Ligation and splicing of peptides and proteins.

Abstract

Steadily but unmistakably, we have entered the period that later historians would characterize as the biotechnological revolution. Led by the discovery of recombination DNA methods in the 1970s, biology has forged a deeper understanding of cellular processes, posted new frontiers through genetic engineering and genomic sequencing, and spurred major expansions in molecular medicine and biochemical sciences, particularly in structures and functions of peptides and proteins. Significant progress has already been achieved in structure determination, folding processes and de novo design of artificial proteins. In parallel, impressive advances in analytical methods have enabled detection and analysis of extremely minute amounts of biomolecules: DNA by PCR methods; peptides and proteins by mass spectrometry. In functional analyses of peptide and proteins, chemists and biologists have found novel synthetic solutions in meeting demands of the biotechnological revolution. These include new methods to produce proteins, both in vivo and in vitro, in large quantity as well as in diversity, the ability of simultaneously producing hundreds to millions of molecules chemically and biologically using methods such as phage-display libraries, yeast hybridization, and peptide and chemical libraries. On another synthetic front, the challenge of chemically preparing complex peptides, proteins and novel biomolecules is met by the development of new methodology. New synthetic methods are necessary to meet demands, not only for validation of new protein targets which are now numbered in the thousands due to genomic sequencing efforts, but also for the creation of new molecules in medicinal, functional, and nano-biomolecules. A biochemically-inclined approach to this solution has been centered on semi-synthesis by exploiting protein building blocks derived from natural or recombinant DNA in synthetic peptide-protein ligation. Such ligation chemistry is regiospecific, often performed in aqueous solutions and does not require a protection scheme or an enthalpic agent. These characteristics are also desirable for total synthesis, particularly the ability to form amide bonds of peptides and proteins. However, ligation chemistry has been often limited to nonamide bonds, and it is only in recent years that significant breakthroughs have been achieved in ligating amide bonds without protecting groups. These breakthroughs are built upon the work of many laboratories. For example, entropic activation required in amide ligation has been conceptualized and vigorously pursued by Daniel Kemp for nearly two decades. Methods developed by Theodore Wieland, Bruce Merrifield, James Blake and Donald Yamashiro, as well as many others, also contributed to the realization of amide-ligation methods. A key aspect of entropic activation is through the proximity-driven acyl shift of an ester to an amide. The acyl exchange of ester and amide bonds is also a key step in many enzymatic processes of making and breaking amide bonds. The recently discovered intein splicing is an outstanding example in which a protein is spliced through a series of intramolecular acyl exchanges of amides and esters to form two new proteins. This mechanism is exploited by several laboratories, including Muir and Xu, to trap the spliced intermediate as a thioester that can be used as building blocks for amide-ligation. With this advance, a major limiting factor of size in total- and semi-syntheses of proteins is removed. More importantly, the ester-amide acyl exchange has become the focal reaction integrating research in chemical synthesis, intein splicing and enzymatic processing. This issue of Peptide Science entitled Ligation and Splicing of Peptides and Proteins focuses on recent advances in the chemical and biochemical aspects of ligation, intein-splicing, and intein- mediated ligation. To provide a framework of this new area of chemistry, Tam, Yu, and Miao describe an overview of ligation chemistry, including history, recent advances and unpublished work of their laboratory. Three articles by Evans and Xu; Ayers, Blaschke, Camarero, Cotton, Holford and Muir; and Lew, Mills and Paulus describe the background, advances, and cutting-edge research in intein-mediated protein ligation of native proteins and those containing unnatural amino acids. Finally, two articles—by Beligere and Dawson and Yao and Chmielewski—provide, respectively, exciting progress in the use of chemical ligation for the synthesis of a three-zinc-finger protein, and its elegant application as a pH-tunable peptide ligase. From these six articles, I hope readers will share our enthusiasm for ligation chemistry towards both old and new synthetic problems of peptides and proteins.