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An operationally simple, scalable, and chemoselective method for the direct phosphorylation of alcohols using a P(V)-approach based on the Ψ-reagent platform is disclosed. The method features a broad substrate scope of utility in both simple and complex settings and provides access to valuable phosphorylated alcohols that would be otherwise difficult to obtain.

Phosphate linkages govern life as we know it. Their unique properties provide the foundation for many natural systems from cell biology and biosynthesis to the backbone of nucleic acids. Phosphates are ideal natural moieties; existing as ionized species in a stable P(V)-oxidation state, they are endowed with high stability but exhibit enzymatically unlockable potential. Despite intense interest in phosphorus catalysis and condensation chemistry, organic chemistry has not fully embraced the potential of P(V) reagents. To be sure, within the world of chemical oligonucleotide synthesis, modern approaches utilize P(III) reagent systems to create phosphate linkages and their analogs. In this Outlook, we present recent studies from our laboratories suggesting that numerous exciting opportunities for P(V) chemistry exist at the nexus of organic synthesis and biochemistry. Applications to the synthesis of stereopure antisense oligonucleotides, cyclic dinucleotides, methylphosphonates, and phosphines are reviewed as well as chemoselective modification to peptides, proteins, and nucleic acids. Finally, an outlook into what may be possible in the future with P(V) chemistry is previewed, suggesting these examples represent just the tip of the iceberg., Phosphorus(V) is a key building block for life. This Outlook draws inspiration from Nature for the development of reagents that harness its unique reactivity and augments popular P(III)-based methods.

Controlled site-specific bioconjugation through chemical methods to native DNA remains an unanswered challenge. Herein, we report a simple solution to achieve this conjugation through the tactical combination of two recently developed technologies: one for the manipulation of DNA in organic media and another for the chemoselective labeling of alcohols. Reversible adsorption of solid support (RASS) is employed to immobilize DNA and facilitate its transfer into dry acetonitrile. Subsequent reaction with P(V)-based Ψ reagents takes place in high yield with exquisite selectivity for the exposed 3′ or 5′ alcohols on DNA. This two-stage process, dubbed SENDR for Synthetic Elaboration of Native DNA by RASS, can be applied to a multitude of DNA conformations and sequences with a variety of functionalized Ψ reagents to generate useful constructs., The development of synthetic elaboration of native DNA by reversible adsorption of solid support (SENDR) is presented, and its utility is demonstrated in multiple examples relevant to the fields of biology through chemistry.

Copyright © 2020 American Chemical Society, This Communication reports the first general method for rapid, chemoselective, and modular functionalization of serine residues in native polypeptides, which uses a reagent platform based on the P(V) oxidation state. This redox-economical approach can be used to append nearly any kind of cargo onto serine, generating a stable, benign, and hydrophilic phosphorothioate linkage. The method tolerates all other known nucleophilic functional groups of naturally occurring proteinogenic amino acids. A variety of applications can be envisaged by this expansion of the toolbox of site-selective bioconjugation methods., Financial support for this work was provided by Bristol-Myers Squibb, the NIH (GM-118176), the Marie Skłodowska-CurieGlobal Fellowships (749359-EnanSET to N.M.P) within the European Union’s Research and Innovation Framework Programme (2014−2020), FCT Portugal (IF/00624/2015), and the Royal Society (URF\R\180019). D.F. was supported by the National Center for Advancing Translational Sciences,National Institutes of Health, through Grant UL1 TR002551 and Linked Award TL1 TR002551.

Artificial Melanosome for Transdermal Delivery In article number 2103503 by Uk‐Jae Lee, Nathaniel S. Hwang, Byung‐Gee Kim, and co‐workers, light‐triggered in situ biosynthesis of artificial melanin in native skin is proposed and demonstrated using photoactivatable tyrosinase. Photoactivatable tyrosinase is synthesized with site‐specific incorporation of o‐nitrobenzyl tyrosine and then encapsulated into lipid nanoparticles as an artificial melanosome for transdermal delivery. This biomimetic system can recapitulate the biosynthetic analogs of naturally occurring melanin. [Image: see text]

An operationally simple, scalable, and chemoselective method for the direct phosphorylation of alcohols using a P(V)-approach based on the PSI-reagent platform is disclosed. The method features a broad substrate scope of utility in both simple and complex settings and provides access to valuable phosphorylated alcohols that would be otherwise difficult to access.

Herein we report the development of an efficient cellular system for the in vivo biosynthesis of Tyr-analogs and their concurrent incorporation into target proteins by the residue-specific approach. This system makes use of common phenol derivatives and the tyrosine phenol lyase machinery to create various tyrosine analogues that impart desired properties on the target proteins. Biosynthesized 2-fluorotyrosine was incorporated into three industrially important enzymes which resulted in enhanced thermostability.

The two main strategies for enzyme engineering, directed evolution and rational design, have found widespread applications in improving the intrinsic activities of proteins. Although numerous advances have been achieved using these ground-breaking methods, the limited chemical diversity of the biopolymers, restricted to the 20 canonical amino acids, hampers creation of novel enzymes that Nature has never made thus far. To address this, much research has been devoted to expanding the protein sequence space via chemical modifications and/or incorporation of noncanonical amino acids (ncAAs). This review provides a balanced discussion and critical evaluation of the applications, recent advances, and technical breakthroughs in biocatalysis for three approaches: (i) chemical modification of cAAs, (ii) incorporation of ncAAs, and (iii) chemical modification of incorporated ncAAs. Furthermore, the applications of these approaches and the result on the functional properties and mechanistic study of the enzymes are extensively reviewed. We also discuss the design of artificial enzymes and directed evolution strategies for enzymes with ncAAs incorporated. Finally, we discuss the current challenges and future perspectives for biocatalysis using the expanded amino acid alphabet.

Site-selective chemical bioconjugation reactions are enabling tools for the chemical biologist. Guided by a careful study of the selenomethionine (SeM) benzylation, we have refined the reaction to meet the requirements of practical protein bioconjugation. SeM is readily introduced through auxotrophic expression and exhibits unique nucleophilic properties that allow it to be selectively modified even in the presence of cysteine. The resulting benzylselenonium adduct is stable at physiological pH, is selectively labile to glutathione, and embodies a broadly tunable cleavage profile. Specifically, a 4-bromomethylphenylacetyl (BrMePAA) linker has been applied for efficient conjugation of complex organic molecules to SeM-containing proteins. This expansion of the bioconjugation toolkit has broad potential in the development of chemically enhanced proteins.

The intriguing structure of tagetitoxin (1), a long-standing challenge in natural product synthesis, has been the subject of multiple revisions and has been confirmed through total synthesis. The route commences from a renewable furan starting material and features a number of unusual transformations (such as rearrangements, bromocyclization, and P(V)-based phosphate installation) to arrive at the target in 15 steps. As the route was designed to enable access to both enantiomers, the absolute configuration of the natural product could be assigned using a bioassay on (+)-1 and (-)-1.

Site-selective chemical protein ligation reactions are enabling tools for chemical biology. Herein, we employ a physical organic study to refine the selenomethionine (SeM) benzylation as a practical protein bioconjugation strategy. SeM is readily introduced through auxotrophic expression and exhibits unique nucleophilic properties that allow it to be selectively modified even in the presence of cysteine. The resulting benzylselenonium adduct is stable at physiological pH, selectively labile to glutathione and embodies a broadly tuneable reactivity profile. Guided by a mechanistic analysis of the reaction, a 4-bromomethylphenylacetyl linker is identified for efficient conjugations of complex organic molecules to SeM containing proteins. This optimized benzyl linker exhibits a rate constant of 3x10-1 M-1s-1, facilitating efficient conjugation at micromolar concentrations. The selenonium conjugate is further advanced through a linker that can be selectively photo-locked or reductively cleaved on demand. This tool-kit of selenonium forming reagents have broad potential in the development of chemically enhanced proteins.

The controlled, site-specific ligation of molecules to native DNA remains an unanswered challenge. Herein, we report a simple solution to achieve this ligation through the tactical combination of two recently developed technologies: One for the manipulation of DNA in organic media, and another for the chemoselective labeling of alcohols. Reversible Adsorption of Solid Support (RASS) is employed to immobilize DNA and facilitate its transfer into dry acetonitrile. Subsequent ligation with P(V)-based Ψ reagents takes place in high yield with exquisite selectivity for the exposed 3’ or 5’ alcohols on DNA. This two-stage process, dubbed SENDR for Synthetic Elaboration of Native DNA by RASS, can be applied to a multitude of DNA conformations and sequences with a variety of functionalized Ψ reagents to generate useful constructs. Such entities can address numerous longstanding challenges, including the selective single coupling of DNA to proteins, ASOs, and functional small molecules, and also can allow the synthesis of doubly-labeled congeners for novel probe constructs including ones of potential interest to COVID-19 research. Finally, a prototype for the industrialization of SENDR in a kit format is presented.

DNA encoded libraries (DEL) have shown promise as a valuable technology for democratizing the hit discovery process. Although DEL provides relatively inexpensive access to libraries of unprecedented size, their production has been hampered by the idiosyncratic needs of the encoding DNA tag relegating DEL compatible chemistry to dilute aqueous environments. Recently reversible adsorption to solid support (RASS) has been demonstrated as a promising method to expand DEL reactivity using standard organic synthesis protocols. Here we demonstrate a suite of on-DNA chemistries to incorporate medicinally relevant and C-S, C-P and N-S linkages into DELs, which are underrepresented in the canonical methods.

DNA Encoded Libraries have shown promise as a valuable technology for democratizing the hit discovery process. Although DEL provides relatively inexpensive access to libraries of unprecedented size, their production has been hampered by the idiosyncratic needs of the encoding DNA tag relegating DEL compatible chemistry to dilute aqueous environments. Recently Reversible Adsorption to Solid Support (RASS) has been demonstrated as a promising method to expand DEL reactivity using standard organic synthesis protocols. Here we demonstrate a suite of on-DNA chemistries to incorporate medicinally relevant and C–S, C–P and N–S linkages into DELs, which are underrepresented in the canonical methods.

Facile synthesis of C-terminal thioesters is integral to native chemical ligation (NCL) strategies for chemical protein synthesis. We introduce a new method of mild peptide activation, which leverages solid-phase peptide synthesis (SPPS) on an established resin linker and classical heterocyclic chemistry to convert C-terminal peptide hydrazides into their corresponding thioesters via an acyl pyrazole intermediate. Peptide hydrazides, synthesized on established trityl chloride resins, can be activated in solution with stoichiometric acetyl acetone (acac), readily proceed to the peptide acyl pyrazoles. Acyl pyrazoles are mild acylating agents and are efficiently exchanged with an aryl thiol, which can then be directly utilized in NCL. The mild, chemoselective, and stoichiometric activating conditions allow this method to be utilized through multiple sequential ligations without intermediate purification steps.

DNA Encoded Libraries have proven immensely powerful tools for lead identification. The ability to screen billions of compounds at once has spurred increasing interest in DEL development and utilization. Although DEL provides access to libraries of unprecedented size and diversity, the idiosyncratic and hydrophilic nature of the DNA tag severely limits the scope of applicable chemistries. It is known that biomacromolecules can be reversibly, non-covalently adsorbed and eluted from solid supports, and this phenomenon has been utilized to perform synthetic modification of biomolecules in a strategy we have described as reversible adsorption to solid support (RASS). Herein, we present the adaptation of RASS for a DEL setting, which allows reactions to be performed in organic solvents at near anhydrous conditions opening previously inaccessible chemical reactivities to DEL. The RASS approach enabled the rapid development of C(sp(2))-C(sp(3)) decarboxylative cross-couplings with broad substrate scope, an electrochemical amination (the first electrochemical synthetic transformation performed in a DEL context), and improved reductive amination conditions. The utility of these reactions was demonstrated through a DEL-rehearsal in which all newly developed chemistries were orchestrated to afford a compound rich in diverse skeletal linkages. We believe that RASS will offer expedient access to new DEL reactivities, expanded chemical space, and ultimately more drug-like libraries.

Herein, we present the adaptation of reversible adsorption to solid support (RASS) for a DEL setting, which allows reactions to be performed in organic solvents at near anhydrous conditions opening previously inaccessible chemical reactivities to DEL. The RASS approach enabled the rapid development of C(sp2)-C(sp3) decarboxylative cross-couplings with broad substrate scope, an electrochemical amination (the first electrochemical synthetic transformation performed in a DEL context), and improved reductive amination conditions. We believe that RASS will offer expedient access to new DEL reactivities, expanded chemical space, and ultimately more drug-like libraries.

Amino-γ-lactam (Agl) bridged dipeptides, commonly known as Freidinger lactams, have been shown to constrain peptide backbone topology and stabilize type II' β-turns. The utility of these links as peptide constraints has inspired new approaches to their incorporation into complex peptides and peptoids, all of which require harsh reaction conditions or protecting groups that limit their use on unprotected peptides and proteins. Herein, we employ a mild and selective alkylation of selenomethionine in acidic aqueous solution, followed by immobilization of the alkylated peptide on to bulk reverse-phase C18 silica and base-induced lactamization in DMSO. The utilization of selenomethionine, which is readily introduced by synthesis or expression, and the mild conditions enable selective backbone engineering in complex peptide and protein systems.

For over 20 years, native chemical ligation (NCL) has played a pivotal role in enabling the total synthesis and semisynthesis of increasingly complex peptide and protein targets. Classical NCL proceeds by the chemoselective reaction of two unprotected polypeptide chains in near-neutral pH aqueous solution and is made possible by the presence of a thioester moiety on the C-terminus of the N-terminal peptide fragment and a natural cysteine residue on the N-terminus of the C-terminal peptide fragment. The reaction yields an amide bond adjacent to cysteine at the ligation site, furnishing the native protein backbone in a traceless manner. This protocol will highlight a number of recent and powerful advances to the methodology and will outline their particular uses, facilitating their continued application in the synthesis of challenging protein targets.

© 2016 The Royal Society of Chemistry. Peptoid nanosheets can be broadly functionalized for a variety of applications. However, they are susceptible to degradation when exposed to chemical or mechanical stress. To improve their strength, photolabile monomers were introduced in order to crosslink the nanosheet interior. Photo-crosslinking produced a more robust material that can survive sonication, lyophilization, and other biochemical manipulations.