Here, we report the synthesis of 3'-deoxyapionucleoside 3''-triphosphates (apioNTPs) and the analysis of their property as substrate of enzymatic polymerization. We established the large scale synthetic route of 3-deoxy-D-apiose from D-galactose, and regio- and stereo-selective glycosylation procedures. Resulting 3'-deoxyapionucleosides were then converted into their 3''-triphosphates. We carried out primer-extension reactions and found that Therminator polymerase, a variant of 9 degrees N DNA polymerase, was an efficient enzyme for incorporation of apioNTPs to synthesize DNA-dependent 3'-deoxy-D-apiose nucleic acids (apioNAs).