A photoaffinity probe for 5-hydroxyeicosanoid dehydrogenase suitable for radioiodination

Article abstract of DOI:10.1016/S0040-4039(01)00699-2

5-Hydroxy eicosanoid dehydrogenase (5h-dh) is a key enzyme responsible for the biosynthesis of 5-oxo-ETE, a potent eosinophil chemoattractant. To facilitate the identification and characterization of 5h-dh we have synthesized a photoaffinity ligand 7, des

Full text of DOI:10.1016/S0040-4039(01)00699-2

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 4445–4448
A photoaffinity probe for 5-hydroxyeicosanoid dehydrogenase
suitable for radioiodination
Seongjin Kim,a Yurdanur Adiyaman,a Goutam Saha,a William S. Powellb and Joshua Rokacha,*
aClaude Pepper Institute and Department of Chemistry, Florida Institute of Technology, 150 W. University Blvd, Melbourne,
FL 32901, USA
bMeakins–Christie Laboratories, Department of Medicine, McGill University, 3626 St-Urbain St., Montreal,
QC, Canada H2X 2P2
Received 20 March 2001; revised 26 April 2001; accepted 27 April 2001
Abstract—5-Hydroxy eicosanoid dehydrogenase (5h-dh) is a key enzyme responsible for the biosynthesis of 5-oxo-ETE, a potent
eosinophil chemoattractant. To facilitate the identification and characterization of 5h-dh we have synthesized a photoaffinity
ligand 7, designed to bind to the enzyme, and shown it to be an excellent substrate for 5h-dh. We have also synthesized a
precursor 34, containing the photoaffinity label and a trimethyl tin group that can readily be displaced by iodide and will be
suitable for radioiodination with 125I. © 2001 Elsevier Science Ltd. All rights reserved.
5-Hydroxyeicosanoid dehydrogenase (5h-dh) is a key
enzyme involved in the transformation of 5-HETE to
5-oxo-ETE.1 5-oxo-ETE is the most potent eosinophil
chemotactic factor among lipid mediators.2,3 We have
proposed that 5-oxo-ETE may be a causative factor in
diseases such as asthma involving the infiltration of
eosinophils.4 5-oxo-ETE exerts its action by activating
a dedicated receptor.5–7 We have reported recently the
first total synthesis of 5-oxo-ETE and tritiated and
deuterated 5-oxo-ETE.8,9
of 5-oxo-ETE. It is also a potential target for inhibiting
the formation of 5-oxo-ETE and preventing the infiltra-
tion of eosinophils into inflammatory sites (Scheme 1).
We have described recently the design and synthesis of
a non-radioactive model of a 5-HETE photoaffinity
ligand that was prepared by coupling the v-amino
10
derivative 4 with a benzoic acid derivative 5 (Scheme
2).
The purpose of this synthesis was to confirm the valid-
ity of our assumptions in the design of this ligand. We
wanted to know how well the cold iodo azido probe 7
The 5h-dh, which has not been identified, is an essential
element in the understanding of the biological activity
CO2H
5h-dh
CO2H
CO2H
5-oxo-ETE
Receptor
5-LO
Biological
Activities
O
OH
keto-
reductase
1
AA
5-oxo-ETE
3
2
5-(S)-HETE
Scheme 1.
HO2C
N3
I
CO2Me
N
CO2H
CO2Me
O
O
5
OTBDPS
OH
OTBDPS
I
I
N
H
NH2
H
6
7
4
N3
N3
Scheme 2.
Keywords: 5-HETE; 5-oxo-ETE; v-functionalized-5-HETE; 5h-dh.
* Corresponding author.
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)00699-2

4446
S. Kim et al. / Tetrahedron Letters 42 (2001) 4445–4448
use a phenyl substituted trimethyl tin derivative as a
precursor to radioactive iodine. This group has been
used previously by some other investigators.11,12 This is
an interesting precursor to radioactive iodine in that the
trimethyl tin group itself can be introduced by substi-
tuting an iodo benzene derivative. The disadvantage of
the 125I is that its half-life is very short, ꢀ11 days,
hence the radio probe might have to be prepared
several times. In addition, because 125I is a strong
g-emitter, extensive precautions must be taken during
the preparation and use of radioiodinated compounds.
It was therefore essential that (1) the radiolabel be
introduced as late as possible in the synthesis, hopefully
the last step, and (2) the precursor be reasonably stable
in order to last as long as possible.
is recognized by 5h-dh and how good a substrate it is
before we embarked on the more difficult task of the
design and synthesis of a radiophotoaffinity probe and
the design of the appropriate precursor for the intro-
duction of the radiolabel. The better the substrate (or
inhibitor), the less we have to contend with non-specific
binding. We are reporting here: (1) that 7 is a very good
substrate for 5h-dh and hence appears to be a good
target for introducing 125I, and (2) the design and total
synthesis of a precursor to the radiophotoaffinity label
and its conversion to 7.
We have incubated 7 with neutrophils and a new
product was detected by HPLC. The UV of this
product, l280 is consistent with the formation of a
dienone. In order to complete the characterization of
the enzymatic product, we have performed the chemical
oxidation of 7 using the Dess–Martin reagent (Scheme
3). The synthetic standard and the enzymatic product
were compared on RP-HPLC and were found to be
identical.10
The benzoic acid derivative 12 has been prepared in
excellent yield as shown in Scheme 4. We first
attempted to introduce the trimethyl tin group directly
on acid 5 but with limited success (15% yield).
We planned to introduce 12 into the photoaffinity
probe, which would provide an acceptor group for 125I.
This could be accomplished by first coupling it with the
v-amino derivative 4 or 33 (Schemes 5 and 6), followed
by replacement of the trimethyl tin group with 125I in
the presence of chloramine-T. An alternative possibility
would be to introduce the radioactive iodine first, e.g.
13, and then couple it with the appropriate v-amino
Furthermore, in preliminary studies 7 was compared to
5-HETE, the natural substrate for 5h-dh and found to
be almost as good at three different concentrations.
We selected 125I as our radio tracer as it has a much
higher specific activity than tritium for example. In
order to introduce the desired radiolabel, we elected to
PMNS
CO2H
O
CO2H
O
O
Dess-
Martin
OH
I
I
N
N
H
H
8
7
N3
N3
Scheme 3.
MeO2C
SnMe3
HO2C
H2N
I
MeO2C
I
HO2C
N3
I
c
a
b
11
10
N3
d
N3
9
5
Na125
I
Chloroamine-T
e
f
HO2C
N3
SnMe3
MeO2C
125I
12
13
N3
Scheme 4. Reagents and conditions: (a) NaNO2, NaN3, HCl, H2O, 0°C, 1.5 h, 88%; (b) CH2N2, ether, rt, 90%; (c) (Sn(Me)3)2,
Pd[Ph3]4, dioxane, 50°C, 5 h, 80%; (d) NaI, chloriamine-T, THF, pH 7.4 buffer, rt, 20 min, 89%; (e) t-BuOK, H2O, THF, 0°C,
99%; (f) (Sn(Me)3)2, Pd[Ph3]4, dioxane, 50°C, 5 h, 15%.
CO2Me
O
TBAF or HF
OH
HO2C
N3
H
SnMe3
CO2Me
N
H
N3
CO2Me
O
15
OTBDPS
OTBDPS
12
SnMe3
TBAF, HF,
N
H
N3
CO2Me
NH2
89%
14
O
4
OH
SnMe3
.
or BF3 Et2O
N
H
16
N3
Scheme 5.

S. Kim et al. / Tetrahedron Letters 42 (2001) 4445–4448
4447
a)
O
OHC
Ph3P
OHC
CHO
20
HO
OH
CO2Me
9-Steps
CO2Me
HO
HO
OBz
21
OBz
18
19
a
D-Arabinose
Br-Ph3P+
b)
OHC
OH
b
HO
OH
c
HO
OTBDMS
OTBDMS
d
22
25
23
24
OH
PPh3I
I
e
f
OTBDMS
OTBDMS
OBz
OTBDMS
26
28
27
PPh3I
c)
CO2Me
CO2Me
OHC
g
h
+
i
CO2Me
OBz
OTBDMS
OBz
OBz
21
OTBDMS
HO2C
OH
28
29
30
SnMe3
CO2Me
I
CO2Me
CO2Me
CO2Me
OBz
O
N3
OBz
12
OBz
j
k
SnMe3
N3
N
H
l
NH2
32
31
33
34
N3
CO2Me
CO2H
O
O
OH
OBz
m
I
I
n
N
H
N
H
7
35
N3
N3
Scheme 6. Reagents and conditions: (a) Benzene, reflux, 5 h, 54%; (b) TBDMSCl, Im, THF, rt, 3 h, 83%; (c) PCC, Al2O3, CH2Cl2,
rt, 3 h, 60%; (d) LiHMDS, HMPA, THF, −78°C to rt, 45%, a few percent of the trans isomer formed; (e) Ph3P, I2, Im, CH2Cl2,
0°C to rt, 97%; (f) Ph3P, CH3CN, 68%; (g) LiHMDS, HMPA, THF, −98°C to −78°C, 56%; (h) DDQ, H2O, THF, rt, 2 h, 99%;
(i) Ph3P, I2, Im, CH2Cl2, 0°C to rt, 92%; (j) NaN3, DMSO, 90°C, 97%; (k) Ph3P, H2O, THF, rt, 6 h, 75%; (l) EDCI, HOBT,
NaHCO3, 1:1 CH2Cl2:CH3CN, rt, 8 h, 84%; (m) NaI, chloroamine-T, THF, pH 7.4 buffer; (n) NaOH, rt, 2 h, HPLC.
derivative. However, this would be impractical because
it would require a greater number of steps with radioio-
dinated intermediates.
advantage being that the hydrolysis of the methyl ester
and benzoyl group can be performed in one pot at the
end of the synthesis. The obvious disadvantages are its
lability and ability to survive the synthesis considering
that we have several basic reactions to perform. In fact,
before completing our successful synthesis shown in
Scheme 6, we performed some not so successful con-
struction (vide infra).
At first, we decided to use the v-amino derivative 4 and
perform the sequence shown in Scheme 5. The conden-
sation of 4 and 12 with EDCI in 1:1 CH3CN and
CH2Cl2 proceeded smoothly in 89% yield. However, the
deprotection of TBDPS was unsuccessful and resulted
in the loss of the trimethyl stannyl group, as shown in
Scheme 5.
The one-pot transformation of 34 to 7 has been
achieved successfully. The iodo derivative 7 prepared
by this method (Scheme 6) and the one prepared by the
direct coupling of the iodo benzoic acid derivative 5
with 4 (Scheme 2)10 are identical as shown by RP-
HPLC (mBondapak C18, 20% 0.1 M aqueous ammo-
nium acetate, 80% methanol, 1 ml/min, at 240 nm),
NMR, etc.
The structural identity of 15 is supported by NMR and
mass spectral data.
We then completely redesigned the synthesis in order to
have a protecting group on the 5-hydroxyl group,
which is easier to remove and does not involve a naked
nucleophile in the deprotection step. We elected to
protect the 5-hydroxy with a benzoyl group 19;13 the
As mentioned, we initially attempted the following
construction (Scheme 7). However, the transformation
OHC
CO2Me
CO2Me
CO2Me
BrPh3P
OTBDMS
CO2Me
OBz
OBz
36
OBz
OTBDMS
37
38
OBz
39
21
OH
CHO
BrPh3P
OTBDMS
CO2Me
40
OBz
OTBDMS
29
Scheme 7.

4448
S. Kim et al. / Tetrahedron Letters 42 (2001) 4445–4448
R1
R1
O
Ph3P
R2
O
O
R1
loss of benzoate
+
decomposition
R1
R2
O
O-
Ph
O
O
O
O
Ph
O
Ph
R3
R3
O
PPh3
Ph
PPh3
41 R1=(CH2)3CO2Me
R2=(CH2)2OTBDMS
42 R1=(CH2)3CO2Me
43 R1=(CH2)3CO2Me
R3=(CH2)8OTBDMS
45
37
29
R2=CH2CHCH(CH2)8OTBDMS
Scheme 8.
of 39 to 29 occurs in dismal yields. It is interesting to
note that the Wittig reaction between 21 and 36 and 21
and 28 works reasonably well, whereas the one between
39 and 29 does not.
3. O’Flaherty, J. T.; Kuroki, M.; Nixon, A. B.; Wijkander,
J.; Yee, E.; Lee, S. L.; Smitherman, P. K.; Wykle, R. L.;
Daniel, L. W. J. Immunol. 1996, 157, 336–342.
4. Stamatiou, P.; Hamid, Q.; Taha, R.; Yu, W.; Issekutz, T.
B.; Rokach, J.; Khanapure, S. P.; Powell, W. S. J. Clin.
Invest. 1998, 102, 2165–2172.
5. Powell, W. S.; Gravel, S.; MacLeod, R. J.; Mills, E.;
Hashefi, M. J. Biol. Chem. 1993, 268, 9280–9286.
6. Norgauer, J.; Barbisch, M.; Czech, W.; Pareigis, J.;
Schwenk, U.; Schroder, J. M. Eur. J. Biochem. 1996, 236,
1003–1009.
7. O’Flaherty, J. T.; Taylor, J. S.; Thomas, M. J. J. Biol.
Chem. 1998, 273, 32535–32541.
8. Khanapure, S. P.; Shi, X. X.; Powell, W. S.; Rokach, J.
J. Org. Chem. 1998, 63, 337–342.
9. Khanapure, S. P.; Shi, X. X.; Powell, W. S.; Rokach, J.
J. Org. Chem. 1998, 63, 4098–4102.
10. Saha, G.; Khanapure, S. P.; Powell, W. S.; Rokach, J.
Tetrahedron Lett. 2000, 41, 6313–6317.
It is possible that if the betaine intermediate 43 is
formed, the oxygen anion is in close proximity to the
benzoyl group, whereas in the two other cases it is not
(Scheme 8).
In summary, the synthesis of the radioprecursor 34 is
described.14 The transformation of this precursor to the
desired derivative 7 is accomplished in a one-pot reac-
tion using chloramine-T/NaI, followed by hydrolysis.
The preparation of the radiolabeled probe will be
effected using chloramine-T/Na125I in an analogous
manner. The preparation of the radioprobe will be
published elsewhere.
11. Perrier, H.; Prasit, P.; Wang, Z. Tetrahedron Lett. 1994,
35, 1501–1502.
Acknowledgements
12. Demin, P. M.; Kochev, D. M.; Perrier, H.; Pace-Asciak,
C. R.; Pivnitsky, K. K. Mendeleev Commun. 1997, 90–91.
13. Khanapure, S. P.; Manna, S.; Rokach, J.; Murphy, R. C.;
Wheelan, P.; Powell, W. S. J. Org. Chem. 1995, 60,
1806–1813.
We wish to acknowledge NIH support under grant
DK-44730 (J.R.); NSF for an AMX-360 NMR instru-
ment (grant CHE-90-13145); and MRC of Canada
under grant MT-6254 (W.S.P.).
14. The NMR data of compound 34: 1H NMR (360 MHz,
CDCl3) l 8.25 (s, 1H), 8.06 (d, J=7.4 Hz, 2H), 7.60 (m,
2H), 7.45 (t, J=7.7 Hz, 2H), 7.49 (br, 1H), 7.17 (d,
J=7.6, 1H), 6.65 (dd, J=15.0 and 11.7 Hz, 1H), 5.97 (t,
11.0 Hz, 1H), 5.71 (dd, J=15.1 and 6.6 Hz, 1H), 5.59 (m,
1H), 5.47–5.30 (m, 3H), 3.67 (s, 3H), 3.47 (m, J=6.9,
2H), 2.92 (t, J=6.5, 2H), 2.38 (m, 2H), 2.02 (m, 2H), 1.76
(m, 2H), 1.60 (m, 4H), 1.28 (m, 12H), 0.31 (s, 9H).
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