Supporting Information

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1 upporting Information Fluorescence Probe for Lysophospholipase C/PP6 Activity and a Potent PP6 Inhibitor Mitsuyasu Kawaguchi,, Takayoshi kabe, hinichi kudaira ζ, Kenjiro anaoka,, Yuuta Fujikawa,, Takuya Terai,, Toru Komatsu,, irotatsu Kojima,, Junken Aoki ζ, and Tetsuo agano,,, * Graduate chool of Pharmaceutical ciences, The University of Tokyo, ongo, Bunkyo-ku, Tokyo , Japan. CRET, JT, anbancho-blg, 5 anbancho, Chiyoda-ku, Tokyo, 12-75, Japan. Chemical Biology Research Initiative, The University of Tokyo, ongo, Bunkyo-ku, Tokyo , Japan. ζ Graduate chool of Pharmaceutical ciences, Tohoku University, 6-3 Aoba Aramaki, Aoba-ku, endai, Miyagi , Japan. * Correspondence: ( ): tlong@mol.f.u-tokyo.ac.jp (T..); (tel) ; (fax)

2 upporting Data cheme 1. ynthesis of TG-mPC analogs. Reactant and reagents: (a) (3-bromopropyl)trimethylammonium bromide or (5-bromopentyl)trimethylammonium bromide, DIEA, DMF, 5 C, 74% and 3 %, respectively; (b) 2-diethylaminoethyl bromide hydrobromide, DIEA, DMF, 5 C, 4%; (c) 1--Cbz-2-bromoethylamine, DIEA, DMF, 5 C, crude; (d) Pd/C, 2, C 2 Cl 2 /Me, rt, 24%; (e) benzyl bromoacetate, DIEA, DMF, 5 C, crude; (f) Pd/C, 2, C 2 Cl 2 /Me, rt, 55%; (g) methyl iodide or propyl iodide, Cs 2 C 3, DMF, 5 C, 15% and 3%, respectively. 2

3 cheme 2. ynthesis of T11. Reactant and reagents: (a) thiourea, Et, reflux, 85%. a 3 Boc b, c CF 3 C 2 3 T12 d CF 3 C 2 T13 e 2 CF 3 C T14 f CF 3 C g 2 CF 3 C T15 cheme 3. ynthesis of T Reactant and reagents: (a) Boc 2, DIEA, Et, 6 C to rt, 9%; (b) thiourea, Et, 85 C, crude; (c) TFA/C 2 Cl 2, C, 25% in 2 steps; (d) thiourea, Et, 85 C, 72%; (e) thiourea, Et, 85 C, 76%; (f) Ac 2, pyridine, rt, 7%; (g) thiourea, Et, 85 C, 37%. 3

4 a T16 CF 3 C CF 3 b F 3 C T17 CF 3 C c d T18 T19 CF 3 C CF 3 C e f R 1 R 1 R 1 =Me:T2 R 1 =Et:T21 R 1 =Bu:T22 n n=1:t23 n=2:t24 CF 3 C CF 3 C g CF 3 C T25 cheme 4. ynthesis of T Reactant and reagents: (a) 1-phenylthiourea, Et, 85 C, 5.7%; (b) 1-(3,5-bis(trifluoromethyl)phenyl)thiourea, Et, 85 C, 1%; (c) 1-(naphthalen-1-yl)thiourea, Et, 85 C, 56%; (d) 1-methylthiourea, Et, 85 C, 48%; (e) 1,3-dimethylthiourea or 1,3-diethylthiourea or 1,3-dibutylthiourea, Et, 85 C, 23, 48, 31%, respectively; (f) imidazolidine-2-thione or tetrahydropyrimidine-2-thione, Et, 85 C, 75, 65%, respectively; (g) 1,1,3-trimethylthiourea, Et, 85 C, 54%. 4

5 (A) extracellular space intracellular space molecules substrates expression sites functions C PP6 PP4 lysophopholipids unknown brain kidney ubiquitous unknown unknown C PP5 unknown brain unknown PP7 lysophopholipids small intestine unknown C PP1 nucleotides ubiquitous multifunction 1 C PP2/ATX lysophopholipids ubiquitous multifunction 2 C PP3 unknown ubiquitous basophil marker (B) LD catalytic LD domain (C) Enzyme ucleotide LPC pp-tmp pppc TG-mPC ucleotide pppc cleavage site pp-tmp PP1 PP2 PP3 P PP4 PP5 Lysophosphatidylcholine (LPC) PP6 PP7 PP6 PP2 Figure 1. (A) tructures and functions of PP6 and other PP family members. The domain structure and membrane orientation of seven PP members and their natural substrates, expression sites, and physiological functions are shown. PP6 and PP7 are anchored to plasma membrane by GPI and PP2 is secreted extracellularly, while the others are transmembrane proteins. 1 Inhibition of bone calcification and insulin metabolism. 2 Vasculature and neural tube formation in embryos, tumor progression, and development of neuropathic pain. LD = nuclease-like domain, LD = somatomedin-b-like domain, ATX = autotaxin. (B) tructures of native substrates, nucleotide, lysophosphatidylcholine (LPC), and synthetic absorption-based substrate, pp-tmp and pppc. Black arrows indicate the cleavage site by PP6, and gray arrows indicate the cleavage site by PP2. (C) Reactivity of various substrates, shown in (B), and TG-mPC towards recombinant PP1-7. For synthetic substrates, pppc and pp-tmp, reactivity was evaluated in terms of absorption increase at 45 nm. 5

6 Absorbance min min Wavelength (nm) Fluorescence Intensity (a.u.) Wavelength (nm) 6 min 2 min Figure 2. Changes of absorption and fluorescence spectra of TG-mPC (2 M) in the presence of PP6, measured every 5 min. Reactions were performed in PP buffer (1 mm Tris-Cl (p 9.), containing 5 mm acl, 5 mm MgCl 2, and.5% Triton-X1) at 37 C. Excitation wavelength was 49 nm a) TG-mPC Absorbance (a.u.) b) 2-Me-4-Me TG c) After Enzymatic Reaction d) Mixture of b) and c) Time (min) Figure 3. PLC charts showing enzymatic reaction of TG-mPC with PP6 to afford 2-Me-4-Me TG: a) a solution of TG-mPC, b) a solution of 2-Me-4-Me TG, c) reaction mixture after enzymatic reaction with PP6, d) mixed solution of b) and c). PLC analysis: eluent A (.1% TFA 2 ) and eluent B (.1% TFA, 8% acetonitrile, 2% 2 ), A/B = 8/2 A/B = /1 (3 min). For all spectra, the chromatogram was monitored at 49 nm. 6

7 a) min TG-mPC Absorbance (a.u.) b) 2 min c) 6 min d) 12 min e) 18 min f) 36 min 2-Me-4-Me TG Time (min) Figure 4. PLC charts showing enzymatic reaction of TG-mPC with PP2 to afford 2-Me-4-Me TG: a)-f) illustrate the chromatograms at, 2, 6, 12, 18, 36 min after the start of enzymatic reaction with PP2. PLC analysis: A/B = 8/2 A/B = /1 (2 min). For all spectra, the chromatogram was monitored at 49 nm. 7

8 Fluorescence Intensity (a.u.) PPs ALP Time (sec) TG-mPhos + ALP TG-mPC + PP2 + ALP TG-mPC + ALP TG-mPC + PPs + ALP Figure 5. Reactivity of TG-mPC towards PPs and alkaline phosphatase (ALP). Time courses of enzymatic reaction of TG-mPhos and TG-mPC with PPs and ALP are shown. Reactions were performed in PP buffer at 37 C. Excitation and emission wavelengths were 49 nm and 51 nm, respectively. PPs, PP1, 2, 3, 4, 5, 7, and ALP was added at 1 min and 1 min, respectively. TG-mPhos reacted with ALP and showed a fluorescence increment. TG-mPC did not react with ALP and also showed no reactivity with PP1, 2, 3, 4, 5, and 7, because no fluorescence increment was observed following the addition of ALP. Fluorescence Intensity (a.u.) PP6 1 M FA Figure 6. Fluorescence intensity of enzymatic reaction mixture of TG-mPC with PP6 in the presence or absence of formaldehyde (FA, 1 M) is shown. Reactions were performed under the same conditions as in Figure 5. The results are mean ±.D. (n = 3). 8

9 TG-mPC3C 12 PP1 PP2 PP3 PP4 PP5 PP6 PP TG-mPEE 1 PP1 PP2 PP3 PP4 PP5 PP6 PP7 5 1 Fluorescence Intensity (a.u.) TG-mPC5C PP1 PP2 PP3 PP4 PP5 PP6 PP TG-mPhos 1 PP1 PP2 PP3 PP4 PP5 PP6 PP PP1 PP2 PP3 PP4 PP5 PP6 PP TG-mPA 2 PP1 PP2 PP3 PP4 PP5 PP6 PP TG-mPPr TG-mPMe TG-mPEA 5 PP1 PP2 PP3 PP4 PP5 PP6 PP7 6 3 PP1 PP2 PP3 PP4 PP5 PP6 PP Time (sec) Figure 7. Reactivity of TG-mPC analogs towards PPs. Time course of enzymatic reaction of TG-mPC3C, TG-mPC5C, TG-mPEE, TG-mPEA, TG-mPhos, TG-mPA, TG-mPMe, and TG-mPPr (1 M) with various recombinant PPs. PPs were added at 2 min. Bold black and brown lines indicate PP6 and PP2, respectively. Reactions were performed in PP buffer at 37 C. Excitation and emission wavelengths were 49 nm and 51 nm, respectively. 9

10 (A) PP6 v (M/min) v (M/min) v (M/min) (B) PP2 v (M/min) TG-mPC (M) TG-mPC (M) v (M/min) TG-mPC 3 C (M) CPF4 (M) pppc (M) Figure 8. Kinetic study of various substrates with PP2 and PP6. (A) Michaelis-Menten plots and fitting curves of TG-mPC, TG-mPC 3 C, and pppc with PP6. (B) Michaelis-Menten plots and fitting curves of TG-mPC and CPF4 with PP2. The initial velocity, v, was measured from the changes of fluorescence intensity (at 51 nm for TG-mPC and TG-mPC 3 C, or at 38 nm for CPF4) or absorbance (at 45 nm for pppc). 1

11 Figure 9. Fluorescence microscopic imagingg of PP6 activity in living cells with TG-mPC. ela cells expressing PP6 and mcherry weree incubated in B at 37 C, then TG-mPC (final 1 M) ) was added. Confocal fluorescence and DIC images were captured beforee and at 3 min and 1 hr after addition of TG-mPC. An arrowhead shows an PP6 (+) cell and fluorescence increments off this cell and its surrounding media were observed immediately after addition of TG-mPC. After 1 hr, the whole fluorescence, not only that of cells but also that of the medium, was increased. This result indicates that 2-Me-4-M TG, whichh is generated by the enzymatic reaction of TG-mPC with PP6, is not well-retained intracellularly. o, we think that furtherr modifications of TG-mPC are still needed to obtain superior PP6 imaging probes, e.g., with betterr intracellularr retention of the fluorophore. R1 11

12 Cl TPC-14 TPC-15 TPC-16 Cl TPC-17 Cl Cl TPC-18 TPC-19 TPC-2 TPC TPC-22 TPC-23 TPC-24 TPC TPC-26 TPC-27 TPC-28 TPC-29 Figure 1. tructures of TPC-14 to TPC-29 shown in Table 3 (hit compounds in the PP6 inhibitor screening). 12

13 IC 5 = 6.7 M IC 5 = 4.3 M compound7 TPC-14 (M) compound14 TPC-15 (M) compound8 TPC-17 (M) IC 5 = 5.5 M IC 5 = 5.6 M IC 5 = 11.1 M compound8 TPC-18 (M) compound2 TPC-2 (mm) (M) compound9 TPC-21 (M) IC 5 = 9.8 M IC 5 = 4.5 M IC 5 = 5. M compound16 TPC-22 (M) compound3 TPC-23 (M) compound6 TPC-24 (M) IC 5 = 16.7 M IC 5 = 11.1 M IC 5 = 5.9 M compound1 TPC-25 (M) compound13 TPC-26 (M) TPC-27 compoun4 (M) IC 5 = 3.9 M compound11 (M) TPC-28 (M) Inihibition (%) IC 5 = 16.2 M Figure 11. Inhibition plots and curves and IC 5 values of hit compounds in the primary screening. The results are mean ±.D. (n = 6). IC 5 values of TPC-16, 19 and 29 could not be determined. 13

14 Reactivity TPC-15 Reactivity TPC-18 Reactivity TPC-2 Reactivity TPC-21 Reactivity TPC-23 Reactivity TPC-26 1 TPC-28 Reactivity Figure 12. electivity test of hit compounds towards recombinant PPs. The concentration of each compound was 5 M. The results are mean ±.D. (n = 3). Fluorescence Intensty (a.u.) units/ml 2 units/ml Time (sec) Figure 13. Cross-reactivity of TG-mPC with acetylcholinesterase (AchE). Time course of enzymatic reaction of TG-mPC (1 M) with AchE (.4, 2 units/ml). AchE was added at 2 min. Reactions were performed in PB (p 7.4) at room temperature. Excitation and emission wavelengths were 49 nm and 51 nm, respectively. 14

15 1 T11 (1 M) istamine Receptor Figure 14. ff-target inhibition by T11. Inhibitory activity of T11 (1 M) towards histamine receptors 1-4 (1-4), inducible (i), constitutive (c), and acetylcholinesterase (AchE). The results are means of duplicate determinations. This experiment was performed by Cerep (France). LPC 11 Zn 2+ Zn 2+ ydrolysis er 71 ot hydrolysis And inhibition er 71 active center of PP6 Figure 15. Proposed inhibition mechanism of T11 towards PP6. The active center of PP6 is shown. er 71 is a catalytic center of PP6 and Zn 2+ is located close to er 71. In the case of LPC, the choline moiety is probably trapped by negatively charged amino acid and the phosphate group chelates Zn 2+. Then, the phosphate ester is hydrolyzed by er 71, i.e. lysoplc activity, and the phosphorylcholine is released. n the other hand, the choline moiety of T11 is trapped similarly and the thiourea moiety chelates Zn 2+. owever, the thiourea moiety cannot be hydrolyzed by lysoplc activity, and as a result, T11 inhibits PP6 activity. Because of the similarity between LPC and T11, T11 is a competitive inhibitor, as shown in Figure 8B. 15

16 IC 5 (M) IC 5 (M) 4.1 M ~2 M 4.4 M >2 M TPC-11 ~1 M >2 M Figure 16. tructure-activity relationship (AR) of TPC-11. Determined IC 5 values are shown. 16

17 ynthesis synthesis of 2 To a solution of dibenzyl phosphate 1 (556 mg, 2. mmol), ac 3 (673 mg, 8. mmol), and n-bu 4 4 (69 mg,.2 mmol) in distilled water (1 ml), C 2 Cl 2 was added at C. The reaction mixture was stirred for 1 min, then chloromethyl chlorosulfate (263 L, 2.6 mmol) in C 2 Cl 2 (7 ml) was added dropwise. The whole was vigorously stirring at C for 3 min, then stirred at room temperature for 2 hr, washed with brine, dried over a 2 4, and evaporated. The residue was purified by column chromatography on silica gel (eluent: C 2 Cl 2 ) to obtain 2 (435 mg, 1.33 mmol), yield 67%. 1 -MR (3 Mz, CDCl 3 )5.1 (d, 4, J = 7.9 z), 5.62 (d, 2, J = 15.6 z), 7.36 (m, 9); 13 C-MR (75 Mz, CDCl 3 ) 69.8, 69.9, 73.4, 73.5, 128., 128.6, 128.7, 135.1, 135.2; RM (EI + ): calcd for [M+a] +, ; found, synthesis of 4 To a solution of 2-Me-4-Me TG (5 mg,.15 mmol) and Cs 2 C 3 (15 mg,.46 mmol) in DMF (5 ml), 2 (12 mg,.3 mmol) in DMF (2 ml) was added dropwise The reaction mixture was stirred at room temperature for 13 hr under Ar, then evaporated. The residue was dissolved in C 2 Cl 2, washed with api buffer (p 9.2) and brine, dried over a 2 4, and evaporated. The residue was purified by column chromatography on silica gel (eluent: C 2 Cl 2 /Me = 97/3) to obtain 4 (4 mg,.64 mmol), yield 43%. 1 -MR (3 Mz, CD 3 D) 1.98 (s, 3), 3.87 (s, 3), 5.4 (d, 4, J = 8.6 z), 5.8 (d, 2, J = 14.1 z), 6.43 (d, 1, J = 2. z), 6.58 (dd, 1, J = 2., 9.6 z), (m, 6), 7.2 (d, 1, J = 2.4 z), (m, 1); 13 C-MR (75 Mz, CDCl 3 ) 19.9, 55.3, 69.6, 69.7, 88.1, 88.2, 13.1, 15.9, 111.6, 113.5, 116., 116.3, 119.5, 124.3, 127.4, 127.8, 128.5, 128.6, 129.7, 13.3, 13.7, 135.1, 135.2, 137.8, 148.8, 153.9, 158.7, 16.2, 16.4, 185.8; M (EI + ): 645 [M+a] + synthesis of 5 (TG-mPhos) To a solution of 4 (4 mg,.64 mmol) in C 2 Cl 2 /Me (3 ml/7 ml), 1% Pd/C was added. The reaction mixture was vigorously stirred at room temperature for 1 hr under 2. Pd/C was removed by filtration, then triethylamine acetate (TEAA) buffer (5 ml) was added, and the mixture was evaporated to remove C 2 Cl 2 and Me. To the residual liquid, chloranil (excess) in C 2 Cl 2 (2 ml) was added. The reaction mixture was stirred at room temperature for 3 min. After removal of C 2 Cl 2, the residual liquid was purified by reversed-phase PLC to obtain TG-mPhos (6.9 mg,.15 mmol), yield 24%. 1 -MR (3 Mz, CD 3 D): 1.3 (t, 9, J = 7.3 z), 2.3 (s, 3), 3.19 (q, 6, J = 7.3 z), 3.9 (s, 3), 5.7 (d, 2, J = 1.2 z), 6.5 (d, 1, J = 2.2 z), 6.63 (dd, 1, J = 1.5, 9.5 z), (m, 2), (m, 4), 7.43 (d, 1, J = 2.1 z); 13 C-MR (1 Mz, CD 3 D): 187.5, 164.5, 162.4, 161.6, 156.2, 155.3, 139.1, 133., 131.6, 131.3, 129.6, 125.4, 119.5, 117.1, 116.8, 116.5, 112.8, 15.5, 14.3, 88.9, 88.9, 55.9, 47.7, 2., 9.2; RM (EI - ): calcd for [M-] -, ; found, synthesis of 6 (TG-mPC) To a solution of TG-mPhos (3. mg,.68 mmol) and diisopropylethylamine (DIEA, 3.5 L,.2 mmol) in DMF (1 ml), bromocholine bromide (3.3 mg,.14 mmol) was added. The reaction mixture was stirred at 5 17

18 C for 24 hr, then evaporated. The residue was purified by reversed-phase PLC to obtain TG-mPC (.6 mg,.11 mmol), yield 17%. 1 -MR (3 Mz, CD 3 D): 2.1 (s, 3), 3.17 (s, 9), (m, 2), (m, 2), 5.74 (d, 2, J = 13.2 z), 6.49 (d, 1, J = 1.5 z), 6.63 (dd, 1, J = 1.5, 9.5 z), (m, 2), (m, 4), 7.43 (d, 1, J = 2.2 z); 13 C-MR (1 Mz, CD 3 D): 187.4, 164., 162.4, 161.5, 156.2, 155.1, 139.1, 133.1, 131.6, 131.6, 129.8, 125.3, 119.8, 117.2, 117.1, 116.2, 112.9, 15.6, 14.2, 89.2, 89.1, 67.3, 6.6, 6.6, 55.9, 54.6, 54.6, 2.; RM (EI + ): calcd for [M] +, ; found, ynthesis of TG-mPC 3 C To a solution of TG-mPhos (4. mg,.9 mmol) and DIEA (16L,.16 mmol) in DMF (1 ml), (3-bromopropyl)trimethylammonium bromide (1 mg,.38 mmol) was added. The reaction mixture was stirred at 4 C for 48 hr, then evaporated. The residue was purified by reversed-phase PLC to obtain TG-mPC 3 C (3.6 mg,.66 mmol), yield 74%. 1 -MR (3 Mz, CD 3 D): 2.3 (s, 3), (m, 2), 3.1 (s, 9), (m, 2), (m, 5), 5.7 (d, 2, J = 12.5 z), 6.48 (d, 1, J = 2. z), 6.61 (dd, 1, J = 2., 9.5 z), 7. (dd, 1, J = 2.6, 8.3 z), 7.5 (d, 1, J = 2.6 z), (m, 2), (m, 2), 7.41 (d, 1, J = 2.4 z); RM (EI + ): calcd for [M] +, ; found, ynthesis of TG-mPC 5 C To a solution of TG-mPhos (3.1 mg,.7 mmol) and DIEA (1L,.1 mmol) in DMF (1 ml), (5-bromopentyl)trimethylammonium bromide (1 mg,.35 mmol) was added. The reaction mixture was stirred at 4 C for 48 hr, then evaporated. The residue was purified by reversed-phase PLC to obtain TG-mPC 5 C (1.2 mg,.21 mmol), yield 3%. 1 -MR (3 Mz, CD 3 D): (m, 2), (m, 2), (m, 2), 2.3 (s, 3), 3.1 (s, 9), (m, 5), 5.69 (d, 2, J = 12.3 z), 6.48 (d, 1, J = 2. z), 6.62 (dd, 1, J = 2., 9.7 z), 7.1 (dd, 1), 7.5 (d, 1), (m, 2), (m, 2), 7.41 (d, 1, J = 2.2 z); RM (EI + ): calcd for [M] +, ; found, ynthesis of TG-mPEE To a solution of TG-mPhos (2.5 mg,.56 mmol) and DIEA (1L,.1 mmol) in DMF (1 ml), 2-(diethylamino)ethyl bromide hydrobromide (1 mg,.38 mmol) was added. The reaction mixture was stirred at 45 C for 48 hr, then evaporated. The residue was purified by reversed-phase PLC to obtain TG-mPEE (1.2 mg,.22 mmol), yield 4%. 1 -MR (3 Mz, CD 3 D): 1.28 (t, 6, J = 7.1 z), 2.4 (s, 3), (m, 4), 3.9 (s, 3), (m, 2), 5.73 (d, 2, J = 13. z), 6.48 (d, 1, J = 2. z), 6.61 (dd, 1, J = 2., 6.7 z), 7. (dd, 1, J = 2.4, 7.9 z), 7.5 (d, 1, J = 2.4 z), (m, 2), (m, 2), 7.41 (d, 1, J = 2.4 z); RM (EI + ): calcd for [M+] +, ; found, ynthesis of TG-PEA To a solution of TG-mPhos (3. mg,.68 mmol) and DIEA (1L,.1 mmol) in DMF (1 ml), benzyl 2-bromoethylcarbamate (12 mg,.46 mmol) was added. The reaction mixture was stirred at 5 C for 48 hr, then evaporated. The residue was purified by reversed-phase PLC to obtain TG-mPEACbz (2.3 mg). To a 18

19 solution of TG-mPEACbz (2.3 mg) in C 2 Cl 2 /Me (1 ml/1 ml), 1% Pd/C was added. The reaction mixture was stirred at room temperature for 3 hr under 2. Pd/C was removed by filtration, then TEAA buffer (4 ml) was added, and the mixture was evaporated to remove C 2 Cl 2 and Me. To the residual liquid, chloranil (excess) in C 2 Cl 2 (2 ml) was added. The reaction mixture was stirred at room temperature for 3 min. After removal of C 2 Cl 2, the residual liquid was purified by reversed-phase PLC to obtain TG-mPEA (.8 mg,.16 mmol), yield 24%. 1 -MR (3 Mz, CD 3 D): 2.4 (s, 3), (m, 2), 3.9 (s, 3), (m, 2), 5.73 (d, 2, J = 12.5 z), 6.48 (d, 1, J = 2.2 z), 6.62 (dd, 1, J = 2., 9.5 z), (m, 6), 7.41 (d, 1, J = 3. z) ynthesis of TG-mPMe To a solution of TG-mPhos (4.6 mg,.1 mmol) and Cs 2 C 3 (8.1 mg,.25 mmol) in DMF (1 ml), methyl iodide (.6 L,.1 mmol) was added. The reaction mixture was stirred at room temperature for 48 hr, then evaporated. The residue was purified by reversed-phase PLC to obtain TG-mPMe (.7 mg,.15 mmol), yield 15%. 1 -MR (3 Mz, CD 3 D): 2.3 (s, 3), 3.51 (d, 3, J = 11. z), 3.89 (s, 3), 5.69 (d, 2, J = 12.1 z), 6.49 ( d, 1, J = 2. z), 6.61 (dd, 1, J = 2., 9.7 z), (m, 6), 7.41 (d, 1, J = 2.4 z); RM (EI - ): calcd for [M-] -, ; found, ynthesis of TG-mPPr Et 3 salt To a solution of TG-mPhos (3. mg,.7 mmol) and Cs 2 C 3 (8.1mg,.25 mmol) in DMF (1 ml), propyl iodide (.6 L,.9 mmol) was added. The reaction mixture was stirred at room temperature for 24 hr, then evaporated. The residue was purified by reversed-phase PLC to obtain TG-mPPr Et 3 salt (1. mg,.21 mmol), yield 3%. 1 -MR (3 Mz, CD 3 D):.86 (t, 3, J = 5.4 z), 1.31 (t, 9, J = 7.3 z), (m, 2), 2.4 (s, 3), 3.2 (q, 6, J = 7.3 z), (m, 2), 3.9 (s, 3), 5.71 (d, 2, J = 9. z), 6.54 (d, 1, J = 1.6 z), 6.65 (dd, 1, J = 1.6, 7.2 z), 7.1 (dd, 1, J = 1.6, 6.3 z), 7.6 (d, 1, J = 1.6 z), (m, 4), 7.44 (d, 1, J = 1.8 z); 13 C-MR (1 Mz, CD 3 D): 186.9, 164.6, 162.5, 161.6, 156.5, 156., 139.1, 133.2, 131.7, 131.6, 129.4, 125.4, 119.6, 117.2, 117.1, 116.7, 112.9, 15.5, 14.2, 89., 88.9, 68.5, 68.5, 55.9, 47.9, 25., 24.9, 2., 1.6, 9.2; RM (EI - ) : calcd for [M-] -, ; found, ynthesis of TG-mPA To a solution of TG-mPhos (4.7 mg,.11 mmol) and Cs 2 C 3 (11.4 mg,.35 mmol) in DMF (1 ml), benzyl 2-bromoacetate (3.9 mg,.17 mmol) was added. The reaction mixture was stirred at room temperature for 48 hr, then evaporated. The residue was purified by reverse-phase PLC to obtain TG-mPABz (4. mg). To a solution of TG-mPABz (4. mg) in C 2 Cl 2 /Me (1 ml/1 ml), 1% Pd/C was added. The reaction mixture was stirred at room temperature for 3 hr under 2. Pd/C was removed by filtration, then TEAA buffer (4 ml) was added, and the mixture was evaporated to remove C 2 Cl 2 and Me. To the residual liquid, chloranil (excess) in C 2 Cl 2 (2 ml) was added. The reaction mixture was stirred at room temperature for 3 min. After removal of C 2 Cl 2, the residual liquid was purified by reversed-phase PLC to obtain TG-mPA (3. mg,.6 mmol), yield 55%. 1 -MR (3 Mz, CD 3 D): 2.3 (s, 3), 3.89 (s, 3), 4.25 (d, 2, J = 8.8 z), 5.75 (d, 2, J = 12.1 z), 6.49 ( d, 1, J = 2. z), 6.61 (dd, 1, J = 2., 9.7 z), 6.99 (dd, 1, J = 2.4,

20 z), 7.4 (d, 1, J = 2.4 z), (m, 2), (m, 2), 7.41 (d, 1, J = 2.2 z); RM (EI + ): calcd for [M] +, ; found, ynthesis of T11 To a solution of (3-bromopropyl)trimethylammonium bromide (261 mg, 1 mmol) in dry Et (1 ml), thiourea (76 mg, 1 mmol) was added. The reaction mixture was stirred at 85 C for 12 hr under Ar, then evaporated. The residue was purified by reversed-phase PLC to obtain T11 (246 mg,.85 mmol), yield 85%. 1 -MR (3 Mz, DM): (m, 2), 3.11 (s, 9), 3.22 (t, 2, J = 7.3 z), (m, 2); RM (EI + ): calcd for [M] +, ; found, ynthesis of T12 To a solution of 3-bromopropylamine hydrobromide (436 mg, 2. mmol), and DIEA (7 L, 4. mmol) in dry Et (1 ml), Boc 2 (65 mg, 3. mmol) was added. The reaction mixture was stirred at 6 C for 1 hr and at room temperature for 3 hr, then evaporated. The residue was purified by column chromatography on silica gel to obtain 3-(Boc-amino)propyl bromide (43 mg, 1.8 mmol), yield 9%. To a solution of 3-(Boc-amino)propyl bromide (237 mg, 1 mmol) in dry Et (1 ml), thiourea (76 mg, 1. mmol) was added. The reaction mixture was stirred at 85 C for 12 hr under Ar, then evaporated. The residue was dissolved in C 2 Cl 2 (5 ml) and cooled to C, then TFA (5 ml) was added. The reaction mixture was stirred for 1 hr at C, then evaporated. The residue was purified by reversed-phase PLC to obtain T12 (62 mg,.25 mmol), yield 25%. 1 -MR (3 Mz, CD 3 D): (m, 2), 3.1 (t, 2, J = 7.2 z), 3.31 (t, 2, J = 7.3 z) ynthesis of T13 To a solution of (5-bromopropyl)trimethylammonium bromide (286 mg, 1. mmol) in dry Et (1 ml), thiourea (76 mg, 1. mmol) was added. The reaction mixture was stirred at 85 C for 12 hr under Ar, then evaporated. The residue was purified by reversed-phase PLC to obtain T13 (24 mg,.72 mmol), yield 72%. 1 -MR (3 Mz, CD 3 D): (m, 2), (m, 4), 3.18 (s, 9), 3.21 (t, 2, J = 7.3 z), (m, 2); RM (EI + ): calcd for [M] +, ; found, ynthesis of T14 To a solution of (3-bromopropyl)triethylammonium bromide (261 mg, 1. mmol) in dry Et (1 ml), thiourea (76 mg, 1. mmol) was added. The reaction mixture was stirred at 85 C for 12 hr under Ar, then evaporated. The residue was purified by reversed-phase PLC to obtain T14 (252 mg,.76 mmol), yield 76%. 1 -MR (3 Mz, CD 3 D): 1.33 (t, 9, J = 7.3 z), (m, 2), (m, 8); RM (EI + ): calcd for [M] +, ; found, ynthesis of T15 To a solution of 1,3-dibromopropane (42 mg, 2. mmol) in Ac 2 (1 ml), pyridine (155 L, 1.9 mmol) was added. The reaction mixture was stirred at room temperature for 2 hr, then evaporated. The residue was purified by reversed-phase PLC to obtain (3-bromopropyl)pyrimidium trifluoroacetate (392 mg, 1.4 mmol), 2

21 yield 7%. 1 -MR (3 Mz, DM): (m, 2), 3.59 (t, 2, J = 6.6 z), 4.73 (t, 2, J = 7.3 z), 8.18 (t, 2, J = 6.2 z), 8.63 (t, 1, J = 8.1 z), 9.12 (d, 2, J = 6.2 z); LRM (EI + ): 2 [M] +. To a solution of (3-bromopropyl)pyrimidium bromide (279 mg, 1. mmol) in dry Et (1 ml), thiourea was added. The reaction mixture was stirred at 85 C for 12 hr under Ar, then evaporated. The residue was purified by reversed-phase PLC to obtain T15 (113 mg,.37 mmol), yield 37%. 1 -MR (3 Mz, CD 3 D): (m, 2), 3.32 (t, 2, J = 7.3 z), 4.82 (t, 2, J = 8.1 z), 8.16 (t, 2, J = 6.2 z), 8.64 (t, 1, J = 8.1 z), 9.9 (d, 2, J = 6.2 z); RM (EI + ): calcd for [M] +, ; found, ynthesis of T16 To a solution of (3-bromopropyl)trimethylammonium bromide (261 mg, 1. mmol) in dry Et (1 ml), 1-phenylthiourea (152 mg, 1. mmol) was added. The reaction mixture was stirred at 85 C for 12 hr under Ar, then evaporated. The residue was purified by reversed-phase PLC to obtain T16 (21 mg,.57 mmol), yield 5.7%. 1 -MR (3 Mz, CD 3 D): (m, 2), 3.22 (s, 9), 3.34 (t, 2, J = 7.3 z), (m, 2), (m, 2), (m, 3); RM (EI + ): calcd for [M] +, ; found, ynthesis of T17 To a solution of (3-bromopropyl)trimethylammonium bromide (261 mg, 1. mmol) in dry Et (1 ml), 1-(3,5-bis(trifluoromethyl)phenyl)thiourea (288 mg, 1. mmol) was added. The reaction mixture was stirred at 85 C for 12 hr under Ar, then evaporated. The residue was purified by reversed-phase PLC to obtain T17 (5 mg,.1 mmol), yield 1%. 1 -MR (3 Mz, CD 3 D): (m, 2), 3.22 (s, 9), 3.33 (t, 2, J = 7.3 z), (m, 2), 7.97 (s, 2), 8.2 (s, 1); RM (EI + ): calcd for [M] +, ; found, ynthesis of T18 To a solution of (3-bromopropyl)trimethylammonium bromide (261 mg, 1. mmol) in dry Et (1 ml), 1-(naphthalen-1-yl)thiourea (22 mg, 1. mmol) was added. The reaction mixture was stirred at 85 C for 12 hr under Ar, then evaporated. The residue was purified by reversed-phase PLC to obtain T18 (232 mg,.56 mmol), yield 56%. 1 -MR (3 Mz, CD 3 D): (m, 2), 2.23 (s, 9), (m, 4), (m, 4), (m, 1), (m, 2); RM (EI + ): calcd for [M] +, ; found, ynthesis of T19 To a solution of (3-bromopropyl)trimethylammonium bromide (261 mg, 1. mmol) in dry Et (1 ml), 1-methylthiourea (9 mg, 1. mmol) was added. The reaction mixture was stirred at 85 C for 12 hr under Ar, then evaporated. The residue was purified by reversed-phase PLC to obtain T19 (145 mg,.48 mmol), yield 48%. 1 -MR (3 Mz, CD 3 D): (m, 2), 3.4 (s, 3), 3.2 (s, 9), 3.28 (t, 2, J = 7.3 z), (m, 2); RM (EI + ): calcd for [M] +, ; found, ynthesis of T2 To a solution of (3-bromopropyl)trimethylammonium bromide (261 mg, 1. mmol) in dry Et (1 ml), 21

22 1,3-dimethylthiourea (14 mg, 1. mmol) was added. The reaction mixture was stirred at 85 C for 12 hr under Ar, then evaporated. The residue was purified by reversed-phase PLC to obtain T2 (73 mg,.23 mmol), yield 23%. 1 -MR (3 Mz, CD 3 D): (m, 2), 3.5 (s, 3), 3.11 (s, 3), 3.21 (s, 9), 3.23 (t, 2, J = 7.3 z), (m, 2); RM (EI + ): calcd for [M] +, ; found, ynthesis of T21 To a solution of (3-bromopropyl)trimethylammonium bromide (261 mg, 1. mmol) in dry Et (1 ml), 1,3-diethylthiourea (132 mg, 1. mmol) was added. The reaction mixture was stirred at 85 C for 12 hr under Ar, then evaporated. The residue was purified by reversed-phase PLC to obtain T21 (165 mg,.48 mmol), yield 48%. 1 -MR (3 Mz, CD 3 D): 1.3 (t, 6, J = 7.3 z), (m, 2), 3.25 (s, 9), 3.35 (t, 2, J = 7.3 z), (m, 6); RM (EI + ): calcd for [M] +, ; found, ynthesis of T22 To a solution of (3-bromopropyl)trimethylammonium bromide (261 mg, 1. mmol) in dry Et (1 ml), 1,3-dibutylthiourea (132 mg, 1. mmol) was added. The reaction mixture was stirred at 85 C for 12 hr under Ar, then evaporated. The residue was purified by reversed-phase PLC to obtain T22 (124 mg,.31 mmol), yield 31%. 1 -MR (3 Mz, CD 3 D):.99 (t, 6, J = 7.2 z), (m, 4), (m, 4), (m, 2), 3.19 (s, 9), 3.34 (t, 2, J = 7.4 z), (m, 6); RM (EI + ): calcd for [M] +, ; found, ynthesis of T23 To a solution of (3-bromopropyl)trimethylammonium bromide (261 mg, 1. mmol) in dry Et (1 ml), imidazolidine-2-thione (12 mg, 1. mmol) was added. The reaction mixture was stirred at 85 C for 12 hr under Ar, then evaporated. The residue was purified by reversed-phase PLC to obtain T23 (237 mg,.75 mmol), yield 75%. 1 -MR (3 Mz, CD 3 D): (m, 2), 3.25 (s, 9), (m, 2), (m, 2), 4. (s, 4); RM (EI + ): calcd for [M] +, ; found, ynthesis of T24 To a solution of (3-bromopropyl)trimethylammonium bromide (261 mg, 1. mmol) in dry Et (1 ml), tetrahydropyrimidine-2-thione (116 mg, 1. mmol) was added. The reaction mixture was stirred at 85 C for 12 hr under Ar, then evaporated. The residue was purified by reversed-phase PLC to obtain T24 (215 mg,.65 mmol), yield 65%. 1 -MR (3 Mz, CD 3 D): (m, 2), (m, 2), 3.21 (s, 9), (m, 2), (m, 6); RM (EI + ): calcd for [M] +, ; found, ynthesis of T25 To a solution of (3-bromopropyl)trimethylammonium bromide (261 mg, 1. mmol) in dry Et (1 ml), 1,1,3-trimethylthiourea (118 mg, 1. mmol) was added. The reaction mixture was stirred at 85 C for 12 hr under Ar, then evaporated. The residue was purified by reversed-phase PLC to obtain T25 (178 mg,.54 mmol), yield 54%. 1 -MR (3 Mz, CD 3 D): (m, 2), 3.17 (t, 2, J = 8.1 z), 3.19 (s, 9), 3.28 (s, 3), 3.38 (brs, 6), (m, 2); RM (EI + ): calcd for [M] +, ; found,

23 upporting Reference (R1) Izumi,., Urano, Y., anaoka, K., Terai, T., and agano, T. J. Am. Chem. oc. 29, 131, Complete references (17) akasaki, T., Tanaka, T., kudaira,., irosawa, M., Umemoto, E., tani, K., Jin,., Bai, Z., ayasaka,., Fukui, Y., Aozasa, K., Fujita,., Tsuruo, T., zono, K., Aoki, J., and Miyasaka, M. Am. J. Pathol. 28, 173, (3) Gierse, J., Thorarensen, A., Beltey, K., andshaw-pierce, E., Cotres-Burgos, L., all, T., Johnston, A., Murphy, M., emirovskiy,., gawa,., Pegg, L., Pelc, M., Prinsen, M., chnute, M., Wendling, J., Wene,., Weinberg, R., Wittwer, A., Zweifel, B., Masferrer, J. J. Pharmacol. Exp. Ther. 21, 334, (44) ausmann, J., Kamtekar,., Christodoulou, E., Day, J. E., Wu, T., Fulkerson, Z., Albers,. M., van Meeteren, L. A., ouben, A. J., van Zeijl, L., Jansen,., Andries, M., all, T., Pegg, L. E., Benson, T. E., Kasiem, M., arlos, K., Kooi, C. W., myth,.., vaa,., Bollen, M., Morris, A. J., Moolenaar, W.., and Perrakis, A. at. truct. Mol. Biol. 211, 18,