Facilitated phospholipid flip-flop using synthetic steroid-derived translocases

Article abstract of DOI:10.1021/ja026022y

Cholate esters with phenylurea groups at the 7α- and 12α-positions are highly effective promoters of phosphatidylcholine translocation across vesicle and cell membranes. The urea groups are essential for strong binding of the highly polar phosphate portio

Full text of DOI:10.1021/ja026022y

Published on Web 04/19/2002
Facilitated Phospholipid Flip-Flop Using Synthetic Steroid-Derived
Translocases
Timothy N. Lambert,† J. Middleton Boon,† Bradley D. Smith,*,† M. Nieves Pe´rez-Paya´n,‡ and
Anthony P. Davis*,‡,§
Department of Chemistry and Biochemistry and Walther Cancer Research Center, UniVersity of Notre Dame,
Notre Dame, Indiana 46556, Department of Chemistry, Trinity College, Dublin 2, Ireland, and School of Chemistry,
UniVersity of Bristol, Cantock's Close, Bristol, BS8 1TS, UK
Received February 25, 2002
It is well-established that lateral diffusion of a phospholipid
through one leaflet of a bilayer membrane is very rapid, whereas
translocation of a phospholipid from one side of the bilayer to the
other (a process also known as flip-flop) is quite slow.1 In most
animal cells, phospholipid translocation is controlled by membrane-
bound proteins called translocases.2 Some of these translocases are
energy-consuming, active-transport systems, which means they
produce transmembrane phospholipid distributions that are not
symmetric. These asymmetric distributions control a range of
important cellular functions such as signal transduction, membrane
fusion, blood coagulation, and cell apoptosis.3 We are attempting
to develop low-molecular-weight synthetic translocases that promote
phospholipid translocation and thus alter transmembrane phospho-
lipid distributions. Synthetic translocases should be useful as
pharmaceuticals or as chemical tools for biological membrane
research.4 Previously, we have reported that tris(2-aminoethyl)amine
(tren)-derived translocases can facilitate phosphatidylcholine trans-
location across synthetic vesicle and erythrocyte membranes.5
Mechanistic studies indicate that these synthetic translocases form
hydrogen-bonded complexes with the highly polar phosphate
portion of the zwitterionic phosphocholine headgroup which
promotes diffusion across the lipophilic interior of the membrane.5d
This current contribution describes the development of a new series
of significantly more active synthetic translocases derived from
cholic acid.6
Cholate derivatives 1-5 were prepared using previously reported
methods.7 Phospholipid translocation was monitored via the well-
established 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)/dithionite
quenching assay which uses probe phospholipid containing an NBD
group in one of its acyl chains.5,8 Exo-labeled vesicles were prepared
by addition of a small aliquot of NBD-lipid (0.5 mol % of total
phospholipid) in ethanol to a solution of unlabeled 1-palmitoyl-2-
oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles. The NBD-
lipid readily inserts into the outer monolayer of the fluid-phase
vesicles. Upon treatment with sodium dithionite (Na2S2O4), the
NBD fluorescence is quenched due to reduction of the NBD nitro
group. Vesicle membranes are effectively impermeable to dithionite;
therefore, only NBD-phospholipid located in the outer leaflet is
chemically quenched. At any given time, the fraction of probe
located in the outer monolayer can be determined from the drop in
fluorescence intensity when a portion of the vesicles is subjected
to dithionite quenching.
All translocation measurements were conducted in 5 mM TES/
100 mM NaCl buffer at pH 7.4 and 25 °C with 120 nm unilamellar
vesicles prepared by membrane extrusion.5
The inward translocation of PC-NBD across vesicle membranes
composed of POPC and 5 mol % of one of the “membrane-
anchored” translocase candidates 1-5 is illustrated in Figure 1. It
is clear that the bis(phenylurea) derivatives 1 and 2 are very efficient
translocases (they are approximately an order of magnitude more
active than our previously reported tren-derived systems5) with
translocation half-lives of ∼15 min and expected final equilibrium
states of 60% PC-NBD probe in the outer monolayer. The failure
of control compounds 3-5 to facilitate any phospholipid trans-
location suggests that 1 and 2 do not simply disrupt the bilayer
structure and generate local “flip sites”.9 Moreover, vesicle leakage
experiments show that large fluorescent dyes (e.g., calcein) are
retained inside POPC vesicles containing compounds 1-5 (5 mol
%) for at least 12 h. Concentration studies with bis-urea 1 indicate
that observed PC-NBD translocation rate constants have a first-
order dependence on translocase concentration. The membrane-
anchored translocases 1-5 were also evaluated for their abilities
to translocate PE-NBD and PS-NBD across POPC vesicle mem-
branes. With compounds 1 or 2 (5 mol %) the order of facilitated
translocation is PC-NBD (t1/2 ≈ 15 min) > PE-NBD (t1/2 ≈ 50
min) . PS-NBD (t1/2 > 180 min), whereas compounds 3-5 do
not improve inward translocation of any phospholipid probe.
* To whom correspondence should be addressed. E-mail: smith.115@nd.edu.
† University of Notre Dame.
‡ Trinity College Dublin.
§ Present address, University of Bristol.
9
5276
J. AM. CHEM. SOC. 2002, 124, 5276-5277
10.1021/ja026022y CCC: $22.00 © 2002 American Chemical Society

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
of charge via the Internet at http://pubs.acs.org.
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