Multiple phosphate-linked nucleotide couplings via 5′ silyl ether protection in the phosphite triester and phosphoramidite approaches

Article abstract of DOI:10.1016/j.tetlet.2013.04.043

Phosphite triester and phosphoramidite coupling methodologies are described for performing fourfold solution phase installations of standard deoxynucleotides onto a single cavitand template molecule. The methodologies here are based on 5′ silyl ether protection of the appropriate nucleoside or nucleoside phosphoramidite. Synthesis of a novel water-soluble species incorporating four covalently-linked guanine nucleotides is shown.

Full text of DOI:10.1016/j.tetlet.2013.04.043

Tetrahedron Letters 54 (2013) 3207–3209
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Tetrahedron Letters
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Multiple phosphate-linked nucleotide couplings via 50 silyl ether protection
in the phosphite triester and phosphoramidite approaches
⇑
Grant A. L. Bare, John C. Sherman
Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1
a r t i c l e i n f o
a b s t r a c t
Article history:
Phosphite triester and phosphoramidite coupling methodologies are described for performing fourfold
solution phase installations of standard deoxynucleotides onto a single cavitand template molecule.
The methodologies here are based on 50 silyl ether protection of the appropriate nucleoside or nucleoside
phosphoramidite. Synthesis of a novel water-soluble species incorporating four covalently-linked guan-
ine nucleotides is shown.
Received 27 March 2013
Accepted 11 April 2013
Available online 19 April 2013
Keywords:
Nucleotide
Ó 2013 Elsevier Ltd. All rights reserved.
Phosphoramidite
Phosphite triester
Template
Introduction
as native phosphates for work in polar solvents or water are not
incorporated directly.6 Reactions capable of installing several
Template-assembled synthetic systems based on G-quartets
and other nucleobase quartet motifs are increasingly becoming uti-
lized to mimic biologically relevant structures and activities. In
these systems, a template molecule is covalently decorated with
nucleosides1 or oligonucleotide strands2 to reveal unconventional
topologies and functions typically afforded from preorganized
G-quartet and higher order G-quadruplex assemblies. G-quadru-
plex DNA is an essential and widely studied nucleic acid secondary
structure owing to its localization in human telomeres and onco-
genes and its putative role as a target for anticancer drug design.3
For instance, single quartet templated systems have resulted in
G-quartet-based G-quadruplex ligands,1b–d a cation-free G-quar-
tet,1f and nanotube construction,1g while template-assembled
G-quadruplex designs have revealed receptors for G-quadruplex
ligands2a,2d and aptamers with anti-HIV activity.2b Expanding be-
yond guanine to other nucleobases has demonstrated the utility
of template-assembled synthesis in stabilized U-quadruplex,
U-quartet, and i-motif DNA structures.4
Linkage chemistries in these templated systems have been lim-
ited to only a few types including triazole production through cop-
per(I) catalyzed azide alkyne cycloadditions (CuAAC), oxime and
amide formation, and solid-phase DNA syntheses. The CuAAC
owing in part to its bioorthogonal nature have found a widespread
application in bioconjugation reactions and other modifications of
nucleic acids and nucleosides.5 However, one disadvantage of
CuAAC mediated triazole formation is that solubilizing groups such
guanine nucleotides at one time have not been described despite
the attention that has been given to template-assembled synthetic
G-quartet (TASQ) compounds.
We report here methodologies inspired by the phosphite
triester and phosphoramidite approach toward DNA synthesis for
performing fourfold phosphate-linked nucleotide couplings onto
an individual substrate via a natural 30 linkage in the solution
phase. In these methodologies, conventional 50 alcohol dimethoxy-
trityl (DMT) protection has been replaced by silyl ether protection
with TBS. The scope of the phosphoramidite linkage methodology
is adapted in order to accommodate four non-standard functional-
ized nucleobases. Furthermore, synthesis of an unprecedented
water-soluble species incorporating four phosphate-linked guan-
ine nucleotides is reported.
Results and discussion
Suitability of phosphorus(III) reagents to carry out multiple
nucleotide forming reactions was explored. A fourfold coupling
reaction was performed by modification of the phosphite triester
DNA synthetic methodology (Scheme 1).7 In situ conversion of
base-protected 50 TBS guanosine 1a8 to its activated 30 phospho-
rus(III) intermediate and coupling to alcohol functionalized [4]
cavitand template 29 were carried out using 2,2,2-trichloroethyl-
phosphorodichloridite as a coupling reagent in the presence of
2,6-lutidine in cooled THF solution. Minimization of steric bulk
and avoidance of acid-catalyzed depurination side-reactions moti-
vated the selection of the TBS group in place of the standard DMT
group for 50 alcohol protection.10 Initial attempts with 50 DMT
⇑
Corresponding author. Tel.: +1 604 822 2305; fax: +1 604 822 2847.
E-mail address: sherman@chem.ubc.ca (J.C. Sherman).
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.04.043

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G. A. L. Bare, J. C. Sherman / Tetrahedron Letters 54 (2013) 3207–3209
Scheme 2. Synthesis of phosphoramidites 5a–d.
and acetylacetone in pyridine.11 Ammonia treatment in a water/
methanol mixture followed by fluoride cleavage facilitated
deblocking of the respective base isobutyryl and 50 silyl ether pro-
tecting groups. Purification on C18 reversed-phase silica afforded
phosphate-linked conjugate 4a in 16% yield over four steps as a
n-tetrabutylammonium salt (Fig. 1).
Standard phosphoramidite based internucleotide bond forming
reactions were also investigated as a potential way to carry out mul-
tiple nucleotide coupling reactions with guanine and other bases.12
A new series of 2-cyanoethyl,-N,N-diisopropylamino phosphorami-
dite reagents of the canonical deoxynucleosides 5a–d with standard
N-isobutyryl or N-Bz base protection and 50 TBS protection were
prepared from deoxynucleosides 1a–d (Scheme 2).13,8 Phosphityla-
tion was performed in THF at room temperature, and synthesis in
77–94% yield of the moisture and air sensitive phosphoramidites
5a–d as diastereomeric pairs was confirmed by the appearance of
two singlets at approximately 150 ppm in the 31P NMR spectrum
after work-up. These reagents were used to perform fourfold cou-
pling reactions on template 2 in the presence of 5-(ethylthio)-tetra-
zole activator in THF or CH2Cl2 solvent (Scheme 3). 5-(Ethylthio)-
tetrazole is noted to have greater solubility in THF solution and im-
proved activity as a catalyst in the phosphoramidite reaction over
1H-tetrazole.14 Equivalents and purification steps were in confor-
mance to the preceding phosphite triester method. Simplified two
step ammonia and TBAF deblocking and purification gave the phos-
phate-linked conjugates 4a–d in up to 40% yield after three steps. In
addition, a route toward compounds 6a and 6c that remained pro-
tected at their base position was achieved by selective phosphate
deblocking with triethylamine instead of ammonia treatment, and
this route is anticipated to facilitate additional base functionaliza-
tion by corresponding substitution of the isobutyryl and Bz base-
protecting groups in the starting material.
Scheme 1. Phosphite triester synthesis of tetra-guanine 4a.
Figure 1. Structure of tetra-guanine 4a.
Installation of the 50 TBS protecting group in place of routine 50
DMT protection and subsequent TBAF cleavage proved to be
advantageous as it provided a convenient route to incorporate a
n-tetrabutylammonium counterion into the phosphate-linked con-
jugates 4a–d, 6a, and 6c. Tetrabutylammonium salts 4a–d and 6a
were found to have millimolar solubility in methanol and in water,
while 6c was found to have millimolar solubility in methanol only.
For guanine conjugate 4a, other counterions could not be found to
impart solubility in water. Imparted water-solubility is an essential
property of molecular assemblies inspired by or used to model
Nature and becomes increasingly difficult at the less negative
charge dense single guanine tetrad level with respect to G-quadru-
plex DNA. Furthermore, tetraalkylammonium counterions may be
beneficial when utilized in NMR characterization studies as chem-
ical exchange between counterions and substrate molecules that
can potentially limit observation of exchangeable proton NMR sig-
nals is avoided. All high molecular weight tetra-coupled nucleotide
target compounds 4a–d, 6a, and 6c were characterized extensively
nucleotide couplings followed by complete deprotection were
unsuccessful. Any presence of partially substituted template prod-
ucts was found to be problematic for subsequent purifications and
reactions. Thus, four equivalents of 1a per alcohol function were ob-
served to be sufficient to achieve complete conversion of starting
material 2 to the fully coupled and protected intermediate conju-
gate, and the presence of starting material 2 or any mono-, di-, or
tri-coupling intermediates could not be detected by TLC after sev-
eral hours of reaction progress. After iodine oxidation, the fully pro-
tected fourfold coupled phosphate triester diastereomeric mixture
3a was purified on silica gel.
Coupling and deblocking reactions were carried out in succes-
sion without isolation or characterization of the intermediates
due to the problematic generation of diastereomeric mixtures
when asymmetric phosphate triesters are formed and due to poor
solubilities of intermediate deblocked compounds. Phosphate
trichloroethyl protecting groups were removed with Zn/Cu couple

G. A. L. Bare, J. C. Sherman / Tetrahedron Letters 54 (2013) 3207–3209
3209
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2013.04.
043.
References and notes
1. (a) Nikan, M.; Patrick, B. O.; Sherman, J. C. ChemBioChem 2012, 13, 1413; (b)
Haudecoeur, R.; Stefan, L.; Denat, F.; Monchaud, D. J. Am. Chem. Soc. 2013, 135,
550; (c) Xu, H.-J.; Stefan, L.; Haudecoeur, R.; Vuong, S.; Richard, P.; Denat, F.;
Barbe, J.-M.; Gros, C. P.; Monchaud, D. Org. Biomol. Chem. 2012, 10, 5212; (d)
Stefan, L.; Guedin, A.; Amrane, S.; Smith, N.; Denat, F.; Mergny, J.-L.; Monchaud,
D. Chem. Commun. 2011, 47, 4992; (e) Murat, P.; Gennaro, B.; Garcia, J.; Spinelli,
N.; Dumy, P.; Defrancq, E. Chem. Eur. J. 2011, 17, 5791; (f) Nikan, M.; Sherman, J.
C. Angew. Chem., Int. Ed. 2008, 47, 4900–4902; (g) Sidorov, V.; Kotch, F. W.; El-
Khouedi, M.; Davis, J. T. Chem. Commun. 2000, 2369.
2. (a) Murat, P.; Bonnet, R.; Van der Heyden, A.; Spinelli, N.; Labbe, P.; Monchaud,
D.; Teulade-Fichou, M.; Dumy, P.; Defrancq, E. Chem. Eur. J. 2010, 16, 6106; (b)
Oliviero, G.; Amato, J.; Borbone, N.; D’Errico, S.; Galeone, A.; Mayol, L.; Haider,
S.; Olubiyi, O.; Hoorelbeke, B.; Balzarini, J.; Piccialli, G. Chem. Commun. 2010, 46,
8971; (c) Oliviero, G.; Borbone, N.; Amato, J.; D’Errico, S.; Galeone, A.; Piccialli,
G.; Varra, M.; Mayol, L. Biopolymers 2009, 91, 466; (d) Murat, P.; Cressend, D.;
Spinelli, N.; Van der Heyden, A.; Labbe, P.; Dumy, P.; Defranq, E. ChemBioChem
2008, 9, 2588; (e) Napoli, L. D.; Fabio, G. D.; Messere, A.; Montesarchio, D.;
Musumeci, D.; Piccialli, G. Tetrahedron 1999, 55, 9899.
Scheme 3. Phosphoramidite synthesis of tetra-nucleotides 4a–d, 6a, and 6c.
3. (a) Neidle, S. Curr. Opin. Struct. Biol. 2009, 19, 239; (b) Franceschin, M. Eur. J. Org.
Chem. 2009, 2225; (c) De Cian, A.; Lacroix, L.; Douarre, C.; Temime-Smaali, N.;
Trentesaux, C.; Riou, J.; Mergny, J. Biochimie 2008, 90, 131.
4. (a) Bonnet, R.; Murat, P.; Spinelli, N.; Defrancq, E. Chem. Commun. 2012, 48,
5992; (b) Hui, B. W.-Q.; Sherman, J. C. ChemBioChem 2012, 13, 1865; (c) Hui, B.
W.-Q.; Sherman, J. C. Chem. Commun. 2012, 48, 109.
5. (a) Singh, Y.; Murat, P.; Defrancq, E. Chem. Soc. Rev. 2010, 39, 2054; (b) Amblard,
F.; Cho, J. H.; Schinazi, R. F. Chem. Rev. 2009, 109, 4207; (c) Gramlich, P. M. E.;
Wirges, C. T.; Manetto, A.; Carell, T. Angew. Chem., Int. Ed. 2008, 47, 8350.
6. Nikan, M.; Bare, G. A. L.; Sherman, J. C. Tetrahedron Lett. 2011, 52, 1791.
7. Letsinger, R. L.; Lunsford, W. B. J. Am. Chem. Soc. 1976, 98, 3655.
8. Eisenhuth, R.; Richert, C. J. Org. Chem. 2009, 74, 26.
using 1H, 31P, 13C, and 1H–1H COSY NMR, and MALDI methods suit-
able for hydrid organic molecules between small molecule and oli-
gonucleotide size.15 Full signal assignments in the 1H NMR spectra
were made with assistance from 1H–1H COSY cross-peaks.
Conclusions
In summary, we have shown the application of the atypical phos-
phite triester DNA synthesis approach and of a new class of 50 TBS
protected phosphoramidites for the construction of multiply-cou-
pled phosphate-linked nucleotide compounds. The unprecedented
synthesis reported here of a water-soluble tetra-coupled guanine
nucleotide template species is of particular interest to the field of
G-quartet/G-quadruplex chemistry, and may find application in
the synthesis of model systems of human telomere structure or of
artificial anticancer ligand receptors. In addition, multiple nucleo-
tide coupled templates may aid in the discovery of unknown four
motif nucleobase structures in Nature or in the design of new func-
tional supramolecular systems.
9. Mezo, A. R.; Sherman, J. C. J. Org. Chem. 1998, 63, 6824.
10. Khorana, H. G. Pure Appl. Chem. 1968, 17, 349.
11. Adamiak, R. W.; Biala, E.; Grzeskowiak, K.; Kierzek, R.; Kraszewski, A.;
Markiewicz, W. T.; Stawinski, J.; Wiewiorowski, M. Nucleic Acids Res. 1977, 4,
2321.
12. (a) Consoli, G. M. L.; Granata, G.; Galante, E.; Di Silvestro, I.; Salafia, L.; Geraci, C.
Tetrahedron 2007, 63, 10758; (b) Consoli, G. M. L.; Granata, G.; Galante, E.;
Cunsolo, F.; Geraci, C. Tetrahedron Lett. 2006, 47, 3245; (c) Sinha, N. D.; Biernat,
J.; McManus, J.; Koster, H. Tetrahedron Lett. 1984, 12, 4539.
13. Ogilvie, K. K. Can. J. Chem. 1973, 51, 3799.
14. Wright, P.; Lloyd, D.; Rappa, W.; Andrus, A. Tetrahedron Lett. 1993, 34, 3373.
15. Characterization data of tetra-guanine 4a: 1H NMR (400 MHz, CD3OD) d 7.90 (s,
4H, H-8), 7.10 (s, 4H, aryl), 6.20 (t, J = 7.0 Hz, 4H, CH-10), 5.66 (d, J = 7.0 Hz, 4H,
H
out), 5.03 (br, 4H, CH-30), 4.74 (t, J = 8.0 Hz, 4H, CH-a), 4.21 (br, 4H, CH-40), 4.09
(d, J = 7.0 Hz, 4H, Hin), 4.00 (m, 8H, CH2-d), 3.82 (m, 8H, CH2-50), 3.24 (m, 32H,
N(CH2CH2CH2CH3)4), 2.66 (m, 4H, CHa-20), 2.42 (m, 12H, CH2-b, CHb-20), 1.80
(overlap, 8H, CH2-c), 1.76 (s, 12H, methyl), 1.67 (m, 32H, N(CH2CH2CH2CH3)4),
1.43
(m,
32H,
N(CH2CH2CH2CH3)4),
1.03
(t,
d
J = 7.0 Hz,
159.4, 155.1, 154.7,
48H,
Acknowledgment
N(CH2CH2CH2CH3)4); 13C NMR (101 MHz, CD3OD)
152.3, 138.6, 138.4, 125.3, 119.2, 118.4, 99.8, 88.4, 86.1, 76.9, 65.7, 63.8,
59.5, 40.1, 37.0, 29.7, 26.2, 24.8, 20.7, 14.0, 10.0; 31P NMR (162 MHz, CD3OD) d
2.3 (s); MS (MALDI-TOF) calculated for C88H103N20O36P4 (MꢀH)ꢀ: 2139.6;
found: 2142.0.
This work was financially supported by the Natural Sciences
and Engineering Research Council of Canada (NSERC).