Fluorescence quenchers for hydrazone and oxime orthogonal bioconjugation

Article abstract of DOI:10.1021/bc300344b

We describe the synthesis and properties of new fluorescence quenchers containing aldehyde, hydrazine, and aminooxy groups, allowing convenient bioconjugation as oximes or hydrazones. Conjugation to oligonucleotides proceeded in high yield with aniline as catalyst. Kinetics studies of conjugation show that, under optimal conditions, a hydrazine or aminooxy quencher can react with aldehyde-modified DNA to form a stable hydrazone or oxime adduct in as little as five minutes. The resulting quencher-containing DNAs were assessed for their ability to quench the emission of fluorescein in labeled complements and compared to the commercially available dabcyl and Black Hole Quencher 2 (BHQ2), which were conjugated as phosphoramidites. Results show that the new quenchers possess slightly different absorbance properties compared to dabcyl and are as efficient as the commercial quenchers in quenching fluorescein emission. Hydrazone-based quenchers were further successfully incorporated into molecular beacons and shown to give high signal to background ratios in single nucleotide polymorphism detection in vitro. Finally, aminooxy and hydrazine quenchers were applied to quenching of an aldehyde-containing fluorophore associated with living cells, demonstrating cellular quenching within one hour.

Full text of DOI:10.1021/bc300344b

Article
pubs.acs.org/bc
Fluorescence Quenchers for Hydrazone and Oxime Orthogonal
Bioconjugation
Pete Crisalli, Armando R. Hernandez, and Eric T. Kool*
́
Department of Chemistry, Stanford University, Stanford, California 94305-5080, United States
S
* Supporting Information
ABSTRACT: We describe the synthesis and properties of new
fluorescence quenchers containing aldehyde, hydrazine, and
aminooxy groups, allowing convenient bioconjugation as oximes
or hydrazones. Conjugation to oligonucleotides proceeded in high
yield with aniline as catalyst. Kinetics studies of conjugation show
that, under optimal conditions, a hydrazine or aminooxy quencher
can react with aldehyde-modified DNA to form a stable hydrazone
or oxime adduct in as little as five minutes. The resulting quencher-containing DNAs were assessed for their ability to quench the
emission of fluorescein in labeled complements and compared to the commercially available dabcyl and Black Hole Quencher 2
(BHQ2), which were conjugated as phosphoramidites. Results show that the new quenchers possess slightly different absorbance
properties compared to dabcyl and are as efficient as the commercial quenchers in quenching fluorescein emission. Hydrazone-
based quenchers were further successfully incorporated into molecular beacons and shown to give high signal to background
ratios in single nucleotide polymorphism detection in vitro. Finally, aminooxy and hydrazine quenchers were applied to
quenching of an aldehyde-containing fluorophore associated with living cells, demonstrating cellular quenching within one hour.
the uncatalyzed condensations possess slow reaction kinetics,
the development of aniline catalysis to greatly enhance reaction
rate has allowed for a variety of useful applications.24−26
Surprisingly, although these bond formations have been used to
attach fluorophores to many molecules of interest,25,27 we are
aware of no reports of the use of oxime or hydrazone chemistry
to attach a quencher to a fluorescently labeled molecule. Such
an approach may be desirable for a variety of reasons. First, the
mild chemistry allows conjugations in molecules that may have
otherwise sensitive reactive groups. Second, this synthetic
approach can allow for temporal control of when a quencher
will be introduced into a system, yielding quenching after some
fluorescence analysis has taken place. Further, if the reactive
groups are placed in direct conjugation, formation of either
oxime or hydrazone can result in direct electronic changes of
the quencher itself (such as redshifting), which has been
observed with hydrazone formation involving dyes.28,29 Finally,
since hydrazone/oxime chemistry can proceed intracellu-
larly,27,29 this offers the possibility of performing quenching
experiments directly in cells.
INTRODUCTION
■
Fluorescence quenchers have garnered a wide variety of uses in
assays for biomolecular detection and analysis.1−11 Potent
fluorescence quenching is often attained by placing the
quencher molecule in close spatial proximity to the fluorophore
to allow for contact quenching, where the fluorophore
undergoes direct electronic interactions with the quencher
moiety.11−16 The resulting emission can be greatly reduced
compared to the “on” state of the fluorophore separated from
the quencher, which is important for lowering background
signals. Many different fluorescence quenchers have been
designed, including the widely used and commercially available
Dabcyl (4-((4-(dimethylamino)phenyl)azo)benzoic acid),
Black Hole Quenchers (BHQ),17 the QSY series,18 and
BlackBerry Quenchers.19 Other quenchers with broadened
absorbance properties have been designed, based on such
scaffolds as azaphthalocyanine,20,21 azulene,22 cyanine,8 or
napthalimide.23
Despite their broad use, however, these quenchers are limited
by their chemistry of conjugation. Chemical attachment is
nearly always done by one of two irreversible approaches: as a
phosphodiester linkage via automated DNA synthesis, or as an
amide link via reaction of carboxylate and amine. Development
of new bioorthogonal means of attaching quencher molecules
could allow for increased ease and speed of conjugation, enable
conjugation of complex molecules with multiple functional
groups, and provide greater freedom for design of multiplex
assays.
Here, we report the preparation and properties of multiple
new hydrazine, aminooxy, and aldehyde-containing fluores-
cence quenchers (Figure 1) that can easily be conjugated to
aldehyde, aminooxy, or hydrazide modified molecules (such as
DNA) (Figure 2) via the stable formation of oximes or
hydrazones. The quenching efficiencies of these molecules are
comparable to or slightly better than a commercially available
Hydrazone and oxime chemistry has been advanced in recent
years as a bioorthogonal means of attaching a variety of labels
to molecules of interest, even in cellular conditions. Although
Received: June 27, 2012
Revised: August 20, 2012
Published: August 23, 2012
© 2012 American Chemical Society
1969
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Figure 1. Aldehyde-, hydrazine-, and aminooxy-modified quenchers prepared for this study.
dabcyl quencher, but the new compounds offer a convenient
bioorthogonal means of attaching the quencher moiety. We
find that with aniline catalysis a quencher can form a stable
DNA hydrazone adduct in high yield in as little as 5 min.
Finally, we present preliminary experiments showing evidence
of fluorescence quenching within living cells by cell-permeable
quencher groups.
amidite, 3-formylindole phosphoramidite and 3′-Fluorescein
CPG were purchased from Glen Research. 5′-BHQ2
phosphoramidite was purchased from Biosearch Technologies.
1
Instrumentation. H and 13C NMR spectra were recorded
on a Varian 500 MHz NMR spectrometer and internally
referenced to the residual solvent signal; J values are reported in
Hz. Oligonucleotide sequences were synthesized on an ABI 394
DNA synthesizer using standard reagents and phosphorami-
dites. Analytical and semipreparative high-performance liquid
chromatography was performed on a LC-CAD Shimadzu liquid
chromatograph, equipped with a SPD-M10A VD diode array
detector and a SCL 10A VP system controller using reverse-
phase C18 columns (Grace ProSphere C18−300 10 μ for
semipreparative scale HPLC, Alltech Platinum C18−100A 5 μ
EXPERIMENTAL SECTION
■
Chemicals and Reagents. Chemicals were purchased from
Sigma-Aldrich or TCI America and used without further
purification. Solvents were purchased from Acros Organics and
used as received. DNA phosphoramidites and solid supports
were purchased from Glen Research. 5′-Dabcyl phosphor-
1970
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10.09 (s, 1H). 13C NMR (125 MHz, CDCl3): 44.9, 113.0,
113.2, 123.3, 123.8, 124.8, 125.5, 127.2, 128.0, 130.8, 133.6,
136.8, 142.7, 155.6, 156.8, 191.8. ESI MS (Calc M+H =
304.14): 304.33.
4-((4-(Dimethylamino)-2-methoxyphenyl)diazenyl)-
benzaldehyde (3). 3 was purified by column chromatography
over silica gel eluting with dichloromethane to provide a dark
red solid.
1H NMR (500 MHz, CDCl3): 3.13 (s, 6H), 4.04 (s, 3H),
6.19 (d, 1H, J = 2 Hz), 6.35 (dd, 1H, J = 9.5 Hz, 2 Hz), 7.84 (d,
1H, J = 9.5 Hz), 7.91−7.96 (m, 4H), 10.03 (s, 1H). 13C NMR
(125 MHz, CDCl3): 40.4, 56.2, 94.3, 105.1, 118.4, 122.7, 130.8,
134.0, 135.8, 155.0, 157.6, 160.2, 191.9. ESI MS (Calc M+H =
284.14): 284.33.
1-(4-((4-(Dimethylamino)phenyl)diazenyl)phenyl)-
ethanone (4). 4 was recrystallized from ethanol to provide a
red solid.
1H NMR (500 MHz, CDCl3): 2.64 (s, 3H), 3.10 (s, 6H),
6.75 (d, 2H, J = 9 Hz), 7.87−7.91 (m, 4H), 8.06 (d, 2H, J = 8.5
Hz). 13C NMR (125 MHz, CDCl3): 26.9, 40.3, 111.5, 122.2,
125.6, 129.4, 136.9, 143.7, 153.0, 156.0, 197.7. ESI MS (Calc M
+H = 268.14): 268.33.
Figure 2. Aldehyde, hydrazide, and aminooxy phosphoramidites
employed for oxime and hydrazone formation on DNA.
1-(4-((4-(Diethylamino)-2-methoxyphenyl)diazenyl)-
phenyl)ethanone (5). 5 was recrystallized from ethanol to
provide a dark red solid.
for analytical HPLC). DNA concentrations were determined on
a Cary 100 Bio UV−visible spectrophotometer at 90 °C by
measuring absorbance at 260 nm. Fluorescence measurements
were performed on a Fluorolog 3 Jobin Yvon fluorescence
spectrometer. ESI MS was determined by the Vincent Coates
Foundation Mass Spectrometry Laboratory, Stanford Univer-
sity Mass Spectrometry. Oligonucleotide masses were deter-
mined by the Stanford University Protein and Nucleic Acid
Facility using a Perspective Voyager-DE RP Biospectrometry
MALDI-TOF mass-spectrometry instrument using a 3-
hydroxypicolinic acid/diammonium hydrogen citrate matrix.
Synthetic Procedures. Compounds 1 and 10 were
prepared according to published procedures.30,31 Compound
6 is commercially available. Compounds 2−5 were prepared
analogously to 1, substituting the appropriate aniline for N,N-
dimethylaniline in 2, 3, and 5 or using 4-aminoacetophenone in
place of 4-aminobenzaldehyde for 4 and 5. Representative
reaction conditions and purification procedures are provided
below. Hydrazide and aminooxy phosphoramidites were
prepared according to published procedures.32−34
Representative Conditions for Synthesis of 2−5. 0.454 g 4-
aminobenzaldehyde or 0.507 g 4′-aminoacetophenone (3.75
mmol) was dissolved in 12 mL 1 M HCl and stirred at 0 °C.
Separately, 0.272 g sodium nitrite (3.94 mmol, 1.05 equiv) was
dissolved in 2 mL water and added dropwise. The resulting
solution was stirred at 0 °C for 30 min. A solution of the
corresponding aniline (3.94 mmol, 1.05 equiv) dissolved in 9
mL 1 M HCl was added followed by a solution of 2.15 g
sodium acetate in 15 mL of water. The resulting red−orange
suspension was stirred and allowed to warm to room
temperature over a period of 1 h. The suspension was filtered,
washed with water, and purified as described below to provide
the desired aldehyde or ketone dye.
1H NMR (500 MHz, CDCl3): 1.24 (t, 6H, J = 7 Hz), 2.62 (s,
3H), 3.45 (q, 4H, J = 7 Hz), 4.02 (s, 3H), 6.18 (d, 1H, J = 3
Hz), 6.31 (dd, 1H, J = 9.5 Hz, 3 Hz), 7.81−7.85 (m, 3H), 8.03
(d, 2H, J = 6.5 Hz). 13C NMR (125 MHz, CDCl3): 12.8, 26.8,
45.0, 56.2, 93.9, 104.9, 118.6, 122.2, 129.4, 133.5, 136.4, 152.7,
156.7, 160.3, 197.7. ESI MS (Calc M+H = 326.19): 326.33.
tert-Butyl 2-(4-((4-(ethoxycarbonyl)phenyl)diazenyl)-
phenyl)-2-phenylhydrazinecarboxylate (14). 315 mg of tert-
butyl 2-(4-nitrophenyl)-2-phenylhydrazinecarboxylate35 was
dissolved in 10 mL MeOH. A catalytic amount of 10%
palladium on carbon was added and the solution stirred at
room temperature overnight under a hydrogen atmosphere,
after which TLC showed consumption of starting materials.
The resulting solution was filtered through Celite and washed
with methanol and the filtrate concentrated to afford the crude
amino product. The crude amino product was dissolved in 7
mL toluene and treated with 256 mg ethyl 4-nitrosobenzoate36
and 282 μL acetic acid. The resulting solution was stirred at 90
°C overnight under an argon atmosphere. TLC indicated the
formation of a bright orange product. Volatiles were removed in
vacuo and the crude material purified by silica column
chromatography eluting with 5:2 Hexanes/EtOAc to afford
440 mg of a bright orange solid (quantitative). An analytically
pure sample was obtained by recrystallization from methanol.
1H NMR (500 MHz, CDCl3): 1.33 (br s, 3H), 1.42 (t, 3H, J
= 7 Hz), 1.51 (br s, 6H), 4.40 (q, 2H, J = 7 Hz), 7.11−7.19 (m,
3H), 7.31−7.40 (m, 4H), 7.88 (m, 4H), 8.16 (d, 2H, J = 8.5
Hz). 13C NMR (125 MHz, CDCl3): 14.4, 28.1, 28.3, 61.2, 81.8.
115.6, 115.9, 122.3, 122.5, 123.2, 124.9, 125.5, 129.5, 130.6,
131.3, 144.6, 147.0, 149.9, 154.7, 155.5, 166.3. ESI MS (Calc M
+H = 461.22): 461.33.
tert-Butyl 2,2-bis(4-nitrophenyl)hydrazinecarboxylate
(15). 300 mg of tert-butyl 2-(4-nitrophenyl)-
diazenecarboxylate35 was dissolved in 10 mL methanol. To
this solution, 240 mg 4-nitrophenylboronic acid (1.2 equiv) and
18 mg (0.075 equiv) copper acetate monohydrate were added.
The resulting solution was stirred overnight at 45 °C. Volatiles
were removed in vacuo and the resulting solid purified by
4-((4-(Dimethylamino)naphthalen-1-yl)diazenyl)-
benzaldehyde (2). 2 was purified by column chromatography
over silica gel eluting with chloroform to provide a red solid.
1H NMR (500 MHz, CDCl3): 3.05 (s, 6H), 7.07 (d, 1H, J =
8 Hz), 7.57 (dd, 1H, J = 7 Hz, 1.5 Hz), 7.65 (dd, 1H, J = 7 Hz,
1.5 Hz), 7.94 (d, 1H, J = 8.5 Hz), 8.02−8.04 (m, 2H), 8.08−
8.11 (m, 2H), 8.22 (d, 1H, J = 8 Hz), 9.02 (d, 1H, J = 9 Hz),
1971
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column chromatography eluting with 3:1 hexanes/EtOAc →
2:1 hexanes/EtOAc to afford 390 mg (87%) of the desired
product as a bright yellow solid.
NMR (125 MHz, CDCl3): 26.6, 28.1, 37.1, 38.5, 50.5, 76.5,
83.4, 112.4, 116.3, 129.3, 149.2, 158.0, 168.9. ESI MS (Calc M
+H = 338.21): 338.27.
1H NMR (500 MHz, CDCl3): 1.30 (br s, 3H), 1.51 (br s,
6H), 7.31 (d, 4H, J = 9 Hz), 8.22 (d, 4H, J = 9 Hz). 13C NMR
(125 MHz, CDCl3): 28.0, 28.3, 83.3, 119.1, 125.7, 143.4, 149.5.
ESI MS (Calc M+H = 375.13): 373.42.
Boc(aminooxy) BHQ1 (19). 183 mg of 18 was dissolved in
10 mL tetrahydrofuran. 271 mg of Fast Corinth V salt was
added followed by 2 mL MeOH to aid in solubility. The red−
brown suspension was stirred at room temperature overnight
and slowly became a dark red solution. Volatiles were removed
and the crude material was purified by column chromatography
eluting with 1:1 Hexanes/EtOAc → 1:3 Hexanes/EtOAc to
afford 47 mg (13%) of a dark red solid.
tert-Butyl 2-(4-((4-(ethoxycarbonyl)phenyl)diazenyl)-
phenyl)-2-phenylhydrazinecarboxylate (16). 300 mg of 15
was dissolved in 10 mL MeOH. A catalytic amount of 10%
palladium on carbon was added and the solution stirred at
room temperature overnight under a hydrogen atmosphere
(balloon), after which TLC showed consumption of starting
materials. The resulting solution was filtered through Celite and
washed with methanol and the filtrate concentrated to afford
the crude diamino product. The crude diamino product was
dissolved in 7 mL toluene and treated with 515 mg ethyl 4-
nitrosobenzoate36 and 270 μL acetic acid. The resulting
solution was stirred at 90 °C overnight under an argon
atmosphere. TLC indicated the formation of a bright orange
product. Volatiles were removed in vacuo and the crude
material purified by column chromatography eluting with 3:1
hexanes/EtOAc → 2:1 hexanes/EtOAc to afford 297 mg (58%)
of a bright red foam. An analytically pure sample was obtained
by recrystallization from methanol.
1H NMR (500 MHz, CDCl3): 1.47 (s, 9H), 1.92 (m, 2H),
2.51 (s, 3H), 2.70 (s, 3H), 3.10 (s, 3H), 3.39 (m, 2H), 3.54 (t,
3H, J = 7.5 Hz), 4.01 (s, 3H), 4.35 (s, 2H), 6.75 (d, 2H, J = 9
Hz), 7.39 (s, 1H), 7.47 (d, 1H, J = 6.5 Hz), 7.57 (d, 2H, J = 8
Hz), 7.66 (d, 1H, J = 8 Hz), 7.70 (s, 1H), 7.91 (d, 2H, J = 9
Hz), 8.41 (br s, 1H). 13C NMR (125 MHz, CDCl3): 16.9, 21.4,
27.0, 28.2, 36.8, 38.8, 50.3, 56.4, 76.6, 83.6, 99.3, 111.4, 119.0,
119.1, 124.4, 126.0, 133.6, 133.6, 141.6, 149.5, 151.1, 169.0. ESI
MS (Calc M+H = 649.31): 649.31.
Boc(aminooxy) BHQ2 (20). 230 mg of 18 was dissolved in
15 mL tetrahydrofuran. 854 mg of Fast Black K salt was added
followed by 3 mL MeOH to aid in solubility. The red-brown
suspension was stirred at room temperature for 2 h and slowly
became a dark purple solution. Volatiles were removed and the
crude material was purified by column chromatography eluting
with 1:1 hexanes/EtOAc → EtOAc to afford 237 mg (53%) of
a dark purple solid.
1H NMR (500 MHz, CDCl3): 1.33 (br s, 3H), 1.43 (t, 6H, J
= 7.5 Hz), 1.53 (br s, 6H), 4.41 (q, 4H, J = 7.5 Hz), 7.36 (d,
4H, J = 9 Hz), 7.91−7.96 (m, 8H), 8.18 (d, 4H, J = 8.5 Hz).
13C NMR (125 MHz, CDCl3): 14.4, 28.1, 28.4, 61.3, 82.4,
119.5, 122.5, 124.8, 130.6, 131.8, 148.1, 148.5, 155.3, 166.2. ESI
MS (Calc M+H = 637.28): 637.42.
tert-Butyl 2-((2-(4-((4-(dimethylamino)phenyl)diazenyl)-
phenylsulfonamido)ethyl)amino)-2-oxoethoxycarbamate
(17). 113 mg of Boc(aminooxy)acetic acid was dissolved in 6
mL CH2Cl2. To this solution, 125 mg EDCI hydrochloride and
88 mg HOAt were added followed by 230 μL triethylamine.
188 mg of N-(2-aminoethyl)-4-((4-(dimethylamino)phenyl)-
diazenyl) benzenesulfonamide37 was added and the resulting
suspension stirred overnight at room temperature. Solvents
were eliminated and the crude solid was purified by column
chromatography eluting with 3% MeOH in CH2Cl2 to afford
116 mg (38%) of a bright orange solid.
1H NMR (500 MHz, CDCl3): 1.47 (s, 9H), 1.93 (m, 2H),
3.11 (s, 3H), 3.40 (m, 2H), 3.55 (t, 3H, J = 7.5 Hz), 4.05 (s,
3H), 4.09 (s, 3H), 4.34 (s, 2H), 6.76 (dd, 2H, J = 7 Hz, 2 Hz),
7.46 (s, 1H), 7.50 (s, 1H), 7.64 (s, 1H), 7.93 (dd, 2H, J = 7 Hz,
2 Hz), 8.04 (dd, 2H, J = 7 Hz, 2 Hz), 8.37 (dd, 2H, J = 7 Hz, 2
Hz), 8.43 (br s, 1H). 13C NMR (125 MHz, CDCl3): 27.0, 28.2,
36.8, 38.9, 50.2, 56.8, 76.6, 83.5, 99.9, 101.0, 111.5, 123.8,
124.8, 126.3, 142.1, 144.5, 146.9, 148.4, 150.9, 152.0, 153.7,
156.5, 158.2, 169.1. ESI MS (Calc M+H = 651.29): 651.25.
DNA Conjugation. Removal of Protecting Groups. For
aminooxy modified DNA, the phthalimide group was removed
selectively with hydrazine as recommended in the literature.34
For hydrazide modified DNA, the trityl group was removed on
the DNA synthesizer using alternating 10 s cycles of
deprotection reagent (3% trichloroacetic acid in dichloro-
methane) and dichloromethane washes for 3 min. Commer-
cially available 3-formylindole utilizes no protection of the
aldehyde. For compounds 7−9 and 11−13, 20 mg of boc-
protected precursor 14−17 or 19−20 was dissolved in 1 mL of
1:1 dichloromethane/trifluoroacetic acid and shaken at room
temperature for 2 h to provide the free hydrazine or aminooxy
compound for conjugation. Volatiles were removed in vacuo
and the crude product was used for DNA labeling without
further purification.
1H NMR (500 MHz, CDCl3): 1.48 (s, 9H), 3.12 (s, 6H),
3.19 (t, 2H, J = 5 Hz), 3.40 (t, 2H, J = 5 Hz), 4.28 (s, 2H), 5.71
(br s, 1H), 6.75 (d, 2H, J = 9.5 Hz), 7.74 (s, 1H), 7.88−7.90
(m, 4H), 7.95 (d, 2H, J = 7 Hz), 8.25 (br s, 1H). 13C NMR
(125 MHz, CDCl3): 28.2, 38.5, 40.5, 43.6, 76.5, 83.8, 111.5,
122.7, 125.8, 128.0, 139.7, 143.6, 153.1, 155.6, 158.1, 169.8. ESI
MS (Calc M-H = 519.20): 519.19.
tert-Butyl 2-((3-(methyl(phenyl)amino)propyl)amino)-2-
oxoethoxycarbamate (18). 300 mg of Boc(aminooxy)acetic
acid was dissolved in 10 mL CH2Cl2. To this solution, 451 mg
of EDCI hydrochloride, 0.657 mL Et3N, and 0.515 mL N-
methyl-N-phenylpropane-1,3-diamine were added and the
resulting solution stirred overnight at room temperature. The
reaction was diluted with CH2Cl2, washed twice with water and
once with brine. The organic layer was dried over MgSO4,
filtered, and concentrated, then purified by silica column
chromatography eluting with 3:2 EtOAc/hexanes to afford 230
mg (43%) of a yellow oil.
DNA Labeling. A solution of approximately 25 mM of
desired label in 750 μL 9:1 dimethylformamide/water was
added to the DNA CPG. 7.5 μL of aniline was added and the
resulting solution was shaken at room temperature overnight.
The CPG was rinsed 4 × 500 μL dimethylformamide and 4 ×
500 μL acetonitrile. Labeled DNA was cleaved using standard
deprotection conditions (concentrated aqueous ammonia, 55
°C, overnight) and purified by HPLC. Purity of the collected
product was assessed by analytical HPLC.
1H NMR (500 MHz, CDCl3): 1.46 (s, 9H), 1.82−1.88 (m,
2H), 2.94 (s, 3H), 3.34−3.42 (m, 4H), 4.32 (s, 2H), 6.67−6.70
Fluorescence Experiments. Fluorescence of the hybrid-
ization buffer (10 mM MgCl2, 70 mM Tris·Borate, pH 7.55)
(m, 3H), 7.20−7.24 (m, 2H), 7.53 (s, 1H), 8.25 (br s, 1H). 13
C
1972
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alone was first determined and subtracted from all subsequent
fluorescence data. Fluorescein-labeled 20mer was added to the
buffer solution to a final concentration of 30 nM. The
fluorescence of the solution was measured in triplicate
(excitation 494 nm and emission 517 nm, slits both 2 nm)
and averaged, subtracting background buffer emission. A
concentrated solution of quencher labeled DNA was added
to bring the final volume of quencher strand to 150 nM. The
oligonucleotides were allowed to hybridize until the fluo-
rescence emission remained constant, then the fluorescence
spectrum was taken in triplicate and averaged, subtracting
background buffer emission. Quenching efficiency was
calculated at the emission maxima by dividing the quenched
fluorescence by the initial fluorescence, multiplying by 100,
then subtracting from 100. All experiments were performed in
triplicate and averaged.
Molecular beacon experiments were performed with the
same hybridization buffer and a 100 nM concentration of
molecular beacon. 200 nM of either the match, mismatch or
deletion sequence were added and the change in fluorescence
monitored. The beacon sequence was 5′-Quencher-(3-
formylindole)-GCG-AGA-AGT-TAA-GAC-CTA-TGC-TCG-
C-FAM-3′, where the underlined regions represent the stem
region.4 Match sequence (5′-CAT-AGT-TCT-TAA-CTT-3′),
mismatch sequence (5′-CAT-AGT-TGT-TAA-CTT-3′), and
deletion sequence (5′-CAT-AGT-T_T-TAA-CTT-3′) were
purified by PolyPakII cartridges (Glen Research).4
Kinetics Experiments. A concentrated solution of 4-nitro-
phenylhydrazine in dimethylformamide (final concentration
200 μM) was added to a buffered solution (100 mM NH4OAc,
pH 4.5) containing 100 μM aldehyde modified DNA and either
0 mM or 100 mM aniline to a total volume of 200 μL. Twenty-
five microliter samples were taken and immediately injected
into an HPLC at 5 min, 80 min, 150 min, and 210 min. Yield
was determined by dividing the integrated area of the product
by the total integrated area of both the product and the starting
material. The yield at time = 0 was assumed to be zero.
Cellular Experiments. CCL-2 HeLa cells (ATCC, Manassas,
VA) were grown at 37 °C (5% CO2) in Dulbecco’s modified
Eagles’ medium (DMEM, GIBCO)supplemented with 10%
fetal bovine serum (FBS, GIBCO), 100 μg/mL streptomycin
(GIBCO), and 100 U/mL penicillin (GIBCO)until 60−80%
confluency was reached. In preparation for in vivo experiments,
cells were trypsinized, suspended in DMEM + 10% FBS,
transferred to 8-chambered microscope slides (Lab-Tek II, 500
μL cell suspension containing ca. 10 000 cells per chamber),
and incubated for 24 h. The following stock solution was
prepared in DMEM (without phenol red indicator, GIBCO)
supplemented with 100 mM sodium pyruvate: 10 μM 7-
diethylamino-3-formylcoumarin. The following four stock
solutions were prepared in Hanks’ buffered saline solution
(HBSS, GIBCO): 100 μM dabcyl, 100 μM dabcyl + 10 mM
aniline, 100 μM 8 or 11, and 100 μM 8 or 11 + 10 mM aniline.
To each 8-chambered microscope slide, media was removed,
cells were washed with phosphate buffered saline (PBS,
GIBCO) solution (2×), and fluorophore-containing media
was added (500 μL). Each slide contained a control chamber
with DMEM (without phenol indicator) only. After 1 h of
incubation, media was removed, cells were washed with PBS
(2×) buffer, and quencher-containing media was added (500
μL). The control chamber and an additional chamber in each
slide contained HBSS only. Slides were incubated at 1, 2, or 3 h.
After incubation period, media was removed, cells were washed
with PBS (2×) buffer, removable chamber walls were detached,
and the slide was prepared for fluorescence microscopy.
Fluorescence microscopy was performed on a Nikon Eclipse
E800 epifluorescence microscope equipped with a Spot RT
Slider camera with an excitation filter of 400−440 nm and
emission cutoff filter of greater than 470 nm. Camera and image
settings were the same for all images.
RESULTS
■
Synthesis of Aldehyde, Hydrazine, and Aminooxy
Quencher Derivatives. Both compounds 1 and 10 have been
reported in the literature, but surprisingly have not been
applied as fluorescence quenchers despite the obvious structural
similarity to the commonly used quencher dabcyl.30,31
Compound 10 has only sparsely been employed in detecting
reducing sugars via hydrazone formation.31 Synthetically, 2−5
were easily prepared using procedures analogous to that for the
synthesis of 1 (Scheme S1).30 Absorption properties of the free
compounds in methanol are provided in Table 1.
Table 1. Absorption Properties of Free Aldehyde or Boc
Protected Hydrazine Dyes in Methanol
dye
absorption maxima
εmax (L mol−1 cm−1
)
ε260 (L mol−1 cm−1
)
1
449 nm
451 nm
464 nm
443 nm
476 nm
439 nm
412 nm
383 nm
437 nm
29 300
22 300
42 100
40 800
41 400
40 200
35 900
28 800
55 600
9000
2
12 400
11 300
12 200
9000
3
4
5
10
14
15
16
14 000
19 000
7300
24 500
Preparation of compounds 7−9 required a different approach
(Scheme S2), particularly in the case of compounds 8 and 9 as
no compounds containing both an aryl hydrazine and diazo
bonds were found in a literature survey. However, the reported
ability to cross-couple aryl boronic acids to asymmetric Boc-
protected diazo compounds afforded a simple preparation of
nitroarenes.35 While the nitroarene precursor for preparation of
14 is known, preparation of dinitro compound 15 was realized
by the analogous reaction of tert-butyl 2-(4-nitrophenyl)-
diazenecarboxylate with 4-nitrophenylboronic acid in the
presence of catalytic copper(II).35 Reduction of the nitro
group followed by reaction with ethyl 4-nitrosobenzoate36 in
the presence of acetic acid easily afforded the desired azo
containing hydrazine precursors 14 and 16. Absorption
properties of the Boc-protected dyes in methanol can be
found in Table 1.
Preparation of quenchers 11−13 required a different
approach, particularly as no dye containing an aminooxy
group attached directly to the aryl ring could be prepared.
Consequently, Boc(aminooxy)acetic acid was attached via
simple linkers to dabcyl and Black Hole Quencher 1 and 2
scaffolds, as shown in Scheme S3. As no changes in the
chromophore were made by this synthetic approach, the
photophysical properties of these molecules were assumed to
be the same as the parent dyes.17
Based upon literature reports regarding possible problems
with the stability of free hydrazines and, to a lesser extent, free
aminooxy compounds,25,38,39 we chose to store 7−9 and 11−
13 as the Boc protected forms. The Boc group provides for a
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Figure 3. Absorption spectra of quenchers conjugated to DNA. (a) Oxime-labeled aminooxy DNA. (b) Bis(oxime)-labeled aminooxy DNA. (c)
Hydrazone-labeled hydrazide DNA. (d) Hydrazone-labeled aldehyde DNA.
bench stable derivative and is easily cleaved with trifluoroacetic
acid in dichloromethane to provide the free hydrazine or
aminooxy compound for subsequent conjugation. MALDI-
TOF data and spectral properties are consistent with the proper
deprotection and labeling of DNA as the desired hydrazones or
oximes.
Labeling of DNA. Conjugation experiments were per-
formed by reaction of the dye with the modified DNA attached
to the solid support in a DMF/water mixture in the presence of
aniline at room temperature. Subsequent deprotection of the
DNA was performed under standard aqueous ammonia
conditions at 55 °C. Attachment of dyes to DNA via oxime
formation (by reaction of aldehyde dyes with aminooxy DNA)
was, qualitatively, higher yielding and provided more stable
dyes than formation of hydrazone linkages, consistent with the
greater stability of oximes versus hydrazones.40 However, use of
diphenylhydrazine derivatives 7−9 provided a higher yield of
labeled DNA when compared to simple labeling with dabsyl
hydrazide 10, indicating that the diphenylhydrazine derivatives
may be preferable to dabsyl hydrazide in terms of higher yield
and stability. Oxime-labeled DNA was found to be stable for at
least six months after storage at −80 °C, while dabsyl
hydrazone labeled DNA did display a small degree of
degradation and may require repurification after prolonged
storage. Both hydrazones and oximes were stable at room
temperature in biologically relevant pHs utilized for hybrid-
ization experiments and at pH 4.5 used for kinetics experi-
ments.
Experiments showed that aldehyde derivatives of the
quencher dyes provided much higher yields of the desired
labeled DNA than did their methyl ketone counterparts. For
example, attempted hydrazone formation with dyes 4 and 5 by
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reaction with hydrazide modified DNA showed no observable
product by HPLC, while only a low yield was obtained when
forming an oxime with these dyes. Similarly, no bis(oximes)
were obtained with the methyl ketone dyes. In contrast,
formation of hydrazones or oximes with aldehyde dyes
provided the desired DNA adduct as the major product (save
for compound 2, which did not form a stable hydrazone) as
confirmed by the spectral properties and MALDI-TOF results.
This is consistent with literature reports of the equilibrium
constant for benzaldehyde-oxime and hydrazone formation
being orders of magnitude higher than formation of the
corresponding acetophenone oximes or hydrazones.41
Dyes 8 and 9 containing the ethyl ester protecting group
provided a complication in labeling DNA, as the deprotection
conditions employed (concentrated aqueous ammonia)
resulted in two different products, one being the free carboxylic
acid (8a, 9a) and the other a primary amide (8b, 9b). It is likely
that using different deprotection conditions (e.g., 0.05 M
K2CO3 in MeOH) would provide only the carboxylic acid
product. However, as discussed below, the absorption and
quenching properties of the dyes are sufficiently similar that the
differences in the two products are relatively unimportant.
Absorption Properties of Labeled DNAs. Hydrazones
and Oximes of Aldehyde Quenchers. Both hydrazones and
oximes of aminooxy or hydrazide modified DNAs with 1
display similar properties to the commercially available amide-
linked dabcyl quencher. Absorption maxima are only slightly
red-shifted for the simple dabcyl derivative 1 (less than 10 nm)
and display slightly higher extinction coefficients (Figure 3,
Table 2). Comparison of the 1-(dimethylamino)-naphthalene
Interesting changes in the absorption spectrum are observed
when two dyes are placed in close proximity via use of the
bis(aminooxy) linker on DNA (Figure 3b, Table 2). In the case
of two incorporations of 1, the absorption maximum shifts to
457 nm, an approximately 20 nm blue shift relative to a single 1
DNA oxime. This is likely the result of a dye−dye interaction in
the small linker. This shift is accompanied by the expected 2-
fold increase in the molar extinction coefficient. Similar results
are seen with the bis(3) oxime DNA, with a similar blue shift to
469 nm (versus 493 nm for the mono 3 oxime DNA) and
increase in the absorbance. Interestingly, the bis(2) oxime
displayed only a small blue shift in absorption maximum (from
445 nm for the mono-oxime to 438 nm for the bis-oxime).
Hydrazones of Hydrazine-Based Quencher Dyes. The
hydrazine dyes reacting with aldehyde-modified DNA provided
interesting changes in spectral properties (Figure 3d, Table 3).
Table 3. Absorption Properties of Hydrazone and Oxime
Quenchers on DNA, Formed by Reaction of Hydrazine or
Aminooxy Based Dyes with Aldehyde-Modified DNA
dye
absorption maxima
Δwλ 1/2 max
Dabcyl
BHQ2
473 nm
589 nm
464 nm
441 nm
493 nm
495 nm
484 nm
495 nm
481 nm
482 nm
542 nm
586 nm
406−519 nm (113 nm)
514−651 nm (137 nm)
404−515 nm (111 nm)
391−495 nm (104 nm)
431−538 nm (107 nm)
431−551 nm (120 nm)
421−549 nm (128 nm)
426−553 nm (127 nm)
419−529 nm (110 nm)
417−527 nm (110 nm)
461−605 nm (144 nm)
520−650 nm (130 nm)
6 DNA Hydrazone
7 DNA Hydrazone
8a DNA Hydrazone
8b DNA Hydrazone
9a DNA Hydrazone
9b DNA Hydrazone
10 DNA Hydrazone
11 DNA Oxime
Table 2. Absorption Properties of Oxime or Hydrazone
Quenchers on DNA, Formed by Reaction of Carbonyl Based
Dyes with Aminooxy or Hydrazide Modified DNA
12 DNA Oxime
13 DNA Oxime
dye
absorption maxima
Δwλ 1/2 max
Most valuably, we find that the formation of a hydrazone from
the hydrazine dye results in increased conjugation and a general
red shift in the optical absorption properties. Comparison of
dye 10 with both products of dyes 8 and 9 shows that the latter
dyes are red-shifted by approximately 15 nm, with absorption
maxima of approximately 495 nm versus 481 nm for 10.
Moreover, comparison of the absorption of the Boc-protected
dyes 14−16 in methanol to the labeled DNA also exhibits the
red-shift upon conjugation (although some solvatochromicity
likely explains part of the red-shift). While dye 14, for example,
absorbs maximally at 412 nm, attachment to DNA in the form
of quencher conjugates 8a and 8b yield an absorption
maximum of approximately 495 nm, an 80 nm red-shift. The
color changes can be readily detected by the naked eye: while
dye 15 is bright yellow as a free molecule in solution (absorbing
maximally at 383 nm in MeOH), upon hydrazone formation
with DNA an orange color is obtained, with an absorption
maximum of 441 nm. Even simple 4-nitrophenylhydrazine
reacts with aldehyde-modified DNA to shift the absorption
maximum from below 400 to 461 nm upon hydrazone
formation.
Dabcyl
473 nm
477 nm
486 nm
445 nm
493 nm
499 nm
473 nm
493 nm
457 nm
438 nm
469 nm
406−519 nm (113 nm)
408−526 nm (118 nm)
417−532 nm (115 nm)
392−527 nm (135 nm)
428−536 nm (108 nm)
432−535 nm (103 nm)
405−516 nm (111 nm)
428−536 nm (108 nm)
401 nm−513 nm (112 nm)
384−520 nm (136 nm)
411−523 nm (112 nm)
1 DNA Oxime
1 DNA Hydrazone
2 DNA Oxime
3 DNA Oxime
3 DNA Hydrazone
4 DNA Oxime
5 DNA Oxime
Bis (1) Oxime
Bis (2) Oxime
Bis (3) Oxime
moiety as in the oxime of 2 to dabcyl, however, shows a marked
blue shift in the absorption maximum of approximately 30 nm
with a decrease in the extinction coefficient, spectral properties
that are generally not desirable in a fluorescence quencher.
Introduction of a methoxy group into the dimethylaniline ring
in the form of 3 provides for a red shift of approximately 25 nm
and induces a greater increase in the extinction coefficient at
the absorbance maximum, both desirable properties. Oximes of
acetophenone dyes 4 and 5 provide similar spectral properties
to their aldehyde counterparts 1 and 3, with the DNA oxime of
4 possessing very similar properties to the commercially
available dabcyl phosphoramidite. Absorption maxima and
other spectral properties for these dye conjugates can be found
in Table 2 and the spectra of the quenchers on DNA are
provided in Figure 3.
Surprising similarities are noted between the hydrazones of
dyes 8 and 9 in labeled DNA, despite the fact that 9 is a dimeric
chromophore while 8 is a monomer. In methanol, protected
derivatives 14 and 16 display distinctly different absorption
properties, with 14 having an absorption maximum of 412 nm
and an extinction coefficient of 35 900, while 16 absorbs
maximally at 437 nm with ε = 55 600. When attached to DNA
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Figure 4. DNA-based assay used to assess fluorescence quenching.
as a hydrazone, however, all derivatives of 8 and 9 display
approximately the same absorption spectra in the visible region
(Figure 3d). This is likely due to steric crowding that shifts one
of the aromatic rings in 9 out of conjugation.
The oxime-labeled DNA derivatives of aminooxy dabsyl
(11), aminooxy BHQ1 (12), and aminooxy BHQ2 (13)
provided, as expected, similar properties to their parent dyes, as
linkers were used for attachment to DNA without any change
in the dye scaffold proper (Figure 3e).
Quenching by Oxime and Hydrazone Dye Conjugates.
Quenching experiments with the DNA−dye conjugates were
performed using fluorescein-labeled DNA complements,
monitoring the decrease in emission at 517 nm upon
hybridization of the two DNA strands. The experimental
duplexes were designed to yield contact quenching (by placing
dye quencher and fluorophore together) as opposed to
resonance energy transfer (RET), as the former is the mode
of quenching most often employed in analytical experiments
and provides greater quenching efficiency.1−11,15 DNA
sequences previously used in other quencher studies were
employed here to allow for best comparison with published
results and to provide for the simplest control of dye location
via attachment to the 5′ end of one strand quenching a
fluorophore on the 3′ end of the opposite strand (Figure
4).15,23
dyes with fluorescein were observed and can be found in the
Supporting Information (SI) file. As expected, the ground-state
complexes between the hydrazone- and oxime-labeled
quenchers and fluorescein are similar to the ground-state
complex formed between dabcyl and fluorescein, indicative of
the same contact quenching mechanism.
Consistent with their lack of spectral advantage (above), the
bis(dye) oximes of 1−3 provided a negligible increase in
quenching efficiency compared to the monodye labeled DNA
derivatives despite having two chromophores. These results
show that a second quencher moiety attached in this way does
not provide a benefit in quenching efficiency versus a
monomeric dye.
The hydrazones formed by reaction of a hydrazine dye with
aldehyde-modified DNA again give similar quenching results as
the commercially available dabcyl phosphoramidite (Table 5).
Table 5. Quenching Efficiency (Fluorescein, λem = 517 nm)
of Hydrazone and Oxime Labeled DNA Formed by Reaction
of Hydrazine or Aminooxy Quencher with Aldehyde
Modified DNA
dye
quenching efficiency
Dabcyl
BHQ 2
96.7 0.8%
96.6 0.3%
95.6 0.5%
96.6 0.1%
96.5 0.2%
96.7 0.1%
93.5 0.2%
96.3 0.3%
96.7 0.6%
96.1 0.1%
96.4 0.1%
96.5 0.2%
6 DNA Hydrazone
7 DNA Hydrazone
8a DNA Hydrazone
8b DNA Hydrazone
9a DNA Hydrazone
9b DNA Hydrazone
10 DNA Hydrazone
11 DNA Oxime
Quenching experiments confirmed that the new dyes are as
efficient at quenching fluorescein as the commercially available
dabcyl and BHQ 2 phosphoramidites (Table 4), confirming
Table 4. Quenching Efficiency (Fluorescein, λem = 517 nm)
of Oxime or Hydrazone Labeled DNA Formed by Reaction
of Carbonyl Based Quencher with Aminooxy or Hydrazide
Modified DNA
12 DNA Oxime
dye
quenching efficiency
13 DNA Oxime
Dabcyl
96.7 0.8%
97.0 0.5%
96.3 0.3%
95.0 0.2%
96.8 0.2%
97.1 0.3%
96.3 0.2%
94.9 0.2%
97.3 0.2%
96.1 0.4%
97.2 0.2%
1 DNA Oxime
1 DNA Hydrazone
2 DNA Oxime
3 DNA Oxime
3 DNA Hydrazone
4 DNA Oxime
5 DNA Oxime
Bis (1) Oxime
Bis (2) Oxime
Bis (3) Oxime
Except for 9a as a DNA hydrazone, all derivatives display
quenching efficiencies of greater than 96%, statistically similar
to the value for dabcyl. The nitroaryl quenchers as (6 and 7)
also provide excellent quenching efficiencies of greater than
96% in each case.
Like the hydrazone conjugates, the oxime linkages (11−13)
also showed similar quenching properties as the parent dyes.
Statistically speaking, oximes 11 and 13 exhibited the same
quenching efficiency as the parent dabcyl or BHQ2 quenchers
(Table 5).
Testing Hydrazone-Linked Quenchers in Common Molec-
ular Probes. The ability to easily synthesize aldehyde-modified
DNA suggested the use of hydrazine quenchers in common
fluorometric assays. As a proof of principle, a molecular beacon
was prepared using commercial 3′-fluorescein and 5′-
formylindole phosphoramidites, placing them in close prox-
imity in the stem region of the beacon.4 Hydrazone formation
was easily achieved and two different molecular beacons created
using dabsyl hydrazide 10 and the newly created dinitrodiphe-
nylhydrazine 7, which served as a surprisingly effective
that attachment via this new approach retains the optical
benefits of these quenchers. Hydrazone and oxime derivatives
of 1 are, statistically speaking, as efficient at quenching
fluorescein as the parent dabcyl with a general value of nearly
97%. Oximes or hydrazones of 3 provide a slight but not
statistically significant increase in quenching efficiency. Two of
the dyes do perform slightly worse than dabcyl (see data for 2
and 5), indicating that these are not preferable for use as
quenchers in future applications. Ground-state complexes of all
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Figure 5. Performance of molecular beacons containing quenchers attached via hydrazone linkages upon exposure to matched, mismatched, and
deletion target DNA sequences. (A) Beacon containing compound 7 as the quencher and (B) Beacon containing compound 10 as quencher.
Figure 6. Kinetics of (a) hydrazone formation or (b) oxime formation between aldehyde modified DNA and 6 (hydrazone) or 11 (oxime) in the
presence or absence of aniline at varying pH.
Figure 7. Treatment of HeLa cells with (a) 7-diethylamino-3-formylcoumarin alone, (b) aldehyde fluorophore, then 3 h incubation with quencher
11 and 10 mM aniline, (c) aldehyde fluorophore, then 3 h incubation with control dabcyl and 10 mM aniline.
quencher of fluorescein in the above DNA hybridization
experiments.
Reaction Kinetics. While all hydrazones and oximes above
were formed via attachment of the dye to DNA on solid
support in a DMF/water mixture, application of quencher
attachment directly in solution is often desirable. In this light,
experiments were performed to assess oxime and hydrazone
formation with dissolved DNAs at pH 4.5 or pH 7 in the
presence or absence of aniline (Figure 6). Consistent with
previous studies, pH 4.5 (100 mM NH4OAc buffer) proved to
be optimal for both hydrazone and oxime formation, while pH
7 (100 mM NaH2PO4) yielded a slower reaction.24−26 Even in
the absence of aniline as a catalyst, HPLC analysis indicated
88% conversion to hydrazone labeled DNA after 3.5 h of
reaction at pH 4.5. In the presence of 100 mM aniline at pH
4.5, both the oxime and hydrazone products could be obtained
Results showed that the molecular beacon containing
quencher 10 increased fluorescence intensity by ca. 27-fold in
the presence of the fully matched sequence, corresponding to a
quenching efficiency of 96.2%, consistent with the DNA
hybridization experiments (Figure 5). Little fluorescence
activation was seen in the presence of a singly mismatched
DNA or a DNA sequence containing a deletion. The hydrazone
conjugate with dye 7 provided yet better results, displaying a
>33-fold increase in fluorescence intensity upon hybridization
with the fully matched sequence, indicative of a quenching
efficiency of approximately 97%, slightly better than found in
the DNA hybridization experiments.
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Figure 8. Treatment of HeLa cells with (a) 7-diethylamino-3-formylcoumarin alone, (b) aldehyde fluorophore, then 3 h incubation with quencher 8
and 10 mM aniline, (c) aldehyde fluorophore, then 3 h incubation with control 10 mM aniline.
in greater than 90% yield after only 5 min of reaction. As
expected, both hydrazone and oxime formation at pH 7 showed
considerably slower kinetics, with the uncatalyzed reaction
showing less than 8% yield for hydrazone formation after 3.5 h
and oxime formation being less than 1% after this time.
Addition of aniline to the pH 7 buffer provided a strong
increase in reaction rate, providing the hydrazone in over 90%
yield after 3.5 h, while the oxime formation attained
approximately 60% yield after this time.
Tests of Intracellular Quenching. To further test the
efficiency and bioorthogonality of oxime and hydrazone
formation in quenching of a fluorophore, HeLa cells were
labeled by incubation with the aldehyde-containing fluorophore
7-diethylamino-3-formylcoumarin.42 Hydrazone derivatives of
this fluorophore have been used previously for intracellular
detection of Cu(II)43,44 and cysteine.45 Without any quencher
treatment, the bright fluorescence of the aldehyde fluorophore
could easily be observed by epifluorescence microscopy (Figure
7a). However, upon treating the cells with aminooxy quencher
11 for 3 h in the presence of 10 mM aniline, considerable
fluorescence quenching was observed (Figure 7b). Treatment
of the cells with the parent non-aminooxy-containing quencher
dabcyl and 10 mM aniline showed only a small degree of
quenching (Figure 7c), confirming the necessity of the
aminooxy group in 11 for oxime formation and fluorescence
quenching. A similar experiment using a non-aldehyde-
containing control fluorophore did not show strong fluo-
rescence quenching by treatment of cells with 11, again
indicating the importance of oxime formation (Supporting
Information).
in extinction coefficient relative to the parent dye. Surprisingly,
even the carboxylic acid derivative of this dye was apparently
unknown in the literature. Incorporation of the methoxy group
in the dimethylaniline ring of 3 provides for a moderate red-
shift while also increasing the extinction coefficient for the dye
in both methanol and on the DNA adducts. As quenching
efficiency is slightly increased as well, this may be a desirable
dabcyl derivative with improved spectral properties for
quenching fluorescein and other dyes emitting in the same
region.
To our knowledge, dyes 1 through 13 represent the first
examples of aldehyde, ketone, aminooxy, or hydrazine based
fluorescence quenchers that can be utilized for hydrazone
formation with a molecule of interest. Significantly, the
formation of the hydrazone results in a red-shift in the
absorption of the dyes, and oxime or hydrazone formation also
increases the extinction coefficients of the dye when conjugated
to DNA. This is also the case for dyes 1−5; for example, dye 1
shows a lower extinction coefficient than 4 in methanol, but
clearly has a higher extinction coefficient when attached to
DNA as a hydrazone or oxime. These results show the
beneficial direct electronic interactions that occur upon
formation of the dye oxime or hydrazone adducts.
This quencher conjugation approach offers clear advantages
of mild reaction conditions, rapid reaction, and bioorthogon-
ality of hydrazone formation. Standard postsynthetic labeling of
amines with quenchers or other chromophores typically
requires many hours to days of reaction and use of organic
cosolvents to dissolve activated chromophores (typically NHS
esters). In contrast, formation of the DNA hydrazone of 4-
nitrophenylhydrazine (for example) proceeded rapidly in
purely aqueous buffer without even the need for catalysis at
pH 4.5, reaching 87% yield in less than 4 h. Addition of aniline
as catalyst provided formation of either oximes or hydrazones
in as little as 5 min.
The mild, rapid, and selective reaction may have utility
beyond simple formation of biomolecular conjugates. For
example, experiments in which a fluorescence signal needs to be
removed prior to measuring a new signal had previously
required photobleaching techniques which may damage cells or
other analytes present.46−49 The present experiments suggest a
milder approach; rapid attachment of a quencher via
bioorthogonal hydrazone or oxime formation might simplify
such experiments and avoid high-intensity light exposure. In
our preliminary experiments, aminooxy and hydrazine
quenchers were shown to efficiently quench an aldehyde
fluorophore in a cellular environment. Interestingly, this cellular
quenching did not show the need for high amounts of aniline
Cellular quenching via the hydrazine quencher 8 with the
same aldehyde fluorophore showed even stronger fluorescence
quenching, as shown in Figure 8. After 3 h incubation, very
little fluorescence was seen in the treated cells as compared to
untreated cells or those exposed to aniline alone, while the
presence of the cells was easily observed by brightfield
microscopy (Supporting Information).
DISCUSSION
■
Our experiments establish compounds 1−13 as useful new
scaffolds for reactive fluorescence quenchers, offering the
convenience of conjugation via oxime or hydrazone formation.
We find that dyes 2, 4, and 5 are less effective because of
reduced quenching efficiency or poor oxime or hydrazone
formation with DNA. However, dyes 1, 3, and 7−13 offer
excellent properties as quenchers of fluorescein. Dye 3 also
provides a new chromophore based on the dabcyl scaffold that
has both a red-shift in the absorption maximum and an increase
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(16) Johansson, M. K. (2006) Choosing reporter-quencher pairs for
efficient quenching through formation of intramolecular dimers. In
Methods of Molecular Biology, Vol 335, Fluorescent Energy Transfer
Nucleic Acid Probes: Designs and Protocols, Didenko, V. V., Ed.,
Humana Press Inc., Totowa, NJ.
(17) Cook, R. M., Lyttle, M., and Dick, D. Dark quenchers for donor-
acceptor energy transfer. U.S. Patent 7,019,129, March 28, 2006.
(18) Haugland, R. P., Singer, V. L., and Yue, S. L. Xanthene dyes and
their application as luminescence quenching compounds. U.S. Patent
6,399,392, March 4, 2002.
catalysis to quickly attain significant fluorescence quenching at
approximately neutral pH inside the cells. Future experiments
will explore the generality and applications of this approach.
ASSOCIATED CONTENT
* Supporting Information
■
S
NMR spectra of quencher dyes and intermediates, HPLC
purity and MALDI-TOF of DNA strands, and additional
spectral data. This material is available free of charge via the
Internet at http://pubs.acs.org.
(19) Berry, D. A., and Pearson, W. H. Dark quenchers, probes and
other conjugates incorporating the same, and their use. U.S. Patent
7,879,986, February 3, 2006.
AUTHOR INFORMATION
Corresponding Author
*E-mail: kool@stanford.edu. Tel.: 650-724-4741. Fax: 650-725-
0295.
■
̌
(20) Kopecky, K., Novakova, V., Miletin, M., Kucera, R., and Zimcik,
P. (2010) Solid-phase synthesis of azaphthalocyanine-oligonucleotide
conjugates and their evaluation as new dark quenchers of fluorescence.
Bioconjugate Chem. 21, 1872−1879.
̌
(21) Kopecky, K., Novakova, V., Miletin, M., Kucera, R., and Zimcik,
Notes
P. (2011) Synthesis of new azaphthalocyanine dark quencher and
evaluation of its quenching efficiency with different fluorophores.
Tetrahedron 67, 5956−5963.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by the National Institutes of Health
(GM068122 and GM067201).
■
(22) Pham, W., Weissleder, R., and Tung., C. H. (2002) An azulene
dimer as a near-infrared quencher. Angew. Chem., Int. Ed. Engl. 41 (19),
3659−3662.
(23) Crisalli, P., and Kool, E. T. (2011) Multi-path quenchers:
efficient quenching of common fluorophores. Bioconjugate Chem. 22
(11), 2345−2354.
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