The synthesis of 5-iodocytidine phosphoramidite for heavy atom
Rostem J. Irani and John SantaLucia Jr ∗Department of Chemistry, Wayne State University, Detroit, MI 48202, USA
Received 12 April 1999; accepted 20 August 1999
The synthesis of an RNA phosphoramidite of 5-iodocytidine is reported. This heavy atom cytidine derivative
was incorporated into four RNAs. Oligoribonucleotides with 5-iodocytidine will be useful for solving the X-raycrystallographic phase problem and for photochemical cross-linking studies. 1999 Elsevier Science Ltd. Allrights reserved.
The heavy atom method to solve the phase problem in X-ray crystallography has been widely used
for DNA and proteins.1 RNA structure determination has remained a challenge because there are few suitable solutions to the phase problem in oligoribonucleotides.2 To overcome this hurdle we designed and synthesized the 5-iodocytidine phosphoramidite 6 (Scheme 1).
The incorporation of iodine at position 5 in cytidine is suitable for structural studies of short RNAs for
two reasons. First, derivatization of the 5 position of cytidine does not disrupt Watson–Crick hydrogenbonding. Second, the iodine at the 5 position of cytidine protrudes into the major grove and thereforecauses minimal perturbation to A-form duplex structure.
For the synthesis of the 5-iodocytidine phosphoramidite the protecting groups used were the same as
those used in commercially available phosphoramidites with 2 TBDMS protection.3 The use of similarprotecting groups allows easy incorporation of this heavy atom derivative into oligoribonucleotides viaan automated chemical synthesizer.
As shown in Scheme 1, compound 24 was prepared from cytidine by iodination with iodic acid and
iodine.5 Pure white crystals of 5-iodocytidine were obtained after recrystallization from methanol in 53% yield. Inspection of the proton NMR of 2 shows the disappearance of the H5 proton of cytidine and concomitant collapse of the H6 proton to a singlet. This assignment verifies the incorporation of iodine at the desired site. Compounds containing iodine are known to be light sensitive, therefore all the subsequent reactions were performed in the dark. Also, the subsequent reactions involved the use of water sensitive reagents and therefore particular care was taken to assure use of dry glassware and the reactions were conducted under argon.
0040-4039/99/$ - see front matter 1999 Elsevier Science Ltd. All rights reserved. P I I: S 0 0 4 0 - 4 0 3 9 ( 9 9 ) 0 1 6 5 0 - 0
Scheme 1. Scheme for 5-iodocytidine phosphoramidite synthesis. Reagents and conditions: (a) iodine (1.0 equiv.), iodic acid(1.0 equiv.), acetic acid, H2O, and CCl4 (8:3:2), respectively, overnight; (b) TMSCl (3.5 equiv.)/pyridine, 0°C, 30 min, followedby benzoyl chloride (1.1 equiv.), 2 h; (c) DMTCl (1.1 equiv.)/pyridine, 25°C, 4 h; (d) TBDMSCl (1.5 equiv.)/pyridine, imidazole(2.5 equiv.), 6 h; (e) β-cyanoethyl N,N-diisopropylphosphoramidite chloride (1.1 equiv.)/THF, N,N-diisopropylethylamine (4equiv.), and DMAP (0.2 equiv.), overnight, 25°C
Protection of the exocyclic amine was achieved via transient protection of the sugar alcohols as trime-
thylsilyl (TMS) ethers and subsequent reaction of the amine with benzoyl chloride.6 After completion of the reaction, the TMS groups were removed by addition of water. Compound 3 was obtained in 66% yield.
Pyridine was added to compound 37 and evaporated several times to remove any water from the
sample. The tritylation was carried out at room te
7. The only amino acid that does not need to enter the A site before entering the P site on a ribosome during the process of translation is methionine. Methionine is coded for by AUG, the start codon, and therefore it is always the first amino acid in a newly synthesized polypeptide. Since it is the first amino acid, there will be no amino acid before it to form a peptide bond with and hence it n