A new ground state single electron donor for excess electron transfer studies in DNA

Article abstract of DOI:10.1039/b906180k

A new photoinducible single electron donor has been developed, which, when linked to thymidine, is shown to be an efficient ground state reducing agent in DNA; the donor can be activated at wavelengths where standard DNA does not absorb.

Full text of DOI:10.1039/b906180k

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A new ground state single electron donor for excess electron transfer
studies in DNAw
Christian Trindler, Antonio Manetto, Jurgen Eirich and Thomas Carell*
¨
Received (in Cambridge, UK) 30th March 2009, Accepted 23rd April 2009
First published as an Advance Article on the web 8th May 2009
DOI: 10.1039/b906180k
A new photoinducible single electron donor has been developed,
which, when linked to thymidine, is shown to be an efficient
ground state reducing agent in DNA; the donor can be activated
at wavelengths where standard DNA does not absorb.
out of the ground state, without the possibility for charge
recombination.
The synthesis of 1 was performed as depicted in Scheme 2,
by coupling the TBDPS-protected thymidine methyl amine 5
with acid 6 using an HBTU/DIPEA coupling protocol,
followed by deprotection of the silyl groups. Acid 6 was
synthesized from the vinylbromine 8 in 4 steps (Scheme 2).
The key transformations included a palladium-N-heterocyclic-
carbene-catalyzed carbonylative Suzuki–Miyaura coupling to
give 910 and a manganese-catalyzed hydro-hydroxylation11 of
the double bond in 9 to form compound 10 with the hydroxyl
group inserted alpha to the carbonyl group. Phenone 10 was
finally deprotected using TBAF9 followed by a TEMPO
catalyzed oxidation8,12 of alcohol 11 to yield the final
carboxylic acid 6. For later comparison, we also prepared
the expected photolysis products 3 and 4 by coupling the
methylamine thymidine 5 with the corresponding carboxylic
acids using an EDC/HCl/HOBt or a HBTU/DIPEA protocol.
In order to study the photochemical properties of nucleoside
1, we dissolved the compound in H2O :MeOH (4 : 1) to a
concentration of 70 mM and irradiated with a Hg(Xe) arc lamp.z
After defined time intervals the samples were removed from the
irradiated solution and analyzed by reversed phase HPLC
(0–75% in 45 min, 0.1 M NEt3/AcOH in H2O - H2O–CH3CN
20 : 80). After irradiation for 5 min, most of ketone 1 was already
cleaved (Fig. 1) showing that the Norrish type I cleavage is an
extremely efficient process. After 20 min, ketone 1 could no
longer be detected, indicating total conversion.
Excess electron transport (EET) studies in DNA require the
incorporation of photoinducible electron donors into DNA
duplexes. So far, investigations have been performed with
electron donors such as flavins,1 naphthalene diamines,2
stilbene diethers,3 phenothiazines,4 pyrenes,5 platinum(II)-6
or iridium-complexes,7 which inject multiple electrons into
DNA due to fast photocycling of the chromophore during
the experiment. In addition, most of these donors are excited
state donors, which establish a strong driving force for charge
recombination after electron injection. For some time, we have
been interested in finding novel ground state electron donors
to insert into DNA, which (1) inject only a single electron into
the duplex and (2) generate no driving force for charge
recombination. The idea is to inject an electron into DNA,
which is then free to travel through the duplex without feeling
a retractive force. A previously investigated system had the
strong disadvantage that photoinduction required a rather
short initiation wavelength of l o 320 nm, so that selective
triggering of the electron injection event was difficult to
achieve.8 Here we report the development of a new nucleoside
1, which is triggered at l 4 340 nm. The compound establishes
an irreversible single electron injection event and keeps the
driving force for charge recombination to a minimum. Both
features—the lack of a recombination driving force and the
fact that only a single electron is injected—will allow excess
electron transfer reactions through DNA duplexes to be
studied with very high precision, and enable cascade reactions
triggered by a single electron in the DNA duplex to be
characterized.8
The HPLC chromatograms depicted in Fig. 1 show the
detection of two new compounds absorbing at the detection
wavelength of 260 nm. The compound with the shorter reten-
tion time of tR = 22.5 min is clearly the major product. The
Electron transfer is established by photoinduced Norrish
type I cleavage of the phenylketone 1, which gives a highly
reducing ketyl radical 2.8,9 This is either quenched by the
surrounding medium, leading to the b-hydroxy-amide 3, or it
is oxidized by the attached pyrimidine moiety forming the
b-keto-amide 4 (Scheme 1). Since the Norrish type I cleavage
of the phenylketone is irreversible, electron donation occurs
Center for Integrative Protein Science at the Department of
Chemistry and Biochemistry, LMU Munich, Butenandtstrasse 5-13,
D-81377 Munich, Germany.
E-mail: Thomas.Carell@cup.uni-muenchen.de;
Fax: +49 89 2180 77756; Tel: +49 89 2180 77750
w Electronic supplementary information (ESI) available: Syntheses of
compounds 1–13, HPLC and UV chromatograms for 1–3, details
about the DNA synthesis. See DOI: 10.1039/b906180k
Scheme 1 Photocleavage of single electron donor 1 to ketyl radical 2
and its conversion to the products b-keto-amide 3 and b-hydroxyl-
amide 4.
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compound 1 can be safely incorporated, however, ultra-mild
deprotection (0.05 M K2CO3 in MeOH) was necessary to
avoid cleavage of the tertiary amide. Next we irradiated
the DNA single strand to see if the injected electron is able
to hop to the nearby BrdU to cause debromination. This is
indeed the case. MALDI-TOF of the irradiated single strand
clearly shows the debrominated strand with a mass of 3436 Da
(see ESIw).
In summary, we report the synthesis of a new nucleoside 1
and its incorporation into DNA. Upon irradiation at l 4 340 nm,
1 undergoes an efficient Norrish type I cleavage to a ketyl
radical that possesses a sufficient redox potential to reduce the
attached thymine, forming a thymine radical anion. Within
DNA, the radical is able to reduce a nearby 5-Br-dU base and
cause debromination. The advantage of the new system is that
the Norrish type I cleavage occurs at long wavelengths
(l 4 340 nm), so that acceptor-modified DNA strands con-
taining nucleoside 1 can be irradiated without fear of addi-
tional excitation of either the nucleobases or any incorporated
electron acceptors such as 5-Br-dU. For us, the high efficiency
of the Norrish type I cleavage is surprising given the
rather weak absorption of nucleoside 1 at these wavelengths
Scheme 2 Synthesis of 1 and its incorporation into DNA. (a)
PhB(OH)2, PEPPSI-IPr, CsCO3, PhCl, CO (balloon), 80 1C, 4 h,
71% (b) Mn(dpm)3, PhSiH3, O2 (balloon), P(OEt)3, iPrOH, 0 1C, 2 h,
64% (c) TBAF, THF, 25 1C, 20 min, 86% (d) TEMPO, NaClO2,
NaOCl, buffer (pH 6.8), CH3CN, 35 1C, 24 h, 88% (e) HBTU,
DIPEA, DMF, 45 1C, 20 h, 81% (f) HF, pyridine, 22 1C, 18 h, 98%
(g) DMT-OTf, 3 A MS, pyridine, 22 1C, 5 h, 89% (h) P(NiPr2)2OCE,
iPr2NH2-tetrazolate, CH2Cl2, 0 1C, 2.5 h, 75%.
ꢂ1
(e340 nm = 37 M cmꢂ1). These studies show that 1 can be
incorporated into DNA and the electron injection into DNA
occurs efficiently.
This work was supported by the Deutsche Forschungs-
gemeinschaft (DFG: SFB 749 and Excellence cluster CiPSM),
by the European Community’s Marie Curie Research Training
Network (CLUSTOXDNA) and by Novartis.
Notes and references
z Photolysis was carried out with an ORIEL 6295H mercury(xenon)
arc lamp; 1000 W, 340 nm long-pass filter (Russian C3C17). The
nucleosides were dissolved in H2O : MeOH (4 : 1) to 70 mM, and prior
to the irradiations the solutions were degassed by bubbling a steady
stream of argon through for 40 min.
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Fig. 1 HPLC chromatograms at 260 nm showing the time depen-
dency of the photocleavage of donor 1 (42.3 min) to b-keto-amide 4
(22.5 min) and b-hydroxy-amide 3 (24.2 min).
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minor product has a retention time of tR = 24.2 min. In order
to study if the major product is compound 4, generated by
quenching of the ketyl radical 2 by electron donation to the
dT-base, or compound 3, which is expected to form after
Hꢁ (H++eꢂ) transfer from the solvent (Scheme 1), we
co-injected these compounds along with the irradiation
mixture into the HPLC and measured mass spectra of the
HPLC peaks. The result of this study was that irradiation of
compound 1 led predominately to formation of compound 4
due to electron transfer to the thymine base.
To show that the electron donor 1 can be inserted in
oligonucleotides, we prepared the phosphoramidite 12 from
7 by standard procedures. Solid phase synthesis was used to
generate a DNA strand 50-GCGTTTBrdUXGAC-30 featuring
5-Br-dU (BrdU) as an electron acceptor next to the donor
1 (X). Detailed analysis of the DNA strand showed that
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This journal is The Royal Society of Chemistry 2009
3584 | Chem. Commun., 2009, 3583–3584