Infrared Spectroscopic Study of C7 Intramolecular Hydrogen Bonds in Peptides*

CH. PULLA RAO, P. BALARAM, and C. N. R: RAO,? Solid S ta te and Structural Chemistry U n i t , Indian Insti tute of Science, Bangalore-

560012, India

Synopsis

The ir-spectra in the N-H stretching region of Piv-Pro-NHMe and Boc-Pro-NHMe have been studied in carbon tetrachloride and chloroform solutions over a wide range of concen- trations. Based on the concentration dependence of the N-H stretching bands, i t has been shown that the characteristic N-H stretching band due to the C7 intramolecular hydrogen bond is around 3335 cm-'. Intermolecular hydrogen bonding also occurs to a small extent in these peptides, giving rise to a slight concentration dependence of the N-H stretching bands. The band around 3335 cm-* need not necessarily be due to C7 hydrogen bonds alone as proposed by Tsuboi et al. or to intermolecular hydrogen bonding alone as proposed by Maxfield et al.; this conclusion is supported by studies on Boc-Leu-NHMe, which undergoes only intermolecular hydrogen bonding We have shown that 2-Aib-Aib-OMe and Z-Aib- Ala-OMe form C7 intramolecular hydrogen bonds in addition to C5 intramolecular hydrogen bonds. The present studies also show that all the peptides studied exist in more than one conformation in solution.

INTRODUCTION

The importance of intraresidue interactions in determining conforma- tions of amino acid residues in proteins has been pointed out in the litera- ture.'-3 In order to understand the intraresidue interactions, experi- menta14-12 and theoretical13-16 studies have been carried out on blocked single residues by several workers. Infrared spectroscopy is one of the important techniques employed to investigate conformations of simple model peptides. Of the several ir studies of blocked amino acids carried out in carbon tetrachloride and chloroform solutions, particular mention must be made of the work of Tsuboi et al.,7 Avignon et a1.8 and Maxfield et al.17 Tsuboi et al.7 examined the ir spectra of five acetyl amino acid N-methylamides in carbon tetrachloride solutions a t low concentrations, where intermolecular hydrogen bonding was negligible, and they inter- preted the results in terms of the presence of the C7 intramolecular hy- drogen-bonded structure along with the extended form; the C7 hydrogen- bonded structure was assigned a N-H stretching frequency of 3360-3330 cm-l. Avignon et a1.8 studied possible conformations (Cs-extended; C7-

* Contribution No. 156 from the Solid State and Structural Chemistry Unit. + To whom all correspondence should be addressed.

Biopolymers, Vol. 22,2091-2104 (1983) G 1983 John Wiley & Sons, Inc. CCC 0006-3525/83/092091-14$02.40

2092 RAO, BALARAM, AND RAO

folded) of blocked glycine, alanine, and serine by employing ir spectroscopy in dilute carbon tetrachloride solutions and assigned characteristic N-H stretching bands due to C5 (3420 f 7 cm-l), C7 (3340 f 30 cm-I), and free N-H species. The C5 conformation is associated with (&,b) N (170°, 170") and the C7 conformation with (&$) N (SO", SO"). Burgess and Scheragal8 have also considered the y-conformation with ($$) N (-60", 140").

Recently, Maxfield et aI.l7 have carried out an excellent study of the N-H stretching bands of N-acetyl-N-methyl-L-amino acids, where the amino acids are glycine, alanine, and leucine in dilute solutions of chloro- form and carbon tetrachloride. These workers assign the band at 3450 cm-l to the unperturbed N-H stretching vibration and a band around 3435 cm-l to conformations where the spatial proximity of the CP atom perturbs the N-H stretching frequency. A band in the region 3300-3370 cm-' was assigned to intermolecularly hydrogen-bonded species in view of its marked concentration dependence (disappearing at low concentra- tions around 10-jM). A band in the region 3405-3430 cm-' has generally been assigned to the C j conf~rmation.~J~ The studies of Maxfield et al.17 point out that considerable flexibility exists in the blocked single residues, as predicted by conformational energy calculations.13-16

In view of the differences in the assignment of the 3300-3370 cm-l band by different workers, we considered it important to examine the ir spectra of a few model peptides likely to form C7 intramolecular hydrogen bonds. For this purpose, we have investigated two blocked proline amides, Piv- Pro-NHMe and Boc-Pro-NHMe, whose conformations would only permit C7 intramolecular hydrogen bonds (being particularly favored by the presence of proline) besides intermolecular hydrogen bonds as shown in Fig. 1. We have also examined the spectra of Boc-Leu-NHMe, Z-Aib- Aib-OMe, and Z-Aib-Ala-OMe, which can, in principle, form C5 and C7, as well as intermolecular hydrogen bonds; the studies of the last two pep- tides were prompted by a recent paper of Peterson et al.,lg who suggested the absence of C5 and C7 hydrogen bonds in such Aib-containing peptides. We have studied the spectra of all these peptides in carbon tetrachloride and chloroform solutions over a wide range of concentrations to delineate inter- and intramolecular hydrogen bonds.20

EXPERIMENTAL

All the peptides studied were synthesized and purified in this laboratory. The ir spectra were recorded with a Perkin-Elmer 580 spectrophotometer using cells of different path lengths. For higher path lengths (1.0 cm and above), prematched cells made of fused silica were used. For smaller path lengths, prematched NaCl windows were used.

The intensities of the N-H stretching bands were evaluated in several ways. The apparent extinction coefficient, f a , was obtained by the rela- tion

IR STUDY OF C7 HYDROGEN BONDS 2093

CH3

i"' H / N \ c

II 0

I

Fig. 1. Schematic representation of (a) the intramolecular C7 hydrogen bond and (b) the intermolecular hydrogen bond formed by Piv-Pro-NHMe.

where c is the concentration in mol L-1; 1, the path length in cm, I0 the percent transmission of the incident beam (background position), and I the percent transmission at the band position. Wherever band shapes were favorable, we have estimated the apparent integrated intensity, I a , by the formula21

1 I0 I" = - In - (Aul/z) cl I

where Aq/p is the bandwidth. We also obtained the normalized areas of bands A , by measuring the band area and dividing it with the concentration and the path length. In the figures where we have shown the concentration dependence of N-H stretching band intensities, the units employed are as follows: c, mol I,-1; I, cm; Au1/2) cm-l, cat L mol-I cm-' x I" , L mol-I cm-2 X and A , L mol-l cm-l, but the values shown in the fig- ures are relative.

2094 RAO, BALARAM, AND RAO

I. 3 x 1 0 - l ~ .0.012 cm 2.6 x 1f2M .O.OEl cm

7.0 x 10-3M, 0.061 cm

2.6 x 10-3M. 0.25 cm

1 .L x M , 0.35 cm

7 . 0 ~ 16'M .O.LS cm

9.2 x lO-'U ,10.0 cm

l I . I * ' .

3500 3300 r/(cm-')

Fig. 2. The ir spectra in the N-H stretching region of Piv-Pro-NHMe in CCll solution. Solution concentration ( M ) and path length (cm) are given at the right.

RESULTS AND DISCUSSION

Piv-Pro-NHMe

In Fig. 2 we show the N-H stretching bands of Piv-Pro-NMe in carbon tetrachloride solution over a wide range of concentrations (-10-5-10-1M). We clearly see two distinct bands at 3460 and 3337 cm-1 over the entire concentration range. The 3460-cm-l band is clearly due to the free N-H stretching vibration, while the 3337-cm-l band is due to a hydrogen-bonded species. Since the 3337-cm-' band is present even at 2 X 1OP5M concen- tration, we feel that it is likely to be due to the C7 intramolecular hydro- gen-bonded species (Fig. 1). However, the concentration dependence of the intensities of these two bands indicates that the 3337-cm-l band may also have some contribution from intermolecular hydrogen bonding. In Fig. 3 we show the N-H stretching bands of Piv-Pro-NHMe in chloroform solution. While the free N-H stretching band at 3455 cm-1 is more intense in chloroform solution compared to that in carbon tetrachloride solution as expected, the band due to the hydrogen-bonded species a t 3335 cm-1 is present over the entire concentrations range (-10-4-10-1M). The

IR STUDY OF C7 HYDROGEN BONDS 2095

1 .1 x 161M,0.012crn

5.5 x r0"M. OD61 cm

2.8 x d2M, 0.061 cm

-2

-3 1.4 x 10 M , 0.061 cm

6.9 x 10 M , 0.25 cm

3.c x 10-3M,0.35 cm

-4 1.0 x 10 M I 9.0 cm

- 3300 3500 V( cm-I)

Fig. 3. The ir spectra in the N-H stretching region of Piv-Pro-NHMe in CHC13 solu- tion.

concentration dependence of the intensities of these bands suggests that here again the 3335-cm-l band may have contributions from both the C7 intramolecular hydrogen bond and intermolecular hydrogen bonding.

We sought to obtain further information on the nature of the hydro- gen-bonded species present in solution from the intensities of N-H stretching bands. The intensity (both in terms of the apparent extinction coefficient, f a , and the normalized area of the band, A) of the free N-H stretching band (3460 cm-l) of Piv-Pro-NHMe in carbon tetrachloride solution is essentially constant up to -10-2M [Fig. 4(a) and (b)] and then decreases. This behavior suggests that intermolecular hydrogen bonding may become significant a t relatively high concentrations ( 1OP2M or more). We have plotted the normalized area of the 3337 cm-I band as a function of concentration in Fig. 4(c), taking into account any uncertainties that may arise from band shape. We see that the area does not change significantly with concentration. The ratios of intensities of the free (3460 cm-I) and hydrogen-bonded (3337 cm-l) N-H stretching bands f a (free)/€ a (bonded), as well as A(free)/A(bonded), show only a slight decrease with increasing concentration [Fig. 4(d)].

We have also examined the variation of the intensity of the free N-H stretching band in chloroform solution against concentration, the intensity being measured in terms of c a, A , and the apparent integrated intensity,

2096 RAO, BALARAM, AND RAO

t o o o ( P O o O o o o I 0.2 1

O I I 1 I t " 1 1 1 I 1

0 w LoL I A A A A

20

- c -3 -2 -1 log c

Fig. 4. Concentration dependence of the N-H stretching band intensities of Piv-Pro- NHMe (in CC14 solution): (a) Apparent extinction coefficient, ca, of the free N-H stretching band a t 3460 cm-I; (b) normalized area, A , of the free N-H stretching band at 3460 cm-'; (c) normalized area of the H-bonded N-H stretching band at 3337 cm-l; (d) ratio of the in- tensity of the free N-H stretching band a t 3460 cm-' and the intensity of the hydrogen- bonded N-H stretching band (A, f a ; 0, A ) . Representative error bars are indicated.

I". The intensity of this band decreases with increasing concentration as in carbon tetrachloride. The intensity of the hydrogen-bonded band at 3335 cm-l shows only a slight decrease with increasing concentration, and the ratios of intensities of the free and hydrogen-bonded N-H stretching bands show negligible variation.

These intensity data can be understood as follows. If there were only intermolecular hydrogen bonding, the intensity of the free N-H stretching band should decrease drastically with increasing concentration accompa- nied by an increase in intensity of the hydrogen-bonded N-H stretching band. If only the intramolecular hydrogen bond were present, we would normally expect the intensity of the bonded N-H stretching band to re- main essentially constant with increasing concentration unless the intra- molecular hydrogen bond changed into an intermolecular one. If both intramolecular and intermolecular hydrogen bonds were present, the in- tensity of the hydrogen-bonded N-H stretching band, as well as the ratio of intensities of the free and bonded N-H stretching bands, would have shown a decrease or an increase with increasing concentrations depending on the relative values of intrinsic intensities as well as the proportions of

IR STUDY OF' C7 HYDROGEN BONDS 2097

8 x 1 i 2 M , 0.012cm

4 x i 2 M , 0.012 cm 2 x rO2M , 0.016 cm

I x G*M, 0.mIcm

5 x 1 0 M , 0.25cm -3

2.5xfi3MM,0.25cm

2 . 5 ~ 1 6 ~ M , X ) . O c m

1.25x%'MM, X).Ocm

6.25 x 165M,10.0cm

I , ' . , .

3500 3300 v [ cm-')

Fig. 5. The ir spectra in the N-H stretching region of Boc-Pro-NHMe in CC14 solu- tion.

intra- and intermolecular species. Based on these observations, we propose that the 3337-cm-l hydrogen-bonded band is mainly due to the C7 intra- molecular hydrogen bond with a small contribution from the intermolecular hydrogen bond formed by the extended species (see Fig. 1). These results suggest that both the C7 intramolecular hydrogen bond and the intermo- lecular hydrogen bond in Piv-Pro-NHMe give rise to an N-H stretching band around the same position. This observation implies that the band around 3335 em-' need not necessarily be due to C7 hydrogen bond alone as proposed by Tsuboi e t al.7 or to the intermolecular hydrogen bonding alone as proposed by Maxfield et al.17 The present study shows that both the C7 and extended species of Piv-Pro-NHMe are present in solution.

Boc-Pro-NHMe

We have also studied the N-H stretching bands of Boc-Pro-NHMe both in carbon tetrachloride (Fig. 5) and chloroform solutions. The spectra clearly show the existence of a single free band around 3450 cm-1,

2098 RAO, BALARAM, AND RAO

4 6 1 IO-'M. 0 01 cm

23 1 IO-'M, 001 crn

2 57 I lo-%, 0 25 cm I 285% 10-3M. 045 cm

64251 10'4M, 50 cm

2 304 1 lo-%, 50 cm

77 1 0 - ~ ~ , 0 0 6 1 ~

I 152 1 10-4M. 50cm

576 x lo-%, lOOcm

/ * I *

3500 3300

v k m - ' ) Fig. 6. The ir spectra in the N-H stretching region of Boc-Leu-NHMe in CCld solu-

tion.

suggesting the likely presence of one isomer and a hydrogen-bonded band around 3350 cm-l. The hydrogen-bonded N-H stretching band continues to be present even at low concentrations (-6 X 10-5M in carbon tetra- chloride and -2 X 10-4M in chloroform) in this peptide as well. We have examined the intensities of both the free and the bonded N-H stretching bands as functions of concentration in carbon tetrachloride and chloroform. We see a small decrease in the apparent extinction coefficient of the free N-H stretching band above 10-2M concentration in carbon tetrachloride solution just as in Piv-Pro-NHMe. Both the apparent integrated intensity and the normalized area of the free N-H stretching band, however, show negligible variation with concentration. The intensity of the hydrogen- bonded peak also does not vary with much concentration in carbon tetra- chloride solution as also the intensity ratios of the free and bonded N-H stretching bands. In chloroform solution, the intensity of the N-H stretching bands shows little or no variation. These results suggest that there is slightly less intermolecular hydrogen bonding in Boc-Pro-NHMe than in Piv-Pro-NHMe. The C7 intramolecular hydrogen-bonded species is undoubtedly the predominant one. If indeed the cis as well as trans urethane bond isomers of Boc-Pro-NHMe coexisted, this conclusion would be unaffected. Our studies on Z-Pro-NHMe also show the C7 hydrogen- bonded species to be prominent in this peptide; the blocking group seems to have marginal effect on the conformation. The results of the present study on Piv-Pro-NHMe and Boc-Pro-NHMe seem to establish that C7

IR STUDY OF C7 HYDROGEN BONDS 2099

-2 5.19 x x) M , OMZcm

-3 6 . U x x) M ,O.O6lcm 3.24 r 6 3 ~ . 0 . b m

1.62 x lG3 M , 045m

Fig. 7. The ir spectra in the N-H stretching region of Boc-Leu-NHMe in CHClB solu- tion.

intramolecular hydrogen bonds can occur along with intermolecular hy- drogen bonds in these peptides because of the coexistence of both confor- mations (Fig. 1).

Boc-Leu-NHMe

The above discussion on the nature of the hydrogen-bonded species based on N-H stretching band intensities finds justification from our studies on Boc-Leu-NHMe. This peptide shows only the presence of intermo- lecular hydrogen bonds both in carbon tetrachloride and chloroform so- lutions, as can be seen in Figs. 6 and 7. We clearly see that the band around 3340 cm-l (position is concentration dependent) due to the hydrogen- bonded species disappears completely at lower concentrations in both carbon tetrachloride and chloroform. Accordingly, in carbon tetrachloride, the intensity of the hydrogen-bonded N-H stretching band increases markedly with the increasing concentration of the peptide, accompanied

2100 RAO, BALARAM, AND RAO

c e 3

a a-

n,

50

0 O ( c ) Bonded N-H bond

& 6

“ t 1

lens 1 , * I 8 > > l

-5 -4 -3 -2 -I

log c Fig. 8. Concentration dependence of N-H stretching band intensities of Boc-Leu-NHMe

(in CC14): (a) A (0) of the free N-H stretching band; (b) t o for the free N-H stretching band; (c) A of the H-bonded N-H stretching band (d) ea of the H-bonded N--H stretching band and (e) ratio of A (0) and eQ (A) of the free and bonded N-H stretching bands.

by a decrease in the intensity of the free N-H stretching band (Fig. 8). Such a variation in intensity is also seen in chloroform. This behavior is exactly that expected for a purely intermolecularly hydrogen-bonded system. The ratios of the intensities of the free and bonded N-H stretching bands also show the expected decrease with increasing concen- tration. According to Cung et a1.,l1 among the possible intermolecularly hydrogen-bonded species, the centrosymmetric dimer is favored in the case of heterochiral (L- and D-) isomers. Since we find the C=O stretching band shifts from 1676 cm-l (at high concentrations) to 1690 cm-l on dilution, with the urethane C=O stretching band (-1702 cm-l) unaffected, we are tempted to suggest that the hydrogen-bonded species is acyclic.

Z-Aib-Aib-OMe

This peptide can exist in two possible conformations (Fig. 9), one of which has two C5 intermolecular hydrogen bonds while the other has one C7 in- tramolecular hydrogen bond, leaving one N-H bond free. The ir spectra of this compound in the N-H stretching region are shown in Fig. 10 for a wide range of concentrations (-10-5-10-2M) in carbon tetrachloride. The spectra show four distinct bands around 3440,3415,3400, and 3325

IR STUDY OF C7 HYDROGEN BONDS 2101

Fig. 9. Possible conformations of 2-Aib-Aib-OMe.

cm-l. The intense band at 3440 cm-l is clearly due to the free N-H stretching vibration. The bands around 3415 and 3400 cm-l can be as- signed to the two C5 hydrogen bonds shown in Fig. 9. Accordingly, in- tensities of these two bands (both and A) do not vary much with con- centration. The band a t 3325 cm-l can arise from the C7 hydrogen bond shown in Fig. 9. This conformation also permits intermolecular hydrogen

I

- 3 8 . 6 ~ 10 M, 0.2cm

1.7 x 10 M , i.Ocm -3

-L 8 . 6 ~ 10 M, 1.0cm

-L k . 3 x 10 M, 2.0cm

-5 8.6X10 M,lO.Ocm

1 1 1 1 1 1 1

3450 3250 ( c m-’1

Fig. 10. The ir spectra in the N-H stretching region of 2-Aib-Aib-OMe in CC14 solu- tion.

2102 RAO, BALARAM, AND RAO

\CH3

Fig. 11. Conformations of Z-Aib-Ala-OMe.

bonding of the N-H bond not involved in intramolecular hydrogen bonding. We, however, find no separate band due to intermolecular hy- drogen bonding in the ir spectrum. Furthermore, the intensity ( c a or A ) of the 3325-cm-' band does not show any significant change with concen- tration, and the band persists even at -10-5M (Fig. 10). The ratios of the apparent extinction coefficients of the 3440- and 3325-cm-' bands are es- sentially constant over the entire concentration range. This behavior suggests that the band at 3325 cm-l is almost entirely due to the C7 hy- drogen-bonded species. Accordingly, the intensity of N-H stretching band at 3440 cm-l is not altered appreciably over a wide range of concen- trations. It is indeed interesting that the free N-H stretching band in C7 conformation does not undergo any self-association. The present study dearly establishes that Z-Aib- Aib-OMe exists in both the conformations shown in Fig. 9 in solution phase.

The ir spectrum of Z-Aib-Aib-OMe recorded in chloroform shows N-H stretching bands that are quite different from those observed in carbon tetrachloride. Thus, we see only an intense, free N-H stretching band at 3435 cm-l and a weaker one at 3400 cm-l, which can be ascribed to the C5 hydrogen bond. It appears that in chloroform solution, the conforma- tion involving the C7 hydrogen bond is unstable. Such solvent dependence of C7 hydrogen bonds is interesting.

Z-Aib-Ala-OMe This peptide can exist in three possible conformations [Fig. ll(a)-(c)].

In (a) it would have one C5 hydrogen bond and one free N-H bond, while

IR STUDY OF C7 HYDROGEN BONDS 2103

9.265 x f i 3 M , 0.2 cm 1 .853 x a 3 h 4 , 1.0cm

4.6325 x id' M , 2.Ocm

2.5 x X?M, 19.0 cm

1 * 3 t a . I

3450 3250 v( cm-1)

Fig. 12. The ir spectra in the N-H stretching region of 2-Aib-Ala-OMe in CCll solu- tion.

in (b) it can have two C5 hydrogen bonds; in (c) it can have one C7 hydrogen bond and one free N-H bond. Of these three conformers, (b) with C5 hydrogen bonds is unlikely to be present, since this would involve a rela- tively free rotation 4 of the alanine residue compared to Aib, thereby in- creasing the separation between the nitrogen and carbonyl oxygen of the alanine residue. The ir spectrum of this peptide in the N-H stretching region (Fig. 12) is simpler than that of Z-Aib-Aib-OMe. We see three N-H stretching bands around 3440,3400,3325 cm-l. The intense band at 3440 cm-' is due to the free N-H stretching vibration, while the band around 3400 cm-' is likely to be due to the C5 hydrogen bond, the intensity of the latter being constant over the entire concentration range. The band at 3325 cm-l is present even at the lowest concentration (-2.5 X 10-5M) studied, indicating that it is likely to be due to the C7 hydrogen bond, as in Fig. Il(c). However, this band, as well as the free N-H stretching band at 3440 cm-l, shows a slight concentration dependence, suggesting that intermolecular

2104 RAO, BALARAM, AND RAO

hydrogen bonding contributes marginally to the 3325-cm-l band. Based on these observations, we conclude that at least two conformations involving C5 and C7 hydrogen bonds coexist in this peptide.

In chloroform solution, Z-Aib-Ala-OMe shows an intense band at 3430 cm-* due to the free N-H stretching vibration and a weaker band at 3400 cm-' due to C5 hydrogen-bonded N-H stretching vibration. A weak feature around 3350 cm-l due to the C7 hydrogen bond is barely observed. I t appears that the conformation involving the C7 hydrogen bond is not stable in chloroform solution in this peptide as well, as in the case of Z- Aib- Aib-OMe.

We thank the U.S. National Institutes of Health for support of this research (Grant 01- 136-N).

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13. Ramachandran, G. N. & Sasisekharan, V. (1968) Adu. Protein Chem. 23,283-438. 14. Lewis, P. N., Momany, F. A. & Scheraga, H. A. (1973) Isr. J . Chem. 11,121-152. 15. Zimmerman, S. S., Pottle, M. S., NBmethy, G. & Scheraga, H. A. (1977) Macromolecules

16. Pullman, B. & Pullman, A. (1974) Adu. Protein Chem. 28,347-526. 17. Maxfield, F. R., Leach, S. J., Stimpson, E. R., Powers, S. P. & Scheraga, H. A. (1979)

18. Burgess, A. W. & Scheraga, H. A. (1973) BiopoEymers 12,2177-2183. 19. Paterson, Y., Rumsey, S. M., Benedetti, E., NBmethy, G. & Scheraga, H. A. (1981) J .

20. Rao, C. N. R. (1963) Chemical Applications of Infrared Spectroscopy, Academic Press,

21. Ramsay, D. A. (1952) J. Am. Chem. SOC. 74,72-80.

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Received June 18,1982 Accepted March 9,1983