Development of diacyltetrol lipids as activators for the C1 domain of protein kinase C

Article abstract of DOI:10.1039/c2mb05452c

The protein kinase C (PKC) family of serine/threonine kinases is an attractive drug target for the treatment of cancer and other diseases. Diacylglycerol (DAG), phorbol esters and others act as ligands for the C1 domain of PKC isoforms. Inspection of the crystal structure of the PKCδ C1b subdomain in complex with phorbol-13-O-acetate shows that one carbonyl group and two hydroxyl groups play pivotal roles in recognition of the C1 domain. To understand the importance of two hydroxyl groups of phorbol esters in PKC binding and to develop effective PKC activators, we synthesized DAG like diacyltetrols (DATs) and studied binding affinities with C1b subdomains of PKCδ and PKC. DATs, with the stereochemistry of natural DAGs at the sn-2 position, were synthesized from (+)-diethyl l-tartrate in four to seven steps as single isomers. The calculated EC₅₀ values for the short and long chain DATs varied in the range of 3-6 μM. Furthermore, the fluorescence anisotropy values of the proteins were increased in the presence of DATs in a similar manner to that of DAGs. Molecular docking of DATs (1b-4b) with PKCδ C1b showed that the DATs form hydrogen bonds with the polar residues and backbone of the protein, at the same binding site, as that of DAG and phorbol esters. Our findings reveal that DATs represent an attractive group of C1 domain ligands that can be used as research tools or further structurally modified for potential drug development.

Full text of DOI:10.1039/c2mb05452c

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Cite this: Mol. BioSyst., 2012, 8, 1275–1285
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PAPER
Development of diacyltetrol lipids as activators for the C1 domain of
protein kinase Cw
Narsimha Mamidi, Sukhamoy Gorai, Rakesh Mukherjee and Debasis Manna*
Received 2nd November 2011, Accepted 6th January 2012
DOI: 10.1039/c2mb05452c
The protein kinase C (PKC) family of serine/threonine kinases is an attractive drug target for the
treatment of cancer and other diseases. Diacylglycerol (DAG), phorbol esters and others act as
ligands for the C1 domain of PKC isoforms. Inspection of the crystal structure of the PKCd C1b
subdomain in complex with phorbol-13-O-acetate shows that one carbonyl group and two
hydroxyl groups play pivotal roles in recognition of the C1 domain. To understand the
importance of two hydroxyl groups of phorbol esters in PKC binding and to develop effective
PKC activators, we synthesized DAG like diacyltetrols (DATs) and studied binding affinities with
C1b subdomains of PKCd and PKCy. DATs, with the stereochemistry of natural DAGs at the
sn-2 position, were synthesized from (+)-diethyl L-tartrate in four to seven steps as single isomers.
The calculated EC50 values for the short and long chain DATs varied in the range of 3–6 mM.
Furthermore, the fluorescence anisotropy values of the proteins were increased in the presence of
DATs in a similar manner to that of DAGs. Molecular docking of DATs (1b–4b) with PKCd
C1b showed that the DATs form hydrogen bonds with the polar residues and backbone of the
protein, at the same binding site, as that of DAG and phorbol esters. Our findings reveal that
DATs represent an attractive group of C1 domain ligands that can be used as research tools or
further structurally modified for potential drug development.
in the cancer field have been a subject of intensive research and
drug development.17
Introduction
Mammalian cells contain many structurally distinct forms of
The PKC isoforms are activated by DAGs and transmit their
sn-1,2-diacyl-glycerols (DAGs) that differ with respect to the
type and degree of unsaturation of their fatty acyl groups.1–3
signal by phosphorylating specific proteins. The PKC protein
family consists of at least eleven known isoforms, categorized
Under equilibrium conditions, mammalian cells generally contain
into conventional (calcium, DAG and phospholipid-dependent),
low concentration of DAGs.1,4 When cells are stimulated by
novel (calcium-independent, but DAG and phospholipids
various stimuli, hydrolysis of phosphatidylinositol-4,5-bisphosphate
dependent) and atypical (calcium and DAG-independent)
subgroups.16,18 Other families of signaling proteins like Ras
(PtdIns(4,5)P2) catalyzed by phosphatidylinositol-specific phos-
pholipase C (PLC) rapidly produces DAGs at the plasma
guanyl nucleotide-releasing proteins (RasGRPs), chimerins,
membrane (PM) that act as second messengers to activate
protein kinase D (PKD), Unc-13 scaffolding proteins, myotonic
protein kinase C (PKC) and other proteins.5–9 The other
dystrophy kinase-related Cdc42-binding kinases (MRCK), and
product of PtdIns(4,5)P2 hydrolysis, inositol-1,4,5-triphosphate
((1,4,5)IP3) releases Ca2+ from ER which also activates various
proteins, including Ca2+-dependent PKC isoforms.5–15 PKC is
the DAG kinases b and g also share the C1 domain with PKC
isoforms.9,16,19 The DAG molecules selectively interact with
proteins containing a C1 domain. This interaction induces their
a family of serine/threonine kinases that is a major cellular
translocation to the discrete subcellular compartments. For
receptor for DAG/tumor promoting phorbol esters. PKC
some of the C1 domain containing proteins, such translocation
leads to activation.16,20,21 The sizes of the C1 domains are small,
isoenzymes play an important role in pathology of several
diseases such as cancer, diabetes, stroke, heart failure and
Alzheimer’s disease.6,11,13,16 Therefore, PKC isoforms particularly
ligand binding site. Also, the number of C1 domain containing
overall structures are highly conserved and there is only one
proteins is considerably smaller than the number of protein kinases
and the catalytic domains of PKC isoforms are substantially
homologous. Several studies already reported that the regulatory
domain of PKCs may have independent biological functions.
Therefore, C1 domains have become an attractive target in
designing selective PKC ligands.15,21–23
Department of Chemistry, Indian Institute of Technology Guwahati,
Assam 781039, India. E-mail: dmanna@iitg.ernet.in;
Fax: +91 03 61258 2350; Tel: +91 03 61258 2325
w Electronic supplementary information (ESI) available: Docking
results, copies of 1H and 13C NMR spectra. See DOI: 10.1039/
c2mb05452c
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Several classes of structurally complex and rigid ligands such
as phorbol esters,24 bryostatins,25 teleocidins,26 aplysiatoxins,27
ingenols,28 and others that target the DAG binding C1 domain
have been previously reported. Most of these C1 domain
ligands are from nature or derivatives of natural products.
Recently developed novel PKC isoforms specific indolactam29
and benzolactam30 derivatives are also structurally complex, so
modification and large-scale production may be unfeasible.
Many of these reported compounds have very strong affinity
for C1 domains in comparison with the flexible, natural DAG
agonists. The family of conformationally constrained DAG
lactones overcomes the spread in potency between the natural
product ligands and DAG.31–35 Therefore, there is a clear and
unmet need to design simple surrogates, whose structure could
be easily modified to achieve higher specificity and selectivity
among the C1 domains of the PKC isoforms. Recently reported
simple isophthalic acid derivatives15,21,23 have shown stronger
affinity for the C1 domain of PKC isoforms. Some of the
C1 domain ligands such as N-benzyladriamycin-14-valerate
(AD 198),36 calphostin C37 and resveratrol38 are already in
clinical trials for the treatment of different types of cancer.
In this study, we describe the design, synthesis and binding
properties of a novel class of tetrol based lipids, diacyltetrols
(DATs), to the C1b subdomains of PKCd and PKCy. DATs
interact with the C1b subdomains of PKC isoforms through
the DAG or phorbol ester binding site and forms hydrogen
bonds with the residues at the activator binding site. The
results show that the positions of two hydroxyl groups play an
important role in recognizing C1 domains of PKC isoforms.
Fig. 1 Structure of diacylglycerols and diacyltetrols (1–4) used for
the present study.
as there in natural DAGs at the sn-2 position. The tetrol based
lipids 1,4-dihydroxy-diacyltetrol (1), 2,3-dihydroxy-diacyltetrol
(2), 1,3-dihydroxy-diacyltetrol (3) and 1,2-dihydroxy-diacyltetrol
(4) were synthesized in four to seven steps from the starting
material (Fig. 1). DATs with long (palmitic acid) and short chain
(octanoic acid) fatty acids were synthesized in order to study the
impact of hydrophobicity on the binding affinity.
Synthesis
The synthesis was accomplished via a modified reported reaction
protocol and the products obtained in moderate to high yields
were characterized by NMR, mass, IR and melting temperature
analysis. We initially prepared DAT analogs 1a and 1b in order
to investigate the role of two hydroxymethyl groups within the
same molecule in binding with the C1b subdomains of PKC.
Lipids 1a and 1b were prepared in six steps from (+)-diethyl
L-tartrate as shown in Scheme 1. Introduction of cyclopentylidene
acetal41,42 into diol of (+)-diethyl L-tartrate (5) and subsequent
ester reduction of fully protected tartrate analog 6 by 2.2 equiv.
of lithium aluminium hydride43 resulted in bishydroxymethyl
compound 7 in 78% yield (two steps).42 Next, dibenzylation44
of compound 7 and careful removal of the cyclopentylidene acetal
under mild acidic conditions furnished diol 9 in 68% yield
(two steps). The acyl chains were then introduced into 9 using a
standard DCC-mediated coupling reaction45 with readily available
palmitic acid and octanoic acid to produce 10a and 10b,
respectively, in 85–90% yield. Finally, the benzyl groups were
removed using 0.1 equiv. of 10% Pd–C under hydrogenation
conditions46 to access DAT analogs 1a and 1b in 90% yield.
Results and discussion
We selected tetrol based unnatural lipids for the development
of PKC modulators. The structures of C1 domain binding
natural product ligands revealed the presence of more than
one hydroxyl group. For example, the phorbol esters interact
with the C1 domain through two hydroxyl groups including
the other functionalities.39,40 The X-ray structure of phorbol-
13-O-acetate bound PKCd C1b shows that the hydroxyl
groups attached to C-20 and C-4, including the carbonyl group
on C-3, form hydrogen bonds with the protein residues.40 This
leads us to develop diacyltetrols (DATs) containing two of the
phorbol ester pharmacophores, the hydroxyl and the carbonyl
functionalities within the same molecule. The DATs have very
high structural similarity to the natural diacylglycerols (DAGs).
The lipids 1, 3 and 4 contain at least one hydroxymethyl group
just as DAGs where as compound 2 have only two hydroxyl
groups. The positions of the hydroxyl groups were varied to get
more insight on which two hydroxyl groups would be better
anchored to the binding pocket of the C1 domain. The presence
of an additional hydroxyl group also gives us the opportunities
for further development of PKC isoforms specific and selective
tetrol based ligands.
The lipids were synthesized from commercially available
(+)-diethyl L-tartrate. The C2 symmetric diethyl L-tartrate
was used to avoid mixing of enantiomeric products during
synthesis. The choice of stereochemistry of lipids is crucial, as
the diversity of stereochemistry of these structures complicates the
specificity. The (+)-diethyl-L-tartrate provides the stereochemistry
Scheme 1 Synthesis route to 1,4-dihydroxy-diacyltetrol (1).
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major products. Regioselective protection of the mixture of 14
and 15 via acetonide formation41,43,48 provided fully protected
16 in 44% yield (two steps) and unprotected 15 in 43% yield
(two steps). The acyl chains were installed into 15 using the
standard DCC-mediated coupling reaction45 with palmitic acid to
yield 17a and octanoic acid to yield 17b. Deprotection of benzyl
groups of 17a and 17b using 0.1 equiv. of 10% Pd–C under
hydrogenation conditions46 yielded DAT analogs 3a and 3b in
77–91% yield. After the removal of benzyl groups from 16 under
mentioned hydrogenation conditions46 the acyl chains were
installed into 18 using the DCC-mediated coupling reaction45 to
afford 19 in 81–84% yield. Finally, careful deprotection of the
acetonide group of 19 using 80% acetic acid (aq) in dichloro-
methane produced DAT analogs 4a and 4b in 75% yield.
Scheme 2 Synthesis route to 2,3-dihydroxy-diacyltetrol (2).
To understand the importance of two hydroxymethyl groups
in binding with the C1b subdomains of PKC, we prepared
compound 2a and 2b having two hydroxyl groups. The DAT
analogs 2a and 2b were synthesized from the bishydroxymethyl
intermediate 7 in two steps (Scheme 2). The bishydroxymethyl
compound 7 was subjected to a DCC-mediated coupling reaction45
with palmitic acid and octanoic acid to yield 11a and 11b
respectively, in 80–85% yield. Removal of the cyclopentylidene
acetal under mild acidic conditions resulted in compounds 2a
and 2b in 82% yield.
Binding of DATs with PKC C1b
Intrinsic fluorescence is widely used as a tool to monitor changes
in protein structures and to draw inferences regarding local
structure and dynamics. The intrinsic fluorescence of the PKC
C1b subdomains is due to the presence of a single tryptophan
(Trp-252 in delta, and Trp-253 in theta) and tyrosine residues
(Tyr-236 and Tyr-238 in delta, Tyr-249 and Tyr-251 in theta)
and the change in their conformation or microenvironment
caused by ligands can be detected by fluorescence spectroscopy.
The binding affinity of diacylglycerols (DAGs) and diacyltetrols
(DATs) to C1b subdomains of PKC was assessed in vitro by a
fluorescence quenching method under similar experimental
conditions and the EC50 value was measured using the Hill
equation for each of the compounds. Fig. 2 shows the repre-
sentative plot of fluorescence quenching data for PKCd C1b
in the presence of different concentrations (0.09–35 mM) of
1,2-dioctanoyl-sn-glycerol (diC8), 1b, 2b, 3b and 4b. The
measured EC50 values (Table 1) revealed that compound 4 with
different chain lengths bind to the C1b subdomains with highest
affinity (3.08–4.85 mM) and other compounds had comparable
binding affinity for both the proteins (Table 1). The EC50 values
of diC8 and diC16 are in accordance with the reported results.49
Compound 4b shows 3.1 fold and 1.7 fold stronger binding affinity
To gain more information about the importance of both
hydroxyl and hydroxymethyl groups in their interactions with
the C1b subdomains of PKC, we prepared compounds 3 and 4
containing one hydroxyl and one hydroxymethyl group at
1,3 and 1,2 positions respectively (Schemes 3 and 4). For the
synthesis of DAT analogs 3 and 4, the precursor 5 was first
monoprotected with the benzyl group using 1.5 equiv. of silver
oxide in anhydrous dichloromethane47 and further ester reduction
by 2.5 equiv. of lithium aluminium hydride produced triol 13
in 63% yield (two steps). Benzylation of triol 13 using 1.5 equiv.
of silver oxide in anhydrous dichloromethane47 resulted in a
non-separable mixture of 1,2-diol (14) and 1,3-diol (15) as
Scheme 3 Synthesis route to (2S,3S)-2,4-bis (benzyloxy)butane-
1,3-diol (15) and (S)-4-((S)-1,2-bis(benzyloxy)ethyl)-2,2-dimethyl-1,3-
dioxolane (16).
Fig. 2 Binding of ligands with PKCd C1b. Representative plot of
fluorescence intensity of PKCd C1b (2 mM) in buffer (50 mM Tris,
150 mM NaCl, 50 mM ZnSO4, pH 7.4) in the presence of varying
concentration of diC8 (’), 1b ( ), 2b ( ), 3b ( ) and 4b ( ), where
F and F0 are fluorescence intensities in the presence and absence of the
ligand, respectively. Solid lines indicate the fit using Hill equation.
Scheme 4 Synthesis route to 1,3-dihydroxy-diacyltetrol (3) and
1,2-dihydroxy-diacyltetrol (4).
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Table 1 EC50 values for the binding of DAGs and DATs with the
PKCd C1b and PKCy C1b proteinsa at room temperature
bonded to the backbone amide proton and the carbonyls of the
C1b subdomain. The DATs could also be involved in additional
hydrogen bonding with the side chain of polar amino acid
of PKCd C1b protein. The C-28 and C-35 hydroxyl groups
of 1b were hydrogen bonded to the backbone amide proton of
Gly-253, Thr-242 and the carbonyl of Ser-240. The C-33 ester
group of 1b formed a hydrogen bond with the side chain amine
proton of Gln-257 (Fig. S1A, ESIw). In the case of 2b the C-29
and C-33 hydroxyl groups were hydrogen bonded to the
backbone carbonyl groups of Ser-240, Thr-242 and Leu-251.
Another hydrogen bond was observed for the C-26 ester group
of 2b with the backbone amide proton of Gly-253 (Fig. S1B,
ESIw). The C-28 hydroxyl group of 3b was hydrogen bonded
to the backbone amide proton of Thr-242 and carbonyl of
Leu-251. The C-33 hydroxyl group of 3b was hydrogen bonded
to the side chain amine proton of Gln-257 and backbone
carbonyl of Tyr-238 (Fig. S1C, ESIw). The C-28 hydroxyl
group of 4b was hydrogen bonded to the backbone carbonyls
of Leu-251 and Gly-253. The C-32 hydroxyl group and C-37
ester group of 4b were hydrogen bonded to the backbone
amide proton of Thr-242 and the side chain amine proton of
Gln-257, respectively (Fig. 3C). The experimental binding
results and docking score values obtained from the models
do not always corroborate (Table S1, ESIw). This difference
indicates that both proteins and compounds can undergo
conformational changes under experimental conditions.
To understand the importance of hydrophobicity of DATs
in their binding with C1b subdomains similar binding analyses
were performed with the long chain DATs. Both proteins
showed a similar pattern of dependence on the hydrophobicity
of the compounds 1–4 (Table 1). In each case, the affinity
increased with the hydrophobicity, with compound 4a
(XLOGP3 = 13.42) having strongest affinity (3.08 mM). For
compound 2a (XLOGP3 = 13.42) and 2b (XLOGP3 = 4.49),
although there is a distinct difference in hydrophobicity, the
difference in binding affinity among them is very small for both
the proteins. This could be due to the binding orientations
with the C1b subdomains through the hydroxyl groups. The
octanol–water partition coefficients (log P) for all DATs were
calculated according to the program XLOGP3. The molecular
docking analysis indicates that the overall high binding affinity
of compounds 3 and 4 is probably associated with the position
of hydroxyl and hydroxymethyl groups, helping them in
anchoring to the bottom of the binding site, which may not
be possible for compounds 1 and 2. Thus, the binding affinity
values of DATs highlight the importance of ligand hydrophobicity
and binding orientation, in a similar manner to those reported
for C1 domain ligands.51 It is also proposed that the hydrophobic
amino acids such as Met-239, Phe-243, Leu-250, and Leu-254 for
PKCd C1b, surrounding the ligand binding site of the C1
domain, interact with the hydrophobic moiety of the C1 domain
ligands. The hydrophobic moiety of compounds may interact
with the membrane lipid bilayer, because the PKC isoforms
interact with the membrane through the C1 domain for
activation and regulation of other cellular pathways.
EC50/mM
Compound
PKCd C1b
PKCy C1b
XLOGP3
DAG16
1a
2a
3a
4a
DAG8
1b
2b
3b
4b
6.68
4.29
4.92
3.72
3.34
15.39
5.89
5.29
5.07
4.85
7.59
3.70
4.63
3.21
3.08
6.12
4.15
4.98
4.03
3.59
14.04
13.42
13.42
13.42
13.42
5.11
4.49
4.49
4.49
4.49
a
Protein, 2 mM in buffer (50 mM Tris, 150 mM NaCl, 50 mM
ZnSO4, pH 7.4).
than diC8 for PKCd-C1b and PKCy-C1b protein, respectively.
The binding results also indicate that DATs have higher
binding affinity for PKCy C1b as compared to the PKCd
C1b, possibly due to the additional hydrogen bonds. Overall,
the DATs have better binding affinity for the C1b subdomains
than DAGs. This could be due to the presence of an additional
hydroxyl group. The molecular models of ligand bound protein
showed that there were three possible hydrogen bonds among
DAG and PKCd C1b, whereas four hydrogen bonds were
possible among DATs and PKCd C1b.
Modeling experiments using the crystal coordinates of the
C1b subdomain of PKCd in complex with phorbol-13-O-acetate
(1PTR)40 revealed the possible binding orientation when diC8
and DATs were docked into the empty C1b subdomain (Fig. 3
and Fig. S1, ESIw). The model structures suggests that hydroxyl
and carbonyl groups of the diC8 are mainly responsible for its
interaction with the C1b subdomain as reported earlier.50 The
diC8 hydroxyl group (C-28) was hydrogen bonded to the
backbone amide proton of Thr-242 and carbonyl of Leu-251.
The C-37 carbonyl group formed a hydrogen bond with the
backbone amide proton of Gly-253 (Fig. 3B). Our docked models
showed that DATs were anchored to the binding site in a
similar fashion, as phorbol esters and DAGs (Fig. 3A and B).
The hydroxyl and carbonyl groups of DATs were also hydrogen
Fig. 3 Structures of the ligand bound PKCd C1b subdomain.
(A) Crystal structure of phorbol 13-O-acetate bound PKCd C1b;
(B) modeled structure of DAG8 docked into PKCd C1b; (C) modeled
structure of a short chain diacyltertol derivative (4b) docked into
PKCd C1b. The modeled structures were generated using the Molegro
Virtual Docker, version 4.3.0. The oxygen atoms and nitrogen atoms
are shown in red and blue, respectively. The dotted line indicates
possible hydrogen bonds.
In order to obtain more information about ligand–protein
interactions, we performed steady-state fluorescence anisotropy
measurements of the proteins in the absence or presence of DATs
and DAGs at room temperature. The degree of anisotropy of
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Table 2 Anisotropya values of DATs and DAGs in the presence and
absence of the PKCd and PKCy C1b proteins at room temperature
Thus, the fluorescence binding assay confirms that C1b
subdomains interact with DATs in a lipid free system, as well
as DAT containing liposomes. The results also indicate that
the long chain DATs can differentially influence the activation
and membrane translocation properties of PKCy and PKCd.
The affinity differences between the proteins are solely because
of the differences in the residues and surface area of the
activator binding pockets. The activating effect of DATs could
be lower than the phorbol esters or other natural compounds
under similar experimental conditions. The protein binding
measurements also suggest that the C1b subdomains interact
differentially with the DATs and their interactions can specifically
modulate the PKC isoforms.
Compound
Bufferb
PKCd C1b
PKCy C1b
0.05793 (0.0056)
0.10282 (0.0061)
0.10434 (0.0064)
0.10971 (0.0058)
0.11389 (0.0049)
0.12106 (0.0054)
0.09777 (0.0063)
0.10339 (0.0053)
0.10014 (0.0055)
0.10027 (0.0052)
0.10459 (0.0049)
0.05529 (0.0032)
0.07268 (0.0037)
0.08161 (0.0049)
0.07754 (0.0035)
0.08333 (0.0043)
0.09531 (0.0058)
0.10693 (0.0051)
0.10959 (0.0072)
0.11008 (0.0089)
0.11620 (0.0078)
0.12634 (0.0089)
c
DAG16
1ac
2ac
3ac
4ac
c
DAG8
1bc
2bc
3bc
4bc
a
b
Values in the parentheses indicate standard deviations. Protein,
2 mM in buffer (20 mM Tris, 160 mM NaCl, 50 mM ZnSO4, pH 7.4).
DAG, 1, 2, 3 and 4, 20 mM; protein, 2 mM in buffer (20 mM Tris,
c
Conclusion
160 mM NaCl, 50 mM ZnSO4, pH 7.4).
We described here the synthesis of a novel group of compounds,
diacyltetrols (DATs), and their binding properties with the C1b
subdomains of PKCy and PKCd. Synthesis of these tetrol lipids
from the scratch provided the ability to generate pure samples
of a single lipid structure and freedom to explore the role of
hydroxyl and hydroxymethyl groups at different positions. The
results showed that DATs interact with the C1b subdomain of
PKC isoforms through the diacylglycerol (DAG) or phorbol
ester binding site and forms hydrogen bonds with the residues
at the activator binding site. Compound 4b with hydroxymethyl
and hydroxyl groups at 1 and 2 positions, respectively, showed
better binding affinity among the DATs. Therefore, DAT
molecules can be further used as research tools or lead
compounds in PKC based drug development. Undoubtedly,
further studies are needed to understand fully the importance
of DATs in activation and regulation of PKC isoenzymes.
Nevertheless, the present study demonstrates that a combination
of experimental and computational approaches can provide
valuable insight into the interaction of DATs with C1b sub-
domains of PKC isoforms.
pure PKCy C1b protein increases from 0.05529 in buffer to
0.10959, 0.11008, 0.11620 and 0.12634 upon interactions with
10-fold excess of ligands 1b, 2b, 3b and 4b respectively
(Table 2). Under similar experimental conditions the degree
of anisotropy of pure PKCd C1b protein increases from
0.05793 in buffer to 0.10339, 0.10014, 0.10027 and 0.10459 in
the presence of compounds 1b, 2b, 3b and 4b, respectively
(Table 2). Similar increase in anisotropy was observed for
proteins in the presence of DAGs.49 Although the changes in
anisotropy values were different for the compounds, this
experiment suggested that the presence of DATs increases
the rigidity of the surrounding environment of the protein in
a similar manner to that of DAGs.
To investigate the binding properties of the C1b sub-
domains of PKC isoforms to long chain DAT containing
liposomes, we performed tryptophan fluorescence quenching
assay (Fig. S2, ESIw). The results indicate that 4a has good
binding affinity for the C1b subdomain of PKCd and other
compounds have comparable binding affinity (Table 3) under
the similar experimental conditions. The liposome binding
assay for compound 4a also shows 3.6 fold and 2.4 fold stronger
binding affinity than diC16 for PKCd-C1b and PKCy-C1b,
respectively. The liposome binding results suggest that the long
chain DATs interact with the C1b subdomain of PKC isoforms
in a similar manner to that of DAG. The competitive binding
assay showed that DATs interact with the C1b subdomains
through the DAG/phorbol ester binding site (Fig. S3, ESIw).
Experimental section
General methods
All reagents were purchased from Sigma, USA and Merck,
India, and used directly without further purification. Dry
solvents were obtained according to the reported procedures.
Column chromatography was performed using 60–120 mesh
silica gel. Reactions were monitored by TLC on silica gel
60 F254 (0.25 mm). 1H NMR and 13C NMR spectra were
recorded at 400 MHz and at 100 MHz, respectively, by using a
Varian AS400 spectrometer. Coupling constants (J values)
were reported in Hertz (Hz). The chemical shifts were shown
in parts per million (ppm) downfield from tetramethylsilane,
using residual chloroform (d = 7.24 in 1H NMR, d = 77.23 in
13C NMR), as an internal standard. Multiplicities were
reported as follows: s (singlet), d (doublet), t (triplet), m (multiplet)
and br (broadened). Infrared (IR) spectra were recorded in
KBr or neat using a Perkin-Elmer Spectrum One FT-IR
spectrometer from 4000 to 450 cmꢀ1. Melting points were
Table 3 EC50 values for the binding of DATs (1a–4a) and DAG16
containing liposomesa with the PKCd C1b and PKCy C1b proteinsb at
room temperature
EC50/nM
Compound
PKCd C1b
PKCy C1b
1a
2a
3a
4a
0.76
0.80
0.44
0.35
1.25
0.81
0.89
0.58
0.48
1.16
diC16
a
Liposome composition, PC/PS/Ligand16 (80 ꢀ x : 20 : x, where
determined using Buchi B-545 melting point apparatus and are
¨
uncorrected. Mass spectra were recorded using a WATERS
MS system, Q-tof premier, and data analyzed by using the
b
x = 0–10). Protein, 0.5 mM in buffer (50 mM Tris, 150 mM NaCl,
50 mM ZnSO4, pH 7.4).
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built-in software. Optical rotations were measured on a Perkin-
Elmer instruments model 343-Polarimeter at room temperature.
1,2-Dipalmitoyl-sn-glycerol (diC18), 1,2-dioctanoyl-sn-glycerol
(diC8), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-
(7-nitro-2-1,3-benzoxadiazol-4-yl) were purchased from Avanti
Polar Lipids. Ultrapure water (Milli-Q system, Millipore, USA)
was used for the preparation of buffer.
yielded 6 (5.6 g, 81%) as a yellow coloured oil. Rf 0.30 (EtOAc/
Hexane, 1 : 4): 1H NMR (400 MHz, CDCl3): dppm 4.65 (s, 2H),
4.20 (q, J = 7.2 Hz, 4H), 1.93–1.88 (m, 2H), 1.82–1.75 (m, 2H),
1.67–1.62 (m, 4H), 1.24 (t, J = 7.2 Hz, 6H); 13C NMR (100 MHz,
CDCl3): dppm 169.1, 122.7, 76.6, 61.2, 36.2, 23.0, 13.6; FT-IR
(KBr) n 2946, 2867, 1705, 1639, 1450, 1376, 1320, 1059 cmꢀ1
.
[(2S,3S)-3-(hydroxymethyl)-1,4-dioxaspiro[4.4]nonan-2-yl]-
methanol (7)43. Protected diester 6 (1.59 g, 5.8 mmol) in
anhydrous THF (3.5 mL) was added dropwise to a suspension
of lithium aluminium hydride (479.5 mg, 12.75 mmol) in
anhydrous THF (20 mL) at 0 1C under a N2 atmosphere.
Stirring was continued at room temperature for 1 h and then
the solution was refluxed for additional 6 h. After completion
of the reaction (monitored by TLC), the temperature was
lowered to 0 1C and the reaction was cautiously quenched
by addition of 1 mL of deionized water, followed by 1 mL of
10% NaOH and 2 mL of deionized water. The mixture was
warmed to room temperature, and stirred until the gray color
disappeared. The reaction mixture was filtered in a sintered
glass crucible using Celite. Resulting solution was dried over
anhydrous Na2SO4 and concentrated under reduced pressure.
Column chromatography with silica gel and a gradient solvent
system of 10–40% ethyl acetate to hexane yielded 7 (1.05 g,
96%) as a colourless oil. Rf 0.32 (EtOAc/Hexane, 1 : 2);
1H NMR (400 MHz, CDCl3): dppm 4.24–4.20 (m, 1H),
4.14–4.03 (m, 2H), 3.88–3.77 (m, 2H), 3.67–3.63 (m, 1H),
2.07 (s, 2H), 1.88–1.78 (m, 4H), 1.67–1.64 (m, 4H); 13C NMR
(100 MHz, CDCl3): dppm 119.1, 78.4, 62.4, 37.1, 23.3; FT-IR
(KBr) n 3445, 2928, 1634, 1403, 1260, 1084 cmꢀ1; HR-MS
m/z (%): calcd. for C9H16O4Na+ [M + Na]+: 211.0946, found:
211.0951.
General procedure for the preparation of DAT esters (I)45.
Palmitic acid/octanoic acid (2.2 equiv.), dicyclohexylcarbodiimide
(2.2 equiv.) and N,N-dimethylaminopyridine (0.1 equiv.) were
added to a solution of protected diol (1.0 equiv.) in anhydrous
dichloromethane (8 mL) under a N2 atmosphere. Stirring was
continued for 12 h at room temperature. After completion of the
reaction (monitored by TLC), the reaction mixture was filtered
and washed (3ꢁ) with dichloromethane. The filtrate was concen-
trated under reduced pressure and the column chromatography
was performed with silica gel and a gradient solvent system of
0–10% ethyl acetate to hexane to yield corresponding esters. The
average yields were 77% to 85%.
General procedure for the deprotection of benzyl groups
(II)46. To a solution of benzyl ether containing compounds
(1 equiv.) in 10 mL of ethyl acetate/ethanol (3 : 1 ratio) was
added 10% Pd–C (0.1 equiv.) and stirring was continued
under a H2 (80 psi) atmosphere, at room temperature for
8–9 h. After completion of the reaction (monitored by TLC),
the catalyst (Pd–C) was filtered off through a pad of Celite and
the solvent was removed under reduced pressure. The crude
reaction mixture was purified by column chromatography
with silica gel and a gradient solvent system of 5–25% of ethyl
acetate to hexane to provide diol derivatives. The average
yields were 77% to 98%.
(2S,3S)-2,3-bis[(benzyloxy)methyl]-1,4-dioxaspiro[4.4]nonane
(8). Sodium hydride (60% in mineral oil, 282.24 mg, 11.76 mmol)
was first taken in a flame-dried round-bottom flask under a
N2 atmosphere and washed thrice with anhydrous hexane to
remove mineral oil. Diol 7 (1 g, 4.89 mmol) in anhydrous THF
(5 mL) was added dropwise to a stirred suspension of NaH in
anhydrous THF (15 mL) at 0 1C and stirred for 1 h. Then
benzyl bromide (1.4 mL, 11.76 mmol) was added dropwise to the
reaction mixture and stirred overnight at room temperature.44
The reaction was quenched by a saturated solution of
ammonium chloride and the residue was extracted with diethyl
ether (3 ꢁ 20 mL). The organic layer was dried over anhydrous
Na2SO4 and concentrated under reduced pressure. Column
chromatography with silica gel and a gradient solvent system
of 0–10% ethyl acetate to hexane yielded 8 (1.5 g, 83%) as a
colorless oil. Rf 0.42 (EtOAc/Hexane, 1:6); 1H NMR (400 MHz,
CDCl3): dppm 7.25–7.15 (m, 10H), 4.46 (s, 4H), 3.90–3.89
(m, 2H), 3.52–3.46 (m, 4H), 1.75–1.72 (m, 4H), 1.57–1.56
(m, 4H); 13C NMR (100 MHz, CDCl3): dppm 137.9, 128.2,
127.5, 119.4, 77.2, 73.3, 70.6, 37.2, 23.4; FT-IR (KBr) n 3031,
2930, 2870, 1637, 1453, 1334, 1259, 1205, 1102, 907, 799,
736, 698, 604 cmꢀ1; HR-MS m/z (%): calcd. for C23H28O4Na+
[M + Na]+: 391.1880, found: 391.1881.
General procedure for the deprotection of the cyclopentylidene
and dimethyl acetal groups (III)42,43. To a stirring solution of
protected tetrols (1 equiv.) in dichloromethane (10 mL), 3–5 mL
of 80% acetic acid was added and stirring was continued at 40 1C
for 4–5 h. After completion of the reaction (monitored by TLC),
the solvent was removed under reduced pressure to yield a
residue. The residue was further dissolved in dichloromethane
(10 mL) and washed with saturated solution of sodium bicarbonate
(3ꢁ). The organic layer was dried over anhydrous Na2SO4 and
concentrated under reduced pressure. Purification by silica gel
column chromatography and a gradient solvent system of
5–20% ethyl acetate to hexane yielded diol derivatives. The
average yields were 75% to 82%.
2,3-Diethyl(2R,3R)-1,4-dioxaspiro[4.4]nonane-2,3-dicarboxylate
(6)41,42. To a stirring solution of (+)-diethyl-L-tartrate (5.2 g,
25.22 mmol) in toluene (50 mL) p-toluenesulfonic acid (480 mg,
2.52 mmol) and cyclopentanone (5.03 mL, 63.05 mmol) were
added respectively. The solution was refluxed for 14 h and the
azeotrope was collected in a Dean–Stark trap. The reaction
mixture was then cooled to room temperature and reaction
mixture was quenched by adding 20% solid sodium bicarbonate.
After stirring for another 30 min at room temperature, the
reaction mixture was filtered and concentrated under reduced
pressure. The purification by silica gel column chromatography
and a gradient solvent system of 0–20% ethyl acetate to hexanes
(2S,3S)-1,4-bis(benzyloxy)butane-2,3-diol (9). Using the general
procedure (III), starting from compound 8 (600 mg, 1.63 mmol)
compound 9 (406.6 mg, 82%) was isolated as colorless oil.
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Rf 0.37 (EtoAc/Hexane, 1 : 2); 1H NMR (400 MHz, CDCl3):
dppm 7.29–7.22 (m, 10H), 4.47 (s, 4H), 3.81–3.79 (m, 2H),
3.55–3.50 (m, 4H), 2.09 (s, 2H); 13C NMR (100 MHz, CDCl3):
dppm 137.7, 128.2, 127.6, 127.5, 73.1, 71.5, 70.4; FT-IR (KBr)
n 3304, 3029, 2919, 2862, 1638, 1454, 1386, 1362, 1261, 1090, 949,
907, 804, 750, 734, 696, 610 cmꢀ1; HR-MS m/z (%): calcd. for
C18H22O4Na+ [M + Na]+: 325.1410, found: 325.1420.
2.40–2.31 (m, 4H), 1.65–1.61 (m, 4H), 1.28–1.23 (m, 16H),
0.85 (m, 6H); 13C NMR (100 MHz, CDCl3): dppm 173.2, 73.3,
62.4, 34.4, 34.2, 31.8, 29.2, 29.15, 29.1, 29.0, 25.1, 24.8, 22.7,
14.2; FT-IR (KBr) n 3434, 2961, 2926, 2854, 1733, 1638,
1461, 1261, 1095, 1024 cmꢀ1; HR-MS m/z (%): calcd. for
C20H38O6Na+ [M + Na]+: 397.2561, found: 397.2562.
[(2S,3S)-3-[(hexadecanoyloxy)methyl]-1,4-dioxapiro[4.4]nonan-
2-yl]methyl hexadecanoate (11a). Using the general procedure (I),
starting from compound 7 (300 mg, 1.6 mmol) and palmitic acid
(900 mg, 3.51 mmol) compound 11a (857 mg, 80%) was isolated
as a white solid. Mp: 98–99 1C; Rf 0.35 (EtOAc/Hexane, 1 : 6);
(2S,3S)-1,4-bis(benzyloxy)-3-(hexadecanoyloxy)butan-2-yl hexa-
decanoate (10a). Using the general procedure (I), starting from
compound 9 (200 mg, 0.662 mmol) and palmitic acid (373.4 mg,
1.46 mmol) compound 10a (437.5 mg, 85%) was isolated as a
white solid. Mp: 95–97 1C; Rf 0.45 (EtOAc/Hexane, 1 : 9);
1
[a]2D0 = ꢀ17.3 (c 0.1, CH2Cl2); H NMR (400 MHz, CDCl3):
1
[a]2D0 = ꢀ9.2 (c 0.1, CH2Cl2); H NMR (400 MHz, CDCl3):
dppm 4.20–4.08 (m, 2H), 3.93–3.78 (m, 2H), 3.62–3.59 (m, 2H),
2.35–2.26 (m, 4H), 1.89–1.83 (m, 4H), 1.76–1.56 (m, 8H), 1.18
(br s, 48H), 0.82–0.78 (m, 6H); 13C NMR (100 MHz, CDCl3):
dppm 173.2, 120.4, 76.0, 63.9, 49.8, 37.4, 36.1, 34.2, 32.9, 32.1,
31.0, 29.84, 29.8, 29.6, 29.56, 29.5, 29.4, 29.3, 26.5, 25.7, 25.5,
25.0, 24.9, 23.7, 22.8, 21.0, 14.3; FT-IR (KBr) n 2918, 2849,
1702, 1627, 1463, 1310, 1295, 1088, 940 cmꢀ1; HR-MS m/z (%):
calcd. for C41H76O6Na+ [M + Na]+: 687.5534, found:
687.5535.
dppm 7.27–7.19 (m, 10H), 5.30 (m, 2H), 4.38 (dd, J = 12 Hz,
J = 12 Hz, 4H), 3.47 (m, 4H), 2.23–2.20 (m, 4H), 1.52–1.50
(m, 4H), 1.18 (m, 48H), 0.81 (t, J = 5.8 Hz, 6H); 13C NMR
(100 MHz, CDCl3): dppm 173.2, 137.8, 128.5, 127.9, 73.3, 70.7,
62.4, 35.9, 34.2, 33.5, 32.7, 32.1, 31.0, 29.8, 29.6, 29.5, 29.4,
29.2, 26.4, 25.7, 25.6, 25.5, 24.9, 24.9, 22.8, 14.2; FT-IR (KBr)
n 2926, 2854, 1711, 1639, 1458, 1263, 1160, 1108, 1026, 790,
713, 697 cmꢀ1; HR-MS m/z (%): calcd. for C50H82O6Na+
[M + Na]+: 801.6004, found: 801.6005.
(2S,3S)-4-hexadecanoloxy-2,3-dihydroxybutyl hexadecanoate
(2a). Using the general procedure (III), starting from compound
11a (500 mg, 0.7523 mmol) compound 2a (370 mg, 82%) was
isolated as a white solid. Mp: 95–96 1C; Rf 0.36 (EtOAc/Hexane,
1 : 7); [a]2D0 = ꢀ10.0 (c 0.1, CH2Cl2); 1H NMR (400 MHz,
CDCl3): dppm 4.24–4.13 (m, 4H), 3.80 (m, 2H), 2.68 (br s, 2H),
2.36–2.30 (m, 4H), 1.60–1.58 (m, 4H), 1.23 (br s, 48H),
0.87–0.84 (m, 6H); 13C NMR (100 MHz, CDCl3): dppm
174.4, 69.8, 65.5, 34.5, 34.4, 32.1, 29.9, 29.9, 29.7, 29.6, 29.5,
29.3, 25.1, 22.9, 14.3; FT-IR (KBr) n 3435, 2955, 2917, 2849,
1736, 1638, 1463, 1383, 1178, 1094 cmꢀ1; HR-MS m/z (%): calcd.
for C36H70O6Na+ [M + Na]+: 621.5065, found: 621.5065.
(2S,3S)-3-(hexadecanoyloxy)-1,4-dihydroxybutan-2-yl hexa-
decanoate (1a). Using the general procedure (II) starting from
compound 10a (250 mg, 0.321 mmol) and 10% Pd–C (3.81 mg,
0.0321 mmol), compound 1a (171 mg, 90%) was isolated as a
white solid. Mp: 97–98 1C; Rf 0.36 (EtOAc/Hexane, 1:7);
1
[a]2D0 = ꢀ8.0 (c 0.1, CH2Cl2); H NMR (400 MHz, CDCl3):
dppm 5.29 (m, 2H), 3.77–3.70 (m, 4H), 2.12–2.11 (m, 2H),
1.92–1.86 (m, 2H), 1.71–1.61 (m, 4H), 1.25 (br s, 48H), 0.88
(m, 6H); 13C NMR (100 MHz, CDCl3): dppm 173.4, 73.3, 62.4,
35.9, 34.2, 33.5, 32.7, 32.0, 30.9, 29.8, 29.6, 29.5, 29.4, 29.2,
26.4, 25.7, 25.6, 25.5, 24.9, 24.8, 22.8, 14.2; FT-IR (KBr) n 3448,
3301, 2919, 2850, 1733, 1638, 1549, 1463, 1384, 1248, 1121 cmꢀ1
;
HR-MS m/z (%): calcd. for C36H70O6Na+ [M + Na]+:
[(2S,3S)-3-[(octanoyloxy)methyl]-1,4-dioxapiro[4.4]nonan-2-yl]-
methyl octanoate (11b). Using the general procedure (I), starting
from compound 7 (300 mg, 1.6 mmol) and octanoic acid
(506.18 mg, 3.51 mmol) compound 11b (600 mg, 85%) was
isolated as colourless oil. Rf 0.32 (EtOAc/Hexane, 1 : 7);
621.5065, found: 621.5064.
(2S,3S)-1,4-bis(benzyloxy)-3-(octanoloxy)butan-2-yl octanoate
(10b). Using the general procedure (I), starting from compound 9
(200 mg, 0.662 mmol) and octanoic acid (211 mg, 1.46 mmol)
compound 10b (313 mg, 85%) was isolated as colorless syrup.
Rf 0.37 (EtOAc/Hexane, 1 : 9); [a]2D0 = ꢀ6.7 (c 0.1, CH2Cl2);
1H NMR (400 MHz, CDCl3): dppm 7.25–7.18 (m, 10H), 5.29
(m, 2H), 4.38 (dd, J = 12, J = 12 Hz, 4H), 3.47 (m, 4H),
2.28–2.19 (m, 4H), 1.57–1.49 (m, 4H), 1.20 (m, 16H), 0.80
(m, 6H); 13C NMR (100 MHz, CDCl3): dppm 173.2, 137.8,
128.5, 127.9, 73.3, 70.7, 68.3, 34.4, 34.2, 31.8, 29.2, 29.15, 29.1,
29.0, 25.1, 24.8, 22.7, 14.2; FT-IR (KBr) n 2926, 2855, 1717, 1457,
1263, 1160, 1107, 1026, 804, 746, 713 cmꢀ1; HR-MS m/z (%):
calcd. for C34H50O6Na+ [M + Na]+: 577.3505, found: 577.3501.
1
[a]2D0 = ꢀ7.3 (c 0.1, CH2Cl2); H NMR (400 MHz, CDCl3):
dppm 4.20–4.11 (m, 4H), 3.94–3.93 (m, 2H), 2.30–2.25 (m, 4H),
1.77–1.76 (m, 4H), 1.61–1.55 (m, 8H), 1.22 (br s, 16H),
0.82–0.79 (m, 6H); 13C NMR (100 MHz, CDCl3): dppm 174.1,
120.4, 75.9, 63.9, 49.9, 37.4, 35.9, 34.2, 32.7, 31.8, 31.0, 29.8,
29.2, 29.0, 26.4, 25.6, 25.4, 24.9, 32.6, 22.7, 20.9, 14.1; FT-IR
(KBr) n 2918, 2849, 1703, 1627, 1463, 1310, 1295, 1088, 941 cmꢀ1
;
HR-MS m/z (%): calcd. for C25H44O6Na+ [M + Na]+:
463.3030, found: 463.3031.
(2S,3S)-2,3-dihydroxy-4-(octanoloxy)butyloctanoate (2b). Using
the general procedure (III), starting from compound 11b (500 mg,
1.135 mmol) compound 2a (349 mg, 82%) was isolated as a
colourless oil. Rf 0.35 (EtOAc/Hexane, 1 : 4); [a]2D0 = ꢀ9.2 (c 0.1,
CH2Cl2); 1H NMR (400 MHz, CDCl3): dppm 4.21–4.11 (m, 4H),
3.85–3.78 (m, 2H), 2.93 (br s, 2H), 2.36–2.24 (m, 4H), 1.60–1.57
(m, 4H), 1.25 (br s, 16H), 0.86–0.82 (m, 6H); 13C NMR
(100 MHz, CDCl3): dppm 174.2, 69.7, 65.4, 34.4, 34.3, 31.8,
29.2, 29.1, 25.0, 22.8, 14.2; FT-IR (KBr) n 3449, 3300, 2928,
(2S,3S)-1,4-dihydroxy-3-(octanoloxy)butan-2-yl octanoate (1b).
Using the general procedure (II), starting from compound 10b
(250 mg, 0.451 mmol) and 10% Pd–C (5.34 mg, 0.0451 mmol),
compound 1b (152 mg, 90%) was isolated as a colourless oil.
Rf 0.30 (EtOAc/Hexane, 1 : 4); [a]2D0 = ꢀ11.0 (c 0.1, CH2Cl2);
1H NMR (400 MHz, CDCl3): dppm 5.36–5.32 (m, 1H),
5.20–5.16 (m, 1H), 3.70–3.56 (m, 2H), 3.51–3.42 (m, 2H),
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2854, 1746, 1637, 1547, 1446, 1375, 1229, 1117, 1050 cmꢀ1
;
afforded a mixture (700 mg, 76%) of 3,4-bis(benzyloxy)
HR-MS m/z (%): calcd. for C20H38O6Na+ [M + Na]+:
butane-1,2-diol (14) and 2,4-bis(benzyloxy) butane-1,3-diol
1
(15) as colourless oil. Rf 0.30 (EtOAc/Hexane, 2 : 1); H NMR
397.2561, found: 397.2561.
(400 MHz, CDCl3): dppm 7.29–7.18 (m, 20H), 4.45 (s, 4H), 5.54
(dd, J = 11.6, J = 11.6 Hz, 4H), 3.91–3.89 (m, 1H), 3.77–3.72
(m, 3H), 3.64–3.57 (m, 4H), 3.55–3.47 (m, 4H), 2.51 (br s, 2H),
1.91 (br s, 2H); 13C NMR (100 MHz, CDCl3): dppm 138.0, 137.9,
137.74, 137.7, 128.2, 127.9, 127.8, 127.74, 127.67, 127.62, 127.56,
127.53, 127.5, 78.6, 78.1, 73.2, 73.1, 72.6, 72.5, 72.4, 72.0, 70.8,
70.3, 69.5, 63.3, 61.0; FT-IR (KBr) n 3437, 2926, 2855, 1637,
1455, 1261, 1188, 1119, 802, 741, 698 cmꢀ1; HR-MS-m/z (%):
calcd. for C18H22O4Na+ [M + Na]+: 325.1410, found:
325.1397.
(2R,3R)-diethyl 2-(benzyloxy)-3-hydroxysuccinate (12)47. To
a solution of (+)-L-diethyl tartrate (5, 1 g, 4.85 mmol) in
anhydrous dichloromethane (20 mL), silver oxide (Ag2O, 1.7 g,
7.27 mmol) and benzyl bromide (634 ml, 5.34 mmol) were added
and stirred at room temperature for 8 h under a N2 atmosphere.
After that the reaction mixture was filtered off through a pad of
Celite. The solvent was removed under reduced pressure.
The purification by silica gel column chromatography and a
gradient solvent system of 5–10% ethyl acetate to hexane
afforded 12 (1300 mg, 90%) as a slight yellow colored oil.
Rf 0.35 (EtOAc/Hexane, 1 : 4); 1H NMR (400 MHz, CDCl3):
To a solution of mixture 14 and 15 (700 mg, 2.32 mmol) in
anhydrous THF (20 mL) were added p-toluenesulfonic acid
(44 mg, 0.232 mmol) and 2,2-dimethoxy propane (341 ml,
2.784 mmol) under a N2 atmosphere at room temperature.
The reaction mixture was stirred at 65 1C for 3 h. The reaction
mixture was quenched by addition of anhydrous K2CO3
(200 mg, 1.5 mmol). The solid residue was removed by
filtration and washed with diethyl ether (3 ꢁ 5 mL). Solvent
was removed under reduced pressure and purified by silica gel
column chromatography (elution with ethyl acetate/hexane
5–15%) to furnish 16 (400 mg, 50%) as colourless oil and
recover 15 (300 mg, 43%) as colourless oil. (2S,3S)-2,4-
Bis(benzyloxy)butane-1,3-diol (15): Rf 0.30 (EtOAc/Hexane,
2 : 1); [a]2D0 = ꢀ4.1 (c 0.1, CH2Cl2); 1H NMR (400 MHz,
CDCl3): dppm 7.35–7.28 (m, 10H), 5.20 (s, 2H), 4.80 (d, J =
11.2 Hz, 2H), 4.48 (d, J = 11.6 Hz, 2H), 4.12–4.10 (m, 2H),
3.91–3.81 (m, 4H); 13C NMR (100 MHz, CDCl3): dppm 137.2,
135.6, 128.8, 128.7, 128.6, 128.4, 128.3, 78.8, 76.9, 73.0, 67.1,
63.5; FT-IR (KBr) n 3430, 2925, 2855, 1628, 1455, 1383, 1261,
1188, 1120, 803, 746, 698 cmꢀ1; HR-MS m/z (%): calcd.
for C18H22O4Na+ [M + Na]+: 325.1410, found: 325.1397.
(S)-4-((S)-1,2-Bis(benzyloxy)ethyl)-2,2-dimethyl-1,3-dioxolane
(16): Rf 0.34 (EtOAc/Hexane , 1 : 1); [a]2D0 = ꢀ19.5 (c 0.1,
CH2Cl2); 1H NMR (400 MHz, CDCl3): dppm 7.29–7.19
(m, 10H), 5.23(s, 2H), 4.64 (dd, J = 12 Hz, J = 12 Hz, 2H),
4.23–4.18 (m, 1H), 3.93–3.90 (m, 1H), 3.72–3.68 (m, 1H),
3.64–3.61 (m, 1H), 3.51–3.44 (m, 2H), 1.35 (s, 3H), 1.29
(s, 3H); 13C NMR (100 MHz, CDCl3): dppm 138.2, 128.3,
127.8, 127.7, 109.2, 79.5, 76.5, 75.6, 72.7, 68.3, 63.3, 26.3, 25.3;
FT-IR (KBr) n 2925, 2852, 1637, 1462, 1381, 1262, 1100, 1020,
801, 669 cmꢀ1; HR-MS m/z (%): calcd. for C21H26O4Na+
[M + Na]+: 365.1723, found: 365.1722.
d
ppm 7.33–7.22 (m, 5H), 4.86 (d, J = 12 Hz, 2H), 4.59 (s, 1H),
4.21 (d, J = 12Hz, 1H), 4.34–4.28 (m, 2H), 4.13–4.02 (m, 2H),
3.39 (bs, 1H), 1.32 (t, J = 7 Hz, 3H), 1.16 (t, J = 7 Hz, 3H);
13C NMR (100 MHz, CDCl3): dppm 173.1, 169.3, 137.8, 128.6,
128.4, 128.2, 128.1, 78.3, 72.9, 72.3, 62.0, 61.5, 14.2, 14.0;
FT-IR (KBr) n 3450, 2930, 1732, 1634, 1455, 1384, 1265, 1015,
861, 740, 700 cmꢀ1; HR-MS m/z (%): calcd. for C15H20O6Na+
[M + Na]+: 319.1152, found: 319.1100.
(2S,3S)-3-(benzyloxy)-1,2,4-triol (13)43. The suspension of
lithium aluminium hydride (413 mg, 10.98 mmol) in anhydrous
THF (15 mL) was prepared in a flame-dried two neck round
bottom flask under a N2 atmosphere at 0 1C. Freshly prepared
solution of compound 12 (1.3 g, 4.39 mmol) in anhydrous THF
(10 mL) was added dropwise via a syringe over 30 min. The
mixture was stirred at the same temperature for 1 h and stirring
was continued for an additional 6 h at 65 1C. Then the reaction
mixture was cooled to 0 1C and quenched by dropwise addition
of 1 mL of deionized water, 1 mL of 10% NaOH and 2 mL of
deionized water in turn with constant stirring for 45 min. The
reaction mixture was filtered off through a sintered glass crucible
using Celite. Resulting solution was dried over anhydrous
Na2SO4 and then concentrated under reduced pressure. The
residue was purified by silica gel column chromatography
(40–70% of ethyl acetate and hexane) to furnish 13 (656 mg,
70%) as a white solid. Mp: 77–78 1C [ref. 1 75.3–76.6 1C];
Rf 0.20 (EtOAC); [a]2D0 = ꢀ27.3 (c 0.1, CH2Cl2); 1H NMR
(400 MHz, CDCl3): dppm 7.31–7.22 (m, 5H), 5.17 (br s, 3H),
4.51 (dd, J = 11.6, J = 11.6 Hz, 2H), 3.85–3.38 (m, 6H);
13C NMR (100 MHz, CDCl3): dppm 137.9, 128.7, 128.2, 79.3,
72.7, 72.1, 63.4, 60.7; FT-IR (KBr) n 3403, 2938, 1638, 1454,
1213, 1050, 911, 742, 700 cmꢀ1; HR-MS m/z (%): calcd. for
C11H16O4Na+ [M + Na]+: 235.0941, found: 235.0942.
(2S,3S)-1,3-bis(benzyloxy)-4-(hexadecanoyloxy)butan-2-yl hexa-
decanoate (17a). Using the general procedure (I), starting from
compound 15 (200 mg, 0.662 mmol) and palmitic acid (373 mg,
1.456 mmol) compound 17a (400 mg, 77%) was isolated as a
white solid. Mp: 88–90 1C; Rf 0.36 (EtOAc/Hexane, 1 : 9);
(2S,3S)-2,4-bis(benzyloxy)butane-1,3-diol (15) and (S)-4-((S)-1,2-
bis(benzyloxy)ethyl)-2,2-dimethyl-1,3-dioxolane (16)41,43,47,48
.
Silver oxide (Ag2O, 1.479 g, 4.597 mmol) and benzyl bromide
(400.5 ml, 3.371 mmol) were added to a solution of (2S,3S)-3-
(benzyloxy)-1,2,4-triol (13, 650 mg, 3.065 mmol) in anhydrous
dichloromethane under a N2 atmosphere and the stirring was
continued for 8 h. After completion of the reaction (monitored
by TLC), the reaction mixture was filtered off through a pad of
Celite. The solvent was removed under reduced pressure.
The purification by silica gel column chromatography and a
gradient solvent system of 30–50% ethyl acetate to hexane
1
[a]2D0 = ꢀ13.1 (c 0.1, CH2Cl2); H NMR (400 MHz, CDCl3):
dppm 7.26–7.16 (m, 10H), 5.28–5.24 (m, 1H), 5.19–5.15 (m, 1H),
4.56 (dd, J = 12, J = 11.6 Hz, 2H), 4.43 (s, 2H), 4.27–4.15
(m, 1H), 4.10–4.01 (m, 1H), 3.85–3.67 (m, 1H), 3.59–3.51
(m, 1H), 2.26–2.13 (m, 4H), 1.51 (m, 4H), 1.18 (br s, 48H),
0.78 (m, 6H); 13C NMR (100 MHz, CDCl3): dppm 173.5, 173.4,
138.1, 128.6, 128.1, 128.03, 128.01, 127.9, 75.5, 73.4, 73.2, 69.4,
62.4, 34.4, 34.2, 34.1, 32.1, 29.9, 29.6, 29.5, 29.4, 29.3, 29.2,
c
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26.3, 25.4, 25.1, 24.9, 24.9, 22.8, 14.2; FT-IR (KBr) n 2921,
2846, 1732, 1638, 1455, 1109, 905, 801, 733, 696 cmꢀ1; HR-MS
m/z (%): calcd. for C50H82O6Na+ [M + Na]+: 801.6004,
found: 801.6004.
13C NMR (100 MHz, CDCl3): dppm 109.2, 76.3, 72.2, 65.5,
63.4, 26.2, 25.1; FT-IR (KBr) n 3446, 2950, 2087, 1637, 1409,
1084, 1042 cmꢀ1; HR-MS m/z (%): calcd. for C7H14O4Na+
[M + Na]+: 185.0784, found: 185.0786
(2S,3S)-4-(hexadecanoyloxy)-1,3-dihydroxybutan-2-yl hexa-
decanoate (3a). Using the general procedure (II), starting from
compound 17a (300 mg, 0.385 mmol) and 10% Pd–C (4.6 mg,
0.0385 mmol), compound 3a (210 mg, 91%) was isolated as a
white solid. Rf 0.40 (EtOAc/Hexane, 1 : 7); mp: 96–97 1C;
(1S)-1-[(4S)-2,2-dimethyl-1,3-dioxan-4-yl]-2-(hexadecanoyloxy)-
ethyl hexadecanoate (19a). Using the general procedure (I),
starting from compound 18 (70 mg, 0.4318 mmol) and palmitic
acid (243.6 mg, 0.95 mmol) compound 19a (224 mg, 81%) was
isolated as a white solid. Rf 0.37 (EtOAc/Hexane, 1 : 7); mp:
1
1
[a]2D0 = ꢀ5.7 (c 0.98, CH2Cl2); H NMR (400 MHz, CDCl3):
86–88 1C; [a]2D0 = ꢀ7.0 (c 0.1, CH2Cl2); H NMR (400 MHz,
d
ppm 5.36–5.24 (m, 2H), 4.32–4.22 (m, 1H), 4.18–4.08 (m, 1H),
CDCl3): dppm 5.13–5.09 (m, 1H), 4.26–4.17 (m, 2H), 4.11–4.06
(m, 1H), 3.98–3.94 (m, 1H), 3.73–3.69 (m, 1H), 2.30–2.20 (m, 4H),
1.57–1.51 (m, 4H), 1.36 (s, 3H), 1.28 (s, 3H), 1.19 (m, 48H), 0.81
(m, 6H); 13C NMR (100 MHz, CDCl3): dppm 173.5, 173.4, 110.0,
74.5, 70.7, 65.6, 62.9, 34.4, 34.2, 34.1, 32.1, 29.9, 29.7, 29.54, 29.5,
29.3, 29.2, 26.3, 25.4, 25.1, 25.0, 24.9, 22.7, 14.3; FT-IR (KBr)
3.92–3.79 (m, 1H), 3.67–3.59 (m, 1H), 2.35–2.24 (m, 4H),
1.59–1.56 (m, 4H), 1.26 (br s, 48H), 0.87 (m, 6H); 13C NMR
(100 MHz, CDCl3): dppm 173.5, 173.3, 76.4, 72.7, 65.5, 61.6,
34.5, 34.4, 34.34, 34.3, 32.1, 29.9, 29.8, 29.6, 29.54, 29.5, 29.3,
25.1, 25.0, 22.9, 14.3; FT-IR (KBr) n 3451, 2922, 2849, 1656,
1519, 1461, 1383, 1261, 1224, 1083 cmꢀ1; HR-MS m/z (%):
calcd. for C36H70O6Na+ [M + Na]+: 621.5065, found:
621.5066.
n 2918, 2850, 1737, 1637, 1463, 1384, 1169, 1099, 1073 cmꢀ1
;
HR-MS m/z (%): calcd. for C39H74O6Na+ [M + Na]+:
661.5378, found: 661.5377.
(2S,3S)-1,3-bis(benzyloxy)-4-(octanoyloxy)butan-2-yl octanoate
(17b). Using the general procedure (I), starting from compound
15 (200 mg, 0.6619 mmol) and octanoic acid (210 mg,
1.456 mmol) compound 17b (300 mg, 82%) was isolated as a
colourless oil. Rf 0.40 (EtOAc/Hexane, 1 : 7); [a]2D0 = ꢀ26.1
(c 0.1, CH2Cl2); 1H NMR (400 MHz, CDCl3): dppm 7.25–7.18
(m, 10H), 5.27–5.25(m, 1H), 5.17–5.16 (m, 1H), 4.57 (dd, J =
12 Hz, J = 11.6 Hz, 2H), 4.44 (s, 2H), 4.24–4.16 (m, 1H),
4.10–4.02 (m, 1H), 3.84–3.72 (m, 1H), 3.58–3.52 (m, 1H),
2.27–2.13 (m, 4H), 1.53–1.49 (m, 4H), 1.19 (br s, 16H), 0.80
(m, 6H); 13C NMR (100 MHz, CDCl3): dppm 173.5, 173.4,
138.1, 128.6, 128.2, 128.1, 128.0, 127.8, 76.5, 75.6, 73.4, 69.6,
62.4, 34.5, 34.43, 34.4, 34.29, 33.2, 31.8, 29.9, 29.2, 29.1, 25.1,
25.0, 22.8, 14.2; FT-IR (KBr) n 2922, 2854, 1732, 1637, 1455,
1383, 1113, 733, 698, 630 cmꢀ1; HR-MS m/z (%): calcd. for
C34H50O6Na+ [M + Na]+: 577.3500, found: 577.3501.
(2S,3S)-1-(hexadecanoyloxy)-3,4-dihydroxybutan-2-yl-hexa-
decanoate (4a). Using the general procedure (III), starting from
compound 19a (150 mg, 0.235 mmol) compound 4a (105 mg,
75%) was isolated as a white solid. Rf 0.36 (EtOAc/Hexane,
1 : 9); mp: 97–98 1C; [a]2D0 = ꢀ12.7 (c 0.1, CH2Cl2); 1H NMR
(400 MHz, CDCl3): dppm 5.23–5.19 (m, 1H), 4.36–4.27 (m,
2H), 4.21–4.16 (m, 1H), 4.08–4.04 (m, 1H), 3.83–3.79 (m, 1H),
2.40–2.30 (m, 4H), 1.67–1.61 (m, 4H), 1.29 (br s, 48H), 0.91
(m, 6H); 13C NMR (100 MHz, CDCl3): dppm 173.5, 173.4,
74.5, 70.7, 65.6, 62.9, 34.4, 34.2, 34.1, 32.1, 29.9, 29.6, 29.54,
29.5, 29.3, 29.2, 26.3, 25.4, 25.1, 25.0, 24.9, 22.8, 14.3; FT-IR
(KBr) n 3430, 2918, 2850, 1638, 1550, 1380, 1245, 1116 cmꢀ1
;
HR-MS m/z (%): calcd. for C36H70O6Na+ [M + Na]+:
621.5065, found: 621.5064.
(1S)-1-[(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]-2-(octanoyloxy)-
ethyl octanoate (19b). Using the general procedure (I), starting
from compound 18 (70 mg, 0.4318 mmol) and octanoic acid
(137 mg, 0.95 mmol) compound 19b (150 mg, 84%) was isolated
as a colourless oil. Rf 0.36 (EtOAc/Hexane, 1 : 7); [a]2D0 = ꢀ8.3
(2S,3S)-1,3-dihydroxy-4-(octanoyloxy)butane-2-yl octanoate
(3b). Using the general procedure (II), starting from compound 17b
(250 mg, 0.451 mmol) and 10% Pd–C (5.3 mg, 0.0451 mmol),
compound 3b (160 mg, 77%) was isolated as colourless oil.
Rf 0.34 (EtOAc/Hexane, 1 : 7); [a]2D0 = ꢀ15.1 (c 0.1, CH2Cl2);
1H NMR (400 MHz, CDCl3): dppm 5.26–5.16 (m, 2H),
4.27–4.17 (m, 1H), 4.11–4.03 (m, 1H), 3.87–3.72 (m, 1H),
3.61–3.53 (m, 1H), 2.28–2.13 (m, 4H), 1.53–1.49 (m, 4H),
1.19 (br s, 16H), 0.80 (m, 6H); 13C NMR (100 MHz, CDCl3):
dppm 173.2, 173.1, 74.4, 70.5, 65.4, 62.7, 34.2, 34.0, 31.6, 29.0,
28.4, 28.3, 26.1, 25.2, 24.9, 24.7, 22.6, 14.4; FT-IR (KBr)
n 3449, 2955, 2926, 2851, 1733, 1705, 1640, 1463, 1382, 1235,
1197, 1109, 1096 cmꢀ1; HR-MS m/z (%): calcd. for C20H38O6Na+
[M + Na]+: 397.2561, found: 397.2560.
1
(c 0.1, CH2Cl2); H NMR (400 MHz, CDCl3): dppm 5.20–5.16
(m, 1H), 4.33–4.24 (m, 2H), 4.18–4.13 (m, 1H), 4.05–4.02 (m, 1H),
3.80–3.76 (m, 1H), 2.38–2.28 (m, 4H), 1.66–1.58 (m, 4H), 1.43
(s, 3H), 1.35 (s, 3H), 1.29 (m, 16 H), 0.88 (m, 6H); 13C NMR
(100 MHz, CDCl3): dppm 173.2, 173.1, 109.7, 74.4, 70.5, 65.4, 62.7,
34.2, 34.0, 31.6, 29.03, 29.0, 28.9, 26.1, 25.2, 24.9, 24.8, 22.6, 14.0;
FT-IR (KBr) n 2922, 2856, 1738, 1642, 1455, 1382, 1222, 1161,
1108, 1071, 964 cmꢀ1; HR-MS m/z (%): calcd. for C23H42O6Na+
[M + Na]+: 437.2874, found: 437.2885.
(2S,3S)-3,4-dihydroxy-1-(octanoyloxy)butane-2-yl octanoate
(4b). Using the general procedure (III), starting from compound 19a
(150 mg, 0.235 mmol) compound 4a (105 mg, 75%) was
isolated as a colourless oil. Rf 0.35 (EtOAc/Hexane, 1 : 4);
(S)-1-((S)-2,2-dimethyl-1,3-dioxolan-4yl)ethane-1,2-diol (18).
Using the general procedure (II), starting from compound 16
(300 mg, 0.876 mmol) and 10% Pd–C (10.37 mg, 0.0876 mmol),
compound 18 (139 mg, 98%) was isolated as a colourless oil.
Rf 0.35 (3 : 1); [a]2D0 = ꢀ27.3 (c 0.1, CH2Cl2); 1H NMR (400 MHz,
CDCl3): dppm 4.18–4.11 (m, 1H), 4.06–4.02 (m, 1H), 3.84–3.80
(m, 1H), 3.65–3.58 (m, 3H), 1.42 (s, 3H), 1.38 (s, 3H);
1
[a]2D0 = ꢀ22.0 (c 0.1, CH2Cl2); H NMR (400 MHz, CDCl3):
dppm 5.23–5.19 (m, 1H), 4.36–4.28 (m, 2H), 4.20–4.16 (m, 1H),
4.08–4.04 (m, 1H), 3.83–3.80 (m, 1H), 2.40–2.30 (m, 4H),
1.67–1.64 (m, 4H), 1.31 (br s, 16H), 0.90 (m, 6H); 13C NMR
(100 MHz, CDCl3): dppm 179.1, 173.1, 74.4, 70.6, 65.4, 62.8,
c
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34.1, 33.9, 31.6, 29.7, 29.01, 29.0, 28.9, 26.0, 25.1, 24.9, 24.8,
24.7, 22.5, 13.9; FT-IR (KBr) n 3451, 2926, 2855, 1745, 1633,
1456, 1372, 1229, 1165, 1047 cmꢀ1; HR-MS m/z (%): calcd.
for C20H38O6Na+ [M + Na]+: 397.2561, found: 397.2560.
proteins in the presence of compounds are the mean values
of three individual determinations. The degree r of anisotropy
in the tryptophan fluorescence of the proteins was calculated
using the following equation at the peak of the protein
fluorescence spectrum, where IVV and IVH are the fluorescence
intensities of the emitted light polarized parallel and perpendicular
to the excited light, respectively, and G = IVH/IHH is the
instrumental grating factor.
Purification of the PKCd C1b and PKCh C1b subdomains
The PKCd C1b and PKCy C1b subdomains fused with glutathione
S-transferase (GST) were expressed in BL21 cells and purified
as reported earlier.52,53 The plasmids were a generous gift from
Prof. Alexandra Newton (University of California, San Diego,
USA). The protein expression was induced by the addition of
250 mM of isopropyl 1-thio-b-D-galactopyranoside and cells
were harvested by centrifugation (5000ꢁg for 10 min at 4 1C)
after 14 h of incubation at 24 1C. The resulting pellet was
resuspended in 20 mL of 20 mM Tris buffer, pH 8, containing
160 mM NaCl, 50 mM phenylmethylsulfonyl fluoride (PMSF),
2 mM dithiothreitol (DTT), and 0.1% Triton X-100. After
sonication for 10 cycles the solution was centrifuged for 30 min
(48 000ꢁg at 4 1C). The supernatant was filtered and 500 ml of the
glutathione S-transferase Tag resin was added. After incubating
this mixture on ice for 1 h with mild shaking at 80 rpm, it was
poured onto a column pre-rinsed with 20 mL of 20 mM Tris
buffer, pH 8, containing 160 mM KCl. The non-specifically
bound proteins were washed off with 100 mL of 20 mM Tris
buffer, pH 8, containing 160 mM KCl, and 1 unit of thrombin in
cleavage buffer was added to cleave the glutathione S-transferase
tag and the column was stored at 4 1C for 12 h. The protein was
then eluted in five fractions using 500 ml of 20 mM Tris buffer,
pH 8, containing 160 mM KCl. Proteins were further purified by
fast performance liquid chromatography (Akta Purifier) using a
Superdext 75 column (GE Healthcare Biosciences), a mobile
phase of 50 mm Tris, 100 mm NaCl, pH 7.4, and a flow rate of
ðIVV ꢀ GIVH
Þ
r ¼
ðIVV þ 2GIVH
Þ
Liposome binding assay
The membrane binding of PKC C1b subdomains was analyzed
by fluorescence quenching measurements. The protein (500 nM)
was added to PC/PS/Ligand16 (80 ꢀ x: 20: x, where x = 0–10)
liposomes prepared by an extrusion method,53 and the emission
spectra of protein were measured with the excitation wavelength
of 295 nm. Fluorescence intensity data, (F0 ꢀ F)/F0, were
plotted against the liposome containing ligand concentrations
to generate the binding isotherms, where F and F0 represent
the fluorescence intensity at 338 nm in the presence and in the
absence of ligand containing liposomes, respectively. The EC50
values were calculated for all curves by fitting with the Hill
equation using OriginLab software.
Molecular docking
Molecular docking was performed using the crystal structure of
PKCd C1b subdomain complexed with phorbol-13-O-acetate
(Protein Data Bank code: 1PTR).40 The hydrogen as well as the
side chains of Lys 234, Arg 273 and Glu 274 not visible in
the X-ray structure was subsequently added to the protein. The
energy minimized three-dimensional structure of ligands was
prepared by using the GlycoBioChem PRODRG2 Server
(http://davapc1.bioch.dundee.ac.uk/prodrg/). The GROningen
MAchine for Chemical Simulations (GROMACS) library of
three-atom combination geometries employing a combination
of short molecular dynamics simulations and energy minimiza-
tions was utilized for the conversion of 2D molecular structures
to 3D structures. Ligand-protein docking was performed using
the Molegro Virtual Docker software, version 4.3.0 (Molegro
ApS, Aarhus, Denmark).54 The binding site was automatically
detected by the docking software and restricted within spheres
with radius of 15 A. During virtual screening, the following
parameters were fixed: number of runs 10, population size 50,
crossover rate 0.9, scaling factor 0.5, maximum iteration 2000
and grid resolution 0.30. The docked results were evaluated on
the basis of moledock and re-rank score. The poses were
exported and examined with PyMOL software.
0.5 mL minꢀ1
.
Fluorescence studies
Fluorescence measurements were performed on a Fluoromax-4
spectrofluorometer using 10 mm path length quartz cuvettes
with the slit width of 20 nm at room temperature. An excitation
wavelength of 295 nm was applied to selectively excite the
tryptophan residues in protein. Proper background corrections
were made to avoid the contribution of buffer. For fluorescence
titration, protein (2 mM) and varying concentration of ligands
(0.67–40 mM for DATs of octanoic acid and 0.099–13 mM for
DATs of palmitic acid) were incubated in a buffer solution
(50 mM Tris, 150 mM NaCl, 50 mM ZnSO4, pH 7.4) at room
temperature. Fluorescence intensity data, (F0 ꢀ F)/F0, were
plotted against the ligand concentration to generate the binding
isotherms, where F and F0 represent the fluorescence intensity at
350 nm in the presence and in the absence of ligand respectively.
The EC50 values were calculated for all curves by fitting with the
Hill equation using OriginLab software. Fluorescence anisotropy
measurements were performed in the same fluorimeter using
parallel and perpendicular polarizers. The tryptophan fluores-
cence of the proteins was recorded over 300–500 nm wave-
lengths by exciting at 295 nm using an excitation slit width of
2 nm. The anisotropy values were averaged over an integration
time of 10 s, and a maximum number of five measurements
were made for each sample. All anisotropy values of the
Acknowledgements
The authors acknowledge their sincere gratitude to the
Department of Chemistry, Central Instrumentation Facility,
Indian Institute of Technology Guwahati, India. The authors
are also thankful to the BRNS (2009/20/37/5/BRNS/3331)
and DBT (BT/PR13309/GBD/27/244/2009), Govt. of India,
for financial support.
c
1284 Mol. BioSyst., 2012, 8, 1275–1285
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31 V. E. Marquez and P. M. Blumberg, Acc. Chem. Res., 2003, 36,
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