Large-scale chemoenzymatic synthesis of blood group and tumor-associated poly-N-acetyllactosamine antigens

Poly-N-acetyllactosamines (pLNs) are common terminal sugars of many N- and O-linked glycan structures present in glycoproteins and glycolipids. Utilizing various glycosyltransferases, we developed new and efficient chemoenzymatic methods for the synthesis of pLNs in gram-scale. Specifically, the use of sialyltransferases and fucosyltransferases enabled us to synthesize and purify 24 blood group and tumor-associated pLN derivatives with alpha-(2-->3)- and alpha-(2-->6)-linked sialic acid, as well as with alpha-(1-->2)- and alpha-(1-->3)-linked fucose. All synthesized derivatives were linked to a short 2-azidoethyl spacer for further modification.     

Rare sugars and sugar-based synthons by chemo-enzymatic synthesis

The unique catalytic potential of the fungal enzyme pyranose oxidase was demonstrated by preparative conversions of a variety of carbohydrates, and by extensive chemical characterization of the reaction products with NMR spectroscopy. The studies revealed that POx not only oxidizes most substrates very efficiently but also that POx possesses a glycosyl-transfer potential, producing disaccharides from beta-glycosides of higher alcohols. Although most substrates are oxidized by POx at the C-2 position, several substrates are converted into the 3-keto-derivatives. On the basis of these products, strategies are developed for the convenient production of sugar-derived synthons, rare sugars and fine chemicals by combining biotechnical and chemical methods.     

Large-scale manufacturing of all four 2'-deoxynucleosides via novel strategies including a chemo-enzymatic process

A chemical synthesis of 2-deoxyribose-1-phosphate 2 and its enzymatic conversion into purine 2'-deoxynucleosides (dNus) are shown. Besides the chemo-enzymatic process for purine dNus, a modified process for practical dC preparation is also established. Consequently, a series of practical manufacturing processes of all four dNus have been realized via novel strategies.     

In situ synthesis of artificial lipids

Lipids constitute one of the most enigmatic family of biological molecules. Although the importance of lipids as basic units of compartmental structure and energy storage is well-acknowledged, deciphering the biosynthesis and precise roles of specific lipid species has been challenging. To better understand the structure and function of these biomolecules, there is a burgeoning interest in developing strategies to produce noncanonical lipids in a controlled manner. This review covers recent advances in the area of in situ generation of synthetic lipids. Specifically, we report several approaches that constitute a powerful toolbox for achieving noncanonical lipid synthesis. We describe how these methodologies enable the direct construction of synthetic lipids, helping to address fundamental questions related to the cell biology of lipid biosynthesis, trafficking, and signaling. We envision that highlighting the current advances in artificial lipid synthesis will pave the way for broader interest into this emerging class of biomimetic molecules.     

Chemoenzymatic synthesis and hydrogelation of amylose-grafted xanthan gums

This paper reports the chemoenzymatic synthesis of an amylose-grafted xanthan gum. An amine-functionalized maltooligosaccharide was chemically introduced to xanthan gum by condensation with its carboxylates using a condensing agent to produce a maltooligosaccharide-grafted xanthan gum. Then, a phosphorylase-catalyzed enzymatic polymerization of glucose 1-phosphate from the graft chain ends on the xanthan gum derivative was performed, giving an amylose-grafted xanthan gum. Furthermore, the product formed a gel with an ionic liquid, which was converted into a hydrogel with high water content by replacement of the ionic liquid with water. The ionically cross-linked hydrogel was also provided by soaking the primary formed hydrogel in FeCl₃ aqueous solution. The mechanical properties of the resulting hydrogels were evaluated by compressive testing.     

Chemoenzymatic synthesis of the sialyl Lewis X glycan and its derivatives

A combination of recombinant FKP and α-(1→3)-fucosyltransferase allows the facile synthesis of the sialyl Lewis X tetrasaccharide glycan and its derivatives in excellent yield. In this system, the universal fucosyl donor, guanidine 5′-diphosphate-β-l-fucose (GDP-fucose), or its analogues can be generated in situ by cofactor recycling using pyruvate kinase.     

Chemoenzymatic synthesis of the sialyl-alpha-(2-->3')-lactosamine trisaccharide with a 3-aminopropyl group as a spacer at the reducing end

The trisaccharide, 3-aminopropyl 5-acetamido-3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosylonic acid-(2-->3)-beta-D-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranoside has been synthesized chemoenzymatically for the first time. First, the acceptor, 3-aminopropyl beta-D-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranoside was synthesized in a conventional chemical manner, and then it was coupled with CMP-sialic acid using alpha-(2-->3)-(N)-sialyltransferase to afford the desired trisaccharide by an enzymatically stereocontrolled manner.     

Glycosyltransferase-catalyzed synthesis of bioactive oligosaccharides

Mammalian cell surfaces are all covered with bioactive oligosaccharides which play an important role in molecular recognition events such as immune recognition, cell-cell communication and initiation of microbial pathogenesis. Consequently, bioactive oligosaccharides have been recognized as a medicinally relevant class of biomolecules for which the interest is growing. For the preparation of complex and highly pure oligosaccharides, methods based on the application of glycosyltransferases are currently recognized as being the most effective. The present paper reviews the potential of glycosyltransferases as synthetic tools in oligosaccharide synthesis. Reaction mechanisms and selected characteristics of these enzymes are described in relation to the stereochemistry of the transfer reaction and the requirements of sugar nucleotide donors. For the application of glycosyltransferases, accepted substrate profiles are summarized and the whole-cell approach versus isolated enzyme methodology is compared. Sialyltransferase-catalyzed syntheses of gangliosides and other sialylated oligosaccharides are described in more detail in view of the prominent role of these compounds in biological recognition.     

Chemoenzymatic synthesis of 2-azidoethyl-ganglio-oligosaccharides GD3, GT3, GM2, GD2, GT2, GM1, and GD1a

We have synthesized several ganglio-oligosaccharide structures using glycosyltransferases from Campylobacter jejuni. The enzymes, alpha-(2-->3/8)-sialyltransferase (Cst-II), beta-(1-->4)-N-acetylgalactosaminyltransferase (CgtA), and beta-(1-->3)-galactosyltransferase (CgtB), were produced in large-scale fermentation from Escherichia coli and further characterized based on their acceptor specificities. 2-Azidoethyl-glycosides corresponding to the oligosaccharides of GD3 (alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-), GT3 (alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-), GM2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), GD2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), GT2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), and GM1 (beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-) were synthesized in high yields (gram-scale). In addition, a mammalian alpha-(2-->3)-sialyltransferase (ST3Gal I) was used to sialylate GM1 and generate GD1a (alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-) oligosaccharide. We also cloned and expressed a rat UDP-N-acetylglucosamine-4'epimerase (GalNAcE) in E. coli AD202 cells for cost saving in situ conversion of less expensive UDP-GlcNAc to UDP-GalNAc.     

Chemoenzymatic synthesis of CMP-N-acetyl-7-fluoro-7-deoxy-neuraminic acid

7-Fluoro sialic acid was prepared and activated as cytidine monophosphate (CMP) ester. The synthesis started with d-glucose, which was efficiently converted into N-acetyl-4-fluoro-4-deoxy-d-mannosamine. Aldolase catalyzed transformation yielded the corresponding fluorinated sialic acid which was activated as CMP ester using three different synthetases in the presence as well as in the absence of pyrophosphatase which possesses inhibitory properties. Finally, conditions were optimized to perform a one-pot reaction starting from fluorinated mannosamine, which yielded the 7-fluoro-7-deoxy-CMP-sialic acid by incubation with three enzymes.     

Recent advances in glycan synthesis

Carbohydrates play important roles in life science, but their synthesis is always hampered by their complicated chemical structures. Scientists have never stopped trying to solve the problem of glycan synthesis from various aspects. Here a brief overview of recent progress in glycan synthesis, including chemical approaches, chemoenzymatic approaches, and automated synthesis, will be discussed, focusing on the efficiency of new glycosylation methods, the stereoselectivity of coupled products, and their applications in the assembly of complex glycan chains.     

Recent advances in the chemoenzymatic synthesis of bioactive natural products

The field of organic chemistry has recently witnessed a rapid rise in the use of chemoenzymatic strategies for the synthesis of complex molecules. Under this paradigm, biocatalytic methods and contemporary synthetic methods are used synergistically in a multistep approach toward a target molecule. In light of the unparalleled regioselectivity and stereoselectivity of enzymatic transformations and the reaction diversity of contemporary organic chemistry, chemoenzymatic strategies hold enormous potential for streamlining access to important bioactive molecules. This review covers recent demonstrations of chemoenzymatic approaches in chemical synthesis, with special emphasis on the preparation of medicinally relevant natural products.     

Chemoenzymatic synthesis of (S)-duloxetine using carbonyl reductase from Rhodosporidium toruloides

A chemoenzymatic strategy was developed for (S)-duloxetine production employing carbonyl reductases from newly isolated Rhodosporidium toruloides into the enantiodetermining step. Amongst the ten most permissive enzymes identified, cloned, and overexpressed in Escherichia coli, RtSCR9 exhibited excellent activity and enantioselectivity. Using co-expressed E. coli harboring both RtSCR9 and glucose dehydrogenase, (S)-3-(dimethylamino)-1-(2-thienyl)-1-propanol 3a was fabricated with so far the highest substrate loading (1000 mM) in a space-time yield per gram of biomass (DCW) of 22.9 mmol L⁻¹ h⁻¹ g DCW⁻¹ at a 200-g scale. The subsequent synthetic steps from RtSCR9-catalyzed (S)-3a were further performed, affording (S)-duloxetine with 60.2% overall yield from 2-acethylthiophene in >98.5% ee.     

Large-scale synthesis of peptides

Recent advances in the areas of formulation and delivery have rekindled the interest of the pharmaceutical community in peptides as drug candidates, which, in turn, has provided a challenge to the peptide industry to develop efficient methods for the manufacture of relatively complex peptides on scales of up to metric tons per year. This article focuses on chemical synthesis approaches for peptides, and presents an overview of the methods available and in use currently, together with a discussion of scale-up strategies. Examples of the different methods are discussed, together with solutions to some specific problems encountered during scale-up development. Finally, an overview is presented of issues common to all manufacturing methods, i.e., methods used for the large-scale purification and isolation of final bulk products and regulatory considerations to be addressed during scale-up of processes to commercial levels.     

Recent developments in lipase-catalyzed synthesis of polymeric materials

In the past three years, enzymatic polymerization has dramatically developed and provided many successful examples in the construction of functional polymeric materials. In this review, the lipase-catalyzed synthesis of polymeric materials is systematically summarized, focusing on the synthesis of complex and well-defined polyesters. Exploration of novel biocatalysts and reaction media is described, with particular emphasis on the enzymes obtained via immobilization or protein engineering strategies, green solvents and reactors. Enzymatic polyester synthesis is then discussed with regard to the different reaction types, including ring-opening polymerization, polycondensation, combination of ring-opening polymerization with polycondensation, and chemoenzymatic polymerization. Using enzymatic polymerization, many polymeric materials with tailor-made structures and properties have been successfully designed and synthesized. Finally, recent developments in catalytic kinetics and mechanistic studies through the use of spectroscopy, mathematics and computer techniques are introduced. Overall, the review demonstrates that lipase-catalyzed synthesis of polymeric materials could be a promising platform for green polymer chemistry, and will be potential to produce biodegradable and biocompatible polymers.     

Enzymatic synthesis of oligosaccharides from branched cyclodextrins

6'-alpha-Maltosyl-maltotriose and 6'-alpha-D-glucosyl-maltotriose were prepared from Novamyl degradation of 6-O-alpha-maltosyl-alpha-cyclodextrin and 6-O-alpha-D-glucosyl-alpha-cyclodextrin, respectively. NMR spectroscopy was used to elucidate their structural identities, in a combination of COSY experiments. Further, a mechanism for the degradation was proposed based on the Novamyl active site geometry.     

Facile synthesis of novel mutual derivatives of nucleosides and pyrimidines by regioselectively chemo-enzymatic protocol

We established a facile regioselectively chemo-enzymatic synthesis procedure for the preparation of mutual derivatives of nucleosides and pyrimidines by sequential Markovnikov addition and acylation. Firstly, pyrimidine derivatives containing vinyl ester group were synthesized from pyrimidines and divinyl esters through Markovnikov addition catalyzed by K₂CO₃ in DMSO at 80 °C, and the yields were ranged from 50% to 87%. Then regioselective acylation of ribavirin and cytarabine with pyrimidine vinyl ester was catalyzed by CAL-B (immobilized lipase from Candida antarctica) in anhydrous acetone. Reaction conditions of enzymatic acylation including enzyme resource and solvents were optimized. A series of mutual derivatives of nucleosides and pyrimidines were synthesized successfully and characterized with NMR, IR, and HRMS. This chemo-enzymatic protocol involving sequential Markovnikov addition and acylation provided a novel way of synthesizing complicated functional compounds regioselectively which was hard to be achieved either by chemical or by enzymatic methods.     

Synthesis of a spacer-containing disaccharide fragment of Bordetella pertussis lipopolysaccharide

The disaccharide 2-(p-aminophenyl)ethyl 4-O-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-2,3-diacetamido-2 ,3-dideoxy-alpha-D-mannopyranoside uronate, which is assumed to be a partial structure of the Bordetella pertussis polysaccharide, was synthesized starting from D-glucose and D-glucosamine, respectively. The major synthetic transformations were conversion of D-glucosamine into the donor ethyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-1-thio-beta-D-glucopyranoside and conversion of glucose, by a sequence involving 2,3-epoxide formation/opening, nucleophilic triflate displacement in the 3-position, and necessary protecting group manipulations, into the acceptor 2-(p-trifluoroacetamidophenyl)ethyl 6-O-benzyl-2,3-diazido-2,3-dideoxy-alpha-D-mannopyranoside. Coupling of the donor and acceptor units promoted by dimethyl(methylthio)sulfonium triflate followed by selective oxidation of the 6'-position and deprotection gave the target disaccharide.