Part A: Psalmopeotoxin I and II-anti-malarial peptides extracted from Psalmopoeus cambridgei: synthesis and thermal stability of their mutants Part B: Functionalized chromans from ortho-Quinone methides via cycloaddition reactionsPakornwit Sarnpitak
( M.Sc. )
Psalmopeotoxin I and II (PcFK1 & PcFK2) were recently isolated and characterized from tarantula’s venom, Psalmopoeus cambridgei. These two peptides have good plasmodial activity against intra-erythrocytic stage of Plasmodium falciparum in vitro (IC50 ~ 1 μM), but do not exhibit any toxicity besides other structurally similar neurotoxins. Previously, both PcFK wild-types were chemically synthesized and thermal stability was studied. The thermal stability of these peptides was reported in terms of mid-point of transition temperature (Tm) from Circular Dichroism (CD) studies. Tm of PcFK1 and PcFK2 were 18.3˚C and 29.5˚C, respectively. Based on thermal unfolding studies, both PcFKs were not thermally stable. The objective of this study is to determine the effect of mutations of PcFK1 and PcFK2 on the thermal stability whether positively or negatively. The designed mutations were based on the conserved-residues of PcFK1 and PcFK2 which are Val10, Pro13, and Pro17; these residues were mutated to alanine. These mutants were manually synthesized by Solid Phase Peptide Synthesis (SPPS) using Fmoc chemistry, regioselective disulfide bond formation and orthogonal thiol protecting group strategy. Thermal stability was studied by Circular Dichroism spectroscopy (CD), fraction unfolding (fu) curve was determined using baseline extrapolation method and mid-point of transition temperature (Tm) was calculated from fraction unfolding curve. Tm of some mutants were higher than their wild-types. The synthesis of benzopyrans (chromans) and some oxadecalins could be accomplished by the cycloaddition reactions of o-QMs with various dienophiles. The o-QMs could be generated by the previously reported method using p-TsOH immobilized on silica (PTS-Si). However, the scope of substrates including dienophiles was limited due to the use of strong acidic PTS-Si which is required in stoichiometric amount. Therefore, to overcome these problems, PtCl4 had been developed to generate o-QMs, catalytically. For the stynthesis of C2-C3 substituted chromans, the scope of dienophiles were expanded from styrene and its derivatives to electron-rich, electron-poor olefins, some selected alkynes, and less-activated olefins. These dienophiles gave chroman products in moderate to excellent yields. Interestingly, some simple alkenes and cycloalkenes also furnished the desired products with moderate yields. The diastereoselectivities for C2-C4 substituted were up to > 99:1, but the yields were lower than C2-C3. Unfortunately, C2-C3-C4 substituted chromans were not obtained. UV spectroscopy was used to determine the coordination between styrene and PtCl4 and revealed that there was a peak, possibly belonging to the complex which was formed. Increasing the ratio of styrene to PtCl4 resulted in bathochromic shift (red shift) and the peaks of styrene disappeared. 1H-NMR was used to understand the mechanism by monitoring the cycloaddition reaction of o-QMs with styrene and the decomposition of precursor with PtCl4. The results suggested that the OAc and MOM group did not leave at the same time, with the MOM group being cleaved first, followed by the generation of o-QMs by the loss of acetate group.