Development of a new thiol site-specific prosthetic group and its conjugation with [Cys40]-exendin-4 for in vivo targeting of insulinomas

Article abstract of DOI:10.1021/bc400084u

A new tracer, N-5-[¹⁸F]fluoropentylmaleimide ([ ¹⁸F]FPenM), for site-specific labeling of free thiol group in proteins and peptides was developed. The tracer was synthesized in three steps (¹⁸F displacement of the aliphatic tosylate, di-Boc removal by TFA to expose free amine, and incorporation of the free amine into a maleimide). The radiosynthesis was completed in 110 min with 11-17% radiochemical yield (uncorrected), and specific activity of 20-49 GBq/μmol. [¹⁸F]FPenM showed comparable labeling efficiency with N-[2-(4-[¹⁸F] fluorobenzamido)ethyl]maleimide ([¹⁸F]FBEM). Its application was demonstrated by conjugation with glucagon-like peptide type 1 (GLP-1) analogue [cys⁴⁰]-exendin-4. The cell uptake, binding affinity, imaging properties, biodistribution, and metabolic stability of the radiolabeled [ ¹⁸F]FPenM-[cys⁴⁰]-exendin-4 were studied using INS-1 tumor cells and INS-1 xenograft model. Positron emission tomography (PET) results showed that the new thiol-specific tracer, [¹⁸F]FPenM-[cys ⁴⁰]-exendin-4, had high tumor uptake (20.32 ± 4.36%ID/g at 60 min postinjection) and rapid liver and kidney clearance, which was comparable to the imaging results with [¹⁸F]FBEM-[cys⁴⁰]-exendin-4 reported by our group.

Full text of DOI:10.1021/bc400084u

Article
pubs.acs.org/bc
Development of a New Thiol Site-Specific Prosthetic Group and Its
Conjugation with [Cys40]‑exendin‑4 for in Vivo Targeting of
Insulinomas
Xuyi Yue,†,‡ Dale O. Kiesewetter,† Jinxia Guo,†,‡ Zhongchan Sun,† Xiaoxiang Zhang,† Lei Zhu,†,‡
Gang Niu,† Ying Ma,† Lixin Lang,† and Xiaoyuan Chen*,†
†National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), 31 Center Drive,
Bethesda, Maryland 20892, United States
‡Center for Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University, Xiamen, 361005, China
S
* Supporting Information
ABSTRACT: A new tracer, N-5-[18F]fluoropentylmaleimide ([18F]FPenM), for
site-specific labeling of free thiol group in proteins and peptides was developed. The
tracer was synthesized in three steps (18F displacement of the aliphatic tosylate, di-
Boc removal by TFA to expose free amine, and incorporation of the free amine into
a maleimide). The radiosynthesis was completed in 110 min with 11−17%
radiochemical yield (uncorrected), and specific activity of 20−49 GBq/μmol.
[18F]FPenM showed comparable labeling efficiency with N-[2-(4-[18F]-
fluorobenzamido)ethyl]maleimide ([18F]FBEM). Its application was demonstrated
by conjugation with glucagon-like peptide type 1 (GLP-1) analogue [cys40]-
exendin-4. The cell uptake, binding affinity, imaging properties, biodistribution, and
metabolic stability of the radiolabeled [18F]FPenM-[cys40]-exendin-4 were studied
using INS-1 tumor cells and INS-1 xenograft model. Positron emission tomography
(PET) results showed that the new thiol-specific tracer, [18F]FPenM-[cys40]-
exendin-4, had high tumor uptake (20.32 4.36%ID/g at 60 min postinjection) and rapid liver and kidney clearance, which was
comparable to the imaging results with [18F]FBEM-[cys40]-exendin-4 reported by our group.
widely used 18F-prosthetic group for labeling peptides and
INTRODUCTION
■
proteins by conjugation of amine groups under mildly basic
Receptor targeting with radiolabeled peptides is an important
conditions (pH 8) and temperatures less than 40 °C.12−17
and successful method for the diagnosis and treatment of
Sometimes peptide labeling with [18F]SFB gives poor yield due
neuroendocrine tumors.1 The most prominent clinically
to the steric hindrance of amino groups or unanticipated
applied peptides for in vivo imaging of human tumors are the
somatostatin analogues.2 The overexpression of many peptide
chemoselectivity and regioselectivity due to abundance of free
amino groups in protein.18,19 Furthermore, a stoichiometric
receptors in cancers, compared to their relatively low density in
normal organs, provides the rationale for in vivo molecular
excess of valuable protein or peptide is often required to
achieve sufficient radiochemical yields.
imaging and targeted radionuclide therapy. Among the available
Alternative 18F-prosthetic groups for peptide labeling are
positron emitters for peptide labeling, fluorine-18 (18F) is most
maleimide-containing compounds that provide site-specific
used due to its nuclear decay properties and chemical
labeling of free thiol groups. Peptides and proteins can be
properties. The nuclide decays with a relatively low positron
engineered to incorporate or expose one cysteine residue to
emission energy of 0.64 MeV, which provides a short tissue
range (0.46 mm) and thus the potential for high image
provide a site for specific labeling.20,21 There are several reports
using maleimides as prosthetic groups for 18F PET study
resolution. Its half-life (109.8 min) is long enough to allow
(Figure 1). The recently used 18F-prosthetic groups for thiol
complex, multistep radiosynthesis and allows image acquisitions
functions include [18F]FPPD and [18F]DDPFB developed by
for two or more hours postinjection. Labeling of organic
Shiue et al.;22 aminooxy-aldehyde condensation series such as
[18F]FBABM,23 [18F]FBAM,24 and [18F]FBOM,25 which
contained an aldehyde-reactive aminooxy group and a thiol-
reactive maleimide group via readily available 4-[18F]-
molecules with 18F is amply described in the literature.3−6
Only a few examples for direct labeling of peptides or other
macromolecules have been reported,7−11 due to the harsh
reaction conditions typically employed for 18F incorporation.
To circumvent this drawback, a prosthetic group carrying the
radioisotope is prepared and subsequently conjugated to a
reactive functionality of macromolecule under mild conditions.
N-Succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) is the most
Received: February 13, 2013
Revised: May 7, 2013
Published: June 10, 2013
© 2013 American Chemical Society
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Figure 1. 18F-labeled thiol site-specific prosthetic groups.
fluorobenzaldehyde. [18F]FDG-MHO, another aminooxy-alde-
hyde condensation agent for thiol-specific labeling based on
With the exceptions of [18F]FDG-MHO, [18F]AlF-NODA-
MPAEM, and [18F]FBSM, the above-described radiofluorina-
tions have been limited to nucleophilic aromatic substitutions.
Activated aromatic rings bearing both a good leaving group and
a strong electron-withdrawing component, especially located
para to the leaving group are required during radiolabeling.
However, heteroaromatic and aliphatic nucleophilic substitu-
tions only require a good leaving group and the reactions are
usually performed with dried no-carrier-added [18F]fluoride
under basic conditions.27,33 Here we developed a new aliphatic
maleimide-based prosthetic group based on aliphatic-nucleo-
philic substitution. It was used for labeling GLP-1 receptor
targeted peptide [cys40]-exendin-4 as an example (Figure 2).
[18F]FDG was developed by Wuest et al.26 Dolle’
́
s group
reported the preparation of a heteroaromatic [18F]maleimide,
1-[3-(2-[18F]fluoropyridine-3-yloxy)propyl]pyrrole-2,5-dione
([18F]FPyME) in three steps with 17−20% uncorrected
radiochemical yield.27 N-[2-(4-[18F]fluorobenzamido)ethyl]-
maleimide ([18F]FBEM) is among the most often used
prosthetic groups for site-specific radiolabeling of the free
sulfhydryl groups of peptides and proteins. Two groups
reported its synthetic methodology, either from previously
reported [18F]SFB precursor28 or through more efficient direct
condensation of 4-[18F]fluorobenzoic acid with N-(2-
aminoethyl)maleimide29 with high specific activity. The other
[18F]FBEM analogue, 4-[18F]fluorobenzylamidopropionyl mal-
eimide ([18F]FBAPM) was synthesized through four radio-
labeling steps with 55% decay-corrected yield in 70 min and
specific activity of 50−70 GBq/μmol. The route features highly
efficient reduction of nitrile group using borohydride/transition
metal catalyst method.30
Recently, several investigators reported novel bifunctional
prosthetic groups containing a maleimide group and a moiety
for incorporation of [18F]fluoride in a single step. Inkster et al.
reported sulfonyl fluoride-based prosthetic compounds as
potential 18F labeling agents under aqueous conditions. One
of the prosthetic groups, 4-N-maleimidobenzenesulfonyl [18F]-
fluoride ([18F]FBSM), was prepared in moderate yield, but the
solvent and base selection during the preparation have a
profound influence on the yield, and the exemplified 18F-
peptide showed evidence of proteolytic defluorination and
modification during incubation with mouse serum.31 McBride
et al., in an extension of their highly successful chelation of
[18F]aluminum fluoride, developed [18F]AlF-NODA-MPAEM.
This prosthetic group was conjugated to antibody Fab′
fragment at room temperature in high yield without much
loss of immunoreactivity. The method is useful for heat labile
compounds32 (Figure 1).
Figure 2. N-[18F]fluoroalkylmaleimide prosthetic groups.
Our group developed two new exendin-4 analogues by
incorporation of a cysteine residue to either the C- or N-
terminus of exendin-4, then site-specifically labeled with
maleimide-containing prosthetic group [18F]FBEM34 or
[18F]AlF-NOTA-MAL.35 Results showed that both
[18F]FBEM-[cys40]-exendin-4 and [18F]AlF-NOTA-MAL-
[cys40]-exendin-4 have high tumor uptake but exhibit
significantly different kidney uptake and clearance. In order
to develop a more rapid radiochemical synthesis of a
maleimide-based prosthetic group and to better understand
the effect of the prosthetic group on the imaging profile of
peptides, we describe here a new thiol-specific prosthetic agent,
N-5-[18F]fluoropentylmaleimide ([18F]FPenM) based on ali-
phatic nucleophilic substitution and its conjugation with
[cys40]-exendin-4. This new bifunctional agent has the lowest
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molecular weight among the maleimide-based tracers reported
and, thus, would be expected to have a minimal effect on
binding affinity compared to the parent exendin-4. The cell
uptake, biodistribution, and imaging properties of [18F]FPenM-
[cys40]-exendin-4 are described.
1-(2-Fluoroethyl)-1H-pyrrole-2,5-dione, 2. Fluoroethyl-
amine hydrochloride (110 mg, 2.2 mmol) was dissolved in a
saturated aqueous solution of sodium bicarbonate (4 mL). The
solution was cooled to 0 °C and stirred for 10 min before
adding N-(methoxycarbonyl)maleimide 1 (310 mg, 2 mmol).
The resulting solution was stirred at 0 °C for 10 min and at
room temperature for an additional 20 min and the reaction
was monitored by TLC. The mixture was directly subjected to
silica gel flash chromatography using hexane/EtOAc (3/1) as
the eluent to afford compound 2 (150 mg, 1.05 mmol, 53%
yield) as a colorless solid. 1H NMR (300 MHz, CDCl3) δ 6.72
(s, 2H), 4.58 (t, J = 5.1 Hz, 1H), 4.42 (t, J = 4.8 Hz, 1H), 3.84
(t, J = 5.1 Hz, 1H), 3.76 (t, J = 5.1 Hz, 1H); 13C NMR (75.5
MHz, CDCl3) δ 170.5, 134.4, 80.5 (d, J = 171.4 Hz), 38.2 (d, J
= 21.9 Hz); 19F NMR (282 MHz, CDCl3) δ −224.7 ∼ −225.2
(m); MS (APCI) 144.0 [M + H]+; HRMS (APCI): Calcd for
C6H7NO2F, 144.0461 [M + H]+, found 144.0457.
EXPERIMENTAL SECTION
■
Reagents and Instrumentation. Unless otherwise stated,
all chemicals were obtained from commercial sources and used
without further purification. Analytical thin layer chromatog-
raphy (TLC) was performed on Merck precoated silica gel 60
F254 plates with visualization by ultraviolet (UV) irradiation at
λ = 254 nm or staining with KMnO4. Purifications were
performed by silica gel chromatography. The water used in the
experiments with [cys40]-exendin-4 (FW 4289.69) was
deionized from Milli-Q Integral Water Purification System.
[Cys40]-exendin-4 was prepared by solid-phase peptide syn-
thesis (CS Bio, Menlo Park, CA, USA). The [cys40]-exendin-4
sequence is His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-
Lys-Gly-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-
Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-
Cys-NH2.
Typical Procedure for the Synthesis of Compounds 2-
[Bis(tert-butoxycarbonyl)amino]ethoxy(4-methylphen-
yl)-dioxo-λ6-sulfane 5a, 3-[Bis(tert-butoxycarbonyl)-
amino]propoxy(4-methylphenyl)-dioxo-λ6-sulfane 5b,
and 5-[Bis(tert-butoxycarbonyl)amino]pentoxy(4-meth-
ylphenyl)-dioxo-λ6-sulfane 5c. Commercial di-tert-butyl
iminodicarboxylate (1 equiv) was dissolved in ethanol and
treated with 1.1 equiv of 40% aqueous n-Bu4N+OH− at room
temperature for 1 h. The solvents were removed and the
residue dried by repeated evaporation with acetonitrile. To the
solution of the resulting salt in acetonitrile was added the bis-
tosylate of ethylene glycol (1.1 equiv). The resulting solution
was stirred at room temperature and monitored by TLC until
the starting material was completely consumed. The reaction
solution was diluted with water and extracted with CH2Cl2. The
organic layer was dried and evaporated. The residue was
subjected to flash chromatography using hexane/EtOAc (4/1)
as the eluent to give the title compound 5.
Mass Spectrometry. LC/MS analysis was conducted on a
Waters LC-MS system (Waters, Milford, MA) that included an
Acquity UPLC unit coupled to the Waters Q-Tof Premier high
resolution mass spectrometer. An Acquity BEH Shield RP18
column (150 × 2.1 mm) was employed for chromatography.
Elution was achieved with isocratic 2 mM ammonium formate,
0.1% formic acid, and 5% CH3CN at 0.35 mL/min. The entire
column elute was introduced into the Q-Tof mass spectro-
meter. Ion detection was achieved in ESI mode using a source
capillary voltage of 3.5 kV, source temperature of 110 °C,
desolvation temperature of 200 °C, cone gas flow of 50 L/Hr
(N2), and desolvation gas flow of 700 L/Hr (N2).
1
Compound 5a. Colorless solid, 2.44 g, yield 64%. H NMR
Chemistry. Compounds 1,36 4a,37 4b,38 4c,39 and 5a40
(Scheme 1) are known compounds and were prepared
according to literature procedures. Their physical and
spectroscopic data were in agreement with those previously
reported.
(300 MHz, CDCl3) δ 7.72 (d, J = 6.6 Hz, 2H), 7.30 (d, J = 8.1
Hz, 2H), 4.07 (t, J = 5.7 Hz, 2H), 3.83 (t, J = 5.7 Hz, 2H), 2.38
(s, 3H), 1.42 (s, 18H).
Compound 5b. Colorless solid, 1.32 g, yield 62%. 1H NMR
(300 MHz, CD3OD) δ 7.81 (d, J = 8.4 Hz, 2H), 7.47 (d, J = 8.1
Hz, 2H), 4.08 (t, J = 6.3 Hz, 2H), 3.64 (t, J = 6.6 Hz, 2H), 2.47
(s, 3H), 1.91 (t, J = 7.2 Hz, 2H), 1.53 (s, 18H); 13C NMR (75.5
MHz, CDCl3) δ 153.9, 146.6, 134.6, 131.3, 129.2, 84.1, 69.9,
29.9, 28.4, 21.7; MS (APCI) 447.2 [M + NH4]+; HRMS
(APCI): Calcd for C20H35N2O7S, 447.2165 [M + NH4]+, found
447.2166.
Scheme 1. Synthesis of N-2-Fluoroethylmaleimide 2, N-3-
Fluoropropylmaleimide 8b, N-5-Fluoropentylmaleimide
Standards 8c, Respectively
1
Compound 5c. Liquid, 0.41 g, yield 45%. H NMR (300
MHz, CD3OD) δ 7.92 − 7.89 (m, 2H), 7.56 (d, J = 7.8 Hz,
2H), 4.18 − 4.14 (t, J = 6.3 Hz, 2H), 3.67 − 3.61 (m, 2H), 2.57
(s, 3H), 1.83 − 1.72 (m, 2H), 1.70 − 1.65 (m, 2H), 1.64 (s,
18H), 1.57 − 1.41 (m, 2H); 13C NMR (75.5 MHz, CDCl3) δ
154.0, 146.3, 134.6, 131.2, 128.9, 83.5, 71.8, 47.0, 29.5, 29.4,
28.5, 23.7, 21.8; MS (APCI) 475.2 [M + NH4]+; HRMS
(APCI): Calcd for C22H39N2O7S, 475.2478 [M + NH4]+, found
475.2478.
Typical Procedure for the Synthesis of 1-(3-Fluoro-
propyl)-1H-pyrrole-2,5-dione 8b and 1-(5-Fluoropentyl)-
1H-pyrrole-2,5-dione 8c. To a test tube with a stirring bar
was added K222 (1 equiv) in acetonitrile (1 mL), and potassium
fluoride (2 equiv). The solvents were evaporated under a
stream of argon while being heated in a 100 °C oil bath. Three
portions of acetonitrile (1 mL) were added successively and
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each evaporated in turn. Then tosylate substrate 5b (or 5c) (1
equiv) in acetonitrile (1 mL) was added. The reaction solution
was heated at 100 °C for 20 min. The solvent was removed
under a stream of argon. The residue was treated with TFA (1
mL) at room temperature for 10 min. After evaporation of TFA
with argon flow, the reaction was placed in 0 °C ice water and
saturated sodium bicarbonate (4 mL) and N-
(methoxycarbonyl)maleimide (2 equiv) were added succes-
sively. The solution was stirred at 0 °C for 20 min and then at
room temperature for 10 min, then subjected to silica gel
chromatography directly using hexane/EtOAc (4/1) as the
eluent to afford compound 8b (or 8c).
solution was cooled to room temperature and diluted with
CH2Cl2 (200 μL); the mixture was loaded onto a short plug of
silica gel and eluted with another portion of CH2Cl2 (400 μL)
into a 13 × 100 mm test tube. The isolated yield for this
radiolabeling step was calculated by dividing the amount of
activity in the eluent by the total initial activity used. The eluate
was evaporated to dryness under a stream of argon at room
temperature. Trifluoroacetic acid (TFA, 200 μL) was added to
the residue and allowed to stand for 5 min. The TFA was
evaporated with a stream of argon. The reaction tube was then
cooled in an ice bath and the following reagents were added in
sequence: saturated NaHCO3 (450 μL); and N-
(methoxycarbonyl)maleimide (9.3 mg, 60 μmol) in THF
(150 μL). Five min later, the solution was diluted with 0.1%
TFA in H2O (0.3 mL), and subjected to semipreparative HPLC
directly (Phenomenex Luna 5 μm C18 column, 250 × 10 mm;
flow rate 4.0 mL/min; solvent A, 0.1% TFA in water; solvent B,
0.1% TFA in acetonitrile; using isocratic with 30% solvent B,
UV detection at 210 nm, and online radioactivity detection,
product tR = 26.1 min). The collected fraction (typically 19−
28% uncorrected yield) was diluted with 10 mL water and the
product was trapped on a Sep-Pak C18 plus cartridge (360 mg
sorbent per cartridge). The cartridge was washed with H2O (3
mL) and hexane (2 mL), and the product was eluted with 10%
EtOH in CH2Cl2 (1.5 mL). The solvent was removed under
argon flow. The three-step radiolabeling was completed in an
average time of 110 min with 11−17% uncorrected yield.
For quality control, the concentrated sample was analyzed by
analytical HPLC (Phenomenex Luna 3 μm C18 column, 150 ×
4.6 mm; flow rate 1.0 mL/min; solvent A, 0.1% TFA in water;
solvent B, 0.1% TFA in acetonitrile), using isocratic elution
with 35% solvent B; product eluted at 8.1 min. Specific activity
was estimated at 210 nm of wavelength by comparison with a
standard curve.
Compound 8b. Colorless solid, 28% yield for three steps, 1H
NMR (300 MHz, CDCl3) δ 6.73 (s, 2H), 4.56 (t, J = 5.7 Hz,
1H), 4.41 (t, J = 5.7 Hz, 1H), 3.70 (t, J = 6.9 Hz, 2H), 2.08 −
2.04 (m, 1H), 1.99 − 1.95 (m, 1H); 13C NMR (75.5 MHz,
CDCl3) δ 170.9, 134.4, 81.8 (d, J = 166.1 Hz), 34.8 (d, J = 21.0
Hz), 29.6 (d, J = 78.0 Hz); 19F NMR (282 MHz, CDCl3) δ
−220.9 ∼ −221.4 (m); MS (APCI) 158.1 [M + H]+; HRMS
(APCI): Calcd for C7H9NO2F, 158.0617 [M + H]+, found
158.0612.
1
Compound 8c. Liquid, 62% yield for three steps, H NMR
(300 MHz, CDCl3) δ 6.71 (s, 2H), 4.53 (t, J = 6.0 Hz, 1H),
4.37 (t, J = 6.0 Hz, 1H), 3.56 (t, J = 7.2 Hz, 2H), 1.81 − 1.60
(m, 4H), 1.48 − 1.28 (m, 2H); 13C NMR (75.5 MHz, CDCl3)
δ 171.0, 134.3, 84.0 (d, J = 164.6 Hz), 37.8, 30.1 (d, J = 78.0
Hz), 28.4, 22.7 (d, J = 21.0 Hz); 19F NMR (282 MHz, CDCl3)
δ −218.4 ∼ −218.9 (m); MS (APCI) 186.1 [M + H]+; HRMS
(APCI): Calcd for C9H13NO2F, 186.0930 [M + H]+, found
186.0927.
General Procedure for the Synthesis of Nonradioac-
tive Fluoroalkylmaleimido-[cys40]-exendin-4. [Cys40]-ex-
endin-4 was dissolved in degassed phosphate-buffered saline
(PBS), N-2-fluoroethylmaleimde, or N-5-fluoropentylmalei-
mide in EtOH was added in one portion and incubated for 1
h, then the mixture was subjected to semipreparative HPLC
chromatography to give N-2-fluoroethylmaleimido-[cys40]-
exendin-4 and N-5-fluoropentylmaleimido-[cys40]-exendin-4,
respectively. For semipreparative HPLC, a Vydac C18 protein
column (9.4 × 250 mm) and a gradient elution profile were
used with 0.1% trifluoroacetic acid (TFA) in water (solvent A)
and 0.1% TFA in CH3CN (solvent B). The elution profile was
isocratic at 25% solvent B for 5 min, then a gradient to 55%
solvent B over 20 min, and finally to 90% B over the next 5
min. The major peak at about 17 min was collected and
lyophilized. For analytical HPLC, a Vydac C18 protein column
(4.6 × 250 mm) was used with a gradient elution profile using
water (solvent A), CH3CN (solvent B), 1 mM EDTANa2, and
1% TFA in water (solvent C). The gradient used was 65%
water, 25% acetonitrile, 10% solvent C for 5 min, then a linear
gradient to 35% water, 55% acetonitrile, 10% solvent C by 25
min, and a linear gradient to 90% acetonitrile, 10% solvent C at
30 min.
Typical Procedure for the Synthesis of Compounds
[18F]8a, [18F]8b, and [18F]8c. To a test tube was added K222
(4.5 mg, 12 μmol in 100 μL acetonitrile), K2CO3 (60 μL of 0.1
M in water, 6 μmol), and [18F]fluoride (59 − 77 mCi). The
solvents were evaporated under a stream of argon while being
heated in a 100 °C heating block. Three portions of acetonitrile
(200 μL) were added successively and each evaporated in turn;
the drying procedure took about 10 min. Then tosylate 5 (2.5
− 3.0 mg, 6.0 μmol) in acetonitrile (200 μL) was added. The
reaction solution was heated at 100 °C for 10 min. The reaction
Radiochemical Synthesis of [18F]FPenM-[cys40]-exen-
din-4. To the above [18F]FPenM (8−11 mCi) was added
ethanol (10 μL) and the tube was vortexed. [Cys40]-exendin-4
(100−200 μg) in 100 μL 0.1% sodium ascorbate in degassed
PBS was added, and the solution was vortexed followed by
incubation for 30 min at room temperature. Aqueous TFA
(0.1% TFA, 100 μL) was added and the solution was injected
onto a semipreparative HPLC system (as described above for
nonradioactive fluoroalkylmaleimido-[cys40]-exendin-4 with the
exception that the first gradient went only to 45% B over 30
min). The elution time of the radioactive product was about
29.3 min. The fraction containing [18F]FPenM-[cys40]-exendin-
4 was diluted to 20 mL with water and the solution passed
through a C18 BondElut cartridge (100 mg). The product that
remained on the cartridge was eluted with 1 mL of 10 mM HCl
in ethanol. The ethanol was concentrated on a rotary
evaporator to about 100−200 μL. A portion of this remaining
volume was used for determination of radiochemical purity.
The remaining volume was diluted in PBS for in vitro and in
vivo studies. For quality control, the same analytical HPLC
condition as preparation of nonradioactive fluoroalkylmaleimi-
do-[cys40]-exendin-4 was used. HPLC-MS 1119.4 [M + 4H]4+;
deconvolves to 4474.0. Calculated exact mass:
C
196H299FN52O63S2 (4474.128).
Mouse Serum Stability Study. [18F]FPenM-[cys40]-
exendin-4 (50 μCi) was mixed with 200 μL freshly harvested
mouse serum and a 50 μL aliquot was removed at 0 min. The
remainder was incubated at 37 °C and additional aliquots of 50
μL were removed at 30 and 150 min. The aliquots were mixed
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with an equal volume of CH3CN and centrifuged. A portion of
the supernatant was subjected to radioHPLC analysis using an
online radioactivity detector.
static PET images were acquired at 30 and 60 min postinjection
(p.i.; n = 4/group), respectively. For blocking experiment, 75
μg [cys40]-exendin-4 was injected 10 min prior to the injection
of [18F]FPenM-[cys40]-exendin-4. The images were recon-
structed using a three-dimensional ordered-subset expectation
maximization (3D OSEM) algorithm without correction for
attenuation or scattering. For each scan, regions of interest
(ROIs) were drawn over the tumor and major organs using
Inveon Workplace 3.0 on decay-corrected whole-body coronal
images. The radioactivity concentrations within the tumors,
muscle, liver, and kidneys were obtained from mean pixel values
within the multiple ROI volume and then converted to MBq/
mL/min using the calibration factor determined for the Inveon
PET system. These values were then divided by the
administered activity to obtain (assuming a tissue density of
1 g/mL) an image-ROI-derived percent injected dose per gram
(%ID/g).
Biodistribution Studies and Metabolite Analysis.
Immediately after PET imaging, the tumor-bearing mice were
sacrificed and dissected. Blood, tumor, major organs, and
tissues were collected and wet-weighed. The radioactivity in the
wet whole tissue was measured with a γ-counter (Wallach
Wizard, PerkinElmer). The results were expressed as
percentage of injected dose per gram of tissue (%ID/g) for a
group of 4 animals. For each mouse, the radioactivity of the
tissue samples was calibrated against a known aliquot of the
injected radiotracer. Values were expressed as mean SD. For
a blocking study, tumor xenografted mice were pretreated with
75 μg [cys40]-exendin-4 10 min prior to injection of the
corresponding [18F]FPenM-[cys40]-exendin-4. A subset of the
collected tissues (homogenized tumor and kidneys, plasma, and
urine) was extracted with a volume of CH3CN equal to the
weight of the sample. The mixtures were centrifuged and the
supernatant separated from the pellet. Supernatants were
analyzed by HPLC using online radioactivity detection or via
collection of 1 min fractions and subsequent γ-counting.
Cell Culture. INS-1 cell line was cultured in RPMI-1640
medium (GIBCO) containing 10% (v/v) fetal bovine serum
(GIBCO) supplemented with penicillin (100 μg/mL),
streptomycin (100 μg/mL), nonessential amino acids (100
μM), and sodium pyruvate (1 mM) at 37 °C with 5% CO2.
Animal Model. All animal studies were conducted in
accordance with the principles and procedures outlined in the
Guide for the Care and Use of Laboratory Animals and were
approved by the Institutional Animal Care and Use Committee
of Clinical Center, NIH. The INS-1 xenograft tumor models
were developed in 5 to 6-week-old female athymic nude mice
(Harlan Laboratories) by injection of 5 × 106 cells in the left or
right shoulder. Tumor growth was monitored using caliper
measurements of perpendicular axes of the tumor. The tumor
volume was estimated by the formula: tumor volume = a ×
(b2)/2, where a and b were the tumor length and width,
respectively, in millimeters. The mice underwent small-animal
PET studies when the tumor volume reached 100−300 mm3
(3−4 weeks after inoculation).
Receptor Binding Assay. The GLP-1 receptor binding
assay was performed to determine binding affinities of FEtM-
[cys40]-exendin-4, FPenM-[cys40]-exendin-4, FBEM-[cys40]-ex-
endin-4, and exendin-4. GLP-1 analogs were dissolved in
DMSO and diluted by cell binding buffer (1 mM CaCl2, 5 mM
MgCl2, 0.5% BSA in RPMI medium) to the desired
concentrations immediately before usage. At 80% confluence,
the cultured cells were scraped off and suspended in binding
buffer at a final concentration of 2 × 106 cells/mL. The cells
were aliquoted into a 96 well plate (1 × 105 INS-1 cells/well)
and incubated with 125I-GLP-1(7−36) (0.02 μCi/well,
PerkinElmer, Inc.) in binding buffer in the presence of different
concentrations of GLP-1 analogs at room temperature for 2 h.
The final volume per well is 200 μL. After incubation, the plate
was washed three times with PBS containing 0.1% BSA, and the
radioactivity was measured by γ-counting. The IC50 values were
calculated by nonlinear regression analysis using the GraphPad
Prism computer-fitting program (GraphPad Software, Inc., San
Diego, CA). Each data point is a result of the average of
duplicate wells.
Cell Uptake and Efflux Studies. For cell uptake, INS-1
cells were seeded into 24-well plates at a density of 1 × 105 cells
per well and incubated with 37 kBq (1 μCi) per well of
[18F]FPenM-[cys40]-exendin-4 in 500 μL of serum free cell
culture medium at 37 °C for 15, 30, 60, and 120 min. The cells
were then washed three times with chilled PBS and lysed with
200 μL 0.1 M NaOH. For efflux studies, about 37 kBq (1 μCi)
per well of [18F]FPenM-[cys40]-exendin-4 were first incubated
with INS-1 cells in 24-well plates for 1 h at 37 °C. The cells
were washed three times with chilled PBS and allowed to stand
with fresh buffer at 37 °C. At various time points (0, 30, and 60
min), the medium was removed and the cells washed three
times with chilled PBS. The cells were then lysed with 200 μL
0.1 M NaOH. The cell suspensions were collected and
measured in a γ-counter. Each data point is an average of
triplicate values.
RESULTS
■
Synthesis. N-2-Fluoroethylmaleimide 2 was prepared from
commercially available 2-fluoroethylamine hydrochloride with a
straightforward strategy (Scheme 1). N-methoxycarbonylmalei-
mide 1, was subjected to reaction with 2-fluoroethylamine
hydrochloride in saturated sodium bicarbonate solution to give
fluoroethylmaleimide standard 2.41 The condensation was
performed at 0 °C, and worked up immediately upon
completion as indicated by thin layer chromatography.
Extended reaction time at room temperature resulted in
hydrolytic decomposition of the maleimide.
The N-3-fluoropropylmaleimide and N-5-fluoropentylmalei-
mide were obtained from similar procedures (Scheme 1). First,
commercially available 1,3-propanediol 3b and 1,5-pentanediol
3c were converted into the corresponding ditosylate 4b and 4c,
respectively. SN2 displacement of one tosylate of 4b and 4c
with di-tert-butyl iminodicarboxylate afforded compounds 5b
and 5c, respectively, according to the reported procedure.40
With 5b and 5c in hand, fluorine incorporation was achieved by
displacement of the remaining tosylate with K222/KF.
Deprotection of the Boc functional group with TFA at room
temperature afforded the free fluoroalkylamine. The conjuga-
tion between resulting primary amine intermediate and N-
methoxycarbonylmaleimide proceeded smoothly under aque-
ous conditions as described above. N-3-Fluoropropylmaleimide
8b and N-5-fluoropentylmaleimide 8c were obtained in 28%
MicroPET Imaging. PET scans and image analysis were
performed using an Inveon microPET scanner (Siemens
Medical Solutions). [18F]FPenM-[cys40]-exendin-4 (∼100
μCi) was administered via tail vein injection while the animal
was under isoflurane anesthesia. Five-minute and ten-minute
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Scheme 2. 18F Radiolabeling of Tosylate Substrates 5a, 5b, 5c
and 62% yield, respectively, in three steps after silica gel
chromatography. Compounds 2 and 8b are colorless solids
while 8c is light yellow liquid. The conjugation of 2 and 8c with
[cys40]-exendin-4 proceeded smoothly in PBS buffer with 0.1%
sodium ascorbate as the antioxidant.
The three synthesized compounds 5a, 5b, and 5c with
different aliphatic chains (ethyl, propyl, and pentyl, respec-
tively) were evaluated for labeling with 18F (Scheme 2). For
substrate 5a, the radiofluorination proceeded in low yield (20−
30%, decay uncorrected), perhaps due to the volatility of the
resulting product. However, when propyl analogue 5b and
pentyl analogue 5c were employed as the substrates, the
fluorine incorporation in the first step typically afforded 50%
and 65% decay uncorrected yields, respectively. After
deprotection of the amine with TFA, the resulting fluoroalkyl-
amine salt was subjected to reaction with N-methoxycarbo-
nylmaleimide. The optimized conditions for this exchange
reaction employed saturated NaHCO3/THF (3/1, v/v) as
solvent and 0 °C as the reaction temperature. A 10-fold excess
of N-methoxycarbonylmaleimide 1 (60 μmol) relative to
substrate 5b or 5c (6 μmol) was used to shorten the reaction
time to 5 min. Poor trapping efficiency was observed for
product [18F]8b, but product [18F]8c with its longer aliphatic
chain was readily purified and isolated from HPLC eluate. The
overall yield of the three-step production of [18F]8c based on
initial [18F]fluoride was 11−17% (uncorrected) after purifica-
tion and isolation by HPLC. The entire synthesis procedure
was completed in an average time of 110 min with product
specific activity of 20−49 GBq/μmol. [18F]FPenM was chosen
as the prosthetic group for coupling with [cys40]-exendin-4
according to the typical procedure.34
Receptor Binding Assay. IC50 values of exendin-4, FBEM-
[cys40]-exendin-4, FEtM-[cys40]-exendin-4, and FPenM-
[cys40]-exendin-4 against 125I-GLP-1 in INS-1 cells are shown
in Figure 3. Of the two modified tracers synthesized, FPenM-
[cys40]-exendin-4 has comparable IC50 (1.11 nM) with parent
exendin-4 (0.98 nM), which also showed almost the same
binding affinity as FBEM-[cys40]-exendin-4 (1.10 nM). FEtM-
[cys40]-exendin-4 exhibited slightly lower binding affinity (1.57
nM) compared to parent exendin-4 and FBEM-[cys40]-
exendin-4. These results indicated that labeling [cys40]-
exendin-4 with our newly developed FPenM did not reduce
binding affinity toward INS-1 cells.
■
Figure 3. Competitive binding assay (IC50) of exendin-4 (red ),
40
FBEM-[cys40]-exendin-4 (black ), FEtM-[cys ]-exendin-4 (brown-
▼
40
⧫
●
ish yellow ), and FPenM-[cys ]-exendin-4 (purple ).
Cell Uptake and Clearance. The total cell uptake kinetics
of [18F]FPenM-[cys40]-exendin-4 with INS-1 tumors cells are
shown in Figure 4. The cell uptake of [18F]FPenM-[cys40]-
exendin-4 showed 0.62 0.03%AD at 0.5 h, increasing to 0.83
0.06%AD after 1 h, then the uptake reached a plateau. The
cell efflux was performed after 60 min incubation, and the cell
retention was reduced to 0.89 0.08%AD at 30 min and 0.72
0.06%AD at 60 min. The specificity of the radiotracer was
confirmed by minimum cell uptake of [18F]FPenM-[cys40]-
exendin-4 (<0.1%AD) in the presence of blocking dose of
exendin-4 (1 μM).
Imaging and Biodistribution in INS-1 Xenograft. High
tumor uptake was apparent by microPET imaging (Figure 5) at
30 min postinjection of [18F]FPenM-[cys40]-exendin-4 (21.30
4.55%ID/g), and remained virtually unchanged (20.32
4.36% ID/g) at 60 min after tracer injection. In contrast, kidney
uptake of [18F]FPenM-[cys40]-exendin-4 was high (34.41
4.59%ID/g) at 30 min and was significantly reduced by 60 min
postinjection (11.30
2.41%ID/g). Similar clearance was
Mouse Serum Stability. Serum stability, as analyzed by
radioHPLC, showed that [18F]FPenM-[cys40]-exendin-4 was
stable in mouse serum up to 2.5 h at 37 °C, with only a
negligible quantity of polar component produced (Figure S1),
which may be due to the oxidation of the methionine in [cys40]-
exendin-4.34 We found that the polar component could be
minimized by adding EDTANa2 to the analytical HPLC solvent
as metal chelator during analysis. The extraction efficiency from
the mouse serum was around 85% based on the supernatant
fraction we injected for online radio-HPLC analysis.
observed from liver (3.36 0.02%ID/g at 30 min vs 1.41
0.02%ID/g at 60 min). Negligible tracer uptake was observed in
the muscle. Injection of a blocking dose of [cys40]-exendin-4
prior to tracer injection resulted in significant reduction of
tumor uptake at 30 min (2.65 0.63%ID/g), but kidney and
liver uptake showed slight reduction, indicating receptor
specific uptake of the tracer to INS-1 tumor. Quantification
of the PET data at the 1 h time point showed high tumor-to-
kidney (1.80) and tumor-to-liver (8.43) ratios. The specific
tumor uptake of [18F]FPenM-[cys40]-exendin-4 was further
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18
40
○
▲
Figure 4. Total cellular uptake (a, , blue) and efflux (b, , green) of [ F]FPenM-[cys ]-exendin-4.
Figure 5. Representative PET images of [18F]FPenM-[cys40]-exendin-4 (100 μCi) in INS-1 mouse xenograft models at 30 and 60 min post
injection: (a) control; (b) INS-1 with a blocking dose of 75 μg [cys40]-exendin-4 administered 10 min prior to the radiotracer; (c) time course of
uptake by selected tissues determined by quantification of PET ROIs; (d) time course of uptake by selected tissues determined by quantification of
PET ROIs following blocking dose.
confirmed by biodistribution, which showed tumor-to-kidney
and tumor-to-liver ratios of 1.75 and 28.14, respectively (Figure
6), and significant blocking was observed by injection of
[cys40]-exendin-4 prior to tracer administration. Negligible
uptake was observed in the bone, indicating the stability of the
tracer to defluorination. The blocking dose resulted in a
reduction in uptake in the kidneys, pancreas, and lung.
The metabolic fate of the [18F]FPenM-[cys40]-exendin-4 in
tumor, kidneys, and urine were investigated by analysis of
samples extracted following biodistribution studies in INS-1
Figure 6. Biodistribution of [18F]FPenM-[cys40]-exendin-4 (100 μCi)
tumor-bearing mice. The extracts from organs were analyzed by
radio-HPLC and then compared to parent [18F]FPenM-
at 1 h measured by radioactive counting of dissected tissues with or
without [cys40]-exendin-4 (75 μg) blocking dose prior to tracer
[cys40]-exendin-4. The resulting radio-HPLC chromatograms
injection.
indicated that urine contained only polar radioactive metabo-
lites and the kidneys contained a mixture of parent [18F]-
FPenM-[cys40]-exendin-4 and polar radioactive metabolites;
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however, the tumor contained predominately parent peptide
(Figure S2).
later time point were further confirmed by ex vivo
biodistribution study. INS-1 tumor uptake of 18F-FBEM-
exendin-4 is 30.27
used in this study is 33.21
5.44% ID/g,34 while that of the probe
DISCUSSION
4.79% ID/g. There is no
■
significant difference as to tumor uptake with these two tracers.
There was negligible uptake in bone, indicating the stability of
the tracer to defluorination. Tracer uptake in pancreas and lung
were both high and can be blocked by injection of unlabeled
[cys40]-exendin-4. This is consistent with the results reported
by our group,34 which is due to the fact that GLP-1 receptor is
mainly expressed in the pancreatic islet cell, the intestine, lung,
kidneys, breast, and brainstem. These results suggest that
[18F]FPenM-[cys40]-exendin-4 specifically binds to GLP-1R
expressing organs.47
We have developed a new thiol-specific prosthetic agent,
[18F]FPenM, based on aliphatic nucleophilic substitution. No
additional activating functional groups are required as in the
case of homoaromatic fluorination. Because the maleimide ring
is not stable under typical nucleophilic radiofluorination
conditions, a three-step radiolabeling procedure was employed:
(1) 18F incorporation by SN2 displacement on an aliphatic
tosylate substrate; (2) TFA acidolysis of amine protecting
group; (3) incorporation of [18F]fluoroalkylamine into a
maleimide via transamidation. Three tosylate substrates 5a,
5b, and 5c, with different lengths of aliphatic chains and hence
different lipophilicity, were screened for 18F labeling.
We showed by radioHPLC that [18F]FPenM-[cys40]-
exendin-4 was stable in mouse serum over 2.5 h. An ex vivo
metabolite study, conducted by tissue extraction, showed that
most radioactivity in tumor was intact parent compound, but
the kidneys contained a great deal of polar metabolites and the
urine contained only polar metabolites. Thus the tumor has
very little retention of radioactive metabolites whether they
were produced or not.
GLP-1R is an important target, both for insulinoma and for
imaging pancreatic beta cells, which also present GLP-1R. Beta
cell transplantation currently is used for treatment of patients
with type I diabetes. Since GLP-1 receptor agonists, such as
exendin-4 analogs, can selectively bind to GLP-1R, they can be
potentially used for detection of insulinoma and to monitor the
survival of transplanted beta cells. However, immunological
rejections limit the survival of the transplant. 18F labeled
exendin-4 could potentially be used to monitor the viability and
mass of transplanted beta cells.41 The utility of the exendin-4
analogs depends on achieving high signal to background ratio,
especially in the abdomen where most insulinomas are found.
In addition, beta cell transplantation is often conducted in the
liver.48,49 Our results indicated specific uptake of [18F]FPenM-
[cys40]-exendin-4 in tissues containing GLP-1R. Uptake is
problematic in the kidneys, but the majority of the gastro-
intestinal tract and liver had very low background radioactivity.
Thus it may prove useful as a PET imaging agent for
insulinoma and pancreas imaging.
Substrate 5a exhibited very low yield in the fluorination step,
possibly due to the volatility of the resulting product. However,
reaction of substrates 5b and 5c afforded higher yield for this
labeling step. The subsequently produced fluoromaleimides 8a
and 8b presented with an unanticipated problem.
Poor trapping efficiency was obtained for products [18F]8a
and [18F]8b using solid-phase extraction of the crude reaction
mixtures directly or from fractions following semipreparative
HPLC. However, product [18F]8c, with its longer aliphatic
chain, was much more efficiently isolated from the HPLC
eluate. The high trapping efficiency for the most lipophilic [18F]
8c led us to apply this to the synthesis of [18F]FPenM-[cys40]-
exendin-4.
We prepared authentic samples of FEtM-[cys40]-exendin-4
and FPenM-[cys40]-exendin-4 in order to evaluate the binding
affinity relative to GLP-1. The binding affinities of exendin-4,
FPenM-[cys40]-exendin-4, and FBEM-[cys40]-exendin-4 were
about 1 nM and not significantly different from each other.
These affinities are higher than previously reported NOTA-
MAL-[cys40]-exendin-4 (2.84 nM)35 and [Lys40(natGa-
DOTA)]exendin-3 (5.7 nM).41 [18F]FPenM-[cys40]-exendin-4
showed quite low uptake into INS-1 tumor cells. However, the
efflux of radiolabeled compound was also quite slow. This cell
uptake and efflux behavior was similar to that reported for
FBEM-[cys40]-exendin-4.34
In INS-1 tumor xenograft model, microPET imaging of
[18F]FPenM-[cys40]-exendin-4 showed high tumor uptake at
early time points (30 min), and remained almost the same at 60
min after tracer injection. The high tumor uptake can be
significantly blocked by injection of unlabeled [cys40]-exendin-4
prior to radiolabeled tracer administration, which indicated
highly specific targeting toward INS-1 tumor of the labeled
peptide. Kidney uptake was higher (33%ID/g) than tumor at
30 min, but had cleared by a factor of 3 (11%ID/g) by 60 min.
This kidney retention is similar to that of [18F]FBEM-[cys40]-
exendin-4 and much lower compared to [18F]AlF-NOTA-
[cys0]-exendin-435 and [Lys40(68Ga-DOTA)]-exendin-3,42
which showed 63%ID/g and 120%ID/g, respectively, at 60
min postinjection. We reported that kidney uptake of [18F]AlF-
NOTA-MAL-[cys40]-exendin-435 increased throughout the
course of a 60-min experiment. Evaluation of kidney uptake
in non-tumor-bearing mice using another exendin-4 analogue
[18F]AlF-NOTA-[cys0]-exendin-4, in which NOTA is attached
at the N-terminus of the peptide, also showed high and
sustained kidney uptake in 60 min. High kidney uptake has
been reported for all radiometal chelate exendin ana-
logues.43−46 The tumor-to-kidney and tumor-to-liver ratios at
CONCLUSION
■
A new prosthetic group, [18F]FPenM, for site-specific labeling
of free thiol group in proteins and peptides was developed. To
the best of our knowledge, this is the first example of
maleimide-containing prosthetic group synthesized based on
aliphatic nucleophilic substitution and it has the smallest
molecular weight among the developed prosthetic agents. Since
the maleimido group was incompatible with harsh radio-
fluorination conditions, three synthetic steps were required for
the prosthetic group synthesis. Thus, we achieved no
improvement in yields or synthetic simplification compared
to [18F]FBEM. The application of [18F]FPenM was demon-
strated in the synthesis and evaluation of [18F]FPenM-[cys40]-
exendin-4 for imaging GLP-1R. The tracer has comparable
imaging results with [18F]FBEM-[cys40]-exendin-4 developed
by our group, showing high tumor-to-liver and tumor-to-kidney
uptake ratios for GLP-1R imaging. INS-1 tumor xenografts,
pancreas, and lung showed specific uptake, indicating that
[18F]FPenM-[cys40]-exendin-4 may be useful as pancreatic β-
cell imaging agent. The developed tracer has higher binding
affinity than NOTA-MAL-[cys40]-exendin-4 and [Lys40(natGa-
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DOTA)]exendin-3, superior tumor targeting, and more rapid
kidney clearance, showing it is a promising imaging probe for in
vivo targeting of insulinomas.
ASSOCIATED CONTENT
■
S
* Supporting Information
Serum stability of [18F]FPenM-[cys40]-exendin-4. Metabolite
analysis of [18F]FPenM-[cys40]-exendin-4 in the tumor,
kidneys, and urine. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: Shawn.Chen@nih.gov.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the intramural research program of
the National Institute of Biomedical Imaging and Bioengineer-
ing, National Institutes of Health. The authors acknowledge the
NIH Warren Grant Magnuson Clinical Center’s PET Depart-
ment for radioisotope production. Lei Zhu was partially
supported by the Center for Neuroscience and Regenerative
Medicine (CNRM) through Henry Jackson Foundation.
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