Expanding the chemical toolbox for the synthesis of large and uniquely modified proteins (original) (raw)

Contemporary Methods for Peptide and Protein Synthesis

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.

Through the looking glass - a new world of proteins enabled by chemical synthesis

Journal of Peptide Science, 2012

Chemical ligation'the regioselective and chemoselective covalent condensation of unprotected peptide segmentshas enabled the synthesis of polypeptide chains of more than 200 amino acids. An efficient total chemical synthesis of the insulin molecule has been devised on the basis of a key ester-linked intermediate that is chemically converted to fully active human insulin. Enzyme molecules of defined covalent structure and with full enzymatic activity have been prepared and characterized by high-resolution X-ray crystallography. A 'glycoprotein mimetic' of defined chemical structure and with a mass of 50,825 Da, has been prepared and shown to have full biological activity and improved pharmacokinetic properties. D-Protein molecules that are the mirror images of proteins found in the natural world have been prepared by total chemical synthesis. Racemic protein mixtures, consisting of the D-enantiomers and L-enantiomers of a protein molecule, form highly ordered centrosymmetric crystals with great ease; this has enabled the determination of the crystal structures of recalcitrant protein molecules. A protein with a novel linear-loop covalent topology of the peptide chain has been designed and synthesized and its structure determined by facile crystallization as the quasi-racemate with the D-form of the native protein molecule. We have developed an optimized total chemical synthesis of biologically active vascular endothelial growth factor-A; total synthesis of the mirror-image protein will be used to systematically develop D-protein antagonists of this important growth factor. The total chemical synthesis of proteins is now a practical reality and enables access to a new world of protein molecules.

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.

Chemical Protein Synthesis by Solid Phase Ligation of Unprotected Peptide Segments

Journal of the American Chemical Society, 1999

In this paper we describe "solid phase chemical ligation" (SPCL), the application of the principles of polymer-supported organic synthesis to the construction of large polypeptide chains for the total chemical synthesis of proteins. In this method, each building block used is an unprotected peptide segment of 20 or more amino acids. These are consecutively reacted by chemical ligation, the chemoselective reaction of the unprotected peptide segments from aqueous solution, to make the polymer-supported target polypeptide. In a final step, the assembled full-length target polypeptide is released from the aqueous-compatible polymer support.

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.

Chemical Synthesis of Proteins Containing 300 Amino Acids

Chemical Research in Chinese Universities, 2020

Chemical synthesis of proteins containing up to 300 amino acids may cover 30%-50% of all the proteins encountered in biomedical studies and may provide an alternate approach to the usually used recombinant expression technology, vastly expanding the chemical space of the latter. In the present review article, we tried to survey the recent progresses made for more rapid synthesis of increasingly long peptides and more efficient ligation of multiple peptide segments. The developments of seminal methods by many research groups have greatly contributed to the recent breakthroughs in the successful total synthesis of a number of functionally important proteins , such as oligoubiquitins, bacterial GroEL/ES chaperones, and mirror-image DNA polymerases. Through these studies, a potential bottleneck has also been recognized for the chemical synthesis of large proteins, namely, how to ensure that each peptide segment from a large protein avoids unfavorable aggregation when dissolved in aqueous solution. Many new methods, such as removable backbone modification(RBM) strategy have been developed to overcome this bottleneck, while more studies need to be carried out to develop more effective and less costly methods that ultimately, may lead to fully automatable chemical synthesis of customized proteins of 300 amino acids bearing any artificial designs.

A Helping Hand to Overcome Solubility Challenges in Chemical Protein Synthesis

Journal of the American Chemical Society, 2016

Although native chemical ligation (NCL) and related chemoselective ligation approaches provide an elegant method to stitch together unprotected peptides, the handling and purification of insoluble and aggregation-prone peptides and assembly intermediates create a bottleneck to routinely preparing large proteins by completely synthetic means. In this work, we introduce a general new tool, Fmoc-Ddae-OH, N-Fmoc-1-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-3-[2-(2-aminoethoxy)ethoxy]-propan-1-ol, a hetero-bifunctional traceless linker for temporarily attaching highly solubilizing peptide sequences (helping hands) onto insoluble peptides. This tool is implemented in three simple and nearly quantita-tive steps: (i) on-resin incorporation of the linker at a Lys residue ε-amine, (ii) Fmoc-SPPS elongation of a desired solubil-izing sequence, and (iii) in-solution removal of the solubilizing sequence using mild aqueous hydrazine to cleave the Ddae linker after NCL-based assembly. Successful int...