pubs.acs.org/joc
the NPE chromophore has only weak absorption above
Caged Ceramide 1-Phosphate Analogues: Synthesis
and Properties
350 nm, cages with more attractive photophysical pro-
perties have been developed. For example, derivatives of
coumarins3 and quinolines4 are rapidly uncaged using two-
photon activation with IR laser light in tiny excitation
volumes and may be used in vivo, since the photolytic
conditions are noninvasive and permit spatial and temporal
control of the uncaging process.
Ravi S. Lankalapalli,† Alberto Ouro,‡ Lide Arana,‡
‡
Antonio Gomez-Munoz, and Robert Bittman*
,†
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†Department of Chemistry and Biochemistry, Queens College
of The City University of New York, Flushing, New York
11367-1597, and ‡Department of Biochemistry and Molecular
Biology, University of the Basque Country, Bilbao, Spain
The phosphorylated sphingolipid metabolites sphingosine
1-phosphate (S1P) and ceramide 1-phosphate (C1P) are
mediators of a multitude of cellular activities.5 Most of the
activities of S1P arise from its action as an extracellular
signaling molecule following binding to a family of five
G-protein coupled receptors at the cell surface.6 In contrast
to S1P, C1P functions as a lipid messenger mainly at the
intracellular level.7 Natural C1P is not taken up readily by
cells in culture unless it is dispersed in organic solvents such
as EtOH/dodecane (49:1), which may allow incorporation of
other extracellular compounds. C1P regulates diverse cellu-
lar functions, including arachidonic acid release, mast cell
degranulation, Ca2þ mobilization, translocation of lipid-
metabolizing enzymes, vesicular trafficking, cell prolifera-
tion, and cell survival.8 Our investigations of cellular res-
ponses to intracellular C1P have been limited to the addition
of unnatural short-chain amide analogues of C1P, such as N-
acetyl- and N-octanoyl-C1P, as no caged C1P derivative has
yet been reported. In fact, the only caged sphingolipid
derivatives that have been reported are S1P analogues that
contain the NPE chromophore.9
Received September 29, 2009
Sphingolipid phosphate analogues bearing 7-(diethylamino)-
coumarin (DECM) and 4-bromo-5-hydroxy-2-nitrobenzhy-
dryl (BHNB) groups in a photolabile ester bond were
synthesized. The ability of the “caged” ceramide 1-phosphate
analogues to release the bioactive parent molecule upon
irradiation at 400-500 nm was demonstrated by stimulation
of macrophage cell proliferation.
We report here the synthesis of caged S1P (1, 2) and C1P (1a,
2a) analogues in which the phosphate headgroup is esterified to
a photolabile 7-(diethylamino)coumarin (DECM) or 4-bromo-
5-hydroxy-2-nitrobenzhydryl (BHNB) group (Figure 1). We
(3) For references about coumarin-4-yl methyl esters of nucleotides and
amino acid derivatives, see: Shembekar, V. R.; Chen, Y.; Carpenter, B. K.;
Hess, G. P. Biochemistry 2007, 46, 5479 and references therein.
(4) For references about the use of 8-bromo-2-hydroxyquinoline deriva-
tives as cages, particularly for carboxylic acids, see: (a) Davis, M. J; Kragor,
C. H.; Reddie, K. G.; Wilson, H. C.; Zhu, Y.; Dore, T. M. J. Org. Chem.
2009, 74, 1721. (b) An, H.-Y.; Ma, C.; Nganga, J. L.; Zhu, Y.; Dore, T. M.;
Phillips, D. L. J. Phys. Chem. A 2009, 113, 2831.
“Caging” is a strategy employed in biochemistry, neuro-
biology, and physiology for the in vitro investigation of the
cellular activity of cell-impermeable bioactive molecules.1
The ionic groups in charged, hydrophilic molecules are
temporarily masked by a covalent link to a photolabile
moiety, thereby facilitating cell delivery and bypass of cell-
surface receptors. The bioactive molecule is released in the
cytosol on photolysis using light that does not damage
cellular components. The first caged biomolecules reported,
the intracellular messengers cyclic-AMP2a and ATP,2b in-
corporated o-nitrobenzyl phosphate esters as the photolabile
moiety. Subsequently, the 1-(2-nitrophenyl)ethyl (NPE)
group and derivatives containing additional hydroxy or
methoxy substituents were used as caging addends.1b,2c Since
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(5) (a) Gomez-Munoz, A.; Steinbrecher, U. P. Recent Res. Dev. Lipids
2004, 7, 65. (b) Kihara, A.; Mitsutake, S.; Mizutani, Y.; Igarashi, Y. Prog.
Lipid Res. 2007, 46, 126. (c) Hannun, Y. A.; Obeid, L. M. Nat. Rev. Molec.
Cell Biol. 2008, 9, 139.
(6) S1P signaling also appears to take place by nonreceptor mechanisms
when intracellularly generated S1P binds to unidentified cytosolic targets: (a)
Van Brocklyn, J. R.; Lee, M. J.; Menzeleev, R.; Olivera, A.; Edsall, L.; Cuvillier,
O. J. Cell Biol. 1998, 142, 229. For a recent review of dual activities of S1P, see:
Pyne, N. G.; Long, J. S.; Lee, S. C.; Loveridge, C.; Gillies, L.; Pyne, S. Adv.
Enzyme Regul. 2009, 49, 214.
(7) Addition of exogenous C1P is not an ideal method for increasing the
intracellular C1P concentration. C1P may interact with a cell-surface recep-
tor, triggering effects independent of C1P intracellular accumulation. For a
report of C1P-mediated cell migration via interaction with a putative plasma
membrane receptor, see: Granado, M. H.; Gangoiti, P.; Ouro, A.; Arana, L.;
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Gonzalez, M.; Trueba, M.; Gomez-Munoz, A. Cell. Signalling 2009, 21, 405.
(8) (a) Chalfant, C. E.; Spiegel, S. J. Cell Sci. 2005, 118, 4605. (b)
Gangoiti, P.; Granado, M. H.; Wang, S. W.; Kong, J. K.; Steinbrecher, U.
(1) For recent reviews of caged biomolecules, see: (a) Mayer, G.; Heckel,
A. Angew. Chem., Int. Ed. 2006, 45, 4900. (b) Ellis-Davies, G. C. R. Nat.
Methods 2007, 4, 619. (c) Ellis-Davies, G. C. R. Chem. Rev. 2008, 108, 1603.
(2) (a) Engels, J.; Schlaeger, E.-J. J. Med. Chem. 1977, 20, 907. (b)
Kaplan, J. H.; Forbush, B., 3rd; Hoffman, J. F. Biochemistry 1978, 17,
1929. (c) Nerbonne, J. M.; Richard, S.; Nargeot, J.; Lester, H. A. Nature
1984, 310, 74.
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P.; Gomez-Munoz, A. Cell. Signalling 2008, 20, 726.
(9) For references on NPE derivatives of sphingosine and S1P, see: (a)
Zehavi, U. Chem. Phys. Lipids 1997, 90, 55. (b) Qiao, L.; Kozikowski, A. P.;
Olivera, A.; Spiegel, S. Bioorg. Med. Chem. Lett. 1998, 8, 711. (c) Scott, R. H.;
Pollock, J.; Ayar, A.; Thatcher, N. M.; Zehavi, U. Methods Enzymol. 2000,
312, 387.
8844 J. Org. Chem. 2009, 74, 8844–8847
Published on Web 10/26/2009
DOI: 10.1021/jo902076w
r
2009 American Chemical Society