10262 J. Am. Chem. Soc., Vol. 122, No. 42, 2000
Oyelere and Strobel
by Arabshahi and Frey20 were unsuccessful. A modification of
the procedure in which the initial thiophosphorylation step was
performed in a reaction mixture containing 0.5 equiv of
trioctylamine, 1.2 equiv of collidine, and PSCl3 in triethyl
phosphate at 0 °C for 30 min followed by warming to room
temperature within 15 min gave about 20-25% of n6CTPRS
4a. However, only a trace amount of f 5CTPRS 4b was obtained
and no ΨiCTPRS 4c and CTPRS 4d were formed by using this
protocol.
to inhibit E. coli RNA polymerase.26 The incorporation of ΨiC
into mammalian DNA and RNA has been inferred by detection
of radioactivity in DNA and RNA components of cells grown
in the presence of radioactive ΨiC.27 However, no report exists
on the direct use of ΨiC triphosphate as a substrate for RNA
polymerase in vitro.
The C nucleotide analogues f 5CTPRS, n6CTPRS, and
ΨiCTPRS were individually tested both for their ability to
sustain transcription in the absence of CTP and for specific and
uniform transcriptional incorporation in the presence of CTP.
Plasmid DNA pUCL-21G414, which has a T7 RNA polymerase
promoter, was digested with EarI for use as a template in runoff
transcription reactions. Not unexpectedly, we found that f 5-
CTPRS could completely replace the parent nucleotide cytidine
without a noticeable loss in transcription efficiency (data not
shown). Similarly, n6CTPRS was also able to support transcrip-
tion in the absence of cytidine, which is in contrast to that
reported for the uridine derivative.26 The fidelity of analogue
incorporation was determined by iodine sequencing of the 5′-
end radiolabeled RNA transcripts. The RNA cleavage pattern
was compared to that of an RNA containing CRS. Within the
limits of detection (∼0.5% infidelity), both analogues were
incorporated exclusively at C’s and the extent of incorporation
at each position was essentially equivalent to that of CRS (data
not shown).
For this reason we turned to the salicyl phosphoramidite
approach, described by Ludwig and Eckstein,21 for the synthesis
of the target thiotriphosphates. This route requires nucleoside
starting materials with protection of the base and the 2′- and
3′-OH groups of the sugar. However, the low yields and multiple
purification steps accompanying the syntheses of these protected
nucleosides precluded the use of the standard nucleoside
protecting groups. For this application, the ideal protecting group
is one that permits a quantitative and rapid protection of the
exocyclic amine and the 2′- and 3′-OH groups in a single step,
as well as being a group that can be easily deprotected under
reaction conditions that are compatible with the thiotriphosphate
bonds. N,N-Dialkylformamide dialkylacetals meet these condi-
tions and their reaction with nucleosides has been described.22
Recently, Jones and co-workers reported the use of 2′,3′-O-
dimethylaminomethylene protected nucleoside to effect a 5′-
selective tritylation reaction despite the inherent instability of
2′,3′-O-dimethylaminomethylene group under such conditions.23
The third nucleotide used in this study, ΨiC, is a C-linked
nucleoside that exists as an equilibrium mixture of two
tautomeric forms in aqueous solution (Figure 4).28 NMR
experiments and theoretical calculations have shown that the
N3-H tautomer is favored over the N1-H form by about 2-9
kcal mol-1, depending on the solvent polarity.29 The N3-H
tautomer creates an “unnatural” donor-donor-acceptor hydro-
gen bonding face for base pairing, which might have a
deleterious effect on the ability of this analogue to be recognized
by the polymerase during transcription. Despite this possibility,
ΨiCTPRS served as an efficient substrate for T7 polymerase
leading to the formation of full-length RNA transcripts at a level
comparable to that of CRS. Iodine sequencing of 5′-end-labeled
RNA transcripts containing either 5% or 100% incorporation
of ΨiCRS and CRS confirmed that ΨiCRS is incorporated
exclusively at C’s in the transcript and that the extent of
incorporation at individual positions is the same as CRS (Figure
5). The high efficiency and fidelity of ΨiCRS incorporation
suggests that pairing to the G in the template shifts the
tautomeric equilibrium toward the less stable N1-H form (Figure
4), which creates a hydrogen bonding pattern similar to that of
cytidine. Such a tautomeric equilibrium shift has been observed
in degenerate nucleosides and oligonucleotides containing these
nucleosides.30
Encouraged by this report, we investigated the use of such
protected nucleosides as starting materials in the synthesis of
5′-O-(1-thio)triphosphates. The reaction of N,N-dimethylfor-
mamide dimethylacetal with nucleosides 1b-d, using the
general procedures described by Jones and co-workers,23 gave
protected nucleosides 2b-d in quantitative yield. After solvent
evaporation, crude 2b-d were subjected to the salicyl phos-
phoramidite chemistry following the Ludwig and Eckstein
protocol21 to give crude 1-thiocyclotriphosphates 3b-d. Aque-
ous hydrolysis of 3b-d led to the formation of 5′-O-(1-thio)-
triphosphates with a concomitant deprotection of the 2′,3′-O-
dimethylaminomethylene group. The exocyclic amidine group
was deprotected by treatment with NH4OAc in NH4OH24 to give
4b-d (Figure 3), which were isolated in relatively good yields
by chromatography on DEAE-Sephadex. All 5′-O-(1-thio)-
triphosphates synthesized were characterized by 31P NMR, UV,
and electrospray MS.
Incorporation of C nucleotide analogues by T7 RNA
polymerase. To be used in a nucleotide analogue interference
mapping (NAIM) assay, nucleotide analogues must be uniformly
and accurately incorporated into large RNA transcripts by
enzymatic runoff transcription in vitro. It is known that
halogenated nucleosides are readily incorporated into DNA and
RNA when cells are grown in media containing these nucleo-
sides or their bases and some of them have been directly
employed in in vitro transcription reactions.25 Although n6C
triphosphate has not been tested in transcription, the related
pyrimidine nucleotide 6-azauridine triphosphate has been shown
Approximately 5% incorporation of the phosphorothioate
tagged analogues is sufficient to conduct a NAIM analysis for
an RNA the size of the group I intron (approximately 400
nucleotide). Nucleotide analogue to CTP ratios needed to
achieve this incorporation level were determined for the three
(26) (a) Scheit, K. H. Nucleotide Analogues: Synthesis and Biological
Function; John Wiley & Sons: New York, 1980; p 73. (b) Rada, B.;
Doskocil, J. Pharmac. Ther. 1980, 9, 171-217.
(20) Arabshahi, A.; Frey, P. A. Biochem. Biophys. Res. Commun. 1994,
204, 150-155.
(27) Zedeck, M. S. Biochem. Pharmacol. 1979, 28, 1440-1443.
(28) Birnbaum, G. I.; Watanabe, K. A.; Fox, J. J. Can. J. Chem. 1980,
58, 1633-1638.
(21) Ludwig, J.; Eckstein, F. J. Org. Chem. 1989, 54, 631-635.
(22) Zemlicka, J. Collect. Czech. Chem. Commun. 1963, 28, 1060-1062.
(23) Zhang, X.; Abad, J.-L.; Huang, Q.; Zeng, F.; Gaffney, B. L.; Jones,
R. A. Tetrahedron Lett. 1997, 38, 7135-7138.
(29) Kan, L.; Lin, W.-C.; Yadav, R. D.; Shih, J. H.; Chao, I. Nucleosides
Nucleotides 1999, 18, 1091-1093.
(30) (a) Kierdaszuk, B.; Stolarski, R.; Shugar, D. FEBS Lett. 1983, 158,
128-130. (b) Abdul-Masih, M. T.; Bessman, M. J. J. Biol. Chem. 1986,
261, 2020-2026. (c) Kamiya, H.; Murata-Kamiya, N.; Lin, P. K. T.; Brown,
D. M.; Ohtsuka, E. Nucleosides Nucleotides 1994, 13, 1483-1492.
(24) McBride, L. J.; Kierzek, R.; Beaucage, S. L.; Caruthers, M. J. J.
Am. Chem. Soc. 1986, 108, 2040-2048.
(25) For a review, see: Morris, S. M. Mut. Res. 1993, 297, 39-51.