Synthesis of pentafluorophenyl, 2,4,5-trichlorophenyl and pentachlorophenyl esters of Fmoc-amino acids using Fmoc-amino acid chlorides as intermediates

The synthesis of pentafluorophenyl,2,4,5-trichlorophenyl and pentachlorophenyl esters ofFmoc-amino acids has been accomplished via Fmoc-amino acidchlorides as intermediates. A two phase system with a mildin-organic base (3% NaHCO₃) in the aqueous layer was used. The esterification reaction was clean and complete in about2–3 hr. All the esters prepared have been obtained in goodyield and are fully characterised.     

Solid-phase synthesis of bombesin by continuous flow procedure using Fmoc-amino acids

Bombesin has been synthesized by the continuous flow solid-phase procedure on the derivatized Kieselguhr-supported polydimethylacrylamide resin. Preformed Fmoc-amino acid symmetrical anhydrides (Met, Leu, and Arg) and Fmoc-amino acid active esters were used for amine acylation. The Mtr and the Pmc groups have been alternatively used for masking the side chain function of Arg-3. The progress of the synthesis was monitored by different analytical methods including quantitative solid-phase Edman degradation. Cleavage from the resin and simultaneous formation of the C-terminal amide function were achieved with a methanolic ammonia solution yielding indistinguishable crude peptides which have been purified by HPLC and fully characterized. Preliminary pharmacological experiments indicated that the activity of the synthetic peptides is similar to that previously measured for other synthetic bombesins. For comparison bombesin has also been prepared by solid-phase synthesis on 4-methyl benhydrylamine resin using the Boc chemistry. The results of the two strategies are discussed and compared.     

Synthesis of Fmoc-amino acid chlorides assisted by ultrasonication, a rapid approach

The chloride derivatives of all common Fmoc-amino acids lacking polar side chains as well as a number of benzyl based side chain protection have been prepared using the corresponding amino acid and thionyl chloride assisted by ultrasound has been described. The protocol is simple, efficient and rapid. All the acid chlorides prepared have been obtained in good yield and purity.     

Synthesis of Fmoc-amino acid chlorides assisted by ultrasonication,a rapid approach

Summary The chloride derivatives of all common Fmoc-amino acids lacking polar side chains as well as a number of benzyl based side chain protection have been prepared using the corresponding amino acid and thionyl chloride assisted by ultrasound has been described. The protocol is simple, efficient and rapid. All the acid chlorides prepared have been obtained in good yield and purity.     

Synthetic strategy for side chain mono-N-alkylation of Fmoc-amino acids promoted by molecular sieves

A new synthetic strategy to alkylate amino groups under mild conditions has been developed. It utilizes only 4 ? molecular sieves as base in order to promote the N-alkylation reaction, in presence of the appropriate alkyl halide. The methodology was validated by the simple and efficient side-chain N-alkylation of o-Ns-protected Fmoc-amino acid. One of them was introduced as building block into a peptide sequence, thus allowing the preparation of site-specific alkylated peptide molecules.     

Fmoc-amino acid chlorides in solid phase synthesis of opioid peptides.

Fmoc-amino acid chlorides were employed in the solid phase synthesis of the opioid peptides [Leu]enkephalin, [Leu]enkephalin amide, and dermorphin. The conventional polystyrene-based Merrifield resin or Wang's resin served as solid support. A binary salt of either triethylamine or diisopropylethylamine in the presence of 1-hydroxybenzotriazole or pivalic acid was used for acylation. The coupling rates were quite fast, being comparatively faster when 1-hydroxybenzotriazole was used along with triethylamine or diisopropylethylamine. The peptides obtained in good yields showed, after purification, biological and spectral properties identical with those of the natural peptides.     

Synthesis of Fmoc-amino acid fluorides via DAST, an alternative fluorinating agent

Summary DAST [(diethylamino)sulfur trifluoride] has shown to be an excellent alternative fluorinating agent for synthesis of Fmoc-amino acid fluorides. The use of DAST is advantageous relative to other fluorinating agents, such as cyanuric fluoride, since handling and isolation of the acid fluorides are facile.     

2-Chlorotrityl chloride resin. Studies on anchoring of Fmoc-amino acids and peptide cleavage

The esterification of 2-chlorotrityl chloride resin with Fmoc-amino acids in the presence of DIEA is studied under various conditions. High esterification yields are obtained using 0.6 equiv. Fmoc-amino acid/mmol resin in DCM or DCE, in 25 min, at room temperature. The reaction proceeds without by product formation even in the case of Fmoc-Asn and Fmoc-Gln. The quantitative and easy cleavage of amino acids and peptides from 2-chlorotrityl resin, by using AcOH/TFE/DCM mixtures, is accomplished within 15-60 min at room temperature, while t-butyl type protecting groups remain unaffected. Under these exceptionally mild conditions 2-chlorotrityl cations generated during the cleavage of amino acids and peptides from resin do not attack the nucleophilic side chains of Trp, Met, and Tyr.     

Nmr and computer-aided modeling studies of the interactions between a cyclic hexapeptide and the two enantiomers of some Boc- and Fmoc-amino acids

The cyclic hexapeptide cyclo[-Pro¹-Gly²-Glu³(OBzl) -Pro⁴-Phes⁵-Leu⁶-] ( 1 ) was modeled and synthesized to be used for chiral discrimination studies. Total correlated spectroscopy and nuclear Overhauser effect spectroscopy spectra of the cyclic hexapeptide 1 in CDCl₃ showed the presence of three stereoisomers: two dominant stereoisomers 1a and 1b that exchanged chemically with each other, and a minor stereoisomer 1c (4%) that exchanged exclusively with the stereoisomer 1b . Of the two dominant stereoisomers, only 1a interacted specifically with t-butyloxycarbonyl (Boc-) and 9-flourenylmethyloxycarbonyl (Fmoc-) amino acids in CDCl₃. The interaction site of la when complexing with the derivatized amino acids was the chain segment Phe⁵-Leu⁶. The Phe⁵ NH and Leu⁶ NH protons are contiguous and solvent exposed. Their nmr signals shifted strongly downfield with the addition of Boc- or Fmoc- amino acids to the peptide solution. Thus, both NH protons hydrogen bond to the amino acids, forming a two-point hydrogen-bonding complex. The peptide stereoisomer 1b did not interact specifically with the Boc- and Fmoc-amino acids because of the lack of two contiguous and solvent-exposed peptidic NH protons that seem to be needed for specific interactions of the cyclic hexapeptide 1 with the Boc- and Fmoc-amino acids. A clear difference in the interaction of 1a with D - and L -enantiomers of BocTrp and Fmoc-Trp was observed with nmr spectroscopy. Docking models and molecular mechanics calculations together with nmr observations showed that the NH proton of the indole ring of the Boc-L -Trp and the Fmoc-L- Trp hydrogen bonded to the Pro¹ carbonyl group. In this three-point hydrogen-bonding complex, the indole ring becomes locked underneath the Leu residue. The nmr signals of all the Leu⁶ protons (except for Leu NH) shifted strongly upfield owing to the shielding effect of the indole aromatic ring currents. The indole NH of the D -enantiomer did not hydrogen bond to the Pro¹ carbonyl group because the formation of such a three-point hydrogen-bonding complex was thermodynamically unfavorable. © 1993 John Wiley & Sons, Inc.     

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

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.     

1-(t-Butyldimethylsilyloxy) benzotriazole (TBDMS-OBt): A new and novel reagent for the synthesis of peptides

Synthesis and use of 1-(t-butyldimethylsilyloxy)benzotriazole (TBDMS-OBt) in the coupling of Fmoc-amino acid chlorides to amino free amino acid esters in homogeneous solution phase is described. The coupling required no addition of base and was fast and racemization free. Work up and isolation of products were easy. Yield, purity and ¹H NMR analysis of peptides, synthesised by this method, were satisfactory.     

1-(t-Butyldimethylsilyloxy) benzotriazole (TBDMS-OBt): A new and novel reagent for the synthesis of peptides

Summary Synthesis and use of 1-(t-butyldimethylsilyloxy)benzotriazole (TBDMS-OBt) in the coupling of Fmoc-amino acid chlorides to amino free amino acid esters in homogeneous solution phase is described. The coupling required no addition of base and was fast and racemization free. Work up and isolation of products were easy. Yield, purity and¹H NMR analysis of peptides, synthesised by this method, were satisfactory.     

Proteinogenic amino acids labelled with 15N and/or 13C for application in peptide synthesis: A short review with a comprehensive list of published derivatives

A review is given of the literature dealing with the most common protected derivatives of ¹⁵N- and/or ¹³C-labelled amino acids of interest in peptide synthesis. The list contains all such Boc-, Z- and Fmoc-amino acids as well as published methyl, ethyl, t-butyl and benzyl esters.     

Preparation of N-9-fluorenylmethoxycarbonylamino acid chlorides from mixed anhydrides by the action of hydrogen chloride.

N-9-Fluorenylmethoxycarbonyl-(Fmoc) amino-acid chlorides have been prepared by reaction of hydrogen chloride on purified mixed Fmoc-amino acid-monoalkyl carbonic acid anhydrides in dichloromethane. The products partially undergo subsequent conversion to the corresponding esters due to the presence of the liberated alcohol, the extent depending on the nature of the alkyl group. Esterification occurred to 5-20% when the alkyl group was isopropyl. Anhydrides of monoisopropenyl carbonic acid which liberate acetone instead of an alcohol gave products uncontaminated with ester. The three components in a reaction mixture could be determined as the reaction progressed by normal phase high-performance liquid chromatography of aliquots, which had been freed of excess hydrogen chloride, on a mu Porasil (underivatized silica) column using tert.-butanol-hexane (1.5:98.5) as solvent.     

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.     

Homologation of alpha-amino acids to beta-amino acids using Fmoc-amino acid pentafluorophenyl esters.

The homologation of alpha-amino acids to beta-amino acids by the two-step Arndt-Eister method is achieved by using Fmoc-alpha-amino acid pentafluorophenyl esters for the acylation of diazomethane, synthesizing the key intermediates Fmoc-aminoacyldiazomethanes as crystalline solids in good yields and purity.     

Straight phase separation of prostaglandin methyl esters on lipophilic gels

A series of straight phase gel chromatography systems have been developed for the separation of prostaglandin methyl esters. Using the methyl esters of prostaglandins B₂, E₂, F₂α and F₂β, the basic relationships between elution volume and the polarities of the gel, the solvent system (heptane-chloroform mixtures), and the prostaglandin have been determined. The separation of prostaglandin methyl esters with slight differences in structure has been demonstrated. Examples include oxo and hydroxy prostaglandins, hydroxy epimers, double bond isomers, prostaglandins of varying α- and ω-chain length, and 1- and 2- (5,6 cis double bond) series prostaglandins. In view of the general advantages of liquid-gel chromatography, it is suggested that these systems may be useful for isolation and purification in a number of areas in the prostaglandin field.     

Biological activity of some synthetic gibberellin glucosyl esters

Four gibberellin (GA₁, GA₃, GA₄ and GA₃₇) glucosyl esters were synthesized and found to be as active as their respective free acids in the rice seedling bioassay. The rapid hydrolysis of the glucosyl esters in rice seedlings was demonstrated by feeding experiments with glucosyl esters of [³H]GA₁ and [³H]GA₄.     

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'-dimethoxybenzhydryl (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]-methylamin 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.     

Purification and Characterization of an Esterase from Micrococcus sp. YGJ1 Hydrolyzing Phthalate Esters

An esterase hydrolyzing phthalate esters has been purified from Micrococcus sp. YGJ1. The enzyme, a monomeric protein (M_r=56 kDa) with a pI of 4.0, hydrolyzes various aliphatic and aromatic carboxylesters. The medium chain (C₃-C₄) esters are the most preferred substrates. The enzyme is inhibited by HgCl₂ and p-chloromercuribenzoate but not by phenylmethyl-sulfonyl fluoride.