The synthesis of the octapeptide, benzyloxycarbonyl-(&#945;-aminoisobutyryl-L-prolyl)4-methyl ester [Z-(Aib-Pro)4-OMe] and an analysis of its solution conformation is reported. The octapeptide is shown to possess three strong intramolecular hydrogen bonds on the basis of studies of the solvent and temperature dependence of NH chemical shifts and rates of hydrogen-deuterium exchange. 13C studies are consistent with a structure involving only trans Aib-Pro bonds, while ir experiments support a hydrogen-bonded conformation. The Aib 3, 5, and 7 NH groups are shown to participate in hydrogen bonding. A 310 helical conformation compatible with the spectroscopic data is suggested. The proposed conformation consists of three type III &#946;-turns with Aib and Pro at the corners and stabilized by 4&#8594; 1 intramolecular hydrogen bonds

Balaram, P.

Venkatachalapathi, Y. V.

English

Publications of the IAS Fellows

X-Pro Peptides: Synthesis and Solution 
Conformation of Benzyloxycarbonyl-(Aib-Pro)d- 
Structure 
Methyl Ester. Evidence for a Novel Helical 
Y. V. VENKATACHALAPATHI and P. BALARAM,* Molecular 
Biophysics Uni t ,  Indian Institute of Science, Bangalore 560 012, India 
Synopsis 
The synthesis of the octapeptide, benzyloxycarbonyl-(c-aminoisobutyryl-~-prolyl~~-methyl 
ester [Z-(Aib-Pro)4-OMe] and an analysis of its solution conformation is reported. The oc- 
tapeptide is shown to possess three strong intramolecular hydrogen bonds on the basis of 
studies of the solvent and temperature dependence of NH chemical shifts and rates of hy- 
drogen-deuterium exchange. '"C studies are consistent with a structure involving only trans 
Aib-Pro bonds, while ir experiments support a hydrogen-bonded conformation. The Aib 
3, 5, and 7 NH groups are shown to participate in hydrogen bonding. A 310 helical confor- 
mation compatible with the spectroscopic data is suggested. The proposed conformation 
consists of three type 111 p-turns with Aib and Pro a t  the corners and stabilized by 4 - 1 in- 
tramolecular hydrogen bonds. 
INTRODUCTION 
The conformational analysis of proline-containing peptides has been the 
subject of considerable attention. As part of a systematic program to ex- 
amine the conformational characteristics of peptide sequences containing 
Pro at  every alternate position, we have chosen to  synthesize oligopeptide 
sequences of the type (X-Pro),. The preceding paper described the so- 
lution and solid-state conformation of benzyloxycarbonyl-(Aib-Pro)2- 
methyl ester.l In this report we describe the synthesis and conformation 
of benzyloxycarbonyl-(Aib-Pro),-methyl ester [Z-(Aib-Pro)4-OMe] in 
organic solvents. a-Aminoisobutyric acid (Aib) was chosen as the X res- 
idue since the conformational freedom of peptides containing this amino 
acid is greatly It was therefore expected that a sequential 
arrangement of Aib and Pro would lead to well-defined solution confor- 
mations even in short peptides. Small acyclic peptides containing Aib have 
been shown to adopt 0-turn and 310 helical conformations in solution and 
in the solid Recent interest in the conformations of Aib-containing 
peptides has been stimulated by the widespread occurrence of the amino 
acid as a constituent of alamethicin and related membrane-active pep- 
tides.a14 The stereochemical constraints resulting from the gem dimethyl 
* To whom correspondence should be addressed 
Biopolymers, Vol. 20, 1137-1145 (1981) 
0 1981 John Wiley & Sons, Inc. CCC 0006-3525/81/061137-09$01.00 
1138 VENKATACHALAPATHI AND BALARAM 
groups a t  the C" atom make the introduction of Aib residues into peptides 
an attractive method for the generation of stereochemically defined acyclic 
peptides. Examples of this approach in generating biologically active 
analogs of enkephalins have been reported.15J6 In this paper a novel helical 
conformation stabilized by three strong 4 - 1 intramolecular hydrogen 
bonds is established for the octapeptide Z-(Aib-Pro)4-OMe in solution. 
EXPERIMENTAL METHODS 
Synthesis of Benzyloxycarbonyl-( cu-aminoisobutyryl-L-prolyl)d- 
methyl Ester [ Z-(Aib-Pro)4-OMe], 1 
2- Aib-Pro- Ai 6- Pro- OH 
2-Aib-Pro-Aib-Pro-OMe (1.35 g)' was dissolved in 10 ml CH30H and 
5 ml of 2N NaOH was added. After standing for 8 hr at room temperature, 
30 ml of water was added and the mixture extracted with ethyl acetate. 
The aqueous layer was acidified with 2N HC1 and extracted with ethyl 
acetate (4 X 20 ml). The organic layer was dried and evaporated to yield 
the tetrapeptide acid as a chromatographically homogeneous (TLC, silica 
gel, 85% CHC13/10% CH30H/5% CH3COOH) solid. Yield, 1.1 g (80%); mp 
108-109°C. [ c Y ] ~  = -43.6" ( C  = 0.37, CH30H). 
HzN-Aib-Pro-Aib- Pro-OMe 
2-Aib-Pro-Aib-Pro-OMe (1.4 g) was taken in 10 ml ethanol. Palladium 
black (0.1 g) and cyclohexene (6 ml) were added and the mixture refluxed 
for 6 hr on a water bath.17 The catalyst was filtered and washed with 
ethanol. The filtrate and washings were combined and evaporated to yield 
an oil (0.8 g), which was used for further coupling without purification. 
Z-(Aib-Pro)4-OMe (1) 
HzN-Aib-Pro-Aib-Pro-OMe (0.8 g) was dissolved in 5 ml dimethyl- 
formamide, cooled in an ice bath, and 2-Aib-Pro-Aib-Pro-OH (1.1 g), 1- 
hydroxybenzotriazole (0.27 g), and DCC (0.41 g) were added successively. 
The mixture was stirred in an ice bath for 2 hr and overnight a t  room 
temperature. The dicyclohexylurea was filtered off and washed with ethyl 
acetate. The combined filtrate and washings were extracted with H20, 
1N HC1,lN NaHC03, and H2O and dried. Evaporation of ethyl acetate 
yielded a solid which was dissolved in minimum volume of CH$N and 
filtered to remove insoluble material. Evaporation of CH&N resulted in 
a white shicing solid, which was chromatographically homogeneous (TLC, 
silica gel, 5% CH30H/CHC13). Yield, 0.5 g (40%); mp 125-127°C. [a]: 
ANAL.: Calcd. for  C44H64011N8: C, 60,O; H, 7.27; N, 12.72. Found: C ,  59.57; H, 7.52; 
= -27.9" ( C  = 1.4, CH30H). 
N. 12.66. 
X-PRO PEPTIDES: NOVEL HELICAL STRUCTURE 1139 
‘H-nmr (270 MHz, CDC13): 7.80, s, 7.64, s, 7.64, s, 3H, Aib (3,5,7) NH; 
7.36, m, 5H, phenyl; 5.48, s, lH,  Aib (1) NH; 5.12, q, 2H, benzyl CH2; 4.59, 
4.31, m, 4H, Pro (2,4,6,8) C”H; 3.72, m, 8H, Pro (2,4,6,8) OH,; 3.69, s, 3H, 
OCH3; 1.86, 1.92, m, 16H, Pro (2,4,6,8) COHz, C”fH2; 1.56,1.52, 1.50, 1.47, 
1.45, 1.41, s, 24H, Aib (1,3,5,7) COH3. 
NMR and IR Studies 
lH and l3CC-nmr spectra were recorded on a Bruker WH-270 FT nmr 
spectrometer a t  the Bangalore NMR Facility a t  270 and 67.89 MHz, re- 
spectively, as described ear1ier.l The ir spectra were recorded in CHC13 
solution (-3-4 mM) on a Perkin-Elmer 580 spectrometer using a vari- 
able-path-length cell with NaCl windows.3 
RESULTS 
The low-field regions of the 270-MHz lH-nmr spectra of the octapeptide 
1 in CDCls and (CD&SO are shown in Fig. 1. The high-field NH resonance 
a t  5.48 6 in CDCl3 may be unambiguously assigned to the urethane NH of 
Aib ( l ) . l ~ ~ J ~  The remaining three singlets a t  7.67,7.68, and 7.84 6 corre- 
I I I I 
8 7 6 5 
6 
Fig. 1. The 270-MHz ‘H-nmr spectra of compound 1, low-field region: CDC13 (top) 
(CD&SO (bottom). Small unidentified peaks correspond to diastereomeric peptide im- 
purities. 
1140 VENKATACHALAPATHI AND BALARAM 
a 
0 50 1c 
X OMSO 
Fig. 2. Dependence of N H  chemical shifts on solvent composition in CDCl:I/(CD:&SO 
mixtures. 
spond to the Aib(3), (5), and (7) NH groups. A unique assignment of these 
signals on the basis of the available data is not possible. The 8.11 6 singlet 
in (CD:&SO is assigned to Aib(1) NH using spectra recorded in 
(CD:+)$3O/CDC1:3 mixtures of varying composition. Solvent titration,I8 
variable-temperature studies of NH chemical shifts,Ig and hydrogen- 
deuterium (H-D) exchange experiments20 have been carried out to evaluate 
the solvent accessibility of the Aib NH protons in 1. 
Figure 2 shows the change in chemical shifts of the NH protons of 1 as 
a function of (CD:&3O concentration in CDCl:{/(CD:J2SO mixtures. While 
the Aib(1) NH shows a large downfield shift between 0 and 20% (CD:&SO, 
the Aib(3), (5), and (7) NH groups are relatively unaffected. This suggests 
that the latter are not accessible to (CD:J,SO, a strongly hydrogen bond 
accepting solvent, whereas the Aib(1) NH is exposed. Table I summarizes 
the relevant nmr parameters for the octapeptide 1. Studies of the tem- 
TABLE I 
Chemical Shifts, Temperature Coefficients, and H-D Exchange Half-Lives of NH Protons 
in Compound 1 
Aib 1 5.48 (5.19, 6.06) 8.1 1 5.8 x lo-" 130 min 19 min 
Aih 3 7.64 (7.61,7.68) 7.55 1.6 X > 3  days >70 min 
1.0 X lo-" > 3  days >70 min 
Aib 7 7.80 (7.79,7.83) 7.79 1.5 X lo-,' > 3  days >70 min 
r" Aib 5 7.64 (7.61,7.68) 1.15 
a T h e  N H  resonances of Aib(3), (5), and (7) are arbitrarily assigned. 
Chemical shifts in CIlC1:3 a t  different concentrations. Values in the  column correspond 
to  10 m M  peptide. Values in parentheses are  for 1.5 and 43 m M  peptide. 
(CD:1)2SO. 
t 1/2, half-life for the  first-order H-D exchange process. 
X-PRO PEPTIDES: NOVEL HELICAL STRUCTURE 1141 
perature dependence of NH chemical shifts in (CD&SO yield a high d 6 / d T  
value of 5.22 X 10-3 ppm/"C for the Aib(1) NH. The other three NH 
groups have very low temperature coefficients of 1.49 X lo-", 1.03 X lo-", 
and 1.61 x ppm/"C. Solvent-exposed peptide hydrogens generally 
yield dG/dT values >4 x 10-3 ppm/°C.21 Rates of H-D exchange were 
measured in CDC13/D2O and (CD3)2SO/D20 mixtures. In the former 
system, Aib(1) NH exchanged relatively fast ( t 1 / 2  - 130 min), while the 
other three NH groups exchanged slowly ( t l / ~ ?  > 3 days). In the 
(CD:$)2SO/D20 system, the rates of exchange were considerably greater, 
but again the Aib( 1) NH yielded a much lower t 1/2 value (19 min) as com- 
pared to the Aib(3), (5) and (7) NH groups (>70 min). 
The results described above strongly suggest that the Aib(3), (5), and 
(7) NH groups are involved in intramolecular hydrogen bonds. In order 
to rule out the possibility of intermolecular association, the concentration 
dependence of the NH chemical shifts was determined in CDCl3. Over the 
range 1.5-43 mM the Aib(3), (5), and (7) NH groups showed very small 
changes in chemical shifts (Table I). The Aib(1) NH, however, moved to 
lower fields over this range. Thus, while peptide association may affect 
the urethane NH parameters, it appears that the other NH groups are 
unaffected. All nmr experiments described earlier were carried out in the 
lower concentration range of -5 mM. 
Figure 3 showed the 67.89-MHz IT-nmr  spectrum of 1 in CDC13. The 
chemical shifts of the various carbons are summarized in Table 11. The 
chemical shifts of the CP (28.5-29.26) and C' (24.1-24.86) resonances of the 
Pro residues clearly indicate that all Aib-Pro bonds are in the trans con- 
formation. This is in agreement with earlier studies that suggest that 
whenever Pro is preceded by a sterically bulky group, the trans form is 
f a v ~ r e d . ~ ~ - ~ ~  The and Cy resonances in 1 appear a t  higher and lower 
0 
V 
n 
4 
.- 
0 I 
1 I I I 1  I I I I 1 I 
180 170 160 ' 70 60 50 LO 30 20 
6 
Fig. 3. The 67.89-MHz 13C-nmr spectrum of 1 in CDClz. Starred peak is the CH2 resonance 
of ethanol. 
1142 VENKATACHALAPATHI AND BALARAM 
TABLE 11 
l3Chemical Shifts (6, ppm) in Z-(Aib-Pro)r-OMe (1) 
C "  C@ C Y  CA c=o 
Aib Pro Aib Pro Pro Pro AibandPro Others 
57.3 62.8 28.2 29.2 24.8 48.6 173.7 156.9 (urethane CO) 
56.7" 62.4 26.4a 28.7 24.2 48.2 172.8 136.6 
56.9 62.5 26.7 29.0 24.6 48.3 173.0a 51.7 (OCH3) 
172.7 128.7a Phenyl 
172.3a 128.5 ring 1 60.7 26.2 28.5 24.1 47.8 25.9" 25.4 171.8 127.ga 
67.0 (Z-CH?) 
a Peaks correspond to two carbon resonances in intensity. 
fields, respectively, than the values reported in model peptides. The C@ 
and Cy chemical shifts compare well with the values reported in pivaloyl- 
Pro-NHMe (CP, 27.26; Cy, 25.76)25 and pivaloyl-Pro-Pro-Ala-NHMe (CP, 
27.2, 29.66; Cy, 24.6, 26.76).23 This trend in CP and Cy chemical shifts is 
also observed in 2-(Aib-Pro)z-OMe and in alamethicin fragmenk2* Three 
Ccy Pro resonances in 1 occur a t  relatively low field (62.4,62.5, and 62.86), 
while one appears at higher field (60.76). In 2-Aib-Pro-OMe, the C" Pro 
resonance is at 60.9. The shift to lower field by -26 of the Pro C" reso- 
nances in 1 tends to support a helical folded structure.24 In Z-Aib-Pro- 
NHMe and 2-Aib-Pro-Aib-Ala-OMe, where type I11 0-turn and incipient 
310 helical structures have been demonstrated in both solutions2-* and the 
solid state,2.5 the Ccy Pro peaks occur at 62.4 and 63.86.24 The low-field 
position of the Pro C" resonance in oligopeptide fragments of alamethicin 
has also been noted.24 
The NH stretching bands (VNH) in the ir spectra of octapeptide 1 and 
2-(Aib-Pro)z-OMe in dilute CHC13 solution are shown in Fig. 4. In the 
tetrapeptide two bands corresponding to vNH(free) a t  3436 cm-l and 
vNH(bonded) at 3340 cm-l are observed. The presence of the band at 3340 
cm-l argues for the occurrence of intramolecular hydrogen-bonded con- 
formations.3 Z-(Aib-Pro)2-OMe has been shown to adopt a type I P-turn 
conformation in solution and solid state, stabilized by an intramolecular 
4 - 1 hydrogen b0nd.l It can be seen in Fig. 4 that in the octapeptide 1, 
two bands are observed at 3432 and 3300 cm-'. The uNH(bonded) peak 
(3300 cm-l) is significantly more intense than in Z-(Aib-Pro)z-OMe, while 
the vNH(free) intensities are comparable in the two peptides. This suggests 
that both the additional NH groups in 1 participate in the formation of 
intramolecular hydrogen bonds. 
DISCUSSION 
Aib-containing peptides have been shown to be stereochemically rigid, 
resulting in the population of only a very limited range of conformational 
states in solution. This has allowed interpretation of spectral data, ob- 
X-PRO PEPTIDES: NOVEL HELICAL STRUCTURE 1143 
I I I 
3500 3400 900 3200 
3 Icm-'l 
10-"M. (b) Z-(Aib-Pro)4-OMe ( I ) ,  3.3 X 10WM. 
tained on small Aib peptides, in terms of only single, well-defined confor- 
mations. Excellent correlations have been obtained between nmr, ir, and 
x-ray studies. The tendency of Aib residues to occur in 310 helical con- 
formations ($ - f60', + - f30') has also been e~tablished.l-~ In octa- 
peptide l the alternation of Aib and L-Pro (where 4 is restricted to -60') 
is expected to result in a restricted range of permissible conformations. The 
nmr and ir results, outlined above, suggest that 1 predominantly adopts 
a structure in solution in which an all-trans peptide backbone is folded to 
yield three strong intramolecular hydrogen bonds involving the Aib(3), (5), 
and (7) NH groups. This is supported by the low solvent and temperature 
dependence of these NH chemical shifts and their slow rates of H-D ex- 
change. A conformation compatible with the spectroscopic data is shown 
in Fig. 5. 
The proposed structure is an approximately 310 helical conformation,26 
with alternate 4 - 1 hydrogen bonds interrupted by the Pro residues. In 
an idealized 310 helical model, unfavorable steric contacts exist between 
the CO groups of Aib(l), (3), and (5) and the C6H2 groups of Pro(4), (6), and 
(8) ,  respectively. These may be relieved by slight distortions introduced 
by nonplanar peptide units and deviations of Aib and Pro 4, Ic/ values from 
those for a perfect 310 helix. The X2-Pro3 P-turn conformations (type 111) 
shown in Fig. 5 have already been observed for z-Aib-pr~-NHMe,~ Z -
Aib-Pro-Aib-Ala-OMe,2 and 2-Aib-Pro-Aib-Pro-OMel in the solid state. 
Fig. 4. UNH bands in the ir spectra of peptides in CHC13. (a) Z-(Aib-Pro)s-OMe, 4.2 X 
1144 VENKATACHALAPATHI AND BALARAM 
Fig. 5. Schematic representation of the intramolecularly hydrogen-bonded conformation 
of 1. Atoms involved in unfavorable steric contacts are shaded [CO groups of Aib(l), (3). and 
(5), and C*H2 of Pro(4), (6) and (811. 
For L-Pro to be accommodated in the approximately 310 helical confor- 
mation, $pro should be restricted to the region -10" to -60". Theoretical 
 calculation^^^ suggest energy minima for L-Pro in the vicinity of $ - -50". 
A population of conformers with $ - -50" has also been suggested for 
poly(L-proline) in trifluoroethanol.28 Further confirmation of the 310 
helical structure suggested for 1 must await crystal-structure determination. 
Attempts to obtain suitable single crystals have thus far proved fruit- 
less. 
This research was supported by the Indian National Science Academy 
References 
1. Venkatachalapathi, Y. V., Nair, C. M. K., Vijayan, M. & Balaram, P. (1981) Biopoly- 
2. Nagaraj, R., Shamala, N. & Balaram, P. (1979) J .  Am. Chem. Soc. 101,16-20. 
3. Rao, Ch. P., Nagaraj, R., Rao, C. N. R. & Balaram, P. (1980) Biochemistry 19,425- 
4. Rao, Ch. P., Nagaraj, R., Rao, C. N. R. & Balaram, P. (1979) FERS Lett. 100, 244- 
5. Prasad, B. V. V., Shamala, N., Nagaraj, R., Chandrasekaran, R. & Balaram, P. (1979) 
6. Shamala, N., Nagaraj, R. & Balaram, P.  (1978) J .  Chem. Soc. Chem. Comn~un., 996- 
7. Prasad, B. V. V., Shamala, N., Nagaraj, R. & Balaram, P. (1980) Acta Crystallogr., Sect. 
8. Martin, D. R. & Williams, R. J .  P. (1976) Riochern. J .  153,181-190. 
9. Pandey, R. C., Carter Cook, J. Jr .  & Rinehart, K. L. Jr. (1977) J .  Am. Chern. Soc. 99, 
10. Jung, G., Konig, W. A,, Liebfritz, D., Ooka, T., Janko, K. & Boheim, G. (1976) Riochirn. 
11. Pandey, R. C., Carter Cook, J. J r .  6: Rinehart, K. L. Jr. (1977) J .  Am. Chem. SOC. 99, 
12. Pandey, R. C., Carter Cook, J. Jr .  & Rinehart, K. L. Jr. (1977) J .  Am. Chem. Soc. 99, 
13. Bruckner, H., Konig, W. A,, Greiner, M. & Jung, G. (1979) Angew. Chem. Int.  Ed .  18, 
14. Fujita, T., Takaishi, Y. & Shimomoto, T. (1979) J .  Chem. Soc. Chcm. Commun., 
15. Nagaraj, R. & Balaram, P. (1978) FERS Lett. 96,273-276. 
mers 20,1123-1136. 
431. 
248. 
Niopolymers 18, 1635-1646. 
997. 
R 36,107-110. 
8469-8483. 
Riophys. Acta 433,164-181. 
5203-5205. 
5205-5206. 
476-477. 
413-414. 
X-PRO ' PEPTIDES: NOVEL HELICAL STRUCTURE 1145 
16. Nagaraj, R., Sudha, T. S., Shivaji, S. & Balaram, P. (1979) FEBS Lett. 106, 271- 
17. Anantharamaiah, G. M. & Sivanandaiah, K. M. (1977) J .  Chem. Soc., Perkins Trans. 
18. Pitner, T. P. & Urry, D. W. (1972) J .  Am. Chem. Soc. 94,1399-1400. 
19. Kopple, K. D., Ohnishi, M. & Go, A. (1969) J .  Am. Chem. Soc. 91,4264-4272. 
20. Stern, A., Gibbons, W. A. & Craig, L. C. (1968) Pruc. Natl. Acad. Sci. USA 61, 734- 
21. Hruhy, V. J. (1974) Chemistry and Biochemistry of Amino Acids, Peptides and Pro- 
22. Nishihara, H., Nishihara, K., Uefuji, T. & Sakota, N. (1975) Bull. Chem. Soc. Jpn. 48, 
23. Venkatachalapathi, Y. V. & Balaram, P. (1979) Nature 281,83-84. 
24. Nagaraj, R. (1980) Ph.D. thesis, Indian Institute of Science, Bangalore. 
25. Nagaraj, R., Venkatachalapathi, Y. V. & Balaram, P.  (1980) Int. J .  Pept. Protein Res. 
26. Donohue, J. (1953) Proc. Natl. Acad. Sci. USA 39,470-478. 
27. Madison, V. (1977) Biopolymers 16,2671-2692. 
28. Clark, D. S., Dechter, J. J. & Mandelkern, L. (1979) Macromolecules 12,626-633. 
274. 
I ,  490-491. 
741. 
teins, Vol. 3, Weinstein, B., Ed., Dekker, New York, pp. 1-188. 
553--555. 
16,291-298. 
Received August 19,1980 
Accepted November 19,1980 


X-Pro peptides: synthesis and solution conformation of benzyloxycarbonyl-(Aib-Pro)<SUB>4</SUB>methyl ester. Evidence for a novel helical structure

Indian Academy of Sciences

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X-Pro peptides: synthesis and solution conformation of benzyloxycarbonyl-(Aib-Pro)<SUB>4</SUB>methyl ester. Evidence for a novel helical structure

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