Methylthiomethyl, a new carboxyl protecting group in peptide synthesis

N alpha-protected amino acid methylthiomethyl esters (MTM) were obtained in good yields under mild conditions using the "ButBr/Me2SO" reagent. Selective removal of the N-protecting group was achieved in HCl/anhydrous ethyl ether and the MTM ester hydrochlorides were successfully used in the synthesis of dipeptides.     

Novel base-labile protecting groups for 5'-hydroxy function in solid-phase oligonucleotide synthesis

The 6-(levulinyloxymethyl)-3-methoxy-2-nitrobenzoyl (LMMoNBz) and 2-(levulinyloxymethyl)-5-methoxy-4-nitrobenzoyl (LMMpNBz) groups were developed as novel base-labile protection for the 5'-hydroxy function in solid-phase oligonucleotide synthesis. A comparative study of the LMMoNBz, LMMpNBz and 2-(levulinyloxymethyl)-5-nitrobenzoyl (LMNBz) protecting groups for oligonucleotide synthesis proved strong feasibility for the LMMoNBz group.     

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.     

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.     

Removal of acid-labile protecting or anchoring groups in the presence of polyfluorinated alcohol: Application to solid-phase peptide synthesis

We describe herein a new method for cleaving from resin and removing acid-labile protecting groups in solid-phase peptide synthesis in the presence of a polyfluorinated alcohol (either trifluoroethanol, TFE, or hexafluoroisopropanol, HFIP). It was shown that 0.1 M HCl in hexafluoroisopropanol or trifluoroethanol removes the acid-labile protecting groups commonly used in Fmoc SPPS for the protection of amino acid side-chains, such as t-butyl ester and ether, Boc, trityl, and Pbf groups including the most acid-resistant p-hydroxymethylphenoxyacetyl group (HMPA), p-benzyloxy benzyl ester (Wang resin), Rink amide, and peptide amide linker (PAL). The addition of 5–10% of a hydrogen-bonding solvent was shown to considerably retard or even fully inhibit the reaction. However, nonhydrogen-bonding solvents, such as dichloromethane, do not slow down the reaction.     

Facile solid-phase synthesis of C-terminal peptide aldehydes and hydroxamates from a common Backbone Amide-Linked (BAL) intermediate.

C-Terminal peptide aldehydes and hydroxamates comprise two separate classes of effective inhibitors of a number of serine, aspartate, cysteine, and metalloproteases. Presented here is a method for preparation of both classes of peptide derivatives from the same resin-bound Weinreb amide precursor. Thus, 5-[(2 or 4)-formyl-3,5-dimethoxyphenoxy]butyramido-polyethylene glycol-polystyrene (BAL-PEG-PS) was treated with methoxylamine hydrochloride in the presence of sodium cyanoborohydride to provide a resin-bound methoxylamine, which was efficiently acylated by different Fmoc-amino acids upon bromo-tris-pyrrolidone-phosphonium hexafluorophosphate (PyBrOP) activation. Solid-phase chain elongation gave backbone amide-linked (BAL) peptide Weinreb amides, which were cleaved either by trifluoroacetic acid (TFA) in the presence of scavengers to provide the corresponding peptide hydroxamates, or by lithium aluminum hydride in tetrahydrofuran (THF) to provide the corresponding C-terminal peptide aldehydes. With several model sequences, peptide hydroxamates were obtained in crude yields of 68-83% and initial purities of at least 85%, whereas peptide aldehydes were obtained in crude yields of 16-53% and initial purities in the range of 30-40%. Under the LiAlH4 cleavage conditions used, those model peptides containing t-Bu-protected aspartate residues underwent partial side chain reduction to the corresponding homoserine-containing peptides. Similar results were obtained when working with high-load aminomethyl-polystyrene, suggesting that this chemistry will be generally applicable to a range of supporting materials.     

An acid-labile anchoring linkage for solid-phase synthesis of C-terminal peptide amides under mild conditions

A series of polymer-supported benzylamides substituted with one to three alkoxy groups in the ring positions were prepared and shown to give carboxamides upon treatment with acid. Based on the initial screening, the bis(o-methoxy)-p-alkoxybenzylamide anchoring linkage was selected for a detailed evaluation of its suitability for solid-phase synthesis of C-terminal peptide amides. The handle derivative 5-[(2' or 4')-Fmoc-aminomethyl-3',5'-dimethoxyphenoxy]valeric acid (1) was prepared in seven facile steps [purification of intermediates unnecessary; overall yield 15% for crystalline product, which is a mixture of positional isomers], and was quantitatively coupled onto amino group-containing supports by use of N,N'-dicyclohexylcarbodiimide plus 1-hydroxybenzotriazole in N,N-dimethylformamide. Stepwise elaboration of peptide chains proceeded smoothly with both N alpha-9-fluorenyl-methyloxycarbonyl (Fmoc) and N alpha-dithiasuccinoyl (Dts) amino acids, and final cleavage of tert.-butyl side-chain protecting groups and of the anchoring linkage occurred readily in trifluoroacetic acid-dichloromethane (7:3) at 25 degrees. The methodology was demonstrated by the syntheses of H-Trp-Asp-Met-Phe-NH2 (tetragastrin) and H-Tyr-Gly-Gly-Phe-Met-NH2 (methionine-enkephalinamide), both with high yields and purities.     

N,O-bisFmoc derivatives of N-(2-hydroxy-4-methoxybenzyl)-amino acids: Useful intermediates in peptide synthesis

2-Hydroxy-4-methoxybenzyl-amino acid residues inhibit interchain association in solid phase peptide synthesis. They are easily introduced through their N,O-bisFmoc derivatives. Preparation of a range of these derivatives is described.     

Use of the excluded protecting group (EPG) method for peptide synthesis.

The excluded protecting group (EPG) method has been used for the solution synthesis of several peptides including Merrifield's Model Tetrapeptide, linear antamanide and an analogue of magainin-1, [Ala(19), Asn(22)]magainin-1. In the approach reported, the C-terminal amino acid is esterified to the 2-position of cholestane as the [2s,3s]iodohydrin ester and the penultimate amino acid added to the aminoacyl-steroid as the Fmoc-pentafluorophenyl-ester. The Fmoc group is removed with Et(2)NH/DMF ( approximately 15% v/v) and, after evaporation to approximately 10 mL, the solution chromatographed on Sephadex LH-20 in DMF. The dipeptidyl-steroid elutes as the free amine well separated from other reaction mixture components. Fractions containing the dipeptide, as determined by counting and TLC, are pooled and reacted with the next Fmoc-amino acid-pentafluorophenyl ester in the sequence. Repetition of the deprotection/purification/reaction cycle yields the fully protected peptide.On completion of the synthesis, the cholestane iodohydrin ester is selectively removed by treatment with Zn degrees /AcOH to yield the peptide with intact alpha-amino and side chain protecting groups. Global deprotection is achieved with HF. All intermediates from the syntheses reported were characterized. The magainin analogue was shown to have full biologic activity. The Fmoc iodohydrin esters of 16 of the 20 proteogenic amino acids have been prepared and characterized for use as the C-terminal amino acids in other EPG syntheses.     

Application of acetamidomethyl and 9-fluorenylmethyl groups for efficient side protection of penicillamine in solid-phase peptide synthesis

Synthesis of S-acetamidomethyl and S-fluorenylmethyl derivatives of penicillamine is described. Both groups are completely stable to all the usual reagents in solid-phase peptide synthesis, including the HF cleavage step, and show an excellent degree of orthogonality to each other. Treatment of the protected peptides Ac-L-Pen(X)-L-Pro-D-Val-L-Cys(X)-NH2 with thallium (III) trifluoroacetate or iodine for X = Acm or piperidine/DMF (1:1) for X = Fm induced with good yield the formation of the intramolecular disulfide bridge. This cyclic peptide appears to assume a type II beta-turn conformation in d6-DMSO as evidenced by 1H-NMR spectra.     

Cyclic diacyl groups for protection of the N6-amino group of deoxyadenosine in oligodeoxynucleotide synthesis.

Three kinds of substituted phthaloyl groups and a succinyl group were introduced onto the N6-amino function of deoxyadenosine derivatives. Among them, the succinyl group was found to be the most effective for prevention of depurination upon detritylation in acidic media and the most stable in basic media. Protection of the N6-amino function of 5'-O-dimethoxytrityldeoxyadenosine and introduction of a succinate linker into the 3'-hydroxyl were achieved simultaneously by a one-step reaction with succinic anhydride. A tetradeoxyribonucleotide, dTpTpTpA containing a 3'-terminal deoxyadenosine was successfully synthesized on a polystyrene support via the phosphotriester method.     

Allyl and allyloxycarbonyl groups as versatile protecting groups in nucleotide synthesis

Allyl group serves as a useful protecting group for an protection of sugar hydroxyls and amino and imide moieties of by brief treatment with a palladium catalyst and a variety of nucleophiles at room temperature.     

New hydrogenation catalyst: An advantageous method for the removal of hydrogenolysable protecting groups in peptide synthesis

Removal of some commonly used protecting groups in peptide synthesis by catalytic transfer hydrogenation employing ammonium formate and magnesium is described. This method is equally competitive with other methods in deblocking most of the commonly used protecting groups in peptide synthesis. tert-Butyl derived and base labile protecting groups were completely stable under these conditions. The use of ammonium formate and magnesium makes this a rapid, low-cost alternative to palladium and reduces the work-up to a simple filtration and extraction operation.     

New hydrogenation catalyst: An advantageous method for the removal of hydrogenolysable protecting groups in peptide synthesis

Summary Removal of some commonly used protecting groups in peptide synthesis by catalytic transfer hydrogenation employing ammonium formate and magnesium is described. This method is equally competitive with other methods in deblocking most of the commonly used protecting groups in peptide synthesis.tert-Butyl derived and base labile protecting groups were completely stable under these conditions. The use of ammonium formate and magnesium makes this a rapid, low-cost alternative to palladium and reduces the work-up to a simple filtration and extraction operation.     

Thio-alpha-amino acids (S-acids) as a carboxyl component in peptide synthesis catalysed by papain

Peptide synthesis catalysed by papain was studied using thio-alpha-amino acids (S-acids) as a carboxyl component. It was found, for example, that with Z-AlaSH (pK 2.70) the maximal yield of the peptide Z-AlaValNH2 was obtained at pH 8-8.5. A two-fold excess of Z-AlaSH furnished peptides with yields close to 100%. Thio-amino acids with bulky side groups, for example, Z-IleSH, Z-Asp(OBu')SH, gave peptides with a low yield. Papain interacts with Z-AlaSH better than do bromelain or ficin.     

Improved approach for anchoring N alpha-9-fluorenylmethyloxycarbonylamino acids as p-alkoxybenzyl esters in solid-phase peptide synthesis

Several Fmoc-amino acids have been esterified by use of N,N-dimethylformamide dineopentyl acetal to 2,4,5-trichlorophenyl 3'-(4'-hydroxymethyl-phenoxy)propionate, and the resultant handle derivatives were purified and then quantitatively coupled onto aminomethyl supports. Compared to literature methodology, the present procedure is preferred because: (i) extra steps to selectively protect and liberate the carboxyl of the handle are circumvented; and (ii) the additional methylene group spacer reflecting substitution of a propionyl group for an acetyl group in the handle changes the electronic parameters of the resultant p-alkoxybenzyl ester sufficiently so that the rates of acidolytic cleavage of the anchoring linkage are 2- to 3-fold increased and useful improvements in yields can be achieved.     

Application of arylsulphonyl side-chain protected arginines in solid-phase peptide synthesis based on 9-fluorenylmethoxycarbonyl amino protecting strategy.

One of the main problems still hampering solid-phase peptide synthesis using orthogonal protection strategies based on the 9-fluorenylmethoxycarbonyl amino protecting group is the difficult removal of currently used arginine arylsulphonyl guanidino protecting groups. Poor acid liability of 4-methoxy-2,3,6-trimethylbenzenesulphonyl-protected arginine has led to the popularity of the newer 2,2,5,7,8- pentamethylchroman-6-sulphonyl guanidino protecting group. This group was initially believed to have liability to trifluoroacetic acid, the reagent commonly used to simultaneously deprotect peptides and detach them from the synthesis resin, comparable to tert.-butyl and trityl type protecting groups used for the protection of other peptide side-chain functionalities. In a comparison of three established cleavage/deprotection mixtures we have shown that this is not always the case, particularly in multiple arginine peptides. We have found that only hard-acid deprotection with trimethylsilyl bromide reliably removed both arylsulphonyl guanidino protecting groups from a variety of arginine-containing peptides.