Multi-path quenchers: Efficient quenching of common fluorophores

Article abstract of DOI:10.1021/bc200424r

Fluorescence quenching groups are widely employed in biological detection, sensing, and imaging. To date, a relatively small number of such groups are in common use. Perhaps the most commonly used quencher, dabcyl, has limited efficiency with a broad range of fluorophores. Here, we describe a molecular approach to improve the efficiency of quenchers by increasing their electronic complexity. Multi-Path Quenchers (MPQ) are designed to have multiple donor or acceptor groups in their structure, allowing for a multiplicity of conjugation pathways of varied length. This has the effect of broadening the absorption spectrum, which in turn can increase quenching efficiency and versatility. Six such MPQ derivatives are synthesized and tested for quenching efficiency in a DNA hybridization context. Duplexes placing quenchers and fluorophores within contact distance or beyond this distance are used to measure quenching via contact or FRET mechanisms. Results show that several of the quenchers are considerably more efficient than dabcyl at quenching a wider range of common fluorophores, and two quench fluorescein and TAMRA as well as or better than a Black Hole Quencher.

Full text of DOI:10.1021/bc200424r

Article
pubs.acs.org/bc
Multi-Path Quenchers: Efficient Quenching of Common Fluorophores
Pete Crisalli and Eric T. Kool*
Department of Chemistry, Stanford University, Stanford, California 94305-5080, United States
S
* Supporting Information
ABSTRACT: Fluorescence quenching groups are widely employed in
biological detection, sensing, and imaging. To date, a relatively small number
of such groups are in common use. Perhaps the most commonly used
quencher, dabcyl, has limited efficiency with a broad range of fluorophores.
Here, we describe a molecular approach to improve the efficiency of quenchers
by increasing their electronic complexity. Multi-Path Quenchers (MPQ) are
designed to have multiple donor or acceptor groups in their structure, allowing
for a multiplicity of conjugation pathways of varied length. This has the effect
of broadening the absorption spectrum, which in turn can increase quenching
efficiency and versatility. Six such MPQ derivatives are synthesized and tested
for quenching efficiency in a DNA hybridization context. Duplexes placing
quenchers and fluorophores within contact distance or beyond this distance are used to measure quenching via contact or FRET
mechanisms. Results show that several of the quenchers are considerably more efficient than dabcyl at quenching a wider range of
common fluorophores, and two quench fluorescein and TAMRA as well as or better than a Black Hole Quencher.
quencher are in sufficiently close proximity to allow for direct
electronic interaction of the excited state of the fluorophore
with the quencher molecule.18−21 Consequently, absorbed light
at the excitation wavelength will primarily be nonradiatively
transferred as heat to the surrounding environment, with only a
limited amount of energy released as fluorescence. When the
distance between the fluorophore and the quencher is
increased, generally to the range 20−100 Å, the alternative
mechanism of FRET quenching is observed.12,14,18 In this
mechanism, quenching efficiency is dependent upon the
orientation of the fluorophore and quencher, the distance
between them, and the overlap of the emission spectrum of the
fluorophore and the absorption spectrum of the quencher.12,14
To take advantage of these mechanisms, it is desirable to design
quenchers with broadened absorbance spectra to allow for a
greater range of fluorophore emissions that can be quenched by
the FRET mechanism, while maintaining strong contact
quenching.12,14
To address this issue, we have designed a novel set of
quenchers that have multiple electronic conjugation pathways.
By altering the existing scaffolds of common quenchers to
increase the number of electron donor and acceptor groups, a
series of Multi-Path Quenchers (MPQs) has been designed that
provides for broader absorbance properties, in turn allowing for
fluorescence quenching of a considerably wider range of
fluorophores for each given quencher (Figure 1). Five new
quenchers based on modifications to the dabcyl structure as
well as one based upon modification of a Black Hole Quencher
scaffold have been prepared and tested in a DNA context for
INTRODUCTION
■
Fluorescence quenchers are employed in a wide variety of
fluorometric assays, particularly for detection of nucleic acids,
reporting on enzymatic activity, and detecting other molecules
of interest with turn-on responses.1−11 However, for optimal
performance, delicate matching of fluorophore and quencher is
often needed, imposing limits on molecular designs and
complicating applications.12−14 For example, in order to cover
the emission spectrum from violet to near-infrared, multiple
different quenchers are needed, which can be an obstacle in
assays designed to detect multiple analytes in one sample.14,15
4-(Dimethylamino)-azobenzene-4′-carboxylic acid (Dabcyl),
perhaps the most commonly used fluorescence quencher, is
generally limited to quenching of fluorophores that emit in the
violet to green region of the visible spectrum (approximately
390 to 520 nm).11,16 To cover fluorophores that emit further to
the red, a variety of other quenchers have been developed, such
as the Black Hole Quenchers (BHQ)16 and QSY quenchers;17
however, coverage of a broad spectrum still requires the use of
multiple quenchers. In the BHQ series, for example, a total of
four different compounds are necessary to provide for
quenching of all commonly used wavelengths in fluorescence
assays.13 QSY quenchers are even more limited in quenching
ability as a result of their narrow absorbance spectra.12,14
Recently, some nonfluorescent cyanine-based quenchers have
been reported that do have broadened spectral properties and
provide for efficient quenching in caspase assays.8 However,
these quenchers display strong spectral overlap only with near-
IR emitting fluorophores and may not quench other
fluorophores as effectively.
Most commonly, quenching of fluorophores occurs by one of
two mechanisms: contact quenching and FRET quench-
ing.12−14,18,19 In contact quenching, the fluorophore and
Received: August 1, 2011
Revised: October 14, 2011
Published: October 28, 2011
© 2011 American Chemical Society
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Figure 1. Structures of Multi-Path Quenchers (MPQs) and commercially available quenchers used in this study.
quenching efficiency, for comparison with previous studies.3
We find that this strategy yields broadened absorption spectra
and enhances efficiency over a wider range of wavelengths as
compared with a common quencher such as dabcyl.
Oligonucleotide masses were determined by the Stanford
University Protein and Nucleic Acid Facility using a Perspective
Voyager-DE RP Biospectrometry MALDI-TOF mass-spec-
trometry instrument using a 3-hydroxypicolinic acid/diammo-
nium hydrogen citrate matrix.
EXPERIMENTAL PROCEDURES
Synthetic Procedures. Methyl 3-(6-Amino-1,3-dioxo-
1H-benzo[de]isoquinolin-2(3H)-yl)propanoate (1). 0.196 g
(0.81 mmol) of 4-nitronaphthalic anhydride23,24 and 0.107 g
(1.5 eq, 1.21 mmol) of β-alanine were refluxed in 4 mL ethanol
for 3 h. The resulting orange suspension was cooled to room
temperature, diluted with water, and filtered to provide 0.185 g
(73%) of the nitro intermediate. The intermediate was
suspended in 3 mL methanol, and 0.7 mL concentrated HCl
and 0.569 g (5.1 equiv, 3 mmol) tin(II) chloride dihydrate was
added and the solution was heated to reflux for 2 h. Cooling to
room temperature and dilution with water followed by filtration
■
Chemicals and Reagents. Chemicals were purchased
from Sigma-Aldrich and used without further purification.
Solvents were purchased from Acros Organics and used as
received. UltraMild DNA phosphoramidites and solid supports
were purchased from Glen Research. BHQ2 phosphoramidite
and 3′-Quasar 670 CPG were purchased from Biosearch
Technologies. AlexaFluor 350 succinimidyl ester was purchased
from Invitrogen. 5′-Fluorophore-modified oligonucleotides
were purchased from Biosearch Technologies (FAM,
TAMRA, Quasar 670) or Stanford University Protein and
Nucleic Acid Facility (Cy3) and purified by HPLC.
1
provided 0.125 g (71%) of a bright yellow−brown solid. H
1
Instrumentation. H and 13C NMR spectra were recorded
NMR (DMSO-d6, 400 MHz): 8.61 (1H, d, J = 8.4), 8.41 (1H,
d, J = 7.6), 8.18 (1H, d, J = 8.4), 7.64 (1H, dd, J = 7.6), 7.46
(2H, brs, NH), 6.83 (1H, d, J = 8.4), 4.24 (2H, t, J = 7.2), 3.57
(3H, s), 2.61 (2H, t, J = 7.2). 13C NMR (DMSO-d6, 100
MHz): 32.3, 35.3, 51.5, 107.3, 108.2, 119.3, 121.6, 124.0, 129.4,
129.7, 131.0, 134.0, 152.8, 162.7, 163.7, 171.4. ESI MS: M + H+
= 299.58 (Calc 299.10).
on a Varian 400 MHz NMR or a Varian 500 MHz NMR
spectrometer and internally referenced to the residual solvent
signal; J values are reported in Hz. Oligonucleotides sequences
were synthesized on an ABI 394 DNA synthesizer using
UltraMild reagents and phosphoramidites. ESI-MS analysis was
performed by the Stanford University Mass Spectroscopy
Facility. 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). DNA
concentrations were determined on a Cary 100 Bio UV−vis
spectrophotometer at 90 °C. Fluorescence measurements were
performed on a Fluorolog 3 Jobin Yvon fluorophotospec-
trometer equipped with an external temperature controller.
General Procedure for MPQ Methyl Ester Synthesis.25 1.5
mL concentrated H2SO4 was cooled to 0 °C in an ice−water
bath. 0.249 g (1.05 equiv, 3.6 mmol) sodium nitrite was slowly
added. The viscous suspension was heated to 65 °C until
complete dissolution occurred, and then cooled to 0 °C. 1.02 g
(3.04 mmol) of 1 was added in portions over 30 min. Two
milliliters acetic acid was added to aid in solubility. After stirring
at room temperature for 3 h, a solution of 3.4 mmol coupling
partner in 0.65 mL acetic acid was added dropwise at 0 °C. The
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resulting suspension was stirred for 1 h, then diluted with
saturated sodium acetate and stirred at 0−10 °C for 3 h. The
resulting solution was filtered, washed with warm water, and
crystallized in ethanol to afford the azo dye methyl ester 2.
solubility). NMR and ESI-MS indicate that this compound
degrades to the naphthalic anhydride during prolonged storage.
ESI MS: M + H+ = 467.50 (Calc 467.17).
1
MPQ3 (3c). (80% yield) H NMR (CDCl3, 500 MHz):
1
MPQ1 Methyl Ester (2a). (50% yield) H NMR (CDCl3,
8.62−8.66 (3H, m), 8.12 (1H, dd, J = 8.5, 2.5), 7.80 (1H, dd, J
= 7.0), 7.41 (1H, d, J = 9.5), 6.55 (1H, dd, J = 9.5, 2.5), 5.99
(1H, d, J = 2.5), 4.51 (2H, t, J = 7), 3.52 (4H, q, J = 7), 2.79
(2H, t, J = 7), 1.31 (6H, t, J = 7). 13C NMR (spectrum could
not be obtained due to poor solubility). ESI MS: M + H+ =
461.33 (Calc 461.18).
400 MHz): 9.25 (1H, d, J = 7.2), 8.63−8.67 (2H, m), 8.04 (2H,
d, J = 7.6), 7.96 (1H, d, J = 8.4), 7.83 (1H, dd, J = 7.2), 6.80
(2H, d, J = 7.6), 4.52 (2H, t, J = 7.4), 3.71 (3H, s), 3.17 (6H, s),
2.80 (2H, t, J = 7.4). 13C NMR (CDCl3, 100 MHz): 32.7, 36.2,
40.4, 51.9, 111.6, 112.3, 121.8, 122.3, 126.5, 126.8, 131.0, 131.6,
132.1, 144.7, 152.2, 153.5, 164.0, 164.4, 171.9. ESI MS: M + H+
= 431.42 (Calc 431.17).
1
MPQ5 (3d). (87% yield) H and 13C NMR data could not
be obtained as a result of very poor solubility in common NMR
solvents. ESI MS: M + H+ = 494.42 (Calc 494.18).
1
MPQ2 Methyl Ester (2b). (62% yield) H NMR (CDCl3,
MPQ4 (3e). A solution of 0.270 g (1.05 equiv, 3.90 mmol)
NaNO2 in 2 mL H2O was added dropwise to a mixture of 0.510
g (1.0 equiv, 3.72 mmol) 4-aminobenzoic acid in 12 mL 1 M
HCl at 0 °C. After 30 min, a solution of 0.657 g (1.05 equiv,
3.90 mmol) of 1-H-perimidine in 9 mL 1 M HCl was added,
and the resulting suspension stirred at 0 °C for 2 h. The
suspension was diluted with a solution of 2.15 g NaOAc in 15
mL H2O, stirred at room temperature for 2 h, then filtered and
washed with warm water to give a dark purple solid.
Crystallization from ethanol afforded 0.916 g (78%). A complex
1H NMR spectrum results from tautomerization in the
perimidine scaffold (see Supporting Information). 13C NMR
data could not be obtained as a result of poor solubility in
common NMR solvents. ESI MS: M + H+ = 317.25 (Calc
317.10).
3-(2-Methyl-2,3-dihydro-1H-perimidin-2-yl)propan-1-ol
(4). 6.19 g (39 mmol) 1,8-diaminonaphthalene was added to a
solution of 4.35 mL (1.1 equiv, 43 mmol) 5-hydroxy-2-
pentanone and 0.089 g (0.01 equiv, 0.4 mmol) p-
toluenesulfonic acid monohydrate in 15 mL ethanol. The
solution was stirred at 60 °C for 90 min, whereupon the
solution had solidified. The resulting solid was cooled to room
temperature, filtered, washed with ethanol, and crystallized
from aqueous ethanol to yield a pale gray solid (4.11 g, 43%).
1H (DMSO-d6, 400 MHz): 7.07 (2H, dd, J = 7.8), 6.83 (2H, d,
J = 8.4), 6.34 (2H, d, J = 7.2), 6.29 (2H, brs, NH), 4.37 (1H, t,
OH), 3.30 (2H, m), 1.53−1.62 (4H, m), 1.29 (3H, s). 13C
NMR (DMSO-d6, 100 MHz): 26.5, 27.1, 36.9, 61.2, 65.5,
103.6, 111.5, 114.0, 127.0, 134.2, 141.8. ESI MS: M + H+ =
243.50 (Calc 243.15).
MPQ6 (5). 0.291 g (1.20 mmol) of 4 was dissolved in a
solution of 20 mL 1:1 THF/acetone stirred at 0 °C. 0.600 g
(1.2 equiv, 1.44 mmol) Fast Corinth V was added in portions
to give a dark blue solution, which was stirred at 0 °C for 1 h.
The reaction was diluted with water and extracted 3 times with
CH2Cl2. The combined organic extracts were washed once with
water, once with brine, dried (MgSO4), filtered, and the solvent
removed in vacuo. Column chromatography (5% MeOH in
CH2Cl2 containing 0.5% Et3N) afforded 0.094 g (14%) of a
dark purple solid. 1H NMR (CDCl3, 400 MHz): 8.19 (1H, d, J
= 8.0), 7.84 (1H, d, J = 8.8), 7.66−7.69 (3H, m), 7.40−7.47
(3H, m), 6.53−6.56 (2H, m), 4.02 (3H, s), 3.66 (2H, t, J =
6.0), 2.74 (3H, s), 2.50 (3H, s), 1.88−1.92 (2H, m), 1.75−1.80
(2H, m), 1.52 (3H, s). 13C NMR (CDCl3, 100 MHz): 17.0,
21.3, 27.2, 27.6, 29.8, 38.6, 56.3, 62.7, 99.0, 112.8, 119.2, 124.4,
133.6, 141.3, 143.6, 147.4. ESI MS: M + H+ = 554.42 (Calc
554.25).
400 MHz): 9.34 (1H, d, J = 8.8), 9.11 (1H, d, J = 8.0), 8.67−
8.70 (2H, m), 8.22 (1H, d, J = 8.4), 8.10−8.14 (2H, m), 7.89
(1H, dd, J = 8.0), 7.69 (1H, dd, J = 8.0), 7.59 (1H, dd, J = 7.6),
7.11 (1H, d, J = 8.4), 4.54 (2H, t, J = 7.4), 3.71 (3H, s), 3.11
(6H, s), 2.81 (2H, t, J = 7.4). 13C NMR (CDCl3, 100 MHz):
32.7, 36.3, 44.8, 52.0, 112.8, 112.9, 114.0, 122.6, 123.8, 125.0,
125.6, 127.2. 127.6, 131.0, 131.7, 132.0, 143.3, 152.0, 156.4,
163.9, 164.3, 167.7, 171.9. ESI MS: M + H+ = 481.92 (Calc
481.19).
1
MPQ3 Methyl Ester (2c). (52% yield) H NMR (CDCl3,
400 MHz): 8.59−8.63 (3H, m), 8.09 (1H, d, J = 8.4), 7.78 (1H,
dd, J = 7.8), 7.38 (1H, d, J = 9.6), 6.53 (1H, dd, J = 9.6, 2.6),
5.97 (1H, d, J = 2.6), 4.50 (2H, t, J = 7.4), 3.70 (3H, s), 3.51
(4H, q, J = 7.2), 2.78 (2H, t, J = 7.4), 1.30 (6H, t, J = 7.2). 13
C
NMR (CDCl3, 100 MHz): 13.1, 32.7, 36.2, 45.6, 51.9, 98.1,
110.8, 111.6, 122.7, 126.9, 128.6, 131.6, 132.6, 154.9, 163.8,
171.9. ESI MS: M + H+ = 475.42 (Calc 475.20).
1
MPQ5 Methyl Ester (2d). (20% yield) H NMR (CDCl3,
400 MHz): 9.34 (1H, d, J = 8.4), 8.66−8.68 (2H, m), 8.40 (d,
1H, J = 8.4), 8.19 (d, 1H, J = 8.4), 8.09 (d, 1H, J = 8.0), 7.84
(dd, 1H, J = 7.6), 7.50 (dd, 1H, J = 7.6), 6.62 (d, 1H, J = 7.2),
6.55 (d, 1H, J = 8.4), 4.53 (t, 2H, J = 7.2), 3.71 (s, 3H), 2.81 (t,
2H, J = 7.2), 1.61 (s, 6H). 13C NMR: (spectrum could not be
obtained due to poor solubility). ESI MS: M + H+ = 508.42
(Calc 508.20).
General Procedure for Methyl Ester Hydrolysis. 500 mg
MPQ methyl ester was dissolved in anhydrous THF (at a
concentration of 0.085 M). To this solution, 2.02 equiv
potassium trimethylsilanolate was added, and the resulting
solution stirred overnight at room temperature, at which point
TLC indicated consumption of starting material. The solvent
was removed in vacuo and the resulting solid dissolved in a
minimal amount of water. Hydrochloric acid (1 M) was added
to precipitate the free carboxylic acid, which was filtered,
washed with water, and dried. The product 3 was used for DNA
conjugation without further purification. All free acids were
found to be poorly soluble in common deuterated solvents.
1
MPQ1 (3a). (80% yield) H NMR (CDCl3, 500 MHz):
9.26−9.35 (1H, m), 8.64−8.96 (2H, m), 7.96−8.05 (3H, m),
7.84−7.91 (1H, m), 6.80 (2H, d, J = 9.0), 4.54 (2H, t, J = 7.0),
3.18 (s, 6H), 2.87 (2H, t, J = 7.0). NMR indicates some
degradation to naphthalic anhydride during prolonged storage.
13C NMR: (spectrum could not be obtained due to poor
solubility). ESI MS: M + H+ = 417.33 (Calc 417.16).
MPQ2 (3b). (36% yield) 1H NMR (CDCl3, 500 MHz): 9.45
(1H, d, J = 8.5), 9.12 (1H, d, J = 7.5), 8.71−8.75 (2H, m), 8.24
(1H, d, J = 8.0), 8.14−8.18 (2H, m), 7.96 (1H, dd, J = 8.0),
7.73 (1H, dd, J = 7.0, 1.5), 7.63 (1H, dd, J = 7.0, 1.5), 7.14 (1H,
dd, J = 8.5), 4.59 (2H, t, J = 7.0), 3.17 (6H, s), 2.91 (2H, t, J =
7.0). 13C NMR: (spectrum could not be obtained due to poor
MPQ6 Phosphoramidite (6). 0.164 g (0.296 mmol) MPQ6
was dissolved in 2.5 mL anhydrous acetonitrile, 0.15 mL (3
equiv, 0.861 mmol) N,N-diisopropylethylamine was added and
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a
Scheme 1. Synthesis of Multi-Path Quenchers
a
(a) β-alanine, EtOH, (73%). (b) SnCl2, MeOH, HCl, (71%). (c) (i) NaNO2, H2SO4; (ii) aniline derivative; (iii) NaOAc (20−62%). (d) KOTMS,
THF (36−87%). (e) (i) NaNO2, HCl; (ii) 1H-perimidine, NaOAc. (f) 5-Hydroxy-2-pentanone, pTsOH (43%). (g) Fast Corinth V, 1:1 THF/
acetone, 0 °C (14%). (h) 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite, DIPEA.
the solution was stirred at room temperature under argon. 0.10
mL (1.5 equiv, 0.448 mmol) 2-cyanoethyl N,N-diisopropyl-
chlorophosphoramidite was added and the resulting solution
was stirred at room temperature under argon for 2 h. The
solution was concentrated and purified by column chromatog-
raphy (2:3 EtOAc/Hexanes → 2:1 EtOAc/Hexanes, containing
0.5% Et3N) to yield 0.036 g product (13%). 31P NMR
displayed two peaks at 148.61 ppm and 148.55 ppm, and the
product was used for DNA synthesis without further character-
ization.
DNA Conjugation. TAMRA-, Fluorescein-, and Quasar
670-conjugated DNA sequences were synthesized using their
respective 3′ CPGs, cleaved, and purified by HPLC using a
gradient elution of 0.05 M TEAA and acetonitrile. AlexaFluor
350 succinimidyl ester and ATTO 590 succinimidyl ester were
reacted with 3′-amino modified DNA. Cy3-modified DNA was
synthesized using Cy3 Phosphoramidite (Glen Research) with
5′ to 3′ synthesis. Quencher-labeled oligonucleotides were
synthesized with a 5′-amino-modifier 5 appended to the 5′-
terminus. The monomethoxy-trityl protecting group was
removed on the synthesizer using alternating 10 s cycles of
deprotection reagent (3% trichloroacetic acid in DCM) and
DCM washes for 3 min. The solid support was added to a
suspension containing 35 mM quencher-COOH, 35 mM 1-
hydroxy-7-azabenzotriazole (HOAt), 35 mM 2-(7-Aza-1H-
benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate (HATU), and 150 mM diisopropylethylamine
(DIPEA) in 750 μL anhydrous DMF. Solutions were shaken
at room temperature and protected from light for 16 h, then
rinsed with 4 × 500 μL DMF and 4 × 500 μL AcCN. The
labeled DNA was cleaved from the solid support using
UltraMild conditions (0.05 M K2CO3 in MeOH) at room
temperature for 4 h, filtered to remove CPG, diluted with
water, and purified by reverse-phase HPLC.
Fluorescence Experiments. A solution of 30 nM of
fluorescently labeled 20mer was created in a total volume of
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Figure 2. Absorption spectra (normalized to absorbance at 260 nm) of quenchers on (a) 20mer and (b) 15mer oligonucleotide sequences. MPQ1
(yellow), MPQ2 (magenta), MPQ3 (brown), MPQ4 (cyan), MPQ5 (green), MPQ6 (black), Dabcyl (orange), BHQ2 (purple).
Table 1. Spectral Properties of Quenchers Conjugated at the 5′-Terminus of 20mer and 15mer Oligonucleotides
20mer
15mer
quencher
λmax
557 nm
Δλ(half max)
λmax
560 nm
Δλ(half max)
MPQ1
MPQ2
MPQ3
MPQ4
MPQ5
MPQ6
Dabcyl
BHQ2
481−618 (137 nm)
522−676 (154 nm)
461−574 (113 nm)
467−606 (139 nm)
447−689 (242 nm)
463−731 (268 nm)
405−520 (115 nm)
503−649 (146 nm)
481−619 (138 nm)
446−640 (194 nm)
478−571 (93 nm)
469−611 (142 nm)
524−723 (199 nm)
463−736 (273 nm)
405−519 (114 nm)
507−652 (145 nm)
614 nm
521 nm
524 nm
529 nm
539 nm
551 nm
503 nm, 623 nm
536 nm, 656 nm
472 nm
646 nm
537 nm, 657 nm
472 nm
580 nm
581 nm
800 μL hybridization buffer (10 mM MgCl2, 70 mM
Tris·borate, pH 7.55) at 37 °C. The fluorescence of the
solution was taken in triplicate (using published excitation and
emission values with excitation and emission slits of 2 and 5
nm, respectively) and averaged. A concentrated solution of
quencher labeled DNA was added to bring the final volume to
150 nM. The oligonucleotides were allowed to hybridize until
the fluorescence emission remained constant, then the
fluorescence spectrum was taken in triplicate and averaged.
Quenching efficiency was calculated at the emission maxima by
dividing the quenched fluorescence by the initial fluorescence,
multiplying by 100, then subtracting from 100.14 All experi-
ments were performed in triplicate and averaged.
carboxylic acid moiety that could easily be attached to amino-
modified DNA by HATU/HOAt coupling. DNA synthesis
utilized UltraMild protecting groups to avoid hydrolysis of the
naphthalimide core under regular ammonia or ammonia/
methylamine cleavage/deprotection. MPQ6 was prepared by
the addition of dihydroperimidine 4 to Fast Corinth V salt in a
cold solution of 1:1 THF/atone, and readily converted to a
phosphoramidite for DNA coupling, again with UltraMild
conditions. DNA sequences were selected to maintain
consistency with previous oligonucleotide-based experiments
for comparing fluorescence quenchers.14 For comparison with
the new quenchers, we prepared analogous DNA conjugates of
dabcyl and of BHQ2, possibly the two most widely used
quenchers. BHQ2 was selected instead of BHQ1 based upon
the better performance of BHQ2 against a variety of
fluorophores in previous studies.14 For testing the quenchers,
we prepared DNA conjugates of common fluorescent dyes with
varied emission properties, emitting from the blue to the far red
(442 to 662 nm).
RESULTS
■
Quencher Design. The new quencher structures are given
in Figure 1. These were designed with the standard diazo dye
framework analogous to the common dabcyl quencher, but
with larger conjugated multifunctional donors or acceptors.
The purpose of the design was to provide multiple conjugated
pathways from donor to acceptor groups, thus potentially
broadening the absorption spectra of the dyes. The first five
(MPQ1−5) were built on the standard azobenzene framework,
while the sixth (MPQ6) was designed as a multipathway variant
of the bis-azobenzene framework common to BHQ quenchers.
Synthesis. As shown in Scheme 1, reaction of β-alanine
with 4-nitronaphthalic anhydride and subsequent reduction of
the nitro group easily afforded 1 which could be diazotized to
allow for formation of MPQs by reaction with a variety of
anilines. Similar derivatives of these compounds were reported
earlier for use as disperse dyes, and the properties of the
compounds synthesized are consistent with those previously
reported.25,26 Hydrolysis of the methyl ester afforded a
Spectral Properties. Absorption data for the six MPQs
conjugated to DNAs are given in Figure 2 and Table 1. The
data reveal that, as designed, the MPQ dyes have considerably
broadened absorbance spectra compared to their parent
quencher molecules (dabcyl and BHQ). Save for MPQ3, all
other quenchers containing the naphthalimide core show
significantly increased Δλ1/2
values when compared to
max
dabcyl, validating the design strategy. This is particularly the
values are
case for MPQ2 and MPQ5, whose Δλ1/2
considerably greater and even further red-shifted than the other
dyes.
max
For the naphthalimide derivatives, where the electron
acceptor has been altered to incorporate a second conjugation
pathway, we noted some differences in spectral properties
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Figure 3. DNA hybridization formats used for determining (a) contact quenching (b) mixed mechanism quenching and (c) FRET quenching
efficiencies of MP quenchers.
a
Table 2. Contact Quenching Efficiencies of Fluorophore/Quencher Pairs
Alexa 350
FAM
Cy 3
TAMRA
Atto 590
Quasar 670
quencher
λem = 442 nm
λem = 517 nm
λem = 563 nm
λem = 580 nm
λem = 624 nm
λem = 662 nm
MPQ1
MPQ2
MPQ3
MPQ4
MPQ5
MPQ6
Dabcyl
BHQ2
73.6 ± 0.7%
72.4 ± 0.1%
74.7 ± 0.9%
72.1 ± 1.2%
69.7 ± 3.5%
71.6 ± 1.8%
75.7 ± 0.7%
77.0 ± 0.7%
95.4 ± 0.2%
94.9 ± 0.4%
96.5 ± 0.2%
96.0 ± 0.2%
93.7 ± 0.1%
95.2 ± 0.2%
96.0 ± 0.2%
96.5 ± 0.2%
98.0 ± 0.4%
97.6 ± 0.2%
97.3 ± 0.2%
97.6 ± 0.3%
96.8 ± 0.4%
98.2 ± 0.6%
97.4 ± 0.1%
98.9 ± 0.3%
86.0 ± 0.7%
87.8 ± 0.4%
86.8 ± 0.5%
88.8 ± 1.0%
85.7 ± 0.4%
87.2 ± 0.2%
87.3 ± 0.6%
87.8 ± 0.2%
96.3 ± 0.2%
96.1 ± 0.1%
96.6 ± 0.2%
96.2 ± 0.7%
95.1 ± 0.2%
96.4 ± 0.2%
95.7 ± 0.3%
97.4 ± 0.3%
96.1 ± 0.3%
97.0 ± 0.2%
93.3 ± 0.4%
94.7 ± 0.2%
95.4 ± 0.3%
95.6 ± 0.8%
83.9 ± 0.4%
98.2 ± 0.2%
a
Results are provided as the average of three experiments with standard deviation shown. See Figure 3a for structural context.
between the 20mer and 15mer quencher−oligonucleotide
conjugates. As anticipated, the parent quencher dabcyl shows
negligible change in spectral properties, maintaining an
absorbance maxima of 472 nm and a Δλ1/2 max of 115 nm in
both sequences. Both MPQ1 and MPQ3, however, display
slight red-shifts upon moving from the 20mer oligonucleotide
to a different 15mer sequence context. MPQ2, in contrast,
shows a considerable blue shift and significant broadening on
going from the 20mer to the 15mer DNA.
Altering the electron donating group also provides for some
interesting and broadened spectral properties. As seen with
MPQ4, a considerable red shift is observed in the absorbance
maximum, while an almost 35 nm increase in Δλ1/2 max is
observed compared to dabcyl. Much like MPQ1−3, this
quencher also shows a change when moved from the 20mer to
a 15mer, with the Δλ1/2 max increasing and the absorbance
maximum again shifting to the red.
same result, indicating that the addition of the new conjugation
pathway may reduce the extinction coefficient of each
individual peak.
The most obvious benefit of the added conjugation is
observed in a comparison of MPQ6 with its parent BHQ1.
While BHQ1 displays an absorption maximum at 534 nm and
generally quenches fluorophores that emit in the range from
480 to 580 nm,16 MPQ6 displays considerably broadened
properties. MPQ6 shares one conjugation pathway with BHQ1,
giving rise to an absorbance maximum at 537 nm, but the
alternative conjugation pathway inherent to the dihydroper-
imidine scaffold adds an absorption at 656 nm as well,
extending the range of quenching available to the dye greatly
(vide infra). Thus, MPQ6 has a Δλ1/2 max ranging from 465 to
735 nm (270 nm total), considerably greater than BHQ2 (146
nm) and in fact greater than the quenching ranges of BHQ1
and BHQ2 combined.
MPQ5 and MPQ6, incorporating the dihydroperimidine
moiety, displayed the most distinct spectral properties. Whereas
all other quenchers possess a single broadened absorbance
maximum, MPQ5 and MPQ6 display two maxima. MPQ5, like
the other quenchers, exhibits a slightly altered absorbance
spectrum as the sequence of the oligonucleotide changes, while
MPQ6, like BHQ2, maintains approximately the same
spectrum on the two DNAs. For these last two quenchers,
the formation of a red-shifted absorbance maximum comes with
a moderate cost to the extinction coefficient. When compared
to MPQ1, MPQ5 clearly displays a lower extinction coefficient
at the λmax, both on DNA and in ethanol (see Supporting
Information). Comparison of BHQ2 and MPQ6 yields the
Fluorescence experiments confirmed that these quenchers
are completely nonemissive, as excitation of solutions
containing the DNA-labeled quenchers at 100 nM concen-
tration showed no measurable emission above that of buffer
(see Supporting Information). Extinction coefficients were
determined at the absorbance maximum of each compound in
ethanol and can be found in the Supporting Information; note
that they differ slightly from the absorbance maxima found
when the quenchers are placed on DNA in Figure 2 and Table
1.
Quenching Efficiency. Quenching efficiency was deter-
mined by the use of DNA hybridization as a means of
controlling fluorophore−quencher distance (Figure 3).14 As
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a
Table 3. Mixed Mechanism Quenching Efficiencies of Fluorophore/Quencher Pairs
Alexa 350
FAM
Cy 3
TAMRA
Atto 590
Quasar 670
quencher
λem = 442 nm
λem = 517 nm
λem = 563 nm
λem = 580 nm
λem = 624 nm
λem = 662 nm
MPQ1
MPQ2
MPQ3
MPQ4
MPQ5
MPQ6
Dabcyl
BHQ2
66.4 ± 0.9%
66.6 ± 3.1%
66.0 ± 0.1%
62.2 ± 2.5%
58.7 ± 1.2%
64.8 ± 0.8%
70.4 ± 3.2%
71.6 ± 0.8%
90.7 ± 0.4%
89.1 ± 0.3%
89.1 ± 0.4%
84.1 ± 0.2%
77.4 ± 1.8%
87.8 ± 0.8%
87.1 ± 0.3%
91.9 ± 1.1%
85.6 ± 0.4%
84.0 ± 0.7%
83.5 ± 1.0%
85.1 ± 0.6%
80.2 ± 0.3%
92.9 ± 0.4%
77.8 ± 0.9%
90.8 ± 0.6%
79.7 ± 0.7%
79.6 ± 0.2%
77.9 ± 0.3%
79.1 ± 0.9%
75.5 ± 0.6%
80.6 ± 0.3%
64.3 ± 0.7%
81.2 ± 0.3%
93.6 ± 1.0%
91.3 ± 0.8%
89.5 ± 1.1%
92.6 ± 0.6%
90.0 ± 1.1%
95.2 ± 0.4%
80.7 ± 1.7%
95.4 ± 0.5%
90.2 ± 0.5%
91.1 ± 0.3%
78.7 ± 0.5%
82.8 ± 1.3%
85.9 ± 0.7%
93.5 ± 0.3%
66.5 ± 3.7%
94.6 ± 0.1%
a
Results are provided as the average of three experiments with standard deviation shown. See Figure 3b for structural context.
a
Table 4. FRET Quenching Efficiences of Fluorophore/Quencher Pairs
FAM
Cy 3
TAMRA
Atto 590
Quasar 670
quencher
MPQ1
λem = 517 nm
λem = 563 nm
λem = 580 nm
λem = 624 nm
λem = 662 nm
53.1 ± 1.4%
50.3 ± 0.2%
60.2 ± 1.3%
47.3 ± 1.4%
50.4 ± 0.9%
41.3 ± 0.9%
54.3 ± 1.0%
29.2 ± 0.7%
62.4 ± 0.7%
60.7 ± 2.2%
59.6 ± 0.9%
56.9 ± 1.9%
64.4 ± 1.2%
52.1 ± 2.8%
63.5 ± 1.8%
53.2 ± 1.2%
77.1 ± 0.5%
74.9 ± 1.4%
74.1 ± 0.4%
73.7 ± 0.6%
78.6 ± 0.4%
71.1 ± 0.8%
80.4 ± 1.2%
71.4 ± 1.1%
45.6 ± 1.5%
43.9 ± 0.8%
29.1 ± 0.9%
33.4 ± 2.1%
57.6 ± 0.6%
26.8 ± 1.9%
60.0 ± 1.3%
23.3 ± 1.4%
28.9 ± 0.8%
33.5 ± 1.2%
22.2 ± 0.5%
25.6 ± 2.9%
43.0 ± 0.5%
22.5 ± 3.1%
40.4 ± 2.8%
23.9 ± 1.5%
MPQ2
MPQ3
MPQ4
MPQ6
Dabcyl
BHQ2
No Quencher
a
Results are provided as the average of three experiments with standard deviation shown. See Figure 3c for structural context.
such, fluorophores were attached to the 3′ end of the sequence
5′-CCG-TAT-TAT-ATG-TTT-AAA-AA-3′ and quenchers were
attached to the 5′ end of complementary 15mer or 20mer
sequences. Use of a 20mer context (placing dye and quencher
immediately adjacent to one another at the duplex terminus)
allowed for analysis of contact quenching. The selected 15mer
sequence had been previously reported to allow for
examination of FRET quenching,14 but the observation of
ground state complex formation between quencher-labeled
15mers and fluorophore containing 20mers indicates a strong
degree of contact quenching as well, consistent with other
quencher studies and indicating a mixed mechanism of
quenching for the sequences shown in Figure 3b (see
Supporting Information).27 To allow for analysis of quenching
entirely by FRET, 5′ fluorophore labeled sequenches were used
(see Figure 3c), separating the quencher and fluorophore by 15
bp of duplex structure. Contact quenching data would be most
relevant for molecular applications where dye and quencher are
in very close proximity, whereas FRET data may be most
predictive of applications in which the two are held at some
distance. Since FRET efficiency depends on spectral overlap,
one expects that FRET quenching performance would be more
strongly dependent on absorption characteristics than contact
quenching, providing a better means of examining the newly
created quenchers.
Quenching efficiencies were measured as fraction (percent)
loss of emission intensity upon hybridization to the fluorophore
of interest, as displayed in Table 2 (contact quenching), Table
3 (mixed mechanism quenching), and Table 4 (FRET
quenching). We also compared the fluorescence ratios
(fluorescence in the unquenched state divided by fluorescence
in the quenched state) of the quenchers against each
fluorophore (Figure 3), which better illustrates the differences
among the dyes, and which is often the most important factor
in a fluorometric assay.
Contact and Mixed Mechanism Quenching. As antici-
pated, quenching of AlexaFluor 350 (λ em = 442 nm) is poor for
the MPQ series (Figure 3a), with dabcyl having a greater
quenching efficiency (75.7% contact, 70.4% FRET) than those
prepared and tested. This is consistent with the fact that dabcyl
is the most blue-shifted quencher of those assayed and has the
greatest spectral overlap with AlexaFluor 350. However, the
quenchers of the MPQ series (particularly MPQ3) nearly equal
dabcyl in contact quenching of this dye. BHQ2 is slightly
superior to either of these quenchers in performance with this
blue coumarin fluorophore.
The data show enhanced performance of the red-shifted
MPQ quenchers as the emission wavelength of the fluorophore
increases. For fluorescein (λ em = 517 nm, Figure 4b), it is
readily observed that MPQ3 is as efficient as even BHQ2 in
contact quenching, while four of the novel quenchers (MPQ1,
MPQ2, MPQ3, and MPQ6) all display superior quenching
abilities in a mixed mechanism context. At the emission of Cy3
(λem = 563 nm), only MPQ5 performs less efficiently than
dabcyl in terms of contact quenching, with all others displaying
superior performance to the common quencher. These results
begin to show the red-shifted properties of the new quenchers
created, as the parent dabcyl is no longer able to efficiently
quench Cy3 emission, while the new dyes display considerably
better quenching properties. Moreover, MPQ6 is particularly
intriguing, as it is equals (contact quenching) or even surpasses
(mixed mechanism quenching) BHQ2 in quenching of Cy3
emission.
As emission shifts further red to TAMRA (λ em = 580 nm), all
MPQ quenchers display markedly greater quenching efficiency
than dabcyl (Figure 4d). We note that TAMRA (unique among
the fluorophores tested here, as it is poorly quenched in
general14) is relatively poorly quenched by all quenchers
including dabcyl and BHQ2, an effect observed previously.
Moreover, MPQ4 is as good at contact quenching of TAMRA
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Figure 4. Fluorescence quenching ratios for contact (blue) and mixed mechanism (red) quenching with (a) AlexaFluor 350, (b) Fluorescein, (c) Cy
3, (d) TAMRA, (e) ATTO 590, and (f) Quasar 670.
as even BHQ2, while all but MPQ3 and MPQ5 are
approximately as efficient in mixed mechanism quenching as
BHQ2. Again, MPQ6 offers promising results, as it displays
comparable quenching properties to BHQ2. At the emission of
Atto 590 (λem = 624 nm), all quenchers still perform markedly
better than the parent dabcyl (Figure 4e), with MPQ5 again
serving as the single exception. Although not operating by an
entirely FRET mechanism, the advantages of the red-shifted
absorbance properties become clearer as the relatively small
five-base increase in length between quencher and fluorophore
greatly affects the quenching ability of dabcyl with considerably
smaller effect on the ability of the MPQs to quench the
emission of Atto 590. Quasar 670 (a Cy5 variant, λem = 662
nm) was the furthest-red emitting fluorophore tested. At this
wavelength, the limits of certain MPQs begin to become
evident, although contact quenching overall remains highly
efficient (up to 97%; see Tables 2 and 3 and Figure 4f).
FRET Quenching. To properly assess quenching based
entirely on a FRET mechanism, hybridization of 5′-quencher
labeled sequenches to 5′-fluorophore labeled sequences was
carried out (Figure 3c).27 As a result of the poor spectral
overlap of all new quenchers with the emission of the short-
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Bioconjugate Chemistry
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wavelength AlexaFluor 350 (λ em = 442 nm), FRET quenching
of this fluorophore was not examined. Similarly, with the poor
quenching efficiency of MPQ5, particularly in mixed mecha-
nism quenching studies (Table 2) and the low extinction
coefficient of this compound relative to the others synthesized,
the quencher was not used in FRET quenching studies.
FRET quenching studies were complicated by the inherent
nucleobase quenching by the 3′-dG on the quencher-containing
strand. To account for this, an oligonucleotide sequence lacking
a 5′ quencher was synthesized and served as a control for
quenching due only to the 3′-dG (Table 4). Although such
quenching was negligible for fluorescein, Atto 590, and Quasar
670, the 3′-dG was found to strongly quench both Cy3 and
TAMRA emission (Table 4). Quenching of TAMRA by
guanosine is well-known and unsurprising,28,29 but quenching
of Cy3 in this context was unexpected, as previous studies had
indicated an activation of Cy3 fluorescence by guanine.14,28
In general terms, the FRET results (Table 4) confirm the
expanded quenching properties of the new dyes, with all new
quenchers performing better than dabcyl for all fluorophores
tested. MPQ1 and MPQ2 were even capable of quenching Atto
590 to a moderate degree, while the parent dabcyl showed, as
expected, negligible quenching of the red-emitting dye,
indicating the ability to red-shift the absorbance properties
and quenching abilities. MPQ6 again provides superior results,
as it was able to quench all fluorophores as well as or even
better than BHQ2 in an entirely FRET context. Whereas
BHQ1 (the parent compound of MPQ6) is a poor FRET
quencher of red-emitting fluorophores such as Quasar 670 and
Atto 590 as a result of negligible spectral overlap,27
incorporation of the dihydroperimidine subunit in MPQ6
gives rise to a greatly broadened absorbance spectrum and
results in a compound with FRET quenching efficiency better
than BHQ2, as in the case of Quasar 670.
new quencher (MPQ6) that also displays quenching efficiency
over a wide wavelength range.
All five quenchers derived from the dabcyl structure (MPQs
1−5) display significantly enhanced quenching of all
fluorophores emitting beyond fluorescein, in both contact
and FRET quenching. As such, they should be considered as
excellent alternatives to dabcyl in any assay that relies on
quenching of a fluorophore emitting above approximately 520
nm and even as far as 624 nm, as shown by the ability of MPQ1
and MPQ2 to effectively quench the emission of Atto 590 in
the FRET mode. MPQ3 appears to be an excellent quencher
specifically for fluorescein, as it performed better than other
quenchers for this fluorophore in both contact and FRET
quenching. Further, MPQ6 uniquely serves as a competitive
alternative to BHQ2 for both contact and FRET quenching of
all fluorophores used in this study, particularly those emitting
between approximately 560 and 670 nm. As the FRET
quenching of this compound is approximately equal to or
greater than BHQ2 for Fluorescein, Cy3, TAMRA, Atto 590,
and Quasar 670, it may have its greatest utility in FRET-based
fluorescence assays.
To our knowledge, the only previous similar approach in
quencher design (i.e., taking advantage of increased electronic
complexity) employed an azulene dimer, but was limited to
quenching only in the near-IR region with limited efficiency.30
An alternative design utilizing an azaphthalocyanine structure
displayed a broadened absorbance spectrum, but has only been
fully assayed to date in quenching of FAM and Cy5.27
Experiments further displayed the ability of the azophthalocya-
nine dye to quench a larger set of fluorophores (ranging in
emission from 517 nm to 703 nm), but only in a contact
quenching setting, without exploration of FRET quenching of
the expanded set of fluorophores.31 The use of the perimidine
scaffold has been shown before to proffer a red-shift to
fluorescent dyes,32 but the current work is the first case of the
use of perimidine for altering the properties of fluorescence
quenchers.
One interesting aspect of the spectral properties of some the
new Multi-Path Quenchers is their environmental sensitivity.
MPQs 1−5 undergo moderate to significant changes upon a
change in nearly DNA sequence. For all but MPQ4, this is
likely the result of the incorporation of the naphthalimide core,
which is well-known to be environmentally sensitive itself.22,33
In addition, the 4-aminonaphthalimide core has been employed
in DNA sensors due to its inherent environmental sensitivity,
providing a further explanation for the changes observed here.34
As use of the 4-aminonaphthalimide core has shown environ-
mental dependence in different DNA settings, it is likely that
such is the same explanation for the derived quenchers, but a
further exploration is necessary to allow for an accurate
conclusion of the mechanism creating the different absorbance
spectra for MPQs 1−5. It seems possible, however, that one
might in the future take advantage of this property in the design
of “smart quenchers”, where the environment of the quencher
could be part of the design, either blue-shifting or red-shifting
the absorbance spectrum in response to structure or changes in
environment. Further investigation is needed to explore this
possibility.
DISCUSSION
■
Overall, the data show that several MPQ dyes outperform their
parent dye (dabcyl) in performance in a contact quenching
format with all of the fluorophores tested except the most blue-
shifted fluorophore (Alexa 350). As for quenching by the FRET
mechanism, which is most relevant to the present electronic
design concept, all of the MPQ quenchers perform better than
dabcyl with all of the fluorophores. This confirms that
broadening of the absorbance spectrum of the quencher can
confer superior performance. While most of the new dyes do
not exceed the quenching ability of the advanced BHQ2
compound, a number of MPQ quenchers equal this quencher's
performance with specific fluorescent labels, and thus could be
considered as serious alternatives to this molecule.
The chief intent of the molecular design of MPQs was to
broaden the efficient quenching range of the parent quenchers.
The data show that this approach is successful: several of the
MPQs give >95% contact quenching and excellent FRET
quenching of fluorophores as far to the blue as fluorescein and
as far red as Quasar 670, while the parent dabcyl compound
quenches none of the five fluorophores significantly in the
FRET mode and only one better than 95% in the contact
mode. Thus, we conclude that incorporation of the
naphthalimide core does result in the desired increase in
generality and a greater range of fluorescence quenching
beyond that of dabcyl. Moreover, incorporation of the
dihydroperimidine moiety into the BHQ1 scaffold affords a
ASSOCIATED CONTENT
* Supporting Information
■
S
NMR spectra of quencher dyes and intermediates, HPLC
purity and MALDI-TOF of quencher and fluorophore
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donor-acceptor energy transfer. U.S. Patent 7,019,129, March 28,
2006.
substituted DNAs, and additional spectral data. This material is
available free of charge via the Internet at http://pubs.acs.org.
(17) Haugland, R. P., Singer, V. L., Yue, S. L. Xanthene dyes and
their application as luminescence quenching compounds. U.S. Patent
6,399,392, March 4, 2002.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: kool@stanford.edu. Tel.: 650-724-4741. Fax: 650-725-
0295.
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ACKNOWLEDGMENTS
■
This work was supported by the National Institutes of Health
(GM068122 and GM067201).
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