Solid-phase peptide synthesis by fragment condensation: Coupling in swelling volume

Summary The condensation of short peptides to resin-bound fragments was examined with respect to high coupling yields with only a small molar excess of a peptide in the reaction solution. The best results were achieved by the addition of reactants (C-unprotected peptide, DIC, and HOBt) dissolved in a so-called swelling volume of an appropriate solvent to a dry resin with an attached N-deprotected peptide chain. Each coupling step was followed by the end-capping of unreacted resin-bound peptide with 2,4-dinitrofluorobenzene. The substituted dinitroaniline chromophore formed in this reaction made the detection and separation of deletion peptides easy. Both conventional and ‘swelling volume’ methods were compared on parallel syntheses of the HIV-1 protease C-terminal 78–99 fragment. The yields of the isolated heneicosapeptide were 21 and 81% in favor of the ‘swelling volume’ procedure.     

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.     

An alternative solid phase peptide fragment condensation protocol with improved efficiency.

The success of solid phase peptide synthesis is often limited by the aggregation of the growing peptide chains on the resin. Working from the results of a study of model coupling reactions in solution between Z-Gly-Phe-OH and H-Phe-OBzl, we have achieved higher efficiency in the repetitive solid phase fragment condensation of VGVAPG, in a 3:1 chloroform-phenol solvent system, using diisopropylcarbodiimide (DIC) as coupling agent, and a combination of 3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HODhbt) and its tetrabutyl ammonium salt as additive, than in DMF with DIC and HODhbt alone.     

Solid-phase acyl donor as a substrate pool in kinetically controlled protease-catalysed peptide synthesis

Recently we have demonstrated the advantage of solid- phase substrate pools mainly in equilibrium controlled protease-catalysed peptide syntheses. The extension of this approach to protease-catalysed acyl transfer reactions will be presented. The model reaction was systematically investigated according to both the influence of solid phases present in the system on enzyme activity as well as nucleophile concentration on peptide yield. The key parameter for obtaining high peptide yield via acyl transfer is the ratio between aminolysis and hydrolysis. We combined high nucleophile concentrations with solid-phase acyl donor pools. This approach enabled us to supply ester substrate and nucleophile in equimolar amounts in a high-density media without the addition of any organic solvent. Several multi-functional di- to tetrapeptides were obtained in moderate to high yields. ©1997 European Peptide Society and John Wiley & Sons, Ltd.     

Solid-phase peptide synthesis by ion-paired alpha-chymotrypsin in nonaqueous media

Solid-phase synthesis of dipeptides in low-water media was achieved using AOT ion-paired alpha-chymotrypsin solubilized in organic solvents. Multiple solvents and systematic variation of water activity, a(w), were used to examine the rate of coupling between N-alpha-benzyloxycarbonyl-L-phenylalanine methyl ester (Z-Phe-OMe) and leucine as a function of the reaction medium for both solid-phase and solution-phase reactions. In solution, the observed maximum reaction rate in a given solvent generally correlated with measures of hydrophobicity such as the log of the 1-octanol/water partitioning coefficient (log P) and the Hildebrand solubility parameter. The maximum rate for solution-phase synthesis (13 mmol/h g-enzyme) was obtained in a 90/10 (v/v) isooctane/tetrahydrofuran solvent mixture at an a(w) of 0.30. For the synthesis of dipeptides from solid-phase leucine residues, the highest synthetic rates (0.14-1.3 mmol/h g-enzyme) were confined to solvent environments that fell inside abruptly defined regions of solvent parameter space (e.g., log P > 2.3 and normalized electron acceptance index <0.13). The maximum rate for solid-phase synthesis was obtained in a 90/10 (v/v) isooctane/tetrahydrofuran solvent mixture at an a(w) of 0.14. In 90/10 and 70/30 (v/v) isooctane/tetrahydrofuran environments with a(w) set to 0.14, seven different N-protected dipeptides were synthesized on commercially available Tentagel support with yields of 74-98% in 24 h.     

Convergent solid-phase synthesis of hirudin.

Hirudin variant 1 (HV1), a small protein consisting of 65 amino acids and three disulfide bonds, was synthesized by using Fmoc-based convergent methods on 2-chlorotrityl resin (CLTR). The linear sequence was assembled by the sequential condensation of 7 protected fragments, on the resin-bound 55-65 fragment. The conditions of fragment assembly were carefully studied to determine the most efficient synthetic protocol. Crude reduced [Cys(16, 28)(Acm)]-HV1 thus obtained was easily purified to homogeneity by RP-HPLC. Disulfide bridges were successfully formed by a two-step procedure, involving an oxidative folding step to form Cys(6)-Cys(14) and Cys(22)-Cys(39) linkages, followed by iodine oxidation to form the Cys(16)-Cys(28) bond. The correct disulfide bond alignment was established by peptide mapping using Staphylococcus aureus V8 protease at pH 4.5.     

Stereospecificity of peptide synthesis by means of phosphorus derivatives: a model of peptide synthesis in molecular evolution

A hypothesis is presented to explain the prebiotic formation of optically pure oligo- and polypeptides from racemic amino acids. Stereospecific condensation reactions favouring the formation of isotactic stereosequences (l-l and d-d blocks) are a basic requirement of this hypothesis. Since phosphorus derivatives such as polyphosphates or nucleic acid imidazolides were postulated to be prebiotic condensing reagents, a variety of peptide syntheses by means of phosphorus derivatives was investigated. Dipeptides and tripeptides were prepared from N-protected d,l-amino acids or d,l-amino acid esters, and d,l-leucine and d,l-valine were subjected to condensation polymerizations. The stereosequences were analysed by means of ¹³C n.m.r. spectroscopy. More than 80% of all condensations were more or less stereospecific and in all cases isotactic sequences were predominant. In the case of poly(d,l-leucines), ¹³C n.m.r. cross-polarization/magic angle spinning (CP/MAS) spectra revealed the formation of α-helical blocks.     

Solid-phase peptide synthesis in highly loaded conditions

The use of very highly substituted resins has been avoided for peptide synthesis due to the aggravation of chain-chain interactions within beads. To better evaluate this problem, a combined solvation-peptide synthesis approach was herein developed taking as models, several peptide-resins and with peptide contents values increasing up to near 85%. Influence of peptide sequence and loading to solvation characteristics of these compounds was observed. Moreover, chain-chain distance and chain concentration within the bead were also calculated in different loaded conditions. Of note, a severe shrinking of beads occurred during the α-amine deprotonation step only when in heavily loaded resins, thus suggesting the need for the modification of the solvent system at this step. Finally, the yields of different syntheses in low and heavily loaded conditions were comparable, thus indicating the feasibility of applying this latter “prohibitive” chemical synthesis protocol. We thought these results might be basically credited to the possibility, without the need of increasing molar excess of reactants, of carrying out the coupling reaction in higher concentration of reactants - near three to seven folds - favored by the use of smaller amount of resin. Additionally, the alteration in the solvent system at the α-amine deprotonation step might be also improving the peptide synthesis when in heavily loaded experimental protocol.     

Development of the efficient preparation method for thermoresponsive elastin-like peptides using liquid-phase synthesis combined with fragment condensation strategy

Elastin-like peptides (ELPs) are synthetic peptides that mimic the characteristic hydrophobic amino acid repeat sequences of elastin and exhibit temperature-dependent reversible self-assembly properties. ELPs are expected to be used as temperature-responsive biomolecular materials across diverse industrial and research fields, and there is a requirement for a straightforward method to mass-produce them. Previously, we demonstrated that phenylalanine-containing ELP analogs, namely, (FPGVG)_n, can undergo coacervation with short chains (n = 5). The Fmoc solid-phase peptide synthesis method is one strategy used to synthesize these short ELPs. However, owing to its low reaction efficiency, an efficient method for preparing ELPs is required. In this study, efficient preparation of ELPs was investigated using a liquid-phase synthesis method with a hydrophobic benzyl alcohol support (HBA-tag). Because HBA-tags are highly hydrophobic, they can be easily precipitated by the addition of poor solvents and recovered by filtration. This property allows the method to combine the advantages of the simplicity of solid-phase methods and the high reaction efficiency of liquid-phase methods. By utilizing liquid-phase fragment condensation with HBA-tags, short ELPs were successfully obtained in high yield and purity. Finally, the temperature-dependent response of the ELPs generated through fragment condensation was assessed using turbidity measurements, which revealed a reversible phase transition. Consequently, the ELPs exhibited a reversible phase transition, indicating successful synthesis of ELPs via fragment preparation with tags. These findings provide evidence of the potential for mass production of ELPs using this approach.     

Polypeptide synthesis using an expressed peptide as a building block for condensation with a peptide thioester: application to the synthesis of phosphorylated p21Max protein(1-101).

An expressed peptide proved to be useful as a building block for the synthesis of a polypeptide via the thioester method. A partially protected peptide segment, for use as a C-terminal building block, could be prepared from a recombinant protein; its N-terminal amino acid residue was transaminated to an alpha-oxoacyl group, the side-chain amino groups were then protected with t-butoxycarbonyl (Boc) groups, and. finally, the alpha-oxoacyl group was removed. On the other hand, an O-phosphoserine-containing peptide thioester was synthesized via a solid-phase method using Boc chemistry. These building blocks were then condensed in the presence of silver ions and an active ester component. During the condensation, epimerization at the condensation site could be suppressed by the use of N,N-dimthylformamide (DMF) as a solvent. Using this strategy, a phosphorylated partial peptide of the p21Max protein, [Ser(PO3H2)2.11]-p21Max(1-101), was successfully synthesized.     

N,O-bisFmoc derivatives of N-(2-hydroxy-4-methoxybenzyl)-amino acids: Useful intermediates in peptide synthesis

2-Hydroxy-4-methoxybenzyl-amino acid residues inhibit interchain association in solid phase peptide synthesis. They are easily introduced through their N,O-bisFmoc derivatives. Preparation of a range of these derivatives is described.     

A Dde resin based strategy for inverse solid-phase synthesis of amino terminated peptides, peptide mimetics and protected peptide intermediates.

This report describes a Dde resin based attachment strategy for inverse solid-phase peptide synthesis (ISPPS). This attachment strategy can be used for the synthesis of amino terminated peptides with side chains and the carboxyl terminus either protected or deprotected. Amino acid t-butyl esters were attached through their free amino group to the Dde resin. The t-butyl carboxyl protecting group was removed by 50% TFA, and inverse peptide synthesis cycles performed using an HATU/TMP based coupling method. Protected peptides were cleaved from the resin with dilute hydrazine. Side chain protecting groups could then be removed by treatment with TFMSA/TFA. The potential of this approach was demonstrated by the synthesis of several short protected and unprotected peptides in good yield and with low epimerization. Its potential for peptide mimetic synthesis was demonstrated by the synthesis of two peptide trifluoromethylketones.     

Coloured peptides: synthesis,properties and use in preparation of peptide sub-library kits

Several methods were developed for the solid-phase synthesis (SPPS) of coloured peptides and peptide libraries. At first a bifunctional red compound, 4-(4-(N-ethyl-N-(3-(tert-butyloxycarbonyl)aminopropyl)amino)phenylazo)benzoic acid (Boc-EPAB), was coupled with chloromethyl resin to obtain a new solid support suitable for SPPS using Boc chemistry. Peptides synthesized on this coloured resin had the chromophore at their C-termini. N-terminally coloured peptides were synthesized on a traditional solid support, coupled with chromophoric carboxylic acid before cleavage. A model pentapeptide, Phe-Ala-Val-Leu-Gly, and its ten derivatives were synthesized and their properties studied. It was found that the presence of chromophores decreases the water solubility of peptides. However, insertion of solubilizing tags (penta-lysine sequences or polyoxyethyl chains) into the molecule of any coloured derivative resulted in enhancement of the solubility. The RP-HPLC hydrophobicity indexes (φ₀) of the coloured peptides were also determined because φ₀ values are closely related to their water solubility. A coloured pentapeptide library was synthesized using the portioning-mixing method. Each component of this library contained the red azo dye (EPAB) and the penta-lysine tag. Before the last coupling step the samples were not mixed. All of the 19 sub-libraries obtained after cleavage were readily soluble in water, giving intense red solutions. The effect of chromophore (EPAB) and/or penta-lysine solubilizing tag on the biological activity was also studied. Potencies of the bovine neurotensin 8–13 fragment and its different coloured and penta-lysine derivatives were compared in isolated longitudinal muscle strips of guinea pig ileum. It was shown that the hexapeptide with penta-lysine tag had almost the same activity as the 8–13 fragment itself. The activity of the EPAB-derivative was found to be rather low. However, the presence of the solubilizing tag in the coloured hexapeptide compensated the negative effect of the chromophore. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Assessment of new 6-Cl-HOBt based coupling reagents for peptide synthesis. Part 1: Coupling efficiency study

The efficiency of a series of well-known coupling reagents (TBTU, HATU, and PyBOP) and of new in situ activating reagents (TCTU, HCTU, and DMTMM) was compared by synthesizing the 65–74 fragment of the Acyl Carrier Protein (H-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly-NH₂₎, containing `a difficult sequence', as a test peptide, in a multiple peptide synthesizer. The longer sequence rMOG(35–55) was also synthesized. It was clear that the aminium salts are more efficient than the phosphonium salt (PyBOP) and that the new 6Cl-HOBt based reagents (HCTU and particularly TCTU) are very efficient, while DMTMM appeared to be not suitable for SPPS.     

Assessment of new 6-Cl-HOBt based coupling reagents for peptide synthesis. Part 1: Coupling efficiency study

Summary The efficiency of a series of well-known coupling reagents (TBTU, HATU, and PyBOP) and of newin situ activating reagents (TCTU, HCTU, and DMTMM) was compared by synthesizing the 65–74 fragment of the Acyl Carrier Protein (H-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly-NH₂), containing ‘a difficult sequence’, as a test peptide, in a multiple peptide synthesizer. The longer sequence rMOG(35–55) was also synthesized. It was clear that the aminium salts are more efficient than the phosphonium salt (PyBOP) and that the new 6Cl-HOBt based reagents (HCTU and particularly TCTU) are very efficient, while DMTMM appeared to be not suitable for SPPS.     

Heterogeneous catalytic transfer hydrogenation in peptide synthesis

International Journal of Peptide Research and Therapeutics -     

How to predict product yield in kinetically controlled enzymatic peptide synthesis

The published theory of kinetically controlled enzymatic peptide synthesis needed experimental verification. We carried out the measurement of the peptide yield and estimation of the key parameters alpha and beta for papain-catalyzed synthesis of Mal-L-Phe-L-Ala-LLeuNH(2) from Mal-L-Phe-L-AlaOMe and L-LeuNH(2). The experimental results demonstrate that this theory adequately describes the real process. (c) 1992 John Wiley & Sons, Inc.     

Synthesis and purification of self‐assembling peptide‐oligonucleotide conjugates by solid‐phase peptide fragment condensation

Peptide‐oligonucleotide conjugates (POCs) are interesting molecules as they covalently combine 2 of the most important biomacromolecules. Sometimes, the synthesis of POCs involves unexpected difficulties; however, POCs with self‐assembling propensity are even harder to synthesize and purify. Here, we show that solid‐phase peptide fragment condensation combined with thiol‐maleimide or copper‐catalyzed azide‐alkyne cycloaddition click chemistries is useful for the syntheses of self‐assembling POCs. We describe guidelines for the selection of reactive functional groups and their placement during the conjugation reaction and consider the cost‐effectiveness of the reaction. Purification is another important challenge during the preparation of POCs. Our results show that polyacrylamide gel electrophoresis under denaturing conditions is most suitable to recover a high yield of self‐assembling POCs. This report provides the first comprehensive study of the preparation of self‐assembling POCs, which will lay a foundation for the development of elegant and sophisticated molecular assemblies.     

Comparison of procedures for directly obtaining protected peptide acids from peptide-resins.

The preparation of small-sized protected peptide acids related to cholecystokinin and gomesin was attempted using peptide-Kaiser oxime resins (KOR) as starting materials. For comparison, peptide-2-Cl-trityl resin (CLTR) was also employed. While peptide detachment from KOR was achieved through the oxime ester bond hydrolysis mediated by DBU, hydroxide ion or Ca(+2) ion, peptide release from CLTR was accomplished through acid-catalysed hydrolysis of the peptide-resin ester linkage. Amino acid analysis of the peptide-resins before and after peptide release allowed the calculation of the reaction yields. RP-HPLC and LC/ESI-MS of the resulting crude peptides allowed estimation of their quality. The data collected indicated that: (i) among the procedures used for peptide displacement from KOR, the one employing DBU was the most efficient since it furnished all model protected peptide acids with the highest quality in a very short time; (ii) although slow, Ca(+2)-assisted peptide detachment from KOR was selective and was suitable for generating high-quality protected peptide acids containing up to five residues; (iii) even though the protocols employed for peptide release from CLTR have shown to be appropriate, the one employing 1% TFA/DCM was the most productive; (iv) in terms of product quality, DBU-catalysed peptide detachment from KOR was similar to TFA-catalysed peptide release from CLTR although the latter was more productive. These findings are relevant to peptide chemists in general, but especially to those interested in preparing acyl donors for convergent peptide syntheses by the t-Boc chemistry.