New and convenient synthesis of a tritiated photoactivatable nicotinic agonist: [3H]-AC5

Article abstract of DOI:10.1002/jlcr.614

A new synthesis of a tritiated photosensitive nicotinic agonist ([³H]-AC5), which uses [³H2] as the source of radioactivity, is described. Several improvements have been introduced to establish a reliable five-step synthesis displayi

Full text of DOI:10.1002/jlcr.614

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 943–953.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.614
Research Article
New and convenient synthesis of a tritiated
photoactivatable nicotinic agonist: [3H]–AC5
A. Mourot1, F. Kotzyba-Hibert1,*, E. Doris2 and M. Goeldner1
1
!
Laboratoire de Chimie Bioorganique, UMR 7514 CNRS, Faculte de
!
Pharmacie, Universite Louis Pasteur Strasbourg, BP 24, 67401 Illkirch, Cedex,
France
2
!
Service de Marquage Moleculaire et de Chimie Bioorganique, CEA/Saclay,
91191 Gif sur Yvette, Cedex, France
Summary
A new synthesis of a tritiated photosensitive nicotinic agonist (½3Hꢀ2AC5),
which uses [3H2] as the source of radioactivity, is described. Several
improvements have been introduced to establish a reliable five-step synthesis
displaying satisfactory overall yields (45%) and requiring a single purification
(HPLC) in the final step. This photosensitive radiolabelled probe will enable us
to undertake a time-resolved photolabelling study on the nicotinic acetylcho-
line receptor in order to analyze at the molecular level the allosteric transitions
occurring after agonist stimulation. Copyright # 2002 John Wiley & Sons, Ltd.
Key Words: dynamic photoaffinity labelling; aryldiazonium salts; nicotinic
acetylcholine receptor; radiolabelled synthesis
Introduction
nAChRs are pentameric allosteric transmembrane proteins that belong
to the superfamily of the ligand-gated ion channels1 (LGIC). Exposure
*Correspondence to: F. Kotzyba-Hibert, Laboratoire de Chimie Bioorganique, UMR 7514 CNRS,
!
Faculte de Pharmacie, Universite Louis Pasteur Strasbourg, BP 24, 67401 Illkirch, Cedex, France.
E-mail kotzyba@bioorga.u-strasbg.fr
!
!
Contract/grant sponsor: Ministere de l’Enseignement Superieur, Naturalia
Contract/grant sponsor: CNRS
Copyright # 2002 John Wiley & Sons, Ltd.
Received 16 April 2002
Accepted 13 May 2002

944
A. MOUROT ET AL.
Figure 1. Structure of photoactivatable agonist ½3Hꢀ-AC5.
to agonists such as acetylcholine lead to at least three transitory states: a
rapid (ms) opening of the channel, causing the depolarization of the
membrane, followed by subsequent fast (ms–s) and slow (s–min)
processes of desensitization.2 This molecular phenomenon plays a key
role in the regulation of the synaptic transmission.
These conformational transitions can be probed at the molecular level
by using photoactivatable radiolabelled cholinergic ligands. The
photolabelling experiments require the combination of rapid mixing
procedures to flash photolysis. The experiments are analyzed at different
mixing times, by quantification of the radioactivity incorporation into
the receptor subunits and subsequently into the labelled amino acids
after microsequencing (for review, see ref.3). Recently, the photosensi-
tive partial agonist [3H]-DCTA4 was used in our laboratory to probe the
conformational reorganization occurring within the ACh binding site
after rapid mixing with the Torpedo receptor. It allowed to analyze, in
particular, the slow desensitization process.5
(
The recent description of the crystal structure at 2.7 A resolution of a
water-soluble protein (AChBP) that has significant structural homology
with the extracellular part of the nAChR,6 prompted us to reinvestigate
the time-resolved photolabelling experiments using a formerly described
photosensitive nicotinic agonist AC5.7 This molecule possesses out-
standing binding and photochemical properties. Due to its flexible
(
chemical structure and length (14 A in the extended conformation,
Figure 1) and referring to AChBP structure,6 it should permit the
mapping of inter-subunit areas in the neighbourhood of the ACh
binding site, during these conformational transitions.8 Indeed, AC5
diazonium moiety is probably located in a region of nAChR where
significant topological changes occur during state transitions, as shown
by fluorescence experiments with Dns-C6-Choline.9 The described
Copyright # 2002 John Wiley & Sons, Ltd.
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NEW AND CONVENIENT SYNTHESIS
945
synthesis of the tritiated AC5 molecule7 used [3H]–CH3I as the
radioactive source which allowed a low yield synthesis of the
½3Hꢀ-AC5 molecule with high specific radioactivity. The actual non-
availability of the tritiated methyl iodide precursor compelled us to
define a new synthetic strategy to proceed with this work.
The present article describes a five-step synthesis of ½3Hꢀ-AC5 using
[3H2] as the radiolabel source. This synthesis is fully reproducible with
high overall radiochemical yields (about 45%) and requires only a single
HPLC purification in the final step.
Results and discussion
Given the large amount of nAChR in the electropaque of Torpedo
marmorata and the radiolytic sensitivity of chemical and biochemical
materials with high specific radioactivity, we preferred not to exceed
1 Ci/mmol for this photoprobe. This specific radioactivity was sufficient
to obtain good labelling patterns to identify the labelled amino acids.
Accordingly, we synthesized the intermediate ½3Hꢀ-2 using a classical
reductive amination10 of the phenylene diamine derivative 111
(Scheme 1) to incorporate one tritium atom per molecule. This radio-
active precursor was stored in MeOH at ꢁ208C and checked by HPLC
for its purity before use. The N-methyl derivatives 2 and ½3Hꢀ-3 were
quantitatively transformed to the N-chlorocarbamoyl derivative ½3Hꢀ-2
by treatment with phosgen12 and this last was used directly without
further purification (Scheme 2).
In our search for an efficient synthetic pathway, we transformed the
brominated spacer 4 to its iodinated derivative 7 after subsequent
protection and deprotection of the amine moiety (Scheme 3). This
iodinated spacer 7 could be directly and efficiently coupled to the ½3Hꢀ-3
Scheme 1.
Copyright # 2002 John Wiley & Sons, Ltd.
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A. MOUROT ET AL.
Scheme 2.
precursor forming compound ½3Hꢀ-8. The iodo-substituent was quanti-
tatively converted by treatment with N(Me)3 in anhydrous toluene to its
quaternary ammonium salt ½3Hꢀ-9 and isolated as a precipitate in the
reaction mixture. This precursor ½3Hꢀ-9 was taken up in water and
lyophilized. The replacement of the bromo-substituent in 4 by the iodo
derivative 7 allowed, therefore, interesting synthetic improvements: first,
the coupling yields to form ½3Hꢀ-8 were enhanced (98% instead of 50%
maximum with the bromo compound); second, due to the precipitation
of the iodide ammonium salt, the formation of ½3Hꢀ-9 occurred with
quantitative yields and allowed a convenient handling of the reaction
product. This improved synthesis of ½3Hꢀ-9 enabled us to use the crude
reaction product for the final diazotization step without intermediate
HPLC purification, preventing the degradation of moist-sensitive
intermediates (½3Hꢀ-3, 7 and ½3Hꢀ-8).
Diazotization must be operated in the dark, and gave ½3Hꢀ-AC5 as the
main product (60% yield, retention time 15 min with gradient I, Table 1)
in the presence of a secondary compound that was probably the
hydrolytic product of the ester (retention time 14 min with gradient I).
To optimize this purification step, we used new eluting conditions
(gradient II).
Characterization of ½3Hꢀ-AC5 (Figure 2) was achieved by HPLC and
UV analyses, and the product was stored as aliquots (50 ml) in aqueous
solution (385 mM, 0.83 Ci/mmol) in the dark at ꢁ808C.
Experimental section
General
Retention times on HPLC for the successive intermediate and the final
1
compound are listed in Table 1. H-NMR and 13C-NMR spectra of
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NEW AND CONVENIENT SYNTHESIS
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A. MOUROT ET AL.
Table 1. Retention time (Rt) on reversed phase 10 m-Hyperbond C18 for
intermediates and final compounds in the synthesis of ½3Hꢀ-AC5 and of the non-
labelled AC5. Compounds were analyzed under the following conditions: Gradient
I=0–60% B in 40 min; 60–100% B in 5 min. Gradient II=Isocratic 5% B.
A=H2O/TFA 0.1%; B=Acetonitrile
Rt (min)
Compounds
Gradient I
Gradient II
2
3
21
35
18
7 and ½3Hꢀ7
8 and ½3Hꢀ8
39
29
15
9 and ½3Hꢀ9
AC5 and ½3HꢀAC5
21
unlabelled compounds were recorded in CDCl3, CD3OD or D2O at 200
and 300 MHz, on Bruker VPC 200 and 300 instruments, d are given in
ppm. Mass spectra analysis of unlabelled compounds were performed
on a Mariner ESI-Tof instrument from Applied Bio-System/Perkin-
Elmer.
Analyses and purifications of both labelled and non-labelled
compounds were made on a reversed phase 10 m-Hyperbond C18
Bondex HPLC (Table 1) under the following gradient conditions: (I)
100% A to 60% B in 40 min, 60% B to 100% B in 5 min; (II) isocratic
5% B (A ¼ H2O/TFA 0.1%, B=acetonitrile).
A Packard Tri-carb 2100 TR liquid scintillation counter was used for
quantification of radioactivity. Typically, 10 ml of sample were added to
1.5 ml of Packard emulsifier-safe and the solution was counted for
2 min. UV-visible spectra were recorded on a Biotek Uvikon XL.
Toluene was distilled on sodium, CH2Cl2 was distilled on calcium
hydride and acetone was distilled on calcium sulfate.
Analytical HPLC injections of the crude products ½3Hꢀ-8, ½3Hꢀ-9 and
½3Hꢀ-AC5 allowed us to characterize (UV spectra) and quantify
(radioactivity counting) each product.
Syntheses
A general synthesis was developed for the unlabelled compound (AC5)
to assess the synthetic methodology as well as the HPLC purification
procedure.
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NEW AND CONVENIENT SYNTHESIS
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10
20
Time (min)
30
40
35000
30000
25000
20000
15000
10000
5000
0
0.9
0.7
0.5
0.3
0.1
200
300
λ (nm)
400
500
1
5
10
15
20
(a)
Fraction number
0.5
0.45
0.4
DO
362 nm
0.4
0.3
0.2
0.1
0.35
0.3
0
200
Time (s)
400
600
0.25
0.2
0.15
0.1
0.05
0
200
250
300
350
400
450
500
(b)
λ (nm)
Figure 2. (a) reversed-phase HPLC analysis and purification of ½3Hꢀ-AC5. Upper
part: 100 ml of ½3Hꢀ-AC5 (38 mM in water) were injected on the C18 column and
eluted with gradient I (see Table 1). UV detection was monitored at 229 nm; lower
part: fractions were collected and counted (10 ml aliquots) for radioactivity (UV
spectral characteristics of fraction 8 are shown here). (b) Spectral changes
associated with the photolysis of probe AC5 (15 mM in water, 108C) irradiated at
364.6 nm (incident light energy: 70 mV). The initial absorbance at 362 nm
decreases with a concomitant increase of new peaks (isobestic points (arrows) at
238, 270 and 277 nm). A half-time of 28 s was calculated according to a pseudo-
first-order equation: Dot=Do0eð-kapp:x); t1=2 ¼ ln 2=kapp. AC5 is stable in the
dark in water at 108C (t1=2 > 48 h, data not shown)
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A. MOUROT ET AL.
The syntheses of N-terbutoxycarbonyl-para-phenylenediamine 111,
N-methyl-N0-terbutoxycarbonyl-para-phenylenediamine 213, N-([3H]-
methyl)-N0-terbutoxycarbonyl-para-phenylenediamine ½3Hꢀ-210, N-
([3H]-methyl)-N-chloroformyl-N0-terbutoxy carbonyl-para-phenylene-
diamine ½3Hꢀ-312 and 6-amino-[(2-bromo)-ethyl)]-hexanoate chlorhy-
drate 47 were previously reported.
For ½3Hꢀ-3 synthesis, ½3Hꢀ-2 (2.15 mmol, 14 mCi, 6.5 Ci/mmol) was
isotopically diluted with 2 (12.9 mmol) to give a mixture ½3Hꢀ-2/2 whose
specific activity corresponds theorically to 0.93 Ci/mmol.
N-terbutoxycarbonyl-6-amino-[(2-bromo)-ethyl)]-hexanoate 5
%
To a solution of 4 (2 g, 7.3 mmol) in 20 ml ethanol were added 2 ml
(8.7 mmol, 1.2 equivalents) of di-tert-butyl-dicarbonate and 1.12 ml
(8 mmol, 1 eq) of Et3N. The reaction was stirred at room temperature
for 5 h. After evaporation of the solvent, the residue was taken up in
150 ml CH2Cl2 and the organic layer was washed with water and dried
(Na2SO4). The resulting oil was purified by chromatography on silica
gel (pentane/ether: 8/2) to afford 2.2 g (6.6 mmol, 90% yield) of yellow
oil.
1H-NMR (CDCl3):d ¼ 1:2621:70 (m, 15H, 3CH2, 3CH3); 2.27–2.35
(t, 2H, J ¼ 7:3 Hz, CH2); 3.01–3.12 (q, 2H, J ¼ 6:6 Hz, CH2); 3.43–3.50
(t, 2H, J ¼ 6:1 Hz, CH2); 4.30–4.37 (t, 2H, J ¼ 6:1 Hz, CH2); 4.60 (br s,
1H, NH).
N-terbutoxycarbonyl-6-amino-[(2-iodo)-ethyl)]-hexanoate 6
%
To a solution of 5 (1.43 g, 4.2 mmol) in 20 ml of freshly distilled acetone
was added 5.2 g (34.6 mmol, 8.2 eq) of anhydrous NaI and the mixture
was heated under reflux for 45 h. After evaporation of the solvent, the
residue was taken up in 150 ml CH2Cl2 and washed with 150 ml H2O.
The organic layer was dried with anhydrous Na2SO4. After evaporation
of the solvent, the residue obtained was chromatographed on silica gel
(heptane/ether: 7/3) to afford 1.51 g (3.9 mmol, 93% yield) of yellow oil.
1H-NMR (CDCl3):d ¼ 1:3221:70 (m, 15H, 3CH2, 3CH3); 2.31–2.36
(t, 2H, J ¼ 7:5 Hz, CH2); 3.07–3.14 (q, 2H, J ¼ 6:2 Hz, CH2); 3.26–3.31
(t, 2H, J ¼ 6:6 Hz, CH2); 4.29–4.35 (t, 2H, J ¼ 6:9 Hz, CH2); 4.55 (br s,
1H, NH).
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NEW AND CONVENIENT SYNTHESIS
951
6-amino-[(2-iodo)-ethyl)]-hexanoate 7
%
1.51 g (3.9 mmol) of 6 was dissolved in 10 ml of freshly distilled CH2Cl2.
10 ml of anhydrous TFA were added drop-wise at 08C to this solution.
The reaction was stirred for 30 min at 08C and then warmed up to 208C
for 45 min. The mixture was cooled to ꢁ788C and lyophilized to avoid 7
from exposure to heat. The yellow oil was taken up twice in 10 ml of
CHCl3 and evaporated under vacuum to eliminate residual TFA. The
residue was taken up in MeOH and recrystallized in isopropanol/hexane
to afford 1.45 g of compound 7 (3.63 mmol, 93% yield) as a yellow-
white solid.
1H-NMR (CDCl3):d ¼ 1:3021:40 (m, 2H, CH2); 1.54–1.65 (m, 4H,
2CH2); 2.25–2.32 (t, 2H, J ¼ 7:2 Hz, CH2); 2.84–2.91 (t, 2H, J ¼ 7:1 Hz,
CH2); 3.19–3.26 (t, 2H, J ¼ 6:7 Hz, CH2); 4.29–4.35 (t, 2H, J ¼ 6:7 Hz,
CH2); 7.8 (br s, 2H, NH2).
13C-NMR (CDCl3):d ¼ 1:48, 24.6, 26.2, 31.2, 34.1, 40.4, 64.9 and
173.7.
MS (ESI, positive ion 130 eV): calculated for (C8H17NO2I)+:286.13;
found: 286.03.
(2-iodo)-ethyl-6-N-[N0-([3H]-methyl)-para-amino-terbutoxy-
carbonyl-phenylurea]-hexanoate ½3Hꢀ-8
4 mg (14 mmol, 13.3 mmol, 13.3 mCi) of ½3Hꢀ-3 and 7.2 mg (18 mmol, 1.3
equivalent) of 7 were dissolved in 300 ml of freshly distilled CH2Cl2 and
5 ml (36.1 mmol, 2.6 equivalents) of Et3N were added. The reaction was
stirred for 7 h at room temperature and monitored by HPLC (gradient
I) until completion. 1 ml of CH2Cl2 was then added and the mixture was
extracted with 1 ml H2O. The aqueous layer was extracted again with
1 ml CH2Cl2 and the organic layers were pooled, dried over Na2SO4 and
evaporated. The residue was taken up in 2 ml of anhydrous CH2Cl2 and
analyzed with HPLC gradient I: the mixture contained 10.4 mCi of
½3Hꢀ-8 (80% radiochemical yield).
(2-trimethylammonium)-ethyl-6-N-N0-([3H]-methyl)-para-amino-terbu-
toxycarbonyl-phenylurea]-hexanoate iodide ½3Hꢀ-9
6 mg (11.2 mmol, 10.4 mCi) of ½3Hꢀ-8 were dissolved in 300 ml of
anhydrous toluene saturated with Me3N and the reaction was stirred
at room temperature for 24 h. The solvent was removed under vacuum
and the residue was taken up in the 300 ml of toluene freshly saturated
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952
A. MOUROT ET AL.
with Me3N and stirred for 70 h. The washing procedure was repeated
twice and the solvent was then removed under reduced pressure. The
residue was treated with 2 ml of toluene and extracted with 2 ml of
water. The aqueous layer contained 10.2 mCi of ½3Hꢀ-9 (98% radio-
chemical yield). The crude product was then lyophilized and taken up in
4 ml of MeOH (2.55 mCi/ml, 3 mM) and stored at ꢁ208C.
(2-trimethylammonium)-ethyl-6-N-[N0-([3H]-methyl)-para-diazonium-
phenylurea]-hexanoate trifluoroacetate ½3Hꢀ-AC5
450 ml (1.3 mmol, 1.1 mCi) of the methanolic ½3Hꢀ-9 solution was dried
under vacuum and cooled to 08C before dropwise addition of 300 ml of
cold TFA. Stirring the mixture for 30 min at 158C allowed full
deprotection of the amino group. The mixture was cooled to ꢁ108C
and handled in the dark. NaNO2 (1.2 eq) in aqueous solution (8.3 mg/
ml) was added portionwise over a 30 min period. Completion of the
diazotization was monitored by UV spectroscopy. The mixture was then
lyophilized twice with 1 ml H2O to remove excess of TFA before HPLC
purification using gradient II. Purification step gave ½3Hꢀ-AC5 with
purity >95% (as confirmed by final HPLC and UV analyses, Figure 2a)
with good radiochemical yield (60%).
½3Hꢀ-AC5 was then stored in aliquots of aqueous solutions (385 mM)
in the dark at ꢁ808C until its use.
Spectral properties of AC5: lmax ¼ 362 nm, e362 ¼ 23400 Mꢁ1 cmꢁ1
½3Hꢀ-AC5 specific activity measured: SA=0.83 Ci/mmol.
.
Conclusion
The present article describes a reliable synthesis of the photosensitive
nicotinic agonist ½3Hꢀ-AC5. Despite a five-step synthesis from the
radioactive precursor ½3Hꢀ-2, ½3Hꢀ-AC5 was obtained with good overall
yield (44%). Its synthetic precursor ½3Hꢀ-9, is chemically and photo-
chemically stable, offering a convenient way to store the compound for
longer time-periods.
The photoactivatable agonist ½3Hꢀ-AC5 has already proven its efficacy
in labeling specifically the nAChR from Torpedo marmorata.7 This
photolabelling efficacy does ensure sufficient radioactivity incorporation
into the nAChR to permit a molecular analysis of the labelling patterns
during conformational transitions. This work is presently in progress.
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NEW AND CONVENIENT SYNTHESIS
953
Acknowledgements
"
The authors thank the Ministere de l’Enseignement Superieur,
!
Naturalia and the CNRS for financial support.
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J Label Compd Radiopharm 2002; 45: 943–953