Excess electron transfer in flavin-capped DNA-hairpins

Article abstract of DOI:10.1002/1099-0690(200210)2002:19<3281::AID-EJOC3281>3.0.CO;2-I

DNA hairpins with an electron-acceptor molecule in the head region have recently allowed Lewis and Wasielewski to gain important insight into the hole conductivity of double helical DNA. In light of our current interest in deciphering the excess electron

Full text of DOI:10.1002/1099-0690(200210)2002:19<3281::AID-EJOC3281>3.0.CO;2-I

FULL PAPER
Excess Electron Transfer in Flavin-Capped DNA-Hairpins
Christoph Behrens,[a] Matthias Ober,[a] and Thomas Carell*[a]
Keywords: DNA / Electron transfer / Flavin / Hairpin / Model compound
DNA hairpins with an electron-acceptor molecule in the
ate/phosphoramidite protocol. We describe here that all
head region have recently allowed Lewis and Wasielewski three flavin-cap molecules yield stable DNA hairpins. UV,
to gain important insight into the hole conductivity of double
helical DNA. In light of our current interest in deciphering
the excess electron transport properties of double helical
fluorescence and melting temperature studies showed that
one of the three flavins is stacked in the intended fashion.
Initial studies show that the flavin in the reduced and depro-
DNA, we evaluate in this report three different flavin molec- tonated state can indeed inject an electron into the hairpin.
ules for their ability to form a stable DNA hairpin cap. The
intention was to construct novel DNA hairpins in which the
flavin chromophore is perfectly stacked on top of the final
base pair. A solid-phase synthesis protocol was devised for
the incorporation of the three flavin-H-phosphonates into oli-
gonucleotides using a mixed phosphoramidite/H-phosphon-
This flavin-capped DNA hairpin allows a detailed investi-
gation of the excess electron transfer capabilities of double
helical DNA.
( Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany,
2002)
Introduction
We are currently investigating how excess electrons as
negative charges move through DNA.[12,13] In this context,
γ-irradiation studies suggest that excess electrons might hop
through DNA as well.[14Ϫ18] Sevilla and co-workers pro-
posed a thermally activated excess electron hopping mech-
anism at temperatures above Ϫ70 °C, while below this tem-
perature superexchange is believed to dominate.
Hairpin-shaped oligonucleotides are DNA strands that
possess a self-complementary stem and a loop region,
known as the ‘‘head’’ of the hairpin.[1Ϫ3] DNA hairpins are
stably folded oligonucleotides with a sharp, concentration-
independent melting point. This feature has made DNA
hairpins ideal model systems for oligonucleotide structure
and function. As such, hairpin DNA molecules containing
a non-nucleotide electron donor or acceptor in the ‘‘head’’
region have been used as model systems to investigate fun-
damental questions related to the electron-transfer capabil-
ities of DNA. Lewis and Wasielewski have used hairpins
with a stilbene electron acceptor ‘‘head’’ to measure the
critical β-value of electron transfer from guanines to the
stilbene head. This β-value describes the distance depend-
ence of the superexchange electron transfer through
DNA.[4Ϫ8] The DNA hairpin experiments revealed a β-
value of 0.7, which was at that time in sharp contrast to
other reports placing the β-value well below 0.1.[9] The hair-
pin model study therefore laid the foundation for our cur-
rent understanding of hole transfer through DNA. It is now
well accepted that positive holes do not move through DNA
by superexchange alone. Giese and Schuster were among
the first to propose that long-range hole movement through
a DNA duplex is best described by a hopping mechanism,
which is far less distance-dependent than a superexchange
transfer.[10,11]
Studies by us with defined donor-DNA-acceptor model
compounds at room temperature showed recently that an
excess electron can move surprisingly efficiently over a dis-
tance of up to 25 A.[12] These studies were possible because
˚
a flavin in its reduced and deprotonated redox state could
be inserted into DNA, where it functions as a strong elec-
tron donor. This unusual donor possesses a very negative
reduction potential of at least Ϫ2.6 V,[19,20] which is low
enough to achieve efficient single-electron reduction of pyr-
imidines and cyclobutane pyrimidine dimers. These dimer
acceptors undergo rapid cycloreversion upon single-elec-
tron reduction, which allows rapid monitoring of the elec-
tron capture event.[21Ϫ23] The same flavin donor used in our
experiments is also found in nature. The enzyme DNA
photolyase repairs UV-induced mutagenic cyclobutane pyr-
imidine dimer DNA lesions by single electron donation.
This process rescues many organisms from UV-induced
cell death.[24,25]
In order to gain further insight into the mechanism of
long-range excess electron transfer, the synthesis of defined
DNA hairpins with a flavin donor in the ‘‘head’’ region and
a cyclobutane pyrimidine dimer electron acceptor in the
stem is required. These hairpins are structurally much
better defined then the double strands used in our previous
studies. This is important for precise studies, because elec-
[a]
Fachbereich Chemie, Philipps-Universität Marburg,
Hans-Meerwein-Strasse, 35032 Marburg, Germany
Fax: (internat.) ϩ49-(0)6421/282-2189
E-mail: carell@mailer.uni-marburg.de
Eur. J. Org. Chem. 2002, 3281Ϫ3289  WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002 1434Ϫ193X/02/1019Ϫ3281 $ 20.00ϩ.50/0
3281

C. Behrens, M. Ober, T. Carell
FULL PAPER
tron-transfer rates depend not only on the distance between From these studies we could narrow down the number of
the donor and the acceptor but are also influenced by the possible flavin cap molecules that fulfil the above require-
orientation of the chromophores relative to each other. In ments. The three most promising calculated hairpin molec-
structurally better defined hairpin donor-DNA-acceptor ules 1؊3, together with the structures of the flavin-cap mo-
systems, the electron-transfer processes can be studied with lecules, are depicted in Figure 1. The flavin cap of hairpin
much higher accuracy and without any interference because 1 contains two C6-chains at N(3) and N(10) of the flavin.
of incomplete duplex formation.
From our modeling, depicted in Figure 1, we concluded
Herein we report the design and the synthesis of stable that the hairpin is formed but the chain length is clearly on
flavin-capped DNA hairpins in which the flavin stacks on the long side, resulting in a slightly too-flexible connection,
top of the last base pair in the hairpin. We also report an which would then disturb the efficient stacking of the flavin.
initial electron injection study. These novel hairpins open The flavin-cap molecule in 3 has two C3 chains at N(3)
the door for a deeper mechanistic investigation of excess and N(10) and, in our series, is the smallest solution. The
electron transfer processes in DNA.
calculations predict perfect stacking. Slight disturbance of
the hairpin helix structure was, however, also noted. A
shorter chain length resulted in a marked increase in duplex
destabilization. The flavin-cap molecule used to build up
hairpin 2 features one C6 chain at N(10) and one C3 chain
at N(3). This flavin gave the best modeling results. We calcu-
lated almost perfect stacking of the flavin on top of the
final base pair and almost no helix disturbance.
Results and Discussion
Design and Synthesis of Flavin-Capped DNA Hairpins
The critical point for the synthesis of a stably folded fla-
vin-capped DNA hairpin is the length of the two chains
connecting the flavin with the DNA. These chains have to
Based on the modeling results we rejected all other pos-
be long enough to avoid any disruption of the B-form stem sible flavin capping molecules and concentrated on the syn-
structure. If, on the other hand, the chains are too long thesis of the three most promising hairpin molecules 1؊3,
then the strictly required stacking of the flavin on top of using the synthetic strategy depicted in Scheme 1. The start-
the final base pair, needed for efficient charge injection into ing point for the synthesis was dimethyldinitrobenzene (4),
the base stack, may not occur. In order to find the optimal which was reacted with either 1-aminopropan-3-ol (5) or 1-
chain length, we first performed detailed molecular amino-hexan-6-ol (6). The nitro groups of the products
modeling studies using the SYBYL (Tripos version 6.8) were subsequently hydrogenated and the resulting amines
software package and the force field MMFF94s. All calcu- were added, without prior isolation, to a solution con-
lations were performed in the gas phase and also in water. taining alloxan and boric acid in acetic acid following a
Figure 1. Calculated (SYBYL, Tripos software version 6.8, force field MMFF94s in the gas phase) structures of the three flavin-capped
DNA hairpins 1؊3 and of the three flavin capping molecules
3282
Eur. J. Org. Chem. 2002, 3281Ϫ3289

Excess Electron Transfer in Flavin-Capped DNA-Hairpins
FULL PAPER
Scheme 1. Synthesis of the flavin H-phosphonates 16؊18 used for the preparation of the flavin capped DNA hairpins 1؊3; a) pyridine,
19 h reflux; b) H2, Pd/C; c) HOAc, B(OH)3, alloxan, room temp.; d) DMTrCl, pyridine, room temp. 93Ϫ98%; e) Cs2CO3, DMF, 16 h,
67Ϫ84%; f) PCl3, triazole, N-methylmorpholine, 43Ϫ48%
flavin synthesis protocol developed by Kuhn and co- from the solid support material and of all nucleobase pro-
workers.[26,27] Subsequent protection of the hydroxyl groups tecting groups was performed at the end of the synthesis by
of the flavin products 7 and 8 with dimethoxytrityl chloride shaking of the solid support material in a solution made up
provided the flavins 9 and 10 as yellow powders. These flav- of three parts ammonia (25%) in water and one part eth-
ins possess excellent solubility in most organic solvents. anol at 20 °C for 16 h. All oligonucleotides were sub-
Subsequent alkylation of both flavins 9 and 10 at N(3) with sequently purified by reversed-phase HPLC on a C18
either 1-iodopropan-3-ol (11) or 1-iodo-hexan-6-ol (12), us- Nucleosil reversed-phase column and characterized by
ing Cs2CO3 as the base in DMF, provided the three flavin MALDI-Tof mass spectrometry.[13,31] We frequently ob-
products 13؊15. Final conversion of the hydroxyl groups served two DNA strands, corresponding to oligonucleotides
into the H-phosphonates gave the three flavin H-phosphon- obtained by cleavage of the phosphate backbone at the 5Ј
ate target compounds 16؊18, ready for the incorporation position to the flavin base, after deprotection rather than
into oligonucleotides.
the desired oligonucleotide. We reasoned that this cleavage
The synthesis of the oligonucleotide hairpins 1؊3 was is caused by incomplete oxidation of the H-phosphonate on
performed on an Expedite 8900 DNA synthesizer using a the solid support followed by cleavage of the H-phosphon-
mixed phosphoramidite/H-phosphonate/phosphoramidite ate under the basic deprotection conditions. In all future
coupling protocol.[28Ϫ31] This complicated protocol is re- syntheses of flavin-containing DNA we therefore pumped
quired because flavin phosphoramidites are efficiently oxid- two different oxidizing solution through the cartridge for
ized under aerobic conditions.[32,33] A complete H-phos- an additional 15 min after completion of the DNA syn-
phonate protocol is also not applicable because only H- thesis. We first pumped a solution made up of 0.1  I2 in
phosphonates with standard protecting groups are commer- MeCN/H2O with N-methylmorpholine as the base. As a se-
cially available. These require harsh deprotection condi- cond oxidizing solution 0.1  I2 in MeCN/H2O with NEt3
tions, which were found to be incompatible with the base- as the base was used. This two step oxidation protocol fully
labile flavin molecule. Deprotection of flavin-containing oli- converted the H-phosphonate into the phosphate, which
gonucleotides requires mild conditions, which forced us to then allowed us to get the flavin-containing oligonucleo-
use very base-labile nucleotide protecting groups. These are tides in good to excellent yields without any observable
only available for phosphoramidite chemistry.
cleavage at the original H-phosphonate side.
Synthesis of the DNA hairpins 1؊3 starts with standard
phosphoramidite chemistry. After cleavage of the DMT-
protecting group with dichloroacetic acid at the 3Ј end to
the flavin, the synthesizer was programmed to pump simul-
taneously the flavin H-phosphonate and adamantoyl acid
chloride. Coupling of the flavin building blocks 16؊18 was
Characterization and Stability Studies of the Flavin-Capped
Hairpins 1؊3
In order to prove that the flavin chromophore survived
extended to about 10 min (as opposed to 2 min for standard the complicated synthesis and deprotection chemistry we
phosphoramidite chemistry). After coupling of the flavin measured UV/Vis and fluorescence spectra of the obtained
we oxidized the H-phosphonate to the phosphate using iod- DNA strands 1؊3. Two representative spectra are depicted
ine in H2O/MeCN/lutidine. The DMT group of the flavin in Figure 2; they were measured with DNA strand 2. Sim-
was subsequently cleaved and the DNA synthesis was con- ilar data, however, were recorded for all three DNA strands
tinued using phosphoramidite chemistry, even in the pres- 1؊3. All spectra are very similar to that of riboflavin itself,
ence of the unprotected phosphate. Cleavage of the DNA showing that the chromophore system of the flavin is not
Eur. J. Org. Chem. 2002, 3281Ϫ3289
3283

C. Behrens, M. Ober, T. Carell
FULL PAPER
Figure 3. UV/Vis melting curve of the three flavin-capped DNA
hairpins 1؊3 (cDNA ϭ 3 ϫ 10Ϫ6  in 0.01  Tris-buffer, 150 m
NaCl, pH 7.4), measured at 260 nm
ity is too high, which inhibits perfect stacking (Tm ϭ 41
°C). All these results are in full agreement with our calcula-
tions, which predicted that the flavin with one C3 and one
C6 chain would be the best cap of an oligonucleotide struc-
ture.
This DNA hairpin 2 was further investigated by CD spec-
troscopy. The obtained CD spectrum is depicted in Fig-
ure 4. It shows the typical curve for a B duplex, which un-
derpins once more that the flavin cap does not disturb the B
conformation of the oligonucleotide stem. The data prove,
furthermore, that the oligonucleotide 2 is stably folded.
Figure 2. a) UV/Vis spectrum of the flavin-capped hairpin 2
(cDNA ϭ 3 ϫ 10Ϫ6  in 0.01  Tris-buffer, 150 mmol NaCl, pH 7.4);
b) fluorescence spectrum of the flavin-capped hairpin 2 (cDNA ϭ 3
ϫ 10Ϫ6  in 0.01  Tris-buffer, 150 m NaCl, pH 7.4), excitation
at 366 nm; similar spectra were recorded for the hairpins 1 and 3
as well.
modified during DNA synthesis, oxidation and deprotec-
tion.
The purity of the final oligonucleotides was confirmed
by HPLC showing a single sharp peak without shoulders
(data not shown). All three mass spectra are in full agree-
ment with the calculated molecular masses of the oligonu-
cleotides 1؊3 (see Exp. Sect.). All these data together prove
that this mixed protocol allows embedding of a flavin cofac-
tor into oligonucleotides.
Figure 4. CD spectrum (averaged from 10 independent measure-
ments) of the flavin-capped molecular hairpin 2 (cDNA ϭ 3 ϫ 10Ϫ6
 in 0.01  Tris-buffer, 150 m NaCl, pH 7.4)
If the flavin is stacking on top of the final base pair one
In order to investigate if the three DNA strands 1؊3 would expect the fluorescence intensity of the flavin chrom-
form stable hairpins at room temperature and to investigate ophore to be modulated. In fact, both fluorescence reduc-
potential stability differences, we measured all three melting tions and fluorescence increases are frequently observed if
points. In agreement with the calculations, which predicted fluorescent aromatic ring systems stack in or on top of
that all three molecules 1؊3 should form a stable hairpin, DNA base pairs.[34] In order to investigate this question fur-
we observe for all three oligonucleotides a concentration- ther we measured a fluorescence melting curve for hairpin
independent melting point (Figure 3), although the absolute 2 (Figure 5). The obtained fluorescence melting curve is fas-
values were different. The highest melting point was meas- cinating and clearly proves that the flavin in hairpin 2 stacks
ured for the hairpin 2 (Tm ϭ 43 °C) containing the flavin on top of the final A-T base pair. The fluorescence intensity
with one C3 and one C6 chain. The hairpin 3 featuring the of a flavin alone decreases with increasing temperature. At
flavin with two C3 chains is strongly destabilized (Tm ϭ 31 the melting temperature of the hairpin, however, we observe
°C) indicating that the chain length is much too short to against this trend a strong fluorescence increase. After com-
bridge the two oligonucleotide strands efficiently. Hairpin plete melting the temperature-dependent fluorescence in-
1, which is capped with the flavin having two C6 chains is tensity decrease continues. From the spectrum it is clearly
slightly less stable than 2 possibly because the chain flexibil- evident that the flavin fluorescence is reduced upon folding
3284
Eur. J. Org. Chem. 2002, 3281Ϫ3289

Excess Electron Transfer in Flavin-Capped DNA-Hairpins
FULL PAPER
this solution into four small glass tubes stoppered with a
rubber septum. After purging the solutions for 10 min with
nitrogen, a sodium dithionite solution was added into each
vial to achieve flavin reduction. The yellow color immedi-
ately vanished confirming successful reduction of the flavin.
These four vials were finally irradiated (366 nm) at T ϭ 5
°C for 0 min, 15 min, 30 min and 60 min, respectively. The
assay solutions were finally exposed to air and shaken to
rapidly re-oxidise the flavin. The solutions were then ana-
lysed by ion-exchange chromatography (Nucleogel SAX
column, Gradiant: 0.2  NaCl to 1  NaCl over 35 min).
The HPL-chromatograms obtained are shown in Figure 6.
It is clearly evident that at 0 min of irradiation just the fla-
vin hairpin 19 is eluting, with a retention time of 23 min.
Further irradiation causes a clear decrease of the DNA
hairpin 19 and the appearance of a peak for the DNA
strand 20, which elutes at about 16 min. This DNA strand
is the hairpin cleaved at the thymine dimer site. This experi-
ment clearly proves that the flavin cap can inject a single
electron into the hairpin, which triggers the cycloreversion
reaction due to capturing of the electron by the dimer
acceptor.
Figure 5. Fluorescence melting curve of the flavin-capped DNA
hairpin 2 (cDNA ϭ 3 ϫ 10Ϫ6  in 0.01  Tris-buffer, 150 m NaCl,
pH 7.4); excitation at 366 nm, emission observed at 520 nm
of the oligonucleotide into the hairpin structure. This result,
together with our calculations, are, for us, a clear proof that
the flavin stacks on top of the final base pair. This stacking
reduces the fluorescence intensity.
Excess Electron Transfer Studies
In order to investigate if the flavin is able to inject elec-
trons into the DNA hairpin we prepared for an initial study
the hairpin molecule 19 (Figure 6). Compound 19 contains
a pyrimidine dimer building block with an opened back-
bone next to the flavin cap. Upon single-electron reduction,
this dimer will induce a strand break as recently described
by us.[12,35] Hairpin 19 is stable as long as the flavin molecule
exists in the oxidized state. For the electron injection experi-
ment, we prepared a solution of hairpin 19 in Tris-buffer
(10 m Tris, cDNA ϭ 20 µ, pH ϭ 7.4) and added 50 µL of
Conclusions
Our goal was the synthesis of flavin H-phosphonate,
which can be used to create a flavin cap for the construction
of DNA hairpin molecules. Particular emphasis was placed
in our design on the ability of the flavin cap to stack per-
fectly on top of the final base pair. This is required if the
flavin is to act as an efficient charge injector into DNA, as
required for electron-transfer studies through DNA. Com-
puter modeling was performed first of all and three promis-
ing flavin H-phosphonates 16؊18 were selected that were
calculated to give stably folded hairpin molecules (1؊3).
These three flavin molecules, one with two C3 chains (16),
one with two C6 chains (18) and one with one C3 and one
C6 chain (17) were synthesized and incorporated into oli-
gonucleotides using a mixed phosphoramidite/H-phos-
phonate/phosphoramidite coupling protocol. The structure
and stability of the three flavin-containing oligonucleotides
1؊3 were investigated. It was found that all three flavins
allow the formation of oligonucleotide hairpins. The C3/
C6 chain flavin H-phosphonate 17, however, gives the most
stable hairpin. Further experiments prove that this flavin
stacks perfectly on top of the final base pair. The flavin H-
phosphonate 17 is therefore perfectly suited for the con-
struction of a series of flavin-containing DNA hairpins for
excess electron transfer studies through DNA. In order to
study if the flavin cap is indeed injecting excess electrons
into DNA, we synthesized the flavin-capped hairpin 19,
containing in addition a cyclobutane thymine dimer, which
undergoes rapid cycloreversion upon electron capture re-
sulting in a strand break. Irradiation of this hairpin con-
taining the flavin in the reduced, and hence electron donat-
ing, state indeed gave rapid strand cleavage. This experiment
showed that flavin-capped hairpins are perfectly suited to
Figure 6. Flavin-capped cyclobutane pyrimidine dimer containing
DNA hairpin 19 and cleavage into 20 upon electron injection into
the DNA strand by the flavin in the reduced state; depiction of the
HPL chromatograms obtained during an irradiation study
Eur. J. Org. Chem. 2002, 3281Ϫ3289
3285

C. Behrens, M. Ober, T. Carell
FULL PAPER
MeOH/NEt3, 10:1:0.2) ϭ 0.61. M.p. 233 °C (decomp.). IR (KBr):
ν˜ ϭ 3447 w cmϪ1, 3160 w, 3107 w, 3031 m, 2954 m, 2835 w, 1706 m,
1652 s, 1609 m, 1576 s, 1516 s, 1502 s, 1464 m, 1250 s, 1174 m, 1155
study excess electron transfer in DNA. Further synthesis of
other flavin-capped, dimer-containing hairpins and investi-
gation of the excess electron transfer process are now un-
der way.
1
s, 1075 m, 1031 m, 825 m. H NMR (200 MHz, CDCl3): δ ϭ 2.10
(m, 2 H, CH2), 2.40 (s, 3 H, CH3), 2.42 (s, 3 H, CH3), 3.27 (d, J ϭ
5.6 Hz, 2 H,CH2O), 3.71 (s, 6 H, OCH3), 4.75 (t, J ϭ 8.0 Hz, 2 H,
CH2N), 6.75 (d, J ϭ 8.4 Hz, 4 H, CHaromatic), 7.15Ϫ7.45 (m, 9 H,
CHaromatic), 7.65 (s, 1 H, CHflavin), 7.99 (s, 1 H, CHflavin) ppm. 13C
NMR (75 MHz, CDCl3): δ ϭ 19.5, 21.5, 27.9, 43.6, 55.6 (2 C),
60.8, 86.5, 113.6 (4 C), 116.1, 127.4, 128.3 (4 C), 130.3 (4 C), 131.2,
133.1, 135.4, 136.2 (2 C), 136.3, 137.5, 145.3, 148.8, 150.1, 155.6,
158.9 (2 C), 159.9 ppm. MS (FD): m/z ϭ 602 [Mϩ], 303 [DMTϩ].
HR-MS ESI calcd. for C36H34N4O5 ϩ Naϩ: 625.2427; found
625.2421.
Experimental Section
General: Reagents and solvents were reagent grade and used with-
out further purification. Anhydrous Na2SO4 was used as the drying
agent after aqueous workup. Evaporation and concentration in va-
cuo was done at H2O aspirator pressure. All reactions were per-
formed in standard glassware. Degassing of solvents was accomp-
lished by N2 purging for at least 4 min. Column chromatography
(CC): Silica gel-H from Fluka. TLC: glass or aluminium sheets
covered with silica gel 60 F254 from Merck, visualization by UV
light. M.p.: Büchi SMP-20 apparatus; uncorrected. UV/Vis spectra:
Varian Bio100 spectrophotometer at room temperature; λmax in nm
(ε in Ϫ1cmϪ1). Melting points were measured using a Cary tem-
perature controller and a sample transport accessory with a multi-
cell block and a temp. gradient of 0.5 °C/min. Oligonucleotides
were dissolved in a Tris buffer (10 m Tris, 150 m NaCl, pH 7.4).
CD spectra were recorded on a JASCO J-810 at 10 °C. IR spectra
(cmϪ1) were recorded in KBr and measured in cmϪ1, with a Bruker
IFS 25 Fourier transform infrared spectrophotometer. Fluores-
cence spectra were measured on a JASCO FP750 Fluorimeter,
single monochromator with Ϯ5 nm, 150 Hg/Xe lamp, in 1 cm
quartz cuvettes at room temperature. Melting points were measured
using a JASCO temperature controller. Temp. gradient 0.5 °C/min.
Mass spectra were recorded with a Bruker Flex III Maldi-Tof mass
spectrometer (matrix: 2,4,6-trihydroxyacetophenone, 0.5  in H2O/
diammonium citrate/0.1  in H2O) and on a Finnigan TSQ 700.
Oligonucleotide synthesis was performed using an Expedite 8900
Gene synthesizer. Pac amidites were purchased from Pharmacia or
Glen Research. A controlled-pore glass bead support was pur-
chased from Perseptive. Acetonitrile for the oligonucleotide syn-
thesis was obtained from Roth. All solvents were stored for 12 h
10-{6-[Bis(4-methoxyphenyl)phenylmethoxy]hexyl}-7,8-dimethyl-
10H-benzo[g]pteridine-2,4-dione (10): The flavin alcohol 8 (0.100 g,
0.29 mmol) was dissolved in pyridine (9 mL) and stirred with mo-
˚
lecular sieves (4 A) for 1 h. 4,4Ј-Dimethoxytrityl chloride (168 mg,
0.50 mmol) was added and the reaction mixture was stirred at room
temp. for 16 h. Methanol (1.0 mL) was added and the reaction
mixture was further stirred for 1 h. The solvent was evaporated
and the solid residue was dissolved in chloroform and filtered. The
organic phase was washed with a satd. NaHCO3 solution and twice
with H2O. After drying with Na2SO4 and filtration the solvent was
evaporated and the crude product was purified by flash chromato-
graphy with CHCl3/MeOH/NEt3 (20:1:0.2) as eluent. Compound
10 (0.176 g, 0.27 mmol, 93%) was obtained as a yellow solid. Rf
(CHCl3/MeOH 20:1): 0.40. M.p. 155Ϫ156 °C. IR (NaCl): ν˜ ϭ 3481
w cmϪ1, 3030 m, 2933 m, 2862 w, 2834 w, 1772 m, 1718 s, 1684 s,
1
1576 s, 1507 s, 1458 m, 1247 s, 1173 m, 1031 m, 826 m, 735 m. H
NMR (200 MHz, CDCl3): δ ϭ 1.35Ϫ1.55 (m, 4 H, CH2), 1.58 (m,
J ϭ 6.4 Hz, 2 H, CH2), 1.7Ϫ1.9 (m, 2 H, CH2), 2.37 (s, 3 H, CH3),
2.46 (s, 3 H, CH3), 2.98 (t, J ϭ 6.2 Hz, 2 H,CH2O), 3.71 (s, 6 H,
OCH3), 4.59 (t, J ϭ 7.6 Hz, 2 H, CH2N), 6.74 (d, J ϭ 7.0 Hz, 4
H, CHaromatic), 7.1Ϫ7.4 (m, 10 H, CHaromatic), 7.99 (s, 1 H, CHflavin
)
ppm. 13C NMR (50 MHz, CDCl3): δ ϭ 19.3, 21.4, 25.9, 26.5, 26.9,
29.7, 45.0, 54.9 (2 C), 62.9, 85.4, 112.9 (4 C), 115.0, 126.3, 127.4 (2
C), 127.9 (2 C),128.9, 129.7 (4 C), 130.8, 132.6, 134.7, 136.0, 136.3
(2 C), 145.1, 148.0, 149.6, 155.1, 158.1 (2 C), 159.4 ppm. MS (FD):
m/z ϭ 644 [Mϩ], 342 [Mϩ Ϫ DMT], 303 [DMTϩ]. HR-MS ESI
calcd. for C39H40N4O5 ϩ Naϩ: 667.2896; found 667.2863.
˚
over 4 A molecular sieves prior to oligonucleotide assembly. HPLC
was performed with a Merck-Hitachi Lachrome HPLC system with
a flow of 1 mL/min using a Nucleosil RP18 (250 mm ϫ 4 mm, 100
˚
˚
A /5 µm or 250 mm ϫ 4 mm, 120 A/3 µm) column from
MachereyϪNagel. HPLC-grade solvents were purchased from Rie-
del de Haen. All oligonucleotides were detected at 260 nm. For
analytical oligonucleotide HPLC chromatography a linear gradient
was used. Preparative HPLC was performed with a Nucleosil RP
10-{3-[Bis(4-methoxyphenyl)phenylmethoxy]propyl}-3-(3-hydroxy-
propyl)-7,8-dimethyl-10H-benzo[g]pteridine-2,4-dione (13): 1-Iodop-
ropan-3-ol was prepared according to the literature.[37] The flavin
9 (150 mg, 0.25 mmol) was dissolved in dry DMF (5.3 mL) and
˚
C18 column (250 mm ϫ 10 mm, 100 A/7 µm). Solvent system: A ϭ
˚
stirred with molecular sieves (4 A) for 2 h. To this solution, Cs2CO3
0.1
 NEt3/HOAc in H2O, B ϭ 0.1  NEt3/HOAc in
(117 mg, 0.36 mmol) was added and the mixture was stirred for
15 min at room temp. 1-Iodopropan-3-ol (11; 0.15 mL, 1.56 mmol)
H2O:CH3CN (1:4).
10-{3-[Bis(4-methoxyphenyl)phenylmethoxy]propyl}-7,8-dimethyl- was added dropwise and the mixture was stirred for 16 h at room
10H-benzo[g]pteridin-2,4-dione (9): For the analytical data of com-
pounds 5؊8 see refs.[33,36] The flavin alcohol 7 (0.194 g, 0.65 mmol)
was dissolved in pyridine (15 mL) and stirred with molecular sieves
temp. The reaction mixture was then filtered, diluted with chloro-
form (150 mL) and the organic phase was washed once with satur-
ated NaHCO3 solution and twice with water. After drying over
Na2SO4 the solvent was evaporated and the crude product was
˚
(4 A) for 1 h. 4,4Ј-Dimethoxytrityl chloride (372 mg, 1.10 mmol)
was added and the reaction mixture was stirred at room temp. for purified by flash chromatography with CHCl3/MeOH/NEt3
16 h. Methanol (2.0 mL) was added and the reaction mixture was
stirred for a further hour. The solvent was evaporated and the solid
residue was dissolved in chloroform and filtered. The organic phase
(30:1:0.2) as eluent. Compound 13 (0.120 g, 0.18 mmol, 73%) was
isolated as a yellow oil. Rf (CHCl3/MeOH/NEt3, 15:1:0.2): 0.61. IR
(NaCl): ν˜ ϭ 3471 m, 3057 w, 3034 w cmϪ1, 2954 m, 2932 m, 2876
was washed once with satd. NaHCO3 solution and twice with H2O. w, 2837 w, 1703 s, 1657 s, 1607 m, 1584 s, 1548 s, 1510 s, 1462 m,
After drying with Na2SO4 and filtration the solvent was evaporated
and the crude product was purified by flash chromatography with
1251 s, 1200 s, 1069 m, 1032 s, 911 s, 828 s, 727 s. 1H NMR
(200 MHz, CDCl3): δ ϭ 1.93 (m, 2 H, CH2CH2CH2OH), 2.15 (m,
CHCl3/MeOH/NEt3 (20:1:0.2) as eluent. Compound 9 (0.398 g, 2 H, CH2CH2CH2O), 2.44 (s, 3 H, CH3), 2.47 (s, 3 H, CH3), 3.32
0.64 mmol, 98%) was obtained as a yellow solid. Rf (CHCl3/
(t, J ϭ 5.0 Hz, 2 H, CH2O), 3.52 (m, 3 H, CH2OH ϩ OH), 3.77
Eur. J. Org. Chem. 2002, 3281Ϫ3289
3286

Excess Electron Transfer in Flavin-Capped DNA-Hairpins
FULL PAPER
(s, 6 H, OCH3), 4.23 (t, J ϭ 6.2 Hz, 2 H, CH2N), 4.81 (t, J ϭ (t, J ϭ 7.6 Hz, 2 H, NCH2), 6.75 (d, J ϭ 9.0 Hz, 4 H, CHaromatic),
7.2 Hz, 2 H, CH2N), 6.80 (d, J ϭ 9.0 Hz, 4 H, CHaromatic), 7.1Ϫ7.4 (m, 10 H, CHaromatic), 7.99 (s, 1 H, CHflavin) ppm. 13C
7.20Ϫ7.35 (m, 7 H, CHaromatic), 7.40 (d, J ϭ 8.0 Hz, 2 H, CHarom-
atic). 7.71 (s, 1 H, CHflavin), 8.05 (s, 1 H, CHflavin) ppm. 13C NMR 27.5, 29.9, 32.5, 41.7, 44.6, 55.1 (2 C), 62.7, 63.1, 85.6, 112.9 (4 C),
(50 MHz, CDCl3): δ ϭ 19.9, 21.9, 28.0, 31.2, 38.8, 43.2, 55.6 (2 C), 115.0, 126.5, 127.6 (2 C), 128.0 (2 C), 130.0 (4 C), 130.9, 132.6,
NMR (50 MHz, CDCl3): δ ϭ 19.4, 21.5, 25.2, 26.0, 26.5, 26.7, 27.0,
59.0, 60.8, 86.9, 113.6 (4 C), 116.0, 124.1, 127.3 (2 C), 128.3, 130.3 134.9, 135.6, 136.4, 136.5 (2 C), 145.2, 147.5, 148.4, 155.7, 158.2 (2
(2 C), 131.5 (4 C), 133.1, 135.6, 136.3 (2 C), 137.3, 145.3, 148.6, C), 159.9 ppm. MS (ESI): m/z ϭ 783 [Mϩ ϩ K], 767 [Mϩ ϩ Na],
149.0, 150.2, 156.7, 158.9 (2 C), 161.0 ppm. HR-MS ESI calcd. for 745 [Mϩ ϩ H]. HR-MS ESI calcd. for C45H52N4O6 ϩ Naϩ:
C39H40N4O6 ϩ Naϩ: 683.2846; found 683.2852.
767.3785; found 767.3756.
10-{6-[Bis(4-methoxyphenyl)phenylmethoxy]hexyl}-3-(3-hydroxy- H-Phosphonate 16: Dichloromethane (4.8 mL), N-methylmorpho-
propyl)-7,8-dimethyl-10H-benzo[g]pteridine-2,4-dione (14): The fla- line (1.76 mL, 18.9 mmol) and triazole (371 mg, 5.4 mmol) were
vin 10 (200 mg, 0.31 mmol) was dissolved in dry DMF (7.0 mL) added to a solution (2 ) of PCl3 in dichloromethane (0.8 mL).
˚
and stirred with molecular sieves (4 A) for 2 h. Cs2CO3 (156 mg,
0.48 mmol) was added to this solution and the mixture was stirred
for 15 min at room temp. 1-Iodopropan-3-ol (11; 0.20 mL,
After stirring for 30 min at room temp. the solution was cooled to
0 °C and a solution of the flavin 13 (120 mg, 0.18 mmol) dissolved
in dichloromethane (3.6 mL) was added dropwise. After stirring
2.09 mmol) was added dropwise and the mixture was stirred for for 30 min the mixture was allowed to warm to room temp. and
16 h at room temp. The reaction mixture was then filtered, diluted
with chloroform (200 mL) and the organic phase was washed once
with saturated NaHCO3 solution and twice with water. After dry-
ing over Na2SO4 the solvent was evaporated and the crude product
was purified by flash chromatography with CHCl3/MeOH/NEt3
(30:1:0.2) as eluent. Compound 14 (0.185 g, 0.26 mmol, 84%) was
isolated as a yellow oil. Rf (CHCl3/MeOH/NEt3, 20:1:0.2): 0.49. IR
triethylammonium bicarbonate (TEAB) buffer (1 , 22 mL) was
added. Stirring was continued for 30 min, then the organic phase
was diluted with dichloromethane (100 mL) and the salts were ex-
tracted with TEAB buffer (100 mL, 1 mol/L). The organic phase
was dried with Na2SO4 and the solvent was evaporated. The crude
product was purified by flash chromatography with CHCl3/MeOH/
NEt3 (10:1:0.2) as eluent. To remove additional salts, the product
(NaCl): ν˜ ϭ 3463 m cmϪ1, 3057 w, 3034 w,2935s, 2864 m, 2837 m, was again dissolved in dichloromethane and the solution extracted
1745 m, 1702s, 1656 s, 1607 m, 1584 s, 1555 s, 1510 s, 1462 m, with TEAB buffer. The H-phosphonate 16 (64 mg 0.08 mmol, 43%)
1445 m, 1249 s, 1178s, 1082 m, 1033 s, 911 m, 828 m, 728 s. 1H was obtained as a yellow oil. Rf (CHCl3/MeOH/NEt3, 10:1:0.2) ϭ
NMR (200 MHz, CDCl3): δ ϭ 1.4Ϫ1.5 (m, 4 H, CH2), 1.58 (m,
0.27. IR (NaCl): ν˜ ϭ 3054 s cmϪ1, 3033 s, 2955 m, 2935 m, 2864 s,
J ϭ 6.0 Hz, 2 H, CH2), 1.78 (m, 2 H, CH2), 1.90 (m, J ϭ 6.0 Hz, 2838 s, 2238 m, 1704 m, 1660 s, 1651 s, 1585 s, 1552 s, 1505 m,
1
2 H, CH2), 2.37 (s, 3 H, CH3), 2.47 (s, 3 H, CH3), 2.99 (t, J ϭ 1455 m, 1250 s, 1203 m, 1178 m, 1063 m, 991 m, 829 s, 727 m. H
6.0 Hz, 2 H, CH2O), 3.37 (t, J ϭ 6.5 Hz, 1 H, OH), 3.50 (q, J ϭ NMR (200 MHz, CDCl3): δ ϭ 1.11 [t, J ϭ 7.2 Hz, 9 H,
6.0 Hz, 2 H, CH2OH), 3.70 (s, 6 H, OCH3), 4.20 (t, J ϭ 6.0 Hz, CH3(NEt3)], 1.95Ϫ2.10 (m, 4 H, CH2), 2.38 (s, 3 H, CH3), 2.41 (s,
2 H, CH2N), 4.59 (t, J ϭ 8.0 Hz, 2 H, CH2N), 6.73 (d, J ϭ 9.0 Hz, 3 H, CH3), 2.72 [q, J ϭ 7.2 Hz, 6 H, CH2(NEt3)], 3.26 (t, J ϭ
4 H, CHaromatic), 7.1Ϫ7.3 (m, 7 H, CHaromatic), 7.31 (s, 1 H, 4.8 Hz, 2 H, CH2), 3.73 (s, 6 H, OCH3), 3.91 (q, J ϭ 7.0 Hz, 2 H,
CHflavin), 7.35 (d, J ϭ 8.0 Hz, 2 H, CHaromatic), 8.00 (s, 1 H,
CH2), 4.10 (t, J ϭ 7.6 Hz, 2 H, OCH2), 4.69 (t, J ϭ 7.2 Hz, 2 H,
NCH2), 6.75 (d, J ϭ 9.0 Hz, 4 H, CHaromatic), 6.80 (d, J ϭ 614 Hz,
CHflavin) ppm. 13C NMR (50 MHz, CDCl3): δ ϭ 19.5, 21.7, 26.2,
26.8, 27.1, 29.9, 30.7, 38.3, 44.8, 55.1 (2 C), 58.5, 63.1, 85.7, 112.9 1 H, PH), 7.1Ϫ7.4 (m, 9 H, CHaromatic), 7.64 (s, 1 H, CHflavin), 7.98
(4 C), 115.1, 123.7, 126.5, 127.6 (2 C), 128.1 (2 C), 129.9 (4 C),
(s, 1 H, CHflavin) ppm. 13C NMR (50 MHz, CDCl3): δ ϭ 9.5 (3 C),
130.9, 132.7, 135.1, 136.5 (2 C), 136.8, 145.2, 148.1, 149.8, 156.3, 19.3, 21.2, 27.3, 29.0, 39.4, 42.3, 45.5 (3 C), 55.0 (2 C), 60.2, 61.6,
158.2 (2 C), 160.6 ppm. MS (ESI): m/z ϭ 741 [Mϩ ϩ K], 439 [Mϩ
86.4, 113.0 (4 C), 115.4, 126.7, 127.8 (4 C), 129.7 (4 C), 130.9,
ϩ K Ϫ DMT], 303 [DMTϩ]. HR-MS ESI calcd. for C42H46N4O6 132.4, 134.8, 135.5, 135.7 (2 C), 136.3, 144.7, 147.5, 148.4, 155.4,
ϩ Naϩ: 725.3315; found 725.3318.
158.4 (2 C), 159.6 ppm. 31P NMR (81 MHz, CDCl3): δ ϭ 5.19
ppm. MS (ESI, negative): m/z ϭ 825 [MϪ], 723 [MϪ Ϫ HNEt3],
421[MϪ Ϫ HNEt3 Ϫ DMT].
10-{6-[Bis(4-methoxyphenyl)phenylmethoxy]hexyl}-3-(6-hydroxy-
hexyl)-7,8-dimethyl-10H-benzo[g]pteridine-2,4-dione
(15):
1-
Iodohexan-6-ol was prepared according to ref.[38] The flavin 10
H-Phosphonate 17: Dichloromethane (7.2 mL), N-methylmorpho-
(250 mg, 0.39 mmol) was dissolved in dry DMF (8.0 mL) and
stirred with molecular sieves (4 A) for 2 h. Cs2CO3 (209 mg, added to a solution (2 ) of PCl3 in dichloromethane (1.26 mL).
line (2.78 mL, 29.8 mmol) and triazole (588 mg, 8.6 mmol) were
˚
0.64 mmol) was added to the solution and the mixture was stirred
for 15 min at room temp. 1-Iodohexan-6-ol (12; 0.25 mL, 0 °C and a solution of the flavin 14 (190 mg, 0.27 mmol) dissolved
1.96 mmol) was added dropwise and the mixture was stirred for in dichloromethane (3.6 mL) was added dropwise. After stirring for
16 h at room temp. The reaction mixture was then filtered, diluted 30 min the mixture was allowed to warm to room temp. and TEAB
After stirring for 30 min at room temp. the solution was cooled to
with chloroform (200 mL) and the organic phase was washed once
with saturated NaHCO3 solution and twice with water. After dry-
ing over Na2SO4 the solvent was evaporated and the crude product
was purified by flash chromatography with CHCl3/MeOH/NEt3
(50:1:0.2) as eluent. Compound 14 (0.194 g, 0.26 mmol, 67%) was
buffer (22 mL, 1 mol/L) was added. Stirring was continued for 30
min, then the organic phase was diluted with dichloromethane (100
mL) and extracted with TEAB buffer (100 mL, 1 mol/L). The or-
ganic phase was dried with Na2SO4 and the solvent was evapor-
ated. The crude product 17 was purified by flash chromatography
isolated as a yellow oil. Rf (CHCl3/MeOH, 15:1): 0.58. IR (NaCl): with CHCl3/MeOH/NEt3 (10:1:0.2) as eluent. To remove surplus
ν˜ ϭ 3471 m cmϪ1, 3057 s, 3034 s, 2999 m, 2935 s, 2861 s, 1704 m,
salts, the product was again dissolved in dichloromethane and the
1660 s, 1607 m, 1585 s, 1545 s, 1510 s, 1462 m, 1250 s, 1178 m, salts were extracted with TEAB buffer. The H-phosphonate 17
1069 m, 1033 s, 911 m, 829 m, 732 s. 1H NMR (200 MHz, CDCl3): (108 mg 0.08 mmol, 46%) was obtained as a yellow oil. Rf (CHCl3/
δ ϭ1.30Ϫ1.90 (m, 16 H, CH2), 2.37 (s, 3 H, CH3), 2.45 (s, 3 H, MeOH/NEt3, 10:1:0.2): 0.34. IR (NaCl): ν˜ ϭ 3054 w cmϪ1, 3033
CH3), 2.99 (t, J ϭ 6.2 Hz, 2 H, CH2), 3.57 (t, J ϭ 6.4 Hz, 2 H, w, 2935 m, 2864 m, 2838 w, 2238 m, 1741 w, 1704 s, 1660 s, 1651 s,
CH2), 3.72 (s, 6 H, OCH3), 4.04 (t, J ϭ 6.4 Hz, 2 H, CH2OH), 4.54
1607 m, 1584 s, 1552 s, 1510 m, 1455 m, 1299 m, 1250 s, 1211 s,
Eur. J. Org. Chem. 2002, 3281Ϫ3289
3287

C. Behrens, M. Ober, T. Carell
FULL PAPER
1050 s, 921 m, 831 m, 727 s. 1H NMR (200 MHz, CDCl3): δ ϭ 1.26 respectively. The cartridge was washed with MeCN/pyridine (1:1)
[t, J ϭ 7.0 Hz, 9 H, CH3(NEt3)], 1.35Ϫ1.50 (m, 4 H, CH2), 1.55 prior to the coupling. The coupling time was 10Ϫ15 min. Capping,
(m, J ϭ 7.0 Hz, 2 H, CH2), 1.7Ϫ1.85 (m, 2 H, CH2), 1.99 (m, J ϭ oxidation and detritylation were the same as for standard phos-
7.6 Hz, 2 H, CH2), 2.37 (s, 3 H, CH3), 2.46 (s, 3 H, CH3), 2.97 [q, phoramidite chemistry.
J ϭ 7.2 Hz, 8 H, CH2(NEt3) ϩ CH2], 3.72 (s, 6 H, OCH3), 3.93
DNA Purification: For the deprotection and purification of the oli-
(q, J ϭ 6.8 Hz, 2 H, OCH2), 4.15 (t, J ϭ 6.6 Hz, 2 H, NCH2), 4.58
gonucleotides, the solid support was suspended in concentrated
(br. t, 2 H, NCH2), 6.74 (d, J ϭ 8.8 Hz, 4 H, CHaromatic), 6.80 (d,
25% NH3 in H2O/EtOH (3:1; 1 mL) and shaken for 16 h at 20 °C
in an Eppendorf thermo shaker to effect deprotection and cleavage.
The solvent was evaporated and the residue suspended in H2O and
purified by HPLC (C18-RP, 0Ϫ40% B in 35 min, A ϭ 0.1  Triethy-
lammonium acetate in H2O, B ϭ 0.1  Triethylammonium acetate
in H2O/80% AcCN). DNA-containing fractions were evaporated
to dryness and redissolved in H2O. Laser desorption mass spectra
(MALDI) (positive ions detected): calcd. for 1: 2908.9; found
2912.0; calcd. for 2 2866.9; found 2870.7; calcd. for 3: 2824.8; found
2826.6; calcd. for 19: 4657.2; found 4654.6.
J ϭ 608 Hz, 1 H, PH), 7.1Ϫ7.4 (m, 9 H, CHaromatic), 7.98 (s, 1 H,
CHflavin), 8.08 (s, 1 H, CHflavin) ppm. 13C NMR (50 MHz, CDCl3):
δ ϭ 8.9 (3 C), 19.3, 21.5, 26.0, 26.7, 27.0, 29.1, 29.9, 39.5, 44.6,
45.8 (3 C), 55.1 (2 C), 61.8, 63.1, 85.6, 112.9 (4 C), 115.0, 126.5,
127.5 (2 C), 128.1 (2 C), 129.9 (4 C), 130.9, 132.7, 134.9, 136.4,
136.5 (2 C), 145.2, 146.7, 147.6, 148.4, 155.5, 158.2 (2 C), 159.8
ppm. 31P NMR (81 MHz, CDCl3): δ ϭ 4.7 ppm. MS (ESI,
negative): m/z ϭ 765 [MϪ Ϫ HNEt3], 565 [MϪ Ϫ DMT], 463 [MϪ
Ϫ HNEt3 Ϫ DMT].
H-Phosphonate 18: Dichloromethane (6.6 mL), N-methylmorpho-
line (0.95 mL, 10.2 mmol) and triazole (201 mg, 2.9 mmol) were
added to a solution (2 ) of PCl3 in dichloromethane (0.43 mL).
After stirring for 30 min at room temp. the solution was cooled to
0 °C and a solution of the flavin 15 (65 mg, 0.09 mmol) dissolved
in dichloromethane (2.0 mL) was added dropwise. After stirring for
30 min the mixture was allowed to warm to room temp. and TEAB
buffer (7 mL, 1 mol/L) was added. Stirring was continued for 30
min, then the organic phase was diluted with dichloromethane (100
mL) and the salts were extracted with TEAB buffer (100 mL, 1
mol/L). The organic phase was dried with Na2SO4 and the solvent
was evaporated. The crude product was purified by flash chromato-
graphy with CHCl3/MeOH/NEt3 (10:1:0.2) as eluent. To remove
surplus salts, the product was again dissolved in dichloromethane
and the organic phase was extracted with TEAB buffer. The H-
phosphonate 18 (40 mg, 0.04 mmol, 48%) was obtained as a yellow
oil. Rf (CHCl3/MeOH/NEt3, 10:1:0.2): 0.27. IR (NaCl): ν˜ ϭ 3054
w cmϪ1, 3034 w, 2933 m, 2859 m, 2237 w, 1734 m, 1705 s, 1649 m,
1607 s, 1583 s, 1508 s, 1460 m, 1298 m, 1248 s, 1212 s, 1187 m, 1056
Acknowledgments
This work was supported by the Volkswagen Foundation, the
Fonds der Chemischen Industrie and the Deutsche Forschungsge-
meinschaft.
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s, 1032 s, 988 m, 829 m, 727 s. H NMR (200 MHz, CDCl3): δ ϭ
1.14 [t, J ϭ 7.0 Hz, 9 H, CH3(NEt3)], 1.3Ϫ1.9 (m, 16 H, CH2),
2.36 (s, 3 H, CH3), 2.45 (s, 3 H, CH3), 2.77 [q, J ϭ 7.3 Hz, 6 H,
CH2(NEt3)], 2.99 (t, J ϭ 6.3 Hz, 2 H, OCH2), 3.71 (s, 6 H, OCH3),
3.76 (q, J ϭ 7.3 Hz, 2 H, OCH2), 4.01 (t, J ϭ 6.6 Hz, 2 H, NCH2),
4.65 (br. t, 2 H, NCH2), 6.74 (d, J ϭ 8.6 Hz, 4 H, CHaromatic), 6.76
(d, J ϭ 610 Hz, 1 H, PH), 7.1Ϫ7.4 (m, 10 H, CHaromatic), 7.97 (s,
1 H, CHflavin) ppm. 13C NMR (50 MHz, CDCl3): δ ϭ 9.7 (3 C),
19.4, 21.6, 25.7, 26.1, 26.7, 26.8, 27.1, 27.8, 29.9, 30.7, 41.9, 44.6,
45.6 (3 C), 55.2 (2 C), 63.2, 63.6, 84.5, 112.9 (4 C), 115.0, 126.6,
127.6 (2 C), 128.1 (2 C), 129.9 (4 C), 130.9, 132.7, 135.0, 136.3,
136.6 (2 C), 145.3, 147.0, 147.4, 148.4, 155.7, 158.3 (2 C), 159.9
ppm. 31P NMR (81 MHz, CDCl3): δ ϭ 5.1 ppm. MS (ESI): m/z ϭ
807 [MϪ Ϫ HNEt3], 505 [MϪ Ϫ HNEt3 Ϫ DMT].
R. L. Letsinger, R. Sanishvili, A. Joachimiak, V. Tereshko, M.
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Oligonucleotide Synthesis: Oligonucleotide synthesis was performed
on an Expedite 8900 DNA Synthesizer, connected to an IBM com-
patible PC. Synthesis of the oligonucleotides was performed by us-
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tions were made up according to the manufacturer’s protocol. The
phosphoramidite (0.1  in MeCN) and 1H-tetrazole (0.5  in
MeCN) solutions were equal in concentration to those used for the
synthesis of natural oligodeoxynucleotides. Average coupling yields
monitored by on-line trityl assay were generally in the range of
95Ϫ99%. All syntheses were run in the trityl-off mode.
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Int. Ed. 1999, 38, 2752Ϫ2755.
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The concentrations used for H-phosphonate chemistry were 0.13 
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3288
Eur. J. Org. Chem. 2002, 3281Ϫ3289

Excess Electron Transfer in Flavin-Capped DNA-Hairpins
FULL PAPER
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