Improved approach for anchoring N alpha-9-fluorenylmethyloxycarbonylamino acids as p-alkoxybenzyl esters in solid-phase peptide synthesis

Several Fmoc-amino acids have been esterified by use of N,N-dimethylformamide dineopentyl acetal to 2,4,5-trichlorophenyl 3'-(4'-hydroxymethyl-phenoxy)propionate, and the resultant handle derivatives were purified and then quantitatively coupled onto aminomethyl supports. Compared to literature methodology, the present procedure is preferred because: (i) extra steps to selectively protect and liberate the carboxyl of the handle are circumvented; and (ii) the additional methylene group spacer reflecting substitution of a propionyl group for an acetyl group in the handle changes the electronic parameters of the resultant p-alkoxybenzyl ester sufficiently so that the rates of acidolytic cleavage of the anchoring linkage are 2- to 3-fold increased and useful improvements in yields can be achieved.     

Application of N,N-dimethylformamide dineopentyl acetal for efficient anchoring of N alpha-9-fluorenylmethyloxycarbonylamino acids as p-alkoxybenzyl esters in solid-phase peptide synthesis

The attachment of Fmoc-amino acids onto p- alkoxybenzyl alcohol resins via DCC-DMAP coupling suffers from two different problems: formation of dimers and racemization. The use of N,N-dimethylformamide dineopentyl acetal for the preparation of Fmoc- aminoacyloxybenzyl handles is the basis of a safe and efficient anchoring method that avoids both problems.     

Solid-phase peptide synthesis using nanoparticulate amino acids in water.

Solid-phase peptide synthesis has many advantages compared with solution peptide synthesis. However, this procedure requires a large amount of organic solvents. Since safe organic solvent waste disposal is an important environmental problem, a technology based on coupling reaction of suspended nanoparticle reactants in water was studied. Fmoc-amino acids are used widely, but most of them show low solubility in water. We prepared well-dispersible Fmoc-amino acid nanoparticles in water by pulverization using a planetary ball mill in the presence of poly(ethylene glycol). Leu-enkephalin amide was prepared successfully using the nanoparticulate Fmoc-amino acid on a poly(ethylene glycol)-grafted Rink amide resin in water.     

Solid phase synthesis without repetitive acidolysis. Preparation of leucyl-alanyl-glycyl-valine using 9-fluorenylmethyloxycarbonylamino acids.

The utility of repetitive nonhydrolytic base cleavage of alpha-amino protective groups in solid phase peptide synthesis is shown by a preparation of the model tetrapeptide leucyl-alanyl-glycyl-valine on a p-benzyloxybenzyl ester polystyrene--1% divinylbenzene resin support. Nalpha-9-Fluorenylmethyloxycarbonyl (Fmoc: Carpino & Han, 1970, 1972) amino acids were coupled by the symmetrical anhydride procedure, followed by Fmoc group cleavage using 50% piperidine in methylene chloride. Quantitative removal of the Fmoc-tetrapeptide from the solid support was effected by treatment with 55% trifluoroacetic acid in methylene chloride. Homogeneous free tetrapeptide was obtained in 87% overall yield. The procedure is proposed to offer advantages over present solid phase methods which use acidolysis for repetitive alpha-amino group deblocking.     

Solid-phase synthesis of pseudo-complementary peptide nucleic acids

Pseudo-complementary peptide nucleic acid (pcPNA) is a DNA analog in which modified DNA bases 2,6-diaminopurine (D) and 2-thiouracil (U(s)) 'decorate' a poly[N-(2-aminoethyl)glycine] backbone, together with guanine (G) and cytosine (C). One of the most significant characteristics of pcPNA is its ability to effect double-duplex invasion of predetermined DNA sites inducing various changes in the biological and the physicochemical properties of the DNA. This protocol describes solid-phase synthesis of pcPNA. The monomers for G and C are commercially available, but the monomers for D and U(s) need to be synthesized (or can be ordered to custom synthesis companies). Otherwise, the procedure is the same as that employed for Boc-strategy synthesis of conventional PNA. This protocol, if the synthesis of D and U(s) monomers is not factored in, takes approximately 7 d to complete.     

Solid-phase synthesis of chemotactic peptides using alpha-azido acids.

Four chemotactic peptides, For-Met-Xxx-Phe-OMe, with an alpha,alpha-disubstituted amino acid at position 2 have been synthesized by the azido acid method [Meldal M, Juliano MA, Jansson AM. 1997. Azido acids in a novel method of solid-phase peptide synthesis. Tetrahedron Lett. 38: 2531-2534] on solid-phase, and were tested for biological activity. Dipropylglycine in the central position (Xxx) was found to be as active as the natural chemotactic peptide for chemotactic activity toward human neutrophils. Higher yields were obtained than previously reported solution-phase syntheses of chemotactic peptides, and EEDQ was used successfully for the difficult solid-phase formylation of amino groups.     

Solid-phase peptide synthesis using tert.-butyloxycarbonylamino acid pentafluorophenyl esters

Boc-amino acid pentafluorophenyl esters have been successfully used in solid-phase peptide synthesis. The coupling rates and the purity of the products are comparable to those with the symmetrical anhydrides. These active esters require modified Merrifield resin, polar medium for coupling and in some cases, base catalysis.     

Solid-phase synthesis of two glycopeptides containing the amino acid sequence 5 to 9 of somatostatin

2,3,4,6 Tetra-O-acetyl-1-N-[N-(tert-butyloxycarbonyl)-l-aspart-4-oyl]-d-glucopyranosylamine and 2-acetamido-3,4,6-tri-O-acetyl-1-N-[N-(tert-butyloxycarbonyl)-l-aspart-4-oyl]-2-deoxy-d-glucopyranosylamine were introduced, respectively, by the solid-phase procedure in the amino acid sequence 5 to 9 of somatostatin. The two resulting glycopeptides β-d-Glcp-(1→4)- and β-d-GlcpNAc-(1→4)-Asn-Phe-Phe-Trp-Lys-OH were homogeneous on examination by t.l.c. and l.c., and their structures were confirmed by m.s. of the N-acetyl, permethyl derivatives.     

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.     

Conversion of big pancreatic somatostatin without peptide bond cleavage into somatostatin tetradecapeptide

Urea treatment of the big form of somatostatin obtained from rat pancreas resulted in a conversion into the small form of somatostatin. Further dissociation does not occur with mercaptoethanol. The results indicate that the existence of big somatostatin is dependent upon the formation of a non-covalent bond of the tetradecapeptide with another peptide fragment.     

Solid-phase synthesis of somatostatin and glucagon-selective analogs in gram quantities

Somatostatin (SS) and two glucagon selective analogs [D -Cys^l4]-SS and [D -Trp⁸, D -Cys¹⁴]-SS have been synthesized in gram quantities by the solid-phase procedure. A general modification of Monahan and Gilon's procedure for esterification of the first protected amino acid onto the chloromethylated resin as well as a general protocol for solid-phase peptide synthesis on Beckman 990 automatic synthesizer are described. A new general procedure for disulfide formation, which involves the adaptation of the “high-dilution” principle to the ferricyanide oxidation and the optimization of the sequence of purification steps as applied to somatostatin and its analogs, yields highly purified peptides (≥ 199% pure) as checked by reverse-phase high-pressure liquid chromatography—which is shown to be a highly sensitive, resolutive, and quantitative analytical tool for evaluation of the homogeneity of peptides.     

Solid-phase synthesis of reactive peptide crosslinker by selective deprotection

An effective and simple strategy for preparing peptide crosslinkers is described. An MMP-13 degradable peptide QPQGLAK-NH(2) was prepared on the solid-phase using Fmoc chemistry. The peptide crosslinker was synthesized on-bead by the coupling reaction between acrylic acid and the amine groups of glutamine and lysine residues. The synthetic procedure employed the acid-labile Fmoc-Lys (Mtt)-OH and base-labile Fmoc-AA-OH derivatives. Selective deprotection, of -Mtt and -Fmoc groups on-bead, freed the amine end-groups on glutamine and lysine residues for coupling reaction with acrylic acid while maintaining the peptide attached to the resin. Subsequent cleavage from the resin yielded a peptide crosslinker with two unsaturated acrylate end-groups with high yield and purity. This method can be generally employed for the synthesis of a wide range of peptides with one or more reactive groups for grafting in the fabrication of biomimetic scaffolds in tissue engineering applications.     

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.     

Solid-Phase synthesis of peptide nucleic acids

Peptide nucleic acids (PNA) were synthesized by a modified Merrifield method using several improvements. Activation by O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate in combination with in situ neutralization of the resin allowed efficient coupling of all four Boc-protected PNA monomers within 30 min. HPLC analysis of the crude product obtained from a fully automated synthesis of the model PNA oligomer H-CGGACTAAGTCCATTGC-Gly-NH₂, indicated an average yield per synthetic cycle of 97.1%. N¹-benzyloxycarbonyl-N6³-methylimidazole triflate substantially outperformed acetic anhydride as a capping reagent. The resin-bound PNAs were successfully cleaved by the ‘low–high’ trifluoromethanesulphonic acid procedure.     

Solid-phase synthesis of oligoribonucleotides using 9-fluorenylmethoxycarbonyl (Fmoc) for 5''-hydroxyl protection.

Efficient solid-phase synthesis of a series of oligoribonucleotides of up to 20 residues is described that utilises the 9-fluorenylmethoxycarbonyl group (Fmoc) for 5'-protection and 4-methoxytetrahydropyran-4-yl (Mthp) for 2'-protection of ribonucleotide monomers and a phosphoramidite coupling procedure. The Fmoc group is removed after each coupling step by treatment with 0.1M DBU in acetonitrile. Oligoribonucleotides are isolated in 2'-protected form in good yield and shown to be readily and efficiently deprotected by mild acidic treatment.     

Solid-phase peptide synthesis: from standard procedures to the synthesis of difficult sequences

This protocol for solid-phase peptide synthesis (SPPS) is based on the widely used Fmoc/tBu strategy, activation of the carboxyl groups by aminium-derived coupling reagents and use of PEG-modified polystyrene resins. A standard protocol is described, which was successfully applied in our lab for the synthesis of the corticotropin-releasing factor (CRF), >400 CRF analogs and a countless number of other peptides. The 41-mer peptide CRF is obtained within approximately 80 working hours. To achieve the so-called difficult sequences, special techniques have to be applied in order to reduce aggregation of the growing peptide chain, which is the main cause of failure for peptide chemosynthesis. Exemplary application of depsipeptide and pseudoproline units is shown for synthesizing an extremely difficult sequence, the Asn(15) analog of the WW domain FBP28, which is impossible to obtain using the standard protocol.     

Solid-phase synthesis of peptide nucleic acid (PNA) monomers and their oligomerization using disulphide anchoring linkers

A new simple solid-phase method has been developed for synthesizing Boc-protected peptide nucleic acid (PNA) monomers. An immobilized backbone 3 was built on Expansin® resin using an ester disulphide handle: 2-hydroxypropyl-dithio-2′-isobutyric acid (HPDI). The base acetic acids of thymine 5 , Z-cytosine 9 , Z-adenine 12 , and 6-O-benzyl guanine 17 were prepared and coupled to the immoblized backbone. The HPDI handle was cleaved under mild conditions by cyanolysis or assisted hydrolysis with tris(2-carboxyethyl)phosphine (TCEP) to give undamaged PNA monomers. These monomers were coupled to form oligomers by solid-phase method with another disulphide linkage: aminoethyldithio-2-isobutyric acid (AEDI) grafted on an amino-functionalized TentaGel® resin, using in situ neutralization and TBTU as activating reagent. Final cleavage of the AEDI linker gave PNA bearing a cysteamide residue that could be useful for optimizing PNA properties. Oligomers of up to 16 residues long were assembled. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Amino-acids condensations in the preparation of N alpha-9-fluorenylmethyloxycarbonylamino-acids with 9-fluorenylmethylchloroformate

Synthesis of N alpha-9-fluorenylmethyloxycarbonyl (Fmoc) amino-acids by reaction of free amino-acids (glycine and alanine) with 9-fluorenylmethylchloroformate leads to formation of small amounts of Fmoc-dipeptide which are difficult to eliminate by crystallization. The alternative way to prepare Fmoc-amino-acids by reacting the Fmoc-chloride first with sodium azide and then with the free amino-acid eliminates this side reaction, at least for glycine and alanine.     

Hydrophobic tag-assisted liquid-phase synthesis of a growth hormone-inhibiting peptide somatostatin

Hydrophobic tag-assisted liquid-phase peptide synthesis technique and disulfide bond formation have been well-combined, leading to the efficient and practical preparation of a growth hormone-inhibiting peptide somatostatin. Intramolecular disulfide bond formation has successfully been carried out even under relatively high concentrations, enabling the effective peptide modifications in preparative scale.