Margaret M. Kayser

Cloning and Characterization of a Gene Cluster Involved in Cyclopentanol Metabolism in Comamonas sp. Strain NCIMB 9872 and Biotransformations Effected by Escherichia coli-Expressed Cyclopentanone 1,2-Monooxygenase

ABSTRACT Cyclopentanone 1,2-monooxygenase, a flavoprotein produced by Pseudomonas sp. strain NCIMB 9872 upon induction by cyclopentanol or cyclopentanone (M. Griffin and P. W. Trudgill, Biochem. J. 129:595-603, 1972), has been utilized as a biocatalyst in Baeyer-Villiger oxidations. To further explore this biocatalytic potential and to discover new genes, we have cloned and sequenced a 16-kb chromosomal locus of strain 9872 that is herein reclassified as belonging to the genus Comamonas. Sequence analysis revealed a cluster of genes and six potential open reading frames designated and grouped in at least four possible transcriptional units as (orf11-orf10-orf9)-(cpnE-cpnD-orf6-cpnC)-(cpnR-cpnB-cpnA)-(orf3-orf4 [partial 3′ end]). The cpnABCDE genes encode enzymes for the five-step conversion of cyclopentanol to glutaric acid catalyzed by cyclopentanol dehydrogenase, cyclopentanone 1,2-monooxygenase, a ring-opening 5-valerolactone hydrolase, 5-hydroxyvalerate dehydrogenase, and 5-oxovalerate dehydrogenase, respectively. Inactivation of cpnB by using a lacZ-Kmr cassette resulted in a strain that was not capable of growth on cyclopentanol or cyclopentanone as a sole carbon and energy source. The presence of σ54-dependent regulatory elements in front of the divergently transcribed cpnB and cpnC genes supports the notion that cpnR is a regulatory gene of the NtrC type. Knowledge of the nucleotide sequence of the cpn genes was used to construct isopropyl-β-thio-d-galactoside-inducible clones of Escherichia coli cells that overproduce the five enzymes of the cpn pathway. The substrate specificities of CpnA and CpnB were studied in particular to evaluate the potential of these enzymes and establish the latter recombinant strain as a bioreagent for Baeyer-Villiger oxidations. Although frequently nonenantioselective, cyclopentanone 1,2-monooxygenase was found to exhibit a broader substrate range than the related cyclohexanone 1,2-monooxygenase from Acinetobacter sp. strain NCIMB 9871. However, in a few cases opposite enantioselectivity was observed between the two biocatalysts.

ASYMMETRIC OXIDATIONS AT SULFUR CATALYZED BY ENGINEERED STRAINS THAT OVEREXPRESS CYCLOHEXANONE MONOOXYGENASE

Recombinant strains of bakers yeast (Saccharomycescerevisiae) and Escherichiacoli expressing cyclohexanone monooxygenase from Acinetobacter sp. NCIB 9871 have been used as whole-cell biocatalysts for oxidations of several sulfides, dithianes and dithiolanes to the corresponding sulfoxides. The enantio- and diastereoselectivities of these reactions compare favorably with oxidations catalyzed by the purified monooxygenase or the parent microorganism (a class II pathogen). The facility of handling yeast reactions makes these biotransformations an attractive alternative route to optically pure sulfoxides.

‘Designer yeast’: a new reagent for enantioselective Baeyer–Villiger oxidations

The catalytic repertoire of bakers yeast has been expanded to include enantioselective Baeyer–Villiger oxidations. To create this catalyst, the Acinetobacter sp. cyclohexanone monooxygenase gene was inserted into a yeast expression vector and this was used to create a ‘designer yeast’ that performed oxidation reactions. Whole cell-mediated Baeyer–Villiger reactions were carried out on a 1.0 mmol scale and several cyclic ketones were converted in 20–30 h into the corresponding lactones in isolated yields of 60–83%. Under the reaction conditions, ketone reduction constituted only a minor side-reaction. Oxidation of prochiral 4-substituted cyclohexanones produced lactones with very high enantioselectivities.

Baeyer-Villiger oxidations of representative heterocyclic ketones by whole cells of engineered Escherichia coli expressing cyclohexanone monooxygenase

Whole cells of an Escherichia coli strain overexpressing Acinetobacter sp. NCIB 9871 cyclohexanone monooxygenase (CHMO; E.C. 1.14.13.22) have been used for the Baeyer-Villiger oxidation of representative heterocyclic six-membered ketones to probe the potential impact of nitrogen, sulfur and oxygen on the chemoselectivity of these reactions. The fact that all of these heterocyclic systems were accepted as substrates by the enzyme and gave normal Baeyer-Villiger products broadens the synthetic utility of the engineered E. coli strain and emphasizes the chemoselectivity achievable with enzymatic oxidation catalysts.

Monooxygenase-catalyzed Baeyer-Villiger oxidations: CHMO versus CPMO

Abstract Cyclopentanone monooxygenase (CPMO) from Comamonas sp. NCIMB 9872 expressed in E. coli was evaluated as a potential new bioreagent for Baeyer–Villiger oxidations of 4-alkoxy- and halo-substituted cyclohexanones (10 examples). The results were compared with those obtained in oxidations catalyzed by an engineered E. coli strain expressing cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. CPMO was found to have modest to good stereoselectivity and broader substrate acceptability than CHMO. The stereoselectivities of the two enzymes were generally opposite. It appears, therefore, that the two engineered strains can be useful and complementary reagents for enantioselective Baeyer–Villiger oxidations of certain prochiral ketones.

Application of yeast-catalyzed reductions to synthesis of (2R,3S)-phenylisoserine

Abstract A simple synthesis of (2R,3S)-phenylisoserine, a precursor of the C-13 side chain of Taxol®(paclitaxel), utilising yeast-catalyzed reduction to generate a second chiral centre is reported. This short enantioselective series of transformations can be readily adapted to large scale production of a variety of N-substituted paclitaxel analogues.

Reduction of cyclic anhydrides. II. Factors affecting regioselectivity of attack on the carbonyl group by metal hydrides

The reduction of unsymmetrically substituted cyclic anhydrides with metal hydrides often leads to the preferential formation of one of the two possible lactones. In the light of recent experimental findings and theories concerning metal hydride addition to the carbonyl function, the electronic and steric factors influencing regioselectivity of cyclic anhydride reductions are discussed and an explanation for the observed patterns is proffered. Similar considerations may be extended to predict the major lactonic products in the reductions of various other unsymmetrical cyclic anhydrides.

CD‐sensitive Zn‐porphyrin tweezer host‐guest complexes, part 2: Cis‐ and trans‐3‐hydroxy‐4‐aryl/alkyl‐β‐lactams. A case study

This article describes an application of the host-guest chiral recognition approach called tweezer methodology for the determination of the absolute configuration of 3-hydroxy-beta-lactams. These substrates represent challenging cases due to their chemical reactivity, the presence of multiple stereogenic centers and several functional groups which offer various possibilities of binding to the Zn-porphyrin host. OPLS-2005, the force field used in this work to predict the interporphyrin twist, modeled correctly the host-guest complexation mechanism and revealed conformational details of the bound substrates. The computational study also suggested that in cases where an increase in the magnitude of the stereodifferentiation and an intense experimental CD are observed, the bound conformation of the conjugates are hydrogen bonded. The present investigation provides evidence that when the tweezer method is assisted by the OPLS-2005 based computational approach, it can be successfully applied to the configurational and conformational elucidation of multi-functional compounds with multiple stereogenic centers.

Reduction of ketones by sodium borohydride in the absence of protic solvents. Inter versus intramolecular mechanism

Abstract Acetone, acetophenone and benzophenone react with sodium borohydride in the absence of protic solvent to give the corresponding tetraalkoxyborates. In view of theseresults, the possibility of the 4-centre mechanism for these reductions is discussed.

Synthesis of Enol Lactones Under a Solid/Liquid Phase Transfer Witting Reaction

Abstract A variety of enol lactones can be prepared through simple and inexpensive solid/liquid phase Witting condensations of cyclic anhydrides with phosphonium salts.