The ratio of mannitol to sucrose and the protein concentration have an impact on the T(g)' and may therefore influence the primary drying temperature. The protein inhibits both the nucleation and growth of mannitol crystals and this effect seems to be concentration dependent. The presence of the protein and the protein concentration dictate the processing conditions, i.e., annealing time, annealing temperature, and primary drying temperature.

The presence of protein as well as the processing conditions (annealing temperature and time, primary and secondary drying temperatures) influenced the physical form of mannitol in the final lyophile. The protein promoted formation of delta-mannitol while inhibiting the formation of mannitol hemihydrate. Since the physical form of mannitol was greatly influenced by the presence of protein, it will be prudent to conduct the preliminary lyophilization cycle development studies in the presence of the protein. If mannitol hemihydrate is formed during annealing, its dehydration may require high secondary drying temperature.

No abstract

We describe a strategy for the incorporation of a 2'-C-branched ribonucleoside, 2'-C-beta-methylcytidine, into oligonucleotides via solid-phase synthesis using phosphoramidite derivatives. 4-N-Benzoyl-2'-C-beta-methylcytidine (2b) was synthesized by coupling persilylated 4-N-benzoylcytosine with 1,2,3,5-tetra-O-benzoyl-2-C-beta-methyl-alpha-(and beta)-D-ribofuranose (1) in the presence of SnCl(4) in acetonitrile, followed by selective deprotection with NaOH in pyridine/methanol. The 3'- and 5'-hydroxyl groups were blocked as a cyclic di-tert-butylsilanediyl ether 3 by treatment with di-tert-butyldichlorosilane/AgNO(3) in DMF. The 2'-hydroxyl group was then protected as a tert-butyldimethylsilyl ether 4a by treatment with tert-butylmagnesium chloride followed by addition of tert-butyldimethylsilyl trifluoromethanesulfonate in THF. As an alternative to 2'-silyl protection, the corresponding 2'-O-tetrahydropyranyl ether 4b was prepared by treatment of 3 with 4,5-dihydro-2H-pyran in the presence of a catalytic amount of 10-camphorsulfonic acid in methylene chloride. The di-tert-butylsilanediyl groups of 4a and 4b were removed by treatment with pyridinium poly(hydrogen fluoride) to afford 5a and 5b, respectively. Protection of the 5'-hydroxyl group as a dimethoxytrityl ether and phosphitylation of the 3'-hydroxyl group by the standard procedure gave the phosphoramidite derivatives 7a and 7b. Both 7a and 7b could be used to incorporate 2'-C-beta-methylcytidine into oligonucleotides efficiently via standard solid-phase synthesis, but the tetrahydropyranyl group of 7b was more readily removed from oligonucleotides than the tert-butyldimethylsilyl group of 7a. Oligonucleotides containing 2'-C-beta-methylcytidine undergo base-catalyzed degradation analogous to natural RNA.