Jianwen Fang

Aziridine synthesis in protic media by using lanthanide triflates as catalysts

Abstract Ln(OTf)3-catalyzed aziridine synthesis from imines and diazo compounds was studied in a variety of protic media. The reactions proceeded readily under mild conditions and were highly selective, affording predominantly cis aziridines. The imines used were typically those derived from aromatic aldehydes and aromatic amines, with either electron-donating or electron-withdrawing substituents. N-benzyl aryl aldimines and imines derived from aromatic amines and hindered aliphatic aldehydes were also found to work well. Among the three diazo compounds examined, ethyl diazoacetate (EDA) and 3-nitrophenyl diazomethane were effective reagents for the aziridination. However, the reactions of ethyl diazoacetoacetate failed to give any desired product. Six lanthanides triflates as well as Sc(OTf)3 and Y(OTf)3 were tested as catalyst in the aziridination reaction. The formation of the by-products was also discussed.

Efficient chemoenzymatic synthesis of globotriose and its derivatives with a recombinant α-(1→4)-galactosyltransferase

A truncated alpha-(1-->4)-galactosyltransferase (LgtC) gene from Neisseria meningitidis was cloned. The recombinant glycosyltransferase was expressed in Escherichia coli BL21 (DE3) strain with high specific activity (5 units/mg protein). Its acceptor specificity was carefully characterized. Then the purified enzyme was utilized in highly efficient syntheses of globotriose and a variety of alpha-(1-->4)-galactosylated derivatives as potential antibacterial agents.

Changing the Donor Cofactor of Bovine α1,3-Galactosyltransferase by Fusion with UDP-galactose 4-Epimerase MORE EFFICIENT BIOCATALYSIS FOR SYNTHESIS OF α-Gal EPITOPES

Two fusion enzymes consisting of uridine diphosphogalactose 4-epimerase (UDP-galactose 4-epimerase, EC 5.1.3.2) and α1,3-galactosyltransferase (EC 2.4.1.151) with an N-terminal His6 tag and an intervening three-glycine linker were constructed by in-frame fusion of the Escherichia coli galEgene either to the 3′ terminus (f1) or to the 5′ terminus (f2) of a truncated bovine α1,3-galactosyltransferase gene, respectively. Both fusion proteins were expressed in cell lysate as active, soluble forms as well as in inclusion bodies as improperly folded proteins. Both f1 and f2 were determined to be homodimers, based on a single band observed at about 67 kDa in SDS-polyacrylamide gel electrophoresis and on a single peak with a molecular mass around 140 kDa determined by gel filtration chromatography for each of the enzymes. Without altering the acceptor specificity of the transferase, the fusion with the epimerase changed the donor requirement of α1,3-galactosyltransferase from UDP-galactose to UDP-glucose and decreased the cost for the synthesis of biomedically important Galα1,3Gal-terminated oligosaccharides by more than 40-fold. For enzymatic synthesis of Galα1,3Galβ1,4Glc from UDP-glucose and lactose, the genetically fused enzymes f1 and f2 exhibited kinetic advantages with overall reaction rates that were 300 and 50%, respectively, higher than that of the system containing equal amounts of epimerase and galactosyltransferase. These results indicated that the active sites of the epimerase and the transferase in fusion enzymes were in proximity. The kinetic parameters suggested a random mechanism for the substrate binding of the α1,3-galactosyltransferase. This work demonstrated a general approach that fusion of a glycosyltransferase with an epimerase can change the required but expensive sugar nucleotide to a less expensive one.

Ytterbium(III) trifluoromethanesulfonate catalyzed solid phase aza Diels-Alder reaction and subsequent facile adduct release

Abstract Ytterbium(III) trifluoromethanesulfonate has been demonstrated to catalyze the solid phase aza Diels-Alder reaction of an aldehyde, a diene, and immobilized benzylamine. The [4+2] adducts were cleaved from solid support efficiently using a ‘trace-less’ release methodology. The piperidine derivatives were obtained in excellent to reasonable yields with high levels of purity.

Chemical and enzymatic synthesis of glycoconjugates 3: Synthesis of lactosamine by thermophilic galactosidase catalyzed galactosylation on a multigram scale

Abstract A library of thermophilic glycosidases was screened for lactosamine preparation from a transgalactosylation reaction between lactose and glucosamine hydrochloride. Two enzymes showed excellent regioselectivity toward glucosamine hydrochloride. The product could be synthesized on a multigram scale and was easily separated and purified by subsequently eluting through cation and anion exchange resin columns.

Production of α‐Galactosyl Epitopes via Combined Use of Two Recombinant Whole Cells Harboring UDP‐Galactose 4‐Epimerase and α‐1,3‐Galactosyltransferase

α‐Galactosyl epitopes (or α‐Gal, oligosaccharides with a terminal Galα1,3Gal sequence) are a class of biologically important oligosaccharides in great demand in bulk quantities for basic and clinical studies on preventing hyperacute rejection in pig‐to‐primate organ xenotransplantaion. A truncated bovine α‐1,3‐galactosyltransferase, the key enzyme responsible for the biosynthesis of the terminal structure of α‐Gal, was cloned and overexpressed previously. The acceptor specificity was further studied in the present paper, and lactose and galactose derivatives were found to be good acceptors. To develop a more proficient reaction process, we report herein an example of an efficient enzymatic synthesis of α‐Gal oligosaccharides catalyzed by the combination of two recombinant Escherichia coli whole cells harboring the genes of a UDP‐galactose 4‐epimerase and the α‐1,3‐galactosyltransferase, respectively. Using lactosyl azide (LacN3) as the acceptor for the glycosyltransferase, the combined use of the two recombinant cells efficiently produced α‐Gal epitope Galα1,3LacN3 in 60−68% yield.

Conformational analysis of an α-galactosyl trisaccharide epitope involved in hyperacute rejection upon xenotransplantation

alpha-Galactosyl epitopes are carbohydrate structures bearing an alpha-Gal-(1-->3)-Gal terminus (alpha-Gal epitopes). The interaction of these epitopes on the surface of animal cells with anti alpha-Gal antibodies in human serum is believed to be the main cause in antibody-mediated hyperacute rejection in xenotransplantation. In this paper, conformational analysis of an N-linked alpha-D-Galp-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp trisaccharide epitope was conducted in terms of each monosaccharide residue conformation, primary hydroxymethyl group configuration, and interglycosidic conformations. Selective 2D J-delta INEPT experiments have been carried out at three different temperatures to evaluate three-bond, long-range 13C-1H coupling constants for the crucial alpha-(1-->3) linkage. The NMR experimental data were complemented by theoretical calculations. The flexibility and dynamics of the trisaccharide have been studied by Metropolis Monte Carlo simulations. Ensemble-averaged three-bond, long-range 13C-1H coupling constants and nuclear Overhauser effects were in good agreement with the experimental data. The alpha-(1-->3) glycosidic linkage has shown a restricted flexibility as indicated by NMR spectroscopy and molecular modeling.

Frontal Affinity Chromatography Coupled to Mass Spectrometry: An Effective Method for Kd Determination and Screening of α‐Gal Derivatives Binding to Anti‐Gal Antibodies (IgG)

Frontal affinity chromatography with mass spectrometric detection (FAC/MS) was developed as an effective method for rapid determination of Kd values for α‐Gal derivatives binding to human anti‐Gal IgG antibodies. Using this method, Kd values for 23 α‐Gal compounds were determined for the first time, including an α‐Gal terminated N‐linked oligosaccharide which mimics a single N‐glycoform present on the surface of animal cells. A mixture of eight α‐Gal derivatives, a model for an α‐Gal compound library, was successfully screened against this anti‐Gal IgG using FAC/MS. The analyte breakthrough sequence, indicated by the ion chromatogram, reflected the magnitude of the Kd values, confirming its potential application in the screening of new α‐Gal derivatives and mimetics. Ten α‐Gal derivatives were designed and synthesized chemically or enzymatically. Among the compounds analyzed, trivalent compound 26 demonstrated the strongest binding affinity to anti‐Gal IgG with a Kd value of 3.1 µM. The α‐Gal terminated N‐linked oligosaccharide 28 had a Kd value of 8.6 µM.