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S1 Supporting Information 18 F-Fluorination of Unactivated C-H Bonds in Branched Aliphatic Amino Acids: Direct Synthesis of Oncological Positron Emission Tomography Imaging Agents Matthew B. Nodwell, Hua Yang, § Milena Čolović, Zheliang Yuan, †,§ Helen Merkens, Rainer E. Martin,* François Bénard,* , Paul Schaffer,* , § Robert Britton* ,Department of Chemistry, Simon Fraser University, Burnaby, BC, Canada, V5A 1S2 § Life Sciences Division, TRIUMF, Vancouver, BC, Canada, V6T 2A3 Department of Molecular Oncology, BC Cancer Agency, Vancouver, BC, Canada, V5Z 1L3 ¥ Medicinal Chemistry, Roche Pharma Research and Early Development (pRED), Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Grenzacherstrasse 124, CH-4070 Basel, Switzerland

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S1

Supporting Information

18F-Fluorination of Unactivated C-H Bonds in Branched

Aliphatic Amino Acids: Direct Synthesis of Oncological

Positron Emission Tomography Imaging Agents

Matthew B. Nodwell,† Hua Yang,§ Milena Čolović, ‡ Zheliang Yuan, †,§ Helen

Merkens, ‡ Rainer E. Martin,*,¥ François Bénard,*, ‡ Paul Schaffer,*, § Robert

Britton*,†

†Department of Chemistry, Simon Fraser University, Burnaby, BC, Canada, V5A 1S2 §Life Sciences Division, TRIUMF, Vancouver, BC, Canada, V6T 2A3 ‡Department of Molecular Oncology, BC Cancer Agency, Vancouver, BC, Canada, V5Z 1L3 ¥ Medicinal Chemistry, Roche Pharma Research and Early Development (pRED), Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Grenzacherstrasse 124, CH-4070 Basel, Switzerland

S2

1) General Experimental

All reactions were carried out with commercial solvents and reagents that were

used as received. Concentration and removal of trace solvents was done via a

Büchi rotary evaporator using dry ice/acetone condenser, and vacuum applied

from an aspirator or Büchi V-500 pump. Nuclear magnetic resonance (NMR)

spectra were recorded using deuterium oxide or acetonitrile-d3. Signal positions

(δ) are given in parts per million from tetramethylsilane ( 0) and were measured

relative to the signal of the solvent (1H NMR: CD3CN: 1.94, D2O: 4.79; 13C

NMR: CD3CN: 118.26). 19F NMR spectra are referenced to trifluoroacetic acid in

D2O -76.6. 183W NMR spectra are referenced to 2.0 M Na2WO4 in H2O. Coupling

constants (J values) are given in Hertz (Hz) and are reported to the nearest 0.1

Hz. 1H NMR spectral data are tabulated in the order: multiplicity (s, singlet; d,

doublet; t, triplet; q, quartet; quint, quintet; m, multiplet), coupling constants,

number of protons. NMR spectra were recorded on a Bruker Avance 600 equipped

with a QNP or TCI cryoprobe (600 MHz) or a Bruker 500 (500 MHz). Where

necessary, N,N-dimethylformamide (DMF) or 1,3,5-tris(trifluoromethyl)benzene

was added to the crude reaction mixtures and used as an internal standard. Yields

were then calculated following analysis of 1H NMR spectra. Preparative RP-HPLC

was performed on an Agilent 1200 series instrument with a SiliCycle SiliaChrom

dtC18 semipreparative column (5 um, 100Å, 10 x 250 mm) with a flow rate of 5

mL/min, or a Phenomenex Gemini-NX C18 preparative column (5um, 110Å, 50 x

30 mm) with a flow rate of 15 mL/min. Analytical HPLC was carried out on an

Agilent 1200 series HPLC system equipped with a diode array detector (DAD) and

Raytest GABI Star scintillation detector. RadioTLC was carried out using BioScan

system 200 Image Scanner. A PerkinElmer Wizard 2480 gamma counter was used

for cell uptake studies.

S3

Supporting Figure 1: Reactor configuration A: 15 W F15T8/BLB lamp and vial used

in the fluorination described in this work.

S4

Supporting Figure 2: Reactor configuration B: 15 W F15T8/BLB lamp and PTFE

reaction tube (Hamilton, 3 m length, 0.7 mm inner diameter (ID), 1.9 mm outer

diameter (OD), ~2.5 mL total volume) used in the fluorination and radiofluorination

described in this work.

S5

Supporting Figure 3: Reactor configuration C: 8 x 9W UV curing lamps in “box”

configuration with air cooling used in the fluorination described in this work.

S6

2) Chemistry

Entry Solvent Photocatalyst

Fluorine

source [leucine] Reactor configuration

Eq.

Fluorine

source

%

conversion Time

1 3:1 MeCN:H2O Tetracyanobenzene (10%) NFSI 0.1 M A 3 0 18h

2 3:1 MeCN:H2O Tetracyanobenzene (10%) Selectfluor 0.1 M A 3 0 18h

3 3:1 MeCN:H2O Anthraquinone (5%) NFSI 0.1 M Vial, 13 W fluorescent bulb 3 0 18h

4 3:1 MeCN:H2O Anthraquinone (5%) Selectfluor 0.1 M Vial, 13 W fluorescent bulb 3 0 18h

5 3:1 MeCN:H2O TiO2 anatase nanopowder (170 wt%) NFSI 0.1 M A, rapid stirring 3 2% 18h

6 3:1 MeCN:H2O TiO2 anatase nanopowder (170 wt%) Selectfluor 0.1 M A, rapid stirring 3 8% 18h

7 3:1 MeCN:H2O Sodium polytungstate (5%) NFSI 0.1 M B 3 0 18h

8 3:1 MeCN:H2O Ammonium paratungstate (5%) NFSI 0.1 M B 3 22% 18h

9 3:1 MeCN:H2O Sodium dodecaphosphotungstate (5%) NFSI 0.1 M B 3 0 18h

10 3:1 MeCN:H2O Sodium dodecaphosphotungstate (5%) Selectfluor 0.1 M B 3 0% 18h

11 1:1 Benzene:H2O TBADT (5%) NFSI 0.25 M A, rapid stirring 3 0 18h

12 1:1 Benzene:H2O NaDT (5%) NFSI 0.25 M A, rapid stirring 3 0 18h

13 MeCN TBADT (5%) NFSI 0.375 M A, rapid stirring 3 28% 18h

14 20:1 MeCN:H2O TBADT (5%) NFSI 0.375 M A, rapid stirring 3 85% 18h

15 7:1 MeCN:H2O TBADT (5%) NFSI 0.375 M A, rapid stirring 3 85% 18h

16 3:1 MeCN:H2O TBADT (5%) NFSI 0.1 M A 3 12% 1h

17 3:1 MeCN:H2O TBADT (10%) NFSI 0.1 M A 3 5% 1h

18 3:1 MeCN:H2O NaDT (5%) NFSI 0.1 M A 3 23% 1h

19 3:1 MeCN:H2O NaDT (5%) NFSI 0.1 M B 3 60% 40 min

20 3:1 MeCN:H2O NaDT (5%) NFSI 0.1 M B 1.5 52% 40 min

Supporting Table 1: Full optimization table for [19F]fluorination of L-leucine.

Reactor configurations are shown in Supporting Figures 1, 2 and 3

Preparation of Sodium Decatungstate

Sodium decatungstate (Na4W10O32.xH2O.yCH3CN) was prepared using the

method of Hill et al.[1] 183W NMR (20.8 MHz, CH3CN, relative to 2 M Na2WO4 in

H2O) –21.2 (s, 8W), -167.6 (s, 2W).

S7

Preparation of leucine-containing dipeptides, H-PheLeu-OH and H-TyrLeu-

OH

Peptide synthesis was carried out on Wang resin using standard Fmoc-based

SPPS protocols. Briefly, Fmoc deprotection was carried out with 20% piperidine

in DMF, and couplings were carried out with 2 eq. Fmoc amino acid, 2 eq.

benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 2

eq ethyl cyano(hydroxyimino)acetate (Oxyma Pure) in DMF and initiated by the

addition of 4 eq. N,N-diisopropylethylamine. Coupling was carried out for 1h at

room temperature. After the final coupling and deprotection, the resin was washed

with DMF, CH2Cl2 and shrunk with MeOH. The peptide was cleaved from the resin

with neat TFA, precipitated into Et2O and purified by RP-HPLC as described above

eluting with solvent A (0.1% TFA in H2O) and solvent B (0.1% TFA in CH3CN) on

a gradient of 2% 100% solvent B over 15 min (15 mL/min flow rate) to yield H-

PheLeu-OH.TFA and H-TyrLeu-OH.TFA as lyophilized powders.

H-PheLeu-OH.TFA:

1H NMR (600 MHz, D2O): 0.79 (d, J = 6.4 Hz, 3H), 0.83 (d, J = 6.6 Hz 3H), 1.52

(m, 3H), 3.10 (dd, J = 14.2, 7.4 Hz, 1H), 3.19 (dd, J = 14.2, 7.1 Hz 1H), 4.19 (t, J

= 7.1 Hz 1H), 4.27 (t, J = 7.2 Hz 1H), 7.22 (m, 2H), 7.32 (m, 3H); 13C NMR (150

MHz, D2O): 20.7, 22.0, 24.3, 36.8, 39.5, 51.9, 54.2, 128.0, 129.1, 129.4, 133.5,

168.8, 175.8

HRMS (ESI+) calcd for C15H22N2O3H+ 279.1709, found 279.1712

H-TyrLeu-OH.TFA:

1H NMR (600 MHz, D2O): 0.77 (d, J = 6.6 Hz, 3H), 0.82 (d, J = 6.6 Hz, 3H), 1.43

(m, 1H), 1.55 (m, 2H), 3.06 (m, 2H), 4.13 (t, J = 7.1 Hz, 1H), 4.27 (t, J = 7.5 Hz,

1H), 6.79 (d, J = 9.0 Hz, 2H), 7.08 (d, J = 8.6 Hz, 2H); 13C NMR (150 MHz, D2O):

20.6, 21.9, 24.2, 35.9, 39.2, 51.6, 54.2, 115.8, 125.3, 130.9, 155.2, 169.0, 175.5

HRMS (ESI+) calcd for C15H22N2O4H+ 295.1658, found 295.1657

S8

Direct Fluorination protocols:

General procedure A

A TFA or HCl salt of the substrate (0.1 M final concentration), sodium

decatungstate (NaDT, 5 mol%) and N-fluorobenzenesulfonimide (NFSI, 3 eq) were

dissolved in 800 L 3:1 CH3CN:H2O with sonication. The resulting solution was

filtered and loaded onto a PTFE reaction tube (Hamilton, 3 m length, 0.7 mm ID,

1.9 mm OD, ~2.5 mL total volume) wrapped around a 15W F15T8/BLB bulb

(Reactor configuration B, Supporting Figure 2). Irradiation was carried out for 40

min, and then the solution was removed via syringe, and the PTFE reaction tube

washed with 2 mL CH3CN. 1,3,5-Tris(trifluoromethyl)benzene was added to the

crude reaction mixture and yields were determined via analysis of the 1H NMR

spectra. The solution was diluted with CH3CN, and loaded onto a strong cation

exchange cartridge (Silicycle SCX resin, 500 mg). After loading, the cartridge was

washed with CH3CN (10 mL), then H2O (10 mL). The fluorinated product/starting

material mixture was eluted with ~4 mL 150 mM NaHCO3 (1 mL/min approximate

flow rate) acidified with HOAc to pH = 5 and lyophilized. Pure samples of 4-FL, 5-

FBAHL, 5-FHL, 3-FV and 3-FI suitable for characterization could be obtained by

preparative RP-HPLC as described above eluting with solvent A (0.1% TFA in

H2O) and solvent B (0.1% TFA in CH3CN) on a gradient of 2% 30% solvent B

over 30 min (5 mL/min flow rate) to yield 4FL.TFA, 5-FBAHL.TFA, 5-FHL.TFA, 3-

FV.TFA or 3-FI.TFA as lyophilized powders.

General procedure B

A TFA salt of the substrate (75-100 mol), NaDT (2 mol %) and NFSI (3 eq) were

dissolved in 1.8 mL 2:1 CD3CN:D2O and placed in an NMR tube. The tube was

then suspended in a UV reactor (Reactor configuration C, Supporting Figure 3).

Irradiation was carried out with compressed air cooling for a period of time

indicated below. After this time, 10 L (53.7 mol) of 1,3,5-

tris(trifluoromethyl)benzene was added the the crude reaction mixture, and yield

S9

was calculated by analysis of the 1H NMR spectrum. The solution was then diluted

with H2O, filtered, and the the product was purified by RP-HPLC as described

above eluting with solvent A (0.1% TFA in H2O) and solvent B (0.1% TFA in

CH3CN) on a gradient of 2% 30% solvent B over 15 min (15 mL/min flow rate)

to yield the fluorinated products as lyophilized powders.

4-FL.TFA: General Procedure A

L-leucine.HCl (15.1 mg, 90 mol), NaDT, (11 mg, 4.5 mol, 5 mol %) and NFSI,

(85 mg, 270 mol). Yield (40 min) = 32 %

1H NMR (600 MHz, D2O): = 1.50 (d, J = 22.8 Hz, 3H), 1.52 (d, J = 22.3 Hz, 3H),

2.35 (m, 2H), 4.26 (m, 1H); 13C NMR (150 MHz, D2O): = 23.7 (d, J = 24 Hz), 26.6

(d, J = 23.4 Hz), 39.8 (d, J = 20.4 Hz), 50.0, 96.7 (d, J = 161 Hz), 115.8 (q, J = 290

Hz), 162.5 (q, J = 35 Hz), 172.2; 19F NMR (470 MHz, D2O): = -138.7

HRMS (ESI+) calcd for C6H12FNO2H+ 150.0930, found 150.0907

5-FBAHL.TFA: General Procedure A

L--homoleucine.TFA: (15 mg, 58 mol), NaDT: (7.0 mg, 2.9 mol, 5 mol %) and

NFSI: (55 mg, 174 mol). Yield (40 min) = 36 %

1H NMR (600 MHz, D2O): = 1.36 (d, J = 22.8 Hz, 3H), 1.38 (d, J = 22.4 Hz, 3H),

1.99 (m, 2H), 2.66 (dd, J = 17.5, 7.3 Hz, 1H), 2.73 (dd, J = 17.5, 5.5 Hz, 1H), 3.88

(m, 1H); 13C NMR (150 MHz, D2O): = 24.1 (d, J = 24.1 Hz), 26.5 (d, J = 23.4 Hz),

37.0, 41.6 (d, J = 19.4 Hz), 44.8, 96.8 (d, J = 161.6 Hz), 115.8 (q, J = 295.6 Hz),

162.5 (q, J = 34.5 Hz), 173.9; 19F NMR (470 MHz, D2O): = -138.7

HRMS (ESI+) calcd for C7H14FNO2H+ 164.1087, found 164.1015

S10

5-FHL.TFA: General Procedure A

L-homoleucine.TFA (11 mg, 43 mol), NaDT (5.2 mg, 2.2 mol, 5 mol %), and

NFSI (41 mg, 129 mol). Yield (40 min) = 74 %

1H NMR (600 MHz, D2O): = 1.31 (overlapping doublets, J = 22.8 Hz, 6H), 1.70

(m, 2H), 1.96 (m, 2H), 3.88 (m, 1H); 13C NMR (150 MHz, D2O): = 24.2 (d, J = 5.2

Hz), 24.9 (d, J = 23.4 Hz), 25.1 (d, J = 23.6 Hz), 35.0 (d, J = 23.2 Hz), 53.1, 97.0

(d, J = 162.1 Hz), 115.8 (q, J = 292.7 Hz), 162.5 (q, J = 35.5 Hz), 172.5; 19F NMR

(470 MHz, D2O): = -136.5

HRMS (ESI+) calcd for C7H14FNO2H+ 164.1087, found 164.1020

3-FV.TFA: General Procedure A

L-valine.TFA (17.4 mg, 75 mol), NaDT (9.2 mg, 3.8 mol, 5 mol%), NFSI (72.5

mg, 230 mol). Yield (40 min) = 26 %

1H NMR (600 MHz, D2O): 1.38 (d, J = 22.5 Hz, 3H), 1.55 (d, J = 23.6 Hz, 3H),

3.91 (d, J = 10.7 Hz, 1H); 13C NMR (150 MHz, D2O); 21.4 (d, J = 23.8 Hz), 25.2

(d, J = 23.2 Hz), 61.3 (d, J = 21.1 Hz), 94.7 (d, J = 173.1 Hz), 170.1 (d, J = 10.1

Hz); 19F NMR (470 MHz, D2O): = -141.6

HRMS (ESI+) calcd for C5H10FNO2H+ 136.0774, found 136.0776

3- and 4-FI.TFA: General Procedure B

L-isoleucine.TFA (22 mg, 89 mol), NaDT (10.8 mg, 4.5 mol), NFSI (84 mg, 267

mol). Yield (40 min) = 12.1 % (3-FI, 1:1 mixture of diastereomers), 13.6 % (4-FI,

1:1 mixture of diastereomers)

This reaction produced a complex mixture of 3-FI, 4-FI and isoleucine which could

not be separated by flash chromatography. Attempts at HPLC purification

produced a 1:1 diastereomeric mixture of 3-FI, which is reported here.

S11

1H NMR (600 MHz, D2O): 0.90 (m, 6H), 1.33 (d, J = 23.0 Hz, 3H), 1.49 (d, J =

23.9 Hz, 3H), 1.58 (m, 1H), 1.82 (m, 3H), 3.94 (m, 2H); 13C NMR (150 MHz, D2O);

6.51 (d, J = 5.9 Hz), 6.71 (d, J = 6.5 Hz), 18.9 (d, J = 24.4 Hz), 21.3 (d, J = 22.5

Hz), 27.3 (d, J = 23.8 Hz), 30.6 (d, J = 21.8 Hz), 59.7 (d, J = 19.3 Hz), 61.2 (d, J =

19.6 Hz), 96.9 (d, J = 176.0 Hz), 96.8 (d, J = 176.0 Hz), 170.0, 173.9; 19F NMR

(470 MHz, D2O): -150.5, -152.6

HRMS (ESI+) calcd for C6H12FNO2H+ 150.0930, found 150.0932

H-PheLeu(F)-OH: General Procedure B

H-PheLeu-OH.TFA (23.5 mg, 60 mol), NaDT (3 mg, 1.2 mol), NFSI (57 mg, 180

mol). Yield (6 h) = 48 %.

1H NMR (600 MHz, D2O): 1.36 (overlapping doublets, J = 22.6 Hz, 6H), 2.03 (m,

1H), 2.21 (m, 1H), 3.13 (m, 1H), 3.23 (dd, J = 14.2, 6.9 Hz, 1H), 4.23 (m, 1H), 4.50

(m, 1H), 7.26 (m, 2H), 7.36 (m, 3H); 13C NMR (150 MHz, D2O): 25.1 (d, J = 23.5

Hz), 25.6 (d, J = 23.8 Hz), 36.2, 40.7 (d, J = 22.4 Hz), 49.8, 53.7, 96.4 (d, J = 162.4

Hz), 115.8 (q, J = 292.7 Hz), 127.5, 128.7, 129.0, 133.1, 162.5 (q, J = 35.5 Hz),

168.0, 174.7; 19F NMR (470 MHz, D2O): = -136.5

HRMS (ESI+) calcd for C15H21FN2O3H+ 297.1614, found 297.1622

H-TyrLeu(F)-OH: General Procedure B

H-TyrLeu-OH.TFA (20.5 mg, 50 mol), NaDT (2.4 mg, 1 mol), NFSI (47 mg, 150

mol). Yield (6 h) = 24 %.

1H NMR (600 MHz, D2O): 1.31 (d, J = 22.9 Hz, 3H), 1.32 (d, J = 22.5 Hz, 3H),

1.98 (m, 1H), 2.14 (m, 1H), 3.01 (dd, J = 14.6, 7.4 Hz, 1H), 3.15 (dd, J = 14.4, 6.4

Hz, 1H), 4.14 (m, 1H), 4.32 (m, 1H), 6.81 (d, J = 8.8 Hz, 2H), 7.11 (d, J = 8.7 Hz,

2H); 13C NMR (150 MHz, D2O): 24.9 (d, J = 23.8 Hz), 25.9 (d, J = 24.1 Hz), 35.3,

41.2 (d, J = 20.7 Hz), 51.2, 53.8, 96.7 (d, J = 162.2 Hz), 115.3, 124.8, 130.4, 154.6,

167.7, 176.6; 19F NMR (470 MHz, D2O): = -135.7

HRMS (ESI+) calcd for C15H21FN2O4H+ 313.1564, found 313.1577

S12

Preparation of sodium dibenzenesulfonamide

Dibenzenesulfonamide (1.07 g, 3.6 mmol) and NaOH (130 mg, 3.2 mmol) were

combined in water (~100 mL) until dissolved. The reaction mixture was then

washed with 3 x EtOAc. The aqueous layer was then frozen and lyophilized to

yield sodium dibenzenesulfonamide (960 mg, 3.0 mmol, 83 % yield) as a white

solid. The 1H and 13C NMR spectra recorded on this material matched that

reported previously.[2]

3) Radiochemistry

Production of [18F]F2 gas

[18F]F2 gas was produced on TRIUMF’s TR13 cyclotron via the 18O(p,n)18F nuclear

reaction in an aluminium-body target using two proton irradiations. First [18O]O2

was loaded into the target to ~270 psi and irradiated with 25 A of 13 MeV protons

for 5-10 minutes. The gas was removed under reduced pressure and cryogenically

trapped for recycling. F2 gas (3 % in Ar) was filled into the target to 14 psi and

topped with Ar to 290 psi. The target was then irradiated for 2-5 min with 20 A of

13 MeV protons. The target was emptied to the chemistry lab carried by Ar.

Synthesis of [18F]N-fluorodibenzenesulfonamide ([18F]NFSI)

Sodium dibenzenesulfonamide (20 mg, 62 mol) was dissolved in 800 L of 3:1

CH3CN:H2O and placed in a conical vial. [18F]F2 produced in the cyclotron target

was then passed through the solution over a period of ~15 min. The waste gas

was trapped by saturated KI solution. Typically 1-2 GBq was trapped in the

reaction mixture. The resulting solution was then passed through a SepPak

(Waters tC18 SepPak Plus Long Cartridge). The cartridge was washed with 10 mL

H2O followed by 600 L CH3CN. [18F]NFSI was then eluted from the SepPak

cartridge in 1.2 mL CH3CN. Typically, 21 ± 8 mol of purified NFSI with an activity

S13

of 0.2-0.5 GBq is produced from this process. The amount of NFSI generated in

each reaction was calculated following HPLC analysis of the reaction mixture and

comparison with a calibration curve prepared from NFSI.

Synthesis of 4-[18F]FL, 5-[18F]FHL, 5-[18F]FBAHL and 3-[18F]FV

The [18F]NFSI solution was added to a slurry of the substrate (L-leucine.HCl =10

mg (55 mol), -aminohomoleucine.TFA = 13 mg (50 mol), homoleucine.TFA =

12 mg (46 mol) and valine.TFA = 12mg (52 mol) and sodium decatungstate

(NaDT, 5 mg, 2.0 mol) in 200-400 L H2O and mixed briefly. The solution was

then loaded onto the photoreactor described above and in Supporting Figure 1 and

irradiated for 40 min. After this time the solution was removed and the photoreactor

was washed with CH3CN (5 mL). The resulting solution was loaded onto a

preconditioned strong cation exchange cartridge (Silicycle, 500 mg resin) and the

cartridge was washed with CH3CN (10 mL) followed by H2O (10 mL). 4-[18F]FL, 5-

[18F]FBAHL, 5-[18F]FHL or 3-[18F]FV were then eluted from the cartridge with 1

mL aliquots of 150 mM NaHCO3, yielding a mixture of fluorinated product and

starting material. The bulk of the activity was typically eluted in the 4th and 5th 1 mL

aliquot. Analytical HPLC was carried out on Phenomenex Monolithic C18 analytical

column (4.6 × 100 mm column, 1 mL/min) using a gradient of 2% solvent A (0.1%

TFA in H2O) and 98% solvent B (0.1% TFA in CH3CN) to 100% solvent B over 30

min or on a Phenomenex Luna C18 (4.6 x 100 mm, 1 mL/min) using a gradient of

100% solvent A (0.1% TFA in H2O) to 100% solvent B (0.1% TFA in CH3CN) over

15 min. RadioTLC analysis was carried out in BuOH:H2O:HOAc (12:5:3), followed

by ninhydrin staining and radioTLC detection. To determine the enantiopurity of 4-

[18F]FL, the above reaction was carried out simultaneously with both DL-leucine

and L-leucine and the final products were analyzed by Chiral HPLC eluting on a

Phenomenex D-Penicillamine 4.6 × 100 mm column, 1 mL/min, isocratic, 20%

EtOH and 80% 1 mM aq. CuSO4.

The radiochemical yield (RCY) is reported as a percentage and represents the

total activity present in the purified 18F-labeled amino acid divided by the total

activity present in the purified [18F]NFSI x 100.

S14

Determination of Specific Activity

To determine the specific activity (SA) of 4-[18F]FL, 5-[18F]BAHL, 5-[18F]HL and 3-

[18F]VL the purified product mixtures were eluted from the ion exchange column in

1 mL fractions. Each fraction was counted, then the whole sample was allowed to

decay at –20 oC. After ~100 h, the fractions were then lyophilized to dryness. Each

entire dried fraction was then taken up in D2O and N,N-dimethylformamide (5 l,

65 mol) was added as an internal standard. After thorough mixing the 1H and 19F

NMR spectra were recorded. Amounts of 4-FL, 5-FBAHL, 5-FHL and 3-VL were

determined by analysis of the 1H NMR spectra (see below). Specific activity was

then determined by correlating the amount of fluorinated product in each fraction

to its activity. SA measurements were determined via at least three independent

experiments. As the radiofluorination process described in this report does not

remove any unreacted amino acid, we have also calculated the effective SA – a

number which takes into account the amounts of fluorinated amino acid as well as

amounts of parent amino acid. These numbers are presented in Supporting Table

2:

Radiotracer Effective SA (MBq/mol)

4-[18F]FL 3.82 ± 0.91

5-[18F]FBAHL 2.2 ± 0.50

5-[18F]FHL 4.92 ± 1.47

3-[18F]FV 0.43 ± 0.40

Supporting Table 2: Effective SA values of radiotracers isolated during this study.

These values take into account amounts of both fluorinated amino acid as well as

parent, unreacted amino acid present in the purified product. All experiments were

repeated ≥ 3 times.

S15

Supporting Figure 4: RadioTLC scan of crude leucine [18F]fluorination reaction

Supporting Figure 5: a) TLC analysis of purified 4-[18F]FL/leucine mixture

visualized with ninhydrin stain and radiodetection. b) Radiodetected chiral HPLC

analysis of purified 4-[18F]FL derived from L-leucine and DL-leucine

18F-

[18F]NFSI 4-[18F]FL

S16

Supporting Figure 6: Radiodetected HPLC traces for radiosynthesis of 4-[18F]FL

Supporting Figure 7: Radiodetected HPLC traces for radiosynthesis of 5-

[18F]FBAHL

S17

Supporting Figure 8: Radiodetected HPLC traces for radiosynthesis of 5-[18F]FHL

Supporting Figure 9: Radiodetected HPLC traces for radiosynthesis of 3-[18F]FV

S18

4) Biological studies

Cell uptake studies

The LNCaP and PC-3 cell lines were obtained from ATCC. MCF-7 were obtained

as a gift from Dr. C.K. Osborne (Houston, TX). All cell lines were authenticated by

short tandem repeat (STR) profiling. Briefly, the cells were seeded in 24-well plates

until approximately 90% confluence, and a fixed amount (74 kBq) of radioactive

tracer (4-[18F]FL or 5-[18F]FHL) was added to each well. The cells were incubated

with the radiotracer at 37 °C for 20, 40 and 60 minutes with gentle agitation.

Blocking experiments were performed using 10 mM 2-amino-2-

norbornanecarboxylic (BCH), a blocker of L-amino acid transport. Following

incubation, each well was washed with ice-cold Hepes buffer. Replicate wells were

used for cell counting. The cells were lysed with 1 M NaOH. The activity in

supernatant, washes and cell lysates was measured using a PerkinElmer Wizard

2480 gamma counter. The activity is reported as the percentage of incubated

activity, and cellular uptake was normalized to cell number.

Biodistribution studies

All animal experiments were conducted in accordance with the guidelines

established by the Canadian Council on Animal Care and approved by the Animal

Care Committee of the University of British Columbia. In vivo biodistribution studies

were performed in healthy mice (n=4 each) to evaluate normal organ uptake of 4-

[18F]FL, 5-[18F]FHL and 5-[18F]FBAHL. Biodistribution studies were also obtained

in immunocompromized mice (NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ) bearing human

glioma (U-87, obtained from ATCC) or prostate cancer cell lines (PC-3) to evaluate

tumor accumulation of 5-[18F]FHL. For tumor bearing mice, 5 x 106 cells were

injected subcutaneously in the dorsal flank of the mice. The tumors were grown to

a diameter of approximately 5-7 mm prior to the biodistribution study. The animals

were lightly sedated with isoflurane, and 1-2 MBq of 18F-labeled radiotracer was

administered intravenously via the caudal lateral tail vein. The radioactivity in the

syringe was measured before and after injection to ensure accurate determination

S19

of the amount injected. The animals quickly recovered from sedation and were

allowed to roam free in the cage during the uptake period. At 15 (U-87 tumors,

n=3) and 60 minutes (U-87, n=4 and PC-3, n=5), the animals were sedated with

isoflurane, sacrificed by CO2 asphyxiation and their blood was collected by cardiac

puncture. The organs were harvested, rinsed with saline, blotted dry, and weighed.

Radioactivity in each organ was measured using a PerkinElmer Wizard 2480

gamma counter, calibrated using a standard curve of 18F. Organ uptake data is

reported as the percentage of injected dose per gram of tissue (%ID/g).

PET/CT imaging

Dynamic PET/CT acquisitions were performed in a distinct set of mice to obtain

representative images and follow the kinetics of uptake in normal organs (4-

[18F]FL) as well as normal organs and tumors (5-[18F]FHL). The mice were sedated

with isoflurane inhalation, a catheter was placed in the caudal lateral tail vein, and

the mice were placed in a preclinical PET/CT scanner (Siemens Inveon). A low-

dose CT scan was performed using 40 kV X-rays at 500 µA. Following CT imaging,

the list-mode dynamic acquisition was started, and the radiotracer was injected

(3.2 – 3.4 MBq). Dynamic scanning was continued for 60 minutes. In addition to

dynamic images, static images were reconstructed at 50-60 minutes using an

iterative reconstruction algorithm (3D-OSEM/MAP).

S20

Supporting Figure 10: Uptake of 4-[18F]FL in LNCaP, PC-3, and MCF-7 cells at 20,

40 and 60 min in the presence (+) and absence (-) of the LAT-1 inhibitor BCH

(10mM). 74 kBq was added to each well, and tracer uptake is normalized to cell

number

S21

Supporting Figure 11: Uptake of 5-[18F]FHL (9) in LNCaP, PC-3, U-87 and MCF-7

cells at 20, 40 and 60 min in the presence (+) and absence (-) of the LAT-1 inhibitor

BCH (10mM). 74 kBq was added to each well, and tracer uptake is normalized to

cell number

S22

Supporting Figure 12: Uptake of 5-[18F]FHL (9) in LNCaP, PC-3, U-87 and MCF-7

cells at 20, 40 and 60 min in the presence (+) and absence (-) of the LAT-1 inhibitor

BCH (10mM). 74 kBq was added to each well, and tracer uptake is normalized to

protein content

S23

Supporting Figure 13: Whole-body maximum intensity projection images overlaid

on CT of the biodistribution of 5-[18F]FHL at 50-60 minutes for the U-87 tumor

xenografts (a and b) and for the PC-3 tumor xenografts (c and d). Cross sectional

images are shown in the insets. Each image represents an individual mouse, and

a red arrow indicates tumor position.

S24

Supporting Figure 14: Biodistribution in selected organs of 5-[18F]FHL at 1 h post

injection in NRG mice bearing U-87 (left bars, purple) and PC-3 (right bars, green)

xenograft tumors.

S25

Supporting Figure 15: Time-activity curves of the accumulation of 5-[18F]FHL in

selected organs, showing rapid accumulation in the U-87 tumor and pancreas

peaking shortly before 10 minutes, followed by a slow progressive efflux of the

amino acid.

S26

5) NMR spectra

Supporting Figure 16: 1H NMR spectrum (600 MHz) of H-PheLeu-OH.TFA in D2O

S27

Supporting Figure 17: 13C NMR spectrum (150 MHz) of H-PheLeu-OH.TFA in D2O

S28

Supporting Figure 18: 1H NMR spectrum (600 MHz) of H-TyrLeu-OH.TFA in D2O

S29

Supporting Figure 19: 13C NMR spectrum (150 MHz) of H-TyrLeu-OH.TFA in D2O

S30

OH

O

NH2

FTFA

Supporting Figure 20: 1H NMR spectra (600 MHz) of 4-FL.TFA in D2O

S31

Supporting Figure 21: 13C NMR spectrum (150 MHz) of 4-FL.TFA in D2O

S32

Supporting Figure 22: 19F spectrum (470 MHz) of 4-FL.TFA in D2O

TFA = -76.6

4-FL = -138.7

S33

Supporting Figure 23: 1H NMR spectra (600 MHz) of 5-FBAHL.TFA in D2O

S34

Supporting Figure 24: 13C NMR spectrum (150 MHz) of 5-FBAHL.TFA in D2O

S35

Supporting Figure 25: 19F NMR spectrum (470 MHz) of 5-FBAHL.TFA in D2O

TFA = -76.6

5-FBAHL = -138.7

S36

Supporting Figure 26: 1H NMR spectra (500 MHz) of 5-FHL.TFA in D2O

S37

Supporting Figure 27: 13C NMR spectrum (150 MHz) of 5-FHL.TFA in D2O

S38

Supporting Figure 28: 19F NMR spectrum (470 MHz) of 5-FHL.TFA in D2O

TFA = -76.6

5-FHL = -136.5

S39

Supporting Figure 29: 1H NMR spectra (600 MHz) of 3-FV.TFA in D2O

S40

Supporting Figure 30: 13C NMR spectrum (150 MHz) of 3-FV.TFA in D2O

S41

Supporting Figure 31: 19F NMR spectrum (470 MHz) of 3-FV.TFA in D2O

TFA = -76.6

3-FV = -141.6

S42

Supporting Figure 32: 19F NMR spectrum (470 MHz) of diasteromeric mixture of

crude 3- and 4-FI.TFA in 3:1 CD3CN:D2O

S43

Supporting Figure 33: 1H NMR spectra (600 MHz) of diasteromeric mixture of 3-

FI.TFA in D2O

S44

Supporting Figure 34: 19F NMR spectrum (470 MHz) of diasteromeric mixture of 3-

FI .TFA in D2O

S45

Supporting Figure 35: 19F NMR spectrum (470 MHz) of diasteromeric mixture of 3-

FI.TFA in D2O

TFA = -76.6

3-FI = -150.5, -152.6

S46

Supporting Figure 36: Timepoints of direct H-PheLeu-OH fluorination as measured

by 1H NMR (500 MHz) in 3:1 CD3CN:D2O (Direct fluorination protocol B). # = H-

PheLeu-OH, $ = H-PheLeu(F)-OH, * = 1,3,5-tris(trifluoromethyl)bezene as internal

standard

t = 0h

t = 1h

t = 2h

t = 3h

t = 4h

t = 6h

#

#

#

#

#

#

$

$

$

$

$ *

S47

Supporting Figure 37: Kinetics of H-PheLeu-OH to H-PheLeu(F)-OH conversion

via direct fluorination protocol B (see above). % Conversion is measured by

analysis of the 1H NMR spectra shown in Supporting Figure 36

0

10

20

30

40

50

60

0 1 2 3 4 5 6 7

% c

on

vers

ion

of

H-P

heL

eu-O

H t

o H

-Ph

eLeu

(F)-

OH

Time (h)

Kinetics of H-PheLeu(F)-OH formation

S48

Supporting Figure 38: 1H NMR spectra (600 MHz) of H-PheLeu(F)-OH.TFA in D2O

S49

Supporting Figure 39: 13C NMR spectra (150 MHz) of H-PheLeu(F)-OH.TFA in

D2O

S50

Supporting Figure 40: 19F NMR spectrum (470 MHz) of H-PheLeu(F)-OH.TFA in

D2O

TFA = -76.6

H-PheLeu(F)-OH = -136.5

S51

Supporting Figure 41: Timepoints of direct H-TyrLeu-OH fluorination as measured

by 1H NMR (500 MHz) in 3:1 CD3CN:D2O. # = H-TyrLeu-OH, $ = H-TyrLeu(F)-

OH, * = 1,3,5-tris(trifluoromethyl)bezene as internal standard

t = 0h

t = 1h

t = 2h

t = 3h

t = 4h

t = 6h

#

#

#

#

#

#

$

$

$

$

$ *

S52

Supporting Figure 42: Kinetics of H-TyrLeu-OH to H-TyrLeu(F)-OH conversion via

direct fluorination protocol B (see above). % Conversion is measured by analysis

of the 1H NMR spectra shown in Supporting Figure 41

0

5

10

15

20

25

0 1 2 3 4 5 6 7

% c

on

vers

ion

of

H-T

yrLe

u-O

H t

o H

-Tyr

Leu

(F)-

OH

Time (h)

Kinetics of H-TyrLeu(F)-OH formation

S53

Supporting Figure 43: 1H NMR spectra (600 MHz) of H-TyrLeu(F)-OH.TFA in D2O

S54

Supporting Figure 44: 13C NMR spectra (150 MHz) of H-TyrLeu(F)-OH.TFA in D2O

S55

Supporting Figure 45: 19F NMR spectrum (470 MHz) of H-TyrLeu(F)-OH.TFA in

D2O

TFA = -76.6

H-TyrLeu(F)-OH = -135.7

S56

Supporting Figure 46: Typical 1H NMR spectrum (500 MHz, D2O) of purified 4-

[18F]FL/leucine mixture after ~100 h decay at –20 oC

S57

Supporting Figure 47: Typical 19F NMR spectrum (470 MHz, D2O) of purified 4-

[18F]FL/leucine mixture after ~100 h decay at –20 oC

4-FL = -138.7

S58

Supporting Figure 48: Typical 1H NMR spectrum (500 MHz, D2O) of purified 5-

[18F]FBAHL/-homoleucine mixture after ~100 h decay at –20 oC

S59

Supporting Figure 49: Typical 19F NMR spectrum (470 MHz, D2O) of purified 5-

[18F]FBAHL/-homoleucine mixture after ~100 h decay at –20 oC

TFA = -76.6

5-FBAHL = -137.9

S60

Supporting Figure 50: Typical 1H NMR spectrum (500 MHz, D2O) of purified 5-

[18F]FHL/homoleucine mixture after ~100 h decay at –20 oC. Doubling of

resonances for methyl groups ( = 1.29) are due to the pH of the elution solvent

(~8.5) (see below)

S61

Supporting Figure 51: 1H NMR spectrum (500 MHz, D2O) of the above mixture

following the addition of HOAc to pH ~4.

S62

Supporting Figure 52: Typical 19F NMR spectrum (470 MHz, D2O) of purified 5-

[18F]FHL/homoleucine mixture after ~100 h decay at –20 oC and following addition

of HOAc to pH ~4.

5-FHL = -136.2

S63

Supporting Figure 53: Typical 1H NMR spectrum (500 MHz, D2O) of purified 3-

[18F]FV/valine mixture after ~100 h decay at –20 oC. Doubling of resonances for

methyl groups ( = 0.85, ~1.4) are due to the pH of the elution solvent (~8.5) (see

below)

S64

Supporting Figure 54: 1H NMR spectrum (500 MHz, D2O) of the above mixture

following the addition of HOAc to pH ~4.

S65

Supporting Figure 55: Typical 19F NMR spectrum (470 MHz, D2O) of purified 3-

[18F]FV/valine mixture after ~100 h decay at –20 oC and following addition of TFA

to pH ~4.

TFA = -76.6

3-FV = -141.6

S66

Supporting Figure 56: 183W NMR spectrum (20.8 MHz, CH3CN) of the sodium

decatungstate (NaDT) used in this study.

S67

6) References:

[1] R. F. Renneke, M. Pasquali, C. L. Hill, J. Am. Chem. Soc. 1990, 112, 6585-

6592.

[2] F. Buckingham, A. K. Kirjavainen, S. Forsback, A. Krzyczmonik, T. Keller,

I. M. Newington, M. Glaser, S. K. Luthra, O. Solin, V. Gouverneur, Angew.

Chem. Int. Ed. 2015, 54, 13366-13369.

Full author list for reference 26 (main text):

[3] J. Barretina, G. Caponigro, N. Stransky, K. Venkatesan, A.A. Margolin, S.

Kim, C. J. Wilson, J. Lehár, G. V. Kryukov, D. Sonkin, A. Reddy, M. Liu, L.

Murray, M. F. Berger, J. E. Monahan, P. Morais, J. Meltzer, A. Korejwa, J.

Jané-Valbuena, F. A. Mapa, J. Thibault, E. Bric-Furlong, P. Raman, A.

Shipway, I. H. Engels, J. Cheng, G. K. Yu, J. Yu, P. Aspesi, M. de Silva, K.

Jagtap, M. D. Jones, L. Wang, C. Hatton, E. Palescandolo, S. Gupta, S.

Mahan, C. Sougnez, R. C. Onofrio, T. Liefeld, L. MacConaill, W. Winckler,

M. Reich, N. Li, J. P. Mesirov, S. B. Gabriel, G. Getz, K. Ardlie, V. Chan,

V. E. Myer, B. L. Weber, J. Porter, M. Warmuth, P. Finan, J. L. Harris, M.

Meyerson, T. R. Golub, M. P. Morrissey, W. R. Sellers, R. Schlegel & L. A.

Garraway, Nature 2012 483, 603-607.