J. CHEM. SOC. PERKIN TRANS. I 1989 Structural Studies of FR900359, a Novel Cyclic Depsipeptide from Ardisia crenata Sims (Myrsinaceae) Akira Miyamae," Mamoru Fujioka, Shigetaka Koda, and Yukiyoshi Morimoto Analytical Research Laboratories, Fujisa wa Pharmaceutical Co., Ltd., 1-6'2-chome, Kashima, Yodoga wa-ku, Osaka 532,Japan The molecular structure and absolute configuration of FR900359, a novel cyclic depsipeptide from Ardisia crenata sims, has been determined by a combination of X-ray crystallographic analysis and g.c./m.s. study of the diastereomeric derivatives of its constituents. There are five intramolecular hydrogen bonds (or short contacts) in the FR900359 molecule. All the imino nitrogen and hydroxy oxygen atoms having a proton-donating ability efficiently participate in the hydrogen bond network.The FR900359 molecule contains two cis peptide bonds, in a conformation which can take part in the hydrogen bonds. This hydrogen bond network contributes to the stabilization of the overall structure of FR900359; constituents not restrained by this network are considered to be flexible. Since the N-methyldehydro-L-alanine residue falls within the unstable region of a Ramachandran (cp-y) plot, it is vulnerable to nucleophilic attack and may, therefore, be involved in the biological activity of FR900359. FR900359 is a novel cyclic depsipeptide isolated from a methanol extract of the whole plant of Ardisia crenata sims (Myrsinaceae) as a result of a continuing search for potentially therapeutic compounds.It inhibits platelet aggregation in vitro and ex vivo in rabbits, decreases blood pressure, and shows dose-related hypotensive action in anaesthetized normotensive rats.' It is also cytotoxic in cultured rat fibroblasts and myelo- cytic leukemia cells.' Efforts at structural determination combined chemical methods with 'H-and I3C-n.m.r. spectroscopic and mass spectrometric studies of the fragments after partial hydrolyses, and resulted in all the FR900359 constituents and their p -0 H-Le u (40)(41) N-Prop--P-OH-Leu sequences being identified by M. Fujioka et aL2 FR900359 seemed to have a novel amino acid constituent; N,O-di- methylthreonine (as yet unreported in Nature), and an un-common amino acid, N-methyldehydroalanine (previously found only in a microcystis toxin from the blue-green alga, Microcystis aeruginose 3).However, the presence of these novel constituents had been only indirectly confirmed because of their susceptibility to hydrolysis. Re~ently,~ an X-ray crystallo- graphic study established unambiguously the structure of FR900359. This is discussed here, together with the absolute configuration and the conformational characteristics of the compound. N,O-M e2-T h r (34Ic N -Ac-@-O H-Leu l3 Figure 1. Chemical structure and atomic numbering of FR900359 molecule used in the X-ray investigation 874 J. CHEM. SOC. PERKIN TRANS. I 1989 Table 1. Fractional co-ordinates with e.s.d.s in parentheses X 0.412 O(5) 0.372 5(7) 0.309 9(6) 0.259 9(6) 0.230 l(4) 0.244 3(8) 0.202 8(6) 0.256 2(4) 0.248 2(5) 0.184 l(5) 0.209 l(4) 0.2 18 5(4) 0.247 l(5) 0.212 2(4) 0.249 3(5) 0.336 l(4) 0.370 6(4) 0.433 8(5) 0.467 6(5) 0.489 6(4) 0.442 O(5) 0.475 3(6) 0.372 4(6) 0.273 4(4) 0.278 5(6) 0.305 8(5) 0.244 7(7) 0.193 7(8) 0.136 4(9) 0.225 5(9) 0.278 7(7) 0.170 4( 9) 0.128 6(6) 0.101 5(11) 0.123 5(12) 0.324 2(7) Y 0.936 6(8) 0.884 8(13) 0.964 3( 1 1) 0.887 9(10) 0.795 2(6) 0.684 O( 12) 0.596 2(9) 0.558 9(7) 0.585 4(8) 0.669 l(8) 0.734 4(6) 0.851 7(8) 0.901 9(8) 0.836 2(7) 0.775 l(8) 0.786 5(9) 0.717 6(6) 0.758 6(8) 0.674 7(9) 0.732 O(8) 0.810 9(10) 0.885 2( 12) 0.778 7( 11) 1.027 7(7) 1.146 7(9) 1.202 l(7) 1.200 O( 10) 0.957 2( 12) 0.877 l(14) 1.040 9( 14) 0.654 8(8) 0.491 5(12) 0.428 l(9) 0.320 O( 16) 0.536 2(16) 0.489 7(10) z 0.906 3(5) 0.97 1 6( 10) 1.003 O(8) 1.061 9(7) 0.997 5(4) 1.028 2(8) 0.956 6(7) 0.885 9(5) 0.787 7(6) 0.747 4(6) 0.664 l(5) 0.665 3(6) 0.571 3(7) 0.482 O(5) 0.41 7 9(6) 0.425 2(6) 0.509 5(5) 0.563 9(6) 0.645 5(7) 0.739 3(6) 0.774 5(7) 0.866 7(7) 0.995 4(9) 0.920 8(5) 0.916 O(7) 0.986 8(6) 0.818 2(8) 1.095 6(9) 1.134 4(11) 1.181 3(12) 1.106 4(6) 1.011 4(9) 0.936 l(7) 0.964 O( 15) 1.092 5(12) 0.924 2(8) X 0.291 5(3) 0.1 11 O(5) 0.039 5(5) 0.023 O(7) -0.026 9( 6) 0.121 4(3) 0.135 O(5) 0.135 8(6) 0.152 5(5) 0.240 4(5) 0.275 3(3) 0.265 7(5) 0.225 l(7) 0.352 l(6) 0.121 7(4) 0.047 5(5) 0.005 5(4) 0.019 O(6) -0.042 9(9) 0.205 l(3) 0.234 6(7) 0.127 7(5) 0.215 8(3) 0.365 l(6) 0.462 8(3) 0.485 l(6) 0.558 l(6) 0.379 7(3) 0.527 2(6) 0.486 l(6) 0.479 3(7) 0.444 6(8) 0.418 6(7) 0.427 5(8) 0.460 3(7) Y 0.544 8(6) 0.599 2(8) 0.674 7( 10) 0.716 7(14) 0.602 l(14) 0.559 2(5) 0.443 3(9) 0.370 6(7) 0.423 6(8) 0.414 4(8) 0.519 5(6) 0.403 O(9) 0.301 3(12) 0.381 2(13) 0.509 7(7) 0.51 1 7(10) 0.436 6(8) 0.607 l(12) 0.578 5(27) 0.911 9(6) 1.031 8(9) 0.835 l(10) 0.715 6(6) 0.741 3(12) 0.851 8(6) 0.567 4(9) 0.682 6( 11) 0.827 3(7) 0.980 1 (1 3) 1.085 3(11) 1.190 3(14) 1.282 7( 13) 1.274 4( 13) 1.174 l(12) 1.080 3(12) Z 0.731 3(4) 0.713 2(6) 0.699 l(8) 0.805 l(10) 0.652 l(9) 0.610 8(4) 0.595 8(7) 0.658 l(6) 0.488 O(6) 0.490 O(7) 0.533 4(4) 0.386 3(7) 0.325 9(8) 0.394 7(9) 0.418 3(5) 0.387 5(7) 0.417 3(6) 0.315 l(9) 0.246 4( 18) 0.737 O(4) 0.561 2(8) 0.465 4(7) 0.352 5(4) 0.327 O(7) 0.550 8(5) 0.622 5(8) 0.801 8(8) 0.732 O(5) 0.840 8(9) 0.788 l(9) 0.838 l(11) 0.782 7( 13) 0.682 4(12) 0.635 6(11) 0.685 9(9) Figure 2.Stereographic view of the molecular conformation of FR900359 (ORTEP 11) Results and Discussion The molecule has 11 asymmetric carbon atoms. The alanine An X-ray crystallographic study identified FR900359 as a cyclic unit was assigned the L-configuration by a g.c./m.s.study, since depsipeptide with a 22-membered ring backbone and a side the retention time for the diastereoisomeric derivative of the chain (see Figure 1). The backbone is composed of one lactic alanine residue with N-trifluoroacetyl-L-proline corresponded to acid (3-Ph-Lac) and six amino acid residues (Ala, N-Me-Ala, N-that for the diastereoisomeric derivatives of authentic L-alanine Me-deH-Ala, P-OH-Leu, N-Ac-P-OH-Leu and N,O-Me,-Thr) with N-trifluoroacetyl-L-proline. The remaining optically active linked by amide or ester bonds. The side chain (N-Prop-P-OH- carbon atoms were assigned from the three-dimensional struc- Leu) is bound to the backbone by an ester bond. ture as determined by X-ray crystal analysis. The results were as J.CHEM. SOC. PERKIN TRANS. I 1989 Table 2. Bond lengths (A) with e.s.d.s in parentheses O(1 )-C(2) 1.325( 18) O(1kC(22) C(2)-C(3) 1.534(20) C(2)-O(23) C(3)-C(4) 1.531(17) C(3)-C(24) C(4)-0(5) 1.433( 13) C(4)-C(28) 0(5)-C(6) 1.3 53( 16) C(6)-C(7) C(6)-O(31) 1.203(20) C(7)-N(8) C(7)-C(32) 1.551(20) N(8)-C(9) N(8)-C(36) 1.488( 14) C(9)-C( 10) C(9)-O( 37) 1.232(12) C(10)-N( 1 1) C( 1 0)-C( 38) 1.553( 13) N( 1 1)-C( 12) C( 12)-C( 13) 1.527( 13) C( 12)-O(56) C( 13)-N( 14) 1.493( 13) C( 1 3 )-C( 57) N(14)-C( 15) 1.339( 12) N(14)-C(58) C(15)-C(16) 1.544(13) C(15)-O(59) C( 16)-N( 1 7) 1.459(12) C( 16)-C( 60) N( 17)-C( 18) 1.355(11) C( 18)-C( 19) C(18)-O(61) 1.207(11) C(19)-N(20) C( 19)-C(62) 1.313(15) N(20)-C(21) N(20)-C(63) 1.5 11(1 6) C(2 1)-C(22) C( 2 1 )-O( 64) 1.205( 14) C(22)-C(65) N( 24)-C(25) 1.367( 13) C(25)-O( 26) C(25)-C(27) 1.5 12( 16) C(28)-C(29) C(28)-C( 30) 1.555(21) C(32)-O( 3 3) C(32)-C( 3 5) 1.5 3 3( 2 7) O(3 3)-C( 34) C( 38)-C( 39) 1.534( 15) C(38)-O(42) C(39)-C(40) 1.562(20) C(39)-C(4 1) 0(42)-C(43) 1.368(11) C(43)-O(44) C(43)-C(45) 1.53 3( 1 4) C(45)-C(46) C(45)-N(51) 1.426( 12) C(46)-O(47) C(46)-C(48) 1.5 18( 14) C(48)-C(49) C(48)-C(50) 1.551(1 8) N(5 1)-C( 52) C( 52)-O( 53) 1.234( 15) C(52l.4254) C( 54)-C(55) 1.396( 33) C(65)-C(66) C(66)-C( 67) 1.389(20) C(66)-C( 71) C(67)-C(68) 1.398(23) C(68)-C(69) C(69)-C( 70) 1.327(22) C(70)-C(71) follows: five chiral centres, C(4), C(22), C(32), C(38), and C(46), took R-configurations, while the other six chiral centres, C(3), C(7), C(lO), C(13), C(16), and C(45), which were all a-carbons of 1.426( 17) amino acid residues, took S-configurations.Consequently, 1.255(20) FR900359 has been named (3S,4RY7S,10S,13S,16SY22R)-3-1.418(15) acetamido-22-benzyl- lo-{ R-1-(2SY3R)-3-hydroxy-4-methyl-2-1.531( 18) propionamidopentanoyloxy-2-methylpropyl)-4-isopropyl-7-1.524( 19) (R-l-methoxyethyl)-19-methylene-8,13,14,16,2O-pentamethyl-1.482(14) 1,5-dioxa-8,11,14,17,20-penta-azacyclodocosane-2,6,9,~2,15,-1.346( 13) 18,21-heptaone.1.541 (1 3) 1.458(11) The final fractional atomic co-ordinates for non-H atoms are 1.352( 12) listed in Table 1 and the bond lengths, bond angles, and selected 1.230( 1 1) torsion angles are listed in Tables 2, 3, and 4, respectively.The 1.507( 16) stereoview of the molecular conformation of FR900359 drawn 1.496( 14) with the ORTEP I1 program is presented in Figure 2. 1.214( 12) There are five short distances suggesting the existence of 1.524( 17) intramolecular hydrogen bonds in the molecule, as shown in 1.530( 13) Figure 3 and Table 5. The hydrogen bond between NH(24) of N-1.436( 13) 1.359(15) Ac-0-OH-Leu and O(56) of N-Me-Ala, with a distance of 1.564( 18) 2.95( 1) A and an angle of 163(10)O, crosses the depsipeptide ring 1.493( 2 1) diametrically, and possibly stabilizes the loose-fitting ring 1.200( 14) structure. The other hydrogen bonds (or short contacts) are 1.507(22) formed between Ala or 0-OH-Leu of the depsipeptide ring and 1.392(19) the N-Prop-P-OH-Leu side chain, i.e., O(59) NH(51)1.394(23) 3.08(1) A, 142(10)0, NH(17) O(47) 2.87(1) A, 158(10)O,1.481(11) NH(11) O(47) 3.32(1) A, 138(9)O, and O(37) OH(47)1.524(20) 2.66(1) A, 166(10)O, which are important in fixing the 1.179( 14) 1.567( 13) exocyclic N-Prop-0-OH-Leu side chain to the depsipeptide 1.446( 1 1) backbone.There are five atoms in all which have proton 1.55 1(1 8) donating ability in the FR900359 molecule, i.e., the imino 1.340( 14) nitrogen atoms, N(11), .N(17), N(24), and N(51) and the 1.511(18) hydroxy oxygen atom, O(47). It is noteworthy that all these five 1.539(20) atoms efficiently participate in the network of the intramolecular 1.400(19) hydrogen bonds (or short contacts) especially the O(47) atom,1.378(23) which both donates a proton, H(47), to O(37) and accepts two 1.365(20) protons from N(11) and N(17), and may play an important role as the key station in this network. Figure 3.Intramolecular hydrogen bonds or short contacts of FR900359 876 J. CHEM. SOC. PERKIN TRANS. I 1989 Table 3. Bond angles (") with e.s.d.s in parentheses Table 4. Selected torsion angles (") C(22)-0(1)-C(2) 124.3(11) O(1)-C(2)-C(3) 111.9(12) 13 Amino acid residue O(l)-C(2)-0(23) 128.1(1 4) C( 3)-C( 2)-O( 23) 118.8( 13) C( 2)-c(3)-c(4) 106.6( 11) C( 2)-C (3)-N( 24) 112.0( 11) cp w 0 x2,I x2'2 C(4 j-c(3)-~(24) 116.9( 10) C(3)-C(4)-0(5) 107.9(9) Ala -135 74 -10 --C(3)-C(4)-C(28) 1 1 1.7( 10) O(5)-C(4)-C(28) 108.5(9) N-Me-Ala -122* 37 178 --C(4)-O( 5)-C(6) 11 7.9(9) O(5)-C(6)-C(7) 111.5(11) P-OH-Leu -133 149 6 37t -76 O(5)-C(6)-0(3 1) 126.0( 14) C(7)-C(6)-0(3 1) 122.2( 13) N-Prop-P-OH-Leu -11 3 -23 -691--50 C(6)-C(7)-N( 8) 107.1 (10) C(6)-C(7)-C(32) 1 12.9( 1 1) N,O-Me,-Thr -113" 88 --65 173 N(8)-C(7)-C( 32) 1 12.q 10) C( 7)-N(8)-C(9) 123.9( 8) N-Ac-p-OH-Leu -117 52 -67.t -53 C(7)-N( 8)-C( 36) 119.1(8) C( 9)-N( 8)-C( 3 6) 116.9(8) N-Me-deH- Ala 33* -135 -176 --N( 8)-C(9)-C( 10) 118.6(8) N(8)-C(9)-0(37) 120.9(9) C( 10)-C(9)-0( 37) 120.6(8) C(9)-C( 10)-N( 1 1) 108.1(7) 2 Amido and ester bonds C(9)-C(lO)-C(38) 110.3(7) N(l l)-C(lO)-C(38) 110.8(7) C(22)-C(21)-N(2O)-C(19): -170C( 10)-N( 1 1)-C( 12) 1 23.2(7) N( 1 1)-C( 12)-C( 13) 114.6(8) 3-Ph-Lac N(l l)-C(12)-0(56) 1 22.1 (8) C( 13)-C( 12)-O(56) 123.3(8) N-Prop-P-OH-Leu C(45)-N(5 1)-C( 52)-C(54) : 179 C( 12)-C( 13)-N( 14) 109.1(8) C(12)-C( 13)-C(57) 112.7(8) N-Prop-P-OH-Leu C(45)-C(43)-0(42)-C(38) : 174 N(14)-C(13)-C(57) 112.3(8) C( 1 3)-N( 14)-C( 15) 126.1(8) N,O-Me,-Thr C( 7)-C( 6)-O( 5)-C (4) : 175 C(13)-N(14)-C(58) 116.5(8) C(15)-N(14)-C(58) 117.4(8) N-Ac- p-OH-Leu c(3)-c(2)-0( 1)-C(22) : 178 N(14)-C(15)-C(16) 118.5(8) N(14)-C(15)-0(59) 121.4(9) o:torsion angle of C;-Ci-Ni + l-C;+,C( 16)-C( 15)-O(59) 120.0(8) C( 15)-C( 16)-N( 17) 109.7( 8) 9:torsion angle of Ni-C:-Ci-Oi -180"C(15)-C( 16)-C(60) 110.9(9) N(17)-C(16)-C(60) 1 12.1(9) w:torsion angle of Hi-Ni-CP-Ci -180"C( 16)-N( 17)-C( 18) 119.0(7) N(17)-C(18)-C(19) 114.0(7) x2*l:torsion angle of N,-C;-C,P-O!N(17)-C(18)-0(61) 124.8(8) C(19)-C(18)-0(61) 12 1.3(8) C(18)-C(19)-N(20) 113.0(8) C( 18)-C( 19)-C(62) 120.2(9) x2., :torsion angle of Ni-CP-C,P-CY N(20)-C( 19)-C(62) 125.8(9) C(19)-N(20)-C(21) 119.3(9) * For N-methyl amino acid residues, the methyl carbon atom C(19)-N(20)-C(63) 1 16.2(9) C(2 l)-N(20)-C(63) 123.4(9) has replaced the Hi atom.t For P-OH-Leu, Oi is the 0-atom N(20)-C(21)-C(22) 1 16.2( 10) N( 20)-C(2 1 )-O( 64) 1 2 1.1 (10) bonded to the C(p)-atom. C(22)-C(2 1 )-O(64) 1223 11) O(l)-C(22)-C(2 1) 106.0( 10) O(1)-C(22)-C( 65) 108.8( 11) C(2 1)-C(22)-C(65) 113.6(11) C( 3)-N(24)-C( 25) 1 2 1.3(9) N(24)-C(25)-0(26) 120.8( 10) Table 5.Intra-molecular hydrogen bonds or short contacts N(24)-C(25)(27) 114.9(9) O(26)-C( 25)-C( 27) 124.3(10) D = donor, A = acceptor, the lengths (A) and angles (") with e.s.d.s in C(4)-C( 28)-C(29) 1 11.2( 11) C( 4)-C (2 8)-C( 3 0) 108.0( 11) parentheses C(29-C(28)-C( 30) 108.9(12) C(7)-C( 32)-0(3 3) 104.7( 12) C( 7)-c(32)-c(3 5) 110.0( 14) 0(33)-C(32)-C( 35) 113.7( 14) Atoms d(D *. A) d(H * * A) +(D-H * * * A) C( 32)-O( 33)-C(34) 116.1(13) C(1 O)-C(38)-C(39) 113.8(8) N(l 1)-H(11) ** O(47) 3.32(1) 2.45(13) 138(9)C( 10)-C( 38)-O(42) 104.3( 7) C(39)-C( 38)-O(42) 104.3(8) N( 17)-H( 17) * O(47) 2.87( 1) 1.97(11) 158(10)C( 38)-C( 39)-C( 40) 107.4( 10) C(38)-C(39)-C(41) 1 09.9 (10) 0(47)-H(47) -O(37) 2.66(1) 1.63( 12) 166(10)c(40)-c( 39)-C( 4 1 ) 109.2( 11) C(38)-0(42)-C(43) 118.7(7) N(24)-H(24) O(56) 2.95( 1) 1.99(12) 163( 10) O(42 j-C(43)-O( 44) 124.5(10) 0(42)-C(43)-C(45) 109.9( 8) N(51)-H(51) * * O(59) 3.08(1) 2.24(12) 142(10)0(44)-C(43)--C(45) 125.5( 10) C(43)-C(45)-C(46) 107.6( 8) C(43)-C(45)-N(5 1) 114.6(8) C(46)-C(45)-N(5 1) 11 1.6(8) C(45)-C(46)-0(47) 109.6(7) C(45)-C(46)-C(48) 112.8(8) 180 -0(47)-C(46)-C(48) 106.5(8) C(46)-C(48)-C(49) 112.1(9) -0 p-OH-LeuC(46)-C(48)-C( 50) 109.9(9) C(49)-C(48)-C( 50) 108.6( 10) C(45)-N(5 1)-C(52) 120.6(8) N(5 l)-C(52)-0(53) 120.0( 1 1) -N(5 1 )-C(52)-C( 54) 1 17.3( 10) 0(53)-C(52)-C(54) 122.7(11) 120 C( 52)-c(54)-c( 55) 116.1(16) C( 22)-C( 65)-C( 66) 113.6( 12) -A!~, 0 N,O-Me2 -T hr C(65)-C( 66)-C( 67) 121 .l( 13) C( 65)-C( 66)-C( 7 1 ) 120.5(12) C(67)-C(66)-C( 7 1 ) 118.2(13) C(66)-C(67)-C(68) 11 7.0( 14) 60 -N-Ac-P-0 H-Leu C(67)-C( 68)-C(69) 123.4( 16) C(68)-C(69)-C( 70) 1 18.6( 15) h -o00 vC(69)-C(7O)-C(7 1) 120.8( 14) C(66)-C(7 1)-C(70) 122.0(13) N-Me-ALa 3.0--0 #-Prop -p -0H-Leu The FR900359 molecule contains two cis peptide bonds, -60-between Ala and N-Me-Ala, and between P-OH-Leu and N,O-Me,-Thr (Table 4); generally a cis conformation is less favoured energetically than its trans counterpart.6 In the case of the -120 0FR900359 molecule, the O(59) and O(37) atoms involved in N-Me-deH -Ala these cis peptide bonds can face the proton donating atoms, N(5 1) and 0(47), respectively, to take part in the intramolecular -180' ' ' ' I ' ' ' ' I I ' -180 -120 -60 0 60 120 180hydrogen bonds (or short contacts).The cis peptide bond occurs very rarely in Nature except at the terminal nitrogen of cp ("1 proline and tyrosine residues, but in FR900359 these intra- Figure 4. 9-w plot for crystalline FR900359 cp and w are torsion angles molecular interactions must contribute to overall molecule about the C(a)-N and C-C(a) bond, respectively stabilization. The cp-and y-angles of Table 4 are also summarized in Figure be impossible because the methylene carbon C(62) deviates 4,8which shows that all (cp, y)-values other than that for N-Me- from the normal P-carbon position of the amino acid. deH-Ala lie in the P-region. For N-Me-deH-Ala, steric in- However, it seems that C(62) is influenced by the Ni+land 0,-1 hibition between Cp and N, + I, and between Cf and Oi would atoms because the distance between C(62) N( 17) is 2.95( 1) 8, J.CHEM. SOC. PERKIN TRANS. I 1989 Figure 5. Crystal packing diagram of FR900359 viewed along the b axis (PLUTO) and between C(62) O(64) is 3.89( 1) A. The (cp, y)-value for the N-Prop-P-OH-Leu side chain indicates that it lies in the a-helix region. The N,O-Me,-Thr, 3-Ph-Lac and N-Me-deH-Ala residues excluded from the hydrogen bond network are considered to be flexible because most atoms in these residues have larger thermal parameters. The N-Me-deH-Ala residue may be especi- ally unstable or reactive since it falls in the unstable region of the 9-w plot and its methylene group is susceptible to nucleophilic attack such as ammonolysis.2 It may thus be inferred to be involved in the biological activity of FR900359.Figure 5 shows the molecular packing diagrams of FR900359 viewed along the b axis.' Most methyl groups and the phenyl ring face outwards from the centre of the molecule, so that its surface can be considered to be hydrophobic. With no inter- molecular hydrogen bonds in the FR900359 crystal, the packing force should be due solely to van der Waals contacts. Experimental FR900359 was prepared as described in ref. 2. Crystals were obtained from hexane solution as transparent prisms, crystal size for X-ray diffraction study was 0.10 x 0.20 x 0.25 mm. Crystal Data.-C,9H75N70, 5, M = 1002.17, monoclinic, space group P2,, Z = 2, a = 17.81 3(2), b = 11.444(1), c = 13.443(1) A, P = 96.42(2)", I/ = 2 723.1(5) A3 by least-squares refinement using the automatic setting angles of 20 reflections, h(Cu-K,) = 1.5418 A, D, = 1.222 g cmP3, ~(CU- K,) = 0.7146 mm-'.Data Collection.-The intensity of 4 886 independent reflec- tions up to 28 = 130deg;, surveyed by the 28-0 scan method, were collected on a Rigaku AFC-5 diffractometer with graphite monochromated Cu-K, radiation. Corrections were applied for Lorentz and polarization factors, but not for absorption and extinction. Structure Determination and Refinement.-The structure was solved by a combination of a direct method using the MULTAN 84 program" and a weighted Fourier technique, and refined by a block-diagonal matrix least-squares method.' The positions of all hydrogen atoms were determined from a difference Fourier synthesis; although some of the methyl hydrogen atoms were not successfully assigned to the peak-top of the Fourier map, they were assigned to reasonable positions.They were included in the last cycle of refinement. The final R value was 0.089 for 4 179 reflections with F, 2 30(F0). Throughout the refinements, a unit weight was given to the intensity of each reflection. The atom scattering factors cited in International Table for X-ray Crystallography vol. IV. * All computations were carried out on FACOM M-150F or FACOM S-3300 computers. Absolute Conjiguration of the A lanine Unit.-An evacuated and sealed tube containing FR900359 (200pg)in constant boiling HCl (Pierce, USA; sequanal grade; 0.5 ml) was heated at 110"C for 20 h.The hydrolysate was evaporated to dryness at reduced pressure, suspended in 10HCl(0.5 ml) in anhydrous butanol (Regis, USA), sonicated at room temperature for 15 min and heated at 90deg;C for 30 min in a sealed vial. The mixture was evaporated to dryness under reduced pressure, treated with N-trifluoroacetyl-L-prolyl chloride (0.2 ml) in chloroform (Regis, USA) at room temperature for 30 min, and was blown down under a stream of nitrogen. The residue was dissolved in chloroform (0.2 ml) and an aliquot of the solution was injected into g.c./m.s. For comparison, ca. 50 pg of D,L-alanine and L-alanine were converted into their respective N-trifluoroacetyl- L-prolyl-butyl ester derivatives, and used as authentic samples.A Shimadzu QP-1000 GC mass spectrometer operated at 70 eV and equipped with a capillary column (Chromato Packing Centre, Japan: ULBON HR-54, 0.32 mm x 25 m) was employed. The column temperature was raised from 90 to 250 "C at a rate of 10 "C/min. Acknowledgements The authors are grateful to Prof. Takaji Fujiwara, Faculty of Science, Shimane University, for his helpful suggestions throughout this work. They are also indebted to Dr. Keizo Yoshida, Exploratory Research Laboratories, Fujisawa Pharm. Co., Ltd., for providing specimens for this work. References 1 Central Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., unpublished work. 2 M.Fujioka, S. Koda, Y. Morimoto, and K. Biemann, J. Org. Chem., 1988, 53, 2820. 3 D. P. Botes, C. C. Viljoen, H. Kruger, P. L. Wessels, and D. H. Williams, Toxicon, 1982, 20, 1037. 4 A. Miyamae, M. Fujioka, S. Koda, and Y. Morimoto, in 'Peptide Chemistry 1986,' ed. T. Miyazawa, Protein Research Foundation, Osaka 1987, p. 135. 5 C. K. Johnson, ORTEP 11, Report ORNL-TM-5138, Oak Ridge National Laboratory, Tennessee, 1976. 6 G. E. Schulz and R. H. Schirmer, 'Principles of Protein Structure,' Springer-Verlag GmbH amp; Co. KG, 1979, ch. 2. 7 S. D. Jolad, J. J. Hoffmann, S. J. Torrance, R. M. Wiedhopf, J. R. Cole, S. K. Arora, R. B. Bates, R. L. Gargiulo, and G. R. Kriek, J. Am. Chem. Soc., 1977,99,8040. 8 J. C. Kendrew, W. Klyn, S. Lifson, T. Miyazawa, G. Nemethy, D. C. Phillips, G. N. Ramachandran, and H. A. Sheraga, IUPAC- IUB Commission on Biochem., J. Mol. Biol., 1970, 52, 1. 878 J. CHEM. SOC. PERKIN TRANS. I 1989 9 S. Motherwell, PLUTO, A Program for Plotting Molecular and 11 T. Ashida, HBLS V, The Universal Crystallographic Computing Crystal Structure, Univ. of Cambridge, England, 1978. System, Osaka, p. 55, The Computation Centre, Osaka University. 10 P. Main, M. M. Woolfson, L. Lessinger, G. Germain, and J. P. 12 lsquo;International Tables for X-Ray Crystallography,rsquo; vol. 111, KynochDeclercq, MULTAN 84, A System of computer programs for the Press, Birmingham, 1962. Automatic Solution of Crystal Structures from X-ray Diffraction Data, Univ. of York,England, 1984. Received 28th June 1988; Priper 8/02561 D
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