Toward catalytically active oligonucleotides: Synthesis of a flavin nucleotide and its incorporation into DNA

Article abstract of DOI:10.1021/ol005739s

(Matrix presented) The synthesis of a vitamin B₂-derived flavin-nucleotide is described. A combined H-phosphonate/phosphoramidite protocol was developed for the first incorporation of flavin coenzymes into a DNA stack. The coenzyme-DNA is predi

Full text of DOI:10.1021/ol005739s

ORGANIC
LETTERS
2000
Vol. 2, No. 10
1415-1418
Toward Catalytically Active
Oligonucleotides: Synthesis of a Flavin
Nucleotide and Its Incorporation into
DNA
Anja Schwo1gler and Thomas Carell*
Department of Chemistry, Philipps-UniVersity Marburg, D-35032 Marburg, Germany
carell@mailer.uni-marburg.de
Received February 29, 2000
ABSTRACT
The synthesis of a vitamin B2-derived flavin-nucleotide is described. A combined H-phosphonate/phosphoramidite protocol was developed for
the first incorporation of flavin coenzymes into a DNA stack. The coenzyme−DNA is predicted to have novel biosensing and catalytic properties.
Coenzymes are small organic compounds which catalyze
chemical reactions within the active site of coenzyme-
dependent enzymes. The protein environment strictly controls
the catalytic properties of the coenzyme by specific hydrogen
bonding, π-stacking, and electrostatic forces.1 These non-
covalent interactions fine-tune the chemical reactivity of the
coenzyme and enable the catalysis of specific biochemical
reactions. Redox coenzymes such as FAD,2 for example,
require adjustment of the redox potential, which varies
between different flavoproteins by several hundreds of
millivolts. In this sense, the protein environment acts as a
molecular device able to change the catalytic properties of
the embedded coenzyme.
tion of coenzymes into DNA or RNA deserves particular
attention.4 In such an oligonucleotide environment, specific
hydrogen bonding and π-stacking contacts to nucleobases
in proximity of the coenzyme could strongly modulate the
catalytic properties.
If the coenzyme is able to function as an informational
nucleobase in an oligonucleotide stack, its properties could
become programmable by the choice of the counter base and
the oligonucleotide sequence.5 The potential to regulate the
redox properties of coenzymes such as FAD within DNA
or RNA could than be exploited for the design of bioana-
lytical devices.6
The FAD redox coenzyme contains as its central unit the
riboflavin moiety 1 depicted in Figure 1. This riboflavin
Because of the recent interest in developing novel bio-
catalysts based on oligonucleotide structures,3 the incorpora-
(4) (a) Berlin, K.; Jain, R. K.; Simon, M. D.; Richert, C. J. Org. Chem.
1998, 63, 1527-1535. (b) Sakthivel, K.; Barbas, C. F., III. Angew. Chem.,
Int. Ed. 1998, 37, 2872-2875.
(5) For a discussion of a coenzym RNA world, see: (a) White, H. B. J.
Mol. EVol. 1976, 7, 101-104. (b) Benner, S. A.; Ellington, A. D.; Tauer,
A. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 7054-7058.
(6) Niemeyer, C. M.; Blohm, D. Angew. Chem., Int. Ed. 1999, 38, 2865-
2869.
(1) (a) Niemz, A.; Rotello, V. M. Acc. Chem. Res. 1999, 32, 44-52. (b)
Rotello, V. M. Curr. Op. Chem. Biol. 1999, 3, 747-751.
(2) (a) Bruice, T. C. Acc. Chem. Res. 1980, 13, 256-262. (b) Walsh, C.
Acc. Chem. Res. 1980, 13, 148-155. (c) Ghisla, S.; Massey, V. Eur. J.
Biochem. 1980, 181, 1-17.
(3) Trawick, B. N.; Daniher, A. T.; Bashkin, J. K. Chem. ReV. 1998, 98,
939-960.
10.1021/ol005739s CCC: $19.00 © 2000 American Chemical Society
Published on Web 04/21/2000

Scheme 1
Figure 1. Hydrogen-bonding capabilities of riboflavin 1 in the
oxidized and reduced state: A, hydrogen bond acceptor; D,
hydrogen bond donor. Definition of the Hoogsteen and the Watson-
Crick side of the flavin heterocycle.
possesses a hydrogen-bonding pattern (ADA) similar to that
of the nucleobases thymidine or uridine at the “Watson-
Crick” face of the flavin heterocycle. Changing the redox
state of the coenzyme, however, influences the hydrogen-
bonding pattern at the “Hoogsteen side”, which switches from
an AA pattern to a DA situation (Figure 1). Targeting this
“Hoogsteen side” of the coenzyme with an opposite base is
one potential possibility to modulate the redox and the
fluorescence properties of flavin coenzymes in an oligo-
nucleotide world.7
To achieve incorporation of a flavin unit into a base stack,
we designed the benzylidene-protected riboflavin building
block 2, in which a 2′,4′-bridging benzylidene rigidifies the
flexible ribityl chain of riboflavin. Computer modeling
suggests that the diastereoisomer 2, containing an all-
equatorial substituted 1,3-dioxane ring, forces of the flavin
nucleus in the DNA double helix with the “Hoogsteen side”
toward the helix axis as the hydrogen-bonding face. The
phenyl ring points in the all-equatorial diastereoisomer away
from the double helix and avoids steric interference with the
base-pairing process. Cleavage of the benzylidene group in
the DNA strand allows for the synthesis of riboflavin
(vitamin B2) containing oligonucleotides. The synthesis of
the monoprotected building blocks 2 and 3 is depicted in
Scheme 1. Reaction of riboflavin 1 with an excess of
benzaldehyde dimethyl acetal furnished bis-benzylidene-
protected riboflavin 4 as a mixture of three diastereoisomers.
Careful treatment of this mixture with a formic acid/acetic
acid/water solution allowed deprotection of the 3′,5′-bridging
benzylidene group and furnished monoprotected riboflavin
compound 3 as a single diastereoisomer. Reaction of
riboflavin 1 with just 1 equiv of benzaldehyde dimethyl acetal
gave a mixture of the 3′,5′- and the 2′,4′-monoprotected
riboflavin derivatives. The desired 2′,4′-benzylidene protected
compound 2 was separated by chromatography and again
obtained as a single diastereoisomer, which, however, was
not identical with 3. NOE experiments were performed in
order to clarify the structures of compounds 2 and 3.
In compound 2, irradiation at the resonance frequency of
the C(6′)H induces a strong NOE of the C(2′)H and of the
C(4′)H as expected for an all-equatorial substituted 1,3-
dioxane ring system. In contrast, diasteroisomer 3 showed a
strong signal intensity enhancement of C(3′)H, which proves
for this diatereoisomer an axial orientation of the phenyl ring
at C(6′). For compound 2 we observe two C(1′)H broad and
unresolved signals, which indicates hindered rotation of the
flavin relative to the six-membered ring. This observation
proves the desired rigidifying effect of the benzylidene group.
Conversion of the desired “all equatorial” 2′,4′-ben-
zylidene-protected riboflavin 2 into the 5′-DMT 3′-H-
phosphonate 5, ready for incorporation into oligonucleotides
by machine-assisted oligonucleotide synthesis, was possible
using standard procedures. Because of the known rapid
oxidation of a flavin phosphoramidite,8 which excludes
incorporation of such derivatives into oligonucleotides using
standard phosphoramidite chemistry, a mixed phosphora-
(7) For the incorporation of related alloxazin nucleobases, see: (a)
Pfleiderer, W.; Ro¨sler, A. HelV. Chim. Acta 1997, 80, 1869-1881. (b) Wang,
Z.; Rizzo, C. Org. Lett. 2000, 2, 227-230. (c) Chanteloup, L.; Thuong, N.
T. Tetrahedron Lett. 1994, 35, 877-880.
(8) For the connection of a flavin to the 5′-end of an oligonucleotide,
see: (a) Frier, C.; De´cout, J.-L.; Fontacave, M. J. Org. Chem. 1997, 62,
3520-3528. (b) Frier, C.; Mouscadet, J.-F.; De´cout, J.-L.; Auclair, C.;
Fontecave, M. Chem. Commun. 1998, 2457-2458.
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Org. Lett., Vol. 2, No. 10, 2000

midite/H-phosphonate/phosphoramidite protocol was devel-
oped with flavin H-phosphonate 5 (Scheme 2).
Scheme 2
To this end, all nucleotides at the 3′- and 5′-end of the
incorporated flavin were coupled as PAC-protected nucle-
otides9 using standard phosphoramidite chemistry. After
DMT deprotection of the nucleotide to the 3′-side of the
flavin, the synthesizer was programmed to pump simulta-
neously flavin H-phosphonate 5 and adamantyl acid chloride
as the activating reagent.
After complete coupling, the H-phosphonate was oxidized
with iodine to give an unprotected phosphodiester linkage.
The phosphoramidite synthesis was continued after cleavage
of the DMT protecting group of the flavin building block in
the DNA strand.
The flavin-embedded DNA strands were subsequently
cleaved from the support and fully deprotected with ammonia
in a water/ethanol mixture at 50 °C for 6 h; purification was
performed by reversed phase HPLC. Figure 2A shows the
HPLC trace of a crude flavin-DNA sample, directly after
cleavage from the solid support. Clearly evident is the high
purity of the synthesized raw material. The inset shows the
HPLC trace of the purified flavin-containing DNA strand.
From the online trityl cation assay, indicating a coupling yield
of the flavin H-phosphonate of 99%, and based upon these
HPLC results, we calculated an incorporation yield of >98%.
Figure 2. (A) HPL chromatogram of crude S1 (260 nm). Inset:
HPL chromatogram of purified S1. (B) UV spectrum of S3. Inset:
MALDI-Tof MS of S3. (C) fluorescence spectra. (a) S1 (MS: calcd
4434, found 4436). (b) S3 (MS: calcd 3611, found 3613). MS for
S2: calcd 2897, found 2898.
Figures 2B and 2C show the UV spectrum of oligonucle-
otide S3 and the fluorescence spectra of S1 and S3 as
examples. All spectra are identical with spectra recorded from
riboflavin, proving the presence of the intact flavin chromo-
(9) PAC amidites are commercially available from Pharmacia.
Org. Lett., Vol. 2, No. 10, 2000
1417

phor in the DNA environment. Additional support for the
presence of the benzylidene-protected flavin building block
was derived from MALDI-Tof mass spectra, which show
as the exclusive signals the correct mass peaks of the
benzylidene-protected flavin oligonucleotides. The inset in
Figure 2B depicts as an example the MALDI-Tof spectrum
of S3. These mass spectrometric data together with the HPLC
results show that the benzylidene group is not cleaved during
the DNA synthesis cycle, which includes treatment of the
resin with dichloroacetic acid to achieve DMT deprotection.
Short treatment, however, of the benzylidene-protected
flavin-containing oligonucleotide with formic acid caused
rapid cleavage of the benzylidene group and furnished the
unprotected riboflavin-containing DNA strands. HPLC moni-
toring of the deprotection reaction proved a clean educt to
product conversion.
The fluorescence spectrum of the flavin inside the oligo-
nucleotide environment was found to be strongly environ-
ment dependent (Figures 2Ca and 2Cb). We observe a strong
flavin fluorescence at 520 nm only if we exclude guanosine
(G) nucleobases in close proximity to the flavin. The
fluorescence of the flavin is almost fully quenched in strands
such as S3, possibly due to an electron transfer from the G
residue to the photoexcited flavin and formation of a G
radical cation (G•+).10
surprisingly stable. We observe no DNA cleavage if the
flavin-containing oligonucleotides are handled in the labora-
tory under normal conditions. This surprising fact now allows
detailed investigation of the pairing properties of a riboflavin
and of the benzylidene-rigidified flavin nucleobase. It also
enables studying of their catalytic and fluorescence properties
in the oligonucleotide environment.12 Initial melting point
studies indicate that the rigidified flavin nucleoside indeed
stabilizes DNA duplexe and participates in the hydrogen-
bonding process.
In conclusion, we describe a straightforward synthesis of
a riboflavin-derived H-phosphonate, containing a benzylidene-
rigidified ribityl backbone. Using a combined phosphora-
midite/H-phosphonate/phosphoramidite coupling protocol,
the building block could be incorporated into oligonucleo-
tides with a synthesis yield of >98%. High-yielding depro-
tection of the benzylidene group is possible in the DNA
strand. Within an oligonucleotide environment, a strong
modulation of the redox and of the fluorescence properties
of the flavin coenzyme is predicted, due to π-stacking and
hydrogen-bonding interactions in the base stack.
Acknowledgment. This work was supported by a grant
from the Volkswagen Stiftung.
OL005739S
Although several reports describe light-induced DNA
strand breaks in the presence of a flavin11 (possibly due to
initial G•+ formation), the flavin-embedded DNA strands are
(11) (a) Peak, J. G.; Peak, M. J.; MacCoss, M. Photochem. Photobiol.
1984, 39, 713-716. (b) Kuratomi, K.; Kobayashi, Y. Biochim. Biophys.
1977, 476, 207-217. (c) Burgstaller, P.; Famulok, M. J. Am. Chem. Soc.
1997, 119, 1137-1138. (d) Kino, I.; Saito, I.; Sugiyama, H. J. Am. Chem.
Soc. 1998, 120, 7373-7374. (e) Kasai, H.; Yamaizumi, Z.; Berger M.;
Cadet, J. J. Am. Chem. Soc. 1992, 114, 9692-9694.
(12) For the incorporation of the flavin cofactor into in vitro selected
oligonucleotides, see: (a) Lauhon, C. T.; Szostak, J. W. J. Am. Chem. Soc.
1995, 117, 1246-1257. (b) Burgstaller, P.; Famulok, M. Angew. Chem.,
Int. Ed. Engl. 1995, 34, 1189-1193.
(10) (a) Ito, K.; Inoue, S.; Yamamoto, K.; Kawanishi, S. J. Biol. Chem.
1993, 268, 13221-13227. (b) Seidel, C. A. M.; Schulz, A.; Sauer, M. H.
M. J. Phys. Chem. 1996, 100, 5541-5553. (c) See also: Meggers, E.;
Michel-Beyerle, M. E.; Giese, B. J. Am. Chem. Soc. 1998, 120, 12950-
12955. (d) Ly, D.; Sanii, L.; Schuster, G. B. J. Am. Chem. Soc. 1999, 121,
9400-9410.
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