Hylthio1H-tetrazole (3), and 5-ethylthio-1H-tetrazole (4), among others, have found favor as “turbo” activators. Ethylthiotetrazole is especially popular4, 5 for use in RNA synthesis, where the coupling step is made more sluggish by the presence of the bulky silyl protecting group on the adjacent 2′-position. A major drawback to the acidity of tetrazole and the quest for more acid versions has been
Trevealed in a study on CN larger scale synthesis.6 N This study revealed that CN N tetrazole is sufficiently H acidic to deprotect to a DCI small extent the trityl 4,5-Dicyanoimidazole group in the monomer solution, leading to a small amount of dimer formation. Coupling of the dimer phosphoramidite leads to the presence of longer oligos in the crude product mixture. DCI The synthesis group at NeXstar Technology Products, working on the preparation of highly modified RNA derivatives, found that the use of tetrazole as activator led to very low yields of fulllength products. Addition of 0.1M methylimidazole (NMI) to the activator solution as a buffer to tetrazole’s acidity led to much higher yield of full-length product.7 This result indicates that tetrazole is, in fact, slightly too acidic and led to a search for an alternative activator. Novel Monomers As can be seen from the mechanism of activation in Scheme Cytofectin GSV 1, the rate determining step is Preparing Antisense Oligos the nucleophilic displacement of the diisopropylamino TAMRA CPG group from the protonated intermediate.97281-47-5 web This led to the search for a less acidic but more nucleophilic activator and the discovery of 4,5dicyanoimidazole (DCI) (5) as the first true alternative activator to tetrazole and its derivatives.34450-16-3 Formula DCI offers the following advantages over tetrazole: DCI is less acidic with a pKa of 5.PMID:30969560 2, compared with a pKa of 4.8 for tetrazole (which is the same as acetic acid). DCI is more soluble than tetrazole allowing a 1.1M solution in acetonitrile, while tetrazole is saturated at 0.5M in acetonitrile. DCI is more nucleophilic than tetrazole and doubles the coupling rate relative to tetrazole. The biggest difference between DCI and tetrazole manifests itself at larger scales with a low monomer excess. For example, a 34mer oligoribonucleotide, including 2′-fluoropyrimidine residues, was prepared on a 1 mmole scale with 2 equivalents of monomer using 0.45M tetrazole, 0.45M tetrazole + 0.1M NMI, or 1M DCI as activator. No full length product was detected with tetrazole activation, while a low yield (13%) of product was observed with the activator containing NMI. With DCI the full-length product was observed in 54% yield. This is a remarkable demonstration of the increased effectiveness of DCI. Our studies with DCI show that 0.25M is the optimal concentration for routine small-scale synthesis ( 15 ole), using normal synthesis cycles. We are therefore providing solutions at that concentration but we will also offer the raw material so that researchers can prepare more or less concentrated solutions should they desire. Distribution of DCI by Glen Research is done in collaboration with NeXstar Technology Products. References:
(1) O. Dahl, J. Nielsen, and B.H. Dahl, Nucleic Acids Res., 1987, 15, 1729-1742. (2) S. Berner, K. Muhlegger, and H. Seliger, Nucleic Acid Res., 1989, 17, 853-864. (3) B.S. Sproat, A.I. Lamond, B. Beijer, P. Neuner, and U. Ryder, Nucleic Acids Res., 1989, 17, 3373. (4) B. Sproat, et al., Nucleosides and
PREPARING OLIGONUCLEOTIDES FOR ANTISENSE EXPERIMENTS
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