‘U— - ————v———-—7——» 7 7 r I .‘ ...v _“_ --.‘.__--_ ._-_-‘.._.-. ‘.'". .- .-¢-o- os*----~-.¢ .‘Q. QQ-sc~- - » . - ~~»----_ ‘- -- -— -.. _ 'ci—.~-..0——q-.'....“‘...g"-"‘-“'-"‘-VV"'“‘j‘l'fiw‘v—v"“““““““ PHYSICOCHEMICAL STUDIES OF CONVULSANT TET-RAZOLES Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY ALFRED J. SMETANA 1977 ABSTRACT PHYSICOCHEMICAL STUDIES OF CONVULSANT TETRAZOLES By Alfred J. Smetana Cyclopolymethylenetetrazoles are known for their stimulating effect on the central nervous system. In sufficient doses, they are capable of inducing epileptic convulsions. The general formula for cyclopolymethylenetetrazoles is shown below. (CH2 q__ fix" > Cyclopolymethylenetetrazole Trimethylenetetrazole (n = 3) is soluble in water up to 1.4 molal. As the number of carbons in the polymethylene chain increases, the water solubility decreases, but reaches a maximum with pentamethylenetetra- zole (n = 5). This compound is soluble in water up to 5.0 molal. As "n" is increased further, the water solubility decreases very rapidly. Previous studies have indicated that trimethylenetetrazole and pentamethylenetetrazole (PMT) form dimers in aqueous solution. In this work, the dimerization of PMT has been studied using carbon-13 nuclear magnetic resonance and vapor pressure osmometry. These tech- niques have confirmed the existence of dimers of PMT in aqueous solu- tion. However, none of these techniques provided quantitative data regarding the dimerization equilibria. Alfred J. Smetana In an attempt to correlate physicochemical properties of a series of convulsant tetrazoles with their pharmacological activities, the interactions of tetrazoles with biological models has been investigated on three levels, a) protein and enzyme, b) membrane, and c) the receptor level. Ultraviolet differential absorption bands have been recorded when these drugs were added to solutions of'a-chymotrypsin, ovalbumin, lysozyme, pepsin, and human serum albumin at various pH's. It appears that these drugs are capable of inducing conformational changes in the proteins which are measurable using the difference absorption technique. However, fluorescence emission properties of the proteins remain unchanged upon addition of these drugs to protein solutions. Fluorescent probes were introduced to protein solutions and membrane preparations of E. coli and phosphatidyl choline vesicles, but none of the drugs had a dramatic effect on the emission properties of the probes. The total brain content of acetylcholine (ACh) appears to vary inversely with the amount of nervous activity. Central nervous system stimulants like PMT decrease the brain content of ACh. Therefbre, the interaction between tetrazoles and the ACh-receptor and its ion conductance modulator were considered using equilibrium dialysis and radioactive labeling techniques. These drugs were found to be incapable of blocking the binding of radiolabelled ACh to the ACh-receptor. The crystal structures of trimethylenetetrazole and PMT have been determined. There is some evidence for the presence of dimers of these compounds in the crystalline state, which may be related to the unusually high solubility of these compounds in water. PHYSICOCHEMICAL STUDIES OF CONVULSANT TETRAZOLES By 1’ ) Alfred J: Smetana A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Chemistry 1977 ACKNOWLEDGMENTS The author wishes to thank Professor Alexander I. Popov for his guidance, encouragement, and friendship throughout this study. He also wishes to thank Professor M. Ashraf El—Bayoumi for his many helpful discussions, encouragement, and enthusiasm. Gratitude is also extended to the Department of Chemistry, Michigan State University, and the National Science Foundation for financial aid. Special thanks is given to Mr. John G. Hoogerheide fbr his many helpful discussions and the appropriate atmosphere for chemical research which he created. Deep appreciation is extended to my Mom, Dad, and sister for their prayers, encouragement, and constant support. Finally, I would like to thank Dianne for her typing, love, patience, and understanding. To her and my family, I dedicate this thesis. ii TABLE OF CONTENTS Chapter I. HISTORICAL ......................... TETRAZOLES ........................ II. DISCUSSION OF TECHNIQUES .................. VAPOR PRESSURE OSMOMETRY ................. ULTRAVIOLET SPECTROSCOPY 0F PROTEINS ........... FLUORESCENCE SPECTROSCOPY ................ Proteins ........................ Membranes ....................... ACETYLCHOLINE RECEPTOR AND ITS ION CONDUCTANCE MODULATOR ......................... III. EXPERIMENTAL ........................ REAGENTS. . ....................... CRYSTAL PREPARATION ................... INSTRUMENTS ....................... ACETYLCHOLINE RECEPTOR AND ITS ION CONDUCTANCE MODULATOR ........................ IV. RESULTS AND DISCUSSION ................... NUCLEAR MAGNETIC RESONANCE. VAPOR PRESSURE OSMOMETRIC. AND X-RAY CRYSTALLOGRAPHIC STUDIES OF CONVULSANT TETRAZOLES ........................ SPECTROSCOPIC STUDIES OF THE INTERACTIONS 0F TETRAZOLES WITH BIOLOGICAL MODELS ............ V. SUGGESTIONS FOR FUTURE WORK ................ LIST OF REFERENCES ........................ m Page l 2 l3 l4 18 24 24 31 33 35 37 37 38 39 4O 58 82 84 Table TO LIST OF TABLES Page Physical and Pharmacological PrOperties of Some Cyclopolymethylenetetrazoles .............. 4 Solubilities of Some Cyclopolymethylenetetrazoles. . . . ll Chromophores of Proteins: Approximate Location, Intensity, and Assignments of Singlet-Singlet Absorption Bands .................... 2l Fluorescent Probes of Polarity ...... . ...... 28 13c Chemical Shifts (ppm) of PMT in 020 Referenced to TMS ......................... 42 13C Chemical Shifts (ppm) of PMT in CCl4 Reférenced to TMS . . . . . . . . . . . . . . ..... . . . . . . 44 Vapor Pressure Osmometry, Calibration Data for NaCl in H20 ......................... 47 Vapor Pressure Osmometry Measurements and Analysis for PMT in H20 ....................... 47 Dimerization of PMT in H20 ............... 53 Alternate Treatment of the Dimerization of PMT in H20 (54) ....... . . . . . ........... 53 iv Figure 0501-500 10 ll 12 13 I4 15 LIST OF FIGURES Aromatic amino acids . . . . . ............. Excited state processes. Straight arrows denote process in which a photon is emitted or absorbed; wavy arrows denote radiationless transitions ......... 0.... ....... The 13C nmr spectrum of PMT in D2 The 13C nmr spectrum of PMT in CCl4. . . . ....... The l80 MHz proton nmr spectrum of PMT in 020 ...... Vapor pressure osmometry calibration curve for NaCl in H20, "constant K" method. . . . ........... Vapor pressure osmonetry calibration curve for NaCl in H20, "variable K" method ............... Vapor pressure osmometry plot for PMT in H20 ...... Plot of the nunber average molecular weight of PMT vs. the concentration of PMT . . . . . ........... Crystal structure of PMT . . . ...... . . . . . . . Crystalline lattice of PMT . . . . . . . . . . ..... Overlap of tetrazole rings in PMT. . . . . . . . . . . . Crystal structure of trimethylenetetrazole ....... Crystalline lattice of trimethylenetetrazole . . . . . . Overlap of tetrazole rings in trimethylenetetrazole. . . Page 26 4] 43 46 48 49 50 52 55 56 57 59 60 61 Figure l6 17 18 19 20 21 22 23 24 25 26 27 Page 5M Difference absorption spectrum of 10' a-chymotrypsin at pH 7. [PMT]/[a-chymotrypsin]: a = 0, b = 5, and c = 8. . . . . . . .......... 53 M Difference absorption spectrum of 10' a-chymotrypsin at pH 7. [8-tert-butyl PMT]/[o-chymo- trypsin]: a = 0, b = 3, and c = 5 ........... 64 Divided cells used for difference absorption studies . . 65 Difference absorption Spectrum of 10-5 M_a-chymotrypsin at pH 7 with divided cells. [8-tert-buty1 PMTJ/[a-chymo- trypsin]: a = 0, b = 4, and c = 5 ........... 66 Fluorescence spectrum of a-chymotrypsin at pH 7 ..... 69 Fluorescence spectrum of'a-chymotrypsin at pH 3 ..... 70 Fluorescence spectra of ANS-a-chymotrypsin at pH 3.6 and 7.0. . . . . . . . . . . . . ...... . ..... 71 The successive red shifts of the emission maximum of ANS-a-chymotrypsin at pH 2.4, 3.6, 4.75, 7.0, and 8.0. . 72 Fluorescence Spectrum of ANS-a-chymotrypsin using an excitation wavelength of 290 nm. . ...... . . . . . 74 Fluorescence spectrum of ANS in H20 (lower trace) and ANS-E. coli in H20 (upper trace) . ........... 77 Fluorescence spectrum of AS-PC vesicles in TRIS buffer at pH 7.4 using an excitation wavelength of 311 nm . . . 80 Fluorescence spectrum of AS-PC vesicles in TRIS buffer at pH 7.4 using an excitation wavelength of 384 nm . . . 81 vi CHAPTER I HISTORICAL TETRAZOLES Tetrazoles and substituted tetrazoles have been studied for many years and by numerous investigators because of their interesting physiological and physiocochemical properties. Tetrazole, the parent compound, has four nitrogens and one carbon in a ring numbered as shown in I. Tetrazoles may exist in two tautomeric forms I and II.(l,2) H H 12...!5 2.! s". \N/ 3 I H 111:2!5 2 / \ 4 H—N\N/N 3 II A theoretical calculation based on dipole Imment contributions has shown that 97% of an equilibrium mixture of I and II exists in the form I.(3) The hydrogen atoms in the 1- and 5-positions may be substituted with aliphatic or aromatic groups to yield 1,5-disubstituted tetrazoles. Over 400 menbers of this class of nitrogen heterocycles have been synthesized and characterized.(4) A special class of 1,5-disubstituted tetrazoles is the cyclopolymethylenetetrazoles in which a polynethylene 1,5-disubstituted tetrazole (CH )In (1.3.2 2 N/ \\ 4 \N/ 3 cyclopolymethylenetetrazol e 3 chain fbrms a second ring fused to the tetrazole ring. The cyclopoly- methylenetetrazoles and their derivatives are known for their ability to induce epileptic convulsions. The convulsant activity varies with the number of methylene groups and the nature and position of the substituent group.(5) The convulsant activity and some physical preperties of various tetrazoles are presented in Table 1. As Table 1 illustrates, the convulsant activity is sensitive to the aliphatic ring size. A monotonic increase in convulsant activity results as the number of methylene groups in the alkyl ring is increased. Of all the cyclopolymethylenetetrazoles, only pentamethy- lenetetrazole (PMT) has been used clinically. The convulsions resulting from the effect of PMT are used as a model fOr petit mal epilepsy. For this reason, PMT has been employed fbr screening anti-convulsant drugs. Pentamethylenetetrazole has been used in patent medicines as a respiratory and cardiac stimulant, in higher doses as an analeptic in barbiturate overdoses, and in veterinary medicine in hastening the recovery of animals from anesthesia.(6) Gross and Featherstone (7) studied the pharmacological activity of a number of tetrazoles and concluded that the activity of the drug depends on the substitution of the tetrazole ring. Bulky groups in the 5-position cause a decrease in activity, while large groups in the l-position usually increase convulsant activity. In the case of PMT, introduction of a substituent in the 8-position resulted in an increase in analeptic activity only when the substituent is small or closely packed as in the cases of methyl and tert-butyl groups. Numerous investigations have been made in an effort to correlate the activity of substituted tetrazoles with their-physicochemical .Paevcu mg» ea agave: scan mo segmopvx Log opo~meump ea msagmrppre mo were: cw :m>pm up amount web .mesnpum mo msounaxm “nee; mca mmzeu on xgemmwumc opo~meumu yo pesosa answers mnu mm «women acemp=>=ou saspcws mghe 8 E 3.3. 235322235853-83 m M: mm .2: 28332... 152.35.. 53-238 2833 8 33% 2826322268885 2% 32., 8 mm 42 2823351622.... e B: 32.. A Z a .8, 23238525238 on 2 8.82 eSNebseeeifigzeez S we 8 .Nfl 2033382235er 8 8 E a: 28282035235“. 8N A : 2 4.2 epfigfieeeifigebfl 82 o: N F 5: 289532323: E 3 88 8.; 2.8.. 23m: 382:: acampz>=oo Eaemcmz acmupmz eupzuopo: mmFoNeeumumcu—zgumsxfioaopuxu meow mo mwvugmaosa peopmopoumELegm ecu .euvmxzm .p m_ae» 5 properties. Schueler gt_al, (8) examined the ultraviolet (uv) spectra of a number of substituted tetrazoles. It was found that compounds of moderate activity and which have alkyl groups substi- tuted on the ring, generally showed little or no absorption down to 220 nm. Compounds which had aryl substituents, which exert a depressant effect, showed absorption bands near 290 and 225 nm, with the 225 nm band having a much larger absorptivity. A decrease in the depressant activity of the series produced a hypochromic effect on the 225 nm band, while an increase in the stimulant activity produced a hypochromic effect on both bands. The authors claim there is some correlation between uv absorption of the tetrazoles and their pharmacological activities. Dister (9) observed that the distribution ratio (organic/aqueous) for PMT, substituted PMT, and 1,5-dialkyltetrazoles is larger in basic media and concluded that these substituted tetrazoles are extremely weak bases, despite the facts that the tetrazole itself is a weak acid (1) with an aqueous Ka of 1.54 x 10'5 and all S-substituted tetrazoles behave as weak acids.(lO) Popov and Holm (11) found that even 10% aqueous solutions of PMT exhibit a neutral pH. These workers titrated PMT in glacial acetic acid with perchloric acid and showed that PMT acts as a weak base in this solvent. Golton (12) studied the distribution of PMT between aqueous and carbon tetrachloride layers as a function of pH and determined that the protonization constant far m in water is m 10'”. Pepov and Marshall (13) measured the proton affinity of PMT, substituted PMT, and l,5-dialkyltetrazoles in fbrmnc acid and determined the pr of these solutes to have a value around 2. In 6 general, the most active compound tested was the most basic, and the least active was the least basic. Erlich and Popov (l4) determined basicity constants for six cyclopolymethylenetetrazoles varying from trimethylenetetrazole to undecamethylenetetrazole in formic acid solutions. These solutes acted as fairly strong bases (pr «:2), even though, as already mentioned, cyclopolymethylenetetrazoles have no detectable proton affinity in aqueous solution. Popov and Holm (15) determined the dipole moments of PMT, 8-tert- butyl PMT, and 8-sec-butyl PMT in benzene to be 6.14 D, 6.20 D, and 6.18 0, respectively. Since 8-tert-butyl PMT is at least an order of magnitude more active than either PMT or 8-sec-butyl PMT, they concluded there was no correlation between the physiological activity of these compounds and the magnitude of their dipole moments. Buchanan gt_al, (16) investigated the surface activity of PMT in a study of air-solution surface tension isotherms. They observed that central nervous system stimulants prefer the aqueous bulk phase while drugs which exhibit depressant action collect at the air-solution interface. It_has also been shown that PMT can emulsify human cell membranes.(l7) The membrane becomes*weakened, ruptured, and is then ufiscible with PMT, as this solute is soluble in lipid substances. Then, PMT diffuses rapidly through the membranes. The effects of several cyclopolymethylenetetrazoles on ion trans- port in toad bladder membranes were studied by Gross and Hoodbury.(18) They observed a good correlation between the convulsant activity of the drug and the increase of the short circuit current produced in the isolated toad bladder. They concluded that the cyclopolymethylene- tetrazoles affect the potassium ion transport across the membrane. 7 One possible explanation for this action is that the tetrazoles have an effect within the membrane. For this to be true, the tetrazole must first pass through or into the menbrane. One theory governing the transport of substances through membranes requires the dissolution of the substance in the membrane. The investigation of this phenomenon involves studying the partition- ing of the substance between two layers, the aqueous solution and the lipid membrane. Distribution studies of biologically active compounds between aqueous solutions and lipid solvents have been performed,(19) but not on the tetrazoles series with the exception of the work done by Baum.(20) He studied the distribution of trimethyltetrazole, PMT, heptamethylenetetrazole, 8-sec-butyl PMT, and Batert-butyl PMT between water and carbon tetrachloride. No correlations could be drawn between pharmacological activity and the distribution ratios fbr these systems, but it is interesting to note that these data indicate the formation of at least dimers if not higher aggregates of PMT in both water and carbon tetrachloride. The investigation of the solution self- association of PMT was not concluded and is therefbre considered in part of this thesis. Nuclear magnetic resonance (nmr) has been used as a very sensitive probe to the chemical environment of the nucleus under examination. Proton nmr and carbon-13 nmr serve as probes to study the concentration dependence of the PMT signals in an effort to study the solution self- association of these molecules. Vapor pressure osmometry, a technique which has been used to measure colligative properties of solutions, is also used to examine this proposed molecular association of PMT. 8 Brubaker (21) prepared and characterized two fbrms of the solid complex bis(S-aminotetrazolato)copper(II). Complexes between copper and tetrazole, S-phenyltetrazole, and l-ethyltetrazole all have 20, but no complex was observed formation constants on the order of 10 between Cu(II) and 1,5-dimethyltetrazole and was interpreted as indicating that a replaceable ring hydrogen is necessary for this complexation reaction to occur. Popov and Holm (11) prepared the silver complexes of several tetrazoles. In one experiment, the slow evaporation of the aqueous solution led to the formation of a solid complex with the formula Ag(PMT)2N03. This reaction demonstrated that the loss of a ring proton is not necessary for the complexation to occur and that the coordination is probably through a ring nitrogen. Popov gt_al, (22) measured spectrOphotometrically the formation constants of the PMT complexes with 101, IBr, and I2 in carbon tetra- chloride solutions in order to study the donor properties of this particular tetrazole. It was shown that the fbrmation constant fer the ICl-PMT conplex is three orders of magnitude larger than the fbrmation constant for the benzene-1C1 complex. They state that this implies complexation through a ring nitrogen. vBaenziger gt_al, (23) used x-ray crystallographic techniques to investigate the [Cl-PMT complex. They found that the tetrazole acts as a monodendate ligand and the iodine monochloride is bonded to the 4-nitrogen of the ring. Baenziger and Schultz (24) also studied Zn(II) complexes with tetrazoles and found that the complexation occurs through the 4-nitrogen. vaughn et_gl: (25) studied the iodine monochloride complexes of 7-methyl-, 9-sec-butyl-, and 9-tert-butyl PMT and fOund that these complexes were only slightly stronger than the corresponding complex for the unsubstituted tetrazole (PMT). D'Itri (26) determined formation constants for the reaction between silver ion and cyclo- polymethylenetetrazoles in aqueous solution. Although the convulsant activity of the tetrazoles varied in both cases, it did not correlate with the donor properties of the tetrazoles in the first case, or with the formation constants which remained constant at «:103 in the latter case. Other complexes of tetrazoles have also been studied: CuCl-PMT,(27) HgC12.PMT,(28) and CdClZ-PMT.(29) Since both the solubilities and dissociation constants of these compounds are small in aqueous solution, they have been used in precipitation reactions for the quantitative determination of PMT. The conditions of the reactions used in these gravimetric determinations must be rigorously controlled, however, so that the exact composition of the precipitate is known, as the ratio of PMT to metal can vary, usually from one to two. Another drawback is that the solubility of the complexes limits the analysis of PMT at low concentration levels. Beyrich and Schlaak (30) determined PMT in the presence of other drugs at concentration levels of 0.3 to 3.0 parts per thousand, by titrating PMT with perchloric acid-glacial acetic acid mixtures in a vessel also containing benzene and acetic anhydride. Daoust (31) described a spectroscopic method for the determination of PMT in pharmacological preparations. Pentamethylenetetrazole was precipitated and isolated as the CuCl-PMT complex which was then 10 dissolved in nitric acid. Tetraethylenepentamine was used to complex the copper and the absorbance of this solution was measured. The relative standard deviation was reported to be 8% at the 400 parts per billion level. Continuous wave proton nuclear magnetic resonance was used by Turczan and Goldwitz (32) to measure the concentration of PMT in pharmaceutical preparations. The detection limits were very poor, and this method required at least 3% by weight of PMT fer reliable results. Haywood et_al, (33) investigated the use of thin layer chroma- tography in the determination of neutral drugs and fOund that PMT could not be determined by this method due to its lack of ultra- violet absorption near 254 nm. However, Guven (34) found that PMT could be spotted with a mixture of 10% copper sulfate and 2% ammonia solution which gave blue spots upon drying. In 1962, Kawamoto (35) used gas chromatography in the determina- tion of PMT in aqueous solutions. He used a phenylmethylsilane column with a thermal conductivity detector. The lowest concentrations of PMT detected were on the order oflOOO parts per million. These analyses suffered from a drifting baseline which made peak area determinations quite difficult. More recent investigations have utilized flame ionization detectors.(36-38) Marcucci gt_gl, (36) determined brain levels of PMT and could observe down to 50 ng quantities of this drug. Stewart and Story (37) used a column packed with 5% polyethylene glycol 20,000 on 800/100 mesh diatomaceous earth and could detect as little as 1 ppm of PMT in biological fluids. Baum g1:_a_l_. (39) extended this method and analyzed 11 cyclopolymethylenetetrazoles and their mixtures. As a result of this investigation, a gas chromatographic method for the routine determina- tion of cyclopolymethylenetetrazoles at the 50-100 ppm level was developed; concentrations of less than 10 ppm can be determined. Baum (20) also determined the solubilities of several cyclopoly- methylenetetrazoles in water. These data are shown in Table 2. Table 2. Solubilities of Some Cyclopolymethylenetetrazoles Molal Solubility Tetrazole Solubility in g/ml Trimethylenetetrazole 1.4 m 0.16 Pentamethylenetetrazole 5.0 0.69 Heptamethylenetetrazole 0.18 0.031 8-sec-butylpentamethylenetetrazole 0.0052 0.0010 8-tert-butylpentamethylenetetrazole 0.0029 0.00057 As the number of carbon atoms increases, it is not unexpected that the water solubility decreases, with the glaring exception of PMT. This compound is soluble up to 5 m. There is already evidence that PMT forms dimers in solution. If the crystalline lattice consists of dimers, then it requires less energy to break down the lattice and solvate dimers than it would in the case of monomers only. Therefbre, crystals of these compounds have been grown for x-ray crystallographic analyses in order to better understand the solubility characteristics of these compounds. 12 Quantitative determination of the interactions between PMT and Li+ (40) and Na+ (41) have been made using the alkali metal nmr technique. This technique has proved to be an extremely sensitive probe to the chemical environment of the metal ion in solution. There was no detectable interaction between PMT and these ions in water, probably because of the strong solvating capabilities of this solvent. However, in nitromethane, a solvent with a fairly high dielectric constant, but much poorer solvating ability, l:l complexes were observed. The formation constant fbr Li+-PMT and Na+-PMT were m 3.5 and'y 0.75, respectively. Examination of the sequence of events involved in synaptic neural transmission (42-44) has led to the definition of three levels of investigation to study possible correlations between physicochemical properties of cyclopolymethylenetetrazoles and their effect on the central nervous system a) protein or enzyme level, b) membrane level, and c) specific receptor level. If the series of convulsant tetrazoles interact with proteins or enzymes, then it may be possible for them to shut down an enzyme which acts to inhibit the transmission of neural signals. One may look also at the membrane level. If these durgs can interact with a membrane, by perhaps inducing conformation changes in the membrane proteins, permeability changes any result, changing the flow of ions such as Na+, K+ and Cl' through the membrane. Finally, a part of this thesis considers the interactions of tetrazoles with a specific neural receptor--the acetylcholine receptor. CHAPTER II DISCUSSION OF TECHNIQUES 13 VAPOR PRESSURE OSMOMETRY Vapor pressure osmometry (VPO) is a dynamic method used to measure the vapor pressures of solutions. The temperature difference between a drop of pure solvent and a drop of solution (solvent plus the solute under study) is measured. Both drops are in a thermostated atmosphere saturated with solvent vapor. The osmometer is first calibrated with solutes of known activity and then sample measurements are made. The phenomenon which led to the development of VPO was discovered by A. V. Hill (45) in 1929. Hill made calorimetric measurements on nuscle contractions and traced some anomalous heat effects back to the condensation of water vapor onto the muscle sample. In VPO, the solution drop has a lower vapor pressure, slower rate of evaporation, and therefore a slightly higher temperature than the solvent drop. Hill's apparatus has been modified with the use of thermocouples (46) and thermistors (47) replacing thermopiles. Recently, a vapor pressure osmometer has been modified enabling measurements of temperature differences m 6 x 10'6 °C.(48) The vapor pressure lowering power of the solute provides the basis for the above mentioned temperature difference and makes VPO a measure of colligative properties. The number average molecular weight of a solute nay be obtained (49) and therefOre, VPO has been used extensively in polyner'research. Use of the appropriate factors fer the solute used to calibrate the instrument may lead to the calculation of osmotic (50) and activity (51) coefficients of the solute. The vapor pressure osmometer has also been examined as an analytical instrument.(52,53) 14 15 VPO has been used in solution studies of self-association in both water and nonaqueous solvents.(54,55) By changing the thermostat temperature, the thermodynamics of the association reaction way be examined.(56) Kirsch and Simon (57) have used VPO to calculate Bjerrum formation curves for some complexation reactions. It has even been suggested that a modified vapor pressure osmometer be used as a detector for liquid chromatography.(58,59) The actual measurement made in a vapor pressure osmometry experi- ment is the resistance (AR) added to the sample leg of a Wheatstone bridge in order to rebalance the bridge. Prior to making any measure- ments, the instrument is zeroed using a drop of pure solvent on each of the thermistor beads. The following relationship exists between AR and the concentration (C) of the solute: + a C + aZC2 + a C3 (1) AR = a0 1 3 The first term, a0, is called the zero point displacement and is normally equal to zero. The last term in equation 1 approaches zero as low concentrations (0.01 - 0.1 M) are normally used. Deletion of these two terms yields equation 2 AR=ac+a§2 (a 1 which may be rearranged DID ” = a1 + a2C (3) Therefbre, a plot of gg-vs. C should yield a straight line, the y-intercept of which has been shown to equal %r-, where K is the n 16 calibration constant which is dependent upon the instrumental conditions and the solvent, and Mb is the number average molecular'weight of the solute.(60) Sodium chloride, sucrose.and dextrose are used as calibration standards in the case of aqueous solutions, M8 fer these solutes is known, and then the calibration constant may be calculated. For these determinations in nonaqueous solvents, calibration solutes such as benzil or biphenyl are commonly used. The method described thus far is termed the "constant K“ method, as a single calibration constant is used. It yields the solute number average molecular weight at zero concentration. The'"variab1e K" method provides the number average molecular weight as a function of solute concentration according to the following procedure: 1) Plot gg-vs. AR (CM = solute molar concentration) for the M reference solute. This serves as the calibration curve. 2) Use AR at a given sample concentration and find the correspond- in '93 9 CM from the calibration curve. 3) Divide each AR by the corresponding éB-read from the calibra- M tion curve. This provides a series of apparent solute molarities. 4) Divide each apparent molarity (CA) by the actual concentration in grams/liter (CH)’ E5. = "”195 = J— (4) CH 9 Mn 5) Plot %r'VS. CH' This plot illustrates the profile of number n average molecular weights; the y-intercept yields %r-at zero n concentration. 17 Coetzee and Lok have used VPO to study the dimerization of car- boxylic acids in nonaqueous solvents.(54) They used the fbllowing formalism to calculate dimer formation constants. (5) The left-hand side of equation 5 represents the degree of association (for dimerization), MB is the measured number average molecular weight of the solute at a given solute concentration, and M is the monomer molecular weight. The dimer fbrmation constant (K1,2) may be calcu- lated using equation 6, - 2 x,,2 - f/tsz(1 - f) l (6) where CM is the solute analytical concentration in moles/liter. Another derivation nay be applied to the calculation of dimer formation constants using VPO data. If P represents the monomer, equation 7 expresses the equilibrium, 2? «5* P2 (7) and the dimer formation is calculated using equation 8. K,,2 = [Pan/[P12 (a) This treatment does not apply any activity corrections, because only molecules are involved in the reaction, and there is no separation of charge. The analytical concentration (CM) of P in solution is shown in equation 9. cM = [P] + 2[P2] (9) 18 Vapor pressure osmometry effectively measures the number of particles (N) in solution. Equation 10 represents the total number of solute partitles in solution. N = NP + NP2 (10) Both sides of equation 10 nay be divided by some arbitrary volume element, V, N/V = (NP + NP2)/V (11) which leads to CA = [P] + [P2] (12) where CA is the measured apparent solute molarity previously described. It may be shown that and [p] = 2cA - cM (14) Then. 2 (is) K1,2 = (Cr ' cAmch ' VP) ULTRAVIOLET SPECTROSCOPY OF PROTEINS Absorption spectra of proteins, to a first approximation, may be considered to be a sum of the spectra of the amino acids of which they are composed. Proteins are commonly recognized by their absorption near 260 and 280 nm, which is characteristic of the side-chain 19 chromophores of the amino acid residues phenylalanine, and tyrosine and tryptophan. respectively. Figure 1 shows the structures of these amino acids. Table 3 (61,62) serves as a reference in which the location and intensity of absorption bands of some chromophores of proteins are given. These data are only approximate and are intended for quick reference. A Since there is usually strong overlapping of bands, ultraviolet absorption of proteins can furnish only gross structural information. Use of the differential technique however, provides a much more sensi- tive method of detecting small discrete changes in the environment of particular chromophores. In this technique, two solutions of the same protein in different environments are compared directly at identical concentrations. One solution serves as the sample and the other as the reference or blank, thereby cancelling out the common features of their spectra. Only those transitions which have been displaced with respect to each other because of alterations in the environment of the chromophore are displaced as either positive or negative differential bands. These bands are recorded on instruments capable of measuring small differences (0 - 0.01 absorbance units) in the absorbance. Absorption spectra of aromatic amino acid residues in proteins nay be affected by a number of factors which include the polarity of the environment, the polarizability of the solvent, the presence of charges or dipoles in the vicinity of the chromophore, and the formation of hydrogen bonds. These interactions may lead to shifts in both position and intensity of the absorption bands. For example, Yanari and Bovey (63) have shown that the spectra of indole, phenol, and benzene undergo a red shift when the refractive index of the solvent 20 PHENYLALANINE CHZCHCOOH NHZ CHngCOOH \\ NHZ TRYPTOPHAN I N H TYROSINE HO CHzeHCOOH NH2 Figure l. Aromatic amino acids. 21 Table 3. Chromophores of Proteins: Approximate Location, Intensity, and Assignments of Singlet-Singlet Absorption Bands Chromophore Residues Location (nm) log emax Assignment Phenyl Phenylalanine 188 4.80 n + n* ' 206 3.90 261 2.35 Phenolic Tyrosine 193 4.70 n-+ n* 222 3.90 270 3.16 Phenolic (Tyrosine)' 235 3.97 n-+ n* 287 3.41 Indole Tryptophan 195 4.30 w +'n* 220 4.53 280 3.70 286 3.30 Infldazole Histidine 211 3.78 n +~n* 22 is increased. Also, in general, when an aromatic side chain is transferred from an aqueous to a hydrophobic medium, the absorption band shifts to the red with an increase in the absorptivity. Some workers (62,64-66) have examined the general principles of ultraviolet difference spectroscopy and the types of information which can be obtained. Ultraviolet difference spectroscopy of proteins and enzymes has gained its widest applications in studies of general confbrmation changes which occur'when, for example, the pH is varied over a wide range, and in investigations of the dissociation behavior of ionizable chromophores, especially tyrosines. Following are several exanples which shall serve to demonstrate the type and degree of insight which new be obtained into changes in the environment of particular chromo- phores which occur*when a perturbant is added or*when the enzyme interacts with certain ligands or inhibitors. Tryptophans and tyrosines have served as the specific residues which have been used most extensively as markers of conformation changes. When the environment of these residues is perturbed, they yield difference absorption bands between 270 and 300 nm. More specifically, the unionized tyrosine difference absorption bands occur at about 278 and 287 nm, while tryptophan bands are at about 284 and 292 nm, with possibly a weak band near 275 nm.(62,67,68) Below the pH of tyrosine ionization, the presence of a difference band at 292 nm usually indicates the involvement of tryptophans, and similar bands at shorter wavelengths may result from either tyrosines or tryptophans, the complete interpretation of which would require more information. 23 Laskowskigt_al, (64,67,69-71) have used ultraviolet difference spectroscopy to study enzyme topography in a technique known as solvent perturbation spectrosc0py. This method is based on the principle that spectral bands undergo small shifts when the polarity of the environ- ment is changed. For example, if an absorbing group is present on the surface of the enzyme in aqueous solution, addition of some non- aqueous component to the solution results in slight alterations in its absorption spectrum. On the other hand, if the same absorbing group is present inside the hydrophobic interior of the protein, addition of this other solvent component does not alter the absorption spectrum, provided the additive doesn't alter the conformation of the protein. In the actual experiment, a difference spectrum is measured between a solution of the protein in aqueous solution containing 20% of an inert perturbant like ethylene glycol, glycerol, sucrose, methanol, or polyethylene glycol, and the same solution without the perturbant. The appearance of a difference spectrum indicates that absorbing species are in some contact with the additive. The extent of group exposure can then be calculated from the ratio of the differential peak intensities with those obtained with a fully unfblded protein, where it is assumed that all groups are exposed. If perturbants of different sizes are used, this technique may be used as a probe of surface topography of the protein. Some data have led to inconclusive results, where chemfical modification has often provided the answers to these questions. For example, in the case of Cllymti‘ypSinogenmxidation of three tyrosines with N-bromosuccin- imide eliminated the solvent perturbation difference spectrum, with 24 no further changes upon oxidation of the remaining residues.(67) It seems reasonable to conclude that in chynotrypsinogen, three trypto- phans are totally exposed and five are completely buried. Difference spectroscopy has also been used as a probe to examine chymotrypsin-substrate interactions. Benmouyal and Trowbridge (72) determined the difference spectra between a-chymotrypsin and its conplexes with several ligands. The difference spectra obtained when p-toluenesulfonylargi nine methyl ester and acetyl phenylalanine ethyl ester were used as ligands are very conplicated and quite different in character. Since neither ligand absorbs at wavelengths above 275 nm, the difference spectra must reflect changes in the environment of the enzyme chromophoric residues. Both the positions and sigms of the difference bands are different for the two substrates, which appears to indicate that their binding to the enzyme induces nonidentical local structural perturbations. Burr and Koshland (73) observed similar effects in some experiments on the binding of substrate to a-chymotrypsi n which had been chemically nodified by the incorporation of a chromophoric reporter group. The utility of the uv difference absorption technique lies in its potential in revealing whether or not the environment of absorbing chromophores is changed upon the addition of some perturbant to the solution. In this investigation, the perturbants are the tetrazole molecules themselves . FLUORESCENCE SPECTROSCOPY 212531."; X-ray crystallography has revealed the structures of nuuerous proteins. However, other techniques are required as the x-ray 25 crystallographic method is essentially a static method, and also, not all proteins can be crystallized. For these reasons, fluorescence spectroscopy of proteins with the use of fluorescent probes has been considered by numerous investigators. Fluorescent probes have been used to establish the degree of polarity of a particular region of a protein, measure distances between groups in a protein, determine the flexibility of a protein, and measure the rate of very rapid conforma- tional transitions. The last two utilizations require nanosecond light pulses and relaxation methods in the nanosecond range and will not be discussed further. Figure 2 serves as a diagram describing the excited state processes involved in emission spectroscopy. A molecule is excited from the ground state (So) to an upper electronic state (S2). It then relaxes very rapidly from S2 to the lowest excited state (S1) without emitting a photon. From here, a number of processes may occur: a) fluorescence, a transition from S1 to So, accompanied by emdssion of a photon; b) internal conversion, a return to S0 without emission of radiation; or c) intersystem crossing, a transition to an excited triplet state (T1) in which the electron spins are no longer paired, as in the singlet states. The molecule may return to the ground state by emitting a photon via phosphorescence, or it may return without emitting radiation. Also, S1 and T1 may transfer their excitation energy to other chromophores or participate in photochemical reactions, which are not shown in Figure 3. The time scale of these events must also be considered. The life- time of the excited state S1 is typically on the order of a few nano- seconds, whereas T1 usually has a lifetime between a millisecond and 26 __z 52 '1 51 Intersystem Crossing .1 T1 C .2 U r: 0 0 O U c: C: U W O 0 2 'P U r— 43 en «3 O E 2 E E O 8 0 m m 4-, o 2 E .5. E L 3”! So Figure 2. Excited state processes. Straight arrows denote process in which a phdton is emitted or absorbed; wary arrows denote radiationless transitions. 27 several seconds. Due to the long phosphorescence lifetime, this process is often quenched, and phosphorescence is seldomly observed, except in rigid media. Three types of fluorescent chromophores in proteins have been defined. The intrinsic chromophores are the aromatic side chains of phenylalanine, tyrosine, and tryptophan residues. Some proteins contain a fluorescent coenzyme such as reduced nicotinamide-adenine dinucleotide, flavin-adenine dinucleotide, or pyridoxal phosphate. Examinations of these coenzymic chromophores have provided information on the structures and interactions of several proteins.(74) However, not all proteins have intrinsic or fluorescent coenzymic chromophores in the specific location of interest in a given protein. Weber (75) introduced an approach where an extrinsic fluorescent probe is inserted into the protein of interest. A few requirements should be met by the extrinsic chromophore. First the fluorescent probe is to be bound to the protein at a unique location, for example, the active site. Secondly, the fluorescent properties of the probe must be sensitive to the structure and dynanncs of its environment. Lastly, the insertion of this chromophore should not appreciably change the features of the protein which are being studied. Weber and Laurence (76) discovered a number of polycyclic aromatic compounds which are mostly nonfluorescent in water, but which become highly fluorescent upon binding to proteins, specifically to serum albumfin. Table 4 shows the structures of a few fluorescent probes and some of the proteins which have been studied using them. These compounds 28 Table 4. Fluorescent Probes of Polarity PROBE PROTEIN . serum albumin _ apomyoglobin H-N SO ' '3 apohemogl obi n l-anilino-B-naphthalene sulfbnate (ANS) CH3 chymotrypsinogen chymotrypsin H-N 3 2-p-toluidinyl-6-naphthalene sulfonate (TNS) SO 8 N(CH3)2 human serum albumin 8 SOZNHCHZCOOH Dansyl glycine (76.77) (77) (77) (88) (88) (90) 29 and related chromophores can be used as sensitive probes of the polarity of their environment. Studies of the ANS-apomyoglobin conplex exami ned the emission characteristics of ANS.(77) Apomyoglobin is myoglobin minus its heme. group. High resolution X-ray studies have shown that apomyoglobin has a highly nonpolar hene-binding site. It is therefore not unexpected that ANS binds to apomyoglobin stoichiometrically to a specific site with a dissociation constant A: 10's. Addition of hemin led to the displacement of ANS, which seems to suggest that ANS and heme bind to the sane site or to sites that substantially overlap one another. Similar results were obtained for the interactions between ANS and apohenoglobin (hemoglobin minus its heme group) which also has a highly nonpolar site for the heme group. Further studies of the fluorescence properties of ANS in various nonaqueous sol vents have illustrated the envi ronnent polarity depen- dence of the ANS enrission.(77) In a series of alcohols, as the polarity of the solvent decreased, the fluorescence quantum yield increased and the wavelength of maxinum emission shifted towards the blue. (Polarity may be defined in terms of dielectric constant or dipole moment. Here, a more polar solvent is one which has a larger dipole moment.) For exanple, in ethylene glycol, the quantum yield was 0.15 and the wave- length of maximm emission was 484 nm, whereas in n-octanol, a much less polar sol vent, the corresponding values were 0.63 and 464 nm respectively. The same type of effect was observed for the case of ethanol -water mixtures. The polarity dependence of the emission wavelength of ANS results from a reorientation of the solvent shell around the chromophore when 30 it is excited. Fluorescent groups which have higher dipole nonents in the excited state than in the ground state show this effect.(78) The more dipolar, excited state of ANS interacts with a polar solvent so as to further align the solvent dipoles, whereas the solvent shell of a nonpolar solvent is less disturbed. A lower energy photon is emitted by ANS in polar solvents, because some of the solvation energy of the excited state is lost when the chromophore returns to the ground state. Therefore, the emission is shifted to the red in a polar solvent. The inportance of solvent relaxation is supported by the finding that 2-p-toluidinylnaphthalene-G-sulfonate (TNS) fluorescence in ice resem- bles that in nonaqueous solvents.(79) Although the probe is surrounded by polar residues, they are not free to relax during the lifetime of the excited state. Fluorescent probes like ANS have been used to study several pro- teins. The degree of polarity of the active sites of apomyoglobin,(77) apohemoglobin,(77) and carbonic anhydrase (80) have been shown to be highly nonpolar. Serum albumin,(81) antibody to the dinethylami no- naphthalene-S-sulfonyl group (anti dansyl antibody),(82) and alcohol dehydrogenase (83) have moderately nonpolar binding sites, and chyno- trypsin has a highly polar active site.(84) N-arylaminonaphthalene sulfonate dyes have been shown to bind at a site removed from the active sites of chymotrypsin,(85) pepsin,(86) and yeast alcohol dehydrogenase. (87) Secondly, the binding of substrates and coenzymes to proteins can be followed if a fluorescent probe is located at or near the active site. The probe may be displaced by the substrate or its fluorescence characteristics may be altered upon the formation of a ternary 31 complex,(85,88) fer example. Also, conformation changes which influence the catalytic process have been detected.(85,88) Hhen dansyl glycine (see Table 4) binds to human serum albumin, the quantum yield for dansyl glycine increases five fold while the fluorescence emission maximum shifts from 580 to 480 nm.(89) A fluorescence titration suggested that there is a single hydrophobic binding site and perhaps other much more polar ones. The association constant fer dansyl glycine and this single hydrophobic site is 4.6 x 105. Several anionic drugs such as phenylbutazone,(90) flufenamic acid,(9l) and dicoumarol (89) can competitively displace dansyl glycine from its binding site on human serum albumin. This technique then, provides not only a convenient method for monitoring drug interactions with human serum albumin, but also gives information on the hydro- phobic nature of the binding sites. Membranes Fluorescent probes have been used to study interactions of drugs, cations, and other ligands with membrane systems. It has been shown, for example, that the binding of ANS to intact mitochondria or isolated mitochondrial membranes is accompanied by both a marked shift in the ffluorescence emission maximum of the dye to shorter’wavelength and an increase in fluorescence quantum yield.(92-95) The addition of oligo- mycin, succinate, uncouplers of oxidative phosphorylation, or ATP produces changes in the fluorescence of membrane-bound ANS. The addition of either butacaine (a local anesthetic) or Ca2+ to ANS- labeled rat liver mitochondria also causes an increase in the fluorescence quantum yield.(93) 32 Christian (96) used ANS to exannne the interaction of some pheno- thiazine derivatives with synaptosomal membranes. These drugs act as central nervous system tranquilizers. Titration of the ANS-membrane complex with chloropromazine indicated that the drug alters the membrane structure in such a way as to create additional ANS binding sites. When chloropromazine sulfoxide was used as the neuroperturbant the sane general effect was noted but to a much smaller degree. These studies show that these tranquilizers are definitely capable of altering the neuronal menbrane structure. Kasai gt_al, (97) observed that ANS binds to membranes from the electric organ of the electric eel. The affinity of ANS fer the mem- brane increased significantly in the presence of Ca2+. Flaxedil and d-tubocurarine, two drugs which are inhibitors of neuromuscular trans- mission, also increase the affinity of the membranes fbr ANS. It appears that there is some correlation between these changes in fluorescent properties and the pharmacological activity of these drugs. One of the major difficulties in interpreting data from membrane systems containing ANS is that the precise location of the fluorescent label is unknown. Several workers have suggested that ANS is bound to nenbrane phospholipids.(98-100) Therefore, it appears that the changes in ANS fluorescence may represent changes not only in the menbrane protein conformation, but also in the phospholipid environment of the dye. Probes specific for nenbrane studies have been synthesized by Haggoner and Stryer (101) which were fbund to be specifically incor- porated into phospholipid bilayer vesicles. Three of these probes are anthroyl stearic acid, dansyl phosphatidylethanolamine, and octadecyl- naphthalamine sulfonic acid and were found to specifically bind in 33 the hydrocarbon region, glycerol layer, and at the aqueous interface of the bilayer, respectively. Fluorescent probes can also be applied to energy transfer studies. Energy absorbed by one chromophore can be transferred to another at some distance away; fluorescent probes can be inserted as a measure of the distance between the probe and another absorbing chromophore, fer example. There are three types of energy transfer: singlet-singlet, triplet-singlet, and triplet-triplet. For singlet-singlet transfer, Fbrster (102) has proposed that the transfer occurs by a resonance interaction of the dipole pair between the energy donor and acceptor chromophores. He provides a quantitative treatment used to calculate the distance at which the singlet-singlet transfer is 50% efficient. This theory has been tested by Stryer and Haugland (103) and has shown excellent agreement with their'results where oligomers of poly- L-proline served as spacers of defined length to separate donor and acceptor by distances ranging from 12 to 46 A. ACETYLCHOLINE RECEPTOR AND ITS ION CONDUCTANCE MODULATOR Two closely coupled proteins, the acetylcholine (ACh) receptor and the ion conductance modulator (ICM) are suggested to constitute the unit that regulates the ACh-induced ionic conductance of postsynaptic mem- branes at motor endplates. Upon reaction with ACh, the receptor acti- vates the ICM into an open conformation allowing Na+ and K+ ions to flow along their chemical gradients Na+ (out +-in) and K+ (in + out), thus leading to membrane depolarization and the formation of an endplate potential. In cell free preparations the receptor is identified by the specific binding of’a variety of cholinergh: ligands,(lO4-107) all of 34 which are known to activate or inhibit the ACh-receptor function in physiological preparations. The ICM is identified by binding of histrionicotoxins, which block ion conductances and modulate endplate currents without direct effects on the ACh-receptor.(108-1ll) Membrane preparations from the electric organs of Torpedo ocellata are highly enriched in both proteins (0.6 nmoles/mg protein of receptor sites and 1.2 nmoles/mg protein of ICM sites). The receptor and the ICM proteins are identified by the binding of [3H]ACh and [3H]perhydro- histrionicotoxin, respectively, using the method of equilibirum dialysis.(lll) Drugs which interact with the ACh-receptor, whether activators or inhibitors, displace [3HJACh, and those which interact with the ICM displace [3H]perhydrohistrionicotoxin. The interaction between tetrazoles and the ACh-receptor and the ICM is examined using these techniques. CHAPTER I II EXPERIMENTAL 35 REAGENTS Dimethyltetrazole was prepared according to Markgraf.(112) Trimethylenetetrazole (Aldrich) was purified by recrystallizing about 10 grams of the tetrazole from a solvent mixture of 50 ml of carbon tetrachloride and 10 m1 of ethanol. Pentamethylenetetrazole (Aldrich) was used without further purification. Baum (20) prepared and purified 8-sec-butylpentamethylenetetrazole and 8-tert-buty1pentamethylene- tetrazole. The other cyclopolymethylenetetrazoles were prepared and purified according to D'Itri.(26,ll3) Proteins which were used include a-chymotrypsin (Aldrich), oval- bumin (Aldrich), lysozyme (Aldrich), pepsin (Worthington), and human serum albumin (Sigma). The fluorescent probes l-anilino-B-naphthalene sulfbnate, 2-p-toluidinylnaphthalene-G-sulfenate (Sigma), and dansyl glycine (Sigma) were used without further purification. N-acetyl-L-tyrosine ethyl ester nonohydrate (Aldrich) was used to measure the activity of a—chymotrypsin.(ll4) The 3-(trimethylsilyl)- l-propanesulfbnic acid, sodium salt hydrate (DSS) (Aldrich) was used as an internal reference for aqueous proton nuclear magnetic resonance measurements. Distilled water, deuterium oxide (Columbia Organic Chemicals), and carbon tetrachloride (Mallinckrodt) were used without further purifica- tion. Phosphate buffers were made to be pH 3, 7, and 11 by dissolving the appropriate salts in distilled water to yield 0.1 M_phosphate buffers measured to be pH 2.92, 6.70, and 10.84, respectively. Samples of the outer membrane of E. coli were prepared and provided by McGroarty.(115) Phosphatidyl choline vesicles were prepared using Haung's method.(ll6) An aliquot of a chlorofOrm solution containing 36 37 12-(9-anthroyl)-stearic acid, a fluorescent probe, was added to phos- phatidyl choline (from hen egg yolk) in chloroform. A 0.1% aqueous dispersion consisting of the phosphatidyl choline and probe was formed by adding 10 ml of buffer and swirling the mixture. The buffer was 0.1 M in NaCl and 0.01 M in tris(hydroxynethyl)aminonethane at pH 7.4. The dispersion was sonicated for 1 hour under nitrogen. The sonicated mixture was centrifuged to give a sufficiently clear solution for fluorescence spectroscopy. This sample was provided by El -Bayouni.(ll7) CRYSTAL PREPARATION Crystals of trimethylenetetrazole and pentamethylenetetrazole were grown from dilute ether solutions, from which the solvent was permitted to evaporate slowly. Crystals of 8-tert-butylpentamethylenetetrazole were grown from aqueous solution. After slow evaporation, clear, well-defi ned crystals remained. INSTRUFENTS Varian A56/600 and CFT-ZO spectrometers were used to record proton nmr and carbon-l3 nmr spectra, respectively. A Mechrolab Model 302 vapor pressure osmometer was operated at 37°C. The instrument manual (118) provided the operating procedure. x-ray crystallographic analyses were carried out by Dr. Donald Ward and his staff.(ll9) A Cary 15 UV-VIS spectrophotometer was used to record uv spectra using 1 cm quartz cells. The balance pots were adjusted each day to match cells and balance the circuitry of the instrument. Aminco-Kiers and Aminco-Bownan spectrophosphorimeters fabricated by the Anerican Instrument Company were used to record emission spectra. 38 4 M_solution of quinine sulfate in 0.1 N_sulfuric acid served A 1 x 10- as an external reference to monitor xenon lamp intensity. ACETYLCHOLINE RECEPTOR AND ITS ION CONDUCTANCE MODULATOR The examination of the interactions between various tetrazoles and the acetylcholine receptor and its ion conductance modulator was carried out by Eldefrawi.(120) A membrane preparation from the electric organs of Torpedo ocellata was used. The receptor and the ICM proteins were identified by the binding of [3HJACh and [3H]perhydrohistrionico- toxin, respectively, using the method of equilibrium dialysis at 21°C fer 4 hours.(lll) Tetrazoles were tested at «'10'4M_by placing the drug in the dialysis medium along with the radiolabeled ligand. CHAPTER IV RESULTS AND DISCUSSION 39 A NUCLEAR MAGNETIC RESONANCE,.VAPOR PRESSURE OSMOMETRIC, AND X-RAY CRYSTALLOGRAPHIC STUDY OF CONVULSANT TETRAZOLES Nuclear Magnetic Resonance Carbon-13 nmr has been used as a probe to examine the concentration dependence of the PMT chemical shifts in 020 and CCl4 in order to study the solution dimerization of PMT. A coaxial capillary containing DMSO served as an external reference (020 solutions) with the signals referenced to TMS. Figure 3 shows the 13C nmr spectrum of PMT in 020. Table 5 shows these data and the cor- responding numbering of the signals. The changes in the chenfical shifts as a function of PMT concentration are quite small. The largest change observed was for signal #5, where the change in the chemical shift in going from 0.1 M to 5 H PMT was only A: 0.6 ppm upfield. If there were no association of these molecules in solution, one would expect no concentration dependence of the chemical shifts, pro- vided that the bulk magnetic susceptibility of the solvent stays fairly constant. Even if the bulk magnetic susceptibility of the solvent changes over this large concentration range, each signal should be shifted by exactly the same amount. Small changes in the 13C resonances nay be indicative of the fermation of dimers.(121) If the dimers form by an electrostatic attraction or overlapping of the tetrazole rings, the largest change observed should be in signal #5, the carbon nucleus closest to the tetrazole rings. Carbon-l3 nmr spectra were also recorded fer a series of solutions of PMT in cc14. Figure 4 shows the 13c nmr spectrum of PMT in cc14. Table 6 shows these data. The solvent served as an internal reference, and the position of this peak remained quite constant. The largest 40 41 due 5. Eu .3 53.53% .2... amp 2:. .m 25m: 953 him .536 o cm 9 8 8a a 9:. 8H g i e e i q J t e . 1 4J ll ‘11 m m eKz/JN m/ _ e e e S A a m w 2 A omzo 42 e m e \z/ Am_m 2,: c_ P N om.e~ me.m~ eo.mm .e.om ~m.~e em.om eo.mm_ oo.m mm.e~ em.m~ ee.A~ Nm.om me.~e em.om m_.em_ me.m _m.e~ me.mN me.A~ Fm.om Ne.~¢ oo.pm m~.mmp eo.m om.e~ em.m~ em.- _m.om Ne.~e so._m em.mm_ m~.p ow.s~ om.mN me.a~ om.om oe.~e m_._m Am.mmp oo.P om.s~ m~.m~ ma.e~ ms.om em.~e mp._m mm.emp Ne.o om.e~ eN.m~ me.e~ pm.om mm.~e m_._m em.mm_ om.o mm.e~ mm.m~ me.e~ Fm.om Ne.~e m~.Pm ee.mm, e~.o om.e~ m~.mN ee.e~ Pm.om mm.~e ep.pm ------ N_.o m A e e omzo o_ m “my a m m z = z a < z e H m eepeeeeeeeeeu mze e» eeeeeeeeem owe es axe to Aseev mee_em .eeseeeu u .m epeee m_. 43 1c: 8 9 op .38 5 En. mo Eaguuflm .5... amp 2:. .e 2:3... eae tam .5266 8 § 8 at: 9: 8H 4 q J I. J" M ‘ Bu 44 m~.m~ mp.e~ mm.m~ ~e.om oe.om em.~m em.~m_ _o.m em.m~ m_.e~ ee.m~ oo.Fm oe.om me.em ow.emp No.4 e~.m~ m_.eN ee.m~ o_._m oe.om me.~m p~.em_ em.~ em.m~ om.e~ ee.m~ PF._m ~m.om me.~e .m.~m_ pm._ mm.m~ m~.em s~.mm ow._m om.om ee.em _e.eme _o._ mm.m~ mm.e~ e~.m~ om.Pm o~.om me.em Fu.em_ ~m.o mm.m~ mw.e~ ee.m~ om._m P~.om me.em -.~m_ Pm.o ae.m~ mm.e~ Ne.mN oe.Pm o~.om ee.em .P.Am_ w~.o cm.m~ mm.e~ em.m~ em.~m P..om ee.em ------ o..o m A m e op e_uo m amv e u m z 2 z A < z u H m ee_eeeeeeeeou mze ea eeeeeeeeem e_uu es Fae co Aseev esteem Fee_eeeu o .e speak 2 45 changes in the chemical shifts were on the order of 0.6 ppm for both signals #5 (downfield) and #6 (upfield). It is also interesting to note that at low concentrations («:0.1 M) of PMT, signal #5 disappears, probably due to the lack of both Nuclear Overhauser Enhancement and the lack of an efficient relaxation mechanism. Proton nmr has also been used to study this effect in 020. These spectra were quite complicated, and exact assignments of the signals were difficult to make using a 60 MHz instrument. The 180 MHz (Bruker WH-lBO) proton nmr spectrum of PMT in 020 is shown in Figure 5 with the assignments of the signals. This spectrum illustrates the com- plicated splitting pattern, which renders these data only qualitative. The chemical shifts measured from the centers of the multiplets rela- tive to internal 055 are a) 4.60, b) 3.15, c) 1.96, and d) 1.79 ppm. The proton nmr spectra in CCl4 were not recorded as the addition of TMS to the solutions resulted in the fbrmation of a precipitate. Vapor Pressure Osmometry Vapor pressure osmometry was then used to examdne the solution self-association of PMT in water. The osmometer was calibrated using NaCl. The calibration data and calibration curves are shown in Table 7 and Figures 6 and 7, respectively; CM (moles/liter) is the analytical concentration of the solute, CW is the solute concentration in (grams/ liter), and AR (ohms) is the resistance added to the sample leg of the Wheatstone bridge in theiosmometer. Figure 6 was constructed using the "constant K" method, and Figure 7 is the calibration curve for the “variable K" method. Table 8 and Figure 8 show the data and the graphical analysis for PMT in H20; CA is the measured or apparent solute concentration in 46 .omo 5 En so .5383 .5... :893 ~12 of 2: .m 6.53... 9a.: E6 .2866 ad 3 3 3 Q: 9m d . II d i i i 31 3:... mmo a co: 47 Table 7. Vapor Pressure Osmometry, Calibration Data for NaCl in H20 CM W AR AR/CM AR/Cw 0.0154 0.90 1.21 78.6 1.34 0.0240 1.40 1.89 78.8 1.35 0.0308 1.80 2.60 84.4 1.44 0.0488 2.85 4.71 96.5 1.65 0.0642 3.75 6.28 97.8 1.67 0.0847 4.95 8.87 104.7 1.79 0.0898 5.25 9.15 101.9 1.74 0.1018 5.95 11.05 108.6 1.86 Table 8. Vapor Pressure Osmometry Measurements and Analysis fOr PMT in H20 CM CH AR AR/CM AR/q” CA R; 0.0130 1.80 1.02 78.46 0.570 0.0129 139 0.0206 2.85 1.28 62.14 0.450 0.0160 178 0.0300 4.15 1.80 60.00 0.434 0.0221 187 0.0514 7.10 2.75 53.52 0.387 0.0327 217 0.0586 8.10 3.28 55.97 0.405 0.0383 212 0.0807 11.15 4.12 51.05 0.370 0.0468 238 0.0901 12.45 4.87 54.05 0.391 0.0538 231 0.1020 14.10 5.47 53.63 0.388 0.0593 238 48 .6058. _.v_ 28.28.. .0? E Sez L8 628 539528 tum—=28 2:395 63> .o «.53... as ea “5 6:388 85 86 3.0 86 o 56 mad 36 J a A 11 d J 4 8 d I ‘ ‘1 d 8a 49 6058. .2 Santa? .om: 5 So: it 9:3 5.59533 \Cumsgmo 953m...“ .89; .5 8:3“. Amie «3 mm 3 fl NH 2 S m m m m m a m N H 1 d — ¢ ¢ 4 a 1 u d 1 11 n J #Jéo .ou: 5 En sou, poi xbmsosmo 8:393 33> .m 953“. QESEZEV E; “a zofiézwoéu S m m m m m a m N H o «q I I ‘ 1 ‘ 15-1 £5 £3 fill H21 J A m6 No 23% Sl moles/liter. Examination of Table 8 and Figure 9 show that the number average molecule weight (fig) has a value essentially equal to that of the monomer (l38.l7) at low PMT concentration, and as the concentration ' increases, the molecular weight of the dimer (276.34) is approached. This is not unexpected, because the osmometer responds equally to each particle in solution. These data seem to fairly conclusively indicate the existence of dimers in a solution of PMT in water. It was then of interest to use these data to calculate the value of the dimer formation constant at the temperature used (37°C). Tables 9 and 10 show the results for this calculation using the two methods described previously. Both methods provide essentially the same results. One notes immediately, however, the extreme variation in the value of the dimer formation constant (K1,2); f is defined as the degree of association fOr dimerization. A data smoothing process was applied where the data used in the calculation of the dimer fbrmation constant were taken from the smooth curve in Figure 9, rather than the experimental points themselves. This did not yield any reduction in the variation in the value of the dimer fbrmation constant, however. Although the data appear to be trustworthy, a satisfactory value of the dimer fbrmation constant has not been obtained. The analysis indicates that the equilibrium may be more complicated than a simple dimerization. Also, Solie (60) has indicated that the accuracy of the VP0 data is the limiting factor in this type of an analysis. However, these data certainly show that there is some association of PMT molecules in aqueous solution. 52 .Em “3 558.2828 93 .m> Ea mo 2983 $2822. 32m; .895: m5 .3 uoE .m 953... 3 En. “6 225.5828 3.0 86 mod 36 86 mod 86 mod 86 86. o ‘1 E f . 8N 53 Table 9. Dimerization of PMT in H20 CM CA 1,2 0.0130 0.0129 0.0001 0.0128 0.6 0.0206 0.0160 0.0046 0.0114 35.4 0.0300 0.0221 0.0079 0.0142 39.2 0.0514 0.0327 0.0187 0.0140 95.4 0.0586 0.0383 0.0203 0.0180 62.7 0.0807 0.0468 0.0339 0.0129 203.7 0.0901 0.0538 0.0363 0.0175 118.5 0.1020 0.0593 0.0427 0.0166 155.0 Table l0. Alternate Treatment of the Dimerization of PMT in H20 (54) cM Mn f Kl’z 0.0130 139 0.018 0.7 0.0205 178 0.445 35.0 0.0300 187 0.525 39.0 0.0514 217 0.727 95.1 0.0585 212 0.594 53.4 0.0807 238 0.840 202.0 0 0901 231 0.805 118.0 0.1020 238 0.839 158.0 X-Ray Crystallography Crystallographic studies of PMT complexes with iodine chloride and Zn(II) showed that PMT acts as a monodendate ligand and coordi- nates through the 4-nitrogen of the tetrazole ring.(23,24) In the case of a silver complex, AgN03-2PMT, a monodendate tetrazole was coordinated to the silver atom via the 4-nitrogen, and bridging tetra- zoles were linked to the silver atom via the 3- and 4-nitrogens.(ll2) It was of interest to us to determine the crystal structure of the free ligand to see if there were any changes in the configuration of the molecule upon complexation and also if the lattice structure would yield any reasons f0r the unusually high solubility of PMT in water. Figure l0 illustrates the PMT crystal structure. The space group of the PMT lattice is PZI/n, and its cell parameters are a = l3.3l0(6), b = 8.409(3), and c = 6.589(2), where the number in parentheses fOl- lowing the dimension in A is the estimated standard deviation applying to the least significant digit. The a- and y-angles are 90° and B = 94.72(3). Carbon hydrogen bond distances are all around l A. It is interesting to note that the bond distances in the tetrazole ring do not indicate strictly single and double bonds, but rather, a n-system seems to be apparent. Careful examination of Figure ll illustrates the possibility of dimer fOrmation in the crystalline lattice of PMT. The 5-membered tetrazole rings in PMT lie on top of one another (see Figure l2) where the parallel planes defined by the two tetrazoles are separated by 3.71 K. The lattice structure indicates that there is some intermolecular association in the crystals. Therefore, the dissolution process may not require a breakdown of the entire crystalline lattice to individual 54 55 (i) 0 1.521(6) 1.505(6) ,3 \ l.520(8) 1.510(5) O 0 ‘~ ~ 0 1.473(4) 1.475(5) 1") 322 3 N 1.343(4) 1.338(4) N N 1.301(4) ,—~\ .328(4) N Figure l0. Crystal structure of PMT. 56 Figure ll. Crystalline lattice of PMT. 57 .Ea 5 35m $8858. .3 3296 .2 5.52... 58 PMT molecules. In addition it has been shown that dimers of PMT exist in aqueous solution. Therefore, it seems plausible that the dimeriza- tion of PMT in the crystalline state and in solution may be related to its high solubility in water. The crystal structure of trimethylenetetrazole was also determined. This structure is still being refined, but Figure 13 shows the bond distances for this molecule at the present time. The space group is also P21/n, and its cell parameters are a = 7.768(2), b = 12.388(3), and c = 6.689(l), with a = y = 90° and B = 102.02(2). Cryoscopic measurements have indicated there is some self- association of trimethylenetetrazole in water. Therefbre, this tetra- zole may be solvated as dimers to some extent, which may help explain why this compound is soluble in water up to 1.4 m. The melting points of trimethylenetetrazole (110°C) and PMT (60°C) also show the same type of trend. The packing of the molecules in the trimethylenetetra- zole and PMT crystals is similar, but their melting points differ by 50°C. The higher melting point of trimethylenetetrazole indicates that it requires more energy to break down the crystalline lattice than it does in the case of PMT. Therefore, it is not unexpected that PMT is much more soluble in water than trimethylenetetrazole. Other crystals of cyclopolymethylenetetrazoles are currently being grown and analyzed to provide a more representative picture of the solid state-solubility properties of these compounds. SPECTROSCOPIC STUDIES OF INTERACTIONS OF TETRAZOLES WITH BIOLOGICAL MODELS A series of commercially available proteins has been selected. Microliter aliquots of various tetrazoles have been added to the 59 1.519(7) 1.712(12) 1.620(6) 1.496(6) 1.371(5) 1.398(5) 1.452(5) 1 343(5) Figure 13. Crystal structure of trimethylenetetrazole. 6O Figure 14. Crystalline lattice of trimethylenetetrazole. 61 .mpogbmumcmifimetu 5 35.. 3323» we $225 .2 9:5: 62 protein solutions at various pH's. These solutions have been studied using ultraviolet and fluorescence spectroscopy. Four tetrazoles which range in convulsant activity are used in this study. Two of them (pentamethylenetetrazole and 8-sec-butyl- pentamethylenetetrazole, 8-sec-butyl PMT) are of moderate activity; one (dimethyltetrazole, DMT) is not acitve; and the last one (8-tert- butylpentamethylenetetrazole, 8-tert-butyl PMT) is the most active compound under study. Addition of each of these tetrazoles to protein solutions of 10'5 H_lysozyme at pH 7 (phosphate buffer) resulted in a negative difference absorption band at‘b 285 nm. The interaction became easily noticeable when there was a lO-fold excess of drug to protein. Even though the range of convulsant activity of the tetrazoles varied tremendously, the same type of positive interactions with lysozyme were recorded. The above four tetrazoles were also added to the so1utions of ovalbumin at pH 7, but precipitates formed immediately upon addition of the tetrazoles to the protein solutions. Solutions of’o-chymotrypsin were also prepared to which the series of tetrazoles was added. Figure 16 shows the difference absorption 5 M solution of spectrum which resulted when PMT was added to a 10' o-chymotrypsin at pH 7. Figure 17 shows the difference absorption spectrum which resulted when 8-tert-buty1 PMT was added to a different solution of a-chymotrypsin at pH 7. (These spectra were smoothed to make figure construction easier.) A positive interaction was also observed when DMT was added to a-chymotrypsin at pH 7. 63 "mcwmnbugéouuH—AEE .N za an qubugzsouam 9.5 Ee§m> GP- buffer buffer buffer .EJJ .LJ... SAMPLE REFERENCE Figure 18. Divided cells used for difference absorption studies. added to a solution of a-chymotrypsin at pH 7. The differential band is essentially the same as the one observed earlier (see Figure 17) when conventional cells were used. So it would seem that the difference absorption spectra recorded at pH 7 are a result of some conformational 66 .m u u 25 .e .1. a .o "In "mfmqbugéuuflkhi 33-2313 .mZmu 3.33.6 5.5 N In an :wmaxgugxsord z mug we 5:36QO 5398mm... 8:89—85 .2 95m: 2 No.9 25 56.94: . 8.? n V om 1’ opm com CNN omm W 4 q ,1 fl 4 . 3 (‘1‘.11 oo o M ’1 W 3 4 po.o 1 No.0 67 change in the protein induced by the addition of tetrazole to the solutions. Positive interactions between a-chymotrypsin and PMT were also observed at pH 3 and pH 11. The same types of differential bands observed previously result, and the interaction appears to be indepen- dent of pH, at least over the range studied. Addition of PMT to an o-chymotrypsin solution at pH 3 resulted in the formation of a precipi- tate, but still showed the same difference band at ~1285 nm. The mag- nitude of the differential band at pH 7 increased with increasing tetrazole concentration. However, even with a very large excess of the drug (> 500/1) the magnitude of the dflfferential band remained on the order of'm 0.02 absorbance units. These data do not show whether the observed spectra1 change is a result of a reaction between the drug and the protein as a whole, or an interaction between the tetrazoles and a specific aromatic amino acid or acids. Therefbre, solutions of phenylalanine, tyrosine, and tryptophan were prepared and titrated with PMT at pH 7. There was no observed interaction between PMT and these three single amino acids. This seems to indicate that the tetrazole is interacting with some specific site of a-chymotrypsin. A similar effect was observed by Tulinsky (123) when crystals of a-chymotrypsin were permitted to equili- brate with PMT in solution. PMT appeared to penetrate the crystal and bind to a site of a—chynotrypsin. Further experiments were conducted to examine these drug inter- actions more specifically. In an effort to determine the proximity of PMT to the active site, it was important to determine whether o-chymotrypsin maintained its catalytic activity in the presence of 68 PMT. This was done using N-acetyl-L-tyrosine ethyl ester monohydrate (ATEE), a substrate for which a-chymotrypsin is specific. The absor- bance at 237 nm is monitored as a function of time.(1l4) There was no change in the rate of this hydrolysis reaction even with a 200/1 excess of PMT to a-chymotrypsin. The conclusion is that the interaction is certainly removed from the active site. Fluorescence spectroscopy provides a much more sensitive method of probing protein conformation. The emission spectrum of a-chymo- trypsin was recorded in the presence and the absence of PMT. Figures 20 and 21 show the emission spectra of o-chymotrypsin at pH 7 and 3, respectively. Addition of PMT to these solutions neither changed the fluorescence intensity nor shifted the emission wavelength, which is surprising because ultraviolet difference absorption spectra indicated that some interaction was taking place. The xenon lamp intensity was monitored with an acidic solution of quinine sulfate which had its enfission maximum at 480 nm. A hydrophobic fluorescent probe, l-anilino-8-naphthalene sulfonate (ANS), was then used to examine the effect of the drug on the emission characteristics of the probe. Figure 22 (124) shows the fluorescence spectra of the ANS-o-chymotrypsin complex at pH 3.6 and 7.0. Figure 23 (124) shows the successive red shifts as the pH of the solution is increased. A solution of a probe-protein complex may be excited at'9 280 mm where the aromatic side chains of the amino acid residues of the protein absorb or at a longer wavelength where the complex absorbs. When a 10'5 H_solution of'o-chymotrypsin with excess ANS at pH 3 is excited at 290 nm, two emission bands result: 346 nm and 495 nm which 69 .n :5 pm Satugafiua .3 23.53% 35083:: .8 9:5: $5 gs. 228:6 ) 83 8. 8m 1 8m kw ‘1 J i d 1 lg E 5 MISNELNI 3393383801131 mum 7O .m :a no 53?..5555 .3 53.58% 358203... .5 Ear. 9.5 $9994: 2235 . B: Q! Rm 8m RN dl d 1 Nmm MISNEJNI BDNBSBHGTH 71 pH 3.6 RELATIVE FLUORESCENCE INTENSITY 7m EMISSION WAVELENGm (MI) Figure 22. Fluorescence spectra of ANS-a-chymotrypsin at pH 3.6 and 7.0 72 o.m can Q.“ .m~.¢ .m.m .e.~ :a an Emaxbgacunaéz,‘ co 32.3.2. caEmEm 05 we 3:5 3.. 33333 «E. .2 95m: 35 5.995, 5825 8m 8m 8. 8: J I] 4 I‘ll (aAlmau-mu) wsuamx mm 73 correspond to the emission of the aromatic side chains of the amino acid residues of the protein and the probe, respectively. Excitation at 390 nm yields only one emission band at'» 49l nm which corresponds to the emission of the probe. Addition of PMT to the ANS-a-chymotrypsin solution at pH 3 also resulted in the fOrmation of a precipitate, therefbre, it is not surprising that a large scattering peak became apparent. Figure 24 shows the emission spectrum of ANS-a-chymotrypsin at pH 3 using an excitation wavelength of 290 nm. Addition of sufficient PMT O» 60 pl), such that the PMT/a-chymotrypsin mole ratio was'» 20, resulted in the fbllowing fluorescence changes. With an excitation wavelength of 290 nm, the protein emission intensity remained fairly constant, but the probe emission intensity doubled. The same effect was observed when the excitation wavelength was 390 nm, where there was no protein emission. Closer examination of Figure 24 indicates that there is efficient energy transfer from the aromatic side chains of the amino acid residues of the protein to the ANS molecule. The environment of the probe does not appear to be changing that drastically or else one would have expected to observe a change in the emission wavelength of the probe upon addition of PMT. Only fluorescence intensity changes were observed, however. A number of possible explanations for the increased intensity of the ANS emission have been proposed: l) the micro-environment of the ANS molecule has become less polar, 2) ANS has binded more strongly to a-chymotrypsin, and/or 3) addition of PMT induces some confbrmational change in o-chymotrypsin so as to bind more ANS molecules. 74 .5: cam mo 535352 5.5333 ca 2.5: Snafugxguuvlmé mo 5330me 853863... .cm 8:3". 9.5 15.99,: 228$ 80 So omm 8m cm: 95 Rm 8m I q .1 d. d‘ C d 3m m3 MISNELNI SDNBGSSHCIT'H 75 The same experiment was repeated at pH 7. There were no observed changes in either the weaker fluorescence intensity or emission wave- lengths as excess PMT was added to a solution of ANS-a-chymotrypsin at this pH. Another fluorescent probe, 2-p-toluidinylnaphthalene-6-sulfonate (TNS), was then used fbr the examination of this drug-proteininter- action. The Z-p-toluidinylnaphthalene-G-sulfbnate non-competitively inhibits the hydrolysis of ATEE by a-chymotrypsin. Therefore, TNS does not bind at the active site. The maximum emission intensity is observed at pH 7.8 and TNS binds to a-chymotrypsin with a dissociation constant of 5 x l0'4.(85) Two different excitation wavelengths were applied in this study. Excitation at 280 nm of a solution of TNS- a-chymotrypsin at pH 7 to which PMT has been added produced a slight increase in the intensity of the two emission bands at 36l nm and 468 nm. Excitation at 450 nm yielded one emission band at 469 nm, the intensity of which increased slightly in the presence of a 20-fbld excess of PMT. These increases were on the order of 20%. The fact that excitation at 280 nm yielded two emission bands indicates that there is efficient energy transfer between aromatic side chains and TNS. Pepsin also served as a model protein with which a drug-protein relationship could be studied. The ultraviolet difference absorption spectra at pH 3, 7, and ll all showed small decreases (m 0.0l absor- bance units) in the absorbance at4a285 nm noticeable with a drug-to- protein mole ratio of'w 10. The emission spectrum of pepsin at pH 7 was independent of the presence of PMT. Excess dansyl glycine was added to a lo"5 N pepsin solution at pH 7, and the emission spectrum 76 of the dansyl glycine-pepsin complex remained unchanged upon addition of 8-sec-butyl PMT. The excitation wavelength of the complex was 328 nm with an emission wavelength of 56l nm. Human serum albumin (HSA) served as the protein closest to a real biological system in this investigation. By using the difference absorption technique, positive interactions between PMT and HSA were recorded at pH 3 and 7 but not ll. The emission spectrum of the protein at pH 7 did not change upon addition of PMT. Excess dansyl glycine was added to another solution of HSA at pH 7. This solution was titrated with 8-sec-butyl PMT and no changes were observed in the emission of the probe. This solution was excited at 352 nm, and the wavelength of maximum fluorescence intensity was Sll nm. It is of interest to explain why 8-sec-butyl PMT was added to the latter solution instead of PMW. Since 8-sec-butyl PMT is much less soluble than PMT in water, there should be a higher probability for an interaction between this tetrazole and hydrophobic binding sites of the protein. This result was not observed, however. Under the same polarity considerations, DMSO was used as the solvent, with no attempt made to control the acidity of the solution. Addition of 8-sec-butyl PMT to HSA in DMSO did not change the emission spectrum of the protein alone or of the dansyl glycine probe when it was added in excess to another HSA solution to which 8-sec-butyl PMT was also added. Exanfination at the membrane level was also defined as a part of this investigation. Figure 25 shows the emission spectrum of ANS in water (lower trace). The upper trace shows the emission of ANS in the presence of the outer membrane of E. coli. When PMT and 77 4325 3.53 cm: 5 :8 mimz< 25 A32» 3.8: cu: 5 mz< 3 5238"". 3589.8: .3 2.5.; 9.5 52595. 223:6 8m 8m 8m 8: d fi d) mme (BAIlV'IBU-NO‘J) MISNElLNI EDNBDSMTH 78 8-tert-butyl PMT are added in sufficient quantity to saturate the solution, the upper trace remained unchanged. These drugs appear to have no effect on the probe. Phosphatidylcholine (PC) vesicles were used as another membrane model. These vesicles were labeled with lZ-(9-anthroyl)-stearic acid (AS), the formula of which is shown below. The emission properties 0 H OH @©@ AS Probe of this probe in PC vesicles show that the fluorescent group of AS is in a benzene-like environment in terms of polarity. It has been inferred (l0l) that the fluorescent moiety of AS is in the hydrocarbon region of the PC bilayer. The vesicles may be pictured as fellows, PC Vesicle where the open circles represent the polar regions of the bilayer and the lines adjoining them the nonpolar hydrocarbon sections of the fatty acid. The AS label is found in this hydrocarbon region. Therefbre, it should be particularly sensitive to fluidity changes in the membrane which may result when a perturbant is added to the solution. 79 Fluorescence changes in the emission of the probe can be indicative of changes in the membrane permeability to ions important in synaptic transmission. A single emission band is obtained at W 460 nm with an excitation wavelength of either 3ll or 384 nm, see Figures 26 and 27, reSpectively. A substantial scattering peak is observed for these vesicles, as the solutions are slightly turbid. A slow increase in the concentration of 8—sec-butyl PMT from a very dilute solution to saturation (vortexing fbr two minutes and sonication for ten minutes at 30°C each time) did not significantly change the emission spectrum of the AS-PC vesicle complex, but increased the magnitude of the scattering peak. Although the drug does not appear to have any effect on the probe, it nay have passed rapidly through the membrane, establishing an equilibrium between the inner and outer environments of the vesicles, where the exchange is fast and the fluorescent probe remains unaffected. The total brain content of acetylcholine appears to vary inversely with the amount of nervous activity.(43) Central nervous system stimulants like PMT decrease brain content of acetylcholine. Therefbre, the interaction between three tetrazoles and the acetylcholine receptor and its ion conductance modulator was examined by Eldefrawi.(l20) DMT (not active), PMT (convulsant), and 8-tert-butyl PMT (very convulsant) all at'» l0"4 N_were feund to be incapable of blocking the binding of [3H]ACH (at 10'6 N) or [3H]perhydrohistrionicotoxin (at 4 x 10'8 MD to the ACh receptor and its ICM, respectively.(125) These results seem to indicate that these tetrazoles do not have their effect on the central nervous system via an interaction with the ACh-receptor or its ICM. .E: Ppm mo cpmcmpm>mz cowumuwuxm cm mcwm: ¢.n In um Lumean mHmh cw mmpuvmm> unim< to Eaguumam mucmummcoa—m 35 5289.? 223—5 8m 93 1 * m—m ome .8 2:3... SM d AlISNalNI BDNBDSSHOHTd 8) it...“ Lama BF F .5: «mm 3 5333? :o_gmuwoxm co mcwma ¢.~ In an Loewsn mHmF c? mmpuvmm> oaim< mo Ezguuwam mucmummgoapu .NN weaned Es Eozflmzi 2235 ALISNELNI 33N33838001$ mwm “me CHAPTER V SUGGESTIONS FOR FUTURE WORK 82 83 Crystal structures of higher members of the cyclopolymethylene- tetrazoles should be determined. Solubilities of heptamethylene- tetrazole, 8-sec-butyl PMT, and 8-tert-butyl PMT in water are known, and further, these three tetrazoles do not appear to associate in aqueous solution. Therefore, it would be interesting to see if the packing of the molecules in these crystals is significantly different from those of trimethylenetetrazole and PMT. With these additional crystal structures, a better understanding of the large water solu- bilities of trimethylenetetrazole and PMT may be obtained. Aryl-tetrazoles which act as central nervous system depressants may be fluorescent.. If they are fluorescent, the drug can be used as a probe of its own chemical environment, without the use of an extrinsic fluorescent probe. Changes in the emission properties of the drug should be indicative of any changes in the chemical environ- ment of the drug in solution. It has been reported that PMT can emulsify human cell membranes,(l7) but such effects were not observed in this research. One could prepare whole cells or vesicles from E. coli, fbr example, place them in a solution of’wio.l !_PMT, and measure the absorbance of the solution as a function of time while the solution is being stirred. If the cells are being emulsified, then the absorbance of the solution should decrease as a result of decreased scattering. LI ST 0F REFERENCES N \1 OT 01 b w o o o o o 10. 11. 12. 13. 14. 15. 16. 17. LIST OF REFERENCES E. Olivera-Mandalla, Gazz. Chim. Ital., $4, 174 (1914). 0. W.)Moore and A. G. Whittaker, J. Amer. Chem. Soc., 82, 5007 1960 . J. B. Lounsbury, J. Phys. 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