Photocaged 5-(hydroxymethyl)pyrimidine nucleoside phosphoramidites for specific photoactivatable epigenetic labeling of DNA

Article abstract of DOI:10.1021/acs.orglett.0c03462

5-Hydroxymethylcytosine and uracil are epigenetic nucleobases, but their biological roles are still unclear. We present the synthesis of 2-nitrobenzyl photocaged 5-hydroxymethyl-2′-deoxycytidine and uridine 3′-O-phosphoramidites and their use in automated

Full text of DOI:10.1021/acs.orglett.0c03462

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pubs.acs.org/OrgLett
Letter
Photocaged 5‑(Hydroxymethyl)pyrimidine Nucleoside
Phosphoramidites for Specific Photoactivatable Epigenetic Labeling
of DNA
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Aswathi Chakrapani, Viola Vankova Hausnerova, Olatz Ruiz-Larrabeiti, Radek Pohl, Libor Krasny,*
and Michal Hocek*
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ABSTRACT: 5-Hydroxymethylcytosine and uracil are epigenetic nucleobases, but their biological roles are still unclear. We present
the synthesis of 2-nitrobenzyl photocaged 5-hydroxymethyl-2′-deoxycytidine and uridine 3′-O-phosphoramidites and their use in
automated solid-phase synthesis of oligonucleotides (ONs) modified at specific positions. The ONs were used as primers for PCR to
construct DNA templates modified in the promoter region that allowed switching of transcription through photochemical uncaging.
xidized congeners of 5-methylcytosine, i.e., 5-hydrox-
Oymethylcytosine (5hmC) or 5-formylcytosine (5fC), are
not only intermediates in active demethylation of DNA, but
they are also epigenetic modifications of DNA,1,2 which
regulate gene expression by influencing binding of tran-
scription factors and RNA polymerases (RNAP) to DNA3 and
modulating chromatin properties.4 To the contrary, the
biological role of another minor DNA base, 5-hydroxymethy-
luracil (5hmU), is not yet fully understood,5 although it was
found in stem cells6 or in some types of cancer.7 5hmU has
also been found in certain bacteriophages8 or parasites.9 We
have recently reported10 a transcription study with bacterial
RNA polymerases (RNAP) of DNA templates containing
5hmU or 5hmC. The study revealed that the presence of these
epigenetic modifications can significantly enhance transcription
(by up to 3.5 times), depending on the promoter. We found
that it is the nontemplate strand of the promoter region where
the presence of these modifications has the strongest effect.10
Subsequently, we also developed11 an artificial switch for
transcription. Enzymatic incorporation of photocaged (2-
nitrobenzyl, NB) derivatives of 5hmU or 5hmC 2′-
deoxyribonucleoside triphosphates (dNTPs) resulted in
caged DNA templates that were not compatible with
transcription, because of the presence of the bulky nitrobenzyl
groups in the major groove. However, their irradiation by UV
or visible light (up to 400 nm) removed the photocaging
groups and released the hmU/hmC-modified DNA, which
became transcriptionally active.11 Unfortunately, the enzymatic
synthesis of DNA from base-modified dNTPs12 is difficult to
be used for site-specific incorporation of one single or several
modification(s).13 Such site-specifically modified DNAs would
be useful for deeper structural and functional studies of the
effect of these epigenetic modifications. Several protected
5hmC14,15 and 5hmU14,16 2′-deoxyribonucleoside phosphor-
amidites suitable for automated solid-phase synthesis of
oligonucleotides17 are known in the literature, but their
photocaged analogues have not been described yet. Consid-
ering the general importance of DNA photocaging18 and the
specific need of site-specific photocaging of a single nucleobase
in the major groove of DNA, we set out to design and
synthesize photocaged 5hmU and 5hmC 2′-deoxyribonucleo-
side phosphoramidites and to study their use in solid-phase
synthesis of photocaged oligonucleotides.
The synthesis of the photocaged phosphoramidites started
from thymidine, which was converted to tert-butyldimethylsilyl
(TBDMS) protected 5-[(2-nitrobenzyl)oxymethyl]-2′-deoxy-
Received: October 16, 2020
https://dx.doi.org/10.1021/acs.orglett.0c03462
© XXXX American Chemical Society
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uridine (1) via a published four-step sequence (Scheme 1).19,20
Intermediate 1 was then transformed to photocaged hmC (2a)
dimethylacetal (DBF-DMA) in anhydrous dimethylformamide
(DMF).22 This gave the corresponding amidine derivative 4a
in good yield, which was further converted to the
phosphoramidite 5a (85% yield) by treatment with 2-
cyanoethyl-N,N-diisopropylchlorophosphoramidite (CEOP-
(Cl)NiPr2), in analogy to the literature protocol.17 In the
same way, the photocaged hmU nucleoside 3b was trans-
formed to the desired phosphoramidite 5b in 74% yield.
The photocaged building blocks 5a and 5b were then used
in the synthesis of oligo-2′-deoxyribonucleotides (ONs) aimed
for the construction of specifically modified DNA templates for
the study of bacterial transcription. In previous works, we used
a 339 mer DNA template containing the 38 bp-long Pveg
promoter for the study of the influence of major-groove
modifications on transcription with RNAP from E. coli.10,11,23
Those fully modified templates were constructed using PCR
with modified dNTPs. For specific single-point modifications
in the nontemplate strand of the promoter region, we
previously designed the synthesis through PCR using modified
forward primer.11 In order to access the important regions of
the promoter, we decided to truncate the template to a 222mer
DNA containing only a core promoter flanked by only two
nucleotides at the 5′-end (Figure 1A). This shortened 222mer
template has retained ∼90% of the activity of the original 339
mer construct. Based on a structural study24 and on our
previous experience,25 we identified specific sites of the core
promoter, mainly in the −35 region of the nontemplate strand,
as important for the interaction with the RNAP. Therefore, we
designed photocaged ONs (ON2−ON5) containing one or
several photocaged hmU or hmC bases in the −35 region
(Figure 1B) and, for comparison, ONs containing these
modifications outside of that region (ON6−ON8). ONs
ON2−ON8 were synthesized on an automated DNA
synthesizer using phosphoramidite building blocks 5a and/or
5b. The synthesis proceeded under standard conditions, giving
the desired 20- or 21-nt photocaged ONs with efficient
incorporation of the modified phosphoramidites, compared to
their natural counterparts.
Scheme 1. Synthesis of the Nitrobenzyl-Photocaged
a
Nucleoside Phosphoramidites
The photocaged ONs (ON2−ON8), along with the natural
ON1, were then used as forward primers for the PCR reaction
on the Pveg plasmid, along with a natural reverse primer (for
the 222mer template) and natural dNTPs (Figure 1C). In all
cases, full-length 222mer DNA amplicons (222DNA1 and NB-
222DNA2−NB-222DNA8) modified in the promoter region
were obtained efficiently and after isolation were used as
templates for in vitro transcription experiments with E. coli
RNAP. The photocaged DNA templates NB-222DNA2−NB-
222DNA8 then were irradiated by a 3-W 400-nm photodiode
for 10 or 30 min in the presence of additives DTT and sodium
azide (in analogy to previous works11,20,21) to release the
uncaged DNA containing unprotected bases hmU and/or
hmC (hm-222DNA2−hm-222DNA8; see Figure 1D) and the
uncaged templates were also used for the transcription
experiments, which were performed as reported previ-
ously.10,11,23
The results of the in vitro transcription experiments are
summarized in Figure 1E. The natural DNA template
(222DNA1) yielded about the same transcription before and
after irradiation. DNA templates containing the photocaged
bases in the −35 region (NB-222DNA2−NB-222DNA5),
which is critical for interaction with RNAP, displayed
significantly lower transcription (14%−78%), whereas tran-
scription of templates modified outside the −35 region (NB-
a
Reagents and conditions: (i) TBDMSCl, imidazole, DMF, 95%; (ii)
NBS, azobisisobutyronitrile (AIBN), benzene; (iii) diisopropylethyl-
amine (DIPEA), H2O, DMF, 27%; (iv) 2-nitrobenzyl bromide,
AgOTf, 2,6-di-tert-butylpyridine, DCM, 40%; (v) TIPSCl, DMAP,
Et3N, DCM; (vi) NH3 (g), dioxane, 80%; (vii) Et3N·3HF, THF,
40%−50%; (viii) DMTrCl, DMAP, pyridine; 50%−70%; (ix) DBF-
DMA, DMF, 76%; and (x) CEOP(Cl)NiPr2, DIPEA, DCM, 70%−
85%.
and hmU 2′-deoxyribonucleosides (2b) also, using known
procedures.20,21 Subsequent dimethoxytritylation of 2a and 2b
gave the 5′-O-4,4′-dimethoxytrityl (DMTr) protected inter-
mediates 3a and 3b in 68% and 50% yields, respectively.
Various protecting group strategies were then attempted for
the transient protection of the amino group of hmC in
compound 3a prior to converting it to the corresponding
phosphoramidite. First, we tried to introduce benzoyl or acetyl
groups, but we obtained low yields and/or side acylation at 3′-
OH. Therefore, the dibutylformamidine protecting group was
attached through the reaction of 3a with dibutylformamide-
B
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Figure 1. (A) Sequence of the DNA template. (B) Sequences of the modified photocaged ONs. (C) PCR construction of DNA templates
containing photocaged pyrimidines in the promoter region. (D) Photochemical uncaging of the templates. (E) Relative yield of transcription with
E. coli RNAP from either photocaged (blue) or uncaged (orange) DNA templates. The bars show averages from at least five independent
experiments, the error bars show the standard deviation ( SD). Transcription from the nonirradiated nonmodified template was set as 1. The
numbers below the graph correspond to construct numbers as shown in panel (B).
222DNA6−NB-222DNA8) was comparable to the unmodi-
fied control. The lowest transcription was observed from the
template containing NB-U at the −35 position (NB-
222DNA2, 33%) and the template with a combination of
three photocaged pyrimidines (NB-222DNA5, 14%). After
irradiation and uncaging of the hydroxymethylpyrimidine
bases, the transcription of the out-of-region-modified DNA
templates was unchanged (hm-222DNA6−hm-222DNA8),
but the transcription of the in-the-region-modified uncaged
templates (hm-222DNA2−hm-222DNA5) was restored to
the level of the unmodified control. In other words, the
transcription was switched on.
In conclusion, we have developed the synthesis of 2-
nitrobenzyl-photocaged 2′-deoxyribonucleoside 3′-phosphor-
amidite derivatives from 5hmU and 5hmC nucleobases. These
phosphoramidite building blocks were used for automated
synthesis of oligonucleotides containing the photocaged
pyrimidine bases bearing epigenetic modifications at specific
positions. We used this approach for the synthesis of
photocaged primers to obtain DNA templates modified in
the promoter region. We synthesized several templates
containing one or more photocaged pyrimidines within and
outside the −35 region of the Pveg promoter and tested them
in in vitro transcription with RNAP from E. coli. As expected,
the presence of photocaged pyrimidines within the −35 region
significantly (though not completely) inhibited the tran-
scription, whereas their presence outside of this region did
not affect the transcription. After irradiation with visible light
(400 nm), the release of hmU or hmC bases was achieved and
the transcription reached the level of unmodified template.
The most significant effect was observed for T/hmU at
position −35 and for the combination of three pyrimidines in
the −35 region. Interestingly, we have not observed an increase
in transcription, compared to the natural template, in any case.
Since in our previous works, we have shown that the templates
fully modified with hmU or hmC exert increased transcription
(up to 350% or 250%, respectively),10,11 it seems that a
combination of several hydroxymethylpyrimidines at several
positions in the promoter is needed for the enhancement. The
new photocaged phosphoramidites 5a and 5b will enable
further, more-detailed studies of the influence of pyrimidines
with epigenetic modifications on transcription with both
bacterial and eukaryotic RNA polymerases, to decipher the
biological roles of those epigenetic modifications and to
develop optical switches for transcription.11,18
C
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2015, 115, 2225−2239. (c) Carell, T.; Kurz, M. Q.; Mu
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ller, M.;
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.0c03462.
■
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Full experimental part with synthetic procedures,
characterization of compounds, detailed gels of PCR
and transcription experiments (PDF)
FAIR data, including the primary NMR FID files, for
compounds 3a, 3b, 4a, 4b, 5a, and 5b (ZIP)
AUTHOR INFORMATION
Corresponding Authors
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Libor Krasny − Laboratory of Microbial Genetics and Gene
Expression, Institute of Microbiology, Czech Academy of
Sciences, CZ-14220 Prague, Czech Republic; Phone: +420
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2015, 523, 621−625. (c) Perera, A.; Eisen, D.; Wagner, M.; Laube, S.
Michal Hocek − Institute of Organic Chemistry and
Biochemistry, Czech Academy of Sciences, 16000 Prague,
Czech Republic; Department of Organic Chemistry, Faculty
of Science, Charles University, CZ-12843 Prague, Czech
Republic; orcid.org/0000-0002-1113-2047;
Phone: +420 220183324; Email: hocek@uochb.cas.cz
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Authors
Aswathi Chakrapani − Institute of Organic Chemistry and
Biochemistry, Czech Academy of Sciences, 16000 Prague,
Czech Republic; Department of Organic Chemistry, Faculty
of Science, Charles University, CZ-12843 Prague, Czech
Republic
Viola Vankova Hausnerova − Laboratory of Microbial
Genetics and Gene Expression, Institute of Microbiology,
Czech Academy of Sciences, CZ-14220 Prague, Czech
Republic
Olatz Ruiz-Larrabeiti − Laboratory of Microbial Genetics and
Gene Expression, Institute of Microbiology, Czech Academy of
Sciences, CZ-14220 Prague, Czech Republic
Radek Pohl − Institute of Organic Chemistry and
Biochemistry, Czech Academy of Sciences, 16000 Prague,
Czech Republic
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.0c03462
(5) Olinski, R.; Starczak, M.; Gackowski, D. Enigmatic 5-
hydroxymethyluracil: Oxidatively Modified Base, Epigenetic Mark
or Both? Mutat. Res., Rev. Mutat. Res. 2016, 767, 59−66.
(6) Pfaffeneder, T.; Spada, F.; Wagner, M.; Brandmayr, C.; Laube, S.
K.; Eisen, D.; Truss, M.; Steinbacher, J.; Hackner, B.; Kotljarova, O.;
Schuermann, D.; Michalakis, S.; Kosmatchev, O.; Schiesser, S.;
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Funding by the European Regional Development Fund; OP
RDE (No. CZ.02.1.01/0.0/0.0/16_019/0000729 to A.C.) and
by the Czech Science Foundation (20-00885X to M.H. and 20-
12109S to L.K.) is gratefully acknowledged. M.H. also thanks
the Czech Academy of Sciences for the Praemium Academiae
personal award. O.R-L. was supported by a postdoctoral grant
(No. POS_2019_1_0033) from the Basque Government. We
Steigenberger, B.; Raddaoui, N.; Kashiwazaki, G.; Muller, U.; Spruijt,
̈
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C. G.; Vermeulen, M.; Leonhardt, H.; Schar, P.; Muller, M.; Carell, T.
Tet Oxidizes Thymine to 5-hydroxymethyluracil in Mouse Embryonic
Stem Cell DNA. Nat. Chem. Biol. 2014, 10, 574−581.
(7) Djuric, Z.; Heilbrun, L. K.; Simon, M. S.; Smith, D.; Luongo, D.
A.; LoRusso, P. M.; Martino, S. Levels of 5-hydroxymethyl-2’-
deoxyuridine in DNA from blood as a marker of breast cancer. Cancer
1996, 77, 691−696.
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thank Dr. I. Barvik (Charles University) for discussions on
structural aspects of RNAP.
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