Side-chain oligo- and polyglutamylation represents an important posttranslational modification in tubulin physiology. The particular number of glutamate units is related to specific regulatory functions. In this work, we present a method for the synthesis of building blocks for the Fmoc synthesis of peptides containing main chain glutamic acid residues that carry side-chain branching with oligo-glutamic acid. The two model peptide sequences CYEEVGVDSVEGEG-E(E x)-EEGEEY and CQDATADEQG-E(E x)-FEEEEGEDEA from the C-termini of mammalian α1- and β1-tubulin, respectively, containing oligo-glutamic acid side-chain branching with lengths of 1 to 5 amino acids were assembled in good yield and purity. The products may lead to the generation of specific antibodies which should be important tools for a more detailed investigation of polyglutamylation processes. © 2010 Werner Tegge et al.2010 Edde, B., Rossier, J., Le Caer, J.-P., Desbruyeres, E., Gros, F., Denoulet, P., Posttranslational Glutamylation of -tubulin (1990) Science, 247 (4938), pp. 83-85Janke, C., Rogowski, K., Van Dijk, J., Polyglutamylation: A fine-regulator of protein function? Protein Modifications: Beyond the usual suspects Review Series (2008) EMBO Reports, 9 (7), pp. 636-641Hammond, J.W., Cai, D., Verhey, K.J., Tubulin modifications and their cellular functions (2008) Current Opinion in Cell Biology, 20 (1), pp. 71-76Van Dijk, J., Miro, J., Strub, J.-M., Lacroix, B., Van Dorsselaer, A., Edde, B., Janke, C., Polyglutamylation is a post-translational modification with a broad range of substrates (2008) Journal of Biological Chemistry, 283 (7), pp. 3915-3922Wolff, A., De Nechaud, B., Chillet, D., Mazarguil, H., Desbruyeres, E., Audebert, S., Edde, B., Denoulet, P., Distribution of glutamylated α and β-tubulin in mouse tissues using a specific monoclonal antibody, GT335 (1992) European Journal of Cell Biology, 59 (2), pp. 425-432Paturle-Lafanechre, L., Eddé, B., Denoulet, P., Van Dorsselaer, A., Mazarguil, H., Le Caer, J.P., Wehland, J., Job, D., Characterization of a major brain tubulin variant which cannot be tyrosinated (1991) Biochemistry, 30 (43), pp. 10523-10528Armstrong, A., Brackenridge, I., Jackson, R.F.W., Kirk, J.M., A new method for the preparation of tertiary butyl ethers and esters (1988) Tetrahedron Letters, 29 (20), pp. 2483-2486Salvador, L.A., Elofsson, M., Kihlberg, J., Preparation of building blocks for glycopeptide synthesis by glycosylation of Fmoc amino acids having unprotected carboxyl groups (1995) Tetrahedron, 51 (19), pp. 5643-5656Tegge, W., Frank, R., Peptide synthesis on Sepharose beads (1997) Journal of Peptide Research, 49 (4), pp. 355-362