Synthetic and conformational studies on dehydrophenylalanine containing model peptides

Int. J . Peptide Protein Res. 33, 1989, 103-109

Synthetic and conformational studies on dehydrophenylalanine containing model peptides

PARAMJEET KAURI, K. UMA', P. BALARAM* and V.S. CHAUHAN'

'Department of Chemistry, University of Delhi, Delhi and 'Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India

Received 24 March, accepted for publication 17 June 1988

Four model dipeptides containing a Z-dehydrophenylalanine residue (A'Phe) at the C-terminal, Boc-X- A'Phe-NHMe (X = Ala (l), Gly (2), Pro (3), and Val (4)), have been synthesised and their solution conformations investigated by 270 MHz 'H n.m.r. and i.r. spectroscopy. N.m.r. studies on these peptides clearly show the presence of intramolecularly hydrogen bonded structures in CHC13 solutions while such structures appear to be absent in the corresponding saturated peptides. This conclusion is also supported by i.r. studies. Studies of the nuclear Overhauser effect provided evidence for the occurrence of a significant population of /I-turn structures in solvents like CDC13 and (CD3)2S0. The observed NOES are consistent with a major contribution from Type I1 /-turn structure in CDC13, while in (CD3)*S0 solutions there is evidence of a partially extended structure also.

K e j ~ words: 8-turn; dehydrophenylalanine; nuclear Overhauser effects

The introduction of a$-dehydroamino acid residues into peptides affords a means of limiting both side chain and backbone flexibility at specific sites along the peptide chain (1-3). Theoretically, computed conformational energy maps for the Z-dehydrophenylalanine (A'Phe) residue suggest that energy minima exist that correspond to commonly observed peptide conformations in the helical and extended strand regions (4-6). Furthermore, the accommodation of AZPhe at either corner position (i + 1 or i + 2) of the various p-turn structures appears energetically feas- ible (7-10). Experimental evidence on the conformational preferences of A' Phe residues has been limited (7-1 2). We describe in this report the conformational analysis in solution, by n.m.r. methods, of a series of model peptides of the type Boc-X-A'Phe-NHMe, where X = Ala (l), Gly (2), Pro (3), or Val (4), and present a comparison of the results with those on the saturated counterparts.

EXPERIMENTAL PROCEDURES

Synthesis of pept ides The dehydropeptides 1-4 were synthesised according to the procedure given in Fig. 1. Boc amino acids were prepared by standard procedures. T.1.c. was carried

CH3 0 H3C-(- 0-C-NH

II

H3

57 0 II

5H-C- NH-FH- C- OH CH3 GH-OH

Ac20-AcONa

1 - FIGURE 1 out on silica gel G in the following solvent systems: A)

CHC1,:MeOH (9: I), B) nBuOH:AcOH:H,O (4: 1: I), Synthesis of dehydropeptide 1.

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P. Kaur et al.

C) nBuOH:AcOH:pyridine:H20 (4: 1 : 1 :2), D) CHC1,:AcOH:MeOH (10: 1 :2) and E) Et0AC:pyridine:AcOH:H,O ( 5 : 5 : 1 :3). The final peptides were each shown to yield a single peak by HPLC on a Lichrosorb RP-18 column (4mm x 250mm, particle size, lOpm), using a methanol/water gradient (55- 75% MeOH in 20min; flow rate 0.8 mLmin-I; detec- tion 226nm).

All ' H n.m.r. spectra were recorded on a WH-270 FT n.m.r. spectrometer at the Sophisticated Instru- ments Facility, Indian Institute of Science. Difference NOE experiments were carried out as described earlier (13). Peptide concentrations range from 10 to 2OmgmL-'.

Boc- V~-LX- Phe (b-0 H) -OH (7) A solution of Boc-Val-OH (3.0g, 13.8mmol) and N-methylmorpholine (1.52 mL, 13.8 mmol) in THF (25 mL) was chilled, isobutylchloroformate (1.8 mL, 13.8 mmol) added and the mixture stirred for 20min. A precooled solution of DL-Phe(B-OH)-OH (3.75 g, 20.7 mmol) in aqueous NaOH (1 N, 20.7 mL) was then added and the mixture stirred for 2 h at 0" and overnight at room temperature. The reaction mixture was evaporated in vucuo to remove THF and the remaining aqueous solution washed with ethyl acetate ( 1 x 25mL), acidified with solid citric acid to pH 3 and extracted with ethyl acetate (3 x 20mL). The combined extracts were washed with water, dried over anhydrous Na2S0, and evaporated in vucuo to give 7 as an oil. Yield 4.5g; Rf 0.5.

Boc- Val-AzPhe oxazolone (8) A solution of the dipeptide 7 (4.4g, 11.56mmol) in acetic anhydride (33 mL) and anhydrous sodium acetate (1.23 g, 15.03 mmol) was stirred overnight at room temperature. The reaction mixture was poured over crushed ice with stirring and the resulting pre- cipitate was filtered, washed with 5 % NaHCO, solution, water and dried in I'UCUO over PzOj to give 8. Yield 4.0g (88%); m.p. 114-1 15"; Rf 0.8, RF 0.9, Rf 0.87; (a); - 77.2' ( c 0.96, MeOH). Anal. calc. for C,,H,,0,N2 (344.40): C 66.26, H 7.02, N 8.13. Found: C 66.23, H 7.04, N 8.10.

Boc- Val-A'Phe-NHCH, (4) To a solution of 8 (0,75g, 2.2mmol) in dichloro- methane (15mL) was added Et,N (1.52mL, 1 1 .O mmol) and methylamine hydrochloride (0.74 g, 1 1 .O mmol). The reaction mixture was stirred for 6 h, washed with 5% NaHCO, solution, water, 10% citric acid solution, water and dried over anhydrous NazSO,. The solvent was removed in vucuo to give crude 4, which was purified by column chromatog- raphy (1.6cm x 30cm) over silica gel 60 (Merck, 70-230 mesh size) using CHCI,MeOH as eluent followed by recrystallization from ethyl acetate-petro- leum ether. Yield, 0.26g (31%); m.p. 108-109"; Rf

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0.65, R; 0.9, RF 0.8; HPLC retention time, 14.5min; (2): - 58.3' (c 1.0, MeOH). Anal. calc. for C,,H,,O,N, (375.46): C 63.97, H 7.79, N 11.19. Found: C 64.10, H 7.83, N 11.10.

'H n.m.r. (270 MHz, (CD,),S0)6: 9.56 (A'Phe NH, lH, S); 7.84 (NHMe, IH, br); 7.57, 7.34 (A'Phe aromatic protons, 5H, m); 7.09 (A'Phe C,H, lH, S); 7.08 (Val NH, lH, d); 3.8 (Val CaH, IH, t); 2.67 (NHCH,, 3H, d); 1.95 (Val C,H, lH, m); 1.42 (Boc CH,, 9H, S); 0.89 (Val C, H,, 6H, d).

Boc-Ala-A'Phe-NHCH, (1) 1 was prepared from Boc-Ala-A'Phe oxazolone (9) on 2.37 mmol scale according to the procedure given for 4. Yield 0.44g (54%); m.p. 210-212"; Rf 0.6, Rf! 0.96, RF 0.77; HPLC retention time, 10.3 min; (a): - 70.4" (c 0.9, MeOH). Anal. calc. for C,,H,,O,N, (347.4): C 62.23, H 7.25, N 12.10. Found: C 62.31, H 7.22, N 12.12.

' H n.m.r. (270 MHz, (CD,),S0)6: 9.57 (A'Phe NH, lH, S); 7.76 (NHMe, lH, br); 7.57, 7.38 (A'Phe aromatic protons, 5H m); 7.4 (Ala NH, lH, d); 7.27 (A'Phe C,H, lH, S); 4.02 (Ala CaH, lH, m); 2.68 (NHCH,, 3H, d); 1.42 (Boc CH,, 9H, S); 1.23 (Ala

Boc-Gly-A'Phe-NHMe (2) 2 was prepared from Boc-Gly-A"Phe oxazolone (14) by a procedure similar to that for 4 on 3.3 mmol scale. Yield, 0.7 g (64.2%); m.p. 206-208"; Rf 0.5, Rf! 0.8, RF 0.8; HPLC retention time, 9.7min. Anal. calc. for C,,H,,O,N, (333.38): C 61.24, H 6.95, N 12.61. Found: C 61.41, H 7.05, N 12.73.

' H n.m.r. (270 MHz, (CD,),S0)6: 9.49 (A'Phe NH, lH, S); 7.86 (NHMe, IH, q); 7.58, 7.36 (A'Phe aromatic protons, 5H, m); 7.16 (A'Phe C,H, lH, S); 3.59 (Gly CH,. 2H, d); 2.65 (NHCH,, 3H, d); 1.40 (Boc CH,, 9H, S).

CpH,, 3H. d).

Boc-Pro-A'Phe-NHMe (3) It was prepared from Boc-Pro-A'Phe oxazolone (1 5) on a 2.92 mmol scale by a procedure similar to that for 4. Yield 0.62g (56.8%); m.p. 209-210'; RF 0.87, RF 0.9; (x ) : + 3.8' (c 0.9, MeOH). Anal. calc. for CzoH,,O,N, (373.44): C 64.32, H 7.29, N 11.25. Found: C 64.19, H 7.44, N 11.29.

' H n.m.r. (270 MHz, (CD,),S0)6: 9.68 (A'Phe NH trans, 1H. S); 9.49 (A'Phe NH cis, lH, S); 7.96 (NHCH, cis, lH, br); 7.78 (NHCH, trans, IH, br); 7.59, 7.39 (A'Phe aromatic protons, 5H, m); 6.75 (A7Phe C,{H, lH, S); 4.3-4.15 (Pro C,H, lH, m); 2.7 (Pro C,H2, 2H, m); 2.62 (NHCH?, 3H, d); 2.23-2.1 (Pro C,H,, 2H, m); 1.95-1.84 (Pro C,H,, 2H, m); 1.45 (Boc CH, trans, 9H, m); 1.38 (Boc CH, cis, 9H, S).

Boc- Val-Phe-NHMe (5) A solution of Boc-Val-OH (l.24g, 5.7mmol) and N- methylmorpholine (0.63 mL, 5.7 mmol) in THF

Dehydrophenylalanine peptides

peptide 3 (X = Pro) an additional conformer is observed in (CD,)2S0 corresponding to the cis orientation about the urethane linkage. In CDCl, solutions, no cis conformers could be detected, in agreement with an earlier report (8).

Difference NOE experiments were carried out by irradiation of various NH groups in peptides 1 to 4, in (CD,),SO. Studies were also carried out in CDCI, or CDC1, containing 5% (CD,),SO, for peptides 1,3 and 4. Table 2 summarizes the results obtained. Peptide 2 was not examined in CDC1, because of limited solubility. 1.r. studies of peptides 1 and 6 were carried out in CHCI, solutions over the concentration range 0.53- 4.8 mmol.

From Table 1 it is seen that the lowest solvent shift on going from CDCI, to (CD3)zSO is observed for the methylamide NH in peptides I , 3, and 4 (0.28- 0.84ppm). The Ah values for the remaining two NH groups in these peptides range from 0.99 ppm to 2.2 ppm. The reduced solvent perturbation observed for methylamide NH is consistent with its involvement in an intramolecular hydrogen bond suggestive of a significant population of p-turn conformers. In peptides 1 to 4, the lowest dd/dT value observed is again for the methylamide NH, indicative of a comparative- ly greater degree of shielding as compared to the other NH groups. Using these two parameters as a crude indicator, the extent of population of p-turn conformers may be given as X = Pro (trans conformer) (3) > X = Ala (1) > X = Gly (2) > X = Val (4). This order is in agreement with a generally expected tendency for these residues to occur at the position i + 1 of b-turn conformers (16). In the case of peptide 3, high dd/dT values are observed for both NH groups in the cis conformer, in (CD,),SO. This is readily ra- tionalized since the orientation of the carbonyl group in the cis conformer precludes intramolecular hydrogen bonding. The comparison of the saturated analogues 5 and 6 with their unsaturated counterparts 4 and 1 re- vealed much higher Ad (1.47ppm) and dS/dT (5.7 x lO-,pprn" K-I) values as compared to those for the unsaturated peptides (viz. 0.84ppm and 4 x 10-3ppmoK-'). These results suggest that in-

(1 5 mL) was chilled, isobutylchloroformate (0.75 mL, 5.7 mmol) added, and the mixture stirred for 20 min. A precooled solution of H-Phe-OH (1.42 g, 8.55 mmol) in aqueous NaOH (1 N, 8.6mL) was then added and the mixture stirred for 2 h at 0" and overnight at room temperature. The reaction mixture was worked up as described for 7 to give Boc-Val-Phe-OH as an oil weighing 1.7 g, Rf 0.57. The oil was dissolved in THF (20mL), cooled to - lo", followed by addition of N-methylmorpholine (0.52 mL, 4.7 mmol) and isobutylchloroformate (0.62 mL, 4.7 mmol). After 10 min, a precooled solution of methylamine hydrochloride (0.94 g, 14.0mmol) in THF-water (3:2), neutralised with Et,N (1.96 mL, 14.0 mmol), was added with vigorous shaking. After 2 h, the solvent was removed in vacuo and the residue was taken up in ethyl acetate (25 mL), washed with 5% NaHCO, solution, water and dried over anhydrous Na,SO,. The solvent was removed in vacuo to give 5, which was crystallized from ethyl acetate-pet. ether. Yield 1.1 g (62.5%); m.p. 170-171"; Rf 0.64, RF 0.86; (a): - 40.3" (c 1.0, MeOH). Anal. calc. for C,,H,,O,N, (377.47): C 63.63, H 8.28, N 11.13. Found: C 63.70, H 8.22, N 11.05.

'H n.m.r. (270 MHz, CDCI,)h: 7.35-7.28 (Phe aromatic protons, 5H, m); 6.58 (Phe NH, lH, d); 6.43 (NHMe, IH, br); 4.93 (Val NH, lH, d); 4.66 (Phe C,H, lH, m); 3.88 (Val C,H, IH, m); 3.12-3.02 (Phe C,)H,, 2H, m); 2.76 (NHCH,, 3H, d); 1.39 (Boc CH,, 9H, S); 0.91 (Val C, H,, 6H, d).

Boc-Ala-Phe-NHCH, (6) Compound 6 was prepared on a 4.6mmol scale by a procedure similar to that for 5. Yield 0.92g (58%); m.p. 157-158"; Rf 0.76, RF 0.84; (a): - 37.7" (c 1.0, MeOH). Anal. calc. for C,,H2,04N3 (349.42): C 61.87, H 7.79, N 12.03. Found: C 61.85, H 7.55, N 12.02.

' H n.m.r. (200 MHz, CDC1,)h: 7.36-7.24 (Phe aromatic protons, 5H, m); 6.64 (Phe NH, lH, d); 6.44 (NHMe, IH, br); 4.96 (Ala NH, lH, d); 4.68 (Phe C,H, lH, m); 4.04 (Ala C,H, IH, m); 3.16-3.0 (Phe C,H,, 2H, m); 2.75 (NHCH,, 3H, d); 1.36 (Boc CH,, 9H, S); 1.28 (Ala C,H,, 3H, d).

RESULTS AND DISCUSSION

The assignment of NH resonances in peptides 1 to 4 is straightforward, with the X NH appearing as a doublet (triplet in the case of Gly), the A'Phe NH as a broad singlet and methylamide NH as a quartet. (Fig. 2a shows a representative 'H n.m.r. spectrum of 4 in CDCI,.) The chemical shifts in CDCI, and (CD,), SO and temperature coefficients measured in (CD,),SO are given in Table 1. The n.m.r. parameters for the NH groups in saturated peptide analogues Boc-Val-Phe-NHMe (5) and Boc-Ala-Phe-NHCH, (6), are also shown for comparison. In the case of

sr

, I I I 8.0 7 0 6 0 5 0 4.0 3 0

b ( P P m )

FIGURE 2 a) 270MHz 'H n.rn.r. spectrum of peptide 4 in (CD,),SO. b) Difference NOE spectra obtained by irradiation of A'Phe NH resonance.

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N.m.r. parametersfor Boc-X-A' Phe NHMe and Boc-X-Phe-NHMe (NH resonances only) ~~

Peptide and Parameter Peptide and Parameter resonance resonance

CDCI, (CD,)2S0 dh/dT CDC1, (CD,),SO dS/d T (PPm) (PPm) pprnK-' x 10' (ppm) (ppm) PpmK-' x 10'

(CD, 12 so (CD, )2 so X = Ala (1) X NH A'Phe NH NHMe

X NH A'Phe NH NHMe X = Pro (3) A'Phe NH NHMe

X = Gly (2)

5.76 7.4 8.58 9.57

7.76 b

7.35 9.49 7.48 d

7.47 9.68 (9.49) 7.50 7.78 (7.96)

7.3 6.3 3.0

6.3 5.5 3.75

5.3 (5.5) 2.8 (6.5)

X = Val (4) X NH 5.09 A'Phe NH 7.59 NHMe 7.00 Boc-Val-Phe-NHMe (5) X NH 4.93 Phe NH 6.58 NHMe 6.43 Boc-Ala-Phe-NHMe (6) X NH 4.96 Phe NH 6.64 NHMe 6.44

7.08 9.56 7.84

6.75 7.90 7.90

7.28

7.92

e

7.3 5.8 4.0

5.7 4.0 5.7

8.4 3.2 5.5

"These parameters are for 95% CDCI, - 5% (CD,),SO. bNHMe resonance overlaps with aromatic peaks. 'Poorly soluble in CDCI,. dValues in parentheses are for the cis isomer, which corresponds to 40% of mixture. 'Phe NH resonance overlaps with aromatic peaks.

TABLE 2 NOES observed in peptides Boc-X-ALPhe-NHMe (1-4) in (CD,),SO"

Resonance irradiated: X NH ALPhe NH NHMe

Peptide Resonance NOE Resonance NOE Resonance NOE observed (Yo) observed ("/.I observed (Yo)

X = Ala (1) X C"H b (4.2)' X C'H 9.9 (12.1) A'Phe NH 3.2 (d) AZPhe NH b (1.2) X NH b (2.5) A'Phe 5.7

NHMe Cp H 2.6 (d) NHMe 4.2

X = Gly (2 ) e,f X C'H 2.5 A'Phe NH 3.2 NHMe 3.1 A' Phe 6.6

Cp H NHMe 4.9

NHMe 3.3 A' Phe 2.7 Cp H NHMe 3.2

A'Phe NH 0 (2.5) X NH 0 (2.2) A'Phe 8.2 Cp H NHMe 4.3

X = Pro (3) - X C'H 12.9 (g) AZPhe NH 2.8 (g)

X = Val (4) X C H 2.2 (3.6) X C'H 10.8 (10.9) A'Phe NH 3.2 (h)

"All NOE experiments are done at 293 K. Values in parentheses correspond to NOE values in CDC1,. bAla NH overlaps with aromatic region in (CD,),SO. 'Peptide 1 was studied in a 95:5% mixture of CDCI, and (CD,)?SO. dNHMe overlaps with aromatic region in CDC1,. 'Gly NH overlaps with aromatic region in (CD,),SO. 'Boc Gly A'Phe NHMe is insoluble in CDCI,. gA'Phe NH and NHMe are under the aromatic region in CDCI,. hIrradiated and observed resonances are very close.

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troduction of the CI, P-double bond stabilizes folded, p-turn conformations.

Fig. 3 shows the NH stretching region of the i.r. spectra of Boc-Ala-A'Phe-NHCH, (1) and the corresponding saturated peptide 6 in CHCI, solutions over the concentration range of 0.53-4.8mM. At the three concentrations studied (0.53 mM, I .6 mM, 4.8 mM), two bands are observed for 1 at - 3350 and 3420cm-', corresponding to hydrogen bonded [vNH (hb)] and free [vNH ( f ) ] NH groups, respectively. The band at 3350 cm-' is largely concentration independent and is observed even at 0.53 mM. This band may, therefore, be assigned to an intramolecularly hydrogen-bonded NH group. A plot of the ratio, vNH (hb)/vNH (f) shows no dependence on concentration (plot not shown) suggesting that the peptide is not. aggregated over the concentration range, 0.53-4.8 mM. For peptide 6, over the same concentration range, in CHCI, solutions two absorption bands are observed at - 3413 and - 3443 cm-', no band corresponding to hydrogen bonded NH group was observed. The observed bands at 3413 and 3443cm-' can only be attributed to non hydrogen bonded NH groups. Therefore, it can be concluded that for 1, intramolecularly hydrogen bonded structures may in fact predo- minate in CHCl, solutions while such structures seem to be absent in the corresponding saturated counter- part 6.

Nuclear Overhauser efSect studies In both solvents, peptides 1 to 4 show appreciable


NOES between Ca(X)H and AZPhe NH protons. (See Fig. 2b for a representative difference NOE spectrum.) In addition, NOES are also observed between the AZPhe NH and methylamide NH protons in (CD,),SO. The Ca(X)H c) NH NOE is smaller in magnitude for the X = Gly (2) peptide, because the C"H protons relax primarily by mutual dipolar relaxa- tion. The observation of such C;+'H t* N,+2H and N,+,H t* N,+,H NOES is characteristic of Type I1 p-turn conformations (Fig. 4a) having the X and AZPhe residues at positions i + 1 and i + 2, respectively. Such conformations would be characterized by conformational angles of dx N - 60", t,hx - 120", AZPhe N 80" and $kPhe - 0".

In (CD,),SO solutions, an appreciable NOE is also observed between the AZPhe C5H and methylamide NH protons. This NOE suggests a short AZPhe CBH- - -NHMe distance ( < 3 A"), which can be obtained only for $A'Phe values - 120" (9). The observation of such an NOE is then clearly indicative of conformational heterogeneity involving a population of partially extended structures at the AZPhe residue (Fig. 4b, 4c).

The AZPhe C5H c-) NHMe NOE is absent in CDCl, solutions suggesting that breakage of the intramolecular 4 + 1 hydrogen bond and reorientation about AZPhe is facilitated by solvation of the peptide in strongly hydrogen bond accepting solvents like (CD,),SO. The magnitudes of the A'Phe CBH t, NHMe NOE follow the order X = Pro < X = Ala < X = Gly < X = Val, a feature

30 3( I , , #

3 3400 3:

FIGURE 3 The i.r. spectra (NH stretching bands) in CHCI, at various peptide concentrations indicated next to the tracings: a) Boc-Ala- A'Phe-NHMe (1) and b) Boc-

00 Ala-Phe-NHMe.

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P. Kaur et al.

FIGURE 4 Proposed structures for Boc-Val- A'Phe-NHMe a) p-turn conformation, b) partially extended conformation, and c) fully extended conformation.

H n

which is consistent with the tendency of these residues to stabilize 8-turn conformers when occurring in the i + 1 position (16, 17).

In CDCl, solutions, a small, but reproducible, NOE is observed between the X NH and AZPhe NH groups in peptides 1 and 4. This NH group is absent in 3 (X = Pro) and the experiment could not be carried out in 2 because of limited solubility. Such N,H t+ Ni+ ,H NOEs are expected for 4x - - 60', $x - - 30' and would correspond to residue i + 1 in a helical conformation or at the i + 1 position of a Type I(II1) fi-turn (16, 17). Such Type I 8-turn conformations would require $AzPhe - - 90" and $d'Phe - O', which is stereochemically possible for the achiral residue, dehydrophenylalanine.

The above n.m.r. studies of peptides of the type Boc-X-A' Phe-NHMe provide evidence for the occurrence of a significant population of 8-turn structures in solvents like CDCl, or (CD,),SO. The observed NOEs are consistent with a major contribution from Type I1 8-turn structures in CDCl,, with some evidence for the population of Type I (111) /&turns also. In (CD,)2S0 solutions, solvation destabilizes the 8- turn conformation and clear evidence is available for partially extended structures (+AZPhe - 120'). It is not possible to distinguish unambiguously between the population of partially extended and fully extended peptide conformations from the available data (Fig. 4b and 4c).

The effect of the X residue on the stabilization of 8-turn conformations is clearly demonstrated by a comparison of the n.m.r. parameters for the NHMe NH group in the various peptides and also by a comparison of the A'Phe CBH c* NHMe NOE in (CD,),SO solutions. As expected, Pro at residue i + 1 has an appreciable influence in stabilizing 8-turn conformations, while Val appears to have the least in-

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fluence. These results suggest that although AZPhe residue does facilitate chain reversals, the local conformations at the A'Phe residue can be influenced by environmental factors. This information may prove helpful in designing analogues of peptide hormones, particularly those containing phenylalanine. This expectation is borne out by recent studies on a chemo- tactic peptide analogue (18) and a cyclic (7, 8) pentapeptide.

ACKNOWLEDGEMENTS

This research was supported by a grant from the Department of Science and Technology, Government of India. P.K. acknowledges the receipt of a fellowship from UGC. K.U. is the recipient of a fellowship from the CSIR.

REFERENCES

I . Spatola, A.F. (1983) in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins (Weinstein, B., ed.), Vol. VII, pp. 267-357, Marcel Dekker, New York Stammer, C.H. (1 982) Op. cir. Vol. VI, pp. 33-74 Noda. K., Shimohigashi, Y. & Izumiya, N. (1983) in The Peptides, Analysis, Synthesis. Biology (Gross, E. & Meien- hofer, J., eds.), Vol. 5 . , pp. 285-339, Academic Press, New York

4. Ajo. D., Granozzi, G., Tondello, E. & Del Pro, A. (1980) Biopoljwers 19, 469475

5. Ajo. D., Busetti, V. & Granozzi, G . (1982) Tetrahedron 38, 3329-3334

6. Ajo, D.. Casarin, M. & Granozzi, G. (1982) b. Mol. Struct. 86,

7. Bach, A.C. I1 & Gierasch, L.M. (1985) J . Am. Chem. Soc. 107,

8. Bach, A.C. I1 & Gierasch, L.M. (1986) Biopolymers 25, 5175- 5191

9. Chauhan, V.S., Sharma, A.K., Uma, K., Paul, P.K.C. & Balaram, P. (1987) Int. J . Pepfide Protein Res. 29, 126-133

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Uma, K., Chauhan, V.S., Kumar,A. &Balaram, P. (1988)Int. J . Peptide Protein Res. 31, 349-358 Aubry, A,, Allier, F., Boussard, G. & Marraud, M. (1985) Biopolymers 24, 639-646 Pieroni, O., Montagnoli, G., Fissi, A., Merlino, S. & Ciardelli, F. (1975) J . Am. Chem. Soc. 97, 6820-6826

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18.

Kishore, R., Raghothama, S . & Balaram, P. (1987) Bio- polymers 26, 873-89 I Shimohigashi, Y., Stammer, C.H., Costa, T. & Vonvoigtlan- der, P.F. (1983) Int. J . Peptide Protein Res. 22, 489-494 Fisher, G.H., Berryer, P., Ryan, J.W., Chauhan, V.S. & Stammer, C.H. (1981) Arch. Biochem. Biophys. 211, 269-275


Smith, J.A. & Pease, L.G. (1980) CRC Crit. Rev. Biochem. 8, 3 15-399 Rose, G.B., Gierasch, L.M. & Smith, J.A. (1985) Adv. Protein Cliem. 31, 1-109 Chauhan, V.S., Kaur, P., San, N., Uma, K., Jacob, J. & Balaram, P. (1988) Tetrahedron 44, 2359-2366

Address: Dr. V.S. Chauhan Department of Chemistry University of Delhi Delhi- 1 10007 India

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Synthetic and conformational studies on dehydrophenylalanine containing model peptides

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