Stepwise Solid-phase Peptide Synthesis Research Articles

Incorporation of pseudoproline monomer (Fmoc-Thr[ψMe,Mepro]–OH) facilitates efficient solid-phase synthesis of difficult peptides

Solid-Phase Peptide Synthesis (SPPS) is a convenient and efficient method by which to prepare peptides. However, the sequences of compounds classified as “difficult” show aggregation of growing peptide chains and their synthesis is extremely challenging by stepwise SPPS. In this context, the concourse of pseudoproline derivatives helps to break the aggregation and facilitates the synthesis. Although pseudoproline moieties are traditionally incorporated as dipeptides, the possibility of using only the monomer adds flexibility to the synthesis. Herein, the introduction of the most hindered β-branched Thr[ψMe,Mepro] derivative is studied in terms of how difficult is its acylation is by the following amino acid and the impact of its use on the final purity of two well-known ”difficult peptides”.

Synthesis of MUC1 Peptide Backbone Bearing Sialyl‐Tn Antigen for Structural and Functional Studies With Endogenous Lectins

ObjectiveTo synthesize the tumor associated analogs of MUC1 for use in structural and functional studies between sialyl‐Tn (sTn) antigen with macrophage galactose‐specific lectin (MGL).AbstractMucin 1 (MUC1) is a heavily glycosylated transmembrane protein, altered in both expression and glycosylation pattern in human carcinomas of the epithelium. The presence of incomplete or truncated glycan structures, often capped by sialic acid, commonly known as tumor‐associated carbohydrate antigens (TACAs), on the cell surface is a well‐known cancer biomarker. Accumulating evidence suggests that TACAs expression is associated with tumor escape from immune defenses through interaction with endogenous carbohydrate binding proteins (lectins). These interactions often lead to creation of a protumor microenvironment, favoring tumor initiation, progression, metastasis, and immune evasion. Macrophage galactose binding lectin (MGL) is a C‐type lectin receptor found on antigen‐presenting cells (APCs) which facilitates the uptake of carbohydrate antigens for antigen presentation, modulating the immune response homeostasis, autoimmunity, and cancer. Considering the crucial role of tumor‐associated forms of MUC1 with MGL in tumor immunology, a thorough understanding of this interaction is essential for it to be exploited for cancer vaccine strategies. The specific aim of this research is to synthesize structurally well‐defined chemical probes, mono and multiple glycosylated MUC1 glycopeptide models carrying the Tn or sTn, that allow for the control of the complexity of the chemical space of the multivalent ligands. For this purpose, a concise scheme was developed for the large synthesis of the sTn antigen building block. Thiophenyl glycoside donors, in the presence TfOH/NIS or TMSOTf/NIS as promoter system, were used for the galactosylation and sialylation steps of the amino acid building block synthesis, respectively. We explored the effect of activator, temperature, solvent, and excess equivalent of sialic acid thioglycoside donor on sialylation reaction. The most favorable reaction conditions improved the desired α‐stereoselectivity, decreased the competing 2,3‐elimination reaction on the sialyl donor during activation, leading to overall increase in the reaction yield. This building block was used in the stepwise solid‐phase peptide synthesis of MUC1 glycopeptide models. The secondary structure of synthesized peptides was determined by circular dichroism (CD) spectroscopy.

Synthesis of O-Acyl Isopeptides: Stepwise and Convergent Solid-Phase Synthesis.

The O-acyl isopeptide method was developed for the preparation of difficult sequence-containing peptides, whose hydrophobic nature hampers both peptide chain construction on resin and purification with HPLC after deprotection. In the O-acyl isopeptide method, the target peptide is synthesized in an O-acyl isopeptide form, which contains an O-acyl isopeptide bond instead of the native N-acyl peptide bond at a hydroxy group-containing amino acid residue, such as Ser or Thr. The hydrophilic O-acyl isopeptide can be isolated, e.g., as a lyophilized TFA salt. The target peptide can be quantitatively obtained by a final O-to-N intramolecular acyl migration reaction with exposure to neutral conditions. Additionally, the O-acyl isopeptide is important as a hydrophilic precursor peptide for biological peptide assays that are difficult to handle. This chapter describes the synthesis of such O-acyl isopeptides by stepwise and convergent Fmoc solid-phase peptide synthesis.

One-Pot Peptide Ligation-Oxidative Cyclization Protocol for the Preparation of Short-/Medium-Size Disulfide Cyclopeptides.

Native chemical ligation (NCL) employing the N-methylbenzimidazolinone (MeNbz) linker readily provided the linear precursor of a 16-mer peptide that is difficult to obtain by stepwise solid-phase peptide synthesis. NCL and the workup conditions were improved toward a protocol that allows for quantitative removal of the 4-hydroxymercaptophenol additive and subsequent formation of the disulfide bridge in the NCL cocktail by oxidation in air, tolerated by the presence of tris(hydroxypropyl)phosphine.

Mambalgins, snake peptides against inflammatory and neuropathic pain through inhibition of ASIC channels

Mambalgins are 57-amino acid peptides isolated from mamba venom. They produce potent analgesic effects in mice against inflammatory pain upon central intrathecal (i.t.), and peripheral local (i.pl.) injections, through inhibition of different ASICs subtypes and involvement of opioid-independent pathways. They produce fewer side effects than morphine and no apparent toxicity. We now show that mambalgins also have an opioid-independent effect on both thermal and mechanical pain upon systemic intravenous (i.v.) administration and are effective against neuropathic pain by i.v., i.t. and i. pl. injections. By combining the use of knockdown and knockout animals, we show the critical involvement of peripheral ASIC1b-containing channels in the i.v. effects of mambalgins against inflammatory pain. The potent analgesic effect on neuropathic pain involves two different mechanisms depending on the route of administration, a naloxone-insensitive and ASIC1a-independent effect associated with i.v. injection, and an ASIC1a-dependent and partially naloxone-sensitive effect associated with i.t. injection. We have done in collaboration with the CEA iBiTecS Institute in Gif-sur-Yvette, the full stepwise solid-phase peptide synthesis of mambalgin-1, solved its 3D crystal structure, mapped the pharmacophore, and identified the binding site and the inhibitory mechanism on ASIC1a channels. These findings identify new roles for ASICs in pain pathways, and mambalgins as new potential analgesics against inflammatory and chronic neuropathic pain.

Evaluation of Lipopeptides as Toll-like Receptor 2 Ligands.

Peptide-based vaccines are considered to be the next generation of modern immunizations, as they are safe, easy to produce and well-defined. However, due to their weak immunogenic effect, it is important to first develop an appropriate adjuvant for peptide-based vaccines. The aim of this work was to synthesize a series of four adjuvanting moieties as alkyne derivatives, incorporating dipalmitoyl serine (DPS), 1,3-diglyceride (DG), two hexadecane lipoamino acids (diLAA), and 2,3-dipalmitoyl-S-glycerylcysteine (Pam2Cys). Next aim was to synthesize and attach the azide derivative of biotinylated J14 peptide (model B-cell epitope) to the alkynes through copper- catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC) reaction. Final aim was to test the ability of the final biotin labeled conjugates to directly interact with in vitro expressed TLR2 and 8 using AlphaScreen proximity assay. All of the peptides were synthesized by manual stepwise solid phase peptide synthesis (SPPS) on rink amide MBHA resin using HATU/DIPEA Fmoc-chemistry. The target compounds were synthesized in a solution phase using CuAAC reaction. Pam2Cys analogue bound to TLR2 as expected. Analogues of DPS and C16-LAA showed also affinity to TLR2, while it did not bind to the control protein (TLR8), demonstrating ability of the DPS and C16-LAA to be recognized by TLR2. Four alkyne derivatives of lipids were successfully synthesized and coupled to a biotinylated J14 peptide to give a series of self-adjuvanting ligands. These ligands showed different affinity to TLR2 upon testing by AlphaScreen assay. The DPS derivative showed the most promising affinity in comparison to the standard TLR2 agonist, Pam2Cys.

Mambalgin-1 Pain-relieving Peptide, Stepwise Solid-phase Synthesis, Crystal Structure, and Functional Domain for Acid-sensing Ion Channel 1a Inhibition

Mambalgins are peptides isolated from mamba venom that specifically inhibit a set of acid-sensing ion channels (ASICs) to relieve pain. We show here the first full stepwise solid phase peptide synthesis of mambalgin-1 and confirm the biological activity of the synthetic toxin both in vitro and in vivo. We also report the determination of its three-dimensional crystal structure showing differences with previously described NMR structures. Finally, the functional domain by which the toxin inhibits ASIC1a channels was identified in its loop II and more precisely in the face containing Phe-27, Leu-32, and Leu-34 residues. Moreover, proximity between Leu-32 in mambalgin-1 and Phe-350 in rASIC1a was proposed from double mutant cycle analysis. These data provide information on the structure and on the pharmacophore for ASIC channel inhibition by mambalgins that could have therapeutic value against pain and probably other neurological disorders.

Lipopeptide Nanoparticles: Development of Vaccines against Hookworm Parasite.

Necator americanus (hookworm) infects over half a billion people worldwide. Anthelminthic drugs are commonly used to treat the infection; however, vaccination is a more favorable strategy to combat this parasite. We designed new B-cell peptide epitopes based on the aspartic protease of N. americanus (Na-APR-1). The peptides were conjugated to self-adjuvanting lipid core peptide (LCP) systems via stepwise solid-phase peptide synthesis (SPPS) and copper catalyst azide-alkyne cycloaddition (CuAAC) reactions. The LCP vaccine candidates were able to self-assemble into nanoparticles, were administered to mice without the use of additional adjuvant, and generated antibodies that recognized the parent epitope. However, only one LCP derivative was able to produce a high titer of antibodies specific to Na-APR-1; circular dichroism analyses of this compound showed a β-sheet conformation for the incorporated epitope. This study provides important insight in epitope and delivery system design for the development of a vaccine against hookworm infections.

Increased Diversity of the HLA-B40 Ligandome by the Presentation of Peptides Phosphorylated at Their Main Anchor Residue

Human leukocyte antigen (HLA) class I molecules bind peptides derived from the intracellular degradation of endogenous proteins and present them to cytotoxic T lymphocytes, allowing the immune system to detect transformed or virally infected cells. It is known that HLA class I-associated peptides may harbor posttranslational modifications. In particular, phosphorylated ligands have raised much interest as potential targets for cancer immunotherapy. By combining affinity purification with high-resolution mass spectrometry, we identified more than 2000 unique ligands bound to HLA-B40. Sequence analysis revealed two major anchor motifs: aspartic or glutamic acid at peptide position 2 (P2) and methionine, phenylalanine, or aliphatic residues at the C terminus. The use of immobilized metal ion and TiO2 affinity chromatography allowed the characterization of 85 phosphorylated ligands. We further confirmed every sequence belonging to this subset by comparing its experimental MS2 spectrum with that obtained upon fragmentation of the corresponding synthetic peptide. Remarkably, three phospholigands lacked a canonical anchor residue at P2, containing phosphoserine instead. Binding assays showed that these peptides bound to HLA-B40 with high affinity. Together, our data demonstrate that the peptidome of a given HLA allotype can be broadened by the presentation of peptides with posttranslational modifications at major anchor positions. We suggest that ligands with phosphorylated residues at P2 might be optimal targets for T-cell-based cancer immunotherapy.

Total Chemical Synthesis of a Heterodimeric Interchain Bis-Lactam-Linked Peptide: Application to an Analogue of Human Insulin-Like Peptide 3

Nonreducible cystine isosteres represent important peptide design elements in that they can maintain a near-native tertiary conformation of the peptide while simultaneously extending the in vitro and in vivo half-life of the biomolecule. Examples of these cystine mimics include dicarba, diselenide, thioether, triazole, and lactam bridges. Each has unique physicochemical properties that impact upon the resulting peptide conformation. Each also requires specific conditions for its formation via chemical peptide synthesis protocols. While the preparation of peptides containing two lactam bonds within a peptide is technically possible and reported by others, to date there has been no report of the chemical synthesis of a heterodimeric peptide linked by two lactam bonds. To examine the feasibility of such an assembly, judicious use of a complementary combination of amine and acid protecting groups together with nonfragment-based, total stepwise solid phase peptide synthesis led to the successful preparation of an analogue of the model peptide, insulin-like peptide 3 (INSL3), in which both of the interchain disulfide bonds were replaced with a lactam bond. An analogue containing a single disulfide-substituted interchain lactam bond was also prepared. Both INSL3 analogues retained significant cognate RXFP2 receptor binding affinity.

Disulfide Bond Formation with Polymer-Supported Reagent

Oxidizing reagents for disulfide formation of peptides are prepared from poly(ethylene glycol)polyacrylamide (PEGA) via the stepwise solid-phase peptide synthesis (method 1) or the oligomerization of N-carboxy anhydride derivatives (method 2). The oxidation of peptides 1 and 2 was carried out with resins 1a-d and 2 to give the corresponding disulfides 1′ and 2′ in 90-100% yield.

Strategies and Limitations in Dendrimeric Immunogen Synthesis. The Influenza Virus M2e Epitope as a Case Study

Dendrimeric platforms such as multiple antigen peptides (MAPs) are regarded as one of the most efficacious approaches for antigenic presentation. Originally described as available by stepwise solid-phase peptide synthesis (SPPS), MAPs have also been prepared by chemical (thioether, oxime, hydrazone) ligation of appropriately functionalized tetra- or octavalent polylysine scaffolds with the peptide antigen to be multiply displayed. In this work, the advantages and limitations of two of the most frequent methods of MAP preparation, namely, chemoselective thioether ligation in solution, and all-solid-phase synthesis, have been tested in the case of a particularly troublesome epitope model, the ectodomain of protein M2 from influenza virus (M2e). The strong tendency of M2e to self-associate is a serious inconvenient for conjugation in solution, which as a result fails to produce the target MAPs with the specified number of M2e copies. In contrast, the fully stepwise SPPS approach is shown to be quite practical, especially when 6-aminohexanoic acid spacer units providing increased internal flexibility are inserted at each branching point.

Role of a Cytoplasmic Dual-function Glycosyltransferase in O2 Regulation of Development in Dictyostelium

In the social amoeba Dictyostelium, a terminal step in development is regulated by environmental O(2). Prolyl 4-hydroxylase-1 (P4H1) was previously implicated in mediating the O(2) signal, and P4H1-null cells require elevated O(2) to culminate. The E3-ubiquitin ligase adaptor Skp1 is a P4H1 substrate, and here we investigate the function of PgtA, a dual function beta3-galactosyltransferase/alpha2-fucosyltransferase that contributes the 2nd and 3rd sugars of the pentasaccharide cap formed on Skp1 hydroxyproline. Although pgtA-null cells, whose Skp1 contains only a single sugar (N-acetylglucosamine or GlcNAc), show wild-type O(2) dependence of culmination, cells lacking AgtA, an alpha3-galactosyltransferase required to extend the trisaccharide, require elevated O(2) as for P4H1-null cells. Skp1 is the only detectable protein modified by purified PgtA added to pgtA-null extracts. The basis for specificity of PgtA was investigated using native Skp1 acceptor glycoforms and a novel synthetic peptide containing GlcNAcalpha1,4-hydroxy(trans)proline. Cysteine-alkylation of Skp1 strongly inhibited modification by the PgtA galactosyltransferase but not the fucosyltransferase. Furthermore, native and synthetic Skp1 glycopeptides were poorly galactosylated, not processively fucosylated, and negligibly inhibitory, whereas the fucosyltransferase was active toward small substrates. In addition, the galactosyltransferase exhibited an atypical concentration dependence on UDP-galactose. The results provide the first evidence that Skp1 is the functional target of P4H1 in O(2) regulation, indicate a gatekeeper function for the beta3-galactosyltransferase in the PgtA dual reaction, and identify an unexpected P4H1-dependent yet antagonistic function for PgtA that is reversed by AgtA.

Solid‐Phase Synthesis of Peptide Libraries Combining α‐Amino Acids with Inorganic and Organic Chromophores

The synthesis of two series of peptidic chains composed of bis(terpyridine)ruthenium(II) acceptor units and organic chromophores (coumarin, naphthalene, anthracene, fluorene) by stepwise solid-phase peptide synthesis (SPPS) techniques is described. The first series of dyads comprises directly amide linked chromophores, while the second one possesses a glycine spacer between the two chromophores. All dyads were studied by UV/Vis and NMR spectroscopy, steady-state luminescence, luminescence decay and electrochemistry, as well as by DFT calculations. The results of these studies indicate weak electronic coupling of the chromophores in the ground state. Absorption spectra of all dyads are dominated by metal-to-ligand charge-transfer (MLCT) bands around 500 nm. The bichromophoric systems, especially with coumarin as organic chromophore, display additional strong absorptions in the visible spectral region. All complexes are luminescent at room temperature ((3)MLCT). Efficient quenching of the fluorescence of the organic chromophore by the attached ruthenium complex is observed in all dyads. Excitation spectra indicate energy transfer from the organic dye to the ruthenium chromophore.

Toward the Development of Prophylactic and Therapeutic Human Papillomavirus Type-16 Lipopeptide Vaccines

Four lipid-core peptide systems were synthesized using stepwise solid-phase peptide synthesis, incorporating a sequence from the human papillomavirus type-16 (HPV-16) E7 protein (E744-62), for the purpose of developing vaccines against HPV-16 associated cervical cancer. d-Mannose was conjugated to the vaccine in order to investigate whether the targeting of dendritic cell mannose receptors would improve vaccine efficacy. The ability of the vaccines to clear or reduce the size of HPV-16 associated tumors was assessed in C57BL/6 (H-2b) mice using the TC-1 HPV-16 tumor model. Overall, significant reductions in the size of TC-1 tumors were observed in the mouse model, with the conjugation of mannose to these vaccines demonstrated to clear or reduce the size of TC-1 tumors to a greater extent than non-mannose-containing vaccines (37 out of 40 versus 21 out of 30 tumors cleared, respectively).

A technique for the synthesis of highly-pure, mono-epitopic, multi-valent lipid core peptide vaccines

The synthesis of lipid core peptide (LCP) vaccines using stepwise solid-phase peptide synthesis commonly results in products which are difficult to purify to homogeneity. A new technique for synthesizing highly-pure, mono-epitopic, multi-valent LCP-systems using native chemical ligation is presented. Various conditions were assessed for ligating four copies of a thioester-modified 88/30 serotype group A streptococcal peptide antigen onto an LCP-system featuring four cysteine residues. Overall, the vaccine was synthesized in high purity (>99%), and high yield (90%) when the ligation reaction was performed in the presence of 1% sodium dodecyl sulfate and at elevated temperatures (37 °C).

Solid-phase synthesis of backbone-cyclized β-helical peptides

This paper describes the synthesis and purification of two 22-residue cyclic peptides, cyclo{[( l-Val- d-Val) 4-( l-Val- d-Pro-Gly)] 2-} 3 and cyclo{[( d-Leu- l-Leu) 4-( d-Leu- l-Pro-Gly)] 2-} 4, that were designed to fold into double-stranded antiparallel β-helical structures. Due to intramolecular hydrogen bonding and the conformational constraints imposed by the two reverse-turn segments ( d-Pro-Gly and l-Pro-Gly, respectively), the linear precursors to 3 and 4 (lin- 3 and lin- 4) were expected to adopt preorganized conformations that would bring the N and C termini close together and thereby favor ring closure. Precursors lin- 3 and lin- 4 were constructed by stepwise Boc solid-phase peptide synthesis using the commercially available alkanesulfonamide ‘safety-catch’ linker and cyclized head-to-tail via the method of cleavage-by-cyclization. The crude cyclic peptides were highly hydrophobic and contained minor impurities that could not be removed solely by reversed-phase HPLC (RP-HPLC); however, two-step purification—first by RP-HPLC with i-PrOH/water gradients, followed by gel-permeation chromatography (GPC) on Sephadex LH-20 with CHCl 3/MeOH—afforded both peptides in pure form (≥95% by 1H NMR) and in acceptable yield (23%). Subsequent 1H NMR experiments supported the expected structures of 3 and 4. The successful formation of the 66-membered rings of 3 and 4 is consistent with the notion of conformational preorganization in the linear precursors; furthermore, the protocols for synthesis and purification described should prove useful for preparing additional cyclic β-helical peptides, including longer peptides and peptides having polar residues.

Total chemical synthesis of the B1 domain of protein L from Peptostreptococcus magnus

Reported here is a native chemical ligation strategy for the total chemical synthesis of the B1 domain of protein L. A synthetic construct of this 76 amino acid protein domain was prepared by the chemoselective ligation of two unprotected polypeptide fragments, one containing an N-terminal cysteine residue and one containing a C-terminal thioester moiety. The polypeptide fragments utilized in the ligation reaction were readily prepared by stepwise solid phase peptide synthesis (SPPS) methods for Boc-chemistry. The milligram quantities of protein required for conventional biophysical studies were readily accessible using the synthetic protocol described here. The folding properties of the synthetic protein L construct were also determined and found to be very similar to those of a similar wild-type protein L constructs prepared by recombinant-DNA methods. This work facilitates future unnatural amino acid mutagenesis experiments on this model protein system to further dissect the molecular basis of its folding and stability.

The lipid core peptide system in vaccine delivery

Lipid core peptide (LCP)-based group A streptococcal (GAS) vaccines incorporating numerous GAS M-protein epitopes have been synthesized in high purity. The LCP system incorporates adjuvant, carrier, and antigens into a single well-characterized construct. A tetra-epitopic vaccine, incorporating a highly conserved GAS M-protein C-terminal epitope and three strain-specific epitopes from the variable GAS M-protein N-terminal region, was synthesized using stepwise solid-phase peptide synthesis. Immunization of mice with this construct yielded high IgG antibody responses without the use of traditional adjuvants.

Synthesis and conformational analysis of Id2 protein fragments: impact of chain length and point mutations on the structural HLH motif

The Id proteins are negative regulators of several basic-helix-loop-helix (HLH) transcription factors, including the ubiquitous E factors and the tissue-specific myogenin-regulating factors. Id1 through Id4 contain highly identical HLH domains but different N- and C-terminal extensions. Beside the heterodimerization with the parent HLH factors, Id2 was shown to additionally interact with the retinoblastoma protein and to be overexpressed in neuroblastoma. Thus, Id2 represents an interesting target for cancer therapy based on the inhibition of protein-protein interactions. Here we present the synthesis and circular dichroism (CD) analysis of peptides derived from point mutations and N-/C-terminal truncations of Id2. The helix character of the HLH domain (residues 36-76) was reduced upon substitution of Met39/-62 and Cys42 with Nle and Ser, respectively, suggesting a structural role of these side chains. The largest sequence that could be obtained by stepwise solid-phase peptide synthesis (SPPS) with Fmoc strategy spanned the entire HLH motif (with Cys42 replaced by Ser) and part of the C-terminus (residues 77-110). This 75-residue long fragment was less helical than the isolated HLH domain and had propensity to aggregate, which was correlated with the presence of the flanking residues C-terminal to helix-2. By CD analysis of an equimolar mixture of the sequence 36-110 with the N-terminus 1-35, noncovalent interactions between the two peptides were detected, which, however, changed upon aging. In contrast, the mixture of the HLH sequence 36-76 with the N-terminus was characterized by a stabilized helix structure that was maintained also upon aging. Presumably, the N-terminal region interacted with the folded HLH motif in a specific manner, whereas only unspecific, weak contacts occurred with the partly unfolded HLH domain and/or the immediate flanking residues 77-110.