Synthesis of guanosine and deoxyguanosine phosphoramidites with cross-linkable thioalkyl tethers for direct incorporation into RNA and DNA

Article abstract of DOI:10.1080/15257770903368385

We describe the synthesis of protected phosphoramidites of deoxyriboguanosine and guanosine derivatives containing a thiopropyl tether at the guanine N2 (7a,b) for site-specific crosslinking from the minor groove of either DNA or RNA to a thiol of a prote

Full text of DOI:10.1080/15257770903368385

Nucleosides, Nucleotides and Nucleic Acids, 28:1076–1094, 2009
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770903368385
SYNTHESIS OF GUANOSINE AND DEOXYGUANOSINE
PHOSPHORAMIDITES WITH CROSS-LINKABLE THIOALKYL
TETHERS FOR DIRECT INCORPORATION INTO RNA AND DNA
Xiaorong Hou, Gang Wang, Barbara L. Gaffney, and Roger A. Jones
Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey,
Piscataway, New Jersey, USA
We describe the synthesis of protected phosphoramidites of deoxyriboguanosine and guanosine
2
derivatives containing a thiopropyl tether at the guanine N2 (7a,b) for site-specific crosslinking from
the minor groove of either DNA or RNA to a thiol of a protein or another nucleic acid. The thiol is
initially protected as a tert-butyl disulfide that is stable during oligonucleotide synthesis. While the
completed oligonucleotide is still attached to the support, or after purification, the tert-butyl thiol
can readily be removed or replaced by thioethylamine or 5-thio-2-nitrobenzoic acid, which have more
favorable crosslinking rates.
Keywords DNA; RNA; guanosine; thioalkyl tether; disulfide crosslink
INTRODUCTION
Useful methods for site-specific disulfide crosslinking of nucleic acid
fragments to themselves or to proteins were developed by Verdine[1] and
Glick.[2] These approaches have proven to be particularly valuable for
enhancing structural studies because otherwise heterogeneous populations
can thereby be constrained into functionally relevant positions.[3–8] Disul-
fide crosslinked complexes of oligonucleotides have also helped to under-
stand enzymatic reactions,[9–11] probe ribozyme function,[12,13] increase
stability,[14–19] delineate folding pathways and dynamics,[20–23] and prepare
conjugates.[24]
Received 21 May 2009; accepted 25 September 2009.
This work was supported by NIH GM66671.
Address correspondence to Roger A. Jones, Department of Chemistry and Chemical Biology, 610
Taylor Road, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, USA. E-mail:
r.jones@rutgers.edu
1076

Guanosine Thioalkyl Tethers
1077
The “convertible nucleoside” approach was developed by Verdine for
introducing a thioalkyl tether in DNA[25] and RNA[26] following oligonu-
cleotide synthesis. Among other sites, the method was used for the N2 of
guanosine by displacement of a 2-fluoro in a modified inosine.[27] This
postsynthetic displacement strategy is particularly valuable when a variety
of linkers are to be used with a given oligonucleotide, but suffers from
having a low yield step at the end of the synthesis. When large amounts
of a single tethered oligonucleotide are required, direct incorporation of a
modified nucleoside already containing the tether may be more efficient.
This approach was developed by Glick for the N3 of uridine/thymidine and
the C5/2ꢁO of cytidine.[28–30] We report here procedures for the synthe-
sis of protected phosphoramidites of deoxyriboguanosine and guanosine
derivatives containing a thiopropyl tether at the guanine N2 (7a,b), for
crosslinking from the minor groove of either DNA or RNA to proteins
or other nucleic acids. Although the tether described here contains three
methylenes, a shorter or longer one could easily be used. Further, the
thiol is initially protected as a tert-butyl disulfide, which was shown by
Glick to be stable during oligonucleotide synthesis.[30] While the completed
oligonucleotide is still attached to the support, or after purification, the
tert-butyl thiol can readily be removed or replaced by other disulfides that
have more favorable crosslinking rates.
RESULTS AND DISCUSSION
In our procedure (Scheme 1), the thioalkylamine disulfide is introduced
directly into the nucleoside by displacement of a 2-fluoro, rather than after
oligonucleotide synthesis as in Verdine’s strategy.[27] Preparation of the
deoxyribo- and ribo-2-fluoroinosine intermediates (4a,b) in good yield re-
quires prior protection of the O6 by a Mitsunobu alkylation. Rather than the
p-nitrophenylethyl group that needs an additional deprotection step,[27] we
have used the acid-labile trimethylsilylethyl group described by Harris.[31]
It is sufficiently stable to survive synthesis of the amidite, but is removed
without consequence during the detritylation steps of oligonucleotide syn-
thesis. We introduce it here with transient protection of the hydroxyls.[32]
Deoxyguanosine (1a) or guanosine (1b) is first treated with trimethylsilyl
imidazole to give 2a,b, followed by reaction with trimethylsilylethanol, diiso-
propylazodicarboxylate, and triphenylphosphine. After removal of the sugar
trimethylsilyl (TMS) groups in dilute NH3, purification by normal phase
chromatography gives 3a in 88% yield from 1a, and crystallization gives
3b in 91% yield from 1b. The subsequent nonaqueous diazotization using
tert-butyl nitrite and pyridine•HF, first reported by Robins,[33] converts the
amino to a fluoro and gives 4a,b in 85–88% yield.

1078

Guanosine Thioalkyl Tethers
1079
For the deoxyribo compound, protection of the 5ꢁ hydroxyl with
the standard dimethoxytrityl group gives 5 in 67% yield and provides
a convenient lipophilic handle for workup after the subsequent dis-
placement of the fluoro with 3-aminopropyl-tert-butyl disulfide, made
from its hydrochloride 10, whose synthesis is described below. This
displacement takes place in pyridine over 3 days at 60◦C to give 6
in 81% yield. Synthesis of the final phosphoramidite uses 2-cyanoethyl
(N,N,N ꢁ,N ꢁ-tetraisopropyl)phosphordiamidite activated with pyridinium
trifluoroacetate[34] and gives 7a in 61% yield.
For the ribo compound, we found it necessary for displacement of
the fluoro to be done before 2ꢁ protection with the tert-butyldimethylsilyl
(TBS) group to avoid cleavage of the TBS by the released fluoride, and
8 is produced in 92% yield. We next introduce the TBS group at the 2ꢁ
hydroxyl, following a procedure reported by Beigelman that uses temporary
protection of the 3ꢁ and 5ꢁ hydroxyls as a di-tert-butylsilylene.[35] Subsequent
dimethoxytritylation gives 9 in 85% yield from 8, and the final phospho-
ramidite, 7b, is made as for 7a in 80% yield.
Synthesis of the tert-butyl disulfide protected thiopropylamine hy-
drochloride (10) is shown in Scheme 2 and starts with treatment of
3-chloropropylammonium chloride (11) with sodium thiosulfate to give
12, followed by treatment with I2 to give 13, as described by Doi and
Musker.[36] We find that purification of the crude disulfide (13) using
a falling film distillation apparatus with toluene as the refluxing solvent
gives a much better yield (80%) than standard vacuum distillation. Re-
duction with dithiothreitol (DTT) then gives the free thioalkylamine (14).
Protection as the tert-butyl disulfide is done by a modified version of a
procedure described by Ellman,[37] using diisopropyl-1-(tert-butylthio)-1,2-
hydrazinedicarboxylate (15) made from diisopropylazodicarboxylate and
tert-butyl thiol according to Wu¨nsch et al.[38]
Using standard phosphoramidite chemistry with 7a at single positions
and commercial amidites elsewhere, we have made a DNA 19mer (16a)
and a DNA 20mer (16b), each with one deoxyguanosine containing a
tert-butyl disulfide protected thiopropyl tether at the N2. The couplings for
the modified amidite 7a gave yields similar to those with the commercial
amidites. Following standard high performance liquid chromatography
(HPLC) purification, desalting, and conversion to the sodium form, we
obtained 56 µmol of the pure DNA 19mer in 80% yield. The DNA 20mer
was left on the support for further modification.
The tert-butyl thiol protecting group is not an efficient leaving group
for crosslinking. Therefore, we have developed procedures to conveniently
remove it by reduction to give the free thiol (17a, DNA), or exchange it
to other disulfides, such as the moderately reactive 2-thioethylamine (19a,
DNA)[4,15] or the even more reactive 5-thio-2-nitrobenzoic acid (TNB) (18a,
DNA) (Scheme 3).[39,40] The TNB disulfide has been shown to be the

1080

Guanosine Thioalkyl Tethers
1081
SCHEME 3
most reactive of these three alternatives and, as a consequence, may not
display sufficient selectivity when several crosslinking sites are available.[40]
However, for cases requiring forced crosslinking to a single site, the high
reactivity of TNB may be desirable.[39] Although the thioethylamine is often
convenient, occasionally it may be too unreactive.[40] The free thiol requires
air oxidation to crosslink with another thiol.[9,14,20,27]
For DNA, the free thiol 17a is generated by treatment of 16a with
dithiothreitol (DTT) at pH 8 for 3 hours at 40◦C, followed by HPLC
purification. The TNB disulfide, 18a, is made by treating the free thiol, 17a,
with commercially available 5,5ꢁ-dithiobis(2-nitrobenzoic acid) at pH 8 for
30 minutes at room temperature, followed by HPLC purification. Although
the ethylamine disulfide, 19a, can be made by a similar two-step method,
we found it can be prepared more conveniently and in higher yield in
a one-flask procedure by adding a mixture of cysteamine hydrochloride
(2-aminoethanethiol hydrochloride) and cystamine dihydrochloride (2,2ꢁ-
diaminodiethyl disulfide dihydrochloride, both commercially available), to

1082
X. Hou et al.
16a at pH 8 for 2 hours at 40◦C. Most of the product forms directly from
the aminoethanethiol, but the small amount of free thiol that is generated
is converted to 19a by reaction with cystamine. HPLC purification then gives
pure 19a.
We also found that the tert-butyl thiol protecting group can be ex-
changed to the aminoethanethiol while the DNA is still attached to the solid
phase. Support containing the DNA 20mer is shaken at room temperature
with an aqueous solution of cysteamine hydrochloride and cystamine di-
hydrochloride at pH 8. After 2 days, the excess reagents are easily washed
away, and the DNA can then be cleaved from the support, deprotected, and
purified as usual, to give pure 19b.
We also made a RNA 23mer, 16c, with one guanosine containing a
tert-butyl disulfide protected thiopropyl tether, using 7b. Using the same
methods described for DNA, 16c can be converted to the free thiol, 17c,
the TNB disulfide, 18c, and the ethylamine disulfide, 19c.
CONCLUSION
We have demonstrated that direct synthesis of both DNA and RNA
fragments containing guanine N2 thioalkyl tethers is an efficient approach
to the preparation of cross-linkable oligonucleotides. The tether is pro-
tected as a stable tert-butyl disulfide during oligonucleotide synthesis and is
subsequently converted to more reactive groups for crosslinking to proteins
or other nucleic acids. For many applications, the ethylamine disulfide may
be ideal, but either the TNB group or the free thiol may be appropriate
under some circumstances. This approach is particularly well suited to
experiments requiring large amounts of oligonucleotides with a given
linker.
EXPERIMENTAL
General Methods
Analytical reverse phase HPLC was carried out on a Waters Alliance
system using XTerra MS or Atlantis dC18 columns, with 0.1M triethylammo-
nium acetate (TEAA, pH = 6.8) and CH3CN gradients. A Waters Micromass
LCZ spectrometer was used for ESI-MS. Semi-preparative HPLC of the
oligonucleotides was performed on a Waters Nova-Pak HR C18 19 × 300
mm column (Waters Corporation, Milford, MA, USA). Oligonucleotides
were analyzed by anion exchange HPLC using a Dionex NucleoPac PA-100
column on a Biocad Sprint Perfusion Chromatography System (Perseptive
Biosystems, Framingham, MA, USA) using gradients of LiClO4 with 0.02 M
CH3COOLi in 10% CH3CN in water. Pure oligonucleotides were charac-
terized by ESI-MS, using MassLynx software to deconvolute the spectra of

Guanosine Thioalkyl Tethers
1083
multiply charged ions. The amounts of DNA and RNA were measured on
an AVIV 14DS UV spectrophotometer at 260 nm using calculated extinction
coefficients. 1H NMR spectra were acquired either on a Varian Mercury 300
MHz or a Varian Unity 400 MHz NMR spectrometer (Varian, Palo Alto,
CA, USA). 31P NMR spectra were acquired on the Varian Mercury and
referenced to neat phosphoric acid at 0.0 ppm.
O6-(Trimethylsilylethyl)-2ꢁ-deoxyguanosine (3a). 2ꢁ-Deoxyguanosine hy-
drate, 1a, (1.43 g, 5.0 mmol) was dried by co-evaporation with 1,4-dioxane
and then suspended in 70 mL 1,4-dioxane. Trimethylsilyl imidazole (2.0 mL,
13.6 mmol, 2.7 equiv.) was added by syringe to the stirred suspension under
N2. This protection step was complete in 30 minutes. Triphenylphosphine
(6.55 g, 25.0 mmol, 5.0 equiv.), trimethylsilylethanol (3.6 mL, 22.5 mmol,
5.0 equiv.), and diethylazodicarboxylate (3.97 mL, 25.2 mmol, 5.0 equiv.)
were added to the reaction mixture under N2, which turned a clear orange.
This Mitsunobu reaction was complete in 1 hour. A mixture of 20 mL
concentrated aqueous NH3 and 35 mL water was added to the reaction
mixture, which was stirred overnight. Water (∼200 mL) was added, and the
solution was extracted three times with methylene chloride. The combined
organic layers were concentrated and purified by normal phase chromatog-
raphy using a gradient of 0–10% methanol in methylene chloride. Fractions
with pure product were combined, concentrated to a foam, and dried in
a vacuum desiccator over P2O5 to give 1.62 g of 3a (4.41 mmol, 88%
from 1a). The mass of 3a was confirmed by ESI-MS in negative mode
as m/z (M-H+CH3COOH) 426.28 (calculated for C15H24N5O4Si·C2H4O2:
1
426.52). UV λmax 248, 281 nm. H NMR (DMSO) δ 8.05 (s, 1H), 6.34 (s,
2H), 6.20 (t, J = 7.0 Hz, 1H), 5.28–5.25 (m, 1H), 5.01 (t, J = 5.4 Hz,
1H), 4.51–4.47 (m, 2H), 4.37–4.31 (m, 1H), 3.83–3.80 (m, 1H), 3.59–3.47
(m, 2H), 2.62–2.53 (m, 1H), 2.23–2.17 (m, 1H), 1.14–1.10 (m, 2H),
0.05 (s, 9H).
2-Fluoro-O6-(trimethylsilylethyl)-2ꢁ-deoxyinosine (4a). At –30◦C under
argon, 10 mL anhydrous pyridine, 5 mL anhydrous toluene and 11 mL
70% HF·pyridine (423 mmol, 211 equiv.) were mixed well and then added
to 3a (0.74 g, 2 mmol). Tert -butyl nitrite (0.8 mL, 6.7 mmol, 3.4 equiv.)
was added dropwise by syringe to the stirred reaction mixture maintained
at −30◦C. This nonaqueous diazotization was complete in 2 hours. The
excess HF was then quenched by slowly pouring the reaction mixture into a
stirred solution of 30 g K2CO3 in 50 mL water in an ice bath. The solution
was extracted three times with ethyl acetate. The combined organic layers
were concentrated and purified by normal phase chromatography using a
gradient of 0–10% methanol in methylene chloride. Fractions with pure
product were combined, concentrated to a foam, and dried in a vacuum
desiccator over P2O5 to afford 0.63 g of 4a (1.7 mmol, 85%). The mass of

1084
X. Hou et al.
4a was confirmed by ESI-MS in negative mode as m/z (M-H+CH3COOH)
429.15 (calculated for C15H22FN4O4Si·C2H4O2: 429.49). UV λmax 255 nm.
1H NMR (CDCl3) δ 8.00 (s, 1 H), 6.36–6.32 (m, 1H), 4.93–4.91 (m, 1H),
4.82–4.79 (m, 1H), 4.72–4.67 (m, 2H), 4.22–4.19 (m, 1H), 4.01–3.96 (m,
1H), 3.86–3.78 (m, 1H), 2.98–2.90 (m, 1H), 2.81–2.76 (m, 1H), 2.42–2.35
(m, 1H), 1.29–1.24 (m, 2H), 0.10 (s, 9H).
5ꢁ-O-(4,4ꢁ-Dimethoxytrityl)-2-fluoro-O6-(trimethylsilylethyl)-2ꢁ-deoxyino-
sine (5). To compound 4a (0.75 g, 2.0 mmol) dried by co-evaporation
with pyridine, and dissolved in 30 mL anhydrous pyridine, was added
4, 4ꢁ-dimethoxytrityl chloride (0.87 g, 2.56 mmol, 1.3 equiv.) under N2,
and the mixture was stirred for 3 hours. The reaction was quenched with
aqueous methanol followed by 3 × 50 mL extractions with ethyl ether.
The combined organic layers were concentrated and purified by normal
phase chromatography using a gradient of 0–5% methanol in methylene
chloride (containing 0.5% pyridine). Fractions with pure product were
combined, concentrated to a foam, and dried in a vacuum desiccator over
P2O5 to afford 0.91 g of 5 (1.35 mmol, 67%). The mass of 5 was confirmed
by ESI-MS in negative mode as m/z (M-H+CH3COOH) 731.46 (calculated
1
for C36H40FN4O6Si·C2H4O2: 731.86). H NMR (CDCl3) δ 8.00 (s, 1H),
7.42–6.75 (m, 13H), 6.38 (t, J = 6.7 Hz, 1H), 4.72–4.62 (m, 3H), 4.16–4.09
(m, 1H), 3.79 (s, 6H), 3.50–3.30 (m, 2H), 2.82–2.68 (m, 1H), 2.61–2.45 (m,
1H), 1.32–1.20 (m, 2H), 0.11 (s, 9H).
5ꢁ-O-(4,4ꢁ-Dimethoxytrityl)-N2-(tert-butyldithiopropyl)-O6-(trimethylsily-
lethyl)-2ꢁ-deoxyinosine (6). 3-Aminopropyl-tert-butyl disulfide hydrochloride
(10, 0.691 g, 3.20 mmol, 4.0 equiv.) was dissolved in 10 mL aqueous 1M
NaOH and then extracted three times with 30 mL methylene chloride.
The combined organic layers were concentrated and mixed with 25 mL
anhydrous pyridine and 0.2 mL triethylamine. Compound 5 (0.54 g,
0.80 mmol) in 20 mL pyridine was added under N2. After being stirred
at 60◦C for 3 days, the reaction mixture was concentrated to an oil and
purified by normal phase chromatography using a gradient of 25–60%
ethyl acetate (containing 0.5% pyridine) in hexane. Fractions with pure
product were combined, concentrated to a foam, and dried in a vacuum
desiccator over P2O5 to afford 0.54 g of 6 (0.65 mmol, 81%). The mass of
6 was confirmed by ESI-MS in negative mode as m/z (M-H+CH3COOH)
890.55 (calculated for C43H56N5O6S2Si·C2H4O2: 891.20). UV λmax 235,
1
285 nm. H NMR (CDCl3) δ 7.65 (s, 1H), 6.79–7.42 (m, 13H), 6.30 (t,
J = 6.7 Hz, 1H), 4.87 (br s, 1H), 4.66 (br s, 1H), 4.60–4.52 (m, 2H),
4.11–4.07 (m, 1H), 3.78 (s, 6H), 3.50–3.39 (m, 3H), 3.36–3.30 (m, 1H),
2.90–2.80 (m, 1H), 2.78–2.70 (m, 2H), 2.49–2.41 (m, 1H), 2.30–2.26
(m, 1H), 2.00–1.90 (m, 2H), 1.33 (s, 9H), 1.27–1.20 (m, 2H), 0.09
(s, 9H).

Guanosine Thioalkyl Tethers
1085
5ꢁ-O-(4,4ꢁ-Dimethoxytrityl)-N2-(tert-butyldithiopropyl)-O6-(trimethylsily-
lethyl)-3ꢁ-O-[(2-cyanoethoxy)-(N,N-diisopropylamino)]phosphinyl-2ꢁ-deoxy-
guanosine (7a). Under argon, 2-cyanoethyl (N,N,Nꢁ,Nꢁ-tetraisopropyl)
phosphordiamidite (0.36 mL, 1.13 mmol, 1.6 equiv.) was added by syringe
to 6 (0.58 g, 0.70 mmol) in 10 mL methylene chloride. Pyridinium
trifluoroacetate (0.24 g, 1.24 mmol, 1.8 equiv.) dissolved in 15 mL
methylene chloride was then added by syringe to the stirred mixture
in an ice bath. The reaction was complete in 4 hours. The solution was
then concentrated and purified by normal phase chromatography using a
gradient of 15–60% ethyl acetate (containing 0.5% pyridine) in hexane.
Fractions with pure product were combined, concentrated to a foam, and
dried in a vacuum desiccator over P2O5 to afford 0.44 g of 7a (0.43 mmol,
61%). The mass of 7a was confirmed by ESI-MS in positive mode as m/z
(M+H) 1033.44 (calculated for C52H75N7O7PS2Si: 1033.39). UV λmax 233,
285 nm. 31P NMR (CDCl3) δ 149.2, 149.5.
O6-(Trimethylsilylethyl)-guanosine (3b). Guanosine hydrate, 1b (1.42
g, 4.7 mmol) was dried by co-evaporation with 1,4-dioxane and then
suspended in 70 mL 1,4-dioxane. Trimethylsilyl imidazole (2.2 mL, 15
mmol, 3.2 equiv.) was added by syringe to the stirred suspension under
N2. This protection step was complete in 1 hour. Triphenylphosphine
(6.5 g, 25 mmol, 5.3 equiv.), trimethylsilylethanol (7.2 mL, 50 mmol, 10.6
equiv.) and diisopropylazodicarboxylate (4.9 mL, 31 mmol, 6.6 equiv.) were
added to the reaction mixture under N2, which turned a clear orange.
This Mitsunobu reaction was complete in 2 hours. A mixture of 20 mL
concentrated aqueous NH3 and 35 mL water was added to the reaction,
which was stirred overnight. Water (∼200 mL) was added, and the solution
was extracted three times with methylene chloride. The combined organic
layers were concentrated and 20 mL dioxane was added. The solution was
then concentrated to 15 mL and left overnight to allow the triphenylphos-
phine oxide to crystallize. After the triphenylphosphine oxide was removed
by filtration, the solution was concentrated again and the product was
crystallized from 20 mL ethyl acetate and methylene chloride (9:1) to afford
1.65 g of 3b (4.30 mmol, 91%). The mass of 3b was confirmed by ESI-
MS in negative mode as m/z (M-H) 382.36 (calculated for C15H24N5O5Si:
382.47). UV λmax 248, 282 nm.1H NMR (DMSO) δ 8.06 (s, 1H), 6.35
(s, 2H), 5.76 (d, J = 6.0 Hz, 1H), 5.38 (d, J = 6.0 Hz, 1H), 5.11 (d, J
= 4.8 Hz, 1H), 5.09 (t, J = 5.2 Hz, 1H), 4.55–4.42 (m, 3H), 4.10–4.07
(m, 1H), 3.89–3.86 (m, 1H), 3.65–3.45 (m, 2H), 1.16–1.10 (m, 2H), 0.06
(s, 9H).
2-Fluoro-O6-(trimethylsilylethyl)-inosine (4b). At –30◦C under argon,
8 mL anhydrous pyridine, 3 mL anhydrous toluene and 11 mL 70%
HF·pyridine (423 mmol, 235 equiv.) were mixed well and then added to

1086
X. Hou et al.
3b (0.69 g, 1.8 mmol). Tert -butyl nitrite (0.70 mL, 5.9 mmol, 3.3 equiv.) was
added dropwise by syringe to the stirred reaction mixture, maintained at
−30◦C. This non-aqueous diazotization was complete in 2 hours. The excess
HF was then quenched by slowly pouring the reaction mixture into a stirred
solution of 30 g K2CO3 in 50 mL water in an ice bath. The solution was
extracted three times with 100 mL ethyl acetate. The combined organic
layers were concentrated and purified by normal phase chromatography
using a gradient of 0–10% methanol in methylene chloride (with 0.5%
triethylamine). Fractions with pure product were combined, concentrated
to a white solid, and dried in a vacuum desiccator over P2O5 to afford 0.610
g of 4b (1.58 mmol, 88%). The mass of 4b was confirmed by ESI-MS in
negative mode as m/z (M-H) 385.40 (calculated for C15H22FN4O5Si: 385.44).
UV λmax 256 nm. 1H NMR (CDCl3) δ 7.81 (s, 1H), 6.23–6.16 (br s, 1H), 5.77
(d, J = 7.6 Hz, 1H), 5.13–5.07 (br s), 5.03–4.95 (br, 1H), 4.68–4.50 (m, 3H),
4.00–3.90 (m, 1H), 3.85–3.76 (m, 1H), 3.38–3.31 (m, 1H), 3.17–3.06 (m
1H), 1.43–1.32 (m, 2H), 0.12 (s, 9H).
N2-(tert-Butyldithiopropyl)-O6-(trimethylsilylethyl)-guanosine (8). 3-Ami-
nopropyl-tert-butyl disulfide hydrochloride (10) (0.86 g, 4.0 mmol, 2.0
equiv.) was dissolved in 10 mL aqueous 1M NaOH and then extracted three
times with 30 mL methylene chloride. The combined organic layers were
concentrated and mixed with 20 mL anhydrous pyridine and 1 mL triethy-
lamine. Compound 4b (0.77 g, 2.0 mmol) in 20 mL pyridine was added
under N2. After being stirred at 60◦C for 3 days, the reaction mixture was
concentrated to an oil and purified by normal phase chromatography using
a gradient of 0–8% methanol (containing 0.5% pyridine) in methylene
chloride. Fractions with pure product were combined, concentrated to a
foam, and dried in a vacuum desiccator over P2O5 to afford 1.01 g of 8 (1.85
mmol, 92%). The mass of 8 was confirmed by ESI-MS in negative mode
as m/z (M-H+CH3COOH) 604.54 (calculated for C22H38N5O5S2Si·C2H4O2:
604.83). UV λmax 253, 292 nm. 1H NMR (CDCl3) δ 7.30 (s, 1H), 5.63 (d, J =
7.6 Hz, 1H), 5.25 (br s, 1H), 5.00 (br s, 1H), 4.64–4.32 (m, 4H), 3.95–3.91
(m, 1H), 3.77–3.71 (m, 1H), 3.65–3.54 (m, 1H), 3.48–3.36 (m, 1H), 2.77 (t,
J = 7.0 Hz, 2H), 2.03–1.92 (m, 2H), 1.34 (s, 9H), 1.38–1.18 (m, 2H), 0.11
(s, 9H).
2ꢁ-tert-Butyldimethylsilyl-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-N2-(tert-butyldithio-
propyl)-O6-(trimethylsilylethyl)-guanosine (9). Compound 8 (1.09 g,
2.0 mmol) was dissolved in 30 mL pyridine and di-tert-butylsilyl
ditriflate (0.72 mL, 2.0 mmol, 1.0 equiv.) was added dropwise over
5 minutes with stirring at 0◦C. The solution was stirred at 0◦C for
30 minutes, and imidazole (0.68 g, 10 mmol, 5.0 equiv.) was then
added. The mixture was stirred for 5 minutes at 0◦C and then for 25
minutes at room temperature. tert-Butyldimethylchlorosilane (0.362 g,

Guanosine Thioalkyl Tethers
1087
2.40 mmol, 1.2 equiv.) was added and the reaction was stirred at
60◦C for 2 hours. The solution was concentrated to an oil and
used in the next step without further purification. The mass of the
crude 2ꢁ-tert-butyldimethylsilyl-3ꢁ,5ꢁ-di-tert-butylsilylene intermediate was
confirmed by ESI-MS in positive mode as m/z (M+H) 800.62 (calculated
for C36H70N5O5S2Si3: 801.36).
Hydrogen fluoride·pyridine (1 mL, 38.5 mmol, 19.2 equiv.) was chilled
and carefully diluted with cold pyridine (8 mL). The resulting solution was
added slowly to a stirred solution of the intermediate described above in 10
mL dichloromethane at 0◦C. After 1 hour, the reaction mixture was washed
twice with saturated aqueous NaHCO3. It was then concentrated to an oil
and used in the following reaction without further purification.
The oil formed above was dried by co-evaporation with pyridine, and
dissolved in 30 mL anhydrous pyridine, 4,4ꢁ-dimethoxytrityl chloride (0.745
g, 2.2 mmol, 1.1 equiv.) was added at 0◦C under N2, and the reaction was
stirred at 0◦C overnight. The mixture was poured into aqueous NaHCO3,
and then extracted three times with methylene chloride. The combined
organic layers were concentrated and purified by normal phase chromatog-
raphy using a gradient of 0–5% acetone in methylene chloride (containing
0.5% pyridine). Fractions with pure product were combined, concentrated
to a foam, and dried in a vacuum desiccator over P2O5 to afford 1.63 g of 9
(1.7 mmol, 85% from 8). The mass of 9 was confirmed by ESI-MS in negative
mode as m/z (M-H) 960.64 (calculated for C49H70N5O7S2Si2: 961.41). UV
1
λmax 236, 284 nm. H NMR (CDCl3) δ 7.70 (s, 1H), 7.47–6.78 (m, 13H),
5.88–5.83 (d, J = 5.2 Hz, 1H), 5.07 (br s, 1H), 4.64–4.54 (m, 3H), 4.39–4.32
(m, 1H), 4.23–4.19 (m, 1H), 3.78 (s, 6H), 3.52–3.24 (m, 3H), 2.78–2.74 (m,
1H), 2.65–2.55 (m, 2H) 1.86–1.76 (m, 2H), 1.32 (s, 9H), 1.34–1.22 (m, 2H),
0.85 (s, 9H), 0.09 (s, 9H), 0.01 (s, 3H), –0.15 (s, 3H).
2ꢁ-O-(tert-Butyldimethylsilyl)-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-N2-(tert-butyldi-
thiopropyl)-O6-(trimethylsilylethyl)-3ꢁ-O-[(2-cyanoethoxy)-(N,N-diisopropy-
lamino)]phosphinyl guanosine (7b). Under argon, 2-cyanoethyl
(N,N,Nꢁ,Nꢁ-tetraisopropyl)phosphordiamidite (0.32 mL, 1.0 mmol, 2.0
equiv.) was added by syringe to 9 (0.48 g, 0.50 mmol) in 10 mL methylene
chloride. Pyridinium trifluoroacetate (0.20 g, 1.0 mmol, 2.0 equiv.)
dissolved in 15 mL methylene chloride was then added by syringe to the
stirred mixture in an ice bath. The reaction was complete in 4 hours.
This solution was then concentrated and purified by normal phase
chromatography using a gradient of 10–50% ethyl acetate (containing
0.5% pyridine) in hexane. Fractions with pure product were combined,
concentrated to a foam, and dried in a vacuum desiccator over P2O5 to
afford 0.47 g of 7b (0.40 mmol, 80%). The mass of 7b was confirmed
by ESI-MS in positive mode as m/z (M+H) 1164.04 (calculated for

1088
X. Hou et al.
C58H89N7O8PS2Si2: 1163.65). UV λmax 233, 285 nm. 31P NMR (CDCl3) δ
149.5, 151.7.
3,3ꢁ-Dithiobispropylamine (13). Five batches of crude reagent were
prepared individually as described here and then purified together. 3-
Chloropropylamine hydrochloride (11) (2.8 g, 21.5 mmol) and sodium
thiosulfate (3.44 g, 21.8 mmol, 1.01 equiv.) were dissolved in 80 mL
aqueous methanol (50%) and refluxed overnight to afford the thiosulfate
intermediate 12. Iodine (2.7 g, 10.6 mmol, 0.49 equiv.) in methanol was
slowly added to the refluxing solution through an addition funnel over
10 hours. The reaction mixture was concentrated, dissolved in 15 mL 6 N
aqueous NaOH, and then extracted three times with methylene chloride.
The methylene chloride layers were concentrated under vacuum. The five
batches of crude reagent were then combined and placed in the addition
funnel of a falling film distillation apparatus (Aldrich, Milwaukee, WI, USA),
with toluene as the refluxing solvent. The product 13 (7.75 g, 43 mmol,
80%) was collected as a clear liquid. 1H NMR (CDCl3) δ 2.29 (t, J = 7.1 Hz,
4H), 2.14 (t, J = 6.8 Hz, 4H), 1.38 (br, 4H), 1.22 (m, 4H).
3-Aminopropylthiol hydrochloride (14). Dithiothreitol (2.15 g, 13.9
mmol, 1.3 equiv.) was added to an aqueous solution of 13 (1.95 g, 10.8
mmol). Concentrated HCl (1.7 mL) was added to adjust the pH to 6. The
reaction mixture was shaken at room temperature for 2 days, concentrated
to an oil, and used below without purification.
Diisopropyl-1-(tert-butylthio)-1,2-hydrazinedicarboxylate (15). 2-Methyl-
2-propanethiol (4.5 mL, 40 mmol) and diisopropyl azodicarboxylate (8.0
mL, 40.6 mmol, 1.0 equiv.) were dissolved in 100 mL anhydrous ethyl
ether under N2. Sodium methoxide (0.2 mL) in methanol was added. The
orange color disappeared after 5 minutes, and the reaction mixture was
concentrated to an oil and used below without purification.
3-Aminopropyl-tert-butyl disulfide hydrochloride (10). Compound 14
was dissolved in 50 mL argon-degassed DMF and then added dropwise to a
solution of 15 in 5 mL anhydrous triethylamine and 50 mL argon-degassed
DMF. After the reaction was stirred for 1 day, 25 mL DMF was added, the
mixture was stirred overnight, and 1.0 mL triethylamine was then added.
The reaction mixture was filtered and concentrated to an oil. The crude
product was precipitated in ethyl ether acidified with HCl and purified by
normal phase chromatography using a gradient of methanol in methylene
1
chloride to afford 2.96 g of 10 (13.7 mmol, 64%). H NMR of free amine
(CDCl3) δ 8.0 (br, 2H), 3.16 (t, J = 7.2 Hz, 2H), 2.82 (t, J = 7.0 Hz, 2H),
2.19 (m, 2H), 1.32 (s, 9H).

Guanosine Thioalkyl Tethers
1089
DNA Synthesis (16a, 16b). The DNA 19mer, d[CAGTCCCTGTT-
CGG(G∗)CGCC], 16a, and DNA 20mer, d[ACAGTCCCTGTTCGG(G∗)-
CGCC], 16b, where G∗ is deoxyguanosine with the tert-butyl disulfide
protected thiopropyl tether, were both synthesized using commercial phos-
phoramidites on an Amersham Oligopilot II synthesizer (Pharmacia, Mil-
waukee, WI, USA). The modified phosphoramidite 7a, along with standard
deoxynucleoside phosphoramidites and deoxycytidine solid support (de-
oxycytidine with acetyl protection) from Glen Research (Sterling, VA, USA),
were used. The scale of the syntheses was 65–70 µmol. Three equivalents
of 0.1 M solutions of amidites in anhydrous CH3CN were used in each
coupling, along with a mixture of 0.22 M pyridinium trifluoroacetate and
0.11 M N -methyl imidazole as activators. After synthesis, the DNA 19mer was
cleaved from the support and deprotected in concentrated aqueous NH3. It
was then purified by reverse phase HPLC using 0.1 M TEAA and CH3CN,
first with the DMT group on, and again after its removal, to give 56 µmol
(80%) pure 16a. The mass of the DNA 19mer with the tert-butyl disulfide
protected tether (16a) was confirmed by ESI-MS as 5916 (calculated 5919).
The tert-butyl thiol protecting group was converted to other forms as
described below. The mass of a small sample of the DNA 20mer with the
tert-butyl disulfide protected tether (16b) was confirmed by ESI-MS as 6232
(calculated 6232). For most of the DNA 20mer, the tert-butyl thiol protecting
group was converted to other forms prior to removal from the support, as
described below. The calculated extinction coefficient for the DNA 19mer
is 164,000 M−1 cm−1, and that for the DNA 20mer is 178,000 M−1 cm−1.
DNA 19mer with propylthiol tether (17a). Dithiothreitol (0.0117 g,
0.076 mmol, 178 equiv.) was dissolved in 2 mL water, and the pH was
adjusted to 8 with concentrated aqueous NH3. Purified DNA 19mer with
the tert-butyl disulfide protected tether (16a, 70 OD) was dissolved in this
solution and heated at 40◦C for 3 hours. After cooling, it was purified by
reverse phase HPLC using 0.1 M TEAA and CH3CN. Fractions containing
pure 17a were lyophilized and desalted by reverse phase HPLC using 0.1 M
+
NH4HCO3 and CH3CN. The NH4 form of 17a was converted to the Na+
form on a Dowex X50 column, with a final yield of 45 OD (64%). The mass
of 17a was confirmed by ESI-MS as 5827 (calculated 5831).
DNA 19mer with nitrobenzoic acid disulfide tether (18a).
5,5ꢁ-Dithiobis(2-nitrobenzoic acid) (0.121 g, 0.305 mmol, 626 equiv.)
was dissolved in 200 mL 0.1 M potassium phosphate (pH = 8). Purified
DNA 19mer with the proplylthiol tether (17a, 80 OD) was dissolved in 1 mL
of this solution. After 30 minutes at room temperature, it was purified by
reverse phase HPLC using 0.1 M TEAA and CH3CN. Fractions containing
pure 18a were lyophilized and desalted by reverse phase HPLC using 0.1 M
+
NH4HCO3 and CH3CN. The NH4 form of 18a was converted to the Na+

1090
X. Hou et al.
form on a Dowex X50 column, with a final yield of 55 OD (69%). The mass
of 18a was confirmed by ESI-MS as 6026 (calculated 6028).
DNA 19mer with ethylamine disulfide tether (19a), one-step method.
Cysteamine hydrochloride (2-aminoethanethiol hydrochloride, 0.069
g, 0.61 mmol, 1000 equiv.) and 0.028 g cystamine dihydrochloride
(2,2ꢁ-diaminodiethyl disulfide dihydrochloride, 0.12 mmol, 200 equiv.) were
dissolved in 1 mL water, and the pH was adjusted to 8 with concentrated
aqueous NH3. Purified DNA 19mer with the tert-butyl disulfide protected
tether (16a, 100 OD) was dissolved in this solution and heated at 40◦C for
2 hours. After cooling, it was purified by reverse phase HPLC using 0.1 M
TEAA and CH3CN. Fractions containing pure 19a were lyophilized and
desalted by reverse phase HPLC using 0.1 M NH4HCO3 and CH3CN. The
NH4+ form of 19a was converted to the Na+ form on a Dowex X50 column,
with a final yield of 75 OD (75%). The mass of 19a was confirmed by ESI-MS
as 5903 (calculated 5906).
DNA 19mer with ethylamine disulfide tether (19a), two-step method.
Cystamine dihydrochloride (0.0137 g, 0.061 mmol, 100 equiv.) was dissolved
in 2 mL of 0.1 M potassium phosphate (pH = 8). Purified DNA 19mer
with the propylthiol tether (17a, 100 OD) was dissolved in 1 mL of this
solution and heated at 40◦C for 1 hour. After cooling, it was purified by
reverse phase HPLC using 0.1 M TEAA and CH3CN. Fractions containing
pure 19a were lyophilized and desalted by reverse phase HPLC using 0.1 M
+
NH4HCO3 and CH3CN. The NH4 form of 19a was converted to the Na+
form on a Dowex X50 column, with a final yield of 70 OD (70%).
DNA 20mer with ethylamine disulfide tether (19b). After DNA
synthesis, cysteamine hydrochloride (2-aminoethanethiol hydrochloride,
0.170 g, 1.50 mmol, 258 equiv.) and 0.330 g cystamine dihydrochloride
(2,2ꢁ-diaminodiethyl disulfide dihydrochloride, 1.47 mmol, 253 equiv.) were
dissolved in 4.0 mL water, and the pH was adjusted to 8 with concentrated
aqueous NH3. This solution was then added to 0.2 g of the solid support
with the fully protected DNA 20mer attached (29 µmol/g) in a 50 mL
centrifuge tube that was then shaken at room temperature for 2 days. The
supernatant was removed after centrifugation, and the solid support was
washed with water three times. Concentrated aqueous NH3 was then added
to the solid support to cleave the DNA and deprotect it, leaving the DMT
group attached. After 2 days at room temperature, the mixture was filtered
and the support washed with 25 mL additional concentrated aqueous NH3.
The filtrates were combined and concentrated in a SpeedVac. The crude
product was then purified twice by reverse phase HPLC using 0.1 M TEAA
and CH3CN, before and again after detritylation using 0.4 M acetic acid
at pH 3.5 for 20 minutes. Fractions containing pure 19b were lyophilized

Guanosine Thioalkyl Tethers
1091
and desal+ted by reverse phase HPLC using 0.1 M NH4HCO3 and CH3CN.
The NH4 form of 19b was converted to the Na+ form on a Dowex X50
column, with a final yield of 340 OD (33%). The mass of 19b was confirmed
by ESI-MS as 6218 (calculated 6219).
RNA 23mer Synthesis (16c). The RNA 23mer, [AGCAGUGGCG(G∗)-
CCGAACAGGGAC] (16c), where G∗ is guanosine with the tert-butyl
disulfide protected thiopropyl tether, was synthesized by the standard
phosphoramidite method on
a
58 µmol scale. The modified
phosphoramidite 7b, along with standard phosphoramidites with 2ꢁ-tom
protection (cytidine with acetyl protection) from Glen Research, were used.
The procedures were similar to those described above for DNA, except
that 6 equivalents of amidites were used. After the synthesis, 2.0 g of the
solid support with the RNA attached (27.6 µmol/g) was treated with 5 mL
40% aqueous methylamine at 65◦C for 10 minutes to partially deprotect
it and cleave it from the support. After filtration, the support was rinsed
with 50 mL aqueous ethanol, and the combined filtrates were concentrated
in a Speed-Vac. The RNA was desilylated using a mixture of 9.4 mL
1-methylpyrrolidinone (NMP), 6.3 mL triethylamine trihydrofluoride
(TEA·3HF), and 4.7 mL dry triethylamine (TEA) at 65◦C for 2 hours.[41]
A fluoride scavenger (isopropoxytrimethylsilane, 30 mL) was used,[42] and
the RNA was isolated by ethyl ether precipitation. It was then purified twice
by reverse phase HPLC using 0.1 M TEAA and CH3CN, before and again
after detritylation using 0.4 M acetic acid at pH 3.5 for 20 minutes. Fractions
containing pure 16c were lyophilized and desalted by reverse phase HPLC
using 0.1 M NH4HCO3 and CH3CN. The NH4+ form of 16c was converted to
the Na+ form on a Dowex X50 column, with a final yield of 1850 OD (15%).
The mass of 16c was confirmed by ESI-MS as 7664 (calculated 7665). The
calculated extinction coefficient for the RNA 23mer is 229,000 M−1 cm−1.
RNA 23mer with propylthiol tether (17c). Dithiothreitol (0.0374 g,
0.242 mmol, 278 equiv.) was dissolved in 2 mL water, and the pH was
adjusted to 8 with concentrated aqueous NH3. Purified RNA 23mer with
the tert-butyl disulfide protected tether (16c, 200 OD) was dissolved in this
solution and heated at 40◦C for 3 hours. After cooling, it was purified by
reverse phase HPLC using 0.1 M TEAA and CH3CN. Fractions containing
pure 17c were lyophilized and desalted by reverse phase HPLC using 0.1 M
+
NH4HCO3 and CH3CN. The NH4 form of 17c was converted to the Na+
form on a Dowex X50 column, with a final yield of 160 OD (80%). The
mass of 17c was confirmed by ESI-MS as 7575 (calculated 7577).
RNA 23mer with nitrobenzoic acid disulfide tether (18c). 5,5ꢁ-
Dithiobis(2-nitrobenzoic acid) (0.25 g, 0.63 mmol, 1800 equiv.) was
dissolved in 200 mL 0.1 M potassium phosphate (pH = 8). Purified RNA

1092
X. Hou et al.
23mer with the propylthiol tether (17c, 80 OD) was dissolved in 1 mL of
this solution. After 30 minutes at room temperature, it was purified by
reverse phase HPLC using 0.1 M TEAA and CH3CN. Fractions containing
pure 18c were lyophilized and desalted by reverse phase HPLC using 0.1 M
+
NH4HCO3 and CH3CN. The NH4 form of 18c was converted to the Na+
form on a Dowex X50 column, with a final yield of 40 OD (50%). The mass
of 18c was confirmed by ESI-MS as 7772 (calculated 7774).
RNA 23mer with ethylamine disulfide tether (19c), one-step method.
Cysteamine hydrochloride (2-aminoethanethiol hydrochloride, 0.20
g, 1.76 mmol, 8080 equiv.) and 0.050 g cystamine dihydrochloride
(2,2ꢁ-diaminodiethyl disulfide dihydrochloride, 0.22 mmol, 1020 equiv.)
were dissolved in 1 mL water, and the pH was adjusted to 8 with
concentrated aqueous NH3. Purified RNA 23mer with the tert-butyl
disulfide protected tether (16c, 50 OD) was dissolved in this solution and
heated at 40◦C for 2 hours. After cooling, it was purified by reverse phase
HPLC using 0.1 M TEAA and CH3CN. Fractions containing pure 19c were
lyophilized and desalted by reverse phase HPLC using 0.1 M NH4HCO3
+
and CH3CN. The NH4 form of 19c was converted to the Na+ form on a
Dowex X50 column, with a final yield of 35 OD (70%). The mass of 19c was
confirmed by ESI-MS as 7650 (calc 7652).
RNA 23mer with ethylamine disulfide tether (19c), two-step method.
Cystamine dihydrochloride (0.0187 g, 0.083 mmol, 190 equiv.) was dissolved
in 2 mL of 0.1 M potassium phosphate (pH = 8). Purified RNA 23mer
with the propylthiol tether (17c, 100 OD) was dissolved in 1 mL of this
solution and heated at 40◦C for 1 hour. After cooling, it was purified by
reverse phase HPLC using 0.1 M TEAA and CH3CN. Fractions containing
pure 19c were lyophilized and desalted by reverse phase HPLC using 0.1 M
+
NH4HCO3 and CH3CN. The NH4 form of 19c was converted to the Na+
form on a Dowex X50 column, with a final yield of 55 OD (55%).
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