Synthesis of oligodeoxynucleotides containing 6-N-([13C]methyl)adenine and 2-N-([13C]methyl)guanine

Article abstract of DOI:10.1039/a700366h

Oligonucleotides containing 6-N-([^l3C]methyl)adenine and 2-N-([¹³C]methyl)guanine have been prepared for NMR studies using the deprotection step to introduce the [¹³C]methylamine group. For this purpose, the use of 2′-deoxy-6-O-(pentafluorophenyl)inosine 1 and 2′-deoxy-2-fluoro-6-O-[2-(4-nitrophenyl)-ethyl]inosine 2 as precursors of the N-methylated nucleosides is described. Preliminary NMR characterization of the ¹³C-labelled oligonucleotides shows that the ¹³C chemical shift of the methyl group in N-methylguanine is sensitive to duplex formation, making it a useful local probe.

Full text of DOI:10.1039/a700366h

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Synthesis of oligodeoxynucleotides containing
6-N-([13C]methyl)adenine and 2-N-([13C]methyl)guanine
Mechtild Hofmann,a Montse Acedo,b Patricia Fagan,c David Wemmer,c
Ramon Eritja *,a and Antonio R. Díaza
a
European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany
b Department of Molecular Genetics, CID-CSIC, Jordi Girona 18–26, 08034 Barcelona, Spain
c Department of Chemistry, University of California at Berkeley, Berkeley, California 94720,
USA
Oligonucleotides containing 6-N-([13C]methyl)adenine and 2-N-([13C]methyl)guanine have been prepared
for N M R studies using the deprotection step to introduce the [13C]methylamine group. For this purpose,
the use of 2Ј-deoxy-6-O-(pentafluorophenyl)inosine 1 and 2Ј-deoxy-2-fluoro-6-O-[2-(4-nitrophenyl)-
ethyl]inosine 2 as precursors of the N-methylated nucleosides is described. Preliminary N M R
characterization of the 13C-labelled oligonucleotides shows that the 13C chemical shift of the methyl
group in N-methylguanine is sensitive to duplex formation, making it a useful local probe.
described for the preparation of oligonucleotides containing
Introduction
modified adenines. Recently, use of 6-(methylthio)purine has
been described for the preparation of 6-substituted purines
including 6-N-methyladenine.20 We have selected 2Ј-deoxy-6-O-
(pentafluorophenyl)inosine 1 (Scheme 1) as precursor of the
One of the problems found during the structural elucidation of
DNA–protein interactions is the complexity of the spectra. The
introduction of 15N and 13C in combination with special pulse
sequences allows the selective observation of specific base
pairs.1 Although a large effort has been devoted to the prepar-
ation of oligodeoxynucleotides having 15N labels,1 there are few
examples of 13C-labelled oligodeoxynucleotides at specific sites.
A thymidine phosphoramidite labelled at C-6 with 13C has been
prepared and incorporated into a synthetic hairpin oligo-
nucleotide.2 NMR relaxation measurements showed a higher
internal motion in the loop region than in the duplex.2,3 Also,
oligonucleotides labelled with 13C at the thymine methyl groups
have been prepared to study hydrophobic contacts between
DNA and the DNA-binding domain of the rat glucocorticoid
receptor.4 The analysis revealed a hydrophobic contact between
a labelled thymine group and the methyl groups of a valine resi-
due. Base-pairing properties of 2Ј-deoxy-5-(methoxymethyl)-
uridine have also been studied by NMR spectroscopy using a
heptanucleotide containing a 13C label at the exocyclic position
of this analogue.5
Target C-13 labelled nucleoside
Nucleoside precursor
F
F
F
F
F
NH-13CH3
N
O
N
N
N
N
N
1, Ipf
N
N
OCH2CH2C6H4NO2-p
O
N
N
N
N
N-H
N
N
NH-13CH3
N
F
2, If
Recently, the development of nucleoside derivatives that
can be transformed into different nucleoside analogues during
deprotection has been described.6–10 This method, named ‘the
convertible nucleoside approach,’ has been used to prepare
oligodeoxynucleotides containing 15N labels at exocyclic
positions.10–12 In the present communication we describe the
preparation of oligodeoxynucleotides containing N-methyl-
adenine and N-methylguanine residues with a 13C label at the
methylamino exocyclic position. The methodology described
here uses a modified deprotection protocol to incorporate the
13C label and to our knowledge is the first time that 13C labels
have been incorporated in the amino groups involved in base-
pairing.
Scheme 1 Nucleoside derivatives used in this work for the preparation
of oligonucleotides containing 6-N-([13C]methyl)adenine and 2-N-
([13C]methyl)guanine
N-methyladenine residue because previous studies have shown
that this derivative is stable to oligonucleotide synthesis con-
ditions and reacts readily with amines even in the presence of
water without detectable hydrolysis.12
2Ј-Deoxy-6-O-(pentafluorophenyl)inosine 12 1 was prepared
following previously described protocols. Overnight treatment
of 2Ј-deoxy-6-O-(pentafluorophenyl)inosine with aq. methyl-
amine (prepared by addition of triethylamine to aq. CH3NH2ؒ
HCl) at 60 ЊC yielded the desired 2Ј-deoxy-N-methyladenosine
as the only nucleoside product that was isolated and
characterized.
Results and discussion
When amines are used for the deprotection of synthetic oligo-
nucleotides, approximately 10–20% of the N-benzoylcytidine
residues react with the amine, yielding the corresponding 4-N-
alkylcytidine6 derivatives. This side reaction can be minimized
by use of other protected derivatives of cytidine.21,22 Among
them, tert-butylphenoxyacetyl23 (ExpediteTM ) protective groups
were selected. A short oligonucleotide containing 3 cytosines
Preparation of oligonucleotides containing
N6-([13C]methyl)adenine
Previous reports have shown that 6-chloropurine and 6-
fluoropurine 2Ј-deoxyriboside react with amines to produce
adenine adducts at nucleoside13–17 and oligonucleotide18,19
levels. On the other hand, 6-O-aryl-dI derivatives8,9,12 have been
J. Chem. Soc., Perkin Trans. 1, 1997
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Fig.
1
Enzymic digestion of purified 6-N-([13C]methyl)adenine
dodecamer {5Ј-GCGAA*TTCGCGC-3Ј where A* is 6-N-([13C]-
Fig.
2
Enzymic digestion of purified 2-N-([13C]methyl)guanine
dodecamer {5Ј-CACCG*ACGGCGC-3Ј where G* is 2-N-([13C]-
methyl)adenine}
methyl)guanine}
(5Ј-CCCAT-3Ј) was prepared using tert-butylphenoxyacetyl
phosphoramidites. At the end of the synthesis, the support was
treated overnight at 60 ЊC with aq. methylamine (prepared by
addition of triethylamine to an aq. CH3NH2ؒHCl). The result-
ing oligonucleotide was purified by HPLC and analysed by
enzyme digestion. Only dC, dA and T were found. The product
resulting from the attack of methylamine on protected cytidine,
4-N-methyl-dC, was not detected. An authentic sample of 4-N-
methyl-dC was prepared from 4-O-ethyl-dU to determine the
position at which 4-N-methyl-dC elutes.
The dodecamer having the recognition sequence of E. coli
methylase (5Ј-GCGAIpfTTCGCGC-3Ј with Ipf being 6-O-penta-
fluorophenyl-dI) was prepared using tert-butylphenoxyacetyl
and 6-O-pentafluorophenyl-dI 12 phosphoramidites. Treatment
of the support with aq. [13C]methylamine overnight at 60 ЊC
yielded a main product that was purified by HPLC using stand-
ard protocols. We used 250 mg of [13C]methylamine hydro-
chloride per 2 µmols of oligonucleotide, which is approximately
90 times the amount needed to remove the protecting groups
and perform the substitution. Enzyme digestion showed the
presence of the four natural nucleosides together with 6-N-
methyl-dA in the expected proportions (see Fig. 1).
solution of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) solution
in acetonitrile at room temperature for 15 min to remove the
NPE group. Under these conditions loss of oligonucleotide by
cleavage of the succinyl bond is negligible12,29 and the majority
of the NPE groups at position 6 in the guanine are removed.
Treatment of the support with aq. [13C]methylamine overnight
at 60 ЊC yielded a main product that was purified by HPLC
using standard protocols. Enzyme digestion showed the pres-
ence of dC, dG and dA together with 2-N-methyl-dG in the
expected proportions (Fig. 2). Moreover, mass spectral analysis
of the purified product by matrix-isolated laser-desorption
time-of-flight mass spectrometry (MALDI-TOF) gave the
expected molecular mass (Found: Mϩ, 3631.2, expected M,
3631). In this oligonucleotide we used 63 mg of [13C]methyl-
amine hydrochloride per 4 µmols of oligonucleotide, which is 10
times the amount needed for removal of the protecting groups.
The cost of the 13C label in this preparation is lower than the
cost of the chemicals needed to produce the oligonucleotide.
The 1D proton spectrum at 600 MHz of the dodecamer
revealed a homogeneous product with sharp signals. To confirm
the labelled methyl group was on the guanine moiety a 2D het-
eronuclear multiple quantum filtered coherence (HMQC)30
spectrum was recorded in which the carbon signals are
indirectly detected via the proton resonances of the coupled
spins. This inverse spectrum of the oligonucleotide showed
very clearly two antiphase satellites only at a centred resonance
frequency of δ[1H] 2.58 split by a coupling constant of 137 Hz
for the C᎐H coupling (data not shown).
A proton-decoupled carbon spectrum of R5 showed a
carbon signal at δ[13C] 34.37, which was assigned to the 13CH3
group of 2-N-methyl-G (see Fig. 3), also confirmed by a gated
decoupled spectrum (quadruplet at the same frequency). The
change of the chemical shift of the 13CH3 group of 2-N-methyl-
G on comparing the spectrum of R5 dodecamer with the spec-
trum of the two annealed complementary oligonucleotides
(δ[13CH3] = 32.84, ∆δ 1.53 ppm) reveals a hybridization of the
two strands.
In summary, we have shown that oligonucleotides carrying
6-N-([13C]methyl)adenine and 2-N-([13C]methyl)guanine bases
can be easily prepared using the phosphoramidites of 6-O-
pentafluorophenyl-dI and 2-fluoro-6-O-NPE-dI together with a
special deprotection protocol using [13C]methylamine. Under
these conditions, oligonucleotides are deprotected and released
from the solid support and the modified nucleoside is converted
into thedesired 6-N-([13C]methyl)adenineand 2-N-([13C]methyl)-
Preparation of oligonucleotides containing
2-N-([13C]methyl)guanine
For the introduction of 13C label at position 2 of guanine, 2Ј-
deoxy-2-fluoroinosine was selected. This nucleoside and its
protected derivatives have been used as precursors of 2-amino
substituted guanine derivatives at the nucleoside and oligo-
nucleotide level.13,14,19,24–27 2Ј-Deoxy-2-fluoro-6-O-[2-(4-nitro-
phenyl)ethyl (NPE)]inosine12 2 (Scheme 1) was prepared as
previously described. Treatment of compound 2 with aq.
methylamine at 60 ЊC gave the desired 2-N-methyl-dG as the
major product. Small amounts (less than 10%) of NPE-
protected 2-N-methyl-dG were also observed, but the hydrolysis
product (dG) was not detected. When treatment with methyl-
amine was performed at room temperature for 16 h, two
products were obtained: NPE-protected 2-N-methyl-dG and
2-N-methyl-dG, together with small amounts of starting
material (data not shown). This indicates that the displacement
of fluoride by methylamine is easier than the removal of the
NPE group.
The dodecamer 5Ј-CACCIfACGGCGC-3Ј (R5)28 with If = 2-
fluoro-6-O-NPE-dI, was prepared using tert-butylphenoxy-
acetyl and 2-fluoro-6-O-NPE-dI 12 phosphoramidites. After the
assembly of the sequence, the support was treated with a 0.5 
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J. Chem. Soc., Perkin Trans. 1, 1997

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Columns: Nucleosil 120C18, 250 × 10 mm and C4-Vydac, 250 ×
10 mm. Flow rate: 3 ml minϪ1. A 20–80% B linear gradient in
30 min for oligonucleotides containing the DMT group and a
5–50% B linear gradient in 30 min for oligonucleotides without
the DMT group.
NMR measurements
The R5 sample contained 4.6 mM (60 O.D. units at 260 nm) of
the purifed product in 0.6 ml of D2O at pH 6.8. The duplex
sample consisted of 1.2 mM (16 O.D. units) of each dodecamer
(R5 and its complementary) in 0.6 ml of D2O at pH 6.8.
The 1D-proton and the HMQC spectra were recorded on a
Bruker 600 MHz spectrometer, the chemical shifts were refer-
enced to D2O at δ 4.75. Carbon spectra were recorded on a
Bruker AM 250 MHz spectrometer with proton decoupling.
The spectra were recorded at 298 K, TD = 16K, with a spectral
width of 14 000 Hz corresponding to 220 ppm and an acquisi-
tion time of 0.59 sec, zero filling to 64K points after Gaussian
multiplication. The methyl carbon of the acetate at δC 29.5 ppm
was used as internal reference in both samples.
Conversion of 2Ј-deoxy-6-O-(pentafluorophenyl)inosine into
2Ј-deoxy-6-N-methyladenosine
2Ј-Deoxy-6-O-(pentafluorophenyl)inosine12 (0.37 mmol) was
dissolved in 2 ml of water and methylamine hydrochloride (7.4
mmol) and triethylamine (7.4 mmol) were added. The solution
was heated at 60 ЊC overnight. The resulting solution was con-
centrated to dryness and the residue was purified on silica gel
column chromatography with a 5–10% MeOH gradient in
DCM. Yield of product 0.2 g (52%). UV spectrum as previ-
ously described.34 TLC (10% MeOH–DCM) Rf 0.26; HPLC
(conditions A): one peak at tR 16.2 min. The product migrated
together with commercially available 2Ј-deoxy-6-N-methyl-
adenosine (Sigma); δH (CDCl3; 200 MHz) 8.3 (1 H, s, H-2), 7.9
(1 H, s, H-8), 6.4 (1 H, m, H-1Ј), 6.3 (1 H, m, OH-3Ј), 5.1 (1 H,
m, OH-5Ј), 4.4 (1 H, m, H-3Ј), 4.3 (1 H, m, H-4Ј), 3.9 (2 H, m,
H2-5Ј), 3.1 (3 H, s, NCH3), 2.9 (1 H, m, H-2Ј) and 2.5 (1 H, m,
H-2Ј); δC(CDCl3; 75 MHz) 155.3, 152.0, 146.0, 139.2, 122.1,
121.7, 89.3, 87.2, 72.1, 63.0, 40.4 and 13.3 (NCH3).
Fig. 3 13C NMR spectra of (a) dodecamer 5Ј-CACCG*ACGGCGC-
3Ј where G* is 2-N-([13C]methyl)guanine and (b) duplex formed by 5Ј-
CACCG*ACGGCGC-3Ј and 3Ј-GTGGCTGCCGCG-5Ј. The peak at
δC 29.5 is the methyl carbon of acetate used as a reference.
guanine residues. Modification of C residues described 6 for
4-N-benzoyl dC is avoided by using tert-butylphenoxyacetyl-
protected phosphoramidites.23 The method is simple and effi-
cient and the introduction of the label is at the end of the
synthetic process. The introduction of the methyl group does
not disturb the formation of the duplex as shown in previous
reports.31,32 Preliminary NMR characterization showed that the
13CH3 group in 2-N-([13C]methyl)guanine moved upfield upon
duplex formation indicating that this type of labelling could be
used to monitor duplex formation as well as protein–DNA
interactions.
Preparation of 2Ј-deoxy-4-N-methylcytidine
2Ј-Deoxy-5Ј-DMT-4-O-ethyluridine33 (40 mg, 0.07 mmol) was
treated with a solution of methylamine hydrochloride (3.6
mmol) and triethylamine (3.6 mmol) in water (2 ml). ACN was
added until a clear solution was obtained. The solution was
heated at 55 ЊC overnight and concentrated to dryness. The
residue was treated with aq. 80% acetic acid for 15 min at room
temperature. The solution was concentrated to dryness and the
residue was purified on silica gel and eluted with a 10–30%
MeOH gradient in DCM. Yield of product 17 mg (90%). Phys-
ical data and UV spectra as previously described.31,35 TLC (20%
MeOH–DCM) Rf 0.13; HPLC (conditions A): one peak at
tR 9.7min;δH (CD3OD;200MHz)7.8(1H, d, J 6.3, H-6), 6.3(1H,
t, J 6.4, H-1Ј), 6.0 (1 H, d, J 6.3, H-5), 4.4 (1 H, m, H-3Ј), 4.0 (1
H, m, H-4Ј), 3.8 (2 H, m, H2-5Ј), 2.3 (2 H, m, H2-2Ј) and 2.9 (3 H,
s, NCH3).
Experimental
Abbreviations: ACN: acetonitrile, DCM: dichloromethane,
DMT: 4,4Ј-dimethoxytrityl, dIf: 2Ј-deoxy-2-fluoroinosine, dIpf:
2Ј-deoxy-6-O-(pentafluorophenyl)inosine, pyr: pyridine. 2Ј-
Deoxy-5Ј-O-(4,4Ј-dimethoxytrityl)-6-O-(pentafluorophenyl)-
inosine 3Ј-O-(2-cyanoethyl)-N,N-diisopropylphosphoramid-
ite,12 2Ј-deoxy-5Ј-O-(4,4Ј-dimethoxytrityl)-2-fluoro-6-O-[2-(4-
nitrophenyl)ethoxycarbonyl]inosine 3Ј-O-(2-cyanoethyl)-N,N-
diisopropylphosphoramidite12 and 5Ј-O-(4,4-dimethoxytrityl)-
4-O-ethylthymidine33 were prepared as described. ExpediteTM
(tert-butylphenoxyacetyl) phosphoramidites were from PerSep-
tive Biosystems. 99% [13C]Methylamine hydrochloride was pur-
chased from Cambridge Isotope Laboratories, Inc.
Conversion of 2Ј-deoxy-2-fluoro-6-O-[2-(4-nitrophenyl)ethyl]-
inosine into 2Ј-deoxy-2-N-methylguanosine
2Ј-Deoxy-2-fluoro-6-O-[2-(4-nitrophenyl)ethyl]inosine12 (150
mg, 0.35 mmol) was dissolved in 1 ml of 20% methylamine (5
mmol) in water. The solution was incubated at 60 ЊC overnight.
The resulting solution was evaporated to dryness. The residue
was dissolved in 5% MeOH in DCM containing 1% triethyl-
amine and was purified on a silica gel column eluted with a 10–
60% MeOH gradient in DCM containing 1% triethylamine.
Fractions containing 2Ј-deoxy-2-N-methylguanosine (eluted
between 40 and 60% MeOH) were pooled and evaporated to
dryness to yield the title compound (40 mg, 40%). Physical and
chromatographic data as described previously.36 TLC (30%
HPLC conditions
In all cases solvent A was 20 mM triethylammonium acetate
(pH 7.8) and solvent B was a 1:1 mixture of water and
acetonitrile. For analytical runs the following conditions were
used. Columns: Nucleosil 120C18, 250 × 4 mm, flow rate: 1 ml
minϪ1 and Radial Pak C18, flow rate 2 ml minϪ1. (A) 5–95% B
linear gradient in 40 min. (B) 5–50% B linear gradient in 20 min.
For semipreparative runs the following conditions were used:
J. Chem. Soc., Perkin Trans. 1, 1997
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MeOH in DCM) Rf 0.41; HPLC (conditions A): one peak at
tR 13.6 min; δC[(CD3)2SO; 250 MHz] 156.9 (C-6), 153.2 (C-2),
150.5 (C-4), 135.7 (C-8), 116.6 (C-5), 87.5 (C-4Ј), 82.6 (C-1Ј),
70.6 (C-3Ј), 61.8 (C-5Ј), 39.5 (C-2Ј) and 27.4 (CH3).
and NATO Collaborative Research Grant 900554. We thank
Dr Nanda D. Sinha for for the generous gift of tert-
butylphenoxyacetyl-protected phosphoramidites.
References
Oligonucleotide syntheses
The sequences A: 5Ј-CCCAT-3Ј, B: 5Ј-GCGAIpfTTCGCGC-3Ј
and C: 5Ј-CACCIfACGGCGC-3Ј were prepared on an Applied
Biosystems automatic DNA synthesizer using tert-butyl-
phenoxyacetyl 2-cyanoethyl phosphoramidites and the modi-
fied phosphoramidite of 2Ј-deoxy-5Ј-O-DMT-6-O-(penta-
fluorophenyl)inosine. Sequence A was prepared on a 1 µmol
scale without the DMT at the end, sequence B on a 2 µmol scale
without DMT, and sequence C on a 4 µmol scale with the last
DMT group.
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Oligonucleotide supports were deprotected with aq. methyl-
amine overnight at 60 ЊC. Cold methylamine hydrochloride was
used for sequence A and [13C]methylamine hydrochloride was
used for sequences B and C.
For sequences A and B the following protocol was used:
Approx. 50–100 mol equiv. of methylamine hydrochloride were
dissolved in water (50 mg of CH3NH2ؒHCl in 0.5 ml for
sequence A and 250 mg of [13C]CH3NH2ؒHCl in 1 ml for
sequence B) and the solution was added to the support followed
by an equimolar amount of triethylamine with respect the
amount of CH3NH2ؒHCl (75 mg for sequence A and 374 mg for
sequence B). The reaction mixtures were kept overnight in a
screw-cap vial tightly closed inside the oven at 60 ЊC.
For sequence C the following protocol was used: The DMT-
oligonucleotide support was treated with 0.5 M DBU in ACN
at room temperature for 15 min. Afterwards, the support was
washed successively with ACN, 1% Et3N solution in ACN (to
remove DBU salts) and again with acetonitrile and was dried
under vacuum. 10 Mol equiv. of [13C]CH3NH2ؒHCl (0.92
mmol, 63 mg) were dissolved in 0.5 ml of water, and 0.13 ml of
Et3N (0.92 mmol) were added. This solution was added to the
oligonucleotide support and the mixture was treated at 60 ЊC
for 16 h. After the treatment, the solutions were concentrated to
dryness and the products were purified by reversed-phase
HPLC (see conditions above). All syntheses presented a major
peak that was collected and analysed by snake venom phos-
phodiesterase and alkaline phosphatase digestion followed by
HPLC analysis of the nucleosides (conditions B, see above; and
Figs. 1 and 2). Yield (O.D. units at 260 nm) : Pentamer A (1 µmol
synthesis): 35 O.D.; dodecamer B (2 µmol): 60 O.D.; dodecamer
C (4 µmol) : 170 O.D. Mass determination of the HPLC-purified
product using MALDI showed a molecular peak at Mϩ, 3631.18
as expected for the labelled oligonucleotide (expected mass 3631
amu).
The complementary strand of R5 (5Ј-GCGCCGTCGGTG-
3Ј) was synthesized on a 1 µmol scale using standard proto-
cols and the last DMT group was left on during deprotection
with ammonia. This oligonucleotide group was purified from
truncated sequences using COPTM (Cruachem Ltd., Scotland)
cartridges.
In order to avoid the triethylammonium signals in the NMR
spectrum purified oligonucleotides were passed through a
Dowex 50W (Naϩ-form) column. Remaining acetate ions were
used as NMR reference. An amount of 16 O.D. units of each
strand were annealed at 65 ЊC and cooled slowly to form the
duplex before the carbon spectrum was recorded.
34 A. Coddington, Biochim. Biophys. Acta, 1962, 59, 472.
35 I. Wempen, R. Duschinsky, L. Kaplan and J. J. Fox, J. Am. Chem.
Soc., 1961, 83, 4755.
36 J. Boryski and T. Ueda, Nucleosides, Nucleotides, 1985, 4, 595.
Paper 7/00366H
Received 15th January 1997
Accepted 6th March 1997
Acknowledgements
This work was supported by funds from CICYT (PB92–0043)
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