscauserchanges in the protonlflfiispectra of the ethidium similar to those found for ethidium intercalation in oligonucleotides. Ethidium has been shown to form a 1:1 and a 2:1 complex with both unstructured 5'GMP and 2'GMP (3:. Results V.A). The equilibrium constants for the formation of the 1:1 complex A -1 for both 5'GMP and 2'GMP is very large, >10 M The equilibrium constant for the 2:1 (nucleotidezethidium l for both nucleotides (of. Results complex) is 10-20M- V.A). The only other equilibrium constant reported for the binding of ethidium to a nucleotide is that of ethidium binding to 2'deoxyadenosine by 1H NMR by Jordan and 225 .oumcnmonalomonfin m poempump on» song wcaocopxo mafia on» can pass soemppou a acomonaop moawcmpoop one .mzo.m new mzu.m mo opsuosaumlmaom cofiusaom +0 on» now Hooos comonopa one \n .- u/ .50 madman @o l T \— nllll.v - Ir - 0.8V L < l 7 _\ _/ coworkersl77. The K -1 association value they determined is 3.0 x 102 M It is not unexpected that the K association of ethidium binding to guanylic acids is larger than that to adenylic acids since ethidium has been found to prefer guanosine regions of oligonucleotides and DNA. In addition to the equilibrium constants for ethidium binding to unstructured 2'GMP and 5'GMP, we have measured the AH and AS of the formation of both the 1:1 and the 2:1 unstructured ethidium complexes, to our knowledge the first determination of thermodynamic parameters be- tween this intercalator and a mononucleotide. The values for 1:1 complex are AH = -19.78i0.96 Kcal/mole and AS1 l = -53.3¢6.6 eu, for the 2:1 complex they are AH2 = 0.07 i0.26 Kcal/mole and AS2 3.9 1.6 eu. The AH is in agree- 1 ment with the theoretically calculated value of AH = -l2.3 to -2A Kcal/mole for minihelicesl26, and the values of Sturgi11183, AH = -20il Kcal/mole and TAS = -A Kcal/mole based on several calorimetric experiments for Etd inter- calated into tRNAPhe under conditions of incomplete saturae tion. LePecq and Paoletti116 have found that both aH and AS are positive at low ionic strength, but both are negative at high ionic strength for the binding of ethidium bromide to DNA at different sodium ion concen- tration. Extremely large ethidium methyl resonance shifts are observed when ethidium is added to a guanylic acid which contains solution self-structure. '7 O \Y a Y a GMP’ and na92'GflP (gg. nesults J.E) the amount 01 solu- t10n self-structure was increased upon addition of ethidium. R ‘ 97 i w Varsnall has reported a decrease in the amount of n25'- datum is suspect because integration of the H( In the case of Na25'- difficult due to the overlapping of several resonances. The binding of ethidium to tetramers which may be present in solution cannot be neglected. available to determine f ethidium bind Ho ent in solution. It seems likely that to stacked tetramer it would bind to tetra is possible that tetramers containing b stack because of the lower net negative association constants for the binding 0 the model given by Equations F 1 + ‘0 fi - P'lQD + .&Q 4- 2'3.) ..v~ U 2 ‘J a C ' : ‘ q 7 ‘ ‘ 0- ils da.a re suspect, s-nce the rcde- . No evid nce to 4i}- .4 9; 0'1.- £12.: '1- “0 0 4-1 -421 O w-y- .L...L / .\ / -' .. A..- «I.-- :- Y‘- :a \H (A) \/ date is 228 (3) - (A) and (23) - (25). A satisfactory fit was ob- tained with this model for the binding of ethidium to Nag- 2'GMP. The equilibrium constants for the formation of a 1:1 and 2:1 ethidium nucleotide complex were 1.9i0.1 x 10A M-1 and 5.8:0.6 x 102 M-l, respectively. A satisfactory fit was not obtained for ethidium bind- ing to K 2'GMP, Na25'GMP, and K25'GMP, probably due to the 2 large number of variables present. It is clear from the dependence of the ethidium methyl chemical shift on the nucleotide/ethidium mole ratio (cf. Figures 6A to 67) that the order of Kassociation for ethidium to solution self- structure is K25'GMP > Na25'GMP 2 K22'GMP > Na22’GMP. A fit to the model represented by Equations (3) — (A) and (23) - (25) may be obtained if the integrated concentra— tions of structure are used as a known variable, to eliminate some unknown variables from the equations used in the KIN- FIT program. VII. Amino Proton Exchange in the Absence of Solution Self-Structure Analysis of proton NMR line widths of the amino proton resonances can give detailed data on the rates of proton exchange between the amino protons and the solvent. In water, the amino proton resonances are broad and sensitive to temperature; at higher temperatures they broaden rapidly and become unobservable above 50°-60°C. The observed rate constants differ from those for "normal" amino groups and reinforce the argument that a significant amount of imide character may account for the relatively slow proton ex- change rates. For imides, it is well established that proton exchange with water occurs at slower rates than maximum (diffusional) because imide pK's lie outside the range in which hydronium-hydroxyl catalysis is efficientl79. It is obvious that the reaction is pseudo first order in amino proton. The ka at 25°C is the same within the limits of error for a 12-fold increase in concentration (3:. Table 2A) of Na22'GMP. The reaction rates and activa- tion parameters are the same for TMA25'GMP, Na22'GMP and TMA+ inhibited TMA22'GMP. The reaction rates and activation parameters are independent of nucleotide, concentration of nucleotide, or counterion. These results are consistent 133. o O o With the work of McConnell and coworkers13 ’ The mechanism of amino proton exchange in guanylic acids has ‘2 been discussed extensively in the literaturelro’l33. CONCLUSIONS Self-association is a general property of guanylic acids which do not have the N(7), 0(6) acceptor positions and N(l), NH2 donor positions blocked for hydrogen bond- ing. A size selective metal ion coordination is involved in the self-structuring process. The ordering of the structure directing ability of the metal ions is K+ > Rb+ > Na+ > Cs+ > Li+ 3 TMA+. The position and charge of the phosphate play an important role in the stability and type of structures formed. Thus solution self-structures are small soluble fragments, probably involving limited stack- ing of tetramer units. The size of the stacked tetramers are limited by interplate phosphate repulsions. The phos- phates also help stabilize structures by interplatlet hydrogen bonding. The order of structure forming tendencies of guanylic acids is guanosine > 3'GMP > 5'GDP > 5'GMP (pH=5) > 5'GMP (pH-7-8) > 2'GMP. The nucleotides are also able to interact with each other forming hydrogen bonded mixed species in solution. The tetramethylammonium ion was found to be a non- structure directing, structure inhibiting ion for 2'GMP, 3'GMP, and 5'GDP. Since TMA+ is a non-structure directing, non-structure inhibiting ion in 5'GMP, TMA+ most likely 230 231 is sterically blocking the formation or stacking of tetramers in 2'GMP, 3'GMP, and 5'GDP. In some reactions of guanylic acids it may not be desirable to conduct the reaction under conditions complicated by gel formation if an alkali metal ion is present. Replacing the alkali metal ion with TMA+ would eliminate the problem of gel formation and solution self-structuring. A catalyzed amino proton exchange reaction occurs in Na22'GMP and Na25'GMP, which may be due to the formation of tetramers. A mechanism involving a water bridge between a phosphate of an adjacent nucleotide and the amino proton not involved in tetramer formation has been postulated. A solution structure must be involved as the catalyst in the amino exchange reaction because the amino protons of Na22'GMP were found to exchange at a slow rate at tempera- tures where no solution self-structure is present. The fast exchange of the amino proton(s) occurs before the onset of structure formation. It is not known if this amino proton exchange is important in biological systems. Ethidium was found to form a 1:1 and a 2:1 complex with both structured and unstructured 2'GMP and 5'GMP with association constants>103. The large equilibrium constants are consistent with ethidium preferring to intercalate in guanosine rich regions of DNA and RNA. Equilibrium con- stants for the ethidium binding to solution self-structured Na22'GMP were determined assuming a stoichiometry of Nau- (2'GMP)8. Equilibrium constants could not be determined 232 with other stoichiometries. This supports the postulate that Na 2'GMP solution self- tructure is an octamer unit. 2 APPENDIX 1 PROGRAMS USED ON THE ASPECT 2000 ON THE BRUKER WMZSO AND PROGRAMS USED IN KINEIT 1. Programs Used on the Aspect 2000 on the Bruker WM250 1.1. The Gated Decoupling Program To produce a spectrum with normal coupling pattern but containing the NOE effect the following program was used on the Bruker-WM250 1 ZE 2 HG 3 D1 A D0 5 D2 6 GO=2 7 EXIT Define 02, the decoupler frequency, D1 and D2 delays before executing AU. Typical values used are DP = 8H-25H (0.1 W-l.lW) 01 = 3-5 sec. D2 = 0.002 sec. 1.2. Inversion-Recovery Tl Program To obtain estimates of T1 the following Inversion- recovery T1 program was used 233 23A 1 ZE 2 D1 3 P1 A V0 5 GO=2 9 EXIT Define D1=5le, P1=180°, PW=90°, VD list as desired, and set NE=no. of experiments. When transforming, do the largest FID first and set AI=1 before typing FT. Use PK for phasing the other spectra. Typical values used on the 5 mm proton probe are Pl=9.0 usec PW=A.5 usec. VD=0.01 - 1.0 sec. 1.3.1. KINFIT Programs Used in Ethidium Bindinggto Guanylic Acids The following programs are the equations used for ethidium binding to guanylic acids. This method of de- termining equilibrium constants is based on the method 8A of ropov and coworkers . 1.3.1.1. Determination of the equilibrum constants for the formation of a 1:1 and a 2:1 complex between un- structured nucleotide and ethidium. pp (237) - (239). 1.3.1.2. Determination of AH and AS for the formation of a 1:1 and a 2:1 complex between unstructured guanylic acids and ethidium. pp (2A0) - (2A3). 1.3.1.3. Determination of the equilibrium constants for the formation of a 1:1 and a 2:1 complex between struc- tured guanylic acids and ethidium. pp (2AA) - (2A7). 1.3.2. Calculation of Activation Parameters for Amino Proton Exchangg with Solvent 1.3.2.1. Determination of E and A for amino proton a exchange with solvent. pp (2A8) — (250). 1.3.2.2. Determination of AH# and A85 for amino proton exchange with solvent. pp (251) - (253). SUBSIDIZED UJ’0UZ NJ~2OUUH o< nan O—G 06v 2H2 ENG. 010 Gwyn PDP thm 0. 0V) 0(x—c 0&2 A>zv 2>O :00x CID-U spa-tax xx. 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CC OCC’QC‘tOCCC 222222222222222222 OGOGGGGGGGOOGGGGGG UUUUUUUUUUUUUUUUUU GO ID 20 oNEo'l’ UUU: 2hr UUUUU'U 3 D :3 3 3 2 :3 3 :3 U II II vvr-ZUUUZ’Z OIZZZZZZUZZZZZZZZZZZZZ (Janwd-Hun)"zm—«mz~¢W«n—tanmznawflznawunnx (Jacunwa~ ULndDhnnzhcm-DFH-Dhfiab:w-Dhfi3h:H-D EGNQ UVUZZHUU-l‘fl-ZWPZPZP—Zor-Zl-ZP-ZP-ZI-Zi-ZD-C UXXOO'IUCHUOUUOUO‘UOKHOUOUOUOUOUOUZ UXXUUDO—U‘J-‘KUZ¢U¢U¢U~¢U¢U¢U¢UCUKUKU U U U U U U 0 CCCCC 0000000 In '0 ”O‘DOU‘OMNIO N fldHI-O *EOROO *EOF Q. and 000 u-unth 000 O O O ncc undo-I 000 000 000 000 o o o 000 0000 000M? 0H00 016.130 000 000 o a I 000 dado-tau O O o O 0000 fins-no nun—«1 o o o o ncm—n «gm-on coco 00000 000005}. 252 «Ito-0d o o o 000 ”'00 000‘ O O 0 0mm «iv-OH 000 000 000 000 C O O 000 0000 00b") ”#00 annn 000 000 O I 0 000 HHHCH O O O O 0000 nun-«4 c—unc o o o o OIONC FOG-40'0"! 0000 0000 0000 Inc-In mm: o o 0 «mo «no-o 000 H0004 C O O O 0000 0000 #0100 o O o o cummn '1de 0000 00000 00000 Fido-Gd O O O O 0000 00—0 0000‘ o o o 0 NF??? and—0H 0000 0000 0000 0000 O O O O 0000 00000 00000 ONHOO‘ onnnw 0000 0000 O O O O 0000 dunno-0'1 o o o o 0 00000 «awn Nnmow. C O. Q. Nnncm “dd—IF. 00000 00000 0 0000 00 tH0000d000¢~00002006coon-060000.«400000 "“256 0000000000: 00000JH0000 0M0"): 0:000“ ¢€°Dd0 GIN-3‘02 MAO-'0 3 t‘Jv-IP)\\I 0 O 0 O 0000 c000“ msnc .4000“ a; co... 0 00000 N N ( 700.0014 2 ”NW”? SON—400‘ tonnnxmnnnthnnnzsmnnnzhmnnmzmmnnnw out—"n was-o o o o o o 000 00 l 000 on 070” 0c 0 o o .o o nun mun «to-om dd 000 00 000 00 000 00 000 00 o o o o o 000 00 u-n c-o 0 0 000.0 00C) 0d00‘ 00C Ottnd ON-O onnn 0'0!) 000 00 000 00 O O O O O 000 00 N Add run—1 o o o o o o 000 000 000‘ 0004 «"00 UH-«n o o o o o o Inna cunln 041-004 . MG!!! 000 000 0000 0000 0000 0000 o 0 Oz 0 0 .1 0000 coco m N N~m~< amp-x pmez 0002 #001 roe-40 0 0001-! 00010 coo»; CC‘aCu-d o o o o o 0 o o 0 0000 0000 00000 00004. O 0 o o I 00000 0000—. g30f>0¢ O O O 0 0 0000-0 0000 C30C30 I 0 O 0 00000 on 00000 D ‘3! )000 o o o o o 0 00000 APPENDIX 2 AMINO PROTON EXCHANGE TIEORY AN THE PROGRAM LORENT 2.1. Amino Proton Exchange Theory The following is taken from ReferenceslflB and 185. Consider a two proton exchange in which there are sites with different local fields giving a resonance with two components A and B shifted by +6w/2 and ~6m/2 from their average angular frequency. The relative intensities of these components are directly proportional to the proton fractions PA and PB contributing to each component. The process in question interchanges protons between sites A and B, so if the protons NA and NB at each site * * are labeled NA and NE at some instant it- dNA /dt (A2-l) -k w * A”A it dNB /dt -k N , (A2-2) where RAPA = kBPB. The average lifetime of protons at each site is therefore ll H \ 7? II A T/PB and TB = l/kB = T/PA, where T = TA B/(TA+TB). When averaged over the proton interchange, the total magnetization M is that given by (A2-3) 253 25M iwlM0C(TA+T )+T T.(a PQ+GPPA)] M = ~ B A B .q ._, .4 :1 , (AZ-3) (1+aATA)(l+aBTB)-l where ”1 is the applied rf field, MO is the static nuclear magnetization at thermal equilibrium, and “A = (l/Tz) - i(Am+5w/2) and a8 = (l/T2) -i(Aw-dw/2). This assumes the resonance absorption is plotted at.a constant static mag- netic field as a function of the different Aw between the applied radiofrequency and the frequency at the center of the two resonance components. In the absence of exchange effects or overlap, the width of each component at half- maximum is 2/T2; this includes all contributions to the line width such as field inhomogeneities and implies a Lorentzian line shape, 6w is the separation of the com- ponents assuming no exchange and no overlap of the com- ponents. The expansion of Equation (A2-3) with retention of only the imaginary part v, gives wlMO[(l+T/T2)P+QRJ v = (A2—u) P2+R2 where P = TE(l/T22)-(Aw)2+(6w/2)2]+l/T2 Q = TEAw-(ow/2)(PA-PB)] R = Aw[l+(2r/T2)l+(5w/2)(PA—PB) 255 This program pp (258) - (263) calculates theoretical NMR line shapes for the case of general two site exchange. The equations used are those given in 2.1. £256 TITLE: LORENT.FTN - GENERATE AND PLOT NHR LINES FOR GENERAL THO SITE EXCHANGE AUTHOR: T V ATKINSON DEPARTMENT OF CHEMISTRY MICHIGAN STATE UNIVERSITY EAST LANSING. MI 48824 DATE: 23-APR-BO THIS PROGRAM CALCULATES THEORETICAL NMR LINE SHAPES FOR THE CASE OF GENERAL THO SITE EXCHANGE. THE EQUATIONS USED ARE THOSE GIVEN IN ROGERS AND HOODBREY. J.PHYS.CHEM.. 66.340 (1962). THIS PROGRAM HAS ADAPTED FROM THE DECEMBER 1969 PROGRAH OF D.A.CASE. THIS VERSION ALLOHS GRAPHICS VIA THE MSU VECTOR PACKAGE. PARAMETER DICTIONARY (INPUT HILL BE PROMPTED) IDENT A STRING OF UP TO 72 CHARACTERS OF IDENTIFYING INFO. XMARG . THE DISTANCEICM) BETHEEN THE EDGE OF THE PLOT AND THE LEFT SIDE DATA AREA _ XLNGTH THE HORIZONTAL DIMENSION(CM) OF THE DATA PLDTTING AREA YMARG THE DISTANCE(CN) DETHEEN THE BOTTOM OF THE PLOT AND THE ' BOTTOM OF THE DATA AREA YLNGTH ATHE VERTICAL DIMENSION(CM) OF THE DATA PLOTTING AREA NPNTS NUHDER OF POINTS TO BE CALCULATED OMEGAL FREGUENCY. The command list is: EX 'Return to calling program (i.e. FTNMR Program) Ex may not be used if "BICON" was started at its starting address using the computer's switch register but only after a ”USER-FUNCTION-CALL" (.02) M0 Call Disk Monitor. This command must not be used unless either the "DIABLO' or the “FLOPPY” disk monitor is ready in core: Else disaster may result! Data Block Commands: .25 Clear Data Block defined by .51, .ST .51 Size of block, in words (not in lK-units!) .ST Start of block. In words starting at begin of data memory .SH Number of times data are divided by 2 before transmission to the other CPU.- This may be useful if it is desired to feed data to a l6-bit system in single precision. etc. Note that it is not possible to multiply data before transmission and the bruker system cannot accept data larger than its 20-bit word length permits. 2656 Decimal-Integer Transfer Commands - These commands use the .ST/.ST limits .HP Punch data on high speed punch .PT Punch data on TTY punch (Iwrite them onto paper) .HR Read data from high speed reader .RT Read data from TTY reader (or from keyboard) .Rc Receive data via the RS-232 interface .XM Transmit data via the RS-232 interface During data output, the limits .ST/.ST are used. During data input, the first data word is deposited at .ST and no more.than SI words are accepted from the other device. If loading less than SI words. the "E“-character must appear at the end of the data set. ‘ The memory of the Aspect is in the following format. 32K spectra - 24000 64000 l00000 124000 0 Program memory Real Spectra Imaginary Spectra ' Parameters ] l6K spectra . 0 24000 40000 4&000 64000 Program memory Real Spectra J Parameters Imaginary Spectra . To determine the address of a data point place the cursor on the data point and use the E command (see Bruker Aspect 2000 manual). This number is decimal and must be converted to octal and added (octal) to 24000. The spectra must be in the form wanted when transmitted. (e,g. transformed and phased). With the spectra in memory call BICON and transmit portion of spectra wanted. The POP ll/03 end of the link is tended by the Program NCDC which runs under the operating system RTll. If the POP ll/03 of CEM 838 is being used the floppy disks used to store data must be formated with a physical interleaving of one sector between sequential numbered sectors. The POP ll/03 is initialized as per the CEN 838 experiment RTl. Once RT ll is running and a properly initialized floppy is in drive 0Xl:. RUN NCDC. The answer to the first question is “carriage return”. At this time the ll/03 is listening to the Aspect. Type a "J" and NCDC will go into 2657 command mode. Enter a 'GLCL' request the flow of information from the serial link into a file. NCDC will then prompt your entering a file name to receive the infor- mation. Next type an “R CR ” to return to listening mode. At the end of a trans- fer, go to command mode, type "6 CR “. When a file specification is requested, type a ' CR ". This procedure is necessary to close the data file properly. (More information can be found in the documentation file for the program CDC.) 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