u . nth-HIP A. v» 5,31...“ 1.: .. bl» V: . w. . .1 .. his I_'~‘ :' .' 'T, . ' ,,‘ ’ :U'w l L.‘“fi‘:’m [II to,” lulu! {iii} 5.1.51... Ink. I. 11.. I. « son'v‘n'v. VII I I'll!!! In}. .r. 3 06h"! ~ . vn - . x .. RJHflJhMVHHUAa u. : 3 .. -4 .A 91: :5... . . J ... m. a . ~ . .r‘Z. . (”on {I 3 9| ”3".” uifl‘l l I: H!" J v I." ot a 1 . .uWU. s It. I}. - 1 . 0 mt nlnrx! ‘0... "Q- I‘lhfl! v ‘ VAV oil}... I L n 3 I(. .. ml,“ l" .ml’flfl. lll'qlwluu'lz I 5.1.! .v In 1!.ll'lo‘lr'l‘l ; ‘ "It! dtfitfé’o!v II . u 4!”le ”1.0”“. lg: v. .3334: 1 o . o. ‘lr’lflqtnf ”44!: v! . ‘ .Ot, v I... 4 :71. - . (unflamufltherdL. . r.» TW" ;; "f‘:fi":":”':‘;""" ‘ I/flgiiliiQW/o/mzflfl/Im W _.... m... E 01“. .5 r! j — -——‘ -— -v.4.,~ ;-j ‘- v-“Wl ‘ 4? r 4 L ___ - ._. _ _ i t w ‘W A... —-v-‘. .. “-wnv' This is to certify that the dissertation entitled Factors Influencing The C=N Stretching Frequency in Neutral and Protonated Schiff's Bases presented by Juan Lopez-Garriga has been accepted towards fulfillment of the requirements fo 4;» fl . degree in g @4446 M 4&3, /j/«< MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 MSU LIBRARIES n RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. PACTDIS INFLUENCING TI! GIN STIBTCIING FIEQUINC! IN NEUTIAL AND PIOIDNAIED’SCIIFF'S BASES. by JUAN LOPEZ-GAIIIGA A DISSEIIIIION enhaitted to liehigea Stete university in pertiel fulfill-eat e! the require-ease for the degree of DOCTOI.OF PBILOSOPEI Depettlent of Che-Jetty 1986 l C .2 w 9 U- 4 ABSTRACT Famous INFLUENCING THE C=N snE'rcmNc FREQUENCY IN NEUTRAL AND Pao'mNmn scnIFF's BASES. by JUAN LOPEZ GARRIGA The C=N stretching frequency has been studied in a series of aromatic Schiff's bases. their protonated derivatives and their reaction products with other Lewis acids. Protonation, deuteration or reaction Vith BF3 increases the C=N stretching frequency in a range from 1 cl"1 to 80 cal."1 . Linear polyene Schiff's bases show sinilar behavior: an increase in the C=N frequency of 30 on."1 is observed upon conpleration of trans-retinal Schiff's base with BFB’ The .g‘nitnd of the incrgggg in the ChN vibrational node is dependent on the extent of conjugation in the aromatic systen. on the nature of the substituent. and on the strength of the Lewis acid. In the NIR spectra of the protonated and conplesed species a downfield chenical shift of the protons nearby the C=N bond is observed which suggests that the nitrogen electronegativity increases in the reaction products relative to the free Schiff's base. These observations. plus the similarities in the behavior of Schiff's bases and nitriles. suggest that rehybridisation at the Schiff's base nitrogen occurs on the reaction of its lone pair with Lewis acids to increase the 0-H bond order. Ab initio calculations on the Schiff's base. nethylinine, support this idea as the C=N bond length decreases and the C=N stretching force constant increases by 0.51 Idyn/A upon protonation. Normal coordinate analysis of this species. of the model structure CH3HC=NCH3, and of their protonated and denterated derivatives are reported here which show that an increase in the stretching force constant of this nagnitude leads to an increase of ~ 30 chl in the frequency of the CbN stretching vibration. Analogous normal coordinate calculations were also carried out for the BF3 addition product which show that a similar increase in C=N stretching force constant upon conplexation is likely. The results indicate that rehybridisation effects, in particular. an increase in the s orbital contribution from the protonated nitrogen to the sp2 hybrid orbital in the Schiff's base linkage. are primary responsible for the increase in the C=N stretching frequency upon conplexation of a Schiff's base by a Lewis acid. To: Carnen Gonzilez, Marti Ldpez. Juan Francisco Ldpez, Teresa Garriga. Rebeca Gonzdlez, liguel Lopez. Francisca Hours and larti Garriga. ii ACKNOWLEDGNENTS I am deeply grateful to Professors Gerald T. Babcock and James F. Harrison for their contributions to this research and to my graduate education. I wish to thank to Professor Gearge E. Leroy and flak-Ryum Nam for their discussions and help with the set of the normal coordinate analysis programs. I would also like to express my gratitude to all my companions in the chemistry building and in particular to those who have share their friendship. time and knowledge with me. So to Jerry, Jim. Aileen Alvarado. Asaad Salehi, Jose Centeno, Demetrios Ghanotakis. Dwight Lillie. Tbny Oertling, Ivan Rodriguez. Robert Kean, Brigette Barry, Harold Fonda. Nam. Pat Iaroney, Pat Callahan, Brian 'ard. and Iargy Lynch. I like to thank to the personal of the chemistry shops for their assistance and patience in the development of the apparatus and equipment used during the course of this research. So to. Russ Geyer, Richard lenke. Carlton Iatters, Keki listry, Nanfred Langer. Scott Bankroff. Ron Rass. Thomas Clarke . Scott Sanderson and Iarti Rabb. In particular I like to thank Ron Bass. Tbny. Jose and Bob for their care and concern of our lasers system and to Dwight for his patience and help with the computer system. I would also like to thank to my family and friends who in some instance of my walk. they have teach me with their thoughts. words and actions where my feet can go. Finally, I am in debt with my brothers iii Franki. Ially. Paco Pepe, Luis, Roberto, L.. Billy, Chiqui. Nieto and Albert who in different occasions save my life from the magnificent sea. iv TABLE OF CONTENTS PAGE LIST OF TABLES .................................................. viii LIST OF FIGURES.................................................. 1 CHAPTER 1. INTRODUCTION............................................... 1 A. Importance of the Schiff's Base Bond (C=N) in Biological Systems. ................................... l B. Optical and Vibrational Properties of Chromophores Containing the C=N Bond................................ 13 C. Previous Nechanism to Account for the Anomalous Increase in the C=N Stretching Frequency Upon Protonation of Schiff's Bases.......................................... 16 D. Description of the Iork to be Presented................. 18 CHAPTER 2. MATERIALS, INSTRUIENTATION AND NETHODS. A. Iaterials............................................... 21 B. Synthesis of Imines..................................... 21 C. Synthesis of Ketimines.................................. 24 D. Schiff's Bases: Lewis Acid Derivatives.................. 24 E. Instrumentation and Calculations........................ 26 1. Ab initio..................................... 28 2. Normal Coordinate Analysis.................... 31 CHAPTER 3. SPECTROSCOPIC STUDIES. A. Introduction............................................ 32 B. spoctIO'copic Ra.n1t'...0.0.0.000...OOOOOOOOOOOOOOOOOOOO 36 1. Imines........................................ 2. Ketimines..................................... C. Discusion............................................... 1. C=N stretching frequencyzNeutral Schiff's base. 2. C=N stretching frequency:Schiff's bases/Lewis acid complexes................................. 3. Absorption maximum of trans-retinylidene-nr butylamine:Lewis acid complexes................ CHAPTER 4. AB INITIO CALCULATION. A. Introduction............................................. B. Theoretical Details...................................... C. Results.................................................. D. Discussion............................................... CHAPTER 5. NORMAL COORDINATE ANALYSES. 36 $4 60 60 67 72 75 77 78 93 A. IntIOductionOOOOCOOO00....O...I...OOOOOOOOOOOOOOOOOOOOOOO. 96 B. Numerical Calculations and lethylimine and lethylenr mania ion force ficld'.O...0.00.00.00.00...OOOOOOOOOOOO 98 C. Force Field of CH3CH=NCH3 and derivatives................ 102 D. Results of the vibrational Analyses...................... 108 1. Nethylimine and Hethylenimmonium ion Vibrational Frequencies....................... 108 2. C33CH=NCH3 and Derivatives Vibrational Froqncnci......O...I.0.000000COOOOOOOOOOOOOOOO 108 E. Di.cu"ion00000000000000......I...OOOOOOOOOOOOOOOIOOOOOOO 120 vi CHAPTER 6. SUNNARI AND FUTURE WORK. A. C. D. Introduction............................................. Summary.................................................. 1. Spectroscopic Studies......................... 2. Ab initio Studies............................. 3. Normal Coordinate Analyses Studies............ Conclusion............................................... Future 'OIkOOOOOOOOOOOOOOOOOIOOOOCOOOOOOOOOOOIOOOOOOOOOOO LIST OF WCESOOOIOOO...0.0.0.0000...OOOOOOOOOOOCOOOIOOOOOOOOO vii 124 126 126 126 127 128 129 135 LIST OF TABLES TABLE PAGE 1. Frequency assigments for Nrbensylidene-n-butylamine (BnBI), and protonated (BnBIH+)and deuterated (BnBID+)derivatives....... 39 2. Frequency assigments for 2-naphtylidene-nrbutylamine (NAPnBI). and protonated (NAPnBIH+), and deuterated (NAPnBID+) derivatives..................................................... 47 3. Chemical shift for carbonyl and imine protons of aromatic Schiff's bases.................................................. 51 4. Absorption maxima, c-c and C=N stretching frequencies of tran-retinylidene-nrbutylamine and Lewis acid derivatives....... 57 5. Carbonyl and imine stretching frequency of polyene and aromatic compounds.............................................. 63 6. Changes in the C-N stretching frequency upon complexation with Lewis acids................................................ 68 7. lethylimine potential curve. Tbtal energy for the C=N stretching motion............................................... 81 8. Nethylenimmonium ion potential curve. Tbtal energy for the C=N stretching motion.......................................... 82 9. Calculated force constants for the C-N and C-N stretching motion.......................................................... 85 10. Nethylimine: Electron distribution.............................. 87 11. lethylenimmonium ion: Electron distribution..................... 87 12. Electron distribution of the (7' bond system.................... 90 13. F matrix elements for methylimine.............................. 100 14. F matrix elements for methylenimmonium ion..................... 101 viii 15. 16. 17. 18. 19. 20. 21. Symmetry coordinates for in-plane vibrations of methylen- immonium ion................................................... Diagonal and off-diagonal force constants of some imines....... F matrix elements for the model structures..................... Iathylimine calculated frequencies of in-plane vibrations...... Hethylenimmonium ion calculated frequencies of in-plane vibrations..................................................... Calculated C=N stretching frequency of methylimine dariv.tiv°s0O...0..0.0...OO0......IOOOOOOOOOOOOOOCOOOOOOOOOOOOO Calculated C=N stretching frequency of model structures........ ix 103 105 106 109 110 111 112 FIGURE LIST OF FIGURES PAGE Bleaching scheme of the visual pigment rhodopsin. The 1l-cis retinal Schiff's base. in rhodopsin. absorbs a photon and is converted in picosecond to bathorhodopsin, which contains a distorted Cll'clz trans chromophore......... 5 Photochemical scheme of bacteriorhodopsin. The light-adapted bacteriorhodopsin (38553), contains an all-trans retinal chromOphore attached to a lysine group. of the protein, through a protonated Schiff’s ............................... 7 The condensation reaction illustrates other function of a Schiff's base, the activation of carbon via an enamine intanedi‘tOeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 12 lodel structures............................................. 23 Schematic Raman spectrometer................................. 27 Schematic spinning cell...................................... 30 Raman spectra of N-bensylidene-nrbutylamine (BnBI) and protonated (BnBIH+) and deuterated (BnBIDI) derivatives in mC13 .OIntion.O....0.000000000000000000000000000000000000 38 Raman spectra of N-bensylidene-nrbutylamine (BnBI) and protonated (BnBIHI) and BF3 (BnBIBF3) derivatives in DRSO solution............................................. 42 Raman spectra of 2-naphthylidene-n-butylamine (NapBI) and protonated (NapBIH+) and deuterated (NapBID+) derivatives in mols OOOOOOIOOOOOOOOOOOOOOOOOOOCOOOOOOOOOOOOOOOOOOOOOOOOO 44 10. 11. 12. 13. 14. 15. 16. 17. Raman spectra of 2-naphthylidene-n-butylamine (a) and 3C13 (b) and BF3 (c) derivatives in DNSO solution.......... 46 250-NIR spectra of bensaldehyde (a). N-benxylidene-nrbutyl- amine (b) and its protonated (c) and deuterated (d) derivatives in CDC13........................................ so Resonance Raman spectra of retinylidene-nrbutylamine (RnB) and protonated (RnBH+) and BF3 (RnBBF3) derivatives in DHSO solution................................ 53 Resonance Raman and absorption (inset) spectra of trans- retinal Schiff's base and Lewis acid derivatives in CHZC12° (a) Trans-retinylidene-n-butylamine. (b) BCl3 complex. (c) BBr3 complex. (d) BF3 coupler. (e) HClOg complex. (f) HCl complex................. 56 Raman spectra of -( -phenylbensylidene-n-butylamine (a) and protonated (b) and deuterated (c) derivatives in methanol ‘OIntionOOOOOIOOOO00....0.00......0.00000COOOOOOOOOCOOOOOOOO 59 Raman spectra of benzephenone (BRO). Ol’Phenylbenxylidene- amine (RBI) and protonated (BBIH+) and BF3 (BBIBF3) doriv.tiv°‘ in ”ISO...OOI...O.COO...OOOOIOOOOOCOOCOOOOOOOOOO 62 Effect of increasing the aromatic ring conjugation on the characteristic group frequency of aldehydes (GD). Schiff's bases (:1) and protonated Schiff's bases (II)............... 65 Ab initio potential curves for the C-N stretching motion. (a) CBNSCF methylimine SCF . (b) C'NSCF methylenimmonium ion SCF. (c) C'NGVB methylimine GVB. (d) c-NGVB methylenimmonium ion GVB. calculations...................... 80 xi 18. 19. 20. 21. 22. 23. 24. 25. Geometry of methylimine (a) and methylenimmonium ion (b).... 83 5' system.“ system and total charge distribution for methylimine............................................. 88 Cr system,‘1"system and total charge distribution for methylenimmonium ion.................................... 89 s-p and p electron distribution of the G'and Tr systems in the C-N bond of methylimine and methylenimmonium ion..... 92 Geometry and internal coordinates employed for the normal coordinate analyses of methylimine and methylimmonium ion... 99 Geometry and internal coordinates employed for the normal coordinate analyses of the model CH3CH=NCH3 gnd its protonated and BF3 derivatives............................. 104 Difference between the C-N stretching frequency in the protonated and neutral Schiff's base CH3CH-NCH{3}. as a function of the force constant of the principal modes which contributed to the potential energy distribution..... 115 Difference between the C=N stretching frequency in the BF3 and neutral Schiff's base C83CH=NCH3, as a function of the force constant of the principal modes which contributed to the potential energy distribution........... 119 xii CHAPTER 1 INTRODUCTION A. Importance of the Schiff's Base Bond. C=N, in Biological Systems. Nitrogen and carbon are able to change their hybridisation readily to form different kinds of bonds. It is not surprising. therefore. that their interrelationship plays a number of important roles in biological systems. The reaction between a carbonyl group of an aldehyde (1) with the amino group of a primary amine (3) leads to the formation of one of these peculiar nitrogen-carbonrbonds.the Schiff's base (3) linkage: H\ H\ /R /C=O + HzN Ra: /C=N +HZO R R S In IN us The Schiff's base (C=N) bond. has attracted interest because of its occurrence in rhodopsin, bacteriorhodopsin and its photocycle derivatives1’17 and in pyridoxal enzyme systems.18'22 letalloporphyrin and metallochlorin Schiff's bases have been synthesised recently and the possibility that these occur in vivo has been raised.23'27 The fact that Schiff's base linkages are versatile in their physical 1 and chemical preperties no doubt accounts for their importance in biological catalysis. The C-N bond, for example. is fairly labile and can be hydrolised and reformed readily (1). Protonation of the C=N nitrogen in a Schiff's base containing H r ,+ \ H‘” «H\ /R /C=N e a /C=N R 1 4% R h 1.. J l ‘i H20 H :C=O + HZNR+H+ R chromophore generally leads to a marked red-shift in the chromOphore absortion spectrum. This reaction is of importance in controlling the Optical properties of the retinal Schiff's base (2;) in the visual Pithent. rhodopsin.1'10 A1l-trans-retinylidene-nrbutylamine. ll-cis-retinylidene-n-butylamine. The primary amino acid sequence of bovine rhodopsin has been determinedzs"30 and it has been established that a lysine group of the protein is bound to the retinal moeity via a Shiff's base linkage.31 After the absortion of light, the 11-cis retinal protonated Schiff's base (£2) chrom0ph0r0 in rhodopsin32'33 photoisomerizes to a red shifted intermediate. bathorhodopsin, with a distorted all-trans retinal configuration.34'35 Following the formation of this primary photoproduct, a series of thermal events occur (see Figure 1) which initiate a change in the photoreceptor cell membrane that leads to a transmitted signal to the brain through the corresponding synaptic processes.1-10'35 BacteriorhodOpsin (ER) is another retinal-protein and it function as a photochemical proton-pump in the purple membrane of Ha10bacteris.11-17’36-38 Under normal illumination conditions, bacteriorhodopsin is in its light-adapted form. a protonated retinal Schiff's base with an absorption maximum at 560 nm.1'2'35 Absorption of light by the bacteriohodopsin retinal prosthetic group converts this light-adapted 33568 form to a red-shifted intermediate K610 which absorbs maximally at 610 nm. This species decays thermally to BRSGS’ throught a series of intermediates. one of them a deprotonated Schiff's base. i.e. N112, (see Figure 2). This light-driven protonation/deprotonation sequence of the Schiff's base nitrogen is the key step in the mechanism 0f th° proton pump action of bacteriorhodopsin.11-17'3“-38 Rany naturally ocurring porphyrinoid compounds contain a ketone or a formyl as functional groups as in the case of chlorOphyl ; (g) in the Figure 1. Bleaching scheme of the visual pigment rhodopsin (from ref. 35). The ll-cis retinal Schiff's base. in rhodOpsin. absorbs a photon and is converted in picosecond to bathorhodopsin, which contains a distorted Cll'clz tggn; chromophore. At physiological temperatures bathorhodopsin decays through a series of intermediates to all-trans retinal and the apoprotein. opsin. ‘\\\I2 \\ 4. \NH— 4. \ NH- RHODOPSIN (493 nm) ISORHODOPSIN (490 nm) A x /’ A BATHORHODOPSIN (543 nm) l> -I40°C LUMI _J .1 <1 § 1 z : if. g METAI a: m 0‘! .3 1 .3 . I Z O? METAII ‘L—opsm + ALL-TRANS RETINAL——~ WAG Figure l Figure 2. Photochemical scheme of bacteriorhodopsin. The light-adapted bacteriorhodopsin (BR563). contains an all-trans retinal chromophore attached to a lysine group. of the protein. through a protonated Schiff's base. Photoisomerization about the C13-C14 bond forms the R610 intermediate, which cycles in ~ 10 msec back to 33568 through L550. "412 and 0640' From reference 35. 2 psec /\ K (6|O nm) L (550 nm) < IO Cpset/CH NOW?) p.586 + H HSCHS 0433-130435 CH3 7 ll '33 I5 I/R \ 8013 in BR (568 nm) M (4:2 nm) R BR (560 nm) H“ O (640 nm) Figure 2 photosynthetic systems or of heme ; (1) in cytochrome oxidase. It is generally observed that the spectral properties of the in vivo chromOphores are, as in the case of rhodopsin and bacteriorhodopsin, red shifted relative to the in vitro chromophore.23’27 / / ”0" CH3 =CH use 2 O=C CH3 H 2 9": CH2 Q”? 'cOOH 000" 7 For example. cytochrome oxidase catalyses the oxidation of ferrocytochrome g and the four electron reduction of dioxygen to water through the centers cytochrome ; and cytochrome 33. Each cytochrome center contains a heme group and a proteinrbound copper ion, and because of their different chemical environments they are denoted heme ; and copper g and heme ;, and copper 33 respectively.41"2 In addition to its redox chemistry. cytochrome oxidase is involved in proton translocation from the inside to the outsite of the membrane.39—42 Such proton pumping action has been postulated to be associated with the cytochromelg 42 and in particular with the heme a chromOphore.39’4o'43 Heme‘; is center the main absorbing chromophore of the redox active center. cytochrome g, in the mitocondrial enzyme. cytochrome oxidase. and is low-spin in both its Fe2+ and Fe3+ states in the protein. Compared to low spin heme g model compounds. red-shifted visible absorption spectra of 10 nm and 17 nm for the oxidized (i.e. 598 nm and 588 nm) and reduced (604 nm and 588 mm) species. respectively are observed.39'4o From an historical point of view. it was originally thought that the red-shifted absorption maximum of the heme a chromophore was produced by a protonated Schiff's base formed between the carboxyl group of heme ; (1) and an amino group from the protein. In analogy to bacteriorhodopsin. a protonation/ deprotonation step of the nitrogen Schiff's base following the redox chemistry of that center was postulated to be inyolved in the proton pump activity 0f th° enzym¢,44t45 However. spectroscopic studies have shown that a protonated Schiff's base is unlikely to be present in heme.g 7' and that the proton pumping activity of the enzyme may be attributed to redox-linked activity at a hydrogen bond involving the carbonyl group of heme ; and a nearby amino acid side chain.39'4°'43 Nevertheless. the former observation has led to the suggestion that the red-shift observed for chlorophyll in vivo may originate. at least in part. from similar phenomena. i.e.. from formation and protonation of Schiff's base linkages in the protein enviroment.23-25 The chlorOphylls in the reaction centers of the photosyntetic apparatus in alga and green plants. with absorption maximum at 700 nm (P700) and 680 nm (P680) and located in photosystem I and in the photosystem II. respectively. show red-shifted absorption spectra relative to monomeric chlorophyl ;. This spectral shift has been attributed to dimers or to higher order tBSIOS'tO8 0f chlorophqug.46'48 However. partly because in the photopigment rhodopsin a protonated Schiff's base is directly involved in the regulation of the Optical absorption wavelength maximum. the dimer lO hypothesis has been challenged and a protonated Schiff's base has been preposed 3‘ source 0f the P700 and P680 red shifted absorption maximum.23-25 In addition to the above proteins. there is a set of enzymatic systems in which the prostetic group and the catalytic function depend explicitaly on a protonated Schiff's base. For instance. the coenzyme. pyridoxal phosphate (8). plays an important role in the metabolic interconversion of amino acids.18'22'49'50 R I OH 3 o-R-o \ O '- 0 K; CH 3 R 3.. The protonation reaction of the Schiff's base group (C=N) of the coenzyme produces the key intermediate which can lead to transamination (2). or the interchange reaction. of an amino group of the protein amino acid by a carboxylic group. The reaction mechanism of the enzyme. aspartate amino transferase49.a classical example of transamination. together with other transaminases involving Schiff's bases. has been extensively discused in a recent book by Christen and Ietzler.’o - + - \ I l ,0 \ I u / C . aC- —C""C \ H20 0/ H \\O ion-'1- 0 H H O 11 Another group of protonated Schiff's base-related enzymes involves a ketimine (19). instead of an imine (1). Schiff's base. This condensation reaction leads to the formation of an enamine intermediate (11.11) during the catalytic cycle of a particular process. Such an intermediate. for instance. is involved in the decarboxylation reaction of acetoacetate by the enzyme acetoacetate decarboxylase52'53. and in the aldol condesation and reverse cleaveage reaction of the RCHO group by the enzymes. aldolase and transaldolase.respectively.5"57 The condensation reaction is shown in Figure 3. H,C\ .,R‘.H+ +1.01% (“R HHFQCP'R (3:: -———--€> (3: PNH /' ‘\ R’ ‘H R’ \H R H 19 u 1.2. A similar mechamisn is present when the pyridoxal cofactor Schiff's base is involved in the decarboxylation reaction of aspartate and in the interconvertion (1g) and/or degradation of aminoacids such as serine. threonine, cysteine. tryptophan. and cystathionine.5°'51 R >530” H,\C/ C o; C HAT/C 0:" 4.. H\ /N\ l2. chz-o-P Ho-o-P M- -En A C=N H- En éH,-OH H- -C-H (3 H b 0/ meg/W H C-O- P H.(l3-O-P ”20"c'3= ~NH -En 0-0 ”Ob.” 6—:Hoc-H+HN- En+H* “'9'0H H- 0-0 H R R. Figure 3. The condensation reaction illustrates other function of a Schiff's base. the activation of carbon via an enamine intermrdiate. P and En represent the phosphate group and aldolase enzyme respectively. 13 The above survey of biological systems has indicated the importance of the protonated Schiff's bases in the biological kindom. In the next section. the optical and vibrational spectroscopic properties of chromOphores involving the C-N group will be discussed. B.Optical and Vibrational Properties of Chromophores Contaning the C=N Group. Visual excitation is initiated by photon absorption in the rhodopsin pigment. This chromophore-protein complex consists of a protonated retinal Schiff's base bound to an apOprotein. cpsin. The 11-cis-retinal protonated Schiff's base. formed from nrbutylamine and ll-cis-retinal. has it absorption maximum at 440 nm in methanol‘l, whereas the bovine rhodOpsin pigment (see Figure 1) absorbs at 500 nm. Honig. Nakanishi and 61.66 eo-workers proposed to call this 1_ vivo red-shift from the 1g vitro absorption maximum. the "cpsin shift” (in omIl). For instance. the opsin shift for cattle opsin would be 2700 cm-1 [22700 cm{-1} (i.e 440 nm) - 20000 cm-1 (i.e. 500 nm)]. Similarly. the opsin shift in bacteriorhodopsin is ~ 5100 cm-1 since the absortion maximum of the protein is at 560 nm.74 The deve10pment of this opsin shift. or wavelength regulation. is caused by the protein enviroment and constitutes one of the central problems in the visual research. Numerous models have been proposed to account for the opsin shifts in rhodopsin. bacteriorhopsin and their various photocycle 61.66.74.95-101 Blatz et 60.95.102 intermediates. al. in their early studies of retinal Schiff's base models. concluded that upon protonation 14 of the retinal imine the partially positive charge on the nitrogen depends on the separation between the center of charge of the cation and anion (counter ion effect). In this way. the fractional charge on the Schiff's base nitrogen not engaged in the ionic bond polarizes the _ system to produce a resulting excitation energy lower than the unprotonated Schiff's base species. To explain the opsin shift in rhodopsin and bacteriorhodopsin. Nakanishi et al.61'66 proposed that. in addition to the counter ion effectloz. a negative charge in the vicinity of carbons 11 and 12 (see Figure 1) can account for the ~ 2700 cm-1 opsin shift of rhodopsin. They also preposed61'66 that for bacteriorhodopsin a negative charge near carbon 5 can account for the 5000 cm”1 cpsin shift. However. recent data from lathies and his group7‘. and others in the field103 suggested that the bacteriorhodopsin shift can be partitioned into a 1200 cm.-1 shift due to ground state configurational changes and a 3900 cm.’1 ehift due to a weak hydrogen bond between the Schiff's base proton and an electronegative group in the protein.74 These observations on the bacteriorhodopsin shift suggest that the enviroment at the protonated retinal Schiff's base moiety plays an important role in regulating the absorption maxima of the photopigments. 15 Schiff's base (11) and protonated Schiff's base (11) C=N vibrational modes have been studied for at least the past three decades.58'59 4} R R H7 \ C=N —H—+ C=N Part of the interest in these species derives from the observation of functionally significant Schiff's base linkages in biological systems. for example. in pyridoxal euzymesls"22 and. more recently. in bacteriorhodopsin. rhodopsin and related visual cycle intermediates and models.1'17 In rhodopsin. the retinal chromophore is bound to the opsin protein moiety through a Schiff's base linkage and resonance Raman spectroscopy has been used extensively to monitor changes in the C=N stretching frequency during the rhodopsin photocycle. For example the C-N stretching mode in the neutral species occurs at 1620 can"1 . This increases to 1655 chl upon protonation and to 1630 chlupon deuteration.1'10 In bacteriorhodopsin. smaller frequency changes. presumably originating in the protonated Schiff's base. occur at stages in the photocycle and suggest different degrees of protein-chromophore interaction for the various intermediates.35 Thus, the decrease in the GIN stretching frequency from the protonated L550 intermediate (RC_N=1544 cm'l) to the "412 intermediate (JC.N-l632 cm'l) has been used as an indicator of the proton pump cycle of bacteriorhodopsin.2ind”.35 Analogously. pyridoxal Schiff's bases show the same trend in the l6 absortion spectra and C=N stretching frequency. For instance. at pH=13 the pyridoxal-valine Schiff's base complex shows an absortion maximum at 385 nm and a C-N stretching frequency at 1630 chl. Upon protonation at pH89. the absorption maximum red shifts to 415 nm and the C-N stretching frequency increase to 1643 cm'l. Deuteration at the nitrOgen shifts this vibrational frequency to 1635 cmfl. Similarly. the pyridoxal enzyme. aspartate aminotransferase. show a C=N stretching frequency of 1649 cm-1 at pH=5 and this mode decreases to 1617 cm"1 upon deuteration.21'22 23 on the Schiff's bases formed between Iork from this laboratory formylated meta110porphyrins or metallochlorins and primary amines shows that upon protonation of these aromatic Schiff's base there is an increase in the C=N stretching frequency as well. For example. for the Schiff's base of Ni(II) porphyrin a the C-N stretching vibration occurs at 1639 cm’l. 1650 cm"1 and 1640 cm"1 . respectively. for neutral. protonated and deuterated species. Accompanying the protonation reaction. there is a marked optical red-shift in the visible region23’25 as also occurs in the polyene case.1-lfl"6°"66 C. Previous lechanisms for the Anomalous Increases in the C=N Stretching Frequency Upon Protonation of Schiff's Bases. Ihile the origin of the optical red-shift has been explored 26.27 60-66 theoretically in both aromatic heterocycle and polyene cases the mechanism underlying the increase in the characteristic Schiff's base frequency upon protonation remains obscure. Both the resonance structures (16.11) and the increase in the reduced mass of the nitrogen 17 suggest that the C-N vibrational frequency should decrease. In the visual pigments and their model compounds the frequency increase associated with the C=N stretching mode upon protonation has been attributed to the interaction between the C=N stretching mode and the C=N-H bending mode.4'5'8'n'13 The presence of this new internal coordinate. (i.e. the C=N-H bending). is reflected in the C=N vibrational frequency by the C=N-H (bending) and C-NlC-N-H (stretch-bend) interaction force constants. As pointed out by Iarcus et al.13. however. the stretch-bend interaction model can not account for the increase in the C-N vibrational mode which occurs when retinal Schiff's base derivatives are methylated instead of protonated. Increases in the unsaturated carbon-nitrogen stretching vibrational frequency upon reaction with a Lewis acid have been reported for other systems. For example. I.R studies on ketimines (R;ch-NR) indicgt. thgt the increase in the frequency of the C=N stretching mode when the nitrogen lone pair is shared with a Lewis acid substituent (e.g. 8+, BF3 or BCl3) results from an increase in the C=N bond order.67 18 Similarly. when nitriles (1Q) react with Lewis acids such as BF3 (;2) sad ac13, the CEN stretching mode increases in frequency. This behavior has also been attributed to an increase in the CeN stretching force constant and to a decrease in the CEN bond distance.68'72 Because of the structural analogies between these species and the Schiff's base system. these results suggest that a similar mechanism may occur for the Schiff's base C=N group. D. Description of the Work to be Presented. In the work described here. absorption. nuclear magnetic resonance and Raman spectroscopic studies have been carried out on aromatic and retinal Schiff's bases and protonated. deuterated. BBr3, gels and 3p3 complexed derivatives. The aromatic Schiff's bases serve as simple models for the aromatic metalloporphyrin systems and protonation of the above systems also allows us to compare the behavior of the C=N group in aromatic vs linear polyene Schiff's bases. The BF3, BCl3 and 33,3 adducts are very useful complexes for studying the behavior of the C-N stretching frequency when the nitrogen lone pair is encumbered by a Lewis acid other than the proton. Complexation of trans-retinal Schiff's base 19 (1;) with a general Lewis acid. such as BF3, should remove the C=N-H bending interaction effects (i.e. the C=N-H bending is substituted by the C=N-B motion) on the C=N stretching frequency while maintaining delocalization of the Tfsystem and thus provide a means by which to test the C=N stretch-C=N—H bend interaction or mechanical coupling hypothesis. Absorption spectroscopy was useful in the characterization of the Schiff's bases and their Lewis acid derivatives. The magnetic technique (NIH) was employed to observe the effect of the protonation on the atoms near the C=N group. Raman and resonance Raman spectrocopy were used to determine the frequencies of the vibrational modes associated with the Schiff's base models and. in particular. to note the behavior of the C=N stretching frequency upon changes in the conjugation. protonation . and Lewis acid adduct formation. The spectroscopic results show73'76 that upon protonation of the Schiff's bases. there is a marked red shift in the absorption maximum. the near by protons of the imine group shift downfield and the C=N stretching frequency increases. Similar trends in the absorption maxima and changes in the DIN stretching frequency are observed upon the reaction of the free Schiff's base with BF3, 8013 and 88:3. Iith this work in mind. ab initio electronic structure calculation for methylimine and its protonated derivative at the Generalized Valence Bond77 (GVB) and Self Consistent Fields78 (SCF) levels were carried out. The GVB calculations show [73] that upon protonation of methylimine there 20 is a reorganization of the electronic character of the nitrOgen lone pair in such a way that the C=N bond order increases (we call this effect the rehybridization model) and surprisingly. the nitrogen appears to be partially negatively charged. Finally. numerical calculations within the normal coordinate analysis framework. were conducted for the models CHZ-NH and cnsncsncns and their protonated. deuterated and BF3 derivatives to explore the predictions of the stretch/bend interation model in light of a restricted set of force constants and to determine the effect of the increase in the C=N bond order (rehybridization model) on the C=N stretching frequency. The results of these analyses indicate that the electron density redistribution (i.e. rehybridization of the C=N bond) which increases the C-N stretching force constant upon protonation of Schiff's bases plays a major role in determining the increase in the C=N stretching frequency. CHAPTER 2 MATERIALS. INSTRUMENTATION AND METHODS A. Materials Benzaldehyde. trans-retinal. methylamine. n-butylamine. tert-butylamine. benzophenone. bromo-bencene. benzonitrile and the Lewis acids. BC13. BBr3 and BF3. were obtained from Aldrich Chemical Co. 2-naphtha1dehyde and 9-anthra1dehyde were obtained from Alfa Products. The solvents used. CH2C12. HCCl3 and DHSO (dimethyl sulfoxide). were freshly distilled and kept in a dry nitrogen atmosphere over 5A.molecular sieves. Benzaldehyde was purified by distillation in vacuum. 2-naphthaldehyde and 9-anthra1dehye were re-crystallized from a methanol-water mixture. All the other starting materials were used with no further purification. B. Synthesis of Imines N-benylidene-n-bntylamine (Figure 4a). 2-naphthylidene-nrbutylamine (Figure 4b). and 9-anthry1idene-nrbutylamine (Figure 4c) were prepared by reaction of the appropriate aldehyde in 4h reflux with dry benzene containing an excess of nebutylamine. Following completion. excess n-butylamine and benzene were removed by vacuum evaporation. N-benzylidene-nrbutylamine was purified by distillation in a vacuum. while 2-naphthalidene Schiff's base was purified by a sublimation technique. Retinylidene-n—butylamine (Figure 4d) was prepared by two different routes. One employed the same procedures as for the aromatic 21 22 Figure 4. a. N-benzylidene-nrbutylamine. b. 2-naphthylidene-nrbutylamine. c. 9-anthrylidene-nrbutylamine. d. All-trans-retinylidene-n-butylamine. e. oC-phenylbenzylidene-n—butylamine. f. af-phenylbenzylideneamine. CH3 CH3 CH3 Figure 4 CH3 ll l3 l5 24 imines. The other involved combining the corresponding retinal isomer with an excess of nrbutylamine in ethanol. This solution was mixed at 0 °C for 2hr under a stream of nitrogen and freeze-dried.14 The two preparative techniques gave product which produced the same spectroscopic results. C. Synthesis of Ketimines of-phenylbonzy1idene-n-bntylsnineso (Figure 4e) was obtained as the reaction product between benzOphenone and the amine with aluminum chloride as catalytic agent.oprhenylbenzylideneamine (Figure 4f) was prepared by means of a reaction between a bromo-benzene-magnesium complex (Grignard reagent) and benzonitrile.81 The Grignard-nitrile complex was prepared by the dropwise addition of 0.45 mole of the nitrile to a ' stirred Grignard reagent prepared from 0.50 mole of the corresponding halide and 0.51g of magnesium in 300 ml of anhydrous ether. followed by a 24 hr. reflux. After cooling to room temperature. the complex was decomposed by the dropwise addition of 100 m1 of anhydrous methanol (40 min). This last reaction was very vigorous. Completion of the decompositon gave a slurry solid. The slurry was filtered. and the filtrate was vacuum destilled. The ketimine was collected between 125-130 °c. D. Schiff's bases: Lewis acid Derivatives Protonated and deuterated derivatives were obtained by adding equivalent amounts of dry HC1(g) or DCl(g) to the corresponding imine 25 solutions. by bubbling the acid through a solution of the Schiff's base in other until precipitation was completed. or by bubbling the acid in the solvent and then adding equivalents amounts of the acidified solvent to the Schiff's base solution. The products obtained following the three methods gave identical spectroscopic results. The aromatic and polyene Schiff's bases Lewis acids derivatives (BF3. BC13) were prepared by reacting a stoichiometric amount of the Lewis acid with the particular imine (i.e. benzaldehyde. 2-nahtha1dehyde and retinal Schiff's base) in dimethyl sulfoxide (DMSO). The trans-retinal Schiff's base: Lewis acid complexes in CH2C12. were prepared by adding to the retinal Schiff's base solution in CH2C12 an equivalent amount of 0.001M solution of the corresponding Lewis acid (8013. BBr3. BF3) in CHZClz. All reactions were carried out after degassing solvents and imine solutions in a nitrogen enviroment. The transfer of the Lewis acid to the solvents or to the Schiff's base solutions was carried out in a dry nitrogen environment in dry. preheated 82 glassware. The solids were washed with ether and dried and stored in a dry nitrogen atmosphere. The formation of the Schiff's bases from its parent aldehydes was. in general. indicated by a blue shift of the absorption spectra while the formation of the Schiff's base complex was indicated. relative to the free base. by a red shift of the spectrum.23-25'6O-66 26 E. Instrumentation and Calculations Optical spectra were recorded with a Perkin Elmer Lambda 5. UV/Vis spectrOphotometer. Concentration of the neutral. protonated and Lewis acid (BF3. BBr3. BC13) Schiff's base complexes were typically 1.0-5.0 x 10-4! in methylene chloride. chloroform or dimethyl sulfoxide for the optical measurements. For the 250-IHz NMR spectra. benzaldehyde and its corresponding imine were prepared in deuterated chloroform: protonated and deuterated derivatives were obtained by adding equivalents amounts of dry HCl or DCl gas. In the Raman experiments. the incident laser frequency was directed through the bottom of a clear cuvette or spinning cell that contained the sample. The scattered light was collected at 90° to the incident beam (see Figure 5). focused and passed through a polarization scrambler. Following dispersion by the double monochromator (Spex 1401). the various light frequencies were selectevely focused upon a cooled photomultiplier (RCA C31034). Photon counting electronics were used and the scattered intensity versus frequency was displayed on a chart recorder. Static and spinning cell arrangements were used for the experiment. Raman spectra of the aromatic Schiff's bases and their derivatives (~ 0.1-0.25 M in the different solvents) were obtained by using two different laser excitation frequencies: >‘ex-647.1 mm (from a krypton ion spectra Physics model 164-11) for 2-naphthalidene and 9-anthralidene derivative and. Xex-514.5 nm (from an argon ion Spectra Physics model 164) for the other samples. Resonance Raman spectra of the retinal LENS h.) 27 DOUBLE PM, I | MONOCHROMATOR — AVERAGER Puntou AND —“ couursa PLOTTER Figure 5. Schematic Raman Spectrometer 28 Schiff's base derivatives were recorded by using 441.6 nm excitation from a helium-cadmium laser. A scan speed of 50 cm'llmin. a time constant of 2.5 sec and 5 chl spectral resolution were used in recording the Raman spectra on a Spex 1401 double monochromator. For recording the Raman spectra of the light sensitive retinal Schiff's bases and derivative. a spinning cell system. consisting of a quartz cylindrical cell. spinning on a precision ball bearing (Model 00-450-082885 from Detroit Ball Bearing Co.) and connected by a 0—ring ( 568-026 from Detroit Ball Bearings Co.) to a motor ( 5.25 v and 4500 rpm model 1950 from C and H Sales Co.) (see Figure 6). was deveIOped and constructed at Michigan State University. 1. Ab 131112 Calculations. The 1b initio C=N bond distance and C-N stretching force constant were determined for methylimine and methylenimmonium ion under the SCF (Self Consistent Field)78 and GVB (Generalize Valence Bond)77 formalisms. All calculations were carried out by using the Argonne National Laboratory collection of Electronic Structure Codes (QUEST-164). In particular. the integrals were done by using the program ARGOS written by Pitzer83 and the GVB calculations were done by using the program GVB 164 written by R. Bair.84 The calculations were done on an FPS 164 attached array processor. The details of the calculations and results will be discussed in Chapter 4. 29 Figure 6. Schematic Spinning Cell a. Plataform supporting system. b. 5.25 v motor. c. Quartz spinning cell. d. Ball bearing. e. 0-ring. f. Distance in mm. 3O 10 r 26 i -—— 23 -——-~ I. J gig it—J l 87 Figure 6 2. Normal Coordinate Analysis. In-plane vibrational frequencies and the corresponding potential energy distribution for methylimine. for a hypothetical methylenimmonium ion and for the model structure CH3CH=NCH3 and the corresponding isotopically substituted derivatives were calculated by using the Shimanouchi normal coordinate analysis programs.”'86 For the specific details of the calculations and results see Chapter 5. CHAPTER 3 SPECTROSCOPIC STUDIES A. Introduction. Vibrational spectroscopic studies provide a powerful means of elucidating donor-acceptor interactions through a comparison of the vibrational frequencies of the complex with those of free acid and base. The present study is centered on the specific effects that these interactions can have on the optical absorption and vibrational preperties of the Schiff's bases upon complexation of the nitrogen lone pair electrons of the C=N linkage with Lewis acids such as H+. BF3. BC13. and BBr3. The interest in the optical and vibrational preperties of the Schiff's bases and their complexed derivatives derives from the important role that the C=N linkage plays in biological systems (see Chapter 1). in organic syntheses via the cyclization of nracyliminium ions (12) of naturally occuring alkaloids87, and in the nature of intramolecular electron transfer in olefin-N-heteroaromatic salt systems.88 Most of our present knowledge on the spectroscopic properties of the 32 33 chromOphores containing the C=N bond comes from the pyridoxalls"22 and retinal Schiff's bases model systems and from the biologically occurring pyridoxal enzymes and photopigments. rhodopsin and bacteriorhodopsin.1-17 Also. scattered studies on porphyrin Schiff's bases and aromatic ketimines are present in the literature.23'25"7 However. a systematic investigation of saturated. unsaturated and aromatic Schiff's bases and their Lewis acid complexes (i.e. H+. BF3. BC13 and BBr3) has not appeared. Rather. most reports deal with compounds specific to the problem at hand. Thus. the C=N stretching frequency for saturated aldimines was assigned by Fabian et al.58'59 to the 1665-1674 cm.-1 range. This work was extended by Steele89 compouds with frequencies in the to 1665-1680 cm-l. Aromatic aldimines of the type C6H5-CH8N-R present the C=N stretch in the 1658-1629 cm."1 region and in the 1637-1626 cm'1 range when the R group is substituted by a phenyl groupsg. Iitkop et al.18 in a study of possible pyridoxal models indicates a 1639-1626 cm."1 range for aromatic Schiff's base models. A further reduction in the vibrational frequency of this mode is obtained when the number of substituent phenyl rings increases.89 In general. these reports show that the region over which the C=N stretching frequency occurs in uncomplexed Schiff's bases is relatively extensive and that the physical and chemical environment of the C-N group. including factors such as the presence of electron donating or electron withdrawing substituents. conjugation effects. resonance effects and hydrogen bonding. contribute to the C-N stretching frequency variations. 34 More extensive insight into the spectral changes of a system containing the C=N chromophore comes from resonance Raman vibrational studies of the retinal Schiff's bases and their derivatives. These studies have helped to develop an understanding of the relationship between the vibrational properties of these species and the different conformations that the polyene can assume in rhodopsin. bacteriorhodopsin and their respective photocycle intemediates. Heyde et a112, Mathies et a17'35 and other workers4’15'90 recognized that the vibrational spectrum of retinal Schiff's base isomers provides a characteristic vibrational fingerprint region (i.e. 1100 - 1400 cm’l) unique to its molecular structure. For example. conversion of all-trans retinal to its Schiff's base causes a 50 chI increase ( see 1;. Chapter 1) of the C14-C15 stretching frequency (i.e. from 1111 car1 to 1161 cmfl). In addition to the shift of the C14-C15 normal mode. other C-C frequencies in retinal Schiff's base and protonated species have been found to be altered?5 In this regard. the use of 2H and 13C isotopic derivative has been key to assessing the C-C. c-c and C-H vibrational modes. More detailed discussion of the trans-retinal Schiff's base and protonated derivatives vibrations can be found in the extensive work by Mathies et alg'11'36 The identification of the all-trans-retinal vibrational frequencies has been used as the reference point to distinguish the presence of other isomers. i.e. 9-cis. ll-cis and l3-cis retinal protonated Schiff's bases in the photOpingments intermediates. It appears. as discused in Chapter 1. that co-ordination of Schiff's bases (i.e.C=N) with Lewis acid acceptor like H+ or BF3 increases the C=N stretching frequeny. For example. reaction of alkene ketimines with the 35 above Lewis acids increases the C=N stretching frequency from 1610 chl to 1672 cmfl. Similarly. aromatic ketimines show increases of the order of 7-64 cm.1 upon protonation67 and protonation of retinal Schiff's bases produces a 35 cm.1 increases in the same vibrational mode i.e. from 1620 cm-1 to 1655 cm-1.1-17 This increase in the C=N vibrational frequency is surprising because it might be expected that co-ordination would cause. in analogy to carbonyl compounds91"93 of the general type RHCOX. a lowering of the C=N bond order and hence lengthening of the C=N bond. with a consequent decrease in the Schiff's base (C=N) stretching frequency. From this point of view. as indicated in Chapter 1. the C=N frequency increases associated with the visual pigments. and Schiff's bases in general. have been attributed to the interaction between the C=N stretching mode and the C=N-H bending vibration.4'5'8'11'13 However. the fact that increases in the C=N stretching frequency are observed for a large variety of substituted Schiff's bases (e.g.aromatic. alkene. polyene and porphyrin) Lewis acid complexes and that the corresponding C=N-H or C=N-B bending motion are expected to be at very different vibrational frequencies leads us to the idea of testing the stretch-bend interaction model. In order to achive this task. we have studied the vibrational properties of the C=N stretching motion in a set of aromatic imines and ketimines as a function of increasing the ring size substituents and in terms of the interaction between the Schiff's base bond and the Lewis acids 11*, 3123. BC13 and BBr Similarly. we have 3. studied the effects of Lewis acid complexation of the nitrogen lone pair electrons (in retinal Schiff's bases) on the absorption maximum and C=N stretching frequency of the model chromOphore. In principle. 36 complexation of the retinal Schiff's bases with Lewis acids. other than the proton. can help to determine whether the cptical absorption red shift in the protonated species is a unique prOpertiy of the protonated complexes (due. for example. to the presence of a counter ion or to mechanical coupling) or is a general property that follows the encounter of the nitrogen lone pair with any Lewis acid. The results of this study and the effects on the C=N stretching frequency in the complexed aromatic and polyene Schiff's bases are below. B. Spectroscopic Results. 1 Imines. Figure 7 shows Raman spectra of N-benzylidene-n-butylamine Schiff's base and its protonated and deuterated derivatives in chloroform. The work on benzaldehyde104 provides assigments for the ring and related vibrational motions (see Table 1). Thus. the band at 1696 cm-1. assigned to C-0 stretching frequency in benzaldehyde. is shifted upon Schiff's base formation to 1646 chl and is assigned to the C=N stretching frequency.1°5'106 Upon 1 and a new protonation. the frequency of this mode increases to 1680 ch band appears at 1425 en'1 in Raman and at 1418 en’1 in IR; this band is absent in the unprotonated and deuterated Schiff's base derivatives and is assigned to the C=N-H bending motion. Deuteration of the Schiff's base increases the C-N frequency mode to 1660 chl. The highest frequency shoulder in the protonated and deuterated species is due to decomposition products. Complexation of benzylidene-n-butylamine with BF3. in dimethyl sulfoxide (DMSO) solution. increases the frequency of the C=N normal mode to 1690 cm'l. Spectra of the parent aldehyde and the 37 Figure 7. Raman spectra of N-benzylidene-n-butylamine (BnBI) and protonated (BnBIH+) and deuterated (BnBID+) derivatives in CHC13 solution. The highest frequency shoulder in the protonated and deuterated species is due to decomposition products. 38 ZZZ” Algsualu]: uouloa IOOO 900 |600 I500 I400 I300. '200 ”00 A (cm") I700 Figure 7 Table 1 Frequency (0:71) Assign-eats to: N-bonzylideas-arbutylnnino (BnBI). and Protonated (BnBIH) and Dontoratod (DnBID) Derivntivos. BnBI 10025 1025m 1060w 1114w 1159w 1170m 1222vs 1221 1293w 1310w 1340w 1378 1452w 1495m 1588w l602vs 16465 BnBIH 10025 1030m 1060w 1121w 1169m 1194m 12255 1228 1294w 1306w 1312w 1334w 1346w 1425m 1451w 1497w 1603vs 16805 BnBID 10025 1030m 1060w 1121w 1168m 1194m 12205 122E 1243m 1294w 1306w 1311w 1334w 1351w 1459w 1497w 1602vs 16605 Assignments 12 v19a chain solvent v9a solvent * ¢-C=N-R. * Cfizwag chain CH aldehyde C=N-H bending v18b 18? 8a 8a (CCC C=N 40 neutral and protonated Schiff's bsse species in this solvent are also shown in Figure 8. The C=N stretching frequency is sensitive to the nature of the amine used in forming the Schiff's base. For the benzaldehyde system, substitution of the nrbutyl group by a methyl group shifts the C=N stretching frequency to 1650 cmfl. The small 4 cm.—1 increase in the C=N vibrationsl frequency of this species may be due to the small mass of the methyl group relative to the nrbutyl derivative. Upon protonation, this mode increases to 1684cm—1. The corresponding frequencies for tert-butyl substitution are 1640 cm’1 and 1666 cm.1 , respectively (spectra not shown). The Raman spectra of 2-naphthslidene-nrbutylamine and its protonated and deuterated Schiff's bases are shown in Figure 9. The C-N stretch increases from 1643 cm"1 (in the neutral species) to 1675 Ol-l upon protonation and to 1655 cm.1 in the deuterated derivative. Similar to the protonated benzaldehyde Schiff's base derivative. the protonated 2-naphthylidene-nrbutyl amine derivative shows a band at 1420 cm”1 in the Raman spectrum which is absent in the spectra of the unprotonated and deuterated species. The Lewis acid (BFS. 8013) complexes of the above species also show an increased C=N vibrational frequency (to 1683 or 1681 1. see Figure 10). Table 2 shows the assignments for the observed 107 cm- vibrational frequencies; the work on naphthalene and naphthaldehydes108 have been used for the main assignments. We have also recorded vibrational spectra for Schiff's base species in a series of anthracene derivatives (spectra not show). Protonation of 41 Figure 8. Raman spectra of N-benzylidene-nrbutylamine (BnBI) and protonated (BnBIH+) and BF3 (BnBIBFs) derivatives in DISO solution. The solvent peaks are denoted by ‘. 42 NNQI 9‘». I hon-l hum. I N2... 09... C I300 I400 I500 I600 I700 A (cm") |200 ”00 Figure 8 43 Figure 9. Raman spectra of 2-naphthy1idene-n-butylamine (NapBI) and protonated (Nspnnm and deuterated (NapBID+) derivatives in CBC13 solution. 44 6K” IZH Sb" 91.” 98” ZJZI QQZI OVEI .29 l 96 GBEI 0’91 999I __ "9| Q Q U z GLQI 009I 029I '-'-'==:: Ebgl AusuawI unmoa I500 I400 I300 I200 II00 l000 AIcm'I) I600 I700 Figure 9 45 Figure 10. lsman spectra of 2-naphthylidene-n-butylamine (a) and 3C13 (b) and BF3 (c) derivatives in DISO solution. The solvent peaks are denotes by ‘. 46 "no" ouc— 22 “III\ :2 U «con 1 «av— can— 1500 1600 1700 1400 1200 1300 A (cm-1) 1100 Figure 10 47 Table 2 Frequency (cm-1) Assignments for 2-Naphtylidene-u-butylamine (WI). and Protonated (mm) and Deuterated (NAPnBID) Derivatives. NAPn BI NAPnBIH NAPnBID Assignments 1019m 1019w 1019w a1 1121w 1143w 1143w 1143w all 1170w 1170w ll78m a1 1185m 1185m 1217m 1217m 1217m solvent (CHC13) 1230m 1230m al 1265w skeletal 1276w 1277w a1 1340w 1344m a1 1373m 1379m 1374m a; 13845 13895 13895 a1 13953 a1 14205 C=N-H bending 1440m 1444m 1444m a1 1468m 1471m 1470m a1 1511w 1511w 1510w a1 1579w 1579w 1579w a1 1600w 1600w 1600w a1 16305 16285 16265 a1 16435 16755 16555 C=N stretching 48 9-anthrylidene-n-butylamine increases the C=N stretching frequency from 1644 cufl to 1663 cm’l. The NIH spectra recorded for benzaldeyde Schiff's bases and their protonated and deuterated forms are presented in Figure 11. Table 3 collects the NMR chemical shift data for the H(a) andNI-CH2 protons of N-benzylidene-nrbutylamine, 2-naphthylinc-urbutylamine and 9-anthrylidene- n-butylamine and their prptonated and deuterated forms. The data indicate. in agreement with similar studies13’109 . that a downfield shift of the H(a) anddPan protons occurs upon protonation or deuteration of the aromatic Schiff's bases. The splitting of the H(a) proton into a doublet and of thedE-CH2 into a quartert is due to the H(b) proton present on the imine nitrogen. As expected. these feature are absent in the unprotonated and deuterated Schiff's bases where the (Ha) proton gives a singlet and thew-CH2 protons appear as a triplet. Linear polyene Schiff's bases are well-known to exhibit behavior analogous to that observed for the aromatic Schiff's bases: upon protonation the C=N stretching frequency increases.1-17 The Raman data in Figure 12 show that the analogy extends to the retinal Schiff's base:BF3 complex as well. Figure 12a shows the spectrum of the neutral schiff's base in DMSO; the O-N stretch occurs at 1623 cm'l. Protonation (Figure 12b) increases this mode to 1654 cmfl. In the BF3 complexed retinal Schiff's base derivative (Figure 12c) the C=N stretching mode occurs at 1656 cm-1. In addition, a series of trans-retinal Schiff's base: Lewis acid complexes (BF3. BCls. BBr3) were prepared and characterized in a less polar solvent. i.e. CHZCIZ' As expected, strong optical absorption 49 Figure 11. 250-NIR spectra of benzaldehyde (a). N-benzylidene- nrbutylamine (b) and its protonated (c) and deuterated (d) derivatives in 00013. 50 a” oceans I: a N M n O h a O 6— — d4aaa— add—Janad...<_.aa.d.1.fi4q..... 35/ \e \Z../ G uIUuIUoHL C.— U ADVI/ Table 3 Chemical Shift for Carbonyl and Imine Protons of the Kind: a H OCH R Ha OCH R / a\ / 2 \ 4 2 ArC C=N C=N \ / / \ 0 Ar Ar H (D) b Ar Substituent H a-CH __ _a_ 2 05115 3‘ Carbonyl 10.08 Imine 8.30(s) 3.6S(t) Prot. Imine 9.38(d) 4.10(q) Deut. Imine 9.38(5) 4.10(t) C10H8 Carbonyl 10.04(5) Imine 8.30(s) 3.60(t) Prot. Imine 8.8 (5) 3.95(b) Deut. Imine 8.7 (s) 3.95(t) C14H10 Carbonyl 11.32(s) Imine 9.28(s) 3.86(t) Prot. Imine 9.75(b) 4.29(b) r - Propyl group; s = singlet; d= doublet; t = triplet; q = quartet; b = broad. a. ZSO-Ifiz. b. GO-Iflz NNR. 52 Figure 12. Resonance Raman spectra of reinylidene-nrbutylamine (RnB) and protonated (RnBH+) and BF3 (RnBBFa) derivatives in DISO solution. The solvent peaks are denoted by ‘. 53 900 A (cm") Figure 12 05 b- ‘2 I’O N ‘2 I 8 RnBH(b) ,0 T g 9 N) 52 - * I 5* " I N) 7' I [c a: I 8 RnBBF3(c) = .. I0 ' 8 '3‘“ <0 '0 co - N0 n .. ._ 1 ._00 (D 0* - - a: - u) [s O) I 1 1 1 1 1 1 1 I000 II00 I200 I300 I400 I500 I600 I700 54 red-shifts are observed for these species (Figure 13). The absorption maximum for protonated Schiff's base complexes is solvent dependent. as has been pointed out by Flats and co-workers.6°'95'102 Similar solvent dependencies are observed for the Schiff's base: general Lewis acid complexes (compare for example. the extent of the red-shift in the BF3 and HCl adducts in CHZClz and in DISO in Table 4). This supports the idea that the physical phenomena underlying the behavior of protonated and general Lewis acid complexed Schiff's bases are similar. The corresponding resonance Raman data indicate, however. that the C=N stretching frequency in the Schiff's base increases by an amount similar to that observed upon protonation. For example. BF3 (Figure 13d) increases the C=N stretching frequency by 31 cmfl, while in the B0103 complex (Figure 13a) an increase of 30 cm"1 is seen. The other Lewis acid ¢OIP10188 (BC13. BBr3 and 801) show comparable increase in the C=N stretching frequency (Figure 13). The small increase in (C=N) for the Schiff'8 bI80’BC13 complex, is similar to the observed trend68 for aromatic nitriles upon complexation with BF3 and BCl3. At this point it is useful to mention that the possibility that a retinal Schiff's base: HF complex was formed instead of the retinal Schiff's base: BF3 co-plcx can be ruled out since the HF complexes in C014 and CBCl3 solutions have absorption maximum at 447 nm and 468 nm. respectively.102 In these solvents, the absorption maximun for the BF3 complexes appears at 456 nm and 480 nm (Table 4), respectively. 2. Ketiminies. Figures 14 shows high frequency Raman spectra for thed-phenylbenzyl idene-n-butylamine system, (C6115 )20-N(CH2 ) 30113 . For the neutral species (Figure 14a). the C=N stretching frequency appears at 55 Figure 13. Resonance Raman and absorption (inset) spectra of trans- retiual Schiff's base and Lewis acid derivatives in CBZClz. (a) Trans-ratinylidene-nrbutylamine. (b) 8013 complex. (c) BBr3 complex. (d) BF3 complex. (e) FClO4 complex. (f) BCl complex. 56 2.0 - I578 d) 0) I6 I 0) -I622 Absorbonce Wavelength (n m) 38 SB BCI3 $B= BBr3 SB= BF3 SB= HCI04 SB= HCI #09099 Figure 13 I65I 4 1 I500 I580 I660 A rm" Thble 4 57 Absorption laxima, C-C and C-N stretching Frequencies‘ of trans-retinylidene-nrbutylamine and Lewis Acid Derivatives. Retinal Schiff’s Base Lewis Acid Schiff’ 5 Base BCla(c) (C) BBra BF3(C) HClOs HCl(C) BF3(d) HC1(d) BF3(e) BF3(e) (C) 452 458 477 476 456 441 440 456 480 §21222£_ CH2C12 CH2C12 CHzClz CH2C12 CH2C12 CH2012 (C83)280 (CH3)2S0 CCle CHCla .0 b. c. d. Absorption.maximum in nm. Stretching frequencies in cm7 From Reference 97. This work (spectra not shown). 1 58 Figure 14. Raman spectra of -phenylbenzylidene-nrbutylamine (a) and protonated (b) and deuterated (c) derivatives in methanol solution. 59 .Unmz.llllnHHH m5. 0.0. mmm. \II \II \II o b C 36:25” 5531 l550 I700 AIcm") Figure 14 60 1618 cm'l; upon protonation (Figure 14b) or deuteration (Figure 14c). this mode is shifted to 1636 cm."1 or to 1616 cmfl. respectively. Figure 15 shows Raman spectra for a second ketimine systemd'phenylbensylidene amine. (C635)20-NH. The spectrum of the parent ketone. benxOphenone, is shown in Figure 15a. The C=N stretching occurs at 1600 cm.”1 and the C=N-B bending mode appears at 1364 cm."1 in the neutral Schiff's base (Figure 15b). These assigments are based on the isotope studies carried out by Datin et a1.110 Protonation (Figure 15c) increases the C=N stretching mode to 1661 cm."1 and in the BF3 complex (Figure 15d) this mode is shifted to 1679 cm’l. C. Discussion. 1. C-N Stretching Frequency: Neutral Schiff's Bases. Thble 5 summarizes aldehyde and neutral Schiff's base functional group stretching frequencies for a number of linear polyenes and aromatic species. For both classes of compounds. conjugation effects are apparent as increasing the number of double bonds leads to a decrease in the C-0 and C=N stretching frequency. This trend is summarised in Figure 16, where the 0-0 frequency in aromatic aldehydes and the C-N frequency in the corresponding neutral Schiff's base formed by reaction with nrbutylamine are plotted as functions of the number of double bonds in the aromatic system. The slepes of the two plots show that conjugation effects are more pronounced for the aldehyde group than for the neutral Schiff's base. most likely the result of the stronger electron withdrawing character of the carbonyl. For example. increasing the resonance system from benzaldehyde to a formylated metalloporphyrin brings about a change 61 Figure 15. Raman spectra of benzOphenone (BBO).CXLphenylbensylidene- amine (RBI) and protonated (8813+) and BF3 (BBIBF3) derivatives in DISO. The solvent peaks are denoted by ‘. 62 cow. man. bIBBI mhw... 0N0. I700 I I200 I300 I400 I500 l600 I I00 A(cm") Figure 15 63 .5» concuouom .u and ccaououcm .n «H concuomcm .« “and codenamed .A «can couscous: .m “on sensuous: .u «now codenamed .e “*9“ OOHOHOMOH av «MHO’ .M—FH .0 «NH OOGOHONQ‘ an nHflH Onion—ONO“ o. mama aammuoozuouanmuuo maoa anaemaamoozuoaammeov mesa w.uouo~am=eov mmoa “.m. aauxsmuoa Hz IHI. coca m. caa>nmuon «+50 amoa rammoovzuumammoov 555a umcasmasuan-cum:moaa5unu:<-m ma Rama amusemeamunua< 5 NH mama oocwamamunnsclocooflaanuAQMZIN qwoa 0.006%:06Hmnusnmz a oaoa uocaamasusnusnmcmnaasucumuz .1w1 qmoa v.umessmeauucmm omoa a myocaoanco Nae: mnoa namcauoutmcmue maoa puaasmaaxmn +amcaumuum=mue whoa momomamuuxoommo 33 ca 38 mozumoa amuumooamuamo 82 meow awn-Bommu N m m m mmoa : A movzozumoamoumuo no coca monomoumu mu Roma smammuomuzumomUNAmmoo maaa monommo Aanaov zno vasonaoo Aauaov 010 65509800 «announce emu-aou< un- caehuom no hcncndcum meaneucuam ecu-H one abnopuao n awash 64 Figure 16. Effect of increasing the aromatic ring conjugation on the characteristic group frequency of aldehydes (0). Schiff's bases (0) and protonated Schiff's bases (I). 820m 3950 .1 as. w _. n: o m .w m mm v m _ a _ a a a _ a a .o. 2.5128 .2 uzmuézpza mzmaqurfiz mzm~zum zuo I1?! . D D IT Izuo + o no u m o o 10¢®_ .10mm. 1000. 10km. 10mm. 10mm. 109t “330‘? 66 1 whereas the corresponding in the C=0 stretching frequency of 28 cm- shift in the Schiff's base analogs is only 7 cm'l. Similar effects are apparent in the cptical spectroscopy of these two clases of compounds23'25, that is. the cptical spectra of aromatic aldehydes are more strongly red-shifted than those of the corresponding neutral Schiff's bases. When the alkyl amine is replaced by an aromatic amine in the Schiff's base linkage. there is a decrease in the C=N frequency although this effect is still fairly small; for the benzaldehyde system. for example there is a 12 cm."1 difference in the C-N mode for the Schiff's bases formed from nebutylamine and aminobenzene. A further reduction in the frequency of the c-N stretch (to 1616 cmfl) is observed as the number of substituent phenyl rings is increased to three. These trends indicate that conjugation effects play a role in determining the C=N stretching frequency but that the dependence is fairly weak. In comparision with the aromatic compounds. it is apparent from Table 5 that the linear polyene Schiff's bases show a much stronger relationship between C=N frequency and number of double bonds. Similar behavior has been reported for the neutral nitrile system where the CIN frequency shows a more pronounced dependence on extent of conjugation in linear unsaturated systems than in analogous aromatic species.113 For the nitrile system. these conjugation effects were suggested to be the determining factor in accounting for the frequency differences between these two clases of compounds. i.e.. the interaction of the C-NTforbital with the‘Tfsystem of the aromatic ring is smaller than its interaction 67 with that of the linear. unsaturated systems. The same phenomenon appears to be in effect in the Schiff's base systems and thus we expect the extent of conjugation to be higher for the C-N group in the linear polyene Schiff's bases than in the aromatic imine Schiff's bases. with the consequent decrease in the C=N stretching frequency of the former. 2. C'N+ Stretching Frequency: Schiff's Base/Lewis Acid Complexes. Table 6 collects data on various Schiff's base: Lewis acid adducts which were studied in the present work or described by other workers. The dependence 0f thO C=N+ stretching frequency on the size of the conjugated system in protonated aromatic Schiff's bases is shown in Figure 16. The same type of dependence as noted above for aromatic aldehydes and neutral Schiff's bases is observed: as the number of double bonds increases the C=N+ stretching frequency decreases. loreover. the slope is steeper than in the neutral Schiff's base compounds. probably reflecting the effect of the increased electronegativity of the protonated substituent on the conjugated system. The data in Table 6 show that the increase in C-N frequency upon complexation with BF3 is comparable to that observed upon protonation. Ioreover. for both of these Lewis acids (8+. BF3) the largest shifts arise for compounds in which the Schiff's base nitrOgen is substituted only by protons whereas the smallest shift occurs for the fully phenyl substituted species. These observations indicate that a common mechanism is likely to be responsible for the increase in the C=N stretching frequency in both cases. 68 Table 6 Changes in the GIN Stretching Frequency Upon Complexation Iith Lewis Acids. Is.) + + (b) (C) Complex vcu “can vCND ”ans Avu+ Av0+ AVA Gama puncuc4u9(d) 1646 1680 1660 1690 34 20 43 1425 thcncaug(d) 1618 1636 1616 18 -2 thcuu(d) 1600 1661 1679 61 79 1364 NaphCNC489(d) 1643 1675 1655 32 12 1420 AntCNC4H9(d) 1644 1663 19 PhHCNMe 1658 1695 1712 37 54 PhHCNPh(e) 1634 1672 1673 38 39 PhZCNMe(e) 1634 1669 1661 35 27 PhZCNPh(e) 1616 1623 1621 7 s suzcnu(e) 1610 1670 1672 60 62 Ni-porphyrin 3(f) 1639 1650 1640 11 1 (g) M412 1620 (8)* Rhodopsin 1655 1630 35 10 as (h) 1642 1625 22 ' 5 1350 570 (h) . 8:603 1641 1623 21 3 1346 an (i) 1633 1621 13 1 1349 578 Retinylidene-n- ‘3’ 1622 1655 1630 33 8 butylamine Retinylidene-n- (d) 1623 1654 1656 31 33 butylamine v Vcfiu v + a. C - N stretching frequency, protonated ( ), deuterated ( CND) and BF3 complex (VCNA). b. AVH+ ' “cfia ‘ '“cn Frequency in cm-l. v + - D V c. C - N - R bending mode (GCNH) CND CN; Av- ‘tNA ' ‘tu d. This work; e. Reference 67; 1’. Reference 23; g. Reference 1; h. Reference 9; 69 As noted in the introduction to this Chapter and also in Chapter 1. the increase in frequency upon protonation is counterintuitive. For liner polyene Schiff's bases a C=N stretch: C-N-H bend model has been invoked to explain this phenomenon. This model has difficulty. however. in accounting for the increase in C-N stretching frequency observed upon methylation. as pointed out by larcus et al.13 It is also unable to explain the similarity in behavior for protonated Schiff's bases and Schiff's bl8¢=BF3 addition complexes reported here. The experiments in Figures 12 and 13 show that this similarity. which we have studied most extensively in the aromatic systems. is also observed in linear polyene systems: protonated retinal and the retinal: BF3 complex in DISO show increases in C=N stretching frequencies. relative to the neutral Schiff's base (1623 cm’l) of 31 cm."1 and 33 cm'1 . respectively. Table 4 summarizes the absorption maximum. and the c-N and C-C stretching frequency data for the retinal Schiff's bases. its protonated and Lewis acid-complexed species in various solvents. The similarities in these properties for the aromatic and retinal Schiff's bases. as well as their solvent dependencies (see also ref. 60.95.102). demonstrate that the absorption red-shift. the increase in C-N vibrational frequency. and the decrease in C-C stretching frequency are general properties of Lewis acid Schiff's base reactions. In agreement with previous resultls'ls. the cthylenic (C=C) frequency of the retinal Schiff's base: Lewis acid complexes shows a stronger correlation with the magnitide of the absorption red shift than the C=N stretching frequency. For example. the difference between the absorption maximum values for the BF3 and 33:3 complexes is 19 nm. The corresponding differences in their c-c and c-N 7O stretching frequencies are 6 cm"1 and 2 cm'l. respectively. which indicates that the C-C stretching force constant is more sensitive to changes in the TT system than is the C=N stretching frequency. This suggests. in turn. that the chages associated with the CIC and C=N stretching frequencies upon reaction with Lewis acids are regulated by more than a single mechanism. Complexation of trans-retinal Schiff's base with general Lewis acids. such It BF3. should remove the C-N—H bending interaction effects on the C=N stretching frequency while maintaining delocalization of the system and thus provide a means by which to test the mechanical coupling (stretch-bend interaction) hypothesis. Our results for such complexes show (see Figure 12-13 and Table 4) that there is little influence of stretch— bend coupling in determining the c-N stretching frequency; rather electronic effects which strengthen the C=N force constant upon complexation appear to be responsible for the observed frequency increase.73 The ketimine data provide another example of the difficulties of the stretch-bend interaction model. The Raman data in Figure 15 show that ci-phenylbenzylideneamine110 (21) in its neutral form has a C=N stretching frequency at 1600 cm"1 and a c-N bending vibration at 1364 cmfl. 0 + G W” C=N ——+ C=N \H H 9 _z__1_ L0 2.2. 71 One might expect in the stretch-bend model that these modes would interact in the neutral species to drive the C=N stretch to higher frequencies. However. the C=N stretch at 1600 cm."1 is lower than the C=N mode in sI-phenylbenxylidene-n-butylamine (11) by 18 cm'l. 0 CN \ 0 “‘9 <3} 306’ 3 Ioreover. protonation of (21) to form (2;) results in a 61 cm,-1 increase in the C-N stretching mode to 1661 cm71. and BF3 substitution increases the frequency of this mode by 79 cm'l. These observations suggest that the change in the electronic enviroment of the nitrogen upon protonation or reaction with a Lewis acid. rather than mechanical coupling. is likely to be critical in determining the increase in the c-N stretching frequency. An analogy nmy be drawn to nitrile systems where the increase in the CIN stretching frequency which usually accompanies reaction with a Lewis acid (e.g. F+, BF3. 8013) has been explained68 by suggesting that the GEN stretching force constant increases upon complexation. The results of the analysis above indicate that the behavior of the nitrogen lone pair is involved in determining the properties of the c-N 72 vibrational mode; changes in the electronic environment of this lone pair upon protonation or reaction with a Lewis acid affect the electron density distribution in the C=N linkage. The later statement is supported by the NIR data shown in Figure 11 and Table 3 which suggest. in agreement with previous results23'109 that an increase in the electronegativity of the nitrogen occurs upon protonation of Schiff’s bases. The protonation of a Schiff's base seems to be analogous to the situation which occurs when a proton is brought up to NH to give NH;. The lone pair electrons forming the new N—B bond will not stay unaltered in their 8p2 hybrid orbital. neither will they be equally shared between N and 11.115 Fe have explored the possibility that a similar phenomenon occurs for Schiff's bases upon protonation by carrying out ab initio calculations at the GVB level for methylimine and protonated methylimine. The calculations are given in the following Chapter and show that a decrease in C=N bond length occurs upon protonation. Accompanying this. there is an increase of 0.51 mdyn/A in the C=N stretching force constant. 3. Absorption Maximum of trans-retinylidene-nrbutylamine: Lewis acid complexes. A general introduction to the problem and importance. of the absorption maximum red shift of the protonated retinal Schiff's base model was given in Chapter 1. (see also references 60,95,102). It was indicated that the bathochromic shift of the protonated species. relative to the unprotonated derivative. was regulated by the separation between 73 the center of charge in the cation (i.e. C-N+) and snion (X' ). This was called the anion effect since the interatomic distance between the positive and negative centers controls the excitation energy102 (or energy gap between the ground and excited states) of the cation and thus. the polyene‘TTsystem delocalization. Flats at ‘1.60.95.102 noted that solvent plays a major role in the determination of the absorption maximum shift of the cation. For instance. NFretinylidene-nrbutylammonium chloride has an absorption maximum at 442 nm in C3308. but the ssme salt shows its absorption maximum at 469 nm in CHC13. A solvent like methanol is said to be a leveling solvent. since it leveled the action of the anion (i.e. different anions show very similar absorption maxima). It has been prOposed60'95'102 that in methanol the N-retinylidene salts. are fully disociated and the cation might be expected to absorb at a lower energy (long wavelength). However. because of the strong interaction between the electron-rich oxygen and the polarixable nitrogen. a significant charge is still localized in the nitrogen with the consequent increase of the retinylic cation excitation energy relative to a less polar solvent like HCC13. The same trends in absorption maximum as a function of the solvent are reported in Table 4. For example. the protonated retinal Schiff's base chloride species has an absorption maximum at 456 nm in CHzCl2 but only 440 in DISO. Analogously. the BF3 complex shows absorption maxima at 477 nm and 441 nm in these two solvents. respectively. In other solvents like C014 or C3013 the retinal Schiff's base:BF3 complexes also behaves as the protonated analog. For example. the absorption maximum for the BF3 complex ion CBCl3 is red shifted relative to its absorption 74 3411‘“! in CC14, The constancy and similarity between the red shifted absortion maximum of the retinal Schiff's bases: Lewis acid complexes (i.e. BF3. BCla. BBr3) and protonated retinal Schiff's bases indicates that the presence of salt (i.e. cation-anion pair) is not unique in inducing a significant red shift in the absorption maxima of the complexed chromophore. This is turn indicates that the solvent dependency in both cases is likely to be governed by a similar mechanism in which the counter ion may play only a limited role. However. at this point more work is needed in this area in order that this hypothesis can be further developed. CHAPTER 4 AB INITIO CALCULATIONS A. Introduction. An interesting aspect of the Schiff's base protonation reaction (and reactions with Lewis' bases in general) is the observation that the C=N stretching frequency increases.73-36 The molecular mechanism underlying this increase is not well understood. In Chapters 1 and 3. we point out an analogy between the vibrational properties of Schiff's bases and nitriles. In nitriles. the observed decrease of the CEN bond length and the accompanying increase in the CEN vibrational frequency upon reaction with a Lewis acid has been interpreted in terms of an increase in the bond order of the CEN linkage.68"72 This interpretation suggests that a similar effect could be responsible for the increase in the C=N stretching frequency in Schiff's bases upon reaction with Lewis acids. lethylimine. the simplest Schiff's base. and its protonated derivative provide a model system which can be used to study the electronic changes in the C=N bond when the nitrogen lone pair is encumbered by a proton. These species are difficult to deal with experimentally and only a few reports of their vibrational prOperties have appeared. Iilliganllé. in infrared spectrocopic studies of the photolysis of methyl aside. assigned the frequency of the DIN stretching mode of methylimine It 1628 08-1. Confirmation of methylimine as a photolysis product was obtained by Moore et al}17 in a study of diasomethane which showed that the C=N stretching vibration was observed 75 76 at 1642 cm-1. The difference in frequency between this result and that reported by Milligan's earlier work116 may have been due to the presence of hydrogen cyanide which ocurred as a second product in the matrix prepared by loore et a1.117 Curiously. substitution of the hydrogens in methylimine by fluoride. i.e. perfluoromethanimine118 . increases the C=N vibrational mode to 1740 cm.-1 despite the increase in mass of the substituents. Theoretical work on the vibrational frequencies in the methylimine system has also been done. Beginning with the results of Hoore et a1117, 119 used ab initio methods and a small basis set to calculate Botschwina the force field of methylimine. For methylenimmoniun ion (protonated methylimine). calculations of neither the C=N stretching force constant nor of the contribution of the s and p orbitals to the sp2 hybrid forming the C=N bond have appeared. However. Fades at ‘1120’ using SCF calculations and a PRDDO geometry. estimated that the C=N-H bond angle increases by approximately 10 degrees and that the C=N bond length increases by 0.019 A upon protonation. In the same work the vibrational frequencies for methylimine and methylenimmonium ion were calculated. but for neither molecules were attempts made.to assign the frequencies or estimate the force constants of the C=N stretching mode. Kollman and co-workers121. in SCF calculations of the electronic structure of CHZNH2' indicated that the nitrogen appears to be partially negatively charged. Vith this previous work in mind we have carried out ab initio electronic structure calculations for methylimine and methylenimmoniun ion. at the Generalized Valence Bond (GVBI77 and Self Consistent Field 77 (SCF) levels.78 The GVB results show an increase in the nitrOgen s character contributing to the C=N sigma bond. an increase of 0.51 mdyn/A in the C=N force constant. a slight decrease in the C=N bond length and a decrease in the carbon electronic charge when methylimine is protonated. 8. Theoretical Details. In the GVB calculations the 12 valence electrons of methylimine were represented by 6 electron pairs. each of which was represented by two natural orbitals. The assignment of the electron pairs to the molecular structure is given as: H/Ix a $1. where b1. b2. b3. and 1 represent the CH's. the NH and the nitrogen lone pair. respectively and the 0‘ and 'I’I'correspond to the particular bonds between nitrogen and carbon. For the protonated methylimine an NH bond (b4) replaces the lone pair 1. The expansion basis was the Hurinaga122 9s5p set on both C and N and the Dunning123 4s set on each H. These were augmented with polarisation functions (d's for C and N'((FO.75 and 0.80. respectively) and p's for each H (WI-1.0)). the resulting basis was (9s5p1d/4slp) and was contracted 7? to [3s2pls/2slp] by using the general contraction of Raffenetti.124 Total charge distribution as well as the per cent s and p character in the N contibution to the C=N bond were calculated from the natural orbitals of the GVB wave function by using the lulliken Population Analysis.125 To calculate the C=N stretching force constant we use the following geometry Optimization procedure. As a starting point. we fixed all geometric parameters for both methylimine and methylenimmoniun ion at the Eades et ‘1120' PRDDO optimized geometry values and varied the C=N distance by 0.025 au (0.01323 A) symmetrically about the initial minimum energy geometry. This calculations gives the energy at a given C=N distance and the new equilibrium geometry. For both molecules the 1126 resulting potential energy curves were fit to a 4th order polynomia in (R’Req) where Re is the calculated C=N bond length. and the force 4 constant for the C=N streching mode was determined from the coefficient of the quadratic term in this expansion. All calculations were carried out by using the Argonne National Laboratory Collection of Electronic Structure Codes (QUEST-164). In particular. the integrals were by done 83 and the GVB calculations were using the program ARGOS written by Pitzer done by using the program GVB 164 written by R. Hair.84 The calculations were done on an FPS 164 attached array processor. C. Results. Calculated potential curves at the GVB and SCF level for the C=N and C=N* stretching modes are show in Figure 17 (see also Tables 7 and 8 respectively). Figure 18 show the calculated GVB equilibrium geometry 79 Figure 1?. Ab initio potential curves for the C-N stretching motion. (a) C-N’scF methylimine SCF . (b) C'NSCF methylenimmonium ion SCF. (c) C=ch3 methylimine GVB. (d) C-NGVB methylenimmonium ion GVB. calculations. 80 S chem; 3255.0 ozom Zlo . uozfima ozom zlo an»... cum; on»; can; mum; 8.9.. 9a.. a; 3"; con; an“: on»; con; 0...”. an“; 3.”, 3.".— on“; 3.“; 8m; x m>aZH o 1. $53. ““3200 e a. ., _ 864m 5 .32.”: w 5.2.0 . a3 (016+)! 81 Thble 7 lethylimine Potential Curve‘l'Z). Total Energy for the c-N Stretching lotion. RC=N ESCF(+94.0 au) EGVB(+94.0 au) 1.222 —o.05033 -0.l4801 1.236 -0.05155 —o.1so31 1.249 -0.05216 -0.15201 1.262 -0.05218(3’ -o.15315 1.275 -0.05168 -o.15379 1.289 -0.05064 -o.15395(3’ 1.302 -o.04924 -0.15367 1.315 -o.04729 -o.1529s 1.328 -o.04512 -0.15196 1.341 -o.04250 -o.1soss 1. Energy in hartrees and bond length in A. 2. Points closest to the equilibrium geometry used for a quadratic polynomial fit. 3. Computed point nearest the equilibrium geometry. 82 Thble 8 Iethylenimmonium ion Potential Curve‘l). Total Energy for the C=N Stretching lotion. 303" Escp(+94.0 au) EGVB(+94.0 au) 1.242 -0.406378 -0.506805 1.255 -0.407058 -0.508163 1.268 -o.407179(1) -o.sos968 1.282 -o.406776 -o.509238‘2’ 1.29s -0.405888 -0.509043 1.308 -o.404572 -o.sos417 1.321 -0.402836 -o.507373 1. See footnotes in Table 7. 2. Computed point nearest the equilibrium geometry. 83 cccuacn a“ ccaudc coop use 4 ma Assoc” moon .Apv “no“ aumnoaamncahmucav voucuououn one any camamuhmuca we huucaoca .mn cunmum 3: 62Y4%.gy9 :3 2.11an or. ./ ACAAv/Y 84 for methylimine and the protonated derivative. The protonated species shows a potential minimum at an energy lower than the unprotonated one. This is in agreement with previous calculations in which a small basis set and the SCF formalismlzo'121 were used. The GVB calculation predicts that upon protonation of methylimine. the C-N bond will be slightly shorter than in the unprotonated one (1.282 A and 1.289 A. respectively). Opposite to this trend. the SCF calculation predicts. in agreement with the SCF calculation of Eades ct aldzo, that the bond distances of the protonated specie (1.268 A) will be slightly longer than the neutal species (1.262 A). The fitted potencial surfaces were used to determine the coefficients of the quadratic terms which in turn give the force constant for the C=N and C=N+ stretching modes. These are shown in Table 9. 'e estimated the anharmonicity corrections and found them to be insignificant. The protonated species at the GVB level shows an increase in the C'N+ stretching force constant. relative to the C-N stretching force constant. of 0.51 mdyn/A. while the SCF level shows a decrease of 0.52 mdyn/A; in addition. the values at the SCF level are higher than at the GVB level. It is generally recognized that SCF calculations overestimate force constants by between 104014.127"131 Since the GVB wave function contains a more appropriate mixture of ionic and convalent terms and separates to the correct asympototic products. the GVB force constants should be more reliable. 85 T4510 9 C416 1 tedII) Force Constants‘2) for the C-NI3) C=N* 4 Stretching Notion. Force constant C=N C=N+ kSCF 13.77 13.25 1. From a polynomial fit of the GVB and SCF potential curves (see text for details). 2. Quadratic valence force constant in mdyn/A. 3. Nethylimine. 4. Nethylenimmonium ion. 86 One may think of the GVB wave function as being the SCF function plus various correction terms. IGVB> = I SCF>+|Corr. Terms The "correction terms” correct for the wrong distance dependent behavior of the SCF. i.e.. when bond lengths are changed. We are not too concerned with the SCF predicted trend upon protonation since we realize it is a much less complete function than the GVB. When one has two approximate wave functions and one is considerably less approximate than the other it seems prudent to trust the predictions of the more complete functions over those of the less complete functions. Note also that the GVB is equivalent to an SCF with limited configuration interactions. The GVB contains electron correlation while the SCF does not. Tables 10 and 11 present the Nulliken Population Analysis for both the protonated and unprotonated methylimine species calculated at the minimum energy geometry. Figures 19 and 20 show the corresponding contribution of the GandTrsystems to the total electron distribution. while the sp electron distribution to various bonds in the 6’system is shown in Table 12. Figure 21 indicates the change in the sp character of the nitrogen when methylimine is protonated. Under the same circumstances. Figure 21 also shows the electron distribution in the'TT systems in the CbN and C=N+ bonds. 87 Table 10 Nethylimine: Electron Distribution. Orbitaln Atom so. pU do p,‘T <17 6 1 Carbon 3.19 1.98 0.05 0.89 0.01 5.22 0.90 Nitrogen 3.58 2.60 0.02 1.08 0.01 6.20 1.09 Hi 0.89 0.01 -—- 0.00 —— 0.90 0.00 Hz 0.88 0.01 -— 0.00 -—- 0.89 0.00 Ha 0.78 0.02 —- 0.00 -— 0.80 0.00 TOTAL 14.01 1.99 Table 11 lethylenimmonium ion: Electron Distribution. Orbitala Atom so p0 do pTr dn 6 1 Carbon 3.23 2.03 0.04 0.61 0.02 5.30 0.63 Nitrogen 3.42 2.41 0.03 1.35 0.01 5.86 1.36 H1 0.77 0.01 —-- 0.00 -— 0.78 0.00 Hz 0.77 0.01 —- 0.00 —- 0.78 0.00 Ha 0.61 0.02 —- 0.00 -—— 0.63 0.00 84 0.61 0.02 —-- 0.00 -—-- 0.63 0.00 TOTAL 13.98 1.99 88 cad-3.3:! uou 533:5:an cauca—c "cue... one acucthEcuahe b .oa can»: .81. \.I mud- Z I U mI\ . Nee/f o~.o+ o_.o+ c9020 .28. 2.0+ m N o 05 \I I’ll/Z I stxx 2 I U/ nI/ O O . . nI\ 8.01.1 60.0.. 96+ 80+ 9.0+ 839$ .F 529$ b 89 n3 financiancaha—ucn «on dong—«nun: cmucmc ~33 vac flcuchc Price?»- b .ou cues:— I NN.O+J . oI NI 8 0+ /-.o.. \ Z I U MI\ NO.O+/_I find... NN 0+ + r .. I a .. a 500+ -o+ e N e u IPO O\I I/ one: I n 37- IUcS. n \2 I U/ I, O I I :6. f f . . Fen? 80+ 1 + ono- 50+ 1 + I 6233 .2. £295.. b 9O Table 12 Electron Distribution of the Bond System‘ Bond‘ 5) Atom 0rbital‘ c > /Atom( 9 ’ Methyl imine Methyleni-oniun Ion s/C 0.32 0.33 Carbon p/C 0.57 0.61 5/81 0.14 0.07 Hi-C 5/81 0.81 0.75 Hydrogen(1) s/C 0.09 0.12 p/C 0.11 0.12 s/C 0.31 0.33 Carbon p/C 0.58 0.61 s/Hz 0.13 0.07 Hb-C s/Hz 0.80 0.75 Hydrogen(2) s/C 0.09 0.12 p/C 0.12 0.12 a/C 0.33 0.29 Carbon p/C 0.51 0.49 C-N s/N 0.31 0.42 Nitrogen P/N 0.58 0.52 Cont. Thble 12 N-Ha N—(lone- pair) N-Hs Nitrogen Hydrogen(3) Nitrogen Lone-Pair Nitrogen Hydrogen(4) 91 s/N p/N s/Ha s/Ha s/N p/N s/N p/N s/N p/N s/N p/N s/Hs s/Ha e/N p/N 0.20 0.69 0.11 cas>c> 2388 0.41 0.51 0.57 0.50 0.33 0.62 0.07 0.61 0.13 a. Calculated from Nulliken Papulation Analysis. b. See Figure 18 for the particular geometry. c. s and p stands for s and p orbitals. respectively. d. C. N and H stands for the carbon. nitrogen and hydrogen atoms involved in the particular bond. 92 6 system .. .1 + 042N (343%., H3 H.\ {0.52 Nz/H3 :2? N/S /C N 0 33 c. H2 ( 029 Cs \H4 {0.51% L. o. 49 Cp (0) (b) 77' system +4.0 —OH3 H.\O O/H3 /C— N H/QQ H. H. L 0 870 0.92 N p p 0 66 CD 0.97 Np {.OI4ND 007 CD { ° { 0.32 Np 0.0309 I0) (6) 1+ Figure 21. s-p and p electron distribution of the (7' and ‘Tr systems in the C-N bond of methylimine and methylenimmonium ion. 93 D. Discussion. Further insight into the mechanism responsible for the increase in the C=N stretching force constant upon protonation may be gleaned from the detailed electron distribution predicted by the GVB functions used in this work. If the nitrogen atom in methylimine did not use its 2s electrons in the bonding to the H or Cflz group we would expect a CNH angle of 90° and no nitrogen 2s character in either the C-N or N-H sigma bonds. The calculated value of 111.9° for the CNH angle in CHZNH reflects the extent to which the nitrogen 2s electrons participate in the bonding and from Figure 21 we see that the GVB calculations allot 0.31 electrons from the nitrogen 2s to this C-N bond. When methylimine is protonated at the nitrogen lone pair. the GNU angle increases further to 122.9°and the calculated number of nitrogen 2s electrons in the C-N bond increases to 0.42. This enhanced nitrogen 2s character in the C-N bond is reflected in a smaller bond length in the positively charged ion relative to the neutral species and in the increase in the stretching force constant. The calculations above indicate that it is possible to attribute the increase in the C=N stretching force constant in methylenimmonium species to a change in the electronic environment of the c-N bond upon protonation of methylimine. This increase in the c-N stretching force constant for the protonated species translates into an increase in the c-N stretching frequency of 30 on,"1 (see next Chapter) and suggests that the same kind of mechanism may be responsible for the observable increase in the ChN stretching frequency in protonated or Lewis acid-complexed 94 Schiff's bases. Several studies have suggested that when one protonates a polyene Schiff's base the C=N bond order decreases resulting in a corresponding decrease in the C=N force constant."5'8'u'13 If the encumbered lone pair mechanism (rehybridization model) is to be dominant for such systems then the change in the bond order and component of the C=N force constant must be smaller than these earlier studies predict. Note. however. that numerical experiments within the normal coordinate analysis model. presented in the next Chapter. suggest that the increase in the C-N stretching frequency observed upon protonation in a variety of molecules can not be reproduced with a ”sensible” set of interaction force constants when there is a decrease of 0.3 mdyn/A in the C-N force constant e The electronic structure of the methylenimmonium ion shows (see Figure 20) that the nitrogen appears to be partially negatively charged and the carbon carries partial positive charge. Since the charge of the system is +1. the hydrogens bear the rest of the positive charge. lulliken atomic populations tend to be basis set dependent. which may indicate that the above charge distribution in methylenimmonium ion is not necessary correct. However. a similar charge distribution was obtained by Kollman and co-workers121 by using a STD-3G and double Zeta basis set. Noreover. Birge et al.64 in their semi-empirical calculations on the sis-trans isomerisation of rhodopsin. indicated that the INDO-CISD atomic charges of a ll-retinal Schiff's base show that the nitrogen retains a negative charge (despite the fact that the chromophore carries 95 a net positive charge) and that the Schiff's base carbon is more positively charged than the nitrogen atom. Thus. our results suggest. in agreement with Dirge et al.‘4 that the common approach of assigning nitrogen a +1 core charge will overestimate the TT potential in semi-empirical calculation of retinal Schiff's bases or Schiff's bases in general. CHAPTER 5 NORMAL COORDINATE ANALYSES A. Introduction. Resonance Raman and Fourier Transform infrared spectroscopy have been used to study the mechanism of excitation and photochemical properties of the retinal Schiff's base chromophore in rhodopsin. bacteriorhodopsin and related photopigments.1'17 Comparison of the vibrational spectra of the retinal Schiff's base model with the pigment spectra has been used to examine the interaction between the retinal chromophore and the protein. For example. the C-N stretching mode in the 1620-1655 cm"1 region has been used to identify the state of protonation of the retinal Schiff's bases. A vibrational frequency 25 cm"1 lower upon deuteration has helped to determine that in rhodopsin the Schiff's base is protonated.2'35’-132 The finger print region of the retinal-based proteins. between 1100.1400 efl'l. often correlates with the isomeric form of the chromophore. Isotopic substitution in this case has helped to distiguish between the particular vibrations of the isomeric forms.2'11'3 However. the environment of the protein can affect the vibrational frequencies of the retinal chromophore. Moreover. the C=N stretching frequency of the protonated Schiff's base species is higher that the non-protonated species. Thus. normal mode calculation have been used to predict the vibrational spectral changes of the chromophore upon interaction with the protein. In particular. the normal mode calculations have been used to emphasize that. upon protonation of the retinal Schiff's bases (or Schiff's bases in general). the C-N stretching 96 97 force constant decreases. The increase of the C=N stretching frequency. relative to the unprotonated Schiff's base. is attributed to the coupling between the C=N stretching and C=N-H bending motions.4'5'8'u'13 For more details see section C in Chapter 1. As discussed in Chapter 3. the stretch-bend model can not account for the experimental increase in the C=N stretching frequency when aromatic imines and retinal Schiff's bases are reacted with the general Lewis acid BF3. 8013. HBr3 or when ketimines are protonated. In the preceding Chapter we have shown that upon protonation of methylimine there is a reorganization of the electronic environment around the C-N bond in such way that the C-N stretching force constant increases and we called this effect the rehybridization model.73’76 To analyze the implications of the rehybridization model on the Schiff's bases vibrational frequency.00_N, we have carried out normal coordinate analyses for methylimine and its protonated dervatives and for the model structures CH3CH-NCH3 and its protonated and BF3 analogs. We have also carried out vibrational analyses for the CH3cn=Nncn3 structure in which the O-N stretching force constant is allowed to decrease upon protonation. This latter calculation allows us to explore the predictions of the stretch/bend interaction model in light of a restricted set of force constants. For both types of force fields. we systematically varied the force constants of the modes which can influence the C-N stretching frequency. which allows an evaluation of the various contributions to the observed behavior of the C-N stretching mode. 98 The results of these analyses indicate. in agreement with the rehybridization lede173'76. that the electron density redistribution which occurs upon protonation plays a mayor role in determining the c-N stretching frequency. the stretch bend interaction is prominent to a much lesser extent. B. Numerical Calculations and Methylimine and Methylenimmonium Ion Force Fields. In-plane vibrational frequencies and the corresponding potential energy distribution for methylimine and for a hypothetical methylenimmonium ion have been calculated. The ab initio geometry determined for methylimine and methylenimmonium ion120 (with the exception of the C=N bond distance. see below). and the force field calculated for methylimine119 were used. The methylenimmonium force field was constructed from the methylimine force field. The C-H cis force constants of the neutral species were used for the two C-H bonds of methyleinimmonium owing to the greater lengths of these bonds in the protonated form and the C2v symmetry of the ion. The C-N bond distance and the C-N stretching force constant for methylimine and protonated methylimine were substituted with our ab initio results. Figure 22 shows the geometries and Table 13 and 14 summarize the force fields. The Shimanouchi pretrelss’86 was used to calculate the frequencies and the potential energy distribution in terms of an internal basis set (see Figure 22). In order to check consistency of the C-H modes in the potential energy distribution. the in-plane 5A1 and 482 vibrational frequencies of the protonated methylimine have been calculated. first. by 99 (1’4/[289 % c \Q “9.6 H I0 H H%R.QK’/ C=N r3 /% I’27, H/Y ' H Id Figure 22. Geometry and internal coordinates employed for the normal coordinate analyses of methylimine and methylimmonium ion. The molecules were assumed to be planar. Bond lengths in A. Bond angles in degrees. 100 Table 13 F. Iatrir Elements for lethylimine... R . r1 r2 r3 0 B Y 11.14‘a) 0.087 0.231 0.259 0.682 0.345 0.479 5.736 0.040 -0.030 0.277 —0.o75 0.086 4.923 0.048 -0.067 -o.026 —0.181 4.994 . 0.025 -0.169 0.003 0.882 -0.021 0.078 1.061 0.458 1.046 .itretghing force constants - mdyn/A; bending force constants - mdyn Irad ; . stretching‘bending interactions force constants - mdyn/rad. .tForce constant from Reference 119. From Reference 73. 101 .mu cnpefi «a encuumnducv can uncu come mmm.o Hmo.ou ovo.H who.o mmv.o ovo.H mmv.o who.o HNO.OI Nmm.o nnm.o moo.ou omo.o Hwa.on omn.m 500.01 mmo.oa Hma.on mmo.o ovo.o mmm.v mmo.o HmH.ou omo.on 500.01 Ono.on moo.o mmm.v HwH.OI mmo.o mh0.01 hhm.o mvo.o Om0.01 ovo.o omn.m mmo.o mom.o mom.o moo.o hmo.o Hmm.o Hmm.o omo.o Amvmm.aa 3 r m 8 on ma mu an m non IdanoaamncuMAucx uou euacacua nmuumx em ea cane? 102 means of a symmetrical coordinate basis set (see Table 15) and second. by using an internal basis set (see Figure 22). The potential energy distribution of the C-H bonds were the same in both coordinate systems. C. Force Fields for the Model CH3CH=NCH3 and Its Derivatives. Vibrational analyses also have been carried out for the Schiff's bases. CH3CH=NCH3. and for its protonated. deuterated and BF3-complexed derivatives. The geometry and force field for 2-propaneimine133. allylimine134. ethylideneimine135. propargylimine136. methylimine and methylenimmonium ion114'121 were employed to construct the geometry and force field for the normal mode calculation. see Figure 23. The c-N bond lengths were taken from the ab initio calculations reported in the previous Chapter. The molecules were assumed to be planar and the CH3 4.8 and the BF3 groups were employed. as in other studies . as point Table 16 presents the main diagonal and off-diagonal force constants for a series of imines.133"136 A combination of these force fields with the force constants associated with the N-C motions in N-methylacetamide137 was used to construct an initial force field for the C83CH=NCH3 Schiff's base and its protonated species. Semi-refined force fields for the unprotonated and protonated models were obtained (see Table 17) from a fit of the initial force field to the frequencies of the C=N stretch. C=N-H bend and ring Schiff's base (DHCINR) motion in N-benrylidene-nrbutylamine and its protonated and deuterated derivatives ( see Figures 7-8 and Table l in Chapter 3). For the C=N/C=N-H 103 Table 15 Symetry Coordinates for In-Plane Vibrations of Methyleni-onium Ion A1 B2 S=R 1 1 8+: (r +r) S-= (r-r) 14 fi— 1 4 14 [2" 1 4 + l _ 1 S = (r+r) S = (r-r) 23 E 2 3 23 '6' 2 3 1 _ 1 5+: (01+00) S = (“‘00) 0“” 0“” 72' [2— + 1 _ 1 = (8+Y) S = (B-Y) 104 .cccuucv am acumen econ .< n« unuunca econ .ueacao ca o» acnuuuc cucn acacccaoa cAH .cc>«uc>«ucu «an an. eoaaaocoua .aa ea. muuznnomnu aouoa one no .o.aa.a. cucnaeuooc acauon cnu «on echoaolc ecu-cavuooc nuanced“ vac huucioco .nu cucmam .aun concucucu ccm .c .euucuu ccuou no uqcucu uou «mu cu mm" cccccucucu ccm .a .cuccucnoc ccuou Anxav uauvccplunuvccn vac Apv unquccn ca» uou nucu\< aha: «Apxcv unanncplmcucuuc one new ecuxnhml «Ac\cv ndadcucuucluc0Acucumc no. any mc«Acucuue cAu «on mucacu hcccnvcuu unaccucuua 210 emu :0 coacucuu«m .d .v .moon can moan-accncc cad cu coauupauucoc cucnuvuooc ~cduoud« none! .nouuoa«uue«m hmucnc neauncuom .nmm .c no [a .a .pa candy macaw cocoa no ecu-e cacao-accacu .e a000= ao-zoa .120000.= .a0-000 .az-ovao a00_ 0a00002-000 AQVON szovo .Aolzvz .AZUIVN .AUIUVOF .AZIUvzm Coop + Hmmzm-onmu a000, a02000.0 .ao-200 .120000.= .10-000_ .az-0000 =00, .a002-0000 a0000 aozooa .a0-200 .a20000 .10-000 .az-0000 000a .a002-0000 A000m 102000 .a0-zo0 .a20000 .10-000a .az-0000 000. 40002-0000 a0000 120:0: am-000 .10-0005 12-0000 000. 020_-000 .azomoa .az-ooa .a0-000 .12-0000 000. 02-000 Prsovo< comm 0.1500210 nonsuoscum .cecucuccuum acme: «o mononucum unamcucuum Zlu «a cue-H 113 similar to that reported recently for a series of simple imines.133'138 In the protonated species the C=N stretching frequency occurs at 1680 cm'”1 with an 89% contribution from the C=N stretching force constant. Other contributions involve the same modes present in the nonprotonated species plus a 5% contribution from the C=N-H bending motion. The contribution from the bending motion was expected since the force field for the protonated model was Optimized to reproduce a C=N-H 1 as observed experimentally bending frequency between 1425 and 1420 cm- for N-benzylidene-nrbutylamine and 2-naphthylidene-nrbutylamine (see Figures 7 and 9). Other molecules. such as allylimine (CHZ-Cfl—cnsum) in which the C=N-H bending motion occurs at 1368 cm'l. show no involvement of the bending mode in the C=N stretching frequency.133"136 Because the normal coordinate analyses for the neutral and protonated Schiff's bases are reasonably well-constrained by the force constant values in Table 17. we have carried out further calculations to investigate the behavior of the C-N linkage. Figure 24 shows the difference between the C=N stretching frequency in the protonated and neutral Schiff's bale. CH3CH=NCH3. as a function of the force constants of the principal modes which contribute to the PED. As noted in the Methods section. the calculations were carried out for both rehydriration model and stretch/bend interation model scenarios. For the former we used our calculated increase of 0.5 mdyn/A in the C=N force constant upon protonation (Case A). denoted as [8+(4)]); for the latter we allowed the C=N force constant to decrease by 0.3 mdynlA upon protonation (Case R. denoted as [H+(*)]). Also shown are calculations for the deuterated 114 Figure 24. Difference between the C=N stretching frequency in the protonated and neutral Schiff's base CH3CH=NCH3. as a function of the force constant of the principal modes which contributed to the potential energy distribution. 115 vs cuenmm 20.540005 woozéaxazéa 20.540005 woofzémzzéa no.0 no.0 A000 30 00.0 00.0 30 9.0- Aavfl _ _ _ . a _ aId - a a _ . .e . O + c 0wN1 V c c a 0.0.1 3.0. . H . 1 03.1. . . - EL... . 4 . -0! 1nd Q AV . a C Q o 1 W 4 100 .. 4 . 03.. 10- 4 - I C O R. 4 . - a. 4 -000 U a 20.40 1m. 4 4 4 n __ a - W. :0 +0 4 . a n 1 . .¢n_( 20.1.. . . . -00.. 0252mm ADVI1Z-U IUkmmkonvI-Z 00.. 00.. no.0 no.0 0.0 0.0 v.0 0.0 .I 0 u a _ _ h 1‘- q . . . .m 0 O O . 00.- . . 0.0.- e m” Aqv+T*c c a .. .. - ”V o e e e 4 m“ HV 4 . ‘ . ho.n~ H 10¢ m n - mm 4 3.0 . . . r1- . - . n. 4 4 . 3+: . .. -000 d. 27.04 4 -.0v~ N m m 1 1 ”H m m n m Aev+I m Rev +I I . . -000. -01. ([100] 4- [(01HN-o] aw ([N-o] 71- [(0)HN'3]’1)V 116 Schiff's base for the rehybridezation nodel. case A. [D+(+)]. Indicgtod in each panel is the value used for the particular force constant from our constrained normal coordinate analysis (Tables 16-17). From the slapes of the curves near these values. these plots provide an indication I! to hOI thedkgu frequency is influenced by the force constants of the various relevant nodes. For both cases A and B, Figures 24a and 24b show that the N-B stretch and C=N/N-B stretch/stretch interaction force constants are unlikely to lead to the observed increase inath upon protonation. Figures 24c and 24d show that the dependence of C=N on the C=N-B bending and C=N/C=N—B stretch/bend interaction force constants is lore pronounced. For case B. [B+({)], for exanple. Figure 24d shows that ‘é=N increases by 68 cm"1 when a force constant of 0.03 Idyu/rad is used for the c-N/C-N—B stretch/bend interaction tern. Figure 24c shows for Case B that a bending force constant of 1.15 ndyn A/rad leads to an increase of 31 cm"1 in the C=N stretching frequency. These observations suggest that a small stretch/bend interaction tern in conjunction with a relatively high bending force constant could account for the 35 cn'l increase in the C=N stretchin frequency in the case where the stretching force constant is assuled to decrease upon protonation. However. in allylinine, (Cflz-CB-CflsNB). the simplest form of an alkene-imine conjugated1T’systen, the bending force constant is 0.86 ndyn Alrad and the stretch/bend interaction force constant is reported to be 0.58 Idyn/rad.6‘b These values appear to be typical for this class of conpounds (see Table 16). Figure 24 shows that a stretch/bend interaction force constant in this range is unable to account for the increase iuJEBN in the Case B [B+({)] situation. If the force constant increases upon protonation. as our calculations indicate. then a 117 stretch/bend interation of this magnitude reproduces the observed C=N frequency (Figure 24d) under conditions when the other force constants are within constraints imposed by Table 17. This suggests that the increased C=N force constant we calculated for the protonated Schiff's base plays a more important role in increasing1a-N thgn does the stretch/bend interaction force constant. Similar conclusions regarding the bending force constant are indicated by Figure 24c. For Case A. Figure 24 also shows that the deuterium isotope shift for the protonated Schiff's base is not linear with a change in force constants of the C=N-B fragment. This is apparent for all four of the force constants in Figure 21. but the difference in slapes for the [E’Q )] and [D+(§)] plots at their calculated. Table 17 values. is most pronounced for the N-B(D) stretch (Figure 24a) and the C=N/N—B(D) stretch/stretch (Figure 24b) interaction force constant. Analogous calculations for the BF3 derivative shown in Figure 25. indicate that the dependencies ofihgu on C=N-BF3 vibrational parameters are generally attenuated relative to those of the C-N-B(D) group in Figure 24. The C=N/N-BF3 stretch/stretch interaction. which is somewhat similar to the C-C/C-CB stretch/stretch interaction that increases the C-C frequency in methylated linear polyenes.6'35 did produce a comparable Shift in4%.N ( 7 cm-1 when varied from 0.73 to 0.03 mdyn/A) but this appears to be too modest to account for both a decrease in C-N force constant and a 50-80 cm71 increase inlk.N. The attenuated depencence on C'N'BF3 parameters contrasts with the more pronouncedflk.N increase upon BF3 complex formation (e.g. for benzaldehyde protonation increases C=N 118 Figure 25. Difference between the C=N stretching frequency in the BF3 and neutral Schiff's base CB3CB=NCH3. as a function of the force constant of the principal modes which contributed to the potential energy distribution. 119 'T :3 ma annulus: 2.... 3.... 3.... a... 8... b h . . - . _ ac. I fit! ud- 1 Teal flV i... can. 4.... 8.... .m. ._ 2.... 3m. 3.. 1 q - u d 4 . . . 1'. 6 n ouunam .uzhmldF—m n... 8.... 8.... 2... an... 2.... 2... 2.... .1 q . u u u u 6N- . + I... . . . . 116 nv . 1n. 119.4. K N K llo‘ a «num x x u .adu .mF—m mane mama an? an“ news no — 1| 1 1 . . .eul * . tic—I 3.. . . [I.Guv Ilseufl Inf-6” “flew «nun u x 4 u 11.0» x x 120 by 35 °‘-1. whereas BF3 complex formation leads to an increase of 45 cm'l). Thus. even though there is uncertainty in the BF3 force field. .3 noted in the Iethods section. a reasonable set of force constants that includes an increase in the C=N stretching force constant provides agreement with the experimental data. We conclude. therefore. that the Case A scenario most likely applies to these adducts as well. D. Discussion. For methylimine and methylenimmonium ion the results show that an increase in the C=N stretching force constant from 11.14 mdyn/A to 11.64 mdyn/A increases the C=N stretching frequency upon protonation by 28 cm"1 and upon deuterstion by 10 cm."1 (see Table 20). For the model structure. CH3CH8NCB3, and its derivatives, the same increase of the C=N stretching force constant increases the C=N mode by 35 cm”1 and shifts the deuterium substituted species by 21 cm71. These values are typical of those observed experimentally (Table 6). Ioreover, the calculated frequencies in Table 21 for various isotopically substituted imines follow the same trend as those measured experimentally by Isthies and co-workers11 and Bagley et al!‘ in their extensive study of protonated retinal Schiff's bases. Their results11 show that deuteration of the Schiff's base nitrogen prOduccs I 23 cI-l shift in the C=N stretching frequency and we calculated a 21 cmfl. decrease in close agreement with our experimental results. Deuteration at the carbon of the protonated retinal is observed11'16 to decrease the C=N stretching frequency between 12-15 cmTl. and Table 21 shows that the calculated decrease for this substitution is 16 cmfl. Substitution by 15N produces a 14 cmfldecrease 121 in the C=N stretching frequency in trans-retinylidene-n-butylamine.11 In their calculations. lathies and co-workers11 obtained an 18 cm."1 shift for the 15H substituted protonated species; we calculated a 20 cm."1 shift for both the unprotonated and protonated derivatives. The shift observed upon deuteration of the Schiff's base nitrogen has been used as an argument in support of the stretch/bend interaction model.4'5'8'u'13 But. as Figure 24 indicates. this shift is also dependent upon the N-H (stretch). C=N/N-B (stretch/stretch) and C=N-B (bend) force constant and suggests that their inclusion in the force field representing a protonated Schiff's base. in as constrained a manner as possible. is necessary. The calculations summarized in Figure 24 and the recent results collected in Table 21 also provide some insight into previous force fields which were used and the normal coordinate analysis which resulted for Schiff's base species. For example, Aton et al.‘ used a relatively low value for the C-N stretching force constant (8.1 mdyn/A) and a negative stretch/bend interaction force constant (-0.2 mdyn/rad) to produce a protonated retinal Schiff's base C=N stretching frequency of 1659 cm'l. It is apparent from Figure 24d that the negative interaction force constant will compensate for the low stretching force constant and push the stretching frequency up. Similarly. Kakitani et al.8 calculated c-N stretching frequencies of 1659 cm."1 and 1657 cm”1 for the protonated retinal Schiff's base and for rhodopsin, respectively. For the protonated retinal model they used a CIN stretching force constant of 9.7 mdyn/A and a stretch/bend interaction constant of 0.2 mdyn/rad. The GIN-B bending force constant was used as a parameter to fit the C-N 122 stretching frequency and they obtained a value of 0.6 mdyn A/rad. For rhodopsin, they used a smaller C=N stretching force constant (9.3 mdyn/A) and a negative stretch/bend interaction force constant (-0.1 mdyn/rad); The bending force constant (0.5 mdyn A/radz) was again optimized to reproduce the 1657 cm"1 rhodopsin C=N stretching frequency. Figure 24c indicates that the difference in bending force constants in these two calculations. 0.1 mdyn A/rad. will not strongly influence the results. Our calculations suggest, however. that the 0.4 mdyn/A decrease in the stretching force constant used by Kakitani et al.8 for rhodopsin relative to the protonated nodel Schiff's base is compensated by the decrease (by 0.3 mdyn/rad) they assumed in the stretch/bend interaction force constant and that this leads to the similarity in calculated values oft£BN in the two compounds. In our normal coordinate analysis of the methyl-substituted Schiff's base we used a C=N/C=N-B stretch bend interaction force constant of 0.53 mdyn/rad. a value that is similar to the interacion terms compiled in Table 16. Tb provide some physical insight into this positive value, it is useful to note that the values in Table 16 are for neutral Schiff's bases. Upon protonation we expect a change in C=N-B bond angle and N-B bond length. Hills141 notes that changes of interbond angle 4111 produce changes in hybridization for the central atom owing to orbital following of the bending coordinate. For situations in which bond angle increases lead to greater a content in the bond and hence to shorter bondlengths, a positive value for the stretch/bend interaction force constant is expected. This appears to be the case for the Schiff's base linkage as Eades et al.120 have calcualted that the C=N-B bond angle increases by 123 11° and the N—B bond length decreases by 0.008 A upon protonation of methylimine. In our own calculations we find an increase in nitrogen s character in the N-B bond in methylimine upon protonation.73 These arguments suggest that the GIN/C-N-B interaction force constant will be positive and relatively large in the protonated species. in agreement with the value we have used. These arguments also indicate that hydrogen bonding to the protonated Schiff's base. to the extent that it alters the hybridization at the nitrogen, the C=N-B bond angle and the C=N snd N-B bond lengths. will have strong effects on the c-N stretching frequency. A recent example of such a situation may be the work on pyridoxal Schiff's basesn'zz which shows a C-N stretching frequency at 1646 cur1 and a vibration at 1465 cm"1 with contributions from the GIN-B bending mode. In this particular case, the imine proton is intramolecularly hydrogen bonded to a nearby oxygen which constrains the system and likely perturbs the force constants associated with the GIN-B group. In conclusion. the results of our analysis above indicate that the behavior of the nitrogen lone pair is involved in determining the properties of the C-N vibrational mode; changes in the electronic environment of this lone pair, upon protonation or reaction with a Lewis acid. affect the electron density distribution in the GIN linkage. CHAPTER 6 SUllARI AND FUTURE RESEARCH A. Introduction. A.main effort in research on visual and related systems is directed toward understanding the conditions that the retinal Schiff's base chromophore experience in the protein to regulate the optical absorption properties of the photopigments. The lack of any detailed X—ray crystallographic information about rhodopsin and bacteriorhodopsin has prevented the identification of the charged amino acid near the retinal Schiff's base chromophore. In this regard. the original point charge model suggested by Nakanishi et al.61'103 has been undermined by new data presented by lathies et al.94 on the ”opsin shift" of retinal Schiff's base models in bacteriorhodopsin. Their results show that much of the "opsin shift" in bacteriorhodopsin (contrary to the original indications) is due to chromophore-protein interactions near the Schiff's base end of the chromcphore. In addition. no crystallographic information is available for Schiff's bases and protonated analogs that indicate if the GIN bond is longer or shorter for the Schiff's base complexed species than for the uncomplexed derivative. In other words. no bond length data is available at present for retinal Schiff's base models that can show the state of the c-N bond order in the protonated photopigments. relative to the unprotonated species. It is important to know the state of the c-N bond upon protonation. because a protonated retinal Schiff's base is the fundamental 124 photopigment in both rhodopsin and bacteriorhodopsin. Furthermore, a protonated retinal Schiff's base is the chromophore present in almost all the intermediates of the rhodopsin and bacteriorhodopsin photocycles. Thus. to understand the primary changes of the electronic structure upon protonation of the GIN moiety is s key step for the understanding of the wavelenght regulation processes in vivo. Analogously. a protonated aromatic Schiff's base has been found to be the key intermediate in the reaction mechanisms of a series of metabolic coenzymes (i.e. pyridoxal phosphate) which play an important role in the interconversion of amino scids. In these cases. as well. changes in the electronic structure upon protonation of the GIN moeity are key to the catalytic action of such coenzymes as discussed in more detail in Chapter 1. If crystalographic information were available for Schiff's bases sud Lewis acid derivatives. it should be possible to determine if the increase in the GIN stretching frequency upon complexation of the nitrogen electron lone pair is attributable to the stretch-bend coupling modelbfi's'u'13 (i.e. longer bond length and a weaker GIN stretching force constant upon Schiff's base protonation) or to the rehybridization model suggested in this thesis 73'76 (i.e. shorter bond length. stronger GIN stretching force constant). Spectroscopic studies on models Schiff's bases offer a good method for understanding the GIN bond. and the insight obtained may easily be extended to rationalixing the preperties of the covalent linkage between the retinal and the protein. As a direct consequence. determination of the properties of the GIN bond can lead to s further understanding of the 126 mechanism of wavelength regulation by the naturally-occuring photopigments. In the preceding Chapters. the application of optical. Inman and resonance Raman spectroscopies. normal coordinate anslysis and ab initio calculations hed proved to be very useful in providing detailed information about optical. vibrational. structural end electronic changes that are experienced by the GIN chromophore upon its complexation with general Lewis scid. for example. H+, BF3, 3013 and 33:3 . B.Summmary. l. Spectroscopic Studies. The spectroscopic studies. see Chapter 3. have shown that the basic operating mechanism which produces changes in the electronic structure of the protonated retinal Schiff's base and of retinal Schiff's base-Lewis acid complexes are very similar. This conclusion is based on the experimental results which show that. relative to the free species. protonation or complexation of retinal Schiff's bases with the Lewis acid BF3, BC13 and BBr3 lead to similar chromophore responses as follows. a) Similar red shifts in the absorption maximum of the chromophore are observed. b) The GIN stretching frequency increases by approximately the same order of magnitude. c) The GIC stretching frequency is inversionally proporional to the absorption absorption maximun for both kinds of complexes. (d) Similar conclusion apply for the protonated and general Lewis acid complexed aromatic Schiff's bases. 2. Ab initio Studies. The ab initio calculations at the GVB level (see Chapter 4). have indicated that upon protonation of methylimine the following occur. a) There is reorganization in the electronic structure 127 of the GIN bond such that the nitrogen s orbital contributing to the sp2 hybrid GIN bond increases. b) The electron charge distribution calculation show. though the chromophore carries a +1 charge. that the nitrogen appear to be partially negatively charged while the carbon and hydrogen carry the rest of the positive charge. c) These electronic changes in the protonated methylimine. relative to the unprotonated species. lead to a decrease in the GIN bond length and to an increase in the GIN stretching force constant of the protonated derivative relative to the uncomplexed analog. 3.Normal Coordinate Analyses Studies. Normal coordinate analyses indicated (see Chapter 5) the following. s) The calculated increase in the GIN stretching force constant upon protonation leads to a 30 cm”1 in the GIN stretching frequency. This indicates that the GIN stretching frequency increase observed upon protonation or reaction of Lewis acid with the Schiff's bases is primarily due to the increase in the GIN stretching force constant. b) The calculations summarised in Figure 24 provide some insight into previous force fields used to calculate the GIN stretching frequency for protonated retinal Schiff's bases. The results show that the vibrational calculations which support the strechrbend coupling model used low or negative GIN/GIN-B (stretch-bend) interaction force constants which can compensate for the decrease in the GIN stretching force constant (assumed upon protonation) and push the GIN stretching frequency up. However. recent studies (see Chapter 5) indicate. in agreement with our rehybridization model. that the GIN/GIN-B interaction force constant is positive and possibly relatively large. c) Numerical calculation.within the normal mode analysis show that the 128 deuterium shift associated with the decrease in the GIN stretching frequency upon substitution of the Schiff's base hydrogen by deuterium is dependent to some extent on the N-B(D) stretch. GIN-E(D) bend. GIN/N-B(D) (stretch-stretch) and GIN-B(D) (stretch-bend) interaction force constants. These force constants therefore need to be taken into consideration for any well restricted normal coordinate anslysis of Schiff's bases. Furthermore. the nonlinearity of this shift (see Figure 24) indicates that the deuterium shift attributed by the stretch-bend model to the decrease in coupling between the GIN stretch and GIN-D bend can be explained. within the rehybridization formalism. by the increase in mass of the GIN-D center relative to the GIN-H moiety. C. Conclusions. The results presented here show that adduct formation between Schiff's bases and Lewis acids (i.e. H+. BF3.BC13. BBr3) lesds to GIN stretching frequency increases because the GIN bond rehybridixes such that the GIN stretching force constant increases. As a result of the rehybridization. the nitrogen and carbon in the GIN group carry partisl negative and positive charges. respectively. flow the increase in the GIN bond order and charge separation in the GIN bond in the protonated retinal Schiff's bases ere used by the photopigments (rhodopsin and bacteriorhodopsin) in conjuction with local environmentsl effects to regulate the changes in the absorption maximum of the retinal chromophore sre not clear yet. The interplay between these factors constitute a very good field of research and form the basis for the future work described below. 129 D. Future 'ork. 1. Introduction. Our long-term objective is to determine how the charge distribution of the protonated Schiff's base. in particular. the charge separation in the GIN bond. is altered sud transmitted to the rest of the retinal moiety. The presence of a positive or negative charge near the GIN group or by the orientation and strength of possible hydrogen bonds between the imine hydrogen and a nearby counter ion are reasonable candidates for this process. An understanding of the contribution of a particular charge distribution within the GIN bond to the absorption maximum of the protoneted retinal Schiff's base chromophore and how variation in the former can lead to variation in the latter is a major goal. Insight into the magnitude of this contribution to the ”opsin shift" in rhodopsin and bacteriorhodopsin due to the interaction of the protein with the Schiff's base end of the chromophore will be gained. These goals csn be addressed by using X—ray crystallographic studies on all-trans-retinylidene-nrbutylamiue and 2-nephylidene-nrbutylamine and their protonated and BF3 derivatives to determine the change in the GIN bond distance upon complexation of the free Schiff's base with Lewis acid. In addition. ab initio calculations and vibrational studies can be used to obtain the geommetry. electronic charge density distribution. force constants and frequency changes that occur upon protonation or BF3 reaction of the allylimine. (CflzICB-CBINB) and its methylated analog (CflzIGB-CBINCB3). Resonance Reman scattering and 13C and 15N nuclear magnetic studies can be used to determine the vibrational frequencies and 130 charge density distribution upon complexation of the retinal Schiff's bases with Lewis acids of the general kind BF3 and BFZX. Vibrational analysis at the general valence force field86 level (GVFF) can be used in 'conjunction with the allylinine results to estimate the force constants and vibrational frequencies of the retinal Schiff's base. Comparison of these vibrationsl frequencies with those from the retinal Schiff's base in models and photOpigments1'17 can give insight into the net effect of the protein on the retinal Schiff's base chromophores. 2. Specific Aims and Nethods. The specific aims are the following: i. To obtain X rays crystallographic data for the GIN bond in all-trans retinylidene-nrbutylanine. 2-naphtylidene-nrbutylamine and their protonated and BF3 derivatives. ii. Ab initio electron density. force constant and frequency calculations for the imine CEZIGB-GBIN-R (where BIB. CB3) and its protonated and BF3 analogs. Isotopic derivative can also be used to assign the particular vibrations. Comparation of the electron density. force constsnt and frequency of the complexed and uncomplexed species will allow us to determine the effect of nitrogen lone pair occupancy on the electronic environment and normal modes of the imine CBZIGl—CBINR. For the protonated allylimine derivative. calculation of the absorption maximum of the chromophore as a function of the change in distance and orientation between the imine proton (GIN-B) hydrogen bonded to s counter 131 ion (negatively charged) csn be useful in predicting the influence of the strength of hydrogen bonded counter ion to the ebsorption maximum of the retinsl chromophore. Furthermore. the above calculations will be the starting point for calculating changes in the absorption maximum of the allylimine nodel as a function of the distance between the protonated Schiff's base nitrogen and a positive or negative point charge. iii. A synthetic method can be developed to prepare the conplexes [CHZICH-CEINE] and [CflzICB-CHINRBFsl. which will ullow us to study the CIC. G-C and GIN vibrational modes in a more localized enviroment than in retinal Schiff's bases. 1c achive this goal a matrix isolation technique1‘2'14‘ can be develOped to study the experimental vibrstional frequencies of the free imine and its complexes. Comparison of both, systems will give insight into the effect of lone pair occupancy and electron density on the vibrational frequencies of the imine normal modes. iv. The study of the retinal Schiff's base-BF3 complexes can be extended to determine the behavior of the absorption maxima and associated vibrational frequencies for a vsriety of solvents different than those presented in Table 4. 13C and 15N magnetic resonance information can be obtained for the Schiff's base and BF3 complex. A comparison of these results with those present in the literature94'145"148 for protonated retinal Schiff's base should sllow us to establish the role of the counter-ion in determining the absortion maximum of the protonated Schiff's bases. 132 v. A synthetic method to preparate retinal Schiff's base-BFZX complex that can incorporate perturbation to the nitrogen lone pair Schiff's base of the GIN bond can be developed. In principle. the synthetic method will consist in the preparation and isolation of the condensation product formed upon reaction of BF3 with l9-diketones.149'15° i.e. / / H,C\ + BF —> HC ’31:: + HF N The isolation of the BFZX complex from the BF product must be the first step in the sequence. since any trace of BF will not allow s clean reaction between BFZX and the retinal Schiff's base. If the isolation of the BFZX product is satisfactory. the next step will be to determine whether the spectroscopic chsracteristics of this new retinal Schiff's base- Bsz complex resemble those of the protonated retinal Schiff's base. If this is the case. the B group that initialy will be e CBS group. will be substituted by electron donating. electron withdrawing. positively and negstively charged groups. The effects of these substituents on the absorption maximum and vibrational properties of the complex will be used to understand the "opsin shift” in the photopigments rhodopsin and bacteriorhodopsin. 133 vi. 13G and 15N nuclesr magnetic resonance studies of the above retinal Schiff's base-Lewis acid complexes will provide information about the electron density distribution in GIN bond in the retinal system under different conditions of nitrogen lone pair occupancy. vii. Absorption and resonance Baman spectroscopy will provide information about the relation between a particular change in the electronic structure of the chromophore and the GIN. GIG and G-G vibrational frequencies. viii. A vibrational analysis with the GVFF method will be used in combination with the information obtained from the CEZIGBICBIN-R system and the data present in the litersture1'17 to deduce the effect of protonation and BF3 reaction on the retinal Schiff's bsse system and to construct sn accurate force field for the Schiff's base end of the retinal chromophore. This force field then will be used to provide information on the protein-induced changes in the vibrstional frequency and force constants of the retinal chromophore due to protein interactions at the Schiff's base. ix. 'ith the above information on the structure of the unprotonated. protonated sud BF3 complex retinal Schiff's base models for the chromophore-protein intersction for rhodopsin and bacteriorhodopsin will be proposed and evalusted. x. Finally. it can be proposed to study the experimental behavior observed upon hydrogen bond formation or protonation of s chromophore 134 containing the groups. GIO. CIN and GIN. For example. chromophores containing the carbonyl (GIG) group usually show a decresse in the GI0 stretching frequency and a red shift in the absorption maximum on hydrogen bond formation. This behavior is contrary to the trend of the imines sud nitriles which show an increase in the GIN and GIN stretching frequency respectively. and a red shift in the absorption maximum. Note. however. that the experimental behavior of the absorption maxima is in all the cases the same. Ab initio calculations can be used to study the effect that electron lone pair occupancy csn have in the electronic characteristics and properties of the GIO. C=N and GIN bonds in ketones (RR'GIO) nitriles (RGIN). and imines (GIN) respectively. A comparative ab initio study of formaldehyde and cyanide with methylimine and allylimine will help to distinguish between the effect of having one lone pair (nitrogen) or two lone pair (oxygen) on the GI! force constant and GI! stretching frequency. This comparstive study will indicate if the rehybridization model. suggested to explain the observed increase in frequency for protonated Schiff's bases. can be extended to explain the experimental trends in the vibrational frequencies for the carbonyl and nitrile containing chromophores. LIST OF REFERENCES 10. 11. 12. 13. 14. 15. LIST OF REFERENCES Ottolenghi. I. Adv. Photochem. 1980. 12. 97-200. Isthies. R. A. in ”Spectroscopy of Biological Iolecules" Sandorfy. C.; Theophanides. T. Ed. D. Reidel Publishing Company. Dordrech/ Boston/Lancaster. 1983; pp. 303-328. Argade. P. V.; Rothschild. I. 1. Biochen. 1983. 22. 3460-3466. Aton. B.; Doukas. A. 0.; Narva. D.; Calleder. R. B.; Dinur. U.; Honig. B. Biophys. J. 1980. 29. 79-94. Smith. S. 0.; Pardoen. 1. A.; Nulder. P. P. 1.; Curry. B.; Lugtenburg. 1.; Mathies. R. Biochem. 1983. 22. 6141-6148. Smith. S. 0.; lyers. A. B.; Pardoen. 1. A.; Iinkel. C.; Mulder. P. P. 1.; Lugtenburg. 1.; Mathies. R. Proc. Natl. Aged. Sci. USA 1984. 81. 2055-2059. Schiffmiller. R.; Callender. R. B.; Isddell. I. B.; Govindje E. R.; Ebrey. T. 0.; Rakitani. B.; Nakanishi. K. Photochen. Photobiol. 1985. 41.653-567. Kakitani. R.; Rakitani. T.; Rodman. R.; Honig. B.; Callender. R. J. Phys. Chem. 1983. 87. 3620-3628. Iassig. 0.; Stockburg. R.; Gartner. I.; 0esterhelt. D.; Towner.P. J. Ranan Spect. 1982. 12. 287-294. Deng. R.; Pande. C.; Callender. R. R.; Ebrey. T.G. Photochen. Photobiol. 1985. 41. 467-470. Smith. S. 0.; Iyers. A. B.; Mathies. R. A.; Pardoen. 1. A.; 'inkel. C.; van den Berg. B. I. R.; Lugtenburg. 1. Biophys. J. 1985. 47. 653-664. Heyde. N. B.; Gill. D.; Rilponen. R. 0.; Rimai. L. 1. Am. Chem. Soc. 1971. 93. 6776-6780. Marcus. N. A.; Lemley. A. T.; Lewis. A. 1. Reman‘Spect. 1979. 8. 22-25. Aton. B.; Douhas. A. 0.; Callender. R. R.; Becher. B.; Ebrey. T. 0. Biochem. 1977. 16. 2995-2998. Cookingham. R. B.; Lewis. A.; Lemley. A. T. Ibid.l978. 17. 4699-4711. 135 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 136 Bagley. K. A.; Balogh-Nair. V.; Croteau. A. A.; Dollinger. 6.; Ebrey. T. 0.; Eisenstein. L.; Bong. N. R.; Nakanishi. R.; Vittitow. 1. Ibid.l985. 24. 6055-6071. Gerwert. R.; Siebert. F. EIBO 1. 1986. 5. 805-811. Iitkop. B.; Boiler. T.'. 1. Am. Chen. Soc. 1954. 76. 5589-5597. Karube. T.; Ono. T.; Iatsushima. T.; Ueda. Y. Chem. Pharm. Bull. 1978. 26. 2642-2648. Ledbetter. 1. I. 1. Phys. Chem. 1982. 86. 2449-2451. Benecky. I. 1.; CoOpeland. R. A.; Rhys. T. R.; Lobenstine. E. I.; Reva. R. P.; Pascsl. 1r.. R. A.; Spiro. T. G. 1. Biol. Chen. 1985. 260. 11663-11670. Benecky. I. 1.; Copeland. R. A.; Rava. R. P.; Feldhaus. R.; Scott. R. D.; Nezler. G. N.; Netsler. D. B.; Spiro. G. T. Ibid. 1985. 260. 11671-11678. 'ard. B.; Callahan. P. R.; Young. R.; Babcock. 0. T.; Chang. C. R. 1. Am. Chem. Soc. 1983. 105. 634-636. Hanson. L. R.; Chang. C. R.; Isrd. B.; Callahan. P. R.; Babcock. G. T.; Read. 1. D. Ibid. 1984. 106. 3950-3958. 'urd. B.; Chang. C. R.; Young. R. Ibid. 1984. 106. 3943-3950. laggiora. L. L.; Naggiora. G. N. Photochem. Photobiol. 1984. 39. 847-849. Petke. 1. D.; Naggiora. G. I. 1. An. Chem. Soc. 106. 3129-3133. Bargrave. P. A.; NcDowell. 1. B.; Curtis. D. R.; Iong. 1. R.; Joszczsk. B.; Fong. S. L.; lohanc Rec. 1. R.; Argus. P. Biop. Struc. Koch. 1983. 9. 235-241. Drstx. E. A.; Rargrave. P. A. rends in Biochem. Sci; 1983. 8. 128-134. Ovchinnikov et a1. Biorg. Rhim. 1982. 8. 1011-1017. 0seroff. A. R.; Callender. A. R. Biochem. 1974. 13. 4243-4248. Nethies. R.; Freedman. T. B.; Stryer. L. 1. Iol. Biol. 1977. 109. 367-373 'ald. 0. Nature (London).1968. 219. 800-807. Yoshixawa. T.; Wald. 6. Ibid. 1963. 197. 1279-1286. Nathies R. A.; Smith. S. 0.; Palings. I. Tb be published in "Biological Applications of Raman Spectroscopy.” 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 137 Stoeckenius. '.; Lozier. R. B.; Bogomolni. R. A. Biochin. Biophys. Acta 1979. 505. 215-278. Ovchinnikov. Yu. A.; Abdulsev. N. 0.; Fligina. N. Yu.; Riselev. A. V.; Lobanev. N. A. FEBS Lett. 1979. 100. 219-224. Rhorana. B. 6.; Gerber. G. B.; Berlihy. ww. C.; Gray. C. P.; Anderess. R. 1.; Nihei. R.; Briemsnn. R. Proc. Natl. Acad. Sci. USAJ1979. 76. 5040-5050. Callahan. P. l.; Babcock. G. T. Biochem. 1983. 22. 452-461. Babcock. G. T.; Callahan. P. N. Ibid. 1983. 22. 2314-2319. Iikstrom. R.; Krab. k.; Saraste. I. in ”Cytochrome Oxidase A Synthesis.” Academic Press. London. 1981. Iikstrom. N. K. F. Nature (London).1977. 266. 271-273. Callahan. P. N. PhD Thesis. Nichigan State University. 1983. 0ndrias. N. R.; Babcock. G. T. Biochem. Biophys. Res. Commun. 1980. 93. 29-35. Babcock. G. T.; Callshan. P. R.; 0ndrias. N. R.; Salmeen. 1. Biochem. 1981.20. 959-966. Rate. 1. 1.; Norris. 1. R.; Shipman. L. L.; Thurnsuer. I. C.; 'asielewski. N. R. Annu. Rev. Biophys. Bioengineer. 1978. 7. 393-434. nggiora. 0. N._;nt. 1. Quantum Chem. 1979. 16. 331-352. Sauer. K. Aggy. Rev. Phys. Chem. 1979. 30. 155-178. Karpeisky. N. Ys.; Ivanov. V. I. Ngture,1966. 40. 493-496. Christen. P.; Netxler. D. B. in ”Transaminases". John 'iley and Sons. Inc. New York. 1985. Fersht. A. in "Enzyme Structure and Mechanism." Freeman and Compsny. New York. 1985: pp. 69-75. Zerner. B.; Goutts. S. R.; Lederer. F.; Iaters. B. B.; 'estheimer. Fe no 3100110.. 1966' 5' 813-8160 'arren. 8.; Zerner. B.; Iestheimer. F. B. Ibid. 1966. 5. 817-823. Grazi. B.; Cheng. T.; Borecker. B. L. Biochem. Biophys. Res. Commun. 1962. 7. 250-256. Speck. 1. C.; Rowley. Jr. P. T.; Rorecker. B. L. 1. Am. Chem. Soc 1963 . 85 . 1012-1013 . ' 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 138 Horecker. B. L.; Tsolas. 0.; Lai. C. Y. The Enzyges. 1972. 7. 213-219. Tsolas. 0.; Horecker. B. L. The Enzzge . 1972. 7. 259-264. Fabian. N. 1.; Legrand. R.; Poirier. P. Bull. Soc. Chim. Fr. 1956. 1499-1509. Fabian. N. 1.; Legrand. N. 1219, 1956. 1641-1643. Blatz. P. B.; Nohler. 1. H. Biochem. 1975. 14. 2304-2309. Sheves. R.; Nakanishi. R. 1. Am. Chem. Soc. 1983. 105. 4033-4039. Baasov. T.; Sheves. N. 1219. 1985. 107. 7524-7533. Kakitani. H.; Kekitani. T.; Rodman. B.; Honig. B. Photochem. Photobiol. 1985. 41. 471-479. Birge. R. R.; Hubbard. L. N. 1. Am. Chem. Soc. 1980. 102. 2195-2205. Freedman. K. A.; Becker. R. S. Ibid. 1986. 108. 1245-1251. Nakanishi. R.; Balogh-Nair. V.; Arnaboldi. R.; Tsujimoto. R.; Honig. B. Ibid. 1980. 102. 7945-7947. Samuel. B.; Snaith. R.; Summerford. C.; Vade. R. 1. Chem. Soc. (A) 1970. 2019-2022. Coerver. H. 1.; Curran. C. 1. Am. Chem. Soc. 1958. 80. 3522-3523. Figeys. H. P.; Geerlings. P.; Berckmans. D; Alsenoy. C. V. 1. Chem. Soc. Faraday Trans. (2) 1981. 77. 721-740. Horac. N.; Vitek. A. "Interpretaion and Processing of Vibrational Spectra" 1978. John Iiley. New York. pp. 302-303. Swanson. B.; Shriver. D. F.; Ibers. 1. A. Inorg. Chem. 1969. 8. 2182-2189. Gerrard. '.; Lappert. N. F. Pysxora. B.; Wallis. 1. I. 1. Chem. Ldpez-Garriga. 1. 1.; Hanton. 8.; Babcock. G. T.; Harrison. 1. F. 1. Am. Chem. Soc. 1986 Accepted for publication. Lopez-Garrige. 1. 1.; Babcock. G. T.; Harrison. 1. F. Biophys. 1. 1985. 47. 96s. Lopez-Gsrriga. 1. 1.; Babcock. G. T.; Harrison. 1. F. 1. Am. Chem. Soc. Accepted for publication. Lepex-Garriga. 1. 1.; Babcock. G. T.; "Tenth International Conference on Raman Spectroscopy”. Eugene. OR. 1986. 77. 78. 79. 80. 81. 82. 83. 85. 86. 87. 89. 90. 91. 92. 93. 94. 95. 96. 97. 139 Goddard. III. I. A.; Dunning. 1r.. T. H.; Hunt. w. 1.; Hey. P.J. Acc. Chem. Res. 1973. 6. 368-376. Hehre. I. 1.; Radom. L.; Schleyer. P. vR.; Pople. J. A. "Ab Initio Nolecular Orbital Theory" John Viley and Sons. Inc. New York. 1986. Santerre. G. R.; Hansrote. 1r.. G. 1.; Crowell. T. I. J. Am. Chem. Soc. 1958. 80. 1254-1257. Layer. R. '. Chem. Rev. 1963. 63. 489-510. Pickard. P. L. Tolbert. T. L. J. Org. Chem. 1961. 26. 4886-4888. Lane. C. F.; Kramer. G. V. Aldrichimics Acta. 1977. 10. 11-18. The ARGOS integral program was developed by R. I. Pitxer (Ohio State University). The GVB-164 program was written by R. Blair (Argonne National Leboratory) Shimanouchi. T. Computer Programs for Normal Coordinete Treatment of Polyathcmic Nolecules. Tokyo University. Tokyo. Japan. 1969. Nsm. H. B.; Leroi. G. E. Spectrochim. Acta,1985. 41A. 67-73. Hart. D. L.; Kauai. I. J. Am. Chem. Soc. 1983. 105. 1255-1263. Yoon. U. C.; Quillen. S. L.; Nariano. P. S.; Swanson. R.; Stavinoha. 1. L.; Bay. B. Ibid. 1985. 105. 1204-1218. Patai. S. "The Chemistry of the Carbon Nitrogen Double Bonds” 1970 Interscience Pub. New York. Cap 1. Lewis. A. Phil. Trans. R. Soc. Lond. A. 1979. 293. 315-327. Susz. B. P.; Cooke. I. gglv. Chim. Acts 1954. 37. 1273. Suez. B. P.; Chalandon. P. Ibid.. 1958. 41. 1332. Lappert. I. F. J. Chem. Soc. 1962. 542. Lugtenburg. 1.; Nuradin-waeykowska. I.; Heeremens. C.; Pardoen. 1. A.; Harbison. G. S.; Herxfeld. 1.; Griffin. R. C.; Smith. 8.0. J. Am. Chem. Soc. Accepted for publication. Rr0pf. A.; Hubbard. R. Ann. N. Y. Acad. Sci. 1958. 74. 266. Irving. C. 8.; Byers. G. F.; Leermakers. P. A. Biochemistry 1970. 9. 858-862. B1utz. P. E.; Iohler. J. R. Ibid. 1972. 11. 3240-3242. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 140 Ebrey. T. C.; Honig. B. 0. Rev. Biophys. 1975. 8. 129-159. Vaddel. I. H.; Schaffer. A. N.; Becker. R. S. 1. Am. Chem. Soc. 1977. 99. 8456-8460. 'arshel. A. Proc. Natl. Acad. Sci. USA 1978. 75. 2558-2563. Ishikawa. R.; Yoshihara. T.; Suzuki. H. J. Phys. Soc. Jpn. 1980. 49. 1505-1511. Blatz. P. B.; Nohler. J. A.; Navangul. H. V. Biochem. 1972. 11. 848-855. Honig. B.; Dinur. U.; Nakanishi. R.; Balogh-Nsir. V.; Gawinowicz. I. A.; Arnaboldi. N.; lotto. N.G. 1. Am. Chem. Soc. 1979. 101. 7084-7086. Zwarich. R.; Smolarek. 1.; Goodman. L. J. Nolecular Spec. 1971. 38. 336-357. Perry. I. A. V.; Robinson. P. 1.; Sainsburry. P. 1.; Ialler. N.J. J. Chem. Soc. (B). 1970. 700-703. Patai. S. "The Chemistry of Carbon Nitrogen Double Bond." 1970. Interscience Publishers. New York. Chapters 1 and 4. Grodcsik. A.; lubinyi. N.; Fogarasi. G. 1. Nol. Struct. 1982. 89. 63-70 0 Sharma. 0. P.; Singh. S. N.; Singh. R. D. Indian 1. Phys. 1974. 48. 494-503. Sharma. 0. N.; Roels. O. A. J. Org. Chem. 1973. 38. 3648-3651. Datin. A. P.; Lebas. 1. I. Spectrochim. Acta. 1969. 25A. 169-185. Yanovskaya. L.A.; Rryshtal. G. V.; Yekovlev. I. P.; Rucherov. V.F.; Simkin. B. Y.; Bren. V. A.; Ninkin. V. 1.; Osipov. 0.; Thmakaova. I. A._Tetrahedrom 1973. 29. 2053-2064. Favrot. 1.; Vbcelle. D.; Sandorfy. G. Photochem. Photobiol. 1979. 30. 417-421. Besnainou. S.; Thomas. B.; Bratov. S. J. No1. Spect. 1966. 21. 113-124. Alshuth. T.; Stockburger. N.; Hegemann. P.; 0esterhelt. D. Eggs Lett. 1985. 179. 55-59. Brown. R. D.; Penfold. A. Trans. Faraday Soc. 1957. 53. 397-402. "11113811. Do 80 Jo Ch... PhY" 1961' 35. 1‘91’1‘97e Noore. C. B.; Pimentel. G. G. Ibid.l965. 43. 63-70. 118. 119. 120. 121. 122. 123. 124. 125. 126. 127. 128. 129. 130. 131. 132. 133. 134. 135. 136. 137. 141 Christen. D.; Oberhammer. B.; Hbmmaker.R. N.; Chang. S. C.; DesNarteau. D. D. J. Am. Chem. Soc. 1982. 104. 6186-6190. Botschwina. P. Chem. Phys. Letters 1974. 29. 580-584. Bedes. R. A.; Veil. D. A.; Elenberger. I. R.; Fsrneth. I. F.; Dixon. D. A.; Douglas. Jr. C. H. J. Am. Chem. Soc. 1981. 103. 5372-5377. Kollman. P. A.; Trager. I. F.; Rothenberg. 8.; Villiams. J. E. 1. Am. Chem. Soc. 1973. 95. 458-463. Huzinaga. S. J. Chem. Phys. 1965. 42. 1293-1320. Dunning. Jr.. T. H. H. Ibid. 1970. 53. 2823-2833. Raffenetti. R. C. Ibid. 1973. 58. 4452-4458. Nulliken. R. S. ibid. 1955. 23. 1833-1840. Alvsrado. A.; Harrison. 1. Gurfit Program. Nichigan State University. East Lansing. NI. Fogarasi. G.; Pulay. P. Acts Chim. Acad. Sci. Hung. 1981. 108. 55-73 0 Dunning. Jr.. T. B.; Pitzer. R. N.; Aung. S. J. Chem. Phys. 1972. 57. 5044-5051. “ 'ahlgren. U.; Pacsnsky. 1.; Bagus. P. S. Ibid. 1975. 63. 2874-2881. Binkley. 1. 8.; Frisch. N. 1.; Schaeffer III. B. F. Chem. Phys. utte 1986' 126. 1-60 Yamaguchi. Y.; Schaefer III. R. F. 1. Chem. Phys. 1980. 73. 2310-2318. 0seroff. A. R.; Callender. R. H. Biochemistry 1974. 13. 4243. Inamori. Th; masda. Y.; Tsuboi. N.; koga. Y.; Rondo. S. 1. lol. Spect. 1985. 109. 256-268. Hamsda. Y.; Tsuboi. N.; Natsusawa. T.; Yamanouchi. K.;Kuchitsu.R Ibid. 1984. 105. 453-464. Hashiguchi. R.; Hemads. Y.; Tsuboi. N.; Roga. Y.; Rondo. 8.; Ibid. 1984. 105. 81-92. Hamsda. Y.; Tsuboi. N.; Takeo. B.; Natsumura. C. Ibid. 1984. 106. 175-185. Sugawara. Y.; Hirakswa. A. Y.; Tsuboi. N. Ibid. 1984. 108. 206-214. 142 138. Botschwina. P.; Nachbaur. B.; Rode. B. H. Chen. ghys. Letters 1976. 41. 486-489. . 139. Laswick. p. 11.; Taylor. R. c. 1. Mel. Struc. 1976. 34. 197-218. 140. Cyvin. B. N.; Cyvin. S. 1.; Hargittai. N.; Hargittai. I. Z. A1101". “1‘. C1103. 1978. ‘40g 111-1180 141. Hills. 1. N. Spectrochimics Acta. 1963. 19. 1585-1594. 142. Swanson. 1.; Jones L. H. "Vibrational Spectra and Structure” Elsevire Science Pub. 1983. vol. 12. pp. 1-63. 143. Redington R.L. 121g. pp. 323-391. 144. Jodl. H.J. {219. 1984. vol 13. pp. 285-416. 145. Allen. N.; Roberts. 1. D. Can, J. Chem. 1981. 59. 451-458. 146. Allen. N.; Roberts. 1. D. J. Org. Chem. 1979. 45.130-135. 147. Harbison. G. 8.; Herzfeld. 1.; Griffin. R. G. Bioghemigtgy; 1983. 22. 5-12. 148. Nateescu. G. D.; Abrahsmson. E. V.; Shriver. J. V.; Capan. V.; Nuccio. D.; Igbal. N.; Vaterhous. V. in "Spectroscopy of - Biological Nolecules" Sandorfy. C.; TheOphanides. T. Ed. D. Reidel Publ. Com. Dordrech/Boston/Lancarter. 1983; pp 257-290. 149. Niedenzu. R.; Dawson. J. in "Boron-Nitrogen Compounds" Academic Press. Inc. Springer-Verlag Ed. Berlin/Heidelberg/New York 1965. 150. Gerrard. V. in "The Orgsnic Chemistry of Boron" Academic Press. London. New York. 1961.