C O M M U N I C A T I O N S
phosphate portion of the headgroup and apparently cannot be
replaced by simple amide, alcohol, or amine moieties. A future
goal is to develop cholate-derived translocases with increased
activity for phosphatidylserine and phosphatidylethanolamine. Our
previous success with the tren-derived translocases,5c plus the
knowledge that naturally occurring binders of phosphatidylserine13
and phosphatidylethanolamine14 are known, augurs well for this
effort.
Acknowledgment. This work was supported by the National
Institutes of Health (GM 59078), the Walther Cancer Research
Center, the University of Notre Dame, the European Commission,
and the EPSRC (GR/R04584/01).
Supporting Information Available: Synthetic procedures, UV
titrations, and translocation data (PDF). This material is available free
Figure 1. Percent exo PC-NBD in the outer leaflet of POPC vesicles (25
µM) containing 5 mol % of 1 ([), 2 (O), 3 (0,), 4 (×), or 5 (4) in 5 mM
TES/100 mM NaCl buffer at pH 7.4, 25 °C. Each point represents the
average of three separate experiments with an uncertainty of (4 percentage
units.
References
(1) (a) Rothman, J. E.; Dawidowicz, E. A. Biochemistry 1975, 14, 2809-
2816. (b) Moss, R. A. Pure Appl. Chem. 1994, 66, 851-858. (c) Kornberg,
R. D.; McConnell, H. M. Biochemistry 1971, 10, 1111-1120.
(2) Daleke, D. L.; Lyles, J. V. Biochim. Biophys. Acta 2000, 1486, 108-
127.
(3) (a) Boon, J. M.; Smith, B. D. Med. Res. ReV. 2002, 22, 251-281. (b)
Fadok, V. A.; de Cathelineau, A.; Daleke, D. L.; Henson, P. M.; Bratton,
D. L. J. Biol. Chem. 2001, 276, 1071-1077. (c) Bevers, E. M.; Comfurius,
P.; Dekkers, D. W. C.; Harmsma, M.; Zwaal, R. F. A. Biol. Chem. 1998,
379, 973-986. (d) Bevers, E. M.; Comfurius, P.; Dekkers, D. W. C.;
Harmsma, M.; Zwaal, R. F. A. Lupus 1998, 7, S126-S131. (e) Bratton,
D. L.; Fadok, V. A.; Richter, D. A.; Kailey, J. M.; Guthrie, L. A.; Henson,
P. M. J. Biol. Chem. 1997, 272, 26159-26165.
(4) Recently, one of our tren-derived translocases was used as a biological
tool in the study of the peroxisome proliferator-activated receptor γ
(PPARγ). Davies, S. S.; Pontsler, A. V.; Marathe, G. K.; Harrison, K.
A.; Murphy, R. C.; Hinshaw, J. C.; Prestwich, G. D.; Hilaire, A. St.;
Prescott, S. M.; Zimmerman, G. A.; McIntyre, T. M. J. Biol. Chem. 2001,
276, 16015-16023.
(5) (a) Boon, J. M.; Smith, B. D. J. Am. Chem. Soc. 2002, 124, 11924-
11925. (b) Boon, J. M.; Smith, B. D. J. Am. Chem. Soc. 2001, 123, 6221-
6226. (c) Boon, J. M.; Shukla, R.; Smith, B. D.; Licini, G.; Scrimin, P.
Chem. Commun. 2002, 260-261. (d) Boon, J. M.; Lambert, T. N.; Smith,
B. D.; Beatty, A. M.; Ugrinova, V.; Brown, S. N. J. Org. Chem. 2000,
67, 2168-2174..
In an effort to gain structural insight, a series of NMR titration
studies were conducted.5b The binding of POPC to 1 or 2 in CDCl3
is too strong (>105 M-1) to be determined quantitatively by NMR;
however, the complexed-induced changes in chemical shift are
consistent with the 1:1 supramolecular complex I.10 In the case of
bis(amide) 3, a relatively weak POPC association constant of 44
M-1 was obtained under the same conditions. Attempts to measure
binding of phosphatidylethanolamine and phosphatidylserine by
NMR titration methods were hampered by self-aggregation of these
aminophospholipids, although qualitatively it appeared that binding
to the translocases was quite weak. This conclusion was confirmed
by a series of UV titration studies in 99:1 CHCl3/CH3OH. Addition
of POPC to translocase 1 produces an increase in translocase
absorbance, and an association constant of (1.2 ( 0.1) × 105 M-1
was extracted using nonlinear methods.11,12 In contrast, addition of
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) or
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) to 1 pro-
duced such slight changes in absorbance that association constants
could not be determined reliably. We tentatively attribute this very
weak binding to inter- or intramolecular hydrogen bonding involv-
ing the cationic ammonium and anionic phosphate (and carboxylate)
residues.
(6) For recent examples of cholic acid derived supramolecular devices, see:
(a) Janout, V.; Staina, I. V.; Bandyopadhya, P.; Regen, S. L. J. Am. Chem.
Soc. 2001, 123, 9926-9927. Bandyopadhya, P.; Janout, V.; Zhang, L.-
H.; Regen, S. L. J. Am. Chem. Soc. 2001, 123, 7691-7696. (b) Goto, C.;
Yamamura, M.; Satake, A.; Kobuke, Y. J. Am. Chem. Soc. 2001, 123,
12152-12159. (c) Vandenburg, Y. R.; Smith, B. D.; Pe´rez-Paya´n, M.
N.; Davis, A. P. J. Am. Chem. Soc. 2000, 122, 3252-3253. De Muynck,
H.; Madder, A.; Farcy, N.; De Clerq, P. J.; Pe´rez-Paya´n, M. N.; O¨ hberg,
L. M.; Davis, A. P. Angew. Chem., Int. Ed. 2000, 39, 145-148. (d) Guan,
Q.; Li, C.; Schmidt, E. J.; Boswell, J. S.; Walsh, J. P.; Allman, G. W.;
Savage, P. B. Org. Lett. 2000, 2, 2837-2840. (e) Cheng, Y.; Suenaga,
T.; Still, W. C. J. Am. Chem. Soc. 1996, 118, 1813-1814.
(7) (a) Davis, A. P.; Pe´rez-Paya´n, M. N. SynLett 1999, 991-993. (b) Ayling,
A. J.; Pe´rez-Paya´n, M. N.; Davis, A. P. J. Am. Chem. Soc. 2001, 123,
12716-12717.
(8) (a) McIntyre, J. C.; Sleight R. G. Biochemistry 1991, 30, 11819-11827.
(b) Moss, R. A.; Bhattacharya, S. J. Am. Chem. Soc. 1995, 117, 8688-
8689.
(9) For synthetic translocases that operate by generating local “flip sites”,
see: (a) Bhattacharya, S.; Moss, R. A.; Ringsdorf, H.; Simon, J. Langmuir
1997, 13, 1869-1872. (b) Kol, M. A.; De Kroon, A. I. P. M.; Rikjers, D.
T. S.; Killian, J. A.; De Kruijff, B. Biochemistry 2001, 40, 10500-10506.
(10) Addition of POPC to 1 or 2 induces all four urea protons to undergo
large downfield movements in chemical shift (∼2 ppm) and substantial
changes in the cholate N-phenyl signals. The sharp titration end point
clearly indicates a 1:1 stoichiometry, but we presently cannot rule out an
alternative complex structure described in Bu¨hlmann, P.; Nishizawa, S.;
Xiao, K. P.; Umezawa, Y. Tetrahedron 1997, 53, 1647-1654.
(11) Xie, H.; Yi, S.; Wu, S. J. Chem. Soc., Perkin Trans. 2 1999, 2751-2754.
(12) A similar association constant has been obtained with an alternative
synthetic phosphatidylcholine receptor. Magrans, J. O.; Ortiz, A. R.;
Molins, M. A.; Lebouille, P. H. P.; Sa´nchez-Quesada, J.; Prados, P.; Pons,
M.; Gago, F.; De Mendoza, J. Angew. Chem., Int. Ed. Engl. 1996, 35,
1712-1715.
For employment as a pharmaceutical or as a reagent for cell
biology,4 a synthetic translocase needs to be formulated so that it
can be delivered to a biological sample. With this goal in mind we
prepared the partially water-soluble, methyl cholate bis(phenylurea)
6 and evaluated its ability to promote PC-NBD translocation across
erythrocyte cell membranes. As shown in the Supporting Informa-
tion, cholate-derived 6 is significantly more effective as a phos-
phatidylcholine translocase than our previously reported tren-derived
sulfonamide system.
In summary, cholate bis(phenylureas) are highly effective
promoters of phosphatidylcholine translocation across vesicle and
cell membranes. They are approximately an order of magnitude
more active than our previously reported tren-derived systems.5 The
urea side chains are essential for strong binding of the highly polar
(13) (a) Williamson, P.; Van Den Eijnde, S.; Schlegel, R. A. Methods Cell
Biol. 2001, 66, 339-364. (b) Rauch, J.; Janoff, A. S. Lupus 1996, 5, 498-
502.
(14) Aoki, O.; Uenaka, T.; Aoki, J.; Umeda, M.; Inoue, K. J. Biochem. 1994,
116, 291-297.
JA026022Y
9
J. AM. CHEM. SOC. VOL. 124, NO. 19, 2002 5277