Methods for polyamine derivatization with a panel of extrinsic fluorophores followed by HPLC with fluorescence and UV absorption detection have been developed. Four fluorophores were examined using polyamines and aminoglycosides. o-Phthalaldehyde (OPA) and fluorescamine are selective fluorophores that only react with primary amines; 9- fluorenylmethyl chloroformate (FMOC Cl) and dansyl chloride react with both primary and secondary amines. Reaction and HPLC conditions were optimized with each of the above fluorophores using a series of model mono- and diamines and then applied to natural and semi-synthetic polyamines. The amines that have been investigated are natural di- and polyamines: putrescine, cadaverine, spermidine, spermine, thermospermine, aminoglycosides: kanamycin, paramomycin, neomycin, and synthetic polyamine conjugates e.g. N⁴,N⁹-dioleoylspermine, N¹-cholesteryl spermine carbamate. The resultant derivatives were confirmed by off-line high resolution electrospray ionization mass spectrometry (HR ESI MS). The results show that the synthesis of polyamine derivatives in quantitative yield depends on the time of reaction, the temperature and the ratio of fluorophore reagent. Linearity of derivatization was calculated and regression coefficients ranged from 0.968 to 0.999 with good reproducibility. HR ESI MS analysis of the reaction products demonstrated complete derivatization of both primary and secondary amino groups with dansyl and FMOC fluorescence derivatives and of primary amine groups for OPA and fluorescamine derivatives. Under the ionization conditions used the dansyl derivatives showed, in addition to monovalent ions [M+H]⁺, divalent cations [M+2H]²⁺ because this chromophore contains a basic amine that can be easily protonated. FMOC derivatives gave prominent [M+Na]⁺ ions. The OPA derivatization reaction is rapid, but the products have poor stability. The derivatization with fluorescamine gave multiple products with glucosamine due to the presence of a chiral centre in the fluorophore. The relative quantum yields of the polyaminefluorophore derivatives were examined to determine the effect of intramolecular fluorescence quenching. Dansylation is the fluorescent derivatization method of choice.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:500693 |
| Date | January 2009 |
| Creators | Buranaphalin, Sawanya |
| Contributors | Blagbrough, Ian ; Rowan, Michael |
| Publisher | University of Bath |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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