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Highly selective and controllable pyrophosphate induced anthracene-excimer formation in water

Feihu Huang and Guoqiang Feng*

Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, P.R. China, [email protected]

1. General Experimental Details.

Starting materials were purchased from commercial suppliers and were used without further purification. 9,10-bisaminomethylanthracene1 and 2-bromomethyl-6-hydroxymethylpyridine2 were prepared according the previously published methods. All solvents were purified by the most used methods before use. N-(2-hydroxyethyl)piperazine-N’-(2-ethane-sulfonic acid) (HEPES) was used to prepare buffer solution and all solutions were prepared with using distilled water that had been passed through a Millipore-Q ultrapurification system. NMR spectra were measured on Varian Mercury 400 and 600 instruments. UV-vis spectra and fluorescent spectra were recorded on an Agilent Cary 100 UV-vis spectrophotometer and an Agilent Cary Eclipse fluorescence spectrophotometer, respectively. Both UV-vis and fluorescence spectrophotometer are equipped with a temperature controller. Standard quartz cuvettes with a 10 mm lightpath are used for all UV-vis spectra and fluorescent spectra measurements.

2. Synthesis of ZnL2

Synthesis of L2: To a solution of 11 (236 mg, 1.00 mmol) and 22 (810 mg, 4.01 mmol) in anhydrous

CH3CN (30 mL) was added K2CO3 (621 mg, 4.50 mmol). The reaction mixture was then heated to reflux for 12 hours, and the solvent was evaporated under reduced pressure. To the residue was added dichloromethane (60 mL), and the organic phase was washed with water and brine followed by drying

over MgSO4. After removal of the solvent under reduced pressure, the crude product was purified by silica gel column (eluent: dichloromethane /methanol = 15:1 (v/v)) to give a yellow powder (576 mg, 1 80% yield). Mp 118-120 ºC. H NMR (600 MHz, CDCl3) δ 8.45 (d, J = 6.3 Hz, 4H), 7.44–7.49 (m, 8H),

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7.13 (d, J = 7.6 Hz, 4H), 6.95 (d, J = 7.6 Hz, 4H), 4.65 (s, 8H), 4.63 (s, 4H), 3.89 (s, 8H). 13C NMR

(150 MHz, CDCl3): δ 158.0, 136.7, 134.1, 130.9, 130.2, 125.4, 125.0, 121.9, 118.7, 63.9, 60.1, 51.4. IR -1 (KBr) νmax (cm ): 3393 (br s, OH), 2855, 1595 (s), 1577 (s), 1454 (s), 1354, 1153, 1067, 995, 789, 760, + + + 703 ; ESI-MS: m/z found 721.4 (M + H ); HR-MS Calc. for C44H45N6O4 (M + H ) 721.3497, found 721.3496.

OH HO

N N OH OH N N N N

1 H NMR spectrum of L2 in CDCl3.

OH HO

N N OH OH N N N N

13 C NMR spectrum of L2 in CDCl3.

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Intens. +MS, 11.2min (#417) x107 OH HO 721.4 N N 6 OH OH N N N N H+

L2 + + 2 M+H :C44H45N6O4 Mass: 721.3

0 200 300 400 500 600 700 800 m/z ESI-MS spectrum of L2

x10 1 +ESI Scan (11.987 min) Frag=160.0V WorklistData23.d 721.3496 1.2 OH HO N N OH OH 1.1 N N N N H+ 1

L2 0.9 + Chemical Formula: C44H45N6O4 Exact Mass: 721.34968 0.8

0.7 722.3533 0.6

0.5 0.4

0.3

0.2 0.1

0 717 718 719 720 721 722 723 724 725 726 727 728 Counts (%) vs. Mass-to-Charge (m/z)

HR-MS spectrum of L2

Synthesis of ZnL2: To a solution of L2 (36 mg, 0.05 mmol) in 5 mL of MeOH, was added

Zn(NO3)2·6H2O (33 mg, 0.11 mmol), and the mixture was stirred for 1 h at rt. After concentrating under reduced pressure, the obtained solid was recrystallised from MeOH to give ZnL2. 1H NMR

(400 MHz, D2O): 8.44(4H, br, PyH), 7.68 (4H, d, J = 4.8 Hz, PyH), 7.47 (4H, dd, J = 4.8 Hz, PyH),

6.95 (4H, d, ArH), 6.37 (4H, br, ArH), 5.11 (4H, br, 2CH2), 4.61 (4H, br, 2CH2), 4.52 (4H, br,

2CH2), 4.24 (4H, br, 2CH2), 4.09 (4H, br, 2CH2). Crystal of ZnL2 suitable for X-ray diffraction studies was obtained from a solution of ZnL2 in a mixture of MeOH and water after addition of - NaCl. Each complex molecule contains two CH3OH and two NO3 as counter ions. The total 2+ - formula of ZnL2 crystal is C46H52Cl2N8O12Zn2 (C44H44Cl2N6O4Zn2 2CH3OH2NO3 ), M = 1110.62. CCDC 949746 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

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Crystal data for ZnL2:

Table 1. Crystal data and structure refinement for mo_111219a_0m. Identification code mo_111219a_0m Empirical formula C23 H26 Cl N4 O6 Zn Formula weight 555.30 Temperature 100(2) K Wavelength 0.71073 Å Crystal system Monoclinic Space group P2(1)/n Unit cell dimensions a = 14.6762(14) Å = 90° b = 8.8145(8) Å = 92.436(2) ° c = 17.7485(16) Å  = 90° Volume 2293.9(4) Å3 Z 4 Density (calculated) 1.608 Mg/m3 Absorption coefficient 1.237 mm-1 F(000) 1148 Crystal size 0.16 x 0.12 x 0.10 mm3 Theta range for data collection 1.76 to 26.00°. Index ranges -18<=h<=18, -10<=k<=10, -19<=l<=21 Reflections collected 15921 Independent reflections 4489 [R(int) = 0.0465] Completeness to theta = 26.00 99.6 % Absorption correction None Max. and min. transmission 0.8863 and 0.8267 Refinement method Full-matrix least-squares on F2 Data / restraints / parameters 4489 / 10 / 334 Goodness-of-fit on F2 1.092 Final R indices [I>2sigma(I)] R1 = 0.0402, wR2 = 0.1055 R indices (all data) R1 = 0.0591, wR2 = 0.1194 Extinction coefficient 0.0030(6) Largest diff. peak and hole 0.834 and -0.465 e.-3

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Fig. S1. The crystal structure of ZnL2

D2O OH HO

N Zn2+ Zn2+ N HO OH N N N N

ZnL2

CH3OH

1 H NMR spectrum of ZnL2 in D2O

References: 1. (a) Gassensmith,J. J.; Arunkumar, E.; Barr, L.; Baumes, J. M.; DiVittorio, K. M.; Johnson, J. R.; Noll, B. C.; Smith, B. D. J. Am. Chem. Soc. 2007, 129, 15054–15059. (b) Gunnlaugsson, T.; Davis, A. P.; O’Brien, J. E.; Glynn, M. Org. Lett. 2002, 4, 2449–2452. (c) Gunnlaugsson, T.; Davis, A. P.; O’Brien, J. E.; Glynn, M. Org. Biomol. Chem. 2005, 3, 48–56. 2. Thiabaud, G.; Guillemot, G.; Schmitz-Afonso, I.; Colasson, B.; Reinaud, O. Angew. Chem., Int. Ed. 2009, 48, 7383–7386.

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3. Fluorescence spectra changes of ZnL2 (5 M) upon addition of different anions

NewFunction2 ( 60 60 Model User) y=I0+m0*0.5*((x +H+k)-sqrt((x+H 50 50 Equation +k)*(x+H+k)-4*x 0M *H)) Reduced 1.26151 40 40 Chi-Sqr Adj. R-Square 0.99535 [ATP] Value Standard Error 30 30 B I0 57.95 0 B m0 -10.54487 0.10257 250M B H50 Intensity 20 20 B K 11.55594 0.49788 Intensity (430nm) 10 10

0 0 400 450 500 550 600 650 700 0 50 100 150 200 250 Wavelength(nm) [ATP] M

Fig. S2 Left: Fluorescent titration of complex ZnL2 (5 M) upon the addition of ATP (0-250 M) in pH = 7.2, 10 mM HEPES buffer solution at 25˚C; Right: The saturation curve of

fluorescent intensity changes at 430 nm, λex = 380 nm, dex = 5 nm, dem = 2.5 nm. Red line is the curve

fitting using a 1:1 binding mode, and the Ka between ZnL2 and ATP was determined to be about 8.65 × 104 M-1 (R2 = 0.99535) under this condition.

60 60 NewFunction2 (U Model ser) y=I0+m0*0.5*((x+ 50 0M 50 H+k)-sqrt((x+H+k Equation )*(x+H+k)-4*x*H))

40 [Citrate] 0.11823 40 Reduced Chi-Sqr

Adj. R-Square 0.99954 30 30 Value Standard Error 100M C I0 56.267 0 C m0 -10.72848 0.04086 Intensity 20 C H50 20 C k 3.99468 0.07019 Intensity (430nm) 10 10

0 0 400 450 500 550 600 650 700 0 20 40 60 80 100 Wavelength(nm) [Citrate] M

Fig. S3. Left: Fluorescent titration of complex ZnL2 (5 M) upon the addition of citrate (0-100 M) in pH = 7.2, 10 mM HEPES buffer solution at 25˚C; Right: The saturation curve of

fluorescent intensity changes at 430 nm, λex = 380 nm, dex = 5 nm, dem = 2.5 nm. Red line is the curve

fitting using a 1:1 binding mode, and the Ka between ZnL2 and citrate was determined to be about 2.51 × 105 M-1 (R2 = 0.99954) under this condition.

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60 [ADP] 0-50M 50

30 Intensity 20

0 400 450 500 550 600 650 700 Wavelength (nm) Fig. S4. Fluorescent titration of complex ZnL2 (5 M) upon the addition of ADP (0-50 M)

in pH = 7.2, 10 mM HEPES buffer solution at 25˚C, λex = 380 nm, dex = 5 nm, dem = 2.5 nm.

90 75 80 500M 70 70 60 [AMP] 50 65 Model NewFunction2 (User) 40 Equation y=I0+m0*0.5*((x+H+k)-sqrt((x+H+k)*(x+H+k)-4*x*H))

Intensity 0M Reduced Chi-Sq 0.30857 30 Adj. R-Square 0.99355 60 Value Standard Error B I0 55.43 0 20 Intensity (430nm) B m0 4.46597 0.10101 BH 5 0 10 55 B k 72.92758 5.0157 0 400 450 500 550 600 650 700 0 100 200 300 400 500 Wavelength(nm) [AMP] M Fig. S5. Left: Fluorescent titration of complex ZnL2 (5 M) upon the addition of AMP (0-500 M) in pH = 7.2, 10 mM HEPES buffer solution at 25˚C; Right: The saturation curve of

fluorescent intensity changes at 430 nm, λex = 380 nm, dex = 5 nm, dem = 2.5 nm. Red line is the curve

fitting using a 1:1 binding mode, and the Ka between ZnL2 and AMP was determined to be about 1.37 × 104 M-1 (R2 = 0.99355) under this condition.

60 ZnL2 (5 M) + other anions

30 Intensity

0 400 450 500 550 600 650 700 Wavelength(nm)

3− Fig. S6. Fluorescent titration of complex ZnL2 (5 M) upon the addition of other anions (PO4 , 2− − − − − − − − 2− − 2− − − − − − HPO4 , H2PO4 , F , Cl , Br , I , ClO4 , AcO , CO3 , HCO3 , SO4 , HSO4 , NO3 , N3 , NO2 , SCN , 2− 2− S2O3 , S2O7 , 50 M of each was used) in pH = 7.2, 10 mM HEPES buffer solution, λex = 380 nm, dex

= 5 nm, dem = 2.5 nm.

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4. Fluorescence spectra changes of ZnL2 (50 M) upon addition of different anions

250

NewFunction2 (U 210 Model ser) y=I0+m0*0.5*((x+ 200 H+k)-sqrt((x+H+k 0 M Equation 180 )*(x+H+k)-4*x*H))

5.12723 150 [ATP] Reduced Chi-Sqr 150 Adj. R-Square 0.99798 Value Standard Error 500 M B I0 212.6 0 100 120 B m0 -3.2185 0.02327 Intensity B H500 B k 16.04892 0.74069 90

50 Intensity (430nm)

60 0 400 450 500 550 600 650 700 0 100 200 300 400 500 Wavelength(nm) [ATP] M Fig. S7. Left: Fluorescent titration of complex ZnL2 (50 M) upon the addition of ATP (0-500 M) in pH = 7.2, 10 mM HEPES buffer solution at 25˚C; Right: The saturation curve of

fluorescent intensity changes at 430 nm, λex = 380 nm, dex = dem = 2.5 nm. Nonlinear least-squares 4 -1 2 fitting the fluorescent titration curve gave Ka between ZnL2 and ATP is 6.23 × 10 M (R = 0.99798) under this condition.

250 250

NewFunction2 ( Model 200 User) 200 y=I0+m0*0.5*((x 0 M +H+k)-sqrt((x+H Equation +k)*(x+H+k)-4*x* [Citrate] H)) 150 150 Reduced 7.17612 Chi-Sqr Adj. R-Square 0.99774 1000 M Value Standard Error 100 B I0 213.334 0 100 B m0 -3.79406 0.02605 Intensity B H500 B k 14.55429 0.72015

50 Intensity (430nm) 50

0 0 400 450 500 550 600 650 700 0 200 400 600 800 1000 Wavelength(nm) [Citrate] M Fig. S8. Left: Fluorescent titration of complex ZnL2 (50 M) upon the addition of citrate (0-1000 M) in pH = 7.2, 10 mM HEPES buffer solution at 25˚C; Right: The saturation curve of

fluorescent intensity changes at 430 nm, λex = 380 nm, dex = dem = 2.5 nm. Nonlinear least-squares 4 -1 2 fitting the fluorescent titration curve gave Ka between ZnL2 and citrate is 6.87 × 10 M (R = 0.99774) under this condition.

250

200

150 [ADP] 0-500M

100 Intensity

0 400 450 500 550 600 650 700 Wavelength(nm) Fig. S9. Fluorescent titration of complex ZnL2 (50 M) upon the addition of ADP (0-500M)

in pH = 7.2, 10 mM HEPES buffer solution at 25˚C, λex = 380 nm, dex = dem = 2.5 nm.

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300

250

200 [AMP] 0-1200M

150

Intensity 100

0 400 450 500 550 600 650 700 Wavelength(nm)

Fig. S10. Fluorescent titration of complex ZnL2 (50 M) upon the addition of AMP (0-1200 M)

in pH = 7.2, 10 mM HEPES buffer solution at 25˚C, λex = 380 nm, dex = dem = 2.5 nm.

300

250 ZnL2 (50 M) + other anions

200

150

Intensity 100

0 400 450 500 550 600 650 700 Wavelength(nm)

3− Fig. S11. Fluorescent titration of complex ZnL2 (50 M) upon the addition of other anions (PO4 , 2− − − − − − − − 2− − 2− − 2− 2− − HPO4 , H2PO4 , F , Cl , Br , I , ClO4 , AcO , CO3 , HCO3 , SO4 , HSO4 , S2O7 , S2O3 , SCN , − − − NO3 , N3 , NO2 , 500 M of each) in pH = 7.2, 10 mM HEPES buffer solution at 25˚C, λex = 380 nm,

dex = dem = 2.5 nm.

Table S1 Summary of the apparent association constants (Ka) of receptors ZnL1 and ZnL2 to some anion species. -1 Anions Ka (M ) between receptor and anions ZnL1a ZnL2b ZnL2b (when 5 M was used) (when 50 M was used) PPi Not reported 5.39 × 105 7.68 × 105 5 c c NaH2PO4 2.9 × 10 - - ATP 4.0 × 105 8.65 × 104 6.23 × 104 ADP 1.6 × 105 -c -c AMP 9.1 × 103 1.37 × 104 -c Citrate Not reported 2.51 × 105 6.87 × 104 a: Literature values (A. Ojida, Y. Mito-oka, K. Sada, I. Hamachi, J. Am. Chem. Soc., 2004, 126, 2454-2463, see b c page 2456). : Experimental values in this work, see above figures. : Ka was not determined due to too small changes of fluorescence. Note: Much higher ion strength conditions (50 mM HEPES, 50 mM NaCl, pH 7.2) were

used in the literature for the Ka measurement between ZnL1 and ATP, ADP and AMP. Take this factor into

consideration, from the Ka values of ATP between ZnL1 and ZnL2, introducing -CH2OH groups to ZnL1 may decrease its binding affinities to phosphate anions.

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5. The fluorescence colour changes of ZnL2 upon addition of different anions

Fig. S12. Fluorescence colour (under 365 nm light) changes of ZnL2 (50 µM) in 10 mM aqueous HEPES buﬀer solution (pH = 7.2) with 500 µM of different anions (from left to right: none, PPi, ATP, 3− 2− − − − − − − − 2− − 2− − Citrate, ADP, AMP, PO4 , HPO4 , H2PO4 , F , Cl , Br , I , ClO4 , AcO , CO3 , HCO3 , SO4 , HSO4 , 2− 2− − − − − S2O7 , S2O3 , SCN , NO3 , N3 , NO2 ).

6. The effect of concentration of ZnL2-PPi on anthracene-excimer formation

300 270 240 210 [ZnL2-PPi] 180 150 120 Intensity 90 60 30 0 400 450 500 550 600 650 700 Wavelength(nm)

Fig. S13. Top: Fluorescent spectra of ZnL2-PPi (5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75

and 80 µM) in 10 mM HEPES buffer solution (pH = 7.2) at 25˚C with λex = 380 nm, dex = 5 nm, dem = 2.5 nm. Down: Emission color changes of ZnL2-PPi under UV light of 365 nm. The concentration of ZnL2-PPi from left to right: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 and 80 µM, respectively.

7. The effect of buffer concentration and salts on PPi induced anthracene-excimer formation

(a) (b) (c) [HEPES buffer] = 100 mM 250 [HEPES buffer] = 20 mM 250 [HEPES buffer] = 50 mM 250

[PPi] 200 [PPi] 200 [PPi] 0-100 M 200 0-100 M 0-250 M

150 150 150 Intensity Intensity Intensity 100 100 100

50 50 50

0 0 0 400 450 500 550 600 650 700 400 450 500 550 600 650 700 400 450 500 550 600 650 700 Wavelength(nm) Wavelength(nm) Wavelength(nm) Fig. S14. The effect of buffer concentration on PPi induced anthracene-excimer formation. HEPES

buffer concentration (a) 20 mM; (b) 50 mM; (c) 100 mM. All experiments were measured at 25˚C and [ZnL2] = 50 µM. These experiments indicate that the excimer formation is more difficult at higher buffer concentrations.

(b) 300 (c) (a) [NaNO ] = 10 mM [NaNO ] = 50 mM [NaNO ] = 100 mM 250 3 3 300 3 250 250 200 200 [PPi] ZnL2 200 [PPi] 0-500 M 150 ZnL2 + 1 eq PPi 0-500 M ZnL2 + 2 eq PPi 150 150 Intensity Intensity 100 Intensity 100 100

50 50 50

0 0 0 400 450 500 550 600 650 700 400 450 500 550 600 650 700 400 450 500 550 600 650 700 Wavelength(nm) Wavelength(nm) Wavelength(nm)

Fig. S15. The effect of presence of salts on PPi induced anthracene-excimer formation. NaNO3 concentration (a) 10 mM; (b) 50 mM; (c) 100 mM. All experiments were measured in 10 mM HEPES buffer (pH 7.2) at 25˚C and [ZnL2] = 50 µM. These experiments indicate that the excimer formation is tending to be hindered at higher salts concentrations.

8. The effect of temperature (T) on PPi induced anthracene-excimer formation

(a) 200 (b) 200

160 20C 160 20C

T T 120 120

90C 90C 80 80 Intensity Intensity

40 40

0 0 400 450 500 550 600 650 700 400 450 500 550 600 650 700 Wavelength(nm) Wavelength(nm)

160

120

80 Intensity (480 nm)

20 30 40 50 60 70 80 90 100 T (℃ )

Fig. S16. (a) The fluorescence spectra changes of ZnL2-PPi (50 µM) as temperature increased from 20˚C to 90˚C. (b) The fluorescence spectra changes of ZnL2-PPi (50 µM) as temperature decreased from 90˚C to 20˚C. (c) A plot of fluorescence intensity changes of ZnL2-PPi (50 µM) at 480 nm as a function of temperature increase () or decrease (). All spectra were measured in

10 mM HEPES buffer solution (pH = 7.2) with λex = 380 nm, dex = 5 nm, dem = 2.5 nm.

9. Job’s plot examined between ZnL2 and PPi, ATP and citrate

(a) 100

40 - I (430nm) 0 I

0 0.0 0.2 0.4 0.6 0.8 1.0 [PPi]/([PPi]+[ZnL2])

45 (b) (c) 40 60

35 50 30 40 25 - I - I 0 0 I 20 I 30

15 20 10 10 5 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 [ATP]/([ATP]+[ZnL2] [Citrate]/([Citrate]+[ZnL2]

Fig. S17. Job's plot examined between ZnL2 and PPi (a), ATP (b) and citrate (c) in 10 mM HEPES buffer (pH = 7.2) solution at 25˚C. All experiments were explored at [PPi, ATP or

citrate] + [ZnL2] = 50 M and with λex = 380 nm, dex = 2.5 nm, dem = 2.5 nm.

10. The 31P NMR and Mass studies

PPi

PPi + ZnL2

ZnL2+PPi ZnL2

31 Fig. S18. Left: P NMR spectra of PPi, and a mixture of PPi and ZnL2 (1 mM of each) in D2O. Right: The fluorescence color of ZnL2 (1mM) under 365 nm in the presence and absence of PPi in the NMR tube.

[L2 +H]+

[2ZnL2 +2PPi+2H]2+

1025

1027 1023

1026 1024 1029 1028 1030 1031 1032

1020 1022 1024 1026 1028 1030 1032 1034 1036 1038 Isotopic Distributions Calculated

Fig. S19. Top: Mass spectrum of a mixture of ZnL2 and PPi (50 µM each). The peak at m/z = 1023 2+ 2+ corresponds to [C88H90N12O22P4Zn4] (= [2ZnL2 + 2PPi + 2H] , which Mass was calculated as 2046).

Bottom: Isotopic distribution calculated for C44H45N6O11P2Zn2 using a program in www.chemcalc.org.

Elemental Composition Report Single Mass Analysis Tolerance = 5.0 PPM / DBE: min = -1.5, max = 50.0 Isotope cluster parameters: Separation = 1.0 Abundance = 1.0% Monoisotopic Mass, Odd and Even Electron Ions 3 formula(e) evaluated with 1 results within limits (up to 50 closest results for each mass)

Minimum: -1.5 Maximum: 200.0 5.0 50.0 Mass Calc. Mass mDa PPM DBE Score Formula

1023.1177 1023.1204 -2.7 -2.7 26.5 1 C44H45N6O11P2Zn2

Z. Abdulkarim hf-10-6-2 37williamsN05 106 (1.767) AM (Cen,4, 80.00, Ar,5000.0,1053.60,1.00); Cm (3:238) 1053.6033 100 1025.1250 6.84e4 1009.5787

% 1004.6189 1048.6520

0 m/z 1000 1010 1020 1030 1040 1050 1060

Fig. S20. High Resolution Mass spectrum of a mixture of ZnL2 and PPi (50 µM each). The peak at

m/z = 1023.1177 corresponds to formula C44H45N6O11P2Zn2, which was calculated as 1023.1204.