A convenient synthesis of the cytidyl 3′-terminal monomer for solid-phase synthesis of RNG oligonucleotides

Article abstract of DOI:10.1016/j.tetlet.2012.05.055

Replacing the phosphodiester backbone of RNA with positively charged guanidinium linkages has been shown to enable RNA oligomers to overcome electrostatic repulsion and bind double-stranded DNA in a triplex with high affinity. Ribonucleotide monomers with

Full text of DOI:10.1016/j.tetlet.2012.05.055

Tetrahedron Letters 53 (2012) 3792–3794
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Tetrahedron Letters
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A convenient synthesis of the cytidyl 30-terminal monomer for
solid-phase synthesis of RNG oligonucleotides
Ahmed M. Awad a, , Michael J. Collazo a, Kathrinna Carpio a, Christina Flores a, Thomas C. Bruice b
⇑
a Chemistry Program, California State University Channel Islands, One University Drive, Camarillo, CA 93012, USA
b Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Replacing the phosphodiester backbone of RNA with positively charged guanidinium linkages has been
shown to enable RNA oligomers to overcome electrostatic repulsion and bind double-stranded DNA in
a triplex with high affinity. Ribonucleotide monomers with the ability to form guanidinium linkages have
been synthesized for the generation of ribooligonucleotides with guanidinium linkages (RNGs) through
solid-phase synthesis. We report herein an efficient method for the synthesis of N4-benzoyl-20-O-(tert-
butyldimethylsilyl)-50-N-(4-monomethoxytritylamino)-30-O-succinyl-50-deoxycytidine, a new monomer
required for the solid-phase synthesis of cytidyl RNG oligonucleotides.
Received 19 April 2012
Revised 5 May 2012
Accepted 10 May 2012
Available online 17 May 2012
Keywords:
Nucleotide-50-amino
Cytidine
Ó 2012 Elsevier Ltd. All rights reserved.
Oligonucleotide
Ribonucleic guanidine (RNG)
Solid-phase synthesis
Mitsunobu reaction
Synthetic oligonucleotides have been proposed as agents for se-
quence specific gene regulation by either antisense or RNAi tech-
nologies.1 The efficacy of these oligonucleotides in vitro has been
evaluated based upon their ability to exhibit a high affinity for
DNA/RNA, maintain specific base-pairing, intracellular delivery
capability, and resist degradation by nucleases.2 In addition, triplex
forming oligonucleotides bound to duplex DNA have been shown
to display normal Watson–Crick base-pairing.3 Chemical modifica-
tions of the pentose sugar as well as those of the phosphate link-
ages between nucleosides have tended to impact the affinity of
oligonucleotides to their target nucleic acids, but have not yet been
shown to affect the specificity of the hydrogen bonding that occurs
between bases.4 In previous experiments, oligonucleotides with
negatively charged phosphate backbones have bound to duplex
DNA poorly due to electrostatic repulsion, and were only effective
in chains rich in adenine and guanosine, limiting the genes that
could be targeted.5
ribonucleic acids are able to bind and silence genes at the tran-
scriptional level without requiring methylation of the DNA.8
Ribooligonucleotides have been proposed as therapeutic agents
for transcriptional control, yet deoxyoligonucleotides have been
focused on, partly due to their stability. Ribooligonucleotides are
more sensitive to degradation by nucleases9 and the propensity
of the 20-hydroxyl to perform
a nucleophilic attack on the
phosphate backbone of RNA limits their use without chemical
modifications. However, RNG oligonucleotides possess stabilizing
characteristics not found in RNA. Guanidinium linkages are
resistant to nucleases.10 In addition, guanidine linkages between
The chemical modification of nucleotides to incorporate posi-
tively charged guanidinium linkages (Fig. 1A) has been observed
to increase the binding affinity between duplex DNA and deoxynu-
cleic guanidine (DNG) oligomers containing the bases adenosine,
cytidine, and thymidine.6 In addition, oligomers of uridyl and
adenyl ribonucleic guanidine (RNG U and A, respectively) have
been reported.7 The discovery of antigene RNA demonstrated that
⇑
Corresponding author. Tel.: +1 805 437 2794; fax: +1 805 437 8895.
E-mail address: ahmed.awad@csuci.edu (A.M. Awad).
Figure 1. (A) Guanidinium linkage in RNG (B) 50-amino-RNA.
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.05.055

A. M. Awad et al. / Tetrahedron Letters 53 (2012) 3792–3794
3793
Scheme 1. Reagents and conditions: (a) (i) TMSCl, pyridine; (ii) BzCl; (iii) aq NH4OH; (b) 2,2-dimethoxypropane, TsOHꢀH2O, acetone; (c) Mitsunobu reaction, tetrachloroph
thalimide, PPh3, THF, 15 h reflux; (d) 75% aq TFA; (e) TBDMSCl, AgNO3, pyridine, DMF; (f) ethylenediamine, THF; (g) MMTrCl, Et3N, CH2Cl2; (h) succinic anhydride, Et3N,
CH2Cl2.
ribonucleic monomers should stabilize oligonucleotide backbones
against nucleophilic attack from the 20-hydroxyl.7
solvent was removed under reduced pressure, and ether was added
to induce further precipitation of compound 1 in a 93% yield.
Compound 2 was produced by Mitsunobu reaction according to
the previously published method.12,13 To a suspension of 1 (22.2
mmol), PPh3, (27.75 mmol) and tetrachlorophthalimide (26.64 m
mol) in anhydrous THF (250 mL), diisopropyl azodicarboxylate
(26.64 mmol) was added dropwise over 15 min and the mixture
was refluxed for 15 h. The solvent was removed under reduced
pressure and the crude mixture was purified by silica gel column
chromatography (50% EtOAc in hexanes) to afford 2 in an 80%
yield. The 20-, 30-hydroxyl groups were then deprotected by stirring
compound 2 (17.0 mmol) in aqueous trifluoroacetic acid (75%,
100 mL) at RT for 2 h. Compound 3 was obtained in an 89% yield.
The low solubility of 3 in organic solvents allowed the precipitation
and isolation of the pure compound from the reaction mixture.
More convenient was direct conversion of the crude of 2 to a pure
precipitate of compound 3. Protection of the 20-hydroxyl group
with TBDMS was performed to allow for further reactions on the
30-hydroxyl group. To a solution of 3 (9.3 mmol) in dry DMF
(80 mL), pyridine (37.1 mmol) and AgNO3 (11.77 mmol) were
added. After stirring for 10 min, tert-butyldimethylsilyl chloride
(11.61 mmol) was added and the reaction mixture was stirred at
RT for 24 h. The mixture was filtered through Celite, washed with
ethanol, and the filtrate was concentrated in a vacuum. The crude
product was purified by silica gel column chromatography (40%
EtOAc in hexanes) to give compound 4.14 This reaction failed when
THF was used as a solvent, likely due to the low solubility of 3 in
THF. The 50-amino nucleoside 5 was generated upon treatment of
4 (2.0 mmol) with ethylenediamine (2.4 mmol) in anhydrous THF
(25 mL). The crude of 5 was directly converted to pure compound
6 in an overall yield of 82%. Compound 6 was purified by silica gel
column chromatography pre-washed with 2% Et3N (33% ethyl
Triplex-forming antigene agents with increased affinity for
duplex DNA, enable the regulation of gene expression at the
transcriptional level and require a miniscule concentration of oligo-
nucleotidesincomparisontothatwhichwouldberequiredto inhibit
the expression of multiple mRNA molecules. While previous
experiments have demonstrated the selectivity of DNG, the interac-
tion between template DNA and RNG oligonucleotides has not yet
been observed, as RNG monomers for solid-phase synthesis exists
only for adenyl RNG and uridyl RNG.7 Our experiment provides a
convenient, high-yield, synthesis of the 30-terminal monomer
N4-benzoyl-20-O-(tert-butyldimethylsilyl)-50-N-(4-monomethoxytr
itylamino)-30-O-succinyl-50-deoxycytidine7 (Scheme1), whichmay
be incorporated into the solid-phase synthesis of cytidyl RNG oligo-
nucleotides. In addition, this monomer can be also used for the solid-
phase synthesis of P30? N50 phosphoramidate ribooligonucleotides
(50-amino-RNA, Fig. 1B) with 50-O-nucleotides replaced by 50-N-
nucleotides.11
For large scale preparations, it was more convenient to perform
some reactions directly on the crude from the previous reaction,
which did not affect the overall yield. High-yields and pure com-
pounds were obtained and confirmed by gravimetric analysis,
1
TLC, HRMS, and H NMR spectroscopy. Protection of the 20- and
30-hydroxyl groups of the pentose ring was performed before run-
ning the Mitsunobu reaction on the 50-hydroxyl group. To a stirred
suspension of N4-benzoylcytedine (30.0 mmol) in acetone (250
mL), p-toluenesulfonic acid monohydrate (3.0 mmol) and an excess
of 2,2-dimethoxypropane (60 mL) were added. After 24 h reflux,
the mixture was filtered to remove the unreacted starting material.
The filtrate was concentrated under vacuum and the residue was
dissolved with EtOAc, washed with 5% NaHCO3 and brine. The

3794
A. M. Awad et al. / Tetrahedron Letters 53 (2012) 3792–3794
acetate in hexanes). Esterification of the 30-OH with succinic anhy-
dride afforded the loading monomer 7. Succinic anhydride
(3.0 mmol) was added to a mixture of 6 (0.6 mmol) and Et3N
(6.0 mmol) in anhydrous CH2Cl2 (15 mL) and the mixture was stir-
red for 20 h. The solvent was removed under reduced pressure and
the residue was dissolved in CH2Cl2, washed with 5% citric acid,
then H2O and brine, dried over anhydrous Na2SO4, and concen-
trated in a vacuum. The crude mixture was purified by silica col-
umn chromatography pre-washed with 2% Et3N (45% EtOAc in
hexanes) to afford compound 7 in a 76% yield.15
A common initial step in the solid-phase synthesis of oligonu-
cleotides is to covalently attach the 30 moiety of a modified pentose
sugar on the first nucleoside to controlled pore glass (CPG), which
contains long chain alkylamines.16 The modifications we have
incorporated enable our loading monomer to be joined to a solid
support17 and form guanidinium linkages with additional syn-
thetic nucleotides.18
11. Tomizu, M.; Negoro, Y.; Osaki, T.; Orita, A.; Ueyama, Y.; Nakagawa, O.;
Imanishi, T. Nucleosides Nucleotides Nucleic Acid 2007, 26, 893.
12. For the Mitsunobu reaction incorporating tetrachlorophthalimide: (a) Jia, Z. J.;
Kelberlau, S.; Olsson, L.; Anilkumar, G.; Fraser-Reid, B. Synlett 1999, 565; (b)
Tetzlaff, C. N.; Schwope, I.; Bleczinski, C. F.; Steinberg, J. A.; Richert, C.
Tetrahedron Lett. 1998, 39, 4215.
13. Diisopropyl azodicarboxylate was chosen to prevent the reaction from favoring
the formation of a side product containing a hydrazylmethyl group. See:
Swamy, K. C.; Kumar, N. N.; Balaraman, E.; Kumar, K. V. Chem. Rev. 2009, 109,
2551.
14. The 47% yield is likely due to the formation of a side product in which the 30-
hydroxyl on the sugar receives the modification.
15. Our products have remained stable in dry anoxic environments.
16. Pon, R. T.; Yu, S. Nucleic Acids Res. 2004, 32, 623.
17. The incorporation of 30-succinyl linker enables a molecule to be tethered to
controlled pore glass for the solid-phase synthesis of ribooligonucleotides. See:
(a) Sharma, P.; Sharma, A. K.; Malhotra, V. P.; Gupta, K. C. Nucleic Acids Res.
1992, 20, 4100; (b) Reddy, P. M.; Bruice, T. C. J. Am. Chem. Soc. 2004, 126, 3736.
18. Kearney, P. C.; Fernandez, M.; Flygare, J. A. J. Org. Chem. 1998, 63, 196.
19. Spectral
data
for
selected
compounds:
N4-Benzoyl-20,30-O-
isopropylidenecytidine (1) HRMS (ESI) m/z Calcd for C19H21N3O6 (M+Na)+
410.1322. Found 410.1311. 1H NMR (400 MHz, DMSO-d6): 11.29 s, 1H (NH);
8.30 d, 1H, J = 7.4 (H-6); 8.02 d, 2H, J = 6.8 (BzH); 7.62–7.50 m, 3H (BzH); 7.35
d, 1H, J = 7.2 (H-5); 5.85 d, 1H, J = 1.6 (H-10); 5.12 t, 1H, J = 5.0 (OH-50); 4.90 dd,
1H, J = 4.6, 1.6 (H-30); 4.76 q, 1H, J = 3.0 (H-20); 4.22 d, 1H, J = 3.2 (H-40); 3.65–
3.56 m, 2H (H-50); 1.48 s, 3H (CH3); 1.28 s, 3H (CH3). N4-Benzoyl-50-
tetrachlorophthalimido-20,30-O-isopropylidene-50-deoxycytidine (2) HRMS
(ESI) m/z Calcd for C27H20N4O7Cl4 (M+H)+ 653.0164. Found 653.0189 (base
peak of 4 Cl isotopes). 1H NMR (400 MHz, CDCl3): 11.62 s, 1H (NH); 7.94 d, 1H,
J = 7.0 (H-6); 7.67 d, 2H, J = 7.0 (BzH); 7.62–7.48 m, 4H (BzH and H-5); 5.54 d,
1H, J = 1.6 (H-10); 5.27 dd, 1H, J = 7.2, 1.6 (H-30); 5.05 q, 1H, J = 3.0 (H-20); 4.48
m, 1H (H-40); 4.30–4.00 m, 2H (H-50); 1.54 s, 3H (CH3); 1.35 s, 3H (CH3). N4-
Benzoyl-50-tetrachlorophthalimido-50-deoxycytidine (3) HRMS (ESI) m/z Calcd
for C24H16N4O7Cl4 (M+Na)+ 634.9665. Found 634.9667 (base peak of 4 Cl
isotopes). 1H NMR (400 MHz, DMSO-d6): 11.29 s, 1H (NH); 8.25 d, 1H, J = 7.8
(H-6); 8.02 d, 2H, J = 7.2 (BzH); 7.66–7.47 m, 3H (BzH); 7.41 d, 1H, J = 7.6 (H-5);
5.74 d, 1H, J = 3.0 (H-10); 5.59 br s, 1H (OH-30); 5.29 br s, 1H (OH-20); 4.20–4.16
m, 2H (H-50); 4.13–3.97 m, 3H (H-20,H-30,H-40). N4-Benzoyl-20-O-(tert-
butyldimethylsilyl)-50-tetrachlorophthalimido-50-deoxycytidine (4) HRMS
(ESI) m/z Calcd for C30H30N4O7SiCl4 (M+Na)+ 749.0530. Found 749.0522 (base
peak of 4 Cl isotopes). 1H NMR (400 MHz, CDCl3): 8.18 d, 1H, J = 7.6 (H-6); 8.91
d, 2H, J = 7.0 (BzH); 7.65–7.47 m, 4H (BzH and H-5); 5.64 d, 1H, J = 1.2 (H-10);
4.42 dd, 1H, J = 1.2, 3.8 (H-20); 4.32–4.16 m, 1H (H-30); 4.12–4.07 m, 1H (H-40);
4.03–3.88 m, 2H (H-50); 0.92 s, 9H (SiC(CH3)3); 0.25 s, 3H (SiCH3); 0.17 s, 3H
(SiCH3). N4-Benzoyl-20-O-(tert-butyldimethylsilyl)-50-amino-50-deoxycytidine
(5) LRMS (ESI) m/z Calcd for C22H32N4O5Si (M+Na)+ 461. Found 461. N4-
Benzoyl-20-O-(tert-butyldimethylsilyl)-50-N-(4-monomethoxytritylamino)-50-
deoxycytidine (6) HRMS (ESI) m/z Calcd for C42H48N4O6Si (M+Na)+ 755.3235.
Found 755.3242. 1H NMR (400 MHz, CDCl3): 7.91 d, 1H, J = 7.6 (H-6); 7.75 d,
2H, J = 7.8 (BzH); 7.61–7.22 m, 16H (ArH, BzH and H-5); 6.87 d, 2H, J = 8.8
(ArH); 5.78 d, 1H, J = 1.2 (H-10); 4.25 dd, 1H, J = 1.2, 3.6 (H-20); 4.17–4.13 m, 1H
(H-30); 4.10–4.06 m, 1H (H-40); 3.80 s, 3H (MMTr-OCH3); 3.73–3.53 m, 2H (H-
50); 0.94 s, 9H (SiC(CH3)3); 0.30 s, 3H (SiCH3); 0.17 s, 3H (SiCH3). N4-Benzoyl-20-
O-(tert-butyldimethylsilyl)-50-N-(4-mono-methoxytritylamino)-30-O-succinyl-
50-deoxycytidine (7) HRMS (ESI) m/z Calcd for C46H52N4O9Si (M+Na)+
833.3576. Found 833.3585. 1H NMR (400 MHz, CDCl3): 8.69 d, 1H, J = 7.6 (H-
6); 7.97 d, 2H, J = 8.4 (BzH); 7.58–7.20 m, 16H (ArH, BzH and H-5); 6.86 d, 2H,
J = 8.8 (ArH); 5.78 d, 1H, J = 1.2 (H-10); 4.88 dd, 1H, J = 4.8, 4.0 (H-30); 4.43 dd,
1H, J = 4.4, 1.2 (H-20); 4.40–4.36 m, 1H (H-40); 3.78 s, 3H (MMTr-OCH3); 3.64–
3.25 m, 2H (H-50); 2.57 s, 4H (CO(CH2)2); 0.90 s, 9H (SiC(CH3)3); 0.21 s, 3H
(SiCH3); 0.07 s, 3H (SiCH3).
In conclusion, we have confirmed the synthesis of N4-benzoyl-
20-O-(tert-butyldimethylsilyl)-50-N-(4-monomethoxytritylamino)-
30-O-succinyl-50-deoxycytidine by means of high resolution
mass-spectroscopy and proton nuclear magnetic resonance.19
Our method provides an efficient means of producing an RNG
(and also a P30? N50 phosphoramidate) monomer with high-yields
and high purity.
Acknowledgments
Funding was provided by the Chemistry Program at CSU Chan-
nel Islands. We thank Scott Duffer and the University of California
Santa Barbara for assistance in obtaining spectral data.
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