Preparation of the very acid-sensitive Fmoc-Lys(Mtt)-OH Application in the synthesis of side-chain to side-chain cyclic peptides and oligolysine cores suitable for the solid-phase assembly of MAPs and TASPs

N ^α-9-Fluorenylmethoxycarbonyl-N^ε-4-methyltrityl-lysine, [Fmoc-Lys(Mtt)-OH], was prepared in two steps from lysine, in 42% overall yield. The N^ε-Mtt function can be quantitatively removed upon treatment with 1% TFA in dichloromethane or with a 1:2:7 mixture of acetic acid/trifluoroethanol/dichloromethane for 30 min and 1 h at room temperature, respectively. Under these conditions, groups of the tert-butyl type and peptide ester bonds to TFA-labile resins, such as the 2-chlorodiphenylmethyl- and the Wang-resin, remained intact. The utility of the new derivative in peptide synthesis has been exemplified with the synthesis of a cyclic cholecystokinin analog. As an example of further application, five types of lysine cores suitable for the solid-phase synthesis of one, two or three epitopes containing antigenic peptides or template-assembled synthetic proteins have been synthesized on Merrifield, Wang and 2-chlorodiphenylmethyl resin. © Munksgaard 1995.     

Multiple continuous-flow solid-phase peptide synthesis Synthesis of an HIV antigenic peptide and its omission analogues

Multiple continuous-flow solid-phase peptide synthesis was performed on a standard polystyrene-based resin under low-pressure conditions using a simple manually operated synthesizer. Stable-flow resin-packed columns were prepared in small polypropylene flow reactors, adjustable for volume. The concurrent synthesis of 10 peptides was carried out in flow reactors concatenated together; solvents and reactants were passed through this set of columns using moderate overpressure. One decapeptide, H-Val-Tyr-Tyr-Arg-Asp-Ser-Arg-Asn-Pro-Leu-NH₂, containing an antigenic determinant of the p31 protein product of the pol gene of the human immunodeficiency virus, and its nine omission analogues were synthesized.     

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 lability 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 lability 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.     

Convergent solid-phase peptide synthesis

The solid-phase synthesis, photolytic detachment from the solid support and purification in solution, of a fully-protected octapeptide containing a methionine residue (protected as the sulphoxide) is described. Protection of methionine in this manner avoids problems associated with the oxidation of this residue during the photolysis. The peptide has been purified by medium pressure liquid chromatography using solvent mixtures containing a high proportion of dimethylformamide in order to avoid precipitation of the peptide on the column.     

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.     

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.     

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 β-turn conformation in d₆-DMSO as evidenced by ¹H-NMR spectra.     

Use of a new sonically cleavable and acido-labile handle for solid-phase peptide amide synthesis

A new handle usable for solid-phase peptide amide synthesis was designed. New releasing conditions of the peptide using sonication allowed much shorter reaction times at lower TFA concentrations.     

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.     

The 2,4-dinitrophenyl group for protection of hydroxyl function of tyrosine during solid-phase peptide synthesis

The facile thiolytic cleavage of the O-2,4-dinitrophenyl (Dnp) tyrosine bond was applied to the solid-phase synthesis of the 22-amino acid residue peptide H-Asp-Ala-Val-Tyr -Thr-Gly-Leu-Asn-Thr-Arg-Asn-Gln-Glu-Thr-Tyr -Glu-Thr-Leu-Lys-His-Glu-Lys-OH, corresponding to positions 62-83 in the chain of the type 1 receptor for Fc_ε, domains expressed on the rat mucosal-type mast cells (line RBL-2H3). A method for the spectrophotometric determination of insoluble O-Dnp as well as of unprotected phenolic moieties of tyrosine was developed. It is based on monitoring S-Dnp-2-mercaptoethanol, produced upon O-Dnp thiolysis by 2-mercaptoethanol. © Munksgaard 1995. Dedicated to the memory of Dr. Susumu Funakoshi, a dear friend and a leader in peptide chemistry.     

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 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.     

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).     

Sulfur protection with the 3-nitro-2-pyridinesulfenyl group in solid-phase peptide synthesis

The 3-nitro-2-pyridinesulfenyl(Npys) group has been used successfully for side chain protection of cysteine during the stepwise solid-phase synthesis of Lys⁸-vasopressin(LVP) on benzhydrylamine resin. The versatility and limitations of this group have been evaluated by comparison of this synthesis with a parallel control synthesis using the 3,4-dimethylbenzyl(DMB) group and with a synthesis utilizing a combination of both groups. The Npys group was found to be stable to TFA as reported and, in addition, was found to be stable to HF:anisole(9:1) for 45 min at 0°, but not when thiol was present in either reagent. Furthermore, compatibility of the Npys group with the Boc-benzyl synthetic tactic in solid-phase peptide synthesis was demonstrated. LVP with full biological activity was obtained after purification by gel filtration and reverse-phase HPLC.     

Aggregation of resin-bound peptides during solid-phase peptide synthesis

Nonrandom incomplete aminoacylation of a pendent peptide chain on an insoluble polymeric support during solid-phase peptide synthesis is sequence-dependent and is caused by aggregation of peptide chains, manifested by a decreased swelling capacity. The volume of the swollen peptidyl-resin after each coupling during the syntheses of 87 sequence unrelated peptides was measured, and for each amino acid an aggregation parameter, 〈P_a〉, was derived that reflects the propensity of the swollen volume of peptidyl-resin to decrease during peptide synthesis. These aggregation parameters were used to predict potentially difficult sequences.     

Application of the allyloxycarbonyl protecting group for the indole of Trp in solid-phase peptide synthesis*

The synthesis and stability of allyloxycarbonyl (Aloe) indole-protected Trp derivatives and their application in solid-phase peptide synthesis are reported. The study shows that the Aloe protection on the indole moiety is suitable for orthogonal protection in the Fmoc/tBu strategy if the Fmoc group is cleaved with DBU. Several tryptophan-containing peptides have been synthesized including dynorphin A-(1-13), which has been intensively studied with respect to side reactions during the final TFA cleavage procedure. The results demonstrate the protective function of the Aloe group on the Trp during final deprotection. Furthermore, it could be demonstrated that Trp(Aloe)-containing peptides can be isolated and that the Aloe group can then be removed in a second step. The synthesis of phosphorylated delta sleep inducing peptide (P-DSIP) using the global phosphorylation approach provides another example in which Trp indole protection by Aloe prevents the formation of oxidative side products.     

In situ generation of Fmoc-amino acid chlorides using bis-(trichloromethyl)carbonate and its utilization for difficult couplings in solid-phase peptide synthesis

Abstract: This paper reports procedures for the straightforward in situ generation of Fmoc-amino acid chlorides using bis-(trichloromethyl)carbonate (BTC) and their utilization for difficult couplings during solid-phase peptide synthesis. The BTC-mediated coupling of all Fmoc-protected proteinogenic amino acids to a large variety of N-alkylated amino acid-peptidyl-resin was studied. The majority of the couplings proceeded with quantitative conversion and without racemization. The utilization of BTC-mediated coupling for facile solid-phase synthesis of backbone cyclic peptides is presented.     

Comparison of 55% TFA/CH2Cl2 and 100% TFA for Boc group removal during solid-phase peptide synthesis

Two parallel syntheses of 40 C-terminal amide peptides, ranging in length from 4 to 20 residues, have been carried out using simultaneous multiple peptide synthesis. All synthetic steps, other than the removal of the Boc group, were performed simultaneously under identical experimental conditions. The two sets of peptides were deprotected with either 55% TFA/DCM for 30 min or 100% TFA for 5 min. The purity of the peptides obtained when deprotecting with 55% TFA/DCM was, on average, 9% higher than with 100% TFA. The major impurity obtained during synthesis when 100% TFA was used for Boc removal corresponded to the omission of the second amino-acid residue added. Volumetric measurements of the swelling of the resin in the different deprotection solvents were carried out. These showed that the omission analogs generated are probably due to insufficient swelling of the resin, resulting in limited solvent transfer of 100% TFA into the resin and, in turn, incomplete Boc removal.     

Incomplete Fmoc deprotection in solid-phase synthesis of peptides

During solid-phase peptide synthesis of homo-oligopeptides containing leucine or alanine using the Fmoc strategy, we have observed ineffective N-α-deprotection with piperidine in a sequence-dependent manner. Incomplete deprotection was found to be associated with subsequent slow or incomplete amino acid coupling. Optimization of the deprotection step was carried out by varying the experimental conditions e.g. deprotection time, temperature, solvents and addition of chaotropes. Coupling and deprotection steps have been investigated using color monitoring, as well as FAB MS and HPLC for product analysis. The phenomena of difficult coupling and deprotection steps in the investigated systems have been demonstrated to have the same physical chemical origins, p-sheet formation. © Munksgaard 1994.     

Further studies into the Boc/solid-phase synthesis of Ser(P)- and Thr(P)-containing peptides

The Ser(P)-containing peptide corresponding to phospholamban 11-19, Ac-Ala-Ile-Arg-Are-Ala-Ser(P)-Thr-Ile-Glu-NH₂, was prepared by the use of Boc-Ser(PO₃Ph₂)-OH in Boc/solid-phase peptide synthesis followed by HF cleavage of the peptide from the polystyrene resin and subsequent platinum-mediated hydrogenolytic cleavage of the phenyl phosphate groups. A study of the HF deprotection step showed that extensive dephosphorylation of the Ser(PO₃Ph₂)-residue occurred using three commonly used HF conditions and gave rise to large quantities of the Ser-containing peptide. The subsequent study of model peptide systems under standard HF conditions established firstly that the extent of dephosphorylation was dependent on the HF-contact time, and secondly that the Ser(PO₃Ph₂) residue underwent dephosphorylation at a slightly higher rate than the Thr(PO₃Ph₂) residue. © Munksgaard 1994.