Synthesis of Oligonucleotides Containing the N6-(2-Deoxy-α,β-d-erythropentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy?dG) Oxidative Damage Product Derived from 2′-Deoxyguanosine

Article abstract of DOI:10.1002/chem.201905795

N⁶-(2-Deoxy-α,β-d-erythropentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy?dG) is a major DNA lesion produced from 2′-deoxyguanosine under oxidizing conditions. Fapy?dG is produced from a common intermediate that leads to 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-OxodGuo), and in greater quantities in cells. The impact of Fapy?dG on DNA structure and function is much less well understood than that of 8-OxodGuo. This is largely due to the significantly greater difficulty in synthesizing oligonucleotides containing Fapy?dG than 8-OxodGuo. We describe a synthetic approach for preparing oligonucleotides containing Fapy?dG that will facilitate intensive studies of this lesion in DNA. A variety of oligonucleotides as long as 30 nucleotides are synthesized. We anticipate that the chemistry described herein will provide an impetus for a wide range of studies involving Fapy?dG.

Full text of DOI:10.1002/chem.201905795

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Oligonucleotide Synthesis |Hot Paper|
Synthesis of Oligonucleotides Containing the N6-(2-Deoxy-a,b-d-
erythropentofuranosyl)-2,6-diamino-4-hydroxy-5-
formamidopyrimidine (Fapy·dG) Oxidative Damage Product
Derived from 2’-Deoxyguanosine
Haozhe Yang, Joel A. Tang, and Marc M. Greenberg*[a]
Abstract: N6-(2-Deoxy-a,b-d-erythropentofuranosyl)-2,6-di-
amino-4-hydroxy-5-formamidopyrimidine (Fapy·dG) is
due to the significantly greater difficulty in synthesizing oli-
gonucleotides containing Fapy·dG than 8-OxodGuo. We de-
scribe a synthetic approach for preparing oligonucleotides
containing Fapy·dG that will facilitate intensive studies of
this lesion in DNA. A variety of oligonucleotides as long as
30 nucleotides are synthesized. We anticipate that the
chemistry described herein will provide an impetus for a
wide range of studies involving Fapy·dG.
a
major DNA lesion produced from 2’-deoxyguanosine under
oxidizing conditions. Fapy·dG is produced from a common
intermediate that leads to 7,8-dihydro-8-oxo-2’-deoxyguano-
sine (8-OxodGuo), and in greater quantities in cells. The
impact of Fapy·dG on DNA structure and function is much
less well understood than that of 8-OxodGuo. This is largely
Introduction
DNA oxidation plays a prominent role in the etiology and
treatment of diseases, as well as aging. Consequently, how in-
dividual damaged nucleotides are recognized by polymerases,
glycosylases and other proteins involved in DNA repair is fun-
damentally important.[1] In addition, recent evidence suggests
that damaged nucleotides may also play a role in regulating
transcription.[2] As the most readily oxidized native nucleotide,
2’-deoxyguanosine modifications have been of significant in-
terest.[3] Of these, 7,8-dihydro-8-oxo-2’-deoxyguanosine (8-Ox-
odGuo), the two-electron oxidation product resulting from
formal hydroxyl radical addition to the C8-position of dG
(C8Add, Scheme 1) is the most well studied lesion.[4] The corre-
sponding 2,6-diamino-4-hydroxy-5-formamidopyrimidine de-
Scheme 1. Formation of Fapy·dG and 8-OxodGuo.
Although formed as a consequence of oxidative stress,
Fapy·dG is at the same oxidation state as dG. In fact, cycliza-
tion and dehydration of Fapy·dG is a possible biosynthetic
pathway to the native nucleotide.[6] Fapy·dG is believed to
result from one electron reduction of C8Add; whereas 8-OxodG-
uo formation requires a second oxidation step (Scheme 1).[7]
The dependence of the relative levels of Fapy·dG and 8-Ox-
odGuo on the environment in which C8Add is produced is gen-
erally consistent with this mechanism.[8] 8-OxodGuo formation
is favored under O2 rich conditions, but Fapy·dG is detected at
higher levels in cells. The preferential formation of Fapy·dG
over 8-OxodGuo in cells is attributed to lower O2 concentration
than in a test tube and the presence of reducing agents in this
environment. However, recently it was reported that Fapy·dG
formation in the test tube required exhaustive O2 depletion in
solution.[9] Fapy·dG and 8-OxodGuo have also been proposed
as part of complex lesions in DNA as a result of their formation
of nucleotide peroxyl radicals.[10] However, the subsequent par-
titioning of the peroxyl radical adduct(s) is expected to follow
the same dependence as C8Add on O2.
rived from dG (Fapy·dG) is believed to also arise from C8Add
,
and under some conditions is produced in greater amounts
than 8-OxodGuo.[5] However, the effects of Fapy·dG on bio-
chemical processes in the test tube and in cells, as well as nu-
cleic acid structure, are less well understood. Obtaining knowl-
edge about the effects of Fapy·dG is hindered due to the rela-
tive difficulty in synthesizing oligonucleotides containing this
lesion. We wish to report a significant advance in synthesizing
oligonucleotides containing Fapy·dG that will facilitate studies
on this important DNA modification.
[a] Dr. H. Yang, Dr. J. A. Tang, Prof. Dr. M. M. Greenberg
Department of Chemistry, Johns Hopkins University
3400 N. Charles St. Baltimore, MD 21218 (USA)
E-mail: mgreenberg@jhu.edu
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under:
https://doi.org/10.1002/chem.201905795.
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Inferential support for the biological significance of Fapy·dG
is that not only is it recognized by the proteins involved in re-
pairing guanine oxidation (GO) products, but it is repaired in a
manner to guard against introducing mutations.[3] For instance,
the bacterial (Fpg) and human glycosylases (hOGG1) that selec-
tively recognize 8-OxodGuo opposite dC versus dA act similarly
on Fapy·dG. Fpg hydrolyzes the glycosidic bond of the lesion
in a Fapy·dG:dC base pair almost 20-times more efficiently
than when opposite dA.[11] hOGG1 is even more selective, dis-
criminating against a Fapy·dG:dA base pair by more than 40-
fold.[12] The bacterial glycosylase, MutY, incises a mismatched
dA opposite Fapy·dG more rapidly than from a dG:dA mispair,
but 4-fold more slowly than from a duplex containing 8-Ox-
odGuo:dA.[11,13] The relevance of a Fapy·dG:dA base pair is
born-out by mutagenesis studies. Fapy·dG bypass in E. coli, as
well as mammalian cells, gives rise to G to T transversions. The
frequency of dA misincorporation opposite Fapy·dG was con-
sistently lower in E. coli than when single-stranded plasmids
containing 8-OxodGuo were replicated.[14] In mammalian cells
(COS-7, HEK293T), Fapy·dG mutagenicity resulting from dA mis-
incorporation, was much more comparable to that of 8-Ox-
odGuo, and in some instances was greater.[15] Insight into the
structural basis for Fapy·dG mutagenicity and recognition by
repair enzymes have been limited to a carbacyclic analogue
(carba-Fapy·dG), due to the difficulty in synthesizing oligonu-
cleotides containing Fapy·dG.[16] Fapy·dG recognition by trans-
lesion synthesis polymerases and other repair enzymes is also
not well understood. This too could be due to synthetic limita-
tions that make it difficult for many investigators to obtain oli-
gonucleotide substrates containing Fapy·dG.
Scheme 2. Isomerization of Fapy·dG.
tion time. Oligonucleotides containing the pyranose isomer
were removed by reverse-phase HPLC and in some instances
this additional purification step was not reported. Another
strategy involved post-synthetic reduction of a 5-nitro group,
followed by formylation.[21] The strong electron withdrawing
nitro group prevents isomerization to the pyranose isomer
when the 5’-hydroxyl group is revealed during oligonucleotide
synthesis. The strategy developed in our group utilizing dinu-
cleotide phosphoramidites to prevent isomerization to the
pyranose isomer also made use of the 5-nitro substituent.[22]
This strategy provided oligonucleotides without any concern
for pyranose formation or the need for additional purification.
However, the syntheses of the dinucleotides were lengthy, cou-
pling yields on solid-phase support were modest (50–70%)
and/or required double-coupling of the Fapy·dG phosphorami-
dite. Finally, the ability to synthesize any oligonucleotide se-
quence requires preparing 4 dinucleotides, of which only two
were reported.
Solid-phase synthesis of oligonucleotides containing
Fapy·dG is challenging due to the availability of the N6-lone
pair electrons, which facilitates reversible ring opening of the
deoxyribose ring (Scheme 2).[17] This results in epimerization
and Fapy·dG exists as a mixture of a- and b-anomers in duplex
DNA.[18] When present as a nucleoside exposure of the primary
hydroxyl results in preferential isomerization to the pyranose
form of Fapy·dG. Possible formation of the pyranose isomer
within DNA was a major consideration when designing a
method for synthesizing oligonucleotides containing Fapy·dG.
Isomerization is avoided by synthesizing carbacyclic (carba-
Fapy·dG) or C-nucleoside (b-C-Fapy·dG) analogues, but these
model compounds may not completely recapitulate the struc-
ture of the native lesion.[19]
Results and Discussion
Strategic overview
We postulated that oligonucleotides containing Fapy·dG could
be prepared via solid-phase synthesis using a “reverse” phos-
phoramidite approach in which the dimethoxytrityl protecting
group was at the 3’-hydroxyl position and the 5’-hydroxyl con-
tained the phosphoramidite (1, Scheme 3). This approach
avoids exposing the 5’-hydroxyl group during oligonucleotide
synthesis, preventing isomerization to the pyranose isomer.
The oligonucleotides would be synthesized on a solid support
in the 5’- to 3’-direction, instead of the more common 3’- to 5’-
direction. In addition, a single phosphoramidite could be used
Rizzo used conventional 3’- to 5’-solid-phase phosphorami-
dite chemistry to synthesize oligonucleotides containing N5-al-
kylated Fapy·dG variants.[20] The authors minimized isomeriza-
tion to the pyranose form during acidic deprotection of the
primary hydroxyl group by decreasing the detritylation reac-
Scheme 3. Strategy for the synthesis of Fapy·dG phosphoramidite 1.
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to synthesize any oligonucleotide sequence. The synthesis of
the phosphoramidite takes advantage of the 5-nitro substitu-
ent to enable exposing the 5’-hydroxyl without incurring isom-
erization (Scheme 2).[22] In addition, an unusual and important
aspect of this strategy includes introducing the formamide
group in the presence of the phosphoramidite (2). The phos-
phoramidite component is usually introduced in the final step
of a synthesis due to its lability. The viability of this aspect of
the strategy was established by examining the compatibility of
a commercially available thymidine phosphoramidite with rep-
resentative hydrogenation (Pd/CaCO3, H2) and formylation
(formic acetic anhydride) conditions used to convert the nitro
group into a formamide prior to embarking upon the synthe-
sis.
Retrosynthetically, 10 was only 3 transformations and 2 puri-
fications from phosphoramidite 1 (Scheme 5). We initially tried
to prepare the corresponding b-cyanoethyl phosphoramidite
(12a, Scheme 5). Phosphitylation of 10 was accomplished in
modest yield (50%). However, attempts to reduce and formyl-
ate 11 a provided complex mixtures from which it was not pos-
sible to isolate 12a. Switching to the corresponding O-methyl
phosphoramidite (11 b) was less fruitful. Despite a variety of re-
action conditions, 11 b was contaminated with significant
amounts of product(s) in which the phosphorous had under-
gone hydrolysis to the organophosphite.
We speculated that the ability to isolate 11 a in which the
phosphorous is substituted with an electron withdrawing b-ni-
trile group but not the more electron rich O-methyl phospho-
rous (11 b) was associated with the pyrimidine nitro-substitu-
ent. We postulated that the nitro substituent that is vital for
preventing rearrangement to the pyranose, increased the acidi-
ty of the N3-proton sufficiently to facilitate hydrolysis at phos-
phorous in 11 b. Our solution to this challenge was to remove
the NH-proton by derivatizing the O4-position. Initially, a
benzyl protecting group was introduced at the O4-position of
9 via a Mitsunobu reaction en route to 13 (Scheme 6). O-
Methyl phosphoramidite formation proceeded in good yield
(65%), consistent with the above hypothesis concerning the
acidity of the N3-proton. However, subsequent reduction and
formylation again yielded a complex product mixture. In addi-
tion, the benzyl group was not removed, despite experiment-
ing with several hydrogenolysis conditions. Subsequently, the
diphenyl carbamoyl (DPC) group, which has been used as an
oxygen protecting group in the nitrogen heterocycles of nu-
cleosides, was employed.[24]
Synthesis of phosphoramidite 1
The syntheses of dinucleotide phosphoramidites employed for
preparing oligonucleotides containing Fapy·dG provided useful
information for the synthesis of 1.[22] The current endeavor also
provided an opportunity to improve transformations that were
common to the two methods. For instance, carefully control-
ling the temperature of the electrophilic nitration reaction pro-
vided 5 in greater yield than previously obtained (90% versus
50%). The synthesis of 4 was also improved by starting from
Hoffer’s sugar (6, Scheme 4), which can be prepared in large
quantities.[23] The a- and b-anomers of 7 were formed in a 3:1
ratio (a:b) and were separable. However, there was no advan-
tage to doing so and in practice 7 was used as a mixture. Fol-
lowing removal of the p-toluoyl groups, 8 was carried on to 9
via 3 as a mixture of anomers as previously described.[22a,b] Dif-
ficulty in obtaining 10 via desilylation of 9 resulted from loss
of the phenoxyacetyl group (Pac) during purification. This
problem was resolved by eliminating the use of triethylamine,
which is often used during chromatography to guard against
adventitious detritylation.
It was important to utilize well purified 9 to obtain good
yields of 14, but the hydrophobicity of the DPC group also im-
proved the ease of its chromatographic purification. The pro-
tected pyrimidine (14) was carried on to the nitro phosphor-
amidite (16), with the desilylation (15) and phosphitylation
steps proceeding in good yield. Anomers of 15 were separated
Scheme 4. Synthesis of intermediate 9: i) BF3·Et2O, TMSN3, CH2Cl2; ii) NaOMe, MeOH; iii) TBDMSCl, pyridine; iv) DMTCl, pyridine; v) Pd/CaCO3, H2, EtOH; vi) DI-
PEA, 5, EtOH; vii) Phenoxyacetic acid, PyBOP, DIPEA, CH2Cl2; viii) Et3N·3HF, THF.
Scheme 5. Attempts to overcome phosphoramidite instability: i) 2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite, S-ethyltetrazole, DIPEA, CH2Cl2;
ii) N,N-diisopropylmethylphosphoramidic chloride, DIPEA, CH2Cl2; iii) Pd/C, H2, DIPEA, THF; iv) Acetic formic anhydride, pyridine, THF.
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Scheme 6. Synthesis of phosphoramidite 1 using a diphenylcarbamoyl protecting group: i) BnOH, PPh3, DEAD, THF; ii) Et3N·3HF, THF; iii) N,N-diisopropylme-
thylphosphoramidic chloride, DIPEA, CH2Cl2; iv) Pd/C, H2, DIPEA, ethyl acetate; v) Pivalic formic anhydride, ethyl acetate; vi) Diphenylcarbamoyl chloride, Et3N,
pyridine.
but only the major isomer was carried forward. The target
phosphoramidite (1) was obtained, albeit in low yield (20%)
when using the previously reported hydrogenation and formyl-
ation conditions.[22] Fortuitously, the DPC protecting group was
cleaved during hydrogenation.
rotation.[22] However, the corresponding proton for the major,
1
more polar isomer was less evident in the H NMR spectrum. A
peak accounting for ꢀ0.7 protons was evident further upfield
at d 7.66, which is close to the region at which the aromatic
protons resonate. One possibility was that the resonance for
the formamide proton of other rotamers of this anomer were
obscured by the large number of aromatic protons. Although
mass spectrometry was compatible with its identification as an
isomer of 1, confirmatory evidence for the presence of the
The low yield of the one pot hydrogenation and formylation
reaction is attributed to proclivity of the phosphoramidite in 1
to hydrolyze during reaction and even flash chromatography
on silica gel. Hence, the difficulty of preparing acetic formic an-
hydride (AFA) free of acetic acid posed a significant problem
when preparing 1.[25] Attempts to prepare and distill pure AFA
yielded inconsistent results. Consequently, a variety of formyl-
ation methods were examined using model compound 9.
Transfer hydrogenation and formylation using ammonium for-
mate was unsuccessful.[26] In addition, formylation using formic
acid and carbodiimide or formic acid, imidazole and oxalyl
chloride did not provide the expected formamide product. The
overall process was significantly improved by carrying out the
hydrogenation reaction in the more hydrophobic ethyl acetate
instead of THF, and substituting pivalic formic anhydride (PFA)
for AFA. PFA was prepared under solvent free conditions and
distilled under high vacuum at 08C.[27] The anhydride product
obtained contains only trace of acids even stored for weeks.
Reactions using PFA proved to be more reproducible than
those with AFA. We attributed this to the greater ease of purifi-
cation of PFA by distillation, as well as by deleting pyridine
from the reaction mixtures.[22a,b] These conditions provided less
complicated reaction mixtures, which facilitated separation
and purification of the anomers of 1. The Fapy·dG phosphor-
amidite (1) is very sensitive to the acidity of silica gel. However,
1 could be purified by conventional silica gel flash chromatog-
raphy using a mixture of acetone and dichloromethane con-
taining 0.1% triethylamine. This proved to be more reliable in
our hands than an automated flash chromatography system
(Combiflash Rf +) equipped with a less reactive cyano end-
capped silica gel column, from which 1 could be eluted using
ethyl acetate/hexane (50%–100%).
1
formamide group was obtained via a H-13C HSQC experiment,
which showed the expected correlation between the form-
amide proton (d=7.66 ppm) and the formyl carbon signal at
d=158 ppm (Figure S35). A similar correlation was observed
for the formamide proton of the minor anomer (Figure S32). If
the major isomer is a-1, the upfield shift of the formyl proton
in the major isomer could be attributed to shielding by the p-
electrons of the aromatic ring(s). However, NOE experiments
aimed at providing confirmation of this hypothesis were am-
biguous.
Synthesis and deprotection of oligonucleotides containing
Fapy·dG using 1
Initial conditions for synthesizing oligonucleotides containing
Fapy·dG using
1 utilized previous reports as a starting
point.[22a,b] Detritylation and oxidation were carried out using
trichloroacetic acid and tBuOOH, respectively. Following cou-
pling of 1 (0.1m), capping with phenoxyacetic anhydride and
1-methylimidazole was replaced with pivalic anhydride/luti-
dine/THF. Coupling conditions of 1 using 4,5-dicyanoimidazole
as activator were optimized by synthesizing a 15mer consisting
of thymidines and a single Fapy·dG (17) on a (“reverse”) 3’-
dimethoxytrityl thymidine support containing a succinate link-
age between the 5’-hydroxyl and long chain alkylamine. On-
line dimethoxytrityl cation monitoring indicated that 1 coupled
from 70–80% when a 15 min reaction time was used. Separate
anomers of 1 were initially used, but no difference in coupling
yields was detected (data not shown). Consequently, an
anomeric mixture of 1 is routinely used in oligonucleotide syn-
thesis, which also facilitates purifying the phosphoramidite.
The Fapy·dG phosphoramidite (1) was much more sensitive to
water in the acetonitrile used to dissolve it than commercially
available phosphoramidites. To avoid hydrolysis, activated mo-
Characterization of the anomers of 1 was challenging. The
overall yield of 1 from 16 was 55% and the more polar isomer
was favored ꢀ2-fold over the less polar anomer. The diagnos-
tic formamide proton was evident as a mixture of rotamers in
1
the H NMR at d 8.65–8.69 for the minor anomer, which is con-
sistent with reported Fapy·dG derivatives and their restricted
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lecular sieves were added to the solution of 1 after filtering
and were present throughout oligonucleotide synthesis. After
demethylating the Fapy·dG phosphate triester with disodium
2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (0.2m,
30 min, 258C),[28] 17 was deprotected and cleaved from the
solid-phase support using K2CO3 (50 mm) in MeOH (258C, 4 h).
The gel purified product was characterized by ESI-MS (Fig-
ure S1). The coupling yield of 1, as well as its synthesis was a
marked improvement over the corresponding dinucleotide
phosphoramidites employed previously.[22]
Having established satisfactory coupling conditions for 1,
conditions for synthesizing oligonucleotides containing
Fapy·dG and all 4 native nucleotides using reverse phosphor-
amidites were explored. To make the synthesis of Fapy·dG con-
taining oligonucleotides accessible to the greatest number of
scientists possible, we sought to maximize the use of commer-
cially available phosphoramidites and solid-phase synthesis
supports. Reverse dT and fast deprotecting dCAc, dAPac and
dGiPrac phosphoramidites are compatible with the K2CO3 condi-
tions used to deprotect 17.[22a,b] However, the latter two re-
verse phosphoramidites are not commercially available. Re-
verse dimethylformamidine protected dG (dGdmf) and the re-
spective solid-phase support are commercially available. This
required establishing mutually compatible conditions for
Fapy·dG and native phosphoramidite deprotection, as well as
oligonucleotide cleavage from the solid-phase supports.
when it was present at the 3’-terminus in 19. Fortunately, we
also determined that conditions reported for the deprotection
and cleavage of oligonucleotides containing N5-methyl
Fapy·dG (0.1m NaOH, 258C, 12 h)[20] were compatible with
Fapy·dG, the phosphoramidites of native nucleotides em-
ployed, as well as UnyLinkerTM support. Oligonucleotides 18,
19 and 26–29 were successfully obtained in comparable yields
as when deprotected with tert-butylamine. However, 26 re-
quired 16 h to achieve complete deprotection of the 12 dGdmf
groups, while complete deprotection of 26 was achieved with
tert-butylamine at 408C for 8 h. Finally, it should be noted that
the deprotection methods employed here were unable to de-
protect commercially available dABz.
Nucleobase deprotection conditions were tested using 17
and an oligonucleotide containing dA, dC, dG and dT (18). Oli-
gonucleotide 18 was prepared on commercially available Uny-
LinkerTM solid-phase synthesis support. Use of this support ob-
viates the need for users to independently synthesizing LCAA-
CPG containing reverse dAPac. Furthermore, any conditions suf-
ficient for cleaving oligonucleotides from UnyLinkerTM will be
sufficient for removing oligonucleotides linked via the stan-
dard, more labile succinate group. dGdmf was incompletely de-
protected using K2CO3 in MeOH, and remnants of the UnyLin-
kerTM were retained at the 5’-terminus. Saturated aqueous am-
monia solution in ethanol (3:1, 158C, 10 h), which is used to
deprotect oligonucleotides containing carba-Fapy·dG com-
pletely deprotected dGdmf within 18 but cleaved at Fapy·dG in
17.[16a] Oligonucleotide 17 was also cleaved at Fapy·dG when
treated with concentrated NH4OH at room temperature over-
night or with ammonia/methyl amine (1:1, v/v, 558C, 1 h).[29]
Fapy·dG also underwent cleavage when subjected to diisopro-
pylethylamine (10% by volume) and b-mercaptoethanol
(0.25m) in MeOH (258C, 2 h).
Treating 17 with tert-butylamine in H2O (1:3 v/v) under the
recommended conditions (608C, 4 h) produced significant
amounts of cleavage at Fapy·dG.[30] However, subjecting 17
and 18 to these conditions at 408C for 8 h provided good
yields of completely deprotected oligonucleotides. The general
utility of this method was demonstrated by synthesizing a
series of oligonucleotides on UnyLinkerTM support that contain
Fapy·dG and the 4 native nucleotides (20–26) (Figures S4–10).
In addition, 27–29 (Figures S11–13) were synthesized on sup-
ports containing the appropriate 5’-terminal reverse nucleo-
side. However, Fapy·dG was cleaved during the deprotection
Conclusions
Fapy·dG is a biologically interesting DNA lesion formed from a
common intermediate as the most well studied DNA lesion, 8-
OxodGuo. Moreover, Fapy·dG is formed in greater quantities
than the latter under some O2 deficient conditions. Structural
and biochemical knowledge of Fapy·dG, as well as its effects
on replication and transcription in cells is limited due to diffi-
culties in synthesizing oligonucleotides containing it. Conse-
quently, research on the formamidopyrimidines has lagged
behind that of 8-OxodGuo. We describe the implementation of
a synthetic strategy using reverse phosphoramidites that is a
marked improvement over previously reported methods for
synthesizing oligonucleotides containing Fapy·dG. The process
is robust, enabling the synthesis of oligonucleotides containing
Fapy·dG suitable for biochemical and structural studies. The
approach should also be compatible with synthesizing oligo-
nucleotides containing N5-alkylated formamidopyrimidines.
The synthesis of phosphoramidite 1 is a significant improve-
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ment over previous methods, in terms of its effectiveness and
ease of implementation. The availability of 1 and its compati-
bility with readily available solid-phase oligonucleotide synthe-
sis reagents will facilitate research on this biologically signifi-
cant family of DNA lesions by the research community.
Preparation of 2-deoxy-a,b-d-[erythro-]pentofuranosyl azide
(8)[21,22b]
Sodium methoxide (1.2 g, 22.7 mmol) was added to a solution of 7
(9 g, 22.7 mmol) in dry MeOH (70 mL). The reaction was stirred at
408C for 2 h, at which time Amberlite GC50-H+ ion exchange resin
(3.5 g) was added. The reaction mixture was stirred for another
30 min at room temperature. The resin was filtered off and the sol-
vent was removed under vacuum. The residue was purified by
flash chromatography (5% À10% MeOH in DCM) to afford 8
1
(3.1 g, 80%, a/b=3:2) as a white solid. H NMR (400 MHz, CDCl3) d
Experimental Section
5.68 (dd, J=3.4, 1.0 Hz, 1H), 5.59 (t, J=4.6 Hz, 0.5H), 4.51–4.67 (m,
0.5H), 4.30–4.25 (m, 2H), 4.51–4.00 (q, J=4.0 Hz, 0.5H), 3.82–
3.65(m, 3H), 2.23–1.93(m, 3H). The chemical shifts of b-isomers are
consistent with the reported data.
General methods
Triethylamine, pyridine, diisopropylamine, tert-butylamine, 2,6-luti-
dine, ethyl acetate and dichloromethane were distilled from CaH2
under Ar. Methanol was dried over molecular sieves 3 ꢁ and dis-
tilled under Ar. THF was distilled from Na under Ar. Pivalic anhy-
dride was distilled under vacuum. All other reagents were pur-
chased from commercial sources and used without further purifica-
tion unless otherwise stated. Nuclear magnetic resonance spectra
were acquired on Bruker 400 MHz for 1H, 101 MHz for 13C and
162 MHz for 31P. High resolution ESI mass spectra for HRMS of syn-
thesized molecules were recorded on a Waters Acquity/ Xevo-G2
UPLC-MS system in positive mode. MALDI-TOF spectra were re-
corded on a Bruker AutoFlex III MALDI-TOF/TOF mass spectrometry
in negative mode. Low resolution ESI mass spectrometry was car-
ried out on a Thermo Finnigan Surveyor LCQ Fleet instrument. Oli-
gonucleotide synthesis was carried out on an ABI-394 synthesizer.
N-Phenoxyacetyl protected 3’-dimethoxytrityl 5’-b-cyanoethyl 2’-
deoxyadenosine phosphoramidite (dAPac) was synthesized as previ-
ously described.[22a] The requisite “reverse” 5’-b-cyanoethyl phos-
phoramidites for thymidine (dT) and N,N-dimethylformamidine 2’-
deoxyguanosine (dGdmf) were obtained from Glen Research. Re-
verse phosphoramidite, N-acetyl 2’-deoxycytidine (dCAc) as well as
Universal UnyLinkerTM Support were purchased from Chemgenes.
All other commercially available oligonucleotide synthesis reagents
were purchased from Glen Research. HP Cyano RediSep Rf gold
chromatography column (15.5 g) was purchased from Teledyne
ISCO.
Preparation of 6-[2’-deoxy-3’-O-dimethoxytrityl-5’-O-tert-bu-
tyl(dimethyl)silyl-a/b-d-ribofuranose-1’-yl]amino-4-(diphe-
nylcarbamoyl)oxy-5-nitro-2-(phenoxyacetyl)aminopyrimidine
(14)
Compound 9[22b] (1 g, 1.2 mmol) was coevaporated with pyridine
(3ꢂ3 mL) and then dissolved in pyridine (5 mL). Diphenylcarbam-
oyl chloride (340 mg, 1.4 mmol. 1.2 equiv.) was added, followed by
Et3N (183 mg, 1.8 mmol) The reaction was stirred in the dark for
40 min and then partitioned into a mixture of 5% NaHCO3/ EtOAc
(1:1, 50 mL). The organic phase was washed with brine, dried over
Na2SO4, filtered and concentrated to dryness under vacuum. The
residue was purified by flash chromatography (Hexane/EtOAc=
4:1- 3:1) to provide 14 as a mixture of anomers (1 g, more polar
1
isomer/less polar isomer=5:1, 80%) as a foam. (The H NMR inte-
gration of the anomeric mixture follows. The integration is report-
ed such that a single proton for the major anomer is normalized to
1
1.) H NMR (400 MHz, CDCl3): d 9.20–9.22 (d, J=8 Hz, 1H), 8.66 (br
s, 1H), 6.81–7.46 (m, 35H), 6.34–6.36 (m, 0.2H), 6.17 (t, J=8 Hz,
1H), 4.77–4.79 (m, 2.4H), 4.33–4.35 (m, 1H), 4.27(s, 1H), 3.77 (s,
8H), 3.32–3.36 (m, 2.4H), 1.93 (m, 1H), 0.78 (s, 12H), À0.09–
À0.06(d, J=12 Hz, 8H). 13C-NMR (101 MHz, CDCl3): d 166.8, 161.6,
158.6, 157.5, 157.1, 155.3, 149.6, 145.0, 136.4, 136.2, 130.3, 130.2,
130.1, 129.8, 129.2, 128.3, 128.2, 128.1, 127.9, 126.9, 122.3, 116.9,
114.9, 114.8, 113.4, 113.3, 113.2, 88.0, 87.2, 83.2, 77.4, 77.3, 77.1,
76.8, 75.5, 68.1, 63.6, 55.2, 38.7, 31.6, 25.3, 20.7, 18.3, 1.0, À5.3,
À5.6. HRMS (ESI-TOF) m/z [M+Na]+ calcd for C57H60N6O11SiNa
1055.3982, found: 1055.3960.
Preparation of 2-deoxy-3,5-bis[O-(p-toluoyl)]-a,b-d-erythro-
pentofuranosyl azide (7)[31]
Preparation of 6-(2’-Deoxy-3’-O-dimethoxytrityl-a,b-d-ribo-
furanose-1’-yl)amino-4-(diphenylcarbamoyl)oxy-5-nitro-2-
(phenoxyacetyl)aminopyrimidine (15)
Hoffer’s chloro sugar[23] (6, 10 g, 25.7 mmol) was suspended in
DCM (50 mL) added with BF3·Et2O (360 mg, 2.6 mmol) and tri-
methylsilyl azide (3.8 g, 31 mmol) at 08C. After 30 min, the reaction
was brought to RT and stirred for 5 h. The reaction mixture was di-
luted with DCM (200 mL), washed with H2O, brine and dried over
Na2SO4. The organic solution was removed by evaporation and res-
idue containing both a- and b-anomers was directly used for next
step without purification. For characterization purposes, the mix-
ture was purified by flash chromatography (5% À10% EtOAc in
Hexane) to afford 7 (9 g, a/b=3:1, 70%) as a white solid. a-
anomer: 1H NMR (400 MHz, CDCl3) d 7.98 (d, J=8.4 Hz, 2H), 7.91
(d, J=8.4 Hz, 2H), 7.22–7.27 (m, 4H), 5.70–5.71 (m, 1H), 5.48–5.51
(m, 1H), 4.71 (m, 1H), 4.50–4.63 (m, 2H), 2.52–2.59 (m, 1H), 2.41 (d,
J=3.2 Hz, 6H), 2.21–2.42 (m, 1H); b-anomer: 1H NMR (400 MHz,
CDCl3) 7.99 (d, J=8.4 Hz, 2H), 7.89 (d, J=8.4 Hz, 2H), 7.20–7.24 (m,
4H), 5.70 (t, J=5.2 Hz, 1H), 5.56–5.59 (m, 1H), 4.52–4.60 (m, 3H),
2.38–2.42 (m, 8H). The spectra of the a- and b-isomers are consis-
tent with the reported data.
Compound 14 (870 mg, 0.84 mmol) was coevaporated with tolu-
ene/DCM (1:1, 2ꢂ2 mL) and then dissolved in THF (10 mL).
Et3N·3HF (1.5 mL, 8.4 mmol,) was added and the reaction was
stirred at room temperature overnight. The mixture was diluted
with ethyl acetate and was washed with saturated NaHCO3, and
brine. The organic layer was dried over Na2SO4, filtered and evapo-
rated to dryness under vacuum. The residue was purified by flash
chromatography (Hexane/EtOAc=2:1-1:1) to provide two isomers
of 15 (670 mg, 87%, more polar isomer/less polar isomer=4:1) as
1
a foam. Less polar anomer: H NMR (400 MHz, CDCl3) d 8.93–8.91
(d, J=8 Hz, 1H), 7.18 (br s, 1H), 6.81–7.46 (m, 28H), 6.34–6.36 (m,
1H), 4.80–4.81 (m, 2H), 4.35–4.36 (m, 1H), 4.11–4.13 (m, 1H), 3.94
(s, 6H), 3.15–3.53 (m, 2H). 13C NMR (101 MHz, CDCl3): d 167.2,
161.5, 158.7, 157.4, 155.4, 149.5, 145.3, 136.5, 136.4, 130.3, 130.2,
129.8, 129.3, 128.3, 128.0, 127.0, 122.2, 116.7, 114.9, 113.3, 87.1,
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86.2, 82.9, 77.5, 77.2, 76.8, 74.8, 68.2, 62.7, 55.2, 40.9, 21.1, 14.2;
More polar anomer 1H NMR (400 MHz, CDCl3) d 9.23 (d, J=8 Hz,
1H), 8.77 (br s, 1H), 6.82–7.46 (m, 28H), 6.24 (t, J=7.2 Hz, 1H),
4.67–4.77 (m, 2H), 4.32 (d, J=6.8 Hz, 1H), 4.23 (s, 1H), 3.77 (s, 6H),
3.21–3.53 (m, 2H), 1.88–1.91 (m, 1H). 13C NMR (101 MHz, CDCl3) d
176.0, 172.0, 166.3, 163.5, 162.5, 161.8, 160.1, 154.4, 149.7, 141.1,
140.9, 135.0, 134.9, 134.5, 133.0, 132.9, 131.8, 127.1, 121.7, 119.6,
118.2, 91.8, 87.9, 82.3, 81.9, 81.6, 80.3, 72.8, 67.5, 65.2, 60.0, 43.9,
25.8, 19.0. HRMS (ESI-TOF) m/z [M+Na]+ calcd for C51H46N6NaO11
941.3117, found: 941.3099 (less polar), 941.3095 (more polar).
(162 MHz, CD3CN) d 148.92, 148.60. More polar isomer: 1H NMR
(400 MHz, CD3CN) d 7.66 (s, 0.7H), 7.46–7.51 (m, 2H), 7.30–7.40 (m,
10H), 7.21–7.26 (m, 2H), 7.00–7.05 (m, 3H), 6.87–6.90 (m, 5H),
6.36–6.41 (m, 1H), 5.93–6.01 (m, 1H), 4.79 (s, 2H), 4.19–4.31 (m,
2H), 3.77 (s, 6H), 3.32–3.51 (m, 4H), 3.21–3.27 (m, 3H), 2.76–2.84
(m, 1H), 1.69–1.81 (m, 1H), 0.98–1.12 (m, 12H). 31P NMR (162 MHz,
CD3CN) d 148.86, 148.64. HRMS (ESI-TOF) m/z [M+H]+ calcd for
C40H43N5O11P 800.2691, found: 800.2684 (less polar isomer),
800.2678 (more polar isomer). (Please note that the mass corre-
sponds to the hydrolyzed organophosphite product)
Oligonucleotide synthesis, deprotection, and characteriza-
tion
Preparation of 6-(2’-deoxy-3’-O-dimethoxytrityl-a,b-d-ribo-
furanose-1’-yl)amino-4-(diphenylcarbamoyl)oxy-5-nitro-2-
(phenoxyacetyl)aminopyrimidine 5’-methyl-N,N’-diisopropyl
phosphoramidite (16)
The incorporation of Fapy·dG (1) into oligonucleotides with reverse
b-cyanoethyl phosphoramidites was carried out on 1 mmol scale
using 4,5-dicyanoimidazole (0.25m in acetonitrile) as activating
agent. Acetonitrile solutions of phosphoramidites synthesized in
our laboratory (1, dAPac) were filtered using 0.45 mm syringe filters.
The coupling of 1 is extremely sensitive to water. Thus, molecular
sieves were added to the solution after filtering and kept in the
bottle throughout oligonucleotide synthesis. The wait time for the
coupling of Fapy·dG phosphoramidite was extended to 900 s,
while for other phosphoramidites the wait time was 180 s. After
coupling 1, commercially available phenoxyacetic anhydride (Cap
A mix) and N-methyl imidazole (Cap B mix) solutions were replaced
by pivalic anhydride/2, 6-lutine/THF (1:1:8, v/v/v). Capping was car-
ried out 60 s at all steps during oligonucleotide synthesis. tBuOOH
(1m in toluene) was used for oxidation (40 s). Following solid
phase synthesis, the methyl group was removed from the Fapy·dG
phosphate triester using disodium 2-carbamoyl-2-cyanoethylene-
1,1-dithiolate trihydrate (0.2m in DMF, 0.3 mL) for 30 min at room
temperature.[22a,b] The resin was then rinsed successively with
MeOH, THF and CH2Cl2, vacuum dried and treated with aq. NaOH
(0.1m) at room temperature for 12 h. The deprotection time was
extended to 16 h for oligonucleotides containing large numbers of
dG (e.g. 28). The solution was neutralized (per pH paper) with
AcOH (5% in MeOH, ꢀ0.5 vol. of the NaOH solution), concentrated
to dryness and purified using 20% denaturing polyacrylamide gel
electrophoresis. Alternatively, the resin was incubated with tBuNH2/
H2O (1:3, v/v) at 408C for 8 h and neutralized (per pH paper) with
glacial acetic acid (ꢀ0.1 vol. of the tBuNH2 solution). The isolated
oligonucleotides were characterized by MALDI-TOF MS or ESI-MS
(Figures S1–S13).
The major isomer of compound 15 (270 mg, 0.29 mmol) was co-
evaporated with toluene (2ꢂ10 mL) and dried in vacuo for 2 h
before it was dissolved DCM (5 mL) at 08C. DIPEA (185 mg,
1.5 mmol) was then added, followed by diisopropylmethylphos-
phonamidic chloride (110 mg, 0.53 mmol). After 10 min, the reac-
tion was brought to room temperature and stirred for 1.5 h, at
which time it was diluted with DCM (2 mL). The resulting solution
was washed with 5% NaHCO3 and brine, dried over Na2SO4, filtered
and concentrated to dryness under vacuum. The residue was puri-
fied by flash chromatography (Hexane/EtOAc=2:1) to provide 16
1
(250 mg, 80%) as a foam. H NMR (400 MHz, CDCl3): d 9.20–9.22 (d,
J=8 Hz, 1H), 6.82–7.38 (m, 28H), 6.16–6.22 (m, 1H), 4.77–4.80 (d,
J=8 Hz, 2H), 4.43–4.37 (dd, J=16, 8 Hz, 1H), 4.33–4.28 (m, 1H),
3.74 (s, 6H), 3.42–3.46 (m, 2H), 3.26–3.31 (m, 3H), 1.12–1.13 (m,
14H). 31P NMR (162 MHz, CDCl3): d 149.0, 149.1. HRMS (ESI-TOF)
m/z [M+H]+ calcd for C58H63N7O12P 1080.4267, found: 1080.4252.
Preparation of 6-(2’-deoxy-3’-O-dimethoxytrityl-a,b-d-ribo-
furanose-1’-yl)amino-5-formamido-2-(phenoxyacetyl)amino
pyrimidine-3H-4-one 5’-methyl-N,N’-diisopropyl phosphor-
amidite (1)
Phosphoramidite 16 (200 mg, 0.17 mmol), DIPEA (180 mg,
1.3 mmol) and 10% palladium on carbon (160 mg) in ethyl acetate
(4 mL) were placed in a pressure tube. The reaction mixture was
pressurized with H2 and vented ten times and then stirred under
H2 (60 psi) for 3 h. The reaction atmosphere is exchanged with Ar
by freeze-pump-thaw degassing. The solution was cooled to 08C.
Pivalic formic anhydride (50 mg, 0.36 mmol) was added and the re-
action mixture was stirred at 08C for 1 h. The reaction mixture was
diluted with 5 mL of EtOAc and centrifuged at 10000 RPM for
3 min. The solution was carefully removed and the solid was
washed with 5 mL EtOAc. After centrifuging, the EtOAc solutions
were combined, washed with 5% NaHCO3, brine and dried over
Na2SO4. The solution was filtered and evaporated to dryness under
vacuum. The residue was purified by flash chromatography using
DCM/Acetone=5:1 and 0.1% Et3N until the less polar isomer
eluted, then 2:1 DCM/Acetone to elute the more polar isomer to
provide the individual anomers of 1 (90 mg, 55%, more polar/less
polar=2:1). Note: Because of tailing, fractions eluted from the
column were dilute. Column fractions (ꢀ6 mL) were concentrated
to ꢀ1 mL prior to TLC analysis. Less polar isomer: 1H NMR
(400 MHz, CD3CN) d 8.65–8.69 (m, 1H), 8.55–8.58 (m, 1H), 7.44–
7.46 (m, 2H), 7.33–7.36 (m, 10H), 7.24–7.27 (m, 1H), 7.00–7.05 (m,
3H), 6.89 (d, J=8.4 Hz, 5H), 6.03–6.04 (m, 1H), 5.96 (q, J=8.4 Hz,
1H), 4.72 (s, 2H), 4.24–4.30 (m, 1H), 4.12 (,m, 1H), 3.78 (s, 6H),
3.32–3.49 (m, 4H), 1.72–1.84 (m, 2H), 0.98–1.10 (m, 12H), 31P NMR
Acknowledgements
We are grateful for support of this research from the National
Institute of Environmental Health Studies (ES-027558). We
thank Professor Carmelo Rizzo for helpful discussions and Pro-
fessor Rebekka Klausen for providing access to the Combiflash
Rf + apparatus.
Conflict of interest
The authors declare no conflict of interest.
Keywords: DNA damage · DNA oxidation · DNA repair ·
formamidopyrimidines · oligonucleotide synthesis
Chem. Eur. J. 2020, 26, 1 – 9
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Manuscript received: December 23, 2019
Version of record online: && &&, 0000
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FULL PAPER
Biochemical, cellular and structural
studies on Fapy·dG have been ham-
pered due to difficulties in synthesizing
oligonucleotides containing this lesion.
A robust method for synthesizing oligo-
nucleotides containing Fapy·dG is re-
ported that will greatly facilitate investi-
gations on the biological and structural
effects of Fapy·dG (see scheme).
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Oligonucleotide Synthesis
H. Yang, J. A. Tang, M. M. Greenberg*
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Synthesis of Oligonucleotides
Containing the N6-(2-Deoxy-a,b-d-
erythropentofuranosyl)-2,6-diamino-4-
hydroxy-5-formamidopyrimidine
(Fapy·dG) Oxidative Damage Product
Derived from 2’-Deoxyguanosine
Chem. Eur. J. 2020, 26, 1 – 9
www.chemeurj.org
9
ꢀ 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers! ÞÞ