3206 J. Am. Chem. Soc., Vol. 119, No. 14, 1997
Jain et al.
X-ray Diffraction. Single crystals of peptide 1 were grown by
controlled evaporation of the peptide (C43H51N5O8, Mw ) 765.9) in
aqueous methanol solution at 4 °C. A colorless crystal mounted on a
glass fiber was used for X-ray diffraction experiments. The crystals
belong to orthorhombic space group P212121, a ) 11.624(2), b )
L-Val residues. This demonstrates the remarkable conforma-
tional consequences produced by consecutive occurrence of
∆Phe residues in peptides.
Experimental Procedures
17.248(2), c ) 21.532(2)Å, V ) 4216(1) Å3, Z ) 4, dc ) 1.18 g cm-3
.
Three-dimensional X-ray intensity data were collected on an Enraf-
Nonius CAD4 diffractometer with Ni filtered Cu KR radiation (λ )
1.5418 Å) up to a Bragg angle of 65° using ω-2θ scan method. A
total of 4083 unique reflections were collected of which 3213 had |Fo|
> 4σ|Fo|. No significant variation was observed in the intensities of
three standard reflections monitored at regular intervals during data
collection implying the electronic and crystal stability. Lorentz and
polarization corrections were applied to the data, and no absorption
correction was made (µ ) 6.3 cm-1). The structure was solved by
direct methods using the computer program SHELXS867 and refined
Synthesis. Boc-Val-DL-Phe(â-OH)-OH (2). To a solution of Boc-
Val-OH (4.0 g, 18.43 mmol) in tetrahydrofuran (20 mL) at -10 °C,
N-methylmorpholine (2.02 mL, 18.43 mmol) and isobutylchloroformate
(2.4 mL, 18.43 mmol) were added. The reaction mixture was stirred
for 10 min. A precooled solution of DL-Phe(â-OH)-OH (3.34 g, 18.43
mmol) in aqueous NaOH (1 N, 18.4 mL, 18.43 mmol) was added to
the reaction mixture. The mixture was stirred for 2 h at 0 °C and
overnight at room temperature. After completion (checked by TLC)
reaction mixture was concentrated in Vacuo, and residue was taken in
water. Maintaining the pH of the aqueous layer to 8-9, it was washed
once with ethyl acetate. Aqueous layer was then acidified with solid
citric acid to pH 3, and the resulting oil was extracted three to four
times with ethyl acetate. Combined organic extract was washed with
saturated NaCl, dried over sodium sulfate, and evaporated to give 2 as
2
on |F| using all 4083 reflections by full-matrix least-squares procedures
using the computer program SHELXL93.7 All the hydrogen atoms
were fixed using stereochemical criteria and used only for structure
factor calculations. The conventional R-factor R1 based on |F|’s for
3213 reflections with |Fo| > 4σ|Fo| is 3.64% and 5.45% for all 4083
1
an oily product (yield ) 80%). Rf [CHCl3-CH3OH (9:1)] ) 0.5; H
2
data. The weighted R-factor wR2 based on |Fo| is 9.79% for all 4083
NMR (270 MHz, CDCl3) δ (ppm): 7.2 (5H, m, aromatic protons); 6.6
(1H, br, NH DL-Phe(â-OH)); 5.2 (1H, br, NH Val); 4.8 (1H, br, CRH
DL-Phe(â-OH)); 4.1 (1H, m, CRH Val); 1.4 (9H, s, 3 × CH3 Boc);
1.0 (6H, d, 2 × CH3 Val).
2
2
data {w ) 1/[σ2 |Fo| + (0.0482 + P)2 + 0.7 + P], P ) (max(|Fo| ,0)
2
+ 2 + |Fc| )/3}. The maximum and minimum residual electron density
in the final difference fourier map are 0.15 and -0.16 eÅ-3
,
respectively.
Boc-Val-∆Phe-Azlactone (3). A solution of dipeptide 2 (6.20 g,
16.31 mmol) in acetic anhydride (40 mL) and anhydrous sodium acetate
(1.734 g, 21.15 mmol) was stirred for 20 h at room temperature. The
reaction mixture was then poured over crushed ice, and the precipitate
was filtered, washed with 5% NaHCO3 and water, and dried under
vacuum. The azlactone was crystallized from acetone/water to get the
pure compound (yield ) 75%). Mp ) 114-115 °C; Rf [CHCl3-CH3-
OH (9:1)] ) 0.8, Rf [butanol-acetic acid-H2O (4:1:1)]) 0.9; Rf
Spectroscopic Studies. 1H NMR spectra were recorded on a Bruker
400 MHz FT NMR at Sophisticated Instruments Facility, Indian Institute
of Science. Spectral width of 10 ppm was used for both one- and two-
dimensional spectra. All chemical shifts are expressed as δ(ppm)
downfield from internal reference tetramethylsilane. Spectra were
recorded at concentration of 5 mg/mL. Two-dimensional COSY8a and
ROESY8b spectra were recorded in CDCl3 at room temperature using
standard procedures. Short mixing time (200-300 ms) was used in
the ROESY experiment in order to minimize spin-diffusion effects.
Data block sizes were 1024 addresses in t2 and 512 equidistant t1 values.
Circular dichroism (CD) measurements were carried out on a JASCO
500 spectropolarimeter equipped with a data processor 500 N. A 1
mm path length cell was used. The spectra were recorded in three
different solventsschloroform, methanol, and trifluoroethanol. The
spectra were normalized for concentration and path length to obtain
the mean residue ellipticity after base line correction. The theoretical
CD calculations were carried out on the basis of the exciton chirality
method. For ∆Phe chromophore, only the low energy π-π* transition
at 280 nm was considered, and the calculations were carried out as
reported earlier.9
1
[CHCl3-CH3OH (9:1)] ) 0.5; HNMR (270 MHz, CDCl3) δ (ppm):
7.3-7.1 (5H, m, aromatic protons); 5.1 (1H, br, NH Val); 4.1 (1H, br,
CRH Val); 2.1 (1H, m, CâH Val); 1.35 (9H, s, 3 × CH3 Boc); 1.0 (6H,
d, 2 × CH3 Val).
Boc-Val-∆Phe-∆Phe-∆Phe-Val-OMe (1). DL-Phe(â-OH)-OH (1.4
g, 7.54 mmol) dissolved in 1 N NaOH (12 mL) and acetone (20 mL)
was added to a solution of azlactone 3 (2 g, 5.8 mmol) in acetone, and
the reaction mixture was stirred at room temperature. After 24 h, the
reaction mixture was neutralized by adding 1 N HCl (12 mL). Solvent
was removed under vacuum, and the residue was dissolved in ethyl
acetate, washed with water, dried over sodium sulfate, and evaporated
to yield Boc-Val-∆Phe-DL-Phe(â-OH)-OH (4) (single spot on TLC).
Tripeptide 4 was used in the next step with no further purification and
(2.8 g, 5.3 mmol) reacted with anhydrous sodium acetate (0.48 g, 5.88
mmol) and acetic anhydride (25 mL) for 48 h at room temperature.
The reaction was worked up as before to yield Boc-Val-∆Phe-∆Phe-
azlactone (5) in pure form. Peptide 5 (2 g, 4.1 mmol) was also reacted
in acetone (20 mL) with DL-Phe(â-OH)-OH (0.817 g, 4.5 mmol)
dissolved in 1 N NaOH (10 mL, 0.018 g, 4.5 mmol) for 50 h at room
temperature. Boc-Val-∆Phe-∆Phe-DL-Phe(â-OH)-OH (6) was obtained
after usual workup and was azlactonized using acetic anhydride (20
mL) and sodium acetate (0.345 g, 4.2 mmol) to yield Boc-Val-∆Phe-
∆Phe-∆Phe-azlactone (7), which showed a single spot on TLC.
To a solution of peptide 7 (0.914 g, 1.44 mmol) in dichloromethane
was added HCl‚Val-OMe (0.365 g, 2.2 mmol) and triethylamine (0.3
mL, 2.2 mmol). The reaction mixture was stirred for 80 h. The reaction
mixture was then washed with NaHCO3 solution, 5% citric acid
solution, and water and dried over sodium sulfate. The solvent was
removed under reduced pressure to give pentapeptide 1, which was
recrystallized from methanol and water. Yield ) 60%. Mp ) 212-
214 °C, Rf [CHCl3-CH3OH (9:1)] 0.63, Rf [butanol-acetic acid-H2O
(4:1:1)] 0.97; the molecular mass of the pentapeptide determined by
Results
Crystal Structure. The bond lengths and bond angles are
listed in Tables 1 and 2, respectively. All bond lengths and
bond angles are normal except those corresponding to the three
∆Phe residues. The CRdCâ bond length in the three ∆Phe
residues are 1.331(4), 1.324(5), and 1.326(5) Å, respectively,
which corresponds to classical CdC double bond. The N-CR
[1.418(4), 1.419(3), and 1.421(4) Å] and CR-C′ [1.490(5),
1.500(5), and 1.501(5) Å] bond distances in ∆Phe residues are
slightly shorter than the corresponding bonds in saturated
residues (1.45 and 1.53 Å, respectively10), as seen in other ∆Phe
peptides.5g-t This shortening is probably due to the extended
conjugation of the ∆Phe ring electrons and the remaining part
of the residue.
(7) Sheldrick, G. M. Acta.Crystallogr. 1990, A46, 467.
(8) (a) Aue, W. D.; Bartholdi, E.; Ernst, R. R. J. Chem. Phys. 1976, 64,
2229. (b) Bothner-By, A. A.; Stephens, R. L.; Lee, J.; Warren, C. D.;
Jeanloz, R. W. J. Am. Chem. Soc. 1984, 106, 811-813.
(9) Inai, Y.; Ito, T.; Hirabayashi, T.; Yokota, K. Biopolymers 1993, 33,
1173.
(10) Benedetti, E. In Chemistry and Biochemistry of Amino Acids,
Peptides and Proteins; Weinstein, B., Ed.; Dekker: New York, 1982, pp
105.
1
ES-MS was 766.4 (calculated molecular mass ) 765.904); H NMR
(270 MHz, CDCl3) δ (ppm): 9.1 (1H, s, NH ∆Phe4); 8.79 (1H, s NH
∆Phe3); 7.75 (1H, s, NH ∆Phe2); 7.65 (1H,d, NH Val5); 7.55-7.2
(18 H, m, aromatic + CâH protons of ∆Phe2, ∆Phe 3 and ∆Phe4);
4.81 (1H, d, NH Val1); 4.55 (1H, m, CRH Val5); 4.12 (1H, m, CRH
Val1); 2.3 (1H, m, CâH Val1); 1.95 (1H, m, CâH Val5); 1.2 (9H, s, 3
× CH3 Boc); 1.1 (6H, dd, CγH Val1); 0.9 (6H, dd, CγH Val1).