In order for facilitating the synthesis of oligosaccharides, transglycosylation reactions mediated by glycoside hydrolases have been studied in various contexts. In this study, we examined the transglycosylating activity of a Golgi endo ‐ α‐mannosidase. We prepared various glycosyl donors and acceptors, and recombinant human Golgi endo‐α‐mannosidase and its various mutants were expressed. The enzyme was able to mediate transglycosylation from α‐glycosyl‐fluorides. Systematic screening of various point mutants revealed that the E407D mutant had excellent transglycosylation activity and extremely low hydrolytic activity. Substrate specificity analysis revealed that minimum motif required for glycosyl acceptor is Manα1– 2Man. The synthetic utility of the enzyme was demonstrated by generation of a high‐mannose‐type undecasaccharide (Glc1Man9GlcNAc2). 相似文献
α,β‐Dehydroamino acid derivatives proved to be a novel substrate class for ene‐reductases from the ‘old yellow enzyme’ (OYE) family. Whereas N‐acylamino substituents were tolerated in the α‐position, β‐analogues were generally unreactive. For aspartic acid derivatives, the stereochemical outcome of the bioreduction using OYE3 could be controlled by variation of the N‐acyl protective group to furnish the corresponding (S)‐ or (R)‐amino acid derivatives. This switch of stereopreference was explained by a change in the substrate binding, by exchange of the activating ester group, which was proven by 2H‐labelling experiments. 相似文献
Certain enzymes of the glycoside hydrolase family 20 (GH20) exert transglycosylation activity and catalyze the transfer of β‐N‐acetylglucosamine (GlcNAc) from a chitobiose donor to lactose to produce lacto‐N‐triose II (LNT2), a key human milk oligosaccharide backbone moiety. The present work is aimed at increasing the transglycosylation activity of two selected hexosaminidases, HEX1 and HEX2, to synthesize LNT2 from lactose and chitobiose. Peptide pattern recognition analysis was used to categorize all GH20 proteins in subgroups. On this basis, we identified a series of proteins related to HEX1 and HEX2. By sequence alignment, four additional loop sequences were identified that were not present in HEX1 and HEX2. Insertion of these loop sequences into the wild‐type sequences induced increased transglycosylation activity for three out of eight mutants. The best mutant, HEX1GTEPG, had a transglycosylation yield of LNT2 on the donor that was nine times higher than that of the wild‐type enzyme. Homology modeling of the enzymes revealed that the loop insertion produced a more shielded substrate‐binding pocket. This shielding is suggested to explain the reduced hydrolytic activity, which in turn resulted in the increased transglycosylation activity of HEX1GTEPG. 相似文献
Glycosynthases—retaining glycosidases mutated at their catalytic nucleophile—catalyze the formation of glycosidic bonds from glycosyl fluorides as donor sugars and various glycosides as acceptor sugars. Here the first glycosynthase derived from a family 35 β‐galactosidase is described. The Glu→Gly mutant of BgaC from Bacillus circulans (BgaC‐E233G) catalyzed regioselective galactosylation at the 3‐position of the sugar acceptors with α‐galactosyl fluoride as the donor. Transfer to 4‐nitophenyl α‐D ‐N‐acetyl‐glucosaminide and α‐D ‐N‐acetylgalactosaminide yielded 4‐nitophenyl α‐lacto‐N‐biose and α‐galacto‐N‐biose, respectively, in high yields (up to 98 %). Kinetic analysis revealed that the high affinity of the acceptors contributed mostly to the BgaC‐E233G‐catalyzed transglycosylation. BgaC‐E233G showed no activity with β‐(1,3)‐linked disaccharides as acceptors, thus suggesting that this enzyme can be used in “one‐pot synthesis” of LNB‐ or GNB‐containing glycans. 相似文献
We developed a fluorescence‐quenching‐based assay system to determine the hydrolysis activity of endo‐β‐N‐acetylglucosaminidases (ENGases). The pentasaccharide derivative 1 was labeled with an N‐methylanthraniloyl group as a reporter dye at the non‐reducing end and with a 2,4‐dinitrophenyl group as a quencher molecule at the reducing end. This derivative is hydrolyzed by ENGase, resulting in an increase in fluorescence intensity. Thus, the fluorescence signal is directly proportional to the amount of the tetrasaccharide derivative, hence allowing ENGase activity to be evaluated easily and quantitatively. Using this system, we succeeded in measuring the hydrolysis activities of ENGases and thus the inhibitory activities of known inhibitors. We confirmed that this assay system is suitable for high‐throughput screening for potential inhibitors of human ENGase that might serve as therapeutic agents for the treatment of N‐glycanase 1 (NGLY1) deficiency. 相似文献
A stereoselective dicyanative 5‐exo‐ and 6‐endo‐cyclization using various enynes has been investigated. The mode of cyclization is critically controlled by the structure of the substrates. For example, N‐allyl derivatives prefer 5‐exo‐cyclization, while methacryloyl amides are transformed to the corresponding lactams with tetra‐substituted carbons at the alpha‐position via 6‐endo‐cyclization. Both reactions include syn‐cyanopalladation to carboncarbon triple bonds in the initial step, and sequential cyclization followed by reductive elimination in one operation enables the construction of the highly functionalized nitrogen heterocycles. The scope of suitable substrates and a proposed mechanism are also described. 相似文献
The highly catalytic asymmetric α‐hydroxylation of 1‐tetralone‐derived β‐keto esters and β‐keto amides using tert‐butyl hydroperoxide (TBHP) as the oxidant was realized by a chiral N,N′‐dioxide‐magnesium ditriflate [Mg(OTf)2] complex. A series of corresponding chiral α‐hydroxy dicarbonyl compounds was obtained in excellent yields (up to 99%) with excellent enantioselectivities (up to 98% ee). The products were easily transformed into useful building blocks and the precursor of daunomycin was achieved in an asymmetric catalytic way for the first time. 相似文献
Highly modular chiral amino diol derivatives have been used as organocatalysts in the enantioselective α‐chlorination of cyclic β‐keto esters. Optimization of the catalyst structure and the reaction conditions has allowed the synthesis of optically active α‐chlorinated products with high enantioselectivities (up to 96% ee) using inexpensive commercially available N‐chlorosuccinimide (NCS) as the chlorine source under mild conditions. 相似文献
Compound 20 , a pseudoenantiomer of β‐isocupreidine (β‐ICD), was synthesized from quinine employing a Barton reaction of nitrosyl ester 13 and acid‐catalyzed cyclization of carbinol 18 as key steps. The Baylis–Hillman reaction of benzaldehyde, p‐nitrobenzaldehyde, and hydrocinnamaldehyde with 1,1,1,3,3,3‐hexafluoroisopropyl acrylate (HFIPA) using 20 as a chiral amine catalyst was found to give the corresponding S‐enriched adducts in high optical purity (>91% ee) in contrast to the β‐ICD‐catalyzed reaction which affords R‐enriched adducts. This result suggests that compound 20 can serve as an enantiocomplementary catalyst of β‐ICD in the asymmetric Baylis–Hillman reaction of aldehydes with HFIPA. 相似文献
The highly enantioselective organo‐co‐catalytic aza‐Morita–Baylis–Hillman (MBH)‐type reaction between N‐carbamate‐protected imines and α,β‐unsaturated aldehydes has been developed. The organic co‐catalytic system of proline and 1,4‐diazabicyclo[2.2.2]octane (DABCO) enables the asymmetric synthesis of the corresponding N‐Boc‐ and N‐Cbz‐protected β‐amino‐α‐alkylidene‐aldehydes in good to high yields and up to 99% ee. In the case of aza‐MBH‐type addition of enals to phenylprop‐2‐ene‐1‐imines, the co‐catalytic reaction exhibits excellent 1,2‐selectivity. The organo‐co‐catalytic aza‐MBH‐type reaction can also be performed by the direct highly enantioselective addition of α,β‐unsaturated aldehydes to bench‐stable N‐carbamate‐protected α‐amidosulfones to give the corresponding β‐amino‐α‐alkylidene‐aldehydes with up to 99% ee. The organo‐co‐catalytic aza‐MBH‐type reaction is also an expeditious entry to nearly enantiomerically pure β‐amino‐α‐alkylidene‐amino acids and β‐amino‐α‐alkylidene‐lactams (99% ee). The mechanism and stereochemistry of the chiral amine and DABCO co‐catalyzed aza‐MBH‐type reaction are also discussed. 相似文献
A series of sialyl‐Lewis X tetrasaccharide analogs 1a — d was prepared using a combined chemical and enzymatic approach. Sialic acid analogs 5b , c were obtained from 2‐azido mannose 4c or 2‐deoxy mannose 4b and pyruvate by an aldolase reaction and converted to the protected thioglycosides 3b , c that served as sialyl donors for the Lewis X acceptor trisaccharide 2 . The resulting sialyl‐Lewis X tetrasaccharides 8a — c were deprotected by deacylation and saponification of the methyl ester. Debenzylation was achieved by careful transfer hydrogenation in the presence of formic acid or ammonium formate as a hydrogen source. Three sialyl‐Lewis X derivatives 1a — c were thus obtained and the parent compound 1a was further modified by alkaline hydrolysis of the two acetamides to give the lyso sialyl‐Lewis X derivative 1d . The four sialyl‐Lewis X tetrasaccharides 1a — d were tested for their binding affinity to E and P‐selectin with the lyso sialyl‐Lewis X derivative 1d showing the highest inhibitory potency for both lectins. 相似文献
The phenylalanine aminomutase (PAM) from Taxus chinensis catalyses the conversion of α‐phenylalanine to β‐phenylalanine, an important step in the biosynthesis of the N‐benzoyl phenylisoserinoyl side‐chain of the anticancer drug taxol. Mechanistic studies on PAM have suggested that (E)‐cinnamic acid is an intermediate in the mutase reaction and that it can be released from the enzyme's active site. Here we describe a novel synthetic strategy that is based on the finding that ring‐substituted (E)‐cinnamic acids can serve as a substrate in PAM‐catalysed ammonia addition reactions for the biocatalytic production of several important β‐amino acids. The enzyme has a broad substrate range and a high enantioselectivity with cinnamic acid derivatives; this allows the synthesis of several non‐natural aromatic α‐ and β‐amino acids in excellent enantiomeric excess (ee >99 %). The internal 5‐methylene‐3,5‐dihydroimidazol‐4‐one (MIO) cofactor is essential for the PAM‐catalysed amination reactions. The regioselectivity of amination reactions was influenced by the nature of the ring substituent. 相似文献
De‐N‐acetylases of β‐(1→6)‐D ‐N‐acetylglucosamine polymers (PNAG) and β‐(1→4)‐D ‐N‐acetylglucosamine residues in peptidoglycan are attractive targets for antimicrobial agents. PNAG de‐N‐acetylases are necessary for biofilm formation in numerous pathogenic bacteria. Peptidoglycan de‐N‐acetylation facilitates bacterial evasion of innate immune defenses. To target these enzymes, transition‐state analogue inhibitors containing a methylphosphonamidate have been synthesized through a direct Staudinger–phosphonite reaction. The inhibitors were tested on purified PgaB, a PNAG de‐N‐acetylase from Escherichia coli, and PgdA, a peptidoglycan de‐N‐acetylase from Streptococcus pneumonia. Herein, we describe the most potent inhibitor of peptidoglycan de‐N‐acetylases reported to date (Ki=80 μM ). The minimal inhibition of PgaB observed provides insight into key structural and functional differences in these enzymes that will need to be considered during the development of future inhibitors. 相似文献
A novel enzymatic production system of optically pure β‐hydroxy α‐amino acids was developed. Two enzymes were used for the system: an N‐succinyl L ‐amino acid β‐hydroxylase (SadA) belonging to the iron(II)/α‐ketoglutarate‐dependent dioxygenase superfamily and an N‐succinyl L ‐amino acid desuccinylase (LasA). The genes encoding the two enzymes are part of a gene set responsible for the biosynthesis of peptidyl compounds found in the Burkholderia ambifaria AMMD genome. SadA stereoselectively hydroxylated several N‐succinyl aliphatic L ‐amino acids and produced N‐succinyl β‐hydroxy L ‐amino acids, such as N‐succinyl‐L ‐β‐hydroxyvaline, N‐succinyl‐L ‐threonine, (2S,3R)‐N‐succinyl‐L ‐β‐hydroxyisoleucine, and N‐succinyl‐L ‐threo‐β‐hydroxyleucine. LasA catalyzed the desuccinylation of various N‐succinyl‐L ‐amino acids. Surprisingly, LasA is the first amide bond‐forming enzyme belonging to the amidohydrolase superfamily, and has succinylation activity towards the amino group of L ‐leucine. By combining SadA and LasA in a preparative scale production using N‐succinyl‐L ‐leucine as substrate, 2.3 mmol of L ‐threo‐β‐hydroxyleucine were successfully produced with 93% conversion and over 99% of diastereomeric excess. Consequently, the new production system described in this study has advantages in optical purity and reaction efficiency for application in the mass production of several β‐hydroxy α‐amino acids.
A series of 1,5‐dideoxy‐1,5‐imino‐(l )‐ribitol (DIR) derivatives carrying alkyl or functionalized alkyl groups were prepared and investigated as glycosidase inhibitors. These compounds were designed as simplified 4‐epi‐isofagomine (4‐epi‐IFG) mimics and were expected to behave as selective inhibitors of β‐galactosidases. All compounds were indeed found to be highly selective for β‐galactosidases versus α‐glycosidases, as they generally did not inhibit coffee bean α‐galactosidase or other α‐glycosidases. Some compounds were also found to be inhibitors of almond β‐glucosidase. The N‐alkyl DIR derivatives were only modest inhibitors of bovine β‐galactosidase, with IC50 values in the 30–700 μm range. Likewise, imino‐l ‐ribitol substituted at the C1 position was found to be a weak inhibitor of this enzyme. In contrast, alkyl substitution at C5 resulted in enhanced β‐galactosidase inhibitory activity by a factor of up to 1000, with at least six carbon atoms in the alkyl substituent. Remarkably, the ‘pseudo‐anomeric’ configuration in this series does not appear to play a role. Human lysosomal β‐galactosidase from leukocyte lysate was, however, poorly inhibited by all iminoribitol derivatives tested (IC50 values in the 100 μm range), while 4‐epi‐IFG was a good inhibitor of this enzyme. Two compounds were evaluated as pharmacological chaperones for a GM1‐gangliosidosis cell line (R301Q mutation) and were found to enhance the mutant enzyme activity by factors up to 2.7‐fold. 相似文献