In-situ
investigation
of
mammalian
inorganic
polyphosphate
localization using novel selective fluorescent probes JC-D7 and JCD8
Plamena R. Angelova3,*, Bikram Keshari Agrawalla2,*, Pia A. Elustondo1, Jacob
Gordon1, Toshikazu Shiba4, Andrey Y. Abramov3, Young-Tae Chang2,#, Evgeny V.
Pavlov1,#.
1
Dalhousie University, Halifax, NS, Canada, 2National University of Singapore,
3
Institute of Neurology, University College London, London, UK, 4Regenetiss, Japan
Contents
General conditions.
Synthesis of JC-D7 and JC-D8
Chart S1
: Building block structure of JC dye library
Scheme S1 : Synthetic scheme of JC-D7 and JC-D8
Figure S1
: HR-MS (ESI) spectra of JC-D7
Figure S2
: HR-MS (ESI) spectra of JC-D7
Figure S3
: 1H NMR spectra of JC-D7
Figure S4
:13C NMR spectra of JC-D7
Figure S5
: 1H NMR spectra of JC-D8
Figure S6
:13C NMR spectra of JC-D8
Quantum yield measurement
Equation S1 : Quantum yield measurement
Figure S7
: Standard curve and equation
Figure S8
: Fluorescence response in different buffer condition.
Figure S9
: pH dependency of the probes.
General Conditions.
All the chemicals (building block aldehydes plus others) and solvents were purchased
from Sigma Aldrich, Alfa Aesar, Fluka, MERCK or Acros, and used without further
purification. 2-chlorotrityl chloride polystyrene resin (100-200 mesh, 1% DVB crosslinking) was purchased from BeadTech. Polyphosphate-60 (Polyp-60 in average have
sixty phosphate residues) used in the experiment was provided by Dr. T. Shiba
(Regenetiss Co. Ltd.). All in vitro screening was done in 20 mM HEPES buffer pH 7.4.
Normal phase purifications were carried out using Merck Silica Gel 60 (particle size:
0.040-0.063 mm, 230-400 mesh). Analytical characterization was performed on an
HPLC-MS (Agilent-1200 series) with a DAD detector and a single quadrupole mass
spectrometer (6130 series) with an ESI probe. Analytical method, unless indicated
otherwise: eluents: A: H2O (0.1% HCOOH), B: ACN (0.1% HCOOH), gradient from 5 to
95% B in 10 min; C18(2) Luna column (4.6 × 50 mm 2, 5 mm particle size). 1 H-NMR
and 13 C-NMR spectra were recorded on BrukerAvance 300 NMR spectrometer, and
chemical shifts are expressed in parts per million (ppm).
Chart S1. Aldehyde building blocks for benzimidazolium dye library (JC). R-CHO, R=
the fragment above.
Synthetic scheme of JC-D7 and JC-D8
Scheme S1. Synthesis of JC-D7 and JC-D8: (a) triethylorthoacetate, catalytic amount of
p-toluenesulfonic acid (H+), toluene, reflux; (b) KOH, MeI, acetone; (C) NaI, Acetonitryl,
80 °C; (d) 4, HATU, DIPEA, 30% DMF/DCM; (e) 1-napthaldehyde or 4-methyl-1napthaldehyde,pyrrolidine, NMP; (f) 5% TFA/DCM.
Synthetic procedure up to the key intermediate 4 has been explained earlier by (Wang
et al;, JACS, 2006; ref-1d). Resin 4 (10 mg, 1 eq) was added to two individual
containers containing, (4-methyl-1-naphthaldehyde; 10eq) and (1-naphthaldehyde; 10
eq) separately. To both 1-methyl-2-pyrrolidinone (300 uL) solution and pyrrolidine (2 uL)
was added and the reactions were stirred under N2 atmosphere at R.T. for 24 h. The
resin was filtered and washed with DMF (5 times), alternatively dichloromethane and
methanol (5 times), dichloromethane (5 times) and dried in vacuum.
Resins 5 and 6 (10 mg) were suspended in 2% trifluoroacetic acid/dichloromethane
cleavage cocktail solution (0.5 mL) and shook for 15 min. The resins was filtered off and
washed with dichloromethane (1 mL) and methanol (1 mL). The solution was collected
and evaporated until dry to obtain the Benzimidazolium dyes JC-D7andJC-D8.
JC-D7: 1H NMR (300 MHz, MeOD4) δ 8.34 (d, 1H, J=12 Hz), 8.30 (d, 1H, J=9.3 Hz),
8.19 (s, 1H), 8.14 (m, 1H), 8.09 (s, 1H), 7.92 (dd, 2H, J=4.5, 7.8 Hz), 7.64 (td, 1H, J=
4.9, 1.2 Hz), 7.57 (td, 1H, J=4.9, 1.2 Hz), 7.21 (t, 1H, J=4.5 Hz), 6.98 (d, 1H, J=12 Hz),
6.93 (d, 1H, J=7.2 Hz), 4.12 (s, 1H), 4.09 (s, 1H), 3.51 (s, 3H), 3.35 (t, 2H, J=6 Hz), 3.16
(m, 4H), 2.96 (t, 2H, J=6 Hz), 2.07 (t, 2H, J=7.2 Hz), 1.91 (m, 6H).
13
C NMR (75 MHz,
MeOD4) δ 176.97, 151.82, 147.71, 135.29, 132.89, 132.75, 132.71, 132.53, 132.18,
131.92, 130.21, 129.01, 128.34, 127.42, 126.83, 124.62, 116.23, 112.14, 47.79, 46.62,
40.82, 38.14, 36.41, 33.51, 29.30, 27.02, 25.92, 25.03. HRMS (ESI): m/zcalcd
(C28H31Cl2N4O): 509.1869; found: 509.1847.
JC-D8: 1H NMR (300 MHz, MeOD4) δ 8.42 (d, 1H, J=5.4 Hz), 8.41 (d, 1H, J=12 Hz),
8.18 (s, 1H), 8.13 (m, 1H), 7.64 (m, 3H), 7.08 (d, 1H, J=7.5 Hz), 6.93 (d, 1H, J=12 Hz),
6.82 (d, 1H, J=7.2 Hz), 4.11 (broad, 2H), 3.52 (s, 3H), 3.35 (t, 2H, 6Hz), 3.16 (m, 4H),
2.96 (t, 2H, J=5.7 Hz), 2.59 (s, 3H), 2.07 (t, 2H, J=7.5 Hz), 1.92 (m, 4H).
13
C NMR (75
MHz, MeOD4) δ 177.167, 152.22, 148.11, 140.16, 134.46, 133.08, 132.87, 132.43,
132.11, 131.29, 128.83, 128.42, 127.76, 127.46, 126.53, 125.40, 116.40, 111.62, 47.95,
46.82, 41.02, 38.34, 36.62, 33.71, 29.51, 27.22, 26.16, 25.23, 19.93.HRMS (ESI):
m/zcalcd (C29H33Cl2N4O): 523.2026; found: 523.2042.
Supplementary Figure S1.HR-MS (ESI) of JC-D7
Supplementary Figure S2. HR-MS (ESI) of JC-D8
Supplementary Figure S3.JC-D7 1H NMR
Supplementary Figure S4.JC-D713C NMR
Supplementary Figure S5.JC-D81H NMR
Supplementary Figure S6.JC-D713C NMR
Quantum-Yield Measurements.
Quantum yields were calculated by measuring the integrated emission area of the
florescent spectra, and referring them to the area measured for Fluorescein in 0.1 N
NaOH (Φ = 0.95) with excitation at 450 nm30,31. Quantum yield of the JC compounds
were then calculated using the equation below, where F represents the area of
fluorescent emission, η is the reflective index of the solvent, and the Abs is the
absorbance and excitation wavelength selected for standards and samples. Emission
was integrated from 450 nm to 650 nm.
Supplementary Equation S1. Quantum yield measurement
Primary screening.
Benzimidazolium compounds were transferred to Greiner 96 well black polypropylene
plates (final concentration as 10 µM) and tested with 0.5 µM, 1 µM, 5 µM and 10 µg/mL
polyP (60). Control intensity was measured in10 mM HEPES buffer (pH = 7.4).
Fluorescent spectra were recorded on a SpectraMax M2 fluorescent plate reader with
excitation at 390 nm (cutoff: 420 nm), emission 450 to 700 nm.
Fluorescence intensity (A.U.)
1.0
0.8
0.6
0.4
0.2
JC-D7 (535nm)
JC-D8 (505nm)
0.0
0
10
20
30
40
50
60
70
80
PolyP (ug/mL)
JC-D7
JC-D8
Equation line
Kd
Standard Error
28.84382
0.40451
23.50841
0.57602
y = A2 + (A1-A2)/(1 + (x/x0)^p)
Supplementary Figure S7: Standard curve and equation
Fluorescence based fractional saturation curve of JC-D7 and JC-D8. Values were
represented as means (n=3) of the fluorescence fold increase after incubation with the
polyp, and fitted to the nonlinear binding curve by the logistic equation (GraphPad Prism
8.0) to estimate the dissociation constant KD: 28.84 ± 0.4 and 23.5 ± 0.5 for JC-D7 and
JC-D8 respectively.
350
Fluorescence Intensity (A.U.)
A)
300
JC-D7 20 mM HEPES buffer
JC-D7 150 mM KCl buffer
250
200
150
100
50
0
0
0.5
1
2
4
2
4
PolyP in µg/mL
500
Fluorescence Intensity (A.U.)
B)
450
JC-D8 20 mM HEPES buffer
400
JC-D8 150 mM KCl buffer
350
300
250
200
150
100
50
0
0
0.5
1
PolyP in µg/mL
Supplementary Figure S8: Fluorescence response in different buffers in physiological
pH condition (pH:7.4).
JC-D7 and JC-D8 fluorescence for PolyP was tested in 20 mM HEPES (4-(2hydroxyethyl)-1-piperazineethanesulfonic acid ) buffer at pH 7.4 and 150 mM KCl
(Potassium chloride) buffer at pH 7.4. We found that both the probes, JC-D7 and JC-D8
show reduced fluorescence intensity in 150 mM KCl buffer solutions, in comparison with
HEPES buffer.
Relative Fluorescence Intensity (RFU)
A)
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
B)
Relative Fluorescence Intensity (RFU)
pH02
pH03
pH04
pH05
pH06
pH07
pH08
pH09
pH10
pH11
10uM
10uM
10uM
10uM
10uM
10uM
10uM
10uM
10uM
10uM
JC-D7
JC-D7
JC-D7
JC-D7
JC-D7
JC-D7
JC-D7
JC-D7
JC-D7
JC-D7
pH02
pH03
pH04
pH05
pH06
pH07
pH08
pH09
pH10
pH11
1.4
1.2
1
0.8
0.6
0.4
0.2
0
10uM
10uM
10uM
10uM
10uM
10uM
10uM
10uM
10uM
10uM
JC-D8
JC-D8
JC-D8
JC-D8
JC-D8
JC-D8
JC-D8
JC-D8
JC-D8
JC-D8
Supplementary Figure S9: pH dependency of the probes.
pH response of JC-D7 and JC-D8 were tested in wide range of pH (pH-2 to pH-11). The
various pH solution were prepared using 20 mM HEPES buffer along with the pH
adjustment with concentrates Sodium hydroxide ( NaOH) and hydrochloric acid (HCl).
Both the probes JC-D7 and JC-D8 do not show any significant pH dependency in the
pH range of 2-11; and the fluorescence response remain stable.

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