A convenient and versatile synthesis of

Laurdan-PS and sulfonated-Laurdan

probes.
Abstract

 The novel Laurdan-PS and sulfonated-Laurdan probes synthesized as cellular probes to study
functional lipid rafts in live cells and the role of lipid rafts in signal transduction.
 Synthesis of Laurdan-PS probe was achieved through covergent synthetic approach from three
suitably protected fragments:
 Phoshphato serine
 Isopropylidene glycerol
 C-Laurdan
 H- phosphonato coupling of pgosphato serine with glycerol acceptor followed by esterification to
C-Laurdan provided the protected optically pure Laurdan –PS probe and final deprotection was
successfully achieved by acid treatment
 On the other hand sulfonated –Laurdan molecules were synthesized in one step from M-Laurdan.
STRUCTURE
INTRODUCTION

 The lipid raft hypothesis postulates that the plasma membrane has a liquid order and
disordered domain.
 Liquid order domain enriched in sphingolipids, cholesterol, and specific protein, whereas
liquid disordered domain enriched in unsaturated glycerophospholipid.
 The liquid order domain are also called lipid rafts.
 The liquid order domain serve as a platform for various cellular process such as signal
transduction, membrane trafficking, pathogen invasion, cholesterol homeostasis,
neurodegenerative disease.
Model membrane by various models

 The traditional method to study lipid raft is based on the observation of detergent-
resistant membranes, composed mainly of sphingomyelin , other saturated
phospholipids, cholesterol, and some membrane proteins.
 This technique was criticized , because it uses detergents that may induce phase
separation and affect the partitioning of membrane proteins to given phase.
 A variety of other technique have been developed to study the phase behaviour of
the model membrane.
 The most direct method to study lipid raft is based on monitoring lipid composition
of the cell membrane with mass spectrometry. This method works under ultra-high
vacuum and with freeze-dried samples.
Model membrane by various methods

 Atomic force microscopy provides a nanosomic resolution for imaging of membrane in

aqueous phase, and can clearly distinguish domain of liquid order phase and disorder
phase.
 AFM technique works well only in model membrane immobilized on surface.
 Nuclear magnetic resonance provides direct information on the order of the lipid head
groups and fatty acid acyl chain, but it is limited to model membranes.
 Currently optical technique are used to study lipid raft in live cells. However, optical
microscopy require the use of fluorescent probes, because biological are poor in intrinsic
fluorescent species system.
Fluorescent Probes

 The fluorescent membrane probes can be classified in three main classes.

 The first class includes the probes that label component of the cell membrane.
 The second class include the probe that based on the selective partitioning of the dye to
either liquid order phase or disorder phase.
 The third class include the environment sensitive probes that can directly distinguish the
liquid order and disorder phase due to difference in their intrinsic properties.
 Fluorescent probe are widely used to reveal and characterize lipid domain, because
fluorescent probe directly incorporated into the cells at sufficient low concentration and
minimal disturbance to the membrane organization
Fluorescent Probes

 One of the most commonly used probes is laurdan and its derivatives. Laurdan and other
related fluorescent molecules containing 2-hydroxy-6-dodecanoyl naphthalene are
environment –sensitive dyes which shows a large stokes shift correlated to the polarity of
the surrounding medium.
 Kim et al synthesized and characterized C-laurdan, which shows greater sensitivity to
change in lipid order in bilayers, but labels the plasma membrane of cells more efficiently
than laurdan.
 Here we report the synthesis of sulfonated laurdan analogue probes and laurdan PS probe
for the study of lipid raft in cell membrane.
 SYNTHESIS

Scheme 1 depict the retrosynthesis of target molecule

1. One disconnection is envisioned to give two main
building blocks: the serine fragment 5,which is
commercially available, and the second one is
phosphonate fragment 6. The key H-phosphonate 6
was further disconnected to give alcohol 7 and C-
Laurdan8: esterification between 7 and 8, followed by
deprotection of PMB, and H-phosphonate preparation
were planned. The alcohol 7 could be synthesized
from commercially available Isopropylidene
glycerol9 through the series of protection and
deprotection.
 SYNTHESIS

Scheme 2. Synthesis of Laurdan-PS probe.

SYNTHESIS

 In the first approach, the synthesis of Laurdan-PS probebegan from protected alcohol
10 (Scheme 2), which was synthesized from Isopropylidene glycerol9by using a
reported method.RefThe alcohol 10 was subjected to benzylation to produce 11 in
90%, followed by TBDMS deprotection by using TBAF in 90% yield of 7. The key
intermediate H-phosphonate 6 was achieved in three steps from 7, which includes
the DIC mediated esterification with C-Laurdan in 85%, PMB deprotection with
DDQ in 85%, and H-phosphonate preparation by using PCl 3, Imidazole in 71%
yield. Then the 6 was coupled to commercially available fragment 5 by using PivCl,
pyridine in 80% of 14. The final deprotection was tried with Pd/C and Pd(OH) 2under
positive H2 atmosphere in MeOH/H2O, but we didn’t achieve the target molecule.
Later we have tried other reagents BF3.OEt2, Me2S, and BCl3for debenzylation but
we didn’t find the target molecule. In another hand we changed our synthetic route
as follows.
SYNTHESIS

Scheme 3 depicts our second

retrosynthetic approach towards the
synthesis ofLaurdan-PS probe1.A
convergent assembly of subunits was
planned for the target Laurdan-PS probe
(1) from phosphonato serine15,
Isopropylidene glycerol9, and C-
Laurdan8. The key intermediate 15 can
be synthesized from BOC-Serine
16through the PMB protection and H-
phosphonate preparation. The
Isopropylidene glycerol9was
commercially available. While C-
Laurdan8can be synthesized from β-
Napthol by a reported method
SYNTHESIS

Scheme 4. Synthesis of Laurdan-PS probe.

SYNTHESIS

 Reagents and Conditions: a) PMBOH, DIC, DMAP, 0 oC, 75%; b) PCl3, Imidazole, Et3N,
DCM, 0 oC, 78%; c) 9, PivCl, Pyridine, I2, H2O, 75%; d) pTSA, MeOH, 12 h, 92%; e) 8, DIC,
DMAP, DCM, 4 h, 72%; f) TFA, DCM, 1 h, 65%.
 In the second approach, the synthesis of Laurdan-PS probe (Scheme 4) was commenced from
BOC-serine. PMB protection was achieved by DIC coupling of BOC-serine with PMBOH in
75% yield. The synthesis of the most challenging phosphodiester 18 was achieved from 16 in
two steps, including H-phosphonate 17preparation using PCl3, imidazole in 78%, and PivCl
mediated coupling of 17 with 9 in 75% yield. Then the 18 was subjected to pTSA mediated
ketal cleavage to 19 in 92%yield. The fully protected Laurdan-PS probe20 was achieved from
19 in 72% by DIC mediated regioselective coupling with 8. The final deprotection was
achieved by treating 20 with 15% TFA in DCM to obtain the target molecule Laurdan-PS
probe1 in quantitative yield, which was confirmed by NMR and HRMS.
Synthesis

Scheme 5. Synthesis of sulfonated-Laurdan probes.

SYNTHESIS

 Reagents and Conditions: a)Sodium-4-bromoethane-1-sulfonate, Na 2CO3, DMF, 10 h,

N2,26% b)Sodium-3-bromopropane-1-sulfonate, Na2CO3, DMF, 10h, N2,23%*9
 c) Sodium-2-bromopropane-1-sulfonate, Na2CO3, DMF, 10 h, N2, 14%
SYNTHESIS

 Here we have synthesized different sulfonated-Laurdan probes having variation in their

chain length. The synthesis of sulfonated-Laurdan probes were started from M-Laurdan
(Scheme 5) which is synthesized from β-napthol by using reported method. REFthe
sulfonated-Laurdan probe22 was achieved by treating M-Laurdan with sodium-4-
bromobutane-1-sulfonate and Na2CO3 in the yield. In the other hand the sulfonated-
Laurdan probe 23 was achieved by treating M-Laurdan with Sodium-3-bromopropane-1-
sulfonate in the presence of Na2CO3 in the yield. While the other sulfonated-Laurdan
probe 24 was synthesized from M-Laurdan by treating with Sodium-2-bromobutane-1-
sulfonate and Na2CO3 in the yield.
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