Novel chemoselective 18F-radiolabeling of thiol-containing biomolecules under mild aqueous conditions

Article abstract of DOI:10.1039/c6cc01982j

We report a novel prosthetic group based on a heterocyclic methylsulfone derivative for the rapid, stable, and chemoselective ¹⁸F-labeling of thiol-containing (bio)molecules under mild aqueous reaction conditions. Compared to established maleimide approaches, the new methodology displays some clear advantages for imaging probe development.

Full text of DOI:10.1039/c6cc01982j

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Chem. Commun., 2016, DOI: 10.1039/C6CC01982J.
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DOI: 10.1039/C6CC01982J
Chemical Communications
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Novel Chemoselective 18F‐Radiolabeling of Thiol‐Containing
Biomolecules under Mild Aqueous Conditions
Aristeidis Chiotellis,a Filippo Sladojevich,a Linjing Mu,a Adrienne Müller‐Herde,a Ibai E. Valverde,b
Received 00th January 20xx,
Accepted 00th January 20xx
Vladimir Tolmachev,c Roger Schibli,a Simon M. Ametamey,a Thomas L. Mindt a,b,d,
*
DOI: 10.1039/x0xx00000x
www.rsc.org/
We report a novel prosthetic group based on a heterocyclic abundant in proteins, which permits conjugation chemistry in a
methylsulfone derivative for the rapid, stable, and chemoselective more controlled fashion. In addition, proteins and peptides can
18F‐labeling of thiol‐containing (bio)molecules under mild aqueous be engineered to contain a free thiol group for site‐specific
reaction conditions. Compared to established maleimide labeling. As a consequence, a number of cysteine‐selective
approaches, the new methodology displays some clear advantages conjugation methods have been reported[3] of which the
for imaging probe development.
prosthetic group approach based on maleimide chemistry
remains the most widely used strategy for the 18F‐labeling of
thiol‐containing (bio)molecules. This is due to fast reaction
kinetics, high selectivity, and the possibility of employing
aqueous reaction conditions at room temperature and neutral
pH. However, the Michael‐type addition of thiols to maleimides
without chiral auxiliaries results inevitably in the formation of
racemic product mixtures (Scheme 1), which can impede with
obtaining approval from regulatory authorities for clinical
applications of the radioconjugate, in particular those derived
from small molecules. In addition, the suitability of the thiol‐
maleimide condensation adduct in bioconjugates has recently
been questioned because of its potential instability under
physiologically relevant (e.g., reducing) conditions.[4] Clearly,
there is a need for new bioconjugation approaches which
provide an alternative to maleimide chemistry for the rapid,
stable, as well as chemo‐ and/or site‐selective (radio)labeling of
cysteine residues.[3, 5]
The development of targeted imaging agents involves the
incorporation of a reporter probe into a molecule of interest in
a chemo‐ and/or site‐specific manner so that the biological
properties of the vector (e.g., target specificity and affinity) are
not compromised. In the case of radionuclides, fast reaction
kinetics are required at the very low concentration levels of
radioactive substrates due to radioactive decay. In addition,
mild aqueous reaction conditions become necessary when
sensitive biologics (e.g., proteins) are employed. Such stringent
requirements can make the development of radiotracers a
challenging endeavour.
Due to its favourable physical properties, fluorine‐18 (18F; +
0.635 MeV, 97% abundance, t1/2 = 109.7 min) is the most
frequently used radionuclide in nuclear medicine for imaging by
positron emission tomography (PET).[1] The incorporation of a
18F label into peptides and proteins is most commonly achieved
by modifications of lysine (Lys) or cysteine (Cys) residues using
a prosthetic group approach.[2] Prosthetic groups are small,
bifunctional molecules, which can be readily radiolabeled with
18F and subsequently conjugated selectively to the molecule of
interest. In comparison to Lys, Cys residues are found less
a. Center for Radiopharmaceutical Sciences ETH‐PSI‐USZ, Institute of
Pharmaceutical Sciences ETH, Vladimir‐Prelog Weg 4, CH‐8093 Zurich,
Switzerland; e‐mail: t.mindt@gmx.ch
b. Division of Radiopharmaceutical Chemistry, University of Basel Hospital,
Petersgraben 4, CH‐4031 Basel, Switzerland
Scheme 1. 18F‐Radiolabeling of (bio)molecules (R) via different thiol reactive prosthetic
groups (A) maleimide derivatives and (B) phenyloxadiazole methylsulfones; squiggled
lines represent different linkers or chemical entities for incorporation of the 18F label.
c. Department of Immunology, Genetics and Pathology, Medical Radiation Science
Program,, Rudbeck Laboratory, Uppsala University, SE‐75181 Uppsala, Sweden
d. Ludwig Boltzmann Institute for Applied Diagnostics, General Hospital of Vienna,
Währinger Gürtel 18‐20, A‐1090 Vienna, Austria
Electronic Supplementary Information (ESI) available: experimental details,
characterisation of intermediates, and final compounds by NMR, MS, and HPLC. See
DOI: 10.1039/x0xx00000x
In 2013, the group of late Barbas III reported a Julia‐Kocieński‐
like reagent for the chemoselective labeling of thiol containing
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proteins with a fluorescent probe by nucleophilic aromatic reported for 18F‐labeled maleimide derivatives.[7] [18F]FPOS 4a
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substitution.[6] They showed that
a
phenyloxadiazole was obtained in sufficient radiochemicalDpOuIr:i1t0y.1s0o39t/hCa6tCiCt0c1o9u82ldJ
methylsulfone reacts rapidly and selectively in aqueous media be used for subsequent reactions with thiol substrates with or
at neutral pH and room temperature in the presence of other without purification by HPLC. Non‐radioactive 19F‐analogue 4b
potential nucleophiles with the sulfhydryl group of cysteine for the syntheses of cold reference compounds 5b
residues of various model compounds as well as proteins. The Supporting Information) was prepared by the reaction of
conjugates, obtained as single isomers, were more stable under alcohol with DAST.
‐8b (see
2
physiological conditions in comparison to the corresponding With [18F]FPOS 4a in hand, we investigated its reactivity with
fluorescent analogues obtained by maleimide chemistry. With model compounds n‐dodecylthiol and the amino acid
the goal to develop this new chemistry in the context of AcCysOMe in aqueous medium (PBS, pH 7.4) which contained
radiotracer synthesis, we set out to investigate the utility of some organic solvents for solubilizing the substrates and
heterocyclic methylsulfone derivatives (Scheme 1) for the 18F‐ reagents (Table 1, entries 1‐2). Complete conversion of crude
radiolabeling of thiol‐containing (bio)molecules.
The phenyloxadiazole methylsulfone scaffold offers different Thioether adducts 5a and 6a were obtained as single products
synthetically accessible positions for the incorporation of an 18
and isolated by HPLC purification (0.6‐0.8 GBq; not optimized).
[18F]FPOS 4a was observed (
‐HPLC) after 15 min at 37 °C.
F
label. For this work, we developed a prosthetic group bearing Their identity as well as those of the other radiolabeled
an appropriately functionalized alkoxy side chain attached to products listed in Table 1 was confirmed by HPLC comparison
the aromatic moiety, which enables fluorination by aliphatic with the corresponding non‐radioactive 19F‐reference
nucleophilic substitution (Scheme 2). Thus, fluorination compounds (Supporting Information).
precursor, nosylate
hydroxybenzoate
approx. 20% (Supporting Information).[6] Compound
as a white solid, was found stable upon storage at ‐25 °C for a example of a protein. Both biomolecules are currently under
3
, was prepared from commercial methyl 4‐ We next turned our attention to Cys‐containing, tumor‐
in a total of 6 steps and good overall yield of targeting molecules. For proof of concept, we chose a derivative
, isolated of the peptide bombesin (BBN) and the affibody ZHER2:2395 as an
1
3
time period of >6 months.
investigations in radiolabeled form as diagnostic probes and/or
therapeutics in nuclear oncology.[8] Specifically, we used a
stabilized analogue of the minimum binding sequence of BBS,
[Nle14]BBN(7–14), a peptide with high affinity and specificity
towards the gastrin‐releasing peptide receptor, which is over‐
expressed by, e.g., prostate, breast, and small‐cell lung
cancer.[9] The peptide was elongated N‐terminally with a (
spacer[10] and N
‐acetylated Cys to provide test compound
AcCys(
Ala)3[Nle14]BBN(7‐14)NH2 (Table 1, entries 3‐5). The
Ala)3


affibody ZHER2:2395 is a non‐immunoglobulin‐based, small
targeting protein (approx. 7 kDa) with high specificity and sub‐
nanomolar affinity towards the human epidermal growth factor
receptor‐2 (HER2), a phosphoglycoprotein expressed in high
incidence by breast, lung, and ovarian cancer as well as B‐cell
acute lymphoblastic leukemia. For the study described herein,
an analogue of the protein bearing a Cys residue at the C‐
terminus, ZHER2:2395‐Cys (Table 1, entries 6‐7), was used.[11]
Scheme 2. Synthesis of fluorinated phenyloxadiazole methylsulfones and their
conjugation to thiol containing model‐ and biomolecules. a) Ns‐Cl, DMAP, CH2Cl2, rt, 60%;
b) for 18F: nosylate 3, [18F]KF‐K2.2.2, CH3CN/tBuOH (1:1), 85 °C, 10 min; c) for 19F: alcohol
2, DAST, CH2Cl2, rt, 40%; d) for 18F: [18F]FPOS 4a, 0.1‐20 mM thiol substrates in mixtures
of PBS (0.2 M, pH 7,4) and different organic solvents (10‐65% v/v), 37‐55 °C, 15 min; e)
for 19F: [19F]FPOS 4b, thiol substrates in mixtures of PBS (0.2 M, pH 7,4) and THF (50%
v/v) or THF/DMSO (each 10% v/v), 2 h, rt, 65‐93%.
Fluorine‐18 labeling of AcCys(
HER2:2395‐Cys was carried out with purified [18F]FPOS 4a to avoid
competing reaction of excess nosylate with the thiol

Ala)3[Nle14]BBN(7‐14)NH2 and
Z
3
substrates. In addition, the reactions were performed at a
concentration range typically reported for the radiolabeling of
these molecules (Table 1). 18F‐labeling of BBN derivative with
[18F]FPOS 4a proceeded smoothly under the reaction conditions
described above for the model compounds yielding, after HPLC
purification, product 7a with a specific activity (As) of 53
GBq/μmol and a d.c. RCY of 33% (Table 1, entry 3). Lowering the
amount of the peptide substrate by a factor of four was possible
but required slightly elevated reaction temperature (50 °C) for
Radiofluorination of nosylate
3 (6 mol, 10 mM) was
accomplished with [18F]KF‐K2.2.2 in a mixture of CH3CN and
tBuOH (1:1) at 85 °C for 10 min. After HPLC purification, 18F‐
labeled prosthetic group 4a (termed [18F]FPOS; fluoro
phenyloxadiazole methylsulfone) was obtained with an activity
of 6‐16 GBq in a radiochemical yield (RCY) of 27±6% (n=7) decay
corrected (d.c.) from end of bombardment (EOB). Unlike
reported maleimide‐based prosthetic groups that require
multistep synthesis due to their sensitivity to the basic reaction
complete conversion of [18F]FPOS
4 within 15 min (Table 1,
entries 4‐5). Reaction of ZHER2:2395‐Cys with [18F]FPOS 4a in PBS
at 37 °C for 15 min resulted only in a very low conversion of the
prosthetic group 4a (approx. 5%) which could be improved at
50 °C to 40% as estimated by radio‐TLC (Table 1, entries 6‐7).
conditions applied for labeling reactions with [18F]KF‐K2.2.2
[18F]FPOS 4a was obtained ready for conjugation chemistry in a
single step and with a RCY comparable or improved to those
,
2 | J. Name., 2012, 00, 1‐3
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Table 1. Investigated thiol-containing substrates and conditions of their reaction with [18F]FPOS 4a.
Entry
Thiol substrate / Product
n-Dodecylthiol / 5a
Amount / Concentration [a]
17 mol/12 mM
Temperature / Time
37 °C/15 min
37 °C/15 min
37 °C/15 min
37 °C/15 min
50 °C/15 min
37 °C/15 min
50 °C/15 min
Conversion of [18F]FPOS 4a
quant.[b], [c]
1
2
3
4
5
6
7
AcCysOMe / 6a
28 mol/20 mM
quant.[b], [c]
AcCys(Ala)3[Nle14]BBN(7-14)NH2 / 7a
AcCys(Ala)3[Nle14]BBN(7-14)NH2 / 7a
AcCys(Ala)3[Nle14]BBN(7-14)NH2 / 7a
ZHER2:2395-Cys / 8a
1.6 mol/3.5 mM
0.4 mol/0.9 mM
0.4 mol/0.9 mM
0.14 mol/0.14 mM
0.14 mol/0.14 mM
quant.[b], [d]
ca. 33% [b], [d]
quant.[b], [d]
<5%[d], [e]
ZHER2:2395-Cys / 8a
ca. 40%[d], [e]
[a] solvent systems (% v/v); entries 1‐2: 35% PBS (0.2 M, pH 7.4), 65% CH3CN/tBuOH (1:1); entries 3‐5: 45% PBS (0.2 M, pH 7.4), 45% CH3CN, 10% DMSO; entries 6‐7: 90% PBS 0.2
M/Na ascorbate 0.02 M (pH 7.4), 10% EtOH; [b] determined by ‐HPLC; [c] reaction with crude [18F]FPOS 4a; [d]reaction with HPLC purified [18F]FPOS 4a; [e] estimated by radio‐
TLC.
Neither longer reaction time nor higher temperature led to a characteristics as a PET imaging agent for the diagnosis of HER2‐
significant improvement of the conversion rate of [18F]FPOS 4a
.
positive tumors which warrants further investigations.
Based on literature data, elevated temperature appears to be a
prerequisite for the modification of the Cys‐modified affibody
presumably by making the C‐terminus more accessible as the
[13]
result of reversible partial unfolding of the protein.[12]
In
contrast to the studied affibody, it is likely that increased
temperature are not necessary for the modification of other
proteins with more exposed Cys residues.[6] Despite the relative
low conversion of [18F]FPOS 4a, [18F]ZHER2:2395‐Cys 8a was
isolated after purification using a size exclusion cartridge within
a total synthesis time 160 min and up to 17 GBq/µmol (n=3),
which is among the highest As reported for 18F‐labeled formats
of the affibody. [7a, 12‐14]
The tumor targeting properties of the radiolabeled affibody
[18F]ZHER2:2395‐Cys 8a was studied in vivo by PET/CT imaging of
CD1 nude mice bearing HER2‐positive SCOV3 xenografts (see
Supporting Information for details). Nude mice with HER2‐
negative RAMOS xenografts were used as a control. Images
were acquired 2 h and 4 h post injection of the radiotracer (7‐
20 MBq/15 µg per mouse; n=2 for each mouse model). Already
2 h post injection, the 18F‐labeled affibody has cleared from the
blood pool and showed a markedly high accumulation in HER2‐
positive xenografts resulting in a favorably high tumor‐to‐
background ratio (Figure 1). Negligible uptake of radioactivity
was observed in the RAMOS‐cell derived tumors of the control
group hence proving the specificity of the radiolabeled protein
towards HER2 receptors. No accumulation of radioactivity was
found in the bones thus demonstrating the in vivo stability of
the radiotracer. Albeit used as a model protein for the current
study, [18F]ZHER2:2395‐Cys 8a exhibits very promising in vivo
Figure 1. Representative 3‐dimensional maximal intensity projections of PET/CT scans
performed with HER2‐positive SKOV3 (A) and HER2‐negative RAMOS (B) tumor‐bearing
mice 2 hours p.i. of [18F]ZHER2:2395‐Cys 8a (5‐10 MBq, 15 µg). The standardized uptake
value (SUV, color bar) is scaled for both animals. Tu: tumors; Gb: gall bladder; Ki: kidneys,
Int: intestine; Ub: urinary bladder.
In summary, we have developed an alternative to the
maleimide‐based conjugation strategy for the chemoselective
18F‐radiolabeling of peptides and proteins via modifications of
Cys residues. The novel prosthetic group based on a 18F‐labeled
derivative of phenyloxadiazole methylsulfone ([18F]FPOS) is
prepared in a single step from the corresponding nosylate
precursor in high radiochemical yield and purity. [18F]FPOS
reacts selectively and fast (within minutes) with thiol‐containing
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substrates in aqueous medium (pH 7.4) at low to moderate
temperature providing uniform radioconjugates with high
specific activities that are stable in vivo. The new methodology
presented herein meets all criteria of an effective and
chemoselective radiolabeling strategy and thus, may have the
potential to become the method of choice for the development
of radiotracers derived from thiol‐containing (bio)molecules.
We are currently investigating applications of the novel
radiolabeling strategy to different diagnostic and/or
therapeutic radionuclides as well as other peptides and proteins
of medical interest.
View Article Online
DOI: 10.1039/C6CC01982J
We thank Affibody AB (Solna, Sweden) for the affibody
ZHER2:2395‐Cys, Dr. M. Behe (Paul Scherrer Institute, Villigen,
Switzerland) for providing xenografted mice, and C. Keller (ETH
Zurich, Switzerland) for technical assistance with animal
experiments.
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