4,4-difluoro-4-bora-3a,4a-diaza-s-indacene as a bright fluorescent label for DNA

Article abstract of DOI:10.1021/jo102519k

4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) as a fluorescent label can be incorporated into DNA by two conceptually different ways: the non-nucleosidic DNA base surrogate Bo exhibits high brightness but no preferential base-pairing properties, whereas the BODIPY-modified uridine BodU has reduced quantum yields but shows preferred Watson-Crick base pairing with adenine.

Full text of DOI:10.1021/jo102519k

NOTE
pubs.acs.org/joc
4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene as a Bright Fluorescent
Label for DNA
Thomas Ehrenschwender and Hans-Achim Wagenknecht*,†
Institute for Organic Chemistry, University of Regensburg, Universit€atsstrasse 31, 93053 Regensburg, Germany
S Supporting Information
b
ABSTRACT: 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)
as a fluorescent label can be incorporated into DNA by two
conceptually different ways: the non-nucleosidic DNA base
surrogate Bo exhibits high brightness but no preferential base-
pairing properties, whereas the BODIPY-modified uridine BodU
has reduced quantum yields but shows preferred Watson-Crick
base pairing with adenine.
4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) represents
an excellent fluorescent label in chemical biology.1 The BOD-
IPYs combine several advantages, mainly nanosecond lifetimes,
fluorescence with sharp and narrow peaks, high quantum
yields,2,3 reasonable stability under physiological conditions,4
and the possibility for synthetic tuning.5-9 BODIPYs are mainly
used for protein labeling and attached using a linker with a
reactive anchor group at the end.10,11 In a similar approach, DNA
can be labeled with BODIPY at the 50-end; however, terminal
labeling limits the versatile applicability.10,12 Two years ago we
presented the synthesis and optical properties of a variety of
BODIPY-modified 20-deoxyuridines.13 Moreover, we could
incorporate one of these labels into oligonucleotides and show
that the DNA polymerase-catalyzed nucleotide incorporation
opposite to BODIPY-modified 20-deoxyuridine (BodU) in tem-
plates follows Watson-Crick selectivity and gets bypassed for
further elongation.14 In a synthetically completely different
approach, chromophores can be incorporated as DNA base
surrogates to promote intercalation of the dye.15,16 Herein, we
compare both synthetic approaches, DNA base modifications
and DNA base substitutions, for the BODIPY dye. We present
the optical properties of BodU-modified DNA and compare it
with BODIPY as DNA base surrogate (Bo). For the latter
modification the synthetic procedure is reported as well.
The synthesis of BodU and its synthetic incorporation into
oligonucleotides using automated phosphoramidite chemistry
followed our published procedures.13,14 For the Bo-type mod-
ification (S)-3-amino-1,2-propanediol was used as an acyclic
linker and substitute for the 20-deoxyriboside between the
phosphodiester bridges. This substitute provides high chemical
stability and conformational flexibility for the chromophore to
intercalate. The synthesis of the corresponding Bo-type phos-
phoramidite 1 started with the assembly of the chromophore
(Scheme 1). The dipyrromethane was formed from the aldehyde
2 and 3-ethyl-2,4-dimethylpyrrole (3) by condensation under
acidic conditions. Subsequently, the intermediate compound was
oxidized to the dipyrromethene with p-chloranil and treated with
BF3 OEt2, yielding the BODIPY derivative 4 (64%). The chro-
3
mophore 4 was tethered to the DMT-protected (S)-3-amino-
1,2-propanediol 5 via a carbamate function, which facilitated the
synthesis of the corresponding DNA building block, because it is
not necessary to protect this linker functionality. Treatment of 4
and 5 with 1,10-carbonyldiimidazole gave 6 in good yield (79%).
The synthesis of the phosphoramidite 1 as the Bo-type DNA
building block and its incorporation into oligonucleotides were
accomplished by standard procedures.15
For both types of DNA modifications, BodU and Bo, two
representative modified oligonucleotides, DNA1/DNA2 and
DNA3/DNA4, were prepared (Chart 1). The corresponding
DNA double strands expose the chromophore to two different
variations in the neighborhood: (i) the dye was placed either in
an A-T environment (DNA1 and DNA3) or next to G-C base
pairs (DNA2 and DNA4) and (ii) within each duplex set the base
opposite to the chromophore site was varied (e.g., DNA1Y with
Y = C, T, A, G).
To study the influence of the BodU and Bo modifications on
the DNA double helix, we determined the thermal stability of all
modified duplexes by measuring the melting temperatures (Tm)
at 260 nm and comparing them with published values of the
corresponding unmodified duplexes (Table 1).16 For the Bo-
type modification, three major results can be drawn from these
data: (i) the melting temperatures of all modified duplexes lie in
the fairly narrow ranges of 55-56 °C (DNA1Y) and 62-64 °C
(DNA2Y), (ii) all modified duplexes show a strong destabiliza-
tion by 4.7-6.8 °C compared to the unmodified duplexes,16
and (iii) Bo as a DNA base substitution does not exhibit any
preferred pairing with the “counterbase”. On the other hand,
the BodU modification induces also strong destabilization,
between 3.5 and 12.5 °C; however, the destabilizing effect is
Received: December 20, 2010
Published: March 07, 2011
r
2011 American Chemical Society
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The Journal of Organic Chemistry
NOTE
Scheme 1. Synthesis of the Bo-phosphoramidite 1
our results from primer extension experiments previously
published.14
The quantum yields of the fluorescence of both types of
BODIPY modifications in single-stranded oligonucleotides are
remarkably high and lie within the ranges of 39-46% (BodU)
and 80-90% (Bo). Such high quantum yields are typical for
this chromophore and make it, as mentioned in the introduc-
tory part, an excellent fluorescent label. The shapes of the
fluorescence spectra are also very similar. The situation changes
dramatically if the BodU-modified single strands DNA1 and
DNA2 are hybridized with the corresponding counterstrands
(Figure 1). As a result, the fluorescence is quenched signifi-
cantly and quantum yields between <0.01 and 0.06 are obtained
in the double strands DNA1Y and DNA2Y. The lowest
quantum yields are obtained with the duplexes that exhibit
the highest Tm values, which could give a hint as to what causes
the dramatic fluorescence quenching. In the case of a Watson-
Crick-type base pairing of BodU with adenine the BODIPY-
phenyl moiety is possibly twisted much more significantly due
to steric interactions with the DNA groove. The charge transfer
contribution in the system is thereby enhanced and the emis-
sion is reduced (similar to a TICT state).17 In DNA duplexes
with the wrong counterbases (C, T, and G) some conforma-
tional flexibility allows regaining some fluorescence, and the
quantum yields are slightly higher. The UV/vis absorption
spectra support this interpretation. The spectra of the single
strands DNA1 and DNA2 show the typical BODIPY sharp peak
at 527 nm, whereas the absorption of the corresponding duplexes
exhibits significantly flattened peaks, indicating a changed struc-
tural scenario (see Supporting Information).
In contrast to the BodU-modified double strands, the fluor-
escence intensities of the Bo-modified duplexes, DNA3Y and
DNA4Y, are maintained much more efficiently (Figure 1). The
quantum yields lie in the applicable range of 0.46-0.69. Since the
Bo chromophore does not exhibit any preferred base in the
complementary strand, the quantum yields are quite similar within
each duplex set. A small amount of quenching is observed both in
the single strand DNA4 and in the duplexes DNA4Y, which is
probably due to stacking interactions with the adjacent guanines.
For biological applications, however, the brightness of a dye is much
more meaningful than the quantum yield. Therefore, we calculated
the brightness of the Bo label in DNA and compared it to the well-
known fluorescein label, representatively for DNA3. TheBo-labeled
oligonucleotides exhibit a brightness of about 55,000 M-1 cm-1 in
the single strand and 44,000 M-1 cm-1 in the double strand
DNA3A, whereas the corresponding fluorescein-labeled DNA
shows only a brightness of approximately 20,000 M-1 cm-1 in
the single strand and 18,000 M-1 cm-1 in the double strand. This is
a remarkable result, since it shows that the BODIPY dye is a much
brighter fluorescent label in DNA than the routinely applied
fluorescein, at least in the given sequence.
Chart 1. Structure of BodU and Bo Modifications in Oligo-
nucleotides and Sequences of DNA1Y-DNA4Y (Y = C, T, A,
G)
In conclusion, we have shown that BODIPY as a fluorescent
label can be incorporated into DNA by two conceptually
different ways: the BODIPY-modified uridine BodU exhibits
preferred Watson-Crick base pairing with adenine in the
counterstrand but shows dramatically reduced quantum
yields, whereas the non-nucleosidic DNA base surrogate Bo
exhibits high quantum yields but no preferential base-pairing
properties. Most importantly, the Bo label in DNA3A has
significantly higher brightness compared to that of standard
fluorescein, which makes the BODIPY label a useful tool for
chemical bioanalytics. Further investigations may focus on
the lowest with A as the complementary base (-5.0 °C in
DNA1A and -3.5 °C in DNA2A). This result indicates that
the 20-deoxyuridine of BodU maintains its preferred binding
to adenine despite the BODIPY modification. This supports
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NOTE
Table 1. Melting Temperatures (Tm), Quantum Yields (Φ),
and Brightness (B) of DNA1Y-DNA4Y
added to the deep red solution. After 30 min BF3 OEt2 (31.5 mL, 251
3
mmol) was added and the mixture was stirred for another 6 h. The
reaction mixture was extracted three times with H2O (50 mL). The
organic phase was dried over Na2SO4, and the solvent was removed.
After purification by chromatography on silica gel (CH2Cl2/acetone
20/1) 4 was obtained as a red solid in 64% yield. 1H NMR (CD2Cl2, 600
MHz): δ 7.18 (d, 2H, J = 8.7 Hz), 7.03 (d, 2H, J = 8.7 Hz), 4.17 (t, 2H,
J = 6.1 Hz), 3.85 (t, 2H, J = 5.9 Hz), 2.49 (s, 6H), 2.33 (q, 4H, J = 7.6 Hz),
2.06 (tt, 2H, J1 = 6.1 Hz, J2 = 5.9 Hz), 1.36 (s, 6H), 0.99 (t, 6H, J = 7.6
Hz). 13C NMR (CD2Cl2, 150 MHz): δ 159.9, 153.8, 141.0, 139.1, 133.2,
131.5, 129.9, 128.1, 115.4, 66.0, 60.3, 32.5, 17.3, 14.8, 12.6, 12.0. 19F
NMR (CD2Cl2, 300 MHz): δ -145.4 (d), -145.7 (d). HRMS (EI-MS):
calcd for C26H33BF2N2O2 [Mþ] 454.2603, found 454.2610.
Synthesis of 6. To a solution of 4 (100 mg, 0.22 mmol) in absolute
DMF (5 mL) was added 1,10-carbonyldiimidazole (54.0 mg, 0.33
mmol), and the mixture was stirred for at 35 °C for 6 h under argon.
DMT-protected (S)-3-amino-1,2-propanediol (5; 175 mg, 0.44 mmol)
was added, and the solution was stirred at 35 °C overnight. The solvent
was removed, and the product was purified by chromatography on silica
gel (CH2Cl2/acetone 15/1), yielding 79% of 6 as a red foam. 1H NMR
(CD2Cl2, 400 MHz): δ 7.43 (m, 2H), 7.35-7.20 (m, 7H), 7.18 (d, 2H,
J = 8.5 Hz)), 7.01 (d, 2H, J = 8.5 Hz), 6.85 (m, 4H), 5.03 (m, 1H), 4.24
(t, 2H, J = 6.3 Hz), 4.09 (t, 2H, J = 6.3 Hz), 3.85 (m, 1H), 3.78 (s, 6H),
3.37 (m, 1H), 3.20-3.10 (m, 3H), 2.49 (s, 6H), 2.32 (q, 4H, J = 7.7 Hz),
2.12 (tt, 2H, J1 = 6.3 Hz, J2 = 6.3 Hz), 1.36 (s, 6H), 0.99 (t, 6H, J = 7.7
Hz). 13C NMR (CD2Cl2, 150 MHz): δ 159.8, 159.1, 157.5, 153.8, 145.3,
141.0, 139.1, 136.2, 133.2, 131.5, 130.4, 129.9, 128.4, 128.3, 128.2, 128.1,
127.2, 115.4, 113.5, 86.6, 70.7, 65.4, 65.1, 62.1, 56.6, 44.6, 29.4, 17.4,
14.8, 12.6, 12.0. 19F NMR (CD2Cl2, 300 MHz): δ -145.4 (d), -145.7
(d). HRMS (EI-MS): calcd for C51H58BF2N3O7 [Mþ] 873.4336, found
873.4327.
Tm (°C)a
ΔTm (°C)a,b
Φc
Bd
BodU
-12.5
-12.5
-5.0
DNA1C
DNA1T
DNA1A
DNA1G
DNA2C
DNA2T
DNA2A
DNA2G
50.0
50.0
57.5
50.5
60.5
59.3
64.5
60.5
0.05
0.03
<0.01
0.01
0.06
0.04
0.02
0.03
1280
720
120
-12.0
-7.5
250
2350
1650
850
-8.7
-3.5
-7.5
1330
Bo
DNA3C
DNA3T
DNA3A
DNA3G
DNA4C
DNA4T
DNA4A
DNA4G
55.7
55.8
55.8
55.7
63.3
62.3
62.1
62.0
-6.8
-6.7
-6.7
-6.8
-4.7
-5.7
-5.9
-6.0
0.69
0.65
0.69
0.68
0.47
0.49
0.47
0.46
36400
35600
38200
38400
22100
22800
22000
23400
a Conditions: 2.5 μM DNA, λ 260 nm, 20-90 °C, interval 0.7 °C/min,
2.5 μM duplex in 10 mM Na-Pi buffer, 250 mM NaCl, pH 7.0. b In
comparison to the unmodified references: Tm = 62.5 °C for DNA1A/
DNA3A and Tm = 68.0 °C for DNA2A/DNA4A, each with T instead of
BodU or Bo, respectively.16 c For DNA3 and DNA3A by photon
counting; for all others indirectly by using DNA3A as a standard (λex
510 nm). All quantum yields were also supported by measurements
Synthesis of 1. 6 (0.19 g, 0.21 mmol) was dissolved in absolute
CH2Cl2 (3 mL) under argon. EtN(iPr)2 (73 μL, 0.42 mmol) and
2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (85 μL, 0.38 mmol)
were added, and the solution was stirred at room temperature for 2 h. The
organic phase was washed with saturated aqueous NaHCO3 and driedover
Na2SO4. The solvent was removed, and the product was dried under
vacuum, yielding 1 as a red solid. The product was used immediately for
oligonucleotide synthesis. 31P NMR (CDCl3, 121 MHz): δ 150.1, 149.8.
Synthesis of DNA1-DNA4. Oligonucleotides were prepared on
a synthesizer using standard phosphoramidite chemistry. For the Bo and
BodU phosphoramidites the coupling time was enhanced from 96 to
500 s. Commercially available reagents and CPG (1 μmol) were used.
After preparation the trityl-off oligonucleotide was cleaved off the resin
and deprotected by treatment with concentrated NH4OH at 45 °C for
12 h. The oligonucleotide was dried and purified by HPLC on an
RP-C18 column using the following conditions: A = NH4OAc buffer
(50 mM; pH 6.5); B = MeCN. The oligonucleotides were lyophilized
and quantified by their absorbance at 260 nm on a photometer. Duplexes
were formed by heating to 90 °C (15 min) followed by slow cooling.
UV/vis (ε260 (M-1 cm-1)/yield (%)): 158 900/32.4 (DNA1), 164 100/
41.8 (DNA2), 151 200/12.4 (DNA3), 156 00/12.5 (DNA4). ESI-MS
(m/z): Mþ calcd for DNA1 5513, found 1837.9 [M/3]3-, 1378.2 [M/
using fluorescein as a standard. d B = Φ ε.
3
Figure 1. Fluorescence of BodU-modified DNA1Y/DNA2Y (left) and
Bo-modified DNA3Y/DNA4Y (right). Conditions: 2.5 μM in Na-Pi
buffer at pH 7, 250 mM NaCl, 20 °C, excitation at 510 nm. All other
optical spectra are shown in the Supporting Information.
non-natural counterbases to the Bo unit, such as pyrene and
perylene.
4]4-; Mþ calcd for DNA2 5563, found1854.7 [M/3]3-, 1390.8 [M/4]4þ
;
M
þ calcd for DNA3 5478, found 1825.7 [M/3]3-, 1368.9 [M/4]4þ; Mþ
calcd for DNA4 5528, found 1842.2 [M/3]3-, 1381.4 [M/4]4þ
.
’ EXPERIMENTAL SECTION
Synthesis of 4. 4-(3-Hydroxypropoxy)benzaldehyde (2; 2.40 g,
13.2 mmol) was dissolved in absolute CH2Cl2 (120 mL) under argon.
3-Ethyl-2,4-dimethylpyrrole (3; 4.06 g, 33 mmol) was added, and the
mixture was stirred at room temperature for 15 min. Trifluoroacetic acid
(0.2 mL) was added, and the solution was stirred at room temperature
overnight. p-Chloranil (3.25 g, 13.2 mmol) was added, and the mixture
was stirred for another 6 h. Triethylamine (31.3 mL, 224 mmol) was
’ ASSOCIATED CONTENT
S
Supporting Information. Text and figures giving experi-
b
mental procedures, data and images of 1H/13C/19F NMR spectra
for 4 and 6, the 31P NMR spectrum for 1, MS analysis for 4 and 6,
HPLC analysis of DNA1-DNA4, and absorption/emission
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The Journal of Organic Chemistry
NOTE
spectra of DNA1-DNA4 and DNA1Y-DNA4Y. This material
2004, 69, 744–751. (c) Wanninger-Weiss, C.; Wagenknecht, H.-A. Eur.
J. Org. Chem. 2007, 64–71. (d) Wagner, C.; Wagenknecht, H.-A. Org.
Biomol. Chem. 2008, 6, 48–50. (e) Holzhauser, C.; Berndl, S.; Menacher,
F.; Breunig, M.; G€opferich, A.; Wagenknecht, H.-A. Eur. J. Org. Chem.
2010, 1239–1248.
is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
(16) Berndl, S.; Herzig, N.; Kele, P.; Lachmann, D.; Li, X.; Wolfbeis,
O.; Wagenknecht, H.-A. Bioconjugate Chem. 2009, 20, 558–564.
(17) Grabowski, Z. R.; Rotkiewicz, K.; Rettig, W. Chem. Rev. 2003,
103, 3899–4031.
Corresponding Author
*E-mail: Wagenknecht@kit.edu.
Present Addresses
†Karlsruhe Institute of Technology, Institute for Organic Chem-
istry, Fritz-Haber-Weg 2, 76131 Karlsruhe, Germany.
’ ACKNOWLEDGMENT
Financial support by the Deutsche Forschungsgemeinschaft
and the University of Regensburg is gratefully acknowledged. T.
E. is grateful for a elite fellowship of the State of Bavaria. We
thank Hartmut Yersin and his group for photon-counting
measurements.
’ REFERENCES
(1) (a) Karolin, J.; Johansson, L. B.-A.; Strandberg, L.; Ny, T. J. Am.
Chem. Soc. 1994, 116, 7801–7806. (b) Yee, M.-C.; Fas, S. C.; Stohlmeyer,
M. M.; Wandless, T. J.; Cimprich, K. A. J. Biol. Chem. 2005, 280,
29053–29059. (c) Johnson, I. D.; Kang, H. C.; Haugland, R. P. Anal.
Biochem. 1991, 198, 228–237. (d) Ferguson, C. G.; Bigman, C. S.;
Richardson, R. D.; van Meeteren, L. A.; Moolenaar, W. H.; Prestwich,
G. D. Org. Lett. 2006, 8, 2023–2026. (e) Peters, C.; Billich, A.; Ghobrial,
M.; H€ogenauer, K.; Ullrich, T.; Nussbaumer, P. J. Org. Chem. 2007,
72, 1842–1845.
(2) See review: Loudet, A.; Burgess, K. Chem. Rev. 2007, 107, 4891–
4932.
(3) See review: Ulrich, G.; Ziessel, R.; Harriman, A. Angew. Chem.,
Int. Ed. 2008, 47, 1184–1201.
(4) Zheng, Q.; Xu, G.; Prasad, P. N. Chem. Eur. J. 2008, 14, 5812–
5819.
(5) (a) Rohand, T.; Qin, W.; Boens, N.; Dehaen, W. Eur. J. Org.
Chem. 2006, 4658–4663. (b) Br€oring, M.; Kr€uger, R.; Link, S.; Kleeberg,
C.; K€ohler, S.; Xie, X.; Ventura, B.; Flamigni, L. Chem. Eur. J. 2008,
14, 2976–2983. (c) Baruah, M.; Qin, W.; Vallee, R.; Beljonne, D.;
Rohand, T.; Dehaen, W.; Boens, N. Org. Lett. 2005, 4377–4380.
(6) (a) Kollmannsberger, M.; Gareis, T.; Heinl, S.; Daub, J. Angew.
Chem., Int. Ed. 1997, 36, 1333–1335. (b) Trieflinger, C.; Rurack, K.;
Daub, J. Angew. Chem., Int. Ed. 2005, 44, 2288–2291.
(7) Wan, C.-W.; Burghart, A.; Chen, J.; Bergstr€om, F.; Johansson,
L. B.-Å.; Wolford, M. F.; Kim, T. G.; Topp, M. R.; Hochstrasser, R. M.;
Burgess, K. Chem. Eur. J. 2003, 9, 4430–4441.
(8) Gabe, Y.; Ueno, T.; Urano, Y.; Kojima, H.; Nagano, T. Anal.
Bioanal. Chem. 2006, 386, 621–626.
(9) Goeb, S.; Ziessel, R. Org. Lett. 2007, 9, 737–740.
(10) Meng, Q.; Kim, D. H.; Bai, X.; Bi, L.; Turro, N. J.; Ju, J. J. Org.
Chem. 2006, 71, 3248–3252.
(11) (a) Worthington, A. S.; Burkart, M. D. Org. Biomol. Chem. 2006,
4, 44–46. (b) Sculimbrene, B. R.; Imperiali, B. J. Am. Chem. Soc. 2006,
128, 7346–7352.
(12) (a) Cornelius, M.; Woerth, C. G. T. C.; Kliem, H.-C.; Wiessler,
M.; Schmeiser, H. H. Electrophoresis 2005, 26, 2591–2598. (b) Shinya,
K.; Takahiro, K.; Kazutaka, Y.; Masaki, T.; Toyokazu, T.; Yoichi, K.;
Ryuichiro, K. Nucleic Acids Res. 2001, 29, e34. (c) Bi, L.; Kim, D. H.; Ju, J.
J. Am. Chem. Soc. 2006, 128, 2542–2543.
(13) Ehrenschwender, T.; Wagenknecht, H.-A. Synthesis 2008, 3657–
3662.
(14) Wanninger-Weiss, C.; Di Pasquale, F.; Ehrenschwender, T.;
Marx, A.; Wagenknecht, H.-A. Chem. Commun. 2008, 1443–1445.
(15) (a) Wagner, C.; Wagenknecht, H.-A. Org. Lett. 2006, 8, 4191–
4194. (b) Huber, R.; Amann, N.; Wagenknecht, H.-A. J. Org. Chem.
2304
dx.doi.org/10.1021/jo102519k |J. Org. Chem. 2011, 76, 2301–2304