Oligonucleosides with a nucleobase-including backbone; synthesis and self-association of novel dinucleotide analogues.

Article abstract of DOI:10.1039/b300932g

The synthesis and self-association of protected oxymethylene-bridged UA analogues are described.

Full text of DOI:10.1039/b300932g

Oligonucleosides with a nucleobase-including backbone; synthesis and
self-association of novel dinucleotide analogues
Andrew J. Matthews, Punit K. Bhardwaj and Andrea Vasella*
Laboratory for Organic Chemistry, ETH-Hönggerberg, HCI, CH-8093 Zürich, Switzerland.
E-mail: vasella@org.chem.ethz.ch; Fax: +41 1632 1136; Tel: +41 1632 5130
Received (in Cambridge, UK) 23rd January 2003, Accepted 26th February 2003
First published as an Advance Article on the web 12th March 2003
The synthesis and self-association of protected oxymethy-
lene-bridged UA analogues are described.
hydroxymethylated 3. The C(8)-hydroxymethylated adenosine
7 was prepared via a similar route from protected adenosine 5.
Treatment of 3 with mesyl chloride gave the chloromethylated
4 (64% from 1). 4,4A-Dimethoxytritylation of 7 yielded 8, which
was then desilylated to 9 (59% from 5). The ether 10 (Scheme
2) was prepared by alkylation of alcohol 9 with the chloride 4;
N-debenzoylation gave the amine 11. Detritylation or desilyla-
tion of 11 led to the monoalcohols 12 and 13, respectively,
which were further deprotected to give diol 14. Acid hydrolysis2
of 14 yielded the oxymethylene dimer 15.‡
We are studying oligonucleotide analogues with a nucleobase-
including backbone (Fig. 1, A), to determine whether the
structural differentiation between nucleobase and backbone in
DNA, RNA, and their analogues (Fig. 1, B), is a prerequisite for
the formation of stable homo- and/or heteroduplexes.1†
According to the chemical shift for H–C(2A),7 the protected
uridine 2 prefers an anti conformation (dH–C(2A) = 4.70 ppm),
whilst the C(6)-substituted derivatives 3, 4 and 10–13 prefer a
syn conformation (d = 5.19–5.40 ppm). Likewise, the protected
adenosine 6 prefers the anti conformation (dH–C(2A) = 5.32
ppm), whilst the C(8)-substituted derivatives 7–13 prefer a syn
conformation (d = 5.70–6.03 ppm).
The 1H-NMR spectra of 11–13 in CDCl3 are characterised by
a concentration dependent downfield shift of the uridine H–
N(3), evidencing an intermolecular hydrogen bond. Association
constants Ka (Table 1),8 and the thermodynamic parameters
DH° and DS° in CDCl3 were calculated for 11–13 from the
d(H–N(3)) concentration and temperature dependence (Fig. 3).§
The d(H–N(3)) of diol 14 in CDCl3 was almost concentration
independent (12.96–12.65 ppm from 33–1 mM), whilst the fully
deprotected dimer 15 was insufficiently soluble to evidence
association. Other solvent systems are under investigation. The
data for 11 and 12 highlight the contribution of the lipophilic
dimethoxytrityl group, but even the Ka value obtained for the
Fig. 1 Schematic representation of (A) oligonucleotide analogues with a
nucleobase including backbone, and (B) oligonucleotides and analogues
with a structural differentiation of nucleobase and backbone.
Tetrameric analogues of type A, derived from ethynediyl-
linked adenosine and uridine, showed no evidence for homo-
pairing, and a similar uridine hexamer did not hetero-pair with
a complementary RNA strand.2 Modeling suggested that an anti
conformation of these analogues is a prerequisite for pairing,
whilst NMR analysis of an adenosine dimer showed that a syn
conformation is preferred.3 Modeling studies also suggested
that oxymethylene-bridged oligomers (Fig. 2), should pair in the
syn conformation (Watson–Crick type hydrogen bonding), so
far only known to occur in Z-DNA.4 We have hence prepared
the corresponding self-complementary UA dimer.
2A,3A-O-Isopropylideneuridine (1) was protected as the TIPS
ether 2 (Scheme 1). Deprotonation with LDA,5 followed by
formylation with DMF and reduction with NaBH46 gave C(6)-
Fig. 2 Oxymethylene bridged oligonucleotide analogues.
Scheme 1 Reagents and conditions: i. TIPSCl, imidazole, CH2Cl2, rt, 98% (2), 96% (6); ii. a) LDA (5 eq.), THF, 278 °C; b) DMF, 278 °C to rt; c) AcOH,
EtOH, NaBH4, rt, 70% (3), 74% (7); iii. MsCl, C5H5N, 0 °C to rt, 94%; iv. DMTrCl, EtN(iPr)2, CH2Cl2, 50 °C, 94%; v. TBAF, THF, rt, 89%.
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Scheme 2 Reagents and conditions: i. NaH, DMF:THF (2+1), 0 °C, 60%; ii. NH4OH, MeOH, 95%; iii. HCO2H, MeNO2, rt, 84% (12), 82% (14); iv. TBAF,
THF, rt, 91% (13), 85% (14); v. HCO2H, H2O (8:2), rt, 75%.
Table 1 Association constants and thermodynamic parameters for the
dimers 11–13 in CDCl3
5.75 (d, J = 1.0, H–C(1A/II)); 5.36 (s, H–C(5/II)); 5.34 (dd, J = 6.3, 3.8, H–
C(3A/I)); 5.27 (dd, J = 1.0, 6.3, H–C(2A/II)); 4.87 (dd, J = 6.3, 4.5, H–C(3A/
II)); 4.55, 4.41 (AB, J = 11.8, 2 H–C(10/I)); 4.44, 4.03 (AB, J = 13.3, 2
H–C(7/II)); 4.31 (ddd, J = 3.8, 5.3, 4.9, H–C(4A/I)); 4.15 (ddd, J = 4.4, 5.4,
7.1, H–C(4A/II)); 3.85 (dd, J = 10.5, 5.4, H–C(5Aa/II)); 3.83 (dd, J = 10.5,
7.1, H–C(5Ab/II)); 3.79 (s, MeO); 3.65 (dd, J = 10.5, 5.3, H–C(5Aa/I)); 3.63
(dd, J = 10.5, 4.9, H–C(5Ab/I)); 1.55, 1.55, 1.42, 1.41 (4s, Me2C); 1.01–0.96
(m, (Me2CH)3–Si). 13C-NMR (75 MHz, CDCl3): 164.07 (s, C(2/II)); 158.50
(s, C(8/I)); 155.61 (s, C(6/I)); 151.91 (d, C(2/I)); 150.74 (s, C(6/II)); 150.24
(s,C(4/II)); 150.10 (s, C(4/I)); 148.81 (s, arom. C); 144.00 (s, arom. C);
135.08 (s, arom. C); 130.02 (d, arom. C); 128.10 (d, arom. CH); 127.81 (d,
arom. CH); 126.90 (d, arom. CH); 118.45 (s, C(5/I));113.59 (s, C(Me)2/I);
113.26 (s, C(Me)2/II); 113.17 (d, arom. CH);103.71 (d, C(5/II)); 91.39 (d,
C(1A/II)); 90.00 (d, C(1A/I)); 89.65 (d, C(4A/II)); 87.50 (s, CAr3);86.74 (d,
C(4A/I)); 84.37 (d, C(2A/II)); 83.57 (d, C(2A/I)); 82.26 (d, C(3A/II)); 81.67 (d,
C(3A/I)); 69.80 (t, C(5A/I)); 68.08 (t, C(7/II)); 64.50 (t, C(5A/II)); 59.27 (t,
C(10/I); 55.23 (q, 2 3 MeO); 27.36, 27.36, 25.76, 25.76 (4q, Me2C); 17.95
(q, Me2CH)3Si); 11.99 (d, Me2CH)3Si). HR-MALDI-MS: 303 (100%,
[DMTr]+); 1114.492 (23%, [M + Na]+; calc. 1114.4936). IR (CHCl3):
3488w, 3185w, 2993m, 2943m, 2866m, 2840w, 1712s, 1636m, 1608m,
1509s, 1446m, 1383m, 1157m, 1068s, 1036m, 882m, 831m.
Ka (M21 a
)
2DH° (kcal mol21
)
2DS° (e.u.)b
11
12
13
966
277
3222
15.8
21.8
24.4
40.4
63.7
64.8
a Determined at 22 °C, uncertainty in Ka estimated at 15%. b e.u. = entropy
units (1 e.u. = 1 cal per (mol.K)).
§
NMR was performed at 295 K on a Varian Gemini300 spectrometer
(300 MHz) in CDCl3 passed through aluminium oxide immediately prior to
use. Experiments started at the highest concentration, with stepwise
replacement of 0.2 ml of the 0.7 ml solution with 0.2 ml pure CDCl3. The
data were analysed graphically and by nonlinear least-squares fitting.8
Thermodynamic parameters were determined by van’t Hoff analysis. The
uridyl dH–N(3) was monitored between 50 and 230 °C at a fixed
concentration (between 20–80% of saturation). Linear fits of data collected
below 0 °C were poor.
Fig. 3 Concentration dependence of d(H–N(3)) for dimers 11, 12 and 13 in
CDCl3 at 295 K.
detritylated 12 compares favourably with that determined for
3A,5A-di-O-acetyl-2A-deoxyuridine with a 2A-deoxyadenosine
derivative (70 M21).9 The high Ka value for 13, and the inability
to dissociate 14 appreciably in CDCl3 correlate with a
downfield shift of H–O(5A) as compared to 1 (Dd ≈ 1.0 ppm),
and are rationalised by the formation of a C(5A)O–H…ONC(2)
intramolecular hydrogen bond.
Watson–Crick type base pairing is suggested by a cross-peak
between the hydrogen bonded imino H–N(3) and the adenine
H–C(2) in a 2D-NOESY experiment on associated dimer 11.9
These results support the contention that a structural
differentiation of nucleobases and backbone is not required for
pairing. We are now investigating the details of base pairing,
stacking, and hydrogen bonding.
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We thank The Royal Society (A. J. M.), the Swiss National
Science Foundation and F. Hoffmann-La-Roche AG, Basel for
generous support.
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Notes and references
† For the sake of simplicity, we have designated these analogues as
‘oligonucleotide analogues with a nucleobase-including backbone’, while,
strictly speaking, these systems do not possess a ‘backbone’.
‡ All new compounds showed satisfactory NMR, IR, and MS data. 11: I =
adenosyl unit; II = uridyl unit. H-NMR (500 MHz, CDCl3): 13.09 (br s,
H–N(3/II)); 8.37 (s, H–C(2/I)); 8.37–7.49 (m, 2 arom. H); 7.47–7.38 (m, 4
arom. H); 7.32–7.21 (m, 3 arom. H); 6.90 (br s, 2 H–N(6/I)); 6.86–6.83 (m,
4 arom. H); 6.22 (d, J = 1.3, H–C(1A/I)); 5.88 (dd, J = 1.3, 6.3, H–C(2A/I));
1
9 A. Dunger, H-H. Limbach and K. Weisz, J. Am. Chem. Soc., 2000, 122,
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