Chemoenzymatic Synthesis Research Articles

Activity and Biocatalytic Potential of an Indolylamide Generating Thioesterase.

The chemical synthesis of N-acyl indoles is hindered by the poor nucleophilicity of indolic nitrogen, necessitating the use of strongly basic reaction conditions that encumber elaboration of highly functionalized scaffolds. Herein, we describe the total chemoenzymatic synthesis of the bulbiferamide natural products by the biochemical activity reconstitution of a nonribosomal peptide synthetase assembly line-derived (NRPS-derived) thioesterase that neatly installs the macrocyclizing indolylamide. The enzyme represents a starting point for biocatalytic access to macrocyclic indolylamide peptides and natural products.

An Efficient Chemoenzymatic Synthesis of Chenodeoxycholic Acid from its Allo‐Epimer through Stereoselective Hydrogenation of 3‐Oxo‐chol‐Δ1,4‐dien‐24‐oate Scaffold

An Efficient Chemoenzymatic Synthesis of Chenodeoxycholic Acid from its <i>Allo</i>‐Epimer through Stereoselective Hydrogenation of 3‐Oxo‐chol‐Δ^1,4‐dien‐24‐oate Scaffold

Chemo-enzymatic synthesis of NPN cofactor taking advantage of ADP-ribosyl cyclase and LarC cyclometallase promiscuous activities

The nickel-pincer nucleotide cofactor (NPN) is a widespread organometallic cofactor required for lactate racemase (LarA) and for α-hydroxy acid racemases and epimerases of the LarA superfamily. Its biosynthesis, which starts with nicotinic acid adenine dinucleotide (NaAD), requires three enzymes: LarB, LarC, and LarE, and can be performed in vitro with purified enzymes. Nevertheless, as LarE and LarC are single turnover enzymes, the in vitro NPN biosynthesis requires huge amounts of enzymes (particularly 2 equivalents of LarE), which hampers the study of NPN and of NPN-dependent enzymes. By using adenosine diphosphate (ADP)-ribosyl cyclase (ARC), we exchanged the nicotinamide moiety in NAD+ with synthetic pyridine-3,5-bisthiocarboxylic acid in order to synthesize the novel intermediate pyridinium-3,5-bisthiocarboxylic acid adenine dinucleotide (P2TAD). The latter could be produced at a multimilligram scale allowing its characterization by Nuclear Magnetic Resonance (NMR) and mass spectrometry. Interestingly, P2TAD could directly be used by LarC in order to generate the NPN cofactor, bypassing both LarB and LarE. Globally, a new chemoenzymatic route towards NPN was developed via the intermediate P2TAD, which should facilitate the biochemical and biotechnological investigations on NPN binding enzymes.

A Kinetic Trapping Approach for Facile Access to 3FaxNeu5Ac and a Photo-Cross-Linkable Sialyltransferase Probe.

Sialic acid (Neu5Ac) is installed onto glycoconjugates by sialyltransferases (STs) using cytidine monophosphate-Neu5Ac (CMP-β-d-Neu5Ac) as their donor. The only class of cell-active ST inhibitors are those based on a 3FaxNeu5Ac scaffold, which is metabolically converted into CMP-3FaxNeu5Ac within cells. It is essential for the fluorine to be axial, yet stereoselective installation of fluorine in this specific orientation is challenging. Sialic acid aldolase can convert 3-fluoropyruvate and 2-acetamido-2-deoxy-d-mannopyranose (ManNAc) to 3FNeu5Ac, but stereocontrol of the fluorine in the product has not been possible. We hypothesized that the 3Fax kinetic product of a sialic acid aldolase reaction could be trapped by coupling with CMP-sialic acid synthetase to yield CMP-3FaxNeu5Ac. Here, we report that highly active CMP-sialic acid synthetase and short reaction times produce exclusively CMP-3FaxNeu5Ac. Removal of CMP from CMP-3FaxNeu5Ac under acidic conditions unexpectedly led to 3-fluoro-β-d-Neu5Ac 2-phosphate (3FaxNeu5Ac-2P). Alkaline phosphatase successfully converted 3FaxNeu5Ac-2P to 3FaxNeu5Ac, enabling stereochemically controlled access to 3FaxNeu5Ac, which is effective in lowering the sialoglycan ligands for Siglecs on cells. Moreover, our kinetic trapping approach could be used to access CMP-3FaxNeu5Ac with modifications at the C5, C9, or both positions, which enabled the chemoenzymatic synthesis of a photo-cross-linkable version of CMP-3FaxNeu5Ac that selectively photo-cross-linked to ST6GAL1 over two other STs.

A Green Chemo-Enzymatic Approach for CO2 Capture and Transformation into Bis(cyclic carbonate) Esters in Solvent-Free Media.

A sustainable approach for CO2 capture and chemo-enzymatic transformation into bis(cyclic carbonate) esters from CO2, glycidol, and organic anhydrides under solvent-free conditions has been demonstrated. The chemo-enzymatic process is based on two consecutive catalytic steps, which can be executed through separated operations or within a one-pot combo system, taking advantage of the synergic effects that emerge from integrating ionic liquid (IL) technologies and biocatalysts. In a first step, lipase-catalyzed transesterification and esterification reactions of different diacyl donors (e.g., glutaric anhydride, succinic anhydride, dimethyl succinate, etc.) with glycidol in solvent-free under mild reaction conditions (70 °C, 6 h) produce the corresponding diglycidyl ester derivatives in up to 41% yield. By a second step, the synthesis of bis(cyclic carbonate) esters was carried out as a result of the cycloaddition reaction of CO2 (from an exhausted gas source, 15% CO2 purity) on these diglycidyl esters, catalyzed by the covalently attached 1-decyl-2-methylimidazolium IL (supported ionic liquid-like phase, SILLP), in solvent-free condition, leading up to 65% yield after 8 h at 45 °C and 1 MPa CO2 pressure. Both key elements of the reaction system (biocatalyst and SILLP) were successfully recovered and reused for at least 5 operational cycles. Finally, different metrics have been applied to assess the greenness of the solvent-free chemo-enzymatic synthesis of bis(cyclic carbonate) esters here reported.

“Novel chemo-enzymatic synthesis, structural elucidation and first antiprotozoal activity profiling of the atropoisomeric dimers of trans-8-Hydroxycalamenene”

Leishmaniasis and malaria are two debilitating protozoan diseases affecting millions globally, particularly in tropical and subtropical regions. Current therapeutic options face significant challenges due to emerging drug-resistant strains, necessitating the discovery of novel antiprotozoal agents. This study explores, for the first time, the antiprotozoal potential of calamenenes and their dimers, naturally occurring sesquiterpenes found in essential oils, through a novel chemo-enzymatic synthesis approach. Using the laccase from Trametes versicolor, atropoisomeric dimers of (−)- and (+)-8-trans-hydroxycalamenene were synthesized from commercially available (−)- and (+)-menthol. Structural elucidation was achieved via 2D-NMR spectroscopy, electronic circular dichroism, and DFT calculations. In vitro profiling against Leishmania spp and drug-resistant Plasmodium falciparum revealed that calamenene dimers exhibited significantly higher antiprotozoal activity compared to their monomeric counterparts, highlighting the potential of dimeric terpenoids as promising antiprotozoal agents. This work lays the foundation for developing novel antimalarial drugs based on calamenene scaffolds, encouraging further interactome studies to optimize their pharmacological properties.

Chemoenzymatic synthesis with the Pasteurella hyaluronan synthase; production of a multitude of defined authentic, derivatized, and analog polymers

AbstractHyaluronan (HA; [‐3‐GlcNAc‐1‐beta‐4‐GlcA‐1‐beta]n), an essential matrix polysaccharide of vertebrates and the molecular camouflage coating in certain pathogens, is polymerized by “HA synthase” (HAS) enzymes. Three HAS classes have been identified with biotechnological utility, but only the Class II PmHAS from Pasteurella multocida Type A has been useful for preparation of very defined HA polymers in vitro. Two general chemoenzymatic strategies with different size products are possible: (1) repetitive step‐wise extension reactions by sequential addition of a single monosaccharide from a donor UDP‐sugar onto an acceptor (or “primer”) comprised of a short glycosaminoglycan chain (e.g., HA di‐, tri‐ or tetrasaccharide) or an artificial glucuronide yielding homogeneous oligosaccharides in the range of 2 to ~20 monosaccharide units (n = 1 to ~10), or (2) “one‐pot” polymerization reactions employing acceptor‐mediated synchronization with stoichiometric size control yielding quasi‐monodisperse (i.e., polydispersity approaching 1; very narrow size distributions) polysaccharides in the range of ~7 kDa to ~2 MDa (n = ~17 to 5000). In either strategy, acceptors containing non‐carbohydrate functionalities (e.g., biotin, fluorophores, amines) can add useful moieties to the reducing termini of HA chains at 100% efficiency. As a further structural diversification, PmHAS can utilize a variety of unnatural UDP‐sugar analogs thus adding novel groups (e.g., trifluoroacetyl, alkyne, azide, sulfhydryl) along the HA backbone and/or at its nonreducing terminus. This review discusses the current understanding and recent advances in HA chemoenzymatic synthesis methods using PmHAS. This powerful toolbox has potential for creation of a multitude of HA‐based probes, therapeutics, drug conjugates, coatings, and biomaterials.

Chemoenzymatic Synthesis of Aromatic Vinyl Monomers from Biorenewable 5-Hydroxymethyl-2-Furfural

Chemoenzymatic Synthesis of Aromatic Vinyl Monomers from Biorenewable 5-Hydroxymethyl-2-Furfural

Divergent synthesis of amino acid-linked O-GalNAc glycan core structures.

O-GalNAc glycans, also known as mucin-type O-glycans, are primary constituents of mucins on various mucosal sites of the body and also ubiquitously expressed on cell surface and secreted proteins. They have crucial roles in a wide range of physiological and pathological processes, including tumor growth and progression. In addition, altered expression of O-GalNAc glycans is frequently observed during different disease states. Research dedicated to unraveling the structure-function relationships of O-GalNAc glycans has led to the discovery of disease biomarkers and diagnostic tools and the development of O-glycopeptide-based cancer vaccines. Many of these efforts require amino acid-linked O-GalNAc core structures as building blocks to assemble complex O-glycans and glycopeptides. There are eight core structures (cores one to eight), from which all mucin-type O-glycans are derived. In this protocol, we describe the first divergent synthesis of all eight cores from a versatile precursor in practical scales. The protocol involves (i) chemical synthesis of the orthogonally protected precursor (3 days) from commercially available materials, (ii) chemical synthesis of five unique glycosyl donors (1-2 days for each donor) and (iii) selective deprotection of the precursor and assembly of the eight cores (2-4 days for each core). The procedure can be adopted to prepare O-GalNAc cores linked to serine, threonine and tyrosine, which can then be utilized directly for solid-phase glycopeptide synthesis or chemoenzymatic synthesis of complex O-glycans. The procedure empowers researchers with fundamental organic chemistry skills to prepare gram scales of any desired O-GalNAc core(s) or all eight cores concurrently.

Advances in the chemoenzymatic synthesis of gamma-aminobutyric acid derivatives

Gamma-aminobutyric acid (GABA) derivatives are a class of effective inhibitory neurotransmitters for treating neurodegenerative diseases, with an immense market demand. Chemical methods are currently the main synthetic strategies for GABA derivatives, facing challenges such as complex processes, low yields, low atom economy, and environmental burden. In recent years, chemoenzymatic synthesis of GABA derivatives has garnered increasing attention because of the high atom economy, high yields, and environmental friendliness. This article reviews the latest advances in the chemical synthesis and chemoenzymatic synthesis of GABA derivatives. Furthermore, it introduces the progress in the industrial synthesis of representative GABA derivatives such as gabapentin, pregabalin, and brivaracetam and prospects the future development of GABA derivatives.

Overcoming a Conceptual Limitation of Industrial ε-Caprolactone Production via Chemoenzymatic Synthesis in Organic Medium.

The multi-10.000 tons scale manufactured chemical ε-caprolactone attracts high industrial interest due to its favorable biodegradability properties. However, besides being of petrochemical origin yet, its production has a conceptual limitation that is the difficult extraction of this highly water-soluble monomer from the water phase resulting from the aqueous solution of H2O2 applied as reagent. In this contribution, we report a chemoenzymatic cascade starting from bio-based phenol, which makes use of O2 instead of H2O2 and runs in pure organic medium, thus requiring only simply decantation and distillation as work-up. In a first step, phenol is hydrogenated quantitatively to cyclohexanol under solvent-free conditions with a Ru-catalyst. After simple removal of the heterogenous catalyst, cyclohexanol is converted to ε-caprolactone in a biocatalytic double oxidation with very high yields just requiring O2 as reagent. This biocatalytic process proceeds in pure organic medium, thus avoiding tedious extraction to isolate the highly water-soluble ε-caprolactone and enabling a substantially simplified work-up by only centrifugal separation of lyophilized whole cells and solvent removal. This oxidation is accomplished using a tailor-made recombinant whole-cell catalyst containing an alcohol dehydrogenase and a cyclohexanone monooxygenase mutant.

From dairy waste to value-added bio-based surfactants

Cheese whey permeate, the main waste stream of dairy industry, was used as a starting material for the production of bio-based surfactants (SFAEs). Specifically, the first step in the sustainable chemoenzymatic synthesis of n-butyl 6-O-palmitoyl-D-glycosides (Fischer glycosylation followed by enzymatic esterification) was optimized by a chemometric study. The surfactancy of the prepared isomeric mixtures was deeply investigated in terms of static and dynamic interfacial tension and emulsifying capability over time.

Concise and Modular Chemoenzymatic Total Synthesis of Bisbenzylisoquinoline Alkaloids

The bisbenzylisoquinoline alkaloids (bisBIAs) have attracted tremendous attention from the synthetic community due to their diverse and intriguing biological activities. Herein, we report the convergent and modular chemoenzymatic syntheses of eight bisBIAs bearing various substitutes and linkages in 5‐7 steps. The gram‐scale synthesis of various well‐designed enantiopure benzylisoquinoline monomers was accomplished via an enzymatic stereoselective Pictet–Spengler reaction, followed by regioselective enzymatic methylation or chemical functionalizations in a sequential one‐pot process. A modified intermolecular copper‐mediated Ullmann coupling enabled the concise and efficient total synthesis of five different linear bisBIAs with either head‐to‐tail or tail‐to‐tail linkage. A biomimetic oxidative phenol dimerization selectively formed the sterically hindered, electron‐rich diaryl ether bond, and subsequent intramolecular Suzuki–Miyaura domino reaction or Ullmann coupling facilitated the first enantioselective total synthesis of three macrocyclic bisBIAs, including ent‐isogranjine, tetrandrine and O‐methylrepandine. This study highlights the great potential of chemoenzymatic strategies in the total synthesis of diverse bisBIAs and paves the way to further explore the biological functions of these natural products.

Concise and Modular Chemoenzymatic Total Synthesis of Bisbenzylisoquinoline Alkaloids

The bisbenzylisoquinoline alkaloids (bisBIAs) have attracted tremendous attention from the synthetic community due to their diverse and intriguing biological activities. Herein, we report the convergent and modular chemoenzymatic syntheses of eight bisBIAs bearing various substitutes and linkages in 5‐7 steps. The gram‐scale synthesis of various well‐designed enantiopure benzylisoquinoline monomers was accomplished via an enzymatic stereoselective Pictet–Spengler reaction, followed by regioselective enzymatic methylation or chemical functionalizations in a sequential one‐pot process. A modified intermolecular copper‐mediated Ullmann coupling enabled the concise and efficient total synthesis of five different linear bisBIAs with either head‐to‐tail or tail‐to‐tail linkage. A biomimetic oxidative phenol dimerization selectively formed the sterically hindered, electron‐rich diaryl ether bond, and subsequent intramolecular Suzuki–Miyaura domino reaction or Ullmann coupling facilitated the first enantioselective total synthesis of three macrocyclic bisBIAs, including ent‐isogranjine, tetrandrine and O‐methylrepandine. This study highlights the great potential of chemoenzymatic strategies in the total synthesis of diverse bisBIAs and paves the way to further explore the biological functions of these natural products.

A Bioorthogonal Precision Tool for Human N-Acetylglucosaminyltransferase V.

Correct elaboration of N-linked glycans in the secretory pathway of human cells is essential in physiology. Early N-glycan biosynthesis follows an assembly line principle before undergoing crucial elaboration points that feature the sequential incorporation of the sugar N-acetylglucosamine (GlcNAc). The activity of GlcNAc transferase V (MGAT5) primes the biosynthesis of an N-glycan antenna that is heavily upregulated in cancer. Still, the functional relevance and substrate choice of MGAT5 are ill-defined. Here, we employ protein engineering to develop a bioorthogonal substrate analog for the activity of MGAT5. Chemoenzymatic synthesis is used to produce a collection of nucleotide-sugar analogs with bulky, bioorthogonal acylamide side chains. We find that WT-MGAT5 displays considerable activity toward such substrate analogues. Protein engineering yields an MGAT5 variant that loses activity against the native nucleotide sugar and increases activity toward a 4-azidobutyramide-containing substrate analogue. By such restriction of substrate specificity, we show that the orthogonal enzyme-substrate pair is suitable to bioorthogonally tag glycoproteins. Through X-ray crystallography and molecular dynamics simulations, we establish the structural basis of MGAT5 engineering, informing the design rules for bioorthogonal precision chemical tools.

Chemoenzymatic Synthesis of DNP-Functionalized FGFR1-Binding Peptides as Novel Peptidomimetic Immunotherapeutics for Treating Lung Cancer.

Receptor-binding peptides are promising candidates for tumor target therapy. However, the inability to occupy "hot spots" on the PPI interface and rapid metabolic instability are significant limitations to their clinical application. We investigated a new strategy in which an FGFR1-binding peptide (Pep1) was site-specifically functionalized with the dinitrophenyl (DNP) hapten at the C-terminus. The resulting Pep1-DNP conjugates retained FGFR1 binding affinity and exhibited a similar potency in inhibiting FGF2-dependent cell proliferation, comparable to that of native Pep1 in vitro. In addition, three conjugates could recruit anti-DNP antibodies onto the surface of cancer cells, thereby mediating the CDC efficacy. In vivo pharmacokinetic studies and antitumor studies demonstrated that optimal conjugate 9 exhibited significantly prolonged half-lives and improved antitumor efficacy without prominent toxicity compared to those of native Pep1. This is a general and cost-effective approach for generating peptidomimetic immunotherapeutics with multiple antitumor mechanisms that may have broad applications in cancer therapy.

Chemoenzymatic Synthesis of Fluorinated L-α-Amino Acids

Chemoenzymatic Synthesis of Fluorinated <scp>L</scp>-α-Amino Acids

Chemoenzymatic Synthesis of Plant‐Derived Kavalactone Natural Products by Dynamic Resolution Using a Biosynthetic O‐Methyltransferase Tailoring Enzyme

Biosynthetic enzymes have enormous potential for the chemoenzymatic synthesis of natural products and other bioactive compounds. Methyltransferases are promising tools for the selective enzymatic modification of complex structures. This paper describes the production, purification and biochemical characterization of the O‐methyltransferase JerF, which catalyzes unique 4‐methoxy‐5,6‐dihydropyranone formation in jerangolid A biosynthesis. Isolation problems had hitherto prevented detailed studies on JerF and were solved by the production of the maltose‐binding protein fusion protein. The differentiation of JerF between styryl‐substituted dihydropyrandion enantiomers was investigated. In combination with a spontaneous racemization occurring with this type of substrates, a new enzymatic dynamic kinetic resolution was observed, which was used for the enantioselective chemoenzymatic synthesis of kavalactone natural products and new derivatives. In combination with an HMT‐based SAM regeneration system, (+)‐kavain, (+)‐11,12‐dimethoxykavain and (+)‐12‐fluorokavain were prepared in 3‐4 steps on the 100 µmol scale with overall yields of 37‐57% and ees of 70‐86%. A mutational study based on an AlphaFold 2 model provided indications for active site residues with an influence on the performance of the enzyme that could be targets for engineering. This example illustrates how the exceptional enzymatic activities and specificities of biosynthetic enzymes can be exploited for the development of new synthesis approaches.

Intensified, Kilogram-Scaled, and Environment-Friendly: Chemoenzymatic Synthesis of Bio-Based Acylated Hydroxystyrenes.

Lignin-derived styrene derivatives are versatile building blocks for the manufacture of biobased polymers. As shown previously, phenol-protected hydroxystyrenes are accessible under industrially sound conditions (>100 g L-1, >95% yield) by subjecting biogenic phenolic acids to enzymatic decarboxylation and base-catalyzed acylation in nonaqueous media (wet cyclopentyl methyl ether, CPME). Herein, we demonstrate the production of 1 kg of 4-acetoxy-3-methoxy-styrene in a 10 L reactor and present practical adjustments to the up- and downstream processing that warrant a straightforward process and high isolated yields. Additionally, an environmental assessment is conducted, starting with a thorough E factor analysis to identify the sources that contribute most to the environmental burden (solvent and downstream processing). Also, the total CO2 production of the process is studied, including contributions from energy use and the treatment of generated wastes. The energy impact is evaluated through thermodynamic analysis, and the environmental footprint contributions by wastes-organic and aqueous fractions-are assessed based on CO2 emissions from solvent incineration and wastewater treatment, respectively. Overall, the holistic assessment of the process, its optimization, scale-up, product isolation, and environmental analysis indicate the feasibility of multistep chemoenzymatic reactions to deliver high-volume, low-value chemicals from biorefineries.

Chemoenzymatic Synthesis of Sulfated N-Glycans Recognized by Siglecs and Other Glycan-Binding Proteins.

Sulfated N-glycans are present in many glycoproteins, which are implicated in playing important roles in biological recognition processes. Here, we report the systematic chemoenzymatic synthesis of a library of sulfated and sialylated biantennary N-glycans and assess their binding to Siglecs and glycan-specific antibodies that recognize them as glycan ligands. The combined use of three human sulfotransferases, GlcNAc-6-O-sulfotransferase (CHST2), Gal-3-O-sulfotransferase (Gal3ST1), and keratan sulfate Gal-6-O-sulfotransferase (CHST1), resulted in asymmetric and symmetric branch-selective sulfation of the GlcNAc and/or Gal moieties of N-glycans. The extension of the sugar chain using α-2,3- and α-2,6-sialyltransferases afforded the sulfated and sialylated N-glycans. These synthetic glycans with different patterns of sulfation and sialylation were evaluated for binding to selected Siglecs and sulfoglycan-specific antibodies using glycan microarrays. The results confirm previously documented glycan-recognizing properties and further reveal novel specificities for these glycan-binding proteins, demonstrating the utility of the library for assessing the specificity of glycan-binding proteins recognizing sulfated and sialylated glycans.