Efficient method for site-specific 18F-labeling of biomolecules using the rapid condensation reaction between 2-cyanobenzothiazole and cysteine

Article abstract of DOI:10.1021/bc300273m

An efficient method based on a rapid condensation reaction between 2-cyanobenzothiazole (CBT) and cysteine has been developed for ¹⁸F-labeling of N-terminal cysteine-bearing peptides and proteins. An ¹⁸F-labeled dimeric cRGD ([¹⁸F]CBTRGD₂) has been synthesized with an excellent radiochemical yield (92% based on radio-HPLC conversion, 80% decay-corrected, and isolated yield) and radiochemical purity (>99%) under mild conditions using ¹⁸F-CBT, and shown good in vivo tumor targeting efficiency for PET imaging. The labeling strategy was also applied to the site-specific ¹⁸F-labeling of a protein, Renilla lucifierase (RLuc8) with a cysteine residue at its N-terminus. The protein labeling was achieved with 12% of decay-corrected radiochemical yield and more than 99% radiochemical purity. This strategy should provide a general approach for efficient and site-specific ¹⁸F-labeling of various peptides and proteins for in vivo molecular imaging applications.

Full text of DOI:10.1021/bc300273m

Article
pubs.acs.org/bc
Efficient Method for Site-Specific 18F‑Labeling of Biomolecules Using
the Rapid Condensation Reaction between 2‑Cyanobenzothiazole
and Cysteine
Jongho Jeon,‡ Bin Shen,‡ Liqin Xiong, Zheng Miao, Kyung Hyun Lee, Jianghong Rao,*
and Frederick T. Chin*
Molecular Imaging Program at Stanford, Department of Radiology, Stanford University, 1201 Welch Road, Stanford, California
94305−5484, United States
S
* Supporting Information
ABSTRACT: An efficient method based on a rapid condensation reaction between 2-cyanobenzothiazole (CBT) and cysteine
has been developed for 18F-labeling of N-terminal cysteine-bearing peptides and proteins. An 18F-labeled dimeric cRGD
([18F]CBTRGD2) has been synthesized with an excellent radiochemical yield (92% based on radio-HPLC conversion, 80%
decay-corrected, and isolated yield) and radiochemical purity (>99%) under mild conditions using 18F-CBT, and shown good in
vivo tumor targeting efficiency for PET imaging. The labeling strategy was also applied to the site-specific 18F-labeling of a
protein, Renilla lucifierase (RLuc8) with a cysteine residue at its N-terminus. The protein labeling was achieved with 12% of
decay-corrected radiochemical yield and more than 99% radiochemical purity. This strategy should provide a general approach
for efficient and site-specific 18F-labeling of various peptides and proteins for in vivo molecular imaging applications.
thiol groups, respectively;8−11 (3) [18F]fluoroazide and [18F]-
fluoroalkyne that can react with a biomolecule equipped with
INTRODUCTION
■
Positron emission tomography (PET) is a non-invasive
molecular imaging technique with excellent sensitivity.1 In the
an alkyne and an azide group, respectively, through copper-
catalyzed click chemistry.12−16 In many cases, originally
past two decades, various biomolecules have been radiolabeled
designed 18F-prosthetic groups require lengthy synthetic
for PET imaging studies of receptor activity in living subjects
and for disease diagnostics including tumor detection.2−5 As
procedures, relatively harsh reaction conditions, or difficult
remote controls, which often result in poor radiochemical yield
(RCY) and difficulty in purification. Recently, to overcome
fluorine-18 (18F) can be easily produced in high quantities on a
medical cyclotron and has an ideal half-life of 110 min for
these problems, fast and specific ligation methods such as
imaging subjects, it is one of the commonly used radioisotopes
copper-free click reaction17,18 and the [4 + 2] inverse electron
for producing many PET tracers. Direct 18F-fluorination,
demand Diels−Alder reaction between tetrazine structure and
however, requires harsh reaction conditions such as high
temperature, high pH, and harmful reagents (e.g., fluorine gas)
that are not suitable for most biomolecules. Therefore, an
trans-cyclooctene19−21 have been applied to the 18F-labeling of
biomolecules.
indirect 18F-labeling strategy is generally applied where the 18F
We have previously reported a versatile bioorthogonal
is first introduced into a small organic molecule (so-called 18F-
prosthetic group) followed by subsequent coupling to a specific
conjugation using 2-cyanobenzothiazole (CBT) that can rapidly
react with a cysteine moiety.22,23 The observed second-order
rate constant for this reaction was determined to be 9.19 M−1
functional group (i.e., −NH2, −CO2H, or −SH) in the
biomolecules. Well-established 18F prosthetic groups include
the following: (1) 18F-labeled benzaldehyde for labeling
aminooxy groups via formation of an oxime bond;6,7 (2) 18F-
labeled activated ester and maleimide for labeling amino and
Received: May 23, 2012
Revised: July 23, 2012
Published: July 31, 2012
© 2012 American Chemical Society
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a
Scheme 1. Synthesis of Tosylated CBT 3 and a Condensation Reaction between 18F−4 and Free Cysteine
a
Reagents and conditions: (a) pyridine−HCl, 200 °C, 2 h, 75%; (b) ethylene glycol di-tosylate, K2CO3, DMF, room temperature, 8 h, 53%; (c) 2-
fluoroethyl 4-methylbenzenesulfonate, K2CO3, DMF, room temperature, overnight, 70%; (d) K[18F]F, 18-Crown-6, K2CO3, MeCN, 90 °C, 10 min,
20% (decay-corrected to end of bombardment, isolated yield); (e) L-cysteine, TCEP·HCl, NaHCO3, MeOH/H2O, room temperature, 1 min, 95%.
s−1. This condensation reaction enables rapid and site-specific
fluorescent labeling of target proteins in vitro and at the surface
of live cells without the need of catalysts under ambient
conditions. Its rapid reaction rate along with biocompatibility
makes this CBT−cysteine condensation reaction attractive for
radiolabeling of biomolecules such as peptides and proteins for
PET imaging applications. Herein, we describe a facile and
efficient 18F-labeling method using 18F-fluorinated-2-cyanoben-
zothiazole (18F-CBT) as a novel prosthetic group and its
application to radiolabeling of a dimeric cRGD peptide for in
vivo cancer targeted PET imaging. We further demonstrated the
site-specific 18F-labeling of a cysteine-bearing protein using 18F-
CBT, and evaluated its biodistribution in a living mouse with
PET imaging.
sample was taken from the crude mixture and the reaction was
quenched with 10% AcOH aqueous solution. After 20 min, the
conversion yield was 92% determined by analytical HPLC. The
reaction was quenched by adding 10% AcOH aqueous solution
and then the crude product was purified by a semipreparative
HPLC to give [18F]CBTRGD2 9 with 80% RCY (decay-
corrected to the end of synthesis). [Phenomenex Gemini
column: 10 × 250 mm, 5 μ, 5 mL/min, and eluent gradient: 0−
50 min 10−50% (0.1% TFA containing MeCN in 0.1% TFA
containing H2O), Rt = 34.4 min.] The specific radioactivity was
1.3 0.15 Ci/μmol.
Radiolabeling of Cys−RLuc8 (11). 18F−4 (10.7 mCi, 7.5
μL) in DMSO solution was added to a solution of Cys−RLuc
11 (5 nmol) in PBS buffer (150 μL, pH = 7.5 with 2 mM
TECP), and stirred at 37 °C for 30 min. After the reaction, the
crude mixture was diluted with PBS buffer until total volume
was up to 1 mL. The crude mixture was directly loaded onto a
NAP-10 column, which was pre-conditioned with elution buffer
(PBS, pH = 7.4). The crude solution (1 mL) was allowed to
enter into the column completely, and then, 1.5 mL of elution
buffer (PBS, pH = 7.4) was added into the column to collect
EXPERIMENTAL SECTION
■
18F-Labeling of Tosylated CBT (3). [18F]-Fluoride (1000
mCi) was prepared by proton bombardment of 2.5 mL [18O]
enriched water target via the 18O(p,n)18F nuclear reaction. The
[18F]-fluoride was then trapped onto a Sep−Pak QMA
cartridge. 18-Crown-6/K2CO3 solution (1 mL, 15:1 MeCN/
H2O, 16.9 mg of 18-Crown-6, 4.4 mg of K2CO3) was used to
elute the [18F]-fluoride from QMA cartridge into a dried glass
reactor. The resulting solution was azeotropically dried with
sequential MeCN evaporations at 90 °C. A solution of
compound 3 (2 mg in 1 mL of anhydrous MeCN) was
added to the reactor and heated at 90 °C for 10 min. After
cooling to 30 °C, 0.05 M HCl (2.5 mL) was added to quench
the reaction mixture and prevented basic hydrolysis of the
product 18F−4. The crude mixture was then purified with a
semipreparative HPLC (Phenomenex Gemini column: 10 ×
250 mm, 5 μ, 3 mL/min, and eluent gradient: 0−3 min 40%
(0.1% TFA containing MeCN in 0.1% TFA containing H2O);
3−35 min 40−100% (0.1% TFA containing MeCN in 0.1%
TFA containing H2O), Rt = 21.0 min. The collected 18F−4 was
diluted with H2O (20 mL) and passed through a C18 cartridge.
The trapped 18F−4 was eluted out with Et2O (2.5 mL). The
Et2O was removed by helium stream and used for next reaction.
The isolated radiochemical yield of 18F−4 was ca. 20% (140−
150 mCi, decay-corrected to end of bombardment).
the product [18F]CBT−RLuc 12 (12
0.7%, n = 3, decay-
corrected to end of synthesis, isolated yield). Overall reaction
and purification steps were completed within 40 min. The
specific radioactivity was 262 mCi/μmol.
microPET Imaging of U87MG Tumor Xenografts in
Mice. Image analyses were carried out using a microPET R4
rodent model scanner (Siemens Medical Solutions). By tail-
vein injection, each mouse was administered approximately 100
μCi of [18F]CBTRGD2 under isoflurane anesthesia. At 0.5, 1,
and 2 h post-injection (n = 3 group), 5 min static PET scans
were acquired. For the blocking experiments, the tumor-bearing
mice were co-injected with 20 mg/kg mouse body weight of
cRGD2 7 and [18F]CBTRGD2. PET images were acquired at
0.5, 1, and 2 h post-injection (n = 3 each group). For each scan,
regions of interest (ROIs) were drawn over the tumor, liver,
and kidney by using vendor software (ASI Pro 5.2.4.0; Simens
Medical Solutions) on decay-corrected whole-body coronal
images. The maximum radiochemistry concentration (accumu-
lation) within a tumor or an organ was obtained from mean
pixel values within the multiple ROI volume, which were
converted to percent injected dose per gram (%ID/g).
Radiosynthesis of [18F]CBTRGD2 (9). cRGD dimer 8 (1.2
mg) was dissolved in DMF (200 μL) containing 2 equiv of
TCEP·HCl and 15 equiv of DIPEA. The resulting solution was
added to 18F−4 (40 mCi) in DMF (200 μL) at room
temperature. At different time points (1, 5, 10, and 20 min), the
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however, an 18F−byproduct was mainly observed on radio−
HPLC chromatograph instead of the desired 18F−4 (Figure
1b), which might be due to the hydrolysis of the cyano group
on CBT under this condition. Indeed, stability tests with
tosylated CBT 3 and fluorinated CBT (19F−CBT) showed that
the CBT compounds were hydrolyzed under K222/K2CO3
condition within 1 min and also unstable in the presence of
Cs2CO3 or tetrabutylammonium bicarbonate (SI Figure S1).
K222 contains nucleophilic nitrogen atoms that could attack the
cyano group of 3 and cause facile hydrolysis under this
common radiofluorination condition. To discover alternate 18F-
labeling conditions, we tested another PTC, and to our delight,
the CBT precursor was stable for 10 min in the presence of 18-
Crown-6/K+ complex at 90 °C. Tosylated CBT 3 (2 mg) in
MeCN was then added to the anhydrous 18-Crown-6/K+/
[18F]F− complex, and the labeling reaction was carried out at 90
°C for 10 min. Radio−HPLC chromatograph indicated
byproduct 18F−5 was suppressed under these conditions
(Figure 1c). Automated syntheses have been accomplished
with up to 1000 mCi of radioactivity, and the expected 18F−4
was obtained at a 20% RCY (decay-corrected to end of
bombardment, isolated yield). Analytical HPLC revealed that
the radiochemical/chemical purity of 18F−4 was more than
99%.
RESULTS AND DISCUSSION
■
Synthesis of CBT Prosthetic Group and Its 18F-
Labeling Procedure. To carry out 18F-labeling of the CBT
structure, tosylated CBT 3 was prepared from commercially
available 6-methoxy-CBT 1 (Scheme 1). The methyl group in
compound 1 was removed using pyridine hydrochloride at 200
°C. The resulting hydroxyl−CBT derivative was converted to
tosylate 3 by reacting with excess amount of ethylene glycol
ditosylate. A 19F-analogue, 19F−4, was synthesized using 2-
fluoroethyl 4-methylbenzenesulfonate as a reference for HPLC
characterization of 18F−4.
18F-labeling of tosylate 3 was first performed with traditional
phase transfer catalyst (PTC) Kryptofix 222 (K222) (Figure 1);
Condensation Reaction between 18F−4 and Free L-
Cysteine. The efficiency of condensation reaction between the
prepared 18F−4 and free cysteine was subsequently investigated
(Scheme 1). The reaction was carried out in MeOH/H2O (1:1,
1 mL) at room temperature. TCEP·HCl (tris(2−carboxyethyl)-
Figure 1. (a) 18F-Fluorination reaction of tosylated CBT 3. (b)
Radiochromatography of the crude product using K222. (c) Radio-
chromatography of the crude product using 18-crown-6.
a
Scheme 2. 18F-Labeling of cRGD2 Peptide Analogue Using 18F−4
a
Reagents and conditions: (a) N-Boc-Cys(Trt) succinimidyl ester; (b) TFA, triisopropylsilane, DCM, 79% over 2 steps; (c) 18F−4, DIPEA,
TCEP·HCl, room temperature, 20 min, 80% (decay-corrected to end of synthesis, isolated yield).
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phosphine hydrochloride, 2 equiv) was added to the reaction
mixture for preventing undesired oxidation of the thiol group
and pH was adjusted to 7.0−7.5 using aqueous NaHCO3. The
condensation reaction was quenched by adding 0.05 M aqueous
HCl, and the conversion yield in the reaction was estimated by
analytical HPLC. As shown in SI Figure S2, 18F−4 was
converted to 18F−6 in more than 95% yield within 1 min.
Given that 18F−4 was fully converted to 18F−6 in a few minutes
at room temperature, the condensation reaction should be
appropriate for 18F-labeling of biomolecules.
nude mice by static small animal imaging PET scans (Figure 2).
PET imaging results showed that the tumor site was clearly
18F-Labeling of a Dimeric cRGD Peptide. We selected a
dimeric cRGD peptide for our labeling because cRGD peptides
have been shown to target αvβ3 integrin expression in
tumors.24,25 A number of 18F prosthetic groups have been
employed for labeling cRGD, some of which are currently in
clinical trials such as [18F]Galacto−RGD26 and [18F]-
FPPRGD2.27,28 However, most of these 18F labeling chemistries
require multistep syntheses that resulted in low radiochemical
yield and could be challenging to routinely produce for clinic
use.26−30 Since 18F-CBT could be easily obtained by direct 18F/
tosylate substitution and the condensation reaction between
18F-CBT and cRGD proceeds readily at room temperature, this
novel 18F-prosthetic group could provide more benefits over
those previously reported. In detail, N-terminal cysteine was
introduced to cRGD dimer 7 using N-Boc-Cys(Trt)
succinimidyl ester in 2 steps (Scheme 2). cRGD derivative 8
was then reacted with 18F−4 in anhydrous DMF at room
temperature. The conversion yield reached 92% after
incubating 20 min with the 18F-labeling agent at room
temperature as determined by analytical radio-HPLC (SI
Figure S3). After the labeling reaction was finished, the crude
product was purified by semipreparative HPLC. The observed
RCY of the 18F-labeled product 9 was 80% (decay-corrected to
end of synthesis), and the radiochemical purity was more than
99%. The final product [18F]CBTRGD2 9 was obtained in an
isolated overall yield of 7.5 2.7% (decay-uncorrected) with a
total synthesis time of 100 min (Table 1). Specific radioactivity
Figure 2. Representative small-animal PET images of U87MG tumor-
bearing (right shoulder) mice. (a) Whole body coronal images at 30,
60, and 120 min after i.v. injection of [18F]CBTRGD2. (b) Whole
body coronal images at 30, 60, and 120 min after co-injection of
[18F]CBTRGD2 with cRGD2 peptide (blocking). (T: U87MG tumor,
K: kidney).
visualized with good tumor-to-background contrast within 30
min and the clearance of tracer uptake was observed at 120 min
(Figure 2a). PET quantification results demonstrated that
tumor-to-background (or muscle) ratios were 4.5 0.83 (30
min), 5.4
0.72 (60 min), and 6.3
1.1 (120 min). The
tumor uptake of [18F]CBTRGD2 was 5.62
1.15%, 4.25
0.82%, and 3.35 0.71% at 30, 60, and 120 min, respectively
(Table 2). A blocking experiment where the tracer was
Table 2. Uptake of [18F]CBTRGD2 in U87MG Tumor,
Kidney, and Liver Derived from PET Quantification (%ID/
g, n = 3)
organ
tumor
time
[18F]CBTRGD2
blocked with cRGD2
30 min
60 min
120 min
30 min
60 min
120 min
30 min
60 min
120 min
5.62 1.15
4.25 0.82
3.35 0.71
5.46 1.10
3.99 1.08
2.26 0.67
3.64 0.76
2.84 0.76
2.15 0.53
1.62 0.10
1.26 0.13
0.49 0.02
2.76 0.85
1.26 0.23
0.81 0.04
1.34 0.06
0.70 0.08
0.32 0.01
Table 1. Comparison of Radiochemical Procedure
[18F]FPPRGD2
[18F]CBTRGD2
kidney
liver
Radiosynthetic steps
Total synthesis time
4
2
180 min
100 min
a
RCY (n = 3)
3.5 1.8%
7.5 2.7%
Specific radioactivity
0.97 0.06 Ci /μmol
1.3 0.15 Ci/μmol
a
Decay-uncorrected, isolated yield.
of the final product was 1.3 0.15 Ci/μmol. It is important to
note that, during the HPLC purification of crude [18F]-
CBTRGD2, all the impurity, reagent, and starting material have
distinct retention time from the product (>10 min); and no
other peaks appeared around the product peak on the HPLC
chromatograph at UV 254 nm (SI Figure S4). In comparison to
[18F]FPPRGD2 as previously reported in clinical studies, two
impurities were identified to have retention times close to that
of the final product (<1 min) and are difficult to remove.27
Therefore, CBT-based 18F labeling chemistry could offer the
following advantages: (1) higher RCY; (2) fewer synthetic
steps and shorter synthesis time; and (3) easier HPLC
purification.
coinjected with cRGD dimer (20 mg/kg) showed significantly
reduced tumor uptake of the tracer (1.62 0.10%, 1.26
0.23%, and 0.81 0.04% at 30, 60, and 120 min, respectively),
suggesting that [18F]CBTRGD2 specifically bound integrin αvβ3
receptor (Figure 2b). We also carried out a comparison
experiment with tracer 9 and [18F]FPPRGD2 in the same mice.
PET imaging results showed that the specific tumor uptake of
[18F]CBTRGD2 was 50% higher than that of [18F]FPPRGD2 at
30 min (SI Figure S5 and Table S1). [18F]CBTRGD2 also
showed higher uptakes in organs such as liver and kidney than
[18F]FPPRGD2, likely due to the increased lipophilicity from
the aromatic structure in 18F-CBT.
Recently, the [4 + 2] inverse electron demand Diels−Alder
reaction was applied to 18F-labeling of a cRGD peptide.31 The
rate of this conjugation method was faster than the other
reactions described, but this ligation unfortunately created a
In Vivo Evaluation of [18F]CBTRGD2 with U87MG
Tumor Xenograft Model. Tumor targeting efficiency of
[18F]CBTRGD2 9 was evaluated in U87MG tumor bearing
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a
Scheme 3. 18F−Labeling of Cys−RLuc8 11 Using 18F−4
a
Reagents and conditions: (a) TEV protease (100 μg of protein/10 units of enzyme), 30 °C, 6 h; (b) 18F−4, 37 °C, 30 min, 12 0.7% (decay-
corrected to end of synthesis, isolated yield).
mixture of isomers. Moreover, the ligation product from the
reaction between tetrazine and trans-cyclooctene was large and
hydrophobic, and thus, large amounts of radioactivity could be
detected in normal organs. Considering that hydrophobic
derivatization of isotope labeled tracers often spoils their
desired in vivo pharmacokinetics properties, a relatively small
hydrophobic part used in the CBT−cysteine reaction may have
a less adverse effect on the biodistribution of the final tracer.
Site-Specific 18F-Labeling of N-Terminal Cysteine
Bearing RLuc8. We next investigated site-specific 18F-labeling
of a protein using 18F−4. Several 18F−prosthetic groups
targeting lysine8,9,32−34 and cysteine35−37 residues have been
applied to protein labeling by means of amidation, conjugate
addition, and so on, but these methods normally result in a
mixture of randomly labeled proteins. Moreover, non-site-
specific labeling of proteins often resulted in decreased
biological activity. For example, 18F-labeled anti-carcinoem-
bryonic agent diabody using N-succinimidyl-4-[18F]-
fluorobenzote ([18F]SFB) showed lower immunoreactivity
compared with nonlabeled protein.33 Therefore, site-specific
labeling of an amino acid residue away from the active site of
the protein is highly desirable. Additionally, an efficient reaction
under mild conditions is preferred for 18F-labeling of protein.
For this study, the bioluminescent protein Renilla luciferase
(RLuc8) was used as a model protein. The peptide substrate of
tobacco etch virus (TEV) protease was fused at the N-terminus
region of RLuc8 10 to generate N-terminal cysteine (Scheme
3). TEV protease was added to the purified fusion protein to
cleave the peptide substrate. After the reaction, it was purified
with Ni−NTA agarose by using 6×His tag in front of the TEV
protease sequence to provide the N-terminal cysteine RLuc
(Cys−RLuc) 11. During this procedure, uncleaved RLuc8 10
and TEV protease with 6×His tag could be separated from the
product. Cys−RLuc 11 was used in the next step without
furthur purification. In the 18F-labeling reaction, 18F−4 was
added to Cys−RLuc 11 (5 nmol) in phosphate buffered saline
(PBS, pH = 7.5 with 2 mM TECP), and then the labeling
reaction proceeded at 37 °C for 30 min. The purification was
accomplished by size exclusion chromatography (NAP−10
column) eluted with PBS (pH = 7.4) to afford [18F]CBT−
RLuc 12 with 262 mCi/μmol of specific activity. The observed
RCY was 12 0.7% (n = 3, decay-corrected to end of synthesis,
isolated yield) and radiochemical purity was more than 99% as
determined by radio−TLC analysis (SI Figure S6). In
comparison, the same labeling reaction with RLuc8 10, which
did not contain the required N-terminal cysteine group for
coupling, provided no 18F-labeled product. Therefore, we can
conclude that 18F-labeling of 11 with 18F−4 is site-specific.
After the labeling reaction, the bioluminescent property of
RLuc8 remained unchanged. The observed bioluminescent
intensities of Cys−RLuc 11 and the purified product from the
18F-labeling reaction were nearly the same (SI Figure S7).
To evaluate our new imaging probe in a living subject, we
then carried out a PET imaging experiment by injecting
[18F]CBT−RLuc 12 (ca. 100 μCi) in non-tumor-bearing mice.
PET images of different time points (20, 60, and 100 min)
showed that primary radioactivity uptake was observed in the
renal system within 20 min of injection and then subsequently
cleared into bladder by 100 min (Figure 3). Based on PET
Figure 3. Whole body coronal images at 20, 60, and 100 min after i.v.
injection of [18F]CBT−RLuc8 (K: kidney).
quantification of the collected images, nonspecific liver
accumulation was not observed (Table 3). These results were
correlated well with a previous biodistribution study using 124I−
labeled RLuc8.38
Among various 18F-precursors, 18F-fluorobenzaldehyde
([18F]FBA)39,40 targeting an aminooxy group and 18F-
maleimide containing prosthetic groups41,42 reacting with a
thiol have been developed as site-specific labeling agents for
proteins. They normally provided good 18F-labeling results in
terms of RCY and purity for each target protein. However, the
labeling reaction using [18F]FBA has to be done under aqueous
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was used for site-specific 18F-labeling of RLuc8, the 18F-labeled
protein showed little alteration in its biodistribution as revealed
by PET imaging in healthy mice. We anticipate that this
method could be generally applied to afford efficient and site-
specific 18F-labeling of peptides, proteins, or antibodies that
contain an N-terminal cysteine.
Table 3. Uptake of [18F]CBT−RLuc8 in Kidney and Liver
Derived from PET Quantification (%ID/g, n = 3)
organ
time
[18F]CBT−RLuc8
kidney
20 min
60 min
100 min
20 min
60 min
100 min
20 min
60 min
100 min
17.3 1.28
7.94 1.07
2.98 0.32
7.18 0.94
3.15 0.30
1.61 0.19
1.24 0.03
1.02 0.02
0.50 0.04
liver
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis and characterization of compounds, stability test of 2-
cyanobenzothiazole (CBT) structures, radiochemical proce-
dures for peptide and protein labeling and in vivo microPET
imaging. This material is available free of charge via the Internet
at http://pubs.acs.org.
muscle
acidic condition (pH < 4.5) that would potentially abolish the
bioactivity of pH-sensitive enzymes or proteins. In the case of
maleimide-containing prosthetic groups, their syntheses always
require an additional coupling step because the maleimide
structure is quite labile under the required basic 18F-labeling
conditions that often results in lengthy synthesis times and
decreased RCYs. Moreover, these site-specific methods
involved relatively complicated chemical ligation reactions or
protein engineering procedures along with a couple of
purification steps to generate a single reactive functional
group on the protein. Compared with these methods, the N-
terminal cysteine residue can be easily produced by a simple
enzymatic cleavage with TEV protease and additional
modification and purification steps are unnecessary for the
18F-labeling reaction. Using this procedure, an N-terminal
cysteine group can be readily prepared in a fusion protein.
Therefore, our method has just three steps in total for protein
labeling, (1) protein modification with TEV protease, (2)
radiosynthesis of 18F−4, and (3) a condensation reaction
between 18F−4 and N-terminal cysteine protein under neutral
condition; and it provides a useful and general protocol for
efficient 18F-labeling of various biomolecules.
AUTHOR INFORMATION
Corresponding Author
*E-mail addresses: jrao@stanford.edu, chinf@stanford.edu.
■
Author Contributions
‡These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part by an IDEA award from
Department of Defense Breast Cancer Research Program
(W81XWH−09−1−0057) and the NCI ICMIC@Stanford
(1P50CA114747−06).
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CONCLUSIONS
■
This study demonstrated a highly efficient 18F-labeling strategy
of biomolecules such as peptide ligands and proteins based on a
rapid condensation reaction of 18F−4. The tosylated CBT
derivative used for preparing 18F−4 can be easily synthesized
from a commercially available precursor in two simple steps.
The fast and specific reactivity of 18F−4 toward a free cysteine
residue provides excellent RCYs under ambient temperature.
The fast and specific reactivity of 18F−4 toward a free cysteine
residue provides excellent RCYs under ambient temperature.
Depending on the cysteine-containing molecules, the labeling
can proceed efficiently in a number of different solvent systems,
as demonstrated in DMF, MeOH/water, and PBS buffer
solutions. An N-terminal cysteine could be introduced into the
target molecules by chemical synthesis or enzymatic processing
of specific peptide sequence fused to the target protein. Using
this convenient labeling method, [18F]CBTRGD2 has been
prepared in a much shorter synthesis time with a higher RCY
compared to the PET tracer currently under clinical trial,
[18F]FPPRGD2, and has shown better tumor targeting
efficiency in mice than [18F]FPPRGD2 prepared by a different
prosthetic group, 4-nitrophenyl 2-18F-fluoropropionate (18F−
NFP). The increased accumulation of [18F]CBTRGD2 in livers
may be due to the lipophilic structure of the CBT group, which
should be ameliorated with modifications of the CBT structure
that increase its hydrophilicity. On the other hand, when 18F−4
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