In the present work, we have developed a highly efficient temperature-dependent chemo-enzymatic methodology for the regioselective synthesis of novel esters of glycerol, G1 tri-glycerol dendrons and related esters for the first time using 4-nitrophenyl 2-(tert-butoxycarbonyl)acetate (Boc-gly-Ph-pNO2) (2) as the acylating agent. This methodology offers efficient and controlled loading of amino acid (glycine) on polyhydroxy compounds.