Fmoc Strategy Research Articles

Total synthesis, purification, and characterization of human [Phe(p-CH2SO 3Na)52, Nle32,53,56, Nal55]-CCK20-58, [Tyr52, Nle32,53,56, Nal55]-CCK-58, and [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK-58.

The synthesis of [Phe(p-CH2SO3Na)52, Nle32,53,56 Nal55]-CCK20-58, [Tyr52, Nle32,53,56, Nal55]-CCK-58 and of [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK-58 using the (9-fluorenylmethyloxy)-carbonyl (Fmoc) strategy on a 2,4-DMBHA resin is described. The crude peptide preparations were extremely complex when analyzed by RP-HPLC, capillary zone electrophoresis (CZE), and ion-exchange chromatography (IE-FPLC). We found that the most effective strategy for purification included cation-exchange chromatography followed by a RP-HPLC desalting step. The highly purified peptides (purity greater than 90%) were characterized by RP-HPLC, size exclusion HPLC (SEC), IE-FPLC, CZE, mass spectrometry, amino acid analysis, and Edman sequence analysis (for [Tyr52, Nle32,53,56, Nal55]-CCK-58). The results demonstrate the applicability of the 2,4-DMBHA resin for Fmoc solid-phase synthesis of long peptides amides (58 residues in length in this case) as well as the efficacy of an FPLC/RP-HPLC approach for the purification of very long, heterogeneous crude peptides, allowing a true assessment of the biological properties of these analogs to be carried out. [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK20-58 was less than 1% as potent as CCK-8 while [Tyr52, Nle32,53,56, Nal55]-CCK-58 and [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK-58 were inactive at the doses tested (< 0.01%).

Cyclic disulfide analogues of the complement component C3a Synthesis and conformational investigations

The flexible C-terminal region of the anaphylatoxic peptide C3a was reported to contain the receptor binding site. To elucidate the receptor binding conformation of the C-terminus, as well as to examine a synthetic approach to potential C3a-antagonists, 26 cyclic disulfide bridged C3a analogues were synthesized. Solid phase peptide synthesis was performed on different polymeric supports by individual peptide synthesis, with Fmoc strategy, and simultaneous multiple peptide synthesis, using Boc and Fmoc strategies. Both strategies gave open-chain peptides in comparable yields. Syntheses using the Boc strategy employed the HF-labile 4(methoxy)benzyl group (Mob) for beta-thiol protection of cysteine; in contrast, the TFA-stable protecting groups, acetamidomethyl (Acm) and trityl (Trt), were chosen for syntheses employing Fmoc strategy. Ring closure reactions by iodine oxidation were carried out starting from protected (Acm/Acm, Trt/Acm) or unprotected dithiols. The resulting cyclic C3a analogues were characterized by HPLC, amino acid analysis, and FAB-MS. Conformational investigations using CD spectroscopy and theoretical structural investigations by means of molecular dynamics calculations revealed that slight variations in sequence result in pronounced conformational consequences. The potential of cyclic C3a analogues to activate or to desensitize guinea pig platelets, a standard test system for biological activities of anaphylatoxic peptides like C3a, revealed relatively low activities for cyclic peptides (< 0.1% C3a activity). N-terminal acylation with cationic, arginine-rich sequences like YRRGR- led to amplified biological effects. Three of the synthesized peptides, namely CAALCLAR (P1), YRRGRCGGLCLAR (P5) and YRRGRAhxCGGLCLAR (P8), point in the direction of C3a antagonists.

One-pot Deprotection and Coupling of Peptides by the Fmoc Strategy

Abstract 9-Fluorenylmethyloxycarbonyl (Fmoc) group can be quickly removed with tetrabutylammonium fluoride hydrate (TBAF·xH2O) in the presence of a large excess of phenylmethanethiol or 1-octanethiol. Subsequent oxidation of the thiol with bis(1-methyl-1H-tetrazol-5-yl) disulfide simultaneously inactivated the TBAF and enabled one-pot peptide bond elongation by the Fmoc strategy.

Application of tetrabutylammonium salt of N-(9-fluorenylmethoxycarbonyl)cysteic acid for solid phase peptide synthesis: Preparation of endothelin antagonistic cyclic pentapeptides

Abstract According to the Fmoc strategy, highly potent endothelin antagonistic cyclic pentapeptides possessing cysteic acid residue(s) were prepared in solid phase peptide synthesis. Use of a tetrabutylammonium salt overcame low solubility of N-(9-fluorenylmethoxycarbonyl)cysteic acid in a conventional solvent such as DMF.

2-phenyl isopropyl esters as car☐yl terminus protecting groups in the fast synthesis of peptide fragments

The esters of Fmoc aminoacids derived from 2-phenylisopropanol have been prepared by using 2-phenylisopropyltrichloroacetimidate 1. They prevent diketopiperazine formation during amino deprotection of dipeptides and can be cleaved in the presence of Boc and O-Bu t. They are thus well suited for the protection of C-terminus when a Fmoc strategy is used to build up peptide fragments.

Synthesis of (2S,3S)-2-amino-3-methylpent-4-ynoic acid, a precursor amino acid for the preparation of tritium- or deuterium-labelled peptide in the isoleucine residue

The synthesis of (2S,3S)-2-amino-3-methylpent-4-ynoic acid (Amp)1, its incorporation into a peptide chain, and the preparation of a deuterium-labelled peptide in the isoleucine residue are reported. Amino acid 1 was synthesized in three steps from 3-chlorobut-1-yne. The optical purity of amino acid 1 was determined by HPLC with a chiral stationary-phase column and was found to be more than 99%(ee). Tyr-Amp-Leu was synthesized by solid-phase synthesis based on Fmoc strategy. The tripeptide was deuteriated catalytically to yield Tyr-[2H]lle-Leu. The distribution of deuterium was investigated by fast-atom-bombardment mass spectrometry (FAB-MS) and 13C NMR spectroscopy, which confirmed that the deuterium was located entirely at the isoleucine residue.

Peptide synthesis: synthesis of the cysteine-containing peptides of biological and pharmaceutical interest, α-h-ANF and h-big endothelin

The biologically important vasoactive peptides, α-h-atrial natriuretic factor (ANF) and h-Big endothelin (Big ET) have been synthesised using the solid-phase Fmoc strategy with reference to the choice of protecting group for cysteine residues. Selective disulfide bond formation was seen to be problematical in the synthesis of Big ET but use of the acetamidomethyl (Acm) group, removal with the silver ions and oxidation using the mixed disulfide method proved successful.

S-2,4,6-trimethoxybenzyl (Tmob): a novel cysteine protecting group for the N.alpha.-(9-fluorenylmethoxycarbonyl) (Fmoc) strategy of peptide synthesis

The S-2,4,6-trimethoxybenzyl (Tmob) group can be introduced onto sulfhydryl functions form the corresponding alcohol, with acid catalysis, and is in turn removed rapidly by treatment with 30% trifluoroacetic acid-di-chloromethane in the presence of phenol, thioanisole, and water (5% each) or 6% trifluoroacetic acid-di-chloromethane in the presence of triethylsilane or triisopropylsilane (0.5%). The appropriate cysteine derivative was prepared and applied with other N α -Fmoc protected amino acids to the solid-phase syntheses of several model peptides

Solid-Phase Synthesis of Steroidogenesis-Activator Polypeptide under Continuous Flow Conditions

Abstract 4-(Hydroxymethyl)phenoxymethyl copoly(styrene–1% divinylbenzene) resin beads loosely packed in a glass column were satisfactorily employed as a solid support for synthesis of a triacontapeptide, steroidogenesis-activator polypeptide, under the Fmoc strategy. Peptide sythesis was carried out in a continuous DMF flow without generation of any considerable back pressure. Change of volume of the resin bed in the column was monitored in the course of peptide elongation. After cleavage from the resin, the product was purified on HPLC to give the desired triacontapeptide in an overall yield of 39% based on the C-terminal amino acid linked to the resin.

Solid-phase synthesis and crystallization of monellin, an intensely sweet protein.

Monellin, a sweet protein, consists of two noncovalently associated polypeptide chains, the A chain of 44 amino acid residues and the B chain of 50 residues. Two different primary structures have been reported for each of the A and B chains. The A and B chains corresponding to one of the reported monellin structures were synthesized by the stepwise solid-phase method using the Fmoc strategy in overall yields of 14.1% and 5.6%, respectively. The characterization of the synthetic peptides by HPLC, FAB-MS, amino acid analysis and sequencing fully supported the expected structures. The individual synthetic A and B chains were not sweet. Combination of the two chains, and subsequent HPLC purification gave monellin in a yield of 53.9 %. The synthetic monellin had a distinct, lingering sweet taste (4000 times sweeter than sucrose) and was crystallized by a vapor diffusion method. The synthetic product was identical to natural monellin by HPLC, but not by tryptic mapping. These results indicate that the reported structure for monellin differs slightly from that of natural monellin.

Use of the Npys thiol protection in solid phase peptide synthesis. Application to direct peptide-protein conjugation through cysteine residues.

The protection of the thiol function of cysteine with the 3-nitro-2-pyridylsulfenyl (Npys) group has been successfully applied in the solid phase synthesis of nine peptides. A reexamination of the chemical stability of the protecting group has shown that, while Npys is essentially suitable for standard Boc/benzyl synthesis conditions, it is inadequate for the Fmoc strategy. Its proven stability to "high" HF acidolysis can not be extended to "low-high" conditions without significant thiol deprotection. On the other hand, the Npys group is quite compatible with standard photolytical cleavage conditions. The stability of Npys to HF and its thiol-activating character allow its application in peptide-carrier protein conjugation reactions by specific coupling through cysteine residues in the peptide.

Highly potent and small neuropeptide Y agonist obtained by linking NPY 1–4 via spacer to α-helical NPY 25–36

Analogues of neuropeptide Y (NPY) containing small N- and C-terminal segments linked via flexible spacer arms were found to exhibit receptor binding affinity constants almost as high as NPY as well as post- and presynaptic NPY-agonistic activities. One of the most active analogues contains N-terminal NPY segment 1–4 linked via ε-aminocaproic acid (Aca) to the C-terminal partially α-helical peptide amide segment 25–36. NPY 1–4-Aca-25–36 is the first highly potent NPY agonist, which is of considerably reduced size in comparison to the native hormone. The analogues are accessible by solid-phase synthesis using Fmoc strategy.

Synthesis of A7,B7-dicarbainsulin, an analogue with a noncleavable bond between A- and B-chain. II. Synthesis of the A-chain segments.

As part of the total synthesis of [A7,B7-L,L-2,7-diaminosuberoyl]-des-(B26-B30)-insulin B25-amide, an insulin analogue containing a non-cleavable bond between A- and B-chain, the chemical synthesis of the A-chain segments is described. The N-terminal sequence A(1-6), Boc-Gly-Ile-Val-Glu(OBut)-Gln-Cys(SBut)-NH-NH2, was synthesized in solution. The middle segment A(8-16), Ddz-Thr(But)-Ser(But)-Ile-Cys(SBut)-Ser(But)-Leu-Tyr- (But)-Gln-Leu-NH-NH2, was obtained by solid phase synthesis according to the Fmoc strategy. The C-terminal segment A(17-21), Bpoc-Glu(OBut)-Asn-Tyr-Cys(Acm)-Asn-OBut, was prepared in solution.

ChemInform Abstract: Solid Phase Synthesis of Peptide Aminoalkylamides Development and Application of New Linking Agents.

Abstract A new method for the preparation of peptide aminoalkylamides by solid phase synthesis using a modified Fmoc strategy is described. Key step is the synthesis of compound 6 as acid labile linker.

Solid-phase synthesis of protected peptide fragments using a trialkoxy-diphenyl-methylester resin.

A trialkoxy-diphenyl-methylester and amide linkage for solid-phase peptide synthesis with Fmoc strategy is described. Protected peptide esters can be smoothly cleaved with weak acid, resulting in fragments with intact side-chain protection. Mild acidic cleavage of the corresponding peptide amide resins yields peptide amides.

Preparation and application of new acid labile anchor groups for the synthesis of peptide amides by FMOC solid phase synthesis

The synthesis and application of new linkage agents for the preparation of peptide amides using a modified Fmoc strategy is described.

Solid phase synthesis of peptide aminoalkylamides development and application of new linking agents

A new method for the preparation of peptide aminoalkylamides by solid phase synthesis using a modified Fmoc strategy is described. Key step is the synthesis of compound 6 as acid labile linker.

Synthesis and biological activities of photoaffinity labeling analogues of substance P.

Substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-MetNH2, SP) is an undecapeptide with important properties as a neurotransmitter and with other functions. No specific antagonists and no long-acting analogues of this peptide hormone are known to date. In order to reach these goals, analogues of SP have been prepared which contain potential affinity, as well as photoaffinity labeling functions, suitable for irreversible attachment to SP receptors. We report here the synthesis of SP analogues which have the Phe residues in positions 7 or 8 replaced with (4'-NO2)Phe, (4'-NH2)Phe, (4'-N2+)Phe, and (4'-N3)Phe. Some of these peptides are used for photoaffinity labeling studies using various bioassays. The synthesis of the (NO2)Phe-containing peptide was carried out on solid phase using Nle instead of Met and the Boc strategy up to residue 4; the remaining amino acids were added using an Fmoc strategy. The protected undecapetide was cleaved by ammonolysis, purified by chromatography on silica gel with chloroform/methanol and deprotected afterwards. The amino, diazonium, and azido peptides were obtained in this sequence by chemical modification of the nitro peptides. On guinea pig ileum the modified peptides in position 8 had close to maximal activity, whereas modifications in position 7 produced some reduced activity, especially the nitro modification. No diazonium peptide produced any irreversible effects on guinea pig ileum. Photoinactivation studies were carried out on strips of guniea pig trachea, but no irreversible effects have been observed, neither permanent stimulation nor permanent inactivation. The biological activities and effects are discussed in view of the molecular properties of the synthesized analogues.