Fmoc Solid-phase Peptide Synthesis Research Articles

Development of Clarstatin, a Novel Drug Lead for the Therapy of Autoimmune Uveitis

We describe the design, synthesis, and activity of a potent thiourea-bridged backbone cyclic peptidomimetic known as Clarstatin, comprising a 5-amino acid sequence (Q/D)1-(R/K)2-X3-X4-A5-(Gln/Asp)1-(Arg/Lys)2-AA3-AA4-Ala5-based on a motif called “shared epitope (SE)”, specifically present in specific alleles of the HLA-DRB1 gene. This SE binds to a particular site within the proline reach domain (P-domain) of the cell surface-calreticulin (CS-CRT). CS-CRT is a multifunctional endoplasmic reticulum (ER) calcium-binding protein that is located on the cell surface of T cells and triggers innate immune signaling, leading to the development of inflammatory autoimmune diseases. The development of Clarstatin was based on the parent peptide W-G-D1-K2-S3-G4-A5- derived from the active region of the SE. Following the design based on the cycloscan method, the synthesis of Clarstatin was performed by the Fmoc solid phase peptide synthesis (SPPS) method, purified by HPLC to 96% homogeneity, and its structure was confirmed by LC-MS. Clarstatin reduced calcium levels in Jurkat lymphocyte cultures, ameliorated uveitis in vivo in the experimental autoimmune uveitis (EAU) mice model, and was safe upon acute toxicity evaluation. These findings identify Clarstatin as a promising lead compound for future drug development as a novel class of therapeutic agents in the therapy of uveitis.

Leveraging Hydrazide as Protection for Carboxylic Acid: Suppression of Aspartimide Formation during Fmoc Solid-Phase Peptide Synthesis.

Despite numerous optimizations in peptide synthesis, the formation of aspartimide remains a significant side reaction that needs to be addressed. Herein, we introduce an approach that utilizes hydrazide as a carboxylic-acid-protecting group to reduce the formation of aspartimide. The aspartic acid hydrazide effectively suppressed the formation of aspartimide, even under microwave conditions, and was readily converted to native aspartic acid using CuSO4 in an aqueous medium.

Synthesis of Asp-based lactam cyclic peptides using an amide-bonded diaminodiacid to prevent aspartimide formation.

Asp-based lactam cyclic peptides are considered promising drug candidates. However, using Fmoc solid-phase peptide synthesis (Fmoc-SPPS) for these peptides also causes aspartimide formation, resulting in low yields or even failure to obtain the target peptides. Here, we developed a diaminodiacid containing an amide bond as a β-carboxyl-protecting group for Asp to avoid aspartimide formation. The practicality of this diaminodiacid has been illustrated by the synthesis of lactam cyclic peptide cyclo[Lys9,Asp13] KIIIA7-14 and 1Y.

A Novel Dual-labeled Peptide for Multimodal Imaging of EGFR with L858R Mutation.

The development of molecular imaging agents targeting epidermal growth factor receptor (EGFR) with L858R mutation may help with the selection of non-small cell lung carcinoma (NSCLCL) patients who may benefit from EFGR tyrosine kinase inhibitor (TKI) therapy. In this study, we developed 99mTc STHHYYP-GHEG-ECGK-tetramethylrhodamine (STHHYYP-ECGK-TAMRA) to target EGFR with L858R mutation in NSCLC tumors and verified its probability as a molecular imaging agent. Fmoc solid-phase peptide synthesis was used to synthesize STHHYYP-ECGKTAMRA. 99mTc labelled STHHYYP-ECGK-TAMRA was prepared. Gamma imaging, fluorescent imaging and biodistribution were performed in murine models bearing NCI-H1975 and NCI-H1650 tumors. The binding affinity value (Kd) of 99mTc STHHYYP-ECGK-TAMRA was estimated to be 130.6 ± 29.2 nM in NCI-H1975 cells. The gamma camera images showed a substantial uptake of 99mTc STHHYYP-ECGK-TAMRA in the NCI-H1975 tumor. The % injected dose/gram of the NCI-H1975 tumor tissue was 2.77 ± 0.70 and 3.48 ± 1.01 at 1 and 3 h, respectively. Specific binding of 99mTc STHHYYP-ECGK-TAMRA to L858R-mutated EGFRpositive NCI-H1975 cells and tumors was demonstrated in in vivo and in vitro studies. The results suggest that 99mTc STHHYYP-ECGK-TAMRA is a good candidate agent for dualmodality imaging targeting EGFR with L858R mutation.

Suppression of alpha-carbon racemization in peptide synthesis based on a thiol-labile amino protecting group

In conventional solid-phase peptide synthesis (SPPS), α-amino groups are protected with alkoxycarbonyl groups (e.g., 9-fluorenylmethoxycarbonyl [Fmoc]). However, during SPPS, inherent side reactions of the protected amino acids (e.g., α-C racemization and aspartimide formation) generate by-products that are hard to remove. Herein, we report a thiol-labile amino protecting group for SPPS, the 2,4-dinitro-6-phenyl-benzene sulfenyl (DNPBS) group, which is attached to the α-amino group via a S–N bond and can be quantitatively removed in minutes under nearly neutral conditions (1 M p-toluenethiol/pyridine). The use of DNPBS greatly suppresses the main side reactions observed during conventional SPPS. Although DNPBS SPPS is not as efficient as Fmoc SPPS, especially for synthesis of long peptides, DNPBS and Fmoc are orthogonal protecting groups; and thus DNPBS SPPS and Fmoc SPPS can be combined to synthesize peptides that are otherwise difficult to obtain.

Design of the peptide C-terminus protecting group and application for the peptide fragment coupling reaction

Herein, the design and synthesis of the C-terminus protecting groups of peptides and application for peptide fragment coupling reaction are described. We prepared ψ-Wang handle (1) and 5 analogs (2–6), and each peptide-handle was synthesized by Fmoc-solid phase peptide synthesis. Additionally, the fragment coupling reactions between the protected peptides and the peptide-handles were performed to afforded the corresponding peptides (20, 23, 25 and 27). This strategy is beneficial for the synthesis of protected peptides with long sequences.

Development of the efficient preparation method for thermoresponsive elastin-like peptides using liquid-phase synthesis combined with fragment condensation strategy.

Elastin-like peptides (ELPs) are synthetic peptides that mimic the characteristic hydrophobic amino acid repeat sequences of elastin and exhibit temperature-dependent reversible self-assembly properties. ELPs are expected to be used as temperature-responsive biomolecular materials across diverse industrial and research fields, and there is a requirement for a straightforward method to mass-produce them. Previously, we demonstrated that phenylalanine-containing ELP analogs, namely, (FPGVG)n , can undergo coacervation with short chains (n = 5). The Fmoc solid-phase peptide synthesis method is one strategy used to synthesize these short ELPs. However, owing to its low reaction efficiency, an efficient method for preparing ELPs is required. In this study, efficient preparation of ELPs was investigated using a liquid-phase synthesis method with a hydrophobic benzyl alcohol support (HBA-tag). Because HBA-tags are highly hydrophobic, they can be easily precipitated by the addition of poor solvents and recovered by filtration. This property allows the method to combine the advantages of the simplicity of solid-phase methods and the high reaction efficiency of liquid-phase methods. By utilizing liquid-phase fragment condensation with HBA-tags, short ELPs were successfully obtained in high yield and purity. Finally, the temperature-dependent response of the ELPs generated through fragment condensation was assessed using turbidity measurements, which revealed a reversible phase transition. Consequently, the ELPs exhibited a reversible phase transition, indicating successful synthesis of ELPs via fragment preparation with tags. These findings provide evidence of the potential for mass production of ELPs using this approach.

Dipropyleneglycol Dimethylether, New Green Solvent for Solid-Phase Peptide Synthesis: Further Challenges to Improve Sustainability in the Development of Therapeutic Peptides.

In recent years, peptides have gained more success as therapeutic compounds. Nowadays, the preferred method to obtain peptides is solid-phase peptide synthesis (SPPS), which does not respect the principles of green chemistry due to the large number of toxic reagents and solvents used. The aim of this work was to research and study an environmentally sustainable solvent able to replace dimethylformamide (DMF) in fluorenyl methoxycarbonyl (Fmoc) solid-phase peptide synthesis. Herein, we report the use of dipropyleneglycol dimethylether (DMM), a well-known green solvent with low human toxicity following oral, inhalant, and dermal exposure and that is easily biodegradable. Some tests were needed to evaluate its applicability to all the steps of SPPS, such as amino acid solubility, resin swelling, deprotection kinetics, and coupling tests. Once the best green protocol was established, it was applied to the synthesis of different length peptides to study some of the fundamental parameters of green chemistry, such as PMI (process mass intensity) and the recycling of solvent. It was revealed that DMM is a valuable alternative to DMF in all steps of solid-phase peptide synthesis.

Amphiphilic Cell-Penetrating Peptides Containing Arginine and Hydrophobic Residues as Protein Delivery Agents

The entry of proteins through the cell membrane is challenging, thus limiting their use as potential therapeutics. Seven cell-penetrating peptides, designed in our laboratory, were evaluated for the delivery of proteins. Fmoc solid-phase peptide synthesis was utilized for the synthesis of seven cyclic or hybrid cyclic-linear amphiphilic peptides composed of hydrophobic (tryptophan (W) or 3,3-diphenylalanine (Dip) and positively-charged arginine (R) residues, such as [WR]4, [WR]9, [WWRR]4, [WWRR]5, [(RW)5K](RW)5, [R5K]W7, and [DipR]5. Confocal microscopy was used to screen the peptides as a protein delivery system of model cargo proteins, green and red fluorescein proteins (GFP and RFP). Based on the confocal microscopy results, [WR]9 and [DipR]5 were found to be more efficient among all the peptides and were selected for further studies. [WR]9 (1-10 µM) + protein (GFP and RFP) physical mixture did not show high cytotoxicity (>90% viability) in triple-negative breast cancer cells (MDA-MB-231) after 24 h, while [DipR]5 (1-10 µM) physical mixture with GFP exhibited more than 81% cell viability. Confocal microscopy images revealed internalization of GFP and RFP in MDA-MB-231 cells using [WR]9 (2-10 μM) and [DipR]5 (1-10 µM). Fluorescence-activated cell sorting (FACS) analysis indicated that the cellular uptake of GFP was concentration-dependent in the presence of [WR]9 in MDA-MB-231 cells after 3 h of incubation at 37 °C. The concentration-dependent uptake of GFP and RFP was also observed in the presence of [DipR5] in SK-OV-3 and MDA-MB-231 cells after 3 h of incubation at 37 °C. FACS analysis indicated that the cellular uptake of GFP in the presence of [WR]9 was partially decreased by methyl-β-cyclodextrin and nystatin as endocytosis inhibitors after 3 h of incubation in MDA-MB-231 cells, whereas nystatin and chlorpromazine as endocytosis inhibitors slightly reduced the uptake of GFP in the presence of [DipR]5 after 3 h of incubation in MDA-MB-231. [WR]9 was able to deliver therapeutically relevant proteins (Histone H2A) at different concentrations. These results provide insight into the use of amphiphilic cyclic peptides in the delivery of protein-related therapeutics.

Unlocking the Potential of the Antimicrobial Peptide Gomesin: From Discovery and Structure–Activity Relationships to Therapeutic Applications

Gomesin is a cationic antimicrobial peptide which is isolated from the haemocytes of the Brazilian tarantula Acanthoscurria gomesiana and can be produced chemically by Fmoc solid-phase peptide synthesis. Gomesin exhibits a range of biological activities, as demonstrated by its toxicity against therapeutically relevant pathogens such as Gram-positive or Gram-negative bacteria, fungi, cancer cells, and parasites. In recent years, a cyclic version of gomesin has been used for drug design and development as it is more stable than native gomesin in human serum and can penetrate and enter cancer cells. It can therefore interact with intracellular targets and has the potential to be developed as a drug lead for to treat cancer, infectious diseases, and other human diseases. This review provides a perspective on the discovery, structure-activity relationships, mechanism of action, biological activity, and potential clinical applications of gomesin.

A novel fluorescence sensor for uranyl ion detection based on a dansyl-modified peptide

It is of great significance to sensitively and selectively detect uranyl ion (UO22+) in environmental and biological samples due to the high risks of UO22+ to human health. However, such suitable sensors are still scarce. A novel fluorescence sensor based on a dansyl-modified peptide, Dansyl-Glu-Glu-Pro-Glu-Trp-COOH (D-P5), was efficiently synthesized by Fmoc solid phase peptide synthesis. As the first linear peptide-based fluorescence sensor for UO22+, D-P5 exhibited high selectivity and sensitivity to UO22+ over 27 metal ions (UO22+, Cr3+, Cu2+, Ba2+, Hg2+, Pb2+, Co2+, Ag+, Fe3+, Ca2+, K+, Mg2+, Mn2+, Na+, Ni2+, Cd2+, Zn2+, Al3+, Dy3+, Er3+, Gd2+, Ho3+, La3+, Lu3+, Pr3+, Sm3+, Tm3+) by a turn-off fluorescence response in 10 mM HEPES buffer (pH 6.3). The effects of anions such as S2−, NO3–, SO42− CO32–, HCOO–, antioxidant ascorbic acid and 4-nitrophenyl acetate on the selectivity for UO22+ detection were also studies. D-P5 sensor could be used for detecting UO22+ in a good linear relationship with concentration in the range of 0–8.0 μM with a low limit of detection of 83.2 nM. Furthermore, the interaction of the sensor with UO22+ was characterized by ESI-MS, IR, XPS and ITC measurements. The 1:1 binding stoichiometry between the sensor and UO22+ was measured by the job’s plot and further verified by ESI-MS. The binding constant of the sensor with UO22+ was calculated to be 9.8 × 104 M−1 by modified Benesi-Hildebrand equation. ITC results showed that theΔHθ andΔSθ for the interaction of D-P5 with UO22+ were −(7.167 ± 1.25) kJ·mol−1 and 66.5 J·mol−1·K−1, respectively. Time-resolved fluorescence spectroscopy indicated that the mechanism of fluorescence quenching of D-P5 by UO22+ ion was static quenching process. In addition, this sensor displayed a good practicality for UO22+ detection in lake water sample without tedious sample pretreatment.

Preparation and Application of an Inexpensive α-Formylglycine Building Block Compatible with Fmoc Solid-Phase Peptide Synthesis.

α-Formylglycine (fGly) is a rare residue located in the active site of sulfatases and serves as a precursor to pharmaceutically relevant motifs. The installation of fGly motifs into peptides is currently challenging due to degradation under the acidic and nucleophile-rich conditions accompanying resin cleavage during solid-phase peptide synthesis. We report the synthesis of acid- and nucleophile-tolerant α-formylglycine building blocks from vitamin C and use them to prepare callyaerin A, a macrocyclic peptide containing an fGly-derived motif.

Fmoc-SPPS-compatible p-methoxyphenacyl-modified glutamic for the synthesis of photocaged peptides

The development of photocaged glutamic acid (Glu) contributes to the time-resolved control of peptide and protein activities. Here, we efficiently synthesized the Glu with the side chain modified by photosensitive p-methoxyphenacyl group. This photocaged Glu can be introduced into peptide through standard 9‑fluorenylmethoxycarbonyl (Fmoc) solid phase peptide synthesis (SPPS), and can be re-exposed the Glu side-chain carboxy group by removing the photosensitive group under light irradiation. We prepared caged Endothelin-1 (ET-1) analogues with p-methoxyphenacyl-modified Glu and successfully achieved decaging.

Selective thiazoline peptide cyclisation compatible with mRNA display and efficient synthesis.

Peptide display technologies are a powerful method for discovery of new bioactive sequences, but linear sequences are often very unstable in a biological setting. Macrocyclisation of such peptides is beneficial for target affinity, selectivity, stability, and cell permeability. However, macrocyclisation of a linear hit is unreliable and requires extensive structural knowledge. Genetically encoding macrocyclisation during the discovery process is a better approach, and so there is a need for diverse cyclisation options that can be deployed in the context of peptide display techniques such as mRNA display. In this work we show that meta-cyanopyridylalanine (mCNP) can be ribosomally incorporated into peptides, forming a macrocycle in a spontaneous and selective reaction with an N-terminal cysteine generated from bypassing the initiation codon in translation. This reactive amino acid can also be easily incorporated into peptides during standard Fmoc solid phase peptide synthesis, which can otherwise be a bottleneck in transferring from peptide discovery to peptide testing and application. We demonstrate the potential of this new method by discovery of macrocyclic peptides targeting influenza haemagglutinin, and molecular dynamics simulation indicates the mCNP cross-link stabilises a beta sheet structure in a representative of the most abundant cluster of active hits. Cyclisation by mCNP is also shown to be compatible with thioether macrocyclisation at a second cysteine to form bicycles of different architectures, provided that cysteine placement reinforces selectivity, with this bicyclisation happening spontaneously and in a controlled manner during peptide translation. Our new approach generates macrocycles with a more rigid cross-link and with better control of regiochemistry when additional cysteines are present, opening these up for further exploitation in chemical modification of in vitro translated peptides, and so is a valuable addition to the peptide discovery toolbox.

Total synthesis, structure elucidation and expanded bioactivity of icosalide A: effect of lipophilicity and ester to amide substitution on its bioactivity.

The first total synthesis of icosalide A, an antibacterial depsipeptide that is unique in that it contains two lipophilic beta-hydroxy acids, has been achieved by following Fmoc solid-phase peptide synthesis in combination with solution-phase synthesis. The ambiguity in the absolute stereochemistry of icosalide A has been resolved by synthesizing the reported structures and other relevant diastereomers of icosalides and comparing their NMR data. NMR-based structure elucidation of icosalide A revealed a well-folded structure with cross-strand hydrogen bonds similar to the anti-parallel beta-sheet conformation in peptides and displayed a synergistic juxtaposition of the aliphatic sidechains. 12 analogues of icosalide A were synthesized by varying the constituent lipophilic beta-hydroxy acid residues, and their biological activities against Bacillus thuringiensis and Paenibacillus dendritiformis were explored. Most of these icosalide analogues showed an MIC of 12.5 μg mL-1 against both bacteria. Swarming inhibition by icosalides was least in B. thuringiensis (8.3%) compared to that in P. dendritiformis (33%). Furthermore, this is the first report of icosalides showing assured inhibitory action (MIC between 2 and 10 μg mL-1) against the active stage of Mycobacterium tuberculosis and cancer cell lines such as HeLa and ThP1. This study could help optimize icosalides for anti-TB, antibacterial, and anti-cancer activities.

Modular Synthesis of Unnatural Peptides via Rh(III)-Catalyzed Diastereoselective Three-Component Carboamidation Reaction.

Herein we report a modular peptide ligation methodology that couples dioxazolones, arylboronic acids, and acrylamides to construct amide bonds in a diastereoselective manner under mild conditions, facilitated by Rh(III) catalysis. By converting the C-terminus of one peptide into a dioxazolone and the N-terminus of a second peptide into an acrylamide, the two pieces can be bridged by an arylboronic acid to construct unnatural phenylalanine, tyrosine, and tryptophan residues at the junction point with diastereoselectivity for their corresponding d-stereocenters. The reaction exhibits excellent functional group tolerance with a large substrate scope and is compatible with a wide array of protected amino acid residues that are utilized in Fmoc solid phase peptide synthesis. The methodology is applied to the synthesis of six diastereomeric proteasome inhibitor analogs, as well as the ligation of two 10-mer oligopeptides to construct a 21-mer polypeptide with an unnatural phenylalanine residue at the center.

Chemical Synthesis of Proteins with Base-Labile Posttranslational Modifications Enabled by a Boc-SPPS Based General Strategy Towards Peptide C-Terminal Salicylaldehyde Esters.

Chemical synthesis of proteins bearing base-labile post-translational modifications (PTMs) is a challenging task. For instance, O-acetylation and S-palmitoylation PTMs cannot survive Fmoc removal conditions during Fmoc-solid phase peptide synthesis (SPPS). In this work, we developed a new Boc-SPPS-based strategy for the synthesis of peptide C-terminal salicylaldehyde (SAL) esters, which are the key reaction partner in Ser/Thr ligation and Cys/Pen ligation. The strategy utilized the semicarbazone-modified aminomethyl (AM) resin, which could support the Boc-SPPS and release the peptide SAL ester upon treatment with TFA/H2 O and pyruvic acid. The non-oxidative aldehyde regeneration was fully compatible with all the canonical amino acids. Armed with this strategy, we finished the syntheses of the O-acetylated protein histone H3(S10ac, T22ac) and the hydrophobic S-palmitoylated peptide derived from caveolin-1.

Solid-Phase-Based Synthesis of Lactazole-Like Thiopeptides.

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

Cyclic N-locked indolicidin analogues with antimicrobial activity: Effect of ring size and fatty acid acylation

Novel antimicrobial drugs are in high demand due to the increasing emergence of multi-drug resistant bacteria. In recent years, much attention has been given to natural occurring antimicrobial peptides (AMPs) such as indolicidin (ILPWKWPWWPWRR-NH2). In this study, 19 cyclic N-locked indolicidin analogues (3–21) were synthesized successfully by standard 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS) combined with a convenient on-resin N-terminus to lysine side-chain cyclization via intramolecular halide substitution using bromoacetic acid. The effect of the ring size on antimicrobial activity and cytotoxicity was initially investigated and subsequently, the effect of fatty acid acylation was explored. We observed that a peptide macrocycle consisting of seven residues was optimal for Gram-positive antibacterial activity. Circular dichroism (CD) spectra of peptide macrocycles (3–13) showed that certain positions of ring-closure induced a conformation seen under sodium dodecyl sulfate (SDS) conditions where Trp side-chains can display exciton coupling. The far-UV CD was consistent with a backbone turn, a conformation with no evidence of helical structure. SAXS data on peptides mixed with lipid vesicles indicate a similar membrane interaction, when comparing the cyclic and linear indolicidin. The most potent analogue identified was 7, which showed antibacterial activity against Gram-positive bacteria (MIC against Staphylococcus aureus ATCC29213 ​= ​12.5 ​μg/mL and Staphylococcus epidermidis HJ56 ​= ​6.3 ​μg/mL). In addition, viable HaCaT cells after 200 ​μg/mL treatment with 7 was 84.3%. The results show that N-locked cyclization of linear antimicrobial peptides provides a method to reduce cytoxicity; although it may affect the antimicrobial activity.

Fluorescence spectral detection of copper(II), mercury(II) and zinc(II) based on a multifunctional peptide-based sensor

A fluorescence peptide-based sensor (Dansyl-His-Thr-Glu-His-Trp-NH2, D-P5) was synthesized by Fmoc solid phase peptide synthesis, using dansyl group as the fluorophore. The sensor exhibited different fluorescence responses and sensitive detections for copper(II), mercury(II) and zinc(II) ions as a multifunctional sensor. The detailed investigation suggested that the limits of detection were 37.6 nmol⋅L−1, 37.8 nmol⋅L−1 and 59.4 nmol⋅L−1 for copper(II), mercury(II) and zinc(II) ions, respectively, and the highly sensitive sensor was not influenced by other species. The sensor exhibited a strong binding ability to copper(II), mercury(II) and zinc(II) ions with the binding constants of 3.23 × 105 L⋅mol−1, 6.37 × 105 L⋅mol−1 and 6.0 × 104 L⋅mol−1, respectively. The binding stoichiometries of the sensor to copper(II), mercury(II) and zinc(II) ions measured by Job's plot were all 1:1. Furthermore, The sensor has been successfully used for the detections of copper(II), mercury(II) and zinc(II) in actual water samples.