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When alpha-aceto[1,3,5-13C3]lactate (2-hydroxy-2-methyl-3-oxo[1,3,5-13C3]butanoate) was incubated with a cell-free system prepared from Salmonella typhimurium, the valine produced was labelled in the C-4 pro-S position. This result proves that during the tertiary ketol rearrangement catalysed by the reductoisomerase of the isoleucine-valine pathway, the methyl group transfer is to the re face of the trigonal centre at C-3 of alpha-acetolactate.

1. 1. In addition to the known 2R,3R- and 2R, 3S-2,3-dihydroxy-3-methylpentanoic acids (DHI), the 2S,3S- and 2S,3R-isomers were prepared. 2S-2,3-Dihydroxy-3-methylbutanoic acid (DHV) was also prepared in addition to the known 2R-isomer. 2. 2. The six dihydroxy acids were examined for their ability to promote the growth of isoleucine-valine ( ilv )-requiring strains of Salmonella typhimurium and to serve as substrates fro the α,β-dihydroxyacid dehydratase of the same organism. 3. 3. Only 2R,3R-2,3-dihydroxy-3-methylpentanoic and 2R-2,3-dihydroxy-3-methylbutanoic acids supported growth of the ilv strains of S. typhimurium . 4. 4. α,β-Dihydroxyacid dehydratase utilized the three isomers with the 2R-configuration as substrates but not those with the 2S-configuration. 5. 5. In an additional growth study that utilized the 3R- and 3S-isomers of 3-methyl-2-oxopentanoic acid, the α-keto acid analogue of isoleucine, only the 3S-isomer supported growth. 6. 6. It is concluded that the mechanism of action of the dehydratase is stereospecific in that the proton that is attached to C-3 of the substrate occupies the same stereochemical position as the departing hydroxyl group (Fig. 6).

(2RS,3S,4S)-[2-14C,4-3H]Isoleucine and (2S,3S,4R)-[U-14C,4-3H]isoleucine have been prepared by stereospecific syntheses. The addition of these substrates to Streptomyces cacaoi led to the isolation of polyoxins from which 3-ethylidene-L-azetidine-2-carboxylic acid was isolated by hydrolysis. The pro-R hydrogen at C-4 of isoleucine was lost and the pro-S hydrogen was retained in the biosynthesis of 3-ethylidene-L-azetidine-2-carboxylic acid. These results indicate that the enzymatic dehydrogenation of isoleucine to form 3-ethylidene-L-azetidine-2-carboxylic acid involves the antiperiplanar elimination of the hydrogen at C-3 and the pro-R hydrogen at C-4.

The crystal structure of axillarine hydrobromide ethanol solvate has been determined from analysis of photographic X-ray data. The crystal system is orthorhombic, space group P212121, with unit cell dimensions a = 9.89, b = 11.23, c = 20.83 A. The structure was determined by means of Patterson and Fourier syntheses, and refined by the method of least-squares to an R index of 9.4%. The structural features of axillarine are noted and compared with those of several other pyrrolizidine alkaloids. The NMR. chemical shift difference between the signals due to the non-equivalent C(9) protons in alkaloids derived from retronecine is also discussed.

[U-14C]-L-Threonine and [U-14C]-L-isoleucine were specifically incorporated into monocrotalic acid (III) the necic acid component of the pyrrolizidine alkaloid monocrotaline (I) in Crotalaria spectabilis and Crotalaria retusa.

Feeding experiments have shown that two five-carbon units derived from isoleucine are specifically incorporated into senecic acid (IV) in Senecio magnificus. The specific incorporation of the methyl carbon atom of methionine into C-3 of serine in pea seedlings has been demonstrated. The significance of this result in relation to necic acid biosynthesis is discussed.

Of the four stereoisomers of isoleucine, only L-isoleucine is efficiently incorporated into senecic acid, the necic acid component of the pyrrolizidine alkaloid senecionine.

Aquick, simple applicable protocol for the reduction of protein disulfide bonds for the purposes of mass spectrometry has been established. The method utilises the chemical reducing agent dithiothreitol. Proteins with various numbers of disulfide bonds per molecule were chosen for the study to demonstrate the general applicability of the method. The results obtained under controlled conditions (concentration of reagents, pH, temperature) showed that a five-minute treatment at 70°C with 10 mM dithiothreitol in 5 mM ammonium acetate buffer pH 5.5 was sufficient for complete reduction of disulfide bonds in all investigated proteins (α-lactalbumin, lysozyme, ribonuclease, oxytocin and wheat germ agglutinin). The progress of disulfide bond reduction was observed by electrospray ionisation and Fourier transform ion cyclotron resonance mass spectrometry. Circular dichroism was used to monitor conformational changes of reduced proteins and of their unreduced counterparts undergoing the same heat treatment.

Analysis by tritium n.m.r. has indicated that the incorporation or the pro-S methyl group of ‘chiral methyl valine’[as (1)] into the C-3′ exocyclic methylene group of cephalosporin C (5) in Cephalosporium acremonium occurs with at least 70% stereospecificity overall; in contrast, the tritium n.m.r. spectra obtained after the incorporation of pro-R methyl groups of opposite configuration into the C-2 methylene group of cephalosporin C were virtually identical, indicating that at some stage there is a common stereochemically identical intermediate.

The reductoisomerase of Salmonella typhimurium has a requirement for the 2S-isomer of acetolactate (III, R = Me) while the αβ-dihydroxy-acid dehydratase has a requirement for the 2R configuration but is not stereoselective with respect to the configuration at C-3.

The first example is reported of the use of vibrational infrared circular dichroism to define the absolute configuration of a compound not previously described: the stereosepcifically deuteriated acetoin produced on incubation of racemic acetoin with acetolactae decarboxylase.

2-Ethoxycarbonylbuta-1,3-dienetricarbonyliron (1) was resolved by enantioselective hydrolysis, catalysed by pig liver esterase, to give the enantiomers of the corresponding acid in a state of high optical purity.

Methylmethacrylate, the monomeric component of the polymethylmethacrylate cement used in orthopedic surgery, has been shown to undergo hydrolysis to methacrylic acid during hip replacement operations. Circulating levels of methacrylic acid were comparable with those of methylmethacrylate. Concentrations of both methylmethacrylate and methacrylic acid normally lay in the range 0--15 micrograms/cc. No correlation could be discerned between changes in the concentrations of methylmethacrylate and methacrylic acid, and changes in blood pressure.

The (–)-erythro- and (–)-threo-isomers of 2,3-dihydroxy-3-methylpentanoicacid (I) have been synthesised. The (–)-erythro-isomer has been shown to have the absolute configuration 2R,3R and to be identical with the naturally occurring (–)-acid, a precursor of isoloucine (II), and with the esterifying acid of the pyrrolizidine alkaloid Strigosine.

Catalytic hydrogenation with H3H in the presence of Wilkinson's catalyst of (2RS)-(E)-[4-2H]-2-acetylamino-3-methylbut-3-enoic acid gave a mixture of (2SR, 3SR, 4RS)-[4-3H2H]-N-acetylvaline [(8) and (9)] and 2SR, 3RS, 4SR-[4-3H2H]-N-acetylvaline [(6) and (7)] in the radio 19:1 by 3H n.m.r. spectroscopy of the derived valines; a similar reduction with 2H2 of 1,5-(Z)-4, 7-DIAZA-7-[1-4-(nitrobenzyloxycarbonyl)-2-methylprop-2-enyl]-3-phenoxymethyl-2-thiabicyclo[3.2.0]hept-3-en-6-one gave a mixture of dideuterio-isomers (11) and (12), in the ratio 3 : 7 by 1H n.m.r. spectroscopy of the derived valines.

Five-carbon intermediates of the pathway of isoleucine metabolism, 2-methylbutanoic acid, angelic acid (5), and 2-methyl-3-oxobutanoic acid, were not specifically incorporated into senecic acid (1), the necic acid component of senecionine (2) in Senecio magnificus. The mechanism of the coupling of isoleucine-derived units during necic acid biosynthesis was investigated through incorporation experiments with [6-3H]- and [4-3H2]-isoleucine (3) in S. magnificus and S. isatideus. 2-Amino-3-[3H2]methylenepentanoic acid (β-[3H2]methylenenorvaline)(13) was incorporated specifically into senecic acid (1).

Incorporation experiments with isoleucine (1) and (2)-aminobutanoic acid [as (20)], stereospecifically labelled with tritium at C-4 and C-3 respectively, have shown that the ethyl migration step during biosynthesis of isoleucine (1) in Senecio species takes place with retention of configuration. It was also shown that coupling of two molecules of isoleucine (1) to give the ten-carbon necic acids of the senecic (2) type takes place with loss of the C-4 pro-S and retention of the C-4 pro-R hydrogen from both components. A new synthesis of βγ-unsaturated amino-acids has been developed which provides for control of the configuration of the olefinic system.

Methods have been devised for the selective esterification of retronecine (I) to give analogues of the naturally occurring hepatotoxic pyrrolizidine alkaloids. Selective esterification at C-9 of retronecine (I) with simple acids was achieved by using NN′-dicyclohexylcarbodi-imide. Selective esterification and esterification with αβ-unsatur-ated and α-hydroxy-αα-dialkyl acids was achieved through the intermediacy of N-acylimidazoles.

Two synthetic routes to the enantiomers of acetoin (2) of high optical purity have been devised. One starting from (S)-3-methylbut-3-en-2-ol (9) led to (S)-(+)-acetoin (13). The other, starting from (2R,3R)-butane-2,3-diol gave (R)-(–)-acetoin (16).

Di-n-butyltin oxide efficiently catalyses the rearrangement of 3-hydroxy-2-oxocarboxylic acid esters into the corresponding 2-hydroxy-3-oxo-esters, in a reaction that simulates the reversible rearrangement catalysed by the enzyme reductiosomerase.

Acetolactate decarboxylase catalyses the decarboxylation of both enantiomers of 2-ethyl-2-hydroxy-3-oxobutanoate to isomeric ketois of high optical purity and both enantiomers of α-acetolactate (2-hydroxy-2-methyl-3-oxobutanoate) to acetoin (3-hydroxybutan-2-one) of high optical purity.

When [1,3,5-13C3]-α-acetolactate {[1,3,5-13C3]-2-hydroxy-2-methyl-3-oxobutanoate}(1′) was incubated with a cell-free system from Salmonella typhimurium, the valine (5′) produced was labelled in the C-4 pro-S position, proving that during the tertiary ketol rearrangement catalysed by the enzyme reductoisomerase, the methyl group transfer is to the re face of the trigonal centre at C-3 of α-acetolactate (1′).