Independent Photochemical Generation and Reactivity of Nitrogen-Centered Purine Nucleoside Radicals from Hydrazines

Article abstract of DOI:10.1021/acs.orglett.7b03368

Photochemical precursors that produce dA^￠ and dG(N₂-H)^￠ are needed to investigate their reactivity. The synthesis of two 1,1-diphenylhydrazines (1, 2) and their use as photochemical sources of dA^￠ and dG(N₂-H)^￠ is presented. Trapping studies indicate production of these radicals with good fidelity, and 1 was incorporated into an oligonucleotide via solid-phase synthesis. Cyclic voltammetric studies show that reduction potentials of 1 and 2 are lower than those of widely used "hole sinks", e.g., 8-oxodGuo and 7-deazadGuo, to investigate DNA-hole transfer processes. These molecules could be useful (a) as sources of dA^￠ and dG(N₂-H)^￠ at specific sites in oligonucleotides and (b) as "hole sinks" for the study of DNA-hole transfer processes.

Full text of DOI:10.1021/acs.orglett.7b03368

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX-XXX
Independent Photochemical Generation and Reactivity of Nitrogen-
Centered Purine Nucleoside Radicals from Hydrazines
Liwei Zheng,† Lu Lin,‡ Ke Qu,‡ Amitava Adhikary,‡ Michael D. Sevilla,‡ and Marc M. Greenberg*,†
†Department of Chemistry, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, United States
‡Department of Chemistry, Oakland University, Rochester, Michigan 48309, United States
S
* Supporting Information
ABSTRACT: Photochemical precursors that produce dA•
and dG(N2−H)• are needed to investigate their reactivity.
The synthesis of two 1,1-diphenylhydrazines (1, 2) and their
use as photochemical sources of dA• and dG(N2−H)• is
presented. Trapping studies indicate production of these
radicals with good fidelity, and 1 was incorporated into an
oligonucleotide via solid-phase synthesis. Cyclic voltammetric studies show that reduction potentials of 1 and 2 are lower than
those of widely used “hole sinks”, e.g., 8-oxodGuo and 7-deazadGuo, to investigate DNA−hole transfer processes. These
molecules could be useful (a) as sources of dA• and dG(N2−H)• at specific sites in oligonucleotides and (b) as “hole sinks” for
the study of DNA−hole transfer processes.
Scheme 1. Neutral Purine Radical Formation
ucleic acid oxidation is an important and complex
1,2
Ncollection of chemical processes.
Valuable mechanistic
information concerning how individual chemotherapeutic
agents damage DNA, which could lead to more effective
therapy, was gleaned by examining their reactivity with nucleic
acids.3−5 A variety of tools have also been used to understand
the more chemically diverse nucleic acid damage pathways
induced by γ-radiolysis.6 Independent generation of reactive
intermediates involved in nucleic acid oxidation is useful for
understanding how these biopolymers are damaged.7 We wish
to report the generation of the nitrogen-centered radicals, 2′-
deoxyadenosin-N6-yl radical (dA•) and 2′-deoxyguanosin-N2-yl
radical (dG(N2−H)•), via photodissociation of the correspond-
ing hydrazines (1 and 2).
Photochemistry has proven to be very useful for
independently generating nucleic acid radicals in nucleosides,
as well as in DNA and RNA.6,7 Most of the nucleic acid radicals
studied via photochemical generation are C-centered radicals
and the Norrish Type I photocleavage reaction of ketones has
been used most frequently to generate them, although aryl
sulfides and aryl selenides have also been employed.8−10
Nucleobase π-radicals (Scheme 1) also play important roles in
nucleic acid oxidation, such as in electron transfer, and the
direct (dG(N1−H)•, dG(N2−H)•) and indirect (dA•) effects of
ionizing radiation.6,11,12 Furthermore, dG(N1−H)• and dG-
(N2−H)• are irreversibly formed from deprotonation of the dG
radical cation involved in DNA charge transfer.13 Progress in
understanding the reactivity of these radicals, particularly within
the biopolymers, has lagged behind that of C-centered radicals.
A small number of approaches for generating neutral purine
radicals have been described.14,15 However, some are
incompatible with solid-phase oligonucleotide synthesis, and
photolysis of others does not provide high mass balances.16,17
Herein, we describe an alternative method to produce dA•
involving photodissociation of 1, and the application of this
approach to provide the first example of dG(N2−H)•
generation.
We speculated that photolysis of hydrazines could result in
cleavage of the relatively weak N−N bond. Furthermore, we
anticipated that the use of aryl hydrazines would enable us to
carry out photolysis at wavelengths of light (350 nm) that
would minimize nucleic acid degradation. Condensed phase
spectroscopy experiments demonstrate that photoexcited
tetraphenyl hydrazine dissociates to form two diphenyl aminyl
radicals.18−21 Although other tetraaryl hydrazines have been
shown to undergo photodissociation, examples of differently
Received: October 28, 2017
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.7b03368
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Letter
substituted hydrazines are rare, and studies on the reactivity of
diffusively free aminyl radicals derived from them are
lacking.21,22 Cage recombination appears to be a major pathway
in these examples. N,N-Diphenyl hydrazines were also
attractive as precursors because the concomitantly produced
diphenyl aminyl radical (Ph2N•) was expected to be less
reactive than many radicals due to the low BDE of the N−H
bond in Ph2NH (<85 kcal/mol).23,24
Hydrazine 1 had a λmax = 267 nm (ε = 1.81 0.01 × 104
M−1 cm−1) in H2O, but its absorption tailed into the >300 nm
region at the concentration (100 μM) at which it was present
during photolysis. Hydrazine 1 was completely consumed after
photolysis (Rayonet photoreactor equipped with λmax = 350 nm
lamps) for 4 h. 2′-Deoxyadenosine (dA) was the major product
detected under all irradiation conditions (Table 1). Thymidine
Table 1. Reactivity of dA• from Irradiated 1
a
% yield
a
[1] (μM)
red. (mM)
dA
64
10
% mass bal
100
100
100
100
100
50
2
1
2
1
2
1
1
9
1
1
1
1
1
1
1
73
71
65
81
78
80
84
2
1
3
1
2
2
1
Fe2+ (10)
65
57
72
67
72
75
6
8
BME (10)
PhSH (100)
PhSH (10)
PhSH (10)
PhSH (10)
10
11
8
25
9
a
Average std dev of three experiments.
was used as an internal standard in all experiments and was
shown to be stable to the reaction conditions (Figure S9). The
only other molecule detected was formal recombination
product 10 (Scheme 3).
We set out to prepare 1 via nucleophilic aromatic
substitution, which has been used to prepare less highly
substituted hydrazine N6-purines.25,26 We initially chose
Lakshman’s O6-(benzotriazol-1-yl)-2′-deoxyinosine (3).27 Re-
action of 3 with phenyl hydrazine (10 equiv) provided the
expected N,N′-disubstituted hydrazine (4) in 99% yield. In
contrast, no reaction was observed between 3 and 1,1-diphenyl
hydrazine (6, 10 equiv) or its hydrochloride salt under a variety
of conditions. These included temperatures as high as 80 °C
and the presence of Cs2CO3 and/or DABCO in either THF,
MeOH, or dimethoxyethane (80 °C). Reacting 6 with 7, which
readily reacts with variety of arylamines,28,29 did not yield
desired product. Similarly, sulfonate 8, which undergoes
nucleophilic aromatic substitution when activated by
DABCO,30 also did not react with 6.
Following these unsuccessful efforts, we turned our attention
to Buchwald−Hartwig amination chemistry. A variety of 6-
substituted purines and N6-amino-substituted adenosines have
been prepared from 6-bromopurine 7 using Pd catalysts31,32
Use of Pd(OAc)2 and BINAP ligand gave only trace amounts of
the desired product (5). However, switching to Xantphos as
ligand and Pd2dba2 as catalyst produced 5 in 48% yield
(Scheme 2).33 Desilylation provided the candidate photo-
chemical hydrazine precursor 1.
Scheme 3. Product Formation from Photolysis of 1
Product 10 was initially identified by LC/MS analysis in the
photolysate of 1. It exhibited (Figure 1a) a molecular ion
identical to that of 1, but in contrast to the hydrazine (Figure
S1), fragmentation of the diphenylamino group was not
observed. Attempted synthesis of 10 via a variety of Pd
coupling conditions of the protected 8-bromo-2′-deoxyadeno-
sine with diphenylamine was unsuccessful.34 Consequently,
Scheme 2. Diphenylhydrazine Synthesis
Figure 1. Mass spectrometric identification of recombination product
10 formed from photolysis of (a) 1 and (b) 8-2H-1.
B
DOI: 10.1021/acs.orglett.7b03368
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Letter
support for the assignment of 10 was obtained via LC/MS
analysis of photolyzed 8-2H-1. The deuterated hydrazine was
prepared from 1 via Et3N-catalyzed D2O exchange.35 The
recombination product formed from 8-2H-1 (Figure 1b) lacked
deuterium, consistent with the proposed structure (10). Yields
of 10 were roughly estimated by synthesizing 11 and measuring
its extinction coefficient at 260 nm.34 This value was compared
to that of 1 (whose response factor was independently
measured) to approximate its yield by HPLC (Table 1).
(dG(N2−H)•). dG(N2−H)• represents one of the two one-
electron oxidized tautomers of dG (Scheme 1).40 The pKa
values of the N1- and N2-protons of the guanine radical cation
(G•+) in dG are believed to be ∼3.9 and ∼4.7, respectively.12,40
From the closeness of these pKa values, it is expected that
deprotonation from N1 as well as N2 of G•+ should be
competitive with each other. dG(N1−H)• is believed to be the
major neutral purine radical formed upon deprotonation,
following one-electron oxidation of the nucleoside dG and in
ssDNA.40,41 However, in dsDNA, owing to the base pairing,
G•+ deprotonation occurs from N1 and N2 to produce
dG(N1−H)• and dG(N2−H)•.42 Therefore, independent
generation of dG(N1−H)• and dG(N2−H)• could help the
study of the properties of these radicals and their role in DNA
damage.
The requisite hydrazine (2) was prepared from the O6-
benzyl-protected, disilylated dG (9, Scheme 2).43 The Pd
coupling reaction proceeded in slightly higher yield (63%) than
the analogous reaction did for the preparation of 1. Precursor 2
was obtained following desilylation and hydrogenolysis.
Hydrazine 2 is slightly more readily oxidized than the
adenine analogue (1). Cyclic voltammetry indicated that the
reduction potential for 2 was 0.77 V vs NHE (Figure 2, Figure
S3). Photoconversion of 2 was qualitatively less efficient than
that for 1, requiring 8 h photolysis for almost complete
conversion instead of 4 h. Importantly, photolysis of 2 provided
strong evidence for the formation of dG(N2−H)• (Table 2).
The major product from photolysis of 1 was dA. Overall, the
effects of reductants on the yields of dA are similar to those
observed when dA• was generated from 11.15 For instance, β-
mercaptoethanol (BME) was an inefficient trap compared to
ferrous ion and thiophenol (PhSH), presumably due to polarity
mismatching, as previously discussed.15 Furthermore, there was
an inverse relationship between dA yield and concentration of
1. Studies on 12 attributed these observations to dA• reduction
by the photochemical precursor. Evidence in support of
hydrazine 1 serving as a viable reductant for dA• is obtained
from cyclic voltammetry (Figure 2, Figure S2). The observed
Table 2. Reactivity of dG(N2−H)• from Irradiated 2
a
a
[2] (μM)
red. (mM)
% yield dG
% mass bal
100
100
100
100
50
54
67
69
68
74
78
3
3
1
2
3
1
54
73
75
74
74
82
3
1
1
2
3
1
Fe2+ (10)
BME (10)
PhSH (10)
PhSH (10)
PhSH (10)
25
a
Average std dev of three experiments.
Comparisons of the yield of dG and mass balance in
photolyzates of 2 under various conditions with the
corresponding mass balances and dA yields from 1 (Table 1)
indicate subtle differences in the two systems. For instance, the
yield of dG (and mass balance) in the absence of exogenous
reducing agent was closer to 50%, which would be the
maximum yield if 2 is the sole reducing agent of dG(N2−H)•.
This could reflect slower electron transfer within the respective
radical pair formed upon photodissociation and correlates with
the smaller thermodynamic driving force for dG(N2−H)•
reduction. The mass balances are modestly higher from the
photolyses of 2 than from 1 in the presence of Fe2+, BME, or
PhSH. This, too, could be a consequence of greater
thermodynamic stability of dG(N2−H)• compared to dA•,
which results in slower reactions with its respective precursor
and Ph2N•.
Figure 2. Reduction potentials of nucleoside radical cations versus
NHE in CH3CN.
reduction potential illustrates that 1 is more readily oxidized
than dG, the most electron rich native nucleotide, 7-
deazadGuo, and even 8-oxo-7,8-dihydro-2′-deoxyguanosine
(8-oxodGuo), a DNA lesion that is susceptible to further
oxidation (Figure 2, Figures S3−5).36−39 However, the yield of
dA in the absence of any exogenous reductant is >50%,
indicating that a species other than 1 must reduce dA•. We
tentatively suggest that in addition to providing 10, the radical
pair also undergoes electron transfer (Scheme 3). Finally, the
yield of 10 is more or less independent of reductant or its
concentration, and substrate concentration, suggesting that it
could result from a radical pair process. However, due to the
expected stability of Ph2N•,23 we cannot rule out selective
reaction of diffusively free aminyl radical with dA•.
Hydrazines 1 and 2 are compatible with solid-phase
oligonucleotide synthesis conditions. This was demonstrated
by introducing 1 into an oligonucleotide (13) via its
corresponding phosphoramidite. The TM of a dodecameric
The success of 1 in generating dA• led us to apply the same
strategy for generating 2′-deoxyguanosin-N2-yl radical
C
DOI: 10.1021/acs.orglett.7b03368
Org. Lett. XXXX, XXX, XXX−XXX

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Letter
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.7b03368.
■
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AUTHOR INFORMATION
Corresponding Author
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*E-mail: mgreenberg@jhu.edu. Tel: 410-516-8095. Fax: 410-
516-7044.
ORCID
Amitava Adhikary: 0000-0001-9024-9579
Michael D. Sevilla: 0000-0001-8799-5458
Marc M. Greenberg: 0000-0002-5786-6118
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We are grateful for generous financial support from the
National Institute of General Medical Science (GM-054996) to
M.M.G. A.A. and M.D.S. acknowledge financial support from
the National Cancer Institute (CA-045424) and the Oakland
University Research Excellence Funds (CBR). L.Z. thanks
Johns Hopkins University for the Glen E. Meyer ’39
Fellowship.
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DOI: 10.1021/acs.orglett.7b03368
Org. Lett. XXXX, XXX, XXX−XXX