Electronic Supplementary Information A far-red fluorescent … · 2017-12-07 · A far-red fluorescent probe based on a phospha-fluorescein ... Fig. S3 Determination of acid dissociation

Electronic Supplementary Information

for

A far-red fluorescent probe based on a phospha-fluorescein scaffold for cytosolic calcium imaging

Hiroaki Ogasawara, Marek Grzybowski, Riho Hosokawa, Yoshikatsu Sato, Masayasu Taki,*

Shigehiro Yamaguchi*

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2017

S1

Contents

1. Experimental Details S2

Scheme S1 S3

2. Photophysical Properties S8

3. Electrochemical Properties S9

4. Cell Culture Experiments S9

5. References S11

Fig. S1 Absorption and emission spectra of CaPF-1 S12

Fig. S2 Determination of dissociation constant for Ca2+ (Kd) S12

Fig. S3 Determination of acid dissociation constant (pKa) S13

Fig. S4 pH dependence of CaPF-1 S13

Fig. S5 Metal ion selectivity of CaPF-1 S14

Fig. S6 Chemical Stability of CaPF-1 in 10 mM GSH (reduced form) solution S14

Fig. S7 Cyclic voltammogram of POF-Me2 S15

Fig. S8 Costaining with CaPF-1-AM and Calcein S15

Fig. S9 Costaining with CaPF-1-AM and ER Tracker Red S15

Fig. S10 Evaluation of cytotoxicity using MTT assay S16

Table S1 Fluorescence quantum yields of CaPF-1 S16

6. NMR Spectra S17

S2

1. Experimental Details

General. 1H, 13C{1H}, and 31P{1H} NMR spectra were recorded with a JEOL ECZ 400

spectrometer equipped with a SuperCOOL probe in CDCl3 or methanol-d4 (400 MHz for 1H,

and 162 MHz for 31P). 13C{1H} NMR spectra (150 MHz) of the compounds were recorded with

a JEOL ECA 600 II spectrometer equipped with an UltraCOOL probe. The chemical shifts in 1H NMR spectra are reported in d ppm using the signals of CHCl3 (7.26 ppm) or methanol (3.31

ppm) as an internal standard and those in 13C NMR spectra are reported using the solvent

signals of CDCl3 (77.16 ppm), methanol-d4 (49.00 ppm), DMSO-d6 (39.52 ppm) as an internal

standard. The chemical shifts in 31P NMR spectra are reported using H3PO4 (0.00 ppm) as an

external standard. Mass spectra were measured with an Exactive Mass Spectrometer (Thermo

scientific) with the ionization methods of electrospray ionization (ESI). Reversed phase column

chromatography was performed using LC-Forte/R (YMC) equipped with a reversed phase silica

gel column (YMC-DispoPack AT ODS 12g, YMC). Preparative reversed phase HPLC was

performed on an YMC Triart C18 (10 × 250 mm) column (YMC) using Delta 600 system

(Waters). Anhydrous dichloromethane, THF, and DMF were purchased from Kanto Chemicals

and further purified by Glass Contour Solvent Systems. 5-Methyl-5'-nitro BAPTA,1

5-amino-5'-methyl BAPTA tetramethyl ester,2 and bis(t-butyldimethylsiloxy)-phospha-xanthone

1,3 and POF-Me24 were prepared according to the literature methods. All other chemicals were

purchased from commercial suppliers and used without further purification.

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A. Phospha-fluorescein based calcium probe CaPF-1

B. Membrane permeable CaPF-1-AM

Scheme. S1 Synthesis of CaPF-1 and its acetoxymethyl ester CaPF-1-AM. Reagents and

conditions: (a) i) 4-tert-butoxycarbonyl-2,6-dimethoxyphenyllithium, THF, –78 °C then rt, ii)

10% TFA, CH2Cl2, rt, 50 min, 63% for 2 steps; (b) 50% TFA, CH2Cl2, rt, 1 h, 74%; (c) i)

5-amino-5'-methyl BAPTA tetramethyl ester, HATU, HOBt·H2O, DMF, 40 h, ii) MeOH, NaOH

aq. 4.5 h, 17% for 2 steps; (d) bromomethyl acetate, Et(iPr)2N, CH2Cl2, 3 d, 49%; (e) H2, Pd/C,

EtOAc, rt, 25 h, 54%; (f) 3, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]py-

ridinium 3-oxid hexafluoro-phosphate (HATU), 1,2,3-benzotriazol-1-ol monohydrate

(HOBt·H2O), DMF, 15 h, 56% (based on 3).

P

O

O PhTBDMSO OTBDMS

PO Ph

HO O

OMeMeO

COOR1

PO Ph

HO O

OMeMeO

HN O

N(CH2CO2H)2

2

CaPF-1

R1 = tBuR1 = H3

(b)

1

N(CH2CO2H)2O O

(c)(a)

NH2

N(CH2CO2Me)2 N(CH2CO2Me)2O O

5-amino-5'-methyl BAPTA tetramethyl ester

NO2

N(CH2CO2R2)2 N(CH2CO2R2)2O O

R2 = HR2 = AM6

(d)

NH2

N(CH2CO2AM)2 N(CH2CO2AM)2O O

PO Ph

HO O

OMeMeO

HN O

N(CH2CO2AM)2

CaPF-1-AM

N(CH2CO2AM)2O O

(e) (f)

S4

tert-Butyl 4-bromo-3,5-dimethoxybenzoate.5 4-Bromo-3,5-dimethoxybenzoic acid (5.22 g,

20.0 mmol), DCC (5.36 g, 26.0 mmol) and DMAP (244 mg, 2.00 mmol) were dissolved in 60

mL of dichloromethane under a nitrogen atmosphere. The solution of tert-butanol (3.85 g, 52.0

mmol) in 20 mL of dichloromethane was added and the resulting mixture was stirred for 3 days

at room temperature. The white precipitate was removed by filtration and the filtrate was

concentrated under reduced pressure. The mixture was purified by silica gel column

chromatography (hexane/CH2Cl2 3/2 to 1/1) to give 3.34 g (10.5 mmol, 53%) of 4 as white solid. 1H NMR (400 MHz, CDCl3) d 7.20 (s, 2H), 3.95 (s, 6H), 1.61 (s, 9H). 13C{1H} NMR (125 MHz,

CDCl3) d 165.3, 157.0, 132.2, 106.3, 105.6, 81.9, 56.7, 28.3.

POF tert-butyl ester 2. To a solution of tert-butyl 4-bromo-3,5-dimethoxybenzoate (567 mg,

1.79 mmol) in anhydrous THF (8.0 mL) was added sec-BuLi (1.05 M in hexane and

cyclohexane, 1.6 mL, 1.68 mmol) at –78 °C over 13 min and the mixture was stirred for 3 h at

the same temperature. A solution of bis(t-butyldimethylsiloxy)-phospha-xanthone 1 (203 mg,

0.360 mmol) in anhydrous THF (2.0 mL) was added dropwise at –78 °C over 13 min. The

reaction mixture was allowed to warm to room temperature and further stirred for 17 h. After

removal of the solvent under reduced pressure, 5.0 mL of a 10/1 CH2Cl2/TFA mixed solution

was added to the mixture followed by stirring for 50 min at room temperature. An aqueous

solution of NaOH was added to the solution and the mixture was extracted with ethyl acetate

three times. The combined organic layer was washed with 1 M HCl aqueous solution and brine,

dried over Na2SO4, filtered, and concentrated under reduced pressure. The obtained red solid

was subjected to reversed-phase column chromatography (C18) eluted with a linear gradient of

20–40% CH3CN in an aqueous solution of 5 mM (NH4)2CO3. The fraction was acidified with 2

M HCl aqueous solution and extracted with ethyl acetate three times. The combined organic

layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced

pressure to afford 127 mg (0.228 mmol, 63 %) of 2 as a red solid. 1H NMR (400 MHz,

CD3OD): d 7.79–7.71 (m, 2H), 7.59 (dt, J = 1.9, 8.2 Hz, 1H), 7.56–7.48 (m, 2H), 7.43 (dd, J =

1.2, 9.2 Hz, 2H), 7.23 (d, J = 2.4 Hz, 1H), 7.19 (d, J = 2.4 Hz, 1H), 7.06 (dd, J = 6.2, 9.2 Hz,

2H), 6.59 (dd, J = 1.8, 11.6 Hz, 2H), 3.82 (s, 3H), 3.81 (s, 3H), 1.66 (s, 9H). 13C{1H} NMR (150

MHz, CD3OD): d 166.4 (s, C), 159.2 (s, C), 158.8 (s, C), 150.8 (d, JCP = 8.6 Hz, C), 139.0 (br,

C), 137.5, (d, JCP = 89.0 Hz, C), 136.5 (s, C), 133.98 (s, CH), 133.97 (d, JCP = 109.1 Hz, C),

131.1 (d, JCP = 11.4 Hz, CH), 130.3 (d, JCP = 12.9 Hz, CH), 127.7 (d, JCP = 5.7 Hz, C), 125.2 (br,

CH), 118.9 (s, C), 106.0 (s, CH), 105.9 (s, CH), 83.1 (s, C), 56.8 (s, CH3), 56.7 (s, CH3), 28.4 (s,

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CH3). One aromatic CH peak is overlapped. 31P{1H} NMR (162 MHz, CD3OD): d 10.2. HRMS

(ESI): m/z calcd. for C32H29O7PNa: 579.1549 ([M+Na]+); found. 579.1541.

POF carboxylic acid 3. A solution of 2 (18.5 mg, 33.2 µmol) in a 1/1 (v/v) mixture of

CH2Cl2/TFA (2.0 mL) was stirred at room temperature for 1 h. All the volatiles were removed

under reduced pressure. The obtained red solid was subjected to reversed phase HPLC

(YMC-Triart C18, 10 × 250 mm) using a linear gradient of 20–80% CH3CN in water containing

0.1% TFA. The fraction was lyophilized to give 12.3 mg (24.6 µmol, 74%) of 3 as a red powder. 1H NMR (400 MHz, CD3OD): d 7.80–7.71 (m, 2H), 7.65–7.57 (m, 1H), 7.57–7.49 (m, 4H),

7.21 (br d, J = 14.8 Hz, 2H), 7.08 (dd, J = 6.4, 9.6 Hz, 2H), 6.61 (br s, 2H), 3.83 (s, 3H), 3.82 (s,

3H). 13C{1H} NMR (150 MHz, DMSO-d6): d 166.8 (s, C), 157.9 (d, J = 35.9 Hz, C), 157.4 (s,

C), 157.0 (s, C), 147.3 (d, J = 5.9 Hz, C), 134.0 (d, J = 106.4 Hz, C), 133.9 (s, C), 132.4 (s, CH),

129.4 (d, J = 10.1 Hz, CH), 129.2 (d, J = 12.9 Hz, CH), 120.3 (br, CH), 118.3 (br, CH), 117.3 (s,

C), 116.3 (br, CH), 105.2 (d, J = 8.7 Hz, CH), 56.5 (s, CH3), 56.2 (s, CH3). One aromatic C peak

and one aromatic CH peak are overlapped). 31P{1H} NMR (162 MHz, CDCl3): d 10.2. HRMS

(ESI): m/z calcd. for C28H20O7P: 499.0947 ([M–H]–); found. 499.0949.

CaPF-1. A solution of 3 (23.5 mg, 47.0 µmol), 5-amino-5'-methyl BAPTA tetramethyl ester

(134 mg, 239 µmol), HATU (317 mg, 833 µmol), and HOBt·H2O (167 mg, 1.24 mmol) in

anhydrous DMF (9.0 mL) was stirred for 40 h at room temperature. After removal of the solvent,

the mixture was subjected to reversed phase column chromatography (YMC-DispoPack AT

ODS 12g) using a linear gradient of 20–80% CH3CN in water containing 0.1% TFA. The

fractions containing the target compound were collected and concentrated under reduced

pressure followed by addition of water and extraction with ethyl acetate three times. The

combined organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated

under reduced pressure. The obtained red solid was dissolved in a mixture of THF (1.5 mL) and

1 M NaOHaq (6.0 mL) and the resulting solution was stirred for 4.5 h at room temperature. The

reaction mixture was acidified with 1 M HCl aqueous solution and extracted with ethyl acetate

four times. The combined organic layers were concentrated under reduced pressure. The crude

mixture was subjected to reversed phase column (YMC-DispoPack AT ODS 12 g) using a

linear gradient of 20–100% CH3CN in water containing 0.1% TFA. The lyophilized compound

was further purified with reversed phase HPLC (YMC-Triart C18, 10 × 250 mm) eluted with a

linear gradient system of 20–80% CH3CN in water containing 0.1% TFA. The fraction was

S6

lyophilized to afford 8.1 mg (8.20 µmol, 17%) of CaPF-1 as a red solid. 1H NMR (400 MHz,

CD3OD): d 7.76 (dd, J = 7.2, 13.2 Hz, 2H), 7.61 (t, J = 7.2 Hz, 1H), 7.58–7.51 (m, 3H), 7.46 (d,

J = 8.0 Hz, 2H), 7.29 (d, J = 9.2 Hz, 1H), 7.24 (d, J = 2.0 Hz, 1H), 7.20 (d, J = 2.0 Hz, 1H),

7.11 (dd, J = 6.2, 9.4 Hz, 2H), 7.02 (d, J = 8.4 Hz, 1H), 6.90–6.86 (m, 2H), 6.71 (d, J = 8.0 Hz,

1H), 6.61 (d, J = 9.2 Hz, 2H), 4.50–4.35 (m, 4H), 4.07 (s, 4H), 4.01 (s, 4H), 3.87 (s, 3H), 3.86 (s,

3H), 2.30 (s, 3H), 13C{1H} NMR (150 MHz, CD3OD): d 175.8 (s, C), 175.6 (s, C), 167.7 (s, C),

159.3 (s, C), 159.0 (s, C), 152.1 (s, C), 152.0 (s, C), 150.8 (d, J = 7.2 Hz, C), 139.6 (s, C), 139.0

(br, CH), 137.7 (s, C), 137.6 (br, CH), 137.4 (s, C), 134.8 (d, J = 54.6 Hz, C), 134.0 (s, CH),

133.9 (d, J = 109.2 Hz, C), 131.1 (d, J = 10.1 Hz, CH), 130.3 (d, J = 13.1 Hz, CH), 128.0 (s, C),

127.8 (s, CH), 125.1 (br, CH), 122.8 (s, CH), 120.5 (s, CH), 120.2 (s, CH), 117.8 (s, C), 115.4

(d, J = 5.7 Hz, CH), 108.7 (s, C), 104.7 (s, CH), 104.6 (s, CH), 101.3 (s, C), 68.5 (s, CH2), 68.1

(s, CH2), 57.0 (s, CH3), 56.8 (s, CH3), 56.1 (s, CH2), 55.8 (s, CH2), 21.1 (s, CH3). One aromatic

CH carbon are overlapped. 31P{1H} NMR (162 MHz, CD3OD): d 10.2. HRMS (ESI): m/z calcd.

for C51H45N3O16P: 986.2537 ([M–H]–); found. 986.2501.

Compound 6. A solution of 5-methyl-5'-nitro BAPTA (136 mg, 0.254 mmol), acetoxymethyl

bromide (0.14 mL, 1.52 mmol), and diisopropylethylamine (0.27 mL, 1.52 mmol) in dry

dichloromethane (3.0 mL) was stirred at room temperature for 3 d under a nitrogen atmosphere.

After addition of 0.1 M HCl aqueous solution, the organic layer was separated, and the aqueous

layer was extracted with dichloromethane two times. The combined organic layer was washed

with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure.

The obtained mixture was subjected to reversed phase column chromatography

(YMC-DispoPack AT ODS 12g) using a linear gradient of 20–100% CH3CN in water

containing 0.1% TFA. The fraction was lyophilized to afford 102 mg (0.123 mmol, 49%) of 6 as

yellow viscous oil. 1H NMR (400 MHz, CDCl3): d 7.85 (dd, 1H, J = 8.6, 2.6 Hz), 7.78 (d, 1H, J

= 2.6 Hz), 6.84 (d, 1H, J = 8.0 Hz), 6.77–6.69 (m, 3H), 5.65 (s, 4H), 5.62 (s, 4H), 4.41–4.26 (m,

8H), 4.14 (s, 4H), 2.28 (s, 3H), 2.08 (s, 6H), 2.07 (s, 6H). 13C NMR (150 MHz, CDCl3): d 170.2,

169.6, 169.5, 169.4, 150.6, 149.0, 144.8, 141.6, 136.5, 133.3, 122.6, 120.6, 118.3, 116.7, 115.2,

108.4, 79.7, 79.3, 68.0, 67.0, 53.7, 53.5, 21.1, 20.8. One methyl peak is overlapped. HRMS

(ESI): m/z calcd. for C35H41N3O20Na: 846.2181, ([M+Na]+); found 846.2167.

Compound 5. A suspension of 6 (50.4 mg, 61.2 µmol) and 10% Pd/C(14.3 mg, 14.1 µmol) in

ethyl acetate (10 mL) was stirred at room temperature for 25 h under a hydrogen atmosphere.

S7

The reaction mixture was filtered through the plug of glass filter, and the filtrate was

concentrated under reduced pressure. The obtained mixture was subjected to reversed phase

column chromatography (YMC-DispoPack AT ODS 12g) using a linear gradient of 20–80%

CH3CN in water containing 0.1% TFA. The fraction was lyophilized to afford 30.1 mg (33.2

µmol, 54%) of 5 as a TFA salt (colorless solid). 1H NMR (400 MHz, CDCl3): d 7.05 (br s, 1H),

6.92 (br d, J = 7.2Hz, 1H), 6.84–7.78 (m, 2H), 7.73–6.68 (m, 2H), 5.94 (br s, 2H), 5.60 (s, 4H),

5.59 (s, 4H), 4.34–4.18 (m, 4H), 4.13 (s, 4H), 4.10 (s, 4H), 2.27 (s, 3H), 2.06 (s, 6H), 2.03 (s,

6H). 13C{1H} NMR (150 MHz, CDCl3): d 170.6, 170.1, 170.0, 169.7, 151.1, 150.5, 138.8, 135.8,

133.7, 125.9, 122.2, 120.2, 120.1, 115.9, 114.2, 108.9, 79.53, 79.47, 67.9, 66.9, 53.7, 53.5, 21.1,

20.73, 20.69. HRMS (ESI): m/z calcd. for C35H43N3O18Na: 816.2439 ([M+Na]+); found

816.2427.

CaPF-1-AM. A solution of 3 (26.1 mg, 52.2 µmol), 5 (125 mg, 138 µmol), HATU (101 mg,

265 µmol), and HOBt•H2O (35.8 mg, 265 µmol) in anhydrous DMF (1.5 mL) was stirred for 15

h at room temperature. After addition of 0.2 M HCl aqueous solution, the aqueous layer was

separated and extracted with ethyl acetate three times. The combined organic layer was washed

with 0.2 M HCl aqueous solution, brine, dried over Na2SO4, filtered, and concentrated under

reduced pressure. The crude mixture was subjected to reversed phase column (YMC-DispoPack

AT ODS 12g) where the eluent was gradually changed from 40% CH3CN to 100% CH3CN with

0.1% TFA. The fraction containing CaPF-1-AM was concentrated under reduced pressure

followed by addition of water and extracted with ethyl acetate three times. The combined

organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under

reduced pressure. The obtained red solid was further purified with reversed phase HPLC, where

the eluent was gradually changed from 40% CH3CN to 100% CH3CN with 0.1% TFA.

Lyophilization of the obtained product gave 37.5 mg (29.4 µmol, 56%) of CaPF-1-AM as a red

powder. 1H NMR (400 MHz, methanol-d6) d 7.76 (dd, J = 6.6 Hz, 12.8 Hz, 2H), 7.61 (t, J = 6.6

Hz, 1H), 7.57-7.51 (m, 3H), 7.45 (d, J = 8.0 Hz, 2H), 7.30–7.17 (m, 3H), 7.11 (dd, J = 6.2, 9.4

Hz, 2H), 6.93 (d, J = 8.0 Hz, 1H), 6.84 (s, 1H), 6.78 (d, J = 8.0 Hz, 1H), 6.69 (d, J = 8.0 Hz,

1H), 6.61 (br, 2H), 5.63 (s, 4H), 5.62 (s, 4H), 4.41–4.31 (m, 4H), 4.21 (s, 4H), 4.16 (s, 4H), 3.87

(s, 3H), 3.86 (s, 3H), 2.28 (s, 3H), 2.05 (s, 6H), 2.02 (s, 6H). 31P{1H} NMR (162 MHz, CDCl3):

d 10.1. HRMS (ESI): m/z calcd. for C63H61N3O24P: 1274.3383 ([M–H]–); found. 1274.3368.

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2. Photophysical Properties

Photophysical measurements. HEPES buffer solutions (50 mM, pH 7.4, 50 mM KCl)

containing 1 mM Ca(NO3)2 or 1 mM EGTA were prepared. UV-vis absorption spectra of 10 µM

CaPF-1 in HEPES buffers (prepared from a stock solution of 10 mM CaPF-1 in DMSO) were

recorded using a Shimadzu UV-3150 spectrometer with a resolution of 1.0 nm (Fig. S1).

Emission spectra of 1 µM CaPF-1 in HEPES buffers (prepared from a stock solution of 1 mM

CaPF-1 in DMSO) were recorded using HORIBA FluoroMax-4 spectrometer with a resolution

of 1.0 nm (Fig. S1). Absolute fluorescence quantum yields were determined with a Hamamatsu

photonics PMA-11 calibrated integrating sphere system.

Determination of calcium dissociation constant (Kd). A series of HEPES-buffered solutions

(50 mM, pH 7.4, 50 mM KCl, 0.1% DMSO, 1 µM CaPF-1) containing various amounts of

Ca(NO3)2·4H2O (ranging between 0 mM to 10 mM) and 10 mM EGTA were prepared. The

concentration of free Ca2+ ([Ca2+]free) was calculated using literature method.6 The fluorescence

intensity at 663 nm (I663) was plotted against [Ca2+]free and the experimental data were analyzed

by non-linear least square curve fitting using the following equation (eq 1):

(eq 1)

where I0 and I∞ represent the initial and final fluorescence intensities at 663 nm, respectively

(Fig. S2).

Determination of acid dissociation constant (pKa). A series of pH-buffered solutions was

prepared with 0.1 M citric acid/0.1 M sodium citrate for pH 4–5.5 and 0.1 M Na2HPO4/0.1 M

NaH2PO4 for pH 5.8–8.2. 2 µL of a stock solution of CaPF-1 in DMSO (10 mM) was added to

each pH solution (2 mL) and the absorption spectra were measured (Fig. 2b). The absorbance at

635 nm (A635) was plotted against the pH values. The data was analyzed by curve fitting using

the following equation (eq 2):

(eq 2)

I663 = I0 Kd + I∞ [Ca2+]freeKd + [Ca2+]free

A635= 10–pH A0 + 10–pKa A∞10–pH + 10–pKa

S9

where A0 and A∞ represent the initial and final absorbance values at 635 nm, respectively (Fig.

S3).

pH dependent fluorescence properties. A series of pH-buffered solution (50 mM KCl) was

prepared with 50 mM MES for pH 5.5–6.5 and 50 mM HEPES for pH 7.0–9.0 with (1 mM

Ca(NO3)2) or without Ca2+ (10 mM EGTA). 2 µL of a stock solution of CaPF-1 in DMSO (1

mM) was added to each pH solution (2 mL) and the emission spectra were measured. The

relative fluorescence intensities (F/F0) were plotted against the pH value (Fig. S4). The

fluorescence quantum yields of CaPF-1 in pH 4.0 and 7.4 solutions containing 1 mM Ca(NO3)2

or 0.5 mM EGTA (Ca2+-free) were measured (Table 1 and Table S1).

Metal ion selectivity. HEPES buffer solutions (50 mM, pH 7.4) of 1 µM CaPF-1 containing 1

mM Ca(NO3)2 or 0.5 mM EGTA (Ca2+-free) were prepared. The pH values were adjusted by

adding Me4NOH·5H2O. To each solution, NaCl (100 mM), KCl (10 mM), MgCl2 (10 mM and 1

mM), or ZnCl2 (0.6 mM) was added and the emission spectra were recorded.

Chemical stability in 10 mM GSH solution. HEPES buffer solutions (50 mM, pH 7.4) of 1

µM CaPF-1 containing 10 mM GSH (reduced form) were prepared. The time course of

absorption spectra in the solution were recorded (Fig. S6a). The relative absorbance at 635 nm

(A/A0) was plotted against each time point (Fig. S6b).

3. Electrochemical Properties

Cyclic voltammetry. Cyclic voltammetry (CV) was performed on an ALS/chi-617A

electrochemical analyzer. The CV cell consisted of a glassy carbon electrode, a Pt wire counter

electrode, and an Ag/AgCl (in 3 M NaCl) reference electrode (0.21 V vs. SHE). A solution of 5

mM POF-Me2 in 0.1 M phosphate buffer (pH 10.0) was prepared and nitrogen gas was bubbled

for 3 min before measurement (Fig. S7).

4. Cell Culture Experiments

HeLa cells (RIKEN Cell Bank, Japan) were cultured in Dulbecco’s modified Eagle’s medium

(DMEM, Sigma) containing 10% fetal bovine serum (FBS, Gibco) and 1%

Antibiotic-Antimycotic (AA, Sigma) at 37 °C in a 5% CO2/95% air incubator. Cells (5 × 104)

were seeded in glass-bottom dishes three days before imaging. Immediately before use, a

S10

solution of 10 mM CaPF-1-AM in DMSO was mixed with the 20 wt% Pluronic F-127 solution

in DMSO at a ratio of 1:4. The pre-mixed solution was then diluted with HBSS to prepare a

final CaPF-1-AM concentration of 2 µM. The solution was then vortexed for 1 min and

centrifuged (20 min, 15000 rpm). The supernatant was used for cell staining.

Co-staining experiments. The incubation medium was removed from the cells, and the cells

were further incubated with 2 µM CaPF-1-AM and 0.2 µM Calcein-AM in HBSS containing

0.2% DMSO and 0.04% Pluronic F-127 for 30 min in a CO2 incubator. After cells were rinsed

with HBSS three times and the dish was filled with 2 mL of HBSS, fluorescence images were

obtained with a FV10i-DOC confocal laser-scanning microscope (OLYMPUS) (Fig. S8), where

LD lasers of 635 nm and 473 nm were used for the excitation of CaPF-1 and Calcein,

respectively. The cells were also co-stained with ER Tracker Red (Invitrogen) in the similar

manner for Calcein except the excitation wavelength of 559 nm (Fig. S9).

Multi-color imaging experiments. After removal of the incubation medium from the culture

dish, the cells were washed with HBSS three times. The cells were simultaneously stained with

2 µM CaPF-1-AM, 0.2 µM Hoechst (Dojindo), and 0.2 µM MitoTracker Green FM (Invitrogen)

in HBSS containing 0.2% DMSO and 0.04% Pluronic F-127 for 30 min at 37 °C. After the

incubation medium was removed, the cells were rinsed with HBSS three times and the dish was

filled with 2 mL of HBSS. For multi-color imaging, a confocal microscope (TCS SP8 STED

3X; Leica) equipped with a 20x objective lens and a HyD detector was used. The confocal

fluorescence images were obtained under the excitation at 405 nm (diode laser), 473 nm (white

light laser), and 633 nm (white light laser) for Hoechst (lem = 420–460 nm), MitoTracker Green

FM (lem = 490–540 nm), and CaPF-1 (lem = 638–795 nm), respectively. Each image was

recorded with a line average of 4 and a frame average of 4 (Fig. 3).

Fluorescence imaging of histamine-induced Ca2+ oscillation in HeLa cells. The incubation

medium was removed from the dish and the cells were incubated with 2 µM CaPF-1-AM in

HBSS containing 0.2% DMSO and 0.04% Pluronic F-127 for 1 h. The cells were rinsed with

HBSS three times, filled with 2 mL of HBSS, and further incubated for 30 min in a CO2

incubator. For histamine stimulation, 1 mL of histamine dihydrochloride (Wako) in HBSS (3

µM) was added to the medium (2 mL) to give the final concentration of 1 µM. For fluorescent

time-lapse imaging, a confocal microscope (TCS SP8 STED 3X; Leica) equipped with a 20x

S11

objective lens was used. The CaPF-1 was excited with 633 nm (white light laser) and the

fluorescence was collected between 638-795 nm with a HyD detector (gain = 50) using a

resonant scanning mode at 12 kHz. Each image was obtained from 6-line average and 3 times

frame accumulation and the frame rate is 1.04 s. The images were processed with Fiji software.

Cytotoxicity evaluation. HeLa cells were seeded into a flat-bottomed 96-well plate (1 × 104

cells/well) and incubated in DMEM containing 10% FBS at 37 °C in a 5% CO2/95% air

incubator for 24 h. The medium was then replaced with a culture medium containing various

concentrations of CaPF-1-AM (0, 1, 2, 5, and 10 µM) in HBSS (0.4% DMSO and 0.04%

Pluronic F-127). After the cells were incubated for 3 h at 37 °C, the medium was removed and

the cells were washed with PBS three times. MTT reagent (final concentration, 0.5 mg/mL) in

PBS was added to each well, and the plates were incubated for another 4 h in a CO2 incubator.

Excess MTT tetrazolium solution was then removed and the cells were once washed with PBS.

After the formazan crystals were solubilized in DMSO (100 µL/well) for 30 min at room

temperature, the absorbance of each well was measured by SpectraMax i3 (Molecular Devices)

with an excitation at 535 nm (Fig. S10).

5. References 1. R. Pethig, M. Kuhn, R. Payne, E. Adler, T.-H. Chen and L. F. Jaffe, Cell Calcium, 1989, 10,

491–498. 2. T. Egawa, K. Hanaoka, Y. Koide, S. Ujita, N. Takahashi, Y. Ikegaya, N. Matsuki, T. Terai, T.

Ueno, T. Komatsu and T. Nagano, J. Am. Chem. Soc., 2011, 133, 14157–14159. 3. A. Fukazawa, S. Suda, M. Taki, E. Yamaguchi, M. Grzybowski, Y. Sato, T. Higashiyama

and S. Yamaguchi, Chem. Commun., 2016, 52, 1120–1123. 4. M. Grzybowski, M. Taki and S. Yamaguchi, Chem. –Eur. J., 2017, DOI:

10.1002/chem.201703456 5. E. Kawaminami, T. Takahashi, T. Kanayama, Y. Fukuda, H. Kaizawa, Y. Kondoh, R. Seo,

K. Kuramoto, K. Take, K. Sakamoto, Patent applications WO2011/037192 A1 (Substituted amide compound).

6. D. M. Bers, C. W. Patton and R. Nuccitelli, In Calcium in Living Cells, Elsevier, Burlington, 2010, 1, 1–26.

S12

Fig. S1 Absorption (dashed line; 0.5% DMSO) and emission (solid line; 0.1% DMSO) spectra of CaPF-1 in 50 mM HEPES buffer (pH 7.4). Red and blue lines represent the spectra of Ca2+-free and Ca2+-bound forms observed with the presence of 0.5 mM EGTA and 1 mM Ca2+, respectively. The emission spectra were measured with the excitation wavelength at 580 nm.

Fig. S2 Plots of the fluorescence intensity at 663 nm (I663) as a function of [Ca2+]free with a best-fit curve for the dissociation constant of Kd = 273 ± 5 nM.

Fluo

resc

ence

inte

nsity

500Wavelength/ nm

600 700 800300 400

Ca2+-bound

Ca2+-free 663636

Mol

ar a

bsor

ptiv

ity ε/

104 M

–1 c

m–1

4.0

3.0

1.0

0.0

5.0

2.0

[Ca2+]free / µM0.0001 0.001 0.01 0.1 1 10 100

Kd(Ca2+) = 273 ± 5 nM

I 663

S13

Fig. S3 Plots of the absorbance at 635 nm (Abs635) as a function of pH values with a best-fit curve for the acid dissociation constant of pKa = 5.9.

Fig. S4 Plots of the fluorescence intensity ratio (F/F0) at 663 nm as a function of pH values for 1 µM CaPF-1. Closed circle and diamond represent the Ca2+-free and Ca2+-bound forms, respectively.

pH3 4 5 6 7 8 9

pKa = 5.9

Abs

635

pH86 7 9

F/F 0

Ca2+-freeCa2+-bound

0

5

10

15

S14

Fig. S5 Fluorescence response of CaPF-1 (1 µM) to various metal ions (100 mM Na+, 100 mM K+, 1 mM Mg2+, 10 mM Mg2+, or 0.6 mM Zn2+) in 0.1 M HEPES buffer (pH 7.4) containing 0.5 mM EGTA. Blue and red bars represent the fluorescence intensity ratio (F/F0) of CaPF-1 in the presence and absence of 1 mM Ca(NO3)2, respectively.

Fig. S6 (a) Absorption spectral change of CaPF-1 (1 µM) in 0.1 M HEPES buffer (pH 7.4) containing 10 mM GSH (reduced form). (b) Plot of relative absorbance (A/A0) at 635 nm versus each time point.

F/F 0

0

5

10

15

Na+ K+ Mg2+ Mg2+ Zn2+

100 100 1 10 0.6 (mM)Na+ K+ Mg2+ Mg2+ Zn2+

100 100 1 10 0.6Ca2+

1

S15

Fig. S7 Cyclic voltammogram of POF-Me2 at a scan rate of 0.1 V s–1 in pH 10 phosphate buffer. The potential vs. Ag/AgCl (in 3 M NaCl) was converted to vs. SHE by adding 0.21 V.

Fig. S8 Co-staining experiment of HeLa cells with Calcein and CaPF-1. The cells were loaded with Calcein-AM (0.2 µM) and CaPF-1-AM (2 µM) at 37 °C for 30 min. Fluorescence images of Calcein (a) and CaPF-1 (b) were observed by the excitation at 559 nm and 635 nm, respectively. A merged image (c) was obtained from (a) and (b). Scale bar: 10 µm.

Fig. S9 Co-staining experiment of HeLa cells with ER Tracker Red and CaPF-1. The cells were loaded with ER Tracker Red (0.1 µM) and CaPF-1-AM (2 µM) at 37 °C for 30 min. Fluorescence images of Calcein (a) and CaPF-1 (b) were observed by the excitation at 559 nm and 635 nm, respectively. A merged image (c) was obtained from (a) and (b). Scale bar: 20 µm.

Potential / V vs SHE–0 –0.2 –0.4 –0.6 –0.8

E1/2 = –0.35 V

0.2

P OHOPhO

POF-Me2

S16

Fig. S10 Cell viability of CaPF-1-loaded HeLa cells determined by MTT assay. The cells were incubated with CaPF-1-AM (0, 2, 5, and 10 µM) in HBSS containing 0.4% DMSO and 0.04% Pluronic F-127 in a CO2 incubator for 4 h. The results are expressed as percentages of the dye-free controls. All data are presented as mean standard deviation (n = 16). Table S1 Fluorescence quantum yields of CaPF-1 in pH 4.0 and 7.4 buffered-solutions containing 0.5 mM EGTA (Ca2+-free) or 1 mM Ca(NO3)2 (Ca2+-bound).

CaPF-1FF

(Ca2+-free)FF

(Ca2+-bound)

pH4.0 >0.01 >0.01

pH7.0 ~0.02 0.22

S17

Fig. S11 1H NMR spectrum of tert-Butyl 4-bromo-3,5-dimethoxybenzoate (400 MHz, CDCl3).

Fig. S12 13C{1H} NMR spectrum of tert-Butyl 4-bromo-3,5-dimethoxybenzoate (125 MHz,

CDCl3).

(Mill

ions

)0

10.0

20.0

30.0

X : parts per Million : Proton

8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0

7.

260

7.

199

3.

946

1.

608

1.

544

9.00

6.16

2.02

abun

danc

e0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

20.0

22.0

24.0

26.0

28.0

30.0

X : parts per Million : Carbon13

180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0

165

.257

157

.012

132

.237

106

.254

105

.553

81.

854

77.

419

77.

160

76.

910

56.

705

28.

283

S18

Fig. S13 1H NMR spectrum of 2 (400 MHz, CD3OD).

Fig. S14 13C{1H} NMR spectrum of 2 (150 MHz, CD3OD).

(Mill

ions

)0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

11.0

12.0

13.0

14.0

15.0


9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0

7.52

47.

516

7.43

97.

419

7.41

67.

231

7.22

57.

191

7.18

57.

079

7.06

37.

055

7.04

06.

602

6.59

86.

579

6.57

3

4.84

1

3.81

53.

807

3.31

83.

315

3.31

03.

305

3.30

2

1.65

58.

81

6.05

2.00

2.00

1.96

1.94

1.89

1.02

0.99

0.95

(Mill

ions

)0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7


7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6

7.77

57.

757

7.75

47.

741

7.72

57.

722

7.59

27.

587

7.57

37.

569

7.54

47.

535

7.52

47.

520

7.51

67.

439

7.41

97.

416

7.23

17.

225

7.19

17.

185

7.07

97.

063

7.05

57.

040

6.60

26.

598

6.57

96.

573

8.81

6.05

2.00

2.00

1.96

1.94

1.89

1.02

0.99

0.95

Filename = OG_761-1-5.jdfAuthor = deltaExperiment = proton.jxpSample_Id = OG_761Solvent = METHANOL-D4Creation_Time = 30-JUL-2016 14:33:52Revision_Time = 20-JUN-2017 17:27:36Current_Time = 20-JUN-2017 17:28:45

Comment = single_pulseData_Format = 1D COMPLEXDim_Size = 13107Dim_Title = ProtonDim_Units = [ppm]Dimensions = XSpectrometer = JNM-ECZ400S/L1

Field_Strength = 9.389766[T] (400[MHz]X_Acq_Duration = 1.63577856[s]X_Domain = 1HX_Freq = 399.78219838[MHz]X_Offset = 5[ppm]X_Points = 16384X_Prescans = 1X_Resolution = 0.61132969[Hz]X_Sweep = 10.01602564[kHz]X_Sweep_Clipped = 8.01282051[kHz]Irr_Domain = ProtonIrr_Freq = 399.78219838[MHz]Irr_Offset = 5[ppm]Tri_Domain = ProtonTri_Freq = 399.78219838[MHz]Tri_Offset = 5[ppm]Clipped = FALSEDecimation_Reg = r: 624( 623),g: 47Scans = 4Total_Scans = 4

Relaxation_Delay = 5[s]Recvr_Gain = 46Temp_Get = 27.3[dC]X_90_Width = 8.85[us]X_Acq_Time = 1.63577856[s]X_Angle = 45[deg]X_Atn = 1.2[dB]X_Pulse = 4.425[us]Irr_Mode = OffTri_Mode = OffComment_1 = *** Pulse ***Comment_111 = *** presat_time ***Comment_201 = *** obs_dante_presatuComment_202 = *** irr_ preaturationComment_203 = *** tri_ preaturationComment_7 = *** Pulse Delay ***Dante_Loop = 500Dante_Presat = FALSEGet_90 = pulse_service::get_90Get_Atn = pulse_service::get_atGet_Freq = pulse_service::get_frGet_Gamma = pulse_service::get_gaGet_Probe_Parameter = probe_service::get_prGet_Spin = pulse_service::get_spInitial_Wait = 1[s]Presat_Time = 5[s]Presat_Time_Flag = FALSERelaxation_Delay_Calc = 0[s]Relaxation_Delay_Temp = 5[s]Repetition_Time = 6.63577856[s]

abun

danc

e0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0


220.0 210.0 200.0 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0 -10.0

166.

425

159.

167

158.

842

150.

818

150.

761

136.

504

133.

976

131.

113

131.

037

130.

357

130.

271

127.

676

127.

638

118.

915

106.

037

105.

941

83.1

05

56.8

4256

.679

49.4

3149

.287

49.1

4449

.000

48.8

5648

.713

48.5

79

28.3

85

abun

danc

e 00.

1


139.0 138.0 137.0 136.0 135.0 134.0 133.0 132.0 131.0 130.0 129.0 128.0 127.0 126.0 125.0

138.

965

137.

777

137.

184

136.

504

134.

330

133.

976

133.

603

131.

113

131.

037

130.

357

130.

271

127.

676

127.

638

125.

167

abun

danc

e-0

.02

00.

020.

040.

060.

080.

10.

12


166.0 165.0 164.0 163.0 162.0 161.0 160.0 159.0 158.0 157.0 156.0 155.0 154.0 153.0 152.0 151.0

166.

425

159.

167

158.

842

150.

818

150.

761

Filename = OG_762_carbon-4-6.jdfAuthor = deltaExperiment = carbon.jxpSample_Id = OG_762Solvent = METHANOL-D4Creation_Time = 30-JUL-2016 15:20:06Revision_Time = 20-JUN-2017 18:06:47Current_Time = 20-JUN-2017 18:06:58

Comment = single pulse decoupled gatData_Format = 1D COMPLEXDim_Size = 26214Dim_Title = Carbon13Dim_Units = [ppm]Dimensions = XSite = JNM-ECA600IISpectrometer = DELTA2_NMR

Field_Strength = 14.09636928[T] (600[MHz])X_Acq_Duration = 0.69206016[s]X_Domain = 13CX_Freq = 150.91343039[MHz]X_Offset = 100[ppm]X_Points = 32768X_Prescans = 2X_Resolution = 1.44496109[Hz]X_Sweep = 47.34848485[kHz]X_Sweep_Clipped = 37.87878788[kHz]Irr_Domain = ProtonIrr_Freq = 600.1723046[MHz]Irr_Offset = 5[ppm]Clipped = FALSEScans = 256Total_Scans = 256

Relaxation_Delay = 2[s]Recvr_Gain = 60Temp_Get = 26.3[dC]X_90_Width = 8.5[us]X_Acq_Time = 0.69206016[s]X_Angle = 30[deg]X_Atn = 12.2[dB]X_Pulse = 2.83333333[us]Irr_Atn_Dec = 18.4[dB]Irr_Atn_Noe = 18.4[dB]Irr_Noise = WALTZIrr_Pwidth = 73[us]Decoupling = TRUEInitial_Wait = 1[s]Noe = TRUENoe_Time = 2[s]Repetition_Time = 2.69206016[s]

S19

Fig. S15 31P NMR spectrum of 2 (162 MHz, CD3OD).

Fig. S16 1H NMR spectrum of 3 (400 MHz, CD3OD).

(Tho

usan

ds)

010

0.0

200.

030

0.0

400.

050

0.0

600.

070

0.0

800.

0

X : parts per Million : Phosphorus31

300.0 200.0 100.0 0 -100.0 -200.0 -300.0

10.1

79

Filename = OG_761-2-5.jdfAuthor = deltaExperiment = single_pulse_dec.jSample_Id = OG_761Solvent = METHANOL-D4Creation_Time = 30-JUL-2016 14:38:Revision_Time = 20-JUN-2017 17:20:Current_Time = 20-JUN-2017 17:20:

Comment = single pulse decouData_Format = 1D COMPLEXDim_Size = 26214Dim_Title = Phosphorus31Dim_Units = [ppm]Dimensions = XSpectrometer = JNM-ECZ400S/L1

Field_Strength = 9.389766[T] (400[MX_Acq_Duration = 0.23068672[s]X_Domain = 31PX_Freq = 161.83469309[MHz]X_Offset = 0[ppm]X_Points = 32768X_Prescans = 2X_Resolution = 4.33488326[Hz]X_Sweep = 142.04545455[kHz]X_Sweep_Clipped = 113.63636364[kHz]Irr_Domain = ProtonIrr_Freq = 399.78219838[MHz]Irr_Offset = 5[ppm]Clipped = FALSEDecimation_Reg = r: 44( 43),g: 28Scans = 32Total_Scans = 32

Relaxation_Delay = 2[s]Recvr_Gain = 66Temp_Get = 27.2[dC]X_90_Width = 25[us]X_Acq_Time = 0.23068672[s]X_Angle = 30[deg]X_Atn = 6[dB]X_Pulse = 8.33333333[us]Irr_Atn_Dec = 23.475[dB]Irr_Atn_Dec_Calc = 23.475[dB]Irr_Atn_Dec_Default_Calc = 23.475[dB]Irr_Atn_Noe = 23.475[dB]Irr_Dec_Bandwidth_Hz = 4.7826087[kHz]Irr_Dec_Bandwidth_Ppm = 11.96303566[ppm]Irr_Dec_Freq = 399.78219838[MHz]Irr_Dec_Merit_Factor = 2.2Irr_Decoupling = TRUEIrr_Noe = TRUEIrr_Noise = WALTZIrr_Offset_Default = 5[ppm]Irr_Pwidth = 0.115[ms]Irr_Pwidth_Default = 0.115[ms]Irr_Pwidth_Default_Calc = 0.115[ms]Irr_Pwidth_Temp1 = 0.115[ms]Irr_Wurst = FALSEComment_1 = *** Pulse ***Comment_102 = *** irr_decouplingComment_110 = *** noe_time ***Comment_7 = *** Pulse Delay **Get_90 = pulse_service::getGet_Atn = pulse_service::getGet_Freq = pulse_service::getGet_Gamma = pulse_service::getGet_Probe_Parameter = probe_service::getGet_Spin = pulse_service::getInitial_Wait = 1[s]Noe_Time = 2[s]Noe_Time_Flag = FALSERelaxation_Delay_Calc = 0[s]Relaxation_Delay_Temp = 2[s]Repetition_Time = 2.23068672[s]

(Mill

ions

)0

1.0

2.0

3.0

4.0

5.0

6.0

7.0


9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0

7.78

07.

763

7.75

87.

748

7.72

67.

546

7.52

97.

508

7.09

67.

081

7.07

37.

056

6.60

8

4.90

64.

833

3.82

83.

821

3.31

83.

315

3.31

03.

305

3.30

2

6.03

3.93

2.08

2.00

1.83

1.77

1.07

(Tho

usan

ds) 0

100.

020

0.0

300.

040

0.0


8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2

7.78

07.

763

7.75

87.

748

7.72

97.

726

7.61

97.

604

7.59

97.

586

7.58

27.

555

7.54

67.

529

7.51

87.

508

7.22

87.

191

7.09

67.

081

7.07

37.

056

6.60

8

6.03

3.93

2.08

2.00

1.83

1.77

1.07

Filename = OG_1033_proton-1-4.jdAuthor = deltaExperiment = proton.jxpSample_Id = OG_1033Solvent = METHANOL-D4Creation_Time = 27-JUL-2017 01:12:32Revision_Time = 27-JUL-2017 13:40:08Current_Time = 27-JUL-2017 13:40:30


Field_Strength = 9.389766[T] (400[MHz]X_Acq_Duration = 1.63577856[s]X_Domain = 1HX_Freq = 399.78219838[MHz]X_Offset = 5[ppm]X_Points = 16384X_Prescans = 1X_Resolution = 0.61132969[Hz]X_Sweep = 10.01602564[kHz]X_Sweep_Clipped = 8.01282051[kHz]Irr_Domain = ProtonIrr_Freq = 399.78219838[MHz]Irr_Offset = 5[ppm]Tri_Domain = ProtonTri_Freq = 399.78219838[MHz]Tri_Offset = 5[ppm]Blanking = 2[us]Clipped = FALSEDecimation_Reg = r: 624( 623),g: 47Scans = 4Total_Scans = 4

Relaxation_Delay = 5[s]Recvr_Gain = 56Temp_Get = 27.1[dC]X_90_Width = 9.5[us]X_Acq_Time = 1.63577856[s]X_Angle = 45[deg]X_Atn = 1.2[dB]X_Pulse = 4.75[us]Irr_Mode = OffTri_Mode = OffComment_1 = *** Pulse ***Comment_111 = *** presat_time ***Comment_201 = *** obs_dante_presatuComment_202 = *** irr_ preaturationComment_203 = *** tri_ preaturationComment_7 = *** Pulse Delay ***Dante_Loop = 500Dante_Presat = FALSEDecimation_Rate = 0Get_90 = pulse_service::get_90Get_Atn = pulse_service::get_atGet_Freq = pulse_service::get_frGet_Gamma = pulse_service::get_gaGet_Probe_Parameter = probe_service::get_prGet_Spin = pulse_service::get_spInitial_Wait = 1[s]Presat_Time = 5[s]Presat_Time_Flag = FALSERelaxation_Delay_Calc = 0[s]Relaxation_Delay_Temp = 5[s]Repetition_Time = 6.63577856[s]

S20

Fig. S17 13C{1H} NMR spectrum of 3 (150 MHz, DMSO-d6).

Fig. S18 31P NMR spectrum of 3 (162 MHz, CD3OD).

abun

danc

e0

0.1

0.2

0.3


210.0200.0190.0180.0170.0160.0150.0140.0130.0120.0110.0100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0 -10.0 -20.0

166.

788

157.

357

157.

021

147.

361

147.

322

129.

446

129.

379

129.

255

129.

169

120.

283

118.

330

117.

305

105.

184

105.

126

56.4

5856

.228

40.0

5639

.932

39.7

9839

.654

39.5

2039

.376

39.2

4239

.099

(tho

usan

dths

)0

10.0


160.0 150.0

166.

788

158.

046

157.

807

157.

357

157.

021

147.

361

147.

322

(tho

usan

dths

)0

10.0

20.0

30.0

40.0

50.0


134.0 133.0 132.0 131.0 130.0 129.0

134.

320

133.

889

133.

611

132.

424

129.

446

129.

379

129.

255

129.

169

(tho

usan

dths

)0

10.0

20.0


120.0 110.0

120.

283

118.

330

117.

305

116.

319

105.

184

105.

126

Filename = OG_821_Carbon-3-7.jdfAuthor = deltaExperiment = carbon.jxpSample_Id = OG_821Solvent = DMSO-D6Creation_Time = 2-OCT-2016 09:44:35Revision_Time = 19-JUN-2017 10:11:19Current_Time = 19-JUN-2017 10:12:43



Relaxation_Delay = 2[s]Recvr_Gain = 50Temp_Get = 30[dC]X_90_Width = 8.5[us]X_Acq_Time = 0.69206016[s]X_Angle = 30[deg]X_Atn = 12.2[dB]X_Pulse = 2.83333333[us]Irr_Atn_Dec = 25[dB]Irr_Atn_Noe = 25[dB]Irr_Noise = WALTZIrr_Pwidth = 0.155[ms]Decoupling = TRUEInitial_Wait = 1[s]Noe = TRUENoe_Time = 2[s]Repetition_Time = 2.69206016[s]

(Tho

usan

ds)

010

.020

.030

.040

.050

.060

.070

.080

.090

.0


300.0 200.0 100.0 0 -100.0 -200.0 -300.0

10.1

52

Filename = OG_1033_single_pulAuthor = deltaExperiment = single_pulse_dec.jSample_Id = OG_1033Solvent = METHANOL-D4Creation_Time = 27-JUL-2017 01:16:Revision_Time = 27-JUL-2017 13:34:Current_Time = 27-JUL-2017 13:34:


Field_Strength = 9.389766[T] (400[MX_Acq_Duration = 0.23068672[s]X_Domain = 31PX_Freq = 161.83469309[MHz]X_Offset = 0[ppm]X_Points = 32768X_Prescans = 4X_Resolution = 4.33488326[Hz]X_Sweep = 142.04545455[kHz]X_Sweep_Clipped = 113.63636364[kHz]Irr_Domain = ProtonIrr_Freq = 399.78219838[MHz]Irr_Offset = 5[ppm]Blanking = 5[us]Clipped = FALSEDecimation_Reg = r: 44( 43),g: 28Scans = 186Total_Scans = 186

Relaxation_Delay = 2[s]Recvr_Gain = 66Temp_Get = 27.1[dC]X_90_Width = 25[us]X_Acq_Time = 0.23068672[s]X_Angle = 30[deg]X_Atn = 6[dB]X_Pulse = 8.33333333[us]Irr_Atn_Dec = 22.86[dB]Irr_Atn_Dec_Calc = 22.86[dB]Irr_Atn_Dec_Default_Calc = 22.86[dB]Irr_Atn_Noe = 22.86[dB]Irr_Dec_Bandwidth_Hz = 4.7826087[kHz]Irr_Dec_Bandwidth_Ppm = 11.96303566[ppm]Irr_Dec_Freq = 399.78219838[MHz]Irr_Dec_Merit_Factor = 2.2Irr_Decoupling = TRUEIrr_Noe = TRUEIrr_Noise = WALTZIrr_Offset_Default = 5[ppm]Irr_Pwidth = 0.115[ms]Irr_Pwidth_Default = 0.115[ms]Irr_Pwidth_Default_Calc = 0.115[ms]Irr_Pwidth_Temp1 = 0.115[ms]Irr_Wurst = FALSEComment_1 = *** Pulse ***Comment_102 = *** irr_decouplingComment_110 = *** noe_time ***Comment_7 = *** Pulse Delay **Decimation_Rate = 0Get_90 = pulse_service::getGet_Atn = pulse_service::getGet_Freq = pulse_service::getGet_Gamma = pulse_service::getGet_Probe_Parameter = probe_service::getGet_Spin = pulse_service::getInitial_Wait = 1[s]Noe_Time = 2[s]Noe_Time_Flag = FALSERelaxation_Delay_Calc = 0[s]Relaxation_Delay_Temp = 2[s]Repetition_Time = 2.23068672[s]

S21

Fig. S19 1H NMR spectrum of CaPF-1 (400 MHz, CD3OD).

Fig. S20 13C{1H} NMR spectrum of CaPF-1 (150 MHz, CD3OD).

(Mill

ions

)0

1.0

2.0

3.0

4.0

5.0


9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0

7.78

77.

769

7.75

57.

737

7.56

07.

552

7.54

17.

532

7.46

67.

446

7.24

37.

238

7.20

37.

199

7.13

17.

116

7.10

87.

092

6.88

1

4.83

5

4.42

04.

399

4.07

33.

867

3.85

9

3.31

93.

315

3.31

03.

307

3.30

2

2.29

6

7.68

6.00

3.74

3.10

3.06

2.20

2.17

2.07

2.05

2.02

1.27

1.15

1.14

1.13

0.98

0.96

(Tho

usan

ds)

-10.

00

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.

011

0.0

120.

0


7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6

7.78

77.

769

7.75

57.

737

7.62

47.

605

7.59

07.

560

7.55

27.

541

7.53

27.

523

7.51

57.

466

7.44

6

7.29

77.

274

7.24

37.

238

7.20

37.

199

7.13

17.

116

7.10

87.

092

7.03

07.

009

6.89

96.

881

6.72

56.

705

6.61

86.

596

7.68

6.00

3.74

3.10

3.06

2.20

2.17

2.07

2.05

2.02

1.27

1.15

1.14

1.13

0.98

0.96

(Tho

usan

ds) 0

100.

020

0.0

300.

040

0.0


4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7

4.42

04.

399

4.07

3

4.01

0

3.86

73.

859

7.68

6.00

3.74

3.10

3.06

2.20

2.17

2.07

2.05

2.02

1.27

1.15

1.14

1.13

0.98

0.96

Filename = OG_801-1-4.jdfAuthor = deltaExperiment = proton.jxpSample_Id = OG_801Solvent = METHANOL-D4Creation_Time = 10-SEP-2016 21:13:07Revision_Time = 19-JUN-2017 03:55:01Current_Time = 19-JUN-2017 03:56:55


Field_Strength = 9.389766[T] (400[MHz]X_Acq_Duration = 1.63577856[s]X_Domain = 1HX_Freq = 399.78219838[MHz]X_Offset = 5[ppm]X_Points = 16384X_Prescans = 1X_Resolution = 0.61132969[Hz]X_Sweep = 10.01602564[kHz]X_Sweep_Clipped = 8.01282051[kHz]Irr_Domain = ProtonIrr_Freq = 399.78219838[MHz]Irr_Offset = 5[ppm]Tri_Domain = ProtonTri_Freq = 399.78219838[MHz]Tri_Offset = 5[ppm]Clipped = FALSEDecimation_Reg = r: 624( 623),g: 47Scans = 4Total_Scans = 4

Relaxation_Delay = 5[s]Recvr_Gain = 46Temp_Get = 27.4[dC]X_90_Width = 9.5[us]X_Acq_Time = 1.63577856[s]X_Angle = 45[deg]X_Atn = 1.2[dB]X_Pulse = 4.75[us]Irr_Mode = OffTri_Mode = OffComment_1 = *** Pulse ***Comment_111 = *** presat_time ***Comment_201 = *** obs_dante_presatuComment_202 = *** irr_ preaturationComment_203 = *** tri_ preaturationComment_7 = *** Pulse Delay ***Dante_Loop = 500Dante_Presat = FALSEGet_90 = pulse_service::get_90Get_Atn = pulse_service::get_atGet_Freq = pulse_service::get_frGet_Gamma = pulse_service::get_gaGet_Probe_Parameter = probe_service::get_prGet_Spin = pulse_service::get_spInitial_Wait = 1[s]Presat_Time = 5[s]Presat_Time_Flag = FALSERelaxation_Delay_Calc = 0[s]Relaxation_Delay_Temp = 5[s]Repetition_Time = 6.63577856[s]

(tho

usan

dths

)0

10.0

20.0

30.0

40.0

50.0

60.0


220.0 210.0 200.0 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0 -10.0

175.

770

175.

598

167.

679

159.

292

158.

976

152.

092

152.

034

134.

014

131.

123

131.

056

130.

386

130.

299

122.

793

120.

160

115.

372

115.

334

108.

708

104.

725

104.

591

101.

336

68.5

4268

.130

56.9

8556

.842

56.0

7655

.808

21.0

80

(tho

usan

dths

)0

1.0

2.0

3.0


170.0 160.0 150.0

175.

770

175.

598

167.

679

159.

292

158.

976

152.

092

152.

034

150.

770

150.

722

(tho

usan

dths

)-1

.00

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0


141.0 139.0 137.0 135.0 133.0 131.0 129.0 127.0 125.0

139.

597

138.

955

137.

682

137.

567

137.

423

134.

982

134.

618

134.

273

134.

014

133.

545

131.

123

131.

056

130.

386

130.

299

127.

982

125.

100

(tho

usan

dths

)-1

.00

1.0

2.0

3.0

4.0

5.0


120.0 110.0

122.

793

120.

514

120.

160

117.

776

115.

372

115.

334

108.

708

104.

725

104.

591

101.

336

Filename = OG_803_Carbon-1-8.jdfAuthor = deltaExperiment = carbon.jxpSample_Id = OG_803Solvent = METHANOL-D4Creation_Time = 11-SEP-2016 14:50:19Revision_Time = 21-JUN-2017 21:34:51Current_Time = 21-JUN-2017 21:35:40



Relaxation_Delay = 2[s]Recvr_Gain = 50Temp_Get = 26.2[dC]X_90_Width = 8.5[us]X_Acq_Time = 0.69206016[s]X_Angle = 30[deg]X_Atn = 12.2[dB]X_Pulse = 2.83333333[us]Irr_Atn_Dec = 18.4[dB]Irr_Atn_Noe = 18.4[dB]Irr_Noise = WALTZIrr_Pwidth = 73[us]Decoupling = TRUEInitial_Wait = 1[s]Noe = TRUENoe_Time = 2[s]Repetition_Time = 2.69206016[s]

S22

Fig. S21 31P NMR spectrum of CaPF-1 (162 MHz, CD3OD).

Fig. S22 1H NMR spectrum of 6 (400 MHz, CDCl3).

(Tho

usan

ds)

010

.020

.030

.040

.050

.060

.070

.0


300.0 200.0 100.0 0 -100.0 -200.0 -300.0

10.1

52

Filename = OG_801-2-2.jdfAuthor = deltaExperiment = single_pulse_dec.jSample_Id = OG_801Solvent = METHANOL-D4Creation_Time = 10-SEP-2016 21:18:Revision_Time = 19-JUN-2017 03:58:Current_Time = 19-JUN-2017 03:59:


Field_Strength = 9.389766[T] (400[MX_Acq_Duration = 0.23068672[s]X_Domain = 31PX_Freq = 161.83469309[MHz]X_Offset = 0[ppm]X_Points = 32768X_Prescans = 4X_Resolution = 4.33488326[Hz]X_Sweep = 142.04545455[kHz]X_Sweep_Clipped = 113.63636364[kHz]Irr_Domain = ProtonIrr_Freq = 399.78219838[MHz]Irr_Offset = 5[ppm]Clipped = FALSEDecimation_Reg = r: 44( 43),g: 28Scans = 256Total_Scans = 256

Relaxation_Delay = 2[s]Recvr_Gain = 66Temp_Get = 27.3[dC]X_90_Width = 25[us]X_Acq_Time = 0.23068672[s]X_Angle = 30[deg]X_Atn = 6[dB]X_Pulse = 8.33333333[us]Irr_Atn_Dec = 22.86[dB]Irr_Atn_Dec_Calc = 22.86[dB]Irr_Atn_Dec_Default_Calc = 22.86[dB]Irr_Atn_Noe = 22.86[dB]Irr_Dec_Bandwidth_Hz = 4.7826087[kHz]Irr_Dec_Bandwidth_Ppm = 11.96303566[ppm]Irr_Dec_Freq = 399.78219838[MHz]Irr_Dec_Merit_Factor = 2.2Irr_Decoupling = TRUEIrr_Noe = TRUEIrr_Noise = WALTZIrr_Offset_Default = 5[ppm]Irr_Pwidth = 0.115[ms]Irr_Pwidth_Default = 0.115[ms]Irr_Pwidth_Default_Calc = 0.115[ms]Irr_Pwidth_Temp1 = 0.115[ms]Irr_Wurst = FALSEComment_1 = *** Pulse ***Comment_102 = *** irr_decouplingComment_110 = *** noe_time ***Comment_7 = *** Pulse Delay **Get_90 = pulse_service::getGet_Atn = pulse_service::getGet_Freq = pulse_service::getGet_Gamma = pulse_service::get

(Mill

ions

)0

1.0

2.0

3.0

4.0

5.0

6.0


9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0

7.86

17.

855

7.84

07.

833

7.78

67.

780

7.26

0

6.85

06.

830

6.76

06.

737

6.71

96.

702

5.65

15.

621

4.39

34.

385

4.33

04.

322

4.28

44.

142

2.35

02.

283

2.08

52.

070

13.1

3

8.75

7.73

4.14

3.68

3.03

1.06

1.00

0.94

Filename = OG_1016_proton-1-4.jdAuthor = deltaExperiment = proton.jxpSample_Id = OG_1016Solvent = CHLOROFORM-DCreation_Time = 20-JUN-2017 08:42:39Revision_Time = 20-JUN-2017 15:15:26Current_Time = 20-JUN-2017 15:15:59




S23

Fig. S23 13C{1H} NMR spectrum of 6 (150 MHz, CDCl3).

Fig. S24 1H NMR spectrum of 5 (400 MHz, CDCl3).

abun

danc

e0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3


220.0 210.0 200.0 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0 -10.0 -20.0

170.

198

169.

633

169.

547

169.

422

150.

598

149.

028

144.

825

141.

646

136.

504

133.

345

122.

583

120.

649

118.

322

116.

665

115.

191

108.

412

79.6

7879

.305

77.3

7177

.160

76.9

4967

.968

66.9

82

53.6

9253

.529

21.0

8120

.774

abun

danc

e-0

.02

-0.0

10

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.1

0.11

0.12

0.13

0.14

0.15


170.2170.1170.0169.9169.8169.7169.6169.5169.4169.3

170.

198

169.

633

169.

547

169.

422

abun

danc

e 00.

10.

2


80.3 80.1 79.9 79.7 79.5 79.3 79.1

79.6

78

79.3

05

abun

danc

e0

0.1

0.2

0.3


21.2 21.0 20.8 20.6

21.0

81

20.7

74

abun

danc

e 00.

1


54.0 53.9 53.8 53.7 53.6 53.5 53.4 53.3

53.6

92

53.5

29

Filename = OG_943_Carbon-1-6.jdfAuthor = deltaExperiment = carbon.jxpSample_Id = OG_943Solvent = CHLOROFORM-DCreation_Time = 3-APR-2017 12:22:06Revision_Time = 20-JUN-2017 15:21:59Current_Time = 20-JUN-2017 15:23:10


Field_Strength = 14.09636928[T] (600[MHz])X_Acq_Duration = 0.69206016[s]X_Domain = 13CX_Freq = 150.91343039[MHz]X_Offset = 100[ppm]X_Points = 32768X_Prescans = 4X_Resolution = 1.44496109[Hz]X_Sweep = 47.34848485[kHz]X_Sweep_Clipped = 37.87878788[kHz]Irr_Domain = ProtonIrr_Freq = 600.1723046[MHz]Irr_Offset = 5[ppm]Clipped = FALSEScans = 207.0Total_Scans = 207.0


(Mill

ions

)0

1.0

2.0

3.0


9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0

7.26

07.

054

6.93

06.

908

6.82

76.

815

6.80

76.

794

6.71

06.

691

6.65

4

5.93

9

5.60

25.

592

4.28

94.

222

4.16

84.

159

4.15

44.

131

4.10

2

2.27

32.

064

2.03

212

.00

8.37

7.77

3.86

2.91

2.72

1.96

1.81

0.73

0.66

Filename = OG_1061_proton-2-26.jAuthor = deltaExperiment = proton.jxpSample_Id = OG_1061Solvent = CHLOROFORM-DCreation_Time = 6-AUG-2017 21:13:03Revision_Time = 6-AUG-2017 23:38:41Current_Time = 6-AUG-2017 23:38:41




S24

Fig. S25 13C{1H} NMR spectrum of 5 (150 MHz, CDCl3).

Fig. S26 1H NMR spectrum of CaPF-1-AM (400 MHz, CD3OD).

abun

danc

e0

1.0

2.0

3.0


220.0 210.0 200.0 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0 -10.0 -20.0

170.

571

170.

074

170.

007

169.

729

151.

135

150.

541

138.

783

135.

758

133.

718

125.

943

122.

152

120.

189

120.

065

115.

871

114.

224

108.

891

79.5

3579

.468

77.3

7177

.160

76.9

4967

.853

66.9

34

53.6

7353

.510

21.0

8120

.726

20.6

88

abun

danc

e0

0.1

0.2


170.0

170.

571

170.

074

170.

007

169.

729

abun

danc

e-0

.02

-0.0

10

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.1

0.11


121.0 120.0

120.

189

120.

065

abun

danc

e0

0.1

0.2

0.3


79.7 79.6 79.5 79.4 79.3 79.2

79.5

35

79.4

68

abun

danc

e0

0.1

0.2

0.3


54.0 53.0

53.6

73

53.5

10

abun

danc

e0

0.1

0.2

0.3


21.2 21.1 21.0 20.9 20.8 20.7 20.6

21.0

81

20.7

2620

.688

Filename = OG_1062_carbon-1-5.jdfAuthor = deltaExperiment = carbon.jxpSample_Id = OG_1062Solvent = CHLOROFORM-DCreation_Time = 6-AUG-2017 22:03:14Revision_Time = 6-AUG-2017 23:58:55Current_Time = 7-AUG-2017 00:01:27




(Mill

ions

)0

1.0

2.0

3.0

4.0

5.0

6.0

7.0


9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0

7.77

17.

738

7.55

27.

541

7.53

47.

462

7.44

27.

241

7.20

27.

134

7.11

87.

110

7.09

56.

940

6.83

86.

793

6.77

4

5.62

85.

622

4.84

4

4.37

04.

211

4.15

73.

867

3.85

9

3.31

83.

315

3.31

03.

305

3.30

1

2.28

22.

047

2.02

1

7.21

5.78

5.76

5.50

4.25

4.07

3.88

3.29

3.09

2.91

2.04

2.00

1.87

1.52

1.34

1.28

1.14

1.13

0.95

(Tho

usan

ds)

-10.

00

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0


7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5

7.78

77.

771

7.75

57.

738

7.60

57.

590

7.55

27.

541

7.53

47.

524

7.52

07.

462

7.44

2

7.27

47.

257

7.24

17.

202

7.13

47.

118

7.11

07.

095

6.94

06.

919

6.83

86.

793

6.77

4

6.70

26.

682

6.61

66.

599

7.21

5.78

5.76

5.50

4.25

4.07

3.88

3.29

3.09

2.91

2.04

2.00

1.87

1.52

1.34

1.28

1.14

1.13

0.95

(Tho

usan

ds)

010

0.0

200.

030

0.0

400.

0


4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8

4.37

04.

351

4.21

1

4.15

7

3.86

73.

859

7.21

5.78

5.76

5.50

4.25

4.07

3.88

3.29

3.09

2.91

2.04

2.00

1.87

1.52

1.34

1.28

1.14

1.13

0.95

Filename = OG_CaPF1_AM_proton-1-Author = deltaExperiment = proton.jxpSample_Id = OG_CaPF1_AMSolvent = METHANOL-D4Creation_Time = 7-MAY-2017 23:19:47Revision_Time = 19-JUN-2017 04:44:41Current_Time = 19-JUN-2017 04:45:28



Relaxation_Delay = 5[s]Recvr_Gain = 46Temp_Get = 25.7[dC]X_90_Width = 9.5[us]X_Acq_Time = 1.63577856[s]X_Angle = 45[deg]X_Atn = 1.2[dB]X_Pulse = 4.75[us]Irr_Mode = OffTri_Mode = OffComment_1 = *** Pulse ***Comment_111 = *** presat_time ***Comment_201 = *** obs_dante_presatuComment_202 = *** irr_ preaturationComment_203 = *** tri_ preaturationComment_7 = *** Pulse Delay ***Dante_Loop = 500Dante_Presat = FALSEDecimation_Rate = 0Get_90 = pulse_service::get_90Get_Atn = pulse_service::get_atGet_Freq = pulse_service::get_frGet_Gamma = pulse_service::get_gaGet_Probe_Parameter = probe_service::get_prGet_Spin = pulse_service::get_spInitial_Wait = 1[s]Presat_Time = 5[s]Presat_Time_Flag = FALSERelaxation_Delay_Calc = 0[s]

S25

Fig. S27 31P NMR spectrum of CaPF-1-AM (162 MHz, CD3OD).

(Tho

usan

ds)

010

.020

.030

.040

.050

.060

.0


300.0 200.0 100.0 0 -100.0 -200.0 -300.0

10.1

25

Filename = OG_CaPF1_AM_singleAuthor = deltaExperiment = single_pulse_dec.jSample_Id = OG_CaPF1_AMSolvent = METHANOL-D4Creation_Time = 7-MAY-2017 23:24:Revision_Time = 19-JUN-2017 04:35:Current_Time = 19-JUN-2017 04:36:


Field_Strength = 9.389766[T] (400[MX_Acq_Duration = 0.23068672[s]X_Domain = 31PX_Freq = 161.83469309[MHz]X_Offset = 0[ppm]X_Points = 32768X_Prescans = 4X_Resolution = 4.33488326[Hz]X_Sweep = 142.04545455[kHz]X_Sweep_Clipped = 113.63636364[kHz]Irr_Domain = ProtonIrr_Freq = 399.78219838[MHz]Irr_Offset = 5[ppm]Blanking = 5[us]Clipped = FALSEDecimation_Reg = r: 44( 43),g: 28Scans = 771Total_Scans = 771

Relaxation_Delay = 2[s]Recvr_Gain = 66Temp_Get = 24.9[dC]X_90_Width = 25[us]X_Acq_Time = 0.23068672[s]X_Angle = 30[deg]X_Atn = 6[dB]X_Pulse = 8.33333333[us]Irr_Atn_Dec = 22.86[dB]Irr_Atn_Dec_Calc = 22.86[dB]Irr_Atn_Dec_Default_Calc = 22.86[dB]Irr_Atn_Noe = 22.86[dB]Irr_Dec_Bandwidth_Hz = 4.7826087[kHz]Irr_Dec_Bandwidth_Ppm = 11.96303566[ppm]Irr_Dec_Freq = 399.78219838[MHz]Irr_Dec_Merit_Factor = 2.2Irr_Decoupling = TRUEIrr_Noe = TRUEIrr_Noise = WALTZIrr_Offset_Default = 5[ppm]Irr_Pwidth = 0.115[ms]Irr_Pwidth_Default = 0.115[ms]Irr_Pwidth_Default_Calc = 0.115[ms]Irr_Pwidth_Temp1 = 0.115[ms]Irr_Wurst = FALSEComment_1 = *** Pulse ***Comment_102 = *** irr_decouplingComment_110 = *** noe_time ***Comment_7 = *** Pulse Delay **Decimation_Rate = 0Get_90 = pulse_service::getGet_Atn = pulse_service::get

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