Fmoc-O-trityl-L-serine

We demonstrated recently that selective side-chain modification of functional cysteine-rich (Tat(21-40)) and arginine-rich (Tat(53-68)) domains of the HIV-1 Tat protein blocks pathogenic activities of these peptides while retaining their immunological characteristics. In the present study, we have synthesized a multiple-peptide conjugate system comprising modified Tat(21-40) and Tat(53-68) peptides (HIV-1-Tat-MPC). Immunization of mice with this highly homogeneous 10.7 kDa HIV-1-Tat-MPC synthetic construct induced an effective immune response in mice. The antibodies generated against HIV-1-Tat-MPC efficiently suppressed Tat-induced viral replication and significantly reduced HIV-associated cytopathic effects in human monocytes. These results indicate that epitope-specific antibodies directed against functional sites of Tat protein using non-pathogenic peptides inhibit HIV pathogenesis. The HIV-1-Tat-MPC, therefore, has potential for the development of a safe, effective, and economical therapeutic vaccine to reduce the progression of HIV infection.

Multiple antigen peptides (MAP) consist of lysine residue cores with branching peptide arms and have been demonstrated to be efficient immunogens as well as useful antigens for ELISA. Synthesis of diepitope MAPs with two different branching peptides has previously been described using combined Boc and Fmoc chemistry. Here, the synthesis of a tetrameric diepitope MAP based on Fmoc chemistry is described. A lysine core was synthesized with N alpha- and N epsilon-amino groups othogonally protected by Fmoc and a recently described protection group, Dde, respectively. On the N alpha-amino groups, a sequence from the Plasmodium falciparum antigen Pf332 was synthesized with a capped N-terminus. After removal of Dde, a sequence from the P. falciparum circumsporozoite protein was synthesized on the core. Amino acid analysis of the MAP displayed equimolar amounts of the two peptide sequences, indicating the reliability of the protection group Dde. In ELISA, antibodies specific for either of the two malarial sequences reacted with the MAP. The major advantages of this approach for synthesis of diepitope MAPs are that only a panel of Fmoc-amino acid derivatives is required and that the more complicated cleavage procedure for Boc chemistry can be avoided.

A chemical ligation procedure has been developed for the synthesis of oligoribonucleotides carrying a trisubstituted pyrophosphate (tsp) linkage in place of a single phosphodiester. Good yields of tsp were obtained when a single 2'-deoxynucleoside 5' to the tsp was used in the ligation reaction. A tsp linkage was found to be reasonably stable to hydrolysis but cleaved by treatment with ethylenediamine or lysine to give phosphoamidate adducts. A model human immunodeficiency virus type 1 (HIV-1) TAR RNA duplex containing an activated tsp was able to bind to HIV-1 Tat protein with only 3-fold reduced affinity and to a Tat peptide (residues 37-72) with identical affinity compared to that of an unmodified duplex. Tsps incorporated at sites previously identified as being in close proximity to Tat protein were able to cross-link to Tat peptide (37-72) to form a covalent phosphoamidate conjugate. Endopeptidase cleavage followed by MALDI-TOF (matrix-assisted laser desorption ionization time of flight) mass spectrometric analysis provided strong evidence that a TAR duplex containing a tsp replacing the phosphate at 38-39 had reacted specifically with Lys51 in the basic region of Tat peptide (37-72). The new chemical cross-linking method may be generally useful for identifying lysines in close proximity to phosphates in basic RNA-binding domains of proteins.