6
314
Y. Li et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6313–6316
Figure 1. Compared to previously reported radiofluoridation of bor-
3
,12,42,43
onates,
radiochemical yields herein appeared high (ꢁ40%).
18
ꢀ
Hence, without separation of the F-ArBF
3
from the arylboronic
acid precursor, the labeling reaction was quenched to elevate the
pH to pH ꢁ8, and directly used for the subsequent click reaction.
3
2
The click reaction between a P-labeled, azide-bearing oligonu-
ꢀ
cleotide and the alkynyl-ArBF
3
2 was first optimized in terms of
reaction time and copper concentration (see supporting informa-
tion). The RNase A treatment of the oligonucleotide product from
the click reaction as shown in Lane 6 in Figure S6 implies the
compatibility of the relatively fragile ribonucleotide residue in
the oligonucleotide with this copper(I) catalyzed click reaction.
Scheme 1. Structures of alkyne compounds.
information as to target access and serum stability.22–24 As PET
imaging can provide high resolution images inside deep tissue,
considerable attention has recently been devoted to radiolabeling
18
Optimal conditions were then applied to F-labeling. Generally,
0
32
the 5 -azidohexyl- P-oligonucleotide along with Cu(II)-TBTA/so-
dium ascorbate was added directly to the quenched reaction fol-
2
2
18
ꢀ
oligonucleotides. Besides the radiolabeling of oligonucleotides
lowing the radiosynthesis of F-alkynylArBF
was then incubated at rt for an additional 30 min and precipitated
with 3% LiClO /acetone to remove most organic impurities. The
pellet was then dissolved in H O and loaded onto a 10% polyacryl-
3
2. The mixture
with radiometals such as 1 In and Ga, several groups have
11
25
68
26
1
8
1,16,19,27–33
radiolabeled oligonucleotides with F-fluoride.
How-
4
ever, most of these labeling procedures require elevated tempera-
tures (70–120 °C), leading to the formation of thermolytic
degradation products. Therefore, milder labeling conditions have
been developed, including one-pot-two-step methods such as cop-
per(I) catalyzed dipolar [2+3] cycloaddition reactions.4
2
amide gel for electrophoresis following which, the gel was re-
moved, covered with a plastic film and allowed to expose a
phosphorimager screen for varying periods of time. To provide
,29,34–36
31
internal electrophoretic standards, a P-labeled oligonucleotide
To examine labeling of an oligonucleotide with 18F-ArBF
3ꢀ
,
that lacked the azide functionality (Lane 5) and a ‘control reaction’
with a non-boronated alkyne analogue 3 (Lane 3) was subjected to
the click reaction as shown in Figure 2. Autoradiographic exposure
was varied over different time intervals such that short exposures
herein we initiated our study based on the copper(I) catalyzed click
reaction. To minimize the unwanted influence/toxicity of copper
3
7
on oligonucleotides, the TBTA ligand (tribenzyltristriazole amine)
was added to facilitate the click reaction.3
8,39
Hence the terminal
18
only registered F-decay whereas longer intervals or ones ac-
1
8
alkyne and azide functionalities were introduced to the arylboron-
ate and oligonucleotides respectively. Briefly, alkynylarylboronic
acid 1 (Scheme 1) was prepared via an acid deprotection of the
quired >1200 min following F-labeling, would only register sig-
3
2
nals resulting from decay of the long lived P-phosphorus.
3
2
The different decay rates between
P-phosphorus and
1
8
1
,8-diaminonaphthalene protected precursor, which was obtained
F-fluorine clearly demonstrated that the click reaction resulted
from a simple amide formation reaction from the corresponding
in a doubly isotopically labeled oligonucleotide. First, from the gel
image, no dramatic difference was observed among the various
click reactions. This was probably due to the short separation
time and slight structural difference of the newly produced oligo-
nucleotides. However, it was indeed quite obvious that the
borimidine and propargyl amine (Scheme S1).9
,11
Similarly, the
non-boronated analogue 3,6-difluorbo-N-(prop-2-yn-1-yl)benz-
amide 3, to mimic the protodeboronation counterpart of 1 or 2,
0
0
was prepared. 5 -Hydroxyl oligonucleotides (ON1: 5 -HO-
0
0
0
18
GCGTGCCrCGTCTGTT-3 and ON2: 5 -HO-GCGTGCCCGTCTGTT-3 )
were first 5 -phosphorylated with ATP- P in the presence of a
small amount of ATP-
do-(PEG) -amine using Knorre s protocol to afford the 5 -azido-
oligonucleotides (Fig. S1). The solution containing the Cu(II)-TBTA
F-related reaction mixtures (Lanes 4 and 5) showed very strong
0
31
autoradiographic intensity compared to the oligonucleotides
32
32
cꢀ
P, followed by derivatization with azi-
labeled only with P-phosphate (Lanes 1, 2, 3 and 6). Moreover,
0
40
0
32
with the passage of time, the signal of P-phosphorus on the
2
1
8
oligonucleotides predominated as that of the F-fluoride had
dissipated as seen in Lane 5. Even for Lane 4, which corresponded
to the dual isotope labeling, the radioactivity decreased dramati-
cally from A to D in Figure 2.
complex was prepared according to a prior report.4
tion of alkynylboronic acid 1 was undertaken in a volume of
.5 L in 60:40 THF:water containing alkynylarylboronic acid 1
100 nmol), HCl (6
at room temperature for 30 min, and then quenched with 5% NH4-
OH/50% aq EtOH (25 L). The quenched reaction (2 L) was further
diluted with the quenching buffer for HPLC analysis as shown in
1 18
F-fluorida-
6
(
l
18
l
mol), 19F-fluoride (500 nmol), and F-fluoride
To further verify these results, a click reaction was carried out
0
31
3
on the all-DNA oligonucleotide 5 -N - P-ON2 and radio-HPLC
l
l
was used to analyze the reaction products. From the radioactive
HPLC traces shown in Figure 3, a new radioactive peak appeared
3
2
2
1
1
00
50
00
50
00
18/19 F
F
HO
B
F
B
F
F
F
OH
F
18/19F-KHF
HCl, THF
2
O
O
HN
HN
1
2
0
5
10
15
20
25
30
t (min)
Figure 1. A radio-HPLC chromatogram of the 18F-fluoridation of alkynylarylboronic acid (1). The radiochemical yield (RCY) was 34% based on the radio-HPLC.
Li, Ying
