Synthesis and Characterization of a [3-15N]-Labeled Cis-Syn

Thymine Dimer-Containing DNA Duplex

Hussam M. Bdour, Jeff Lung-Fa Kao, and John-Stephen Taylor*
Department of Chemistry, Washington UniVersity, St. Louis, Missouri 63130

[email protected]

ReceiVed NoVember 22, 2005

Cis-syn thymine dimers are the major photoproducts of DNA and are the principal cause of mutations
induced by sunlight. To better understand the nature of base pairing with cis-syn thymine dimers, we
have synthesized a decamer oligodeoxynucleotide (ODN) containing a cis-syn thymine dimer labeled at
the N3 of both T’s with 15N by two efficient routes from [3-15N]-thymidine phosphoramidite. In the
postsynthetic irradiation route, an ODN containing an adjacent pair of [3-15N]-labeled T’s was irradiated
and the cis-syn dimer-containing ODN isolated by HPLC. In the mixed building block route, a mixture
of cis-syn and trans-syn dimer-containing ODNs was synthesized from a mixture of [3-15N]-labeled thymine
dimer phosphoramidites after which the cis-syn dimer-containing ODN was isolated by HPLC. The N3-
nitrogen and imino proton signals of an 15N-labeled thymine dimer-containing decamer duplex were
assigned by 2D 1H-15N heterocorrelated HSQC NMR spectroscopy, and the 15N-1H coupling constant
was found to be 1.8 Hz greater for the 5′-T than for the 3′-T. The larger coupling constant is indicative
of weaker H-bonding that is consistent with the more distorted nature of the 5′-base pair found in solution
state NMR and crystallographic structures.

Introduction and less capable of H-bonding with an A in the complementary

Cis-syn cyclobutane thymine dimers are the major UV strand than the 3′-T. In support of this conclusion, the shift of
photoproducts of DNA and have been correlated with mutations the N3 imino proton of the 5′-T of the cis-syn thymine dimer-
and skin cancer.1-5 These products arise via a [2 + 2] containing DNA duplexes was more upfield than that of the
cycloaddition between the 5,6 double bonds of two adjacent 3′-T, though some or all of this upfield shift has been attributed
thymidines in DNA (Scheme 1) and change the local structure to shielding effects of the neighboring A, which also causes a
and properties of the DNA. Two-dimensional NMR studies on large upfield shift on the methyl group of the 5′-T.10 Intensive
DNA duplexes containing a cis-syn thymine dimer have revealed theoretical studies have also been carried out on cis-syn dimer
perturbations to the structure and H-bonding of the duplex at containing duplexes with similar conclusions with regard to the
the site of dimer.6-10 In particular, the 3D structures derived distorted nature of the 5′-T, but with differing conclusions as
from the 2D NOE data indicated that the 5′-T was more distorted to the extent of DNA bending.11-16 Most recently, a crystal

* Corresponding author. Tel: (314) 935-6721. (7) Kemmink, J.; Boelens, R.; Koning, T.; van der Marel, G. A.; van
(1) Cadet, J.; Vigny, P. In Bioorganic Photochemistry; Morrison, H., Boom, J. H.; Kaptein, R. Nucleic Acids Res. 1987, 15, 4645-4653.
Ed.; John Wiley & Sons: New York, 1990; Vol. 1, pp 1-272. (8) Taylor, J.-S.; Garrett, D. S.; Brockie, I. R.; Svoboda, D. L.; Telser,
(2) Taylor, J.-S. Acc. Chem. Res. 1994, 27, 76-82. J. Biochemistry 1990, 29, 8858-8866.
(3) Begley, T. P. In ComprehensiVe Natural Products Chemistry; Poulter, (9) Kim, J.-K.; Patel, D.; Choi, B.-S. Photochem. Photobiol. 1995, 62,
C. D., Ed.; Elsevier: New York, 1999; Vol. 5, pp 371-399. 44-50.
(4) Sinha, R. P.; Hader, D. P. Photochem. Photobiol. Sci. 2002, 1, 225- (10) McAteer, K.; Jing, J.; Kao, J.; Taylor, J. S.; Kennedy, M. A. J.
236. Mol. Biol. 1998, 282, 1013-1032.
(5) Pfeifer, G. P.; You, Y.-H.; Besaratinia, A. Mutat. Res. 2005, 571, (11) Rao, S. N.; Keepers, J. W.; Kollman, P. Nucleic Acids Res. 1984,
19-31. 12, 4789-4807.
(6) Kemmink, J.; Boelens, R.; Koning, T. M. G.; Kaptein, R.; van der (12) Raghunathan, G.; Kieber-Emmons, T.; Rein, R.; Alderfer, J. L. J.
Marel, G. A.; van Boom, J. H. Eur. J. Biochem. 1987, 162, 37-43. Biomol. Struct. Dyn. 1990, 7, 899-913.

10.1021/jo0524167 CCC: $33.50 © 2006 American Chemical Society

1640 J. Org. Chem. 2006, 71, 1640-1646 Published on Web 01/13/2006
15N-Labeled Cis-Syn Thymine Dimer

SCHEME 1

structure was obtained of a cis-syn thymine dimer-containing yeast pol η would suggest that Hoogsteen base pairing is not
duplex decamer which corroborates the distorted nature of the involved,19 but this remains to be verified by other methods.
base pair with the 5′-T that was deduced from the NMR and 15N NMR has proven to be very useful for studying

theoretical studies.17 protonation, tautomerization, and hydrogen bonding to nitrogen

What remains unresolved about DNA damaged by pyrimidine and for determining the relative orientations of NH bond vectors
dimer formation, however, is the extent to which the 5′- and in DNA through analysis of residual dipolar coupling.26-37 15N-
3′-T’s can hydrogen bond to complementary and mismatched labeling also greatly facilitates the assignment and editing of
bases in a DNA duplex and in the active site of a polymerase. proton, 13C and 15N spectra. A [3-15N]-labeled thymine dimer
The latter question is of particular interest, given recent results would therefore be especially useful for probing the nature of
indicating that insertion opposite the more distorted 5′-T of a H-bonding interactions between the 5′- and 3′-T's of thymine
dimer by polymerase η (eta) proceeds with greater selectivity dimer and complementary and mismatched bases in a duplex
than opposite the 3′-T.18,19 Pol η is a member of the Y family and in the active site of a polymerase or repair enzyme by
of DNA damage bypass polymerases and is the enzyme solution and/or solid-state NMR. The most straightforward
responsible for the almost error-free bypass of thymine dimers method to prepare a [3-15N]-labeled thymine dimer-containing
in mammalian cells.20,21 Loss of pol η activity in humans has duplex would be either to irradiate an 15N-labeled precursor
been linked to the genetic disease Xeroderma pigmentosum that ODN that was prepared by automated DNA synthesis (postsyn-
results in increased sensitivity to sunlight and a much higher thetic irradiation route) or to use an 15N-labeled thymine dimer
incidence of skin cancer.22-24 Unfortunately, it has not yet been phosphoramidite (building block route). Though uniformly 15N-
possible to crystallize either yeast or human pol η with DNA, labeled phosphoramidites are now commercially available
but a crystal structure of the related Y family polymerase Dpo4 (Spectra Stable Isotopes), they are very expensive ($750-$1200/
revealed the surprising result that insertion opposite the 5′-T µmol) and because they are uniformly labeled complicate
occurs via Hoogsteen base pairing, whereas insertion opposite spectral analysis. Recently, a [3-15N]-thymidine phosphoramidite
the 3′-T occurs via a normal Watson-Crick base pairing.25 has been synthesized from [3-15N]-3′,5′-O-(1,1,3,3-tetraisopro-
Recent results showing that 7-deaza-adenosine is inserted with pyldisiloxan-1,3-dyl)thymidine and used to study base pairing
about the same efficiency as adenosine opposite the 5′-T by to a complementary A in a DNA duplex.34

(13) Miaskiewicz, K.; Miller, J.; Cooney, M.; Osman, R. J. Am. Chem. (26) Wang, C.; Gao, H.; Gaffney, B. L.; Jones, R. A. J. Am. Chem. Soc.
Soc. 1996, 118, 9156-9163. 1991, 113, 5486-5488.
(14) Spector, T. I.; Cheatham, T. E., III; Kollman, P. A. J. Am. Chem. (27) Goswami, B.; Gaffney, B. L.; Jones, R. A. J. Am. Chem. Soc. 1993,
Soc. 1997, 119, 7095-7104. 115, 3832-3833.
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339. M.; Wuthrich, K. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 14147-14151.
(16) Danilov, V. I.; Les, A.; Alderfer, J. L. J. Biomol. Struct. Dyn. 2001, (29) De Los Santos, C. Compr. Nat. Prod. Chem. 1999, 7, 55-80.
19, 179-191. (30) Wohnert, J.; Dingley, A. J.; Stoldt, M.; Gorlach, M.; Grzesiek, S.;
(17) Park, H.; Zhang, K.; Ren, Y.; Nadji, S.; Sinha, N.; Taylor, J. S.; Brown, L. R. Nucleic Acids Res. 1999, 27, 3104-3110.
Kang, C. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 15965-15970. (31) Kojima, C.; Ono, A.; Kainosho, M. J. Biomol. NMR 2000, 18, 269-
(18) McCulloch, S. D.; Kokoska, R. J.; Masutani, C.; Iwai, S.; Hanaoka, 277.
F.; Kunkel, T. A. Nature 2004, 428, 97-100. (32) Liu, A.; Majumdar, A.; Hu, W.; Kettani, A.; Skripkin, E.; Patel, D.
(19) Hwang, H.; Taylor, J. S. Biochemistry 2005, 44, 4850-4860. J. J. Am. Chem. Soc. 2000, 122, 3206-3210.
(20) Yang, W. FEBS Lett. 2005, 579, 868-872. (33) Pervushin, K.; Fernandez, C.; Riek, R.; Ono, A.; Kainosho, M.;
(21) Prakash, S.; Johnson, R. E.; Prakash, L. Annu. ReV. Biochem. 2005, Wuthrich, K. J. Biomol. NMR 2000, 16, 39-46.
74, 317-353. (34) Ishikawa, R.; Kojima, C.; Ono, A.; Kainosho, M. Magn. Reson.
(22) Carty, M. P.; Glynn, M.; Maher, M.; Smith, T.; Yao, J.; Dixon, K.; Chem. 2001, 39, S159-S165.
McCann, J.; Rynn, L.; Flanagan, A. Biochem. Soc. Trans. 2003, 31, 252- (35) Laxer, A.; Major, D. T.; Gottlieb, H. E.; Fischer, B. J. Org. Chem.
256. 2001, 66, 5463-5481.
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(24) Lehmann, A. R. FEBS Lett. 2005, 579, 873-876. (37) Leppert, J.; Urbinati, C. R.; Hafner, S.; Ohlenschlager, O.; Swanson,
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Nature 2003, 424, 1083-1087. 1183.

J. Org. Chem, Vol. 71, No. 4, 2006 1641

 Bdour et al.

SCHEME 2 SCHEME 3

In this paper, we report two efficient routes for the synthesis

of ODNs containing cis-syn [3-15N]-thymine dimers from
[3-15N]-thymidine phosphoramidite (Scheme 2). In the postsyn-
thetic irradiation route, an ODN containing a [3-15N]-labeled
TT site was synthesized from [3-15N]-thymidine phosphor-
amidite and then irradiated in the presence of a sensitizer to
produce the cis-syn dimer which is isolated by HPLC. In the
building block route, a mixture of cis-syn and trans-syn [3-15N]-
prepare d(CGAA[3-15N]T(cis-syn)[3-15N]TAAGC) by the
thymine dimer-containing ODNs was synthesized from a
postsynthetic irradiation and mixed building block routes. This
mixture of [3-15N]-labeled thymine dimer phosphoramidites after
sequence was chosen to allow direct comparison of H-bonding
which the cis-syn containing ODN was isolated by HPLC. We
and structural data obtained from solution NMR studies of its
also report the assignment and analysis of the 15N shifts and
duplex form with that of the crystal structure.17
coupling constants of a [3-15N]-thymine dimer-containing
Postsynthetic Irradiation Route. For the postsynthetic
decamer duplex leading to the conclusion that the 5′-T is less
irradiation route, phosphoramidite 7 was used to incorporate
hydrogen bonded than the 3′-T.
[3-15N]-labeled thymidine into the central two nucleotides of
10-mer ODN using solid-phase automated DNA synthesis on a
Results and Discussion 1 µmol scale. Following automated synthesis, the ODN was
Both the postsynthetic and mixed building block routes to released from the support, completely deprotected with con-
the [3-15N]-labeled thymine dimer-containing ODNs proceeded centrated ammonia, and purified by reversed-phase chromatog-
from the [3-15N]-thymidine phosphoramidite 7, which was raphy. The ODN was then irradiated with a 450 W medium-
prepared in six steps from thymidine (Scheme 3) by adapting pressure Hannovia mercury lamp for 1 h in the presence of the
routes used for the synthesis of [3-15N]-uridine and its triplet sensitizer acetophenone to produce the 15N-labeled cis-
derivatives.38-40 Thus, thymidine 1 was first acetylated with syn thymine dimer-containing ODN as the major product (Figure
acetic anhydride to give 2, which was then nitrated at N3 to 1). To help confirm the identity of the cis-syn thymine dimer-
give 3.41,42 Treatment of 3 with 15NH4Cl (1.3 equiv), KOH, and containing ODN, the chromatographic behavior of the [3-15N]-
Et3N in CH3CN-H2O for 6 days at room temperature afforded
4 together with partially and fully deacetylated products. To
increase the yield of 4, the mixture of products was reacetylated
in situ prior to chromatographic isolation. Compound 4 was then
deacetylated with saturated ammonia in methanol to give
[3-15N]-labeled thymidine 5. The 5′-hydroxyl group of 5 was
then protected by reaction with 4,4′-dimethoxytrityl chloride
to afford 6 which was then converted to the phosphoramidite 7
by standard procedures. Phosphoramidite 7 was then used to

(38) Ariza, X.; Bou, V.; Vilarrasa, J. J. Am. Chem. Soc. 1995, 117, 3665-
3673.
(39) Ariza, X.; Farras, J.; Serra, C.; Vilarrasa, J. J. Org. Chem. 1997,
62, 1547-1549.
(40) Ariza, X.; Vilarrasa, J. J. Org. Chem. 2000, 65, 2827-2829.
(41) Ariza, X.; Bou, V.; Vilarrasa, J.; Tereshko, V.; Campos, J. L. Angew. FIGURE 1. HPLC analysis of the acetophenone-sensitized irradiation
Chem. 1994, 106, 2535-2537. products of d(CGAA[3-15N]T[3-15N]TAAGC). Abbreviations: c,s, cis-
(42) Gorchs, O.; Hernandez, M.; Garriga, L.; Pedroso, E.; Grandas, A.; syn photoproduct-containing ODN; SM, starting material (ODN). See
Farras, J. Org. Lett. 2002, 4, 1827-1830. the Experimental Section for details.

1642 J. Org. Chem., Vol. 71, No. 4, 2006

15N-Labeled Cis-Syn Thymine Dimer

FIGURE 2. HPLC analysis of the major product isolated from the

irradiation of d(CGAA[3-15N]T[3-15N]TAAGC). Panel A: the major FIGURE 3. HPLC analysis of the products from the mixed building
product from irradiation. Panel B: authentic cis-syn dimer-containing block approach. Panel A: products of the mixed building block
decamer. Panel C: co-injection of the authentic product and that from approach. The two minor products with retention times greater than
the major irradiation product. See the Experimental Section for details. the major product are most likely the trans-syn-I and trans-syn-II
products. Panel B: authentic sample of cis-syn dimer-containing ODN.
labeled ODN, d(CGAA[3-15N]T(cis-syn)[3-15N]TAAGC) was Panel C: co-injection of the authentic and synthetic ODNs. See the
compared to an authentic sample of d(CGAAT(cis-syn)TAAGC) Experimental Section for details.
that was prepared from a pure cis-syn dimer building block and
whose crystal structure has been solved17 (Figure 2). Co- three stereoisomers have to be separated since deprotection
injection of both samples resulted in the appearance of single removes the chirality at phosphorus (Ren, Y., Ph.D. Thesis,
coeluting peak. Washington University, St. Louis, December, 2000). Thus,
Mixed Building Block Route. Our original synthesis of a [3-15N]-thymidine phosphoramidite 7 was coupled with [3-15N]-
cis-syn thymine dimer phosphoramidite building block required labeled thymidine to produce the dinucleotide DMT-TpT 8
six steps from a noncommercially available phosphoramidite. (Scheme 4). This dinucleotide was irradiated with a 450-W
It also required a tedious and low-yielding chromatographic medium-pressure Hanovia mercury lamp in the presence of the
separation of six cis-syn, trans-syn-I, trans-syn-II stereoisomers triplet sensitizer acetophenone for 3 h to give a mixture of cis-
that result from irradiation of a diastereomeric mixture of RP syn, trans-syn-I, and trans-syn-II cyclobutane thymine dimers
and SP methyl phosphate esters of 3′-TBDMS-thymidylyl- 9. The mixture was not separated, but phosphorylated to give
(3′f5′)thymidine.43 More recently, we have developed a three- the mixed phosphoramidite building block 10. The mixed
step mixed building block approach from commercially available building block was used to incorporate the mixture of thymine
T phosphoramidite in which the stereoisomers are not separated dimer isomers into the same 10-mer ODN prepared by the
until after DNA synthesis and deprotection. At this point, only postsynthetic irradiation. The resulting mixture of [3-15N]-
labeled cis-syn, trans-syn-I, and trans-syn-II dimer-containing
(43) Taylor, J.-S.; Brockie, I. R.; O’Day, C. L. J. Am. Chem. Soc. 1987, ODNs were separated by a reverse phase chromatography. The
109, 6735-6742. major peak was identified as the cis-syn thymine dimer-

J. Org. Chem, Vol. 71, No. 4, 2006 1643

 Bdour et al.

SCHEME 4

containing ODN by its coelution with a sample of authentic double bond, it is likely that part of the large upfield shift of
ODN (Figure 3). the N3H of the 5′-T relative to the 3′-T is due to shielding by
NMR Spectroscopy. A complementary DNA duplex was the neighboring base pair has been previously suggested.10
prepared, and the 15N shifts were assigned by correlation with None-the-less, it appears from the increase in the 15N-1H
the imino protons which have previously been assigned by 2D coupling of the 5′-T of the dimer relative to the 3′-T that the
NOE at 5 °C in the closely related dodecamer sequence 5′-T is less H-bonded than the 3′-T and could explain in part
d(GCACGAAT[c,s]TAAG)‚d(CTTAATTCGTGC).10 In the the 1.5 kcal/mol destabilization caused by cis-syn dimer
dodecamer sequence, which shares the same d(GAAT[c,s]- formation.45 The greater selectivity in the insertion of A opposite
TAAG) core, the more upfield proton at δ 11.87 was assigned the 5′-T than the 3′-T by polymerase η19 would therefore appear
to the 5′-T whereas the more downfield proton at δ 12.93 was
assigned to the 3′-T. Figure 4 shows the HSQC spectrum
collected at 25 °C in which the more downfield proton at δ
12.72 assigned to the 3′-T correlates with the more upfield
nitrogen at δ 153.38. Likewise, the more upfield proton at δ
11.68 assigned to the more distorted 5′-T correlates with the
more downfield nitrogen at δ 155.05.
The coupling constant for the 15N-1H of the 5′-T was found
to be 89.09 Hz (Figure 5) and is 1.8 Hz greater than that of
87.27 Hz observed for the 3′-T. The larger coupling constant is
consistent with weaker H-bonding for the 5′-T than for the 3′-T
based on a recent study of the correlation between 15N-1H
coupling constants and H-bonding strength for a series of
substituted uridines in duplex DNA.44 In that study the theoreti-
cally calculated H-bonding energy was found to linearly
correlate with the 15N-1H coupling constant as well as with
the pKa and the chemical shift of the imino proton. From their
linear fit parameters an increase of 1.8 Hz in the N-H coupling
constant of the 5′-T compared to the 3′-T corresponds to a loss
of 0.39 kcal/mol in the strength of the H-bond. When using
their linear fit parameters for proton chemical shift, the decrease
of 1.06 ppm in chemical shift of the N3H of the 5′-T corresponds
to a 0.76 kcal/mol loss in H-bond strength which is about two
times larger than that calculated from the difference in the N-H
coupling constant data. Though we do not yet know how well
any of these parameters apply to thymines with a saturated 5,6 FIGURE 4. Section of the proton-coupled 15N-1H HSQC spectrum
of d(CGAA[3-15N]Td[3-15N]TAAGC)‚d(GCTTAATTCG) showing the
(44) Ishikawa, R.; Ono, A.; Kainosho, M. Nucleic Acids Res. Suppl. 2003, shift correlations and coupling constants for the N3H bonds of the
3, 57-58. thymine dimer.

1644 J. Org. Chem., Vol. 71, No. 4, 2006

15N-Labeled Cis-Syn Thymine Dimer

CDCl3) δ 1.94 (s, 3H, C5CH3), 2.12 (s, 3H, OCOCH3), 2.14 (s,
3H, OCOCH3), 2.18 (m, 1H, H2′), 2.47 (m, 1H, H2′′), 4.24 (m,
1H, H4′). 4.37 (m, 2H, H5′ and H5′′), 5.22 (m, 1H, H3′), 6.33 (t,
J ) 7.1 Hz, 1H, H1′), 7.28 (s, 1H, ArH), 8.95 (d, J ) 90.6 Hz, 1H,
15NH); 15N NMR (90 MHz, CDCl ) δ 155.9; 13C NMR (75.4 MHz,
3
CDCl3) δ 12.0, 20.3, 20.4, 36.8, 64.1, 74.7, 82.2, 85.0, 110.8, 135.8,
150.9, 164.0, 170.4; HRMS (FAB) calcd for C14H18N15NO7 (M +
Li)+ 334.1244, found 334.1239.
[3-15N]-Thymidine (5). Ammonia (7 N) in MeOH (10 mL) was
added to a solution of [3-15N]-3′,5′-di-O-acetylthymidine (4, 985
mg, 3.01 mmol) in MeOH (10 mL) and stirred for 6 h at room
temperature. The solvent was then removed under vacuum, and
the remaining acetamide was removed by heating in a vacuum at
100 °C for 2 h to yield 5 (731 mg, 100%): 1H NMR (300 MHz,
DMSO-d6) δ 1.77 (s, 3H, C5CH3), 2.07 (m, 2H, H2′ and H2′′),
3.66 (m, 2H, H5′ and H5′′), 3.76 (s, 1H, C4′-OH), 4.23 (s, 1H,
C3′-OH), 5.03 (m, 1H, H4′), 5.24 (m, 1H, H3′), 6.16 (t, J ) 6.8
Hz, 1H, H1′), 7.70 (s, 1H, ArH), 11.29 (d, J ) 97.2 Hz, 15NH);
13C NMR (75.4 MHz, DMSO-d ) δ 12.2, 61.3, 70.4, 83.7, 87.2,
6
109.3, 136.1, 150.4, 163.6, 163.8; HRMS (FAB) calcd for
C10H14N15NO5 (M + Li)+ 250.1051, found 250.1045.
FIGURE 5. Section of the 1D 1H spectrum of d(CGAA[3-15N]Td
[3-15N]TAAGC)‚d(GCTTAATTCG) at 0.11 Hz digital resolution from [3-15N]-Dimethoxytritylthymidine (6). 4,4′-Dimethoxytrityl chlo-
which the coupling constants were determined. ride (10221 g, 3.61 mmol) was quickly added to a solution of 5
(731 mg, 3.01 mmol) in 15 mL of pyridine and the solution stirred
overnight at room temperature. The pyridine was removed under
not to be associated with H-bonding strength, but more likely vacuum to yield the crude product as a brown gum which was
due to the constrained nature of the 5′-T in the active site of redissolved in EtOAc and washed with saturated NaCl. The organic
the polymerase as previously proposed.46 A more definitive layer was dried over Na2SO4 and evaporated under reduced pressure
answer, however, will require a study of the H-bonding in a to give a brown gum which was purified by flash column
complex between pol η and a thymine dimer-containing template chromatography using 80% EtOAc in hexane to give 6 (1.183 g,
primer. 72%): 1H NMR (300 MHz, acetone-d6) δ 1.47 (s, 3H, C5CH3),
2.34 (m, 2H, H2′ and H2′′), 3.38 (d, J ) 3.3 Hz, 2H, H5′ and
H5′′), 3.79 (s, 6H, ArOCH3), 4.04 (m, 1H, H4′), 4.60 (m,1H, H3′),
Conclusion
6.36 (t, J ) 7.2 Hz, 1H, H1′), 6.83-7.57 (m, 13H, ArH), 7.63 (s,
We have described the use of a [3-15N]-thymidine phos- 1H, ArH5), 9.9 (d, J ) 91.2 Hz, 1H, 15NH); 13C NMR (75.4 MHz,
phoramidite to prepare a [3-15N]-labeled cis-syn thymine dimer- acetone-d6) δ 12.1, 41.0, 55.4, 64.6, 72.3, 85.3, 87.0, 87.2, 111.0,
113.8, 127.6, 128.6, 128.8, 130.8, 136.3, 136.5, 136.6, 145.7, 151.3,
containing oligodeoxynucleotide by two routes. The presence
159.5, 159.5, 164.5, 164.7; HRMS (FAB) calcd for C31H32N15NO7
of the 15N label greatly facilitated the assignment of the 15N (M + Li)+ 552.2355, found 552.2351.
shifts and 15N-1H coupling constants of the dimer from which [3-15N]-Dimethoxytritylthymidine Phosphoramidite (7). To a
we concluded that the 5′-T is only slightly less strongly solution of 6 (550 mg, 1.01 mmol) in anhydrous CH3CN were added
H-bonded than the 3′-T. The availability of the [3-15N]-labeled 4,5-dicyanoimidazole (179 mg, 1.52 mmol) and 2-cyanoethyl
thymine dimer opens the door to study the sequence context tetraisopropylphosphorodiamidite (642 µL, 2.02 mmol) at room
dependence of base pairing of thymine dimers in DNA duplexes, temperature. The reaction mixture was stirred for 0.5 h and then
as well as in the active site of DNA polymerases and repair extracted with EtOAc and washed with saturated aqueous NaHCO3.
enzymes by both solution and solid-state NMR. The organic layer was further washed with brine, dried over
anhydrous Na2SO4, and concentrated. The residue was purified by
flash column chromatography after neutralizing the silica gel with
Experimental Section Et3N using 60% EtOAc in hexane to give 7 (658 mg, 87%): 31P
[3-15N]-3′,5′-Di-O-acetylthymidine (4). Water (15 mL), CH3- (121.5 MHz, acetone-d6 referenced to trimethyl phosphate TMP)
CN (15 mL), Et3N (2.40 mL, 17.3 mmol), and a solution of 3′,5′, δ 145.78, 145.85; HRMS (FAB) calcd for C40H49N315NO8P (M +
di-O-acetyl-3-nitrothymidine in 30 mL of acetonitrile (3, 4.927 g, Li)+ 752.3405, found 752.3407.
13.26 mmol)41 were added sequentially to a tightly sealed 250-mL DMT-[3-15N]T-PO(OCH2CH2CN) [3-15N]T (8). To [3-15N]-
round-bottom flask containing 15NH4Cl (940 mg, 17.25 mmol) and labeled thymidine (5, 278 g, 1.14 mmol) and 4,5-dicyanoimidazole
KOH (85%, 1.050 g, 15.92 mmol). After being stirred vigorously (90 mg, 0.76 mmol) in 2 mL of anhydrous DMF was added
for 6 days, the solution was first saturated with NaCl and then dropwise a 2 mL solution of DMT-[3-15N]dT phosphoramidite (7,
extracted with CH2Cl2. The aqueous layer was evaporated, and the 284 mg, 0.38 mmol) in anhydrous DMF at room temperature. After
residue was triturated with CH2Cl2-MeOH (9:1). The combined 5 min, t-BuOOH (209 µL, 5-6 M solution in nonane, 1.25 mmol)
organic layers were dried over Na2SO4 and evaporated. The was added. After 10 min at room temperature, the reaction mixture
resulting residue was dissolved in pyridine (30 mL), and acetic was extracted with ethyl acetate and washed with saturated NaHCO3
anhydride (50 mmol) was added. After the mixture was stirred for aqueous solution. The organic layer was further washed with brine,
3 days, 4 mL of methanol was added and the solvent evaporated. dried over anhydrous Na2SO4, and concentrated. The residue was
The residue was purified by column chromatography with ethyl purified by column chromatography after neutralizing the silica gel
acetate as the eluant to give 4 (3.037 g, 70%): 1H NMR (300 MHz, with Et3N and eluted with 10% methanol in ethyl acetate to afford
the title compound (558 mg, 70%) as a yellowish solid: TLC
(methanol/ethyl acetate, 5:95) Rf 0.25; 31P (121.5 MHz, acetone-
(45) Jing, Y.; Kao, J. F.-L.; Taylor, J.-S. Nucleic Acids Res. 1998, 26,
3845-3853. d6, referenced to TMP as an external reference) δ -4.5 and -4.7;
(46) Sun, L.; Zhang, K.; Zhou, L.; Hohler, P.; Kool, E. T.; Yuan, F.; MS (+ESI); HRMS calcd for C40H49N315NO8P (M + Na)+
Wang, Z.; Taylor, J. S. Biochemistry 2003, 42, 9431-9437. 926.2774, found 926.2783.

J. Org. Chem, Vol. 71, No. 4, 2006 1645

 Bdour et al.

DMT-[3-15N]T[d]PO(OCH2CH2CN)[3-15N]T (9): To a Pyrex Synthesis of d(CGAA[3-15N]Td[3-15N]TAAGC). The title

immersion well photochemical reactor were added compound 8 (400 oligodeoxynucleotide was synthesized from the mixed thymidine
mg, 0.44 mmol), 400 mL of acetonitrile/H2O (1:1), and acetophe- dimer building block 10. The desired cis-syn product was purified
none (0.4 mL, 3.42 mmol). The solution was purged with argon and analyzed (Figure 3) under standard conditions and used for
for 40 min and continued while the mixture was irradiated with a the preparation of the NMR sample. The cis-syn, trans-syn-I, and
450 W medium-pressure Hanovia mercury lamp for 3 h. Solvents trans-syn-II eluted at 24.4, 26.1, and 27.0 min, respectively.
were evaporated under reduced pressure, and the residue was Preparation of the Duplex DNA NMR Sample. A solution of
subjected to silica gel flash chromatography after neutralizing the d(CGAA[3-15N]Td[3-15N]TAAGC)‚d(GCTTAATTCG) (3.5 mM)
silica gel with Et3N and eluted with 10% MeOH in ethyl acetate to was prepared by mixing approximately equimolar amounts of the
afford a mixture of dimers 9 (300 mg, 0.33 mmol, 75%) as a two ODNs and then using HPLC with detection at 260 nm to
yellowish solid: 31P NMR (121.5 MHz, acetone-d6, referenced to determine the ratio of the two strands from (area % of the
TMP as an external reference) -4.6, -4.8, -5.2, -7.5, -7.8, and complementary strand/molar extinction coefficient of the comple-
-8.2 ppm; HRMS (FAB) calcd for C44H48N315N2O14P (M + H)+ mentary strand)/(area% of the dimer strand/ molar extinction
904.2954, found 904.3325. coefficient of the dimer strand). The ratio was adjusted to ∼1.0 by
DMT-[3-15N]T[d]PO(OCH2CH2CN)[3-15N]T-P(OCH2CH2CN)- addition of the appropriate strand, and then made up to a final
(NiPr2) (10). To 5 mL of an anhydrous acetonitrile solution of the volume of 0.2 mL in 10% D2O (99.96%) 90% ddH2O containing
photoproduct mixture 9 (300 mg, 0.33 mmol) and 4,5-dicyanoimi- 100 mM NaCl, 10 mM sodium phosphate buffer (pH 7), and 0.01%
dazole (58 mg, 0.49 mmol) was added 2-cyanoethyl tetraisopro- NaN3. The final pH of the solution was 5.7. 15N NMR was
pylphosphorodiamidite (210 µL, 0.66 mmol) at room temperature. referenced to external 15NH4Cl at δ 23.6.47
The reaction mixture was stirred for 0.5 h under argon and then 15N NMR Spectroscopy. NMR experiments were performed at
extracted with ethyl acetate. The organic layer was washed with 25 °C on a 600 MHz spectrometer with 3 mm gradient probe (600
saturated aqueous NaHCO3 and brine, dried over Na2SO4, and MHz 1H, 60.8 MHz 15N). Proton chemical shifts were measured in
concentrated, and the residue was purified by column chromatog- parts per million (ppm) downfield from an internal TSP standard.
raphy after neutralizing the silica gel with Et3N and eluted with Proton spectra were obtained at a spectral width of 14000 Hz and
80% ethyl acetate in hexane to afford the photoproduct mixture 10 collected into 32K data points with a hard-pulse WATERGATE
(232 mg, 0.21 mmol, 64%) as a yellowish solid: 31P NMR (121.5 sequence for water suppression.48 Proton-nitrogen coupling con-
MHz, acetone-d6, referenced to TMP as an external reference) stants were measured directly from the splitting of the proton
146.15, 146.08, 146.03, 145.92, 145.85, 145.78, 145.71, 145.50, resonance signals with a 14000 Hz spectral width collecting into
-4.54, -4.63, -7.53, -8.19. 128K data points (digital resolution 0.11 Hz). A two-dimensional
ODN Synthesis and HPLC Analysis and Purification. Oli- 1H-15N HSQC experiment was collected with spectral width of
godeoxynucleotides were synthesized on an automated DNA 14000 and 10000 Hz along the F2 (1H) and F1 (15N) dimensions,
synthesizer on a 1 µmol scale. The ODNs were cleaved from the respectively. A proton-coupled 1H-15N HSQC spectrum was
support with concentrated ammonium hydroxide (30% aqueous, obtained with a 1500 Hz in F2 and a 5000 Hz in F1 dimension.
1.5 mL) and fully deprotected by heating the solution at 55 °C in The 90° 1H pulse width was 6.8 µs and the 90° 15N pulse width
a sealed tube for 8 h. The samples were dried in a Speedvac, was 32 µs. A data matrix with 256 complex points in F1 and 4096
dissolved in ddH2O, and purified and analyzed by HPLC on an complex points in F2 with 16 scans per t1 value was collected using
XTerra MS C18 2.5 µM 10 × 50 mm column at a flow rate of 1 pulse sequence of Kay and co-workers.49
mL/min with UV detection at 260 nm. Unless otherwise indicated,
standard conditions for purification and analysis were 5 min of
100% buffer A (5% acetonitrile/0.05 M triethylammonium acetate Acknowledgment. Supported by the NIH (RO1-CA40463)
TEAA, pH ∼ 7), followed by a 50 min gradient of 100% buffer A with assistance of the Washington University Mass Spectrometry
to 80% buffer A, 20% buffer B (50% acetonitrile, 0.05 M TEAA, Resource (Grant No. P41RR0954).
pH ∼ 7).
Synthesis of d(CGAA[3-15N]Td[3-15N]TAAGC) by Irradia- Supporting Information Available: 1H and assorted 13C, 15N,
tion of d(CGAA[3-15N]T[3-15N]TAAGC). d(CGAA[3-15N]T[3- and 31P NMR spectra of compounds 4-10. This material is
15N]TAAGC) was synthesized using phosphoramidite 7 and purified available free of charge via the Internet at http://pubs.acs.org.
by HPLC under standard conditions. Dimer formation was induced
JO0524167
by irradiating d(CGAA[3-15N]T[3-15N]TAAGC) (300 µM) and
acetophenone (30 mM) in 15% CH3CN in ddH2O with Pyrex
filtered light for 1 h at 0 °C. HPLC analysis (Figure 1) was carried (47) Wishart, D. S.; Bigam, C. G.; Yao, J.; Abildgaard, F.; Dyson, H.
out using 5 min 100% solvent A (10% MeOH, 90% 75 mm J.; Oldfield, E.; Markley, J. L.; Sykes, B. D. J. Biomol. NMR 1995, 6, 135-
140.
phosphate buffer, pH ∼7) followed by a 30 min gradient of 100% (48) Sklenar, V.; Piotto, M.; Leppik, R.; Saudek, V. J. Magn. Reson. A
solvent A to 100% solvent B (40% MeOH, 60% phosphate buffer, 1993, 102, 241-245.
pH ∼7). The cis-syn containing ODN was then purified and (49) Kay, L. E.; Keifer, P.; Saarinen, T. J. Am. Chem. Soc. 1992, 114,
analyzed (Figure 2) under standard conditions. 10663-10665.

1646 J. Org. Chem., Vol. 71, No. 4, 2006