Synthesis and characterization of the native anticodon domain of E. coli tRNA(Lys): Simultaneous incorporation of modified nucleosides mnm5s2U, t6A, and pseudouridine using phosphoramidite chemistry

Article abstract of DOI:10.1021/jo000338b

The anticodon domain of E. coli tRNA(Lys) contains the hypermodified nucleosides mnm⁵s²U and t⁶A at positions 34 and 37, respectively, along with a more common ψ at position 39. The combination of these three nucleotides represents one of the most extensively modified RNA domains in nature. 2-Cyanoethyl diisopropylphosphoramidites of the hypermodified nucleosides mnm⁵s²U and t⁶A were each synthesized with protecting groups suitable for automated RNA oligonucleotide synthesis. The 17 nucleotide anticodon stem-loop of E. coli tRNA(Lys) was then assembled from these synthons using phosphoramidite coupling chemistry. Coupling efficiencies for the two hypermodified nucleosides and for pseudouridine phosphoramidite were all greater than 98%. A mild deprotection scheme was developed to accommodate the highly functionalized RNA. High coupling yields, mild deprotection, and efficient HPLC purification allowed us to obtain 1.8 mg of purified RNA from a 1 μmol scale RNA synthesis. Our efficient synthetic protocol will allow for biophysical investigation of this rather unique tRNA species wherein nucleoside modification has been shown to play a role in codon-anticodon recognition, tRNA aminoacyl synthetase recognition, and programmed ribosomal frameshifting. The human analogue, tRNA(Lys,3), is the specific tRNA primer for HIV-1 reverse transcriptase and has a similar modification pattern.

Full text of DOI:10.1021/jo000338b

J . Org. Chem. 2000, 65, 5609-5614
5609
Syn th esis a n d Ch a r a cter iza tion of th e Na tive An ticod on Dom a in
of E. coli tRNALys: Sim u lta n eou s In cor p or a tion of Mod ified
Nu cleosid es m n m 5s2U, t6A, a n d P seu d ou r id in e Usin g
P h osp h or a m id ite Ch em istr y
Mallikarjun Sundaram, Pamela F. Crain, and Darrell R. Davis*
Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah, 84112-5820
davis@adenosine.pharm.utah.edu
Received March 8, 2000
The anticodon domain of E. coli tRNALys contains the hypermodified nucleosides mnm5s2U and t6A
at positions 34 and 37, respectively, along with a more common ψ at position 39. The combination
of these three nucleotides represents one of the most extensively modified RNA domains in nature.
2-Cyanoethyl diisopropylphosphoramidites of the hypermodified nucleosides mnm5s2U and t6A were
each synthesized with protecting groups suitable for automated RNA oligonucleotide synthesis.
The 17 nucleotide anticodon stem-loop of E. coli tRNALys was then assembled from these synthons
using phosphoramidite coupling chemistry. Coupling efficiencies for the two hypermodified
nucleosides and for pseudouridine phosphoramidite were all greater than 98%. A mild deprotection
scheme was developed to accommodate the highly functionalized RNA. High coupling yields, mild
deprotection, and efficient HPLC purification allowed us to obtain 1.8 mg of purified RNA from a
1 µmol scale RNA synthesis. Our efficient synthetic protocol will allow for biophysical investigation
of this rather unique tRNA species wherein nucleoside modification has been shown to play a role
in codon-anticodon recognition, tRNA aminoacyl synthetase recognition, and programmed ribosomal
frameshifting. The human analogue, tRNALys,3, is the specific tRNA primer for HIV-1 reverse
transcriptase and has a similar modification pattern.
The anticodon domain of E. coli tRNALys contains
hypermodified nucleosides at the wobble position (mnm5s2-
U34) and at the conserved purine immediately adjacent
to the anticodon (t6A37) in addition to a pseudouridine
(ψ39) at the base of the stem.1 These modifications are
critically important for correct function of this tRNA in
protein synthesis as undermodified tRNAs have poor or
altered mRNA binding,2,3 and are not properly recognized
by the cognate lysyl-tRNA aminoacyl synthetase.4,5 An-
ticodon modification also seems to be functionally im-
portant in programmed frameshifting required for ex-
pression of certain bacterial genes,6,7 and modification is
required for proper function of human tRNALys,3 as the
primer for HIV reverse transcriptase.8 In an attempt to
explain the novel biochemical properties of this tRNA, a
model of the tRNA anticodon domain has been proposed
where the two hypermodified nucleosides interact di-
rectly.9 However, the stem-loop shown in Figure 1 is
F igu r e 1. Secondary structure of the 17 nucleotide anticodon
domain of E.coli tRNALys and structures for the hypermodified
nucleosides.
* To whom correspondence should be addressed. Tel: 801-581-7006.
(1) Sprinzl, M.; Horn, C.; Brown, M.; Ioudovitch, A.; Steinberg, S.
Nucleic Acids Res. 1998, 26, 148-153.
(2) Von Ahsen, U.; Green, R.; Schroeder, R.; Noller, H. RNA 1997,
3, 49-56.
constrained in a manner distinctly different from the
model pentamer, and extensive biochemical and genetic
studies indicate the functional tRNA anticodon domain
includes the entire loop and at least the first two base
pairs of the stem.10,11
(3) Ashraf, S.; Sochacka, E.; Cain, R.; Guenther, R.; Malkiewicz, A.;
Agris, P. RNA 1999, 5, 188-194.
(4) Cusack, S.; Yaremchuk, A.; Tukalo, M. EMBO J . 1996, 15, 6321-
6334.
(5) Tamura, K.; Himeno, H.; Asahara, H.; Hasegawa, T.; Shimizu,
M. Nucleic Acids Res. 1992, 20, 2335-2339.
(6) Farabaugh, P.; Bjo¨rk, G. EMBO J . 1999, 18, 1427-1434.
(7) Mejlhede, N.; Atkins, J .; Neuhard, J . J . Bacteriol. 1999, 181,
2930-2937.
(8) Isel, C.; Lanchy, J .-M.; Le Grice, S. F. J .; Ehresmann, C.;
Ehresmann, B.; Marquet, R. EMBO J . 1996, 15, 917-924.
(9) Agris, P. F.; Guenther, R.; Ingram, P. C.; Basti, M. M.; Stuart,
J . W.; Sochacka, E.; Malkiewicz, A. RNA 1997, 3, 420-428.
The mnm5s2U and t6A nucleosides have previously
been incorporated into an RNA pentamer using the
(10) Yarus, M. Science 1982, 218, 646-652.
(11) Dao, V.; Guenther, R.; Malkiewicz, A.; Nawrot, B.; Sochacka,
E.; Kraszewski, A.; J ankowska, J .; Everett, K.; Agris, P. Proc. Natl.
Acad. Sci. U.S.A. 1994, 91, 2125-2129.
10.1021/jo000338b CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/08/2000

5610 J . Org. Chem., Vol. 65, No. 18, 2000
Sundaram et al.
phosphotriester method in solution.9 The nucleoside
phosphoramidite of the mnm5U nucleoside lacking the
2-thio group has also been reported,12 but no details were
provided about the RNA yields or physical characteriza-
tion of the RNA product. The amine protecting strategy
we employed was the same as reported previously for the
mnm5U and mnm5s2U nucleosides.12-14 The phosphora-
midite method has been used recently to synthesize a
tRNAfMet anticodon stem-loop containing t6A at position
37.15 The protecting strategy employed was similar to
that which we report with the exception that a base-labile
2-(4-nitrophenyl)ethyl group was used to protect the side
chain carboxylate. The general synthetic strategy we
describe will allow for biochemical study and high-
resolution structural determination of tRNALys as well
as other tRNA isoacceptors where either mnm5s2U or t6A
is found. Preliminary NMR data indicate the modified
anticodon domain is structurally distinct from both
unmodified tRNALys and the modified pentamer.9,16
Sch em e 1a
For compatibility with the standard RNA phosphora-
midite chemistry, the two functional group challenges of
mnm5s2U are the sulfur at position 2 and the secondary
amine side chain. Sulfur was introduced into the nucleo-
base of 2′,3′-isopropylidineuridine via H2S treatment
of the 5′,2-anhydro nucleoside formed under Mitsunobo
conditions to give 3.17 The 5-methylaminomethyl side
chain was elaborated in a series of high-yielding steps
and then protected as the base labile trifluoroacetate.17,18
The 2′,3′-acetonide was removed with mild acid to gener-
ate the N-protected nucleoside 5 (Scheme 1).18 Synthesis
of the protected phosphoramidite 6 was then accom-
plished using typical RNA phosphoramidite procedures.19
a
Key: (i) Ph3P, DEAD, 1,4-dioxane, 95%; (ii) H2S, pyridine,
90%; (iii) (CHO)n, 0.5 M NH3, 90%; (iv) TMS-Cl, 1,4-dioxane; (v)
CH3NH2, methanol, 60%; (vi) TMS-Cl, pyridine; TFAA, 70%; (vii)
20% acetic acid, 95%; (viii) DMT-Cl, pyridine, 90%; (ix) TBS-Cl,
imidazole, pyridine, 70%; (x) [(CH3)2CH]2NP(Cl)OCH2CH2CN,
DMAP, DIEA, THF, 90%.
Sch em e 2a
The hypermodified nucleoside t6A presents several
challenges for incorporation into oligonucleotides via
phosphoramidite chemistry. The secondary hydroxyl and
the carboxylate require protection, and the carbamoyl
linkage is relatively base-sensitive, requiring some care
if one chooses to use the commercial A, C, and G RNA
phosphoramidites having base labile amine protecting
groups. The t6A nucleoside was synthesized according to
literature procedures.20 Compound 9 was obtained by
TBS-OTf treatment21 of the sugar-protected t6A nucleo-
side, then selective hydrolysis of the labile TBS-ester. The
carboxylate was protected as the stable TMSE alkyl
ester22 followed by selective removal of the acetates20 to
give 10 (Scheme 2). Elaboration of 10 to phosphoramidite
11 proceeded in high yield by the same general procedure
as for 6 to give an overall yield of 2.2% from adenosine.
a
Key: (i) (CH3CO)2O, pyridine, 95%; (ii) ethylchloroformate,
pyridine, 75%; (iii) L-threonine, pyridine, 90%; (iv) TBS-OTf, Et3N,
CH2Cl2, 95%; (v) 2 M NH3/methanol, 95%; (vi) DCC, TMSE-OH,
pyridine, 91%; (vii) 2 M NH3/methanol, 90%; (viii) DMT-Cl,
pyridine, 90%; (ix) TBS-Cl, imidazole, pyridine, 75%; (x) [(CH3)2CH]2-
NP(Cl)OCH2CH2CN, DMAP, DIEA, THF, 90%.
(12) Agris, P. F.; Malkiewicz, A.; Kraszewski, A.; Everett, K.;
Nawrot, B.; Sochacka, E.; J ankowska, J .; Guenther, R. Biochimie 1995,
77, 125-134.
(13) Malkiewicz, A.; Sochacka, E. Tetrahedron Lett. 1983, 24, 5387-
5390.
(14) Malkiewicz, A.; Sochacka, E. Tetrahedron Lett. 1983, 24, 5391-
5394.
The fully modified anticodon domain in Figure 1 was
synthesized using commercial PAC-protected phosphora-
midites (Glen Research) and standard coupling chemis-
try19 with the exception of tBuOOH oxidation.23-25 Trityl
assays indicated >98% coupling for all phosphoramidites;
this was verified by HPLC and MALDI/MS of the crude,
deprotected RNA. To avoid reaction of the secondary
(15) Boudou, V.; Langridge, J .; van Aerschot, A.; Hendrix, C.; Millar,
A.; Weiss, P.; Herdewijn, P. Helv. Chim. Acta 2000, 83, 152-161.
(16) Durant, P. C.; Davis, D. R. J . Mol. Biol. 1999, 285, 115-131.
(17) Malkiewicz, A. J .; Nawrot, B.; Sochacka, E. Z. Naturforsch.
1987, 42b, 360-366.
(18) Ikeda, K.; Tanaka, S.; Mizuno, Y. Chem. Pharm. Bull. 1975,
23, 2958-2964.
(19) Damha, M. J .; Ogilvie, K. K. Methods Mol. Biol. 1993, 20, 81-
114.
(20) Hong, C. I.; Chheda, G. B. In Nucleic Acid Chemistry; Towsend,
L. B., Tipson, R. S., Eds.; Wiley: New York, 1972; pp 661-664.
(21) Mander, L. N.; Sethi, S. P. Tetrahedron Lett. 1984, 25, 5953-
5956.
(23) J ager, A.; Engels, J . Tetrahedron Lett. 1984, 25, 1437-1440.
(24) Hayakawa, Y.; Uchiyama, M.; Noyori, R. Tetrahedron Lett.
1986, 27, 4191-4194.
(22) Seiber, P. Helv. Chim. Acta 1977, 60, 2711-2716.
(25) Kumar, R. K.; Davis, D. R. J . Org. Chem. 1995, 60, 7726-7727.

Synthesis of the Anticodon Domain of E. coli tRNALys
J . Org. Chem., Vol. 65, No. 18, 2000 5611
U35 and U36, a key feature of a “U-turn” structure.33,35
The chemical shift of this peak is rather unique and is
downfield of a merely slow exchanging imino, consistent
with our interpretation of the imino-phosphate interac-
tion. The published spectra of tRNAPhe have an almost
identical resonance and we have unambiguously assigned
the imino spectrum of a tRNALeu anticodon domain which
has a strong peak for the U33-NH to phosphate interac-
tion. These analogous studies, the chemical shift argu-
ments, and the exchange behavior argue in favor of our
assignment for tRNALys, but the broad peak has pre-
vented us from obtaining direct NOE confirmation.
Additional support for our assignment and interpretation
comes from the 31P NMR spectrum (Figure 2b) which
shows a downfield shifted phosphorus assigned to the
U33-p-mnm5s2U34 phosphate.36-38 Earlier work from our
laboratory showed that in the unmodified tRNALys anti-
codon, no U-turn is present.16 These results clearly show
that the hypermodified nucleosides in tRNALys have a
dramatic effect on the structure of this tRNA. In addition
to the imino protons, we have used standard NMR
experiments32 to completely assign the base and sugar
protons, including stereospecific assignments for some of
the H5′, H5′′ protons. A number of NOE cross-peaks
supporting the U-turn structure39 are seen in the 2D
NOESY spectrum (Supporting Information). The efficient
chemical synthesis methodology we have presented will
enable us to pursue the high-resolution NMR structure
of the anticodon domain and to investigate the structural
effects of the individual mnm5s2U and t6A hypermodified
nucleosides.
F igu r e 2. (a) 1H NMR imino spectrum of the native anticodon
stem-loop of E. coli tRNALys; (b) 31P NMR spectrum showing
the downfield resonance characteristic of a “U-turn” between
U33 and mnm5s2U34. Sample conditions were 1.0 mM RNA
in 0.25 mL of 10 mM sodium phosphate, pH 6.8 containing 50
mM NaCl, 50 mM KCl, 10 mM MgCl2, and 5% deuterium
oxide.
amine of mnm5s2U with acrylonitrile generated during
base-deprotection26 we used a two-step deprotection
procedure wherein the column bound RNA is treated first
with tert-butylamine and then with NH3/ethanol.27 After
ammonia treatment the RNA solution was dried, then
treated with Et3N‚HF to remove the TBS ethers and the
TMS-ethyl ester.28 The fully deprotected RNA was then
purified by HPLC.29 MALDI-MS was used to verify that
the purified oligonucleotides had the correct molecular
weight. The presence of the major and modified nucle-
otides was verified by digesting the RNA to mononucleo-
sides and then analyzing the digest by LC/MS.30,31
Exp er im en ta l Section
Gen er a l Meth od s. Melting points are uncorrected. 1H and
31P NMR spectra were collected in deuterated solvents on
1
either 200 or 500 MHz spectrometers. H chemical shifts are
1
reported relative to tetramethylsilane at 0.0 ppm and refer-
enced to the residual proton signal of the deuterated solvent.
31P chemical shifts are relative to external phosphoric acid at
0.0 ppm. Analytical thin-layer chromatography (TLC) was
carried out on Merck silica gel 60F-254 plates. Column
chromatography was carried out with Merck silica gel 60 (230-
400 mesh). Chemical reagents were purchased from Aldrich
except where noted and used without further purification.
Spectral grade acetonitrile (Baxter) was used for HPLC.
Instrumentation and sample preparation for MALDI MS and
ESI LC/MS are described below. Elemental analysis was done
by Galbraith Laboratories (Knoxville, TN).
The H NMR spectrum in Figure 2a shows the down-
field, imino spectral region where N-H bound protons
in slow exchange with water resonate.32 There are a
number of distinctive features evident for the tRNALys
hairpin. The three strong peaks at 13.23, 12.38, and 11.75
are from the central base pairs of the stem. The most
downfield resonance is for the N3-H proton of ψ39,
which forms a Watson-Crick base pair with A31.16 The
resonance at 10.64 is the ψ39 N1-H proton, while the
peaks at 9.94, and 8.85 are the amide and H6 protons of
1
t6A. Resonances are seen in both the H and 31P NMR
5′-O-(4,4′-Dim eth oxytr ityl)-5-(N-tr iflu or oacetyl)m eth yl-
a m in om eth yl-2-th iou r id in e (5a ). 2′,3′-O-Isopropylidene-2-
thiouridine (3) was synthesized from 2′,3′-O-isopropylideneu-
ridine (2) (Aldrich) by the method of Malkiewicz et al.17
5-Methylaminomethyl-2-thiouridine was synthesized as de-
scribed by Ikeda et al.18 and the secondary amino side chain
protected as the trifluoroacetate to give the N-protected
nucleoside 5.17 Intermediate 5 (1.0 g, 2.5 mmol) was dissolved
in 25 mL of dry pyridine and 4,4′-dimethoxytrityl chloride (1.05
g, 3.12 mmol, 1.25 equiv) added to the stirred solution. The
reaction was continued for 12 h and then diluted with 75 mL
of CH2Cl2 and washed with 2 × 20 mL of 5% NaHCO3 and 20
mL of saturated NaCl. The organic layer was dried over
sodium sulfate and solvent removed under reduced pressure.
spectra that indicate a stable structure reminiscent of
1
the yeast tRNAPhe crystal structure.33,34 In the H spec-
trum a weak peak is seen at 11.10 ppm for the U33
N3-H proton hydrogen bonded to the phosphate between
(26) Griffey, R. H.; Monia, B. P.; Cummins, L. L.; Freier, S.; Greig,
M. J .; Cuinoso, C. J .; Lesnik, E.; Manalili, S. M.; Mohan, V.; Owens,
S.; Ross, B. R.; Sasmor, H.; Wancewicz, E.; Weiler, K.; Wheeler, P. D.;
Cook, P. D. J . Med. Chem. 1996, 39, 5100-5109.
(27) Sinha, N. D.; Beirnat, J .; McManus, J .; Koster, H. Nucleic Acids
Res. 1984, 12, 4539-4557.
(28) 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.
(29) Sproat, B.; Colonna, F.; Mulla, B.; Tsou, D.; Andrus, A.; Hampel,
A.; Vinayak, R. Nucleosides Nucleotides 1995, 14, 255-273.
(30) Crain, P. F. Methods Enzymol. 1990, 193, 782-790.
(31) Pomerantz, S. C.; McCloskey, J . A. Methods Enzymol. 1990,
193, 796-824.
(32) Varani, G.; Tinoco, I. Quart. Rev. Biophys. 1991, 24, 479-532.
(33) Quigley, G. J .; Rich, A. Science 1976, 194, 794-806.
(34) J ack, A.; Ladner, J . E.; Klug, A. J . Mol. Biol. 1976, 108, 619-
649.
(35) Clore, G.; Gronenborn, A.; Piper, E.; McLaughlin, L.; Graeser,
E.; Van Boom, J . Biochem J . 1984, 221, 737-751.
(36) J ucker, F. M.; Pardi, A. RNA 1995, 1, 219-222.
(37) Legault, P.; Pardi, A. J . Magn. Reson. B 1994, 103, 82-86.
(38) Schweisguth, D. C.; Moore, P. B. J . Mol. Biol. 1997, 267, 505-
519.
(39) Schweisguth, D.; Moore, P. J . Mol. Biol. 1997, 267, 505-519.

5612 J . Org. Chem., Vol. 65, No. 18, 2000
Sundaram et al.
The residual pyridine was removed by coevaporation with dry
toluene, and the resulting foam carried forward.
equiv) and triethylamine (5.6 mL, 37.2 mmol, 5 equiv) in 100
mL of dry dichloromethane.21 The reaction mixture was stirred
for 4 h, at which time TLC indicated that both the secondary
hydroxyl and the carboxyl group were converted to their TBS
derivatives. The reaction mixture was diluted with 50 mL of
dichloromethane and washed with cold, 5% sodium bicarbon-
ate. The organic layer was dried over anhydrous sodium
sulfate and solvent removed under reduced pressure. The
residue was triturated with dry ether, separating the soluble
product from insoluble triethylammonium triflate. The ether
solution was then dried over anhydrous sodium sulfate. The
ether was removed under reduced pressure and the dry
product treated with 25 mL of 2 M ammonia in methanol for
1 min to remove the labile TBS-ester. The ammonia solution
was concentrated under reduced pressure to give 4.3 g of the
dry product 9 (95% yield). This crude material was carried to
the next step without purification.
5′-O-(4,4′-Dim eth oxytr ityl)-2′-O-(ter t-bu tyld im eth ylsi-
lyl)-5-(N-tr iflu or oa cetyl)m eth yla m in om eth yl-2-th iou r i-
d in e. The 5′-DMT nucleoside (1.0 g, 1.42 mmol, 1.0 equiv) was
dissolved in 12 mL of dry pyridine under Ar atmosphere. To
this were added imidazole (0.22 g, 3.55 mmol, 2.5 equiv) and
tert-butyldimethylsilyl chloride (0.238 g, 1.78 mmol, 1.25
equiv). The reaction mixture was stirred for 24 h, at which
time the starting material was converted to a nearly equimolar
mixture of 2′ and 3′ TBS products. The reaction was diluted
with 75 mL of CH2Cl2 and extracted with 5% sodium bicarbon-
ate. The organic layer was dried over sodium sulfate and
solvent removed under reduced pressure. The 2′ TBS isomer
was isolated by flash chromatography on silica gel using CH2-
Cl2: ethyl acetate (19:1) to yield 0.5 g, 44% of a white foam.
The 2′ and 3′ isomers have Rf’ s of 0.4 and 0.3 respectively on
silica gel TLC. A 2D COSY NMR experiment was used to
confirm the identities of each isomer from the correlations from
H2′ to H2′OH for the 3′ TBS isomer and from the H3′ to H3′OH
for the 2′ TBS isomer. The 3′ TBS compound could be isomer-
ized to an equimolar mixture of 2′ and 3′ isomers by stirring
in methanol with a trace of triethylamine. This isomerization
and chromatography was repeated twice in order to obtain
additional quantities of the pure 2′ TBS isomer for an overall
yield of 70%: mp 96-98 °C; 1H NMR (DMSO-d6) δ 0.1 (s, 3H),
0.2 (s, 3H), 0.85 (s, 9H), 2.5 (d, 3H), 3 (d, 2H), 3.2 (s, 3H), 3.3
(s, 3H) 5.0 to 4.3 (m, 5H), 6.6 (d, 1H), 6.90 (d, 4H), 7.30 (m),
7.8 (s, 1H), 12.8 (s, 1H); HRMS (FAB) MH+ calcd for
(2,3,5-Tr i-O-a cetyl-N-[(9-â-D-Ribofu r a n osyl-9H-p 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
Tr im eth ylsilyleth yl Ester (9a ). Compound 9 (2.5 g, 3.82
mmol, 1 equiv) was dissolved in 20 mL of anhydrous pyridine.
DCC (987 mg, 4.8 mmol, 1.25 equiv) was added, and the
mixture was stirred at room temperature for 5 min. To this
was added 2-trimethylsilylethanol (700uL, 4.8 mmol, 1.25
equiv) and the reaction mixture stirred for 18 h, at which time
TLC indicated the reaction was complete.22 The reaction
mixture was diluted with 100 mL ethyl acetate and washed
with saturated sodium bicarbonate. The organic layer was
dried over sodium sulfate and solvent removed under reduced
pressure. The crude product was purified by flash chromatog-
raphy on silica gel using EtOAc/CH2Cl2 (1:1) to yield 2.6 g
(91%) of the protected nucleoside as a white foam: mp 96-98
C
C
40H48N3O8Si1S1F3 816.2962, obsd 816.2932. Anal. Calcd for
40H48N3O8Si1S1F3: C, 58.95; H, 5.81; N, 5.15. Found: C,
58.78; H, 6.22; N, 4.91.
1
°C; H NMR (Me2SO-d6) δ 0.0 (s, 9H), 0.2 (s, 3H), 0.3 (s, 3H),
5′-O-(4,4′-Dim eth oxytr ityl)-2′-O-(ter t-bu tyld im eth ylsi-
lyl)-5-(N-tr iflu or oa cetyl)m eth yla m in om eth yl-2-th iou r i-
din e-3′-(cyan oeth yl)-N,N-diisopr opylph osph or am idite (6).
The 5′-DMT, 2′-TBS nucleoside (0.60 g, 0.74 mmol, 1.0 equiv)
was dissolved in 12 mL of dry THF under Ar atmosphere. To
this were added DMAP (0.018 g, 0.14 mmol, 0.2 equiv) and
diisopropylethylamine (0.316 mL, 1.62 mmol, 2.2 equiv). The
solution was stirred while adding 2-cyanoethyl N,N-diisopro-
pylphosphonamidic chloride (0.328 mL, 1.48 mmol, 2.0 equiv).
After 30 min, a white precipitate formed, and the stirring was
continued for 3 h. The reaction was followed by taking a 50
µL aliquot of the reaction mixture, removing the solvent,
oxidizing for 3 min with tBuOOH/toluene, followed by evapo-
ration and then silica gel TLC. The amidite product is found
at the origin while the unreacted starting material is unaf-
fected by the oxidation procedure. This protocol is very helpful
since the starting material and products migrate with identical
Rf in the solvent systems we investigated. The reaction was
quenched by adding 100 mL of ethyl acetate, and then
extracting with saturated NaHCO3 followed by saturated
NaCl. The organic layer was separated, dried over anhydrous
sodium sulfate and the solvent was removed under reduced
pressure. The residue was purified by flash chromatography
on silica gel using CH2Cl2/acetonitrile (18:1) to yield 0.47 g
(64%) of compound 6 as a white foam. The material exists as
1:1 ratio of stereoisomers about phosphorus and two chemical
shifts are observed for some of the NMR resonances. The
secondary shifts are indicated in parentheses: mp 88-90 °C;
1H NMR (acetone-d6): δ 0.1(0.2) (s, 3H), 0.3(0.4) (s, 3H), 0.85-
(0.9) (s, 9H), 4.7 to 2.5 (m, 18Η), 7.6 to 6.6(m, 14H), 7.9(8.0)
(s, 1H); 31P NMR (acetone-d6) 155.79, 155.57; HRMS (FAB)
MH+ calcd for C49H66N5O9Si1S1F3P1 1016.4040, obsd 1016.3991.
Anal. Calcd for C49H66N5O9Si1S1F3,P1: C, 57.97; H, 6.35; N,
6.89. Found: C, 57.59, H; 6.49; N, 5.98.
0.8 (s, 9H), 1.0 (d, 4H), 1.2 (d, 3H), 1.95 (s, 3H), 2.0 (s, 3H),
2.05 (s, 3H), 5.0 to 4.3 (m, 5H), 5.65 (t, 1H), 6.05 (t, 1H), 6.3
(d, 1H), 8.4 (s, 1H), 8.65 (s, 1H), 9.85 (d, 1H), and 10.05 (s,
1H); HRMS (FAB) MH+ calcd for C32H52N6O11Si2 753.3311,
obsd 753.3285.
N-[(9-â-D-Ribofu r a n osyl-9H-p u r in -6-yl)ca r ba m oyl)]-O-
ter t-bu tyld im eth ylsilyl-L-th r eon in e Tr im eth ylsilyleth yl
Ester (10). The triacetyl protected nucleoside (4.6 g, 6.1 mm,
1 equiv) was dissolved in 100 mL of 2 M ammonia in methanol
and the solution stirred for 6 h at room temperature and then
evaporated to dryness under reduced pressure. Crude yield:
3.6 g (95%). The reaction was quite clean as judged by thin-
layer chromatography, and for preparative purposes this ma-
terial was carried forward without purification. Analytical
samples were prepared by flash chromatography on silica gel
using EtOAc/methanol (98:2): mp 102-104 °C; 1H NMR
(Me2SO-d6): δ 0.0 (s, 9H), 0.2 (s, 3H), 0.3 (s, 3H), 0.8 (s, 9H),
1.0 (d, 4H), 1.2 (d, 3H), 5.0 to 4.3 (m, 5H), 5.65 (t, 1H), 6.05 (t,
1H), 6.3 (d, 1H), 8.4 (s, 1H), 8.65 (s, 1H), 9.85 (d, 1H), and
10.05 (s, 1H); MS (FAB) MH+ 627.2, calcd for C26H46N6O8Si2
627.9.
5′-O-(4,4′-Dim eth oxytr ityl)-N-[(9-â-D-r ibofu r a n osyl-9H-
p u r in -6-yl)ca r ba m oyl)]-O-ter t-bu tyld im eth ylsilyl-L-th r e-
on in e Tr im eth ylsilyleth yl Ester (10a ). Protected nucleoside
10 (1 g, 1.6 mmol, 1 equiv) was dissolved in 20 mL of
anhydrous pyridine under Ar atmosphere. 4,4′-Dimethoxytrityl
chloride (750 mg, 2.2 mmol, 1.4 equiv) was added, and the
mixture was stirred at room temperature for 5 h. The reaction
mixture was diluted with 100 mL of CH2Cl2 and washed with
saturated sodium bicarbonate. The organic layer was dried
over sodium sulfate and solvent removed under reduced
pressure. The crude product was purified by flash chromatog-
raphy on silica gel using EtOAc/MeOH (20:1) to yield 1.4 g
(95%) of the 5′-DMT nucleoside as white foam: 1H NMR
(Me2SO-d6) δ 0.0 (s, 9H), 0.2 (s, 3H), 0.3 (s, 3H), 0.8 (s, 9H),
1.0 (d, 4H), 1.2 (d, 3H), 3.2 (s, 3H), 3.3 (s, 3H) 5.0 to 4.3 (m,
5H), 5.65 (t, 1H), 6.05 (t, 1H), 6.3 (d, 1H), 6.90 (d, 4H), 7.30
(m, 9H), 8.4 (s, 1H), 8.65 (s, 1H), 9.85 (d, 1H), and 10.05 (s,
1H); MS (FAB) MH+ 928.4, calcd for C47H64N6O10Si2 929.0.
(2,3,5-Tr i-O-a cetyl-N-[(9-â-D-Ribofu r a n osyl-9H-p u r in -
6-yl)ca r ba m oyl])-L-th r eon in e. The modified nucleoside t6A
was synthesized as the 2′, 3′, 5′-triacetate according to
literature procedures.20 1H NMR spectra were consistent with
that previously reported and mass spectrometry was used to
confirm the identity of the starting material.
5′-O-(4,4′-Dim eth oxytr ityl)-2′-O-(ter t-bu tyld im eth ylsi-
lyl)-N-[(9-â-D-r ib ofu r a n osyl-9H -p u r in -6-yl)ca r b a m oyl)]-
O-ter t-bu tyld im eth ylsilyl-L-th r eon in e Tr im eth ylsilyleth -
yl Ester (10b). The 5′-DMT nucleoside 10a (1.4 g, 1.5 mm, 1
(2,3,5-Tr i-O-a cetyl-N-[(9-â-D-Ribofu r a n osyl-9H-p 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
(9). tert-Butyldimethylsilyl triflate (7.6 mL, 29.6 mm, 4 equiv)
was added to a solution of t6A triacetate (4 g, 7.4 mmol, 1

Synthesis of the Anticodon Domain of E. coli tRNALys
J . Org. Chem., Vol. 65, No. 18, 2000 5613
F igu r e 3. Ion- exchange HPLC of the native E. coli tRNALys stem-loop. HPLC was done at 60 °C using a 1 × 15 cm Pharmacia
Resource Q column. The mobile phase was 0.02 M ammonium acetate and the RNA eluted with a gradient of 20-600 mM LiClO4.
(a) The crude material obtained using the standard, mild deprotection conditions commonly used for PAC protected RNA. There
are multiple “full-length” species corresponding to material where the t6A side chain has been lost, and where the methylamino
side chain is adducted with acrylonitrile; (b) an analytical (10 µg) injection of crude material after deprotection with the optimized
protocol; (c) an analytical trace of the pooled, lyophilized, and dialyzed RNA used for NMR.
equiv.) was dissolved in 25 mL of anhydrous pyridine under
Ar atmosphere. To this were added imidazole (260 mg, 4.2 mm,
2.8 equiv) and tert-butyldimethylsilyl chloride (281 mg, 2.1
mm, 1.4 equiv). The reaction mixture was stirred for 24 h, at
which time the starting material was converted to a nearly
equimolar mixture of 2′ and 3′ TBS products. The reaction
mixture was diluted with 100 mL of CH2Cl2 and washed with
saturated sodium bicarbonate. The organic layer was dried
over sodium sulfate and solvent removed under reduced
pressure. The crude product was purified by flash chromatog-
raphy on silica gel using EtOAc/CH2Cl2 (6:1) to yield 703 mg
(45%) of the 5′-DMT, 2′-TBS nucleoside as a white foam. The
2′ and 3′ isomers have a Rf’s of 0.4 and 0.2 respectively on
silica gel TLC with EtOAc/CH2Cl2 (6:1) as the eluting solvent.
The identities were confirmed by 2D COSY NMR as described
for the mnm5s2U nucleoside. The 3′ TBS compound can be
isomerized to an equimolar mixture of 2′ and 3′ isomers by
5′-O-(4,4′-Dim eth oxytr ityl)-2′-O-(ter t-bu tyld im eth ylsi-
lyl)-N-[(9-â-D-r ib ofu r a n osyl-9H -p u r in -6-yl)ca r b a m oyl)]-
O-ter t-bu tyld im eth ylsilyl-L-th r eon in etr im eth ylsilyleth -
yl Ester 3′-(Cyan oeth yl N,N-diisopr opylph osph or am idite)
(11). 5′-DMT, 2′-TBS-t6A (550 mg, 0.53 mmol, 1 equiv) was
dissolved in 10 mL of anhydrous THF under Ar atmosphere.
To this were added DMAP (13 mg, 0.11 mmol, 0.2 equiv) and
diisopropylethylamine (186 µL, 1.06 mmol, 2 equiv). The
reaction mixture was stirred for 5 min before adding 2-cya-
noethyl N,N-diisopropylphosphoramidic chloride (237 µL, 1.06
mmol, 2 equiv). After 10 min a white precipitate was formed,
and the stirring continued for 3 h. The reaction was followed
by taking a 50 µL aliquot of the reaction mixture, removing
the solvent, oxidizing for 3 min with tBuOOH/toluene, followed
by evaporation and then silica gel TLC. The oxidized amidite
product is found at the origin while the unreacted starting
material is unaffected by the oxidation procedure. This protocol
is helpful since the starting material and the products migrate
with identical RF in the solvent systems that we investigated.
The reaction mixture was diluted with 50 mL of dichlo-
romethane and washed with 5% sodium bicarbonate. The
organic layer was dried over sodium sulfate and solvent
removed under reduced pressure. The crude product was
purified by flash chromatography on silica gel using EtOAc:
CH2Cl2 (6:1) to yield 526 mg (80%) of compound 11 as a white
foam. The material exists as a 1:1 ratio of stereoisomers about
stirring in methanol with
a trace of triethylamine. The
isomerization and chromatography was repeated twice to
obtain additional, pure 2′-TBS material: mp 94-96 °C; 1H
NMR (Me2SO-d6) δ -0.2 (s, 3H), -0.1 (s, 3H), 0.0 (s, 9H), 0.2
(s, 3H), 0.3 (s, 3H), 0.75 (s, 9H), 0.85 (s, 9H), 1.0 (d, 4H), 1.2
(d, 3H), 3.2 (s, 3H), 3.3 (s, 3H), 5.0 to 4.3 (m, 5H), 5.65 (t, 1H),
6.05 (t, 1H), 6.3 (d, 1H), 6.90 (d, 4H), 7.30 (m, 9H), 8.4 (s, 1H),
8.65 (s, 1H), 9.85 (d, 1H), and 10.05 (s, 1H); HRMS (FAB) MH+
calcd for C53H79N6O10Si3 1043.5166, obsd 1043.5175.

5614 J . Org. Chem., Vol. 65, No. 18, 2000
Sundaram et al.
phosphorus and two chemical shifts are observed for some of
the resonances. The secondary shifts are indicated in the
parenthesis: 1H NMR (acetone-d6) δ -0.2 (s, 3H), -0.1 (s, 3H),
0.0 (s, 9H), 0.2 (s, 3H), 0.3 (s, 3H), 0.75 (s, 9H), 0.85 (s, 9H),
1.0 (d, 4H), 1.2 (d,3H), 3.2 (s, 3H), 3.3 (s, 3H), 4.3 to 3.8 (m,
14H), 5.0 to 4.3 (m, 5H), 5.6 (5.65) (t, 1H), 5.8 (5.85) (t, 1H),
6.7 (d, 1H), 6.90 (d, 4H), 7.30 (m, 9H), 8.8 (8.85) (s, 1H),
8.9 (8.95) (s, 1H), and 10.55 (d, 1H); 31P NMR (acetone-d6)
154.74, 153.15. HRMS (FAB) MH+ calcd for C62H97N8O11Si3P1
1243.6244, obsd 1243.6143. The purity was verified by 1H
NMR as shown in Figure S8 (Supporting Information).
Sa m p le P r ep a r a tion for MALDI. All matrix compounds
used in this work were purchased from Sigma Chemical Co.
and were used without further purification. Matrix and co-
matrix solutions were prepared as follows. 0.04 g of 6-aza-2-
thiothymine (ATT matrix) was added to 500 µL of 50% ACN
and mixed on a Vortex mixer for 30 s. Ammonium citrate co-
matrix solution was prepared by dissolving 0.023 g of am-
monium citrate in 1 mL of water. Oligonucleotide concentra-
tions for most of the samples were ∼10 pmol/µL. For each
spectrum, 0.5 µL of the sample solution was spotted onto a
gold plated sample well and allowed to air-dry. A 0.5 µL aliquot
of co-matrix solution was added to the dry sample and allowed
to air-dry. A 0.5 µL aliquot of matrix solution was then added
to the sample/co-matrix mixture in the sample well. After the
sample had again dried, the MALDI probe was inserted into
the mass spectrometer.
MALDI Ma ss Sp ect r om et r y. MALDI spectra were
obtained on a reflectron MALDI/TOF mass spectrometer
(Model: Perseptive Voyager-DE STR, PE Applied Biosystems
Co., Foster City, CA) equipped with a delayed extraction ion
source, and a pulsed linear detector. A nitrogen laser at 337
nm (3 ns pulse width) was used to desorb the ions in the source
region. The time-of-flight data were either externally cali-
brated or mass converted using ion peaks of known masses.
Background pressure within the instrument was less than 1
× 10-7 Torr as measured by a Bayard-Alpert ion gauge located
below the source.
Com bin ed Liqu id Ch r om a togr a p h y/Ma ss Sp ectr om -
etr y (LC/MS) An a lysis of th e Nu cleosid e Con ten t of
Oligon u cleotid e 1. The fully modified oligonucleotide 1 was
digested to nucleosides using nuclease P1, phosphodiesterase
I, and alkaline phosphatase.30 The resulting nucleoside mix-
ture was analyzed by LC/MS using a HP 1090 liquid chro-
matograph interfaced to a Fisons Quattro II triple quadrupole
mass spectrometer (Manchester, U.K.) using electrospray
ionization. As shown in Figure S2, the HPLC elution monitored
at 260 nm indicates that the RNA oligonucleotide contains the
4 major nucleosides as well as ψ (3.78 min), mnm5s2U (13.89
min), and t6A (19.89 min). The seven nucleosides in this sample
elute with the correct retention times and their identities were
verified by mass spectrometry as they eluted.31
Oligon u cleotid e Syn th esis. The oligoribonucleotides were
synthesized on an Applied Biosystems 394 oligoribonucleotide
synthesizer on a 1 µmole scale using 0.05 M acetonitrile
solutions of PAC amidites (PAC A, isopropyl PAC G, and acetyl
C) from Glen Research. The concentrations of t6A and mnm5s2U
amidites were 0.12 M each. The phosphoramidites were
coupled for 25 min. The normal I2/H2O oxidation solution was
used up until the mnm5s2U nucleoside and then 2 × 5 min
oxidation cycles with 10% tBuOOH in ACN was used from that
point forward.
RNA Dep r otection a n d P u r ifica tion . The CPG-bound
RNA sequences were transferred from the column to a screw
cap glass vial, to which was added 1 mL of 10% tert-butylamine
in dry pyridine.27 The solution was stirred at rt for 1 h. The
solvent was removed in vacuo, and the residue was treated
with 3 mL of NH4OH/ethanol (3:1 v/v) at rt for 1 h and at 55
°C for 4 h. The supernatant was decanted, the support material
washed with an additional 1.0 mL of NH4OH/ethanol, and the
combined washings were lyophilized on a Speed-Vac concen-
trator. The dried material was dissolved in 1 mL of neat Et3N‚
3HF (Aldrich) and stirred at room temperature for 9-12 h.28,40
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. The
precipitated RNA was recovered by centrifugation and dried
under reduced pressure in a Speed-Vac concentrator. The
residue was then dissolved in 1 mL of 1 M tetrabutylammo-
nium fluoride in THF and stirred at rt for 12-18 h.19 The
solution was passed through a NAP-25 (Pharmacia) size
exclusion column to remove the tetrabutylammonium coun-
terions. The RNA-containing eluate (as monitored by UV
absorption at 260 nm) was lyophilized and then purified by
anion exchange HPLC (Figure 3).29
Ack n ow led gm en t. This research was supported by
the NIH (GM55508 and GM29812). NMR, MS, and
oligonucleotide facilities are supported by NIH grants
CA42014 and RR06262. We thank Sherri Manalili for
advice regarding amino-containing nucleosides. The
HIV-1 virion electron micrograph on the cover was
provided by Wes Sundquist, and the tRNAPhe figure was
made from PDB file 1TRA with the program RIB-
BONS.41
Fractions containing full length material as judged by HPLC
were collected, lyophilized, and then dialyzed against 2 × 1 L
of 0.1 M NaCl, 0.1 mM EDTA, and then against 2 × 1 L of
deionized water. The dialyzed material was lyophilized to yield
1.8 mg of RNA, a yield of 32% based on a theoretical column
loading of 1 micromole.
Su p p or tin g In for m a tion Ava ila ble: MALDI-MS spec-
trum, LC/MS nucleoside analysis, and 2D NOESYs for oligo-
nucleotide 1. 1H NMR spectra for 5a , 5b, 10a , and 11. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(40) Wincott, F.; DiRenzo, A.; Shaffer, C.; Grimm, S.; Tracz, D.;
Workman, C.; Sweedler, D.; Gonzalez, C.; Scaringe, S.; Usman, N.
Nucleic Acids Res. 1995, 23, 2677-2684.
J O000338B
(41) Carson, M. J . Appl. Crystallogr. 1991, 24, 958-961.