Chemo-enzymatic Pathway Research Articles

A chemo-enzymatic pathway to expand cellooligosaccharide chemical space through amine bond introduction

Enzymatic functionalization of oligosaccharides is a useful and environmentally friendly way to expand their structural chemical space and access to a wider range of applications in the health, food, feed, cosmetics and other sectors. In this work, we first tested the laccase/TEMPO system to generate oxidized forms of cellobiose and methyl β-D-cellobiose, and obtained high yields of novel anionic disaccharides (>60 %) at pH 6.0. Laccase/TEMPO system was then applied to a mix of cellooligosaccharides and to pure D-cellopentaose. The occurrence of carbonyl and carboxyl groups in the oxidation products was shown by LC-HRMS, MALDI-TOF and reductive amination of the carbonyl groups was attempted with p-toluidine a low molar mass amine to form the Schiff base, then reduced by 2-picoline borane to generate a more stable amine bond. The new grafted products were characterized by LC-HRMS, LC-UV-MS/MS and covalent grafting was evidenced. Next, the same procedure was adopted to successfully graft a dye, the rhodamine 123, larger in size than toluidine. This two-step chemo-enzymatic approach, never reported before, for functionalization of oligosaccharides, offers attractive opportunities to anionic cellooligosaccharides and derived glucoconjugates of interest for biomedical or neutraceutical applications. It also paves the way for more environmentally-friendly cellulose fabric staining procedures.

A sustainable preparative-scale chemo-enzymatic synthesis of 6-hydroxy-5,7-dimethoxy-2-naphthoic acid (DMNA) from sinapic acid

ABSTRACT Naturally occurring sinapic acid is a valuable synthon for many high-value applications (e.g. cosmetic, pharmaceutic) that can be found in numerous plants, especially Brasiccacea species (e.g. rapeseed, mustard). It has been demonstrated that alkaline extraction of matter rich in sinapic acid resulted in the conversion of sinapic acid into 6-hydroxy-5,7-dimethoxy-2-naphtanoic acid (DMNA). Although DMNA could be a potential alternative to petro-based naphtanoic acid used in polymers, untill now, no reliable (bio)synthetic procedure existed to synthesize it at the preparative-scale. Herein, starting from sinapic acid, we describe a very simple one-pot two-step chemo-enzymatic pathway to DMNA – and reaction intermediaries bislactone (BL) and thomasidioic acid (TA) – at gram scale using a simple filtration as purification step. Green metrics – Process Mass Index and EcoScale – have been determined to assess the sustainability of this new process. Finally, the antiradical activities of DMNA, BL and TA have been evaluated.

Chemical and chemoenzymatic routes to bridged homoarabinofuranosylpyrimidines: Bicyclic AZT analogues.

Conformationally restricted diastereomeric homoarabinofuranosylpyrimidines (AZT analogue), i.e., (5′R)-3′-azido-3′-deoxy-2′-O,5′-C-bridged-β-ᴅ-homoarabinofuranosylthymine and -uracil had been synthesized starting from diacetone ᴅ-glucofuranose following chemoenzymatic and chemical routes in 34–35% and 24–25% overall yields, respectively. The quantitative and diastereoselective acetylation of primary hydroxy over two secondary hydroxy groups present in the key nucleoside precursor was mediated with Lipozyme® TL IM in 2-methyltetrahydrofuran following a chemoenzymatic pathway. Whereas, the protection of the primary hydroxy over the lone secondary hydroxy group in the key azido sugar precursor was achieved using bulky tert-butyldiphenylsilyl chloride (TBDPS-Cl) in pyridine in 92% yield following a chemical synthetic pathway. The chemoenzymatic method was found to be superior over the chemical method in respect of the number of synthetic steps and overall yield of the final product.

Convergent Chemoenzymatic Strategy to Deliver a Diversity of Shigella flexneri Serotype-Specific O-Antigen Segments from a Unique Lightly Protected Tetrasaccharide Core.

Progress in glycoscience is strongly dependent on the availability of broadly diverse tailor-made, well-defined, and often complex oligosaccharides. Herein, going beyond natural resources and aiming to circumvent chemical boundaries in glycochemistry, we tackle the development of an in vitro chemoenzymatic strategy holding great potential to answer the need for molecular diversity characterizing microbial cell-surface carbohydrates. The concept is exemplified in the context of Shigella flexneri, a major cause of diarrhoeal disease. Aiming at a broad serotype coverage S. flexneri glycoconjugate vaccine, a non-natural lightly protected tetrasaccharide was designed for compatibility with (i) serotype-specific glucosylations and O-acetylations defining S. flexneri O-antigens, (ii) recognition by suitable α-transglucosylases, and (iii) programmed oligomerization following enzymatic α-d-glucosylation. The tetrasaccharide core was chemically synthesized from two crystalline monosaccharide precursors. Six α-transglucosylases found in the glycoside hydrolase family 70 were shown to transfer glucosyl residues on the non-natural acceptor. The successful proof of concept is achieved for a pentasaccharide featuring the glucosylation pattern from the S. flexneri type IV O-antigen. It demonstrates the potential of appropriately planned chemoenzymatic pathways involving non-natural acceptors and low-cost donor/transglucosylase systems to achieve the demanding regioselective α-d-glucosylation of large substrates, paving the way to microbial oligosaccharides of vaccinal interest.

First Total Synthesis of (β-5)-(β-O-4) Dihydroxytrimer and Dihydrotrimer of Coniferyl Alcohol (G): Advanced Lignin Model Compounds.

To investigate lignin degradation, scientists commonly use model compounds. Unfortunately, these models are most of the time simple β-O-4 dimers and do not sufficiently mimic the wide complexity of lignin structure (i.e., aliphatic side chains and robust C-C bonds). Herein, we present a methodology to access advanced lignin models through the first synthesis of two trimers of monolignol G—possessing side-chains and both robust β-5 bond and labile β-O-4 bond—via a chemo-enzymatic pathway. Key steps were (1) the C-C coupling via laccase-mediated oxidation, (2) the C-O coupling via a simple SN2 between a phenolate and a bromoketoester, and (3) a modified Upjohn dihydroxylation or a palladium-catalyzed hydrogenation. (β-5)-(β-O-4) dihydroxytrimer and dihydrotrimer of coniferyl alcohol (G) were obtained in good global yield, 9 and 20%, respectively, over nine steps starting from ferulic acid.

Expanding the Scope of Asinger Chemistry towards Enantiomerically Pure Secondary Amines and β-Aminothiols through Chemoenzymatic Derivatization of 3-Thiazolines

A proof of concept for a novel approach towards enantiomerically highly enriched acyclic secondary amines and β-aminothiols as non-cyclic target molecules when starting from 3-thiazolines as heterocycles is presented. Starting from 2,2,4,5,5-pentamethyl-3-thiazoline, we demonstrated this chemoenzymatic pathway to both of these types of amine molecules, which were isolated as urea derivatives with a non-optimized yield of up to 20%. As a substrate, 2,2,4,5,5-pentamethyl-3-thiazolidine, which was obtained with an enantiomeric excess (ee) of 99% in a biotransformation from the corresponding 3-thiazoline according to a recently developed protocol, was used. For the reductive desulfurization of this substrate leading to a sulfur-free secondary amine, in situ formed Ni2B turned out to be a suitable reducing reagent. However, when using lithium aluminum hydride as a reducing agent, β-aminothiol was obtained.

Probing the Substrate Promiscuity of Isopentenyl Phosphate Kinase as a Platform for Hemiterpene Analogue Production.

Isoprenoids are a large class of natural products with wide-ranging applications. Synthetic biology approaches to the manufacture of isoprenoids and their new-to-nature derivatives are limited due to the provision in nature of just two hemiterpene building blocks for isoprenoid biosynthesis. To address this limitation, artificial chemo-enzymatic pathways such as the alcohol-dependent hemiterpene (ADH) pathway serve to leverage consecutive kinases to convert exogenous alcohols into pyrophosphates that could be coupled to downstream isoprenoid biosynthesis. To be successful, each kinase in this pathway should be permissive of a broad range of substrates. For the first time, we have probed the promiscuity of the second enzyme in the ADH pathway-isopentenyl phosphate kinase from Thermoplasma acidophilum-towards a broad range of acceptor monophosphates. Subsequently, we evaluate the suitability of this enzyme to provide unnatural pyrophosphates and provide a critical first step in characterizing the rate-limiting steps in the artificial ADH pathway.

An Artificial Pathway for Isoprenoid Biosynthesis Decoupled from Native Hemiterpene Metabolism.

Isoprenoids are constructed in nature using hemiterpene building blocks that are biosynthesized from lengthy enzymatic pathways with little opportunity to deploy precursor-directed biosynthesis. Here, an artificial alcohol-dependent hemiterpene biosynthetic pathway was designed and coupled to several isoprenoid biosynthetic systems, affording lycopene and a prenylated tryptophan in robust yields. This approach affords a potential route to diverse non-natural hemiterpenes and by extension isoprenoids modified with non-natural chemical functionality. Accordingly, the prototype chemo-enzymatic pathway is a critical first step toward the construction of engineered microbial strains for bioconversion of simple scalable building blocks into complex isoprenoid scaffolds.

Chemo-enzymatic pathways toward pinene-based renewable materials

Chemo-enzymatic conversion of underutilized forest-waste products into multi-functional polymers.

Chemo-Enzymatic Synthesis and Free Radical Polymerization of Renewable Acrylate Monomers from Cellulose-Based Lactones

A chemo-enzymatic pathway involving a Candida antarctica type B lipase was developed to produce (S)-γ-hydroxymethyl-α,β-butenolide methacrylate (HBO-m) and (S)-γ-hydroxymethyl-γ-butyrolactone methacrylate (2H-HBO-m) from (S)-γ-hydroxymethyl-γ-butyrolactone (HBO), a biobased molecule obtained from cellulose-derived levoglucosenone. The acrylated monomer was then copolymerized through a free radical process with methacrylamide and methylene-γ-valerolactone, a green intermediate. Finally, methylene-tetrahydropyranyl (S)-γ-hydroxymethyl-γ-butyrolactone (M-THP-2H-HBO), synthesized via α-methylenation of 2H-HBO, was copolymerized with methyl methacrylate to access copolyacrylates exhibiting high glass transition temperatures.

Synthesis of novel 3′-azido-3′-deoxy-α-L-ribo configured nucleosides: A comparative study between chemical and chemo-enzymatic methodologies

GRAPHICAL ABSTRACT

Chemo-enzymatic Synthesis of Clickable Xylo-oligosaccharide Monomers from Hardwood 4-O-Methylglucuronoxylan.

A chemo-enzymatic pathway was developed to transform 4-O-methylglucuronic acid (MeGlcpA) containing xylo-oligosaccharides from beechwood into clickable monomers capable of polymerizing at room temperature and in aqueous conditions to form unique polytriazoles. While the gluco-oligosaccharide oxidase (GOOX) from Sarocladium strictum was used to oxidize C6-propargylated oligosaccharides, the acid-amine coupling reagents 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDAC) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) were employed and compared for their ability to append click functionalities to carboxylic acid groups of enzyme-treated oligosaccharides. While DMT-MM was a superior coupling reagent for this application, a triazine side product was observed during C-1 amidation. Resulting bifunctional xylo-oligosaccharide monomers were polymerized using a Cu(I) catalyst, forming a soft gel which was characterized by 1H NMR, confirming the triazole product.

A Chemoenzymatic Route to a Class of Sucrose Esters

Sucrose esters are the most developed carbohydrate esters and are used in the food, cosmetic, and pharmaceutical industries. Herein, we introduce a novel chemoenzymatic pathway for the synthesis of β‐d‐fructofuranosyl‐(2,1)‐α‐d‐uronic acid derivatives, a new class of sucrose esters.

Preparation of new β-D-xyloside- and β-D-xylobioside-based ionic liquids through chemical and/or enzymatic reactions

Several tetraalkylphosphonium and tetraalkylammonium salts containing xyloside- and xylobioside-based anionic moieties have been prepared. Two stereoselective routes have been developed: i) a chemical pathway in four steps from D-xylose, and ii) a chemoenzymatic pathway directly from biomass-derived xylans. These salts displayed interesting properties as ionic liquids. Their structures have been correlated to their thermal properties (melting, glass transition and decomposition temperatures).

Epoxy resins without BPA

A biobased bisphenol can be used to prepare renewable epoxy-amine resins, eliminating the need to use potenially-toxic bisphenol A, chemists report (ChemSusChem 2017, DOI: 10.1002/cssc.20d1601595). BPA is a petroleum-based endocrine disruptor and has been banned by the Food & Drug Administration for use in children’s product packaging. Epoxies, with their hardy chemical and thermal properties, are ubiquitous, used in materials such as electronics and insulators. Scientists have been avidly looking for substitutes for BPA in epoxies. A team led by Florent Allais of AgroParisTech has found that syringaresinol, a naturally occurring, non-endocrine-disrupting bisphenol found in plants such as Syringa patula and Magnolia thailandica, can be used to produce epoxy-amine resins. The researchers developed a synthesis of syringaresinol via a chemo-enzymatic pathway. Though the epoxy-amine resins based on syringaresinol have mechanical and thermal stabilities that are close to that of BPA-produced resins, the author...

Chemoenzymatic pathways for the synthesis of biologically active natural products

Chemoenzymatic pathways for the synthesis of biologically active natural products

Recent advances and challenges in the heterologous production of microbial nitrilases for biocatalytic applications.

The aim of this study is to review the current state of and highlight the challenges in the production of microbial nitrilases as catalysts for the mild hydrolysis of industrially important nitriles. Together with aldoxime dehydratase, the nitrile-hydrolyzing enzymes (nitrilase, nitrile hydratase) are key enzymes in the aldoxime-nitrile pathway which is widely distributed in bacteria and fungi. The availability of nitrilases has grown significantly over the past decade due to the use of metagenomic and database-mining approaches. Databases contain plenty of putative enzymes of this type, whose overproduction may improve the spectrum and the industrial utility of nitrilases. By exploiting this resource, the number of experimentally verified nitrilases has recently increased to several hundred. We especially focus on the efficient heterologous expression systems that are applicable for the overproduction of wild-type nitrilases and their artificial variants. Biocatalyst forms with industrial potential are also highlighted. The potential industrial applications of nitrilases are classified according to their target products (α-hydroxy acids, α- and β-amino acids, cyano acids, amides). The emerging uses of nitrilases and their subtypes (cyanide hydratases, cyanide dihydratases) in bioremediation is also summarized. The integration of nitrilases with other enzymes into artificial multienzymatic and chemoenzymatic pathways is considered a promising strategy for future applications.

Optically Active β-Methyl-δ-Valerolactone: Biosynthesis and Polymerization

Chemo-enzymatic pathways were developed to prepare optically enriched (+)-β-methyl-δ-valerolactone and (−)-β-methyl-δ-valerolactone. Anhydromevalonolactone, synthesized by the acid-catalyzed dehydration of bioderived mevalonate, was transformed to (+)-β-methyl-δ-valerolactone with 76% ee and 69% conversion using the mutant enoate reductase, YqjM(C26D, I69T). With the same substrate but a different enoate reductase (OYE2), we obtained the other enantiomer ((−)-β-methyl-δ-valerolactone) with higher selectivity and yield (96% ee and a 92% conversion). The enzyme-docking program LibDock was used to help explain the origin of the divergent enatntioselectivity of the two reductases, and complementary in vitro experiments were used to determine the turnover number and Michaelis constant for each. Finally, the effect of the enantiopurity of the β-methyl-δ-valerolactone monomer on the properties of the corresponding polyester was investigated. Like atactic poly((±)-β-methyl-δ-valerolactone), the isotactic polymer was determined to be amorphous with a low softening temperature (−52 °C).

GH13 amylosucrases and GH70 branching sucrases, atypical enzymes in their respective families.

Amylosucrases and branching sucrases are α-retaining transglucosylases found in the glycoside-hydrolase families 13 and 70, respectively, of the clan GH-H. These enzymes display unique activities in their respective families. Using sucrose as substrate and without mediation of nucleotide-activated sugars, amylosucrase catalyzes the formation of an α-(1→4) linked glucan that resembles amylose. In contrast, the recently discovered branching sucrases are unable to catalyze polymerization of glucosyl units as they are rather specific for dextran branching through α-(1→2) or α-(1→3) branching linkages depending on the enzyme regiospecificity. In addition, GH13 amylosucrases and GH70 branching sucrases are naturally promiscuous and can glucosylate different types of acceptor molecules including sugars, polyols, or flavonoids. Amylosucrases have been the most investigated glucansucrases, in particular to control product profiles or to successfully develop tailored α-transglucosylases able to glucosylate various molecules of interest, for example, chemically protected carbohydrates that are planned to enter in chemoenzymatic pathways. The structural traits of these atypical enzymes will be described and compared, and an overview of the potential of natural or engineered enzymes for glycodiversification and chemoenzymatic synthesis will be highlighted.

Quantification of sialic acids in red meat by UPLC-FLD using indoxylsialosides as internal standards.

Herein we describe a UPLC-FLD-based method for the quantification of the sialic acid content of red meat, using a synthetic neuraminic acid derivative as an internal standard. X-Gal-α-2,6-N-propionylneuraminic acid was synthesized via a chemoenzymatic pathway and its hydrolytic stability was characterized. Known quantities of this compound were incubated with samples of red meat under sialic acid-releasing conditions. The released sialic acids were derivatized, analyzed by UPLC-FLD, and the Neu5Ac/Neu5Gc content of the meat sample was determined by comparison with the internal standard. A number of red meats were analyzed by this method with the following results (Neu5Ac μg/g tissue, Neu5Gc μg/g tissue ± s.d.): pork (68±3, 15.2±0.7), beef (69±8, 36±5), lamb (46±2, 33±1), rabbit (59±2, 0.4±0.4), and hare (50±4, 1±1). We envisage that this methodology will find application in investigating the health effects of dietary Neu5Gc. Graphical abstract ᅟ.