Synthesis and glycosidase inhibition of 3,4,5-trihydroxypiperidines using a one-pot amination-cyclisation cascade reaction.

Trihydroxypiperidines are a therapeutically valuable class of iminosugar. We applied a one-pot amination-cyclisation cascade reaction to synthesise 3,4,5-trihydroxypiperidine stereoisomers in three steps from commercially available pentoses and in excellent overall yields. Using our methodology, the yields of the syntheses of meso-1, meso-2 and 3L are the highest reported to date. The synthetic methodology was readily extended to the three-step synthesis of N-alkyl derivatives by replacing the ammonia nitrogen source with a primary amine. The trihydroxypiperidines and N-alkyl analogues were screened for enzyme inhibitory activity using Fabrazyme (Fabry disease), GCase (Gaucher's disease), Agrobacterium sp. ß-glucosidase, and Escherichia coli ß-galactosidase. N-Phenylethyl 3,4,5-trihydroxypiperidine (N-phenylethyl-1-(3R,4R,5S)-piperidine-3,4,5-triol) showed good inhibitory activity of Fabrazyme (Ki = 46 µM). This activity was abolished when the N-phenylethyl group was removed or replaced with a non-aromatic alkyl chain.

Novel pyrrolidine-alkylamino-substituted dicyanoisophorone derivatives as near-infrared fluorescence probe for imaging ß-amyloid in vitro and in vivo.

BACKGROUND: The formation of amyloid-ß (Aß) plaques is one of the key neuropathological hallmarks of Alzheimer's disease (AD). Near-infrared (NIR) probes show great potential for imaging of Aß plaques in vivo and in vitro. Dicyanoisophorone (DCIP) based Aß probes have attracted considerable attention due to their exceptional properties. However, DCIP probes still has some drawbacks, such as short emission wavelength (<650 nm) and low fluorescence intensity after binding to Aß. It is clear that further modification is needed to improve their luminescence efficiency and sensitivity. RESULTS: We designed and synthesize four novel pyrrolidine-alkylamino-substituted DCIP derivatives (6a-d) as imaging agents for ß-amyloid (Aß) aggregates. Compound 6c responds better to Aß aggregates than the other three compounds (6a, 6b and 6d) and its precursor DCIP. The calculated detection limit is to be as low as 0.23 µM. Compound 6c shows no cytotoxicity in the tested concentration for SH-SY5Y and HL-7702 cells. Additionally, compound 6c is successfully applied to monitor Aß aggregates in live SH-SY5Y cells and APP/PS1 transgenic mice. The retention time in the transgenic mice brain is much longer than that of age-matched wild-type mice. SIGNIFICANCE: The results indicates that compound 6c had an excellent ability to penetrate the blood-brain barrier and it could effectively distinguish APP/PS1 transgenic mice and wide-type mice. This represents its promising applications for Aß detection in basic and biomedical research.

Biocatalysis enables the scalable conversion of biobased furans into various furfurylamines.

Biobased furans have emerged as chemical building blocks for the development of materials because of their diverse scaffolds and as they can be directly prepared from sugars. However, selective, efficient, and cost-effective scalable conversion of biobased furans remains elusive. Here, we report a robust transaminase (TA) from Shimia marina (SMTA) that enables the scalable amination of biobased furanaldehydes with high activity and broad substrate specificity. Crystallographic and mutagenesis analyses provide mechanistic insights and a structural basis for understanding SMTA, which enables a higher substrate conversion. The enzymatic cascade process established in this study allows one-pot synthesis of 2,5-bis(aminomethyl)furan (BAMF) and 5-(aminomethyl)furan-2-carboxylic acid from 5-hydroxymethylfurfural. The biosynthesis of various furfurylamines, including a one-pot cascade reaction for BAMF generation using whole cells, demonstrates their practical application in the pharmaceutical and polymer industries.

Reaction Discovery Using Spectroscopic Insights from an Enzymatic C-H Amination Intermediate.

Engineered hemoproteins can selectively incorporate nitrogen from nitrene precursors like hydroxylamine, O-substituted hydroxylamines, and organic azides into organic molecules. Although iron-nitrenoids are often invoked as the reactive intermediates in these reactions, their innate reactivity and transient nature have made their characterization challenging. Here we characterize an iron-nitrosyl intermediate generated from NH2OH within a protoglobin active site that can undergo nitrogen-group transfer catalysis, using UV-vis, electron paramagnetic resonance (EPR) spectroscopy, and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) techniques. The mechanistic insights gained led to the discovery of aminating reagents─nitrite (NO2-), nitric oxide (NO), and nitroxyl (HNO)─that are new to both nature and synthetic chemistry. Based on the findings, we propose a catalytic cycle for C-H amination inspired by the nitrite reductase pathway. This study highlights the potential of engineered hemoproteins to access natural nitrogen sources for sustainable chemical synthesis and offers a new perspective on the use of biological nitrogen cycle intermediates in biocatalysis.

Stereospecific Enzymatic Conversion of Boronic Acids to Amines.

Boronic acids and esters are highly regarded for their safety, unique reactivity, and versatility in synthesizing a wide range of small molecules, bioconjugates, and materials. They are not exploited in biocatalytic synthesis, however, because enzymes that can make, break, or modify carbon-boron bonds are rare. We wish to combine the advantages of boronic acids and esters for molecular assembly with biocatalysis, which offers the potential for unsurpassed selectivity and efficiency. Here, we introduce an engineered protoglobin nitrene transferase that catalyzes the new-to-nature amination of boronic acids using hydroxylamine. Initially targeting aryl boronic acids, we show that the engineered enzyme can produce a wide array of anilines with high yields and total turnover numbers (up to 99% yield and >4000 TTN), with water and boric acid as the only byproducts. We also demonstrate that the enzyme is effective with bench-stable boronic esters, which hydrolyze in situ to their corresponding boronic acids. Exploring the enzyme's capacity for enantioselective catalysis, we found that a racemic alkyl boronic ester affords an enantioenriched alkyl amine, a transformation not achieved with chemocatalysts. The formation of an exclusively unrearranged product during the amination of a boronic ester radical clock and the reaction's stereospecificity support a two-electron process akin to a 1,2-metallate shift mechanism. The developed transformation enables new biocatalytic routes for synthesizing chiral amines.

Dual magnetization and amination of cellulosic chains for the efficient adsorption of heavy metals.

Compounds functionalized with hydroxyl and amino groups were found to have good potential for the adsorption of different ions. In this work, a new system of cellulosic chains was amended with amine substitutions and bonded to a magnetic core of NiFe2O4@SiO2 to form NiFe2O4@SiO2-cellulose-NH2 system. The prepared sample showed suitable magnetic separation and was characterized via XRD, FT-IR, SEM, EDS, and TGA-DTA analyses. The adsorption potential of NiFe2O4@SiO2-cellulose-NH2 system has been investigated on the heavy metals (Cd, Ni, and Pb) removal from a synthetic wastewater environment. The results show that the magnetic property created by the magnetic core increased the recycling potential of the adsorbent and the magnetic core has a positive effect on the absorption potential of the polymer. The adsorption removal of Cd(II), Ni(II), and Pb(II) ions was studied using NiFe2O4@SiO2-cellulose-NH2 systems in different pH, temperatures, metal ion concentrations, and adsorbent dosages. The maximum adsorption capacities of single heavy metal ions were obtained as 406.44 mg/g (for Cd(II) ions), 411.63 mg/g (for Ni(II) ions), and 414.68 mg/g (for Pb(II) ions) under optimized conditions as pH = 6.5, ion concentration: 500 mg/L, adsorbent dosage: 1.2 g/L and room temperature.

Cu-Catalyzed Amination of Base-Sensitive Aryl Bromides and the Chemoselective N- and O-Arylation of Amino Alcohols.

We report a general and functional-group-tolerant method for the Cu-catalyzed amination of base-sensitive aryl bromides including substrates possessing acidic functional groups and small five-membered heteroarenes. The results presented herein substantially expand the scope of Cu-catalyzed C-N coupling reactions. The combination of L8, an anionic N1,N2-diarylbenzene-1,2-diamine ligand, along with the mild base NaOTMS leads to the formation of a stable yet reactive catalyst that resists deactivation from coordination to heterocycles or charged intermediates. This system enables the use of low catalyst and ligand loadings. Exploiting the differences in nucleophile deprotonation in C-O and C-N coupling reactions catalyzed by Cu·L8 we developed a method to chemoselectively N- and O-arylate a variety of amino alcohol substrates. Employing NaOt-Bu as the base resulted exclusively in C-O coupling when the amino alcohols featured primary alcohols and more hindered amines or aniline groups. Utilizing NaOTMS enabled the ability to override the steric-based selectivity of these reactions completely and exclusively promoted C-N coupling regardless of the structure of the amino alcohol. The ability to invert the observed chemoselectivity is distinct from previously described methods that require protecting group manipulations or rely entirely on steric effects to control reactivity. These results substantially improve the scope of Cu-catalyzed C-N coupling reactions using N1,N2-diarylbenzene-1,2-diamine ligands and introduce a new chemoselective method to arylate amino alcohols.

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.

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.

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.

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.

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.

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.

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.