Chemoenzymatic Approach Research Articles

A Sustainable Chemo-Enzymatic Approach to the Synthesis of Liraglutide

A Sustainable Chemo-Enzymatic Approach to the Synthesis of Liraglutide

Bioorthogonal Chemistry: Enzyme Immune and Protein Capture for Enhanced LC-MS Bioanalysis.

Immunocapture liquid chromatography-mass spectrometry (IC-LC-MS) bioanalysis has become an indispensable technique across various scientific disciplines, ranging from drug discovery to clinical diagnostics. While traditional immunocapture techniques have proven to be effective, they often encounter limitations in sensitivity, specificity, and compatibility with MS analysis. Chemoenzymatic immunocapture and protein capture (IPC) offers a promising solution, combining the high specificity of antibodies or proteins with the versatility of enzymatic and chemical modifications. This Review explores the foundational principles of chemoenzymatic IPC and examines various modification strategies including bioorthogonal click-chemistry, enzymatic-tagging, and HaloTag/CLIP-tag. Recent advancements in chemoenzymatic IPC techniques have significantly expanded their applicability to a diverse range of biomolecules including small molecules, peptides, RNAs, and proteins. This Review focuses on improvements in analytical performance achieved through these innovative approaches. Moreover, we discuss the broad applications of chemoenzymatic immunocapture in drug discovery, clinical diagnostics, and environmental analysis and explore its potential for future advancements in bioanalysis. We propose a novel solid-phase chemoenzymatic IPC assay (SCEIA) that effectively utilizes bioorthogonal click chemistry and chemoenzymatic approaches for efficient IPC and target analyte release. In summary, chemoenzymatic IPC represents a transformative paradigm shift in IC-LC-MS bioanalysis. By overcoming the limitations of traditional IPC techniques, this approach paves the way for more robust, sensitive, and versatile analytical workflows.

Chemoenzymatic Synthesis of Phosphosaccharides from Haemophilus parasuis Strains ER-6P (Serovar 15) and Nagasaki (Serovar 5).

Herein, we describe a chemoenzymatic and diversity-oriented approach for the first syntheses of octasaccharide repeating units of the capsular polysaccharides of Haemophilus parasuis serovar 15 and serovar 5. The synthetic method features efficient enzymatic assembly of sialyl galactose or N-acetyl-galactosamine building blocks, highly stereoselective chemical construction of α-type H-phosphonate, and the β-stereospecific 1,3-glycosylation reaction of a rare sugar donor.

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.

Engineering an Epoxide Hydrolase for Chemoenzymatic Asymmetric Synthesis of Chiral Triazole Fungicide (S)- and (R)-Flutriafol.

Flutriafol, a globally utilized triazole fungicide in agriculture, is typically applied as a racemic mixture, but its enantiomers differ in bioactivity and environmental impact. The synthesis of flutriafol enantiomers is critically dependent on chiral precursors: 2,2-bisaryl-substituted oxirane [(2-fluorophenyl)-2-(4-fluorophenyl)oxirane, 1a] and 1,2-diol [1-(2-fluorophenyl)-1-(4-fluorophenyl)ethane-1,2-diol, 1b]. Here, we engineered a Rhodotorula paludigensis epoxide hydrolase (RpEH), obtaining mutant Escherichia coli/RpehH336W/L360F with a 6.4-fold enhanced enantiomeric ratio (E) from 5.5 to 35.4. This enabled a gram-scale resolution of rac-1a by E. coli/RpehH336W/L360F, producing (S)-1a (98.2% ees) and (R)-1b (75.0% eep) with 44.3 and 55.7% analytical yields, respectively. As follows, chiral (S)-flutriafol (98.2% ee) and (R)-flutriafol (75.0% ee) were easily synthesized by a one-step chemocatalytic process from (S)-1a and a two-step chemocatalytic process from (R)-1b, respectively. This chemoenzymatic approach offers a superior alternative for the asymmetric synthesis of flutriafol enantiomers. Furthermore, molecular dynamics simulations revealed insight into the enantioselectivity improvement of RpEH toward bulky 2,2-bisaryl-substituted oxirane 1a.

Chemoenzymatic Labeling, Detection and Profiling of Core Fucosylation in Live Cells.

Core fucosylation, the attachment of an α-1,6-linked-fucose to the N-glycan core pentasaccharide, is an abundant protein modification that plays critical roles in various biological processes such as cell signaling, B cell development, antibody-dependent cellular cytotoxicity, and oncogenesis. However, the tools currently used to detect core fucosylation suffer from poor specificity, exhibiting cross-reactivity against all types of fucosylation. Herein we report the development of a new chemoenzymatic strategy for the rapid and selective detection of core fucosylated glycans. This approach employs a galactosyltransferase enzyme identified fromCaenorhabditis elegansthat specifically transfers an azido-appended galactose residue onto core fucose via a β-1,4 glycosidic linkage. We demonstrate that the approach exhibits superior specificity toward core fucose on a variety of complex N-glycans. The method enables detection of core fucosylated glycoproteins from complex cell lysates, as well as on live cell surfaces, and it can be integrated into a diagnostic platform to profile protein-specific core fucosylation levels. This chemoenzymatic labeling approach offers a new strategy for the identification of disease biomarkers and will allow researchers to further characterize the fundamental role of this important glycan in normal and disease physiology.

Chemoenzymatic Oxidation of Labdane and Formal Synthesis of Nimbolide.

In nature, basic terpene skeletons are produced and subsequently undergo enzymatic or nonenzymatic oxidative transformations, leading to diverse structural variations. To date, thousands of natural products featuring a variety of oxidation patterns have been isolated solely from the labdane family. This work describes a strategy for the comprehensive introduction of oxidation states into the labdane core by employing a combination of enzyme library screening, directed evolution, and sequential chemical oxidation processes. Furthermore, we showcase the functional viability of our chemoenzymatic approach by accomplishing a formal synthesis of nimbolide, highlighting its potential for streamlining the synthesis of complex natural products.

Template-dependent DNA ligation for the synthesis of modified oligonucleotides

Chemical modification of DNA is a common strategy to improve the properties of oligonucleotides, particularly for therapeutics and nanotechnology. Existing synthetic methods essentially rely on phosphoramidite chemistry or the polymerization of nucleoside triphosphates but are limited in terms of size, scalability, and sustainability. Herein, we report a robust alternative method for the de novo synthesis of modified oligonucleotides using template-dependent DNA ligation of shortmer fragments. Our approach is based on the fast and scaled accessibility of chemically modified shortmer monophosphates as substrates for the T3 DNA ligase. This method has shown high tolerance to chemical modifications, flexibility, and overall efficiency, thereby granting access to a broad range of modified oligonucleotides of different lengths (20 → 120 nucleotides). We have applied this method to the synthesis of clinically relevant antisense drugs and ultramers containing diverse modifications. Furthermore, the designed chemoenzymatic approach has great potential for diverse applications in therapeutics and biotechnology.

Diazirine-AIOC-NAADP, a Clickable-Photoactive NAADP Analog for Sea Urchin NAADP Binding Proteins.

Calcium ions (Ca2+) play a vital role as intracellular messengers, regulating essential cellular processes. Nicotinic acid adenine dinucleotide phosphate (NAADP) serves as a potent second messenger, responsible for releasing Ca2+ in both mammals and echinoderms. Despite identification of two human NAADP receptor proteins, their counterparts in sea urchins remain elusive. Sea urchin NAADP binding proteins are important due to their unique identities and NAADP binding properties which may illuminate new signaling modalities in other species. Consequently, the development of new photoactive and clickable NAADP analogs with specificity for binding targets in sea urchin egg homogenates is a priority. We designed and synthesized diazirine-AIOC-NAADP, a photoactive and "clickable" NAADP analog, to specifically label and identify sea urchin NAADP receptors. This analog, synthesized using a chemo-enzymatic approach, induced Ca2+ release from sea urchin egg homogenates at low-micromolar concentrations. The ability of diazirine-AIOC-NAADP to mobilize Ca2+ in cultured human cells was investigated by microinjection of the probe into U2OS cells. Microinjected NAADP elicited a robust Ca2+ release, but even 6000-fold higher concentrations of diazirine-AIOC-NAADP were unable to release Ca2+. Our results indicate that our new probe is specifically recognized at low concentration by sea urchin egg NAADP receptors but not by the NAADP receptors in a human cultured cell line.

Merging Organolithium Chemistry and Stereoselective Biocatalysis: Transformation of Aromatic Nitriles into Chiral Alcohols

AbstractThe combination of RLi‐mediated organic transformations (under air and at room temperature) with a subsequent stereoselective biocatalytic reaction is for the first time presented. Most of the previous asymmetric chemoenzymatic routes have been limited to the concomitant or sequential combination of transition metals/organocatalysts with enzymes. However, the use of polar organometallic reagents (RLi) in the design of these stereoselective hybrid protocols has been totally neglected, as far as we are concerned. Thus, in this work, the combination of organolithium chemistry and asymmetric biocatalysis is described for the first time in a one‐pot fashion. The chemoenzymatic approach converts a series of nitriles into chiral alcohols consisting of two steps, where the key item was the finding of suitable conditions to adapt the reactivities of organolithiums and alcohol dehydrogenases (ADHs) in the same recipient. The organolithium addition occurred with total chemoselectivity (no side reactions were observed) under neat conditions and room temperature leading to the corresponding imines, which were hydrolyzed using a buffer, and adjusting the pH for the subsequent ADH action. Commercial and made in house overexpressed ADHs allowed to produce chiral alcohols with excellent selectivities and good overall yields. The different behavior displayed for the reductive enzymes in the presence of diethyl ether clearly influenced in the decision to let the organolithium solvent evaporate under open‐air conditions before developing the bioreduction step. Our results demonstrate the importance of the fine orchestration of the reaction conditions for the development of efficient hybrid chemoenzymatic cascades without the need of intermediate isolation/purification steps or compartmentalization of the different synthetic systems.

Chemoenzymatic synthesis of sialyl-α2,3-lactoside–functionalized BSA conjugate inhibits influenza infection

Influenza remains a global public health threat, and the development of new antivirals is crucial to combat emerging drug-resistant influenza strains. In this study, we report the synthesis and evaluation of a sialyl lactosyl (TS)–bovine serum albumin (BSA) conjugate as a potential multivalent inhibitor of the influenza virus. The key trisaccharide component, TS, was efficiently prepared via a chemoenzymatic approach, followed by conjugation to dibenzocyclooctyne-modified BSA via a strain-promoted azide-alkyne cycloaddition reaction. Biophysical and biochemical assays, including surface plasmon resonance, isothermal titration calorimetry, hemagglutination inhibition, and neuraminidase inhibition, demonstrated the strong binding affinity of TS-BSA to the hemagglutinin (HA) and neuraminidase (NA) proteins of the influenza virus as well as intact virion particles. Notably, TS-BSA exhibited potent inhibitory activity against viral entry and release, preventing cytopathic effects in cell culture. This multivalent presentation strategy highlights the potential of glycocluster-based antivirals for combating influenza and other drug-resistant viral strains.

Investigation of N-Glycan Functions in Receptor for Advanced Glycation End Products V Domain through Chemical Glycoprotein Synthesis.

The receptor for advanced glycation end products (RAGE) plays a crucial role in inflammation-related pathways and various chronic diseases. Despite the recognized significance of N-glycosylation in the ligand-binding V domain (VD) of RAGE, a comprehensive understanding of the site-activity and structure-activity relationships is lacking due to the challenges in obtaining homogeneous glycoprotein samples through biological expression. Here, we combined chemical and chemoenzymatic approaches to synthesize RAGE-VD and its congeners with Asn3-glycosylation by incorporating precise N-glycan structures. Evaluation of these samples revealed that, in comparison to other RAGE-VD forms, α2,6-sialylated N-glycosylation at the Asn3 site results in more potent inhibition of HMGB1-induced nuclear factor-κB (NF-κB) expression in RAGE-overexpressing cells. Hydrogen/deuterium exchange-mass spectrum analysis revealed a sialylated RAGE-VD-induced interaction region within HMGB1. Conversely, Asn3 N-glycosylation in VD has negligible effects on RAGE-VD/S100B interactions. This study established an approach for accessing homogeneously glycosylated RAGE-VD and explored the modulatory effects of N-glycosylation on the interactions between RAGE-VD and its ligand proteins.

Enantioselective Synthesis of Secondary Amines by Combining Oxidative Rearrangement and Biocatalysis in a One-Pot Process.

This contribution describes the development of chemoenzymatic one-pot processes, which combine an oxidative rearrangement and a biotransformation catalyzed by an imine reductase (IRED), for the synthesis of highly enantiomerically enriched secondary amines, such as an aryl-substituted pyrrolidine and a benzazepine. The benefits of this chemoenzymatic one-pot approach include high overall conversions (up to >99%), high enantiomeric excesses (up to >99% ee), and a straightforward synthetic approach toward secondary amines without the need to isolate the formed intermediate. For the initial chemical reaction, namely, the oxidative rearrangement, PhI(OAc)2 in methanol is used as a non-natural reagent, whereas the enzymatic step requires only stoichiometric amounts of d-glucose along with catalytic amounts of IRED, glucose dehydrogenase (GDH), and the cofactor NADPH. This methodology, demonstrating the compatibility of a "classic" organic synthesis using a non-natural, highly reactive reagent and a subsequent biocatalytic step, can be applied for different amines as substrates, thus making this concept a versatile tool in synthetic organic chemistry in general and for enantioselective synthesis of heterocyclic secondary amines in particular.

Chemoenzymatic synthesis of (1R,3R)-3-hydroxycyclopentanemethanol: An intermediate of carbocyclic-ddA

The synthesis of carbocyclic-ddA, a potent antiviral agent against hepatitis B, relies significantly on (1R,3R)-3-hydroxycyclopentanemethanol as a key intermediate. To effectively produce this intermediate, our study employed a chemoenzymatic approach. The selection of appropriate biocatalysts was based on substrate similarity, leading us to adopt the CrS enoate reductase derived from Thermus scotoductus SA-01. Additionally, we developed an enzymatic system for NADH regeneration, utilising formate dehydrogenase from Candida boidinii. This system facilitated the efficient catalysis of (S)-4-(hydroxymethyl)cyclopent-2-enone, resulting in the formation of (3R)-3-(hydroxymethyl) cyclopentanone. Furthermore, we successfully cloned, expressed, purified, and characterized the CrS enzyme in Escherichia coli. Optimal reaction conditions were determined, revealing that the highest activity occurred at 45°C and pH 8.0. By employing 5mM(S)-4-(hydroxymethyl)cyclopent-2-enone, 0.05mM FMN, 0.2mM NADH, 10μM CrS, 40μM formic acid dehydrogenase, and 40mM sodium formate, complete conversion was achieved within 45min at 35°C and pH 7.0. Subsequently, (1R,3R)-3-hydroxycyclopentanemethanol was obtained through a simple three-step chemical conversion process. This study not only presents an effective method for synthesizing the crucial intermediate but also highlights the importance of biocatalysts and enzymatic systems in chemoenzymatic synthesis approaches.

Welding PROxAb Shuttles: A Modular Approach for Generating Bispecific Antibodies via Site-Specific Protein-Protein Conjugation.

Targeted protein degradation is an innovative therapeutic strategy to selectively eliminate disease-causing proteins. Exemplified by proteolysis-targeting chimeras (PROTACs), they have shown promise in overcoming drug resistance and targeting previously undruggable proteins. However, PROTACs face challenges, such as low oral bioavailability and limited selectivity. The recently published PROxAb Shuttle technology offers a solution enabling the targeted delivery of PROTACs using antibodies fused with PROTAC-binding domains derived from camelid single-domain antibodies (VHHs). Here, a modular approach to quickly generate PROxAb Shuttles by enzymatically coupling PROTAC-binding VHHs to off-the-shelf antibodies was developed. The resulting conjugates retained their target binding and internalization properties, and incubation with BRD4-targeting PROTACs resulted in formation of defined PROxAb-PROTAC complexes. These complexes selectively induced degradation of the BRD4 protein, resulting in cytotoxicity specifically to cells expressing the antibody's target. The chemoenzymatic approach described herein provides a versatile and efficient solution for generating antibody-VHH conjugates for targeted protein degradation applications, but it could also be used to combine antibodies and VHH binders to generate bispecific antibodies for further applications.

Engineering Styrene Oxide Isomerase Towards Enhanced Chemoenzymatic Synthesis of (S)‐Flurbiprofen

AbstractThe conventional synthesis of (S)‐flurbiprofen, a valuable non‐steroidal anti‐inflammatory drug (NSAID), involves inefficient chiral resolution with low‐yielding multiple reaction steps, or asymmetric synthesis with low enantioselectivity and/or regioselectivity. Here, we developed high‐yielding, enantioselective, and sustainable synthesis of (S)‐flurbiprofen via two chemoenzymatic approaches from a simple alkene: (A) enzymatic epoxidation‐isomerization‐oxidation cascade and Pd‐catalyzed arylation; and (B) chemical epoxidation, enzymatic isomerization‐oxidation cascade, and Pd‐catalyzed arylation. Styrene oxide isomerase (SOI)‐catalyzed enantio‐retentive Meinwald rearrangement was the key step in the two pathways, but SOI suffers from low activity. SOI was engineered via two rounds of iterative saturation mutagenesis and screening with a three‐enzyme cascade assay, generating a double‐mutant SOIDM (N99A/D95A) with 5.5‐fold improvement in catalytic efficiency (kcat/KM). In Pathway A, cascade biotransformation of 3‐fluoro‐4‐bromo‐α‐methylstyrene with engineered E. coli strain expressing SOIDM, styrene monooxygenase (SMO) and aldehyde dehydrogenase (ALDH), and subsequent Pd‐catalyzed arylation afforded (S)‐flurbiprofen in 98% yield and 91% ee. In Pathway B, chemical epoxidation (Sharpless dihydroxylation and ring‐closing), cascade conversion using an engineered E. coli strain expressing SOIDM, ALDH and NADH oxidase (NOX), and Pd‐catalyzed arylation produced (S)‐flurbiprofen in 83% yield and 98% ee. Compared to conventional synthesis, the developed methods reduced reaction steps, minimized waste generation and improved enantioselectivity or overall yield.

Chemoenzymatic Synthesis of N,N,N‐Trimethyl‐D‐Glucosamine Chitotriomycin and Its Analogues

Comprehensive SummaryN,N,N‐Trimethyl‐D‐glucosamine (TMG)‐chitotriomycin, a naturally occurring chitin related oligosaccharide, is a specific β‐N‐acetylhexosaminidases (HexNAcases) inhibitor for insects and fungi. Although TMG‐chitoriomycin holds great promise as a novel class insecticide and fungicide, the limited accessibility of TMG‐chitotriomycin prevents its further biological evaluation. We report herein a simple and eco‐friendly chemoenzymatic approach for the efficient synthesis of TMG‐chitotriomycin and its analogues. In this strategy, the readily available chitosan was enzymatically hydrolyzed and chemically N‐acetylated to afford the chitooligosaccharides ranging from disaccharide to hexasaccharide. These chitooligosaccharides were selectively deacetylated by two different chitin deacetylases followed by chemical N‐trimethylation to obtain the desired TMG‐chitotriomycin and a total of 13 TMG‐chitotriomycin analogues in the longest linear sequence of 4 steps in over 12% total yields.

Three-component systems represent a common pathway for extracytoplasmic addition of pentofuranose sugars into bacterial glycans

Cell surface glycans are major drivers of antigenic diversity in bacteria. The biochemistry and molecular biology underpinning their synthesis are important in understanding host-pathogen interactions and for vaccine development with emerging chemoenzymatic and glycoengineering approaches. Structural diversity in glycostructures arises from the action of glycosyltransferases (GTs) that use an immense catalog of activated sugar donors to build the repeating unit and modifying enzymes that add further heterogeneity. Classical Leloir GTs incorporate α- or β-linked sugars by inverting or retaining mechanisms, depending on the nucleotide sugar donor. In contrast, the mechanism of known ribofuranosyltransferases is confined to β-linkages, so the existence of α-linked ribofuranose in some glycans dictates an alternative strategy. Here, we use Citrobacter youngae O1 and O2 lipopolysaccharide O antigens as prototypes to describe a widespread, versatile pathway for incorporating side-chain α-linked pentofuranoses by extracytoplasmic postpolymerization glycosylation. The pathway requires a polyprenyl phosphoribose synthase to generate a lipid-linked donor, a MATE-family flippase to transport the donor to the periplasm, and a GT-C type GT (founding the GT136 family) that performs the final glycosylation reaction. The characterized system shares similarities, but also fundamental differences, with both cell wall arabinan biosynthesis in mycobacteria, and periplasmic glucosylation of O antigens first discovered in Salmonella and Shigella. The participation of auxiliary epimerases allows the diversification of incorporated pentofuranoses. The results offer insight into a broad concept in microbial glycobiology and provide prototype systems and bioinformatic guides that facilitate discovery of further examples from diverse species, some in currently unknown glycans.

Modular chemoenzymatic synthesis of ten fusicoccane diterpenoids.

Fusicoccane diterpenoids display intriguing biological activities, including the ability to act as modulators of 14-3-3 protein-protein interactions. However, their innate structural complexity and diverse oxygenation patterns present enormous synthetic challenges. Here we develop a modular chemoenzymatic approach that combines de novo skeletal construction and late-stage hybrid C-H oxidations to achieve the synthesis of ten complex fusicoccanes in 8-13 steps each. A convergent fragment coupling strategy allowed rapid access to a key tricyclic intermediate, which was subjected to chemical and enzymatic C-H oxidations to modularly prepare five oxidized family members. We also conceived a complementary biomimetic skeletal remodelling strategy to synthetically access five rearranged fusicoccanes with unusual bridgehead double bonds. This work may facilitate future investigation into the biological activities of the fusicoccanes and also inspire the implementation of similar hybrid strategies to provide family-level synthetic solutions to other natural product scaffolds.