Structure/activity study of tris(2-aminoethyl)amine-derived translocases for phosphatidylcholine

Article abstract of DOI:10.1021/jo016416s

Sulfonamide and amide derivatives of tris(aminoethyl)amine (TREN) are known to facilitate phospholipid translocation across vesicle and erythrocyte membranes; that is, they act as synthetic translocases. In this report, a number of new TREN-based translocases are evaluated for their abilities to bind phosphatidylcholine and translocate a fluorescent phosphatidylcholine probe. Association constants were determined from ¹H NMR titration experiments, and translocation half-lives were determined via 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)/dithionite quenching assays. A rough correlation exists between translocase/phosphatidylcholine association constants and translocation half-lives. The tris-sulfonamide translocases are superior to the tris-amide versions because they associate more strongly with the phospholipid headgroup. The stronger association is due to the increased acidity of the sulfonamide NHs as well as a molecular geometry (as shown by X-ray crystallography) that is able to form tridentate complexes with one of the phosphate oxygens. Two fluorescent translocase analogues were synthesized and used to characterize membrane partitioning properties. The results indicate that the facilitated translocation of phospholipids by TREN-derived translocases is due to the formation of hydrogen-bonded complexes with the phospholipid headgroups. In the case of zwitterionic phosphatidylcholine, it is the neutral form of the translocases that rapidly associates with the phosphate portion of the phosphocholine headgroup. Complexation masks the headgroup polarity and promotes diffusion of the phospholipid-translocase complex across the lipophilic interior of the membrane.

Full text of DOI:10.1021/jo016416s

2168
J . Org. Chem. 2002, 67, 2168-2174
Str u ctu r e/Activity Stu d y of Tr is(2-a m in oeth yl)a m in e-Der ived
Tr a n sloca ses for P h osp h a tid ylch olin e
J . Middleton Boon, Timothy N. Lambert, Bradley D. Smith,* Alicia M. Beatty,
Vesela Ugrinova, and Seth N. Brown
Department of Chemistry and Biochemistry and the Walther Cancer Research Center,
University of Notre Dame, Notre Dame, Indiana 46556
smith.115@nd.edu
Received December 30, 2001
Sulfonamide and amide derivatives of tris(aminoethyl)amine (TREN) are known to facilitate
phospholipid translocation across vesicle and erythrocyte membranes; that is, they act as synthetic
translocases. In this report, a number of new TREN-based translocases are evaluated for their
abilities to bind phosphatidylcholine and translocate a fluorescent phosphatidylcholine probe.
1
Association constants were determined from H NMR titration experiments, and translocation half-
lives were determined via 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)/dithionite quenching assays. A
rough correlation exists between translocase/phosphatidylcholine association constants and trans-
location half-lives. The tris-sulfonamide translocases are superior to the tris-amide versions because
they associate more strongly with the phospholipid headgroup. The stronger association is due to
the increased acidity of the sulfonamide NHs as well as a molecular geometry (as shown by X-ray
crystallography) that is able to form tridentate complexes with one of the phosphate oxygens. Two
fluorescent translocase analogues were synthesized and used to characterize membrane partitioning
properties. The results indicate that the facilitated translocation of phospholipids by TREN-derived
translocases is due to the formation of hydrogen-bonded complexes with the phospholipid
headgroups. In the case of zwitterionic phosphatidylcholine, it is the neutral form of the translocases
that rapidly associates with the phosphate portion of the phosphocholine headgroup. Complexation
masks the headgroup polarity and promotes diffusion of the phospholipid-translocase complex
across the lipophilic interior of the membrane.
In tr od u ction
tripodal tris(amido benzo-15-crown-5) conjugate has been
used to extract and transport NaTcO4 through a liquid
organic membrane.6 TREN tris-urea and tris-thiourea
derivatives have also been synthesized and shown to bind
a range of anions in highly polar organic solvents.7,8
Although the metal coordination chemistry of tris(2-
aminoethyl)amine (TREN) derivatives has been studied
extensively,1 less is known about their anion recognition
properties.2 Reinhoudt and co-workers were the first to
characterize anion receptors based on the TREN scaffold.3
In 1993, they reported that TREN tris-sulfonamides and
tris-amides have a binding preference in organic solvents
Our research group has examined a number of TREN
derivatives as synthetic receptors for phospholipid head-
groups. In particular, we have previously reported that
compounds 1 and 2 facilitate the translocation or “flip-
flop” of fluorescent phosphatidylcholine probes across the
membranes of surface differentiated vesicles and red
blood cells (Figure 1).9,10 We have proposed that these
synthetic translocases form hydrogen-bonded complexes
with the phosphate portion of the phosphocholine head-
group, which decreases headgroup polarity and promotes
diffusion across the lipophilic interior of the bilayer
membrane (Figure 2). With this contribution we provide
of H2PO4 > HSO4 > Cl-. More recently, tris-amide
receptors have been employed in combination with crown
ethers as dual host systems for the enhanced extraction
of CsNO3 into organic solvents,4 and in aiding the detec-
tion of nitrates by MicroITIES analysis.5 Similarly, a
-
-
* To whom correspondence should be addressed. Phone: (219) 631
8632. fax: (219) 631 6652.
(1) (a) Liao, C.-F.; Lai, J .-L.; Chen, J .-A.; Chen, H.-T.; Cheng, H.-
L.; Her, G.-R.; Su, J .-K.; Wang, Y.; Lee, G. H.; Leung, M.-K.; Wang,
C.-C. J . Org. Chem 2001, 66, 2566-2571. (b) Prodi, L.; Montalti, M.;
Zaccheroni, N.; Dallavalle, F.; Folesani, G.; Lanfranchi, M.; Corradini,
R.; Pagliari, S.; Marchelli, R. Helv. Chim. Acta 2001, 84, 690-706. (c)
Prodi, L.; Bolleta, F.; Montalti, M.; Zaccheroni, N. Eur. J . Inorg. Chem.
1999, 455-460. (d) Tecilla, P.; Tonellato, U.; Veronese, A.; Felluga,
F.; Scrimmin, P. J . Org. Chem. 1997, 62, 7621-7628. (e) Cernerud,
M.; Adolfsson, H.; Moberg, C. Tetrahedron: Asymmetry 1997, 8, 2655-
2662. (f) Schrock, R. R. Acc. Chem. Res. 1997, 30, 9-16. (g) Verkade,
J . G. Acc. Chem. Res. 1993, 26, 483-489.
(4) Kavallieratos, K.; Danby, A.; Van Berkel, G. J .; Kelly, M. A.;
Sachleben, R. A.; Moyer, B. A.; Bowman-J ames, K. Anal. Chem. 2000,
72, 5258-5264.
(5) Qian, Q.; Wilson, G. S.; Bowman-J ames, K.; Girault, H. H. Anal.
Chem. 2001, 73, 497-503.
(6) Beer, P. D.; Hopkins, P. K.; McKinney, J . D. Chem. Commun.
1999, 1253-1254.
(7) Raposo, C.; Almaraz, M.; Martin, M.; Weinrich, V.; Mussons, M.
(2) For reviews of anion recognition, see: (a) Gale, P. A.; Beer, P.
D. Angew. Chem., Int. Ed. 2001, 40, 486-516. (b) Schneider, H. J .;
Yatsimirski, A. Principles and Methods in Supramolecular Chemistry;
Wiley: Somerset, 2000. (c) Antonisse, M. M. G.; Reinhoudt, D. N. J .
Chem. Soc., Chem. Commun. 1998, 443-447. (d) The Supramolecular
Chemistry of Anions; Bianci, A., Bowman-J ames, K., Carcia-Espana,
E., Eds.; VCH: Weinheim, 1997; pp 355-420.
L.; Alcazar, V.; Caballero, M. C.; Moran, J . R. Chem. Lett. 1995, 759-
760.
(8) Xie, H.; Yi, S.; Wu, S. J . Chem. Soc., Perkin Trans. 2 1999, 2751-
2754.
(9) Boon, J . M.; Smith, B. D. J . Am. Chem. Soc. 1999, 121, 11924-
11925.
(10) Boon, J . M.; Smith, B. D. J . Am. Chem. Soc. 2001, 123, 6221-
6226.
(3) Valiyaveettil, S.; Engbersen, J . F. J .; Verboom, W.; Reinhoudt,
D. N. Angew. Chem., Int. Ed. Engl. 1993, 32, 900-901.
10.1021/jo016416s CCC: $22.00 © 2002 American Chemical Society
Published on Web 03/14/2002

TREN-Derived Translocases
J . Org. Chem., Vol. 67, No. 7, 2002 2169
Sch em e 1
F igu r e 1. Phospholipid translocation or flip-flop.
F igu r e 2. Proposed supramolecular complex between TREN-
sulfonamide translocases and the phosphocholine headgroup.
structure/function data that further support our hypoth-
esis. Specifically, we describe the synthesis and structure
of a series of related TREN derivatives and correlate the
structural information with phosphatidylcholine binding
constants, membrane partitioning values, and phospho-
lipid translocation rates. Although the focus of this report
is on phosphatidylcholine recognition, our results help
explain the more general anion-binding properties of
TREN-derived receptors. In particular, a comparison of
the X-ray crystal structures of sulfonamide 1 and amide
2 shows why the sulfonamide 1 is a much better anion
binder.
amide) 7 was substantially lower (7%), although this
reaction was only run once. Compound 3 was synthesized
(66% yield) by reacting triethanolamine with a slight
excess of the corresponding acyl chloride. The fluorescent
derivatives 8 and 9 were prepared in a convergent
manner (Scheme 1). Treatment of TREN (10, 10 molar
equiv) with di-tert-butyl dicarbonate (1 molar equiv) at
-78 °C in CH2Cl2 under high-dilution conditions,11 fol-
lowed by warming to room temperature and stirring
overnight, gave the desired mono-Boc-protected TREN
derivative 11 in 65% yield after careful chromatog-
raphy.1d,12 Subsequent treatment of 11 with toluenesulfo-
nyl chloride gave 12, which was deprotected with 50%
TFA to give 13 as its bis-trifluoroacetate salt. Compound
13 was coupled with 4-chloro-7-nitrobenzo-2-oxa-1,3-
diazole (NBD-Cl) to give 8 or with 5-(dimethylamino)-1-
naphthalenesulfonyl chloride1b (DANS-Cl) to give 9.
P h osp h a tid ylch olin e Bin d in g in CDCl3. The ability
of compounds 1-7 to complex with 1-palmitoyl-2-oleoyl-
sn-glycero-3-phosphocholine (POPC) in CDCl3 at 25 °C
was evaluated using 1H NMR spectroscopy. Titration
isotherms were generated by adding aliquots of POPC
to solutions of the corresponding receptor, and association
constants were extracted by fitting the curves to a 1:1
binding model using iterative computer methods.13 The
validity of the 1:1 binding model was confirmed in the
case of 1 with a J ob plot (data not shown). As discussed
previously,10 the complexed-induced shifts (along with the
X-ray structural data described below) are consistent
with the hydrogen-bonded complex shown in Figure 2.
The order of observed association constants with POPC
was found to be 6 [(9.1 ( 1.4) × 103 M-1] > 5 [(2.5 ( 0.4)
× 103 M-1] ≈ 4 [(2.2 ( 0.3) × 103 M-1] ≈ 1 [(2.1 ( 0.3) ×
103 M-1] > 7 [(6.4 ( 0.9) × 102 M-1] > 2 [(2.0 ( 0.3) ×
101 M-1] . 3 (<1 M-1). Broadly speaking, the association
constants correlate with the acidity of the receptor NH
groups. For example, all of the aryl sulfonamides have a
greater affinity than that of the propyl sulfonamide 4,
Resu lts a n d Discu ssion
(11) J acobsen, A. R.; Makris, A. N.; Sayre, L. M. J . Org. Chem. 1987,
52, 2592-2594.
Syn th esis of Recep tor s 1-9. Compounds 1, 2, and
4-6 were prepared in 45-70% yield by reacting TREN
(10), with a slight excess of the corresponding aryl
sulfonyl or acyl chloride. The yield for tris(propylsulfon-
(12) Valente, S.; Gobbo, M.; Licini, G.; Scarso, A.; Scrimin, P. Angew.
Chem., Int. Ed. 2001, 40, 3899-3902.
(13) Hughes, M. P.; Smith, B. D. J . Org. Chem. 1997, 62, 4492-
4501.

2170 J . Org. Chem., Vol. 67, No. 7, 2002
Boon et al.
F igu r e 3. Crystal structure of sulfonamide 1‚H2O with 50%
ellipsoid probability. Only sulfonamide and water hydrogen
atoms are shown for clarity. Dashed lines indicate hydrogen
bonds.
F igu r e 4. Crystal structure of (1 + H)+:(dibenzyl phosphate)-
ionic complex with 50% ellipsoid probability. Only relevant
hydrogen atoms are shown. Included CHCl3 has been omitted
for clarity. Dashed lines indicate hydrogen bonds.
which in turn binds better than amide 2; ester 3 has no
discernible binding ability. The higher affinity displayed
by sulfonamide 6 relative to the other aryl sulfonamides
can be attributed to its lower pKa.14 The binding constants
for 1, 4, and 5 are within the experimental error. On the
basis of acidity, a lower binding constant is predicted for
5 because the order of electron-donating ability is phe-
noxy > methoxy > methyl.14 It is possible that there are
additional weak interactions between the receptor and
the phospholipid headgroup. For example, π-stacking of
the phenoxy rings in 5 with the cationic choline portion
may compensate for the weakened hydrogen bonding to
the phosphate group.15
The crystal structure of the protonated form of sul-
fonamide 1 complexed with dibenzyl phosphate is shown
in Figure 4. Again, the secondary sulfonamides adopt the
characteristic conformation 14.19 In this case, only one
sulfonamide NH and the protonated tertiary amine are
hydrogen-bonded to one of the phosphate oxygens (NH‚‚‚
O distances of 1.94 and 1.77 Å, respectively). While this
ionic structure is not so relevant to the translocation of
zwitterionic phosphatidylcholine (which we believe in-
volves a neutral translocase/phospholipid complex), it is
relevant to the facilitated translocation of anionic phos-
pholipids such as phosphatidylglycerol,9 which most
likely requires a protonated translocase/phospholipid
complex.
X-r a y Cr ysta llogr a p h y.16 The crystal structure of
tris-sulfonamide 1‚H2O is shown in Figure 3. The three
secondary arylsulfonamides adopt their characteristic
conformation 14, where the nitrogen lone pair and the
Acyclic secondary amides are well-known to prefer a
Z conformation about the amide C-N bond,20 a prefer-
ence that is seen in the crystal structure of tris-amide 2
(Figure 5).21 Because of this conformational constraint,
the putative tripodal “anion binding pocket” in receptor
2 is effectively closed as a result of the steric bulk of the
three aromatic rings, which are oriented edge-to-face with
each other. While the amide E/ Z conformational equi-
librium can be altered by noncovalent interactions,22 it
is an energetically costly process.20 Thus, it appears that
aromatic TREN tris-amides such as 2 cannot bind anions
in the same tridentate fashion as the analogous tris-
sulfonamide 1, a point that has not previously been noted
in the literature.3-6 We conclude that tris-amide 2 has a
relatively weak affinity for phosphatidylcholine for two
reasons: (1) the amide hydrogen atoms are less acidic,
aromatic p-orbital lie in a plane that bisects the O-S-O
internuclear angle.17,18 The overall molecular geometry
of 1 is roughly a C3-symmetric tripodal arrangement with
all three NHs hydrogen-bonded to a water molecule
(NH‚‚‚O distances are 2.43, 2.48 and 2.18 Å), which is
also hydrogen-bonded to a sulfonyl oxygen in the same
molecule (SO‚‚‚H distance of 1.89 Å). Overall, the solid-
state structure of 1 provides a clear visual picture of its
“anion binding pocket" and supports the supramolecular
complex proposed in Figure 2.
(14) For Hammet and Taft equations for pKa values of arylsulfon-
amides and accompanying σ° values refer to: (a) Dauphlin, G.;
Kergomard, A. Bull. Soc. Chim. Fr. 1961, 3, 486. (b) Perrin, D. D.;
Dempsey, B.; Serjeant, E. P. pKa Prediction for Organic Acids and
Bases; Chapman and Hall: London, 1981.
(15) For an example of π-stacking with the choline portion of the
headgroup, see: Magrans, J . O.; Ortiz, A. R.; Molins, M. A.; Lebouille,
P. H. P.; Sanchez-Quesada, J .; Prados, P.; Pons, M.; Gago, F.; de
Mendoza, J . Angew. Chem., Int. Ed. Engl. 1996, 35, 1712-1715.
(16) X-ray data for 1‚H2O, 2 and the [(1 + H)+:(dibenzyl phosphate)-
‚CHCl3] complex can be retrieved from the Cambridge Crystallographic
Data Center using deposition numbers CCDC 175856 (1), CCDC
175858 (2), and CCDC 175857 [(1 + H)+:(dibenzyl phosphate)-‚CHCl3].
(17) (a) Beddoes, R. L.; Dalton, L.; J oule, J . A.; Mills, O. S.; Street,
J . D.; Watt, C. I. F. J . Chem. Soc. Perkin Trans. 2 1986, 787-797. (b)
Menziani, M. C.; Cocchi, M.; De Benedetti, P. G. THEOCHEM 1992,
256, 217-229.
(19) Torsion angles:18 Φ ) 77.8°, 115.8°, 79.4° and ψ ) 75.0°, 62.9°,
53.2°.
(20) The Z conformation of N-methylacetamide is more stable than
the E conformation by 2.1-2.5 kcal/mol. Furthermore, the free energy
barrier to rotation about the OC-N bond in N-methylacetamide is 21.3
kcal/mol. (a) Eliel, E. E.; Wilen, S. H.; Doyle, M. P. Basic Organic
Stereochemistry; Wiley-Interscience: New York, 2001; p 392. (b)
Stewart, W. E.; Siddall, T. H. Chem. Rev. 1970, 70, 517-551.
(21) For related TREN-based tris-amide X-ray crystal structures,
see: (a) Danby, A.; Seib, L.; Alcock, N. W.; Bowman-J ames, K. Chem.
Commun. 2000, 973-974. (b) Byrne, J . J .; Chavant, P. Y.; Averbuch-
Pouchot, M.-T.; Vallee, Y. C. R. Seances Acad. Sci., Ser. IIc 1998, 1,
75.
(22) For examples of hydrogen bonded stabilization of E secondary
amides, see: (a) Deetz, M. J .; Fahey, J . E.; Smith, B. D. J . Phys. Org.
Chem. 2001, 14, 463-467. (b) Beijer, F. H.; Sijbesma, R. P.; Vekemans,
J . A. J . M.; Meijer, E. W.; Kooijman, H.; Speck, A. L. J . Org. Chem.
1996, 61, 6371-6380. (c) Perni, G. J .; Kilburn, J . D.; Essex, J . W.;
Mortshire-Smith, R. J .; Rowley, M. J . Am. Chem. Soc. 1996, 118,
10220-10227. (d) Bairaktari, E.; Mierke, D. F.; Mammi, S.; Peggion,
E. J . Am. Chem. Soc. 1990, 112, 5383-5384.
(18) The torsion angles Φ [C(2)-C(1)-S-N] and ψ [C(1)-S-N-H]
are used to describe the conformation of a secondary arylsulfonamide
skeleton,17b and torsion angles of Φ ≈ 90° and ψ ≈ 90° are generally
observed. For tris-sulfonamide 1, the torsion angles are Φ ) 97.5°,
84.8°, 79.4° and ψ ) 94.0°, 69.5°, 76.3°, consistent with the literature.

TREN-Derived Translocases
J . Org. Chem., Vol. 67, No. 7, 2002 2171
translocation (flip), were prepared by addition of a small
aliquot of PC-NBD (0.5 mol % of total phospholipid) in
ethanol to a solution of unlabeled POPC vesicles (25 mM).
The PC-NBD readily inserts into the vesicle outer mono-
layer, and upon treatment with sodium dithionite
(Na2S2O4), the NBD fluorescence is quenched as a result
of reduction of the NBD nitro group. Vesicle membranes
are effectively impermeable to dithionite, therefore only
PC-NBD located in the outer leaflet is chemically
quenched. Lysis of the vesicles with detergent allows the
dithionite to gain access to the remaining PC-NBD, and
thus the percent exo PC-NBD can be computed. The
inward translocation experiment starts with 100% exo
PC-NBD and progresses to an equilibrium value of
around 60%. The translocation half-life is the time taken
to reach 80% exo PC-NBD. All translocation measure-
ments were conducted at pH 7.4 and 25 °C with 120 nm
unilamellar vesicles that were prepared by membrane
extrusion.
F igu r e 5. Crystal structure of amide 2 with 50% ellipsoid
probability. Only amide hydrogen atoms are shown for clarity.
We have already shown that sulfonamide 1 is more
effective than amide 2 (and the control ester 3) at
translocating PC-NBD across surface differentiated
vesicles.9 In the absence of translocase the half-life for
inward PC-NBD translocation is many hours, but in the
presence of compound 1 (38 µM) the half-life is about 5
min.9 A goal of this present study was to measure the
relative abilities of translocases 1-7 to transport PC-
NBD across the membranes of surface differentiated
vesicles and to determine how the translocation cor-
relates with the POPC binding constants. On the basis
of the binding constants, 3-(trifluoromethyl)phenyl sul-
fonamide 6 was expected to be the most effective trans-
locase. However, it facilitated PC-NBD translocation by
approximately the same amount as tolyl sulfonamide 1
(uncertainty for the measured translocation half-lives is
around (33%). Unfortunately, sulfonamides 4 and 5
suffered from poor aqueous solubility, and reproducible
translocation data could not be obtained. Propyl sulfon-
amide 7 is water-soluble up to 100 µM but is nonetheless
ineffective at PC-NBD translocation; this behavior is
likely due to its poor phosphocholine binding ability. As
previously reported, amide 2 is also ineffective at PC-
NBD translocation.9 In all cases, carboxyfluorescein
leakage experiments showed that the translocases do not
induce vesicle leakage.
Although there is a rough correlation between trans-
locase/phosphatidylcholine association constants and PC-
NBD translocation half-lives, the trend is not exact. It
appears that other factors such as membrane partitioning
values, membrane diffusion constants, etc. may also be
influential.
F lu or escen t Tr a n sloca se An a logu es. In an effort
to gain some insight into how the TREN sulfonamides
interact with vesicle membranes, two fluorescent ana-
logues, 8 and 9, were prepared (Scheme 1) and used in
binding and partitioning experiments. A fluorescence
resonance energy transfer (FRET) assay was performed
to determine how quickly mono-NBD derivative 8 (38 µM)
comes in contact with the headgroup region of POPC
vesicles (25 µM) containing 0.6% of the energy acceptor
Rhodamine-PE (Rh-PE). The fluorescence emission pro-
F igu r e 6. 1H NMR titration curves for sulfonamide 1 (9, CH2-
NH), amide 2 (b, CH2NH), and ester 3 ([, CH2O) in 9:1 CD3-
OD/D2O.
and (2) it cannot achieve tridentate complexation with
the phosphate portion of the headgroup.
/
p Ka Mea su r em en ts. Previously, pKa values for the
protonated forms of compounds 1-3 were obtained by a
potentiometric method in 9:1 CH3OH/H2O and found to
be 1 (4.4), 2 (5.7), and 3 (4.2).10 We have now verified
1
these measurements with H NMR titrations in 9:1 CD3-
OD/D2O (Figure 6), and the numbers are in good agree-
ment: 1 (4.00 ( 0.01), 2 (5.52 ( 0.02), 3 (3.75 ( 0.01).
These values suggest that compounds 1-3 exist prima-
rily in their free base form at neutral pH and indicate
that during the phospholipid translocation assay (pH )
7.4) a smaller fraction of sulfonamide 1 is protonated, as
compared to amide 2. This is another reason why
sulfonamide 1 is a better translocase of zwitterionic
phosphatidylcholine than amide 2 (translocation involves
a neutral translocase/phospholipid complex). It also
explains why amide 2 is a better translocase of anionic
phosphatidylglycerol than sulfonamide 1 (translocation
involves a protonated translocase/phospholipid complex).9
In w a r d Tr a n sloca tion of P h osp h olip id s. Phospho-
lipid translocation was monitored via the well-established
7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)/dithionite quench-
ing assay, which uses a phosphatidylcholine probe con-
taining an NBD group in one of its acyl chains (PC-
NBD).9,10,23,24 Exo-labeled vesicles, used to measure inward
(23) Moss, R. A.; Battacharya, S. J . J . Am. Chem. Soc. 1995, 117,
8688-8689.
(24) (a) McIntyre, J . C.; Sleight, R. G. Biochemistry 1991, 30, 11819-
11827. (b) Balch, C.; Morris, R.; Brooks, E.; Sleight, R. G. Chem. Phys.
Lipids 1994, 70, 205-212.

2172 J . Org. Chem., Vol. 67, No. 7, 2002
Boon et al.
penetration of dithionite into the vesicles. We conclude
that the initial binding of the translocases to the mem-
brane surface is relatively rapid and that subsequent
movement across the membrane, as either the free
species or the translocase-phospholipid complex, is sig-
nificantly slower.
The mono-dansyl sulfonamide 9 was prepared and used
to gain information concerning the equilibrium position
of the translocases within the membrane. The fluores-
cence emission wavelength of the dansyl chromophore is
known to be sensitive to solvent polarity, and dansyl
derivatives are widely used to obtain membrane location
information.25,26 In this case, the fluorescence emission
spectra of 9 in a number of solvents (chosen to mimic
the different polarities of the bilayer regions) were
acquired and the observed λmax values plotted against
known solvent polarity values (ET).27 A value of λmax
)
510 nm was obtained for mono-dansyl 9 (5 mol %) in
POPC vesicles (25 mM), which corresponds to a polarity
value of ET ) 52 (ethanol-like polarity). This suggests
that 9 resides, on average, in the mid-polar/headgroup
region of bilayer membranes.
Su m m a r y
The results in this paper support our hypothesis that
the facilitated translocation of phospholipids by TREN-
derived translocases is due to the formation of hydrogen-
bonded complexes with the phospholipid headgroups. In
the case of zwitterionic phosphatidylcholine, it is the
neutral form of the translocase that rapidly associates
with the phosphate portion of the phosphocholine head-
group (Figure 2). This hydrogen-bonded complex masks
the headgroup polarity and promotes diffusion of the
phospholipid-translocase complex across the lipophilic
interior of the membrane. The tris-sulfonamide translo-
cases (such as 1) are superior to the tris-amide versions
(such as 2) because they associate more strongly with the
phospholipid headgroup. The stronger association is due
to the increased acidity of the tris-sulfonamide NHs as
well as a molecular geometry that is able to form a
tridentate complex with one of the phosphate oxygens.
In the case of facilitated translocation of anionic phos-
pholipids, it is the protonated form of the translocase that
binds to the phosphate portion of the headgroup, produc-
ing a complex that is analogous to that shown in Figure
4. These results provide structural insight into the
supramolecular chemistry behind the facilitated trans-
location process; however, a number of kinetic questions
remain that will require additional mechanistic study.
F igu r e 7. Fluorescence resonance energy transfer assays. (A)
Fluorescence emission of POPC/Rh-PE (0.6%) vesicles (25 µM)
with (solid line) or without (dashed line) addition of mono-
NBD 8 (38 µM). (B) Time drive spectrum of POPC/Rh-PE
(0.6%) vesicles (25 µM) with mono-NBD 8 (38 µM) added at t
) 50 s and detergent added at t ) 350 s.
file in Figure 7a shows that the excitation energy of the
NBD chromophore in 8 (excitation 470 nm, emission 530
nm) is transferred to the Rh-PE molecules, resulting in
a Rh-PE emission peak at 590 nm. Furthermore, the
time-drive spectrum in Figure 7b demonstrates that 8
moves rapidly (within seconds) into close contact with
the headgroup portion of the phospholipid bilayer. Dis-
ruption of the membrane by detergent addition at t )
350 s leads to immediate loss of the energy transfer.
A qualitative measure of the extent that the translo-
cases partition across the membrane was gained by
conducting dithionite quenching experiments. If mono-
NBD derivative 8 binds only to the external leaflet of the
bilayer or remains in the surrounding aqueous media,
then treatment with dithionite will quench the fluores-
cence. On the other hand, if 8 diffuses into the inner
leaflet of the bilayer or enters the aqueous vesicle
interior, then no quenching will be observed. We find that
when 8 (38 µM) is added to a solution of unlabeled POPC
vesicles (25 µM), roughly 20% of 8 is partitioned inside
the vesicles and protected from dithionite reduction. The
fluorescence intensity for a vesicle/8 mixture treated with
dithionite gradually decreases over time (drops to 5%
after 1 h), indicating that the protected probe 8 slowly
diffuses out of the vesicles and into the outer aqueous
environment. Control experiments with vesicles contain-
ing PC-NBD indicate that these results are not due to
Exp er im en ta l Section
Gen er a l Syn th etic P r oced u r e for Tr a n sloca ses 4-7.
Compounds 1-3 were prepared as previously described.9,28
Compounds 4-7 were prepared in a manner similar to the
method of Reinhoudt and co-workers.3 In general, a solution
of tris(2-aminoethyl)amine 10 (3.68 mmol) in CH2Cl2 or THF
(50 mL) was added dropwise to a solution of the corresponding
sulfonyl chloride (12.08 mmol) and triethylamine (12.08 mmol)
in CH2Cl2 or THF (5-50 mL) at room temperature under an
atmosphere of N2. The reaction mixture was then stirred at
(25) Chen, R. F. Arch. Biochem. Biophys. 1967, 120, 609-620.
(26) Abel, E.; Maguire, G. E. M.; Murillo, O.; Suzuki, I.; De Wall, S.
L.; Gokel, G. W. J . Am. Chem. Soc. 1999, 121, 9043-9052.
(27) Reichardt, C. Solvents and Solvent Effects in Organic Chemistry,
2nd ed.; Verlag Chemie, Weinheim: 1988; p 366.

TREN-Derived Translocases
J . Org. Chem., Vol. 67, No. 7, 2002 2173
room temperature for 12 h, after which time it was gravity
filtered to remove the white precipitate (triethylammonium
chloride). The solvent was removed in vacuo to give the crude
product, which was purified by column chromatography using
silica gel.
the solvent was removed in vacuo. The resulting residue was
extracted with CH2Cl2 and filtered to remove the suspended
solids, and the solvent was removed in vacuo to give 0.331 g
of crude product. Column chromatography (SiO2) with ethyl
acetate/hexane gradient elution gave the product 12 (0.241 g,
64%) as a viscous oil: 1H NMR (500 MHz, CDCl3) δ 1.43 (s,
9H), 2.38 (t, J ) 5.8 Hz, 2H), 2.41 (s, 6H), 2.46 (t, J ) 5.0 Hz,
4H), 2.86-2.92 (m, 4H), 3.02 (dt, J ) 5.8 and 5.5 Hz, 2H), 5.12
(t, J ) 5.5 Hz, 1H), 5.89 (t, J ) 5.2 Hz, 2H), 7.29 (d, J ) 8.0
Hz, 4H), 7.78 (d, J ) 8.0 Hz, 4H); 13C NMR (125 MHz, CDCl3)
δ 21.5, 28.4, 38.4, 40.8, 53.8, 54.3, 79.5, 127.1, 129.7, 137.0,
143.2, 156.6; MS(FAB+) exact mass calcd for C25H38N4O6S2 [M
+ H]+ 555.2311, found 555.2297.
Tr is[2-(4-m eth oxy(ph en ylsu lfon am ido))eth yl]am in e (4).
Column chromatography with 0-5% CH3OH/CHCl3 gradient
elution, followed by recrystallization from CHCl3, gave 4 as a
white solid (45%): mp 156-157 °C; 1H NMR (500 MHz, CDCl3)
δ 2.50 (t, J ) 5.0 Hz, 6H), 2.92 (q, J ) 6.5 Hz, 6H), 3.86 (s,
9H), 5.71 (t, J ) 6.2 Hz, 3H), 6.99 (d, J ) 9.0 Hz, 6H), 7.87 (d,
J ) 9.0 Hz, 6H); 13C NMR (75 MHz, DMSO-d6) δ 40.2, 53.0,
55.6, 114.4, 128.6, 132.2, 162.1; MS(FAB+) mass calcd for
24H36N4O9S3 [M + H]+ 658, found 658.
N,N-Bis[2-(4-m eth yl(p h en ylsu lfon a m id o))eth yl]-N-(2-
a m in oet h yl)a m in e b is(t r iflu or oa cet a t e) Sa lt (13). To a
solution of 12 (0.118 g, 0.213 mmol) in CH2Cl2 (6 mL) at 0 °C
under N2 was added TFA (6 mL) dropwise over 2-3 min. The
reaction was stirred at 0 °C for 45 min after which time the
solvents were removed in vacuo to give 13 (0.144 g, 100%) as
a hygroscopic pale white solid: 1H NMR (500 MHz, CD3OD) δ
2.42 (s, 6H), 2.72 (t, J ) 6.2 Hz, 4H), 2.87 (t, J ) 6.0 Hz, 2H),
2.93 (t, J ) 6.2 Hz, 4H), 3.07 (t, J ) 6.0 Hz, 2H), 7.38 (d, J )
7.5 Hz, 4H), 7.73 (d, J ) 8.5 Hz, 4H); 13C NMR (125 MHz,
C
Tr is[2-(4-ph en oxy(ph en ylsu lfon am ido))eth yl]am in e (5).
The requisite 4-phenoxybenzenesulfonyl chloride was prepared
in 31% overall yield in two steps following known proce-
dures.29,30 Column chromatography with 0-5% CH3OH/CHCl3
gradient elution, followed by recrystallization from CHCl3,
1
gave 5 as a white solid (70%): mp 52-56 °C; H NMR (500
MHz, CDCl3) δ 2.58 (t, J ) 5.0 Hz, 6H), 3.02 (q, J ) 5.8 Hz,
6H), 5.78 (t, J ) 6.5 Hz, 3H), 7.03 (d, J ) 8.5 Hz, 6H), 7.06 (d,
J ) 8.0 Hz, 6H), 7.20 (t, J ) 7.5 Hz, 3H), 7.20 (t, J ) 7.5 Hz,
3H), 7.38 (t, J ) 8.0 Hz, 6H), 7.87 (d, J ) 9.0 Hz, 3H); 13C
NMR (75 MHz, CDCl3) δ 40.8, 54.1, 117.7, 120.0, 124.6, 129.3,
130.0, 133.3, 155.1, 161.2; MS(FAB+) mass calcd for C42H42N4O9-
S3 [M + H]+ 843, found 843.
1
CD3OD) δ 21.6, 36.7, 40.3, 52.3, 54.8, 117.6 (q, J CF ) 288.9
2
Hz), 128.3, 131.1, 138.0, 145.4, 162.1 (q, J CF ) 36.2 Hz); MS-
(FAB+) exact mass calcd for C20H30N4O4S2 [M + H]+ 455.1787,
found 455.1786.
N,N-Bis[2-(4-m eth yl(p h en ylsu lfon a m id o))eth yl]-N-[2-
(7-n itr oben zo-2-oxa -1,3-dia zole-4-a m in o)-eth yl]a m in e (8).
To a solution of 13 (0.142 g, 0.207 mmol) in CH3OH (10 mL)
at 0 °C under N2 was added KHCO3 (0.133 g, 1.32 mmol). The
resulting solution was stirred at 0 °C for 5 min then at room
temperature for 10 min after which time a solution of NBD-
Cl (0.056 g, 0.281 mmol) in CH3OH (10 mL) was added
dropwise over 5 min. The reaction was stirred at room
temperature for 22 h after which time the solvent was removed
in vacuo. The residue was extracted with ethyl acetate and
concentrated in vacuo. and the product was purified with
column chromatography (Al2O3) using 0.5-1.0% CH3OH /ethyl
acetate gradient elution to give 8 (0.065 g, 51%) as a yellow-
green oil: 1H NMR (500 MHz, CD3CN) δ 2.37 (s, 6H), 2.52 (t,
J ) 6.0 Hz, 4H), 2.70 (t, J ) 6.2 Hz, 2H), 2.83 (t, J ) 6.0 Hz,
4H), 3.43 (br s, 2H), 5.58 (s, 2H), 6.25 (s, 1H), 7.30 (d, J ) 8.0
Tr is[2-(3-tr iflu or om eth yl(p h en ylsu lfon a m id o))eth yl]-
a m in e (6). Column chromatography with 0-5% CH3OH/
CHCl3 gradient elution, followed by recrystallization from
CHCl3, gave 6 (69%): mp 121-124 °C; 1H NMR (500 MHz,
CDCl3) δ 2.63 (t, J ) 5.0 Hz, 6H), 3.09 (q, J ) 5.5 Hz, 6H),
5.98 (t, J ) 6.0, 3H), 7.70 (t, J ) 8.2, 3H), 7.84 (d, J ) 8.0 Hz,
3H), 8.16 (d, J ) 6.8 Hz, 3H), 8.17 (s, 3H); 13C NMR (75 MHz,
1
3
CDCl3) 40.8, 54.1, 122.9 (q, J CF ) 270.0 Hz), 123.8 (q, J CF
)
)
3
2
3.8 Hz),129.0 (q, J CF ) 3.2 Hz), 130.1, 130.3, 131.4 (q, J CF
32.5 Hz), 141.6; MS(FAB+) mass calcd for C27H27F9N4O6S3 [M
+ H]+ 771, found 771.
Tr is[2-(pr opylsu lfon am ido)eth yl]am in e (7). Column chro-
matography with ethyl acetate gave the product 7 (7%): 1H
NMR (300 MHz, CDCl3) δ 1.05 (t, J ) 7.5 Hz, 9H), 1.79-1.92
(m, 6H), 2.61 (t, J ) 5.2 Hz, 6H), 3.05 (t, J ) 8.0 Hz, 6H), 3.22
(q, J ) 6.2, 6H), 5.71 (t, J ) 6.3 Hz, 3H); 13C NMR (75 MHz,
CDCl3) δ 13.1,17.4, 40.9, 54.4, 55.4; MS(FAB+) mass calcd for
Hz, 5H), 7.66 (d, J ) 8.0 Hz, 4H), 8.46 (d, J ) 4.5 Hz, 1H); 13
C
NMR (125 MHz, CD3CN) δ 20.2, 40.6, 41.0, 51.3, 53.0, 98.9,
122.3, 126.4, 129.3, 136.9, 143.2, 144.0, 144.2, 144.4; MS(FAB+)
exact mass calcd for C26H31N7O7S2 [M + H]+ 618.1805, found
618.1824.
C
15H36N4O6S3 [M + H]+ 465, found 465.
N,N-Bis(2-am in oeth yl)-N-[2-(ter t-bu tylcar bam oyl)eth yl]-
a m in e (11). To a solution of tris-(2-aminoethyl)amine (10)
(2.93 g, 20.0 mmol) in CH2Cl2 (300 mL) at -78 °C under N2
was added a solution of di-tert-butyl dicarbonate (0.444 g, 2.03
mmol) in CH2Cl2 (100 mL) dropwise over 1 h. The reaction
mixture was allowed to warm to room temperature and stirred
overnight after which time the solvent was removed in vacuo.
Column chromatography, eluting with a ternary eluent (10:
4:1) of CHCl3/MeOH/concentrated aqueous NH4OH gave the
product 11 (0.324 g, 65% based on Boc2O) as a viscous oil: 1H
NMR (500 MHz, CDCl3) δ 1.43 (s, 9H), 1.58 (s, 4H), 2.52 (t, J
) 6.0 Hz, 4H), 2.55 (t, J ) 5.8 Hz, 2H), 2.75 (t, J ) 6.0 Hz,
4H), 3.18 (unresolved t, 2H), 5.34 (br s, 1H); 13C NMR (125
MHz, CDCl3) δ 28.4, 38.8, 39.6, 54.1, 57.1, 78.8, 156.3; MS-
(FAB+) exact mass calcd for C11H26N4O2 [M + H]+ 247.2134,
found 247.2130.
N,N-Bis[2-(4-m eth yl(p h en ylsu lfon a m id o))eth yl]-N-[2-
(ter t-bu tylca r ba m oyl)eth yl]a m in e (12). To a mixture of 11
(0.168 g, 0.684 mmol) and K2CO3 (0.292 g, 2.11 mmol) in CH3-
CN (10 mL) at room temperature under N2 was added a
solution of toluenesulfonyl chloride (0.322 g, 1.69 mmol) in
CH3CN (5 mL) dropwise over 5 min. The reaction mixture was
allowed to stir at room temperature for 23 h after which time
N,N-Bis[2-(4-m eth yl(p h en ylsu lfon a m id o))eth yl]-N-[2-
(5-(d im et h yla m in o)-1-n a p h t h a len esu lfon a m id o)et h yl]-
a m in e (9). A mixture of 13 (0.148 g, 0.217 mmol) and K2CO3
(0.24 g, 1.74 mmol) in CH3CN (10 mL) was allowed to stir
under N2 at room temperature for 15 min. A solution of dansyl
chloride (0.109 g, 0.404 mmol) in CH3CN (10 mL) was then
added dropwise over 15 min. The reaction was allowed to stir
for 19 h after which time it was filtered, and the solvent
removed in vacuo. The product was purified by column
chromatography (SiO2) with ethyl acetate/hexanes gradient
elution to give 9 (0.126 g, 84%) as a light yellow semisolid:
1H NMR (500 MHz, CDCl3) δ 2.38 (s, 6H), 2.38-2.46 (m, 6H),
2.82-2.92 (m, 6H), 2.87 (s, 6H), 5.90 (t, J ) 6.0 Hz, 2H), 6.07
(t, J ) 5.8 Hz, 1H), 7.13 (d, J ) 7.5 Hz, 1H), 7.25 (d, J ) 8.0
Hz, 4H), 7.51 (t, J ) 7.5 Hz, 2H), 7.79 (d, J ) 8.0 Hz, 4H),
8.24 (dd, J ) 7.5 and 1.0 Hz, 1H), 8.33 (d, J ) 9.0 Hz, 1H),
8.52 (d, J ) 8.0 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 21.7,
41.1, 41.3, 45.6, 54.4, 54.7, 115.4, 119.3, 123.4, 127.4, 128.7,
129.5, 129.7, 129.9, 130.0, 130.4, 135.2, 137.1, 143.4, 152.0;
MS(FAB+) exact mass calcd for C32H41N5O6S3 [M + H]+
688.2297, found 688.2316.
1H NMR Titr a tion s. P OP C Bin d in g. Translocases 4-7
were titrated with POPC. Four different NMR tubes each
contained a solution of translocase in CDCl3 (5 mM, 750 µL).
Small aliquots of POPC stock solution (0.375 M) were added,
(28) Chen, D.; Motekaitis, R. J .; Murase, I.; Martell, A. E. Tetrahe-
dron 1995, 51, 77-88.
(29) Suter, C. M. J . Am. Chem. Soc. 1931, 53, 1112-1116.
(30) Tamura, Y.; Watanbe, F.; Nakatani, T.; Yasui, K.; Fuji, M.;
Komurasaki, T.; Tsuzuki, H.; Maekawa, R.; Yoshioka, T.; Kawada, K.;
Sugita, K.; Ohtani, M. J . Med. Chem. 1998, 41, 640-649.
1
followed by the acquisition of a H NMR spectrum. Concentra-
tions and equivalents were adjusted to give the optimum

2174 J . Org. Chem., Vol. 67, No. 7, 2002
Boon et al.
change in Weber r values (0.2-0.8).31 Titration isotherms were
generated for the sulfonamide NHs. Fitting the data to a 1:1
binding model using an iterative curve-fitting method yielded
association constants and maximum change in chemical shift.13
p Ka Mea su r em en ts. Translocases 1-3 were dissolved in
9:1 CD3OD/D2O (1 mM, 750 mL) in three different NMR tubes.
To each tube, 4 equiv of DCl were added to fully protonate
the tertiary amine. Chemical shifts of the NCH2 and NCH2CH2
protons were monitored upon the addition of small aliquots
(1-3 mL) of NaOD stock solution (0.1 M). A pD reading was
also recorded after each addition. The changes in chemical shift
were plotted against the pD values, and the pKa values were
extracted from a curve fitting procedure (Figure 6).
an appropriate amount of 5 mM N-tris[hydroxymethyl]methyl-
2-aminoethanesulfonic acid (TES, pH 7.4) and 100 mM NaCl.
Multilamellar vesicles were generated using a Vortex mixer;
use of a Pyrex glass bead ensured complete lipid removal from
the flask wall. The multilamellar vesicles were extruded to
form large unilamellar vesicles with a hand-held Basic Li-
posoFast device purchased from Avestin, Inc, Ottawa, Canada.
The vesicles were extruded 29 times through a 19-mm poly-
carbonate Nucleopore filter with 100-nm diameter pores.
NBD-Lip id Tr a n sloca tion Assa y. Excitation was set at
470 nm, and fluorescence emission was measured at 530 nm
using a 515 nm filter. Exo-labeled vesicles were generated
upon addition of an ethanolic solution of NBD-lipid to a 35
mL solution of unlabeled liposomes (25 µM) at room temper-
ature in TES buffer. To each vesicle solution, translocase (38
µM) was added from THF stock solutions. (Control experi-
ments showed that THF does not induce flip-flop on its own.)
At varying time intervals, 3-mL samples were removed and
assayed for transbilayer movement. The assay consisted of a
dithionite injection (60 mM) at t ) 50 s, and a Triton X-100
injection (0.5%) at t ) 150 s. Total assay time was 200 s. The
amount of probe located in the outer monolayer was calculated
according to the following equation: % Exo Probe ) (Fi - Ff)/
Fi, where Fi and Ff are the intensities just prior to the additions
of dithionite and Triton X-100, respectively. All % exo probe
values contain (5% error. Experiments were repeated at least
three times, and the values reported represent averages.
Ca r boxyflu or escein Lea k a ge Assa y. Excitation was set
at 495 nm, and fluorescence emission was measured at 520
nm using an open filter. Vesicles encapsulating carboxyfluo-
rescein were freeze-thawed 10 times prior to extrusion. The
fluorescence of a 3-mL sample of vesicles (25 µM) in TES buffer
was monitored. After 50 s, an aliquot of translocase (38 µM)
was added. Detergent (0.5% Triton X-100) was added at t )
250 s. Total assay time was 300 s. A constant fluorescent
intensity up until the point of detergent addition indicated no
vesicle leakage.
Mon o-NBD 8/Dith ion ite P r otection Assa y. The prepara-
tion was analogous to that described above for the NBD-lipid
translocation assay, with the exception that unlabeled phos-
pholipids were used. In addition, for select samples, Triton
X-100 was not added until t ) 3500 s instead of t ) 150 s.
Sin gle-Cr ysta l X-r a y Diffr a ction An a lysis.16 Crystal
data were collected and integrated using a Bruker Apex
system, with graphite monochromated Mo KR (λ ) 0.71073
Å) radiation at 100 K (170 K for sulfonamide 1). The structures
were solved by direct methods using SHELXS-97 and refined
using SHELXL-97 (Sheldrick, G. M., University of Go¨ttingen).
Crystals of 1‚H2O deposited directly from the eluent after
column chromatography with ethyl acetate. Non-hydrogen
atoms were refined anisotropically; full matrix least squares
refinement on F2 converged with wR2 ) 0.1170, R1 ) 0.0424
[5562 reflections with I > 2σ(I)], goodness of fit ) 1.014. The
hydrogen atoms in the structure were found in difference
Fourier maps and refined isotropically. Crystallographic sum-
mary for 1‚H2O: triclinic, Mr ) 626.79, Z ) 2 in a cell of unit
dimensions a ) 9.4007(6) Å, b ) 11.5963(7) Å, c ) 14.5787(9)
Å, R ) 92.1160(10)°, â ) 95.6880(10)°, γ ) 105.3620(10)°, V )
1521.6(2) Å3, Fcalc ) 1.368 Mg m-3, F(000) ) 664.
Crystals of the ionic complex [(1 + H)+:(dibenzyl phosphate)-]‚
CHCl3] were obtained in the following way. A solution of
sulfonamide 1 (0.250 g, 0.411 mmol) and dibenzyl phosphate
(0.114 g, 0.410 mmol) in CHCl3 (ca. 1-2 mL) in a small vial
was carefully layered with ethyl acetate (ca. 5-8 mL) and
allowed to sit loosely capped for 2 days after which time
crystals suitable for analysis were obtained. Non-hydrogen
atoms were refined anisotropically; full matrix least squares
refinement on F2 converged with wR2 ) 0.1410, R1 ) 0.0545
[8281 reflections with I > 2σ(I)] goodness of fit ) 1.037. The
unit cell contained one tertiary ammonium ion, one phosphate
ion, and one 2-fold disordered CHCl3 solvent molecule (relative
site occupancies 59%/41%). One of the phosphate benzyl rings
was also disordered (51%/49% site occupancies). Hydrogen
atom positions were located from difference Fourier maps,
except for the hydrogen atoms located on the disordered benzyl
rings and disordered solvent molecule which were placed at
idealized positions with the appropriate site occupancy factor.
Crystallographic summary for [(1 + H)+:(dibenzyl phosphate)-]‚
CHCl3]: triclinic, Mr ) 1006.38, Z ) 2 in a cell of unit
dimensions a ) 10.7898(13) Å, b ) 15.1014(18) Å, c ) 16.812(2)
Å, R ) 114.089(2)°, â ) 91.449(2)°, γ ) 105.462(2)°, V )
2382.1(5) Å3, Fcalc ) 1.403 Mg m-3, F(000) ) 1052.
Mon o-NBD 8/Rh -P E F RET Assa y. Excitation was set at
470 nm, and fluorescence emission was measured at 590 nm
using a 515 nm filter. An aliquot of 8 (38 µM) was added to a
stirring solution of POPC/Rh-PE (0.6%) vesicles (25 µM) in
TES buffer at t ) 50 s. Triton X-100 (0.5%) was added at t )
350 s.
Mon o-Da n syl 9 Solva tion Stu d y. Excitation was set at
340 nm, and an emission scan was obtained from 400 to 600
nm using an open filter. Spectra were acquired for solutions
of 9 (38 µM) in 3 mL of THF, CH2Cl2, BuOH, EtOH, and
MeOH. A spectrum was acquired of POPC vesicles (25 µM)
extruded in the presence of 9 (5 mol %), in TES buffer, pH
7.4.
Amide 2 was recrystallized from ethyl acetate with a few
drops of chloroform. Full matrix least squares refinement on
F2 converged with wR2 ) 0.1410, R1 ) 0.0705 [2823 reflections
with I > 2σ(I)], goodness of fit ) 1.100. All non-hydrogen atoms
were refined anisotropically, except for the phenyl and methyl
carbon atoms which were refined isotropically. Hydrogen atom
positions were placed at idealized positions, except for the
amide hydrogen atoms, which were located from difference
Fourier maps. A riding model with fixed thermal parameters
[uij ) 1.2Uij(eq) for the atom to which they are bonded], was
used for subsequent refinements. Crystallographic summary
for amide 2: monoclinic, Mr ) 500.63, Z ) 4 in a cell of unit
dimensions a ) 14.391(3) Å, b ) 12.212(3) Å, c ) 15.413(3) Å,
Ack n ow led gm en t. This work was supported by the
National Institutes of Health (GM 59078), the Walther
Cancer Research Center (Postdoctoral Fellowship to
T.N.L) and the University of Notre Dame (Molecular
Biosciences and George M. Wolf Fellowships to J .M.B.).
S.N.B. and V.U. acknowledge support from a DuPont
Young Professor Grant and the Dow Innovation Recog-
nition Program.
R ) 90o, â ) 94.033(5)°, γ ) 90°, V ) 2701.9(10) Å3, Fcalc
)
Su p p or tin g In for m a tion Ava ila ble: NMR spectra of
compounds 8 and 9 (4 pages). This material is available free
of charge via the Internet at http://pubs.acs.org. This material
is contained in libraries on microfiche, immediately follows this
article in the microfilm version of the journal, and can be
ordered from the ACS; see any current masthead page for
ordering information.
1.231 Mg m-3, F(000) ) 1072.
F lu or escen ce Assa ys. Vesicle P r ep a r a tion . Lipids were
dissolved in chloroform and were then dried in vacuo for at
least 1 h. Hydration was performed at room temperature with
(31) Wilcox, C. S. In Frontiers in Supramolecular Organic Chemistry
and Photochemistry; Schneider, H.-J ., Durr, H., Eds.; VCH: Weinham,
1991; pp 123-143.
J O016416S