New hydrogenation catalyst: An advantageous method for the removal of hydrogenolysable protecting groups in peptide synthesis

Removal of some commonly used protecting groups in peptide synthesis by catalytic transfer hydrogenation employing ammonium formate and magnesium is described. This method is equally competitive with other methods in deblocking most of the commonly used protecting groups in peptide synthesis. tert-Butyl derived and base labile protecting groups were completely stable under these conditions. The use of ammonium formate and magnesium makes this a rapid, low-cost alternative to palladium and reduces the work-up to a simple filtration and extraction operation.     

Novel and Efficient Transfer Hydrogenolysis of Protected Peptides Using Recyclable Polymer-Supported Hydrogen Donor

Palladium catalyzed transfer hydrogenolysis of protected peptides using a recyclable polymer-supported formate as hydrogen donor affords pure hydrogenolyzed products without the need for any chromatographic purification steps and provides a facile method for the clean and efficient removal of some of the commonly used protecting groups in peptide synthesis.     

Tryptophan reduction and histidine racemization during deprotection by catalytic transfer hydrogenation of an analog of the luteinizing hormone releasing factor

(D-Trp)6-LHRH:pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-GlyNH2 was prepared by solid-phase peptide synthesis using the nitro group to protect the guanidine side chain of the arginyl residue. Removal of the side-chain protecting groups was carried out by catalytic transfer hydrogenation (CTH) using palladium acetate/ammonium formate or palladium on charcoal/formic acid. We show in this paper that this deprotection method induces i) reduction of the tryptophan residue and ii) epimerization at the histidine level (with palladium acetate/ammonium formate). Despite the formation of significant amounts of reduced peptide, CTH enabled us to obtain (D-Trp)6-LHRH in relatively good yield.     

New hydrogen donor: a facile method for the removal of hydrogenolysable protecting groups in Peptide synthesis

Removal of some commonly used protecting groups in peptide synthesis by catalytic transfer hydrogenation employing hydrazinium monoformate and 10%Pd on carbon is described. This method is equally competitive with other methods in deblocking most of the commonly used protecting groups in peptide synthesis. tert-Butyl derived and base labile protecting groups were completely stable under these conditions. This is more effective than hydrazine or formic acid.     

Application of arylsulphonyl side-chain protected arginines in solid-phase peptide synthesis based on 9-fluorenylmethoxycarbonyl amino protecting strategy.

One of the main problems still hampering solid-phase peptide synthesis using orthogonal protection strategies based on the 9-fluorenylmethoxycarbonyl amino protecting group is the difficult removal of currently used arginine arylsulphonyl guanidino protecting groups. Poor acid liability of 4-methoxy-2,3,6-trimethylbenzenesulphonyl-protected arginine has led to the popularity of the newer 2,2,5,7,8- pentamethylchroman-6-sulphonyl guanidino protecting group. This group was initially believed to have liability to trifluoroacetic acid, the reagent commonly used to simultaneously deprotect peptides and detach them from the synthesis resin, comparable to tert.-butyl and trityl type protecting groups used for the protection of other peptide side-chain functionalities. In a comparison of three established cleavage/deprotection mixtures we have shown that this is not always the case, particularly in multiple arginine peptides. We have found that only hard-acid deprotection with trimethylsilyl bromide reliably removed both arylsulphonyl guanidino protecting groups from a variety of arginine-containing peptides.     

Removal of acid-labile protecting or anchoring groups in the presence of polyfluorinated alcohol: Application to solid-phase peptide synthesis

We describe herein a new method for cleaving from resin and removing acid-labile protecting groups in solid-phase peptide synthesis in the presence of a polyfluorinated alcohol (either trifluoroethanol, TFE, or hexafluoroisopropanol, HFIP). It was shown that 0.1 M HCl in hexafluoroisopropanol or trifluoroethanol removes the acid-labile protecting groups commonly used in Fmoc SPPS for the protection of amino acid side-chains, such as t-butyl ester and ether, Boc, trityl, and Pbf groups including the most acid-resistant p-hydroxymethylphenoxyacetyl group (HMPA), p-benzyloxy benzyl ester (Wang resin), Rink amide, and peptide amide linker (PAL). The addition of 5–10% of a hydrogen-bonding solvent was shown to considerably retard or even fully inhibit the reaction. However, nonhydrogen-bonding solvents, such as dichloromethane, do not slow down the reaction.     

Facile preparation of the N‐acetyl‐glucosaminylated asparagine derivative with TFA‐sensitive protecting groups useful for solid‐phase glycopeptide synthesis

In this study, a novel N‐acetyl‐glucosaminylated asparagine derivative was developed. This derivative carried TFA‐sensitive protecting groups and was derived from commercially available compounds only in three steps. It was applicable to the ordinary 9‐fluorenylmethoxycarbonyl (Fmoc)‐based solid‐phase peptide synthesis (SPPS) method, and the protecting groups on the carbohydrate moiety could be removed by a single step of TFA cocktail treatment generally used for the final deprotection step in Fmoc‐SPPS. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Why peptide synthesis continues to remain a formidable challenge

Striking advances have been made in the synthesis of biologically active peptides. The synthesis of fully active crystalline bovine ribonuclease A by Yajima and Fujii [(1981) Biopolymers 20 , 1855–1863] represents an exciting milestone. The main factors contributing to this success include sequential coupling of many small segments and a sophisticated multistage protocol for the final acidolytic cleavage of all protecting groups. However, syntheses of large peptides and proteins and even of many medium-sized peptides continue to present formidable challenges. In peptide coupling the challenge is to join large peptide segments efficiently and in good yields. For solid-phase synthesis the challenge is application of polar supports and alternative N^α-protection. With protecting groups the challenge is to achieve quantitive cleavage at the end of synthesis. Concerning racemization the challenge is to develop sensitive and rapid procedures for measuring racemization during coupling. For purification the challenge is to improve the existing procedures and to search for novel alternative separation principles.     

Synthesis of disulfated peptides corresponding to the N‐terminus of chemokines receptors CXCR6 (CXCR61–20) and DARC (DARC8–42) using a sulfate‐protecting group strategy

Tyrosine sulfation is a post translational modification that occurs on integral membrane and secreted proteins, and is required for mediating crucial biological processes. Until recently the synthesis of sTyr peptides, especially those containing multiple sTyr residues, were among the most challenging peptides to prepare. We recently described an efficient strategy for Fmoc‐based solid phase synthesis of sTyr peptides in which the sulfate group in the sTyr residue(s) is protected with a DCV group (FmocTyr(SO₃DCV)OH, 1 ). After cleavage of the peptide from the support the DCV group is removed by hydrogenolysis. Here we demonstrate that sTyr peptides containing Met or Trp residues can be prepared using our sulfate‐protecting group strategy by preparing peptides corresponding to residues 1–20 of chemokine receptor CXCR6 and 8–42 of chemokine receptor DARC. Removing the DCV groups at the end of the syntheses was readily achieved, without any reduction of the indole ring in Trp, by performing the hydrogenolysis in the presence of triethylamine. These conditions were found to be particularly efficient for removing the DCV group and superiour to our original conditions using H₂, ammonium formate, Pd/C. The presence of Met was found not to interfere with the removal of the DCV group. The use of pseudoproline dipeptides and N‐backbone protection with the 2,4‐dimethoxybenzyl group were found to be very effective tactics for preventing aggregation and aspartimide formation during the synthesis of these peptides. We also report an alternative and more cost effective synthesis of amino acid 1 . Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Microwave‐assisted cleavage of Alloc and Allyl Ester protecting groups in solid phase peptide synthesis

Orthogonal protection of amino acid side chains in solid phase peptide synthesis allows for selective deprotection of side chains and the formation of cyclic peptides on resin. Cyclizations are useful as they may improve the activity of the peptide or improve the metabolic stability of peptides in vivo. One cyclization method often used is the formation of a lactam bridge between an amine and a carboxylic acid. It is desirable to perform the cyclization on resin as opposed to in solution to avoid unwanted side reactions; therefore, a common strategy is to use –Alloc and –OAllyl protecting groups as they are compatible with Fmoc solid phase peptide synthesis conditions. Alloc and –OAllyl may be removed using Pd(PPh₃)₄ and phenylsilane in DMF. This method can be problematic as the reaction is most often performed at room temperature under argon gas. It is not usually done at higher temperatures because of the fear of poisoning the palladium catalyst. As a result, the reaction is long and reagent–intensive. Herein, we report the development of a method in which the –Alloc/–OAllyl groups are removed using a microwave synthesizer under atmospheric conditions. The reaction is much faster, allowing for the removal of the protecting groups before the catalyst is oxidized, as well as being less reagent–intensive. This method of deprotection was tested using a variety of amino acid sequences and side chain protecting groups, and it was found that after two 5‐min deprotections at 38°C, all –Alloc and –OAllyl groups were removed with >98% purity. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Design, synthesis and catalytic activity of a serine protease synthetic model

Summary The design, synthesis and catalytic properties of a cyclic branched peptide carrier that possesses the catalytic triad residues of the serine proteases is reported. The synthesis of the peptide model was totally completed on solid support using three different orthogonal amino protecting groups. Hydrolytic activity measurements against Suc-Ala-Ala-Ala-pNA substrate showed that it is hydrolysed by the peptide model to a small extent. Despite this small hydrolytic activity, it is the first time, to our knowledge, that hydrolysis of such a substrate is reported by an enzyme model compound. Contrary, this enzyme model peptide showed considerable activity against the Boc-Ala-pNP substrate (k _cat =0.414 min⁻¹ andK _m =0.228 mm). These results suggest that the designed carrier brings in appropriate contact the catalytic triad residues (Ser, His, Asp) resulting in the obtained hydrolytic activity.     

Conformation studies on oligoalanines, substance P, and the myoglobin sequence (66-73). Circular dichroism of polyethyleneglycol-bound peptides]

The conformation of polyethylene glycol-bound peptides, synthesized by the liquid-phase method, was investigated. This marcromolecular C-terminal protecting group is transparent in the visible and the ultraviolet range to 190 nm and solubilizes peptides in many different solvents. The CD spectra of the polymer-bound myoglobin sequence 66–73 and of the biologically active undecapeptide “substance P” were measured in each step of the synthesis. In both examples the formation of a secondary structure during the growth of the peptide chain was found. In the hydrophobic octapeptide containing the myoglobin sequence 66–73, the influence of either the blocked or the free N-terminal amino group on the conformation was observed. The blocked octapeptide in trifluoroethanol showed a higher degree of α-helix contribution than in its free state. The conformation of the polyethylene glycol-bound nona- and decaalanine in trifluoroethanol and water was determined. The peptide with a free amino end group has β-conformation in trifluoroethanol as well as in water. The corresponding N-Boc-protected derivatives show helical structure. The amino end group has a decisive influence on the formation of β-structure. The method of CD investigation of polymer-bound peptide sequences during the peptide synthesis in solution enables one to determine the influence of protecting groups and the chain end of a peptide on its conformation. It is also possible to study the relationship between the secondary structure, the chain length, and the kinetic of the coupling reaction in different solvents. Since the crystallization method for the liquid-phase peptide synthesis allows one to synthesize peptides in very short time, a new method of studying peptide conformations is opened.     

Incorporation of N‐amidino‐pyroglutamic acid into peptides using intramolecular cyclization of α‐guanidinoglutaric acid

N‐terminal modification of peptides by unnatural amino acids significantly affects their enzymatic stability, conformational properties and biological activity. Application of N‐amidino‐amino acids, positively charged under physiological conditions, can change peptide conformation and its affinity to the corresponding receptor. In this article, we describe synthesis of short peptides, containing a new building block—N‐amidino‐pyroglutamic acid. Although direct guanidinylation of pyroglutamic acid and oxidation of N‐amidino‐proline using RuO₄ did not produce positive results, N‐amidino‐Glp‐Phe‐OH was synthesized on Wang polymer by cyclization of α‐guanidinoglutaric acid residue. In the course of synthesis, it was found that literature procedure of selective Boc deprotection using TMSOTf/TEA reagent is accompanied by concomitant side reaction of triethylamine alkylation by polymer linker fragment. It should be mentioned that independently from cyclization time and coupling agent (DIC or HCTU), the lactam formation was incomplete. Separation of the cyclic product from the linear precursor was achieved by HPLC in ammonium formate buffer at pH 6. HPLC analysis showed N‐amidino‐Glp‐Phe‐OH stability at acidic and physiological pH and fast ring opening in water solution at pH 9. The suggested method of N‐amidino‐Glp residue formation can be applied in the case of short peptide chains, whereas synthesis of longer ones will require fragment condensation approach. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and mass spectrometry analysis of quaternary cryptando‐peptidic conjugates

The bicyclic amines in the form of cryptands, the crown ether analogs, were used in the synthesis of cryptando‐peptidic conjugates with simultaneous formation of quaternary ammonium nitrogen moiety. A series of model cryptando‐peptidic conjugates at the peptide N‐terminus was efficiently prepared by the standard Fmoc solid phase synthesis. Tandem mass spectrometric analysis of the obtained conjugates has shown the specific fragmentation pattern during MS/MS experiment. The obtained cryptandic quaternary ammonium group undergoes the Hofmann elimination during collision‐induced dissociation fragmentation followed by the ethoxyl group elimination. The presented quaternization of cryptands by iodoacetylated peptides is relatively easy and compatible with standard solid‐phase peptide synthesis. Additionally, the applicability of such peptide derivatives and their isotopologues selectively deuterated at the α‐carbon in the quantitative LC‐MS analysis was analyzed. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Systematic evaluation of the dihydrogen-oxidising and NAD+-reducing soluble [NiFe]-hydrogenase from Ralstonia eutropha H16 as a cofactor regeneration catalyst

Abstract

The oxygen-tolerant NAD⁺-reducing soluble hydrogenase (SH) from Ralstonia eutropha H16 has been described as a promising catalyst for cofactor regeneration in biocatalysed reductions. In this study, the actual potential of this enzyme for application in technical synthesis was evaluated. An overproduced, purified version of the enzyme was coupled to the carbonyl reductase from Candida parapsilosis (CPCR), where it allowed an almost quantitative conversion of the model substrate; total turnover numbers (TTN: n_product/n_enzyme) of up to 143,666 were achieved. This was distinctly superior to the commonly used NADH regenerating enzyme formate dehydrogenase (FDH) from Candida boidinii. In a systematic quantitative approach, maximum activity for NAD⁺ reduction was observed at 35 °C and pH 8, which corresponds to that of native SH. The half-life of the enzyme under these conditions was 5.3 hours. In the presence of sodium salts, distinct inhibitory effects were observed while ammonium and potassium ions increased the enzyme stability. Overall, a high but not unusual sensitivity of SH for changes in temperature, pH and mechanical stress in a reactor was found. Technical application in chemical synthesis can therefore be considered a feasible goal.     

High-throughput synthesis of conopeptides: a safety-catch linker approach enabling disulfide formation in 96-well format.

Conotoxins exhibit a high degree of selectivity and potency for a range of pharmacologically relevant targets. The rapid access to libraries of conotoxin analogues, containing multiple intramolecular disulfide bridges for use in drug development, can be a very labor intensive, multi-step task. This work describes a high-throughput method for the synthesis of cystine-bridged conopeptides.Peptides were assembled on a peptide synthesizer employing the Fmoc solid-phase strategy using a safety-catch amide linker (SCAL). Side-chain protecting groups were removed on solid phase before SCAL activation with ammonium iodide in TFA, finally releasing the peptide into the TFA solution. Disulfide bond formation was performed in the cleavage mixture employing DMSO.This improved method allows mixtures of oxidized peptides to be obtained in parallel directly from a peptide synthesizer. A single HPLC purification of the resulting crude oxidized material produced peptides of > 95% purity.     

Isolation and characterization of a marine methanogenic bacterium from the biofilm of a shiphull in Los Angeles harbor

A marine mesophilic, irregular coccoid methanogen, which shows close resemblance toMethanococcus sp., was isolated from the biofilm of shiphulls docked in Los Angeles harbor. Hydrogen plus carbon dioxide or formate served as substrates for methanogenesis in a mineral salt medium. The isolate did not use acetate and methanol as sole source of carbon and energy. The organism had an optimal pH range of 6.8–7.0 and a temperature optimum of 37°C. Elevated levels of sodium chloride were required for optimum growth. Optimum levels of total sulfide and magnesium chloride for growth were 1.0mm and 10mm respectively. The isolate used ammonia as nitrogen source. The concentration of 30mm ammonium chloride supported maximum growth of the isolate.     

Preparation of polymer-bound trityl-hydrazines and their application in the solid phase synthesis of partially protected peptide hydrazides

Summary Polymer-bound N-tritylhydrazines 4 were easily prepared by reacting polymeric tritylchlorides 3 with hydrazine. Subsequently, compounds 4 have been successfully applied to the solid phase synthesis of partially protected peptide hydrazides using 1-hydroxybenzotriazolyl esters of Fmoc- or Trt-amino acids. The synthesized peptide hydrazides can be quantitatively split off from the resins by mild acidic treatment, while the benzyl- and tert-butyl protecting groups remain unaffected.     

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.     

Chemo-enzymatic synthesis of a glycosylated peptide containing a complex <Emphasis Type="Italic">N</Emphasis>-glycan based on unprotected oligosaccharides by using DMT-MM and Endo-M

For chemo-enzymatic synthesis of a glycosylated peptide, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) was used for the synthesis of a N-acetylglucosaminyl peptide and a pseudoglycopeptide by solid-phase peptide synthesis without the requirement of protecting groups on the carbohydrate. We also performed transglycosylation of an N-glycan to the N-acetylglucosaminyl peptide using endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) to synthesize a glycopeptide containing a complex N-glycan.