Amination of naringinase to improve citrus juice debittering using a catalyst immobilized on glyoxyl-agarose.

A naringinase complex was chemically aminated prior to its immobilization on glyoxyl-agarose to develop a robust biocatalyst for juice debittering. The effects of amination on the optimal pH and temperature, thermal stability, and debittering performance were analyzed. Concentration of amino groups on catalysts surface increased in 36 %. Amination reduced the ß-glucosidase activity of naringinase complex; however, did not affect optimal pH and temperature of the enzyme and it favored immobilization, obtaining α-l-rhamnosidase and ß-d-glucosidase activities of 1.7 and 4.2 times the values obtained when the unmodified enzymes were immobilized. Amination favored the stability of the immobilized biocatalyst, retaining 100 % of both activities after 190 h at 30 °C and pH 3, while its non-aminated counterpart retained 80 and 52 % of α-rhamnosidase and ß-glucosidase activities, respectively. The immobilized catalyst showed a better performance in grapefruit juice debittering, obtaining a naringin conversion of 7 times the value obtained with the non-aminated catalyst.

Stereoselective and controllable C3-Amination or C1,C3-di-amination of 2-nitroglycals.

Precise and selective modification of carbohydrates is a critical strategy in producing diverse carbohydrate derivatives for exploiting their functions. We disclosed a simple, efficient, and highly regioselective and stereoselective protocol to controllable amination of 2-nitroglycals under mild conditions in 5 min. A range of 3-amino-carbohydrates including 3-arylamino-2-nitro-glycals and 1,3-di-amino-carbohydrate derivatives were obtained in good to excellent yield with excellent stereoselectivity. The produced 3-amino-2-nitro-glycals can be used as a precursor for further transformation.

Protein Tyrosine Amination: Detection, Imaging, and Chemoproteomic Profiling with Synthetic Probes.

Protein tyrosine nitration (PTN) by oxidative and nitrative stress is a well-known post-translational modification that plays a role in the initiation and progression of various diseases. Despite being recognized as a stable modification for decades, recent studies have suggested the existence of a reduction in PTN, leading to the formation of 3-aminotyrosine (3AT) and potential denitration processes. However, the vital functions of 3AT-containing proteins are still unclear due to the lack of selective probes that directly target the protein tyrosine amination. Here, we report a novel approach to label and enrich 3AT-containing proteins with synthetic salicylaldehyde (SAL)-based probes: SALc-FL with a fluorophore and SALc-Yn with an alkyne tag. These probes exhibit high selectivity and efficiency in labeling and can be used in cell lysates and live cells. More importantly, SALc-Yn offers versatility when integrated into multiple platforms by enabling proteome-wide quantitative profiling of cell nitration dynamics. Using SALc-Yn, 355 proteins were labeled, enriched, and identified to carry the 3AT modification in oxidatively stressed RAW264.7 cells. These findings provide compelling evidence supporting the involvement of 3AT as a critical intermediate in nitrated protein turnover. Moreover, our probes serve as powerful tools to investigate protein nitration and denitration processes, and the identification of 3AT-containing proteins contributes to our understanding of PTN dynamics and its implications in cellular redox biology.

Chemoenzymatic total synthesis of rotigotine via IRED-catalyzed reductive amination.

A short and chemoenzymatic synthesis of rotigotine using an IR-36-M5 mutant is reported. Focusing on the residues that directly contact the 2-tetralone moiety, we applied structure-guided semi-rational design to obtain a double-mutant F260W/M147Y, which showed a good isolated yield and S-stereoselectivity >99% toward 2-aminotetralin synthesis. Furthermore, the utility of this biocatalytic protocol was successfully demonstrated in the enantioselective synthesis of rotigotine via enzymatic reductive amination as the key step.

Amination and crosslinking of acetone-fractionated hardwood kraft lignin using different amines and aldehydes for sustainable bio-based wood adhesives.

Hardwood kraft lignin from the pulping industry is burned or discarded. Its valorization was conducted by subjecting fractionation, amination with ethylenediamine, diethylenetriamine, and monoethanolamine, and crosslinking with formaldehyde or glyoxal to obtain bio-based wood adhesives. Acetone-soluble and insoluble hardwood kraft lignin were prepared and subjected to amination and then crosslinking. Fourier transform infrared, 13C NMR, 15N NMR, and X-ray photoelectron spectroscopy results revealed successful amination with amide, imine, and ether bonds and crosslinking of all samples. Hardwood kraft lignin aminated with diethylenetriamine/ethylenediamine and crosslinked using glyoxal exhibited excellent results in comparison with samples crosslinked using formaldehyde. Acetone-insoluble hardwood kraft lignin aminated and crosslinked using diethylenetriamine and formaldehyde, respectively, exhibited excellent adhesion strength with plywood, satisfying the requirements of the Korean standards. The amination and crosslinking of industrial waste hardwood kraft lignin constitute a beneficial valorization method.

Chemoenzymatic Synthesis of Selegiline: An Imine Reductase-Catalyzed Approach.

(R)-Homobenzylic amines are key structural motifs present in (R)-selegiline, a drug indicated for the treatment of early-stage Parkinson's disease. Herein, we report a new short chemoenzymatic approach (in 2 steps) towards the synthesis of (R)-selegiline via stereoselective biocatalytic reductive amination as the key step. The imine reductase IR36-M5 mutant showed high conversion (97%) and stereoselectivity (97%) toward the phenylacetone and propargyl amine substrates, offering valuable biocatalysts for synthesizing alkylated homobenzylic amines.

Reductive amination of ω-conotoxin MVIIA: synthesis, determination of modification sites, and self-assembly.

Peptide drugs have disadvantages such as low stability, short half-life and side effects, which limit their widespread use in clinical practice. Therefore, peptide drugs can be modified to improve these disadvantages. Numerous studies have shown that alkyl-modified peptide drugs can self-assemble to prolong the duration of efficacy and/or reduce side effects. However, the commonly used solid-phase synthesis method for alkyl-modified peptides is time-consuming. To overcome this, a simple reductive amination reaction was employed, which can directly graft the alkyl chain to the peptide sequence and effectively avoid stepwise synthesis from C- to N-terminal with amino acids. In this study, ω-conotoxin MVIIA was used as the peptide drug, while myristic aldehyde was used as the alkylating agent. To obtain the maximum productivity of modified peptides, the molar ratio of peptide MVIIA to myristic aldehyde in the reductive amination reaction was optimized. Furthermore, the peptide modification sites in this reaction were confirmed by secondary mass spectrometry analysis. Besides, alkyl-modified peptide MVIIA was able to form micelles by self-assembly and improved stability in serum, which was related to our previous work where myristoylated peptide MVIIA micelles can improve the drug stability. Finally, this study was intended to provide a methodological basis for modifying the alkyl chain of peptide drugs.

Use of Reductive Amination to Produce Capsular Polysaccharide-Based Glycoconjugates.

Reductive amination is a relatively simple and convenient strategy for coupling purified polysaccharides to carrier proteins. Following their synthesis, glycoconjugates can be used to assess the protective capacity of specific microbial polysaccharides in animal models of infection and/or to produce polyclonal antiserum and monoclonal antibodies for a variety of immune assays. Here, we describe a reproducible method for chemically activating the 6-deoxyheptan capsular polysaccharide (CPS) from Burkholderia pseudomallei and covalently linking it to recombinant CRM197 diphtheria toxin mutant (CRM197) to produce the glycoconjugate, CPS-CRM197. Similar approaches can also be used to couple other types of polysaccharides to CRM197 with little to no modification of the protocol.

sp3 -Rich Heterocycle Synthesis on DNA: Application to DNA-Encoded Library Production.

DNA encoded library (DEL) synthesis represents a convenient means to produce, annotate and store large collections of compounds in a small volume. While DELs are well suited for drug discovery campaigns, the chemistry used in their production must be compatible with the DNA tag, which can limit compound class accessibility. As a result, most DELs are heavily populated with peptidomimetic and sp2 -rich molecules. Herein, we show that sp3 -rich mono- and bicyclic heterocycles can be made on DNA from ketochlorohydrin aldol products through a reductive amination and cyclization process. The resulting hydroxypyrrolidines possess structural features that are desirable for DELs and target a distinct region of pharmaceutically relevant chemical space.

Intramolecular C-H bond amination catalyzed by myoglobin reconstituted with iron porphycene.

C-H bond amination is an effective way to obtain nitrogen-containing products. In this work, we demonstrate that myoglobin reconstituted with iron porphycene (rMb(FePc)) catalyzes intramolecular C(sp3)-H bond amination of arylsulfonyl azides to yield corresponding sultam analogs. The total turnover number of rMb(FePc) is up to 5.7 × 104 for the C-H bond amination of 2,4,6-triisopropylbenzenesulfonyl azide. Moreover, rMb(FePc) exhibits higher selectivity for the desired C-H bond amination than the competing azide reduction compared to native myoglobin. Kinetic studies reveal that the kcat value of rMb(FePc) is 4-fold higher than that of native myoglobin. Furthermore, H64A, H64V and H64I mutants of rMb(FePc) enhance the turnover number (TON) and enantioselectivity for the C-H bond amination of 2,4,6-triethylbenzenesulfonyl azide. The present findings indicate that iron porphycene is an attractive artificial cofactor for myoglobin toward the C-H bond amination reaction.

Palladium N-Heterocyclic Carbene-Catalyzed Aminations: An Outline.

Amination reactions play a pivotal role in synthetic organic chemistry, facilitating the generation of nitrogen-containing scaffolds with broad applications in drug synthesis, material production, polymer formation, and the generation of amino acids and peptides. Amination offers the potential to fine tune the properties of natural products and produce functional materials for various applications. Palladium N-heterocyclic carbene (Pd-NHC) emerges as an innovative and highly effective catalyst in this context. Under favorable reaction conditions, this robust and simple catalyst efficiently facilitates the synthesis of a diverse range of compounds with varying complexity and utility. Pd-NHC complexes exhibit significant σ-electron donating potential, enhancing the ease of the oxidative addition process in their mechanistic pathway. Their steric topography further contributes to a rapid reductive elimination. These complexes demonstrate remarkable stability, a result of the strong Pd-ligand bond. The wide variety of Pd-NHC complexes has proven highly efficient in catalyzing reactions across a spectrum of complexities, from simple to intricate. The domain of aminations catalyzed by Pd-NHC has undergone significant diversification, presenting new opportunities, particularly in the realms of material chemistry and natural product synthesis. This review outlines the advancements in Pd-NHC-catalyzed amination reactions, covering literature up to date.

Micelle-Promoted Reductive Amination of DNA-Conjugated Amines for DNA-Encoded Library Synthesis.

DNA-encoded libraries (DELs) have become a leading technology for hit identification in drug discovery projects as large, diverse libraries can be generated. DELs are commonly synthesised via split-and-pool methodology; thus, chemical transformations utilised must be highly efficient, proceeding with high conversions. Reactions performed in DEL synthesis also require a broad substrate scope to produce diverse, drug-like libraries. Many pharmaceutical compounds incorporate multiple C-N bonds, over a quarter of which are synthesised via reductive aminations. However, few on-DNA reductive amination procedures have been developed. Herein is reported the application of the micelle-forming surfactant, TPGS-750-M, to the on-DNA reductive amination of DNA-conjugated amines, yielding highly efficient conversions with a broad range of aldehydes, including medicinally relevant heterocyclic and aliphatic substrates. The procedure is compatible with DNA amplification and sequencing, demonstrating its applicability to DEL synthesis.

Discovery and biocatalytic characterization of opine dehydrogenases by metagenome mining.

Enzymatic processes play an increasing role in synthetic organic chemistry which requires the access to a broad and diverse set of enzymes. Metagenome mining is a valuable and efficient way to discover novel enzymes with unique properties for biotechnological applications. Here, we report the discovery and biocatalytic characterization of six novel metagenomic opine dehydrogenases from a hot spring environment (mODHs) (EC 1.5.1.X). These enzymes catalyze the asymmetric reductive amination between an amino acid and a keto acid resulting in opines which have defined biochemical roles and represent promising building blocks for pharmaceutical applications. The newly identified enzymes exhibit unique substrate specificity and higher thermostability compared to known examples. The feature that they preferably utilize negatively charged polar amino acids is so far unprecedented for opine dehydrogenases. We have identified two spatially correlated positions in their active sites that govern this substrate specificity and demonstrated a switch of substrate preference by site-directed mutagenesis. While they still suffer from a relatively narrow substrate scope, their enhanced thermostability and the orthogonality of their substrate preference make them a valuable addition to the toolbox of enzymes for reductive aminations. Importantly, enzymatic reductive aminations with highly polar amines are very rare in the literature. Thus, the preparative-scale enzymatic production, purification, and characterization of three highly functionalized chiral secondary amines lend a special significance to our work in filling this gap. KEY POINTS: • Six new opine dehydrogenases have been discovered from a hot spring metagenome • The newly identified enzymes display a unique substrate scope • Substrate specificity is governed by two correlated active-site residues.

Metal-free visible light mediated direct C-H amination of benzoxazole with secondary amines.

An efficient visible light mediated, eosin Y catalyzed direct C-H oxidative amination of benzoxazoles with secondary amines has been developed, which providing a straightforward, green, and environmentally benign access to a wide variety of substituted benzoxazole-2-amines under mild reaction conditions. The biological studies such as drug-likeness and molecular docking are also carried out on the molecule.

Fabrication of self-assembled micelles based on amphiphilic oxidized sodium alginate grafted oleoamine derivatives via Schiff base reduction amination reaction for delivery of hydrophobic food active ingredients.

The application of hydrophobic ß-carotene in the food industry are limited due to its susceptibility to light, high temperature, pH value, and other factors, leading to poor stability and low bioavailability. To address this problem, we adopt a more green and environmentally friendly reducing agent, 2-methylpyridine borane complex (pic-BH3), instead of traditional sodium borohydride, to achieve the simple green and efficient synthesis of amphiphilic oxidized sodium alginate grafted oleoamine derivatives (OSAOLA) through the reduction amination reaction of Schiff base. The resultant OSAOLA with the degree of substitution (DS) of 7.2 %, 23.6 %, and 38.8 % were synthesized, and their CMC values ranged from 0.0095 to 0.062 mg/mL, indicating excellent self-assembly capability in aqueous solution. Meanwhile, OSAOLA showed no obvious cytotoxicity to RAW 264.7 cells, thus revealing good biocompatibility. Furthermore, ß-carotene, as the hydrophobic active ingredients in foods was successfully encapsulated in the OSAOLA micelles by ultrasonic-dialysis method. The prepared drug-loaded OSAOLA micelles could maintain good stability when stored at room temperature for 7 d. Additionally, they were able to continuously release ß-carotene and exert long-term effects in pH 7.4 PBS at 37 °C, effectively improving the bioavailability of ß-carotene, which exhibited tremendous application potential in functional food and biomedical fields.

Biocatalytic reductive aminations with NAD(P)H-dependent enzymes: enzyme discovery, engineering and synthetic applications.

Chiral amines are pivotal building blocks for the pharmaceutical industry. Asymmetric reductive amination is one of the most efficient and atom economic methodologies for the synthesis of optically active amines. Among the various strategies available, NAD(P)H-dependent amine dehydrogenases (AmDHs) and imine reductases (IREDs) are robust enzymes that are available from various sources and capable of utilizing a broad range of substrates with high activities and stereoselectivities. AmDHs and IREDs operate via similar mechanisms, both involving a carbinolamine intermediate followed by hydride transfer from the co-factor. In addition, both groups catalyze the formation of primary and secondary amines utilizing both organic and inorganic amine donors. In this review, we discuss advances in developing AmDHs and IREDs as biocatalysts and focus on evolutionary history, substrate scope and applications of the enzymes to provide an outlook on emerging industrial biotechnologies of chiral amine production.

Redox-Mediated Amination of Pyrogallol-Based Polyphenols.

Flavonoids are known to covalently modify amyloidogenic peptides by amination reactions. The underlying coupling process between polyphenols and N-nucleophiles is assessed by several in vitro and in silico approaches. The coupling reaction involves a sequence of oxidative dearomatization, amination, and reductive amination (ODARA) reaction steps. The C6-regioselectivity of the product is confirmed by crystallographic analysis. Under aqueous conditions, the reaction of baicalein with lysine derivatives yields C-N coupling as well as hydrolysis products of transient imine intermediates. The observed C-N coupling reactions work best for flavonoids combining a pyrogallol substructure with an electron-withdrawing group attached to the C4a-position. Thermodynamic properties such as bond dissociation energies also highlight the key role of pyrogallol units for the antioxidant ability. Combining the computed electronic properties and in vitro antioxidant assays suggests that the studied pyrogallol-containing flavonoids act by various radical-scavenging mechanisms working in synergy. Multivariate analysis indicates that a small number of descriptors for transient intermediates of the ODARA process generates a model with excellent performance (r=0.93) for the prediction of cross-coupling yields. The same model has been employed to predict novel antioxidant flavonoid-based molecules as potential covalent inhibitors, opening a new avenue to the design of therapeutically relevant anti-amyloid compounds.

Reductive amination cascades in cell-free and resting whole cell formats for valorization of lignin deconstruction products.

The selective introduction of amine groups within deconstruction products of lignin could provide an avenue for valorizing waste biomass while achieving a green synthesis of industrially relevant building blocks from sustainable sources. Here, we built and characterized enzyme cascades that create aldehydes and subsequently primary amines from diverse lignin-derived carboxylic acids using a carboxylic acid reductase (CAR) and an ω-transaminase (TA). Unlike previous studies that have paired CAR and TA enzymes, here we examine multiple homologs of each of these enzymes and a broader set of candidate substrates. In addition, we compare the performance of these systems in cell-free and resting whole-cell biocatalysis formats using the conversion of vanillate to vanillyl amine as model chemistry. We also demonstrate that resting whole cells can be recycled for multiple batch reactions. We used the knowledge gained from this study to produce several amines from carboxylic acid precursors using one-pot biocatalytic reactions, several of which we report for the first time. These results expand our knowledge of these industrially relevant enzyme families to new substrates and contexts for environmentally friendly and potentially low-cost synthesis of diverse aryl aldehydes and amines.

Reshaping the Substrate Binding Pocket of ß-Amino Acid Dehydrogenase for the Synthesis of Aromatic ß-Amino Acids.

By reshaping the substrate-binding pocket of ß-amino acid dehydrogenase (ß-AADH), some variants were obtained with up to 2560-fold enhanced activity toward the model substrates (S)-ß-homophenylalanine and (R)-ß-phenylalanine. A few aromatic ß-amino acids were prepared with >99% ee and high isolated yields via either kinetic resolution of racemates or reductive amination of the corresponding ß-keto acids. This work expands the catalytic capability of ß-AADHs and highlights their practical application in the synthesis of pharmaceutically relevant ß-amino acids.

Directed evolution of P411 enzymes for amination of inert C-H bonds.

Functionalizing inert C-H bonds selectively is a formidable task due to their strong bond energy and the difficulty of distinguishing chemically similar C-H bonds. While enzymatic oxygenation of C-H bonds is ubiquitous and well established, there is currently no known natural enzymatic process for direct nitrogen insertion. Instead, nature typically relies on pre-oxidized compounds for nitrogen incorporation. Direct biocatalytic C-H amination methods developed in the last few years are only selective for activated C-H bonds that contain specific groups such as benzylic, allylic, or propargylic groups. However, we recently used directed evolution to generate cytochrome P411 enzymes (engineered P450 enzymes with axial ligand mutation from cysteine to serine) that directly aminate inert C-H bonds with high site-, diastereo-, and enantioselectivity. Using these enzymes, we demonstrated the regiodivergent desymmetrization of methylcyclohexane, among other reactions. This chapter provides a comprehensive account of the experimental protocols used to evolve P411s for aminating unactivated C-H bonds. These methods are illustrative and can be adapted for other directed enzyme evolution campaigns.