Journal of Molecular Structure, 3 18 (1994) 1 - 13 ~22-28~194~$07.00 0 1994 - Elsevier Science B.V. All rights reserved

Infrared matrix isolation studies Molecular structure of proline

of amino acids.

I.D. Reva*, S.G. Stepanian, A.M. Plokhotnichenko, E.D. Radchenko, G.G. Sheina, Yu.P. Blagoi

Institute for Low Temperature Physics and Engineering, Ukrainian Academy of Sciences, 47, Lenin Avenue, Kharkov, 310164, Ukraine

(Received 8 April 1993)

Abstract

IR spectra of proline and deuteroproline isolated in low temperature Ar matrixes have been obtained. It is shown that in the isolated state proline exists in the molecular form. The spectra are interpreted using normal coordinate analysis. It is found that band splitting in the spectra is caused by the occurrence of two proline conformations. The structures of these conformations differ in the position of the COOH group with respect to pyrrolidine ring. The conformations are found to be stabilized by the intramolecular hydrogen bond. It has been shown that conformational equilibrium results in the splitting of most bands in the IR spectra of proline and deuteroproline. This splitting is maximal for C=O stretching bands (23cm-‘). The structure and relative energies of the conformations are determined by the AM1 quantum chemical method.

Introduction

The ~onfo~ational mobility of the complex organic molecules is one of the most important questions in studies of molecular structure. Natural amino acids are of special interest because their structure and conformational mobility deter- mine the variety and functional specificity of pro- teins as well as of natural and synthetic polypeptides. Although there is extensive struc- tural information about amino acids, it seems impossible to use it for solving the tasks of compu- ter simulation of peptide chain structures. The rea- son is that this information was mainly obtained for the condensed state (crystals and solutions) where molecules are in the zwitterion form. As shown below, this form is not characteristic for

* Corresponding author.

SSDI 0022-2860(93)07907-E

the polypeptide chains:

0 II

(H,N)+-CH-(COO)- H,N-CH-C-OH

k k

zwitterion molecular form

0 II P 0

II *H-CH-C-NH-CH-C-NH-CH-C-

I RI

peptide chain

where R is an amino acid residue. For this reason, the study of the structural pecu-

liarities of the molecular forms of amino acids and their conformers, and their ability to participate in inter- and intramolecular bonding is of the greatest interest. A new approach to such studies is based

2 I.D. Reva et al./J. Mol. Strut. 318 (1994) I-13

on theoretical [l] and experimental [2] works which reveal that amino acids, when isolated (gas phase, noble gas matrixes), should be in the molecular, but not the zwitterion, form. The significant conforma- tional mobility was predicted theoretically [3-71 even for the simplest amino acids - glycine, alanine and serine - and was found experi- mentally for glycine [&lo]. For example, Jensen and Gordon [7] considered 16 conformations of glycine, the smallest of amino acids. The conforma- tional variety of amino acids is determined to a large extent by mutual effects of atoms of amino and carboxylic groups capable of intramolecular H-bonding. Among the natural amino acids the exception is proline, whose imino group is fixed rigidly in the pyrrolidine ring, and the conforma- tional mobility of the N-H bond with respect to the carboxyl group is limited. However, the pyrro- lidine cycle can restrict the mobility of the car- boxylic fragment in its own right. Perhaps this is responsible for the specific function of proline in the ,& and r-turns of polypeptide chains [l 11.

Theoretical and experimental studies on the struc- ture of proline [12-151 predict a simpler set of conformers compared to other amino acids. There- fore, proline was chosen as one of the first objects for investigation in the conducted cycle of studies on the structure of isolated amino acids. The spectra of the molecular form of amino acids are obtained by matrix isolation which, combined with IR spectroscopy, allows one to study the finest structural peculiarities of the molecules.

Experimental

The liquid helium cryostat of filling type used for matrix IR spectroscopy is described in ref. 16. A modernized Specord IR-75 spectrophotometer (Carl Zeiss, Jena) was used to record IR spectra. The spectrophotometer was protected by dry nitro- gen flow to keep out atmospheric water and CO* vapours. The sample was prepared by simul- taneous deposition of the matrix gas and the com- pound to be investigated was deposited onto the cooled CsI window. Argon (99.99% purity) was

used as a matrix gas, which was cooled down to the liquid nitrogen temperature before deposition. This provided a stable vapour pressure over the solid Ar and a constant matrix gas flow during the deposition. Low temperature quartz micro- balances permit the required concentrations of the compound in the matrix to be achieved with an accuracy of f 3%. Sample preparation, includ- ing the selection and stabilization of the deposition conditions, the stabilization of the optical window temperature and the control of quartz micro- balances, as well as recording of spectra, were carried out using the original program set for an IBM AT computer. To increase the signal-to-noise ratio, spectra were accumulated in different fre- quency ranges. Some of the bands having complex spectral structures were divided into Gauss com- ponents, so that the frequency and intensity of each component could be determined.

The conditions of sample preparation were the following. The CsI window temperature was kept at 17 K during the deposition (this provided the formation of the matrix with minimum scattering and sufficient rigidity). The sample was then cooled down to 12K to prevent overheating by the spectrometer beam during recording of spectra. DL-proline (“Reanal”) was purified further by driving off crystalline hydrate water and easily volatile impurities by means of a one-hour exposure in a Knudsen cell at 140°C. It should be noted that a small CO2 impurity was nevertheless present in the samples. The sublimation tempera- ture was in the range from 145 to 157°C. To pre- vent evaporation of the surface layer of the Ar matrix during annealing, the matrix was protected by a Kr film deposited over it. Deuteration was performed by double recrystallization of proline from a heavy water solution.

Calculations

The semiempirical quantum chemical AM1 method [17] was used to determine the structure and relative energies of proline conformations.

I.D. Reva et al&J. Mol. Strut. 318 (1994) l-13

This method is effective in studying H-bonded systems. Calculations were made with complete optimization of all geometric parameters. Point- by-point optimization was carried out for two torsion angles, N-C,-C=O and O=C-O-H. Two types of possible conformation were con- sidered. In the first case the carboxylic group was set on the same side of the ring as N-H bond (cis conformations). In the second case the carboxyl group and N-H bond were situated on different sides of the ring (trans conformations). In addition to molecular structures of proline, the zwitterion form was also studied. The formation energies of different H-bonded proline dimers were calculated with complete geometry optimization.

Frequencies and potential energy distributions (PEDs) of the normal vibrations were calculated with the empirical force constants for two most stable conformations of the proline and deutero- proline using the program set developed for an IBM personal computer [18].

Results and discussion

Structure of proline conformers

Table 1 gives the relative energies of six calcu- lated conformers of the proline molecular form and of zwitterion. As can be seen from the table, in the isolated state proline must exhibit only the mole- cular form, which is much more stable than the zwitterion one. As was noted, molecular confor- mers of proline can be divided into two groups

Table 1 Relative energies of the proline conformations I-IV (kcal mol-‘)a

Method I II III IV

AM1 (cis) 0.0 0.5 5.1 37.9 (trans) 4.0 3.3 9.5 -

ST0 3Gb 0.0 3.1 ST0 6-31Gb 0.0 4.5

a Both AM 1 and ab initio calculations give the planar struc- ture of the ring in proline. b From ref. 13.

I

II IV

Fig. 1. Calculated structures of the most stable proline con- formers. I-III, molecular forms; IV, zwitterion. Nitrogen atoms are marked 0.

according to the cis or trans positions of the N-H bond and the carboxyl group with respect to the imaginary plane of the pyrrolidine ring. These calculations predict that cis conformers are

0 60 120 160 240 300 3t 4j n ’ n ’ ’ ’ ’ ’ ’ ’ ’

II II II I ’ I ’ 0 60 120 180 240 300 3

0

i0

Dihedral angle, degrees

Fig. 2. The variation of the potential energy of proline with the N-C,-C=O (a) and O=C-O-H (b) dihedral angles.

4 I.D. Reva ef al./J. Mol. Strucr. 318 (1994) I-13

Table 2 Optimized geometries of the proline conformations I-IV

Parameter I II III IV

N-C, 1.464 1.462 1.462

G-C, 1.547 1.547 1.547

CT-C, 1.522 1.522 1.522

C,-C, 1.537 1.537 1.537 C6-N 1.461 1.461 1.460 c&-c 1.515 1.515 1.519 c=o 1.234 1.232 1.230 c-o 1.361 1.363 1.365 N-H 1.004 1.003 1.005 O-H 0.972 0.912 0.968

Angles (deg) H-N-C, N-C&-C c,-c=o o=c-0 N-C&+ c,-c,-c, C*-c,-Cb C-O-H N-C,-C=O H-N-C,-C H-N-C,+ N-C,-CD-C, c,-c~-c,-c~ O=C-O-H

111.0 111.3 110.8 113.7 116.9 114.6 129.0 121.2 127.2 117.1 117.2 114.2 109.3 109.2 109.3 106.0 106.0 106.0 106.2 106.1 106.2 109.6 109.6 109.6

-15.0 160.0 -5.1 0.4 0.3 0.4

237.1 235.5 236.7 0.1 0.6 0.2 0.1 0.5 0.0 0.0 0.0 180.0

1.505 1.527 1.524 1.532 1.491 1.601 1.260 1.241 1.048 -

105.6 109.5 114.7 130.0 108.3 107.3 107.4 _

5.0 1.5

239.9 0.7

-0.4 -

a Values of C-H bond lengths are 1.12-l .13 A. Values of C-C-H bond angles are 109-I 11”.

more stable than the corresponding trans con- formers (Table 1). Structures of three cis confor- mers as well as the structure of the ~itterion are given in Fig. 1. As the figure shows, conformers differ due to rotation of the carboxylic and hydro- xylic groups around the C,-C and C-O bonds, respectively. The energetically close conformers I and II. are preferred. The difference in energy is 0.5 kcalmol-‘. Each of the conformers is stabilized by the intramolecular interaction between the proton of the imino group and the corresponding oxygen of the carboxyl group. The energy of the barrier between conformations I and II is 1.7 kcal mol-’ (Fig. 2). The calculated value suggests the absence of free rotation of the carboxyl group about the C,-C bond of proline, since under experimental conditions the energy

---‘----’ 1850 1800 1'750 I DO

2’. cm -I V, cm -1

Fig. 3. C=O stretching region: (a) proline in Ar matrix, matrix dilution = l/l 150, (b) deuteroproline in Ar matrix, matrix dilution = l/750.

E = kT is 0.85 kcal mol-i. This reasoning shows that the vapour phase of proline must consist mainly of two conformations - I and II.

X-ray structure analysis of crystalline proline showed that the pyrrolidine ring structure is not planar. The deviation of the C, atom from the

a

O.QO , I I, I I I, ‘ I I, I $ I ( , I,

3709 3500 3300 3100 2900 2

v, cm -1

b

0.05-

0.00, 1 , , I,, I, r, I I I 3000 2800 2600 24

v, cm -1

DO

Fig. 4. X-H (X-D) stretching region: (a) proline, matrix dilution = l/1150; (b) deuteroproline, matrix dilution = l/750.

1.D. Reva et al.lJ. Mol. Struct. 318 (1994) 1-13

T

2/, cm-’

Fig. 5. 1500-400 cm-’ region of the IR spectrum of proline in Ar matrix, matrix dilution = 1 /1150.

plane Cb-N-C,+ in this structure is 0.6A [19]. Therefore we concentrated chiefly on the confor-

mational structure of the pyrrolidine ring. AM1 calculations reveal an almost planar structure of the ring in all the conformations of proline (Table 2). A similar result was obtained from non- empirical ST0 3G and ST0 4-31G calculations

[131.

Interpretation of IR spectra

Similarly to the previously studied glycine [lo] and leucine [20], matrix isolated proline and

deuteroproline occur in the molecular form. This is confirmed by the presence of characteristic bands of C=O stretching vibrations in the range from 1790 to 1760cm-l (Fig. 3) as well as those of OH and OD stretching vibrations with frequencies of 3559 and 2626cm-‘, respectively (Fig. 4). Besides this, the spectra recorded do not exhibit the bands inherent in the spectrum of the proline zwitterion (1611 cm-’ (COO- asym. str.) and 1598cm’

(NH; sciss.) [21]). This means that the matrix does not contain proline molecules in the zwitter- ion form. As follows from calculations of con- former energies, the trapped proline is a mixture

0.15

0.10

0.05

Fig. 6. 1500-400cm-’ region of the IR spectrum of deuteroproline in Ar matrix, matrix dilution = l/750.

6 AL). Reva et al./.& Mol. Stnrct. 318 (1994) I-13

Table 3 Observed and calculated IR spectra of prolinea

Observedb

(“Cd) A Int.

Calculated

Conformer I II

PED

3559 3545 3393 3369 3025 2984 2959

{ 2934 2916 2885

1 2865 2846

(

1795 1789 1781 1766 1488

1 1463 1451 1412 1405

1 1384 1381 1364

0.16 0.07 0.03 0.02 0.03 0.12 0.09 0.05 0.06 0.11 0.04 0.03 sh 0.45 sh 0.52 0.02 0.03 0.05 0.09 0.35 0.56 0.46 0.08

1.72 0.97 0.54 0.50 5.20 2.10 1.98 1.10 1.24 2.28 0.48 0.71

7.22 1784 C=O str (71), C-O str (12)

6.03 0.05 0.29 0.52 2.28 1.40 6.23

1350 1330 1320

1 1294 1286

0.06 0.03 0.04 sh 0.06

1247 0.06

0.83

0.54 0.10 0.12

0.32

0.10

{ 1210 1206

1 1150 1142 1109 1105 1072 1021

0.04 0.05 sh 0.17 0.35 0.35 0.06 0.02

0.13 0.12

2.21 2.77 2.94 0.26 0.12

976 0.02 0.04

3563 3563

3383

O-H str (97)

3379 N-H str (91) N-H str (91)

2993 2993 2957 2957 2929 2929 2894 2894 2890 2890 2837 2837 2828 2831

very broad band C.,Hz str as (60), CBH, str as (30) CpH2 str as @I), C,H2 str as (30) C,H2 str s (74), CBH2 str s (14) C6H2 str as (80), C,H2 str as (17) CpHz str s (78), C,H2 str s (14) CaH2 str s (83) C,-H str (74)

1769 1487 1464 1447

1487 1465 1448 1421

1415

1391

C-O str (72), C-O str (12) C,H2 sciss (49), CBHz sciss (25) CpHz s&s (52), C,H2 sciss (21) CsH2 sciss (55)

I C-O str (21), C-N str (15), C-C str (ll), C,-H bend (12)

N-H bend (48), C,-H bend (14)

1369 C-N str (25), N-H bend (22), CHS twist (20)

1333 1302

CH2 twist (49) CH? twist (47)

1273 1272 1262 1264 1236 1237

1234 1235 1232 1232

1217 1203 1163

1218 1203

1158 1108

1103 1097 1016

1094 1011

CH2 twist (42), C-O str (18) C,-H bend (26), C-N str (10) CH2 wag (31), ring str (28), CH2 twist (18) CH2 wag (57), ring str (35) CH2 wag (34), ring str (22), C,-H bend (14) CHI wag (68), ring str (10) ring str (41), CH, wag (34) C-O str (33), O-H bend (21) C-O str (31), O-H bend (19) O-H bend (37), C-O str (29) O-H bend (39), C-O str (28) CH2 wag (37), CH2 rock (10) ring str (40), CH, wag (20), C,-C str (9)

987 983 ring str (51), CH2 wag (15) 984 974 CHz wag (251, C,-C str (17)

I.D. Reva et al./J. Mol. Strucl. 318 (1994) I-13

Table 3 (continued)

Observedb

A Int.

Calculated

Conformer I II

PED

955 950 916 907

t 899 896

1 884 876 853 844 822

0.02 0.13 0.05 0.23 0.04 0.16 0.04 0.17 0.12 0.51 0.15 0.46 0.16 0.65 0.06 0.42 0.04 0.35 0.04 0.31 0.02 0.06

957 943

ring str (34), N-H bend (12) ring str (29), N-H bend (14)

18)

18)

897 ring breathing (53), CH2 rock

891

838 816

769

858 847

791

ring breathing (54), CH2 rock ring str (27), C,-C str (11) CH2 rock (62) CHr rock (60) ring str (22), ring bend (13), c,-c str (10) N-H bend (29), CHI rock (25) N-H bend (29), CH2 rock (28)

782 0.03 0.46 768 0.09 0.60 751 0.05 0.18 726 0.11 0.85 717

671

ring str (22), ring bend (19), N-H bend (10) ring str (21), ring bend (17), N-H bend (11) O-H tor (20), ring bend (15), C-C=0 bend (13) O-H tor (34}, ring bend (18), C,-C bend (10)

713 0.03 0.58 705

675 0.03 0.26

668 0.04 0.18 663

663

f 628 621

{ 603 597

t 584 576

[ 569 564 560 551

t 546 541

0.02 0.04 0.07 0.04 0.03 sh 0.05 0.03 0.05 0.04 0.04 0.11 0.21

0.09 0.43 0.45 0.24 0.21

631 632

605 597

O=C-0 bend (28), ring bend (13), c=o oop (12) ring bend (44)

0.52 0.16 0.15 0.13 0.17 0.45 0.81

554

431 339 137 94

544

457 319 138 97

ring bend (33) COOH tor (18)

C-C=0 bend (15), C=O oop (2 l), ring bend (12) ring oop (37) C=O oop (22) ring oop (32), O-C=0 bend (19) ring oop (28), COOH tor (24) C&-C oop (55) ring oop (17)

a A, relative peak intensity; Int., relative integral intensity; sh, band shoulder; braces indicate merged bands. b In Ar matrix at 1 I IC (matrix dilution = l/l 150). Abbreviations: as, antisymmet~c; s, symmetric; str, stre~hing; bend, bending; oop, out-of-plane; sciss, scissoring; twist, twisting; wag, wagging; rock, rocking.

8

Table 4

I.D. Revu et al./J. Mol. Struct. 318 (1994) l-13

Observed and calculated IR spectra of deuteroproline’

Observedb

A Int. .

Calculated

Conformer I II

PED

2984 0.12 2.66 2956 0.09 2.11 2934 0.05 0.78 2919 0.04 0.83 2884 0.10 2.56 2857 0.02 0.26 2840 0.03 0.51 2626 0.13 0.74 2617 0.08 0.73 2519 0.01 0.14 2499 0.01 0.22 1779 0.50 6.80 1756 0.39 5.99 1487 0.01 0.02 1461 0.02 0.33 1452 0.04 0.31 1387 0.07 2.39

1344 0.03 0.68 1323 0.11 0.76 1311 0.09 0.46 1301 0.06 0.77

I

1292 1287 1281 1276 1271 1257 1238

0.07 0.43 0.06 0.30 0.12 0.94 0.07 0.26 0.16 1.06 0.06 1.02 0.06 0.55

I

1227

1222

0.16 1.92

0.16 1225

1196 0.03 0.22 1188 0.03 0.20 1178 0.05 0.37 1168 0.08 0.52

1155 0.06 0.81

1139 0.06 1123 0.10 1118 sh 1100 0.05

0.76 1.47

0.34 1106

2993 2957 2929 2895 2890 2837 2828 2628

2993 2957 2929 2895 2890 2838 2828 2628

2503

C,H2 str as (60), C$Iz str as (30) CBHz str as (58), C,Hz str as (30) C,Ht str s (75), C,H, str s (13) CdH2 str as (80), C,HI str as (14) CpH, str s (77), C,Hz str s (14) CbH2 str s (83) C,H str (74) O-D str (93)

2496 N-D str (83) N-D str (83)

1781 1766 1487 1464 1448 1394

1487 1464 1448 1390

1354 1329

1300

C=O str (73), C-O str (13) C=O str (70), C-O str (13) C,Hs sciss (41), CBH2 sciss (23) C0H2 sciss (46) C,H2 s&s (19) CsH2 sciss (54) C-O str (22), C-N str (16), C,-C str (14) C,-H bend (10) C,-H bend (23), C-N str (20) C-N str (30), C&-H bend (26)

ring str (33), CHs twist (27), C-O str (19)

1292 CH2 twist (31), ring str (30)

1280 C,-H bend (28), CH2 twist (24)

1260 CHs twist (42), C,-H bend (13)

1237 1234 1231

1237 1234 1228

1223

ring str (47), CH2 wag (32) CH2 wag (45), ring str (41) C-O str (26), CHz wag (26), ring str (16) CH2 wag (40), ring str (25) C,-H bend (16)

1186 1181

1163

CH2 wag (65), ring str (15) CHs wag (67), ring str (10) ring str (37) CH, wag (25) C-H bend (17) ring str (34), CHZ wag (22), C,-H bend (12)

1157

1115 N-D bend (31) CH2 wag (30)

N-D bend (33) CH2 wag (18), CHI rock (15)

I.D. Reva et al/J. Mol. Struct. 318 (1994) 1-13

Table 4 (continued)

Observedb

A Int.

Calculated

Conformer I II

PED

9

1087 0.02 0.30

1059 0.01 0.12

1038 1019

1 991 984 966 961

0.02 0.16 0.01 0.05 0.15 1.61 0.07 0.32 0.01 0.04 0.01 0.04

1061

1022 996

952

949 0.01 0.07 936 0.02 0.13 927 0.02 0.18

911 0.02 0.31 894 0.05 0.50 876 0.03 0.30 858 0.02 0.15

909

841

816 0.01 0.14 809 763 0.01 0.04 769

754

1 729 722 690 672 662

1 656 646

0.01 0.03 0.02 0.02 0.02 0.03 sh 0.09

0.06 0.19 0.14 0.52 0.30 0.19

693

0.92 655

638 615 0.04

0.02 0.02 0.03 0.02 0.02

0.02 0.02 0.01 0.02

0.64 622

604 597 589 579 570

0.12 0.18 0.22 0.10 0.16

613 598

584

55s 552 547 533

0.10 0.06 0.04 0.09

1067

1028

994

ring str (43) C,--H bend (16), N-D bend (13) ring str (44), N-D bend (15), C, -H bend (15) ring str (SO), CHs wag (15) ring str (52), CH2 wag (11) CHs wag (41), O-D bend (32)

C,-C str (23), ring str (23), CHs wag (10)

926

913

839

810

752

701

590

577

ring str (28), N-D bend (18), CH2 wag (16) ring breathing (38), CH2 rock (25) ring breathing (46), CH2 rock (22)

ring str (34), CH2 rock (IS), O-D bend (10) CH2 rock (40), N-D bend (14) ring str (29), CH2 rock (15), C,-C str (15) ring str (32), CHz rock (18)

ring bend (27), ring str (21) ring bend (29), ring str (20)

N-D bend (24), C=O bend (14), ring bend (18) ring bend (3S), N-D bend (16) O-C=0 bend (25), ring bend (18), COOH tor (11) O-C=0 bend (26), ring bend (25) ring bend (44), N-D bend (17) ring bend (46) ring bend (42) C-O bend (21), N-D bend (17), C&-C bend (11)

10 I.D. Reva et 51./J. Mol. Stwct. 318 (I9941 I-13

Tabie 4 (continued)

Observedb Calculated

A Int. Conformer I II

PED

527 sh 522 0.06 518 sh 510 0.03 450 0.03 430 0.10

0.56

0.08 0.87 0.77

514

435 425 316

136

91

519

452

432 296

136

97

C-C=0 bend (32), ring bend (16) C-C=0 bend (23), C=O oop (13)

O-D tor (35), O-C=0 bend (15) O-D tor (30), O-C=0 bend (16) ring oop (16), C-C=0 bend (16) ring oop (ZO), C,-C bend (ZO), O-C=0 bend (11) ring oop (27), COOD tor (23), c,-c oop (13) C,-C oop (46), ring oop (16)

a A, relative peak intensity; Int. relative integral intensity; sh, band shoulder; braces indicate merged bands. b In Ar matrix at 11 K (matrix dilution = l/500). Abbreviations: as, antisymmetric; s, symmetric; str, stretching; bend, bending; oop, out-of-plane; sciss, scissoring; twist, twisting; wag, wagging; rock, rocking.

of at least two conformers. The IR spectra of these conformers must be different. Differences should arise primarily between frequencies of vibrations of those fragments which are involved differently in intramolecular interactions, such as C=O, C-O and N-H groups. In conformations I and II such groups form different intramolecular H-bonds (Fig. l), N-H --.0--C (I) and N-H . ..O-C (II). In connection with this the analysis of IR spectroscopic regions containing bands of confor- mationally sensitive vibrations is of particular interest.

C=O modes

There are two intensive bands (1789 and 1766cm-‘) in the C=O stretching region of the proline spectrum (Fig. 3). These bands correspond to the C=O stretching vibrations of two proline conformers. The high-frequency band refers to the stretching vibration of the C=O bond non- interacting with the N-H one in conformation II. The formation of the weak intramolecular H-bond in conformation I leads to the low-frequency shift

of the C=O stretching band by 23 cm-‘. The com- pletely similar situation is observed in the case of deuteroproline. Its IR spectrum has bands at 1779 and 1756cm-‘. The value of the low frequency shift of C=O stretching vibrations is equal to that of the proline spectrum, i.e. 23 cm-‘. The assign- ment of these bands is the same as for proline. The analysis of the intensities of C=O stretching vibra- tions of the two conformations showed the com- parable quantities of I and II conformers present in the frozen vapour phase of proline. The integral intensity ratio is 1.19 for proline (Z~~s~/Z,~~) and 1.13 for deuteroproline (Z,~~~/Z~~~~). The bands at 628, 621 cm-t (OCO bending), 541 cm-’ (CC0 bending) in the proline spectrum and 615, 604cm-’ (OCO bending), 522 cm-’ (CC0 bend- ing) in the deuteroproline spectrum correspond to C=O bending vibrations (Figs. 5 and 6). The OCO and CC0 bending modes can be sensitive to con- fo~ational equilib~um in proline. We examined this region of the spectrum using the division of bands into Gauss components and revealed that the bands of the OCO and CC0 bending vibrations have a doublet structure (Tables 3 and 4).

Thus, the matrix contains approximately equal

I.D. Reva et al./J. Mol. Struck 318 (1994) 1-13 11

A

~, I

_- A 2

0.1 _ -_--- --

3

~

o*__ - ---

3600 3550 3500 -1

v, cm

Fig. 7. Changes in the IR spectrum of proline on matrix annealing: (1) initial spectrum; (2) spectrum after matrix annealing at 30K for one hour: (3) difference spectrum (3 = 2 - 1). Matrix dilution = l/l 150.

proportions of the two proline conformations. This result agrees well with quantum chemical calculations of relative energies by the AM1 method (Table 1). In fact, the difference between the conformer energies AE = EII - .EI is 0.5 kcalmol-‘. However, the energy k7’ at evaporation temperature is 0.85 kcal mol-’ . This means that the two proline conformations must be present in the vapour phase in the ratio of 1: 1.8 in terms of the Boltzman distribution of the molecules throughout the available energy levels.

O-H mod&

In the region of OH (OD) stretching vibrations the bands 3559crn-’ in the proline spectrum and 2626 cm-’ in the deuteroproline spectrum are observed (Fig. 4). Deuteration isotopic shift Gus = 1.355 of the OH stretching vibration is characteristic of the OH-bond in carboxylic groups, being 1.356 for formic acid [22] and 1.354 for glycine [lo]. As seen in Fig. 4, the bands of the OH and OD stretching vibrations are split, by 14 and 9 cm-’ respectively. To under- stand the nature of this splitting, we annealed the matrix at 30 K for an hour. Fig. 7 presents spectra of proline before and after annealing of the matrix

and also the differential spectrum. As can be seen from Fig. 7, the intensities of the components are redistributed. Since at the annealing temperature (30K) rotation of the carbonyl group about the C,-C bond in proline is most improbable (the barrier between the two conformations is about 1.7 kcal mol-‘), we believe that the annealing- induced redistribution of the intensities represents a site effect. A similar result is observed in the spectrum of deuteroproline in the range of the OD stretching vibrations.

In the OH-bending region (1200-l 100 cm-‘) two intense bands, at 1142 and 1105 cm-‘, are observed. As Tables 3 and 4 show, the PEDs (in percent) of these vibrations are complex. The energetic contributions of the C-O bond and the COH angle are approximately equai. This result is in good agreement with calculations of the vibrational spectra of formic acid [22]. The IR s~ctrum of formic acid in the Ne matrix has two complex bands in this region, at 1217 cm-’ (PED: 37% OH stretching, 34% COH bending) and 1105cm-’ (PED: 43% COH bending, 39% OH stretching). The PEDs of OD bending vibrations in deuteroproline are essentially different from those in proline. The intense 991 cm-’ band corresponds to the OD bending vibration, the energy contribution of the COD angle being significant (Table 4). This time the band at 1227 cm-’ is assigned to the C-O stretching vibration. In addition to the C-O bond, CCH angles contribute considerably to this vibration. The bands at 675 and 668cm-’ (Fig. 5) in the proline spectrum and at 450 and 43Ocm-* (Fig. 6) in the deuteroproline spectrum correspond to OH (OD) torsion vibrations of the two proline confo~ations. As seen from Tables 3 and 4, these vibrations have different PEDs, which is the cause of the splitting. The differences in the halfwidths and the relative intensities between the bands of the OH and OD torsion vibrations are also due to the differences in the PEDs of these vibrations. The OH torsion vibrations are mixed with the ring bending vibrations and the OD torsion vibrations with the OCO bending vibrations.

12 I.D. Reva et al./J. Mol. Struct. 318 (1994) 1-13

N-H modes

The region of the imino group stretching vibra- tion is shown in Fig. 4. In the case of glycine and ethylamine [lo] we showed that N-H stretching vibrations were of very weak intensity in the IR spectra of these matrix isolated compounds. In spectra of both proline and deuteroproline the bands of the N-H and N-D stretching vibrations are very weak. Weak split bands are registered in the IR spectra of proline and deuteroproline, which are assigned to the N-H (N-D) stretching vibra- tions. Their frequencies are 3393 and 3369 cm-’ in the case of proline, and 2519 and 2499 cm-’ in the case of deuteroproline. Deuteration isotopic shift v(NH)/Y(ND) = 1.346 is common to N-H stretching of glycine [lo] and pyrrolidine [23] in the vapour phase, and supports the validity of such an assignment. Perhaps the band splitting is caused by the participation of the proton of the N-H bond in the intramolecular H-bonding in conformations I and II in two different ways.

There are a few bands in the spectra below 1500cm-‘, which can be assigned to N-H bend- ing. Intense bands at 1384, 782, 768 cm-’ in the proline spectrum and 1123, 1100, 646 cm-’ in the deuteroproline spectrum correspond to N-H (N-D) bending vibrations, All the vibrations men- tioned are of complex PEDs (Tables 3 and 4). Vibrations of the C-H group contribute appreci- ably to the vibrations with the frequencies 1384cm-’ (N-H bending) and 1123, llOOcm_’ (N-D bending). The low-frequency vibrations at 768 and 646cm-’ are mixed with the C-H defor- mation and the cycle bending vibrations. The assignment of N-H bending vibrations is in accordance with the results of studies on IR spec- tra of pyrrolidine in the vapour phase. The corre- sponding frequencies occur at 1418 and 792cm-’ in the pyrrolidine gas phase spectrum [23].

CH, modes

The bands of CH stretching vibrations are

observed in the usual well-known spectral region of 3000-2800 cm-‘. Each CH;! group has two absorption bands: the bands of CH2 antisym- metric stretching have moderate intensity and the less intense bands correspond to CH2 symmetric stretching (Tables 3 and 4). The CH2 stretching frequencies do not change significantly on deutera- tion of O-H and N-H groups (the shift being about 2cm-‘) and have poor confo~ational sensitivity. Most of the bands corresponding to HCH and CCH bending vibrations are in the region from 1500 to 800 cm-‘. (A detailed assign- ment is given in Tables 3 and 4.) The following spectral regions are determined for different C-H deformations: 1500-1450 cm-’ for HCH bending; 1350-1200cm-’ for CH2 twisting; 1250- lOOOcm-’ for CH, wagging; 1100-800 cm-’ for CH2 rocking. In the region below 1200 cm-’ all the CH;! bending vibrations are mixed with the ring stretching and bending vibrations.

Ring modes

The cycle stretching modes are situated in the region from 1100 to 800 cm-‘. The bands assigned to the ring bending are below 800 cm-‘. The forms of the stretching and bending vibrations are determined by the local symmetry of the pyrrolidine ring. We assign the bands in the 900- 880cm-’ region in the proline spectrum and the 911, 894cm-’ bands in the deuteroproline spectrum to the symmetric breathing mode. This is in good agreement with the assignment of the breathing mode in Raman spectra of pyrro- lidine in the vapour phase (902 cm-’ [23]). Perhaps the splitting of the breathing vibration band is caused by the existence of a conformational equilibrium.

Some out-of-plane bending vibrations of the cycle and torsion vibrations of the COOH group are situated below 400cm-‘, i.e. in the region we did not observe. These vibrations can be interpreted on the basis of calculated spectra and experimental data for the pyrrolidine

1231.

I.D. Reva et al./J. Mol. Struct. 318 (1994) l-13 13

Conclusions

This work reports for the first time the IR spectra of proline trapped in an Ar matrix. The peculiari- ties of the isolated proline structure are studied on the basis of experimental IR spectra analysis. It is found that isolated proline, similar to glycine [lo] and leucine 1201, exists in the molecular form. This is evidenced by the OH (OD) stretching bands at 3559 (2626) cm-‘, and by the C=O stretching vibrations in the region from 1790 to 1760 cm-‘, present in the spectra.

The AM 1 semiempirical quantum chemical method was used to determine the structures and relative energies of proline conformations. The cal- culations show that isolated proline may have two conformations. Experiments have revealed an equilibrium between the two proline conformers that are present in the argon matrix in almost equal proportions. Conformational splitting of C=O stretching vibrations proved to be maximal. In the first case the H-bond is formed between the imino group proton and hydroxyl oxygen, whereas the carbonyl group does not take part in H-bonding and the 1789 cm-’ band corresponds to C=O stretching. In the second case the molecule is stabilized by the N-H . . f 0 = C hydrogen bond with the C=O stretching frequency at 1766cm-‘. The conclusion on the occurrence of two proline conformations in the matrix is also supported by the splitting of the N-H stretching band because the N-H group forms the intramolecular H-bond in two different ways.

We acknowledge with thanks the support for this research (Grant No 2/381) by the Ukrainian State Committee of Science and Technology.

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