Synthesis of fused pyran-carbahexopyranoses as glycosidase inhibitors

Synthesis of polyhydroxylated oxabicyclo[4,4,0]decanes, which constitute a new family of annulated carbasugars, has been accomplished in a stereoselective manner by employing readily available 1,2-anhydro-3,4,6-tri-O-benzyl-α-d-glycopyranoses.     

Unusual Role of the 3-OH Group of Oligosaccharide Substrates in the Mechanism of Bacillus 1,3-1,4-β-glucanase

Abstract

In the mechanism of retaining β-glycosidases, the 2-hydroxyl group of the substrate in the monosaccharyl unit involved in catalysis (subsite -1) is beleived to play an important role through hydrogen bonding interactions with protein residues that are optimized at the transition state. Commonly, removal of the 2-OH group of the substrate results in a 10–12 kcal·mol^-1 transition state destabilization. However, this effect seems not to be general as reported here for Bacillus 1,3-1,4-β-glucanase, a family 16 retaining endo-glycosidase. A p-nitrophenol 2-deosxy tetrasaccharide substrate was synthesized to probe the involvement of the 2-OH group in catalysis. Comparative kinetics with wild-type and subsite +1 mutants show that the 2-deoxy analog is a better substrate than the corresponding 2-hydroxy substrate. It is tentatively proposed that the 2-deoxy analog adopts a different conformation upon binding that compensates for the lack of the 2-OH substituent.     

Crystal Structure of the Chlamydomonas Starch Debranching Enzyme Isoamylase ISA1 Reveals Insights into the Mechanism of Branch Trimming and Complex Assembly

The starch debranching enzymes isoamylase 1 and 2 (ISA1 and ISA2) are known to exist in a large complex and are involved in the biosynthesis and crystallization of starch. It is suggested that the function of the complex is to remove misplaced branches of growing amylopectin molecules, which would otherwise prevent the association and crystallization of adjacent linear chains. Here, we investigate the function of ISA1 and ISA2 from starch producing alga Chlamydomonas. Through complementation studies, we confirm that the STA8 locus encodes for ISA2 and sta8 mutants lack the ISA1·ISA2 heteromeric complex. However, mutants retain a functional dimeric ISA1 that is able to partly sustain starch synthesis in vivo. To better characterize ISA1, we have overexpressed and purified ISA1 from Chlamydomonas reinhardtii (CrISA1) and solved the crystal structure to 2.3 Å and in complex with maltoheptaose to 2.4 Å. Analysis of the homodimeric CrISA1 structure reveals a unique elongated structure with monomers connected end-to-end. The crystal complex reveals details about the mechanism of branch binding that explains the low activity of CrISA1 toward tightly spaced branches and reveals the presence of additional secondary surface carbohydrate binding sites.     

The synthesis of novel chromogenic enzyme substrates for detection of bacterial glycosidases and their applications in diagnostic microbiology

The preparation and evaluation of chromogenic substrates for detecting bacterial glycosidase enzymes is reported. These substrates are monoglycoside derivatives of the metal chelators catechol, 2,3-dihydroxynaphthalene (DHN) and 6,7-dibromo-2,3-dihydroxynaphthalene (6,7-dibromo-DHN). When hydrolysed by appropriate bacterial enzymes these substrates produced coloured chelates in the presence of ammonium iron(III) citrate, thus enabling bacterial detection. A β-d-riboside of DHN and a β-d-glucuronide derivative of 6,7-dibromo-DHN were particularly effective for the detection of S. aureus and E. coli respectively.     

Synthesis and glycosidase inhibitory activities of chain-modified analogues of the glycosidase inhibitors salacinol and blintol

The synthesis of chain-modified analogues of the naturally-occurring glycosidase inhibitor, salacinol, and its selenium analogue, blintol is described. The modification consists of a frame shift of the sulfate moiety by one carbon atom in the zwitterionic structures as well as an extension of the acyclic chain to five carbons. The target molecules were synthesized by alkylation of 1,4-anhydro-2,3,5-tri-O-p-methoxybenzyl-4-thio (or seleno)-D-arabinitol at the ring heteroatom by 2,3,5-tri-O-p-methoxybenzyl D- or L-xylitol-1,4-cyclic sulfate, followed by deprotection with trifluoroacetic acid. Two of the four compounds inhibit recombinant human maltase glucoamylase, one of the key intestinal enzymes involved in the breakdown of glucose oligosaccharides in the small intestine, with Ki values of 20+/-4 and 53+/-5 microM.     

Synthesis of aza- and thia-spiroheterocycles and attempted synthesis of spiro sulfonium compounds related to salacinol

The synthesis of aza- and thia-spiroheterocycles and the attempted synthesis of spiro sulfonium compounds related to salacinol are described. The binding of the nanomolar inhibitor swainsonine to Drosophila Golgi alpha-mannosidase II (dGMII) involves a large contribution of interactions between the six-membered ring of the inhibitor and the hydrophobic pocket within the enzyme active site. Salacinol, a naturally occurring sulfonium ion, is one of the active principles in the aqueous extracts of Salacia reticulata that are traditionally used in Sri Lanka and India for the treatment of diabetes. Spiro aza- and thia-heterocycles and a spiro analogue of salacinol were designed with the expectation that the hydrocarbon portions would make hydrophobic contributions to binding. The former sets of compounds were synthesized successfully but the salacinol analogue proved to be elusive. The stereochemistry of the final compounds was determined by means of 1D-NOESY experiments. The aza- and thia-heterocycles were not effective inhibitors of Golgi alpha-mannosidase II or human maltase glucoamylase.     

Synthesis and NMR experiments of (4,5,6-13C)-deoxymannojirimycin. A new entry to 13C-labeled glycosidase inhibitors

The synthesis of (4,5,6-13C)-deoxymannojirimycin is described. The route employed is based on Sharpless asymmetric epoxidation of (1,2,3-13C)(E)-2,4-pentadien-1-ol and uses ring-closing metathesis as a key step. The labeled compound may be easily used for protein-binding experiments using NMR spectroscopic methods.     

Synthesis of pentopyranosyl-containing thiodisaccharides. Inhibitory activity against β-glycosidases

β-(1→4)-Thiodisaccharides formed by a pentopyranose unit as reducing or non reducing end have been synthesized using a sugar enone derived from a hexose or pentose as Michael acceptor of a 1-thiopentopyranose or 1-thiohexopyranose derivatives. Thus, 2-propyl per-O-acetyl-3-deoxy-4-S-(β-d-Xylp)-4-thiohexopyranosid-2-ulose (3) and benzyl per-O-acetyl-3-deoxy-4-S-(β-d-Galp)-4-thiopentopyranosid-2-ulose (11) were obtained in almost quantitative yields. The carbonyl function of these uloses was reduced with NaBH₄ or K-Selectride, and the stereochemical course of the reduction was highly dependent on the reaction temperature, reducing agent and solvent. Unexpectedly, reduction of 3 with NaBH₄–THF at 0 °C gave a 3-deoxy-4-S-(β-d-Xylp)-4-thio-α-d-ribo-hexopyranoside derivative (6) as major product (74% yield), with isomerization of the sulfur-substituted C-4 stereocenter of the pyranone. Reduction of 11 gave always as major product the benzyl 3-deoxy-4-S-(Galp)-4-thio-β-d-threo-pentopyranoside derivative 14, which was the only product isolated (80% yield) in the reduction with K-Selectride in THF at −78 °C. Deprotection of 14 and its epimer at C-2 (13) afforded, respectively the free thiodisaccharides 19 and 18. They displayed strong inhibitory activity against the β-galactosidase from Escherichia coli. Thus, compound 18 proved to be a non-competitive inhibitor of the enzyme (K_i = 0.80 mM), whereas 19 was a mixed-type inhibitor (K_i = 32 μM).     

Glycoside hydrolase family 89 alpha-N-acetylglucosaminidase from Clostridium perfringens specifically acts on GlcNAc alpha1,4Gal beta1R at the non-reducing terminus of O-glycans in gastric mucin

In mammals, α-linked GlcNAc is primarily found in heparan sulfate/heparin and gastric gland mucous cell type mucin. α-N-acetylglucosaminidases (αGNases) belonging to glycoside hydrolase family 89 are widely distributed from bacteria to higher eukaryotes. Human lysosomal αGNase is well known to degrade heparin and heparan sulfate. Here, we reveal the substrate specificity of αGNase (AgnC) from Clostridium perfringens strain 13, a bacterial homolog of human αGNase, by chemically synthesizing a series of disaccharide substrates containing α-linked GlcNAc. AgnC was found to release GlcNAc from GlcNAcα1,4Galβ1pMP and GlcNAcα1pNP substrates (where pMP and pNP represent p-methoxyphenyl and p-nitrophenyl, respectively). AgnC also released GlcNAc from porcine gastric mucin and cell surface mucin. Because AgnC showed no activity against any of the GlcNAcα1,2Galβ1pMP, GlcNAcα1,3Galβ1pMP, GlcNAcα1,6Galβ1pMP, and GlcNAcα1,4GlcAβ1pMP substrates, this enzyme may represent a specific glycosidase required for degrading α-GlcNAc-capped O-glycans of the class III mucin secreted from the stomach and duodenum. Deletion of the C-terminal region containing several carbohydrate-binding module 32 (CBM32) domains significantly reduced the activity for porcine gastric mucin; however, activity against GlcNAcα1,4Galβ1pMP was markedly enhanced. Dot blot and ELISA analyses revealed that the deletion construct containing the C-terminal CBM-C2 to CBM-C6 domains binds strongly to porcine gastric mucin. Consequently, tandem CBM32 domains located near the C terminus of AgnC should function by increasing the affinity for branched or clustered α-GlcNAc-containing glycans. The agnC gene-disrupted strain showed significantly reduced growth on the class III mucin-containing medium compared with the wild type strain, suggesting that AgnC might have an important role in dominant growth in intestines.     

Synthesis, structures and DNA binding of ternary transition metal complexes M(II)L with the 2,2′-bipyridylamine (L = p-aminobenzenecarboxylic acid, M = Ni, Cu and Zn)

New ternary transition metal complexes of formulations [Ni(bpa)(p-AB)Cl]_n · 3nH₂O (bpa = 2,2′-bipyridylamine, p-AB = aminobenzenecarboxylic acid) (1), [Cu(bpa)(p-AB)Cl] · H₂O (2), [Zn(bpa)(p-AB)₂] · H₂O (3) are prepared, their structural features are characterized by crystal structural studies, and their DNA binding propensity has been evaluated by fluorescence and viscosity method. In complex 2 and 3, both bpa and p-AB act as the bidentate N and O-donor ligand, respectively. While in complex 1, p-AB acts as a rare tridentate ligand. In the packing pattern of each complex, π-π interaction in their solid state is also described. The complexes show the competitive inhibition of ethidium binding to DNA, and the DNA binding propensity can be reflected as the relative order: 1 > 2 > 3.