infected intraperitoneally with TCRV (2000 TC50/animal) 1–4 days after birth. Mice were treated with CpG ODN fma1555 or CpG ODN 1555 (50 mg/mouse) on the day of infection (n = 9 for each group of mice) or three days before infection (n = 8 or 7, respectively, for each group of mice). Mice were also treated with a combination of CpG ODN fma1555 (25 mg/mouse) and CpG ODN 1555 (25 mg/mouse) on the day of infection (n = 11) or three days before infection (n = 10). Mice that were left untreated (n = 11) or received an ODN lacking a CpG motif (ODN fma1556, 50 mg/mouse, n = 6) three days before the infection were used as negative controls. The mice were monitored daily but infections were allowed to proceed to their natural outcome in order to assess survival. For each condition, survival was determined from 2 to 4 independent experiments using 2–4 mice per group in each experiment. No obvious delay in development or weight loss was observed in uninfected mice treated with the oligonucleotides. RESULTS AND DISCUSSION The preparation of the deoxyribonucleoside phosphoramidites 1a–d is a prerequisite to solid-phase synthesis of oligonuc- leotides functionalized with the thermolytic 2-(N-formyl-N- methyl)aminoethyl thiophosphate protecting group. These phosphoramidites are obtained from the reaction of suitably protected deoxyribonucleosides (9a–d) with the phosphorodi- amidite 8 and 1H-tetrazole (Scheme 2) essentially as reported by Lee and Moon (41). The crude phosphoramidites are puri- ﬁed by silica gel chromatography using an eluant containing triethylamine to prevent dedimethoxytritylation and hydro- lysis of the phosphoramidite monomers during puriﬁcation given the inherent acidity of silica gel. It is therefore critic- ally important to remove excess triethylamine from the puriﬁed phosphoramidites monomers to avoid neutralization of 1H-tetrazole and poor coupling efﬁciency during solid- phase oligonucleotide synthesis. The removal of residual triethylamine from puriﬁed 1a–d is preferably achieved by dissolving the phosphoramidites in a minimum amount of dry benzene followed by precipitation in a large volume of cold hexane. To ensure complete removal of triethylamine and adventitious moisture, the phosphoramidite precipitate is dis- solved in a substantial amount of dry benzene ( 10 ml/g) and then freeze-dried under high vacuum over an extended period of time (16–24 h). Characterization of the phosphoramidites 1a–d by 31P NMR spectroscopy revealed a noteworthy multiplicity of signals at 25 C. Typically, four signals are observed for 1b and three signals each for 1a, 1c and 1d. However, only two broad 31P NMR signals are seen when either 1b or 1c is heated to 50 C in the NMR probe. These observations are consistent with the notion of 1a–d existing as a mixture of diastereomeric rotamers (Scheme 2) as a consequence of the partial double- bond character of the C–N bond in tertiary amides (11). Like- wise, the achiral phosphinylating reagent 8 also exists as a mixture of two rotamers as evidenced by two 31P NMR signals observed at dP 118.0 and 118.7 p.p.m. The puriﬁed deoxyribonucleoside phosphoramidites 1a–d are used as 0.1 M solutions in anhydrous MeCN in the solid-phase synthesis of CpG ODN fma1555 and ODN Nucleic Acids Research, 2005, Vol. 33, No. 11 3555 fma1556 according to a modiﬁed synthesis cycle. Commercial 2-cyanoethyl deoxyribonucleoside phosphoramidites are also employed under identical conditions in the preparation of CpG ODN 1555, which serves as a positive immunostimulat- ory control oligonucleotide in mice. Modiﬁcations of the standard solid-phase oligonucleotide synthesis cycle include: (i) extending the phosphoramidite coupling time to 180 s; (ii) replacing the iodine oxidation step with a sulfurization step effected by 0.05 M 3H-1,2-benzodithiol-3-one 1,1- dioxide in MeCN over a period of 120 s; (iii) performing the sulfurization step before the capping step to minimize phosphorus oxygenation (33) and (iv) extending the capping reaction time to 60 s to ensure efﬁcient chain termination of shorter than full-length oligonucleotides. Upon completion of the last synthesis cycle, the terminal 50-O-DMTr group is not removed from the oligonucleotide to facilitate its hydrophobic separation from a population of shorter 50-O-acetylated oligonucleotides that failed quantitat- ive chain extension during oligonucleotide synthesis. The solid support is exposed to pressurized ammonia gas to cleave nucleobase protecting groups and release the oligonucleotide from the support. It is important to note that in the case of CpG ODN fma1555 and ODN fma1556, the standard conditions used for nucleobase deprotection (concentrated NH4OH, 55 C, 10 h) were unsatisfactory, as these caused unwanted thermolytic cleavage of the 2-(N-formyl-N- methyl)aminoethyl thiophosphate protecting group to an unacceptable level and resulted in an intractable mixture of oligonucleotides. The 50-O-DMTr-oligonucleotide is eluted off the column along with the shorter oligonucleotides, and is puriﬁed by RP-HPLC using a column packed with a C18 resin, which has a higher afﬁnity for the 50-O-DMTr-oligonucleotide than for the shorter 50-O-acetylated oligonucleotides. The puri- ﬁed 50-O-DMTr-DNA oligonucleotide is then dedimethoxy- tritylated upon treatment with 80% acetic acid and re-puriﬁed by RP-HPLC through a C18 column. The chromatographic proﬁle of CpG ODN fma1555 is shown in Figure 1 and exhib- its a retention time (tR) of 37 min. The shape of the peak corresponding to the puriﬁed oligonucleotide is consistent with that of a complex mixture of rotameric diastereomers (2). Characterization of the puriﬁed CpG ODN fma1555, CpG ODN 1555 and ODN fma1556 by mass spectrometry resulted in the following mass determinations: CpG ODN fma1555 (+ESI-TOF MS): calcd for C203H283N71O89P14S14 (M)+ 6024, found 6023. ODN fma1556 (+ESI-TOF MS): calcd for C205H283N75O89P14S14 (M)+ 6105, found 6103. CpG ODN 1555 (+ESI-TOF MS): calcd for C147H185N57 O75P14S14 (M + 14H)+ 4832, found 4832. The experimental mass determination of each oligonucleotide is in close agree- ment with its expected mass, and thus conﬁrms its identity. A kinetic proﬁle of the thermolytic cleavage of the 2-(N- formyl-N methyl)aminoethyl thiophosphate protecting group is obtained by heating puriﬁed CpG ODN fma1555 and ODN fma1556 to 37 or 90 C in 1· PBS (pH 7.2), and analyzing the deprotection reaction at various time points by RP-HPLC. The half-time of thiophosphate deprotection is estimated to be 73 h at 37 C, and complete deprotection is achieved within 600 h. Fully deprotected CpG ODN fma1555 exhibited a tR (23 min) identical to that of CpG ODN 1555 under identical chroma- tographic conditions (Figure 1). The thermolytic deprotection