18
F-PEG-biotin: Precursor (boroaryl-PEG-biotin) synthesis, 18F-
labelling and an in-vitro assessment of its binding to NeutravidinTM
-trastuzumab pre-treated cells.
Tim A D Smith*, Michael Simpson*+, Richard Cheyne*+, Laurent Trembleau+
John Mallard PET Unit, Aberdeen Biomedical Imaging Centre, School of Medical
Sciences*, School of Computing and Natural Sciences+, University of Aberdeen,
Aberdeen AB25 2ZD
Correspondence:
Dr T A D Smith
Biomedical Physics Building
John Mallard PET Unit
Aberdeen Biomedical Imaging Centre
School of Medical Sciences
University of Aberdeen
Foresterhill. Aberdeen AB25 2ZD
01224 553481
t.smith@abdn.ac.uk
Running title: Synthesis of 18F-PEG-biotin
1
Abstract
In terms of nuclear decay 18F-fluorine is the most ideal PET nuclide but its short t1/2
precludes its use for directly labelling whole antibodies due to their long blood residence
times. Pre-targeted imaging using affinity systems such as NeutravidinTM -biotin facilitate
the application of short-lived nuclides by their attachment to biotin for imaging cell
surface proteins targeted with NeutravidinTM -conjugated antibodies.
Methods
Boroaryl functionalised biotin was prepared with a PEG linker and radiolabelled by
incubation with18F-fluorine in acidified aqueous solution. Cells expressing high (SKBr3),
medium (MDA-MB-453) and low (MDA-MB-468) levels of HER-2 were pre-incubated
with NeutravidinTM -conjugated Trastuzumab, washed, then incubated with 18F-PEGbiotin.
Results
The 18F-fluorination of boroaryl-PEG-biotin was much more efficient than reported for
other versions of boroaryl-biotin. The novel 18F-PEG-biotin was demonstrated to bind to
HER2-expressing cells in-vitro pre-incubated with NeutravidinTM –conjugated
trastuzumab.
Conclusion
2
Biotin can be functionalised with boroaryl and readily 18F-radiolabelled in aqueous
solution and will bind to cells pre-incubated with NeutravidinTM -antibody conjugates.
Keywords
18F-fluorine; PET; NeutravidinTM; breast cancer cells: biotin
3
Introduction
In terms of its nuclear properties 18F is the most ideal PET isotope for imaging due to its
simple decay and emission properties with high (97%) positron abundance and relatively
low positron emission energy (0.635MeV max). In addition, all PET facilities with
cyclotrons produce 18F, which is consequently the most readily available PET isotope.
Currently 2-[18F]fluoro-2-deoxy-D-glucose (FDG), a glucose analogue, is by far the most
widely available PET tracer being utilized in about 90% of all PET investigations
(Coenen et al 2010). However FDG is a non-specific tracer taken up by all glucoseutilizing tissue. In contrast macromolecules such as antibodies selectively accumulate in
tissue expressing specific cell surface molecules. These include the therapeutic antibodies
cetuximab and trastuzumab which target HER-1 and HER-2 expressing tumour tissue
respectively (Rusnak et al 2007).
Monoclonal antibodies and derivatives such as diabodies (Wu et al 1996) have a high
avidity for their target molecules but are too large to be rapidly cleared from the
bloodstream so unsuitable for direct labelling with
110min. A strategy for utilizing
18
18
F which has a half-life of only
F for in-vivo diagnosis which enables the use of high
molecular weight antibodies is to pre-target the antigen of interest with the antibody,
conjugated to NeutravidinTM (a neutral and deglycosylated version of avidin which
exhibits lower levels of non-specific cell binding than does avidin) which has a high
4
affinity for biotin. Several days later after the unbound antibody conjugate has cleared
from the blood (this may be assisted by addition of a clearing molecule), radiolabeled
biotin, which being small and water soluble is rapidly cleared from the blood, is
administered and the patient scanned after 1 or 2 hours. This has been carried out with
nuclides including
68
Ga (Griffiths et al 2004) but is applicable to
18
F. It is also well
established that using pre-targeted imaging improves image quality when compared with
the administration of directly labelled antibodies (Goldenburg et al 2006).
In this paper we describe the synthesis of a model biotin conjugate that is functionalized
with difluoroboroaryl to facilitate
18
F-radiolabelling using an aqueous
18
F-fluorination
procedure. The in-vitro binding of this tracer (18F-PEG-biotin) is then demonstrated using
HER-2 expressing cells pre-incubated with NeutravidinTM-conjugated trastuzumab.
Materials and Methods
Materials
Unless otherwise stated all chemicals used in the synthesis of BA-PEG-Biotin and
fluorination reactions were obtained from Sigma-Aldrich (Poole, UK).
18
F-fluoride was
synthesised by the Aberdeen PET Centre. The source of all other reagents is stated within
the methods section.
5
Analysis
Melting points were recorded on a Gallenkamp melting point apparatus and are
uncorrected. 1H and 13C nuclear magnetic resonance spectra were recorded on a Bruker
AC 250 or a Varian 400 MHz Unity INOVA spectrometer. 11B and 19F nuclear magnetic
resonance spectra were recorded on a Varian 600/54 PS spectrometer in the Department
of Chemistry at Aberdeen University. Mass spectra were obtained on a Waters LCT
Premier XE mass spectrometer using electrospray ionization at the University of
Sheffield. NMR analyses in each of the methods section describing a synthesis are
preceded by e.g. for proton data: δH (solvent, operating frequency). Mass spectroscopy
are preceded by m/z (ESI).
Synthesis of (2-{2-[2-(2-{2-[2-(2-{2-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)pentanoylamino]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethyl)carbamic acid tert-butyl ester (1)
Addition of PEG linker to biotin: A solution of biotin N-hydroxysuccinimide ester (25
mg, 73.2 μmol), O-(2-aminoethyl)-O'-[2-(Boc-amino)ethyl]-hexaethylene glycol (34 mg,
72.6 μmol) and triethylamine (8.0 mg, 11 μL, 78.9 μmol) was stirred in CHCl3 (2 mL) at
room temperature under Ar for 15 hours. The reaction mixture was washed three times
with H2O then dried (MgSO4), filtered and evaporated to leave the product as a white
solid. Analysis of product 1: 50 mg (100 %); δH(250 MHz; CDCl3) 1.40-1.77 (15 H, m),
2.18 (2 H, t, J 7.3), 2.70 (1 H, d, J 12.8), 2.85 (1 H, dd, J 12.5, 4.6), 3.06-3.12 (1 H, m),
6
3.25-3.60 (32 H, m), 4.23-4.28 (1 H, m), 4.43-4.48 (1 H, m), 5.09 (1 H, bs), 5.93 (1 H,
bs), 6.80 (1 H, bs), 6.93 (1 H, bt); δC(62.5 MHz; CDCl3) 25.5, 28.0, 28.2, 28.3, 35.8,
39.0, 40.2, 40.4, 55.6, 60.1, 61.6, 69.8, 69.9, 70.1, 70.2, 70.3, 70.4, 79.0, 155.9, 164.2,
173.3; m/z (ESI) 695.3892 ([M+H]+. C31H59N4O11S requires 695.3901).
Synthesis of 5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoic acid [2-(2{2-[2-(2-{2-[2-(2-amino-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)ethyl]-amide dihydrochloride (2)
Cleavage of the Boc protecting group: A 4 M solution of HCl in dioxane (1.6 mL, 6.40
mmol) was added to a solution of 1 (88 mg, 0.127 mmol) in MeOH (1.6 mL) and the
mixture was stirred for 90 minutes at room temperature under Ar. The mixture was
concentrated in vacuo to leave the product as a colourless oil. Analysis of product 2: 84
mg (99 %); δH(250 MHz; CD3OD) 1.37-1.42 (2 H, q, J 6.1), 1.52-1.67 (4 H, m), 2.26 (2
H, t, J 6.7), 2.76 (1 H, d, J 13.1), 2.97 (1 H, dd, J 12.5, 3.1), 3.18-3.23 (1 H, m), 3.323.37 (4 H, m), 3.60-3.69 (28 H, m), 4.38-4.43 (1 H, m), 4.57-4.62 (1 H, m).
Synthesis of 2,6-Difluoro-4-[(carboxy-methoxyimino)-methyl]-phenylboronic acid
pinacol ester (3)
Oxime formation between the boroaryl group and an amino acid: A mixture of (Ocarboxymethyl)hydroxylamine hemihydrochloride (171 mg, 1.57 mmol), 2,6-difluoro-4formylphenylboronic acid pinacol ester (350 mg, 1.31 mmol) and Hünig’s base (102 mg,
7
137 µL, 0.787 mmol) was stirred in anhydrous acetonitrile (10 mL) containing 3 Å
molecular sieves at room temperature under Ar for 2 hours. The solvent was evaporated
and the residue dissolved in EtOAc. The ethyl acetate was washed once with H2O then
dried (MgSO4), filtered and evaporated to leave a white solid. This was recrystallised
from a mixture of petroleum ether 40/60 and EtOAc (4:1) to leave the product as a white
solid. Analysis of product 3: 228 mg (51 %); m.p. 151-152° C; δH(250 MHz; CDCl3) 1.37
(12 H, s), 4.77 (2 H, s), 7.06 (2 H, d, J 7.6), 8.09 (1 H, s), 10.40 (1 H, bs); δC(62.5 MHz;
CDCl3) 24.7, 70.5, 84.4, 109.8 (d, J 28), 136.5 (t, J 10), 148.5, 166.6 (dd, J 251, 13),
175.4; δB(192 MHz; CDCl3) 29.1; m/z (ESI) 342.1323 ([M+H]+. C15H19BF2NO5 requires
342.1324).
Synthesis of 2,6-Difluoro-4-{[2-oxo-2-(pentafluorophenoxy)-ethoxyimino]-methyl}phenylboronic acid pinacol ester (4)
Activation of the carboxylic acid functional group: EDC hydrochloride (26 mg, 0.136
mmol) and pentafluorophenol (22 mg, 0.120 mmol) were added to a solution of 3 (40 mg,
0.117 mmol) in CHCl3 (1.5 mL) and the mixture was stirred at room temperature under
Ar for 15 hours. The choloroform was evaporated and the residue dissolved in a mixture
of EtOAc and H2O. The layers were separated and the organic portion dried (MgSO4),
filtered and evaporated to leave the product as a colourless oil. Analysis of product 4: 59
mg (100 %); δH(250 MHz; CDCl3) 1.38 (12 H, s), 5.06 (2 H, s), 7.08 (2 H, d, J 6.7), 8.12
(1 H, s); δC(62.5 MHz; CDCl3) 24.7, 70.2, 84.5, 109.8 (d, J 28), 136.2 (t, J 11), 149.0,
165.6, 166.6 (dd, J 251, 14); δB(192 MHz; CDCl3) 29.4.
8
Synthesis of 2,6-Difluoro-4-{[2-oxo-2-(2-{2-[2-(2-{2-[2-(2-{2-[5-(2-oxo-hexahydro1H-thieno[3,4-d]imidazol-4-yl)-pentanamido]-ethoxy}-ethoxy)-ethoxy]-ethoxy}ethoxy)-ethoxy]-ethoxy}-ethylamino)-ethoxyimino]-methyl}-phenylboronic acid
pinacol ester (5)
Attachment of the boroaryl functional group to biotin via a PEG linker: A mixture of 2
(82 mg, 0.123 mmol), 4 (63 mg, 0.124 mmol) and Hünig’s base (32 mg, 43 µL, 0.247
mmol) was stirred in CHCl3 (4 mL) at room temperature under Ar for 15 hours. The
chloroform was washed once with H2O then dried (MgSO4), filtered and evaporated. In
order to remove pentafluorophenol, the residue was dissolved in a minimum volume of
chloroform then precipitated by addition of petroleum ether 40/60, before being subjected
to ultrasonic-assisted extraction. The solution was decanted, leaving the product as a pale
orange, viscous oil. Analysis of product 5: 106 mg (94 %); δH(400 MHz; CDCl3) 1.341.71 (18 H, m), 2.19 (2 H, bs), 2.71 (1 H, bd, J 10.2), 2.86 (1 H, bd, J 9.0), 3.10 (1 H, bs),
3.38-3.65 (32 H, m), 4.30 (1 H, bs), 4.50 (1 H, bs), 4.63 (2 H, s), 5.62 (1 H, bs), 6.43 (1
H, bs), 6.79 (1 H, bs), 6.85 (1 H, bs), 7.05 (2 H, d, J 7.8), 8.11 (1 H, s); δC(100 MHz;
CDCl3) 24.6, 25.5, 28.0, 28.1, 35.7, 38.8, 39.2, 40.4, 55.5, 60.3, 61.8, 69.6, 69.7, 70.0,
70.17, 70.28, 70.32, 70.34, 70.4, 73.4, 84.4, 109.7 (d, J 30), 129.6, 136.5 (t, J 10), 148.8
(t, J 4), 166.5 (dd, J 251, 14), 169.3; δF(564 MHz; CDCl3) -99.5 δB(192 MHz; CDCl3)
29.3; m/z (ESI) 918.4554 ([M+H]+. C41H67BF2N5O13S requires 918.4517).
18F-radiolabelling
9
To a mixture of 5 (BA-PEG-biotin) (1µmole in 10ul of DMSO) and 5ul of glacial acetic
acid was added 10µl of a premixed solution containing 18F-fluoride (20MBq) and 1.5
µmole KHF2 (i.e. equivalent to 3 Fs per boroaryl to ensure all the phenylboronic acid
pinacol ester reacts to form the stable phenyl-BF3 (which will be a mix of 18F and 19F)
derivative. The solution was then incubated at room T for up to 2h. Reaction progress
was monitored by applying 0.5 µl samples to silica TLC plates which were run using an
ethanol:water (95%:5%) mix. HPLC confirmed the labelling efficiency. The product was
purified from the un-reacted 18F-fluoride by applying to a Silica cartridge (Waters UK)
and eluting with 150µl of ethanol. TLC confirmed the absence of 18F-flouride form the
purified product.
HPLC conditions
HPLC analysis was carried out using a Jupitor 5µ C5 silica-based reversed phase 300A
column (250×4.6 mm) (Phenomenex, Macclesfield UK) using a 0-50 % acetonitrile
gradient (balance water) containing 0.15 % trifluoroacetic acid over a period of 40mins at
a flow rate of 1 mL per min. The HPLC system consisted of a Perkin-Elmer series 200
quaternary pump, series 200 autosampler, series 200 UV/Vis detector (set to 210 nm) and
5 channel vacuum degasser. The radioactive detector used was a Berthold Radioflow
Detector LB509. The autosampler was programmed to deliver 50 μL of sample which
had been prepared by adding 5 μL of reaction mixture to 200 μL of PBS and neutralised
with 0.1 M NaOH.
10
Cell binding
SKBr3, MDA-MB-453 and MDA-MB-468 cells (obtained from the American Tissue
Culture Collection (ATCC)) were maintained in 75cm2 tissue culture flasks in
Dulbecco’s Modified Eagles Medium (Gibco UK) containing 10% Foetal Bovine Serum
(Gibco UK). Confluent flasks were trypsinzed with 5ml of trypsin and neutralised with
complete medium then used to set up the 25cm2 flasks. Several days later when the cells
in each flask were about 80% confluent they were used for the determination of targeted
18
F-PEG-biotin binding.
NeutravidinTM conjugation to trastuzumab
Trastuzumab was purified from10mg of Herceptin (Roche) dissolved in 2ml of distilled
water by filtration though an Amicon Ultra centrifugal filter device (Millipore USA) with
a molecular weight cut off 30KDa by centrifuging at 1500g for 20min at room T to
remove salts and buffers that are present in the clinical preparation and may interfere with
the conjugation procedure. PBS (2ml) was then added to the retained trastuzumab and the
filter centrifuged at 5000g for a further 20mins. The washed trastuzumab retained by the
filter was then resuspended in 0.2ml PBS. The concentration of trastuzumab was then
determined by measurement of absorbance at 280nm using a Helios UV
spectrophotometer (Thermo Spectronic UK) using the Extinction coefficient for
antibodies of 210,000 M-1cm-1.
11
Reduction of thiol groups in trastuzumab: To 1mg of trastuzumab was added 1mg of
mercaptoethanol (ME) in PBS pH 7.2 and the solution incubated at 37oC for 90 min. ME
was then removed from the reduced trastuzumab by filtration though an Amicon Ultra
centrifugal filter device (Millipore USA) with a molecular weight cut off 30KDa by
centrifuging at 1500g for 20min followed by 2 washes with 1ml of PBS. The reduced
trastuzumab was then incubated immediately with the commercially available product,
maleimide-activated NeutravidinTM (1mg) (Thermo Scientific UK), in maleimide
conjugation buffer (sodium phosphate (0.1M), EDTA (5mM) pH 7.5) and left overnight
at room T. The thiol reduction procedure and conjugation with maleimide-activated
NeutravidinTM is described in the instruction sheet that accompanies the maleimideactivated NeutravidinTM kit (Thermo Scientific UK) which was adhered to.
Pre-incubation with NeutravidinTM conjugated trastuzumab and incubation with
18F-biotin
NeutravidinTM conjugated trastuzumab (containing 1mg of trastuzumab) supplemented
with 1.5mg of biotin-saturated NeutravidinTM (6 fold excess of NeutravidinTM in
NeutravidinTM conjugated trastuzumab) to reduce non-HER2 binding) was added to 5ml
of medium and incubated with flasks (0.3ml per flask) of MDA-MB-453, MDA-MB468
or SKBr3 cells in triplicate. To each flask was added 0.3ml of medium containing 37KBq
of 18F-PEG-biotin. Incubation was continued for 1h after which the flasks were flushed
3X with 5ml of PBS to remove unbound 18F-PEG-biotin. Cells were removed by
12
trypsinisation with 0.3ml of trypsin and cell associated 18F determined using a well
counter (the cells were not separated from the trypsin/media so any 18F removed by the
trypsin would still be measured). The cells were then dissolved overnight in 0.1ml of
NaOH (1M) then neutralised with 0.1ml of HCl (1M) and protein content determined
using the bicinchoninic acid protein assay kit (Sigma-Aldrich UK). The protein data was
then used to normalise the radioactive binding allowing comparison of data between each
cell type.
Preparation of biotin-saturated NeutravidinTM.
1.5mg of NeutravidinTM was dissolved in 100ul of PBS was added to 0.5mg of biotin (i.e.
a 100 fold molar excess) in 0.5ml of PBS. The mixture was incubated at 37oC for 1h.
Excess biotin was then removed on an Amicon centrifugal filter (30KDa mwt cut off) and
centrifuged at 14,000 g for 10min. This was repeated 4 times after addition of 0.5ml of
PBS. The biotin-saturated NeutravidinTM was then recovered.
Results
Labelling of BA-PEG-biotin with 18F
Figure 1 shows the time course of labelling with 18F of 0.1 µmoles and 0.5 µmoles of
BA-PEG-biotin with 18F. The initial rate of labelling is more rapid with 0.5 µmoles but at
both concentrations the maximum labelling efficiency plateaus at about 65%. The
13
specific activity achieved using 0.1 µmol of precursor is 100MBq/µmol using about
20MBq of 18F-fluroride and could be increased by using higher activity of 18F-fluoride
(potentially GBqs). Figure 2 shows a UV (210nm) absorbance and radio-chromatogram
from 30min incubation of BA-PEG-biotin with 18F-fluoride. A similar sample was also
applied to a TLC silica plate and run using a mobile phase of ethanol:water (95%:5%)
until it reached 70mm. The result shown in figure 3 shows 18F-fluoride at the origin and
the product 18F-PEG-biotin at 35 mm with an Rf of 0.5. Purified and neutralised 18F-PEGbiotin preparations were maintained in media for 0.5h and 2h then applied to a silica TLC
plate using ethanol:water (95%:5%) as the mobile phase. Less than 1% and 5% of the 18F
were in the form of free fluoride after 0.5 and 2h respectively.
Binding of 18F-PEG-biotin to cells pre-incubated with NeutravidinTM conjugated
trastuzumab
Figure 4 shows the binding of 18F-PEG-biotin to SKBr3, MDA-MB-453 and MDA-MB468 cells that have been pre-incubated with NeutravidinTM conjugated trastauzumab.
SKBr3 cells exhibited the greatest binding of 18F activity consistent with its very high
HER-2 expression. MDA-MB-453 cells exhibited lower binding and MDA-MB-468 cells
which only express HER-2 at low levels was associated with the lowest 18F-activity.
Discussion
14
Biotin has been labelled with 99mTc (Kleine et al 2008), 111In (Lazzeri et al 2004) and
64
Cu (Lewis et al 2003) positron emitting isotopes for SPECT and PET imaging and with
cytotoxic beta-emitting nuclides for therapy (Breitz et al 2000). Simpson et al (2011)
recently reported the labelling of biotin with [18F]-FDG. Shoup et al (1994) have
described the conjugation of biotin with mesylate to facilitate 18F-labelling using
nucleophilic substitution. They suggested that 18F-biotin may be useful means of imaging
infection. However 18F-fluorination using nucleophilic substitution requires stringent
conditions including high temperature and the use of organic solvents. More recently
Ting et al (2005) functionalised biotin with a pinacol phenylboronate diester then fluorinated
the compound in aqueous conditions but this group later showed that this compound was
readily defluorinated (Ting et al 2008). To increase the stability of the B-18F bonds the
use of 2,6-difluoro-4-carboxyphenylboronic acid, which include electron withdrawing atoms
linked via ethylenediamine to biotin (Harwig et al 2008) were tested these proved less
readily 18F-fluorinated but the B-18F bond formed was more stable. Thus Ting et al
(2008) labeled tetraphenylpinacolyl arylboronate (0.3µmoles) using 18F over a 159min
incubation period at 37oC and produced only a 15% labeling efficiency (based on product
activity versus free [18F]-fluoride). We produced labeling efficiencies (also based on
product activity versus free [18F]-fluoride) of 50 and 70% after 1h at room temperature
using 0.1 and 0.5 µmoles respectively using a pinacol group linked to biotin via a PEG
linker. PEGylation increases water solubility so this may also have contributed to
improving the 18F-fluorination efficiency.
15
Biotinidase-resistant biotin has been the subject of a series of recent papers using longer
tracer to imaging times or therapy with long-lived radionuclides. This may not be an
issue with short lived 18F where administration to scan times need to be short limiting
exposure of biotin to biotinidases. However the use of biotinidase-resistant biotin is a
potential modification that could be carried out prior to in-vivo application of 18F-PEGbiotin (Pratesi et al 2010).
Avidin has a high pI and is appreciably glycosylated. Both of these characteristics result
in non-targeted interactions with cells (Marttila et al 2000). Although NeutravidinTM,
does not possess carbohydrate moieties and has a neutral pI previous at least one study
(Watlick et al 2004) suggests that non-specific binding can still be a problem so we
included biotin-saturated NeutravidinTM during the incubation of cells with
NeutravidinTM –conjugated trastuzumab to saturate the non-HER-2 NeutravidinTMbinding sites so reducing non-specific binding.
In conclusion, we have synthesised a 2,6 difluoroboroaryl-functionalised biotin molecule
and demonstrated good rates of 18F-radiolabelling with [18F]-fluorine in aqueous solution.
The resultant 18F-PEG-biotin exhibited binding to cells that had been pre-incubated with
a NeutravidinTM -conjugated antibody.
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Acknowledgements
This work was funded by the Breast Cancer Campaign (UK). Mass Spectroscopy data
were obtained by Simon Thorpe at the University of Sheffield.
20
Scheme 1) Pathway for the synthesis of BA-PEG-biotin.
Figure 1) Labelling efficiency (percentage of product associated 18F) with time (h) using
0.1mg (triangles) and 0.5mg (squares) of BA-PEG-biotin (n=3).
Figure 2) HPLC trace of sample taken from a 30min reaction of BA-PEG-biotin with
18
F-fluoride using UV detection at 210 nm (A) and using scintilation detection (B). X-
(axis:Retention time (min)).
Figure 3) TLC trace from sample taken from a 30min reaction of BA-PEG-biotin with
18
F-fluoride and developed using ethanol: water (95%:5%). Distance migrated (in mm)
versus radioactivity (in mV).
Figure 4) Binding of 18F-PEG-biotin to breast tumour cells pre-incubated with
NeutravidinTM-conjugated trastuzumab (black) in the presence of biotin-saturated
NeutravidinTM (n=3 for each cell line).
21
O
HN
NH
O
O
S
H
N
H2N
N
O
O
O
7
O
O
Et3N, CHCl3, 15 h, rt
O
HN
NH
H
N
H
N
S
O
O
1
O
7
O
4 M HCl in dioxane, MeOH, 90 min, rt
O
HN
NH
H
N
NH2 .2HCl
S
O
O
O
2
7
F
B
O
CHCl3, Hunig's base, 15 h, rt
N
F
OPfP
O
4
O
O
F
HN
O
NH
B
O
H
N
S
H
N
N
O
O
7
O
O
5
22
F
Figure 1)
L abelling effic ienc y (% )
100%
80%
60%
40%
20%
0%
0
1
2
3
4
la be lling tim e (h)
23
5
6
Figure 2)
A
B
0
10
20
30
Retention time (min)
24
40
50
Figure 3
C/mm *1000
TLC
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00
0
10
20
30
40
25
50
60
mm
Figure 4)
26