Specificity of Donor Structures for endo‐β‐N‐Acetylglucosaminidase‐Catalyzed Transglycosylation Reactions

To demonstrate the structural specificity of the glycosyl donor for the transglycosylation reaction by using endo‐β‐N‐acetylglucosaminidase from Mucor hiemalis (endo‐M), a series of tetrasaccharide oxazoline derivatives was synthesized. These derivatives correspond to the core structure of an asparagine‐linked glycoprotein glycan with a β‐mannose unit of a non‐natural‐type monosaccharide, including β‐glucose, β‐galactose, and β‐talose in place of the β‐mannose moiety. The transglycosylation activity of wildtype (WT) endo‐M and two mutants, N175Q and N175A, was examined by using these tetrasaccharide donors with p‐nitrophenyl N‐acetylglucosaminide (GlcNAc‐pNp). The essential configuration of the hydroxy group for the transglycosylation reaction was determined. On the basis of these results, the transglycosylation reaction was investigated by using chemically modified donors, and transglycosylated products were successfully obtained.     

Loop Protein Engineering for Improved Transglycosylation Activity of a β‐N‐Acetylhexosaminidase

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, HEX1_GTEPG, 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 HEX1_GTEPG.     

N‐Benzyl Substitution of Polyhydroxypyrrolidines: The Way to Selective Inhibitors of Golgi α‐Mannosidase II

Inhibition of the biosynthesis of complex N‐glycans in the Golgi apparatus influences progress of tumor growth and metastasis. Golgi α‐mannosidase II (GMII) has become a therapeutic target for drugs with anticancer activities. One critical task for successful application of GMII drugs in medical treatments is to decrease their unwanted co‐inhibition of lysosomal α‐mannosidase (LMan), a weakness of all known potent GMII inhibitors. A series of novel N‐substituted polyhydroxypyrrolidines was synthesized and tested with modeled GH38 α‐mannosidases from Drosophila melanogaster (GMIIb and LManII). The most potent structures inhibited GMIIb (K_i=50–76 μm , as determined by enzyme assays) with a significant selectivity index of IC₅₀(LManII)/IC₅₀(GMIIb) >100. These compounds also showed inhibitory activities in in vitro assays with cancer cell lines (leukemia, IC₅₀=92–200 μm ) and low cytotoxic activities in normal fibroblast cell lines (IC₅₀>200 μm ). In addition, they did not show any significant inhibitory activity toward GH47 Aspergillus saitoiα1,2‐mannosidase. An appropriate stereo configuration of hydroxymethyl and benzyl functional groups on the pyrrolidine ring of the inhibitor may lead to an inhibitor with the required selectivity for the active site of a target α‐mannosidase.     

Characterization of a Galactosynthase Derived from Bacillus circulans β‐Galactosidase: Facile Synthesis of D‐Lacto‐ and D‐Galacto‐N‐bioside

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.     

α‐L‐Rhamnosyl‐β‐D‐glucosidase (Rutinosidase) from Aspergillus niger: Characterization and Synthetic Potential of a Novel Diglycosidase

We report the first heterologous production of a fungal rutinosidase (6‐O‐α‐L ‐rhamnopyranosyl‐β‐D ‐glucopyranosidase) in Pichia pastoris. The recombinant rutinosidase was purified from the culture medium to apparent homogeneity and biochemically characterized. The enzyme reacts with rutin and cleaves the glycosidic linkage between the disaccharide rutinose and the aglycone. Furthermore, it exhibits high transglycosylation activity, transferring rutinose from rutin as a glycosyl donor onto various alcohols and phenols. The utility of the recombinant rutinosidase was demonstrated by its use for the synthesis of a broad spectrum of rutinosides of primary (saturated and unsaturated), secondary, acyclic and phenolic alcohols as well as for the preparation of free rutinose. Moreover, the α‐L ‐rhamnosidase‐catalyzed synthesis of a chromogenic substrate for a rutinosidase assay – para‐nitrophenyl β‐rutinoside – is described.

     

Structural Investigation of the Binding of 5‐Substituted Swainsonine Analogues to Golgi α‐Mannosidase II

Golgi α‐mannosidase II (GMII) is a key enzyme in the N‐glycosylation pathway and is a potential target for cancer chemotherapy. The natural product swainsonine is a potent inhibitor of GMII. In this paper we characterize the binding of 5α‐substituted swainsonine analogues to the soluble catalytic domain of Drosophila GMII by X‐ray crystallography. These inhibitors enjoy an advantage over previously reported GMII inhibitors in that they did not significantly decrease the inhibitory potential of the swainsonine head‐group. The phenyl groups of these analogues occupy a portion of the binding site not previously seen to be populated with either substrate analogues or other inhibitors and they form novel hydrophobic interactions. They displace a well‐organized water cluster, but the presence of a C(10) carbonyl allows the reestablishment of important hydrogen bonds. Already approximately tenfold more active against the Golgi enzyme than the lysosomal enzyme, these inhibitors offer the potential of being extended into the N‐acetylglucosamine binding site of GMII for the creation of even more potent and selective GMII inhibitors.     

MgCl2‐supported TiCl4 catalysts containing diethyl norbornene‐2,3‐dicarboxylate internal electron donor for 1‐butene polymerization: Effects of internal electron donor configuration

Three isomeric 5‐norbornene‐2,3‐dicarboxylic acid diethyl ester (NDDE) with endo‐, exo‐, and trans‐configuration have been synthesized and employed as internal electron donors (IED) in 1‐butene polymerization over magnesium chloride supported Ziegler–Natta catalysts. It was found that the configuration of NDDE plays a key role in tuning the catalyst activity, stereospecificity, molecular weight (MW), and polydispersity index (PDI) of resulting poly(1‐butene). The type of catalyst with cis‐5‐norbornene‐endo‐2,3‐dicarboxylic acid diethyl ester as IED shows the highest catalyst activity, while catalyst with trans‐NDDE as IED yields the poly(1‐butene) with the highest MW and the most broad PDI. IR results showed that the NDDE with endo‐, exo‐, and trans‐configuration have different coordination way to MgCl₂, subsequently affecting the catalysts performance. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40758.     

The Molecular Basis of Inhibition of Golgi α‐Mannosidase II by Mannostatin A

Mannostatin A is a potent inhibitor of the mannose‐trimming enzyme, Golgi α‐mannosidase II (GMII), which acts late in the N‐glycan processing pathway. Inhibition of this enzyme provides a route to blocking the transformation‐associated changes in cancer cell surface oligosaccharide structures. Here, we report on the synthesis of new Mannostatin derivatives and analyze their binding in the active site of Drosophila GMII by X‐ray crystallography. The results indicate that the interaction with the backbone carbonyl of Arg876 is crucial to the high potency of the inhibitor—an effect enhanced by the hydrophobic interaction between the thiomethyl group and an aromatic pocket vicinal to the cleavage site. The various structures indicate that differences in the hydration of protein–ligand complexes are also important determinants of plasticity as well as selectivity of inhibitor binding.     

Unusual CC Bond Isomerization of an α,β‐Unsaturated γ‐Butyrolactone Catalysed by Flavoproteins from the Old Yellow Enzyme Family

An unexpected, redox‐neutral C?C bond isomerization of a γ‐butyrolactone bearing an exo‐methylene unit to the thermodynamically more favoured endo isomer (k_cat=0.076 s^?1) catalysed by flavoproteins from the Old Yellow Enzyme family was discovered. Theoretical calculations and kinetic data support a mechanism through which the isomerization proceeds through FMN‐mediated hydride addition onto exo‐Cβ, followed by hydride abstraction from endo‐Cβ′, which is in line with the well‐established C?C bond bioreduction of OYEs. This new isomerase activity enriches the catalytic versatility of ene‐reductases.     

Synthesis of 6‐Kestose using an Efficient β‐Fructofuranosidase Engineered by Directed Evolution

The β‐fructofuranosidase (Ffase) from Schwanniomyces occidentalis (Ffase‐Leu196 variant) was subjected to four cycles of directed evolution to enhance the transglycosylation activity for the synthesis of β‐(2 → 6) linked fructooligosaccharides (FOS). With a 5.5‐fold improvement in fructose transferase activity over the parental type and greater selectivity for the synthesis of 6‐kestose (up to 73% of the total FOS), the mutants doubled FOS synthesis to 168 g L.⁻¹ Whilst the F523V and H510P mutations were located at the C‐terminus of the protein, mutations Q78L and I203L were associated with the acidic catalytic triad where they modified its interactions with the surrounding residues, in turn varying the hydrolase and transferase rates.     

Synthesis and biological activity of medium molecular weight polymers containing 3,6‐endo‐methylene‐1,2,3,6‐tetrahydrophthalimidobutanoyl‐5‐fluorouracil

A new monomer, 3,6‐endo‐methylene‐1,2,3,6‐tetrahydrophthalimidobutanoyl‐5‐fluorouracil (ETBFU), was synthesized by reaction of 3,6‐endo‐methylene‐1,2,3,6‐tetrahydrophthalimidobutanoyl chloride and 5‐fluorouracil. The homopolymer of ETBFU and its copolymers with acrylic acid (AA) or vinyl acetate (VAc) were prepared by photopolymerization using 2,2‐dimethoxy‐2‐phenylacetophenone as an initiator at 25 °C. The synthesized ETBFU and its polymers were identified by FTIR, ¹H NMR and ¹³C NMR spectroscopies. The ETBFU content in poly(ETBFU‐co‐AA) and poly(ETBFU‐co‐VAc) was 43 and 14 mol%, respectively. The apparent number‐average molecular weight (M_n) of the polymers determined by GPC ranged from 8400 to 11 300. The in vitro cytotoxicity of the samples against mouse mammary carcinoma (FM3A), mouse leukaemia (P388), and human histiocytic lymphoma (U937) cancer cell lines decreased in the order 5‐FU ≥ ETBFU > poly(ETBFU) > poly(ETBFU‐co‐AA) > poly(ETBFU‐co‐VAc). The in vivo antitumour activity of the polymers against Balb/C mice bearing sarcoma 180 tumour cells was greater than that of 5‐fluorouracil at all doses tested. © 2000 Society of Chemical Industry     

Silicon Fluorides for Acid‐Base Catalysis in Glycosidations

Adduct formation between alcohols as glycosyl acceptors and phenylsilicon trifluoride (PhSiF₃) as catalyst permits acid‐base‐atalyzed glycosidations with O‐glycosyl trichloroacetimidates as glycosyl donors. In this way, from various glycosyl donors and acceptors 1,2‐trans‐ and some 1,2‐cis‐glycosides could be obtained with high anomeric selectivity. A preference for an intramolecular bimolecular nucleophilic substitution (S_N2‐type) reaction course with concomitant donor and acceptor activation is supported by the results.     

Entrapment of urease in poly(1‐vinyl imidazole)/poly(2‐acrylamido‐2‐methyl‐1‐propanesulfonic acid) network

In this article, urease was immobilized in a conducting network via complexation of poly(1‐vinyl imidazole) (PVI) with poly(2‐acrylamido‐2‐methyl‐1‐propanesulfonic acid) (PAMPS). The preparation method for the polymer network was adjusted by using Fourier transform infrared (FTIR) spectroscopy. A scanning electron microscope (SEM) study revealed that enzyme immobilization had a strong effect on film morphology. The proton conductivity of the PVI/PAMPS network was measured via impedance spectroscopy, under humidified conditions. The basic characteristics (Michealis‐Menten constants, pH_opt, pH_stability, T_opt, T_stability, reusability, and storage stability) of the immobilized urease were determined. The obtained results showed that the PAA/PVI polymer network was suitable for enzyme immobilization. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

A Kinase‐Independent One‐Pot Multienzyme Cascade for an Expedient Synthesis of Guanosine 5′‐Diphospho‐d‐mannose

Biomimetic synthesis routes towards the important natural d ‐mannosyl donor guanosine 5′‐diphospho‐d ‐mannose (GDP‐Man) rely on kinase‐catalyzed nucleotide triphosphate (NTP)‐dependent phosphorylations of d ‐mannose (Man), to give d ‐mannose 6‐phosphate or α‐d ‐mannose 1‐phosphate (αMan 1‐P) as an intermediate product. A GDP‐Man synthesis not requiring the kinase/NTP system would be practical and cost‐effective. Here, we have developed a multienzyme cascade towards GDP‐Man, characterized in that αMan 1‐P was obtained by a diastereoselective phosphatase‐catalyzed phosphorylation of Man. α‐d ‐Glucose 1‐phosphate (αGlc 1‐P), prepared in situ through phosphorylase‐catalyzed conversion of sucrose in the presence of inorganic phosphate, was used as an expedient phosphoryl donor. The incipient αMan 1‐P and guanosine triphosphate (GTP) were converted into GDP‐Man by a highly manno compared to gluco selective nucleotidyltransferase. Pyrophosphatase was additionally required to hydrolyze the pyrophosphate released from the GTP, thus driving the reaction towards GDP‐Man. The enzymatic cascade was operated with the αMan 1‐P and the GDP‐Man formation decoupled from one another (sequential mode) or having all steps run concurrently (simultaneous mode). Detailed time course analysis revealed that kinetic pull due to the constant removal of the intermediate αMan 1‐P in simultaneous‐mode reactions was important to promote phosphorylation of Man from αGlc 1‐P in high efficiency, avoiding loss of sugar 1‐phosphates by hydrolysis. Under optimized conditions for the one‐pot transformation involving four enzymes, 100 mM (67 g L⁻¹) GDP‐Man was prepared from 140 mM sucrose and phosphate, using 400 mM Man as the phosphoryl acceptor. The product was recovered by anion‐exchange and size‐exclusion chromatography in ≥95% purity in about 50% yield (100 mg). These results demonstrate for the first time the practical use of a phosphorylase‐phosphatase combi‐catalyst as an alternative to the canonical kinase for the anomeric phosphorylation of the sugar substrate in nucleoside diphospho‐sugar synthesis. Phosphorylation from inorganic phosphate via the intermediate αGlc 1‐P rather than from NTP, particularly GTP, appears advantageous specifically in cases where the sugar acceptor is a bulk commodity that can be applied in suitable excess to the phosphatase reaction.

     

Synthesis and Structure–Activity Relationship Studies of 2‐(1,3,4‐Oxadiazole‐2(3H)‐thione)‐3‐amino‐5‐arylthieno[2,3‐b]pyridines as Inhibitors of DRAK2

In recent years, DAPK‐related apoptosis‐inducing protein kinase 2 (DRAK2) has emerged as a promising target for the treatment of a variety of autoimmune diseases and for the prevention of graft rejection after organ transplantation. However, medicinal chemistry optimization campaigns for the discovery of novel small‐molecule inhibitors of DRAK2 have not yet been published. Screening of a proprietary compound library led to the discovery of a benzothiophene analogue that displays an affinity constant (K_d) value of 0.25 μM . Variation of the core scaffold and of the substitution pattern afforded a series of 5‐arylthieno[2,3‐b]pyridines with strong binding affinity (K_d=0.008 μM for the most potent representative). These compounds also show promising activity in a functional biochemical DRAK2 enzyme assay, with an IC₅₀ value of 0.029 μM for the most potent congener. Selectivity profiling of the most potent compounds revealed that they lack selectivity within the DAPK family of kinases. However, one of the less potent analogues is a selective ligand for DRAK2 and can be used as starting point for the synthesis of selective and potent DRAK2 inhibitors.     

Catalytic Dicyanative 5‐exo‐ and 6‐endo‐Cyclization Triggered by Cyanopalladation of Alkynes

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 carboncarbon 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.     

Synthesis and Analysis of Specific Covalent Inhibitors of endo‐Xyloglucanases

A series of N‐bromoacetylglycosylamines and bromoketone C‐glycosides were synthesised from complex xyloglucan oligosaccharide (XyGO) scaffolds as specific active‐site affinity labels for endo‐xyloglucanases. Compounds based on XXXG (Xyl₃Glc₄) and XLLG (Xyl₃Glc₄Gal₂) oligosaccharides exhibited strikingly higher affinities and higher rates of irreversible inhibition than known cellobiosyl and new lactosyl disaccharide congeners when tested with endo‐xyloglucanases from two distinct glycoside hydrolase (GH) families. Intact‐protein mass spectrometry indicated that inactivation with XyGO derivatives generally resulted in a 1:1 labelling stoichiometry. Together, these results indicate that XyGO‐based affinity reagents have significant potential as inhibitors and proteomic reagents for the identification and analysis of diverse xyloglucan‐active enzymes in nature, to facilitate industrial enzyme applications.     

Endoplasmic Reticulum (ER)‐Targeted,Galectin‐Mediated Retrograde Transport by Using a HaloTag Carrier Protein

Investigations into metabolic processes within the cell have often relied on genetic methods such as forced expression and knockout or knockdown techniques. An alternative approach would be introducing a molecule into the desired location inside the cell. To translocate compounds from outside cells into the endoplasmic reticulum (ER), we constructed a delivery carrier protein. This comprised N‐terminal galectin‐1 for cell‐surface binding (G1), a protease cleavable sequence (ps), a HaloTag domain for attaching exogenous compounds (Halo), and a C‐terminal KDEL sequence for ER retention. Fluorescently labeled G1‐ps‐Halo‐KDEL passed through the Golgi apparatus and reached the ER. By using Man₉GlcNAc₂‐BODIPY as a cargo compound, the carrier protein was also delivered into the ER with concomitant processing of mannose to Man_5,6, by the ER‐resident α1,2‐mannosidase. G1‐ps‐Halo‐KDEL might serve as a new type of delivery carrier protein to direct compounds into the ER.     

Improved conversion of 6‐endo‐tosyloxybicyclo[2.2.2]octan‐2‐ones into 6‐exo‐acetoxy and 6‐exo‐benzoyloxybicyclo[2.2.2]octan‐2‐ones

The reaction conditions for the conversion of 6‐endo‐tosyloxybicyclo[2.2.2]octan‐2‐one ( 7b ) into 6‐exo‐acetoxy ( 8b ) and 6‐exo‐benzoyloxybicyclo[2.2.2]octan‐2‐one ( 8a ), respectively, were improved. Thus known 6‐endo‐tosyloxy‐bicyclo[2.2.2]octan‐2‐ones (+)‐(1RS,6SR,8SR,11RS)‐11‐[(4‐toluenesulfonyl)oxy]tricyclo[6.2.2.0^1,6]dodecan‐9‐one ( 1a ), 13‐methyl‐15‐oxo‐9β,13b‐ethano‐9β‐podocarpan‐12β‐yl‐4‐toluenesulfonate ( 3a ), and methyl (13R)‐16‐oxo‐13‐[(4‐tolylsulfonyl)oxy]‐17‐noratisan‐18‐oate ( 5 ), were converted,in comparable yields, as previously recorded, but much shorter times, into (+)‐(1RS,6SR,8SR,11SR)‐11‐(benzoyloxy) tricyclo[6.2.2.0^1,6]dodecan‐9‐one ( 2 ), 13‐methyl‐15‐oxo‐9β,13β‐ethano‐9β‐podocarpan‐12α‐yl benzoate ( 4 ), and methyl (13S)‐13‐(benzoyloxy)‐16‐oxo‐17‐noratisan‐18‐oate ( 6 ), respectively.     

Application of chitosan‐entrapped β‐galactosidase in a packed‐bed reactor system

The model enzyme β‐galactosidase was entrapped in chitosan gel beads and tested for hydrolytic activity and its potential for application in a packed‐bed reactor. The chitosan beads had an enzyme entrapment efficiency of 59% and retained 56% of the enzyme activity of the free enzyme. The Michaelis constant (K_m) was 0.0086 and 0.011 μmol/mL for the free and immobilized enzymes, respectively. The maximum velocity of the reaction (V_max) was 285.7 and 55.25 μmol mL^?1 min^?1 for the free and immobilized enzymes, respectively. In pH stability tests, the immobilized enzyme exhibited a greater range of pH stability and shifted to include a more acidic pH optimum, compared to that of the free enzyme. A 2.54 × 16.51‐cm tubular reactor was constructed to hold 300 mL of chitosan‐immobilized enzyme. A full‐factorial test design was implemented to test the effect of substrate flow (20 and 100 mL/min), concentration (0.0015 and 0.003M), and repeated use of the test bed on efficiency of the system. Parameters were analyzed using repeated‐measures analysis of variance. Flow (p < 0.05) and concentration (p < 0.05) significantly affected substrate conversion, as did the interaction progressing from Run 1 to Run 2 on a bed (p < 0.05). Reactor stability tests indicated that the packed‐bed reactor continued to convert substrate for more than 12 h with a minimal reduction in conversion efficiency. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 91: 1294–1299, 2004