Selenomethionine as an expressible handle for bioconjugations.

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.

Evaluating the Catalytic Efficiency of the Human Membrane-type 1 Matrix Metalloproteinase (MMP-14) Using AuNP-Peptide Conjugates.

Interactions of plasmonic nanocolloids such as gold nanoparticles and nanorods with proximal dye emitters result in efficient quenching of the dye photoluminescence (PL). This has become a popular strategy for developing analytical biosensors relying on this quenching process for signal transduction. Here, we report on the use of stable PEGylated gold nanoparticles, covalently coupled to dye-labeled peptides, as sensitive optically addressable sensors for determining the catalytic efficiency of the human matrix metalloproteinase-14 (MMP-14), a cancer biomarker. We exploit real-time dye PL recovery triggered by MMP-14 hydrolysis of the AuNP-peptide-dye to extract quantitative analysis of the proteolysis kinetics. Sub-nanomolar limit of detections for MMP-14 has been achieved using our hybrid bioconjugates. In addition, we have used theoretical considerations within a diffusion-collision framework to derive enzyme substrate hydrolysis and inhibition kinetics equations, which allowed us to describe the complexity and irregularity of enzymatic proteolysis of nanosurface-immobilized peptide substrates. Our findings offer a great strategy for the development of highly sensitive and stable biosensors for cancer detection and imaging.

Recent Advances in Biocatalysis with Chemical Modification and Expanded Amino Acid Alphabet.

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.

Structural characterization of anti-CCL5 activity of the tick salivary protein evasin-4.

Ticks, as blood-sucking parasites, have developed a complex strategy to evade and suppress host immune responses during feeding. The crucial part of this strategy is expression of a broad family of salivary proteins, called Evasins, to neutralize chemokines responsible for cell trafficking and recruitment. However, structural information about Evasins is still scarce, and little is known about the structural determinants of their binding mechanism to chemokines. Here, we studied the structurally uncharacterized Evasin-4, which neutralizes a broad range of CC-motif chemokines, including the chemokine CC-motif ligand 5 (CCL5) involved in atherogenesis. Crystal structures of Evasin-4 and E66S CCL5, an obligatory dimeric variant of CCL5, were determined to a resolution of 1.3-1.8 Å. The Evasin-4 crystal structure revealed an L-shaped architecture formed by an N- and C-terminal subdomain consisting of eight ß-strands and an α-helix that adopts a substantially different position compared with closely related Evasin-1. Further investigation into E66S CCL5-Evasin-4 complex formation with NMR spectroscopy showed that residues of the N terminus are involved in binding to CCL5. The peptide derived from the N-terminal region of Evasin-4 possessed nanomolar affinity to CCL5 and inhibited CCL5 activity in monocyte migration assays. This suggests that Evasin-4 derivatives could be used as a starting point for the development of anti-inflammatory drugs.

An Integrated Cofactor/Co-Product Recycling Cascade for the Biosynthesis of Nylon Monomers from Cycloalkylamines.

We report a highly atom-efficient integrated cofactor/co-product recycling cascade employing cycloalkylamines as multifaceted starting materials for the synthesis of nylon building blocks. Reactions using E. coli whole cells as well as purified enzymes produced excellent conversions ranging from >80 and 95 % into desired ω-amino acids, respectively with varying substrate concentrations. The applicability of this tandem biocatalytic cascade was demonstrated to produce the corresponding lactams by employing engineered biocatalysts. For instance, ϵ-caprolactam, a valuable polymer building block was synthesized with 75 % conversion from 10 mM cyclohexylamine by employing whole-cell biocatalysts. This cascade could be an alternative for bio-based production of ω-amino acids and corresponding lactam compounds.

Serine-Selective Bioconjugation.

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.

RASS-Enabled S/P-C and S-N Bond Formation for DEL Synthesis.

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.

Expanding Reactivity in DNA-Encoded Library Synthesis via Reversible Binding of DNA to an Inert Quaternary Ammonium Support.

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.

Rigid Peptide Macrocycles from On-Resin Glaser Stapling.

Peptide macrocycles are widely utilized in the development of high affinity ligands, including stapled α-helices. The linear rigidity of a 1,3-diynyl linkage provides an optimal distance (7 Å) between ß-carbons of the i,i+4 amino acid side chains, thus suggesting its utility in stabilizing α-helical structures. Here, we report the development of an on-resin strategy for an intramolecular Glaser reaction between two alkyne-terminated side chains by using copper chloride, an essential bpy-diol ligand, and diisopropylethylamine at room temperature. The efficiency of this ligation was illustrated by the synthesis of (i,i+4)-, (i,i+5)-, (i,i+6)-, and (i,i+7)-stapled BCL-9 α-helical peptides using the unnatural amino acid propargyl serine. Overall, this procedurally simple method relies on inexpensive and widely available reagents to generate low molecular weight 23-, 26-, 29-, and 32-membered peptide macrocycles.

Efficient Assembly of Quantum Dots with Homogenous Glycans Derived from Natural N-Linked Glycoproteins.

Coating inorganic nanoparticles with polyethylene glycol (PEG)-appended ligands, as means to preserve their physical characteristics and promote steric interactions with biological systems, including enhanced aqueous solubility and reduced immunogenicity, has been explored by several groups. Conversely, macromolecules present in the human serum and on the surface of cells are densely coated with hydrophilic glycans that act to reduce nonspecific interactions, while facilitating specific binding and interactions. In particular, N-linked glycans are abundant on the surface of most serum proteins and are composed of a branched architecture that is typically characterized by a significant level of molecular heterogeneity. Here we provide two distinct methodologies, covalent bioconjugation and self-assembly, to functionalize two types of Quantum Dots with a homogeneous, complex-type N-linked glycan terminated with a sialic acid moiety. A detailed physical and functional characterization of these glycan-coated nanoparticles has been performed. Our findings support the potential use of such fluorescent platforms to sense glycan-involved biological processes, such as lectin recognition and sialidase-mediated hydrolysis.

Expedient on-resin synthesis of peptidic benzimidazoles.

The benzimidazole moiety is a ubiquitous pharmacophore present in numerous anthelmintic, antibacterial, antiviral, antineoplastic, and antifungal drugs. While the polypharmacology of this heterocycle has spurred the development of numerous solution-phase syntheses, only a handful of disparate and inefficient methods detailing its synthesis on-resin have been reported. Here we report the concise and expedient syntheses of internal and C-terminal peptidic benzimidazoles - an emerging class of peptide deformylase (PDF)-inhibiting antimicrobials. This method benefits from being performed wholly on solid-phase at room temperature resulting in minimal purification and tolerance of temperature-sensitive functionality.

Post-Translational Backbone Engineering through Selenomethionine-Mediated Incorporation of Freidinger Lactams.

Amino-γ-lactam (Agl) bridged dipeptides, commonly known as Freidinger lactams, have been shown to constrain peptide backbone topology and stabilize type II' ß-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.

Leveraging the Knorr Pyrazole Synthesis for the Facile Generation of Thioester Surrogates for use in Native Chemical Ligation.

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.

Conformational Heterogeneity and DNA Recognition by the Morphogen Bicoid.

The morphogenic activity of the Drosophila transcription factor bicoid (Bcd), the first morphogenic protein identified, is controlled by its DNA binding homeodomain. Homeodomains mediate developmental processes in all multicellular organisms, but the Bcd homeodomain appears to be unique as it can bind multiple DNA sequences and even RNA. All homeodomain proteins adopt a three-helix fold, with residues of the third helix mediating recognition of the nucleic acid target via interactions with the major groove. Interestingly, previous studies have revealed that conformational heterogeneity is present in the Bcd residues that interact with bound DNA, suggesting that it may underlie the morphogen's unique polyspecificity. To begin to directly characterize the conformational heterogeneity in the homeodomain, we have introduced C-D bonds within each structural element and characterized their absorptions in the free and bound states, as well as during thermal denaturation. The data reveal that while residues within the first two helices experience unique environments, each environment is well-defined and similar in the presence and absence of bound DNA. In contrast, the data are consistent with residues within the recognition helix adopting multiple conformations, and while the binding of DNA does alter the environments, the conformational heterogeneity is similar in the bound and unbound states. Finally, thermal denaturation studies reveal that the conformational heterogeneity observed in this and previous studies results not from local instability and unfolding, as has been suggested for other transcription factors, but rather from the population of multiple stable conformations within the folded state of the protein. The results have important implications for how Bcd recognizes its different targets to mediate its critical developmental functions.

Concurrent Modulation of Quantum Dot Photoluminescence Using a Combination of Charge Transfer and Förster Resonance Energy Transfer: Competitive Quenching and Multiplexed Biosensing Modality.

An emerging trend with semiconductor quantum dots (QDs) is their use as scaffolds to assemble multiple energy transfer pathways. Examples to date have combined various competitive and sequential Förster resonance energy transfer (FRET) pathways between QDs and fluorescent dyes, luminescent lanthanide complexes, and bioluminescent proteins. Here, we show that the photoluminescence (PL) of QD bioconjugates can also be modulated by a combination of FRET and charge transfer (CT), and characterize the concurrent effects of these mechanistically different pathways using PL measurements at both the ensemble and the single particle level. Peptides were distally labeled with either a fluorescent dye that quenched QD PL through FRET or a ruthenium(II) phenanthroline complex that quenched QD PL through electron transfer. The labeled peptides were assembled around a central CdSe/ZnS QD at different ratios, tuning the relative rates of FRET and CT, which were competitive quenching pathways. The concurrent effects of FRET and CT were predictable from a rate analysis that was calibrated to the isolated effects of each of these pathways. Notably, the dye/QD PL intensity ratio reflected changes in the relative rate of FRET but was approximately independent of CT. In turn, the sum of the QD and dye PL intensities, when adjusted for quantum yields, reflected changes in the relative rate of CT quenching, approximately independent of FRET. The capacity for multiplexed sensing of protease activity was demonstrated using these two orthogonal detection channels. Combined CT-FRET configurations with QDs are thus promising for applications in bioanalysis, sensing, and imaging, and may prove useful in other photonic applications.

Base-catalyzed diastereoselective trimerization of trifluoroacetone.

Amphiphilic fluorocarbons have unique properties that facilitate their self assembly and adhesion to both inorganic and biological substrates. Incorporation of these moieties into valuable constructs typically require complex synthetic routes that have limited their use. Here, the base-catalyzed diastereoselective synthesis of 6-methyl-2,4,6-tris(trifluoromethyl)tetrahydro-2H-pyran-2,4-diol is reported. Trimerization of trifluoroacetone in the presence of 5 mol% KHMDS delivers one of four diastereomers selectively in 81% yield with no column chromatography. Temperature screening revealed the reversibility of this trimerization and the funneling of material into the most thermodynamically stable oxane. Subsequent functionalization with boronic acids is reported.

Adapting the Glaser Reaction for Bioconjugation: Robust Access to Structurally Simple, Rigid Linkers.

Copper-mediated coupling between alkynes to generate a structurally rigid, linear 1,3-diyne linkage has been known for over a century. However, the mechanistic requirement to simultaneously maintain CuI and an oxidant has limited its practical utility, especially for complex functional molecules in aqueous solution. We find that addition of a specific bpy-diol ligand protects unprotected peptides from CuII -mediated oxidative damage through the formation of an insoluble CuII gel which solves the critical challenge of applying Glaser coupling to substrates that are degraded by CuII . The generality of this method is illustrated through the conjugation of a series of polar and nonpolar labels onto a fully unprotected GLP-1R agonist through a linear 7 Šdiynyl linker.

Quantum dot-mediated delivery of siRNA to inhibit sphingomyelinase activities in brain-derived cells.

The use of RNAi to suppress protein synthesis offers a potential way of reducing the level of enzymes or the synthesis of mutant toxic proteins but there are few tools currently available for their delivery. To address this problem, bioconjugated quantum dots (QDs) containing a hydrophobic component (N-palmitate) and a sequence VKIKK designed to traverse across cell membranes and visualize drug delivery were developed and tested on cell lines of brain origin. We used the Zn outer shell of the QD to bind HIS6 in JB577 (W•G•Dap(N-Palmitoyl)•VKIKK•P9 •G2 •H6 ) and by a gel-shift assay showed that siRNAs would bind to the positively charged KIKK sequence. By comparing many peptides and QD coatings, we showed that the QD-JB577-siRNA construct was taken up by cells of nervous system origin, distributed throughout the cytosol, and inhibited protein synthesis, implying that JB577 was also promoting endosome egress. By attaching siRNA for luciferase in a cell line over-expressing luciferase, we showed 70% inhibition of mRNA after 24-48 h. To show more specific effects, we synthesized siRNA for neutral (NSMase2), acid (lysosomal ASMase) sphingomyelinase, and sphingosine kinase 1 (SK1), we demonstrated a dose-dependent inhibition of activity. These data suggest that QDs are a useful siRNA delivery tool and QD-siRNA could be a potential theranostic for a variety of diseases.

Arginine selective reagents for ligation to peptides and proteins.

A new class of arginine-specific bioconjugation reagents for protein labeling has been developed. This method utilizes a triazolyl-phenylglyoxal group on the probe molecule that reacts selectively with the guandinyl group of Arg residues in a protein or peptide. The reaction proceeds in neutral to basic bicarbonate buffers and is selective for arginine residues in peptides and folded proteins. Importantly, the triazolyl-phenylglyoxal group can be introduced into complex molecules containing alkyne groups using CuAAC chemistry, providing a robust approach for the generation of phenylglyoxal reactive groups into molecules to be covalently attached onto the surface of proteins. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Oxime conjugation in protein chemistry: from carbonyl incorporation to nucleophilic catalysis.

Use of oxime forming reactions has become a widely applied strategy for peptide and protein bioconjugation. The efficiency of the reaction and robust stability of the oxime product has led to the development of a growing list of methods to introduce the required ketone or aldehyde functionality site specifically into proteins. Early methods focused on site-specific oxidation of an N-terminal serine or threonine and more recently transamination methods have been developed to convert a broader set of N-terminal amino acids into a ketone or aldehyde. More recently, site-specific modification of protein has been attained through engineering enzymes involved in posttranslational modifications in order to accommodate aldehyde-containing substrates. Similarly, a growing list of unnatural amino acids can be introduced through development of selective amino-acyl tRNA synthetase/tRNA pairs combined with codon reassignment. In the case of glycoproteins, glycans can be selectively modified chemically or enzymatically to introduce aldehyde functional groups. Finally, the total chemical synthesis of proteins complements these biological and chemoenzymatic approaches. Once introduced, the oxime ligation of these aldehyde and ketone groups can be catalyzed by aniline or a variety of aniline derivatives to tune the activity, pH preference, stability and solubility of the catalyst. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.