Heterogeneous catalytic transfer hydrogenation in peptide synthesis

International Journal of Peptide Research and Therapeutics -     

Catalysis in peptide synthesis

Enzyme catalyzed formation of peptide bonds reached practical significance in partial syntheses such as the conversion of pork insulin to human insulin. Catalysis is important also in coupling with active esters. The earlier applied acid-base catalysis was replaced by the use of bifunctional catalysts, 1-hydroxybenzotriazole being an outstanding example: it brought about major improvements in peptide bond formation. Intramolecular catalysis can be discerned in the rapid acylation by certain active esters, e.g., esters of catechol, 4-nitroguaiacol or 8-hydroxyquinoline. The ready formation of symmetrical anhydrides in the reaction of acylamino acids with carbodiimides is best explained by intramolecular catalysis within the O-acylisourea intermediates. Catalysis plays important roles both in introduction and in removal of blocking groups. Preparation of alkyl esters through base-catalyzed transesterification of active aryl esters and the application of this reaction for the anchoring of peptides to polymeric supports are described. Enzyme catalyzed hydrolysis of alkyl esters and hydrolytic fission of the phenylacetyl group from lysine side chains with aid of penicillin amidohydrolase are characteristic examples of the application of catalysis for the removal of blocking groups. Acidolysis of the benzyl groups including the benzyloxcarbonyl group is catalyzed by thioanisole or by 4-methylthiophenol. The catalytic effect of solvents is demonstrated with the cleavage of triphenylmethyl and biphenylyl-isopropyloxycarbonyl groups by 1-hydroxybenzotriazole in trifluoroethanol. The increasing role of catalysis in peptide synthesis and its future application for the solution of fundamental problems, such as amine activation, are discussed.     

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.     

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

Summary 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.     

Facile production of mono-substituted urea side chains in solid phase peptide synthesis

A method is reported for the straightforward generation of urea-containing peptides during Boc solid phase peptide synthesis. Primary amine side chains are converted to mono-alkyl ureas in two steps via an intermediate p-nitrophenyl carbamate. Use of p-methoxybenzyl amine as an ammonia equivalent affords mono-alkyl final products from standard resin cleavage methods, without the need for additional steps. The reaction is highly efficient and applicable to variable length side chains and peptides.     

Advances in Fmoc solid‐phase peptide synthesis

Today, Fmoc SPPS is the method of choice for peptide synthesis. Very‐high‐quality Fmoc building blocks are available at low cost because of the economies of scale arising from current multiton production of therapeutic peptides by Fmoc SPPS. Many modified derivatives are commercially available as Fmoc building blocks, making synthetic access to a broad range of peptide derivatives straightforward. The number of synthetic peptides entering clinical trials has grown continuously over the last decade, and recent advances in the Fmoc SPPS technology are a response to the growing demand from medicinal chemistry and pharmacology. Improvements are being continually reported for peptide quality, synthesis time and novel synthetic targets. Topical peptide research has contributed to a continuous improvement and expansion of Fmoc SPPS applications. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

Facile synthesis of peptide nucleic acids and peptide nucleic acid‐peptide conjugates on an automated peptide synthesizer

Peptide nucleic acids (PNAs) are DNA mimics with a neutral peptide backbone instead of the negatively charged sugar phosphates. PNAs exhibit several attractive features such as high chemical and thermal stability, resistance to enzymatic degradation, and stable binding to their RNA or DNA targets in a sequence‐specific manner. Therefore, they are widely used in molecular diagnosis of antisense‐targeted therapeutic drugs or probes and in pharmaceutical applications. However, the main hindrance to the effective use of PNAs is their poor uptake by cells as well as the difficult and laborious chemical synthesis. In order to achieve an efficient delivery of PNAs into cells, there are already many published reports of peptides being used for transport across the cell membrane. In this protocol, we describe the automated as well as cost‐effective semi‐automated synthesis of PNAs and PNA‐peptide constructs on an automated peptide synthesizer. The facile synthesis of PNAs will be helpful in generating PNA libraries usable, e.g. for high‐throughput screening in biomolecular studies. Efficient synthetic schemes, the automated procedure, the reduced consumption of costly reagents, and the high purity of the products are attractive features of the reported procedure. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Matrix‐assisted peptide synthesis on nanoparticles

We report a new method for multistep peptide synthesis on polymeric nanoparticles of differing sizes. Polymeric nanoparticles were functionalized via their temporary embedment into a magnetic inorganic matrix that allows multistep peptide synthesis. The matrix is removed at the end of the process for obtaining nanoparticles functionalized with peptides. The matrix‐assisted synthesis on nanoparticles was proved by generating various biologically relevant peptides. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Optimization of butanediol dimethacrylate crosslinked polystyrene: A novel polymeric support for solid phase peptide synthesis

Studies leading to optimization of butanedioldimethacrylate-crosslinked polystyrene supports (BDDMA–PS) forsolid phase peptide synthesis are delineated. BDDMA–PScopolymers with different crosslink densities were prepared andfunctionalised with chloromethyl groups. The reactivity of theLys(2-Cl-Z)-OH residue bound to these polymers through a benzylester linkage was investigated by following the kinetics ofacylation by the HOBt active ester of Boc-Alanine. From theresults it was observed that the rate of peptide bond formationwas maximum for a 2% BDDMA crosslinked resin. This resin wascompared with a 2% DVB-crosslinked polystyrene resin (DVB–PS). Synthesis of an extremely insoluble, hydrophobic,antiparallel -sheeted difficult sequencepeptide LMVGGVVIA ( 34–42), C-terminal fragment of -amyloid protein, (1–42), wascarried out on both 2% DVB–PS and 2% BDDMA-crosslinkedpolystyrene supports. The synthesis of the peptide was carriedout using Boc amino acid strategy. Greater extent of swellingof the resino peptide, increased coupling efficiency during theassembly of amino acids and relatively high purity of synthesised peptide were observed in the case of 2% BDDMA–PS polymer.     

Optimization of butanediol dimethacrylate crosslinked polystyrene: A novel polymeric support for solid phase peptide synthesis

Summary Studies leading to optimization of butanediol dimethacrylate-crosslinked polystyrene supports (BDDMA-PS) for solid phase peptide synthesis are delineated. BDDMA-PS copolymers with different crosslink densities were prepared and functionalised with chloromethyl groups. The reactivity of the Lys(2-Cl−Z)−OH residue bound to these polymers through a benzyl ester linkage was investigated by following the kinetics of acylation by the HOBt active ester of Boc-Alanine. From the results it was observed that the rate of peptide bond formation was maximum for a 2% BDDMA crosslinked resin. This resin was compared with a 2% DVB-crosslinked polystyrene resin (DVB-PS). Synthesis of an extremely insoluble, hydrophobic, antiparallel β-sheeted difficult sequence peptide LMVGGVVIA (β 34–42), C-terminal fragment of β-amyloid protein, β (1–42), was carried out on both 2% DVB-PS and 2% BDDMA-crosslinked polystyrene supports. The synthesis of the peptide was carried out using Boc amino acid strategy. Greater extent of swelling of the resino peptide, increased coupling efficiency during the assembly of amino acids and relatively high purity of synthesised peptide were observed in the case of 2% BDDMA-PS polymer.     

Limiting racemization and aspartimide formation in microwave-enhanced Fmoc solid phase peptide synthesis.

Microwave energy represents an efficient manner to accelerate both the deprotection and coupling reactions in 9-fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis (SPPS). Typical SPPS side reactions including racemization and aspartimide formation can occur with microwave energy but can easily be controlled by routine use of optimized methods. Cysteine, histidine, and aspartic acid were susceptible to racemization during microwave SPPS of a model 20mer peptide containing all 20 natural amino acids. Lowering the microwave coupling temperature from 80 degrees C to 50 degrees C limited racemization of histidine and cysteine. Additionally, coupling of both histidine and cysteine can be performed conventionally while the rest of the peptide is synthesized using microwave without any deleterious effect, as racemization during the coupling reaction was limited to the activated ester state of the amino acids up to 80 degrees C. Use of the hindered amine, collidine, in the coupling reaction also minimized formation of D-cysteine. Aspartimide formation and subsequent racemization of aspartic acid was reduced by the addition of HOBt to the deprotection solution and/or use of piperazine in place of piperidine.     

Rapid solid-phase peptide synthesis using thermal and controlled microwave irradiation.

A rapid and efficient microwave-assisted solid-phase synthesis method is described for the preparation of the nonapeptide WDTVRISFK, using conventional Fmoc/Bu(t) orthogonal protection strategy. The synthesis protocol is based on the use of cycles of pulsed microwave irradiation with intermittent cooling of the reaction during the removal of the Fmoc protecting group and during the coupling. The desired nonapeptide was obtained in highest yield and purity by employing MicroKan technology. The chemical reactions were carried out in a single-mode microwave reactor, equipped with a fiber-optic probe to monitor the reaction temperature continuously.     

Monitoring of solid phase peptide synthesis by FT-IR spectroscopy

Aggregation phenomena of growing peptides on the resin have seldom been investigated. We report here how conformations are determined by FT-IR spectroscopy. Therefore the sequence 80–99 of HIV 1-protease was synthesized. After every coupling a resin sample was taken out of the reaction column and a FT-IR spectrum recorded. The results were compared with the UV monitoring obtained from another synthesis of the same peptide. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Novel amino-Li resin for water-based solid-phase peptide synthesis

We report the first application of a novel amino-Li resin to water-based solid-phase peptide synthesis (SPPS) applying the Smoc-protecting group approach. We demonstrated that it is a suitable support for the sustainable water-based alternative to a classical SPPS approach. The resin possesses good swelling properties in aqueous milieu, provides significant coupling sites, and may be applicable to the synthesis of difficult sequences and aggregation-prone peptides.     

Solid-phase synthesis of tailed cyclic RGD peptides using glutamic acid: unexpected glutarimide formation.

To provide multiple conjugating sites on cyclic peptides for their increasing biomedical applications, a tailed cyclic RGD peptide, c[RGDfE(GGGKK-NH(2))] was designed with c(RGDfE) linked through Glu to a tail consisting of a spacer of three Gly residues and a linker of two Lys residues. The spacer is used to increase the mobility and binding ability of the c(RGDfE) ligand, and the linker is used to proved multiple active sites for conjugating other molecules or biomaterials. We found that the sequence of Glu(Gly)-OAll leads to glutarimide formation, which disrupts the formation of cyclic RGD peptides. However, our results show that glutarimide formation is sequence dependent and can be inhibited by incorporating an amino acid like Lys(Boc) with steric hindrance from the protecting group. To prevent glutarimide formation, Ser(tBu) was used to replace the glycine in the GGG spacer adjacent to the residue of Glu, and a tailed cyclic RGD peptide, c[RGDfE(SGGKK-NH(2))] was successfully obtained.     

Disulfide bond polymerization of a cyclic peptide derived from the surface loop 4 of class 1 OMP of Neisseria meningitidis

Summary Two peptides derived from the surface loop 4 of class 1 Outer Membrane Protein (OMP) ofNeisseria meningitidis were synthesized on solid phase using the Boc/Bzl strategy: one containing the entire loop 4 cyclized and the other representing the polymerized cyclic loop 4. To test a more efficient cyclic peptide presentation, in the present study a strategy was developed to obtain polymers of cyclic peptides. In order to obtain the polymeric cyclic peptide, two protecting groups for cysteine were used — Acm and Mob. The Cys(Acm)-protected cyclic peptide was obtained after removing the Mob group. The polymerization reaction was carried out by simultaneous deprotection/oxidation ofS-Acm with iodine. Analysis of the polymeric cyclic peptide in Tris-tricine-SDS-PAGE showed different bands with molecular weights higher than expected for the corresponding monomeric cyclic peptide. Both peptides were used in immunization of four different mouse strains. The antisera raised against the peptides were evaluated by ELISA and Western blotting vs. OMP preparation ofN. meningitidis. The titers raised against the polymerized cyclic peptide were higher than the ones raised against the cyclic peptide. The antisera elicited did not show bactericidal activity. Nevertheless, the antisera elicited against the polymeric cyclic peptide in the CBA/J mouse strain showed opsonic activity. The antibodies raised against the polymeric cyclic peptide were successfully used as probes in Western blotting experiments to verify the display of loop 4 peptide on the surface of filamentous phage M13.     

Polymer-supported biopolymer synthesis: 5. Ultra-high load solid (gel) phase peptide synthesis—the stepwise elaboration of quasi-homogeneous peptide gel networks?

An approach to ultra-high load solid (gel) phase peptide synthesis is described in which a bead-form phenolic core polymer, crosslinked poly[N-{2-(4-hydroxyphenyl)ethyl}-acrylamide], is used as a support matrix at near theoretical maximum loading. Consecutive repeating units of the core polymer carry peptide chains undergling stepwise elongation. Synthesis proceeds through a series of solvated networks, which consist mainly of protected peptide. The solvated networks are deemed to be quasi-homogeneous, insofar as each has a regular covalent framework and each is believed to be uniformly distributed throughout the gel beads. Illustrative synthesis of two fully-protected acylpeptide hydrazide segments, corresponding to dynorphin(6–12) and to β_h-endorphin (18–26), are described.     

Disulfide bond polymerization of a cyclic peptide derived from the surface loop 4 of class 1 OMP of Neisseria meningitidis

Two peptides derived from the surface loop 4 of class1 Outer Membrane Protein (OMP) of Neisseriameningitidis were synthesized on solid phase usingthe Boc/Bzl strategy: one containing the entire loop4 cyclized and the other representing the polymerizedcyclic loop 4. To test a more efficient cyclic peptidepresentation, in the present study astrategy was developed to obtain polymers of cyclic peptides. Inorder to obtain the polymeric cyclic peptide, twoprotecting groups for cysteine were used – Acm andMob. The Cys(Acm)-protected cyclic peptide wasobtained after removing the Mob group. Thepolymerization reaction was carried out bysimultaneous deprotection/oxidation of S-Acmwith iodine. Analysis of the polymeric cyclic peptidein Tris-tricine-SDS-PAGE showed different bandswith molecular weights higher than expected for thecorresponding monomeric cyclic peptide. Both peptideswere used in immunization of four different mouse strains.The antisera raised against the peptides wereevaluated by ELISA and Western blotting vs. OMPpreparation of N. meningitidis. The titersraised against the polymerized cyclic peptide werehigher than the ones raised against the cyclicpeptide. The antisera elicited did not showbactericidal activity. Nevertheless, the antiseraelicited against the polymeric cyclic peptide in theCBA/J mouse strain showed opsonic activity. Theantibodies raised against the polymeric cyclic peptidewere successfully used as probes in Western blottingexperiments to verify the display of loop 4 peptide onthe surface of filamentous phage M13.