Enzyme-based resolution

After the first preparation of enantiomerically pure (27 ,2 7 )-t/zreo-methylphenidate hydrochloride (1) in 1958, it is only recently that a great deal of interest has been demonstrated in the synthesis of this molecule. Various approaches to the preparation of enantiomerically pure (2R,2>R)-(-i-)-t/zreo-methylpheni-date hydrochloride (1) are reviewed. These approaches include synthesis using enantiomerically pure precursors obtained by resolution, classical and enzyme-based resolution approaches, enantioselective synthesis approaches, and approaches based on enantioselective synthesis of 2S, 2 Ryerythro-methylphenidate followed by epimerization at the 2-position. Classical resolution approaches have been successfully upscaled to produce 1 on a multi-kilo-gram scale due to the ready availability of racemic ( )-t/zreo-methylphenidate hydrochloride (10). VVfiiIt some enantioselective approaches are short, they do not provide 1 of the desired enantiomeric purity necessary for drug development. Enantioselective synthesis approaches to produce 1, however, will be-... 

Generally, a distinction can be made between membrane bioreactors based on cells performing a desired conversion and processes based on enzymes. In ceU-based processes, bacteria, plant and mammalian cells are used for the production of (fine) chemicals, pharmaceuticals and food additives or for the treatment of waste streams. Enzyme-based membrane bioreactors are typically used for the degradation of natural polymeric materials Hke starch, cellulose or proteins or for the resolution of optically active components in the pharmaceutical, agrochemical, food and chemical industry [50, 51]. In general, only ultrafiltration (UF) or microfiltration (MF)-based processes have been reported and little is known on the application of reverse osmosis (RO) or nanofiltration (NF) in membrane bioreactors. Additionally, membrane contactor systems have been developed, based on micro-porous polyolefin or teflon membranes [52-55]. 

Recent reviews of enzyme-based dynamic kinetic resolution ... 

Enzyme-based processes for the resolution of chiral amines have been widely reported [2, 3] and are used in the manufacture of pharmaceuticals, for example, BASF s process for chiral benzylic amine intermediates. Scheme 13.1 [4]. The methods used are enantioselective hydrolysis of an amide and enantioselective synthesis of an amide, both of which are kinetic resolutions. For high optical purity products the processes depend upon a large difference in the catalyzed reaction rates of each enantiomer. 

Synaptic neurotransmission in brain occurs mostly by exocytic release of vesicles filled with chemical substances (neurotransmitters) at presynaptic terminals. Thus, neurotransmitter release can be detected and studied by measuring efflux of neurotransmitters from synapses by biochemical methods. Various methods have been successfully employed to achieve that, including direct measurements of glutamate release by high-performance liquid chromatography of fluorescent derivatives or by enzyme-based continuous fluorescence assay, measurements of radioactive efflux from nerve terminals preloaded with radioactive neurotransmitters, or detection of neuropeptides by RIA or ELISA. Biochemical detection, however, lacks the sensitivity and temporal resolution afforded by electrophysiological and electrochemical approaches. As a result, it is not possible to measure individual synaptic events and apply quantal analysis to verify the vesicular nature of neurotransmitter release. 

This strategy was used repeatedly by Mori and co-workers (Brevet and Mori, 1992 Muto and Mori, 2003a, b Nakanishi and Mori, 2005), first using enzyme-based kinetic resolution of an achiral, mc.vo-diacctatc to generate an enantiomerically enriched epoxyalcohol synthon (Scheme 5A), which could be further purified if necessary by dinitroben-zoate derivatization and recrystallization. Conversion of the alcohol to a leaving group,... 

Our biotransformation group (Drs. David Dodds, Alex Zaks, and Brian Morgan) contributed to most of our chiral synthesis projects, although in most cases enzyme-based routes were not selected over chiral induction or classical resolution processes for the short-term needs in API synthesis. This area, however, remains one of huge promise with the prospect of working in water being one of its most appealing attractions. 

The currendy available crystal structures of succinaterquinone oxi-doreductases are those of two prokaryotic quinolrfumarate reductases, both since 1999. The E. coli QFR, determined at 3.3 A (Iverson et al., 1999), belongs to the type D enzymes, and the QFR of W. succinogenes, refined at 2.2 A resolution (Lancaster et al., 1999), is of type B. Three structures of the latter enzyme, based on three different crystal forms, are available. The first two, PDB entries 1QLA and 1QLB (Lancaster et al., 1999), are considerably better defined and more accurate than the structure of the third crystal form, PDB entry 1E7P (Lancaster et al., 2000, 2001). Therefore, the first two crystal forms of W. succinogenes QFR will be used for the description of structural features, and that of the third crystal form will be referred to for comparison. 

Abstract Various approaches to the preparation of enantiomerically pure (2/i,27i)-(+)-Z/irco-mclhyl-phenidate hydrochloride (1) are reviewed. These approaches include synthesis using enantiomerically pure precursors obtained hy resolution, classical and enzyme-hased resolution approaches, enan-tioselective synthesis approaches, and approaches based on enantioselective synthesis of (2S,2 R)-ery-Zftro-methylphenidate followed by epimerization at the 2-position. 

Molecular separation along with simultaneous chemical transformation has been made possible with membrane reactors [17]. The selective removal of reaction products increases conversion of product-inhibited or thermodynamically unfavourable reactions for example, in the production of ethanol from com [31]. Enzyme-based membrane reactors were first conceived 25 years ago by UF pioneer Alan Michaels [49]. Membrane biocatalytic reactors are used for hydrolytic conversion of natural polymeric materials such as starch, cellulose, proteins and for the resolution of optically active components in the pharmaceutical, agrochemical, food and chemical industries. Membrane bioreactors for water treatment were introduced earher in this chapter and are discussed in detail in Chapters 2 and 3. 

A very specialized application of enzyme-based biosensor arrays has been reported for the resolution of pesticide mixtures containing dichlorvos and methylparaoxon, with a three-element array and a flow injection system [41]. The screen-printed, amperometric electrode array was modified with three acetylcholinesterase enzyme variants, one from electric eel and two from Drosophila (fruit fly) mutants, and were used to measure signal inhibition in conjunction with an artificial neural network. Good results down to the low uM range of pesticide concentrations were reported. 

The coupling of enzyme-catalyzed resolution with metal-catalyzed racemization constitutes a powerful DKR methodology for the synthesis of enantioenriched alcohols, amines, and amino acids. In many cases, the metalloenzymatic DKRs provide high yields and excellent enantiopurities, both approaching 100%, and thus provide useful alternatives to the chemical catalytic asymmetric reactions employing transition metals (complexes) or organocatalysts. The wider applications of a metalloenzymatic DKR method, however, are often limited by the low activity, narrow substrate specificity, or modest enantioselectivity of the enzyme employed. The low activities of metal-based catalysts, particularly in the racemization of amines and amino acids, also limit the wider applications of DKR. It is expected that fm-ther efforts to overcome these limitations with the developments of new enzyme-metal combinations will make the metalloenzymatic DKR more attractive as a tool for asymmetric synthesis in the future. 

Enzymatic resolution (Section 7 13) Resolution of a mixture of enantiomers based on the selective reaction of one of them under conditions of enzyme catalysis... 

In many cases only the racemic mixtures of a-amino acids can be obtained through chemical synthesis. Therefore, optical resolution (42) is indispensable to get the optically active L- or D-forms in the production of expensive or uncommon amino acids. The optical resolution of amino acids can be done in two general ways physical or chemical methods which apply the stereospecific properties of amino acids, and biological or enzymatic methods which are based on the characteristic behavior of amino acids in living cells in the presence of enzymes. 

Unprotected racemic amines can be resolved by enantioselective acylations with activated esters (110,111). This approach is based on the discovery that enantioselectivity of some enzymes strongly depends on the nature of the reaction medium. For example, the enantioselectivity factor (defined as the ratio of the initial rates for (3)- and (R)-isomers) of subtiHsin in the acylation of CX-methyl-ben zyl amine with tritiuoroethyl butyrate varies from 0.95 in toluene to 7.7 in 3-methyl-3-pentanol (110). The latter solvent has been used for enantioselective resolutions of a number of racemic amines (110). 

The second group of studies tries to explain the solvent effects on enantioselectivity by means of the contribution of substrate solvation to the energetics of the reaction [38], For instance, a theoretical model based on the thermodynamics of substrate solvation was developed [39]. However, this model, based on the determination of the desolvated portion of the substrate transition state by molecular modeling and on the calculation of the activity coefficient by UNIFAC, gave contradictory results. In fact, it was successful in predicting solvent effects on the enantio- and prochiral selectivity of y-chymotrypsin with racemic 3-hydroxy-2-phenylpropionate and 2-substituted 1,3-propanediols [39], whereas it failed in the case of subtilisin and racemic sec-phenetyl alcohol and traws-sobrerol [40]. That substrate solvation by the solvent can contribute to enzyme enantioselectivity was also claimed in the case of subtilisin-catalyzed resolution of secondary alcohols [41]. 

A review is given of the application of Molecular Dynamics (MD) computer simulation to complex molecular systems. Three topics are treated in particular the computation of free energy from simulations, applied to the prediction of the binding constant of an inhibitor to the enzyme dihydrofolate reductase the use of MD simulations in structural refinements based on two-dimensional high-resolution nuclear magnetic resonance data, applied to the lac repressor headpiece the simulation of a hydrated lipid bilayer in atomic detail. The latter shows a rather diffuse structure of the hydrophilic head group layer with considerable local compensation of charge density. 

Catalytic transformation based on combined enzyme and metal catalysis is described as a new class of methodology for the synthesis of enantiopure compounds. This approach is particularly useful for dynamic kinetic resolution in which enzymatic resolution is coupled with metal-catalyzed racemization for the conversion of a racemic substrate to a single enantiomeric product. 

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