Lipidation and PEGylation Strategies to Prolong the Half-Life of a Nanomolar EphA4 Receptor Antagonist.

The EphA4 receptor tyrosine kinase plays a role in neurodegenerative diseases, inhibition of nerve regeneration, cancer progression and other diseases. Therefore, EphA4 inhibition has potential therapeutic value. Selective EphA4 kinase inhibitors are not available, but we identified peptide antagonists that inhibit ephrin ligand binding to EphA4 with high specificity.

Stretching Peptides to Generate Small Molecule β-Strand Mimics.

Advances in the modulation of protein-protein interactions (PPIs) enable both characterization of PPI networks that govern diseases and design of therapeutics and probes. The shallow protein surfaces that dominate PPIs are challenging to target using standard methods, and approaches for accessing extended backbone structures are limited. Here, we incorporate a rigid, linear, diyne brace between side chains at the to 2 positions to generate a family of low-molecular-weight, extended-backbone peptide macrocycles.

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.

A Shelf Stable Fmoc Hydrazine Resin for the Synthesis of Peptide Hydrazides.

C-terminal hydrazides are an important class of synthetic peptides with an ever expanding scope of applications, but their widespread application for chemical protein synthesis has been hampered due to the lack of stable resin linkers for synthesis of longer and more challenging peptide hydrazide fragments. We present a practical method for the regeneration, loading, and storage of trityl-chloride resins for the production of hydrazide containing peptides, leveraging 9-fluorenylmethyl carbazate. We show that these resins are extremely stable under several common resin storage conditions.

Direct observation of peptide hydrogel self-assembly.

The characterization of self-assembling molecules presents significant experimental challenges, especially when associated with phase separation or precipitation. Transparent window infrared (IR) spectroscopy leverages site-specific probes that absorb in the "transparent window" region of the biomolecular IR spectrum. Carbon-deuterium (C-D) bonds are especially compelling transparent window probes since they are non-perturbative, can be readily introduced site selectively into peptides and proteins, and their stretch frequencies are sensitive to changes in the local molecular environment.

Light-Triggered In Situ Biosynthesis of Artificial Melanin for Skin Protection.

Tyrosinase-mediated melanin synthesis is an essential biological process that can protect skin from UV radiation and radical species. This work reports on in situ biosynthesis of artificial melanin in native skin using photoactivatable tyrosinase (PaTy). The I41Y mutant of Streptomyces avermitilis tyrosinase (SaTy) shows enzymatic activity comparable to that of wild-type SaTy.

Improving the Gastrointestinal Stability of Linaclotide.

High susceptibility to proteolytic degradation in the gastrointestinal tract limits the therapeutic application of peptide drugs in gastrointestinal disorders. Linaclotide is an orally administered peptide drug for the treatment of irritable bowel syndrome with constipation (IBS-C) and abdominal pain. Linaclotide is however degraded in the intestinal environment within 1 h, and improvements in gastrointestinal stability might enhance its therapeutic application.

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).

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.

Correction to "Synthetic Elaboration of Native DNA by RASS (SENDR)".

[This corrects the article DOI: 10.1021/acscentsci.0c00680.

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.

Two for the Price of One: Heterobivalent Ligand Design Targeting Two Binding Sites on Voltage-Gated Sodium Channels Slows Ligand Dissociation and Enhances Potency.

Voltage-gated sodium (Na) channels are pore-forming transmembrane proteins that play essential roles in excitable cells, and they are key targets for antiepileptic, antiarrhythmic, and analgesic drugs. We implemented a heterobivalent design strategy to modulate the potency, selectivity, and binding kinetics of Na channel ligands. We conjugated μ-conotoxin KIIIA, which occludes the pore of the Na channels, to an analogue of huwentoxin-IV, a spider-venom peptide that allosterically modulates channel gating.

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.

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.

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.

In Situ Neutralization Protocols for Boc-SPPS.

Significant effort has been devoted to the optimization of solid-phase peptide synthesis (SPPS) to maximize the process to facilitate the synthesis of a desired peptide sequence, without extensive optimization or resynthesis. Over the last 25 years, a set of synthetic protocols developed by Kent and Alewood has proven to be robust and efficient for Boc/Bzl SPPS and has been widely adopted by the research community. In this chapter, we describe a variation of manual in situ neutralization protocols for Boc-SPPS that are highly effective for the rapid synthesis of peptides with different C-terminal functionalities.

In vivo biosynthesis of tyrosine analogs and their concurrent incorporation into a residue-specific manner for enzyme engineering.

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.

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.

Copying Life: Synthesis of an Enzymatically Active Mirror-Image DNA-Ligase Made of D-Amino Acids.

Our objective is the creation of a mirror-image synthetic biology: that is, to mimic, entirely independent of Nature, a biological system and to re-create it from artificial component parts. Utilizing enantiomeric L-nucleotides and D-amino acids rather than the natural components, we use chemical synthesis toward a basic, self-replicating mirror-image biological system. Here, we report the synthesis of a functional DNA-ligase in the D-enantiomeric conformation, which is an exact mirror-image of the natural enzyme, exhibiting DNA ligation activity on chirally inverted nucleic acids in L-conformation, but not acting on natural substrates and with natural co-factors.

Native Chemical Ligation of Peptides and Proteins.

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.

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.

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.