Recent Tricks in Solid-Phase Peptide Synthesis (original) (raw)
Fundamentals of Modern Peptide Synthesis
ChemInform, 2006
The purpose of this article is to delineate strategic considerations and provide practical procedures to enable non-experts to synthesize peptides with a reasonable chance of success. This article is not encyclopedic but rather devoted to the Fmoc/tBu approach of solid phase peptide synthesis (SPPS), which is now the most commonly used methodology for the production of peptides. The principles of SPPS with a review of linkers and supports currently employed are presented. Basic concepts for the different steps of SPPS such as anchoring, deprotection, coupling reaction and cleavage are all discussed along with the possible problem of aggregation and side-reactions. Essential protocols for the synthesis of fully deprotected peptides are presented including resin handling, coupling, capping, Fmoc-deprotection, final cleavage and disulfide bridge formation. Index Entries: Solid phase peptide synthesis (SPPS); resin; Fmoc SPPS; coupling reagents; protecting groups; anchoring; side reaction.
An Improved Procedure for N- to C-Directed (Inverse) Solid-Phase Peptide Synthesis
Journal of Combinatorial Chemistry, 2000
A method for solid-phase peptide synthesis in the N-to C-direction that delivers good coupling yields and a low degree of epimerization is reported. The optimized method involves the coupling, without preactivation, of the resin-bound C-terminal amino acid with excess amounts of amino acid tri-tert-butoxysilyl (Sil) esters, using HATU as coupling reagent and 2,4,6-trimethylpyridine (TMP, collidine) as a base. For the amino acids investigated, the degree of epimerization was typically 5%, except for Ser(t-Bu) which was more easily epimerized (ca. 20%). Five tripeptides (AA 1 -AA 2 -AA 3 ) with different properties were used as representative model peptides in the development of the synthetic method: Asp-Leu-Glu, Leu-Ala-Phe, Glu-Asp-Val, Asp-Ser-Ile, and Asp-D-Glu-Leu. The study used different combinations of HATU and TBTU as activating agents, N,N-diisopropylethylamine (DIEA) and TMP as bases, DMF and dichloromethane as solvents, and cupric chloride as an epimerization suppressant. The epimerization of AA 2 in the coupling of AA 3 was further reduced in the presence of cupric chloride. However, the use of this reagent also resulted in a decrease in loading onto the resin and significant cleavage between AA 1 and AA 2 . Experiments indicated that the observed suppressing effect of cupric chloride on epimerization in the present system merely seemed to be a result of a base-induced cleavage of the oxazolone system, the key intermediate in the epimerization process. Consequently, the cleavages were most pronounced in slow couplings. An improved synthesis of fully characterized amino acid tri-tert-butoxysilyl (Sil) ester hydrochloride building blocks is presented. The amino acid Sil esters were found to be stable as hydrochlorides but not as free bases. Although only a few peptides have been used in this study, we believe that the facile procedure devised herein should provide an attractive alternative for the solid-phase synthesis of short (six residues or less) C-terminally modified peptides, e.g., in library format.
Journal of the American Chemical Society, 1998
Peptide targets for synthesis are often desired with C-terminal end groups other than the more usual acid and amide functionalities. Relatively few routes exist for synthesis of C-terminal-modified peptidessincluding cyclic peptidessby either solution or solid-phase methods, and known routes are often limited in terms of ease and generality. We describe here a novel Backbone Amide Linker (BAL) approach, whereby the growing peptide is anchored through a backbone nitrogen, thus allowing considerable flexibility in management of the termini. Initial efforts on BAL have adapted the chemistry of the tris(alkoxy)benzylamide system exploited previously with PAL anchors. Aldehyde precursors to PAL, e.g. 5-(4-formyl-3,5-dimethoxyphenoxy)valeric acid, were reductively coupled to the R-amine of the prospective C-terminal amino acid, which was blocked as a tert-butyl, allyl, or methyl ester, or to the appropriately protected C-terminal-modified amino acid derivative. These reductive aminations were carried out either in solution or on the solid phase, and occurred without racemization. The secondary amine intermediates resulting from solution amination were converted to the 9-fluorenylmethoxycarbonyl (Fmoc)-protected preformed handle derivatives, which were then attached to poly-(ethylene glycol)-polystyrene (PEG-PS) graft or copoly(styrene-1% divinylbenzene) (PS) supports and used to assemble peptides by standard Fmoc solid-phase chemistry. Alternatively, BAL anchors formed by onresin reductive amination were applied directly. Conditions were optimized to achieve near-quantitative acylation at the difficult step to introduce the penultimate residue, and a side reaction involving diketopiperazine formation under some circumstances was prevented by a modified protocol for N R-protection of the second residue/ introduction of the third residue. Examples are provided for the syntheses in high yields and purities of representative peptide acids, alcohols, N,N-dialkylamides, aldehydes, esters, and head-to-tail cyclic peptides. These methodologies avoid postsynthetic solution-phase transformations and are ripe for further extension.
Journal of the American Chemical Society, 1998
Peptide targets for synthesis are often desired with C-terminal end groups other than the more usual acid and amide functionalities. Relatively few routes exist for synthesis of C-terminal-modified peptidessincluding cyclic peptidessby either solution or solid-phase methods, and known routes are often limited in terms of ease and generality. We describe here a novel Backbone Amide Linker (BAL) approach, whereby the growing peptide is anchored through a backbone nitrogen, thus allowing considerable flexibility in management of the termini. Initial efforts on BAL have adapted the chemistry of the tris(alkoxy)benzylamide system exploited previously with PAL anchors. Aldehyde precursors to PAL, e.g. 5-(4-formyl-3,5-dimethoxyphenoxy)valeric acid, were reductively coupled to the R-amine of the prospective C-terminal amino acid, which was blocked as a tert-butyl, allyl, or methyl ester, or to the appropriately protected C-terminal-modified amino acid derivative. These reductive aminations were carried out either in solution or on the solid phase, and occurred without racemization. The secondary amine intermediates resulting from solution amination were converted to the 9-fluorenylmethoxycarbonyl (Fmoc)-protected preformed handle derivatives, which were then attached to poly-(ethylene glycol)-polystyrene (PEG-PS) graft or copoly(styrene-1% divinylbenzene) (PS) supports and used to assemble peptides by standard Fmoc solid-phase chemistry. Alternatively, BAL anchors formed by onresin reductive amination were applied directly. Conditions were optimized to achieve near-quantitative acylation at the difficult step to introduce the penultimate residue, and a side reaction involving diketopiperazine formation under some circumstances was prevented by a modified protocol for N R-protection of the second residue/ introduction of the third residue. Examples are provided for the syntheses in high yields and purities of representative peptide acids, alcohols, N,N-dialkylamides, aldehydes, esters, and head-to-tail cyclic peptides. These methodologies avoid postsynthetic solution-phase transformations and are ripe for further extension.
ChemInform, 2009
The most popular way to synthesize peptides is via the solidphase approach, mostly on a research scale, although progress is being made in large-scale production. The most evident example is Fuzeon, a commercial anti-HIV peptide, which is produced in multi-kilograms using a solid support for the synthesis of the fragments. Success in solid-phase peptide synthesis is heavily determined by the solid support. In this review we focus on the evolution of the solid support from the totally polystyrene-based resin used by Merrifield to the most sophisticated ones currently available on the market. These new resins offer access to previously inaccessible compounds as well as the possibility to be used in diverse applications but without losing stability. Moreover, these new supports are easy to handle. The final chapter of the review highlights the complex sequences that are difficult to achieve and the reasons for this. It then concludes by explaining the approaches that have been followed to synthesize such "difficult" peptides.
Cyclohexyloxycarbonyl based orthogonal solid phase peptide synthesis in Boc chemistry
Tetrahedron, 1998
Application of N-cyclohex'yloxTcafoonyl (Choc) protection in Boc chemistry on solid phase provides a new possibility for the preparation of protected peptide fragments. A Choc/OcHex protection scheme allows also the assembly of cyclic lactam peptides linked to the resin through the C-terminus. Choc protection is stable under the 1M TMSOTf-thioanisole/TFA cleavage condition at 0°C, but it is removable by anhydrous HF. We have utilized cyclohexTloxycarbonyl as an orthogonal protecting group for the synthesis of a i) bicyclic epitope peptide of glycoprotein D of HSV 1 on BHA resin and ii) fully protected hexapeptide involved in protein transport on Merrifield resin.
Journal of Combinatorial Chemistry, 2003
Preliminary studies and synthesis development for the preparation of a bicyclic homodetic peptide library have been carried out using orthogonal protection schemes. The best results have been obtained using two Fmoc/tBu-based strategies, in which the first cycle is carried out in the solid phase through side chain functional groups previously protected with Aloc/Al groups. The second cycle is performed either in the solid phase, which requires side chain anchoring of a trifunctional amino acid and Dmb protection for the C-terminus carboxyl group, or in solution, which requires the use of highly labile resins, such as the 2-chlorotrityl (Barlos) resin. Only when the cycles are formed in a ziplike manner, that is, first the small cycle and then the larger ring, is the desired final product obtained.
High Throughput Synthesis of Peptides and Peptidomimetics
ChemInform, 2007
Peptide synthesis has been developed into one of the most efficient synthetic procedures in organic chemistry. The problems of orthogonal functional group protection and amide bond formation without racemization have been developed in a number of ingenious strategies. Optimization, in particular, has been achieved in stepwise solid phase synthesis. This in turn made possible the development of combinatorial synthesis allowing the synthesis of millions of peptide compounds of high purity in a few days. A variety of methodologies and strategies have been developed and continue to be developed to determine structures and to evaluate peptides and peptidomimetics. The development of methods for solid phase synthesis of a variety of organic and inorganic structures using similar strategies as in peptide synthesis are being vigorously pursued. However, existing instrumentation and technology is not sufficient to cover current demands for peptides, and thus new approaches and technologies for cost-effective synthesis of peptide arrays are needed.
Chemical Methods for Peptide and Protein Production
Molecules, 2013
Since the invention of solid phase synthetic methods by Merrifield in 1963, the number of research groups focusing on peptide synthesis has grown exponentially. However, the original step-by-step synthesis had limitations: the purity of the final product decreased with the number of coupling steps. After the development of Boc and Fmoc protecting groups, novel amino acid protecting groups and new techniques were introduced to provide high quality and quantity peptide products. Fragment condensation was a popular method for peptide production in the 1980s, but unfortunately the rate of racemization and reaction difficulties proved less than ideal. Kent and co-workers revolutionized peptide coupling by introducing the chemoselective reaction of unprotected peptides, called native chemical ligation. Subsequently, research has focused on the development of novel ligating techniques including the famous click reaction, ligation of peptide hydrazides, and the recently reported -ketoacid-hydroxylamine ligations with 5oxaproline. Several companies have been formed all over the world to prepare high quality Good Manufacturing Practice peptide products on a multi-kilogram scale. This review describes the advances in peptide chemistry including the variety of synthetic peptide methods currently available and the broad application of peptides in medicinal chemistry.
Solid-phase peptide synthesis using Nα-trityl-amino acids
Letters in Peptide Science, 2001
The preparation of N α -trityl-amino acids is described. Several derivatives of trifunctional amino acids carrying acid-and base-labile side-chain protecting groups and the trityl group at the N α position are prepared for first time. The incorporation of N α -trityl-amino acids into peptide sequences using solid-phase protocols was achieved. The use of the trityl group for the protection of the α-amino group in conjunction with base-labile side-chain protecting groups constitutes a new method for the assembly of peptides in mild conditions.