Optimization of peptide synthesis time and sustainability using novel eco-friendly binary solvent systems with induction heating on an automated peptide synthesizer.

On December 12th, 2023, the European Commission took regulatory action to amend Annex XVII of REACH, imposing restrictions on the use of N,N-dimethylformamide (DMF) within the EU market owing to its high toxicity. Historically, DMF has been widely considered the gold standard for solid-phase peptide synthesis (SPPS). Being urgent to propose alternative solvents, we tested the suitability of non-hazardous neat and mixed solvents. Notably, binary solvent mixtures containing dimethyl sulfoxide as one of the solvent partners demonstrated high efficacy in solubilizing reagents while maintaining the desired swelling characteristics of common resins. A series of binary solvent mixtures were tested in automated SPPS, both at room temperature and high temperature, employing the PurePep® Chorus synthesizer, which enabled controlled induction heating between 25 and 90°C with oscillation mixing. The performances were assessed in challenging peptide sequences, i.e., ACP (65-74), and in longer and aggregating sequences like SARS-CoV-2 RBM (436-507) and ß-amyloid (1-42). Furthermore, as part of the proposed sustainable approach to minimize the utilization of hazardous solvents, we coupled the novel PurePep EasyClean catch-and-release purification technology. This work, addressing regulatory compliance, emphasizes the crucial role of green chemistry in advancing safer and more environmentally friendly practices in SPPS.

Traceless parallel peptide purification by a first-in-class reductively cleavable linker system featuring a safety-release.

Hundreds of peptides can be synthesized by automated parallel synthesizers in a single run. In contrast, the most widely used peptide purification method - high-pressure liquid chromatography (HPLC) - only allows one-by-one processing of each sample. The chromatographic purification of many peptides, therefore, remains a time-consuming and costly effort. Catch-and-release methods can be processed in parallel and potentially provide a remedy. However, no such system has yet provided a true alternative to HPLC. Herein we present the development of a side-reaction free, reductively cleavable linker. The linker is added to the target peptide as the last building block during peptide synthesis. After acidic cleavage from synthetic resin, the linker-tagged full-length peptide is caught onto an aldehyde-modified solid support by rapid oxime ligation, allowing removal of all impurities lacking the linker by washing. Reducing the aryl azide to an aniline sensitizes the linker for cleavage. However, scission does not occur at non-acidic pH enabling wash out of reducing agent. Final acidic treatment safely liberates the peptide by an acid-catalysed 1,6-elimination. We showcase this first-in-class reductively cleavable linker system in the parallel purification of a personalized neoantigen cocktail, containing 20 peptides for cancer immunotherapy within six hours.

Immobilization methods for the rapid total chemical synthesis of proteins on microtiter plates.

The chemical synthesis of proteins typically involves the solid-phase peptide synthesis of unprotected peptide fragments that are stitched together in solution by native chemical ligation (NCL). The process is slow, and throughput is limited because of the need for repeated high performance liquid chromatography purification steps after both solid-phase peptide synthesis and NCL. With an aim to provide faster access to functional proteins and to accelerate the functional analysis of synthetic proteins by parallelization, we developed a method for the high performance liquid chromatography-free synthesis of proteins on the surface of microtiter plates. The method relies on solid-phase synthesis of unprotected peptide fragments, immobilization of the C-terminal fragment and on-surface NCL with an unprotected peptide thioester in crude form. Herein, we describe the development of a suitable immobilization chemistry. We compared (i) formation of nickel(II)-oligohistidine complexes, (ii) Cu-based [2 + 3] alkine-azide cycloaddition and (iii) hydrazone ligation. The comparative study identified the hydrazone ligation as most suitable. The sequence of immobilization via hydrazone ligation, on-surface NCL and radical desulfurization furnished the targeted SH3 domains in near quantitative yield. The synthetic proteins were functional as demonstrated by an on-surface fluorescence-based saturation binding analysis. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Parallel Chemical Protein Synthesis on a Surface Enables the Rapid Analysis of the Phosphoregulation of SH3 Domains.

Analysis of postranslationally modified protein domains is complicated by an availability problem, as recombinant methods rarely allow site-specificity at will. Although total synthesis enables full control over posttranslational and other modifications, chemical approaches are limited to shorter peptides. To solve this problem, we herein describe a method that combines a) immobilization of N-terminally thiolated peptide hydrazides by hydrazone ligation, b) on-surface native chemical ligation with self-purified peptide thioesters, c) radical-induced desulfurization, and d) a surface-based fluorescence binding assay for functional characterization. We used the method to rapidly investigate 20 SH3 domains, with a focus on their phosphoregulation. The analysis suggests that tyrosine phosphorylation of SH3 domains found in Abl kinases act as a switch that can induce both the loss and, unexpectedly, gain of affinity for proline-rich ligands.

Solid phase synthesis of short peptide-based multimetal tags for biomolecule labeling.

We describe an unprecedented solid phase peptide synthesis (SPPS) of short peptide-based multimetal tags designated as elemental tags for the quantification of biomolecules via inductively coupled plasma mass spectrometry (ICP-MS). The macrocyclic chelator 1,4,7,10-tetraazacyclododecane N,N',N″,N‴-tetra acetic acid (DOTA) was attached to the side chain of N-α-(9-fluorenylmethoxycarbonyl)-l-lysine (Fmoc-Lys-OH) and metalated with a lanthanide to provide a building block for Fmoc-based SPPS. Thereby, in contrast to existing strategies for the synthesis of DOTA-peptide conjugates, an already metalated DOTA-amino acid was used as a building block for SPPS. The DOTA-lanthanide complex was stable throughout the whole SPPS, even during the final cleavage in concentrated trifluoroacetic acid. This indicates that the strategy to first metalate the Fmoc-Lys(DOTA)-OH and to utilize the metal coordination to protect the carboxyl groups of DOTA offers an alternative to conventional synthetic routes using tert-butyl protected DOTA. Several small peptides containing up to four metal ions were synthesized, among them peptides carrying defined metal sequences consisting of two different lanthanides. The peptides were N-terminally maleimide-functionalized, thus introducing a moiety for conjugation to thiol-bearing biomolecules. The final objective of this work was the signal enhancement in ICP-MS-based DNA quantification assays. To evaluate the performance of the multimetal peptide tags in assay, they were applied to label thiol-modified 15mer DNA oligonucleotide probes. These served as reporter probes in a model sandwich-type hybridization assay. Thereby, we found that the ICP-MS signal increased linearly with the number of lanthanide ions attached to the reporter probe.

En route to dinitroacetylene: nitro(trimethylsilyl)acetylene and nitroacetylene harnessed by dicobalt hexacarbonyl.

Dinitroacetylene and other nitroacetylenes are attractive stoichiometric precursors to high energy-density materials, but suffer from high reactivity and thermal instability. Herein, we report that nitroacetylenes can be dramatically stabilized in the form of their dicobalt hexacarbonyl complexes. In particular, we describe the syntheses and characterization of the first two transition-metal complexes of nitroalkynes, [µ-1-nitro-2-(trimethylsilyl)ethyne-1,2-diyl]bis(tricarbonylcobalt)(Co-Co) and [µ-1-nitroethyne-1,2-diyl]bis(tricarbonylcobalt)(Co-Co). The chemistry of these compounds reveals their potential as reaction partners in [2+2+2] cyclotrimerizations, furnishing nitroindane, nitrotetralin, and trinitrobenzene products. The X-ray crystal structure of 1,3,5-trinitro-2,4,6-tris(trimethylsilyl)benzene presents a distorted, yet planar, aromatic ring.