Synthetic peptides with selective affinity for apoptotic cells

Article abstract of DOI:10.1039/b514748d

The appearance of anionic phosphatidylserine (PS) in the outer monolayer of the plasma membrane is a universal indicator of the early/intermediate stages of cell apoptosis. The most common method of detecting PS on a cell surface is to use the protein annexin V; however, in certain applications there is a need for alternative reagents. Recent research indicates that rationally designed zinc 2,2′-dipicolylamine (Zn²⁺-DPA) coordination complexes can mimic the apoptosis sensing function of annexin V. Here, a series of fluorescently-labelled, tri-and pentapeptides with side chains containing Zn²⁺-DPA are prepared and shown to selectively bind to anionic vesicle membranes. Fluorescein-labelled versions of the peptides are used to detect apoptotic cells by fluorescence microscopy and flow cytometry. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b514748d

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthetic peptides with selective affinity for apoptotic cells
Kristy M. DiVittorio,a James R. Johnson,a Emma Johansson,b Aaron J. Reynolds,b
Katrina A. Jolliffe*b and Bradley D. Smith*a
Received (in Pittsburgh, PA) 18th October 2005, Accepted 6th March 2006
First published as an Advance Article on the web 30th March 2006
DOI: 10.1039/b514748d
The appearance of anionic phosphatidylserine (PS) in the outer monolayer of the plasma membrane is
a universal indicator of the early/intermediate stages of cell apoptosis. The most common method of
detecting PS on a cell surface is to use the protein annexin V; however, in certain applications there is a
need for alternative reagents. Recent research indicates that rationally designed zinc 2,2ꢀ-dipicolylamine
(Zn2+–DPA) coordination complexes can mimic the apoptosis sensing function of annexin V. Here, a
series of fluorescently-labelled, tri-and pentapeptides with side chains containing Zn2+–DPA are
prepared and shown to selectively bind to anionic vesicle membranes. Fluorescein-labelled versions of
the peptides are used to detect apoptotic cells by fluorescence microscopy and flow cytometry.
is a useful apoptosis sensor, but it has a number of limitations and
Introduction
there is a need for replacement reagents that are cheap, robust,
low-molecular weight, rapidly-binding, membrane-impermeable,
and Ca2+-independent.
Assays for programmed cell death, or apoptosis, are used often
in cell biology research and also in the drug discovery process.
Various intracellular methods for detecting apoptosis have been
reported such as caspase action and nucleic acid fragmentation.1
An attractive extracellular strategy is to monitor the change in
phospholipid distribution across the cell plasma membrane. In
particular, the appearance of anionic phosphatidylserine (PS)† in
the membrane outer monolayer is a universal indicator of the
early/intermediate stages of cell apoptosis and can be detected
before morphological changes can be observed.2–4 The most
common method of detecting PS on a cell surface is to use the Ca2+-
dependent, PS-binding protein annexin V.5–7 For in vitro assays, the
35 kDa protein is typically labelled with a fluorescent dye, whereas
radioactive and MRI contrast agents are employed for in vivo
imaging.8,9 Although it is utilized extensively, the labelled protein
is expensive and moderately unstable. Thus, it is not convenient for
high throughput screening assays in drug discovery. An additional
concern is that approximately 2.5 mM of extracellular Ca2+ is
needed for complete binding. This can lead to false positive results
because most animal cells have a Ca2+ dependent scramblase that
can move PS to the cell surface.10 Furthermore, complete annexin
binding requires incubation times of up to one hour, which is
problematic for kinetic assays.11,12 In short, dye labelled annexin V
We have discovered that rationally designed zinc 2,2ꢀ-
dipicolylamine (Zn2+–DPA) coordination complexes can mimic
the apoptosis sensing function of annexin V. Our designs were
conceived after inspection of the X-ray crystal structure of an
annexin V–glycerophosphoserine complex. The X-ray structure
shows that one phosphoserine head group is coordinated to
two bridging Ca2+ ions that are in turn coordinated to one of
the four canonical binding sites at the protein surface.13 This
three-component assembly picture suggested to us that synthetic
metal coordination complexes with appropriate charge, geometry,
and spatial orientation may bind to the head group of an-
ionic phospholipids preferentially over zwitterionic phospholipids
(Scheme 1).‡
Scheme 1 Dye-labelled, metal coordination complexes can mimic the
protein annexin V and coordinate to a PS head group via the phosphate
and/or carboxylate anion.
aDepartment of Chemistry and Biochemistry, University of Notre Dame,
Notre Dame, IN 46556, USA. E-mail: smith.115@nd.edu; Fax: 574-631-
6652
bSchool of Chemistry, The University of Sydney, Sydney, 2006, NSW,
Australia. E-mail: jolliffe@chem.usyd.edu.au; Fax: 61-2-9351-3329
The breakthrough compound was PSS-380 (phosphatidylserine
sensor 380 absorbance, see Scheme 2) which was successfully used
to detect apoptosis in Jurkat cells.14,15 The utility of PSS-380 as
† Abbreviations: 7AAD: 7-aminoactinomycin D, DHPC: 1,2-dihexanoyl-
sn-glycero-3-phosphocholine, DHPS: 1,2-dihexanoyl-sn-glycero-3-[phospho-
L-serine], DPA: 2,2ꢀ-dipicolylamine, EDC: N-(3-dimethylaminopropyl)-
Nꢀ-ethylcarbodiimide, FITC: fluorescein isothiocyanate, HOBt: 1-hy-
droxybenzotriazole, NBD: 7-nitro-1,3-benz-2-oxa-1,3-diaza-4-yl, NMM:
N-methylmorpholine, POPA: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate,
POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPG: 1-
palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol, POPS: 1-palmitoyl-2-
oleoyl-sn-glycero-3-[phospho-L-serine], PS: phosphatidylserine, TES: N-
tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid, Zn2+–DPA: zinc
2,2ꢀ-dipicolylamine.
‡ The membrane recognition ability of annexin V is due primarily to its
ability to bind Ca2+ cations in a geometrical way that allows simultaneous
metal coordination by a PS head group. The DPA ligand acts in a
functionally similar manner. It can bind Zn2+ cations with high affinity
while leaving a vacant metal coordination site for a PS head group.
The three-component assembly does not work with Ca2+ because Ca2+
association with the DPA ligand is too weak.
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based on a synthetically flexible, peptide architecture. The peptide
design was pursued for a number of reasons. Previous structural
studies suggested that two closely spaced Zn2+–DPA units were
required for selective binding to an anionic membrane surface,
however, this requirement had not been thoroughly tested.18–20
Indeed, we find that highly selective binding of anionic membranes
can be achieved using peptides containing two widely spaced Zn2+–
DPA units. Moreover, reasonably selective binding is observed
with a peptide containing just a single Zn2+–DPA unit. To facilitate
the peptide synthesis we have developed a simple method of
converting the amine terminus of an ornithine side chain into
a DPA unit. The preparation of dye-labelled, zinc coordinated
tripeptides 1–NBD and 1–Fl is described, as well as the analogous
pentapeptides 2–NBD and 2–Fl (Scheme 3). The NBD-labelled
peptides are employed as probes to measure affinity to vesicle
membranes, and the Fl-labelled peptides as fluorescent stains
for detecting cell apoptosis by fluorescence microscopy and flow
cytometry.
Results and discussion
Synthesis
The pentapeptides 2–NBD and 2–Fl were designed with glycine
residues between the two amino acids bearing the DPA ligands,
which allows the two coordinated zinc(II) ions to simultaneously
contact a membrane surface without causing molecular strain. The
central building block is the ornithine derivative 4, with a DPA
side chain. Attempts to prepare this compound by direct alky-
lation of the amino terminus of N-Boc-ornithine, 3,21 were only
partially successful due to difficulties with product purification.
Therefore, we turned to a reductive amination procedure,22 treating
3 with two equivalents of picolylaldehyde in the presence of
sodium triacetoxyborohydride to give 4 in good yield (Scheme 4).
Scheme 2 PS sensors PSS-380 and PSS-480.
a fluorescent stain for PS has been confirmed independently in
publications from other research groups.16,17 Although PSS-380
is a useful fluorescent sensor, its excitation wavelength (380 nm)
is not compatible with the lasers in most flow cytometers. Thus,
we pursued a more flexible second generation design that allowed
conjugation to a range of fluorescent reporter groups.18 This work
led to several new apoptosis sensors such as the fluorescein-linked
PSS-480.19,20 Here, we describe a third generation design that is
Scheme 3 Dye-labelled Zn2+–DPA peptides.
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Treatment of 5 with a 4 M solution of HCl in dioxane gave 6
as the hydrochloride salt, which was then coupled with N-Boc-
glycine to give the tripeptide 8 in good yield (Scheme 5). Boc-
deprotection using the conditions described above gave the amine
9 as the hydrochloride salt, which was treated with FITC in the
presence of triethylamine to give the fluorescein labelled tripeptide
10. Attempts to react 9 directly with 7-chloro-4-nitrobenzofurazan
(NBD-Cl) to give the analogous NBD-labelled tripeptide were
unsuccessful. We considered the probable reason for this to be
steric congestion and so redesigned the compound to contain a
six-carbon alkyl chain between the NBD fluorophore and the
peptide. Therefore, amine 6 was coupled with the known NBD-5-
aminohexanoic acid 1123 to give the NBD-labelled tripeptide 12 in
moderate yield.
A fragment coupling approach was employed to convert 5 into
a pentapeptide. That is, the acid fragment 7 was coupled to amine
6 to yield the tetrapeptide 13 (Scheme 6). Boc-deprotection of 13
using standard conditions gave the amine 14 as the hydrochloride
salt, which was coupled with 11 to give the NBD-labelled
pentapeptide 15 in high yield. Similarly, coupling of amine 14 with
N-Boc-protected hexanoic acid 16 under the standard conditions
described above gave the pentapeptide 17 in good yield. Deprotec-
tion of 17 followed by treatment with FITC, gave the fluorescein
labelled pentapeptide 18 in good yield. The fluorescently labelled
peptides 10, 12, 15 and 18 were then treated with the appropriate
molar equivalents of zinc(II) nitrate to give the zinc-coordinated
peptides 1–Fl, 1–NBD, 2–NBD and 2–Fl, respectively.
Scheme
4
Reagents and conditions: (a) 2-pyridinecarboxaldehyde
(3 equiv.), NaHB(OAc)3 (9 equiv.), CH2Cl2, 85%; (b) EDC·HCl (1.2 equiv.),
HOBt·H2O (1.2 equiv.), NMM (3 equiv.), CH2Cl2–DMF, 95%; (c) 4 M HCl
in dioxane, quant.; (d) NaOH (3 equiv.), MeOH–H2O, 90%.
Vesicle studies
Material prepared via this method was readily purified by column
chromatography on silica gel. The acid 4 is readily incorporated
into peptides using standard solution phase peptide coupling
techniques. Coupling to glycine methyl ester using EDC–HOBt
as the activating agents gave the dipeptide 5 in good yield.
The fluorescent derivatives 1–NBD and 2–NBD were prepared
because previous work had shown how the NBD fluorophore
can be used to measure binding and partitioning into vesi-
cle membranes.18,24 The emission intensity of an NBD-labelled
Zn2+–DPA conjugate increases when it associates with a vesicle
Scheme 5 Reagents and conditions: (a) N-Boc-Gly-OH, EDC·HCl, HOBt·H2O, NMM (3 equiv.), CH3CN, 89%; (b) 4 M HCl in dioxane, quant.;
(c) FITC (2 equiv.), NEt3 (4 equiv.), MeOH, 84%; (d) EDC·HCl (1.8 equiv.), HOBt·H2O (1.8 equiv.), NMM (5 equiv.), CH3CN, 58%.
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Scheme 6 Reagents and conditions: (a) EDC·HCl (1.1 equiv.), HOBt·H2O (1.1 equiv.), NMM (5 equiv.), CH2Cl2–DMF, 80%; (b) 4 M HCl in dioxane,
quant.; (c) 11 (1.5 equiv.), EDC·HCl (2.5 equiv.), HOBt·H2O (2.5 equiv.), NMM (10 equiv.), CH3CN, 82%; (d) EDC·HCl (1.1 equiv.), HOBt·H2O
(1.1 equiv.), NMM (6 equiv.), CH3CN, 78%; (e) Step 1: 4 M HCl in dioxane; Step 2: FITC (1.3 equiv.), NEt3 (10 equiv.), MeOH, 86%.
membrane, which allows titration experiments to be conducted.
Thus, separate samples of 1–NBD and 2–NBD were titrated
with either zwitterionic vesicles composed only of POPC, or
anionic vesicles composed of 1 : 1 mixtures of POPG–POPC,
POPA–POPC, or POPS–POPC (see Scheme 7 for phospholipid
structures). The unilamellar vesicles (100 nm diameter, 25 lM
total phospholipid, pH 7.4) were prepared by standard extrusion
methods. The titration curves in Fig. 1 show that both peptides
have very weak affinities for the zwitterionic vesicles, but moder-
ately strong affinities for the anionic vesicles. The apparent binding
constants are all around 5 × 104 M−1 (Table 1), which are
very similar to those obtained previously with other Zn2+–DPA
Fig. 1 Fluorescence intensity change of: (a) 1–NBD, or (b) 2–NBD;
(1 lM) in the presence of 100 mol% POPC (ꢀ), 1 : 1 POPG–POPC (×), 1 :
1 POPS–POPC (ꢁ) and POPS–POPC (ꢁ) vesicles in buffer (5 mM TES,
Scheme 7 Phospholipid structures.
The Royal Society of Chemistry 2006
100 mM NaCl, pH 7.4) at 25 ◦C.
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Table 1 Binding constants in TES buffer at 25 ◦Ca
anoylphosphatidylserine (DHPS) to a 1 lM solution of 2–NBD
results in no detectable increase in NBD fluorescence intensity.
The vesicle partitioning assay is based on the ability of dithionite
to chemically reduce the NBD fluorophore and quench its
fluorescence. Sodium dithionite cannot cross vesicle membranes,
so addition of the reagent to vesicle dispersions can only quench
fluorophores that are in the external solution or exposed on the
membrane surface. Any NBD-labelled compound that partitions
into the vesicle membrane is protected from immediate quenching
by added sodium dithionite. The results of our NBD protection
assays are shown in Fig. 2. The peptides 1–NBD and 2–NBD were
added to various zwitterionic and anionic vesicle dispersions, and
after standing for 2 h, an aliquot from each sample was treated with
excess sodium dithionite. In the case of 2–NBD, the fluorescence of
all samples was completely and immediately quenched. In other
words, the bis(zinc) 2–NBD does not partition into any of the
vesicle compositions. In the case of 1–NBD, however, there is clear
evidence that anionic vesicles composed of 1 : 1 POPG–POPC
can protect about 40% of the NBD-conjugate from immediate
reaction with the dithionite. The 1–NBD quenching curve in
Fig. 2b shows biphasic kinetics. Upon dithionite addition there is
a sudden drop in fluorescence emission due to rapid reaction with
the exposed NBD groups, followed by a slower rate of quenching
attributed to either transport of the dithionite into the vesicles
or re-exposure of internalized NBD on the membrane surface.
100%
1 : 1
1 : 1 POPS–POPC 1 : 1
Peptide POPC POPA–POPC Ka × 104/M−1b
POPG–POPC
1–NBD <1
2–NBD <1
3.9 0.5
4.7 0.5
2.4 1.5
4.9 0.6
1.8 1.2
4.4 1.2
a 5 mM TES, 100 mM NaCl, pH 7.4. b Average of 3 separate experiments.
complexes.18 In the case of 1–NBD there is some variation in the
change in fluorescence intensity upon vesicle binding, which is
suggestive of differences in the non-covalent interactions of the
monozinc peptide with the vesicle membrane. However, in the case
of 2–NBD the changes in fluorescence intensity upon binding to
anionic vesicles are very similar for all three vesicle compositions.
This suggests that the attractive interaction between the strongly
hydrophilic bis(zinc) peptide and the anionic vesicle membrane
is dominated by electrostatic effects, and is independent of the
specific structure of the phospholipid head-groups. The peptides
1–NBD and 2–NBD only respond to membrane-bound phospho-
lipids because no changes in emission are observed when the titra-
tions are repeated with short acyl chain phospholipids that do not
form a bilayer membrane. For example, addition of a 1 : 1 mixture
(100 lM) of dihexanoylphosphatidylcholine (DHPC) and dihex-
Fig. 2 NBD protection assay. An aliquot (1.25 lM) of 1–NBD (ꢁ) or 2–NBD (×) was added to a 25 lM solution of vesicles in TES buffer, at 25 ◦C. After
2 h, a sample was taken and assayed. Sodium dithionite was added at assay time of 50 s and Triton-X 100 was added at 150 s. (a) 1 : 1 POPA–POPC
vesicles, (b) 1 : 1 POPG–POPC vesicles, (c) 1 : 1 POPS–POPC vesicles, (d) 100 mol% POPC vesicles.
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In other words, the mono zinc 1–NBD not only associates with
the anionic POPG–POPC vesicle membranes but it subsequently
diffuses to the inner membrane surface. In summary, both the
monozinc 1–NBD and the bis(zinc) 2–NBD appear to associate
equally well with anionic vesicle membranes. However, the less
charged 1–NBD appears to interact with the phospholipid head-
groups and in one case (1 : 1 POPG–POPC) there is clear evidence
that it can partition into the vesicle membrane. The more highly
charged 2–NBD stays only at the anionic membrane surface and
its binding interaction is essentially independent of the specific
structure of the phospholipid head-groups.
Cell studies
Fluorescence microscopy was used to determine if the fluorescein-
labelled peptides 1–Fl and 2–Fl could selectively stain apoptotic
Fig. 5 Fluorescence micrographs (600× magnification) of Jurkat cells
treated with camptothecin (10 lM) for 3.5 h to induce apoptosis, then
simultaneously stained with 1–Fl (10 lM) and 7AAD (500 ng mL−1). Phase
contrast image of treated cells is shown in (a). Fluorescence emission due
to 7AAD is shown in (b), fluorescence emission due to 1–Fl is shown in
(c). Overlay of both (b) and (c) onto phase contrast image in (d).
Fig. 3 Fluorescence micrographs (600× magnification) of HeLa cells
treated with camptothecin (10 lM) for 3.5 h to induce apoptosis, then
simultaneously stained with 1–Fl (10 lM) and 7AAD (500 ng mL−1). Phase
contrast image of treated cells is shown in (a). Fluorescence emission due
to 7AAD is shown in (b), fluorescence emission due to 1–Fl is shown in
(c). Overlay of both (b) and (c) onto phase contrast image in (d).
Fig. 6 Fluorescence micrographs (600× magnification) of Jurkat cells
treated with camptothecin (10 lM) for 3.5 h to induce apoptosis, then
simultaneously stained with 2–Fl (10 lM) and 7AAD (500 ng mL−1). Phase
contrast image of treated cells is shown in (a). Fluorescence emission due
to 7AAD is shown in (b), fluorescence emission due to 2–Fl is shown in
(c). Overlay of both (b) and (c) onto phase contrast image in (d).
cells. Separate samples of HeLa and Jurkat cells were treated
first with the anticancer drug camptothecin to induce apoptosis,
and then simultaneously with 1–Fl (or 2–Fl) and the nuclear
stain 7AAD (7-aminoactinomycin D) (Fig. 3–6). Necrotic cells,
as well as those cells in the advanced stages of apoptosis, have
permeabilized membranes and allow 7AAD to stain the cell
nucleus. Healthy cells and those cells in the early to intermediate
stages of apoptosis retain their membrane integrity and exclude
7AAD. This allows cells in early apoptosis to be identified by
selective staining with 1–Fl (or 2–Fl) and exclusion of 7AAD. The
images in Fig. 3–6 indicate that both peptides are quite effective
at selectively staining apoptotic cells. A close comparison of the
staining in Fig. 5–6 suggests that the bis(zinc) 2–Fl is perhaps
slightly better at discriminating between vital and apoptotic cells.
However, in both cases the staining selectivity is very similar to
that obtained with annexin V–FITC. Furthermore, the staining
Fig. 4 Fluorescence micrographs (600× magnification) of HeLa cells
treated with camptothecin (10 lM) for 3.5 h to induce apoptosis, then
simultaneously stained with 2–Fl (10 lM) and 7AAD (500 ng mL−1). Phase
contrast image of treated cells is shown in (a). Fluorescence emission due
to 7AAD is shown in (b), fluorescence emission due to 2–Fl is shown in
(c). Overlay of both (b) and (c) onto phase contrast image in (d).
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of the peptide. Furthermore, it should possible to attach these
peptides to the surfaces of nanoparticles which should enhance
affinity due to multivalent interactions. This may a useful way to
construct novel contrast agents for in vivo imaging of apoptotic
tissue.8,25,26 Another potential application is tumor targeting, since
it is known that PS exposure is increased on the surface of tumor
blood vessels.27
Experimental
General
Unless specified otherwise, all reactions were performed under an
inert atmosphere of nitrogen with dry, freshly distilled solvents un-
der anhydrous conditions and monitored by TLC using aluminium
backed plates coated with Merck Silica Gel 60 F254 and visualised
using either UV light (254 or 366 nm) or a molybdenum staining
reagent [a solution of (NH4)6Mo7O24·4H2O (20 g) and Ce(SO4)2
(0.4 g) in 10% sulfuric acid (400 mL)]. Flash chromatography was
performed on silica gel (Merck silica gel 60, 40–63 lm) at a pressure
of 0.3–0.4 bar and the yields given refer to chromatographically
and spectroscopically (1H NMR) homogenous material. Ratios
of solvent systems for TLC and column chromatography are ex-
pressed in v/v as specified. Reverse phase HPLC (RP-HPLC) was
performed using a Waters apparatus with a tunable absorbance
detector. Analytical RP-HPLC was performed using an Alltech-
Altima C18 column (5 lm, 4.6 mm ID, 250 mm) with a flow rate
of 0.8 mL min−1. Preparative RP-HPLC was performed using
an Alltech-Altima C18 column (10 lm, 22 mm ID, 300 mm)
with a flow rate of 7.0 mL min−1. Retention times given are for
the analytical column. Melting points were determined using a
Gallenkamp melting point apparatus and are reported in degrees
Celsius (uncorrected). Infrared absorption spectra were obtained
using a Shimadzu FTIR-8400S spectrometer as a thin film between
sodium chloride plates. NMR spectra were recorded on a Bruker
Avance DPX 200 or a Bruker Avance DPX 300 spectrometer.
Fig. 7 Flow cytometry histograms illustrating staining of Jurkat cells by
2–Fl. Cells treated with camptothecin (II, 10 lM for 16 h and III, 12.5 lM
for 16 h) exhibit significantly more staining by 2–Fl than do control cells (I,
no camptothecin). About 30% of treated cells were identified as apoptotic
using 2–Fl, while less than 4% of the untreated cells were stained with 2–Fl.
a.u., arbitrary units.
rate is essentially instantaneous which is an improvement over the
sluggish kinetics of annexin V.11,12
The utility of 2–Fl in flow cytometry was demonstrated using
a population of Jurkat cells that were treated with different
concentrations of camptothecin (0 lM, 10 lM, or 12.5 lM for 16
h) to induce apoptosis. The histograms in Fig. 7 indicate that the
fraction of apoptotic cells increases with the dose of camptothecin.
Nearly identical percentages of apoptotic cells were identified in
each group using annexin V–FITC (data not shown).
1
The solvent H and 13C signals, dH 7.26 for residual CHCl3 and
dC 77.0 for CDCl3, dH 3.31 and dC 49.0 for d4-MeOH, dH 2.50
and dC 39.5 for d6-DMSO, were used as internal references. J
values are given in Hz. Low resolution mass spectra were recorded
on a Finnigan LCQ ion trap mass spectrometer (ESI). High
resolution mass spectra were recorded on a BioApex Fourier
Transform Ion Cyclotron Resonance Mass Spectrometer (ESI).
Optical rotations were measured on a Polaar 2001 dual wavelength
polarimeter monitor in a 2.5 dm cell at 22 ◦C using the indicated
spectroscopic grade solvents. Elemental analyses were performed
by Campbell Microanalytical Laboratories. Most reagents were
commercially available from Aldrich, Fluka or Novabiochem and
Conclusions
The fluorescent Zn2+–DPA peptides, 1–Fl and 2–Fl, are able to
identify apoptotic cells using fluorescence microscopy. Further-
more, 2–Fl is an effective reagent for determining the fraction
of apoptotic cells in a cultured population using flow cytometry.
The cationic peptide complexes selectively stain apoptotic cells
because the cell plasma membrane becomes increasingly anionic
due to the appearance of phosphatidylserine. The peptides are
practical alternatives to the 35 KD protein annexin V currently
used in apoptosis assays. The results reported here demonstrate
that the molecular design of synthetic phosphatidylserine sensors
does not necessarily require two closely spaced Zn2+–DPA units.
Indeed, it is reasonable to expect that a wide range of Zn2+–DPA
coordination complexes will have selective affinity for apoptotic
cells. An attractive feature with the peptides reported here is they
have two orthogonal conjugation points. That is, their N-terminus
is labelled with a dye but their C-terminus remains available for
attachment to groups that can fine-tune the recognition properties
used as supplied. NBD-Hex-OH was prepared according to the
23
method of Jurss and Maelicke. All polar lipids were purchased
from Avanti Polar Lipids, Inc. annexin V and 7AAD were obtained
from BD Biosciences.
¨
Synthesis
Boc-Orn(DPA)-OH (4). 2-Pyridinecarboxaldehyde (13.8 g,
129 mmol) was added to a fine suspension of 3 (10.5 g, 45 mmol) in
dichloromethane (400 mL) and the resulting mixture was stirred
for 15 min. Sodium triacetoxyborohydride (40.2 g, 430 mmol)
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was added and the resulting cloudy yellow solution was stirred
for 18 h at ambient temperature under a nitrogen atmosphere.
The reaction was quenched by the addition of aqueous sodium
hydroxide (2 M, 150 mL) then the mixture was acidified to pH 6
by addition of 1 M HCl. The organic phase was separated and
the aqueous phase was extracted with chloroform–isopropanol
(3 : 1, v/v) (2 × 100 mL). The organic phases were combined and
washed with brine (200 mL) then dried with MgSO4. The solvent
was removed under reduced pressure to give a yellow oil which
was purified by column chromatography on silica gel (9 : 1 v/v
chloroform–methanol elution). Concentration of the appropriate
fractions gave 4 as a colourless solid (15.9 g, 85%); mp 128–131 ◦C;
[a]D22 +14.9 (c 1.5 in CHCl3); mmax (NaCl)/cm−1 3417, 3311, 2975,
1660, 1479, 1249, 983, 763; dH (300 MHz, d6-DMSO) 8.45 (d, J
4.3, 2H), 7.74 (dd, J 7.7 and 7.3, 2H), 7.50 (d, J 7.7, 2H), 7.22
(m 2H) 6.31 (br. s, 1H), 3.77 (m, 1H), 3.67 (s, 4H), 2.41 (m, 2H),
1.85–1.49 (m, 4H), 1.35 (s, 9H), OH not observed; dC (75 MHz,
d6-DMSO) 176.2, 159.5, 155.0, 148.7, 136.4, 122.4, 122.0, 77.5,
59.5, 54.4, 53.5, 30.1, 28.2, 22.7; m/z (ESI) 415.2352 (C22H31N4O4
requires 415.2346), 415 [M + H]+ (100%), 437 [M + Na]+ (30%),
315 (38%).
Boc-Orn(DPA)-Gly-OH (7). A solution of NaOH (0.35 g,
8.8 mmol) in H2O (2 mL) was added to a solution of 5 (1.4 g,
2.9 mmol) in methanol (7 mL). The solution was stirred at ambient
temperature for 4 h, then adjusted to pH 7 by the addition of
1 M HCl. The resulting mixture was extracted with chloroform–
isopropanol (3 : 1 v/v, 4 × 50 mL) and the extracts combined, then
the solvent removed under reduced pressure to give the carboxylic
acid 7 as a pale yellow solid (0.46 g, 90%) which was used without
further purification; dH (300 MHz, d6-DMSO) 8.49 (d, J 4.3, 2H),
8.12 (m, 1H), 7.76 (m, 2H), 7.51 (d, J 7.8, 2H), 7.26 (m, 2H),
6.89 (d, J 8.2, 1H), 4.31 (m, 1H), 3.9–3.45 (m, 6H), 2.49 (partially
obscured, 2H), 1.65–1.40 (m, 4H), 1.36 (s, 9H), OH not observed;
m/z (ESI) 494 [M + Na]+ (40%), 472 [M + H]+ (100%).
Boc-Gly-Orn(DPA)-Gly-OMe (8). Boc-Gly-OH (0.12 g,
0.68 mmol) and 6 (0.28 g, 0.68 mmol) were dissolved in acetonitrile
◦
(8 mL) and the solution cooled to 0 C under an atmosphere of
nitrogen. HOBt·H2O (0.10 g, 0.66 mmol) and EDC·HCl (0.13 g,
0.66 mmol) were then added followed by N-methylmorpholine
(0.18 g, 1.8 mmol) and the mixture was warmed to room temper-
ature and stirred for 23 h. Saturated aqueous sodium bicarbonate
(50 mL) was then added and the resulting mixture was extracted
with ethyl acetate (3 × 50 mL). The combined organic phases
were washed with water (50 mL) and brine (70 mL) then dried
(MgSO4). The solvent was removed under reduced pressure to
give a yellow solid which was purified by column chromatography
on silica gel (190 : 9 : 1, v/v chloroform–methanol–aqueous
ammonia elution). Concentration of the appropriate fractions
gave the tripeptide 8 as a pale yellow solid (0.33 g, 89%); mp
Boc-Orn(DPA)-Gly-OMe (5). Acid 4 (6.80 g, 16 mmol) and
HCl.NH2-Gly-OMe (2.40 g, 19 mmol) were dissolved in a mixture
of dichloromethane (40 mL) and dimethylformamide (12 mL). The
solution was cooled to 0 ◦C under an atmosphere of nitrogen, then
HOBt·H2O (2.76 g, 20 mmol) and EDC·HCl (3.45 g, 18 mmol)
were added, followed by N-methyl morpholine (5.4 mL, 49 mmol).
The mixture was warmed to room temperature and stirred for 18 h,
then partitioned between saturated aqueous sodium bicarbonate
(100 mL) and ethyl acetate (100 mL). The aqueous phase was
extracted with ethyl acetate (3 × 100 mL) and the combined
organic phases were washed with water (100 mL) and brine
(2 × 100 mL) then dried (MgSO4). The solvent was removed
under reduced pressure to give a yellow oil which was purified by
column chromatography on silica gel (190 : 9 : 1, v/v chloroform–
methanol–aqueous ammonia elution). Concentration of the ap-
propriate fractions gave 5 as a yellow oil (7.54 g, 95%); [a]2D2 +1.08
(c 13.7, CHCl3); mmax (CHCl3)/cm−1: 3304, 2976, 2951, 2934, 2818,
1750, 1700, 1675, 1591, 1526, 1435, 1366, 1207, 1175; dH (300 MHz,
d6-DMSO) 8.47 (m, 2H), 8.24 (t, J 5.7, 1H), 7.75 (ddd, J 7.7, 7.7
and 1.8, 2H), 7.51 (d, J 7.7 Hz, 2H), 7.23 (m, 2H), 6.90 (d, J 8.0,
1H), 3.93–3.79 (m, 3H), 3.70 (s, 4H), 3.60 (s, 3H), 2.44 (m, 2H),
1.56–1.54 (m, 4H), 1.35 (s, 9H); dC (75 MHz, d6-DMSO) 172.7,
170.2, 159.4, 155.2, 148.7, 136.4, 122.5, 122.0, 79.2, 59.5, 53.9,
53.3, 51.6, 40.6, 29.9, 28.2, 22.9; m/z (ESI) 486.2703 [(M + H)+;
C25H37N5O5 requires 486.2716], 508 [M + Na]+ (75%).
◦
36–42 C (hygroscopic); [a]2D2 +0.72 (c 5.0 in CHCl3); found: C,
56.1; H, 7.0; N, 14.6. C27H38N6O6.2H2O requires C, 56.0; H, 7.3;
N, 14.5; mmax (CHCl3)/cm−1: 3300, 3295, 3055, 3007, 2977, 2950,
2935, 2816, 1755, 1660, 1435, 1367, 1281, 1248, 1207, 1173, 1049,
864, 754; dH (300 MHz, d6-DMSO) 8.47 (dd, J 4.7 and 0.8, 2H),
8.40 (t, J 5.7, 1H), 7.86 (d, J 8.0, 1H), 7.75 (ddd, J 7.8, 7.7 and 1.8,
2H), 7.50 (d, J 7.8, 2H), 7.23 (m, 2H), 6.95 (t, J 5.8, 1H), 4.28 (m,
1H), 3.82 (t, J 5.5, 2H), 3.70 (s, 4H), 3.60 (s, 3H), 3.55 (d, J 5.9,
2H), 2.43 (m, 2H), 1.70–1.40 (m, 4H), 1.36 (s, 9H); dC (75 MHz,
d6-DMSO) 172.0, 170.1, 169.5, 159,4, 155.8, 148.7, 136.4, 122.5,
122.0, 78.0, 59.4, 53.2, 52.0, 51.6, 43.2, 40.4, 30.1, 28.1, 22.5; m/z
(ESI) 543.2940 [(M + H)+; C27H39N6O6 requires 543.2931], 565
[M + Na]+ (100%).
HCl·H2N-Gly-Orn(DPA)-Gly-OMe (9). A solution of HCl in
dioxane (4 M, 2 mL) was added to tripeptide 8 (80 mg, 0.15 mmol)
and the resulting mixture stirred at ambient temperature under
a nitrogen atmosphere for 8 h. The solvent was removed under
reduced pressure and the residue azeotroped with toluene (3 ×
10 mL) to give 9 as a colourless solid (0.15 mmol, quant.) which
was used without further purification; dH (200 MHz, CD3OD) 8.77
(dd, J 5.5 and 1.0, 2H), 8.36 (ddd, J 7.8, 7.7 and 1.6, 2H), 7.95
(d, J 7.8, 2H), 7.83 (m, 2H), 4.36 (s, 4H), 4.05 (m, 2H), 4.00–
3.68 (m, 3H), 3.68 (s, 3H), 2.94 (m, 2H), 1.82 (m, 4H), 2 × NH
and NH3+ not observed; m/z (ESI) 465 [M + Na]+ (15%), 443
[M + H]+ (100%).
HCl·NH2-Orn(DPA)-Gly-OMe (6). A solution of HCl in
dioxane (4 M, 2 mL) was added to dipeptide 5 (1.0 g, 2.1 mmol)
and the resulting mixture stirred at ambient temperature under
a nitrogen atmosphere for 8 h. The solvent was removed under
reduced pressure and the residue azeotroped with toluene (3 ×
10 mL) to give 6 as a colourless solid (2.1 mmol, quant.) which
was used without further purification; dH (300 MHz, d6-DMSO)
9.21 (m, 1H), 8.76 (d, J 4.5, 2H), 8.47 (m, 2H), 8.34 (m, 2H), 7.96
(m, 2H), 7.74 (m, 2H), 4.45 (s, 4H), 4.00–3.75 (m, 3H), 3.62 (s,
3H), 2.90 (m, 2H), 1.8 (m, 4H); m/z (ESI) 386 [M + H]+ (100%).
Fl–Gly-Orn(DPA)-Gly-OMe (10). FITC (0.30 g, 0.77 mmol)
was added to a solution of tripeptide 9 (0.16 g, 0.36 mmol) in
MeOH (2.5 mL) then NEt3 (0.19 mL, 1.4 mmol) was added. The
mixture was stirred at room temperature under an atmosphere
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of nitrogen for 2.5 h in the dark then cooled in an ice bath to
give 10 as a dark red precipitate which was collected by filtration
(0.25 g, 84%). A portion of this precipitate (30 mg) was purified by
preparative HPLC (gradient of 30 to 100% MeOH (0.05% TFA)
in H2O (0.05% TFA) over 30 min; tR 24.6 min) for characterization
and use in assays; dH (200 MHz, CD3OD) 8.55 (2H, d, J 4.2), 8.23
(1H, d, J 1.7), 7.82–7.68 (3H, m), 7.42–7.30 (4H, m), 7.08 (1H, d,
J 8.2), 6.67–6.47 (6H, m), 4.49 (4H, s), 4.45 (1H, m), 4.03 (2H,
ABq, J 16.7), 3.87 (2H, m), 3.61 (3H, s), 3.20 (2H, m), 2.2–1.6
(4H, m), 2 × OH and 4 × NH not observed; m/z
with water (4 × 50 mL), followed by brine (50 mL) then dried
with MgSO4. The solvent was removed under reduced pressure
to give a yellow oil. Subjection of the crude material to column
chromatography on silica gel (190 : 9 : 1, v/v chloroform–
methanol–aqueous ammonia elution) and concentration of the
appropriate fractions gave the tetrapeptide 13 as a pale yellow
◦
solid (0.40 g, 80%); mp 58–62 C; [a]2D2 +0.27 (c 5.7 in CHCl3);
found: C, 59.2; H, 6.8; N, 15.6. C44H58N10O7.3H2O requires C,
59.2; H, 7.2; N, 15.7; mmax (CHCl3)/cm−1: 3295, 3053, 2949, 2934,
2818, 1751, 1684, 1647, 1433, 1366, 1248, 1207, 1171; dH (300 MHz,
CD3OD) 8.43 (m, 4H), 7.77 (m, 4H), 7.61 (m, 4H), 7.26 (m, 4H),
4.33 (m, 1H), 4.00–3.83 (m, 5H), 3.77 (s, 8H), 3.67 (s, 3H), 2.53
(m, 4H), 1.95–1.50 (m, 8H), 1.39 (s, 9H), 4 × NH not observed; dC
(75 MHz, CD3OD) 175.9, 174.7, 171.6, 160.7, 160.6, 149.5, 138.8,
125.1, 123.8, 80.7, 61.0, 56.3, 55.1(9), 55.1(3), 54.5, 52.6, 43.7, 41.9,
30.9, 28.8, 24.3, 24.2; m/z (ESI) 839.4542 [(M + H)+; C44H59N10O7
requires 839.4568], 861 [M + Na]+ (66%).
(ESI) 830.2649 [(M − H+)−; C43H40N7O9S requires 830.2617].
Fl–Gly-Orn(DPA)-Gly-OMe·[Zn(NO3)2] (1–Fl). A solution
of 10 (0.024 mmol) in MeOH (2 mL) and an aqueous solution
of Zn(NO3)2·6H2O (0.024 mmol) were mixed and stirred for 0.5 h.
The solvent was removed and the residue lyophilized to afford the
complex 1–Fl in quantitative yield.
HCl·H2N-Orn(DPA)-Gly-Orn(DPA)-Gly-OMe (14). A solu-
tion of HCl in dioxane (4 M, 2 mL) was added to tetrapeptide 13
(0.13 g, 0.16 mmol) and the resulting mixture stirred at ambient
temperature under a nitrogen atmosphere for 8 h. The solvent was
removed under reduced pressure and the residue azeotroped with
toluene (3 × 10 mL) to give 14 as a colourless solid (0.16 mmol,
quant.) which was used without further purification; dH (200 MHz,
CD3OD) 8.89 (br. s, 4H), 8.61 (br. s, 4H), 8.19 (br. s, 4H), 8.03 (br. s,
4H), 4.38 (s, 8H), 4.40–3.75 (m, 6H), 3.66 (s, 3H), 2.71 (br. s, 4H),
NBD–Hex-Orn(DPA)-Gly-OMe
(12). NBD–5-aminohex-
anoic acid 11 (66 mg, 0.22 mmol) was added to a solution of 6
(74 mg, 0.15 mmol) in CH3CN (2.5 mL) and N-methylmorpholine
(0.12 mL, 1.1 mmol), then EDC·HCl (52 mg, 0.27 mmol) and
HOBt·H2O (36 mg, 0.27 mmol) were added. The resulting solution
was stirred at room temperature for 24 h, then partitioned between
CHCl3–i-PrOH (3 : 1, 20 mL) and sat. aq. NaHCO3 (20 mL).
The aqueous layer was extracted with CHCl3–i-PrOH (3 : 1, 2 ×
20 mL), then the organic fractions were combined and washed
with H2O (2 × 20 mL) and brine (20 mL), dried (MgSO4) and
the solvent was removed under reduced pressure to give a dark
brown oil. Purification by preparative thin layer chromatography
(CHCl3–MeOH–aq. NH3; 140 : 18 : 2) gave 12 as an orange oil
(58 mg, 58%). dH (300 MHz, CD3OD) 8.39 (m, 3H), 7.75 (dt,
J 1.7 and 7.7, 2H), 7.57 (d, J 7.7, 2H), 7.22 (m, 2H), 6.24 (d, J
8.9, 1H), 4.33 (m, 1H), 3.92 (ABq, J 17.6, 2H), 3.74 (s, 4H), 3.66
(s, 3H), 3.47 (br. s, 2H), 2.52 (t, J 6.5, 2H), 2.26 (t, J 7.3, 2H),
1.84–1.59 (br. m, 8H), 1.50–1.42 (m, 2H), 3 × NH not observed;
dC (75 MHz, CD3OD) 178.8, 175.9, 174.9, 160.5, 149.3, 146.5,
145.7, 145.4, 138.6, 124.9, 123.7, 122.8, 99.6, 60.9, 55.1, 54.3, 52.6,
44.7, 41.8, 36.5, 31.0, 29.0, 27.5, 26.4, 24.1, 1 signal obscured or
overlapping; m/z (ESI) 662.3038 [(M + H)+; C32H40N9O7 requires
662.3045], 684 [M + Na]+ (21%).
+
1.87–1.65 (m, 8H), 3 × NH and NH3 not observed; m/z (ESI)
762 [M + Na]+ (100%), 740 [M + H]+ (55%).
NBD–Hex-Orn(DPA)-Gly-Orn(DPA)-Gly-OMe (15). Acid
11 (66 mg, 0.22 mmol) was added to a solution of 14 (110 mg,
0.1 mmol) in CH3CN (2.5 mL) and N-methylmorpholine
(0.12 mL, 1.1 mmol), then EDC·HCl (52 mg, 0.27 mmol)
and HOBt·H2O (41 mg, 0.27 mmol) were added. The resulting
solution was stirred at room temperature for 24 h, then partitioned
between CHCl3–i-PrOH (3 : 1, 20 mL) and sat. aq. NaHCO3
(20 mL). The aqueous layer was extracted with CHCl3–i-PrOH
(3 : 1, 2 × 20 mL), then the organic fractions were combined
and washed with H2O (2 × 20 mL) and brine (20 mL), dried
(MgSO4) and the solvent was removed under reduced pressure
to give a dark brown oil. Purification by preparative thin layer
chromatography (CHCl3–MeOH–aq. NH3; 170 : 27 : 3) gave 15
as an orange oil (83 mg, 82%). dH (300 MHz, CD3OD) 8.40 (m,
5H), 7.75 (m, 4H), 7.57 (m, 4H), 7.23 (m, 4H), 6.26 (d, J 8.9,
1H), 4.34 (m, 1H), 4.19 (m, 1H), 3.98–3.82 (m, 4H), 3.73 (s, 8H),
3.64 (s, 3H), 3.48 (br. s, 2H), 2.52 (m, 4H), 2.23 (t, J 7.2, 2H),
1.89–1.40 (br. m, 14 H), 5 × NH not observed; dC (75 MHz,
CD3OD) 176.0, 175.1, 174.7, 171.5, 171.4, 160.6, 160.5, 149.4,
149.3, 146.5, 145.7, 138.6, 124.9, 123.7, 122.7, 99.6, 60.9, 55.1,
55.0, 54.5, 52.6, 44.6, 43.6, 41.8, 36.4, 30.8, 30.4, 29.0, 28.8, 27.5,
26.3, 24.2, 24.0, 6 signals obscured or overlapping; m/z (ESI)
1037.4690 [(M + Na)+; C51H62N14O9Na requires 1037.4717], 1016
[M + H]+ (100%).
NBD–Hex-Orn(DPA)-Gly-OMe·[Zn(NO3)2] (1–NBD). A so-
lution of 12 (0.030 mmol) in MeOH (2 mL) and an aqueous
solution of Zn(NO3)2·6H2O (0.030 mmol) were mixed and stirred
for 0.5 h. The solvent was removed and the residue lyophilized to
afford the complex 1–NBD in quantitative yield.
Boc-Orn(DPA)-Gly-Orn(DPA)-Gly-OMe (13). Acid 7 (0.28 g,
0.59 mmol) and amine 6 (0.29 g, 0.59 mmol) were dissolved in
a solution of dichloromethane–dimethylformamide (3 : 1, v/v,
8 mL) which was cooled to 0 ◦C under an atmosphere of nitrogen.
HOBt·H2O (0.1 g, 0.68 mmol) and EDC·HCl (0.13 g, 0.68 mmol)
were then added followed by N-methylmorpholine (0.32 mL,
2.95 mmol) before the mixture was warmed to room temperature
and stirred for 23 h. The reaction mixture was partitioned between
saturated aqueous sodium bicarbonate (50 mL) and ethyl acetate
(40 mL). The aqueous phase was extracted with ethyl acetate
(3 × 50 mL) then the organic phases were combined and washed
NBD–Hex-Orn(DPA)-Gly-Orn(DPA)-Gly-OMe·2[Zn(NO3 )2 ]
(2–NBD). A solution of 15 (0.020 mmol) in MeOH (2 mL) and
an aqueous solution of Zn(NO3)2.6H2O (0.040 mmol) were mixed
and stirred for 0.5 h. The solvent was removed and the residue
lyophilized to afford the complex 2–NBD in quantitative yield.
1974 | Org. Biomol. Chem., 2006, 4, 1966–1976
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Boc-Hex-Orn(DPA)-Gly-Orn(DPA)-Gly-OMe
(17). Boc-
Vesicle preparation
Hex-OH 16 (0.035 g, 0.15 mmol) and 14 (0.16 g, 0.16 mmol) were
dissolved in anhydrous acetonitrile, and the resulting solution
was cooled to 0 ◦C under an atmosphere of nitrogen. HOBt·H2O
(0.026 g, 0.17 mmol) and EDC·HCl (0.033 g, 0.17 mmol) were then
added followed by N-methyl morpholine (0.11 mL, 1.0 mmol)
then the mixture was warmed to room temperature and stirred
for 23 h. The reaction mixture was partitioned between saturated
aqueous sodium bicarbonate (50 mL) and ethyl acetate (40 mL).
The aqueous phase was extracted with ethyl acetate (3 × 50 mL)
then the organic phases were combined and washed with water
(4 × 50 mL), followed by brine (50 mL) then dried with MgSO4.
The solvent was removed under reduced pressure to give a yellow
oil. Subjection of the crude material to column chromatography
on silica gel (190 : 9 : 1, v/v chloroform–methanol–aqueous
ammonia elution) and concentration of the appropriate fractions
gave the pentapeptide 17 as a pale yellow foam (0.11 g, 78%); dH
(300 MHz, CD3OD) 8.42 (4H, m), 7.78 (4H, m), 7.60 (4H, dd,
J 6.4 and 7.5), 7.26 (4H, m), 4.33 (1H, m), 4.14 (1H, m), 3.93–
3.84 (4H, m), 3.81 (8H, s), 3.67 (3H, s), 2.99 (2H, t, J 6.8), 2.54
(4H, m), 2.18 (2H, t, J 7.3), 1.87–1.19 (14H, br. m), 1.45 (9H,
s), 5 × NH not observed; dC (75 MHz, CD3OD) 176.3, 175.2,
174.8, 171.5, 171.4, 160.7, 160.6, 158.5, 149.4(4), 149.4(2), 138.7,
125.0, 123.8(7), 123.8(6), 61.0, 60.9, 55.1(3), 55.1(1), 54.6, 52.6,
43.6, 41.8, 41.2, 36.6, 30.8, 30.7, 30.4, 28.2, 27.5, 26.5, 24.3, 24.1,
3 signals obscured or overlapping.
Stock solutions of 10 mM vesicles (either 100 mol% POPC, 1 : 1
POPA–POPC, 1 : 1 POPG–POPC or 1 : 1 POPS–POPC) were
made by rehydration of thin films at room temperature with
TES buffer (5 mM TES, 100 mM NaCl, pH 7.4). The resulting
multilamellar vesicles were extruded to form unilamellar vesicles
with a Basic LiposoFast device purchased from Avestin, Inc. The
samples were extruded 29 times through a 19-mm polycarbonate
Nucleopore filter with 100-nm diameter pores.
NBD protection assay
The excitation was set at 460 nm while the emission was measured
at 530 nm using a 515 nm cut off filter on a Perkin Elmer
Luminescence Spectrometer LS50B. An aliquot of 1–NBD or 2–
NBD (4.5 lL of a 5 mM solution to give a final concentration
1.25 lM) in 1 : 1 methanol–water was added to unilamellar vesicles
(25 lM final concentration) in TES buffer (22.5 mL) at 25 ◦C. After
2 h, a 3 mL sample was withdrawn and treated at assay time t =
50 s, with sodium hydrosulfate (180 lL of 60 mM in 1 M Tris, pH
ca. 10); at t = 150 s, Triton X-100 (20 lL of 20% v/v) was added
to lyse the vesicles. The curves shown in Fig. 2 are typical results
from three separate trials.
Vesicle titrations
The excitation was set at 460 nm while the emission was measured
at 530 nm using a 515 nm cut off filter on a Perkin Elmer
Luminescence Spectrometer LS50B. To a solution of 1–NBD or
2–NBD (1 lM in 3 mL TES buffer, 10 mM) was added aliquots of
vesicles in TES buffer. The change in fluorescence emission that
was induced by the vesicle addition was rapid (complete within
10 s). The resulting fluorescence intensity was plotted against
effective phospholipid concentration (60% of total concentration)
and then fitted to a 1 : 1 binding model using an iterative curve-
fitting method.18,24 Each binding constant is the average of three
independent measurements.
Fl–Hex-Orn(DPA)-Gly-Orn(DPA)-Gly-OMe (18). A solution
of HCl in dioxane (4 M, 2 mL) was added to pentapeptide 17
(0.10 g, 0.11 mmol) and the resulting mixture stirred at ambient
temperature under a nitrogen atmosphere for 8 h. The solvent
was removed under reduced pressure and the residue azeotroped
with toluene (3 × 10 mL) to give the corresponding amine as
a colourless solid (0.11 mmol, quant.) which was used without
further purification.
FITC (28 mg, 0.073 mmol) was added to a solution of this
amine (50 mg, 0.056 mmol) in MeOH (2.5 mL) then NEt3
(0.08 mL, 0.58 mmol) was added. The mixture was stirred at room
temperature under an atmosphere of nitrogen for 2.5 h in the dark
then cooled in an ice bath to give 18 as a dark red precipitate
which was collected by filtration (60 mg, 86%). A portion of this
precipitate (20 mg) was purified by preparative HPLC (gradient of
10 to 60% MeOH (0.05% TFA) in H2O (0.05% TFA) over 30 min;
tR 26.1 min) for characterization and use in assays. dH (200 MHz,
CD3OD) 8.65 (4H, d, J 4.6), 8.25 (1H, d, J 1.6), 7.94 (4H, ddd, J
7.8, 7.7 and 1.5), 7.77 (1H, d, J 6.3), 7.55–7.45 (8H, m), 7.21 (1H,
d, J 8.2), 6.88– 6.64 (6H, m), 4.55 (8H, s), 4.48–4.26 (2H, m), 3.89
(4H, m), 3.67 (3H, s), 3.66 (2H, m), 3.24 (4H, m), 2.29 (2H, t, J
7.0), 2.01–1.22 (14 H, br. m), 2 × OH and 6 × NH not observed;
m/z (ESI) 1241 [(M + H)+ (10%)].
Cell staining and fluorescence microscopy
Jurkat cells were cultured in RPMI 1640, 10% FCS and incubated
at 37 ◦C, 5% CO2. Aliquots of cells were treated with camptothecin
◦
(10 lM final concentration) in growth media for 3.5 h at 37 C,
5% CO2. The cells were spun down and re-suspended in annexin
binding buffer (10 mM HEPES–sodium salt, 2.5 mM CaCl2,
140 mM NaCl, pH 7.4) for experiments in which annexin V was
used, or in growth media for experiments in which annexin V was
not used. Cells were then treated with the appropriate staining
reagents at the indicated concentrations. annexin V–FITC was
used according to the manufacturer’s protocol. All reagents were
added simultaneously. The cell suspensions were mixed thoroughly
◦
by repeated inversion and then incubated 15 min at 37 C. Cells
Fl–Hex-Orn(DPA)-Gly-Orn(DPA)-Gly-OMe·2[Zn(NO3)2] (2–
Fl). A solution of 18 (0.012 mmol) in MeOH (2 mL) and an
aqueous solution of Zn(NO3)2·6H2O (0.024 mmol) were mixed
and stirred for 0.5 h. The solvent was removed and the residue
lyophilized to afford the complex 2–Fl in quantitative yield. m/z
(ESI) 683.1705 [(M–2H+ + 2Zn2+)2+; C66H70N12O11SZn22+ requires
683.1800].
were then centrifuged at 2500 rpm for 2 min, re-suspended
and washed three times in phenol-free RPMI 1640, 10% FCS
growth media. At this point, 200 lL of each cell suspension was
transferred to a 8-well chamber slide for microscopy. The HeLa
cells were cultured in 75 cm2 tissue culture flasks using Eagle’s
minimum essential medium, 10% FCS, at 37 ◦C, 5% CO2 and
passed according to ATCC protocols. For imaging experiments,
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Org. Biomol. Chem., 2006, 4, 1966–1976 | 1975
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the cells were transferred to 8-well chamber slides and allowed to
grow to ca. 70% confluence. Cells were treated with camptothecin
to induce apoptosis (10 lM final concentration) in growth media
for 3.5 h at 37 ◦C, 5% CO2. The rest of the staining procedure
is described above. Fluorescence microscopy was performed
immediately after cell staining on an Axiovert S100 TV microscope
(Carl Zeiss) equipped with filter sets DAPI/Hoechst/AMCA,
FITC/RSGFP/Bodipy/Fluo3/DiO, Cy3 (Chroma). Pictures
were taken using a black and white digital camera and coloured
upon acquisition using Metamorph software version 6.2.
3 R. A. Schlegel and P. Williamson, Cell Death Differ., 2001, 8, 551–
563.
4 W. L. van Heerde, P. G. de Groot and C. P. Reutelingsperger, Thromb.
Haemostasis, 1995, 73, 172.
5 B. Plasier, D. R. Lloyd, G. C. Paul, C. R. Thomas and M. Al Rubeai,
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Flow cytometry
Jurkat cells were cultured as described for fluorescence microscopy.
A 10.0 mL volume of cells was treated with camptothecin (10 lM,
or 12.5 lM final concentration) in growth media for 16.5 h at
37 ◦C, 5% CO2. Cells were spun down and resuspended in annexin
binding buffer for experiments in which annexin V was used, or in
a buffer of 5 mM TES, 145 mM NaCl, pH 7.4 for experiments in
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Acknowledgements
This work was supported by the NIH, and Philip Morris USA
Inc. and Philip Morris International. The Australian group thank
the ARC for funding and for a QEII Fellowship to KAJ.
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