Site of azido substitution in the sugar moiety of azidopyrimidine nucleosides influences the reactivity of aminyl radicals formed by dissociative electron attachment

Article abstract of DOI:10.1021/acs.jpcb.0c08201

In this work, electron-induced site-specific formation of neutral π-type aminyl radicals (RNH·) and their reactions with pyrimidine nucleoside analogs azidolabeled at various positions in the sugar moiety, e.g., at 2′-, 3′-, 4′-, and 5′- sites along with a model compound 3-azido-1-propanol (3AZPrOH), were investigated. Electron paramagnetic resonance (EPR) studies confirmed the site and mechanism of RNH· formation via dissociative electron attachment-mediated loss of N₂ and subsequent facile protonation from the solvent employing the ¹⁵N-labeled azido group, deuterations at specific sites in the sugar and base, and changing the solvent from H₂O to D₂O. Reactions of RNH· were investigated employing EPR by warming these samples from 77 K to ca. 170 K. RNH· at a primary carbon site (5′-azido-2′,5′-dideoxyuridine, 3AZPrOH) facilely converted to a σ-type iminyl radical (R=N·) via a bimolecular H-atom abstraction forming an α-azidoalkyl radical. RNH· when at a secondary carbon site (e.g., 2′-azido-2′-deoxyuridine) underwent bimolecular electrophilic addition to the C5=C6 double bond of a proximate pyrimidine base. Finally, RNH· at tertiary alkyl carbon (4′-azidocytidine) underwent little reaction. These results show the influence of the stereochemical and electronic environment on RNH· reactivity and allow the selection of those azidonucleosides that would be most effective in augmenting cellular radiation damage.

Full text of DOI:10.1021/acs.jpcb.0c08201

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Article
Site of Azido Substitution in the Sugar Moiety of Azidopyrimidine
Nucleosides Influences the Reactivity of Aminyl Radicals Formed by
Dissociative Electron Attachment
Mukesh Mudgal, Thao P. Dang, Adam J. Sobczak, Daniel A. Lumpuy, Priya Dutta, Samuel Ward,
Katherine Ward, Moaadh Alahmadi, Anil Kumar, Michael D. Sevilla, Stanislaw F. Wnuk,*
and Amitava Adhikary*
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ABSTRACT: In this work, electron-induced site-specific formation of neutral π-type aminyl
radicals (RNH·) and their reactions with pyrimidine nucleoside analogs azidolabeled at various
positions in the sugar moiety, e.g., at 2′-, 3′-, 4′-, and 5′- sites along with a model compound 3-
azido-1-propanol (3AZPrOH), were investigated. Electron paramagnetic resonance (EPR)
studies confirmed the site and mechanism of RNH· formation via dissociative electron
attachment-mediated loss of N2 and subsequent facile protonation from the solvent employing
the 15N-labeled azido group, deuterations at specific sites in the sugar and base, and changing
the solvent from H2O to D2O. Reactions of RNH· were investigated employing EPR by
warming these samples from 77 K to ca. 170 K. RNH· at a primary carbon site (5′-azido-2′,5′-
dideoxyuridine, 3AZPrOH) facilely converted to a σ-type iminyl radical (RN·) via a
bimolecular H-atom abstraction forming an α-azidoalkyl radical. RNH· when at a secondary
carbon site (e.g., 2′-azido-2′-deoxyuridine) underwent bimolecular electrophilic addition to
the C5C6 double bond of a proximate pyrimidine base. Finally, RNH· at tertiary alkyl
carbon (4′-azidocytidine) underwent little reaction. These results show the influence of the
stereochemical and electronic environment on RNH· reactivity and allow the selection of those azidonucleosides that would be most
effective in augmenting cellular radiation damage.
INTRODUCTION
■
Neutral aminyl radicals (RNH·) are π-type radicals that are
important in chemical synthesis and in biology.1−6 RNH· plays
an important role in the prototropic equilibria of DNA-base π-
cation radicals (such as guanine cation radical (G·+), cytosine
cation radical (C·+), adenine cation radical (A·+)) that are
involved in charge (hole) transfer processes followed by
localization of damage.4,6 Moreover, ·OH addition to the
C4C5 double bond in purine bases (G, A) followed by water
elimination lead to the same RNH· species (G(N1−H)·,
A(N6−H)·) that are formed via deprotonation of G·+ and A·+.4,6
It is well established that RNH· undergo a wide variety of
reactions that are summarized below.
(A) Tautomerization: π-Type RNH· is shown to undergo
tautomerization to the σ-type RN·:
Also, the deprotonated cytosine π-cation radical (C(N4−H)·)
tautomerizes facilely to the σ-iminyl radical (reaction 1b).17
Received: September 8, 2020
Revised: November 9, 2020
For example, electron paramagnetic resonance (EPR) studies
have established that π-type aminyl radical (U-(5-CCH2)-
NH·) formed from 5-(1-azidovinyl) modified 2′-deoxyuridine
facilely tautomerizes to the σ-type iminyl radical, U-(5-C-
CH3)N· (reaction 1a).7
https://dx.doi.org/10.1021/acs.jpcb.0c08201
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A

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Table 1. Hyperfine Coupling Constants (HFCC) and g Values of Radicals Reported in This Work
hyperfine couplings (G)
compound/type/radical
site(nucleus)
exp
theory
g value (exp)
a
a
π/T(5′-CH2)-NH· (5′-AZT)
β-protons (H5′, H5″)
N5′−H (α-proton)
N5′-coupling
Sum 91
a
a
a
a
(−, −, −30)
(0, 0, 43)
Sum 93.5
(−39.8, 0.4, −26.7)
g∥ = 2.0020 , g⊥ = 2.0043
a
(0, 0, 41.8) G a
b
b
b
π/U-5-CH2-NH· (AmdU)
β-protons (H5, H5)
5/π/dU-5′-CH2-NH·
β-protons (H5′, H5″) (for 5)
N5−H (α-proton)
Sum 90.5
(−, −, −30)
g∥ = 2.0020, g⊥ = 2.0043
b
a
N5′−H (α-proton)
N5/N5′-coupling
1αH
1αH
1Ν
(0, 0, 42.5)
α-azidoalkyl radical
5/π/dU-5′-CH·-N3
(−10.97, −15.03,-5.09)
(−14.46,-20.64,-6.67)
(10.5, −0.5, −1.32)
2.0038 yy
2.0021 zz
2.0043 xx
c
b
b
σ U-5-CHN·
β-proton (H5) b
N5-coupling b
β-proton (H5′) 5
N5′-coupling
N2′-coupling
C2′−H (β-proton)
N2′−H (α-proton)
N3′-coupling
C3′−H (β-proton)
N3′−H (α-proton)
N4′-coupling
N4′−H (α-proton)
82
(71.8, 78.0,72.8)
b
b
(0, 0, 36.5)
82
(−4.2, 0, 37.7)
g∥ = 2.0016, g⊥ = 2.0040
d
5 σ dR-5′-CHN·
(72.9, 78.7, 73.0)
d
(0, 0, 36.5)
(0, 0, 40.5)
51.5
(−4.44, −0.9, 36.6)
1 π dC(C2′)-ND· (π C(C2′)-ND·)
2 π dU(C2′)-ND· (π U(C2′)-ND·)
g∥ = 2.0020, g⊥ = 2.0043
g∥ = 2.0020, g⊥ = 2.0043
e
(*, *, −28)
e
3 π 2′,3′-ddU(C3′)-ND·
(0, 0, 37.5)
41
e
e
(*, *, −28)
e
e
4 π C(C4′)-ND·
(0, 0, 37.5)
(*, *, −28)
g∥ = 2.0020, g⊥ = 2.0043
a
b
c
Taken from ref 8. Taken from ref 7. Employing the B3LYP/6-31G** method, the structure of 5′-CH·-N3 was optimized and HFCCs of the fully
optimized structure of 5′-CH·-N3 was calculated (see the Supporting Information). Linewidth = 3.5 G and a mixed lineshape (Lorentzian/Gaussian
d
= 1) was employed to simulate the experimental spectrum. Employing the B3LYP/6-31G** method, structures of 5′-CHN· of U-5-CHN·
were fully optimized and HFCCs of these optimized structures were calculated (see the Supporting Information). Linewidths as (7, 5, 5) G and
e
lineshape with a mixed Lorentzian/Gaussian = 1 were employed to simulate the experimental spectrum. Taken from ref 8. For simulation, we used
linewidth = 10 G and lineshape with a mixed Lorentzian/Gaussian = 1.
azidoalkyl radical, which undergoes a facile conversion to the σ-
type R′N·:7
R′−CH2−N3
−N2
̇
RNH· ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→ R′−CH−N3 ⎯⎯⎯→ R′−CHN·
− RNH2
(2a)
(C) Transposition of radical site via intramolecular H-atom
transfer, e.g., 1,2 shift, 1,5 shift: Pulse radiolysis of amino acids at
ambient temperature presented evidence of 1,2 H-atom shift in
the ·OH-mediated formation of oxidizing radicals in amino
(B) Intermolecular H-atom abstraction generating C-centered
radicals: It is well established that RNH· can abstract H atoms
from weaker CX−H bonds via a bimolecular pathway to form C-
centered radicals.1−16 For example, RNH· generated from AZT
undergo bimolecular H-atom abstractions from the −CH3
group at C5 of thymine base forming the allylic radical,
dUCH2· (ca. 55%), and from the C5′-site of a proximate AZT
producing the strand break precursor radical, C5′·.8
−
acids, for example, aminyl radical (·NH−CH2−CO2 ) from
−
glycine rearranges to NH−·CH2−CO2 by intramolecular 1,2
H-shift (reaction 3):22,23
1,2 H‐atom
−
−
̇
·NH−CH2−CO2 ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→⎯ NH2−CH−CO2
(3)
shift
EPR studies have presented evidence of facile and intra-
molecular 1,5 H-shift in RNH· in lyxofuranoside leading to the
formation of C5· radical.9
(D) β-Elimination: Pulse radiolysis has shown that the
−
oxidizing aminyl radical (e.g., ·NH−CH2−CO2 ) generated
by the reaction of ·OH with an amino acid (e.g., glycine) leads to
the formation of CO2·− (reaction 4):22,23
In addition, intermolecular H-atom abstraction by RNH· from a
proximate compound (e.g., 5-azidomethyl-2′-deoxyuridine)
having an α-azidoalkyl moiety leads to the formation of an α-
−
•−
·NH−CH2−CO2 → NHCH2 + CO2
(4)
B
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Scheme 1. Synthesis of 2′-Azido-2′-deoxyuridine 2 and Its 5,6-[2H2]-2 and 2′-[15N]-N3-2 Isotopes
(E) Intramolecular cyclization: Dialkylaminyl radicals [RH,
Me; R′H, Me (reaction 5)] are shown to undergo
intramolecular cyclization reactions leading to the formation
of C-centered radicals.10,16
subsequent reactions of RNH· are investigated employing EPR
spectroscopy. A set of [2H]-labeled 2 (C2′, C3′, C4′, C5, and
C6) isotopomers and that of [15N]-azido labeled 2 have led us to
unambiguously assign that in all compounds including the
isotopically substituted ones, the predominant site of electron
attachment is the azido group, and to characterize the resultant
RNH· and its subsequent reactions. The experimentally
obtained hyperfine coupling constant (HFCC) values of
RNH· have been compared with the corresponding theoretically
calculated values for EPR spectral assignment (Table 1). Similar
to our earlier work on the reaction of RNH· from the azido
substitution to a primary carbon at C5 of 5-azidomethyl-2′-
deoxyuridine (AmdU),7 a bimolecular conversion of RNH·
attached to a primary carbon site (5′, 5, 6) to the σ-type iminyl
radical (RN·) involving an α-azidoalkyl radical as inter-
mediate was observed. RNH· from 7 underwent this conversion
as well. Thus, our earlier results from AmdU,7 our results from 5,
6, and 7 in this worked have established the general nature of this
RNH· to RN· bimolecular conversion, i.e., this conversion
happens in any azido compound in which the azido group is
linked to a primary carbon. Bimolecular electrophilic addition of
RNH· attached to the secondary carbon site to the C5C6
double bond of a proximate pyrimidine (uracil and cytosine but
not thymine) base is observed. Finally, a little reaction was
observed in the glassy system from RNH· at tertiary alkyl carbon.
Our results presented in this work demonstrate that the
stereochemical and electronic environment of azido substitution
at specific sites in the sugar moiety significantly affect the
reactivity of RNH·. Azidonucleosides have also been shown to
augment radiation damage in various types of cancer cells.7,35−39
In addition, 2′-azido-2′-deoxyuridine (2) 5′-phosphate is shown
to be a potent inhibitor of ribonucleotide reductases following a
radical chemistry-based inactivation pathway.40−42 Therefore,
the results presented in this work in conjunction with our own
work on azido substitution at C5 in the pyrimidine base7 provide
the basis to choose the type of azidonucleoside that will be
suitable to augment significant radiation damage to tumor cells,
thereby improving the efficacy of tumor radiochemotherapy.
RNH· radical is generated by one-electron oxidation of
primary amines followed by deprotonation of the primary amine
cation radical (RNH2·+).4,17−34 Thus, RNH· becomes the
Brønsted base, RNH2·+ is the conjugate acid, and these two
species (RNH· and RNH2·+) are in prototropic equilibrium if R
is a delocalized structure (see for example, reaction 1b). In
addition, the formation of RNH· via photodissociation11−15 and
via ·OH employing pulse radiolysis4,22,23 have been reported.
We have developed a novel method of site-specific electron-
mediated RNH· generation employing azidonucleosides (re-
action 2).6−9 Our work has established that radiation-produced
prehydrated electron (epre−)-mediated dissociative electron
attachment to azidonucleosides (RN3) in homogeneous super-
cooled glassy systems at 77 K,9 at first, leads to the formation of
highly basic and unstable nitrene anion radical (RN·−) at 77 K
via spontaneous loss of N2 from the azidonucleoside anion
radical (RN3·−). Subsequent facile protonation of RN·−
produces RNH·. Neither RN3·− nor RN·− intermediates were
detected by using electron paramagnetic resonance (EPR)
spectroscopy even at 77 K, and only the EPR spectra of RNH·
were observed.6−9 So far, investigations from our laboratory
have presented evidence of reactions 1−3a. Also, our work has
shown that reaction mechanisms of RNH· depend on the type of
compounds (azidomethyl vs azidovinyl)6,7 and conformation of
the sugar (lyxofuranose vs 2′-deoxyribofuranose).9
In this work, we have employed uracil and cytosine
nucleosides with the azido group substituted at the 2′ (2′-
azido-2′-deoxycytidine (2′-AZdC, 1), 2′-azido-2′-deoxyuridine
(2′-AZdU, 2)), 3′ (3′-azido-2′,3′-dideoxyuridine (3′-AZ-2′,3′-
ddU, 3)); 4′ (4′-azidocytidine (4′-AZC, 4)); and 5′ (5′-azido-
2′,5′-dideoxyuridine (5′-AZ-2′,5′-ddU, 5), 5′-azido-5′-deoxy-
thymidine (5′-AZT, 6)) positions. An azido-substituted alcohol
(3-azido-1-propanol (3AZPrOH, 7)) has also been investigated
as a model of azido group linked to the primary carbon. Thus,
the azido group is attached to the primary carbon, 5′ (5, 6) and
3AZPrOH (7), to the secondary carbon (2′ and 3′), and to the
tertiary carbon (4′) site. RNH· formation via attachment of
radiation-produced prehydrated electrons to these compounds
in γ-irradiated aqueous glassy (7.5 M LiCl) systems and
EXPERIMENTAL SECTION
■
2′-Azido-2′-deoxycytidine (2′-AZdC, 1)43 and 4′-azidocytidine
(4′-AZC, 4)44 were prepared by slight modifications of the
literature protocols. The synthesis and spectroscopic character-
ization of 2′-azido-2′-deoxyuridine (2′-AZdU, 2) and its
derivatives with deuterations at specific sites of the sugar
moiety, e.g., 2′-D-2′-AZdU ([2′-D]-2), 3′-D-2′-AZdU ([3′-D]-
2), and 4′-D-2′-AZdU ([4′-D]-2), or at the base, 5,6-D,D-2′-
C
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AZdU ([5,6-D,D]-2) and 15N incorporated azido group in 2 are
described in the main manuscript (Schemes 1 and 2). The
methodologies, and the details of their synthesis as well as their
coupling with uracil to give uridines 12a−c; their subsequent
conversion to 2,2′-O-anhydro precursors 13a−c are described in
the Supporting Information.
Scheme 2. Synthesis of Ribose Deuterated 2′-Azido-2′-
deoxyuridine isotopes 2′-[2H]-2 (X = D), 3′-[2H]-2 (Y = D),
and 4′-[2H]-2 (Z = D)
2′-Azido-2′-deoxyuridine (2). The stirred solution of 9a (30
mg, 0.13 mmol; see the Supporting Information for the
preparation of 9a) and NaN3 (60 mg, 0.93 mmol) in HMPA
(0.5 mL) was heated at 150 °C. After 30 min, benzoic acid (16
mg, 0.13 mmol) was added and heating was continued for
another 15 min. The cooled reaction mixture was diluted with
H2O and washed with CHCl3. The organic layer was back-
extracted with H2O. The combined aqueous layers was
concentrated and purified on silica gel column (EtOAc/
MeOH, 9:1) or preparative RP-HPLC (H2O/MeCN, 6:1) to
afford 246,47 (20 mg, 56%): 1H NMR (D2O) δ 3.70 (dd, J = 4.3,
12.8 Hz, 1, H5″), 3.82 (dd, J = 2.8, 12.8 Hz, 1, H5′), 4.00 (“ddd”,
J = 2.9, 4.2, 6.0 Hz, 1, H4′), 4.22 (“dd”, J = 4.5, 5.7 Hz, 1, H2′),
4.35 (t, J = 5.9 Hz, 1, H3′), 5.77 (d, J = 8.1 Hz, 1, H5), 5.80 (d, J
= 4.4 Hz, 1, H1′), 7.75 (d, J = 8.1 Hz, 1, H6); MS (ESI−) m/z
268 (MH−).
2′-[15N]-N3-2′-Azido-2′-deoxyuridine (2′-[15N]-N3-2). Treat-
ment of 9a (20 mg, 0.09 mmol) with [15N]-NaN3 (40.5 mg, 0.61
mmol; >97% 15N) as described above gave 2′-[15N]-N3-248 (8
mg, 33%) with identical physical, chemical, and spectroscopic
properties (1H NMR) as 2 except for MS (ESI−) m/z 269 (M −
H−).
5,6-[2H2]-2′-Azido-2′-deoxyuridine (5,6-[2H2]-2). Azidation
of 9b (18 mg, 0.08 mmol; see the Supporting Information for the
preparation of 9b) as described above afforded 5,6-[2H2]-2 (11
mg, 52%) with identical physical, chemical, and spectroscopic
properties as 2 except for the disappearance of H6 peak at 7.75
ppm and the presence of the residual peak (∼ 30%) for H5 at
5.77 ppm (s): MS (ESI−) m/z 270 (MH−).
general experimental methodologies and synthesis of all other
synthetic intermediates (Schemes S1 and S2) are described in
the Supporting Information. Azido compounds 3′-AZ-2′,3′-
ddU (3), 5′-AZ-2′,5′-ddU (5), 5′-AZT (6), 3AZPrOH (7), and
3′-AZT are commercially available. Sodium azide 15N-labeled
(97%) in the terminal position and deuterium-labeled uracil
were purchased from Cambridge Isotope Laboratories.
For EPR studies, methods of preparation of homogeneous
glassy samples of 1 to 7, [2′-D]-2, [3′-D]-2), [4′-D]-2), [5,6-
D,D]-2, and 15N incorporated azido group in 2 in 7.5 M LiCl/
D2O or 7.5 M LiCl/H2O, γ-irradiation and storage of these
glassy samples including the stepwise annealing of these glassy
samples are described in the Supporting Information. An EPR
spectrometer, experimental setup (field calibration employing
Fremy’s salt, microwave power, etc.) for recording the EPR
spectra at 77 K, and methods of theoretical calculations are
described in the Supporting Information.
2′-[2H]-2′-Azido-2′-deoxyuridine (2′-[2H]-2). Azidation of
13a (22 mg, 0.10 mmol) as described above afforded 2′-[2H]-2
(7 mg, 27%) with identical spectroscopic properties as 2 except
for the disappearance of H2′ at 4.22 ppm and simplification of
proton splitting for H1′ at 5.80 ppm (s) and H3′ at 4.35 ppm (d,
J = 6.0 Hz); MS (ESI−) m/z 269 (MH−).
3′-[2H]-2′-Azido-2′-Deoxyuridine (3′-[2H]-2). Azidation of
13b (20 mg, 0.09 mmol) as described above afforded 3′-[2H]-2
(15 mg, 63%) with identical spectroscopic properties as 2 except
for the disappearance of the H3′ peak at 4.35 ppm and
simplification of proton splitting for H2′ at 4.22 ppm (d, J = 4.5
Hz) and H4′ at 4.00 ppm (“dd”, J = 2.9, 4.2 Hz): MS (ESI−) m/z
269 (MH−).
4′-[2H]-2′-Azido-2′-deoxyuridine (4′-[2H]-2). Azidation of
13c (25 mg, 0.11 mmol) as described above afforded 4′-[2H]-2
(20 mg, 67%) with identical spectroscopic properties as 2 except
for the disappearance of the H4′ peak at 4.00 ppm and
simplification of proton splitting for H3′ at 4.35 ppm (d, J = 5.8
Hz), H5′ at 3.82 ppm (d, J = 12.8 Hz), and H5″ at 3.70 ppm (d, J
= 12.8 Hz): MS (ESI−) m/z 269 (MH−).
RESULTS
■
Synthesis. The 2′-azido-2′-deoxyuridine 2 (2′-AZdU) and
its selectively labeled 5,6-[2H2]-2, , 2′-[2H]-2, 3′-[2H]-2, and 4′-
[2H]-2 analogues were prepared by conversion of uridine 8a and
the selectively deuterated uridine analogues 8b and 12a−c to
2,2′-O-anhydrouridine derivatives 9a, 9b, and 13a−c employing
the Hampton and Nichol methodology.45 Subsequent ring-
opening with azide anion [generated from trimethylsilylazide
(TMSN3)/LiF and tetramethylethylenediamine (TMEDA) or
NaN3 in hexamethylphosphoramide (HMPA) in the presence of
benzoic acid] gave desired 2′-azido-2′-deoxyuridine 246,47 and
its deuterium-labeled analogues in uracil 5,6-[2H2]-2 (Scheme
1) or ribose (2′-[2H]-2, 3′-[2H]-2, and 4′-[2H]-2; Scheme 2)
moieties. Ring-opening of 9a with [15N]-NaN3 provided 2′-
[15N]-azido-labeled 2 (Scheme 1).48 The selectively deuterated
ribofuranose precursors 2-[2H]-10a, 3-[2H]-10b (Schemes S1
and S2), and 4-[2H]-11c were prepared following literature
EPR Studies. Characterization of RNH·. Characterization of
the RNH· formed from glassy samples of 2′-azido-2′-
deoxyuridine (2′-AZdU, 2) is presented here as an example.
From our previous assignments of RNH·6−9 (for example,
T(C2′)-14ND· in 3′-AZT8) the major HFCC values in RNH·
from 2 (dU(C2′)-14ND· and its corresponding 15ND· analog
(dU(C2′)-15ND·)) are expected from three sources: the axially
symmetric anisotropic HFCC from the azide N, the isotropic β-
proton coupling owing to H2′, and the αH/aD anisotropic
hyperfine coupling from the exchangeable NH/ND.
D
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Assignment of the Axially Symmetric Anisotropic HFCC
Due to the Aminyl N. The 77 K EPR spectrum (green) of RNH·
(U(C2′)-14ND·) formed via γ-radiation-induced (absorbed
dose = 500 Gy at 77 K) electron attachment in an aqueous
glassy (7.5 M LiCl/D2O (pH ca. 5)) sample of 2 (1 mg/mL)
after visible illumination by using a photoflood lamp at 77 K for
15 min is presented in Figure 1A. The 77 K visible illumination
Figure 1. 77 K EPR spectrum of RNH· (U(C2′)-14ND·) formed via
electron-induced attachment in an aqueous glassy (7.5 M LiCl/D2O)
sample of (A) unlabeled 2, green color, and (B) 15N labeled 2, red color,
after visible illumination of each sample by using a photoflood lamp at
77 K for 15 min to remove the uracil anion radical by photoejection of
the excess electron. The simulated spectra (blue) (for simulation
parameters, see text) are superimposed on the top of the each
experimentally recorded spectrum. The three reference markers (open
triangles) in this figure and in other figures show the position of Fremy’s
salt resonance with the central marker at g = 2.0056. Each of these
markers is separated from each other by 13.09 G.
Figure 2. EPR spectra of the aminyl radicals (dU(C2′)-ND·) found in
matched samples (1 mg/mL of each compound) via radiation-
produced (absorbed dose = 500 Gy at 77 K) one-electron addition at
77 K of the homogeneous glassy solution of 7.5 M LiCl in D2O after
visible illumination of each sample by using a photoflood lamp at 77 K
for 15 min to remove the uracil anion radical by photoejection of the
excess electron of (A) 2′-AZdU (2), (B) 4′-D-2′-AZdU ([4′-D]-2),
(C) 3′-D-2′-AZdU ([3′-D]-2), (D) 5,6-D,D-2′-AZdU ([5,6-D,D]-2),
and (E) 2′-D-2′-AZdU ([2′-D]-2). The extent of β-hyperfine coupling
by H2′ atom and the anisotropic nitrogen hyperfine coupling A∥ in
U(C2′)-ND· of 2′-AZdU are represented by stick diagrams.
was carried out following our work on T(C2′)-14ND·) in 3′-
AZT8; 77 K visible illumination removed line components of
uracil anion radical by photoejection of the excess electron. The
spectrum in Figure 1A shows a large central doublet due to an
isotropic β-proton HFCC value of H2′ at 51.5 G, a triplet from
an axially symmetric anisotropic aminyl 14N (spin = 1) HFCC
(Azz (A∥) = 40.5 G, A⊥ = Axx = Ayy = 0 G), along with g∥ = 2.0020
and g⊥ = 2.0043 (see also Figure 2A and Table 1). The
anisotropic 14N HFCC and the g values agree with those of
aminyl radicals reported in the literature.7−9,17−29 Based on
these results, the green spectrum in Figure 1A is assigned to
U(C2′)-14ND·.
spectrum following our previous works on the isolation of the 77
K RNH· spectrum.7−9,49−51 A small residual peak of the Cl2·−
subtraction is found on the low field end of the red spectrum in
Figure 1B. This does not belong to the experimental
U(C2′)-15ND· spectrum (red). Employing these HFCC and g
values, the U(C2′)-14ND· (green, Figure 1A) spectrum and
U(C2′)-15ND· (red, Figure 1B) spectrum were simulated
employing the following parameters: anisotropic α-D (N-D)
HFCC (4.5, 0, 6) G7−9 and a mixed Lorentzian/Gaussian (1/1)
linewidth of 10 G. The simulated spectra (blue) matched nicely
with corresponding experimentally recorded spectra in Figure
1A,B. These results provided unequivocal assignment of the
axially symmetric anisotropic N HFCC of RNH·.
Sugar Deuteration Studies Establish That the Isotropic β-
Hydrogen HFCC in dU(C2′)-ND· Originates from H2′. To
investigate the origin of β-hydrogen coupling, we have employed
matched homogeneous glassy samples (1 mg/ mL of each
compound) of 2 and its derivatives with deuterium substitution
at specific sites in the deoxyribose sugar (at 2′ ([2′-D]-2), 3′
([3′-D]-2), and 4′ ([4′-D]-2)) and at the C5,C6 sites of uracil
base moiety ([5,6-D,D]-2)). EPR spectra of dU(C2′)-ND·
formed in these samples via γ-radiation-induced (absorbed dose
ca. 500 Gy at 77 K) electron attachment after visible illumination
by using a photoflood lamp at 77 K for 15 min are shown in
Figure 2. Each experimentally recorded spectrum shown in
Figure 2 was obtained after subtraction of the line components
of the Cl2·− spectrum following our previous works on the
isolation of the 77 K RNH spectrum.7−9,49−51 Analyses of the
spectra presented in Figure 2A−E establish that the EPR
spectrum of dU(C2′)-ND· is affected only by deuteration at 2′
Based on our previous work on 1-Me-(C2)-15ND· from
methyl 2-azido-2-deoxy-β-D-ribofuranoside,9 experiments were
carried out using a matched sample of 15N incorporated 2
(where the N atoms in the 2′-azido group of 2 were 15N labeled)
to make this assignment of U(C2′)-14ND· unequivocal; the
results are presented in Figure 1B. Analyses of the
experimentally recorded spectrum (red) in Figure 1B shows
the large central doublet due to an isotropic β-proton HFCC
value of H2′ at 51.5 G and g∥ = 2.0020 and g⊥ = 2.0043 (same as
the green spectrum in Figure 1A). The red spectrum in Figure
1B also shows line components due to an axially symmetric
anisotropic aminyl 15N (spin = 1/2) HFCC (Azz = 56.9 G, Axx =
Ayy = 0 G). Following our assignment of the green spectrum in
Figure 1A to U(C2′)-14ND·, the red spectrum in Figure 1B is
assigned to U(C2′)-15ND·. 15N (spin = 1/2) couplings are found
to be 1.404 times the 14N (spin = 1), and this is equal to the
gyromagnetic ratio of 15N/14N.9,18 We note here that each
experimentally recorded spectrum shown in Figure 1A,B was
obtained after subtraction of the line components of the Cl2·−
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(spectrum in Figure 2E). The total hyperfine splitting, lineshape,
and center of the spectra remain unchanged in spectra presented
in Figure 2A−D. Following our analyses of the green spectrum
in Figure 1A, spectra presented in Figure 2 show a large central
doublet due to an isotropic β-proton HFCC value of H2′ at 51.5
G, a triplet from an axially symmetric anisotropic aminyl N
HFCC (Azz (A∥) = 40.5 G, A⊥ = Axx = Ayy = 0 G), along with g∥ =
2.0020 and g⊥ = 2.0043.
the D2O glasses (green color) because a deuteron coupling is
only 15% (1/6.514) that of a proton in the same environment.
Therefore, the results presented in Figures 1−3 clearly
establish the electron-induced site-specific formation of π-
RND·/π-RNH· (dU(C2′)-ND·)/dU(C2′)-NH·) from 2 and its
various deuterated derivatives [2′-D]-2, [3′-D]-2, [4′-D]-2, and
[5,6-D,D]-2 (Scheme 3). These results further confirm that the
The collapse of the ca. 51.5 G doublet due to deuteration at 2′
([2′-D]-2) in the spectrum in Figure 2E establishes unequiv-
ocally the presence of β-H2′ HFCC in dU(C2′)-ND·. All other
analogs of 2 having deuteration at the C5 and C6 positions in the
uracil ring or at positions other than C2′ in the sugar moiety
show this doublet in the dU(C2′)-ND· spectrum.
Scheme 3. Radiation-Produced Electron-Mediated Site-
Specific Formation of dU(C2′)-NH· from 2
a
Studies in D2O vs H2O Establish the αH-Atom Anisotropic
Hyperfine Coupling of the Exchangeable N−H Proton in
dU(C2′)-ND· (or dU(C2′)-NH·). EPR spectral studies of
radiation-produced electron attachment to matched samples
of 2 in H2O glasses (7.5 M LiCl/H2O) were performed and
compared with the results obtained in D2O glasses (7.5 M LiCl/
D2O, Figure 1).
a
Boxes in the structure of dU(C2′)-ND· indicate those nuclei with
substantial HFCC values. These couplings arise from the axially
symmetric anisotropic azide N, the isotropic β-proton (H2′), and the
anisotropic αH/αD of the exchangeable NH/ND.
These results are shown in Figure 3 below. Comparison of the
linewidth of the green-colored spectrum (in D2O) shown in
HFCC values due to the axially symmetric anisotropic 14N, the
isotropic β-proton (H2′), and the anisotropic αH/αD of the
exchangeable NH/ND contribute to the total hyperfine splitting
of dU(C2′)-ND·.
We note here that these results, along with our previous
results on radiation-produced prehydrated electron-mediated
formation of RNH· and their reactions in 5-azidopyrimidines,7
AZT derivatives,8 and azidopentoses,9 reported predominant
formation of phenylnitrene anion radical due to gas-phase low-
energy electron (LEE, 0 to 1 eV) attachment to phenyl-
azide.8,52,53 Light-induced reduction of aromatic azides to
amines in the presence of CdS and CdSe nanoparticles8,54
showed the facile and site-specific formation of RNH· from a
localized RN3·− (Scheme 3). In addition, pulse radiolysis carried
out in aqueous solution of 3′-AZT at ambient temperature
reports that the solvated (aqueous) electron reacts with 3′-AZT
with a diffusion-controlled rate (k = 1.9 × 1010 M−1 s−1),55
thereby supporting the reaction mechanism and its facile nature
represented in Scheme 3.
Reactions of Aminyl Radicals. RNH· Formed at a Primary
Carbon Site (5′-Azido-2′,5′-dideoxyuridine (5′-AZ-2′,5′-ddU,
5)) Converts Bimolecularly to the σ-Type Iminyl Radical (R
N·) Involving an α-Azidoalkyl Radical Intermediate. Figure 4
presents the EPR spectra (red) showing the formation of RNH·
by prehydrated electron attachment7−9,50 to 5 (5 mg/mL) and
the subsequent reactions of RNH· in supercooled homogeneous
glassy (7.5 M LiCl/D2O) solutions. Electrons were produced by
gamma radiation (absorbed dose = 500 Gy at 77 K). These
spectra are overlapped on our recently published spectra (blue)
of radiation-produced electron-induced formation of π-type
RNH· and its reactions in 5-azidomethyl 2′-deoxyuridine
(AmdU) samples.7 Note that all the EPR spectra presented in
Figure 4 and in subsequent figures are obtained after subtraction
of line components due to the expected low-field resonance from
the matrix radical, Cl2·−, which are produced by scavenging of
radiation-produced holes (reactions 6 and 7).7−9,49−51
Figure 3. EPR spectrum of aminyl radical formed in matched samples
(1 mg/mL of each compound) via radiation-produced (absorbed dose
= 500 Gy at 77 K) one-electron addition by γ-irradiation of 2 (A)
dU(C2′)-ND· (green color) in 7.5 M LiCl (D2O) and (B) dU(C2′)-
NH· (red color) in 7.5 M LiCl (H2O) color after visible illumination of
each sample by using a photoflood lamp at 77 K for 15 min to remove
the uracil anion radical by photoejection of the excess electron. Panel
(A) is the same one shown in Figures 1A and 2A. The simulated spectra
(blue) are superimposed on the top of the experimentally recorded
spectra for comparison. Each experimentally recorded spectrum shown
in Figure 2 was obtained after subtraction of the line components of the
Cl2·− spectrum following our previous works on the isolation of the 77
K RNH· spectrum.7−9,49−51
Figure 3A with that of the red-colored spectrum (in H2O, Figure
3B) clearly establishes that the central doublet observed in the
red-colored spectrum in Figure 3B is due to an extra proton
hyperfine coupling in the aminyl radical in H2O (U(C2′)-NH·)
that is present in the H2O glasses and is missing in the D2O
glasses (dU(C2′)-ND·, green color) as it is an exchangeable
proton. Comparison of these two spectra clearly shows the A∥
(i.e., the Azz) component ca. 22 G due to the α-N−H proton of
the aminyl radical in H2O (U(C2′)-NH·) (red color) is lost in
h+ + Cl− → Cl·
(6)
Cl·+Cl− → Cl2·−
(7)
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the hyperfine structures of the red spectrum in Figure 4B are due
to a single axially symmetric anisotropic nitrogen with hyperfine
coupling constant (HFCC) value as (42.5, 0, 0) G along with
values of g∥ (2.0020) and g⊥ (2.0043) that are typical of a neutral
aminyl radical nitrogen (Table 1). This is manifested by the
excellent agreement of these N HFCC, g∥, and g⊥ values with the
corresponding nitrogen HFCC, g∥, and g⊥ values of T(5′-CH2)-
NH· from 5′-AZT8 and of U-5-CH2-NH· from AmdU.7
The spectra (red, Figure 4A,B) of U-5′-CH2-ND· show a
broad doublet of ca. 90.5 G (see Figure S1), and this is very
similar to the published spectra of (ca. 93.5 G) U-5-CH2-NH·
(blue, Figure 4A,B) from AmdU7 and of (ca. 91 G) T(5′-CH2)-
NH· from 5′-AZT (6).8 As for these similar structures, the
radical-site p orbital on the aminyl-N at C5′-CH2 in U-5′-CH2-
ND· strongly couples with two 5′-CH2− protons that are
attached to C5′ and gives rise to this broad doublet spectrum.
These results show that substitution of the −CH2−N3 moiety
either at the sugar of 5 or at the uracil base of AmdU7 yields
similar π-aminyl radical (U-5′-CH2-ND· vs U-5-CH2-ND·) with
near-identical EPR spectra.
Taking the U·− spectrum from the literature27−29 and the U-
5′-CH2-ND· spectrum (red, Figure 4B) as benchmarks, analysis
of the red spectrum in Figure 4A shows that this spectrum is a
cohort of spectra due to two radicals, i.e., U-5′-CH2-ND· (ca.
80%) and U·− (ca. 20%). Thus, the predominant site of electron
capture in azido compounds used in this work and in our
previous works6−9 is the azido group; thus, the azido group is
even more electron affinic than uracil, the most electron affinic
nucleic acid base. These results, in agreement with our previous
results,6−9 further establish the facile formation of site-specific
neutral π-aminyl radical at 77 K via radiation-produced electron-
mediated addition followed by N2 removal via dissociative
electron attachment mechanism and subsequent protonation
from the surrounding solvent (Schemes 1 and 2).
Progressive annealing of the sample of 5 from ca. 140−160 K
has resulted in the spectra (red) that are presented in Figure
4C,D (also see Figure S1). The red spectrum in Figure 4C is
found to be very similar to that (blue) already reported for
AmdU under equivalent conditions.7 From the similarities
(overall hyperfine splitting, the center of the spectra, and
lineshape) found in the red spectra with those in the blue spectra
in Figure 4B,C, the red spectra in Figure 4B,C are assigned to π-
aminyl radical (U-5′-CH2-ND·). In addition, at the right of both
red and blue spectra in Figure 4B−D, the splitting of large broad
resonances into three-line components is observed. This,
according to our reported analysis of the AmdU sample,7 is
due to unresolved deuterium hyperfine couplings.
Figure 4. Spectra (A−D) in red were obtained from samples of 5′-AZ-
2′,5′-ddU (5) ([5] = 5 mg/mL) after subtraction of the Cl2·−
spectrum7−9,49 from the individual experimentally recorded spectrum.
(A) EPR spectrum (red) after radiation-produced prehydrated one-
electron addition to the same sample of 5 at 77 K in 7.5 M LiCl/D2O.
Spectrum (B, red) was obtained after 15 min photoexcitation by a
photoflood lamp at 77 K. Spectra (C, red) to (F, red) were obtained via
stepwise annealing of the sample for 15 min at 140, 160, 165, and 170 K.
All spectra were recorded at 77 K. Sum of two isotropic β-proton
couplings is assigned to the central doublet (ca. 90.5 G) found in red
spectra (A) to (D); the large central doublet (ca. 82 G) in spectra (D−
F, red) is due to one isotropic β-proton coupling. Meanwhile, the wings
in spectra (A)−(F) show the Azz components of the anisotropic
nitrogen hyperfine coupling. All spectra were recorded at 77 K. The
experimental spectra from AmdU from ref 7 (2.2 mg/mL, blue) are
superimposed in (A)−(F) for comparison.
Figure 4A presents the EPR spectrum (red) of the radicals
formed in the sample of 5 at 77 K. The center of spectrum in
Figure 4A shows the doublet27−29 expected for U·−.
Furthermore, a comparison of this red spectrum with the
already reported spectrum (blue) of the neutral π-aminyl radical
U-5-CH2-ND· from AmdU samples at 77 K7 shows that the red
spectrum has additional line components that match nicely with
those due to U-5-CH2-ND·. Therefore, we assign these line
components to the neutral π-aminyl radical U-5′-CH2-ND· in
the red spectrum in Figure 4A.
The broad line component found at the center of the red
spectra in Figure 4B,C splits into various line components in
Figure 4D as found previously for spectra (blue) of AmdU
samples.7 Following the similarities in the blue and red spectra in
Figure 4D and following the assignment of these line
components in the blue spectrum in Figure 4D to the C-
centered α-azidoalkyl radical U-5-CH·-N3 formed via H-
abstraction by U-5-CH2-ND· from a proximate parent,
AmdU,7 the corresponding line components at the center of
the red spectrum in Figure 4E is assigned to the C-centered α-
azidoalkyl radical U-5′-CH·-N3 formed via H-abstraction by U-
5′-CH2-ND· from a proximate parent, 5 (Scheme 4).
Following our work on AZT,8 photoejection of the electron
from the U·− via visible photoexcitation at 77 K results in the red
spectrum shown in Figure 4B. Comparison of the red spectrum
in Figure 4B with the corresponding one in Figure 4A shows that
the central doublet due to U·− is absent in the red spectrum in
Figure 4B. Therefore, we conclude that the electron has been
completely photoejected from U·− to form U and has reacted
with parent 5 to produce additional U-5′-CH2-ND·. Total
hyperfine splitting of the red spectra in Figure 4A,B is ca. 175.5 G
(see Figure S1) in comparison to that (178.5 G) found in the
blue spectra in Figure 4A,B. Moreover, the U-5′-CH2-ND·
spectrum (red, Figure 4B) from 5 is very similar to the reported
spectrum7 of U-5-CH2-ND· (blue, Figure 4B) from AmdU
obtained via annealing in the dark at ca. 135 K for 15 min. Thus,
Progressive annealing of the sample of 5 from ca. 165−170 K
lead to the spectra (red) in Figure 4E,F. From the similarities of
our reported spectra of AmdU samples7 to the red spectra from
5, it is apparent that, apart from the new line components due to
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Scheme 4. Formation of Neutral π-Aminyl Radical RNH· from 5′-AZ-2′,5′-ddU (5) and 5′-AZT (6) and Its Subsequent
Bimolecular Reactions with 5 or 6, Respectivelya
a
H-atom abstraction from the CH3 group at C5 in 6 by RNH· prevails (ca. 80%) in competition to the H-atom abstraction by the α-azidoalkyl
radical, U-5′-CH·-N3, from 5′-CH2 (ca. 20%) in 6 forming the σ-iminyl radical (RN·). As the allylic radical, U-5-CH2·, is known to undergo
either dimerization or addition to the >CC< double bond,4,33,34 we expect that the allylic radical from 6, 5′-N3-U-5-CH2·, would undergo similar
reactions. However, owing to the absence of the CH3 group in 5, H-atom abstraction by the α-azidoalkyl radical, U-5′-CH·-N3, from 5′-CH2 is the
only reaction taking place, leading to the facile formation of σ-iminyl radical (RN·).
C-centered α-azidoalkyl radical U-5′-CH·-N3 at the center, the
second species begins to be formed at the wings of the spectrum
in Figure 4D and becomes the only radical whose spectra are
observed in Figure 4F (see Figure S2).
Studies employing samples of 5 with different concentrations
(0.5 mg/mL and 5 mg/mL) showed that signal-to-noise ratios of
EPR spectra increase substantially with an increase in
concentration of 5 (Figure S5). These results point out that
the C-centered α-azidoalkyl radical, U-5′-CH·-N3, is formed as
an intermediate via a bimolecular H-atom abstraction by π-U-5′-
CH2-ND· from a proximate 5 (Scheme 4).
Our reported work (Figures 4D and 5 in ref 8) on 5′-AZT (6;
methylazide group at sugar) showed a small observable
conversion of π-RNH· to σ-RN· or σ-T-5′-CHN· (ca.
20%, see Figure S6). It was shown that the H-atom abstraction
reaction from the sterically accessible methyl group at C5 of
thymine base by π-T-5′-CH2-NH· predominates (ca. 80%) in 6,
leading to the formation of allylic radical, dU-CH2· (Scheme 4).
Based on our new finding of the σ-type iminyl radical spectrum
from AmdU7 and from 5, this analysis accounts for the central
multiline pattern from a superposition of the UCH2· and T-5′-
CHN· spectra (see Figure S6). Therefore, we now conclude
that this assignment corrects our earlier assignment of C3′· as
the final radical spectrum to the spectra shown in Figures 4D and
5 from 6 in ref 8.
EPR investigation of the prehydrated electron attachment to
3AZPrOH (7), a simple model of the azido substitution at the
primary carbon site, also showed σ-iminyl radical formation via
the α-azidoalkyl radical intermediate (Figure S7) thereby
proving the general character of the azido substitution at any
primary carbon site which leads to the facile conversion of π-
RNH· to σ-RN· via the α-azidoalkyl radical intermediate.
RNH· Formed at a Secondary Carbon Site. Nature of the
Base (e.g., Cytosine in 2′-AZdC (1) or Uracil in 2′-AZdU (2))
Does Not Affect the Formation of Aminyl Radicals and Their
Subsequent Reactions. γ-Irradiation-mediated π-RNH· ob-
served in matched homogeneous glassy samples (native pD ca.
5) of 2′-AZdU (black, 2) and 2′-AZdC (blue, 1) are chosen as
models of π-RNH· produced at secondary carbons, and
subsequent reactions of these π-RNH· are presented in Figure 5.
The total width of the red and blue spectra in Figure 4E,F are
found to be identical (ca. 155 G), and wings show line
components arising from an axially symmetric anisotropic
nitrogen HFCCs, A∥, of ca. 36.5 G, A⊥ = 0 and g∥ = 2.0016, g⊥ =
2.0040. The large doublet in both the red and blue spectra in
Figure 4F results from a single β-proton coupling of ca. 82 G.
Therefore, following our assignment to the blue spectrum in
Figure 4F to the σ-iminyl radical, U-5-CHN· from AmdU,7
we assign the red spectrum in Figure 4F also to the σ-iminyl
radical, U-5′-CHN· (For its assignment, see Table 1 and the
Supporting Information). These results show that the reactive π-
U-5′-CH2-ND·, abstracts H atom from the parent (5) to form
the α-azidoalkyl radical intermediate, U-5′-CH·-N3. The U-5′-
CH·-N3 promptly undergoes a unimolecular β-N2 elimination
from the azide group to produce the thermodynamically more
stable σ-U-5′-CHN· (Scheme 4 and Figure 8). Based on these
assignments, we conclude that the red spectrum in Figure 4E is a
cohort of spectra due to U-5′-CH·-N3 and U-σ-5′-CHN·
whereas the cohort of spectra due to U-5-CH·-N3 and σ-U-5-
CHN· explains the blue spectrum in Figure 4E. In addition, it
is evident from these assignments that the blue spectrum in
Figure 4D is a cohort of spectra due to U-5-CH2-ND·, U-5-CH·-
N3, and σ-U-5-CHN·.
As expected, because of the lack of an exchangeable proton at
the iminyl nitrogen of U-5′-CHN·, the EPR spectrum of this
radical was the same in 7.5 M LiCl/D2O and 7.5 M LiCl/H2O
(Figure S3) and also in 7.5 M LiBr/H2O (Figure S4). These
results add further support to our assignment of spectra in Figure
4F to the σ iminyl radical, U-5′-CHN·. They also confirm that
irrespective of solvent (D2O or H2O) and the glass (LiCl or
LiBr), the formation of σ-U-5′-CHN· from π-U-5′-CH2-ND·
involves the α-azidoalkyl radical intermediate, U-5′-CH·-N3.
H
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Figure 5A,B, the blue spectrum in Figure 5B is assigned to
dC(C2′)-ND·. Therefore, dC(C2′)-ND· shows a large central
doublet due to an isotropic β HFCC value of 2′-H (ca. 51.5 G),
wing line component due to an axially symmetric anisotropic
14N-atom HFCC (Azz = 40.5 G, Axx = Ayy = 0 G), g∥ = 2.0020 and
g⊥ = 2.0043 (Characterization of RNH· section, Table 1).
Comparison of spectra in Figure 5B with those of Figure 5A
shows that the central doublet owing to U·− (black) or C·−
(blue) is absent in the spectra presented in Figure 5B. This result
demonstrates that the excess electron is completely photo-
ejected either from U·− (black) and from C·− (blue) via
photoexcitation at 77 K and reacts with unreacted parent 1 and 2
also at 77 K to produce additional π-RNH· (dU(C2′)-ND· (or
dC(C2′)-ND·)).
Based on these assignments, spectra shown in Figure 5A are
found to be a cohort of U·− (black) or C·− (blue) (ca. 20%) and
π-RNH· (dU(C2′)-ND·, black (or dC(C2′)-ND·, blue)) (ca.
80%) (see Figure 4A,B).
The spectra shown in Figure 5C−E and Figure S8 are due to
subsequent reactions of dU(C2′)-ND· or dC(C2′)-ND· due to
progressive annealing up to 165 K that caused softening of the
glass and migration of these radicals.
The spectra presented in Figure 5E is due to the final radical
formed via bimolecular reaction of either dU(C2′)-ND· (or
dC(C2′)-ND·) with a proximate unreacted parent 2 or 1. EPR
spectral studies carried out using matched samples of 2 in 7.5 M
LiCl/D2O and in 7.5 M LiCl/H2O glasses (Figure 3 (π-RNH·
identification) and Figure 5E), Figure S9 (final radical spectra))
establishes that the reaction of dU(C2′)-ND· (or dC(C2′)-
ND·) leads to a C-centered radical formation as there is no
observable difference in the spectra in D2O and in H2O glasses.
There are two possible assignments of this C-centered radical.
(A) The radical site could either be at the C5- or C6-site in
uridine (2) or in cytosine (1) via the addition of π-RNH·
(dU(C2′)-ND· or dC(C2′)-ND·) to the C6- or C5-site of the
C5C6 double bond. Alternatively, (B) π-RNH· being a good
H-atom abstractor6−9 (also, see the Introduction and Figure 4),
it might cause H-atom abstraction from the 5′-site of 1 or 2 and
the resulted C5′· has two possibilities: (i) it can be in a
conformation, which has an anisotropic α-H coupling at C5′ and
owing to a significant isotropic β-H coupling at C4′,24 and (ii) it
can undergo a ring-opening reaction leading to an open-chain
conformation of C4′· (similar to our results in azidolyxofurano-
side9) with an anisotropic α-H coupling at C4′ and owing to a
significant isotropic β-H coupling at C3′.
Figure 5. EPR spectra of matched samples of 2′-AZdU (2) (black) and
2′-AZdC (1) (blue) at the native pD (ca. 5) of the homogeneous glassy
solution of 7.5 M LiCl in D2O after subtraction of the Cl2·−
spectrum7−9,49 from the individual experimentally recorded spectrum.
(A) After γ-irradiation (absorbed dose = 500 Gy) produced one-
electron addition at 77 K. (B) After visible illumination of samples
employing a photoflood lamp at 77 K for 15 to 20 min. The central
doublet in both spectra due to the uracil anion radical (U·−) and
cytosine anion radical (C·−) are removed by photoejection of the excess
electron. Spectra (C)−(E) are obtained after subsequent annealing for
ca. 15 min in the dark from ca. 150 to ca. 165 K. All EPR spectra shown
in (A)−(E) were recorded at 77 K. See also Supporting Information,
Figure S8 for additional data.
Figure 5 shows that EPR spectral characteristics (e.g., overall
hyperfine splitting, g value at the center, and lineshape) obtained
from the samples of 1 and 2 are quite identical. Hence, we
conclude that the formation of π-RNH· and their subsequent
reactions are similar in these samples and are not affected by the
base (C (1) or U (2)).
Following spectral assignments shown in Figure 4A, the
central doublets in Figure 5A are assigned to the uracil anion
radical (U·−, black) and cytosine anion radical (C·−, blue).
Based on dU(C2′)-ND· assignment (Characterization of
RNH· section) and on the spectral similarities observed in
As samples of 1 or 2 lead to similar final radical spectra (Figure
5 and Figures S8 and S9), EPR studies were performed using
deuterated derivatives of 2 with deuterations at various specific
positions of the sugar moiety and at the base ([2′-D]-2, [3′-D]-
Scheme 5. Electrophilic Addition of π-RNH· (dC(C2′)-ND·) to the C5C6 Double Bond of the Cytosine Base in 1a
a
The corresponding scheme for electrophilic addition of π-RNH· (dU(C2′)-ND·) to the C5C6 double bond of the uracil base in 2 is shown in
Scheme S3.
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2, [4′-D]-2, and [5,6-D,D]-2 (see Figure 2) to obtain an
unequivocal assignment of the final radical species found in 1 or
Following EPR spectral results presented in Figures 4A,B and
5A,B, we assign the black 77 K spectrum presented in Figure 6A
obtained after γ-irradiation as the cohort of spectra due to π-
RNH· (2′,3′ddU(C3′)-ND·, ca. 80%) and U·− (ca. 20%). After
15 min photoexcitation at 77 K by a photoflood lamp, the excess
electron is completely photoejected from U·−, leading to only π-
RNH· (2′,3′-ddU(C3′)-ND·, black spectrum in Figure 6B)
similar to our findings shown in Figures 4B and 5B. As the azido
group is at the 3′-site in both 3′-AZT and 3, our reported
spectrum (pink) of T(C3′)-ND· from 3′-AZT8 is superimposed
on the black spectrum of 2′,3′-ddU(C3′)-ND·. EPR character-
istics (total hyperfine splitting, g value at the center, and
lineshape) of these two spectra match nicely and further support
our assignment of the black spectrum in Figure 6B to 2′,3′-
ddU(C3′)-ND·. Therefore, β(C3′-H) HFCC, Azz, Axx, Axx
values of the axially symmetric anisotropic N, g∥ and g⊥ values
of 2′,3′-ddU(C3′)-ND· are similar to those of T(C3′)-ND·, i.e.,
for 2′,3′-ddU(C3′)-ND· β(C3′-H) HFCC = ca. 41 G, Azz = 37.5
G, Axx = Ayy = 0 G, g∥ = 2.0020 and g⊥ = 2.0043.
̀
2. EPR spectra of non-deuterated parent 2 vis-a-vis deuterated at
various positions at the sugar moiety (Figures S10 and S11)
show no observable difference. These results establish that
deuterations at the sugar moiety do not help in radical
assignment and rule out the possibility (B) (vide supra).
However, the collapse of the central part of the spectrum
obtained after annealing at ca. 167 K for 1 h in the dark due to
deuteration at C5 and C6 of the uracil base of 2 ([5,6-D,D]-2)
(Figure S12) suggests the formation of the C-centered radical
via electrophilic addition of π-RNH· (dU(C2′)-ND·) to the
C5C6 double bond of the uracil base in 2 (Scheme 5 and
Scheme S1). Due to spectral similarities observed from samples
of 1 and 2 (Figure 5), formation of the similar C-centered radical
occurs in 1 via the same pathway (Scheme 5). These results also
establish that the uracil or cytosine base does not affect the
formation and subsequent reactions of π-RNH·.
3′-Azido-2′,3′-dideoxyuridine (3′-AZ-2′,3′-ddU, 3). Since
C3′ in the 2′-deoxyribose ring is also a secondary carbon in
which the azido group can be attached, EPR studies are
performed employing 3′-AZ-2′,3′-ddU, 3; these results (black
spectra) are subsequently compared with those obtained from 2
(blue spectra) and are presented in Figure 6.
Subsequent annealing of this sample from 3 at ca. 145 K and at
ca. 165 K for 15 min in the dark led to the black spectra in Figure
6C,D. The black spectrum in Figure 6C, owing to its similarities
to the black spectrum in Figure 6B, is assigned to 2′,3′-
ddU(C3′)-ND·. Owing to the similarities in the blue spectra in
Figure 6B,C and in Figures 6C and 5B, the blue spectrum in
Figure 6C is assigned to dU(C2′)-ND· (see Figure 5).
A nice match observed between the blue and black spectra
shown in Figure 6D suggests the formation of the radical species
due to electrophilic addition of 2′,3′-ddU(C3′)-ND· to the
C5C6 double bond of the uracil base of a proximate parent, 3.
This reaction is similar to those observed in the case of π-RNH·
formed from another secondary carbon site (2′) in 1 and 2 and
their derivatives (Scheme 5 and Scheme S1).
Results presented in Figures 5 and 6 establish that, unlike
azidopyrimidine nucleosides in which the azido group is
attached to a primary carbon (−CH2N3) (Reactions of Aminyl
Radicals section) , no observable line components due to σ-R
N· are found in azidopyrimidine nucleosides in which the azido
group is attached to a secondary carbon (>CH−N3). In
addition, our earlier work on π-RNH· on a secondary carbon
site (T(C3′)-ND·/T(C3′)-NH·) formed from electron addition
to 3′-AZT show predominant formation of allylic UCH2· via H-
atom abstraction from the −CH3 group at C5 of the thymine
base.8
RNH· Formed at a Tertiary Carbon Site. Since the 4′-site is a
tertiary carbon site in ribose (RNA) and in 2′-deoxyribose
(DNA), 4′-azidocytidine (4′-AZ-C, 4) has been chosen as the
model to investigate π-RNH· formation at the tertiary carbon
site and reactions of these π-RNH·. These results are compared
with the formation of π-RNH· from 1 and its reactions (Figure
5). These results are presented in Figure 7.
Following EPR spectral results presented in Figures 5AB and
6A,B, we assign the black 77 K spectrum presented in Figure 7A
obtained after γ-irradiation as the cohort of spectra due to π-
RNH· (C(C4′)-ND·, ca. 85%) and C·− (ca. 15%). After 15 min
photoexcitation at 77 K by a photoflood lamp, the excess
electron is completely photoejected from C·−, leading to only π-
RNH· (C(C4′)-ND·, black spectrum in Figure 7B) similar to
our findings shown in Figures 5B and 6B. Comparison of the
black spectrum of C(C4′)-ND· presented in Figure 7B with
those presented in Figures 4, 5, and 6 clearly demonstrates that
C(C4′)-ND· does not have any isotropic β-H HFCC owing to
the attachment of aminyl radical site N to the tertiary carbon
Figure 6. EPR spectra of samples of 2′-AZdU (2) [2 mg/mL] (blue)
and 3′-AZ-2′,3′-ddU (3) 1 mg/mL] (black) at the native pD (ca. 5) of
the homogeneous glassy solution of 7.5 M LiCl in D2O after subtraction
of the Cl2·− spectrum7−9,49 from the individual experimentally recorded
spectrum. (A) After γ-irradiation (absorbed dose = 500 Gy) produced
one-electron addition at 77 K. (B) After visible illumination of samples
employing a photoflood lamp at 77 K for 15 min. The central doublet in
both spectra due to the uracil anion radical (U·−) and cytosine anion
radical (C·−) are removed by photoejection of the excess electron. The
corresponding data of the 3′-AZT sample is taken from ref 8. (C)
Spectra obtained after subsequent annealing for ca. 15 min in the dark
from ca. 145 to ca. 150 K. (D) Spectra obtained after subsequent
annealing for ca. 45 to 60 min in the dark from ca. 165 K. All EPR
spectra shown in (A)−(D) were recorded at 77 K.
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Scheme 6. Schematic Representation of Different Types of
Reactions Undergone by π-RNH· Attached to a Primary, a
Secondary, or a Tertiary Alkyl Carbon in Azidopyrimidine
Nucleosides
Theoretical Calculations: We have calculated the forward
barrier for dissociation of the abasic 5-α-azidoalkyl radical, dR-
5′-CH·-N3, to the σ iminyl radical, dR-5′-CHN· and N2 in the
gas phase employing dR-5′-CH·-N3 as a model (Figure 8 and
Figure 8. Dissociation of (5′-CH·)-N3 to 5′-CHN· and N2 in the gas
phase with a small activation barrier of only 0.9 kcal/mol. The
geometries of these radicals were optimized, and relative energies (kcal/
mol) were calculated by the B3LYP/6-31G** method.
Figure 7. EPR spectra of matched samples of 4′-AZ-C (4, black) and 2′-
AZdC (blue, 1 also shown in Figure 5) at the native pD (ca. 5) of the
homogeneous glassy solution of 7.5 M LiCl in D2O after subtraction of
the Cl2·− spectrum7−9,49 from the individual experimentally recorded
spectrum. (A) After γ-irradiation (absorbed dose = 500 Gy) produced
one-electron addition at 77 K. (B) After visible illumination of samples
employing a photoflood lamp at 77 K for 20 min. The central doublet in
both spectra due to the cytosine anion radical (C·−) is removed by
photoejection of the excess electron. The simulated spectrum of
C(C4′)-ND· is also shown (for simulation parameters, see text).
Spectra (C) and (D) are obtained after subsequent annealing for 15
min in the dark at ca. 155 K and at ca. 165 K. All EPR spectra shown in
(A)−(D) were recorded at 77 K.
Reactions of Aminyl Radicals section) to reduce the computa-
tional cost. The potential energy surface (PES) for the
dissociation of dR-5′-CH·-N3 → dR-5′-CHN· + N2 was
calculated in the gas phase using the B3LYP/6-31G** method,
see Figure 8. The relative energies (kcal/mol) of barrier height
and products with respect to the reactant were calculated from
the total SCF energy of each fragment shown in Figure 8. We
have used the B3LYP/6-31G** optimized geometry of 5′-CH·-
N3 to obtain its HFCCs for accounting of the center of the blue
spectrum in Figure 4D,E. Employing this method, we have also
calculated the HFCCs of other aminyl and iminyl radicals
reported in this work.
The B3LYP/6-31G** level of theory predicts a forward
barrier of 0.9 kcal/mol, which is easily accessible even at the low-
temperature range of our EPR experiments. The overall reaction
is also predicted to be highly favorable with an energy change of
−40.8 kcal/mol. The very similar EPR spectral results from
samples of 5, 6 (this work) and AmdU7 (Figure 4 and Reactions
of Aminyl Radicals section) lead us to conclude that the barriers
for conversions of U-5-CH·-N3 to σ-U-5-CHN·, dU-5′-CH·-
N3 to σ-dU-5′-CHN· would be similar to that calculated for
dR-5′-CH·-N3 (Table 1).
(C4′). This spectrum has been simulated by employing axially
symmetric anisotropic 14N HFCC (37.5, 0, 0) G, anisotropic α-
D (N-D) (4.5, 0, 6) G, a mixed Lorentzian/Gaussian (1/1)
linewidth of 10 G, along with g values of (2.0020, 2.0043,
2.0043). The simulated spectrum (red) of C(C4′)-ND·
obtained by employing these EPR parameters match the
experimental spectrum (black) well shown in Figure 7B.
Subsequent annealing of this sample from 4 at ca. 145 K and at
ca. 165 K for 15 min in the dark led to the black spectra in Figure
7C,D. The black spectrum in Figure 7C, owing to its similarities
to the black spectrum in Figure 7B, is assigned to C(C4′)-ND·.
The blue spectrum in Figure 7C is assigned to dC(C2′)-ND·.
The black spectrum in Figure 7D represents the decay of
C(C4′)-ND·. On the other hand, the blue spectrum in Figure
7D is of the radical species due to electrophilic addition of
dC(C2′)-ND· to the C5C6 double bond of the uracil base of a
proximate parent, 1 (Scheme 5). Thus, a comparison of spectra
in Figure 7D suggests that π-RNH· attached to a tertiary alkyl
carbon (generated from 4) could be less reactive than π-RNH·
attached to primary and secondary alkyl carbons (Scheme 6).
DISCUSSION/CONCLUSIONS
■
Our radiation-chemical results demonstrate the following:
(1) Unequivocal identification of aminyl and iminyl radicals:
Application of 15N isotopic substitution, suitable deuterations at
the sugar moiety along with the collapse of the exchangeable α-
N−H anisotropic HFCC by changing the solvent from H2O to
D2O lead to the unequivocal assignment of electron-mediated
site-specific formation of π-RNH·. These isotopic substitution
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methods also aided the identification of subsequent radical
species, e.g., iminyl radicals.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jpcb.0c08201.
■
sı
(2) Stereochemical structure and electronic environment
affect the formation and reactivity of various types of π-RNH·
generated from azidopyrimidine nucleosides: Despite different
electronic and chemical properties π U-5′-CH2-ND· from 5
(methylazide group in a sugar), π HOCH2-CH2-CH2-ND· from
7 (methylazide group in an alcohol) and π U-5-CH2-ND· from
AmdU (methylazide group at the C5 position of the uracil base)
have similar spectral features and undergo analogous reactions,
i.e., a facile bimolecular H-atom abstraction to form α-azidoalkyl
radicals, which unimolecularly and facilely convert to σ-type R
N·.
However, electron addition to the azido group attached to a
secondary carbon site (2′) in 1 and 2 produces π dC(C2′)ND·
and π dU(C2′)ND·, respectively, with very similar radical
conformations, and these radicals undergo electrophilic addition
to the C5C6 double bond of a proximate base (C (1) or U
(2)). Moreover, 2′,3′-ddU(C3′)-ND· formed via electron
addition to an azido group attached to the 3′-secondary carbon
site in the sugar moiety also undergoes electrophilic addition to
the C5C6 double bond of a proximate base (U (3)). Thus,
while the azido group (and hence the π-RNH· radical) attached
to a primary carbon site results in its subsequent facile
conversion to σ-type RN·, our EPR spectral results establish
that these reactions are not observed in the case of the π-RNH·
radical attached to a secondary carbon site (Scheme 4 vs Scheme
5), which instead undergoes ring addition. In principle,
following the mechanism shown in Scheme 4, π-RNH·
generated at a secondary carbon site might lead to an α-
azidoalkyl radical in the sugar ring via bimolecular H-atom
abstraction from a proximate unreacted parent molecule.
However, restricted conformations of the cyclic ring in the
transition state likely prevent the formation of iminyl radical in
these cases and force the observed electrophilic addition (i.e.,
addition to the C5C6 double bond) pathway.
Synthetic procedures for the preparation and character-
ization of the deuterium labeled nucleosides and their
sugar precursors (Schemes S1 and S2), additional EPR
spectra (Figures S1−S12), addition of aminyl radical to
uracil base in 2 (Scheme S3), and supportive theoretical
calculation data (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Stanislaw F. Wnuk − Department of Chemistry and
Biochemistry, Florida International University, Miami, Florida
33199, United States; orcid.org/0000-0002-3111-3919;
Phone: 001 305 348 6195; Email: wnuk@fiu.edu; Fax: 001
305 348 3772
Amitava Adhikary − Department of Chemistry, Oakland
University, Rochester, Michigan 48309, United States;
orcid.org/0000-0001-9024-9579; Phone: 001 248 370
2094; Email: adhikary@oakland.edu; Fax: 001 248 370
2321
Authors
Mukesh Mudgal − Department of Chemistry and Biochemistry,
Florida International University, Miami, Florida 33199,
United States
Thao P. Dang − Department of Chemistry and Biochemistry,
Florida International University, Miami, Florida 33199,
United States
Adam J. Sobczak − Department of Chemistry and Biochemistry,
Florida International University, Miami, Florida 33199,
United States
Daniel A. Lumpuy − Department of Chemistry and
Biochemistry, Florida International University, Miami, Florida
33199, United States
Priya Dutta − Department of Chemistry, Oakland University,
Rochester, Michigan 48309, United States
Samuel Ward − Department of Chemistry, Oakland University,
Rochester, Michigan 48309, United States
Katherine Ward − Department of Chemistry, Oakland
University, Rochester, Michigan 48309, United States
Moaadh Alahmadi − Department of Chemistry, Oakland
University, Rochester, Michigan 48309, United States
Anil Kumar − Department of Chemistry, Oakland University,
Rochester, Michigan 48309, United States; orcid.org/
0000-0002-9979-7798
Michael D. Sevilla − Department of Chemistry, Oakland
University, Rochester, Michigan 48309, United States;
orcid.org/0000-0001-8799-5458
Because of steric hindrance (i.e., due to the crowded transition
state) and stabilization due to hyperconjugation, π-RNH·
attached to a tertiary carbon site exhibits lower reactivity in
our glassy system at low temperature. Thus, these studies
provide the free radical mechanistic basis for the various types of
reactions (H-atom abstraction by π-RNH· and addition of π-
RNH· to the CC double bond) that were postulated from
product analyses studies by Wagner et al.12
(3) Implications as radiosensitizers in radiochemotherapy of
cancer: Recently, π-RNH· from AmdU (π-RNH· attached to a
primary carbon site) have been shown to significantly augment
radiation damage in EMT6 breast cancer cells.7 EPR spectral
results presented in this work under anoxic conditions predict
that π-RNH· attached to a primary or a secondary carbon site
would be more effective in augmenting radiation damage to
hypoxic cancer cells than π-RNH· attached to a tertiary carbon
site owing to its lower reactivity. Thus, these studies have
exhibited the potential to select those azidoDNA models as
adjuvants that could significantly improve cancer radiother-
apeutic efficacy by causing higher hypoxic cancer cell killing.
These results are of fundamental importance in the chemistry
of aminyl radicals, which have widespread applications in
synthesis, in environmental degradation of pharmaceuticals, in
free radical-mediated damage to DNA and proteins, etc.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jpcb.0c08201
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
M.D.S., A.A., and A.K. thank the National Cancer Institute of the
National Institutes of Health (grant no. RO1CA045424) for
support. A.A. and M.D.S. thank REF, CBR at OU for support.
A.A. thanks the National Science Foundation under grant no.
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(19) Adhikary, A.; Khanduri, D.; Sevilla, M. D. Direct Observation of
the Hole Protonation State and Hole Localization Site in DNA-
oligomers. J. Am. Chem. Soc. 2009, 131, 8614−8619.
(20) Adhikary, A.; Kumar, A.; Munafo, S. A.; Khanduri, D.; Sevilla, M.
D. Prototropic Equilibria in DNA Containing One-electron Oxidized
GC: Intra-duplex vs. Duplex to Solvent Deprotonation. Phys. Chem.
Chem. Phys. 2010, 12, 5353−5368.
CHE-1920110. T.P.D. is grateful to the FIU Graduate School
for her Doctoral Evidence Acquisition fellowship and Kaufmann
Foundation for support and S.F.W. to NIGMS/NCI
(SC1CA138176) grant for support.
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