Introducing an Asp-Pro Linker in the Synthesis of Random One-Bead- One-Compound Hexapeptide Libraries Compatible with ESI-MS Analysis Yordanka Masforrol, † Jeovanis Gil, ‡ Luis Javier Gonza ́ lez, ‡ Yasset Pe ́ rez-Riverol, § Jorge Ferna ́ ndez-de-Cossío, ∥ Aniel Sa ́ nchez, ‡ La ́ zaro Hiram Betancourt, ‡ Hilda Elisa Garay, † Ania Cabrales, † Fernando Albericio, ⊥,#,▽ Hongqian Yang, ▼ Roman A. Zubarev, ▼ Vladimir Besada, ‡ and Osvaldo Reyes Acosta* ,† † Departments of Chemical Synthesis, ‡ Proteomics, and § Bioinformatics, Division of Physical-Chemistry and ∥ Division of Informatics, Center for Genetic Engineering and Biotechnology, PO Box 6162, Havana, Cuba ⊥ Institute for Research in Biomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028, Barcelona, Spain # CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona, Spain ▽ Department of Organic Chemistry, University of Barcelona, Martí i Franque ́ s 1-11, 08028 Barcelona, Spain ▼ Division of Molecular Biometry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17 177 Stockholm, Sweden * S Supporting Information ABSTRACT: A random hexapeptide library (one-bead-one-compound), containing sixteen amino acids (16 6 different sequences) was synthesized on a Tentagel resin previously modified with a dipeptide linker (Asp-Pro). This peptide bond is highly susceptible to cleavage under mild acidic conditions in a salt-free solution prepared with H 2 16 O/H 2 18 O (60/40% v/ v). In the hydrolysis, hexapeptides are released with an additional Asp residue partially labeled with 18 O at the C-terminus. These conditions are fully compatible with ESI-MS analysis and facilitate sequencing by MS, as N- and C-terminal ions can be easily differentiated in MS/MS spectra. The peptides were sequenced manually and also with de novo sequencing programs, and identifying them in a database containing all possible heptapeptide sequences or in a filtered database. The proposed strategy is also compatible with stepwise Edman degradation using either intact beads or the released free peptides. KEYWORDS: peptide library, one-bead-one-compound, tentagel, mass spectrometry, electrospray ionization, de novo sequencing C ombinatorial chemistry has a direct impact on drug discovery, allowing the synthesis and screening of a large number of compounds with potential therapeutic or diagnostic uses. Phage display peptide libraries, together with one-bead- one-compound (OBOC) peptide libraries, 1 have become important tools for understanding the biological basis of molecular recognition. OBOC libraries, in particular, allow for further optimization of lead compounds by simply including non-natural, or D-amino acids and other organic building blocks, thus accelerating the drug discovery process. 2−5 After library screening, the structural elucidation of the selected compounds is a critical step. Edman sequencing has been widely used for this purpose because the beads can be directly analyzed without cleaving off and retrieving the peptide. However, this procedure is time-consuming, expensive, requires a free N-terminus on the peptide and is not compatible with modified amino acids and other organic blocks commonly used during lead optimization. Mass spectrometry has been successfully used for sequencing peptides isolated from OBOC libraries by the combination of “ladder-synthesis”, 6 “ladder-sequencing”, 7 and MALDI-MS analysis. The major disadvantage of ladder-synthesis is that peptide ladders and the full length sequence coexist on the bead surface, which might interfere with biological screening. 8 To overcome this disadvantage, Son et al. 9 reported the application of “ladder synthesis” and bilayer bead concepts to segregate full-length peptides and their truncated variants. The outer layer carries the full-length library compound synthesized at low Received: July 4, 2011 Revised: January 25, 2012 Published: January 25, 2012 Technology Note pubs.acs.org/acscombsci © 2012 American Chemical Society 145 dx.doi.org/10.1021/co200159r | ACS Comb. Sci. 2012, 14, 145−149