Electron-Mediated Aminyl and Iminyl Radicals from C5 Azido-Modified Pyrimidine Nucleosides Augment Radiation Damage to Cancer Cells

Article abstract of DOI:10.1021/acs.orglett.8b03035

Two classes of azido-modified pyrimidine nucleosides were synthesized as potential radiosensitizers; one class is 5-azidomethyl-2′-deoxyuridine (AmdU) and cytidine (AmdC), while the second class is 5-(1-azidovinyl)-2′-deoxyuridine (AvdU) and cytidine (AvdC). The addition of radiation-produced electrons to C5-azido nucleosides leads to the formation of π-aminyl radicals followed by facile conversion to σ-iminyl radicals either via a bimolecular reaction involving intermediate α-azidoalkyl radicals in AmdU/AmdC or by tautomerization in AvdU/AvdC. AmdU demonstrates effective radiosensitization in EMT6 tumor cells.

Full text of DOI:10.1021/acs.orglett.8b03035

Letter
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Electron-Mediated Aminyl and Iminyl Radicals from C5 Azido-
Modified Pyrimidine Nucleosides Augment Radiation Damage to
Cancer Cells
†
‡
†
†
‡
,‡
†
†
‡
§
Zhiwei Wen, Jufang Peng, Paloma R. Tuttle, Yaou Ren, Carol Garcia, Dipra Debnath,
‡
‡
§
Sunny Rishi, Cameron Hanson, Samuel Ward, Anil Kumar, Yanfeng Liu, Weixi Zhao,
§
†
,‡
,†
Peter M. Glazer, Yuan Liu, Michael D. Sevilla,* Amitava Adhikary,* and Stanislaw F. Wnuk*
†
Department of Chemistry and Biochemistry, Florida International University, Miami, Florida 33199, United States
‡
Department of Chemistry, Oakland University, Rochester, Michigan 48309, United States
§
Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut 06520, United States
*
S Supporting Information
ABSTRACT: Two classes of azido-modified pyrimidine nucleosides
were synthesized as potential radiosensitizers; one class is 5-azidomethyl-
2
5
′-deoxyuridine (AmdU) and cytidine (AmdC), while the second class is
-(1-azidovinyl)-2′-deoxyuridine (AvdU) and cytidine (AvdC). The
addition of radiation-produced electrons to C5-azido nucleosides leads to
the formation of π-aminyl radicals followed by facile conversion to σ-
iminyl radicals either via a bimolecular reaction involving intermediate α-
azidoalkyl radicals in AmdU/AmdC or by tautomerization in AvdU/
AvdC. AmdU demonstrates effective radiosensitization in EMT6 tumor
cells.
yrimidine nucleosides modified at C5 (e.g., 5-bromo-2′-
deoxyuridine (AmdU, 4), 5-azidomethyl-2′-deoxycytidine
(AmdC, 6), 5-(1-azidovinyl)-2′-deoxyuridine (AvdU, 12),
and 5-(1-azidovinyl)-2′-deoxycytidine (AvdC, 13) ana-
P
deoxyuridine) are well-investigated as radiosensitizers in
1
cancer therapy. 5-(Phenylselenyl)methyl-2′-deoxyuridine and
12,13
5
-thiocyanato-2′-deoxyuridine were shown to induce reactive
logues
(Schemes 1 and 2) to study the radiation-mediated
2
3
•
benzyl-type or uracil-C5-thiyl radical for DNA interstrand
formation of RNH and its subsequent reactions. Recently,
AmdU has been metabolically incorporated into DNA in living
2
,3
4
cross-linking and apoptosis in cancer cells.
14
3
′-Azido-3′-deoxythymidine (AZT) has shown a significant
cells for click labeling of DNA. Moreover, its 5′-triphosphate
radiosensitization in irradiated human colon cancer, larynx
was found to be the substrate for DNA polymerases and PCR
5
15,16
Herein, we report that π-aminyl RNH•
squamous carcinoma, and malignant glioma cells. Moreover,
amplification.
AZT has been employed as a radiation sensitizer in
generated by radiation-produced electron addition to azido-
nucleosides undergoes conversion to the thermodynamically
6
radiotherapy of tumors for HIV-positive patients. We found
7
•
that the radiation-produced prehydrated electrons leads to the
more stable σ-iminyl radicals (RN ). We also show
•
site-specific formation of a localized π-aminyl radical (RNH )
incorporation of AmdU and AmdC phosphates into DNA
fragments by polymerase-catalyzed reactions and present
evidence that these azidonucleosides can act as radio-
sensitizers.
8
9,10
in azido-substituted nucleosides and sugars.
Our ESR
•
studies show that RNH formed from AZT undergoes
bimolecular H atom abstraction from a C5-methyl group,
•
AmdU 4 was synthesized from thymidine 1 by sequential
generating a thymine allyl radical (dUCH ), or from a sugar
2
•
8
•
silylation, bromination with NBS, displacement of bromide
moiety yielding C5′ . Moreover, RNH and modified base
14,17
with NaN , and desilylation (45% overall, Scheme 1).
radicals can produce sugar radicals which lead to DNA-strand
3
1
a
1c
Treatment of 3 with 2,4,6-triisopropylbenzenesufonyl chloride
TIPBSCl) in the presence of TEA/DMAP followed by
breaks and eventually lead to induction of apoptosis. We
hypothesize that incorporation of azido-modified nucleosides
into genomic DNA would augment radiation-induced damage
in cells owing to the reactions of aminyl radicals under hypoxic
conditions and, therefore, act as potential radiosensitizers.
Owing to the poor incorporation of AZT in genomic
(
displacement of the resulting aryl sulfate with NH OH and
4
deprotection of 5 with TBAF provided AmdC 6 (57% overall).
Received: September 28, 2018
1
1
DNA, in this work, we have employed 5-azidomethyl-2′-
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.8b03035
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Scheme 1. Synthesis of 5-Azidomethyl Pyrimidine
Nucleosides and Their 5′-Triphosphates
Scheme 2. Synthesis of 5-Azidovinyl Pyrimidine
Nucleosides
Figure 1. (A) ESR spectrum (blue) after radiation-produced one-
electron addition to AmdU 4 (2.2 mg/mL) at 77 K (γ-irradiation, 500
Gy) in 7.5 M LiCl/D O in dark. Spectra B−E were obtained via
2
stepwise annealing of the sample for 15 min at 140, 160, 165, and 170
K in the dark. All spectra were recorded at 77 K. The radiation
produced background Cl2• spectrum was subtracted from spectra A
and B for clarity. Three reference triangles show Fremy’s salt
resonances with central marker at g = 2.0056.
−
18
AmdU and AmdC were triphosphorylated to give AmdUTP
7
(76%) and AmdCTP 8 (23%).19
Silver-catalyzed hydroazidation of the protected 5-ethynyl-
Scheme 3. Formation of π-Aminyl Radical from AmdU and
Its Bimolecular Conversion to the σ-Iminyl Radical
2
0
2
′-deoxyuridine 9 with TMSN regioselectively produced α-
3
21
azidovinyl 11 (52%; Scheme 2). Deacetylation of 11 gave
1
3
AvdU 12 (90%). Analogous hydroazidation of the protected
2
2
5
-ethynyl-2′-deoxycytidine 10 followed by deacetylation
provided AvdC 13 (42% overall). Conversation of uracil
23
base in 11 to a cytosine counterpart by treatment of 11 with
TIPBSCl followed by ammonolysis also gave 13 (Supporting
Information (SI)).
Radiation-produced electron addition to 4 allowed us to
•
identify three radicals: (i) π-aminyl radical, π-U-5-CH −ND ;
2
•
(
ii) α-azidoalkyl radical, U-5-CH −N ; and (iii) σ-iminyl
3
•
radical, U-5-CHN (Figures 1 and S1, Scheme 3). The
center of the spectrum in Figure 1A (blue) does not show the
2
4
•−
doublet expected for U , thereby indicating that the azide
moiety of 4 scavenged nearly all radiation-produced electrons
to form the π-aminyl RNH via a dissociative electron
attachment. The initial electron attachment is followed by
rapid protonation of the incipient nitrene anion radical
in the center of spectrum 1C and resolved as a multiplet in
•
spectrum 1D is assigned to a C-centered α-azidoalkyl radical
•
•
(U-5-CH −N ) formed via H-abstraction by RNH from a
3
proximate parent 4 (Scheme 3). In the spectrum in Figure 1C,
the second species begins to form. The total hyperfine splitting
decreases by ca. 8 G from that found in Figure 1A,B (i.e., the
wings line components move in), and this new species
becomes the only radical found in Figure 1E. The spectrum in•
Figure 1E is assigned to the σ-iminyl radical, U-5-CHN
resulting from couplings of one anisotropic nitrogen and one
β-proton. For assignments of radicals contributing to the
spectra in Figure 1D,E and HFCC, see the SI (pages S6 and
(
Scheme 3). The spectrum extends over 178.5 G, and its
hyperfine structure shows line components from the hyperfine
coupling (HFCC) due to single axially symmetric anisotropic
nitrogen [A = ∼42.5 G, A = ∼0] with g and g values that
||
⊥
||
⊥
are typical of aminyl radical nitrogen (Table S1). In U-5-CH −
2
•
ND , the radical-site p-orbital strongly couples with two β-
−
CH2 protons, generating the wide doublet (ca. 93.5 G). The
•
simulated spectrum of U-5-CH −ND (Figure 1A, red; see the
SI for simulation parameters) matches the line components of
the experimental blue spectrum in Figure 1A.
2
S7, Table S1, and Figures S1 and S2). These results show that
•
the reactive π-U-5-CH −ND abstracts an H-atom from the
2
On subsequent stepwise annealing at 140, 160, 165, and 170
K, new line components appear which are assigned to two new
species (Figure 1B−E, blue). The first species initially observed
parent 4 to form the azidoalkyl radical intermediate, U-5-
CH −N . Subsequently, U-5-CH −N promptly undergoes a
unimolecular β-N elimination from the azide group to
•
•
3
3
2
B
DOI: 10.1021/acs.orglett.8b03035
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
•
produce the thermodynamically more stable σ-U-5-CHN
ESR spectra obtained upon progressive annealing (Figure
2B−D, black) show that height of the singlet at the center
increases with concomitant decrease of the line components
(Scheme 3).
•
The π-C-5-CH −ND formation and its subsequent
2
•
conversion to the σ-iminyl radical, C-5-CHN , has also
been observed upon radiation-produced electron attachment
to cytosine counterpart AmdC 6 (Scheme S1, Figure S2, and
Table S1). The α-azidoalkyl radicals generated from photolysis
from the two anisotropic α CH protons. The black
2
spectrum (Figure 2D) is due solely to an axially symmetric
anisotropic nitrogen (for HFCCs see Table S1) and is assigned
•
to the σ-iminyl radical, U-5-C(CH )N . The simulated
3
25
26
of α-azidoacetophenones or γ-azidobutyrophenone as well
as α-azido o-iodoanilides under tin-mediated radical reac-
tions are known to undergo similar conversion to the σ-
iminyl radicals.
A radiation-produced electron addition to the vinyl azide 12
also generates π-RNH , which undergoes facile tautomeriza-
spectrum (Figure 2D, red; Table S1) matches the line
components of the black spectrum. Nearly identical spectra
28
2
7
were obtained from one-electron attachment to AvdC, proving
•
that formation of the π-aminyl radical (C-5-C(ND )CH )
2
and its facile tautomerization to the σ-iminyl radical (C-5-
C(CH )N ) has a general character and occurs independent
•
•
3
tion to thermodynamically more stable σ-iminyl radical (Figure
and Scheme 4). The 77 K ESR spectrum (black, Figure 2A)
of the nucleobase.
2
Increasing the concentration (1 to 5 mg/mL) of 12 has no
effect on the extent of this conversion as observed from spectra
recorded under the same microwave power, modulation, and
gain. From these results, we conclude that the conversion of π-
RNH• to σ-RN observed in 12 and 13 is by
tautomerization; it occurs via facile intramolecular proton
•
transfer from the aminyl group to the double-bonded CH
2
•
•
•
group in the π-RNH . Conversion of π-RNH to σ-RN has
also been observed in one-electron oxidized 1-methylcytosine
2
8
and its derivatives.
In contrast to our previous results in which π-RNH•
produced from one-electron reduction of AZT and azidopen-
8
,9
toses undergoes H atom abstraction reactions, we find that
•
π-RNH from 4, 6, 12, and 13 undergoes conversion to σ-R
•
•
•
N . For 4 and 6, the π-RNH to σ-RN conversion is
bimolecular which, at first, involves H-abstraction to form an
α-azidoalkyl radical followed by a unimolecular decomposition
•
to σ RN (Scheme 3). However, 12 and 13 undergo
•
•
tautomerization of π-RNH to σ-RN (Scheme 4). Owing to
1a
the high free-radical scavenger concentrations in cells, the
Figure 2. (A) ESR spectrum (black) after radiation-produced one-
electron addition to 12 (1 mg/mL) at 77 K (γ-irradiation, 500 Gy) in
•
•
bimolecular conversion of π-RNH to σ-RN from 4 and 6
is unlikely to take place. However, the tautomerization of π-
7
.5 M LiCl/D O in dark. (B−D) Spectra after stepwise annealing at
•
•
2
RNH to σ-RN from 12 and 13 should occur even in cells.
1
35, 150, and 165 K recorded at 77 K. The red spectra in (A) and
•
•−
Further, reactivity of σ-RN from 12 and 13 is far less than
(
D) are the simulated spectra. The background Cl2 spectrum has
•
•
that of a π-RNH . Therefore, it is expected that the π-RNH
been subtracted from spectra A and B for clarity.
from 4 and 6 could augment radiation damage more effectively
•
than the σ-RN from 12 and 13.
Scheme 4. Tautomerization of π Aminyl Radical, Generated
from AvdU, to σ-Iminyl Radical
Incorporation of nucleosides into DNA is important for
1b−d,29
them to exhibit radiosensitization.
Thus, AmdUTP was
successfully incorporated by the E. coli Klenow fragment of
DNA polymerase I (pol I) and human repair DNA polymerase
β (pol β) during DNA leading and lagging strand synthesis and
BER (base excision repair) using an open template, one-
nucleotide gap substrates, and one-nucleotide substrate
containing a 5′-THF (a tetrahydrofuran ring which mimics a
sugar residue; Figure S6, Table S2; see the SI for more details).
Incorporation of AmdUTP in the presence of dATP, dGTP,
and dCTP showed that both polymerases readily inserted
AmdUTP into dsDNA and extended the nucleotide during
DNA replication and BER (Figure S7). AmdU incorporation
can be ligated into duplex DNA during DNA replication and
BER in the presence of LIG I (Figure S8). These results are
consistent with a recent finding that AmdU was efficiently
incorporated into newly synthesized DNA in human cancer
shows line components from single axially symmetric
anisotropic nitrogen HFCCs and from two anisotropic protons
of the CH group in 12 (Table S1). We assign these to the
2
•
π-aminyl radical, U-5-C(ND )CH (Scheme 4). Formation
2
•
of U-5-C(ND )CH results from a dissociative electron
2
attachment pathway similar to that shown in Scheme 3. The
simulated red spectrum (see Table S1 for simulation
parameters) matches line components of the black spectrum
14
cells. The AmdCTP 8 was also incorporated into DNA by
pol β during DNA replication and BER (Table S2, template 2;
Figure S9).
To test our hypothesis that azidonucleosides incorporated
into DNA can act as radiosensitizers, we investigated the
and supports our assignment (Figure 2A). HFCC values for
•
the anisotropic nitrogen and CH α-protons in U-5-C(ND )
2
CH and its optimized geometry are reported in Table S1 and
2
Figure S5.
C
DOI: 10.1021/acs.orglett.8b03035
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
radiation response of EMT6 breast cancer cells to the presence
of 100 μM azido-modified nucleosides in both aerobic and
hypoxic environments in terms of survival fraction (SF). For
survival fraction measurements in aerobic cells, the cultures
were treated with a 100 μM azido compound or vehicle for 48
h. For the measurements in hypoxic cells, hypoxic conditions
were applied for 4 h after a 44 h aerobic incubation with 100
μM azido compounds or vehicles. To investigate the
radiosensitizing effect, cells were irradiated with 7.5 Gy X-ray
during the final few minutes of the 48 h incubation (Figure 3
and Table S3).
ASSOCIATED CONTENT
Supporting Information
■
*
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.8b03035.
Experimental procedures, mechanistic and theoretical
studies, and compound characterization data for all new
compounds (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
*
*
*
E-mail: sevilla@oakland.edu.
E-mail: adhikary@oakland.edu.
E-mail: wnuk@fiu.edu.
ORCID
Zhiwei Wen: 0000-0002-0821-1369
Anil Kumar: 0000-0002-9979-7798
Michael D. Sevilla: 0000-0001-8799-5458
Amitava Adhikary: 0000-0001-9024-9579
Stanislaw F. Wnuk: 0000-0002-3111-3919
Figure 3. Radiosensitizing effect of 100 μM azido-modified
nucleosides on EMT6 cells: (A) normoxic and (B) hypoxic
conditions.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors are thankful for financial support from the NIH
M.D.S., R01 CA045424; Y.L., ES023569; S.F.W.,
SC1CA138176). Z.W. is a recipient of the FIU Dissertation
Year Fellowship. J.P. is grateful for support from the Oversea
Scholarship Program of Shaanxi Normal University.
AmdU showed radiosensitization under both normoxic and
hypoxic environments with sensitization enhancement ratios
■
(
(
(
SER) at 7.5 Gy X-ray (SERSF7.5) of 4.57 and 4.10, respectively
Figure 3, Table S3). These results show that aminyl radicals
generated in AmdU augments radiation damage to cells. In a
•
hypoxic microenvironment, RNH formed from electron
addition to AmdU can be involved in the H atom abstraction
1
a,8,9
1
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■
c
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•
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•
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2
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Org. Lett. XXXX, XXX, XXX−XXX

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E
DOI: 10.1021/acs.orglett.8b03035
Org. Lett. XXXX, XXX, XXX−XXX