Natural glycoproteins are mixtures of multiple glycoforms possessing identical peptide backbones, but differing in composition of glycan parts. This microheterogeneity presents a serious challenge in pharmaceutical applications of glycoproteins, where well-characterized and reproducible structural characteristics are required. Due to the growing importance of biopharmaceutical glycoconjugates, a wide variety of practical approaches to the preparation of glycosylated proteins and oligopeptides have been developed in recent years, with emphasis on strategies for selective glycosylation. This review presents a concise summary of concepts and synthetic approaches currently employed in glycoprotein chemistry.

Attaining higher levels of catalytic activity of enzymes in organic solvents is one of the major challenges in nonaqueous enzymology. One of the most successful strategies for enhancing enzyme activity in organic solvents involves tuning the enzyme active site by molecular imprinting with substrates or their analogues. Unfortunately, numerous imprinters of potential importance are poorly soluble in water, which significantly limits the utility of this method. In the present study, we have developed strategies that overcome this limitation of the molecular-imprinting technique and that thus expand its applicability beyond water-soluble ligands. The solubility problem can be addressed either by converting the ligands into a water-soluble form or by adding relatively high concentrations of organic cosolvents, such as tert-butyl alcohol and 1,4-dioxane, to increase their solubility in the lyophilization medium. We have succeeded in applying both of these strategies to produce imprinted thermolysin, subtilisin, and lipase TL possessing up to 26-fold higher catalytic activity in the acylation of paclitaxel and 17beta-estradiol compared to nonimprinted enzymes. Furthermore, we have demonstrated for the first time that molecular imprinting and salt activation, applied in combination, produce a strong additive activation effect (up to 110-fold), suggesting different mechanisms of action involved in these enzyme activation techniques.

The published applications of combinatorial biocatalysis have continued to expand at a growing rate. This is exemplified by the variety of enzyme catalysts and whole-cell catalysts used for the creation of libraries through a wide range of biocatalytic reactions, including acylation, glycosylation, halogenation, oxidation and reduction. These biocatalytic methods add the capability to perform unique chemistries or selective reactions with complex or labile reagents when integrated with classical combinatorial synthesis methods. Thus, applications towards the production of libraries de novo, the expansion of chemically derived combinatorial libraries, and the generation of novel combinatorial reagents for library synthesis can be achieved. Theoretically, these results illustrate what is already evident from nature: that complex, biologically active, structurally diverse compound libraries can be generated through the application of biocatalysis alone or in combination with classical organic synthesis approaches.

Arylsulfotransferase (AST, EC 2.8.2.22), an enzyme capable of sulfating a wide range of phenol-containing compounds was purified from a Clostridium innocuum isolate (strain 554). The enzyme has a molecular weight of 320 kDa and is composed of four subunits. Unlike many mammalian and plant arylsulfotransferases, AST from Clostridium utilizes arylsulfates, including p-nitrophenyl sulfate, as sulfate donors, and is not reactive with 3-phosphoadenosine-5'-phosphosulfate (PAPS). The enzyme possesses broad substrate specificity and is active with a variety of phenols, quinones and flavonoids, but does not utilize primary and secondary alcohols and sugars as substrates. Arylsulfotransferase tolerates the presence of 10 vol% of polar cosolvents (dimethyl formamide, acetonitrile, methanol), but loses significant activity at higher solvent concentrations of 30-40 vol%. The enzyme retains high arylsulfotransferase activity in biphasic systems composed of water and nonpolar solvents, such as cyclohexane, toluene and chloroform, while in biphasic systems with more polar solvents (ethyl acetate, 2-pentanone, methyl tert-butyl ether, and butyl acetate) the enzyme activity is completely lost. High yields of AST-catalyzed sulfation were achieved in reactions with several phenols and tyrosine-containing peptides. Overall, AST studied in this work is a promising biocatalyst in organic synthesis to afford efficient sulfation of phenolic compounds under mild reaction conditions.

2-Hydroxybiphenyl 3-monooxygenase (HbpA, E.C. 1.14.13.44) was partially purified on a gram scale from recombinant E. coli JM101 by expanded bed adsorption (EBA) chromatography. In order to develop an effective in vitro biotransformation system using isolated HbpA as catalyst, its stability and activity in the presence of different apolar and polar organic solvents was investigated. HbpA showed good stability in 80% (v/v) decanol and 10% (v/v) methanol. Since the reaction catalyzed by HbpA is cofactor dependent, enzymatic NADH cofactor regeneration by yeast alcohol dehydrogenase (YADH) was tested in the presence of 10% (v/v) methanol used both as a cosolvent and substrate for YADH. In an in vitro biotransformation on a 25 ml scale 5.4 mM of 2,2′-dihydroxybiphenyl was hydroxylated to 2,2′,3-trihydroxybiphenyl in 2.5 h and 2,2′,3,3′-tetrahydroxybiphenyl in 23 h at a mean volumetric productivity of 0.43 g/(l h) and 0.05 g/(l h), respectively. Additionally, HbpA and formate dehydrogenase (FDH) were applied in 80% (v/v) decanol to hydroxylate 2-hydroxybiphenyl with a productivity of 0.46 g/(l h) and a total turnover number of 503. In 10% (v/v) aqueous methanol the total turnover number of 30 and enzyme stability for at least 60 h were shown. This demonstrated the feasibility of using isolated HbpA for syntheses in the presence of polar as well as apolar solvents with enzymatic regeneration of reduced cofactors.

A new reaction system suitable for phospholipase D (PLD)-catalyzed transphosphatidylation of alcohols with phosphatidylcholine under anhydrous conditions is reported. The key innovation of the reaction system is a cation-exchange resin serving as a scavenger for choline that forms as a byproduct in the transphosphatidylation reaction. Due to the absence of water in this system, the reaction path dramatically shifts in favor of the target transphosphatidylated product, whereas the undesirable side hydrolysis of phosphatidylcholine is completely suppressed, in contrast to commonly used biphasic water-organic systems. In addition, a salt activation technique is successfully applied to increase the catalytic activity of PLD in this anhydrous system. The new reaction system is successfully used for transphosphatidylation of a wide range of primary, secondary, and aromatic alcohols catalyzed by PLD from Streptomyces sp.

The synthetic potential of amine oxidases was examined in different reaction systems, ranging from aqueous solutions to organic solvents with low water content. Substantial conversion was achieved in biphasic systems, which eliminated the product inhibition observed in the aqueous system. The conversion was particularly high in the more hydrophobic solvents. The use of low water systems was studied using amine oxidase immobilized on celite and pre-equilibrated in a salt hydrate environment to reach a constant water activity. Addition of water in the solvent was shown to be unnecessary, with significant conversion being attained through the water supplied by pre-equilibration of the immobilized enzyme at aw=0.55. The use of organic solvent-containing reaction systems thus presents a convenient method for oxidising poorly water-soluble amines using amine oxidases.

The solvent-free microwave-assisted synthesis of 2,4,5-substituted and 1,2,4,5-substituted imidazoles is reported. Imidazoles are obtained as a result of the condensation of a 1,2-dicarbonyl compound with an aldehyde and an amine using acidic alumina impregnated with ammonium acetate as the solid support.

Biotransformation technologies have enjoyed a renewed interest from researchers and industry because of the progress made in the discovery and design of new, efficient biocatalysts for synthetic applications. Biocatalysis in nonaqueous media, which offers unique capabilities and thus plays a major role in biotransformation technologies, has made tremendous progress in recent years. On average, at least one paper dealing with biocatalysis in organic solvents is published every day. New, remarkable developments have taken place in several key areas of this exciting field during the past year.

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Nature's most potent molecules are produced by enzyme-catalysed reactions, coupled with the natural selection of those products that possess optimal biological activity. Combinatorial biocatalysis harnesses the natural diversity of enzymatic reactions for the iterative synthesis of organic libraries. Iterative reactions can be performed using isolated enzymes or whole cells, in natural and unnatural environments, and on substrates in solution or on a solid phase. Combinatorial biocatalysis is a powerful addition to the expanding array of combinatorial methods for the generation and optimization of lead compounds in drug discovery and development.

A simple combinatorial strategy for sequential regioselective enzymatic acylation of multifunctional lead compounds has been developed and demonstrated using a polyhydroxylated flavonoid, bergenin, as a model. The approach is based on the ability of different enzymes to regioselectively acylate different sites on a lead molecule without affecting other similar functional groups. In sharp contrast to enzymatic acylation, conventional chemical acylation methods showed almost complete lack of regioselectivity. The enzymatic strategy was applied successfully to produce a solution phase combinatorial library of 167 distinct selectively acylated derivatives of bergenin on a robotic workstation in a 96-well plate format. General applicability of the automated combinatorial biocatalysis strategy is discussed.

Biotransformation is a broad and growing field of biotechnology and encompasses both enzymatic and microbial biocatalysis. Progress has been made in research on the key drivers of biotransformations, including the isolation and characterization of microbes and their enzymes from, and their utilization in, extreme environments, the manipulation, alteration, and augmentation of metabolic pathways, and the use of combinatorial biosynthesis and biocatalytic methodologies for new compound development.

A new highly efficient MICROCOS technology (Microwave-assisted Combinatorial Synthesis) for generating combinatorial libraries is described. The technology is applied to the high throughput, automated, one-step, parallel synthesis of diverse substituted pyridines using the Hantzsch synthesis. The advantages of microwave-assisted chemistry for combinatorial synthesis include a broad range of available chemistries, simple reaction setup and product recovery readily amenable to automation, extremely short reaction times, and high product yields.

ADVERTISEMENT RETURN TO ISSUEPREVCommunicationNEXTSynthesis of Water-Soluble Paclitaxel Derivatives by Enzymatic AcylationYuri L. Khmelnitsky, Cheryl Budde, J. Michael Arnold, Alexander Usyatinsky, Douglas S. Clark, and Jonathan S. DordickView Author Information EnzyMed, Inc., 2501 Crosspark Road Oakdale Research Park, Iowa City, Iowa 52242 Department of Chemical Engineering University of California Berkeley, California 94720 Department of Chemical and Biochemical Engineering University of Iowa, Iowa City, Iowa 52242 Cite this: J. Am. Chem. Soc. 1997, 119, 47, 11554–11555Publication Date (Web):November 26, 1997Publication History Received3 September 1997Published online26 November 1997Published inissue 1 November 1997https://pubs.acs.org/doi/10.1021/ja973103zhttps://doi.org/10.1021/ja973103zrapid-communicationACS PublicationsCopyright © 1997 American Chemical SocietyRequest reuse permissionsArticle Views1176Altmetric-Citations77LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-AlertscloseSupporting Info (1)»Supporting Information Supporting Information SUBJECTS:Antineoplastic agents,Organic reactions,Peptides and proteins,Post-translational modification,Reaction products Get e-Alerts

Comparing the behavior of freely suspended and immobilized enzyme in organic media with low water contents can yield insights into interactions among the biocatalyst, solvent, and support that influence protein structure and function. Immobilized chymotrypsin has higher activity than the suspended enzyme (by 1-2 orders of magnitude) in anhydrous organic solvents ranging from nonpolar n-octane to polar acetonitrile. In anhydrous tetrahydrofuran (THF), glass-adsorbed chymotrypsin is ca. 10 times more active than the suspended enzyme, and election spin resonance (ESR) spectra of an active-site spin-label reveal greater local flexibility. Upon adding up to 0.5% v/v water, increased catalytic efficiency of the immobilized enzyme is accompanied by a sharp rise in active-site polarity but no apparent change in active-site conformation or dynamics. Under the same conditions the activity of the suspended enzyme also increases; however, the active-site polarity remains nearly constant while the spin-label reflects increasing molecular flexibility. For both preparations, further changes in protein structure occur as enzyme activity increases with 0.5-7% v/v added water. Computer simulations of the room-temperature ESR spectra suggest that different initial conformational states contribute to the different behavior of the two enzyme systems over the entire range of added water. These findings show that the structural properties of suspended and immobilized enzyme can differ markedly and that these differences are important to enzyme activity in organic media.