Experiments on the protection of the thioether in methionine

The decrease in the solubility of peptides when their methionine residues are replaced by methionine sulfoxides prompted the exploration of an alternative approach to the protection of the thioether in methionine side chains. Alkylation of tert.-butyloxycarbonyl methionine p-nitrophenyl ester with methyl p-toluenesulfonate yielded the crystalline derivative of methionine S -methyl p-toluenesulfonate which could be incorporated into peptide chains. Alternatively, methionine S -methyl p-toluenesulfonate (Mmt) residues could be generated by the action of methyl p-toluenesulfonate on methionine containing peptides. The protecting group remained intact under the conditions of aminolysis and ammonolysis commonly used in peptide synthesis, and it was unchanged after the removal of other blocking groups with trifluoroacetic acid or diethylamine. On treatment with hydrobromic acid in acetic acid the toluenesulfonate anion was replaced by bromide ion, while hydrogenation resulted in the decomposition of the modified methionine side chain. In the process of deprotection Mmt residues could be smoothly converted to methionine residues by thiolysis. Thus, the protecting group functioned well in several respects but an increase in solubility (in dimethylformamide) on alkylation was observed only in a part of the peptide derivatives tested. Therefore, the value of the new approach for the protection of the methionine side chain in peptide synthesis remains to be established.     

Solid-phase synthesis of phosphopeptides

We report the solid-phase synthesis of peptides containing O-phosphoserine. Coupling was with commercially available Fmoc-amino acid pentafluorophenyl esters, with base used at each cycle to cleave Fmoc. Phosphorylation of those serine residues left unprotected on the peptide-resin was achieved with dibenzylphosphochloridate, and finally trifluoroacetic acid was used to remove side-chain protecting groups (including the benzyl groups used for the phosphate), and to cleave the peptide from the resin in the same step. This synthetic strategy enables the preparation of peptides with individual, selectively phosphorylated residues. Alternative approaches to introduce protected phosphate and continue with coupling of further amino acids were less advantageous due to the lability of the phosphate group to base and to steric hindrance.     

3-NITRO-2-PYRIDINESULFENYL (Npys) GROUP

The novel 3-nitro-2-pyridinesulfenyl (Npys) group, which is useful for the protection and the activation of amino and hydroxyl groups for peptide synthesis, is reported. The Npys group is readily introduced by treatment of amino acids with 3-nitro-2-pyridinesulfenyl chloride. The Npys group is easily removed by treatment with very dilute HCl, e.g. 0.1-0.2 N HCl in dioxane, but it is resistant to trifluoroacetic acid and 88% formic acid. Npys is also selectively removed under neutral conditions using triphenylphosphine or 2-pyridinethiol 1-oxide without affecting benzyloxycarbonyl (Z), tert-butyloxycarbonyl (Boc), 2-(4-biphenylyl) propyl(2) oxycarbonyl (Bpoc), 9-fluorenylmethyloxycarbonyl (Fmoc), benzyl (Bzl) or tert-butyl (tBu) protecting groups. The N-Npys and O-Npys groups when activated in the presence of RCOOH by the addition of tertiary phosphine form peptide or ester bonds via oxidation-reduction condensation. The important features of the Npys group are demonstrated through the synthesis of peptides in solution and by solid phase methodology without a formal deprotection procedure. In solid phase synthesis, 4-(Npys-oxymethyl) phenylacetic acid is used as the key intermediate for the introduction of the trifluoroacetic acid resistant 4-(oxymethyl) phenylacetamido linking group to the resin.     

Use of 10% sulfuric acid/dioxane for removal of N-α-tertiary-butyloxycarbonyl group during solid phase peptide synthesis

The hazards and high costs associated with the use of trifluoroacetic acid (TFA) in the removal of the N-α-tertiary-butyloxycarbonyl (Boc) group during solid phase peptide synthesis prompted an examination of alternative acidolytic reagents for α-amino group deprotection. N-α-Boc-glycine and N-α-Boc-isoleucine resins as well as an N-α-Boc-peptide resin were used to test the lability to various deprotection mixtures of both the N-α-Boc resin group as well as the amino acid or peptide-O benzyl ester resin linkage. Of the combinations tried, several were found, including 10% H₂SO₄/dioxane, which gave results roughly comparable to 50% TFA/CH₂Cl₂. Several peptides, 5–10 amino acid residues in length, have been successfully synthesized using the 10% H₂SO₄/dioxane mixture and were found to be comparable in purity to the same peptides prepared using the standard TFA/CH₂Cl₂ method of N-α-Boc removal. Thus, for the peptides examined, 10% H₂SO₄dioxane was found to be an inexpensive, safe, and practical alternative reagent to the more costly and hazardous 50% TFA/CH₂Cl₂ commonly used in the deprotection step of solid phase peptide synthesis.     

Ninhydrin as a reversible protecting group of amino‐terminal cysteine

Abstract: The proximity of the α‐amine and β‐thiol of α‐amino terminal‐cysteine (NT‐Cys) residues in peptides imparts unique chemical properties that have been exploited for inter‐ and intra‐molecular ligation of unprotected peptides obtained through both synthetic and biological means. A reversible protecting group orthogonal to other protection strategies and reversible under mild conditions would be useful in simplifying the synthesis, cleavage, purification and handling of such NT‐Cys peptides. It could also be useful for the sequential ligation of peptides. To this end, we explored tri‐one chemistry and found that ninhydrin (indane‐1,2,3 trione) reacted readily with cysteine or an NT‐Cys‐containing peptide on‐ or off‐resin at pH 2–5 to form Ninhydrin‐protected Cys (Nin‐Cys) as a thiazolidine (Thz). The Thz ring, protecting both the amino and thiol groups in Nin‐Cys, completely avoids the formylation and Thz side reactions found during hydrofluoric acid (HF) cleavage when N‐π‐benzyloxymethyl histidine groups are present. Nin‐Cys is stable during coupling reactions and various cleavage conditions with trifluoroacetic acid or HF, but is deprotected under thiolytic or reducing conditions. These properties enable a facile one‐step deprotection and end‐to‐end‐cyclization reaction of Nin‐Cys peptides containing C‐terminal thioesters.     

Use of the 3-nitro-2-pyridine sulfenyl protecting group to introduce Nε-branching at lysine during solid-phase peptide synthesis I. Application to the synthesis of a peptide template containing two addressable sites

TASPs (template-assembled synthetic peptides) are generated by the covalent attachment of linear peptides to a common peptide backbone, thus generating larger synthetic peptides/proteins with prefolded structure. In this work we present a strategy for the synthesis of a heterotemplate-assembled synthetic peptide containing two addressable sites. This orthogonal protection strategy would allow the selective introduction of different peptide chains via the ε-amino functions of template lysines being protected by either fluorenylmethoxycarbonyl (Fmoc) or 3-nitro-2-pyridine sulfenyl (Npys) groups. The N^α-Boc-N^ε-Npys-l -lysine required for solid-phase peptide synthesis (SPPS) is not readily available at a reasonable cost. To facilitate the more widespread use of this reagent we have compared the two published procedures for synthesizing this protected amino acid and evaluated the suitability of the products for SPPS. Two resin-bound peptides, a tripeptide Ac-G-K-Npys)-G-resin and an octapeptide template Ac-P₁-K₂-K₃-L₄-K_s-K₆-P₇-G₈-resin, were synthesized by SPPS. The ε-amino functions of lysines K₂ & K₆ and K₃ & K₅ of the octapeptide were protected by Fmoc and Npys groups, respectively. Secondly, these peptides were used to evaluate various reagents and reaction conditions for the deprotection of the ε-amino function of lysines bearing the N_ε-Npys protecting group. A procedure for the optimized selective and quantitative deprotection of the Npys group from the ε-amino function of lysine in a resin-bound peptide using 2-mercaptopyridine-N-oxide is described. © Munksgaard 1995.     

Synthesis of γ-carboxyglutamic acid-containing peptides by the Boc strategy

γ-Carboxyglutamic acid (Gla) derivatives having several protecting groups at the γ-carboxyl function were synthesized and examined for their stabilities and removabilities under the conditions used in peptide synthesis by the Boc strategy. Among them, the cyclohexyl (cHx) group of the Gla residue was found to be stable during the synthesis of the protected peptides, but was quantitatively cleaved by the final HF treatment without decarboxylation. Using Boc-Gla(OcHx)₂-OH as a starting material, the synthesis of Gla-containing peptides was achieved by the Boc strategy using a standard HF procedure for the final deprotection.     

Solid-phase peptide synthesis without side-chain hydroxyl protection of threonine

A peptide containing four threonine residues was synthesised by the solid-phase method using fluorenyl-methoxycarbonylamino acid reactive esters or coupling by preactivation with 1-hydroxybenzotriazole and Castro's reagent. In two separate experiments the synthesis was carried out with or without protection of the side-chain hydroxyl group of threonine as the tert-butyl ether. Comparison of the crude peptides after deprotection and detachment from the synthesis resin suggests that side-chain protection of threonine is unnecessary under the synthetic conditions employed.     

Preparation of ‘side‐chain‐to‐side‐chain’ cyclic peptides by Allyl and Alloc strategy: potential for library synthesis

Abstract: Automated and manual deprotection methods for allyl/allyloxycarbonyl (Allyl/Alloc) were evaluated for the preparation of side‐chain‐to‐side‐chain cyclic peptides. Using a standard Allyl/Alloc deprotection method, a small library of cyclic peptides with lactam bridges (with seven amino acids) was prepared on an automatic peptide synthesizer. We demonstrate that the Guibé method for removing Allyl/Alloc protecting groups under specific neutral conditions [Pd(PPh₃)₄/PhSiH₃)/DCM] can be a useful, efficient and reliable method for preparing long cyclic peptides on a resin. We have also manually synthesized a cyclic glucagon analogue containing 24 amino acid residues. These results demonstrated that properly controlled palladium‐mediated deprotection of Allyl/Alloc protecting groups can be used to prepare cyclic peptides on the resin using an automated peptide synthesizer and cyclic peptides with a long chain.     

Solid phase synthesis of bioadhesive analogue peptides with trifluoromethanesulfonic acid cleavage from PAM resin

Several decapeptides related to consensus peptide sequences found in the natural polyphenolic proteins from the sea mussels M. edulis or M. californianus have been synthesized in high yields and purities. The peptides were prepared by solid phase peptide synthesis on PAM support resins utilizing BOC protection strategies. The product peptides were then deprotected and cleaved from the PAM resins with TFMSA in a two-step procedure. The course of selective peptide deprotection with TFMSA was followed by high resolution ¹³C n.m.r. on aliquots of the peptide resin swollen in DMF and optimal deprotection times for batch processing of the peptide resins were chosen based on the n.m.r. results. Our two-step deprotection and cleavage procedure using TFMSA was shown to be at least as good as the usual HF procedure for the class of synthetic peptides described in this paper. N to O rearrangements of threonine and/or serine residues in the synthesized decapeptides were observed in both procedures but were reversible following treatment with ammonium bicarbonate. Coupled HPLC-u.v./vis spectroscopy, HPLC-MS and FAB MS analytical methods were used to characterize the product peptides and to provide substantial amino acid sequence information.     

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

Abstract: 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, [Ala¹⁹, Asn²²]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 Et₂NH/DMF (～15% v/v) and, after evaporation to ～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°/AcOH to yield the peptide with intact α‐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.     

Cysteic- and homocysteic acid-S-(aminoiminomethyl) amides: a new class of modified arginines useful in peptide synthesis

A novel, practical synthesis of the title compounds and their derivatives are described. The protecting groups for amino, guanidino and carboxylic functions of substituted amides of cysteic and homocysteic acid were selected with the aim of making the amino acid derivatives synthons for peptide synthesis both in solution and by the solid phase method. Studying the structure–activity relationship some new kyotorphin, [Leu¹] kyotorphin and MIF-1 analogues, containing the unusual amino acid cysteic acid-S-(aminoiminomethyl) amide (sArg) in position two, have been prepared. It is a very promising compound, a structural analogue of arginine and an efficient antagonist in its metabolism.     

SOLID-PHASE PEPTIDE SYNTHESIS OF SOMATOSTATIN USING MILD BASE CLEAVAGE OF Nα-9- FLUORENYLMETHYLOXYCARBONYLAMINO ACIDS

Experimental details for the “Fmoc solid phase peptide synthesis” of somatostatin are described. The 9-fluorenylmethyloxycarbonyl group was rapidly and quantitatively cleaved by 55% piperidine in dimethylformamide and monitored (u.v.) manually. For a kinetic study, a centrifugal reactor with a photometric control system and reference cell was used at each stage. The symmetrical anhydride coupling reaction was rapid and either acetic anhydride or fluorescamine termination was incorporated to minimize formation of deletion peptides. Anchor-bond cleavage was effected with trifluoroacetic acid which simultaneously removed all the acid labile tert.-butyl side chain protecting groups. Nα-9-fluorenylmethyloxycarbonyl peptides may be obtained by omitting the piperidine deprotection step after the last cycle of synthesis. From several syntheses, analytically pure di-S-protected somatostatin 14-peptide was obtained in 55–60% overall yield. The S-protecting groups were removed and the product was purified by gel filtration to give homogeneous dihydrosomatostatin (91% yield). Oxidation of dihydrosomatostatin with potassium ferricyanide and purification by countercurrent distribution provided analytically pure homogeneous somatostatin.     

Hydrolysis of peptide esters by different enzymes

The combined use in peptide synthesis of the Fmoc-group with methyl, benzyl or p-nitro benzyl esters is not practical because of the elimination of the Fmoc-group under basic conditions and by catalytic hydrogenation. Nevertheless the solution synthesis of peptides requires those combinations in some cases. For this purpose we have investigated enzymatic hydrolysis of some tri and tetrapeptide esters. The hydrolysis were carried out under pH-control. We measured deprotection of the carboxyl group by thermitase, porcine liver esterase, carboxypeptidase A and x-chymotrypsin. The main problems are to suppress proteolytic degradation of the peptide bond and to bring the protected peptides into solution. To solve both problems we used dimethylformamide and dimethylsulfoxide as cosolvents. The ratios between esterolytic and proteolytic activity were estimated under various cosolvent concentrations. Advantages of this method are to avoid side reactions of alkaline instable side chains (e.g. asparagine, glutamine), cleavage of base labile protecting groups and racemization by alkaline saponification. The enzymatic deprotection was followed by HPLC, HPTLC and titration. On a preparative scale this method gives good yields and sufficently pure products.     

New mild acid-labile protecting groups for the guanidino function of Nα-fluorenylmethoxycarbonyl-l-arginine in solid-phase peptide synthesis: 10, 11-dihydro-5H-dibenzo[a,d]. cyclohepten-5-yl, 2-methoxy-10, 11-dihydoro-5H-dibenzo[a,d]. cyclohepten-5-yl and 5H-dibenzo[a,d]. cyclohepten-5-yl groups

10, 11-Dihydro-5H-dibenzo[a, d]. cyclohepten-5-yl [5-dibenzosuberyl]. and 5H-dibenzo[a, d]. -cyclohepten-5-yl [5-dibenzosuberenyl]. groups have been found to be useful protecting groups for the guanidino function of arginine in solid-phase peptide synthesis on Fmoc chemistry. The arginine derivatives ( 4a, b, c ) derivatized with these groups were easily deprotected with mild acid (less than 30 min with 25% trifluoroacetic acid). Tryptophan-containing peptide sequences, two hexapeptides ( 6 ) and (8), were synthesized in good yield by mild acid treatment (50% trifluoroacetic acid in 1 h) of the peptide resins ( 5a, c-f and 7a, c, d ) assembled via 4a, b, c using benzotriazol-1 -yl-oxy-tris-(pyrrolidino)-phosphonium hexafluorophosphate-1-hydroxybenzotriazole mediated coupling.     

Sulfonation of arginine residues as side reaction in Fmoc-peptide synthesis

Several arginine-rich peptides containing the C-terminus of neuropeptide Y (NPY) were prepared by solid phase peptide synthesis using Fmoc chemistry and cleaved from the resin with trifluoroacetic acid (TFA). The products were characterized by fast atom bombardment-MS, LC-thermospray-MS, ion spray-MS/MS, and Edman degradation. The side products could be identified as peptides with sulfonated arginine residues resulting from an unexpected cleavage of Mtr or Pmc protecting groups. The degree of sulfonation depended on the choice and composition of the cleavage solution. Several scavenger mixtures were used and a mixture of thioanisole/thiocresol was found to be the most efficient for suppressing sulfonation. Furthermore treatment with the enzyme arylsulfate-sulfohydrolase desulfonated the peptides yielding the correct sequence.     

Synthesis of O-phosphonotyrosyl peptides

A general method for the synthesis of O-phosphonotyrosyl peptides using solid phase methodology is described. Protected O-phosphonotyrosine derivatives with the general structure Boc-Tyr(R₂PO₃)-OH (R = methyl, ethyl or benzyl) were prepared as potential synthons for the introduction of O-phosphonotyrosine residues into peptide sequences. Using ³¹P n.m.r. spectroscopy, the alkyl phosphate protecting groups (R = methyl or ethyl) were shown to be stable to the coupling, deprotection and neutralization cycles of the Merrifield method of solid phase peptide synthesis. Facile removal of the methyl phosphate protecting groups from the O-phosphonotyrosyl peptide analogue Ac-Tyr(Me₂PO₃)-NHMe was demonstrated using 45% HBr/acetic acid. The O-phosphonotyrosyl heptapeptide H-Leu-Arg-Arg-Ala-PTyr-Leu-Gly-OH was subsequently prepared using solid phase methodology via incorporation of N²-tert-butyloxycarbonyl-O-dimethylphosphonotyrosine.     

Chemical mechanism to account for artifactual formation of shortened peptides with free alpha-amino groups in solid phase peptide synthesis

The formation of terminated peptides with free α-amino groups has often been observed in stepwise solid phase peptide synthesis. This has been attributed to variable accessibility in regions of the swollen crosslinked resin supports. It is now shown that impurities in the amino acid reagents are responsible for these by-products. Thus, sec. -butyloxycarbonylamino acids were isolated from tert. -butyloxycarbonylamino acids after treatment with trifluoroacetic acid under standard deprotection conditions for the removal of the tert. -butyloxycarbonyl (Boc) group. Direct reverse phase HPLC analysis of Boc-amino acids from commercial sources also showed the sec. -Boc-amino acids as impurities present at varying levels. The sec. -Boc group was stable to treatment at room temperature with trifluoroacetic acid in dichloromethane (1:1, v/v) (half-life 7 years), but was removed by HF-anisole under the standard conditions of cleavage and deprotection of assembled peptides. In model syntheses, the level of terminated free peptides corresponded to the level of preexisting sec. -Boc-amino acid impurities present in the Boc-amino acid reagents. Use of Boc-amino acids with no detectable sec. -Boc resulted in negligible levels (< 0.05%) of terminated peptides. The problem is thus readily overcome by the use of pure Boc-amino acid starting materials and is not a reflection of a shortcoming inherent to the polymer supported nature of solid phase syntheses as has been previously suggested.     

Controlled peptide-protein conjugation by means of 3-nitro-2-pyridinesulfenyl protection-activation

The disulfide bond in S-3-nitro-2-pyridinesulfenyl (S-Npys) compounds is stable towards the acid treatment used in solid-phase peptide synthesis, yet the lability of S-Npys-peptides towards nucleophiles enables the conjugation to proteins to proceed under mild conditions. Thus Boc-Cys(Npys)-OH was coupled as N-terminal residue to a resin-linked peptide chain. After deprotection and cleavage from the resin the Npys-cysteinylpeptide was attached to a properly functionalized protein by reaction with a mercapto group. The amount of peptide conjugated to the protein was determined by measuring the amount of 3-nitro-2-thiopyridone liberated. The cysteinylpeptide which was detached from the protein by reduction of the disulfide bond was shown to be identical with the product obtained by reduction of the Npys-cysteinylpeptide.     

Preparation and properties of Nα-Bpoc-amino acid pentafluorophenyl esters

The preparation and properties are reported of several N^x-Bpoc -amino acid pentafluorophenyl esters, including those bearing tert-butyl-, allyl- and trityl-based protecting groups. These derivatives have been used in the solid-phase peptide synthesis of sevral short peptides.