Synthesis of 18F-labelled biotin analogues

Article abstract of DOI:10.1002/jlcr.1913

A one-step ¹⁸F-labelling strategy was used to prepare three labelled analogues of the vitamin biotin, which can be useful as tracers because of biotin's high affinity for avidin. The labelled compounds were obtained in decay-corrected yields of up to 35% and specific radioactivity of 320 GBq/μmol. When evaluated in situ, the analogues showed good affinity for avidin: 60-75% of the radiolabelled compounds were bound to avidin within 5 minutes. The binding was site-specific, as shown by blocking experiments with native biotin. Copyright

Full text of DOI:10.1002/jlcr.1913

Note
Received 17 February 2011,
Revised 15 June 2011,
Accepted 16 June 2011
Published online 1 September 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1913
Synthesis of 18F-labelled biotin analogues
*
Elisabeth Blom, Oleksiy Itsenko, and Bengt Långström
A one-step 18F-labelling strategy was used to prepare three labelled analogues of the vitamin biotin, which can be useful as
tracers because of biotin’s high affinity for avidin. The labelled compounds were obtained in decay-corrected yields of up to
35% and specific radioactivity of 320GBq/mmol. When evaluated in situ, the analogues showed good affinity for avidin:
60–75% of the radiolabelled compounds were bound to avidin within 5minutes. The binding was site-specific, as shown by
blocking experiments with native biotin.
Keywords: 18F; biotin analogues; radiolabelling; avidin; one-step labelling; nucleophilic fluorination
prepared by conjugation of pegylated biotin to [18F]FDG, and
Introduction
this conjugate also showed binding to avidin.15
In the current work, three analogues of biotin that have 18F
labels on alkyl and ethylene glycol side-chains were prepared,
Biotin (4-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-
4-yl]butanoic acid) is a naturally occurring vitamin that is generally
found in its protein bound form. The affinity of biotin for the
and their binding abilities were investigated as a continuation
of our previous work with 68Ga-labelled analogues of biotin.16
protein avidin, a cationic glycoprotein obtained from egg
white, is the highest known (Kd ꢀ 10-15M) for a ligand and protein
in nature.1 The quaternary structure of avidin (Mw ꢀ 66kDa) con-
Experimental
sists of a noncovalent tetramer. The monomer units combine
into the active form,2,3 so each avidin unit is capable of binding
Experimental procedures are described in supplementary material.
four biotin molecules.3,4 The avidin-biotin system has been used
as a tool for various purposes, such as the diagnosis5 of, for
example, tumours6 and for isolation (affinity chromatography).7
The intention of the present work was to use the avidin-biotin
system in combination with positron emission tomography
(PET) imaging to follow the fate of the transplanted islets of
Langerhans. The transplantation of islets isolated from donor
pancreas into a patient with type I diabetes has been studied
for decades, and patients can become independent of insulin
after the transplantation. The efficiency of the procedure is,
however, low; and islets from more than one pancreas are
often needed to achieve independence from external insulin.8
Initial identification of graft rejection has been hard because of
the lack of suitable tools for diagnosis.9
Results and discussion
18F-labelling
The syntheses of the 18F-fluorinated target compounds 4–6 from
the corresponding mesyl compounds 1–3 are outlined in
Scheme 1. The 18F label was placed on different side chains,
alkyl and di(ethylene glycol) to obtain a set of compounds
with different properties, such as size and lipopilicity.17
The labelled compounds 4–6 were synthesised using no-
carrier-added [18F]fluoride in a one-step nucleophilic substitu-
tion (Scheme 1). In short, heating a precursor with [K/K
2.2.2]+18F- in 400 mL acetonitrile at 110ꢁC for 15minutes gave
the corresponding labelled product. The crude product was
purified by semipreparative HPLC, which gave the isolated
product in 10–35% decay-corrected radiochemical yield
(Table 1), within 60minutes after the end of radionuclide pro-
duction. Specific radioactivity was 320GBq/mmol (n=2) for com-
pound 513 at the end-of-synthesis. The radiochemical purity of
product always exceeded 98% after purification. Preliminary
identification in the analysis of the 18F-labelled compounds in
To better understand the fate of the transplanted islets, a
method for imaging in vivo is needed. PET has shown the poten-
tial to monitor islet number, mass and function.10 A useful PET
tracer for determining the fate of islets during transplantation
would have high specific binding and high intracellular stability.
2-[18F]Fluoro-2-deoxy-D-glucose ([18F]FDG) is one tracer that has
been used for this purpose.11,12 We wanted to explore the well-
known high affinity between biotin and avidin to find another
suitable tracer for quantifying the fate of the transplanted islets.
Biotin has already been labelled with 18F. The 18F-labelled biotin
analogue N-(3-[18F]fluoropropyl)-5-[(3aS,4S,6aR)-2-oxohexahydro-
1H-thieno[3,4-d]imidazol-4-yl]pentanamide (5) (Scheme 1) binds
to avidin and has been used for PET imaging of infection.13
Another 18F-labelled analogue, in which the radiolabel sits on
an aromatic moiety attached to biotin, has also been prepared
Uppsala University Department of Biochemistry and Organic Chemistry, Box 576,
Husargatan 3, BMC, SE-751 23 Uppsala, Sweden
* Correspondence to: Bengt Långström, Uppsala University, Department of
Biochemistry and Organic Chemistry, Box 576, Husargatan 3, BMC, SE-751 23
Uppsala, Sweden.
and evaluated in vitro.14 18F-Labelled biotin has also been E-mail: Bengt.Langstrom@biorg.uu.se
J. Label Compd. Radiopharm 2011, 54 681–683
Copyright © 2011 John Wiley & Sons, Ltd.

E. Blom et al.
O
H
O
H
H
H
H
H
H
N
H
N
18F-
N
H
N
H
O
S
O
S
N
H
N
H
O
O
1: n = 1, 2: n = 2
n
18F
S
4: n = 1, 5: n = 2
O
n
O
O
O
O
18F
3
6
S
O
Scheme 1. 18F-labelling reaction to yield compounds 4–6.
7–9 were mesylated using methanesulfonyl chloride in pyridine
to give the desired precursors 1–3 in 11–71% yield (Scheme 2).
Attempts were made to prepare the tosylated alcohols, in
pyridine (with or without DMF as co-solvent) or sodium
hydroxide in water. However, none of these attempts led to
product, one possible reason being the lower reactivity of
para-toluenesulfonyl chloride compared with methanesulfonyl
chloride,18 although a four carbon chain biotin amide tosylate
has been published.19 However, tosylated biotin amide compounds
with shorter chains than four carbons have not been reported. A
plausible reason may be the steric hindrance from internal binding
of the alcohol starting compound.
Table 1. Isolated radiochemical yields of labelling reactions
Compound
RCY (%)*
4
5
6
13Æ2
10Æ3
35Æ3
RCY, radiochemical yield
*Isolated decay-corrected radiochemical yield, calculated from
the amount of radioactivity at the start of synthesis and radio-
activity of LC-purified product. All experiments were performed
at least twice.
The unlabelled reference substances corresponding to
compounds 4–6 were synthesised by refluxing the corresponding
precursors with tetrabutylammonium fluoride in THF.
the liquid chromatography runs was based on the retention
times of the unlabelled references.
Binding to avidin
The influence of fluoro-alkyl or -di(ethylene glycol) substituents
on the binding of biotin to avidin was investigated in situ,
using the same procedure as for the 68Ga-labelled componds16.
Varying amounts of the biotin or its analogues (2–128nmol) were
incubated together with a constant amount of avidin (2nmol).
Samples were withdrawn after 5minutes and analysed by
HPLC to determine the percent consumption of avidin, and
Synthesis of precursors and reference compounds
The precursors 1–3 were synthesised from biotin and the corre-
sponding amino alcohols. The coupling was accomplished using
triphenylphosphine, tetrachloromethane and triethylamine in
dimethylformamide (DMF) (Scheme 2). The obtained alcohols
O
H
O
H
H
H
H
H
H
N
H
N
PPh3 CCl4 Et3N
DMF
N
H
OH
O
S
n
O
S
N
H
N
H
OH
7: n = 1, 8: n = 2
OH
n
H2N
O
O
H2N
OH
9
OH
O
S
O
H
Cl
H
H
N
O
N
H
O
S
N
H
pyridine
H
O
O
1: n = 1, 2: n = 2
S
O
n
O
O
O
3
S
O
Scheme 2. Synthesis of alcohols 7–9 and the mesylated compounds 1–3.
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Copyright © 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 681–683

E. Blom et al.
a 50-fold excess of native biotin before adding any of the
labelled compounds 4–6 (Figure 2). The limited sensitivity of
the ultraviolet and radiodetectors precluded binding studies
at concentration levels corresponding to the KD of the native
biotin-avidin affinity (10-15M).
The binding of the biotin analogues might be high
enough to make them useful in labelling avidin-treated islets
of Langerhans.
100
90
80
70
60
50
40
30
20
10
0
biotin
4
5
6
Conclusion
Three 18F-labelled biotin analogues have been synthesised using
a one-step nucleophilic 18F-fluorination strategy. The labelled
compounds were obtained with a decay-corrected yield up to
35%, and the specific radioactivity for one of the 18F-labelled
compounds was 320GBq/mmol. All analogues could bind to
avidin in situ, as good as or to a slightly higher degree compared
with our previously prepared 68Ga-labelled biotin analogues, and
5–15% lower compared with native biotin. The binding of the
tracers will be further evaluated in vitro and in an in vivo evaluation
of the islet of Langerhans transplantation model.
0
2
4
6
8
10
12
14
16
18
ligand/avidin
Figure 1. Saturation of the binding of biotin and nonradioactive compounds 4–6
to avidin in solution, as determined by HPLC analysis. The incubation was carried
out for 5minutes at room temperature. Data are presented as mean values (n=2).
100
80
Acknowledgement
60
non-blocked
Financial support from The Swedish Science Research Council
and Juvenile Diabetes Research Foundation (JDRF) (BL) is grate-
fully acknowledged. This work was conducted in collaboration
with Uppsala Imanet and Uppsala Applied Science Lab, GE
Healthcare.
40
blocked
20
0
4
5
6
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J. Label Compd. Radiopharm 2011, 54 681–683
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