One-Pot Peptide Ligation–Oxidative Cyclization Protocol for the Preparation of Short-/Medium-Size Disulfide Cyclopeptides

Native chemical ligation (NCL) employing the <i>N</i>-methylbenzimidazolinone (MeNbz) linker readily provided the linear precursor of a 16-mer peptide that is difficult to obtain by stepwise solid-phase peptide synthesis. NCL and the workup conditions were improved toward a protocol that allows for quantitative removal of the 4-hydroxymercaptophenol additive and subsequent formation of the disulfide bridge in the NCL cocktail by oxidation in air, tolerated by the presence of tris­(hydroxypropyl)­phosphine

Chemical Protein Synthesis Using a Second-Generation <i>N</i>‑Acylurea Linker for the Preparation of Peptide-Thioester Precursors

The broad utility of native chemical ligation (NCL) in protein synthesis has fostered a search for methods that enable the efficient synthesis of C<i>-</i>terminal peptide-thioesters, key intermediates in NCL. We have developed an <i>N-</i>acylurea (Nbz) approach for the synthesis of thioester peptide precursors that efficiently undergo thiol exchange yielding thioester peptides and subsequently NCL reaction. However, the synthesis of some glycine-rich sequences revealed limitations, such as diacylated products that can not be converted into <i>N</i>-acylurea peptides. Here, we introduce a new <i>N-</i>acylurea linker bearing an <i>o-</i>amino­(methyl)­aniline (MeDbz) moiety that enables in a more robust peptide chain assembly. The generality of the approach is illustrated by the synthesis of a pentaglycine sequence under different coupling conditions including microwave heating at coupling temperatures up to 90 C, affording the unique and desired <i>N-</i>acyl-<i>N</i>′-methylacylurea (MeNbz) product. Further extension of the method allowed the synthesis of all 20 natural amino acids and their NCL reactions. The kinetic analysis of the ligations using model peptides shows the MeNbz peptide rapidly converts to arylthioesters that are efficient at NCL. Finally, we show that the new MeDbz linker can be applied to the synthesis of cysteine-rich proteins such the cyclotides Kalata B1 and MCoTI-II through a one cyclization/folding step in the ligation/folding buffer

Imidazole-1-sulfonyl Azide-Based Diazo-Transfer Reaction for the Preparation of Azido Solid Supports for Solid-Phase Synthesis

An efficient, standard, mild, and copper-free imidazole-1-sulfonyl azide hydrochloride-based diazo-transfer method was implemented in a set of four resins that cover a broad range of hydrophobicity. The imidazole-1-sulfonyl azide hydrochloride is easily prepared/commercially available, stable upon storage at 4 °C, and proved to be a suitable alternative to triflyl azide for diazo-transfer reactions in amine functionalized resins. We have successfully applied the azido resins for the conjugation of a TFA-labile Wang-type linker using Click Chemistry

Chemical Protein Synthesis Using a Second-Generation <i>N</i>‑Acylurea Linker for the Preparation of Peptide-Thioester Precursors

The broad utility of native chemical ligation (NCL) in protein synthesis has fostered a search for methods that enable the efficient synthesis of C<i>-</i>terminal peptide-thioesters, key intermediates in NCL. We have developed an <i>N-</i>acylurea (Nbz) approach for the synthesis of thioester peptide precursors that efficiently undergo thiol exchange yielding thioester peptides and subsequently NCL reaction. However, the synthesis of some glycine-rich sequences revealed limitations, such as diacylated products that can not be converted into <i>N</i>-acylurea peptides. Here, we introduce a new <i>N-</i>acylurea linker bearing an <i>o-</i>amino­(methyl)­aniline (MeDbz) moiety that enables in a more robust peptide chain assembly. The generality of the approach is illustrated by the synthesis of a pentaglycine sequence under different coupling conditions including microwave heating at coupling temperatures up to 90 C, affording the unique and desired <i>N-</i>acyl-<i>N</i>′-methylacylurea (MeNbz) product. Further extension of the method allowed the synthesis of all 20 natural amino acids and their NCL reactions. The kinetic analysis of the ligations using model peptides shows the MeNbz peptide rapidly converts to arylthioesters that are efficient at NCL. Finally, we show that the new MeDbz linker can be applied to the synthesis of cysteine-rich proteins such the cyclotides Kalata B1 and MCoTI-II through a one cyclization/folding step in the ligation/folding buffer

Detection of HIV-1 Specific Monoclonal Antibodies Using Enhancement of Dye-Labeled Antigenic Peptides

A simple bifunctional colorimetric/fluorescent sensing assay is demonstrated for the detection of HIV-1 specific antibodies. This assay makes use of a short peptide sequence coupled to an environmentally sensitive dye that absorbs and emits in the visible portion of the spectrum. The core peptide sequence is derived from the highly antigenic six-residue epitope of the HIV-1 p17 protein and is situated adjacent to a terminal cysteine residue which enables site-specific fluorescent labeling with Cy3 cyanine dye. Interaction of the Cy3-labeled p17 peptide with monoclonal anti-p17 antibody resulted in an up to 4-fold increase in dye absorption and greater than 5-fold increase in fluorescent emission, yielding a limit of detection as low as 73 pM for the target antibody. This initial study demonstrates both proof-of-concept for this approach and suggests that the resulting sensor could potentially be used as a rapid screening method for HIV-1 infection while requiring minimal equipment and reagents. The potential for utilizing this assay in simple field-portable point-of-care and diagnostic devices is discussed

Rapid Covalent Ligation of Fluorescent Peptides to Water Solubilized Quantum Dots

Water solubilized nanoparticles such as CdSe−ZnS core−shell nanocrystals (quantum dots, QDs) have great potential in bioimaging and sensing applications due to their excellent photophysical properties. However, the efficient modification of QDs with complex biomolecules represents a significant challenge. Here, we describe a straightforward arylhydrazone approach for the chemoselective covalent modification of QDs that is compatible with neutral pH and micromolar concentrations of the peptide target. The kinetics of covalent modification can be monitored spectroscopically at 354 nm in the presence of the QD and average peptide/QD ratios from 2:1 to 11:1 were achieved with excellent control over the desired valency. These results suggest that aniline catalyzed hydrazone ligation has the potencial to provide a general method for the controlled assembly of a variety of nanoparticle-biomolecule hybrids

On the Utility of Chemical Strategies to Improve Peptide Gut Stability

Inherent susceptibility of peptides to enzymatic degradation in the gastrointestinal tract is a key bottleneck in oral peptide drug development. Here, we present a systematic analysis of (i) the gut stability of disulfide-rich peptide scaffolds, orally administered peptide therapeutics, and well-known neuropeptides and (ii) medicinal chemistry strategies to improve peptide gut stability. Among a broad range of studied peptides, cyclotides were the only scaffold class to resist gastrointestinal degradation, even when grafted with non-native sequences. Backbone cyclization, a frequently applied strategy, failed to improve stability in intestinal fluid, but several site-specific alterations proved efficient. This work furthermore highlights the importance of standardized gut stability test conditions and suggests defined protocols to facilitate cross-study comparison. Together, our results provide a comparative overview and framework for the chemical engineering of gut-stable peptides, which should be valuable for the development of orally administered peptide therapeutics and molecular probes targeting receptors within the gastrointestinal tract

On the Utility of Chemical Strategies to Improve Peptide Gut Stability

Inherent susceptibility of peptides to enzymatic degradation in the gastrointestinal tract is a key bottleneck in oral peptide drug development. Here, we present a systematic analysis of (i) the gut stability of disulfide-rich peptide scaffolds, orally administered peptide therapeutics, and well-known neuropeptides and (ii) medicinal chemistry strategies to improve peptide gut stability. Among a broad range of studied peptides, cyclotides were the only scaffold class to resist gastrointestinal degradation, even when grafted with non-native sequences. Backbone cyclization, a frequently applied strategy, failed to improve stability in intestinal fluid, but several site-specific alterations proved efficient. This work furthermore highlights the importance of standardized gut stability test conditions and suggests defined protocols to facilitate cross-study comparison. Together, our results provide a comparative overview and framework for the chemical engineering of gut-stable peptides, which should be valuable for the development of orally administered peptide therapeutics and molecular probes targeting receptors within the gastrointestinal tract

Multidentate Poly(ethylene glycol) Ligands Provide Colloidal Stability to Semiconductor and Metallic Nanocrystals in Extreme Conditions

We present the design and synthesis of a new set of poly(ethylene glycol) (PEG)-based ligands appended with multidentate anchoring groups and test their ability to provide colloidal stability to semiconductor quantum dots (QDs) and gold nanoparticles (AuNPs) in extreme buffer conditions. The ligands are made of a PEG segment appended with two thioctic acid (TA) or two dihydrolipoic acid (DHLA) anchoring groups, bis(TA)-PEG-OCH3 or bis(DHLA)-PEG-OCH3. The synthesis utilizes Michael addition to create a branch point at the end of a PEG chain combined with carbodiimide-coupling to attach two TA groups per PEG chain. Dispersions of CdSe−ZnS core−shell QDs and AuNPs with remarkable long-term colloidal stability at pHs ranging from 1.1 to 13.9 and in the presence of 2 M NaCl have been prepared and tested using these ligands. AuNPs with strong resistance to competition from dithiothreitol (as high as 1.5 M) have also been prepared. This opens up possibilities for using them as stable probes in a variety of bio-related studies where resistance to degradation at extreme pHs, at high electrolyte concentration, and in thiol-rich environments is highly desirable. The improved colloidal stability of nanocrystals afforded by the tetradentate ligands was further demonstrated via the assembly of stable QD−nuclear localization signal peptide bioconjugates that promoted intracellular uptake

Interactions between Redox Complexes and Semiconductor Quantum Dots Coupled via a Peptide Bridge

Colloidal quantum dots (QDs) have a large fraction of their atoms arrayed on their surfaces and are capped with bifunctional ligands, which make their photoluminescence highly sensitive to potential charge transfer to or from the surrounding environment. In this report, we used peptides as bridges between CdSe-ZnS QDs and metal complexes to promote charge transfer between the metal complexes and QDs. We found that quenching of the QD emission is highly dependent on the relative position of the oxidation levels of QDs and metal complex used; it also traces the number of metal complexes brought in close proximity of the nanocrystal surface. In addition, partial bleaching of the absorption was measured for the QD-metal complex assemblies. These proximity driven interactions were further used to construct sensing assemblies to detect proteolytic enzyme activity