Silicon-based building blocks for one-step18F-radiolabeling of peptides for PET imaging

Article abstract of DOI:10.1002/anie.200705854

(Equation Presented) Radio-controlled landing of F: The facile ¹⁸F labeling of biomolecules has been achieved under mild conditions by using a unique silicon-based one-step approach (see scheme). A di-tert-butylsilyl derivative with an aryl lin

Full text of DOI:10.1002/anie.200705854

Communications
DOI: 10.1002/anie.200705854
Labeling with Radioactive Fluorine
Silicon-Based Building Blocks for One-Step 18F-Radiolabeling of
Peptides for PET Imaging
Linjing Mu, Aileen Höhne, P. August Schubiger, Simon M. Ametamey,* Keith Graham,
John E. Cyr, Ludger Dinkelborg, Timo Stellfeld, Ananth Srinivasan, Ulrike Voigtmann, and
Ulrich Klar*
Positron emission tomography (PET) is an important diag-
nostic tool in modern medicine due to its ability to locate and
assess abnormalities in neurology,[1a–e] oncology,[1f–j] and
cardiology.[1k,l] The application of 18F-labeled small bioactive
peptides for diagnostic imaging has emerged as an important
and interesting field in nuclear medicine.[2] However, cur-
rently established 18F-labeling procedures require scrupu-
lously dry, strongly basic reaction conditions at high temper-
ature, which are not suitable for biomolecules such as
peptides and proteins.Therefore, the labeling of peptides
and proteins is usually achieved by using suitable prosthetic
groups labeled with 18F.This approach, however, requires a
multistep reaction sequence and is time-consuming.[3] Owing
to the short half-life (110 min) of 18F and the chemical
properties of biomolecules, a more efficient, one-step method
for site-specific labeling under mild conditions is required.
Based on the high silicon–fluorine bond energy (135 kcal
the corresponding non-radioactive 19F compound, which leads
to relatively low specific radioactivity.Very recently, the same
group used the highly effective labeling reagent p-(di-tert-
butylfluorosilyl)benzaldehyde for coupling to N-terminal
aminooxy (N-AO) derivatized peptides to achieve high
specific activities with a two-step procedure.[7b] Ting et al.
published the carrier-added 18F-labeling of trialkoxysilanes
with multiple fluorine atoms attached to silicon,[8] and the
alkyltetrafluorosilicate was moderately stable in aqueous
media.
A one-step no-carrier-added nucleophilic 18F-fluorination
of biomolecules such as peptides using silicon–fluorine
chemistry is the main goal of our study.For the silicon-
based 18F imaging agent to be effective as a PET probe, the
À
Si F bond needs to be sufficiently stable under physiological
conditions.It is known that the hydrolytic stability of the
silicon–halogen bond is determined by the nature of the
substituents on the silicon atom.Therefore, a series of
bifunctional silicon building blocks were designed and
synthesized, which contained different substituents and leav-
ing groups suitable for fluorination and linkers suitable for
subsequent coupling to a biomolecule.Model fluorosilanes
using non-radioactive fluoride (19FÀ) were also prepared.
These compounds were used for stability studies and as
standard reference compounds.
molÀ1 vs.116 kcalmol À1 for C F) and the experimental results
À
of Whitmore et al.,[4] the concept of exploiting the fluoride
substitution at silicon for the 18F-labeling of biomolecules has
been discussed and tested by different research groups.[5] Up
to now, site-specific 18F-radiolabeling of organosilanes under
mild conditions has been achieved, however, most methods
still require at least a two-step procedure.Recently, Choudhry
et al.evaluated the hydrolytic stability of four model trialkyl-
fluorosilanes and proposed to use the most stable compound
as a building block for the direct 18F-labeling of biomole-
cules.[6] Schirrmacher et al.also reported on the direct radio-
labeling of an organosilicon-modified peptide by an isotope
exchange reaction,[7a] but the product contains predominantly
The amides 3a and 3b were synthesized from commer-
cially available dimethyl- and diisopropylsilylamines 1a and
1b, respectively.Fluorination of 3a and 3b with BF3·OEt2
afforded compounds
(Scheme 1).
I and II as standard references
Scheme 2 depicts the synthetic pathway towards silane
derivatives with an aryl linker.Compound 5a was synthesized
by nucleophilic substitution of diisopropylchlorosilane with
an ate complex generated from 4, isopropylmagnesium
bromide, and nBuLi.Compound 5b was synthesized by
nucleophilic substitution of di-tert-butylchlorosilane with {4-
[2-(tetrahydro-2H-pyran-2-yloxy)ethyl]phenyl}lithium, which
was generated in situ by metal–halogen exchange of bromide
4 with nBuLi.Basic hydrolysis of 5b with KOH in EtOH
yielded the silanol 6.Treatment of compound 5a, 5b, and 6
with toluenesulfonic acid in ethanol gave the alcohols 7, 8a,
and 8b, respectively.Compound 7 was oxidized by means of a
Jones oxidation to give the carboxylic acid 9a.Compound 9b
was obtained by oxidizing 9a with Pd/C in a H2O–CCl4
mixture.The di- tert-butyl-substituted compounds 10a and
10b were obtained in good yields by Jones oxidation of the
crude 8a and 8b directly.Coupling of 10a or 10b with
[*] Dr. L. Mu,[+] A. Höhne,[+] Prof. Dr. P. A. Schubiger,
Prof. S. M. Ametamey
Animal Imaging Center-PET
Center for Radiopharmaceutical Science of ETH, PSI and USZ
ETH-Hönggerberg, D-CHAB IPW HCI H427
Wolfgang-Pauli-Strasse 10, 8093 Zurich (Switzerland)
Fax : (+41)446-331-367
E-mail: simon.ametamey@pharma.ethz.ch
Dr. K. Graham, Dr. J. E. Cyr, Dr. L. Dinkelborg, Dr. T. Stellfeld,
Dr. A. Srinivasan, Dr. U. Voigtmann, Dr. U. Klar
Bayer Schering Pharma AG
Global Drug Discovery, 13342 Berlin (Germany)
Fax : (+49)304-689-2635
E-mail: ulrich.klar@bayerhealthcare.com
[+] These authors contributed equally to the work.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Chemie
H2O–CCl4, and amidation with benzylamine.Fluorination of
11 and 12a with potassium fluoride in the presence of crypt-
222 and acetic acid yielded compounds III and IV.
Scheme 3 depicts the radiolabeling conditions.Good to
high yields of 18F-incorporation were achieved under mild
labeling conditions with acetic acid as an additive (Table 1).In
Scheme 3. 18F-radiolabeling of silicon-based building blocks.
Scheme 1. Synthesis of silane derivatives with alkyl linkers. a) Biphenyl-
4-carbonyl chloride (2), NEt3, dioxane, RT, 1 h for 3a, 61%; 1.5 h for
3b, 55%; b) BF3·OEt2, Et2O, reflux, 15 min for compound I, 63%;
30 min for compound II, 97%.
most cases, the addition of acetic acid enhances the radio-
labeling yield significantly.For example, with acetic acid as an
additive, the 18F-incorporation for the diisopropylsilanol at
658C was 90%, whereas without acetic acid it was only 3%
(Table 1, entries 14, 15).The enhanced yields are probably
due to the protonation of hydroxy and alkoxy groups, which
makes them better leaving groups.It is not surprising that
with hydrogen as the leaving group, the addition of acetic acid
did not have any dramatic effect on the radiochemical yield
(Table 1, entries 22 and 23).Under the same reaction
conditions, the di-tert-butylsilyl model com-
pounds gave a much higher radiolabeling
yield with the hydrogen leaving group than
with the hydroxy leaving group (Table 1,
entries 16–23).Therefore, we decided to use
benzylamine using 1-ethyl-3-(3-di-methyl-aminopropyl)car-
bodiimide hydrochloride (EDC·HCl) as the coupling reagent
provided amides 12a and 12b, respectively.Compound 11 was
obtained from compound 9a in three steps: activation of the
acid with N-hydroxysuccinimide, hydrolysis with Pd/C in
the silane building block for the subsequent
coupling reactions instead of the silanol
building block.As shown in entry 10 in
Table 1, decreasing the amount of the pre-
cursor resulted in a fourfold lower yield of
18F-incorporation at 308C.However, 18F-
incorporation with lower amounts of pre-
cursor could be improved from 19% to 88%
by increasing the reaction temperature from
308C to 908C (Table 1, entries 10, 11).With
the diisopropyl derivatives, a temperature of
308C was sufficient to achieve high 18F-
incorporation, whereas with the di-tert-butyl
derivatives, a relatively higher reaction tem-
perature was required.
The time-dependent hydrolysis of fluo-
rosilane model compounds I to IV was
determined at pH 7.0 and their hydrolytic
half-lives (T1/2) were calculated (I: < 5 min,
II: 12 h, III: 8 h, IV: @ 170 h).[9] The model
fluorosilane compound I (R = Me) is stable
in anhydrous organic solvents such as aceto-
nitrile and diethyl ether, but is readily
hydrolyzed in the presence of water.Intro-
ducing bulkier isopropyl and tert-butyl
groups into the fluorosilanes (compounds
II, III, and IV) significantly increased their
stability towards hydrolytic cleavage.Based
Scheme 2. Synthesis of silane derivatives with aryl linkers. a) 5a: nBuLi, iPrMgBr, THF,
iPr2SiClH, 97%; 5b: nBuLi, THF, tBu2SiClH, 74%; b) 6: KOH, EtOH, 71%; c) 7: pTsOH,
EtOH, 93%; 8a and 8b: TsOH, EtOH; d) 9a: Jones reagent, acetone, 69%; 9b: Pd/C,
H2O, CCl4 from 9a 69%; 10a: from 5b, 1. pTsOH, EtOH, 2. Jones reagent, acetone, overall
yield 80%; 10b: from 6, 1. TsOH, EtOH, 2. Jones reagent, acetone, overall yield 77%;
e) 11: starting with 9a, 1. NHS, EDC·HCl, CH2Cl2, 83%; 2. Pd/C, H2O, CCl4, 78%;
3. BnNH2, CH2Cl2, 76%; 12a and 12b: EDC·HCl, BnNH2, CH2Cl2, 66% for 12a and 76%
for 12b; f) KF/crypt-222, AcOH, THF, 81% for compound III, 98% for compound IV from
12a. THP = tetrahydropyranyl, Bn = benzyl, Ts = toluenesulfonyl.
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Table 1: 18F radiolabeling of various silicon model compounds.[a]
Entry Precursor (mg) T [8C] Acetic acid [mL] Conversion [%][b]
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
3a (5)
3a (5)
3a (5)
3a (5)
3b (5)
3b (5)
3b (5)
3b (5)
3b (3)
3b (1)
3b (1)
11 (5)
30
30
65
65
30
30
65
65
30
30
90
30
30
65
65
30
30
65
65
30
30
65
65
3
0
3
0
3
0
3
0
3
3
3
3
0
3
0
3
0
3
0
3
0
3
0
84
18
92
2
93
24
96
13
79
19
88
53
9
90
3
15
0
23
4
59
24
69
69
11 (5)
11 (5)
11 (5)
Scheme 4. 18F-radiolabeling of silicon tetrapeptides. a) K[18F]F, 5 mg
crypt-222, 1 mgK2CO3, 3 mL AcOH, 1 mg 13a in 0.3 mL DMSO,
15 min, 908C, 53% conversion for 14a; K[18F]F, 5 mg crypt-222,
1 mgK2CO3, 2 mg 13b in 0.3 mL DMSO, 15 min, 658C, 45% conver-
sion for 14b.
12b (5)
12b (5)
12b (5)
12b (5)
12a (5)
12a (5)
12a (5)
12a (5)
we have developed a unique silcon-based method for the
facile 18F-labeling of biomolecules under mild conditions and
using a one-step approach.
[a] 18F-labeling experiments were carried out in 300 mL DMSO for 15 min.
[b] Determined from the radio-HPLC chromatogram: ratio of the radio-
activity area of product to the total radioactivity area.
Received: December 20, 2007
Revised: February 20, 2008
Published online: May 21, 2008
Keywords: fluorine · peptides · PET imaging · radiolabeling ·
silicon
on these current results, steric hindrance at the R position
seems to play a key role in the stabilization of the silicon–
fluorine bond.Interestingly, the linker appeared to have less
steric influence in our model compounds; fluorosilanes with
aryl linkers were not substantially better with regard to
hydrolytic stability than those with alkyl linkers.Of all the
compounds tested, the fluorosilane IV with tert-butyl sub-
stituents and an aryl linker exhibited the best hydrolytic
stability.
To test the practical utility of these new silicon building
blocks, the acids 9b and 10a were coupled to a tetrapeptide by
using standard solid-phase peptide synthesis protocols.[10] The
18F-labeling of these peptides was performed under similar
reaction conditions to those reported for the silicon model
compounds (Scheme 4).Around 50% 18F-incorporation was
achieved after a reaction time of 15 min for compounds 13a
and 13b.The preliminary stability test for the silicon-based
tetrapeptide 13b was very promising.After two hours of
incubation in human plasma, no degradation products were
observed.
A series of bifunctional silicon building blocks with
different linkers, leaving groups, and substituents were
synthesized and labeled with 18F.The hydrolytic stability of
the fluorosilane center appears to depend on steric hindrance
at the silicon center.The most stable building block was
successfully used for the synthesis of a radiolabeled tetrapep-
tide, which showed a hydrolytic stability in the range required
for PET studies.In vivo PET imaging and biodistribution
studies with bioactive peptides labeled with 18F using this new
labeling methodology are currently ongoing.In conclusion,
.
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