Total chemical synthesis of a 304 amino acid K48-linked tetraubiquitin protein.

Recent advances in chemical and semisynthesis of proteins have allowed for the efficient production of naturally occurring proteins, polymer–protein conjugates, and posttranslationally modified proteins for structural and functional analyses. Among these examples are the recent developments of the non-enzymatic preparation of highly homogeneous ubiquitinated peptides and proteins, which are known to be crucial bioconjugates for the studies aiming at deciphering the effect of ubiquitination on cellular processes. In this regard, various research groups, including ours, have reported innovative strategies for constructing the native isopeptide bond and its mimetics, which links the lysine side chain of a protein target to the C-terminus of ubiquitin (Ub). The chemical synthesis of highly homogeneous and naturally occurring ubiquitinated proteins is just beginning to have an impact on our understanding of various systems involving Ub. Of particular interest are the very recent reports on the semiand chemical synthesis of diUb chains through native and nonnative isopeptide bond formation. 6] Notably, our group reported the total chemical synthesis of all seven Lys-linked diUb chains, thus paving the way for studying various aspects of these chains. Unarguably, the diUb analogues have provided useful information on several aspects of Ub biology and will continue to contribute to the field in various studies. In this regard, several structural and biochemical studies have already been performed on the diUb chains linked thorough K63, K48, and more recently on the remaining chains. 6,7] For most of the chains their optimal length for function is still unknown and this will have to be further investigated. For example, in the case of the K48 linkage, a chain of four Ub monomers is required for an efficient substrate recognition by the 26S proteasome. Moreover, for structural information the diUb chains may not fully represent the native structure of the longer chain. Finally, several in vitro studies have shown that most Ub binding domains bind to the Ub monomer, however, increasing the number of Ub units in a specific chain can be used both as a mechanism to increase binding and to enforce cellular specificity of deubiquitinases. Hence, the preparation of longer Ub chains is crucial to address some of the open questions in the field. Earlier efforts involving the use of the enzymatic machinery (E1-E3) so far has been limited mainly to Ub chains linked through K48 and K63, which requires also mutations in the Ub sequence (e.g. Lys/ Arg) and the formation of an isopeptide bond through thialysine. Moreover, these methods are very limited to the natural sequence, thus rendering the introduction of a specific modification in a highly controlled manner unreachable. On the other hand, by using chemical synthesis, virtually unlimited variations could be performed and incorporated in a highly controlled manner into each Ub unit within the specific chain (e.g. specific labeling). This would lead to unraveling of the thus-far unattainable details of Ub biology. Despite previous successes in applying chemical methods to prepare diUb chains, the synthesis of the tetraUb chain is much more challenging and requires new chemical methods to achieve such a formidable task. The tetraUb chain could, in principle, be synthesized through a linear approach or by applying a convergent strategy in which two diUb chains are synthesized separately and then linked to form the tetraUb chain (Scheme 1). The latter approach might be preferable, as we would expect it to proceed faster and give better yield. However, it also entails a challenge in that the Ub4-Ub3 fragment must be prepared in the thioester form to allow ligation to Ub2-Ub1 bearing d-mercaptolysine group. With this in mind, we initially tested the convergent approach wherein we first started to prepare the challenging Ub4-Ub3 thioester (Scheme 2). To achieve this, we applied our recently developed strategy to chemically synthesize Ub thioester to allow the incorporation of the d-mercaptolysine group in the Ub sequence to enable further chain elongation. Thus, Ub3 was first prepared from two fragments, Ub(1-45): Ub-N, and Ub(46-76): Ub3-C, wherein the latter was equipped with the 2-nitrobenzyl-protected N-methylcysteine and the d-mercaptolysine groups in the thiazolidine form. In this case, it was crucial to use the protected Nmethylcysteine group because following Ub3 assembly a treatment with methoxylamine is required to unmask the dmercaptolysine group. Under these reaction conditions (100 mm methoxylamine, pH 4) and when no protection was applied, the nucleophilic methoxylamine was found to attack the partially formed N S acyl-transfer intermediate (25– 30%). Meanwhile, if the protected N-methylcysteine group was used this side reaction was completely avoided. On the other hand, in the case of Ub4, which has no d-mercaptolysine [*] Dr. K. S. A. Kumar, Dr. S. N. Bavikar, L. Spasser, T. Moyal, S. Ohayon, Prof. A. Brik Department of Chemistry Ben-Gurion University of the Negev Beer Sheva 84105 (Israel) Fax: (+ 972)8-6472943 E-mail: abrik@bgu.ac.il Homepage: http://www.bgu.ac.il/~abrik [] These authors contributed equally.