1 Griseofulvin生合成研究における^2H nmrの利用

抄録

Recent development of the proton-decoupled pulsed Fourier transform nmr method have opened the way to employ ^2H nmr to various types of chemical and biological problems. In the elucidation of the biosynthetic pathways of griseofulvin (1) by Penicillium urticae, we demonstrate that ^2H nmr is very powerful nondestructive method to study biosynthetic pathways involving hydrogen. Earlier studies from our laboratories have demonstrated the incorporation of [2-^3H, 2-^<14>C]acetate using the shaking culture of P. urticae. In order to investigate the behavior of hydrogens in detail, sodium [2-^2H_3]acetate was added to the seven-days-old mycelia of the microorganism in the synthetic medium, giving ^2H-enriched griseofulvin for ^2H nmr analysis. In order to perform unambiguous assignment of ^2H signals, a series of selectively deuterated griseofulvin samples were prepared. Figure 1 shows ^2H nmr spectra of deuterated griseofulvin samples, indicating the incorporation of deuterium atoms at 5, 3', 5'α, 6'-methyl, and methoxyls. Those assignments were confirmed from measurements of T_1s and comparison of the peak intensities with those of proton-coupled spectra. The extent of deuterium incorporation was unequivocally obtained from the measurement of peak areas. In addition, seven-days-old mycelia of P. urticae were fermented in the media containing 50% D_2O and cold sodium acetate. ^2H nmr studies show that the deuterium atoms from D_2O were incorporated in the positions anticipated from the results of [2-^2H_3]acetate tracer experiment (Figure 2). As an another application of ^2H nmr, we have investigated the microbial transformation of dehydrogriseofulvin (3) by Streptomyces cinereocrocatus, proving the stereochemistry of the reduction of 3 to griseofulvin and the hydroxylation of the latter to 5'α-hydroxygriseofulvin (4) (Figure 3 and Scheme 1).