Microfluidic radiosynthesis and biodistribution of [18F] 2-(5-fluoro-pentyl)-2-methyl malonic acid

Article abstract of DOI:10.1002/jlcr.3016

Microfluidics technology has emerged as a powerful tool for the radiosynthesis of positron emission tomography (PET) and single-photon emission computed tomography radiolabeled compounds. In this work, we have exploited a continuous flow microfluidic system (Advion, Inc., USA) for the [ ¹⁸F]-fluorine radiolabeling of the malonic acid derivative, [ ¹⁸F] 2-(5-fluoro-pentyl)-2-methyl malonic acid ([¹⁸F]- FPMA), also known as [¹⁸F]-ML-10, a radiotracer proposed as a potential apoptosis PET imaging agent. The radiosynthesis was developed using a new tosylated precursor. Radiofluorination was initially optimized by manual synthesis and served as a basis to optimize reaction parameters for the microfluidic radiosynthesis. Under optimized conditions, radio-thin-layer chromatography analysis showed 79% [¹⁸F]-fluorine incorporation prior to hydrolysis and purification. Following hydrolysis, the [¹⁸F]-FPMA was purified by C18 Sep-Pak, and the final product was analyzed by radio-HPLC (high-performance liquid chromatography). This resulted in a decay-corrected 60% radiochemical yield and ≥98% radiochemical purity. Biodistribution data demonstrated rapid blood clearance with less than 2% of intact [ ¹⁸F]-FPMA radioactivity remaining in the circulation 60 min post-injection. Copyright

Full text of DOI:10.1002/jlcr.3016

Research Article
Received 13 November 2012,
Revised 29 November 2012,
Accepted 30 November 2012
Published online 12 February 2013 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3016
Microfluidic radiosynthesis and
biodistribution of [18F] 2-(5-fluoro-pentyl)-2-
methyl malonic acid†
Gajanan K. Dewkar,a Gobalakrishnan Sundaresan,a
Narottam Lamichhane,a Jerry Hirsch,a Celina Thadigiri,a Thomas Collier,b
Matthew C. T. Hartman,c,d Ganesan Vaidyanthan,e and Jamal Zweita,d
*
Microfluidics technology has emerged as a powerful tool for the radiosynthesis of positron emission tomography (PET)
and single-photon emission computed tomography radiolabeled compounds. In this work, we have exploited a continuous
flow microfluidic system (Advion, Inc., USA) for the [18F]-fluorine radiolabeling of the malonic acid derivative, [18F] 2-(5-
fluoro-pentyl)-2-methyl malonic acid ([18F]-FPMA), also known as [18F]-ML-10, a radiotracer proposed as a potential
apoptosis PET imaging agent. The radiosynthesis was developed using a new tosylated precursor. Radiofluorination was
initially optimized by manual synthesis and served as a basis to optimize reaction parameters for the microfluidic
radiosynthesis. Under optimized conditions, radio-thin-layer chromatography analysis showed 79% [18F]-fluorine incorpora-
tion prior to hydrolysis and purification. Following hydrolysis, the [18F]-FPMA was purified by C18 Sep-Pak, and the final
product was analyzed by radio-HPLC (high-performance liquid chromatography). This resulted in a decay-corrected 60%
radiochemical yield and ≥98% radiochemical purity. Biodistribution data demonstrated rapid blood clearance with less than
2% of intact [18F]-FPMA radioactivity remaining in the circulation 60 min post-injection. Most organs showed low accumula-
tion of the radiotracer, and radioactivity was predominately cleared through kidneys (95% in 1 h). Radio-HPLC analysis of
plasma and urine samples showed a stable radiotracer at least up to 60 min post-injection.
Keywords: microfluidic technology; [18F]-FPMA; [18F]-ML-10; PET radiochemistry
using this microfluidic technology. These include the 18F-labeled
Introduction
compounds [18F]-fludeoxyglucose,1,5–8 30-deoxy-30-[18F]fluorothymi-
dine,9 1-(5-[18F]fluoro-5-deoxy-a-D-arabinofuranosyl)-2-nitroimida-
Recently, microfluidics technology has emerged as a powerful
zole,10 20-[18F]fluoro-20-deoxy-1b-D-arabinofuranosyl-5-iodouracil,11
tool for the radiosynthesis of radiolabeled compounds for
positron emission tomography (PET). The high surface-to-volume
[
18F]fallypride,12 18F]altanserin,13 18F]fluroethyl-di-tosylate,14
[ [
and O-(2-[18F]fluoroethyl)-L-tyrosine.15 The system has also
been utilized for [11C]-carbonylation reactions3 and more
recently for [13N]-labeled radiotracers.4 Radiometal synthesis,
using [64Cu] DOTA-cyclo peptide (RGDfK), has also been
reported using similar microfluidic technology.16
ratios offered by this technology provide many advantages
for more efficient radiochemical synthesis. Compared with
conventional radiochemistry, microfluidic devices allow the
use of smaller amounts of precursor and still achieve high
radiochemical yields (RCYs) with less radiochemical impurities.1
This results in simplification of purification steps and increased
specific activity. Microfluidic devices also offer possibilities for
increased reaction speed, improve reproducibility, and reduce
costs. The application of microfluidic technology to radiochemis-
try has been demonstrated for the radiosynthesis of [11C]-carbon,
[18F]-fluorine, and [13N]-nitrogen PET radiotracers.2–4 Another
important feature of microfluidic radiochemistry is the ability to
vary reaction parameters, during radiosynthesis development,
and hence enable faster optimization of different reaction
conditions. This is particularly advantageous for PET radiochemis-
try with short half-life radionuclides.
aCenter for Molecular Imaging, Department of Radiology, Virginia Common-
wealth University, Richmond, VA 23298, USA
bAdvion, Inc., Ithaca, NY, USA
cDepartment of Chemistry, Virginia Commonwealth University, Richmond, VA,
USA
dMassey Cancer Center, Virginia Commonwealth University, Richmond, VA, USA
eDepartment of Radiology, Duke University School of Medicine, Durham NC,
27710, USA
A continuous flow microfluidic system has been developed
and marketed by Advion, Inc., USA (see Figure 2 for schematic
diagram). Radiochemical reactions at high pressure can be
performed on this system, resulting in faster incorporation of
the radiolabel into the final radiotracer, using a small amount
of precursor. A number of PET radiotracers have been synthesized
*Correspondence to: Jamal Zweit, Center for Molecular Imaging, 1101 E. Marshall
Street, Sanger Hall, Room 8-022, PO Box 980031, Richmond, VA 23298-0031, USA.
E-mail: jzweit@vcu.edu
† Supporting information may be found in the online version of this article.
J. Label Compd. Radiopharm 2013, 56 289–294
Copyright © 2013 John Wiley & Sons, Ltd.

G. K. Dewkar et al.
The fluorine-18 labeled malonic acid derivative, 2-(5-[18F] confirmed by 19F-NMR, which showed the expected multiplet
fluoro-pentyl)-2-methyl malonic acid, has been synthesized pattern and had a chemical shift in the expected region (ꢀ218.88
including its automated ‘TRACERLab’ synthesis17–19 and reported for terminal (CH2F)). The calculated molecular mass for purified 2-
as a potential apoptosis imaging agent.20–23 In this manuscript, (5-fluoro-pentyl)-2-methyl malonic acid was in agreement with
we report on the microfluidic synthesis of this radiotracer, using experimental value. Chemical purity was found to be greater than
a new tosylated precursor and its in vivo biodistribution and 98% as determined by analytical HPLC.
stability profile.
Manual radiosynthesis of [18F] 2-(5-fluoro-pentyl)-2-methyl
Results and discussion
malonic acid
Synthesis [19F] 2-(5-fluoro-pentyl)-2-methyl malonic acid (4)
Scheme 2 represents manual radiosynthesis of [18F] 2-(5-fluoro-
pentyl)-2-methyl malonic acid ([18F]-FPMA) in two steps: (i)
Kryptofix-mediated direct nucleophilic fluorination and (ii) NaOH
base hydrolysis.
Scheme 1 describes the synthesis of the tosylated precursor and
reference standard compound [19F] 2-(5-fluoro-pentyl)-2-methyl
malonic acid (4). The reference non-radioactive compound 4
was synthesized in four steps starting with commercially
available diethyl methylmalonate. Diethyl methylmalonate was
alkylated with 1,5-dibromopentane in presence of NaH to give
alkylated compound 1 in 72% yield using the procedure of Astles
et al.24 Excess 1,5-dibromopentane was used to avoid disubstitu-
tion. The bromide was displaced using commercially available
silver tosylate to give a tosylated precursor 2 in 96% yield. The
tosylate group was exchanged for the required fluoride by a
nucleophilic displacement reaction using tetrabutylammonium
fluoride (TBAF) to give diethyl 2-(5-fluoropentyl)-2-methylmalonate
(3) in 84% yield. Finally, fluoropentyl diethyl malonate ester 3 was
hydrolyzed using lithium hydroxide under mild conditions to
give 2-(5-fluoro-pentyl)-2-methyl malonic acid as a white solid
with 50% overall yield. All the intermediate compounds and
final compound were fully characterized by 1H-NMR, 13C-NMR,
19F-NMR, and ESI-MS analysis. Fluorination of compound 3 was
In optimization studies, radiofluorination of 2 was investigated
as a function of three parameters: mass of precursor, tempera-
ture, and reaction time. The reaction was monitored over an
interval of 5, 10, 15, and 20 min. In the presence of Kryptofix
and K2CO3 in acetonitrile, optimum conditions of 4-mg precur-
sor, 110 ꢁC, and 10 min. reaction time resulted in >90% RCY of
non-hydrolyzed compound 5 as confirmed by radio-thin-layer
chromatography (TLC) analysis. This is comparable with the
value obtained in a previously described method.25 After radiola-
beling, the acetonitrile was evaporated with helium flow under
vacuum, and the product was subjected to base hydrolysis.
Complete hydrolysis was achieved by methanolic sodium hydro-
xide (3 M) in dichloromethane (DCM)/methanol (9:1) for 20 min at
45 ꢁC, which is comparable with the hydrolysis efficiency of
malonic ester derivatives.26 The final product was purified by
solid phase extraction. After evaporation of the solvent, under
vacuum and helium flow, the product was re-dissolved in water
and adjusted to pH 2–3 using 3 M HCl. The reaction mixture was
passed through a C18 Sep-Pak column, and the column was
washed with 10 mL of water to remove free fluoride. The product
was eluted with multiple 0.5 mL ethanol fractions. Greater than
90% of product radioactivity was eluted in fractions 3 and 4.
The overall decay-corrected (yield of final product, 50 min after
introduction of [18F]-fluoride activity) isolated RCY of [18F]-FPMA
was 60%, and the radiochemical purity (RCP) was more than 98%
in 50 min total synthesis time. RCP was confirmed by radio-HPLC.
Co-injections with non-radioactive fluorinated compound gave
the same retention time as [18F]-FPMA = 2.7 min.
Synthesis
Microfluidic radiosynthesis
Scheme 1. Synthesis of cold reference compound [
19F]-FPMA and tosylate
A major goal of this work was to develop an automated microflui-
dic platform for rapid, robust synthesis of [18F]-FPMA. Scheme 2
represents the microfluidic radiosynthesis of [18F]-FPMA carried
precursor of [18F]-FPMA. Reagents and conditions: (a) 1,5-dibromopentane, NaH,
DMF, 50 ꢁC, 12 h, 72%; (b) silver tosylate, ACN, reflux, 12 h, 96%; (c) TBAF, THF,
60 ꢁC, 3 h, 84%; (d) LiOH/H2O THF/MeOH/H2O (6:3:1), rt, 24 h, 86%.
Scheme 2. Synthesis of [18F]-FPMA. Reagents and conditions: (a) K18F/K222, K2CO3, ACN, 110 ꢁC, 10 min condition used for manual synthesis; (b) glass microfluidic device,
K18F/K222, K2CO3, ACN, 190 ꢁC, condition used in automated microfluidic synthesis; (c) 3 mol NaOH, DCM/MeOH (9:1), 45 ꢁC, 20 min; (d) 3 M HCl, in manual and sodium
citrate buffer (pH 2.79) in microfluidic synthesis, was used for pH adjustment (pH 2–3).
www.jlcr.org
Copyright © 2013 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2013, 56 289–294

G. K. Dewkar et al.
out in two steps: (i) Kryptofix-mediated direct nucleophilic in normal female nude mice. The stability of [18F]-FPMA was
fluorination and (ii) NaOH base hydrolysis. Under optimum analyzed by radio-TLC and radio-HPLC at 1, 5, 30, 60, and
conditions of 100 mL ꢂ 2 m reactor loop, 50 mL/min flow rate, 120 min post-administration. The radio-HPLC peak appeared
and 190 ꢁC temperature, radio-TLC analysis showed a non- at the same retention time as the non-radioactive reference
purified RCY approaching 79% (Table 1).
compound, [19F]-FPMA, demonstrating an intact [18F]-FPMA,
Optimized condition/s for hydrolysis were found to be heating 60min post-injection. No radioactivity was detected in 120-min
the reaction to 50 ꢁC for 20 min. The final product was separated samples, which could be attributed to the fast physiological
by C18 Sep-Pak purification method as described earlier. This clearance of the radiotracer from the blood.
resulted in 60% decay-corrected RCY of purified product, in a
total reaction time of 50 min. Radio-HPLC analysis showed 99%
RCP of final product (Figure 5 in Supporting Information).
Experimental
Reagents and instrumentation
Biodistribution and in vivo stability analysis
Figure 1 shows the biodistribution of [18F]-FPMA in female nude
mice. The radiotracer demonstrated rapid blood clearance with
less than 2% of radioactivity remaining in the circulation after
1 h. The blood half-life was determined to be 13 min (Figure 1
inset). Most organs show low accumulation of the radiotracer,
and radioactivity was predominantly cleared through the kidneys
with greater than 95% cleared 1 h after injection.
All reagents and solvents were purchased from Sigma-Aldrich and used
without further purification unless otherwise indicated. All cold reactions
were performed under argon atmosphere in oven-dried glassware. Flash
chromatography was performed employing Sigma-Aldrich 230-400 mesh
60 Å silica gel. TLC (Sigma-Aldrich, Saint Louis, MO) was performed using
silica gel-coated aluminum plates with F-254 indicator (250 mm, 20 cm
20 cm, Whatman). NMR (Piscataway, NJ) spectra (1H-NMR, 13C-NMR,
and 19F-NMR) were obtained using Varian Mercury 300 MHz and Varian
Inova 400 MHz (Sigma-Aldrich, Saint Louis, MO) using tetramethylsilane
as an internal standard. Melting points were determined using a Thomas
Hoover melting point apparatus and are uncorrected. Mass spectra were
recorded on an Orbitrap Velos mass spectrometer. Aqueous [18F]-fluoride
was produced by the 18O(p,n)18F reaction, in a PET tracer cyclotron (GE
Medical Systems, Wausheka, WI, USA), by the irradiation of an isotopically
enriched [18O] water (Rotem Industries, Ber Sheva, Israel) target. For
fluoride trapping, Sep-Pak LightW quaternary methyl ammonium (QMA)
cartridge was used in manual synthesis from Waters Corp. (Milford, MA,
USA) and Micro-SPE cartridges mini-trap used in microfluidic synthesis
from ORTG (TN, USA). Microfluidic radiosynthesis was performed using
NanoTek microfluidic synthesis system Advion. In the final product purifica-
tion, C-18 a Sep-pak cartridge was used from Waters Corp. Radio-HPLC sta-
bility analysis was carried using a Waters HPLC pump (Waters, Model 1525)
equipped with UV detector (Waters, Model 2489) and radiation detector
(Bio-scan, Model B-FC-3300) connected in series. For reversed-phase HPLC,
a WatersW Nova-Pak 4m C18 150mmꢂ 3.9mm column was eluted isocrati-
cally with 25/75 I/Water containing 0.1% TFA at flow rate of 1mL/min.
Radio-TLC was performed on a radio-TLC scanner (BioScan, Model AR/
2000). Radioactivity was measured with dose calibrator Captintec CRC-15
PET. Animals, normal adult athymic female nude mice, were purchased
from Harlan Laboratories, USA.
The in vivo stability of [18F]-FPMA in plasma and urine was
assessed during the first 120min after intravenous administration
Table 1. Optimization of RCY for microfluidic synthesis of
[18F]-diethyl 2-(5-fluoropentyl)-2-methylmalonate. Reaction
conditions: (i) microreactor volume = 16 mL; (ii) volume ratio
(P1:P3 = 1), starting radioactivity = 10–15 mCi
Synthesis
no.
Reactor
P3 flow rate
(mL/min)
Unisolated
yield (%)
temp. (ꢁC)
1
2
3
4
5
6
7
8
100
125
150
175
170
180
180
190
30
30
30
30
50
50
75
50
0
6
23
63
32
78
46
79
Figure 1. Biodistribution of [18F]-FPMA in normal adult female nude mice (n = 3) at 1, 5, 30, 60, and 120 min after injection. Radio tracer uptake in %ID/g was determined
by gamma counting.
J. Label Compd. Radiopharm 2013, 56 289–294
Copyright © 2013 John Wiley & Sons, Ltd.
www.jlcr.org

G. K. Dewkar et al.
Non-radioactive reference compound 2-(5-fluoropentyl)-2-
methylmalonic acid (4)
Synthesis of cold reference compound 2-
(5-fluoropentyl)-2-methylmalonic acid (4)
and intermediate (Scheme 1)
To a stirred solution of fluoropentyl malonate 3 (310mg, 1.5 mmol)
in THF/MeOH/H2O (6:3:1, 15 mL), lithium hydroxide monohydrate
(631 mg, 15mmol) was added. The reaction mixture was stirred
24 h at room temperature. After the reaction was complete, the
organic solvents were removed under reduced pressure. The
aqueous layer was acidified with oxalic acid solution (1mol) to
pH 2–3 and then extracted with diethyl ether (3mLꢂ 25 mL). The
combined organic layers were dried over anhydrous sodium
sulfate and then concentrated under reduced pressure. The
residue was dissolved in chloroform, the undissolved residue was
filtered, and the filtrate was concentrated under reduced pressure
to give reference compound ([19F]-FPMA) 4 (208 mg, 86%) as
a white solid: mp 102–104 ꢁC: 1H-NMR (300MHz) (CDCl3) d
1.32–1.47 (m, 4H), 1.49 (s, 3H), 1.65–1.78 (m, 2H), 1.90–1.94 (m,
2H), 4.37 (t, J = 6.05 Hz, 1H), 4.49 (t, J = 6.05 Hz, 1H) 10.48 (s, 2H);
13C-NMR (75 MHz) (CDCl3) d 20.09, 24.22, 25.59, 25.66, 30.14,
30.40, 35.63, 54.03, 83.04, 85.22, 178.43; 19F-NMR (300 MHz) (CDCl3)
(coupled) d ꢀ218.88 (m); ESI-MS [M+ Na]+ m/z calcd for
C9H15FO4 + Na 229.09, found 229.09. 1H-NMR, 13C-NMR, and 19F-NMR
spectra are shown in Figure 1.
Diethyl 2-(5-bromopentyl)-2-methylmalonate (1)
A solution of diethyl methylmalonate (6.0 g, 34.5 mmol) in dry
dimethylformamide (50 mL) was treated portionwise with
sodium hydride (1.5 g of a 60% dispersion, w/w, in mineral oil,
38 mmol) with cooling. The reaction mixture was stirred for 30 min
at ambient temperature and treated with 1,5-dibrompentane
(11.9 g, 51.8 mmol). The reaction mixture was heated at 50 ꢁC
overnight and quenched by adding 30% ammonium hydroxide
solution. Distilled water (50 mL) was added in to the reaction
mixture and extracted with DCM (3 mL ꢂ 50 mL). The organic layer
was washed with brine, dried over anhydrous sodium sulfate, and
concentrated under reduced pressure. The residue was purified by
flash chromatography using 5% ethyl acetate in hexane to give alkyl
malonate 1 as colorless viscous oil (8.0 g, 72%) (Scheme 1): 1H-NMR
d 1.20–1.33 (m, 2H), 1.25 (t, J= 7.32 Hz, 6H), 1.40 (s, 3H), 1.42–1.51 (m,
2H), 1.81–191 (m, 4H), 3.39 (t, J= 6.73 Hz, 2H), 4.17 (q, J= 7.32 Hz, 4H);
13C-NMR (75 MHz) (CDCl3) d 13.9, 19.7, 23.3, 28.2, 32.3, 33.4, 35.1,
53.4, 60.9, 172.1; ESI-MS [M + Na]+ m/z calcd for C13H23BrO4 + Na
345.07, found 345.08.
Radiosynthesis of [18F] 2-(5-fluoro-pentyl)-2-methyl malonic
acid (Scheme 2)
Diethyl 2-methyl-2-(5-(tosyloxy)pentyl)malonate (2)
To a solution of alkyl malonate 1 (3.2 g 10 mmol) in dry
acetonitrile (30 mL), silver tosylate (3.48 g, 12.5 mmol) was added.
After the reaction mixture was refluxed for 24 h under an argon
atmosphere, the reaction mixture was filtered through celite to
remove the silver salt, and the solvent was evaporated under
reduced pressure. The residue obtained from the solvent
evaporation was purified by flash chromatography using 10%
ethyl acetate in hexane to give tosylate precursor 2 as a colorless
viscous oil (3.9 g, 96%): 1H-NMR (300 MHz) (CDCl3) d 1.18–1.25
(m, 2H), 1.23 (t, J = 7.22 Hz, 6H), 1.26–1.35 (m, 2H), 1.36 (s, 3H),
1.61–1.68 (m, 2H), 1.77–1.81 (m, 2H), 2.45 (s, 3H), 4.00
(t, J = 6.44 Hz, 2H), 4.16 (q, J = 7.22 Hz, 4H), 7.35 (d, J = 8.19 Hz,
2H) 7.78 (d, J = 8.19 Hz, 2H); 13C-NMR (75 MHz) (CDCl3) d
13.7, 19.4, 21.1, 23.2, 25.2, 28.1, 34.8, 53.1, 60.7, 70.1, 127.4,
129.6, 132.8, 144.4, 171.7; ESI-MS [M + Na]+ m/z calcd for
C13H23BrO4 + Na 437.16, found 437.43.
Radiosynthesis of [18F]-FPMA was carried out using tosylate
precursor 2. The QMA cartridge was preconditioned with 10 mL
(0.25 mol) potassium carbonate solution followed by 20 mL
sterile water. [18F]-fluoride was then passed through a QMA
Sep-Pak cartridge (carbonate form) to remove [18O] H2O. The
trapped [18F]-fluoride was eluted with a solution of 0.08 mL of
potassium carbonate (0.25 M) and 0.42 mL of sterile water into
a 10 mL V-shaped reaction vial containing a solution of Kryptofix
12 mg in dry acetonitrile 0.8 mL. The reaction mixture was dried
with the addition of dry acetonitrile (3 ꢂ 1 mL) under vacuum
and helium flow at 80 ꢁC. To the dried [18F]-fluoride, tosylate
precursor 2 (4 mg) in acetonitrile (0.8 mL) was added, and the
reaction mixture was heated at 110 ꢁC for 10 min in the sealed
10-mL V-shaped vial with stirring. The reaction mixture was
cooled for 5 min and the solvent evaporated under vacuum
and helium flow at room temperature. Base hydrolysis was
accomplished by the addition of methanolic NaOH solution
(3 M, 500 mL) in 2 mL of DCM/MeOH (9:1) followed by stirring
for 20 min at 45 ꢁC. The solvent was then evaporated under
vacuum and helium flow at room temperature. Water (1 mL)
was added and the reaction mixture adjusted to pH 2–3 using
3 M HCl. The reaction mixture was passed through a C18 Sep-
Pak column that was preconditioned with 10 mL water, 10 mL
ethanol, and 10 mL water, respectively. The compound was
eluted with ethanol in 6 mL ꢂ 0.5 mL fractions; most of the
activity was observed in fractions 3 and 4. Ethanol was evapo-
rated at 45 ꢁC under vacuum and helium flow, and the residue
was redissolved in PBS. The [18F]-FPMA was analyzed by radio-
HPLC using the following conditions: Waters Nova-Pak 4 m C18
150 mm ꢂ 3.9 mm, CH3CN/H2O 25:75, containing 0.1% TFA at
flow rate 1 mL/min, UV 218 nm. The radiochemical identity
was confirmed by co-injections with non-radioactive standard
fluorinated compound 4 and gave the same retention time as
[18F]-FPMA = 2.7 min.
Diethyl 2-(5-fluoropentyl)-2-methylmalonate (3)
To a solution of tosylate 2 (2.1 g, 5 mmol) in dry THF (20 mL), a
solution TBAF (1 M solution in THF, 1.58 g, 6 mL, 6 mmol) was
added dropwise under argon atmosphere. The reaction mixture
was stirred at 60 ꢁC for 3 h. The reaction mixture was cooled to
room temperature, and the solvent was evaporated under
reduced pressure. The product obtained from the solvent eva-
poration was purified by flash chromatography using 10% ethyl
acetate in hexane to give fluoropentyl malonate 3 as a light
yellow colored viscous oil (1.1 g, 84%): 1H-NMR (300 MHz) (CDCl3)
d 1.24 (t, J = 7.32 Hz, 6H), 1.25–1.34 (m, 2H), 1.40 (s, 3H), 1.36–1.48
(m, 2H), 1.61–1.78 (m, 2H), 1.83–1.89 (m, 2H), 4.17 (q, J = 7.32 Hz,
4H), 4.34 (t, J = 6.15 Hz, 1H), 4.50 (t, J = 6.15 Hz, 1H); 13C-NMR
(75 MHz) (CDCl3) d 13.87, 19.66, 23.79, 25.33, 25.40, 29.89, 30.15,
35.25, 53.43, 60.92, 82.49, 84.68, 172.12; ESI-MS [M + Na]+ m/z
calcd for C13H23FO4 + Na 285.15, found 285.31.
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Copyright © 2013 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2013, 56 289–294

G. K. Dewkar et al.
Automated microfluidic radiosynthesis of [18F] 2-(5-fluoro-
pentyl)-2-methyl malonic acid (Scheme 2)
(10 mCi) and tosylate precursor (4 mg in 750 mL) were loaded in
their respective storage loop in the microfluidic reactor appara-
tus. The [18F]fluorination reaction was optimized in terms of tem-
perature, flow rate, and flow ratio of reactants in the discovery
mode. The temperature was varied from 100 to 190 ꢁC, fluoride
flow rate from 30 to 75 mL/min, and maintained the precursor/
fluoride flow ratio 1:1 as shown in Table 1. Samples were
collected after each discovery run (every ~3 min) and analyzed
on radio-TLC. On the basis of synthesis entry no. 8, we observed
that 190 ꢁC with a P3 flow rate of 50 mL/min flow rate gave a
maximum yield 79% of intermediate 5. Radiochemical yields
were calculated from TLC analysis. Radio-TLC was performed
on silica gel layers (Waters) developed with ethyl acetate/hexane
(3:7 v/v).
The radiosynthesis of [18F]-FPMA was conducted using a NanoTek
Microfluidic Synthesis System (Advion) shown in Figure 2, which
consists of two concentrator modules, one reactor module, and
one base module and was controlled by a standard laptop running
with the NanoTek software 1.4 release [11].27
Preparation of [18F]fluoride ion reagent
Cyclotron-produced [18F]fluoride ion in [18O]water (1.7 ꢃ 0.1 mL)
was passed through a preconditioned (with 500 mL water) small
anion-exchange (MP-1) column by aspirating 3 ꢂ 1 mL; this opera-
tion was also used to dry the lines of water. The trapped 18F fluoride
activity was eluted with a solution containing 30 mg of Krypto-
fix2.2.2 (K222) and 4.5 mg of K2CO3 in 1 mL of 90% CH3CN/water
into a V-vial preheated to 110 ꢁC. Nitrogen flow was applied to
the reactor while delivering the 18F solution to facilitate solvent
evaporation; flow parameters were optimized in order to keep the
volume at a maximum of 300 mL of solution throughout the whole
process, so that activity was collected at the bottom of the 3 mL
V-vial. The solution was then evaporated to dryness. Additionally,
three aliquots of 0.1 mL of anhydrous CH3CN were added dropwise
to the vial for further azeotropic distillation through all of the fluor-
ide drying lines. After this stage, only vacuum was applied for 1 min
at 110 ꢁC and 1 min after stopping the heating. The dried complex
was then reconstituted with 0.50 mL of CH3CN according to the
experimental protocol. The whole drying procedure took 12 min.
Production of [18F] 2-(5-fluoro-pentyl)-2-methyl malonic acid
in microfluidic reactor
The optimized conditions described earlier were implemented in
scaled up batch production of the radiotracer. Dry [18F]FꢀK+-
K222 solution (286 mCi in 500 mL I) and tosylate precursor (4 mg
in 750 mL I) were loaded in their respective storage loop (439-mL
storage loop (P1) for tosylate precursor and a 403-mL loop (P3)
for the K222–fluoride complex) in the microfluidic reactor appara-
tus. Solutions from each loop (50 mL) were infused simultaneously
at 50 mL/min into the reactor (100mm ꢂ 2 m) held at a temperature
of 190 ꢁC. The reaction mixture was collected in concentrator 2
in 2 mL methanolic NaOH solution (0.5mL, 3 M, 500uL) and
DCM/MeOH (9:1) (1.5mL), and this was heated to 45 ꢁC for
20min. After hydrolysis, the solvent was evaporated by blowing
nitrogen, and 2 mL sodium citrate buffer (pH 2.79) was added
through concentrator 2. At a pH between 2 and 3, the reaction
mixture was passed through a pre-conditioned C18 Sep-Pak
Optimization of [18F]-diethyl 2-(5-fluoropentyl)-2-
methylmalonate (5) synthesis in the microreactor
Microfluidic optimization experiments were conducted using column. Unreacted fluoride was washed with water, and the
low activity (10–15 mCi of 18F). Dry [18F]FꢀK+-K222 solution final product was eluted with ethanol. The final product
Figure 2. A schematic diagram of microfluidic apparatus.
J. Label Compd. Radiopharm 2013, 56 289–294
Copyright © 2013 John Wiley & Sons, Ltd.
www.jlcr.org

G. K. Dewkar et al.
[18F]-FPMA was analyzed by radio-HPLC using the same conditions
as described earlier. It is by using the automated microfluidic
synthesis that a 60% RCY and greater than 98% RCP of [18F]-FPMA
were achieved in less than 50 min. This procedure resulted in a
batch yield of 124 ꢃ 5 mCi, 60%.
Conflict of Interest
The authors did not report any conflict of interest.
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Acknowledgement
This work was supported by Virginia Commonwealth University
School of Medicine. We would like to thank Bob Ylimaki, IBA
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www.jlcr.org
Copyright © 2013 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2013, 56 289–294