Photoconjugation of 3-azido-5-nitrobenzyl-[18F]fluoride to an oligonucleotide aptamer

Article abstract of DOI:10.1002/jlcr.565

Photoconjugation techniques are well established in biochemistry and molecular biology applications. We report here the first application of photoconjugation to label an oligonucleotide with positron emitting fluorine-18. ANBF, 3-azido-5-nitrobenzyl fluor

Full text of DOI:10.1002/jlcr.565

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 257–268.
DOI: 10.1002/jlcr.565
Research Article
Photoconjugation of 3-azido-5-nitrobenzyl-
[18F]fluoride to an oligonucleotide aptamer
Christopher W. Lange, Henry F. VanBrocklin and Scott E. Taylor*
Department of Functional Imaging, Lawrence Berkeley National Laboratory,
1 Cyclotron Road MS 55-121, Berkeley, CA 94720, USA
Summary
Photoconjugation techniques are well established in biochemistry and
molecular biology applications. We report here the first application of
photoconjugation to label an oligonucleotide with positron emitting
fluorine-18. ANBF, 3-azido-5-nitrobenzyl fluoride, was tagged with
fluorine-18 in 30–50% yield from the corresponding tosylate. The HPLC
purified [18F]ANBF was photochemically conjugated to a hexylamine
modified 15-base, single stranded, DNA aptamer with up to 20% radiolabeling
efficiency, based on starting [18F]ANBF. Separation by gel exclusion
chromatography and HPLC analysis of the DNA fractions indicated that
the benzyl fluoride was covalently bound to the aptamer. The total
reaction time from the start of radiosynthesis was 135 min. Based on
these results, the feasibility to photolabel oligonucleotide molecules
with fluorine-18 labeled arylazides for PET applications has been
established. Copyright # 2002 John Wiley & Sons, Ltd.
Key Words: fluorine-18; PET; photoconjugation; phenylazide; aptamer
*Correspondence to: S. E. Taylor, Lawrence Berkeley National Laboratory, University of
California, 1 Cyclotron Road, MS 55-121, Berkeley, CA 94720, USA.
E-mail: setaylor@lbl.gov
Contract/grant sponsor: US Department of Energy; Contract/grant number: DE-AC03-76SF00098
Copyright # 2002 John Wiley & Sons, Ltd.
Received 21 August 2000
Revised 21 December 2000
Accepted 28 November 2001

258
C. W. LANGE ET AL.
Introduction
The application of DNA and RNA oligonucleotides as molecular
therapeutic agents is maturing rapidly. Likewise, the utilization of
radiolabeled DNA, RNA or oligonucleotide probes for diagnostic
imaging or evaluation of new therapeutic agents is an area of
current intense interest. Oligonucleotides labeled with beta-
emitting radioisotopes such as tritium, phosphorous-32 and sulfur-35
are well-established tools in cell and molecular biology. Methods
for single photon (technetium-99m, indium-111, iodine-125) labeling
of oligonucleotides were developed in the 1990s.1–4 These methods
are largely based on the labeling strategies designed for proteins
and peptides.5 More recently, fluorine-18 labeled DNA/RNA
based molecules for quantitative imaging using positron emission
tomography (PET) have been reported.6–10 Most attractive for labeling
with short-lived positron emitters are the small (15–50 mer) single-
stranded DNA/RNA oligonucleotides, called aptamers, that are
engineered with high target specificity and to possess favorable
pharmacokinetic profiles.11–13
One reported strategy for fluorine-18 labeling of oligonucleotides
is based on the two-step synthesis of N-(4-[18F]fluorobenzyl)-2-
bromoacetamide followed by S-conjugation to a 30-phosphorotioate
oligonucleotide.9 This procedure, like many of the fluorine-18
labeling schemes for tagging proteins or peptides, is labor-
intensive and time-consuming. We report here on our progress
towards a two-step oligonucleotide labeling method based on
photochemical conjugation of a fluorine-18 labeled molecule to
a 50-amino modified 15-mer. This approach is modeled after
photoaffinity labeling reagents14 that have found great utility
in biochemistry and molecular biology applications such as
tagging proteins, enzymes and receptors with fluorescent dyes or
radioiodine. Initial studies towards photoiodination of monoclonal
antibodies were described by Pandey et al.15 Similarly, two photo-
conjugation reactions for fluorine-18 tagging of proteins using
4-azidophenacyl-[18F]fluoride16 and [18F]-3,5-difluorophenyl azide17
have been reported. These bioconjugates are compared to the new
azido compound described herein.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 257–268

[
18F]OLIGONUCLEOTIDE APTAMER
259
Results and discussion
Preparation of 3-azido-5-nitro-benzylfluoride (ANBF) and [18F]ANBF
The synthetic scheme for the production of the fluorine containing
arylazide photolabeling reagent is shown in Figure 1. The nitro analog
was chosen to facilitate photoconjugation at higher wavelengths to
reduce potential UV damage to the oligonucleotide. Partial reduction of
the dinitrobenzyl alcohol (1) was achieved with ammonium sulfide
following a literature procedure18 yielding the aniline (2). Diazotization
of (2) followed by displacement with azide gave (3) in modest yield.
Direct fluorination with (diethylamino)sulfur trifluoride gave the non-
radioactive 3-azido-5-nitro-benzylfluoride (4). The precursor for radio-
fluorination (5) was prepared by tosylation of the benzyl alcohol (3).
The one-step reaction to produce the fluorine-18 labeled ANBF is
also shown in Figure 1. Tetrabutylammonium [18F]fluoride was
prepared from the cyclotron-produced aqueous [18F]fluoride and was
resolubilized in anhydrous acetonitrile. The acetonitrile solution was
added to the tosylate (5) and the mixture was heated at 808C for 5–10
min. Following reversed-phase semi-preparative HPLC purification,
[18F]ANBF (6) was obtained in 30–50% decay corrected yield 40–50 min
after the start of the synthesis. HPLC analysis confirmed that
[18F]ANBF co-eluted with unlabeled ANBF (4).
The [18F]ANBF yields were superior to the 0–12% production of
fluoroarylazides by 18F-for-19F exchange described by Hashizume
et al.17 and modest compared to the production of 4-azidophenacyl-
[18F]fluoride by Wester et al.16 in 71% radiochemical yield 15 min into
CH2OH
CH2OH
CH2OH
1) NaNO2, H+
2) NaN3
(NH4)2S
CH3OH
O2N
NO2
O2N
NH2
O2N
N3
3
1
2
TsCl, py
CH2Cl2
DAST
CH2Cl2
CH218F
CH2OTs
CH2F
nBu4N+ 18F-
CH3CN
O2N
N3
O2N
N3
O2N
N3
6
5
4
Figure 1. Synthesis of [19F/18F] 3-amino-5-nitro-benzylfluoride (ANBF)
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260
C. W. LANGE ET AL.
the synthesis. The choice of either the tosylate leaving group or the
position of the label may have contributed to our modest radio-
fluorination yields.
Photoconjugation of 3-azido-5-nitro-benzyl[18F]fluoride ([18F]ANBF)
with an amino modified oligonucleotide
Commercially synthesized thrombin aptamer modified at the 50-
terminus with a hexylamine provided a primary amine target for
interaction with the photolabeling reagent as shown in Figure 2. Based
on the previous accounts, heptoazocyclotetraene.16,19,20 of arylnitrene
chemistry the proposed structures for the labeled oligonucleotide (1) is
shown in Figure 2. Photolysis of the arylnitrene promotes ring
expansion to the heptoazocyclotetraene. The hexylamine acts as a
nucleophile and reacts with the intermediate to form a covalent bond.
This is analogous to the case in protein labeling where the terminal
amine of lysine forms a covalent bond with the heptoazocyclotetraene
CH218F
O2N
CH218F
O2N
CH218F
N
N
ν
h
O2N
N3
CH218F
O2N
6
O2N
CH218F
N
N
GGTTGGTGTGGTTGG
H2N
O2N
CH218F
CH218F
O2N
N
Oligo-(H2C)6-HN
N
NH-(CH2)6-Oligo
7
Figure 2. Synthesis of the radiofluorinated aptamer
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[
18F]OLIGONUCLEOTIDE APTAMER
261
intermediate. Given the substitution pattern on the phenyl ring it is
possible to form the two species as shown in Figure 2. No attempt was
made to determine if one or both of the positional isomers constitute the
final product.
Purified [18F]ANBF was extracted from the HPLC solvent using a
C18 SepPak1 and eluted in methanol. The methanol was evaporated
and the aptamer, in a buffer solution, was added to the [18F]ANBF
residue. The photolysis was carried out at 08C for 10 min using a 275 W
mercury lamp. Purification and analysis of the labeled aptamer was
performed using gel exclusion chromatography and reversed-phase
HPLC. The specific activities of the intermediate [18F]ANBF and the
labeled aptamer were not measured.
The labeled aptamer and free [18F]ANBF were easily separated by gel
exclusion chromatography. An eluent profile is shown in Figure 3. The
labeled aptamer eluted shortly after the blue dextran void volume
marker band at about 40% of the column volume with the radioactivity
and DNA absorbance peaks coinciding. Free ANBF was retained by
the column and eluted at 80–90% of the column volume. The
photolabeling efficiency ranged from 7 to 20% (based on starting
ANBF), increasing linearly (R2=0.949) with increasing DNA concen-
tration (see Figure 4). These yields compare favorably with the 15–30%
protein photolabeling yields reported by Wester et al.16 and exceeds the
7% yield achieved by Hashizume et al.17 Wester found that the labeling
efficiency increased as a function of the number of lysines and terminal
amino groups in the protein being labeled. While the radiochemical
15
10
5
2.5
2
uCi 18-F
Abs 260
1.5
1
0.5
0
0
0
4
6
8
10
2
Fraction Number
Figure 3. Gel exclusion chromatogram demonstrating separation of the labeled
aptamer and free [18F]ANBF
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262
C. W. LANGE ET AL.
25
20
15
10
5
0
0
5
10
15
20
25
30
35
Aptamer Concentration (ug/uL)
Figure 4. Radiochemical yield, based on the starting [18F]ANBF, of the labeled
aptamer as a function of aptamer concentration
yield plateau was never reached in our study (Figure 4), we achieved a
comparable yield with far less amino density. Further enhancement of
the photolabeling efficiency might be possible at higher oligonucleotide
concentrations.
The labeled aptamer was analyzed by radio-HPLC. A gradient
solvent system was used to elute the aptamer from the C18 column. The
radioactive peak co-eluted with the UV active oligonucleotide peak. The
retention time for free [18F]ANBF was nearly 30 min longer than the
labeled aptamer. The results from the size exclusion chromatography
and the analytical HPLC indicate that the benzyl fluoride was
covalently bound to the aptamer. Additionally, there was no evidence
on the HPLC of aptamer breakdown as a result of the photochemical
reaction.
The specificity of the ANBF for the amine on the 50-terminus was
indicated by the inability of the ANBF to bind to a oligonucleotide with
the same sequence but constructed without the 50-hexyl amine
modification. The covalent nature of the bond was tested with
[18F]ANBF added in excess to the reaction, and then treated with high
levels of lysine (3 M) after completion of the photoconjugation reaction
in an attempt to displace any charged reaction products off the poly-
anionic backbone of the oligonucleotide molecule. An HPLC compar-
ison of the ANBF–aptamer complex in the presence and absence of
lysine showed that there was no change in either the retention or
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[
18F]OLIGONUCLEOTIDE APTAMER
263
spectral characteristics of the ANBF–aptamer product, strongly
suggesting that the association between the aptamer and labeling agent
was not an ionic interaction.
Experimental
Materials and methods
All chemicals and solvents were obtained from Aldrich Chemical Co.
(Milwaukee, WI), with the exception of HPLC grade CH3CN and
MeOH from J.T. Baker (Phillipsburg, NJ) and were used without
further purification. Flash chromatography was performed using silica
(
gel (60 A, 230–400 mesh) from Aldrich. Melting points were taken on a
Mel-TempTM apparatus and are reported uncorrected. Proton magnetic
resonance spectra were obtained on a Bruker AMX 300 instrument at
300 MHz; chemical shifts are referenced to tetramethylsilane. Purifica-
tion of the radiolabeled compounds was carried out by HPLC (column
and conditions noted below) with a Waters 560 pump and an in-line
LinearTM UV-106 spectrophotometer (254 nm) to detect mass and a NaI
detector and Ortec NIM components to measure radioactivity, or on a
Thermo Separation Products 4000 HPLC system equipped with a photo
diode array (PDA) detector and a column oven.
3-Amino-5-nitrobenzyl alcohol 2 was prepared by reduction of 3,5-
dinitrobenzyl alcohol with ammonium sulfide according to the
procedure of Meindl et al.18
3-Azido-5-nitrobenzyl alcohol (3)
The following reaction was carried out with stirring under reduced light.
In a 100 ml flask, 3-amino-5-nitrobenzyl alcohol (0.97 g, 5.8 mmol) was
added to 12 ml 12 N hydrochloric acid to form a slurry. The mixture was
chilled in an ethylene glycol/dry ice bath (ꢀ158C). Acetic acid (10 ml)
was added, followed by careful addition of sodium nitrite (0.64 g,
9.3 mmol) dissolved in 3 ml of water. The temperature was not allowed
to rise above 108C. The mixture was stirred for 30 min. Sodium azide
(0.65 g, 10 mmol) was dissolved in 3 ml water and added dropwise to the
reaction mixture. The mixture was gradually allowed to warm to room
temperature following the addition of 15 ml of water. A light tan
precipitate was collected by filtration and washed with water. After
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C. W. LANGE ET AL.
drying in vacuo, 0.71 g (64%) of tan powder was recovered: mp 1048C.
1H NMR (CD3OD): d 4.70 (s, 2H, CH2OH), 7.44, 7.74, 8.00 (m, 3H,
aromatic) HRMS(EI): C7H6N4O3 calcd: 194.043990 found: 194.044419.
3-Azido-5-nitro-benzylfluoride (4)
Alcohol 3 (0.26 g, 1.3 mmol) was dissolved in 12 ml dichloromethane.
An excess of (diethylamino)sulfur trifluoride (DAST) (0.72 g, 4.5 mmol)
was dissolved in 4 ml dichloromethane and cooled in a dry ice/ethylene
glycol bath (ꢀ158C). The alcohol solution was added to the cooled
DAST solution over the course of 10 min. The mixture was allowed to
warm to room temperature and stirred for an additional 30 min. The
reaction mixture was poured over 3 g of ice. The organic layer was
separated. The aqueous layer was extracted with 2 ꢁ 5 ml portions of
dichloromethane. The combined organic layers were washed with 1 ml
water and dried over magnesium sulfate. After filtration the solvent was
removed in vacuo. The crystalline residue, recrystallized from dichlor-
omethane/hexane, gave 110 mg (52%) of light brown crystals: mp
1
66–678 C. H NMR (CDCl3): d 5.41 (d, 2H, CH2F, J=47 Hz), 7.27,
7.80, 7.91 (m, 3H, aromatic) HRMS(EI): C7H5FN4O2 calcd: 196.039654
found: 196.040194.
3-Azido-5-nitrobenzyl tosylate (5)
Alcohol 3 (0.25 g, 1.3 mmol) and pyridine (0.60 ml, 7.4 mmol) were
mixed in 10 ml dichloromethane and chilled in an ice bath.
p-Toluenesulfonyl chloride (1.11 g, 5.8 mmol) was dissolved in 10 ml
dichloromethane and added to the stirring alcohol solution via a
syringe. The reaction mixture was placed in the freezer and allowed to
stand for 2 days. The mixture was quenched by adding to 100 ml of
water and extracted with dichloromethane. After removal of solvent by
rotary evaporation, the oily residue was purified by flash chromato-
graphy (20% ethyl acetate/hexane). Removal of solvent gave 0.26 g
(58%) of a viscous oil which crystallized on standing. A portion of this
material was additionally purified by recrystallization (ethyl acetate/
1
hexane) to give a light yellow crystalline solid: mp 73–748C. H NMR
(CDCl3): d 2.46 (s, 3H, Ar-CH3), 5.11 (s, 2H, CH2OTs), 7.22 (m, 1H,
aromatic), 7.35–7.38, (m, 2H, aromatic), 7.79–7.82 (m, 4H, aromatic).
HRMS(FAB/EI): C14H12N4O5S calcd: 348.052842 found: 348.052949.
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[
18F]OLIGONUCLEOTIDE APTAMER
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Production of [18F]-fluoride
[18F]fluoride ion was produced via the 18O(p,n)18F reaction by
irradiation of 18O-enriched water with a 11 MeV proton beam using
the Center for Functional Imaging CTI RDS 111 cyclotron.
3-Azido-5-nitrobenzyl-[18F]-fluoride (6)
The aqueous [18F]-fluoride was added to a vacutainer containing 2 ml of
tetrabutylammonium hydroxide (1 M in water). The water was
azeotropically removed with acetonitrile under a gentle stream of
nitrogen. The residue was dissolved in acetonitrile (2 ꢁ 150 ml) and
transferred to a vial containing the tosylate precursor (5) (1 mg,
2.9 mmol). The mixture was heated to 808C for 5–10 min. Water (300 ml)
was added and the entire solution was injected onto a reversed-phase
HPLC column (Phenomenex Bondclone-10 C18 column, 3.9 ꢁ 300 mm,
mobile phase: 50% CH3CN/H2O, flow rate: 1 ml minꢀ1) for purifica-
tion. [18F]ANBF (6) was separated (Rt=15 min) from unreacted
tosylate (5) (Rt=32 min). The [18F]ANBF fraction was passed through
an activated C18 SepPak1 and the [18F]ANBF was eluted with
methanol (1 ml). The methanol was then evaporated leaving the
[18F]ANBF residue. Purified [18F]ANBF was obtained in 40–50 min
with decay corrected yields ranging from 30 to 50% from the starting
[18F]fluoride ion.
Photoconjugation of [18F]ANBF to the aptamer (7)
All aqueous reaction solutions and buffers used with the aptamer were
prepared with DNAse-free water (Sigma). The thrombin aptamer was
commercially prepared (Genset Corp) as the single-stranded native
deoxyribonucleotide oligomer of the sequence GGTTGGTGT-
GGTTGG.13 The 50-terminus was modified by the attachment of a
hexylamine linker. Immediately prior to use the aptamer was diluted to
the desired final concentration using water and the appropriate amount
of potassium phosphate buffer to obtain a final buffer concentration of
10 mM (pH 7.4). The aptamer in 0.11 ml of buffer was added to the vial
containing the dried [18F]ANBF (6) and mixed thoroughly. The
photolysis was conducted at 08C with a 275 W mercury lamp (General
Electric Model RSM 275; emission max=297 nm) for 10 min. The
reaction vial was kept 10 cm from the lamp surface, and the reaction was
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C. W. LANGE ET AL.
mixed with a magnetic stirrer. The aptamer was purified by gel exclusion
chromatography. The reaction mixture was loaded directly onto a
Exocellulose GF-5 column (Pierce; MW exclusion limit= 5000) and was
eluted with water. A couple of drops of a blue dextran solution
(Pharmacia) was added to visualize the void volume. Fractions (0.5 ml)
were collected and the absorbance was measured at 260 nm on a Perkin
Elmer Lambda 3 UV/VIS spectrophotometer and 18F activity was
determined using a Capintec CRC30 dose calibrator. Purified labeled
aptamer was obtained in 1–7% decay corrected yield (based on starting
[18F]fluoride ion), 135–150 min from the start of synthesis.
The specific binding of the ANBF to the hexylamine bridging group
was examined by repeating the above reactions with an aptamer that
without the amine group attached to the terminus.
The specificity of ANBF for the amine moiety on the 50-terminus was
further examined using non-radioactive ANBF. The reaction was
carried out under the same conditions as with the radiolabeled ANBF,
except a 10-fold excess of the ANBF was added to the reaction.
Following photoconjugation, the reaction was split into two aliquots,
and lysine was added to one of the aliquots to a final concentration of
3 M. HPLC was then carried out to determine if the conjugated ANBF
remained with the aptamer or was displaced by the lysine.
HPLC Conditions
HPLC analysis of the labeled aptamer was carried out using two
different methods. In the first, a Nova-Pak C-18 3.9 ꢁ 300 mm column
(Waters) was used. A linear gradient of 10% CH3CN/0.1 M TEA to
50% CH3CN/0.1 M TEA was used at a rate of 1 ml minꢀ1. The eluent
was analyzed by UV absorbance and radioactivity detection. The
retention time of the labeled aptamer was 10–11 min, while ANBF was
retained for 43 min by this system. The second system utilized a
4 ꢁ 250 mm PA100 DNAPac column (Dionex). A linear gradient of
25 mM Tris (pH 8.0)/200 mM NH4Cl/1% acetonitrile to 25 mM Tris
(pH 8.0)/800 mM NH4Cl/1% acetonitrile was used at 1.5 ml minꢀ1. The
column was maintained at 368C. Eluent was monitored with a PDA
detector over a 210–600 nm wavelength range. The aptamer was
retained for 10 min, the ANBF–aptamer conjugate for 7.5 min, and
free ANBF (both prior and after exposure to UV light) eluted with the
solvent front.
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[
18F]OLIGONUCLEOTIDE APTAMER
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Conclusion
We have prepared a positron labeled arylazide [18F]ANBF and
demonstrated the ability to couple the labeled intermediate with an
amine modified DNA aptamer. The best overall non-decay corrected
yield for the entire reaction sequence was ꢂ3% at 135 min. While
improvement in either the fluorination and/or photolabeling yields are
necessary for this labeling strategy to find widespread applicability, the
feasibility of photolabeling oligonucleotide molecules with a fluorine-18
labeled arylazide has been established.
Acknowledgements
This work was supported by the Director, Office of Science, Office of
Biological and Environmental Research, Medical Science Division of
the US Department of Energy under contract no. DE-AC03-
76SF00098.
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