A safety catch linker for Fmoc-based assembly of constrained cyclic peptides.

A new safety-catch linker for Fmoc solid-phase peptide synthesis of cyclic peptides is reported. The linear precursors were assembled on a tert-butyl protected catechol derivative using optimized conditions for Fmoc-removal. After activation of the linker using TFA, neutralization of the N-terminal amine induced cyclization with concomitant cleavage from the resin yielding the cyclic peptides in DMF solution. Several constrained cyclic peptides were synthesized in excellent yields and purities.     

An efficient, convenient solid-phase synthesis of amino acid-modified peptide nucleic acid monomers and oligomers

An efficient and highly versatile method for the synthesis of amino acid-modified peptide nucleic acid (PNA) monomers is described. By using solid-phase Fmoc techniques, such monomers can be assembled readily in a stepwise manner and obtained in high yield with minimal purification. Protected neutral hydrophilic, acidic, and basic amino acids were coupled to 2-chlorotrityl chloride resin. Following Fmoc removal, innovative conditions for the key step, reductive alkylation with N-Fmoc-aminoacetaldehyde, were developed to circumvent problems encountered with previously reported methods. Activation and coupling of pyrimidine and purine nucleobases to the resulting secondary amines afforded amino acid-modified PNA monomers. The mild reaction conditions utilized were compatible with sensitive and labile functional groups, such as tert-butyl ethers and tert-butyl esters. PNA monomers were obtained in 36-42% overall yield and very high purity, after cleavage and purification. Using standard solid-phase Fmoc chemistry, two of these monomers were incorporated with high coupling efficiency into a variety of modified PNA oligomers, including four tetradecamers designed to target bcl-2 mRNA. Such modified oligomers have the potential to enhance water solubility and cell portability, while maintaining hybridization affinity and promoting favorable biodistribution properties.     

An efficient, asymmetric solid-phase synthesis of benzothiadiazine-substituted tetramic acids: potent inhibitors of the hepatitis C virus RNA-dependent RNA polymerase

An efficient, asymmetric solid-phase synthesis of benzothiadiazine-substituted tetramic acids is reported. Starting from commercially available chiral Fmoc-protected alpha-amino acids loaded onto Wang resin, Fmoc removal, reductive amination followed by amide bond formation, and base-catalyzed cyclization with simultaneous cleavage from the resin provided the desired products. Compounds described are potent inhibitors of the hepatitis C virus RNA-dependent RNA polymerase.     

Synthesis of hydroxydiamines and triamines via reductive cleavage of N–N bond in substituted pyrazolidines

Aliphatic polyamines, being a versatile class of organic compounds, are widely used in many fields of medicine and organic chemistry. However, the general approach to the synthesis of chiral aliphatic polyamines has been still undeveloped. Here, we describe a new method for the synthesis of chiral trifunctional amino compounds, namely hydroxydiamines and triamines. The initial compounds, namely substituted hydroxy- or aminopyrazolidines and pyrazolines, are readily available using convenient stereoselective methods developed earlier by us. The proposed method allows synthesizing of chiral diaminoalcohols and triamines, which are the analogs of a well-known anti-TB drug, namely ethambutol, and cannot be obtained alternatively. The key step of the synthesis is N-N bond cleavage in substituted hydroxy- or aminopyrazolidines and pyrazolines with borane-tetrahydrofuran complex; other known methods for N-N bond cleavage turned out to be ineffective. The main advantage of the proposed method is the retention of a certain configuration of stereocenters in the course of the reaction. Six new chiral diasteomerically pure substituted hydroxydiamines and triamines and the enantiomerically pure triamine with four chiral centers were synthesized and characterized using NMR, IR and mass spectroscopy, as well as elemental analysis.     

Synthesis of a new disulfide Fmoc monomer for creating biologically susceptible linkages in peptide nucleic acid oligomers

Peptide nucleic acids (PNA) are one of many synthetic mimics of DNA and RNA that have found applications as biological probes, as nano-scaffold components, and in diagnostics. In an effort to use PNA as constructs for cellular delivery we investigated the possibility of installing a biologically susceptible disulfide bond in the backbone of a PNA oligomer. Here we report the synthesis of a new abasic Fmoc monomer containing a disulfide bond that can be incorporated into a PNA oligomer (DS-PNA) using standard solid phase peptide synthesis. The disulfide bond survives cleavage from the resin and DS-PNA forms duplexes with complementary PNA oligomers. Initial studies aimed at determining if the disulfide bond is cleavable to reducing agents while in a duplex are explored using UV thermal analysis and HPLC.     

Side-chain anchoring strategy for solid-phase synthesis of peptide acids with C-terminal cysteine

Many naturally occurring peptide acids, e.g., somatostatins, conotoxins, and defensins, contain a cysteine residue at the C-terminus. Furthermore, installation of C-terminal cysteine onto epitopic peptide sequences as a preliminary to conjugating such structures to carrier proteins is a valuable tactic for antibody preparation. Anchoring of N(alpha)-Fmoc, S-protected C-terminal cysteine as an ester onto the support for solid-phase peptide synthesis is known to sometimes occur in low yields, has attendant risks of racemization, and may also result in conversion to a C-terminal 3-(1-piperidinyl)alanine residue as the peptide chain grows by Fmoc chemistry. These problems are documented for several current strategies, but can be circumvented by the title anchoring strategy, which features the following: (a). conversion of the eventual C-terminal cysteine residue, with Fmoc for N(alpha)-amino protection and tert-butyl for C(alpha)-carboxyl protection, to a corresponding S-xanthenyl ((2)XAL(4)) preformed handle derivative; and (b). attachment of the resultant preformed handle to amino-containing supports. This approach uses key intermediates that are similar to previously reported Fmoc-XAL handles, and builds on earlier experience with Xan and related protection for cysteine. Implementation of this strategy is documented here with syntheses of three small model peptides, as well as the tetradecapeptide somatostatin. Anchoring occurs without racemization, and the absence of 3-(1-piperidinyl)alanine formation is inferred by retention of chains on the support throughout the cycles of Fmoc chemistry. Fully deprotected peptides, including free sulfhydryl peptides, are released from the support in excellent yield by using cocktails containing a high concentration (i.e., 80-90%) of TFA plus appropriate thiols or silanes as scavengers. High-yield release of partially protected peptides is achieved by treatment with cocktails containing a low concentration (i.e., 1-5%) of TFA. In peptides with two cysteine residues, the corresponding intramolecular disulfide-bridged peptide is obtained by either (a). oxidation, in solution, of the dithiol product released by acid; (b). simultaneous acidolytic cleavage and disulfide formation, achieved by addition of the mild oxidant DMSO to the cleavage cocktail; or (c). concomitant cleavage/cooxidation (involving a downstream S-Xan protected cysteine), using reagents such as iodine or thallium tris(trifluoroacetate) in acetic acid.     

Synthesis of glyoxylyl peptides using an Fmoc-protected alpha,alpha'-diaminoacetic acid derivative.

The synthesis of glyoxylyl peptides by coupling the masked glyoxylic acid derivative (FmocNH)(2)CHCO(2)H, 1, to a peptidyl resin assembled using Fmoc/tert-butyl chemistry has been described recently. Deprotection and cleavage of the peptide from the solid support using TFA was followed by unmasking of the glyoxylyl group in solution in the presence of DBU. [] The glyoxylyl peptide was thus generated using non-oxidizing conditions by comparison with the method based on the periodic oxidation of a seryl-precursor. However, base treatment of the (FmocNH)(2)CHCO(2)-peptide led to the formation of a byproduct besides the desired glyoxylyl peptide. This paper describes an optimized procedure for unmasking the Fmoc-protected alpha,alpha'-diaminoacetic acid moiety in solution which suppressed byproduct formation. Also presented is a series of experiments that permitted a structure and a mechanism of formation for the byproduct to be suggested.     

Synthesis of cyclic peptides and peptide libraries on a new disulfide linker.

A new cysteine-based disulfide linker for Fmoc solid phase peptide synthesis was developed (Fmoc-Cys(3-mercapto-3-methylbutanoic acid)OPp) that allows the on-resin assembly and side chain deprotection of cyclic peptides. Model peptides and a cyclic peptide library of the structure [a-a-x-x-a-a-c] composed of D-amino acids were assembled and the synthesis and cleavage conditions studied. The best cyclization results were obtained with PyBOP/HOAt/diisopropylethyl amine. Racemization rates of the cysteine in the analyzed model sequences were between 5.2 and 12.3%. Cleavage of the disulfide bond was best carried out with DTT in 50% 2-propanol/100 mM ammonium bicarbonate. The cleaved peptides can be used directly in biological assays.     

Application of gel-phase 19F NMR spectroscopy for optimization of solid-phase synthesis of a hydrophobic peptide from the signal sequence of the mucin MUC1.

This paper describes the manual Fmoc/t-Bu solid-phase synthesis of a difficult nine-residue hydrophobic peptide LLLLTVLTV from one of the signal sequences that flank the tandem repeat of the mucin MUC1. Gel-phase 19F NMR spectroscopy was used as a straightforward method for optimization of the solid-phase synthesis. Different approaches were applied for comparative studies. The strategy based on modified solid-phase conditions using DIC/HOAt for coupling, DBU for Fmoc deprotection, and the incorporation of the pseudo proline dipeptide Fmoc-Leu-Thr(psiMe, Me pro)-OH as a backbone-protecting group was found to be superior according to gel-phase 19F NMR spectroscopy. Implementation of the optimized Fmoc protocol enabled an effective synthesis of signal peptide LLLLTVLTV.     

Heterotrimeric collagen peptides containing functional epitopes. Synthesis of single‐stranded collagen type I peptides related to the collagenase cleavage site

Synthetic collagen peptides containing larger numbers of Gly‐Pro‐Hyp repeats are difficult to purify by standard chromatographic procedures. Therefore, efficient strategies are required for the synthesis of higher molecular weight collagen‐type peptides. Applying the Fmoc/tBu chemistry, a comparative analysis of the standard stepwise chain elongation procedure on solid support with the procedure based on the use of the synthons Fmoc‐Gly‐Pro‐Hyp(tBu)‐OH and Fmoc‐Pro‐Hyp‐Gly‐OH was performed. The crude products resulting from the stepwise elongation procedure and from the use of Fmoc‐Gly‐Pro‐Hyp(tBu)‐OH clearly revealed large amounts of microheterogeneities that result from incomplete imino acid acylation as well as from diketopiperazine formation with cleavage of Gly‐Pro units from the growing peptide chain. Conversely, by the use of the Fmoc‐Pro‐Hyp‐Gly‐OH synthon, the quality of the crude products was significantly improved; moreover, protection of the Hyp side chain hydroxyl function is not required using the Fmoc/tBu strategy. With this optimized synthetic procedure, relatively large collagen‐type peptides were obtained in satisfactory yields as highly homogeneous compounds. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Fmoc/solid-phase synthesis of Tyr(P)-containing peptides through t-butyl phosphate protection.

The synthesis of Tyr(P)-containing peptides by the use of Fmoc-Tyr(PO3Me2)-OH in Fmoc/solid phase synthesis is complicated since, firstly, piperidine causes cleavage of the methyl group from the -Tyr(PO3Me2)-residue during peptide synthesis and, secondly, harsh conditions are needed for its final cleavage. A very simple method for the synthesis of Tyr(P)-containing peptides using t-butyl phosphate protection is described. The protected phosphotyrosine derivative, Fmoc-Tyr(PO3tBu2)-OH was prepared in high yield from Fmoc-Tyr-OH by a one-step procedure which employed di-t-butyl N,N-diethyl-phosphoramidite as the phosphorylation reagent. The use of this derivative in Fmoc/solid phase peptide synthesis is demonstrated by the preparation of the Tyr(P)-containing peptides, Ala-Glu-Tyr(P)-Ser-Ala and Ser-Ser-Ser-Tyr(P)-Tyr(P).     

High-throughput synthesis of conopeptides: a safety-catch linker approach enabling disulfide formation in 96-well format.

Conotoxins exhibit a high degree of selectivity and potency for a range of pharmacologically relevant targets. The rapid access to libraries of conotoxin analogues, containing multiple intramolecular disulfide bridges for use in drug development, can be a very labor intensive, multi-step task. This work describes a high-throughput method for the synthesis of cystine-bridged conopeptides.Peptides were assembled on a peptide synthesizer employing the Fmoc solid-phase strategy using a safety-catch amide linker (SCAL). Side-chain protecting groups were removed on solid phase before SCAL activation with ammonium iodide in TFA, finally releasing the peptide into the TFA solution. Disulfide bond formation was performed in the cleavage mixture employing DMSO.This improved method allows mixtures of oxidized peptides to be obtained in parallel directly from a peptide synthesizer. A single HPLC purification of the resulting crude oxidized material produced peptides of > 95% purity.     

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.     

Silyl linker-based approach to the solid-phase synthesis of Fmoc glycopeptide thioesters

An efficient solid-phase synthesis of Fmoc (glyco)peptide thioesters is described. Fmoc x Ser x OAll and Fmoc x Thr x OAll bound to resin with a silyl ether linker were deallylated by Pd(0) catalysis and condensed with thiophenol, benzyl mercaptane, and ethyl 3-mercaptopropionate by activation with DCC/HOBt. The thioesters were released from the resin either by treatment with CsF-AcOH or by acidic hydrolysis. The effectiveness of this silyl linker strategy is further demonstrated by the synthesis of more complex (glyco)peptide thioesters 25, 26 and 27 involving N-->C and C-->N peptide elongation.     

Solid-phase synthesis of peptides containing phosphoserine using phosphate tert.-butyl protecting group

In this paper, we report the solid-phase synthesis of peptides containing O-phosphonoserine using BOP as coupling reagent. Commercially available Fmoc amino-acids linked to p-alkoxybenzyl resin were used in the first step and Alloc amino acids in the following. Alloc group was removed by catalytic hydrostannolytic cleavage. Acid-labile side-chain protecting groups (including phosphate residue) were used. Thus, both removal of side-chain protecting groups and cleavage of the phosphopeptide from the resin were achieved in one step by treatment with TFA. Alloc serine was phosphorylated by the phosphoramidite method. This strategy enables the preparation of peptides with selectively phosphorylated residue and overcomes problems due to repetitive treatments with TFA and final cleavage with HF.     

Heating and microwave assisted SPPS of C-terminal acid peptides on trityl resin: the truth behind the yield

Despite correct purity of crude peptides prepared on trityl resin by Fmoc/tBu microwave assisted solid phase peptide synthesis, surprisingly, lower yields than those expected were obtained while preparing C-terminal acid peptides. This could be explained by cyclization/cleavage through diketopiperazine formation during the second amino acid deprotection and third amino acid coupling. However, we provide here evidence that this is not the case and that this yield loss was due to high temperature promoted hydrolysis of the 2-chlorotrityl ester, yielding premature cleavage of the C-terminal acid peptides.     

Discovery of novel conformationally restricted diazocan peptidomimetics as inhibitors of interleukin-1beta synthesis

A novel diazocan containing dipeptide mimetic was synthesized via reductive N-N bond cleavage of a pyrazolidino-pyrazolidine using Raney-Ni and evaluated as an ICE inhibitor. This versatile 8-membered ring containing scaffold possesses an N-5 ring nitrogen that was used to explore structure-activity relationships in a cell-based assay measuring inhibition of interleukin-1beta.     

Side Reaction with N-Carboxymethyl Amino Acids in the Synthesis of Backbone Cyclized Peptides

The use of N-carboxymethyl amino acids in the assembly of peptides with backbone cyclization can lead to diketopiperazine formation by intramolecular aminolysis which occurs despite the tert-butyl protection of the carboxy group. This undesired side reaction can be prevented by a very short deprotection time for the Fmoc group, by elongation of the N-carboxyalkyl chain or by forming the backbone (lactam) bridge before Fmoc removal, but not by the use of DBU or additives.     

Convenient synthesis of thioamidated peptides and proteins

The unique physicochemical properties of a thioamide bond, which is an ideal isostere of an amide bond, have not been fully exploited because of the tedious synthesis of thionated amino acid building blocks. Here, we report a purification‐free and highly efficient synthesis of thiobenzotriazolides of Fmoc‐protected and orthogonally protected 20 naturally occurring amino acids including asparagine, glutamine, and histidine. The near‐quantitative conversion to the respective thioamidated peptides on solid support demonstrates the robustness of the synthetic route. Furthermore, the unaltered incorporation efficiency of thiobenzotriazolides from their stock solution till 48 h suggests their compatibility toward automated peptide synthesis. Finally, utilizing an optimized cocktail of 2% DBU + 5% piperazine for fast Fmoc‐deprotection, we report the synthesis of a thioamidated Pin1 WW domain and thioamidated GB1 directly on solid support.     

Evaluation of new base-labile 2-(4-nitrophenylsulfonyl) ethoxycarbonyl (Nsc)-amino acids for solid-phase peptide synthesis.

The 2-(4-nitrophenylsulfonyl)ethoxycarbonyl (Nsc) group is a new base-labile protecting group for solid-phase peptide synthesis, completely interchangeable with the fluorenylmethoxycarbonyl (Fmoc) protecting group, but with certain advantages. In this paper, we report a methodology with Nalpha-Nsc-protected amino acids for the synthesis of some melanotropins important to our research, namely, gamma-melanocyte-stimulating hormone (gamma-MSH), its [Nle3]-analogue, and a cyclic alpha-MSH/beta-MSH hybrid. We developed an efficient protocol for the synthesis of the cyclic MSH analogue that yielded this peptide in >98% purity. The gamma-MSH synthesis, which gave problems with both the Boc and Fmoc strategies, yielded the desired peptide by Nsc-chemistry but was accompanied by side products. Finally, the Nle3-gamma-MSH analogue was synthesized more efficiently using the Fmoc strategy, suggesting that Nsc-chemistry might not be the best methodology for certain sequences.