Preparation of oligomer-free Nα-Fmoc and Nα-urethane amino acids

A variety of N^α-urethane blocked amino acids, in particular 9-fluorenylmethyloxycarbonyl (Fmoc) derivatives, have been synthesized by utilizing intermediate O,N-bis-trimethylsilyl-amino acids, formed in situ by treating an amino acid with trimethylsilylchloride and a base in an aprotic solvent. The intermediate is then reacted with an acylating agent. A general procedure is given which eliminates the oligomerization side reactions normally observed in Schotten-Baumann type methods. Protected amino acids obtained from this procedure are of high purity as judged by t.l.c, HPLC, ion exchange chromatography, and other physical parameters.     

Synthesis of β and γ-fluorenylmethyl esters of respectively Nα-Boc-L-aspartic acid and Nα-Boc-L-glutamic acid

The orthogonal synthesis of N^x-Boc-L-aspartic acid-γ-fluorenylmethyl ester and N^α-Boc-L-glutamic acid-δ-fluorenylmethyl ester is reported. This is a four-step synthesis that relies on the selective esterification of the side-chain carboxyl groups on N^x-CBZ-l -aspartic acid and N^α-CBZ-l -glutamic acid. Such selectivity is accomplished by initially protecting the a-carboxyl group through the formation of the corresponding 5-oxo-4-oxazolidinone ring. Following side-chain esterification, the α-carboxyl and α-amino groups are deprotected with acidolysis. Finally, the α-amino group is reprotected with the t-butyl-oxycarbonyl (Boc) group. Thus aspartic acid and glutamic acid have their side-chain carboxyl groups protected with the base-labile fluorenylmethyl ester (OFm) and their α-amino groups protected with the acid-labile Boc group. These residues, when used in conjunction with N^x-Boc-N^ε-Fmoc-l -lysine, are important in the formation of side-chain to side-chain cyclizations, via an amide bridge, during solid-phase peptide synthesis.     

Facile synthesis of Nα‐protected‐l‐α,γ‐diaminobutyric acids mediated by polymer‐supported hypervalent iodine reagent in water

Abstract: Hofmann rearrangement of N^α‐Boc‐l ‐Gln‐OH mediated by a polymer‐supported hypervalent iodine reagent poly[(4‐diacetoxyiodo)styrene] (PSDIB) in water afforded N^α‐Boc‐l ‐α,γ‐diaminobutyric acid (Boc‐Dab‐OH, 1 ) in 87% yield. N^α‐Z‐derivative (Z‐Dab‐OH, 2 ) was prepared with PSDIB in 83% yield. Since the reaction of N^α‐Fmoc‐Gln‐OH by this procedure did not proceed because of the insolubility of Fmoc‐Gln‐OH in aqueous media, we synthesized Fmoc‐Dab(Boc)‐OH ( 5 ) from 2 in 54% yield. Polymyxin B heptapeptide (PMBH) which contains four Dab residues was successfully synthesized in a solution‐phase synthesis.     

Facile synthesis of orthogonally protected amino acid building blocks for combinatorial N‐backbone cyclic peptide chemistry

Abstract: Protected N^α‐(aminoallyloxycarbonyl) and N^α‐(carboxyallyl) derivatives of all natural amino acids (except proline), and their chiral inverters, were synthesized using facile and efficient methods and were then used in the synthesis of N^α‐backbone cyclic peptides. Synthetic pathways for the preparation of the amino acid building units included alkylation, reductive amination and Michael addition using alkylhalides, aldehydes and α,β‐unsaturated carbonyl compounds, and the corresponding amino acids. The resulting amino acid prounits were then subjected to Fmoc protection affording optically pure amino acid building units. The appropriate synthetic pathway for each amino acid was chosen according to the nature of the side‐chain, resulting in fully orthogonal trifunctional building units for the solid‐phase peptide synthesis of small cyclic analogs of peptide loops (SCAPLs^?). N^α‐amino groups of building units were protected by Fmoc, functional side‐chains were protected by t‐Bu/Boc/Trt and N‐alkylamino or N‐alkylcarboxyl were protected by Alloc or Allyl, respectively. This facile method allows easy production of a large variety of amino acid building units in a short time, and is successfully employed in combinatorial chemistry as well as in large‐scale solid‐phase peptide synthesis. These building units have significant advantage in the synthesis of peptido‐related drugs.     

Synthesis and properties of human β-endorphin-(1–9) and its analogs

Four analogs of human β-endorphin (β_h-EP) were synthesized by the solid-phase method: β_h-EP-(1–9) (I), [D-Ala²]-β_h-EP-(1–9) (II), [Gln⁸]-β_h-EP-(1–9) (III), and [D-Ala², Gln⁸]-β_h-EP-(1–9) (IV). Measurement in a radioreceptor binding assay with use of tritiated β_h-EP as primary ligand gave relative potencies as follows: Met-enkephalin, 100; I, 76; II, 100; III, 200; IV, 200. Two new amino acid derivatives were prepared and used for synthesis of the analogs: N^α-t-butyloxycarbonyl-O-(cyclopentyl) -tyrosine and N^α-t-butyloxycarbonyl-γ-(cyclopentyl)-glutamic acid.     

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.     

Improved method for the synthesis of Nα-9-fluorenylmethyloxycarbonyl-Nδ,ω bis-adamantyloxycarbonyl-L-arginine

A modified method is reported for the prepartion of N^α-9-fluorenylmethyloxycarbonyl-N^δ,ω bis-adamantyloxycarbonyl-L-arginine, giving an overall yield of 60% over three steps based on N^α-benzyloxycarbonyl-L-arginine. Commercially available adamantyl fluoroformate for guanidine function protection of N^α benzyloxycarbonyl-L-arginine, catalytic transfer hydrogenation with formic acid on palladium black for removal of the benzyloxycarbonyl protecting group, and fluorenylmethylsuccinimidyl carbonate for the final synthesis, were introduced to simplify and reduce the cost of preparation of this arginine derivative. The reaction conditions have been accurately studied at each step in order to optimize the yields.     

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.     

Preparation and properties of Nα-trityl amino acid 1-hydroxybenzotriazole esters

N ^α-Trityl amino acid 1-hydroxybenzotriazole active esters, in contrast to HOBt esters of other protected amino acids, exhibit high hydrolytic stability and can be isolated in excellent yields and purity. The derivatives occur in three isomeric forms (an ester=I, two amides=II and III). All active ester forms react at high concentrations within 2 h with various alkyl amino acid esters to produce the corresponding protected dipeptides in high yields. Amorphous active esters on prolonged storage at — 20° rearrange partially towards form III without any sign of decomposition.     

A convenient general method for synthesis of Nα- or Nω-dithiasuccinoyl (Dts) amino acids and dipeptides: application of polyethylene glycol as a carrier for functional purification*

N ^α-Dithiasuccinoyl (Dts) amino acids ( 1 ) needed for solid-phase peptide synthesis have been prepared in good yields and excellent purities by a new method that exploits the solubility properties of polyethylene glycol (PEG; bifunctional with average molecular weight 2000 was found to be optimal). Suitably side-chain protected amino acid derivatives are first reacted with a polymeric xanthate ( 11 ), following which the free α-carboxyl is blocked by silylation and the Dts heterocycle is elaborated in the same pot by reaction with chlorocarbonylsulfenyl chloride ( 4 ). Upon aqueous workup, the polymeric carrier removes any urethane blocked amino acids which arise during the process. Exaggerated conditions were explored to prove the power of this functional purification approach, and mechanisms of formation of polymer-bound urethanes are proposed and supported by solution model studies. The preparation and characterization of the companion N-(iso-propyldithio)carbonyl derivative of proline is also presented.     

Chiral recognition of Nα-protected amino acids and derivatives in non-covalently molecularly imprinted polymers

Highly crosslinked synthetic polymers, selective for various N^α-protected amino acids and derivatives, were prepared by non-covalent molecular imprinting. Methacrylic acid and ethylene glycol dimethacrylate were copolymerized in the presence of the print molecules, which were subsequently extracted from the polymers. The recognition of the polymers for the print molecules and molecules of similar structures was investigated by using the polymers as stationary phases in HPLC. The functional groups of the print molecules interact via hydrogen bonds with the positioned carboxyls of the polymer. It was shown that the N^α-protecting group, the C^α-protecting group and the amino acid side chain are also recognized by the binding sites in the polymer. © Munksgaard 1994.     

Solid-phase synthesis of chicken vasoactive intestinal peptide by a mild procedure using Nα-9-fluorenylmethyloxycarbonyl amino acids

The octacosapeptide amide corresponding to the entire amino acid sequence of chicken vasoactive peptide (VIP) was assembled on a p-benzyloxybenzylamine resin support using the base-labile 9-fluorenylmethyloxycarbonyl as N^α-protecting group, cleaved by mild acid treatment, and purified by gel-filtration and ion-exchange chromatography. The symmetrical anhydride coupling was employed and monitored by two independent methods, and acetic anhydride termination was incorporated to minimize formation of deletion peptides. The homogeneity of the final product, obtained in 18% yield, was assessed by t.l.c., disc electrophoresis, amino-terminal amino acid analysis, and amino acid analyses of acid and enzyme hydrolysates. The purified chicken VIP was shown to be active on gastric acid secretion and on pancreatic blood flow. Previously reported ring closure of the Asp-Asn unit seemed to be at a minimum, owing to the mild basic and acid treatments.     

Susceptibility of glycans to β-elimination in Fmoc-based O-glycopeptide synthesis

order to investigate the possible extent of β-elimination occuring in Fmoc-based continuous-flow solid-phase glycopeptide synthesis, the influence of the pK_b of the base used for N^α-deprotection has been studied. A glycosylated pentapeptide as synthesized using 50%morpholine, 10%, piperidine or 2% DBU, respectively, in DMF for deprotection. The dehydropentapeptide N^α-.Ac-Thr-Thr-δAba-Val-Thr-NH₂, which would be formed in the case of β-elimination, was prepared independently and used as a control in HPLC analysis; however, this product was not formed under any of the deprotection conditions applied. Furthermore, a 23 amino acid long glycopeptide from human intestinal mucin was prepared using 2% DBU as a base for Fmoc cleavage, and similarly no β-elimination was observed. The glycopeptide products were subjected to a prolonged treatment with sodium hydroxide in methanol/water without significant formation of byproducts, and the pure glycopeptides were isolated and characterized by ¹H-NMR spectroscopy.     

Nsc and Fmoc Nα‐amino protection for solid‐phase peptide synthesis: a parallel study

Abstract The 2‐(4‐nitrophenylsulfonyl)ethoxycarbonyl (Nsc) group is an alternative to Fmoc for N^α‐protection in solid‐phase peptide synthesis. Nsc‐amino acids may be particularly suitable for automatic synthesizers, in which the amino acids are stored in solution, and the incorporation of residues prone to racemization such as Cys and His. Owing to the hydrophilicity of the Nsc group, these derivatives are useful for the preparation of protected peptides in convergent solid‐phase peptide synthesis strategies.     

Synthesis of an analogue of α-MSH-(1–10)-decapeptide as a substrate for enzymatic Nα-acetylation

The synthesis of a stable substrate for enzymatic N^α-acetylation is described. To this end an analogue of α-MSH-(1–10)-decapeptide with norleucine instead of methionine at position 4 is prepared. t-Butylation of an intermediate Z-Tyr-OMe leads only to about 75% conversion in a few hours' reaction time, and cannot be carried to completion. The [Nle⁴]-decapeptide is as good a substrate for enzymatic N^α-acetylation as the original decapeptide containing methionine.     

Synthesis of optically pure Cα-methyl-arginine

Optically pure L-(+)-C^α-methyl-arginine and D-(-)-C ^α-methyl-arginine were synthesized. Experimental results indicated that DL- C^α-methyl-arginine methyl ester could be resolved by trypsin, but workup posed a technical difficulty. Chemical resolution at the stage of DL-C^α-methyl-ornithine, followed by selective guanid-ination using N,N′-di-Cbz-S-methylisothiourea and hydrogenolysis provided a effective and practical method for the synthesis of optically pure C^α-methyl-arginine.     

Synthesis of N‐(2‐chloro‐3,4‐dimethoxybenzylideneamino)guanidinium acetate [α‐14C]

¹⁴C‐Labelled N‐(2‐chloro‐3,4‐dimethoxybenzylideneamino)guanidinium acetate has been synthesized as a part of a four‐step procedure which involved decarboxylation of 2‐chloro‐3,4‐dimethoxybenzoic acid by Pb(OAc)₄ to give 2‐chloro‐3,4‐dimethoxy‐1‐iodobenzene, followed by a selective lithiation at the iodine position and electrophilic substitution with N,N‐dimethylformamide [α‐¹⁴C] and final reaction with aminoguanidine bicarbonate. The specific activity was 59 mCi/mmol and the overall yield 49%. Copyright © 2002 John Wiley & Sons, Ltd.     

SYNTHESIS OF THE DODECAPEPTIDE-α MATING FACTOR OF SACCHAROMYCES CEREVISIAE

The synthesis of His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr, the dodeca-peptide α-mating factor from Saccharomyces cerevisiae, and its Ala²-and Cha²-(β-cyclohexylalanine) analogs are reported. Peptides were synthesized in solution using a combination of mixed anhydride and 1-hydroxybenzotriazole accelerated active ester coupling procedures. Dilute methanesulfonic acid (0.1–0.2 M) in methylene chloride-formic acid solution was employed to specifically remove the tert.-butoxycarbonyl group in the presence of the benzyloxycarbonyl group. Free peptides were obtained using catalytic transfer hydrogenation with formic acid as the hydrogen donor followed by mild acidolysis with trifluoroacetic acid. The α-factor and the Cha²-analog exhibited almost equal ability to cause “shmooing” of a-mating types of S. cerevisiae whereas the Ala²-analog exhibited no activity in this assay. These results differ with structure-activity studies reported on the tridecapeptide α-factor.     

Chemical synthesis and receptor binding of catfish somatostatin: a disulfide‐bridged β‐d‐Galp‐(1→3)‐α‐d‐GalpNAc O‐glycopeptide*

Abstract: The glycopeptide hormone catfish somatostatin (somatostatin‐22) has the amino acid sequence H‐Asp‐Asn‐Thr‐Val‐Thr‐Ser‐Lys‐Pro‐Leu‐Asn‐Cys‐Met‐Asn‐Tyr‐Phe‐Trp‐Lys‐Ser‐Arg‐Thr‐Ala‐Cys‐OH; it includes a cyclic disulfide connecting the two Cys residues, and the major naturally occurring glycoform contains d ‐GalNAc and d ‐Gal O‐glycosidically linked to Thr⁵. The linear sequence was assembled smoothly starting with an Fmoc‐Cys(Trt)‐PAC‐PEG‐PS support, using stepwise Fmoc solid‐phase chemistry. In addition to the nonglycosylated peptide, two glycosylated forms of somatostatin‐22 were accessed by incorporating as building blocks, respectively, N^α‐Fmoc‐Thr(Ac₃‐α‐D‐GalNAc)‐OH and N^α‐Fmoc‐Thr(Ac₄‐β‐D‐Gal‐(1→3)‐Ac₂‐α‐D‐GalNAc)‐OH. Acidolytic deprotection/cleavage of these peptidyl‐resins with trifluoroacetic acid/scavenger cocktails gave the corresponding acetyl‐protected glycopeptides with free sulfhydryl functions. Deacetylation, by methanolysis in the presence of catalytic sodium methoxide, was followed by mild oxidation at pH 7, mediated by N^α‐dithiasuccinoyl (Dts)‐glycine, to provide the desired monomeric cyclic disulfides. The purified peptides were tested for binding affinities to a panel of cloned human somatostatin receptor subtypes; in several cases, presence of the disaccharide moiety resulted in 2‐fold tighter binding.     

Chemical synthesis of α-inhibin-92 by the thiocarboxyl segment coupling method

The 92 amino acid residue peptide, α-inhibin-92 (α-IB-92), has been synthesized by the thiocarboxyl segment strategy. Three segments were synthesized by the solid phase method, purified, and characterized: [GlyS³⁴]-α-IB-92-(l-34) (I), CF₃CO-[GlyS⁶⁵]-α-IB-92-(35–65) (II), and Msc-α-IB-92-(66–92) (III). All were reacted with citraconic anhydride followed by removal of the Msc group in III to give Ia, IIa, and IIIa, respectively. Peptide IIIa was coupled to IIa by the silver nitrate/N-hydroxysuccinimide procedure and, after removal of uncoupled segments and the trifluoroacetyl group, Ia was coupled followed again by removal of uncoupled segments. Final deblocking to remove citraconyl groups was accomplished under exceptionally mild conditions in aqueous acetic acid. The synthetic product was identical to natural α-IB-92 in amino acid analysis, HPLC, gel electrophoresis, and tryptic mapping. The synthetic peptide was indistinguishable from natural α-IB-92 in a radioimmunoassay and in an in vitro mouse pituitary assay for measuring suppression of FSH release in the presence of LHRH.