Synthesis and evaluation of fluorogenic substrates for phospholipase D and phospholipase C

Article abstract of DOI:10.1021/ol060773d

Fluorogenic analogues of phosphatidylcholine and lysophosphatidylcholine, DDPB and lysoDDPB, were synthesized by an enzyme-assisted strategy. The analogues were evaluated as substrates for phospholipases C and D and lysophospholipase D. DDPB was cleaved b

Full text of DOI:10.1021/ol060773d

ORGANIC
LETTERS
2006
Vol. 8, No. 12
2575-2578
Synthesis and Evaluation of Fluorogenic
Substrates for Phospholipase D and
Phospholipase C
Tyler M. Rose and Glenn D. Prestwich*
Department of Medicinal Chemistry, UniVersity of Utah, 419 Wakara Way, Suite 205,
Salt Lake City, Utah 84108
Glenn.Prestwich@hsc.utah.edu
Received March 30, 2006
ABSTRACT
Fluorogenic analogues of phosphatidylcholine and lysophosphatidylcholine, DDPB and lysoDDPB, were synthesized by an enzyme-assisted
strategy. The analogues were evaluated as substrates for phospholipases C and D and lysophospholipase D. DDPB was cleaved by bacterial
and plant phospholipase D (PLD) enzymes and represents the first direct fluorogenic substrate for real-time measurement of PLD activity.
Both fluorogenic substrates have potential in screening for PLD and PC
activity in cells.
−PLC inhibitors and for monitoring spatiotemporal changes in PLD
PLC, PLD, and lysoPLD are three phospholipases that
catalyze hydrolysis of phospholipid (PL) headgroups. PLC
catalyzes the hydrolysis of the PL phosphodiester glyceryl
P-O bond to give diacylglycerol and a phosphomonoester.
PLD cleaves the headgroup P-O bond of the phosphodiester
linkage, to produce phosphatidic acid (PA) and an alcohol.
LysoPLD catalyzes the same reaction as PLD but is selective
for lysophospholipids. Most PLDs also catalyze transphos-
phatidylation,1 in which a primary alcohol replaces water as
the cleaving nucleophile.
PLC isozymes selectively hydrolyze PLs with either
inositol or choline headgroups. There are at least 11 different
phosphoinositide-selective PLCs (PI-PLC)2 and two putative
phosphatidylcholine-selective PLCs (PC-PLC)3 in mammals
and one PC-PLC in plants.4
PLD isozymes found in mammals are involved in a
diversity of normal and disease-related biological processes.5-7
The PLD enzymes found in mammals, plants, and some
bacteria are called “HKD PLD” because they share a
consensus amino acid sequence (HxKx4Dx6GG/S) and
employ a common catalytic mechanism involving a covalent
histidine intermediate.8 The “non-HKD PLD” enzymes lack
an HKD consensus sequence and have a catalytic mechanism
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10.1021/ol060773d CCC: $33.50
© 2006 American Chemical Society
Published on Web 05/13/2006

Scheme 1. Synthesis of Didabcyl Intermediate 4
in which metal ions are required to position the PL substrate
Fluorogenic substrates for lysoPLD have recently been
and activate the attacking nucleophile.9 Streptomyces chro-
mofuscus PLD (scPLD) is an archetypical non-HKD PLD.
Similar to the non-HKD PLD, lysoPLD requires metal ions
for catalysis and does not contain an HKD sequence.10 The
lysoPLD autotaxin, a nucleotide pyrophosphatase/phosphodi-
esterase, catalyzes hydrolysis via an active site threonine.11
HKD PLDs can be detected in cell biological studies by
inhibition with exogenous ethanol, 1-propanol, or 1-butanol.
Biochemical assays employ radioactive12 or fluorescent13
PLs, requiring extraction, separation, and detection. Alter-
natively, PLD products can be detected in vitro using direct14
or indirect15-18 methods. There are also indirect assays for
PC-PLC18,19 and lysoPLD;20 in particular, p-nitrophenyl
phosphate analogues have also been used for monitoring non-
HKD PLD and lysoPLD21,22 and PC-PLC23 in vitro.
described.24,25
We report here the synthesis of fluorogenic PC and lysoPC
analogues that contain a fluorescence quencher (dabcyl, a.k.a,
p-methyl red) at each acyl chain terminus and a fluorophore
appended to the PL headgroup through a choline-mimetic
linker. These PL analogues, denoted DDPB and lysoDDPB,
were evaluated in microtiter plate assays as substrates for
lysoPLD, scPLD, PLC, and phospholipase A2 (PLA2), as well
as several commercially available HKD PLD enzymes.
DDPB and lysoDDPB were synthesized efficiently as
illustrated in Schemes 1 and 2. The lysophosphatidylcholine
intermediate 2 containing the shorter dabcyl acyl chain was
obtained by a selective monoacylation of the commercially
available glycerol phosphocholine, 1, followed by installation
of the longer-chain dabcyl quencher at the sn-2 position (4,
Scheme 2. Synthesis of DDPB and LysoDDPB
2576
Org. Lett., Vol. 8, No. 12, 2006

Scheme 1). Although the longer, dodecanoyl acyl chain at
sn-2 improved the lipophilicity of the analogue over com-
pounds with two hexanoyl chains, adding a second C12 linker
at sn-1 afforded a less-soluble analogue that did not exhibit
either improved micelle insertion or in vitro enzyme activity
(data not shown).
The key step was the modification of the headgroup of
intermediate 4 by transphosphatidylation, using Genzyme
PLD(P) and a designed “choline-like” primary alcohol 6,
prepared as shown in Scheme 2. Transphosphatidylation has
been exploited previously26-28 to generate PL analogues with
both natural and unnatural headgroups. In this case, the
headgroup remodeling allowed the installation of a phos-
phodiester linkage bearing an internal quaternary amine
similar to choline as well as a protected primary amine that
could be used for further conjugation reactions (7, Scheme
2). The primary amine was deprotected and allowed to react
with an activated ester of BODIPY-FL (Molecular Probes)
to give the fluorogenic PC analogue DDPB (Scheme 2).
Removal of the sn-2 ester of DDPB by cobra venom PLA2
gave lysoDDPB, a fluorogenic lysoPC analogue.
Mixed micelles containing DDPB or lysoDDPB in Triton
X-100 (reduced) were incubated with commercial PLDs from
various sources for 3 min in buffers near each enzyme’s pH
optimum. Over this period, DDPB (Figure 1a) gave robust
fluorescence increases with the enzyme used for its synthesis,
Genzyme PLD(P), and with scPLD. LysoDDPB (Supporting
Information, Figure S2), over 3 min, gave a signal compa-
rable to that of scPLD but only a very small response to
Genzyme PLD(P). Over 60 min (Figure 1b), DDPB mixed
micelles with HKD PLD from peanut, cabbage, and Strep-
(9) Zambonelli, C.; Roberts, M. F. Prog. Nucleic Acid Res. Mol. Biol.
2005, 79, 133-181.
(10) Gijsbers, R.; Ceulemans, H.; Stalmans, W.; Bollen, M. J. Biol. Chem.
2001, 276, 1361-1368.
Figure 1. Fluorescence evolution (λEx/λEm ) 500/530 nm) during
(a) 3 min incubation of DDPB mixed micelles with PLD from
various sources (blue ( ) peanut PLD; red 9 ) cabbage PLD;
yellow 2 ) Strep. PMF PLD; brown × ) Genzyme PLD(P); O )
scPLD), (b) 60 min incubation of DDPB mixed micelles with PLD
and PLA2 from various sources (blue ( ) peanut PLD; red 9 )
cabbage PLD; brown 2 ) Strep. PMF PLD; green × ) cobra
venom PLA2; O ) bee venom PLA2), and (c) 3 min incubation of
DDPB or lysoDDPB mixed micelles with PC-PLC or PI-PLC
from B. cereus or C. perfringens (red 9 ) B. cereus PC-PLC +
DDPB; blue 2 ) B. cereus PC-PLC + lysoDDPB; ) ) C.
perfingens PC-PLC + DDPB; orange b ) C. perfringens PC-
PLC + lysoDDPB; brown + ) B. cereus PI-PLC + DDPB; green
rectangle ) B. cereus PI-PLC + lysoDDPB).
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542-550.
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tomyces PMF (BIOMOL) evolved fluorescence to a degree
approaching that observed in 3 min with scPLD, but bee
and cobra venom sPLA2 did not generate a fluorescent signal
over the same time period, consistent with the retention of
an intramolecular quencher even after cleavage of the sn-2
quenched acyl chain. A longer incubation time with lyso-
DDPB mixed micelles (Supporting Information, Figure S3)
only amplified fluorescence from Genzyme PLD(P). These
results support the idea that lysophospholipids are substrates
for scPLD14 but are not substrates for HKD PLD.29,30
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Org. Lett., Vol. 8, No. 12, 2006
2577

Next, mixed micelles of the PC analogues were tried as
substrates for PC-PLC, from Bacillus cereus and Clostrid-
ium perfringens, and PI-PLC, from B. cereus (Figure 1c).
Over 3 min, DDPB gave an overwhelmingly large fluores-
cence response with B. cereus PC-PLC and virtually none
with B. cereus PI-PLC or C. perfringens PC-PLC. Lyso-
DDPB gave a small but detectable signal with B. cereus PC-
PLC but none with the other PLC tested.
DDPB and lysoDDPB were both designed to be PC-like,
amphiphilic probes that could be used in living cells by
insertion into lipid bilayers; neither is freely soluble in water.
DDPB was further engineered to have fluorescence quench-
ers at the end of each acyl chain to prevent fluorescence
increases resulting from cellular PLA2 or PLA1 activity.
Indeed, no fluorescent signal was observed even after
incubation of DDPB with venom PLA2 at 37 °C for 1 h
(Figure 1c).
We anticipated that lysoDDPB would be accepted as a
fluorogenic substrate by lysoPLD, but neither lysoDDPB-
nor DDPB-containing mixed micelles produced fluorescence
when assayed in the presence of FBS, an abundant source
of lysoPLD activity,31 even after 60 min incubation. On the
other hand, the commercial lysoPLD substrates pNP-TMP32
and FS-325 both generated significant UV and fluorescence
signals, respectively (Supporting Information, Figure S4).
LysoDDPB was assayed with 50% FBS at 37 °C, but no
fluorescence increase was observed even when the concen-
tration of lysoDDPB was quadrupled and the FBS concen-
tration was increased to 80% (data not shown). Incubation
of lysoDDPB-containing micelles with up to 2 µg of venom
from Loxosceles reclusa, another known source of lysoPLD,33
failed to show a fluorescence increase (data not shown).
The inability of lysoPLD to hydrolyze lysoDDPB was
unexpected, considering the close structural similarity among
lysoPC, lysoDDPB, and FS-3 (Supporting Information,
Figure S4). The phosphate linker of FS-3, despite its lack of
a quaternary amine, is nearly twice the length of that in
lysoDDPB. This extra length may give the bulky fluorescent
group of FS-3 flexibility to avoid unfavorable interactions
in the binding site. Increasing the chain length between the
phosphate and the fluorophore in future fluorogenic ana-
logues of lysoPC could test this supposition.
Given the well-established importance of the HKD PLDs
and the growing importance of PC-PLC in cell signaling,
DDPB and lysoDDPB have the potential to be valuable tools
in cellular and molecular biology. The utility of these
fluorogenic substrates with mammalian HKD PLD has been
observed and will be reported elsewhere.34 Both DDPB and
lysoDDPB are cell permeant (data not shown) and may be
applicable for cell-based assays, as previously described for
PLA2.35,36 Further studies examining the use of DDPB and
lysoDDPB in living systems are merited and will be reported
in due course.
Acknowledgment. We thank C. Ferguson (Echelon
Biosciences, Inc., an Aeterna Zentaris company) for provid-
ing a gift of FS-3. The Center for Cell Signaling, a Utah
Center of Excellence (1997-2002), and the NIH (Grants
HL070231 and NS29632 to G.D.P.) provided financial
support.
Supporting Information Available: Figures S2, S3, and
S4, full experimental details, and NMR and MS spectroscopic
data for the characterization of DDPB and lysoDDPB and
their intermediates. This material is available free of charge
via the Internet at http://pubs.acs.org.
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OL060773D
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