Peptide Synthesis Protocols

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.

Peptide Synthesis, 2019

2016

How to cite Complete issue More information about this article Journal's homepage in redalyc.org Scientific Information System Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Non-profit academic project, developed under the open access initiative

Peptides, 1992

Current Organic Chemistry, 2001

This review describes current methods for peptide and protein syntheses, largely from a strategic point of view. The solid-phase method is useful for the rapid preparation of peptides. Two major synthetic strategies have been adopted by this method, namely, the Boc and Fmoc strategies. At the final stage of the Boc solidphase method, a protected peptide resin is treated with a strong acid to obtain a free peptide. On the other hand, in the Fmoc solid-phase method, a free peptide is obtained by treating a protected peptide resin with a weak acid. Both solid phase methods are quite useful for the preparation of peptides with molecular weights in the vicinity of five thousand. Ligation methods were developed to overcome the molecular weight barrier existing in a solid phase method. Building blocks used for ligation are prepared by the solid phase method, or more recently by biological methods. All the current ligation methods that produce a native peptide bond use peptide C-terminal thiocarboxylic acids or thioesters as building blocks. Blake et al. developed a selective activation method of the C-terminal carbonyl group by the combination of thiocarboxylic acid and silver ions. Based on this approach, a thioester method was developed, in which partially protected peptide thioesters are used as building blocks. Subsequently, a new ligation method was developed using peptide thioesters, in which protecting group is no longer necessary. The discovery of protein splicing phenomenon added a biological route to the preparation of peptide thioesters. A partially protected peptides segment can be also derived from an expressed peptide segment. Polypeptides with a molecular weight of more than 10 thousand can be routinely synthesized.

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.

2015

The peptides used in this work encompass the transmembrane domain of M2 and correspond to residues Ser22 to Leu46: SSDPLVVAASIIGILHLILWILDRL [1]. The peptides were synthesized using standard solid-phase N-(9-fluorenyl methoxycarbonyl) chemistry, cleaved from the resin with trifluoroacetic acid, and lyophilized. Each peptide was labelled using 1-13C=18O amino acids, using 13C labelled amino acids precursors (Cambridge Isotopes Laboratories, Andover, MA) at positions indicated in Fig. 1a using procedures descried elsewhere in detail [2, 3]. Peptide purification 0.5 mg of the crude synthesis was dissolved in 0.75 ml of trifluoroacetic acid, and

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.

Biopolymers, 2000

Recent advances in the areas of formulation and delivery have rekindled the interest of the pharmaceutical community in peptides as drug candidates, which, in turn, has provided a challenge to the peptide industry to develop efficient methods for the manufacture of relatively complex peptides on scales of up to metric tons per year. This article focuses on chemical synthesis approaches for peptides, and presents an overview of the methods available and in use currently, together with a discussion of scale-up strategies. Examples of the different methods are discussed, together with solutions to some specific problems encountered during scale-up development. Finally, an overview is presented of issues common to all manufacturing methods, i.e., methods used for the large-scale purification and isolation of final bulk products and regulatory considerations to be addressed during scale-up of processes to commercial levels.

2015

La sintesis de peptidos, dentro del campo de la quimica organica, sufrio un giro significativo promovido por el desarrollo del revolucionario descubrimiento en 1963 llevado a cabo por el Dr. Merrifield. Este avance sintetico se conoce con el nombre de sintesis de peptidos en fase solida (SPPS) y a partir de su aparicion, diferentes grupos de investigacion en este campo han invertido esfuerzos en el desarrollo de mejoras dirigidas a este estrategia. De modo que se han disenado nuevos grupos protectores para los amino acidos (AAs), sintesis de resinas (o soportes polimericos) sobre los que hacer crecer la cadena peptidica, diferentes agentes de acoplamiento para la incorporacion eficaz de los AAs, entre otros. A pesar de la gran diversidad de nuevos descubrimientos enfocados en este sector, todavia existe una gran demanda de nuevos metodos sinteticos para la sintesis de ciertas secuencias peptidicas complejas. En la presente tesis, y en base al diseno de metodologias de sintesis que c...