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Lipophosphoglycans (LPGs) are found on the surface of Leishmania, a protozoan parasite, and are immunologically important. Herein, disaccharide 1-phosphate repeating units of LPGs were successfully synthesized on a solid support with high anomeric purity using a disaccharide α-1-phosphoramidite building block. To enhance solubility in the reaction solvent, hydroxy-protecting groups in the form of para-t-butylbenzoyl were introduced to the building block. The saccharide chain was elongated via stable glycosyl boranophosphate linkages, followed by the conversion of inter-sugar linkages to phosphodiester counterparts using an oxaziridine derivative. The addition of a silylating reagent post-reaction with the oxaziridine derivative efficiently facilitated the conversion of boranophosohodiesters to phosphodiesters. This method enabled the α-selective synthesis of up to 15 repeating units, marking the longest homogeneous repeating units of LPGs synthesized chemically. Given the chain length equivalence to native LPGs, the method developed herein holds promise for advancing anti-Leishmanial pharmaceuticals and vaccines., (© 2024 The Author(s). Chemistry - A European Journal published by Wiley-VCH GmbH.)

This protocol describes the synthesis of long oligonucleotides (up to 401-mer), their isolation from complex mixtures using the catching-by-polymerization (CBP) method, and the selection of error-free sequence via cloning followed by Sanger sequencing. Oligo synthesis is achieved under standard automated solid-phase synthesis conditions with only minor yet critical adjustments using readily available reagents. The CBP method involves tagging the full-length sequence with a polymerizable tagging phosphoramidite (PTP), co-polymerizing the sequence into a polymer, washing away failure sequences, and cleaving the full-length sequence from the polymer. Cloning and sequencing guided selection of error-free sequence overcome the problems of substitution, deletion, and addition errors that cannot be addressed using any other methods, including CBP. Long oligos are needed in many areas such as protein engineering and synthetic biology. The methods described here are particularly important for projects requiring long oligos containing long repeats or stable higher-order structures, which are difficult or impossible to produce using any other existing technologies. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Long oligo synthesis Support Protocol 1: Synthesis of polymerizable tagging phosphoramidite (PTP) Support Protocol 2: Synthesis of 5'-O-Bz phosphoramidite Basic Protocol 2: Catching-by-polymerization (CBP) purification Basic Protocol 3: Error-free sequence selection via cloning and sequencing., (© 2024 Wiley Periodicals LLC.)

Chemical protein synthesis (CPS), in which custom peptide segments of ~20-60 aa are produced by solid-phase peptide synthesis and then stitched together through sequential ligation reactions, is an increasingly popular technique. The workflow of CPS is often depicted with a "bracket" style diagram detailing the starting segments and the order of all ligation, desulfurization, and/or deprotection steps to obtain the product protein. Brackets are invaluable tools for comparing multiple possible synthetic approaches and serve as blueprints throughout a synthesis. Drawing CPS brackets by hand or in standard graphics software, however, is a painstaking and error-prone process. Furthermore, the CPS field lacks a standard bracket format, making side-by-side comparisons difficult. To address these problems, we developed BracketMaker, an open-source Python program with built-in graphic user interface (GUI) for the rapid creation and analysis of CPS brackets. BracketMaker contains a custom graphics engine which converts a text string (a protein sequence annotated with reaction steps, introduced herein as a standardized format for brackets) into a high-quality vector or PNG image. To aid with new syntheses, BracketMaker's "AutoBracket" tool automatically performs retrosynthetic analysis on a set of segments to draft and rank all possible ligation orders using standard native chemical ligation, protection, and desulfurization techniques. AutoBracket, in conjunction with an improved version of our previously reported Automated Ligator (Aligator) program, provides a pipeline to rapidly develop synthesis plans for a given protein sequence. We demonstrate the application of both programs to develop a blueprint for 65 proteins of the minimal Escherichia coli ribosome., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Solid-phase peptide synthesis (SPPS) is the prevailing method for synthesizing research peptides today. However, SPPS is associated with a significant environmental concern due to the utilization of hazardous solvents such as N,N-dimethylformamide (DMF) or N-methylpyrrolidone, which generate substantial waste. In light of this, our research endeavors to identify more environmentally friendly solvents for SPPS. In this study, we have assessed the suitability of five green solvents as alternatives to DMF in microwave assisted SPPS. The solvents evaluated include Cyrene, ethyl acetate, 1,3-dioxolane, tetrahydro-2-methylfuran, and N-Butylpyrrolidinone (NBP). Our investigation encompassed all stages of the synthesis process, from resin swelling, dissolution of reagents, culminating in the successful synthesis of five diverse peptides, including the challenging ACP 65-74, Peptide 18A, Thymosin α1, and Jung-Redemann peptide. Our findings indicate that NBP emerged as a strong contender, performing on par with DMF in all tested syntheses. Furthermore, we observed that combinations of NBP with either ethyl acetate or tetrahydro-2-methylfuran demonstrated excellent results. This research contributes to the pursuit of more sustainable and environmentally conscious practices in peptide synthesis., (© 2024 European Peptide Society and John Wiley & Sons Ltd.)

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 β-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., (© 2024 European Peptide Society and John Wiley & Sons Ltd.)

The negligible cytotoxicity of anion surface-linked dendrons makes glutamic acid-based dendrons a potential candidate for materials and biological applications. Despite the inherent drawbacks of the conventional solution phase synthesis of glutamic acid-based dendrons, there have been no advancements in these protocols. Herein, we demonstrate the first-ever convergent solid phase synthesis of dendrons, up to fourth generation, having glutamic acid branching points produced by preactivation of dicarboxylic acid groups with N -hydroxysuccinimide and simultaneous coupling with amine groups of two growing peptide chains, with excellent yields (30-70%). In addition to the general advantages, such as the easy workup, a final single purification step, and an overall short synthesis duration, the convergent solid phase synthesis allowed us to chemically synthesize glutamic acid branching-based dendrons that cannot be accessed by standard divergent solid phase synthesis. This method has also been validated for its application in synthesizing hard-to-achieve Janus peptide dendrimers in a single stretch on a solid support. Our work corroborates the efficacy of controlled -COOH activation to accomplish an atypical solid phase synthesis of diverse glutamic acid dendrons in a convergent fashion. This is the first example of a Janus peptide dendrimer being synthesized on a solid support, utilizing both convergent and divergent approaches simultaneously.

Self-promoted glycosylations with trichloroacetimidate glycosyl donors are demonstrated on solid-phase-anchored peptides orthogonally deprotected and tosylcarbamoylated on the side-chains of cysteine and serine, respectively. The donor scope included glucosyl as well as mannosyl trichloroacetimidates, carrying benzyl, acetyl, or isopropylidene protecting groups. Isopropylidene groups were found to be removed under the acidic conditions used for release of the neoglycopeptides from the solid support, yielding neoglycopeptides with unprotected hydroxyl groups. Glycosylation of a peptide containing a carbamoylated tyrosine was attempted as well, but the desired neoglycopeptide could not be synthesized due to thermal instability of the carbamate., (© 2024 The Authors. ChemPlusChem published by Wiley-VCH GmbH.)

The synthesis of linker-payloads is a critical step in developing antibody-drug conjugates (ADCs), a rapidly advancing therapeutic approach in oncology. The conventional method for synthesizing cathepsin B-labile dipeptide linkers, which are commonly used in ADC development, involves the solution-phase assembly of cathepsin B-sensitive dipeptides, followed by the installation of self-immolative para -aminobenzyl carbonate to facilitate the attachment of potent cytotoxic payloads. However, this approach is often low yield and laborious, especially when extending the peptide chain with components like glutamic acid to improve mouse serum stability or charged amino acids or poly(ethylene glycol) moieties to enhance linker hydrophilicity. Here, we introduce a novel approach utilizing late-stage desulfurization chemistry, enabling safe, facile, and cost-effective access to the cathepsin B-cleavable linker, Val-Ala-PABC-MMAE, on resin for the first time.

Semaglutide is currently the most promising antidiabetic drug, especially for the treatment of type 2 diabetes mellitus, due to its excellent efficacy in glycemic control and weight loss. However, the production of semaglutide remains high cost, and high yield, low cost, and high purity still remains a challenge. Herein, we reported a convenient and high-yield strategy for the preparation of semaglutide through fragmented condensation coupling, involving solid-phase peptide synthesis of tetrapeptide and on-column refolding and on-column enzyme cleavage based inclusion body expression of Lys 26 Arg 34 GLP-1 (11-37) with fused protein tags in an X-Y-D4K-G pattern. The optimized N-terminal protein tag significantly boosts inclusion body expression level, while on-column refolding and on-column enzyme cleavage avoid precipitation, enhancing efficiency and yield together with one-step purification. The successful preparation of semaglutide is expected to achieve large-scale industrial production with low cost, high yield and high purity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Organoboron compounds have a wide range of applications in numerous research fields, and methods to incorporate them in biomolecules are much sought after. Here, on-resin chemical syntheses of aliphatic and vinylogous peptide boronic acids are presented by transition metal-catalyzed late-stage hydroboration of alkene and alkyne groups in peptides and peptoids, for example on allyl- and propargylglycine residues, using readily available chemicals. These methods yield peptide boronic acids with much shorter linkers than previously reported on-resin methods. Furthermore, the methods are regio- and stereoselective, compatible with all canonical amino acid residues and can be applied to short, long, and in part even "difficult" peptide sequences. In a feasibility study, the protected peptide vinylboronic acids are further derivatized by the Petasis reaction using salicylaldehyde derivatives. The ability of the obtained peptide boronic acids to reversibly bind to carbohydrates is demonstrated in a catch-release model experiment using a fluorescently labeled peptide boronic acid on cross-linked dextran beads. In summary, this highlights the potential of the target compounds for drug discovery, glycan-specific target recognition, controlled release, and diagnostics., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

In this protocol, we describe how to perform the photo-isomerization of cyclic peptides containing an unsaturated β-amino acid. This process triggers the formation or disassembly of cyclic peptide nanotubes under appropriate light irradiation. Specifically, we start by describing the solid-phase synthesis of the cyclic peptide component. We also present a technique for performing isomerization studies in solution and how to extend it to microfluidic aqueous droplets. For complete details on the use and execution of this protocol, please refer to Vilela-Picos et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The escalating resistance of agricultural pests to chemical insecticides necessitates the development of novel, efficient, and safe biological insecticides. Conus quercinus , a vermivorous cone snail, yields a crude venom rich in peptides for marine worm predation. This study screened six α-conotoxins with insecticidal potential from a previously constructed transcriptome database of C. quercinus , characterized by two disulfide bonds. These conotoxins were derived via solid-phase peptide synthesis (SPPS) and folded using two-step iodine oxidation for further insecticidal activity validation, such as CCK-8 assay and insect bioassay. The final results confirmed the insecticidal activities of the six α-conotoxins, with Qc1.15 and Qc1.18 exhibiting high insecticidal activity. In addition, structural analysis via homology modeling and functional insights from molecular docking offer a preliminary look into their potential insecticidal mechanisms. In summary, this study provides essential references and foundations for developing novel insecticides.

The first drugs discovered using DNA-encoded chemical library (DEL) screens have entered late-stage clinical development. However, DEL technology as a whole still suffers from poor chemical purity resulting in suboptimal performance. In this work, we report a technique to overcome this issue through self-purifying release of the DEL after magnetic bead-based synthesis. Both the first and last building blocks of each assembled library member were linked to the beads by tethers that could be cleaved by mutually orthogonal chemistry. Sequential cleavage of the first and last tether, with washing in between, ensured that the final library comprises only the fully complete compounds. The outstanding purity attained by this approach enables a direct correlation of chemical display and encoding, allows for an increased chemical reaction scope, and facilitates the use of more diversity elements while achieving greatly improved signal-to-noise ratios in selections.

The rise of antimicrobial resistance and multi-drug resistant pathogens has necessitated explorations for novel antibiotic agents as the discovery of conventional antibiotics is becoming economically less viable and technically more challenging for biopharma. Antimicrobial peptides (AMPs) have emerged as a promising alternative because of their particular mode of action, broad spectrum and difficulty that microbes have in becoming resistant to them. The AMPs bacitracin, gramicidin, polymyxins and daptomycin are currently used clinically. However, their susceptibility to proteolytic degradation, toxicity profile, and complexities in large-scale manufacture have hindered their development. To improve their proteolytic stability, methods such as integrating non-canonical amino acids (ncAAs) into their peptide sequence have been adopted, which also improves their potency and spectrum of action. The benefits of ncAA incorporation have been made possible by solid-phase peptide synthesis. However, this method is not always suitable for commercial production of AMPs because of poor yield, scale-up difficulties, and its non-'green' nature. Bioincorporation of ncAA as a method of integration is an emerging field geared towards tackling the challenges of solid-phase synthesis as a green, cheaper, and scalable alternative for commercialisation of AMPs. This review focusses on the bioincorporation of ncAAs; some challenges associated with the methods are outlined, and notes are given on how to overcome these challenges. The review focusses particularly on addressing two key challenges: AMP cytotoxicity towards microbial cell factories and the uptake of ncAAs that are unfavourable to them. Overcoming these challenges will draw us closer to a greater yield and an environmentally friendly and sustainable approach to make AMPs more druggable., (© 2024 The Authors. Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.)

Ribonucleic acids (RNAs) play a vital role in living organisms. Many of their cellular functions depend critically on chemical modification. Methods to modify RNA in a controlled manner-both in vitro and in vivo-are thus essential to evaluate and understand RNA biology at the molecular and mechanistic levels. The diversity of modifications, combined with the size and uniformity of RNA (made up of only 4 nucleotides) makes its site-specific modification a challenging task that needs to be addressed by complementary approaches. One such approach is solid-phase RNA synthesis. We discuss recent developments in this field, starting with new protection concepts in the ongoing effort to overcome current size limitations. We continue with selected modifications that have posed significant challenges for their incorporation into RNA. These include deazapurine bases required for atomic mutagenesis to elucidate mechanistic aspects of catalytic RNAs, and RNA containing xanthosine, N 4 -acetylcytidine, 5-hydroxymethylcytidine, 3-methylcytidine, 2'-OCF 3 , and 2'-N 3 ribose modifications. We also discuss the all-chemical synthesis of 5'-capped mRNAs and the enzymatic ligation of chemically synthesized oligoribonucleotides to obtain long RNA with multiple distinct modifications, such as those needed for single-molecule FRET studies. Finally, we highlight promising developments in RNA-catalyzed RNA modification using cofactors that transfer bioorthogonal functionalities., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Molecularly imprinted polymers (MIPs) are established artificial molecular recognition platforms with tailored selectivity towards a target molecule, whose synthesis and functionality are highly influenced by the nature of the solvent employed in their synthesis. Steps towards the "greenification" of molecular imprinting technology (MIT) has already been initiated by the elaboration of green MIT principles; developing MIPs in a solvent-free environment may not only offer an eco-friendly alternative, but could also significantly influence the affinity and expected selectivity of the resulting binding sites. In the current study the first solvent-free mechanochemical synthesis of MIPs via liquid-assisted grinding (LAG) is reported. The successful synthesis of the imprinted polymer was functionally demonstrated by measuring its template rebinding capacity and the selectivity of the molecular recognition process in comparison with the ones obtained by the conventional, non-covalent molecular imprinting process in liquid media. The results demonstrated similar binding capacities towards the template molecule and superior chemoselectivity compared to the solution-based MIP synthesis method. The adoption of green chemistry principles with all their inherent advantages in the synthesis of MIPs may not only be able to alleviate the potential environmental and health concerns associated with their analytical (e.g., selective adsorbents) and biomedical (e.g., drug carriers or reservoirs) applications, but might also offer a conceptual change in molecular imprinting technology.

The loading (i.e., substitution) of solid supports for oligonucleotide synthesis is an important parameter in large-scale manufacturing of oligonucleotides. Several key process parameters are dependent on the substitution of the solid support, including the number of phosphoramidite nucleoside equivalents used in the coupling step. For dimethoxytrityl (DMTr)-loaded solid supports, the substitution of the resin is determined by quantitatively cleaving the DMTr protecting group from the resin under acidic conditions and then analyzing the DMTr cation extinction by UV/vis spectroscopy. The spectrometric measurement can be performed at 409 nm or the global extinction maximum of 510 nm. The substitution is then calculated based on the Lambert-Beer law analogously to the substitution determination of Fmoc-substituted resins. Below, the determination of the molar extinction coefficient at 510 nm in a solution of 10% dichloroacetic acid in toluene and subsequent determination of the DMTr loading of DMTr-substituted resins is reported. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Determination of the molar extinction coefficient at 510 nm in DCA Deblock solution Basic Protocol 2: Substitution determination of DMTr-substituted resins by cleavage of the DMTr cation., (© 2024 Bachem AG. Current Protocols published by Wiley Periodicals LLC.)

This study presents an efficient method for on-resin dimer generation through self-condensation of 3,3-dimethoxypropionic acid-modified molecules, resulting in 2-pyridones. The approach demonstrated remarkable versatility by producing homodimers of peptides, peptoids, and non-peptidic ligands. Its ease of application, broad utility, and mild reaction conditions not only hold significance for peptide and peptoid research but also offer potential for the on-resin development of a wide range of bivalent ligands., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

While most FDA-approved peptide drugs are cyclic, robust cyclization chemistry of peptides and the deconvolution of the cyclic peptide sequences using tandem mass spectrometry render cyclic peptide drug discovery difficult. In this chapter, the protocol for the successful synthesis of tetrazine-linked cyclic peptide library in solid phase, which shows both robust cyclization and easy sequence deconvolution, is described. The protocol for the linearization and cleavage of cyclic peptides from the solid phase by simple UV light irradiation, followed by accurate sequencing using tandem mass spectrometry, is described. We describe the troubleshooting for this dithiol bis-arylation reaction and for the successful cleavage of the aryl cyclic peptide into linear form. This method for efficient solid-phase macrocyclization can be used for the rapid production of loop-based peptides and screening for inhibition of protein-protein interactions, by using the covalent inverse electron-demand Diels Alder reaction to supplement the non-covalent interaction between a protein and its peptide binder, isolating highly selective peptides in the process., (Copyright © 2024. Published by Elsevier Inc.)

Synthetic peptides are important as drugs and in research. Currently, the method of choice for producing these compounds is solid-phase peptide synthesis. Here, we describe the scope and limitations of Fmoc solid-phase peptide synthesis. Furthermore, we provide a detailed protocol for Fmoc peptide synthesis., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

N-alkylated glycine residues are the main constituent of peptoids and peptoid-peptide hybrids that are employed across the biomedical and materials sciences. While the impact of backbone N-alkylation on peptide conformation has been extensively studied, less is known about the effect of N-amination on the secondary structure propensity of glycine. Here, we describe a convenient protocol for the incorporation of N-aminoglycine into host peptides on solid support. Amide-to-hydrazide substitution also affords a nucleophilic handle for further derivatization of the backbone. To demonstrate the utility of late-stage hydrazide modification, we synthesized and evaluated the stability of polyproline II helix and β-hairpin model systems harboring N-aminoglycine derivatives. The described procedures provide facile entry into peptidomimetic libraries for conformational scanning., (Copyright © 2024. Published by Elsevier Inc.)

Conjugation to carrier proteins is necessary for peptides to be able to induce antibody formation when injected into animals together with a suitable adjuvant. This is usually performed by conjugation in solution followed by mixing with the adjuvant. Alternatively, the carrier may be adsorbed onto a solid support followed by activation and conjugation with the peptide by solid-phase chemistry. Different reagents can be used for conjugation through peptide functional groups (-SH, -NH2, -COOH), and various carrier proteins may be used depending on the peptides and the intended use of the antibodies. The solid phase may be an ion exchange matrix, from which the conjugate can subsequently be eluted and mixed with adjuvant. Alternatively, the adjuvant aluminum hydroxide may be used as the solid-phase matrix, whereupon the carrier is immobilized and conjugated with peptide. The resulting adjuvant-carrier-peptide complexes may then be used directly for immunization., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

This work catalogued oligonucleotide sequences and sequence compositions based on the overall yield of full-length product obtained by the phosphoramidite chemistry-based solid phase synthesis. In total, 76 sequences with different dinucleotide and trinucleotide repeats were synthesized, and the fully-deprotected products were analyzed by denaturing anion exchange HPLC. Overall, sequences containing more 2'-deoxyadenosine residues were obtained in relatively lower yields, likely due to the relative ease of 2'-deoxyadenosine to undergo depurination during the detritylation reaction. Furthermore, dinucleotide steps, such as d(CG)/d(GC) and d(AG)/d(GA), likely contribute the overall lower yields of full-length products as well.

Through systematic optimization of halopyridinium compounds, we established a peptide coupling protocol utilizing 4-iodine N -methylpyridinium ( 4IMP ) for solid-phase peptide synthesis (SPPS). The 4IMP coupling reagent is easily prepared, bench stable, and cost-effective. Employing 4IMP in the SPPS process has showcased remarkable chemoselectivity and efficiency, effectively eliminating racemization and epimerization. This achievement has been substantiated through the successful synthesis of a range of peptides via the direct utilization of commercially available amino acid substrates for SPPS.

Peptide stapling is a robust strategy for generating enzymatically stable, macrocyclic peptides. The incorporation of biologically relevant tags (such as cell-penetrating motifs or fluorescent dyes) into peptides, while preserving their binding interactions and enhancing their stability, is highly sought after. Despite the unique opportunities offered by tryptophan's indole scaffold for targeted functionalisation, its utilisation in peptide stapling has been limited as compared to other amino acids. Herein, we present an approach for peptide stapling using the tryptophan-mediated Petasis reaction. This method enables the synthesis of both stapled and labelled peptides and is applicable to both solution and solid-phase synthesis. Importantly, the use of the Petasis reaction in combination with tryptophan facilitates the formation of stapled peptides in a straightforward, multicomponent fashion, while circumventing the formation of undesired by-products. Furthermore, this approach allows for efficient and diverse late-stage peptide modifications, thereby enabling rapid production of numerous conjugates for biological and medicinal applications., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

A method for automated solid-phase synthesis of oligo(disulfide)s was developed. It is based on a synthetic cycle comprising removal of a protecting group from a resin-bound thiol followed by treatment with monomers containing a thiosulfonate as an activated precursor. For ease of purification and characterization, the disulfide oligomers were synthesized as extensions of oligonucleotides on an automated oligonucleotide synthesizer. Six different dithiol monomer building blocks were synthesized. Sequence-defined oligomers of up to seven disulfide units were synthesized and purified. The sequence of the oligomer was confirmed by tandem MS/MS analysis. One of the monomers contains a coumarin cargo that can be released by a thiol-mediated release mechanism. When the monomer was incorporated into an oligo(disulfide) and subjected to reducing conditions, the cargo was released under near-physiological conditions, which underlines the potential use of these molecules in drug delivery systems., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The development of peptide-based pharmaceutics is a hot topic in the pharmaceutical industry and in basic research. However, from the research and development perspective there is an unmet need for new, alternative, solid-phase peptide synthesizers that are highly efficient, automated, robust, able to synthetize peptides in parallel, inexpensive (to obtain and operate), have potential to be scaled up, and even comply with the principles of green chemistry. Moreover, a peptide synthesizer of this type could also fill the gap in university research, and therefore speed the advancement of peptide-based pharmaceutical options. This paper presents a Tecan add-on peptide synthesizer (TaPSy), which has operational flexibility (coupling time: 15-30 min), can handle all manual synthesis methods, and is economical (solvent use: 34.5 mL/cycle, while handling 0.49 mmol scale/reactor, even with ≤3 equivalents of activated amino acid derivatives). Moreover, it can carry out parallel synthesis of up to 12 different peptides (0.49 mmol scale in each). TaPSy uses no heating or high pressure, while it is still resistant to external influences (operating conditions: atmospheric pressure, room temperature 20-40 ˚C, including high [>70%] relative humidity). The system's solvent can also be switched from DMF to a green and biorenewable solvent, γ-valerolactone (GVL), without further adjustment. The designed TaPSy system can produce peptides with high purity (>70%), even with the green GVL solvent alternative. In this paper we demonstrate the optimization path of a newly developed peptide synthesizer in the context of coupling reagents, reaction time and reagent equivalents applying for a synthesis of a model peptide. We compare the results by analytical characteristics (purity of raw material, crude yield, yield) and calculated overall cost of the syntheses of one mg of crude peptide using a specified set of reaction conditions., Competing Interests: Declaration of Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Electrochemical transformations provide enticing opportunities for programmable, residue-specific peptide modifications. Herein, we harness the potential of amidic side-chains as underutilized handles for late-stage modification through the development of an electroauxiliary-assisted oxidation of glutamine residues within unprotected peptides. Glutamine building blocks bearing electroactive side-chain N,S-acetals are incorporated into peptides using standard Fmoc-SPPS. Anodic oxidation of the electroauxiliary in the presence of diverse alcohol nucleophiles enables the installation of high-value N,O-acetal functionalities. Proof-of-principle for an electrochemical peptide stapling protocol, as well as the functionalization of dynorphin B, an endogenous opioid peptide, demonstrates the applicability of the method to intricate peptide systems. Finally, the site-selective and tunable electrochemical modification of a peptide bearing two discretely oxidizable sites is achieved., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

As peptides gained interest as new drugs in the past years, their synthetic routes had been the subject of review and improvement. Fmoc/tBu-based solid-phase peptide synthesis (SPPS) is the most convenient technique, and the implementation in continuous flow has allowed for single-pass coupling and deprotection reactions. The focus of this research is to evaluate the relationship between undesired solvent-promoted reactions and final crude purity, by studying volume changes of a variable bed flow reactor through the synthesis. Based on the temperature, typical solvents for SPPS such as dimethylformamide (DMF) or N-methyl-2-pyrrolidone (NMP) can cause unwanted Fmoc removal during wash steps. It was found that for every millilitre of DMF used at 80°C, up to 1 μmol of Fmoc-protected peptide is deprotected, leading to additional impurities. This effect can, however, be minimised by adding additives such as HOBt, which reduces such unwanted deprotection to just 0.1 μmol/ml., (© 2022 European Peptide Society and John Wiley & Sons Ltd.)

A strategy for the synthesis of de novo discovered lactazole-like thiopeptides is reported. The approach revolves around a convergent and scalable preparation of the central triheterocyclic amino acid and its utilization in Fmoc solid-phase peptide synthesis for modular peptide chain assembly. A technique for preparing C-terminally functionalized thiopeptides for biological studies is also described. The syntheses of 11 TNIK-inhibitor thiopeptides and 6 of their derivatives in multimilligram quantities highlight the practical utility of the developed protocols.

The hybrid peptides consisting of α and β-amino acids show great promise as peptidomimetics that can be used as therapeutic agents. Therefore, the development of new unnatural amino acids and the methods of their incorporation into the peptide chain is an important task. Here, we described our investigation of the possibility of 5-amino-3-methyl-isoxazole-4-carboxylic acid (AMIA) application in the solid phase peptide synthesis. This new unnatural β-amino acid, presenting various biological activities, was successfully coupled to a resin-bound peptide using different reaction conditions, including classical and ultrasonic agitated solid-phase synthesis. All the synthesized compounds were characterized by tandem mass spectrometry. The obtained results present the possibility of the application of this β-amino acid in the synthesis of a new class of bioactive peptides.

Peptides of importance to both academia and industry are mostly synthesized in the solid-phase mode using a two-dimensional scheme. The so-called Fmoc/ t Bu strategy, where the groups are removed by piperidine and TFA, respectively, is currently the method of choice for peptide synthesis. However, as the molecular diversity of cyclic and branched peptides becomes a challenging interest, a high level of orthogonal dimensionality is required, such as through triorthogonal protection schemes. Here we present a fourth category of orthogonal protecting groups that are stable under cleavage conditions, including the TFA treatment that removes the t Bu-based groups. At the end of the synthetic process and upon some chemical manipulation, the groups in this fourth category were removed with TFA. This new concept of protecting groups could facilitate the synthesis and manipulation of difficult peptides.

Fmoc and Boc group are the two main groups used to protect the α-amino function in Solid-Phase Peptide Synthesis (SPPS). In this regard, the use of the Mmsb linker allows the combination of these two groups. Peptide-O-Mmsb-Resin is stable to the piperidine and trifluoroacetic acid (TFA) treatment used to remove Fmoc and Boc, respectively. The peptide is detached in a two-step protocol, namely reduction of the sulfoxide to the sulfide with Me 3 SiCl and Ph 3 P, and then treatment with TFA. The advantage of this strategy has been demonstrated by the following: preparation of peptide with no diketopiperazine formation in sequences prone to this side reaction; on-resin cyclization without the concourse of common organic reagents such as Pd(0) but of difficult use in a biological laboratory; and on-resin disulfide formation in a total side-chain unprotected peptide. The use of Mmsb linker together with Msib (4-(methylsulfinyl)benzyl) and Msbh (4,4'-bis(methylsulfinyl)benzhydryl) described in the accompanying manuscript add a fourth dimension to the SPPS protecting group scheme.

Thiol groups are suitable handles for site-selectively modifying, immobilizing or cyclizing individual peptides or entire peptide libraries. A limiting step in producing the thiol-functionalized peptides is the chromatographic purification, which is particularly laborious and costly if many peptides or even large libraries are to be produced. Herein, we present a strategy in which thiol-functionalized peptides are obtained in >90% purity and free of reducing agent, without a single chromatographic purification step. In brief, peptides are synthesized on a solid support linked via a disulfide bridge, the side-chain protecting groups are eliminated and washed away while the peptides remain on resin, and rather pure peptides are released from the solid support by reductive cleavage of the disulfide linker. Application of a volatile reducing agent, 1,4-butanedithiol (BDT), enabled removal of the agent by evaporation. We demonstrate that the approach is suited for the parallel synthesis of many peptides and that peptides containing a second thiol group can directly be cyclized by bis-electrophilic alkylating reagents for producing libraries of cyclic peptides.

Chemoselective construction of naphthoxazoles (NapOx) via a three-component annulation reaction enables proline selective labeling of peptides in solution or in solid-phase synthesis. The fluorogenic peptides possess low cytotoxicity, efficient cell membrane permeability and excellent bioimaging potential for biomedical applications.

There is considerable interest in the development of libraries of non-peptidic macrocycles as a source of ligands for difficult targets. We report here the solid-phase synthesis of a DNA-encoded library of several hundred thousand thioether-linked macrocycles. The library was designed to be highly diverse with respect to backbone scaffold diversity and to minimize the number of amide N-H bonds, which compromise cell permeability. The utility of the library as a source of protein ligands is demonstrated through the isolation of compounds that bind Streptavidin, a model target, with high affinity., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

As a commercially available and orthogonally protected amino acid building block, Fmoc-Dab(Mtt)-OH showed abnormally poor coupling efficiency during solid-phase peptide synthesis (SPPS). Herein, we reveal that Fmoc-Dab(Mtt)-OH undergoes rapid lactamization under a series of conditions with various coupling reagents. Although the complete incorporation of Fmoc-Dab(Mtt)-OH can be achieved using a multi-time and preincubation-free protocol with the coupling reagent DEPBT, alternative orthogonally protected building blocks are suggested to be used for avoiding such a costly and tedious procedure.

Formation of amide bonds is of immanent importance in organic and synthetic medicinal chemistry. Its presence in "traditional" small-molecule active pharmaceutical ingredients, in linear or cyclic oligo- and polypeptidic actives, including pseudopeptides, has led to the development of dedicated synthetic approaches for the formation of amide bonds starting from, if necessary, suitably protected amino acids. While the use of solid supported reagents is common in traditional peptide synthesis, similar approaches targeting amide bond formation in continuous-flow mode took off more significantly, after a first publication in 2006, only a couple of years ago. Most efforts rely upon the transition of traditional approaches in flow mode, or the combination of solid-phase peptide synthesis principles with flow chemistry, and advantages are mainly seen in improving space-time yields. This Review summarizes and compares the various approaches in terms of basic amide formation, peptide synthesis, and pseudopeptide generation, describing the technological approaches and the advantages that were generated by the specific flow approaches. A final discussion highlights potential future needs and perspectives in terms of greener and more sustainable syntheses., (© 2022 The Authors. ChemSusChem published by Wiley-VCH GmbH.)

Despite numerous reports on catalytic amide bond formation (ABF), these methods have thus far had a minimal impact on the universal fluorenylmethoxycarbonyl (Fmoc)/ t -Bu solid-phase peptide synthesis (SPPS) methodology. We now report a proof-of-principle Fmoc/ t -Bu SPPS in which both couplings and Fmoc deprotections were catalyzed by readily available reagents in an inexpensive green solvent. Couplings were carried out with >99% stereoselectivity, employing 1.1 equiv of Fmoc amino acids (AAs), using diisopropylcarbodiimide (DIC) as a coupling agent and 1-hydroxy-1,2,3-triazole-5-carboxylic acid ethyl ester (HOCt) (TON ∼ 30) as a catalyst, while Fmoc deprotections were carried out using 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU) (TON ∼ 7), facilitating synthesis of a model pentapeptide in 95% HPLC purity while also enabling minimization of solvent washing.

Macrocycles represent attractive candidates in organic synthesis and drug discovery. Since 2014, nineteen macrocyclic drugs, including three radiopharmaceuticals, have been approved by FDA for the treatment of bacterial and viral infections, cancer, obesity, immunosuppression, etc. As such, new synthetic methodologies and high throughput chemistry (e.g., microwave-assisted and/or solid-phase synthesis) to access various macrocycle entities have attracted great interest in this chemical space. This article serves as an update on our previous review related to macrocyclic drugs and new synthetic strategies toward macrocycles ( Molecules , 2013 , 18 , 6230). In this work, I first reviewed recent FDA-approved macrocyclic drugs since 2014, followed by new advances in macrocycle synthesis using high throughput chemistry, including microwave-assisted and/or solid-supported macrocyclization strategies. Examples and highlights of macrocyclization include macrolactonization and macrolactamization, transition-metal catalyzed olefin ring-closure metathesis, intramolecular C-C and C-heteroatom cross-coupling, copper- or ruthenium-catalyzed azide-alkyne cycloaddition, intramolecular S N Ar or S N 2 nucleophilic substitution, condensation reaction, and multi-component reaction-mediated macrocyclization, and covering the literature since 2010.

Unlike solid phase synthesis of peptides, synthesis of oligosaccharides by solid phase methods is lagging behind owing to inherent challenges faced while executing glycosidations. In this communication, silver-assisted gold-catalyzed glycosidations are found to be excellent for solid phase oligosaccharide synthesis. Glycosidations under catalytic conditions, one time coupling with four equivalents of donor, reactions in less than 30 min at 25 °C and on-resin deprotection of silyl and benzoate protecting groups are the salient features. Photocleaved glycans possess a protected amino functionality that can be utilized for bioconjugation. The versatility of this approach is established by synthesizing linear and branched pentaarabinofuranosides.

Chemical synthesis can provide hydrophobic proteins with natural or man-made modifications (e.g. S-palmitoylation, site-specific isotope labeling and mirror-image proteins) that are difficult to obtain through the recombinant expression technology. The difficulty of chemical synthesis of hydrophobic proteins stems from the hydrophobic nature. Removable backbone modificaiton (RBM) strategy has been developed for solubilizing the hydrophobic peptides/proteins. Here we take the chemical synthesis of a S-palmitoylated peptide as an example to describe the detailed procedure of RBM strategy. Three critical steps of this protocol are: (1) installation of Lys6-tagged RBM groups into the peptides by Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis, (2) chemical ligation of the peptides, and (3) removal of the RBM tags by TFA (trifluoroacetic acid) cocktails to give the target peptide., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

A novel synthetic approach to thioamide-substituted peptides is reported. It provides a practical tool for the chemical biology study of peptides and proteins by replacing a carbonyl oxygen atom of an amide bond by an sp 2 -hybridized sulfur atom to precisely introduce a thioamide bond Ψ[CS-NH] into a peptide backbone. The α-thioacyloxyenamide intermediates, originating from ynamide coupling reagent and proteinogenic amino monothioacids, are proved to be novel effective thioacylating reagents in both the solution and solid phase peptide syntheses. Herein, we describe the detailed synthesis protocol for site-specifically incorporating a thioamide bond at 19 of 20 proteinogenic amino acid residues (except for His) of a peptide backbone in a racemization/epimerization-free manner., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Peptides bearing C-terminal thioester and selenoester functionalities are essential precursors for the chemical synthesis of larger proteins using ligation chemistry, including native chemical ligation (NCL) and diselenide-selenoester ligation (DSL). The use of a side-chain anchoring thioesterification or selenoesterification approach offers a robust method to access peptide thioesters or peptide selenoesters in excellent yields and in high purity. Importantly, this methodology overcomes solubility issues and epimerization of the C-terminal amino acid residue that can occur using solution-phase approaches. Detailed methods for the solid-phase synthesis of peptide thioesters and selenoesters using a side-chain anchoring approach are outlined in this article., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Considering our interest in the use of peptides as potential target-specific drugs or as delivery vectors of metallodrugs for various biomedical applications, it is crucial to explore improved synthetic methodologies to accomplish the highest peptide crude purity in the shortest time possible. Therefore, we compared "classical" fluorenylmethoxycarbonyl (Fmoc)-solid phase peptide synthesis (SPPS) with ultrasound(US)-assisted SPPS based on the preparation of three peptides, namely the fibroblast growth factor receptor 3(FGFR3)-specific peptide Pep1 (VSPPLTLGQLLS-NH 2 ) and the novel peptides Pep2 (RQMATADEA-NH 2 ) and Pep3 (AAVALLPAVLLALLAPRQMATADEA-NH 2 ), which are being developed aimed at interfering with the intracellular protein-protein interaction(PPI) RANK-TRAF6. Our results demonstrated that US-assisted SPPS led to a 14-fold ( Pep1 ) and 4-fold time reduction ( Pep2 ) in peptide assembly compared to the "classical" method. Interestingly, US-assisted SPPS yielded Pep1 in higher purity (82%) than the "classical" SPPS (73%). The significant time reduction combined with high crude peptide purity attained prompted use to apply US-assisted SPPS to the large peptide Pep3 , which displays a high number of hydrophobic amino acids and homooligo-sequences. Remarkably, the synthesis of this 25-mer peptide was attained during a "working day" (347 min) in moderate purity (approx. 49%). In conclusion, we have reinforced the importance of using US-SPPS towards facilitating the production of peptides in shorter time with increased efficacy in moderate to high crude purity. This is of special importance for long peptides such as the case of Pep3 .

The stable and metastable phase diagrams between the sinister and the rectus ibuprofen enantiomers were established by means of thermal analysis and X-ray powder diffraction experiments as a function of temperature. The results obtained allow proving for the first time the existence, for the stable system, of a solid solution by mixing the racemic ibuprofen with one of its enantiomers for low concentration of the enantiomer. Since the rectus ibuprofen is a non-active pharmaceutical agent which can be partially bio-converted into the sinister enantiomer, the present work offers a new approach for scalemic mixtures preparation in order to improve the benefit/risk ratio related to ibuprofen solid dosage form administration., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Protease chemiluminescent probes exhibit extremely high detection sensitivity for monitoring activity of various proteolytic enzymes. However, their synthesis, performed in solution, involves multiple synthetic and purification steps, thereby generating a major limitation for rapid preparation of such probes with diverse substrate scope. To overcome this limitation, we developed a general solid-phase-synthetic approach to prepare chemiluminescent protease probes, by peptide elongation, performed on an immobilized chemiluminescent enol-ether precursor. The enol-ether precursor is immobilized on a 2-chlorotrityl-chloride resin through an acrylic acid substituent by an acid-labile ester linkage. Next, a stepwise elongation of the peptide is performed using standard Fmoc solid-phase peptide synthesis. After cleavage of the peptide-enol-ether precursor from the resin, by hexafluoro-iso-propanol, a simple oxidation of the enol-ether yields the final chemiluminescent dioxetane protease probe. To validate the applicability of the methodology, two chemiluminescent probes were efficiently prepared by solid-phase synthesis with dipeptidyl substrates designed for activation by aminopeptidase and cathepsin-B proteases. A more complex example was demonstrated by the synthesis of a chemiluminescent probe for detection of PSA, which includes a peptidyl substrate of six amino acids. We anticipate that the described methodology would be useful for rapid preparation of chemiluminescent protease probes with vast and diverse peptidyl substrates.

Microwave-assisted solid-phase synthesis method was simple, convenient, and fast, and herein adopted to produce nitrogen-doping carbon dots (N-CDs) in only 3 min. The N-CDs possessed high fluorescence quantum yield up to 15.9% with satisfactory stability to the environmental pH, ionic strength, and ultraviolet radiation. Particularly, the N-CDs had excellent dispersibility in both water and water-compatible organic solvents with similar fluorescence properties. Sunitinib, a small-molecule tyrosine inhibitor effective for some solid tumors, was found to quench the fluorescence of N-CDs in these media via the inner-filter effect. Hence, it was convenient to combine the proper sample pretreatment with the N-CD probe for sensing sunitinib avoiding the medium incompatibility problem. For rat plasma sample, salting-out liquid-liquid extraction was employed to minimize the sample matrix and concentrate the target sunitinib from aqueous to acetonitrile. The fluorescence detection of sunitinib was then achieved in acetonitrile by the addition of the proper amount of N-CDs. The method provided a good linearity of 0.1 μg/mL to 7 μg/mL with a limit of detection of 30 ng/mL, which met the requirement of the therapeutic drug monitoring of sunitinib. The developed method was potential for on-site detection of sunitinib., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

An atom-economic N-to-C-directed solid-phase peptide synthesis is reported that uses benzyl (Bn) or (benzhydryl-carbamoyl)-methyl (BcM) esters of amino acids as the building blocks, which facilitate efficient hydrazinolysis, convenient conversion to acyl azide, and robust amidation with the next amino acid ester. This method is free of coupling reagents and free of protection on the side-chain OH, CO 2 H, CONH2, etc., therefore exhibiting a significantly improved atom economy compared to those of BOC- or Fmoc-based C-to-N-directed approaches.

Solid-phase synthesis of cyclic, branched or side-chain-modified peptides typically involves introduction of a residue carrying a temporary side-chain protecting group that undergoes selective on-resin removal. In particular, N α -Fmoc-N ε -(4-methyltriphenylmethyl) (Mtt)-protected lysine and its shorter analogues are commercially available and extensively used in this context. Nevertheless, rapid reliable methods for on-resin removal of Mtt groups in the presence of tert-butyloxycarbonyl (Boc) groups are needed. Current commonly used conditions involve low concentrations (1-3%) of trifluoroacetic acid (TFA) in dichloromethane, albeit adjustment to each specific application is required to avoid premature removal of Boc groups or cleavage from the linker. Hence, a head-to-head comparison of several deprotection conditions was performed. The selected acids represent a wide range of acidity from TFA to trifluoroethanol. Also, on-resin removal of the N-(4-methoxytriphenylmethyl) (Mmt) and O-trityl groups (on serine) was investigated under similar conditions. The mildest conditions identified for Mtt deprotection involve successive treatments with 30% hexafluoroisopropanol (HFIP) or 30% perfluoro-tert-butanol [(CF 3 ) 3 COH] in dichloromethane (3 × 5 or 3 × 15 min, respectively), while 30% HFIP, 30% (CF 3 ) 3 COH, or 10% AcOH-20% trifluoroethanol (TFE) in CH 2 Cl 2 (3 × 5 min) as well as 5% trichloroacetic acid in CH 2 Cl 2 (3 × 2 min) enabled Mmt removal. Treatment with 1% TFA with/without 2% triisopropylsilane added (3 × 5 min), but also prolonged treatment with 30% (CF 3 ) 3 COH (5 × 15 min), led to selective deprotection of an O-Trt group on a serine residue. In all cases, the sequences also contained N-Boc or O-tBu protecting groups, which were not affected by 30% HFIP or 30% (CF 3 ) 3 COH even after a prolonged reaction time of 4 h. Finally, the optimized conditions involving HFIP or (CF 3 ) 3 COH proved applicable also for selective deprotection of a longer resin-bound peptide [i.e., Ac-Gly-Leu-Leu-Lys(Mtt)-Arg(Pbf)-Ile-Lys(Boc)-Ser(tBu)-Leu-Leu-RAM-PS] as well as allowed for an almost complete deprotection of a Dab(Mtt) residue., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)