Molecular and Crystal Structures of Aib-Containing Oligopeptides Boc-Leu,-Aib-Leu,-OBzl and Boc- ( Leu ,-Aib) ,-OBzl

K. OKUYAMA, Y. SAGA, M. NAKAYAMA, and M. NARITA

Faculty o i Technology, Tokyo University of Agriculture and Technology, Koganei Tokyo 184, japan

SYNOPSIS

Sample peptides Boc-Leu,-Aib-Leu,-OBzl and Boc- ( Leu,-Aib )z-OBzl, were crystallized by the solvent-evaporation method. Both crystals are monoclinic, with space group of P21 and Z = 2. The cell parameters are a = 16.580(7), b = 21.105(7), c = 11.583(4) A, and 0 = 104.90(3)" (Boc-Leu,-Aib-Leu,-OBzl),anda = 15.247(9), b = 19.04( l ) ,c = 16.311(9) A, and 0 = 117.10( 1 ) O [ Boc- (Le~, -Aib)~-OBzl] . Crystal structures were solved by the direct method and refined to R values of 0.096 (the former peptide) and 0.112 (the latter). Peptide backbones fold into a right-handed a-helix, except for the C-terminal Aib residue in Boc-( Leu4-Aib)z-OBzl. Both peptide molecules are stabilized by six (the former) or seven (the latter) intramolecular ( 5 -+ 1 ) hydrogen bonds, and arranged in the head-to- tail fashion, which makes an infinite column. In this column, one (the former) or two (the latter) intermolecular hydrogen bonds link the neighboring molecules. In the case of Boc- Leu,-Aib-Leu,-OBzl, the solvent molecule N,N-dimethylformamide was found in the dif- ference Fourier map. There was a hydrogen bond between peptide and solvent molecule. Along the lateral direction, only hydrophobic contacts were observed between adjacent peptide molecules.

I NTRO D U CTI 0 N

The a-aminoisobutyric acid (Aib) residue is mainly contained in the peptides that form membrane channels such as alamethicin and suzukacillin. It is known that these channels are formed by the as- sociation of several helical units. Because of the steric hindrance between the carbonyl oxygen and the two methyl moieties linked to a C, atom, the torsion angles of an Aib residue are severely re- stricted in the narrow regions, that is, a right- and left-handed a- or 310-helix regions. Therefore, the peptides containing the Aib residues commonly adopt the above a- or 3,,)-helical conformations.' In particular, the 310-helical conformation is found in the relatively short peptides and also in the peptide segments with high contents of the Aib r e ~ i d u e . ~ - ~ By utilizing the ability of Aib residue to promote helical folding of peptides, the improvement of the

Biupolymers. Vol : < I , 975-98.5 (1991) t> 1991 John Wilev & Sons, Inc. CCC 0006-3525/91/080975-11$04.00

solubility of the protected peptide fragment was ex- pected.' In fact, conformational analysis by ir spec- troscopies indicated that Boc- ( Leu4-Aib)2-OBzl had helical conformations ( 310- or a-helices) in dichlo- romethane solution.8 The helical type ( a or 310) is very important since the distribution of side chains along the helical rod and the radius of the helix is fairly different from each other. In order to study relationships between the content of Aib residue and its sequence in the fragment peptide and the helical conformation, we have analyzed several crystal structures of Aib-containing pep tide^.^-" In this pa- per, we will discuss molecular and crystal structures of the Aib-containing oligopeptides Boc-Leu,-Aib- Leu4-OBzl and Boc- ( Le~~-Aib )~-OBzl .

EXPERIMENTAL

Both oligopeptides were prepared by stepwise elon- gation and fragment condensation methods. The procedures have been described in detail elsewhere.' The powder sample of Boc-Leu4-Aib-Leu4-OBzl was

975

976 OKUYAMA ET AL.

Table I Crystal Data and Details of Experiments and Analyses

Compound

Boc-Leu4-Aib-Leu,-OBzl DMF Boc-(Leu,-Aib)y-OBzl

Formula weight Crystal system Space group a (A) b (A) c (A) B ("1 Cell volume (A3) 2 DCa1, ( g - ~ m - ~ ) Do~,s (g-cm-') Radiation p(ca1cd) (cm-') Crystal size (mm3) Scan mode Scan width ("; in w ) Scan speed ( O ; in w ) min-' 28 range (") No. of reflections with Fo 2 0 No. of reflections for R

R R,

1271.7 Monoclinic P2, 16.580 (7) 21.105 (7) 11.583 (4) 104.90 (3) 3917 (1) 2 1.143 1.16 CuKcv 5.0 0.3 X 0.3 X 0.3 28 - w

1.2 + 0.14 tan fl 6

5930 4603 Fo 2 3u ( F o ) 0.096 0.084

4.0-120

1283.7 Monoclinic p21 15.247 (9) 19.04 (1) 16.311 (9) 117.10 (1) 4215 (5) 2 1.011 1.03 CuKcv 5.0 0.5 X 0.5 X 0.3 28 - w 1.8 + 0.14 tan 8 6

5524 4406 Fo 2 20 (Fo) 0.112 0.096

4.5-120

Table I1 for Nonhydrogen Atoms of Boc-Leu4-Aib-Leu4-OBzl and B~c-(Leu,-Aib)~-OBzl, with Estimated Standard Deviations in Parentheses'

Fractional Coordinates and Equivalent Isotropic Temperature Factors

Boc-Leu,-Aib-Leu,-OBzl Boc-(Leu4-Aib)2-OBzl

Atom (lo4) Y (lo4) (lo4) Be, (A') (lo4) Y (lo4) (lo4) Be, (-4')

Boc c1 c 2 c3 c4 01 c5 0 2

Leu1 N C a C' 0 Cb C r C61 C62

Leu2 N CCI

7531 (12) 7393 (11) 8791 (9) 7861 (10) 7776 (5) 7041 (9) 6362 (6)

7149 (7) 6465 (8) 5728 (8) 4994 (6) 6837 (9) 6213 (12) 6523 (12) 6256 (18)

5924 (7) 5236 (9)

1596 (1 1) 2783 (12) 2331 (0) 2263 (12) 2398 (9) 2468 (10) 2349 (8)

2688 (9) 2993 (10) 2537 (11) 2691 (8) 3231 (10) 3630 (13) 3631 (13) 4337 (14)

1942 (9) 1516 (10)

13535 (16) 13855 (16) 13863 (13) 13359 (14) 12090 (8) 11311 (13) 11476 (9)

10243 (10) 9395 (12) 8922 (12) 8836 (9) 8356 (12) 7420 (18) 6226 (16) 7852 (22)

8522 (10) 7930 (13)

9.9 (8) 10.0 (8) 9.3 (7) 7.2 (7) 6.7 (4) 5.7 (5) 7.2 (4)

5.6 (4) 5.6 (5) 5.6 (6) 7.0 (4) 6.3 (6)

10.5 (8) 10.9 (9) 17.1 (15)

5.6 (4) 5.8 (5)

1534 (13) 1946 (13) 2337 (13) 804 (11) 906 (6)

1255 (12) 2069 (6)

613 (8) 916 (10)

1185 (10) 1939 (6)

144 (12) -79 (18)

-923 (19) 793 (19)

599 (8) 722 (1 1)

2667 (0) 1968 (11) 3158 (15) 2537 (12) 2981 (7) 3200 (10) 3087 (7)

3659 (8) 4158 (10) 3777 (9) 3931 (6) 4710 (11) 5244 (13) 5778 (12) 5629 (12)

3316 (8) 2990 (10)

-3152 (11) -2638 (13) -3033 (12) -4151 (10) -2772 (7) -1911 (12) -1249 (6)

-1800 (7) -1065 (9) -141 (9)

577 (5) -1220 (11) -1979 (13) -1996 (15) - 1838 (14)

-114 (7) 777 (10)

13.6 (9) 17.0 (10) 18.6 (12) 14.9 (9) 10.5 (4) 9.4 (7)

11.3 (4)

7.6 (4) 8.4 (6) 7.8 (6) 9.4 (3) 9.6 (7)

14.1 (9) 18.5 (11) 18.5 (11)

9.1 (5) 8.7 (6)

STRUCTURES OF AIB-CONTAINING OLIGOPEPTIDES 977

Table 11 (Continued)

Boc-Leu,-Aib-Leu,-OBzl Boc-(Leu,-Aib)p-OBzl

Atom (lo4) Y (10,) (10~) Be, (A2) (10,) Y (10~) (lo4) Be, (A2)

4819 (10) 4057 (6) 5579 (9) 6048 (10) 6314 (12) 5499 (13)

5336 (7) 4950 (9) 4391 (10) 3754 (6) 5625 (10) 6180 (11) 6810 (12) 5714 (13)

4667 (7) 4145 (9) :3319 (9) 2668 (5) 4658 (10) 4266 (13) 4831 (15) 4195 (13)

3353 (7) 2586 (10) 1975 (10) 1220 (6) 2 157 (10) 2847 (10)

2296 (7) 1769 (11) 1:377 (11) 699 (7)

2:<15 (12) 2136 (14) 3010 (14) 1511 (15)

2306 (18) 1888 (19) '543 (22) 11 39 (18)

1857 (8) 1601 (12) 906 (14)

1245 (10) 1166 (9) 984 (10)

1227 (12) 621 (12)

1705 (13)

1068 (9)

1270 (11) 1086 (9) 546 (10) 20 (12)

-217 (13) -565 (12)

774 (10)

1868 (10) 2356 (10) 2434 (10) 2480 (8) 2959 (10) 3473 (13) 4069 (11) 3253 (12)

2464 (10) 2549 (11) 2011 (12) 2103 (8 ) 3191 (11) 2481 (12)

1431 (10) 878 (11) 858 (11) 640 (9) 205 (10)

19 (12) -551 (12) -365 (12)

391 (15) -265 (16) -844 (18) -328 (15)

1113 (10) 1139 (11) 1607 (14)

8843 (16) 8594 (9) 7259 (13) 6334 (16) 5782 (17) 5437 (17)

9934 (11) 10823 (14) 11247 (13) 11469 (9) 11883 (13) 11543 (18) 12816 (20) 10845 (19)

11379 (11) 11777 (15) 10794 (18) 11159 (9) 12056 (17) 12618 (20) 12768 (20) 13867 (21)

9660 (11) 8698 (14) 8786 (14) 8660 (10) 8774 (17) 7524 (15)

8952 (12) 8953 (16)

10043 (20) 9986 (10) 9242 (18) 7951 (21) 8069 (20) 7254 (21)

8704 (30) 8616 (29) 8562 (30) 7598 (27)

11079 (14) 12163 (18) 12102 (17)

6.5 (6) 7.4 (4) 6.4 (6) 7.9 (7)

10.2 (8) 11.0 (9)

6.1 (4) 6.1 (6) 6.4 (6) 7.3 (4) 6.4 (6)

10.3 (8) 11.6 (9) 12.8 (10)

6.6 (4) 6.7 (6) 6.7 (6) 7.3 (4) 8.6 (7) 9.8 (9)

13.7 (12) 11.6 (9)

6.6 (4) 6.8 (6) 6.7 (5) 7.8 (4) 8.9 (7) 8.5 (7)

6.6 (5) 7.5 (7) 7.4 (7) 9.0 (5) 6.5 (5) 9.3 (6)

10.5 (7) 11.0 (7)

5.9 (8) 7.7 (8)

10.1 (10) 6.5 (8)

7.1 (5) 8.1 (7) 9.6 (9)

1589 (10) 2109 (6) -162 (15) -898 (19) -699 (30)

-1966 (13)

1716 (10) 2531 (12) 3513 (11) 4203 (7) 2629 (20) 1847 (13) 1931 (15) 1960 (16)

2309 (38) 3060 (24) 2744 (26) 2836 (32)

3548 (10) 4428 (10) 4581 (13) ,5452 (6) 4397 (12) 4566 (16) 5738 (16) 4411 (22)

3840 (9) 3939 (11) 4451 (9) 5154 (6) 2890 (10) 4572 (11)

4098 (8) 4443 (11) 5513 (11) 6119 (6) 3796 (10) 4065 (27) 3851 (31) 3323 (34)

2917 (12) 2953 (15) 2549 (18)

5761 (8) 6760 (11) 7489 (11)

2468 (10) 2401 (6) 2633 (13) 3108 (15) 3358 (24) 2635 (18)

2095 (9) 1624 (9) 2097 (12) 1859 (6) 1192 (16) 687 (12) 297 (12)

39 (12)

1279 (33) 724 (22) 340 (18) 193 (24)

2690 (10) 3104 (11) 3408 (9) 3397 (6) 3726 (12) 3508 (14) 3273 (17) 4193 (20)

3652 (8) 3958 (9) 3416 (10) 3532 (6) 4055 (9) 4648 (8)

2762 (9) 2208 (10) 2025 (9) 1898 (6) 1528 (8) 995 (24)

1222 (29) 349 (33)

1554 (11) 1936 (13) 838 (15)

1943 (7) 1730 (9) 2317 (12)

1087 (11) 1941 (6) 716 (11) 759 (20)

1655 (21) 420 (18)

462 (8) 806 (10)

1187 (11) 1910 (6) -28 (16)

-336 (12) -1220 (14)

290 (14)

-49 (36) 28 (23)

-950 (23) 476 (27)

756 ( 8 ) 1180 (11) 2116 (10) 2773 (5) 563 (12)

-238 (20) 256 (20)

-789 (18)

2208 (9) 3062 (8) 3794 (9) 4577 (5) 2946 (9) 3288 (9)

3631 (7) 4311 (10) 4583 (10) 5390 (5) 3981 (10) 4683 (27) 5514 (32) 4189 (36)

4252 (12) 5005 (14) 4372 (16)

3906 (7) 4057 (11) 4658 (10)

8.9 (6) 10.2 (4) 11.6 (8) 18.7 (15) 37.6 (25) 24.6 (17)

9.5 (5) 9.8 (6) 9.6 (7)

10.4 (4) 9.9 (8)

11.9 (5) 15.8 (6) 15.0 (6)

7.0 (19) 8.5 (9) 9.3 (9)

12.2 (12)

9.3 (5) 9.6 (7) 9.2 (7) 9.6 (4)

11.5 (8) 15.8 (13) 15.4 (17) 26.4 (16)

9.0 (5) 8.7 (6) 8.2 (6) 9.6 (3)

11.2 (6) 10.8 (6)

8.0 (4) 9.2 (6) 8.2 (6) 9.7 (4)

11.5 (6) 13.0 (11) 16.4 (15) 18.5 (18)

12.0 (5) 16.6 (7) 19.9 (8)

8.7 (5) 9.7 (7) 9.6 (7)

978 OKUYAMA ET AL.

Table I1 (Continued)

Boc-Leu,-Aib-Leu,-OBzl Boc-( Leu,-Aib),-OBzl

Atom x (10,) Y (lo4) (10,) Be, (A2) (lo4) JJ (10,) (10,) Be, (A2)

0 CP C r C6* C6,

Leu7' CP CY C6, C6,

Leu8 N CCU c' 0 CP C r C61 C6,

Leu9 N CCY C' 0 CP CY C61 C6,

AiblO N CCY C' 0 C& CP2

OBzl 01 c1 c 2 c 3 c 4 c 5 C6 c 7

DMF N1 c1 c 2 c 3 01

385 (10) 2294 (21) 2707 (17) 3330 (20) 2240 (33)

2558 (23) 2277 (19) 2977 (19) 2167 (30)

903 (8) 348 (9)

-388 (10) -1035 (7)

859 (10) 1509 (12) 2054 (13) 1127 (15)

-290 (7) -950 (9)

-1359 (11) -1994 (7) -636 (10) -307 (11)

15 (15) -948 (13)

-873 (7) -1141 (12) -811 (11) -118 (10)

182 (12) -255 (14) -939 (13)

-1192 (12)

-1666 (10) -880 (13)

-2072 (26) -2112 (20) -2826 (8 )

1562 (10) 1371 (19) 651 (15) 867 (17) 137 (25)

1171 (20) 1052 (16) 1289 (15) 392 (27)

2161 (10) 2689 (11) 2736 (10) 3007 (9) 3321 (11) 3384 (13) 3975 (12) 3322 (17)

2521 (9) 2605 (11) 1987 (12) 1953 (9) 2946 (11) 3636 (12) 3910 (12) 4082 (11)

1502 (9) 885 (10) 809 (11)

1153 (11) 1057 (11) 590 (12) 245 (12) 380 (11)

531 (13) 480 (23) -35 (19) 955 (16)

1047 (11)

12686 (15) 13345 (39) 13760 (26) 14828 (30) 14333 (48)

13127 (30) 14289 (29) 15328 (26) 14627 (42)

11470 (11) 11527 (13) 10451 (16) 10532 (9) 11762 (15) 12937 (22) 12871 (23) 14024 (18)

9389 (11) 8298 (14) 7751 (16) 6976 (9) 7315 (14) 7643 (19) 6573 (19) 8072 (20)

8235 (9) 7682 (16) 6555 (14) 6425 (14) 5436 (19) 4535 (17) 4694 (18) 5698 (18)

10400 (15) 11079 (24) 9837 (38)

10217 (26) 9681 (15)

17.2 (8) 5.6 (5) 9.3 (6)

13.9 (7) 10.0 (8)

8.6 (5) 10.3 (6) 12.0 (7) 9.4 (7)

6.4 (5) 6.7 (6) 6.8 (6) 9.3 (5) 7.9 (6)

10.1 (9) 12.2 (12) 18.5 (17)

5.9 (4) 6.1 (6) 7.0 (7) 9.5 (5) 7.4 (6) 9.6 (8)

13.9 (12) 11.4 (10)

7.7 (4) 8.4 (7) 6.5 (6) 7.5 (6) 8.9 (8 ) 9.3 (8) 9.4 (8 ) 8.5 (7)

12.8 (10) 25.8 (26) 29.6 (30) 16.5 (19) 13.1 (7)

8324 (7) 6698 (12) 6405 (15) 7157 (19) 6491 (18)

7588 (25) 7559 (39) 7920 (42)

7222 (8) 7897 (10) 8000 (11) 8738 (6) 7602 (11) 7714 (16) 8744 (19) 7282 (17)

7263 (9) 7346 (12) 8056 (11) 8635 (7) 6333 (12) 5663 (17) 6046 (17) 4577 (13)

7986 (9) 8608 (12) 8580 (14) 9292 (8) 8137 (12) 9682 (10)

7692 (8) 7581 (13) 6521 (16) 5815 (24) 4863 (17) 4609 (31) 5334 (32) 6249 (21)

2132 (7) 1706 (10) 966 (12) 386 (14)

1062 (13)

1505 (20) 1301 (29) 736 (34)

2993 (9) 3560 (9) 3594 (8) 3861 (6) 4235 (10) 4259 (13) 4236 (19) 4982 (13)

3378 (7) 3318 (11) 2749 (13) 2810 (8) 3209 (12) 3830 (14) 4458 (15) 3575 (15)

2128 (10) 1521 (12) 1266 (11) 1074 (10) 882 (10)

1711 (11)

1246 (7) 946 (16)

1062 (13) 1456 (15) 1554 (20) 1321 (31) 871 (41) 707 (25)

5220 (6) 3139 (14) 2706 (15) 3229 (17) 1726 (16)

3107 (25) 2168 (36) 3464 (38)

4513 (7) 5026 (9) 5997 (8) 6610 (6) 4525 (11) 3639 (12) 3836 (17) 3150 (14)

6150 (6) 7091 (10) 7607 (11) 8451 (6) 7080 (11) 6773 (14) 7485 (17) 6523 (14)

7166 (8) 7509 (12) 8385 (12) 9088 (7) 6726 (12) 7689 (11)

8314 (7) 9070 (14) 8966 (13) 8195 (21) 8270 (35) 8790 (31) 9420 (36) 9464 (20)

11.8 (4) 13.7 (9) 16.2 (7) 16.9 (8) 19.7 (8)

11.0 (10) 16.1 (16) 18.5 (19)

7.9 (4) 7.8 (5) 7.4 (6) 9.7 (4) 9.6 (7)

15.8 (11) 26.4 (17) 19.0 (11)

8.4 (5)

10.3 (7) 12.8 (5) 10.8 (8) 15.2 (10) 21.6 (14) 18.6 (13)

10.0 (6) 13.3 (9) 11.7 (8) 18.3 (7) 12.9 (8) 14.4 (9)

12.7 (5) 16.2 (10) 12.9 (9) 19.6 (14) 20.9 (23) 23.3 (26) 47.5 (40) 29.5 (22)

10.2 (7)

a B, = Z Zu, * up,,. Site occupation factors in Leu 6 and 7 of Boc-Leu,-Aib-Leu4-OBzl are 0.6 and 0.5, respectively, and those in Leu 3, 6, and 7 of Boc-(Leu,-Aib)2-OBzl are 0.7.

S T R U C T U R E S OF AIB-CONTAINING OLIGOPEPTIDES 979

dissolved in N,N-dimethylformamide ( DMF) . After about two weeks, the colorless, cubic, single crystals were obtained by slow evaporation of the solvent at room temperature. The cubic single crystals of Boc- ( Leu,-Aib)2-OBzl were grown from a dichloro- methane solution by the similar procedure.

The cell parameters and x-ray diffraction inten- sities of the above two crystals were measured on a four-circle diffractometer (RASA-5RI1, Rigaku Corp.) with graphite monochromated CuK,, radia- tion ( A = 1.5418 A ) . The cell parameters were re- fined by the least-squares fit using 17 reflections in the 20 range of 50"-60" for the former and 25 re- flections in the 20 range of 45"-55" for the latter. Three reflections, monitored every 100 reflections,

Figure 2. Molecular structure of Boc- (Leu,-Aib )?-OBzl together with the atomic numbering in the terminal moi- eties (Boc and OBzl) . Balls with oblique lines denote the atoms with the smaller occupancies (Leu3', Leu6' and Leu7'). Broken lines denote intra- and inter-molecular hydrogen bonds.

Figure 1. Molecular structure of Boc-Leu4-Aib-Leu4- OBzl together with the atomic numbering in the terminal moieties (Boc and OBzl ) . Balls with oblique lines denote the atoms with the smaller occupancies (Leu6' and Leu7'). Broken lines denote intra- and intermolecular hydrogen bonds.

showed no significant intensity deterioration during the data collection for both crystals. The intensity was corrected for Lorentz and polarization factors, but not for absorption. Crystal data and the exper- imental conditions are listed in Table I.

The structures of these crystals were solved by the direct method with the SHELX-86 program.I2 Both structures were refined by the full-matrix least- squares method for all nonhydrogen atoms with iso- tropic thermal factors in the early stage of refine- ments. After several refinement cycles, large thermal factors were observed for some atoms in the Leu side chains. Using the structures without these at- oms, D-Fourier syntheses were performed, which revealed two plausible conformations with different occupancies for the side chains in Leu6 and Leu7 of

980 OKUYAMA ET AL.

Table I11 Torsional Angles (") of Main Chains and Those of Side Chains in Leu Residues"

Leu1 Leu2 Leu3 3' Leu4 Aib5 Leu6 6' Leu7 7' Leu8 Leu9 AiblO

-64 -49

-173 -174 -160

85

-71 -49

-173 -69

-180 -58

- 73 -41

-177 -56

-179 -53

-72 -35

-180 -79 159

-78

-66 -38 180 -63

-174 -52

- 70 -37 176

-74 178 177 178

-74 74

Boc-Leu4-Aib-Leu,-OBzl

-65 -56 -68 -44 -45 -33

-179 -175 180 -173 -88 -178 -176 168 -170

65 - 85 65

Boc-(Leu,-Aib),-OBzl

-65 -54 -65 -41 -50 -46

-179 -173 -177 -75 -174 -90 175 -179 -150

-70 67 -27

-70 -36

-169 -88

-180 -74

-62 -39

-177 -91

-175 -63

-168 -160

85

-180 -167

63

- 103 -29

-174 -64 169

-59

- 75 -28 173

-68 175 - 68

-113 - 16

-62 178

-53

-67 59 -45 45 177

-70 164

-68

Torsional angles of the disordered side chains are listed in the columns for 3', 6'. and 7'. Maximum standard deviations of (d, $, w ) and ( X I , X2, X;) are 4" and 7", respectively.

Boc-Leu4-Aib-Leu4-OBzl, and Leu3, Leu6, and Leu7 of Boc- ( Le~~-Aib )~-OBzl . Including occupancies of every disordered atoms, both structures were refined another several cycles. In both cases, hydrogen at- oms except for those in the above disordered side chains were located a t calculated positions and their coordinates were refined together with their isotropic thermal factors a t the last stage of the analyses. The final R values were 0.096 ( R , = 0.087) for Boc-Leu4- Aib-Leu4-OBzl and 0.112 ( R , = 0.091) for Boc- ( Le~~-Aib )~-OBzl . The final atomic parameters for nonhydrogen atoms of each crystal are listed in Ta- ble 11.

The atomic scattering factors were taken from International Tables for X-Ray Crystallography." Computations were done on an A-70 minicomputer with the help of the CRYSTAN program in RASA- 5RII system (Rigaku Corp.) and on an ACOS 1000 computer a t the Information Processing Center, Tokyo University of Agriculture and Technology.

RESULTS AND DISCUSSION

For the convenience of atom specification, all the residues are numbered in series from the N- to the C-terminus. Therefore, the molecules of Boc-Leu4- Aib-Leu,-OBzl and Boc- ( L e ~ ~ - A i b ) ~ - O B z l are de- noted as Boc-Leul-Leu2-Leu3-Leu4-Aib5-Leu6- Leu7-Leu8-Leu9-OBzl and Boc-Leul-Leu2-Leu3- Leu4-Aib5-Leu6-Leu7-Leu8-Leu9-Aib10-OBzl. The

names of atoms in each residue are those given in the IUPAC-IUB recommendations, l4 while those in the protected groups a t N- and C-termini are shown in Figure 1.

Peptide-Backbone Conformations

Perspective views of both molecules are shown in Figures 1 and 2. There are no unusual bond lengths and bond angles in the main chains of both peptides. Torsional angles in the main chains of these peptides are listed in Table 111, together with those in the side chains of Leu residues. The torsional angles (4, $) indicate that both peptide backbones fold into a right-handed helix, with the exception of the AiblO residue in Boc- ( Le~~-Aib )~-OBzl . The change of helical sense at C-terminal Aib residue is commonly observed in the oligopeptide helices with a bulky protected moieties a t C-terminus.6~9~"~~'5~17 That is, since the 4 and $ values of Aib residue are severely restricted to the right- or left-handed a- (or 310-) helical regions, the helical sense of the C-terminus Aib residue has to be reversed in order to avoid con- tacts between the protected moiety and the rest of the molecule, especially contact between 01 (Boc) and 0 ( Leu7). On the other hand, in the case of Boc-Leu4-Aib-Leu4-OBzl, C-terminal amino acids (Leu8 and Leu9) avoided sterical short contacts by deviating from the helical conformation. The change of the helical sense in the AiblO residue pushed the C-terminal benzyl moiety outside the helical rod

.E C

Figure 3. perpendicular t o the helix axis and ( b ) along the helix axis.

Stereoviews of the packing structure of Boc-Leu,-Aib-Leu4-OBzl ( a ) projected

982 OKUYAMA ET AL.

Figure 4. perpendicular to the helix axis and (b ) along the helix axis.

Stereoviews of the packing structure of Boc- ( Leu4-Aib)?-OBzl ( a ) projected

STRUCTURES OF AIB-CONTAINING OLIGOPEPTIDES 983

further than that in Boc-Leu4-Aib-Leu4-OBzl. The difference between these two conformations a t the C-terminus region was clearly shown in Figures 3b and 4b.

Except for the C-terminal Leu7 and Leu8 in Boc- Leu,-Aib-Leu4-OBzl and AiblO in Boc- ( L e ~ ~ - A i b ) ~ - OBzl, the distribution of 4 and $ in a Ramachandran map coincide well with those reported for the helical peptides, which contain only Aib, Ala, and Leu res- idues.' The mean values of these 4 and $ in the former are (-66", -41") and those in the latter are (-67", -41" ) except for the C-terminal AiblO. Be- cause of the overlapped regions of the allowed tor- sional angles for the a- and 310-helix in the +$ map, it is very difficult to determine the helical type from 4 and $ values. Therefore, the average helical pa- rameters calculated from those of each amino acid residues were used to determine the helical The helical parameters are the unit height h ( the height per residue along the helical axis) and the unit twist B ( the rotational angle per residue around the helical axis). Assuming that each amino acid residue constructs its own helix, the helical param- eters are derived from its bond lengths, bond angles, and torsional angles by the method of Sugeta and Miyazawa." These values for each residue are shown in Tahle IV. The average values excluding the C - terminal residue for Boc-Leu4-Aib-Leu4-OBzl and Boc- ( I , e~~-Aib )~-OBzl are h = 1.55 A, 8 = 95", and h = 1.48 A, B = 98", respectively. Since the helical parameters for the a- and 310-helix are h = 1.5 A, B = loo", and h = 2.0 A, B = 120", respectively, it is obvious that these peptides have an a-helical con- format ion. These results are consistent with the he- lical types expected from the figure, which shows the distribution of helical types with a plot of the total number of residues in a peptide vs the number of Aib residue^.^ That is, the presence of a small fraction of Aib residues in a peptide prefers an a- helix.

Hydrogen-bond lengths and angles for each pep- tide are listed in Table V. The helical structure of

Boc- ( L e ~ ~ - A i b ) ~ - O B z l is stabilized by successive seven 5 + 1 intramolecular hydrogen bonds, which belongs to the a-helix. In the case of Boc-Leu4-Aib- Leu4-OBzl, six intramolecular hydrogen bonds with the same mode were observed. These results agree with those deduced from the helical parameters. It seems that the lengths of hydrogen bonds in both peptides are slightly longer than the standard value (2.9 k 0.1 A)" usually observed in the peptide structures in which no Aib residue is included. Hy- drogen-bond angles, 137" for N (Leu7) . . O (Leu3) and N (Leu8) - * * 0 (Leu4) in Boc-Leu,-Aib-Leu4- OBzl, are significantly deviated from the ideal angle ( 180" ) . These deviations are also observed in some Aib-containing o l ig~pep t ides .~*~""~~

Side-Chain Conformations

Torsional angles of Leu side chains are listed in Ta- ble 111. All side chains without disorder adopt the g- ( t , g- ' ) conformation, with the exception of those of Leul and Leu4 in Boc-Leu4-Aib-Leu4-OBzl, where the t ( t , g) conformation is observed. In the case of disordered side chains, with the exception of Leu6' in Boc- ( Leu4-Aib)*-OBzl, the major conformation is g - ( t , g - ) while the minor one is t ( t , g) . These observations were consistent with the side-chain conformations of Leu residues found in the other oligopeptide crystals." Neither conformation is adopted in Leu6' of Boc-Leu4-Aib-Leu-OBzl.

Crystal Structure

The packing structures of Boc-Leu4-Aib-Leu4-OBzl and Boc- ( Leu4-Aib)2-OBzl viewed along the per- pendicular direction to the helical axis and along the helical axis are shown in Figures 3 and 4. In Figure 3a and Figure 4a, peptide molecules arranged side by side are related by the Z1 symmetry along the b axis. The relations between molecules arranged in a head-to-tail fashion are ( x , y , z ) and ( x + 1, y , z ) in Boc-Leu4-Aib-Leu,-OBzl, and ( x , y , z ) and ( x

Table IV and Boc-(Leu4-Aib)z-OBzl

Helical Parameters for Each Amino Acid in Boc-Leu,-Aib-Leu4-OBzl

Leul Leu2 Leu3 Leu4 Aib5 Leu6 Leu7 Leu8 Leu9 Average

Boc-Leu,-Aib-Leu,-OBzl h (A) 1.54 1.39 1.55 1.47 1.63 1.69 1.85 1.27 1.55 0 ( " 1 91 93 100 97 102 102 96 81 95

h (A) 1.46 1.47 1.40 1.55 1.57 1.47 1.64 1.45 1.33 1.48 0 ("1 88 99 99 95 101 95 102 103 98 98

Boc-( Leu,-Aib),-OBzl

984 OKUYAMA E T AL.

Table V and Boc-(Leu,-Aib),-OBzl"

Hydrogen-Bond Parameters of Boc-Leu,-Aib-Leu4-OBzl

~

Length (A) Angle ( " ) Donor Acceptor N * - a 0 N-H. . * O

Boc-Leu,-Aib-Leu,-OBzl Intramolecular

N (Leu4) N (Aib5) N (Leu6) N (Leu71 N (Leu8) N (Leu9)

Intermolecular N (Leul) N (Leu3)

Intramolecular N (Leu4) N (Aib5) N (Leu6) N (Leu71 N (Leu81 N (Leu9) N (AiblO)

Intermolecular N (Leul) N (Leu2)

Boc-(Leu,-Aib)2-OBzl

0 (Boc) 0 (Leul) 0 (Leu2) 0 (Leu3) 0 (Leu4) 0 (Aib5)

0 (Leu8) 0 (DMF)

0 (Boc) 0 (Leul) 0 (Leu2) 0 (Leu3) 0 (Leu41 0 (Leu5) 0 (Leu6)

0 (Leu8) 0 (Leu9)

2.96 3.14 3.10 3.06 3.11 2.97

3.02 3.13

3.10 2.97 3.11 3.04 2.99 3.08 3.03

2.88 2.96

156 162 154 137 137 156

141 145

159 162 167 160 147 148 148

168 153

a Estimated standard deviations of bond length is less than 2 A.

+ 1, y , z + 1) in B~c-(Leu~-Aib)~-OBzl . One (the former ) or two (the latter ) intermolecular hydrogen bonds are formed between adjacent helices along the helical axis (Table V ) , which results in the infinite columns along their helical axes. In the case of Boc- Leu4-Aib-Leu4-OBzl, the solvent molecule DMF is accommodated in the space between the head and the tail part of the peptide molecules in the same column. The carbonyl oxygen of DMF plays a role as an acceptor for the hydrogen bond with the NH group of Leu3 (Table V ) . There are no hydrogen bonds between adjacent molecules in the lateral di- rection in both cases. Only hydrophobic interactions stabilize the crystal structure in this direction.

Figure 3b and Figure 4b show the arrangement of helix columns running through the [ 1 0 01 di- rection and [ 1 0 11 direction, respectively. In the latter case, one column is surrounded with six an- tiparallel columns and packed in the pseudo-hex- agonal arrangement (Figure 4b). This mode of ag- gregation is frequently observed in crystal structures for many Aib-containing oligopeptides.4~9J0J6~1g~*1~23 On the other hand, since the molecular axis of Boc-

Leu4-Aib-Leu4-OBzl lies on the crystallographic 21 axis parallel to the b direction, the columns are ar- ranged in a square lattice (Figure 3b) in which four nearest molecules are packed in an antiparallel fashion. It is interesting that the mode of aggregation of the columns is quite different from each other, even though the amino acid sequence of these pep- tides is quite similar.

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4. Bosch, R., Jung, G., Schmitt, H. & Winter, W. ( 1985)

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STRUCTURES OF AIB-CONTAINING OLIGOPEPTIDES 985

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678.

Received January 4, 1991 Accepted April 22, I991