Design, synthesis, and analysis of yeast tRNAPhe analogs possessing intra- and interhelical disulfide cross-links

Article abstract of DOI:10.1021/ja960091t

Disulfide cross-links have been site-specifically incorporated into unmodified yeast tRNA^Phe by total chemical synthesis. One cross-link is between positions 1 and 72 in the amino-acid acceptor stem, and it was prepared by replacing G1 and C72

Full text of DOI:10.1021/ja960091t

J. Am. Chem. Soc. 1996, 118, 5207-5215
5207
Design, Synthesis, and Analysis of Yeast tRNAPhe Analogs
Possessing Intra- and Interhelical Disulfide Cross-Links†
Jay T. Goodwin,‡ Scott E. Osborne, Emily J. Scholle, and Gary D. Glick*,§
Contribution from the Department of Chemistry, UniVersity of Michigan,
Ann Arbor, Michigan 48109-1055
ReceiVed January 11, 1996X
Abstract: Disulfide cross-links have been site-specifically incorporated into unmodified yeast tRNAPhe by total
chemical synthesis. One cross-link is between positions 1 and 72 in the amino-acid acceptor stem, and it was prepared
by replacing G1 and C72 with N3-(thioethyl)uridine. A second cross-link is in the central D-region of yeast tRNAPhe
between 11 and 25, and it was synthesized by replacing C11 and C25 with 2′-O-alkylthiol modified cytosine residues.
Air oxidation to form the cross-link at both sites occurs in 12 h and is nearly quantitative. Analysis of the cross-
linked products by native and denaturing PAGE along with Pb(II) cleavage experiments demonstrates that the cross-
linked molecules are monomeric and suggests that the disulfide bridges do not significantly alter the structure of the
modified tRNAs relative to the parent sequence. The finding that cross-link formation between thiol-derivatized
residues correlates with the position of these groups in the crystal structure of native yeast tRNAPhe and that the
modifications apparently do not perturb native structure suggests that this methodology should be applicable to the
study of RNA structure, dynamics, and folding.
Introduction
conformationally homogeneous and thermally more stable than
their wild-type parent sequences. In this report we show that
both inter- and intrahelical disulfide cross-links can be site-
specifically placed into unmodified yeast tRNAPhe, a relatively
large oligoribonucleotide, by total chemical synthesis.5 The
ability to form these cross-links correlates with predictions based
on the crystal structure of native yeast tRNAPhe, and the cross-
links do not appear to significantly alter the structure of the
thiol/disulfide-modified tRNAs relative to the parent sequence.
Collectively, these results demonstrate that the mild and selective
chemistry of disulfide formation is compatible with RNA of
larger and more complex structure.
Although significant progress has been made in elucidating
the folding pathways and structure of ribonucleic acids, our
current understanding of these properties lags behind that for
DNA.1 Indeed, biophysical analysis of RNA can be quite
difficult because oligoribonucleotides often self-associate and/
or equilibrate between several different conformations, particu-
larly at the concentrations required for many high-resolution
biophysical measurements.2 To help circumvent these problems,
an array of chemical and biochemical approaches has been
developed to explore RNA conformation and dynamics. For
example, RNA structure and folding have been probed by
footprinting with base- and phosphate-specific-modifying re-
agents, transition metal complexes, and a broad spectrum of
nucleases.3 Complementary to these experiments has been the
design, synthesis, and site-specific incorporation of residues
possessing modifications to the base and/or sugar-phosphate
backbone.4 These latter experiments have proved particularly
useful for precisely assessing the role of various functional
groups in both the structure and function of RNA.
Results
Design. Yeast tRNAPhe was selected for these studies because
a high-resolution X-ray crystal structure of the native oligomer
is available6 and solution measurements indicate that the
conformation of fully unmodified yeast tRNAPhe is similar to
that of the native molecule.7 Sites for modification and design
of the actual cross-links were guided by the following criteria.
In previous work we described a method to probe for the
presence of helical termini on A-form RNA.4g Specifically,
incorporation of N3-thioethyluridine at both the 3′ and 5′ termini
of duplex RNA, followed by air oxidation, produces intra-helical
disulfide cross-links. RNAs possessing this cross-link are both
(3) For representative examples, see: (a) Heilek, G. M.; Marusak, R.;
Meares, C. F.; Noller, H. F. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 1113-
1116. (b) Vlassov, V. V.; Zuber, G.; Felde, B.; Behr, J.-P.; Giege´, R. Nucleic
Acids Res. 1995, 23, 3161-3167. (c) Matsuo, M.; Yokogawa, T.; Nishikawa,
K.; Watanabe, K.; Okada, N. J. Biol. Chem. 1995, 270, 10097-10104. (d)
Han, H.; Dervan, P. B. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4955-
4959. (e) Burrows, C. J.; Rokita, S. E. Acc. Chem. Res. 1994, 27, 295-
301. (f) Cload, S. T.; Richardson, P. L.; Huang, Y.-H.; Schepartz, A. J.
Am. Chem. Soc. 1993, 115, 5005-5014. (g) Chow, C. S.; Behlen, L. S.;
Uhlenbeck, O. C.; Barton, J. K. Biochemistry 1992, 31, 972-982. (h)
Tullius, T. D. Curr. Opin. Struct. Biol. 1991, 1, 428-434. (i) Celander, D.
W.; Cech, T. R. Biochemistry 1990, 29, 1355-1361. (j) Chow, C. S.; Barton,
J. K. J. Am. Chem. Soc. 1990, 112, 2839-2841. (k) Latham, J. A.; Cech,
T. R. Science 1989, 245, 276-282. (l) Murakawa, G. J.; Chen, C. B.;
Kuwabara, M. D.; Nierlich, D. P.; Sigman, D. S. Nucleic Acids Res. 1989,
17, 5361-5375. (m) Zuckermann, R. N.; Schultz, P. G. Proc. Natl. Acad.
Sci. U.S.A. 1989, 86, 1766-1770. (n) Stern, S.; Moazed, D.; Noller, H. F.
Methods Enzymol. 1988, 164, 481-489. (o) Ehresmann, C.; Baudin, F.;
Mougel, M.; Romby, P.; Ebel, J.-P.; Ehresmann, B. Nucleic Acids Res. 1987,
15, 9109-9128. (p) Romby, P.; Moras, D.; Dumans, P.; Ebel, J. P.; Giege´,
R. J. Mol. Biol. 1987, 195, 193-204. (q) Kean, J. M.; White, S. A.; Draper,
D. A. Biochemistry 1985, 24, 5062-5070. (r) Holbrook, S. R.; Kim, S.-H.
Biopolymers 1983, 22, 1145-1166.
† The first three authors made significant contributions to this research.
‡ Present address: Pharmacia & Upjohn Inc., Kalamazoo, MI 49001.
§ Phone, (313) 764-4548; FAX, (313) 764-8815; e-mail, gglick@umich.edu.
X Abstract published in AdVance ACS Abstracts, May 15, 1996.
(1) For reviews and leading examples, see: (a) Shen, L. X.; Cai, Z.;
Tinoco, I., Jr. FASEB J. 1995, 9, 1023-1033. (b) Pyle, A. M.; Green, J. B.
Curr. Opin. Struct. Biol. 1995, 5, 303-310. (c) Zarrinkar, P. P.; Williamson,
J. R. Science 1994, 265, 918-924. (d) Williamson, J. R. Nature Struct.
Biol. 1994, 1, 270-272. (e) Draper, D. E. Acc. Chem. Res. 1992, 25, 201-
207. (f) Tinoco, I., Jr.; Puglisi, J. D.; Wyatt, J. R. Nucleic Acids Mol. Biol.
1990, 4, 205-226. (g) Wyatt, J. R.; Puglisi, J. D.; Tinoco, I., Jr. BioEssays
1989, 11, 100-106. (h) Crothers, D. M.; Cole, P. E. Conformational
Changes of tRNA. In Transfer RNA; Altman, S., Ed.; MIT Press:
Cambridge, MA, 1978; pp 196-247.
(2) For example, see: (a) Varani, G.; Cheong, C.; Tinoco, I., Jr.
Biochemistry 1991, 30, 3280-3289. (b) Holbrook, S. R.; Cheong, C.;
Tinoco, I., Jr.; Kim, S.-H. Nature 1991, 353, 579-581.
S0002-7863(96)00091-1 CCC: $12.00 © 1996 American Chemical Society

5208 J. Am. Chem. Soc., Vol. 118, No. 22, 1996
Goodwin et al.
First, all of the alkylthiol modifications were designed so as
not to interfere with noncovalent interactions that stabilize the
folded tertiary structure, such as hydrogen bonding and Mg2+
binding. Second, all of the alkylthiol linkers are on the
periphery of the folded tRNA molecule where they should be
accessible to the various reagents required for oxidation,
reduction, and alkylation of the sulfhydryl groups. Third, the
thiols that comprise each cross-link are within 10 Å of each
other so that cross-link formation should proceed relatively
rapidly, and the (entropic) stability conferred by the cross-link
will be maximized.
Two initial sites were chosen based on these criteria. The
first site (I) is between G1 and C72 in the amino-acid acceptor
stem (Figure 1A,B). This site is distal from any tertiary
interactions in the folded structure, and an intrahelical cross-
link bridging these two bases should not grossly perturb native
geometry. A cross-link between these residues can be incor-
porated by replacing G1 and C72 with N3-(thioethyl)uridines
(US).4g Although replacing both G1 and C72 with US can result
in a loss of Watson-Crick hydrogen bonding, due to end-fraying
effects at the termini of duplexes, base-pairing is reduced and
we have found that terminal cross-links are not energetically
unfavorable.4g,8,9 Furthermore, cross-links positioned at helical
termini do not appear to perturb the conformation of adjacent
base pairs.9
not interact with other sites on the folded tRNA. Modeling
studies suggest that replacing C11 and C25 with 2′-O-(thio-
ethyl)cytosine (CS) will afford a cross-link between these two
hydroxyl groups that neither disrupts base-pairing interactions
involving C11 and C25 nor introduces significant structural
perturbations in the parent structure. In addition, we chose site
II because it is not precisely known where along the folding
pathway of yeast tRNAPhe the D-region assembles, and by
measuring the rate at which IItBu and IIXL fold as a function of
temperature, it should be possible to elucidate at what point
this region of tertiary structure forms.1h,10
Monomer Synthesis. Synthesis of the fully protected N3-
(thioethyl)uridine phosphoramidite was conducted as previously
described.4g Briefly, the N3-position of 2′,3′,5′-tris-O-(trimeth-
ylsilyl)uridine was deprotonated with NaH and alkylated with
O-tosyl-S-benzoyl mercaptoethanol.11 Removal of the silyl
groups with aqueous HF afforded 1 in 63% yield from uridine
(Scheme 1). Preparation of the modified nucleoside for solid-
phase synthesis was accomplished by protection of the 5′-
hydroxyl group as a 4,4′-dimethoxytrityl ether, exchange of the
thiobenzoyl to the tert-butyl mixed disulfide,12 and silylation
of the 2′-hydroxyl group. To incorporate US at the 5′-terminus
of site I, the 3′-hydroxyl was activated as a â-cyanoethyl N,N-
diisopropylphosphoramidite using conditions that suppress silyl
group migration.13 For incorporation of US at the 3′-terminus
of site I, intermediate 4 was coupled to controlled pore glass
(CPG, 1000 Å) through a succinate linker. The loading
concentration of the modified monomer, as ascertained by the
trityl cation release assay, was typically 32 µmol/g.14
Synthesis of fully protected CS proceeded from 3′,5′-O-
(tetraisopropyldisiloxane-1,3-diyl)-N4-benzoyl-2′-O-allylcyti-
dine, which was prepared as described previously by Sproat
and co-workers.15 The allyl group was dihydroxylated using
OsO4,16 oxidatively cleaved with NaIO4, and reduced with
NaBH4 to afford the saturated alcohol 10 in good yield (Scheme
2). Mesylation of the primary alcohol followed by displacement
with thiobenzoic acid afforded the thiobenzoyl intermediate 12,
which upon removal of the silyl protecting group with aqueous
HF was exchanged to the tert-butyl mixed disulfide. Protection
of the 5′-hydroxyl and activation of the 3′-hydroxyl following
standard procedures17 provided the desired CS monomer for use
in solid-phase synthesis.
RNA Synthesis and Cross-Link Formation. Solid-phase
synthesis of ItBu and IItBu was conducted using standard
â-cyanoethyl N,N-diisopropylphosphoramidite chemistry as
previously described (the coupling efficiency of Us and Cs is
g98.5%).18 All tRNA syntheses were conducted on a 1 µmol
scale, and each synthesis afforded between 80 and 130 OD260
units of crude material, from which ∼10% of pure RNA could
be isolated by denaturing polyacrylamide gel electrophoresis
The second site for modification (II) is in the central D-region
of yeast tRNAPhe between C11 and C25 (Figure 1C,D). The
X-ray structure of native yeast tRNAPhe indicates that the 2′-
hydroxyl groups on these residues converge to ∼9 Å and do
(4) For representative examples, see: (a) Agrawal, S.; Iyer, R. P. Curr.
Opin. Biotechnol. 1995, 6, 12-19. (b) Agris, P. F.; Malkiewicz, A.;
Kraszewski, A.; Everett, K.; Nawrot, B.; Sochacka, E.; Jankowska, J.;
Guenther, R. Biochimie 1995, 77, 125-134. (c) Gait, M. J.; Grasby, J. A.;
Karn, J.; Mersmann, K.; Pritchard, C. E. Nucleosides Nucleotides 1995,
14, 1133-1144. (d) Nawrot, B.; Malkiewicz, A.; Smith, W. S.; Sierzpu-
towska-Gracz, H.; Agris, P. F. Nucleosides Nucleotides 1995, 14, 143-
165. (e) Sproat, B. S. J. Biotechnol. 1995, 41, 221-238. (f) Fu, D.-J.; Rajur,
S. B.; McLaughlin, L. W. Biochemistry 1994, 33, 13903-13909. (g)
Goodwin, J. T.; Glick, G. D. Tetrahedron Lett. 1994, 35, 1647-1650. (h)
Grasby, J. A.; Gait, M. J. Biochimie 1994, 76, 1223-1234. (i) Ng, M. M.
P.; Benseler, F.; Tuschl, T.; Eckstein, F. Biochemistry 1994, 33, 12119-
12126. (j) Richardson, P. L.; Gross, M. L.; Light-Wahl, K. J.; Smith, R.
D.; Schepartz, A. BioMed. Chem. Lett. 1994, 4, 2133-2138. (k) Beaucage,
S. L.; Iyer, R. P. Tetrahedron 1993, 49, 6123-6194. (l) Fu, D.-J.; Rajur,
S. B.; McLaughlin, L. W. Biochemistry 1993, 32, 10629-10637. (m)
Heidenreich, O.; Pieken, W.; Eckstein, F. FASEB J. 1993, 7, 90-95 and
references therein. (n) Herschlag, D.; Eckstein, F.; Cech, T. R. Biochemistry
1993, 32, 8299-8311. (o) Herschlag, D.; Eckstein, F.; Cech, T. R.
Biochemistry 1993, 32, 8312-8321. (p) Sproat, B. S. Curr. Opin. Biotech-
nol. 1993, 4, 20-28. (q) Tuschl, T.; Ng, M. M. P.; Pieken, W.; Benseler,
F.; Eckstein, F. Biochemistry 1993, 32, 11658-11668. (r) Usman, N.;
Cedergren R. Trends Biochem. Sci. 1992, 17, 334-339. (s) Williams, D.
M.; Pieken, W. A.; Eckstein, F. Proc. Natl. Acad. Sci. U.S.A. 1992, 89,
918-921. (t) Green, R.; Szostak, J. W.; Benner, S. A; Rich, A.; Usman, N.
Nucleic Acids Res. 1991, 19, 4161-4166. (u) Herschlag, D.; Piccirilli, J.
A.; Cech, T. R. Biochemistry 1991, 30, 4844-4854. (v) Pieken, W. A.;
Olsen, D. B.; Aurup, H.; Williams, D. M.; Heidenreich, O.; Benseler, F.;
Eckstein, F. Nucleic Acids Symp. Ser. 1991, 24, 51-53. (w) Piccirilli, J.
A.; Krauch, T.; Moroney, S. E.; Benner, S. A. Nature 1990, 343, 33-37.
(x) Talbot, S. J.; Goodman, S.; Bates, S. R. E.; Fishwick, C. W. G.; Stockley,
P. G. Nucleic Acids Res. 1990, 18, 3521-3528. (y) Caruthers, M. H. Front.
Chem. 1989, 1, 55-61.
(10) Clarke, J.; Fersht, A. R. Biochemistry 1993, 32, 4322-4329 and
references therein.
(11) Glick, G. D. J. Org. Chem. 1991, 56, 6746-6747.
(12) (a) Wu¨nsch, E.; Moroder, L.; Romani, S. Hoppe-Seyler’s Z. Physiol.
Chem 1982, 363, 1461-1464. (b) Bock, H.; Kroner, J. Chem. Ber. 1966,
99, 2039-2051.
(5) The tRNAs are referred to as “unmodified” because none of the post-
transcriptionally modified bases found in native yeast tRNAPhe are present.
(6) Holbrook, S. R.; Sussman, J. L.; Warrant, R. W.; Kim, S.-H. J. Mol.
Biol. 1978, 123, 631-660.
(7) Hall, K. B.; Sampson, J. R.; Uhlenbeck, O. C.; Redfield, A. G.
Biochemistry 1989, 28, 5794-5801.
(8) Osborne, S. E. Ph.D. Thesis, University of Michigan, 1996, pp
38-79.
(9) (a) Wang, H.; Zuiderweg, E. R. P.; Glick, G. D. J. Am. Chem. Soc.
1995, 117, 2981-2991. (b) Cain, R. J.; Zuiderweg, E. R. P.; Glick, G. D.
Nucleic Acids Res. 1995, 23, 2153-2160. (c) Wang, H.; Osborne, S. E.;
Zuiderweg, E. R. P.; Glick, G. D. J. Am. Chem. Soc. 1994, 116, 5021-
5022.
(13) Scaringe, S. A.; Francklyn, C.; Usman, N. Nucleic Acids Res. 1990,
18, 5433-5441.
(14) Schaller, H.; Weimann, G.; Lerch, B.; Khorana, H. G. J. Am. Chem.
Soc. 1963, 85, 3821-3827.
(15) Sproat, B. S.; Iribarren, A.; Beijer, B.; Pieles, U.; Lamond, A. I.
Nucleosides Nucleotides 1991, 10, 25-36.
(16) Dihydroxylation of the C5-C6 double bond is not observed under
these conditions.
(17) (a) Horn, T.; Urdea, M. S. Tetrahedron Lett. 1986, 27, 4705-4708.
(b) Beaucage, S. L.; Caruthers, M. H. Tetrahedron Lett. 1981, 22, 1859-
1862.
(18) Goodwin, J. T.; Ashton, W. A.; Glick, G. D. J. Org. Chem. 1994,
59, 7941-7943 and references therein.

Yeast tRNAPhe Analogs with Disulfide Cross-Links
J. Am. Chem. Soc., Vol. 118, No. 22, 1996 5209
Figure 1. Location and chemical composition of cross-links. (A, top left) Primary sequence and secondary structure of unmodified yeast tRNAPhe
showing position and chemical structure of site I. Note that, for site I, a truncated version of the tRNA lacking the last four bases on the 3′ terminus
was synthesized. (B, top right) Model of yeast tRNAPhe crystal structure showing the site I cross-link (stereoview). The purple sphere is a lead atom
in its binding site, and the yellow spheres are the sulfur atoms that comprise the cross-link. The cross-link was constructed using the Biosym
INSIGHT/DISCOVER software. Molecular mechanics and dynamics calculations show that the alkylthiol linker is fully staggered and the C-S-
S-C dihedral angle is ∼90°. (C, bottom left) Primary sequence and secondary structure of unmodified yeast tRNAPhe showing position and chemical
structure of the site II cross-link. (D, bottom right) Model of yeast tRNAPhe showing the site II cross-link (stereoview). The coloring scheme is the
same as in part B. The fully base-, sugar-, and phosphate-deprotected sequences still possessing the thiol protecting groups are designated ItBu and
IItBu for sites I and II, respectively. The corresponding cross-linked molecules are designated IXL and IIXL
.
(PAGE). Prior to cross-link formation, the tert-butyl protecting
groups on the thiols must be removed. However, we could not
monitor the progress of thiol deprotection because the reduced
and tert-butyl protected tRNAs cannot be resolved by PAGE,
ion-exchange, or reversed-phase HPLC, and the initial protected
tRNAs and reduced products have similar spectral properties.
By contrast, reduced and tert-butyl protected alkylthiol-modified
DNAs can be separated by HPLC.19 Therefore, the tDNA
sequences corresponding to ItBu and IItBu were synthesized and
removal of the thiol protecting groups was followed by reversed-
phase HPLC to select conditions for the quantitative removal
of the thiol protecting groups. On the basis of the results from
these model studies, purified samples of ItBu and IItBu were
reduced by treatment with dithiothreitol (DTT; 200 equiv/
disulfide in 100 mM sodium phosphate, pH 8.3) for 12 h.
Following dialysis to remove excess DTT, a near quantitative
recovery of the reduced tRNAs was obtained, and these
materials were used immediately to form the desired cross-links.
Disulfide bond formation was initiated by heat denaturing
and slowly cooling the reduced oligomers in the presence of
Mg2+ to ensure proper folding.20 The pH was then adjusted to
8.0, and the tRNA solutions were stirred exposed to air to effect
cross-link formation. To follow the progress of disulfide bond
formation, aliquots of each reaction mixture were treated with
7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin
(CPM), which produces a fluorescent adduct with thiols (∼5
pmol of thiol can be detected).21 After 12 h, the fluorescence
signal was at background and the cross-linked tRNAs were
isolated by ethanol precipitation.22 Denaturing PAGE analysis
of the crude reaction mixtures shows a single major band
(∼95%) with only minor amounts of higher molecular weight
products, demonstrating that disulfide bond formation is near
quantitative (Figure 2). Intramolecular cross-links do not form
if the tRNAs are not folded (i.e., in the absence of Mg2+), which
suggests that the thiol linkers are in close proximity in the folded
structure, and that the alkylthiol modifications do not provide
a kinetic or thermodynamic impediment to tertiary folding.
Both IXL and IIXL migrate more slowly than either unmodified
yeast tRNAPhe or their corresponding tert-butyl disulfide
(20) Behlen, L. S.; Sampson, J. R.; DiRenzo, A. B.; Uhlenbeck, O. C.
Biochemistry 1990, 29, 2515-2523.
(21) Parvari, R.; Pecht, I.; Soreq, H. Anal. Biochem. 1983, 133, 450-
456.
(22) Terminal nucleoside analysis of 5′-end-labeled ItBu and IXL along
with OH- footprinting for IItBu and IIXL was used to verify incorporation
of the modified bases and cross-links, respectively. See: (a) Silberklang,
M.; Gillum, A. M.; RajBhandary, U. L. Methods Enzymol. 1979, 59, 58-
109. (b) Butcher, S. E.; Burke, J. M. J. Mol. Biol. 1994, 244, 52-63.
(19) Glick, G. D.; Osborne, S. E.; Knitt, D. S.; Marino, J. P., Jr. J. Am.
Chem. Soc. 1992, 114, 5447-5448.

5210 J. Am. Chem. Soc., Vol. 118, No. 22, 1996
Goodwin et al.
Scheme 1a
Figure 2. PAGE analysis of site I disulfide cross-link formation
reaction (15% polyacrylamide denaturing gel visualized with Stains-
All). Lane 1, purified native yeast tRNAPhe; lane 2, purified ItBu; lane
3, crude IXL after 12 h. Similar results are obtained for formation of
IIXL (data not shown).
cycle whereas IIXL contains a smaller, internal loop. By
contrast, unmodified yeast tRNAPhe and all four thiol-modified
tRNAs run similarly under nondenaturing conditions, which
indicates that IXL and IIXL are monomeric. Furthermore, these
results suggest that the modifications do not significantly alter
the folded structure of the tRNAs relative to the parent sequence.
Further evidence that the modified tRNAs fold properly was
obtained by Pb(II)-induced cleavage, which is a very sensitive
assay of tRNAPhe tertiary structure.20 Specifically, a Pb(II)
binding site between the D- and T-loops forms upon proper
folding of tRNAPhe into its canonical three-dimensional motif
(see Figure 1B,D).23 After binding of Pb(II) in this site, heating
at pH 7 produces cleavage of the phosphodiester backbone,
a (a) TMSCl, Et3N, DMF; (b) NaH, DMF; (c) pTsOCH2CH2SBz,
DMF; (d) HF (aqueous) (63% from a); (e) DMTrCl, pyridine; (f) 1-(tert-
butylthio)-1,2-hydrazinedicarboxmorpholide, LiOH, MeOH (70% from
e); (g) TBDMSCl, imidazole, DMF (65%; 3:4, 3:2); (h) 2-cyanoethyl
N,N-diisopropylphosphoramidochloridite, 2,4,6-collidine, N-methylimi-
dazole, THF (82%); (i) succinic anhydride, DMAP, pyridine; (j)
pentachlorophenol, DCC, DMAP, CH2Cl2 (90% from i); (k) CPG (1000
Å), Et3N, DMF.
Scheme 2a
predominantly between U17 and G18. In the absence of Mg2+
,
none of the tRNAs fold properly and only minor nonspecific
hydrolysis is observed (Figure 4). By contrast, Pb(II) cleavage
of either unmodified yeast tRNAPhe, IXL, or IIXL annealed in
the presence of Mg2+ affords a single major band corresponding
to strand scission between U17 and G18 in the D-loop. The
conclusion that the alkylthiol linkers do not significantly alter
native structure is also supported by UV thermal denaturation
experiments. Specifically, the Tm values for both ItBu and IItBu
are within ∼1 °C of the Tm value of unmodified yeast tRNAPhe
(Figure 5). Furthermore, the shapes of the melting transitions
for both ItBu and IItBu are nearly superimposable with that for
unmodified yeast tRNAPhe, which suggests that the alkylthiol-
modified tRNAs and the parent sequence denature along similar
pathways.
Discussion
In 1966 Lipsett showed that iodine oxidation of the two native
4-thiouridine residues in tRNATyr from Escherichia coli affords
a unique intramolecular disulfide cross-link.24 Although the
potential of these disulfide cross-links as probes of both structure
and function was noted in this early work, neither methods to
synthesize RNA site-specifically labeled with thiol groups nor
sensitive biochemical and structural assays to analyze the cross-
linked products were available. Building on chemistry devel-
oped over the past few years to cross-link various DNA
secondary structures with disulfide bonds,11,19,25 we showed that
disulfide cross-links also can be placed within small RNA
sequences without significantly perturbing native structure.4g
More recently, both Eckstein26 and Verdine27 have described
the use of disulfide cross-links to probe the conformation of
a (a) OsO4, N-methylmorpholine N-oxide, acetone, H2O (90%); (b)
NaIO4, p-dioxane, H2O (97%); (c) NaBH4, MeOH (93%); (d) meth-
anesulfonyl chloride, pyridine (92%); (e) thiobenzoic acid, Et3N, DMF
(76%); (f) HF (aqueous), CH3CN (100%); (g) 1-(tert-butylthio)-1,2-
hydrazinedicarboxmorpholide, LiOH, MeOH, THF (74%); (h) DMTrCl,
Et3N, DMF, DMAP (77%); (i) 2-cyanoethyl N,N-diisopropylphos-
phoramidochloridite, N,N-diisopropylethylamine, CH2Cl2 (84%).
(23) Brown, R. S.; Dewan, J. C.; Klug, A. Biochemistry 1985, 24, 4785-
4801.
(24) (a) Lipsett, M. N. Cold Spring Harbor Symp. Quant. Biol. 1966,
31, 449-455. (b) Lipsett, M. N. J. Biol. Chem. 1967, 242, 4067-4071.
protected precursors by denaturing PAGE (Figure 3). The
observation that IXL migrates slower than IIXL on denaturing
gels can be explained given that IXL is a 72-base-long macro-

Yeast tRNAPhe Analogs with Disulfide Cross-Links
J. Am. Chem. Soc., Vol. 118, No. 22, 1996 5211
Figure 3. PAGE analysis of 32P-end-labeled tRNAs. Left: 15%
polyacrylamide denaturing gel; lane 1, IXL; lane 2, ItBu; lane 3, IIXL
;
lane 4, IItBu; lane 5, unmodified yeast tRNAPhe. Right: 15% polyacryl-
amide nondenaturing gel; lane 1, IXL; lane 2, ItBu; lane 3, IIXL; lane 4,
Figure 5. Representative normalized UV thermal denaturation curves
for unmodified tRNAPhe (s) and IItBu (- - -). Spectra were measured
in pH 7.2 buffer as described in the Experimental Section. The Tm values
for unmodified yeast tRNAPhe and IItBu are 67.3 and 68.3 °C,
respectively. Similar results are obtained with ItBu (data not shown).
The cross-linked tRNAs melt several degrees higher than their tert-
butyl protected counterparts.
IItBu; lane 5, unmodified yeast tRNAPhe
.
used to investigate both the hammerhead ribozyme and an
analog of HIV-1 TAR RNA.28 However, glycol and related
cross-links are necessarily incorporated during solid-phase
synthesis and can only be placed between sites immediately
(and linearly) adjacent in sequence. RNA cross-links have also
been introduced postsynthetically. For example, UV irradiation
of unmodified yeast tRNAPhe yields a pyrimidine-pyrimidine
photoadduct between C48 and U59 in the T-region of the folded
sequence.29 However, formation of this and related cross-links30
generally depends on the fortuitous predisposition of the two
reacting groups and therefore is not a flexible strategy to
examine RNA structure and folding.31 Moreover, only the
disulfide cross-links are easily cleaved (by treatment with
reducing agents), which is an important requirement for several
types of footprinting experiments.32
We have demonstrated that disulfide cross-links can be site-
specifically incorporated into unmodified yeast tRNAPhe and that
cross-link formation between thiol-derivatized loci correlates
with the position of these groups in the crystal structure of native
yeast tRNAPhe. Within the resolution of our assays, the thiol/
disulfide modifications do not alter the structure or folding of
the derivatized tRNAs. In principle, incorporation of disulfide
cross-links as described in this work is not restricted to RNAs
for which high-resolution structural data already exists. Since
disulfide bond formation is mild and highly selective, such cross-
links can be used to verify theoretical models of RNA geometry.
By varying the length of the alkyl thiol linkers, information on
the distance between structural elements can be investigated.
In this regard, incorporating disulfide cross-links is comple-
mentary to other techniques used to study RNA folding and
structure, including transient electric birefringence33 and fluo-
Figure 4. Lead cleavage analysis of 32P-end-labeled tRNAs. The
cleavage reactions with Pb(OAc)2 were conducted at 40 °C for 10 min
as previously described.20 After cleavage reactions involving IXL and
IIXL, the disulfide cross-link was reduced with DTT and the thiol groups
were blocked with N-ethylmaleimide to prevent reoxidation prior to
or during electrophoresis.32 Lead cleavage of ItBu and IItBu affords
similar results (data not shown). Left: IXL; lane 1, RNase T1 ladder;
lane 2, Pb(II) plus 5 mM MgCl2; lane 3, Pb(II) only. Middle: IIXL
;
lane 1, RNase T1 ladder; lane 2, Pb(II) plus 5 mM MgCl2; lane 3, Pb-
(II) only. Right: unmodified yeast tRNAPhe; lane 1, RNase T1 ladder;
lane 2, Pb(II) plus 5 mM MgCl2; lane 3, Pb(II) only. Note that the
reaction products for the three tRNAs were analyzed on different gels,
which gives rise to the small differences in band migration and band
intensities.
the hammerhead ribozyme and to stabilize a small RNA hairpin,
respectively.
(28) (a) Fu, D.-J.; Benseler, F.; McLaughlin, L. W. J. Am. Chem. Soc.
1994, 116, 4591-4598. (b) Ma, M. Y.-X.; McCallum, K.; Climie, S. C.;
Kuperman, R.; Lin, W. C.; Sumner-Smith, M.; Barret, R. W. Nucleic Acids
Res. 1993, 21, 2585-2589. (c) Ma, M. Y.-X.; Reid, L. S.; Climie, S. C.;
Lin, W. C.; Kuperman, R.; Sumner-Smith, M.; Barnett, R. W. Biochemistry
1993, 32, 1751-1758.
Other cross-links, not based on disulfide chemistry, have been
used to examine RNA structure and function. For example,
ethylene glycol-based linkers bridging the 5′- and 3′-terminal
hydroxyl groups on opposing strands of duplex RNA have been
(25) (a) Goodwin, J. T.; Osborne, S. E.; Swanson, P. C.; Glick, G. D.
Tetrahedron Lett. 1994, 35, 4527-4530. (b) Gao, H.; Chidambaram, N.;
Chen, B. C.; Pelham, D. E.; Patel, R.; Yang, M.; Zhou, L.; Cook, A.; Cohen,
J. S. Bioconjugate Chem. 1994, 5, 445-453. (c) Milton, J.; Connolly, B.
A.; Nikforov, T. T.; Cosstick, R. J. Chem. Soc., Chem. Commun. 1993,
779-780. (d) Ferentz, A. E.; Keating, T. A.; Verdine, G. L. J. Am. Chem.
Soc. 1993, 115, 9006-9014. (e) Erlanson, D. A.; Chen, L.; Verdine, G. L.
J. Am. Chem. Soc. 1993, 115, 12583-12584. (f) Ferentz, A. E.; Verdine, G.
L. J. Am. Chem. Soc. 1991, 113, 4000-4002.
(29) Behlen, L. S.; Sampson, J. R.; Uhlenbeck, O. C. Nucleic Acids Res.
1992, 20, 4055-4059.
(30) (a) Woisard, A; Fourrey, J.-L.; Favre, A. J. Mol. Biol. 1994, 239,
366-370. (b) Butcher, S. E.; Burke, J. M. Biochemistry 1994, 33, 992-
999. (c) Downs, W. D.; Cech, T. R. Biochemistry 1990, 29, 5605-5613.
(d) Favre, A.; Thomas, G. Annu. ReV. Biophys. Bioeng. 1981, 10, 175-
200.
(31) A notable exception is the use of circular permutation to cross-link
various sites; see: Nolan, J. M.; Burke, D. H. Pace, N. R. Science 1993,
261, 762-765.
(32) Swanson, P. C.; Glick, G. D. BioMed. Chem. Lett. 1993, 3, 2117-
2118.
(26) Sigurdsson, S. Th.; Tuschl, T.; Eckstein, F. RNA 1995, 1, 575-
583.
(27) Allerson, C. R.; Verdine, G. L. Chem. Biol. 1995, 2, 667-675.

5212 J. Am. Chem. Soc., Vol. 118, No. 22, 1996
Goodwin et al.
rescence resonance energy transfer.34 Furthermore, the ability
to ligate RNA fragments enzymatically opens the way to prepare
very large molecules site-specifically labeled with thiol groups.35
Finally, by analogy to experiments devised for proteins contain-
ing cysteine residues, the ability to place thiol groups in RNA
should facilitate examination of both the conformational flex-
ibility36 and folding pathways1,10 of ribonucleic acids.
CH2Cl2 and washed with H2O and brine. The organic layer was dried
over Na2SO4 and concentrated in Vacuo. The oily residue was purified
by flash chromatography (CH2Cl2:CH3OH, 19:1) to afford 1 as a white
1
foam (26 g, 63% yield): TLC (CH2Cl2:CH3OH, 9:1) Rf ) 0.41; H
NMR (300 MHz, CD3CN) δ 3.30 (2 H, t, J ) 5 Hz, CH2SC(O)Ar),
3.65-3.81 (2 H, 2 dd, J ) 2.5, 10 Hz, 5′, 5′′), 3.96 (1 H, m, 4′), 4.16-
4.19 (4 H, m, 2′, 3′, NCH2), 5.66 (1 H, d, J ) 8.1 Hz, 6), 5.80 (1 H,
d, J ) 3.8 Hz, 1′), 7.43-7.62 (3 H, m, Ar), 7.81 (1 H, d, J ) 8.1 Hz,
5), 7.88 (2 H, d, J ) 7.3 Hz, Ar); 13C NMR (75 MHz, CD3CN) δ 27.4
(CH2SC(O)Ar), 40.9 (NCH2), 62.1 (5′), 70.8 (3′), 75.6 (2′), 85.9 (4′),
91.8 (1′), 102.1 (5), 128.0, 129.9, 134.7, 138.0 (Ar), 140.3 (6), 152.4
(2), 163.9 (4), 192.4 (SC(O)Ar); IR (KBr) ν 3548, 3448, 3086, 3060,
2944, 2901, 1706, 1673, 1655, 1462, 1388, 1286, 1211, 1124, 1079,
919, 813, 779, 695, 647, 569 cm-1; FAB MS (3-NBA/trifluoroacetic
acid) m/z 409 (M+ + 1).
Experimental Section
General. Reagents were purchased from Aldrich Chemical Com-
pany. Controlled-pore glass (1000 Å) was obtained from CPG, Inc.
7-Diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin was ob-
tained from Molecular Probes, Inc. RNase T1 was obtained from United
States Biochemical. CH3CN, CH2Cl2, N,N-diisopropylethylamine,
pyridine, and triethylamine (Et3N) were each distilled from CaH2 under
N2. CH3OH was dried by distillation from Mg(OCH3)2. Tetrahydro-
furan (THF) was distilled from sodium and benzophenone under N2.
N,N-Dimethylformamide (DMF) was dried by storing for 1 week over
activated 4 Å molecular sieves and then decanting under N2 onto fresh
sieves prior to use. Silica gel (32-63 mesh) for flash chromatography
was obtained from ICN Biochemicals. Glass-backed silica gel 60 plates
for thin-layer chromatography (TLC) were precoated with a 0.25-mm-
thick layer of Kieselgel 60F-254 and were obtained from E. Merck.
All reactions were performed at room temperature unless otherwise
noted. 1H and 13C NMR spectra were measured on either a Bruker
AM 360 MHz spectrometer or a Bruker AM 300 MHz spectrometer.
31P NMR spectra were measured on a Bruker AMX 500 spectrometer.
All NMR spectra were measured at ambient probe temperature using
the residual solvent proton peak as an internal reference, except for
31P NMR, which was referenced to trimethyl phosphate (δ ) 0.00).
Infrared spectra (IR) were measured on a Nicolet Model 5D Fourier-
transform spectrophotometer. Bands are reported in reciprocal centi-
meters (cm-1) and were calibrated by comparison with the 1601 cm-1
stretch in polystyrene. Mass spectra (MS) were measured on a VG
Instruments Model 7070 spectrometer with 3-nitrobenzyl alcohol (3-
NBA) as a matrix or by electron ionization (EI). Yields refer to
chromatographically and spectroscopically homogeneous materials.
The unmodified and tert-butyl disulfide-modified tRNAPhe sequences
were synthesized on an Expedite 8909 DNA/RNA synthesizer using
standard â-cyanoethyl diisopropylphosphoramidites purchased from
PerSeptive Biosystems. Synthesis, deprotection, desilylation, and
purification of tRNA sequences was performed as previously de-
scribed.18 Dialysis of tRNA samples was conducted by buffer exchange
(5 mL/min) using a Spectrum microdialyzer fitted with a cellulose ester
membrane (5000 molecular weight cutoff).
5′-O-(4,4′-Dimethoxytrityl)-N3-ethyluridine tert-Butyl Disulfide
(2). Compound 1 (15.0 g, 37 mmol) was coevaporated once from CH3-
CN:pyridine (100 mL, 9:1) and dissolved in pyridine (185 mL), and
4,4′-dimethoxytrityl chloride (15.0 g, 44 mmol, 1.2 equiv) was added
in portions (2.5 g each) over a 6 h period at 4 °C with stirring. The
reaction was allowed to warm to room temperature overnight with
stirring under N2, after which time CH3OH (5 mL) was added. The
mixture was stirred for an additional 10 min, the solvents were removed
in Vacuo, and coevaporation of the residue with CH3CN (100 mL)
yielded a light yellow-orange foam (24.4 g, 93%). 1-(tert-Butylthio)-
1,2-hydrazinedicarboxmorpholide12 (8.31 g, 24 mmol, 1.2 equiv) and
LiOH‚H2O (2.52 g, 60 mmol, 3.0 equiv) were added to the crude
tritylated product (14 g, 20 mmol) in CH3OH (100 mL). The reaction
mixture was stirred under N2 for 12 h, after which the reaction mixture
was concentrated in Vacuo, then dissolved in ethyl acetate (EtOAc),
and washed with brine. The organic layer was dried over Na2SO4 and
concentrated in Vacuo, and the residue was purified by flash chroma-
tography (EtOAc:petroleum ether, step gradient of 2:3 to 3:2) to afford
2 as a white foam (9.7 g, 70% yield): 1H NMR (360 MHz, CD3CN)
δ 1.31 (9 H, s, SS(CH3)3), 2.90 (2 H, t, J ) 6.0 Hz, CH2SS), 3.36 (2
H, m, 5′, 5′′), 3.75 (6 H, s, 2 OCH3), 4.02 (1 H, m, 4′), 4.11 (2 H, m,
NCH2), 4.18 (1 H, m, 2′), 4.32 (1 H, m, 3′), 5.38 (1 H, d, J ) 8.1 Hz,
6), 5.78 (1 H, d, J ) 3.2 Hz, 1′), 6.85-7.44 (13 H, m, Ar), 7.73 (1 H,
d, J ) 8.1 Hz, 5); 13C NMR (90 MHz, CD3CN) δ 30.1 (SSC(CH3)3),
37.2 (CH2SS), 41.0 (NCH2), 48.5 (SSC(CH3)3), 56.0 (OCH3), 63.4 (5′),
70.5 (3′), 75.5 (2′), 83.8 (4′), 87.5 (OC(Ph)3), 91.4 (1′), 101.9 (5), 114.2
(Ar), 128.0, 129.0, 131.1, 136.5, 136.7 (Ar), 139.7 (6), 145.9 (Ar),
152.0 (2), 159.8 (Ar), 163.3 (4); IR (film; NaCl) ν 3452, 2959, 2940,
1708, 1665, 1608, 1509, 1457, 1252, 1177, 1103, 1035, 829, 810, 702
cm-1; FAB MS (3-NBA) m/z 695 (M+ + 1).
5′-O-(4,4′-Dimethoxytrityl)-2′-O-(tert-butyldimethylsilyl)-N3-
ethyluridine tert-Butyl Disulfide (3) and 5′-O-(4,4′-Dimethoxytrityl)-
3′-O-(tert-butyldimethylsilyl)-N3-ethyluridine tert-Butyl Disulfide (4).
Compound 2 (9.5 g, 13.6 mmol) was dissolved in DMF (50 mL).
Imidazole (2.31 g, 34 mmol, 2.5 equiv) and tert-butyldimethylsilyl
chloride (2.58 g, 17.1 mmol, 1.25 equiv) were added, and the mixture
was stirred overnight under N2. The reaction mixture was diluted with
EtOAc, and the mixture was washed with brine and dried over Na2-
SO4. The residue was purified by flash chromatography (petroleum
ether:EtOAc, 9:1) to afford 3 and 4 as white foams (3, 3.9 g, 40%
yield; 4, 2.1 g, 25% yield): Compound 3: TLC (petroleum ether:
N3-(2-Thiobenzoylethyl)uridine (1). Freshly distilled Et3N (83.6
mL, 600 mmol, 6 equiv) was added to a solution of uridine (24.42 g,
100 mmol) in DMF (250 mL) and cooled to 4 °C. Chlorotrimethyl-
silane (42 mL, 330 mmol, 3.3 equiv) was added slowly, and the reaction
mixture was stirred under N2 for 2 h. Salts that had precipitated during
the course of the reaction were removed by filtration under N2, and
the residual salts in the filtrate were triturated with petroleum ether:
diethyl ether (Et2O) (1:1). Sodium hydride (4.40 g, 110 mmol, 1.1
equiv) was added to the crude reaction mixture at 4 °C with stirring
under N2. After hydrogen evolution subsided, O-tosyl-S-benzoylmer-
captoethanol11 (37 g, 110 mmol, 1.1 equiv) was added and the reaction
mixture was stirred at 45 °C overnight. The solution was cooled to
room temperature, and silyl groups were removed by the addition of
HF (5 mL, 48%). After 1 h the reaction mixture was diluted with
1
EtOAc, 4:1) Rf ) 0.30; H NMR (300 MHz, CD3CN) δ 0.12 (6 H, s,
Si(CH3)2), 0.90 (9 H, s, SiC(CH3)3), 1.30 (9 H, s, SSC(CH3)3), 2.88 (2
H, t, J ) 8.0 Hz, CH2SS), 3.38 (2 H, 2 dd, J ) 2.4, 11.0 Hz, 5′, 5′′),
3.74 (6 H, s, 2 OCH3), 4.05 (3 H, m, 4′, NCH2), 4.24 (1 H, m, 2′), 4.31
(1 H, m, 3′), 5.35 (1H, d, J ) 8.2 Hz, 6), 5.83 (1H, d, J ) 4.0 Hz, 1′),
6.84-7.43 (13 H, m, Ar), 7.74 (1 H, d, J ) 8.1 Hz, 5); 13C NMR (75
MHz, CD3CN) δ -4.4 (Si(CH3)2), 18.8 (SiC(CH3)3), 26.3 (SiC(CH3)3),
30.2 (SSC(CH3)3), 37.5 (CH2SS), 41.0 (NCH2), 48.4 (SSC(CH3)3), 55.9
(OCH3), 63.7 (5′), 71.2 (3′), 76.9 (2′), 84.1 (4′), 87.7 (OC(Ph)3), 90.6
(1′), 102.1 (5), 114.1 (Ar), 127.8, 128.8, 128.9, 130.9, 136.3, 136.5
(Ar), 139.3 (6), 145.6 (Ar), 151.7 (2), 159.7 (Ar), 162.8 (4); IR (film;
NaCl) ν 3856, 3546, 2955, 2930, 2857, 2361, 2334, 1710, 1668, 1608,
1509, 1456, 1253, 1177, 1122, 1036, 836 cm-1; FAB MS (3-NBA)
m/z 809 (M+ + 1). Compound 4: TLC (petroleum ether:EtOAc, 4:1)
(33) (a) Leehey, M. A.; Squassoni, C. A.; Friederich, M. W.; Mills, J.
B.; Hagerman, P. J. Biochemistry 1995, 34, 16235-16239. (b) Duckett, D.
R.; Murchie, A. I. H.; Lilley, D. M. J. Cell 1995, 83, 1027-1036.
(34) (a) Gohlke, C.; Murchie, A. I. H.; Lilley, D. M. J.; Clegg, R. M.
Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 11660-11664. (b) Tuschl, T.;
Gohlke, C.; Jovin, T. M.; Westhof, E.; Eckstein, F. Science 1994, 266, 785-
789.
(35) Moore, M. J.; Sharp, P. A. Science 1992, 256, 992-997.
(36) (a) Chervitz, S. A.; Lin, C. M.; Falke, J. J. Biochemistry 1995, 34,
9722-9733. (b) Careaga, C. L.; Sutherland, J.; Sabeti, J.; Falke, J. J.
Biochemistry 1995, 34, 3048-3055. (c) Careaga, C. L.; Falke, J. J. Biophys.
J. 1992, 62, 209-216. (d) Falke, J. J.; Koshland, D. E., Jr. Science 1987,
237, 1596-1600.
1
Rf ) 0.14; H NMR (300 MHz, CD3CN) δ 0.04, 0.05 (6 H,2 s, Si-
(CH3)2), 0.81 (9 H, s, SiC(CH3)3), 1.30 (9 H, s, SSC(CH3)3), 2.88 (1

Yeast tRNAPhe Analogs with Disulfide Cross-Links
J. Am. Chem. Soc., Vol. 118, No. 22, 1996 5213
H, dd, J ) 3.8, 11.1 Hz, 5′), 2.89 (2 H, t, J ) 7.6 Hz, CH2SS), 3.46 (1
H, dd, J ) 2.4, 11.0 Hz, 5′′), 3.74 (6 H, s, 2 OCH3), 4.00 (1 H, m, 4′),
4.10 (3 H, m, 2′, NCH2), 4.33 (1 H, m, 3′), 5.38 (1 H, d, J ) 8.1 Hz,
6), 5.80 (1 H, d, J ) 3.0 Hz, 1′), 6.84-7.42 (13 H, m, Ar), 7.74 (1 H,
d, J ) 8.1 Hz, 5); 13C NMR (75 MHz, CD3CN) δ -4.4, -4.1 (Si-
(CH3)2), 18.7 (SiC(CH3)3), 26.3 (SiC(CH3)3), 30.3 (SSC(CH3)3), 37.5
(CH2SS), 41.0 (NCH2), 48.3 (SSC(CH3)3), 56.0 (OCH3), 63.3 (5′), 72.0
(3′), 75.4 (2′), 84.1 (4′), 87.7 (OC(Ph)3), 92.0 (1′), 102.1 (5), 114.1
(Ar), 127.7, 128.7, 129.0, 131.0, 136.4 (Ar), 139.6 (6), 145.5 (Ar),
151.7 (2), 159.7 (Ar), 162.9 (4); IR (film; NaCl) ν 3869, 2955, 2929,
2858, 2364, 2334, 1710, 1670, 1509, 1456, 1252, 1176, 1116, 1035,
836 cm-1; FAB MS (3-NBA) m/z 809 (M+ + 1).
(succinate CO2); IR (film; NaCl) ν 2956, 2931, 1786, 1751, 1713, 1672,
1608, 1509, 1455, 1390, 1363, 1253, 1229, 1177, 1154, 1107, 1036,
837 cm-1; FAB MS (3-NBA) m/z 1173 (M+ + 1). Anal. Calcd for
C52H59N2O11S2SiCl5: C, 53.96; H, 5.10; N, 2.42. Found C, 54.03; H,
5.15; N, 2.40.
5′-O-(4,4′-Dimethoxytrityl)-3′-O-(tert-butyldimethylsilyl)-N3-
ethyluridine 2′-O-(CPG succinyl) tert-Butyl Disulfide (7). Long-
chain alkyl amino controlled-pore glass (1.0 g, 1000 Å pore size, 100
µmol amino groups/g, 120/200 mesh) was suspended in DMF (4.0 mL)
with 6 (0.58 g, 0.5 mmol, 5 equiv) and Et3N (0.14 mL, 1.0 mmol, 10
equiv). The mixture was gently swirled in the dark for 2 days. The
support was then vacuum filtered and rinsed successively with
DMF (15 mL), CH3OH (50 mL), and Et2O (50 mL), and the residual
solvents were removed in Vacuo. The unreacted amino groups were
acetylated by swirling the support for 1 h with acetic anhydride (0.70
mL, 7.0 mmol, 100 equiv) and DMAP (10 mg, 70 µmol, 1 equiv) in
pyridine (4 mL). The support was rinsed successively with pyridine
(30 mL), CH3OH (90 mL), and Et2O (90 mL), and the residual solvents
were removed in Vacuo. The nucleoside loading concentration was
32 µmol/g.14
3′,5′-O-(Tetraisopropyldisiloxane-1,3-diyl)-N4-benzoyl-2′-O-(2,3-
dihydroxypropyl)cytidine (8). 3′,5′-O-(Tetraisopropyldisiloxane-1,3-
diyl)-N4-benzoyl-2′-O-allylcytidine15 (1.34 g, 2.30 mmol) and N-
methylmorpholine N-oxide (0.27 g, 2.34 mmol, 1.1 equiv) were
dissolved in acetone:H2O (21 mL, 6:1), and OsO4 (5 mg, 21 µmol,
0.01 equiv) was added. The reaction mixture was stirred in the dark
for 2.5 h, after which an aqueous solution of saturated sodium bisulfite
(1 mL) was added to precipitate osmium salts. The solution was
decanted, and the light brown residue was diluted with Et2O and washed
with saturated NaHCO3 and brine. The combined organic layers were
dried over Na2SO4 and concentrated in Vacuo. The yellow residue was
purified by flash chromatography (CH2Cl2:CH3OH, step gradient of
19:1 to 37:3) to afford 8 as a brown foam (1.27 g, 90% yield): TLC
(CH2Cl2:CH3OH, 19:1) Rf ) 0.23; 1H NMR (360 MHz, CDCl3) δ (two
diastereomers) 98-1.11 (28 H, m, 4 (CH3)2CHSi, 4 (CH3)2CHSi), 3.66
(1 H, dd, J ) 5.2, 10.7 Hz, CH(OH)CH2aOH), 3.72-3.76 (2 H, m,
CH2CH(OH)CH2, CH(OH)CH2bOH), 3.88-4.06 (4 H, m, 2′, 5′, OCH2-
CH(OH)), 4.15-4.28 (2 H, m, 3′, 4′), 4.30 (1 H, d, J ) 13.5 Hz, 5′′),
5.83 (1 H, s, 1′), 7.49-7.63 (4 H, m, 5, Ar), 7.90-7.92 (2 H, m, Ar),
8.31-8.34 (1 H, m, 6), 8.94 (1 H, br s, NH); 13C NMR (90 MHz,
CDCl3) δ (two diastereomers) 12.5, 12.6, 12.9, 13.0 ((CH3)2CHSi), 16.8,
16.9, 17.0, 17.3, 17.4, 17.4, 17.6 ((CH3)2CHSi), 59.2 (5′), 63.6, 63.9
(CH(OH)CH2OH), 67.8 (3′), 70.4, 70.5 (OCH2CH(OH)), 73.4, 74.3
(CH2CH(OH)CH2), 82.0 (2′), 83.1, 83.3 (4′), 89.9, 90.2 (1′), 96.3 (5),
127.6, 129.0, 132.8 (Ar), 133.3 (6), 144.3 (2), 162.5 (4); IR (film; NaCl)
ν 3367, 2946, 2868, 1700, 1655, 1617, 1486, 1257, 1126, 1040, 886,
704 cm-1; FAB MS (3-NBA) m/z 664 (M+ + 1).
5′-O-(4,4′-Dimethoxytrityl)-2′-O-(tert-butyldimethylsilyl)-N3-
ethyluridine 3′-O-(â-Cyanoethyl N,N-diisopropylphosphoramidite)
tert-Butyl Disulfide (5). Compound 3 (0.81 g, 1.0 mmol) was dissolved
in THF (3.0 mL), and 2,4,6-collidine (1.0 mL, 7.5 mmol, 7.5 equiv)
and N-methylimidazole (40 µL, 0.5 mmol, 0.5 equiv) were added.
2-Cyanoethyl N,N-diisopropylphosphoramidochloridite (0.56 mL, 2.5
mmol, 2.5 equiv) was then added dropwise with stirring under N2. After
2 h, the reaction mixture was diluted with EtOAc and the mixture
washed with NaHCO3 and brine, dried over Na2SO4, and concentrated
in Vacuo. The residue was purified by flash chromatography (petroleum
ether:EtOAc:Et3N, 80:15:5) to afford 5 as a brittle white foam (0.82 g,
82% yield): TLC (petroleum ether:EtOAc:Et3N, 80:15:5) Rf ) 0.28;
1H NMR (300 MHz, CD3CN) δ (two diastereomers) 0.09, 0.12 (6 H,
2 s, Si(CH3)2), 0.83, 0.85 (9 H, 2 s, SiC(CH3)3), 1.01, 1.14 (12 H, d, J
) 9 Hz, 2 NCH(CH3)2), 1.31 (9 H, s, SSC(CH3)3), 2.45 (2 H, m,
OCH2CH2CN), 2.88 (2 H, m, CH2SS), 3.40 (2 H, m, 5′, 5′′), 3.50-
3.90 (4 H, m, OCH2CH2CN, 2 NCH(CH3)2), 3.75 (6 H, s, 2 OCH3),
4.10 (2 H, m, NCH2CH2SS), 4.20-4.42 (3 H, m, 2′, 3′, 4′), 5.37, 5.39
(1 H, d, J ) 8.1 Hz, 6), 5.84, 5.89 (1 H, d, J ) 6.6 Hz, 1′), 6.83-7.46
(13 H, m, Ar), 7.72, 7.77 (1 H, d, J ) 8.1 Hz, 5); 13C NMR (75 MHz,
CD3CN) δ (two diastereomers) -4.2 (Si(CH3)2), 18.9 (SiC(CH3)3), 21.1
(OCH2CH2CN), 25.0, 25.1, 25.2, 25.3 (NCH(CH3)2), 26.4 (SiC(CH3)3),
30.4 (SC(CH3)3), 37.8 (CH2SS), 41.2 (NCH2CH2SS), 44.1, 44.3, 44.5,
44.6 (NCH(CH3)2), 48.5 (SSC(CH3)3), 56.2 (OCH3), 59.4, 60.0 (POCH2-
CH2CN), 64.0, 64.2 (5′), 73.5, 73.7 (3′), 76.1, 76.5 (2′), 83.9, 84.0
(4′), 88.1 (OC(Ph)3), 90.3, 90.4 (1′), 102.4, 102.5 (5), 114.4 (Ar), 128.1,
129.0, 129.2, 129.3, 131.2, 136.4, 136.5, 136.6 (Ar), 139.3, 139.4 (6),
145.7, 145.8 (Ar), 152.1 (2), 160.0 (Ar), 162.9, 163.0 (4); 31P NMR
(202 MHz, CD3CN) δ (two diastereomers) 147.4, 146.8; IR (KBr) ν
2965, 2931, 2859, 2254, 1712, 1671, 1611, 1509, 1456, 1391, 1365,
1253, 1179, 1037, 979, 836 cm-1; FAB MS (3-NBA) m/z 1009.5 (M+
+ 1). Anal. Calcd for C51H73N4O9PS2Si: C, 60.70; H, 7.28; N, 5.55.
Found: C, 60.77; H, 7.24; N, 5.47.
5′-O-(4,4′-Dimethoxytrityl)-3′-O-(tert-butyldimethylsilyl)-N3-
ethyluridine 2′-O-Pentachlorophenylsuccinate tert-Butyl Disulfide
(6). Compound 4 (1.77 g, 2.2 mmol) was dissolved in pyridine (12.0
mL) under N2 followed by the addition of succinic anhydride (0.69 g,
6.6 mmol, 3.0 equiv) and 4-(dimethylamino)pyridine (DMAP) (0.133
g, 1.1 mmol, 0.5 equiv). After 12 h the reaction mixture was
concentrated in Vacuo and the residue was dissolved in CH2Cl2, washed
with brine, dried over Na2SO4, and concentrated in Vacuo. The crude
succinate was then dissolved in CH2Cl2 (25 mL), and pentachlorophenol
(0.88 g, 3.3 mmol, 1.5 equiv), DMAP (67 mg, 0.55 mmol, 0.25 equiv),
and dicyclohexylcarbodiimide (0.91 g, 4.4 mmol, 2.0 equiv) were added.
After 8 h, petroleum ether was added to precipitate dicyclohexylurea,
and the reaction mixture was filtered and concentrated in Vacuo.
Purification of the residue by flash chromatography (petroleum ether:
EtOAc, 4:1) afforded 6 as a white foam (2.3 g, 90% yield): TLC
3′,5′-O-(Tetraisopropyldisiloxane-1,3-diyl)-N4-benzoyl-2′-O-(formyl-
methyl)cytidine (9). Compound 8 (1.27 g, 1.91 mmol) was dissolved
in p-dioxane:H2O (20 mL, 3:1), and NaIO4 (0.49 g, 2.29 mmol, 1.2
equiv) was added. The reaction mixture was stirred in the dark for
4.5 h and then diluted with Et2O and washed with water. The organic
layer was dried over Na2SO4 and concentrated in Vacuo. Purification
of the residue by flash chromatography (CH2Cl2:CH3OH, 19:1) afforded
9 as a white foam (2.94 g, 97% yield): TLC (CH2Cl2:CH3OH, 37:3)
Rf ) 0.50; 1H NMR (360 MHz, CDCl3) δ 0.98-1.11 (28 H, m, 4
(CH3)2CHSi, 4 (CH3)2CHSi), 3.98-4.07 (2 H, m, 2′, 5′), 4.18-4.31 (3
H, m, 3′, 4′, 5′′), 4.47-4.59 (2 H, m, OCH2CHO), 5.86 (1 H, s, 1′),
7.49-7.63 (4 H, m, 5, Ar), 7.89-7.91 (2 H, m, Ar), 8.37 (1 H, d, J )
7.5 Hz, 6), 8.81 (1 H, br s, NH), 9.81 (1 H, s, CH2CHO); 13C NMR
(90 MHz, CDCl3) δ 12.3, 12.9, 13.0, 13.4 ((CH3)2CHSi), 16.7, 16.9,
17.3, 17.4 ((CH3)2CHSi), 59.2 (5′), 68.1 (3′), 76.2 (OCH2CHO), 81.8
(2′), 83.0 (4′), 89.7 (1′), 96.2 (5), 127.5, 129.0, 132.8 (Ar), 133.2 (6),
144.3 (2), 162.4 (4), 200.7 (CH2CHO); IR (film; NaCl) ν 2946, 2868,
1700, 1667, 1619, 1484, 1253, 1130, 1064, 1040, 886, 703 cm-1; FAB
MS (3-NBA) m/z 632 (M+ + 1).
1
(petroleum ether:EtOAc, 4:1) Rf ) 0.68; H NMR (360 MHz, CD3-
CN) δ -0.07, 0.01 (6 H, s, Si(CH3)2), 0.79 (9 H, s, SiC(CH3)3), 1.29
(9 H, s, SSC(CH3)3), 2.79 (4 H, m, succinate CH2), 3.03 (2 H, m, CH2-
SS), 3.29-3.47 (2 H, m, 5′, 5′′), 3.76 (6 H, s, 2 OCH3), 3.96-4.12 (3
H, m, 4′, NCH2), 4.38 (1 H, m, 3′), 5.43 (2 H, m, 6, 2′), 5.97 (1 H, d,
J ) 4.8 Hz, 1′), 6.21-7.45 (13 H, m, Ar), 7.65 (1 H, d, J ) 8.1 Hz,
5); 13C NMR (90 MHz, CD3CN) δ -4.4, -4.1 (Si(CH3)2), 18.6
(SiC(CH3)3), 26.1 (SiC(CH3)3), 29.4, 29.5 (succinate CH2), 30.2
(SSC(CH3)3), 37.0 (CH2SS), 41.1 (NCH2CH2SS), 48.5 (SSC(CH3)3),
56.0 (OCH3), 63.6 (5′), 71.4 (3′), 76.1 (2′), 85.3 (4′), 88.0 (OC(Ph)3),
88.7 (1′), 102.5 (5), 114.2 (Ar), 128.1, 129.0, 129.1, 131.2, 136.4 (Ar),
139.4 (6), 145.7 (Ar), 152.0 (2), 159.9 (Ar), 163.1 (4), 169.6, 171.8
3′,5′-O-(Tetraisopropyldisiloxane-1,3-diyl)-N4-benzoyl-2′-O-(2-hy-
droxyethyl)cytidine (10). Compound 9 (1.17 g, 1.85 mmol) was
dissolved in CH3OH (19 mL), and NaBH4 (21 mg, 0.56 mmol, 0.3
equiv) was added. The mixture was stirred under N2 in the dark for
90 min and then diluted with Et2O and washed with saturated NaHCO3
and brine. The aqueous layer was washed once with Et2O, and the

5214 J. Am. Chem. Soc., Vol. 118, No. 22, 1996
Goodwin et al.
combined organic layers were dried over Na2SO4 and concentrated in
Vacuo. The residue was purified by flash chromatography (CH2Cl2:
CH3OH, 19:1) to afford 10 as a white foam (1.09 g, 93% yield): TLC
(CH2Cl2:CH3OH, 37:3) Rf ) 0.56; 1H NMR (360 MHz, CDCl3) δ 0.99-
1.10 (28 H, m, 4 (CH3)2CHSi, 4 (CH3)2CHSi), 3.17 (1 H, br s, CH2-
CH2OH), 3.72-3.77 (2 H, m, CH2CH2OH), 3.93-4.03 (4 H, m, 2′, 5′,
OCH2CH2OH), 4.15-4.25 (2 H, m, 3′, 4′), 4.30 (1 H, d, J ) 13.6 Hz,
5′′), 5.83 (1 H, s, 1′), 7.49-7.63 (4 H, m, 5, Ar), 7.89-7.92 (2 H, m,
Ar), 8.35 (1 H, d, J ) 7.5 Hz, 6), 8.92 (1 H, br s, NH); 13C NMR (90
MHz, CDCl3) δ 12.6, 12.9, 13.0, 13.4 ((CH3)2CHSi), 16.8, 16.9, 17.0,
17.3, 17.4, 17.4 ((CH3)2CHSi), 59.3 (5′), 61.7 (CH2CH2OH), 68.0 (3′),
73.1 (OCH2CH2OH), 82.0 (2′), 82.3 (4′), 90.5 (1′), 96.2 (5), 127.5,
129.0, 132.9 (Ar), 133.2 (6), 144.3 (2), 162.4 (4); IR (film; NaCl) ν
2946, 2868, 1699, 1664, 1619, 1485, 1263, 1128, 1074, 1063, 1040,
886, 703 cm-1; FAB MS (3-NBA) m/z 634 (M+ + 1).
3′,5′-O-(Tetraisopropyldisiloxane-1,3-diyl)-N4-benzoyl-2′-O-(2-
(methylsulfonyl)ethyl)cytidine (11). Compound 10 (1.09 g, 1.72
mmol) was dissolved in CH2Cl2 (17 mL) and pyridine (1.4 mL, 17.18
mmol, 10 equiv) and cooled under N2 to 0 °C, and methanesulfonyl
chloride (0.19 mL, 2.41 mmol, 1.2 equiv) was added dropwise. The
mixture was stirred under N2 while gradually warming to room
temperature overnight. The reaction mixture was diluted with Et2O
and washed with saturated NaHCO3 and brine. The organic layer was
dried over Na2SO4 and concentrated in Vacuo, and purification of the
residue by flash chromatography (CH2Cl2:CH3OH, 24:1) afforded 11
as a white foam (1.13 g, 92% yield): TLC (CH2Cl2:CH3OH, 19:1) Rf
) 0.35; 1H NMR (360 MHz, CDCl3) δ 0.97-1.11 (28 H, m, 4 (CH3)2-
CHSi, 4 (CH3)2CHSi), 3.14 (3 H, s, CH3SO2), 3.97-4.01 (2 H, m, 2′,
5′), 4.17-4.21 (4 H, m, 3′, 4′, OCH2CH2OSO2), 4.30 (1 H, d, J )
13.7 Hz, 5′′), 4.46-4.49 (2 H, m, CH2CH2OSO2), 5.82 (1 H, s, 1′),
7.50-7.62 (4 H, m, 5, Ar), 7.89-7.92 (2 H, m, Ar), 8.38 (1 H, d, J )
7.5 Hz, 6); 13C NMR (90 MHz, CDCl3) δ 12.4, 12.9, 13.1, 13.4
((CH3)2CHSi), 16.8, 16.9, 17.1, 17.3, 17.4, 17.4 ((CH3)2CHSi), 37.80
(CH3SO2), 59.3 (5′), 67.9 (3′), 69.0 (CH2CH2OSO2), 69.5 (OCH2CH2-
OSO2), 81.9 (2′), 82.4 (4′), 89.5 (1′), 96.2 (5), 127.6, 129.1, 132.8 (Ar),
133.3 (6), 144.5 (2), 162.4 (4); IR (film; NaCl) ν 2945, 2868, 1664,
1484, 1170, 1128, 1074, 1063, 1040, 885, 704 cm-1; FAB MS (3-
NBA) m/z 712 (M+ + 1).
3′,5′-O-(Tetraisopropyldisiloxane-1,3-diyl)-N4-benzoyl-2′-O-(2-
thiobenzoylethyl)cytidine (12). Compound 11 (1.04 g, 1.46 mmol)
was dissolved in DMF (5.8 mL) and Et3N (2.0 mL, 14.6 mmol, 10
equiv), and thiobenzoic acid (0.34 mL, 2.91 mmol, 2.0 equiv) was
added. The mixture was stirred under N2 in the dark overnight, then
diluted with Et2O, and washed with saturated NaHCO3 and brine. The
organic layer was dried over Na2SO4 and concentrated in Vacuo to give
a dark orange-brown solid. The residue was purified by flash
chromatography (step gradient of 0 to 10% CH3CN in petroleum ether:
EtOAc, 2:1) to afford 12 as a white foam (0.84 g, 76% yield): TLC
(petroleum ether:EtOAc:CH3CN, 6:3:1) Rf ) 0.57; 1H NMR (360
MHz, CDCl3) δ 0.98-1.11 (28 H, m, 4 (CH3)2CHSi, 4 (CH3)2CHSi),
3.35-3.43 (2 H, m, CH2CH2SBz), 3.97-4.02 (2 H, m, 2′, 5′), 4.09-
4.13 (2 H, m, OCH2CH2SBz), 4.18 (1 H, dd, J ) 3.9, 9.6 Hz, 3′), 4.25
(1 H, dd, J ) 2.0, 9.6 Hz, 4′), 4.30 (1 H, d, J ) 13.5 Hz, 5′′), 5.84 (1
H, s, 1′), 7.40-7.62 (7 H, m, 5, Ar), 7.91-7.99 (4 H, m, Ar), 8.37 (1
H, d, J ) 7.4 Hz, 6), 8.97 (1 H, br s, NH); 13C NMR (90 MHz,
CDCl3) δ 12.5, 12.8, 13.1, 13.4 ((CH3)2CHSi), 16.8, 16.9, 17.1, 17.3,
17.4, 17.5 ((CH3)2CHSi), 29.1 (CH2CH2SBz), 59.4 (5′), 67.9 (3′), 69.9
(OCH2CH2SBz), 81.8 (2′), 81.9 (4′), 90.0 (1′), 96.0 (5), 127.2, 127.6,
128.5, 129.0, 133.0 (Ar), 133.2 (6), 137.1 (Ar), 144.6 (2), 162.3 (4),
191.5 (Ar); IR (film; NaCl) ν 2945, 2868, 1699, 1667, 1620, 1489,
1373, 1265, 1126, 1075, 1063, 1040, 691 cm-1; EI MS m/z 754
(M+ + 1).
3′), 5.87 (1 H, s, 1′), 7.33-7.54 (7 H, m, 5, Ar), 7.82-7.94 (4 H, m,
Ar), 8.57 (1 H, d, J ) 7.5 Hz, 6), 9.22 (1 H, br s, NH); 13C NMR (90
MHz, CDCl3) δ 28.8 (CH2SBz), 59.8 (5′), 67.4 (3′), 69.4 (OCH2CH2-
SBz), 82.0 (2′), 84.6 (4′), 90.1 (1′), 96.7 (5), 126.8, 127.2, 127.6, 128.3,
128.6, 128.8, 132.9, 133.1 (Ar), 133.5 (6), 136.7 (Ar), 146.0 (2), 162.6
(4), 191.4 (Ar); IR (film; NaCl) ν 3345, 2928, 1699, 1658, 1617, 1558,
1485, 1379, 1258, 1111, 1067, 913, 705, 689 cm-1; FAB MS (3-NBA)
m/z 512 (M+ + 1).
N4-Benzoyl-2′-O-ethylcytidine tert-Butyl Disulfide (14). Com-
pound 13 (0.25 g, 0.49 mmol) was dissolved in a CH3OH:THF mixture
(3.8 mL, 1:1) and 1-(tert-butylthio)-1,2-hydrazinedicarboxmorpholide12
(0.20 g, 0.58 mmol, 1.2 equiv) and LiOH‚H2O (41 mg, 0.97 mmol,
2.0 equiv) were added. The reaction mixture was stirred under N2 at
0 °C for 45 min, diluted with EtOAc, and washed with 1 N citric acid,
saturated NaHCO3, and brine. The organic layer was dried over Na2-
SO4 and concentrated in Vacuo, and the residue was purified by flash
chromatography (CH2Cl2:CH3OH, 24:1) to afford 14 as a pink foam
(0.18 g, 74% yield): TLC (CH2Cl2:CH3OH, 24:1) Rf ) 0.29; 1H NMR
(360 MHz, CDCl3) δ 1.32 (9 H, s, C(CH3)3), 2.93 (2 H, t, J ) 5.8 Hz,
CH2SS), 3.93-3.99 (2 H, m, 2′, 5′), 4.09-4.18 (3 H, m, 5′′, OCH2-
CH2SS), 4.24-4.30 (1 H, m, 4′), 4.33-4.41 (1 H, m, 3′), 5.84 (1 H, d,
J ) 1.7 Hz, 1′), 7.48-7.62 (4 H, m, 5, Ar), 7.87-7.89 (2 H, m, Ar),
8.48 (1 H, d, J ) 7.5 Hz, 6), 9.01 (1 H, br s, NH); 13C NMR (90 MHz,
CDCl3) δ 29.8 (C(CH3)3), 40.3 (CH2SS), 48.0 (C(CH3)3), 60.3 (5′),
67.8 (3′), 69.0 (OCH2CH2SS), 81.7 (2′), 85.0 (4′), 90.9 (1′), 96.7 (5),
127.6, 129.0, 132.9 (Ar), 133.3 (6), 146.4 (2), 162.5 (4); IR (film; NaCl)
ν 3374, 2960, 2922, 1699, 1648, 1617, 1558, 1487, 1379, 1260, 1110
cm-1; FAB MS (3-NBA) m/z 496 (M+ + 1).
5′-O-(4,4′-Dimethoxytrityl)-N4-benzoyl-2′-O-ethylcytidine tert-Bu-
tyl Disulfide (15). Compound 14 (0.10 g, 0.21 mmol) and DMAP
(13 mg, 0.10 mmol, 0.5 equiv) were dissolved in DMF (0.8 mL) and
pyridine (26 µL, 0.31 mmol, 1.5 equiv), and 4,4′-dimethoxytrityl
chloride (85 mg, 0.25 mmol, 1.2 equiv) was added. The reaction
mixture was stirred under N2 for 6 h, diluted with CH2Cl2, and washed
with saturated NaHCO3 and brine. The organic layer was dried over
Na2SO4 and concentrated in Vacuo. The residue was purified by flash
chromatography (acetone:petroleum ether, 1:1) to afford 15 as a tan
foam (0.13 g, 77% yield): TLC (acetone:petroleum ether, 1:1) Rf )
1
0.23; H NMR (360 MHz, CD3CN) δ 1.30 (9 H, s, C(CH3)3), 2.95 (2
H, t, J ) 6.3 Hz, CH2SS), 3.39-3.44 (2 H, m, OCH2CH2SS), 3.76 (6
H, s, 2 OCH3), 3.90-4.03 (3 H, m, 2′, 5′, 5′′), 4.12-4.19 (1 H, m, 4′),
4.39-4.47 (1 H, m, 3′), 5.84 (1 H, d, J ) 1.7 Hz, 1′), 6.87-6.89 (4 H,
m, Ar), 7.15-7.61 (13 H, m, 5, Ar), 7.92-7.95 (2 H, m, Ar), 8.46 (1
H, d, J ) 7.6 Hz, 6); 13C NMR (90 MHz, CD3CN) δ 30.2 (C(CH3)3),
41.0 (CH2SS), 48.5 (C(CH3)3), 56.0 (OCH3), 62.1 (5′), 68.9 (3′), 70.1
(OCH2CH2SS), 83.3 (2′), 83.6 (4′), 87.7 (OC(Ph)3), 90.2 (1′), 97.2 (5),
114.3, 128.1, 129.1, 129.1, 129.2, 129.7, 131.0, 131.2, (Ar), 133.9 (6),
134.5, 136.6, 137.0, 145.5 (Ar), 145.9 (2), 155.7, 159.8 (Ar), 163.9
(4), 168.2 (Ar); IR (KBr) ν 3392, 2959, 2924, 1700, 1667, 1610, 1553,
1510, 1482, 1377, 1252, 1113, 1033, 704 cm-1; FAB MS (3-NBA)
m/z 798 (M+ + 1).
5′-O-(4,4′-Dimethoxytrityl)-N4-benzoyl-2′-O-ethylcytidine tert-Bu-
tyl Disulfide 3′-O-(â-Cyanoethyl N,N-diisopropylphosphoramidite)
(16). Compound 15 (55 mg, 0.07 mmol) was dissolved in CH2Cl2 (0.3
mL) and N,N-diisopropylethylamine (60 µL, 0.35 mmol, 5 equiv) and
cooled under N2 to 0 °C. 2-Cyanoethyl N,N-diisopropylphosphorami-
dochloridite (23 µL, 0.10 mmol, 1.5 equiv) was added dropwise, and
the reaction mixture stirred under N2 while being warmed to room
temperature. After 2 h the excess chloridate was quenched with CH3-
OH (0.3 mL), and the mixture was concentrated in Vacuo. The residue
was purified by flash chromatography (petroleum ether:acetone, 2:1)
to afford 16 as a white foam (58 mg, 84% yield): TLC (petroleum
N4-Benzoyl-2′-O-(2-thiobenzoylethyl)cytidine (13). Compound 12
(0.37 g, 0.49 mmol) was dissolved in CH3CN (4.3 mL), and HF (0.5
mL, 48%) was added. The reaction mixture was stirred for 7 h, after
which the solution was diluted with Et2O and washed with H2O. The
organic layer was dried over Na2SO4 and concentrated in Vacuo. The
pink residue was purified by flash chromatography (CH2Cl2:CH3OH,
19:1) to afford 13 as a white foam (0.25 g, 100% yield): TLC (CH2-
ether:acetone, 2:1) Rf ) 0.40; H NMR (300 MHz, CD3CN) δ (two
1
diastereomers) 1.05-1.20 (12 H, m, 2 NCH(CH3)2), 1.30 (9 H, s,
C(CH3)3), 2.50-2.67 (2 H, m, OCH2CH2CN), 2.94-3.01 (2 H, m, CH2-
SS), 3.41-3.71 (6 H, m, OCH2CH2SS, OCH2CH2CN, 2 NCH(CH3)2),
3.79 (6 H, s, 2 OCH3), 3.80-4.22 (4 H, m, 2′, 4′, 5′, 5 ′′), 4.43-4.63
(1 H, m, 3), 5.88 (1 H, s, 1′), 6.87-6.92 (4 H, m, Ar), 7.03-7.66 (13
H, m, 5, Ar), 7.91-7.97 (2 H, m, Ar), 8.42-8.55 (1 H, 2 d, J ) 7.6
Hz, 6); 13C NMR (75 MHz, CD3CN) δ (two diastereomers) 21.2, 21.3
(OCH2CH2CN), 24.9, 25.0, 25.2, 25.3 (NCH(CH3)2), 30.3 (C(CH3)3),
41.4 (CH2SS), 44.1, 44.3 (NCH(CH3)2), 48.5 (C(CH3)3), 56.0 (OCH3),
1
Cl2:CH3OH, 19:1) Rf ) 0.21; H NMR (360 MHz, CDCl3) δ 3.25-
3.42 (2 H, m, CH2SBz), 3.91-3.96 (2 H, m, 2′, 5′), 4.05-4.19 (3 H,
m, 5′′, OCH2CH2SBz), 4.20-4.25 (1 H, m, 4′), 4.35-4.37 (1 H, m,

Yeast tRNAPhe Analogs with Disulfide Cross-Links
J. Am. Chem. Soc., Vol. 118, No. 22, 1996 5215
59.3, 59.6 OCH2CH2CN), 61.8, 62.2 (5′), 70.2, 70.4 (3′), 70.8 (OCH2-
CH2SS), 82.2 (2′), 83.1 (4′), 87.8 (OC(Ph)3), 90.8, 91.1 (1′), 97.3 (5),
114.2, 128.1, 129.0, 129.0, 129.3, 129.6, 131.2 (Ar), 133.8 (6), 134.5,
136.6, 136.7, 145.4 (Ar), 145.6 (2), 155.4, 159.8 (Ar), 163.6 (4); 31P
NMR (202 MHz, CD3CN) δ 147.57, 146.51; IR (film; NaCl) ν
2967, 2934, 1708, 1686, 1509, 1510, 1462, 1251, 1180, 1035, 979
cm-1; FAB MS (3-NBA) m/z 998 (M+ + 1). Anal. Calcd for
C52H64N5O9PS2: C, 62.56; H, 6.48; N, 7.02. Found: C, 62.41; H, 6.41;
N, 6.92.
volume of 4 µL) and formamide (4 µL, 80% v/v) containing
bromophenol blue (0.1% w/v) and xylene cyanole (0.1% w/v) tracking
dyes. The samples were loaded onto a denaturing gel (15% polyacryl-
amide, 29:1 acrylamide:bisacrylamide, 8 M urea, 31.0 cm × 38.5 cm
× 0.4 mm) and electrophoresed at constant power (55 W) in TBE until
the xylene cyanole dye had migrated 27 cm. The gel was autoradio-
graphed for 20 h at -78 °C on FUJI RX film using an intensifying
screen.
Native Analytical PAGE. An aliquot of an aqueous solution of
each 32P-end-labeled tRNA (7500 cpm) was concentrated in Vacuo and
dissolved in 5X Tris-HEPES buffer (2 µL, 170 mM Tris, 330 mM
HEPES, 0.5 mM EDTA, 10 mM MgCl2, pH 7.5) and H2O (2.5 µL).
The samples were heated to 80 °C for 2 min and allowed to cool to 45
°C. MgCl2 (0.5 µL, 120 mM) was added, and the samples were
equilibrated at room temperature for about 15 min. Sucrose loading
buffer (5 µL, 25% w/v) was then added, and each sample was placed
on ice for 2 min prior to loading onto a native gel (15% polyacrylamide,
29:1 acrylamide:bisacrylamide; 14 cm × 16 cm × 0.8 mm) which had
been equilibrated at 4 °C for 1 h after polymerization. The samples
were electrophoresed at constant voltage (300 V) for 10 h in 1X Tris-
HEPES buffer at 10 °C using a Hoefer SE600 electrophoresis apparatus
attached to a recirculating water bath, until the xylene cyanole tracking
dye (in a blank lane) had migrated 15 cm.
Disulfide Cross-Link Formation. The requisite tert-butyl disulfide-
modified tRNA (∼3 OD260 units; 130 µg, 5 nmol) was dissolved in
phosphate buffer (50 µL, 100 mM Na2HPO4, pH 8.3), and DTT (54
µg, 200 equiv/disulfide) was added. The mixture was sealed and
incubated at 25 °C for 12 h. The reaction mixture was then diluted
with phosphate buffer (200 µL, 5 mM Na2HPO4, 5 mM NaCl, pH 7.0)
and dialyzed against the same buffer (3 L) at room temperature for 10
h. The reduced and dialyzed tRNA was diluted in phosphate buffer
(pH 7.0) to a final concentration of 10 µM, heated to 70 °C for 1 min,
and allowed to cool to ∼40 °C. At this time MgCl2 (0.1 M) was added
to a final [Mg2+] of 5 mM. After the solution was equilibrated at room
temperature for 30 min, the pH was adjusted to 8.0 with NaOH (0.1
M), and the reaction mixture was stirred at room temperature, exposed
to air. At 6 h intervals, an aliquot (5 µL) of the reaction mixture was
mixed with 5X CPM buffer (1 µL, 250 mM Tris-HCl, 2.6 M NaCl,
0.5 mM EDTA, 0.15% (v/v) Triton X-100, pH 7.5) and CPM (5 µL of
a 0.4 mM CPM stock solution in HPLC-grade i-PrOH).21 The mixture
was incubated at room temperature for 10 min and then diluted with
1% Triton X-100 (489 µL). The fluorescence intensity at 480 nm (λex
) 390 nm) was corrected for background fluorescence, and the amount
of free thiol remaining was quantified on the basis of a calibration
curve constructed for N3-(thioethyl)uridine.4g After 12 h, no free thiol
groups were present and the reaction mixture was either ethanol
precipitated or dialyzed against TE buffer (3 L, 10 mM Tris-HCl, 1
mM EDTA, pH 8.0, 10 h) and stored at -20 °C.
UV Thermal Denaturation. tRNA samples for UV thermal
denaturation experiments were dialyzed against phosphate buffer (3
L, 5 mM Na2HPO4, 5 mM NaCl, pH 7.2, 10 h), and aliquots of tRNA
were diluted with the same degassed buffer (final volume of 1 mL).
tRNA solutions were heat-denatured at 90 °C for 1 min and then cooled
to 10 °C at a cooling rate of 3 °C/min. For experiments with Mg2+ in
the buffer, MgCl2 (100 mM MgCl2 in 5 mM Na2HPO4, 5 mM NaCl,
pH 7.2) was added to a final [Mg2+] of 5 mM after the sample had
cooled to ∼40 °C. The tRNA samples were equilibrated at 10 °C for
30 min prior to thermal denaturation. The heating rate was 0.5 °C/
min with absorbance monitored at 260 nm and a data interval of 0.1
°C. The melting temperature, Tm, was obtained by nonlinear regression
analysis of the denaturation curve as described by Blatt et al.37
32P-Labeling of tRNA. tRNA samples (5 pmol, ∼3 × 10-3 OD260
unit) were dissolved in kinase buffer (10 µL, 70 mM Tris-HCl, 10
mM MgCl2, 0.1 mM EDTA, 5% glycerol, pH 7.5) containing T4
polynucleotide kinase (10 units) and [γ-32P]ATP (10 µCi, 33 pmol,
6000 Ci/mmol). The mixture was incubated at 37 °C for 30 min.
Formamide (10 µL, 80% v/v) containing bromophenol blue (0.1% w/v)
and xylene cyanole (0.1% w/v) tracking dyes was added to each sample,
and the mixture was electrophoresed on a denaturing gel (15%
polyacrylamide, 29:1 acrylamide:bisacrylamide, 8 M urea, 31.0 cm ×
38.5 cm × 0.8 mm) at constant power (55 W) in TBE (90 mM Tris-
borate, 2 mM EDTA, pH 8.3) for 5 h. The labeled tRNA was excised
from the gel and electroeluted into 4X TAE (300 µL, 160 mM Tris,
80 mM acetic acid, 8 mM EDTA, pH 7.5) using a Hoefer SixPac
microelectroeluter at +50 V for 1 h. The eluted tRNA was concentrated
in Vacuo to 50 µL and precipitated with sodium acetate (NaOAc) (50
µL, 3 M, pH 5.5) and ethanol (EtOH) (300 µL, 100% v/v) to remove
residual EDTA and urea. The specific activity (cpm/µL) of each sample
was determined by Cerenkov counting.
Lead(II) Autocleavage. An aliquot of an aqueous solution of each
32P-end-labeled tRNA (7.5 × 104 cpm) was concentrated in Vacuo and
dissolved in native precleavage buffer (4 µL, 15 mM MOPS, 1.5 mM
spermine, pH 7.0) or semidenaturing precleavage buffer (10 µL, 15
mM MOPS, pH 7.0) along with unlabeled carrier tRNA (50 µg), and
heat-denatured at 70 °C for 1 min. The samples were slowly cooled
to ∼40 °C, and MgCl2 (6 µL, 25 mM in native precleavage buffer;
final concentration of 15 mM) was added to the native reaction mixture.
After equilibration at room temperature for 30 min, Pb(OAc)2 (0.2 µL,
10 mM, final concentration of 200 µM) was added. The reaction
mixtures were incubated at 40 °C for 10 min and quenched with NaOAc
(80 µL, 1.5 mM, pH 5.5), and the tRNAs were precipitated with
glycogen (1 µL, 20 µg/µL) and EtOH (250 µL, 100% v/v) at -80 °C
for 15 min. Following centrifugation (16000 g, 4 °C, 30 min) the
supernatants were removed and the pellets gently blotted dry. For lead-
cleavage reactions of disulfide cross-linked tRNAs, the pellets were
dissolved in an aqueous solution of DTT (20 µL, 50 mM) and incubated
at 37 °C for 1 h. The tRNAs were precipitated with NaOAc (80 µL,
1.5 M, pH 5.5), glycogen (1 µL), and EtOH (250 µL, 100% v/v) at
-80 °C for 15 min. The reduced sequences were dissolved in Tris-
HCl (45 µL, 50 mM, pH 8.0) at 37 °C and treated with aliquots of
N-ethylmaleimide (5 µL, 50 mM in H2O) every 30 min for 1.5 h to
block the thiols from reoxidation.32 The reduced, blocked tRNA was
ethanol precipitated, and the pellets were rinsed once with EtOH (80%
v/v), blotted dry, and dissolved in urea loading buffer (5 µL, 9.8 M)
for electrophoresis. To locate the cleavage sites, hydroxide and RNase
T1 cleavage ladders were run in parallel with the lead cleavage lanes.
The sequencing and lead cleavage reactions were electrophoresed on a
denaturing gel (15% polyacrylamide, 29:1 acrylamide:bisacrylamide,
8 M urea, 31.0 cm × 38.5 cm × 0.4 mm) at constant power (55 W) in
TBE, and the product bands were visualized by autoradiography.
Acknowledgment. This work was supported by grants from
the NIH (GM 53861) and NSF (CHE 9357117), an NIH
postdoctoral fellowship to J.T.G. (GM 16133), and an NIH
Molecular Biophysics predoctoral fellowship to E.J.S. (T32 GM
08270). G.D.G. is the recipient of a National Arthritis Founda-
tion Arthritis Investigator Award, an American Cancer Society
Junior Faculty Research Award, a National Science Foundation
Young Investigator Award, a Camille Dreyfus Teacher-Scholar
Award, and a research fellowship from the Alfred P. Sloan
Foundation.
Denaturing Analytical PAGE. An aliquot of an aqueous solution
of each 32P-end-labeled tRNA (7500 cpm) was diluted with H2O (total
(37) Blatt, N. B.; Osborne, S. E.; Cain, R. J.; Glick, G. D. Biochimie
1993, 75, 433-441.
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