Chemoenzymatic Approach Research Articles

Chemoenzymatic Synthesis of Optically Pure l- and d-Biarylalanines through Biocatalytic Asymmetric Amination and Palladium-Catalyzed Arylation

A chemoenzymatic approach was developed and optimized for the synthesis of a range of N-protected nonnatural l- and d-biarylalanine derivatives. Starting from 4-bromocinnamic acid and 4-bromophenylpyruvic acid using a phenylalanine ammonia lyase (PAL) and an evolved d-amino acid dehydrogenase (DAADH), respectively, both enantiomers of 4-bromophenylalanine were obtained and subsequently coupled with a panel of arylboronic acids to give the target compounds in high yield and optical purity under mild aqueous conditions.

Chemo-enzymatic synthesis of imidazolium-tagged sialyllactosamine probes.

Two novel α-linked sialyltrisaccharide imidazolium-type probes (ITags) based on the structures of biologically relevant 6'-sialyllactosamine and 3'-sialyllactosamine were efficiently and stereoselectively prepared using a chemo-enzymatic approach. The apparent kinetic parameters for the enzyme catalyzed transformations with α-2,3-sialyltransferase (α-2,3-ST) and α-2,6-sialyltransferase (α-2,6-ST) were measured by LC-MS using the ionic probes. This strategy demonstrates the suitability of the ITags to probe glycosyltransferase activity and their versatility in the preparation of sialylated epitopes for glycobiology research.

Efficient two-step chemo-enzymatic synthesis of all-trans-retinyl palmitate with high substrate concentration and product yield.

A new two-step chemo-enzymatic approach for highly efficient synthesis of all-trans-retinyl palmitate is constructed in this study. In the first step, retinyl acetate as starting material was fully hydrolyzed to retinol by potassium hydroxide. In the hydrolysis system, anhydrous ethanol was the best co-solvent to increase the solubility of retinyl acetate. The addition amounts of 5 M potassium hydroxide and anhydrous ethanol were 8 and 10 mL against 10 g retinyl acetate, respectively, and 100 % hydrolysis rate was obtained. In the second step, esterification was catalyzed by immobilized lipase on macroporous acrylic resin AB-8 using the extracted retinol and palmitic acid as substrates in non-aqueous system. After optimization, the parameters of esterification reaction were confirmed as follows: non-aqueous solvent was selected as n-hexane, washing times of extraction solution was four times, retinol concentration was 300 g/L, substrate molar ratio of retinol to palmitic acid was 1:1.1, the amount of immobilized enzyme was 10 g/L, and the esterification temperature was 30 °C. Under the optimal conditions, this protocol resulted in a 97.5 % yield of all-trans-retinyl palmitate in 700-L reactor. After purification, all-trans-retinyl palmitate was obtained with above 99 % of purity and 88 % of total recovery rate. This methodology provides a promising strategy for the large-scale production of all-trans-retinyl palmitate.

Total Synthesis of Solandelactones A and B

AbstractThe total synthesis of solandelactones A and B is presented. The eastern cyclopropyl moiety was prepared following an exceptionally short chemoenzymatic approach whereas enantioselective synthesis of the western side‐chain relied on the application of diastereomerically pure allyl boronates. The natural products solandelactones A and B were isolated in good overall yields following convergence of each eastern and western element by application of the Nozaki–Hiyama–Kishi reaction.

Glycoengineered Monoclonal Antibodies with Homogeneous Glycan (M3, G0, G2, and A2) Using a Chemoenzymatic Approach Have Different Affinities for FcγRIIIa and Variable Antibody-Dependent Cellular Cytotoxicity Activities.

Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain), and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-β-N-acetyl glucosaminidases (ENG’ases), one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2), high-mannose type (Man4-9GlcNAc2), and complex type (Man3GlcNAc3-4) N-glycans. As a result of the cleavage of several ENG’ases (endoS, endoM, endoD, endoH, and endoLL), the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1) were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG’ase mutant (endoS-D233Q), and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2) was performed using SKBR-3 and BT-474 as target cells, and revealed that the glycoform influenced ADCC activity.

Self-Assembled Hybrid Aptamer-Fc Conjugates for Targeted Delivery: A Modular Chemoenzymatic Approach.

Over the past decade, DNA and RNA aptamers have attracted keen research interest due to their ability to specifically bind targets of therapeutic relevance. However, their application is often hampered by a short serum half-life and missing effector functions. Conjugation of aptamers to antibody Fc fragments could improve pharmacokinetics, enable immune effector mechanisms, and provide an option for the introduction of desired payloads (e.g., toxins or fluorescent dyes). We developed a modular scaffold-supported system based on human IgG1 Fc fragments, which allows for its dual functionalization with moieties of interest. In our approach, two bioorthogonal, enzyme-mediated reactions were used in combination with oxime ligation and self-assembly based on PNA-DNA base pairing. Thus, an engineered synthetic peptide nucleic acid (PNA) oligomer was coupled to the C-termini of the Fc dimer upon sequence-specific sortase A-mediated transpeptidation. Hybridization of the resulting Fc-PNA conjugate with a tailored DNA aptamer that binds cancer-related hepatocyte growth factor receptor (c-MET) led to a hybrid construct which showed strong and specific binding to c-MET and was readily internalized by c-MET-overexpressing cells. To install an additional orthogonally addressable site, aldehyde tag technology was applied followed by oxime ligation with an aminooxy-bearing fluorescent dye as model cargo. Delivery of fluorescent probe specifically to c-MET-overexpressing cells was confirmed by flow cytometry. Our approach can provide access to engineered aptamer-Fc conjugates with desired target specificity and cytotoxic payloads.

Advances in the Development of Site-Specific Antibody-Drug Conjugation.

Antibody-drug conjugates (ADCs) showed strong anticancer efficacy in the clinic. However, the current conventional technologies generate conjugates with undefined attachment sites and heterogeneous profiles containing different sub-populations, leading to potential off-target toxicity. In order to reduce the variability and heterogeneity associated with the ADCs generated using conventional technologies, several site-specific antibody-drug conjugation strategies were developed for the next generation of ADCs. These strategies include cysteine-targeted conjugation by engineering a free cysteine into the antibody or by placing a thiol bridge on cysteines in hinge disulfides. Glutamine-targeted conjugation was also demonstrated by coupling the drug-linker to glutamine residues through an engineered glutamine tag or a native glutamine, as well as an additionally introduced glutamine residue in aglycosylated antibody mutant using microbial transglutaminase. The site-specific conjugation of drug-linker to antibody carbohydrates was developed either through metabolic engineering or a chemo-enzymatic approach. Other amino acids, such as unnatural amino acids or amino acid derivatives introduced through protein engineering, have also been shown to be efficient targets for site-specific conjugation. The sitespecific ADCs with homogeneous profiles and well-defined conjugation sites were obtained using these second generation ADC methods and showed potent in vitro cytotoxicity and strong in vivo antitumor activity. These results suggest that newly developed site-specific conjugation technologies can potentially be applied in producing the next generation ADC for cancer treatment in the clinic with high therapeutic index.

Using Strictosidine Synthase to Prepare Novel Alkaloids

The Pictet-Spenglerase strictosidine synthase (STR) has been characterized as the central enzyme in the biosynthesis of around 2000 monoterpenoid indole alkaloids in plants. In the light of a high therapeutic value and huge scaffold diversity these alkaloids represent, STR as an enzyme has attracted great attentions in recent years, intending to be utilized in the formation of new interesting alkaloids with unusual substitution pattern or even with novel scaffolds. For outlining the application potential that STR possesses, together with insight into the reaction mechanism catalyzed by STR, strategies and methods for exploring the applicability of STR have been updated in this article by taking R. serpentina STR (RS-STR) and C. roseus. STR (CR-STR) as representative models, followed by introducing the latest released complex structures of RS-STR with new substrates. Examples provided here, including substrate scaffold tailoring, X-ray crystal complex structure comparison, protein engineering and biosynthetic pathway reprogramming, pave the way to finally construct novel alkaloids libraries by chemo-enzymatic approaches. Keywords: Alkaloids, application strategies, protein engineering, strictosidine synthase, substrates, X-ray complex structures.

A new chemo-enzymatic approach to the stereoselective synthesis of the flavors tetrahydroactinidiolide and dihydroactinidiolide

The alcohols cis-5-hydroxy-cyclogeraniol and cis-α-epoxy-cyclogeraniol can be easily prepared with high diastereoisomeric purity starting from ethyl-α-cyclogeraniate. A few straightforward chemical transformations allow their conversion into tetrahydroactinidiolide and dihydroactinidiolide, two relevant natural flavors of carotenoid origin. The two starting C10 alcohols can also be obtained in enantioenriched form by means of an optimized lipase-mediated resolution procedure. The fractional crystallization of the obtained enantioenriched tetrahydroactinidiolide proved to be very efficient, allowing the preparation of both tetrahydroactinidiolide and dihydroactinidiolide in enantiopure form (ee >98%). Finally, a comprehensive study on the oxidation of tetrahydroactinidiolide to dihydroactinidiolide was accomplished by devising a new selenium-free chemical process for this type of transformation.

Chemoenzymatic Synthesis of (36)S Isotopologues of Methionine and S-Adenosyl-L-methionine.

Substrates containing isotope labels at specific atoms are required for transition-state analysis based on the measurement of multiple kinetic isotope effects.(36)S-labeled l-methionine and S-adenosyl-l-methionine were synthesized from elemental sulfur using a chemoenzymatic approach with >98% (36)S enrichment. This method provides access to previously inaccessible sulfur isotope-labeled substrates for sulfur kinetic isotope effect studies.

Chemoenzymatic approach to optically active 1,4-dihydropyridine derivatives

A series of racemic (2-methyl)propanoyloxymethyl 4-aryl-6-chloro-5-methanoyl-2-methyl-1,4-dihydropyridine-3-carboxylates [(±)-5a–h] have been prepared by a four step sequence including a multicomponent Hantzsch process and a Vilsmeier–Haack reaction. The subsequent resolution of (±)-5a–h was carried out by means of lipase-catalyzed hydrolysis, the most adequate enzymes being lipases from Candida rugosa (CRL) and Candida antarctica (CAL-B). The moderate to high enantioselectivities values (E up to >200) obtained in most cases allowed us to obtain the corresponding optically active 1,4-dihydropyridine derivatives with high enantiomeric excesses (ee≥94%) and yields.

O‐GlcNAc Modification of Runx2 Links Nutrient Metabolism with Osteogenesis

The runt-related transcription factor 2 (Runx2) is the master switch controlling osteoblast differentiation and formation of the mineralized skeleton. The post-translational modification (PTM) of Runx2 by phosphorylation, ubiquitinylation, and acetylation modulates its activity, stability, and interactions with transcriptional co-regulators and chromatin remodeling proteins downstream of osteogenic signals. We have found that Runx2 is O-GlcNAc modified within the N-terminal activation domain and the proline/serine/threonine (PST)-rich domain, regions which are known to regulate Runx2 transcriptional activity. A chemoenzymatic labeling approach, employed to measure the stoichiometry of Runx2 O-GlcNAcylation in osteogenic bone marrow derived mesenchymal stem cells (BMMSCs), demonstrated approximately 40% of Runx2 was O-GlcNAc modified after pharmacological inhibition of O-GlcNAcase (OGA) with Thiamet G. In MC3T3-E1 preosteoblasts, Thiamet G treatment resulted in increased Runx2 transcriptional activity which was dependent on Mek1/2 activity. Furthermore, osteogenic differentiation in the presence of Thiamet G, enhanced basal and potentiated BMP2/7-induced activity of the early bone marker, alkaline phosphatase (ALP). These studies suggest that elevated O-GlcNAc modification in osteogenic stem cells and in osteoblasts potentiates Runx2 activity.

Chemoenzymatic preparation of musky macrolactones

A chemoenzymatic approach to some musk macrolactones has been explored by the optimization of macrolactonization catalyzed by Candida antarctica lipase B (Novozym 435). This fast and high yield optimized methodology represents a large improvement to previously reported results. The methodology was applied to the preparation of 16-hexadecanolide, exaltolide, ambrettolide and (15R)-15-hexadecanolide.

Synthesis and microarray-assisted binding studies of core xylose and fucose containing N-glycans.

The synthesis of a collection of 33 xylosylated and core-fucosylated N-glycans found only in nonmammalian organisms such as plants and parasitic helminths has been achieved by employing a highly convergent chemo-enzymatic approach. The influence of these core modifications on the interaction with plant lectins, with the human lectin DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Nonintegrin), and with serum antibodies from schistosome-infected individuals was studied. Core xylosylation markedly reduced or completely abolished binding to several mannose-binding plant lectins and to DC-SIGN, a C-type lectin receptor present on antigen presenting cells. Employing the synthetic collection of core-fucosylated and core-xylosylated N-glycans in the context of a larger glycan array including structures lacking these core modifications, we were able to dissect core xylose and core fucose specific antiglycan antibody responses in S. mansoni infection sera, and we observed clear and immunologically relevant differences between children and adult groups infected with this parasite. The work presented here suggests that, quite similar to bisecting N-acetylglucosamine, core xylose distorts the conformation of the unsubstituted glycan, with important implications for the immunogenicity and protein binding properties of complex N-glycans.

Quantum Chemical Calculations and Experimental Validation of the Photoclick Reaction for Fluorescent Labeling of the 5' cap of Eukaryotic mRNAs.

Bioorthogonal click reactions are powerful tools to specifically label biomolecules in living cells. Considerable progress has been made in site-specific labeling of proteins and glycans in complex biological systems, but equivalent methods for mRNAs are rare. We present a chemo-enzymatic approach to label the 5’ cap of eukaryotic mRNAs using a bioorthogonal photoclick reaction. Herein, the N7-methylated guanosine of the 5’ cap is enzymatically equipped with an allyl group using a variant of the trimethylguanosine synthase 2 from Giardia lamblia (GlaTgs2). To elucidate whether the resulting N2-modified 5’ cap is a suitable dipolarophile for photoclick reactions, we used Kohn–Sham density functional theory (KS-DFT) and calculated the HOMO and LUMO energies of this molecule and nitrile imines. Our in silico studies suggested that combining enzymatic allylation of the cap with subsequent labeling in a photoclick reaction was feasible. This could be experimentally validated. Our approach generates a turn-on fluorophore site-specifically at the 5’ cap and therefore presents an important step towards labeling of eukaryotic mRNAs in a bioorthogonal manner.

Construction of a high-mannose-type glycan library by a renewed top-down chemo-enzymatic approach.

A comprehensive method for the construction of a high-mannose-type glycan library by systematic chemo-enzymatic trimming of a single Man9-based precursor was developed. It consists of the chemical synthesis of a non-natural tridecasaccharide precursor, the orthogonal demasking of the non-reducing ends, and trimming by glycosidases, which enabled a comprehensive synthesis of high-mannose-type glycans in their mono- or non-glucosylated forms. It employed glucose, isopropylidene, and N-acetylglucosamine groups for blocking the A-, B-, and C-arms, respectively. After systematic trimming of the precursor, thirty-seven high-mannose-type glycans were obtained. The power of the methodology was demonstrated by the enzymatic activity of human recombinant N-acetylglucosaminyltransferase-I toward M7-M3 glycans, clarifying the substrate specificity in the context of high-mannose-type glycans.

Facile chemoenzymatic synthesis of biotinylated heparosan hexasaccharide.

A biotinylated heparosan hexasaccharide was synthesized using a one-pot multi-enzyme strategy, in situ activation and transfer of N-trifluoroacetylglucosamine (GlcNTFA) to a heparin backbone significantly improved the synthetic efficiency. The biotinylated hexasaccharide could serve as a flexible core to diversify its conversion into heparan sulfate isoforms with potential biological applications and therapeutics.

A chemoenzymatic approach toward the preparation of site-specific antibody–drug conjugates

An efficient chemical synthesis of UDP-N-azidoacetylgalactosamine (UDP-GalNAz) is presented, while the value of this molecule was demonstrated through its attachment to an antibody Fc domain. Thus, the antibody was first degalactosylated, which was followed by loading of the UDP-GalNAz with a recombinant galactosyltransferase. This engineered Azide-Fc-N-glycan antibody was subsequently ‘clicked’ by a strain-promoted alkyne-azide cycloaddition reaction for site-specific attachment of a fluorescent probe. The principles detailed will allow for the facile preparation of chemically defined homogeneous antibody–drug conjugates (ADCs).

Impact of structural differences in hyperbranched polyglycerol–polyethylene glycol nanoparticles on dermal drug delivery and biocompatibility

Polyglycerol scaffolds and nanoparticles emerged as prominent material for various biomedical applications including topical drug delivery. The impact of slight structural modifications on the nanoparticles’ properties, drug delivery potential, and biocompatibility, however, is still not fully understood.Hence, we explored the influence of structural modifications of five structurally related polyglycerol-based nanoparticles (PG–PEG, SK1–SK5) on dermal drug delivery efficiency and biocompatibility. The PG–PEG particles were synthesized via randomly and controlled alkylated chemo-enzymatic approaches resulting in significantly varying particle sizes and interactions with guest molecules. Furthermore, we observed considerably improved dermal drug delivery with the smallest particles SK4 and SK5 (11nm and 14nm) which also correlated with well-defined surface properties achieved by the controlled alkylated synthesis approach. The consistently good biocompatibility for all PG–PEG particles was mainly attributed to the neutral surface charge. No irritation potential, major cytotoxicity or genotoxicity was observed. Nevertheless, slightly better biocompatibility was again seen for the particles characterized by alkyl chain substitution in the core and not on the particle surface.Despite the high structural similarity of the PG–PEG particles, the synthesis and the functionalization significantly influenced particle properties, biocompatibility, and most significantly the drug delivery efficiency.

Protecting‐Group‐Free Diastereoselective Total Synthesis of (±)‐6‐epi‐Cleistenolide and Chemoenzymatic Synthesis of (–)‐6‐epi‐Cleistenolide

AbstractA short, efficient, practical, and protecting‐group‐free diastereoselective total synthesis of (±)‐6‐epi‐cleistenolide (1) has been achieved in five steps in 60 % overall yield. The use of a chemoenzymatic approach also gave (–)‐6‐epi‐cleistenolide (1) (>99.9 % ee). The Achmatowicz reaction, chemoselective oxidation of a hemiacetal, diastereoselective 1,3‐anti reduction of a β‐hydroxy ketone, and enzymatic resolution of a 1,3‐diol are the key features of this linear total synthesis. The synthetic strategy demonstrated in this paper could be extended for an asymmetric total synthesis of (–)‐cleistenolide (1) and related biologically active natural products.