Vibrational studies, normal-coordinate analysis, and infrared VCD of

Reinhard Schweitzer-Stenner, Fatma Eker, Qing Huang, and Kai Griebenow , Piotr A. Mroz and Pawel M. Kozlowski. The Journal of Physical Chemistry B 200...
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548

J. Phys. Chem. 1992,96, 548-554

decrease from 1.880 A in the gauche conformer to 1.861 A in the trans conformer and that conformer bands were observed for the PS stretch in the Raman spectra. No significant change in the PO bond distance was evident in the ab initio structures of CH,CH2P(0)Fz, and similarly, no conformer bands for the PO stretch in the vibrational spectra were observed. This study represents another one in our investigation of a series of ethyl-substituted phosphines. The ab initio results, although not in total agreement with the experimental findings, are reasonable, given the size and the addition of heavy atoms in CH3CH2P(0)F2as opposed to CH3CH2PH2. A study of the corresponding compounds where the lone pair is used for bonding to the borane group would be of interest since the change in

bonding for these molecules would not be as drastic as those for the corresponding oxygen- or sulfur-containing molecules. Acknowledgment. We gratefully acknowledge the partial financial support of this study by National Science Foundation Grant CHE-83-11279. Also we thank Dr. C. G. James for recording the initial infrared spectrum. Registry NO. CH,CH,P(O)F,, 753-98-0. Supplementary Material Available: Listings of a b initio predicted force constants for trans-ethylphosphonic difluoride and gauche-ethylphosphonic difluoride (2 pages). Ordering information is given on any current masthead page.

Vibrational Studies, Normal-Coordinate Analysis, and Infrared VCD of Alanylalanine in the Amide I11 Spectral Region M. Diem,* 0. Lee, and G.M. Robertst Department of Chemistry, City University of New York, Hunter College, 695 Park Avenue, New York, New York 10021 (Received: May 20, 1991; In Final Form: September 17, 1991)

Detailed vibrational assignments and normal coordinate calculations for L-alanyl-L-alanine and L-alanyl-palanine and several deuteriated isotopomers in the spectral region between 1200 and 1700 cm-' are reported. In this spectral region, the peptide amide I, 11, and I11 vibrations occur. Large infrared vibrational circular dichroism (VCD) intensities are reported for the amide I11 vibration. These studies were undertaken to obtain a description correlating the atomic displacements to strong VCD intensities. We demonstrate that large VCD signals occur in either delocalized, coupled C-H/N-H deformations, or in C*-H deformations which are similar to those which produce large VCD in alanine itself.

Introduction Vibrational spectroscopy has been used for the determination of peptide solution and solid phase conformation. In particular, observed frequencies of the amide I11 vibration have been utilized as a qualitative probe for the solution conformation of peptides and proteins in Raman and infrared spectroscopies.' This is possible since the amide 111 vibration exhibits frequency shifts which depend on the secondary structure. Lordl proposed a quantitative correlation between the conformational angle 9 and the frequency of the amide TI1 vibration; however, for most purposes the correlation between the amide I11 frequencies and the secondary structure remained purely qualitative. The qualitative character of this correlation is partially due to the poor understanding of the nature of the amide I11 vibration. We have found over the past years that this vibration is a complex, delocalized motion of C-H and N-H deformation coordinates, quite different from the description developed originally by Miyazawa et aL2 A reevaluation of this vibration via detailed normal coordinate calculations will be a major part of this publication. The vibrational assignment which underlies the normal-mode calculations is based on our previous Raman ~ t u d i e s . ~ In addition, new VCD features in the amide 111vibration will be presented. Previously, amide I11 VCD had been reported for L-Ala-L-Ala in H 2 0by us4 and in D 2 0 by Freedman et aL5 In order to discuss the origin of the amide 111VCD, vibrational data in the 1200-1400-~m-~region of a number of small peptides will be presented and interpreted. The results presented here suggest that large VCD signals are observed in delocalized vibrations composed of N-H and C-H deformation coordinates, or in C-H deformation vibrations which resemble those modes in alanine which produce large VCD effects6 'Present address: Biophysical Research Division and Department of Physics, The University of Michigan, Ann Arbor, MI 48109.

0022-3654/92/2096-548$03.00/0

Experimental Methods All VCD results discussed were obtained on the Hunter College dispersive VCD instrument, which has been operative since 1987. Its design and performance were described in detail.' Infrared data were obtained simultaneously with the VCD data via the VCD spectrometer. Undeuteriated peptide samples were obtained commercially (Sigma Chemical Co., Chemical Dynamics or Research Plus). Peptides were checked for purity via NMR and Raman spectroscopies. All alanyl peptides, which are deuteriated at the alanine 2-carbon, were synthesized and purified in-house via solid-phase peptide synthetic method^.^ Deuteriation of any labile protons was achieved by lyophilization of the samples from DzO. Samples, dissolved in either H20or D20, were contained between CaFz plates separated by 15- or 25-pm spacers. Concentrations were 0.5 M. Due to difficulties in reproducing the sample path length exactly, results are given in units of absorbance AU, rather than molar extinction coefficients. Computational Procedures The purpose of the normal-coordinate calculations presented here is to derive a force field and atomic displacement vectors ( 1 ) Lord, R. C. Appl. Spectrosc. 1977, 31, 187. (2) Miyazawa, T.; Shimanouchi, T.; Mizushima, S.J. Chem. Phys. 1958, 29, 611. ( 3 ) Oboodi, M. R.; Aha, C.; Diem, M. J . Phys. Chem. 1984, 88, 501. (4) Roberts, G. M.; Lee, 0.; Callienni, J.; Diem, M. J . Am. Chem. Soc. 1988, 110, 1749. ( 5 ) Freedman, T. B.; Chernovitz, A. C.; Zuk, W. M.; Paterlini, M. G.; Nafie, L. A. J. Am. Chem. SOC.1988, 110,6970. (6) Diem, M. J . Am. Chem. Soc. 1988, 110,6967. (7) Diem, M.; Roberts,G. M.; Lee, 0.;Barlow, A. Appl. Specrrosc. 1988, 42, 20.

0 1992 American Chemical Society

The Journal of Physical Chemistry, Vol. 96, No. 2, 1992 549

Vibrational Studies of Alanylalanine Me

0

0

Me

0

Figure 1. Atomic positions and structure of alanylalanine. All bond lengths are given in units of A. Bond angles were assumed to be 109' for sp3 and 120° for sp2-hybridized atoms.

which represent all the vibrational features of alanylalanine and its deuteriated isotopomers. This entails reproducing the observed spectra and the complex pattern of mixing of deformation coordinates between 1200 and 1350 cm-I, which leads to a better understanding of the origin of the VCD spectra. For the calculations, the molecule was defined as shown in Figure 1. The methyl and the -NH3+ groups are represented as point masses to simplify the computational procedures. The -COT group was assumed to lie in the plane of the peptide linkage. All bond lengths are also indicated in Figure 1. The calciilations were carried out for six isotopically substituted species, and one diastereomericform, with a final number of 47 force constants. We opted for an empirical (Urey-Bradley* type) force field, rather than a quantum mechanically derived one, because the amount and quality of the vibrational spectral data allows even an empirical force field to represent the molecular vibrations adequately. In the Urey-Bradley force field utilized, the nonbonded force constants involving interactions extending over more than two bonds, Le., the cis interactions, were ignored. Calculations were performed using normal-coordinate programs described in detail el~ewhere.~These programs were originally developed by Schachtschneider,Ioand subsequently adapted to execute on personal computers and modified to be particularly suitable for asymmetric molecule^.^ Computations were carried out on an IBM-XT compatible computer, equipped with a coprocessor board (Definicon, Inc, Model DSI-20), incorporating 16 MHz, 32-bit Motorola 68020/6888 1 processors, and operating in the IBM DOS environment. This computer allowed a typical normal-coordinate computation for alanyl alanine to be carried out in about 10 s.

Results and Discussion 1. The Vibrations of the Peptide Linkage. The early normal-coordinate analyses of the peptide linkage, by Miyazawa et a1.,2 were carried out on the peptide model N-methylacetamide H CH3-C-N

II

I

-CH3

0

The characteristic peptide vibrations were identified and named amide I-VI1 and amide A and B. Later, it was found1*''that the frequencies of some vibrations shift as the peptide undergoes conformational changes, and that these frequency shifts can be correlated with secondary structure, such as sheets and CY helices. For the remainder of this discussion, only the amide 1-111 vibrations are of interest. The amide I vibration, observed at 1653 cm-I, was assigned by Miyazawa et a1.2 to the C = O stretching vibration. Upon deuteriation of the N-H hydrogen the mode shifts to 1642 cm-I. The amide I1 vibration is observed at 1567 an-',and shifts to 1472

cm-' upon deuteriation. This band was found2to be largely due to the N-H in-plane deformation, with a much smaller contribution from the C-N stretching coordinate. The amide I11 region was assigned2to consist of the N-H deformation and, to a lesser extent, of the stretching of the C-N bond. This latter assignment, however, appears to be correct only for the particular model, N-methylacetamide. Real peptides incorporate methine hydrogens on the adjacent CY carbon, which have deformation vibrations nearly degenerate with the N-H def~rmation.~ In these systems, the amide I11 vibration consists of coupled N-H/C-H deformation motions. This observation was first reported3by us in the course of detailed Raman studies of alanine dipeptides. 2. Infrared Spectra and Vibrational Assignments in the Amide III Region. In these studies, we noted that the "amide 111" vibration, which had been described as a N-H deformation coupled with the C-N stretching motion, disappears or becomes much weaker when either of the two protons marked with an (*) were substituted for with deuterium atoms in L-Ala-L-Ala (I). One * * H

H

NHj-C

I

I

I

H

I I

-C-N-C-C02-

II

CH3 0

CH3

I expects that the "amide I11 vibration" shifts drastically upon deuteriation of the amide proton. However, the disappearance of this vibration upon deuteriation of the methine hydrogen was totally unexpected. Thus, we postulated3 that the C-H deformation at the adjacent CY carbon in the fragment H

I

H

N-C

I

contributes to the amide I11 motion. This hypothesis was found to be correct through a detailed vibrational study involving six isotopically substitutes alanyl dipeptides, to be described in the following. Before we discuss this vibrational assignment further, a paragraph on the vibrations of alanine in the same spectral region is in order. Alanine shows two C-H deformations,I2at 1358 and 1306 cm-'. This is typical for asymmetric molecules with a single methine hydrogen. In a symmetric molecule such as chloroform, there is only one doubly degenerate C-H deformation vibration. In alanine, this degeneracy is lifted, and consequently, two vibrations are observed in the Raman and IR spectra.I2 These vibrations show opposite signs in VCD,6 and shift toward lower frequency (1343 and 1295 cm-') if alanine is deuteriated, or otherwise substituted, at the N atom. For differentiation, these two vibrations will be henceforth referred as C-HI1 and C-HI, respectively. We now turn to the vibrational assignment of the isotopically substituted alanyl dipeptides in the amide I11 region. In this discussion, the coupling of certain deformation coordinates is often difficult to establish. However, for some modes there is a distinct absence of coupling, which presents a more easily discernible method to follow these vibrations between the isotopically substituted analogues. The infrared absorption spectra of these peptides are shown in Figure 2, and the observed frequencies and intensities are summarized in Table I. The spectrum of L-Ala-d,-L-Ala-d, (11)

I

II

CH3 0

AH3

I1 (8) Urey, H. C.; Bradley, C. A. Phys. Rev. 1931, 38, 1969. (9) Barlow, A.; Diem, M. J. Chem. Educ. 1991, 68, 35. (10) Schachtschneider, J. H. 'Vibrational Analysis of Polyatomic Molecules. V. and VI." Shell Development Company Report, Emeryville, CA, 1964. ( 1 1) Thomas, G. J., Jr. In Vibrational Spectra and Structure; Durig, J., Ed.;Elsevier Science Publishers: Amsterdam, The Netherlands, 1975; Vol. 3, 239.

in H 2 0 (not shown) exhibits only one deformation vibration3in the 1200-1400-cm-' region at 1336 cm-'. This vibration disappears when the N-H group is deuteriated. Thus, it can be assigned (12) Diem, M.; Polavarapu, P. L.; Oboodi, M. R.; Nafie, L. A. J. Am. Chem. SOC.1982,104, 3329.

Diem et al.

550 The Journal of Physical Chemistry, Vol. 96, No. 2, 1992 TABLE I: Frequencies, IR Absorption, and VCD Intensities Alanylalanine in the Amide 111 Region L-Ala-d,-L-Ala L-Ala-d,-L-Ala L- Ala+ Ala L- Ala+ Ala in H20 (I) in D20 (V) in D20 (111) in H20 (IV) obs IR VCD obs IR VCD obs IR VCD obs IR VCD freq, int, int, AU freq, int, int, AU freq, int, int, AU freq, int, int, AU cm-' AU X lo5 cm-I AU X lo5 cm-' AU X lo5 cm-l AU X lo5

1355 1346" 1330

1279

0.22

4.4

-1.75 1340

0.03

1325

0.06

0.01

-2.7

0.13

0.29

1311

0.05

1274

0.03

-0.33 1.23

1329

0.17

1305 1276

0.06 0.10

-0.9

-0.7

assignment CN-H" AmIIIj C-H/N-Hb Cc-H" AmII12 C-HIN-H~

0.9 2.85

1302c 1266c 1281

1.8

0.15

CN-HI Cc-H' AmIII'

" Raman frequencies, very weak in absorption. bCoupledN-H/Cc-H" deformation, cf. text. Band positions derived via band decomposition,cf. ref 3. 0 molecule in the 1250-1400-~m-~region show three vibrations at 4 1274, 1311, and 1346 cm-l (Figure 2, peaks D, E, and F). The lowest one of them (D) agrees in frequency with the Cc-H1 mode AxlO' C (B) in I11 and is assigned accordingly. The other two peaks B / \ / 2 observed, a t 1346 and 1311 cm-I, are due to two coupled vibrational states of the N-H (1 336 cm-l) and the C-HI1 (1330 an-') deformation coordinates. The two resulting vibrational states split apart and appear as a polarized ( 1311 cm-I) and a depolarized 2 b F (1345 cm-I) peak in the Raman ~ p e c t r a .The ~ corresponding I \ Ax10 infrared spectrum (Figure 2, trace b) shows the 1311-cm-l member clearly, whereas the higher frequency peak at 1346 cm-* is ob1 served only as a weak shoulder. In L-Ala-L-Ala in D 2 0 (V), four C-H deformations are ob-

A I\

ti

D

2 AxlO'

CH3 0 1

3

H

I I I ND~$-C -c-N-C-CO,I II I

CH3

V

L

AxlO' 2

1 1270

1330 1390 Wavenumber (l/cm)

1450

Figure 2. Infrared absorption spectra of (a) L-Ala-d,-L-Ala (111) in D20, (b) L-Ala-d,-L-Ala (IV) in HzO; (c) L-Ala-L-Ala (V) in DzO (d) L-

Ala-L-Ala (I) in HzO. The invariant peaks at ca. 1370 and 1410 cm-l are the methyl symmetric deformation and the carboxylate symmetric stretching vibrations. The dashed traces in (b) and (c) represent the corresponding Raman spectra (cf. ref 3). unequivocally to the unperturbed N-H deformation motion. In L-Ala-d,-L-Ala in D 2 0 (111), the two C-terminal alanyl D ND3*-C

I

D

I

H

-C-N-C-CO,-

II

I

CH3 0

I I

CH3

111

deformations are observed at 1279 and 1330 cm-' (Figure 2, peaks R and C). These vibrations will be henceforth referred to as the Cc-H1 and the Cc-HI1 modes, respectively, where the subscript C refers to the C-terminal alanyl residue. If 111is dissolved in H 2 0 instead of D20,it will exchange the labile protons, and species (IV) is obtained The spectra of this D

H

I

I

H

NH3+-C -C-N-C-CO,-

I

II

CH3 0

I

I

CH3

rv

served at 1276, 1305, 1329, and 1355 cm-l (Figure 2, peaks G, H, I, and J). We assigned the C-H deformation of the C-terminal alanine residue to the peaks observed at 1330 (CC-H") and 1279 cm-l (Cc-H1). Thus,the two C-H deformations of the N-terminal alanyl residue are assigned to vibrations at 1355 ((&-HI1) and 1305 cm-' (CN-H1), at somewhat higher frequencies than in N-deuteriated alanine itself. Due to the N-D group, with a vibrational frequency that differs greatly from those of the C-H deformation vibrations, little coupling occurs, and the four C-H deformations are observed virtually unperturbed. For L-Ala-L-Ala in H 2 0 (I), Le., the entirely undeuteriated species, the spectrum in this region is drastically different. This region was previously assigned to consist of two weakly coupled modes K (the Cc-H' deformation at 1266 cm-I) and M (the C r H ' deformation at 1303.cm-I) and three highly coupled modes L, N, and 0. The modes K and M were only observed via a band deconv~lution.~ The three other modes, to be discussed in detail later, occur at 1281, 1325, and 1340 cm-', and have been designated the AmIII', AmII12, and AmII13 modes. They are highly coupled modes consisting of the N-H, the CC-H", and the C r H " deformation coordinates. We now turn to the discussion of the vibrational spectra of the diastereomeric molecule, L-Ala-D-Ala. The IR spectra of the diastereomers L-Ala-L-Ala (solid trace) and L-Ala-D-Ala (broken trace) in H 2 0between 1250 and 1450 cm-' are shown in Figure 3. The subtle differences observed in the Raman3spectra of these diastereomers also appear in the IR spectrum: This difference is a more distinct splitting between the AmII12 and the AmII13 modes at 1325/1340 an-,.These changes are interpreted in terms of differences in coupling geometries of vibrational coordinates. These differences are comparable to slight conformational changes in a peptide, since the geometry between interacting groups varies similarly in both cases. However, vibrational techniques are less sensitive to these changes than VCD, and we attribute the large changes in the VCD spectra, to be discussed below, to differences

The Journal of Physical Chemistry, Vol. 96, No. 2, 1992 551

Vibrational Studies of Alanylalanine

TABLE II: Observed and Calculated Frequencies (in cm-') for L-Ala-L-Ah and Various Isotopomen Ala-dl-Ala-dl Ala-d,-Ala-d, Ala-d,-Ala Ala-d,-Ala in DzO in H20 (11) in DzO(111) in H20(IV) obs calc obs calc obs calc obs calc amide I 1663.0 1678.4 1680.0 1682.4 1664.0 1678.4 1680.0 1682.5 COI- asym str 1584.0 1585.8 1584.0 1591.1 1590.0 1588.3 1584.0 1592.5 amide I1 1478.4 1450.3 1570.0 1576.0 !479.0 1451.0 1570.0 1578.1 C02- sym str 1406.0 1410.7 1406.0 1411.1 1408.0 1412.4 1407.0 1412.9 amide I11 1336.0 1314.9 1346.0 1333.5 1311.0

1330.0 1279.0

" Frequencies found via a band decomposition (cf. ref 3

1

1 0

-1 -2

t------

3 Ax10 2

1

1270

1330

1390

1450

Wovanumber (l/cm)

Figure 3. Infrared absorption (bottom) and VCD (top) spectra of L-

Ala-L-Ala in HzO (solid traces) and L-Ala-D-Alain H20(dashed traces)

(from ref 4). in the coupling of the three coordinates. The resulting new description of the amide I11 vibration as a coupled C-H/N-H deformation is able to account for the large frequency shifts the amide I11 vibration experiences when the conformation, or secondary structure, of the peptide is varied. 3. Normal-Coordinate Calculations. The goal of this analysis is to provide a force field which adequately describes the atomic motions of the vibrations in the 1200-1500-~m-~ range in order to describe the coupling of coordinate which give rise to the large observed VCD, and to obtain a pictorial description of these vibrations. In calculation with as many variables as in the prwnt one, there will be some degree of indeterminacy. The result of this ambiguity is that there is more than one force field which reproduces the observed frequencies within a few percent error. In selecting the force field which we believe is best, certain criteria were established: 1. The refined force constants must be close to those reported for alanine and the peptide moiety. Thus, force constants of alanine,I2based on a detailed vibrational study of five isotopomers, were used wherever possible. The force constants for the peptide linkage were transferred from previous studies by Gupta and Gupta.13 These numbers are less reliable since they were based on solid-stateinfrared spectra of undeuteriated alanyl alanine only. (13) Gupta, M. K.; Gupta, V. D. Indian J. Biochem. Biophys. 1978, 15, 41 3.

1274.0

calc 1681.5 1588.3 1455.4 1412.4

1355.0 1305.0 1329.0 1276.0

1343.2 1284.0 1329.7 1278.3

1273.8

Ala-Ala in H 2 0 (I) obs calc 1680.0 1584.0 1570.0 1407.O 1345.0' 1325.0" 1281.0"

1685.6 1592.7 1578.4 1413.0 1355.1 1330.7 1307.8

1302.0"

1284.0

1266.0"

1273.3

3).

AA~~O:

-3

1329.8 1278.4

obs 1665.0 1592.0 1483.0 1407.0

1312.2

CN-H def Cc-H def

Ala-Ala in D20 (V)

2. Differences between observed and calculated frequencies should be less than 1%. However, since some interaction force constants (the cis interactions force constants) were omitted, the force field is not expected to reproduce all vibrations equally well. Therefore, obtaining a perfect frequency fit was considered less important than fulfilling requirement 3, discussed next. 3. There should be agreement between the calculated motions of the molecule and the interpretation of the observed spectra. This requirement may produce a biased force field since it depends greatly on the interpretation of the vibrational spectra. For this reason, a large number of isotopomer vibrations must be available for input into the calculations. However, we believe that our approach is valid and produces a better description of the atomic motion than the frequently practiced method of fitting a force field to the vibrational spectrum of one isotopomer and basing the interpretation of mixing patterns on such results. The degree of coupling is one of the major aspects where normal-coordinate calculations can be entirely misleading, since a good frequency fit, which is usually employed as the criterion for the quality of a force field, may not represent the vibrational motion at all. Thus, detailed isotopic data need to be analyzed for the exact behavior of the vibrations under consideration, and subsequently, force constants need to be perturbed to reproduce both frequencies and the character of a vibration. 4. The calculations should reproduce the frequencies observed in the amide I11 region, as well as the frequencies above and below that region. The higher frequency vibrations (3300-1500 cm-') had been assigned in our previous vibrational analysis.' In the skeletal stretching region just below the amide I11 region, no reliable assignment has yet been made since the C-D deformations mix with or overlap the skeletal stretches in unpredictable ways, and it is impossible to distinguish all the stretching motions involved. To succeed with this assignment, an entirely new set of nitrogen- and carbon-substituted isotopomers is required. Thus, we found it straightforward, albeit time consuming, to fit the vibrations above and in the amide I11 region, but impossible to fit the ones below that region. However, the force constants belonging to the skeletal stretches have little or no effect on the frequencies calculated for the amide I11 region. Calculated and observed vibrational frequencies for L-alanylL-alanine and five isotopomers in the amide I to amide I11 regions are tabulated in Table I1 to show the extent of agreement between observed and calculated frequencies. The assignments were verified by inspection of the potential energy distribution (PED)I4 and the displacement vector associated with each mode. Table I11 summarizes the final set of force constants utilized for the computations. Details of the computations may be found elsewhere.14 The agreement between the force constants transferred from similar molecules and the final set is remarkable, as indicated above. We now turn to the discussion of the displacement vectors for the normal vibrations in the amide I11 region. The displacement (14) Robcrts, G. M. Ph.D. Dissertation, City University of New 1990.

York,

552 The Journal of Physical Chemistry, Vol. 96, No. 2, 1992

Diem et al.

,-

Vib. 0

Vib. C

Vib. 8

Vlb. A

h

Vib.

Vib. F

Vib. E

Vib. H

G h

n

u Vib.

U

Vib.

M

V1b.L

Vib. K

Vib. J

I

Vib. N

Vib. 0

Fimve 4. Atomic disdacement vectors for the vibrations in the 1200-1400-~m-~ region. Capital letters in Figure 2 correspond to the modes marked in-Figure 4. For definition of atoms, see Figure 1.

vectors, displayed in Figure 4 and henceforth referred to,as the s vectors, depict the magnitude of all atomic displacement vectors relative to each o t h q as in all vibrational computations, the overall amplitude of the displacements is unknown. For normal modes of vibration, for which only one major displacement coordinate is shown, the phase of this coordinate is indeterminate as well; i.e., the motion of the atom may be drawn as an increasing or decreasing intemal coordinate. Only displacement components, for which the absolute value is larger than 0.1 A, are depicted. A complete listing of the s vectors has been p~b1ished.I~ In L-Ala-d+Ala-d, in H 2 0 (11) there is one vibration in the region of interest. This vibration was referred to above as the 'unperturbed" N-H deformation vibration. Its s vector, Figure 4A, reveals that it consists mostly of the N-H in plane deformation, and some contribution from the C-N stretching coordinate. This is in agreement with the findings of Miyazawa:* with both methine hydrogens exchanged for deuterium, the surroundings of the peptide linkage precludes any coupling between N-H and C-H deformation coordinates. Thus, the vibration is, indeed, best described as a mixture of the N-H deformation and the C-N stretching motion. The two Cc-H deformation vibrations for L-Ala-dl-L-Ala in D20(111) are shown in Figure 4, B and C. These vibrations are nearly pure C-H deformations, are approximatelyorthogonal and exhibit large, bisignate VCD spectra (vide infra). For L-Ala-d,-L-Ala in H 2 0 (IV), the three modes observed in the spectra are depicted in Figures 4D-F. The two higher frequency modes, E and F, were assigned above to be due to the mixing of the N-H and the Cc-H" deformation. Their displacement vectors form symmetric (F) and antisymmetric (E) coupled states. The remaining mode D is similar to mode B in (111) and is consequently referred to as Cc-H'. In L-Ala-L-Ala in D20(V), the four separate methine deformation modes depicted in Figure 4 G J originate from two CC-H and two CN-H deformations. The calculations confirm that these four vibrations do interact very little. Inspection of Figure 4, G and I, reveals that the deformations G and I of the Cc-H residue occur as group vibrations which are approximately orthogonal, as are the two deformation modes of the C r H hydrogen, H and

J. Substantial coupling between these modes is prevented by the presence of the N-D group, which has a very different vibrational frequency. Of the five modes in ~-Ala-~-Ala in H 2 0 (I), two are uncoupled C-H deformations: mode K (Cc-H1), calculated to occur at 1273 cm-I, and mode M ((&-HI), calculated to occur at 1284 cm-l. Both these modes fall under the broad and intense peak centered around 1280 cm-'and were observed only via band decomposition? The displacement vectors of mode K is very similar to the s vector of the C r H ' deformation G in (V). S i l y , mode M is virtually identical with mode H. The remaining three modes L, N, and 0 are heavily coupled. All three have contributions from the methine deformations ( C r H " and CN-H"). AmIII', mode L, is the most delocalized mode and has contributions from the N-H in-plane deformation, and C-N stretch, and the two methine deformations. It thus represents the most highly mixed of all the modes described so far. We believe that the s vector is close to the true description because this mode disappears upon deuteriation of methine or amide proton. AmII13, mode 0,also has a large contribution from the N-H in-plane deformation, coupled mostly to the CN-H" motion. AmIIIZ, mode N, consists mostly of the Cc-H" deformation, coupled to the C r H " deformation with a negligible contribution from the N-H deformation. Upon substitution of a wAla for a L-Ala residue at the Cterminal position in alanyl dipeptide, the observed and calculated vibrational frequencies in the amide I11 region undergo small frequency shifts3 The difference between calculated frequencies for L-Ala-L-Ala and L-Ala-D-Ala are within in the error of the calculations. Thus, any computational results must be sought in the s vectors, rather than the frequencies. When the s vectors of an enantiomeric pair are compared, their absolute values are identical. This is not necessarily true for diastereomers, and two cases may occur: A "pure" vibration, where the motion of a single atom completely dominates the atomic displacements, exhibits s vector components for which the absolute value is almost completely identical for the diastereomers. Such is the case for the amide A vibration, which consists almost entirely

The Journal of Physical Chemistry, Vol. 96, No. 2, 1992 553

Vibrational Studies of Alanylalanine

TABLE In: Refmed Set of Urey-Bradley Force Co118tants' no. final value 8.40 1 2 6.500 3 4 5 6 7 8 9

IO 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

5.100 2.400 1.800 4.150 2.400 7.450 2.700 0.450 0.300 0.500 0.430 0.720 0.360 0.370 0.720 0.400 0.720 0.353 0.500 0.219 0.500 0.185 0.429 0.400 0.130 0.499 0.778 0.545 0.387 0.300 0.580 0.053 1.ooo 0.700 1.400 0.550 0.550 0.878 0.499 0.387 0.500 0.480 1.ooo 3.100 1.850

init value*

definition

8.400* 6.350* 4.778 2.500 1.800 4.101 3.010 7.355 2.500 0.750* 0.219 0.500 0.398 0.720 0.368 0.353 0.720 0.4 18 0.720 0.353 0.500 0.219 0.420* 0.090. 0.529 0.400* 0.130 0.264 0.580 0.387 0.490 0.778 0.499 0.053 1.075 0.700 2.500 0.6 19 0.500* 0.778 0.499 0.387 0.264 0.580 1.075 3.184 1.800

Cc=O str (peptide linkage) Cc-N str (peptide linkage) N-H str N C * str C*-m str C*-H str C * C c str Co-O str C*-a str M c - N bend (peptide linkage) O-Cc-C* bend CcC*-m bend Cc-C*-H bend C*-Cc-N bend mC*-H bend H-C*-a bend mC*-a bend H-NC* bend N-C*-m bend N-C*-H bend N-C*-Co bend C*-Co-O bend Cc=O wag N-H wag Co-O wag Cc-N-C* bend O-Co-O bend Cc-C*-m tet Cc-C*-H tet Cc-C*-a tet m*C*-H tet mC*-a tet H-C*-a tet rho C*-Cc-O gem C*-Cc-N gem N-Cc-O gem (peptide linkage) H-N-Cc gem (peptide linkage) C*-N-Cc gem N-C*-m tet N-C*-H tet N-C*-Co tet mC*-Co tet H-C*-Co tet O-Co-C* gem O.-Co-O gem C*-Cc str

L-Ala-L-Ala L-Ala-D-Ala Figure 5. Atomic displacementvectors of the vibrations of L-Ala-L-Ala

(left) and L-Ala-D-Ala(right): AmIII' (top), AmII12(middle), AmIII' (bottom). For definition of atoms, see Figure 1. 4

4

~

0 5 2

0 -2 -4 4

1 '

h

Ax10

2

Units: stretching and nonbonded interaction constants mdyn/A; bending, wagging, and intermolecular tension constants mdyn/rad. Abbreviations: Cc, carbonyl carbon; Co, carboxylate carbon; C*, chiral carbon; m, methyl group; a, NH3+group. bInitialvalues marked * are from ref 13; all others one from ref 12.

Ala-d,-L-Alain D20(dashed) and L-Ala-d,-L-Ala in H20(solid). The latter spectra were scaled by a factor of 2 to allow for a better comparison with the spectra in D20.

of the N-H stretch. For other vibrations, which do not include any significant contribution from the atoms attached to the chiral carbons (as for example amide I), the s vectors are still almost identical. In complex vibrations, such as the Am111 modes, however, many atoms participate in the motion, and the differences between diastereomeric forms become significant and involve all atoms which take part in the coupling. Thus, the differences are now much larger and need not be mirror images for the diastereomeric forms. A comparison of the displacement vectors for the three amide I11 vibrations between L-Ala-L-Ala and L-Ala-D-Ala is shown in Figure 5 . The AmIII' mode, (Figure 5, top) shows interesting differences between L-Ala-L-Ala and L-Ala-D-Ala: In both cases, the mode consists of a coupled N-H/Cc-H" motion, with smaller contributions of the CN-H" deformation and the C-N stretching motions. Yet, the difference in the sign of the VCD (vide infra) signal must be due to the fact that the relative geometries vary between the forms, since the displacement vectors have the same phase relationship with each other. This appears to be the case in the other two amide I11 vibrations as well, which differ mostly

in the arrangement of the displacement components between the L-L and the L-D forms. 4. Amide Region VCD. After discussion of the normalcoordinate analysis, which established the coupling of the deformation coordinates in the 1200-1400-cm~~region, we may attempt to interpret the VCD signals observed in this region. L-Ala-d,-L-Ala in D 2 0 (111), shown in Figure 6 (dashed trace), shows VCD features nearly identical with t h c a of alanine in DzO, since it has only one methine hydrogen and there is no perturbing N-H vibration. The two methine deformation modes in (111) occur at lower frequencies than those in alanine, but they are separated in both cases by approximately 50 cm-I. The similarity between the VCD of alanine and (111) is striking and leads to the conclusion that uncoupled C-H groups give rise to similar VCD patterns, regardless of their immediate vicinity. However, if coupling to a neighboring C-H or N-H deformation coordinate is possible, the observed VCD spectra are changing drastically: L-Ala-d,-L-Ala in H 2 0 (IV), shown in Figure 6 (solid trace), shows quite a different VCD spectrum than (111). It exhibits the low-frequency, positive signal of the Cc-H* deformation, and only very small negative VCD at 1330 cm-', where

Wavenumber (1 /cm)

Figure 6. Infrared absorption (bottom) and VCD (top) spectra of L-

554 The Journal of Physical Chemistry, Vol. 96, No. 2, 1992

1

-3

A

4

Ax10

2.1

il Wavenumber (l/cm)

Figure 7. Infrared absorption (bottom) and VCD (top) spectra of LAla-L-Ala in D20(solid traces) and L-Ala-o-Ala in D20(dashed traces).

molecule (111) shows large negative intensity. The other two vibrations of (IV) in this region are the highly coupled combination modes E and F. If the coupling was dipolar in nature, one would expect a distinct couplet for these modes at 1311 and 1346 cm-'. However, very small VCD signals are observed at these two frequencies. Thus, mechanical coupling of the C-H and N-H deformation is likely, which changes the nature of the vibrations significantly but does not create VCD intensity in the way a dipole-dipole coupling would. This is confirmed indirectly by the large decrease in VCD intensity at 1330 cm-I: In the absence of coupling, the unperturbed CC-H1I mode shows large negative VCD intensity which disappears when coupling between this Cc-H" mode and the N-H deformation occurs. The VCD spectra of L-Ala-L-Ala in DzO are shown in Figure 7. Our VCD data, collected at about 6 cm-' resolution and excellent S / N ratio, demonstrate clearly that the sign patterns observed in L-alanine are maintained in L-Ala-L-Ala in D20: the low-frequency 1305 cm-I (CN-H1) and 1276 cm-' (Cc-H') deformations both show positive VCD (the 1305-cm-' peak is very weak in absorption, but shows clearly in the Raman spectrum). The high-frequency deformations at 1355 cm-I ( C r H " ) and 1329 cm-l (Cc-H") both show negative VCD. Thus, both residues exhibit a positive/negative couplet: the C-terminal residue at 1276/1329 cm-l and the N-terminal residue at 1305/1355 cm-I. These observed VCD spectra are in direct agreement with the earlier vibrational assignment and observed alanine VCD. These data were originally reported by Freedman et al.,s but we do not agree with their interpretation of the data. The comparison of the spectra in D 2 0 (Figure 7) with thaw in H 2 0(Figure 3) shows significant changes upon deuteriationof the amide proton, which was not realized in the previous work due to lower signal quality.5 Although there is an overall similarity in the positive/negative pattern observed between 1250 and 1400 cm-' for

Diem et al. L-Ala-L-Ala in HzO and DzO, even a casual inspection of Figure 3 and 7 reveals that this similarity is purely coincidental, since the infrared transitions giving rise to the VCD in this region are totally different. Since our VCD data were taken at much better S / N ratios, the features of all four C-H deformation vibrations can be clearly distinguished. We now turn to the discussion of the VCD spectrum of LAla-D-Ala in D 2 0 (Figure 7). Here, one finds that the two C-H deformations of the C-terminal residue switch sign. Thus, the peak at 1276 (CC-HI) and the one at 1329 cm-' (Cc-H") are negative/positive, whereas in L-Ala-L-Ala, they were positive/ negative. The CN-H' vibration at 1305 cm-l remains unchanged in sign and magnitude. The remaining vibration, the CN-H" vibration at 1355 cm-I, is very strong in absorption but shows little VCD. In fact, we believe that its small, negative VCD signal is masked by the positive VCD of the Cc-H" peak. These VCD results are in agreement with the vibrational assignment and normal-coordinate results which suggest uncoupled C-H deformation oscillators. Finally, we turn to the VCD spectra of the totally undeuteriated species, L-Ala-L-Ala and L-Ala-D-Ala, which have been reported earliera4Figure 3 shows the IR and VCD spectra of these two species in H 2 0 . The dominant VCD features of both diastereomers are located under the peaks at 1280, 1325, and 1340 cm-I. They are sensitive to the chirality of the acid terminal alanine residue, for they change sign between L-Ala-L-Ala and L-Ala-DAla. L-Ala-L-Ala (Figure 3, solid trace) shows a large positive peak corresponding to the AmIIII mode at 1280 cm-', and a smaller negative feature corresponding to AmII12at 1325 cm-l with little intensity in the AmII13 mode. The spectrum of L-Ala-&Ala in HzO (Figure 3, dashed trace) shows reversal of the VCD features, with large negative intensity for the AmIII' and a smaller positive feature for AmII13at 1340 cm-l and no intensity in the AmII12 mode. In fact, it was this observation which first suggested to us that the amide I11 VCD can be interpreted in terms of coupled vibrations involving the N-H and two C-H deformations.

Conclusion We have demonstratedthat the vibrational features of a number of isotopically substituted peptides in the amide 111 region can be interpreted in terms of extensive coupling of vibrational coordinates. These results are in agreement with a previous Raman analysis of the amide I11 vibrational region. VCD adds an additional spectroscopic technique to probe molecular vibrations in an extremely sensitive way and may be valuable to deduce molecular conformation. A force field, which is very similar to the one developed for alanine, reproduces the vibrational frequencies for L-alanyl+ alanine and its isotopomers in the region from 3500 to 1200 cm-I. We have s u d d in analyzing the amide I11 region in a manner which is consistent with the observed spectra. The calculations have further supported the view that this region consists of a complex coupling of the N-H hydrogen deformation with the deformations of the two adjacent methine protons and that the region changes drasticallyif any of the three protons is deuteriated. Acknowledgment. Support of this research from the National Institutes of Health (GM 28619) and several City University of New York Faculty Research Awards is gratefully acknowledged.