Synthesis and characterization of photoaffinity probe of acetogenin, a strong inhibitor of mitochondrial complex i

Article abstract of DOI:10.1016/j.tetlet.2011.03.149

Acetogenins are valuable inhibitor probes to get an insight into the structural and functional properties of mitochondrial NADH-ubiquinone oxidoreductase (complex I). We synthesized a photoreactive acetogenin mimic ([¹²⁵I]DANA) which retained a strong inhibitory activity. The preliminary photoaffinity labeling with bovine heart submitochondrial particles revealed that [¹²⁵I]DANA binds to the ND1 subunit among 45 different subunits with high specificity.

Full text of DOI:10.1016/j.tetlet.2011.03.149

Tetrahedron Letters 52 (2011) 3090–3093
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis and characterization of photoaffinity probe of acetogenin,
a strong inhibitor of mitochondrial complex I
⇑
Shuhei Yamamoto, Masato Abe, Sayo Nakanishi, Masatoshi Murai, Hideto Miyoshi
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Acetogenins are valuable inhibitor probes to get an insight into the structural and functional properties of
mitochondrial NADH-ubiquinone oxidoreductase (complex I). We synthesized a photoreactive acetoge-
nin mimic ([125I]DANA) which retained a strong inhibitory activity. The preliminary photoaffinity labeling
with bovine heart submitochondrial particles revealed that [125I]DANA binds to the ND1 subunit among
45 different subunits with high specificity.
Received 8 March 2011
Revised 24 March 2011
Accepted 31 March 2011
Available online 7 April 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Acetogenins
Photoaffinity labeling
Radioiodination
Mitochondrial complex I
Respiratory enzyme
More than 400 annonaceous acetogenins have been isolated
from the plant family Annonaceae since the discovery of uvaricin
in 1982.1,2 Acetogenins have very potent and diverse biological
effects such as antitumor, antimalarial, and pesticidal activities;1,2
hence a variety of molecular probes have been developed for
biochemical and medicinal researches.3 The diverse biological
activities of acetogenins are thought to be attributable to the
inhibition of mitochondrial NADH-ubiquinone oxidoreductase
(complex I),4,5 which is the most complicated multisubunit
enzyme in the respiratory system.6,7 There are few structural sim-
ilarities between acetogenins and traditional complex I inhibitors
such as rotenoids and piericidins. Considering the unusual struc-
tural characteristics as well as the very strong inhibitory effect of
acetogenins, detailed analysis of the action mechanism of these
inhibitors is expected to provide valuable insights into the struc-
tural and functional futures of the enzyme.
On the basis of photoaffinity labeling study using a photoreac-
tive mimic of
D
lac-acetogenins ([125I]DAA, Fig. 1), whose inhibition
manner differs from that of natural acetogenins,8,9 we suggested
that the hydroxylated bis-THF ring moiety of natural acetogenins
binds at the third matrix-side loop connecting the fifth and sixth
transmembrane helices in the ND1 subunit of bovine complex
I.10 This idea is very important to know the role of the subunit in
the supposed dynamic interaction between the hydrophilic and
membrane arms of complex I.6,7,11 However, since [125I]DAA has
no
c-lactone ring, which is a common structural component of
⇑
Corresponding author. Tel.: +81 75 753 6119; fax: +81 75 753 6408.
E-mail address: miyoshi@kais.kyoto-u.ac.jp (H. Miyoshi).
Figure 1. Structure of the compounds discussed in the present study.
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.03.149

S. Yamamoto et al. / Tetrahedron Letters 52 (2011) 3090–3093
3091
numerous natural acetogenins, the above proposition needs to be
directly verified using a natural acetogenin-type photoaffinity
probe. We here synthesized a photoreactive natural acetogenin
mimics [diazinylated natural acetogenin (DANA) and its 125I-la-
beled derivative [125I]DANA, see Figure 1] which have a small
photoreactive diazirine group, in place of a hydroxy group, at-
tached to the bis-THF moiety. We expected that DANA might sus-
tain the strong inhibitory activity because structure–activity study
revealed that a lack of both hydroxy groups adjacent to the bis-THF
ring results in a drastic decrease in the inhibitory activity, but the
presence of either sufficiently sustains strong activity at the nano-
molar level.12 We also conducted preliminary photoaffinity label-
ing with bovine heart submitochondrial particles (SMP) to
determine whether [125I]DANA is an adequate photoaffinity probe.
The synthetic procedures of DANA and [125I]DANA are outlined
in Scheme 1. The key intermediate 1 was synthesized by the proce-
dure reported previously.13 The epoxide 1 was opened by the treat-
ment with 1-lithium-4-(tert-butyldiphenylsilyloxy)-1-butylide in
the presence of Et2AlCl,14 and subsequent catalytic hydrogenation
on 10% Pd/C afforded 2. After acetylation of both hydroxyl groups
of 2, deprotection of MOM ether and sequential tosylation pro-
vided 3. After hydrolysis of the acetyl group of 3 to give epoxide,
protection of a free hydroxyl group by MOMCl afforded 4. Opening
of the epoxide 4 with lithium(trimethylsilyl)acetylide in the pres-
ence of Et2AlCl and sequential desilylation provided 5. Sonogashira
Oxidation of the hydroxy group of 7 with Dess–Martin period-
inane gave 8 quantitatively. After desilylation of TBDPS ether of
8, introduction of 4-iodophenyl into 8 by Mitsunobu reaction16
and sequential deprotection of MOM ether provided 9. The car-
bonyl group of 9 was converted to diazirine (10) by the treatment
with hydroxylamine-O-sulfonic acid in liquid ammonia followed
by oxidation by iodine in 57% yield (2 steps).17 Oxidation of the sul-
fide moiety of 10 with mCPBA and sequential thermal elimination
of the resultant sulfoxide15 provided DANA.18 Conversion of the
carbonyl group to diazirine after the formation of
-lactone ring resulted in significant decomposition of the ring
moiety.
With DANA in hand, we determined its inhibitory activity
against complex I in bovine heart SMP (30 g of protein/mL)
a,b-unsaturated
c
l
according to the reported method,9 using bullatacin as a control.
The IC50 values of DANA and bullatacin were 1.2 ( 0.20) and 0.90
( 0.11) nM, respectively, indicating that DANA retains the strong
inhibitory activity irrespective of displacement of the hydroxy
group with a diazirine group. Consequently we next examined
the synthesis of 125I-labeled DANA to enable photoaffinity labeling
experiment.
To prepare radioiodinated pharmaceuticals, organotin com-
pounds have been extensively exploited as precursors for subse-
quent iododestannylation.19 We tried, therefore, stannylation of
DANA by reacting with hexabutylditin in the presence of
Pd(CH3CN)2Cl2 or Pd(Ph3P)4 in anhydrous HMPA,20 whereas a yield
of the reaction was disappointingly low (5–10%). 1H NMR spectra
cross-coupling of 5 with the c-lactone moiety 6, which was synthe-
sized according to the reported method,15 and hydrogenation of
the resultant enyne by Wilkinson’s catalyst afforded 7.
of the major products indicated that the c-lactone as well as the
Scheme 1. Reagents and conditions: (a) (1) 3-Butyn-1-tert-butyldiphenylsilylether, n-BuLi, Et2AlCl (10 equiv), dry toluene, 0 °C, 30 min, (2) H2, 10% Pd/C, EtOH, rt, 1 h, 64% (2
steps); (b) (1) AcCl, DMAP, CH2Cl2, rt, 1 h, (2) BF3ꢀEt2O, CH3SCH3, ꢁ20 °C, 1 h, 3) TsCl, DMAP, TEA, CH2Cl2, 35 °C, 16 h, 85% (3 steps); (c) (1) K2CO3, MeOH, rt, 3 h, (2) MOMCl,
DIPEA, CH2Cl2, 35 °C, 16 h, 72% (2 steps); (d) (1) trimethylsilylacetylene, n-BuLi, Et2AlCl (10 equiv), toluene, 0 °C, 20 min, (2) K2CO3, MeOH, rt, 2 h, 91% (2 steps); (e) (1)
(PPh3)4Pd, CuI, dry TEA, rt, 1 h, (2) H2, (PPh3)3RhCl, dry benzene, rt, 18 h, 77% (2 steps); (f) Dess–Martin periodinane, CH2Cl2, 30 °C, 2 h, 99%; (g) (1) TBAF, THF, 50 °C, 2 h, (2) 4-
iodophenol, DIAD, PPh3, THF, 0 °C, 1 h, (3) 4% AcCl/MeOH, CH2Cl2, 30 °C, 4 h, 47% (3 steps); (h) (1) liq. NH3, HONHSO3H, MeOH, CH2Cl2, rt, 10 h, (2) I2, TEA, MeOH, 0 °C, 30 min,
57% (2 steps); (i) (1) mCPBA, CH2Cl2, ꢁ40 °C, 1 h, (2) toluene, 60 °C, 2 h, 77% (2 steps); (j) bis(neopentylglycolato)diboron, PdCl2(dppf)2, KOAc, DMSO, 60 °C, 20 min, 38%; (k)
[
125I]NaI, chloramine-T, 50% H2O/THF, rt, 10 min, 47%.

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S. Yamamoto et al. / Tetrahedron Letters 52 (2011) 3090–3093
diazirine-ring was decomposed. Although several conditions were
explored in various molar ratios of DANA/hexabutylditin and com-
binations of solvents, the yield could not be improved. Having
failed to prepare the tributyltin precursor, we decided to change
the synthetic strategy.
may be the ND1 subunit in the membrane arm. To verify this, we
carried out immnoprecipitation using the anti-bovine ND1 anti-
body according to the previous method (Fig. 2B).9,10 The results
unambiguously revealed that the radiolabeled protein is the ND1
subunit. Thus, [125I]DANA turned out to be an excellent probe to
identify the binding site of the bis-THF motif of acetogenins in
the ND1 subunit. Identification of the cross-linked region in the
subunit is currently underway in our laboratory.
Survey of literatures revealed that arylboronate esters have
been rarely used to prepare radioiodinated aryl iodides;21 namely,
arylboronate esters can be converted to radioiodinated aryl iodides
using labeled NaI in the presence of chloramine-T. However, to the
best of our knowledge, this procedure has not yet applied to com-
plicated chemicals such as pharmaceuticals and natural products.
We synthesized the boronate precursor (11) by the palladium-cat-
alyzed coupling of DANA and bis(neopentylglycolato)diboron,21 al-
beit in moderate yield (38%).18 As introduction of iodine into 11
using a cold 125 mM NaI solution by the catalysis of chloramine-
T appeared to be successful in sufficient yield (ꢂ80%) by HPLC anal-
ysis, we proceeded to the preparation of [125I]DANA from 11 using
Acknowledgment
The rabbit anti-ND1 antibody was a generous gift from Dr. Tak-
ao Yagi (The Scripps Research Institute, La Jolla, CA 92037, USA).
Supplementary data
125I]NaI as a radioisotope donor.22 The radiochemical yield from
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2011.03.149.
[
the initial [125I]NaI was 47%, and the specific radioactivity was
ꢂ2,000 Ci/mmol. Radiochemical purity was examined by TLC and
HPLC, and determined to be over 99%. Thus our results corrobo-
rated that arylboronate ester can be used as a precursor of radioio-
dinated aryl iodide. Taking into account the fact that organotin
precursor for the radioiodination is not necessarily available due
to some sort of synthetic difficulties (a recent example of such a
difficulty in organotin preparation, see Ref. 23), the application of
this procedure might be further appreciated.
References and notes
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10. Kakutani, N.; Murai, M.; Sakiyama, N.; Miyoshi, H. Biochemistry 2010, 49, 4794–
4803.
Finally we carried out preliminary photoaffinity labeling with
bovine heart SMP. The photoaffinity labeling was performed
according to the procedures described previously.9,10 The complex
I photocross-linked by 6 nM [125I]DANA, which was isolated by
electroelution after Blue Native-PAGE, was subjected to SDS–PAGE.
Although bovine heart mitochondrial complex I is composed of
different 45 subunits,6,7 a single protein band (ꢂ30 kDa) was
cross-linked by [125I]DANA with high specificity ( Fig. 2A). The
cross-linking was completely blocked by 50-fold bullatacin (data
not shown), indicating competitive behavior between the two
inhibitors. The apparent molecular mass suggests that this protein
11. Sinha, P. K.; Torres-Bacete, J.; Nakamaru-Ogiso, E.; Castro-Guerreo, N.;
Matsuno-Yagi, A.; Yagi, T. J. Biol. Chem. 2009, 284, 9814–9823.
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Bioorg. Med. Chem. Lett. 2004, 14, 779–782.
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Chem. Soc. 1991, 113, 9369–9371; (b) Motoyama, T.; Yabunaka, H.; Miyoshi, H.
Bioorg. Med. Chem. Lett. 2002, 12, 2089–2092.
14. Fried, J.; Lin, C.-H.; Ford, S. H. Tetrahedron Lett. 1969, 18, 1379–1381.
15. Makabe, H.; Tanaka, A.; Oritani, T. J. Chem. Soc. Perkin Trans. 1 1994, 1975–
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17. MacKinnon, A. L.; Garrison, J. L.; Hedge, R. S.; Taunton, J. J. Am. Chem. Soc. 2007,
129, 14560–14561.
18. Spectral data for DANA: a colorless waxy oil; ½a D25
ꢃ
+13.2 (c, 0.11, EtOH); 1H NMR
(400 MHz/CDCl3) d 7.53 (d, J = 9.0 Hz, 2H), 6.98 (d, J = 0.8 Hz, 1H), 6.66 (d,
J = 9.0 Hz, 2H), 4.99 (qd, J = 6.8, 1.7 Hz, 1H), 3.98 (dt, J = 6.6, 6.6 Hz, 1H), 3.91
(dt, J = 1.5, 6.5 Hz, 2H), 3.84 (ddt, J = 5.8, 5.8, 5.8 Hz, 1H), 3.80 (m, 1H), 3.70 (m,
1H), 3.38 (m, 1H), 2.44 (br d, 1H), 2.26 (t, J = 7.5 Hz, 2H), 2.01–1.87 (m, 3H),
1.87–1.70 (m, 3H), 1.70–1.38 (m, 17H, including a doublet at 1.41, J = 6.8 Hz,
3H), 1.38–1.13 (m, 12H), 1.08–0.95 (m, 2H); 13C NMR (125 MHz/CDCl3) d
174.00, 159.06, 148.96, 138.23 (2C), 134.40, 117.01 (2C), 83.12, 82.41, 81.58,
81.52, 79.26, 78.98, 77.49, 74.00, 68.05, 33.41, 29.70, 29.50, 29.42, 29.40, 29.34,
29.30, 29.24, 29.18, 28.83, 28.42, 27.81, 27.72, 27.48, 26.14, 25.46, 25.26, 23.61,
19.31; MS (ESI) m/z 709.3 [(MꢁN2)+H]+, 737.4 [M+H]+, 759.3 [M+Na]+; HR-MS
(ESI) m/z calcd for C36H53IN2O6Na [M+Na]+ 759.2841; found 759.2846, calcd for
C
36H52IN2O6 [MꢁH]ꢁ 735.2876; found 735.2906. Compound 11: a colorless
waxy oil; 1H NMR (400 MHz/CDCl3) d 7.72 (d, J = 8.6 Hz, 2H), 6.98 (d, J = 1.5 Hz,
1H), 6.87 (d, J = 8.7 Hz, 2H), 4.99 (qd, J = 6.8, 1.7 Hz, 1H), 3.99 (m, 1H), 3.98 (t,
J = 6.5 Hz, 2H), 3.84–3.78 (m, 2H), 3.75 (s, 3H), 3.74 (m, 1H), 3.71 (m, 1H), 3.38
(m, 1H), 2.41 (br s, 1H), 2.26 (t, J = 8.0 Hz, 2H), 1.99–1.87 (m, 3H), 1.87–1.76 (m,
3H), 1.76–1.37 (m, 17H, including a doublet at 1.41, J = 6.8 Hz, 3H), 1.37–1.13
a
singlet at 1.02, 6H); 13C NMR
Figure 2. Photoaffinity labeling of bovine submitochondrial particles (SMP). (A) The
complex I photocross-linked by [125I]DANA (15 nM [125I]DANA at 1.5 mg of protein/
mL) was isolated from the preparative BN gel by electroelution. Then the isolated
(m, 12H), 1.08–0.95 (m, 8H, including
(125 MHz/CDCl3) d 171.76, 161.43, 149.00, 135.56 (2C), 134.45, 125.00, 113.77
(2C), 108.91, 83.16, 81.56, 81.49, 78.99, 77.51, 74.02, 72.33 (2C), 67.69, 33.41,
31.97, 30.75, 29.72, 29.50, 29.48, 29.40, 29.34, 29.26, 29.24, 28.81, 28.42, 27.80,
27.71, 27.47, 26.21, 25.48, 25.25, 23.61, 22.01 (2C), 19.31; MS (ESI) m/z 677.4
[(MꢁN2ꢁH2O)+H]+; HRMS (ESI) m/z calcd for C41H62BN2O7 [(MꢁH2O)+H]+
705.4645; found 705.4622, calcd for C41H62BO7 [(MꢁN2ꢁH2O)+H]+ 677.4583;
found 677.4575.
complex I was analyzed by SDS–PAGE on a 12.5% Laemmli’s gel (equivalent to 50 lg
of SMP protein/lane), followed by CBB staining (left) and autoradiography (right).
(B) For the identification of the 30 kDa protein cross-linked by [125I]DANA, the
isolated complex I was subjected to immunoprecipitation with the bovine ND1
antibody as described in Refs. 9 and. 10

S. Yamamoto et al. / Tetrahedron Letters 52 (2011) 3090–3093
3093
19. Azizian, H.; Eaborn, C.; Pidcock, A. J. Organomet. Chem. 1981, 215, 49–58.
20. (a) Beletskaya, I. P. J. Organomet. Chem. 1983, 250, 551–564; (b) Deng, H.;
Gifford, A. N.; Zvonok, A. M.; Cui, G.; Li, X.; Fan, P.; Deschamps, J. R.; Flippen-
Anderson, J. L.; Gateley, S. J.; Makriyannis, A. J. Med. Chem. 2005, 48, 6386–
6392.
the resulting mixture was extracted with chloroform (100
combined organic layer was concentrated using vacuum-centrifugal
evaporator. The crude product was dissolved in 90% MeOH/H2O (50 ll) and
subjected to HPLC (Shimadzu LC-10 AS) purification using a C18 column
(COSMOSIL 5C18-MS-II, 4.6 ꢄ 150 mm, Nacalai Tesque) at a flow rate of 0.8 ml/
min with isocratic 95% MeOH/water system as an eluent. The fraction was
l
l ꢄ 3). The
21. (a) Kabalka, G. W.; Akula, M. R.; Zhang, J. Nucl. Med. 2002, 29, 841–843; (b)
Kabalka, G. W.; Akula, M. R.; Zhang, J. Nucl. Med. 2003, 30, 369–372.
collected every 30 s (400 ll) and the radioactivity was measured by c-counting
22. To a solution of 11 (1.0 mM in 50% THF/H2O, 25
plastic-tube was added [125I]NaI (Perkin Elmer, NEZ033A, 37 MBq, 10
radioiodination was initiated by adding freshly prepared aqueous chloramine-
T (10 mg/ml, 20 l), and the mixture was incubated at room temperature for
10 min. The reaction was terminated with aqueous NaHSO3 (10%, 100 l), and
l
l) in screw-capped 1.5 ml
system (COBRA™ II, Packard). The strong radioactive fractions, corresponding
to a retention time of DANA (14.7 min), were combined and concentrated using
vacuum-centrifugal evaporator (Fig. S1).
ll). The
l
23. Ramesh, C.; Nayak, T. K.; Burai, R.; Dennis, M. K.; Hathaway, H. J.; Sklar, L. A.;
Prossnitz, E. R.; Arterburn, J. B. J. Med. Chem. 2010, 53, 1004–1014.
l