C5 Substituted Amidites and Supports

Epigenetic Research Studies:

ChemGenes Corporation Now Offers Various Phosphoramidite monomers for Epigenetic Re­search Studies. Epigenetics is defined as heritable changes in gene activity and expression that occur without alteration in DNA sequence.¹ It is one of the fastest-growing research areas of science and has now become a cen­tral issue in biological studies.

• Figure 1 shows epigenetic pathway of Cytidine. In human cells, S-adenosylmethioninedependent DNA methyl transferases² catalyze the C-5 methylation of cytidine to generate 5-methyl cytidine (5m-dC).
•  Tahiliani et. al. found that the 2-oxoglutarate and Fe(II)-dependent hydroxylase TET catalyzes the con­version of 5-mdC to 5-hydroxymethylcytidine (5hm-dC) in human cell lines.³
• Ito and co-workers showed that Tet enzymes are able to convert 5hm-dC to 5-formyl-dC (5f-dC),⁴ and also observed the presence of 5f-dC in mouse embryonic stem cells and various mouse organ tissues.
• Research in this area using these nucleosides as part of oligonucleotides can help explain how cells car­rying identical DNA differentiate into different cell types, and how they maintain differentiated cellular states.⁵

Figure 1: Epigenetic pathway of Cytidine.

• Availability of these phosphoramidite monomers enables incorporation of these modified monomers into synthetic oligonucleotides for use as research tools to help researchers definitively determine these modifications.
• These epigenetic bases have functional groups at the 5-position that must be protected, which dictates the deprotection conditions of oligos containing these modifications, and compatibility with other modi­fications.

Figure 2: Chemical structures of various phosphoramidite monomers for epigenetic research and development.

5-Methyl Cytidine (5m-dC):

• One of the more commonly identified epigenetic modifications is the 5-methyl cytidine (5m-dC).
• De­pending on its location in a DNA, 5m-dC modification is involved in a variety of biological roles, from protection against restriction enzymes to gene regulation.
• In eukaryotes, 5m-dC has been associated with the regulation of transcriptional activity.⁶
• The presence of 5m-dC in CpG islands of promoter regions of genes can result in inhibition of transcription.⁷ Aberrant DNA methylation patterns are linked to diseases including cancer.⁸

5-Hydroxymethyl modifications:

• Recently, presence of 5-hydroxymethyl-dC in Purkinje neurons has been reported. 5hm-dC has been characterized as the ‘sixth base’ in human DNA
• Lack of these TET enzymes yields malfunctioning stem cells providing a link between formation of the base 5hm-dC and cellular development.⁹ To facilitate the biochemical investigation of 5hm-dC dependent biological processes, supply of a very pure 5hm-dC amidite monomer is needed.
• ChemGenes offers both 5-hydroxymethylcytidine (Figure 2, 5hm-rC ANP-6425) and 5-hydroxymeth­yl-2′-deoxy cytidine phosphoramidite (Figure 2, 5hm-dC ANP-6424) monomers

5-Formyl DNA modifications:

• There is strong evidence that DNA demethylation occurring via a Tet-mediated enzymatic pathway in­volving 5-formyl-dC as a key intermediate. So, 5-formyl-dC modified oligos can serve as research tools for the DNA demethylation process.
• 5-Formyl-2′-dU is formed in a DNA as a result of ionization radiation and several other means such as gamma-ray irradiation and oxidative damages.¹⁰
• ChemGenes now offers 5-(1,2-Diacetyloxyethyl)-dU-3′-phosphoramidite (Figure 2, ANP-6067) and 5-Formyl-dC-3′-phosphoramidite (Figure 2, ANP-6423) monomers.
• In the 5-(1,2-diacetyloxyethyl)-dU-3′-phosphoramidite monomer (ANP-6067) the aldehyde is masked as a protected 1,2-diol. After oligonucleotide synthesis, 1,2-diol is converted to an aldehyde by oxidation.
• In the 5-formyl-dC-3′-phosphoramidite monomer, the aldehyde is not protected, but the 4-amino group is protected with an acetyl group. We offer labile deprotecting amidites for the synthesis of DNA with this monomer.

References:

1. Bird, A. Nature 2007, 447, 396; 2. Goll, M. G. et. al. Annu. Rev. Biochem. 2005, 74, 481;

3. Tahiliani, M.et. al. Science 2009, 324, 930;

4. Ito, S. et. al. Science 2011, 333, 1300.;

5. Jaenisch, R. et. al. Cell 2008, 132, 567;

6. Rottach, A.et. al. Cell Biochem. 2009, 108, 43;

7. Attwood, J. T. et. al. Cell. Mol. Life Sci. 2002, 59, 241;

8. Smith, S. S. et. al. J. Mol. Biol. 2000, 302, 1;

9. Ito, S. et. al. Nature 2010, 466, 1129;

10. a) Bjelland, S.et. al. Biochemistry 1995, 34,14758. b) Sugiyama, H.et. al. Tet. Lett. 1996, 9067-9070.