Supplementary Methods Synthesis overview 4 a-c C 5 TBS d H 2 TBS 6 H 2 H 2 (Boc) 2 e f-h i Br TBS TBS TBS 7 8 9 j,k (Boc) 2 l H 2 H H 10 fmk (1) Scheme 1. Reagents and conditions: a. malononitrile, MeH, H 2, ah (91% yield); b. triethyl orthoformate, acetic anhydride; c. ah, DM, then 3-(t-butyldimethylsilyloxy)propyl iodide (70% yield over 2 steps); d. H 3, MeH (61% yield); e. - bromosuccinimide, DM (99% yield); f. α-(ethoxyvinyl)tin, Pd[P(Ph) 3 ] 4, toluene; g. 1 HCl, TH; h. imidazole, t-butyldimethylsilyl chloride, DM (76% yield over 3 steps); i. (Boc) 2, DMAP, TH (78% yield); j. LDA, TH, then -fluorobenzenesulfonimide; k. 1 HCl, TH (60% yield over 2 steps); l. 1 HCl, dioxane (70% yield).
(Boc) 2 a (Boc) 2 b,c H 2 B H H HBoc H H H 10 11 fmk-bdipy (2) Scheme 2. Reagents and conditions: a. carbonyldiimidazole, DIPEA, CH 2 Cl 2, then - Boc-1,4-diaminobutane (69% yield); b. TA, CH 2 Cl 2 ; c. BDIPY L-HS, DIPEA, DM (71% yield over 2 steps). (Boc) 2 a-c H 2 H H 10 fmk-pa (3) Scheme 3. Reagents and conditions: a. methanesulfonyl chloride, DIPEA, CH 2 Cl 2 ; b. propargylamine (neat); c. TA, CH 2 Cl 2 (33% yield over 3 steps).
Procedures General. 1 H and 13 C MR spectra were recorded on a Varian 400 spectrometer at 400 and 100 MHz, respectively. Chemical shifts were reported as parts per million (ppm) downfield from an internal tetramethylsilane standard (δ = 0.0 for 1 H MR) or from solvent references. Low-resolution electrospray ionization mass spectra (ESI + -MS) were recorded on a Waters Micromass ZQ 4000 spectrometer. LC/MS (MS: ESI + ) was performed on a Waters AllianceHT LC/MS with a flow rate of 0.2 ml min -1 (monitored at 210 nm and 260 nm) using an Xterra MS C18 column (Waters). High-resolution electron impact mass spectra (HRMS) were recorded on a Micromass VG70E spectrometer by David Maltby at the University of California-San rancisco Biomedical Mass Spectrometry Center. or air- and water-sensitive reactions, glassware was oven- or flame-dried prior to use and reactions were performed under argon. Dichloromethane, dimethylformamide, methanol, tetrahydrofuran, toluene, and diisopropylamine were dried using the solvent purification system manufactured by Glass Contour, Inc. (Laguna Beach, CA). All other solvents were of ACS chemical grade (isher) and used without further purification unless otherwise indicated. Commercially available starting reagents were used without further purification. 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sindacene-3-propionyl)amino)hexanoic acid, succinimidyl ester (BDIPY L-HS) was purchased from Invitrogen and used without further purification. Analytical and preparative thin layer chromatography were performed with silica gel 60 254 glass plates (EM Science). lash chromatography was conducted with 230-400 mesh silica gel (Selecto Scientific). High performance liquid chromatography (HPLC) was performed on a Prostar 210 (Varian) with a flow rate of 10 ml min -1 (monitored at 210 nm and 260 nm) using a CMBI-A C18 preparatory column (Peeke Scientific).
C TBS 5 -alkyl pyrrole iminoether 5. To a solution of malononitrile (3.24 g, 48.9 mmol) in 6:1:1 MeH/48% aq. (w/w) ah/h 2 (56 ml) was added 2-phthalimido-4 -methylacetophenone 1 (4) (10.5 g, 37.6 mmol). The reaction mixture was stirred at room temperature for 1 h after which the product precipitated out of solution. The solid was collected by filtration, washed with H 2, CH 2 Cl 2, and hexanes to give 6.7 g (91% yield) of the desired pyrrole as a brown solid: R f 0.70 (10% MeH/CH 2 Cl 2 ); 1 H MR (400 MHz, CD 3 D) δ 7.409 (d, J = 7.6 Hz, 2H), 7.08 (d, J = 7.6 Hz, 2H), 6.36 (s, 1H), 4.83 (br s 2H), 2.27 (s, 3H). To a solution of the pyrrole (5 g, 25.3 mmol) in triethyl orthoformate (30 ml) was added acetic anhydride (0.5 ml) and the mixture was refluxed for 1 h. After cooling to room temperature, the solvent was removed in vacuo. The crude pyrrole iminoether was azeotropically dried with toluene (2 x 10 ml) and carried on directly to the next step. To a suspension of ah (60% in oil, 0.9 g, 23.4 mmol) in 20 ml of DM at room temperature was added a solution of the crude pyrrole iminoether in 10 ml of DM. After stirring for 30 min, 3-(t-butyldimethylsilyloxy)propyl iodide 2 (7.0 g, 23.4 mmol) was added over 10 min. After stirring for an additional 3 h, the solvent was removed in vacuo and the residue was purified by flash chromatography (10-50% ethyl acetate/hexanes) to afford 5 g (70% yield) of -alkyl pyrrole iminoether 5 as a brown oil: R f 0.8 (4:1 hexanes/ethyl acetate); 1 H MR (400 MHz, CDCl 3 ) δ 8.43 (s, 1H), 7.47 (d, J = 8.0 Hz, 2H), 7.16 (d, J = 8.0 Hz, 2H), 6.62 (s, 1H), 4.32 (q, J = 8.0 Hz, 2H), 3.96 (t, J = 7.2 Hz, 2H), 3.59 (t, J = 5.6 Hz, 2H), 2.33 (s, 3H), 1.91-1.82 (m, 2H), 1.37 (t, 3H), 0.88 (s, 9H),
0.03 (s, 6H); 13 C MR (100 MHz, CDCl 3 ) δ 158.2, 144.1, 136.8, 130.5, 129.7, 126.2, 125.0, 119.0, 118.5, 115.6, 63.4, 59.7, 43.1, 33.4, 26.1, 21.4, 18.5, 14.2, -5.4. H 2 TBS 6 Pyrrolo[2,3-d]pyrimidine 6. -alkyl pyrrole iminoether 5 (18.0 g, 42.3 mmol) was dissolved in MeH (100 ml) and transferred to 350 ml sealed-tube reaction vessel. Argon gas was then bubbled through the solution for 15 min. The reaction vessel was submerged in a dry ice/meh bath and H 3 gas was bubbled through the solution for 15 min. The reaction vessel was quickly sealed with a Teflon screw cap (fitted with a rubber -ring) and allowed to warm to room temperature. The reaction was stirred for 4 days at 50 C and then submerged in an ice bath. The H 3 gas was slowly released. The solvent was removed in vacuo and the crude product purified by flash chromatography (3:2 ethyl acetate/hexanes with 1% Et 3 ) to give 10.1 g (61% yield) of 6 as a light-brown solid: R f 0.41 (1:1 hexanes/ethyl acetate); 1 H MR (400 MHz, CDCl 3 ) δ 8.32 (s, 1H), 7.37 (d, J = 8.4 Hz, 2H), 7.26 (d, J = 8.0 Hz, 2H), 6.92 (s, 1H), 5.1 (br s, 2H), 4.34 (t, J = 6.8 Hz, 2H), 3.65 (t, J = 6.0 Hz, 2H), 2.41 (s, 3H), 2.10-2.05 (m, 2H), 0.91 (s, 9H), 0.05 (s, 6H); 13 C MR (100 MHz, CDCl 3 ) δ 156.9, 151.9, 150.6, 136.8, 132.0, 129.7, 128.7, 123.2, 115.8, 101.3, 59.8, 41.5, 33.0, 25.9, 21.1, 18.2, -5.4; ESI + - MS 419 [M+a] +, 397 [M+H] +.
H 2 Br TBS 7 Bromide 7. To a solution of 6 (1.5 g, 3.78 mmol) in DM (20 ml) was added - bromosuccinimide (0.74 g, 4.16 mmol) and the mixture stirred for 24 h protected from light. The reaction was diluted with ether (100 ml) and washed with water (3 x 200 ml). The combined aqueous fractions were extracted with ether (3 x 50 ml). The combined organic fractions were dried over anhydrous a 2 S 4, filtered, and concentrated in vacuo to give 1.77 g (99% yield) of bromide 7 as a brown solid: R f 0.59 (100% ethyl acetate); 1 H MR (400 MHz, CDCl 3 ) δ 8.21 (s, 1H), 7.35 (d, J = 8.0 Hz, 2H), 7.30 (d, J = 8.0 Hz, 2H), 5.0 (br s, 2H), 4.41 (t, J = 6.8 Hz, 2H), 3.73 (t, J = 6.0 Hz, 2H), 2.43 (s, 3H), 2.1 (m, 2H), 0.91 (s, 9H), 0.06 (s, 6H); ESI + -MS 500 [M+2+a] +, 497 [M+a] +, 477 [M+2] +, 475 2+.
H 2 TBS 8 Methylketone 8. To a solution of bromide 7 (1.46 g, 3.07 mmol) in toluene (20 ml) was added α-(ethoxyvinyl)tributyltin (1.24 ml, 3.68 mmol). Argon gas was bubbled through the solution for 10 min. Tetrakis(triphenylphosphine)palladium (355 mg, 0.307 mmol) was quickly added and mixture was refluxed for 16 h. The solvent was removed in vacuo and the crude product was dissolved in TH (10 ml) and 1 HCl (3 ml). After stirring for 12 h at room temperature, the reaction mixture was diluted with ethyl acetate (50 ml) and washed with saturated ahc 3 (200 ml). The crude product was extracted from the organic fraction with 1 HCl (2 x 150 ml). The acidic aqueous fraction was brought to ph~8 with ah and then extracted with ethyl acetate (2 x 150 ml). The combined organic fractions were dried over a 2 S 4, filtered, and concentrated in vacuo to give 850 mg (85% yield) of the TBS-deprotected ketone as an off-white solid. The crude product was used in the next reaction without further purification. To a solution of the crude ketone (850 mg, 2.62 mmol) in DM (15 ml) was added imidazole (450 mg, 6.60 mmol) followed by t-butyldimethylsilyl chloride (474 mg, 3.14 mmol). After stirring for 12 h at room temperature, the reaction mixture was poured into 150 ml of 10% citrate buffer [ph 4.0] and extracted with ethyl acetate (200 ml). The organic fraction was washed with brine (1 x 200 ml), dried over a 2 S 4, filtered, and concentrated in vacuo. Purification by flash chromatography (1:1 ethyl acetate/hexanes) provided ketone 8 (876 mg, 76% yield over 3 steps): R f 0.70 (100% ethyl acetate); 1 H MR (400 MHz, CDCl 3 ) δ 8.28 (s, 1H), 7.28 (s, 4H), 5.10 (br s, 2H), 4.60 (t, 2H), 3.66 (t,
2H), 2.41 (s, 3H), 1.94 (s, 3H), 0.85 (s, 9H), 0.05 (s, 6H); 13 C MR (100 MHz, CDCl 3 ) δ 192.0, 158.8, 154.9, 151.0, 138.8, 131.7, 130.1, 129.9, 129.7, 123.9, 102.0, 61.1, 41.3, 33.8, 30.9, 25.9, 21.3, 18.3, -5.4; ESI + -MS 439.1 [M+H] +. (Boc) 2 TBS 9 Diboc methylketone 9. To a solution of methylketone 8 (780 mg, 1.78 mmol) in TH (10 ml) was added (Boc) 2 (1.16 g, 5.33 mmol). DMAP (44 mg, 0.36 mmol) was quickly added and the mixture was stirred for 2.5 h at room temperature. The reaction mixture was diluted with ethyl acetate (150 ml) and washed with 0.5 HCl (200 ml) followed by brine (200 ml). The organic fraction was dried over a 2 S 4, filtered, and concentrated in vacuo. Rapid purification by flash chromatography (1:5 ethyl acetate/hexanes) gave 884 mg (78% yield) of diboc methylketone 9: R f 0.50 (4:1 ethyl acetate/hexanes); 1 H MR (400 MHz, CDCl 3 ) δ 8.51 (s, 1H), 7.17 (s, 4H), 4.70 (t, 2H), 3.64 (t, 2H), 2.37 (s, 3H), 1.98 (s, 3H), 1.23 (s, 18H), 0.85 (s, 9H), 0.05 (s, 6H); 13 C MR (100 MHz, CDCl 3 ) δ 194.0, 153.9, 153.7, 153.1, 149.9, 138.1, 134.1, 129.9, 129.3, 121.3, 113.8, 83.2, 60.7, 41.5, 33.6, 31.2, 27.8, 25.9, 21.3, 18.0, -5.4; ESI + -MS 639.2 [M+H] +.
(Boc) 2 H 10 Diboc fluoromethylketone 10 (diboc fmk). A solution of TH (3 ml) and diisopropylamine (0.32 ml, 2.28 mmol) was cooled to 78 C. n-butyllithium (2.48 M in hexanes, 0.657 ml, 1.63 mmol) was added and the reaction mixture was warmed to 0 C. After 30 min, the reaction was cooled to 78 C and 9 (800 mg, 1.25 mmol) in TH (2.5 ml) was added dropwise. The reaction mixture was warmed to 0 C over 20 min, then cooled to 78 C. -fluorobenzenesulfonimide (631 mg, 2 mmol) in TH (2 ml) was added dropwise and the reaction mixture was warmed to room temperature. After 25 min, saturated H 4 Cl (1 ml) was added and the reaction mixture was poured into saturated H 4 Cl (150 ml) and extracted with ethyl acetate (100 ml). The organic fraction was dried over a 2 S 4, filtered, and concentrated in vacuo. The crude product was dissolved in 3:1 TH/1 HCl (12 ml). After 3 h at room temperature, the reaction mixture was diluted with ethyl acetate (100 ml) and washed with saturated ahc 3 (100 ml) followed by brine (100 ml). The organic fraction was dried over a 2 S 4, filtered, and concentrated in vacuo. Purification by flash chromatography (4:1 ethyl acetate/hexanes) afforded diboc fmk 10 (408 mg, 60% yield over 2 steps) as an off-white solid: R f 0.50 (100% ethyl acetate); 1 H MR (400 MHz, CDCl 3 ) δ 8.91 (s, 1H), 7.24 (d, J = 8 Hz, 2H), 7.20 (d, J = 8 Hz, 2H), 4.70 (t, 2H), 4.60 (d, J H = 47 Hz, 2H), 3.70 (t, 1H), 3.41 (m, 2H), 2.42 (s, 3H), 2.05 (m, 2H), 1.29 (s, 18H); 13 C MR (100 MHz, CDCl 3 ) δ 189.4, 155.2, 154.5, 154.0, 149.8, 139.2, 131.1, 129.7, 129.4, 128.8, 123.5, 114.2, 85.1, 83.4 (d, J C = 20 Hz), 58.2, 41.0,
32.9, 27.7, 21; LC/MS (LC: gradient 0-100% MeC [0.1% HC 2 H] over 30 min, 0.2 ml/min flow rate, MS: ESI + ): retention time, 15.12 min; purity, 95%; 543.1 [M+H] +. H 2 H fmk (1) mk (1). Diboc fmk 10 (46 mg, 0.085 mmol) was dissolved in 1 HCl in dioxane (1 ml) and stirred for 2 h at 0 C, and 3 h at room temperature. The solvent was removed in vacuo and the crude product was partitioned between CH 2 Cl 2 (10 ml) and saturated ahc 3 (10 ml). The organic fraction was washed with brine (200 ml), dried over a 2 S 4, filtered, and concentrated in vacuo. Purification by flash chromatography (5% MeH in CH 2 Cl 2 ) afforded fmk (19 mg, 70% yield) as an off-white solid: 1 H MR (400 MHz, CDCl 3 ) δ 8.37 (s, 1H), 7.38 (d, J = 8 Hz, 2H), 7.34 (d, J = 8 Hz, 2H), 4.95 (br s, 2H), 4.72 (t, J = 5.6 Hz, 2H), 4.62 (d, J H = 47 Hz, 2H), 3.46 (m, 2H), 2.49 (s, 3H), 2.10 (m, 2H); HRMS Calcd for C 18 H 19 4 2 342.1492, found 342.1502; HPLC (gradient 30-100% MeH over 15 min, 10 ml min -1 flow rate): retention time, 10.3 min; purity > 99%.
(Boc) 2 H 11 HBoc Carbamate 11. To a solution of diboc fmk 10 (59 mg, 0.11 mmol) in CH 2 Cl 2 (1 ml) was added DIPEA (0.015 ml, 0.085 mmol), followed by carbonyldiimidazole (14 mg, 0.085 mmol). After stirring for 2 h at room temperature, -Boc-1,4-diaminobutane (26 mg, 0.14 mmol) was added. After 3 h, additional -Boc-1,4-diaminobutane (16 mg, 0.085 mmol) was added and the reaction was stirred for 2 h at room temperature. The reaction mixture was diluted with ethyl acetate (30 ml) and washed with 0.5 HCl (10 ml) followed by brine (30 ml). The organic fraction was dried over a 2 S 4, filtered, and concentrated in vacuo. Purification by flash chromatography (3:2 ethyl acetate/hexanes) gave 57 mg (69% yield) of carbamate 11: R f 0.50 (100% ethyl acetate); 1 H MR (400 MHz, CDCl 3 ) δ 8.94 (s, 1H), 7.27 (d, J = 8 Hz, 2H), 7.23 (d, J = 8 Hz, 2H), 4.84 (m, 2H), 4.66-4.63 (d, J H = 47 Hz, 2H), 4.13 (m, 2H), 3.06 (m, 4H), 2.46 (s, 3H), 2.16 (m, 2H), 1.42 (s, 9H), 1.42 (m, 4H), 1.31 (s, 18H); LC/MS (LC: gradient 0-100% MeC [0.1% HC 2 H] over 30 min, 0.2 ml min -1 flow rate, MS: ESI + ): retention time, 17.69 min; purity, > 95%; 757.2 [M+H] +.
H 2 B H H H fmk-bdipy (2) fmk-bdipy (10). To a solution of carbamate 11 (20 mg, 0.026 mmol) in CH 2 Cl 2 (0.3 ml) was added TA (0.3 ml) at 0 C. After slowly warming the reaction mixture to RT over a period of 2 h, solvent was removed in vacuo. A portion of the crude bocdeprotected product (5.6 mg, 0.0084 mmol) was dissolved in DM (0.1 ml). BDIPY L-HS (5 mg, 0.01 mmol) was added followed by DIPEA (3.9 mg, 0.03 mmol). After stirring for 12 h at room temperature, the reaction mixture was concentrated in vacuo. Purification by preparative thin layer chromatography (10:1 CH 2 Cl 2 /MeH) afforded fmk- BDIPY (5 mg, 71% yield over 2 steps) as an off-white solid: 1 H MR (400 MHz, CDCl 3 ) δ 8.35 (s, 1H), 7.37 (d, J = 8 Hz, 2H), 7.31 (d, J = 8 Hz, 2H), 7.10 (s, 1H), 6.89 (d, 1H), 6.29 (d, 1H), 6.12 (s, 1H), 5.86 (m, 1H), 5.78 (m, 1H), 4.94 (m, 2H), 4.75 (t, J = 6.8 Hz, 2H), 4.62 (d, J H = 47 Hz, 2H), 4.08 (m, 2H), 3.20 (m, 6H), 2.62 (t, J = 7.2 Hz, 2H), 2.55 (s, 2H), 2.48 (s, 3H), 2.26 (s, 3H), 2.13 (m, 4H), 1.59-1.24 (m, 12H); LC/MS (LC: gradient 0-100% MeC [0.1% HC 2 H] over 30 min, 0.2 ml min -1 flow rate, MS: ESI + ): retention time, 12.42 min; purity, > 95%; 844.3 [M+H] + ; HRMS Calcd for C 43 H 54 3 9 5 844.4293, found 844.4283.
H 2 mk-pa (3). To a solution of diboc fmk 10 (75 mg, 0.14 mmol) in CH 2 Cl 2 (3 ml) was added DIPEA (0.057 ml, 0.317 mmol) at 0 C. Methanesulfonyl chloride (1 M in CH 2 Cl 2, 0.17 ml, 0.17 mmol) was added and the reaction mixture was stirred at 0 C. After 30 min, the reaction mixture was diluted with ethyl acetate and washed with saturated H 4 Cl (10 ml) followed by brine (10 ml). The organic fraction was dried over a 2 S 4, filtered, and concentrated in vacuo to give 82 mg (96% yield) of the crude mesylate, which was used in the next reaction without purification. To the crude mesylate (57 mg, 0.092 mmol) was added propargylamine (0.5 ml) and the reaction mixture was stirred for 30 h at 40 C. The solvent was removed in vacuo and the crude product was dissolved in CH 2 Cl 2 (1 ml) and TA (1 ml). After stirring for 2 h at room temperature, the solvent was removed in vacuo. Purification by preparative HPLC (gradient 0-65% acetonitrile, 0.1 % TA, over 23 min, 10 ml min -1 flow rate) afforded fmk-pa (4.4 mg, 35% yield): 1 H MR (400 MHz, CDCl 3 ) δ 11.1 (br s, 1H), 8.31 (s, 1H), 7.44 (d, J = 8 Hz, 2H), 7.31 (d, J = 8 Hz, 2H), 5.42 (br s, 2H), 4.76 (t, 2H), 4.67 (d, J H = 47 Hz, 2H), 3.92 (s, 2H), 3.24 (m, 2H), 2.58 (s, 1H), 2.50 (s, 3H), 2.41 (m, 2H); LC/MS (LC: gradient 0-100% MeC [0.1% HC 2 H] over 30 min, 0.2 ml min -1 flow rate, MS: ESI + ): retention time, 9.28 min; purity, >95%; 380.0 [M+H] +. HRMS Calcd for C 21 H 23 5 380.1886, found 380.1878. H fmk-pa (3)
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