Synthesis and application of a 5′-aldehyde phosphoramidite for covalent attachment of DNA to biomolecules

Article abstract of DOI:10.1021/jo060723m

We recently reported the use of covalently attached DNA as a structural constraint for rational control of macromolecular conformation. Reductive amination was employed to attach each strand of the duplex DNA constraint to RNA, utilizing an aldehyde tethered to the 5′-terminus of the DNA. Here we describe the synthesis of a thymidine phosphoramidite that has the 5′-tethered aldehyde masked as a 1,2-diol. We also describe optimized reductive amination conditions for linking 5′-aldehyde-DNA with 2′-amino-2′-deoxy-RNA. These procedures should be generally applicable for attaching DNA to biomolecules.

Full text of DOI:10.1021/jo060723m

internally into short RNA oligonucleotides via solid-phase
synthesis,6,7 and oligonucleotides can be assembled into larger
RNA molecules by ligation.8-10 Here we have used 2′-amino-
2′-deoxy-RNA (abbreviated 2′-NH2-RNA) as the amine reaction
partner. For generation of aldehydes on DNA, a few examples
have been demonstrated at specific locations such as the 5′- or
the 3′-termini,11-19 an internal 2′-position,20 a 1′-position of an
abasic site,21 and the nucleobases.22 The termini of a DNA strand
are appropriate modification sites because modifications at an
internal 2′-position or at a nucleobase may unintentionally
influence DNA duplex formation, which is the basis of the
constraint approach.1,2 Similarly, modifications are preferably
made without alteration to the deoxyribose ring. We therefore
focused on the termini to introduce DNA aldehydes.
Synthesis and Application of a 5′-Aldehyde
Phosphoramidite for Covalent Attachment of
DNA to Biomolecules
Chandrasekhar V. Miduturu and Scott K. Silverman*
Department of Chemistry, UniVersity of Illinois at Urbanas
Champaign, 600 South Mathews AVenue, Urbana, Illinois 61801
scott@scs.uiuc.edu
ReceiVed April 4, 2006
Automated solid-phase DNA synthesis utilizes a variety of
reaction conditions and reagents, some of which are incompat-
ible with an unprotected aldehyde.23 A 1,2-diol (glycol) is a
stable precursor of an aldehyde, because the 1,2-diol can be
cleaved oxidatively to the aldehyde using NaIO4 after oligo-
nucleotide synthesis. Utilizing this or other strategies, aldehydes
have been generated both at the 5′- and 3′-ends of DNA in
various ways (see structures A-G).12-18 To minimize the
exposure of the modified nucleoside to the solid-phase synthesis
conditions and for ease of synthesis of oligonucleotides in the
conventional 3′f5′ direction, installation of the aldehyde is
preferred at the 5′- rather than the 3′-end (i.e., not B or E). In
all of the published examples A-G, after reductive amination
the tether that connects the DNA to the amine-bearing biomol-
ecule was either relatively long and flexible (A-D) or short
and rigid (E-G). A long tether would probably be a poor choice
for connecting a structural constraint because flexibility could
We recently reported the use of covalently attached DNA
as a structural constraint for rational control of macromo-
lecular conformation. Reductive amination was employed to
attach each strand of the duplex DNA constraint to RNA,
utilizing an aldehyde tethered to the 5′-terminus of the DNA.
Here we describe the synthesis of a thymidine phosphora-
midite that has the 5′-tethered aldehyde masked as a 1,2-
diol. We also describe optimized reductive amination con-
ditions for linking 5′-aldehyde-DNA with 2′-amino-2′-deoxy-
RNA. These procedures should be generally applicable for
attaching DNA to biomolecules.
(6) Pieken, W. A.; Olsen, D. B.; Benseler, F.; Aurup, H.; Eckstein, F.
Science 1991, 253, 314-317.
(7) Jin, S.; Miduturu, C. V.; McKinney, D. C.; Silverman, S. K. J. Org.
Chem. 2005, 70, 4284-4299.
(8) Moore, M. J.; Sharp, P. A. Science 1992, 256, 992-997.
(9) Moore, M. J.; Query, C. C. Methods Enzymol. 2000, 317, 109-123.
(10) Purtha, W. E.; Coppins, R. L.; Smalley, M. K.; Silverman, S. K. J.
Am. Chem. Soc. 2005, 127, 13124-13125.
(11) Kremsky, J. N.; Wooters, J. L.; Dougherty, J. P.; Meyers, R. E.;
Collins, M.; Brown, E. L. Nucleic Acids Res. 1987, 15, 2891-2909.
(12) Agrawal, S.; Christodoulou, C.; Gait, M. J. Nucleic Acids Res. 1986,
14, 6227-6245.
(13) Skrzypczynski, Z.; Wayland, S. Bioconjugate Chem. 2003, 14, 642-
652.
(14) Jones, D. S.; Hachmann, J. P.; Osgood, S. A.; Hayag, M. S.; Barstad,
P. A.; Iverson, G. M.; Coutts, S. M. Bioconjugate Chem. 1994, 5, 390-
399.
(15) Podyminogin, M. A.; Lukhtanov, E. A.; Reed, M. W. Nucleic Acids
Res. 2001, 29, 5090-5098.
(16) Urata, H.; Akagi, M. Tetrahedron Lett. 1993, 34, 4015-4018.
(17) Oberhuber, M.; Joyce, G. F. Angew. Chem., Int. Ed. 2005, 44, 7580-
7583.
The DNA constraint strategy as applied to control RNA
folding requires covalent attachment of DNA oligonucleotides
to an RNA macromolecule.1,2 To enable site-specific attachment
of DNA, the coupling reaction must use functional groups that
are compatible with those found naturally in DNA and RNA.
Several linkage reactions have been explored for attaching DNA
oligonucleotides to small organic molecules and large biomol-
ecules, such as reductive amination and formation of oximes,
hydrazones, and disulfides.3-5 Among these approaches, reduc-
tive amination via the formation of an imine (Schiff base) and
its subsequent reduction with a borohydride reagent is particu-
larly desirable, because the linkage has no stereoisomers and is
chemically stable.
(18) Kodama, T.; Greenberg, M. M. J. Org. Chem. 2005, 70, 9916-
9924.
Reductive amination requires both amine and aldehyde
reaction partners. Primary amino groups can be incorporated
(19) Haralambidis, J.; Lagniton, L.; Tregear, G. W. Bioorg. Med. Chem.
Lett. 1994, 4, 1005-1010.
(1) Miduturu, C. V.; Silverman, S. K. J. Am. Chem. Soc. 2005, 127,
10144-10145.
(2) Miduturu, C. V.; Silverman, S. K. Angew. Chem., Int. Ed. 2006, 45,
1918-1921.
(3) Thuong, N. T.; Asseline, U. In Current Protocols in Nucleic Acid
Chemistry; Wiley: New York, 2000; Unit 4.2.
(4) Greenberg, M. M. In Current Protocols in Nucleic Acid Chemistry;
Wiley: New York, 2000; Unit 4.5.
(5) Zatsepin, T. S.; Stetsenko, D. A.; Gait, M. J.; Oretskaya, T. S.
Bioconjugate Chem. 2005, 16, 471-489.
(20) Kachalova, A. V.; Zatsepin, T. S.; Romanova, E. A.; Stetsenko, D.
A.; Gait, M. J.; Oretskaya, T. S. Nucleosides, Nucleotides Nucleic Acids
2000, 19, 1693-1707.
(21) Tilquin, J. M.; Dechamps, M.; Sonveaux, E. Bioconjugate Chem.
2001, 12, 451-457.
(22) Tre´visiol, E.; Renard, A.; Defrancq, E.; Lhomme, J. Tetrahedron
Lett. 1997, 38, 8687-8690.
(23) Caruthers, M. H.; Barone, A. D.; Beaucage, S. L.; Dodds, D. R.;
Fisher, E. F.; McBride, L. J.; Matteucci, M.; Stabinsky, Z.; Tang, J. Y.
Methods Enzymol. 1987, 154, 287-313.
10.1021/jo060723m CCC: $33.50 © 2006 American Chemical Society
Published on Web 06/17/2006
5774
J. Org. Chem. 2006, 71, 5774-5777

SCHEME 1. Synthesis of Phosphoramidite 7
SCHEME 2. Synthesis of 5′-Aldehyde-DNA from
Phosphoramidite 7 via 5′-Diol-DNA
ether.24 Allylation of the 5′-OH of 2 with 2 equiv of NaH and
1 equiv of allyl bromide afforded compound 3 (2 equiv of NaH
were necessary to deprotonate the 5′-OH because the uracil NH
is more acidic). Asymmetric dihydroxylation (AD)25 of the
alkene 3 using AD-mix â afforded 1,2-diol 4 as an inseparable
2:1 mixture of diastereomers (absolute configurations were not
determined). Although it was unnecessary to control the
stereoselectivity of this dihydroxylation reaction because the
new stereocenter would ultimately become the aldehyde carbon,
AD-mix â was used for ease of reaction setup and purification.
Diol 4 was treated with excess benzoyl chloride and triethyl-
amine in refluxing methylene chloride to provide the trisben-
zoylated nucleoside 5. Although N3-benzoyl protection is not
required for use of the phosphoramidite in solid-phase synthesis,
such protection is acceptable because the N3-benzoyl group is
readily removed by routine NH4OH treatment. Catalytic hy-
drogenation of 5 to 6 followed by phosphitylation afforded the
phosphoramidite 7 in an overall yield of 31% starting from
thymidine.
compromise the integrity of the constraint. In this report, an
new short tether for connecting DNA and RNA (structure H)
was designed and implemented. This was achieved via the
synthesis of thymidine nucleoside phosphoramidite 7 that has
its reactive aldehyde functionality masked as a 1,2-diol.12-14,16-19
Compound 7 was utilized as the final phosphoramidite during
standard solid-phase DNA synthesis, with coupling yield >95%
(using standard conditions except the coupling time was
increased from 1.5 to 10 min). Treatment with NH4OH to cleave
the DNA from the solid support and to remove the benzoyl
and nucleobase protecting groups afforded the desired DNA with
a 5′-diol modification (5′-diol-DNA; Scheme 2).The 5′-diol-
DNA was oxidized to 5′-aldehyde-DNA using NaIO4. Following
oxidative cleavage to the aldehyde, the excess NaIO4 must be
removed completely, because excess NaIO4 would oxidize the
1,2-diol of the 3′-terminal ribose of the RNA during the
reductive amination reaction. Sephadex G-25 size-exclusion
columns were used for desalting the DNA following oxidation
with NaIO4. This approach was found to be effective in the
recovery of 5′-aldehyde-DNA from the column while removing
essentially all NaIO4.
Reductive amination reactions are generally performed under
acidic conditions to favor protonation of the intermediate imine,
which is subsequently reduced to an amine using sodium
cyanoborohydride.26 These reactions are usually performed with
excess amine and a limiting amount of the aldehyde. However,
in the case of coupling modified DNA and RNA, a large excess
of the 2′-NH2-modified RNA is prohibitive, whereas excess
DNA can more readily be used. In our initial experiments,
typical reductive amination yields were only 15-20% even with
a 1000× excess of 5′-aldehyde-DNA (data not shown). Reduc-
tive amination reactions involving organic molecules have been
The target phosphoramidite 7 was synthesized from thymidine
in eight steps (Scheme 1). Thymidine 1 was converted to benzyl
ether 2 in a three-step procedure starting with the protection of
the 5′-OH as the triphenylmethyl ether followed by benzylation
of the 3′-OH and subsequent deprotection of the triphenylmethyl
(24) Griffin, B.; Todd, A. J. Chem. Soc. 1958, 1389-1393.
(25) Sharpless, K. B.; Amberg, W.; Bennani, Y. L.; Crispino, G. A.;
Hartung, J.; Jeong, K.-S.; Kwong, H.-L.; Morikawa, K.; Wang, Z.-M.; Xu,
D.; Zhang, X.-L. J. Org. Chem. 1992, 57, 2768-2771.
(26) Borch, R. F.; Bernstein, M. D.; Durst, H. D. J. Am. Chem. Soc.
1971, 93, 2897-2904.
J. Org. Chem, Vol. 71, No. 15, 2006 5775

polyacrylamide gel electrophoresis (PAGE) purification. The
product identity was confirmed by MALDI mass spectrometry
(see Experimental Section).
Some reductive amination sites may be desirable within
regions of RNA that have considerable secondary or tertiary
structure. Acylation reactions of 2′-NH2 groups in both RNA
and DNA are dependent on local flexibility of the nucleic acid;
for example, a decreased acylation rate was observed when the
2′-NH2 group was present in a duplex secondary structure or
involved in a tertiary contact.29 To demonstrate that the reductive
amination approach for DNA attachment works well for
structured RNAs, we used the 160-nucleotide P4-P6 RNA
domain of the Tetrahymena group I intron RNA as the 2′-NH2-
RNA partner (Figure 1B).1,2 With the 2′-NH2 at two different
sites in P4-P6 (either U107 or A114), in both cases a
preparatively useful yield of RNA-DNA product was obtained.
The reductive amination yield was higher at 45 °C than at 25
°C, and increasing the Ni2+ concentration from 2.5 to 25 mM
was modestly effective at improving the yield. Most important
for a high yield of reductive amination product using the P4-
P6 RNA was inclusion of a “disruptor” DNA oligonucleotide
that is complementary to a substantial portion of the RNA
sequence, thereby leaving the 2′-NH2-RNA within a single-
stranded region.
FIGURE 1. Reductive amination between 2′-NH2-RNA and 5′-
aldehyde-DNA: (A) Shown are reactions performed with 5′-32P-labeled
RNA and unradiolabeled 5′-aldehyde-DNA oligonucleotides, each of
which were 15 nt in length (NaOAc pH 5.0, 10 mM NaCNBH3, 2.5
mM NiCl2 if present, 25 °C). In each set of lanes, the three timepoints
were 0, 3, and 6 h (20% PAGE). (B) Shown are reactions performed
with 5′-32P-labeled P4-P6 RNA (160 nt) and unradiolabeled 15-nt 5′-
aldehyde-DNA (NaOAc pH 5.0, 10 mM NaCNBH3, 2.5 mM NiCl2,
45 °C). In each set of lanes, the four timepoints were 0, 1.5, 3, and 6
h (8% PAGE). For 2′-NH2 at P4-P6 nucleotide U107 and A114 without
disruptor but with 25 mM NiCl2, the yields were improved to 21%
and 20%, although slight degradation was observed (data not shown).
In conclusion, a new DNA phosphoramidite 7 was synthe-
sized that permits the preparation of 5′-aldehyde-DNA in which
the aldehyde carbon is connected via a single methylene group
to the 5′-oxygen of the DNA (structure H). A convenient
protocol for the oxidative cleavage of the 1,2-diol to the
aldehyde was developed using NaIO4, for which the excess was
removed by size-exclusion chromatography. The covalent
attachment of 5′-aldehyde-DNA to 2′-NH2-RNA was achieved
by reductive amination, resulting in a short 5-atom tether
connecting the ribose rings of the DNA and the RNA. The
optimized reaction conditions (including Ni2+ concentration,
temperature, and inclusion of disruptor DNA oligonucleotide)
were dependent on the 2′-NH2-RNA reaction partner. The
synthesis of the DNA phosphoramidite 7 and the development
of the reductive amination protocol expand our ability to attach
DNA (and perhaps small molecules with aldehyde functional
groups) to biomolecules such as RNA.
improved by using Lewis acid catalysts to activate the alde-
hyde.27 Therefore, we tested a number of divalent transition
metal cations such as Mn2+, Fe2+, Co2+, Ni2+, Cu2+, and Zn2+
(as their chloride and sulfate salts) in the reductive amination
between the 5′-aldehyde-DNA and the 2′-NH2-RNA. Because
RNA is susceptible to degradation in the presence of high
concentration of metal ions, the concentration of the metal ion
and the temperature of the reaction were carefully optimized.
The addition of 2.5 mM NiCl2 (50× relative to DNA) at 25 °C
was found to be optimal for increasing the reductive amination
yield with minimum RNA degradation (Figure 1A, compare
first two sets of lanes). No effect was observed when Ni2+ was
replaced with monovalent metal ions such as Na+ and K+, even
at 100 mM (data not shown). The mechanism of this Ni2+
enhancement of the reductive amination reaction was not
pursued in detail.28 Plausibly, Ni2+ may act as a Lewis acid to
activate the 5′-aldehyde on the DNA for imine formation, which
is thought to be rate-determining.26 In addition, divalent metal
ions such as Ni2+ could induce some tertiary structure in the
RNA, which could indirectly enhance the rate of imine
formation. If imine reduction is instead rate-determining, then
Ni2+ must interact with the imine to increase its electrophilicity.
To corroborate that the reductive amination occurs between
the 2′-NH2-RNA and the 5′-aldehyde-DNA, control reactions
were performed with substrates lacking one of the reactive
functional groups. No reaction was observed when the RNA
lacked the 2′-NH2 or when the DNA lacked the 5′-aldehyde
(Figure 1A, last two sets of lanes). Importantly, these experi-
ments demonstrated that the exocyclic amine groups on the
nucleobases do not react appreciably under our reductive
amination conditions. The reductive amination reaction between
2′-NH2-RNA and 5′-aldehyde-DNA could be performed on the
preparative (5 nmol) scale, with ∼50% isolated yield after
Experimental Section
5′-O-Allyl-3′-O-benzylthymidine (3). A 60% dispersion of
sodium hydride in mineral oil (0.68 g, 17.1 mmol) was added to a
solution of 224 (2.58 g, 7.7 mmol) in 20 mL of THF. The suspension
was stirred at 60 °C for 2 h, and allyl bromide (0.67 mL, 7.7 mmol)
was added at room temperature. The mixture was stirred at room
temperature overnight and quenched with methanol (1 mL). The
mixture was diluted with 50 mL of EtOAc, and the organic layer
was washed with saturated NaHCO3 (50 mL). The aqueous layer
was extracted with EtOAc (2 × 25 mL). The combined organic
extracts were washed with saturated aqueous NaCl (50 mL), dried
over Na2SO4, and concentrated under vacuum. The resulting oil
was purified via silica gel chromatography with 0-35% EtOAc in
CH2Cl2 as the eluant to produce 2.64 g (92%) of 3 as a pale yellow
1
syrup: Rf 0.35 (3:1 CH2Cl2/EtOAc); for H and 13C NMR, see
Supporting Information. EI-HRMS: M+ calcd for C20H24N2O5,
372.1685; found, 372.1688.
5′-O-(2,3-Dihydroxypropyl)-3′-O-benzylthymidine (4). AD-
mix-â [20.1 g, containing 0.03 mmol K2OsO2(OH)4] dissolved in
(27) Mattson, R. J.; Pham, K. M.; Leuck, D. J.; Cowen, K. A. J. Org.
Chem. 1990, 55, 2552-2554.
(28) The reductive amination proceeds well with 2′-NH2-C and A in
addition to U. Reactions with 2′-NH2-A are faster and require little or no
Ni2+ for optimal reactivity (data not shown).
(29) Chamberlin, S. I.; Weeks, K. M. J. Am. Chem. Soc. 2000, 122, 216-
224.
5776 J. Org. Chem., Vol. 71, No. 15, 2006

70 mL of water and 70 mL of t-BuOH was stirred at room
temperature to produce two clear phases. The reaction mixture was
cooled to 0 °C, and a syrup of 3 (2.64 g, 7.12 mmol) dissolved in
20 mL of t-BuOH and 20 mL of water was added at once. The
heterogeneous slurry was stirred vigorously at 4 °C overnight. The
reaction was quenched by the addition of sodium sulfite (21 g).
The mixture was allowed to warm to room temperature and was
stirred for 30-60 min. The mixture was diluted with 75 mL of
EtOAc and, after separation of the layers, the aqueous layer was
washed with EtOAc (2 × 50 mL). The combined organic extracts
were washed with saturated aqueous NaCl (75 mL), dried over
Na2SO4, and concentrated under vacuum. The resulting paste was
purified via silica gel chromatography with 0-5% MeOH in CH2Cl2
as the eluant to produce 2.56 g (89%, 2:1 ratio of unassigned
TCCG-3′ (Figure 1A); 5′-TTGACCCGCTCTCCT-3′ (Figure 1B).
MALDI-MS (two sequences): calcd 4616.9, found 4612.3 and calcd
4528.9, found 4531.4. For oxidation to the 5′-aldehyde-DNA, to a
solution of 5′-diol-DNA (6 nmol) in 200 mM sodium phosphate
buffer, pH 7.0, was added NaIO4 (1000× relative to DNA, added
from an 0.2 M aqueous stock solution). The 50 µL sample was
incubated at room temperature for 30 min, and excess NaIO4 was
removed with a Sephadex G-25 mini spin column (Amersham).
The eluted material was evaporated to dryness, and the 5′-aldehyde-
DNA (assumed 6 nmol) was used immediately in the reductive
amination reaction. MALDI-MS for 5′-aldehyde-DNA (two se-
quences) calcd 4584.9, found 4587.6 and calcd 4496.9, found
4494.9.
1
diastereomers as estimated from H NMR) of 4 as a white foam:
Analytical-Scale Reductive Amination Reactions. 2′-NH2-RNA
(5 pmol, supplemented with a trace amount of 5′-32P-labeled RNA)
and 5′-aldehyde-DNA (500 pmol) were mixed in a total volume of
10 µL containing 100 mM NaOAc pH 5.0, 10 mM NaH2PO4 (as a
general 5′-phosphatase inhibitor), 2.5 mM NiCl2, and 10 mM
NaCNBH3 at 25 °C (the NaCNBH3 was added last). For Figure
1A, the RNA sequence was 5′-GGAAUUGCGGGAAAG-3′ (the
2′-NH2-uridine is underlined). For Figure 1B, the RNA sequence
was that of the P4-P6 RNA30 with a 2′-NH2 at either U107 or
A114, prepared by splint ligation using T4 DNA ligase as
described.1 When included, the disruptor DNA was complementary
to P4-P6 nucleotides 175-225. Aliquots of 2 µL were withdrawn
at appropriate timepoints and quenched onto a solution of 80%
formamide, 1× TB [89 mM each Tris and boric acid (pH 8.3)],
and 50 mM EDTA containing 0.25% each bromophenol blue and
xylene cyanol. The quenched samples were electrophoresed on 20%
or 8% PAGE and imaged on a phosphorimager.
1
Rf 0.6 (1:9 MeOH/CH2Cl2); for H and 13C NMR, see Supporting
Information. EI-HRMS: M+ calcd for C20H26N2O7, 406.1740;
found, 406.1737.
5′-O-(2,3-Di-O-benzoylpropyl)-N3-benzoyl-3′-O-benzylthymi-
dine (5). A portion of 4 (0.20 g, 0.49 mmol) was coevaporated
from pyridine (2 mL) and dissolved in CH2Cl2 (2 mL). Et3N (0.70
mL, 5.07 mmol) and benzoyl chloride (0.57 mL, 5.01 mmol) were
added, and the mixture was heated at reflux for 1 h. After cooling
to room temperature, CH2Cl2 (20 mL) and saturated NaHCO3 (20
mL) were added. After separation of the layers, the aqueous layer
was washed with CH2Cl2 (2 × 15 mL). The combined organic
extracts were washed with aqueous NaCl (30 mL), dried over
Na2SO4, and concentrated under vacuum. The resulting paste was
purified via silica gel chromatography with 0-10% EtOAc in CH2-
Cl2 to afford 0.36 g (100%) of 5 as a syrup: Rf 0.90 (1:9 EtOAc/
1
CH2Cl2); for H and 13C NMR, see Supporting Information. EI-
HRMS: M+ calcd for C41H38N2O10, 718.2526; found, 718.2517.
5′-O-(2,3-Di-O-benzoylpropyl)-N3-benzoylthymidine (6). A
portion of 10% Pd-C (0.5 g, 0.45 mmol) was added to a solution
of 5 (0.35 g, 0.48 mmol) in MeOH (4 mL). The flask was evacuated
and placed in an atmosphere of hydrogen for 45 min. It was
observed that the N3-benzoyl group was partially removed under
these conditions if the reaction time was prolonged (>90 min). The
mixture was filtered, and the residue was purified via silica gel
chromatography with 0-30% EtOAc in CH2Cl2 to afford 0.26 g
(85%) of 6 as a pale yellow foam: Rf 0.15 (1:9 EtOAc/CH2Cl2);
for 1H and 13C NMR, see Supporting Information. EI-HRMS: M+
calcd for C34H32N2O10, 628.2056; found, 628.2063.
Preparative-Scale Reductive Amination Reactions. 2′-NH2-
RNA (5 nmol; sequence from above) was mixed with 5′-aldehyde-
DNA (10 nmol, combined from two preparations as above; sequence
same as for Figure 1B as listed above) in a total volume of 167 µL
containing 100 mM NaOAc (pH 5.0) 2.5 mM NiCl2, and 10 mM
NaCNBH3 at 25 °C for 4 h (the NaCNBH3 was added last). The
colorless solution was quenched onto a 300 µL solution of 80%
formamide, 1× TB, and 50 mM EDTA containing 0.25% each
bromophenol blue and xylene cyanol, and the product was purified
by 20% PAGE. A typical yield was 2.5 nmol of the reductive
amination product. MALDI-MS for RNA-DNA product: calcd,
9399.0; found, 9394.1.
5′-O-(2,3-Di-O-benzoylpropyl)-N3-benzoylthymidine-3′-O-(2-
cyanoethyl-N,N-diisopropyl)phosphoramidite (7). A portion of
6 (0.15 g, 0.24 mmol) was coevaporated with pyridine (2 mL) and
dissolved in CH2Cl2 (2.0 mL). 4,5-Dicyanoimidazole (DCI, 31 mg,
0.26 mmol) and 2-cyanoethyl N,N,N′,N′-tetraisopropylphospho-
rodiamidite (0.83 mL, 0.26 mmol) were added. The cloudy mixture
was stirred for 4 h at room temperature and partitioned between
CH2Cl2 (20 mL) and saturated aqueous NaHCO3 (20 mL). After
separation of the layers, the aqueous layer was extracted with CH2-
Cl2 (2 × 20 mL). The combined organic extracts were washed with
aqueous NaCl (40 mL), dried over Na2SO4, and concentrated under
vacuum. The resulting paste was purified via chromatography on
Et3N-washed silica gel with 1:2 to 1:1 CH2Cl2-hexanes containing
1% Et3N to afford 7 (0.16 g, 79%) as a pale yellow foam: Rf 0.50
Acknowledgment. This research was supported by the
Burroughs Wellcome Fund (New Investigator Award in the
Basic Pharmacological Sciences), the March of Dimes Birth
Defects Foundation (Research Grant No. 5-FY02-271), the
National Institutes of Health (Grant GM-65966), the American
Chemical Society Petroleum Research Fund (Grant 38803-G4),
and the UIUC Department of Chemistry (all to S.K.S.). S.K.S.
is the recipient of a fellowship from The David and Lucile
Packard Foundation.
1
(1:1 CH2Cl2/hexanes containing 1% Et3N); for H and 13C NMR,
see Supporting Information. 31P NMR (202 MHz, acetone-d6) δ
149.68, 149.58, 149.24, 149.15. FAB-HRMS: [M+H]+ calcd for
C43H50N4O11P, 829.3213; found, 829.3210.
Supporting Information Available: General experimental
procedures; tabulation of 1H and 13C NMR spectral peaks for 3-7;
images of 1H NMR spectra for 3-7; and image of 31P NMR
spectrum for 7. This material is available free of charge via the
Internet at http://pubs.acs.org.
Preparation and Oxidation of 5′-Diol-DNA Oligonucleotides.
Each DNA oligonucleotide modified with a 5′-diol was synthesized
using phosphoramidite 7 and purified by 20% PAGE. During solid-
phase synthesis, the standard coupling conditions were used except
for an increase in the coupling time from 1.5 to 10 min; the coupling
yield was >95% based on appearance of the abort band on PAGE.
DNA sequences (5′-T modified as a diol): 5′-TTGACCATGAT-
JO060723M
(30) Murphy, F. L.; Cech, T. R. Biochemistry 1993, 32, 5291-5300.
J. Org. Chem, Vol. 71, No. 15, 2006 5777