Development of a photoaffinity label for respiratory syncytial virus inhibitors

Article abstract of DOI:10.1002/jlcr.743

The syntheses of tritiated and radio-iodinated respiratory syncytial virus inhibitor photoaffinity labels are reported. ³H-1-Isopropyl-3-(1-[2-(3-methyl-3H-diazirin-3-yl)-ethyl] -1H-benzoimidazol-2-yl)methyl)-1,3-dihydrobenzoimidazol-2-one (1),

Full text of DOI:10.1002/jlcr.743

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 1105–1116.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.743
Research Article
Development of a photoaffinity label for
respiratory syncytial virus inhibitors
Douglas D. Dischino1,*, Christopher W. Cianci2, Rita Civiello3,
Mark Krystal2, Nicholas A. Meanwell3, Hiromi Morimoto4,
Philip G. Williams4 and Kuo-Long Yu3,y
1 Department of Chemical Synthesis, Bristol-Myers Squibb, Wallingford,
CT 06492, USA
2 Department of Virology, Bristol-Myers Squibb, Wallingford, CT 06492, USA
3 Department of Chemistry, Bristol-Myers Squibb, Wallingford, CT 06492, USA
4 The National Tritium Labeling Facility and Structural Biology Division,
Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
Summary
The syntheses of tritiated and radio-iodinated respiratory syncytial virus
3
inhibitor photoaffinity labels are reported. H-1-Isopropyl-3-(1-[2-(3-methyl-
3H-diazirin-3-yl)-ethyl]-1H-benzoimidazol-2-yl)methyl)-1,3-dihydrobenzoimid-
azol-2-one (1), was prepared in two steps by the initial tritiation of
N-isopropenyl-2-benzimidazolone followed by coupling to 2-chloromethyl-
1-[2-(3-methyl-3H-diazirin-3-yl)ethyl]-1H-benzoimidazole. The mono and
diiodo-radio-iodinated derivatives of 1-(4-hydroxybenzyl)-3-(1-[2-(3-methyl-
3H-diazirin-3-yl)-ethyl]-2,3-dihydro-1H-benzo-imidazol-2-ylmethyl)-1,3-dihydro-
benzoimidazol-2-one (2, 3), were prepared by the radio-iodination of the
corresponding phenol with Na125I and iodogen. Copyright # 2003 John
Wiley & Sons, Ltd.
Key Words: photoaffinity label; radioiodination; RSV inhibitor
*Correspondence to: D. D. Dischino, Department of Chemical Synthesis, Bristol-Myers Squibb,
5 Research Parkway, Wallingford, CT 06492-7600, USA. E-mail: douglas.dischino@bms.com
y Current address: Discovery Chemistry Research & Technology, Eli Lilly & Company, Lilly
Corporate Center, Drop Code 0520 Indianapolis, IN 46285, USA.
Copyright # 2003 John Wiley & Sons, Ltd.
Received 16 May 2003
Revised 9 June 2003
Accepted 24 June 2003

1106
D. D. DISCHINO ET AL.
Introduction
Respiratory syncytial virus (RSV) has been recognized as a leading
cause of virus-induced lower respiratory tract disease in infants, and
children.1–3 Continuing studies have also shown that RSV is a
significant etiology agent in other populations especially the elderly
and the immunocompromised.4–8
This manuscript describes the design and synthesis of a photoaffinity
label that was utilized to confirm the identity of the virus protein
targeted by this class of RSV inhibitors and to label the specific amino
acid residues intimately involved in the binding interaction. The results
have delineated a binding site for this class of compounds and provided
insight into the mode of action at a molecular level.9
The design of the photoaffinity probe was predicated on the
convergence of structure-activity relationships (SAR) developed
throughout the program with the physicochemical properties of a
suitable labeling element. The diazirine moiety was selected as the
preferred photo-activated element because of the powerful labeling
associated with carbene derivatives and that the SAR developed
indicated tolerance for small heterocycles at the terminus of the alkyl
side chain.10 As a consequence, the diazirine derivative 8 was identified
as a structural element that met these specifications and was readily
O
N
T
N
N
T
N
N
N
1
O
O
N
N
N
N
125I
OH
N
N
N
N
OH
125I
125I
N
N
N
N
2
3
Figure 1. Structures of photoaffinity probes 1, 2 and 3
Copyright # 2003 John Wiley & Sons, Ltd.
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DEVELOPMENT OF A PHOTOAFFINITY LABEL
1107
derivatized at a late stage to afford either the tritiated derivative 1 or the
radio-iodinated derivatives 2 and 3 (Figure 1).
Results and discussion
3H-labeled-1-Isopropyl-3-(1-[2-(3-methyl-3H-diazirin-3-yl)-ethyl]-1H-
benzo-imidazol-2-yl)methyl)-1,3-dihydrobenzoimidazol-2-one (1) was
prepared in two steps as shown in Scheme 1. Mesylation of
N
N
N
N
N
t-BuOK
MsCl
+
OH
MsO
HO
N
H
6
4
5
N
N
N
N
OH
Cl
SOCl2
N
N
N
N
8
7
T
T
N
N
NaH
DMF
T2, Pd/C
EtOAc
O
8
O
+
N
H
N
H
9
10
O
N
N
T
N
N
T
N
N
1
3
Scheme 1. Synthesis of H-labeled 1-Isopropyl-3-(1-[2-(3-methyl-3H-diazirin-3-
yl)-ethyl]-1H-benzoimidazol-2-yl)methyl)-1,3-dihydrobenzoimidazol-2-one, (1)
Copyright # 2003 John Wiley & Sons, Ltd.
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1108
D. D. DISCHINO ET AL.
3,3-azobutanol11 (4) provided 5 which was exposed to 2-benzimidazo-
lemethanol (6) in the presence of potassium tert-butoxide to afford the
N-alkylated derivative 7. Chlorination of the alcohol moiety of 7 using
thionyl chloride provided the benzimidazole derivative 8. N-isoprope-
nyl-N-benzimidazolone (9) was tritiated under standard conditions to
yield approximately 7 Ci of 3H-labeled N-isopropyl-N-benzimidazolone
(10). Alkylation of the benzimidazolone 10 with the diazirine 8 was
accomplished by using sodium hydride as the base in DMF as solvent to
3
afford the H-labeled photoaffinity probe 1.
The specific activity of 1 was found to be 150 Ci/mmol. This value
3
implies 5.2 tritium atoms per molecule. H NMR analysis of this
material clearly indicated extensive labeling in the methyl position of the
isopropyl group. The ratio of the methyl tritium to the methine tritium
was 11.9:1 in the proton-decoupled spectrum, and 12.0:1 in the proton-
coupled spectrum. (Proton decoupling can differentially change the
tritium signal intensity through NOE if not carefully done). Theoreti-
cally, these values should be 1:1, however unequal addition of tritium
across double bonds is a well known phenomenon. Williams and co-
3
workers have published a detailed account of the use of modern H
NMR techniques to analyze these complex mixtures.12
Binding studies with 1 demonstrated that the label was associated
with the RSV F protein, but the specific activity of the 3H material was
insufficient to conduct subsequent mapping studies. These results
prompted us to prepare a radio-iodinated photoaffinity label and
thereby take advantage of the higher specific activity available with
mono or di-radio-iodinated probes.
The 125I labeled radio-iodinated phenols were synthesized as depicted
in Scheme 2. Ethyl 2,3-dihydro-2-oxo-1H-benzimidazole-1-carboxylate
(11) was alkylated with 4-(chloromethyl)phenyl acetate using t-
butylimino-tri(pyrrolidino)phosphorane (BTPP) as the base in the
presence of tetrabutylammonium bromide. The crude product was
doubly deprotected by exposure to 4 N sodium hydroxide in methanol
and the liberated phenol 12 protected as the tert-butyldimethylsilyl ether
13. Alkylation of 13 with the diazirine derivative 8 using BTPP as the
base followed by removal of the silyl protecting group with TBAF in
THF provided phenol 14. Radio-iodination of the 14 was accomplished
by treatment with Na125I and iodogen. Binding studies were conducted
with both 2 and 3 and demonstrated that both labels behaved similarly
and were associated with the RSV F protein. Subsequent mapping
studies were then conducted with 3 to take advantage of the higher
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1105–1116

DEVELOPMENT OF A PHOTOAFFINITY LABEL
1109
O
O
O
O
N
OEt
HN
N
HN
N
c
HN
a,b
Si
O
OH
11
13
12
O
N
N
N
f
Cl
N
N
d, e
+
13
N
OH
N
N
N
N
14
8
O
O
N
N
N
125I
OH
N
N
N
N
+
125I
OH
N
125I
N
N
N
N
3
2
Reagents: a, 4-(chloromethyl)phenyl acetate, BTPP, (Bu)4N+Brꢀ, CH2Cl2; b,
NaOH, CH3OH; c, t-butyldimethylsilyl chloride, imidazole, DMF; d, BTPP,
CH2Cl2; e, TBAF; f, Na125I, iodogen.
Scheme 2. Synthesis of 125I labelled 2 and 3
specific activity of 3. The results of these studies have recently been
reported in part and a complete manuscript will be published
elsewhere.9
Materials and methods
Na125I was obtained from both Nordion International Inc and DuPont/
NEN. All other reagents were obtained from Aldrich unless otherwise
noted and were either ACS grade or the highest quality material
commercially available. HPLC purification and analysis was performed
on a Rainin Dynamax HPLC system consisting of two SD-200 pumps, a
Rainin UV-I detector and an INUS g-RAM radioactive flow-through
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1110
D. D. DISCHINO ET AL.
detector. A Biodex Medical Systems AtomlabTM 100 Dose Calibrator
and a Wallac Model 1409 liquid scintillation counter were used for
radioactivity measurements.
High performance liquid chromatography
Method A. In this method samples are loaded on to an RxCN column
(4.6 ꢁ 250 mm) with a mobile phase of 10% CH3CN and 90% 0.1%
TFA at a flow rate of 1.0 ml/min. The UV-1 detector was set at 265 nm.
In this system, N-isopropenyl-2-benzimidazolone (9) has an Rt of
approximately 28.7 min and N-isopropyl-2-benzimidazolone (10) has an
Rt of approximately 33.5 min.
Method B. In this method samples are loaded on to an RxCN column
(4.6 ꢁ 250 mm) with a mobile phase of 45% CH3CN and 55% 0.1%
TFA at a flow rate of 1.0 ml/min. The UV-1 detector was set at 265 nm.
In this system, N-isopropyl-2-benzimidazolone (10) has an Rt of
approximately 4.7 min, 2-chloromethyl-1-[2-(3-methyl-3H-diazirin-3-
yl)ethyl]-1H-benzoimidazole (8) has an Rt of approximately 7.1 min
and the desired product 1 has an Rt of approximately 12.6 min.
Method C. In this method samples are loaded onto a Jupiter C18
(
column (4.6 ꢁ 250 mm, 5 m 300 A) with a mobile phase of 35% CH3CN
and 65% 0.1% TFA at a flow rate of 1.0 ml/min. The UV-1 detector
was set at 271 nm. In this system, 2 has an Rt of approximately 24 min
while 3 has an Rt of approximately 37 min.
Experimental
Methanesulfonic acid 2-(3-methyl-3H-diazirin-3-yl)ethyl ester, 5: To a
stirred solution of alcohol 411 (1.01 g, 10 mmol) in dichloromethane
(15 ml) at 08C was added methanesulfonyl chloride (1.2 g, 10.5 mmol)
followed by triethylamine (1.52 g, 15 mmol). After stirring for 2 h at 08C
the reaction was diluted with water and the product extracted with
diethyl ether. Purification by flash column chromatography gave 0.95 g
(49% yield) of 5 as an oil which was used in the next step.
{1-[2-(3-Methyl-3H-diazirin-3-yl)-ethyl-1H-benzoimidazol-2-yl}-
methanol, 7: To a solution of 2-benzimidazolemethanol (6) (Aldrich,
1.0 g, 6.8 mmol) in anhydrous THF (20 ml) was added potassium t-
butoxide (1 M in THF, 6.8 ml, 6.8 mmol) at room temperature. After
stirring for 10 min, mesylate 5 (1 g, 5.2 mmol) was added and the
Copyright # 2003 John Wiley & Sons, Ltd.
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DEVELOPMENT OF A PHOTOAFFINITY LABEL
1111
suspension was stirred at room temperature for 18 h. As the reaction
was incomplete, the mixture was heated to 708C for 5 h. The solvent was
evaporated. The residue was diluted with aqueous saturated sodium
bicarbonate and extracted with ethyl acetate. The organic layer was
dried (MgSO4), filtered, and evaporated. Flash column chromatography
(gradient, 1:1 EtOAc/hexane to 2:1 EtOAc/hexane to 10:1 EtOAc/
1
methanol) gave 642 mg (41% yield) of 7 as a white solid. H NMR
(CDCl3) d 0.99 (s, 3H), 1.86–1.91 (m, 2H), 3.97 (br s, 1H), 4.10–4.22 (m,
2H), 4.95 (s, 2H), 7.23–7.31 (m, 3H), 7.68–7.71 (m, 1H). IR (KBr, cmꢀ1)
3153, 1469, 1038, 748. MS m/e 231 [M+H]+. Anal. Calcd. for
C12H14N4O: C, 62.59; H, 6.13; N, 24.33; Found C, 62.43; H, 6.09; N, 24.50.
2-Chloromethyl-1-[2-(3-methyl-3H-diazirin-3-yl)ethyl]-1H-benzo-
imidazole, 8: To a solution of alcohol 7 (230 mg, 1 mmol) in
dichloromethane was added thionyl chloride (238 mg, 2 mmol). The
reaction mixture was stirred at room temperature for 30 min before
evaporating the solvent. The residue was diluted with ethyl acetate,
washed with aqueous saturated sodium bicarbonate, dried (MgSO4),
filtered and the solvent removed by evaporation. The crude product was
purified by flash column chromatography to give 210 mg (85% yield) of
1
8 as an oil which solidified upon standing. H NMR (CDCl3) d 1.02 (s,
3H), 1.92–1.97 (m, 2H), 4.18–4.23 (m, 2H), 4.94 (s, 2H), 7.28–7.36 (m,
3H), 7.75–7.79 (m, 1H). IR (KBr, cmꢀ1) 1467, 753. MS m/e 249
[M+H]+. Anal. Calcd. for C12H13ClN4: C, 57.95; H, 5.27; N, 22.53;
Found C, 57.68; H, 5.24; N, 22.60.
1-Isopropenyl-2-benzimidazolone, 9: Compound 9 was prepared as
described in the literature.13 1H NMR (CDCl3) d 2.23 (s, 3 H), 5.23
(s, 1 H), 5.39 (d, J=1.0 Hz, 1 H), 7.05–7.11 (m, 4 H), 9.69 (s, 1 H). IR
(KBr, cmꢀ1) 1694, 1479, 1386, 740. MS m/e 175 [M+H]+.
3H-N-isopropyl-2-benzimidazolone, 10: A sample of N-isopropenyl-2-
benzimidazolone (9), (10.2 mg, 58.5 mm) was dissolved in EtOAc (1.5 ml)
and the solution frozen in liquid N2, and then thawed under vacuum to
degas the solution prior to tritiation. The process was repeated twice
prior to the addition of the 10% Pd/C catalyst (15.9 mg). The tritiation
was allowed to proceed under 740 mm of T2 gas for 6 h. After 6 h, the
excess tritium was returned to the uranium bed and the solvent removed
under vacuum. Labile tritium was removed by dissolving the residue in
3
EtOAc (1 ml) and evaporating under vacuum (2x). H-N-isopropyl-2-
benzimidazolone, (10), (7.5 Ci, 150 Ci/mmol) was then dissolved in
EtOAc (7 ml) for use in the next reaction. The radiochemical purity of
10, was >99% (HPLC Method A).
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D. D. DISCHINO ET AL.
3H-1-Isopropyl-3-(1-[2-(3-methyl-3H-diazirin-3-yl)-ethyl]-1H-benzo-
imidazol-2-ylmethyl)-1,3-dihydrobenzoimidazol-2-one, 1: The following
procedure was conducted under red light. A solution of 10 in EtOAc,
(2 Ci, 16.7 mm, 1.85 ml) was evaporated to near dryness under a stream
of N2 and then placed under vacuum for 10 min. To this was then added
DMF (0.5 ml) and the solution transferred to an amber colored 5 ml
reaction vial and to this was added NaH (60%, 6.0 mg, 14.2 mm). The
mixture was stirred for 0.5 h, after which time 8, (4 mg, 16 mm) was
added and the reaction allowed to stir at room temperature for 16 h.
The reaction was quenched with saturated NaHCO3, rinsed with H2O
and extracted with Et2O. The Et2O layer was dried by passage through
a short column of MgSO4. The solution was concentrated to dryness
under a stream of N2 and immediately dissolved in 5 ml of HPLC
mobile phase (45% CH3CN and 55% 0.1% TFA). A 100 mCi sample of
crude 1 was purified via HPLC (Method B) to yield 1 (70 mCi). No
radioactive impurities were found in the final purified product. The
specific activity was determined via HPLC. In this procedure, a
radioactive sample was applied to the column, and the mass was
determined by comparison of the UV absorbance to a standard curve,
and the total radioactivity was measured via liquid scintillation
counting. The specific activity of 1 was found to be 150 Ci/mmol.
3
Stability studies of the H-photoaffinity label 1 were conducted in
EtOH and HPLC mobile phase at 08C for 2 weeks in amber colored
screw top reaction vials. At the end of 2 weeks, it was found that
4 radiolabeled impurities representing 9% of the total radioactivity were
found in the sample stored in EtOH, whereas no radiolabeled impurities
were found in the sample stored in the HPLC mobile phase. Samples of
1 for subsequent biological studies were thus kept in the HPLC mobile
phase until immediately prior to the intended use when the mobile phase
was removed by lyophilization and then immediately redissolved in
EtOH.
1-[4-(tert-Butyl-dimethylsilanyloxy)-benzyl]-1,3-dihydro-benzoimida-
zol-2-one, 13: A mixture of 1114 (1.55 g, 7.5 mmol), 4-(chloro-
methyl)phenyl acetate (1.52 g, 8.25 mmol), BTPP (Fluka, t-butylimino-
tri(pyrrolidino)phosphorane, 2.81 g, 9.0 mmol), and tetrabutylammonium
bromide (2.9 g, 9.0 mmol) in dichloromethane (20 ml) was stirred at
room temperature for 2 h. The solvent was evaporated off and the
residue was dissolved in ethyl acetate, washed with water, dried
(MgSO4), filtered and evaporated. This residue was dissolved in
methanol (30 ml) and to this solution was added 4 N NaOH (5 ml).
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DEVELOPMENT OF A PHOTOAFFINITY LABEL
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The mixture was stirred at room temperature for 30 min. The methanol
was evaporated off and the aqueous layer acidified with concentrated
HCl and extracted with ethyl acetate. The combined extracts were dried
(MgSO4), filtered and evaporated. Purification of 12 was attempted
using flash column chromatography (gradient 1:1 EtOAc/hexanes to
straight EtOAc to 10:1 EtOAc/MeOH) and gave 680 mg of the 12
containing 30% of an impurity (structure below). The product was used
in the next step without further purification.
O
O
HN
N
HN
N
OH
O
12
desired product (major)
OH
side product (minor)
The crude phenol 12 (680 mg), tert-butyldimethylsilyl chloride
(467 mg, 3.10 mmol, 1.1 eq), and imidazole (561 mg, 8.49 mmol, 3 eq)
were stirred together in DMF (10 ml) at 758C for 18 h. The reaction was
incomplete. After cooling to room temperature, additional tert-
butyldimethylsilyl chloride (467 mg, 3.10 mmol) and imidazole
(561 mg, 8.49 mmol) were added and the reaction mixture was stirred
at room temperature for 1 h. The mixture was diluted with ethyl acetate,
washed with water, dried over MgSO4, filtered, and evaporated. The
residue was purified by flash column chromatography (gradient, 1:4
EtOAc/hexanes to 1:1 EtOAc/hexanes) to give 335 mg of 13 as a white
1
solid. MS m/e 355 [M+H]+. H NMR (CDCl3) d 0.17 (s, 6H), 0.96
(s, 9H), 5.01 (s, 2H), 6.77–6.80 (m, 2H), 6.87–6.90 (m, 1H), 6.98–7.11
(m, 3H), 7.21 (d, J=8.7 Hz, 2H), 9.34 (s, 1H). Anal. Calcd. for
C20H26N2O2Si: C, 67.76; H, 7.39; N, 7.90, Found: C, 67.39; H, 7.28; N,
7.63.
1-(4-Hydroxybenzyl)-3-{1-[2-(3-methyl-3H-diazirin-3-yl)-ethyl]-2,3-
dihydro-1H-benzoimidazol-2-ylmethyl}-1,3-dihydro-benzoimidazol-2-one,
14: A mixture of 8 (216 mg, 0.87 mmol), 13 (308 mg, 0.87 mmol),
and BTPP (Fluka, t-butylimino-tri(pyrrolidino)phosphorane, 352 mg,
1.13 mmol) was stirred in dichloromethane (5 ml) for 1 h at room
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1114
D. D. DISCHINO ET AL.
temperature. The solvent was evaporated off and the residue diluted
with ethyl acetate, washed with water, brine, dried (MgSO4), filtered
and evaporated. Flash column chromatography (gradient, 1:4 EtOAc/
hexane to 1:1 EtOAc/hexane) gave the coupling product, as the silyl
ether which was immediately used in the next step. MS m/e 567
[M+H]+.
To the silyl ether was added 1.3 ml of 1 M TBAF (tetrabutylammo-
nium fluoride) solution in tetrahydrofuran. The mixture was stirred at
room temperature for 1 h, diluted with ethyl acetate and diethyl ether,
washed with water, dried (MgSO4), filtered and evaporated. The residue
was purified by flash column chromatography (gradient, 1:1 EtOAc/
hexanes to straight EtOAc) to give 232 mg (59%) of 14 as a white solid.
1H NMR (CDCl3) d 1.00 (s, 3H), 1.55 (t, J=7.8 Hz, 2H), 4.43 (t,
J=7.8 Hz, 2H), 5.00 (s, 2H), 5.60 (s, 2H), 6.81 (d, J=8.1 Hz, 2H), 6.89–
6.92 (m, 1H), 6.99–7.02 (m, 2H), 7.21 (d, J=8.1 Hz, 2H), 7.31–7.55 (m,
3 H), 7.50–7.60 (m, 1H), 7.84–7.89 (m, 1H). IR (KBr, cmꢀ1) 2925, 1708,
749.
1-(4-Hydroxy-3,5-diiodo-benzyl)-3-{1-[2-(3-methyl-3H-diazirin-3-yl)-
ethyl]-2,3-dihydro-1H-benzoimidazol-2-ylmethyl}-1,3-dihydrobenzoimi-
dazol-2-one, 3a: To a solution of iodogen (19.2 mg, 0.0444 mmol) in
CHCl3 (1 ml) was added a pH 7 phosphate buffer (5 ml) and an aqueous
solution of KI (24 mg, 0.144 mmol dissolved in 0.3 ml of H2O) at 08C.
The solution was stirred for 10 min followed by the addition of 14
(20 mg, 0.044 mmol) in a mixture of CHCl3 (5 ml) and DMF (0.3 ml).
After stirring at 08C for 15 min, the mixture was diluted with CH2Cl2
(40 ml). The organic layer was separated and washed with an aqueous
Na2S2O5 solution (5%, 5 ml), H2O (10 ml), dried (MgSO4), filtered and
concentrated on a rotary evaporator. The residue was triturated in hot
CH3OH, cooled to RT, and filtered to yield 3a (23 mg, 74%) as a white
powder. MS m/e 705 [M+H]+.
125I labeled 1-(4-Hydroxy-3-iodobenzyl)-3-(1-[2-(3-methyl-3H-diazir-
in-3-yl)-ethyl]-2,3-dihydro-1H-benzoimidazol-2-ylmethyl)-1,3-dihydro-
benzo-imidazol-2-one, 2 and 125I labeled 1-(4-Hydroxy-3-iodobenzyl)-3-
(1-[2-(3-methyl-3H-diazirin-3-yl)-ethyl]-2,3-dihydro-1H-benzoimidazol-
2-ylmethyl)-1,3-dihydrobenzo-imidazol-2-one, 3: The following procedure
was conducted under red light. Into a 5 ml conical reaction vial was
added 15 ml of a solution containing 1 mg/ml of 14 dissolved in 40%
DMF/EtOH, 10 ml of a solution of 2 mg/ml of iodogen dissolved in
EtOH, 15 ml of NaHPO4 buffer 0.05 M, pH 8.0) and 10–15 ml of Na125I.
The solution was then allowed to stir at room temperature for 10 min
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DEVELOPMENT OF A PHOTOAFFINITY LABEL
1115
prior to quenching the reaction with 15 ml of a solution of Na2S2O5
(9 mg/ml). The sample was purified by HPLC (Method C). Each
product was then concentrated in a Savant Speedvac and redissolved in
EtOH. The specific activity of 2 and 3 were assumed to be 2100 and
4200 Ci/mmol, respectively, based upon the specific activity of the
Na125I used in these experiments.
Radio-iodination experiments (n=4) were completed with routine
radiochemical yields of 2+3 ranging from 45 to 60%. In addition,
relative yields of 2 and 3 were comparable with 3 usually being prepared
in slightly higher yields. In a typical radio-iodination experiment
starting with 2.7 mCi of Na125I, we obtained 0.7 mCi of 2 and 0.8 mCi of
3 following HPLC purification.
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