Cyclohexanone monooxygenase (E. C. 1.14.13.22) from Acinetobacter sp. NCIB 9871 has been expressed in baker's yeast (Saccharomyces cereVisiae) to create a general reagent for asymmetric Baeyer-Villiger oxidations. This "designer yeast" approach combines the advantages of using purified enzymes (single catalytic species, no overmetabolism, etc.) with the benefits of whole-cell reactions (experimentally simple, no cofactor regeneration necessary, etc.). The yeast reagent was used to systematically examine a series of 2-, 3-, and 4-substituted cyclohexanones (R ) Me, Et, n-Pr, i-Pr, allyl, n-Bu), almost all of which were oxidized to the corresponding -caprolactones in good yields and high enantioselectivities (typically g 95%). Mesomeric 4-substituted cyclohexanones were oxidized to -caprolactones in g 92% ee. The engineered yeast strain also effected kinetic resolutions of 2-substituted cyclohexanones with enantioselectivity values g 200 for substituents larger than methyl. The behavior of 3-substituted cyclohexanones depended upon the size of the substituent. The engineered yeast strain cleanly converted the antipodes of 3-methyl-and 3-ethylcyclohexanone to divergent regioisomers. On the other hand, for cyclohexanones with larger substituents (n-Pr, allyl, n-Bu), both antipodes were oxidized by the enzyme to a single regioisomer. In these cases, the observed enantioselectivities were due to a combination of a modest preference for one enantiomer by the enzyme and an unfavorable conformational preequilibrium required prior to binding of the less-favored antipode, a phenomenon we refer to as substrate-assisted enantioselectivity.

Abstract:The biological activity of midkine, a cytokine implicated in neurogenesis and tumorigenesis, is regulated by its binding to glycosaminoglycans (GAGs) such as heparin and chondroitin sulfate (CS). To better understand the molecular recognition of GAG sequences by this growth factor, we have studied here the interactions between synthetic chondroitin sulfate-like tetrasaccharides and midkine using different techniques. First, a synthetic approach for the preparation of CS-like oligosaccharides in the sequence GalNAc-GlcA was developed. A fluorescence polarization competition assay was then employed to analyse the relative binding affinities of the synthetic compounds and revealed that midkine interacts with CS-like tetrasaccharides in the micromolar range. The 3D structure of these tetramers was studied in detail by a combination of NMR experiments and molecular dynamics simulations. Saturation transfer difference (STD) NMR experiments indicate that the CS tetrasaccharides bind to midkine in an extended conformation, with similar saturation effects along the entire sugar chain. These results are compatible with docking studies suggesting an interaction of the tetrasaccharide with midkine in a folded structure. Overall, our study gives valuable information on the interaction between midkine and well-defined, chemically synthesized CS oligosaccharides and these data can be useful for the design of more active compounds that modulate the biological function of this protein.

Die Richtung und der Grad der Enantioselektivität von Baeyer‐Villiger‐Reaktionen, die durch eine Cyclohexanon‐Monooxygenase katalysiert und durch Luft vermittelt werden (z. B. die Desymmetrisierung von 1) lassen sich durch gerichtete Evolution steuern.

54 -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-␤-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.Pseudomonas sp. strain NCIMB 9872, which is capable of growth on 0.1% cyclopentanol as sole carbon source, was isolated from a freshwater stream in Illinois by P. J. Chapman some three decades ago. Trudgill and coworkers (27) used this strain as a prototype organism to establish the biochemical pathway of cyclopentanol metabolism shown in Fig. 1. For reason of utility as a Baeyer-Villiger (BV) biocatalyst and its mechanistic aspects (2,8,13,58,71), the second biochemical step catalyzed by cyclopentanone 1,2-monooxygenase (CPMO) has been most extensively studied in this organism. The conversion of cyclopentanone to 5-valerolactone is NADPH and flavin adenine dinucleotide (FAD) dependent, and CPMO has been purified to near homogeneity. It is a tetramer of subunit molecular weight 50,000 to 54,000 (8,66). Assay of the FAD/ protein ratio gave values of 2 to 4 molecules of FAD bound to each tetrameric enzyme molecule (28,66). An N-terminal 29-amino-acid sequence of CPMO has been determined (8).A chemical BV oxidation is the transformation of ketones into esters or of cyclic ketones into lactones by peracids, such as 3-chloroperbenzoic acid. This century-old reaction (for reviews, see references 54 and 63) continues to attract interest not only in broadening the spectrum of applications (ranging from the synthesis of steroids, antibiotics, and pheromones to the synthesis of monomers for polymerization, etc.) but also in developing oxidants that are more chemoselective and efficient and thus result in more product than waste (20).The biological BV reactions catalyzed by BV monooxygenases (BVMOs) offer the prospect of eco-efficient chemical transformations in whole cells or improved expression systems via cloning. Moreover, BVMOs can provide products in optically active form which are difficult to obtain by other strategies (for reviews, see references 55, 61, 71, and 74). Notable ste-* Corresponding author. Mailing address:

Here, we present a novel approach for the chemical synthesis of chondroitin and dermatan sulfate oligosaccharides. A key point of this strategy is the preparation and use of an N-trifluoroacetyl galactosamine building block containing a 4,6-O-di-tert-butylsilylene group. Glycosylation reactions proceeded in good yields (74-91%) with our protecting group distribution. Using this approach, we have synthesized, for the first time, a chondroitin/dermatan sulfate-like tetrasaccharide that contains both types of uronic acids, D-glucuronic and L-iduronic acid. Moreover, we have employed a fluorescence polarization competition assay to evaluate the interactions between the synthesized oligosaccharides and FGF-2 (basic fibroblast growth factor). Our results show that this method, using standard instrumentation and minimal sample consumption, is a powerful tool for the rapid analysis of the glycosaminoglycan affinity for proteins in solution.

This paper outlines the design and execution of the first mini-evolution of cyclopentanone monooxygenase (CPMO). The methodology described is a relatively inexpensive and rapid way to obtain mutant enzymes with the desired characteristics. Several successful mutants with enhanced enantioselectivities were identified. For example, mutant-catalyzed oxidation of 4-methoxycyclohexanone gave the corresponding lactone with 92% entantiometric excess (ee) compared to the 46% ee achieved with wild-type cyclohexanone monoxygenase (WT-CHMO). The original design of the mini-evolution and the following evaluation of mutants can provide valuable insights into the active site's construction and dynamics and can suggest other catalytically profitable mutations within the putative active site.

No abstract

The direction and degree of enantioselectivity in the cyclohexanone monooxygenase catalyzed air‐mediated Baeyer–Villiger reactions, such as the desymmetrization of 1, can be controlled by directed evolution.