Ammonium-Bearing Dinuclear Copper(II) Complex: A Highly Selective and Sensitive Colorimetric Probe for Pyrophosphate Wenxiang Yu, Jian Qiang, Jun Yin, Srinivasulu Kambam, Fang Wang, Yong Wang, and Xiaoqiang Chen*, State Key Laboratory of Materials-Oriented Chemical Engineering, College of Chemistry and Chemical Engineering, anjing Tech University, anjing 210009, P.R. China. Key Laboratory of Pesticide and Chemical Biology, Ministry of Education, College of Chemistry, Central China ormal University, Wuhan 430079, P.R. China. *E-mail: chenxq@njtech.edu.cn 1
Materials and Instruments Unless otherwise noted, materials were obtained from commercial suppliers and were used without further purification. 4-Methyl-2,6-bis((pyridin-2-ylmethylamino)methyl)phenol was prepared according to the previous report (Bioorganic & Medicinal Chemistry Letters, 2008, 18, 109 113). Buffer solution (HEPES 10 mm, ph 7.0) was prepared using deionized water. 1 H MR and 13 C MR spectra were recorded at 300 K on a Bruker Avance DRX 500 FT MR spectrometer, operating at operating at 500 or 300 MHz for 1 H MR spectra and 75 MHz for 13 C MR spectra. Mass spectra were obtained using a Waters Micromass Q-Tof micro mass spectrometer. UV absorption spectra were obtained on α-1860a UV/VIS Spectrometer. Mass spectra of Cu-L a and Cu-L b were obtained using LCQ Fleet Mass Spectrometer. The IR spectra were obtained using Thermo Scientific icolet is10 FT-IR Spectrometer. Preparation of solutions for absorption study Stock solutions (10 mm) of pyrocatechol violet (PV) and anions PPi, a 2 HPO 4, CH 3 COOH 4, a 2 SO 4, a 2 CO 3, af, acl, abr, KI and MgCl 2 in distilled water were prepared. In a typical experiment, test solutions were prepared by placing 60 μl of PV stock solution into a test tube, diluting the solution to 3 ml with HEPES buffer, and adding an appropriate buffer of complex and anions. Absorption spectra were measured immediately. PPase Assays In a typical experiment, the stock solutions of Cu-L b -PV and PPi were added the HEPES buffer (10mM, ph7.0), and the final concentration are 20 μm and 200 μm, respectively. After incubated for 5 min at room temperature, the reaction processes were monitored by UV-Vis spectrameter under three catalytic conditions: (a) only PPase (10 units); (b) only Mg 2+ (20 μm); (c) PPase (10 units) Mg 2+ (20 μm). Synthesis O OH O OH O OH OH OH Cl OH H 2 O CH3 COOH,110 o C,5h Pht HMT/TFA,74 o C 2,20h,HCl abh 3 C/ZnCl 2 Pht MeOH, 2,r.t.,12h Pht SOCl 2 /CH 2 Cl 2 Pht 1 2 3 4 H OH H Pht OH Pht OH HO 1) 2 H 4,EtOH,r.t,12h H 2 OH H 2 OH HO O Pht= CH 2 Cl 2,r.t,Et 3 2)aOH(aq.),r.t,45min O 5 Scheme S1. Synthesis of compound L b. L b 2-(3-hydroxy benzyl)xylylenimine-1,3-diketone (1) 2
A mixture of 2-aminomethyl phenol (2.5 g) and phthalic anhydride (3.125 g) in acetic acid (75 ml) was stirred at 110 o C for 5 h. Solvent was evaporated and the residue left was washed with water to give 2-(3-hydroxy benzyl)xylylenimine-1,3-diketone as a yellow solid (5.6 g, 95%). 1 H MR (500 MHz, CD 3 OD) δ: 4.74(s, 2H), 6.66(t, 1H, J=9.5 Hz), 6.82(t, 2H, J=18.2 Hz), 7.13(t, 1H, J=15.7 Hz), 7.79(m, 2H), 7.86(m, 2H); 13 C MR (75 MHz, (CD 3 ) 2 SO) δ: 41.22, 114.52, 118.37, 123.66, 130.07, 131.96, 135.02, 138.44, 158.00, 168.10. TOF MS m/z = 252.0664 [M + H] +, calc. for C 15 H 11 O 3 = 252.0661. 4-((1,3-dioxo-2,3-dihydro-1H-inden-2-yl)methyl)-2-hydroxybenzaldehyde (2) A mixture of compound 1 (5 g, 20 mmol) and methenamine (5.5 g, 40 mmol) in trifluoroacetic acid was refluxed for 20 h. After the completion of the reaction, 50 ml HCl (4 M) was added, and stirred for 40 min. The solution was dissolved in CH 2 Cl 2 and washed with water. The organic layer was separated and dried over a 2 SO 4, and the solvent was evaporated under reduced pressure. The product was purified by silica gel chromatography with CH 2 Cl 2 as the eluent to obtained the product (1.54 g, 28%). 1 H MR (300 MHz, CDCl 3 ) δ: 4.85(s, 2H), 6.97(s, 1H), 7.05(d, 1H, J=9.3), 7.52(d, 1H, J=7.9 Hz), 7.75(m, 2H), 7.89(m, 2H), 9.85(s, 1H), 11.00(s, 1H). 13 C MR (75 MHz, (CD 3 ) 2 SO) δ: 40.60, 115.34, 116.86, 118.29, 121.52, 123.22, 129.38, 131.47, 134.52, 136.02, 145.27, 160.78, 167.56, 190.76. TOF MS m/z = 280.0610 [M + H] +, calc. for C 16 H 11 O 4 = 280.0623. 2-(3-hydroxy-4-(hydroxymethyl)benzyl)-2H-indene-1,3-dione (3) Dried ZnCl 2 (0.48 g, 3.48 mmol), abh 3 C (0.89 g, 14.4 mmol) and 2 (1.08 g, 3.48 mmol) were added to anhydrous methanol (20 ml). The mixture was reacted over night, the color of the solution changed from yellow to white, then stopped the reaction. The solution was evaporated under reduced pressure to remove the half of solvent, then hydrochloric acid (2 mol/l, 40 ml) was added, and continued to stir for 45 min resulting in white solid separated out. After filtered, washed with water, then dried in vacuum to give the desired product as a white solid (0.98 g, 82%). 1 H MR (300 MHz, CDCl 3 ) δ: 4.78(s, 2H), 4.81(s, 2H), 6.91(d, 1H, J=10.3 Hz), 6.92(s, 1H), 6.99(d, 1H, J=7.6 Hz), 7.72(m, 2H), 7.85(m, 2H). 13 C MR (75 MHz, (CD 3 ) 2 SO) δ: 39.50, 57.95, 113.31, 117.76, 123.17, 127.51, 131.47, 134.56, 135.80, 154.22, 167.60. TOF MS m/z = 282.0770 [M + H] +, calc. for C 16 H 13 O 4 = 282.0780. 2,2'-(3,3'-(2-hydroxy-5-methyl-1,3-phenylene)bis(methylene)bis((pyridin-2-ylmethyl)azanediyl)bis(methyl ene)bis(3-hydroxy-4,1-phenylene))bis(methylene)diisoindoline-1,3-dione (5) 3 (0.25 g, 0.88 mmol) were dissolved in 20 ml of SOCl 2 and stirred ambient temperature over night under nitrogen. Excess of SOCl 2 was removed under reduced pressure. The resulting solid separated, dried over a 2 SO 4 and the solvent was evaporated to give 0.26 g (96%) of the product 4. A solution of 4 (0.5 g, 1.66 mmol), 3
4-Methyl-2,6-bis((pyridin-2-ylmethylamino)methyl)phenol (0.26 g, 0.75 mmol) and Et 3 in CH 2 Cl 2 (10 ml) was stirred at ambient temperature for two days. The mixture was diluted with CH 2 Cl 2 and washed with brine (2 10 ml). The organic was dried over a 2 SO 4 and solvent was evaporated. Purified on silica gel using CH 2 Cl 2 /CH 3 OH (100/1.5) as eluent to give the desired product (0.25 g, 33%). 1 H MR (500 MHz, CDCl 3 ) δ: 2.16(d, 3H, J= 12.3 Hz), 3.67(s, 2H), 3.71(s, 2H), 3.77(s, 2H), 4.72(s, 4H), 4.86(d, 1H, J=15.8 Hz), 6.77(s, 2H), 6.78(d, 2H, J=8.3 Hz), 6.92(d, 2H, J=8.0 Hz), 7.20(t, 4H, J=8.4 Hz), 7.67(m, 8H), 7.81(m, 4H), 8.55(d, 2H, J=4.5 Hz). 13 C MR (75 MHz, (CD 3 ) 2 SO) δ: 20.64, 40.01, 53.87, 55.35, 58.55, 114.72, 118.23, 122.96, 123.54, 129.99, 130.66, 131.97, 134.99, 148.98, 153.69, 157.00, 158.11, 168.07. TOF MS m/z = 879.3518 [M + H] +, calc. for C 53 H 46 6 O 7 = 879.3533, m/z = 902.3401 [M + a] +, calc. for C 53 H 46 6 O 7 a= 902.3431. 2,2'-(2-hydroxy-5-methyl-1,3-phenylene)bis(methylene)bis((pyridin-2-ylmethyl)azanediyl)bis(methylene)bis( 5-(aminomethyl)phenol) (L b ) 5 (0.2 g, 2.27 nmol) was suspended in 10 ml of EtOH and hydrazine hydrate (0.18 g) was added. The solution was refluxed for 2 hours and stirred at ambient temperature over night. The solvent was evaporated under reduced pressure and the resulting solid treated with 30 ml of aoh (2 M). After stirring for 45 min, the basic solution was adjusted to ph 3 with 2 M HCl, then filtrated and the filtrate was adjusted back to ph 9, after refiltrated then afforded (0.98 g, 70%) of the solid product. 1 H MR (500 MHz, D 2 O) δ: 2.11(s, 3H), 3.96(s, 4H), 4.38(m, 14H), 6.75(d, 4H, J=19.4 Hz), 6.85(d, 4H, J=6.1 Hz), 7.05(d, 4H, J=11.0 Hz), 7.25(d, 4H, J=7.5 Hz), 7.32(s, 8H), 7.76(s, 4H), 8.31(s, 2H). 13 C MR (75 MHz, D 2 O) δ: 17.18, 40.27, 46.85, 53.24, 53.77, 54.14, 113.26, 115.15, 116.41, 118.60, 124.82, 125.87, 130.15, 130.54, 132.79,134.06, 142.11, 142.36, 143.48, 149.77, 153.02. TOF MS m/z = 619.3397 [M + H] +, calc. for C 37 H 42 6 O 3 = 619.3424. The preparation of Cu-L a and Cu-L b : L a (5.6 mg, 0.01 mmol) was suspended in 5 ml H 2 O. Then the solution of Cu(ClO 4 ) 2 6H 2 O (7.4 mg, 0.02 mmol) in 5 ml H 2 O was added to the solution of L a. After that, heat the mixed solution at 50 o C for 1 h and the light yellow solution Cu-L a was obtained. Similarly, the light yellow solution of Cu-L b was obtained. Mass for Cu-L a : 683.42, [Cu-L a ] +, C 35 H 33 Cu 2 4 O 3 ; 701.25, [Cu-L a +H 2 O] +, C 35 H 35 Cu 2 4 O 4 ; 719.25, [Cu-L a +2H 2 O] +, C 35 H 37 Cu 2 4 O 5 ; 783.25, [Cu-L a +HClO 4 ] +, C 35 H 34 ClCu 2 4 O 7. Mass for Cu-L b : 777.50, [Cu-L b +2OH - ] +, C 37 H 43 Cu 2 6 O 5 ; 813.25, [Cu-L b +2OH - +2H 2 O] +, C 37 H 47 Cu 2 6 O 7. 4
Figure S1. 1 H MR spectrum of compound 1 in CD 3 OD. Figure S2. 13 C MR spectrum of compound 1 in (CD 3 ) 2 SO. 5
1 SAMPLE-131031-02 72 (1.335) 100 252.0664 TOF MS ES- 3.13e3 % 253.0689 0 121.0412 165.0256 228.1093 254.0722 310.0246 376.0996 398.0845 454.0610 m/z 100 150 200 250 300 350 400 450 Figure S3. High-Resolution ESI-MS spectrum of compound 1. Figure S4. 1 H MR spectrum of compound 2 in CDCl 3. 6
Figure S5. 13 C MR spectrum of compound 2 in (CD 3 ) 2 SO. 2 SAMPLE-131031-02 206 (3.822) 100 280.0614 TOF MS ES- 488 % 281.0487 171.0160 312.0826 231.0156 365.0220 414.0705 0 m/z 160 180 200 220 240 260 280 300 320 340 360 380 400 420 Figure S6. High-Resolution ESI-MS spectrum of compound 2. 7
Figure S7. 1 H MR spectrum of compound 3 in CDCl 3. Figure S8. 13 C MR spectrum of compound 3 in CDCl 3 (CD 3 ) 2 SO. 8
3 SAMPLE-131031-02 334 (6.199) 100 282.0770 TOF MS ES- 4.90e3 % 0 283.0682 171.0215 280.0619 284.0755 340.0435 409.2947 565.1287 100 150 200 250 300 350 400 450 500 550 m/z Figure S9. High-Resolution ESI-MS spectrum of compound 3. Figure S10. 1 H MR spectrum of compound 5 in CDCl 3. 9
Figure S11. 13 C MR spectrum of compound 5 in (CD 3 ) 2 SO. 4 SAMPLE-131031-01 456 (8.496) 100 879.3518 TOF MS ES+ 1.44e3 % 880.3574 901.3371 902.3401 903.3436 698.3300 819.4164 868.4188 727.2947 935.3028 973.4457 0 m/z 700 750 800 850 900 950 1000 Figure S12. High-Resolution ESI-MS spectrum of compound 5. 10
Figure S13. 1 H MR spectrum of compound L b in D 2 O. Figure S14. 13 C MR spectrum of compound L b in D 2 O. 11
5 SAMPLE-131031-01 625 (11.646) 100 619.3405 TOF MS ES+ 7.06e3 % 620.3474 0 616.3340 617.3280 618.3421 621.3452 622.3367 623.2684 m/z 616.00 618.00 620.00 622.00 Figure S15. High-Resolution ESI-MS spectrum of compound L b. 12
Figure S16. The mass spectra of Cu-L a : 683.42, [Cu-L a ] +, C 35 H 33 Cu 2 4 O 3 ; 701.25, [Cu-L a +H 2 O] +, C 35 H 35 Cu 2 4 O 4 ; 719.25, [Cu-L a +2H 2 O] +, C 35 H 37 Cu 2 4 O 5 ; 783.25, [Cu-L a +HClO 4 ] +, C 35 H 34 ClCu 2 4 O 7. 13
Figure S17. The mass spectra of Cu-L b : 777.50, [Cu-L b +2OH - ] +, C 37 H 43 Cu 2 6 O 5 ; 813.25, [Cu-L b +2OH - +2H 2 O] +, C 37 H 47 Cu 2 6 O 7. 14
Figure S18. Color changes of PV (20 μm) in the presence of complex Cu-L b (20 μm) and complex Cu-L b (20 μm) in the HEPES buffer (10 mm, ph 7.0). Figure S19. Job plot of the combination between PV and the complex Cu-L a or Cu-L b. Total concentration of the PV and the complex Cu-L a or Cu-L b is 20 μm. The spectra were measured in the HEPES buffer (10 mm, ph 7.0). Determination of the association constants for PV/PPi with complex Cu-L a and Cu-L b L is the ligand (complex Cu-L a or Cu-L b ). P is the pyrocatechol violet (PV). I is the inions. L + P K a LP [LP] K a = [L][P] (1) Initial concentration [L] 0 [P] 0 At equilibrium [L] 0 - [LP] [P] 0 - [LP] [LP] From [LP] = K a ([L] 0 - [LP])([P] 0 - [LP]) We can ge the concentration of LP: [ LP] 2 ([ ] [ ] a) [ ] [ L] [P] + [L] + 1/ K P + L + 1/ k 4 P = 2 0 0 a 0 0 0 0 (2) The concentration of P: [P] = [P] 0 - [LP] (3) The observed absorption intensity at 444 nm is the sum of the absorption intensity of P: 15
A = a[p] + A 0 (a is the absorption coefficient of P) (4) Equations (2), (3), (4) are used to the nonlinear fitting the titration date. Origin 8.0 software was used to fit above nonlinear plot to give the K a value. L + I For indicator displacement approach: K b LI [LI] K b = (5) [L][I] LP + I [ LP][ I] K LI + P a k = = (6) k [L I][ P] K b Initial concentration [LP] 0 [I] 0 At equilibrium [LP] 0 -[P] [I] 0 -[P] [P] [P] From k[p] 2 = ([LP] 0 -[P])( [I] 0 -[P]) We can get the concentration of P: [ P] 2 ( ) 4[ ][ ] [ ] [ ] [ ] [ LP] 0 + I LP + I LP I 0 0 0 0 = (7) 2(1 k) Equations (3), (4), (7) are used to the nonlinear fitting the titration date. Origin 8.0 software was used to fit above nonlinear plot to give the k value. Finally, we get the K a and k, so we can obtain the K b according to the equation (5) and (6). 16
(a) 0.40 K a = 3.54*10 5 M -1 0.32 Equation y = y0+a*sqrt((0.00002+x +1/ka)^2-0.00008*x)+a*(0.00002-x-1/ka) Absorption 0.24 Adj. R-Sq 0.99218 Value a 5320.22518 y0 0.15987 ka 354083.21932 0.16 0.00000 0.00002 0.00004 0.00006 0.00008 [Cu-L a ] (b) 0.4 K a = 1.90*10 7 M -1 0.3 Equation y = y0+a*sqrt((0.00002+x +1/ka)^2-0.00008*x)+a*(0. 00002-x-1/ka) Absorption 0.2 Adj. R-Squa 0.98566 Value a 5890.05567 y0 0.11708 ka 1.89833E7 0.1 0.00000 0.00001 0.00002 0.00003 0.00004 0.00005 [Cu-L b ] Figure S20. Curve fitting of the titration date. (a) Titration of Cu-L a (0-80 μm) to PV (20 μm). (b) Titration of Cu-L b (0-40 μm) to PV (20 μm). Figure S21. The color of the ensemble Cu-L a -PV (20 μm) with or without the addition of various anions (200 μm); from left to right : no anion, PPi, HPO 2-4, Ac -, SO 2-4, CO 2-3, F -, Cl -, Br -, and I -. 17
(a) 0.33 K a = 3.54*10 5 M -1, k = 32.57, K b = 1.09*10 4 M -1 Absorption 0.30 0.27 Equation y = y0+m*(0.00002+xsqrt((0.00002-x)^2+0.0 0008*k*x))/(1-k) Adj. R-Squ 0.94652 Value y0 0.23611 m 3293.522 k 32.57094 0.24 0.0000 0.0002 0.0004 0.0006 0.0008 0.0010 [PPi] (b) 0.35 Ka = 1.90*10 7 M -1, k=3.302, K b =5.75*10 6 M -1 Absorption 0.30 0.25 0.20 0.15 Equation y = y0+m*(0.00002+xsqrt((0.00002-x)^2+0.0 0008*k*x))/(1-k) Adj. R-Squ 0.99045 Value y0 0.15617 m 4908.9851 k 3.30219 0.10 0.0000 0.0002 0.0004 0.0006 0.0008 0.0010 [PPi] Figure S22. Curve fitting of the titration date. (a) Titration of PPi (0-900 μm) to ensemble Cu-L a -PV (20 μm); (b) Titration of PPi (0-900 μm) to ensemble Cu-L b -PV (20 μm). 18
Figure S23. The IR spectra of Cu-L b and Cu-L b in the presence of 10 equiv PPi. 19
Figure S24. The change in the absorbance of ensembles Cu-L b -PV (20 μm) mixing with PPi (200 μm) under the following catalytic conditions: (a) PPase (10 units) only; (b) Mg 2+ (20 μm) only; (c) PPase (10 units) and Mg 2+ (20 μm) at different time: 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 90, and 120 min. 20