Chromogenic substrate from 4-nitro-1-naphthol for hydrolytic enzyme of neutral or slightly acidic optimum pH: 4-Nitro-1-naphthyl-β-d- galactopyranoside as an example

Article abstract of DOI:10.1016/j.bmcl.2012.11.120

At pH from 5.5 to 7.6, absorptivity of 4-nitro-1-naphthol at 450 nm is over 2.1-fold of that of para-nitrophenol at 405 nm and over 9.6-fold of that of ortho-nitrophenol at 415 nm. On 4-nitro-1-naphthyl-β-d-galactopyranoside at pH 7.4, catalytic efficiency of Escherichia coli β-d-galactosidase is 3-fold of that on para-nitrophenyl-β-d-galactopyranoside and about 40% of that on ortho-nitrophenyl-β-d-galactopyranoside, and produces a lower quantification limit of penicillin G by enzyme-linked-immunoabsorbent-assay. Hence, 4-nitro-1-naphthol is favorable to prepare chromogenic substrates of hydrolytic enzymes of neutral or slightly acidic optimum pH.

Full text of DOI:10.1016/j.bmcl.2012.11.120

Bioorganic & Medicinal Chemistry Letters 23 (2013) 646–649
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Bioorganic & Medicinal Chemistry Letters
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Chromogenic substrate from 4-nitro-1-naphthol for hydrolytic enzyme
of neutral or slightly acidic optimum pH: 4-Nitro-1-naphthyl-b-D-
galactopyranoside as an example
Jizheng Dang , Hongbo Liu , Xiaolan Yang , Yi Zhang, Yanling Xie, Yuanli Li, Jun Pu, Juan Liao,
⇑
Yonghua Yuan, Fei Liao
Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing
Medical University, Chongqing 400016, China
a r t i c l e i n f o
a b s t r a c t
Article history:
At pH from 5.5 to 7.6, absorptivity of 4-nitro-1-naphthol at 450 nm is over 2.1-fold of that of para-nitro-
Received 4 October 2012
Revised 28 November 2012
Accepted 30 November 2012
Available online 8 December 2012
phenol at 405 nm and over 9.6-fold of that of ortho-nitrophenol at 415 nm. On 4-nitro-1-naphthyl-b-
galactopyranoside at pH 7.4, catalytic efficiency of Escherichia coli b- -galactosidase is 3-fold of that on
para-nitrophenyl-b- -galactopyranoside and about 40% of that on ortho-nitrophenyl-b- -galactopyrano-
side, and produces a lower quantification limit of penicillin G by enzyme-linked-immunoabsorbent-
assay. Hence, 4-nitro-1-naphthol is favorable to prepare chromogenic substrates of hydrolytic enzymes
of neutral or slightly acidic optimum pH.
D-
D
D
D
Keywords:
4-Nitro-1-naphthol
Chromogenic substrate
Hydrolytic enzymes
Ó 2012 Elsevier Ltd. All rights reserved.
b-D-Galactosidase
Hydrolytic enzymes like phosphatases and glycosidases play
1-naphthol (4NNP) is a better chromogen. After ionization, the
absorbance peaks of pNP, oNP and 4NNP center on 405, 415, and
about 458 nm, respectively. The apparent absorptivity of 4NNP at
460 nm is 1.03–1.07 times of that at 450 nm from pH 5.5 to 9.0.
At pH from 7.6 to 9.0, the apparent absorptivity of 4NNP at
450 nm is 1.7–2.1 times of that of pNP at 405 nm while 7.9–9.6
times of that of oNP at 415 nm (Fig. 1). At pH from 5.5 to 7.6, the
apparent absorptivity of those three chromogens becomes smaller,
but relative differences of their apparent absorptivity grow larger.
Predominantly, at pH from 5.5 to 6.6, pNP has the apparent absorp-
tivity below 2.7 (mM cm)ꢀ1 at 405 nm and oNP has the apparent
absorptivity below 0.70 (mM cm)ꢀ1 at 415 nm, but 4NNP displays
the apparent absorptivity over 13.0 (mM cm)ꢀ1 at 450 nm; the
apparent absorptivity of 4NNP at 450 nm is 7.5–21 times of that
of pNP at 405 nm, and 33–66 times of that of oNP at 415 nm. At
pH from 6.6 to 7.6, the apparent absorptivity of 4NNP at 450 nm
is 2.1–7.5 times of that of pNP at 405 nm, and is 9.6–33 times of
that of oNP at 415 nm. From the changes of the apparent absorptiv-
ity to pH values, pKa of 4NNP is approximated to be slightly below
6.0 while that of pNP is about 7.3; pKa of 4NNP may be lower than
that of methyl purple.10 Hence, 4NNP is a chromogen better than
pNP and oNP to prepare chromogenic substrates of hydrolytic en-
zymes with maximum activities at neutral or slightly acidic pH.
The specific activity of a hydrolytic enzyme on its chromogenic
substrate is also a concern for the sensitivity to quantify the en-
zyme, which is more pronounced for an enzyme as an ELISA label.
In open databases, there are no records of glycosides of 4NNP as
important roles in bio-recognition and signal transduction, and
also are important tools for bio-processing and bio-analyses
including enzyme-linked-immunoabsorbent-assay (ELISA).1–9 The
sensitive quantification of the activities of those hydrolytic en-
zymes is thus a pivotal work and the estimation of their activities
from reaction curves is always favored. At pH over 8.0, para-nitro-
phenol (pNP) and ortho-nitrophenol (oNP) are ionized into anions
possessing strong absorbance peaks at wavelengths over 400 nm
where the unionized pNP and oNP have negligible absorbance.
For hydrolytic enzymes, thus, chromogenic substrates from pNP
and oNP are widely used to record their reaction curves by absor-
bance. In practice, the sensitivity to quantify the activity of a
hydrolytic enzyme is proportional to the specific activity of the en-
zyme on a chromogenic substrate and the absorptivity of the chro-
mogenic product. Some hydrolytic enzymes show their maximum
activities at neutral or even slightly acidic pH values which greatly
reduce the ionization of pNP and oNP into anions and thus their
apparent absorptivity. Therefore, new chromogens are still needed
to prepare chromogenic substrates of hydrolytic enzymes exhibit-
ing the maximum activities at neutral or slightly acidic pH.
For preparing chromogenic substrates of hydrolytic enzymes
with maximum activities at neutral or slightly acidic pH, 4-nitro-
⇑
Corresponding author. Tel.: +86 23 68485240; fax: +86 23 68485111.
E-mail addresses: liaofeish@cqmu.edu.cn, liaofeish@yahoo.com (F. Liao).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.120

J. Dang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 646–649
647
4NNPG is prepared via the following four reactions
(Scheme 1).10,14 (a) Full acetylation of b-
-galactose. In detail,
5.0 g -galactose, 2.4 g anhydrous sodium acetate and 24 mL acetic
D
D
anhydride are mixed in a flask under room temperature. After reac-
tion for 2.5 h under refluxing, the solution is diluted with 200 mL
ice-water and stirred for 2 h; the precipitates are collected via fil-
tration and are extracted with 100 mL water under stirring for
1 h. Via filtration, the insoluble precipitates are finally collected
and re-crystallized with ethanol to give the white solids of b-
actose-1,2,3,4,6-O-pentaacetate with a melting point of 145 °C. (b)
Bromination of the acetylated b- -galactose. To 2.0 g b- -galact-
D-gal-
D
D
ose-1,2,3,4,6-O-pentaacetate in 7.0 mL acetic acid in ice-water
bath, 33% HBr in 2 mL acetic acid was added drop-by-drop. After
reaction for 2 h, the solution is neutralized with saturated sodium
bicarbonate solution and then extracted with CH2Cl2. The organic
layer is washed twice with saturated sodium bicarbonate solution
and then with water, and dried over anhydrous sodium sulfate. The
removal of CH2Cl2 yields solid residuals that are re-crystallized
with ether/petroleum ether (5:1) to give white solids with a melt-
ing point of about 80 °C. (c) Displacement of bromide. In detail,
Figure 1. Changes of absorptivity of 4NNP, pNP and oNP to pH values. Buffers are
sodium phosphate at 0.10 M from pH 5.5 to 7.5, and Tris–HCl at 0.10 M from pH 8.0
to 9.0.
0.46 g 2,3,4,6-O-tetraacetyl-a-D-galactopyranosyl bromide, 0.22 g
glycosidase substrates; the sole record of a 4NNP derivative as a
potential chromogenic substrate of hydrolytic enzyme is 4-nitro-
1-naphthylphosphate (4NNPP), but its hydrolysis by phosphatases
has not been tested yet.11 Calf intestine alkaline phosphatase
(CIAP) is an ELISA label.5,6 At pH 8.5 in 50 mM Tris–HCl, CIAP has
4NNP (Alfa Aesar), 0.156 g anhydrous potassium carbonate, and
10.0 mL acetone are mixed and refluxed for 5 h. After the removal
of solvent, the residuals are dissolved in CH2Cl2 and extracted
twice with 10 mL solution of 1.0 M NaOH. After being dried, CH2Cl2
is removed to give solid residuals with a melting point of about
138 °C. (d) Deacetylation. To 0.56 g 1-(4-nitro-1-naphthyl)-
a Michaelis–Menten constant (Km) of (7.7 2.0)
lM for 4NNPP
while a Km of (35 5) M for para-nitrophenylphosphate; its spe-
l
2,3,4,6-O-tetraacetyl-b-
D-galactopyranoside in 3 mL methanol,
cific activity at 0.15 mM 4NNPP is about 40% of that on 4.0 mM
para-nitrophenylphosphate. Thus, the sensitivity to quantify CIAP
by the absorbance at 450 nm with 0.15 mM 4NNPP is comparable
to that by the absorbance at 405 nm with 4.0 mM 4-nitrophenyl-
phosphate. These results indicate that the use of 4NNPP for CIAP
assay just reduces the cost of substrate and alleviates of the poten-
tial interference from contaminants in 4NNPP. CIAP has an opti-
mum pH over 8.5 that mitigate the significance of 4NNPP, but
most glycosidases have their maximum activities at neutral or
slightly acidic pH. To quantify such glycosidases, their chromo-
genic substrates from 4NNP should be much favorable when glyco-
200 L sodium methoxide at 1.0 M in methanol is added for reac-
l
tion at 25 °C for 30 min. The solution was kept at <ꢀ10 °C in a
refrigerator for 10 h; the precipitates are collected and re-crystal-
lized trice with methanol–water (3:1), yielding 4NNPG with a
melting point of about 228 °C. The molar yield from D-galactose
is slightly over 3%. The resulting 4NNPG has the purity about 93%
based on the quantity of 4NNP released by the action of a purified
BGAL (Sigma–Aldrich, G4155).
4NNPG structure is confirmed by NMR and element formula-
tion. 1H NMR data are (Brucker Avance 500 MHz, DMSO): d 8.578
(d, 1H, J = 9.0 Hz, ArH), d 8.517 (d, 1H, J = 8.5 Hz, ArH), d 8.432 (d,
1H, J = 9 Hz, ArH), d 7.868 (t, 1H, J = 7.5 Hz, ArH), d 7.742 (t,
1H,J = 8.0 Hz, ArH), d 7.3105 (d, 1H, J = 8.5 Hz, ArH), d 5.22 (d, 1H,
J = 7.5 Hz, GAl-CH), d 3.84 (d, 1H, J = 7.5 Hz, GAl-CH), d 3.79 (s,
1H, GAl-CH), d 3.75 (t, 1H, J = 6.0 Hz, GAl-CH), d 3.60 (t, 1H, J = 5–
6 Hz, GAl-CH), 3.53 (t, 2H, J = 8.0 Hz, GAl-CH), d 4.7–5.7 (m, 4H,
GAl-OH). 13C NMR data are: d 157.968 (s, 1C, Ar–C), d 139.249 (s,
1C, Ar–C), d 130.261(s, 1C, Ar–C), d 127.1 (s, 1C, Ar–C), d 126.8 (s,
1C, Ar–C), d 125.8 (s, 1C, Ar–C), d 125.0 (s, 1C, Ar–C), d 123.1 (s,
sidases have sufficient specific activities. Escherichia coli b-
galactosidase (BGAL) is a practical ELISA label; b- -galactosides of
methyl purple and other large chromogens are effective substrates
of BGAL.6–10,12,13 Therefore, 4-nitro-1-naphthyl-b-
-galactopyrano-
side (4NNPG) was prepared for the first time and compared against
para-nitrophenyl-b- -galactopyranoside (pNPG, Alfa Aesar) and
D-
D
D
D
ortho-nitrophenyl-b-
D
-galactopyranoside (oNPG, Alfa Aesar) as
BGAL substrates.
Scheme 1. Synthesis of 4-nitro-1-naphthyl-b-
D-galactopyranoside and its hydrolysis by BGAL.

648
J. Dang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 646–649
1C, Ar–C), d 122.6 (s, 1C, Ar–C), d 106.8 (s, 1C, Ar–C), d 101.1 (s, 1C,
GAl-C), d 76.0 (s, 1C, GAl-C), d 73.0 (s, 1C, GAl-C), d 70.2 (s, 1C, GAl-
C), d 67.9 (s, 1C, GAl-C), d 60.2 (s, 1C, GAl-C). By ESI-HRMS, m/z cal-
culated for [M+Na]+ of 4NNPG is 374.0846 while that determined is
374.0848.
phosphate buffer at pH 7.4. The activated penicillin G solution of
50 L is then mixed with 0.44 mg BGAL in 250 L of the sodium
l
l
phosphate buffer at pH 7.4 for reaction at 4 °C for 120 min. Modi-
fied BGAL is then passed through a Sephadex G25 column
(10 mm ꢁ 150 mm) equilibrated and eluted with the sodium phos-
phate buffer at pH 7.4; the first peak in a total of 2.0 mL is collected
based on the absorbance at 280 nm. After chromatographic purifi-
cation, the penicillin G-modified BGAL acts as the tracer; it has a
specific activity of about 100 U/mg on 0.20 mM 4NNPG at 25 °C
and a concentration of about 11 U/mL in the elution. Competitive
ELISA of penicillin G is performed as follows (Scheme 2).
BGAL activity is measured in 0.10 M sodium phosphate buffer at
pH 7.4 and 25 °C, unless stated otherwise, using Shanghai Mapada
UV-1600 PC ultraviolet spectrophotometer. One unit of BGAL activ-
ity is defined as that to release one micromole chromogen per min.
Recorded with Shimadzu UV 2550, 4NNPG displays an absorbance
peak around 375 nm; BGAL hydrolyzes 4NNPG to produce 4NNP,
with clear increases in absorbance around 458 nm (Fig. 2). After
the correction of the effects of pH on absorptivity, the optimum
pH for the actions of BGAL on 4NNPG, pNPG and oNPG is consis-
tently about 7.0; there is about 30% increase in the specific activity
of BGAL on 4NNPG after temperature is increased from 25 to 37 °C;
the optimum temperature of BGAL on 4NNPG is about 50 °C, con-
sistent with those on oNPG and pNPG.
The complexes of the tracer and mouse anti-penicillin antibody
(Abcam ab15070) are captured by goat anti-mouse IgG polyclonal
antibodies immobilized on wells (Pierce 15134). The anti-penicillin
antibody is diluted by 1:500 and 20 lL of the diluted antibody
solution is employed in each well to saturate the capture antibod-
ies. The quantity of the tracer is optimized via step-by-step in-
crease till the change of absorbance by the action of the tracer
bound in the absence of penicillin G is over 0.080 after reaction
By Lineweaver–Burk plot analysis of the response of initial rates
to substrate concentrations, BGAL possess Km of (66 5)
4NNPG (n = 3), about one-third of that for pNPG and less than
l
M for
for 40 min at 25 °C. Finally, 20
lized with 4NNPG, which basically saturate anti-penicillin anti-
body; 50 L of the same elution of the tracer with a total activity
lL of the elution of the tracer is uti-
one-fifth of that for oNPG, but comparable to that for b-
D
-galacto-
l
side of methyl purple.10 The specific activity of BGAL on 0.20 mM
4NNPG is about 140 U/mg at 25 °C, corresponding to a maximum
catalytic activity of about 175 U/mg and a turnover number of
about 2.0 ꢁ 104 minꢀ1. This turnover number of BGAL on 4NNPG
of 0.32 U on 6.0 mM pNPG is used with pNPG, which completely
saturate anti-penicillin antibody. The binding ratio is calculated
as the activity of the tracer bound in the presence of penicillin G
at a given level divided by the activity of the tracer bound in the
absence of penicillin G. The detection limit is the lowest quantity
of penicillin G to produce a change of absorbance over trice the
random variation of absorbance by the microplate reader within
40 min. The response of binding ratios to logarithmic quantities
of penicillin G in wells is plotted. The lower limit and upper limit
of quantification are the smallest and largest quantity of penicillin
G in wells within the linear part of such a response, respectively.
The low Km of BGAL for 4NNPG makes it reasonable to compare
the use of 0.20 mM 4NNPG against the use of 6.0 mM pNPG. Com-
petitive ELISA indicates the effectiveness of 4NNPG as a chromo-
genic substrate of BGAL. The uses of 4NNPG and pNPG to
quantify the tracer yield similar response curves of binding ratios
to logarithmic quantities of penicillin G in wells and consistent
recoveries of penicillin G (Fig. 3). The estimation of the binding ra-
tios over 30% displays the coefficients of variation below 3% (n >5).
Clearly, there is more rapid decrease of the binding ratios to loga-
rithmic quantities of penicillin G with 4NNPG, which should be due
primarily to the use of a smaller quantity of the tracer. The random
is about twice of that on b-D
-galactoside of methyl purple,10 about
three times of that on pNPG and about 40% of that on oNPG. How-
ever, considering the absorptivity, the sensitivity to quantify BGAL
activities on 0.20 mM 4NNPG by the absorbance change rates at
450 nm is about six times of that on 6.0 mM pNPG, about three
times of that on 6.0 mM oNPG. Conventional microplate readers
provide filters for light sources at 405 nm. If the absorbance change
rate of BGAL on 6.0 mM oNPG is quantified at 405 nm, the sensitiv-
ity to quantify BGAL on 0.20 mM 4NNPG by absorbance change
rate at 450 nm is four times higher. Hence, the use of 4NNPG to
quantify BGAL is advantageous over the uses of pNPG and oNPG.
To test the potential application of 4NNPG for quantifying BGAL
as an ELISA label, BGAL (Sigma–Aldrich, G4155) is conjugated to
penicillin G for competitive ELISA of penicillin G. In brief, 0.73 g
penicillin G is activated with 0.40 g 1-ethyl-(3-dimethylamino-
propanyl)-carbadiimide for 30 min at 25 °C in 2.0 mL N,N0-dimeth-
ylformamide containing 0.30 g 1-hydroxyl-benzotriazole; the
resulting yellow solution is diluted by 1:14 with 0.10 M sodium
Figure 2. Absorbance spectra during BGAL action on 4NNPG in 0.10 M sodium phosphate at pH 7.4.

J. Dang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 646–649
649
Scheme 2. Competitive ELISA of penicillin G with BGAL and 4NNPG.
hydrolytic enzyme will produce an isoabsorbance wavelength
where the chromogenic substrate and product have the same
absorptivity. For the new format of ELISA of much higher effi-
ciency, the action of one enzyme label on its chromogenic sub-
strate should produce the longest isoabsorbance wavelength
where the chromogenic product of the other enzyme can be quan-
tified with sufficient sensitivity. The action of BGAL on 4NNPG pro-
duces the longest isoabsorbance wavelength of 400 nm where pNP
can be quantified by absorbance (Fig. 2); the combination of BGAL
on 4NNPG and a-D-glucosidase on para-nitrophenyl-a-D-glucoside
is effective for this new format of ELISA (data unpublished). 4NNP
has excellent stability, small size, large apparent absorptivity at pH
below 7.6 and simple structure for preparing chromogenic sub-
strates of hydrolytic enzymes. Taken together, 4NNP is a better
chromogen to prepare chromogenic substrates of hydrolytic en-
zymes with maximum activities at neutral or slightly acidic pH;
the combination of a chromogenic substrate of 4NNP and that of
pNP for two enzyme labels may facilitate the application of dual-
enzyme simultaneous assay in one reaction solution to ELISA for
much higher efficiency.
Figure 3. Responses of binding ratios to logarithmic quantities of penicillin G. Milk
(drink) is centrifuged at 12,000 rpm for 15 min and filtered through 0.50
lm
membrane to remove insoluble substances. Penicillin G is added to the filtrate for
known final levels. Other operations follow conventional guides. For competitive
binding, reaction buffer is 0.10 M sodium phosphate at pH 7.4. Biotek ELX 800 is
used to record absorbance at 405 nm with pNPG and at 450 nm with 4NNPG. The
binding ratios are determined at least in triplicate with coefficients of variation <3%.
Acknowledgments
With 4NNPG, the linear part gives B/B0 = 93 ꢀ 18 ꢁ log(
v
), R2 >0.994; with pNPG,
the linear part gives B/B0 = 100 ꢀ 15 ꢁ log(
v
), R2 >0.999.
This project was supported by ‘863’-High-Technology Program
(No. 2011AA02A108), the program for New Century Excellent Tal-
ent in University (NCET-09-0928), National Natural Science Foun-
dation of China (No. 81071427) and Natural Science Foundation
Project of CQ (CSTC2011BA5039, CSTC2012JJA081).
variation of absorbance for continuous record of BGAL reaction
curve within 40 min with Biotek ELX800 is about 0.002. The detec-
tion limit of penicillin G with 4NNPG corresponds to a binding ratio
of 95% for less than 0.3 ng/well while that with pNPG corresponds
to a binding ratio of 92% for about 1.0 ng/well. The linear response
of binding ratios to logarithmic quantities of penicillin G gives the
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upper limit of quantification of about 2.2 lg/well with either sub-
strate, but the lower limits of 9 ng/well and 27 ng/well with
4NNPG and pNPG, respectively. Based on the detection limit and
quantification limit of penicillin G by ELISA, the use of 4NNPG as
BGAL substrate should be advantageous over the use of oNPG.
Therefore, for quantifying BGAL as an ELISA label, 4NNPG is a bet-
ter chromogenic substrate.
In practice, the toughest challenge to ELISA is how to enhance
its efficiency. Conventional microplate readers already can swiftly
alter detection wavelengths to quantify the absorbance of one
solution simultaneously at multiple wavelengths. Dual-enzyme
simultaneous assay in one reaction solution via concurrent quanti-
fication of the absorbance of two chromogens at two wavelengths
may be feasible and applicable to ELISA for much higher efficiency.
This new format of ELISA requires a pair of enzyme labels, a pair of
chromogenic substrates with special spectral properties and par-
ticular methods to process data. A chromogenic substrate of a