STABLE ISOLATED SYMMETRICAL ANHYDRIDES OF Nα-9-FLUORENYLMETHYLOXYCARBONYLAMINO ACIDS IN SOLID-PHASE PEPTIDE SYNTHESIS:

The synthesis and isolation of symmetrical anhydrides of N^α-9-fluorenylmethyloxycarbonyl (Fmoc) amino acids using water soluble carbodiimide is described. These compounds were used in a solid phase peptide synthesis of methionine-enkephalin on a p-benzyloxybenzyl ester polystyrene 1% divinylbenzene resin support. Homogeneous free pentapeptide was obtained in 42% overall yield. The Fmoc amino acid symmetrical anhydrides were stable during prolonged storage (2 years at 0°) and offer advantages over present “Fmoc solid phase” methods which use anhydrides formed in situ.     

SOLID-PHASE PEPTIDE SYNTHESIS USING MILD BASE CLEAVAGE OF NαFLUORENYLMETHYLOXYCARBONYLAMINO ACIDS,EXEMPLIFIED BY A SYNTHESIS OF DIHYDROSOMATOSTATIN

N ^α-9-Fluorenylmethyloxycarbonyl (Fmoc) amino acids will be of advantage in solid phase peptide synthesis. The Fmoc-group is quantitatively cleaved by mild base (piperidine). This permits the use of tert-butyl-type side chain blocking and of peptide-to-resin linkage cleavable by mild acidolysis. Side reactions arising from repetitive acid deprotection and final HF cleavage in contemporary solid phase synthesis are avoided. Fully bioactive and homogeneous dihydrosomatostatin was obtained in 53% overall yield.     

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.     

FACTORS THAT INFLUENCE ACYL PROLINE CLEAVAGE BY SODIUM IN LIQUID AMMONIA

The reductive cleavage of aminoacyl proline bonds by sodium in liquid ammonia was compared by a simple chromatographic technic for various protected and unprotected analogs of the C-terminal tetrapeptide of the insulin B-chain, -Thr-Pro-Lys-Ala-OH. The literature suggests that the Thr-Pro bond in this peptide is especially sensitive to Na-NN ₃, compared to various other acyl proline bonds. The present study shows that this is true regardless of whether the sidechains, the α-amino, and the α-carboxyl groups are protected or free when they are introduced into liquid ammonia; there is no difference in the amount of reductive Thr-Pro cleavage despite the different numbers of protons released into liquid ammonia by the functional groups of the residues surrounding the Thr-Pro bond. The C-terminal tetrapeptide and its blocked derivatives, prepared by solid phase synthesis, were reduced by Na-NH ₃ to the same extent as similarly synthesized B-chains and natural B-chain. Thr-Pro cleavage could be minimized by sharply limiting the amount of sodium and the time of exposure of the peptide to it. The results are therefore useful in planning synthetic approaches to the B-chain. The Ser-Pro bond shares the lability of the Thr-Pro bond to Na-NH ₃ reduction, whereas the Val-Pro bond is only half as labile.     

4-Chloromethylphenoxyacetyl polystyrene and polyamide supports for solid-phase peptide synthesis

Two functionalised supports for the solid-phase synthesis of peptides under mild reaction conditions were prepared: 4-chloromethylphenoxyacetamidomethylcopoly (styrene-1%-divinylbenzene) and 4-chloromethylphenoxyacetyl-norleucylpoly(dimethylacrylamide). They were devised in order to avoid the danger of racemization which exists during base-catalyzed esterification of the first protected amino acid to the 4-alkoxybenzyl alcohol resins formerly employed in combination with N^α-9-fluorenylmethoxycarbonyl and tert.-butyl side-chain protecting groups. Esterification of N^α-protected amino acids to the new resins can be achieved easily and without significant levels of racemization by means of their caesium salts, while cleavage from the supports is possible by treatment with trifluoroacetic acid. The 4-chloromethylphenoxyacetyl polystyrene resin was tested by the synthesis of Leu-enkephalin which was cleaved, at the end of the synthesis, from the solid support in 91% yield by 60% trifluoroacetic acid in methylene chloride, and was shown to be more than 99% pure by ion-exchange chromatography and reverse phase high pressure liquid chromatography.     

A cleavage method which minimizes side reactions following Fmoc solid phase peptide synthesis

The success of solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl (Fmoc) amino acids is often limited by deleterious side reactions which occur during TFA peptide-resin cleavage and side-chain deprotection. The majority of these side reactions modify susceptible residues, such as Trp, Tyr, Met, and Cys, with TFA-liberated side-chain protecting groups and linkers. The purpose of this study was to assess the relative effectiveness of various scavengers in suppressing these side reactions. We found that the cleavage mixture 82.5% TFA: 5% phenol: 5% H₂O : 5% thioanisole : 2.5% EDT (Reagent K) was maximally efficient in inhibiting a great variety of side reactions. Synthesis and cleavage of 10 peptides, each containing 20-50 residues, demonstrated the complementarity of Fmoc chemistry with Reagent K for efficient synthesis of complex peptides.     

Solution phase synthesis of Saccharomyces cerevisiae a-mating factor and its analogs

The solution phase synthesis of the Saccharomyces cerevisiae a-mating factor and nonfarnesylated and nonmethylated a-factor analogs are reported. The a-factor, a lipopeptide with the sequence Tyr-Ile-Ile-Lys-Gly-Val-Phe-Trp-Asp-Pro-Ala-Cys(S-Farnesyl)OCH₃ was synthesized by the condensation of the amine terminal protected decapeptide with the carboxyl terminal farnesylated dipeptide using benzotriazol-l-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP reagent) as the coupling agent. The synthesis of the decapeptide involved 5 + 5 fragment coupling with the BOP reagent and the successful application of 9-fluorenylmethyl ester(OFm) and 9-fluorenylmethoxycarbonyl(Fmoc) groups for the protection of Asp and Lys side chains and Tyr α-amine and of phenacyl esters (OPa) for α-carboxyl protection. The OFm and Fmoc groups tolerated repeated couplings and were completely stable to zinc powder in acetic acid, a condition under which the OPa group was removed. The synthesis of the nonfarnesylated a-factor was accomplished by the coupling of the decapeptide with tetrapeptide (Ala-CysOCH³)₂ followed by the deprotection of the OFm and Fmoc groups with piperidine and the cleavage of the disulfide bond with zinc powder in acetic acid. The nonmethylated a-factor was prepared by 10 + 2 fragment coupling using OFm protection of the dipeptide carboxyl group followed by removal of all protecting groups with piperidine. Attempts to saponify a-factor were not successful. The synthetic nonfarnesylated and nonmethylated a-mating pheromones were 100-1000 times less active than the a-factor, indicating that although the methyl ester and the farnesyl group are not essential for biological activity, they are necessary for high potency.     

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

The synthesis is 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(PO₃ Me₂)-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(PO₃^tBu₂)-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).     

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.     

Synthesis of biologically active analogs of the dodecapeptide α-factor mating pheromone of Saccharomyces cerevisiae

A number of dodecapeptides with the sequence YIIKGVFWDPAC were synthesized using solid phase peptide synthesis. The purity of the crude cleavage product was found to be directly related to the cysteine protecting group and the conditions employed for cleavage of the peptide from the resin. When 4-methyl-benzyl cysteine was used, complete deprotection was only achieved with low-high HF conditions at temperatures of 10°-25°, whereas milder conditions could be used for dodecapeptides containing ethyl cysteine or acetamidomethyl cysteine. In several syntheses the biological activity of the crude cleavage product greatly exceeded the biological activity of a purified major peptide component. The high activity found in the crude cleavage peptide was probably due to minor peptide side products in which the cysteine sulfur was alkylated by hydrophobic species during HF treatment. Two dodecapeptides, YIIKGV-FWDPAC and YIIKGFWDPAC(Ethyl), had significant α-factor activity against MATα strains of Saccharomyces cerevisiae. These peptides represent the first synthetic analogs with α-factor activity.     

Synthesis of S-alkyl and C-terminal analogs of the Saccharomyces cerevisiae a-factor

The a-mating factor of Saccharomyces cerevisiae, Tyr-Ile-Ile-Lys-Gly-Val-Phe-Trp-Asp-Pro-Ala-Cys(far-nesyl)OCH₃, and 10 analogs modified at the cysteine side chain and/or the terminal carboxyl were synthesized using a combination of solid phase and solution phase methodologies. The strategy of synthesis involved the condensation of an amine terminal protected decapeptide with a carboxyl terminal S-alkylated dipeptide ester or amide using benzotriazoI-1-yloxy-tris(dimethylamino)-phosphonium hexafluoro-phosphate as the coupling agent. The protected decapeptide was assembled on a PAM-resin using 9-fluorenylmethoxycarbonyl (Fmoc) for the protection of the Tyr α-amine and Lys ω-amine and 9-fluorenyl-methyl ester (OFm) for the protection of the Asp β-carboxyl. Premature loss of the OFm group from the HF cleavage was observed at 0-2°, whereas no loss occurred when the cleavage reaction was conducted at – 5°. In contrast to these results, the OFm group in Asp(OFm) was partially removed by HF at – 5° and was completely stable to HF only at – 20°. The S-alkylated dipeptide esters were prepared, in yields from 64% to 88%, via thioalkylation of amine protected or unprotected dipeptide esters using potassium fluoride dihydrate as the base. The use of a tertiary amine as the base for thiohexadecanylation resulted in low reactivity.     

Synthesis of hydrazinopeptides using solid-phase N-electrophilic amination: extension to the Fmoc/tert-butyl strategy and chemistry of the N–N bond in strong acid media

Abstract: The synthesis of hydrazinopeptides using solid-phase N-electrophilic amination was extended to the Fmoc/tert-butyl strategy. Both Boc/benzyl and Fmoc/tert-butyl strategies led to the isolation of by-products arising from the partial instability of the N–N bond during the final cleavage and deprotection step. Two paths of decomposition have been shown: the cleavage of the N–N bond leading to the regeneration of the amine and a Hofmann-type elimination yielding original dianisyl adducts. Our data suggest that the Fmoc/tert-butyl strategy is better suited for the synthesis of hydrazinopeptides.     

Solid‐phase precipitation and extraction,a new separation process applied to the isolation of synthetic peptides

Abstract: A new method for separation and purification is described. The process, referred to as solid‐phase precipitation and extraction (SPPE), was developed and applied to postcleavage isolation of synthetic peptides. The technique uses normal approaches of chromatography and solid‐phase extraction sorbents with a precipitation or drying procedure so that the sorbent becomes a support matrix for thin‐film deposition of the compounds of interest. This procedure causes precipitated compounds of interest to be trapped on the large surface area or in the pores of the matrix so that by‐products and impurities can be removed by strong wash solvents. In application to solid‐phase peptide synthesis chemistry, by‐products from the cleavage and deprotection are selectively extracted from the crude sample mixture under mild conditions. In comparison to the ether precipitation method used in peptide chemistry, the SPPE process provides isolated products that are 14–17% (w/w) higher purity.     

Evaluation of new base‐labile2‐(4‐nitrophenylsulfonyl)ethoxycarbonyl (Nsc)‐amino acids for solid‐phase peptide synthesis

Abstract: 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 N^α‐Nsc‐protected amino acids for the synthesis of some melanotropins important to our research, namely, γ‐melanocyte‐stimulating hormone (γ‐MSH), its [Nle³]‐analogue, and a cyclic α‐MSH/β‐MSH hybrid. We developed an efficient protocol for the synthesis of the cyclic MSH analogue that yielded this peptide in > 98% purity. The γ‐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‐γ‐MSH analogue was synthesized more efficiently using the Fmoc strategy, suggesting that Nsc‐chemistry might not be the best methodology for certain sequences.     

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.     

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.     

Efficient Fmoc/solid-phase synthesis of Abu(P)-containing peptides using Fmoc-Abu(PO3Me2)-OH

The synthesis of the two 4-phosphono-2-aminobutanoyl-containing peptides, Leu-Arg-Arg-Val-Abu(P)-Leu-Gly-OH.CF₃CO₂H and Ile-Val-Pro-Asn-Abu(P)-Val-Glu-Glu-OH.CF₃CO₂H was accomplished by the use of Fmoc-Abu(PO₃Me₂)-OH in Fmoc solid-phase peptide synthesis. The protected phosphoamino acid, Fmoc-Abu(PO₃Me₂)-OH, was prepared from Boc-Asp-O'Bu in seven steps, the formation of the C—P linkage being effected by the treatment of Boc-Asa-O'Bu with dimethyl trimethylsilyl phosphite. Peptide synthesis was performed using Wang Resin as the polymer support with both peptides assembled by the use of PyBOP® for the coupling of Fmoc amino acids and 20%, piperidine for cleavage of the Fmoc group from the Fmoc-peptide after each coupling cycle. Cleavage of the peptide from the resin and peptide deprotection was accomplished by the treatment of the peptide-resin with 5%, thioanisole/TFA followed by cleavage of the methyl phosphonate group by 1 M bromotrimethylsilane/l M thioanisole in TFA.     

Chain-length dependence for secondary structure formation of homo-oligopep tides fromε-tert.-butyloxycarbonyl-L-lysine with a lipophilic C-terminal group

A solid-state and solution analysis of the homo-oligopeptides from ε-tert.-butyloxycarbonyl-L-lysine with p-oxymethylbenzylcholestan-3β-yl succinate as C-terminal group, using infrared absorption and circular dichroism, is described. The occurrence of intermolecular β-structure is seen in the solid state and in solvents of low polarity, e.g. methylene chloride, for peptides of intermediate size (from pentamer to decamer). Conversely, the eicosapeptide exhibits a high percentage of α-helical structure both in the solid state and in 2, 2, 2-trifluoroethanol. The influence of the C-terminal group on the conformational preferences of the ε-blocked homo-oligolysines in the solid state and in organic solvents appears negligible.     

Novel C-terminal gastrin antagonists Synthesis and biological activity

The C-terminal tetrapeptide, Trp-Met-Asp-Phe-NH₂, is a full agonist of gastrin, but des-Phe analogues, including Boc-Trp-Met-Asp-NH₂, are antagonists. To ascertain the minimum structural requirement for an antagonist, we used conventional solution phase methodology to synthesize analogues with further modifications including removal of the α-amino group of Trp, conversion of the indole to a phenyl ring, and methylation of amide bonds. These analogues were tested for their effect on pentagastrin-stimulated acid release in dogs surgically prepared with a gastric fistula. When infused intravenously at a dose of 20pmolkg^?1h^?1, the peptides significantly inhibited acid secretion. The extent of inhibition ranged from 12% to 60%. Thus, tripeptide analogues based on the C-terminal sequence of gastrin act as potent and specific antagonists of gastrin-stimulated acid secretion.     

PREPARATION AND PROPERTIES OF Nα-9-FLUORENYLMETHYLOXYCARBONYLAMINO ACIDS BEARING TERT.-BUTYL SIDE CHAIN PROTECTION

The preparation is described of several Nα-9-fluorenylmethyloxycarbonylamino acids and derivatives bearing tert.-butyl type side-chain protection of amine, carboxyl, guanido, hydroxyl, imidazol, and sulfhydryl functionalities. Physicochemical properties of these compounds have been determined. Cleavage of the Fmoc group by various amines appears to depend on the base strength and steric hindrance. Premature deblocking of Fmoc group by amine on solid support is very slow and may be negligible under the conditions of solid-phase synthesis.