Solid phase peptide synthesis in water VI: evaluation of water-soluble coupling reagents for solid phase peptide synthesis in aqueous media

Solid phase peptide synthesis requires large amounts of organic solvents, the safe disposal of which is an important environmental issue. Peptide synthesis, if performed in water and using less or nontoxic reagents, circumvents the disposal problem. Our ultimate aim is to develop an "environment-friendly" solid phase peptide synthesis (SPPS) methodology. Previously, we showed that SPPS in water is feasible. To perform SPPS in water, the coupling reagent must be water-soluble and maintain its reactivity in water. For this report, we tested the efficacy of the water-soluble coupling reagents, 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), towards SPPS in water. We successfully synthesized Leu-enkephalin amide on a solid support suspended in aqueous 50% EtOH using DMT-MM and 2-(4-sulfophenylsulfonyl)ethoxycarbonylamino acids.     

Peptide synthesis catalyzed by polyethylene glycol-modified chymotrypsin in organic solvents

Chymotrypsin modified with polyethylene glycol was successfully used for peptide synthesis in organic solvents. The benzene-soluble modified enzyme readily catalyzed both aminolysis of N-benzoyl-L-tyrosine p-nitroanilide and synthesis of N-benzoyl-L-tyrosine butylamide in the presence of trace amounts of water. A quantitative reaction was obtained when either hydrophobic or bulky amides of L- as well as D-amino acids were used as acceptor nucleophiles, while almost no reaction occurred with free amino acids or ester derivatives. The acceptor nucleophile specificity of modified chymotrypsin as a catalyst in the formation of both amide and peptide bonds in organic solvents was quite comparable to that in aqueous solution as well as to that of the leaving group in hydrolysis reactions. By contrast, the substrate specificity of modified chymotrypsin in organic solvents was different from that in water since arginine and lysine esters were found to be as effective as aromatic amino acids to form the acyl-enzyme with subsequent synthesis of a peptide bond.     

Non-coded amino acids as acyl donor substrates for peptide bond formation catalyzed by thermoase in toluene

The peptide bond formation of N-protected non-coded amino acids having different structures as acyl donor substrates that is catalyzed by thermoase in organic media was investigated. In these reactions, N-protected l--non-coded amino acids, including l-Orn, l-Cit, -aminobutyric acid (l--Abu) and phenylalanine homologues, were used as the acyl donors and phenylalanine derivatives were used as the acyl acceptors. This kind of enzymatic reactions cannot be carried out in an aqueous buffer due to the rigid specificity of proteases to coded amino acids in water. The results demonstrated that the substrate specificity of proteases could be broadened in organic solvents. In addition, the factors that influenced these protease-catalyzed reactions, including structures of the substrates, water content and the bases used, were systematically studied. Our work provided important evidence for broadening the application of protease in organic synthesis.     

Peptide synthesis by proteases in organic solvents: medium effect on substrate specificity.

The substrate specificities of alpha-chymotrypsin and subtilisins for peptide synthesis in hydrophilic organic solvents were investigated. Chymotrypsin exhibited high specificity to aromatic amino acids as acyl donors, while subtilisin Carlsberg and subtilisin BPN' were specific to aromatic and neutral aliphatic amino acids, in accordance with the S1 specificities of the enzymes for peptide hydrolysis in aqueous solutions. On the contrary, chymotrypsin exhibited higher specificities to hydrophilic amino acid amides as acyl acceptors (nucleophiles) for peptide synthesis with N-acetyl-L-tyrosine ethyl ester, in contrast to the S1' specificity for peptide hydrolysis and peptide synthesis in aqueous solutions. Furthermore, nucleophile specificity changed with the change in water-organic solvent composition; the increase in water content led to increase in relative reactivity of leucinamide to that of alaninamide. It was also found that protection of the carboxyl group of alanine by amidation is much preferable to protection by esterification in terms of reactivity as nucleophiles.     

The applicability of subtilisin Carlsberg in peptide synthesis.

The synthesis of peptide bonds catalysed by subtilisin Carlsberg was studied in different hydrophilic organic solvents with variable H2O concentration. Z-Val-Trp-OMe and Z-Ala-Phe-OMe were used as acyl donors, and a series of amino acid derivatives, di- and tripeptides of the general structure Xaa-Gly, Gly-Xaa, Gly-Gly-Xaa (Xaa represents all natural L-amino acids except cysteine) and other peptides were used as nucleophiles. A comparative study of the enzymatic synthesis in aqueous DMF (50%, v/v) and acetonitrile containing 10% (v/v) of H2O demonstrated that the yields of peptide products were higher in most cases when acetonitrile with low H2O concentration was used. The acylation of weak nucleophiles was improved in organic solvents with very low H2O concentration (2%). The reactions in anhydrous Bu(t)-OH proceeded with substantially lower velocity. Generally, the restricted nucleophile specificity of the enzyme for glycine and hydrophilic amino acid residues in P1' position, as well as numerous side reactions, limit the utilization of subtilisin in peptide synthesis, especially in the case of the segment condensations. Contrary to the published data, we have proved that proline derivatives were not acylated in any media with the help of subtilisin Carlsberg. Effective ester hydrolysis of a protected nonapeptide corresponding to the N-terminal sequence of dicarba-eel-calcitonin catalysed by subtilisin was achieved.     

Unusual specificity of polyethylene glycol-modified thermolysin in peptide synthesis catalyzed in organic solvents

Summary Polyethylene glycol-modified thermolysin was found to efficiently catalyze peptide synthesis in organic solvents. As in aqueous media, the reaction occurred through a rapid equilibrium random bireactant mechanism. However, the substrate specificity of modified thermolysin was actually changed since hydrophilic as well as acidic amino acids were better carboxyl group donors than hydrophobic residues, contrary to what is observed in both the enzyme-catalyzed synthesis and hydrolysis of peptide bonds in water.     

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.     

Peptide synthesis ‘in water’ by a solution‐phase method using water‐dispersible nanoparticle Boc‐amino acid

Regulatory pressure has compelled the chemical manufacturing industry to reduce the use of organic solvents in synthetic chemistry, and there is currently a strong focus on replacing these solvents with water. Here, we describe an efficient in‐water solution‐phase peptide synthesis method using Boc‐amino acids. It is based on a coupling reaction utilizing suspended water‐dispersible nanoparticle reactants. Using this method, peptides were obtained in good yield and with high purity. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Microwave-assisted Boc-solid phase peptide synthesis of cyclic cysteine-rich peptides.

In this study we describe the first protocols for the synthesis of cystine-rich peptides in the presence of microwave radiation with Boc-solid phase peptide synthesis (SPPS). This method is exemplified for macrocyclic peptides known as cyclotides, which comprise approximately 30 amino acids and incorporate a cystine knot arrangement of their three disulfide bonds. However, the method is broadly applicable for a wide range of peptides using Boc-SPPS, especially for SPPS of large peptides via native chemical ligation. Microwave radiation produces peptides in high yield and with high purity, and we were able to reduce the time for the assembly of approximately 30 mer peptide chains to an overnight reaction in the automated microwave-assisted synthesis.     

Amino acid deletion products resulting from incomplete deprotection of the Boc group from Npi-benzyloxymethylhistidine residues during solid-phase peptide synthesis.

In peptide synthesis, the use of N(alpha)-tert-butyloxycarbonyl-N(pi)-benzyloxymethylhistidine [Boc-His(pi-Bom)] raises the problem of the Bom group generating formaldehyde during the hydrogen fluoride (HF) cleavage reaction. This can lead to modification of the functional groups on amino acids in the peptide chain. Besides this side reaction, the failure of N(alpha)-Boc deprotection from the His(pi-Bom) residue occurs during TFA treatment for the standard solid-phase peptide synthesis (SPPS) even in the case of a non 'difficult sequence'. This gives amino acid deletion products generated at the N-terminus of the His(pi-Bom) residues. Reviewing the removability of the Boc group on amino acid derivatives showed that the group on the His(pi-Bom) residue was much more resistant under the deprotecting conditions than expected. To circumvent this problem, special precautions, i.e. prolonged deprotection steps and/or increased concentrations of TFA, should be taken for a successful SPPS.     

Making Ends Meet: Microwave-Accelerated Synthesis of Cyclic and Disulfide Rich Proteins Via In Situ Thioesterification and Native Chemical Ligation

The development of synthetic methodologies for cyclic peptides is driven by the discovery of cyclic peptide drug scaffolds such as the plant-derived cyclotides, sunflower trypsin inhibitor 1 (SFTI-1) and the development of cyclized conotoxins. Currently, the native chemical ligation reaction between an N-terminal cysteine and C-terminal thioester group remains the most robust method to obtain a head-to-tail cyclized peptide. Peptidyl thioesters are effectively generated by Boc SPPS. However, their generation is challenging using Fmoc SPPS because thioester linkers are not stable to repeated piperidine exposure during deprotection. Herein we describe a Fmoc-based protocol for synthesizing cyclic peptides adapted for microwave assisted solid phase peptide synthesis. The protocol relies on the linker Di-Fmoc-3,4-diaminobenzoic acid, and we demonstrate the use of Gly, Ser, Arg and Ile as C-terminal amino acids (using HBTU and HATU as coupling reagents). Following synthesis, an N-acylurea moiety is generated at the C-terminal of the peptide; the resin bound acylurea peptide is then deprotected and cleaved from the resin. The fully deprotected peptide undergoes thiolysis in aqueous buffer, generating the thioester in situ. Ultimately, the head-to-tail cyclized peptide is obtained via native chemical ligation. Two naturally occurring cyclic peptides, the prototypical Möbius cyclotide kalata B1 and SFTI-1 were synthesized efficiently, avoiding potential branching at the diamino linker, using the optimized protocol. In addition, we demonstrate the possibility to use the approach for the synthesis of long and synthetically challenging linear sequences, by the ligation of two truncated fragments of a 50-residue long plant defensin.     

Surfactant-protease complex as a novel biocatalyst for peptide synthesis in hydrophilic organic solvents*

The peptide synthesis from N-acetyl-L-phenylalanine ethyl ester with alaninamide catalyzed by a surfactant-protease complex has been performed in anhydrous hydrophilic organic solvents. Proteases derived from various sources were converted to surfactant-coated complexes with a nonionic surfactant. The surfactant-subtilisin Carlsberg (STC) complex had a higher enzymatic activity than the other protease complexes and the initial reaction rate in tert-amyl alcohol was 26-fold that of STC lyophilized from an optimum aqueous buffer solution. Native STC hardly catalyzed the same reaction. The addition of water to the reaction medium activated the lyophilized STC, however, the reaction rate was much lower than that of the STC complex, and a hydrolysis reaction preferentially proceeded. The STC complex exhibited a high catalytic activity in hydrophilic organic solvents (e.g. tertiary alcohol). The addition of dimethylformamide as a cosolvent improved the solubility of amino acid amides and further activated the STC complex due to the water mimicking effect. When hydrophilic amino acid amides were employed as an acyl acceptor, the peptide formation proceeded efficiently compared to that using hydrophobic substrates. The surfactant-STC complex is a powerful biocatalyst for peptide synthesis because the STC complexes display a high catalytic activity in anhydrous hydrophilic organic solvents and did not require the excess amount of water. Thus the side (hydrolysis) reaction is effectively suppressed and the yield in the dipeptide formation is considerably high.     

Tools for investigating peptide-protein interactions: peptide incorporation of environment-sensitive fluorophores via on-resin derivatization

This protocol presents the peptide incorporation of environment-sensitive fluorophores derived from the dimethylaminophthalimide family. The procedure utilizes anhydride precursors of 4-dimethylaminophthalimide (4-DMAP) or 6-dimethylaminonaphthalimide (6-DMN), whose syntheses are described in a related protocol from these authors. In this protocol, the fluorophores are directly incorporated after solid-phase peptide synthesis (SPPS) via on-resin derivatization of peptides prepared using commercially available diamino acids, which are Alloc-protected on the side-chain amino group. The time required to complete the procedure depends on the size and number of peptides targeted. As an alternative to this approach, the corresponding fluorescent amino acids can be obtained in an Fmoc-protected form for convenient use as building blocks in SPPS. This option is described in a related protocol by these authors.     

Preventing aspartimide formation in Fmoc SPPS of Asp‐Gly containing peptides — practical aspects of new trialkylcarbinol based protecting groups

In our efforts to develop a universal solution to the problem of aspartimide formation in Fmoc SPPS, we investigated the application of our new β‐trialkylmethyl protected aspartic acid building blocks to the synthesis of peptides containing the Asp‐Gly motif. The N^α‐Fmoc aspartic acid β‐tri‐(ethyl/propyl/butyl)methyl esters were used in the synthesis of the classic model peptide scorpion toxin II (VKDGYI), and their effectiveness in minimising aspartimide formation during extended piperidine treatments was evaluated. Furthermore, we compared their efficacy against that of the commonly used approach of adding acids to the Fmoc deprotection solution. Finally, we applied our aspartic acid building blocks to the stepwise Fmoc SPPS of teduglutide, a human GLP‐2 analogue, whose synthesis is made challenging by extensive aspartimide formation. In all experiments, our approach led to almost complete reduction of aspartimide formation with accompanied suppression of aspartic acid epimerisation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Thermodynamic parameters monitoring the equilibrium shift of enzyme-catalyzed hydrolysis/synthesis reactions in favor of synthesis in mixtures of water and organic solvent

The main strategy developed to shift the equilibrium state of a hydrolase-catalyzed hydrolysis/synthesis reaction consists in reducing water activity by addition of organic solvents in the reaction medium. We have used several mixtures of water and 1,4-butanediol, ranging from pure water to pure 1,4-butanediol, to study the hydrolysis/synthesis reaction of the N-Cbz-L-tryptophanyl-glycineamide dipeptide, catalyzed by alpha-chymotrypsin. In the presence of 1,4-butanediol, alpha-chymotrypsin also catalyzed the esterification reaction between this diol and N-Cbz-L-tryptophan; this ester hydrolysis/synthesis reaction has thus also been examined. The dipeptide and ester equilibrium concentrations increase when the water content of the reaction medium is decreased. Using our experimental data, we have determined the equilibrium constants of the hydrolysis/synthesis equilibria involving the nonionized forms of the protected amino acids, the estimated values of which are Ksp = 8 10(5) for the dipeptide and Kse = 78 for the ester respectively. They are true thermodynamic equilibrium constants, each related to a single, well-defined reaction equilibrium and with water activity being taken into account. If an organic solvent is added to the reaction medium these equilibria can be shifted towards synthesis by decreasing the water activity but also by modifying the ionization/neutralization equilibrium constant of the ionizable groups. These two effects depend both on the water content and on the nature of the organic solvent used, and, in particular, on its dielectric constant. Because of the importance of this parameter in our study, we discuss using it as an indicator to select an appropriate organic solvent to perform an enzyme-catalyzed synthesis.     

Benzylprotected aromatic phosphonic acids for anchoring peptides on titanium

The development of biocompatible coatings is an ongoing issue. Mimicking the physiological adhesion process of osteoblasts to the extracellular matrix improves cell adhesion of osteoblasts in vitro and results in improved and earlier osseous integration of implants in vivo. Titanium, an often used material in implant surgery, can be easily coated by peptides bearing phosphonic acid groups. We report here, the synthesis of benzyl protected phosphonic acids suitable for solid-phase peptide synthesis (SPPS), which can be easily deprotected with TFA.     

Peptidyl molecular imaging contrast agents using a new solid-phase peptide synthesis approach

A versatile method is disclosed for solid-phase peptide synthesis (SPPS) of molecular imaging contrast agents. A DO3A moiety was derivatized to introduce a CBZ-protected amino group and then coupled to a polymeric support. CBZ cleavage with Et2AlCl/thioanisole was optimized for SPPS. Amino acids were then coupled to the aminoDOTA-loaded resin using conventional stepwise Fmoc SPPS to create a product with DOTA coupled to the C-terminus of the peptide. In a second study, the DO3A moiety was coupled to a glycine-loaded polymeric support, and amino acids were then coupled to the amino-DOTA-peptide-loaded resin using SPPS to incorporate DOTA within the peptide sequence. The peptide-(Tm3+-DOTA) amide showed a paramagnetic chemical exchange saturation transfer (PARACEST) effect, which demonstrated the utility of this contrast agent for molecular imaging. These results demonstrate the advantages of exploiting SPPS methodologies through development of unique DOTA derivatives to create peptide-based molecular imaging contrast agents.     

Tools for investigating peptide-protein interactions: peptide incorporation of environment-sensitive fluorophores through SPPS-based 'building block' approach

This protocol presents the synthesis and peptide incorporation of environment-sensitive fluorescent amino acids derived from the dimethylamino-phthalimide family. The procedure uses anhydride precursors of 4-dimethylaminophthalimide (4-DMAP) or 6-dimethylaminonaphthalimide (6-DMN), whose syntheses are described in a related protocol by these authors. In this study, the corresponding fluorescent amino acids can be readily obtained in Fmoc-protected form for convenient use as building blocks in solid phase peptide synthesis (SPPS). The time required to complete the procedure depends on the size and the number of peptides targeted. Alternatively, the chromophores can be incorporated directly after SPPS via on-resin derivatization of peptides, which is an option described in a related protocol by these authors.     

Synthesis of N-protected peptide alcohols catalyzed by subtilisin or alpha-chymotrypsin in organic solvents

A series of N-protected peptide alcohols were synthesized using amino alcohols with unprotected hydroxy groups as amino components by the catalysis of subtilisin or alpha-chymotrypsin in organic solvents. N-protected aromatic amino acid esters were more suitable as acyl donors for subtilisin. The influences of different N-protecting groups, organic solvents, and content of water on synthesis of N-protected peptide alcohols were systematically studied.     

Native and modified subtilisin Karlsberg as an effective catalyst of peptide bond formation in organic media

The activity and stability of native subtilisin Karlsberg and subtilisin 72 and their complexes with sodium dodecyl sulfate (SDS) in organic solvents were studied. The kinetic constants of the hydrolysis of specific chromogenic peptide substrates Z- ALA-Ala-Leu-pNA and Glp-Ala-Ala-Leu-pNA by the subtilisins were determined. It was found that the subtilisin Karlsberg complex with SDS in anhydrous organic solvents is an effective catalyst of peptide synthesis with multifunctional amino acids in positions P1 and P'1 (Glu, Arg, and Asp) containing unprotected side ionogenic groups.