Nucleic Acids Research, 2005, Vol. 33, No. 11 3551 Scheme 1. Deoxyribonucleoside phosphoramidites functionalized with thermolytic groups for phosphorus protection. phosphate/thiophosphate protecting group may serve as potential therapeutic oligonucleotide prodrugs in vivo on the basis of the anticipated sluggish removal of the phosphate/ thiophosphate protecting group at 37 C. Oligonucleotide prodrugs have been the focus of intense scrutiny in recent years in an effort to facilitate cellular uptake of antisense oligonucleotides and provide these biomolecules with increased resistance to hydrolytic nucleases. Thus, mask- ing the phosphodiester groups of oligonucleotides with acyl- thioethyl (17–21), acyloxymethyl (22) and 4-acyloxybenzyl (23,24) protecting groups or with groups derived from bis(hydroxymethyl)-1,3-dicarbonyl compounds (25,26) that would expectedly be cleaved upon reaction with intracellular enzymes, offers a viable solution to the notoriously poor cellular delivery of negatively charged oligonucleotide drugs. From this perspective, oligonucleoside phosphorothioates functionalized with the 2-(N-formyl-N-methyl)aminoethyl group for thiophosphate protection are likely to exhibit the characteristics of oligonucleotide prodrugs in that they are uncharged and, thus, inherently resistant to the hydrolytic activity of nucleases. A distinctive feature of this class of modiﬁed oligonucleotides lies in that esterases or other intra- cellular enzymes are not required for prodrug to drug conver- sion. Only a 37 C environment is necessary to thermolytically convert oligonucleoside 2-(N-formyl-N-methyl)aminoethyl phosphorothioate triesters to functional oligonucleoside phosphorothioate diesters. Synthetic single-stranded DNA oligonucleoside phosphoro- thioates containing unmethylated CpG motifs (CpG ODNs) have been selected as a model to evaluate whether thermo- lytic CpG ODNs can be administered as prodrugs in vivo. Scheme 2. Synthesis of the phosphinylating reagent 8 and preparation of deoxyribonucleoside phosphoramidites (1a–d) from suitably protected deoxyribonucleosides (9a–d). (i) bis(N, N-diisopropylamino)chlorophosphine, N,N-diisopropylethylamine, CH2Cl2, 25 C, 2 h; (ii) 8, 1H-tetrazole, MeCN, 25 C, 2–16 h; (iii) silica gel chromatography. Typically, CpG ODNs are rapidly internalized by immune cells (B cells, macrophages, dendritic cells and monocytes) (27,28) and localize to endocytic vesicles where they inter- act with Toll-like receptor 9. This interaction triggers an immunostimulatory cascade that is characterized by B-cell proliferation, dendritic cell maturation, natural killer cell activation, and the secretion of a variety of cytokines, chemo- kines and polyreactive immunoglobulins (27,29). Such a pro-inﬂammatory and Th1-biased immune response improves resistance of the host to infectious pathogenic microorgan- isms. In this regard, numerous studies indicate that adminis- tration of CpG ODNs can act alone to improve the response to parasitic, bacterial and viral infections in animal models (30–32). In this study, we report the synthesis and character- ization of a CpG ODN functionalized with thermolytic 2-(N- formyl-N-methyl)aminoethyl phosphorothioate triesters and assess for the ﬁrst time its immunostimulatory and immuno- protective properties in vivo. MATERIALS AND METHODS 2-(N-Formyl-N-methyl)aminoethan-1-ol (7) This compound (Scheme 2) was prepared from the reaction of 2-(methylamino)ethanol (Aldrich) with ethyl formate (Aldrich) as described earlier (2).