Synthetic peptides for Plasmodium vivax malaria sero-epidemiology. Application of Fmoc-polyamide and displacement chromatography

The immunodominant epitope of Plasmodium vivax, one of the major causative agents of malaria in man, consists of the tandem repetitions of a nonapeptide sequence, AspArgAlaAsp/AlaGlyGlnProAlaGly, with Asp (variant d) or Ala (variant a), in the fourth position. Synthetic peptides corresponding to the P. vivax epitope, containing a different number of nonapeptide sequences, were prepared by solid-phase synthesis according to the Fmoc-polyamide method. Three peptides, containing 1, 2, and 4 copies of the d variant, were assembled on the gel polymer; none of these peptides, however, was suitable for P. vivax sero-epidemiology. A 45-peptide containing both the d and a variants, ddaad, was prepared by continuous-flow Fmoc-polyamide (flow-polyamide). Among the cleavage procedures evaluated for the removal of the five Mtr groups only TFMSA/TFA/ 1,2-ethanedithiol (1:89:10 by vol.) brought deblocking to completion; a substantial level of impurities originated, however, from these procedures. The product was purified by reversed-phase displacement chromatography, a technique only recently applied to peptides, which shows distinct advantages over conventional, linear elution chromatography. In a single experiment, 107 mg of the crude mixture were loaded onto an analytical column (250 × 4 mm), obtaining in purified form 85% of the desired material present in the sample. An ELISA test base on the ddaad peptide was developed and is being applied to the sero-epidemiology of P. vivax malaria.