Stereoselective nucleoside deuteration for NMR studies of DNA

Article abstract of DOI:10.1080/15257770.2010.494650

A procedure has been elaborated for stereoselective deuterium substitution of one of the diastereotopic 5'-protons in 2'-deoxynucleotides. The synthetic scheme uses the reduction of the 5-oxosugar derivative with deuterated Alpine-Borane. The resulting de

Full text of DOI:10.1080/15257770.2010.494650

Nucleosides, Nucleotides and Nucleic Acids, 29:562–573, 2010
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2010.494650
STEREOSELECTIVE NUCLEOSIDE DEUTERATION FOR NMR
STUDIES OF DNA
Mark Lukin and Carlos de los Santos
Department of Pharmacological Sciences, Stony Brook University, Stony Brook,
New York, USA
A procedure has been elaborated for stereoselective deuterium substitution of one of the di-
2
astereotopic 5ꢁ-protons in 2ꢁ-deoxynucleotides. The synthetic scheme uses the reduction of the 5-
oxosugar derivative with deuterated Alpine-Borane. The resulting deuterosugar is converted into
pyrimidine nucleosides and incorporated into DNA using standard protocols. Comparison of two-
dimensional NMR spectra of the fully protonated and partially deuterated duplexes allowed us to
assign diastereotopic 5ꢁ protons, increasing the number of experimental restraints used for structure
determination.
Keywords Deuterated nucleosides; oligonucleotides; solution state NMR; DNA lesions
INTRODUCTION
The 5ꢁ deoxyribose protons generate multiple inter- and intra-nucloetide
nOe contacts and scalar coupling interactions that can provide valuable
structural information about key sugar-phosphate backbone angles in nu-
cleic acids. However, the usage of such data poses a serious challenge, since
the 4ꢁ and 5ꢁ protons form a non-first order ABC spin system that is hard to
assign unambiguously. Chemical shift based rules that allow the assignment
of 5ꢁ and 5ꢁꢁ resonances in canonical B-form DNA are hardly applicable for
non-standard DNA structures or for duplexes bearing modified nucleotides.
In addition, the spectral range where 5ꢁ sugar protons resonate is very nar-
row, so the 5ꢁ proton signals have severe overlap. As a result, examples of
efficient and reliable utilization of structural information derived from the
nucleotide 5ꢁ proton resonances are scarce.
Received 28 January 2010; accepted 14 May 2010.
This research was supported by NIH Grants CA47995 and ES004068. We are grateful to Dr. Robert
Rieger, Stony Brook University’s proteomic facilities (supported by the shared instrumentation grant,
NIH/NCRR 1 S10 RR023680-1) for collection of HRMS spectra. We also are grateful to Drs. Mike Goger,
Shibani Bhattacharya, and Kaushik Dutta from New York Structural Biology Center for assistance with
NMR experiments.
Address correspondence to Mark Lukin, Department of Pharmacological Sciences, Stony Brook
University, Stony Brook, NY, 11794-8651, USA. E-mail: lukin@pharm.stonybrook.edu
562

Stereoselective Nucleoside Deuteration for NMR of DNA
563
Selective deuteration as a method for masking resonances in the NMR
spectra of nucleic acids has become popular during last decades.[1] In this
regard, simplification of signals from the 5ꢁ-methylene group can be achieved
by the selective deuteration of one of its hydrogens. This could be done by
either conversion of this group into a chiral synthon (e.g., α-halogen[2] or α-
selenophenyl[3] ether), followed by a reduction with a deuteride ion donor,
or oxidation to the corresponding 5ꢁ aldehyde, followed by stereospecific
reduction.[4]Although the first approach allows the usage of more general
reducing agents, it is usually more laborious requiring many synthetic steps
that frequently have insufficient stereospecificity. Therefore, a number of
approaches have been used for the reduction of deuterated 5ꢁ-oxosugar
derivatives by chiral hydride donors, including (−)-isobornyloxymagnesium
bromide,[4] chloro(diisopinocampheyl)borane,[5] or AlpineBorane.[6]
Alternatively, non-deuterated 5ꢁ-oxosugars were reduced with deuter-
ated isobornyloxymagnesium bromide or deutero-AlpineBorane,[7] with the
latter approach successfully utilized for preparation of 5ꢁ-deuterated ribonu-
cleosides.[8] Since the preparation of deuterated AlpineBorane is straight-
forward following hydroboration of α-pinene with in situ generated deutero-
borabicyclononane (d-BBN),[7] we utilized this approach for our structural
study of damaged DNA.
RESULTS AND DUSCUSSION
Although the synthesis of 5ꢁ-monodeuterated ribonucleosides can be
successfully performed starting from 5ꢁ-oxonucleosides, the same approach
worked poorly for the preparation of 2ꢁ-deoxynucleotides, presumably due
to the insufficient stability of 5ꢁ-oxo-2ꢁ-deoxynucleosides (data not shown).
Therefore we decided to use a sugar precursor for our synthesis. The start-
ing material for the synthesis (Scheme 1), 3-O-t-butyldimethylsilyl-2-deoxy-D-
erythro-pentofuranoside (1) was obtained in three steps from deoxyribose.[9]
Oxidation of the 5-hydroxyl with DMSO/oxalyl chloride yielded the alde-
hyde (2), which was immediately subjected to asymmetric reduction by B-3-
pinanyl-9-borabicyclo[3.3.l]nonane (3) (Alpine-Borane-d) without previous
purification. Alpine-Borane-d was prepared by hydroboration of R-(+)-α-
pinene with B-deutero-BBN generated in situ by treatment of commercially
available B-methoxy-9-BBN with lithium aluminium deuteride. Treatment
of the aldehyde with Alpine-Borane-d yielded the deuterated deoxyriboside
in almost quantitative yield. The deuterated sugar (4) was converted into
the di-p-toluyl ester (6) and then into the α-chlorosugar (7) with 54% over-
all yield according to the standard procedure (Scheme 1).[10] Coupling of
the chlorosugar with bis-trimethylsilyl thymine or uracyl in standard condi-
tions[11] produced protected thymidine or 2ꢁ-deoxyuridine (8a,b), respec-
tively, compounds that were further converted into 5ꢁ-dimethoxytrithylated

564
M. Lukin and C. del los Santos
D
1)
B
3
1) TlCl/Py
2) TBAF
3) TlCl/Py
D
H
HO
O
O
O
HO
2) MeCHO, H2O2, NaOH
DMSO/(COCl)2
OMe
O
OMe
OMe
TBSO
TBSO
TBSO
TlO
1
2
4
D
D
H
D
H
H
TlO
TlO
O
TlCl/Py
HCl/AcOH
O
O
OMe
OMe
Cl
HO
TlO
TlO
5
7
6
Bis-trimethylsilyl thymine
or
Bis-trimethylsilyl uracyl
D
H
B
XO
O
YO
8a : B=Thymine, X=Tl, Y=Tl
8
b : B=Uracyl, X=Tl, Y=Tl
NH3/MeOH
DMTCl/Py
9a : B=Thymine, X=H, Y=H
9
b : B=Uracyl, X=H, Y=H
10a: B=Thymine, X=DMT, Y=H
10
b : B=Uracyl, X=DMT, Y=H
SCHEME 1 Synthetic procedure used for preparation of partially deuterated nucleosides.
nucleosides (10a,b). High-resolution MS analysis indicated 97.7% deu-
terium incorporation and (Figure 1, right panel), whereas 1H-NMR spectra
demonstrated disappearance of the 5ꢁHR proton (configuration at C-5ꢁ was
determined according to the mechanism of reduction as proposed by Mid-
land et al.7) with enantiomeric ratio greater than 95% (Figure 1, left panel).
Proton decoupled 13C-NMR spectra of the deuterated derivatives contained
a triplet signal typical for deuterium-carbon spin-spin coupling (JD,C = 19.5
Hz) that served as additional evidence for deuterium incorporation (Fig-
ure 2). Since conversion of deoxyuridine to deoxycytidine is straightfor-
ward[11], this method can be readily extended to the preparation of deuter-
ated dC derivatives.
Subsequent phosphoramidite preparation and solid phase synthesis of
deuterated oligonucleotides were performed following standard protocols
without modifications.[12]
Implementation of this approach was vital for structure determination
of a damaged undecamer duplex containing at its center the dA lesion of
aristolochic acid II paired to thymidine. Aristolochic acids (AAs) are a family
of naturally occurring nitroaromatic compounds that, upon metabolic acti-
vation to a reactive carbenium cation, react with the exocyclic amino groups

565

566
M. Lukin and C. del los Santos
FIGURE 2 Fragments of proton decoupled 13C-NMR spectra of methyl-5–2H-3,5-O-bis-p-toluoyl-2-α,β-D-
erythro-pentofuranoside (left), 5ꢁ-2H-5ꢁ-dimethoxytritylthymidine (center), and 5ꢁ-2H-5ꢁ-dimethoxytrityl-
2ꢁ-deoxyuridine (right). The signals of C-5 (C-5ꢁ) with carbon-deuterium splitting are marked with aster-
isks.
of adenine and guanine forming highly mutagenic lesions.[13] In order to
elucidate molecular mechanisms of lesion repair and mutagenesis, it is nec-
essary to know the three-dimensional structure of duplex DNA containing
AA lesions (Figure 3). NMR studies of the lesion containing duplex demon-
strated that the thymidine complementary to the AA adduct assumed an
extrahelical orientation. As a result, it exhibited a limited number of nOe
contacts with adjacent nucleosides and its conformation poorly defined in
the final refined structure. To overcome this problem, we prepared the du-
plex containing a stereoselectively deuterated thymidine residue opposite
the AA lesion. Comparison of the NOESY spectra of the fully protonated
and partially deuterated duplexes (Figure 4, left) allowed us to assign both
FIGURE 3 The oligonucleotide duplex bearing an ALII lesion used in the present study.

Stereoselective Nucleoside Deuteration for NMR of DNA
567
FIGURE 4 Left) Superimposed regions of two-dimensional 1H-NOESY spectra of DNA duplexes con-
taining an aristolochic acid II generated lesion. The spectrum of the non-deuterated duplex is shown in
red and that of the 5ꢁ-monodeuterated duplex is in blue. Right) Close up view of the lesion site structure
after energy minimization of the damageded duplex. Distances corresponding to the cross-peaks visible
only on the deuterated duplex’s spectrum are shown as dashed lines. The 5ꢁꢁ-Deuterium is shown in
green.
diastereotopic 5ꢁ protons unequivocally, increasing the number of experi-
mental restraints used for structure determination. Energy minimized three-
dimensional structure of the duplex (Figure 4, right)[14] demonstrated that
the nOe distances derived from the 5ꢁ and 5ꢁꢁ protons provided valuable
information for defining the thymidine glycosidic χ and sugar γ torsion
angles, as well as the relative localization of the thymine and the aristolochic
acid residues in the duplex.
CONCLUSION
We developed a simple and robust scheme for the preparation of stere-
oselectively deuterated 5ꢁ-pyrimidine nucleosides, in quantities suitable for
chemical synthesis of oligonucleotides in amounts needed for NMR stud-
ies. We demonstrated that the use of the stereoselective deuterium labeling
lead to considerable simplification of NMR spectra and facilitated access
to otherwise inaccessible information used for determination of the duplex
conformation. A manuscript describing the complete NMR structure de-
termination of duplex DNA having an AAII-dA adduct will be submitted
elsewhere.

568
M. Lukin and C. del los Santos
EXPERIMENTAL
General Methods
NMR spectra were recorded on Varian Inova (400, 500, or 600 MHz, Var-
ian Inc., CA, USA) and Brucker Avance (600 or 800 MHz, Bruker BioSpin,
MA, USA) spectrometers. Chemical shifts are reported in parts per mil-
lion (ppm) relative to the residual proton signal (7.26 ppm) of deuterated
chloroform or to the central carbon peak (77.0 ppm) for proton and car-
bon spectra, respectively. Analytical thin layer chromatography (TLC) was
performed with E. Merck precoated TLC plates, silica gel 60F-254, layer
thickness 0.25 mm. Flash chromatography separations were performed on
E. Merck Kieselgel 60 (230–400) mesh silica gel.
Methyl-5–2H-5-O-p-toluoyl-3-O-tert-butyldimethylsilyl-2-α,β-D-erythro-
pentofuranoside (4)
A mixture of dimethyl sulfoxide (71 mL, 100 mmol) in 20 mL of dry
CH2Cl2 was added drop wise to a cooled (−55◦C) solution of oxalyl chlo-
ride (40 mL, 46 mmol) and dry dichloromethane (120 mL) under nitrogen.
The reaction mixture was stirred at −50◦C for 30 minutes and methyl-3-O-
tert-butyldimethylsilyl 2ꢁ-deoxyribofuranoside (7.39 g, 30 mmol) in CH2Cl2
(50 mL) was added drop wise via syringe during a period of 30 minutes
under vigorous stirring. After complete addition, the reaction mixture was
further stirred at −55◦C for one more hour, time after which 30 mL of dry
triethylamine were added drop wise via syringe and the mixture allowed
to warm to room temperature. Saturated sodium bicarbonate (200 mL)
was added, the organic layer removed, subsequently washed with saturated
sodium bicarbonate, brine, and later dried over Na2SO4 and filtered through
Celite. The solvent was removed under reduced pressure and the resulting
2
aldehyde was dissolved in 50 mL of THF and H-AlpineBorane (18.3 mL,
60 mmol), prepared according to the Midland’s procedure from (+)-α-
pinene and deutero-BBN[7] was added under atmosphere of nitrogen. The
reaction was stirred overnight at room temperature, quenched by addition of
6 mL acetaldehyde, stirred for an additional hour, followed by dropwise addi-
tion of 6 mL THF, 60 mL of 1M NaOH and 20 mL of 30% H2O2 with vigorous
stirring and cooling in an ice bath. Saturated sodium bicarbonate (200 mL)
and ethyl acetate (200 mL) were added, organic layer separated, washed
with brine, dried over Na2SO4, and evaporated. The transparent viscous oil
residue was dissolved in 150 mL of acetonitrile and washed with hexane (2 ×
100 mL), the acetonitrile layer was separated, evaporated, co-evaporated
with pyridine (2 × 50 mL), dissolved in anhydrous pyridine (100 mL) fol-
lowed by addition of p-toluoyl chloride (6 mL, 45 mmol). The reaction was
left 5 hours at room temperature, partitioned between sodium bicarbonate

Stereoselective Nucleoside Deuteration for NMR of DNA
569
and ethyl acetate, the organic layer was separated, washed with sodium bi-
carbonate (3 × 200 mL), water (2 × 200) and brine, dried over Na2SO4,
evaporated to dryness and co-evaporated with toluene. Flash chromatogra-
phy of the residue (hexane–EtOAc 1:1) gave methyl-5–2H-5-O-p-toluoyl-3-
O-tert-butyldimethylsilyl-2-deoxyribofuranoside as a semitransparent viscous
oil (5.1 g, 12.9 mmol, 43% from 1). NMR showed that the desired product
was still contaminated with low polar compounds that were separated out
at the next step. 1H-NMR, anomeric mixture (500 MHz, CDCl3) δ 7.98–7.95
(m, 2H, ArH), 7.23–7.28 (m, 4H, ArH), 5.08–5.04 (m, 1H, H1 α+β), 4.53
(m, 1H, H4 α+β) 4.46 (m, 1H, H5 α+β), 4.21 (q, 0.5H, H3 α or β), 4.14
(m, 0.5H, H3 α or β), 3.41(s, 1.5H, OCH3 α or β), 3.32 (s, 1.5H, OCH3
α or β), 2.48 m, 1H, 2.42 (s, 3H, ArCH3 α or β), 2.24–2.08 (m, 1H, H2ꢁα+β)
1.90–1.86 (m, 1H, H2 α+β) 0.89 (s, 9H, t-Bu), 0.06 (s, 6H, SiCH3). 13C-NMR
(125.7 MHz, CDCl3) δ166.7, 143.9, 130.4, 130.0, 129.9, 129.7, 129.4, 129.3,
129.2, 127.5, 105.2, 104,6, 84.0, 81.1, 72.5, 72.0, 64.8 (t, JC,D = 19.6 Hz), 63.6
(t, JC,D = 19.6 Hz), 55.3, 55.1, 42.3, 42.0, 33.6, 31.7, 27.4, 25.9, 25.6, 23.1,
21.9, 18.14, −4.3, −4.4, −4.6.
Methyl-5–2H-5-O-p-toluoyl-2-α,β-D-erythro-pentofuranoside (5)
A 1.0 M solution of TBAF in THF (40 mL) was added to methyl-5–2H-5-
O-p-toluoyl-3-O-tert-butyldimethylsilyl-2-deoxyribofuranoside (4.24 g, 11.64
mmol) and the homogeneous solution was left overnight at room tempera-
ture. The mixture was poured into a saturated sodium bicarbonate solution,
extracted with ethyl acetate, the organic layer was washed with saturated
sodium bicarbonate, dried over Na2SO4, and evaporated to dryness. Flash
chromatograpy of resulting oily residue (methanol, 0–5% in methylene chlo-
ride) gave 2.6 g of methyl-5–2H-5-O-p-toluoyl-2-deoxyribofuranoside (10.35
mmol, 89%) as a yellowish transparent oil. 1H-NMR, anomeric mixture (500
MHz, CDCl3) δ 7.96 (d, 1H, ArH), 7.90 (d, 1H, ArH), 7.24 (d, 2H, ArH),
5.14 (d, 0.5H, H1 β or α), 5.10 (d, 0.5H, H1 α or β), 4.55 (m, 1H, H4α+β),
4.43–4.39(m, 1H, H5α+β), 4.32–4.18(m, 1H, H3 α+β), 1H, 3.40 (s, 1.5H,
OCH3 α or β), 3.32 s, 1.5H, OCH3 α or β), 2.41 (s, 3H, ArCH3), 2.32–2.08
(m, 2H, H2 α+β). 13C-NMR (125.7 MHz, CDCl3) δ166.8, 166.6, 144.1, 144.0,
130.0, 129.9, 129.4, 129.3, 127.4, 127.2, 105.7, 105.3, 85.2, 83.9, 73.3, 72.6,
65.1 (t, JC,D = 19.1 Hz), 64.3 (t, JC,D = 19.1 Hz), 55.2, 41.8, 41.1, 21.8.
Methyl-5–2H-3,5-O-bis-p-toluoyl-2-α,β-D-erythro-pentofuranoside (6)
N-methylimidazole (100 µl) were added to methyl-5–2H-5-O-p-toluoyl-2-
deoxyribofuranoside (2.27 g, 9.22 mmol) dissolved in 20 mL of anhydrous
pyridine and p-toluoyl chloride (1.32 mL, 10 mmol). The mixture was left
overnight at room temperature. Methanol (5 mL) was added and the re-
action mixture evaporated, the residue dissolved in 100 mL of methylene
chloride, washed with saturated sodium bicarbonate (3 × 200 mL), brine,

570
M. Lukin and C. del los Santos
dried over Na2SO4, and evaporated in vacuo. The residue (3.5 g, 8.67 mmol,
95%) was used without further purification. A sample for NMR study was
1
additionally purified by crystallization from methanol. H-NMR, anomeric
mixture (500 MHz, CDCl3) δ 7.98 (m, 2H, ArH), 7.94 (d, 2H, ArH), 7.23 (m,
4H, ArH), 5.60 (t, 0.53H, H1β), 5.41 (m, 0.47H, H1α), 5.19–5.24 (m, 0.5H,
H4 α or β) 5.19 (m, 0.5H, H4 α or β), 4.36–4.61 (m, 2H, H3+H5 α+β),
3.43 (s, 1.5H, OCH3 α or β) 3.37 (s, 1.5H, OCH3 β or α), 2.58–2.52 (m,
2H, H2 α+β) 2.42 (s, 3H, ArCH3) 2.40 (s, 3H, ArCH3), 2.57–2.22 (m, 2H,
H2 α+β). 13C-NMR (125.7 MHz, CDCl3) δ 166.7, 166.5, 166.3, 144.2, 144.1,
144.0, 130.0, 129.9, 129.3, 127.3, 105.9, 105.3, 82.0, 81.1, 75.6, 74.8, 65.0 (t,
JC,D = 19.5 Hz), 64.2 (t, JC,D = 19.5 Hz), 39.5, 21.9.
5ꢀ-2H-3ꢀ,5ꢀ-bis-O-toluolyl-thymidine (8a) and 5ꢀ-2H-3ꢀ,5ꢀ-bis-O-
toluolyl-2ꢀ-deoxyuridine (8b)
Methyl 5-;2H -3,5-bis-O-toluolyl-2-deoxyriboside (6) (1.91 g, 5.18 mmol)
was dissolved in 15 mL of glacial acetic acid—CH2Cl2 (1:1), the mixture
was cooled at 0◦C and a stream of dry HCl was passed through the reaction
mixture. After 1 hour the white precipitate was filtered off, washed with
small amount of ice cold ether, dried in vacuo over KOH and immediately
converted into corresponding nucleosides. Yield 1.45 g, (3.88 mmol, 75%
from 6).
Thymine (0.38 g, 3 mmol) was refluxed overnight in a mixture of
chlorotrimethylsilane (5 mL) and hexamethyldisilazane (15 mL). The so-
lution was allowed to cool and the volatile materials were evaporated in
vacuo. The residue was dissolved in 20 mL of anhydrous CH2Cl2, the chloro
sugar 7 (0.84 g, 2.25 mmol) added, and the reaction mixture was stirred
for 6 hours, time after which MeOH (1 mL) was added and the excess
thymine removed by filtration. The filtrate was evaporated, and the residue
was recrystallized from EtOAc, yielding 0.81 g (1.69 mmol) of 5ꢁ-2H -3ꢁ,5ꢁ-
1
bis-O-toluolylthymidine (8a, 75% from 7). H-NMR (500 MHz, CDCl3) δ
8.55 (1H, br s, NH), 7.97–7.93 (4H, m, ArH), 7.30–7.27 (5H, m, ArH+6H),
6.49–6.45 (1H, dd, J = 5.3Hz, 8.1 Hz, H1ꢁ), 5.64 (1H, m, H3ꢁ), 4.78 (1H, d,
J = 2.8 Hz, H5ꢁ), 4.53 (1H, m, H4ꢁ), 2.73–2.69 (1H, ddd, J = 14.3Hz, 5.3
Hz, 1.3 Hz, H2ꢁ or H2ꢁꢁ), 2.50 (3H, s, ArCH3) 2.44 (3H, s, ArCH3), 2.33–2.29
(1H, m, H2ꢁꢁ or H2ꢁ), 1.64 (3H, s, CH3).
13C-NMR (125.7 MHz, CDCl3) δ 166.3, 163.5, 150.4, 144.8, 130.1, 129.8,
129.7, 129.5, 126.8, 126.5, 111.9, 85.2, 83.0, 75.1, 64.1 (br. t), 38.3, 21.9, 12.3.
Uracyl (0.28 g, 2.5 mmol) was refluxed overnight in a mixture of
chlorotrimethylsilane (5 mL) and hexamethyldisilazane (15 mL). The so-
lution was allowed to cool and the volatile materials were evaporated in
vacuo. The residue was dissolved in 20 mL of anhydrous CH2Cl2, the chloro
sugar 7 (0.59 g, 1.57 mmol) was added, and the reaction mixturestirred for
6 hours, time after which MeOH (1 mL) was added and the excess of uracil

Stereoselective Nucleoside Deuteration for NMR of DNA
571
was removed by filtration. The filtrate was evaporated and the residue was
recrystallized from EtOAc, yielding 0.57 g (1.22 mmol) of 5ꢁ-2H-3ꢁ,5ꢁ-bis-O-
toluolyl-2ꢁ-deoxyuridine (8b, 78% from 6). 1H-NMR (500 MHz, CDCl3) δ 8.5
(1H, br s, NH), 7.97 (4H, m, ArH), 7.91 d, 4H, 7.54 d, 1H, 7.29–7.25 m,
4H, 6.4 t, 1H, 5.6 m, 2H, 4.7 m, 1H, 4.5 m, 1H, 2.78–2.74 m, 1H, 2.44 s, 6H,
2.34–2.30 m, 1H. 13C-NMR (125.7 MHz, CDCl3) δ 166.3, 162.7, 150.2, 144.9,
139.0, 130.0, 129.9, 129.7, 129.6, 129.5, 126.7, 126.5, 103.1, 85.6, 83.2, 74.8,
63.6 (br. t), 38.6.
5ꢀ-2H-5ꢀ-dimethoxytritylthymidine (10a)
5ꢁ-2H-3ꢁ,5ꢁ-O-bis-p-toluoylthymidine (8a, 0.66 g, 1.38 mmol) was added
to 100 mL of saturated methanolic ammonia solution and the mixture was
stirred overnight at room temperature. When the nucleotide was fully de-
protected (TLC, chloroform-methanol 10:1), the mixture was evaporated
in vacuo. The residue was co-evaporated with pyridine (2 × 10 mL) and
dissolved in 10 mL of dry pyridine. Dimetoxytrityl chloride (0.68 g, 2 mmol)
was added and the reaction mixture left overnight at room temperature.
After disappearance of the starting nucleoside (TLC, chloroform-methanol
9:1), 150 mL of saturated sodium bicarbonate was added and the reaction
mixture extracted with EtOAc (2 × 100). Combined organic extracts were
washed with sodium bicarbonate, brine, dried over over Na2SO4, evaporated
in vacuo and co-evaporated with toluene (2 × 10 mL). Flash-chromatography
of the resulting residue (metanolic gradient, 0–5% in CH2Cl2 + 1% triethy-
lamine) gave the final product, 5ꢁ-2H-5ꢁ-dimethoxytritylthymidine (10a) as
yellowish foam (0.61 g, 1.12 mmol, 81% from 8a). 1H-NMR (500 MHz, CDCl3
+ triethylamine) δ 7.61 (1H, s, H6), 7.40 (1H, d, J = 7.2 Hz, ArH), 7.30 (6H,
m, ArH), 7.20 (7H, m, ArH) 6.84 (4H, d, J = 9.1 Hz, ArH), 6.43 (1H, ψt,
J = 6.1 Hz, H1ꢁ), 4.56 (1H, m, H3ꢁ), 4.07 (1H, m, H4ꢁ), 3.78 (6H, s, OCH3),
3.50 (H1, d, J = 3.0 Hz, H5ꢁ), 2.45–2.41 (1H, m, H2ꢁ or H2ꢁꢁ), 2.33–2.75 (1H,
m, H2ꢁꢁ or H2ꢁ), 1.43 (3H, s, CH3). 13C-NMR (125.7 MHz, CDCl3 + triethy-
lamine) δ 164.3, 158.9, 150.8, 144.6, 135.9, 135.7, 135.6, 130.3, 128.3, 128.2,
127.3, 113.5, 111.4, 86.4, 85.0, 72.39, 64.5 (br. t.), 55.4, 46.2, 41.2, 12.0, 11.4.
HRMS [M-Na]+ Calc. m/z 568.2164, obs. m/z 568.2159 (mass tolerance
10 ppm).
5ꢀ-2H-5ꢀ-dimethoxytrityl-2ꢀ-deoxyuridine (10b)
5ꢁ-2H -3ꢁ,5ꢁ-O-bis-p-toluoyl-2ꢁ-deoxyuridine (8b, 0.54 g, 1.16 mmol) was
added to 100 mL of saturated methanolic ammonia solution and the mix-
ture was stirred at room temperature overnight. When the nucleotide was
fully deprotected (TLC, chloroform-methanol 10:1), the mixture was evap-
orated in vacuo, the residue co-evaporated with pyridine (2 × 10 mL) and
dissolved in 10 mL of dry pyridine. Dimetoxytrityl chloride (0.64 g, 1.9 mmol)

572
M. Lukin and C. del los Santos
was added and the reaction mixture was left overnight at room temperature.
After disappearance of the starting nucleoside (TLC, chloroform-methanol
9:1), 150 mL of saturated sodium bicarbonate was added and the reaction
mixture extracted with EtOAc (2 × 100). Combined organic extracts were
washed with sodium bicarbonate, brine, dried over Na2SO4, evaporated in
vacuo, and co-evaporated with toluene (2 × 10 mL). Flash-chromatography
of the resulting residue (metanolic gradient, 0–5% in CH2Cl2 + 1% tri-
ethylamine) gave the final product, 5ꢁ-2H-5ꢁ-dimethoxytrityl-2ꢁ-deoxyuridine
1
(10b) as yellowish foam. (0.48 g, 0.9 mmol, 78% from 8b) H-NMR (500
MHz, CDCl3 + triethylamine) δ 7.81 (1H, d, J = 8.1 Hz, H6), 7.41 (1H, d,
J = 7.2Hz, ArH), 7.28–7.22 (m, 8H, ArH) 6.84 (4H, d, J = 8.71 Hz, ArH),
6.31 (1H, ψt, J = 6.1 Hz, H1ꢁ), 5.39 (1H, d, J = 8.1 Hz, H5), 4.54 (1H,
m, H3ꢁ), 4.01 (1H, m, H4ꢁ), 3.75 (6H, s, OCH3), 3,45 (1H, d, J = 3.1 Hz,
H5ꢁ), 2.46–2.41 (1H, m, H2ꢁ or H2ꢁꢁ), 2.27–2.22 (1H, m, H2ꢁꢁ or H2ꢁ). 13C-
NMR (125.7 MHz, CDCl3+ triethylamine) δ 163.4, 159.0, 150.5, 144.5, 140.4,
135.6, 135.5, 130.3, 130.2, 128.4, 128.2, 127.4, 113.5, 102.4, 87.2, 86.2, 85.2,
71.5, 62.5 (br. t), 55.5, 46.3, 41.5, 11.5. HRMS [M-Na]+ Calc. m/z 554.2008,
obs. m/z 554.2004 (mass tolerance 10 ppm)
Preparation of Phosphoramidite and Oligonucleotide Synthesis
Conversion of 10a into corresponding phosphoramidite and subse-
quent solid phase synthesis of the deuterated oligonucleotide (the bot-
tom strand of duplex in Figure 3) were performed following standard
protocols without modifications.[12] 5ꢁ-2H-5ꢁ-dimethoxytritylthymidine (10a,
65 mg, 0.119 mmol) was co-evaporated with dry toluene. Diisopropylam-
monium tetrazolide (10mg, 0.059 mmol) was added, followed by addi-
tion of 2 ml of dry dichloromethane under argon atmosphere. Bis-(N-
diisopropylamino)-2-cyanoethoxy phosphine (27 ul, 0.13 mmol) was added
and the mixture stirred overnight under argon atmosphere. After addition of
dichloromethane (5 mL), and the mixture was extracted with 2% potassium
carbonate and brine, dried over Na2SO4, evaporated in vacuo, co-evaporated
with dry acetonitrile and used immediately for the oligonucleotide synthesis.
Solid phase synthesis was performed on ABI 380 DNA synthesizer at 10 umol
scale. Incorporation of the deuterated residue was done via manual injection
of the amidite-activator mixture directly into the column. Coupling time was
increased to 2 minutes, coupling efficiency was 92%. The oligonucleotide
was deprotected and purified according to standard protocols.[14] ESI [M-
H+] Cald m/z 3372.3, obs. 3373.27 1.0. Synthesis of the AAII modified
oligonucleotide (top) strand has been reported elsewhere.[15] Details of the
two-dimensional NMR experiments, as well as full assignment of the duplex’
proton resonances will be reported elsewhere.[16]

Stereoselective Nucleoside Deuteration for NMR of DNA
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