Glen Report 34-12: New Product — DEACM Caged-dG

As detailed in the previous article, the photocaging of oligonucleotides is a powerful method for structural and kinetic investigations. For this reason, we introduced NPOM Caged-dT (Figure 1) years ago1 to give our customers the ability to perform such experiments. The NPOM is an o-nitrobenzyl group that is attached to the base pairing face of thymine. Upon irradiation with UV light at 365 nm, the NPOM is readily removed to give native thymine. Oligonucleotides containing NPOM Caged-dT have been successfully used in a whole host of applications including DNAzymes,2 antisense oligonucleotides,3 microRNA logic gates4 and more recently, CRISPR.5 With the help of Dr. Heckel and his team, we are happy to now provide a second option for synthesizing photocaged oligonucleotides, DEACM Caged-dG (Figure 1). This will give our customers access to another nucleobase as well as a very different photoremovable protecting group.Figure 1

Figure 1. Photocaged Phosphoramidites

In our hands, we found DEACM Caged-dG relatively straightforward to use in oligonucleotide synthesis. For insertion, a three minute coupling time was required using tetrazole as the activator. For deprotection, both the coumarin and the 6-position of dG are susceptible to reaction with nucleophiles. For example, treatment with ammonium hydroxide and prolonged heating will convert the guanine to a diaminopurine. For this reason, the phosphoramidite is provided with the UltraMILD protecting group, iPr-Pac, to facilitate UltraMILD synthesis and deprotection. Possibly due to steric hindrance from the DEACM, deprotection of the iPr-Pac group requires more time. For a 7 nt sequence with a single insertion of DEACM-dG, deprotection required three and six hours, respectively, for ammonium hydroxide and potassium carbonate (50 mM in MeOH). For a 12 nt sequence with three insertions, both conditions required overnight treatment. In all cases, the potassium carbonate treatment always gave cleaner chromatograms. Therefore, in general use, an UltraMILD synthesis and potassium carbonate deprotection (50 mM in MeOH) for 6-17 h at room temperature is recommended. Those who prefer a standard synthesis can still obtain relatively good results by using ammonium hydroxide at room temperature overnight (dmf-dG required) or AMA at room temperature for two hours (Ac-dC required) for deprotection. Heat should be avoided, both during deprotection and processing.

We also found that the DEACM group is relatively stable to ambient lighting. We placed aliquots of the yellow phosphoramidite solid in the lab exposed to standard fluorescent lighting during the day and subsequently analyzed solutions of them by reverse phase HPLC. We found that the purity of the phosphoramidite was reduced by only 0.4 % per day over a three day period. Even though DEACM can be released with visible light, we will fortunately not have to work in the dark. We would like to thank Dr. Heckel and his team for their excellent overview of DEACM-dG, their scientific input as well as their review of this article.

References

  1. The Glen Report, 2011, 23.2, 7.
  2. H. Lusic, D.D. Young, M.O. Lively, and A. Deiters, Org Lett, 2007, 9, 1903-6.
  3. D.D. Young, H. Lusic, M.O. Lively, J.A. Yoder, and A. Deiters, Chembiochem, 2008, 9, 2937-40.
  4. J. Hemphill, and A. Deiters, J Am Chem Soc, 2013, 135, 10512-8.
  5. Y. Liu, et al., Science, 2020, 368, 1265-1269.

Product Information

DEACM Caged-dG-CE Phosphoramidite

NPOM Caged-dT-CE Phosphoramidite