Quantification of azide groups on a material surface and a biomolecule using a clickable and cleavable fluorescent compound

Article abstract of DOI:10.1039/C8RA09421G

Here, we propose a novel method for quantifying azide groups on a solid surface and a protein using a clickable and cleavable fluorescent compound. The clickable and cleavable fluorescent compound conjugates with the azide groups on the material surface a

Full text of DOI:10.1039/C8RA09421G

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Quantification of azide groups on a material
surface and a biomolecule using a clickable and
Cite this: RSC Adv., 2019, 9, 4621
cleavable fluorescent compound†
*
Rika Sakai, Hiroki Iguchi and Tatsuo Maruyama
Received 15th November 2018
Accepted 29th January 2019
Here, we propose a novel method for quantifying azide groups on a solid surface and a protein using
a clickable and cleavable fluorescent compound. The clickable and cleavable fluorescent compound
conjugates with the azide groups on the material surface and the fluorophore is then liberated into the
solvent via a cleavage reaction, which can be simply quantified with a conventional fluorometer.
DOI: 10.1039/c8ra09421g
rsc.li/rsc-advances
surfaces of polymeric materials vary depending on the envi-
ronment. Therefore, it is of great importance to quantify the
Introduction
clickable moieties on the outermost surface in a practical
environment (e.g., aqueous solution) for surface modication
based on click chemistry.
A bioorthogonal reaction is a chemical reaction that can be
selectively and reliably performed in a complex biological envi-
ronment without non-favorable interactions and interference
with the biological system.1–3 Huisgen 1,3-dipolar cycloaddition
between an azide and an alkyne (the azide/alkyne cycloaddition
click reaction), which is one of the most famous bioorthogonal
reactions, proceeds in a high yield under mild aqueous condi-
tions without byproducts.4–6 This chemistry has been applied to
a number of biomolecules and synthetic compounds to create
multifunction-integrated molecular systems. This click reaction
has also been used to immobilize biomolecules, ligands, and
polymers on substrate surfaces7–11 for microarray preparation,12
fabrication of biomolecule-conjugated nanoparticles13–16 and
uorescent labeling of living cells.17–19
In this study, we aimed to develop a method for quantica-
tion of the azide groups present on the outermost surface in an
aqueous environment. We synthesized a clickable and cleavable
uorescent compound that conjugates with the azide groups on
the substrate surface via Huisgen 1,3-dipolar cycloaddition
(Fig. 1). Hydrolysis of the ester bond in the uorescent
compound liberates the uorophore into the bulk solution,
which allows the azide groups on a substrate surface to be
quantied with a conventional uorometer. The present
methodology can be also applied to the quantication of azide
groups conjugated with a protein.
When click chemistry is applied to sensitive analytical and
biological devices, quantication of the reactive sites plays an
important role in controlling the amount of azide groups (or
alkyne groups) present on a substrate surface, because the
surface density of the functional molecules affects the perfor-
mance of the substrate.20,21 However, to our knowledge, there
have been no reports of quantication methods for clickable
moieties (azide and alkyne groups) on substrate surfaces.
Conventional surface analysis methods, such as X-ray photon
spectroscopy (XPS), Fourier transform infrared spectroscopy
and secondary-ion mass spectrometry are qualitative and
provide information about the elements or functional groups
not only on the outermost surface but also those at a certain
depth (e.g., 10 nm for XPS) from the surface.22,23 These methods
require a high vacuum environment or dry atmosphere, and the
Experimental
Materials
2,4-Dihydroxybenzaldehyde, 2,2-dimethyl-1,3-dioxane-4,6-dione,
piperidinium acetate, 2-hydroxy-4-methoxybenzaldehyde, prop-
argyl-PEG4-alcohol, and azidobenzoic acid were purchased from
Tokyo Chemical Industry (Tokyo, Japan). K2CO3, propargyl-
Department of Chemical Science and Engineering, Graduate School of Engineering,
Kobe University, 1-1 Rokkodai, Nada-ku, Kobe 657-8501, Japan. E-mail: tmarutcm@
crystal.kobe-u.ac.jp
† Electronic supplementary information (ESI) available: The experimental
procedures, and additional results. See DOI: 10.1039/c8ra09421g
Fig. 1 Schematic of quantification of the azide groups on substrate
surfaces using the clickable and cleavable fluorescent compound.
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bromide, CuSO4, b-casein from bovine milk, rhodamine B iso-
thiocyanate (RITC), and Tris–EDTA buffer solution (pH 7) were
purchased from Sigma-Aldrich (St. Louis, MO). DBCO-s-s-amine
was purchased from Conju-Probe LLC (San Diego, CA). The
other chemicals were purchased from Wako Pure Chemical
Industries, Ltd. (Osaka, Japan). NH2-PEG-resin particles (10 mm)
were purchased from Watanabe Chemical Industries (Hir-
oshima, Japan). Slide-A-Lyzer G2 dialysis cassettes (20k MWCO,
15 mL) and micro BCA protein assay were purchased from
Thermo Fisher Scientic (Waltham, USA). High-quality deionized
(DI) water (>15 MU cm) produced by an Elix-5 system (Millipore,
Molsheim, France) was used in the experiments.
Synthesis of 7-methoxycoumarin-3-carboxylic acid. 2-
Hydroxy-4-methoxybenzaldehyde (50 mmol), 2,2-dimethyl-1,3-
dioxane-4,6-dione (50 mmol), and piperidinium acetate (1
mmol) were dissolved in dehydrated ethanol (25 mL). The
mixture was stirred at room temperature for 10 min and
reuxed for 3 h. The reaction mixture was cooled to room
temperature, and then further cooled in an ice bath for 30 min.
The precipitate was collected by ltration and washed with
ethanol three times. The product was dried overnight in
a vacuum chamber.
Synthesis of C^C–EG3–CMRN. Propargyl-PEG4-alcohol (0.2
mmol) was added to a solution of 7-methoxycoumarin-3-
carboxylic acid (0.4 mmol), 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide, hydrochloride (WSC, 0.24 mmol) and N,N-
dimethyl-4-aminopyridine (DMAP, 0.2 mmol) in DMF (1 mL).
The solution was stirred at room temperature for 3 h. The
product was puried by HPLC. Aer evaporation and freeze-
drying, the product was obtained as a yellow powder.
1H-NMR (500 MHz, CDCl3): d (ppm) 2.41 (t, 1H), 3.70–3.73
(m, 8H), 3.84 (t, 2H), 3.91 (s, 3H), 4.19 (d, 2H, J ¼ 2.2 Hz), 7.88 (t,
2H, J ¼ 9 Hz), 6.82 (s, 1H), 6.90 (dd, 2H, J ¼ 2.5, 8.5 Hz), 7.51 (d,
1H, J ¼ 8.9 Hz), 8.53 (s, 1H). DART-TOF/MS (m/z): [M + H]+ calcd
for C20H23O8, 391.1; found, 391.2. Yield: 21%.
Click reaction of C^C–EG3–CMRN. A solution of C^C–EG3–
CMRN (2.5 mmol) and azidobenzoic acid (2.5 mmol) in DMSO
(0.4 mL) was mixed and stirred with an aqueous solution (1.6
mL) of CuSO4 (6 mmol) and sodium ascorbate (30 mmol) at 37 ꢂC
for 3 h. The solution was analyzed as described above (click
reaction of C^C–EG3–CMRN).
1H-NMR (500 MHz, DMSO-d6): d (ppm) 3.54–3.61 (m, 8H),
3.71 (t, 2H), 3.88 (s, 3H), 4.34 (t, 2H, J ¼ 2.2 Hz), 4.61 (s, 2H), 6.98
(dd, 1H, J ¼ 2.5, 8.8 Hz), 7.01 (s, 1H),7.82 (d, 1H, J ¼ 8.6 Hz), 7.98
(d, 2H, J ¼ 8.5 Hz), 8.09 (d, 2H, J ¼ 8.9 Hz), 8.69 (s, 1H), 8.86 (s,
1H). Yield: 96%.
Cleavage. NaOH solution (0.1 M, 0.5 mL) was added to the
solution aer the click reaction of C^C–EG3–CMRN with azi-
dobenzoic acid. The mixture was stirred at 37 ꢂC for 3 h and
analyzed by HPLC.
1H-NMR (500 MHz, CDCl3): d (ppm) 3.97 (s, 3H), 6.93 (d, 1H, J
¼ 2.2 Hz), 7.03 (dd, 1H, J ¼ 2.6, 8.9 Hz), 7.65 (d, 1H, J ¼ 8.8 Hz),
8.86 (s, 1H), 12.18 (s, 1H). DART-TOF/MS (m/z): [M + H]+ calcd
for C11H9O5, 221.0; found, 221.0. Yield: 83%.
Synthesis of C^C–CMRN. Propargyl bromide (0.4 mmol)
was added to a dehydrated DMF solution (7 mL) containing
7-methoxycoumarin-3-carboxylic acid (0.2 mmol) and K2CO3
(0.6 mmol). The solution was stirred at room temperature for
24 h. Crude C^C–CMRN was obtained as a yellow powder aer
ltration and evaporation. A HPLC system (LC-20AT, Shimadzu,
Kyoto, Japan) equipped with an Inertsil ODS-3 column (10 mm
ꢀ 250 mm, GL Science, Tokyo, Japan) was used to purify C^C–
CMRN. The mobile phase was water containing 0.1 wt% tri-
uoroacetic acid (TFA) and acetonitrile containing 0.1 wt% TFA
and the ow rate was 5 mL minꢁ1. The eluted compounds were
observed by measuring the absorbance at 350 nm. The gradient
started at 30% acetonitrile and 70% water, and the proportion
of acetonitrile increased to 100% over 30 min. Isocratic 100%
acetonitrile was maintained for 10 min.
Azidation of the resin particles. The resin particles had
65 mmol gꢁ1 amino groups on their surfaces. A dispersion of
amino-resin particles (3.8 mmol amino groups), azidobenzoic
acid (380 mmol), and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-
methylmorpholinium chloride (DMT-MM, 75 mmol) was
mixed in DMSO (3 mL) and stirred at 37 ꢂC for 24 h to conjugate
azidobenzoic acid with the amino groups on the resin particles.
The resin particles were washed four times with methanol (6
mL) and water (6 mL).
Quantication of the azide groups on the resin particles using
C^C–CMRN. Azidated resin particles (0, 0.1875, 0.375, or 0.75
mg) were dispersed in water (1.8 mL) containing CuSO4 (1 mmol)
and sodium ascorbate (4 mmol). The dispersion was added to
1 mM C^C–EG3–CMRN in DMSO (0.1 mL) and shaken at 40 ꢂC
for 24 h. Aer centrifugation, the supernatant was removed and
the resin particles were washed three times with methanol and
water. The resin particles were dispersed in NaOH aqueous
solution (1 M, 0.2 mL) and then shaken at 37 ꢂC for 24 h to liberate
CMRN from the resin particles. Aer centrifugation, HCl solution
(1 M, 0.2 mL) and phosphate buffer (0.1 M, pH 7.4, 2 mL) were
added to neutralize the supernatant. The uorescence intensity of
the neutralized solution was measured with a uorescence spec-
trophotometer. The excitation and emission wavelengths were
335 and 406.5 nm, respectively. The excitation and emission band
widths were 5 nm. The standard curve of a liberated uorophore
in phosphate buffer is shown in Fig. S2a.† Quantication was
performed in triplicate, unless specied otherwise.
1H-NMR (500 MHz, CDCl3): d (ppm) 3.91 (s, 3H), 4.93 (s, 2H),
6.83 (s, 1H), 6.91 (dd, 1H, J ¼ 2.5, 8.8 Hz), 7.52 (d, 1H, J ¼ 8.8
Hz), 8.58 (s, 1H), DART-TOF/MS (m/z): [M + H]+ calcd for
C
14H11O5, 259.1; found, 259.1. Yield: 12%.
Click reaction of C^C–CMRN. A DMSO solution (0.4 mL)
containing 4 mmol C^C–CMRN and 4 mmol azidobenzoic acid
was mixed and stirred with an aqueous solution (1 mL) of
0.4 mmol CuSO4 and 2 mmol sodium ascorbate at 25 ꢂC in
a water bath for 3 h. This solution was evaporated and analyzed
by HPLC (Fig. S1†). The HPLC fractions of the reaction products
were collected and lyophilized.
1H-NMR (500 MHz, DMSO-d6): d (ppm) 3.96 (s, 3H), 5.57 (s,
2H), 6.83 (s, 1H), 6.91 (dd, 1H, J ¼ 2.5, 8.8 Hz), 7.52 (d, 1H, J ¼
8.5 Hz),7.90 (d, 2H, J ¼ 8.8 Hz), 8.25 (d, 2H, J ¼ 9.2 Hz), 8.09 (d,
2H, J ¼ 8.9 Hz), 8.69 (s, 1H), 8.86 (s, 1H).
4622 | RSC Adv., 2019, 9, 4621–4625
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Quantication of the azide groups on the resin particles using
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strain-promoted (Cu-free) click chemistry (Fig. S3†). Azidated resin
particles (0.1875, 0.375, or 0.75 mg) were dispersed in 10 mM
HClaq (1.4 mL). The dispersion was added to 0.6 mL tetrahydro-
furan (THF) containing 1 mM DBCO-s-s-NH2 and shaken at 40 ꢂC
for 12 h. Aer centrifugation, the supernatant was removed and
the resin particles were washed three times with THF/10 mM HCl
aqueous solution (1/1, v/v) and once with phosphate buffer
(50 mM, pH 8). The resin particles dispersed in phosphate buffer
(50 mM, 1 mL) were added to phosphate buffer (50 mM, 2 mL)
containing 1 mM RITC and 10 vol% trimethylamine (TEA) and
ꢂ
then shaken at 40 C for 3 h. The supernatant was removed and
phosphate buffer (50 mM, 2 mL) containing 1ꢂmM RITC and
10 vol% TEA was added again and shaken at 40 C for 3 h. Aer
centrifugation, the supernatant was removed and the resin parti-
cles were washed with methanol/10 mM NaOH aqueous solution
(1/1, v/v), methanol/10 mM HCl aqueous solution (1/1, v/v) and
phosphate buffer (50 mM, pH 8). Aer centrifugation, phosphate
buffer (50 mM, pH 8, 2 mL) containing 4 mM tris(2-carboxyethyl)
phosphine were added to liberate the uorophore. The uores-
cence intensity of the solution was measured with a uorescence
spectrophotometer. The excitation and emission wavelengths were
495 and 515 nm, respectively. The excitation and emission band
widths were 5 nm.
Quantication of the azide groups on b-casein by TNBS. b-
Casein was dissolved in 0.1 M NaHCO3 buffer (pH 8) at
concentrations of 20–200 mg mLꢁ1 to construct the standard
curve. A solution of 0.01% (w/v) TNBS (0.25 mL) was added to
sample solutions (0.5 mL) containing 100 mg mLꢁ1 azidated b-
casein or b-casein, and the solutions were then incubated at
37 ꢂC for 2 h. Aer the reaction, 10% SDS aqueous solution (0.25
mL) and 1 M HCl aqueous solution (0.125 mL) were added to
the sample solution to quench the TNBS reaction. The absor-
bance of the solutions was measured at 335 nm.
Fig.
2
(a) Synthesis of the clickable and cleavable fluorescent
compounds C^C–CMRN and C^C–EG3–CMRN. (b) HPLC profiles of
C^C–EG3–CMRN after the click reaction and after subsequent
hydrolysis.
clickable part and the ester bond acts as the cleavable part in
hydrolysis. C^C–CMRN has no spacer between the ester bond
and alkyne, but C^C–EG3–CMRN has an oligo (ethylene glycol)
spacer. The click reactivities of C^C–CMRN and C^C–EG3–
CMRN were investigated in a homogeneous solution. Azido-
benzoic acid was used as a model azide compound and the click
reaction was performed with a copper catalyst in a mixture of
water/dimethylformamide (DMF) (5/1, v/v). High-performance
liquid chromatography (HPLC) measurements of C^C–CMRN
indicate simultaneous synthesis of compound 10 (at a retention
time (RT) of 15.3 min) and production of 7-methylcoumarin-3-
carboxylic acid (Fig. S1†), indicating that hydrolysis of the
ester bond is probably due to chelation of a Cu ion at the
neighboring site. HPLC measurements of C^C–EG3–CMRN
reveal that the peak at RT ¼ 14.6 min disappears and a peak at
RT ¼ 7.7 min appears (Fig. 2b). 1H-NMR analysis of these
fractions reveals that the peaks at RT ¼ 14.6 and 7.7 min
correspond to C^C–EG3–CMRN and compound 1 (the product
of the click reaction), respectively. These results indicate that
C^C–EG3–CMRN can be used as a substrate in the azide/alkyne
click reaction and that the reaction proceeds in high yield
(>96%).
Quantication of the azide groups on azidated b-casein.
Azidated casein (50 nmol) was dissolved in 25 mM NaHCO3
buffer (pH 8, 1.2 mL) containing CuSO4 (2.4 mmol) and sodium
ascorbate (12 mmol). C^C–EG3–CMRN (5 mmol) in DMSO (0.4
mL) was added to the solution and the solution was stirred at
ꢂ
37 C for 3 h. Aer the reaction, excess C^C–EG3–CMRN and
Cu(I) catalyst were removed by dialysis for 2 days. NaOH solu-
tion (1 M, 0.5 mL) was added to the solution (0.5 mL) and the
solution was then shaken at 40 ꢂC for 2 h to liberate CMRN
uorophore from b-casein. HCl solution (1 M, 0.5 mL) and
NaHCO3 buffer (1 M, pH 8, 2 mL) were added to neutralize the
solution. The uorescent intensity of the solution was
measured with a uorescent spectrometer. The excitation and
emission bands were 335 and 406.5 nm, respectively. The
standard curve of a liberated uorophore in carbonate buffer is
shown in Fig. S2b.† Quantication was performed in triplicate,
unless specied otherwise.
Next, we investigated hydrolysis of the ester bond of C^C–
EG3–CMRN. NaOH aqueous solution was added to the solution
containing compound 1 to give a nal NaOH concentration of
0.1 M, and the mixture was then stirred at 37 ꢂC for 3 h. HPLC
measurements show that the peak of compound 1 (at RT ¼ 7.7
min) completely disappears and
a
new peak of
7-methylcoumarin-3-carboxylic acid appears at RT ¼ 5.4 min
(Fig. 2b). These results demonstrate that compound 1, which is
the product of the C^C–EG3–CMRN/azidobenzoic acid click
reaction, can be hydrolyzed by aqueous NaOH to liberate its
uorophore.
Results and discussion
First, we designed and synthesized two different types of click-
able and cleavable uorescent compounds: C^C–CMRN and
C^C–EG3–CMRN (Fig. 2a). The alkyne group acts as the
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We then attempted to quantify the azide groups on resin solubility is very low. We conjugated DBCO-s-s-amine with azide
particles using C^C–EG3–CMRN. In a previous study, we re- groups on resin surfaces and then the resultant amino groups
ported that the resin particles have 65 nmol mgꢁ1 amino were conjugated with rhodamine B isothiocyanate (RITC). The
groups.24 Before quantication, we produced azide groups on the disulde bond was cleaved by a reducing agent (TCEP) to liberate
resin particles by the condensation reaction between azido- the uorophore, which was measured using a uorometer.
benzoic acid and the amino groups on the resin particles (azi- Although the liberated uorophore increased linearly with the
dation, Fig. 3a). The azide groups on the resin particle surfaces amount of azidated resin particles (Fig. S5†), we detected only 4.2
were quantied using C^C–EG3–CMRN. Azidated resin particles nmol azide groups per 1 mg resin. The remarkably low amount of
(0, 0.19, 0.38, or 0.75 mg) were dispersed in water (1.8 mL) con- azide groups quantied might be explained by the following
taining CuSO4 (1 mmol) and sodium ascorbate (4 mmol). The possible reasons. (i) The sterically bulky structure of DBCO-s-s-
dispersion was mixed with dimethyl sulfoxide (DMSO, 0.1 mL) amine affected its conjugation with an azide group on
containing 1 mM C^C–EG3–CMRN and shaken at room a surface, leading to a low conjugation ratio. (ii) A mixture of
temperature for 24 h. Aer washing the resin particles, the resin THF/water, which was used to dissolve DBCO-s-s-amine for a Cu-
particles were dispersed in NaOH aqueous solution (nal free click reaction, altered the surface composition of resin,
concentration of 0.1 M) to liberate the CMRN uorophore from resulting in the inhibition of the azide group quantication.
the resin particles. Aer centrifugation and neutralization, the These results suggest that
a relatively small uorescent
uorescence intensity of the solution was measured with compound, which has water solubility and avoids steric
a conventional uorescence spectrophotometer. There is high hindrance, is appropriate for the quantication of azide groups
linearity between the amount of particles and the uorescence on a surface. The following experiments were carried out using
intensity of the liberated uorophore (Fig. 2b). The quantied C^C–EG3–CMRN.
value of the azide groups on the resin particles is 55 nmol mgꢁ1
,
In the last decade, clickable moieties have oen been intro-
which is comparable with the amount of amino groups on the duced to biomolecules (e.g., proteins and nucleic acids) to create
resin particles. The slightly smaller value for the azide groups is functionally engineered biomolecules and immobilize them on
because of the yield of azidation of the amino groups on the resin various surfaces.26–30 Therefore, we then investigated quantica-
particles. These results suggest successful quantication of the tion of azide groups introduced to a protein. b-Casein was chosen
azide groups on the resin particles using the proposed method. as a model protein and azide groups were introduced to the
The standard curve (Fig. S4†) shows that the limit of detection of amino groups of b-casein by N-hydroxysuccinimide ester chem-
the present method was 5 nmol mlꢁ1, which means that azide istry (Fig. 4a). b-Casein has 12 amino groups.31 The TNBS
groups of 5 pmol on a surface was detected when using 1 mL method32,33 shows that the azidation procedure conjugates 86%
aqueous solution of liberated uorophore.
of the amino groups with azidobenzoic acid. Azidated b-casein
While the above investigations employed Cu(I) as a catalyst for (50 nmol) in an aqueous buffer (pH 7.0, 1.2 mL) containing
1,3-dipolar cycloaddition of azide and alkyne, a Cu-free click CuSO4 (2.4 mmol) and sodium ascorbate (12 mmol) was mixed
reaction (strain-promoted azide–alkyne cycloaddition) using with DMSO (0.4 mL) containing C^C–EG3–CMRN (5 mmol) and
a cycloalkyne has attracted a broad range of attention because of then shaken at 37 ꢂC for 3 h. Aer dialysis to remove excess
no use of a catalyst.25 We then attempted to quantify the azide C^C–EG3–CMRN and the Cu catalyst, NaOH aqueous solution
groups on resin surfaces using a Cu-free click reaction (Fig. S3†). was added to the solution to liberate the CMRN moiety from
Although a dibenzocyclooctyne (DBCO) derivative is a useful azidated b-casein.
functional molecule for a Cu-free click reaction, its water
Fluorescence measurement of the solution shows 410 nmol
CMRN moieties per milligram of casein (Fig. 4b), meaning that
81% of the amino groups of b-casein are azidated. The obtained
azidation ratio is comparable with the result of the TNBS
method.
Fig. 3 (a) Schematic of azidation of the amino groups on the resin
particles. (b) Quantification of the azide groups on the resin particles Fig. 4 (a) Schematic of azidation of the amino groups on b-casein. (b)
using C^C–EG3–CMRN.
Quantification of the azide groups on b-casein.
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In conclusion, we have proposed a novel approach to quantify
the azide groups on a material surface and on a protein using
a clickable and cleavable uorescent compound (C^C–EG3–
CMRN). This is the rst time that quantication of the azide
groups on a solid material and a biomolecule has been ach-
ieved. Quantitative analysis of the clickable moieties on
a surface will play an important role in surface chemistry and
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Conflicts of interest
There are no conicts to declare.
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Acknowledgements
We thank Prof. A. Mori for his technical help. This work was
supported by Special Coordination Funds for Promoting
Science and Technology, Creation of Innovation Centers for
Advanced Interdisciplinary Research Areas (Innovative Bio-
production Kobe), MEXT, Japan and also by JSPS KAKENHI
(16H04577, 18K18976 and 18H04556).
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