A copper(ii)-dipicolylamine-coumarin sensor for maltosyltransferase assay

Article abstract of DOI:10.1039/c9dt01339c

A Cu(ii)-[di(2-methylpyridyl)methylamino]coumarin fluorescence turn-on sensor (Cu-1b) is designed to detect phosphate ions with K_ass = 1.4 × 10⁵ M^-1 in HEPES buffer. Cu-1b is applied to probe the GlgE-catalyzed maltose-tra

Full text of DOI:10.1039/c9dt01339c

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DOI: 10.1039/C9DT01339C
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Copper(II)−dipicolylamine−coumarin sensor for
maltosyltransferase assay
Wei-Li Lee,a,‡ Tse-Wei Hsu,a,‡ Wei-Cheng Hung,a and Jim-Min Fang*ab
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
A Cu(II)−[di(2-methylpyridyl)methylamino]coumarin fluorescence that Pi in extracellular environment (pH 7.4) exists as
turn-on sensor (Cu-1b) is designed to detect phosphate ion with equilibrium of hydrogen phosphate (HPO42−) and dihydrogen
−
Kass = 1.4 × 105 M–1 in HEPES buffer. Cu-1b is applied to probe the phosphate (H2PO4 ).
OH
GlgE catalyzed maltose-transfer reaction of α-maltose-1-
O
GlgE cat.
maltose transfer
HO
HO
phosphate to α-1,4-glucan with concomitant release of phosphate
ion in Mycobacterium tuberculosis.
OH
O
OH
(Glc)n+2
+
HO
P
O
O
O
(Glc)n
O
HO
O
OH
Phosphate ion
Tuberculosis (TB) is a serious infectious disease caused by
Mycobacterium tuberculosis (Mtb), which has persistently
evolved to multi- and extensively-drug resistant strains. The
maltosyltransferase GlgE is recently validated as an appealing
anti-TB target.1 GlgE uses α-maltose-1-phosphate (M1P) as the
substrate for elongation of α-1,4-glucan with concomitant
release of phosphate ion (Pi).2 Inhibition of GlgE causes
accumulation of M1P, and triggers the self-poisoning of Mtb.
GlgE is essential for the survival of Mtb, but absent in human
body. The activity of GlgE can be monitored by quantitative
analysis of the released Pi (Figure 1). An MESG-based
phosphate assay kit is commonly applied to quantify the
released Pi by colorimetric method.3−5 MESG is converted by
purine nucleoside phosphorylase (PNP) in the presence of Pi to
give AMMP with the absorption band shifting from 330 nm to
360 nm. The difference of 360-nm absorbance between MESG
substrate and AMMP product varies depending on the pH of
media.5 Thus, the MESG-based phosphate assay may give
inaccurate results under certain conditions due to pH change
in the enzymatic reactions. Though the aminium ion in MESG is
a crucial moiety for acting as a good PNP substrate, the
positively charged moiety also causes thermal instability of
nucleoside, especially in basic media.5 To overcome the
limitation on using MESG-based phosphate assay, we thus
exploited a new fluorescent turn-on sensor Cu-1b that can
selectively detect Pi over M1P in buffer solution. It is noted
O
P
O
O
α-Maltose-1-phosphate
PNP,
MESG
(M1P)
S
N
H3C
O
N
S
H3C
N
O
HO
O
P
O
N
O
NH
NH2
+
N
NH2
HO
N
N
O
OH OH
Ribose-1-phosphate
MESG
max = 330 nm
AMMP
max = 360 nm

OH OH

Figure 1. GlgE catalyzed elongation of α-1,4-glucan with α-maltose-1-phosphate in M.
tuberculosis. The released phosphate ion is quantified using an MESG assay kit via the
PNP catalyzed phospho-transfer reaction.
Our designed ligand 1b comprises a coumarin fluorophore
that is attached to a di(methylpyridylmethyl)amine (mDPA)
moiety for chelation with Cu(II) ion as the binding site of Pi
(Figure 2, R = Me). The fluorescence of 1b will be dimmed by
chelation with metal ion, and turned bright when the chelation
strength of mDPA−metal motif is attenuated by addition of
negatively charged Pi. The sensor operated by internal charge
transfer (ICT) is expected to be sensitive to Pi and less
susceptible to solvent effect. Furthermore, Cu-1b complex
having methyl substituents at the ortho position of each
pyridine ring6 is expected to provide steric hindrance to
disfavor the binding with bulky M1P. To demonstrate the
important role of the methyl substituents, the related
Cu(II)−DPA−coumarin complex (Cu-1a) was also examined.
Various molecular fluorescent sensors have been devised
to detect phosphate ion, diphosphate ion (PPi) and biological
phosphates.7−13 Metal complexes appear to be good
candidates for operation in water and biological media. The
chemosensors bearing Zn2+−DPA or Cu2+−DPA motif have been
a. Department of Chemistry, National Taiwan University, Taipei 106, Taiwan. E-mail:
jmfang@ntu.edu.tw
b. The Genomics Research Centre, Academia Sinica, Taipei 115, Taiwan.
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: Experimental procedures,
UV-vis, fluorescence, ESI–MS and NMR spectra, Job plots and X-ray
crystallographic data (CCDC 1876683). See DOI: 10.1039/x0xx00000x
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exploited to detect various anions,14−16 for example, Yoon and Glass and coworkers have devised a ditopic sensor, which
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coworkers have shown that some Cu2+−DPA−coumarin features a coumarin aldehyde for iminium formation to assist
DOI: 10.1039/C9DT01339C
complexes can selectively bind with PPi over Pi, AMP, ADP and the Zn2+−DPA unit in chelation with phosphoserine.19 Hamachi
ATP.17 Kikuchi and coworkers have designed a chemosensor and coworkers have demonstrated a glycosyltransferase assay
which contains an azacycle linked to coumarin for by utilizing antracene-9,10-bis(DPA−Zn2+) complex,20 which
complexation with Cd2+ ion to probe PPi in HEPES buffer.18 selectively binds uridine diphosphate (UDP) over galactosyl-
Nonetheless, this sensor exhibits much weaker affinity to Pi.18 UDP.

O
O
O
O
O
O
Mn+
N
N
N
H2PO4
N
N
N
N
N
N
R
R
R
Mn+
R
Mn+
R

1a R = H
1a
R = H
M-
M-
H2PO4
1b R = Me
1b R = Me
R
SensorPi complex,
Turn-on fluorescence
Metal sensor,
Dim fluorescence
DPACoumarin ligand
Figure 2. Ligand 1b derived from merge of di(methylpyridylmethyl)amine (mDPA) with coumarin, and the corresponding DPA−coumarin ligand 1a. Fluorescence dims on formation
of metal−ligand complex, and turns bright on binding with negatively charged phosphate ion via an ICT mechanism.
Scheme 1 shows the synthetic procedures of ligands 1a and
1b. The alkylation reaction of m-silyloxyaniline (2) with two
equivalents of 2-(bromomethyl)pyridine (3a) was accompanied
by in situ removal of the silyl protecting group to give the
DPA−phenol product 4a. Using m-aminophenol for direct
alkylation with 3a (2 equiv) was complicated by O-alkylation to
give an inferior yield of 4a (< 30%). By a similar procedure,
alkylation of m-silyloxyaniline with 2-bromomethyl-6-
methylpyridine (3b) afforded 4b, which underwent Pechmann
condensation with ethyl acetoacetate to give ligand 1b.
a
N
OH
+
Br
N
R
N
H2N
OTMS
(2 equiv)
R
N
2
3a
3b
R = H
R = Me
Figure 3. UV-vis titration curve of ligand 1b (10 μM solution in MeOH) upon
incremental additions of Cu2+ ions (10 mM stock solution in water).
4a
4b
R = H
R
R = Me
b
c
N
O
O
O
O
N
N
Cu2+
N
R
R
N
N
–
2 NO3
R
R
1a
1b
1a
Cu- R = H
1b
Cu- R = Me
R = H
R = Me
Scheme 1. Synthesis of DPA−coumarin ligands (1a and 1b) and their copper(II)
complexes. Reagents and reaction conditions: (a) NaOH(aq), CTAC, rt, 24−48 h; 4a, 55%;
NaHCO3(aq), CTAC, rt, 24−48 h; 4b, 66%. (b) ethyl acetoacetate, 70% H2SO4(aq), rt, 3 h; 1a,
94%; 1b, 90%. (c) Cu(NO3)2, MeOH, H2O, 30 min. CTAC = cetyltrimethylammonium
chloride.
Ligand 1b exhibited an absorption maximum at 358 nm and
a strong fluorescence emission at 442 nm in MeOH solution.
The broad fluorescence band with a large Stokes shift
Figure 4. ORTEP drawing of Cu-1b complex in the dimeric form [H2O•Cu-1b]2 (CCDC
1876683); thermal ellipsoids drawn at the 50% probability level.
indicated that an ICT mechanism involved in the fluorescence
22
emission.21,
After screening various metal ions
(supplementary Figure S1), Cu2+ ion was found to cause the
most prominent chelation-enhanced quenching effect in
fluorescence,23, 24 Formation of Cu-1b complex also showed a
significant blue shift of absorption band from 358 nm to 328
nm (Figure 3).
The UV-vis titration curves exhibited an isosbestic point at
339 nm, indicating the complex of Cu-1b was formed in 1:1
stoichiometry that was also confirmed by Job plot
(supplementary Figure S2D). In the ESI–HRMS spectrum, a
2 | J. Name., 2012, 00, 1-3
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signal at m/z 510.0949 was attributable to the [(Cu-1b)•NO3]+ m/z 566.8617 attributable to the sodiated PiV•i(eCwuA-rt1icble)Oniloinne
ion. The X-ray diffraction of the crystal obtained from aqueous [C24H24CuN3O6P+Na]+ (supplementary FigDuOreI: S106.)1039/C9DT01339C
MeOH indicated the existence of a dimeric [H2O•Cu-1b]2
To cope with GlgE assay that is generally performed in
buffer solution, we also studied the photochemical properties
of Cu-1b sensor in HEPES buffer. Ligand 1b and Cu-1b complex
exhibited the absorption bands at 364 and 334 nm in HEPES
buffer (pH 7.4), respectively. Upon addition of phosphate ion,
formation of the Pi•(Cu-1b) complex was characterized by
showing the isosbestic point at λabs = 343 nm (Figure 5A). The
binding of phosphate ion to Cu-1b also enhanced the
fluorescence intensity at 448 nm (Figure 5B). The 1:1
stoichiometry of Pi•(Cu-1b) complex in HEPES buffer was
verified by Job plot (supplementary Figure S4C). The Kass value
of (1.4 ± 0.2) × 105 M–1 in HEPES buffer was determined from
the fluorescence titration (supplementary Figure S4A). The
changes of fluorescence intensity on binding of Cu-1b with Pi
at low concentrations (e.g. 1−10 μM) exhibited a good linear
relationship, and are suitable for quantitation of Pi in
physiological condition (supplementary Figure S4B). The limit
of detection (LOD) is 2.08 × 10−6 M. It was noted that the
intensity of absorption and fluorescence throughout the
titration process of Cu-1b with Pi could not revert to the
original absorption or fluorescence intensity of free ligand 1b
at λabs = 364 nm and λem = 448 nm, respectively. Thus, addition
of Pi to Cu-1b likely forms the Pi•(Cu-1b) complex, rather than
abstracts Cu2+ ion from Cu-1b to release free ligand 1b.
complex with two water molecules coordinated to the two
Cu(II) centers (Figure 4).
In comparison, the UV-vis titration curves of ligand 1a with
Cu2+ ion did not exhibit an isosbestic point (supplementary
Figure S2A). The Job plot showed the maximum at 0.4 molar
ratio (supplementary Figure S2B), which could be ascribed to
the combination of Cu-1a and 1a-Cu-1a complexes at
equilibrium. As the 1:1 stoichiometry of Cu-1b was clearly
defined, it was chosen to investigate the binding events with Pi
and M1P. Upon incremental addition of Pi to Cu-1b in MeOH
solution, the absorption at 358-nm gradually increased at the
expense of the 328-nm absorption of Cu-1b (supplementary
Figure S3A). As that depicted in Figure 2, coordination of
phosphate ion reduced the mDPA−Cu2+ chelation strength, and
thus caused a bathochromic shift of the absorption band. The
binding of Cu-1b with Pi also rendered appreciable
enhancement of the fluorescence at 442 nm, which reached
saturation upon addition of 1.5 equiv Pi (supplementary Figure
S3B). The association constant (Kass) was calculated to be (6.2 ±
0.1) × 106 M–1 in MeOH based on 1:1 stoichiometry for the
Pi•(Cu-1b) complexation. The 1:1 complex of Pi•(Cu-1b) was
supported by the ESI−HRMS analysis, which showed a signal at
Figure 5. Titration curves of sensor Cu-1b (10−5 M) in HEPES buffer (pH 7.4): (A & B) Incremental additions of Pi; and (C & D) incremental additions of M1P. (E) Fluorescence
enhancement by addition of Pi to a solution of M1P (1.0 × 10−4 M) and maltotetraose (1.25 × 10−4 M) in HEPES buffer (20 mM, pH 7.53, containing 10 mM MgCl2). (F) Detection of
GlgE catalyzed reaction in HEPES buffer (20 mM, pH 7.53, containing 10 mM MgCl2) by fluorescence enhancement. λex = 343 nm, λem = 448 nm.
The Cu-1b sensor was insensitive to halide, acetate and In contrast, enhanced fluorescence was observed by addition
sulfate ions in HEPES buffer, but bound with diphosphate ion of Pi (1.0 × 10−4 M) to an HEPES solution containing M1P (1.0 ×
(supplementary Figure S5). To our anticipation, Cu-1b could 10−4 M), maltotetraose (1.25 × 10−4 M) and Cu-1b (1.0 × 10−5
not effectively bind with M1P as no appreciable change of the M) (Figure 5E). Compared with the strong binding of Pi, the
absorption or fluorescence was observed (Figure 5C and 5D). access of M1P bearing a bulky maltosyl group to Cu-1b would
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be blocked by the methyl substituents on the pyridine rings.
When M1P (1.0 × 10−4 M) and maltotetraose (1.0 × 10−4 M)
were incubated with GlgE (47.5 μg) and Cu-1b (1.0 × 10−5 M) in
HEPES buffer for 18 h at 37 C, the fluorescence at 448 nm
increased as Pi was released from the enzymatic reaction
(Figure 5F).
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In summary, we have developed a coumarin based
Cu(II)−mDPA sensor (Cu-1b) using ICT as the sensing
mechanism for selective detection of phosphate ion in HEPES
buffer with an association constant of 1.4 × 105 M–1. Upon
binding with phosphate ion, the dim sensor Cu-1b turns bright.
Our experimental results indicate that the sensing event is
consistent with formation of Pi•(Cu-1b) complex, rather than
abstraction of Cu2+ ion from Cu-1b to release free ligand 1b.
The methyl substituents introduced to the ortho-positions of
the pyridine rings in the DPA moiety not only enhance the
metal–ligand binding strength but also provide steric
hindrance to prevent the access of bulky maltose-1-phosphate.
We also demonstrate that the Cu(II)−mDPA−coumarin sensor
acts as a robust and sensitive sensor to probe the GlgE
catalyzed reaction. This fluorescence turn-on assay may
facilitate the screening of GlgE inhibitors for discovery of new
anti-TB drugs.
24 R. Bergonzi, L. Fabbrizzi, M. Licchelli and C. Mangano, Coord.
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This research was supported by Academia Sinica and
Ministry of Science and Technology [MOST 103-2113-M-002-
017-MY3, MOST 106-0210-01-15-02, and MOST 107-0210-01-
19-01]. We thank Mr. Tzu-Hsien Tseng at the Department of
Chemistry, National Chung Hsing University for X-ray
crystallographic analysis.
Conflicts of interest
There are no conflicts to declare.
Notes and references
‡ W.-L. L and T.-W. H contributed equally.
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DOI: 10.1039/C9DT01339C
Table of Contents
OH
O
HO
HO
OH
OH
O
(Glc)n
O
HO
O
OH
O
O
O
O
N
N
N
N
O
P
O
O
H3C
H3C
O
Cu2+
Cu2+
α-Maltose-1-phosphate
N
N
O
GlgE cat.
maltose transfer
P
O
H3C
H3C
OH
1b
Cu- sensor
Dim fluorescence
SensorPi complex,
Turn-on fluorescence
Binding
O
O
(Glc)n+2
+
HO
P
O
A fluorescence turn-on sensor (Cu-1b) is designed for selective detection of the phosphate
ions released from GlgE catalyzed maltose-transfer reaction.