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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
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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
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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.
S35
Supporting Figure 25: 19F NMR spectrum (470 MHz) of 5-FBAHL.TFA in D2O
TFA = -76.6
5-FBAHL = -138.7
S42
Supporting Figure 32: 19F NMR spectrum (470 MHz) of diasteromeric mixture of
crude 3- and 4-FI.TFA in 3:1 CD3CN: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
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
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
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[1] R. F. Renneke, M. Pasquali, C. L. Hill, J. Am. Chem. Soc. 1990, 112, 6585-
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[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.
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Mahan, C. Sougnez, R. C. Onofrio, T. Liefeld, L. MacConaill, W. Winckler,
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V. E. Myer, B. L. Weber, J. Porter, M. Warmuth, P. Finan, J. L. Harris, M.
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