Visible-Light-Driven Decarboxylative Borylation: Rapid Access to α- and ß-Amino-boronamides.

In this study, we described a two-step process involving an efficient visible-light-induced decarboxylative borylation of α- and ß-amino redox-active esters with bis(catecholato)diboron, followed by transamination with 1,8-diaminonapthalene (DANH2). A series of boronamides were obtained in moderate to excellent yields in this one-pot procedure. The photochemical process proved to be very efficient even when conducted under flow conditions with shorter reaction durations and scalable synthesis of DAN boronates.

Production of l-Methionine from 3-Methylthiopropionaldehyde and O-Acetylhomoserine by Catalysis of the Yeast O-Acetylhomoserine Sulfhydrylase.

l-Methionine is an essential bioactive amino acid with high commercial value for diverse applications. Sustained attentions have been paid to efficient and economical preparation of l-methionine. In this work, a novel method for l-methionine production was established using O-acetyl-homoserine (OAH) and 3-methylthiopropionaldehyde (MMP) as substrates by catalysis of the yeast OAH sulfhydrylase MET17. The OAH sulfhydrylase gene Met17 was cloned from Saccharomyces cerevisiae S288c and overexpressed in Escherichia coli BL21. A 49 kDa MET17 was detected in the supernatant of the recombinant E. coli strain BL21-Met17 lysate with IPTG induction, which exhibited the biological activity of l-methionine biosynthesis from OAH and MMP. The recombinant MET17 was then purified from E. coli BL21-Met17 and used for in vitro biosynthesis of l-methionine. The maximal conversion rate (86%) of OAH to l-methionine catalyzed by purified MET17 was achieved by optimization of the molar ratio of OAH to MMP. The method proposed in this study provides a possible novel route for the industrial production of l-methionine.

Production of Methionol from 3-Methylthiopropionaldehyde by Catalysis of the Yeast Alcohol Dehydrogenase Adh4p.

Methionol is a sulfur-containing aroma compound that contributes to the flavors of fermented foods. In this work, a novel method for methionol production was established using 3-methylthiopropionaldehyde (MMP) and alcohol dehydrogenase (ADH). First, expression of seven ADH genes was analyzed in yeast fermentation added with MMP. Only ADH4 displayed an evident increased expression in response to MMP. ADH4 was then overexpressed in Saccharomyces cerevisiae S288c and Escherichia coli BL21. The recombinant yeast strain S4 produced more methionol than control strain in MMP fermentation. Furthermore, 0.55 g/L 42 kDa Adh4p was prepared from E. coli by induced expression and purification. A fed-batch catalysis system was finally established to produce methionol from MMP by Adh4p. In 10 h of continuous catalysis, the conversion rate of MMP remained 80-95%, and a final yield of 0.2 g/L methionol was achieved. This work proposed a novel method for methionol production by enzymatic catalysis with a potential application prospect in industry.

Novel Method for l-Methionine Production Catalyzed by the Aminotransferase ARO8 from Saccharomyces cerevisiae.

The aminotransferase ARO8 was proved to play an efficient role in conversion of l-methionine into methionol via the Ehrlich pathway in Saccharomyces cerevisiae in our previous work. In this work, the reversible transamination activity of ARO8 for conversion of α-keto-γ-(methylthio) butyric acid (KMBA) into l-methionine was confirmed in vitro. ARO8 was cloned from S. cerevisiae S288c and overexpressed in Escherichia coli BL21. A 2-fold higher aminotransferase activity was detected in the recombinant strain ARO8-BL21, and ARO8 was detected in the supernatant of ARO8-BL21 lysate with IPTG induction by SDS-PAGE analysis. The recombinant ARO8 was then purified and used for transforming KMBA into l-methionine. An approximately 100% conversion rate of KMBA into l-methionine was achieved by optimized enzymatic reaction catalyzed by ARO8. This work fulfilled l-methionine biosynthesis catalyzed by the aminotransferase ARO8 using glutamate and KMBA, which provided a novel method for l-methionine production by enzymatic catalysis with the potential application prospect in industry.

Detailed Investigation of the Immunodominant Role of O-Antigen Stoichiometric O-Acetylation as Revealed by Chemical Synthesis, Immunochemistry, Solution Conformation and STD-NMR Spectroscopy for Shigella flexneri†3a.

Shigella flexneri 3a causes bacillary dysentery. Its O-antigen has the {2)-[α-d-Glcp-(1→3)]-α-l-Rhap-(1→2)-α-l-Rhap-(1→3)-[Ac→2]-α-l-Rhap-(1→3)-[Ac→6]≈40 % -ß-d-GlcpNAc-(1→} ([(E)ABAc CAc D]) repeating unit, and the non-O-acetylated equivalent defines S. flexneri X. Propyl hepta-, octa-, and decasaccharides sharing the (E')A'BAc CD(E)A sequence, and their non-O-acetylated analogues were synthesized from a fully protected BAc CD(E)A allyl glycoside. The stepwise introduction of orthogonally protected mono- and disaccharide imidate donors was followed by a two-step deprotection process. Monoclonal antibody binding to twenty-six S. flexneri types 3a and X di- to decasaccharides was studied by an inhibition enzyme-linked immunosorbent assay (ELISA) and STD-NMR spectroscopy. Epitope mapping revealed that the 2C -acetate dominated the recognition by monoclonal IgG and IgM antibodies and that the BAc CD segment was essential for binding. The glucosyl side chain contributed to a lesser extent, albeit increasingly with the chain length. Moreover, tr-NOESY analysis also showed interaction but did not reveal any meaningful conformational change upon antibody binding.

Design of alpha-transglucosidases of controlled specificity for programmed chemoenzymatic synthesis of antigenic oligosaccharides.

Combined with chemical synthesis, the use of biocatalysts holds great potential to open the way to novel molecular diversity. We report in vitro chemoenzymatic pathways that, for the first time, take advantage of enzyme engineering to produce complex microbial cell-surface oligosaccharides and circumvent the chemical boundaries of glycochemistry. Glycoenzymes were designed to act on nonnatural conveniently protected substrates to produce intermediates compatible with a programmed chemical elongation. The study was focused on the synthesis of oligosaccharides mimicking the O-antigen motif of Shigella flexneri serotypes 1b and 3a, which could be used for the development of multivalent carbohydrate-based vaccines. A semirational engineering approach was successfully applied to amylosucrase, a transglucosidase that uses a low cost sucrose substrate as a glucosyl donor. The main difficulty was to retain the enzyme specificity toward sucrose, while creating a new catalytic function to render the enzyme able to regiospecifically glucosylate protected nonnatural acceptors. A structurally guided library of 133 mutants was generated from which several mutants with either completely new specificity toward methyl alpha-l-rhamnopyranoside or a tremendously enhanced one toward allyl 2-acetamido-2-deoxy-alpha-d-glucopyranoside acceptors were isolated. The best variants were used to synthesize glucosylated building blocks. They were then converted into acceptors and potential donors compatible with chemical elongation toward oligosaccharide fragments of the O-antigens of the two targeted serotypes. This is the first report of a successful engineering of an alpha-transglycosidase acceptor binding site that led to new specificities. It demonstrates the potential of appropriate combinations of a planned chemoenzymatic pathway and enzyme engineering in glycochemistry.

Efficient synthesis of six tri- to hexasaccharide fragments of Shigella flexneri serotypes 3a and/or X O-antigen, including a study on acceptors containing N-trichloroacetylglucosamine versus N-acetylglucosamine.

Six tri- to hexasaccharide fragments of the {2)-[alpha-D-Glcp-(1-->3)]-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->3)-[Ac-->2]-alpha-L-Rhap-(1-->3)-beta-D-GlcpNAc-(1-->}(n) polymer ([(E)AB(Ac)CD](n)) were synthesized as their propyl glycosides. All targets share the (E)AB sequence. Following a thorough investigation on the use of N-trichloroacetylglucosamine- versus N-acetylglucosamine-containing tri- and tetrasaccharide acceptors, the successful strategy was based on an efficient combination of the trichloroacetimidate chemistry, a trichloroacetyl used as permanent N-protection, and an allyl aglycon as temporary and/or permanent anomeric protection of selected building blocks. Use of an EAB intermediate orthogonally protected at 2(A) provided both the trisaccharide target and acceptor 12, the condensation of which with a chain terminator D followed by full deprotection, gave tetrasaccharide D(E)AB. Alternatively, stepwise glycosylation of 12 with a D donor compatible with a selective deblocking at position 3(D) and a 2-O-acetyl C donor following exposure of OH-3(D) led to a pentasaccharide, which was partially and fully deprotected into free (Ac)CD(E)AB and CD(E)AB, respectively. Furthermore, chain elongation of the common D(E)AB acceptor with a 2(B)-O-levulinoyl rhamnobiose donor BC and subsequent partial or total deprotection of the resulting hexasaccharide provided B(Ac)CD(E)AB and BCD(E)AB, respectively. All of the synthesized oligosaccharides are parts of the O-antigen of Shigella flexneri 3a, a prevalent serotype. Moreover, the non-O-acetylated fragments are also parts of the S. flexneri serotype X O-antigen.