The configuration of the double bond in pseudomonic acid A (1 a) is shown to be € by comparison of the spectroscopic properties of its methyl ester (1 b) with those of methyl isopseudomonate A (2) obtained from (1 b) by photolysis. Ozonolysis of methyl pseudomonate A (1 b) afforded the crystalline ketone (3a). An X-ray analysis of the o-bromophenylhydrazone derivative of ketone (3a) confirmed the structural assignment and provided the absolute stereochemistry a t each of the eight chiral centres. Pseudomonic acid A. therefore, may be formulated as (4). 9-(4-[5S-(2S,3S-epoxy-5Shydroxy-4S-methylhexyl)-3R,4R-dihydroxytetrahydropyran-2S-yl] -3met h y I b u t -2 (€ )en o y I oxy} no n a no i c a c i d .

By suitable protection and deprotection, the 9-hydroxynonanoic acid side-chain of pseudomonic acid A (1 a), a naturally occurring antibiotic, was cleaved in a highly efficient one-pot reaction to the allylic acid (3a). 4-[5S-(2S.3S-epoxy-5shydroxy-4s-methylhexyl) -3R.4R-di hydroxytetrahydropyran -2S-ylI -3-methylbut-2 ( E ) -enoic acid. We have ascribed the trivial name, monic acid A, to this allylic acid. Esters of monic acid A were readily prepared from the free acid (3a) and also from the ketone (2) which could be condensed with phosphonoacetates.PSEUDOMONIC ACID A, a naturally occurring antibiotic produced by fermentation of a strain of Pseudomonas fluorescens, was assigned the gross structure (la) by general protein binding appears to correlate with lipoyhilicity one approach to reducing the extent of binding was to attempt the replacement of this C-9 acidic alcohol with shorter chain alcohols. In other words, we sought to effect a transesterification of pseudomonic acid A (la).In our first approach to simple esters of the allylic acid (3a), to which we have given the trivial name monic acid A, we utilised the readily available ketone triol (2). Reaction of (2) with the Wadsworth-Emmons reagent, triethyl phosphonoacetate (4a), in a stirred suspension of sodium hydride in tetrahydrofuran, provided a complex mixture in which both ethyl monate A (3c) and ethyl 1 2 isomonate A (5a) were present as shown by h p.1.c.

Spin-lattice and transverse fluorine relaxation rates have been determined for the title enzyme derivative at pH 7. The data have been analyzed to provide an estimate of the rotational correlation time (rc) near the trifluoromethylbenzenesulfonyl group and the correlation time (r¡) for internal rotation of trifluoromethyl. The major part of the fluorine relaxation is due to proton-fluorine dipole-dipole interactions. Specific deuteration experiments show that these interactions predominantly involve protons of the enzyme and solvent.

p-Trifluoromethylbenzenesulfonyl-alpha-chymotrypsin, an analog of tosylchymotrypsin, has been prepared and shown to be stable enough to permit fluorine nuclear magnetic resonance experiments. Up to four distinct trifluoromethyl resonances can be observed for the modified protein at 94.1 MHz even when the enzyme derivative is prepared from protein which has been purified by several methods. The resonances observed appear to represent proteins which are grossly similar as regards molecular size and the ability to bind and hydrolyze substrates, but nonetheless distinctive enough in the active-site region to produce appreciable chemical-shift effects.

Fluorine and deuterium NMR relaxation studies have been used to examine the motion of the 4-fluorophenyl ring attached to the active site of [(4-fluorophenyl)sulfonyl]-alpha-chymotrypsin at pH 4. Analysis of the results indicates that rotation about the 2-fold axis of this ring is reasonably rapid, though not as fast as in tosylchymotrypsin. Two-dimensional (2D) nuclear Overhauser effects (NOEs) were used to suggest the shifts of those protons of the enzyme close enough to the fluorine nucleus to lead to relaxation; important proton-fluorine dipolar relaxation contributions arise from protons with shifts of 7.4 +/- 0.3 ppm and between 4.0 and 5.4 ppm. Specific deuteration permits the assignment of the first of these to the protons ortho to the fluorine while serine-189, cysteines-191 and -220, and methionine-192 are suggested as possible bearers of the other protons. The fluorine chemical shift effect observed for the native conformation of this protein is 9 ppm downfield of the shift observed with the denatured protein; this large shift may be the result of van der Waals interactions between the fluorine and one or more of the protons whose signals appear in the 2D NOE experiments.