8696-8701 Utilizing electrostatic interactions to facilitate F-18 radiolabeling of poly(amido)amine (PAMAM) dendrimers

Article abstract of DOI:10.1039/c4ob01616e

The development of methods for the facile conjugation and radiolabeling of poly(amido)amine (PAMAM) dendrimers would be of great benefit in evaluating biomedical applications of these intriguing molecularly defined polymers. Two anionic N-hydroxysuccinimide (NHS) esters (7 and 10) were developed and radio-labeled with fluorine-18 using Cu(I)-catalyzed click reactions. The radiolabeling of a primary amine-terminated PAMAM generation-6 (G6) dendrimer with [18F]7 or [18F]10 was complete in water or methanol within 5 min at room temperature. This highly efficient conjugation reaction benefits from a high, localized concentration of these NHS esters on the surface of PAMAM dendrimers, due to the electrostatic attraction between the anionic NHS esters and the positively-charged PAMAM dendrimers. The large medium effect (pH, salt, solvent) observed for these conjugation reactions is consistent with this mechanism. This novel strategy of utilizing electrostatic interactions provides a novel, facile, and efficient method for the conjugation and radiolabeling of PAMAM dendrimers that also has potential for radiolabeling other appropriate nanoparticles.

Full text of DOI:10.1039/c4ob01616e

Organic &
Biomolecular Chemistry
View Article Online
View Journal | View Issue
PAPER
Utilizing electrostatic interactions to facilitate
F-18 radiolabeling of poly(amido)amine
(PAMAM) dendrimers†
Cite this: Org. Biomol. Chem., 2014,
12, 8696
Dong Zhou,*a Sung Hoon Kim,b Vincent M. Carroll,b Carmen S. Dencea and
John A. Katzenellenbogenb
The development of methods for the facile conjugation and radiolabeling of poly(amido)amine (PAMAM)
dendrimers would be of great benefit in evaluating biomedical applications of these intriguing molecularly
defined polymers. Two anionic N-hydroxysuccinimide (NHS) esters (7 and 10) were developed and radio-
labeled with fluorine-18 using Cu(I)-catalyzed click reactions. The radiolabeling of a primary amine-termi-
nated PAMAM generation-6 (G6) dendrimer with [18F]7 or [18F]10 was complete in water or methanol
within 5 min at room temperature. This highly efficient conjugation reaction benefits from a high, loca-
lized concentration of these NHS esters on the surface of PAMAM dendrimers, due to the electrostatic
attraction between the anionic NHS esters and the positively-charged PAMAM dendrimers. The large
medium effect (pH, salt, solvent) observed for these conjugation reactions is consistent with this mech-
anism. This novel strategy of utilizing electrostatic interactions provides a novel, facile, and efficient
method for the conjugation and radiolabeling of PAMAM dendrimers that also has potential for radiolabel-
ing other appropriate nanoparticles.
Received 29th July 2014,
Accepted 8th September 2014
DOI: 10.1039/c4ob01616e
www.rsc.org/obc
cularly defined polymers by positron emission tomographic
(PET) imaging. In other work yet to be published, we have
Introduction
Poly(amido)amine (PAMAM) dendrimers have been explored in found that the distribution phase of 18F labeled PAMAM dendri-
a variety of biomedical applications, including as a platform for mers targeted to estrogen-responsive vascular tissues was com-
targeted delivery of therapeutic agents and for imaging plete within 2 hours, indicating that 18F is an appropriate
purposes.1–8 The functional groups on the surface of these struc- radiotracer for such species. However, radiolabeling of nano-
turally well-defined PAMAM dendrimers, especially in their particles with 18F, in general, is challenging, especially in terms
whole integer generations (i.e. NH2 groups), allow for of achieving high specific activity (SA), due to the short half-life
functionalization of these dendrimers with multiple modalities. of 18F (t1/2 = 109.8 min) and the generally slow kinetics of the
Among many reported strategies,9–15 N-acylation with N-hydroxy- labeling reaction under conventional conjugation conditions.
succinimide (NHS) esters is a commonly used method for conju- Besides utilizing fast and specific coupling reactions,12–15
gation reactions with amine-terminated PAMAM dendrimers.
another strategy to achieve fast kinetics is to have a mechanism
PAMAM dendrimers have been labeled with different radio- for co-localizing the radiolabeling reagent with the target sub-
isotopes (3H, 111In, 88Y, 177Lu, 125I, 99mTc, etc.)16–20 for bio- strate.21 PAMAM dendrimers of even integer generations are ter-
distribution and pharmacokinetic studies, but up to now not minated in primary amines and are positively charged on their
with fluorine-18. Fluorine-18, in a large quantity and with high surface at physiological pH; this surface positive charge may
specific activity, is easily available from most medical cyclo- enable a high concentration of negatively charged substances to
trons, and radiolabeling of PAMAM dendrimers with 18F become localized on their surface. In this paper, we report the
would provide a very useful tool for biomedical studies invol- facile conjugation and labeling reactions between cationic
ving tracing of the distribution of these interesting, mole- PAMAM dendrimers and anionic NHS reagents.
aWashington University Medical School, Mallinckrodt Institute of Radiology,
Results and discussion
Saint Louis, MO 63110, USA. E-mail: zhoud@mir.wustl.edu; Fax: +314-362-9940;
Tel: +314-362-9072
Conjugation of PAMAM with NHS-fluorescein (1)
bDepartment of Chemistry, University of Illinois, Urbana, IL 61801, USA
As a model for the coupling reaction of PAMAM G6 dendri-
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4ob01616e
mers, we first carried out a reaction with NHS-fluorescein (1)
8696 | Org. Biomol. Chem., 2014, 12, 8696–8701
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
reported.22 Nevertheless, our further studies of the reaction
between the PAMAM G6 dendrimer and fluorescein itself
under the same conditions showed no coupling, indicating
that covalent bond formation between PAMAM G6 and 1 had
taken place in the above reaction. We presume that the initial
reversible electrostatic uptake of anionic 1 on the cationic
surface of the PAMAM G6 dendrimers results in a high, loca-
lized concentration of 1 on the dendrimer surface, driving the
conjugation reaction of the NHS ester in 1 with NH2 groups on
the periphery of the dendrimer. This model reaction demon-
strated the feasibility of fast, covalent radiolabeling of cationic
PAMAM dendrimers with 18F-labeled anionic NHS esters by
capitalizing on the concentrating effect of these electrostatic
interactions.
Fig. 1 Conjugation of G6 PAMAM and NHS-fluorescein (1).
Synthesis and radiosynthesis of anionic NHS reagents
Two anionic NHS esters (7 and 10) were developed, as shown
(see Fig. 1) in methanol, without addition of buffer or in Scheme 1. Compound 7 is a derivative of sulfo-NHS esters,
additional bases and using 4.6% or 46% molar equivalents of which contains an anionic sulfonate group. Compound 10 is a
1 relative to the 256 NH2 groups on the periphery of this den- derivative of an NHS ester that contains a second COOH
drimer. According to the HPLC analysis, within 5 min at room group. Both [18F]7 and [18F]10 were radiosynthesized from the
temperature the reaction with 4.6% of 1 was essentially com- alkyne precursors 6 and 9, respectively, using a Cu(I)-catalyzed
plete and that with 46% molar equivalent of 1 was also nearly click reaction with 2-[18F]fluoroethyl azide, according to the
complete, with exclusive formation of the fluorescein conju- methods described in our prior publication on the radiosynth-
gation product of the PAMAM (see ESI†).
esis of [18F]4,23 which is a NHS analogue of [18F]7. A benzoic
Electrostatic uptake of fluorescein itself, which is anionic, acid derivative [18F]5 was also generated from [18F]4 by
into PAMAM dendrimers, which are cationic, has been hydrolysis.
Scheme 1 Synthesis and radiosynthesis of NHS and sulfo-NHS esters of various 18F-labeled benzoic acids. Reaction conditions: (a) NHS-OH, DCC, DMF;
(b) 2-fluoroethyl azide/2-[18F]fluoroethyl azide, CuSO4, Na ascorbate, BPDS, DMF; (c) NaOH; (d) sulfo-NHS-OH, DCC, DMF; (e) TSTU, Bu4NOH, MeCN.
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 8696–8701 | 8697

View Article Online
Paper
Organic & Biomolecular Chemistry
Radiolabeling of PAMAM with anionic NHS reagents
Medium effects
The conjugation reactions of PAMAM G6 with different PAMAM dendrimers have a well-defined molecular architec-
18F-labeled reagents were carried out in a volume of 500 µL, ture; pH and salt concentration, however, have great effects on
which is a practical volume for radiolabeling. The radiochemi- the conformation of PAMAM dendrimers, and one can
cal yields were determined by radio-TLC (see ESI†). Analytical presume that they greatly affect the accessibility and reactivity
HPLC chromatographs of a conjugation of PAMAM G6 with of surface NH2 groups: at low pH, PAMAM dendrimers exhibit
[18F]7 and its purified product using a Sephadex G-25 column an open and extended conformation due to electrostatic repul-
is shown in the ESI.† As shown in Table 1, the conjugation sion between the dendrimer branches;24 by contrast, at pH
reactions with anionic NHS esters, [18F]7 and [18F]10, were values higher than 9, the dendrimers adopt a compact structure
complete within 5 min at room temperature, whereas and in that results from hydrogen bonds between branches.25 High salt
sharp contrast, the reactions with the neutral NHS ester [18F]4 concentrations and solvent dielectric properties also have an
and [18F]SFB were slow. As expected, no reaction occurred with impact on PAMAM dendrimer conformations and potentially
the simple anionic benzoic acid reagent [18F]5. The difference also on dendrimer reactivity towards charged reagents, such as
in reaction rates between anionic and neutral NHS esters was those we have been studying.26 Therefore, we examined the
also demonstrated by reactions with polyethylenimine effect of solvent and reaction medium on the conjugation reac-
(Table 1, note c). These results suggest that the fast conju- tions of PAMAMs with [18F]4 and [18F]7 (Table 3).
gation reactions and covalent bond formation take place as a
In a variety of solvents, the conjugation reaction with [18F]4
result of the anionic NHS esters pre-localized on the surface of was slower than that with [18F]7. Water was the ideal solvent
the cationic PAMAM dendrimers, due to the electrostatic for both [18F]4 and [18F]7, but there was no reaction with the
attraction between the species of opposite charge.
reagent, [18F]4, in methanol, in sharp contrast to that with
The high efficiency of this method was further probed by anionic [18F]7 in methanol. In 0.1% TFA, there was no reaction
the reactions shown in Table 2. In these studies, the amount with either reagent: at pH 1, the NH2 groups on the dendrimer
of PAMAM G6 in the labeling reactions was gradually reduced. would be fully protonated; the sulfonate group in [18F]7 would
Nevertheless, radiochemical yields in excess of 90% were also be protonated, and being neutral, would no longer be
achieved within 5 min with as low as 4 µg of PAMAM dendri- undergoing electrostatic interactions at the dendrimer surface,
mers in the 500 µL reaction volume. Only with 0.4 µg, which is thereby interrupting the covalent reaction. Notably, when the
an extremely low amount, the reaction was slow, but it still pro- labeling was conducted in 1 M aqueous NaCl, yields for both
ceeded with time. The minimal amount of PAMAM dendri- [18F]4 and [18F]7 were significantly reduced. Because the reac-
mers required for this type of coupling is ideal for producing tivity of both the neutral and anionic reagents is reduced, the
radiolabeled dendrimers in high specific activity.
salt effect is likely due to electrostatic screening that reduces
the local concentration of [18F]7 on the surface of the dendri-
mer and enables the PAMAM dendrimer to adopt a more
compact morphology; the latter would reduce the reactivity of
[18F]4 as well as [18F]7. The conjugation reactions in 0.1 M
aqueous NaHCO3 proceeded very slowly, presumably because
the dendrimer surface would be less positive at elevated pH.
No reaction was observed in DMF. In brief, solvents and
the pH and ionic strength of the medium have a great impact
on the outcome of the conjugation reactions of PAMAM
dendrimers.
Table 1 Summary of radiochemical yields (%) of PAMAM G6 with 18F
labeled substratesa
NHS
[
18F]4
[
18F]5
[
18F]7c
[
18F]10
[
18F]SFBb,c
5 min (%)
15 min (%) 71.3 9.6
28.1 5.1
0
0
95.3 0.9 95.2 0.3
93.5 0.9 96.3 0.3 76.9 1.2
41 3.7
a G6 PAMAM 20 µg in 500 µL water, radiochemical yields were
determined by radioTLC (silica TLC/MeOH), n = 3. b In 500 µL
methanol, 2.1 0.2% (5 min) and 5.0 2.0% (15 min). c With 50 µg
polyethylenimine (branched, average Mw
∼ 25 000) in 500 µL
Specific activity
methanol: 95.4 0.8% (5 min), 96.1 0.5% (15 min) for [18F]7 and
11.3 0.7% (5 min), 24.6 2.5% (15 min) for [18F]SFB.
The specific activity (SA) of radiolabeled PAMAM dendrimers
is dependent on how many NHS substrates become conjugated
on each dendrimer and the SA of radiolabeled reagent itself.
In the above diagnostic reactions, only about 1/30 to 1/50 of
the final HPLC-purified products [18F]4 and [18F]7 were used
for each reaction. The low stoichiometric ratio of the
18F-labeled reagent to the PAMAM dendrimer in these cases
would have a significant impact on the outcome of the conju-
gation reactions, ensuring high yields of activity incorporation
but giving the final conjugated product in less than optimal SA
because only a limited number of reagents would become
attached to each dendrimer. Therefore, we examined these
conjugation reactions using a large amount of [18F]4 and [18F]7;
Table 2 Summary of radiochemical yields (%) with different amounts of
PAMAM G6a
400
5
40
5
4
5
0.4
5
PAMAM (µg)
Time (min)
15
15
15
15
[
[
18F]7 (%)
92
91
92
91
92
91
92
91
90
91
90
91
17
8
31
18
18F]10 (%)
a Radiochemical yields were determined by radioTLC (silica TLC/
MeOH), n = 1.
8698 | Org. Biomol. Chem., 2014, 12, 8696–8701
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
Table 3 Medium effects on conjugation yields (%) of PAMAM G6 with NHS and sulfo-NHS estersa
Solvent
Time
Water
0.1% TFA
1 M NaCl
0.1 M NaHCO3
MeOH
DMF
[
18F]4
18F]7
5 min
15 min
5 min
28.1 5.1
71.3 9.6
95.2 0.5
93.5 0.9
0
0
0
0
5.4 0.5
9.7 1.3
26.8 1.4
37.3 1.4
6.1 0.3
12.5 0.2
11.1 0.2
24.5 0.8
0
0
0
0
0
0
[
93.1 0.9
93.3 0.9
15 min
a PAMAM G6 20 µg in 500 µL solvent, radiochemical yields were determined by radioTLC (silica TLC/MeOH), n = 3.
under these conditions, the reagent [18F]7 still gave high multiplet. Elemental analysis (C, H, N) were performed by
labeling efficiency (94% at 5 min and 95% at 15 min), while Atlantic Microlab, Inc., Norcross, GA. High performance liquid
the efficiency of labeling with [18F]4 was very much less (5% at chromatography (HPLC) was performed with an ultraviolet
5 min and 13% at 15 min). When half of the final [18F]7 was detector and a well-scintillation NaI (Tl) detector and associ-
used to conjugate with 20 µg PAMAM dendrimers in methanol, ated electronics for radioactivity detection. An Agilent SB-C18
over 90% incorporation was achieved within 5 min; this 250 × 9.4 mm 5 µ semi-preparative column (A) and an Agilent
afforded a product having two [18F]7 molecules conjugated per SB-C18 250 × 4.6 mm 5 µ analytical column (B) were used for
dendrimer, determined by comparing the SA of radiolabeled preparation and analysis respectively. [18F]Fluoride was pro-
dendrimers with the SA of [18F]7. In principle, the stoichio- duced at Washington University by the 18O(p,n)18F reaction
metric ratio of [18F]7 to PAMAM dendrimer could be increased through proton irradiation of enriched (95%) [18O] water in the
so that multiple copies of the radiolabeled reagent per dendri- RDS111 cyclotron. Radio-TLC was accomplished using a
mer are incorporated; this would result in a radiolabeled Bioscan AR-2000 imaging scanner (Bioscan, Inc., Washington,
dendrimer having a SA multiple fold higher than that of the DC).
radiolabeled reagent itself.
PAMAM dendrimers provide an excellent platform for
surface functionalization with a variety of moieties that are
Synthesis of 5
A solution of 2-fluoroethyl azide (1.6 mmol) in DMF (3.5 mL)
was added into a 10 mL screw-cap tube containing methyl
4-(prop-2-yn-1-yloxy)benzoate (0.2 g, 1.05 mmol), followed by
addition of Cu(I)/BPDS (100 µL from a mixture of 5 mg
CuSO4·5H2O in 50 µL water, 15 mg sodium ascorbate in 50 µL
water, and 6 mg BPDS 100 µL in 4 : 1 water–DMF). The reaction
was stirred at room temperature for 2 h, and then at 50 °C for
2 h to complete the reaction. The reaction mixture was diluted
with water (50 mL), and then passed through a Waters Oasis®
HLB cartridge (1 g), which was further rinsed with water (3 ×
10 mL). The product was eluted with methanol (7 mL), and
NaOH (0.18 g, 4.5 mmol) in water (7 mL) was added to the
above solution. The reaction mixture was stirred at room temp-
erature for 24 h, and then was diluted with water (100 mL).
The pH was adjusted to pH = 1 using 1 N HCl solution.
The formed white solids were filtered, rinsed with water
(3 × 10 mL), and dried at room temperature to afford 5 (0.21 g)
useful for many biomedical applications. However, using pre-
viously reported strategies, it is not easy to control the ligand/
PAMAM ratio,27 which is important for biological studies. The
method reported here, utilizing electrostatic interactions to
amplify the reactivity, not only provides a facile method for
radiolabeling, but also would enable efficient conjugation of
other interesting ligands having biological activity in a process
that allows for accurate control of the ligand/PAMAM ratios
and easy purification. Because surface charge is common in
nanoparticles, this strategy of harnessing electrostatic inter-
actions to facilitate covalent labeling has the potential for
improving the conjugation and labeling of many of these
macromolecular species.
Experimental
1
in 75% overall yield. H NMR (400 MHz, DMSO-d6) δ 12.64 (s,
All chemicals were obtained from standard commercial
sources and used without further purification. All reactions
were carried out by standard air-free and moisture-free tech-
niques under an inert nitrogen atmosphere with dry solvents
unless otherwise stated. Flash column chromatography was
conducted using silica gel, 60A, “40 Micron Flash” (32–63 µm)
1H), 8.27 (s, 1H), 7.87 (d, J = 8 Hz, 2H), 7.10 (d, J = 8 Hz, 1H),
5.21 (s, 2H), 4.80 (dt, J = 4, 48 Hz, 2H), 4.70 (dt, J = 4, 28 Hz,
2H); 13C NMR (200 MHz, DMSO-d6) δ 167.38, 162.01, 142.81,
131.76, 125.66, 123.68, 114.93, 82.35 (d, J = 1.67 Hz), 61.61,
50.50 (d, J = 0.19 Hz); m.p. 179.5–180.5 °C.
1
(Scientific Adsorbents, Inc.). Routine H and 13C NMR spectra
Synthesis of 6
were recorded at 400 MHz on an Agilent Technologies spectro-
meter. All chemical shifts were reported as parts per million A mixture of N-hydroxysulfosuccinimide sodium salt (0.47 g,
(ppm) downfield from tetramethylsilane (TMS). All coupling 2.16 mmol), DCC (0.48 g, 2.33 mmol), and the benzoic acid
constants (J) are given in hertz (Hz). Splitting patterns are typi- derivative 228 (0.37 g, 2.10 mmol) in DMF (5 mL) was stirred
cally described as follows: s, singlet; d, doublet; t, triplet; m, for 24 h at room temperature. After white solids were removed
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 8696–8701 | 8699

View Article Online
Paper
Organic & Biomolecular Chemistry
by filtration, the reaction mixture was acidified with 0.5 N HCl purification (TR = 14 min). Yield, 36%. 1H NMR (400 MHz,
(5 mL), diluted with water (80 mL), and then purified by solid DMSO-d6) δ 13.55 (br, 1H), 8.26 (s, 1H), 8.15 (s, 1H), 7.91 (s,
phase extraction using a Waters Oasis® HLB cartridge (3 g). 1H), 7.87 (s, 1H), 5.34 (s, 2H), 4.76 (dt, J = 4, 44 Hz, 2H), 4.74
The collected product was purified again using the HLB car- (dt, J = 4, 28 Hz, 2H), 2.87 (s, 4H); 13C NMR (200 MHz, DMSO-
tridge, and the final product was obtained as a white solid d6) δ 170.62, 166.12, 161.45, 159.11, 142.65, 133.98, 126.63,
(97 mg) after lyophilization. 1H NMR (400 MHz, DMSO-d6) 125.68, 123.17, 122.65, 120.13, 82.32 (d, J = 1.67 Hz), 62.32,
δ 8.04 (d, J = 8 Hz, 2H), 7.18 (d, J = 8 Hz, 2H), 4.94 (d, J = 2 Hz, 50.51 (d, J = 0.19 Hz), 26.00; m.p. 207.0–209.0 °C. Anal. Calcd
2H), 4.00 (m, 1H), 3.65 (t, J = 2 Hz, 1H), 3.20 (br, 1H), 2.88 (m, for C17H15FN4O7: C, 50.25; H, 3.72; N, 13.79. Found: C, 49.88;
1H); 13C NMR (200 MHz, DMSO-d6) δ 169.41, 165.94, 163.06, H, 3.84; N, 13.43.
132.78, 116.12, 79.53, 78.77, 56.76, 56.42, 31.42; m.p. 150 °C,
decomposition. Anal. Calcd for C16H15FN4O8S·0.5H2O: C,
42.58; H, 3.57; N, 12.41. Found: C, 42.72; H, 3.47; N, 12.52.
General procedure of Cu(I)-catalyzed click labeling using
2-[18F]fluoroethyl azide
To a 10 mL screw-cap Pyrex tube containing ∼1 mg alkyne pre-
cursor was added 2-[18F]fluoroethyl azide in DMF (250 µL)
according to the literature, followed by addition of Cu(I)/batho-
phenanthroline disulfonate (BPDS) (50 µL from a mixture of
5 mg CuSO4·5H2O in 50 µL water, 15 mg sodium ascorbate in
50 µL water, and 6 mg BPDS 100 µL in 4 : 1 water–DMF). The
tube was shaken and then allowed to settle for 5–10 min to
complete the reaction. 0.1% TFA or 10% MeCN–90% water–
0.1% TFA (up to 4 mL) was added to the reaction mixture for
HPLC purification using an Agilent SB-C18 column (5 µm 250
× 9.4 mm) with a flow rate of 4 mL min−1 and UV at 240 nm
and the specified mobile phase for each compound.
Synthesis of 7
General method of Cu(I)-catalyzed click reaction: a solution of
2-fluoroethyl azide (0.23 mmol) in DMF (0.5 mL) was added to
a 10 mL screw-cap tube containing the sulfo-NHS alkyne
derivative 6 (20 mg, 0.073 mmol), followed by addition of Cu(I)/
BPDS (100 µL from a mixture of 5 mg CuSO4·5H2O in 50 µL
water, 15 mg sodium ascorbate in 50 µL water, and 6 mg BPDS
100 µL in 4 : 1 water–DMF). The reaction was complete within
5 min according to HPLC analysis. The reaction mixture was
diluted with 0.2% TFA (4.5 mL), and purified by reversed
phase HPLC to afford the final product as a white solid
(15 mg) (HPLC column: Nucleosil, C18 250 × 16 7 µ; mobile
phase: 17% MeCN–83% water–0.1% TFA; flow rate: 8 mL
General procedure of conjugation and radiolabeling of
PAMAM dendrimers
min−1; retention time: 15 min). H NMR (400 MHz, DMSO-d6)
1
δ 8.30 (s, 1H), 8.03 (d, J = 8 Hz, 2H), 7.25 (d, J = 8 Hz, 2H), 5.30
(s, 2H), 4.80 (dt, J = 4, 44 Hz, 2H), 4.70 (dt, J = 4, 28 Hz, 2H),
3.98 (m, 1H), 3.20 (br, 1H), 2.88 (m, 1H); 13C NMR (200 MHz,
DMSO-d6) δ 169.45, 165.95, 142.46, 132.85, 125.82, 116.01,
82.33 (d, J = 1.67 Hz), 61.94, 56.78, 50.52 (d, J = 0.19 Hz), 31.44;
mp 240 °C decomposition. Anal. Calcd for C14H11NO8S·3H2O:
C, 41.28; H, 4.21; N, 3.44. Found: C, 41.47; H, 4.02; N, 3.60.
To a 500 µL solution of PAMAM dendrimers in a 1.5 mL micro-
centrifuge tube was added 18F NHS ester in DMF (10 µL). The
reaction mixture was immediately vortexed, and then allowed
to react at room temperature. At a specified time point, an
aliquot of the reaction mixture was analyzed by silica TLC.
Synthesis of 9
Conclusion
A solution of 1 M Bu4NOH in water (0.52 mL) was azeotropi-
cally dried with MeCN (5 × 1 mL) under a flow of N2 at 105 °C.
A suspension of 829 (115 mg, 0.52 mmol) in MeCN (5 mL) was
added to the dried Bu4NOH to form a clear solution, to which
a solution of N,N,N,N-tetramethyl-O-(N-succinimidyl)uronium
tetrafluoroborate (TUSU) (160 mg, 0.53 mmol) in MeCN (1 mL)
was added. The reaction was complete within 5 min according
to HPLC analysis. The reaction mixture was purified by
In conclusion, two anionic NHS esters, 7 and 10, were syn-
thesized, and they were radiolabeled with 18F using a Cu(I)-
catalyzed click reaction with 2-[18F]fluoroethyl azide. The treat-
ment of primary amine-terminated PAMAM G6 dendrimers
with [18F]7 and [18F]10 demonstrated highly efficient conju-
gation and radiolabeling reactions between the anionic NHS
esters and the cationic PAMAM dendrimers, due to a favorable
electrostatic interaction, the extent of which was greatly
affected by solvent and medium effects. The strategy using
electrostatic interactions to facilitate the labeling of macro-
molecules has great potential for improving the conjugation
and labeling efficiency of both PAMAM dendrimers and other
nanoparticles with a variety of interesting modifying groups.
1
reversed phase HPLC to afford 9 as a white solid (105 mg). H
NMR (400 MHz, DMSO-d6) δ 13.63 (br, 1H), 8.17 (s, 1H), 7.87
(s, 1H), 7.81 (s, 1H), 5.01 (d, J = 2 Hz, 2H), 3.66 (t, J = 2 Hz,
1H), 2.87 (s, 4H); 13C NMR (200 MHz, DMSO-d6) δ 169.03,
164.45, 159.79, 156.57, 132.34, 124.96, 121.89, 121.16, 118.60,
78.18, 77.14, 55.04, 24.39; mp 172.0–174.0 °C. Anal. Calcd for
C15H11NO7: C, 56.79; H, 3.50; N, 4.42. Found: C, 56.63; H, 3.52;
N, 4.54.
Acknowledgements
Synthesis of 10
The same method as for 7 was used except that as the mobile We are grateful for support of this research by research and
phase 32% MeCN–68% water–0.1% TFA was used for HPLC training grants from the Department of Energy (ER64671 and
8700 | Org. Biomol. Chem., 2014, 12, 8696–8701
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
DE-SC0005434). We thank Robert Dennett and Brian Wingber- 13 D. Astruc, L. Liang, A. Rapakousiou and J. Ruiz, Acc. Chem.
muehle for the production of [18F]fluoride.
Res., 2012, 45, 630.
14 B. Huang, A. Desai, H. Zong, S. Tang, P. Leroueil and
J. R. Baker Jr., Tetrahedron Lett., 2011, 52, 1411.
15 B. Huang, J. F. Kukowska-Latallo, S. Tang, H. Zong,
K. B. Johnson, A. Desai, C. L. Gordon, P. R. Leroueil
and J. R. Baker Jr., Bioorg. Med. Chem. Lett., 2012, 22,
3152.
References
1 J. R. Baker Jr., Hematology Am. Soc. Hematol. Educ. Program,
2009, 1, 708.
16 V. Biricova, A. Laznickova, M. Laznicek, M. Polasek and
P. Hermann, J. Pharm. Biomed. Anal., 2011, 56, 505.
17 M. R. Tassano, P. F. Audicio, J. P. Gambini, M. Fernandez,
J. P. Damian, M. Moreno, J. A. Chabalgoity, O. Alonso,
J. C. Benech and P. Cabral, Bioorg. Med. Chem. Lett., 2011,
21, 5598.
18 A. Laznickova, V. Biricova, M. Laznicek and P. Hermann,
Appl. Radiat. Isot., 2014, 84, 70.
19 C. Kojima, C. Regino, Y. Umeda, H. Kobayashi and
K. Kono, Int. J. Pharm., 2010, 383, 293.
20 S. Sadekar, A. Ray, M. Janat-Amsbury, C. M. Peterson and
H. Ghandehari, Biomacromolecules, 2011, 12, 88.
21 D. Zeng, N. S. Lee, Y. Liu, D. Zhou, C. S. Dence,
K. L. Wooley, J. A. Katzenellenbogen and M. J. Welch, ACS
Nano, 2012, 6, 5209.
22 J. C. Rainwater and E. V. Anslyn, Chem. Commun., 2010, 46,
2904.
23 D. Zhou, W. Chu, C. S. Dence, R. H. Mach and M. J. Welch,
Nucl. Med. Biol., 2012, 39, 1175.
2 V. Biricova and A. Laznickova, Bioorg. Chem., 2009, 37, 185.
3 V. J. Venditto, C. A. Regino and M. W. Brechbiel, Mol.
Pharm., 2005, 2, 302.
4 J. M. Criscione, L. W. Dobrucki, Z. W. Zhuang,
X. Papademetris, M. Simons, A. J. Sinusas and
T. M. Fahmy, Bioconjugate Chem., 2011, 22, 1784.
5 M. Adlanmerini, R. Solinhac, A. Abot, A. Fabre,
I. Raymond-Letron, A. L. Guihot, F. Boudou, L. Sautier,
E. Vessieres, S. H. Kim, P. Liere, C. Fontaine, A. Krust,
P. Chambon, J. A. Katzenellenbogen, P. Gourdy,
P. W. Shaul, D. Henrion, J. F. Arnal and F. Lenfant, Proc.
Natl. Acad. Sci. U. S. A., 2014, 111, E283.
6 S. M. Bartell, L. Han, H. N. Kim, S. H. Kim,
J. A. Katzenellenbogen, B. S. Katzenellenbogen,
K. L. Chambliss, P. W. Shaul, P. K. Roberson,
R. S. Weinstein, R. L. Jilka, M. Almeida and
S. C. Manolagas, Mol. Endocrinol., 2013, 27, 649.
7 V. Saint-Criq, S. H. Kim, J. A. Katzenellenbogen and
B. J. Harvey, PLoS One, 2013, 8, e78593.
24 F. Tajarobi, M. El-Sayed, B. D. Rege, J. E. Polli and
H. Ghandehari, Int. J. Pharm., 2001, 215, 263.
25 C. Z. Chen, N. C. Beck-Tan, P. Dhurjati, T. K. van Dyk,
R. A. LaRossa and S. L. Cooper, Biomacromolecules, 2000, 1,
473.
26 M. Ma, Y. Cheng, Z. Xu, P. Xu, H. Qu, Y. Fang, T. Xu and
L. Wen, Eur. J. Med. Chem., 2007, 42, 93.
27 D. G. Mullen and M. M. Banaszak Holl, Acc. Chem. Res.,
2011, 44, 1135.
8 W. P. Wong, J. P. Tiano, S. Liu, S. C. Hewitt, C. Le May,
S. Dalle, J. A. Katzenellenbogen, B. S. Katzenellenbogen,
K. S. Korach and F. Mauvais-Jarvis, Proc. Natl. Acad.
Sci. U. S. A., 2010, 107, 13057.
9 K. H. Schlick, J. R. Morgan, J. J. Weiel, M. S. Kelsey and
M. J. Cloninger, Bioorg. Med. Chem. Lett., 2011, 21, 5078.
10 M. Ciolkowski, J. F. Petersen, M. Ficker, A. Janaszewska,
J. B. Christensen, B. Klajnert and M. Bryszewska, Nano-
medicine, 2012, 8, 815.
28 J. I. Gavrilyuk, U. Wuellner, S. Salahuddin, R. K. Goswami,
S. C. Sinha and C. F. Barbas III, Bioorg. Med. Chem. Lett.,
2009, 19, 3716.
11 S. N. Goonewardena, P. R. Leroueil, C. Gemborys,
P. Tahiliani, S. Emery, J. R. Baker Jr. and H. Zong, Bioorg.
Med. Chem. Lett., 2013, 23, 2230.
29 Z. Jia, D. E. Lonsdale, J. Kulis and M. J. Monteiro, ACS
Macro Lett., 2012, 1, 780.
12 A. P. Goodwin, S. S. Lam and J. M. Frechet, J. Am. Chem.
Soc., 2007, 129, 6994.
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 8696–8701 | 8701