Unprotected Peptides Research Articles

Rhodium-Catalyzed Oxidation of Unprotected Peptide Thiols to Disulfides with Oxygen in Water

Unprotected peptide thiols are efficiently oxidized to peptide disulfides in homogeneous water. The oxidation reaction proceeds under ambient pressure of oxygen (1 atm), using a readily available R...

Light-induced primary amines and o-nitrobenzyl alcohols cyclization as a versatile photoclick reaction for modular conjugation

The advent of click chemistry has had a profound impact on many fields and fueled a need for reliable reactions to expand the click chemistry toolkit. However, developing new systems to fulfill the click chemistry criteria remains highly desirable yet challenging. Here, we report the development of light-induced primary amines and o-nitrobenzyl alcohols cyclization (PANAC) as a photoclick reaction via primary amines as direct click handle, to rapid and modular functionalization of diverse small molecules and native biomolecules. With intrinsic advantages of temporal control, good biocompatibility, reliable chemoselectivity, excellent efficiency, readily accessible reactants, operational simplicity and mild conditions, the PANAC photoclick is robust for direct diversification of pharmaceuticals and biorelevant molecules, lysine-specific modifications of unprotected peptides and native proteins in vitro, temporal profiling of endogenous kinases and organelle-targeted labeling in living systems. This strategy provides a versatile platform for organic synthesis, bioconjugation, medicinal chemistry, chemical biology and materials science.

Synthesis of Peptido RNAs from Unprotected Peptides and Oligoribonucleotides via Coupling in Aqueous Solution

Peptido RNAs are hybrid molecules with a phosphoramidate link between the N‐terminus of a peptide and the 5'‐phosphate of an oligoribonucleotide. Such species are formed in spontaneous co‐oligomerizations of amino acids and ribonucleotides in aqueous condensation buffer. To shed light on the properties of these fascinating molecules, a synthetic method for their preparation in monodisperse form is needed. Herein, we report how peptido RNAs can be prepared via solution‐phase coupling of unprotected peptides and oligoribonucleotides in aqueous solution. The preferred protocol uses pre‐activation of the 5'‐phosphate of the RNA as an imidazolide at pH 6.5, followed by precipitation and coupling to the peptide at pH 8 with an organocatalyst. The procedure gave peptido RNAs from water‐soluble peptides and synthetic oligoribonucleotides in up to 68 % yield. The method is convenient and inexpensive and can produce NMR quantities, opening the door to the systematic exploration of the chemistry of peptido RNAs.

Cysteine/Penicillamine Ligation Independent of Terminal Steric Demands for Chemical Protein Synthesis

AbstractThe chemical ligation of two unprotected peptides to generate a natural peptidic linkage specifically at the C‐ and N‐termini is a desirable goal in chemical protein synthesis but is challenging because it demands high reactivity and selectivity (chemo‐, regio‐, and stereoselectivity). We report an operationally simple and highly effective chemical peptide ligation involving the ligation of peptides with C‐terminal salicylaldehyde esters to peptides with N‐terminal cysteine/penicillamine. The notable features of this method include its tolerance of steric hinderance from the side groups on either ligating terminus, thereby allowing flexible disconnection at sites that are otherwise difficult to functionalize. In addition, this method can be expanded to selective desulfurization and one‐pot ligation‐desulfurization reactions. The effectiveness of this method was demonstrated by the synthesis of VISTA (216‐311), PD‐1 (192‐288) and Eglin C.

Cysteine/Penicillamine Ligation Independent of Terminal Steric Demands for Chemical Protein Synthesis.

The chemical ligation of two unprotected peptides to generate a natural peptidic linkage specifically at the C- and N-termini is a desirable goal in chemical protein synthesis but is challenging because it demands high reactivity and selectivity (chemo-, regio-, and stereoselectivity). We report an operationally simple and highly effective chemical peptide ligation involving the ligation of peptides with C-terminal salicylaldehyde esters to peptides with N-terminal cysteine/penicillamine. The notable features of this method include its tolerance of steric hinderance from the side groups on either ligating terminus, thereby allowing flexible disconnection at sites that are otherwise difficult to functionalize. In addition, this method can be expanded to selective desulfurization and one-pot ligation-desulfurization reactions. The effectiveness of this method was demonstrated by the synthesis of VISTA (216-311), PD-1 (192-288) and Eglin C.

β-Turn Mimics by Chemical Ligation.

We report a simple reductive amination protocol to ligate two peptides, while simultaneously installing a β-turn mimic at the ligation junction. This strategy uses commercially available materials, mild chemical conditions, and a chemoselective ligation reaction of unprotected peptide substrates accessed through standard solid phase methods. This system was implemented in a designed β-hairpin system, and biophysical analysis demonstrates effective mimicry of the β-turn.

Selenocystine Peptides Performance in 5‐endo‐dig Reactions

Herein, we present methods for the generation of selenocysteinyl electrophile by weak Lewis acids or oxidants. The electrophilic selenium species were further utilized in 5‐endo‐dig cyclization reactions with 2‐ethynyl phenols, anisoles, and anilines, yielding substituted benzo[b]furans and indoles bearing short selenocysteine‐containing peptides. Copper(II) bromide promoted 5‐endo‐dig cyclization can be successfully applied for protected and unprotected peptides in high yields. Elaborated protocol allows the construction of phenylindeno[1,2‐c]chromene moiety in 5‐endo‐dig/6‐endo‐dig cascade reactions.

Catalytic Chemoselective O-Phosphorylation of Alcohols

Phosphorylation of alcohols is a fundamentally important reaction in both life science and physical science. Product phosphate monoesters play key roles in living organisms, natural products, pharmaceuticals, and organic materials. Most of the chemical methods to date for synthesizing phosphate monoesters, however, require multistep sequences or are limited to specific types of substrates possibly due to harsh conditions. An alternative way to enable the simple production of phosphate monoesters from highly functionalized precursor alcohols is, thus, highly desired. We report herein a catalytic phosphorylation of alcohols with high functional group tolerance using tetrabutylammonium hydrogen sulfate (TBAHS) and phosphoenolpyruvic acid monopotassium salt (PEP-K) as the catalyst and phosphoryl donor, respectively. This method enables the direct introduction of a nonprotected phosphate group to the hydroxy group of a diverse menu of alcohol substrates, including functionalized small molecules, carbohydrates, and unprotected peptides. Nuclear magnetic resonance, mass spectrometric, and density functional theory analyses suggest that an unprecedented mixed anhydride species, generated from PEP-K and TBAHS, acts as an active phosphoryl donor in this reaction. This operationally simple and chemoselective catalytic phosphorylation allows for the efficient production of densely functionalized O-phosphorylated compounds, which are useful in diverse fields including biology and medicine.

A Sulfonium Triggered Thiol-yne Reaction for Cysteine Modification.

We report a facile thiol-yne type reaction triggered by the sulfonium center. After facile propargylation of thiolethers, the resulting sulfonium could undergo facile addition with thiols in aqueous media at ambient temperature. Further applying this reaction in unprotected peptides bearing neighboring methionine and cysteine could enable a facile intramolecular addition to construct cyclic peptides with better stability, good glutathione resistance, and increased cellular uptakes. Also, the propargylated sulfonium may be used as robust and versatile probes to target cysteines containing biomolecules.

Ortho-Phthalaldehyde (OPA)-based chemoselective protein bioconjugation and peptide cyclization.

Ortho-Phthalaldehyde (OPA)-amine reaction and OPA-amine-thiol reaction have been developed to effectively modify native peptides and proteins under the physiological conditions. First, OPA and its derivatives can rapidly and smoothly react with primary amine moieties in peptides and proteins to achieve native protein biconjugations. Furthermore, OPA-alkyne bifunctional linkers can be used for proteome profiling. Second, OPA-amine-thiol three-component reaction has been developed for chemoselective peptide cyclization, directly on unprotected peptides in the aqueous buffer. Moreover, this OPA-guided cyclic peptide can be further modified with the N-maleimide moiety in one pot to introduce additional functionalities. The development of this OPA based chemoselective bioconjugation and peptide cyclization extends the toolbox for protein chemical modification and construction of cyclic peptides.

Selective modification of sulfamidate-containing peptides.

Hybrid peptides whose N-terminal residues are activated in the form of α-methylisoserine-derived cyclic sulfamidates exhibit rich reactivity as electrophiles, allowing site- and stereoselective modifications at different backbone and side chain positions. The unique properties of this scaffold allow the stereocontrolled late-stage functionalization of the peptide backbone by nucleophilic ring opening with fluorescent probes, thiocarbohydrates and tags for strain-promoted azide-alkyne cycloaddition as well as by installing labile N-terminal affinity tags (biotin) and cytotoxic drugs (chlorambucil) for pH-controlled release. Finally, an unexpected base-promoted acyl group migration from the sulfamidate N-terminus allows fast and quantitative intramolecular modification of nucleophilic side chains on the fully unprotected peptides.

Decarboxylative Couplings for Late-Stage Peptide Modifications.

The application of designer peptides in medicinal chemistry, chemical biology, and materials science has generated new interest in synthetic methods for the structural modification of amino acids. Strategies which facilitate the direct diversification of proteinogenic functional groups within unprotected peptide substrates are particularly attractive as they leverage modern solution- and solid-phase protocols-tools which are now both robust and routine-for the synthesis of native peptides. Accordingly, a recent approach to the decarboxylative functionalization of peptidic carboxylic acids, including aspartic/glutamic acid residues and α-carboxylic acids, has proven to be a promising new strategy for peptide modification. This synthetic method merges conventional strategies for the activation of carboxylic acids with transition metal-catalyzed cross-coupling chemistry to forge new C-C bonds for the late-stage introduction of valuable synthetic handles. This chapter details a step-by-step protocol for the activation and nickel-catalyzed decarboxylative alkylation of a simple peptide substrate to highlight the broad utility of this strategy for the synthesis of designer peptides.

Synthesis of Antimicrobial Lipopeptides Using the "CLipPA" Thiol-Ene Reaction.

Cysteine Lipidation on a Peptide or Amino acid (CLipPA) technology provides a facile method for the lipidation of unprotected peptides containing a free thiol group by using a "click" radical-initiated thiol-ene reaction to effect addition to a vinyl ester. The methodology is highly versatile, leading to high conversion rates while maintaining excellent chemoselectivity and tolerance for a large variety of peptide substrates and functional groups. Herein we describe the simple general procedure for the synthesis of a focused library of bioactive S-lipidated antimicrobial peptides via late-stage derivatization using solution-phase CLipPA lipidation.

Aspartic Acid Forming α-Ketoacid-Hydroxylamine (KAHA) Ligations with (S)-4,4-Difluoro-5-oxaproline.

The α-ketoacid-hydroxylamine (KAHA) ligation allows the coupling of unprotected peptide segments. Currently, the most applied hydroxylamine is the 5-membered cyclic hydroxylamine (S)-5-oxaproline, which forms a homoserine ester as the primary ligation product. In order to access native aspartic acid residues at the ligation site, we synthesized a 4,4-difluoro version of this monomer. Upon KAHA ligation, the resulting difluoro alcohol hydrolyzes to an aspartic acid residue with little or no formation of aspartamide. We applied this monomer for the synthesis of the hormone peptides glucagon and an insulin variant, and as well for segment ligation of the peptides UbcH5a and SUMO3.

Site-Selective Modification of α-Amino Acids and Oligopeptides via Native Amine-Directed γ-C(sp3)-H Arylation

Site-selective modification of chemically and biologically valuable α-amino acids and peptides is of great importance for biochemical study and pharmaceutical development. Few methods based on remote C(sp3)-H functionalization of aliphatic side-chains of peptides has been disclosed in recent years. In this report, we developed a novel approach for γ-C(sp3)-H and γ-/δ-C(sp2)-H arylation of α-amino acids with α-hydrogen by native amine-directed C-H functionalization and further realized the γ-C(sp3)-H arylation of N-terminally unprotected peptides.

Late‐Stage Functionalization of Histidine in Unprotected Peptides

AbstractThe late‐stage functionalization (LSF) of peptides represents a valuable strategy for the design of potent peptide pharmaceuticals by enabling rapid exploration of chemical diversity and offering novel opportunities for peptide conjugation. While the C(sp2)−H activation of tryptophan (Trp) is well documented, the resurgence of radical chemistry is opening new avenues for the C−H functionalization of other aromatic side‐chains. Herein, we report the first example of LSF at C2 of histidine (His) utilizing a broad scope of aliphatic sulfinate salts as radical precursors. In this work, the exquisite selectivity for histidine functionalization was demonstrated through the alkylation of complex unprotected peptides in aqueous media. Finally, this methodology was extended for the installation of a ketone handle, providing an unprecedented anchor for selective oxime/hydrazone conjugation at histidine.

Late-Stage Functionalization of Histidine in Unprotected Peptides.

The late-stage functionalization (LSF) of peptides represents a valuable strategy for the design of potent peptide pharmaceuticals by enabling rapid exploration of chemical diversity and offering novel opportunities for peptide conjugation. While the C(sp2 )-H activation of tryptophan (Trp) is well documented, the resurgence of radical chemistry is opening new avenues for the C-H functionalization of other aromatic side-chains. Herein, we report the first example of LSF at C2 of histidine (His) utilizing a broad scope of aliphatic sulfinate salts as radical precursors. In this work, the exquisite selectivity for histidine functionalization was demonstrated through the alkylation of complex unprotected peptides in aqueous media. Finally, this methodology was extended for the installation of a ketone handle, providing an unprecedented anchor for selective oxime/hydrazone conjugation at histidine.

A Threonine-Forming Oxazetidine Amino Acid for the Chemical Synthesis of Proteins through KAHA Ligation.

α-Ketoacid-hydroxylamine (KAHA) ligation allows the coupling of unprotected peptide segments through the chemoselective formation of an amide bond. Currently, the most widely used variant employs a 5-membered cyclic hydroxylamine that forms a homoserine ester as the primary ligation product. In order to directly form amide-linked threonine residues at the ligation site, we prepared a new 4-membered cyclic hydroxylamine building block. This monomer was applied to the synthesis of wild-type ubiquitin-conjugating enzyme UbcH5a (146 residues) and Titin protein domain TI I27 (89 residues). Both the resulting UbcH5a and the variant with two homoserine residues showed identical activity to a recombinant variant in a ubiquitination assay.

Chemoselective Peptide Cyclization and Bicyclization Directly on Unprotected Peptides

Cyclic peptides are drawing wide attention as potential medium-sized modulators of biomolecular interactions with large binding surfaces. Simple but effective peptide cyclization methods are needed to construct cyclic peptide libraries by both peptide and nonpeptide chemists. Herein, we report a highly chemoselective and operation-simple method directly cyclizing unprotected peptides, in which ortho-phthalaldehyde (OPA) is found to react with the lysine/N-terminus and cysteine within one unprotected peptide sequence effectively to form the isoindole-bridged cyclic peptides. This reaction is carried out in the aqueous buffer and features tolerance of diverse functionalities, rapid and clean transformation, and operational simplicity. In addition, OPA peptide cyclization can also be combined with native chemical ligation-mediated cyclization to generate bicyclic peptides. Furthermore, the OPA peptide cyclization product can further react with the N-maleimide moiety in a one-pot manner to introduce additional functional motifs, like a fluorophore probe, biomolecules (e.g., glycan, peptide, or DNA). This OPA-cyclization method extends the toolbox for integrating postcyclization modification and bioconjugation into peptide cyclization with an all-in-one manner strategy.

Templated chemistry for bioorganic synthesis and chemical biology.

In light of the 2018 Max Bergmann Medal, this review discusses advancements on chemical biology–driven templated chemistry developed in the author's laboratories. The focused review introduces the template categories applied to orient functional units such as functional groups, chromophores, biomolecules, or ligands in space. Unimolecular templates applied in protein synthesis facilitate fragment coupling of unprotected peptides. Templating via bimolecular assemblies provides control over proximity relationships between functional units of two molecules. As an instructive example, the coiled coil peptide–templated labelling of receptor proteins on live cells will be shown. Termolecular assemblies provide the opportunity to put the proximity of functional units on two (bio)molecules under the control of a third party molecule. This allows the design of conditional bimolecular reactions. A notable example is DNA/RNA–triggered peptide synthesis. The last section shows how termolecular and multimolecular assemblies can be used to better characterize and understand multivalent protein‐ligand interactions.