Asymmetric hydrogenation of ketimines with minimally different alkyl groups.

Asymmetric catalysis enables the synthesis of optically active compounds, often requiring the differentiation between two substituents on prochiral substrates1. Despite decades of development of mainly noble metal catalysts, achieving differentiation between substituents with similar steric and electronic properties remains a notable challenge2,3. Here we introduce a class of Earth-abundant manganese catalysts for the asymmetric hydrogenation of dialkyl ketimines to give a range of chiral amine products. These catalysts distinguish between pairs of minimally differentiated alkyl groups bound to the ketimine, such as methyl and ethyl, and even subtler distinctions, such as ethyl and n-propyl. The degree of enantioselectivity can be adjusted by modifying the components of the chiral manganese catalyst. This reaction demonstrates a wide substrate scope and achieves a turnover number of up to 107,800. Our mechanistic studies indicate that exceptional stereoselectivity arises from the modular assembly of confined chiral catalysts and cooperative non-covalent interactions between the catalyst and the substrate.

Bile salt hydrolase catalyses formation of amine-conjugated bile acids.

Bacteria in the gastrointestinal tract produce amino acid bile acid amidates that can affect host-mediated metabolic processes1-6; however, the bacterial gene(s) responsible for their production remain unknown. Herein, we report that bile salt hydrolase (BSH) possesses dual functions in bile acid metabolism. Specifically, we identified a previously unknown role for BSH as an amine N-acyltransferase that conjugates amines to bile acids, thus forming bacterial bile acid amidates (BBAAs). To characterize this amine N-acyltransferase BSH activity, we used pharmacological inhibition of BSH, heterologous expression of bsh and mutants in Escherichia coli and bsh knockout and complementation in Bacteroides fragilis to demonstrate that BSH generates BBAAs. We further show in a human infant cohort that BBAA production is positively correlated with the colonization of bsh-expressing bacteria. Lastly, we report that in cell culture models, BBAAs activate host ligand-activated transcription factors including the pregnane X receptor and the aryl hydrocarbon receptor. These findings enhance our understanding of how gut bacteria, through the promiscuous actions of BSH, have a significant role in regulating the bile acid metabolic network.

Enantioconvergent Cu-catalysed N-alkylation of aliphatic amines.

Chiral amines are commonly used in the pharmaceutical and agrochemical industries1. The strong demand for unnatural chiral amines has driven the development of catalytic asymmetric methods1,2. Although the N-alkylation of aliphatic amines with alkyl halides has been widely adopted for over 100 years, catalyst poisoning and unfettered reactivity have been preventing the development of a catalyst-controlled enantioselective version3-5. Here we report the use of chiral tridentate anionic ligands to enable the copper-catalysed chemoselective and enantioconvergent N-alkylation of aliphatic amines with α-carbonyl alkyl chlorides. This method can directly convert feedstock chemicals, including ammonia and pharmaceutically relevant amines, into unnatural chiral α-amino amides under mild and robust conditions. Excellent enantioselectivity and functional-group tolerance were observed. The power of the method is demonstrated in a number of complex settings, including late-stage functionalization and in the expedited synthesis of diverse amine drug molecules. The current method indicates that multidentate anionic ligands are a general solution for overcoming transition-metal-catalyst poisoning.

Multifunctional biocatalyst for conjugate reduction and reductive amination.

Chiral amine diastereomers are ubiquitous in pharmaceuticals and agrochemicals1, yet their preparation often relies on low-efficiency multi-step synthesis2. These valuable compounds must be manufactured asymmetrically, as their biochemical properties can differ based on the chirality of the molecule. Herein we characterize a multifunctional biocatalyst for amine synthesis, which operates using a mechanism that is, to our knowledge, previously unreported. This enzyme (EneIRED), identified within a metagenomic imine reductase (IRED) collection3 and originating from an unclassified Pseudomonas species, possesses an unusual active site architecture that facilitates amine-activated conjugate alkene reduction followed by reductive amination. This enzyme can couple a broad selection of α,ß-unsaturated carbonyls with amines for the efficient preparation of chiral amine diastereomers bearing up to three stereocentres. Mechanistic and structural studies have been carried out to delineate the order of individual steps catalysed by EneIRED, which have led to a proposal for the overall catalytic cycle. This work shows that the IRED family can serve as a platform for facilitating the discovery of further enzymatic activities for application in synthetic biology and organic synthesis.

Aziridine synthesis by coupling amines and alkenes via an electrogenerated dication.

Aziridines-three-membered nitrogen-containing cyclic molecules-are important synthetic targets. Their substantial ring strain and resultant proclivity towards ring-opening reactions makes them versatile precursors of diverse amine products1-3, and, in some cases, the aziridine functional group itself imbues important biological (for example, anti-tumour) activity4-6. Transformation of ubiquitous alkenes into aziridines is an attractive synthetic strategy, but is typically accomplished using electrophilic nitrogen sources rather than widely available amine nucleophiles. Here we show that unactivated alkenes can be electrochemically transformed into a metastable, dicationic intermediate that undergoes aziridination with primary amines under basic conditions. This new approach expands the scope of readily accessible N-alkyl aziridine products relative to those obtained through existing state-of-the-art methods. A key strategic advantage of this approach is that oxidative alkene activation is decoupled from the aziridination step, enabling a wide range of commercially available but oxidatively sensitive7 amines to act as coupling partners for this strain-inducing transformation. More broadly, our work lays the foundations for a diverse array of difunctionalization reactions using this dication pool approach.

Skeletal editing through direct nitrogen deletion of secondary amines.

Synthetic chemistry aims to build up molecular complexity from simple feedstocks1. However, the ability to exert precise changes that manipulate the connectivity of the molecular skeleton itself remains limited, despite possessing substantial potential to expand the accessible chemical space2,3. Here we report a reaction that 'deletes' nitrogen from organic molecules. We show that N-pivaloyloxy-N-alkoxyamides, a subclass of anomeric amides, promote the intermolecular activation of secondary aliphatic amines to yield intramolecular carbon-carbon coupling products. Mechanistic experiments indicate that the reactions proceed via isodiazene intermediates that extrude the nitrogen atom as dinitrogen, producing short-lived diradicals that rapidly couple to form the new carbon-carbon bond. The reaction shows broad functional-group tolerance, which enables the translation of routine amine synthesis protocols into a strategy for carbon-carbon bond constructions and ring syntheses. This is highlighted by the use of this reaction in the syntheses and skeletal editing of bioactive compounds.

Catalysis driven by an amyloid-substrate complex.

Amine modification through nucleophilic attack of the amine functionality is a very common chemical transformation. Under biorelevant conditions using acidic-to-neutral pH buffer, however, the nucleophilic reaction of alkyl amines (pKa ≈ 10) is not facile due to the generation of ammonium ions lacking nucleophilicity. Here, we disclose a unique molecular transformation system, catalysis driven by amyloid-substrate complex (CASL), that promotes amine modifications in acidic buffer. Ammonium ions attached to molecules with amyloid-binding capability were activated through deprotonation due to the close proximity to the amyloid catalyst formed by Ac-Asn-Phe-Gly-Ala-Ile-Leu-NH2 (NL6), derived from islet amyloid polypeptide (IAPP). Under the CASL conditions, alkyl amines underwent various modifications, i.e., acylation, arylation, cyclization, and alkylation, in acidic buffer. Crystallographic analysis and chemical modification studies of the amyloid catalysts suggested that the carbonyl oxygen of the Phe-Gly amide bond of NL6 plays a key role in activating the substrate amine by forming a hydrogen bond. Using CASL, selective conversion of substrates possessing equivalently reactive amine functionalities was achieved in catalytic reactions using amyloids. CASL provides a unique method for applying nucleophilic conversion reactions of amines in diverse fields of chemistry and biology.

A general carbonyl alkylative amination for tertiary amine synthesis.

The ubiquity of tertiary alkylamines in pharmaceutical and agrochemical agents, natural products and small-molecule biological probes1,2 has stimulated efforts towards their streamlined synthesis3-9. Arguably the most robust method for the synthesis of tertiary alkylamines is carbonyl reductive amination3, which comprises two elementary steps: the condensation of a secondary alkylamine with an aliphatic aldehyde to form an all-alkyl-iminium ion, which is subsequently reduced by a hydride reagent. Direct strategies have been sought for a 'higher order' variant of this reaction via the coupling of an alkyl fragment with an alkyl-iminium ion that is generated in situ10-14. However, despite extensive efforts, the successful realization of a 'carbonyl alkylative amination' has not yet been achieved. Here we present a practical and general synthesis of tertiary alkylamines through the addition of alkyl radicals to all-alkyl-iminium ions. The process is facilitated by visible light and a silane reducing agent, which trigger a distinct radical initiation step to establish a chain process. This operationally straightforward, metal-free and modular transformation forms tertiary amines, without structural constraint, via the coupling of aldehydes and secondary amines with alkyl halides. The structural and functional diversity of these readily available precursors provides a versatile and flexible strategy for the streamlined synthesis of complex tertiary amines.

Catalytic asymmetric addition of an amine N-H bond across internal alkenes.

Hydroamination of alkenes, the addition of the N-H bond of an amine across an alkene, is a fundamental, yet challenging, organic transformation that creates an alkylamine from two abundant chemical feedstocks, alkenes and amines, with full atom economy1-3. The reaction is particularly important because amines, especially chiral amines, are prevalent substructures in a wide range of natural products and drugs. Although extensive efforts have been dedicated to developing catalysts for hydroamination, the vast majority of alkenes that undergo intermolecular hydroamination have been limited to conjugated, strained, or terminal alkenes2-4; only a few examples occur by the direct addition of the N-H bond of amines across unactivated internal alkenes5-7, including photocatalytic hydroamination8,9, and no asymmetric intermolecular additions to such alkenes are known. In fact, current examples of direct, enantioselective intermolecular hydroamination of any type of unactivated alkene lacking a directing group occur with only moderate enantioselectivity10-13. Here we report a cationic iridium system that catalyses intermolecular hydroamination of a range of unactivated, internal alkenes, including those in both acyclic and cyclic alkenes, to afford chiral amines with high enantioselectivity. The catalyst contains a phosphine ligand bearing trimethylsilyl-substituted aryl groups and a triflimide counteranion, and the reaction design includes 2-amino-6-methylpyridine as the amine to enhance the rates of multiple steps within the catalytic cycle while serving as an ammonia surrogate. These design principles point the way to the addition of N-H bonds of other reagents, as well as O-H and C-H bonds, across unactivated internal alkenes to streamline the synthesis of functional molecules from basic feedstocks.

A photochemical dehydrogenative strategy for aniline synthesis.

Chemical reactions that reliably join two molecular fragments together (cross-couplings) are essential to the discovery and manufacture of pharmaceuticals and agrochemicals1,2. The introduction of amines onto functionalized aromatics at specific and pre-determined positions (ortho versus meta versus para) is currently achievable only in transition-metal-catalysed processes and requires halogen- or boron-containing substrates3-6. The introduction of these groups around the aromatic unit is dictated by the intrinsic reactivity profile of the method (electrophilic halogenation or C-H borylation) so selective targeting of all positions is often not possible. Here we report a non-canonical cross-coupling approach for the construction of anilines, exploiting saturated cyclohexanones as aryl electrophile surrogates. Condensation between amines and carbonyls, a process that frequently occurs in nature and is often used by (bio-)organic chemists7, enables a predetermined and site-selective carbon-nitrogen (C-N) bond formation, while a photoredox- and cobalt-based catalytic system progressively desaturates the cyclohexene ring en route to the aniline. Given that functionalized cyclohexanones are readily accessible with complete regiocontrol using the well established carbonyl reactivity, this approach bypasses some of the frequent selectivity issues of aromatic chemistry. We demonstrate the utility of this C-N coupling protocol by preparing commercial medicines and by the late-stage amination-aromatization of natural products, steroids and terpene feedstocks.

Synthesis of peptide-siRNA conjugates via internal sulfonylphosphoramidate modifications and evaluation of their in vitro activity.

Conjugates of therapeutic oligonucleotides (ONs) including peptide conjugates, provide a potential solution to the major challenge of specific tissue delivery faced by this class of drugs. Conjugations are often positioned terminal at the ONs, although internal placement of other chemical modifications are known to be of critical importance. The introduction of internal conjugation handles in chemically modified ONs require highly specialized and expensive nucleoside phosphoramidites. Here, we present a method for synthesizing a library of peptide-siRNA conjugates by conjugation at internal phosphorous positions via sulfonylphosphoramidate modifications incorporated into the sense strand. The sulfonylphosphoramidate modification offers benefits as it can be directly incorporated into chemically modified ONs by simply changing the oxidation step during synthesis, and furthermore holds the potential to create multifunctionalized therapeutic ONs. We have developed a workflow using a novel pH-controlled amine-to-amine linker that yields peptide-siRNA conjugates linked via amide bonds, and we have synthesized conjugates between GLP1 peptides and a HPRT1 siRNA as a model system. The in vitro activity of the conjugates was tested by GLP1R activity and knockdown of the HPRT1 gene. We found that conjugation near the 3'-end is more favorable than certain central internal positions and different internal conjugation strategies were compared.

Site-specific N-alkylation of DNA oligonucleotide nucleobases by DNAzyme-catalyzed reductive amination.

DNA and RNA nucleobase modifications are biologically relevant and valuable in fundamental biochemical and biophysical investigations of nucleic acids. However, directly introducing site-specific nucleobase modifications into long unprotected oligonucleotides is a substantial challenge. In this study, we used in vitro selection to identify DNAzymes that site-specifically N-alkylate the exocyclic nucleobase amines of particular cytidine, guanosine, and adenosine (C, G and A) nucleotides in DNA substrates, by reductive amination using a 5'-benzaldehyde oligonucleotide as the reaction partner. The new DNAzymes each require one or more of Mg2+, Mn2+, and Zn2+ as metal ion cofactors and have kobs from 0.04 to 0.3 h-1, with rate enhancement as high as ∼104 above the splinted background reaction. Several of the new DNAzymes are catalytically active when an RNA substrate is provided in place of DNA. Similarly, several new DNAzymes function when a small-molecule benzaldehyde compound replaces the 5'-benzaldehyde oligonucleotide. These findings expand the scope of DNAzyme catalysis to include nucleobase N-alkylation by reductive amination. Further development of this new class of DNAzymes is anticipated to facilitate practical covalent modification and labeling of DNA and RNA substrates.

Caveat fluorophore: an insiders' guide to small-molecule fluorescent labels.

The last three decades have brought a revolution in fluorescence microscopy. The development of new microscopes, fluorescent labels and analysis techniques has pushed the frontiers of biological imaging forward, moving from fixed to live cells, from diffraction-limited to super-resolution imaging and from simple cell culture systems to experiments in vivo. The large and ever-evolving collection of tools can be daunting for biologists, who must invest substantial time and effort in adopting new technologies to answer their specific questions. This is particularly relevant when working with small-molecule fluorescent labels, where users must navigate the jargon, idiosyncrasies and caveats of chemistry. Here, we present an overview of chemical dyes used in biology and provide frank advice from a chemist's perspective.

An Efficient, Amine-Specific, and Cost-Effective Method for TMT 6/11-plex Labeling Improves the Proteome Coverage, Quantitative Accuracy and Precision.

Tandem mass tags (TMT) are widely used in proteomics to simultaneously quantify multiple samples in a single experiment. The tags can be easily added to the primary amines of peptides/proteins through chemical reactions. In addition to amines, TMT reagents also partially react with the hydroxyl groups of serine, threonine, and tyrosine residues under alkaline conditions, which significantly compromises the analytical sensitivity and precision. Under alkaline conditions, reducing the TMT molar excess can partially mitigate overlabeling of histidine-free peptides, but has a limited effect on peptides containing histidine and hydroxyl groups. Here, we present a method under acidic conditions to suppress overlabeling while efficiently labeling amines, using only one-fifth of the TMT amount recommended by the manufacturer. In a deep-scale analysis of a yeast/human two-proteome sample, we systematically evaluated our method against the manufacturer's method and a previously reported TMT-reduced method. Our method reduced overlabeled peptides by 9-fold and 6-fold, respectively, resulting in the substantial enhancement in peptide/protein identification rates. More importantly, the quantitative accuracy and precision were improved as overlabeling was reduced, endowing our method with greater statistical power to detect 42% and 12% more statistically significant yeast proteins compared to the standard and TMT-reduced methods, respectively. Mass spectrometric data have been deposited in the ProteomeXchange Consortium via the iProX partner repository with the data set identifier PXD047052.

α,ß-Desaturation and Formal ß-C(sp3)-H Fluorination of N-Substituted Amines: A Late-Stage Functionalization Strategy Enabled by Electrochemistry.

Incorporation of C(sp3)-F bonds in biologically active compounds is a common strategy employed in medicinal and agricultural chemistry to tune pharmacokinetic and pharmacodynamic properties. Due to the limited number of robust strategies for C(sp3)-H fluorination of complex molecules, time-consuming de novo syntheses of such fluorinated analogs are typically required, representing a major bottleneck in the drug discovery process. In this work, we present a general and operationally simple strategy for site-specific ß-C(sp3)-H fluorination of amine derivatives including carbamates, amides, and sulfonamides, which is compatible with a wide range of functional groups including N-heteroarenes. In this approach, an improved electrochemical Shono oxidation is used to set the site of functionalization via net α,ß-desaturation to access enamine derivatives. We further developed a series of new transformations of these enamine intermediates to synthesize a variety of ß-fluoro-α-functionalized structures, allowing efficient access to pertinent targets to accelerate drug discovery campaigns.

Elucidating and Optimizing the Photochemical Mechanism of Coumarin-Caged Tertiary Amines.

Photoactivatable or "caged" pharmacological agents combine the high spatiotemporal specificity of light application with the molecular specificity of drugs. A key factor in all optopharmacology experiments is the mechanism of uncaging, which dictates the photochemical quantum yield and determines the byproducts produced by the light-driven chemical reaction. In previous work, we demonstrated that coumarin-based photolabile groups could be used to cage tertiary amine drugs as quaternary ammonium salts. Although stable, water-soluble, and useful for experiments in brain tissue, these first-generation compounds exhibit relatively low uncaging quantum yield (Φu < 1%) and release the toxic byproduct formaldehyde upon photolysis. Here, we elucidate the photochemical mechanisms of coumarin-caged tertiary amines and then optimize the major pathway using chemical modification. We discovered that the combination of 3,3-dicarboxyazetidine and bromine substituents shift the mechanism of release to heterolysis, eliminating the formaldehyde byproduct and giving photolabile tertiary amine drugs with Φu > 20%─a 35-fold increase in uncaging efficiency. This new "ABC" cage allows synthesis of improved photoactivatable derivatives of escitalopram and nicotine along with a novel caged agonist of the oxytocin receptor.

Unexpected Cyclization Product Discovery from the Photoinduced Bioconjugation Chemistry between Tetrazole and Amine.

Bioconjugation chemistry has emerged as a powerful tool for the modification of diverse biomolecules under mild conditions. Tetrazole, initially proposed as a bioorthogonal photoclick handle for 1,3-dipolar cyclization with alkenes, was later demonstrated to possess broader photoreactivity with carboxylic acids, serving as a versatile bioconjugation and photoaffinity labeling probe. In this study, we unexpectedly discovered and validated the photoreactivity between tetrazole and primary amine to afford a new 1,2,4-triazole cyclization product. Given the significance of functionalized N-heterocycles in medicinal chemistry, we successfully harnessed the serendipitously discovered reaction to synthesize both pharmacologically relevant DNA-encoded chemical libraries (DELs) and small molecule compounds bearing 1,2,4-triazole scaffolds. Furthermore, the mild reaction conditions and stable 1,2,4-triazole linkage found broad application in photoinduced bioconjugation scenarios, spanning from intramolecular peptide macrocyclization and templated DNA reaction cross-linking to intermolecular photoaffinity labeling of proteins. Triazole cross-linking products on lysine side chains were identified in tetrazole-labeled proteins, refining the comprehensive understanding of the photo-cross-linking profiles of tetrazole-based probes. Altogether, this tetrazole-amine bioconjugation expands the current bioconjugation toolbox and creates new possibilities at the interface of medicinal chemistry and chemical biology.

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.

Copper-Nitroxyl-Catalyzed α-Oxygenation of Cyclic Secondary Amines Including Application to Late-Stage Functionalization.

Cyclic secondary amines are prominent subunits in pharmaceutical compounds. Methods for direct functionalization of N-unprotected/unsubstituted piperidines and related heterocycles have limited precedent despite their potential to impact medicinal chemistry and organic synthesis. Herein, we report a Cu/nitroxyl co-catalyzed method for direct conversion of cyclic secondary amines to the corresponding lactams via aerobic dehydrogenation and oxidative coupling with water. The mild reaction conditions tolerate diverse functional groups, enabling application to molecules that cover broad chemical space. The method is showcased in selective functionalization of building blocks and complex molecules, including late-stage functionalization of bromodomain inhibitors.

Transaminase-Catalyzed Synthesis of ß-Branched Noncanonical Amino Acids Driven by a Lysine Amine Donor.

Transaminases are choice biocatalysts for the synthesis of chiral primary amines, including amino acids bearing contiguous stereocenters. In this study, we employ lysine as a "smart" amine donor in transaminase-catalyzed dynamic kinetic resolution reactions to access ß-branched noncanonical arylalanines. Our mechanistic investigation demonstrates that, upon transamination, the lysine-derived ketone byproduct readily cyclizes to a six-membered imine, driving the equilibrium in the desired direction and thus alleviating the need to load superstoichiometric quantities of the amine donor or deploy a multienzyme cascade. Lysine also shows good overall compatibility with a panel of wild-type transaminases, a promising hint of its application as a smart donor more broadly. Indeed, by this approach, we furnished a broad scope of ß-branched arylalanines, including some bearing hitherto intractable cyclopropyl and isopropyl substituents, with high yields and excellent selectivities.