Synthesis of Phosphatidylserine and Its Stereoisomers: Their Role in Activation of Blood Coagulation

Article abstract of DOI:10.1021/acsmedchemlett.8b00008

Natural phosphatidylserine (PS), which contains two chiral centers, enhances blood coagulation. However, the process by which PS enhanced blood coagulation is not completely understood. An efficient and flexible synthetic route has been developed to synthesize all of the possible stereoisomers of PS. In this study, we examined the role of PS chiral centers in modulating the activity of the tissue factor (TF)-factor VIIa coagulation initiation complex. Full length TF was relipidated with phosphatidylcholine, and the synthesized PS isomers were individually used to estimate the procoagulant activity of the TF-FVIIa complex via a FXa generation assay. The results revealed that the initiation complex activity was stereoselective and had increased sensitivity to the configuration of the PS glycerol backbone due to optimal protein-lipid interactions.

Full text of DOI:10.1021/acsmedchemlett.8b00008

Letter
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
Synthesis of Phosphatidylserine and Its Stereoisomers: Their Role in
Activation of Blood Coagulation
Suman Mallik, Ramesh Prasad, Anindita Bhattacharya, and Prosenjit Sen*
Department of Biological Chemistry, Indian Association for the Cultivation of Science, 2A & 2B Raja S. C. Mullick Road, Jadavpur,
Kolkata-700032, India
S
* Supporting Information
ABSTRACT: Natural phosphatidylserine (PS), which contains two chiral centers,
enhances blood coagulation. However, the process by which PS enhanced blood
coagulation is not completely understood. An efficient and flexible synthetic route has
been developed to synthesize all of the possible stereoisomers of PS. In this study, we
examined the role of PS chiral centers in modulating the activity of the tissue factor (TF)-
factor VIIa coagulation initiation complex. Full length TF was relipidated with
phosphatidylcholine, and the synthesized PS isomers were individually used to estimate
the procoagulant activity of the TF-FVIIa complex via a FXa generation assay. The results
revealed that the initiation complex activity was stereoselective and had increased sensitivity
to the configuration of the PS glycerol backbone due to optimal protein−lipid interactions.
KEYWORDS: Blood coagulation, tissue factor, factor VIIa, phospholipid, phosphatidylserine
lood coagulation is a tightly regulated process.1−3 Any
aberrations in its regulation may lead to pulmonary
B
embolism, ischemic stroke, unstable angina, deep-vein
thrombosis, acute myocardial infarction, and many other
blood-related disorders.4−7 This process is initiated by the
formation of a binary protein complex (TF-FVIIa) between a
transmembrane protein, tissue factor (TF), and circulatory
factor VIIa (FVIIa), followed by a series of protease
reactions.1,8,9 Coagulation is primarily regulated by TF-FVIIa
complex activity. It has been well documented that TF-FVIIa
activity is highly modulated by the composition of the
neighboring phospholipid (PL) environment.10−12 Incorpora-
tion of phosphatidylserine (PS) dramatically enhances TF-
FVIIa complex activity.13−15 However, the detailed molecular
level mechanism responsible for this is not fully understood.
Structurally, PS consists of a polar headgroup comprising a
serine and a phosphate group, a neutral glycerol backbone, and
hydrophobic tails. From a stereochemical point of view, PS has
two chiral centers (as shown in Figure 1), one in the headgroup
serine moiety and the other in the glycerol backbone.16
Naturally occurring PS has the conformation of 1,2-diacyl-sn-
glycero-3-phospho-L-serine. In the present work, we have
provided a synthetic approach for synthesizing different PS
stereoisomers and demonstrated how the configuration of the
PS chiral centers regulates TF-FVIIa activity.
Figure 1. Chemical structure of different PS stereoisomers and their
retrosynthetic analysis.
1); we have planned to synthesize the target molecules (1, 2)
from the corresponding triethylamine H-phosphonate salts
(TEAHP 6 and 8) via H-phosphonates 5 and 7, respectively.
Other targets (3, 4) could also be synthesized with a similar
approach from intermediates 6 and 8 by coupling with D-
A number of approaches (phosphoramidite method, H-
phosphonate method, phosphotrichloride method, alkyl or aryl
dichlorophosphites, etc.)17−21 have been adopted to synthesize
PL. In most of the cases, PLs have been synthesized using 2,3-
isopropylidene-sn-glycerol22 or D-mannitol as the starting
material.23 Our synthetic approach is quite different (Figure
Received: January 9, 2018
Accepted: April 12, 2018
Published: April 12, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.8b00008
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ACS Medicinal Chemistry Letters
Letter
serine. We have developed an efficient route for the preparation
of the PL phosphodiester bond using diphenyl H-phosphonate
instead of widely used PCl3/imidazole. The uniqueness of our
synthesis is the easy separation of the highly stable intermediate
(can be stored for several months) TEAHP 6/8.
Scheme 2. Synthesis of the Intermediate TEAHP (8) with
the Opposite Conformation from the Native Glycerol
Backbone
a
In most earlier studies, PL amine and acid head groups were
protected as Cbz and benzyl ester, respectively, which made it
difficult to produce unsaturated fatty acid-containing PLs, as
deprotection of these groups by hydrogenation would also lead
to saturation of the fatty acid. We have protected the amine and
acid functionalities of the headgroup as Boc and tert-butyl
groups, respectively, which can be easily deprotected under
mild acidic conditions without hampering the unsaturation in
the fatty acid chains. This new synthesis route may provide a
viable route for the synthesis of any type of PL (saturated,
unsaturated, or mixed fatty acids) with high yield and excellent
stereospecificity.
The synthesis of the TEAHP intermediate 6 is shown in
Scheme 1. We initiated our synthesis from known compound 9
a
Reagents and conditions: (a) 2,2-dimethoxypropane, HClO4 and
Scheme 1. Synthesis of Intermediate TEAHP (6) with a
Native Glycerol Backbone Conformation
a
acetone at rt for 2 h, 92%; (b) LiAlH4 and Et2O at 0 °C, 94%; (c)
TrCl, NEt3, TBAI, and CH2Cl2 at rt for 2 h, 86%; (d) SbCl3, H2O, and
CH3CN under reflux for 4 h, 53%; (e) oleic acid, DCC, DMAP, and
CH2Cl2 at 0 °C to rt, 96%; (f) TFA and CH2Cl2 at 0 °C for 0.5 h,
98%; (g) diphenyl phosphite and pyridine for 0.5 h then NEt3/H2O
(1:1) for 1 h at rt, 92%.
was then reduced with LAH to obtain alcohol 15 and further
reacted with TrCl/Et3N/TBAI to give compound 16. Next, the
cyclic acetal in compound 16 was deprotected using SbCl3 in
aqueous acetonitrile to obtain compound 17, which gets finally
transformed into the required compound 8 via the
intermediates 18 and 19 following similar chemistry developed
earlier (Scheme 1).
The completion of the synthesis of molecules 1−4 is
depicted in Scheme 3. TEAHP 6 was subjected to react
separately with acid- and amine-protected L- and D-serine in
the presence of PivCl/pyridine to obtain H-phosphonates 20
and 21, respectively. Next, H-phosphonates 20 and 21 were
oxidized separately to their corresponding phosphates using I2/
pyridine25 and then subjected to global deprotection in the
presence of TFA to obtain 1 and 3, respectively, with very good
yields. These were purified easily using silica gel column
chromatography. Similarly, 2 and 4 were also prepared in high
yields from TEAHP 8 through intermediates 22 and 23,
respectively. All the synthesized stereoisomers are >95%
optically pure (details are provided in the Supporting
Information).
We adopted two different synthetic routes for the two
enantiomers in Schemes 1 and 2 because we wanted to
synthesize all the analogs starting from L-serine. Moreover,
using L-serine as the starting material instead of D-serine is
more cost-effective.
To investigate the role of these newly synthesized PSs and
their stereoisomers in coagulation, we reconstituted full length
TF (flTF) within liposomes containing 80% phosphatidylcho-
line (PC) and 20% phosphatidylserine 1 or its stereoisomers
(2, 3, and 4). For flTF, we cloned human TF into the pET 28a
vector with a His-tag, expressed it in E. coli (BL21 DE3 strain),
and purified it with an established protocol.26 Liposomes were
prepared using a known literature procedure.27 Their formation
and stability were tested using dynamic light scattering
a
Reagents and conditions: (a) (i) H2SO4, H2O, and NaNO2 at 0 °C to
rt for 48 h; (ii) MeOH and SOCl2 at 0 °C to rt, 78%; (b) TrCl, NEt3,
TBAI, and CH2Cl2 at rt for 2 h, 86%; (c) LiAlH4 and Et2O at 0 °C,
94%; (d) oleic acid, DCC, DMAP, and CH2Cl2 at 0 °C to rt, 96%; (e)
TFA and CH2Cl2 at 0 °C for 0.5 h, 98%; (f) diphenyl phosphite and
pyridine for 0.5 h then NEt3/H2O (1:1) for 1 h at rt, 90%.
prepared from cheap and readily available L-serine following a
literature procedure,24 which was subjected to react with TrCl/
Et3N/TBAI to obtain compound 10. The ester functionality in
compound 10 was reduced with LAH to obtain alcohol 11,
which is a glycerol derivative with the same configuration as
naturally occurring PS (1). Next, alcohol 11 was acetylated
further with oleic acid in the presence of DCC to give
compound 12 with a good yield. Compound 12 was then
reacted with TFA to access trityl-deprotected compound 13,
which was further treated sequentially with diphenyl phosphite/
pyridine and Et3N−H2O to yield the TEAHP intermediate 6
with excellent yield (>90%).
The synthesis of the other TEAHP intermediate 8, with an
opposite configuration to compound 6, is described in Scheme
2. Our synthesis commenced with the easily available material
9, which was treated with 2,2-DMP in the presence of HClO4
to yield compound 14. The ester functionality of compound 14
B
DOI: 10.1021/acsmedchemlett.8b00008
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ACS Medicinal Chemistry Letters
Letter
a
Scheme 3. Syntheses of 1−4
a
Reagents and conditions: (a) (S)-tert-butyl 2-((tert-butoxycarbonyl)amino)-3-hydroxypropanoate, PivCl and pyridine for 0.5 h at rt, 90−95%; (b)
(R)-tert-butyl 2-(tert-butoxycarbonyl)amino)-3-hydroxypropanoate, PivCl and pyridine for 0.5 h at rt, 90−98%; (c) (i) I2 and 95% pyridine-H2O at
rt; (ii) TFA and CH2Cl2 at 0 °C, 98%.
measurements. With these liposomes, pro-coagulant activity
was measured by a FXa generation assay as described earlier.28
In this assay, a saturated concentration of lipidated TF (1 nM)
was incubated with a limited concentration of recombinant
FVIIa (10 pM) to make sure that all the FVIIa remained in the
TF-FVIIa complex form (detailed procedure is mentioned in
the Supporting Information). The results show that maximal
pro-coagulant activity was observed when native PS (1) was
introduced in combination with PC. This activity is almost 6-
fold over the activity shown by the only PC containing vesicles
(Figure 2). The observed rate enhancement is consistent with
previous observations.29 Changing the configuration of the
headgroup in native PS from L to D (3) substantially decreases
FXa generation in comparison with native PS (1), but the
activity remained appreciably higher with respect to PC. In
contrast, when the glycerol backbone configuration was
Figure 2. Comparison of the effect of different PSs on FX activation
inverted, keeping the headgroup L-serine intact (2), a further
through the TF-FVIIa complex. The data presented here are the mean
reduction in pro-coagulant activity was observed, and this
SEM (n = 3). Differences are statistically significant at p < 0.05 by
reduction was significant with respect to both 1 and 3. The
the student’s t test. Phosphatidylserine lipids 1, 2, 3, and 4 are denoted
activity of 2 remained almost 2-fold compared to pure PC, but
a complete attenuation of enhanced TF-FVIIa activity due to
PS was observed when both the chiral centers were inverted
(molecule 4). Thus, it is clearly demonstrated that the
configuration of both the headgroup and the glycerol backbone
are the important contributors for enhanced TF-FVIIa complex
activity. These results demonstrate that the activity enhance-
by 1,2-dioleoyl-sn-glycero-3-phospho-L-serine; 2,3-dioleoyl-sn-glycero-
1-phospho-L-serine; 1,2-dioleoyl-sn-glycero-3-phospho-D-serine; and
2,3-dioleoyl-sn-glycero-1-phospho-D-serine, respectively.
ment is more vulnerable to the configuration of the glycerol
backbone than that of the headgroup.
C
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Figure 3. Protein−lipid interaction profile of the flTF-FVIIa complex with 1 and 4 containing a lipid environment. Snapshot of the TF and FVIIa H-
bond-interacting residues with molecules (A) 1 and (B) 4 containing a lipid bilayer and obtained from MD simulation of the binary complex.
Figure 4. Effect of the PC, PC:1, and PC:4 lipid bilayers on catalytic triad (CT) dynamics. Scattered plots for CT residues between the Ser195-
His57 Cβ-atom and between the Asp102-His57 Cβ-atom obtained from a 40 ns simulation of flTF-FVIIa in the (A) PC-, (B) PC:1-, and (C) PC:4-
containing phospholipid environments. The reference values for the CT residues are 6.5 and 5.5 Å for S195:Cβ/H57:Cβ and D102:Cβ/H57:Cβ,
respectively. Localization of the CT residue is shown by the licorice representation (right panel). Lipids 1 and 4 denote 1,2-dioleoyl-sn-glycero-3-
phospho-L-serine and 2,3-dioleoyl-sn-glycero-1-phospho-D-serine, respectively.
Next, to identify the probable reasons for the altered TF-
FVIIa activity in the presence of phospholipids having different
chirality at the atomic level, we adopted a molecular dynamics
(MD) simulation approach. We modeled the flTF-FVIIa
complex embedded in the PC:1 and PC:4 bilayers, as
individually described in the Supporting Information. As
depicted, the difference in the pro-coagulant activity of TF-
FVIIa in the PC:1 and PC:4 phospholipid molecules was
maximal; therefore, we have focused on these two stereo-
isomers (1 and 4) in the MD simulation study. We performed
40 ns simulations for these complexes after equilibration. From
the trajectories, we mapped the protein−lipid interaction
profiles of TF and FVIIa in the PC:1- and PC:4-containing
lipid bilayer systems individually. Interestingly, we found a
significant differential interaction profile for PC:1 and PC:4
with the TF-FVIIa binary complex (Table S2). PC:1 has a
significantly higher degree of interactions with both TF and
FVIIa in comparison with the PC:4 bilayer (Figure 3 and Table
D
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Letter
S2). More precisely, the amine, carboxylate, and phosphate
groups from 1 interact with E183, D180, and E183 from TF,
respectively, whereas the glycerol backbone interacts with the
N184 residue. Changing the headgroup and backbone chirality
completely attenuates the amine, carboxylate, and glycerol
backbone interaction profile in 4; however, the interaction with
the phosphate moiety remains intact. It is well-established that
the FVIIa-GLA domain, comprising ten Gla residues (post-
translationally modified glutamic acid, gamma-carboxyglutamic
acid-rich), plays a critical role in TF-FVIIa proteolytic activity
by interacting with PL.30−32 While analyzing the lipid
interaction profile with FVIIa, we found that Gla6, R9, and
Gla16 residues from the FVIIa-GLA domain interact with the
amine moiety of 1, though these interactions are absent for 4.
Among the several elements considered for efficient catalysis
in serine proteases,33 the most important factor is the
positioning of the catalytic triad (CT) residues (His57,
Asp102, and Ser195) in the FVIIa protease domain. The
distance between Asp102-His57 and Ser195-His57 should
remain optimal for the efficient catalysis by the protease
enzyme.34,35 The distances between the S195:CB-H57:CB and
D102:CB-H57:CB residue pairs in FVIIa in the PC, PC:1 and
PC:4 binary complexes were examined and plotted (Figure 4).
Consistent with our earlier study,3 we found a highly dispersed
pattern for the CT residue scattered profile when TF-FVIIa was
relipidated in PC, which is considered as an almost inactive
state for the binary complex. The TF-FVIIa complex activity
showed a maximum when TF-FVIIa was relipidated in PC:1.
We found that the distances between the complex CT residues
in PC:1 were ordered and confined within the region. This
result indicates that the CT of the binary complex in PC:1
remains in a relatively stabilized form, whereas the CT residues
reside in a fluctuating state when the complex is formed with
PC alone. We and other groups consider that stabilization of
CT is the major guiding factor for showing the enhanced
activity of the binary complex in PC:1.3,36 Interestingly, we
found that when the binary complex is formed in PC:4, the CT
residue distances remain scattered as in the binary complex
embedded in PC. Overall, the data indicate that the chiral
centers in PS play a vital role in guiding the activity of the TF-
FVIIa complex by making proper interactions with TF and the
FVIIa-GLA domain, which allosterically keep the CT residues
stable in the FVIIa protease domain.
In summary, we have proposed a new synthetic route for PL
that is advantageous in many aspects over previously
mentioned procedures. Our synthetic method is efficient for
synthesizing stereospecific PL with high yield. Adopting this
procedure, we were able to synthesize four PS molecules with
all the possible stereoisomers including the natural one. Here,
we established that PS stereochemistry plays a vital role in
regulating the activity of the coagulation initiation complex TF-
FVIIa. Both the chiral centers have significant roles because
alterations to either center abates TF-FVIIa activity. Among the
two chiral centers, the center in the PS glycerol backbone has
more impact on the activity, as inversion of this center reduces
more activity than altering the other chiral center in the
headgroup. Through our computational study, we provide a
probable reason for this altered activity for TF-FVIIa in
different PL environments. We suggest that alterations in the
chiral centers reduce the interactions between the PS head
groups and glycerol backbone with TF and FVIIa, which
allosterically regulates the catalytic activity of the TF-FVIIa
complex. However, there may be alternative explanations
related to the ability of PS to promote factor X binding to
the surface. Our findings provide new insights from the
structural point of view for PL, which will certainly enrich the
understanding of the structure−activity relationship (SAR) as
well as protein−lipid interactions in coagulation at the
molecular level.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.8b00008.
1H, 13C, and 31P NMR spectra of all new compounds,
experimental procedures for their preparation, and
computational methodology (PDF)
AUTHOR INFORMATION
Corresponding Author
*Tel: (+91)3324734971. Fax: (+91)3324732805. E-mail:
bcps@iacs.res.in.
■
ORCID
Prosenjit Sen: 0000-0002-1233-1822
Author Contributions
P.S. designed the research. S.M. and R.P. performed the
research and analyzed the data. A.B. performed the cloning and
purification of TF. P.S., R.P., and S.M. wrote the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Dr. R. K. Goswami and Dr. A. Dey for
insightful discussions and also for their technical assistance.
S.M. and A.B. gratefully acknowledges the support of the SPM
fellowship from the CSIR, Govt. of India. R.P. acknowledges
the DST for the INSPIRE fellowship. This work was supported
by project grants from the Department of Science and
Technology (DST), Govt. of India (DST No-SR/SO/BB-
0125/2012).
ABBREVIATIONS
■
TF, tissue factor; FVIIa, activated factor VII; FXa, activated
factor X; MD, molecular dynamics; PL, phospholipids; PS,
phosphatidylserine; PC, phosphatidylcholine; TEAHP, triethyl-
amine H-phosphonate salts; PS, phosphatidylserine; TrCl,
triphenylmethyl chloride; Boc, tert-butyl carbonate; tBu, tertiary
butyl; DCC, N,N′-dicyclohexylcarbodiimide; LAH, lithium
aluminum hydride
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