Combined Fmoc-Alloc strategy for a general SPPS of phosphoserine peptides; preparation of phosphorylation-dependent tau antisera.

A block method for the solid phase synthesis (SPPS) of serine phosphopeptides has been developed using a combination of Fmoc and Alloc strategies. Alloc-Ser[PO(OCH2CH CH2)2] OH2, prepared in a one pot procedure from Alloc-Ser-OH, was introduced at the N-terminus of a sequence prepared by standard Fmoc-SPPS. Global cleavage of the allyl ester based protecting groups, followed by coupling of a tripeptide fragment, led to the tau phosphopeptide, 1. Using tau phosphopeptides a series of phosphorylation state-dependent antisera to human tau protein have been raised. These antisera are valuable tools for studying the tau protein which is found in an abnormal, hyperphosphorylated form in Alzheimer's disease brain.

Conformations of synthetic beta peptides in solid state and in aqueous solution: relation to toxicity in PC12 cells.

The secondary structures of peptides beta 25-35 (the active toxic fragment) and beta 35-25 (reverse sequence and non-toxic fragment), as well as of the amidated beta (25-35)-NH2 peptide were investigated in aqueous solution and in the solid state by means of Fourier-transformed infrared spectroscopy and circular dichroism spectroscopy. The conformations of the beta 25-35 and beta 35-25 in solid state were identical and contained mostly beta-sheet structures. In solid state the amidated beta (25-35)-NH2 peptide also contained mostly beta-sheet structures. Freshly prepared aqueous solutions of the beta 25-32 (0.5 - 3.8 mM) contained a mixture of beta-sheet and random coil structures. Within 30-60 min incubation at 37 degrees C in water or in phosphate-buffered saline solution (PBS), beta 25-35 was almost fully converted to a beta-sheet structure. Decreasing the temperature from 37 degrees C to 20 degrees C decreased the rate of conversion from random coil to beta-sheet structures, 1-2 h being required for complete conversion. In contrast beta 35-25 in water or in PBS buffer had mostly a random coil structure and remained so for 6 days. The amidated beta(25-35)-NH2 peptide in water (2.7 mM) was also mostly random coil. However, when this peptide (2-2.7 mM) was dissolved in PBS (pH 7.4) or in 140 mM NaCl, a gel was formed and its conformation was mostly beta-sheet. Decreasing the concentration of beta (25-35)-NH2 peptide in 140 mM NaCl aqueous solution from 2 mM to 1 mM or below favored the conversion from beta-sheet structures to random coil structures. The beta 25-35 was toxic to PC12 cells while beta 35-25 was not. The amidated peptide beta (25-35)-NH2 was at least 500-fold less toxic than beta 25-35. Structural differences between these beta peptides in aqueous solutions may explain the difference in their respective toxicities.