Tandem ring-closing metathesis and hydrogenation towards cyclic dinucleotides

Article abstract of DOI:10.1039/b206560f

Cyclic dinucleotides containing a butylene nucleobase-phophotriester connection are synthesised by a tandem ring-closing metathesis and hydrogenation reaction.

Full text of DOI:10.1039/b206560f

Tandem ring-closing metathesis and hydrogenation towards cyclic
dinucleotides
Philip Børsting and Poul Nielsen*
Nucleic Acid Center,† Department of Chemistry, University of Southern Denmark, DK-5230, Odense M,
Denmark. E-mail: pon@chem.sdu.dk
Received (in Cambridge, UK) 8th July 2002, Accepted 13th August 2002
First published as an Advance Article on the web 22nd August 2002
Cyclic dinucleotides containing a butylene nucleobase–
phophotriester connection are synthesised by a tandem ring-
closing metathesis and hydrogenation reaction.
reactions. The synthesis of the dinucleotide 6a has been
described recently.15 Thus, 3a was coupled to the allylphos-
phoramidite 216 followed by oxidation to give the dinucleotide
4a which in an RCM-reaction afforded the cyclic dinucleotide
5a and, after deprotection, 6a as a mixture of stereoisomers‡
(Scheme 1).15 Hereby, we present the similar construction of the
bis-(2A-deoxy)dinucleotide. Thus, the known 5-allyl-2A-deoxy-
uridine 717 was converted to 3b in 64% yield over three steps
using standard protection/deprotection procedures. Subse-
quently, the dinucleotide 4b was synthesised as an epimeric
mixture by coupling of 2 and 3b using 1H-tetrazole as an
activator followed by oxidation of the resulting phosphite in
64% overall yield. Finally, an RCM reaction applying the same
conditions as before,15i.e. the precatalyst 1 in CH2Cl2 afforded
in 58% yield 5b as an epimeric mixture. Thus, MS-data
Conformational restriction of nucleic acid fragments has
attracted a lot of attention due to important potential applica-
tions in therapeutics and diagnostics.1,2 Thus, oligonucleotides
containing nucleoside monomers with bi- and tricyclic carbohy-
drate moieties have demonstrated high affinity for com-
plementary nucleic acids.2–4 Furthermore, conformationally
restricted dinucleotides mimicking tertiary structure ele-
ments5–7 e.g. the anticodon loop in bacterial tRNA7 have been
synthesised and investigated.5–7 Recently, the ring-closing
metathesis (RCM) methodology has shown to be a powerful
tool for introducing conformationally restricting rings into
biomolecules such as peptide structures.8,9 Especially, the
ruthenium based precatalysts such as 1 (Scheme 1) developed
by Grubbs and co-workers10 have broadened the scope of
RCM.11 We have recently applied RCM (and 1) in the
construction of bicyclic nucleosides12 and of conformationally
restricted dinucleotide13–15 and trinucleotide structures15 con-
taining additional unsaturated rings involving phosphortriester
internucleoside linkages.13–15 We hereby present important
synthetic improvements to this RCM based strategy. Thus, the
general lability of the phosphortriesters is hereby explored and
the subsequent synthetic demand for saturated phosphortriester
analogues is solved by a tandem RCM-hydrogenation proto-
col.
1
confirmed the loss of the mass of ethylene, whereas the H
NMR signals verified the conversion of terminal to internal
double bonds.§ Two major products were formed correspond-
ing to the two phosphorus epimers with (E)-configuration of the
2-butene linker in combination with only trace amounts of the
(Z)-configured isomers.§
Whereas the phosphortriester moieties were stable towards
acidic treatment as exemplified by the deprotection of 5a to give
6a, the lability towards basic conditions was proven to be
significant. Thus, when 6a was dissolved in deuterated pyridine,
the solvent acted as a nucleophile towards the phosphate bound
methylene of the 2-butene linker opening the cyclic structure to
give the zwitterionic dinucleotide 8a as the only major product
as judged from NMR. Also MS verified a covalently bound
pyridinium moiety. When 6a was treated with concentrated
aqueous ammonia for 24 h at room temperature, a similar ring-
opening reaction affording 9a was complete. An analogueous
ring-opening reaction as well as contemporary double desilyla-
The dinucleotide structures are synthesised from nucleoside
building blocks containing allyl moieties followed by RCM-
† Nucleic Acid Center is funded by the Danish National Research
Foundation for studies on nucleic acid chemical biology.
Scheme 1 Reagents and conditions: i, a. 1H-tetrazole, CH3CN, b. t-BuOOH, CH3CN, 96% (4a), 64% (4b); ii, 5 mol% 1, CH2Cl2, 40 °C, 47% (5a), 58%
(5b); iii, 90% aq. TFA, 96%; iv, a. DMT-Cl, AgNO3, pyridine, b. TBDMSCl, AgNO3, pyridine, c. p-toluenesulfonic acid, CH2Cl2, MeOH, 0 °C, 64%; v,
pyridine-d5, quant. (8a); 32% aq. NH3, rt., quant. (9a); 20 mol% Pd/C, H2, MeOH, quant. (10a); 32% aq. NH3, 55 °C, quant. (9b).
2140
CHEM. COMMUN., 2002, 2140–2141
This journal is © The Royal Society of Chemistry 2002

tion was seen when 5b was subjected to hot aqueous ammomnia
affording the zwitterion 9b.
oligonucleotides should be straightforward as it has been with
analogous dinucleotides with shorter nucleobase–phosphor-
triester linkages.5,6 The lability of the allylic phosphortriesters
gives, on the other hand, the interesting opportunity of
conjugation to the nucleobase applying other nucleophiles.
Thus, transferring the RCM chemistry to solid phase oligonu-
cleotide synthesis reveals the 2-butene linkage as a platform for
postsynthetic functionalisation19 e.g. towards zwitterionic
DNA.20
In conclusion, the present RCM-based strategy has proven
very convenient towards conformationally restricted cyclic
dinucleotides. Importantly, the precatalyst 1 has proven very
useful also as an efficient and selective hydrogenation catalyst
even towards compounds unobtainable by conventional hydro-
genation methods. We expect the conformational restriction
introduced, and the present RCM methodology in general, to be
a very important tool towards nucleic acid analogues mimicking
tertiary nucleic acid structures.
Since the present significant electrophilic character of the
phosphortriester moiety must at least in part be deduced to its
allylic nature, a hydrogenation of the 2-butene linker was
approached. However, when 6a was subjected to standard
hydrogenation conditions, i.e. atmospheric pressure of hydro-
gen and a palladium catalyst, this led surprisingly not only to the
hydrogenation of the double bond but also to reduction of the
phosphortriester linkage to give the dinucleotide 10a in a
quantitative yield. Palladium hydroxide as a catalyst gave the
same result. A solution to the problem of hydrogenation was
given by a method of Grubbs and co-workers who recently
presented a tandem ring-closing metathesis and hydrogenation
procedure in which catalyst 1 was applied also as a hydro-
genation catalyst.18 Thus, a solution of 4a in CH2Cl2 was
refluxed with 5 mol% of 1 until completion of the RCM reaction
as detected by TLC and then subjected to 1000 psi H2 at 50 °C
overnight to give 11a as an epimeric mixture in 54% (Scheme
2). Thus, while the RCM reaction proceeded in moderate yield,
the hydrogenation was quantitative as indicated by the yield,
which is similar to the yield of the RCM reaction alone, and by
MS and NMR as no presence of the unsaturated compound 5a
could be detected after hydrogenation. 11a was deprotected
using the usual acidic treatment14 to give 12a in 85% yield.¶
Applying the same conditions, 11b was synthesised as an
epimeric mixture in an even higher 63% yield from 4b.
Deprotection gave 12b in quantitative yield.¶ The stability
towards nucleophiles of these saturated phosphortriesters was
examined and, as expected, found to be significantly improved
compared to the unsaturated analogues. Thus, 12a did not react
at all with deuterated pyridine within 24 h, and treatment with
concentrated aqueous ammonia for 24 h at room temperature
resulted in only approximately 10% conversion to 13a accord-
ing to 31P NMR. A harsher treatment of 12a with ammonia at 55
°C for 5 days resulted, however, in complete conversion to 13a.
Similar properties were seen in the 2A-deoxy series as treatment
of 12b with 32% aq. NH3 at 55 °C in 5 days gave complete
conversion to 13b, whereas less than 10% conversion was seen
after 24 h at room temperature.
Notes and references
‡ 6a contains a 10+10+1+1 mixture of the (E,SP), (E,RP), (Z,SP) and (Z,RP)
stereoisomers, respectively.15
§ Selected data: for 4b: 1H NMR (CDCl3) d 5.00–5.41 (4H, m, CHNCH2),
5.84–5.95 (2H, m, CHNCH2); 31P NMR (CDCl3) d 20.61. For 5b: 1H NMR
(CDCl3) d 5.84–5.98 (2H, m, CHNCH); 31P NMR (CDCl3) d 20.65, 2.01;
IR (KBr) 3431, 3065, 1694, 987, 780 cm21
.
¶ Selected data: for compound 12a: 1H NMR (DMSO-d6) d 1.50–1.70 (4H,
m, CH2CH2), 1.76–1.80 (3H, br s, CH3), 2.28–2.43 (4H, m, CH2-U, T-
H2A,2B), 3.60–3.65 (2H, m, T-H5A,5B), 3.98–4.39 (8H, m, T-H4A, U-
H5A,5B,4A,3A,2A, CH2-OP), 4.93–4.99 (1H, m, T-H3A), 5.80 (0.5H, d, J 2.3 Hz,
U-H1A), 5.87 (0.5H, d, J 3.9 Hz, U-H1A), 6.18–6.24 (1H, m, T-H1A), 7.28
(0.5H, s, U-H6), 7.41 (0.5H, s, U-H6), 7.67–7.71 (1H, br s, T-H6),
11.34–11.40 (2H, m, NH); 31P NMR (DMSO-d6) d 20.58, 0.29; IR (KBr)
3419, 1695, 1024, 786 cm21. HiRes MALDI FT-MS: [MNa+] found/calc.
m/z 625.1507/625.1517. For compound 12b: 1H NMR: (DMSO-d6) d
1.50–1.70 (4H, m, CH2CH2), 1.76–1.79 (3H, br s, CH3), 2.10–2.42 (6H, m,
CH2-dU, 2 3 H2A,2B), 3.50–4.40 (9H, m, 2 3 H5A,5B,4B, dU-H3A, CH2-OP),
4.93–4.97 (1H, m, T-H3A), 6.18–6.29 (2H, m, 2 3 H1A), 7.31 (0.5H, s, dU-
H6), 7.43 (0.5H, s, dU-H6), 7.66–7.70 (1H, br s, T-H6), 11.31–11.35 (2H,
m, NH); 31P NMR (DMSO-d6) d 20.71, 0.00; IR (KBr) 3435, 1690, 1022,
In summary, the RCM protocol has proven very efficient in
preparing the conformationally restricted cyclic dinucleotides
5a,b. The potential use of these compounds, however, is
hampered by the high base lability, which is not compatible
with standard solid phase oligonucleotide synthesis applying
base-labile protecting groups and solid support connections.
The saturated counterparts 12a,b are much more stable, and the
standard deprotection conditions usually applied (i.e. conc.
ammonia at rt) afforded only a slow cleavage of the conforma-
tionally restricting rings. Thus, the incorporation of 12a,b into
784 cm21
;
HiRes MALDI FT-MS: [MNa+] found/calc. m/z
609.1584/609,1568.
1 E. T. Kool, Chem. Rev., 1997, 97, 1473.
2 P. Herdewijn, Biochim. Biophys. Acta, 1999, 1489, 167.
3 C. J. Leumann, Bioorg. Med. Chem., 2002, 10, 841.
4 M. Meldgaard and J. Wengel, J. Chem. Soc., Perkin Trans. 1, 2000,
3539.
5 M. Sekine, O. Kurasawa, K. Shohda, K. Seio and T. Wada, Eur. J. Org.
Chem., 2001, 1989.
6 M. Sekine, O. Kurasawa, K. Shohda, K. Seio and T. Wada, J. Org.
Chem., 2000, 65, 6515.
7 M. Seio, T. Wada and M. Sekine, Helv. Chim. Acta, 2000, 83, 162.
8 J. F. Reichwein, C. Versluis and R. M. J. Liskamp, J. Org. Chem., 2000,
65, 6187.
9 H. E. Blackwell, J. D. Sadowsky, R. J. Howard, J. N. Sampson, J. A.
Chao, W. E. Steinmetz, D. J. O’Leary and R. H. Grubbs, J. Org. Chem.,
2001, 66, 5291.
10 M. Scholl, S. Ding, C. W. Lee and R. H. Grubbs, Org. Lett., 1999, 1,
953.
11 T. M. Trnka and R. H. Grubbs, Acc. Chem. Res., 2001, 34, 18.
12 J. Ravn and P. Nielsen, J. Chem. Soc., Perkin Trans. 1, 2001, 985.
13 A. M. Sørensen and P. Nielsen, Org. Lett., 2000, 2, 4217.
14 A. M. Sørensen, K. E. Nielsen, B. Vogg, J. P. Jacobsen and P. Nielsen,
Tetrahedron, 2001, 57, 10191.
15 P. Børsting, A. M. Sørensen and P. Nielsen, Synthesis, 2002, 797.
16 B. Bhat, N. J. Leonard, H. Robinson and A. H.-J. Wang, J. Am. Chem.
Soc., 1996, 118, 10744.
17 J. L. Ruth and D. E. Bergstrom, J. Org. Chem., 1978, 14, 2870.
18 J. Louie, C. W. Bielawski and R. H. Grubbs, J. Am. Chem. Soc., 2001,
123, 11312.
Scheme 2 Reagents and conditions: i, 5 mol% 1, CH2Cl2, 40 °C then 1000
psi H2, 50 °C, 54% (11a), 63% (11b); ii, 90% aq. TFA, 85% (12a), quant.
(12b); iii, 32% aq. NH3, 55 °C, quant. (13a), quant. (13b).
19 S. Dey and T. L. Sheppard, Org. Lett., 2001, 3, 3983.
20 H. Hashimoto, M. G. Nelson and C. Switzer, J. Am. Chem. Soc., 1993,
115, 7128.
CHEM. COMMUN., 2002, 2140–2141
2141