Independent Generation and Reactivity of Thymidine Radical Cations

Article abstract of DOI:10.1021/acs.joc.7b02017

Thymidine radical cation (1) is produced by ionizing radiation and has been invoked as an intermediate in electron transfer in DNA. Previous studies on its structure and reactivity have utilized thymidine as a precursor, which limits quantitative product analysis because thymidine is readily reformed from 1. In this investigation, radical cation 1 is independently generated via β-heterolysis of a pyrimidine radical generated photochemically from an aryl sulfide. Thymidine is the major product (33%) from 1 at pH 7.2. Diastereomeric mixtures of thymidine glycol and the corresponding 5-hydroxperoxides resulting from water trapping of 1 are formed. Significantly lower yields of products such as 5-formyl-2′-deoxyuridine that are ascribable to deprotonation from the C5-methyl group of 1 are observed. Independent generation of the N3-methyl analogue of 1 (NMe-1) produces considerably higher yields of products derived from water trapping, and these products are formed in much higher yields than those attributable to the C5-methyl group deprotonation in NMe-1. N3-Methyl-thymidine is, however, the major product and is produced in as high as 70% yield when the radical cation is produced in the presence of excess thiol. The effects of exogenous reagents on product distributions are consistent with the formation of diffusively free radical cations (1, NMe-1). This method should be compatible with producing radical cations at defined positions within DNA.

Full text of DOI:10.1021/acs.joc.7b02017

Article
pubs.acs.org/joc
Independent Generation and Reactivity of Thymidine Radical Cations
Huabing Sun, Marisa L. Taverna Porro,† and Marc M. Greenberg*
Department of Chemistry, Johns Hopkins University, 3400 N. Charles St., Baltimore, Maryland 21218, United States
S
* Supporting Information
ABSTRACT: Thymidine radical cation (1) is produced by ionizing radiation and has been invoked as an intermediate in
electron transfer in DNA. Previous studies on its structure and reactivity have utilized thymidine as a precursor, which limits
quantitative product analysis because thymidine is readily reformed from 1. In this investigation, radical cation 1 is independently
generated via β-heterolysis of a pyrimidine radical generated photochemically from an aryl sulfide. Thymidine is the major
product (33%) from 1 at pH 7.2. Diastereomeric mixtures of thymidine glycol and the corresponding 5-hydroxperoxides
resulting from water trapping of 1 are formed. Significantly lower yields of products such as 5-formyl-2′-deoxyuridine that are
ascribable to deprotonation from the C5-methyl group of 1 are observed. Independent generation of the N3-methyl analogue of
1 (NMe-1) produces considerably higher yields of products derived from water trapping, and these products are formed in much
higher yields than those attributable to the C5-methyl group deprotonation in NMe-1. N3-Methyl-thymidine is, however, the
major product and is produced in as high as 70% yield when the radical cation is produced in the presence of excess thiol. The
effects of exogenous reagents on product distributions are consistent with the formation of diffusively free radical cations (1,
NMe-1). This method should be compatible with producing radical cations at defined positions within DNA.
Thymidine (1) and N1-methylthymine (2) radical cations
have been generated by two photon photoionization (248 nm)
or γ-radiolysis in solid matrices.7,8,11 The products charac-
terized from γ-radiolysis in solid matrices are indicative of the
O2 deficient conditions in which the experiments were carried
out. Although it is not mentioned, product mixtures formed
from photolysis at 248 nm may be influenced by the short
irradiation wavelength. This issue is avoided by using excited
2-methyl-1,4-napththoquinone to generate 1 via photoinduced
single electron transfer.6 In yet another approach, pulse
radiolysis yields the radical cations following the generation
of a sulfate radical anion from persulfate.4 The sulfate radical
anion yields the radical cation(s) via addition, followed by
sulfate elimination. These pulse radiolysis experiments reveal
that the radical cations react on the microsecond time scale.4
Four major reaction pathways are open to monomeric 1
(Scheme 1). Hydration, C5-methyl deprotonation, and single
electron transfer are possible from NMe-1, but not N3-
deprotonation. Spectroscopic measurements indicating that
N3-deprotonation of 1 was more rapid than hydration or C5-
methyl deprotonation was inconsistent with the aforemen-
tioned photoionization study in which the latter reaction was
the major process detected.4,6 Furthermore, N3-methyl-
deprotonation, but not C5-methyl-deprotonation, was detected
in a time-resolved EPR investigation in which 2 was generated
INTRODUCTION
■
Nucleoside oxidation is a chemical process with important
consequences on human health and is a useful biotechnology
tool. One electron oxidation of the nucleobase is a general
pathway, and much attention has been paid to 2′-
deoxyguanosine reactivity in this manner because it is the
most readily oxidized of the four native nucleosides.1,2
However, some of the most common oxidizing agents such
as γ-radiolysis and photoionization are unselective and ionize
pyrimidines in addition to purines.3 Thymidine radical cation
(1) is formed by ionizing radiation and chemical oxidants (e.g.,
sulfate radical anion and photosensitization).4−8 Radical cation
1 has also been invoked as an important intermediate in DNA
electron transfer within A·T rich substrates.9,10 The structure
and reactivity of 1, its N3-methyl analogue (NMe-1), and 2
have been examined via EPR, product analysis, and pulse
radiolysis.11 We wish to report a method for generating 1 and
NMe-1 for the first time from a photochemical precursor other
than a pyrimidine. This approach is compatible with
biologically relevant conditions and, in contrast to when
using pyrimidines themselves as precursors, enables us to
quantify the products from all major reaction pathways.
Received: August 10, 2017
© XXXX American Chemical Society
A
DOI: 10.1021/acs.joc.7b02017
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
researchers used an alkoxyl group as the leaving group, and
radical cation yields were low. This strategy was also attractive
to us because of our long-term aspiration of applying this
chemistry to oligonucleotides. Phosphate triesters, such as
those present in 6, should be compatible with solid-phase
oligonucleotide synthesis. We postulated that 6 and NMe-6
would serve as precursors to 1 and NMe-1, although the
respective carbocations (9) may also be produced to some
extent upon 350 nm photolysis.14 A benzoyl group was
incorporated at the 5′-position to provide a suitable
chromophore for UPLC detection of photolysis products by
UV−vis absorption. (Please note that for convenience
benzoylated molecules (e.g., 1) are referred to by the same
number as the corresponding nonbenzoylated molecules.)
Radical Cation Precursor Synthesis and Stability. The
phosphorylated aryl sulfides were synthesized in a direct
manner from the previously reported sulfide (10, Scheme 3).14
Scheme 1. Formation and Reactivity of the Thymidine
Radical Cation
from anthraquinone-2,6-disulfonic acid sensitized ionization.5
However, in a different matrix isolation investigation Adhikary
and Sevilla observed that C5-deprotonation occurred prefer-
entially over hydration.11 In addition to these discrepancies,
product studies are compromised because the radical cations
are generated from the nucleobase in each of these
investigations. Radical cation generation from thymidine
prevents detecting one electron reduction, which regenerates
the starting material. The method described herein for
independently generating 1 and NMe-1 from dihydropyr-
imidine precursors does not face this limitation.
a
Scheme 3. Syntheses of Radical Cation Precursors
RESULTS AND DISCUSSION
■
A number of photochemical precursors have been reported for
nucleobase radicals resulting from a hydroxyl radical (HO•) or
hydrogen atom addition to pyrimidine nucleosides.12 Aryl
sulfides have served as useful precursors to HO• radical
adducts.13−16 We envisioned capitalizing on these molecules by
conjoining them with the chemistry largely developed by Crich
and Newcomb to generate thymidine radical cations (1, NMe-
1, Scheme 2).17−20 These investigators established that
appropriately substituted alkyl radicals (7) generate diffusively
free alkene radical cations (1) via β-heterolysis of a
phosphatoxyl group. Formation of the diffusively free radical
cation from the ion pair (8) is facilitated by polar solvents and
strongly influenced by the leaving groups. Crich and Newcomb
made extensive use of phosphate diesters as leaving groups.
Nishimoto and co-workers also employed this approach to
produce thymi(di)ne radical cations.21 However, these
a
Key: (a) (BnO)2P(NiPr2) and S-ethyltetrazole then t-BuOOH, (b)
Et3N·3HF, (c) BzCN, (d) Mel, (e) BzCl.
Phosphorylation using chlorophosphate reagents was unsuc-
cessful. However, more electrophilic P(III) reagents reacted
with the tertiary alcohol, and in situ oxidation with t-BuOOH
provided the desired phosphorylated protected nucleoside
(11). Phosphate triester 11 was carried on to radical cation
precursors for 1 and NMe-1. Methylation was carried out
under mild alkaline conditions using iodomethane. Both 11
and 12 were desilylated and 5′-benzyoylated. Selectivity for the
primary alcohol was not very good. Consequently, benzoyla-
Scheme 2. Photochemical Radical Cation Generation
B
DOI: 10.1021/acs.joc.7b02017
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
tion reactions were carried out to ∼40% conversion to
minimize benzoylation at the 3′- or 3′- and 5′-positions. The
reported yields are based on unrecovered starting material.
Furthermore, in our hands, BzCN was preferred when
preparing 6, but BzCl provided a higher yield of NMe-6.
The half-life of desilylated 11 at 25 °C in a 1:1 mixture of
phosphate buffered (100 mM, pH 6.6) saline (1 M) and
CD3OD was monitored by 31P NMR (Figure 1) and
determined to be 16.2 days. UPLC-MS indicated that it
decomposed by losing a single benzyl group (Figure S1).
Scheme 5. Anticipated Products following C5-
Deprotonation from the Radical Cation
The three products typically associated with C5-methyl
deprotonation of 1 are derived from O2 trapping of 4. The
corresponding hydroperoxides (15, NMe-15) were not
detected by LC-MS, but their hydrolysis products (16, NMe-
16) and reduction product (17, NMe-17) were observed. We
employed an approach developed by Sugiyama and Saito for
preparing oligonucleotides containing 5-formyl-2′-deoxyuridine
in which the aldehyde group is introduced via Pd(0) coupling
to suitably protected 5-iodo-2′-deoxyuridine (Scheme 6).28
Stille coupling reactions were carried out on 5′-benzoyl-5-iodo-
2′-deoxyuridine (18)29 or NMe-18. The latter was prepared by
benzoylating 5-iodo-3-methyl-2′-doxyuridine.30 The vinyl
substituted nucleosides were converted to the aldehyde
products using a one-pot osmylation, periodate oxidation
procedure.31 During the course of the UPLC product analyses
discussed below, we determined that 16 was inseparable from
the internal standard (5′-benzoyl-2′-deoxyuridine, BzU).
Consequently, 16 was quantified as its oxime (20), which
was prepared quantitatively from the aldehyde. The hydrox-
ymethyl nucleosides (17, NMe-17, Scheme 5) were synthe-
sized from the corresponding protected thymidines, following
silyl protection of the 3′-hydroxyl groups (21, NMe-21,
Scheme 7). The hydroxymethyl groups (22, NMe-22) were
introduced via allylic bromination, followed by hydrolysis of
the crude material.32,33
Figure 1. Decomposition of desilylated 11 (X) in CD3OD/phosphate
buffered saline (v/v = 1:1) monitored by 31P NMR.
Independent Synthesis and Analysis of Anticipated
Photochemical Products. The identities of products (and
secondary products) from 1 were established in previous
studies.6,22−24 The syntheses described here are adaptations of
known methods to accommodate the N3-methyl group and/or
the 5′-benzoyl group. Reduction of the radical cations either
directly or following N3-deprotonation of 1 yields the
corresponding thymidine derivative.25 Thymidine glycols are
the most stable products resulting from hydration of 1 and
NMe-1 (Scheme 4). Thymidine hydroperoxides (13, NMe-13)
are unstable; consequently, we anticipated characterizing them
via LC-MS and by taking advantage of their reduction to
thymidine glycols (14, NMe-14). Thymidine glycol 14 was
previously reported, but NMe-14 was obtained by osmylation
of 5′-benzoylated NMe-T in 39% yield as a mixture of two
14
1
isomers in an approximately 92:8 ratio (by H NMR). On
the basis of previous work, the major isomer is ascribed to
5R,6S-NMe-14 and the minor isomer is believed to be 5S,6R-
NMe-14.14,26,27 For quantitative studies, UPLC response
factors were measured for 14 and 5R,6S-NMe-14 using 5′-
benzoyl-2′-deoxyuridine (BzU) as an internal standard. We
used these response factors to approximate those of the other
thymidine glycol diastereomers, as well as the respective
dihydrothymidine hydroperoxides (13, NMe-13) (Figures S3
and S4).
Abasic sites have been reported to be produced from the 2′-
deoxycytidine radical cation but not 1 or NMe-1.34 However,
carbocations formed upon photolysis of the aryl sulfide
precursors are potential alternative sources of abasic sites
(Scheme 2).15 5-Benzoyl-2-deoxyribose (25) was synthesized
Scheme 4. Anticipated Products following Radical Cation Hydration
C
DOI: 10.1021/acs.joc.7b02017
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
a
Scheme 6. Syntheses of the Aldehyde Products and Oxime Derivative
a
Key: (a) Tributyl(vinyl)stannane and Pd(0), (b) OsO4 and NalO4, (c) MeONH2·HCI.
a
Scheme 7. Syntheses of the 5-Hydroxymethyl-2′-deoxyuridine Products
a
Key: (a) TBDMSCI, (b) NBS and AIBN then aq NaHCO3, (c) Et3N·3HF.
from 23 (Scheme 8) but was inseparable from other products
in the photolysates in which 1 was generated.35 Consequently,
photolysis mixtures contained 50% CH3CN by volume due to
solubility limitations of 6. Consequently, photolyses were
carried out in H2O/CH3CN (1:1 v/v), aqueous phosphate
buffer (pH 7.2, 20 mM)/CH3CN (1:1 v/v), or Chelex treated
citrate buffer (pH 5.0, 40 mM)/CH3CN (1:1 v/v). Chelex
treatment was carried out to test for possible trace metal
effects, and the purpose of the lower pH was to carry out
reactions closer to the pKa (3.9) of radical cation 1. Mass
balances ranged from 48−63%. AP sites (26) were formed in
minor amounts. Although it is uncertain, we suggest that they
result from the carbocation (9) that is formed either directly
via heterolysis or as a result of electron transfer within the
radical cage. Products (13, 14, and 27) resulting from
hydration of the pyrimidine π-bond were detected in ∼10%
combined yield in H2O and pH 7.2 buffer. Hydroperoxide 13
was identified by LC-MS, and its reactivity was previously well
characterized by Wagner and Cadet (Figure S2).22 For UPLC
quantitation of 13, the response factor of independently
synthesized 14 versus BzU was used (Figure S4). Product 27
was identified by LC-MS and was consistent with the known
degradation products of thymidine glycol hydroperoxides.22
The response factor of structurally similar 26 was used for
quantifying this molecule by UPLC (Figure S3). Higher yields
of hydration products were detected at a pH of 7.2 than at 5.0,
consistent with a greater concentration of the hydroxide ion.
Products consistent with methyl group deprotonation (16, 17)
of 1 were also detected, but their yields were consistently
lower than those of hydration products.
Scheme 8. Syntheses of 5-Benzoyl-2-deozyribose and Its O-
a
Methyl Oxime
a
Key: (a) BzCI, (b) AcCI, H2O, and CH3CN, (c) MeONH2·HCI.
the respective O-methyl oxime (26) obtained upon reaction of
25 was used to quantify the abasic site formation when 6 was
photolyzed. Methoxyamine is commonly used to functionalize
abasic sites in DNA and prevent their repair.36,37 UPLC
analysis indicated that the reaction of 25 with methoxyamine
hydrochloride yielded 26 (which was independently synthe-
sized from 2-deoxyribose) in 98% yield, indicating that this
method would be suitable for quantifying abasic sites formed
in the photolysis experiments.
Photochemical Generation and Reactivity of 1.
Precursor 6 was photolyzed under aerobic conditions at 350
nm for 0.5 h in three different solvent mixtures (Table 1). All
Table 1. Product Yields from Photolysis of Radical Cation Precursor 6
a
% yield
b
conditions
H2O
13
14
27
16
17
26
T
6
mass balance
3.4 0.6
0.6 0.1
1.5 0.3
4.8 0.1
3.7 0.1
6.7 0.9
4.3 1.1
3.6 0.1
3.2 0.1
2.0 0.1
1.2 0.0
3.9 0.1
1.7 0.3
4.0 0.1
4.4 0.1
30.1 0.5
42.5 0.2
27.3 0.3
36.1 0.8
6.8 0.9
9.2 3.5
5.2 0.5
5.6 0.5
54.8 0.7
63.0 4.8
47.9 2.3
58.1 0.5
c
BME
pH 5.0 (Chelex)
pH 7.2
2.1 0.5
1.9 0.1
2.1 0.2
1.1 0.1
3.6 0.0
1.0 0.1
a
b
Yields and mass balances are the average standard deviation of at least 3 measurements. All experiments were carried out in a 1:1 (v/v) mixture
c
of CH3CN and the solvent listed. [BME] = 10 mM.
D
DOI: 10.1021/acs.joc.7b02017
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
product formation. Hydration of NMe-1 is expected to occur
at C6 (Scheme 1), followed by O2 trapping of the C5-radical
(NMe-5, Scheme 4). Photolysis in H216O/16O2 produced
products with m/z = 433.1207 and 417.1273 corresponding to
the molecular ions for NMe-13 (Figure 2A) and NMe-14
(Figure 3), respectively. Fragmentation within the MS
supported the regioisomeric assignment shown for NMe-13
(Figure 2). The molecular ion fragments released the C5-
carbon containing the methyl group and, importantly, the
hydroperoxide component. When H218O is present during
photolysis (Figure 2B), the observed parent ion increases by 2
Da and the 18O is retained upon fragmentation. In contrast,
when 18O2 is employed (Figure 2C), the parent ion of NMe-
13 increases by 4 Da, as expected for trapping of NMe-5
(Scheme 4), but both 18O atoms are lost upon fragmentation.
The labeling pattern is consistent with the anticipated C6-
hydration, followed by O2 trapping.
Similar analyses were carried out for the thymidine glycol
(NMe-14) formed upon photolysis of NMe-6 (Figure 3),
which was expected to result from reduction of the
hydroperoxide (Scheme 4). The glycol molecular ion increases
by 2 Da when either H218O (Figure 3B) is substituted for H2O
or 18O2 (Figure 3C) is bubbled through the solution, prior to
sealing and photolysis. These observations are consistent with
the aforementioned observations concerning the hydroper-
oxide (NMe-13, Figure 2).
5-Formyl-2′-deoxyuridine (NMe-16) was attributed to C5-
deprotonation from NMe-1, followed by O2 trapping of NMe-
4 to form the peroxyl radical, which is ultimately transformed
into the aldehyde via hydrolysis of the hydroperoxide (NMe-
15, Scheme 5). However, LC-MS analysis of 18O-labeling
experiments (Figure 4) suggests that the formal reduction
product, 5-hydroxymethyl-2′-deoxyuridine (NMe-17), is
formed by two pathways. Isotopic incorporation (m/z ∼
401.12) is observed when either H218O (Figure 4B) or 18O2
(Figure 4C) is employed. The latter is consistent with the
radical pathway, but the incorporation of 18O from H218O is
indicative of nucleophilic substitution. One explanation for the
formation of NMe-17 (Scheme 9) that is consistent with
literature precedent on related molecules involves initial
formation of the carbocation NMe-9 (Scheme 2).14,15,38−42
Phosphate rearrangement, followed by deprotonation, produ-
ces an allylic species analogous to others that undergo
nucleophilic substitution by H2O.
Thymidine was the major product under all conditions, and
its yield increased from 30 to >42% when β-mercaptoethanol
(BME, 10 mM) was included in the photolysis mixture. The
presence of BME also resulted in an increase in yields of
thymidine glycol(s) (14) and 5-hydroxymethyl-2′-deoxyuridine
(17) at the expense of the corresponding thymidine glycol
hydroperoxides (13) and 5-formyl-2′-deoxyuridine (16), a
hydrolysis product of hydroperoxide 15. However, the overall
yields of these products were unaffected by 10 mM BME,
indicating that the thiol did not compete with H2O,
deprotonation, or alkyl radical trapping by O2.
Photochemical Generation and Reactivity of NMe-1.
Mass balances from photolysis of NMe-6 (Table 2) were
typically higher than those determined from 6 and approached
100% when carried out in the presence of BME (10 mM)
(Figure S5). In the absence of thiol (BME), higher yields of
products attributable to H2O trapping of NMe-1 (NMe-13,
NMe-14, 27, 28) accounted for most of the increased mass
balance. In contrast to trapping studies on 1, the combined
yield of water trapping products of NMe-1 were greater than
that of NMe-T under all photolysis conditions except pH 5.0.
Increases in the yields of water trapping products of NMe-1
account for higher mass balances at pH 7.2 and 8.0 than at pH
5.0. We attribute higher relative yields of NMe-1 hydration
products relative to NMe-T to the inability of the radical
cation to yield thymidine via N3-deprotonation, which
produces 3 from 1 (Scheme 1). The elimination of an
intramolecular pathway for the radical cation also explains why
the presence of BME (10 mM) during photolysis has a greater
effect on the thymidine yield from NMe-1 than from 1.
Although the thymidine glycol (NMe-14) analogue was
independently synthesized, the respective hydroperoxides
(NMe-13) and fragmentation products (27, 28) were initially
identified by LC-MS and comparison to the thorough reports
by Wagner and Cadet on the hydroperoxides (Figure S2).22,24
The hydroperoxide assignment is also consistent with
observations made upon treatment of the photolysate with
BME, which decreased the amount of the product assigned to
NMe-13 and increased the yield of NMe-14 (Table 2). 18O-
Isotopic labeling and MS/MS analysis provided additional
structural support and mechanistic information regarding
Overall, the competition (Scheme 1) between the C5-
methyl deprotonation of NMe-1 to yield NMe-4 and hydration
products (NMe-5) is strongly in favor of the latter, consistent
with what is observed for 1. Hydration of NMe-1 is favored
Table 2. Product Yields from Photolysis of Radical Cation Precursor NMe-6
a
% yield
b
conditions
NMe-13
NMe-14
27
28
NMe-16
NMe-17
NMe-T
NMe-6
mass balance
H2O
15.5 1.7
3.8 2.6
4.4 3.7
9.0 0.1
9.5 0.4
20.8 1.9
18.0 0.3
7.7 1.1
13.2 3.5
17.8 0.6
6.5 0.3
6.8 0.1
10.6 1.1
11.2 0.4
7.2 0.2
2.6 0.5
8.4 0.8
3.1 0.2
3.3 0.1
3.3 0.4
3.4 0.3
4.8 1.1
1.8 0.6
5.3 0.6
6.1 0.1
6.4 0.2
3.0 0.5
2.8 0.2
5.4 0.5
1.6 0.4
7.0 0.6
3.8 0.1
3.9 0.1
4.3 0.3
2.4 0.1
3.5 0.3
1.5 0.2
4.5 0.6
3.2 0.2
3.6 0.4
5.9 0.1
6.2 0.5
33.4 0.3
71.4 1.1
33.4 0.3
28.1 0.1
28.6 0.5
32.5 0.4
32.7 0.2
7.3 6.5
6.5 6.7
2.8 0.4
7.6 6.9
7.4 7.1
8.1 7.0
8.8 7.7
84.8 3.0
102.4 7.2
83.7 1.4
67.3 6.3
69.3 6.0
88.5 5.8
85.5 7.0
c
BME
d
BME (after hν)
pH 5.0 (Chelex)
pH 5.0
pH 7.2
pH 8.0
a
b
Yields and mass balances are the average standard deviation of at least 3 measurements. All experiments were carried out in a 1:1 (v/v) mixture
c
d
of CH3CN and the solvent listed. [BME] = 10 mM. [BME] = 100 mM at 37 °C for 1 h.
E
DOI: 10.1021/acs.joc.7b02017
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
Figure 2. ESI-MS spectra of obtained NMe-13 from photolysis of NMe-6 in the presence of (A) H2O/O2, (B) H218O/O2, and H2O/18O2.
Figure 3. ESI-MS spectra of obtained NMe-14 from photolysis of NMe-6 in the presence of (A) H2O/O2, (B) H218O/O2, and H2O/18O2.
Figure 4. ESI-MS spectra of NMe-17 from photolysis of NMe-6 in the presence of H2O/H218O and O2/18O2.
Scheme 9. Possible Non-Radical Cation Mechanism for the Formation of NMe-17
over methyl group deprotonation by at least 5-fold over a
range of pHs and almost 15-fold at pH 8.
yields of NMe-T were greater than 60% in the presence of
either BME or KI and approached 70% when using the latter.
The varying effects of different reductants on the NMe-T
yield support the proposal that freely diffusible NMe-1 is
produced in these reactions. In previous studies, the formation
of freely diffusible olefin radical cations by β-heterolysis of
radicals was dependent upon the radical substituents, the
leaving group in the β-position, and solvent polarity.17−20 The
ability of exogenous reductants to increase the NMe-T yields
suggests that they are reacting with freely diffusible NMe-1.
Further evidence for trapping freely diffusible NMe-1 was
Radical Cation Reduction. A variety of reductants (10
mM) were screened for their ability to reduce NMe-1 (Figure
5 and Table S1). The reaction of NMe-1 with each of these
molecules was expected to be thermodynamically favorable.
Increased yields of the thymidine analogue (NMe-T) were
observed when any one of the reductants in Figure 5 was
present during photolysis. Of the reductants screened, BME
and KI were the most effective at reducing NMe-1.43 The
F
DOI: 10.1021/acs.joc.7b02017
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
could represent product formation that occurs within the ion
pair (NMe-8, Scheme 2).
CONCLUSIONS
■
Thymidine (1) and N3-methyl thymidine (NMe-1) radical
cations were independently generated from precursors other
than (N3-methyl) thymidine for the first time. This enabled us
to more completely determine the product distributions from
the radical cations because their facile reduction produces
thymidine. (N3-Methyl) Thymidine can be produced via direct
reduction (e.g., by thiol) of the corresponding radical cation.
An additional pathway is available to the radical cation derived
from the native nucleoside (1). N3-Deprotonation (3, Scheme
1), followed by formal one electron reduction and protonation,
also yields thymidine. Products resulting from hydration are
formed in the greatest yields from the N3-methylated radical
cation. 5-Formyl-2′-deoxyuridine (NMe-16) resulting from
deprotonation of the methyl group of NMe-1 is formed in a
considerably lower yield over the entire pH range (5−8)
examined. 18O-Labeling studies indicate that the corresponding
5-hydroxymethyl product (NMe-17), which is also formed in a
relatively low yield, arises from the radical cation and via a
second pathway, possibly the carbocation (Schemes 2 and 9).
We find that N3-methyl thymidine is a major product from
NMe-1 and is the major product when suitable reducing
agents, including the biologically relevant thiol or ferrous
agent, are present.
Figure 5. Effect of reductant (10 mM) on the yield of NMe-T from
photolysis of NMe-6. DMS = dimethyl sulfide. TCEP = tris-
(carboxyethyl)phosphine.
gleaned from the examination of the competition of the
reducing agent for NMe-1 with hydration (NMe-13, NMe-14,
27, 28) and deprotonation pathways (NMe-16). NMe-17 is
not included in this analysis because we believe it is produced
from two pathways (per above). Furthermore, given that
NMe-17 is formed in a low yield, we do not believe that it
significantly affects the conclusions drawn from Figure 6. The
two most effective nucleophiles, BME (Figure 6A) and KI
(Figure 6B), were employed. In each instance, the combined
yields of trapping products attributable to hydration and C5-
methyl group deprotonation approached similar limiting
Thymidine is the major product formed upon generation of
1, even in the absence of excess exogenous reducing agent,
presumably via deprotonation from N3 of the radical cation
(Scheme 1). The resulting N3-radical (3) could yield
thymidine by either hydrogen atom abstraction or sequential
reduction and protonation. Product analysis indicates that
deprotonation from N3 is faster than hydration or
deprotonation from the C5-methyl group. Although pulse
radiolysis and anthraquinone photosensitization experiments
indicate that N3-deprotonation of 1 is rapid,4,5 the pathway
was not detectable in product studies where thymidine was the
source of the radical cation.6 Generation of 1 (and NMe-1) in
our hands also indicates that hydration is more rapid than
methyl group deprotonation. 18O-Labeling studies on the
NMe-1 reactivity are consistent with hydration at C6, followed
by O2 trapping (Scheme 4). This mechanism is consistent with
other product and spectroscopic studies but is possibly
inconsistent with the work of Sevilla.4−6,11 However, the latter
was carried out at a greater pH (as high as pH 12) in 7.5 M
LiCl and in the absence of O2. This observation is contrasted
by studies concerning electron transfer in dA·T rich DNA
sequences, where deprotonation from the C5-methyl group of
the thymidine radical cation (1) has been suggested to
predominate.9,44 It is possible that the reactivity of 1 is
different when paired with dA in duplex DNA than when
present as a monomeric nucleoside in solution. Certainly, one
can envision how base pairing would affect the pKa of the
radical cation and base stacking in duplex DNA could influence
the rate of hydration. The independent generation of 1 from a
photochemical precursor (6) will enable us to address this
issue in synthetic oligonucleotides.
values, 12.3
0.4% for BME and 9.0
0.6% for KI
(Supporting Information, Tables S2 and S3). These values
EXPERIMENTAL PROCEDURES
General Methods. THF was distilled over Na/benzophenone.
DCM, TEA, DMF, and pyridine were dried over CaH2. 18O2 gas was
98% enriched, and that of H218O was 97%. All other reagents were
Figure 6. Effect of reductant on product yields from photolysis of
NMe-6. (A) Reductant = BME. (B) Reductant = KI. “Trapping
products” are the summation of yields of NMe-13, NMe-14, NMe-16,
27, and 28.
■
G
DOI: 10.1021/acs.joc.7b02017
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
purchased from commercial sources and were used without further
purification unless noted otherwise. All reactions were carried out
under a positive pressure of an argon atmosphere and monitored by
TLC on silica gel G-25 UV254 (0.25 mm) unless stated otherwise.
Spots were detected under UV light and/or by an ethanolic solution
of p-anisaldehyde. Column flash chromatography was performed with
Silicycle grade 70−230 mesh, 60−200 μm, 60 Å silica. The ratio
between silica gel and crude product ranged from 100:1 to 20:1 (w/
w). BME, KI, and solutions of other reductants were freshly prepared.
Photolysis samples were prepared in a mixture of acetonitrile and
water/buffer (phosphate buffer (20 mM) or Chelex-treated pH = 5
citrate buffer (40 mM)) at a ratio of 1:1 (v/v) containing the
appropriate precursor (100 μM) and 20 μM internal standard 5′-
benzoyl-2′-deoxyuridine (BzU). Photolyses were carried out in Pyrex
tubes in a Rayonet photoreactor fitted with 16 lamps with maximum
output at 350 nm. Samples containing 6 were photolyzed for 30 min,
while samples containing NMe-6 were photolyzed for 45 min.
Photolyzed samples were analyzed using UPLC with an Acquity 1.8
μm C18 UPLC HSS column (100 × 2.1 mm). Detection was carried
out at 230 nm, following separation using water (solvent A) and
acetonitrile (solvent B) with the following linear gradient (0.2 mL/
min): (time (min), % B) 0, 5; 30, 40; 35, 70; 40, 97; 42, 5; 45, 5.
(UP)LC/MS/MS analysis was performed on a UPLC system
equipped with a Q-TOF ESI mass spectrometer using the same
UPLC column and gradient conditions. Response factors (Rf) for
each compound (X) versus 5′-benzoyl-2′-deoxyuridine (BzU) was
calculated using the following formula: ([X]/[BzU]) = Rf(A(X)/
A(BzU)), where [X] is the concentration of compound X and [BzU]
is the concentration of BzU. A(X) and A(BzU) are the areas under
the peaks corresponding to X and BzU.
26.0, 25.8, 22.9, 18.4, 18.0, −4.66, −4.74, −5.3, −5.4; 31P NMR (162
MHz, CDCl3) δ −5.88; IR (NaCl) 1642, 1492, 1454 cm−1; HRMS
(ESI-TOF) m/z [M + H]+ calcd for C44H66N2O11PSSi2 917.3663,
found 917.3657.
Preparation of 12. K2CO3 (69 mg, 0.5 mmol) and MeI (71 mg,
0.5 mmol) were added to a solution of 11 (198 mg, 0.22 mmol) in
DMF (10 mL). The mixture was stirred at room temperature for 7 h.
After quenching with water (40 mL), the mixture was extracted with
EtOAc (3 × 30 mL). The combined organic phases were washed with
water (50 mL), brine (50 mL), dried over Na2SO4, filtered, and then
concentrated under reduced pressure. The residue was purified by
flash chromatography on silica gel (ethyl acetate/dicholoromethane =
1
1:9), affording 12 (160 mg, 0.17 mmol, 80%) as a colorless oil: H
NMR (400 MHz, CDCl3) δ 7.30−7.39 (m, 10H), 7.05 (d, J = 3.0 Hz,
1H), 6.86−6.78 (m, 2H), 6.76 (d, J = 9.0 Hz, 1H), 6.33−6.23 (m,
1H), 5.27 (s, 1H), 5.26−5.07 (m, 4H), 4.32 (dd, J = 3.5, 2.0 Hz, 1H),
3.89−3.78 (m, 1H), 3.78−3.66 (m, 4H), 3.63 (s, 3H), 3.60−3.49 (m,
1H), 2.79 (s, 3H), 2.67−2.48 (m, 1H), 1.86 (ddd, J = 13.3, 5.4, 1.9
Hz, 1H), 1.82 (s, 3H), 0.97−0.82 (m, 18H), 0.17−0.03 (m, 12H);
13C NMR (101 MHz, CDCl3) δ 167.6, 154.9, 153.2, 151.3, 136.2,
136.0, 128.47, 128.45, 128.31, 128.26, 128.0, 127.7, 123.2, 117.5,
117.2, 111.8, 86.3, 85.1, 79.5, 79.4, 72.7, 69.9, 69.3, 63.8, 62.6, 62.5,
56.0, 55.7, 36.3, 27.9, 26.0, 25.8, 23.2, 18.4, 18.0, −4.65, −4.72, −5.3,
−5.4; 31P NMR (162 MHz, CDCl3) δ −5.85; IR (NaCl) 2951, 2857,
1734, 1689, 1458 cm−1; HRMS (ESI-TOF) m/z [M + H]+ calcd for
C45H68N2O11PSSi2 931.3820, found 931.3810.
Preparation of Desilylated 11. Et3N·3HF (290 mg, 1.8 mmol)
was added to a solution of 11 (92 mg, 0.1 mmol) in THF (3.0 mL),
and the mixture was stirred at room temperature overnight. The
solvent was removed under reduced pressure. The residue was
purified by flash column chromatography (methanol/CH2Cl2 = 1:9)
to afford the product desilylated 11 (66 mg, 0.096 mmol, 96%) as a
31P NMR/UPLC-MS Monitoring of Desilylated-11 Decom-
position. Desilylated 11 (5 mg, 7.3 mmol) was dissolved in a
mixture of Chelex-treated PBS buffer (400 μL, pH 6.6, 100 mM
sodium phosphate, 1 M NaCl) and CD3OD (400 μL) at room
temperature. The 31P NMR spectrum was collected at various times.
After 22 days, the sample was dried under reduced pressure and
redissolved in CH3CN for UPLC-MS analysis.
General Procedure for the MeONH2·HCl Trapping Study.
Photolyzed samples were incubated at 37 °C in the presence of
MeONH2·HCl (10 mM) and NaOAc (10 mM) for 1 h. The samples
were neutralized using pH 8.0 PBS buffer (100 mM, 12% volume of
samples) prior to the UPLC analysis.
1
white foam: H NMR (400 MHz, CDCl3) δ 7.44−7.28 (m, 10H),
7.23 (s, 1H), 7.05 (d, J = 3.1 Hz, 1H), 6.89 (dd, J = 9.0, 3.1 Hz, 1H),
6.76 (d, J = 9.0 Hz, 1H), 6.19 (dd, J = 9.0, 5.8 Hz, 1H), 6.03 (s, 1H),
5.40 (dd, J = 11.9, 7.4 Hz, 1H), 5.31 (dd, J = 11.9, 6.6 Hz, 1H), 5.11
(dd, J = 7.7, 1.1 Hz, 2H), 4.45 (d, J = 5.1 Hz, 1H), 4.00 (s, 1H), 3.84
(d, J = 13.7 Hz, 1H), 3.74 (s, 3H), 3.68 (s, 3H), 2.44 (ddd, J = 14.6,
9.1, 5.6 Hz, 1H), 2.05 (s, 1H), 1.83 (s, 3H), 1.67 (ddd, J = 13.7, 5.8,
1.7 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ 167.5, 155.6, 153.1,
150.4, 135.73, 135.65, 135.23, 135.15, 128.7, 128.6, 128.58, 128.53,
128.1, 127.9, 124.0, 117.9, 116.8, 111.7, 86.9, 85.6, 79.34, 72.9, 70.2,
70.1, 62.8, 62.3, 56.0, 55.8, 37.0, 23.0; 31P NMR (162 MHz, CDCl3) δ
−6.33; IR (NaCl) 1698, 1541 cm−1; HRMS (ESI-TOF) m/z [M +
H]+ calcd for C32H38N2O11PS 689.1934, found 689.1928.
General Procedure for the 18O Labeling Study. Photolyses in
H2O/O2, H218O/O2, or H2O/18O2 were carried out side-by-side. For
experiments with H218O, the isotopically labeled solvent was
substituted for H2O, otherwise all other conditions were the same.
For experiments with 18O2, the gas was bubbled through the solution
in an ice bath at a rate of 1 mL/min for 5 min and sealed before
photolysis. The photolyzed samples were evaporated to dryness and
then redissolved in a mixture of acetonitrile and water at a ratio of 1:1
(v/v) with the same volume before LC-MS/MS analysis.
Preparation of Desilylated 12. Et3N·3HF (725.4 mg, 4.5 mmol)
was added to a solution of 12 (233 mg, 0.25 mmol) in THF (3.0
mL). The mixture was stirred at room temperature overnight, and the
solvent was removed under reduced pressure. The residue was
purified by flash column chromatography (methanol/CH2Cl2 = 1:9)
to afford desilylated 12 (146 mg, 0.21 mmol, 83%) as a white foam:
1H NMR (400 MHz, CDCl3) δ 7.39−7.31 (m, 10H), 7.01 (d, J = 3.1
Hz, 1H), 6.85 (dd, J = 9.0, 3.1 Hz, 1H), 6.77 (d, J = 9.0 Hz, 1H), 6.30
(dd, J = 8.7, 6.0 Hz, 1H), 5.96 (s, 1H), 5.44 (dd, J = 11.9, 7.3 Hz,
1H), 5.32 (dd, J = 11.9, 6.5 Hz, 1H), 5.13 (dd, J = 7.6, 1.4 Hz, 2H),
4.53 (dd, J = 3.9, 1.8 Hz, 1H), 3.87 (dd, J = 15.1, 3.1 Hz, 2H), 3.74
(s, 3H), 3.72−3.70 (m, 1H), 3.69 (s, 3H), 2.74 (s, 3H), 2.73−2.63
(m, 1H), 1.87 (ddd, J = 13.8, 6.0, 2.0 Hz, 1H), 1.78 (s, 1H); 13C
NMR (101 MHz, CDCl3) δ 167.3, 155.5, 153.1, 151.5, 135.8, 135.3,
128.6, 128.59, 128.57, 128.5, 128.1, 127.9, 124.0, 117.8, 116.7, 111.8,
86.9, 86.1, 79.0, 72.8, 70.2, 70.1, 62.8, 61.4, 56.0, 55.9, 37.1, 27.8,
23.4; 31P NMR (162 MHz, CDCl3) δ −6.35; IR (NaCl) 3424, 1732,
1685, 1457 cm−1; HRMS (ESI-TOF) m/z [M + H]+ calcd for
C33H40N2O11PS 702.2090, found 702.2092.
Preparation of 11. Dibenzyl N,N-diethyl phosphoramidite (76
mg, 0.24 mmol) was added to a solution of 1014 (131 mg, 0.20
mmol) in CH2Cl2 (2 mL), followed by 5-ethylthiotetrazole (0.25 M
in acetonitrile, 0.24 mmol). The solution was stirred at room
temperature overnight and then cooled to 0 °C with an ice bath. t-
BuOOH (5−6 M in decane, 0.3 mL) was added, and the mixture was
stirred for 1 h at 0 °C. After warming to room temperature, the
mixture was directly subjected to flash column chromatography
(EtOAc/CH2Cl2 = 1:3) to provide 11 (101 mg, 0.13 mmol, 64%) as
1
a white foam: H NMR (400 MHz, CDCl3) δ 7.42−7.28 (m, 10H),
7.07 (d, J = 3.1 Hz, 2H), 6.85 (dd, J = 9.0, 3.1 Hz, 1H), 6.75 (d, J =
9.0 Hz, 1H), 6.17 (dd, J = 9.4, 5.4 Hz, 1H), 5.34 (s, 1H), 5.28−5.03
(m, 4H), 4.29 (dd, J = 3.8, 1.9 Hz, 1H), 3.87−3.76 (m, 1H), 3.74 (s,
3H), 3.69 (dd, J = 10.6, 4.6 Hz, 1H), 3.62 (s, 3H), 3.53 (dd, J = 10.6,
6.8 Hz, 1H), 2.46 (ddd, J = 13.5, 9.5, 5.7 Hz, 1H), 1.86 (s, 3H), 1.72
(ddd, J = 13.4, 5.4, 1.8 Hz, 1H), 0.89 (s, 16H), 0.07 (s, 6H), 0.07 (s,
6H); 13C NMR (101 MHz, CDCl3) δ 155.1, 153.2, 150.0, 136.0,
128.49, 128.46, 128.37, 128.31, 128.0, 127.8, 123.1, 117.6, 117.2,
111.7, 86.3, 84.6, 79.7, 79.6, 72.7, 69.9, 69.4, 63.4, 56.0, 55.7, 36.2,
Preparation of 6. BzCN (8.8 mg, 0.07 mmol) and Et3N (8.5 mg,
0.084 mmol) were added to desilylated 11 (31.6 mg, 0.056 mmol) in
THF (1.0 mL) at −78 °C. The mixture was stirred at −78 °C for 3 h.
After quenching the reaction with MeOH (0.3 mL), the solution was
warmed to room temperature, and the solvent was removed under
reduced pressure. The residue was purified by flash column
H
DOI: 10.1021/acs.joc.7b02017
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
chromatography (MeOH/CH2Cl2 = 1:15) to afford the starting
material (20 mg, 0.0354 mmol) and 6 (6.4 mg, 0.008 mmol, 39%
based on unrecovered starting material) as a white foam: H NMR
reaction. The mixture was extracted with EtOAc (2 × 50 mL). The
combined organic layers were washed with H2O (30 mL) and brine
(30 mL) and then dried over anhydrous Na2SO4. The solvent was
removed under reduced pressure. The residue was purified by column
chromatography on silica gel (ethyl acetate/hexane = 1:1), providing
1
(400 MHz, CDCl3) δ 8.05 (dd, J = 8.4, 1.4 Hz, 2H), 7.56−7.46 (m,
1H), 7.45−7.36 (m, 2H), 7.36−7.27 (m, 10H), 7.16 (s, 1H), 7.06 (d,
J = 3.1 Hz, 1H), 6.85 (dd, J = 9.0, 3.1 Hz, 1H), 6.73 (d, J = 9.1 Hz,
1H), 6.26 (dd, J = 8.1, 6.2 Hz, 1H), 5.44 (s, 1H), 5.21−4.88 (m, 4H),
4.52 (dd, J = 11.8, 6.0 Hz, 1H), 4.42 (dd, J = 11.8, 4.8 Hz, 2H), 4.10
(ddd, J = 5.9, 4.7, 3.5 Hz, 1H), 3.74 (s, 3H), 3.60 (s, 3H), 2.80 (ddd,
J = 14.5, 8.2, 6.7 Hz, 1H), 2.26 (s, 1H), 2.01 (ddd, J = 14.0, 6.2, 3.4
Hz, 1H), 1.84 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 167.3, 166.5,
155.1, 153.2, 150.0, 135.9, 133.3, 129.8, 129.6, 128.50, 128.48, 128.41,
128.35, 128.0, 127.8, 123.1, 117.5, 117.3, 111.9, 84.5, 82.9, 79.6, 72.3,
69.9, 69.4, 64.7, 63.4, 56.1, 55.7, 36.0, 22.8; 31P NMR (162 MHz,
CDCl3) δ −5.77; IR (NaCl) 3397, 1699, 1650, 1541 cm−1; HRMS
(ESI-TOF) m/z [M + H]+ calcd for C39H42N2O12PS 793.2196, found
793.2183.
1
21 (680 mg, 1.48 mmol, 74%) as a white foam: H NMR (400 MHz,
CDCl3) δ 9.28 (s, 1H), 8.09−7.96 (m, 2H), 7.59 (ddd, J = 8.8, 5.4,
1.3 Hz, 1H), 7.46 (td, J = 7.6, 1.7 Hz, 2H), 7.24 (d, J = 1.2 Hz, 1H),
6.30 (t, J = 6.6 Hz, 1H), 4.62 (dd, J = 12.3, 3.3 Hz, 1H), 4.51−4.43
(m, 2H), 4.19 (q, J = 3.6 Hz, 1H), 2.37 (ddd, J = 13.4, 6.2, 3.7 Hz,
1H), 2.13 (dt, J = 13.5, 6.7 Hz, 1H), 1.66 (d, J = 1.1 Hz, 3H), 0.89
(d, J = 0.9 Hz, 9H), 0.09 (s, 6H); 13C NMR (101 MHz, CDCl3) δ
166.2, 134.9, 133.6, 129.5, 129.4, 128.7, 111.2, 85.2, 84.9, 72.0, 63.8,
41.1, 25.70, 25.69, 18.0, 12.3, −4.7, −4.9; IR (NaCl) 1697, 1465
cm−1; HRMS (ESI-TOF) m/z [M + Na]+ calcd for C23H32N2NaO6Si
483.1927, found 483.1922.
Preparation of NMe-21. 5′-Benzoyl-3-methyl-thymidine (432
mg, 1.2 mmol) was reacted with imidazole (245 mg, 3.6 mmol) and
TBDMSCl (271 mg, 1.8 mmol) in DMF (12 mL) as described above
for the preparation of 21. Following flash column chromatography
(ethyl acetate/hexane = 1:2), NMe-21 (460 mg, 0.97 mmol, 81%)
was obtained as a colorless oil: 1H NMR (400 MHz, CDCl3) δ 8.05−
7.94 (m, 2H), 7.61−7.53 (m, 1H), 7.48−7.39 (m, 2H), 7.23 (d, J =
1.2 Hz, 1H), 6.29 (t, J = 6.6 Hz, 1H), 4.60 (dd, J = 12.3, 3.3 Hz, 1H),
4.50−4.41 (m, 2H), 4.18 (q, J = 3.6 Hz, 1H), 3.30−3.27 (m, 3H),
2.36 (ddd, J = 13.5, 6.2, 3.7 Hz, 1H), 2.10 (dt, J = 13.4, 6.6 Hz, 1H),
1.66 (d, J = 0.9 Hz, 3H), 0.91−0.84 (m, 9H), 0.07 (d, J = 0.7 Hz,
6H); 13C NMR (101 MHz, CDCl3) δ 166.1, 163.5, 150.9, 133.5,
132.7, 129.5, 129.4, 128.7, 110.1, 85.9, 84.8, 71.9, 63.7, 41.2, 27.8,
25.69, 25.67, 17.9, 13.0, −4.7, −4.9; IR (NaCl) 1708, 1466 cm−1.
HRMS (ESI-TOF) m/z [M + Na]+ calcd for C24H34N2NaO6Si
497.2084, found 497.2083.
Preparation of 22. N-Bromosuccinimide (107 mg, 0.6 mmol)
and AIBN (8.2 mg, 0.05 mmol) were added to a solution of 21 (230
mg, 0.5 mmol) in benzene (5 mL). The mixture was refluxed for 3 h,
at which time additional N-bromosuccinimide and AIBN (half the
amount of the first addition) were added. The mixture was refluxed
for another 3 h and then cooled to room temperature. After removing
the solvent under a vacuum, aq 5% NaHCO3 (15 mL) was added.
The mixture was stirred at room temperature overnight and then
extracted with EtOAc (2 × 50 mL). The combined organic layers
were washed with H2O (30 mL) and brine (30 mL) and then dried
over anhydrous Na2SO4. After removing the solvent under a vacuum,
the residue was purified by chromatography (ethyl acetate/hexane =
1:1 to 2:1), yielding 22 (52 mg, 0.11 mmol, 22%) as a colorless oil:
1H NMR (400 MHz, CDCl3) δ 9.44 (s, 1H), 8.02 (dt, J = 8.4, 1.6 Hz,
2H), 7.60 (t, J = 7.4 Hz, 1H), 7.52 (s, 1H), 7.51−7.41 (m, 2H),
6.34−6.21 (m, 1H), 4.54 (qd, J = 12.3, 3.9 Hz, 2H), 4.48−4.42 (m,
1H), 4.26−4.17 (m, 2H), 4.12 (dd, J = 10.1, 4.3 Hz, 1H), 2.74 (s,
1H), 2.46−2.34 (m, 1H), 2.22−2.09 (m, 1H), 0.92−0.87 (m, 9H),
0.13−0.06 (m, 6H); 13C NMR (101 MHz, CDCl3) δ 178.0, 166.3,
163.7, 150.0, 136.8, 133.6, 129.6, 129.4, 128.7, 114.1, 85.6, 85.0, 71.8,
63.7, 58.4, 41.2, 29.6, 25.7, 17.9, −4.7, −4.9; IR (NaCl) 3482, 1712,
1468 cm−1; HRMS (ESI-TOF) m/z [M + Na]+ calcd for
C23H32N2NaO7Si 499.1876, found 499.1876.
Preparation of NMe-22. Protected nucleoside NMe-21 (237 mg,
0.5 mmol) was reacted as described above for 21 using the same
amount of reagents, with one exception. Acetonitrile (5 mL) was
added during hydrolysis to help solubilize the bromide intermediate.
Following flash chromatography (ethyl acetate/hexane = 1:1 to 2:1),
NMe-22 (146 mg, 0.30 mmol, 60%) was obtained as a colorless oil:
1H NMR (400 MHz, CDCl3) δ 8.05−7.97 (m, 2H), 7.64−7.56 (m,
1H), 7.50 (s, 1H), 7.49−7.42 (m, 2H), 6.28 (t, J = 6.5 Hz, 1H), 4.54
(qd, J = 12.3, 3.9 Hz, 2H), 4.48−4.40 (m, 1H), 4.28−4.17 (m, 2H),
4.11 (ddd, J = 14.2, 11.2, 6.3 Hz, 1H), 3.30 (s, 3H), 2.76 (s, 1H), 2.41
(ddd, J = 13.5, 6.2, 3.9 Hz, 1H), 2.12 (dt, J = 13.4, 6.6 Hz, 1H),
0.91−0.86 (m, 9H), 0.10−0.07 (m, 6H); 13C NMR (101 MHz,
CDCl3) δ 166.2, 163.2, 150.6, 134.2, 133.6, 129.5, 129.4, 128.7, 113.0,
86.2, 85.0, 71.8, 63.6, 59.3, 41.3, 27.6, 25.7, 17.9, −4.7, −4.9; IR
Preparation of NMe-6. Desilylated 12 (84.24 mg, 0.12 mmol)
was azeotropically dried with pyridine (3 × 2.0 mL) and redissolved
in pyridine (2.0 mL) at 0 °C. Benzoyl chloride (18.6 mg, 0.13 mmol)
was added. The mixture was allowed to warm to room temperature
and stirred for 6 h. After quenching the reaction with MeOH (0.3
mL), the solvent was removed under reduced pressure. The residue
was purified by flash column chromatography (EtOAc/CH2Cl2 = 1:1)
1
to afford NMe-6 (34 mg, 0.042 mmol, 35%) as a colorless oil: H
NMR (400 MHz, CDCl3) δ 8.11−7.98 (m, 2H), 7.48 (d, J = 7.4 Hz,
1H), 7.42−7.27 (m, 12H), 7.04 (d, J = 3.1 Hz, 1H), 6.82 (dd, J = 9.0,
3.1 Hz, 1H), 6.75 (d, J = 9.0 Hz, 1H), 6.35 (dd, J = 7.9, 6.3 Hz, 1H),
5.36 (s, 1H), 5.13 (ddd, J = 30.5, 15.0, 5.0 Hz, 4H), 4.53 (dd, J =
11.8, 5.9 Hz, 1H), 4.48−4.32 (m, 2H), 4.11 (dd, J = 3.7, 1.4 Hz, 1H),
3.73 (s, 3H), 3.61 (s, 3H), 2.93 (ddd, J = 14.4, 7.9, 6.8 Hz, 1H), 2.77
(s, 3H), 2.12 (ddd, J = 14.0, 6.3, 3.6 Hz, 1H), 1.79 (s, 3H); 13C NMR
(101 MHz, CDCl3) δ 167.4, 166.5, 155.1, 153.2, 151.3, 133.2, 129.8,
129.7, 128.5, 128.34, 128.28, 128.0, 127.72, 127.71, 123.3, 117.5,
117.2, 111.9, 85.0, 82.9, 79.4, 79.3, 76.7, 72.3, 69.9, 69.4, 69.3, 64.8,
56.1, 55.7, 36.2, 27.9, 23.2; 31P NMR (162 MHz, CDCl3) δ −5.82; IR
(NaCl) 3407, 1688, 1455 cm−1; HRMS (ESI-TOF) m/z [M + H]+
calcd for C40H44N2O12PS 807.2353, found 807.2340.
Preparation of NMe-14. To a solution of NMe-T (180 mg, 0.5
mmol) in a mixture of THF (4 mL) were added t-butyl alcohol (4
mL), water (1 mL), N-methylmorpholine N-oxide (117 mg, 1 mmol),
and OsO4 (5.1 mg, 0.02 mmol). The yellowish solution was stirred at
45 °C for 36 h. After cooling the solution to 0 °C, sodium sulfite
(252 mg, 2 mmol) was added. The mixture was stirred at 0 °C for 30
min and warmed to room temperature. The solvent was removed, and
the residue was dissolved in ethyl acetate (100 mL). The organic
phase was washed with water (50 mL) and brine (50 mL) and then
dried over with Na2SO4. After concentrating under reduced pressure,
the residue was purified by flash chromatography on silica gel (ethyl
acetate/hexane = 1:1), affording NMe-14 as a mixture of
diastereomers (92:8) in 39% (76 mg) yield as a white foam. Major
1
stereoisomer of NMe-14: H NMR (400 MHz, CDCl3) δ 8.10−8.00
(m, 2H), 7.63−7.56 (m, 1H), 7.50−7.43 (m, 2H), 6.34 (dd, J = 7.2,
6.6 Hz, 1H), 5.01 (s, 1H), 4.60 (dd, J = 12.1, 4.4 Hz, 1H), 4.56−4.45
(m, 2H), 4.14 (dd, J = 7.9, 3.7 Hz, 1H), 3.21 (s, 3H), 2.42 (ddd, J =
17.7, 7.3, 6.6 Hz, 1H), 2.27 (ddd, J = 13.9, 6.3, 3.9 Hz, 1H), 1.35 (s,
3H). Minor stereoisomer of NMe-14: 1H NMR (400 MHz, CDCl3) δ
8.10−8.00 (m, 2H), 7.63−7.56 (m, 1H), 7.50−7.43 (m, 2H), 6.44 (t,
J = 6.8 Hz, 1H), 4.85 (s, 1H), 4.70 (dd, J = 12.2, 5.0 Hz, 1H), 4.45
(d, J = 4.3 Hz, 2H), 4.10 (d, J = 7.2 Hz, 1H), 3.21 (s, 3H), 2.62 (s,
1H), 2.14 (dd, J = 14.1, 6.9 Hz, 1H), 1.32 (s, 3H); 13C NMR (101
MHz, CDCl3) δ 173.5, 166.5, 151.1, 133.5, 129.7, 129.4, 128.6, 84.6,
83.3, 77.3, 77.0, 76.7, 71.7, 71.5, 64.1, 38.5, 28.3, 22.8. IR (NaCl)
3425 (bd), 1679, 1464 cm−1; HRMS (ESI-TOF) m/z [M + Na]+
calcd for C18H22N2NaO8 417.1274, found 417.1265.
Preparation of 21. To a solution of 5′-benzoyl-thymidine (692
mg, 2.0 mmol) in anhydrous DMF (12 mL) were added imidazole
(409 mg, 6.0 mmol) and TBDMSCl (452 mg, 3.0 mmol). The
mixture was stirred at room temperature overnight, and methanol
(0.5 mL), followed by water (30 mL), was added to quench the
I
DOI: 10.1021/acs.joc.7b02017
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
(NaCl) 3474, 1713, 1469 cm−1; HRMS (ESI-TOF) m/z [M + Na]+
calcd for C24H34N2NaO7Si 513.2033, found 513.2031.
NMe-19 (330 mg, 81%) as a white foam: 1H NMR (400 MHz,
CDCl3) δ 8.04−7.97 (m, 2H), 7.63−7.57 (m, 1H), 7.53 (s, 1H), 7.44
(td, J = 7.6, 1.6 Hz, 2H), 6.32 (t, J = 6.5 Hz, 1H), 6.26−6.15 (m,
1H), 5.77 (dd, J = 17.6, 1.4 Hz, 1H), 5.05 (dd, J = 11.4, 1.3 Hz, 1H),
4.63 (t, J = 3.5 Hz, 2H), 4.55−4.49 (m, 1H), 4.30 (dd, J = 7.1, 3.7
Hz, 1H), 3.32 (s, 3H), 2.71 (s, 1H), 2.55 (ddd, J = 13.9, 6.2, 4.0 Hz,
1H), 2.27−2.20 (m, 1H); 13C NMR (101 MHz, CDCl3) δ 166.5,
161.7, 150.2, 133.7, 133.6, 129.6, 129.2, 128.7, 128.2, 115.5, 112.0,
86.2, 84.7, 71.4, 63.9, 41.1, 27.9; IR (NaCl) 3429, 1708, 1464 cm−1;
HRMS (ESI-TOF) m/z [M + H]+ calcd for C19H21N2O6 373.1400,
found 373.1387.
Preparation of 17. Et3N·3HF (102 mg, 0.63 mmol) was added to
a solution of 22 (33 mg, 70 μmol) in THF (2 mL). The solution was
stirred at room temperature overnight. The solvent was removed
under reduced pressure. The residue was purified by flash column
chromatography (methanol in CH2Cl2 5−10%) to afford 17 (21 mg,
1
58 μmol, 83%) as a white foam: H NMR (400 MHz, MeOD) δ
8.06−8.04 (m, 1H), 8.04−8.02 (m, 1H), 7.66 (t, J = 1.0 Hz, 1H),
7.64−7.59 (m, 1H), 7.52−7.46 (m, 2H), 6.30 (t, J = 6.8 Hz, 1H),
4.62−4.52 (m, 2H), 4.52−4.44 (m, 1H), 4.26−4.19 (m, 1H), 4.20−
4.08 (m, 2H), 2.40 (dtd, J = 9.9, 6.5, 3.8 Hz, 1H), 2.29 (ddd, J = 13.8,
7.1, 6.6 Hz, 1H); 13C NMR (101 MHz, MeOD) δ 166.3, 163.6,
150.7, 137.2, 133.1, 129.6, 129.2, 128.3, 114.1, 85.4, 84.6, 71.0, 64.2,
56.4, 39.6; IR (NaCl) 3355, 1657, 1557 cm−1; HRMS (ESI-TOF) m/
z [M + Na]+ calcd for C17H18N2NaO7 385.1012, found 385.1004.
Preparation of NMe-17. The reaction of NMe-22 (128 mg, 0.26
mmol) was carried out as described above for 22, with the exception
that 377 mg (2.34 mmol) of Et3N·3HF was employed. Following
flash chromatography, 91 mg (0.24 mmol, 93%) of NMe-17 was
Preparation of 16. OsO4 (4.0% solution in water, 7.0 mg, 27.5
μmol) and NaIO4 (705.9 mg, 3.3 mmol) were added to a suspension
of 19 (197 mg, 0.55 mmol) in a mixture of dioxane and water (8 mL,
v/v = 3:1) and 2,6-lutidine (118 mg, 1.1 mmol). The mixture was
stirred at room temperature overnight. The solvent was removed, and
the residue was dissolved in EtOAc (80 mL). The mixture was
washed with water (30 mL) and brine (30 mL) and dried over
anhydrous Na2SO4. The solvent was removed under reduced
pressure. The residue was purified by flash column chromatography
(methanol in dichloromethane 2−5%), providing 16 (180 mg, 91%)
1
obtained as a white foam: H NMR (400 MHz, DMSO) δ 8.03−7.94
1
(m, 2H), 7.72−7.61 (m, 1H), 7.60−7.48 (m, 2H), 6.26 (t, J = 6.7 Hz,
1H), 5.51 (d, J = 4.5 Hz, 1H), 4.97 (t, J = 5.4 Hz, 1H), 4.47 (ddd, J =
17.5, 12.0, 4.7 Hz, 2H), 4.40−4.30 (m, 1H), 4.19−4.01 (m, 3H), 3.34
(d, J = 6.1 Hz, 2H), 3.15 (s, 3H), 2.34−2.17 (m, 2H); 13C NMR (101
MHz, DMSO) δ 166.1, 162.0, 150.9, 135.0, 134.0, 129.8, 129.7, 129.3,
113.9, 85.8, 84.3, 70.6, 64.9, 56.8, 27.8; IR (NaCl) 3622, 1698, 1557
cm−1; HRMS (ESI-TOF) m/z [M + Na]+ calcd for C18H20N2NaO7
399.1168, found 399.1160.
as a white foam: H NMR (400 MHz, DMSO) δ 11.80 (s, 1H), 9.67
(s, 1H), 8.33 (s, 1H), 8.03−7.93 (m, 2H), 7.69−7.62 (m, 1H), 7.53−
7.45 (m, 2H), 6.11 (t, J = 6.4 Hz, 1H), 5.54−5.49 (m, 1H), 4.59−
4.51 (m, 1H), 4.48−4.42 (m, 1H), 4.40−4.31 (m, 1H), 4.25−4.18
(m, 1H), 2.39−2.30 (m, 2H); 13C NMR (101 MHz, DMSO) δ 186.4,
166.1, 162.0, 149.9, 146.8, 134.0, 129.7, 129.3, 129.2, 111.1, 87.1,
85.2, 70.6, 64.7; IR (NaCl) 3390, 1701, 1464 cm−1; HRMS (ESI-
TOF) m/z [M + Na]+ calcd for C17H16N2NaO7 383.0855, found
383.0843.
Preparation of NMe-18. 5-Iodo-3-methyl-2′-deoxyuridine4 (736
mg, 2 mmol) was azeotropically dried with pyridine (5 mL × 3) and
redissolved in pyridine (8 mL). The solution was cooled to 0 °C, and
benzoyl chloride (309 mg, 2.2 mmol) was added. The mixture was
stirred at 0 °C for 3 h, at which time methanol (1 mL) was added to
quench the reaction, and the mixture was warmed to room
temperature. The solvent was removed under reduced pressure.
The residue was purified by flash column chromatography (dry
loading, EtOAc/hexane = 1:3 to 2:3 to remove the remaining
pyridine; then methanol in dicholoromethane 2−10%) to yield NMe-
Preparation of NMe-16. Compound NMe-19 (186 mg, 0.5
mmol), 2,6-lutidine (107 mg, 1 mmol), OsO4 (6.4 mg, 25 μmol), and
NaIO4 (641.7 mg, 3 mmol) were subjected to the reaction conditions
described above for the formation of 16 to provide NMe-16 (153 mg,
1
82%) as a white foam: H NMR (400 MHz, DMSO) δ 9.72 (s, 1H),
8.37 (s, 1H), 8.02−7.94 (m, 2H), 7.68−7.62 (m, 1H), 7.53−7.46 (m,
2H), 6.14 (t, J = 6.4 Hz, 1H), 5.52 (dd, J = 8.3, 4.4 Hz, 1H), 4.55
(dd, J = 12.2, 3.4 Hz, 1H), 4.44 (dt, J = 11.9, 4.7 Hz, 1H), 4.40−4.34
(m, 1H), 4.23 (dt, J = 5.5, 3.6 Hz, 1H), 3.17 (s, 3H), 2.42−2.34 (m,
2H); 13C NMR (101 MHz, DMSO) δ 186.7, 166.1, 161.2, 150.1,
144.8, 134.0, 129.7, 129.2, 110.2, 88.1, 85.4, 70.5, 64.7, 27.7; IR
(NaCl) 3415, 1719, 1462 cm−1; HRMS (ESI-TOF) m/z [M + Na]+
calcd for C18H18N2NaO7 397.1012, found 397.1004.
1
18 (0.78 g, 1.65 mmol, 83%) as a white foam: H NMR (400 MHz,
DMSO) δ 8.03 (s, 1H), 8.02−7.96 (m, 2H), 7.73−7.64 (m, 1H), 7.54
(t, J = 7.7 Hz, 2H), 6.15 (t, J = 6.7 Hz, 1H), 5.49 (d, J = 4.3 Hz, 1H),
4.50 (ddd, J = 17.6, 12.1, 4.6 Hz, 2H), 4.37 (td, J = 7.5, 3.7 Hz, 1H),
4.13 (dt, J = 5.4, 3.6 Hz, 1H), 3.19 (s, 3H), 2.37−2.18 (m, 2H); 13C
NMR (101 MHz, DMSO) δ 166.1, 160.1, 150.6, 143.2, 136.6, 134.0,
129.7, 129.4, 124.4, 86.7, 84.8, 70.7, 69.0, 64.8, 29.2; IR (NaCl) 3461,
1708, 1452 cm−1; HRMS (ESI-TOF) m/z [M + Na]+ calcd for
C17H18IN2O6 473.0210, found 473.0208.
Preparation of 20. A mixture containing 16 (48 mg, 0.13 mmol),
MeONH2·HCl (30.1 mg, 0.36 mmol), NaOAc (48.1 mg, 0.59 mmol),
H2O (3.6 mL), and THF (1.2 mL) was stirred at room temperature
for 2 h. The solution was extracted with EtOAc (2 × 30 mL). The
combined organic layers were washed with water (30 mL) and brine
(30 mL) and dried over anhydrous Na2SO4. The solvent was
removed under reduced pressure to quantitatively yield 20 (56 mg,
Preparation of 19. Tributyl(vinyl) stannane (419 mg, 1.32
mmol) was added to a solution of 185 (504 mg, 1.1 mmol) and
Pd(Ph3P)2Cl2 (39 mg, 55 μmol) in anhydrous THF (5 mL). The
solution was refluxed overnight. After cooling the dark mixture to
room temperature, the solvent was removed under reduced pressure
and then dissolved in EtOAc (100 mL). The solution was washed
with H2O (30 mL) and brine (30 mL) and then dried over anhydrous
Na2SO4. After removing the solvent under reduced pressure, the
residue was purified by chromatography (methanol in CH2Cl2 2−
1
100%) as a white foam: H NMR (400 MHz, DMSO) δ 11.70 (s,
1H), 8.01 (s, 1H), 7.98−7.93 (m, 2H), 7.81−7.76 (m, 1H), 7.70−
7.63 (m, 1H), 7.54−7.47 (m, 2H), 6.15 (t, J = 6.6 Hz, 1H), 5.50 (t, J
= 3.8 Hz, 1H), 4.56−4.47 (m, 1H), 4.48−4.40 (m, 1H), 4.41−4.33
(m, 1H), 4.22−4.14 (m, 1H), 3.55 (s, 3H), 2.33−2.23 (m, 2H); 13C
NMR (101 MHz, DMSO) δ 166.1, 161.7, 150.0, 142.0, 137.6, 134.0,
129.7, 129.7, 129.2, 106.2, 86.2, 84.9, 70.8, 65.1, 61.6; IR (NaCl)
3395, 1699, 1460 cm−1; HRMS (ESI-TOF) m/z [M + H]+ calcd for
C18H20N3O7 390.1301, found 390.1298.
1
5%), yielding 19 (336 mg, 85%) as a white foam: H NMR (400
MHz, DMSO) δ 11.46 (s, 1H), 8.02−7.92 (m, 2H), 7.72−7.62 (m,
2H), 7.58−7.48 (m, 2H), 6.25−6.12 (m, 2H), 5.89 (dd, J = 17.6, 2.1
Hz, 1H), 5.49 (d, J = 4.4 Hz, 1H), 5.01 (dd, J = 11.5, 2.1 Hz, 1H),
4.55 (dd, J = 12.0, 3.7 Hz, 1H), 4.50−4.36 (m, 2H), 4.09 (dt, J = 5.4,
3.7 Hz, 1H), 2.32 (dt, J = 13.5, 6.8 Hz, 1H), 2.21 (ddd, J = 13.6, 6.5,
3.8 Hz, 1H); 13C NMR (101 MHz, DMSO) δ 166.1, 162.4, 149.9,
138.2, 134.0, 129.8, 129.7, 129.3, 129.2, 115.0, 111.6, 85.1, 84.4, 70.7,
64.9; IR (NaCl) 3417, 1699, 1458 cm−1; HRMS (ESI-TOF) m/z [M
+ Na]+ calcd for C18H18N2NaO6 381.1063, found 381.1056.
Preparation of 24. BzCl (247 mg, 1.76 mmol) was added to 236
(237 mg, 1.6 mmol) in pyridine (2.0 mL) at 0 °C. The mixture was
allowed to warm to room temperature overnight. After quenching the
reaction with MeOH (0.3 mL), the solvent was removed under
reduced pressure. The residue was purified by flash column
chromatography (EtOAc in hexane 10−25%) to afford 24 as a
mixture of stereoisomers (86:14) in 61% (246 mg) yield as a colorless
1
oil. Major stereoisomer: H NMR (400 MHz, CDCl3) δ 8.03−7.98
Preparation of NMe-19. 5′-Benzoyl-5-iodo-3-methyl-2′-deoxyur-
idine (NMe-18, 519 mg, 1.1 mmol) was subjected to the reaction
conditions described above for the preparation of 19, resulting in
(m, 2H), 7.57−7.52 (m, 1H), 7.47−7.42 (m, 2H), 5.13 (d, J = 4.2 Hz,
1H), 4.37 (ddd, J = 6.2, 4.8, 2.2 Hz, 3H), 4.25 (d, J = 6.2 Hz, 1H),
3.40 (s, 3H), 2.18 (ddd, J = 13.9, 6.2, 4.6 Hz, 1H), 2.06 (ddd, J =
J
DOI: 10.1021/acs.joc.7b02017
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
13.9, 1.4, 0.6 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ 166.4, 133.2,
129.7, 128.5, 105.5, 85.0, 73.1, 64.6, 55.0, 41.0. Minor stereoisomer:
1H NMR (400 MHz, CDCl3) δ 8.10−8.04 (m, 2H), 7.58 (t, J = 1.3
Hz, 1H), 7.42−7.40 (m, 2H), 5.09 (dd, J = 5.3, 1.6 Hz, 1H), 4.55 (td,
J = 6.9, 4.7 Hz, 1H), 4.47 (dd, J = 11.6, 5.1 Hz, 1H), 4.40 (d, J = 2.3
Hz, 1H), 4.19 (dd, J = 10.2, 5.2 Hz, 1H), 3.31 (s, 3H), 2.35−2.26 (m,
1H), 2.14−2.09 (m, 1H); 13C NMR (101 MHz, CDCl3) δ 166.5,
133.1, 129.8, 128.4, 105.1, 83.7, 72.4, 65.3, 55.2, 41.7; IR (NaCl)
3426, 1719, 1455 cm−1; HRMS (ESI-TOF) m/z [M + Na]+ calcd for
C13H17NNaO5 290.1004, found 290.0991.
NMR and MS spectra of new compounds, structures
and response factors of photoproducts or trapping
products, and determination of response factors (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: mgreenberg@jhu.edu. Phone: 410-516-8095. Fax:
410-516-7044.
ORCID
Preparation of 25. Acetyl chloride (471 mg, 6 mmol) was added
to an ice-cold mixture of acetonitrile and water (6 mL, CH3CN/H2O
= 1:99), and the solution was warmed to room temperature and
stirred for 30 min. A solution of 24 (202 mg, 0.8 mmol) in 1,4-
dioxane (2 mL) was added, and the mixture was stirred at room
temperature overnight. The solution was neutralized with a saturated
NaHCO3 solution (15 mL). After extraction with CH2Cl2 (3 × 30
mL), the combined organic phases were washed with water (30 mL)
and brine (30 mL) and then dried over anhydrous Na2SO4. The
solvent was evaporated under reduced pressure. The residue was
purified by flash chromatography on silica gel (EtOAc in hexane 30−
60%) to afford 25 as a mixture of diastereomers (81:19) in 57% (108
Marc M. Greenberg: 0000-0002-5786-6118
Present Address
†Institute of Pharmaceuticals Chemistry and Metabolism,
Faculty of Pharmacy and Biochemistry, University of Buenos
Aires, Buenos Aires, Argentina
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
1
mg) yield as a colorless oil. Major stereoisomer: H NMR (400 MHz,
We are grateful for the support of this research from the
National Institute of General Medical Science (GM-054996).
CDCl3) δ 8.04−8.02 (m, 1H), 8.01−7.98 (m, 1H), 7.61−7.53 (m,
1H), 7.48−7.40 (m, 2H), 5.71−5.58 (m, 1H), 4.60−4.53 (m, 1H),
4.39−4.30 (m, 3H), 3.41 (d, J = 4.5 Hz, 1H), 2.93 (d, J = 8.3 Hz,
1H), 2.19−2.08 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 166.4,
133.3, 129.7, 128.5, 99.5, 85.2, 73.4, 64.6, 41.4. Minor stereoisomer:
1H NMR (400 MHz, CDCl3) δ 8.08−8.07 (m, 1H), 8.05 (d, J = 1.4
Hz, 1H), 7.57 (dddd, J = 8.5, 7.4, 2.8, 1.4 Hz, 1H), 7.47−7.41 (m,
2H), 5.65 (t, J = 4.5 Hz, 1H), 4.63−4.59 (m, 1H), 4.51−4.48 (m,
3H), 4.19 (dd, J = 10.1, 5.1 Hz, 1H), 3.16 (d, J = 2.7 Hz, 1H), 2.31
(ddd, J = 13.5, 6.8, 2.2 Hz, 1H), 2.24−2.19 (m, 1H); 13C NMR (101
MHz, CDCl3) δ 166.7, 133.3, 129.7, 128.5, 98.8, 83.8, 72.4, 65.6,
42.4; IR (NaCl) 3395 (bd), 1709, 1450 cm−1; HRMS (ESI-TOF) m/
z [M + Na]+ calcd for C12H14NaO5 261.0739, found 261.0727.
Preparation of 26. 2-Deoxy-D-ribose (268 mg, 2 mmol) and
MeONH2·HCl (201 mg, 2.4 mmol) were dissolved in pyridine (2
mL). The mixture was stirred at room temperature for 36 h. The
solvent was removed under reduced pressure, and the major product
was collected by flash column chromatography (CH2Cl2/MeOH =
1:1). This material was dissolved in pyridine (3.0 mL) at 0 °C.
Benzoyl chloride (155 mg, 1.1 mmol) was added, and the solution
was allowed to warm to room temperature overnight. After quenching
the reaction with MeOH (0.3 mL), the solvent was removed under
reduced pressure. The residue was purified by flash column
chromatography (methanol in CH2Cl2 2−8%) to provide 26 as a
mixture of stereoisomers (52:48) as a colorless oil in a 19% overall
REFERENCES
■
(1) Steenken, S.; Jovanovic, S. V. J. Am. Chem. Soc. 1997, 119, 617−
618.
(2) Cadet, J.; Douki, T.; Ravanat, J.-L. Acc. Chem. Res. 2008, 41,
1075−1083.
(3) von Sonntag, C. Free-Radical-Induced DNA Damage and Its
Repair; Springer-Verlag: Berlin, Germany, 2006.
(4) Deeble, D. J.; Schuchmann, M. N.; Steenken, S.; von Sonntag, C.
J. Phys. Chem. 1990, 94, 8186−8192.
(5) Geimer, J.; Beckert, D. J. Phys. Chem. A 1999, 103, 3991−3998.
(6) Wagner, J. R.; van Lier, J. E.; Johnston, L. J. Photochem.
Photobiol. 1990, 52, 333−343.
(7) Malone, M. E.; Symons, M. C. R.; Parker, A. W. J. Chem. Soc.,
Perkin Trans. 2 1993, 2, 2067−2075.
(8) Shaw, A. A.; Voituriez, L.; Cadet, J.; Gregoli, S.; Symons, M. C.
R. J. Chem. Soc., Perkin Trans. 2 1988, 2, 1303−1307.
(9) Barnett, R. N.; Joseph, J.; Landman, U.; Schuster, G. B. J. Am.
Chem. Soc. 2013, 135, 3904−3914.
(10) Kanvah, S.; Schuster, G. B. Org. Biomol. Chem. 2010, 8, 1340−
1343.
(11) Adhikary, A.; Kumar, A.; Heizer, A. N.; Palmer, B. J.;
Pottiboyina, V.; Liang, Y.; Wnuk, S. F.; Sevilla, M. D. J. Am. Chem.
Soc. 2013, 135, 3121−3135.
(12) Greenberg, M. M. Adv. Phys. Org. Chem. 2016, 50, 119−202.
(13) Zhang, Q.; Wang, Y. J. Am. Chem. Soc. 2004, 126, 13287−
13297.
1
yield (101 mg). Major stereoisomer: H NMR (400 MHz, CDCl3) δ
8.09−8.03 (m, 2H), 7.62−7.56 (m, 1H), 7.53 (t, J = 5.2 Hz, 1H),
7.49−7.42 (m, 2H), 4.65−4.47 (m, 2H), 4.01−3.91 (m, 1H), 3.90 (s,
3H), 3.88−3.85 (m, 1H), 2.73−2.43 (m, 2H); 13C NMR (101 MHz,
CDCl3) δ 167.3, 148.3, 133.5, 129.8, 129.6, 128.5, 73.1, 69.9, 66.2,
61.8, 32.7. Minor stereoisomer: 1H NMR (400 MHz, CDCl3) δ
8.09−8.03 (m, 2H), 7.62−7.56 (m, 1H), 7.49−7.42 (m, 2H), 6.90
(dd, J = 7.8, 3.9 Hz, 1H), 4.65−4.47 (m, 2H), 4.01−3.91 (m, 1H),
3.88−3.85 (m, 1H), 3.84 (s, 3H), 2.73−2.43 (m, 2H); 13C NMR
(101 MHz, CDCl3) δ 167.3, 148.3, 133.4, 129.8, 129.5, 128.5, 72.7,
69.5, 66.0, 61.7, 29.3; IR (NaCl) 3421 (bd), 1702, 1457 cm−1; HRMS
(ESI-TOF) m/z [M + Na]+ calcd for C13H17NNaO5 290.1004, found
290.0991.
(14) San Pedro, J. M. N.; Greenberg, M. M. Org. Lett. 2012, 14,
2866−2869.
(15) San Pedro, J. M. N.; Greenberg, M. M. J. Am. Chem. Soc. 2014,
136, 3928−3936.
(16) Zhang, Q.; Wang, Y. Chem. Res. Toxicol. 2005, 18, 1897−1906.
(17) Horner, J. H.; Bagnol, L.; Newcomb, M. J. Am. Chem. Soc.
2004, 126, 14979−14987.
(18) Horner, J. H.; Taxil, E.; Newcomb, M. J. Am. Chem. Soc. 2002,
124, 5402−5410.
(19) Newcomb, M.; Miranda, N.; Sannigrahi, M.; Huang, X.; Crich,
D. J. Am. Chem. Soc. 2001, 123, 6445−6446.
(20) Newcomb, M.; Miranda, N.; Huang, X.; Crich, D. J. Am. Chem.
Soc. 2000, 122, 6128−6129.
ASSOCIATED CONTENT
■
(21) Mori, M.; Ito, T.; Kawano, M.; Takao, Y.; Hatta, H.;
Nishimoto, S. Chem. Res. Toxicol. 2008, 21, 958−965.
(22) Wagner, J. R.; van Lier, J. E.; Berger, M.; Cadet, J. J. Am. Chem.
Soc. 1994, 116, 2235−2242.
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.joc.7b02017.
K
DOI: 10.1021/acs.joc.7b02017
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
(23) Bjelland, S.; Eide, L.; Time, R. W.; Stote, R.; Eftedal, I.; Volden,
G.; Seeberg, E. Biochemistry 1995, 34, 14758−14764.
(24) Wagner, J. R.; van Lier, J. E.; Decarroz, C.; Berger, M.; Cadet, J.
Methods Enzymol. 1990, 186, 502−511.
(25) Saudi, M.; Zmurko, J.; Kaptein, S.; Rozenski, J.; Neyts, J.; Van
Aerschot, A. Eur. J. Med. Chem. 2014, 76, 98−109.
(26) Barvian, M. R.; Greenberg, M. M. J. Org. Chem. 1993, 58,
6151−6154.
(27) Iwai, S. Chem. - Eur. J. 2001, 7, 4343−4351.
(28) Sugiyama, H.; Matsuda, S.; Kino, K.; Zhang, Q.-M.; Yonei, S.;
Saito, I. Tetrahedron Lett. 1996, 37, 9067−9070.
(29) Knapp, D. C.; D’Onofrio, J.; Engels, J. W. Bioconjugate Chem.
2010, 21, 1043−1055.
(30) Chirakul, P.; Sigurdsson, S. T. Tetrahedron Lett. 2003, 44,
6899−6901.
(31) Yu, W.; Mei, Y.; Kang, Y.; Hua, Z.; Jin, Z. Org. Lett. 2004, 6,
3217−3219.
(32) Kuang, Y.; Sun, H.; Blain, J. C.; Peng, X. Chem. - Eur. J. 2012,
18, 12609−12613.
(33) Sun, H.; Peng, X. Bioconjugate Chem. 2013, 24, 1226−1234.
(34) Decarroz, C.; Wagner, J. R.; Cadet, J. Free Radical Res.
Commun. 1987, 2, 295−301.
(35) Hamon, N.; Quintiliani, M.; Balzarini, J.; McGuigan, C. Bioorg.
Med. Chem. Lett. 2013, 23, 2555−2559.
(36) Vermeulen, C.; Verwijs-Janssen, M.; Cramers, P.; Begg, A. C.;
Vens, C. DNA Repair 2007, 6, 202−212.
(37) Liu, L.; Nakatsuru, Y.; Gerson, S. L. Clin. Canc. Res. 2002, 8,
2985−2991.
(38) Weng, L.; Horvat, S. M.; Schiesser, C. H.; Greenberg, M. M.
Org. Lett. 2013, 15, 3618−3621.
(39) Hong, I. S.; Greenberg, M. M. J. Am. Chem. Soc. 2005, 127,
10510−10511.
(40) Ding, H.; Majumdar, A.; Tolman, J. R.; Greenberg, M. M. J.
Am. Chem. Soc. 2008, 130, 17981−17987.
(41) Beckwith, A. L. J.; Crich, D.; Duggan, P. J.; Yao, Q. Chem. Rev.
1997, 97, 3273−3312.
(42) Peng, X.; Hong, I. S.; Li, H.; Seidman, M. M.; Greenberg, M.
M. J. Am. Chem. Soc. 2008, 130, 10299−10306.
(43) Meggers, E.; Dussy, A.; Schafer, T.; Giese, B. Chem. - Eur. J.
̈
2000, 6, 485−492.
(44) Ghosh, A.; Joy, A.; Schuster, G. B.; Douki, T.; Cadet, J. Org.
Biomol. Chem. 2008, 6, 916−928.
L
DOI: 10.1021/acs.joc.7b02017
J. Org. Chem. XXXX, XXX, XXX−XXX