Assunta Giordano

Glycosyl Hydrolases and Glycosyltransferases in the Synthesis of Oligosaccharides

Glycobiology and related disciplines have received an enormous interest in recent years as they shed new light on the functional roles of carbohydrates in biological events leading to the understanding of mechanisms of important pathologies and to the development of new therapeutics. Although carbohydrate can be isolated from natural sources, the synthetic strategy plays its own role allowing access to larger quantities of structurally defined material and entry to analogs of naturally occurring structures. Nowadays, the bottleneck in this field is represented by some limitations of the potential of carbohydrate containing molecules because their complex structures make classical chemical synthesis very difficult. The synthesis of oligosaccharides have to face with three main snags: (i) reactivity of the leaving group on the monosaccharide acting as donor; (ii) regioselectivity towards a single hydroxyl group on the acceptor molecule; (iii) stereoselectivity in forming pure anomers of the new glycosidic product. The protecting-group manipulations that are needed for the stereocontrol of the products are demanding procedures thus yields and selectivity are lowered hindering the efficient production of oligosaccharides needed for biological testing. A particular benefit of the renewed interest for sugar chemistry has been the attention of scientific community to the biological methodologies in the production of oligosaccharides, an area which emerged in recent decades. In the carbohydrate field different enzymes were used for diverse purposes but enzymatic strategies for high-yield and stereospecific construction of glycosidic bonds are based on the action of two types of enzymes: glycosyl hydrolases (endo- and exo-glycosidases) and glycosyltransferases. Glycosyl hydrolases in the cells are responsible for the cleavage of glycosidic linkages; the exo-glycosidase are involved for glycan processing during in vivo glycoprotein synthesis. The glycosyltransferases are instead responsible in vivo for the synthesis of most cell-surface glycoconjugates. This review deals with the application of different types of glycosyl hydrolases. Elegant examples concerning the use of genetically modified representatives of glycosyl hydrolases (glycosynthases and thioglycoligases) will be also reported; some recent advances on the use of glycosyltransferases are also included. In compiling this review we were aware of the huge amount of excellent material published in the recent years on this topic, thus we will limit the covering of literature to the last decade. Attention will be devoted to the regioselectivity towards pyranosidic templates and to the yield of reactions. The molecular diversity obtained by the use of enzymes from different biological sources and the biological importance of the compounds synthesized will be focused as well. This review will put in evidence that glycosyl hydrolases and glycosyltransferases for the synthesis of the glycosidic linkages will play a relevant role in the next future as on the bench bio-catalysts for the chemists involved in the synthesis of oligosaccharides of biological interest.

Identification of an Archaeal α-l-Fucosidase Encoded by an Interrupted Gene PRODUCTION OF A FUNCTIONAL ENZYME BY MUTATIONS MIMICKING PROGRAMMED −1 FRAMESHIFTING

The analysis of the complete genome of the thermoacidophilic Archaeon Sulfolobus solfataricusrevealed two open reading frames (ORF), named SSO11867 and SSO3060, interrupted by a −1 frameshift and encoding for the N- and the C-terminal fragments, respectively, of an α-l-fucosidase. We report here that these ORFs are actively transcribed in vivo, and we confirm the presence of the −1 frameshift between them at the cDNA level, explaining why we could not find α-fucosidase activity in S. solfataricus extracts. Detailed analysis of the region of overlap between the two ORFs revealed the presence of the consensus sequence for a programmed −1 frameshifting. Two specific mutations, mimicking this regulative frameshifting event, allow the expression, in Escherichia coli, of a fully active thermophilic and thermostable α-l-fucosidase (EC 3.2.1.51) with micromolar substrate specificity and showing transfucosylating activity. The analysis of the fucosylated products of this enzyme allows, for the first time, assigning a retaining reaction mechanism to family 29 of glycosyl hydrolases. The presence of an α-fucosidase putatively regulated by programmed −1 frameshifting is intriguing both with respect to the regulation of gene expression and, in post-genomic era, for the definition of gene function in Archaea.

SYNTHESIS OF (17R)-17-METHYLINCISTEROL, A HIGHLY DEGRADED MARINE STEROID

Abstract The synthesis of (17 R )-17-methylincisterol, a highly degraded marine steroid, has been achieved starting from vitamin D 2 in 8 steps and in 13% overall yield. Its O-demethyl analog, which is an intermediate in the synthetic sequence, is possibly the true naturally occurring molecule.

Highly Productive Autocondensation and Transglycosylation Reactions with Sulfolobus solfataricus Glycosynthase

Transglycosylation reactions (autocondensation of the substrate or transfer of the glycon donor moiety to different acceptors) with the hyperthermophilic glycosynthase from Sulfolobus solfataricus acting in dilute sodium formate buffer at pH 4.0 are reported; the use of 4‐nitrophenyl β‐glucopyranoside as both donor and acceptor in the self‐transfer reaction and a highly productive reaction with 1.1 M 2‐nitrophenyl β‐glucopyranoside were possible. Interesting effects, governed by the anomeric configuration and lipophilicity of heteroacceptors, on the regioselectivity and yield of reactions were found for the first time with this enzyme and are discussed. The results demonstrate the unexplored synthetic potential of this glycosynthase; the tuning of the reaction conditions and the choice of different donors/acceptors can lead to products of applicative interest.

Synthesis of antifungal N-isoprenyl-indole alkaloids from the fungus Aporpium caryae ☆

Abstract The synthesis of two antifungal alkaloids 1 and 2 is described. It involves the N -isoprenyl-indole brominated key-intermediate 3 prepared by introduction of the isoprenyl group on the indole core itself.

Biochemical characterization of a recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius

The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases (SDRs) superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh gene was heterologously overexpressed in Escherichia coli, and the protein (SaADH) was purified to homogeneity and characterized. SaADH is a tetrameric enzyme consisting of identical 28,978-Da subunits, each composed of 264 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 75°C and a 30-min half-inactivation temperature of ~90°C, and shows good tolerance to common organic solvents. SaADH has a strict requirement for NAD(H) as the coenzyme, and displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and α-keto esters, but is poorly active on aliphatic, cyclic and aromatic alcohols, and shows no activity on aldehydes. The enzyme catalyses the reduction of α-methyl and α-ethyl benzoylformate, and methyl o-chlorobenzoylformate with 100% conversion to methyl (S)-mandelate [17% enantiomeric excess (ee)], ethyl (R)-mandelate (50% ee), and methyl (R)-o-chloromandelate (72% ee), respectively, with an efficient in situ NADH-recycling system which involves glucose and a thermophilic glucose dehydrogenase. This study provides further evidence supporting the critical role of the D37 residue in discriminating NAD(H) from NAD(P)H in members of the SDR superfamily.

Stereochemistry and total synthesis of janolusimide, a tripeptide marine toxin

Abstract The stereochemistry of janolusimide, a lipophilic tripeptide marine toxin, has been fully elucidated by stereoselective synthesis of the lactam component (5S)-3,3′-dimethyl-5-isopropylpyrrolidin-2,4-dione. The peptide was then synthesized in 13 steps and in 0.8% total yield.

Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation

Phosphomannomutase 2 (PMM2) deficiency represents the most frequent type of congenital disorders of glycosylation. For this disease there is no cure at present. The complete loss of phosphomannomutase activity is probably not compatible with life and people affected carry at least one allele with residual activity. We characterized wild‐type PMM2 and its most common hypomorphic mutant, p.F119L, which is associated with a severe phenotype of the disease. We demonstrated that active species is the dimeric enzyme and that the mutation weakens the quaternary structure and, at the same time, affects the activity and the stability of the enzyme. We demonstrated that ligand binding stabilizes both proteins, wild‐type and F119L‐PMM2, and promotes subunit association in vitro. The strongest effects are observed with glucose‐1,6‐bisphosphate (Glc‐1,6‐P2) or with monophosphate glucose in the presence of vanadate. This finding offers a new approach for the treatment of PMM2 deficiency. We propose to enhance Glc‐1,6‐P2 concentration either acting on the metabolic pathways that control its synthesis and degradation or exploiting prodrugs that are able to cross membranes.

Stereoselective synthesis of 4-amino-3-hydroxy-2-methylpentanoic acids: stereochemistry of the amino acid occurring in the marine toxin janolusimide

Abstract Four diastereomers of 4-amino-3-hydroxy-2-methylpentanoic acid, an amino acid constituent of the hexapeptide portion of the antitumor antibiotic bleomycin A 2 , have been stereoselectively synthesized by crotylboration of N -Boc- l -alaninal. The synthesis allowed the assignment of the stereochemistry as 2 R ,3 S ,4 S to the 4-amino-3-hydroxy-2-methylpentanoic acid occurring in the tripeptide marine toxin janolusimide.

Insight into the Stereospecificity of Short-Chain Thermus thermophilus Alcohol Dehydrogenase Showing pro-S Hydride Transfer and Prelog Enantioselectivity

The stereochemistry of the hydride transfer in reactions catalyzed by NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus HB27 was determined by means of (1)H-NMR spectroscopy. The enzyme transfers the pro-S hydrogen of [4R-(2)H]NADH and exhibits Prelog specificity. Enzyme-substrate docking calculations provided structural details about the enantioselectivity of this thermophilic enzyme. These results give additional insights into the diverse active site architectures of the largely versatile short-chain dehydrogenase superfamily enzymes. A feasible protocol for the synthesis of [4R-(2)H]NADH with high yield was also set up by enzymatic oxidation of 2-propanol-d(8) catalyzed by Bacillus stearothermophilus alcohol dehydrogenase.