Synthesis of the tRNALys,3 anticodon stem-loop domain containing the hypermodified ms2t6A nucleoside

Article abstract of DOI:10.1021/jo025826t

The synthesis of a protected form of the hypermodified nucleoside, N-[(9-β-D-ribofuranosyl-2- methylthiopurin-6-yl)carbamoyl]threonine, (ms²t⁶A) is reported. The hypermodified nucleoside was subsequently elaborated to the protected nucleoside phosphophoramidite using a protecting group strategy compatible with standard RNA oligonucleotide chemistry. The phosphoramidite reagent was then used to synthesize the 17-nucleotide RNA hairpin having the sequence of the anticodon stem-loop (ASL) domain of human tRNA^Lys,3, the primer for HIV-1 reverse transcriptase. Introduction of the modification at position 37 of the tRNA ASL modestly decreases the thermodynamic stability of the RNA hairpin as has been seen previously for the prokaryotic t⁶A nucleoside lacking the 2-methylthio substituent. 2D NOESY NMR spectra of the ms²t⁶A containing tRNA ASL indicate that the threonyl side chain adopts a conformation similar to that seen in the solution structure of the analogous t⁶A containing E. coli tRNA^Lys, despite the presence of the bulky methylthio group. This synthetic approach allows for site-specific incorporation of the hypermodified nucleoside and should facilitate future studies directed at understanding the roles of nucleoside modification in modulating the stability and specificity of biologically important RNA-RNA interactions. Our synthesis of the ms²t⁶A containing RNAs demonstrates that this methodology is suitable for obtaining quantities of RNA required for structural studies of the HIV primer tRNA.

Full text of DOI:10.1021/jo025826t

Syn th esis of th e tRNALys,3 An ticod on Stem -Loop Dom a in
Con ta in in g th e Hyp er m od ified m s2t6A Nu cleosid e
Ashok C. Bajji† and Darrell R. Davis*
Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112
davis@adenosine.pharm.utah.edu
Received April 16, 2002
The synthesis of a protected form of the hypermodified nucleoside, N-[(9-â-D-ribofuranosyl-2-
methylthiopurin-6-yl)carbamoyl]threonine, (ms2t6A) is reported. The hypermodified nucleoside was
subsequently elaborated to the protected nucleoside phosphophoramidite using a protecting group
strategy compatible with standard RNA oligonucleotide chemistry. The phosphoramidite reagent
was then used to synthesize the 17-nucleotide RNA hairpin having the sequence of the anticodon
stem-loop (ASL) domain of human tRNALys,3, the primer for HIV-1 reverse transcriptase.
Introduction of the modification at position 37 of the tRNA ASL modestly decreases the
thermodynamic stability of the RNA hairpin as has been seen previously for the prokaryotic t6A
nucleoside lacking the 2-methylthio substituent. 2D NOESY NMR spectra of the ms2t6A containing
tRNA ASL indicate that the threonyl side chain adopts a conformation similar to that seen in the
solution structure of the analogous t6A containing E. coli tRNALys, despite the presence of the bulky
methylthio group. This synthetic approach allows for site-specific incorporation of the hypermodified
nucleoside and should facilitate future studies directed at understanding the roles of nucleoside
modification in modulating the stability and specificity of biologically important RNA-RNA
interactions. Our synthesis of the ms2t6A containing RNAs demonstrates that this methodology is
suitable for obtaining quantities of RNA required for structural studies of the HIV primer tRNA.
In tr od u ction
Interestingly, the integrity of the anticodon/A-loop
interaction is strongly dependent on one or more hyper-
modified nucleosides present in the anticodon stem-loop
(ASL) domain of tRNALys,3. Chemical probing has estab-
lished the presence of a high-affinity interaction in vitro,
and the extensive investigations by Ehresmann and co-
workers of the anticodon/A-loop interaction have shown
that while an unmodified tRNA transcript interacts with
the 18 nucleotide PBS, no interaction is detected between
the unmodified anticodon nucleotides and the A-loop.6
Despite the relatively complex nucleoside structures,
chemical synthesis is the method of choice for site-
specifically introducing hypermodified nucleosides into
RNA oligonucleotides due to the lack of an appropriate
biosynthetic system. The ability to synthesize the tRNA
ASL domain containing single modified nucleosides, or
any combination of the natural modified nucleosides, is
a powerful tool for studying the structural effects of these
nucleosides on the tRNA anticodon.7,8
tRNALys,3 is the specific RNA primer for HIV-1 reverse
transcription.1 The transcription initiation complex in-
volves two specific RNA-RNA interactions between the
human tRNA and the 5′ region of the HIV-1 genomic
RNA. The first is a Watson-Crick RNA duplex involving
the 3′ 18 nucleotides of the tRNA and the complementary
primer-binding site (PBS) of the HIV genome, while the
second is a less well-understood interaction between a
conserved “A-rich loop” in the HIV genome and the
anticodon domain of tRNALys,3 2,3
Genetic studies have
.
shown that the ability to form an anticodon/A-loop
interaction is necessary for stable tRNA primer mainte-
nance, and with the appropriate complementarity to the
anticodon several different tRNAs can be made to serve
as primers.4 The importance of the tRNA anticodon/A-
loop interaction appears to be that this is a recognition
event that helps validate that the reverse transcription
complex is “correct”, allowing DNA synthesis to then
proceed from this “initiation” complex to the “elongation”
complex.5
The t6A family of nucleosides consists of related modi-
fied adenosines at position 37 found adjacent to the
anticodon of tRNAs with U36 as the last codon nucleotide
* To whom correspondence should be addressed. Phone: (801) 581-
7006. Fax: (801) 581-7087.
(5) Isel, C.; Lanchy, J .-M.; Le Grice, S. F. J .; Ehresmann, C.;
Ehresmann, B.; Marquet, R. EMBO J . 1996, 15, 917-924.
(6) Isel, C.; Marquet, R.; Keith, G.; Ehresmann, C.; Ehresmann, B.
J . Biol. Chem. 1993, 268, 25269-25272.
† Current address: Myriad Genetics, Salt Lake City, UT.
(1) Mak, J .; Kleiman, L. J . Virol. 1997, 71, 8087-8095.
(2) Isel, C.; Westhof, E.; Massire, C.; Le Grice, S. F. J .; Ehresmann,
B.; Ehresmann, C.; Marquet, R. EMBO J . 1999, 18, 1038-1048.
(3) Yu, Q.; Morrow, C. D. Virology 1999, 254, 160-168.
(4) Zhang, Z.; Kang, S.; Li, Y.; Morrow, C. D. RNA 1998, 4, 394-
406.
(7) Agris, P. F.; Malkiewicz, A.; Kraszewski, A.; Everett, K.; Nawrot,
B.; Sochacka, E.; J ankowska, J .; Guenther, R. Biochimie 1995, 77,
125-134.
(8) Sundaram, M.; Crain, P. F.; Davis, D. R. J . Org. Chem. 2000,
65, 5609-5614.
10.1021/jo025826t CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/18/2002
5352
J . Org. Chem. 2002, 67, 5352-5358

Synthesis of ms2t6A-Containing RNAs
(Figure 1).9 The adenosine base is derivatized by either
threonine or glycine connected to N6 via a carbamoyl
linkage. The nucleosides can then be further modified by
N6 methylation,10 and by methylthiolation at position 2
of the base,11 but remarkably little is known about their
structural and functional role.12 The highly functionalized
threonine group been proposed to be a specific coordina-
tion site for metal ions,13 but there is some dispute about
this effect.14 Recent measurements of the thermodynamic
effect of t6A modification have shown that t6A has a
modest destabilizing effect on the ASL Tm,15,16 but no
dramatic structural or stability effects were seen upon
titration with magnesium.17 In the context of the tRNALys
ASL, we have shown that t6A37 helps to promote a
canonical U-turn structure,16 which could be important
in lowering the entropic cost of the tRNA binding either
a cognate codon during translation18 or the HIV A-loop.
In a series of classic studies by Grosjean and co-workers,
the kinetic and thermodynamic effects of a wide variety
of tRNA modifications were investigated using temper-
ature jump methods.19 They found that t6A37 has a
stabilizing effect on tRNA-tRNA complexes in the range
of 1-4-fold20 and that the 2-methylthio is likely to have
a somewhat stronger effect, but still in the neighborhood
of 4-fold.21 Unfortunately, since they were limited to
natural tRNAs for these studies, they were restricted in
some cases to using partially complementary tRNAs, and
the combinations available resulted in an inability to
separate the methylthio and threonyl effects within the
same sequence context.
F IGURE 1. Secondary structure of the 17-nucleotide anti-
codon stem-loop domain of human tRNALys,3. The structures
of the modified nucleosides at tRNA positions 37 and 39 are
shown. Native tRNALys,3 also has a hypermodified mcm5s2U
nucleoside at position 34.
E. coli tRNALys ASL containing t6A37 lacking the meth-
ylthio group.16 In the current study, we have now
synthesized the protected ms2t6A nucleoside and subse-
quently elaborated this nucleoside to the phosphoramid-
ite. This phosphoramidite reagent was used in a modified
RNA synthesis protocol with very mild deprotection
conditions to synthesize the tRNALys,3 ASL RNA oligo-
nucleotide. NMR spectra of the ms2t6A37 containing 17
nucleotide tRNALys,3 ASL indicate a nucleoside conforma-
tion similar to that of the free t6A nucleoside and similar
A recent X-ray crystal structure of tRNALys,3 at 3.3 Å
resolution established the overall fold of the tRNA;
however, the resolution was insufficient to accurately
position the threonyl side chain of ms2t6A relative to the
base.22 The deposited coordinates (PDB ID:1FIR) suggest
the side chain adopts a position different from that seen
in the high-resolution X-ray structure of t6A nucleoside23
and also different from what we had determined for the
to that determined for t6A within native E. coli tRNALys
.
Resu lts a n d Discu ssion
The present study describes the efficient synthesis of
N-[[9-(â-D-ribofuranosyl)-2-methylthiopurine-6-yl]carbam-
oyl]-L-threonine nucleoside phosphoramidite (Scheme 1)
and its incorporation into the anticodon stem loop domain
of tRNALys,3 (Figure 1). To the best of our knowledge, the
synthesis of the ms2t6A nucleoside has not been previ-
ously reported. However, numerous studies have been
done on the simpler t6A nucleoside, which lacks the
methylthio group at the 2-position. A standard literature
protocol for synthesizing the t6 nucleoside that we have
used previously involves displacing the ethoxy group of
ethyl [9-(2,3,5-tri-O-acetyl-â-D-ribofuranosyl)-9H-purine-
6-yl]carbamate with L-threonine.8,24 A recent report from
Herdewijn and co-workers25 describes an alternate ap-
proach to the nucleoside where they reinvestigate an
early route to the nucleoside that involved reacting
adenosine triacetate with the protected isocyanate of
L-threonine.26 Recognizing that an advantage of the
isocyanate strategy for making protected phosphoramid-
ites was its convergence, Herdewijn and co-workers
addressed the poor coupling with threonine isocyanate
by inverting the reactant functionalities. They generated
the protected adenosine N6-isocyanate in situ and then
(9) Rozenski, J .; Crain, P. F.; McCloskey, J . A. Nucleic Acids Res.
1999, 27, 196-197.
(10) Kimura-Harada, F.; von Minden, D. L.; McCloskey, J . A.;
Nishimura, S. Biochemistry 1972, 11, 3910-3915.
(11) Yamaizumi, Z.; Nishimura, S.; Limburg, K.; Raba, M.; Gross,
H. J .; Crain, P. F.; McCloskey, J . A. J . Am. Chem. Soc. 1979, 101,
2224-2225.
(12) Bjork, G. R. In tRNA: Structure, Biosynthesis and Function;
Soll, D., RajBhandary, U. L., Eds.; ASM Press: Washington, DC, 1995;
pp 165-205.
(13) Reddy, P. R.; Hamill, W. D.; Chheda, G. B.; Schweizer, M. P.
Biochemistry 1981, 20, 4979-4986.
(14) Varnagy, K.; J ezowska-Bojczuk, M.; Swiatek, J .; Kozlowski, H.;
Sovago, I.; Adamiak, R. W. J . Inorg. Biochem. 1990, 40, 357-363.
(15) Stuart, J . W.; Gdaniec, Z.; Guenther, R. H.; Marszalek, M.;
Sochacka, E.; Malkiewicz, A.; Agris, P. F. Biochemistry 2000, 39,
13396-13404.
(16) Sundaram, M.; Durant, P. C.; Davis, D. R. Biochemistry 2000,
39, 12575-12584.
(17) Sundaram, M. Ph.D. Dissertation, University of Utah, 2000.
(18) Yarian, C.; Marszalek, M.; Sochacka, E.; Malkiewicz, A.;
Guenther, R.; Miskiewicz, A.; Agris, P. F. Biochemistry 2000, 39,
13390-13395.
(19) Grosjean, H.; Houssier, C.; Romby, P.; Marquet, R. In Modifica-
tion and Editing of RNA; Grosjean, H., Benne, R., Eds.; ASM Press:
Washington, DC, 1998; pp 113-133.
(20) Weissenbach, J .; Grosjean, H. Eur. J . Biochem. 1981, 116, 207-
213.
(21) Houssier, C.; Grosjean, H. J . Biomol. Struct. Dyn 1985, 3, 387-
408.
(22) Benas, P.; Bec, G.; Keith, G.; Marquet, R.; Ehresmann, C.;
Ehresmann, B.; Dumas, P. RNA 2000, 6, 1347-1355.
(23) Parthasarathy, R.; Ohri, J .; Chheda, G. Biochemistry 1977, 16,
4999-5007.
(24) Hong, C. I.; Chheda, G. B. In Nucleic Acid Chemistry; Towsend,
L. B., Tipson, R. S., Eds.; Wiley: New York, 1972; pp 661-664.
(25) Boudou, V.; Langridge, J .; van Aerschot, A.; Hendrix, C.; Millar,
A.; Weiss, P.; Herdewijn, P. Helv. Chim. Acta 2000, 83, 152-161.
(26) Schweizer, M. P.; Chheda, G. B.; Baczynskyj, L.; Hall, R. H.
Biochemistry 1969, 8, 3283-3289.
J . Org. Chem, Vol. 67, No. 15, 2002 5353

Bajji and Davis
SCHEME 1. m s2t6A P h osp h or a m id ite Syn th esis
subsequently coupled with the L-threonine derivative to
synthesize protected t6A with a combined 19% yield for
the activation and coupling steps. Although the coupling
yield was still modest, by using threonine with the
protecting groups desired for the final product, the overall
yield to the protected phosphoramidite was approxi-
mately 5% starting from adenosine. We noticed that this
approach was somewhat reminiscent of work by Adamiak
and co-workers where they made a heptamer oligonu-
cleotide corresponding to the t6A containing anticodon
loop of an eukaryotic initiator tRNA using the phospho-
triester approach.27,28 The strategy we envisioned was to
use the high-yielding coupling step of Adamiak, but in
the context of Herdewijn’s relatively labile threonine
nitrophenylethyl carboxylic ester. Since simply generat-
ing the starting 2-methylthioadenosine required several
steps, some improvement over the published methods
was viewed as critical for making the milligram quanti-
ties of RNA required for structural studies.
1 could also be made from commercially available 4,6-
diamino-2-mercaptopyridine in three steps. The nucleo-
side base was then coupled with protected ribose under
Lewis acid conditions without prior base silylation to
yield the 2′,3′,5′-tri-O-benzoyl 2-methylthioadenosine
nucleoside in 48% yield.31 Unfortunately, the nucleoside
tri-O-benzoate was found to be unsuitable since we could
not get complete removal of the benzoyl groups after
threonine coupling without simultaneous t6A side-chain
hydrolysis. Therefore, the benzoyl groups were removed
from compound 2 with sodium methoxide in methanol
and then the sugar hydroxyls reprotected as the acetates.
The logical alternative would be to directly produce the
triacetate by coupling the nucleobase with tetraacetyl
ribose, but the yield for this reaction was poor in our
hands. The [9-(2,3,5-tri-O-acetyl-â-D-ribofuranosyl)-2-
methylthiopurin-6-yl]phenylcarbamate 5 was prepared
in 74% yield by reacting 4 and phenoxycarbonyl tetrazole
in 1,4-dioxane at 35-40 °C for 16 h.28 The carbamate 5
was then combined with the 2-(4-nitrophenyl)ethyl-O-
tert-butyldimethylsilyl-L-threonine ester25 in pyridine at
35-40 °C to give fully protected ms2t6A 6 in 75% yield.
The acetyl groups of 6 were removed by treating with 2
M ammonia in methanol at room temperature for 3 h to
afford 72% yield of the threonine protected nucleoside 7.
By reacting the protected amino acid with the carbamate,
we achieved an overall coupling yield of 55% for protected
ms2t6A compared to 19% reported previously for t6A.25
m s2t 6A Nu cleosid e P h osp h or a m id it e Syn t h esis.
2-Methylthioadenine base 1 was synthesized in four steps
starting from thiourea and malononitrile following lit-
erature procedures in 21% overall yield.29,30 Compound
(27) Adamiak, R. W.; Biala, E.; Grzeskowiak, K.; Kierzek, R.;
Kraszewski, A.; Markiewicz, W. T.; Okupniak, J .; Stawinski, J .;
Wiewiorowski, M. Nucleic Acids Res. 1978, 5, 1889-1905.
(28) Adamiak, R. W.; Stawinski, J . Tetrahedron Lett. 1977, 22,
1935-1936.
(29) Saxena, N. K.; Bhakuni, D. S. Indian J . Chem. 1979, 18B, 348-
351.
(30) Taylor, E. C.; Vogl, O.; Cheng, C. C. J . Am. Chem. Soc. 1958,
81, 2442-2248.
(31) Ojha, L. M.; Gulati, D.; Seth, N.; Bhakuni, D. S.; Pratap, R.
Nucleosides Nucleotides 1995, 14, 1889-1900.
5354 J . Org. Chem., Vol. 67, No. 15, 2002

Synthesis of ms2t6A-Containing RNAs
The acetate deprotection is a critical step since the
carbamoyl linkage is base sensitive, and extended reac-
tion times result in significant degradation of the nucleo-
side. The 5′ hydroxy group of 7 was then protected using
DMT-Cl in pyridine to give the 5′ dimethoxytrityl ether
nucleoside in 63% yield. Treatment of the 5′ DMT
derivative with TBS-Cl in THF in the presence of AgNO3
and pyridine gave the 2′-TBS derivative in 59% yield and
a small amount of the 3′-TBS isomer.32 Reaction of the
2′-TBS-protected nucleoside with 2-cyanoethyl-N,N-di-
isopropylchlorophosphoramidite in THF for 4 h in the
presence of diisopropylethylamine with DMAP catalyst
afforded the target phosphoramidite 8 in 57% yield.33
RNA Syn th esis, Dep r otection , a n d P u r ifica tion .
The modified tRNALys,3 anticodon domain in Figure 1 was
synthesized using commercial PAC-protected phosphor-
amidites (Glen Research) and standard coupling chem-
istry with t-BuOOH oxidation.34,35 The methylthio should
be stable to normal I2/water oxidation, but we have
noticed little decrease in coupling yields when using
t-BuOOH oxidation and therefore used 10% tBuOOH/
acetonitrile for all oxidation steps to establish conditions
that would also be compatible with 2-thiouridine nucleo-
sides. Trityl assays indicated >97% coupling for all the
phosphoramidites; this was verified by HPLC and MALDI/
MS of the fully deprotected RNA. The deprotection was
carried out by first treating the column-bound RNA with
10% DBU in THF for 1.5 h at 40 °C to remove the
nitrophenylethyl group. After DBU/THF treatment, the
supernatant was removed, and the support-bound RNA
was reacted with a solution of 33% ethanolic methyl-
amine for 2 h. This procedure is similar to that of Her-
dewijn and co-workers, and we also found that a two-
step base treatment is necessary to deprotect the RNA
while maintaining the sensitive side chain.25 The RNA
solution was then dried in vacuo and treated with Et3N‚
3HF to remove the TBS ethers. The fully deprotected
RNA was then purified by HPLC,36 and MALDI-MS was
used to verify the identity of the purified oligonucleotide
as previously described.8
Effect of m s2t6A Mod ifica tion on Stem -Loop Sta -
bility. Previous studies from our laboratory and others
have shown that t6A37 slightly destablizes the tRNALys
ASL compared to A37.15,16 Thermal melting studies of the
RNA oligomer in Figure 1 showed that ms2t6A37 causes
a somewhat greater decrease in the Tm; 5 °C compared
to the 2 °C decrease seen for t6A. The imino NMR
spectrum shown in Figure 2 indicates that the effect of
ms2t6A on the base of the anticodon stem is similar to
that seen for t6A. The N3-H imino resonance for ψ39 is
completely melted out indicating that the A31-ψ39 base
pair is destabilized, and the ψ N1-H is also broad
compared to the spectrum of the tRNA ASL with A37
acquired under similar conditions.37 This loss of hydrogen
bonding probably occurs from stacking destabilization,
although a detailed chemical shift analysis of the 2D
F IGURE 2. NMR spectra of the 17-mer RNA shown in Figure
1. The RNA concentration was 1 mM in 25 mM sodium
phosphate buffer, pH 7.0 containing 100 mM NaCl and 10 mM
MgCl2: (a) downfield spectral region showing the hydrogen-
bonded imino protons and side-chain amide resonances; (b)
expanded region of the 2D NOESY spectrum showing the
cross-peaks from the methylthio group diagonal peak at 2.62
ppm. These NOE connectivities indicate that the threonyl side
chain is oriented trans relative to the imidazole ring of
adenosine as shown in Figure 4.
NMR spectra will be necessary to fully characterize these
structural changes.
CD spectra (Figure 3) were also collected in order gain
insight into the structural environment of the hyper-
modified nucleoside. The CD spectrum has an overall
appearance that is consistent with a largely A-form RNA
structure, characterized by the dominant maximum at
260 nm. The additional, long wavelength band at 300 nm
is due to the methylthio group. The temperature depen-
dence of the CD spectrum indicates that although there
is a significant base-stacking loss as the stem melts, the
environment of the methylthio group changes little over
this temperature range. While one should be cautious
about drawing detailed structural conclusions from CD
spectra, this behavior is consistent with our preliminary
2D NMR experiments. The 300 nm ellipticity is a
potential reporter of stacking interactions involving
ms2t6A as the signal could change significantly when the
tRNA ASL forms a complex with a complementary RNA.
Con for m a tion of th e Th r eon yl Sid e Ch a in in
m s2t6A Con ta in in g tRNALys,3 ASL. High-resolution
structures of t6A nucleoside and NMR studies of t6A
containing RNAs have shown that the threonyl side chain
adopts an orientation trans (distal) to the adenosine
imidazole ring that would be stabilized by a potential
(32) Hakimelahi, G. H.; Proba, Z. A.; Ogilvie, K. K. Can. J . Chem.
1982, 60, 1106-1113.
(33) Scaringe, S. A.; Francklyn, C.; Usman, N. Nucleic Acids Res.
1990, 18, 5433-5441.
(34) J ager, A.; Engels, J . Tetrahedron Lett. 1984, 25, 1437-1440.
(35) Kumar, R. K.; Davis, D. R. J . Org. Chem. 1995, 60, 7726-7727.
(36) Sproat, B.; Colonna, F.; Mulla, B.; Tsou, D.; Andrus, A.; Hampel,
A.; Vinayak, R. Nucleosides Nucleotides 1995, 14, 255-273.
(37) Durant, P. C.; Davis, D. R. J . Mol. Biol. 1999, 285, 115-131.
J . Org. Chem, Vol. 67, No. 15, 2002 5355

Bajji and Davis
nucleoside in a tRNA with the same anticodon sequence.
Within the context of a canonical tRNA structure, this
would then orient the large, hydrophobic methylthio
group over the 5′ codon nucleotide of a codon-anticodon
complex. This type of stacking interaction would be
analogous to the 3′ end effect that has been quantitated
by Turner and co-workers39 and explain the methylthio
stabilization of complementary tRNAs.21 Methylthio-
mediated 3′-stacking stabilization is also a possible
explanation for the importance of nucleoside modifica-
tion in stabilizing the HIV A-loop interaction with the
tRNALys,3 anticodon.
Exp er im en ta l Section
Gen er a l Meth od s. Melting points are recorded by the open
capillary method and are uncorrected. Standard nucleoside
phosphoramidite procedures were used to synthesize the
protected phosphoramidite of pseudouridine.33,40 1H and 31P
NMR of phosphoramidites and precursors were acquired in
deuterated solvents on either 200 or 400 MHz spectrometers.
The NMR spectra of the modified 17-mer tRNA ASL were
collected at 500 MHz. 1H chemical shifts are reported relative
to tetramethylsilane at 0.0 ppm and referenced to the residual
proton signal of the deuterated solvents. 31P chemical shifts
are relative to external phosphoric acid at 0.0 ppm. Analytical
thin-layer chromatography (TLC) was carried out on silica gel
60F-254 plates. Flash column chromatography was carried out
with silica gel 60 (230-400 mesh). Chemical reagents were
purchased from commercial suppliers and used without further
purification. Instrumentation and sample preparation for
MALDI MS and ESI LC/MS have been described previously.8
2′,3′,5′-Tr i-O-ben zoyl-2-m eth ylth ioa d en osin e(2). To a
suspension of 2-methylthioadenine 129,30 (10.0 g, 55.18 mmol)
and 1-O-acetyl-2,3,5-tri-O-benzoyl-â-D-ribofuranose (33.40 g,
66.21 mmol) in anhydrous nitromethane (160 mL) was added
SnCl4 (10 mL) dropwise at 0 °C. The temperature of the
reaction mixture was slowly raised to room temperature and
stirring continued for 16 h at room temperature. At the end
of this period, the solvent was evaporated, and the brown
residue was triturated with a saturated solution of NaHCO3
and the solid collected by filtration. The crude product was
purified by flash chromatography using a mixture of dichlo-
romethane and methanol (95:5) to give product 2 (16.58 g, 48%)
F IGURE 3. CD spectrum of a 5 µM solution of the modified
tRNALys ASL. These spectra were collected from 10 to 60 °C
in the NMR buffer absent magnesium. The spectrum shows a
characteristic profile for a predominantly A-form RNA struc-
ture with an additional ellipticity maximum at 300 nm from
the methylthio group.
F IGURE 4. Geometry-optimized model of ms2t6A nucleoside.
hydrogen bond from NH-11 to N1.16,23 Quantum chemical
calculations on 2-methylthio t6A have indicated that
although the bulky methylthio could sterically interfere
with the trans orientation as seen for 2-methylthio N6-
isopentenyladeonsine, in ms2t6A the methylthio orients
away from N1 as shown in Figure 4. This allows for a
hydrogen bond stabilized threonine conformation just as
1
as a pale yellow solid: mp 108-110 °C; H NMR (DMSO-d6,
200 MHz) δ (ppm) 2.56 (s, 3H), 4.72-4.99 (m, 3H), 6.25 (t,
J ) 6.0 Hz, 1H), 6.45-6.60 (m, 4H), 7.39-7.62 (m, 9H), 7.93-
8.01 (m, 6H), 8.10 (s, 1H).
2-Meth ylth ioa d en osin e (3). To a solution of 2 (16.0 g,
25.73 mmol) in methanol (150 mL) was added freshly prepared
sodium methoxide (0.7 g of sodium in 5.0 mL of anhydrous
methanol). The mixture was stirred at 25 °C for 4 h, neutral-
ized with Dowex 50 (H+, 200-mesh), and filtered. The solvent
was evaporated completely, and the crude product was then
triturated with 5% ethyl acetate in petroleum ether and
filtered. The product was washed with diethyl ether and dried
to afford 3 (7.4 g, 92%): mp 222-224 °C (lit. mp41 225-228.5
°C). This product was sufficiently pure to use for the next step
and therefore carried forward without further purification: 1H
NMR (DMSO-d6, 200 MHz) δ (ppm) 2.45 (s, 3H), 3.20-3.74
(m, 4H), 3.85 (b, 1H), 4.29 (bs, 1H), 5.30 (bs, 1H), 5.82 (d, J )
5.8 Hz, 1H), 7.59 (bs, 2H), 8.21 (s, 1H); FABMS (MH+) calcd
for C11H15N5O4S 312.4, obsd 313.22.
seen for t6A.38 In the recent X-ray structure of tRNALys,3
,
the resolution was insufficient to accurately position the
modified nucleoside side chains, but the carbamoyl
threonine conformation was modeled into the structure
with an extended conformation lacking an N11-H to N1
hydrogen bond.22 The 2D NOESY NMR spectrum in
Figure 2b for our synthetic tRNALys,3 ASL shows numer-
ous NOEs from the easily assigned methylthio at 2.62
ppm to protons of the ms2t6A37 nucleoside and also shows
NOEs to assigned protons on adjacent nucleotides. From
this NMR data, it seems clear that the threonine side
chain is oriented trans as seen for t6A in the E. coli
16
tRNALys
and also consistent with the low energy
conformation calculated by Tewari.38
2′,3′,5′-Tr i-O-a cetyl-2-m eth ylth ioa d en osin e (4). A mix-
ture of 2-methylthioadenosine 3 (6.0 g, 19.15 mmol), anhydrous
Our structural studies are still preliminary, but the
thermodynamic and NMR data presented here indicate
that the structural effect of the threonyl group in ms2t6A
containing tRNAs will be similar to the simpler t6A
(39) Turner, D. H.; Sugimoto, N.; Freier, S. M. Annu. Rev. Biophys.
Biophys. Chem. 1988, 17, 167-192.
(40) Hall, K. B.; McLaughlin, L. W. Nucleic Acids Res. 1992, 20,
1883-1889.
(41) Kikugawa, K.; Suehiro, H.; Yanase, R.; Aoki, A. Chem. Pharm.
Bull. 1977, 25, 1959-1969.
(38) Tewari, R. J . Biomol. Struct. Dyn. 1990, 8, 675-686.
5356 J . Org. Chem., Vol. 67, No. 15, 2002

Synthesis of ms2t6A-Containing RNAs
pyridine (50 mL), and acetic anhydride (30 mL) was stirred
at 0 °C for 4 h. The excess reagent and pyridine were removed
under reduced pressure and coevaporated with toluene (2 ×
50 mL). The crude product was crystallized from a mixture of
ethyl acetate and petroleum ether to afford 4 (7.2 g, 86%) as
a colorless solid: mp 159-161 °C; 1H NMR (DMSO-d6, 200
MHz) δ (ppm) 1.94 (s, 3H), 2.05 (s, 3H), 2.09 (s, 3H), 2.47 (s,
3H), 4.14-4.20 (m, 1H), 4.30-4.42 (m, 2H). 5.67 (t, J ) 5.9
Hz, 1H), 6.04 (t, 5.9 Hz, 1H), 6.14 (d, J ) 4.2 Hz, 1H), 7.45 (s,
2H), 8.17 (s, 1H); 13C NMR (DMSO-d6) δ (ppm) 13.69, 20.27,
20.33, 62.55, 69.69, 71.87, 78.72, 80.34, 117.00, 139.35, 149.55,
155.48, 164.93, 169.36, 169.46, 170.01; FABMS (MH+) calcd
for C17H21N5SO7 439.44, obsd 440.0.
N-[[9-(5′-O-(4,4′-Dim eth oxytr ityl)-â-D-r ibofu r a n osyl)-2-
m eth ylth iop u r in -6-yl]ca r ba m oyl]-O-ter t-bu tyld im eth yl-
silyl-L-th r eon in e 2-(4-Nitr op h en yl)eth yl Ester (8). A mix-
ture of 7 (1.34 g, 1.85 mmol) and 4,4′-dimethoxytrityl chloride
(0.81 g, 2.41 mmol) in anhydrous pyridine (10 mL) was stirred
at 25 °C under nitrogen atmosphere for 16 h. At the end of
this period, the solvent was removed under reduced pressure,
and the residue was coevaporated with toluene (2 × 20 mL).
The product was purified by flash chromatography using a
mixture of dichloromethane and methanol (9:1) to give the 5′-
dimethoxytrityl compound (1.2 g, 63%) as a yellow solid: mp
1
104-106 °C; H NMR (DMSO-d6, 400 MHz) δ (ppm) -0.6 (s,
3H), 0.03 (s, 3H), 0.08 (s, 9H), 1.21 (d, 3H, J ) 6.0 Hz), 2.42
(s, 3H), 3.08 (t, 3H, J ) 6.1 Hz), 3.12-3.28 (m, 2H), 3.71 (s,
6H), 4.02-4.10 (m, 1H), 4.21-4.48 (m, 5H), 4.74 (t, 1H, J )
4.7 Hz), 5.23 (d, 1H, J ) 5.0 Hz), 5.58 (d, 1H, J ) 5.2 Hz),
5.96 (d, 1H, J ) 5.1 Hz), 6.72-6.82 (m, 4H), 7.15-7.35 (m,
9H), 7.50 (d, 2H, J ) 7.6 Hz), 8.01 (d, 1H, J ) 7.6 Hz), 8.42 (s,
1H) 9. 21 (d, 1H, J ) 6.8 Hz), 9.99 (s, 1H); MS (FAB) MH+
calcd for C51H61N7O12SiS 1024.2, obsd 1024.1.
[9-(2′,3′,5′-Tr i-O-a cet yl-â-D-r ib ofu r a n osyl)-2-m et h yl-
th iop u r in -6-yl]p h en ylca r ba m a te (5). 2-Methylthioadeno-
sine triacetate 4 (1.4 g, 3.18 mmol) was dissolved in 5 mL of
1,4-dioxane and phenoxycarbonyl tetrazole (1.82 g, 9.56 mmol)28
added to the stirred solution. The solvent was removed in
vacuo and then the suspension/evaporation step repeated twice
before addition of 5 mL of 1,4-dioxane a final time. The thick
slurry was heated at 35-40 °C for 16 h. At the end of this
period, the solvent was removed under reduced pressure and
the crude product was purified by flash chromatography using
a mixture of dichloromethane and ethyl acetate (8:2) to afford
N-[[9-(2′-O-ter t-Bu t yld im et h ylsilyl-5′-O-(4,4′-d im et h -
oxytr ityl)-â-D-r ibofu r a n osyl)-2-m eth ylth iop u r in -6-yl]ca r -
ba m oyl]-O-ter t-bu tyld im eth ylsilyl-L-th r eon in e 2-(4-Ni-
tr op h en yleth yl) Ester (9). To a solution of the 5′-dimeth-
oxytrityl compound 8 (0.4 g, 0.39 mmol) in dry THF (5 mL)
was added AgNO3 (0.079 g, 0.47 mmol), and the reaction
mixture was stirred at room temperature for 30 min. To this
reaction mixture were added TBS-Cl (0.076 g, 0.50 mmol) and
pyridine (50 µL), and stirring was continued at room temper-
ature for 12 h. At the end of this period, the mixture was
filtered through Celite into a saturated solution of NaHCO3
(50 mL) and extracted with dichloromethane (2 × 30 mL). The
organic layer was washed with water (50 mL), dried (Na2SO4),
and filtered, and the solvent was removed under reduced
pressure to give predominantly the 2′-TBS isomer and less
than 10% of the 3′-TBS isomer. The crude mixture was purified
by flash chromatography over silica gel using a mixture of
dichloromethane and ethyl acetate (6:1) to afford the 2′-TBS
isomer (0.26 g, 59%) as a pale yellow solid: mp 96-97 °C; 1H
NMR (DMSO-d6, 400 MHz) δ (ppm) -0.08 (s, 3H), -0.06 (s,
3H), 0.0 (s, 3H), 0.02 (s, 3H), 0.75 (s, 3H), 0.08 (s, 9H), 1.18 (d,
3H, J ) 6.5 Hz), 2.37 (s, 3H), 3.02 (t, 2H, J ) 6.3 Hz), 3.18-
3.25 (m, 1H), 3.68 (s, 3H), 3.71 (s, 3H), 4.08-4.12 (m, 1H),
4.25-4. 40 (m, 4H), 4.42-4.48 (m, 1H), 4.88 (t, 1H, J ) 5.1
Hz), 5.18 (d, 1H, J ) 5.5 Hz), 6.0 (d, 1H, J ) 5.6 Hz), 6.28-
6.70 (m, 4H), 7.15-7.28 (m, 7H), 7.36 (d, 2H, J ) 7.3 Hz), 7.49
(d, 2H, J ) 7.4 Hz), 7.99 (d, 2H, J ) 7.4 Hz), 8.42 (s, 1H), 9.22
(d, 1H, J ) 6.7 Hz), 9.99 (s, 1H); MS (FAB) MH+ calcd
1
5 (1.32 g, 74%). The H NMR spectrum shows that the product
is relatively pure; however, minor peaks are seen at 6.8 and
9.3 ppm indicating a persistent impurity that resisted multiple
attempts to obtain absolutely clean material. Regardless, a
sharp melting point was obtained: mp 120-122 °C; 1H NMR
(DMSO-d6, 200 MHz) δ (ppm) 1.94 (s, 3H), 1.97 (s, 3H), 2.06
(s, 3H), 2.75 (s, 3H), 4.10-4.29 (m, 1H), 4.30-4.45 (m, 2H),
5.68 (t, 1H, J ) 5.4 Hz), 6.05 (t, 1H, J ) 6.0), 6.24 (d, 1H, J )
4.3 Hz), 7.10-7.39 (m, 3H), 7.40-7.52 (m, 2H), 8.50 (s, 1H),
11.20 (s, 1H); MS (FAB) MH+ calcd for C24H25N5O9S 559.5,
obsd 559.9.
N-[[9-(2′,3′,5′-Tr i-O-a cet yl-â-D-r ib ofu r a n osyl)-2-m et h -
ylth iop u r in -6-yl]ca r ba m oyl]-O-ter t-bu tyld im eth ylsilyl-L-
th r eon in e 2-(4-Nitr op h en yl)eth yl Ester (6). To a solution
of 5 (3.07 g, 5.48 mmol) in pyridine (20 mL) was added O-(tert-
butyldimethylsilyl)-L-threonine 2-(4-nitrophenyl)ethyl ester
(4.19 g, 10.97 mmol). The protected amino acid was synthe-
sized as described previously.25 The reaction mixture was
stirred at 35-40 °C for 12 h. At the end of this period, the
solvent was evaporated to dryness, and the residue was
coevaporated with toluene (2 × 15 mL). The pale yellow
residue was purified by flash chromatography using dichlo-
romethane and ethyl acetate (6:1) to afford 6 (3.5 g, 75%) as a
pale yellow solid: mp 67-69 °C; 1H NMR (DMSO-d6, 400 MHz)
δ (ppm) -0.05 (s, 3H), 0.04 (s, 3H), 0.82 (s, 9H), 1.19 (d, 3H,
J ) 6.2 Hz), 1.96 (s, 3H), 2.05 (s, 3H), 2.59 (s, 3H), 3.25 (t, 2H,
J ) 6.4 Hz), 4.18-4.42 (m, 7H), 5.63 (t, 1H, J ) 5.4 Hz), 6.08-
6.13 (m, 1H), 6.25 (d, 1H, J ) 5.5 Hz), 7.56 (d, 2H, J ) 7.5
Hz), 8.09 (d, 2H, J ) 7.5 Hz), 8.51 (s, 1H), 9.82 (d, 1H, J ) 6.5
C
57H75N7O12Si2S 1138.4, obsd 1138.1.
N-[[9-(2′-O-ter t-Bu tyldim eth ylsilyl-3′-(2-cyan oeth yl-N,N-
diisopr opylph osph or am idite)-5′-O-(4,4′-dim eth oxytr ityl)-
â-D-r ib ofu r a n osyl)-2-m et h ylt h iop u r in -6-yl]ca r b a m oyl]-
O-ter t-b u t yld im et h ylsilyl-L-t h r eon in e, 2-(4-Nit r op h en -
yleth yl) Ester (10). The above 5′-DMT, 2′-TBS derivative
(0.24 g, 0.21 mmol) and N,N-dimethylaminopyridine (5.1 mg,
0.042 mmol) were dissolved in dry THF under argon atmo-
sphere. To the reaction mixture were added sequentially
diisopropylethylamine (110 µL, 0.63 mmol) and 2-cyanoethyl-
N,N-diisopropylchlorophosphoramidite (94 µL, 0.42 mmol) with
stirring at 25 °C. After 30 min, a white solid was formed, and
the stirring was continued for an additional 3 h. The reaction
was followed by taking 50 µL of the aliquot of the reaction
mixture, removing the solvent, oxidizing for 5 min with
t-BuOOH/toluene, evaporating the toluene, and analysis by
silica gel TLC. The oxidized amidite product is found at the
origin, while unreacted starting material is not affected by the
oxidation procedure. This protocol is helpful since the starting
material and the products migrate with the identical Rf’s in
the solvent systems that we investigated. Upon completion,
the reaction mixture was extracted with ethyl acetate (50 mL),
washed with 5% sodium bicarbonate solution (25 mL), and
finally with water (2 × 25 mL). The organic layer was
Hz), 10.02 (s, 1H); HRMS (FAB) MH+ calcd for C36H50N7O13
SiS 848.2878, obsd 848.2910.
-
N-[[9-(â-D-Ribofu r a n osyl)-2-m eth ylth iop u r in -6-yl]ca r -
ba m oyl]-O-ter t-bu tyld im eth ylsilyl-L-th r eon in e 2-(4-Ni-
tr op h en yl)eth yl Ester (7). Compound 6 (3.5 g, 4.12 mmol)
was dissolved in 2 M ammonia in methanol (25 mL) and stirred
at room temperature for 2 h. The solvent was removed under
reduced pressure, and the residue was purified by flash
chromatography using a mixture of dichloromethane and
methanol (9:1) to afford 7 (2.15 g, 72%) as an off-white solid:
1
mp 78-79 °C; H NMR (DMSO-d6, 400 MHz) δ (ppm) -0.06
(s, 3H), 0.03 (s, 3H), 0.82 (s, 9H), 1.19 (d, 3H, J ) 6.2 Hz),
2.56 (s, 3H), 3.10 (t, 2H, J ) 6.4 Hz), 3.45-3.48 (m, 1H), 3.60-
3.72 (m,1H), 3.86-3.89 (m, 1H), 4.18-4.44 (m, 5H), 4.55-4.62
(m, 1H), 5.00 (t, 1H, J ) 4.7 Hz), 5.21 (d, 1H, J ) 4.3 Hz),
5.48 (d, 1H, J ) 5.2 Hz), 5.92 (d, 1H, J ) 5.2 Hz), 7.59 (d, 2H,
J ) 7.6 Hz), 8.15 (d, 2H, J ) 7.6 Hz), 8.61 (s, 1H), 9.30 (d, 1H,
J
C
) 6.6 Hz), 10.01 (s, 1H); HRMS (FAB) MH+ calcd for
30H44N7O10SiS 722.2639, obsd 722.2667.
J . Org. Chem, Vol. 67, No. 15, 2002 5357

Bajji and Davis
dried (Na2SO4) and filtered, and the solvent evaporated under
reduced pressure. The mixture was purified by flash chroma-
tography using a mixture of dichloromethane and ethyl acetate
(6:1) to yield the target nucleoside phosphoramidite as a
mixture of diastereomers (0.16 g, 57%): mp 77-81 °C; 31P
(DMSO-d6) δ (ppm) 149.14, 150.30. The 1H and 31P NMR
spectra are included in the Supporting Information and
indicate the product is homogeneous although the existence
of two diastereomers significantly complicates the 1H NMR
spectrum.
RNA Oligon u cleotid e Syn th esis. The oligonucleotide was
synthesized using an Applied Biosystems 394 oligonucleotide
synthesizer on a 1 µmol scale using 0.05 M acetonitrile
solutions of PAC amidites (PAC A, isopropyl PAC G, and acetyl
C) from Glen Research. The concentration of ms2t6A amidite
was 0.12 M. The phosphoramidites were coupled for 25 min.
The normal I2/H2O oxidation solution was replaced with 10%
t-BuOOH in acetonitrile, and 2 × 5 min oxidation cycles were
used for each coupling step.
trations of 5 µM in either sodium cacodylate or sodium
phosphate buffers, pH 7.4. The buffers contained 100 mM
NaCl, 0.1 mM EDTA, and either 0, 5, or 10 mM MgCl2. For
the temperature-dependent UV studies, the temperature was
raised from 10-90 °C in 1° steps with a 1 min equilibration
period between measurements. CD spectra were acquired in
a 1 cm water-jacketed CD cell. The spectra were acquired from
10 to 60 °C in 10° increments. The sample was allowed to
equilibrate for 10 min between measurements.
Molecu la r Mod elin g. A molecular model of ms2t6A was
built using Spartan 5.1.1 (Wavefunction, Inc.). The nucleoside
was constructed with a truncated sugar according to the
specification of Kollman and co-workers in order to ultimately
derive RESP charges for the AMBER force field.43 A prelimi-
nary low energy conformation was obtained using the AM1
force field in Spartan and then the geometry was optimized
by ab initio Hartree-Fock calculations with the 6-31G+ basis
set using Gaussian 98.44
RNA Dep r otection a n d P u r ifica tion . The CPG-bound
RNA was transferred from the column to a screw-cap glass
vial, to which was added 1 mL of 10% DBU in THF (v/v).25
The solution was magnetically stirred at 45 °C for 2 h. The
supernatant was decanted, the support was washed with an
additional 1.0 mL of THF, and the support was dried on a
Speed-Vac concentrator. The dried support was treated with
a solution of 33% ethanolic methylamine (Fluka) for 2 h. The
supernatant was decanted, the support was washed with an
additional 1.0 mL of dimethylamine solution, and the combined
washings were lyophilized on the Speed-Vac. The dried mate-
rial was dissolved in 1 mL of neat Et3N‚3HF and stirred at
room temperature for 9-12 h.42 The reaction was quenched
by adding 0.1 mL of water, and the RNA was precipitated by
adding 10 mL of n-butanol and allowing the solution to stand
at -20 °C for 6 h. After centrifugation, the RNA pellet was
dissolved in 0.5 mL of water and purified by anion-exchange
HPLC using a Pharmacia Resource Q anion-exchange column
(1 × 15 cm) and eluting with a linear gradient of lithium
perchlorate at 50 °C.36 The fractions containing full length
material as judged by HPLC were collected, lyophilized, and
then dialyzed against 3 × 1 L of deionized water. The dialyzed
material was lyophilized on a Speed-Vac and then directly
dissolved in the appropriate buffer for further characterization.
CD a n d UV Sp ectr oscop y. Temperature-dependent CD
and UV spectroscopy were conducted on RNA sample concen-
Ack n ow led gm en t. The work was supported by a
National Institutes of Health grant to D.R.D (GM55508),
by Grant Nos. RR06262 and RR13030 for NMR facili-
ties, and by Grant No. CA42014 to support the RNA
synthesis and mass spectrometry facilities.
Su p p or tin g In for m a tion Ava ila ble: RNA MALDI-MS,
HPLC trace, and NMR spectra for compounds 2, 4-10. This
material is available free of charge via the Internet at
http://pubs.acs.org.
J O025826T
(43) Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Gould, I. R.; Merz, K.
M.; Ferguson, D. M.; Spellmeyer, D. C.; Fox, T.; Caldwell, J . W.;
Kollman, P. A. J . Am. Chem. Soc. 1995, 117, 5179-5197.
(44) Frisch, M. J .; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J . R.; Zakrzewski, V. G.; Montgomery, J . A.;
Stratmann, R. E.; Burant, J . C.; Dapprich, S.; Millam, J . M.; Daniels,
A. D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J .; Barone, V.;
Cossi, M.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.; Ochterski,
J .; Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.; Salvador, P.;
Dannenberg, J . J .; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.;
Foresman, J . B.; Cioslowski, J .; Ortiz, J . V.; Baboul, A. G.; Stefanov,
B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Gomperts, R.;
Martin, R. L.; Fox, D. J .; Keith, T.; Al-Laham, M. A.; Peng, C. Y.;
Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; J ohnson, B.; Chen,
W.; Wong, M. W.; Andres, J . L.; Gonzalez, C.; Head-Gordon, M.;
Replogle, E. S.; Pople, J . A. Gaussian 98, Revision A.9; Gaussian,
Inc.: Pittsburgh, PA, 1998.
(42) Gasparutto, D.; Livache, T.; Bazin, H.; Dupla, A.; Guy, A.;
Khordin, A.; Molko, D.; Roget, A.; Teoule, R. Nucleic Acids Res. 1992,
20, 5159-5166.
5358 J . Org. Chem., Vol. 67, No. 15, 2002