John T. Sime

Non-thiazolidinedione antihyperglycaemic agents. Part 3 : The effects of stereochemistry on the potency of α-methoxy-β-phenylpropanoic acids

Rhizopus delemar lipase catalysed ester hydrolysis of the alpha-methoxy-beta-phenylpropanoate 1 affords the (R)-(+) and (S)-(-) isomers in > 84% enantiomeric excess. Absolute stereochemistry was determined by a single crystal X-ray analysis of a related synthetic analogue. The activity of these two enantiomers on glucose transport in vitro and as anti-diabetic agents in vivo is reported and their unexpected equivalence attributed to an enzyme-mediated stereospecific isomerisation of the (R)-(+) isomer. Binding studies using recombinant human PPARgamma (peroxisomal proliferator activated receptor gamma), now established as a molecular target for this compound class, indicate a 20-fold higher binding affinity for the (S) antipode relative to the (R) antipode.

Isolation of two novel intracellular β-lactams and a novel dioxygenase cyclising enzyme from Streptomyces clavuligerus

Two novel β-lactams, one monocyclic, the other bicyclic, and a dioxygenase enzyme which converts the former to the latter, have been isolated from the mycelium of the clavulanic acid producing organism Streptomyces clavuligerus ATCC 27064.

Facile biocatalytic reduction of the carbon–carbon double bond of 5-benzylidenethiazolidine-2,4-diones. Synthesis of (±)-5-(4-{2-[methyl(2-pyridyl)amino]ethoxy}benzyl)thiazolidine-2,4-dione (BRL 49653), its (R)-(+)-enantiomer and analogues

A novel biotransformation system for the reduction of carbon-carbon double bonds in 5-benzylidenethiazolidine-2, 4-diones, to give the corresponding 5-benzylthiazolidine-2, 4-diones, using whole cells of red yeasts is described. These reduced compounds, which are recovered in good yield, are of potential use in the treatment of non-insulin dependent diabetes mellitus. The mild reaction conditions developed allow reduction of 5-benzylidenethiazolidine-2, 4-diones containing other functionalities which are not compatible with alternative reduction methods. The biocatalytic reduction is enantioselective and the synthesis of R-(+)-5-(4-{2-[methyl(2-pyridyl)amino]ethoxy}benzyl)thiazolidine-2, 4-dione by Rhodotorula rubra CBS 6469 and structure confirmation by X-ray crystallography is detailed. Optimisation of reaction conditions (including immobilisation) for these whole cell reduction systems is described.

Studies on the biosynthesis of clavulanic acid. Part 4. Synthetic routes to the monocyclic β-lactam precursor, proclavaminic acid

Aldol condensation of 3-substituted propionaldehydes with the lithium enolate of ethyl or benzyl (2-oxoazetidin-1-yl)acetate yielded derivatives of proclavaminic acid. The proportion of the threo diastereoisomer in the aldol product could be increased by thermodynamically controlled equilibration with 1,5-diazabicyclo[4.3.0]non-5-ene. In the case of benzyl 5-azido-3-hydroxy-2-(2-oxoazetidin-1-yl)valerate the diastereoisomers were separated and the threo diastereoisomer was resolved by enzymatic hydrolysis of the ester by subtilisin Carlsberg [EC 3.4.21.14]. Catalytic reduction of the unhyorolysed threo enantiomer yielded (2S, 3R)-5-amino-3-hydroxy-2-(2-oxoazetidin-1-yl)valerate which had spectroscopic properties identical with those of natural proclavaminic acid and which was a substrate for clavaminic acid synthase. Two crystalline, derivatives of (2S, 3R)-proclavaminic acid were prepared for X-ray analysis.

Synthesis of isotopically chiral [13C]penciclovir (BRL 39123) and its use to determine the absolute configuration of penciclovir triphosphate formed in herpes virus infected cells

Isotopically chiral [13C]penciclovir (BRL 39123) has been synthesised via a stereospecific hydrolysis catalysed by the lipase from Candida cylindraceae and has been used to determine, by 13C NMR, that the triphosphate of penciclovir formed in herpes simplex type 1 infected cells has (S) stereochemistry with an enantiomeric purity of > 95%.

Chemoenzymatic approach to the synthesis of the antiviral agents penciclovir and famciclovir in isotopically chiral [13C] labelled form

The antiviral agents penciclovir and famciclovir have been synthesised in isotopically chiral [13C] form. The synthesis of (+)-methyl 4-benzyloxy-2-(hydroxymethyl)butanoate 12a by use of enzymatic hydrolysis catalysed by the lipase from Candida cylindracea is described as is the confirmation of the stereochemistry of this intermediate as R by convergent synthetic routes. The butanoate 12a produced by the enzymic reaction was converted into the (–)-α-hydroxymethyl-γ-butyrolactone which was compared with the (S)-(+)-α-hydroxymethyl-γ-butyrolactone synthesised by an alternative, stereodefined route. The products of the enzymic reaction were used as intermediates in the synthesis of the final products.

The roles of clavaminic acid and proclavaminic acid in clavulanic acid biosynthesis

Clavulanic acid samples isolated from fermentations of Streptomyces clavuligerus ATCC 27064 fed with 13C-clavaminic acid or 13C-proclavaminic acid were found to be appropriately labelled, indicating that the title compounds are biosynthetic precursors of clavulanic acid.

Studies on the biosynthesis of clavulanic acid. Part 5. Absolute stereochemistry of proclavaminic acid, the monocyclic biosynthetic precursor of clavulanic acid

Proclavaminic acid (1) was synthesized by a route which indicated its constitution to be (2S,3R)-5-amino-3-hydroxy-2-(2-oxoazetidin-1-yl)valeric acid. The spectroscopic properties of the synthetic material were identical with those of natural proclavaminic acid, and, like the natural product, it was converted into clavaminic acid (2) by clavaminic acid synthase. An efficient synthesis of 3-hydroxyornithine derivatives was devised which allowed the separation of diastereoisomers and the resolution of a threo compound by the acylase [EC 3.5.1.11] from Escherichia coli. The β-lactam ring was subsequently elaborated by Michael addition of a protected 3-hydroxyornithine to acrylic acid followed by ring closure using triphenylphosphine/di-2-pyridyl disulphide. Model reactions were carried out with enantiomerically pure threonine derivatives to confirm that the formation of the β-lactam moiety did not impair the integrity of the α- and β-chiral centres and that the enzymatic deacylation reaction was capable of resolving the α-centre of an α-amino-β-hydroxy acid. The enantiomeric purity of intermediates was determined using HPLC, 1H NMR spectroscopy utilising the chiral solvating reagents (R)- and (S)-1-(9-anthryl)-2,2,2-trifluoroethanol, and chiral GLC techniques.

Synthesis of the novel monocyclic β-lactam proclavaminic acid

Proclavaminic acid has been chemically synthesised and one pure enantiomer separated which was shown to possess identical physicochemical and biochemical properties to the natural material isolated from Streptomyces clavuligerus.

A HIGHLY REGIOSELECTIVE ESTERASE FROM PENICILLIUM FREQUENTANS IMI 92265.

An esterase isolated from Penicillium frequentans IMI 92265 selectively hydrolyses the acetoxyethyl ester in preference to the acetoxymethyl esters in the triacetate substrate 1,4-diacetoxy-2-acetoxymethylbutane (1) which was converted to 4-acetoxy-3-acetoxymethylbutan-l-ol (2) in good yield. The enzyme gave no detectable hydrolysis of l,3-diacetoxy-2-acetoxymethylpropane (4). When immobilised to a cyanogen bromide activated Sepharose resin the enzyme was highly stable and showed no loss of regioselectivity in the hydrolysis of (1). A method is described for the elective isolation of microorganisms which have the ability to hydrolyse (1).