Synthesis and application of acid labile anchor groups for the synthesis of peptide amides by Fmoc-solid-phase peptide synthesis

The preparation and application of a new linker for the synthesis of peptide amides using a modified Fmoc-method is described. The new anchor group was developed based on our experience with 4,4′-dimeth-oxybenzhydryl (Mbh)-protecting group for amides. Lability towards acid treatment was increased dramatically and results in an easy cleavage procedure for the preparation of peptide amides. The synthesis of N-9-fluorenylmethoxycarbonyl-[(5-carboxylatoethyl-2.4-dimethoxyphenyl)-4′-methoxyphenyl]-methyla-mine is reported in detail. This linker was coupled to a commercially available aminomethyl polystyrene resin. Peptide synthesis proceeded smoothly using HOOBt esters of Fmoc-amino acids. Release of the peptide amide and final cleavage of the side chain protecting groups was accomplished by treatment with trifluoroacetic acid-dichloromethane mixtures in the presence of scavengers. The synthesis of peptide amides such as LHRH and C-terminal hexapeptide of secretin are given as examples.     

Cleavage kinetics and anchor linked intermediates in solid phase peptide amide synthesis

Kinetics and cleavage conditions of peptide amide synthesis were studied using the anchor molecules 5-(4′-aminomethyl-3′,5′-dimethoxyphenoxy)valeric acid (4-ADPV-OH) and 5-(2′-aminomethyl-3′,5′-dimethoxyphenoxy)valeric acid (2-ADPV-OH). Unexpectedly the anchor amide alanyl-4-ADPV-NH₂ was isolated and characterized as an intermediate during the cleavage with trifluoroacetic acid (TFA) of alanyl-4-ADPV-alanyl-aminomethyl-polystyrene to yield the alanine amide. As a matter of fact the NH–CH_α bond of the alanyl spacer has to be cleaved to form this intermediate. Using TFA-dichloro-methane (1:9) alanyl-4-ADPV-NH₂ was obtained as a cleavage product in 50% yield within 60min, whereas the isomeric alanyl-2-ADPV-NH₂ was formed more slowly under these mild conditions. At high TFA concentration no difference between the 2- and 4-ADPV anchor was observed in the rate of formation of the free alanine amide. The presence of tryptophan amide in the cleavage mixture resulted in an anchor alkylated tryptophan amide, which remains stable in acidic solution but disappears rapidly in the presence of the resin. A low TFA/high TFA cleavage procedure is recommended for peptide amid synthesis applying the ADPV anchor.     

Preparation of protected peptide amides using the Fmoc chemical protocol

Different resins were examined for their potential use in the solid phase synthesis of protected peptide amides using the 9-fluorenylmethoxycarbonyl (Fmoc) chemical protocol. The model protected peptide amide BocTyr-Gly-Gly-Phe-Leu-Arg(Pmc)NH₂ (1) was synthesized on both the acid-labile 4-(2′,4’-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin (Rink amide resin) (2) and on resins containing the base-labile linker 4-hydroxymethylbenzoic acid. Of the resins examined only the methylbenzhydrylamine resin containing the 4-hydroxymethylbenzoic acid linkage, which was cleaved by ammonolysis in isopropanoll, gave the model peptide 1 in good overall yield (53% including functionalization). Thus the synthesis of protected peptide amides by solid phase synthesis using Fmoc-protected amino acids with t-butyl-type side chain protecting groups is feasible. The choice of peptide-resin linkage and its cleavage conditions, however, are critical to the success of such syntheses. The potential application of this synthetic strategy to the preparation of novel peptide amides is discussed.     

Practical approach to solid-phase synthesis of C-terminal peptide amides under mild conditions based on a photolysable anchoring linkage1

Several 3-nitro-4-(N-protected aminomethyl)benzoic acids, with protection provided by tert.-butyloxycar-bonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc), trifluoroacetyl (Tfa), dithiasuccinoyl (Dts), or phthaloyl (Phth), have been prepared by reproducible routes. Synthesis of Dts-handle 6 illustrates some particularly novel and efficient chemistry, and is preferred over more intricate routes to Boc-handle 3 and Fmoc-handle 4. The five handles were each evaluated for their application to the synthesis of peptide amides. Coupling onto amino-functionalized supports provided a general starting point for peptide chain assembly. The handle amino function was deblocked (Boc, Fmoc, Dts), the C-terminal residue was coupled as its N^x-protected free acid, and ultimately the ortho-nitrobenzylamide anchoring linkage was cleaved photolytic-ally to give the corresponding amide. Starting with handles 3, 4, and 6, several free and protected peptide amides were synthesized.     

4-Sulfobenzyl ester – its use in peptide synthesis

Amino acid 4-sulfobenzyl esters were employed for the synthesis of peptides in solution. They significantly increase the hydrophilicity of protected intermediates as shown by analytical reversed-phase high performance liquid chromatography and counter-current distribution. General methods for working up are described which permit simple and standardized isolations of products. The use of several coupling methods was demonstrated in the synthesis of Z-Val-Gly-OBzl-SO₃NH₄. Ion-exchange chromatography was introduced as a selective purification procedure for protected peptide 4-sulfobenzyl esters. A step-wise synthesis of [Leu⁵]-enkephalin was performed, starting from leucine 4-sulfobenzyl ester. Removal of N-terminal protecting groups produced zwitterionic peptide 4-sulfobenzyl esters. These were readily purified by crystallization from slightly acidic media; no further purification was necessary. The biologically fully active pentapeptide Tyr-Gly-Gly-Phe-Leu was obtained in an overall yield of 30.2%.     

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^α-9-fluorenyl-methyloxycarbonyl (Fmoc) and N^α-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°. 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.     

Design of chimeric peptide ligands to galanin receptors and substance P receptors

Several chimeric peptides were synthesized and found to be high-affinity ligands for both galanin and substance P receptors in membranes from the rat hypothalamus. The peptide galantide, composed of the N-terminal part of galanin and C-terminal part of substance P (SP), galanin-(1-12)-Pro-SP-(5-11) amide, which is the first galanin antagonist to be reported, recognizes two classes of galanin binding sites (K_D(1)<0.1 nM and K_D(2)∼ 6 nM) in the rat hypothalamus, while it appears to bind to a single population of SP receptors (K_D∼ 40 nM). The chimeric peptide has higher affinity towards galanin receptors than the endogenous peptide galanin-(1-29) (KD ∼ 1 nm ) or its N-terminal fragment galanin-(1-13) (K_D∼ 1,nm ), which constitutes the A′-terminus of the chimeric peptide. Galantide has also higher affinity for the SP receptors than the C-terminal SP fragment-(4-11) amide (K_D= 0.4μm ), which constitutes its C-terminal portion. Substitution of amino acid residues, which is of importance for recognition of galanin by galanin receptors, such as [Trp²], in the galanin portion of the chimeric peptide or substitution of ([Phe⁷] or [Met¹¹]-amide) in the SP portion of chimeric peptide both cause significant loss in affinity of the analogs of galantide for both the galanin- and the SP-receptors. These results suggest that the high affinity of the chimeric peptide, galantide, may in part be accounted for by simultaneous recognition/binding to both receptors. In line with this suggestion is the finding that the binding of the chimeric ligands to the galanin receptor is strongly influenced by the presence of SP (1 μm ) or spantide (1 μm ). We have performed the synthesis and binding studies with 11 chimeric peptides, all composed of the N-terminal galanin-(1-13) fragment or of its analogs, linked to the C-terminal portion of SP or its peptide antagonist, spantide. Our results, similar to earlier reports on chimeric peptides, suggest that high-affinity ligands to peptide receptors can be produced by linking biologically active N-terminal and C-terminal portions of peptides via linkers, enabling a) independent recognition of the chimeric peptide by the relevant receptors and b) intramolecular interactions between the joined N- and C-terminal peptide fragments. These two phenomena may also explain why some of the chimeric peptides have higher affinity than the endogenous peptide(s) and why galantide, and some of its analogs presented here, behave(s) as a galanin receptor antagonist(s).     

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

Abstract: 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.     

Solid-phase synthesis of protected peptides using new cobalt(III) ammine linkers†

Cobalt(III) ammine complexes of the type cis-[CoL₄(4-AMB)O-AA-Boc](CF₃SO₃)₂, where L₄= bisethylenediamine (en)₂ or tetraammine (NH₃)₄, and 4-AMB = 4-(aminomethyl)benzoic acid, have been synthesized and used as linkers to polystyrene resins for solid-phase synthesis of protected peptides. Boc/t-Bu-protected [Leu⁵]enkephalin was assembled on the two different Co(III) resins, and then cleaved from the resins by reduction of the Co(III) center in 93–96%; yield. HPLC-purified protected [Leu⁵]enkephalin was obtained in 67–69% overall yield and characterized by amino acid analysis and ¹H NMR. Stepwise synthesis on the Co(en)₂-resin was also used in the assembly of Boc-Asp(OcHex)-Arg(Mts)-Gly-Asp(OcHex)-Ala-Pro-Lys(2Cl-Z)-Gly-OH, a sequence from collagen α1 Type 1. The protected peptide was cleaved from the Co(III) resin in 74% yield, and the HPLC-purified nonapeptide was characterized by amino acid analysis, ¹H NMR and liquid secondary-ion mass spectrometry (LSIMS). New routes are described for the synthesis of isomerically pure Co(III) anchor complexes. The Co(III) resins were found to be compatible with both the tert-butyloxycarbonyl (Boc) and the 9-fluorenylmethoxycarbonyl (Fmoc) N^α-protecting group strategies used in solid-phase peptide synthesis.     

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.     

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.     

Synthesis of peptide amides using Fmoc-based solid-phase procedures on 4-methylbenzhydrylamine resins

A two-step low-high protocol for the efficient synthesis of peptide amides is described. The protocol exploits the efficiency of Reagent K for side-chain deprotection with the capability of the hard acid trifluoromethane-sulfonic acid (TFSMA) for cleavage of the peptide from the benzhydrylamine resin. This procedure has proven to be an effective method for the synthesis of peptide amides. The formation of α-aminosuccinimide (Asu) derivatives were observed with aspartyl-containing peptides as a minor side reaction product of this procedure, but this Asp→Asu rearrangement could be successfully suppressed by employing low temperature conditions. The N- to O-acyl rearrangement of threonine and/or serine residues also only occurred to a minor extent under these synthetic conditions. © Munksgaard 1995.     

4-Sulfobenzyl ester – an anionic protecting group for peptide synthesis

The preparation of the 4-sulfobenzyl esters of 18 amino acid derivatives is described. This carboxyl protecting group was introduced according to Hubbuch et al. (1980). The caesium or dicyclohexylammonium salts of N-terminal protected amino acids were reacted with 4-(bromomethyl)benzenesulfonate (1). After N-terminal deblocking, the amino acid-4-sulfobenzyl esters were isolated as zwitterions. The protecting group was removable by catalytic hydrogenation and by saponification. The 4-sulfobenzyl esters could be easily converted to amides and hydrazides. They were stable to 2 M hydrogen bromide in acetic acid as well as to a 10-fold excess of trifluoromethane sulfonic acid in trifluoro-acetic acid. The behaviours of ⁺H₂-Gly-Phe-Leu-OBzl-SO^-₃ and the corresponding methyl, benzyl and 4-nitrobenzyl esters were compared under various conditions.     

Enhancement of peptide coupling reactions by 4-dimethylaminopyridine

4-Dimethylaminopyridine (DMAP) was found to be useful in the enhancement of peptide coupling reactions mediated by dicyclohexylcarbodiimide or symmetrical anhydrides. In an automated synthesis of the model heptapeptide Boc-Ala-Cle-Ile-Val-Pro-Arg(Tos)-Gly-OCH₂-Resin (Cle, cycloleucine), the efficiencies of various coupling methods such as dicyclohexylcarbodiimide, dicyclohexylcarbodiimide plus 1-hydroxybenzotriazole, and symmetrical anhydride were compared with that of dicyclohexylcarbodiimide plus 4-dimethylaminopyridine. Based on the amino acid composition of the peptide-resin samples and high pressure liquid chromatographic analyses of the protected heptapeptide amide obtained from the ammonolytic cleavage of the peptideresin samples, it was concluded that only dicyclohexylcarbodiimide plus 4-dimethylaminopyridine gave the desired near quantitative couplings in those cycles involving the sterically hindered amino acid residues. Observations were also made that 4-dimethylaminopyridine was a useful additive in a modified symmetrical anhydride method of coupling. In the synthesis of the model tetrapeptide Leu-Ala-Gly-Val on a Pam resin, the anhydride couplings were accelerated by DMAP and the product was equivalent in homogeneity to that obtained by the best previous methods. In addition, no racemization was detectable by a sensitive chromatographic method. There also was no detectable racemization found in a DCC-DMAP coupling of Boc-Ile-OH with H-Val-OCH₂-resin. However, significant racemization was observed during the coupling of Boc-Phe-OH with H-Glu(OBzl)-OCH₂-resin. DMAP is recommended as an additive for coupling hindered amino acids, particularly C^α-substituted residues, where little or no racemization is expected.     

Efficient Fmoc/solid-phase peptide synthesis of O-phosphotyrosyl-containing peptides and their use as phosphatase substrates

A general synthetic method for the efficient preparation of Tyr(P) -containing peptides is described by the use of Fmoc-Tyr(PO₃¹Bu₂) -OH in Fmoc/solid-phase synthesis followed by simultaneous cleavage of the peptide from the resin and peptide deprotection by acidolytic treatment. The applicability of this approach is demonstrated by the synthesis of H-Ser-Ser-Ser-Tyr(P) -Tyr(P) -OH.TFA and the synthesis of the phosphorylated forms of the two physiological peptides, angiotensin II and neurotensin 8–13. In addition, the three phosphorylated peptides were used as substrates in the study of the local specificity determinants of T-cell protein tyrosine phosphatase. In a competition assay using ³²P-radiolabeled [Tyr(P)]⁴-angiotensin II, both un-labeled synthetic [Tyr(P)]⁴-angiotensin II and Ser-Ser-Ser-Tyr(P) -Tyr(P) reduced the release of ³²P and indicated that they efficiently competed as substrates for the phosphatase. Conversely, [Tyr(P)]⁴-neurotensin 8–13 was ineffective as a competitive substrate and indicated that this particular Tyr(P) -containing peptide sequence was not recognized by the enzyme. The marked difference in the recognition of Asp-Arg-Val-Tyr(P) -Ile-His-Pro-Phe and Arg-Arg-Pro-Tyr(P) -Ile-Leu is consistent with the presence of an acidic residue in the -3 position relative to the Tyr(P) residue.     

Synthesis of N-tert.-butoxycarbonyl(α-phenyl)aminomethylphenoxyacetic acid for use as a handle in solid-phase synthesis of peptide α-carboxamides

N-tert.-butoxycarbonyl-aminomethyl(α-phenyl)phenoxyacetic acid was synthesized and found to be suitable for use as a handle in the solid-phase synthesis of peptide α-carboxamides. This handle was prepared with an 82% yield when N-tert.-butoxycarbonyl-(p-hydroxy)benzhydrylamine was treated with excess sodium iodoacetate under alkaline conditions. In stability studies the linkage between the C-terminal amino acid and the handle was found to be resistant to acidolysis in 50% TFA/CH₂Cl₂ (< 1% loss after 10h). Upon treatment for 30min with HF:anisole(9:1) at 0°, 92% cleavage of glycinamide from Glyhandle-resin was obtained. In a test synthesis of a peptide α-carboxamide, pyroglutamylhistidylprolinamide was synthesized in 83% yield. Two other handles, tert.-butoxycarbonyl-aminomethylphenoxyacetic acid and N-tert.-butoxycarbonyl-aminornethylphenyloxymethylphenoxyacetic acid, were also synthesized but found to be unsuitable for carboxamide synthesis under the same conditions of solid-phase synthesis.     

Synthesis and characterization of indolicidin,a tryptophan-rich antimicrobial peptide from bovine neutrophils *

Indolicidin, a novel tryptophan-rich microbicidal tridecapeptide amide isolated originally from granules of bovine neutrophils, has been prepared by optimized manual and automated protocols of stepwise solid-phase synthesis with N^α-9-fluorenylmethyloxycarbonyl (Fmoc) amino acid derivatives. Both standard polystyrene (PS) and polyethylene glycol-polystyrene (PEG-PS) graft supports were used in combination with handles that provide C-terminal peptide amides: 5-(4-Fmoc-aminomethyl-3,5-dimethoxyphenoxy)valeric acid (PAL) or 5-(9-Fmoc-aminoxanthen-2-oxy)valeric acid (XAL). Final deprotection/cleavage was carried out with reagent K, trifluoroacetic acid–phenol–water–thioanisole–1,2-ethanedithiol (82.5:5:5:5:2.5), or reagent B, trifluoroacetic acid–phenol–water–tri(isopropyl)silane (88:5:5:2), and related cocktails. Initial purities as high as 93% were obtained immediately following cleavage. In the largest-scale synthesis carried out, 0.8 g of HPLC-purified indolicidin (> 99% pure) was obtained, representing a 39% overall yield based on C-terminal Arg(Pmc) anchored to PAL-PS-resin. The main synthetic product, and some by-products, were characterized by analytical high-performance liquid chromatography (HPLC), sequencing, and fast atom bombardment mass spectrometry (FABMS). The antimicrobial potencies of natural and synthetic indolicidin, as determined by in vitro antibacterial and antifungal assays, were identical. Further, the reactivities of natural and synthetic peptides with anti-indolicidin antibody were indistinguishable. © Munksgaard 1995.     

Solid phase synthesis of retro-inverso peptide analogues

The solid phase synthesis of a partially modified retro-inverso analogue of the bradykinin potentiating peptide BPP_9a, [gLys⁶, (R,S)-mPhe⁷, Ala⁸] BPP_9a is described. The analogue, which is active in vitro and in vivo, displays prolonged resistance towards cleavage by angiotensin converting enzyme (ACE).     

Synthesis of N,N‐diethyl‐4‐[phenyl‐(piperidin‐4‐ylidene)methyl]‐[carboxy‐14C]benzamide,a potent δ opioid receptor agonist

The synthesis of carbon‐14 labelled N,N‐diethyl‐4‐[phenyl‐(piperidin‐4‐ylidene)methyl]‐benzamide is described. The radioisotope is introduced via an aryllithium reaction with ¹⁴CO₂ to form the labelled acid, which is subsequently transformed into the amide. Copyright © 2002 John Wiley & Sons, Ltd.     

Acid-labile anchoring linkages for solid phase synthesis of C-terminal asparagine peptides using the Fmoc strategy

Two acid-labile substituted benzylamine type anchoring linkages, 4-benzoxy-2,6-dimethoxybenzylamine and 2-benzoxy-4,6-dimethoxybenzylamine, for solid phase synthesis of peptide amides were prepared. The N^a-9-fluorenylmethyloxycarbonyl (Fmoc) amino acids could be easily attached to the resins with DCC/HOBt (loading 0.5–0.6 mmol/g resin). After final removal of the N^a-protecting groups, treatment with TFA (50–95%) yielded amino acid and peptide amides in high purity. As we could show for the synthesis of thymulin (FTS, pGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn), these two resins with anchoring linkages are well suited for the synthesis of C-terminal Asn peptides using protected aspartic acid derivative as starting material.