Enhancement of excess electron transfer efficiency in DNA containing a phenothiazine donor and multiple stable phenanthrenyl base pairs

Article abstract of DOI:10.1002/chem.201301983

EET grown ohm: Excess electron transfer (EET) was observed within a DNA duplex containing π-stacked phenothiazine as an electron donor, phenanthrenes as electron carriers and 5-bromouracil as an electron trap. Increasing the number of phenanthrenyl base pairs increased EET efficiency. Copyright

Full text of DOI:10.1002/chem.201301983

COMMUNICATION
EET grown ohm: Excess electron
transfer (EET) was observed within a
DNA duplex containing p-stacked
phenothiazine as an electron donor,
phenanthrenes as electron carriers and
5-bromouracil as an electron trap.
Increasing the number of pehanthrenyl
base pairs increased EET efficiency.
Electron Transfer
P. Roethlisberger, F. Wojciechowski,
C. J. Leumann* ............... &&&& — &&&&
Enhancement of Excess Electron
Transfer Efficiency in DNA
Containing a Phenothiazine Donor
and Multiple Stable Phenanthrenyl
Base Pairs
Chem. Eur. J. 2013, 00, 0 – 0
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DOI: 10.1002/chem.201301983
Enhancement of Excess Electron Transfer Efficiency in DNA Containing a
Phenothiazine Donor and Multiple Stable Phenanthrenyl Base Pairs
Pascal Roethlisberger,[a] Filip Wojciechowski,[a, b] and Christian J. Leumann*[a]
Electron transport through the p-stacked DNA base pairs
has attracted considerable interest and continues to be in-
tensively studied.[1] A better understanding of photoinduced
negative charge transfer, often called excess electron trans-
fer (EET), could allow to develop DNA nanoscale devices,
such as biological sensors used in, for example, single-nu-
cleotide polymorphism detection.[2] The electron conducting
properties of DNA are determined by the physicochemical
properties of the natural nucleobases and for certain appli-
cations, such as DNA nanowires, enhancement of the con-
ducting properties would be highly desirable. One envi-
sioned way to enhance EET efficiency is to replace the natu-
ral base pairs with non-natural hydrogen bonding partners
or other aromatic residues with favorable conductive prop-
erties.[3]
Figure 1. a) Schematic representation highlighting EET from a photoex-
cited PTZ-b-C-nucleoside (PTZ) placed against an abasic site (f) to a in-
terstrand stacked phenanthrenyl (Phen) base pair and 5-bromouracil
(
BrU); the long arrows represent a phosphodiester backbone; b) an EET
pathway from photoexcited (PTZ*) to three Phen base pairs.
In earlier work we investigated EET through a stacked
phenanthrenyl (Phen–Phen) pair in a DNA duplex contain-
ing 5-(pyren-1-yl)uridine as an electron injector and 5-bro-
mouracil (BrU) as an electron acceptor.[3a] This provided the
first example of electron transfer through a stable, inter-
strand p-stacked, aromatic base pair. However, the reduc-
tion potential of Phen is more negative than that of the
pyrene donor, which excludes electron transfer to occur
through a hopping mechanism. This, however, would be nec-
essary to increase efficiency and distance of EET. Indeed,
we observed lower efficiency through a Phen–Phen pair
compared to an A–T base pair. To remedy and to explore
the scope and limitations of EET through such interstrand
stacked aromatic residues, devoid of hydrogen-bonding ca-
pability, we decided to use a stronger electron donor, such
as phenothiazine (PTZ; Eox*ꢀꢁ2.7 V vs. SCE; Figure 1)[4]
and to use up to three Phen–Phen pairs in between the elec-
tron injector and acceptor in an otherwise similar setup.
PTZ has been used previously as an electron donor in
DNA. Using a flexible, acyclic linker PTZ was placed at the
5’ end of DNA or at internal positions facing the natural
bases/an abasic site.[3b,5] We reasoned that using a rigid PTZ-
b-C-nucleoside the PTZ unit could intercalate well against
an abasic site and stack favorably with a planar Phen resi-
due, thus enabling efficient EET. On the example of duplex-
es containing one or three Phen–Phen pairs we show here
that EET becomes more efficient as compared to duplexes
with the same number of A–T base pairs. Moreover, we
demonstrate for the first time that EET can occur over mul-
tiple Phen–Phen pairs.
Although a PTZ-b-C-nucleoside has previously been syn-
thesized by Grinstaff and co-workers, they were unable to
protect its 5’-hydroxyl with DMT-Cl, and consequently PTZ
was placed at the 5’ end of the DNA.[6] Our attempts at pro-
tecting the 5’-hydroxyl with standard DMT-Cl/pyridine also
failed. Fortunately, the 5’-hydroxyl could be protected with
DMT-triflate,[7] a reagent occasionally used to protect steri-
cally hindered hydroxyl groups.[8]
The DMT protected nucleoside was subsequently phos-
phitylated using standard conditions yielding the corre-
sponding
Scheme S1). PTZ-, Phen-,[9] and 5-bromouracil phos
amidites were used in the synthesis of modified oligodeoxy-
phosphoramidite
(Supporting
Information,
ACHTUNGTRENNUNG
[a] P. Roethlisberger, Dr. F. Wojciechowski, Prof. C. J. Leumann
Department of Chemistry and Biochemistry
AHCTUNGTRENNUNG
University of Bern, Freiestrasse 3, 3012 Bern (Switzerland)
E-mail: leumann@ioc.unibe.ch
nucleotides (ODNs) 1–14 (Table 1). The ODNs were puri-
fied by RP-HPLC and characterized by ESI-MS.
[b] Dr. F. Wojciechowski
Thermal denaturation experiments were performed with
duplexes D1–D9 to judge the stability of DNA containing
PTZ and multiple Phen–Phen pairs. The Tm data are sum-
marized in Table 1. PTZ placed against an abasic site (PTZ–
f, duplex D2) leads to a duplex that is less stable by 5.88C
Present address: Department of Chemistry
University of Konstanz, Universitꢁtsstrasse 10
78457 Konstanz (Germany)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201301983.
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Electron Transfer Efficiency in DNA
COMMUNICATION
Table 1. Sequence and thermal stability (Tm) data for DNA duplexes
1–9.[a]
No.
ss[a]
Duplex sequence
Tm [8C][b]
D1
1
2
5’-GCGATf(A)1AATGCG-3’
3’-CGCTAPtz(T)1BrUTACGC-Fluo
49.0
48.0
51.0
54.8
53.8
49.8
56.3
51.4
57.0
D2
D3
D4
D5
D6
D7
D8
D9
3
4
5
6
7
8
9
10
3
11
12
13
7
13
7
5’-GCGATf
3’-CGCTAPtzCAHTUNGTRENNNUG
ACHTUNGTRENNUNG
5’-GCGATf(A)3AATGCG-3’
3’-CGCTAPtz(T)3BrUTACGC-Fluo
5’-GCGATf
3’-CGCTAPtz
5’-GCGATA(Phen)1 AATGCG-3’
3’-CGCTAT
(Phen)1BrUTACGC-Fluo
5’-GCGATf(Phen)1AATGCG-3’
3’-CGCTAPtz(Phen)1TTACGC-Fluo
5’-GCGATA(Phen)3AATGCG-3’
3’-CGCTAT
(Phen)3BrUTACGC-Fluo
5’-GCGATf(Phen)3AATGCG-3’
3’-CGCTAT
(Phen)3BrUTACGC-Fluo
5’-GCGATf(Phen)3AATGCG-3’
3’-CGCTAPtz(Phen)3TTACGC-Fluo
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
Figure 2. Fluorescence image of a 20% denaturing PAGE showing DNA
strand cleavage of duplex D4 after UV irradiation (365 nm, ~48C). Con-
ditions: 4.0 mm duplex in 10 mm NaH2PO4, 0.15m NaCl, pH 7.0. The
DNAs were exposed to UV light for the indicated time and analyzed
after subsequent piperidine treatment at 908C for 30 min. Lane 1: control
DNA treated with piperidine at 908C for 30 min without UV irradiation;
lane 10: synthesized DNA fragment a; lane 11: fragment b.
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
14
ACHTUNGTRENNUNG
[a] Single-stranded oligonucleotide; [b] c=1.2 mm duplex in 10 mm
NaH2PO4, 0.15m NaCl, pH 7.0; estimated error in Tm = ꢂ0.58C.
products, which do not accumulate with irradiation time, a
low mobility band was observed (Figure 2, fragment c). The
product from this band was isolated from the gel and its
mass was determined by ESIꢁ and MALDI mass spectrome-
try (m/z found: 5297.1; see the Supporting Information). It
could be attributed to a Phen specific intra-strand photore-
action product (Supporting Information, Scheme S2) that
accumulated with irradiation time in duplex DNA and
single strands containing Phen residues. This product occurs
only if BrU is present in the sequence and does not accumu-
late with time to a significant extent, in duplexes D5 or D7,
which lack the PTZ donor (see the Supporting Information).
Since this reaction results from EET, we propose that it
occurs most likely by a radical mechanism between uracil-5-
yl radical and an adjacent Phen residue, and less likely by
way of a cyclobutane adduct[12] (Supporting Information,
Schemes S3 and S4).
than duplex D5 containing an A–T base pair instead. How-
ever, PTZ is accommodated much better in sequences con-
taining three Phen–Phen base pairs; here duplex D4 con-
taining PTZ–f is only ꢁ1.58C less stable than an the duplex
D7 containing an A–T base pair. Further evidence that PTZ
intercalates and provides stability is obtained by difference
of the Tm of duplex D4 (PTZ–f) and duplex D8 (T–f),
which shows that PTZ stabilizes the duplex by +3.48C
when compared to thymine. In general, insertion of a Phen–
Phen pair is consistent with our previously obtained re-
sults.[9] One Phen–Phen pair as a replacement for an A–T
base pair only slightly destabilizes the duplex (DTm =
ꢁ1.08C, D1 and D2) and three Phen–Phen pairs (duplex
D4) stabilize the duplex by +3.88C compared to duplex D3
containing three A–T base pairs.
The effect of excess electron transfer in DNAs with A–T
or Phen–Phen pairs was investigated by selective photoexci-
tation of PTZ using UVA light (lmax =365 nm, Sylvania 8 W,
under N2). In this assay, when migrating electrons encounter
and are captured by BrU, Brꢁ is released and a uracil-5-yl
radical is formed. This radical then abstracts a hydrogen
from a 5’ adjacent deoxyribose neighbor, and after piperi-
dine treatment strand cleavage occurs giving short DNA
fragments.[10] We prepared the expected fragments contain-
ing a 3’-phosphate/5’-fluorescein (Fluo), the tetramer (a: 3’-
O4P-ACGC-Fluo) and the pentamer (b: 3’-O4P-TACGC-
Fluo), and used them as controls and DNA markers. EET
yields are determined by quantifying the formation of frag-
ment a using polyacrylamide gel electrophoresis (PAGE,
Figure 2).
Quantification of the amount of formed cleavage prod-
AHCTUNGTREGuNNUN ct a versus time for duplexes D1–D4 is shown in Figure 3,
and strand cleavage yields after 60 min of irradiation for du-
plexes D1–D4 single strands 2, 4, 6 and 8 are shown in
Figure 4. Substituting one A–T base pair (duplex D1) with a
Phen–Phen pair (duplex D2) slightly decreased the yield
from 7.0 to 5.7%. Increasing the number of A–T base pairs
(duplex D3) decreased the electron transfer efficiency to
2.4%. However, replacing three A–T base pairs with three
Phen–Phen pairs (duplex D4) increased the electron transfer
efficiency to 7.3%. To test if migrating electrons remain
within the DNA p-stack, duplex D4 was saturated with
N2O.[10d,13] Irradiation under a N2O atmosphere maintained
a similar cleavage yield (see the Supporting Information),
providing evidence that strand cleavage did not occur from
solvated electrons present in solution but was due to EET
within the DNA p-stack. Single strand 2 (3.4%) and single
strand 6 (1.2%) containing A–T base pairs exhibited ap-
proximately a 50% decrease in electron transfer efficiency
Selective strand scission was not detected in duplexes D5
and D7 lacking the PTZ electron donor or duplexes D6 and
D9 lacking the BrU. A UV independent cleavage at BrU was
observed without irradiation after piperidine treatment and
is most likely induced by heat.[11] Along with various photo-
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C. J. Leumann et al.
although there exists no rigorous experimental proof for it.
Although the exact reasons for this efficient EET is not evi-
dent from our studies, we can speculate that the six phenan-
threnes present in duplex D4 are interacting/p-stacking in a
way that produces favorable LUMO overlap. Asymmetry in
the overlap of the HOMOs of the DNA bases has been pro-
posed as an explanation for hole-transfer preference in the
5’!3’ direction.[18] By analogy, LUMO orbital overlap has
also been used to rationalize EET transfer efficiencies.[10e,19]
Less favorable LUMO overlap in single strands could ex-
plain why three intrastrand stacked Phen residues are 63%
less efficient in EET than duplex D4 containing three inter-
strand stacked Phen–Phen pairs.
From these experiments we conclude that EET readily
occurs through DNA duplexes containing up to three Phen–
Phen pairs with an efficiency that is superior to duplexes
containing three A–T base pairs instead, despite of the fact
that the distance between injector and acceptor is larger in
the former case. We hypothesize that the reduction potential
of PTZ is high enough to reduce a Phen unit, permitting
EET through a hopping mechanism, much in the same way
as it occurs through long stretches of A–T base pairs. We
have shown previously that the Phen units can be peripher-
ally modified with electron donating or accepting groups
without compromising with the interstrand intercalation rec-
ognition motif.[9] This enables, in the future, the installation
of a LUMO gradient within the stack of Phen units in a
DNA duplex that may suppress back electron transfer.[20]
This may render EET over longer distances even more effi-
cient. Moreover, such DNA assemblies could essentially be
based also on other aromatic units with favorable electronic
conducting properties.
Figure 3. Accumulation of product a, from EET induced DNA strand
cleavage, as a function of time. Conditions as given in the legend of
Figure 2.
Figure 4. DNA cleavage yields for duplexes D1–D4 and single strands
(ss) 2, 4, 6, and 8 after 60 min of UV irradiation.
when compared to the duplex DNAs (Figure 3). Phen–Phen
containing single strands also exhibited a decrease in EET
efficiency. However, single strand 4 (4.1%) containing one
Phen residue was only 28% less efficient than duplex D2
(5.7%) containing a Phen–Phen pair, whereas single strand
8 (2.7%) containing three Phen residues was 63% less effi-
cient than duplex D4 (7.3%) containing three Phen–Phen
pairs.
Both the reduction potentials of Phen (ꢁ2.62 V vs.
NHE)[14] and thymine (ꢁ2.1 V vs. SCE)[15] are high enough
to accept an electron from photoexcited PTZ. Assuming
that electron transfer from PTZ to thymine or Phen is an
exergonic process (Figure 1b), based on the above-men-
tioned reduction potentials, electron transfer should occur
more efficiently to thymine than to Phen. This was observed
in duplex DNAs D1 and D2. However, in duplex D3 con-
taining three A–T base pairs we observed a decrease in
EET efficiency by increasing the distance from one A–T
(3.4 ꢂ) to three A–T base pairs (10.2 ꢂ).[10d,16] On the other
hand, three Phen–Phen pairs (D2) mediate efficient electron
transfer over a distance that is supposed to be twice as long
(20.4 ꢂ). This increase in distance is based on interstrand in-
tercalated Phen residues, a structural model derived from bi-
phenyl containing DNA.[17] This model may also apply here,
Experimental Section
All details are provided in the Supporting Information.
Acknowledgements
Financial Support by the Swiss National Science Foundation (grant-No.:
200020-130373), and the EU for a Marie-Curie Fellowship (F.W.) is grate-
fully acknowledged.
Keywords: DNA recognition
·
electron transfer
·
oligonucleotides · phenanthrene · stacking interactions
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Published online: && &&, 0000
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