si obc jp4 rev...supporting information synthesis of analogs of the radiation mitigator jp4-039 and...

Supporting Information

Synthesis of analogs of the radiation mitigator JP4-039 and

visualization of BODIPY derivatives in mitochondria Marie-Céline Frantz,a Erin M. Skoda,a Joshua R. Sacher,a Michael W. Epperly,b Julie Goff,b Joel S.

Greenberger,b and Peter Wipf*a,c aDepartment of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA, bDepartment of Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15232, USA, cDepartment of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA

Contents General Information ................................................................................................................................................................... S2 2-Benzyl-1,1,3,3-tetramethylisoindoline.................................................................................................................................... S3

1,1,3,3-Tetramethylisoindoline. .................................................................................................................................................. S3

1,1,3,3-Tetramethylisoindolin-2-yloxyl (TMIO, 1)................................................................................................................... S4

5-Amino-1,1,3,3-tetramethylisoindolin-2-yloxyl (5-amino-TMIO, 2). ................................................................................... S4

(S,E)-N-(1,1,3,3-Tetramethyl-2-oxo-isoindolin-5-yl)-5-(tert-butoxycarbonylamino)-7-methyloct-3-enamide (12). .......... S5

9-Benzyl-9-azabicyclo[3.3.1]nonan-3-one (13)......................................................................................................................... S6

9-Benzyl-9-azabicyclo[3.3.1]nonan-3-one oxime (14). ............................................................................................................ S6

tert-Butyl 9-benzyl-9-azabicyclo[3.3.1]nonan-3-ylcarbamate (15). ........................................................................................ S7

3-(tert-Butoxycarbonylamino)-9-azabicyclo[3.3.1]nonane N-oxyl (3-Boc-amino-ABNO, 16). ........................................... S7

3-Amino-9-azabicyclo[3.3.1]nonane N-oxyl (3-amino-ABNO, 6). ......................................................................................... S8

tert-Butyl (S,E)-8-(9-benzyl-9-azabicyclo[3.3.1]nonan-3-ylamino)-2-methyl-8-oxooct-5-en-4-ylcarbamate (18). ............ S8

(S,E)-N-(9-Azabicyclo[3.3.1]nonan-9-oxo-3-yl)-5-(tert-butoxycarbonylamino)-7-methyloct-3-enamide (17). .................. S9

2-Adamantanecarbonitrile. ........................................................................................................................................................ S10

2-Adamantane carboxylic acid. ................................................................................................................................................ S10

5,7-Dibromo-2-adamantane carboxylic acid (19).................................................................................................................... S10

(5,7-Dibromoadamantan-2-yl)-carbamic acid tert-butyl ester (20)........................................................................................ S11

(7-Methylenebicyclo[3.3.1]nonan-3-one-9-yl)-carbamic acid tert-butyl ester (21).............................................................. S11

(7-Methylenebicyclo[3.3.1]nonan-3-one oxime-9-yl)-carbamic acid tert-butyl ester (22). ................................................. S12

(1-Iodomethyl-2-azaadamantan-6-yl)-carbamic acid tert-butyl ester (23). ........................................................................... S13

(1-Methyl-2-azaadamantane-N-oxyl-6-yl)-carbamic acid tert-butyl ester (6-Boc-amino-1-Me-AZADO, 24). ................. S13

6-Amino-1-methyl-2-azaadamantane-N-oxyl (6-amino-1-Me-AZADO, 8).......................................................................... S14

6-Amino-1-methyl-2-azaadamantane (26). .............................................................................................................................. S14

(S,E)-N-(1-Methyl-2-azaadamant-2-oxo-6-yl)-5-(tert-butoxycarbonylamino)-7-methyloct-3-enamide (25)..................... S14

(S,E)-Methyl 5-((tert-butoxycarbonyl)amino)-7-methyloct-3-enoate (27)............................................................................ S15

(S,E)-5,5-difluoro-3-(3-((8-methoxy-2-methyl-8-oxooct-5-en-4-yl)amino)-3-oxopropyl)-7,9-dimethyl-5H-dipyrrolo-

[1,2-c:2',1'-f][1,3,2]diazaborinin-4-ium-5-uide (29). ....................................................................................................... S16

(S,E)-5,5-Difluoro-3-(3-((8-((1-oxo-2,2,6,6-tetramethylpiperidin-4-yl)amino)-2-methyl-8-oxooct-5-en-4-yl)amino)-3-

oxopropyl)-7,9-dimethyl-5H-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-4-ium-5-uide (BODIPY-FL-JP4-039, 30). S16

Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013

S2

(S,E)-5,5-Difluoro-3-(3-((8-((1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl)amino)-2-methyl-8-oxooct-5-en-4-yl)am-ino)-

3-oxopropyl)-7-phenyl-5H-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-4-ium-5-uide

(BODIPY®-R6G-JP4-039, 33). ....................................................................................................................................... S17

X-Ray crystallography data for 25. .......................................................................................................................................... S19

Irradiation survival curves for JP4-039, 30, 33, 12, 17, and 25 in 32Dcl3 cells ................................................................... S29

Visualization of 33 in KM101 cells. ....................................................................................................................................... S31

NMR spectra for new compounds ............................................................................................................................................ S32

General Information. All moisture- and air-sensitive reactions were performed using syringe-septum cap techniques

under an inert atmosphere (N2 or argon) in glassware that was dried in an oven at 140 °C for at least 2 h prior to use.

Reactions carried out at a temperature below 0 °C employed a brine/ice bath or a CO2/acetone bath. All reagents and

solvents were used as received unless otherwise specified. THF and Et2O were distilled over sodium/benzophenone ketyl;

MeOH was distilled over magnesium; benzene, acetonitrile and DME were distilled over CaH2; DMF was distilled and

stored over 4Å molecular sieves; pyridine and triethylamine were distilled over CaH2 and stored over KOH; CH2Cl2 and

toluene were purified using an alumina column filtration system. Analytical thin-layer chromatography (TLC) was

performed on pre-coated SiO2 60 F254 plates (250 µm layer thickness) available from Merck. Visualization was

accomplished by UV irradiation at 254 nm and/or by staining with Vaughn’s reagent (4.8 g (NH4)6Mo7O24•4H2O and 0.2 g

Ce(SO4)2•4H2O in 100 mL of a 3.5 N H2SO4 solution), a KMnO4 solution (1.5 g KMnO4 and 1.5 g K2CO3 in 100 mL of a

0.1% NaOH solution), a ninhydrin solution (2 g ninhydrin in 100 mL EtOH), a PMA solution (5 g phosphomolybdic acid

in 100 mL EtOH), or a p-anisaldehyde solution (2.5 mL p-anisaldehyde, 2 mL AcOH and 3.5 mL conc. aq. H2SO4 in 100

mL EtOH). Flash column chromatography was performed using SiO2 60 (particle size 0.040–0.055 mm, 230–400 mesh, or

Silicycle SiliaFlash® P60, 40-63 µm). Melting points were determined on a Laboratory Devices Mel-Temp II capillary

melting point apparatus fitted with a Fluke 51 II digital thermometer. Optical rotations were determined using a Perkin-

Elmer 241 polarimeter. Infrared spectra were recorded on a Smiths IdentifyIR ATR spectrometer. 1H NMR and 13C NMR

spectra were obtained on a Bruker Avance 300 or 400 instrument at 300/75 MHz or 400/100 MHz, respectively. Chemical

shifts were reported in parts per million (ppm) as referenced to residual solvent. 1H NMR spectra are tabulated as follows:

chemical shift, multiplicity (app = apparent, b = broad, s = singlet, d = doublet, t = triplet, q = quartet, quint = quintuplet,

sext, = sextuplet, m = multiplet), number of protons, coupling constant(s). 13C NMR were obtained using a proton-

decoupled pulse sequence and are tabulated by observed peak. LC-MS analyses were performed on a Shimadzu UFLC

instrument equiped with an Applied Biosystems MDS SCIEX API 2000 mass spectrometer (ESI), under the following

conditions: column: Varian Polaris C18-A (100 x 4.6 mm, 5 µm) equilibrated at 40 °C; buffer A: 0.1% aqueous AcOH,

buffer B: 0.1% AcOH in MeCN; 30 min gradient: 5% buffer B in buffer A for 1 min, then 5 to 95% buffer B in buffer A

over 13 min, then 95% buffer B in buffer A for 4 min, then 95 to 5% buffer B in buffer A over 7 min, then 5% buffer B in

buffer A for 5 min; flow rate: 0.2 mL/min; detection: TIC and/or UV λ = 254/280 nm. Mass spectra were obtained on a

Waters Autospec double focusing mass spectrometer (EI) or a Waters Q-Tof mass spectrometer (ESI), at the University of

Pittsburgh Mass Spectrometry facility. Experimental procedures and spectral details for compounds 11 and JP4-039 have

previously been reported in Org. Lett. 2011, 13, 2318-2321.


S3

NBn

2-Benzyl-1,1,3,3-tetramethylisoindoline.1 An oven-dried 250 mL, three-necked, round-bottom flask was flushed with

nitrogen, and magnesium turnings (3.84 g, 156 mmol) were introduced, that were covered with dry Et2O (9 mL). A

solution of MeI (9.45 mL, 150 mmol) in dry Et2O (80 mL) was then added dropwise via a dropping funnel while stirring

over a period of 50 min. The resulting reaction mixture was then stirred for an additional 30 min, and then concentrated by

slow distillation of solvent until the internal temperature reached 80 °C. The residue was allowed to cool to 60 °C, and a

solution of N-benzylphtalimide (6.00 g, 25.0 mmol) in dry toluene (76 mL) was added dropwise via a dropping funnel

with stirring at a sufficient rate to maintain this temperature. When the addition was complete, solvent was distilled slowly

from the mixture until the temperature reached 108-110 °C. The reaction mixture was refluxed at 110 °C for 4 h, then

concentrated again by further solvent distillation. It was then cooled to rt and diluted with hexanes. The resulting purple

slurry was filtered through Celite and washed with hexanes. The combined yellow filtrate turned dark red-purple after

standing in air overnight. It was then concentrated in vacuo. The resulting purple residue was passed through a short

column of basic alumina (grade I, 70-230 mesh), eluting with hexanes (~1 L), to afford 2.58 g (39%) of the title compound

as a colorless oil which solidified to give a white solid. Representative experimental data are as follows: mp 61.0-61.4 °C

(lit. 63-64 °C from MeOH or EtOH); IR (neat) 3081, 3062, 3047, 3019, 3006, 2978, 2965, 2958, 2918, 2890, 2849, 2820,

1485, 1446, 1379, 1372, 1355, 1319, 1299, 1284, 1265, 1237, 1211, 1195, 1176, 1161, 1140, 1120, 1103, 1073, 1045,

1025, 945, 932, 904, 874, 827, 790, 760, 751, 742, 701, 686 cm-1; 1H NMR (300 MHz, CDCl3) δ 7.48 (app d, 2 H, J = 7.2

Hz), 7.34-7.19 (m, 5 H), 7.18-7.11 (m, 2 H), 4.00 (s, 2 H), 1.31 (s, 12 H); 13C NMR (75 MHz, CDCl3) δ 148.0, 143.6,

128.5, 128.1, 127.0, 126.5, 121.5, 65.4, 46.5, 28.6; EI-MS m/z 265 (M+, 25), 251 (100), 235 (53), 158 (69), 144 (86), 128

(60), 115 (67), 103 (67), 92 (86), 77 (65), 65 (84); HRMS (EI) m/z calcd for C19H23N 265.1830, found 265.1824.

NH

1,1,3,3-Tetramethylisoindoline.1 2-Benzyl-1,1,3,3-tetramethylisoindoline (3 x 621 mg, 7.02 mmol) was dissolved in

AcOH (3 x 11 mL) in 3 separated Parr flasks, then 10% Pd/C (3 x 56.5 mg) was added. The flasks were placed in 3 high

pressure reactors. The reactors were charged with H2 and purged for 5 cycles and was finally pressurized with H2 at 4 bars

(60 psi). After stirring at rt for 3 h, the combined reaction mixtures were filtered through Celite, and the solvent removed

in vacuo. The resulting residue was dissolved in water (5 mL) and the solution neutralized with 2.5 N aq. NaOH (pH

11.5), and extracted with Et2O (3 x 50 mL). The combined organic layers were dried (Na2SO4), filtered and concentrated

in vacuo to yield 1.16 g (95%) of the crude title compound as slightly yellow crystals. Representative experimental data

are as follows: mp 36.0-36.5 °C (lit. 36-38 °C from MeOH/H2O); IR (neat) 3064, 3036, 3013, 2956, 2920, 2861, 1478,

1459, 1441, 1414, 1372, 1359, 1314, 1297, 1241, 1209, 1178, 1165, 1139, 1124, 1105, 1023, 1001, 993, 932, 883, 764,

734, 697 cm-1; 1H NMR (300 MHz, CDCl3) δ 7.30-7.23 (m, 2 H), 7.18-7.11 (m, 2 H), 1.86 (bs, 1 H), 1.48 (s, 12 H); 13C

NMR (75 MHz, CDCl3) δ 148.9, 127.2, 121.6, 62.9, 32.1.

1 Griffiths, P. G.; Moad, G.; Rizzardo, E.; Solomon, D. H. Aust. J. Chem. 1983, 36, 397.


S4

N O

1,1,3,3-Tetramethylisoindolin-2-yloxyl (TMIO, 1).1 To a solution of 1,1,3,3-tetramethylisoindoline (1.46 g, 8.33 mmol)

in a 14:1 mixture of MeOH/MeCN (16.6 mL) were added successively NaHCO3 (560 mg, 6.67 mmol), Na2WO4•2H2O

(83.3 mg, 0.25 mmol) and 30% aq. H2O2 (3.12 mL, 27.50 mmol). The resulting suspension was stirred at rt. After 18 h, a

bright yellow suspension formed and 30% aq. H2O2 (3.00 mL, 26.44 mmol) was added. The reaction mixture was stirred

for 2 days, then diluted with water and extracted twice with hexanes. The combined organic layers were washed with 1 M

aq. H2SO4 and brine, dried (Na2SO4), filtered and concentrated in vacuo to yield 1.55 g (98%) of crude 1 as a yellow

crystalline powder, that was carried on without further purification. Representative experimental data are as follows: mp

122-125 °C (softening point: 108 °C) (lit. 128-129°C from petroleum ether); IR (neat) 2971, 2922, 2859, 1670, 1482,

1465, 1450, 1427, 1372, 1353, 1316, 1295, 1278, 1165, 1120, 1109, 1021, 760, 678 cm-1; EI-MS m/z 268 (100), 252 (30),

239 (43), 191 (M+, 75), 155 (60); HRMS (EI) m/z calcd for C12H17NO 191.1310, found 191.1306.

N OH2N

5-Amino-1,1,3,3-tetramethylisoindolin-2-yloxyl (5-amino-TMIO, 2).2 Conc. H2SO4 (13.5 mL) was added dropwise via

syringe to TMIO (1.34 g, 7.07 mmol, crude) cooled in an ice-water bath, forming a dark-red solution which was then

warmed to 60 °C for 15 min and then cooled to 0 °C. Conc. HNO3 (0.90 mL, 19.1 mmol) was added dropwise via syringe.

When the reaction appeared complete, the yellow-orange solution was heated at 100 °C for 10 min, the color turning to

red-orange. After cooling to rt, the reaction mixture was neutralized by careful addition to ice-cooled 2.5 N aq. NaOH (30

mL, final pH > 12). This aqueous phase was extracted with Et2O until it became colorless and the combined organic layers

were dried (Na2SO4), filtered and concentrated in vacuo to yield 1.64 g (98%) of crude 5-nitro-1,1,3,3-

tetramethylisoindolin-2-yloxyl3 as a yellow-orange powder, that was carried on without further purification.

A flask containing a solution of 5-nitro-1,1,3,3-tetramethylisoindolin-2-yloxyl (1.50 g, 6.38 mmol, crude) in MeOH (75

mL) was purged and filled with argon, then 10% Pd/C (150 mg) was added. The flask was purged and filled 3 times with

H2, and the resulting black suspension was stirred at rt under H2 (1 atm) for 4 h. The reaction mixture was then filtered

through Celite, the Celite washed with MeOH, and the solution concentrated in vacuo to yield 1.38 g of the crude

hydroxylamine as a yellow solid that was carried on without further purification. Representative experimental data are as

follows: 1H NMR (300 MHz, CD3OD) δ 6.89 (d, 1 H, J = 8.1 Hz), 6.62 (dd, 1 H, J = 8.1, 2.1 Hz), 6.54 (d, 1 H, J = 2.1

Hz), 3.35 (s, 2 H), 1.35 (s, 6 H), 1.33 (s, 6 H); 13C NMR (75 MHz, CD3OD) δ 148.1, 147.4, 136.5, 123.3, 116.3, 110.0,

68.3, 68.0, 27.1, 26.8.

To a solution of the crude hydroxylamine (1.38 g, 6.38 mmol) in MeOH (75 mL) was added Cu(OAc)2•H2O (26 mg, 0.128

mmol). The reaction mixture was stirred at rt under air for 1.5 h, the color turning to dark brown. The solvent was then

removed in vacuo, the residue taken up in CHCl3 and a small amount of MeOH to dissolve the insoluble material, and

washed with water. The aqueous phase was extracted twice with CHCl3, and the combined organic layers were washed

with brine, dried (Na2SO4), filtered and concentrated in vacuo. Chromatography of the residue on SiO2 (6:4 to 5:5,

hexanes/EtOAc) afforded 1.13 g (86%, 2 steps) of 2 as a yellow powder. Representative experimental data are as follows:

2 Giroud, A. M.; Rassat, A. Bull. Soc. Chim. Fr. 1979, II, 48. 3 Bolton, R.; Gillies, D. G.; Sutcliffe, L. H.; Wu, X. J. Chem. Soc., Perkin Trans. 2 1993, 2049.


S5

mp 192-194 °C (softening point: 189 °C) (lit. 198 °C from 1:2, benzene/petroleum ether); IR (neat) 3463, 3429, 3368,

3353, 3239, 3049, 2969, 2924, 2857, 1640, 1619, 1588, 1580, 1508, 1495, 1452, 1424, 1372, 1357, 1334, 1314, 1297,

1280, 1247, 1161, 1120, 1068, 1042, 943, 924, 885, 867, 820, 680 cm-1; EI-MS m/z 205 (M+, 75), 190 (90), 175 (58), 173

(68), 160 (100), 144 (65), 130 (63), 115 (43), 91 (35), 77 (36); HRMS (EI) m/z calcd for C12H17N2O 205.1341, found

205.1336.

BocHN

O

NH

N O

(S,E)-N-(1,1,3,3-Tetramethyl-2-oxo-isoindolin-5-yl)-5-(tert-butoxycarbonylamino)-7-methyloct-3-enamide (12). To

a solution of the alcohol 10 (187 mg, 0.728 mmol) in acetone (7 mL) at 0 °C was slowly added a solution of Jones reagent

(2.5 M, 0.73 mL, 1.82 mmol). The resulting dark suspension was stirred at 0 °C for 1 h, then diluted with Et2O and water.

The aqueous phase was separated and extracted with twice Et2O. The combined organic layers were washed with water

(2x) and brine (1x), dried (Na2SO4), filtered and concentrated in vacuo to yield 190 mg (96%) of the crude acid 11 as a

slightly yellow oil, that was carried on without further purification.

To a solution of acid 11 (187 mg, 0.691 mmol, crude) in dry CH2Cl2 (8 mL) at 0 °C were added successively 2 (213 mg,

1.04 mmol), DMAP (93.7 mg, 0.760 mmol), HOBt•H2O (103 mg, 0.760 mmol) and EDCI (162 mg, 0.829 mmol). The

resulting yellowish solution was stirred at rt for 16 h, and then washed with sat. aq. NH4Cl. The aqueous phase was

separated and extracted once with CH2Cl2, and the combined organic layers were washed with 1 N aq. HCl (2x) and sat.

aq. NaHCO3 (1x), dried (Na2SO4), filtered and concentrated in vacuo. Chromatography of the residue on SiO2 (6:4,

hexanes/EtOAc) afforded 221 mg (70%, 67% over 2 steps) of 12 as a pale orange foam: mp 78-79 °C (softening point: 70

°C); [α]D22 +72.2 (c 0.5, CH2Cl2); IR (neat) 3310 (br), 2969, 2926, 2864, 1675, 1618, 1599, 1526, 1493, 1465, 1452, 1429,

1420, 1388, 1362, 1325, 1310, 1280, 1243, 1161, 1118, 1042, 1021, 969, 872, 826 cm-1; EI-MS m/z 458 (M+, 72), 428

(50), 372 (30), 342 (37), 175 (49), 160 (31), 91 (44), 73 (61), 69 (100); HRMS (EI) m/z calcd for C26H40N3O4 458.3019,

found 458.3011.

A sample of this nitroxide (44.8 mg, 0.0977 mmol) was dissolved in dry MeOH (0.9 mL) and L-ascorbic acid (17.4 mg,

0.0977 mmol) was added. The orange solution turned slighly yellow within a few minutes. After stirring at rt for 50 min,

an additional amount of L-ascorbic acid (8.7 mg, 0.0488 mmol) was added, but a complete discoloration of the solution

could not be achieved and a residual amount of starting material was seen on TLC. After stirring for 1.5 h, the solvent was

removed in vacuo. The resulting residue was dissolved in CH2Cl2 and washed with water. The aqueous phase was

extracted twice with CH2Cl2, and the combined organic layers were dried (Na2SO4), filtered and concentrated in vacuo to

yield 42.6 mg (95%) of the corresponding hydroxylamine (contaminated with nitroxide) as a pale yellow foam: 1H NMR

(300 MHz, CD3OD, broad) δ 7.42 (s, 1 H), 7.36 (d, 1 H, J = 5.7 Hz), 7.09 (d, 1 H, J = 6.3 Hz), 6.65 (bs, 1 H), 5.75-5.68

(m, 1 H), 5.68-5.50 (m, 1 H), 4.08 (app bs, 1 H), 3.22-3.00 (m, 2 H), 1.65-1.56 (m, 1 H), 1.52-1.05 (m, 2 s at 1.41 and

1.38, 23 H), 0.91 (d, 6 H, J = 4.5 Hz); 13C NMR (75 MHz, CD3OD) δ 172.3, 158.0, 158.0, 147.0, 142.3, 139.2, 137.1,

124.0, 123.1, 120.8, 115.0, 79.9, 68.5, 68.2, 51.8, 45.3, 41.7, 29.0, 28.9, 26.9, 26.8, 25.9, 23.3, 22.7.


S6

NBn

O

9-Benzyl-9-azabicyclo[3.3.1]nonan-3-one (13).4 A mecanically stirred solution of acetonedicarboxylic acid (3.24 g, 21.5

mmol) and glutaraldehyde (24% in water, 12.7 mL, 32.3 mmol) in H2O (30 mL) was treated at 0 °C with benzylamine (2.0

mL, 17.9 mmol). The resulting pale yellow paste was vigorously stirred at rt under argon for 12 h. The brownish reaction

mixture was then adjusted to pH < 2 with conc. HCl and the resulting clear brown solution heated to 60 °C for 1 h to

complete decarboxylation. After cooling, the mixture was extracted with CH2Cl2 (3 x 30 mL). It was then adjusted to pH >

12 with solid NaOH and extracted with CH2Cl2 (4 x 50 mL). These combined organic layers were dried (Na2SO4), filtered

and concentrated in vacuo. Chromatography of the residue on SiO2 (7:3, hexanes/EtOAc) afforded 13 (2.83 g, 69%) as a

colorless crystalline solid. Representative experimental data are as follows: mp 70.3-70.5 °C (hexanes/EtOAc) (lit. 63-66

°C); IR (neat) 3062, 3025, 2934, 2921, 2874, 2846, 1689, 1493, 1456, 1441, 1415, 1403, 1362, 1338, 1329, 1320,1305,

1288, 1279, 1266, 1219, 1202, 1185, 1133, 1100, 1070, 1029, 1018, 984, 947, 911, 844, 766, 731, 699 cm-1; 1H NMR

(CDCl3, 300 MHz) δ 7.48-7.20 (m, 5 H), 3.91 (s, 2 H), 3.32 (app s, 2 H), 2.74 (dd, 2 H, J = 16.2, 6.6 Hz), 2.26 (d, 2 H, J =

16.2 Hz), 2.08-1.85 (m, 2 H), 1.65-1.40 (m, 4 H); 13C NMR (CDCl3, 75 MHz) δ 211.9, 139.4, 128.6, 128.5, 127.3, 57.2,

53.7, 43.0, 29.5, 16.8; ESI-MS m/z 230 ([M+H]+, 100), 172 (42); HRMS (ESI) m/z calcd for C15H20NO (M+H) 230.1545,

found 230.1550.

NBn

HON

9-Benzyl-9-azabicyclo[3.3.1]nonan-3-one oxime (14). To a solution of ketone 13 (2.40 g, 10.5 mmol) in EtOH (28 mL)

were added NH2OH•HCl (1.48 g, 20.9 mmol) and then 3 N aq. NaOH (7.0 mL, 20.9 mmol). The reaction mixture was

refluxed under argon for 14 h, then diluted with CH2Cl2 and water. The layers were separated and the aqueous phase

extracted with CH2Cl2. The combined organic layers were washed with brine, dried (Na2SO4), filtered and concentrated in

vacuo. The residue was taken up in CH2Cl2, and the insoluble material filtered and rinsed with CH2Cl2 to afford 1.42 g of a

first crop of product as colorless crystals. Chromatography of the mother liquor on SiO2 (8:2 to 4:6, hexanes/EtOAc)

afforded 1.11 g of a second crop as a colorless crystalline solid. The total amount of 14 obtained was 2.53 g (99%): mp

135.4-135.7 °C (CH2Cl2); IR (neat) 3300-2500 (br), 3057, 3157, 2960, 2939, 2921, 2883, 2842, 1644, 1497, 1467, 1456,

1433, 1418, 1389, 1364, 1357, 1348, 1323, 1286, 1266, 1243, 1210, 1197, 1111, 1096, 1079, 1074, 1059, 1005, 980, 964,

952, 928, 900, 841, 760, 747, 723, 693 cm-1; 1H NMR (CDCl3, 300 MHz) δ 7.72 (bs, 1 H), 7.44-7.20 (m, 5 H), 3.88 (s, 2

H), 3.14-3.05 (m, 2 H), 3.03 (d, 1 H, J = 16.2 Hz), 2.73 (dd, 1 H, J = 15.4, 5.9 Hz), 2.38 (dd, 1 H, J = 16.3, 6.8 Hz), 2.25

(d, 1 H, J = 15.3 Hz), 2.05-1.73 (m, 3 H), 1.60-1.44 (m, 3 H); 13C NMR (CDCl3, 100 MHz) δ 160.0, 139.7, 128.6, 128.5,

127.2, 57.0, 51.9, 51.2, 32.0, 29.8, 29.0, 25.9, 17.3; ESI-MS m/z 245 ([M+H]+, 100); HRMS (ESI) m/z calcd for

C15H21N2O (M+H) 245.1654, found 245.1642.

4 Mach, R. H.; Luedtke, R. R.; Unsworth, C. D.; Boundy, V. A.; Nowak, P. A.; Scripko, J. G.; Elder, S. T.; Jackson, J. R.; Hoffman, P. L.; Evora, P. H.; Rao, A. V.; Molinoff, P. B.; Childers, S. R.; Ehrenkaufer, R. L. J. Med. Chem. 1993, 36, 3707.


S7

NBn

BocHN

tert-Butyl 9-benzyl-9-azabicyclo[3.3.1]nonan-3-ylcarbamate (15). A suspension of oxime 14 (1.30 g, 5.34 mmol) and

NiCl2 (69.2 mg, 0.534 mmol) in MeOH (25 mL) was treated at –20 °C with NaBH4 (2.13 g, 53.4 mmol) portionwise. The

resulting dark mixture was vigorously stirred between –20 and –10 °C under argon. After 1.5 h, a solution of NaBH4 (1.70

g, 42.7 mmol) in water (5 mL) was added. The reaction mixture was stirred at the same temperature for 1.5 h, then

warmed to rt. After stirring for 1 h at rt, the mixture was quenched carefully with acetone, then filtered over Celite and the

brown solid rinsed with MeOH. The solution was concentrated in vacuo, and the residue was taken up in water, acidified

with 6 N aq. HCl (pH < 2), extracted twice with Et2O, then basified with solid NaOH (pH > 9) and extracted twice with

CHCl3. An emulsion formed with a white precipitate that was filtered over Celite. The combined chloroformed layers were

washed with brine, dried (K2CO3), filtered and concentrated in vacuo to afford 1.49 g of the crude amine as a pale yellow

oil that was carried on to the next step without further purification.

To a solution of this amine in dry CH2Cl2 (100 mL) were added Et3N (2.25 mL, 16.0 mmol) and then Boc2O (1.29 g, 5.87

mmol) at 0 °C. The reaction mixture was stirred at rt under argon for 18 h, then quenched with sat. aq. NH4Cl and the

aqueous phase extracted with CH2Cl2. The combined organic layers were dried (Na2SO4), filtered and concentrated in

vacuo. The residue was taken up in CH2Cl2, and the insoluble material filtered to afford 481 mg of a first crop of 15 as a

white powder. Chromatography of the mother liquor on SiO2 (9:1 to 5:5, hexanes/EtOAc) afforded 4 other fractions of

different purity for a total amount of 964 mg. The total amount of 15 obtained was ca. 1.44 g (82%, 2 steps). A sample of

15 was recrystallized in EtOH (ca. 4 mL) to afford colorless crystals. 1H NMR showed a 3:1 mixture of stereoisomers: mp

153.6-155.0 °C (EtOH); IR (neat) 3312 (br), 3023, 2988, 2971, 2943, 2911, 2861, 2846, 2826, 2809, 1672, 1532, 1493,

1472, 1450, 1387, 1364, 1316, 1303, 1290, 1282, 1254, 1228, 1185, 1169, 1133, 1068, 1046, 1029, 1018, 874, 837, 766,

736, 699, 671 cm-1; 1H NMR (CDCl3, 300 MHz) δ 7.40-7.18 (m, 5 H), 4.40-4.24 (m, 1 H), 4.24-4.00 (m, 1 H), 3.82 (s, 2

H), 3.07 (bd, 2 H, J = 11.1 Hz), 2.38 (ddd, 2 H, J = 12.0, 12.0, 6.3 Hz), 1.93 (d, 2 H, J = 6.3 Hz), 1.60-1.50 (m, 2 H), 1.46

(s, 9 H), 1.15 (app td, 2 H, J = 11.0, 3.0 Hz), 0.99 (app bd, 2 H, J = 6.6 Hz); 13C NMR (CDCl3, 100 MHz) δ 155.5, 140.7,

128.5, 128.4, 126.9, 79.2, 56.0, 49.4, 42.9, 34.2, 28.7, 24.8, 14.5; ESI-MS m/z 331 ([M+H]+, 100), 275 ([M–t-Bu+H]+, 93);

HRMS (ESI) m/z calcd for C20H31N2O2 (M+H) 331.2386, found 331.2392.

N

BocHN

O 3-(tert-Butoxycarbonylamino)-9-azabicyclo[3.3.1]nonane N-oxyl (3-Boc-amino-ABNO, 16). A flask containing a

suspension of the Bn-protected amine 15 (715 mg, 2.16 mmol) and AcOH (5 drops) in 10:1 EtOH/EtOAc (30 mL) was

purged and filled 3 times with argon, then 10% Pd/C (143 mg) was added. The flask was purged and filled 3 times with

H2, and the resulting black suspension was stirred at 40-50 °C under H2 (1 atm) for 3 h. The reaction mixture was then

filtered through a pad of Celite, the Celite washed with EtOAc and MeOH, and the solution concentrated in vacuo to

afford 591 mg of the crude amine as a white powder, that was carried on to the next step without further purification.

To a suspension of this amine (755 mg, 3.14 mmol) in MeCN (3.1 mL) was added Na2WO4•2H2O (523 mg, 1.57 mmol)

and the mixture was stirred at rt for 30 min. After cooling to 0 °C, UHP (1.52 g, 15.7 mmol) was added and the reaction

mixture was stirred at 0 °C for 1 h, then allowed to warm to rt. After stirring for 3.5 h at rt, water was added and the

resulting mixture was extracted with CHCl3 (3x). The combined organic layers were dried (K2CO3), filtered and

concentrated in vacuo. The residue was taken up in EtOH, and the insoluble material filtered to afford 467 mg of a first


S8

crop of 16 as a yellow powder. Chromatography of the mother liquor on SiO2 (8:2 to 4:6, hexanes/EtOAc) afforded 120

mg of a second crop as a yellow powder. The total amount of 16 obtained was 587 mg (73%, 2 steps): mp 222.8 °C

(sublimation); IR (neat) 3334 (br), 2969, 2947, 2870, 1677, 1525, 1389, 1366, 1351, 1307, 1292, 1273, 1252, 1232, 1225,

1167, 1131, 1116, 1068, 1046, 1036, 1019, 869, 846, 826, 779, 770, 753 cm-1; ESI-MS m/z 298 ([Mred+MeCN+H]+, 30),

278 ([M+Na]+, 63), 275 (54), 263 (100), 257 ([Mred+H]+, 20), 241 ([M–O+H]+, 23), 222 (50), 212 (28); HRMS (ESI) m/z

calcd for C13H23N2O3Na (M+Na) 278.1606, found 278.1593.

A sample of nitroxide 16 (20.0 mg, 0.0783 mmol) was suspended in MeOH (0.8 mL) and L-ascorbic acid (13.9 mg,

0.0783 mmol) was added. The slightly yellow suspension became white within a few seconds. After stirring at rt for 40

min under argon, the solvent was removed in vacuo. The resulting residue was taken up in EtOAc and sonicated. The

insoluble material was filtered, rinsed with EtOAc and MeOH, and dried, to yield 15.0 mg (75%) of the corresponding

hydroxylamine as a white solid. Representative experimental data are as follows: 1H NMR (DMSO-d6, 400 MHz, 2:1

mixture of isomers) δ 7.93 (s, 0.8 H), 7.43 (s, 0.4 H), 6.57 (d, 0.4 H, J = 8.0 Hz), 6.45 (d, 0.7 H, J = 8.8 Hz), 4.40-4.25 (m,

0.8 H), 3.85-3.70 (m, 0.4 H), 3.40-3.20 (m, 1 H), 3.20-3.10 (m, 1 H), 2.23-2.02 (m, 2 H), 2.02-1.80 (m, 2 H), 1.70-1.53 (m,

2 H), 1.37 (s, 9 H), 1.25-1.14 (m, 1 H), 1.08 (app bt, 2 H, J = 11.2 Hz), 0.82 (app bd, 1 H, J = 11.6 Hz); 13C NMR

(DMSO-d6, 100 MHz, mixture of isomers) δ 154.8, 77.3, 77.1, 55.2, 53.3, 40.4, 33.2, 32.2, 30.8, 28.3, 28.1, 23.4, 13.0,

12.3.

N

H2N

O 3-Amino-9-azabicyclo[3.3.1]nonane N-oxyl (3-amino-ABNO, 6). A mixture of Boc-protected amine 3-Boc-amino-

ABNO (16, 200 mg, 0.783 mmol) in a 4.0 N solution of HCl in 1,4-dioxane (3.3 mL, 13.2 mmol) was stirred at 0 °C for 1

h and at rt for an additional 2 h. The suspension was then filtered and the solid rinsed with cold dry Et2O, to give 167 mg

of an off-white powder. It was dissolved in a minimum of 2.5 N aq. NaOH, and extracted with warm CHCl3 (7x). The

combined organic layers were dried (K2CO3), filtered and concentrated in vacuo to afford 110 mg (90%) of a red oil. Upon

high vacuum and/or storage at rt for not more than 1 h, the product started to loose the red color characteristic for a

nitroxide and became pale yellow. Thus, the title compound is unstable.

NBn

NH

ONHBoc

tert-Butyl (S,E)-8-(9-benzyl-9-azabicyclo[3.3.1]nonan-3-ylamino)-2-methyl-8-oxooct-5-en-4-ylcarbamate (18). A

suspension of oxime 14 (400 mg, 1.64 mmol) and NiCl2 (21.2 mg, 0.164 mmol) in MeOH (8 mL) was treated at –15 °C

with NaBH4 (652 mg, 16.4 mmol) portionwise. The resulting dark mixture was vigorously stirred between –15 and 0 °C

under argon. After 1.5 h, a solution of NaBH4 (326 mg, 8.18 mmol) in water (1 mL) was added. The reaction mixture was

stirred from 0 °C to rt. After 1 h, the reaction mixture was quenched carefully with acetone, then filtered over Celite and

the brown solid rinsed with MeOH. The solution was concentrated in vacuo, and the residue was taken up in water,

acidified with 6 N aq. HCl (pH < 2), extracted twice with Et2O, then basified with solid NaOH (pH > 9) and extracted with

CHCl3 (3x). The combined chloroformed layers were dried (Na2SO4), filtered and concentrated in vacuo to afford 416 mg

of the crude amine as a yellow oil that was carried on to the next step without further purification.

To a solution of acid 11 (424 mg, 1.52 mmol, crude) in dry CH2Cl2 (18 mL) at 0 °C were added successively HOBt•H2O

(246 mg, 1.82 mmol), DMAP (206 mg, 1.67 mmol), a solution of this amine (403 mg, 1.59 mmol, crude) in dry CH2Cl2 (2


S9

mL), and EDCI (356 mg, 1.82 mmol). The resulting mixture was stirred at rt under argon for 14 h, then washed with 2.5 N

aq. NaOH (3x). The organic layer was dried (Na2SO4), filtered and concentrated in vacuo. Chromatography on SiO2 (8:2

to 4:6, hexanes/EtOAc) afforded 421 mg (57%, 3 steps) of 18 as a white powder: mp 115.0-115.4 °C; [α]D23 +12.6 (c 1.0,

CH2Cl2); IR (neat) 3338 (br), 3277 (br), 2924, 2867, 1681, 1635, 1519, 1495, 1467, 1452, 1435, 1389, 1364, 1349, 1327,

1307, 1277, 1252, 1163, 1137, 1081, 1060, 1044, 1025, 1010, 993, 964, 930, 874, 842, 760, 742, 703 cm-1; 1H NMR

(CDCl3, 400 MHz) δ 7.39-7.34 (m, 2 H), 7.33-7.27 (m, 2 H), 7.25-7.19 (m, 1 H), 5.95-5.80 (m, 1 H), 5.66 (dt, 1 H, J =

15.2, 7.2 Hz), 5.46 (dd, 1 H, J = 15.4, 6.6 Hz), 4.55-4.45 (m, 1 H), 4.41 (ddddd, 1 H, J = 12.0, 12.0, 8.4, 6.0, 6.0 Hz), 4.04

(app p, 1 H, J = 7.2 Hz), 3.82 (s, 2 H), 3.08 (app bd, 2 H, J = 10.4 Hz), 2.98 (d, 2 H, J = 7.2 Hz), 2.40-2.26 (m, 2 H), 2.06-

1.87 (m, 3 H), 1.73-1.60 (m, 3 H), 1.58-1.49 (m, 1 H), 1.44 (s, 9 H), 1.34 (app td, 2 H, J = 7.3, 1.4 Hz), 1.03-0.92 (m, 2 H),

0.93 (d, 3 H, J = 6.8 Hz), 0.93 (d, 3 H, J = 6.4 Hz); 13C NMR (CDCl3, 100 MHz) δ 170.4, 155.6, 140.6, 137.2, 128.6,

128.4, 126.9, 123.6, 79.5, 56.0, 51.6, 49.3, 49.3, 44.2, 42.0, 40.4, 33.5, 33.4, 28.6, 24.8, 24.7, 22.7, 14.5; ESI-MS m/z 527

(80), 506 ([M+Na]+, 84), 484 ([M+H]+, 67); HRMS (ESI) m/z calcd for C29H45N3O3Na (M+Na) 506.3359, found

506.3336.

N

NH

ONHBoc

O (S,E)-N-(9-Azabicyclo[3.3.1]nonan-9-oxo-3-yl)-5-(tert-butoxycarbonylamino)-7-methyloct-3-enamide (17). A

solution of Bn-protected amine 18 (150 mg, 0.309 mmol) in 5:1 MeCN/H2O (12 mL) was treated with CAN (339 mg,

0.618 mmol). The resulting red reaction mixture was stirred at rt under argon. After 3.5 h, more CAN (170 mg, 0.309

mmol) was added. After 19 h stirring, sat. aq. NaHCO3 (12 mL) was added and stirring was continued for 15 min. The

mixture was then extracted with CHCl3 (3 x 50 mL). The combined organic layers were washed with brine, dried

(Na2SO4), filtered and concentrated in vacuo to yield 144 mg of the crude amine as a brownish residue, that was carried on

to the next step without further purification.

To a suspension of this amine (140 mg, 0.301 mmol) in 1:1 MeCN/MeOH (1.2 mL) was added Na2WO4•2H2O (50.2 mg,

0.151 mmol) and the mixture was stirred at rt for 30 min. After cooling to 0 °C, UHP (146 mg, 1.51 mmol) was added and

the reaction mixture was stirred at 0 °C for 1 h, then allowed to warm to rt and stirred overnight. After 17 h, water was

added and the resulting mixture was extracted twice with CHCl3. The combined organic layers were dried (K2CO3),

filtered and concentrated in vacuo. Chromatography on SiO2 (5:5, hexanes/EtOAc to EtOAc) afforded 84.9 mg (69%, 2

steps) of 17 as orange microcrystals formed from an initial red oil. Representative experimental data are as follows: mp

140.2-141.0 °C (EtOAc); [α]D23 +25.9 (c 1.0, CH2Cl2); IR (neat) 3292 (br), 2949, 2926, 2867, 1690, 1657, 1539, 1469,

1448, 1400, 1389, 1362, 1325, 1308, 1284, 1269, 1247, 1221, 1167, 1116, 1088, 1070, 1040, 1018, 977, 678 cm-1; ESI-

MS m/z 431 ([M+Na]+, 77); HRMS (ESI) m/z calcd for C22H38N3O4Na (M+Na) 431.2760, found 431.2736.

A sample of this nitroxide (10.0 mg, 0.0245 mmol) was dissolved in MeOH (0.3 mL) and L-ascorbic acid (4.4 mg, 0.0245

mmol) was added. The pale orange suspension became colorless within a few seconds. After stirring at rt for 30 min under

argon, the solvent was removed in vacuo. The resulting residue was dissolved in CH2Cl2 with some MeOH and washed

twice with water. The combined aqueous layers were extracted with warm CH2Cl2 (4x), and the combined organic layers

were dried (Na2SO4), filtered and concentrated in vacuo to yield 9.4 mg (94%) of the corresponding hydroxylamine as a

white powder: 1H NMR (CDCl3, 400 MHz, 3:1 mixture of isomers) δ 6.02-5.91 (m, 0.25 H), 5.82 (app bd, 0.75 H, J = 6.0

Hz), 5.71-5.58 (m, 1 H), 5.52-5.41 (m, 1 H), 4.84 (ddddd, 0.75 H, J = 11.6, 11.6, 6.8, 6.8, 6.8 Hz), 4.52 (app bs, 1 H), 4.23


S10

(app o, 0.25 H, J = 6.2 Hz), 4.09-3.97 (m, 1 H), 3.57 (app bd, 1.5 H, J = 9.6 Hz), 3.42 (app bd, 0.5 H, J = 10.0 Hz), 3.00-

2.91 (m, 2 H), 2.51-2.38 (m, 0.6 H), 2.38-2.19 (m, 2 H), 2.05-1.88 (m, 0.5 H), 1.80-1.57 (m, 3 H), 1.56-1.46 (m, 2 H), 1.44

(s, 9 H), 1.34 (app td, 2 H, J = 7.3, 1.8 Hz), 1.38-1.26 (m, 1 H), 1.14 (app bt, 2 H, J = 12.4 Hz), 0.93 (d, 3 H, J = 6.8 Hz),

0.92 (d, 3 H, J = 6.8 Hz); 13C NMR (CDCl3, 100 MHz, mixture of isomers) δ 170.6, 170.4, 155.6, 137.4, 137.3, 123.6,

123.5, 79.4, 55.9, 55.9, 54.1, 51.6, 51.5, 44.2, 40.3, 40.3, 39.7, 33.8, 33.8, 32.5, 31.2, 31.1, 29.9, 28.6, 24.8, 23.6, 22.9,

22.7, 14.3, 13.5, 12.8. CN

2-Adamantanecarbonitrile.5 A 3-5 °C solution of 2-adamantanone (21.0 g, 137 mmol), TosMIC (35.5 g, 178 mmol) and

EtOH (14 mL, 233 mmol) in DME (470 mL) was treated with portionwise addition of solid t-BuOK (39.2 g, 342 mmol),

maintaining the internal temperature below 10 °C. After the addition, the resulting slurry reaction mixture was stirred at rt

for 30 min and then at 35-40 °C for 30 min. The heterogeneous reaction mixture was filtered and the solid washed with

DME. The filtrate was concentrated in vacuo, loaded to a short alumina column (activated, neutral, Brockmann I, 150

mesh, 7 cm thick x 15 cm height), and washed off with a 5:1 mixture of hexanes/CH2Cl2 (~1.5 L). The solution was

concentrated in vacuo to afford 19.0 g (86%) of the title compound as a white powder: mp 170-174 °C (softening point:

~155 °C, sealed tube) (lit. 170-177 °C); IR (neat) 2903, 2851, 2229, 1468, 1450, 1355, 1342, 1239, 1111, 1098, 1073, 988,

976, 818, 807, 792 cm-1; 1H NMR (300 MHz, CDCl3) δ 2.91 (app s, 1 H), 2.23-2.08 (m, 4 H), 2.00-1.80 (m, 4 H), 1.80-

1.66 (m, 6 H); 13C NMR (75 MHz, CDCl3) δ 122.5, 37.2, 36.9, 36.8, 33.3, 30.6, 27.1, 27.0. CO2H

2-Adamantane carboxylic acid. A mixture of 2-adamantanecarbonitrile (18.9 g, 117 mmol) in AcOH (56 mL) and 48%

HBr (224 mL) was stirred at 120 °C overnight. The reaction mixture was cooled at 4 °C, let stand for 4 h, then filtered.

The solid was washed with water and dried under vacuum over silica gel overnight, to yield 20.6 g (98%) of the title

compound as colorless crystals: mp 145.5-145.8 °C (sealed tube) (lit.5 143-144 °C); IR (neat) 3200-2400 (br), 2922, 2915,

2894, 2849, 2630, 2950, 1681, 1468, 1452, 1439, 1418, 1351, 1342, 1332, 1314, 1301, 1277, 1249, 1226, 1176, 1099,

1064, 1049, 984, 941, 926, 911, 831 cm-1; 1H NMR (300 MHz, DMSO-d6) δ 12.09 (s, 1 H), 2.55-2.47 (m, 1 H), 2.20 (app

bs, 2 H), 1.87-1.64 (m, 10 H), 1.60-1.50 (m, 2 H).

CO2H

BrBr

5,7-Dibromo-2-adamantane carboxylic acid (19).6 A vigorously stirred 0 °C solution of AlBr3 (18.9 g, 69.6 mmol),

BBr3 (2.40 g, 9.49 mmol) and Br2 (40 mL) was treated portionwise with 2-adamantane carboxylic acid (5.70 g, 31.6

mmol). Upon completion of the addition, the reaction mixture was stirred at 70 °C for 48 h, then cooled in an ice bath, and

quenched carefully with sat. aq. sodium bisulfite. Stirring was continued at rt overnight. The resultant pale brown

suspension was filtered, the solid washed with water and dried overnight under vacuum at 60 °C to yield 10.95 g (quant.)

of crude 19 as a beige powder. Representative experimental data are as follows: mp 230-232 °C (sealed tube); IR (neat)

5 Oldenziel, O. H.; Wildeman, J.; van Leusen, A. M. Org. Synth. 1977, 57, 8. 6 Rohde, J. J.; Pliushchev, M. A.; Sorensen, B. K.; Wodka, D.; Shuai, Q.; Wang, J.; Fung, S.; Monzon, K. M.; Chiou, W. J.; Pan, L.; Deng, X.; Chovan, L. E.; Ramaiya, A.; Mullally, M.; Henry, R. F.; Stolarik, D. F.; Imade, H. M.; Marsh, K. C.; Beno, D. W. A.; Fey, T. A.; Droz, B. A.; Brune, M. E.; Camp, H. S.; Sham, H. L.; Frevert, E. U.; Jacobson, P. B.; Link, J. T. J. Med. Chem. 2007, 50, 149.


S11

3200-2400 (br), 2956, 2933, 2909, 2861, 1692, 1444, 1431, 1418, 1331, 1319, 1301, 1291, 1284, 1269, 1249, 1217, 1204,

1189, 1150, 1032, 1017, 995, 973, 945, 939, 928, 818, 803, 753, 745, 703, 663 cm-1; 1H NMR (300 MHz, DMSO-d6) δ

12.56 (bs, 1 H), 2.85 (app d, 2 H, J = 12.9 Hz), 2.75-2.55 (m, 2 H), 2.50-2.35 (m, 2 H), 2.35-2.10 (m, 7 H); LC-MS (ESI)

tR 20.4 min, m/z 337 ([M–H]–, 100).

NHBoc

BrBr

(5,7-Dibromo-adamantan-2-yl)-carbamic acid tert-butyl ester (20).6 The reaction was performed in 3 separated

batches. For each of them, a suspension of the acid 19 (7.80 g, 23.1 mmol) in dry toluene (117 mL) was treated

successively with Et3N (3.9 mL, 27.7 mmol) and DPPA (6.20 mL, 27.7 mmol). The resulting brown solution was stirred at

85 °C for 12 h. To a separated flask containing a solution of t-BuOK (5.28 g, 46.1 mmol) in dry THF (312 mL) at 0 °C

was added the resulting isocyanate solution dropwise via a dropping funnel. The reaction mixture was allowed to warm to

rt over 3 h, then quenched with water. The THF was removed in vacuo, and the resulting material was diluted with EtOAc.

The organic layer was washed with 1 N aq. HCl, sat. aq. NaHCO3 and brine, dried (Na2SO4), filtered and concentrated in

vacuo. The crude residue was sonicated in a minimum of anhydrous Et2O, filtered and washed with cold Et2O to yield 20

as a white powder. The 3 mother liquors were then combined and 2 additional filtrations afforded another fraction of 20 as

an off-white powder. Chromatography of the residual mother liquor on SiO2 (9:1, hexanes/EtOAc) afforded a white

powder contaminated with a yellow oil. This residue was taken up in hexanes, filtered, and the white powder washed with

hexanes. This resulting white powder was only an impurity. The mother liquor was concentrated in vacuo and again taken

up in a small amount of hexanes. The solvent was removed from the insoluble white powder with a Pasteur pipet, to

separate the product from the yellow oil. The white powder was dried to yield an additional fraction of 20. The combined

fractions afforded 21.6 g (77%) of 20 as a white powder. A sample of higher purity obtained after chromatography

(recovered starting material from the next step) was used for analysis: mp 187.5-189.5 °C; IR (neat) 3250, 3146, 3125,

3000, 2974, 2959, 2932, 2876, 2862, 1681, 1476, 1457, 1388, 1366, 1345, 1332, 1292, 1275, 1256, 1250, 1230, 1157,

1079, 1042, 1021, 999, 842, 835, 820, 803, 775, 734, 725, 680 cm-1; 1H NMR (300 MHz, CDCl3) δ 4.71 (bd, 1 H, J = 6.3

Hz), 3.75 (app bs, 1 H), 2.86 (s, 2 H), 2.47-2.10 (m, 10 H), 1.45 (s, 9 H); 13C NMR (75 MHz, CDCl3) δ 155.2, 80.2, 60.2,

59.7, 59.0, 51.2, 46.4, 41.5, 38.8, 28.6; EI-MS m/z 411 (M+, 9), 409 (M+, 15), 407 (M+, 8), 354 ([M–t-Bu]+, 67), 352 ([M–

t-Bu]+, 56), 350 ([M–t-Bu]+, 15), 274 ([M–t-Bu–Br]+, 100), 272 ([M–t-Bu–Br]+, 98), 230 ([M–Boc–Br]+, 48), 228 ([M–

Boc–Br]+, 51), 213 ([M–NHBoc–Br]+, 40), 211 ([M–NHBoc–Br]+, 39), 148 ([M–Boc–2Br]+, 41), 132 (54), 131 (76), 117

(43), 105 (50), 93 (52), 91 (80), 79 (65), 77 (68), 65 (37), 59 (79), 56 (87); HRMS (EI) m/z calcd for C15H24Br2NO2

408.0174, found 408.0154.

NHBoc

O (7-Methylene-bicyclo[3.3.1]nonan-3-one-9-yl)-carbamic acid tert-butyl ester (21). The reaction was performed in 3

separated batches. A mixture of 20 (3 x 2.46 g, 18.0 mmol) and 2 N aq. NaOH (3 x 13.5 mL, 81.0 mmol, degassed) in

dioxane (3 x 16.5 mL, degassed) was heated in a stainless steel Parr bomb at 160 °C (20 bar) under argon for 2 h. The

crude mixtures were combined and the dioxane was removed in vacuo. The aqueous residue (pH > 12) was extracted with

CH2Cl2 (3x). The combined organic layers were washed with brine, dried (Na2SO4), filtered and concentrated in vacuo to


S12

afford 3.90 g of crude 9-amino-7-methylene-bicyclo[3.3.1]nonan-3-one9 as a yellow solid, that was carried on without

further purification.

To a solution of this amine (2.98 g, 18.0 mmol, crude) in dry CH2Cl2 (270 mL) was added Et3N (7.60 mL, 54.0 mmol) and

then Boc2O (4.37 g, 19.8 mmol) at 0 °C. The reaction mixture was stirred at rt under nitrogen for 20 h, then quenched with

sat. aq. NH4Cl. The aqueous phase was extracted twice with CH2Cl2, and the combined organic layers were dried

(Na2SO4), filtered and concentrated in vacuo. The crude was taken up in CH2Cl2 but was poorly soluble. A filtration

afforded a white powder. The mother liquor was evaporated and taken up in a 7:3 hexanes/EtOAc mixture. After

sonicaton, the white powder was filtered. This procedure was repeated one more time. Chromatography of the resulting

mother liquor on SiO2 (9:1 to 6:4, hexanes/EtOAc) afforded 964 mg of another fraction as a pale yellow powder. Since the

3 fractions from filtration were impure and still contained Et3N, they were combined, dissolved in CHCl3 (50 mL), washed

with 5% aq. CuSO4 (2 x 20 mL), dried (Na2SO4), filtered and concentrated in vacuo. Chromatography of the residue on

SiO2 (9:1 to 6:4, hexanes/EtOAc) afforded 1.624 g of the major isomer as a white powder, and 2.59 g (42%, 4 steps) of 21

were obtained, while 509 mg (~7%) of starting material 20 (45% yld b.r.s.m.) was recovered. Representative experimental

data are as follows: mp 167.8-182.0 °C; IR (neat) 3271, 3131, 2971, 2930, 2849, 1685, 1478, 1452, 1437, 1407, 1385,

1364, 1351, 1316, 1250, 1239, 1215, 1161, 1142, 1109, 1073, 1064, 1044, 1017, 1008, 904, 857, 783, 773, 729, 710, 688,

682 cm-1; 1H NMR (300 MHz, CDCl3, 4:1 mixture of isomers) δ 4.93 (app bs, 0.2 H), 4.84 (s, 2 H), 4.74 (app bs, 0.8 H),

4.12 (app bs, 0.2 H), 3.96-3.87 (app m, 0.8 H), 2.65-2.12 (m, 10 H), 1.49 and 1.48 (2 s, 9 H); 13C NMR (75 MHz, CDCl3,

4:1 mixture of isomers) δ 209.2, 155.6, 139.7, 139.7, 116.5, 115.9, 80.2, 51.4, 50.5, 47.0, 42.3, 41.0, 35.9, 35.0, 34.4, 28.6;

ESI-MS m/z 288 ([M+Na]+, 100); HRMS (ESI) m/z calcd for C15H23NO3Na (M+Na) 288.1576, found 288.1567.

NHBoc

HON (7-Methylene-bicyclo[3.3.1]nonan-3-one oxime-9-yl)-carbamic acid tert-butyl ester (22). To a solution of ketone 21

(2.53 g, 9.53 mmol) in dry pyridine (18 mL) was added NH2OH•HCl (2.02 g, 28.6 mmol). The reaction mixture was

stirred at rt under argon for 12 h. The solvent was removed in vacuo, and the residue diluted with EtOAc and then water

was added. The layers were separated and the aqueous phase extracted with EtOAc. The combined organic layers were

washed with 5% aq. CuSO4 (8x), brine (2x), dried (Na2SO4), filtered and concentrated in vacuo. Chromatography of the

residue on SiO2 (5:5, hexanes/EtOAc) followed by trituration in Et2O afforded 2.41 g (90%) of 22 as a white powder: mp

60-86 °C; IR (neat) 3265 (br), 2971, 2924, 2833, 1690, 1515, 1508, 1498, 1450, 1437, 1388, 1364, 1249, 1161, 1077,

1062, 1040, 1012, 978, 965, 949, 921, 881, 777, 716 cm-1; 1H NMR (300 MHz, CDCl3, 4:1 mixture of isomers) δ 8.01 (bs,

1 H), 4.93 (app bs, 0.35 H), 4.90-4.70 (m, 0.65 H), 4.79 (s, 1 H), 4.68 (d, 1 H, J = 1.5 Hz), 3.86 (app bs, 1 H), 3.25 (d, 0.2

H, J = 15.9 Hz), 3.09 (d, 0.8 H, J = 16.5 Hz), 2.58-2.19 (m, 8 H), 2.19-1.93 (m, 1 H), 1.47 (s, 9 H); 13C NMR (75 MHz,

CDCl3, 4:1 mixture of isomers) δ 155.5, 141.1, 141.1, 113.2, 112.7, 80.0, 52.2, 51.4, 41.3, 40.5, 37.6, 35.7, 35.1, 34.2,

34.0, 32.6, 32.5, 31.6, 28.6, 24.9; ESI-MS m/z 281 ([M+H]+, 65), 266 (50), 225 ([M–t-Bu]+, 100); HRMS (ESI) m/z calcd

for C15H25N2O3 (M+H) 281.1865, found 281.1855.

NHBoc

HN

I


S13

(1-Iodomethyl-2-azaadamantan-6-yl)-carbamic acid tert-butyl ester (23). To a mixture of oxime 22 (2.35 g, 8.39

mmol) and MoO3 (1.70 g, 11.8 mmol) in dry MeOH (84 mL) at 0 °C under argon was added NaBH4 (3.24 g, 83.9 mmol)

portionwise. The resulting dark brown reaction mixture was stirred at 0-10 °C for 4 h, then quenched with acetone and

filtered through Celite. The Celite was rinsed with acetone, and the filtrate was concentrated in vacuo. The resulting

residue was diluted with water (100 mL) and EtOAc (200 mL), and filtered through Celite to break the emulsion. The

organic layer was separated, washed with a few brine, dried (K2CO3), filtered and concentrated in vacuo to afford ~2.5 g

of crude tert-butyl-3-amino-7-methylenebicyclo[3.3.1]nonan-9-ylcarbamate as a pale brown oily foam, that was carried on

without further purifcation.

To a suspension of this amine (2.23 g, 8.39 mmol, crude) in dry MeCN (42 mL) at 0 °C under argon was added I2 (2.13 g,

8.39 mmol). The reaction mixture was stirred at rt for 3 h and then quenched with sat. aq. NaHCO3 and sat. aq. Na2S2O3

(pH 8). The resulting yellow mixture was extracted with CHCl3 (3x), and the combined organic layers were dried

(K2CO3), filtered and concentrated in vacuo. Chromatography of the residue on SiO2 (EtOAc to 9:1, EtOAc/MeOH)

afforded 1.55 g (47%, 2 steps) of 23 as a dark orange powder: mp 125-138 °C; IR (neat) 3278 (br), 3269, 2967, 2918,

2868, 2853, 1701, 1670, 1541, 1448, 1444, 1431, 1388, 1362, 1351, 1295, 1280, 1269, 1250, 1217, 1200, 1167, 1118,

1086, 1077, 1045, 1012, 995, 891, 771 cm-1; 1H NMR (300 MHz, CDCl3, 3:1 mixture of isomers) δ 4.83 (app bs, 0.65 H),

4.75 (app bs, 0.35 H), 3.82-3.67 (m, 1 H), 3.24 (bs, 1 H), 3.19 (s, 1.5 H), 3.16 (s, 0.5 H), 2.20-2.07 (m, 1 H), 1.98-1.80 (m,

3 H), 1.80-1.60 (m, 6 H), 1.46 (s, 9 H); 13C NMR (75 MHz, CDCl3, 3:1 mixture of isomers) δ 155.5, 79.7, 53.7, 49.0, 47.4,

47.2, 40.9, 36.1, 35.8, 32.4, 31.7, 30.8, 28.6, 23.7, 23.2; EI-MS m/z 392 (M+, 18), 265 ([M–I]+, 68), 209 ([M–I–t-Bu]+, 88),

148 (100), 94 (80); HRMS (ESI) m/z calcd for C15H25IN2O2 392.0961, found 392.0958.

NHBoc

NO

(1-Methyl-2-azaadamantane-N-oxyl-6-yl)-carbamic acid tert-butyl ester (6-Boc-amino-1-Me-AZADO, 24). The

reaction was performed in 2 separated batches. A Parr flask was charged with the iodoalkane 23 (2 x 414 mg, 2.11 mmol),

EtOH (2 x 10 mL) and 20% Pd(OH)2/C (2 x 82 mg). The flask was placed in a high pressure reactor. The reactor was

charged with H2 and purged for 5 cycles and finally pressurized with H2 at 4 bar (60 psi), then heated at 50 °C. After

stirring at 50 °C for 26 h, the reaction mixtures were combined and filtered through Celite, the Celite washed with MeOH,

and the solution concentrated in vacuo to yield a dark reddish powder. The crude was dissolved in CH2Cl2 and washed

with sat. aq. Na2CO3, dried (K2CO3), filtered and concentrated in vacuo to afford 556 mg (99%) of crude (1-methyl-2-

azaadamantan-6-yl)-carbamic acid tert-butyl ester as a yellow solid, that was carried on without further purification.

A mixture of this amine (554 mg, 2.08 mmol, crude) and Na2WO4•2H2O (347 mg, 1.04 mmol) in MeOH (6 mL) was

stirred at rt for 20 min then cooled to 0 °C and treated with 30% aq. H2O2 (2.36 mL, 20.8 mmol). The resulting reaction

mixture was stirred at 0 °C for 30 min then allowed to warm to rt. After 4 h, the reaction mixture was cooled to 0 °C and

treated again with H2O2 (0.70 mL, 6.24 mmol), then allowed to warm to rt. After an additional 6 h stirring at rt, the bright

orange reaction mixture was quenched with sat. aq. NaHCO3 and extracted with CH2Cl2 (3x). The combined organic

layers were dried (Na2SO4), filtered and concentrated in vacuo. Chromatograpy of the residue on SiO2 (7:3 to 5:5,

hexanes/EtOAc) afforded 345 mg (59%, 58% over 2 steps) of 24 as a red solid. Representative experimental data are as

follows: mp 140-150 °C (softening point: 131 °C); IR (neat) 3265, 3138, 2999, 2969, 2922, 1688, 1474, 1448, 1383, 1362,

1286, 1275, 1252, 1163, 1137, 1079, 1066, 1045, 1008, 984, 958, 777, 727, 708 cm-1; ESI-MS m/z 284 (70), 282 ([M+H]+,

100); HRMS (ESI) m/z calcd for C15H26N2O3 (M+H) 282.1943, found 282.1941.


S14

NH2

NO

6-Amino-1-methyl-2-azaadamantane-N-oxyl (6-amino-1-Me-AZADO, 8). A solution of 6-Boc-amino-1-Me-AZADO

(335 mg, 1.19 mmol) in a 4.0 N solution of HCl in 1,4-dioxane (5.0 mL, 20.0 mmol) was stirred at 0 °C for 1 h and at rt

for an additional 2 h. Dioxane was removed in vacuo and the yellow residue was dissolved in 2.5 N aq. NaOH and

extracted with CH2Cl2 until the organic layer became colorless. The combined organic layers were dried (K2CO3), filtered

and concentrated in vacuo to afford 230 mg (quant.) of crude 8 as a red oil: LC-MS (ESI) tR 7.71 min, m/z 183

([Mred+H]+), 166 ([M–O+H]+).

NH2

HN 6-Amino-1-methyl-2-azaadamantane (26). The reaction was performed in 2 separate batches. A Parr flask was charged

with the iodoalkane 23 (2 x 494 mg, 2.52 mmol), EtOH (2 x 12 mL) and 20% Pd(OH)2/C (2 x 99.0 mg). The flask was

placed in a high pressure reactor. The reactor was charged with H2 and purged for 5 cycles and finally pressurized with H2

at 4 bar (60 psi), then heated at 50 °C. After stirring at 50 °C for 22 h, the reaction mixtures were combined and filtered

through Celite, the Celite washed with MeOH and EtOAc, and the solution concentrated in vacuo. The crude was

dissolved in CH2Cl2 and washed with sat. aq. Na2CO3, dried (K2CO3), filtered and concentrated in vacuo to afford 638 mg

(95%) of crude (1-methyl-2-azaadamantan-6-yl)-carbamic acid tert-butyl ester as a dark grey foam, that was carried on

without further purification.

A mixture of (1-methyl-2-azaadamantan-6-yl)-carbamic acid tert-butyl ester (180 mg, 0.676 mmol, crude) in a 4.0 N

solution of HCl in 1,4-dioxane (2.85 mL, 11.4 mmol) was stirred at 0 °C for 1 h and at rt for an additional 2.5 h. Dioxane

was removed in vacuo and the residue was dissolved in 2.5 N aq. NaOH and extracted with CHCl3 (3x). The combined

organic layers were dried (K2CO3), filtered and concentrated in vacuo to afford 113 mg (quant.) of crude 26 as a dark

brown oil, that was carried on without further purification.

NHBoc

NH

ON

O

(S,E)-N-(1-Methyl-2-azaadamant-2-oxo-6-yl)-5-(tert-butoxycarbonylamino)-7-methyloct-3-enamide (25). To a

solution of alcohol 10 (180 mg, 0.699 mmol) in acetone (8 mL) at 0 °C was slowly added a solution of Jones reagent (2.5

M, 0.70 mL, 1.75 mmol). The resulting dark suspension was stirred at 0 °C for 1 h, then diluted with Et2O and water. The

aqueous phase was separated and extracted with Et2O (2x). The combined organic layers were washed with water (2x) and

brine (1x), dried (Na2SO4), filtered and concentrated in vacuo to yield 189 mg (quant.) of the crude acid (11) as a colorless

oil, that was carried on without further purification.

To a solution of this acid (58.7 mg, 0.216 mmol, crude) in dry CH2Cl2 (2.0 mL) at 0 °C were added successively

HOBt•H2O (35.1 mg, 0.260 mmol), DMAP (29.4 mg, 0.238 mmol), a solution of amine 26 (40.0 mg, 0.216 mmol) in dry

CH2Cl2 (0.7 mL), and EDCI (50.0 mg, 0.260 mmol). The resulting mixture was stirred at rt under argon for 15 h, then the

mixture was diluted with CH2Cl2, washed twice with 1 N aq. NaOH, dried (K2CO3), filtered and concentrated in vacuo.

Chromatography of the residue on deactivated SiO2 (EtOAc then 9:1, EtOAc/MeOH + 1% NH4OH) afforded 54.0 mg


S15

(59%) of tert-butyl (S,E)-8-(1-methyl-2-azaadamant-6-ylamino)-2-methyl-8-oxooct-5-en-4-ylcarbamate as a pale yellow

foam.

To a mixture of this amine (54.0 mg, 0.129 mmol) in 1:1 MeCN/MeOH (0.6 mL) was added Na2WO4•2H2O (21.4 mg,

0.0643 mmol), and the resulting mixture was stirred at rt for 20 min. After cooling to 0 °C, UHP (100 mg, 1.03 mmol) was

added and the reaction mixture was stirred under air at 0 °C for 1 h then at rt for 12 h. Water was then added, and the

resulting aqueous mixture was extracted with CHCl3 (2x). The combined organic layers were dried (K2CO3), filtered and

concentrated in vacuo. Flash chromatography of the residue on SiO2 (5:5, hexanes/EtOAc to EtOAc) afforded 35.4 mg

(63%) of 25, 8.6 mg as red crystals (suitable for X-ray) and 26.8 mg as a red-orange foam: mp 154-156 °C (EtOAc); [α]D23

+6.2 (c 1.0, CH2Cl2); IR (neat) 3308 (br), 2948, 2928, 2868, 1677, 1663, 1651, 1646, 1523, 1450, 1387, 1362, 1336, 1325,

1312, 1284, 1245, 1167, 1133, 1116, 1066, 1042, 1023, 1003, 969, 951 cm-1; ESI-MS m/z 457 ([M+Na]+, 63), 401 (51),

318 (33); HRMS (ESI) m/z calcd for C23H42N3O4Na (M+Na) 457.2917, found 457.2884.

A sample of this nitroxide (4.0 mg, 0.00920 mmol) was dissolved in dry MeOH (0.2 mL) and L-ascorbic acid (1.6 mg,

0.00920 mmol) was added. Complete discoloration of the solution occured immediately. After stirring at rt for 15 min, the

solvent was removed in vacuo. The resulting residue was dissolved in CHCl3 and washed with water. The aqueous phase

was extracted once with CHCl3, and the combined organic layers were dried (Na2SO4), filtered and concentrated in vacuo

to yield 5.2 mg (>100%) of the corresponding hydroxylamine as a colorless solidified oil: 1H NMR (400 MHz, CDCl3) δ

6.14 (bs, 1 H), 5.69 (dt, 1 H, J = 14.9, 7.3 Hz), 5.53 (dd, 1 H, J = 15.6, 5.6 Hz), 4.46 (app bs, 1 H), 4.14-4.03 (m, 1 H),

4.03-3.96 (m, 1 H), 3.27 (app bs, 1 H), 3.02 (d, 2 H, J = 7.2 Hz), 2.36-2.20 (m, 2 H), 2.07-1.70 (m, 8 H), 1.70-1.55 (m, 1

H), 1.43 (2 s, 9 H), 1.40-1.20 (m, 2 H), 1.11 (s, 3 H), 0.93 (app d, 6 H, J = 6.4 Hz); 13C NMR (100 MHz, CDCl3) δ 170.5,

155.4, 137.8, 122.8, 79.5, 57.0, 56.3, 52.4, 51.1, 44.4, 42.9, 42.8, 40.4, 35.1, 35.0, 32.1, 31.2, 31.1, 30.7, 30.5, 30.5, 29.9,

29.8, 29.5, 28.6, 29.6, 26.4, 24.9, 23.3, 23.3, 22.9, 22.8, 22.6, 14.3.

NHBoc

O

O

(S,E)-Methyl 5-((tert-butoxycarbonyl)amino)-7-methyloct-3-enoate (27). To a solution of the acid 11, (142 mg, 0.523

mmol, crude) in acetonitrile (8.7 mL, 0.06 M) was added K2CO3 (362 mg, 5.23 mmol), followed by MeI (326 µL, 5.233

mmol). The resulting reaction mixture was stirred at rt under N2 in the dark for 11 h, then quenched with H2O (5 mL) and

extracted with CH2Cl2 (3 x 20 mL). The combined organic portion was washed with brine (5 mL), dried (Na2SO4),

filtered, and concentrated. Chromatography on SiO2 (ISCO, 12 g column, liquid load in CH2Cl2, 0-100% EtOAc/hexanes

gradient, eluted at 50% EtOAc/hexanes) afforded 27 as a colorless oil (159 mg, quant.): [α]D20 –13.0 (c 1.0, CH2Cl2); IR

(CH2Cl2) 3351 (br), 2952, 2932, 2867, 1737, 1692, 1512, 1452, 1435, 1389, 1364, 1327, 1245, 1159, 1118, 1081, 1042,

1016, 967 cm-1; 1H NMR (400 MHz, CDCl3) δ 5.69 (dtd, 1 H, J = 15.5, 6.9, 1.2 Hz), 5.49 (dd, 1 H, J = 15.2, 4.9 Hz), 4.39

(bs, 1 H), 4.23-4.00 (m, 1 H), 3.68 (s, 3 H), 3.07 (d, 2 H, J = 6.9 Hz), 1.72-1.59 (m, 1 H), 1.44 (s, 9 H), 1.39-1.25 (m, 2 H),

0.92 (d, 3 H, J = 6.6 Hz), 0.91 (d, 3 H, J = 6.6 Hz); 13C NMR (100 MHz, CDCl3) δ 172.3, 155.5, 135.6, 122.0, 79.5, 52.0,

50.4, 44.8, 37.7, 28.6, 24.9, 22.9, 22.6; HRMS (ESI) m/z calcd for C18H27N3O4Na (M+Na)+ 308.1838, found 308.1860.


S16

O

OHN O

NNBFF

(S,E)-5,5-Difluoro-3-(3-((8-methoxy-2-methyl-8-oxooct-5-en-4-yl)amino)-3-oxopropyl)-7,9-dimethyl-5H-

dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-4-ium-5-uide (29). To a solution of ester 27 (150 mg, 0.526 mmol) in

CH2Cl2 (8 mL) at 0 °C was added TFA (0.4 mL, 5.26 mmol). The resulting colorless solution was stirred at rt. TLC

analysis showed that the starting material had been consumed after 4 h. The reaction mixture was concentrated, and the

residue was taken up CH2Cl2 and washed with 5% aq. Na2CO3 (pH 9). The aqueous layer was extracted once with CH2Cl2,

and the combined organic portion was dried (K2CO3), filtered, and concentrated to afford 92.2 mg (95%, crude) of amine

as a pale brownish oil, that was carried on without further purification.

A solution of activated ester 28 (BODIPY-FL-NHS, 224 mg, 0.576 mmol) and DIEPA (0.26 mL, 1.49 mmol) in dry

CH2Cl2 (15 mL) was treated with a solution of amine (89.0 mg, 0.480 mmol, crude) in dry CH2Cl2 (5 mL). The resulting

red solution was stirred at rt under N2 for 9 h, then washed with water. The aqueous phase was extracted with CH2Cl2 until

it became almost colorless. The combined organic portion was dried (Na2SO4), filtered, and concentrated.

Chromatography on SiO2 (75:25 to 6:4 to 5:5, hexanes/EtOAc) afforded 180 mg (82%) of 29 as a red, viscous oil that

solidified to give a red crystalline solid: 1H NMR (400 MHz, CDCl3) δ 7.09 (s, 1 H), 6.88 (d, 1 H, J = 4.0 Hz), 6.30 (d, 1

H, J = 4.0 Hz), 6.13 (s, 1 H), 5.58-5.62 (m, 2 H), 5.42 (dd, 1 H, J = 15, 6.0 Hz), 4.48 (dt, 1 H, J = 15, 7.3, 7.3 Hz), 3.67 (s,

3 H), 3.28 (t, 2 H, J = 7.3 Hz), 3.01 (d, 2 H, J = 6.9 Hz), 2.66 (t, 2 H, J = 7.3 Hz), 2.57 (s, 3 H), 2.26 (s, 3 H), 1.44 (m,

1H), 1.31 (ddd, 1 H, J = 7.2, 6.4, 2.4 Hz), 1.26 (ddd, 1 H, J = 7.2, 6.4, 1.6 Hz), 0.85 (dd, 6 H, J = 7.8, 6.6 Hz); 13C NMR

(100 MHz, CDCl3) δ 172.2, 171.0, 160.4, 157.6, 144.1, 135.3, 135.0, 133.6, 128.5, 124.0, 122.1, 120.6, 117.9, 52.0, 48.8,

44.2, 37.8, 36.3, 25.1, 24.8, 23.0, 22.4, 15.1, 11.5; HRMS (APCI) m/z calcd for C24H32N3O3F2BNa (M+Na)+ 482.2402,

found 482.2446.

NH

OHN NO

O

NNBFF

(S,E)-5,5-Difluoro-3-(3-((8-((1-oxo-2,2,6,6-tetramethylpiperidin-4-yl)amino)-2-methyl-8-oxooct-5-en-4-yl)amino)-3-

oxopropyl)-7,9-dimethyl-5H-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-4-ium-5-uide (30). To a vigorously stirred

mixture of ester 29 (34.5 mg, 0.0751 mmol) and pig liver esterase (PLE, 17 U/mg, 7 mg) in acetone (1.5 mL) was added

phosphate buffer (0.05 M, pH 7.0, 2.75 mL). The resulting heterogenous reaction mixture was vigorously stirred at rt

overnight. TLC analysis (1:1 EtOAc:hexanes, KMnO4) after 13 h showed only starting material. More PLE (7 mg) was

added. The reaction mixture was allowed to stir for 10 d, and additional PLE was added after 3, 4, 5, and 7 d. After 10 d,

TLC analysis showed completed consumption of starting material, and the reaction mixture was concentrated. Citric acid

(~pH 3) was added and the reaction mixture was extracted with CH2Cl2 (3 x 10 mL). The combined organic portion was


S17

dried (Na2SO4), filtered, and concentrated to give the crude, red solid (34.6 mg, quant). This solid was carried on to the

next reaction without purification.

To the resulting acid (34.6 mg, 0.0777 mmol) in CH2Cl2 (1 mL) at 0 °C in the dark was added 4-amino TEMPO (20.0 mg,

0.117 mmol) in CH2Cl2 (1 mL) followed by DMAP (10.4 mg, 0.0855 mmol), HOBt-H2O (13.1 mg, 0.0855 mmol), and

EDCI (17.9 mg, 0.0932 mmol). The reaction mixture was allowed to warm to rt as it stirred overnight. The reaction

mixture was washed with H2O (3 mL) and sat. NH4Cl (3 mL), and the aqueous portion was extracted with CH2Cl2 (2 x 15

mL). The combined organic portion was dried (Na2SO4), filtered, and concentrated to give a crude, red oil (63 mg). The

crude material was purified by chromatography on SiO2 (ISCO, 4 g column, liquid load in CH2Cl2, 0-20% MeOH/CH2Cl2

gradient, eluted at 5% MeOH/CH2Cl2) to give a crude, impure orange oil (41 mg). The oil was then purified by

chromatography on SiO2 (ISCO, 4 g column, liquid load in CH2Cl2, 10-100% EtOAc/hexanes gradient, eluted at 100%

EtOAc) to provide 30 as a reddish orange oil (26 mg, 56%): Rf = 0.41 (10% MeOH/CH2Cl2); IR (ATR) 3288, 2954, 2926,

1642, 1605, 1528, 1249, 1133, 1084, 999 cm-1; 13C NMR (100 MHz, CDCl3, δ) 169.6, 168.5, 158.8, 154.3, 142.2, 134.8,

133.2, 131.2, 126.2, 121.9, 121.7, 118.8, 115.5, 48.8, 41.2, 34.5, 31.6, 27.5, 23.3, 22.4, 20.5, 2.3, 13.9, 9.5; HRMS (ESI)

m/z calcd for C32H47BN5O3F2Na (M+Na)+ 621.3638, found 621.3589.

A sample of this nitroxide (14.4 mg, 0.0241 mmol) in MeOH (1 mL) was added L-(+)-ascorbic acid (6.4 mg, 0.036

mmol). After stirring at rt for 2 h, the reaction mixture was concentrated. The resulting residue was dissolved in CH2Cl2

and washed once with water. The aqueous layers was extracted once with CH2Cl2, and the combined organic portion was

washed with brine, dried (Na2SO4), filtered, and concentrated in vacuo to yield the corresponding hydroxylamine as a red

oil (12.2 mg, 85%): 1H NMR (400 MHz, CDCl3) δ 7.10 (s, 1H), 6.87 (d, 1 H, J = 3.9 Hz), 6.64 (d, 1 H J = 7.2 Hz), 6.32

(d, 1 H, J = 3.9 Hz), 6.13 (s, 1 H), 6.03 (d, 1 H, J = 6.3 Hz), 5.54 (dt, 1 H, J = 15, 7.5, 7.5 Hz), 5.35 (dd, 1 H, J = 15, 6.6

Hz), 4.28 (m, 2 H), 3.25 (t, 2 H, 7.4 Hz), 2.89 (dd, 1 H, J = 7.2, 2.1 Hz), 2.69 (t, 1 H, J = 7.4 Hz), 2.55 (s, 3 H), 2.26 (s, 3

H), 2.04 (br s, 1 H), 1.50 (d, 4 H, J = 4.8 Hz), 1.28-1.30 (m, 3 H), 1.25 (s, 12 H), 0.84 (t, 6 H, J = 5.8 Hz).

NH

OHN O

NNBPh FF

NO

(S,E)-5,5-Difluoro-3-(3-((8-((1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl)amino)-2-methyl-8-oxooct-5-en-4-yl)am-

ino)-3-oxopropyl)-7-phenyl-5H-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-4-ium-5-uide (BODIPY®-R6G-JP4-039,

33). To a solution of JP4-039 (125 mg, 0.294 mmol) in MeOH (3 mL) was added ascorbic acid (65 mg, 0.37 mmol). The

orange solution became homogenous in less than 1 min. Stirring was continued 30 min, and the reaction mixture was

concentrated. To the residue was added ethyl acetate (10 mL), water (5 mL), and 5% Na2CO3 (5 mL)). The layers were

separated, and the aqueous layer was extracted with EtOAc. The combined organic portion was dried (MgSO4) and

evaporated, and the crude hydroxylamine was carried on to the next reaction.

To a solution of the crude hydroxylamine in CH2Cl2 (3 mL) at 0 °C was added trifluoroacetic acid (440 µL, 5.89 mmol).

The solution was warmed to rt and stirred 4 h. The solvent and excess acid were evaporated, and the residue was

dissolved in EtOAc (25 mL). The organic portion was washed with saturated Na2CO3 (2 x 5 mL) and brine (5 mL), dried

over MgSO4, and cocentrated to give the free amine as a white solid, which was carried on to the next reaction.


S18

To BODIPY®-R6G-NHS (31) (50.0 mg, 0.114 mmol) was added a solution of the crude amine in CH2Cl2 (2 mL),

followed by diisopropylethylamine (36 µL, 0.21 mmol). The resulting red solution was stirred at rt for 18 h, and the

reaction mixture was concentrated. The residue was dissolved in MeOH (2 mL), and catalytic copper (II) acetate hydrate

was added. The mixture was stirred open to air for 30 min, then poured into water (10 mL). The aqueous mixture was

extracted with EtOAc, and the combined organics were dried over MgSO4 and evaporated. The resulting residue was

purified by chromatography on SiO2 (1-5% MeOH/CH2Cl2) to provide BODIPY-R6G JP4-039 (33) (46 mg, 62%) as a red

semisolid: IR (ATR) 3303, 2958, 2926, 2855, 1653, 1616, 1540, 1463, 1245, 1137, 1085 cm-1; HRMS (ESI) [M+H]+ calcd

for C36H48BF2N5O3 647.3813, found 647.3802. NMR spectra are severely broadened and not useful, so the compound was

characterized as the hydroxylamine after reduction with ascorbic acid in methanol.

Analytical data for hydroxylamine 32: 1H NMR (600 MHz, CDCl3 layered with 5% ascorbic acid in D2O) δ 7.89 (dd, 2

H, J = 7.2, 1.2 Hz), 7.47 (m, 3 H), 7.25 (s, 1 H), 7.13 (d, 1 H, J = 4.2 Hz), 7.03 (d, 1 H, J = 4.2 Hz), 6.65 (d, 1 H, J = 4.2

Hz), 6.41 (d, 1 H, J = 3.6 Hz), 5.53 (m, 1H), 5.31 (dd, 1 H, J = 15.3, 6.9 Hz), 4.20 (m, 2 H), 3.27 (dd, 2 H, J = 6.9, 5.1

Hz), 2.90 (m, 2 H), 2.64 (q, 2 H, J = 6.8 Hz), 1.89 (m, 2 H), 1.47 (m, 1 H), 1.32–1.22 (m, 17 H), 0.83 (dd, 6 H, J = 8.4,

3.6 Hz,); 13C NMR (150 MHz, CDCl3 layered with 5% ascorbic acid in D2O) δ 171.7, 170.5, 161.4, 136.7, 135.1, 132.5,

130.9, 130.0, 129.4, 128.5, 123.8, 12.6, 120.2, 101.3, 56.1, 50.6, 43.3, 40.2, 40.0, 35.9, 29.8, 35.2, 24.6, 22.6, 22.4, 20.4;

HRMS (ESI) [M+H]+ calcd for C36H49BF2N5O3 648.3891, found 648.3896.


S19

X-Ray Crystallography Data for Compound 25.

Table S1. Crystal data and structure refinement for 25.

Identification code mf29229s

Empirical formula C24 H40 N3 O4

Formula weight 434.59

Temperature 223(2) K

Wavelength 0.71073 Å

Crystal system Orthorhombic

Space group P 21 21 21

Unit cell dimensions a = 6.8433(18) Å α= 90°.

b = 18.424(5) Å β= 90°.

c = 20.221(5) Å γ = 90°.

Volume 2549.5(12) Å3

Z 4

Density (calculated) 1.132 Mg/m3

Absorption coefficient 0.077 mm-1

F(000) 948

Crystal size 0.27 x 0.25 x 0.18 mm3

Theta range for data collection 2.21 to 24.99°

Index ranges -8<=h<=8, -21<=k<=21, -24<=l<=24

Reflections collected 20281

Independent reflections 2581 [R(int) = 0.0592]

Completeness to theta = 24.99° 99.9 %

Absorption correction Semi-empirical from equivalents

Max. and min. transmission 0.9863 and 0.9796

Refinement method Full-matrix least-squares on F2

Data / restraints / parameters 2581 / 0 / 288

Goodness-of-fit on F2 1.100

Final R indices [I>2sigma(I)] R1 = 0.0484, wR2 = 0.1258

R indices (all data) R1 = 0.0707, wR2 = 0.1371

Absolute structure parameter ?

Largest diff. peak and hole 0.183 and -0.147 e.Å-3


S20

Table S2. Atomic coordinates (x 104) and equivalent isotropic displacement parameters (Å2x 103)

for MCF292-29 (25). U(eq) is defined as one third of the trace of the orthogonalized Uij tensor.

________________________________________________________________________________

x y z U(eq)

________________________________________________________________________________

O(1) 9677(4) 7238(1) 840(1) 61(1)

N(1) 7414(5) 6362(2) 920(2) 70(1)

C(1) 14877(9) 6353(3) -2903(2) 110(2)

N(2) 3320(5) 5820(2) 2602(2) 72(1)

O(2) 1622(4) 5793(2) 2844(1) 84(1)

C(2) 16937(7) 6357(3) -1884(2) 91(2)

O(3) 15174(4) 4187(1) -520(1) 62(1)

N(3) 14139(4) 5171(2) -1022(1) 54(1)

C(3) 15166(6) 6026(2) -2215(2) 69(1)

O(4) 15157(4) 5257(1) 40(1) 62(1)

C(4) 13312(6) 6131(2) -1813(2) 57(1)

C(5) 13401(5) 5912(2) -1085(2) 49(1)

C(6) 11417(5) 5982(2) -783(2) 52(1)

C(7) 10828(5) 6512(2) -393(2) 50(1)

C(8) 8781(5) 6604(2) -149(2) 54(1)

C(9) 8693(5) 6765(2) 582(2) 49(1)

C(10) 6973(6) 6477(3) 1624(2) 72(1)

C(11) 4995(10) 6830(2) 1712(2) 87(2)

C(12) 4680(11) 7011(2) 2443(2) 103(2)

C(13) 4778(6) 6333(2) 2856(2) 64(1)

C(14) 7051(6) 5790(3) 2022(2) 79(1)

C(15) 6743(7) 5968(4) 2753(2) 94(2)

C(16) 5536(9) 5285(2) 1808(2) 91(2)

C(17) 3594(7) 5605(3) 1908(2) 86(2)

C(18) 3405(7) 6296(4) 1504(2) 109(2)

C(19) 4341(8) 6473(3) 3587(2) 101(2)

C(20) 14845(5) 4906(2) -456(2) 50(1)

C(21) 15660(6) 3735(2) 43(2) 62(1)

C(22) 17630(7) 3947(3) 320(2) 91(2)

C(23) 14055(9) 3773(2) 560(2) 99(2)

C(24) 15751(8) 2982(2) -266(2) 84(1)

________________________________________________________________________________


S21

Table S3. Bond lengths [Å] and angles [°] for MCF292-29 (25).

_____________________________________________________

O(1)-C(9) 1.219(4)

N(1)-C(9) 1.336(4)

N(1)-C(10) 1.471(4)

N(1)-H(1N) 0.93(4)

C(1)-C(3) 1.528(5)

C(1)-H(1A) 0.9700

C(1)-H(1B) 0.9700

C(1)-H(1C) 0.9700

N(2)-O(2) 1.262(4)

N(2)-C(13) 1.466(5)

N(2)-C(17) 1.471(5)

C(2)-C(3) 1.512(7)

C(2)-H(2A) 0.9700

C(2)-H(2B) 0.9700

C(2)-H(2C) 0.9700

O(3)-C(20) 1.349(4)

O(3)-C(21) 1.451(4)

N(3)-C(20) 1.334(4)

N(3)-C(5) 1.462(4)

N(3)-H(3N) 0.91(3)

C(3)-C(4) 1.520(6)

C(3)-H(3A) 0.9900

O(4)-C(20) 1.214(4)

C(4)-C(5) 1.526(5)

C(4)-H(4A) 0.9800

C(4)-H(4B) 0.9800

C(5)-C(6) 1.495(5)

C(5)-H(5A) 0.9900

C(6)-C(7) 1.318(4)

C(6)-H(6A) 0.9400

C(7)-C(8) 1.495(5)

C(7)-H(7A) 0.9400

C(8)-C(9) 1.507(5)

C(8)-H(8A) 0.9800

C(8)-H(8B) 0.9800

C(10)-C(14) 1.501(6)


S22

C(10)-C(11) 1.512(7)

C(10)-H(10A) 0.9900

C(11)-C(18) 1.526(7)

C(11)-C(12) 1.531(6)

C(11)-H(11A) 0.9900

C(12)-C(13) 1.504(6)

C(12)-H(12A) 0.9800

C(12)-H(12B) 0.9800

C(13)-C(15) 1.517(7)

C(13)-C(19) 1.529(5)

C(14)-C(16) 1.458(7)

C(14)-C(15) 1.529(6)

C(14)-H(14A) 0.9900

C(15)-H(15A) 0.9800

C(15)-H(15B) 0.9800

C(16)-C(17) 1.468(7)

C(16)-H(16A) 0.9800

C(16)-H(16B) 0.9800

C(17)-C(18) 1.517(7)

C(17)-H(17A) 0.9900

C(18)-H(18A) 0.9800

C(18)-H(18B) 0.9800

C(19)-H(19A) 0.9700

C(19)-H(19B) 0.9700

C(19)-H(19C) 0.9700

C(21)-C(22) 1.511(6)

C(21)-C(24) 1.522(5)

C(21)-C(23) 1.517(6)

C(22)-H(22A) 0.9700

C(22)-H(22B) 0.9700

C(22)-H(22C) 0.9700

C(23)-H(23A) 0.9700

C(23)-H(23B) 0.9700

C(23)-H(23C) 0.9700

C(24)-H(24A) 0.9700

C(24)-H(24B) 0.9700

C(24)-H(24C) 0.9700


S23

C(9)-N(1)-C(10) 123.3(3)

C(9)-N(1)-H(1N) 121(2)

C(10)-N(1)-H(1N) 116(2)

C(3)-C(1)-H(1A) 109.5

C(3)-C(1)-H(1B) 109.5

H(1A)-C(1)-H(1B) 109.5

C(3)-C(1)-H(1C) 109.5

H(1A)-C(1)-H(1C) 109.5

H(1B)-C(1)-H(1C) 109.5

O(2)-N(2)-C(13) 121.1(3)

O(2)-N(2)-C(17) 118.5(3)

C(13)-N(2)-C(17) 114.9(3)

C(3)-C(2)-H(2A) 109.5

C(3)-C(2)-H(2B) 109.5

H(2A)-C(2)-H(2B) 109.5

C(3)-C(2)-H(2C) 109.5

H(2A)-C(2)-H(2C) 109.5

H(2B)-C(2)-H(2C) 109.5

C(20)-O(3)-C(21) 121.8(2)

C(20)-N(3)-C(5) 122.8(3)

C(20)-N(3)-H(3N) 116.1(19)

C(5)-N(3)-H(3N) 119.4(19)

C(2)-C(3)-C(4) 112.4(3)

C(2)-C(3)-C(1) 110.4(4)

C(4)-C(3)-C(1) 109.2(4)

C(2)-C(3)-H(3A) 108.3

C(4)-C(3)-H(3A) 108.3

C(1)-C(3)-H(3A) 108.3

C(3)-C(4)-C(5) 116.7(3)

C(3)-C(4)-H(4A) 108.1

C(5)-C(4)-H(4A) 108.1

C(3)-C(4)-H(4B) 108.1

C(5)-C(4)-H(4B) 108.1

H(4A)-C(4)-H(4B) 107.3

N(3)-C(5)-C(6) 111.0(3)

N(3)-C(5)-C(4) 110.2(3)

C(6)-C(5)-C(4) 109.6(3)

N(3)-C(5)-H(5A) 108.7


S24

C(6)-C(5)-H(5A) 108.7

C(4)-C(5)-H(5A) 108.7

C(7)-C(6)-C(5) 125.9(3)

C(7)-C(6)-H(6A) 117.0

C(5)-C(6)-H(6A) 117.0

C(6)-C(7)-C(8) 124.6(3)

C(6)-C(7)-H(7A) 117.7

C(8)-C(7)-H(7A) 117.7

C(7)-C(8)-C(9) 112.6(3)

C(7)-C(8)-H(8A) 109.1

C(9)-C(8)-H(8A) 109.1

C(7)-C(8)-H(8B) 109.1

C(9)-C(8)-H(8B) 109.1

H(8A)-C(8)-H(8B) 107.8

O(1)-C(9)-N(1) 122.7(3)

O(1)-C(9)-C(8) 122.6(3)

N(1)-C(9)-C(8) 114.7(3)

N(1)-C(10)-C(14) 112.9(4)

N(1)-C(10)-C(11) 111.1(4)

C(14)-C(10)-C(11) 109.3(3)

N(1)-C(10)-H(10A) 107.8

C(14)-C(10)-H(10A) 107.8

C(11)-C(10)-H(10A) 107.8

C(10)-C(11)-C(18) 109.2(3)

C(10)-C(11)-C(12) 109.5(5)

C(18)-C(11)-C(12) 107.8(4)

C(10)-C(11)-H(11A) 110.1

C(18)-C(11)-H(11A) 110.1

C(12)-C(11)-H(11A) 110.1

C(13)-C(12)-C(11) 110.5(3)

C(13)-C(12)-H(12A) 109.6

C(11)-C(12)-H(12A) 109.6

C(13)-C(12)-H(12B) 109.6

C(11)-C(12)-H(12B) 109.6

H(12A)-C(12)-H(12B) 108.1

N(2)-C(13)-C(12) 108.1(4)

N(2)-C(13)-C(15) 105.7(4)

C(12)-C(13)-C(15) 109.3(4)


S25

N(2)-C(13)-C(19) 108.3(3)

C(12)-C(13)-C(19) 112.8(4)

C(15)-C(13)-C(19) 112.4(4)

C(16)-C(14)-C(10) 110.7(4)

C(16)-C(14)-C(15) 109.0(4)

C(10)-C(14)-C(15) 109.4(4)

C(16)-C(14)-H(14A) 109.2

C(10)-C(14)-H(14A) 109.2

C(15)-C(14)-H(14A) 109.2

C(13)-C(15)-C(14) 110.5(3)

C(13)-C(15)-H(15A) 109.5

C(14)-C(15)-H(15A) 109.5

C(13)-C(15)-H(15B) 109.5

C(14)-C(15)-H(15B) 109.5

H(15A)-C(15)-H(15B) 108.1

C(14)-C(16)-C(17) 110.3(4)

C(14)-C(16)-H(16A) 109.6

C(17)-C(16)-H(16A) 109.6

C(14)-C(16)-H(16B) 109.6

C(17)-C(16)-H(16B) 109.6

H(16A)-C(16)-H(16B) 108.1

N(2)-C(17)-C(16) 110.8(4)

N(2)-C(17)-C(18) 106.1(4)

C(16)-C(17)-C(18) 109.9(4)

N(2)-C(17)-H(17A) 110.0

C(16)-C(17)-H(17A) 110.0

C(18)-C(17)-H(17A) 110.0

C(11)-C(18)-C(17) 109.4(3)

C(11)-C(18)-H(18A) 109.8

C(17)-C(18)-H(18A) 109.8

C(11)-C(18)-H(18B) 109.8

C(17)-C(18)-H(18B) 109.8

H(18A)-C(18)-H(18B) 108.3

C(13)-C(19)-H(19A) 109.5

C(13)-C(19)-H(19B) 109.5

H(19A)-C(19)-H(19B) 109.5

C(13)-C(19)-H(19C) 109.5

H(19A)-C(19)-H(19C) 109.5


S26

H(19B)-C(19)-H(19C) 109.5

O(4)-C(20)-N(3) 125.3(3)

O(4)-C(20)-O(3) 125.0(3)

N(3)-C(20)-O(3) 109.7(3)

O(3)-C(21)-C(22) 110.2(3)

O(3)-C(21)-C(24) 102.1(3)

C(22)-C(21)-C(24) 110.6(4)

O(3)-C(21)-C(23) 110.4(3)

C(22)-C(21)-C(23) 112.3(4)

C(24)-C(21)-C(23) 110.8(4)

C(21)-C(22)-H(22A) 109.5

C(21)-C(22)-H(22B) 109.5

H(22A)-C(22)-H(22B) 109.5

C(21)-C(22)-H(22C) 109.5

H(22A)-C(22)-H(22C) 109.5

H(22B)-C(22)-H(22C) 109.5

C(21)-C(23)-H(23A) 109.5

C(21)-C(23)-H(23B) 109.5

H(23A)-C(23)-H(23B) 109.5

C(21)-C(23)-H(23C) 109.5

H(23A)-C(23)-H(23C) 109.5

H(23B)-C(23)-H(23C) 109.5

C(21)-C(24)-H(24A) 109.5

C(21)-C(24)-H(24B) 109.5

H(24A)-C(24)-H(24B) 109.5

C(21)-C(24)-H(24C) 109.5

H(24A)-C(24)-H(24C) 109.5

H(24B)-C(24)-H(24C) 109.5

_____________________________________________________________

Symmetry transformations used to generate equivalent atoms:


S27

Table S4. Anisotropic displacement parameters (Å2x 103) for MCF292-29 (25). The anisotropic

displacement factor exponent takes the form: -2π2[ h2 a*2U11 + ... + 2 h k a* b* U12 ]

______________________________________________________________________________

U11 U22 U33 U23 U13 U12

______________________________________________________________________________

O(1) 57(2) 68(1) 57(1) 0(1) 4(1) -8(1)

N(1) 76(2) 87(2) 46(2) -15(2) 18(2) -31(2)

C(1) 111(4) 164(5) 54(2) 20(3) 13(3) 6(4)

N(2) 61(2) 104(3) 51(2) 0(2) 11(2) -21(2)

O(2) 57(2) 131(2) 63(2) 28(2) 12(1) -14(2)

C(2) 73(3) 120(4) 79(3) 0(3) 26(3) -8(3)

O(3) 85(2) 51(1) 51(1) -3(1) -15(1) 6(1)

N(3) 66(2) 53(2) 42(2) -9(1) -6(1) 7(1)

C(3) 74(3) 83(3) 49(2) 2(2) 13(2) 3(2)

O(4) 79(2) 61(1) 45(1) -7(1) -9(1) -6(1)

C(4) 58(2) 63(2) 50(2) 1(2) 2(2) 8(2)

C(5) 49(2) 53(2) 45(2) -3(1) 1(2) -1(2)

C(6) 50(2) 56(2) 50(2) -3(2) 2(2) -4(2)

C(7) 54(2) 54(2) 44(2) 1(2) 6(2) -2(2)

C(8) 51(2) 64(2) 47(2) 4(2) 9(2) 2(2)

C(9) 41(2) 57(2) 49(2) 4(2) 5(2) 3(2)

C(10) 72(3) 97(3) 49(2) -21(2) 19(2) -34(2)

C(11) 138(5) 61(2) 61(2) 16(2) 31(3) 24(3)

C(12) 166(6) 73(3) 71(3) -3(2) 56(3) 14(3)

C(13) 61(2) 88(3) 41(2) -4(2) 5(2) -12(2)

C(14) 59(3) 120(4) 57(2) 7(3) 5(2) 27(3)

C(15) 67(3) 168(5) 47(2) 3(3) -7(2) 10(3)

C(16) 143(5) 67(3) 64(3) 8(2) 29(3) 6(3)

C(17) 82(3) 120(4) 57(2) -7(3) 0(2) -46(3)

C(18) 59(3) 221(7) 49(2) -2(3) -5(2) 53(4)

C(19) 106(4) 147(4) 50(2) -18(3) 22(2) -33(4)

C(20) 49(2) 57(2) 42(2) -4(2) -4(2) -4(2)

C(21) 71(3) 60(2) 55(2) 5(2) -6(2) 6(2)

C(22) 84(3) 111(4) 79(3) -7(3) -25(2) 22(3)

C(23) 127(4) 71(3) 99(3) 2(2) 45(3) -8(3)

C(24) 105(3) 63(2) 83(3) 6(2) -3(3) 15(2)

______________________________________________________________________________


S28

Table S5. Hydrogen coordinates (x 104) and isotropic displacement parameters (Å2x 10 3)

for MCF292-29 (25).

________________________________________________________________________________

x y z U(eq)

________________________________________________________________________________

H(1N) 6740(60) 5990(20) 714(19) 72(12)

H(1A) 16055 6285 -3162 164

H(1B) 14602 6868 -2862 164

H(1C) 13790 6114 -3120 164

H(2A) 17116 6138 -1452 137

H(2B) 16744 6875 -1834 137

H(2C) 18086 6268 -2153 137

H(3N) 13850(50) 4837(16) -1341(16) 48(9)

H(3A) 15397 5499 -2266 83

H(4A) 12264 5852 -2024 69

H(4B) 12944 6644 -1836 69

H(5A) 14306 6244 -852 59

H(6A) 10507 5615 -879 62

H(7A) 11761 6856 -259 60

H(8A) 8042 6159 -239 65

H(8B) 8158 7002 -392 65

H(10A) 7970 6813 1804 87

H(11A) 4912 7277 1442 104

H(12A) 5686 7353 2590 124

H(12B) 3402 7242 2501 124

H(14A) 8347 5560 1964 95

H(15A) 6802 5521 3014 113

H(15B) 7787 6291 2906 113

H(16A) 5640 4833 2061 109

H(16B) 5717 5169 1339 109

H(17A) 2570 5255 1776 104

H(18A) 3537 6183 1032 131

H(18B) 2114 6512 1576 131

H(19A) 3074 6705 3628 151

H(19B) 5340 6787 3771 151

H(19C) 4330 6016 3824 151

H(22A) 18607 3919 -27 137


S29

H(22B) 17979 3620 676 137

H(22C) 17569 4440 487 137

H(23A) 14020 4257 750 149

H(23B) 14312 3421 906 149

H(23C) 12806 3667 355 149

H(24A) 16775 2971 -597 126

H(24B) 14506 2871 -472 126

H(24C) 16029 2626 75 126 ________________________________________________________________________________

Irradiation survival curves in 32Dcl3 cells. Procedure: Murine 32D cl 3 cells were suspended at 1 x105 cells and irradiated to doses of irradiation ranging from 0 to 8 Gy using a J. L. Shepherd Mark I cesium irradiator at a dose rate of 72 cGy per min. In some groups, nitroxides were added at a concentration of 10 µM either 1 h before irradiation or 10 min after irradiation. The cells were resuspended in methycellulose containing 10% serum and 15% WEHI-3 conditioned medium as a source of IL3, and incubated at 37 °C for 7 d, at which colonies of >50 cells were counted. The data was analyzed using linear quadratic and single-hit, multi-target models.


S30

Data used to calculate D0 and ñ for Organic and Biomolecular Chemistry manuscript.

Before Irradiation After Irradiation Compound D0 ñ D0 ñ

1.4 1.0 1.7 1.0 1.3 1.0 1.8 1.0

Control

1.3 1.1 1.7 1.2 1.2 1.1 1.3 2.1 1.3 1.0 1.6 1.0

TEMPO

1.3 1.0 1.3 1.7 2.8 1.0 1.3 3.7 2.3 1.0 1.2 3.2

JP4-039

1.7 0.9 1.0 3.6 1.7 1.2 1.3 4.5 2.8 1.4 1.4 3.4

BODOPY®-FL-JP4-

039 3.2 1.2 1.2 5.6 1.3 4.2 1.5 4.0

BODOPY®-R6G-

JP4-039 1.6 2.2 2.1 0.9 1.4 2.0 1.6 1.0 1.2 2.9

TMIO

1.8 1.0 1.2 2.5 4.3 1.0 1.5 1.8 2.7 1.1 1.4 2.4

ABNO

2.3 1.0 1.2 2.1 3.5 1.0 1.5 2.6 2.3 1.1 1.2 4.0

1-Me-AZADO

2.1 0.9 1.3 1.5


S31

A B C

D E F

Visualization of 33 in KM101 cells. Panel A: Untreated KM101 cells. Panel B: KM101 cells incubated with

BODIPY®-R6G-JP4-039 (33, red). Panel C: KM101 cells incubated with BODIPY®-R6G-JP4-039 (33, red)

+ MitoTracker Deep Red FM (grey). Panel D: GFP+ KM101 cells. Panel E: GFP+ KM101 cells incubated with

BODIPY®-R6G-JP4-039 (33, red). Panel F: GFP+ KM101 cells incubated with BODIPY®-R6G-JP4-039 (33,

red) + MitoTracker Deep Red FM (grey).

In Panels C and F, pink colors indicate co-localization of MitoTracker Deep Red FM and BODIPY®-R6G-JP4-

039.


S32

NMR Spectra


9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

1.3095

3.9987

7.1280

7.1387

7.1468

7.1572

7.2212

7.2353

7.2457

7.2538

7.2645

7.2700

7.2920

7.2973

7.3201

7.4621

7.4855

12.1

8

2.00

1.96

3.23

1.91

2.08

NBn


190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

28.64

46.47

65.40

76.83

77.23

77.66

121.54

126.65

127.00

128.14

128.54

143.64

148.05

NBn


9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

1.4764

7.1196

7.1296

7.1401

7.1486

7.1586

7.1708

7.1738

7.2363

7.2393

7.2512

7.2614

7.2698

7.2804

7.2915

11.9

6

1.00

1.95

2.06

NH


190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

32.12

62.92

76.81

77.23

77.66

121.60

127.25

148.91

NH


9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

1.3326

1.3515

3.2995

3.3048

3.3101

3.3153

3.3209

3.3490

4.8922

6.5396

6.5459

6.6079

6.6144

6.6346

6.6411

6.8725

6.8992

5.96

5.98

2.49

1.00

1.00

0.97

N OHH2N

2-H


190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

26.83

27.09

48.30

48.58

48.87

49.15

49.44

49.72

50.00

67.94

68.31

109.98

116.34

123.29

136.50

147.39

148.08

N OHH2N

2-H


9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

0.9053

0.9206

1.3773

1.4123

1.6584

3.0907

3.1521

3.3052

3.3100

4.0807

4.8673

5.6039

5.7337

6.6457

7.0787

7.0990

7.3493

7.3682

7.4233

6.42

23.0

9

1.33

2.01

1.00

1.01

1.02

0.63

0.82

0.84

0.79

12-H

NHBoc

NH

ON OH


190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

22.70

23.29

25.93

26.82

26.87

28.92

28.97

41.67

45.29

48.30

48.58

48.87

49.15

49.43

49.72

50.00

51.80

68.22

68.48

79.95

114.96

120.86

123.11

124.01

137.12

139.17

142.32

146.99

157.96

158.01

172.29

12-H

NHBoc

NH

ON OH


9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

1.4688

1.5056

1.5442

1.9167

1.9602

2.0053

2.2345

2.2887

2.7039

2.7258

2.7581

2.7799

3.3210

3.9135

7.2504

7.2700

7.2952

7.3215

7.3452

7.3694

7.4013

7.4256

4.03

2.06

2.00

2.00

1.99

1.98

5.17

13NBn

O


MCF237!94 CDCl3 exp2 301 02/20/2010

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

16.771

29.549

43.038

53.718

57.253

76.809

77.233

77.656

127.345

128.514

128.587

139.437

211.873

13NBn

O


9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

1.7950

1.8089

1.8259

1.8385

1.8524

1.8707

1.8841

1.9020

1.9203

1.9430

1.9588

1.9850

2.0253

2.0424

2.2221

2.2733

2.3375

2.3595

2.3917

2.4136

2.6975

2.7165

2.7485

2.7681

3.0020

3.0564

3.1040

3.8806

7.2348

7.2483

7.2581

7.2700

7.2813

7.2867

7.3094

7.3137

7.3285

7.3339

7.3504

7.3570

7.3612

7.3877

7.4121

7.7229

3.18

3.27

1.05

1.04

1.04

1.14

1.96

2.00

0.68

4.43

1.01

14NBn

HON


190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

17.31

25.93

29.04

29.79

32.02

51.25

51.93

57.03

76.91

77.23

77.55

127.15

128.49

128.60

139.73

159.99

14NBn

HON


9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

1.0020

1.1019

1.1120

1.1472

1.1853

1.1956

1.4644

1.5259

1.5402

1.5824

1.9190

1.9403

2.3262

2.3460

2.3646

2.3845

2.4037

2.4241

3.0544

3.0910

3.8140

4.1137

4.1352

4.2953

7.1945

7.2172

7.2407

7.2703

7.2955

7.3189

7.3430

7.3664

2.08

2.10

8.98

1.84

2.38

2.00

1.99

2.00

1.11

0.79

1.33

3.32

15NBn

BocHN


190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

14.51

24.79

28.68

34.18

42.94

49.36

55.99

76.91

77.23

77.55

79.23

126.86

128.39

128.50

140.66

155.50

15NBn

BocHN


9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

1.1909

1.2055

1.3672

1.6180

1.8470

1.8556

1.8803

1.9235

1.9502

1.9682

1.9965

2.0543

2.0878

2.1215

2.1570

2.1709

2.4956

2.4999

2.5042

3.1399

3.1595

3.1715

3.2801

3.3296

3.3800

3.7847

3.7968

4.3277

4.3472

6.4362

6.4582

6.5638

6.5842

7.4313

7.9284

0.93

1.75

1.27

9.23

2.44

2.13

2.00

1.19

1.36

0.47

0.80

0.70

0.37

0.42

0.77

16-HN

BocHN

OH


190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

12.34

13.03

23.41

28.08

28.29

30.82

32.20

33.22

38.88

39.09

39.30

39.51

39.72

39.92

40.13

40.45

53.30

55.23

77.12

77.32

154.86

16-HN

BocHN

OH


9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

0.9189

0.9221

0.9354

0.9386

0.9538

0.9709

0.9840

1.0124

1.3264

1.3412

1.3440

1.3593

1.3627

1.4443

1.5071

1.5362

1.6450

1.6621

1.6793

1.6941

1.9184

1.9282

1.9378

1.9514

1.9603

2.3064

2.3220

2.3290

2.3351

2.3508

2.9661

2.9844

3.0626

3.0889

3.8198

4.0224

4.0401

4.0578

4.4034

4.4242

5.4325

5.4493

5.4709

5.4877

5.6434

5.6623

5.6817

7.2195

7.2372

7.2703

7.2789

7.2979

7.3158

7.3578

7.3758

3.19

2.99

2.16

2.10

9.22

1.29

3.49

3.07

2.04

2.00

2.13

2.07

1.00

1.09

0.85

1.01

1.02

0.89

1.02

2.14

2.05

NBn18

NH

ONHBoc


190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

14.55

22.71

24.71

24.84

28.64

33.45

33.53

40.43

42.03

44.26

49.27

49.30

51.55

55.96

76.91

77.23

77.55

79.49

123.65

126.87

128.38

128.55

137.21

140.61

155.58

170.44

NBn18

NH

ONHBoc


9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

0.9155

0.9190

0.9320

0.9355

0.9816

1.1133

1.1437

1.1753

1.2568

1.3224

1.3358

1.3398

1.3542

1.3585

1.4406

1.4974

1.6244

1.6411

1.6580

1.6748

1.6919

1.7158

1.7313

1.7397

2.2299

2.2410

2.2522

2.2852

2.3040

2.3126

2.3309

2.9402

2.9563

2.9740

3.5533

3.5767

3.9953

4.0126

4.0300

4.0473

4.5201

4.8286

4.8481

5.4298

5.4465

5.4580

5.4682

5.4849

5.6102

5.6283

5.6475

5.6665

5.8122

5.8269

7.2700

3.05

2.92

2.04

3.13

9.26

2.18

3.50

0.53

2.28

0.62

2.01

0.49

1.51

0.99

0.26

0.95

0.73

1.00

1.07

0.69

0.25

N17-H

NH

ONHBoc

OH


190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

22.68

22.88

23.64

24.81

28.61

29.89

31.08

31.21

32.55

33.76

33.85

39.67

40.30

40.35

44.21

51.54

51.63

54.10

55.89

55.92

76.91

77.23

77.55

79.45

123.47

123.58

137.26

137.40

155.58

170.41

170.59

N17-H

NH

ONHBoc

OH


9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

1.7282

1.7745

1.8746

1.9162

1.9366

1.9484

2.1366

2.1792

2.9056

7.2697

6.60

4.09

4.00

0.88

CN


190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

26.95

27.11

30.60

33.31

36.84

36.93

37.23

76.81

77.23

77.66

122.53

CN


12 11 10 9 8 7 6 5 4 3 2 1 ppm

1.5336

1.5732

1.6810

1.7372

1.7712

1.8113

2.2002

2.5002

2.5236

12.0851

2.12

10.3

7

2.00

0.75

0.81

CO2H


13 12 11 10 9 8 7 6 5 4 3 2 1 ppm

2.1963

2.2412

2.2508

2.2621

2.2987

2.3308

2.3384

2.4019

2.4428

2.4493

2.4598

2.4942

2.5001

2.5040

2.6530

2.6941

2.7212

2.8272

2.8699

12.5616

6.69

1.81

1.68

2.00

0.33

CO2H

Br

Br 19


9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

1.4513

2.1403

2.1796

2.1839

2.2382

2.2839

2.2944

2.3256

2.3320

2.3645

2.3834

2.8615

3.7516

4.6988

4.7198

7.2703

9.12

10.2

7

2.00

1.01

1.02

NHBoc

Br

Br 20


190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

28.57

38.77

41.55

46.40

51.21

59.00

59.68

60.20

76.81

77.23

77.65

80.16

155.24

NHBoc

Br

Br 20


9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

1.4820

1.4881

2.1519

2.1990

2.2752

2.2908

2.3301

2.3851

2.4415

2.4657

2.5147

2.5735

2.6133

2.6296

3.9196

4.1180

4.7453

4.8451

4.9280

7.2699

9.00

10.2

0

0.79

0.20

0.74

2.05

0.17

NHBoc

O21


200 180 160 140 120 100 80 60 40 20 ppm

28.58

34.37

35.02

35.90

41.05

42.28

46.98

50.47

51.43

76.81

77.23

77.65

80.22

115.88

116.48

139.66

139.74

155.62

209.23

NHBoc

O21


9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

1.4685

2.0073

2.1176

2.1667

2.1772

2.2675

2.3142

2.3412

2.3961

2.4617

3.0655

3.1211

3.2229

3.2764

3.8646

4.6745

4.6796

4.7897

4.9263

7.2697

8.0137

9.07

0.51

0.68

7.83

0.76

0.18

1.00

1.09

0.67

0.32

0.85

NHBoc

HON22


190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

24.93

28.62

31.59

32.54

32.61

34.01

34.20

35.14

35.68

37.63

40.54

41.32

52.16

76.81

77.23

77.65

80.04

112.69

113.17

141.06

141.14

155.54

NHBoc

HON22


9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

1.4596

1.6680

1.7134

1.8740

1.9188

2.1167

3.1619

3.1906

3.2356

3.7677

4.7456

4.8265

7.2701

9.00

6.00

3.43

1.36

0.47

1.33

0.91

0.90

0.33

0.62

NHBoc

23HN

I


190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

23.22

23.66

28.62

30.76

31.74

32.42

35.77

36.14

40.92

47.22

47.39

48.97

53.71

76.81

77.23

77.65

79.71

155.54

NHBoc

23HN

I


9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

1.6989

1.7434

1.7531

1.7626

1.7711

1.8068

1.8155

1.9001

2.0025

2.0118

2.0465

2.0556

2.1572

2.2018

2.2714

2.2812

2.2903

2.3150

2.3246

2.3337

3.1398

3.2988

3.3043

3.3098

3.3152

3.3207

3.4462

3.4604

3.6213

3.6491

4.9032

6.7527

6.7732

0.55

2.30

1.80

9.00

1.15

2.36

2.07

1.25

0.93

0.88

0.94

0.98

0.77

NHBoc

24-H (6-Boc-amino-1-Me-AZADO-H)

NHO


190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

23.79

26.93

28.94

31.51

32.45

32.64

36.08

43.78

55.27

57.20

58.48

80.26

158.19

NHBoc

24-H (6-Boc-amino-1-Me-AZADO-H)

NHO


9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

0.9211

0.9374

1.1129

1.4248

1.4327

1.6116

1.6288

1.6453

1.6622

1.6791

1.6960

1.7691

1.9562

1.9998

2.2729

2.3017

2.3080

3.0071

3.0249

3.2729

3.9912

4.0097

4.0605

4.0739

4.0912

4.1077

4.4645

5.5043

5.5180

5.5428

5.5568

5.6555

5.6731

5.6920

5.7119

5.7289

6.1346

7.2703

6.41

3.50

2.33

9.72

1.74

8.61

2.37

2.00

0.99

1.09

1.16

0.92

1.09

1.10

0.91

NHBoc

NH

ONOH

25-H


190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

26.39

28.55

28.58

29.55

29.85

29.89

30.50

30.54

30.73

31.13

31.19

32.11

34.99

35.08

40.42

42.83

42.89

44.44

51.12

52.43

56.30

56.97

76.91

77.22

77.54

79.52

122.80

137.81

155.43

170.54

NHBoc

NH

ONOH

25-H


9 8 7 6 5 4 3 2 1 ppm

0.824

0.838

0.853

1.250

1.431

1.496

1.507

2.000

2.035

2.067

2.101

2.134

2.170

2.257

2.553

2.679

2.697

2.715

2.896

2.901

2.917

3.254

4.262

4.280

4.295

5.298

5.335

5.352

5.374

5.391

5.519

5.539

5.558

6.022

6.038

6.128

6.312

6.322

6.635

6.654

6.866

6.876

7.095

7.260

9.92

13.3

56.

415.

78

4.10

2.79

2.73

1.71

1.60

1.72

1.88

0.18

1.19

0.88

0.69

0.87

0.81

0.72

0.92

1.00

PC 1.00GB 0LB 0.30 HzSSB 0WDW EMSF 400.1300099 MHzSI 65536P1 9.31 usecNUC1 1H======== CHANNEL f1 ========

D1 1.00000000 secTE 296.0 KDE 6.50 usecDW 60.800 usecRG 114AQ 3.9846387 secFIDRES 0.125483 HzSWH 8223.685 HzDS 2NS 16SOLVENT CDCl3TD 65536PULPROG zg30PROBHD 5 mm PADUL 13CINSTRUM spectTime 13.22Date_ 20110602PROCNO 1EXPNO 3NAME EMF338!009

PROTON CDCl3 C:\Bruker\TOPSPIN wipf 2

NH

OHN NO

O

30-H

NN

BFF

H


190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

1.00

11.3

114

.10

14.9

320

.59

22.2

122

.60

22.6

724

.44

24.8

528

.38

29.3

429

.68

31.9

135

.98

39.5

939

.83

42.2

443

.22

50.5

3

67.3

0

117.

5412

0.53

123.

78

128.

27

133.

3313

5.18

136.

65

144.

06

157.

0516

0.35

170.

6917

2.03

NH

OHN NO

O

30-H

NN

BFF

H


190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

9.51

13.94

20.25

20.48

22.38

23.33

27.53

31.59

34.48

41.20

48.80

115.47

118.75

121.74

121.93

126.17

131.19

133.22

134.77

142.23

154.34

158.81

168.47

169.59

PC 1.40GB 0LB 1.00 HzSSB 0WDW EMSF 100.6381482 MHzSI 32768SFO2 400.2316009 MHzPL13W 0.08816721 WPL12W 0.40393090 WPL2W 11.09959412 WPL13 20.00 dBPL12 13.39 dBPL2 !1.00 dBPCPD2 75.00 usecNUC2 1HCPDPRG2 waltz16======== CHANNEL f2 ========

SFO1 100.6479773 MHzPL1W 51.07626343 WPL1 !1.59 dBP1 10.00 usecNUC1 13C======== CHANNEL f1 ========

TD0 1D11 0.03000000 secD1 3.00000000 secTE 296.7 KDE 6.50 usecDW 20.800 usecRG 203AQ 1.3631988 secFIDRES 0.366798 HzSWH 24038.461 HzDS 4NS 591SOLVENT CDCl3TD 65536PULPROG zgpg30PROBHD 5 mm PABBO BB!INSTRUM spectTime 9.22Date_ 20110411PROCNO 1EXPNO 3NAME EMF338!008c

13 C of EMF338!008

120130140150160170 ppm

1015202530354045 ppm

NH

OHN NO

O

30

NNBFF


The image cannot be displayed. Your computer may not have enough memory to open the image, or the image may have been corrupted. Restart your computer, and then open the file again. If the red x still appears, you may have to delete the image and then insert it again.

O

ONHBoc

27


102030405060708090100110120130140150160170 ppm

22.61

22.88

24.90

28.61

37.74

44.80

50.35

52.03

76.91

77.23

77.55

79.46

121.97

135.60

155.46

172.32

MCF292!93 (A) CDCl3 400B exp3 12/03/2010

O

ONHBoc

27


0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.5 ppm

1.5836

2.2639

2.5792

2.8440

2.8504

3.0746

3.0931

3.1114

3.3755

3.3938

3.4122

6.1376

6.3405

6.3505

6.8956

6.9055

7.1083

7.2698

3.01

3.01

4.02

2.00

2.02

0.98

0.99

0.99

0.99

MCF324!32 CDCl3 400B exp2 02/09/2011

O O

NNBFF

NO O

BODIPY!-FL-NHS (28)


102030405060708090100110120130140150160170180190 ppm

11.552

15.229

23.571

25.801

30.556

76.914

77.231

77.549

116.981

120.955

124.251

128.101

133.494

135.769

144.606

154.928

161.468

168.034

169.229

MCF324!32 CDCl3 400B exp5 02/09/2011

O O

NNBFF

NO O

BODIPY!-FL-NHS (28)


7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

0.8261

0.8430

0.8456

0.8623

1.2367

1.2550

1.2708

1.2745

1.2861

1.2915

1.3072

1.4247

1.4409

1.4573

1.4766

1.6336

1.6484

1.6598

2.2664

2.5747

2.6385

2.6570

2.6752

3.0057

3.0229

3.2573

3.2756

3.2939

3.6704

4.4595

4.4766

4.4943

5.3913

5.4060

5.4300

5.4448

5.5448

5.5475

5.5620

5.5649

5.5791

5.5826

5.5863

5.5927

5.6004

5.6034

5.6178

5.6207

6.1321

6.2918

6.3018

6.8767

6.8867

7.0949

7.2700

6.24

2.35

1.04

1.54

3.10

3.12

2.03

2.02

2.05

3.00

1.00

1.02

1.98

1.01

1.01

1.02

1.02

PROTON CDCl3 C:\Bruker\TOPSPIN wipf 4MCF324!47 CDCl3 400A exp4 02/19/2011

O

OHN O

29

NNBFF


O O

NNBFF

N

O

OBODIPY!-R6G-NHS (31)


NH

OHN O

NNBPh FF

N OH

33-H


si obc jp4 rev...supporting information synthesis of analogs of the radiation mitigator jp4-039 and...

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