Detection of cellular sialic acid content using nitrobenzoxadiazole carbonyl-reactive chromophores

Article abstract of DOI:10.1021/bc200276k

The selective ligation of hydrazine and amino-oxy compounds with carbonyls has gained popularity as a detection strategy with the recognition of aniline catalysis as a way to accelerate the labeling reaction in water. Aldehydes are a convenient functional group choice since there are few native aldehydes found at the cell surface. Aldehydes can be selectively introduced into sialic acid containing glycoproteins by treatment with dilute sodium periodate. Thus, the combination of periodate oxidation with aniline-catalyzed ligation (PAL) has become a viable method for detection of glycoconjugates on live cells. Herein we examine two fluorescent nitrobenzoxadiazole dyes for labeling of glycoproteins and cell surface glycoconjugates. We introduce a novel 4-aminooxy-7-nitro-benz- [2,1,3-d]-oxadiazole (NBDAO) (5) fluorophore, and offer a comparison to commercial dyes including the known 4-hydrazino-7-nitro-benz-[2,1,3-d]- oxadiazole (NBDH) (2) and Bodipy FL hydrazide. We confirm specificity for sialic acid moieties and that both dyes are suitable for in vitro and in vivo labeling studies using PAL and fluorescence spectroscopy. The dyes examined here are attractive labeling agents for microscopy, as they can be excited by a 488 nm laser line and can be made in a few synthetic steps. These carbonyl-reactive chromophores provide a one step alternative to avidin-biotin labeling strategies and simplify the detection of sialic acid in cells and glycoproteins.

Full text of DOI:10.1021/bc200276k

Article
pubs.acs.org/bc
Detection of Cellular Sialic Acid Content Using Nitrobenzoxadiazole
Carbonyl-Reactive Chromophores
Jessie A. Key, Caishun Li, and Christopher W. Cairo*
Alberta Glycomics Centre, Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada
S
* Supporting Information
ABSTRACT: The selective ligation of hydrazine and amino-oxy compounds with carbonyls has gained popularity as a detection
strategy with the recognition of aniline catalysis as a way to accelerate the labeling reaction in water. Aldehydes are a convenient
functional group choice since there are few native aldehydes found at the cell surface. Aldehydes can be selectively introduced
into sialic acid containing glycoproteins by treatment with dilute sodium periodate. Thus, the combination of periodate oxidation
with aniline-catalyzed ligation (PAL) has become a viable method for detection of glycoconjugates on live cells. Herein we
examine two fluorescent nitrobenzoxadiazole dyes for labeling of glycoproteins and cell surface glycoconjugates. We introduce a
novel 4-aminooxy-7-nitro-benz-[2,1,3-d]-oxadiazole (NBDAO) (5) fluorophore, and offer a comparison to commercial dyes
including the known 4-hydrazino-7-nitro-benz-[2,1,3-d]-oxadiazole (NBDH) (2) and Bodipy FL hydrazide. We confirm
specificity for sialic acid moieties and that both dyes are suitable for in vitro and in vivo labeling studies using PAL and
fluorescence spectroscopy. The dyes examined here are attractive labeling agents for microscopy, as they can be excited by a 488
nm laser line and can be made in a few synthetic steps. These carbonyl-reactive chromophores provide a one step alternative to
avidin−biotin labeling strategies and simplify the detection of sialic acid in cells and glycoproteins.
groups that provide sensitive detection;13 however, this strategy
can suffer from incomplete label incorporation. Lectins can be
INTRODUCTION
Sensitive detection of biomolecules by derivatization with
■
large proteins which may suffer from relatively low affinities.
fluorescent dyes is a critical technique used in histology, cell
biology, and biochemistry.1−3 Specific detection of conjugates
Importantly, many lectins or antibodies can perturb cells by
cross-linking receptorslimiting their application in live
relies on chemoselective ligation of functional groups found on
cells.14,15 Selective chemical modification of sialic acids using
PAL presents an attractive alternative.8 However, previous
reports of PAL on cells have relied upon biotin-amino-oxy
conjugates which were then detected by fluorescently labeled
streptavidin. This two-step method required the use of a 60
kDa tetrameric protein for labeling a single carbohydrate
residue (ca. 300 Da).7,16 Such a bulky label could mask other
epitopes on the same cell or macromolecule, and may have
reduced labeling capacity due to molecular crowding.
the biomolecule and the label.4 As a result, strategies to
introduce reactive functional groups into biomolecules have
been developed. Typical strategies include the metabolic
incorporation of synthetic labels and chemical modification of
native biomolecules.4,5 A well-known strategy for labeling of
glycoproteins is the use of sodium periodate to oxidize the
glycerol side-chain of sialic acid to an aldehyde.6 While this
method is more commonly used in vitro, it has recently been
extended to the labeling of glycans in live cells.7,8 Live cell
labeling requires mild conditions and near-quantitative
reactions, which has been achieved by performing the oxidation
reaction at low temperature, and accelerating the conjugation
reaction with an aniline catalyst.8 This method of periodate
oxidation and aniline-catalyzed ligation (PAL) can then be used
to label glycoconjugates for microscopy.
Small molecule fluorophores which contain either hydrazine
or amino-oxy functional groups can be applied to bioorthog-
onal and PAL labeling strategies.17−19 7-Nitro-benz-[2,1,3-d]-
oxadiazoles (NBDs) are a class of small, synthetically accessible,
and environmentally sensitive fluorophores which can be easily
modified for use with PAL. In general, NBDs possess relatively
high fluorescence quantum yields (0.3 for primary amine
Sialic acids are a class of carbohydrates which have important
biological functions in embryogenesis, development, and
immune response.9−11 Methods of detecting sialylation include
metabolic labeling, lectins, and chemical modification.12
Metabolic labeling allows the introduction of unique functional
Received: May 31, 2011
Revised: January 17, 2012
Published: January 30, 2012
© 2012 American Chemical Society
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Bioconjugate Chemistry
Article
derivatives of NBDCl)2 and long excitation and emission
wavelengths when compared to fluorophores of similar size,
such as coumarins.20 Interestingly, the attachment of a
hydrazine functional group to these dyes results in fluorogenic
derivatives which allow direct detection of carbonyl derivatives
upon formation of the corresponding hydrazone. Reported
fluorogenic and fluorescent hydrazino-NBD derivatives include
7-hydrazino-4-nitrobenzo-2-oxa-1,3,-diazole (NBDH),21 4-
(N,N-dimethylaminosulfonyl)-7-hydrazino-benz-2,1,3-oxadia-
zole (DBDH),22 4,4-aminosulfonyl-7-hydrazino-2,1,3-benzoxa-
diazole (ABDH),22 4-(N,N-dimethylaminosulfonyl)-7-N-meth-
ylhydrazino-benz-2,1,3-oxadiazole (MDBDH),23 4-[2-(N,N-
dimethylamino)ethylaminosulfonyl]-7-N-methylhydrazino-
benz-2,1,3-oxadiazole (DAABD-MHz),24 and N-methyl-4-
hydrazino-7-nitrobenzofurazan (MNBDH).25 Several of these
derivatives are commercially available and have found
application in biological assays,26 as well as the detection of
trace carbonyls in air samples such as automobile exhaust.23
We considered that small molecule labels compatible with
PAL should be of interest for fluorescence microscopy and
quantitation of glycoconjugates. To take advantage of the PAL
strategy, the fluorophore should contain a nucleophilic group
that will react with an aldehyde rapidly in water. Although
hydrazines have been explored for this purpose, we chose to
investigate an amino-oxy-containing fluorophore.8 Herein we
describe the synthesis of a novel amino-oxy fluorophore,
NBDAO (5), which has improved sensitivity over existing dyes
for selective labeling of sialic acid glycoconjugates on live cells.
We contrast the properties of NBDAO to a known carbonyl
reactive fluorophore, NBDH (2).21 We first observed labeling
of cells with a fluorescence microplate reader. Additionally,
NBDAO was used to label a glycoprotein in conjunction with
PAL, followed by fluorescence detection in SDS-PAGE. Finally,
we compared the performance of NBDAO to commercially
available hydrazine dyes NBDH and Bodipy FL hydrazide in
fluorescence microscopy and flow cytometry assays. We
conclude that these dyes can be used as an effective one-step
live-cell labeling strategy for glycoconjugates, and that NBDAO
provides the best level of sensitivity among the dyes tested.
Chloro-7-nitrobenz-[2,1,3-d]-oxadiazole (1) (100 mg, 0.5
mmol, 1 equiv) was dissolved in chloroform (50 mL). A 1%
hydrazine solution (0.77 mL hydrazine in 50 mL methanol)
was then added to the solution and allowed to stir at room
temperature for 1 h. A yellow-brown precipitate was formed
1
and isolated without further purification (104 mg, quant.) H
NMR (400 MHz, D2O): δ 7.04 (d, 1H, 3J = 10.5 Hz), 6.37 (d,
1H, 3J = 10.5 Hz); 13C NMR (100 MHz, D2O): δ 147.6, 145.4,
131.4, 121.9, 120.8, 115.1; IR (microscope): ν = 3378, 3278,
3191, 3051, 2984, 2692, 1618, 1542, 1500, 1465, 1213, 1019,
982, 873, 794 cm−1. ES-HRMS calculated for C5H5N5O3Na:
218.0285, observed: 218.0284.
4-(2-Benzylidenehydrazinyl)-7-nitrobenz-[2,1,3-d]-oxadia-
zole (3). 4-Hydrazinyl-7-nitrobenz-[2,1,3-d]-oxadiazole (2) (25
mg, 0.13 mmol, 1 equiv) was dissolved in methanol (10 mL).
Benzaldehyde (0.13 mL, 1.3 mmol, 10 equiv) was added,
turning the reaction mixture a red color. The product gradually
formed as a dark red-black precipitate and was isolated by
vacuum filtration. Additional product was isolated from the
supernatant after flash column chromatography (EtOAc/
hexanes). The precipitate and column fractions were combined
(27 mg, 74%). 1H NMR (400 MHz, (CD3)2CO): 8.62 (d, 1H,
3J = 8.8 Hz), 8.59 (s, 1H), 7.92−7.79 (m, 2H), 7.56−7.39 (m,
3H), 7.31 (d, 1H, 3J = 8.8 Hz); 13C NMR (100 MHz,
(CD3)2CO): δ 149.9, 145.2, 144.1, 141.0, 137.0, 134.9, 131.6,
129.8, 128.4, 126.0, 102.7; IR (microscope): ν = 3486, 3307,
3219, 3144, 3060, 2955, 1603, 1581, 1515, 1446, 1406, 1295,
1117, 997 cm−1. ES-HRMS calculated for C13H10N5O3:
284.0778, observed: 284.0778. Rf = 0.23 (1:3 EtOAc/hexanes).
Ethyl N-7-nitrobenz-[2,1,3-d]-oxadiazol-4-yloxyacetimi-
date (4). Ethyl N-hydroxyacetimidate (35 mg, 0.34 mmol, 3
equiv) was dissolved in H2O (2 mL). Sodium carbonate (100
mg, 0.94 mmol, 7.5 equiv) was added and the reaction mixture
was allowed to stir for 10 min. 4-Chloro-7-nitrobenz-[2,1,3-d]-
oxadiazole (1) (25 mg, 0.125 mmol, 1 equiv) was then added
and allowed to stir at room temperature for approximately 15
min. The resulting brown-yellow precipitate was isolated by
vacuum filtration without further purification (35 mg, quant.).
1H NMR (300 MHz, CDCl3): δ 8.54 (d, 1H, 3J = 8.4 Hz), 7.25
3
3
(d, 1H, J = 8.4 Hz), 4.27 (q, 2H, J = 6.9 Hz), 2.32 (s, 3H),
1.42 (t, 3H, J = 6.9 Hz); 13C NMR (100 MHz, CDCl3): δ
3
METHODS
■
170.2, 154.3, 144.2, 143.9, 134.6, 129.7, 106.2, 64.4, 15.3, 14.5.
IR (microscope): ν = 3133, 3090, 2999, 2988, 1933, 1713,
1645, 1623, 1537, 1451, 1379, 1164, 997, 854 cm−1. ES-HRMS
calculated for C10H10N4O5Na: 289.0543, observed: 289.0541.
Rf = 0.69 (1:3 EtOAc/hexanes).
General Experimental Methods. Reagents were pur-
chased from commercial sources such as Sigma-Aldrich
(Oakville, Ont) and used without additional purification.
Proton (1H) and carbon (13C) NMR spectra were obtained
on Varian 300, 400, 500, or 700 MHz instruments at room
temperature as noted. Deuterated solvents were obtained from
Cambridge Isotope Laboratories (Andover, MA). Mass
spectrometry was performed using an MS50G positive electron
impact instrument from Kratos Analytical (Manchester, UK)
and a Mariner Biospectrometry positive ion electrospray
instrument from Applied Biosystems (Foster City, CA).
Spectroscopy. Absorbance spectra for all compounds were
collected at room temperature with a Hewlett-Packard (Palo
Alto, CA) model 8453 diode array UV−visible spectropho-
tometer or Varian (Walnut Creek, CA) Cary 50 spectropho-
tometer. Fluorescence spectra for all compounds were collected
at room temperature with a Photon Technology International
model MP1 steady-state fluorimeter. Absorbance and fluo-
rescence measurements were taken using NSG Precision Cells
ES quartz cuvettes (190−2000 nm; Farmingdale, NY).
O-(7-Nitrobenzo-[2,1,3-d]-oxadiazol-4-yl)hydroxylamine
(5, NBDAO). Ethyl N-7-nitrobenz-[2,1,3-d]-oxadiazol-4-yloxya-
cetimidate (4) (250 mg, 0.94 mmol, 1 equiv) was dissolved in
1:1 TFA/water (25 mL) and allowed to stir at room
temperature for 2 h. The TFA was removed in vacuo, and
the resulting aqueous solution was concentrated by lyophiliza-
tion. The product was isolated by flash column chromatography
1
with a dichloromethane mobile phase (94 mg, 55%). H NMR
(700 MHz, CDCl3): δ 8.56 (d, 1H, 3J = 8.4 Hz), 7.40 (d, 1H, 3J
= 8.4 Hz), 6.51 (s, 2H). 13C NMR (175 MHz, CDCl3): δ 156.0,
143.9, 143.5, 134.0, 130.1, 105.7; IR (microscope): ν = 3325,
3255, 3167, 3104, 2918, 2851, 1641, 1588, 1537, 1328, 1083,
997, 729 cm−1. ES-HRMS calculated for C6H4N4O4Na:
219.0125, observed: 219.0127. Rf = 0.25 (1:3 EtOAc/hexanes).
Butan-2-one O-7-nitrobenzo-[2,1,3-d]-oxadiazol-4-yl
oxime (6). O-(7-Nitrobenz-[2,1,3-d]-oxadiazol-4-yl)-
hydroxylamine (5) (27 mg, 0.14 mmol, 1 equiv) was dissolved
Synthesis of Dyes and Model Conjugates. 4-
Hydrazinyl-7-nitrobenz-[2,1,3-d]-oxadiazole (2, NBDH). 4-
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Article
Scheme 1. Synthesis of NBDH (2) and NBDAO (5) Carbonyl Reactive Chromophores and Model Hydrazone and Oxime
Ligation Products (3 and 6)
in methylethylketone (MEK) (0.08 mL, 0.83 mmol, 6 equiv).
The reaction was allowed to run at room temperature for 2 h.
Excess MEK was removed in vacuo and the remaining residue
was purified as an inseparable mixture of E/Z isomers (1.36:1)
using flash column chromatography (EtOAc/hexanes) (15 mg,
in a humidified incubator. The growth media was removed and
the plates were washed with Dulbecco’s phosphate buffered
saline (DPBS). Wells were then treated with sodium periodate
(80 μL, 1 mM) or DPBS buffer control (80 μL) for 30 min at 4
°C. The solution was removed by inversion, and the plates were
washed with DPBS (2 × 100 μL). Control and sample wells
were treated with dye for 1 h at 4 °C (NBDH, 2.50 × 10−5 M;
NBDAO, 2.5 × 10−5 M; or Bodipy-FL hydrazide, 2.50 × 10−5
M). Dye solutions were prepared in 1 mL aliquots with or
without aniline (4.38 × 10−4 M) in DPBS (pH 7.4) or 0.1 M
sodium acetate buffer (pH 4.7) as indicated. Fluorescence
measurements were taken on a Molecular Devices M2e plate
reader (Sunnyvale, CA).
1
3
44%). H NMR (400 MHz, CDCl3): δ 8.56 (d, 1H, J = 9.3
3
3
Hz), 7.35 (d, 1H, J = 9.6 Hz), 2.73 (q, 0.90H, J = 7.8 Hz),
3
2.48 (q, 1.29H, J = 7.5 Hz), 2.25 (s, 1.88H), 2.14 (s, 1.38H),
1.26 (m, 5.23H); 13C NMR (125 MHz, CDCl3): δ 169.7, 168.8,
153.98, 153.92, 144.0, 143.8, 134.4, 129.8, 106.6, 29.8, 24.0,
19.3, 15.5, 10.4; IR (microscope): ν = 3113, 3092, 2987, 2987,
2919, 2849, 1633, 1533, 1453, 1369, 1327, 887, 854, 733 cm−1.
ES-HRMS calculated for C10H11N4O4: 251.0775, observed:
251.0778. Rf = 0.67 (1:1 EtOAc/hexanes).
Fluorescence Microscopy. HeLa cells were added to a 6-
well plate (∼10 000 cells/well) containing treated coverglass
(1% (m/v) BSA in TBS), and grown to approximately 60%
confluence. The growth media was removed and the wells were
washed with DPBS. Sample wells received sodium periodate in
DPBS (1 mL, 1 mM) treatment; control wells received DPBS
(2 mL); samples were then incubated for 30 min at 4 °C. Cells
can optionally be fixed at this point by washing with sodium
acetate buffer (0.1 M, pH 4.7; 2 × 2 mL), incubation for 10 min
in a 1:1 solution of sodium acetate buffer and methanol, and
incubation for 10 min in methanol. Fixing solution is then
removed, and the samples are washed with sodium acetate
buffer (2 × 2 mL). The double negative control was treated
with buffer solution, while control and experimental wells were
then treated with dye for 0.5 to 1 h at 4 °C (NBDH, 2.50 ×
10−5 M; NBDAO, 2.50 × 10−5 M; or Bodipy-FL hydrazide,
2.50 × 10−5 M). Dye solutions were prepared in 1 mL aliquots
with aniline (4.38 × 10−4 M) in sodium acetate buffer (0.1 M,
pH 4.7). Each treatment was observed in triplicate. Cells were
washed with buffer after staining, and then visualized using a
Nikon Eclipse Ti inverted fluorescence microscope with 60×
objective (NA 1.49). Images were acquired with a Photometrics
QuantEM 512SC camera.
Flow Cytometry. Jurkat cells were grown in RPMI 1640
media (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum
(FBS) at 37 °C under 5% CO2 to approximately 1 × 106 cells/
mL. The growth media was removed and the cells were washed
with DPBS. Control cell aliquots were incubated in DPBS for 1
h at 37 °C. Sialidase-treated cell aliquots were incubated at 37
°C in the presence of Clostridium perfringens neuraminidase
(0.4 mg/mL; Sigma-Aldrich, St. Louis, MO). Cells were
pelleted by centrifugation (2000 g × 4 min) and washed with
DPBS. Oxidation-treated aliquots received sodium periodate in
Reaction Kinetics. Dye solutions of NBDH (6.88 × 10−5
M) or NBDAO (2.50 × 10−5 M) were prepared in 1 mL
aliquots with or without aniline (4.38 × 10−4 M) in buffer (0.1
M sodium acetate, pH 4.7; or 0.1 M HEPES, pH 7.4). An
aliquot of MEK (10 μL, 1.11 × 10−4 M) was added and the
fluorescence emission of the reaction was monitored for 1500 s
(ex. 480 nm; em. 540 nm).
Glycoprotein Labeling. Fetal calf serum fetuin (1 mg/mL,
0.5 mL aliquots) was treated with 1 mL aliquots of
neuraminidase from Clostriduim perfringens (C. Welchii) (0.1
mg/mL) in sodium acetate buffer (0.1 M, pH 5.4) for 0.5 h at
37 °C. Sodium acetate buffer was removed by ultrafiltration
(Millipore, Billerica, MA) for 15 min at 1600 g, followed by a
water wash (1 mL). Oxidation was performed by treatment
with sodium periodate (300 μL, 0.1 M) for 30 min at 4 °C.
Excess periodate was removed by ultrafiltration for 15 min at
1600 g, followed by a water wash (1 mL). Aliquots of the
protein (20 or 10 μL) were then treated with a labeling solution
(20 μL) consisting of NBDAO (1.25 × 10−5 M) and aniline
(4.38 × 10−4 M) in sodium acetate buffer (0.1 M, pH 4.7) for
0.5−1 h at 4 °C. Labeled proteins were resolved by SDS PAGE
(stacking 4%, resolving 12%). The resulting gels were imaged
using a FujiFilm Fluorescent Image Analyzer FLA-5000
(Fujifilm medical systems USA Inc., Stamford, CT) with 473
nm excitation and a low band-pass filter. Coomassie staining
was used to detect molecular weight standards.
Microplate Assay. HeLa cells were grown in Dulbecco’s
modified Eagle medium (DMEM) with 10% fetal bovine serum
(FBS) at 37 °C under 5% CO2 to approximately 80%
confluency. Cells were trypsinized, and then added to 96 well
plates (∼2000 cells/well) (tissue culture treated; Corning,
Corning, NY). The plate was then incubated for 96 h at 37 °C
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Table 1. Photophysical Properties of NBDH, NBDAO, Hydrazone, and Oxime Conjugates
a
b
c
compound
solvent
λ maxabs [nm]
λ maxem [nm]
ΦF (R6G)
ε absorption coefficient [cm−1 M−1
]
brightness
NBDH (2)
aq. pH 7.4
aq. pH 5.4
aq. pH 4.7
CH3CN
EtOH
271, 374, 494
271, 372, 496
377
na
na
na
na
552
na
0.001
na
14800
2950
10
na
280, 405, 425
334, 470
531, 548
537
na
0.003
0.280
na
56700
110500
67900
52100
630
170
31547
34
hydrazone (3)
aq. pH 7.4
aq. pH 5.4
aq. pH 4.7
CH3CN
EtOH
360, 444
356, 440, 495
379, 506
552
598
555
555
552
541
540
566
538
545
0.006
0.004
0.026
0.038
0.006
0.133
0.054
0.003
0.138
0.160
320
2.5
306, 323, 492
307, 327, 493
291, 381, 470
279, 376, 466
275, 376, 472
390, 465
98000
25600
8000
2501
977
46
NBDAO (5)
oxime (6)
aq. pH 4.7
CH3CN
EtOH
14000
10200
16700
15600
7900
1863
548
42
aq. pH 4.7
CH3CN
EtOH
264, 380, 470
270, 378, 467
2150
1263
a
b
Absorbance maxima are listed with the peak used for fluorescence excitation underlined. Fluorescence quantum yield (ΦF) standard was
c
rhodamine 6G in ethanol measured with excitation at 485 nm. Molar absorption coefficient, ε, measured at 485 nm.
Figure 1. Absorbance and emission spectra in ethanol (excitation 475 nm) of (a) NBDH (2), (b) hydrazone (3), (c) NBDAO (5), and (d) oxime
(6).
DPBS (1 mL, 1 mM); nonoxidized aliquots received DPBS (1
mL). Samples were then incubated for 30−45 min at 4 °C.
Cells were again pelleted by centrifugation (2000 g × 4 min)
and washed three times with DPBS. Unlabeled control aliquots
were treated with buffer solution, while a set of nonoxidized
and oxidized aliquots were treated with dye (100 μL) for 1−1.5
h at 4 °C (NBDAO, 2.50 × 10−5 M; Bodipy-FL hydrazide 2.50
× 10−5 M (Invitrogen, Carlsbad, CA); NBDH 2.50 × 10−5 M).
Dye solutions were prepared in 1 mL aliquots with aniline
(4.38 × 10−4 M) in sodium acetate buffer (0.1 M, pH 4.7).
Cells were pelleted by centrifugation (2000 g × 4 min) and
washed three times with DPBS before flow cytometry. Flow
cytometry was performed on an Accuri C6 cytometer (Ann
Arbor, MI) with 488 nm excitation and 533 nm detection. Each
treatment was observed in triplicate runs with 10 000 events.
RESULTS AND DISCUSSION
■
Fluorophore Synthesis. NBDH (2) was synthesized from
commercially available 7-nitro-4-chlorobenz-2,1,3-oxadiazole
(NBDCl) (1) in chloroform by reaction with 1% hydrazine
as described by Gubitz et al.21 Alternatively, the
NBDH·NH2NH2 salt may be prepared from 7-nitro-4-
fluorobenz-2,1,3-oxadiazole using the method of Uzu and
colleagues.22 A hydrazone derivative, 3, was then generated in
good yield by conjugation with benzaldehyde in methanol at
room temperature (Scheme 1). NBDAO (5) was synthesized
by an initial SNAr of NBDCl (1) with ethyl N-hydroxyaceti-
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Article
Figure 2. Reaction kinetics of (a) NBDH (2) and (b) NBDAO (5) with MEK at pH 4.7. The rates of both reactions are enhanced by the presence
of aniline in the reaction mixture. The excitation wavelength was 485 nm, and emission was monitored at 540 nm for 1500 s.
midate under basic conditions, to generate the ethyl
acetohydroxamate intermediate (4).27 The acetimidate func-
tions as a protected amino-oxy group, and reported
deprotection protocols for aryl substrates involve the use of
strong acids, such as perchloric acid and hydrofluoric acid.27,28
We found that the amino-oxy functional group could be
unveiled in moderate yield by hydrolysis with aqueous
trifluoroacetic acid. Care must be taken at the deprotection
step to avoid alkaline workup conditions. In our hands, basic
solutions lead to decomposition of the amino-oxy compound,
presumably to the corresponding phenolate.29 Additionally, we
found that care should be taken to remove any trace amounts of
ketone or aldehyde contaminants (such as acetone) to avoid
the rapid formation of the oxime. A representative oxime
derivative (6) was generated by reaction with neat MEK at
room temperature.
Fluorophore Characterization. The NBD dyes (2 and 5)
and corresponding hydrazone (3) and oxime (6) conjugates
were characterized to determine their absorbance spectra,
fluorescence emission, molar absorption coefficients, and
fluorescence quantum yields (ΦF) (Table 1). Quantum yield
was determined using a Rhodamine 6G standard in ethanol.
Similar to other NBD derivatives, the photophysical properties
of these compounds were found to be environmentally
sensitive.3,30 Consistent with previous reports, we confirmed
conversion of 2 to 3 was fluorogenic with an 8.5-fold increase in
ΦF and a 15-fold increase in brightness in acetonitrile.
Additionally, we observed that NBDH derivatization was
fluorogenic in water, particularly at low pH. For example, at
pH 5.4 the hydrazone (3) has a 9.2-fold increase in ΦF and 32-
fold increase in brightness relative to the starting hydrazine (2).
However, NBDH and its hydrazone derivative shows less
fluorescence in water relative to ethanol and acetonitrile
solutions.
others. However, we note that several of these values are close
to the lower limit of detection, and therefore may not be
accurate enough to make this determination. We observed that
NBDAO (5) underwent degradation to the corresponding
phenolate in the presence of alkaline water,29 which could
complicate its use in assays which require high pH. In labeling
experiments, we found that this degradation product could be
easily removed by performing a wash step after dye treatment.
Reaction Kinetics. The mechanism of oxime or hydrazone
formation by NBDAO and NBDH is expected to be accelerated
in the presence of aniline, which acts as a nucleophilic catalyst
in acidic media.31,32 Differences in the fluorescent properties of
these dyes and their conjugates could be used as a method to
observe the rate of the reaction directly. To confirm the role of
aniline in these reactions, we observed the kinetics of ligation
for NBDH (2) or NBDAO (5) with MEK in the presence or
absence of aniline using a time-based fluorescence assay (Figure
2). As expected, we found that aniline did increase the rate of
reaction for both dyes at pH 4.7. In the case of NBDH, the rate
acceleration was limited to the early phase of the reaction
(<400 s). In the case of the NBDAO dye, the rate acceleration
was more substantial, with limited formation of the oxime in
the absence of aniline. These findings suggest that NBDAO
may be a more selective labeling agent than NBDH when used
with aniline catalysis. At pH 7.4, we found that aniline did not
increase the rate of reaction for either dye.
Glycoprotein Labeling. To explore the utility of NBDAO
for the detection of glycoconjugates, we tested the ability of the
dyes to specifically label a known glycoprotein in SDS PAGE.
Fetal calf serum fetuin is a commonly used, commercially
available sialoprotein.33,34 Detection of fetuin was performed by
treatment with periodate followed by labeling with NBDAO in
the presence of aniline (Figure 3). Labeling of a band (∼64
kDa) corresponding to the protein was visible by fluorescence
imaging. To demonstrate the specificity of this methodology for
sialic acid, controls were performed using fetuin which was
pretreated with neuraminidase (NEU). As expected, the dye
only labeled the glycoprotein when sialic acid was present, and
only after periodate treatmentconfirming that the labeling
chemistry was specific for sialylated protein. We observed only
minor residual staining in control samples; and factors that may
contribute to background staining could include nonspecific
binding35 and naturally occurring protein oxidation.36
The derivatization of NBDAO (5) to an oxime (6) leads to
an increase in the absorption coefficient of the dye; however,
there is only a slight shift in the emission wavelength (Figure
1). Both NBDAO and its oxime conjugate (6) show a
remarkably increased Stokes shift relative to NBDH, which
should result in improved sensitivity. Both NBDAO and the
oxime derivative have reduced environmental sensitivity when
compared to compounds 2 and 3, respectively, though both
dyes (5 and 6) exhibited a smaller range of brightness among
water, ethanol, and acetonitrile as compared to the hydrazine
and hydrazone derivatives (2 and 3). The conversion of
NBDAO to the oxime appeared to be fluorogenic in certain
solvent environments (ethanol), but was relatively static in
Live Cell Labeling. We tested the ability of NBDH (2) and
NBDAO (5) to detect glycans on live cells using PAL. We
tested HeLa cells cultured in a microplate which were exposed
to periodate, washed, and then treated with one of the
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Figure 3. Fluorescent detection of glycoproteins in gel. Labeling of
fetal calf serum fetuin by PAL was performed with NBDAO (5).
Control lanes used protein which was treated with neuraminidase
(NEU), and protein not treated with periodate as indicated. Molecular
weights are given in kDa.
carbonyl-reactive dyes. Control wells lacked the periodate
oxidation step, but were otherwise identical. After labeling, the
fluorescence of treated cells was compared to that of the
control wells. We found that the mean fluorescence after PAL
with both dyes resulted in specific labeling when performed at
acidic pH. The NBDH dye gave an increase of 1.3-fold at pH
4.7, and the Bodipy FL hydrazide 3.0-fold, while NBDAO gave
a larger increase of 4.3-fold (Figure 4). Labeling by hydrazine
Figure 5. Fluorescence and brightfield images of HeLa cells labeled
with NBDH, Bodipy, and NBDAO at pH 4.7. For each dye, cells were
observed after one of the following treatments: no periodate and no
dye (--); no periodate and dye (-+); and periodate with dye (++).
Fluorescence images (488 nm ex/515 nm em) are shown on the left,
and DIC images of the same field are shown on the right. Scale bars
represent 50 μm. Dye labeling is shown for live and fixed cells (see
Materials and Methods).
treatments. We evaluated labeling conditions at pH 4.7, 5.4, 6.1,
and 7.4. In our hands, pH 4.7 gave the best results for dye
labeling. Fluorescence images of NBDH-treated cells showed
staining which was dependent on the use of both periodate and
dye. The Bodipy FL hydrazide dye showed higher background
than NBDH in samples not treated with periodate (-+), and
provided intense labeling of cells after periodate treatement (+
+). The NBDAO dye provided both low background in
samples not treated with periodate (-+), and bright staining of
cells treated with periodate (++). Additional controls
performed with compound 6 typically showed only background
staining (data not shown). Based on these results, we
concluded that NBDAO was more suited to fluorescence
imaging, and presents a significant improvement over NBDH
and Bodipy FL hydrazide. We also note that PAL labeling
protocols which exploit small-molecule fluorophores should
require fewer steps than affinity-based reagents.8 The data in
Figure 5 suggest that staining by all three dyes in live cells is not
restricted to the cell membrane. These results may indicate that
periodate oxidation and the dyes used here can penetrate to
intracellular compartments. Previous reports have observed
labeling of intracellular compartments by NBD-based dyes37,38
and protein conjugates.39
Flow Cytometry. To determine the applicability of small
molecule dyes for quantitation of cellular sialic acid content, we
examined labeling of Jurkat cells by flow cytometry (Figure 6).
Cells were treated with periodate, followed by a solution of dye
containing aniline. As with the experiments described above,
the oxidation and labeling step was performed at low
temperature. To compare the background staining, we included
samples that were treated without periodate or dye (--),
periodate and no dye (+-), no periodate and dye (-+), and both
periodate and dye (++). We tested NBDAO in the assay, and
compared it to NBDH and Bodipy FL hydrazide. The NBDAO
Figure 4. Fluorescence enhancement, expressed as fold enhancement
over negative control of 96 well plates after PAL treatment with
NBDH (2), Bodipy FL hydrazide, or NBDAO (5) at pH 4.7. Each
point represents at least 8 wells of a representative experiment; error is
shown as the standard deviation. All dyes were used at 2.50 × 10−5 M,
excitation was at 485 nm, and emission was observed at 538 nm.
and aminooxy dyes were similar to control when the labeling
was performed at pH 7.4 (data not shown).
Fluorescence Microscopy. In order to explore the utility
of these dyes for imaging applications, we tested their ability to
label glycoconjugates in fixed and live cells. HeLa cells were
treated with periodate, followed by a solution of dye containing
aniline. Both the oxidation and labeling steps were performed at
low temperature based on the protocol of Zeng et al.8 Cells
were then imaged using fluorescence microscopy and DIC
(Figure 5). To determine the background staining, we included
controls that were treated with no periodate and no dye (--), no
periodate and dye (-+), and periodate and dye (++). We
employed conditions for staining of live and fixed cells for all
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suggesting that this method may be useful to monitor
enzymatic changes to sialoproteins in vitro.
Detection strategies for sialic acid on cells and glycoproteins
currently include the use of lectins, metabolic labels, or
bioconjugation of dyes. In this study, we have examined the
utility of small molecule dyes that contain either hydrazine or
amino-oxy functional groups to label sialic acid based on the
PAL conditions of Zeng et al.8 In previous studies, labeling of
sialic acid by PAL employed a biotin−streptavidin complex;
however, here we have explored the utility of small molecule
dyes for direct detection of sialoforms using fluorescence. We
tested two commercially available dyes, and compared them to
the NBDAO dye reported here. Our synthesis of NBDAO (5)
was carried out in two steps: an SNAr to form the ethyl
acetohydroxamate intermediate (4), followed by acidic
deprotection.27 The synthesis of NBDH (2) was performed
using the method established by Gubitz et al.,21 and this dye is
also commercially available. The kinetics of the reactions
between NBDH or NBDAO with MEK were evaluated using a
time-based fluorescence assay. Increased reaction rates were
observed for both NBDH and NBDAO in the presence of
aniline for both reagents at low pH. Comparison of the
spectroscopic properties of the dyes and their conjugates
initially suggested that the fluorogenic NBDH would have
improved signal-to-noise over NBDAO. However, we found
that NBDAO was superior to NBDH in the plate assay and
microscopy experiments. Importantly, both the NBDH and
Bodipy FL hydrazide dyes had poor sensitivity when compared
to NBDAO in flow cytometry experiments. These observations
may be due to the increased Stokes shift of compound 5 and its
oxime conjugates, or the increased stability of the oxime linkage
formed between NBDAO and the aldehyde.40
SUMMARY
■
We report the synthesis and application of the carbonyl reactive
fluorophore, NBDAO, for the selective labeling of sialic acid
using PAL. We compared the labeling of NBDAO to two
commercially available carbonyl reactive dyes, NBDH and
Bodipy FL hydrazide. We found that NBDH and NBDAO dyes
can be used to follow the kinetics of hydrazone or oxime
formation when combined with an aldehyde substrate.
Importantly, when used in conjunction with aniline catalysis
these dyes are ideal for labeling of glycoproteins, detection of
the sialic acid content of whole cells, and as small-molecule
fluorophore stains of glycans on live cells.41 We demonstrated
that these dyes can be used as a simple procedure for sialic acid
labeling when combined with periodate oxidation as a method
for modifying sialic acids in cells.8,41 As well, we have shown
these dyes can be used as a method for quantitative detection of
sialic acid content by flow cytometry. The NBDAO dye showed
significant staining of sialic acid over background (6.7-fold),
which was superior to the commercially available fluorophores
NBDH (2.0-fold) and Bodipy FL hydrazide (2.9-fold). We
expect this methodology will prove useful for a variety of
applications in glycobiology, particularly in the study of changes
to sialic acid content and subcellular location in live cells. These
dyes may also prove useful in other carbonyl ligation
strategies.42−45
Figure 6. Representative flow cytometry histograms of the relative
fluorescence of Jurkat cells treated with a solution containing only the
fluorophore (-+, solid black line), or the fluorophore after PAL-
treatment of the cells (++, dashed gray line). Dyes shown are (a)
NBDH, (b) NBDAO, and (c) Bodipy FL hydrazide.
and NBDH-treated cells showed minor nonspecific background
staining (-+) which increased the mean fluorescence intensity
(MFI) of cells by approximately 4.5- and 3.0-fold over
untreated controls, respectively (see Supporting Information).
In contrast, the Bodipy FL hydrazide dye gave a larger increase
in background staining (approximately 500-fold). This result
likely indicates that the Bodipy fluorophore is more hydro-
phobic, leading to integration of the dye into the cell
membrane. To determine the fidelity of sialic acid detection
for each dye, we compared cells which were dye treated (-+) to
those which were PAL and fluorophore treated (++). In the
case of the NBDAO dye, cells showed an almost 7-fold increase
in fluorescence after staining. This was the largest increase we
observed, as both commercial dyes (NBDH and Bodipy FL
hydrazide) gave only 2−3-fold increases over background.
Based on these data we concluded that NBDAO gave the best
combination of low background and high signal-to-noise for
sialic acid detection. Jurkat cells that were treated with bacterial
neuraminidase for one hour, followed by PAL and NBDAO,
showed reduced MFI (78 8%) compared to untreated cells,
ASSOCIATED CONTENT
* Supporting Information
Absorption and emission spectra of compounds 2, 3, 5, and 6 in
■
S
water, acetonitrile, and ethanol and flow cytometry histograms.
369
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(17) Rayo, J., Amara, N., Krief, P., and Meijler, M. M. (2011) Live
Cell Labeling of Native Intracellular Bacterial Receptors Using Aniline-
Catalyzed Oxime Ligation. J. Am. Chem. Soc. 133, 7469−7475.
(18) Dilek, O., and Bane, S. (2011) Synthesis and Spectroscopic
Characterization of Fluorescent Boron Dipyrromethene-Derived
Hydrazones. J. Fluoresc. 21, 347−354.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
̈
(19) Dilek, O., and Bane, S. L. (2008) Synthesis of boron
*Tel.: 780 492 0377; fax: 780 492 8231; e-mail: ccairo@
ualberta.ca.
dipyrromethene fluorescent probes for bioorthogonal labeling.
Tetrahedron Lett. 49, 1413−1416.
(20) Key, J. A., Koh, S., Timerghazin, Q. K., Brown, A., and Cairo, C.
W. (2009) Photophysical characterization of triazole-substituted
coumarin fluorophores. Dyes Pigm. 82, 196−203.
Notes
The authors declare no competing financial interest.
(21) Gubitz, G., Wintersteiger, R., and Frei, R. W. (1984)
Fluorogenic labeling of carbonyl compounds with 7-hydrazino-4-
nitrobenzo-2-oxa-1,3,-diazole (NBD-H). J. Liq. Chromatogr. 7, 839−
854.
(22) Uzu, S., Kanda, S., Imai, K., Nakashima, K., and Akiyama, S.
(1990) Fluorogenic Reagents - 4-Aminosulphonyl-7-Hydrazino-2,1,3-
Benzoxadiazole, 4-(N,N-Dimethylaminosulphonyl)-7-Hydrazino-2,1,3-
Benzoxadiazole and 4-Hydrazino-7-Nitro-2,1,3-Benzoxadiazole Hydra-
zine for Aldehydes and Ketones. Analyst 115, 1477−1482.
(23) Jachmann, N., and Karst, U. (2001) Synthesis and application of
4-(N,N-dimethylaminosulfonyl)-7-N-methylhydrazino-2,1,3-benzoxa-
diazole (MDBDH) as a new derivatizing agent for aldehydes. Fresenius
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(24) Santa, T., Al-Dirbashi, O. Y., Ichibangase, T., Rashed, M. S.,
Fukushima, T., and Imai, K. (2008) Synthesis of 4-[2-(N,N-
dimethylamino)ethylaminosulfonyl]-7-N-methylhydrazino-2,1,3-benz
oxadiazole (DAABD-MHz) as a derivatization reagent for aldehydes in
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ACKNOWLEDGMENTS
■
This work was supported by Natural Sciences and Engineering
Research Council of Canada (NSERC) and the Alberta
Glycomics Centre (AGC). We would like to thank the
Canadian Foundation for Innovation for infrastructure support.
The authors would like to thank the University of Alberta
Spectral Services laboratory, and M. R. Richards and N. A.
Romaniuk (UofA) for helpful discussions.
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