Modification Of Peptides Research Articles

N-tert-Butoxycarbonyl-N-(2-(tritylthio)ethoxy)glycine as a Building Block for Peptide Ubiquitination.

N-Boc-N-(2-(tritylthio)ethoxy)glycine has been developed as a building block for peptide ubiquitination, which is fully compatible with solid-phase Fmoc chemistry and common peptide modifications including phosphorylation, methylation, acetylation, biotinylation, and fluorescence labeling. The optimal conditions for peptide cleavage and auxiliary removal were obtained. The utility of this building block in peptide ubiquitination was demonstrated by the synthesis of seven ubiquitinated histone and Tau peptides bearing various modifications. Cys residues were well tolerated and did not require orthogonal protection. The structural integrity and folding of the synthesized ubiquitinated peptides were confirmed by enzymatic deubiquitination of a fluorescently labeled ubiquitin conjugate. The synthetic strategy using this building block provides a practical approach for the preparation of ubiquitinated peptides with diverse modifications.

Design, synthesis, and analysis of macrobicyclic peptides for targeting the Gαi protein.

Bicyclic peptides are important chemical tools that can function, for example, as bioactive ligands switching on/off signaling pathways mediated by guanine nucleotide-binding proteins as bicycles are more broadly applicable. Despite their relevance in medicinal chemistry, the synthesis of such peptides is challenging, and the final yield is highly dependent on the chemical composition and physicochemical properties of the scaffold. We recently discovered novel, state-specific peptide modulators targeting the Gαi protein, namely, GPM-2/GPM-3, by screening a one-bead-two-compound combinatorial library. A more detailed analysis, including sequence alignments and computer-assisted conformational studies based on the hit compounds, revealed the new peptide 10 as a potential macrobicyclic Gαi ligand sharing high sequence similarity to the known Gαi modulators. The Gαs protein was included in this study for comparison and to unravel the criteria for the specificity of modulator binding to Gαi versus Gαs. This work provides in-depth computer-assisted experimental studies for the analysis of novel macrobicyclic, library-derived Gαi protein ligands. The sequence and structural comparison of 10 with the lead compounds GPM-2 and GPM-3 reveals the importance of the size and amino acid composition of one ring of the bicyclic system and suggests features enhancing the binding affinity of the peptides to the Gαi protein.

Photo-induced universal modification of small-diameter decellularized blood vessels with a hemocompatible peptide improves in vivo patency

Decellularized vessels (DVs) have the potential to serve as available grafts for small-diameter vascular (<6 mm) reconstruction. However, the absence of functional endothelia makes them likely to trigger platelet aggregation and thrombosis. Luminal surface modification is an efficient approach to prevent thrombosis and promote endothelialization. Previously, we identified a hemocompatible peptide, HGGVRLY, that showed endothelial affinity and antiplatelet ability. By conjugating HGGVRLY with a phenylazide group, we generated a photoreactive peptide that can be modified onto multiple materials, including non-denatured extracellular matrices. To preserve the natural collagen of DVs as much as possible, we used a lower ultrahydrostatic pressure than that previously reported to prepare decellularized grafts. The photoreactive HGGVRLY peptide could be modified onto DV grafts via UV exposure for only 2 min. Modified DVs showed improved endothelial affinity and antiplatelet ability in vitro. When rat abdominal aortas were replaced with DVs, modified DVs with more natural collagen demonstrated the highest patent rate after 10 weeks. Moreover, the photoreactive peptide remained on the lumen surface of DVs over two months after implantation. Therefore, the photoreactive peptide could be efficiently and sustainably modified onto DVs with more natural collagens, resulting in improved hemocompatibility. Statement of SignificanceWe employed a relatively lower ultrahydrostatic pressure to prepare decellularized vessels (DVs) with less denatured collagens to provide a more favorable environment for cell migration and proliferation. The hemocompatibility of DV luminal surface can be enhanced by peptide modification, but undenatured collagens are difficult to modify. We innovatively introduce a phenylazide group into the hemocompatible peptide HGGVRLY, which we previously identified to possess endothelial affinity and antiplatelet ability, to generate a photoreactive peptide. The photoreactive peptide can be efficiently and stably modified onto DVs with more natural collagens. DV grafts modified with photoreactive peptide exhibit enhanced in vivo patency. Furthermore, the sustainability of photoreactive peptide modification on DV grafts within bloodstream is evident after two months of transplantation.

Combination of cellulose nanofiber and artificial fusion protein for biocementation

Biomineralization occurring in living organisms is mostly controlled by organic macromolecules such as polysaccharides and proteins. Recently, biomineralization has been attracting much attention as a green and sustainable cementation technique including enzyme-induced carbonate precipitation (EICP), where CaCO3 is formed by hydrolysis of urea by urease in the presence of calcium ions. In this study, we have developed a novel hybrid biocementation method combining CaCO3 and cellulose nanofiber (CNF). In nature, matrix proteins work as a binder at the interface of organic and inorganic materials to form hybrid biomaterials. By mimicking the natural system, we designed an artificial fusion protein to facilitate the deposition of CaCO3 on CNF. Calcite-binding peptide (CaBP) and carbohydrate-binding module (CBM) were introduced in the artificial fusion protein CaBP-CBM to connect CaCO3 and cellulose. The addition of CNF in the EICP system resulted in the formation of a number of small particles of CaCO3 compared to a non-additive system. The addition of the fusion protein CaBP-CBM to CNF led to an increase in the size of CaCO3 particles. Furthermore, the combination of CaBP-CBM and CNF provides higher strength of samples in sand solidification. Therefore, introduction of CNF and the fusion protein would be promising for novel biocementation techniques.

Backbone-enabled modification of peptides with benzoquinone via palladium-catalyzed δ-C(sp2)-H functionalization.

Backbone-enabled site-selective modification of peptides with benzoquinone via Pd-catalyzed δ-C(sp2)-H functionalization has been achieved. The amide groups of peptides serve as internal directional groups, facilitating C-H functionalization through a kinetically less favored six-membered palladacycle. This methodology presents novel opportunities for the late-stage site-selective diversification of peptides.

Harnessing a bis-electrophilic boronic acid lynchpin for azaborolo thiazolidine (ABT) grafting in cyclic peptides.

Chemical modifications of native peptides have significantly advanced modern drug discovery in recent decades. On this front, the installation of multitasking molecular grafts onto macrocyclic peptides offers numerous opportunities in biomedical applications. Here, we showcase a new class of borono-cyclic peptides featuring an azaborolo thiazolidine (ABT) graft, which can be readily assembled utilizing a bis-electrophilic boronic acid lynchpin while harnessing the inherent reactivity difference (>103 M-1 s-1) between the N-terminal cysteine and backbone cysteine for rapid and highly regioselective macrocyclization (∼1 h) under physiological conditions. The ABT-crosslinked peptides are fairly stable in endogenous environments, but can provide the linear diazaborine peptides via treatment with α-nucleophiles. This efficient peptide crosslinking protocol was further extended for regioselective bicyclizations and engineering of α-helical structures. Finally, ABT-grafted peptides were exploited in biorthogonal conjugation, leading to highly effective intracellular delivery of an apoptotic peptide (KLA) in cancer cells. The mechanism of action by which ABT-grafted KLA peptide induces apoptosis was also explored.

2-cyanopyridine derivatives enable N-terminal cysteine bioconjugation and peptide bond cleavage of glutathione under aqueous and mild conditions.

Inspired by the chemical reactivity of apalutamide, we have developed an efficient method for N-terminal cysteine bioconjugation with 2-cyanopyridine derivatives. Systematic investigations of various 2-cyanopyridines revealed that 2-cyanopyridines with electron-withdrawing groups react efficiently with cysteine under aqueous and mild conditions. Moreover, the highly reactive 2-cyanopyridines enable the peptide bond cleavage of glutathione. The utility of our method is demonstrated by its application to the cysteine-selective chemical modification of bioactive peptides.

Visible-light mediated selective phosphonylation modification of tryptophan residues in oligopeptides

A direct C2-H phosphonylation strategy driven by visible light for specific modification of tryptophan-containing peptides has been reported, providing a plethora of phosphorylated tryptophan-containing peptides

Intermolecular sulfur atom transfer cascade enabled late-stage introduction of sulfilimines into peptides.

Sulfilimines, a privileged class of -S(iv)[double bond, length as m-dash]N- functional groups found in nature, have been exploited as valuable building blocks in organic synthesis and as pharmacophores in drug discovery, and have aroused significant interest in the chemical community. Nevertheless, strategies for late-stage introduction of sulfilimines into peptides and proteins have still met with limited success. Herein, we have developed a method of introducing biological sulfilimine fragments into peptides by an intermolecular sulfur atom transfer cascade reaction, utilizing hydroxylamine condensed with the acid moieties of peptides and varied diaryl disulfides. It provides a convenient, efficient, metal-free and widely applicable method for late-stage modification and functionalization of peptides at their acid sites both in the homogeneous phase and on-resins in SPPS. Moreover, the modified peptides with sulfilimines have been demonstrated as cleavable linkers for peptide conjugates under reducible conditions, providing unique opportunities in peptide therapeutics development and drug discovery.

Recent Progress in Electrochemical Modification of Amino Acids and Peptides

Recent Progress in Electrochemical Modification of Amino Acids and Peptides

Nontargeted Identification of D-Amino Acid-Containing Peptides Through Enzymatic Screening, Chiral Amino Acid Analysis, and LC-MS.

D-amino acid-containing peptides (DAACPs) in animals are a class of bioactive molecules formed via the posttranslational modification of peptides consisting of all-L-amino acid residues. Amino acid residue isomerization greatly impacts the function of the resulting DAACP. However, because isomerization does not change the peptide's mass, this modification is difficult to detect by most mass spectrometry-based peptidomic approaches. Here we describe a method for the identification of DAACPs that can be used to systematically survey peptides extracted from a tissue sample in a nontargeted manner.

A multicomponent reaction for modular assembly of indole-fused heterocycles.

Indoles are privileged chemical entities in natural products and drug discovery. Indole-fused heterocycles, particularly seven-membered ones, have received increasing attention due to their distinctive chemical characteristics and wide spectrum of bioactivities. However, the synthetic access to these compounds is highly limited. Herein, we report a unique multicomponent reaction (MCR) for modular assembly of indole-fused seven-membered heterocycles. In this process, indole, formaldehyde and amino hydrochloride could assemble rapidly to yield indole-fused oxadiazepines, and another addition of sodium thiosulphate would furnish indole-fused thiadiazepines. The biological evaluation disclosed the promising anticancer activity of these compounds. Furthermore, this MCR could be applicable in the late-stage and selective modifications of peptides. Therefore, this work provides a powerful strategy for indole functionalization and valuable tool for construction of seven-membered heterocycles.

Late-stage peptide modification and macrocyclization enabled by tertiary amine catalyzed tryptophan allylation.

Late-stage modification of peptides could potentially endow peptides with significant bioactivity and physicochemical properties, and thereby provide novel opportunities for peptide pharmaceutical studies. Since tryptophan (Trp) bears a unique indole ring residue and plays various critical functional roles in peptides, the modification methods for tryptophan were preliminarily developed with considerable progress via transition-metal mediated C-H activation. Herein, we report an unprecedented tertiary amine catalyzed peptide allylation via the SN2'-SN2' pathway between the N1 position of the indole ring of Trp and Morita-Baylis-Hillman (MBH) carbonates. Using this method that proceeds under mild conditions, we demonstrated an extremely broad scope of Trp-containing peptides and MBH carbonates to prepare a series of peptide conjugates and cyclic peptides. The reaction is amenable to either solid-phase (on resin) or solution-phase conditions. In addition, the modified peptides can be further conjugated with other biomolecules at Trp, providing a new handle for bioconjugation.

Electrochemistry-enabled residue-specific modification of peptides and proteins.

Selective chemical reactions at precise amino acid residues of peptides and proteins have become an exploding field of research in the last few decades. With the emerging utility of bioconjugated peptides and proteins as drug leads and therapeutic agents, the design of smart protocols to modulate and conjugate biomolecules has become necessary. During this modification, the most important concern of biochemists is to keep intact the structural integrity of the biomolecules. Hence, a soft and selective biocompatible reaction environment is necessary. Electrochemistry, a mild and elegant tunable reaction platform to synthesize complex molecules while avoiding harsh and toxic chemicals, can provide such a reaction condition. However, this strategy is yet to be fully exploited in the field of selective modification of polypeptides. With this possibility, the use of electrochemistry as a reaction toolbox in peptide and protein chemistry is flourishing day by day. Unfortunately, there is no suitable review article summarizing the residue-specific modification of biomolecules. The present review provides a comprehensive summary of the latest manifested electrochemical approaches for the modulation of five redox-active amino acid residues, namely cysteine, tyrosine, tryptophan, histidine and methionine, found in peptides and proteins. The article also highlights the incredible potential of electrochemistry for the regio- as well as chemoselective bioconjugation strategy of biomolecules.

Site-selective S-gem-difluoroallylation of unprotected peptides with 3,3-difluoroallyl sulfonium salts.

Bench-stable 3,3-difluoroallyl sulfonium salts (DFASs), featuring tunable activity and their editable C-β and gem-difluoroallyl group, proved to be versatile fluoroalkylating reagents for site-selective S-gem-difluoroallylation of cysteine residues in unprotected peptides. The reaction proceeds with high efficiency under mild conditions (ambient temperature and aqueous and weak basic conditions). Various protected/unprotected peptides, especially bioactive peptides, are site-selectively S-gem-difluoroallylated. The newly added gem-difluoroallyl group and other functional groups derived from C-β of DFASs are poised for ligation with bio-functional groups through click and radical chemistry. This stepwise "doubly orthogonal" modification of peptides enables the construction of bioconjugates with enhanced complexity and functionality. This proof of principle is successfully applied to construct a peptide-saccharide-biotin chimeric bioconjugate, indicating its great potential application in medicinal chemistry and chemical biology.

Seroprevalence of binding and neutralizing antibodies against 18 adeno-associated virus types in patients with neuromuscular disorders.

High levels of pre-existing antibodies are a major challenge for the application of viral vectors since they can severely limit their efficacy. To identify promising candidates among adeno-associated virus (AAV) based vectors for future gene therapies for the treatment of hereditary neuromuscular disorders (NMDs), we investigated the antibody levels in sera from patients with NMDs against 18 AAV types, including 11 AAVs with wild-type capsids, 5 AAVs with peptide-modified capsids and 2 AAVs with shuffled capsids. With regard to the wild-type capsid AAVs, the lowest binding antibody levels were detected against AAV6, AAV5, AAV12 and AAV9, whereas the highest binding antibody levels were detected against AAV10, AAV8, AAV1, and AAV2. The lowest neutralizing antibody levels against wild-type AAVs were detected against AAV12, AAV5, AAV9, AAV7, AAV8 and AAV10, and the highest neutralizing antibody levels were detected against AAV13, AAV2 and AAV3. Interestingly, the influence of peptide modifications or shuffling of AAV capsids on antibody binding and AAV neutralization seemed to depend on the parental AAV. While the sex of the serum donors had no significant impact on binding or neutralizing antibody levels, we observed a trend to higher binding antibodies in older serum donors against some AAV types and a clear positive correlation of neutralizing antibody titers with the age of the serum donors. The disease status on the other hand did not have a meaningful impact on antibody levels, with no changes in AAV neutralization. Our data indicate that several wild-type or peptide-modified AAV may be good candidates for therapeutic application due to low pre-existing antibody levels, and that the age of potential recipients rather than their health status with regard to NMDs has the biggest impact on vector applicability.

Methionine-enabled peptide modification through late-stage Pd-catalyzed β-C(sp3)-H olefination/cyclization.

We present a method for site-selective diversification of peptides via Pd-catalyzed β-C(sp3)-H olefination/cyclization. In this protocol, the native methionine residue acts as a directing group, enabling site-specific olefination/cyclization of peptides. This chemistry demonstrates broad substrate scope, offering a versatile tool for peptide ligation.

Biomimetic nanoparticles with enhanced rapamycin delivery for autism spectrum disorder treatment via autophagy activation and oxidative stress modulation.

Rationale: Autism spectrum disorder (ASD) represents a complex neurodevelopmental condition lacking specific pharmacological interventions. Given the multifaced etiology of ASD, there exist no effective treatment for ASD. Rapamycin (RAPA) can activate autophagy by inhibiting the mTOR pathway and has exhibited promising effects in treating central nervous system disorders; however, its limited ability to cross the blood-brain barrier (BBB) has hindered its clinical efficacy, leading to substantial side effects. Methods: To address this challenge, we designed a drug delivery system utilizing red blood cell membrane (CM) vesicles modified with SS31 peptides to enhance the brain penetration of RAPA for the treatment of autism. Results: The fabricated SCM@RAPA nanoparticles, with an average diameter of 110 nm, exhibit rapid release of RAPA in a pathological environment characterized by oxidative stress. In vitro results demonstrate that SCM@RAPA effectively activate cellular autophagy, reduce intracellular ROS levels, improve mitochondrial function, thereby ameliorating neuronal damage. SS31 peptide modification significantly enhances the BBB penetration and rapid brain accumulation of SCM@RAPA. Notably, SCM@RAPA nanoparticles demonstrate the potential to ameliorate social deficits, improve cognitive function, and reverse neuronal impairments in valproic acid (VPA)-induced ASD models. Conclusions: The therapeutic potential of SCM@RAPA in managing ASD signifies a paradigm shift in autism drug treatment, holding promise for clinical interventions in diverse neurological conditions.

Mechanistic Insights into Peptide Binding and Deactivation of an Adhesion G Protein-Coupled Receptor.

Adhesion G protein-coupled receptors (ADGRGs) play critical roles in the reproductive, neurological, cardiovascular, and endocrine systems. In particular, ADGRG2 plays a significant role in Ewing sarcoma cell proliferation, parathyroid cell function, and male fertility. In 2022, a cryo-EM structure was reported for the active ADGRG2 bound by an optimized peptide agonist IP15 and the Gs protein. The IP15 peptide agonist was also modified to antagonists 4PH-E and 4PH-D with mutations of the 4PH residue to Glu and Asp, respectively. However, experimental structures of inactive antagonist-bound ADGRs remain to be resolved, and the activation mechanism of ADGRs such as ADGRG2 is poorly understood. Here, we applied Gaussian accelerated molecular dynamics (GaMD) simulations to probe conformational dynamics of the agonist- and antagonist-bound ADGRG2. By performing GaMD simulations, we were able to identify important low-energy conformations of ADGRG2 in the active, intermediate, and inactive states, as well as explore the binding conformations of each peptide. Moreover, our simulations revealed critical peptide-receptor residue interactions during the deactivation of ADGRG2. In conclusion, through GaMD simulations, we uncovered mechanistic insights into peptide (agonist and antagonist) binding and deactivation of the ADGRG2. These findings will potentially facilitate rational design of new peptide modulators of ADGRG2 and other ADGRs.

Enzymatische Peptid‐ und Proteinbromierung: Der BromoTrp Tag

AbstractBioorthogonale Reaktionen von Proteinen und ungeschützten Peptiden sind von hohem Wert für die chemische Biologie. Die Kombination aus enzymatischer Halogenierung und übergangsmetallkatalysierter Kreuzkupplung bietet einen praktikablen Ansatz zur Modifizierung von Proteinen und ungeschützten Peptiden. Durch semirationales Protein Engineering, wurden Varianten der Tryptophan 6‐Halogenase Thal identifiziert, welche die Bromierung von Peptiden mit einem C‐terminalen Tryptophanrest ermöglichen. Das Substratspektrum wurde anhand von Di‐, Tri‐ und Tetrapeptid‐Bibliotheken charakterisiert, und ein optimierter Peptid‐Tag identifiziert, dem wir den Namen BromoTrp‐Tag gegeben haben. Dieser Tag wurde in drei Modellproteine eingeführt. Deren präparative, posttranslationale Bromierung mit nur einem Kultivierungs‐ und Aufreinigungsschritt wurde durch das bromierende E. coli Koexpressionssystem Brocoli ermöglicht. Pd‐katalysierte Suzuki–Miyaura Kreuzkupplung war mittels Pd‐Nanopartikeln als Katalysator bei 37 °C möglich, was das vielversprechende Potential dieser Strategie zum Zweck bioorthogonaler Funktionalisierung und Konjugation demonstriert.