J . Phys. Chem. 1989, 93, 1771-1779
1771
Vibrational Circular Dichroism in the CH-Stretching Region of L-a-Amino Acids as a Function of pH William M. Zuk, Teresa B. Freedman,* and Laurence A. Nafie* Department of Chemistry, Syracuse University, Syracuse, New York 13244-1200 (Received: April 29, 1988; In Final Form: August 15, 1988)
The CH-stretchingvibrational circular dichroism (VCD) spectra of several amino acids (L-alanine and deuteriated isotopomers, L-serine, L-cysteine, and P-chloro-L-alanine)are examined as a function of pH. At neutral and high pH, the VCD spectra of the amino acids studied exhibit a large positive VCD intensity bias which is associated with the C,H methine stretching mode. At low pH the bias is absent and only very weak VCD signals are observed. The VCD spectra are interpreted within the framework of the ring-current mechanism. It is proposed that closed pathways due to a variety of intramolecular interactions in these amino acids can support vibrationally generated ring currents, which give rise to VCD intensity enhancement. The effects of sample solution pH on the intramolecular interactions, the VCD biases, and the amino acid conformations are investigated in detail.
Vibrational circular dichroism (VCD),1-5the differential absorption of left and right circularly polarized infrared radiation during vibrational excitation, has been shown to be extremely sensitive to intramolecular interactions within a wide variety of optically active molecules in solution.613 Many types of molecules, including amino acids6J4 and small peptides,l exhibit VCD intensities for localized vibrational regions that are strongly biased to positive or negative intensity. These biases have not been adequately explained by conventional VCD models based on vibrational displacement of electron densities. Recently, however, a new model of VCD intensity generation has been proposed, the ring-current mechanism for VCD,'V'-'~which can account for the enhanced VCD intensities. The vibrational-ring-current mechanism was originally proposed to explain intense monosignate VCD features observed in the C H stretching region of L-a-amino acids and a-hydroxy acids.6 The mechanism was subsequently extended to the C H stretches in amino acid-transition-metal complexes,E-" phenylethane derivatives,12 and sugars,13 and to other types of vibrational motion. According to this model, intramolecular interactions within these molecules close rings that can support oscillating electronic currents generated by the nuclear vibrational motion. These currents, which occur at constant electron density, give rise to a large magnetic dipole transition moment. Since VCD intensity is proportional to the scalar product of the electric dipole and magnetic dipole moments for a transition, the magnetic dipole transition moment produced by the ring current provides a source of VCD intensity enhancement. Three empirical rules have been defined that de(1)Freedman, T. B.; Nafie, L. A. In Topics in Stereochemistry; Eliel, E. L., Wilen, S. H., Eds.; Wiley: New York, 1987;Vol. 17,pp 113-206. (2)Stephens, P. J.; Lowe, M. A. Annu. Rev. Phys. Chem. 1985,36,213. (3)Keiderling, T.A. Appl. Spectrosc. Rev. 1981,17, 189. (4)Nafie, L. A. In Advances in Infrared and Raman Spectroscopy; Clark, R. J. M., Hester, R. E., Eds.; Wiley-Heyden: London, 1984;Vol. 11, p 49. (5)Polavarapu, P. L. In Vibrational Spectra and Structure; Durig, J. R., Ed.; Elsevier: Amsterdam, 1984;Vol. 13, pp 103-160. (6)(a) Nafie, L. A.; Oboodi, M. R.; Freedman, T. B. J . Am. Chem. SOC. 1983,105, 7449. (b) Oboodi, M.R.; Lal, B. B.; Young, D. A,; Freedman, T. B.; Nafie, L. A. J. Am. Chem. SOC.1985,107, 1547. (7)Nafie, L. A.; Freedman, T. B. J . Phys. Chem. 1986,90, 763. (8)Young, D. A,; Lipp, E. D.; Nafie, L. A. J . Am. Chem. SOC.1985,107, 6205. (9) Young, D. A.; Freedman, T. B.; Lipp, E. D.; Nafie, L. A. J . Am. Chem. SOC.1986,108, 7255. (10)Freedman, T.B.; Young, D. A,; Obocdi, M. R.; Nafie, L. A. J. Am. Chem. SOC.1987, 109, 1551. (11) Young, D. A.; Freedman, T. B.; Nafie, L. A. J. Am. Chem. Soc. 1987, 109, 7674. (12)Freedman, T. B.; Balukjian, G. B.; Nafie, L. A. J . Am. Chem. SOC. 1985,107, 6213. (13)Paterlini, M.G.; Freedman, T. B.; Nafie, L. A. J. A m . Chem. SOC. 1986,108, 1389. - (14)(a) Diem, M.;Gotkin, P. J.; Kupfer, J. M.; Tindall, A. G.; Nafie, L. A. J . A m . Chem. SOC.1977,99, 8103. (b) Diem, M.;Gotkin, P. J.; Kupfer, J. M.; Nafie, L. A. J . Am. Chem. Soc. 1978,100,5644.(c) Diem, M.;Photos, E.; Khouri, H.; Nafie, L. A. J . Am. Chem. SOC.1979,101, 6829.
0022-3654/89/2093-177 1$01.50/0
scribe the direction of electronic current around an intramolecular ring for a given phase of nuclear m o m e n t ~ m , l and - ~ >a~theoretical ~ approach to the ring-current mechanism has been formulated.' The VCD spectra of L-a-amino acids in aqueous solution at neutral pH exhibit strong positive intensity biases associated with the C,H methine stretching mode.6 This bias was attributed to current generated around a ring closed by hydrogen bonding between the amino and carboxylate groups. Our previous studies demonstrated that this bias was absent in L-cysteine hydrochloride and (S)-glycine-C,-dl and that, at neutral pH, the bias in some of the @-substitutedamino acids such as L-serine was much lower than that for alanine.6b These results suggest that additional intramolecular interactions in amino acids are important in generating VCD intensity in the C H stretching region and that these interactions are sensitive to the ionization state of the amino acid. In this report we examine in detail the pH dependence of the CH-stretching VCD spectra in alanine, alanine-2-dl, alanine-33-3-d3, serine, cysteine, and @-chloroalanine. Analyses of these spectra provide further insight into the sources of VCD intensity and detailed information on the solution conformations of these molecules.
Experimental Section The L- and D-enantiomers of alanine, serine, cysteine, and @-chloroalaninewere obtained from commercial sources (Sigma or Aldrich). Sample purities are approximately 98-99%, and no further purification was required. Samples of methyl and methine deuteriated L- and D- or DL-alanine (>98% D) were purchased from MSD Isotopes. All samples were studied as solutions in D20. Neutral samples were dissolved directly in D 2 0 and were exchanged three times with D 2 0 to remove labile hydrogen atoms. The highest and lowest pD samples correspond to the sodium salts and hydrochlorides, respectively, and were prepared by adding excess molar amounts of 1 M N a O H or 1 M HC1, respectively, to the amino acid samples, evaporating under vacuum to remove H 2 0 , adding D 2 0 , and exchanging. These samples were maintained in a nitrogen atmosphere to minimize absorption of atmospheric water. The titrated samples of L-alanine cited below were prepared by adding incremental amounts of HCI or NaOH and subsequently exchanging with DzO. Spectra were obtained by using a variable path length cell equipped with BaFz windows. The VCD spectra were recorded at approximately 14-cm-' resolution on a dispersive grating instrument described previo~sly.'~ All spectra were obtained with a 10-s time constant. The VCD spectrometer was interfaced15 to an IBM instruments C S 9000 Computer System on which all VCD data was recorded. VCD base lines were determined by comparing the VCD spectra of either the L- and D-enantiomersor the L-enantiomer and racemate. (15)Zuk, W. M.Ph.D. Dissertation, Syracuse University, 1986.
0 1989 American Chemical Society
1772 The Journal of Physical Chemistry, Vol. 93, No. 5, 1989
Zuk et al.
I
IO
5-
I: 4
I
4-
*
3-
- neutral ---.pD 5 ........ pD 3
W
3000
Figure 1. Absorption (lower) and VCD (upper) spectra of L-alanine-N-d, in the CH stretching region, titrated to lower pD. Sample concentration 1.7 M in D20 solution; path length 150 pm. (-) neutral pD; (- - -) pD 5, pD 3; (-*-) pD 1.5. (a**)
I
2900
WAVENUMBER
WAVENUMBER
Figure 3. VCD and absorption spectra in the CH stretching region of ~-alanine-2-d~-N-d~ at neutral pD (-), pD 13.5 (---),and pD 1.5 sample concentration 1.7 M in D20; path length 150 pm. (.e.).
TABLE I: Frequencies, Intensities, and Assignments of CH Stretching Modes of L-Alanine-N-d3
1
absorption" freq, t, 10) cm-l cm2 m o P
6
VCD freq, cm-'
104at,10' cm2 mol-'
assignmentb
Neutral pD
I
- neutral
3008 2992
12.9 (sh) 14.7
2952 2921 2895 2839
11.2 2.2 (sh) 4.6 1.0
3049 3010 2993
1.3 5.0 (sh) 7.1
2962 2924 2896 2847
8.2 2.5 3.0 0.8
3011 2990 2966
+2.6 -4.5
+11.8
2915 2882 2852
+1.0 -0.6 +0.2
{
pD 1.5 3052 3005
-0.3 -0.4
2974
+1.1
2921
+1.0
2848
+0.2
WAV EN UMBER
{
ue(CH,) ue(CH3) v(C,H) us(CH3) + 2 X P(CH3) Fermi triad P(CH3) + P(CH3) u~(CO~+ H )v'(CO~H) uS(CH,) UYCH,) u(C,H) uS(CH3)+ 2 X P(CH3) Fermi triad 6"(CHj) + dS(CH3)
pD 13.5
Figure 2. CH stretching absorption and VCD spectra of L-alanine-N-d,, 1.7M in D20 solution, 150-gm path length as a function of pD: (-) neutral pD; ( - - - ) pD 9.5, pD 11.5; (-.-) pD 13.5.
2980 29.0
(e-)
Absorption spectra were recorded at 4-cm-I resolution on a Nicolet 7 199 FT-IR spectrometer. Since all experiments involving the CH stretching region were conducted in D 2 0 solution, VCD and absorption spectra are described as a function of pD instead of p H (pD = p H 0.4), accurate to within 0.2 pD units.
+
Results L-Alanine and Deuteriated Isotopomers. The CH stretching VCD and infrared absorption spectra of L-alanine-N-d3 at neutral pD and at three low pD values are presented in Figure 1. The corresponding spectra for titration to higher pD values are shown in Figure 2. The VCD and absorption spectra of ~-alanine-2dl-N-d3and ~-alanine-3,3,3-d~-N-d~ at neutral, high, and low pD values are compared in Figures 3 and 4. Observed band frequencies, intensities, and normal mode assignments are compiled in Tables 1-111. The ~-alanine-N-d,absorption spectra are a composite of the ~-alanine-2-d,-N-d~ and ~-alanine-3,3,3-d~-N-d~ spectra. The band assignments determined previously for neutral pDI6 can be readily
2940 2912 2888 a
18.4 9.1 (sh) 9.4
2994 297 1 -2910'
+2.4 -5.7 +4.7
fl(CH3) ua(CH3) u(C,H) 2 X P(CH3)
2855
Absorption intensities measured relative to extrapolated base line
for each spectrum. = antisymmetric stretch; uJ = symmetric stretch; d = CH deformation; sh = shoulder. CModeestimated to occur at -2930 cm-l in the absence of overlapping negative bands.
extended to the other pD values. The two, nearly degenerate, antisymmetric methyl stretching modes give rise to the absorption feature near 3000 cm-'. The symmetric methyl stretching fundamental undergoes strong Fermi resonance with'the overtones of the antisymmetric methyl deformation modes, resulting in a Fermi triad consisting of the intense bands near 2950 and 2890 cm-' and a weaker feature near 2910 cm-1.16bThe methine stretch generates a broad, weak band near 2970 cm-' at neutral and low ~
~~~
(16) (a) Diem, M.; Polavarapu, P. L.; Oboodi, M.; Nafie, L. A. J . Am. Chem. SOC.1982, 104, 3329. (b) Lal, B. B.; Diem, M.; Polavarapu, P. L.; Oboodi, M. R.; Freedman, T. B.; Nafie, L. A. J . Am. Chem. Sor. 1982,104, 3336. ( e ) Freedman, T. B.; Diem, M.; Polavarapu,P. L.; Nafie, L. A. J . Am. Chem. SOC.1982, 104, 3343.
VCD in CH-Stretching Region of L-a-Amino Acids
The Journal of Physical Chemistry, Vol. 93, No. 5, 1989 1773
TABLE II: Frequencies, Intensities, and Assignments of CH Stretching Modes of ~ - A l a n i n e - 2 - d ~ - N - d ~
absorptiona freq, t, io3 cm-1 cm2 mol-'
VCD freq, i04At;i03 cm-l cm2 mol-1 Neutral pD
3009 2990
6.1 (sh) 9.1
2949 2918 289 1 2848
5.1 1.3 (sh) 2.6 0.6
3008 2997 2952 2914 2892 2847
4.8 (sh) 5.8 4.5 0.6 2.0 0.5
301 1
+1.2
2949
-1.0
2857
-0.5
2978 2939 2908 2884
22.9 11.4 3.8 (sh) 5.8
2989 2940
+2.5 -2.1
2860
-0.8
3010 2987 2969e 2943
+2.2 -1 .o +1.2 -1.5
2871
-0.8
I
I
I
I
assignmentb
-21
L-Alanine-3,3,3-d3-N-d3
pD 1.5 v8(CH3) 2 X P(CH3)
Fermi triad P(CH3)
pD 13.5
+ P(CH3)
{ p$&,) Fermi triad
Absorption intensities measured relative to extrapolated base line for each spectrum. = antisymmetric stretch; 4 = symmetric stretch; 6 = C H deformation; sh = shoulder. CDueto L-alanine N-d3 impurity in deuteriated sample. TABLE 111: Frequencies, Intenlties, and Assignments of CH Stretching Modes of ~ - A l a n i n e - 3 , 3 , 3 - d ~ - N - d ~
absorptiona freq, e, io3 cm-I cm2 mo1-I
VCD freq, i04Ae, io3 cm-I cm2 moP Neutral pD
3013 2970 2924 2892
3.0 2.5 0.6 0.4
3021 2970
-0.4 +5.2
2888
+1.2
3050 2970
0.9 (sh) 4.0
2970
2975 2936 289 1
4.2 (sh) 11.4 4.2 (sh)
2972 2933 2885
assignmentb
+
IqC02) vS(CO2) G H ) $ ( C o d + 6(CuH) va(C02) 6(C,H)
+
pD 1.5 +0.5
P(CO2) 4CJ-U
+ v"C02)
pD 13.5 +1.1 (sh) +5.0 +1.7
4CJ-U
a Absorption intensities measured relative to extrapolated base line for each spectrum. = antisymmetric stretch; vs = symmetric stretch; 6 = C H deformation; sh = shoulder.
pD, which increases in intensity and shifts to -2930 cm-I at high pD. The methine absorption is not resolved in ~-alanine-N-d,at neutral pD, but a distinct feature due to the methine stretch is observed at 2962 cm-' as the pD is lowered, and at high pD, a shoulder at -2930 cm-I in L-alanine-N-d,, which is absent in ~-alanine-2-d~-N-d,, can be attributed to the methine stretching mode. Weaker features can be ascribed to combination bands, the most prominent being the band near 2845 cm-I due to the methyl symmetric plus methyl antisymmetric deformation combination (fundamentals at 1460 and 1385 cm-I) and the carboxyl or carboxylate symmetric plus antisymmetric stretching combination at 3050 or 3013 cm-I. The latter feature is most evident Combination bands due to the anin ~-alanine-3,3,3-d,-N-d,. tisymmetric COz- stretch plus a methine bend are also observed in some of the spectra. Several general trends are noted in these spectra. As the pD is raised from neutral, the absorption bands shift to lower frequency and increase in intensity by a factor of 2-5. As the pD is lowered, the methyl stretching absorption intensities decrease by a factor of 2, while the methine absorption increases slightly
WAV.ENUMBER
Figure 4. Absorption and VCD spectra of ~-alanine-3,3,3-d,-N-~~ in the C H stretching region. Sample concentration 1.7 M; path length 150 pm. (-) neutral pD; (- - -) pD 13.5; pD 1.5. (-e)
in intensity. The frequencies of all C H stretching modes in neutral alanine and other amino acids are higher than the corresponding frequencies observed in hydrocarbons. This is apparently due to the fact that the C H bonds are in the environment of charged NH3+ and C02- groups. Removing the charge on the amino group has the largest effect on the C H stretching frequencies, at 1037-cm-I decrease in frequency. The effects of changing solution pD on the VCD spectra are quite dramatic. We consider first the spectra of ~-alanine-2dl-N-d3and ~-alanine-3,3,3-d,-N-d,, for which no coupling between methine and methyl modes is present. The methine stretch in ~-alanine-3,3,3-d~-N-d, generates strong, monosignate positive VCD intensity at both neutral and high pD, but since the molar absorptivity for the mode increases when the neutral amine group is formed, the anisotropy ratio A € / € decreases from 2 X lo4 at neutral pD to 2.5 X at pD 13.5. The change in VCD intensity upon formation of the hydrochloride salt, pD 1.5, is even more dramatic. Both the VCD intensity and anisotropy ratio decrease by over a factor of 10 compared to neutral pD; only a very weak, broad VCD signal remains. The positive VCD intensity producing the shoulder at -2890 cm-' at neutral and high pD can be attributed to the combination band of the antisymmetric carboxylate stretch and the lower frequency methine bend, which has gained both absorption and VCD intensity through Fermi resonance with the methine stretch. The VCD spectra of ~-alanine-2-d~-N-d~ exhibit weak features corresponding to the antisymmetric and symmetric methyl stretches at all three pD values. At neutral pD, the frequency splitting of the two antisymmetric methyl stretches is evident in the absorption spectra, and a (+,-) VCD couplet arising from the splitting of the antisymmetric methyl stretching degeneracy in a chiral environment is observed at 3010 and 2987 cm-'. At pD 1.5, the VCD intensity in this region decreases, and the two absorption features are no longer resolved. At both pD 1.5 and pD 13.5, only a positive feature is observed for the antisymmetric stretches, centered 10 cm-' above the absorption maximum. At all three pD values, the symmetric methyl stretching mode contributes negative VCD intensity that is distributed between the highest and lowest members of the Fermi triad roughly in proportion to the absorption intensities. The positive VCD intensity at -2970 cm-I at neutral pD can be attributed to the methine stretch due to some undeuteriated L-alanine-N-d3impurity in the sample. This impurity was previously detected in the Raman spectrum in the methine bending region16aand in our earlier VCD study.'6b In the parent compound, the VCD at low pD is very weak and at high pD the methine stretch is weaker, broader and
Zuk et al.
1774 The Journal of Physical Chemistry, Vol. 93, No. 5, 1989 TABLE I V Frequencies, Intensities, and Assignments of CH Stretching Modes of 8-Chloro-L-alanine-N-d3
absorption" freq,
cm" 3030 2970
io3 cm2 mo1-l e,
7.8 9.3
VCD freq, 1o4aC, 10) cm-' cmz mol-' Neutral pD
.o
3040
-1
2955
+6.0
assignmentb UYCH2) uS(CH2) 4CJ-O
PD 2 3038 2982 2957
8.5 9.3 (sh) 14.0
3032 2980 2945
+1.5 -3.0 +1.5
l\
41
s= i -
ua(CHz) 4CJ-V
12
vs(CHz) " Absorption intensities measured relative to extrapolated base line = antisymmetric stretch; 4 = symmetric for each spectrum. stretch; sh = shoulder. less prominent than at neutral pD. The undeuteriated impurity in ~ - a l a n i n e - 2 - d ~ - Ndoes - d ~ not give rise to distinct features at low or high pD in Figure 3. In L-alanine-N-d3 the methine stretch couples to some extent with each methyl stretch, the largest coupling being with the lower frequency antisymmetric methyl stretch. This coupling has only a minor effect on the frequencies and absorption intensities in this region. In the VCD spectra, the mixing of the methine and antisymmetric methyl stretches gives rise to a coupled oscillator contrib~tion,~' manifested by a (-,+) VCD couplet at -2990, -2966 cm-', superimposed on the intrinsic contributions from the methyl and methine stretching modes. Thus, in the VCD spectrum of ~-alanine-N-d,at neutral pD, the antisymmetric stretch at 2985 cm-' exhibits larger negative VCD intensity than in ~-alanine-2-d,-N-d,,and the methine stretch at 2966 cm-' appears less broad than in ~-aIanine-3,3,3-d,-N-d~due to the cancellation from negative contributions from the antisymmetric methyl stretch at 2985 cm-' and the symmetric methyl stretch at -2945 cm-l. The coupled oscillator contribution results in an increased positive VCD intensity for the methine stretch compared to that in ~-aIanine-3,3,3-d,-N-d,. In the VCD spectra for titrations of L-alanine-N-d3 to both higher and lower pD, Figures 1 and 2, distinct isosbestic points are observed. These are indicative of the equilibrium between two species, the zwitterion and neutral carboxyl forms as the pD is lowered and the zwitterion and the neutral amine forms as the pD is raised. Trends similar to those observed for the two isotopomers are noted in the VCD spectra of titrated L-alanine. As the pD is lowered, the VCD intensity of all the bands is drastically reduced; at pD 1.5 only very weak positive VCD intensity remains, corresponding in frequency to the methine stretch at 2966 cm-' and the methyl symmetric stretch/antisymmetric deformation overtone Fermi triad component at 2910 cm-I. As the pD is raised, the bands shift to lower frequency. Due to the coupling between the methine stretch and lower frequency antisymmetric methyl stretch, the (+,-) VCD couplet for the antisymmetric stretches is observed at all the higher pD values, in contrast to the monosignate feature at pD 13.5 in the VCD spectrum of ~-alanine-2dl-N-d3. The negative VCD intensity contributions from the antisymmetric methyl stretch at -2975 cm-l and symmetric methyl stretch at 2940 cm-' shift the observed maximum of the positive methine stretching VCD contribution from 2933 to 2910 cm-' for the species with a neutral amine group. P-Chloro-L-alanine,L-Serine, and L-Cysteine. In this series of amino acids with a chloro, hydroxyl, or mercapto P-substituent, three rotameric conformers about the C,-C, bond are possible. The C H stretching spectra contain contributions from the methylene antisymmetric stretch, the methine stretch, and the methylene symmetric stretch. Depending on the frequency of the latter mode, Fermi resonance between the symmetric methylene stretch and overtone of the methylene scissors mode may also be present. The coupling between the methine and methylene modes will depend on both the C,-C, conformation and the relative frequencies of the modes.
WAVENUMBER Figure 5. CH stretching VCD and absorption spectra of @-chloro-Lalanine at neutral pD (-) and pD 2 (---). Sample concentration 1.7 M in D,O; path length 150 Fm.
3
n
-' t
L-Serine
~
WAVE NUMBER
Figure 6. VCD and absorption spectra of L-serine-N-d3-O-din the CH stretching region. Spectra recorded at neutral pD (-), pD 13 (---), and for samples 1.7 M in DzO solution with 150-~m path length. pD 2 (-e)
The absorption and VCD spectra of D 2 0 solutions of P-chloro-~-alanine-N-d~ hydrochloride and an equimolar mixture of NaOD and P-chloro-~-alanine-N-d,hydrochloride are compared in Figure 5, and frequencies, intensities, and assignments are given in Table IV. The presence of the chloro substituent causes all the bands to be shifted to higher frequency compared to L-alanine-N-d,. At pD 2, the antisymmetric and symmetric methylene stretches are observed at 3038 and 2957 cm-I, respectively. The methine stretch is assigned to the absorption shoulder at 2982 cm-'. These thcee modes generate a distinct (+,-,+) VCD pattern. In the neutral sample, the methylene modes generate absorption features at -3030 and -2970 cm-I. A single intense positive VCD band is observed at 2954 cm-I, which we assign to the methine stretch. The absorption and VCD spectra of ~-serine-N-d,-O-dat neutral, basic, and acidic pD are presented in Figure 6. Frequencies, intensities, and mode assignments are collected in Table V. The VCD and absorption spectra of L-serine in D 2 0 at neutral pD have been presented previously.6bq1" These measurements have been repeated for this study. The two principal bands in the absorption spectrum at all three pD values result from a strong Fermi resonance interaction between the methylene symmetric stretch and the overtone of the 1475-cm-' scissors vibration. The antisymmetric methylene stretch is not resolved in any of the absorption spectra, but should lie at -2980 cm-'. The methine stretch, also obscured by the methylene symmetric stretching
-
VCD in CH-Stretching Region of L-a-Amino Acids
The Journal of Physical Chemistry, Vol. 93, No. 5, 1989 1775
3030 2960 2899
2960 2905
2.2 3016 2967
-0.2 +3.3
12.9 10.0
2900
+0.5
11.3 7.4
2985 2960 2895
PD 2
-0.8
1::;
2950
2920 2883 2880
+1.3
23.2 17.7
+2.6 +2.4
I
vycoz-)+ vl(c02-) v'(CH2) v(C,H) vl(CHz) + 2 Fermi diad
u(C,H) V'(CH2) + 2 Fermi diad
X
b(CH2)
X
b(CH2)
X
6(CH2)
pD 13.5 2966
,
I
TABLE V: Frequencies, Intensities, and Assignments of CH Stretching Modes of L-Serine-N-d3-O-d absorption" VCD t, io3 freq, i04Ar, 10' freq, cm-' cm2 mol-' cm-l cm2 mol-' assignmentb Neutral pD
v(C,H) S(CH2) + 2 u(C,H) v(C,H) vl(CH2) + 2
- ..--
X
6(CH2)
"Absorption intensities measured relative to extrapolated base line for each spectrum. * v a = antisymmetric stretch; vl = symmetric stretch; 6(CHz) = scissors deformation. Fermi diad in the absorption spectra, should occur at frequencies near those in the alanine spectra. The effects of pD on the absorption spectra of L-serine-N-d3-Od are similar to those observed for L-alanine-N-d3, a decrease in intensity at low pD and a large increase in intensity accompanied by a decrease in frequency at high pD. The strongly biased VCD spectrum at neutral pD is dominated by the positive methine stretching band at 2967 cm-'. The methylene symmetric stretching Fermi diad contributes positive intensity at 2900 cm-' and, presumably, at 2960 cm-'. The integrated intensity of the major positive VCD feature is approximately half that of the methine stretch of ~-alanine-3,3,3-d~-N-d~ at neutral pD. At low pD the VCD intensity is again greatly diminished. The weak (-,+) VCD feature at 2985,2960 cm-l may arise from coupling between the methine and symmetric methylene stretches. At high pD, three positive VCD features are observed. The band at 2920 cm-' can be associated with a methine stretch, since the frequency is near that observed for L-alanine-3,3,3-d3-Nd3 at high pD. The band at 2880 cm-' occurs at a frequency assigned to a component of the Fermi diad involving the symmetric methylene stretch. However, since any VCD intensity due to the symmetric methyl stretch should be. distributed between the components of the Fermi diad in proportion to the absorption intensities and no intense positive VCD band is observed at 2950 cm-I, the 2880-cm-' VCD band cannot be assigned to the symmetric methylene stretch. The third positive feature centered at 2965 cm-' may result from the antisymmetric methylene stretch, but it is unlikely that this mode would have a larger VCD intensity than the symmetric methylene stretch. A plausible explanation for all three positive VCD features in L-serine-N-d2-Odat pD 13 is assignment to the methine stretch in the three C,-C, rotamers. We will consider this assignment in more detail below. The absorption and VCD spectra of L-cysteine-N-d,-S-d at neutral pD, pD 2, and pD 13 are given in Figure 7, and frequencies, peak intensities, and vibrational mode assignments are collected in Table VI. The VCD spectra of L-cysteine-N-d3-S-d at neutral pD and of L-cysteine-N-d3-S-dhydrochloride have been discussed previously.6b Both the absorption and VCD spectra of L-cysteine differ greatly in magnitude from the corresponding spectra of L-serine. Although the absorption peak intensities of cysteine at neutral pD are approximately half those of serine, the maximum VCD intensity for cysteine is about three times larger. The presence of the mercapto substituent increases the frequency of the methylene stretching modes in cysteine, compared to serine, and there is no evidence of Fermi resonance at neutral or low pD. The two intense absorption features at these pD values correspond to the antisymmetric methylene stretch ( 3 0 1 0 cm-I)
WAVENUMBER
Figure 7. CH stretching VCD and absorption spectra of L-cysteine-Nd3-S-d at neutral pD (-), pD 13 (---), and pD 2 Sample concentration 0.83 M in D20; path length 150 pm. (e-).
TABLE VI: Frequencies, Intensities, and Assignments of CH Stretching Modes of L-Cysteine-N-d-S-d absorption" VCD freq, t, io3 freq, i04Ac, io3 cm-' cm2 mol-' cm-' cm2 mol-' assignment* Neutral pD 3010 4.5 3020 -1.8 va(CH2) 2978 +9.0 u(C,H) 2955 6.1 WH2) 3010
6.4
2955
8.2
2940
15.1
3005 2960 2930
PD 2 +1.5 -0.8 +1.5
fl(CH2) v(C,H) vl(CH2)
pD 13 2916 2834
2935
+7.3
2888
+5.0
17.7 5.8
fl(CH2) v(C,H) vl(CH2) + 2 u(C,H) vS(CH,) + 2
X
b(CH2)
X
b(CH2)
Absorption intensities measured relative to extrapolated base line = antisymmetric stretch; vs = symmetric stretch; b(CH2) = scissors deformation. for each spectrum.
and symmetric methylene stretch (2955 cm-I). The methine stretching mode occurs at -2978 cm-I, as seen from the VCD data presented below. At high pD, the S D group (pK, 8.5), along with the ND3+ group, is deprotonated, and the methylene and methine stretching frequencies decrease. We tentatively assign the 2940-cm-' shoulder to the antisymmetric methylene stretch, and the 2916- and 2834-cm-' features to a symmetric methylene stretching Fermi diad. At neutral pD, the antisymmetric methylene stretch in L-cysteine-N-d, gives rise to a negative VCD band at 3020 cm-I. The methine stretch produces an intense VCD feature at 2978 cm-I. The positive shoulder at 2940 cm-' can be ascribed to the symmetric methylene stretch. At pD 2, a weak (+,-,+) VCD pattern is observed that is similar to the @-chloro-L-alanine-N-d3hydrochloride VCD spectrum (Figure 6). The VCD spectrum of Lcysteine-N-d, at pD 13 consists of two intense, positive VCD bands at 2935 and 2888 cm-I. These features may also arise from the methine stretching mode in two different conformations, since it is unlikely that the antisymmetric methylene stretch could contribute such a large positive VCD band. Discussion An interpretation of the pD dependence of the C H stretching VCD spectra of simple L-a-amino acids in D 2 0 solution must account for several general observations: (a) at neutral pD, the methine stretch generates positive VCD intensity with large an-
1776 The Journal of Physical Chemistry, Vol. 93, No. 5, 1989 isotropy ratio; (b) at low pD and in the absence of nuclear coupling, the methine stretching VCD is very weak; (c) at high pD, the methine stretching VCD is positive with a smaller anisotropy ratio than at neutral pD; (d) at neutral and high pD, the anisotropy ratios for the VCD bands in L-serine are smaller than those for L-cysteine, P-chloro-L-alanine, or L-alanine; (e) at low pD, a conservative (+,-,+) VCD pattern is observed for the C H stretching modes in L-cysteine and fl-chloro-L-alanine; a (-,+) pattern is observed in L-serine; (0 at high pD, three broad positive VCD bands in L-serine and two broad positive VCD bands in L-cysteine are observed, none of which correlate with methylene stretches; and (8) (S)-glycine-C-d, at neutral pD is an exception to (a) above, since a strong positive methine stretching VCD band is not observed for this amino acid with no The VCD spectra for a localized vibrational region such as the C H stretching region can be understood as the superposition of two characteristic VCD patterns. Two or more bands of alternating sign with conservative VCD intensity (that is, with no net VCD intensity for the group of bands) originate from the coupling of chirally oriented oscillator^,^^ or from the splitting of two degenerate modes by a chiral environment.'* The intrinsic positive or negative VCD due to a single vibration of an isolated group, such as a methyl, methylene, or methine group, can in some cases be the predominant source of VCD intensity in a region. Such intrinsic VCD contributions may also result in positive or negative VCD bias in a region in which coupling effects are also present. The conservative VCD spectrum at low pD and the strongly positively biased spectrum at neutral pD for fl-chloro-L-alanine in Figure 5 are examples of those two VCD patterns. Conservative VCD patterns can often be adequately explained by the coupled oscillator mechanism.17 We have developed the ring-current mechanism of VCD to explain intense, biased intrinsic VCD contributions in a wide variety of molecules for several types of vibrational motion.'.' Vibrational circular dichroism intensity is proportional to the rotational strength, the scalar product of the electric dipole transition moment, p, and the magnetic dipole transition moment, m. The main premise of the ring-current mechanism is that localized nuclear vibrational motion external to or within a ring closed by covalent or hydrogen bonding can generate electronic current in the ring. This current, oscillating at the vibrational frequency at constant electron density, results in a large magnetic moment contribution but does not contribute to the electric dipole transition moment. The ring-current mechanism also provides a link between the sign of the ring-current-enhanced VCD features and local conformations in the molecule. For three types of vibrational motion, empirical rules, based on a large number and a wide variety of spectra, have been devised that dictate the direction of current flow around a ring generated by a given sense of driving nuclear m o m e n t ~ m . ] * The ~ . ] ~direction of the ring-current magnetic dipole transition moment can be determined from the right-hand rule for positive current flow in a ring (opposite in direction to electron flow). For simple localized nuclear motion, the direction of the electric dipole transition moment can be deduced from the directions of the atomic displacements. Thus, the sign of p m can be predicted for a vibrational mode for various possible local conformations and compared with experiment. For the interpretation of the C H stretching VCD spectra of L-a-amino acids, we require both the direction of positive p for a C H stretch, which has been shown to be C-H for C H contraction,I9 and the direction of ring current generated by the stretching mode of a C H bond external to a ring. According to rule 1 of the ring-current model,'Jz the contraction of a C H bond adjacent to a heteroatom X in a ring results in electron flow preferentially toward the heteroatom, which initiates positive (17) (a) Holzwarth, G.; Chabay, I. J . Chem. Phys. 1972,57, 1632. (b) Sugeta, H.; Marcott, C.; Faulkner, T. R.; Overend, J.; Moscowitz, A. Chem. Phys. Let?. 1976,40, 397. (18) Nafie, L . A.; Polavarapu, P.L.; Diem, M . J . Chem. Phys. 1980,73, 3530. (19) Wiberg, K. 9.;Wendoloski, J. J. J . Phys. Chem. 1984,88, 586.
Zuk et al. current around the ring directed X-C. As originally formulated,6 the positive enhanced VCD for the methine stretch in an L-amino acid is attributed to current around the ring closed by NH-OC hydrogen bonding, as diagrammed in 1.
't
H
m
1
This simple schematic diagram is not sufficient to explain the pH dependence of the VCD spectra reported here or the results In order to understand this data, we for (S)-glycine-C-dl-N-d3.6b must consider in detail the probable conformations for the substituents on the a-carbon and all the possible intramolecular associations. We consider structures in which both the R group and the amine or protonated amine bonds are staggered with respect to C,H. The most stable conformations for the carboxylate group are those that involve intramolecular CO-HN hydrogen bonding. We define the dihedral angle 9 for the carboxyl or carboxylate group with respect to the methine bond as shown in 2.
L
Three possible hydrogen-bonded structures for L-alanine are shown in structures 3a-c, in which one carboxylate oxygen is
3a
y
@
3b
Q
Q
:: : :; a
3c
.
0'
hydrogen bonded to the amino hydrogen trans to C,H (9= 30°) in 3a, to the amino hydrogen gauche to C,H (9= 90°) in 3b, or to both in a bifurcated structure (9 = 60') in 3c. In 3b, and, somewhat more weakly, in 3c, interaction between the other carboxylate oxygen and a methyl CBH bond is possible. Although this is not a "traditional" hydrogen bond, there is evidence from crystal structures20,21and t h e r m o ~ h e m i s t r y , and ~ ~ - from ~ ~ VCD (20) (a) Taylor, R.; Kennard, 0. Acc. Chem. Res. 1984,17, 320. (b) Taylor, R.; Kennard, 0. J . Am. Chem. SOC.1982,104, 5063. (21) Sutor, D. J. J . Chem. SOC.1963, 1105. (22) Meot-Ner (Mautner), M. Acc. Chem. Res. 1984,17, 186. Meot-ner (Mautner), M. J . Am. Chem. SOC.1983,105, 4912.
VCD in CH-Stretching Region of L-a-Amino Acids
The Journal of Physical Chemistry, Vol. 93, No. 5, 1989 1777 0
4a
p
1
900
R >> 0
Figure 8. Stereoprojections of L-alanine depicting the directions of the electric dipole transition moment fi and the ring-current magnetic dipole transition moment m for a methine CH contraction. Three carboxylate orientations are shown. Loop with arrow indicates pathway for positive
ring current. spectra of other m ~ l e c u l e s , ~that ~ ~ interactions ' ~ ~ ' ~ ~ ~ between ~ CH bonds and lone pairs do occur and can play a stabilizing structural role. Ab initio calculations26-28on (CH-X) hydrogen bonds indicate that C H can indeed serve as a proton donor, and, for ionic species, the bonding interaction can be substantial. The rotational strengths for these structures arising from the ring current interpretation are illustrated in Figure 8. For cp = 30°, the methine bond is equatorial to the hydrogen-bonded ring, and p and m are nearly orthogonal. Little ring-current enhancement for the methine stretching vibration is predicted for this conformation. For cp = 60-90°, the methine bond is axial to the hydrogen-bonded ring involving the gauche N H bond, and, furthermore, the CO-HC interaction enlarges the ring-current pathway with a ring segment to which the methine is also axial. Thus, with these orientations of the carboxylate group, p and m have parallel components, and positive ring current enhanced VCD is predicted for the methine stretch in these confomations. The maximum VCD intensity will be at cp = 90'. The importance of the auxiliary C,H-OC ring is seen by comparing the results for ~ - a l a n i n e - N - dand ~ (S)-glycine-2-dlN-d3.6bIn the latter species a deuterium replaces the methyl group in L-alanine, and no 0-CH bonds are present. Three orientations of the carboxylate group are illustrated in 4a-c. Structures 4a and 4b for deuteriated glycine are energetically equivalent; structures with hydrogen bonds to the amino hydrogens gauche or trans to C,H should be equally populated. Structure 4c depicts the bifurcated hydrogen bonded structure; alternatively, a structure similar to 4c, but with a single hydrogen bond to an amino group rotated by 30°, is possible. In both 4b and 4c current generated around the gauche NH-OC ring by the methine stretch will result in a magnetic dipole transition moment with a positive overlap with p. However, no enhanced methine stretching VCD is observed for (S)-glycine-2-dl-N-d3. The magnetic moment due to (23) Grimsrud, E. P.; Kebarle, P. J . Am. Chem. SOC.1973, 95, 7939. (24) de Boer, J. A,; Reinhardt, D. N.; Harkema, S.; van Hummel, G. J.; de Jong, F. J . Am. Chem. SOC.1982, 104, 4073. (25) Ragunathan, N.; Freedman, T. B.; Nafie, L. A. Unpublished results. (26) Vishveshwara, S. Chem. Phys. Letr. 1978, 59, 26. (27) Hirao, K.; Sano, M. Chem. Phys. Lett. 1982, 87, 181. (28) Sreerama, N.; Vishveshwara, S. J . Mol. Struct. (Theochem) 1985, 133, 139.
4b
current in a ring is directly proportional to the area of the ring. Within the framework of the ring-current mechanism, the enlarged, favorably oriented current pathways for L-alanine shown in Figure 8 for 4 = 60' and 90' increase both the magnitude of m and the positive overlap of m and p, compared to deuteriated glycine. It is interesting to note that, in deuteriated glycine, the VCD in the 2850-3050-cm-' region6bis dominated by combination bands which may cancel any weak positive methine contribution. The (-,+) features at 2940, 2888 cm-I arise from the combination of the antisymmetric carboxylate stretch and the two methine bends; the VCD signs are the same as observed for the methine bending f ~ n d a m e n t a l s . Both ~ ~ the symmetric plus antisymmetric carboxylate stretching combination band at 3018 cm-' and the antisymmetric carboxylate stretching fundamenta130 exhibit negative VCD. The large decrease in magnitude of the methine stretching VCD at low pD can also be explained by the carboxyl orientation. When a carboxylate oxygen is protonated at low pD, the carboxylate is no longer charged, the lone pairs of the oxygen are redirected, and the CO-HC interaction depicted in 3b is weakened and no longer favorable. We propose that the conformation of L-alanine at low pD is predominantly 5 ( c p = 30°), with the methine bond
0
0
equatorial to the hydrogen-bonded ring, for which there is no carboxylate-methyl interaction and no ring-current-enhanced VCD intensity. When the pD is raised, we observe a large increase in methine stretching absorption intensity, but not VCD intensity, for Lalanine-3,3,3-d3-N-d3 (Figure 4). The distorted band shapes, which are not identical for absorption and VCD, suggest the presence of multiple conformations. Some possible forms are depicted in 6a-c, in which both the carboxylate orientation and the position of the nitrogen lone pair are varied. The presence of the nitrogen lone pair leads to an increase in methine absorption intensity. Clearly, the position of the nitrogen lone pair will influence the carboxylate orientation. If the carboxylate conformations with cp = 30' or 60' are more favored relative to cp = 90' at high pD compared to neutral pD, a decreased net (29) Freedman, T. B.; Chernovitz, A. C. Zuk, W. M.; Nafie, L. A. J . A . Chem. SOC.1988, 1IO, 6970. (30) Roberts, G. M.; Lee, 0.;Calienni, J.; Diem, M. J . Am. Chem. SOC. 1988, 110, 1749.
1778 The Journal of Physical Chemistry, Vol. 93, No. 5, 1989
Zuk et al. aqueous solution, since the VCD couplet for these two modes has the opposite sign pattern in the two phases. In the Raman spectra,'" the higher frequency antisymmetric stretch is the more intense in the solid, whereas in solution the lower frequency mode is the more intense, also suggestive of differences in the nature of the splitting. The three amino acids with p-substituents included in this study can assume the rotameric forms 1-111. From N M R measure-
I
ring-current enhancement at high pD would result, producing an observed decrease in anisotropy ratio. In ~-alanine-Zd~-N-d~ at neutral pD, the (+,-) features at 3010 and 2987 cm-] are due to a splitting of the degeneracy of the antisymmetric methyl stretches by the chiral environment.18 The symmetric methyl mode exhibits weak, intrinsic negative VCD intensity, which may arise from a structure with cp = 30'. In this orientation, the methyl group is axial to the hydrogen-bonded ring. The in-phase contraction of the methyl CH bonds can generate electronic current around the hydrogen-bonded ring, in a direction dictated by rule 1, resulting in negative ring current enhanced VCD for the symmetric methyl stretch as shown in 7.
+
7
The VCD intensity of this mode does not change much pD and is consistent with some population of this carboxylate conformation at all pD values. We have previously proposed a similar interpretation for the VCD intensity arising from the symmetric methyl stretch of the OCH, group in a- and @-methyl glucoside.'^ The interpretation developed here for L-alanine in solution also allows us to understand the VCD spectrum of polycrystalline ~-alanine-N-d,as a mull in a halocarbon oil.'" The observed VCD bands (with AA X lo5values in parentheses) at 2996 (-l.l), 2985 (+2.8), and 2962 cm-' (-3.5) correspond to strong features in the Raman spectrum assigned to the two antisymmetric methyl stretching modes and the symmetric methyl stretch. The methine stretch is also assigned near 2960 cm-I. The VCD pattern is opposite in sign to that of L-alanine-N-d3 in solution. From the crystal structure of alanine,^' we find cp = 41.4' for the carboxylate orientation, with 0.-HN separations of 2.45 and 2.68 A for the N H bond trans or cis to C,H, respectively; that is, the hydrogen bonding is to the trans N H . With this carboxylate orientation, the methyl group is axial to the CO-HN hydrogen-bonded ring, whereas the methine is nearly equatorial to this ring. The negative VCD observed at 2962 cm-' can thus be assigned to the methyl symmetric stretch, as in structure 7. The methine VCD is not enhanced, and does not contribute to the observed VCD spectrum. The environment of the methyl group in the crystal apparently gives rise to the opposite sense of splitting of the antisymmetric methyl stretching degeneracy compared to (31) Lehmann, M. S.; Koetzle, T. F.;Hamilton, W. D. J . Am. Chem. Soc 1972, 94, 2657.
U
[I1
ments, the rotameric populations of I, 11, and 111 were found to be 28%, lo%, and 62% for serine3*at pD 5 and 45%, 17%, and 38% for cysteine33 at pD 14. No measurements of rotameric populations are available for @-chloroalanine. In the VCD spectra of the amino acid hydrochlorides at pD 2, a (+,-,+) pattern is observed for P-chloro-L-alanine and L-cysteine, corresponding to the antisymmetric methylene, methine, and symmetric methylene stretches, respectively. A (-,+,+) VCD pattern for L-serine at pD 2 can be associated with the methine stretch and the two Fermi resonance components of the methylene symmetric stretch. These spectra exhibit little VCD bias. As we have previously shown@ ,' coupling of the methine and methylene motions will result in a conservative (+,-,+) VCD sign pattern for the antisymmetric methylene, methine, and symmetric methylene stretches of conformer I, VCD bands of the opposite signs for conformer 11, and little or no coupled oscillator VCD for conformer 111, in which the methine and methylene are not chirally oriented. In serine, the highest frequency positive VCD feature that is predominantly antisymmetric methylene stretch is apparently canceled by the central negative component. The VCD spectra are thus consistent with an excess of I over I1 at low pD in all three amino acids. From the ring-current interpretation developed above for alanine, we also deduce a large excess of the cp = 30' carboxyl conformation, since no ring-current enhancement for the methine is observed. At neutral pD, all three @-substitutedamino acids exhibit similar VCD spectra. Each spectrum is dominated by a broad positive band assigned to the methine stretch. Only very weak intensity is associated with the methylene stretches, negative for the antisymmetric stretch and positive for the symmetric methylene stretch. The intense methine stretching VCD band and the much lower anisotropy ratio for this mode in serine compared to cysteine, @-chloroalanine,or alanine-C-d3are readily understood within the ring-current interpretation. As for alanine, the methine stretch is strongly enhanced for conformers I and 111 for carboxylate conformations with cp = 90' and to a smaller extent with cp = 60'. In conformer 11, there is no @-CHin a position to interact with a carboxylate oxygen, and little ring-current enhancement is expected. The strong positive methine stretching VCD band and lack of any VCD corresponding to the methylene stretching/ methine coupling in @-chloro-L-alanineat neutral pD is indicative of a carboxylate conformation with cp near 90°, and a large excess of rotamer 111 and/or nearly equal populations of I and 11, for which the methylene contributions will cancel. In L-serine in conformers I and 111, an additional HO-HN hydrogen-bonded ring can form, as depicted in 8a and 8b. When the methine bond contracts, ring current can be directed around this ring in addition to the ring between the amino and carboxylate groups. This additional ring-current pathway will decrease the magnitude of the magnetic dipole transition moment due to current (32) Kainosho, M.; Ajisaka, K.; Kamisaka, M.; Murai, A. Biochem. Biophys. Res. Commun. 1965.64, 425. Kainosho, M.; Ajisaka, K. J . Am. Chem
SOC.1975, 97, 5630. (33) Fujiwara, S.; Arata, Y. Bull. Chem. SOC.Jpn. 1963, 36, 578.
VCD in CH-Stretching Region of L-a-Amino Acids
The Journal of Physical Chemistry, Vol. 93, No. 5, 1989 1779
10a
in the CO-HN ring, since the amount of current generated around that ring will decrease. In addition, the magnetic dipole transition moment due to current in the HO-HN ring of 8a and 8b (by rule 1) is either orthogonal or antiparallel, respectively, to the electric dipole transition moment for the methine stretch. The effects of the interaction between the hydroxyl and amino groups in L-serine thus serve to decrease the positive ring-current enhancement of the methine stretch compared to L-cysteine and @-chloro-L-alanine, for which any such hydrogen bonding to the &substituent is much weaker, and to L-alanine, which has no 0-substituent. At pD 13, L-serine exhibits three resolved broad positive VCD bands, whereas L-cysteine exhibits two. As noted above, the frequencies and relative intensities of these bands make assignment to methylene stretching modes unlikely. We have previously observed large differences in the methine stretching frequencies in similar molecules, depending on the relative orientations of the methine bond and adjacent substituents. For example, in sugars13 the stretch of a methine bond axial to the pyranose ring at carbon 2, 3, or 4 occurs at 2925 cm-I, whereas the stretching frequency of an equatorial methine bond at carbon 2, 3, or 4 is 2905 cm-'. In L-cysteine at pD 13, we propose that the positive VCD bands at 2935 and 2888 cm-' corresponding to the methine stretches in the two most abundant rotamers, shown in 9a and 9b, with cp n
9a
9b
= 60-90'. The amine orientation(s) and the exact correlation of each band with a specific structure cannot be definitely deduced from the VCD spectra. Clearly, however, the methine environment with respect to the location of the ionized sulfhydryl group and possibly the nitrogen lone pairs will differ considerably in the two conformers, and different stretching frequencies are reasonable. In L-serine, in addition to structures 10a and lob, which correspond to 9a and 9b but include OH-N hydrogen bonding between the hydroxyl and amine groups, an additional hydrogen-bonded form is possible. In 1Oc we show a structure in conformation I1 with a hydrogen bond between the hydroxyl and carboxylate groups, with cp = 60-90°. Again the methine bonds are in different environments in the three structures, and all three forms should exhibit positive ring current enhanced methine stretching VCD. Correlation of the methine stretches for the three possible hydrogen bonded forms loa-c with the three observed VCD bands is reasonable. A structure corresponding to 1Oc at neutral pH is also possible, but conformation I1 was found to be
10b
Q
0
present at low abundance at pD 5.
Conclusions The results of this study provide evidence that the C H stretching vibrational circular dichroism spectra of L-amino acids in aqueous solution are sensitive to pH due to conformational changes. Of particular importance are the orientation of the carboxyl or carboxylate group relative to the methine C,H bond and intramolecular associations, both of which can be altered by changes in pH. When the methine bond is equatorial to a ring closed by CO-eHN hydrogen bonding, little or no methine stretching VCD intensity is observed. Carboxylate orientations that place the methine bond axial to a CO-HN ring result in positive enhanced methine stretching VCD when the carboxylate also interacts with a @-hydrogen. The presence or absence of enhanced methine stretching VCD also correlates with the relative orientations of the methine bond and an adjacent hydrogen-bonded ring for OH-OH rings in sugars13 and OH--NH or HO-HN rings in ephedrine and related molecules.34 Rings closed by the interaction of C H bonds and lone pairs are important in the interpretation of the VCD spectra of amino acid-transition-metal complexes,1° amino alcohols,25and diols.25 The ring-current mechanism of VCD provides a consistent explanation for the sign and relative intensities of the C H stretching VCD features in all the spectra considered in this study, with the exception of a few couplets that can be ascribed to the coupled oscillator mechanism or the splitting of degeneracies in a chiral environment. Although the VCD spectra cannot provide quantitative determination of the relative concentrations of specific conformations, they do provide evidence for the predominant species in solution and for types of intramolecular interactions that are not generally probed by other physical or spectroscopic measurements. Acknowledgment. We acknowledge financial support by grants from the National Institutes of Health (GM-23567) and National Science Foundation (CHE-86-02854). Registry No. Alanine, 56-41-7; alanine-2-dl, 2 1386-65-2; alanine3,3,3-d3,63546-27-0; P-chloroalanine, 2731-73-9; serine, 56-45-1; cysteine, 52-90-4. (34) Freedman, T. B.; Lee, N.-S.; Nafie, L. A. Unpublished results.
