3.1. General
The HPLC analysis of the commercial stevioside showed that the
sample was 97.8% pure.
3.2. Hydrolysis of stevioside
Stevioside (St) 1 was hydrolysed to the corresponding acid 2 by
the use sodium hydroxide in methanol (Scheme 1). The structure of
the obtained product was identified by spectroscopic analysis, IR,
1H NMR, 13C NMR and elemental analysis.
3.3. Synthesis of stevia glycine ethyl ester 3
The obtained acid 2 was allowed to condense with the ethyl
ester of glycine using EDCHCl and the potassium salt of Oxyma
as a coupling reagent in a dichloromethane water mixture to afford
the desired product stevia glycine ethyl ester 3 (Scheme 1).
Recently OxymaPure was introduced, by our research group
(Cherkupally et al., 2013), as an additive for peptide bond formation
through a new formulation in which the N-hydroxylamine
group is replaced by a potassium salt. The complete suppression
of its acidity converts K-Oxyma into the most suitable coupling
choice when peptides are assembled in highly acid-labile
solid-supports. The coupling efficiency and epimerisation reduction
ability are conserved with regard to the parent OxymaPure.
In addition, K-Oxyma displays great solubility in water and a
variety of organic solvents and is safer than classical 1-hydroxybenzotriazole
additives.