Dillon Flood

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… Show more

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. Show less

The 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… Show more

The 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. Show less

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… Show more

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. Show less

In 1992, Brenner and Lerner hypothesized that individual chemical transformations could be encoded in DNA, allowing the rapid synthesis and screening of large collections of small molecules. Since their report, huge investments into the development of the DNA encoded library (DEL) technology have enabled the acceleration of the drug discovery process especially early phase discovery undertakings such as target validation and hit identification. As DEL lies at the nexus between chemistry and… Show more

In 1992, Brenner and Lerner hypothesized that individual chemical transformations could be encoded in DNA, allowing the rapid synthesis and screening of large collections of small molecules. Since their report, huge investments into the development of the DNA encoded library (DEL) technology have enabled the acceleration of the drug discovery process especially early phase discovery undertakings such as target validation and hit identification. As DEL lies at the nexus between chemistry and biology, there is an increasing need to expand the toolboxes of both organic transformations and biological methods. However, the myriad of techniques and reactions already reported can be difficult to digest for practitioners whose expertise resides outside the realm of DEL. This review therefore focuses on a stepwise presentation of DEL from the basic concepts to newest developments. The presentation includes the history, fundamentals, and successes of DEL, different methods for DEL synthesis and affinity selection, the conventional transformations, and finally the latest developments from a synthetic organic perspective. Show less

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… Show more

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. Show less

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, noncovalently adsorbed and eluted from solid… Show more

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, noncovalently 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(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. 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. Show less

For over 20 years, native chemical ligation (NCL) has played a pivotal role in enabling total synthesis and semisynthesis of increasingly complex peptide and protein targets. Classical NCL proceeds by 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… Show more

For over 20 years, native chemical ligation (NCL) has played a pivotal role in enabling total synthesis and semisynthesis of increasingly complex peptide and protein targets. Classical NCL proceeds by 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 a native protein backbone in a traceless manner. This unit highlights a number of recent and powerful advances in the methodology and outlines their particular uses, facilitating application in the synthesis of challenging protein targets. Show less

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… Show more

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. Show less

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… Show more

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. Show less

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.