Safety-Catch Linkers for Solid-Phase Peptide Synthesis.

Solid-phase peptide synthesis (SPPS) is the preferred strategy for synthesizing most peptides for research purposes and on a multi-kilogram scale. One key to the success of SPPS is the continual evolution and improvement of the original method proposed by Merrifield. Over the years, this approach has been enhanced with the introduction of new solid supports, protecting groups for amino acids, coupling reagents, and other tools. One of these improvements is the use of the so-called "safety-catch" linkers/resins. The linker is understood as the moiety that links the peptide to the solid support and protects the C-terminal carboxylic group. The "safety-catch" concept relies on linkers that are totally stable under the conditions needed for both α-amino and side-chain deprotection that, at the end of synthesis, can be made labile to one of those conditions by a simple chemical reaction (e.g., an alkylation). This unique characteristic enables the simultaneous use of two primary protecting strategies: tert-butoxycarbonyl (Boc) and fluorenylmethoxycarbonyl (Fmoc). Ultimately, at the end of synthesis, either acids (which are incompatible with Boc) or bases (which are incompatible with Fmoc) can be employed to cleave the peptide from the resin. This review focuses on the most significant "safety-catch" linkers.

Late-stage meta-C-H alkylation of pharmaceuticals to modulate biological properties and expedite molecular optimisation in a single step.

Catalysed C-H activation has emerged as a transformative platform for molecular synthesis and provides new opportunities in drug discovery by late-stage functionalisation (LSF) of complex molecules. Notably, small aliphatic motifs have gained significant interest in medicinal chemistry for their beneficial properties and applications as sp3-rich functional group bioisosteres. In this context, we disclose a versatile strategy with broad applicability for the ruthenium-catalysed late-stage meta-C(sp2)-H alkylation of pharmaceuticals. This general protocol leverages numerous directing groups inherently part of bioactive scaffolds to selectivity install a variety of medicinally relevant bifunctional alkyl units within drug compounds. Our strategy enables the direct modification of unprotected lead structures to quickly generate an array of pharmaceutically useful analogues without resorting to de novo syntheses. Moreover, productive late-stage modulation of key biological characteristics of drug candidates upon remote C-H alkylation proves viable, highlighting the major benefits of our approach to offer in drug development programmes.

Total Synthesis of Macrocyclolipopeptide Dysoxylactam A.

A highly stereoselective total synthesis of potent multidrug-resistant reverser dysoxylactum A has been accomplished in the longest linear sequences of 20 steps with an overall 10.2% yield. The key steps of this synthesis included Brown's crotylation, Evans alkylation, the Carreira protocol to generate the stereogenic center, and Yamaguchi macrolactonization.

Late-stage guanine C8-H alkylation of nucleosides, nucleotides, and oligonucleotides via photo-mediated Minisci reaction.

Chemically modified nucleosi(ti)des and functional oligonucleotides (ONs, including therapeutic oligonucleotides, aptamer, nuclease, etc.) have been identified playing an essential role in the areas of medicinal chemistry, chemical biology, biotechnology, and nanotechnology. Introduction of functional groups into the nucleobases of ONs mostly relies on the laborious de novo chemical synthesis. Due to the importance of nucleosides modification and aforementioned limitations of functionalizing ONs, herein, we describe a highly efficient site-selective alkylation at the C8-position of guanines in guanosine (together with its analogues), GMP, GDP, and GTP, as well as late-stage functionalization of dinucleotides and single-strand ONs (including ssDNA and RNA) through photo-mediated Minisci reaction. Addition of catechol to assist the formation of alkyl radicals via in situ generated boronic acid catechol ester derivatives (BACED) markedly enhances the yields especially for the reaction of less stable primary alkyl radicals, and is the key to success for the post-synthetic alkylation of ONs. This method features excellent chemoselectivity, no necessity for pre-protection, wide range of substrate scope, various free radical precursors, and little strand lesion. Downstream applications in disease treatment and diagnosis, or as biochemical probes to study biological processes after linking with suitable fluorescent compounds are expected.

Methionine Alkylation as an Approach to Quantify Methionine Oxidation Using Mass Spectrometry.

Post-translational oxidation of methionine residues can destabilize proteins or modify their functions. Although levels of methionine oxidation can provide important information regarding the structural integrity and regulation of proteins, their quantitation is often challenging as analytical procedures in and of themselves can artifactually oxidize methionines. Here, we develop a mass-spectrometry-based method called Methionine Oxidation by Blocking with Alkylation (MObBa) that quantifies methionine oxidation by selectively alkylating and blocking unoxidized methionines. Thus, alkylated methionines can be used as a stable proxy for unoxidized methionines. Using proof of concept experiments, we demonstrate that MObBa can be used to measure methionine oxidation levels within individual synthetic peptides and on proteome-wide scales. MObBa may provide a straightforward experimental strategy for mass spectrometric quantitation of methionine oxidation.

Total synthesis of (6R,12R)-6,12-dimethylpentadecan-2-one, the sex pheromone of Diabrotica balteata LeConte.

Diabrotica balteata LeConte is one of the most important polyphagous agricultural pests. The sex pheromone of this pest was synthesized using Evans asymmetric alkylation, ring-opening reaction of (R)-2-methyloxirane, SN 2 alkylation of secondary tosylate, and coupling of chiral tosylate with Grignard reagent as central strategies. The sex pheromone prepared herein would be useful to control D. balteata.

Synthesis of 1,3,5-Triazepines and Benzo[f][1,3,5]triazepines and Their Biological Activity: Recent Advances and New Approaches.

This review article discusses the recent progress in synthesizing seven-membered ring 1,3,5-triazepine and benzo[f][1,3,5]triazepine derivatives. These derivatives can be either unsaturated, saturated, fused, or separated. This review covers strategies and procedures developed over the past two decades, including cyclo-condensation, cyclization, methylation, chlorination, alkylation, addition, cross-coupling, ring expansions, and ring-closing metathesis. This review discusses the synthesis of 1,3,5-triazepine derivatives using nucleophilic or electrophilic substitution reactions with various reagents such as o-phenylenediamine, 2-aminobenzamide, isothiocyanates, pyrazoles, thiazoles, oxadiazoles, oxadiazepines, and hydrazonoyl chloride. This article systematically presents new approaches and techniques for preparing these compounds. It also highlights the biological importance of benzo[f][1,3,5]triazepine derivatives, which have been used as drugs for treating nervous system diseases. This review aims to provide researchers with the necessary information to create and develop new derivatives of these compounds as quickly as possible.

Copper-Catalyzed Asymmetric Remote C(sp3)-H Alkylation of N-Fluorocarboxamides with Glycine Derivatives and Peptides.

Saturated hydrocarbon bonds are ubiquitous in organic molecules; to date, the selective functionalization of C(sp3)-H bonds continues to pose a notorious difficulty, thereby garnering significant attention from the synthetic chemistry community. During the past several decades, a wide array of powerful new methodologies has been developed to enantioselectively modify C(sp3)-H bonds that is successfully applied in asymmetric formation of diverse bonds, including C-C, C-N, and C-O bonds; nevertheless, the asymmetric C(sp3)-H alkylation is elusive and, therefore, far less explored. In this work, we report a direct and robust strategy to construct highly valuable enantioenriched unnatural α-amino acid (α-AA) cognates and peptides by a copper-catalyzed enantioselective remote C(sp3)-H alkylation of N-fluorocarboxamides and readily accessible glycine esters under ambient conditions. The key to success lies in the optically active Cu catalyst generated through the coordination of glycine derivatives to enantiopure bisphosphine/Cu(I) species, which is beneficial to the single electronic reduction of N-fluorocarboxamides and the subsequent stereodetermining alkylation. More importantly, all types (primary, secondary, tertiary, and even α-oxy) of δ-C(sp3)-H bonds could be site- and stereospecifically activated by the kinetically favored 1,5-hydrogen atom transfer (1,5-HAT) step.

Development of Cyclic N,O-Aminal-Embedded Bis-tetrahydroisoquinoline Analogues as Potential DNA Alkylation Agents.

This work described a novel "functional hybrid" design for bis-tetrahydroisoquinoline (bis-THIQ) analogues as potential DNA alkylation agents by replacing the labile C21-carbinolamine on the bis-THIQ skeleton of ET-743 with a chemically stable cyclic N,O-aminal functionality. In vitro anti-proliferation evaluation has proven that it is a successful approach to deliver new bis-THIQ analogues with common cytotoxicities, among which several exhibited sub-micromolar-range IC50 against the proliferation of human cancer cell lines A549, HepG2, and MDA-MB-231, respectively.

Identification of new multi-substituted 1H-pyrazolo[3,4-c]pyridin-7(6H)-ones as soluble guanylyl cyclase (sGC) stimulators with vasoprotective and anti-inflammatory activities.

Herein, we describe the rational design, synthesis and in vitro functional characterization of new heme-dependent, direct soluble guanylyl cyclase (sGC) agonists. These new compounds bear a 1H-pyrazolo[3,4-c]pyridin-7(6H)-one skeleton, modified to enable efficient sGC binding and stimulation. To gain insights into structure-activity relationships, the N6-alkylation of the skeleton was explored, while a pyrimidine ring, substituted with various C5'-polar groups, was installed at position C3. Among the newly synthesized 1H-pyrazolo[3,4-c]pyridin-7(6H)-ones, derivatives 14b, 15b and 16a display characteristic features of sGC "stimulators" in A7r5 vascular smooth muscle cells in vitro. They strongly synergize with the NO donor, sodium nitroprusside (SNP) in inducing cGMP generation in a manner that requires the presence of a reduced heme moiety associated with sGC, and elevate the cGMP-responsive phosphorylation of the protein VASP at Ser239. In line with their sGC stimulating capacity, docking calculations of derivatives 16a, 15(a-c) on a cryo-EM structure of human sGC (hsGC) in an ΝΟ-activated state indicated the implication of 1H-pyrazolo[3,4-c]pyridin-7(6H)-one skeleton in efficient bonding interactions with the recently identified region that binds known sGC stimulators, while the presence of either a N6-H or N6-methyl group pointed to enhanced binding affinity. Moreover, the in vitro functional effects of our newly identified sGC stimulators were compatible with a beneficial role in vascular homeostasis. Specifically, derivative 14b reduced A7r5 cell proliferation, while 16a dampened the expression of adhesion molecules ICAM-1 and P/E-Selectin in Human Umbilical Vein Endothelial Cells (HUVECs), as well as the subsequent adhesion of U937 leukocytes to the HUVECs, triggered by tumor necrosis factor alpha (TNF-α) or interleukin-1 beta (IL-1ß). The fact that these compounds elevate cGMP only in the presence of NO may indicate a novel way of interaction with the enzyme and may make them less prone than other direct sGC agonists to induce characteristic hypotension in vivo.

Synthesis of Boron-Containing Nucleoside Analogs.

Over the last century, nucleoside-based therapeutics have demonstrated remarkable effectiveness in the treatment of a wide variety of diseases from cancer to HIV. In addition, boron-containing drugs have recently emerged as an exciting and fruitful avenue for medicinal therapies. However, borononucleosides have largely been unexplored in the context of medicinal applications. Herein, we report the synthesis, isolation, and characterization of two novel boron-containing nucleoside compound libraries which may find utility as therapeutic agents. Our synthetic strategy employs efficient one-step substitution reactions between a diverse variety of nucleoside scaffolds and an assortment of n-alkyl potassium trifluoroborate-containing electrophiles. We demonstrated that these alkylation reactions are compatible with cyclic and acyclic nucleoside substrates, as well as increasing alkyl chain lengths. Furthermore, regioselective control of product formation can be readily achieved through manipulation of base identity and reaction temperature conditions.

Extending the Coverage of Lys-C/Trypsin-Based Bottom-up Proteomics by Cysteine S-Aminoethylation.

To improve the coverage in bottom-up proteomics, S-aminoethylation of cysteine residues (AE-Cys) was carried out with 2-bromoethylamine, followed by cleavage with lysyl endopeptidase (Lys-C) or Lys-C/trypsin. A model study with bovine serum albumin showed that the C-terminal side of AE-Cys was successfully cleaved by Lys-C. The frequency of side reactions at amino acids other than Cys was less than that in the case of carbamidomethylation of Cys with iodoacetamide. Proteomic analysis of A549 cell extracts in the data-dependent acquisition mode after AE-Cys modification afforded a greater number of identified protein groups, especially membrane proteins. In addition, label-free quantification of proteins in mouse nonsmall cell lung cancer (NSCLC) tissue in the data-independent acquisition mode after AE-Cys modification showed improved NSCLC pathway coverage and greater reproducibility. Furthermore, the AE-Cys method could identify an epidermal growth factor receptor peptide containing the T790 M mutation site, a well-established lung-cancer-related mutation site that has evaded conventional bottom-up methods. Finally, AE-Cys was found to fully mimic Lys in terms of collision-induced dissociation fragmentation, ion mobility separation, and cleavage by Lys-C/trypsin, except for sulfoxide formation during sample preparation.

H2 O ⋠B(C6 F5 )3 -Catalyzed para-Alkylation of Anilines with Alkenes Applied to Late-Stage Functionalization of Non-Steroidal Anti-Inflammatory Drugs.

Anilines are core motifs in a variety of important molecules including medicines, materials and agrochemicals. We report a straightforward procedure that allows access to new chemical space of anilines via their para-C-H alkylation. The method utilizes commercially available catalytic H2 O ⋅ B(C6 F5 )3 and is highly selective for para-C-alkylation (over N-alkylation and ortho-C-alkylation) of anilines, with a wide scope in both the aniline substrates and alkene coupling partners. Readily available alkenes are used, and include new classes of alkene for the first time. The mild reaction conditions have allowed the procedure to be applied to the late-stage-functionalization of non-steroidal anti-inflammatory drugs (NSAIDs), including fenamic acids and diclofenac. The formed novel NSAID derivatives display improved anti-inflammatory properties over the parent NSAID structure.

High-Density Immobilization of TCEP on Silica Beads for Efficient Disulfide Reduction and Thiol Alkylation in Peptides.

Tris-(2-carboxyethyl)phosphine (TCEP) linked to agarose beads is widely used for reducing disulfide bridges in proteins and peptides. The immobilization of TCEP on beads allows efficient removal after reduction to prevent its reaction with alkylating reagents and thus interference with conjugation reactions. However, a limitation of agarose TCEP is its relatively low reduction capacity per milliliter of wet beads (about 15 µmol/ml), making it unsuitable for the reduction of disulfides from molecules at millimolar concentrations. In this work, we tested the immobilization of TCEP to a range of different solid supports and found that conjugation to silica gel offers TCEP beads with about 8-fold higher reduction capacity (129±16 µmol/ml wet beads). We show that it allows reducing disulfide-cyclized peptides at millimolar concentrations for subsequent cyclization by bis-electrophile linker reagents. Given the substantially higher reduction capacity, the robust performance in different solvents, the low cost of the silica gel, and the ease of functionalization with TCEP, the silica gel-TCEP is suited for reducing disulfide bridges in essentially any peptide and is particularly useful for reducing peptides at higher concentrations.

Oxidation/Alkylation of Amino Acids with α-Bromo Carbonyls Catalyzed by Copper and Quick Access to HDAC Inhibitor.

A facile and efficient method was reported for Cu-catalyzed selective α-alkylation processes of amino acids/peptides and α-bromo esters/ketones through a radical-radical coupling pathway. The reaction displays an excellent functional group tolerance and broad substrate scope, allowing access to desired products in moderate to excellent yields. Notably, this method is distinguished by site-specificity and exhibits total selectivity for aryl glycine motifs over other amino acid units. Furthermore, the practicality of this strategy is certified by the efficient synthesis of the novel SAHA phenylalanine-containing analogue (SPACA).

Itaconate inhibits SYK through alkylation and suppresses inflammation against hvKP induced intestinal dysbiosis.

Hypervirulent Klebsiella pneumoniae (hvKP) is a highly lethal opportunistic pathogen that elicits more severe inflammatory responses compared to classical Klebsiella pneumoniae (cKP). In this study, we investigated the interaction between hvKP infection and the anti-inflammatory immune response gene 1 (IRG1)-itaconate axis. Firstly, we demonstrated the activation of the IRG1-itaconate axis induced by hvKP, with a dependency on SYK signaling rather than STING. Importantly, we discovered that exogenous supplementation of itaconate effectively inhibited excessive inflammation by directly inhibiting SYK kinase at the 593 site through alkylation. Furthermore, our study revealed that itaconate effectively suppressed the classical activation phenotype (M1 phenotype) and macrophage cell death induced by hvKP. In vivo experiments demonstrated that itaconate administration mitigated hvKP-induced disturbances in intestinal immunopathology and homeostasis, including the restoration of intestinal barrier integrity and alleviation of dysbiosis in the gut microbiota, ultimately preventing fatal injury. Overall, our study expands the current understanding of the IRG1-itaconate axis in hvKP infection, providing a promising foundation for the development of innovative therapeutic strategies utilizing itaconate for the treatment of hvKP infections.

One-step site-specific S-alkylation of full-length caveolin-1: Lipidation modulates the topology of its C-terminal domain.

Caveolin-1 is an integral membrane protein that is known to acquire a number of posttranslational modifications upon trafficking to the plasma membrane. In particular, caveolin-1 is palmitoylated at three cysteine residues (C133, C143, and C156) located within the C-terminal domain of the protein which could have structural and topological implications. Herein, a reliable preparation of full-length S-alkylated caveolin-1, which closely mimics the palmitoylation observed in vivo, is described. HPLC and ESI-LC-MS analyses verified the addition of the C16 alkyl groups to caveolin-1 constructs containing one (C133), two (C133 and C143), and three (C133, C143, and C156) cysteine residues. Circular dichroism spectroscopy analysis of the constructs revealed that S-alkylation does not significantly affect the global helicity of the protein; however, molecular dynamics simulations revealed that there were local regions where the helicity was altered positively or negatively by S-alkylation. In addition, the simulations showed that lipidation tames the topological promiscuity of the C-terminal domain, resulting in a disposition within the bilayer characterized by increased depth.

Total Synthesis of the 2,5-Disubstituted γ-Pyrone E1 UAE Inhibitor Himeic Acid A.

The first total synthesis of the E1 ubiquitin-activating enzyme inhibitor, himeic acid A, is reported. A McCombie reaction was used to form the core γ-pyrone via a 6π-electrocyclization. A dioxenone ring-opening/acyl ketene trapping reaction with a primary amide provided the unusual unsymmetrical imide functionality. Other key steps include the use of an Evans auxiliary alkylation (d.r. ≥ 95:5) to install the (S)-2-methyl succinic acid fragment and a cross-metathesis to install the unsaturated side-chain.

Using the Colloidal Method to Prepare Au Catalysts for the Alkylation of Aniline by Benzyl Alcohol.

Using the colloidal method, attempts were made to deposit Au NPs on seven different material supports (TiO2, α and γ-Al2O3, HFeO2, CeO2, C, and SiO2). The deposition between 0.8 and 1 wt% of Au NPs can be generally achieved, apart for SiO2 (no deposition) and α-alumina (0.3 wt%). The resultant sizes of the Au NPs were dependent on the nature as well as the surface area of the support. The catalytic activity and selectivity of the supported Au catalysts were then compared in the alkylation of aniline by benzyl alcohol. Correlations were made between the nature of the support, the size of the Au NP, and the H-binding energy. A minimum H-binding energy of 1100 µV K-1 was found to be necessary for high selectivity for the secondary amine. Comparisons of the TEM images of the pre- and post-reaction catalysts also revealed the extent of Au NP agglomeration under the reaction conditions.

The N-Alkylation of Agelastatin A Modulates Its Chemical Reactivity.

Agelastatin A is a marine alkaloid with potent biological activity. To date, at least 17 different strategies have achieved its total synthesis, along with many analogues. The present study focuses on the acidity stability of some N-methyl derivatives of agelastatin A. The study made use of chemical reactions and spectroscopic acquisitions. The chemical structure of some derivatives can undergo a profound rearrangement. The results could shed light on the mechanism of action of agelastatin A and suggest the preparation of analogues with improved pharmacological efficacy.