SUPPLEMENTARY INFORMATION

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1 SUPPLEMETARY IFRMATI doi: 1.138/nchem658 ucleophilic Catalysis of Acylhydrazone Equilibration for Protein- Directed Dynamic Covalent Chemistry Venugopal T. Bhat, Anne M. Caniard, Torsten Luksch, Ruth Brenk, Doic J. Campopiano * and Michael F. Greaney * School of Chemistry, University of Edinburgh, King s Buildings, West Mains Rd, Edinburgh, EH9 3JJ, UK. College of Life Sciences, University of Dundee, James Black Centre, Dow St, Dundee, DD1 2EH, UK Supporting Information Part A: Synthesis of DCL components S1 Part B: Protein synthesis S6 Part C: DCL HPLC Analyses S9 Part D: Assay Data S18 Part A: Synthesis of DCL components General Methods Melting points are uncorrected. 1 H and 13 C MR spectra were recorded on a Brüker dpx36 (36 MHz) instrument and calibrated to residual solvent peaks: proton (d 6 -DMS 2.49 ppm) and carbon (d 6 -DMS 39.5 ppm). The 1 H data is presented as follows: chemical shift (in ppm on the δ scale), multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet), the coupling constant (J, in Hertz) and integration. The 13 C data is reported as the ppm on the δ scale followed by the interpretation. Electrospray and electron impact high resolution mass spectrometry was performed by the EPSRC ational Mass Spectrometry Service Centre, Swansea, using a Finnigan MAT 9 XLT double focusing mass spectrometer. The data is recorded as the ionisation method followed by the calculated and measured masses. TLC was performed on Merck 6F254 silica plates and visualised by UV light and/or anisaldehyde or potassium permanganate stains. All chemicals were purchased from a chemical supplier and used as received. nature chemistry Macmillan Publishers Limited. All rights reserved.

2 supplementary information doi: 1.138/nchem658 Aldehyde 5: In a 5 ml round bottom flask, 4-chloro-3-nitrobenzaldehyde 1 (5 mg, 2.69 mmol) and H 2 CH H S H 2 CH glutathione (994 mg, 3.23 mmol) were dissolved in a mixture of water/ethanol (15 ml, 1:1). Sodium carbonate (343 mg, 3.23 mmol) was then added and the reaction mixture stirred at room temperature for 48 hours. The yellow coloured solution was washed with diethyl ether (2 15 ml) and the aqueous phase was concentrated to approximately 5 ml followed by purification by H RP-HPLC. The column fractions were pooled together and lyophilized to give the pure compound as a yellow fluffy solid. (66% yield). 1 H MR (D 2, 36 MHz) δ H 9.81 (s, 1H), 8.52 (d, J = 1.74 Hz, 1H), 7.99 (dd, J = 8.5, 1.74 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 4.67 (dd, J = 9.2, 4.74 Hz, 1H), (m,4h) 3.3 (dd, J = 14.3, 9.3 Hz, 1H) (m, 1H) 2.2 (dd, J = 14.69, 7.7 Hz, 1H). 13 CMR (D 2, 9 MHz), 193.3, 176.5, 175.2, 174.3, 171.4, 146., 144., 133.7, 133., (2 signals overlapping), 54.4, 52., 43.7, 33.8, 31.7, HRMS calcd for [C 17 H S] (MH + ) requires m/z ; found (ESI + ). M.p. 2 ºC (darkens), > 28 ºC (decomp.). ppm (t1) nature chemistry 21 Macmillan Publishers Limited. All rights reserved.

3 doi: 1.138/nchem658 supplementary information ppm (t1) General method for the synthesis of hydrazones In a 25 ml round bottom flask the aldehyde (1 eq) and the corresponding hydrazide (1.1eq.) were dissolved in ethanol (1 ml), followed by the addition of a few drops of glacial acetic acid. The resulting precipitate was filtered off, washed with cold ether, and then recrystallised from ethanol to afford the pure hydrazone. General method for the synthesis of Glutathione conjugated hydrazones In a 25 ml round bottom flask the glutathione conjugated aldehyde (1 eq) and the corresponding hydrazide (1.1 eq) were dissolved in a mixture of 7:3 water / acetonitrile (1 ml) followed by the addition of a few drops of glacial acetic acid. The resulting yellow precipitate was filtered off, washed with cold ether and then lyophilized to afford the pure hydrazone. Representative hydrazone data Hydrazone 3c: White solid (81%yield). 1 H MR δ H (s, 1H), 8.52 (s, 1H) 8.41 (s, 1H), 8.8 (d, J = 8.25 Hz, 1H), 7.89 (d, J = 6.82 Hz, 3H), 7.58 (d, J = 8.31 Hz, 2H), 1.34 (s, 9H) 13 CMR (d 6 H -DMS, 9 MHz), 163.2, 155., 147.9, 144., 135., 132.2, 131.5, 13.2, 127.5, 125.6, 125.3, 123.5, 34.7, 3.9. HRMS calcd for [C 18 H 19 Cl 3 3 ] Cl (MH + ) requires m/z 36.16; found (ESI + ). M.p ºC (EtH). 2 nature chemistry Macmillan Publishers Limited. All rights reserved.

5 doi: 1.138/nchem658 supplementary information Cl 2 H Hydrazone 3f: White solid (76% yield). 1 H MR δ H (s, 1H), 8.51 (s, 1H), 8.39 (d, J = 1.96 Hz, 1H), 8.5 (dd, J = 8.45, 2.2 Hz, 1H), 8.1 (d, J =.98 Hz, 1H), 7.87 (d, J = 8.42 Hz, 1H), 7.38 (s, 1H), 6.75 (dd, J = 3.52, 1.74 Hz, 1H). 13 CMR (d 6 -DMS, 9 MHz), 154.3, 147.9, 146.2, 144.2, 134.9, 132.2, 131.5, 125.7, 123.5, 115.5, HRMS calcd for [C 12 H 9 Cl 3 4 ] (MH + ) requires m/z ; found (ESI+). M.p ºC (EtH) ppm (t1) ppm (t1) nature chemistry Macmillan Publishers Limited. All rights reserved.

6 supplementary information doi: 1.138/nchem658 Part B: Protein Synthesis Expression and purification of SjGST The SjGST construct pgex-6p-1 (GE Healthcare) was used to transform E. coli expression strain BL21 (DE3) and grown on LB/ampicillin (1 μg / ml) agar plates. ne colony was used to inoculate 2 ml of LB broth containing ampicillin (1 μg / ml) and grown overnight at 37 C with agitation (25 rpm). The 2 ml overnight culture was used to inoculate 3 L of LB broth and grown to an D 6 of.6 at 37 C and then induced with.1 mm IPTG for 3 to 4 hours at 37 C. Cells were harvested by refrigerated centrifugation at 5 g for 1 immediately after induction. Cell pellets were frozen and stored at -2 C until required. The SjGST expressing pellet was resuspended (4 ml / g wet cell pellet) in buffer binding buffer (5 mm Tris-HCl, ph 7) containing one protease inhibitor cocktail tablet (EDTA-free). The resuspended pellet was sonicated for 15 (3 s on / 3 s off) on ice and was then centrifuged at 27 g for 3 at 4 C to remove insoluble debris. The supernatant was filtered through a.45 μm membrane prior to chromatography at 4 C. The cell lysate was loaded onto a 2 ml GSTPrep FF 16/1 column (GE Healthcare) previously equilibrated with binding buffer. The column was then washed with 5 column volumes of binding buffer before elution using a linear gradient of -1 % elution buffer (5 mm Tris-HCl, 1 mm glutathione, ph 8) over 5 column volumes. Fractions were analysed by SDS-PAGE and those containing sjgst were pooled and applied onto a 32 ml HiPrep Sephacryl S-2 HR column (GE Healthcare) pre-equilibrated with a.1 M potassium phosphate buffer containing 15 mm acl, ph 6.8. The column was then eluted with one column volume of the same buffer. Fractions containing sjgst were pooled and dialysed against 4 L of a.1 M potassium phosphate buffer, ph 6.8 at 4 C. Expression and purification of hgstp1-1 verexpression of His 6 -Tagged hgstp1-1 was carried out by transforg E. coli BL21 (DE3) cells with the pet15b/hgstp1 vector. A single colony was added to 2 ml LB supplemented with ampicillin (1 μg.ml -1 ) and grown overnight at 37 C and 25 rpm. This seed culture was then used to inoculate 3 L of fresh LB medium and grown to D 6 of.6 before induction with isopropyl thio-β- D -glalactoside (IPTG, 1. mm final concentration) for 3 to 4 hours at 37 C and 25 rpm. The cells were then harvested by centrifugation (5 g for 15 at 4 C) and stored at -2 C. Cells overexpressing His 6 -hgstp1-1 were resuspended in binding buffer (5 mm Tris- HCl,.5 M acl, 5 mm imidazole, ph 7, 4 ml per gram of wet cell paste) with one protease inhibitor cocktail tablet (EDTAfree) and disrupted by sonication (15 pulses of 3 s at 3 s intervals) at 4 C. The cell debris were removed by centrifugation at 27 x g for 3 utes at 4 C, after which the supernatant was filtered through a.45 μm membrane prior to chromatography. The cell lysate was loaded onto a 5 ml HisTrap HP column (GE Healthcare) previously equilibrated with the same binding buffer. The column was then washed with 5 column volumes of binding buffer before the bound material was eluted using a linear gradient of -1 % elution buffer (5 mm Tris- HCl,.5 M acl,.5m imidazole, ph 8) over 2 column volumes at 4 C. 6 nature chemistry 21 Macmillan Publishers Limited. All rights reserved.

7 doi: 1.138/nchem658 supplementary information Fractions were analysed by SDS-PAGE and those containing hgstp1-1 were pooled and applied onto a 32 ml HiPrep Sephacryl S-2 HR column (GE Healthcare) pre-equilibrated with a.1 M potassium phosphate buffer containing 15 mm acl, ph 6.8. The column was then eluted with one column volume of the same buffer. Fractions containing hgstp1-1 were pooled and dialysed against 4 L of a.1 M potassium phosphate buffer, ph 6.8 at 4 C. Site-directed mutagenesis, expression and purification of SjGST-Y7F Plasmid pgex-6p-1 was used as a template in the site-directed mutagenesis experiment. The Y7F-For (5 - TCCCCTATACTAGGTTTTTGGAAAATTAAG- 3 ) and Y7F-Rev (5 - CTTAATTTTCCAAAAACCTAGTATAGGGGA- 3 ) oligonucleotides were used to change the TAT codon in the SjGST sequence to TTT, so that the tyrosine ao-acid residue was replaced by a phenylalanine (SjGST-Y7F). Mutations were performed by using Stratagene site-directed mutagnenesis kit. Each reaction contained plasmid DA (5 μl), 1x reaction buffer (5 μl), primer-forward (1 μl), primer-reverse (1 μl), dtp mix (1 μl), distilled water (final volume 5 μl) and Pfu DA polymerase 2.5 u / μl (1 μl). The amplification parameters were as follow: an initial DA denaturation at 95 C for 2 followed by 16 cycles of denaturation at 95 C for 3 sec, annealing at 55 C for 6 s and extension at 68 C for 7. ne microliter (5 U) of DpnI restriction enzyme was added to the PCR product and incubated at 37 C for 2 h. The ligation product was used to transform competent E. coli cells DH5α (DE3). The new mutated plasmid was confirmed by DA sequencing and named pgex-6-p1-y7f. The pgex-6p-1-y7f construct was used to transform E. coli expression strain BL21 (DE3) and grown on LB/ampicillin (1 μg / ml) agar plates. ne colony was used to inoculate 2 ml of LB broth containing ampicillin (1 μg / ml) and grown overnight at 37 C with agitation (25 rpm). The 2 ml overnight culture was used to inoculate 3 L of 2 YT broth and grown to an D 6 of.6 at 37 C and then induced with.1 mm IPTG for 5 to 6 hours at 3 C. Cells were harvested by refrigerated centrifugation at 5 g for 1 immediately after induction. Cell pellets were frozen and stored at -2 C until required. The Y7F SjGST expressing pellet was resuspended (4 ml / g wet cell pellet) in buffer binding buffer (5 mm Tris-HCl, ph 7) containing one protease inhibitor cocktail tablet (EDTA-free). The resuspended pellet was sonicated for 15 (3 s on / 3 s off) on ice and was then centrifuged at 27 g for 3 at 4 C to remove insoluble debris. The supernatant was filtered through a.45 μm membrane prior to chromatography at 4 C. The cell lysate was loaded onto a 2 ml GSTPrep FF 16/1 column (GE Healthcare) previously equilibrated with binding buffer. The column was then washed with 5 column volumes of binding buffer before elution using a linear gradient of -1 % elution buffer (5 mm Tris-HCl, 1 mm glutathione, ph 8) over 5 column volumes. Fractions were analysed by SDS-PAGE and those containing sjgst were pooled and applied onto a 32 ml HiPrep Sephacryl S-2 HR column (GE Healthcare) pre-equilibrated with a.1 M potassium phosphate buffer containing 15 mm acl, ph 6.8. The column was then eluted with one column volume of the same buffer. Fractions containing sjgst were pooled and dialysed against 4 L of a.1 M potassium phosphate buffer, ph 6.8 at 4 C. nature chemistry Macmillan Publishers Limited. All rights reserved.

8 supplementary information doi: 1.138/nchem658 Protein analyses Mass spectrometry analyses of the pure enzymes gave the molecular masses of the Schistosoma japonicum GST as Da, the His 6 - hgstp1-1 as Da and the SjGST-Y7F as Da in good agreement with the predicted values of Da, Da and Da for the monomers, respectively. The concentration of SjGST, hgstp1-1 and SjGST-Y7F were detered using a Bradford assay. The proteins were stored in a.1 M potassium phosphate buffer, ph 6.8 containing 2 % glycerol (v / v) at -2 C. Protein activity under DCL conditions L-Glutathione (reduced) (2 µl, 1mM aqueous), CDB (2 µl, 1mM, DMS) and SjGST (1 µl, 18 µm in KPhos buffer.1 M, ph 6.8) were added to ammonium acetate buffer (95 µl 5mM, ph 7.2). The fraction of glutathione conjugate formed was monitored in every five ute intervals by HPLC over 5. (HPLC conditions: Column: Luna 5 µ C18(2) 3 mm x 4.6 mm, flow rate 1mL -1, wavelength 254nm, temperature 23 o C, gradient H 2 / MeC (.1% TFA) from 95% to 2% over 4 ) SjGST (1 µl, 18 µm in KPhos buffer.1 M, ph 6.8) was added to a mixture of DMS (11 µl),ammonium acetate buffer (84 µl 5mM, ph 6.2) and aniline (2 µl, 1M, DMS) Two samples of these mixtures were incubated at 25 o C over 24 hours and 48 hours respectively. To both the samples were added reduced glutathione (2 µl, 1mM aqueous) and CDB (2 µl, 1mM, DMS) and the fraction of glutathione conjugate formed was measured for five ute intervals (Figure B1). Figure B1: Activity of SjGST under aniline-catalysed DCL conditions. Assessed using GSH-CDB conjugation assay 1.2 Fraction of CDB conjugate form ed Time () o Aniline ph 7.2 Enzyme incubated w ith 2 mm Aniline over 24 hr ph 6.2 Enzyme incubated w ith 2mM aniline over 48hr ph nature chemistry 21 Macmillan Publishers Limited. All rights reserved.

9 doi: 1.138/nchem658 supplementary information Figure B2: Comparative CDB assays between SjGST WT and SjGST Y7F, the mutant catalytic activity was reduced to 1.7 % SjGST Y7F (.15 nm) SjGST (.15 nm).35 [GS-CDB] (mm) Part C: DCL data Time (s) Effect of aniline concentration on DCL equilibration: Single hydrazone formation Aldehyde 4 (2 µl, 5 mm aqueous) and hydrazide 2b (2 µl, 5 mm, DMS) were added to a mixture of DMS (8 µl) and ammonium acetate buffer (88 µl.1m, ph 6.2). The fraction of the hydrazone (5e) formed at room temperature was monitored by HPLC over 8 hours. (HPLC conditions: Column: Luna 5 µ C18(2) 3 mm 4.6 mm, flow rate 1mL -1, wavelength 254 nm, temperature 3 o C, gradient H 2 / MeC (.1% TFA) from 95% to 3% over 4 ). The reaction was then performed under increasing concentrations of aniline (.1-1 mm). The concentrations and fractions were calculated from the integrals of the HPLC signals (254 nm). Figure C1: Formation of hydrazone 5b over time under different aniline concentrations Fraction of Hydrazone formed o Aniline.1 mm Aniline.1 mm Aniline 1 mm Aniline 5 mm Aniline 1 mm Aniline Time (hr) S9 nature chemistry Macmillan Publishers Limited. All rights reserved.

10 supplementary information doi: 1.138/nchem658 Effect of aniline concentration on DCL equilibration: 3 membered DCL Hydrazides 2b, 2e and 2f (3 2 µl,.1 M, DMS), aldehyde 4 (2 µl, 5 mm, aqueous) and aniline (2 µl, 5 mm, DMS) were added to a mixture of DMS (1 µl) and ammonium acetate buffer (88 µl,.1m, ph 6.2). The DCL with an aniline concentration of 1mM was allowed to stand at room temperature with occasional shaking and monitored periodically by HPLC to establish the composition until the relative concentrations of the hydrazones became constant. (HPLC conditions: Column: Luna 5 µ C18(2) 3 mm 4.6 mm, flow rate = 1 ml -1, wavelength 254 nm, temperature 3 o C, gradient H 2 / MeC (.1% TFA) from 95% to 8% over 1.5 then to 6% over 3 and eventually to 3% over 1.5 ). The DCL was then re-generated and analysed over time in the presence of.1 mm and.1 mm aniline concentration, respectively. Figure C2a: DCL with 1 mm aniline f 5b t = hr 6 t = 1 6 t = 6 hr 6 5e Figure C2b: DCL with 1 mm aniline t = hr t = 3 t = 1 hr t = 4 hr nature chemistry 21 Macmillan Publishers Limited. All rights reserved.

11 doi: 1.138/nchem658 supplementary information Figure C2c: DCL with.1 mm aniline t = hr t = 1 hr t = 8 hr t = 24 hr 1 Figure C2d: DCL with.1 mm aniline t = hr t = 2 hr t = 18 hr t = 48 hr nature chemistry Macmillan Publishers Limited. All rights reserved.

12 supplementary information doi: 1.138/nchem658 Scheme C1: Acyl hydrazone DCL based on benzaldehyde 1. Cl 2 CH RHH 2 Aniline, ph Cl H R 1-membered DCL R= Me S t-bu Me Me H 2a 2b 2c 2d 2e S H 2 H Me S 2f 2g 2h 2i 2j Figure C3: MS data for DCL, indicating that all the expected ten acylhydrazones are present. 3c 3e 3d 3b 3h 3j 3f 3i 3a 3g 12 nature chemistry 21 Macmillan Publishers Limited. All rights reserved.

13 doi: 1.138/nchem658 supplementary information Figure C4A. Proof of Reversibility: Addition of excess component (2b) to DCL at equilibrium b t = hr t = 4hr t = 12hr Figure C4B. Proof of Reversibility: Constitute DCL from alternative starting point, hydrazone 3g g t = t = 6 hr t = 18hr nature chemistry Macmillan Publishers Limited. All rights reserved.

14 supplementary information doi: 1.138/nchem658 Figure C5: Control experiment: BSA-templated DCL shows no amplifications. 25 VWD1 A, Wavelength=254 nm (VEU29\VB632D.D) Blank VWD1 A, Wavelength=254 nm (VEU29\VB665A.D) BSA Figure C6: Demonstration of thiophene hydrazone 3g as a GST substrate: Conjugation by SjGST SG 2 5g H S Cl 2 3g H S nature chemistry 21 Macmillan Publishers Limited. All rights reserved.

15 doi: 1.138/nchem658 supplementary information Scheme C2: Acyl hydrazone DCL based on GSH-conjugate aldehyde 3 Figure C7: MS data for DCL, indicating that all the expected ten acylhydrazones are present. 5c 5d 5b 5j 5e 5g 5a 5f 5j 5i nature chemistry Macmillan Publishers Limited. All rights reserved.

16 supplementary information doi: 1.138/nchem658 Figure C8: Control Experiment: BSA-templated DCL shows no amplification Figure C9: Control experiment for GSH conjugate DCL in the presence of excess ethacrynic acid (1 μm) o amplification Blank SjGST EA hgstp1-1 EA nature chemistry 21 Macmillan Publishers Limited. All rights reserved.

17 doi: 1.138/nchem658 supplementary information Figure C1: GSH-conjugate DCL templated by SjGST-Y7F mutant amplifying 5g SG 2 5g H S nature chemistry Macmillan Publishers Limited. All rights reserved.

18 supplementary information doi: 1.138/nchem658 Part D: Assay Data Inhibition assays were carried out on a Perkin Elmer Wallac VICTR2 142 Multilabel counter UVvisible 96 well-plate spectrophotometer. To 36 μl well were added phosphate buffer (255 μl,.1 M, ph 6.8), GST (15 μl,.15 mg.ml -1 ) and inhibitor (15 μl,.2 to 2 μm). The solution was mixed well and after incubation at 25 C for 5, CDB (15 μl, 4 mm) and GSH (15 μl, 4 mm) were added quickly and mixed. Absorbance was measured at 34 nm, 25 C for 5. The K i values with respect to both GSH and CDB were detered. Inhibition kinetics were studied at 8 different, fixed concentrations of either GSH or CDB (8, 16, 31, 63, 125, 25, 5 and 1 μm) with variable concentrations of the inhibitor (2 to 2 μm). Data were collected and analysed using SigmaPlot software. Table 1: IC 5 data (μm) Hydrazone sjgst hgst P ± ± 2 5a 24 ± 1 D 5b 4 ± 2 D 5c 5 ± 3 57 ± 2 5d 26 ± 1 D 5e 36 ± 2 D 5f 61 ± 3 D 5g 22 ± 1 87 ± 3 5h 34 ± ± 8 5i 37 ± 1 84 ± 4 5j 25 ± 1 D Table 2: Ki data (μm) hgst P1-1 SjGST CDB GSH CDB GSH K m (mm).81 ±.4.2 ± ± ±.2 5g K i (μm) ± ± ± ±.23 5c K i (μm) 1.66 ± ± ± ±.27 5g IC 5 (μm) 87 ± 3 22 ± 1 5c IC 5 (μm) 57 ± 2 5 ± 3 18 nature chemistry 21 Macmillan Publishers Limited. All rights reserved.

19 doi: 1.138/nchem658 supplementary information Figure D1: Inhibition of SjGST by 5g using GSH and CDB as substrates. The IC 5 value is the concentration of inhibitor giving 5% inhibition of enzyme activity. Data are the mean ± standard deviation of triplicate experiments. 1 8 % inhibition 6 4 IC 5 = 22. μm [5g] (μm) 1/v (µmol//mg) I = I = 2.5 I = 7.4 I = 22.2 I = 66.7 I = 2 1/v (µmol//mg) I = I = 2.5 I = 7.4 I = 22.2 I = 66.7 I = 2-2 K i 5g = 5.1 μm /[GSH] (mm) K 5g i = 12.8 μm /[CDB] (mm) Figure D2: Inhibition of hgstp1-1 by 5c using GSH and CDB as substrates. 1 8 % inhibition 6 4 IC 5 = 57.3 μm /v (µmol//mg) [5c] (μm) I = I = 2.5 I = 7.4 I = 22.2 I = 66.7 I = 2 1/v (µmol//mg) I = I = 2.5 I = 7.4 I = 22.2 I = 66.7 I = K i 5c = 6.6 μm -2 K i 5c = 1.7 μm /[GSH] (mm) /[CDB] (mm) nature chemistry Macmillan Publishers Limited. All rights reserved.

20 supplementary information doi: 1.138/nchem658 Isothermal Titration Calorimetry Isothermal titration calorimetry (ITC) was carried out in collaboration with Pr. Alan Cooper and the Glasgow Biological Microcalorimetry Facility. The ITC measurements were performed on a VP-ITC calorimeter (Microcal Inc orthampton, USA) at 25 C. SjGST and hgstp1-1 were dialysed against a.1 M potassium phosphate buffer, ph 6.8. The final dialysis buffer was used to make up the ligand solution as well as for instrument calibration and baseline controls. 5c and 5g were dissolved and titrated in 1 % DMS. The concentrations of SjGST and hgstp1-1 (approximately 2 μm) were detered by measuring the absorption at 28 nm and the concentrations of ligands were approximately 3 times higher. The proteins SjGST and hgstp1-1 were placed in the 2 ml sample chamber and compounds 5c or 5g in the syringe. A typical ITC measurement consisted of a first control injection of 1 μl followed by 29 successive injections of 1 μl for 2 s with a 3 interval between each injection. Raw data were collected, corrected for ligand heats dilution, and the peaks generated integrated using RIGI software (Microcal Inc) by plotting the values in microcalories against the molar ratio of injectant to reactant within the cell. Data were fitted using the one single-site binding model. 1 From the dissociation constant K D and the reaction enthalpy value ΔH, the change in free Gibbs energy (ΔG ) and entropy change (ΔS ) can be calculated using the equation ΔG = -RT ln(1/k D ) = ΔH - TΔS where R is the universal gas constant and T the absolute temperature. Figure D3: ITC of SjGST in the presence and in the absence of 5g. Top: Heats of injection of 276 μm 5g into a cell containing 17 μm SjGST. Bottom: Data from the upper panel integrated and plotted as a function of the molar ratio of 5g after subtraction of heats generated by injection of 276 μm 5g into buffer. 1 Arouri, A.; Garidel, P.; Kliche, W.; Blume, A. Eur. Biophys. J., 27, 36, nature chemistry 21 Macmillan Publishers Limited. All rights reserved.

21 doi: 1.138/nchem658 supplementary information Figure D4: ITC of SjGST in the presence and in the absence of 5c. Top: Heats of injection of 64 μm 5c into a cell containing 21 μm SjGST. Bottom: Data from the upper panel integrated and plotted as a function of the molar ratio of 5c after subtraction of heats generated by injection of 64 μm 5c into buffer. Time () µcal/sec kcal/mole of injectant Data: a9tbsj1_dh Model: nesites Chi^2 = ±.1335 K 2.449E5 ±1.12E4 ΔH ±178.8 ΔS Molar Ratio Figure D5: ITC of hgstp1-1 in the presence and in the absence of 5c. Top: Heats of injection of 68 μm 5c into a cell containing 2 μm hgstp1-1. Bottom: Data from the upper panel integrated and plotted as a function of the molar ratio of 5c after subtraction of heats generated by injection of 68 μm 5c into buffer. Time () µcal/sec -2 kcal/mole of injectant Data: a4tbp11_dh Model: nesites Chi^2 = ±.2393 K 3.54E5 ±3.12E4 ΔH E4 ±393.1 ΔS Molar Ratio nature chemistry Macmillan Publishers Limited. All rights reserved.

22 supplementary information doi: 1.138/nchem658 Figure D6: ITC of hgstp1-1 in the presence and in the absence of 5g. Top: Heats of injection of 598 μm 5g into a cell containing 2 μm hgstp1-1. Bottom: Data from the upper panel integrated and plotted as a function of the molar ratio of 5g after subtraction of heats generated by injection of 598 μm 5g into buffer. Time () µcal/sec -1 kcal/mole of injectant Data: a11tpp11_dh Model: nesites Chi^2 = ±.55 K 1.519E5 ±2.383E4 ΔH E4 ±991.2 ΔS Molar Ratio 22 nature chemistry 21 Macmillan Publishers Limited. All rights reserved.

Convenient Synthesis of Nucleoside 5 -Triphosphates for RNA Transcription. Supplemental Materials

Convenient Synthesis of Nucleoside 5 -Triphosphates for RNA Transcription. Supplemental Materials Supplementary Material (ESI) for Chemical Communications This journal is The Royal Society of Chemistry 2010 Convenient Synthesis of ucleoside 5 -Triphosphates for RA Transcription Julianne Caton-Williams,