Efficient synthesis of 5-hydroxymethyl-, 5-formyl-, and 5-carboxyl-2′-deoxycytidine and their triphosphates

Article abstract of DOI:10.1039/c4ra07670b

An efficient P(V)-N activation strategy for the preparation of high-quality 5-hydroxymethyl-, 5-formyl-, and 5-carboxyl-2′-deoxycytidine triphosphates has been developed. The method was also optimized for gram-scale synthesis of the corresponding parent n

Full text of DOI:10.1039/c4ra07670b

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Efficient synthesis of 5-hydroxymethyl-, 5-formyl-,
and 5-carboxyl-20-deoxycytidine and their
Cite this: RSC Adv., 2014, 4, 36036
triphosphates†
a
Qi Sun, Jian Sun,a Shan-Shan Gong,a Cheng-Jun Wang,a Shou-Zhi Pua
Received 6th June 2014
Accepted 6th August 2014
*
b
*
and Fu-De Feng
DOI: 10.1039/c4ra07670b
www.rsc.org/advances
An efficient P(V)-N activation strategy for the preparation of high-
In the past few years, several phosphoramidite-based solid
quality 5-hydroxymethyl-, 5-formyl-, and 5-carboxyl-20-deoxy- phase approaches for the preparation of 5-hmC-, 5-fC-, and 5-
cytidine triphosphates has been developed. The method was also caC-containing oligodeoxynucleotides (ODNs) have been
optimized for gram-scale synthesis of the corresponding parent developed.9 To advance the investigation of the mechanisms
nucleosides from 20-deoxythymidine.
and enzymes related to 5-mC oxidation, longer DNA fragments
with 5-hmC-, 5-fC-, and 5-caC bases are highly desired but hard
to be synthesized by the tedious solid phase methods. More
recently, Carell and his co-workers reported an expeditious
synthesis of long 5-hmC-, 5-fC-, and 5-caC-containing ODNs
(150 bp) from 5-hydroxylmethyl-, 5-formyl-, and 5-carboxyl-20-
deoxycytidine triphosphates (5-HOMedCTP (1), 5-CHOdCTP (2),
and 5-COOHdCTP (3)) by polymerase chain reaction (PCR).10
However, the oxidative modications of cytosine posed a huge
challenge for the preparation of the corresponding triphos-
phates (Fig. 1). Though the reported yield for 5-CHOdCTP was
70%, the disproportional peaks on its 31P NMR spectrum
revealed that the sample was contaminated with a signicant
amount of polyphosphate impurities.10 Similar issues were also
found in the low-yielding synthesis of 5-COOHdCTP (7% yield)
and the protected 5-HOMedCTP (1% yield).10 In this paper, we
report an efficient preparation of the triphosphates of all three
5-MedC oxidation products (1–3) on the basis of the P(V)-N
activation strategy we established for nucleoside polyphosphate
During the past few decades, it has been unravelled that DNA
methyltransferases-mediated methylation of cytosine in
eukaryotic genomes is one of the most important epigenetic
marks for transcriptional gene silencing.1 In mammalian DNA,
cytosines are predominantly methylated within CpG sites, and
the methylation patterns are heritable as stable epigenetic
signals during cell divisions.2 Meanwhile, DNA demethylation
in specic contexts is required for the recovery of cytosine bases,
enabling exible and dynamic regulation of gene expression
during cellular development.3
Recently, a series of 5-methylcytosine (5-mC) oxidation prod-
ucts, 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC),
and 5-carboxycytosine (5-caC) have been detected in mammalian
DNA.4 The ten eleven translocation proteins (TET1-3) were iden-
tied as the corresponding oxidases with molecular oxygen and
2-ketoglutarate as cofactors.5 The new research evidence reported
by He et al.6 and Zhang et al.7 strongly supported that TET-
mediated oxidation of 5-mC leads to active DNA demethylation in
epigenetic programming of cells.8 While thymine-DNA glyco-
sylase (TDG) was determined as an enzyme for 5-caC excision
repair,6 other enzymes involved in either base excision repair
(BER) or decarboxylation pathways remain to be elucidated.
aJiangxi Key Laboratory of Organic Chemistry, Jiangxi Science & Technology Normal
University, 605 Fenglin Avenue, Nanchang, Jiangxi 330013, PR China. E-mail:
sunqi96@tsinghua.org.cn; Tel: +86 791 83805183
bDepartment of Polymer Science & Engineering, College of Chemistry & Chemical
Engineering, Nanjing University, Nanjing, Jiangsu 210093, PR China. E-mail:
fengfd@nju.edu.cn; Tel: +86 25 83592119
† Electronic supplementary information (ESI) available: Experimental procedures
and NMR spectra of intermediates and products are included. See DOI:
10.1039/c4ra07670b
Fig.
1
The 5-RdCTP-based PCR technology for the expeditious
preparation of long 5-hmC-, 5-fC-, and 5-caC-containing ODNs.10
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synthesis.11 The optimized method for gram-scale synthesis of and desilylation for 9/desilylation for 13) gave 5-HOMedC (4) and
the parent nucleosides, 5-HOMedC (4), 5-CHOdC (5), and 5-COOHdC 5-CHOdC (5) in high yields.
(6), from dT is also described.
Due to the presence of the TBS groups and glycosidic bond in
Currently, there are two major synthetic routes for the 5-HOMedC precursor 10, most conventional strong oxidizing
preparation of 5-MedC oxidation derivatives. The one utilizing methods are too harsh to transform the hydroxyl group to
5-iodo-20-deoxycytidine
(
5-IdC) starting material directly carboxylic acid with high chemoselectivity. To obtain 5-COOHdC
installed the 5-formyl or 5-methoxycarbonyl group by the (6), 10 was oxidized with TEMPO/BAIB (0.2/2.5 equiv.) under
Pd-catalyzed Stille reaction.9a–d The reduction of 5-CHOdC (5) mild conditions.17 The subsequent removal of the TBS groups
afforded 5-HOMedC (4).9a,10 However, the cost of 5-IdC and the use with TFA afforded 6 in nearly 70% yield over two steps (Scheme
of pressurized reactor for gaseous CO limited its application. 2). It is noteworthy that the outcomes of TEMPO/BAIB-mediated
Therefore, we employed the other approach starting from dT.4c oxidation are strongly correlated with the amount of BAIB.18
As shown in Scheme 1, 30,50-diTBS-protected dT (7) which When stoichiometric amount of BAIB was applied (TEMPO/
could be easily prepared from dT was brominated with NBS in BAIB (0.2/1.1 equiv.)), 10 could be efficiently transformed into
CCl4, and then treated with potassium acetate to afford the the corresponding aldehyde 13, providing an alternative
acetylated intermediate 8 in 65% yield.12 Aer 8 was deacety- approach to 5-CHOdC (5).
lated with K2CO3, mild oxidation of the protected 5-HOMedU (11)
with activated MnO2 in CH2Cl2 furnished clean conversion to started from acetylated 5-HOMedC intermediate 9 (Scheme 3).
the silylated 5-CHOdU (12) in 94% yield.9e,13 Efficient regioselective desilylation at 50 position was achieved
Our synthetic route to 5-HOMedCTP (1) and 5-CHOdCTP (2)
As Carell mentioned, the major challenge for 5-HOMedCTP (1) by lowering the concentration of TFA (THF/H2O/TFA, 4 : 1 : 1)
synthesis is associated with the 5-hydroxylmethyl group in and reaction temperature (0 C).19 Treatment of 15 with POCl3
ꢀ
5-HOMedC (4), which makes it difficult to selectively phosphory- in PO(OMe)3 followed by sequential deacetylation and desily-
late the OH group at C50 position to access 5-HOMedC 50-mono- lation afforded the 5-HOMedC 50-monophosphate (16) in 64%
phosphate (16).10 To address this issue, we attempted to yield. Our observation that no proton sponge was required for
conduct selective amination at C4 position of 8 to obtain the the phosphorylation of 15 was in agreement with previous
acetylated 5-HOMedC intermediate 9, which could be later
utilized for the synthesis of 16. However, the conventional
POCl3/1,2,4-triazole/methanolic NH3 method only gave 9 in low
yield (12%) with signicant amount of deacetylated product.4c,14
Switching to 28% NH4OH largely reduced the undesired
deacetylation on the 5-hydroxylmethyl group, and afforded 9 in
60% yield.12a,15 To further improve the synthetic efficacy and
simplify the procedures, TsCl was used with 1-methylpiperidine
as the activator to form a more reactive quaternary ammonium
intermediate.16 The in situ aminolysis selectively afforded
5-HOMedC precursor 9 in 72% yield within 30 min. More inter-
estingly, this one-pot reaction with TsCl/1-methylpiperidine/
Scheme 2 Synthesis of 5-COOHdC (6) and 5-CHOdC (5). Reagents and
28% NH4OH exhibited excellent compatibility with the 5-formyl
conditions: (a) TEMPO/BAIB (0.2/2.5 equiv.), CH2Cl2/H2O, 20 ꢀC, 8 h;
group in 12, and yielded the protected 5-CHOdC precursor 13 in
high conversion rate (75%). Final deprotection (deacetylation
(b) TEMPO/BAIB (0.2/1.1 equiv.), CH2Cl2/H2O, 20 ꢀC, 2 h; (c) THF/H2O/
TFA (2 : 1 : 1), 20 ꢀC, 1 h.
Scheme 1 Synthesis of 5-HOMedC (4) and 5-CHOdC (5). Reagents and conditions: (a) NBS, AIBN, CCl4, 80 ꢀC, 1.5 h; (b) CH3COOK, 40 ꢀC, 30 min; (c)
TsCl, N-methylpiperidine, Et3N, 4 h; (d) 28% NH4OH, 30 min; (e) K2CO3/MeOH/H2O, 20 ꢀC, 2 h; (f) THF/H2O/TFA (2 : 1 : 1), 20 ꢀC, 1 h; (g) activated
MnO2, CH2Cl2, 30 ꢀC, 48 h.
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Scheme 3 Synthesis of 5-HOMedCTP (1), 5-CHOdCTP (2), and 5-COOHdCTP (3). Reagents and conditions: (a) THF/H2O/TFA (4 : 1 : 1), 0 ꢀC, 2 h; (b)
POCl3, PO(OCH3)3, 0 ꢀC, 2 h; (c) TFA/H2O (1 : 1), 20 ꢀC, 1 h; (d) K2CO3/MeOH/H2O, 20 ꢀC, 1 h; (e) 2,20-dithiodianiline, PPh3, piperidine, DMSO,
20 ꢀC, 8 h; (f) activated MnO2, MeOH, 50 ꢀC, 24 h; (g) (nBu4N)3HP2O7, DCI, 20 ꢀC, 6 h; (h) TEMPO/BAIB (0.4/2.5 equiv.), tBuOH/CH2Cl2/H2O
(4 : 4 : 1), 20 ꢀC, 48 h.
indicating that the P(V)-N activation method well tolerated the
hydroxylmethyl and formyl modications on cytosine. Ethanol
precipitation followed by ion exchange chromatography affor-
ded 1 and 2 in high isolated yields.
The attempt to oxidize 17 with TEMPO/BAIB (0.2/2.5
equiv.) system only afforded the desired 5-COOHdC 50-phos-
phoropiperidate (19) in low yield (<20%) due to the labile
nature of phosphoropiperidate under even weakly acidic
conditions. Therefore, we directly oxidized 5-HOMedCTP (1)
with TEMPO/BAIB (0.4/2.5 equiv.) to yield 5-COOHdCTP (3). 31
P
NMR tracing results showed that the oxidation process in
monophasic tBuOH/CH2Cl2/H2O (4 : 4 : 1) solvent system21
was smooth and clean. Aer 48 h, 3 was isolated in 78% yield.
The quality of 3 was determined with analytic RP-HPLC along
with 1 and 2. The HPLC traces in Fig. 3 showed that
triphosphates 1–3 prepared by our method were of high
purity (>95%). But it is worth noting that the solvent system
also played a key role in the TEMPO/BAIB oxidation. When
monophasic CH3CN/H2O (1 : 1) or biphasic CH2Cl2/H2O
(1 : 1) was used, the oxidation of 1 was extremely slow.
While the TEMPO/BAIB oxidation of 10 in biphasic
CH2Cl2/H2O (1 : 1) afforded 14 within 8 h, the oxidation in
monophasic tBuOH/CH2Cl2/H2O (4 : 4 : 1) required much
longer time (48 h).
Fig.
synthesis.
2
The stacked 31P NMR tracing spectra of 5-CHOdCTP (2)
Fig. 3 The HPLC traces of 5-HOMedCTP (1), 5-CHOdCTP (2), and
5-COOHdCTP (3).
Conclusions
reports on the synthesis of cytosine-containing nucleoside
monophosphates.20 In the following step, 16 was converted to In summary, we have developed an efficient method for the
5-HOMedC 50-phosphoropiperidate (17) by the redox condensa- preparation of high-quality 5-hydroxylmethyl-, 5-formyl-, and
tion method in excellent yield (94%).11c Treatment of 17 with 5-carboxyl-20-deoxycytidine triphosphates (1–3) on the basis of
activated MnO2 smoothly oxidized 5-hydroxylmethyl group to the P(V)-N activation strategy. The synthesis of the parent
5-formyl group to give 5-CHOdC 50-phosphoropiperidate (18). nucleosides (4–6) were also optimized to provide facile access
Finally, 17 and 18 were subjected to the 4,5-dicyanoimidazole to all three oxidation products of 5-MedC. The P(V)-N activation
(DCI)-promoted P(V)-N activation strategy to synthesize method described in this paper along with the 5-RdCTP-based
5-HOMedCTP (1) and 5-CHOdCTP (2).11a,c 31P NMR tracing experi- PCR technology may greatly facilitate the investigation of
ments showed that both 1 and 2 were obtained with high 5-MedC-related epigenetic regulations and development of
conversion efficacy as exemplied by the reaction of 2 (Fig. 2), regenerative drugs.
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Acknowledgements
We thank the National Natural Science Foundation of China
(no. 21002041 and 21262014), Key Project of Chinese Ministry of
Education (no. 212092), Scientic Research Foundation of
Chinese Ministry of Human Resources and Social Security for
Returned Chinese Scholars (2011), and Research Funds (no.
ky2012zy08 and 2013QNBJRC001) and Startup Funds for PhDs
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