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1187
Table 1
Sequences of oligonucleotides
reaction mixture (Table S1). In the presence of catalytic amount of
divalent copper ion, the major oxidative product of mdC shifted to
fdC and generation of 5-hydroxymethyl-20-deoxycytidine (hmdC)
were not detectable. No significant improvement in yields was
observed by using 30,50-proteced nucleosides, which indicated that
hydroxyl groups on deoxyribose would neither decompose nor
interfere the oxidation.22 Na2S2O8–Cu2+ can be directly applied to
oxidize mC nucleoside and extra synthetic steps of introducing
and removing protection groups can be avoided. The reaction suc-
cessfully proceeded to completion either under microwave assist-
ed heating (80 °C) or under conventional heating (65 °C) within
regular RBF open to air. Reaction time was significantly reduced
from 1 h to 5 min per reaction under microwave-assisted heating.
At the end of reaction, 1 was completely consumed by radical oxi-
dants and 2 was afforded as the only product with detectable UV
absorption either on TLC or by HPLC, which made purification by
silica column chromatography relatively simple. The synthetic
pathway for fC phosphoramidite was developed as shown in
Scheme 1. Since aldehyde can readily survive phosphoramidite
based chemistry of oligonucleotide (ODN) synthesis, formyl group
was not protected in the final fC phosphoramidite. We firstly,
selectively protect the exocyclic amino group of fdC by reacting
with benzoic anhydride in hot ethanol and generate 3 in moderate
yield 65%. Compared to acetyl protecting group, benzoyl function-
ality is chemically more stable during solid phase synthesis of
oligonucleotides, so that ultra-mild synthetic condition required
by N4-acetyl-fC phosphoramidite can be avoided.17 In addition, 3
precipitated from reaction solvent and could be simply isolated
from reaction mixture by centrifugation. Furthermore, N4-protect-
ed fdC can also avoid the by-reactions with dimethoxytrityl chlo-
ride (DMT-Cl) if DMT was introduced to bare nucleoside. The
subsequent DMT protection and 30-phosphitylation were accom-
plished with decent yields of 91% and 73%, respectively. This
synthetic route entails merely four steps to prepare fC
phosphoramidite 5 and is more convenient in the aspect of experi-
ment setup. fC phosphoramidite was then incorporated into
oligonucleotide ODN1 by standard solid phase DNA synthesis with
extended coupling time (5 min) to ensure high synthetic yield.
Decent coupling yield of fC phosphoramidite (78%) was achieved,
according to the trityl graph of solid phase synthesis (data not
shown). ODN1 was cleaved from the resin by incubation in concen-
ODN
Sequencesa
ODN1
ODN2
ODN3
ODN4
ODN5
ODN6
ODN7
50-TTC CAC GfCG CGT TCC TGA CTG ACT C-30
50-TTC CAC GhmCG CGT TCC TGA CTG ACT C-30
50-TTC CAC GCG CGT TCC TGA CTG ACT C-30
30-AAG GTG CGC GCA ACC ACT GAC TGA G-50
30-AAG GTG CAC GCA ACC ACT GAC TGA G-50
30-AAG GTG CCC GCA ACC ACT GAC TGA G-50
30-AAG GTG CTC GCA ACC ACT GAC TGA G-50
a
Epigenetic bases and complementary bases are highlighted in bold and italic.
trated NH4OH at 75 °C for 24 h. Simultaneously deprotection of
natural nucleobases was accomplished during the incubation.
Since fC phosphoramidite 5 uses the common benzoyl protection
as regular cytosine phosphoramidites, no additional deprotection
procedures are required for fC containing oligonucleotide. After
HPLC purification before and after detritylation by 80% acetic acid
at room temperature, the desired oligonucleotide ODN1 (sequence
in Table 1) was obtained and the mass of ODN1 was verified by ESI-
MS (Fig. S2). For further validation of successful incorporation of fC,
ODN1 was submitted to enzymatic digestion to yield free nucleo-
sides and the resultant mixture was analysed by LC–MS. Similar
as literature, 5-formyl-20-deoxycytidine was eluted at the same
retention time as thymidine from C18 column, and thus was not
distinguishable by HPLC analysis (Fig. 1B).16 To confirm the coex-
isting of fdC and dT in HPLC elutes at 16.6 min, the co-elutes were
submitted to LC–MS spectroscopy for both baseline separation and
mass characterization of fC and thymidine. Well resolved mass
spectra of 5-formyl-20-deoxycytidine (Fig. 1C, [M+H]+ m/z for
C
10H14N3O+5 256.0912 and nucleobase fragment C6H6N3O2+
140.0445) and thymidine (Fig. S1, [M+H]+ m/z for C10H15N2O+5
243.0916 and nucleobase fragment C5H7N2O+2 127.0498) were
observed.
The same Na2S2O8–Cu2+ oxidant system were also applied to
prepare hmC phosphoramidite (Scheme 2). Unlike fC phospho-
ramidite, hydroxymethyl group cannot survive the synthetic
reactions of oligonucleotides and needs to be protected in
phosphoramidite. In order to avoid the interference from
N4-exocylic amine during protection of hydroxymethyl group, we
chose thymidine (dT, 6), instead of mdC, as the starting substrate
Scheme 1. Synthetic route for fC phosphoramidite 5 building block. Synthetic yields are indicated under reaction condition of each step.