Differential interaction of an AmB Analogue and ergosterol in enantiomeric membranes

Article abstract of DOI:10.1021/ol1003516

An amphotericin B derivative exhibits differential interaction with vesicles formed from either natural (R)-or unnatural (S)-POPC and ergosterol. This implicates a close association between the polyene macrolide and the phospholipid bilayer and may account for its increased antifungal activity.

Full text of DOI:10.1021/ol1003516

ORGANIC
LETTERS
2010
Vol. 12, No. 8
1772-1775
Differential Interaction of an AmB
Analogue and Ergosterol in
Enantiomeric Membranes
Ock-Youm Jeon and Erick M. Carreira*
Laboratorium fu¨r Organische Chemie, ETH-Zu¨rich, CH-8093 Zu¨rich, Switzerland
carreira@org.chem.ethz.ch
Received February 15, 2010
ABSTRACT
An amphotericin B derivative exhibits differential interaction with vesicles formed from either natural (R)-or unnatural (S)-POPC and ergosterol.
This implicates a close association between the polyene macrolide and the phospholipid bilayer and may account for its increased antifungal
activity.
Amphotericin B (AmB, 1) is a commonly used antifungal
agent for numerous systemic fungal infections.1 It was
originally isolated from fermented cultures of Streptomyces
nodosus.2 In the widely accepted model for its mode of
action, AmB forms ion channels in cell membranes that lead
to efflux of potassium ions (K+) and other small electrolytes.3
It has been suggested that the channels are preferentially
stabilized by ergosterol over cholesterol, accounting for the
selective action against fungal membranes.4 In addition to
its current clinical use as an antifungal agent, there have been
reports that AmB may have therapeutic utility in treatment
of HIV, Creutzfeld Jakob, and other diseases with implica-
tions of other possible modes of action.5 Thus, it is
increasingly important to understand in greater detail the role
of chemistry, biology, and biochemistry of AmB.6
Herein we disclose an unexpected result in which the most
active anlogue of AmB (2, Figure 1) reported to date leads
to K+-efflux in liposomes that is sensitive to the configuration
of the phospholipid which constitutes the liposome, namely
(R)- versus (S)-POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-
phosphocholine). Although previous studies have examined
the use of ent-cholesterol7 on K+-efflux, there have been no
studies on the effect the configuration of the phospholipid
has on channel formation. We believe the observations from
such a study could shed light on the underlying reasons for
the increased activity of AmB derivative 2.
(1) (a) Gallis, H. A.; Drew, R. H.; Pickard, W. W. ReV. Infect. Dis.
1990, 12, 308–329. (b) Hann, I. M.; Prentice, H. G. Int. J. Antimicrob.
Agents 2001, 17, 161–169. (c) Lemke, A.; Kiderlen, A. F.; Kayser, O. Appl.
Microbiol. Biotechnol. 2005, 68, 151–162.
In landmark studies, Rychnovsky examined conductance
in liposomes induced by AmB in the presence of cholesterol
(2) (a) Sternberg, T. H.; Wright, E. T.; Oura, M. A. Antibiot. Annu.
1955, 3, 566–573. (b) Gold, W.; Stout, H. A.; Pagano, J. F.; Donovick, R.
Antibiot. Annu. 1955, 3, 579–586. (c) Vandeputte, J.; Wachtel, J. L.; Stiller,
E. T. Antibiot. Annu. 1955, 3, 587–591.
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D. M.; Manthorpe, J. M.; Wurtz, N. R.; Carreira, E. M. Chem.sEur. J.
2009, 15, 7117–7128.
(3) For a recent review, see: Cereghetti, D. M.; Carreira, E. M. Synthesis
2006, 914–942.
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Kobayashi, G. S. Antimicrob. Agents Chemother. 1974, 5, 377–382. (b)
Herve, M.; Debouzy, J. C.; Borowski, E.; Cybulska, B.; Garybobo, C. M.
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10.1021/ol1003516  2010 American Chemical Society
Published on Web 03/25/2010

Scheme 1. Synthesis of (S)-POPC
Figure 1. Structures of AmB (1) and N,N-bis(3-aminopropyl) AmB
(2).
and ent-cholesterol.8 Interestingly, no conductance was
observed for liposomes incorporating ent-cholesterol at
concentrations in which the corresponding vesicles including
natural cholesterol displayed ion leakage. These results are
consistent with direct interaction between cholesterol and
AmB channels, as suggested by the models for channel
formation wherein sterol participation in stabilization of the
channel is critical.9 The study leads to an intriguing question
of whether cholesterol may also be altering the overall
macroscopic properties of the lipid membrane in addition to
interacting with the channel formed by AmB. These experi-
ments are landmark, as they initiated a new approach
involving the use of enantiomeric lipids as probes for
membrane function.10
myces cereVisiae. Morever, 2 elicits reduced hemotoxicity
(EH50 ) 10 µM for 2 vs 4.0 µM for AmB) and displays fast
K+-efflux above 1.0 µM, similar to AmB. This represents
one of a very select few analogues of AmB that have been
prepared with increased activity.16 However, we have not
been able to discern the underlying reason for its enhanced
activity.
There are numerous studies that document the effect of
various sterols on the membrane and susbsequent activity
of AmB.11,12 However, it is also important to note that AmB
is active with sterol-free phospholipid membranes, leading
to electrolyte efflux.13 Consequently, studies of the role of
the specific role of membrane components, specifically
phospholipids, would warrant investigation.14 Thus, we
embarked on a study involving the effect of phospholipid
stereochemistry on channel properties as assayed by K+-
efflux.
As shown in Scheme 1, optically pure (S)-POPC was
synthesized in six steps from commercially available racemic
benzyl ether of 2,3-epoxy-1-propanol. Hydrolytic kinetic
resolution (HKR)17 with [(R,R)-N,N′-bis(3,5-di-tert-butyl-
salicylidene)-1,2-cyclohexandiaminato(2-)]cobalt(III) ac-
etate complex gave an (S)-benzyl glycidyl ether (3) in
optically pure form (>99%ee).18 Palmitic acid was shown
to participate in opening of (S)-benzyl glycidol epoxide (3)
in the presence of tetraethylammonium bromide (Et4NBr)
to provide 1-O-benzyl-3-O-palmitoyl-sn-glycerol (4) in 64%
yield.19 The secondary hydroxyl group of 1-O-benzyl-3-O-
palmitoyl-sn-glycerol (4) was efficiently acylated with oleic
acid in quantitative yield using 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide hydrochloride (EDCI) and a stochio-
metric amount of N,N-dimethyl-4-aminopyridine (DMAP).20
The O-benzyl protecting group in (5) was removed by the
action of boron trichloride (BCl3) in dry dichloromethane
(CH2Cl2) at -78 °C, affording alcohol 6 in 73% yield.
Recently, we documented a novel analogue of AmB,
namely, N,N-bis(3-aminopropyl) AmB derivative (2), which
possesses notable activity in a variety of fungal assays with
the added benefit of displaying increased therapeutic index.15
Thus, N,N-bis(3-aminopropyl) AmB displays MIC (minimal
inhibitory concentration) ) 0.020 µM compared to AmB
with MIC ) 0.30 µM in yeast assays involving Saccharo-
(8) Belani, J. D.; Rychnovsky, S. D. J. Org. Chem. 2008, 73, 2768–
2773.
(9) Matsumori, N.; Sawada, Y.; Murata, M. J. Am. Chem. Soc. 2006,
128, 11977–11984.
(10) Biellmann, J. F. Chem. ReV. 2003, 103, 2019–2033.
(11) (a) De Kruijff, B.; Demel, R. A. Biochim. Biophys. Acta 1980, 599,
484–492. (b) Brutyan, R. A.; McPhie, P. J. Gen. Physiol. 1996, 107, 69–
(16) Paquet, V.; Volmer, A. A.; Carreira, E. M. Chem.sEur. J. 2008,
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78
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(17) (a) Tokunaga, M.; Larrow, J. F.; Kakiuchi, F.; Jacobsen, E. N.
Science 1997, 277, 936–938. (b) Furrow, M. E.; Schaus, S. E.; Jacobsen,
E. N. J. Org. Chem. 1998, 63, 6776–6777.
(12) Zumbuehl, A.; Stano, P.; Heer, D.; Walde, P.; Carreira, E. M. Org.
Lett. 2004, 6, 3683–3686
(13) Backes, B. J.; Rychnovsky, S. D. Anal. Biochem. 1992, 205, 96–
.
(18) Schaus, S. E.; Brandes, B. D.; Larrow, J. F.; Tokunaga, M.; Hansen,
K. B.; Gould, A. E.; Furrow, M. E.; Jacobsen, E. N. J. Am. Chem. Soc.
2002, 124, 1307–1315.
99.
(14) (a) Barwicz, J.; Tancrede, P. Chem. Phys. Lipids 1997, 85, 145–
155. (b) H1c-Wydro, K.; Kapusta, J.; Jagoda, A.; Wydro, P.; Dynarowicz-
Łatka, P. Chem. Phys. Lipids 2007, 150, 125–135.
(19) Lok, C. M. Chem. Phys. Lipids 1978, 22, 323–337.
(20) Dyer, B. S.; Jones, J. D.; Ainge, G. D.; Denis, M.; Larsen, D. S.;
Painter, G. F. J. Org. Chem. 2007, 72, 3282–3288.
(15) Paquet, V.; Carreira, E. M. Org. Lett. 2006, 8, 1807–1809.
Org. Lett., Vol. 12, No. 8, 2010
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Figure 2.
K+ release from LUV after external addition of N,N-bis(3-aminopropyl) AmB (2): (R)-POPC or (S)-POPC (s), with 13% of
ergosterol (- - -) and with 30 mol % of cholesterol (···): (A) 2 (0.1 µM) with (R)-POPC; (B) 2 (1 µM) with (R)-POPC; (C) 2 (0.1 µM) with
(S)-POPC; (D) 2 (1 µM) with (S)-POPC.
Installation of the choline headgroup was effected by
treatment of 6 with 2-chloro-2-oxo-1,3,2-dioxaphospholane
to give a glycerophosphoryl intermediate, which was then
subjected to a ring-opening substitution reaction by exposure
to excess anhydrous NMe3 to provide (S)-POPC (7).21
differenece in K+-efflux in (S)-POPC versus (R)-POPC
membranes and in the presence or absence of ergosterol or
cholesterol.
In contrast to the results with AmB, for the active
bis(aminopropylene) derivative 2 marked differences were
observed in the K+-efflux experiments involving liposomes
prepared from (R)-POPC or (S)-POPC (Figure 2). Compari-
son of the efflux data at both concentrations tested reveals
differences between the two enantiomeric liposomes: at 0.1
µM, Figure 2 A versus C and at 1 µM, Figure 2 B versus D.
The difference is especially marked for liposomes incorpo-
rating ergosterol (- - -). In this respect, the liposomes
including cholesterol (···) and liposomes with only POPC (s)
display no discernible difference: compare A versus B and
C versus D. The data at the higher concentration is
particularly telling (Figure 2 B vs D). At 1 µM, N,N-bis(3-
aminopropyl) AmB (2) in (R)-POPC liposomes with ergos-
terol displayed full K+ leakage within 10 min, whereas the
cholesterol-containing liposomes plateau at about 70% efflux.
By contrast, in (S)-POPC liposomes efflux proceeds to about
60% in both ergosterol and cholesterol containing membranes
over the course of 10 min. Thus, interestingly, at this higher
The large unilamellar vesicles (LUV) containing either (R)-
POPC or (S)-POPC were prepared according to literature
procedures22 (see the Supporting Information) in the presence
of ergosterol (13%), cholesterol (30%) or absent sterol
altogether. The induced potassium ion (K+) efflux was
measured by K+ selective electrodes in the enantiomeric
LUVs at low (0.1 µM) as well as high (1 µM) concentrations
of AmB or N,N-bis(3-aminopropyl) AmB. The parent poly-
ene macrolide AmB (0.1 or 1 µM) did not show any marked
(21) Menger, F. M.; Chen, X. Y.; Brocchini, S.; Hopkins, H. P.;
Hamilton, D. J. Am. Chem. Soc. 1993, 115, 6600–6608.
(22) (a) Zumbuehl, A.; Martin, S. E.; Hussey, S. L.; Sohrmann, M.;
Clark, D. D.; Peter, M.; Peterson, B. R.; Carreira, E. M. Angew. Chem.,
Int. Ed. 2004, 43, 5181–5185. (b) Paquet, V.; Zumbuehl, A.; Carreira, E. M.
Bioconjugate Chem. 2006, 17, 1460–1463. (c) Szpilman, A. M.; Cereghetti,
D. M.; Wurtz, N. R.; Manthorpe, J. M.; Carreira, E. M. Angew. Chem., Int.
Ed. 2008, 47, 4335–4338. (d) Zumbuehl, A.; Stano, P.; Sohrmann, M.;
Dietiker, R.; Peter, M.; Carreira, E. M. ChemBioChem 2009, 10, 1617–
1620.
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Org. Lett., Vol. 12, No. 8, 2010

concentration the efflux in liposomes constituted from (S)-
POPC in ergosterol containing liposomes substantially
diminished while that of cholesterol containing LUVs
remains effectively unchanged.
for the observed differences between (S)-POPC and (R)-
POPC liposomes and potassium efflux with 2. First, it may
very well be that the phospholipid itself plays a role in
stabilization of the channels. The fact that 2 is more active
and that it incorporates a greater number of protonated
amines may in fact enhance the interaction between the
polyene macrolide and the phospholipid. Alternatively, it may
be that a stronger interaction for ergosterol with (S)-POPC
membranes over (R)-POPC would make it less available to
or less mobile to interact and stabilize channel formation.
The differentiation between these two modes of action
provides stimulus for further investigation.
It has been previously reported by Covey23 that cholesterol
enantioselectively interacts with egg yolk sphingomyelin
(SPM), and this specific interaction influences the physical
properties of the membrane. Thus, in the experiment, SPM
monolayers containing 20-40 mol % of either cholesterol
or ent-cholesterol exhibited different compression behavior,
and this was suggested to be indicative of diastereomeric
sterol-lipid interactions occurring in these monolayers.
However, it should be noted that in a later publication no
significant enantiospecific interactions were observed (dif-
ferential scanning calorimetry, X-ray diffraction, and neutral
buoyant-density experiments) in a lipid bilayer model system
consisting of enantiomeric cholesterol and egg sphingomy-
elin.24
In summary, we document an unusual effect in enantio-
meric liposomes with a novel, potent AmB analog. In
liposomes constituted from (R)-POPC and ergosterol the
observed efflux of K+ is greater than that observed in the
corresonding liposomes generated from the enantiomeric (S)-
POPC and ergosterol. Interestingly, the effect is not observed
for AmB and, consequently, may be indicative of the
observed enhanced activity of 2 (MIC ) 0.020 µM)
compared to AmB (MIC ) 0.30 µM) in yeast. In a broader
sense the experiments delineated herein underscore the
possibilities for the investigation of biologically relevant
processes with additional stereochemical probes beyond
steroids, such as enantiomeric phospholipids.
The results of our investigation indicates that enantiospe-
cific interactions can be observed in a more complex three-
bodied situation involving the phospholipid ((R)- or (S)-
POPC), steroid (cholesterol or ergosterol) and a natural
product analogue (polyene macrolide antibiotic derivative).
Importantly, it suggests a role for the phospholipid compo-
nent of the membrane in the differential stabilization of the
putative channels formed by the polyene antibiotic in
combination with ergosterol. Although there have been
models that focus on the interaction of sterols with the
polyene macrolide channel, the role of the phospholipid itself
is not considerably well investigated.
Acknowledgment. We thank the Swiss National Science
Foundation for support of this research in the form of a grant.
Supporting Information Available: Experimental pro-
cedures and characterization data of all new compounds;
experimental details involving K+-efflux studies with lipo-
somes. This material is available free of charge via the
Internet at http://pubs.acs.org.
In the case at hand, there are two plausible explanations
(23) (a) Lalitha, S.; Kumar, A. S.; Covey, D. F. Chem. Commun. 2001,
1192–1193. (b) Lalitha, S.; Kumar, A. S.; Stine, K. J.; Covey, D. F. J.
Supramol. Chem. 2001, 1, 53–61.
(24) (a) Mannock, A.; McIntosh, T. J.; Jiang, X.; Covey, D. F.;
McElhaney, R. N. Biophys. J. 2003, 84, 1038–1046. We thank a reviewer
for pointing this out.
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