doi:10.1016/j.bmcl.2007.12.049 
Copyright © 2007 Elsevier Ltd All rights reserved.
Synthesis and biological evaluation of trimethyl-substituted cap analogs
Anilkumar R. Kore
, a, 
and Muthian Shanmugasundarama
aAmbion, Inc., An Applied Biosystems Business, Bioorganic Chemistry Division, 2130 Woodward Street, Austin, TX 78744-1832, USA
Received 3 December 2007;
revised 17 December 2007;
accepted 19 December 2007.
Available online 4 January 2008.
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Abstract
The N7-methyl guanosine cap located on the 5′-terminus of mRNAs is important for a number of biochemical processes. A new dinucleoside triphosphate cap analog was synthesized with methyl groups on the N7 of both guanine moieties, as well as the 3′-OH of one of the ribose moieties 
. The function of this trimethylated cap analog was compared with those of three other, less-methylated cap analogs: one omitting the ribose methylation (m7G[5′]ppp[5′]m7G), one omitting the N7 methylation linked to the unmodified ribose 
, and the standard cap analog, m7G[5′]ppp[5′]G. These cap modifications were assayed with respect to their effects on capping efficiency, yield of RNAs during in vitro transcription, and the translational activity of these RNAs upon transfection into HeLa cells. The translational activity was monitored by measuring the luciferase activity of a luciferase-fusion protein produced from the in vitro synthesized RNAs. The RNA capped with the trimethylated analog 
was translated the most efficiently, with 
2.6-fold more activity than the conventional cap (m7G[5′]ppp[5′]G). The other two variants were also more efficient, generating, 2.2 times (for the 
analog) and, 1.6 times (for the m7G[5′]ppp[5′]m7G analog) more luciferase function than the conventional cap.
Graphical abstract
Design, synthesis, and biological evaluation of trimethyl-substituted cap analog 
was reported.
Keywords: Capping efficiency; In vitro transcription; Translation efficiency; Trimethylated cap analog; Luciferase activity; HeLa cells
Scheme 1. Reagents and conditions: (a) POCl3, (OMe)3P, 0 °C, 2 h; (b) imidazole, aldrithiol, PPh3, DMF, rt, 5 h; (c) (Et3NH)3PO4, DMF, rt, 3 h; (d) dimethyl sulfate, water, pH 4.0, rt, 4 h; (e) ZnCl2, DMF, rt, 6 h.
Scheme 2. Reagents and conditions: (a) ZnCl2, DMF, rt, 6 h.
Figure 1. dPAGE gel (20%) showing capping efficiency of compounds 7, 9, and 10 along with conventional cap analog. The control reaction was normal in vitro transcription reaction, in which no cap analog was added. Radiation in the gel bands of interest was quantified by a phosphorimager (GE Healthcare).23
Figure 2. Yield of T7 RNA polymerase transcription reaction with methyl-substituted cap variants.24
Figure 3. Luciferase activity of methylated cap variants from compounds 7, 9, and 10, and conventional mCAP. Translation efficiency of compound 7, 9, and 10, and conventional mCAP-capped luciferase RNA poly(A). Comparison of protein expression from conventional mCAP and methylated cap variants capped luciferase RNAs poly(A). The conventional mCAP, and modified methyl-substituted cap variants capped in vitro transcribed poly(A) tailed luciferase RNA (400 ng) were transfected into HeLa cells. The fold induction of luciferase protein data was normalized to the control reaction, that is, no cap, mRNA poly(A) transfection results.25
Abstract
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