Chemistry of bleomycin. XX. The X-ray structure determination of P-3A Cu(II)-complex a biosynthetic intermediate of bleomycin.

Sir: During a biosynthetic study of bleomycin (BLM), several peptides, structurally related to BLM, were isolated from the culture broth of Streplomyces verticillus. One of them, designated as P-3A, was crystallized from water (NAKATANI et al., unpublished) as an equimolar Cu(II)-complex. Acid hydrolysis of metal-free P-3A gave ~-aminoalanine (amine component V" of BLM), 2-(I-amino-2-carboxyethyl)-6aminopyrimidine-4-carboxylic acid (a demethyl analogue of amine component 112) of BLM, demethyl-II), L-histidine and L-alanine. Terminal group analysis of P-3A showed that the a-amino group of V and the carboxyl group of alanine are free by SANGER's DNP-method and by AKABORI'S hydrazinolysis method, respectively. The 13CNMR spectrum of P-3A31 indicated that it consists of one mole each of the above 4 amino acids. However, the molecular formula of P-3A could not be established definitely from the elemental analysis, P-3A Cu(II)-complex, Found: C, 39.43; H, 5.08; N, 24.26; Cu, 10.37. Metal-free P-3A, Found: C, 43.52; H, 6.02; N, 26.92. In this communication, the structure of P-3A Cu(II)-complex as determined by X-ray crystallographic analysis is presented. The crystals of P-3A Cu(ll)-complex as recrystallized from aqueous solutions were rhombohedral in shape and dark blue in color. As the crystals readily deteriorated in air by losing water of crystallization, they were sealed in glass capillaries prior to exposure to X-rays. A specimen of the approximate dimensions 0.2 x 0.2 x 0.25 mm was mounted on a Philips PW1100 diffractometer and the unit cell dimensions and intensity data were measured with monochromatic (graphite plate) CuKa radiation. The cell dimensions ab17.075(9), c21.289 (I1) A, i-=120°, and the symmetry of the intensity distribution indicated that the LAUE group of the crystal must be 3 m. The space group was at first assumed to be P3,21* taking into account the LAUE group and the absent reflexions, 1=3 in 0 0 1. The density of the crystal measured by the flotation method using benzene carbontetrachloride solutions was 1.47 gcm-3 which showed that the molecular weight for a structure unit in an asymmetric unit is 793 assuming that 6 units are contained in a cell. Although the molecular weight was not known exactly, the most probable formula derived from the elemental analysis was C2oH27N11O6Cu•2H2O, which gave the molecular weight 581 and the calculated weight compositions of C, 38.93; H, 5.06; N, 24.97; 0, 20.74; Cu, 10.30. The difference between 793 and 581 should be due to additional water of crystallization contained in an asymmetric unit, corresponding to 11.8 molecules of water for every one molecule of P-3A Cu-complex. Intensities for about 5100 reflexions, including many crystallographically equivalent reflexions, were measured within a 20 range of 0120' and a total of 1817 hk • l and 1 103 hk• l structure factors were obtained. The crystal structure was solved by the heavy atom method and the entire structure was revealed by the repeated cycles of structure factor-FoURIER and least-squares calculations. Refinement by the block-diagonal least-squares method including anisotropic temperature factors gave an R factor of 0.153 for all the 38 atoms of P-3A Cu-complex and 4 water oxygen atoms. However, the electron density map showed some unusual features for two terminal groups. The Cterminal carboxylic acid group of L-alanine and the "carboxylic acid or carboxamide" side-chain of demethyl-I1 appeared to exhibit unusually large thermal vibrations. The vibrations of the former group could be explained by vibrational motion about the C-C' (carboxyl carbon atom) bond whereas those of the latter group could not be interpreted by such a simple rotational motion. Furthermore, the intermolecular distances between the molecules related by a diad axis exhibited some unusually short contacts between these demethyl-11 side-chains. It was therefore assumed that the symmetry of the space group P3121 was violated in this part of the molecule although no definite deviation of the intensity distribution from that expected for the point group 32 symmetry had been observed. Refinement of the structure in the space group P31 was then carried out including the * The possibility of the alternative space group