Glycosynthases: Mutant Glycosidases for Glycoside Synthesis

2002 ◽  
Vol 55 (2) ◽  
pp. 3 ◽  
Author(s):  
S. J. Williams ◽  
S. G. Withers
Keyword(s):  
Substrate Specificity ◽  
Product Formation ◽  
Protecting Groups ◽  
Rapid Screening ◽  
Widespread Application ◽  
Glycoside Synthesis ◽  
General Methodology ◽  
Glycosyl Donor ◽  
Substrate Variation ◽  
Catalytic Power

Glycosynthases are engineered mutant glycosidases that catalyse the formation of a glycosidic bond from a glycosyl donor and an acceptor alcohol. They are constructed by mutation of the enzymic nucleophile of a retaining glycosidase to a small non-nucleophilic residue. To date, five glycosynthases have been reported capable of synthesizing a range of β-glycosidic linkages. Methods to integrate protecting groups into glycosynthase-mediated glycosylations have been developed that broaden their applicability and enable finer control over product formation. Mutagenesis studies have improved the catalytic power of the original Abg glycosynthase, and a general methodology has been developed that allows the rapid screening of libraries of mutant glycosynthases for catalysts with improved activity. A method for determining aglycon substrate specificity has been developed to define the limits of substrate variation tolerated by a parent glycosidase and thence the derived glycosynthase. Together, these developments portend a bright future for the discovery of new glycosynthases and their widespread application as catalysts to assist in the rapid and efficient assembly of complex glycoconjugates.

scholarly journals A One-Pot Approach to Novel Pyridazine C-Nucleosides

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2341
Author(s):  
Flavio Cermola ◽  
Serena Vella ◽  
Marina DellaGreca ◽  
Angela Tuzi ◽  
Maria Rosaria Iesce
Keyword(s):  
Organic Chemistry ◽  
Research Field ◽  
Modified Nucleosides ◽  
Protecting Groups ◽  
Coupling Reactions ◽  
One Pot ◽  
Pharmacological Interest ◽  
Classical Methodology ◽  
Glycosyl Donor ◽  
Neutral Conditions

The synthesis of glycosides and modified nucleosides represents a wide research field in organic chemistry. The classical methodology is based on coupling reactions between a glycosyl donor and an acceptor. An alternative strategy for new C-nucleosides is used in this approach, which consists of modifying a pre-existent furyl aglycone. This approach is applied to obtain novel pyridazine C-nucleosides starting with 2- and 3-(ribofuranosyl)furans. It is based on singlet oxygen [4+2] cycloaddition followed by reduction and hydrazine cyclization under neutral conditions. The mild three-step one-pot procedure leads stereoselectively to novel pyridazine C-nucleosides of pharmacological interest. The use of acetyls as protecting groups provides an elegant direct route to a deprotected new pyridazine C-nucleoside.

scholarly journals Substrate Recognition Properties of Oligopeptidase B from Salmonella enterica Serovar Typhimurium

2002 ◽  
Vol 184 (12) ◽  
pp. 3329-3337 ◽  
Author(s):  
Rory E. Morty ◽  
Vilmos Fülöp ◽  
Norma W. Andrews
Keyword(s):  
Substrate Specificity ◽  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Site Directed Mutagenesis ◽  
Cloning And Expression ◽  
Preferential Cleavage ◽  
Oligopeptidase B ◽  
Serovar Typhimurium ◽  
Important Virulence Factor ◽  
Catalytic Power

ABSTRACT Oligopeptidase B (OpdB) is a serine peptidase broadly distributed among unicellular eukaryotes, gram-negative bacteria, and spirochetes which has emerged as an important virulence factor and potential therapeutic target in infectious diseases. We report here the cloning and expression of the opdB homologue from Salmonella enterica serovar Typhimurium and demonstrate that it exhibits amidolytic activity exclusively against substrates with basic residues in P1. While similar to its eukaryotic homologues in terms of substrate specificity, Salmonella OpdB differs significantly in catalytic power and inhibition and activation properties. In addition to oligopeptide substrates, restricted proteolysis of histone proteins was observed, although no cleavage was seen at or near residues that had been posttranslationally modified or at defined secondary structures. This supports the idea that the catalytic site of OpdB may be accessible only to unstructured oligopeptides, similar to the closely related prolyl oligopeptidase (POP). Salmonella OpdB was employed as a model enzyme to define determinants of substrate specificity that distinguish OpdB from POP, which hydrolyzes substrates exclusively at proline residues. Using site-directed mutagenesis, nine acidic residues that are conserved in OpdBs but absent from POPs were converted to their corresponding residues in POP. In this manner, we identified a pair of glutamic acid residues, Glu576 and Glu578, that define P1 specificity and direct OpdB cleavage C terminal to basic residues. We have also identified a second pair of residues, Asp460 and Asp462, that may be involved in defining P2 specificity and thus direct preferential cleavage by OpdB after pairs of basic residues.

scholarly journals Substrate Specificities of Hybrid Naphthalene and 2,4-Dinitrotoluene Dioxygenase Enzyme Systems

1998 ◽  
Vol 180 (9) ◽  
pp. 2337-2344 ◽  
Author(s):  
Rebecca E. Parales ◽  
Matthew D. Emig ◽  
Nancy A. Lynch ◽  
David T. Gibson
Keyword(s):  
Escherichia Coli ◽  
Substrate Specificity ◽  
Product Formation ◽  
Small Subunit ◽  
Enantiomeric Purity ◽  
Oxidation Products ◽  
Substrate Specificities ◽  
Enzyme Systems ◽  
Α And Β Subunits ◽  
Dioxygenase Enzymes

ABSTRACT Bacterial three-component dioxygenase systems consist of reductase and ferredoxin components which transfer electrons from NAD(P)H to a terminal oxygenase. In most cases, the oxygenase consists of two different subunits (α and β). To assess the contributions of the α and β subunits of the oxygenase to substrate specificity, hybrid dioxygenase enzymes were formed by coexpressing genes from two compatible plasmids in Escherichia coli. The activities of hybrid naphthalene and 2,4-dinitrotoluene dioxygenases containing four different β subunits were tested with four substrates (indole, naphthalene, 2,4-dinitrotoluene, and 2-nitrotoluene). In the active hybrids, replacement of small subunits affected the rate of product formation but had no effect on the substrate range, regiospecificity, or enantiomeric purity of oxidation products with the substrates tested. These studies indicate that the small subunit of the oxygenase is essential for activity but does not play a major role in determining the specificity of these enzymes.

scholarly journals Minimizing Side Product Formation in Alkyne Functionalization of Ruthenium Complexes by Introduction of Protecting Groups

2020 ◽  
Vol 646 (13) ◽  
pp. 842-848
Author(s):  
Pascal Wintergerst ◽  
Kamil Witas ◽  
Djawed Nauroozi ◽  
Marie‐Ann Schmid ◽  
Ebru Dikmen ◽  
...  
Keyword(s):  
Ruthenium Complexes ◽  
Product Formation ◽  
Side Product ◽  
Protecting Groups

scholarly journals Gold-catalyzed glycosidation for the synthesis of trisaccharides by applying the armed–disarmed strategy

2013 ◽  
Vol 9 ◽  
pp. 2147-2155 ◽  
Author(s):  
Abhijeet K Kayastha ◽  
Srinivas Hotha
Keyword(s):  
Reaction Temperature ◽  
Acidic Medium ◽  
Gold Catalysis ◽  
Protecting Groups ◽  
Benzyl Ethers ◽  
Glycosyl Donor ◽  
Leaving Groups

The synthesis of oligosaccharides is still a challenging task as there is no universal glycosyl donor for the synthesis of all oligosaccharides. The gold catalysis for glycosidation reactions, in which alkynylated glycosides are used, has emerged as one of the versatile options in this regard. A cleavage of the interglycosidic bond that was thought to be due to the higher reaction temperature and the acidic medium was observed during the synthesis of trisaccharides. In addition, a very little percentage of deprotection of benzyl protecting groups at the C-6 position was observed and no deprotection of benzyl ethers in aliphatic molecules was noticed. In order to overcome this fact, a collection of leaving groups that contain an alkynyl moiety were screened. It was found that 1-ethynylcyclohexanyl (Ech) glycosides are suitable for carrying out the glycosidation at 25 °C in the presence of 5 mol % each of AuCl3 and AgSbF6. Subsequently, Ech-glycosides were observed to be suitable for the synthesis of trisaccharides under gold catalysis conditions.

scholarly journals Strategies toward protecting group-free glycosylation through selective activation of the anomeric center

2017 ◽  
Vol 13 ◽  
pp. 1239-1279 ◽  
Author(s):  
A Michael Downey ◽  
Michal Hocek
Keyword(s):  
Biological Process ◽  
Protecting Groups ◽  
Protecting Group ◽  
Research Groups ◽  
Selective Activation ◽  
Chemical Laboratory ◽  
Differential Reactivity ◽  
Glycosyl Donor ◽  
Anomeric Center

Glycosylation is an immensely important biological process and one that is highly controlled and very efficient in nature. However, in a chemical laboratory the process is much more challenging and usually requires the extensive use of protecting groups to squelch reactivity at undesired reactive moieties. Nonetheless, by taking advantage of the differential reactivity of the anomeric center, a selective activation at this position is possible. As a result, protecting group-free strategies to effect glycosylations are available thanks to the tremendous efforts of many research groups. In this review, we showcase the methods available for the selective activation of the anomeric center on the glycosyl donor and the mechanisms by which the glycosylation reactions take place to illustrate the power these techniques.

Synthesis of 6-O-octyl-β-D-galactopyranosyl-(1 → 5)-L-arabinose and comparative 1-deoxyglycitolation of Nα-Z-L-lysine with amphiphilic lipodisaccharides

1990 ◽  
Vol 68 (12) ◽  
pp. 2253-2257 ◽  
Author(s):  
Daniel Cabaret ◽  
Michel Wakselman
Keyword(s):  
Chemical Modification ◽  
Organic Solvents ◽  
Future Study ◽  
Acyl Transfer ◽  
Protecting Groups ◽  
Reductive Alkylation ◽  
Glycoside Synthesis ◽  
Mild Conditions ◽  
Chemical Modification Of Enzymes

A lipodisaccharide possessing a reactive aldopentose unit, 6-O-octyl-β-D-galactopyranosyl-(1 → 5)-L-arabinose 6, was obtained by glycoside synthesis. To avoid a possible intramolecular acyl transfer, benzoyl protecting groups and mild conditions of detritylation were used in the preparation of the furanosyl acceptor. The reductive alkylation of Nα-Z-L-lysine was then studied and compared to that of previously prepared liposaccharides. In this reaction, amphiphilic five-membered hemiacetals are generally more reactive than their six-membered analogues. The newly prepared disaccharide is the most reactive of the series and also the easiest to prepare. Therefore this reagent has been selected for a future study on the chemical modification of enzymes and the use in organic solvents of the biocatalysts obtained. Keywords: liposaccharides, reductive alkylation.

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