Concise procedure for the synthesis of cardiolipins having different fatty acid combinations

Article abstract of DOI:10.1016/j.tetlet.2010.02.071

Production of a wide variety of cardiolipin (CL) analogues is critical for studying molecular mechanism of diverse biological functions of CL in mitochondria. We describe a concise procedure for the synthesis of CL using a phosphoramidite approach, which

Full text of DOI:10.1016/j.tetlet.2010.02.071

Tetrahedron Letters 51 (2010) 2071–2073
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Concise procedure for the synthesis of cardiolipins having different fatty
acid combinations
*
Masato Abe, Shigeki Kitsuda, Shunji Ohyama, Shinya Koubori, 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:
Production of a wide variety of cardiolipin (CL) analogues is critical for studying molecular mechanism of
diverse biological functions of CL in mitochondria. We describe a concise procedure for the synthesis of
CL using a phosphoramidite approach, which allows for the production of diverse CL analogues bearing
linoleic acid(s) at any position on the glycerol backbone.
Received 20 January 2010
Revised 11 February 2010
Accepted 15 February 2010
Available online 18 February 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Cardiolipin (CL, 1 in Scheme 1), a negatively charged phospho-
lipid bearing four fatty acid chains, is a major phospholipid found
in mammalian mitochondria (up to 20–25%) with a multitude of
biological functions (reviewed in Refs. 1,2). For instance, CL is
responsible for regulation of the activity of several mitochondrial
enzymes involved in ATP biosynthesis,3,4 though the precise
molecular mechanism of its regulation remains to be elucidated.
Recently, Kagan and co-workers demonstrated that in the early
stages of apoptosis, cytochrome c (cyt c) bound to the CL-contain-
ing mitochondrial inner membrane acts as a peroxidase that selec-
tively catalyzes CL peroxidation: this event contributes to the
release of cyt c into the cytosol, and initiation of the apoptotic pro-
gram.5,6 Although the fatty acid composition of CL is suggested to
be critical for the formation of the cyt c-CL complex to yield the
peroxidase activity,6 the molecular mechanism has not been suffi-
ciently studied. This is primarily because CL analogues used in the
previous biochemical studies are limited to natural and/or a few
commercially available CL analogues; in the former, the chain moi-
ety is a mixture of various fatty acids, and in the latter, the chem-
ical variation of fatty acid chains is very poor. Therefore, to explore
in detail the molecular mechanisms of both the formation of the
cyt c-CL complex and the induction of peroxidase activity of cyt
c, biochemical studies using structurally variable CL analogues
are needed.
preparation of large quantities owing to use of highly unstable
intermediates or expensive reagents (e.g., Refs. 7c,d).
The phosphoramidite approach, widely exploited in oligonu-
cleotide chemistry (e.g., Ref. 8), described by Ahmad and col-
leagues is an excellent way to obtain large quantities of CL
analogues in high yields.9 Unfortunately, they did not use linoleic
acid as the acyl chain(s), and their procedure does not give asym-
metrically substituted CL analogues. We herein describe a concise
procedure for the synthesis of CL using phosphoramidite chemis-
try, which produces diverse CL analogues bearing linoleic acid(s)
at any position of the four acyl chains on a gram scale. This ap-
proach also allows for the production of CL containing a biophysi-
cal probe (nitroxide spin-label, fluorescent label, etc.) in one of the
four chains.
Several procedures for the synthesis of CL have been reported.7
Previous studies however were not necessarily concerned with
generating structurally diverse CL analogues. For example, some
procedures (e.g., Ref. 7e) for the synthesis of CL bearing only satu-
rated fatty acid chains are not suitable for the synthesis of CL con-
taining linoleic acid(s) (C18:2), which is a major fatty acid of
natural CL in mammalian mitochondria,2 because the cis-1,4-diene
structure in linoleic acid is remarkably degradable under the con-
ditions. In addition, some methods are not feasible for the routine
Scheme 1. Retrosynthetic analysis of cardiolipin bearing four different acyl chains.
* Corresponding author. Tel.: +81 75 753 6119; fax: +81 75 753 6408.
E-mail address: miyoshi@kais.kyoto-u.ac.jp (H. Miyoshi).
R1–R4: fatty acids.
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.02.071

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M. Abe et al. / Tetrahedron Letters 51 (2010) 2071–2073
Retrosynthetic analysis of CL (1) bearing four different acyl
of the cis-1,4-diene structure. Removal of the PMB group with
the frequently used reagent DDQ in wet CH2Cl2 (0.5% water in vol-
ume) at room temperature resulted in low yields of the product
(30–40%). Attempts to improve the yield were unsuccessful and
only led to partial decomposition of the double bonds. We also
examined the protection of 6 with a silyl group (TBS or TBDPS)
and its deprotection from the corresponding 1,2-di-O-acyl-3-O-
silylated glycerol with TBAF, whereas significant migration after
the deprotection occurred under the basic conditions.10
chains (R1–R4) gives two phosphoramidite fragments, 3 and 4,
and a diprotected glycerol fragment, 5 (Scheme 1). The choice of
protection groups for alcohol and phosphate functionalities is crit-
ical in the synthesis of CL bearing an unsaturated acyl chain(s). To
find milder reaction conditions for avoiding the decomposition of
an unsaturated acyl chain(s), we introduced a sensitive linoleic
acid into all four positions, and carefully checked the cis-1,4-diene
structure by 1H and 13C NMR spectroscopy. All procedures for the
reaction and purification were performed with shielding from
light.
Coupling of the 1,2-di-O-acyl-sn-glycerol 11a with bifunctional
N,N-diisopropylmethylphosphonamidic chloride in the presence of
DIPEA (1.2 equiv) afforded the 1,2-diacylglyceryl (N,N-diisopropyl-
amino)phosphoramidite 4, which was used as such for the next
reaction without any purification. Subsequent coupling of 4 with
the TBS-protected glycerol 5 in the presence of 1H-tetrazole
(1.2 equiv) as a promoter of the reaction8 gave a phosphite triester
intermediate, which was oxidized in situ with nBu4NIO4 at À20 °C
to afford intermediate 12 in moderate yield after column purifica-
tion. Oxidation by the frequently used reagent t-BuOOH9,10,12 re-
sulted in slight, but not negligible, decomposition of the double
bonds in the acyl chains. Treatment of 12 with HF-pyridine/pyri-
dine/dry-THF (1:2:5, v/v) for 1.5 h at 0 °C selectively removed the
TBS group of the primary alcohol to afford the phosphatidylglyc-
erol methyl ester 2 in 63% yield along with a byproduct (20%)
due to cleavage of both TBS groups; however, this byproduct was
recycled through reprotection with TBSCl to give 12. Using differ-
ently protected glycerol (2-O-Bn-3-O-TBS-(2S)-sn-glycerol and 3-
O-TBS-2-O-PMB-(2S)-sn-glycerol) in place of 5, we actually pre-
pared the corresponding phosphatidylglycerol methyl esters, and
examined the sequential procedures. As a result, the reason for
selecting the TBS group to protect the secondary alcohol in 12 is
due to its satisfactory removal in a final reaction step l, as de-
scribed later.
PMB protection of the commercially available (S)-1,2-O-isopro-
pylidene-sn-glycerol 6 followed by acid-catalyzed hydrolysis of the
acetal afforded, in good isolated yield, diol 7,7e,10 a key intermedi-
ate for all three fragments (Scheme 2). DCC-mediated esterification
of 7 with 0.4 equiv of linoleic acid selectively afforded the monoes-
ter 9 without migration of the acyl group, and no reacted 7 was
recycled. Subsequent esterification of
9 with linoleic acid
(1.2 equiv) quantitatively afforded the diester 10. Using desired
fatty acids in place of linoleic acid, the diester having different acyl
chains in the sn-1- and sn-2-glycerol positions can be prepared. It
is noteworthy that installation of the alkyl group into the glycerol
building block is extremely difficult once the phosphate moiety has
been constructed.10
Removal of the protection group of the sn-3 position in 1,2-dia-
cyl-sn-glycerol is a delicate step for the synthesis of phospholipids
because of migration of the acyl group from the sn-2 position to the
sn-3 position during the reaction or isolation conditions.10–12 The
acyl chain migration can be determined accurately by 1H
NMR.10–12 Through careful examination of several reaction condi-
tions, we found that the treatment of 10 with 10 equiv of CAN in
CH3CN/H2O (9:1, v/v) at room temperature affords 11a in high
yield without migration of the acyl group and the decomposition
Scheme 2. Reagents and conditions: (a) (i) PMBCl, NaH, DMF, rt, 2 h; (ii) AcOH/H2O (4:1, v/v), 50 °C, 2 h, 85% (two steps); (b) TBSCl, imidazole, CH2Cl2, 70 °C, 2.5 h, 97%; (c)
DDQ, CH2Cl2/H2O (20:1, v/v), rt, 30 min, 88%; (d) R1COOH (0.4 equiv), DCC, DMAP, CH2Cl2, 0 °C, 6 h, 84% (65% of 7 recovered); (e) R2COOH (1.2 equiv), DCC, DMAP, CH2Cl2, rt,
2.5 h, 99%; (f) CAN (10 equiv), CH3CN/H2O (9:1, v/v), rt, 1.5 h, 93%; (g) (i-Pr)2NP(OMe)Cl (1.2 equiv), DIPEA (1.2 equiv), CH2Cl2, rt, 1 h; (h) (i) 5, 1H-tetrazole (1.2 equiv), CH3CN,
rt, 2.5 h; (ii) Bu4NIO4, CH2Cl2, À20 °C to rt, 2 h, 70% (three steps); (i) HF-pyridine/pyridine/THF (1:2:5), rt, 1 h, 63%; (j) (i-Pr)2NP(OMe)Cl (1.2 equiv), DIPEA (1.2 equiv), CH2Cl2,
rt, 1 h; (k) (i) 2, 1H-tetrazole (1.2 equiv), CH3CN, rt, 2.5 h; (ii) Bu4NIO4, CH2Cl2, À20 °C to rt, 2 h, 55% (three steps); (l) (i) NaI (2.5 equiv), 2-butanone, reflux, 1 h; (ii) 1.0 M HCl/
H2O/THF (0.1:1:2, v/v), rt, 13 h, then 25% NH4OH aq, 71% (two steps).

M. Abe et al. / Tetrahedron Letters 51 (2010) 2071–2073
2073
3. Lange, C.; Nett, J. H.; Trumpower, B. L.; Hunte, C. EMBO J. 2001, 20, 6591–6600.
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Zou, M.; Di, P.; Svistunenko, D. A.; Kurnikov, I. V.; Borisenko, G. G. Nat. Chem.
Biol. 2005, 1, 223–232.
With half of the CL molecule (2) in hand, the whole molecule is
constructed as follows. The desired 1,2-diacyl-sn-glycerol (11b)
prepared by the method described above was reacted with N,N-
diisopropylmethylphosphonamidic chloride in the presence of DI-
PEA to give 1,2-diacyl-sn-glyceryl (N,N-diisopropylamino)phos-
phoramidite (3). To this intermediate was added
a CH2Cl2
6. Belikova, N. A.; Vladimirov, Y. A.; Osipov, A. N.; Kapralov, A. A.; Tyurin, V. A.;
Potapovich, M. V.; Basova, L. V.; Peterson, J.; Kurnikov, I. V.; Kagan, V. E.
Biochemistry 2006, 45, 4998–5009.
solution of 2 in the presence of 1H-tetrazole, followed by in situ
oxidation with nBu4NIO4 at À20 °C to afford a phosphotriester of
the CL precursor 13 in a moderate yield. Deprotection of the phos-
phate function in 13 has to be carried out prior to the removal of
the TBS group to avoid migration of the phosphate.9d Therefore,
the methyl groups in 13 were cleaved with sodium iodide
(2.5 equiv) in refluxing 2-butanone. Finally, removal of the TBS
group was accomplished by treating the resultant phosphate in
1.0 M HCl/THF/H2O (0.1:2:1, v/v) without any decomposition of
the double bonds, and converted into the ammonium salt 1 by
treatment with 0.1 M HCl followed by 25% ammonium hydrox-
ide.9c,13 For the synthesis of unsaturated CL [tetraoleoyl (C18:1)-
CL], Ahmad and co-workers used a levulinoyl group as a protection
group of the secondary alcohol of the glycerol bridge in the CL pre-
cursor,9a,d corresponding to 13 in our study, and performed depro-
tection of this group by hydrazinolysis14 with hydrazine in
pyridine. The application of this method, however, to the demethy-
lated 13 resulted in about 10% decomposition of the double bonds.
CL contains two 1,2-diacyl-sn-glycero-3-phosphoryl moieties
linked by a glycerol bridge, one phosphatidyl moiety is in pro-R
and the other is in pro-S position with respect to the central carbon
atom of the glycerol bridge. Obviously, the central carbon atom be-
comes a true chiral center if the two phosphatidyl residues contain
different fatty acids.
7. (a) Ramirez, F.; Ioannou, P. V.; Marecek, J. F.; Dodd, G. H.; Golding, B. T.
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Dale, M. P.; Weintraub, S. T.; Robinson, N. C. Biochemistry 2006, 45, 746–754;
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6923–6925; (c) Lin, Z.; Ahmad, M. U.; Ali, S. M.; Ahmad, I. Lipids 2004, 39, 285–
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Ahmad, I. Tetrahedron 2006, 62, 6990–6997.
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3837–3840.
13. The data for 1: FTIR (ATR): 3211, 3009, 2925, 2854, 1739, 1457, 1377, 1211,
1064 cmÀ1 1H NMR (400 MHz, CD3OD) d 5.39–5.28 (m, 16H), 5.23 (m, 2H),
;
4.45 (dd, J = 12.0, 3.1 Hz, 2H), 4.19 (dd, J = 12.1, 6.9 Hz, 2H), 3.99 (dd, J = 5.5,
5.5 Hz, 4H), 3.95–3.88 (m, 5H), 3.60 (br s, 1H), 2.77 (t, J = 6.3 Hz, 8H), 2.34 (t,
J = 7.4 Hz, 4H), 2.31 (t, J = 7.4 Hz, 4H), 2.06 (dt, J = 7.0, 7.0 Hz, 16H), 1.68–1.56
(m, 8H), 1.43–1.26 (m, 56H), 0.91 (t, J = 6.9 Hz, 12H); 13C NMR (125 MHz,
CD3OD) d 173.50 (2C), 173.18 (2C), 129.64 (4C), 129.57 (4C), 127.82 (4C),
127.25 (4C), 78.16 (2C), 70.80, 66.32 (2C), 63.44 (2C), 62.43 (2C), 33.70 (2C),
33.55 (2C), 31.39 (4C), 29.48 (4C), 29.20 (4C), 29.13 (4C), 29.08 (4C), 28.99 (4C),
28.92 (4C), 26.90 (4C), 25.29 (4C), 24.71 (4C), 22.35 (4C), 13.19 (4C); 31P NMR
References and notes
(162 MHz, CD3OD)
d 0.94 (2P); HRMS (ESI) calcd for C81H141O17P2
(MÀ2NH3ÀH)À 1447.9649; found 1447.9594, calcd for C81H140O17P2
(MÀ2NH3À2H)2À 723.4788; found 723.4750.
1. Hatch, G. M. Biochem. Cell. Biol. 2004, 82, 99–112.
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14. Van Boom, J. H.; Burgers, P. M. J. Tetrahedron Lett. 1976, 17, 4875–4878.