Solid-phase Peptide Synthesis Protocols Research Articles

General Installation of (4H)-Imidazolone cis-Amide Bioisosteres Along the Peptide Backbone.

Imidazolones represent an important class of heterocycles present in a wide range of pharmaceuticals, metabolites, and bioactive natural products and serve as the active chromophore in green fluorescent protein. Recently, imidazolones have received attention for their ability to act as a nonaromatic amide bond bioisotere which improves pharmacological properties. Herein, we present a tandem amidine installation and cyclization with an adjacent ester to yield (4H)-imidazolone products. Using amino acid building blocks, we can access the first examples of α-chiral imidazolones that have been previously inaccessible. Additionally, our method is amenable to on-resin installation which can be seamlessly integrated into existing solid-phase peptide synthesis protocols. Finally, we show that peptide imidazolones are potent cis-amide bond surrogates that preorganize linear peptides for head-to-tail macrocyclization. This work represents the first general approach to the backbone and side-chain insertion of imidazolone bioisosteres at various positions in linear and cyclic peptides.

Solid Phase Synthesis and TAR RNA-Binding Activity of Nucleopeptides Containing Nucleobases Linked to the Side Chains via 1,4-Linked-1,2,3-triazole.

Nucleopeptides (NPs) represent synthetic polymers created by attaching nucleobases to the side chains of amino acid residues within peptides. These compounds amalgamate the characteristics of peptides and nucleic acids, showcasing a unique ability to recognize RNA structures. In this study, we present the design and synthesis of Fmoc-protected nucleobase amino acids (1,4-TzlNBAs) and a new class of NPs, where canonical nucleobases are affixed to the side chain of L-homoalanine (Hal) through a 1,4-linked-1,2,3-triazole (HalTzl). Fmoc-protected 1,4-TzlNBAs suitable for HalTzl synthesis were obtained via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) conjugation of Fmoc-L-azidohomoalanine (Fmoc-Aha) and N1- or N9-propargylated nucleobases or their derivatives. Following this, two trinucleopeptides, HalTzlAAA and HalTzlAGA, and the hexanucleopeptide HalTzlTCCCAG, designed to complement bulge and outer loop structures of TAR (trans-activation response element) RNA HIV-1, were synthesized using the classical solid-phase peptide synthesis (SPPS) protocol. The binding between HalTzls and fluorescently labeled 5'-(FAM(6))-TAR UCU and UUU mutant was characterized using circular dichroism (CD) and fluorescence spectroscopy. CD results confirmed the binding of HalTzls to TAR RNA, which was evident by a decrease in ellipticity band intensity around 265 nm during complexation. CD thermal denaturation studies indicated a relatively modest effect of complexation on the stability of TAR RNA structure. The binding of HalTzls at an equimolar ratio only marginally increased the melting temperature (Tm) of the TAR RNA structure, with an increment of less than 2 °C in most cases. Fluorescence spectroscopy revealed that HalTzlAAA and HalTzlAGA, complementary to UUU or UCU bulges, respectively, exhibited disparate affinities for the TAR RNA structure (with Kd ≈ 30 and 256 µM, respectively). Hexamer HalTzlTCCCAG, binding to the outer loop of TARUCU, demonstrated a moderate affinity with Kd ≈ 38 µM. This study demonstrates that newly designed HalTzls effectively bind the TAR RNA structure, presenting a potential new class of RNA binders and may be a promising scaffold for the development of a new class of antiviral drugs.

Design, Synthesis, and Anticancer Activity of N‐1 Terminal Segment of α‐(2, 5,7‐Tri‐tert‐butylindol‐3‐yl)alanine‐Incorporated Short Peptides

AbstractUsing a minimallised approach,we designed and synthesized N‐terminal modified peptides W‐KRGNKWLA‐CONH2 (P1) and W‐ARGDGGNGRGA‐CONH2 (P2) using Fmoc‐Chemistry solid phase peptide synthesis protocol. High‐performance liquid chromatograms and mass spectrometry (LCMS and analytical HPLC) were employed to determine the purity and molecular weight of the peptide. Surface tension measurements are used to evaluate peptide surface activity. The primary structure of the peptideis anlaysed by NMR spectroscopy. Circular dichroism studies demonstrate that the secondary structure of the peptide changes minimally when it attached to anionic lipid bilayers (POPC : POPG). According to a steady‐state fluorescence experiment, peptides P1 and P2 preferentially interact with anionic (POPC : POPG) lipid bilayers rather than zwitterionic (POPC) lipid bilayers. The peptide P2 interacts preferentially with anionic lipid membranes and has an anticancer effect due to RGD and NGR binding motifs. The cytotoxicity of the MTT experiment demonstrated that both normal and cancerous cells could lower peptide concentrations below their IC50 levels. Molecular docking also supports in‐vitro anticancer research. Both peptides P1 and P2 were non‐toxic to vero cells and appeared to be more promising as it demonstrated wide spectrum anticancer activity.

Antiviral peptides inhibiting the main protease of SARS-CoV-2 investigated by computational screening and in vitro protease assay.

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays an important role in viral replication and transcription and received great attention as a vital target for drug/peptide development. Therapeutic agents such as small-molecule drugs or peptides that interact with the Cys-His present in the catalytic site of Mpro are an efficient way to inhibit the protease. Although several emergency-approved vaccines showed good efficacy and drastically dropped the infection rate, evolving variants are still infecting and killing millions of people globally. While a small-molecule drug (Paxlovid) received emergency approval, small-molecule drugs have low target specificity and higher toxicity. Besides small-molecule drugs, peptide therapeutics are thus gaining increasing popularity as they are easy to synthesize and highly selective and have limited side effects. In this study, we investigated the therapeutic value of 67 peptides targeting Mpro using molecular docking. Subsequently, molecular dynamics (MD) simulations were implemented on eight protein-peptide complexes to obtain molecular-level information on the interaction between these peptides and the Mpro active site, which revealed that temporin L, indolicidin, and lymphocytic choriomeningitis virus (LCMV) GP1 are the best candidates in terms of stability, interaction, and structural compactness. These peptides were synthesized using the solid-phase peptide synthesis protocol, purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and authenticated by mass spectrometry (MS). The in vitro fluorometric Mpro activity assay was used to validate the computational results, where temporin L and indolicidin were observed to be very active against SARS-CoV-2 Mpro with IC50 values of 38.80 and 87.23 μM, respectively. A liquid chromatography-MS (LC-MS) assay was developed, and the IC50 value of temporin L was measured at 23.8 μM. The solution-state nuclear magnetic resonance (NMR) structure of temporin L was determined in the absence of sodium dodecyl sulfate (SDS) micelles and was compared to previous temporin structures. This combined investigation provides critical insights and assists us to further develop peptide inhibitors of SARS-CoV-2 Mpro through structural guided investigation.

Rapid, traceless and facile peptide cyclization enabled by tetrazine-thiol exchange.

Cyclic peptides offer many advantages compared to their linear counterparts, including prolonged stability within the biological environment and enhanced binding affinity. Typically, peptides are cyclized by forming an amide bond, either on-resin or in solution, through extensive use of orthogonal protecting groups or chemoselective ligation strategies, respectively. Here, we show that the chemoselective tetrazine-thiol exchange is a powerful tool for rapid in situ cyclization of peptides without the need for additional activation reagents or extensive protecting group reshuffling. The reaction between N-terminal sulfide-bearing unsymmetric tetrazines and internal cysteines occurs spontaneously within a mildly acidic environment (pH 6.5) and is of traceless nature. The rapidly available unsymmetric sulfide tetrazine building blocks can be incorporated on resin using standard solid-phase peptide synthesis protocols and are orthogonal to trifluoroacetic acid cleavage conditions. The cyclized peptides display high stability, even when incubated with a large excess of free thiols. Due to its traceless and mild nature, we expect that the tetrazine-thiol exchange will be of high value for the in situ formation of cyclic peptide libraries, thus being applicable in drug discovery and development.

Modular synthetic strategy for N/C-terminal protected amyloidogenic peptides.

N/C-terminal protected amyloidogenic peptides are valuable biomaterials. Optimization of the protective structures at both termini is, however, synthetically laborious because a linear sequence of solid-phase peptide synthesis protocol (on-resin peptide assembly/peptide removal from resin/high-performance liquid chromatography purification) is required for the peptides each time the protective group is modified. In this study, we demonstrate a modular synthetic strategy for the purpose of rapidly deriving the N/C-terminal structures of amyloidogenic peptides. The precursor sequences that can be easily synthesized due to a non-amyloidogenic property were stocked as the synthetic intermediates. Condensation of the intermediates with N/C-terminal units in a liquid phase followed by high-performance liquid chromatography purification gave the desired peptides P1-P8. The amyloidogenic peptides that have various N/C-terminal protective structures were therefore synthesized in a labor-effective manner. This method is suggested to be useful for synthesizing amyloidogenic peptides possessing divergent protective structures at the N/C-terminus.

Development of New Leishmanicidal Compounds via Bioconjugation of Antimicrobial Peptides and Antileishmanial Guanidines.

Leishmaniasis refers to a collection of diseases caused by protozoa from the Leishmania genus. These diseases, along with other parasitic afflictions, pose a significant public health issue, particularly given the escalating number of at-risk patients. This group includes immunocompromised individuals and those residing in impoverished conditions. The treatment of leishmaniasis is crucial, particularly in light of the mortality rate associated with nontreatment, which stands at 20-30,000 deaths per year globally. However, the therapeutic options currently available are limited, often ineffective, and potentially toxic. Consequently, the pursuit of new therapeutic alternatives is warranted. This study aims to design, synthesize, and evaluate the leishmanicidal activity of antimicrobial peptides functionalized with guanidine compounds and identify those with enhanced potency and selectivity against the parasite. Accordingly, three bioconjugates were obtained by using the solid-phase peptide synthesis protocol. Each proved to be more potent against intracellular amastigotes than their respective peptide or guanidine compounds alone and demonstrated higher selectivity to the parasites than to the host cells. Thus, the conjugation strategy employed with these compounds effectively contributes to the development of new molecules with leishmanicidal activity.

Design and synthesis of novel N-terminal peptides of integrin and aminopeptidase are new finding for anticancer activity

The short peptides, containing the amino acid sequence asparagine-glycine-arginine (NGR) and arginine-glycine-aspartic acid (RGD), possess the strong binding ability to N (APN/CD13) aminopeptidase receptor and integrin proteins involved in antitumor properties are overexpressed. A novel short N-terminal modified hexapeptides P1 and P2 was designed and synthesized using the Fmoc-chemistry solid phase peptide synthesis protocol. Notably, the cytotoxicity of the MTT assay demonstrated the viability of normal and cancer cells up to lower peptide concentrations. Interestingly, both peptides show good anticancer activities against the four cancer cells and normal cells namely, Hep-2, HepG2, MCF-7, A375, and Vero and compared with standard drugs, doxorubicin and paclitaxel. Additionally, in silico studies were applied to predict the binding sites and binding orientation of the peptides for potential anticancer targets. Steady-state fluorescence measurements showed that peptide P1 exhibits preferential interactions with POPC/POPG anionic bilayers rather than the zwitterionic POPC lipid bilayers and peptide P2, did not show any preferential interaction with lipids bilayers. But impressively, peptide P2 shows anticancer activity due to the NGR/RGD motif. Circular dichroism studies demonstrated that the peptide's secondary structure changes only minimally upon binding to the anionic lipid bilayers.

An identification method to distinguish monomeric sugar isomers on glycopeptides.

A one-step protocol for the automated flow synthesis of protected glycosylated amino acids is described using pumps with open-source controls in overall yields of 21-50%. The resulting glycosylated amino acids could be used directly in solid-phase peptide synthesis (SPPS) protocols to quickly produce glycopeptide standards. Access to a variety of stereoisomers of the sugar enabled the development of an LC-MS/MS protocol that can distinguish between peptides modified with carbohydrates having the same exact mass. This method could definitively identify fucose in an O-glycosylation site on the transmembrane protein, Notch1.

A Series of Dipeptide Derivatives Containing (S)‐5‐Oxo‐pyrrolidine‐2‐carboxylic acid Conjugates: Design, Solid‐Phase Peptide Synthesis, in vitro Biological Evaluation, and Molecular Docking Studies

AbstractUtilizing the solid phase peptide synthesis protocol, we have designed and prepared a novel dipeptide library containing (S)‐5‐oxopyrrolidine‐2‐carboxylic acid moiety. All the newly synthesized dipeptides were characterized by spectroscopic techniques as well as elemental analysis. Furthermore, the in vitro antimicrobial activities of the synthesized dipeptide conjugates were evaluated. Two Gram‐positive (Streptococcus pyogenes and Staphylococcus aureus) and two Gram‐negative bacteria (Escherichia coli and Pseudomonas aeruginosa) were utilized to evaluate the antibacterial activity of the screened derivatives. In contrast to the standard drug Ampicillin, the targeted derivatives exhibited good antibacterial activity. Additionally, two fungi (Candida albicans and Aspergillus niger) were used to evaluate the antifungal activity of the target dipeptides. The targeted derivatives also exhibited good antifungal activity compared to the standard drug Nystatin. In continuous, the molecular docking study of the targeted derivatives was also carried out, which revealed that the dipeptide analogs showed encouraging binding interaction networks with Escherichia coli DNA gyrase B and lanosterol‐14 alpha demethylase resulting in antibacterial and antifungal activities, respectively. Such synthesis, biological evolution, and molecular docking study of peptide derivatives with oxopyrrolidine conjugates open the door for the future development of new therapeutics containing heterocycle and peptide hybrids with potency as antimicrobial agents.

Chemoselective Solution- and Solid-Phase Synthesis of Disulfide-Linked Glycopeptides.

Glycosylation of peptides and proteins is a widely employed strategy to mimic important post-translational modifications or to modulate the physicochemical properties of peptides to enhance their delivery. Furthermore, glycosylation via a sulfur atom imparts increased chemical and metabolic stability to the resulting glycoconjugates. Herein, we report a simple and chemoselective procedure to prepare disulfide-linked glycopeptides. Acetate-protected glycosylsulfenyl hydrazines are shown to be highly reactive with the thiol group of cysteine residues within peptides, both in solution and as part of conventional solid-phase peptide synthesis protocols. The efficiency of this glycosylation methodology with unprotected carbohydrates is also demonstrated, which avoids the need for deprotection steps and further extends its utility, with disulfide-linked glycopeptides produced in excellent yields. Given the importance of glycosylated peptides in structural glycobiology, pharmacology, and therapeutics, the methodology outlined provides easy access to disulfide-linked glycopeptides as molecules with multiple biological applications.

Synthesis of malformin‐A1, C, a glycan, and an aglycon analog: Potential scaffolds for targeted cancer therapy

AbstractImprovement in therapeutic efficacy while reducing chemotherapeutic side effects remains a vital objective in synthetic design for cancer treatment. In keeping with the ethos of therapeutic development and inspired by the Warburg effect for augmenting biological activities of the malformin family of cyclic‐peptide natural products, specifically anti‐tumor activity, a β‐glucoside of malformin C has been designed and synthesized utilizing precise glycosylation and solution phase peptide synthesis. We optimized several glycosylation procedures utilizing different donors and acceptors. The overarching goal of this study was to ensure a targeted delivery of a glyco‐malformin C analog through the coupling of D‐glucose moiety; selective transport via glucose transporters (GLUTs) into tumor cells, followed by hydrolysis in the tumor microenvironment releasing the active malformin C a glycon analog. Furthermore, total synthesis of malformin C was carried out with overall improved strategies avoiding unwanted side reactions thus increasing easier purification. We also report on an improved solid phase peptide synthesis protocol for malformin A1.

A Selenium-based Cysteine Surrogate for Protein Chemical Synthesis.

N-selenoethyl cysteine (SetCys) in the form of its cyclic selenosulfide is a cysteine surrogate, whose reactivity depends on the reducing power of the medium. SetCys does not interfere with the native chemical ligation reaction under mild reducing conditions, that is in the absence of tris(2-carboxyethyl)phosphine (TCEP). In contrast, subjecting SetCys to TCEP results in the spontaneous loss of its N-selenoethyl appendage and thus to its conversion into a Cys residue. Therefore, SetCys can be used for the redox-controlled assembly of peptide segments using NCL. We provide in this protocol detailed procedures for the synthesis of Fmoc-protected SetCys residue and for its incorporation into peptides using standard solid-phase peptide synthesis protocols. We also describe its use for the chemical synthesis of proteins through the redox-controlled assembly of three peptide segments in one-pot.

The application of anisole in greener solid-phase peptide synthesis protocols – Compatibility with green bases in Fmoc removal and new green binary mixture for coupling

As a step toward green Fmoc solid-phase peptide synthesis (SPPS), we explored the compatibility of the green bases morpholine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and NaOH with the green solvent anisole as a replacement for the traditionally used hazardous piperidine in N,N-dimethylformamide (DMF) for the Fmoc removal step. Green Fmoc cleavage protocols were optimized for the Fmoc-Ala-NH-Rink resin model and subsequently verified for other Fmoc-amino acids. Only Gly required a modified NaOH-based protocol with the addition of 5 % water. Furthermore, we evaluated the effects of basic treatment on the notorious aspartimide formation as an undesired side product of SPPS. We also introduced a new green solvent mixture, anisole/dimethyl sulfoxide (DMSO) (4:1) for acylation step, which did not cause racemization. The applicability of these new green protocols was shown during SPPS of the pentapeptides Leu-enkephalin and Aib-enkephalin using polystyrene (PS)-based resins. Additionally, we identified the new sequence-dependent side products that formed after treatment with NaOH in anisole/ethanol (EtOH) (1:1).

Tea Bags for Fmoc Solid-Phase Peptide Synthesis: An Example of Circular Economy.

Peptide synthesis is an area with a wide field of application, from biomedicine to nanotechnology, that offers the option of simultaneously synthesizing a large number of sequences for the purpose of preliminary screening, which is a powerful tool. Nevertheless, standard protocols generate large volumes of solvent waste. Here, we present a protocol for the multiple Fmoc solid-phase peptide synthesis in tea bags, where reagent recycling steps are included. Fifty-two peptides with wide amino acid composition and seven to twenty amino acid residues in length were synthesized in less than three weeks. A clustering analysis was performed, grouping the peptides by physicochemical features. Although a relationship between the overall yield and the physicochemical features of the sequences was not established, the process showed good performance despite sequence diversity. The recycling system allowed to reduce N, N-dimethylformamide usage by 25–30% and reduce the deprotection reagent usage by 50%. This protocol has been optimized for the simultaneous synthesis of a large number of peptide sequences. Additionally, a reagent recycling system was included in the procedure, which turns the process into a framework of circular economy, without affecting the quality of the products obtained.

Synthesis and Structural Stability of α-Helical Gold(I)-Metallopeptidesy

AbstractThe synthesis of hexa- and dodecapeptides functionalized with two Au(I)–phosphine complexes is reported. The high stability of the Au(I)–phosphine bond allowed orthogonal peptide-protecting-group chemistry, even when using hard Lewis acids like boron tribromide. This enabled the preparation of an Fmoc-protected lysine derivative carrying the Au(I) complex in a side chain, which was used in standard Fmoc-based solid-phase peptide synthesis protocols. Alanine and leucine repeats in the metallododecapeptide formed α-helical secondary structures in 2,2,2-trifluoroethanol–H2O and 1,1,1,3,3,3-hexafluoroisopropanol–H2O mixtures with high thermal stability, as shown by temperature-dependent CD spectroscopy studies.

Benzoisothiazolone (BIT): A Fast, Efficient, and Recyclable Redox Reagent for Solid Phase Peptide Synthesis

Solid‐phase peptide synthesis (SPPS), a preferred synthetic procedure, generates by‐products and effluents in multiple equivalents for one equivalent of desired product. Presented herein is the use of a fast and efficient coupling protocol for SPPS using a benzoisothiazolone (BIT), which can be fully recycled. The BIT, as redox activator, works under very mild conditions and generates minimal amount of waste. As a case study, the BIT coupling protocol is applied to the synthesis of side chain of the recently discovered antibiotic, teixobactin.

Leucine-glycine and carnosine dipeptides prevent diabetes induced by multiple low-doses of streptozotocin in an experimental model of adult mice.

Aims/IntroductionPeptides are considered to be quasi‐hormones and effective molecules for regulation of the cells function and prevention of metabolic disorders. Di‐ and tripeptides gastrointestinal absorption ability have been proposed to prevent diabetes progression.Materials and MethodsSmall peptides with different sequences of specific amino acids were synthesized based on a solid phase peptide synthesis protocol, and carnosine (A) and glutathione were examined for the prevention of diabetes induced by multiple low‐doses of streptozotocin in mice.ResultsThe peptides A, Leu‐Gly (D) and Pro‐Pro showed preventive effects on blood glucose elevation and impairment of the signaling and performance of β‐cells. The β‐cell function assessed by immunofluorescence and blood glucose level in mice exposed to diabetes treated by the peptides A and D was similar to the normal mice. The peptide D prevented bodyweight loss caused by diabetes induction. The use of D and A peptides dramatically prevented the incidence of disruption in β‐cells signaling by maintaining the natural balance of intracellular Akt‐2 and cyclic adenosine monophosphate.ConclusionsThe results proved that peptide D (Leu‐Gly), named Hannaneh, inhibits the bodyweight loss caused by diabetes induction. The Hannaneh and carnosine dipeptides, with preservation of normal β‐cell signaling and anti dipeptidyl peptidase‐4 activity, prevented blood glucose increases in mice at risk of diabetes. These dipeptides might be regarded as the pharmaceutical agents for the prevention of diabetes.

Solid-Phase Total Synthesis and Dual Mechanism of Action of the Channel-Forming 48-mer Peptide Polytheonamide B.

Polytheonamide B (1) is a unique peptide natural product because of its extremely complex structure, a channel-forming ability in vitro, and the extremely potent cytotoxicity. The 48-mer sequence of 1 comprises alternating d,l-amino acids and possesses an array of sterically bulky β-tetrasubstituted and hydrogen bond forming residues. These unusual structural features are believed to drive 1 to fold into a 4.5 nm long tube, form a transmembrane ion channel at the plasma membrane, and exert cytotoxicity. Despite its potential biological application, however, multiple substitutions by these unusual residues significantly heightened the synthetic challenges, impeding the solid-phase peptide synthesis (SPPS) of 1. In this study, we first addressed the synthesis problem by extensive optimization of various factors of the SPPS. Adaptation of a new protective group strategy allowed for elongation of a 37-mer peptide on resin, to which an N-terminal 11-mer fragment was condensed. Removal of the 18 protective groups and resin gave rise to 1 in excellent overall yield (4.5%, 76 steps from 17). The SPPS protocol is operationally simple and was proven easily amenable to total synthesis of the fluorescent 48-mer probe 2. Synthetic 1 and 2 were utilized for analysis of their cellular behavior. Reflecting its ion-channel function, the addition of 1 to MCF-7 cells rapidly diminished a potential across the plasma membrane. Furthermore, fluorescence imaging study revealed that 1 and 2 were also internalized into the cells, accumulating in acidic lysosomes and neutralizing the lysosomal pH gradient. These new findings indicated that 1 is capable of exerting two functions upon causing apoptotic cell death of mammalian cells: It induces free cation transport across the plasma as well as lysosomal membranes. The present chemical and biological studies provide valuable information for the design and synthesis of polytheonamide-based molecules with more potent and selective biological activities.

Synthetic α- and β-Ser-ADP-ribosylated Peptides Reveal α-Ser-ADPr as the Native Epimer.

A solid-phase methodology to synthesize oligopeptides, specifically incorporating serine residues linked to ADP-ribose (ADPr), is presented. Through the synthesis of both α- and β-anomers of the phosphoribosylated Fmoc-Ser building block and their usage in our modified solid-phase peptide synthesis protocol, both α- and β-ADPr peptides from a naturally Ser-ADPr containing H2B sequence were obtained. With these, and by digestion studies using the human glycohydrolase, ARH3 (hARH3), compelling evidence is obtained that the α-Ser-ADPr linkage comprises the naturally occurring configuration.