OPTIMIZED STRATEGIES FOR THE SYNTHESIS OF BIOMOLECULES: THE MICROWAVE APPROACH TO COMPLEX PEPTIDES FOR DIAGNOSTIC/PROGNOSTIC IMMUNOASSAYS
Prof. Anna Maria Papini, PhD

Speediness represents a critical factor for the growing demand of synthetic biomolecules, such as peptides, by combinatorial and medicinal chemistry, strongly affecting the process of drug discovery. Conventional Solid-Phase Peptides Synthesis remains the principal strategy for peptide synthesis. Recently, the use of microwave energy was proposed to fulfil the requirement to couple a high-speed technology with an efficient solid-phase synthetic approach.

In our Laboratory, we were attracted by the possibility to improve peptide production, by using microwave energy (LibertyTM Microwave Peptide Synthesizer, CEM), that decreases chain aggregation during the syntheses, improving the coupling rates, and possibly preventing side reactions. In particular we were interested in complex peptide sequences, such as the glycosylated peptide CSF114(Glc), the first immunochemical probe for detecting specific autoantibodies in Multiple Sclerosis patients' sera, useful for the development of a specific diagnostic/prognostic tool [1]. In particular, the main difficulties associated with glycopeptide synthesis are both in obtaining non-commercially available building-blocks, in large scale at a reasonable price, and in the low coupling efficiency of the sterically hindered sugar-bound amino acid derivatives.

Solid-Phase Glycopeptides Synthesis (SPGS) is improved by employing a large excess of glycosylated building blocks orthogonally protected for SPGS. Considering the non trivial protocol to achieve these starting materials, it is important to optimise synthetic strategies to the corresponding glycopeptides in order to obtain them in high yield for research & development phases of the corresponding immunoassays (Toscana Biomarkers Srl - www.toscanabiomarkers.com & EspiKem Srl - www.espikem.com).

The use of microwave irradiation (Discover® BenchMate, CEM) allowed a rapid and high-yield preparation of N-glycosyl amino acids orthogonally protected for SPPS [5]. Moreover, our "peptide-based reverse approach", successfully used in the case of Multiple Sclerosis [6], has been extended to detect biomarkers of other autoimmune diseases (e.g. Rheumatoid Arthritis) [7].

Therefore, there will be a concrete demand of CSF114(Glc), for marketing MS PepKit, as specific diagnostic test for monitoring Multiple Sclerosis patients' disease activity and response to therapy, but also for different complex peptides for the development of peptide-based immunoassays for other autoimmune diseases. This will place the microwave technology in an remarkable position not only as a modern trend, but also as a reliable technological support to overcome problems in the rapid production of complex peptide sequences for the development of diagnostic products particularly useful as "theranostics".

References
[1] F. Lolli, B. Mulinacci, A. Carotenuto, B. Bonetti, G. Sabatino, B. Mazzanti, A.M. D'Ursi, E. Novellino, M. Pazzagli, L. Lovato, M.C. Alcaro, E. Peroni, M.C. Pozo-Carrero, F. Nuti, L. Battistini, G. Borsellino, M. Chelli, P. Rovero, A.M. Papini. Proc. Natl. Acad. Sci. USA (2005) 102, 29, 10273-10278.
[2] F. Rizzolo, G. Sabatino, M. Chelli, P. Rovero and A.M. Papini. Int. J. Pept. Res. Ther. (2007) 13, 1-2, 203-208.
[3] F. Lolli, B. Mazzanti, M. Pazzagli, E. Peroni, M.C. Alcaro, G. Sabatino, R. Lanzillo, V. Brescia Morra, L. Santoro, C. Gasperini, S. Galgani, M.M. D' Elios, V. Zipoli, S. Sotgiu, M. Pugliatti, P. Rovero , M. Chelli, A.M. Papini. J. Neuroimmunol. (2005) 167, 131-137.
[4] "Preparation of glycopeptides for use in the diagnosis or therapeutic treatment of multiple sclerosis". Inventors: A.M. Papini, P. Rovero, M. Chelli, F. Lolli. Applicant: University of Florence, Italy. PCT International application (2003) WO 03000733 A2. Brevetto italiano n. 0001327122 (27/04/2005). Granted USA Patent (02/08/2005).
[5] I. Paolini, F. Nuti, M.C. Pozo-Carrero, F. Barbetti, B. Kolesinska, Z.J. Kaminski, M. Chelli, and A.M. Papini. Tetrahedron Letters (2007) 48, 16, 2901-2904.
[6] M.C. Alcaro, F. Lolli, P. Migliorini, M. Chelli, P. Rovero, and A.M. Papini. Peptides as autoimmune diseases antigenic probes: a peptide-based reverse approach to detect biomarkers of autoimmune diseases. Chemistry Today (2007) 25(3), 14-16.
[7] "Galactosylated peptides, their preparation and use in autoimmune diseases diagnosis" Filing date 16/10/2007. Applicant: Toscana Biomarkers Srl. Inventors: M.C. Alcaro, M. Chelli, F. Lolli, P. Migliorini, I. Paolini, A.M. Papini, P. Rovero. EPC Application (2007) 07118566.4.