Preparation and evaluation of reagents for tagging amino and thiol groups with fluorous stannanes. A convenient method for producing radioiodinated compounds in high effective specific activity

Article abstract of DOI:10.1002/jlcr.1807

Building on the previously reported fluorous labeling strategy (FLS), a new approach for preparing molecular imaging and therapy agents derived from radioiodine in high purity without the need to employ HPLC was developed. A series of novel reagents conta

Full text of DOI:10.1002/jlcr.1807

Research Article
Received 30 March 2010,
Revised 28 May 2010,
Accepted 3 June 2010
Published online 5 August 2010 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1807
Preparation and evaluation of reagents for
tagging amino and thiol groups with fluorous
stannanes. A convenient method for producing
radioiodinated compounds in high effective
specific activityy
Ã
Amanda C. Donovan, and John F. Valliant
Building on the previously reported fluorous labeling strategy (FLS), a new approach for preparing molecular imaging and
therapy agents derived from radioiodine in high purity without the need to employ HPLC was developed. A series of novel
reagents containing a fluorous arylstannane, including fluorous benzaldehydes (1a/b) and an aryl-iodoacetamide (2), were
prepared so that the FLS could be used to label and purify targeting vectors that contain free amines or thiol groups. The
reagents were conjugated to model amines and thiols in generally high yields (79–95%) under mild conditions. The fully
characterized products were radiolabeled with Na[125I] in the presence of iodogen and the products were purified using
fluorous solid-phase extraction to yield the desired iodinated products in high yield (483%) and high effective specific
activity (ESA). The work reported creates a convenient and flexible means of preparing targeted molecular imaging and
therapy agents derived from radioisotopes of iodine in high ESA.
Keywords: fluorous chemistry; molecular imaging; high effective specific activity; radioiodination
the use of fluorous supports.8 In this work, fluorine-rich
substituents are used in place of the polymer in solid-phase
Introduction
Existing methods used to prepare radiopharmaceuticals involve
the use of a large excess of the compound to be labeled
compared with the amount of radionuclide. This can be
problematic in the case of molecular imaging probes1,2 that
are designed to seek out protein targets that have relatively low
expression levels. The large excess of unlabeled ligand can
compete with the labeled form for the limited number of target
sites, consequently the reaction mixture in these cases must be
purified prior to use.2–4
Purification is normally performed using HPLC, which is
acceptable for research studies but is less desirable for routine
clinical production. A number of alternative techniques have
been developed to address the excess ligand issue including
solid-phase labeling. In solid-phase labeling, a precursor is
attached to a cross-linked polymer through an arylstannane.5–7
On addition of radioiodine and an oxidant, which cleaves the
polymer-tin-aryl bond, the desired product is released into
solution and the precursor is removed by filtration. Although
highly effective in removing excess ligand, solid-phase labeling
has a number of challenges including difficulties in conjugating
precursors to the polymer support quantitatively and the
inability to remove any polymer-bound impurities prior to
labeling.
labeling. On reaction with radioiodine and an oxidant, the
fluorous-tin aryl bond is cleaved and the product is separated
from the precursor by passing the reaction mixture through
a fluorine-rich solid-phase extraction (FSPE) cartridge. In FSPE,
the precursor and all fluorous byproducts are retained while
the desired compound is selectively eluted. This type of
chemoselective filtration has been used to produce a library of
iodobenzamides in high yield and high purity in mere minutes
without the need to employ HPLC.8 The fluorous labeling
strategy (FLS) has also been used to produce clinically relevant
radiopharmaceuticals including meta-iodobenzylguanidine and
5-iodo-20-deoxyuridine in high ESA using different isotopes
of iodine.9,10
The FLS has been used predominately for tagging small
molecules. Given the rapidly expanding use of peptides and
biomolecules as targeting vectors, it would be beneficial to have
fluorous-tin synthons for tagging amino and thiol groups found
on these vectors as a means to preparing and purifying the
Department of Chemistry, McMaster University, 1280 Main St. West, Hamilton,
Ont., Canada L8S 4M1
*Correspondence to: John F. Valliant, Department of Chemistry, McMaster
University, 1280 Main St. West, Hamilton, Ont., Canada L8S 4K1.
E-mail: valliant@mcmaster.ca
A new solution phase technique for producing and purifying
radioiodinated compounds in high effective specific activity
(ESA), which addresses these limitations was developed through
ySupporting information may be found in the online version of this article.
J. Label Compd. Radiopharm 2011, 54 65–71
Copyright r 2010 John Wiley & Sons, Ltd.

A. C. Donovan and J. F. Valliant
the gamma detector was 1 s in a 10-ml loop. The elution
conditions were as follows: Method A: Solvent A = CH3CN
containing 0.1% HOAc, Solvent B = H2O containing 0.1% HOAc;
Elution conditions: 60% A 0–2 min; 60–100% A 2–15 min; 100%
A 15–25 min. Method B: Solvent A = CH3CN (0.1 % H3PO4),
Solvent B = H2O (0.1% H3PO4); 0–10 min 23% A; 10–15 min
23–100% A; 15–30 min 100% A. For both the methods, the flow
rate was maintained at 1 ml/min.
Figure 1. Targeted fluorous synthons (R = CH2CH2(CF2)5CF3).
iodinated derivatives. To this end, the synthesis of a series of
new fluorous ‘tagging’ groups and a study of their reactivities
towards model compounds was undertaken along with develop-
ing the method for labeling and purifying the products.
3-(Tris[2-perfluorohexylethyl]stannyl)benzaldehyde (1a)
Under an argon atmosphere, 3-bromobenzaldehyde diethyl
acetal (311 mg, 1.16 mmol) was dissolved in diethyl ether and
cooled to ꢂ1001C. A solution of tert-butyllithium (1.7 M,
1.7 mmol) in pentane was added dropwise via syringe. After
40 min, a solution of tris[2-perfluorohexylethyl]tin bromide
(1.06 g, 856 mmol) in diethyl ether (5 ml) was added dropwise
via cannula. The reaction mixture was stirred at ꢂ1001C for an
additional 40 min, after which it was warmed slowly to room
temperature. After stirring for a minimum of 3 h, the solvent was
removed by rotary evaporation. A colorless oil was isolated by
liquid–liquid extraction using FC-72s (3 ꢁ 5 ml) and dichloro-
methane (3 ꢁ 10 ml), which was subsequently stirred in 20 ml of
1 N HCl for 6 h. Following this, the fluorous components were
extracted into FC-72s (3 ꢁ 10 ml) and was later purified by flash
chromatography (5 % EtOAc in hexanes) to give a colorless oil.
Yield: 440 mg, 35%. TLC(CH2Cl2): Rf = 0.72. 1H NMR (500 MHz,
CDCl3): 10.03 (s, 1H, CHO), 7.89 (m, 2H, Ar-H), 7.65 (m, 1H, Ar-H),
7.59 (m, 1H, Ar-H), 2.33 (m, 6H, SnCH2CH2C6F13), 1.37 (t, J = 8.2 Hz,
6H, SnCH2CH2C6F13). 13C NMR (125MHz, CDCl3): 193.0, 142.9,
138.5, 137.9, 137.2, 132.0, 130.3, 28.4, 0.1. FTIR (KBr, cmꢂ1): 2933,
2852, 1706, 1577.
Benzaldehyde derivatives are an attractive choice for modifying
small molecules and biomolecules, as they are readily introduced
into diverse scaffolds at free amine-sites under mild conditions.
Furthermore, the resulting Schiff-base can be irreversibly reduced
to a stable amine under mild conditions that are compatible with
a number of sensitive functional groups.11 Similarly, iodoaceta-
mides react with a number of functional groups on proteins
including the thioether in methionine, the sulfhydryl group of
cysteine, the e-amine of lysine and N-terminal a-amines.12
Building on this general utility and versatility, methods for
preparing the fluorous benzaldehyde (1) and aryl-iodoacetamide
(2) derivatives were developed (Figure 1).
Experimental section
Reagents and general procedures
All chemicals were purchased from Sigma-Aldrich and Fluorous
Technologies Inc. Compounds 3 and 5 were prepared as
previously reported.9,13 Sodium [125I] iodide with a specific
activity of ꢀ17 Ci/mg was obtained from the McMaster Nuclear
Reactor (Hamilton, Ont.).
4-(Tris[2-perfluorohexylethyl]stannyl) benzaldehyde (1b)
Prepared in the same fashion as described for 1a. Yield: 377 mg,
30%. TLC(CH2Cl2): Rf = 0.71. 1H NMR (500 MHz, CDCl3): 10.02
(s, 1H, CHO), 7.88 (d, J = 8.0 Hz, 2H, Ar-H), 7.59 (m, 2H, Ar-H), 2.33
(m, 6H, SnCH2CH2C6F13), 1.35 (t, J = 8.1 Hz, 6H, SnCH2CH2C6F13);
13C NMR (125MHz, CDCl3): 192.2, 146.1, 137.1, 136.5, 129.3, [118.2,
116.2, 111.1], 27.6, ꢂ1.1; FTIR (KBr, cmꢂ1): 2933, 2831, 1707, 1592.
Caution
125I is radioactive and should only be handled in an appropriately
equipped and licensed facility.
Instrumentation
NMR spectra were recorded using a Bruker DRX 500 spectro-
meter with chemical shifts reported in ppm relative to the
residual proton (1H NMR) or carbon (13C NMR) signal of the
deuterated solvent. Signals for C–F carbon atoms are enclosed
in square brackets. Infrared (IR) spectra were acquired using
a BioRad FTS-40 FT-IR spectrometer. All spectra were recorded
at ambient temperature. Analytical thin-layer chromatograms
(Merck F254 silica gel on aluminum plates) were visualized using
UV light. Purification of products was carried out using Ultrapure
Silica Gel from Silicycle (70–230 mesh). Electrospray ionization
(ESI) mass spectrometry experiments were performed on a
Waters/Micromass Quattro Ultima instrument, and samples were
first dissolved in methanol. High-resolution mass spectrometry
data were obtained using a Waters-Micromass Q-TOF Ultima
Global spectrometer. High-performance liquid chromatography
was performed using a Varian ProStar Model 230 instrument,
fitted with a Varian ProStar model 330 PDA detector, an IN/US
g-RAM gamma detector, and a Star 800 analog interface module.
Reverse-phase chromatography was performed using an Agilent
2-Chloro-N-(3-(tris[2-perfluorohexylethyl]stannyl)-benzyl)
acetamide (6)
To a solution of 3-(tris [2-perfluorohexylethyl]stannyl)benzylamine
(80 mg, 67 mmol) in chloroform was added chloroacetyl chloride
(21 mg, 188 mmol) at 01C, followed by slow addition of NEt3 (500 ml,
3.6 mmol). The resulting mixture was stirred at ambient tempera-
ture overnight. Solvent and excess triethylamine were removed
under reduced pressure; the resulting residue was dissolved in
FC-72s (20 ml) and extracted with water (3 ꢁ 15 ml). The fluorous
layer was dried over anhydrous sodium sulfate. Removal of the
FC-72s gave dark yellow–brown oil, which was purified by silica
gel chromatography where the product was isolated using 10%
ethanol in chloroform. Yield: 62 mg, 78%. TLC (9:1 CHCl3:EtOH):
Rf = 0.70. 1H NMR (500 MHz, CDCl3): 7.41 (m, 1H, Ar-H), 7.31 (m, 3H,
Ar-H), 6.89 (s, 1H, Ar-CH2NH), 4.50 (d, J=5.9Hz, 2H, Ar-CH2NH), 4.10
(s, 2H, CH2Cl), 2.32 (m, 6H, SnCH2CH2C6F13), 1.32 (t, J= 8.2 Hz, 6H,
SnCH2CH2C6F13). 13C NMR (125 MHz, CDCl3): 165.9, 138.0, 137.5,
135.3, 135.2, 129.3, 128.9, [118.3,] 43.8, 42.5, 27.7, ꢂ1.3. HRMS
(QTOF1): C33H22NOF39SnCl [M1H]1 = 1343.9789, found: 1343.9807.
FTIR (KBr, cmꢂ1): 3298, 2943, 1666, 1550.
˚
Zorbax SB-C18 column (4.6 ꢁ 250 mm, 300A–5 mm). The wave-
length for UV detection was set at 254 nm and the dwell time in
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Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 65–71

A. C. Donovan and J. F. Valliant
(m, 4H, Ar-H), 3.85 (s, 3H, OCH3), 3.60 (s, 2H, Ar-CH2-N), 3.10
(s, 4H, N(CH2CH2)2N), 2.67 (s, 4H, N(CH2CH2)2N), 2.32 (m, 6H,
Sn(CH2CH2C6F13)3), 1.32 (t, J = 8.3 Hz, 6H, Sn(CH2CH2C6F13)3). 13C
NMR (125 MHz, CDCl3): 152.3, 141.4, 138.8, 136.6, 136.2, 134.6,
130.5, 128.8, 122.9, 121.0, 118.2, [116.1], 111.3, [111.1], 63.0,
55.3, 53.3, 50.6, 27.7, ꢂ1.5. HRMS (QTOF1): mass calcd for
C42H33F39N2OSn: 1443.1070, found: 1443.1052. FTIR (KBr, cmꢂ1):
3422, 2940, 2817, 1501.
2-Iodo-N-(3-(tris[2-perfluorohexylethyl]stannyl)-benzyl)
acetamide (2)
Compound 6 (45 mg, 33mmol) was dissolved in acetonitrile (5 ml)
and combined with sodium iodide (50 mg, 335 mmol). The
resulting solution was heated at reflux (851C) overnight and then
allowed to cool to ambient temperature. The solvent was
removed and the resulting residue was dissolved in FC-72s and
extracted with water (3 ꢁ 10ml). The fluorous layer was dried
over anhydrous sodium sulfate. Removal of the FC-72s gave an
off-white solid. Yield: 42mg, 86 %. TLC (9:1, CHCl3:EtOH) Rf = 0.67.
1H NMR (500MHz, CDCl3): 7.40 (m, 1H, Ar-H), 7.32 (m, 3H, Ar-H),
6.34 (s, 1H, Ar-CH2NH), 4.47 (d, J = 5.8 Hz, 2H, Ar-CH2), 3.73 (s, 2H,
CH2I), 2.32 (m, 6H, SnCH2CH2C6F13), 1.30 (t, J = 8.2 Hz, 6H,
SnCH2CH2C6F13). 13C NMR (125MHz, CDCl3): 166.7, 138.2, 137.5,
135.2, 129.0, 128.8, [120.3, 118.3, 116.2], 44.3, 27.7, ꢂ1.2 HRMS
(QTOF1) calcd for C33H21INOF39Sn [M1NH4]1: 1452.9411, found:
1452.9417. FTIR (KBr, cmꢂ1): 3281, 3084, 2922, 2857, 1652, 1559.
N-4-(Tris[2-perfluorohexylethyl])stannyl)-1-(2-methoxyphenyl)-
piperazine (8b)
Yield: 150 mg, 83%. TLC (9:1 CHCl3:EtOH): Rf = 0.68. 1H NMR
(500 MHz, CDCl3): 7.42 (d, J=7.3Hz, 2H, Ar-H), 7.34 (d, J=7.6Hz,
2H, Ar-H), 6.99–6.85 (m, 4H, Ar-H), 3.85 (s, 3H, OCH3), 3.59 (s, 2H, Ar-
CH2-N), 3.10 (s, 4H, N(CH2CH2)2N), 2.66 (s, 4H, N(CH2CH2)2N), 2.31
(m, 6H, Sn(CH2CH2C6F13)3), 1.30 (t, J= 8.3 Hz, 6H, Sn(CH2CH2C6F13)3).
13C NMR (125 MHz, CDCl3): 152.3, 141.5, 139.9, 135.9, 134.7, 129.8,
122.9, 121.0, 118.3, 111.2, 62.9, 55.3, 53.4, 50.7, 27.7, ꢂ1.5. HRMS
(QTOF1): mass calcd for C42H33F39N2OSn [M1H]1: 1443.1070,
found: 1443.1066. FTIR (KBr, cmꢂ1): 2943, 2819, 1596, 1502.
Aldehyde conjugation via reductive amination: general
procedure
To a solution of 3- or 4-tris[2-perfluorohexylethyl]stannyl-benzalde-
hyde (1a or 1b) (75 mg, 61 mmol) in chloroform, the amine
(234 mmol) of interest was added. The mixture was stirred for 2 h at
which point sodium triacetoxyborohydride (50 mg, 234 mmol) was
added. The reaction mixture was stirred at ambient temperature
for 16 h prior to removal of the solvent under reduced pressure.
The resulting white residue was suspended in FC-72s (10 ml) and
extracted with 1 M Na2CO3 (20 ml) and twice with H2O (2ꢁ 20ml).
The fluorous layer was dried over anhydrous sodium sulfate and
the solvent was evaporated under reduced pressure to afford
a colorless oil. The desired products were isolated using silica gel
chromatography and 10% ethanol in chloroform.
2-Amino-3-[(3-2-perfluorohexylethylstannyl-benzylcarbamoyl)-
methylsulfanyl] propionic acid (9)
Fluorous iodoacetamide (2) (30mg, 21mmol) was dissolved in a 1:1
mixture of perfluorobutyl methyl ether (PFBME)/methanol (10 ml).
A solution of L-cysteine (10 mg, 82 mmol) in 1 M methanolic KOH
(5 ml) was then added and the resulting homogeneous solution
was stirred for 2 h. The solvent was removed by rotary evaporation
and the product (9) isolated by liquid–liquid extraction using
FC-72s (3 ꢁ 5 ml) and water (15 ml). Yield: 28 mg, 95%. TLC (9:1,
CHCl3:EtOH): Rf =0.1. 1H NMR (500MHz, CD3OD): 7.44 (m, 4H, Ar-H),
4.41 (s, 2H, Ar-CH2NH), 3.36 (m, 1H, SCH2CH(NH2)), 3.26 (d, 2H,
NHC(O)CH2S), 2.99 (m, 1H, CH2SCH2), (1H, CH2SCH2) 2.41 (m, 6H,
SnCH2CH2C6F13), 1.42 (t, J=8.3Hz, 6H, SnCH2CH2C6F13). 13C NMR
(125 MHz, CD3OD): 180.3, 172.5, 140.2, 139.0, 136.4, 136.1, 129.8,
129.5, 56.6, 44.4, 39.8, 29.0, ꢂ0.5. HRMS (QTOF -): mass calcd for
C36H26N2O3F39SSn: 1427.0063 [M–H]-, found: 1427.0048. FTIR (KBr,
window, cmꢂ1): 3401, 1634.
N-Butyl-3(-tris[2-perfluorohexylethy]stannyl)benzylamine (7a)
Yield: 140 mg, 85%. TLC (9:1 CHCl3:EtOH): Rf =0.30. 1H NMR
(500 MHz, CDCl3): 7.37 (m, 4H, Ar-H), 3.79 (s, 2H, Ar-CH2NH), 2.64
(t, J= 7.0 Hz, 2H, NHCH2CH2CH2CH3), 2.31 (m, 6H, SnCH2CH2C6F13),
1.51 (m, 2H, NHCH2CH2CH2CH3), 1.37–1.27 (m, 8H, Sn(CH2CH2C6F13)3,
NHCH2CH2CH2CH3), 0.90 (t, J= 7.3 Hz, 3H, NHCH2CH2CH2CH3). 13C
NMR (125 MHz, CDCl3): 141.0, 136.4, 135.6, 134.5, 129.2, [120.3, 118.2,
116.2, 110.8, 108.6], 54.0, 49.2, 32.1, 27.7, 20.4, 13.8, ꢂ1.5. HRMS
(QTOF1): mass calcd. for C35H28NF39Sn: 1324.0707, found:
1324.0653. FTIR (KBr, cmꢂ1): 3395, 2922, 1646.
General procedure for preparing iodo-arylamines (10, 11)
The desired amine (1.01mmol) was dissolved in chloroform
(10ml). To this was added either 3- or 4-iodobenzylbromide
(100 mg, 337 mmol) followed by triethylamine (363 mg, 36mmol).
The resulting mixture was heated to reflux and stirred overnight.
The reaction solvent was removed under reduced pressure
and the resulting yellow residue was subsequently purified by
using silica gel chromatography (10 % ethanol in chloroform).
N-Butyl-4-tris[2-perfluorohexylethyl])stannyl-benzylamine (7b)
Yield: 132 mg, 80%. TLC (9:1 CHCl3:EtOH): Rf =0.30. 1H NMR (500MHz,
CDCl3): 7.42 (d, J=7.6Hz, 2H, Ar-H), 7.35 (d, J= 7.8 Hz, 2H, Ar-H), 3.83
(s, 2H, Ar-CH2NH), 2.66 (t, J= 7.3 Hz, 2H, NHCH2CH2CH2CH3), 2.30
(m, 6H, SnCH2CH2C6F13) 1.56 (m, 2H, NHCH2CH2CH2CH3), 1.33 (m, 8H,
Sn(CH2CH2C6F13)3, NHCH2CH2CH2CH3), 0.91 (t, J= 7.3 Hz, 3H, NHCH2
CH2CH2CH3). 13C NMR (125 MHz, CDCl3): 136.1, 129.0, [118.3, 116.0,]
53.3, 48.7, 31.5, 27.7, 20.3, 13.8, ꢂ1.5. HRMS (QTOF1): mass calcd for
C35H29NF39Sn: 1324.0699, found: 1324.0653. FTIR (KBr, cmꢂ1): 3395,
2922, 1996, 1436, 1239.
N-Butyl-3-iodobenzylamine (10a)
Yield: 76 mg, 78%. TLC (9:1 CHCl3:EtOH): Rf = 0.5. HPLC:
1
Rt = 6.0 min (Method A). H NMR (500 MHz, CDCl3): 7.72 (s, 1H,
Ar-H), 7.60 (d, J = 7.8 Hz, 1H, Ar-H), 7.30 (d, J = 7.5 Hz, 1H, Ar-H)
7.06 (t, J = 7.7 Hz, 1H, Ar-H), 3.76 (s, 2H, Ar-CH2NH), 2.64
(t, J = 7.2 Hz, 2H, Ar-CH2NH-CH2), 1.61 (s, 1H, Ar-CH2NH) 1.52
(m, 2H, NH-CH2CH2CH2), 1.38 (m, 2H, NH-CH2CH2CH2CH3), 0.94
(t, J = 7.3 Hz, 3H, NH-CH2CH2CH2CH3). 13C NMR (125 MHz, CDCl3):
142.6, 137.2, 136.0, 130.1, 127.4, 94.4, 53.2, 49.0, 32.0, 20.4, 13.9.
HRMS (QTOF1): mass calcd for C11H17NI: 290.0406, found:
290.0413. FTIR (KBr, cm1): 3448, 2962, 2935, 2865, 2796.
N-3-(Tris[2-perfluorohexylethyl]stannyl)-1-(2-methoxyphenyl)-
piperazine (8a)
Yield: 142 mg, 79%. TLC (9:1 CHCl3:EtOH): Rf = 0.70. 1H NMR
(500 MHz, CDCl3): 7.39 (m, 3H, Ar-H), 7.27 (m, 1H, Ar-H), 7.01–6.85
J. Label Compd. Radiopharm 2011, 54 65–71
Copyright r 2010 John Wiley & Sons, Ltd.
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A. C. Donovan and J. F. Valliant
4.44 (d, J = 6.0 Hz, 2H, Ar-CH2), 4.11 (s, 2H, CH2Cl). 13C NMR
(125MHz, CDCl3): 166.0, 139.9, 137.0, 136.8, 130.6, 127.1, 94.7,
43.2, 42.7. HRMS (QTOF1): mass calcd for C9H10NOClI: 309.9496,
found: 309.9505. FTIR (KBr, cmꢂ1): 3272, 3068, 1653, 1555.
N-Butyl-4-iodobenzylamine (10b)
Yield: 76 mg, 78%. TLC (9:1 CHCl3:EtOH): Rf = 0.5. 1H NMR
(500 MHz, CDCl3): 7.64 (d, J = 8.1 Hz, 2H, Ar-H), 7.08 (d,
J = 8.1 Hz, 2H, Ar-H), 3.74 (s, 2H, Ar-CH2NH), 2.60 (t, J = 7.3 Hz,
2H, NH-CH2), 1.48 (m, 2H, NH-CH2CH2), 1.35 (m, 2H, NH-
CH2CH2CH2CH3), 0.91 (t, J = 8.3 Hz, 3H, NH-CH2CH2CH2CH3). 13C
NMR (125 MHz, CDCl3): 140.5, 137.5, 130.3, 92.2, 53.6, 49.3, 32.4,
20.6, 14.2. HRMS (QTOF1): mass calcd for C11H17NI: 290.0406,
found: 290.0417. FTIR (KBr, cmꢂ1): 2956, 2927, 2797.
2-Iodo-N-(3-iodobenzyl)acetamide (13)
3-Iodobenzyl-chloroacetamide (12) (53 mg, 172 mmol) was dis-
solved in acetonitrile (5 ml) and combined with sodium iodide
(258 mg, 1.72 mmol). The resulting solution was heated at reflux
overnight and allowed to cool to room temperature. The solvent
was removed and the resulting residue was purified by using
silica gel chromatography where the product was eluted using
N-(3-Iodobenzyl)-1-(2-methoxyphenyl)-piperazine (11a)
Yield: 131 mg, 95%. TLC (CHCl3): Rf = 0.38. HPLC: Rt = 14.5 min 10 % ethanol in chloroform. Yield: 59 mg, 86%. TLC (CHCl3)
(Method B). 1H NMR (500 MHz, CDCl3): 7.74 (s, 1H, Ar-H), 7.60 Rf = 0.67. 1H NMR (500 MHz, CDCl3): 7.64 (m, 2H, Ar-H), 7.27
(d, J= 7.9 Hz, 1H, Ar-H), 7.33 (d, J = 7.6 Hz, 1H, Ar-H), 7.06 (m, 1H, Ar-H), 7.09 (m, 1H, Ar-H), 6.38 (s, 1H, Ar-CH2NH), 4.41
(t, J = 7.7 Hz,1H, Ar-H), 7.01–6.85 (m, 4H, Ar-H), 3.89 (s, 3H, OCH3), (d, J = 5.9 Hz, 2H, Ar-CH2), 3.75 (s, 2H, CH2Cl). 13C NMR (125 MHz,
3.55 (s, 2H, Ar-CH2-N), 3.13 (s, 4H, N(CH2CH2)2N), 2.68 (s, 4H, CDCl3): 167.0, 140.1, 137.0, 136.8, 130.7, 127.1, 94.7, 43.8. HRMS
N(CH2CH2)2N). 13C NMR (125 MHz, CDCl3): 152.3, 141.4, 140.8, (QTOF1): mass calcd for C9H10NOI2: 401.8825, found: 401.8860.
138.1, 136.2, 130.1, 128.5, 122.9, 121.1, 118.3, 111.2, 94.5, 62.5, 55.4, FTIR (KBr, cmꢂ1): 3271, 1632, 1550.
53.4, 50.7. HRMS (QTOF1): mass calcd for C18H22N2OI: 409.0777,
found: 409.0785. FTIR (KBr, cmꢂ1): 3063, 2935, 2816, 1500, 1241.
2-Amino-3-[(3-iodo-benzylcarbamoyl)-methylsulfanyl] propionic
acid (14)
N-(4-Iodobenzyl)-1-(2-methoxyphenyl)-piperazine (11b)
L-Cysteine (30 mg; 247 mmol) was dissolved in methanolic KOH
Yield: 128 mg, 93%. TLC (CHCl3): Rf = 0.40. 1H NMR (500 MHz, (5ml, 1M) and added to a solution of 13 (100 mg; 250 mmol) in
CDCl3): 7.66 (d, J = 8.2 Hz, 2H, Ar-H), 7.12 (d, J = 8.2 Hz, 2H, Ar-H), methanol (2 ml). The resulting homogeneous mixture was stirred
7.00–6.85 (m, 4H, C6H4OCH3), 3.86 (s, 3H, OCH3), 3.52 (s, 2H, Ar- for 1 h whereupon the reaction solvent was removed by rotary
CH2-N), 3.10 (s, 4H, N(CH2CH2)2N), 2.64 (s, 4H, N(CH2CH2)2N). 13C evaporation. Yield: 95 mg, 97 %. TLC (9:1, CHCl3:EtOH): Rf =0.1.
1
NMR (125 MHz, CDCl3): 152.4, 141.5, 138.1, 137.4, 131.3, 122.9, HPLC: Rt = 11.1 min (Method B). H NMR (500MHz, CD3OD): 7.65
121.1, 118.3, 111.4, 92.4, 62.6, 55.5, 53.4, 50.7. HRMS (QTOF1): (s, 1H, Ar-H), 7.56, (d, J=7.7Hz, 1H, Ar-H), 7.28 (d, J= 7.6Hz, 1H,
mass calcd for C18H22N2OI: 409.0777, found: 409.0795. FTIR (KBr, Ar-H), 7.06 (t, J=7.8Hz, 1H, Ar-H), 4.32 (s, 2H, Ar-CH2), 3.34 (m, 1H,
cmꢂ1): 2936, 2816, 1593.
SCH2CH(NH2)), 3.28 (m, 2H, CH2SCH2), 2.96 (m, 1H, CH2SCH2), 2.75
(m, 1H, CH2SCH2). 13C NMR (125MHz, CD3OD): 180.3, 172.6, 142.6,
137.6, 137.4, 131.4, 127.9, 94.9, 56.6, 49.9, 43.6, 39.8. ESMS(1): 394.1
[M1H]1. (KBr, cmꢂ1): 3417, 2505, 1625, 1592, 1418.
2-Chloro-N-(3-iodobenzyl)acetamide (12)
3-Iodobenzylamine (501mg, 1.85mmol) was dissolved in chloro-
form (15 ml), then NEt3 (500ml, 3.6 mmol) was added. The solution
was then cooled to 01C prior to the addition of chloroacetyl
chloride (313mg, 2.78mmol) and additional NEt3 (3 ml, 21mmol).
The mixture was stirred at room temperature overnight prior to
the removal of solvent and excess triethylamine under reduced
pressure. The resulting residue was purified by silica gel
chromatography where the desired product was eluted using a
10 % ethanol in chloroform. Yield: 348 mg, 61 %. TLC (9:1
CHCl3:EtOH): Rf = 0.70. 1H NMR (500MHz, CDCl3): 7.64 (m, 2H,
Ar-H), 7.27 (m, 1H, Ar-H), 7.09 (m, 1H, Ar-H), 6.94 (s, 1H, Ar-CH2NH),
General radioiodination procedure
Compound 7, 8, or 9 (0.5 mg) was added to a sample vial
previously coated with Iodogens (prepared by evaporating 20 ml
of a solution of Iodogen in CH2Cl2, 1 mg/ml). A solution of 5%
acetic acid in methanol (100 ml) was added followed by sodium
[
125I]iodide (5 ml, 20 mCi/ml, pH 10). After swirling the mixture for
3 min, the reaction was quenched by the addition of aqueous
sodium metabisulfite (Na2S2O5, 10 ml, 44 mg/ml). The reaction
mixture was diluted with water (1 ml) and loaded onto a
FluoroFlashs solid-phase extraction (F-SPE) cartridge which had
been previously activated by washing first with 80:20 (v/v)
solution of methanol–water (6 ml), followed by water (6 ml). The
reaction vial was subsequently rinsed with an additional 3 ml of
water that was also added to the F-SPE cartridge. The cartridge
was eluted with water (6 ml), followed by 80:20 (v/v) solution of
methanol–water (6 ml). Radioactive fractions eluted from the
F-SPE cartridge were analyzed by using HPLC where in all cases,
Scheme 1. Preparation of the fluorous benzaldehyde 1 (R = CH2CH2(CF2)5CF3).
Scheme 2. Preparation of iodoacetamide 2 (R = CH2CH2(CF2)5CF3).
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A. C. Donovan and J. F. Valliant
one radioactive peak was observed where the retention times Conjugation chemistry
matched that for the authentic non-radioactive standards.
Reductive amination is often employed in bioconjugate
N-butyl-3- [125I]iodobenzylamine ([125I]10a): Rt: 6.4 min (Method
A); RCY: 92 %; Radiochemical purity:498 %.
N-(3-[125I]iodobenzyl)-1-(2-methoxyphenyl)piperazine
([125I]11a): Rt: 14.8 min (Method B); RCY: 83 %; Radiochemical
purity:498 %.
chemistry to link aldehyde-containing molecules to a targeting
vector including small molecules, proteins, and peptides that
have free amine functionalities.12 To establish the suitability of
1a/b for reactions of this type and to probe the feasibility of the
conjugation irrespective of the position of the functional group
relative to the fluorous moiety, representative reductive amina-
tion reactions were conducted using n-butylamine (a primary
amine) and 1-(2-methoxyphenyl)piperazine (a secondary amine).
The latter compound is routinely used to prepare radio-
pharmaceuticals for targeting the 5HT1A receptor.15 Compound
1a/b and the desired amine were combined in chloroform in the
presence of an excess of sodium triacetoxyborohydride under
an inert atmosphere (Scheme 3).16 Once again, the reductive
amination products were isolated via liquid–liquid extraction
using FC-72s and water. The resulting oil was subsequently
purified by silica gel chromatography, affording products 7a/b
in 80 and 85% yield, respectively, and 8a/b in 79% and 83%
yield, respectively.
L-Cysteine conjugate ([125I]14): Rt: 11.4 min (Method B); RCY:
86%; Radiochemical purity:498%.
Results and discussion
Preparation of fluorous conjugation synthons
To probe the differing in vivo stabilities of the positional isomers,
the preparation of the first targets (meta and para-fluorous-tin
benzaldehydes 1a/b) was accomplished by combining the
lithium salt of 3- or 4- bromobenzaldehyde diethyl acetal with
tris(perfluorohexylethyl)stannyl bromide (Scheme 1). Note that
to obtain good yields of the product it was essential to use tert-
BuLi instead of n-BuLi. Compound 4a/b was subsequently
isolated from the reaction mixture following liquid–liquid
extraction with perfluorinated hexanes (FC-72s) and dichlor-
omethane. Immediately after isolating 4a/b, the acetal was
stirred in the presence of 1N HCl to effectively remove the
protecting group. Purification via silica gel chromatography
gave compounds 1a or 1b in 30 and 35 % yield, respectively.
The preparation of the iodoacetamide 2 involved conversion
of the fluorous benzylamine 5, whose synthesis has been
previously reported,9 to 6 via treatment with chloroacetyl
chloride (Scheme 2).14 A standard fluorous workup including
liquid–liquid extraction between FC-72s and the reaction
solvent (chloroform) was used to isolate the desired compound.
Following purification by column chromatography, compound 6
was converted to the iodoacetamide 2 via a halogen exchange
reaction. After purification by column chromatography, com-
pound 2 was isolated in 86 % yield.
To probe the reactivity and selectivity of 2, L-cysteine was
chosen as a model ligand system. Ultimately, conjugation was
achieved by utilizing a mixture of methanol and PFBME; a
fluorous solvent which is miscible with most organic solvents
(Scheme 4).17 The two reagents were combined using an excess
of cysteine in methanolic KOH for 1 h, according to a procedure
18
adapted from Armstrong et al.
The desired product 9 was
isolated in high yield (95%) by liquid–liquid extraction using
perfluorinated hexanes (FC-72s) and water; no further purifica-
tion was required.
Preparation of cold iodinated reference standards
Prior to radiolabeling the fluorous conjugates (7a, 8a and 9), the
cold iodinated reference standards were synthesized and
characterized. The non-radioactive iodinated aryl amines were
Scheme 3. Conjugation of 1 with model amines (R = CH2CH2(CF2)5CF3).
Scheme 4. Reaction of 2 with a model thiol (cysteine) (R = CH2CH2(CF2)5CF3).
J. Label Compd. Radiopharm 2011, 54 65–71
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

A. C. Donovan and J. F. Valliant
prepared according to Scheme 5. The 3- and 4-iodobenzyl radiolabeling reactions were not conducted. The average yield
bromides were each treated with two mole equivalents of the (n = 2) of the desired product 10a was 92 % (Table 1).
respective amine; purification by silica gel chromatography gave When the same approach was applied to the other fluorous
compounds 10 and 11 in good yields (78–95%) and the conjugates (8a and 9), the yields exceeded 83% (Table 1)
characterization data for all products were consistent with that and good correlation was observed in all cases between the
predicted for the desired products.
reference standards (UV) and the product observed in the
Compound 13 was prepared from 3-iodobenzylamine which radio-HPLC traces (e.g. Figure 2). In all the experiments,
was converted to the chloro precursor 12 using chloroacetyl there was no evidence of residual fluorous starting materials in
chloride in the presence of base in 61% yield (Scheme 6). Using the HPLC chromatogram, which reconfirms the effectiveness
a similar procedure employed to make the fluorous analog (NaI of F-SPE at removing unreacted starting material. Work in
in acetonitrile), compound 12 was converted to the iodoaceta- progress involves utilizing the synthons developed here and
mide 13 in high yield (86%). This product was subsequently fluorous SPE for radioiodinating peptide and biomolecule
conjugated in nearly quantitative yield to L-cysteine in a 1 M derived vectors in which the weight percent of fluorine is
methanolic KOH solution to give 14 (Scheme 7). Again, greatly reduced.
characterization data for each step were consistent with the
formation of the desired products.
Conclusions
Two new fluorous tagging reagents that can be used to link
arylstannanes to amino and sulfhydryl groups were prepared
and evaluated. The resulting conjugates were radiolabeled
and isolated in high yield in mere minutes without the need for
HPLC purification by employing fluorous solid-phase extraction
(F-SPE). The conjugation and radiolabeling methodology
reported in this work can be applied to the development of
targeted molecular imaging probes using targeting vectors
derived from small molecules, peptides, or possibly even higher
molecular weight proteins.
Radiochemistry
Radioiodination reactions using the fluorous conjugates were
conducted using sodium [125I]iodide and iodogen (Scheme 8).
The radioiodination was performed to show that there were no
interferences or complications due to the interactions between
the substituents on the ring and to determine whether
there were differences between the primary and secondary
amine functionalities during the reaction. As these variables
are unchanged in the positional isomers 7b and 8b, the
Scheme 8. Radioiodination of fluorous conjugates using Na [125I]iodide.
Table 1. Summary of radiochemical yields
Compound
number
Radiochemical yield
(n = 2) (%)
Radiochemical
purity (%)
10a
11a
14
9272
8375
8673
498
498
498
Scheme 5. Preparation of iodinated aryl amine standards.
Scheme 6. Synthesis of compound 13.
Scheme 7. Preparation of iodinated reference standard 14.
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J. Label Compd. Radiopharm 2011, 54 65–71

A. C. Donovan and J. F. Valliant
Figure 2. UV and g-HPLC chromatograms (HPLC Method A) of non-radioactive 10a (top) and F-SPE purified [125I]10a (bottom). The slight difference in retention time is
associated with the fact that the UV and g-detectors are connected in series.
[7] G. Vaidyanathan, D. J. Affleck, K. L. Alston, X.-G. Zhao, M. Hens,
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15, 3430.
[8] A. Donovan, J. Forbes, P. Dorff, P. Schaffer, J. Babich, J. F. Valliant,
J. Am. Chem. Soc. 2006, 128, 3536.
[9] A. C. Donovan, J. F. Valliant, Nucl. Med. Biol. 2008, 35, 741.
[10] J. W. McIntee, C. Sundararajan, A. C. Donovan, M. S. Kovacs,
A. Capretta, J. F. Valliant, J. Org. Chem 2008, 73, 8236.
[11] J. A. Prescher, C. R. Bertozzi, Nat. Chem. Biol. 2005, 1, 13.
[12] G. T. Helmanson, Bioconjugate Techniques, Academic Press, Toronto,
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[14] S. Liu, J. Pietryka, C. E. Ellars, D. S. Edwards, Bioconjugate Chem.
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Acknowledgements
This study was conducted with the support of the Ontario Institute
for Cancer Research through funding provided by the Government
of Ontario as well as Molecular Insight Pharmaceuticals.
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J. Label Compd. Radiopharm 2011, 54 65–71
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