SYNTHESIS AND CONFORMATIONAL STUDIES OF N-GLYCOSYLATED ANALOGS OF THE HIV-1 PRINCIPAL NEUTRALIZING DETERMINANT

The principal neutralizing determinant (PND) of HIV-1 is found in the V3 loop of the envelope glycoprotein. Antibodies elicited by peptides from this region, containing the GlyProGlyArgAlaPhe (GPGRAF) sequence, were able to neutralize diverse HIV-1 isolates [Javaherian et al. (1990) Science 250, 1590-1593]. The GPGR tetrapeptide was predicted to adopt a type II beta-turn conformation. Earlier, we showed that glycosylation of synthetic T cell epitopic peptides at natural glycosylation sites stabilized beta-turns [Otvos et al. (1991) Int. J. Pept. Protein Res. 38, 467-482]. To evaluate the secondary structure modifying effect of the introduction of an N-glycosylated asparagine residue and to find a correlation between conformation and a possible PND potential, a series of glycopeptide derivatives, N(sugar)GPGRAFY-NH2 (4a-f), have been prepared, together with the parent peptides GPGRAFY-NH2 (2) and NGPGRAFY-NH2 (3), by solid-phase peptide synthesis [sugars: (a) beta-D-glucopyranosyl (Glc); (b) beta-D-galactopyranosyl (Gal); (c) Glc-beta(1 --> 4)-Glc; (d) 2-acetamido-2-deoxy-beta-D-glucopyranosyl (GlcNAc); (e) 2-acetamido-2-deoxy-beta-D-galactopyranosyl (GalNAc); (f) GlcNAc-beta(1 --> 4)-GlcNAc; sugars are attached through a beta(1 --> N(beta)) linkage to asparagine (N).] Peptides 2-4 were characterized by amino acid analysis, reversed-phase HPLC, and fast atom bombardment mass spectrometry. Circular dichroism (CD) and Fourier-transform infrared (FT-IR) spectroscopic studies were performed in trifluoroethanol (TFE) and water (D2O was used in FT-IR experiments). Nonglycosylated peptides showed significantly different CD spectra in aqueous and TFE solution. Moreover, a continuous spectral change was observed for all the peptides investigated when going from water to TFE. The chiral contribution of the aromatic side chains and acetamido sugars was also estimated. On the basis of CD and FT-IR evidence, the introduction of an N-glycosylated Asn residue does not destroy but rather stabilizes the suggested type II beta-turn conformation of the PND peptide.