a,P-Dehydro-Amino Acid Residues in the Design of Peptide Structures: Synthesis, Crystal Structure, and Molecular Conformation of Two Homologous Peptides- N-Ac-Dehydro-Phe-~-Leu=0CH, and N-Ac-Dehydro-Phe-NorVal-OCH,

PUSHKAR SHARMA, PUNlT NARULA, and TEJ P. SlNCH

Depdrtrrierit of Biophysics, All India Institute of Medical Sciences, New Delhi 110029, India

SYNOPSIS

The dehydro-residue containing peptides N-Ac-dehydro-Phe-L-Leu-OCH3 ( I ) and N-Ac- dehydro-Phe-NorVal-OCH, (11) were synthesized by the usual workup procedures. The peptides crystallize from their solutions in methanol in space group P6,: ( I ) a = b = 12.528(2) A, c = 21.653(5) A; (11) a = b = 12.532(2) A, c = 21.695(4) A. Thestructures were determined by direct methods. Both peptides adopt similar conformations with @,# of dehydro-Pheasfollows: ( I ) -57.0(5)" and -37.0(5)"; (11) -56.0(5),' and-37.5(5)". The observed data on dehydro-Phe when placed at the ( i + 1) position show that the &J,# values of dehydro-Phe are either -60°, 140" or -60", -30". The conformation of -Boo, 140" can be accommodated only with a flexible residue at the ( i + 2) position while the 4,$ values of -60", -30" are obtained with a bulky residue at the ( i + 2) position as in the present structures. The molecules are packed in a helical way along the c axis. These are held by two strong intermolecular hydrogen bonds involving both NH as donors and acetyl group and dehydro-Phe oxygen atoms as acceptors. 0 1994 John Wiley & Sons, Inc.

INTRODUCTION

The dehydro-amino acids provide one of the most promising tools for designing the backbone confor- mations of peptides.' The studies of synthetic model peptides, in and in the solid ~ t a t e ~ - ~ ' have shown that dehydro-residues induce specific con- formations in ~ept ides .4~ The available data indicate that the backbone of dehydro-Phe adopts one of the three conformations with ~,IJ values of 80", 0", -60", 140", and -60°, -30", and their enantiomeric forms. In most of the structures reported so far, dehydro- Phe is found at the ( i + 2 ) position; thus, the con- formational preferences of dehydro-Phe at the ( i + 2 ) position have been clearly understood to adopt

Biopolymers, Vol. 34, 1243-1249 (1994) G 1994 .John Wiley & Sorib, Iric. CCC 0006-3525/94/091243-07

a conformation with 4 = 80" and 9 = 0" in a tet- rapeptide / tetrapseudopeptide to form a P-turn I1 structure. The number of available structures with a dehydro-Phe at ( i + 1 ) position is still very small. Thus a clear picture has not yet emerged about the preferences of conformations, as well as its specific influence on the resulting peptide structures. Therefore, more sequences with dehydro-Phe at the ( i + 1 ) position have to be analyzed. In view of this, we have synthesized two homologous model dipep- tides-( I ) N-Ac-dehydro-Phe-L-Leu-OCH:i and (11) N-Ac-dehydro-Phe-NorVal-OCH3-with a dehydro- Phe a t the ( i + 1) position to obtain a more gen- eralized picture and to analyze small differences in peptide conformations that might be caused by very slightly different residues a t the ( i + 2 ) position. Here, we report the syntheses, crystal structures, and molecular conformations of two peptides con- taining dehydro-Phe at the ( z + 1 ) position.

1243

1244 SHARMA, NARULA, AND SINGH

EXPERIMENTAL PROCEDURE OCH3. Yield 1.0 g (61%); mp = 188OC; R f ( a ) = 0.84; R f ( b ) = 0.94.

Melting points recorded are uncorrected. Thin layer chromatography was carried out on Silica Gel G in the solvent systems (by volume) ( a ) CHC13 : MeOH ( 9 : 1) and ( b ) n-BuOH : AcOH: H 2 0 ( 4 : 1 : 1 ) .

N- Ac-dehydro-Phe-L-leu-OCH,

N-Ac-dehydro-Phe-OH or a-acetoamido cinnamic acid was coupled with 1,-Leu-OMe by mixed anhy- dride procedure. One gram of N-Ac-dehydro-Phe- OH (4.9 mmol) was dissolved in tetrahydrofuran ( T H F ) and cooled at -10°C in an ice salt bath. One equivalent each of NMM (0.54 mL) and IBCF (0.63 mL) were added. After 30 min a solution of L-Leu- OMe in THF, obtained on neutralizing HCl-L-1,eu- OMe (0.91 g, 5.88 mmol) with NMM (0.65 mL, 5.88 mmol) was added. The reaction mixture was stirred at -10°C for 2 h and then a t room temperature overnight. The T H F was evaporated under vacuo and the product was dissolved in ethyl acetate. The ethyl acetate layer was washed with water, 10% so- dium bicarbonate, and a 5% citric acid, and dried overnight over sodium sulphate in the refrigerator. The ethyl acetate layer was removed under vacuo to give the white solid N-Ac-dehydro-Phe-L-Leu-

N-Ac-dehydro-Phe-1-NorVal-OCH,

This peptide was synthesized using the same method as for I. One gram of N-Ac-dehydro-Phe-OH (4.9 mmol) was coupled with L-NorVal-OCH, [obtained by neutralizing HC1-NorVal-OCH,, (0.88 g, 5.7 mmol) with NMM (0.62 mL) J using NMM (0.52 mL) and IBCF (0.62 mL) . The reaction mixture was worked up as for I. Yield: 1.1 g (65%), mp = 190°C; R f ( a ) = 0.87, R f (b) = 0.96.

The peptide samples were crystallized for struc- ture analysis work from their solutions in methanol a t 24°C under controlled evaporation conditions. The details of crystal data, intensity data collection, and refinement are given in Table I. The Lorentz and polarization corrections were applied. The ab- sorption correction was not applied. The structures were determined by direct methods using the pro- gram SHELXS86 44 and difference Fourier calcula- tions. The coordinates of 24 (I) and 23 (11) non- hydrogen atoms and their thermal parameters were refined anisotropically using a full-matrix structure factor least-squares procedure using I F I values.45

Table I The Details of Crystal Data, Intensity Data Collection, and Refinement

Peptide I Peptide I1

Crystal system Space group a = b (A) c (A) v (A3)

M W

d, ( g d, ( g ~ m - ~ ) F(000) Number of reflections collected Number of unique reflections Rint

Number of observed reflections (I 2 30 IF I ) Mode of data collection w Instrument used Max 28 limit Crystal dimensions (mm3)

Radiation used Final R S (A/u)

Hexagonal P65 12.528(2) 21.653(5) 2943.2(5) 332.4

1.125(4)

3860 3463 0.032 2973

0.04

1.52.5O

1.128(5)

1068

w - 28

Hexagonal

12.532(2) 21.695(4) 2950.5(5) 318.4 1.070(6) 1.075(5) 1020 3756 3244

P65

0.028 2850 w - 28 0.03

Enraf Nonius CAD4 152.5'

0.80 x 0.08 x 0.06 1.00 X 0.05 X 0.04 Graphite monochromated CuKcvX

= 1.5418 A 0.069 0.070 0.88 0.86 0.10 0.08

Table IIa Atomic Coordinates ( X 1 0 4 ) and Their Anisotropic Thermal Parameters ( X 1 0 3 ) of Peptide I

Atom X Y z Ul 1 U'22 u:3:3 u2, U,, Ul2

C, 2441(5) C' 2144(3)

Nl 1389(3) cy 1185(3) Ct 113(3) c: - 1150(4) C;' -1453(4) (2;' -- 265 7 (5) Cl -3582( 5) C;L -3309(5) c:' -2102(5)

0; 2458(3) N, 3 196( 3) C;' 4367(4)

c: 5613(5) C:l U 7 9 ( 7) Cq:! 6420(8)

0; 3538(5)

0 1 2599(3)

c; 2339(3)

C$ 5407(4)

c; 4348(5)

0 2 5335(4) c, 5459(7)

13739(4) 12470(3) 12252(2) 11571(3) 10346(3) 9342(3) 914")

10082 (4) 9805(7) 8644(8) 7731(6) 7972(5)

10262(3) 9501(3)

11010(3) 11067(4) 12389(5) 13074(6) 12650(10) 14437(8) 10370(5) 9947(5)

10264(4) 9668(8)

140(3) 294(2) 748(2) -91(2) --17(2)

24(2) 34(2) -3(3) -7(3) 24(4) 68(5) 77(4)

-36 (2 ) 283(0)

-431(2) -510(2) -540(3)

78(4) 573(4) -48(6)

- 1088(3) -1447(3) -I 126( 2) --1657(5)

Table IIb Atomic Coordinates ( X 1 0 4 ) and Their Anisotropic Thermal Parameters ( a 1 0 3 ) of Peptide I1

Atom X Y 2 U* 1 l J 2 2 [JXi u z 3 UlS u, 2

1281(4) 325(3)

180(3) -842(3) -772(3)

303(4) 1534(3) 2448(5) 2228(7) 1062(9)

64(5) -2073(3) -2961(2)

~ 2 189(3 ) - : 3 3 13 (4) -302 l(5)

-347(3)

-25:i9(6) ~ :3553(10) -3993(5) -3622(5) -5075(4) - 580 1 (8 )

3732(4) 2477(3) 2252(2) 1572(3) 353(3)

-660(3) -850(4)

74(5) -210(7) - 1368(8) -2262(8) -2030(5)

259(3) -506(3) 1006(3 ) 1053(4) 2400(5) 3074(6) 2656(9) 352(5)

-102(5) 246(4)

-374(7)

882(2) 731(2) 269(3)

11 12(2) 1044(2) 989(2) 989(2)

1029(3) 1032(3) 1012(4) 949(6) 951(4)

1062(2) 740(0)

1456(2) 1537(2) 1562(3) 920(5) 463(4)

2092(3) 2463(2) 2141(2) 2686(4)

1246 SHARMA, NARULA, AND SINGH

The z coordinate of 0; was kept fixed during the course of refinement in both structures. The hydro- gen atoms were fixed geometrically and their posi- tions were compared with the difference Fourier maps. They were assigned the final isotropic thermal parameters of the atoms to which they were bonded. These were included in the final cycles of refinement as fixed parameters. The atomic scattering factors used in these calculations were those of Cromer and Mann46 for nonhydrogen atoms, and of Stewart, Davidson, and Simpson 4i for hydrogen atoms. The final positional and anisotropic thermal parameters of nonhydrogen atoms for both the structures are given in Tables IIa and IIb.

RESULTS AND DISCUSSION *

The ORTEP drawings of the peptides I and I1 to- gether with numbering schemes are shown in Figures l a and lb. The dehydro-Phe in both the peptides adopts a conformation comparable with those ob- served for dehydro-Phe while a t ( i + 1) position with a bulky residue a t ( i + 2 ) position. The mol- ecules linked by intermolecular hydrogen bonds are arranged in the form of helices along the c axis.

Molecular Dimensions

The bond lengths and bond angles in both peptides are normal. The most significant influence on the geometry of the peptides caused by the steric factors due to dehydrogenation at the C Oi and C ' atoms can be seen in the opening of the bond angles Cy-Cf-Cy and NI-C;-Cf: ( I ) 131.4(4)' and 125.9(3)"; (11) 131.1(4)" and 125.7(3)". At the same time, reduction in the value of valence angle N,-CY-C; [ ( I ) 114.2(3)"; (11) 114.9(3)"]. The C"- C"distances of 1.306(5) A ( I ) and 1.320(5) A (11) agree well with the standard double bond distance of 1.337 A. These changes in the geometry are typical of those observed in dehydro-residues.'

Conformation of the Peptides

The values of a,/3 in both the peptides are very sim- ilar. The backbone torsion angles of dehydro-Phe

* The listing observed and calculated structure factors for peptides (1) N-Ar-dehydro-Phe-L-Leu-OCH, and (IT) N-Ac-de- hydro-Phe-NorVal-OCH,, and other tables, are available on mi- crofische. Please contact Journal Production Manager, ,John Wiley & Sons, Inc , 605 Third Avenue, New York, NY 10016, USA.

Figure 1. ( a ) T h e perspective view of peptide I and the numhering scheme. ( h ) T h e perspective view of peptide I1 and the numbering scheme.

in various peptides with dehydro-Phe at the ( i + 1) position are listed in Table 111. In the first three examples, the ( i + 2 ) site has relatively flexible res- idues, whereas in the remaining structures, the ( i + 2 ) position is occupied by bulky and rigid residues. As stated earlier, the dehydro-Phe at the ( i + 1) position is found to adopt one of the two confor- mations with @,+close to -60", 140" or -60°, -30". Thus, as seen from Table I11 with flexible residues a t the ( i t 2 ) position (first three sequences), the dehydro-Phe tends to promote a &turn I1 confor- mation while with a rigid residue (sequences 4-9 in Table IV) it tends to generate an alternating right- handed-left-handed helical s t r u ~ t u r e . ~ ~ ' ~ ~ ~

a,P-DEHYDRO-AMINO ACID RESIDUES 1247

Table I11 Conformation of Dehydro-Phe in Peptides Containing Dehydro-Phe at the (i + 1) Position

Peptide 41 $1 @2 *2 Ref.

1. N-Ac-dehydro-Phe-NHCH, 2. N-Ac-dehydro-Phe-L-Ala-OCH, 3. N-Ac-dehydro-Phe-L-Pro-OH 4. N-Ac-dehydro-Phe-L-Leu-OCH3 5. N-Ac-dehydro-Phe-NorVal-OCH, 6. N-Ac-dehydro-Phe-r~-Val-OH 7. N-Ac-dehydro-Phe-rd-Val-L-Val-

8. N-Ac-dehydro-Phe-dehydro-F'he-

9. N-Ac-dehydro-Phe-dehydro-Phe- Gly-OH

-58.3(4) -68.0(1) -5 1.5 (6) -57.0(5) -56.0(5) -60(2)

i) -40.0(1) ii) -40.0(1)

42.9(3)

41.0(3)

148.0(5) 147.0( 1) 135.2(8) -37.0(5) -37.5(5)

-50.0(1)

51.2(4)

-31(2)

-49.0(1)

55.3(3)

-75.0( 1) -69.2(5) 101.7(3) 100.4(2) 5%') 54.0(1) 54.0(1)

~ 55.2(4)

-48.0(3)

46.0(1) 46.0(1)

--49.6( 5 )

-39.1 (3)

17 16 12

p.s. p.s. 30 31 31 7

8 L-Ala-OH

* There are two crystallographically independent molecules in the unit cell. ps.; present structures

Molecular Packing and Hydrogen Bonding

The molecular packings in the unit cells as seen along the a axis are shown in Figure 2a and 2b. The molecules are arranged in the form of helices along the c axis. The molecules are linked with each other by two hydrogen bonds: N1-Hl-OI (symmetry re- lated) = 2.872(4) A ( I ) [LN1-H1-O1 = 161.0(3)0] and 2.872(4) 8, (11) [LN1-Hl-OI = 161.1(2)0];

Figure 2. ( a ) The unit cell packing of peptide I as seen along the b axis. ( b ) The unit cell packing of peptide I1 as seen along the b axis.

N2-H2-0; (symmetry related) = 2.860(4) 8, ( I ) [LN2-H2-O; = 160.7(2)O] and 2.858(4) 8, (11) [ LN2-H2-0; = 160.7 ( 3 ) " ] . I t is noteworthy that the sequences N-Ac-dehydro-Phe-Ala-OH, N-Ac- dehydro-Phe-L-Val-OH, and the present peptides crystallize isomorphously and show identical pack- ings along the c axis.

CONCLUSIONS

1. A dehydro-Phe/Leu/Abu at the ( i + 2 ) position in a three-peptide unit sequence induces a p-turn I1 conformation with an i

2. A dehydro-residue at the ( i + 1) position with a flexible residue such as Gly, Ala at the ( i + 2 ) position produces a P-turn I1 confor- r,lation.' 2,lfi,17,26

3. A dehydro-residue at the ( i + 1 ) position, with a bulky residue such as Leu, Val, etc., a t the ( i + 2 ) position, is accommodated in an alternating right-left helical conforma- tion.30.3 l

4. If the dehydro-residues appear consecutively in a peptide sequence, the resulting confor- mation is an alternating right-handed-left- handed

5 . A sequence containing two or more dehydro- residues, which are separated by one or two saturated residues, results in a 310-helical conf~rrnation.~~~~~~"~~~

6. The dehydro-Ala has only a -CH2 as its

+ 3 + i hydrogen bond.14,20-2~,25,~7,28, 1 A'55.41.42

1248 SHARMA, NARULA, AND SINGH

side chain. The steric effects caused by this are small, which can be released through a small distortion in geometry. The N-C "-C angle in dehydro-Ala is on the order of 110". As a result of this, a planar dehydro-Ala adopts a more preferable extended confor- mation with +,I) on the order of 180°,

7. The constraints introduced by dehydro-Val are much more pronounced than those ob- served in dehydro-Phe, dehydro-Leu, dehy- dro-Pro, dehydro-Abu, and dehydro-Ala. This is because the side-chain branching in de- hydro-Val occurs a t the C" atom itself. As a result of this, dehydro-Val at the ( i + 2 ) po- sition, unlike dehydro-Phe, cannot be ac- commodated in a 8-turn I1 conformation. Therefore, it produces a @-turn I11 confor- mation in a sequence of Xxx-Yyy-dehydro- V a l - Z ~ z . ~ ~

1800.6,10,39

The authors thank CSIR New Delhi and AvH Foundation, Germany, for financial support.

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Received September 1, 1993 Accepted March 28, 1994