Ligand-Phospholipid Conjugation: A Versatile Strategy for Developing Long-Acting Ligands That Bind to Membrane Proteins by Restricting the Subcellular Localization of the Ligand

Article abstract of DOI:10.1021/acs.jmedchem.8b00041

We hypothesized that if drug localization can be restricted to a particular subcellular domain where their target proteins reside, the drugs could bind to their target proteins without being metabolized and/or excreted, which would significantly extend the half-life of the corresponding drug-target complex. Thus, we designed ligand-phospholipid conjugates in which the ligand is conjugated with a phospholipid through a polyethylene glycol linker to restrict the subcellular localization of the ligand in the vicinity of the lipid bilayer. Here, we present the design, synthesis, pharmacological activity, and binding mode analysis of ligand-phospholipid conjugates with muscarinic acetylcholine receptors as the target proteins. These results demonstrate that ligand-phospholipid conjugation can be a versatile strategy for developing long-acting ligands that bind to membrane proteins in drug discovery.

Full text of DOI:10.1021/acs.jmedchem.8b00041

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Ligand-Phospholipid Conjugation: a Versatile Strategy for
Developing Long-Acting Ligands that Bind to Membrane
Proteins by Restricting the Subcellular Localization of the Ligand
Shuhei Kawamura, Yoshihiko Ito, Takatsugu Hirokawa, Eriko Hikiyama, Shizuo Yamada, and Satoshi Shuto
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00041 • Publication Date (Web): 13 Apr 2018
Downloaded from http://pubs.acs.org on April 14, 2018
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LigandꢀPhospholipid Conjugation: a Versatile Strategy for Developing LongꢀActing Ligands that Bind
to Membrane Proteins by Restricting the Subcellular Localization of the Ligand
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Shuhei Kawamura,a Yoshihiko Ito,c Takatsugu Hirokawa,d Eriko Hikiyama,c Shizuo Yamada,c Satoshi Shutoa,b*
aFaculty of Pharmaceutical Sciences and bCenter for Research and Education on Drug Discovery, Hokkaido
University, Kitaꢀ12, Nishiꢀ6, Kitaꢀku, Sapporo 060ꢀ0812, Japan
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bCenter for PharmaꢀFood Research, Department of Pharmacokinetics and Pharmacodynamics, Graduate School of
Pharmaceutical Sciences, University of Shizuoka, 52ꢀ1 Yada, Surugaꢀku, Shizuoka 422ꢀ8526, Japan
dMolecular Profiling Research Center for Drug Discovery (MOLPROF), National Institute of Advanced Industrial
Science and Technology (AIST), 2ꢀ4ꢀ7, Aomi, Koutouꢀku, Tokyo 135ꢀ0064, Japan
Abstract: We hypothesized that if drug localization can be restricted to a particular subcellular domain where
their target proteins reside, the drugs could bind to their target proteins without being metabolized and/or excreted,
which would significantly extend the halfꢀlife of the corresponding drugꢀtarget complex. Thus, we designed
ligandꢀphospholipid conjugates, in which the ligand is conjugated with a phospholipid through a polyethylene
glycol linker, to restrict the subcellular localization of the ligand in the vicinity of the lipid bilayer. Here, we
present the design, synthesis, pharmacological activity, and binding mode analysis of ligandꢀphospholipid
conjugates with muscarinic acetylcholine receptors as the target proteins. These results demonstrate that
ligandꢀphospholipid conjugation can be a versatile strategy for developing longꢀacting ligands that bind to
membrane proteins in drug discovery.
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Introduction
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The pharmacological effects of drugs are generated by the drugꢀtarget complex, and the timeꢀcourse is
governed by both the drug pharmacokinetics, i.e., administration, distribution, metabolism, and excretion, and the
pharmacodynamics, i.e., affinity and kinetics of the drugꢀtarget interactions.1 The concentration of the drugꢀtarget
complex must generally be maintained at a high enough level that the pharmacodynamic effects can continue to
be generated throughout the treatment period.1 Historically, drug candidates have been developed by focusing
primarily on their target binding affinity, however, and therefore many of them have failed in preꢀclinical and
clinical trials due to their poor treatment efficacy due to an inadequate in vivo drugꢀtarget complex concentration,
which is a major problem in drug discovery.2 Here we present a ligandꢀphospholipid conjugation strategy to
remarkably extend the halfꢀlife of the drugꢀtarget complex, which can be a useful approach for addressing this
problem, allowing us to develop highly potent drugs with prolonged pharmacodynamic effects.
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Although the optimization of the pharmacokinetic properties by chemical modification of the lead compounds
is the primary approach to solving this problem,3 extensive efforts are required on a caseꢀbyꢀcase basis, and
furthermore, a long plasma halfꢀlife is potentially promote offꢀtarget toxicity. Another approach is to use
compounds that bind covalently to the target biomolecule4 or compounds with a long target residence time.1
Because the dissociation rate of these compounds is slow, the drugꢀtarget complex continues to be present even
after the elimination of the free compounds from the systemic circulation. The development of covalent inhibitors,
however, is applicable only in limited cases where the reactive amino acid residue of the targets is available,4 and
intentional optimization of the dissociation halfꢀlife is also quite difficult.1c Furthermore, the covalent
modification or tight binding of drugs, which induces conformational change of the target biomolecules,
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sometimes invokes immune responses, causing significant toxicity.1a,4b,5 Thus, there is no versatile approach for
addressing this problem and exhaustive caseꢀbyꢀcase optimization of the pharmacokinetic properties is generally
required, which significantly retards the drug discovery process.
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In cellular systems, subcellular localization of biomacromolecules, such as proteins, is strictly regulated, which
is of vital importance for their organized biological functions in the cell. We hypothesized that modifying a drug
to induce its localization to a particular subcellular domain containing the target protein would result in the drug
working for a longer time without being metabolized and/or excreted. Membrane proteins are one of the most
important drug targets6 including receptors,7 transporters,8 and channels,9 which localize in the membrane by
hydrophobic interactions with the lipid bilayer. Some membrane proteins are anchored to the membrane by
conjugating with nonꢀpeptide hydrophobic anchors, such as glycosylphosphatidylinositol (GPI),10 fatty acids, or
isoprenoids.11 Although a fatty acid or isoprenoid is not hydrophobic enough to stably anchor the conjugated
proteins,11d,12 a GPI anchor, whose phospholipid moiety is more hydrophobic than a fatty acid or isoprenoid,
functions as a very stable anchor.13
Thus, we thought that effective membrane anchoring of small molecules would be possible by conjugating
them to a phospholipid, and devised the ligandꢀphospholipid conjugation strategy as shown in Figure 1.14 The
ligandꢀphospholipid conjugates (LPCs) are expected to anchor the attached ligand to the membrane where they
can avoid being metabolized and/or excreted, which will significantly extend the halfꢀlife of the ligandꢀtarget
complex to prolong the duration of its pharmacodynamic effects. It has been reported that this kind of interaction
between drugs and cell membrane often contribute to prolong ligand residence time relating to the clinical
efficacy.14 In this paper, we present the design, synthesis, pharmacological activity and binding mode analysis of
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LPCs with muscarinic acetylcholine receptors as target proteins, demonstrating that the ligandꢀphospholipid
conjugation strategy can be a versatile methodology for developing longꢀacting ligands that bind to membrane
proteins due to restriction of the ligand subcellular localization in the vicinity of the lipid bilayer.
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Figure 1. The ligandꢀphospholipid conjugation strategy for developing ligands with prolonged activity due to
restriction of the ligand localization: (a) a common ligand interacting with its target membrane protein; (b) a
ligand conjugated with phospholipid through a polyethylene glycol (PEG) linker.
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Results and Discussion
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Design of LPCs. We designed the ligandꢀphospholipid conjugates (LPCs), in which a ligand is associated with a
phospholipid through a PEG linker as shown in Figure 2, to develop a versatile methodology, which is generally
and easily applicable for various ligands that bind to target membrane proteins. A variety of LPCs could be
readily synthesized by amide coupling of ligands bearing a linker moiety with carboxylic acid to the PLꢀPEG
units with a terminal amino group, which could be prepared using a phospholipase D from Streptomyces sp.
(PLDP) catalyzed transphosphatidylation method that we previously developed.15 In the LPCs, the hydrophobic
region of the phospholipid moiety functions as an effective anchor to the membrane, and a hydrophilic and
flexible polyethylene glycol (PEG)16 linker allows for binding of the attached ligand to the target membrane
protein.
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Figure 2. Design and synthetic strategy of LPCs
We selected the muscarinic acetylcholine receptor as a target membrane protein to demonstrate the
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effectiveness of our strategy, because the Xꢀray crystal structures of the M2 and M3 receptors have been solved,
making the rational structureꢀbased drug design (SBDD) possible.17 Furthermore, the highly potent antagonist
with a large fluorescent group was previously reported, enabling the rational ligandꢀbased drug design (LBDD).18
As summarized in Table 1, the fluorescent analogue 1 derived from the muscarinic receptor antagonist tolterodine,
a clinical drug used in the treatment of overactive bladder,19 has significant binding affinity to muscarinic
receptors, comparable to tolterodine,18 suggesting that introduction of the PL (phospholipid) ꢀPEG units to
tolterodine would be achieved without a significant loss of binding affinity to the receptors.
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Table 1. Binding affinity of tolterodine and its fluorescent derivative 1 to muscarinic receptors reported by Jones
and coꢀworkers.17
Ki [nM]
compound
M1
1.4
4.6
M2
2.7
9.0
M3
3.6
M4
3.1
6.3
M5
2.2
tolterodine
10.3
17.5
1
To design LPCs targeting muscarinic receptors, we performed a docking simulation of compound 2, which is a
partial structure of fluorescent compound 1 without the fluorescent group, with the Xꢀray analyzed structure of the
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M3 muscarinic receptor.17b As shown in Figure 3, the terminal structure of compound 2 shown in red is apparently
exposed to the solvent at the receptor surface, indicating that the terminal moiety can be modified without
significantly decreasing the binding affinity. Thus, we designed a series of LPCs 3ꢀ8 targeting muscarinic
receptors by conjugating tolterodine with a phospholipid through various lengths of PEG linkers, as shown in
Figure 4. The impact of the PEGꢀlinker length on the pharmacological activity of LPCs would be investigated by
evaluating these compounds.
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Figure 3. Plausible binding mode of compound 2 to the M3 muscarinic receptor predicted by a docking
simulation: green tube, the predicted binding mode of 2; yellow thin tube, the Xꢀray analyzed binding mode of
tiotropium to the M3 receptor reported by Kruse and coꢀworkers (PDB code, 4DAJ);17b a solvent accessible
surface of the M3 receptor is shown in mesh (a) or solid (b).
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Figure 4. The designed LPCs targeting muscarinic receptors
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Synthesis of the PL-PEG units and LPCs. The designed PLꢀPEG units 9ꢀ14 were synthesized by PLDP (from
Streptomyces sp.) catalyzed transphosphatidylation which is previously developed by us,15 as shown in Scheme 1.
The transphosphatidylation is an enzymatic phosphatidyl group transfer reaction with a twoꢀphase system of
CHCl3ꢀacetate buffer that effectively provides a variety of phospholipid derivatives from a phosphatidylcholine
(phosphatidyl donor) and an alkanol (phosphatidyl acceptor).15 The initial attempt using free amine PEG units as
phosphatidyl acceptors afforded almost none of the transphosphatidylation products and most of the
distearoylphosphatidylcholine (DSPC) remained intact, suggesting that the PLDP was denatured under the
alkaline conditions due to the basic acceptors. Therefore, we used hydrochloride salts of the PEG units as
acceptors, which gave the desired PLꢀPEG units 9ꢀ14 as transphosphatidylation products in excellent yields. Thus,
we efficiently synthesized a series of the key PLꢀPEG units that could be generally used for the synthesis of
various LPCs, by the PLDPꢀcatalyzed transphosphatidylation.15
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Scheme 1. Synthesis of PLꢀPEG units 9ꢀ14 by phospholipase D (PLDP) catalyzed transphosphatidylation
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The synthesis of the tolterodine unit 19 is shown in Scheme 2. The phenolic hydroxyl group of tolterodine was
protected by a benzyl group to yield compound 15. The benzylic methyl group of 15 was oxidized with ceric
ammonium nitrate (CAN) to yield the corresponding aldehyde, which was subsequently reduced to alcohol with
NaBH4 in methanol. The resulting alcohol was successively treated with SOCl2 and NaCN to yield nitrile 16. The
cyano group of 16 was reduced with DIBAL, and the resulting amino group was condensed with carboxylic acid
17 by the mixed anhydride method to afford compound 18. The two benzyl groups of 18 were simultaneously
removed by hydrogenolysis to yield the tolterodine unit 19.
Scheme 2. Synthesis of tolterodine unit 19
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Finally, the tolterodine unit 19 was condensed with each of the PLꢀPEG units 9ꢀ14, as shown in Scheme 3.
Although tolterodine unit 19 was almost insoluble in CHCl3, a mixed solvent of CHCl3 and tꢀBuOH effectively
dissolved it for use in the reaction. The condensation effectively proceeded to afford a series of designed LPCs
3ꢀ8 in high yields. These results suggested that various ligands bearing a linker moiety with a carboxyl group can
be generally and readily derivatized into the corresponding LPCs by condensation with the key PLꢀPEG units.
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Scheme 3. Synthesis of the designed LPCs 3ꢀ8 targeting muscarinic acetylcholine receptors
Pharmacological effects of the LPCs targeting muscarinic receptors. The binding affinity of the synthesized
LPCs 3ꢀ8 to muscarinic receptors in rat brain homogenates was investigated using [3H] Nꢀmethyl scopolamine.20
As summarized in Table 2, the binding affinity of the LPCs to the muscarinic receptors was significantly affected
by the PEGꢀlinker length: as the PEGꢀlinker length increased from n = 1 to n = 9 (LPCs 3ꢀ7), the binding affinity
clearly increased. The LPCs would be initially incorporated into the membrane by the strong but nonꢀspecific
hydrophobic interactions between the phospholipid moiety and the membrane to anchor the attached ligand to the
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membrane (Figure 1b, II). Thereafter, lateral diffusion of the anchor moiety in the membrane would effectively
facilitate the attached ligand to interact with its target protein (Figure 1b, III). Therefore, if the PEGꢀlinker was not
long enough, the hydrophobic region of the phospholipid moiety would have to protrude from the membrane to
place the attached ligand into the binding pocket of the target protein, which would impair the binding affinity, as
clearly shown in LPC 3 (n = 1). On the other hand, LPC 8 (n = 11) showed almost the same binding affinity as
LPC 7 (n = 9), suggesting that when the PEGꢀlinker is longer than the optimal length, the impact of the linker
length on the binding affinity is insignificant probably due to the highly flexible nature of the PEGꢀlinker.16b Thus,
in the design of LPCs, using a long PEGꢀlinker might effectively provide potent LPCs without requiring the
optimization of the linker length. We also synthesized PEGꢀtolterodine 20, having the PEG linker but not the
phospholipid moiety, which showed a binding affinity slightly lower than tolterodine itself, probably because of
the introduced PEGꢀlinker moiety. Importantly, LPC 7 with the same length of PEG linker as 20, had a binding
affinity much higher than 20, and even higher than tolterodine itself, suggesting that the membrane anchoring
effectively improved the binding affinity by localizing the attached ligand close to its target receptor in the
membrane.x
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Table 2. Binding affinity of the LPCs 3ꢀ8, PEGꢀtolterodine 20 and tolterodine to muscarinic receptors in rat brain
homogenates using [3H] Nꢀmethyl scopolamine
a
compound
n
log IC50
[3H] Nꢀmethyl scopolamine binding
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(% of control)a
wash × 1
no wash
wash × 2
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1
3
ꢀ6.91 ± 0.065
ꢀ7.74 ± 0.004
ꢀ7.86 ± 0.036
ꢀ8.15 ± 0.043
ꢀ8.53 ± 0.076
ꢀ8.49 ± 0.074
ꢀ8.20 ± 0.018
66.4 ± 2.1
32.3 ± 3.4
14.5 ± 4.5
4.7 ± 0.6
6.0 ± 2.2
11.2 ± 4.4
9.0 ± 0.8
59.2 ± 2.4
15.5 ± 2.0
5.1 ± 1.1
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5
7
2.1 ± 0.3
9
1.7 ± 0.6
2.1 ± 0.3
11
1.9 ± 0.8
tolterodine
36.2 ± 1.7
93.2 ± 3.8
63.5 ± 4.9
ꢀ7.88 ± 0.082
7.1 ± 0.9
34.9 ± 0.8
20
aBased on at least four experiments
Next, we conducted washꢀout experiments to investigate the duration of the compound binding to the
muscarinic receptors. Rat brain homogenates containing muscarinic receptors were incubated with 100 nM LPCs
3ꢀ8, tolterodine, or PEGꢀtolterodine 20 at 25 °C, and [3H] Nꢀmethyl scopolamine20 binding to the muscarinic
receptors was measured before and after washꢀout with a iceꢀcold buffer solution. As shown in Figure 5, none of
the LPCs showed any decrease in binding after washꢀout regardless of the extent of their binding affinity,
suggesting that the duration of the binding was solely due to the membrane anchoring. In contrast, the binding of
tolterodine and PEGꢀtolterodine 20 decreased drastically after washꢀout, showing drastic effects of the membrane
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anchoring to extend the receptor binding period of the anchored ligand. Interestingly, all LPCs showed increased
binding affinity to the receptors after the first washꢀout compared with those before the washꢀout, suggesting that
the LPCs bind to the receptors in a timeꢀdependent manner after anchoring into the membrane (Figure 1b, II to
III). Thus, the designed LPCs had potent binding affinity to the muscarinic receptors, and importantly, the binding
was not attenuated even after the washꢀout, which was in stark contrast with the drastic attenuation in the parent
compound tolterodine, as expected.21
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Figure 5. [3H] Nꢀmethyl scopolamine binding to muscarinic receptors treated with LPCs 3ꢀ8, PEGꢀtolterodine 20
and tolterodine before and after washꢀout. Data are Based on at least four experiments.
Finally, we investigated the pharmacological effects of LPC 7 in vivo. Tolterodine or LPC 7 was intravesically
instilled to rats, and [3H] Nꢀmethyl scopolamine20 binding to the muscarinic receptors in the bladder was directly
measured by dissecting the bladder 30 min or 24 h after the instillation to calculate the apparent Kd value (Kdapp).
At 30 min after the instillation, both tolterodine and LPC 7 increased the Kdapp value of the [3H] Nꢀmethyl
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scopolamine to a similar extent, showing that both of them effectively bound to the muscarinic receptors in vivo.
Importantly, the Kdapp value of [3H] Nꢀmethyl scopolamine was not significantly decreased even after 24 h in rats
treated with LPC 7, while the value was significantly decreased after 24 h in rats treated with tolterodine. To the
extent that an increase in Kdapp values for radioligands in drugꢀpretreated tissues in the radioreceptor assay
refers generally to competition with the radioligand for same binding sites.22 Therefore, these findings
indicate that LPC 7 exhibits longꢀterm binding to muscarinic receptors even in vivo, compared with its parent
compound tolterodine.
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Figure 6. Kdapp value of [3H] Nꢀmethyl scopolamine binding to the muscarinic receptors in the bladder of rats
treated with tolterodine or LPC 7. The Kdapp value was directly measured by dissecting the bladder 30 min or 24 h
†
after the intravesical instillation of the compounds. *p < 0.05, ***p < 0.001 vs control; p < 0.05 vs tolterodine
(30 min), n.s., not significant, n = 4.
Molecular dynamics analysis of the binding mode of LPCs. The binding mode of LPC 3 and 7 were analyzed
by molecular dynamics (MD) simulations using the Xꢀray structure of the M3 muscarinic receptor17b (see
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supporting information for details). The zꢀcoordinates of the phosphorous atom and ωꢀcarbons of the phospholipid
moiety during the simulation (time step = 0.1 ns, 1000 steps) were plotted for LPC 3 (n = 1) and 7 (n = 9) as
shown in Figure 7a, and the snapshots of the simulations at 20, 50 and 100 ns, along with their initial model, are
shown in Figure 7b (LPC 3, n = 1) and 7c (LPC 7, n = 9), respectively. Both of the systems appeared to reach
equilibrium within 50 ns, and after that, the zꢀcoordinates of the ωꢀcarbons of LPC 3 (C1n1 and C2n1) were around
20 – 30 Å, while those of LPC 7 (C1n9 and C2n9) were around ꢀ5 – 5 Å. This clearly shows that the phospholipid
moiety of the LPC 3 protrudes from the lipid bilayer to have the attached ligand moiety interact with the
muscarinic receptor (Figure 7b), which will significantly deteriorate its binding affinity (Table 2: tolterodine, log
IC50 = ꢀ8.20; LPC 3, log IC50 = ꢀ6.91). On the other hand, the phospholipid moiety of LPC 7 is well embedded in
the lipid bilayer even when the ligand moiety interacts with the muscarinic receptor (Figure 7c), which will
facilitate the ligand binding rather than disrupt it by working just like a “bivalent” ligand (Table 2: tolterodine, log
IC50 = ꢀ8.20; LPC 7, log IC50 = ꢀ8.53).16b
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Figure 7. Design of LPCs targeting muscarinic receptors: (a) structures of tolterodine, tiotropium and compound
1; (b) and (c) plausible binding mode of compound 1 to the M3 muscarinic receptor predicted by a docking
simulation (green tube, the predicted binding mode of 1; yellow thin tube, the Xꢀray analyzed binding mode of
tiotropium to the M3 receptor reported by Kruse and coꢀworkers (PDB code, 4DAJ);10 a solvent accessible surface
of the M3 receptor is shown in mesh (b) or solid (c)); (d) structures of the designed LPCs 2ꢀ7.
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Conclusions
We successfully developed a ligandꢀphospholipid conjugation strategy to develop longꢀacting ligands that bind
to membrane proteins due to restriction of the subcellular localization of the conjugated ligands in the vicinity of
the lipid bilayer. The designed and synthesized LPCs had significantly prolonged target binding not only in vitro
but also in vivo, compared with their parent compound, suggesting that this strategy is practical for actual drug
discovery research. In principle, any type of ligands can be anchored to the outerꢀleaflet of the lipid bilayer by
derivatizing them into the corresponding LPCs, which, except for the intravesically instillation, may be used as
inhalation and ointment drugs. Furthermore, LPCs can be synthesized by a simple amide coupling between ligands
and the designed key PLꢀPEG units, which were efficiently synthesized by PLDPꢀcatalyzed
transphosphatidylation, which we previously developed,15 showing that this is a versatile and readily available
strategy. Importantly, although phospholipid conjugates of proteins or small molecules have been reported to date,
which includes antibodyꢀ or small moleculeꢀphospholipid conjugates for liposome targeting or GPIꢀanchored
proteins or peptides expressed by genetic modification,22 none of the phospholipid conjugates that restrict the
subcellular localization of the ligand for developing longꢀacting ligands was known. Thus, this newly developed
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ligandꢀphospholipid conjugation strategy based on the totally new concept “restricting the ligand subcellular
localization” will be a widely applicable methodology for developing longꢀacting drug candidates that target
membrane proteins.
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EXPERIMENTAL SECTION
General methods and materials
1HꢀNMR spectra were recorded in CDCl3 at ambient temperature unless otherwise noted, at 400 or 500 MHz,
with TMS as an internal standard. 13C NMR spectra were recorded in CDCl3 at ambient temperature unless
otherwise noted, at 100 or 125 MHz. Silica gel column chromatography was performed with silica gel 60 N
(spherical, neutral, 63ꢀ210 ꢀm, Kanto Chemical Co., Inc.). Flash column chromatography was performed with
silica gel 60 N (spherical, neutral, 40ꢀ50 ꢀm, Kanto Chemical Co., Inc.). Celite 545 was purchased from Kanto
Chemical Co., Inc. Pd/C (PE type) was purchased from N.E. Chemcat Co. Pd(OH2)/C was purchased from Tokyo
Chemical Industry Co., Ltd. [NꢀMethylꢀ3H]scopolamine methyl chloride ([3H]NMS, 3.03 TBq/mmol) was
purchased from PerkinElmer Life Sciences, Inc. (Boston, MA). Combustion analysis was performed to confirm
≥95% sample purity (within ±0.4% of the calculated value).
General Procedure for the Preparation of LPCs 3-8. To a solution of tolterodine unit 19 (20.9 mg, 0.0420
mmol, 1.0 equiv) in a mixture of CHCl3 and tꢀBuOH (1.25 ml, 3:2) was added HOAt (6.86 mg, 0.0504 mmol,
1.2 equiv) and EDC･HCl (9.66 mg, 0.0504 mmol, 1.2 equiv). After 5 min, CHCl3 (750 ꢀl), triethylamine (7.02
ꢀl, 0.0504 mmol, 1.2 equiv) and the PLꢀPEG unit (0.0420 mmol, 1.0 equiv) were added and the resulting
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mixture was stirred until consumption of the PLꢀPEG unit was confirmed by TLC analysis. The reaction
mixture was diluted with a mixture of CHCl3 and MeOH (2:1) and the resulting mixture was washed twice with
1 N HCl. The organic layer was dried over Na2SO4 and the solvent was removed under reduced pressure. The
crude product was purified by silica gel column chromatography (CHCl3/ MeOH 20:1ꢀ9:1) and the fractions
containing desired product were concentrated in vacuo. The resulting residue was dissolved in a mixture of
CHCl3 and MeOH (2:1) and washed with 1 N HCl and water. The organic layer was dried over Na2SO4 and the
solvent was removed under reduced pressure. The residue was further purified by trituration with nꢀhexane and
subsequently lyophilized from water to yield the corresponding LPC as a white amorphous solid.
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Preparation and Spectral and Analytical Data of LPC 3. LPC 3 (42.7 mg, 0.0336 mmol, 80%) was prepared
from PLꢀPEG unit 9. [α]22D ꢀ3.86 (c 0.56, CHCl3/ MeOH 2:1); 1HꢀNMR (500 MHz, CDCl3/ CD3OD 3:1) δ 7.60
(br, 1H, amide NH), 7.53 (br, 1H, amide NH), 7.36 (d, J = 7.6 Hz, 2H, aromatic), 7.29 (dd, J = 7.6, 7.6 Hz, 2H,
aromatic), 7.20 (t, J = 7.6 Hz, 1H, aromatic), 7.05 (s, 1H, aromatic), 6.88 (d, J = 7.6 Hz, 1H, aromatic), 6.73 (d,
J = 7.6 Hz, 1H, aromatic), 5.26ꢀ5.16 (m, 1H, glycerol CH), 4.38 (dd, J = 12.4, 3.8 Hz, 1H, glycerol CH2), 4.31
(dd, J = 7.6, 7.6 Hz, 1H, benzyl CH), 4.16 (dd, J = 12.4, 6.7 Hz, 1H, glycerol CH2), 4.05ꢀ3.92 (m, 4H, POCH2
and glycerol CH2), 3.65ꢀ3.58 (m, 2H, OCH2), 3.58ꢀ3.47 (m, 4H, OCH2 and NCH), 3.47ꢀ3.30 (m, 4H,
CONHCH2), 2.97ꢀ2.79 (m, 2H, NCH2), 2.69 (t, J = 6.7 Hz, 2H, benzyl CH2), 2.65ꢀ2.46 (m, 2H, NCH2CH2),
2.35ꢀ2.21 (m, 4H, COCH2), 2.17ꢀ2.02 (m, 4H, NHCOCH2), 1.66ꢀ1.39 (m, 8H, NHCOCH2CH2 and COCH2CH2),
1.34ꢀ1.18 (m, 68H, CH2 and isopropyl CH3), 1.18ꢀ1.07 (m, 2H, CH2), 0.88 (t, J = 6.7 Hz, 3H, CH3), 0.88 (t, J =
6.7 Hz, 3H, CH3); 13CꢀNMR (100 MHz, CDCl3/ CD3OD 3:1) δ 174.5, 174.2, 173.6, 173.2, 152.5, 142.4, 130.1,
129.3, 128.3, 127.8, 126.4, 115.4, 70.2 (d, JCꢀP = 6.7 Hz), 70.1 (d, JCꢀP = 4.8 Hz), 69.1, 64.5 (d, JCꢀP = 5.7 Hz),
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63.1 (d, JCꢀP = 4.8 Hz), 62.4, 54.3, 54.3, 46.2, 41.7, 40.2, 39.0, 35.6, 35.5, 34.1, 34.0, 33.8, 31.6, 29.4, 29.4, 29.3,
29.1, 28.9, 28.8, 27.6, 24.9, 24.8, 24.6, 24.6, 22.4, 18.1, 18.0, 16.5, 16.3, 13.7; 31PꢀNMR (202 MHz, CDCl3/
CD3OD 3:1) δ 3.08; LRMS (ESI) m/z 1292.91 [(M+Na)+]; HRMS (ESI) calcd for C73H128N3O12PNa: 1292.9128
[(M+Na)+], found: 1292.9099; Anal. calcd for C73H128N3O12P･3.5H2O: C, 65.73; H, 10.20; N, 3.15. Found: C,
65.86; H, 9.84; N, 3.13.
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Preparation and Spectral and Analytical Data of LPC 4. LPC 4 (53.6 mg, 0.0394 mmol, 94%) was prepared
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from PLꢀPEG unit 10. [α]20 ꢀ0.15 (c 0.63, CHCl3/ MeOH 2:1); HꢀNMR (500 MHz, CDCl3/ CD3OD 3:1) δ
D
7.51 (br, 1H, amide NH), 7.38 (br, 1H, amide NH), 7.36 (d, J = 7.6 Hz, 2H, aromatic), 7.30 (dd, J = 7.6 Hz, 2H,
aromatic), 7.20 (t, J = 7.6 Hz, 1H, aromatic), 7.03 (s, 1H, aromatic), 6.87 (d, J = 7.6 Hz, 1H, aromatic), 6.73 (d,
J = 7.6 Hz, 1H, aromatic), 5.25ꢀ5.17 (m, 1H, glycerol CH), 4.38 (dd, J = 11.5, 2.9 Hz, 1H, glycerol CH2), 4.31
(t, J = 6.7 Hz, 1H, benzyl CH), 4.19ꢀ4.12 (m, 1H, glycerol CH2), 4.03ꢀ3.90 (m, 4H, POCH2 and glycerol CH2),
3.70ꢀ3.58 (m, 10H, OCH2), 3.58ꢀ3.47 (m, 4H, OCH2 and NCH), 3.43ꢀ3.32 (m, 4H, CONHCH2), 2.95ꢀ2.74 (m,
2H, NCH2), 2.68ꢀ2.60 (m, 2H, benzyl CH2), 2.60ꢀ2.47 (m, 2H, NCH2CH2), 2.36ꢀ2.22 (m, 4H, COCH2),
2.15ꢀ2.04 (m, 4H, NHCOCH2), 1.66ꢀ1.49 (m, 8H, COCH2CH2), 1.38ꢀ1.11 (m, 70H, CH2 and isopropyl CH3),
0.88 (t, J = 6.7 Hz, 3H, CH3), 0.88 (t, J = 6.7 Hz, 3H, CH3); 13CꢀNMR (100 MHz, CDCl3/ CD3OD 3:1) δ 174.3,
174.1, 173.5, 173.1, 152.4, 142.4, 130.0, 129.4, 128.2, 127.7, 127.6, 126.3, 115.2, 70.3 (d, JCꢀP = 7.6 Hz), 70.2,
70.1 (d, JCꢀP = 8.6 Hz), 69.9, 69.7, 69.3, 64.2 (d, JCꢀP = 5.7 Hz), 63.1 (d, JCꢀP = 4.8 Hz), 62.4, 54.2, 46.1, 41.4,
40.3, 38.9, 35.6, 35.5, 34.2, 33.9, 33.7, 31.6, 29.3, 29.2, 29.0, 28.8, 27.9, 25.0, 24.6, 24.5, 22.3, 18.0, 17.9, 16.4,
16.3, 13.6; 31PꢀNMR (202 MHz, CDCl3/ CD3OD 3:1) δ 3.03; LRMS (ESI) m/z 1380.96 [(M+Na)+]; HRMS
(ESI) calcd for C77H136N3O14PNa: 1380.9652 [(M+Na)+], found: 1380.9618; Anal. calcd for C77H136N3O14P･
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3.3H2O: C, 65.21; H, 10.13; N, 2.96. Found: C, 64.90; H, 9.74; N, 2.89.
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Preparation and Spectral and Analytical Data of LPC 5 (56.1 mg, 0.0388 mmol, 92%) was prepared from
PLꢀPEG unit 11. [α]20D 2.58 (c 0.51, CHCl3/ MeOH 2:1); 1HꢀNMR (500 MHz, CDCl3/ CD3OD 3:1) δ 7.51 (br,
1H, amide NH), 7.37 (br, 1H, amide NH), 7.37 (d, J = 7.6 Hz, 2H, aromatic), 7.30 (dd, J = 7.6, 7.6 Hz, 2H,
aromatic), 7.20 (t, J = 7.6 Hz, 1H, aromatic), 7.03 (d, J = 1.9 Hz, 1H, aromatic), 6.89 (dd, J = 7.6, 1.9 Hz, 1H,
aromatic), 6.74 (d, J = 7.6 Hz, 1H, aromatic), 5.26ꢀ5.18 (m, 1H, glycerol CH), 4.39 (dd, J = 12.4, 3.8 Hz, 1H,
glycerol CH2), 4.35ꢀ4.25 (m, 1H, benzyl CH), 4.16 (dd, J = 12.4, 7.6 Hz, 1H, glycerol CH2), 4.02ꢀ3.91 (m, 4H,
POCH2 and glycerol CH2), 3.70ꢀ3.57 (m, 18H, OCH2), 3.57ꢀ3.48 (m, 4H, OCH2 and NCH), 3.43ꢀ3.30 (m, 4H,
CONHCH2), 2.94ꢀ2.79 (m, 2H, NCH2), 2.73ꢀ2.64 (m, 2H, benzyl CH2), 2.64ꢀ2.50 (m, 2H, NCH2CH2),
2.35ꢀ2.23 (m, 4H, COCH2), 2.15ꢀ2.04 (m, 4H, NHCOCH2), 1.66ꢀ1.39 (m, 8H, COCH2CH2), 1.39ꢀ1.14 (m, 70H,
CH2 and isopropyl CH3), 0.89 (t, J = 6.7 Hz, 3H, CH3), 0.89 (t, J = 6.7 Hz, 3H, CH3); 13CꢀNMR (125 MHz,
CDCl3/ CD3OD 3:1) δ 174.2, 173.9, 173.5, 173.1, 152.4, 142.4, 130.1, 129.4, 128.2, 127.8, 127.5, 126.3, 115.3,
70.4 (d, J = 8.4 Hz), 70.2, 70.1, 70.1, 70.0, 70.0, 69.7, 69.3, 64.2 (d, J = 6.0 Hz), 63.1 (d, J = 6.0 Hz), 62.4, 54.1,
46.1, 41.5, 40.3, 38.9, 35.7, 35.6, 34.2, 33.9, 33.8, 31.6, 29.4, 29.3, 29.2, 29.2, 29.0, 29.0, 28.8, 28.8, 28.1, 25.0,
24.6, 24.5, 22.3, 18.0, 17.9, 16.4, 16.3, 13.7; 31PꢀNMR (202 MHz, CDCl3/ CD3OD 3:1) δ 3.03; LRMS (ESI)
m/z 1469.01 [(M+Na)+]; HRMS (ESI) calcd for C81H144N3O16PNa: 1469.0176 [(M+Na)+], found: 1469.0144;
Anal. calcd for C81H144N3O16P･3.0H2O: C, 64.81; H, 10.07; N, 2.80. Found: C, 64.94; H, 9.82; N, 2.76.
Preparation and Spectral and Analytical Data of LPC 6. LPC 6 (59.2 mg, 0.0386 mmol, 92%) was prepared
from PLꢀPEG unit 12. [α]20D 3.97 (c 0.87, CHCl3/ MeOH 2:1); 1HꢀNMR (500 MHz, CDCl3/ CD3OD 3:1) δ 7.45
(br, 1H, amide NH), 7.36 (d, J = 7.6 Hz, 2H, aromatic), 7.30 (dd, J = 7.6, 7.6 Hz, 2H, aromatic), 7.28 (br, 1H,
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amide NH), 7.20 (t, J = 7.6 Hz, 1H, aromatic), 6.99 (d, J = 1.9 Hz, 1H, aromatic), 6.88 (dd, J = 8.6, 1.9 Hz, 1H,
aromatic), 6.74 (d, J = 8.6 Hz, 1H, aromatic), 5.25ꢀ5.17 (m, 1H, glycerol CH), 4.38 (dd, J = 12.4, 2.9 Hz, 1H,
glycerol CH2), 4.31 (t, J = 6.7 Hz, 1H, benzyl CH), 4.15 (dd, J = 12.4, 7.6 Hz, 1H, glycerol CH2), 4.03ꢀ3.91 (m,
4H, POCH2 and glycerol CH2), 3.71ꢀ3.57 (m, 26H, OCH2), 3.57ꢀ3.47 (m, 4H, OCH2 and NCH), 3.43ꢀ3.30 (m,
4H, CONHCH2), 2.94ꢀ2.78 (m, 2H, NCH2), 2.67 (t, J = 6.7 Hz, 2H, benzyl CH2), 2.63ꢀ2.47 (m, 2H, NCH2CH2),
2.36ꢀ2.23 (m, 4H, COCH2), 2.17ꢀ2.02 (m, 4H, NHCOCH2), 1.66 (m, 8H, COCH2CH2), 1.37ꢀ1.17 (m, 70H, CH2
and isopropyl CH3), 0.88 (t, J = 6.7 Hz, 3H, CH3), 0.88 (t, J = 6.7 Hz, 3H, CH3); 13CꢀNMR (100 MHz, CDCl3/
CD3OD 3:1) δ 174.1, 173.9, 173.5, 173.1, 152.5, 142.4, 130.0, 129.4, 128.2, 127.8, 127.8, 127.5, 126.3, 115.4,
70.4 (d, J = 8.5 Hz), 70.1, 70.0, 69.7, 69.3, 64.2 (d, J = 5.6 Hz), 63.1 (d, J = 4.7 Hz), 62.4, 54.1, 46.1, 41.6, 40.4,
38.9, 35.6, 35.6, 34.3, 33.9, 33.8, 31.6, 29.4, 29.3, 29.2, 29.2, 29.0, 29.0, 28.8, 28.8, 28.2, 25.0, 24.6, 24.5, 22.3,
18.0, 16.4, 13.7; 31PꢀNMR (202 MHz, CDCl3/ CD3OD 3:1) δ 3.02; LRMS (ESI) m/z 1557.07 [(M+Na)+];
HRMS (ESI) calcd for C85H152N3O18PNa: 1557.0701 [(M+Na)+], found: 1557.0691; Anal. calcd for
C85H152N3O18P･3.0H2O: C, 64.24; H, 10.02; N, 2.64. Found: C, 64.33; H, 9.77; N, 2.61.
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Preparation and Spectral and Analytical Data of LPC 7. LPC 7 (59.3 mg, 0.0365 mmol, 87%) was prepared
from PLꢀPEG unit 13. [α]20D 5.06 (c 0.98, CHCl3/ MeOH 2:1); 1HꢀNMR (500 MHz, CDCl3/ CD3OD 3:1) δ 7.44
(br, 1H, amide NH), 7.36 (d, J = 7.6 Hz, 2H, aromatic), 7.30 (dd, J = 7.6, 7.6 Hz, 2H, aromatic), 7.26 (br, 1H,
amide NH), 7.20 (t, J = 7.6 Hz, 1H, aromatic), 6.98 (d, J = 1.9 Hz, 1H, aromatic), 6.88 (dd, J = 7.6, 1.9 Hz, 1H,
aromatic), 6.74 (d, J = 7.6 Hz, 1H, aromatic), 5.25ꢀ5.17 (m, 1H, glycerol CH), 4.38 (dd, J = 11.5, 2.9 Hz, 1H,
glycerol CH2), 4.31 (dd, J = 7.6, 7.6 Hz, 1H, benzyl CH), 4.15 (dd, J = 11.5, 6.7 Hz, 1H, glycerol CH2),
4.03ꢀ3.89 (m, 4H, POCH2 and glycerol CH2), 3.71ꢀ3.57 (m, 34H, OCH2), 3.57ꢀ3.48 (m, 4H, OCH2 and NCH),
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3.43ꢀ3.29 (m, 4H, CONHCH2), 2.93ꢀ2.81 (m, 2H, NCH2), 2.71ꢀ2.63 (m, 2H, benzyl CH2), 2.63ꢀ2.47 (m, 2H,
NCH2CH2), 2.35ꢀ2.24 (m, 4H, COCH2), 2.17ꢀ2.03 (m, 4H, NHCOCH2), 1.66ꢀ1.45 (m, 8H, COCH2CH2),
1.40ꢀ1.17 (m, 70H, CH2 and isopropyl CH3), 0.88 (t, J = 6.7 Hz, 3H, CH3), 0.88 (t, J = 6.7 Hz, 3H, CH3);
13CꢀNMR (100 MHz, CDCl3/ CD3OD 3:1) δ 174.1, 173.8, 173.5, 173.0, 152.5, 142.4, 130.0, 129.3, 128.2,
127.9, 127.7, 127.5, 126.3, 115.4, 70.3 (d, J = 8.5 Hz), 70.1, 70.0, 70.0, 69.8, 69.7, 69.3, 64.2 (d, J = 5.6 Hz),
63.1 (d, J = 4.7 Hz), 62.4, 54.1, 54.1, 46.1, 41.7, 40.4, 38.8, 35.6, 35.6, 34.3, 33.9, 33.7, 31.6, 29.4, 29.3, 29.2,
29.2, 29.0, 29.0, 28.8, 28.8, 28.2, 25.0, 24.6, 24.5, 22.3, 18.0, 17.9, 16.3, 13.6; 31PꢀNMR (202 MHz, CDCl3/
CD3OD 3:1) δ 3.15; LRMS (ESI) m/z 1646.12 [(M+Na)+]; HRMS (ESI) calcd for C89H160N3O20PNa: 1645.1225
[(M+Na)+], found: 1645.1204; Anal. calcd for C89H160N3O20P･2.5H2O: C, 64.08; H, 9.97; N, 2.52. Found: C,
64.07; H, 9.81; N, 2.45.
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Preparation and Spectral and Analytical Data of LPC 8. LPC 8 (58.8 mg, 0.0344 mmol, 82%) was prepared
from PLꢀPEG unit 14. [α]20D 4.79 (c 0.89, CHCl3/ MeOH 2:1); 1HꢀNMR (500 MHz, CDCl3/ CD3OD 3:1) δ 7.43
(br, 1H, amide NH), 7.36 (d, J = 7.6 Hz, 2H, aromatic), 7.30 (dd, J = 7.6, 7.6 Hz, 2H, aromatic), 7.25 (br, 1H,
amide NH), 7.20 (t, J = 7.6 Hz, 1H, aromatic), 6.97 (d, J = 1.9 Hz, 1H, aromatic), 6.88 (dd, J = 7.6, 1.9 Hz, 1H,
aromatic), 6.74 (d, J = 7.6 Hz, 1H, aromatic), 5.26ꢀ5.16 (m, 1H, glycerol CH), 4.38 (dd, J = 12.4, 2.9 Hz, 1H,
glycerol CH2), 4.31 (dd, J = 7.6, 7.6 Hz, 1H, benzyl CH), 4.15 (dd, J = 12.4, 6.7 Hz, 1H, glycerol CH2),
4.03ꢀ3.90 (m, 4H, POCH2 and glycerol CH2), 3.72ꢀ3.58 (m, 42H, OCH2), 3.58ꢀ3.47 (m, 4H, OCH2 and NCH),
3.43ꢀ3.29 (m, 4H, CONHCH2), 2.93ꢀ2.83 (m, 2H, NCH2), 2.66 (t, J = 7.6 Hz, 2H, benzyl CH2), 2.63ꢀ2.47 (m,
2H, NCH2CH2), 2.29 (t, J = 7.6 Hz, 2H, COCH2), 2.29 (t, J = 7.6 Hz, 2H, COCH2), 2.17ꢀ2.04 (m, 4H,
NHCOCH2), 1.65ꢀ1.49 (m, 8H, COCH2CH2), 1.38ꢀ1.18 (m, 70H, CH2 and isopropyl CH3), 0.88 (t, J = 6.7 Hz,
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3H, CH3), 0.88 (t, J = 6.7 Hz, 3H, CH3); 13CꢀNMR (100 MHz, CDCl3/ CD3OD 3:1) δ 174.1, 173.8, 173.5, 173.0,
152.5, 142.4, 130.0, 129.3, 128.2, 127.9, 127.7, 127.5, 126.3, 115.4, 70.4, 70.3, 70.1, 70.1, 70.0, 70.0, 69.7,
69.3, 64.2 (d, J = 5.6 Hz), 63.1 (d, J = 4.7 Hz), 62.4, 54.1, 54.1, 46.1, 41.7, 40.4, 38.8, 35.6, 35.6, 34.3, 33.9,
33.7, 31.6, 29.4, 29.3, 29.2, 29.2, 29.0, 29.0, 28.8, 28.8, 28.2, 25.0, 24.9, 24.6, 24.5, 22.3, 18.0, 16.4, 13.7;
31PꢀNMR (202 MHz, CDCl3/ CD3OD 3:1) δ 3.15; LRMS (ESI) m/z 1734.18 [(M+Na)+]; HRMS (ESI) calcd for
C93H168N3O22PNa: 1733.1749 [(M+Na)+], found: 1733.1732; Anal. calcd for C93H168N3O22P･1.5H2O: C, 64.26;
H, 9.91; N, 2.42. Found: C, 64.16; H, 9.83; N, 2.41.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website. Experimental details of
synthesis except for final compounds, biological evaluations, computational simulations, a table listing
combustion analysis data for final compounds (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding author Information
Eꢀmail address: shu@pharm.hokudai.ac.jp
Telephone & fax number: +81ꢀ11ꢀ706ꢀ3769
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Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGEMENTS
This investigation was supported by GrantꢀinꢀAids for challenging Exploratory Research (16K15136) from
Ministry of Education, Culture, Sports, Science and TechnologyꢀJapan.
ABBREVIATIONS USED
CAN, ceric ammonium nitrate; DSPC, distearoylphosphatidylcholine; GPI, glycosylphosphatidylinositol; LBDD,
ligandꢀbased drug design; LPC, ligandꢀphospholipid conjugates; MD, molecular dynamics; PEG, polyethylene
glycol; PLDP, phospholipase D from Streptomyces sp; PL, phospholipid; SBDD, structureꢀbased drug design,
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