Vibrational analysis of the dipeptides containing alanine and serine

VIBRATIONAL SPECTROSCOPY ELSEVIER

Vibrational Spectroscopy8 (1995) 279--291

Vibrational analysis of the dipeptides containing alanine and serine G. Jalsovszky *, S. Holly Central Research Institutefor Chemistry, HungarianAcademy of Sciences, P.O.Box 17, H-1525 Budapest, Hungary

Received 27 April 1994

Abstract

Fourier-transform infrared and Raman spectra of L-Ala-L-Ser, D-Ala-L-Ser and L-Ser-L-Ala were recorded, and significant differences were found between the spectra of these dipeptides. In order to interpret these differences, the normal vibrations of L-Aia-L-Ser, o-Ala-L-Ser, L-Ser-L-Ala and o-Ser-L-Ala were calculated from an empirical general valence force field and molecular geometries calculated by MM2 methods. Initial values of the force constants for the normal coordinate analysis were transferred from the known spectroscopic empirical force fields of alanine, serine and poly (L-alanine). Then, a partial refinement of force constants was performed to improve the fit to the experimental spectra of L-Ala, L-Ser and some of their deuterated analogues. Differences were obtained between the calculated spectra of ELand DE derivatives; the significant differences between the experimental spectra, however, could be reproduced with limited success, suggesting that the latter may be due in part to differences in crystal rather than in molecular structures. Keywords: Infrared spectrometry;Raman spectrometry;Alanine; Dipeptides; Normal coordinate analysis; Serine; Valence force field

1. Introduction

Alanylserine is one of the simplest heterodipeptides which contain two centres of asymmetry, and thus may exist in the form of four optical isomers: L-Ala-L-Ser, D-AIa-L-Ser, o-Ala-D-Ser and L-Ala-D-Ser. Pairs LL-DD and DEED, being the mirror images of one another, have the same vibrational spectra (disregarding now the phenomena of vibrational circular dichroism or Raman optical activity). The LL and DL forms give rise, however, to different vibrational spectra, since the relative positions of bonds are different in them, leading to entirely different stable conformations and thus different couplings between vibrational modes. The vibrational spectra of serylalanine should be different from those of alanylserine, and, again, the spectra * Corresponding author. 0924-2031/95/$09.50 © 1995 Elsevier ScienceB.V. All rights reserved SSDI0924-2031 ( 94 ) 00033-D

of the LL and DL forms should be different from one another. Thus, infrared and Raman spectra may be used to identify the conformation and sequence of oligopeptides forwhich the dipeptides Ala-Ser and Ser-Ala can be used as a simple model. Vibrational spectra of oligo- and polypeptides containing alanine have been extensively analysed including those of alanylalanine [ 1 - 4 ] , tri-L-alanine [ 5 ] and poly (L-alanine) [ 6,7 ]. For alanylalanine, the effect of optical diastereomery on the vibrational spectra and vibrational circular dichroism was also studied [ 3,4]. These investigations were focused on the amide-III mode (s), which are represented by strong bands in the Raman spectrum and show frequency shifts depending on the secondary structures of peptides. The thorough analysis, using the spectra of the various isotopomers of L-Ala-L-Ala and L-Ala-D-Ala, also included normal coordinate calculations [4]. The force field used was,

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however, a Urey-Bradley field with the methyl and NH~- groups represented as point groups to simplify the computational procedures. Since the bending modes of these two groups may be mixed (or overlapped in the spectra) heavily with the amide-III modes, a full normal coordinate analysis appears to be more suitable for our purpose. Serine-containing peptides have been given much less attention than alanine analogues. For serine itself, the only vibrational analysis found by us is the one performed by Susi et al. [8] in 1983. The weak points of this analysis will be discussed below. A series of AIa-X peptides, in which X also includes serine, was studied by Williams et al. [9]. In this study optimized structures and frequencies were calculated for Ala-X peptides using ab initio calculations on geometries and normal mode methods based on empirical force fields. These force fields were constructed by transferring force constants from related molecules; force constant values for the serine side chain were e.g. taken from the paper of Susi et al. [ 8 ]. For the interpretation of spectral differences between the epimeric forms of Ser-Ala and Ala-Ser, the vibrational spectra should be analysed and the infrared and Raman bands assigned to the normal vibrations of the molecules. With this objective a complete normal coordinate analysis of L-Ala-L-Ser, D-Ala-L-Ser, L-Ser--LAla and n-Ser-L-Ala was performed.

Table 1 Force constants for the dipeptides No.

Parameter

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

NCa CaC CN CO NH Calla1 Calla2 CaCbl CaCb2 CbHbl CbHb2 NCaC CaCN CNCa NCO NCaH NCaCb CaNH CaCO CCaHa CCaCb CNH HaCaCb CaCbHbl CaCbHb2 HbCbHbl HbCbHb2 COob NHob CNt CaCt NCat CaCbt 1 CaCbt2 N'H HN'H HN'Ca N'Cat C"O" O"C"O" CaC"O" C"O"ob CaC"t CbOb ObH ObCbHbl CaCbOb CbObH CbObt NCa-NCaHa CaCbl-NCaCb CaCb2-NCaCb

2. Experimental The infrared and Raman spectra of L-Ala-L-Ser, DAla-L-Ser and L-Ser-L-AIa were recorded. Infrared spectra of KBr discs and Nujol mulls were taken on a Nicolet 170SX FT-IR instrument. Raman spectra of the crystalline samples were measured on a Nicolet 950 FT-Raman spectrometer with Nd-YAG laser excitation using 180 ° geometry. Even with near-IR excitation there was a tendency of sample overheating, thus in order to prevent the samples from "burning", relatively low excitation energies (100 mW) were used. The spectra contain several overlapping bands. The frequencies (and relative Raman intensities) listed in Tables 2-4 were obtained by fitting Gauss-Cauchy product functions to the band envelope. The reality of the components was also checked by Fourier selfdeconvolution.

32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

Value 4.523 4.160 6.415 9.882 5.674 4.700 4.780 5.450 4.740 4.800 4.800 0.819 1.033 0.526 1.246 0.765 1.193 0.527 1.246 0.684 1.181 0.527 0.518 0.537 0.646 0.545 0.545 0.587 0.129 0.680 0.037 0.170 0.088 5.434 0.626 0.731 0.486 9.302 2.030 1.200 0.726 0.568 4.247 6.567 0.735 1.648 0.755 0.195 0.627 0.617

Source DK01 DK02 DK03 DK04 DK05 Ser Ala Ser Ala DK08 DK08 DKll DK12 DK13 DK14 DK15 DK16 DK17 DK18 DK19 DK20 DK21 DK22 Ser Ala Ser Ala DK27 DK28 DK31 DK30 DK29 Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ser Ser Ser Ser Ser Ser DK47 DK66 DK66

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Table 1 (continued)

Table 1 (continued) No.

Parameter

51

CN-CO CaC-CO CO--CaCO CO-NCO CaCbl-HaCaCb CaCbl-CCaCb CaCb2-HaCaCb CaCb2--CCaCb NCa-NCaCb CaC-CCaCb CaCbl--CaCbHbl CaCb2-CaCbHb2 CaC-CN CN-NCa NCa--CaC CaC-CaCN CaC-NCaC CN--CaCN CN-CNCa NCa-CNCa NCa-NCaC NCa-CaNH CN-CNH NCO---CNH CaC~CCaHa CaC-CaCO CN-NCO NCa-CCaCb CaCN--CNH NCaC-NCaCb CCaCb-COob CCaHa-COob CaCO-CCaHa NCaHa-NHob NCaC-NHob NCaCb-NHob CaCbHb 1-HaCaCb CaCbHb2-HaCaCb CaC-CaCbl NCa-CaCbl CaC-CaCb2 NCa-CaCb2 CaNH-NCaHa CNCa-NCaHa CaCbHb 1-HaCaCb CaCbHb2-HaCaCb CbHbl--CbHbl CbHb2-CbHb2 CaCbHbl-CaCbHbl ObCbHb 1--ObCbHbl CaCbHb2-CaCbHb2 NCaC--CaNH NHob-CNt

52 53

54 55 56

57 58 59

60 61 62

63 64

65 66 67 68 69 70

Value 0.500 0.450 0.417

0.367 0.353 0.300

0.294 0.251 0.200

0.162 0.150 0.110

0.122 0.100

0.070 0.035 - 0.045 - 0.056 - 0.100 - 0.148

Source

No.

Parameter

Value

Source

DK41 DK40 DK63 DK64 DK68 DK68 DK68 DK68 DK48 DK57 DK70 DK70 DK36 DK37 DK37 DK53 DK52 DK59 DK60 DK44 DK45 DK46 DK62 DK88 DK56 DK54 DK61 DK51 DK77 DK74 DK93 DK91 DK87 DK81 DK76 DK79 DK95 DK95 DK39 DK38 DK39 DK38 DK90 DK78 DK96 DK96 DK42 Ala DK94 Ser Ala DK71 DK100

71 72 73 74 75 76 77 78 79 80

N'H-N'H NCa-HN'H NCa-HN'Ca NCaH-HN'Ca C"O"-C"O" CaC'O"-CaC'O" CaC--C"O" CaC-O"C"O" C"O"--O"C~O " C"O"-CaC"O" C"O"-CaC'O"' x ( - 1) CaCbl-CbOb CbOb--CbObH CbOb-4)bCbH1 CbOb-CaCbOb

0.078 - 0.150 0.294 0.110 1.083 - 0.100 1.440 - 0.519 - 0.135 0.509

Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ser Ser Ser Ser

81 82 83 84

0.136 0.274 0.264 0.715

C, O, N, H: atoms of the peptide link. Ca: C~, carbon; Cb: Ca carbon; C": carboxylic carbon; N': nitrogen of NH~- group; Ob: hydroxy oxygen of Serine; O": oxygen of carboxylate group; CaHal, CbHbl, CaCbl, CaCbHbl and CaCbtl refer to Serine; Calla2, CbHb2, CaCb2, CaCbHb2 and CaCbt2 refer to Alanine. DK: Serial number as denoted by Dwivedi and Krimm [7]; Ala and Ser are force constants as denoted in the text.

3. Calculations S i g n i f i c a n t d i f f e r e n c e s w e r e f o u n d b e t w e e n the spectra o f the t h r e e s u b s t a n c e s , a t t r i b u t a b l e to d i f f e r e n c e s in m o l e c u l a r c o n f o r m a t i o n a n d / o r crystal structure. T o d e c i d e w h i c h r e a s o n is the d o m i n a n t one, the v i b r a tional frequencies and potential energy distributions were estimated by an approximate normal coordinate analysis. T h e e m p i r i c a l g e n e r a l v a l e n c e force field u s e d for the a n a l y s i s w a s t r a n s f e r r e d f r o m r e l a t e d m o l e c u l a r s y s t e m s . T h i s p a p e r r e p o r t s the results o f t h e s e calcul a t i o n s p e r f o r m e d for the i s o l a t e d d i p e p t i d e m o l e c u l e s (i.e. n o H - b o n d effects w e r e t a k e n into a c c o u n t ) w i t h a f o r c e field c o n t a i n i n g 120 p a r a m e t e r s ( 8 4 o f w h i c h are d i f f e r e n t ) . T h e M O L V I B p r o g r a m o f S u n d i u s [ 10] w a s u s e d w i t h a r e d u n d a n t set o f 7 2 v a l e n c e c o o r d i n a t e s to c a l c u l a t e t h e 66 n o r m a l f r e q u e n c i e s o f t h e m o l e c u l e s . N o r m a l v i b r a t i o n s w e r e c h a r a c t e r i z e d b y the largest e l e m e n t s o f p o t e n t i a l e n e r g y d i s t r i b u t i o n ( P E D ) in t e r m s o f i n t e r n a l v a l e n c e c o o r d i n a t e s , e x c e p t for t h e g r o u p s CH2, CH3, NH~- a n d C O O - , w h e r e P E D w a s defined in t e r m s o f local s y m m e t r y c o o r d i n a t e s . T h e f o r c e field w a s c o n s t r u c t e d b y t r a n s f e r r i n g the force c o n s t a n t s o f the p e p t i d e l i n k a n d m a i n c h a i n f r o m

G. Jalsovszky, S. Holly / Vibrational Spectroscopy 8 (1995) 279-291

282

Table 2 Observed and calculated frequencies (in cm 1) of L-Ala-L-Ser Observed

Calculated

IR band centre

Raman band centre Raman relative intensity

3430b 3225 b

3230

0.08

3009 2993 2978 2965

0.48 0.58 0.98 0.51

2943 2923 2885 1681 1657 1632 1606 1556

1.00 0.30 0.59 0.40 0.10 0.08 0.05 0.03

1467 1455 1448 1413 1395 1377 1360 1335 1316 1302 1291 1267 1255 1232 1206 1152

0.33 0.25 0.12 0.41 0.35 0.25 0.11 0.17 0.18 0.10 0.20 0.31 0.48 0.10 0.11 0.07

1141 1117 1093

0.09 0.12 0.30

1036 1015 974 936 889 864 847 802 751

0.12 0.35 0.16 0.39 0.67 0.14 0.10 0.11 0.07

3062 s 3010w 2980w

2890vw 1687 s 1675 sh 1620 vs 1605 sh 1554 vs 1526 sh 1457vw 1414 s 1381 s 1334 vw

1296 w 1270m 1255 w 1235 w 1208 m 1148m 1117 m 1093 m 1082 w 1037m 1019 w 957w 939 w 889 w 848 w 802m

Frequency

Main % contributions to PED in symmetry coordinates

3430 3208 3154 3147 3109 2994 2990 2983 2964 2954 2938 2916 1677 1651 1644 1561 1544 1523 1467 1461 1449 1440

OH(100) NH(99) NHaas (99) NH3as (98) NHass(99) CH3as (90) CH3as (89) CH2as (99) CaHa2 (98) CH2ss(95) CaHal (95) CH3ss (99) FR CO(68) NH3ab (64) NH3ab (67) COOas (86) NH3sb (89) NHipb (37) CHaab(70) CHaab(71) CH2b(80) COOss (55)

1383 1363 1339 1313

CH3as(9) CH3as(9)

CN(9) NH3ab(24) NH3ab(25) COOr (10)

CaCN (5) NH3r2 (6)

Nnipb (5)

CN(30) CH3ab(15) CH3ab(15) COOss (8) COOb (20)

CaC (7) CH3rl (9) CH3r2 (9)

NCa (5)

CaC (10)

CH2b (10)

CH3sb (69) NCaH (47) CaCbl (31) CCaH(29)

HCaCb (11) CCaH (15) CH2w(25) CH3sb(12)

CCaH (8)

CaCb2 (5)

CCaH (12) CaC (9)

HCaCb (8) HCaCb(9)

1288 1281 1254 1231 1210 1186 1168 1149 1130 1093

CbOH (46) NCaH(43) NHipb (24) CH2t (45) CaCbl (30) CaCb2 (32) CH2w(31) NCa (32) NCa (21) NH3r2 (20)

CH2t(36) HCaCb(17) N C a H(10) CbOH (27) NCa (18) NHarl (25) HCaCb(10) CH3rl (28) NH3r2 (12) NCa (15)

CH3sb(10) CCaH (9) CCaH (11) CCaH (13) NH3r2(8) CaCbl (9)

NH3r2(7) CN (8)

CHar2(8) NH3rl (13)

CH2w(8) CH3r2(8)

1031 1003 987 958 889 879 841

CH3r2 (56) CaCb2 (29) CH2r (30) CbO (73) NCa (25) CHzr (33) NCa (31)

NH3r2(17) NH3rl (18) CaC (24) OCCb (6) CH3rl (17) COOb (18) CaCb2 (12)

NH3rl (10) CH3rl (14) CCaCb (7)

Cn3ab (6) Na3r2 (14)

CaC (12) CaC (14) CN (7)

CN (9) CbOt (13) CH3rl (6)

758

C"Oob (31)

COOb (15)

CbOt (12)

COOss (8)

HCaCb (12) CH3sb(5) NCaH(7)

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283

Table 2 (continued) Observed

Calculated

IR band centre

Raman band centre Raman relative intensity

735 m

741 715 677 660 631

667 vw 631 m 607 vw 543 m 483 w 428 w 419 w

540 488 477 422 385 306 296 254 243

0.09 0.08 0.08 0.04 0.04 592 0.08 0.18 0.12 0.30 0.09 0.08 0.06 0.29 0.14

Frequency

Main % contributions to PED in symmetry coordinates

684 671 658 595 CbOt (17) 556 514 504 428 396 353 327 239 236 221 199 163 115 97 57 45 22

CaC (11) COob (35) CbOt (30) CNt (45) CaC (15) N'Cat (20) N'Cat (48) N'Cat (19) OCCb (29) NCaCb (31) C"Oob (11) NCaCb (21) CCaCb (23) CCaCb (32) CaCbt (85) NCaC (24) CaC"t (65) CaCbt (30) CaCbt (43) NHob (35) CaCt (59) NCat (61)

COOr (10) C " O o b(23) COob (25) NHob (20) CNt (12) CaCO (18) HCaCb (9) COOr (19) COOr (16) COOr (11) NCaC (10) NCaCb (20) NCaC (20) NCaCb(13)

OCCb (9) NCaC (6) C " O o b(11) CoOt (6) NHob (8) CaC (12) CCaCb (9) CCaCb (13) CaCbl (9) OCCb (10) CNt (8) NCaC (12) CaCO (11) CaCbt(12)

NCaC (19) NCaCb (7) CNCb (20) CNCb (22) NCaC (25) NCat (19) CaCt (27)

COob (11) OCCb (6) CaC"t(10) CCaCb (5) COob (6) NHob (9)

CNCb (9) COOb (6)

CNt (8) CaCO (5) NCa (10) NCaCb (8) CCaCb (9) CCaCb (8) COOr (6) CNCb (9) CaC't (8) CaCbt (6) CaCN (9) CaCN (5) CNCb (6) NCaC (7)

ss: symmetric stretch; as: antisymmetric stretch; sb: symmetric bend; ab: antisymmetric bend; ipb: in-plane bend; ob: out-of-plane bend; r: rocking; w: wagging; t: twisting/torsion. For atom and valence coordinate notations see notes to Table 1.

poly(L-Ala) [7], those of carboxylate end group from L-Val-Gly-Gly [ 11], those of the ammonium end group from tri-L-Ala [5] and finally those of the CH2OH group from serine [8]. Then, the end-group force constants were refined to reproduce the vibrational frequencies of L-alanine and its three isotopomers [12] (130 observed frequencies, mean frequency error: 10.2 cm-1). The transferred force constants of the CHEOH group proved to be rather poor for two reasons. First, the analysis of Susi et al. [8] was an overlay calculation to reproduce the frequencies of the isotopomers of cysteine, serine and fl-chloroalanine, and thus it cannot be expected to yield the best force field for the CHEOH side group. Second, the force field was fitted to the spectra of DL-serine, which are significantly different from those of L-serine. Such differences may often occur, and are attributed to the different crystal structures of the optically active substance and the racemic compound. With serine, this

difference is well known: in the crystal of L-serine the terminal OH groups partake in an infinite series of weak hydrogen bonds, whereas in DL-serine the OH groups form stronger hydrogen bonds to the more electronegative carboxylate oxygen atoms [ 13,14]. Thus, if force constants are to be transferred, a better choice is to take the force field of the optically active species. Therefore, in parallel to the vibrational analysis of dipeptides, the force field of serine was revised by fitting the force constants to the spectrum of L-serine instead of the racemic compound ( 19 force constant parameters were varied to fit 36 observed frequencies). The final set of force constants used in the dipeptide calculations is shown in Table 1. The force constants taken from poly (L-alanine) are denoted b y " D K " and a serial number corresponding to the numbering of force constants in the paper of Dwivedi and Krimm [ 7]. Force constants denoted b y " A l a " a n d " S e r " are those refined by us as discussed above.

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Table 3 Observed and calculated frequencies (in cm- 1) of D-AIa-L-Ser Observed IR band centre 3410 m 3300 s

2960 vw 2940 w 2860 w 1691 m 1661 s 1603 vs 1545 s 1523 s

Calculated Raman band centre

Raman relative intensity

3301 3254

0.05 0.06

3009

0.18

2987 2969

0.22 0.44

2941 2885

1.00 0.36

1656 1639 1602 1539

0.47 0.15 0.09 0.09

1458

0.38

1450 1418 1390 1365 1354 1324 1302

0.11 0.09 0.14 0.08 0.13 0.08 0.27

1270 1249

0.06 0.40

1215 1164 1145 1126 1109 1062 1033 1000 955 906 892 884 823 760 696 672 643 631

0.15 0.05 0.17 0.11 0.10 0.12 0,29 0,11 0,13 0.09 0.75 0.17 0.21 0.05 0.07 0.03 0.04 0.04

1454 m 1413 s 1396m 1374 w 1356 m 1326 m 1299 m 1274 w 1249 m 1225 m

1147w 1128 m 1107 m 1062 s 1030 m 1014 m 954 m 908 w 892 w 860 w 827w 766 m 673 s 640 s

Frequency

Main % contributions to PED in symmetry coordinates

3430 3208 3153 3147 3109 2993 2991 2983 2964 2954 2938 2915 1686 1651 1643 1561 1546 1539 1466 1462 1449 1440 1406 1367 1339 1319 1288 1267 1237 1226 1204 1170 1160 1138 1136 1104 1039 1000 988 957

OH (100) NH (99) NHaas (83) NH3as (83) NH3ss (99) CH3as (55) CH3as (54) CH2as (99) CaHa2 (98) CH2ss (95) CaHal (95) CH3ss (99) CO (67) NH3ab (67) NH3ab (71) COOas (85) NH3sb (80) NHipb (31) CH3ab (86) CH3ab (89) CH2b (82) COOss (55) CH3sb (60) NCaH (43) CaCbl (31) CCaH (17) CbOH (48) NCaH (50) CH2t (35) CCaH (18) CaCbl (31) CH2w (18) CH2w (18) NH3r2 (39) NCa (17) NCa (27) CH3rl (43) CaCb2 (27) CH2r (30) CbO (72)

898 879 841 761 686 670 658 628

NCa (30) CHEr (35) NCa (24) C"Oob (31) COob (17) C"Oob (14) C"Oob (17) COob (16)

NH3as(16) NHaas(16) CHaas(44) CHaas(44)

CN (12) NH3ab(20) NH3ab(21) COOr (10)

CaCN (5) NH3r2(7)

CN (23) CH3r2(11) CH3rl (10) COOss (7) COOb (20) HCaCb (12) CCaH (13) CH2w(23) NHipb (17) CH2t (37) NHarl (13) CbOH (12) CH2t (13) CbOH (11) CaCb2 (14) CaCb2 (15) CCaH (11) CH3rl (17) NHar2 (9) NH3r2(16) NHarl (20) CaC (23) OCCb (5)

NH3sb (10)

CaC (9)

CaC (10) CCaH (7)

CH2b (8) CaCb2 (6)

CCaH (13) CaC (9)

HCaCb (7) NCaH (8)

CH3sb(9) NCa (9) CbOH (10) HCaCb (8) CaCbl (8) NCa (13) CH3r2(6) CHar2(14) OCCb (8) CH3r2(16) NHar2(13) CCaCb (7)

nCaCb (8) CH2w (7) NHipb (10) NCa (7) NCa (7) HCaCb (9) NI-Iarl (5) NH3rl (13) NH3rl (6) NH3rl (11) CH3r2 (lO)

CaC (12) CbOt (14) NCO (7) CbOt (12) CaC (13) COob (9) CbOt (13) CaCN (8)

CN (8) CaC (13) CaC (6) COOss (8) C"Oob (9) OCCb (9) COOb (7) CaCO (7)

CH3r2 (16) COOb (17) CaCb2 (15) COOb (14) CaCO (13) CbOt (14) COob (16) CbOt (12)

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Table 3 (continued) Observed

Calculated

IR band centre

Ramanband centre Ramanrelative intensity Frequency Main % contributions to PED in symmetry coordinates

618 s

616 598 525 466 452

0.05 0.02 0.07 0.06 0.05

375 317 293 263 229 214 172 136

0.08 0.03 0.10 0.02 0.07 0.06 0.11 0.09

527 m 461 m 420 w

591 583 523 470

CbOt (20) CNt (46) N'Cat (80) NCaCb (27)

CaC (16) NHob (26)

COOb(9)

COob (8)

COOr(16)

NCa (7)

NCO (5)

424 374 344 298 274 230 219 201 166 117 93 58 46 22

OCCb (25) NCO (10) NCaCb (25) CCaCb (34) CaCO (21) CCaCb (27) CaCbt (87) NCaC (29) CaC"t (60) CaCbt (32) CaCbt (40) NHob (38) CaCt (61) NCat (55)

COOr(24) NCaCb(23) C"Oob(9) OCCb(8) NCaCb(13) NCaC (12) NCaCb(10) CCaCb(7) NCaC(16) CaCN(11) NCaCb(14) CaCbt(13)

CaCbl (9) CCaCb(7) NCaC(9) CaC (5) CCaCb(10) CaC"t(9)

NCaC(18) NCaCb(8) CNCb(21) CNCb(26) NCaC(30) NCat (31) CaCt (28)

NCaCb(8)

COob(12) OCCb(8) CaC"t(13) CCaCb(6) COob(6)

CaCN(7) CaCN(5)

NHob(8)

ss: symmetric stretch; as: antisymmetric stretch; sb: symmetric bend; ab: antisymmetric bend; ipb: in-plane bend; ob: out-of-plane bend; r: rocking; w: wagging; t: twisting/torsion. For atom and valence coordinate notations see notes to Table 1. With force constants transferred from related molecules, the choice of geometry is extremely important, mainly for bending modes. In our case a " s t a n d a r d " geometry was chosen for the peptide link [15], whereas CH, carboxylate CO and ammonium NH + bond lengths were taken from the papers wherefrom force constants were transferred [ r ( C H ) = 1 . 0 7 for C~H, 1.09 for the methyl and methylene groups, r ( N ÷ H ) = 1.04, r ( O H ) = 0 . 9 6 , carboxylic r ( C O ) = 1 . 2 5 / k ] . Since experimental geometry was available for L-AIa-L-Ser only [ 16], we decided to use theoretical geometries for all dipeptides investigated. Thus, the dipeptides were constructed from a library of amino acids, the peptide geometry and terminal bond lengths were constrained at the above values, and the dihedral angles were varied to obtain an energy minimum by means of an M M 2 minimisation procedure [ 17]. Although experimental data are available for three molecules only, the normal frequencies and potential energy distributions are given for all the four possible species: L-AIa--L-Ser (Table 2), D-Ala-L-Ser (Table 3), L-Ser-L-AIa (Table 4) and D-Ser-L-Ala (Table 5). As experimental data, both infrared and Raman fre-

quencies are given (the latter followed by the intensity related to the strongest band of the spectrum).

4. Results and discussion With force fields transferred from related molecules and not fitted to the observed frequencies, no precise agreement can be expected between observed and calculated band positions. However, there should be a match between the calculated potential energy distribution, i.e. the character of the vibration, and the intensity of the observed band to which the calculated frequency is assigned. With this in mind, we first attempted to identify the most prominent features of the Raman spectra, and then to analyse the agreement of weaker bands. In this analysis, the main point is to see how the differences found in the experimental spectra are reproduced by the calculation, and how these differences can be used to characterize the conformation of the peptide.

G. Jalsovszky, S. Holly / Vibrational Spectroscopy 8 (1995) 279-291

286

Table 4 Observed and calculated frequencies of L-Ser-L-Ala Observed

Calculated

IR band centre

Raman band centre

Raman relative intensity

3315 s

3317

0.17

3105m 3002 w

3102 3002

0.02 0.44

2985w

2984 2958 2942 2923 2901 2876 1663 1647 1629 1599 1568 1540 1465 1456 1448 1403

0.90 0.79 1.00 0.34 0.19 0.40 0.43 0.06 0.23 0.06 0.07 0.04 0.23 0.49 0.12 0.26

1390 1360 1324 1287 1300 1271 1255

0.32 0.13 0.30 0.17 0.41 0.24 0.31

1167 1132 1116 1087 1063 1044 1006 947 930 897 887

0.28 0.11 0.29 0.13 0.33 0.30 0.16 0.23 0.18 0.31 0.11

758 712 673 646

0.12 0.11 0.10 0.05

2948 w

2875w 1671 vs 1650 sh 1630 s 1606 s 1571 s 1545 sh 1462 sh 1451m 1400s 1359 s 1320 sh 1299 s 1273 sh 1253 s 1168 s 1132w ll16w 1086w 1062 s 1044 w 1008 w 948m 929w 890m 830m 773 w 753m 715m 675 s 648m

Frequency

Main % contributions to PED in symmetry coordinates

3431 3208 3152 3150 3108 2992 2992 2984 2963 2952 2938 2915 FR 1679 1649 1646 1582 1557 1531 1465 1464 1450 1426 1396 1382 1351 1330 1278 1268 1241 1226 1205 1179 1162 1118 1085 1082 1065 1035 970 948 936 888 865 769 755 668 642 600

OH (100) NH (99) NH3as (71) NH3as (71) NH3ss (100) CH3as (96) CH3as (96) CH2as (99) Calla2 (98) CH2ss (93) Calla1 (93) CH3ss (99) CO (68) NH3ab (67) NHaab (68) COOas (82) NH3sb (50) NH3sb (37) CH3ab (86) CHaab (85) CH2b (91) COOss (56) CH3sb (68) CaCbl (26) NCaH (22) NCaH (25) HCaCb (29) NHipb (20) CH2t (79) NCa (24) CaCbl (14) HCaCb (30) CbOH (19) NH3r2 (66) CaCb2 (34) NCa (27) CH3r2 (41) NH3rl (40) CH2r (54) CH3rl (36) CbO (67) COOb (15) CaC (20) C"Oob (33) C"Oob (28) CbOt (19) CaC (21) COOr (27)

NH3as(29) NH3as(29)

CaHal (6) CH2ss(6)

CN (10) NH3ab(24) NH3ab(25) COOr (10) NHipb (16) YHipb (24) CH3r2(11) CH3rl (11)

CaCN (6) NH3r2(6) NH3rl (5)

COOb (22) NCaH (12) CH2w(21) CCaH (14) CCaH (16) CbOH (24) CH2w(8) CCaH (8) NH3rl (14) NH3rl (11) CH3sb(13) C H z w(17) NH3ab(6) CaC (14) CH2w (15) CaCb2 (10) CaCbl (17) CbOt (14) NCa (19) OCCb (9) COOss (10) CN (11) COob (14) COob (25) NCaC (8) COOb (21) NCa (8)

CaC (10) HCaCb (8) CbOH (19) CH3sb(9) NCaH (11) NCaH (20) CN (7)

CN (10) CN (19)

CH2w(11) CbOH (10) CCaH (11) NCa (10) CH2r (5) CHar1 (10) HCaCb (9) CH3rl (9) CbO (9) NHar2(10) CaC (15) NH3rl (6) CaCb2 (9) NCO (8) CCaCb (8) CbOt (8) COob (7) COob (10) CaCN (8)

CaC (7)

CCaH (6) NCaH (7) CCaH (10) CaC (7) CH3rl (8) NCa (9) NCaH (7) CCaH (7) CH3r2(8) CaCb2 (8) CH3ab(5) NCa (7) COOb (5) CaC (9) CaCb2 (7) COOb (7) CCaCb (8) COOb (7) C"Oob(7) NCa (8)

G. Jalsovszky, S. Holly / Vibrational Spectroscopy 8 (1995) 279-291

287

Table 4 (continued) Observed

Calculated

IR band centre

Raman band centre

Raman relative intensity

Frequency

Main % contributions to PED in symmetry coordinates

590 m

595 589 546 482 402 391 384 359 267 249 234 215 184 149 134

0.05 0.04 0.03 0.07 0.23 0.13 0.39 0.13 0.09 0.09 0.16 0.04 0.13 0.46 0.13

583 554 516 477 392 368 350 310 249 243 224 216 183 128 81 55 48 20

CbOt (33) CNt (55) N'Cat ( 81 ) OCCb (27) NCaCb (19) NCaCb (32) CCaCb (21) NCaCb (20) CCaCb (16) NCaC (49) CCaCb (16) CaCbt (89) CaC"t (52) CaCbt (40) CNCb (29) NHob (43) CaCt (43) CaCt (44)

480 m 401 s

CaC (14) NHob (26)

CH2r (13)

CaCO (8)

NCa (15) NHob (16) COOr (18) CaCO (12) NCaCb (16) NCaC (16) NCaCb (20) CaCbt (9)

NCaCb (10) CCaCb (11) CaCN (10) CNCb (12) CCaCb (14) COob (12) NCaCb (6) CNCb (9)

CaCbl (10) HCaCb (8) OCCb (8) C"Oob(10) OCCb (12) CCaCb (7)

CaCbt (16) CaC't (19) CaCbt (14) NCaC (20) NCat (36) NCat (43)

NCaC (7) CNCb (17) CaCN (11) COob (7) NHob (9) NHob (6)

COob (9)

NHob (5) NCaC (6) CCaCb (6)

ss: symmetric stretch; as: antisymmetric stretch; sb: symmetric bend; ab: antisymmetric bend; ipb: in-plane bend; ob: out-of-plane bend; r: rocking; w: wagging; t: twisting/torsion. For atom and valence coordinate notations see notes to Table 1.

4.1. The NH and OH stretching region Band frequencies in this region depend to a great extent on hydrogen bond effects, thus it cannot be (and has not been) expected that an analysis not taking into account these effects will reproduce NH and OH frequencies very well. Without referring to calculations, which predict 3208 c m - 1for the amide-A (NH stretch ) frequency for all the four isomers, it is interesting to note that L-Ser-L-Ala and D-Ala-L-Ser produce sharp bands at 3317 and 3301 cm-1, respectively, whereas L-Ala-L-Ser has a broad amide-A band centred at 3227 c m - 1. The same ambiguity holds for the OH and N ÷ H stretching bands.

2958 cm -a to the same mode of the alanine unit. Degenerate antisymmetric methyl stretching frequencies were calculated for L-Ser-L-Ala and D-Ser-L-AIa, whereas there is a (small) calculated splitting for L-Ala-L-Ser and D-Ala-L-Ser, indicating that the three hydrogens are equivalent in the former and situated in slightly different environments in the latter (the calculated antisymmetric methyl bending frequencies support the same conclusion). Fermi resonance between the overtone of this bending and symmetric methyl stretching is observable in the Raman spectra of all the three molecules: the bands in the 2885-2875 cm-1 region represent one component, the other can be found by deconvolution except for D-Ala-L-Ser.

4.2. The CH stretching region

4.3. The amide-I and amide-H regions

These vibrations give rise to the most prominent bands of the Raman spectrum (unlike infrared, dominated by the broad features due to the N÷H group). As one can see, there are characteristic differences between the spectra. On the basis of our calculations, the strongest band at 2940 c m - 1 is due to the Co~Hstretching of serine, and the next strongest one between 2968 and

The amide-I band is predicted by our calculations to occur in the 1688-1677 cm -1 range, the observed bands lie between 1680 and 1656 cm-1. There is no correlation between calculated and experimental frequencies, indicating that this frequency is influenced to a great extent by hydrogen bonding and intermolecular coupling effects [ 15].

288

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Table 5 Calculated frequencies and potential energy distributions of D-Ser-L-Ala Calculated frequency

Main % contributions to the PED in symmetry coordinates

3430 3208 3153 3148 3108 2992 2992 2984 2963 2953 2940 2916 1688 1652 1646 1560 1540 1521 1465 1463 1461 1443 1397 1359 1332 1300 1289 1256 1240 1220 1203 1193 1175 1125 1097 1086 1069 995 948 945 906 884 851 771 743 699 664 639 624 572 521 510

OH (100) NH (99)

Nn3as (51) NH3as (51) NHass (100) CH3as (61) CH3as (60) CH2as (99) CaHa2 (98) CH2ss (99) CaHal (99) CH3ss (99) CO (66) NH3ab (87) NH3ab (88) COOas (85) NH3sb (91 ) NHipb (29) CH3ab (83) CH3ab (83) CH2b (94) COOss (61) CH3sb (73) NCaH (19) NCaH (19) NCaH (34) CbOH (30) CaCbl (21) NHipb (21) CH2t (34) CH2w (26) HCaCb (26) CH2w (15) NH3rl (73) NH3r2 (40) CaCb2 (31) CH3r2 (40) NHar2 (21) CH3rl (36) CbO (62) CaC (13) CH2r (24) NCa (21) CaC (13) C"Oob (39) NHob (12) CbOt (24) CNt (21) CNt (20) COOr (30) N'Cat (70) N'Cat (15)

NH3as (48) NH3as (48) CH3as (39) CH3as (38)

CN (13) NHar2 (8) NH3rl (6) COOr (10)

CaCN (5)

CN (25) CH3r2 ( 11 ) CH3rl (11)

CaC (14)

NCO (7)

COOb (23) NCaH ( 10 ) CCaH (12) CCaH (17) CH2t (28) NCaH (24) CH2w (20) NCa (14) CbOH (15) CHEt (8) CH2w (15) NHipb (11) NH3ab (5) NCa (18) CaC (16) CaCb2 (19) NCa (18) NCa (17) OCCb (9) CH3r2 (12) NCa (15) CaCbl (9) C"Oob (8) CCaCb (8) OCCb (10) COob (24) COob (21) CaC (15) NCO (9)

CaC (11) HCaCb (9) CaCbl (10) CaCbl (12) HCaCb (12) CaCbl (10) CbOH (20) CCaH (8) NCa (12) CCaI-I (8) CH3sb (13) CN (8)

NHipb (10) CCaH (11) CbOH (11) CH2t (8) CCaH (13) CH3rl (6) CH2w (6) CaCb2 (8) CCaH (10) HCaCb (6)

CCaH (9) NHipb (6)

CH2r (6) CH3r2 (12) Cn3rl (9) CHzr (15) CaC (9)

NCaCb (6) CH3rl (12)

HCaCb (6) CCaH (7)

CaCbl (9)

CaC (7)

COOb (9) CbOt (11) CaC (8) CaCO (7) COOb (7) C"Oob (9) NCa (6) CbOt (15) COOb (14) CNt (6)

CNCb (8) CbO (7) NCa (8) CCaCb (7)

CN (8) COOb (5) COOb (7) COOb (5)

CaCb2 (7) COob (5)

CbOt (8) CCaCb (5) NHob (6) C"Oob (9) CaCN (6)

CNt (8)

COob (6)

NHob (9) CaC (6)

COob (6) CbOt (5)

COOr (14)

NCaCb (9)

CbOt (8)

OCCb (7)

NCa (7)

CaC (6)

CH2w (7) HCaCb (8) CN (6)

CbO (6) CbOH (6)

NCa (7) NCaH (8)

CaCb2 (6)

CH2r (6)

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Table 5 (continued) Calculated frequency

Main % contributions to the PED in symmetrycoordinates

394 379 323 308 268 235 217 216 183 124 92 58 42 20

NCaCb (42) CNt (16) NCaCb (27) NCaC (22) CCaCb (33) OCCb (23) CaCbt (94) NCaC (21) CaC't (49) CaCbt (29) CaCbt (46) NCaC (35) NCat (47) CaCt (46)

CaCN (10) NCaC (10) CCaCb (17) NCaCb (15) NCaCb (19) CCaCb (17)

NCO (7) NCaC (9) CCaCb (16) CaCO (11) CaCN (7) NCaCb (8)

COOr (6) OCCb (7) COOr (6) COOr (9) NCa (5) HCaCb (6)

NCaC (17) NCaC (9) CNCb (22) CCaCb (12) NHob (23) CaCt (43) NCat (44)

CaC"t (12) NCaCb (7) CaC"t (16) CNCb (11) COob (8)

CNCb (12) NCaC (6) CNt (6) CaCN (7) CNCb (6)

CaCO (7) C'Oob (5) NCaC (7)

CaC (7) CNCb (6)

CNCb (5) COob (10)

COOr (5)

CaCt (6)

ss: symmetric stretch; as: antisymmetric stretch; sb: symmetric bend; ab: antisymmetricbend; ipb: in-plane bend; ob: out-of-plane bend; r: rocking; w: wagging;t: twisting/torsion. For atom and valence coordinate notations see notes to Table 1. Vibrations containing a dominant PED contribution from NH in-plane bending were considered as amideII vibrations. In L-Ala-L-Ser, D-AIa-L-Ser and D-SerL-Ala the calculated frequencies are 1523, 1539 and 1521 cm -1, respectively, with distinct NH bending character. For L-Ser-L-AIa the 1531 and 1557 cm -1 normal modes contain amide-II contributions (mixed with NH 3 symmetric bend, which is the dominant contribution for both vibrations). The antisymmetric stretch of the carboxylate ion appears in this region as well. The calculated frequencies ( 1582-1560 c m - 1) are lower than the experimental frequencies ( 1606-1599 c m - ~), and there seems to be no correlation between observed and calculated frequencies, indicating again the role of non-bonded interactions. 4.4. The amide-III region

This is the most favoured region of Raman spectroscopic studies for at least two reasons. First, unlike amide-I and -II modes, amide-III vibrations produce relatively strong Raman scattering, and second, these bands can be related most directly to the conformational features of peptides. This sensitivity is ascribed to a very strong coupling of NH in-plane bending and methine CH deformation modes [ 1 - 4 ] . Diem et al. [1] constructed a coupled oscillator model for the interpretation of this phenomenon. In our case this model is

inapplicable, as it pertains to peptides composed of identical subunits (Ala-Ala in their case). In a more recent paper [4] the same research group reported detailed vibrational analysis on the subject, confirming the above conclusions. In turn, Cheam and Krimm [ 18], using a scaled ab initio force field for the alanine dipeptide, concluded that much of the conformational sensitivity in the amide-III region is due to the CH bending (!) component, and one should consider all amide and CH bending modes when trying to correlate amide-III frequencies with conformation. Ab initio calculations have also shown that the interaction force constants between these modes are not too high, i.e. coupling is mainly kinetic [ 18]. Thus, it is not entirely hopeless to use simplified empirical valence force fields to predict the variation of the frequencies and vibrational modes with molecular conformation. With Ser-Ala and Ala-Ser epimers the situation is even more complex. First, the coupling mechanism is different since the CH groups are not identical. Second, in addition to the methyl deformation of alanine the bands due to the methylene deformation (and COH bending) of serine may also appear in this region. Thus, the results of normal coordinate analysis can be taken as a guide for the separation of the latter deformations from "true amide-III" modes. In this respect one should consider all normal modes to which the PED contribution of NH bend exceeds 4% [9].

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For all molecules we have found four vibrational modes in this region with NH or CH deformation contributions. The first one in the 1367-1351 cm-1 range is predominantly CH bending in character (with a 10% NH contribution for D-Ser-L-Ala). The second vibration appears between 1330 and 1313 cm-1. In o-AlaL-Ser this vibration contains an NH bend contribution, in the others it is practically pure CH deformation. The calculated frequencies of these modes are in a good agreement with the experiment (mean deviation less than 5 cm- 1). The calculated frequencies of the third mode are 1281, 1267 and 1278 c m - 1 for L-AIa-L-Ser, D-AIa--LSer and L-Ser-L-Ala, and the vibrations show strong CH deformation character. The corresponding experimental bands can be found at 1267, 1274 and 1287 c m - 1, i.e. the sequence is not reproduced although the deviations do not exceed 14 c m - 1. The fourth "amide-III" mode, varying between 1268 and 1226 cm 1, shows the strongest NH bend character. The 1226 cm- 1vibration of D-Ala-L-Ser has the least NH bend contribution, which might be the reason for the lower frequency. The corresponding experimental frequencies (the strongest Raman bands in this region) appear between 1300 and 1249 cm-1. The conformational sensitivity of this mode is the most prominent, and in this case it is equally reflected in the observed and calculated frequencies. 4.5. The region below 1200 cm -1

The strongest band in this region appears at 889 cm-1 for L-AIa-L-Ser and at 892 cm-1 for D-AIa--LSer. The corresponding calculated frequencies are 889 and 898 c m - 1, with PEDs showing a strong mixing of NC stretch and methyl rocking. L-Ser-L-Ala has a band of medium intensity in this region, at 897 cm- 1, which turns out to be a carboxylate bending mode. The predicted frequency of methyl rocking in this molecule is 948 cm-1, and indeed at 947 cm-1 there is a band of medium intensity. The change in intensity, together with the calculated PED contributions indicates that in this molecule the coupling between NC stretch and methyl rocking is much weaker.

5. Conclusions Normal coordinate calculations using empirical force fields transferred from related molecules can be

used to produce differences between the vibrational normal frequencies of epimeric dipeptides of different conformation. For certain vibrational modes differences between experimental spectra are reproduced sufficiently well, for other modes this is not the case. It can be stated that the major features of CH stretching and bending modes could be accounted for. Conformation sensitive"amide-III" modes proved to be combinations of NH in-plane-bending and methine CH deformation vibrations, the calculated frequencies of these modes being significantly different for the four molecules investigated. In order to improve agreement between experimental and calculated differences the calculations should be extended to crystals in which intermolecular H-bonds and dipole-dipole couplings can be considered explicitly and/or the calculated frequencies should be compared to data obtained from solution spectra.

Acknowledgements The authors are indebted to Dr. Tom Sundius (University of Helsinki) for the MOLVIB program and for fruitful discussions concerning the application of the program and to Dr. Istvfin Sch6n (Chemical Works of Gedeon Richter Ltd.) for the samples. This research was supported by the National Scientific Research Fund (OTKA) under contract number 1772.

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[ 16] P.G. Jones, L. Falvello and O. Kennard, Acta Crystallogr., B34 (1978) 1939. [17] U. Burkert and N.L. Allinger, Molecular Mechanics, ACS Monograph, Vol. 177, American Chemical Society, Washington, DC, 1982. [18] T.C. Cheam and S. Krimm, J. Mol. Struct. (Theochem), 188 (1989) 15.