3550–3560 Nucleic Acids Research, 2005, Vol. 33, No. 11 doi:10.1093/nar/gki657 Thermolytic CpG-containing DNA oligonucleotides as potential immunotherapeutic prodrugs 1 1 Andrzej Grajkowski, Joao Pedras-Vasconcelos , Vivian Wang , Cristina Aus´ın, Sonja Hess2, Daniela Verthelyi1 and Serge L. Beaucage* Laboratory of Chemistry and 1Laboratory of Immunology, Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Food and Drug Administration, 8800 Rockville Pike, Bethesda, MD 20892, USA and 2Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, The National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA Received February 14, 2005; Revised May 9, 2005; Accepted May 26, 2005 ABSTRACT A CpG-containing DNA oligonucleotide functional- ized with the 2-(N-formyl-N-methyl)aminoethyl thio- phosphate protecting group (CpG ODN fma1555) was prepared from phosphoramidites 1a–d using solid-phase techniques. The oligonucleotide beha- ved as a prodrug by virtue of its conversion to the well-studied immunomodulatory CpG ODN 1555 through thermolytic cleavage of the 2-(N-formyl-N- methyl)aminoethyl thiophosphate protecting group. Such a conversion occurred at 37 C with a half- time of 73 h. The immunostimulatory properties of CpG ODN fma1555 were evaluated in two in vivo assays, one of which consisted of mice challenged in the ear with live Leishmania major metacyclic promastigotes. Local intradermal administration of CpG ODN fma1555 was as effective as that of CpG ODN 1555 in reducing the size of Leishmania lesions over time. In a different infectious model, CpG ODN 1555 prevented the death of Tacaribe- infected mice (43% survival) when administered between day 0 and 3 post infection. Administration of CpG ODN fma1555 three days before infection resulted in improved immunoprotection (60–70% survival). Moreover, co-administration of CpG ODN fma1555 and CpG ODN 1555 in this model increa- sed the window for therapeutic treatment against Tacaribe virus infection, and thus supports the use of thermolytic oligonucleotides as prodrugs in the effective treatment of infectious diseases. INTRODUCTION Over the last few years, our research efforts have focused on the design and development of thermolytic groups for 50-hydroxyl (1) and phosphate (2–7) protection in an attempt to implement a ‘heat-driven’ process (8) for the synthesis of DNA oligonucleotides on microarrays. In sharp contrast to the current methods employed for oligonucleotide synthesis, such a process would involve rapid and efﬁcient thermal removal of protecting groups from oligonucleotides under neutral conditions throughout chain assembly and ﬁnal depro- tection. Speciﬁcally, heat-sensitive phosphate/thiophosphate protecting groups exhibiting unique thermolytic properties have been incorporated into oligonucleotides via phosphor- amidites 1–6 (Scheme 1) employing solid-phase techniques. The deprotection mechanism of each thermolytic phosphate protecting group is consistent with a well-studied intramole- cular cyclodeesteriﬁcation reaction (9–12), which has also been observed by others (13–16). Given the relatively rapid phosphate deprotection kinetics of DNA oligonucleotides that have been prepared using phosphoramidites 4, 5 and 6 (6,7), the thermolytic 3-(2-pyridyl)-1-propyl, 2-[N-methyl-N- (2-pyridyl)]aminoethyl, and 4-methythio-1-butyl groups for phosphate protection may indeed ﬁnd application in the synthesis of oligonucleotides on microarrays considering the mildness of the deprotection conditions used for each group. However, oligonucleotides functionalized with the 2-(N-formyl-N-methyl)aminoethyl group for phosphate/ thiophosphate protection require a much higher temperature (90 C) over a considerably longer period of time ( 3 h) to complete the thermolytic cleavage of this protecting group. Although these conditions are not optimal for the preparation of diagnostic oligonucleotide microarrays, oligonucleotides functionalized with the 2-(N-formyl-N-methyl)aminoethyl *To whom correspondence should be addressed. Tel: +1 301 827 5162; Fax: +1 301 480 3256; Email: beaucage@cber.fda.gov This paper is dedicated to Professor Wojciech J. Stec on the occasion of his 65th birthday. The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors The Author 2005. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. 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