Received: 29 October 2021 Revised: 2 December 2021 Accepted: 3 December 2021

DOI: 10.1002/chir.23406

SPECIAL ISSUE ARTICLE

Chiral 2-phenyl-3-hydroxypropyl esters as PKC-alpha

modulators: HPLC enantioseparation, NMR absolute
configuration assignment, and molecular docking studies

Pasquale Linciano1 | Rita Nasti1,2 | Roberta Listro1 | Marialaura Amadio1 |

Alessia Pascale1 | Donatella Potenza3 | Francesca Vasile3 | Marco Minneci3 |
Jihyae Ann4 | Jeewoo Lee4 | Xiaoling Zhou5 | Gary A. Mitchell5 |
Peter M. Blumberg5 | Daniela Rossi1 | Simona Collina1

1
Department of Drug Sciences, University
of Pavia, Pavia, Italy
Abstract
2
Department of Environmental Science Protein kinase C (PKC) isoforms play a pivotal role in the regulation of numer-
and Policy, University of Milan, Milan, ous cellular functions, making them extensively studied and highly attractive
Italy
drug targets. In our previous work, we identified in racemate 1–2, based on the
3
Department of Chemistry, University of
Milan, Milan, Italy
2-benzyl-3-hydroxypropyl ester scaffold, two new potent and promising PKCα
4
Laboratory of Medicinal Chemistry,
and PKCδ ligands, targeting the C1 domain of these two kinases. Herein, we
College of Pharmacy, Seoul National report the resolution of the racemates by enantioselective semi-preparative
University, Seoul, South Korea HPLC. The attribution of the absolute configuration (AC) of homochirals
5
Laboratory of Cancer Biology and
1 was performed by NMR, via methoxy-α-trifluoromethyl-α-phenylacetic acid
Genetics, Center for Cancer Research,
National Cancer Institute, Bethesda, MD, derivatization (MTPA or Mosher's acid). Moreover, the match between the
USA experimental and predicted electronic circular dichroism (ECD) spectra con-

Correspondence
Daniela Rossi, Department of Drug
Sciences, University of Pavia, Via
Taramelli 12, 27100 Pavia, Italy.
Email: daniela.rossi@unipv.it
firmed the assigned AC. These results proved that Mosher's esters can be prop-
erly exploited for the determination of the AC also for chiral primary alcohols.
Lastly, homochiral 1 and 2 were assessed for binding affinity and functional
activity against PKCα. No significative differences in the Ki of the enantiopure
compounds was observed, thus suggesting that chirality does not seem to play
a significant role in targeting PKC C1 domain. These results are in accordance
with the molecular docking studies performed using a new homology model
for the human PKCαC1B domain.

1 | INTRODUCTION isoform structure and sensitivity to the activators.2 More

Protein kinases C (PKCs) are a family of serine/threonine
kinases and represent important regulators of cellular
functions.1 At present, 10 PKC isoforms have been identi-
fied in mammals. They are classified in conventional
(cPKCs; α, ßI, ßII, and γ), novel (nPKCs; δ, ε, η, and θ),
and atypical (aPKCs; ζ, ι/λ) subfamilies according to
Pasquale Linciano and Rita Nasti have equal contribution.
in detail, cPKCs are sensitive to Ca2+ and diacylglycerol
(DAG); nPKCs are activated solely by DAG, whereas
aPKCs are controlled by other endogenous ligands.3
Moreover, all the PKC isoforms share a high homology
degree in their primary structure (75%–80%) and possess
a conserved overall tertiary structure. The PKCs' single
strain polypeptide chain is composed of four highly con-
served domains: C1–C4.3 The N-terminal end hosts the
regulatory domains C1 and C2. The C1 domain of cPKC

Chirality. 2021;1–16. wileyonlinelibrary.com/journal/chir © 2021 Wiley Periodicals LLC. 1

2 LINCIANO ET AL.
and nPKC can be further divided in two subdomains,
namely, C1a and C1b, presenting the binding motif for
the endogenous activator DAG4 and for the exogenous
activators (i.e., phorbol 12-myristate 13-acetate).5,6 Con-
versely, the C-terminal constitutes the catalytic region of
the kinase and possesses two domains C3 and C4 hosting
the binding site for the ATP and the substrate,
respectively.
In general, the PKC family regulates several biological
functions by phosphorylating a wide variety of substrates.
Individual PKC isoforms are involved in different cellular
processes such as permeability, secretion, contraction,
migration, differentiation, proliferation, survival, apopto-
sis, and metabolism, with even opposing roles for selected
PKC isoforms in the same cell system.7 The complexity of
this picture is further increased because a specific PKC
isozyme function can vary among different cell types due
to the interaction of PKC isoforms with anchoring pro-
teins or other regulatory pathways in the cell.8
Aberrant functions of PKCs have been implicated and
extensively studied in several pathologies like cancer, dia-
betes4 and heart diseases.9 The role of PKC family in the
modulation of both proliferative and apoptotic signaling
clearly makes PKC as an attractive target in cancer, with
pharmacological modulators aimed to either dampen or
trigger PKC-mediated signaling given that the different
PKC isoforms can act as either oncogenes or onco-
suppressors. Mentioning just one recent evidence on a
widespread malignant tumor, PKC isoform α, ε, η, ι, ζ
upregulation has been reported in non-small cell lung
cancer, where PKC overexpression correlates with worse
prognosis in these patients.10
Further, various PKC isoforms are involved in brain
development and memory function,11,12 and PKC alter-
ations have been documented in diverse brain patholo-
gies, such as epilepsy, stroke, and neurodegenerative
diseases, including Alzheimer's disease (AD).13,14 In par-
ticular, PKCα activation mediates the physiological, non-
amyloidogenic, α-secretase-mediated processing of the
amyloid precursor protein (APP), a central element in
AD pathophysiology15; positive effects of PKCα activation
on memory have been reported in vivo in an AD model.16
In addition to APP, downstream PKCα targets include
proteins linked to synaptic remodeling and cognitive pro-
cesses17 and/or endowed with neurotrophic effects.
In vivo evidence suggests also atypical PKC, in particular
the PKC-λ/ι isoform, as novel potential AD target. In con-
trast with the positive effects of cPKC activators, Sajan
et al. reported that PKC-λ/ι favors β-secretase activity and
increases Aβ-peptide and phospho-tau levels in insulin-
resistant diabetic mice, which present a higher
amyloidogenic APP processing and that aPKC inhibitors
corrected a memory impairment in that animal model.18
Besides, several studies reported the involvement of
PKCα and PKCδ in the prevention and/or reparation of
tissue damages.19 In particular, PKCα promotes scarring
by improving cell–cell adhesion as demonstrated by sev-
eral in vivo studies.19–22
According to the literature, PKC isoforms show either
increased or decreased activity depending on the patho-
logical condition. Consequently, both PKC inhibitors and
activators may possess a strong therapeutic potential in
various pathological contexts.
In the past 30 years, the high therapeutic value of
PKC as druggable target for the development of modula-
tors for the treatment of the abovementioned pathologies
and related implications has been widely demonstrated.23
Research in this field has been firstly addressed to
develop ligands able to interact with the catalytic domain
of PKCs, and several classes of compounds have been
reported in literature through the years. However, the
complexity and redundancy of the PKC signaling net-
work demanded the development of PKC isoform-specific
inhibitors and activators; at the beginning, most inhibi-
tors were mainly designed to target the C3 and/or C4
domains, and therefore, they suffered for PKCs' selectiv-
ity issues and improper safety profile. For these reasons,
the research of more selective and safer PKC ligands
recently shifted in targeting the C1 domain of PKC.
Indeed, while the ATP binding site in the catalytic
domain is highly preserved throughout the human
kinome, targeting the regulatory C1 domain greatly
increases the selectivity for PKC over other kinases.
Indeed, besides PKCs, there are only six other protein
families, compared with more than 500 protein kinases
in the human genome, containing a DAG-responsive C1
domain. Throughout the years, several PKC activators
with higher affinity than the natural DAG were
described, and they represent a significant class of PKC
modulators. Example of natural, semisynthetic, and syn-
thetic C1 domain ligands are phorbol esters, bryostatins,
DAG lactones, isophthalates, and the 2-aryl-
3-hydroxypropyl pivalates discovered by our research
team (Figure 1).24–29 From a structural standpoint, most
ligands discovered so far contain one or more stereogenic
centers with a defined stereochemistry, thus suggesting a
potential role of chirality in the interaction with the
protein.
As regards compounds previously proposed by our
research team, namely, 2-phenyl-3-hydroxypropyl esters,
preliminary SAR considerations have been drawn, based
on their affinity toward PKCα and PKCδ (in Figure 2).
Compound 2-[4-(benzyloxy)phenyl]-3-hydroxypropyl
pivalate (rac-1) and the corresponding 2-phenylacetate
ester rac-2 (Figure 2) emerged as the most promising hit
compounds.23 Although they possess a chiral carbon, our
LINCIANO ET AL.
FIGURE 1 Chemical structures of the most representative natural/semisynthetic (A) and synthetic (B) PKC ligands
approach consisted in gaining preliminary SAR informa-
tion on racemates.23 Nevertheless, considering that most
of the PKCα ligands are homochiral and that the investi-
gation of the single enantiomers plays a key role in the
drug discovery not only for academia but also for phar-
maceutical industry,30 studying the role of chirality in the
binding with C1 domain of PKC α is essential for guiding
future investigation.
Because in this initial phase of the drug discovery
process small quantities of both enantiomers are
required for a first screening, in the present work, we
separated racemic 1 and 2 by enantioselective HPLC
using chiral stationary phases (CSPs) and assigned the
absolute configuration (AC) to the enantiomers. The
binding affinity (Ki) of homochiral 1 and 2 at PKCα was
3
assessed in vitro and rationalized by molecular model-
ling studies. Lastly, the functional activity of homochiral
and racemic 1 and 2 upon binding to PKCα was evalu-
ated in cells.
2 | MATERIALS AND METHODS
2.1 | Synthesis
Reagents and solvents were purchased from Sigma-
Aldrich and used as received unless otherwise specified.
The reactions sensitive to moisture or oxygen were per-
formed under nitrogen atmosphere, using glassware
dried in oven at 120
C for at least 1 h before utilization
4 LINCIANO ET AL.
F I G U R E 2 SAR study around the 2-phenyl and 2-benzyl-3-hydroxypropyl pivalate scaffold that led to the identification of compounds
rac-1 and rac-2 as dual ligands of PKCα and PKCδ
and anhydrous solvent. The progression of the reactions
was monitored by thin-layer chromatography (TLC) on
silica gel precoated glass-backed plates (Fluka Kieselgel
60 F254, Merck) and visualized with UV radiation or
with cerium ammonium molybdate or alkaline potas-
sium permanganate stains. Flash chromatography was
performed on Silica Gel 60 (particle size 230–400 mesh).
1
H and 13C nuclear magnetic resonance (NMR) spectra
were recorded on a Bruker Avance 400 MHz spectrome-
ter and Bruker Avance 600 MHz spectrometer at 25
C.
Proton chemical shifts (δ) are reported in ppm and
referred to the solvent residual peak (CDCl3,
δ = 7.26 ppm). Signals are abbreviated as s (singlet), d
(doublet), t (triplet), q (quartet), m (multiplet) and br
(broad). The coupling constant values (J) are reported in
Hz. 13C NMR spectra were recorded with complete pro-
ton decoupling. Carbon chemical shifts (δ) are reported
in ppm and referred to the solvent residual peak (CDCl3,
δ = 77.23 ppm). Compound purity was determined by
high-pressure liquid chromatography (HPLC) coupled
with UV-ESI/MS. The analyses were carried out on an
Acquity UPLC Waters LCQ FLEET system using an ESI
source operating in positive ion mode, controlled by
ACQUITY PDA and 4 MICRO (Waters). Analyses were
run on a Synergi Fusion-RP 80A (0.2 cm
diameter  5 cm length, 4 μm) column, at room tempera-
ture, with gradient elution (Solvent A: acetonitrile con-
taining 0.1% of formic acid; Solvent B: water containing
0.1% of formic acid; gradient: 10% A in B to 100% A in
4 min, followed by isocratic elution 100% A for 3 min,
and return to the initial conditions in 0.5 minutes) at a
flow rate of 0.3 ml/min. All the final compounds had 95%
or greater purity. Optical rotation was measured on a
Jasco photoelectric polarimeter DIP-1000 using a 0.5-dm
cell and a mercury lamp (λ = 405 nm).
2.2 | General procedure for the synthesis
of Mosher's esters 3–6
(R)- or (S)-Methoxy-α-trifluoromethyl-α-phenylacetic acid
(MTPA), at 0
C and under nitrogen atmosphere, was
directly solubilized in an excess of thionyl chloride and a
catalytical amount of DMF was added. The reaction was
stirred in the same conditions for 1 h. The excess of
thionyl chloride was removed under vacuum, and the
obtained (R)- or (S)-MTPAcyl chloride was directly used
in the esterification reaction. (R)- or (S)-MTPAcyl chlo-
ride was solubilized in anhydrous DCM and added
dropwise, at room temperature and under nitrogen atmo-
sphere, to a solution of the homochiral (+)-1 or ()-1
(25 mg, 1 equiv.) in the same solvent. DMAP supported
on resins (10% mol) was added, and the reaction mixture
was stirred in the same conditions for 72 h. Thereafter, it
was filtered on sintered glass filter and the precipitated
rinsed with DCM. The filtrate was concentrated in vacuo,
and the crude obtained was purified over silica gel using
DCM 100% as mobile phase.
2.3 | (S)-2-(4-(Benzyloxy)phenyl)-
3-(pivaloyloxy)propyl-(S)-3,3,3-trifluoro-
2-methoxy-2-phenylpropanoate (3)
36 mg of a light-yellow oil (90% yield). 1H NMR (CDCl3,
400 MHz) δ 7.54–7.42 (m, 4H), 7.45–7.36 (m, 6H), 7.11 (d,
LINCIANO ET AL.
J = 8.6 Hz, 2H), 6.90 (d, J = 8.6 Hz, 2H), 5.06 (s, 2H),
4.64 (dd, J = 11.0, 6.6 Hz, 1H), 4.51 (dd, J = 11.0, 5.8 Hz,
1H), 4.29 (dd, J = 11.1, 6.2 Hz, 1H), 4.21 (dd, J = 11.1,
7.3 Hz, 1H), 3.58 (s, 3H), 3.41–3.35 (m, 1H), 1.17 (s, 9H).
2.4 | (S)-2-(4-(Benzyloxy)phenyl)-
3-(pivaloyloxy)propyl-(R)-3,3,3-trifluoro-
2-methoxy-2-phenylpropanoate (4)
35 mg of a light-yellow oil (85% yield). 1H NMR (CDCl3,
400 MHz) 7.60–7.58 (m, 4H), 7.47–7.38 (m, 6H), 7.12 (d,
J = 8.6 Hz, 2H), 6.91 (d, J = 8.6 Hz, 1H), 5.07 (s, 2H),
4.66 (dd, J = 11.0, 7.3 Hz, 1H), 4.49 (dd, J = 11.0, 5.8 Hz,
1H), 4.30 (dd, J = 11.1, 6.2 Hz, 1H), 4.17 (dd, J = 11.0,
6.6 Hz, 1H), 3.57 (s, 3H), 3.41–3.34 (m, 1H), 1.16 (s, 9H).
2.5 | (R)-2-(4-(Benzyloxy)phenyl)-
3-(pivaloyloxy)propyl-(S)-3,3,3-trifluoro-
2-methoxy-2-phenylpropanoate (5)
39 mg of a light-yellow oil (95% yield). 1H NMR (CDCl3,
400 MHz) 7.55–7.48 (m, 2H), 7.39–7.33 (m, 4H), 7.33–
7.26 (m, 4H), 7.03 (d, J = 8.6 Hz, 2H), 6.82 (d, J = 8.6 Hz
2H), 4.98 (s, 2H), 4.57 (dd, J = 11.0, 6.6 Hz, 1H), 4.36 (dd,
J = 11.0, 5.8 Hz, 1H), 4.21 (dd, J = 11.1, 6.2 Hz, 1H), 4.08
(dd, J = 11.1, 7.3 Hz, 1H), 3.48 (s, 3H), 3.32–3.25 (m, 1H),
1.08 (s, 9H).
2.6 | (R)-2-(4-(Benzyloxy)phenyl)-
3-(pivaloyloxy)propyl-(R)-3,3,3-trifluoro-
2-methoxy-2-phenylpropanoate (6)
34 mg of a light-yellow oil (83% yield). 1H NMR (CDCl3,
400 MHz) δ 7.49 (s, 2H), 7.40–7.36 (m, 4H), 7.27 (ddd,
J = 10.3, 8.2, 4.7 Hz, 5H), 7.03–7.01 (d, J = 8.6 Hz, 2H),
6.81 (d, J = 8.6 Hz, 1H), 4.97 (s, 2H), 4.55 (dd, J = 11.0,
7.3 Hz, 1H), 4.42 (dd, J = 11.0, 5.8 Hz, 1H), 4.18 (dd,
J = 11.1, 6.2 Hz, 1H), 4.12 (td, J = 11.0, 6.6 Hz, 1H), 3.47
(s, 3H), 3.31–3.25 (m, 1H), 1.07 (s, 9H).
2.7 | Analytical and semipreparative
enantioselective HPLC
Enantioselective HPLC runs were conducted on a Jasco
HPLC system equipped a PU-4180 plus quaternary gradi-
ent pump and an MD-2010 plus multiwavelength detec-
tor. Experimental data were acquired and processed by
Jasco ChromoNav Software. Solvent used were HPLC
grade and supplied by Carlo Erba or VWR.
5
2.8 | Experimental electronic circular
dichroism
Experimental electronic circular dichroism (ECD) spectra
were acquired on a Jasco J1500 spectropolarimeter, at
room temperature, using a 1-cm cylindrical quartz
cuvette and in the range 240–300 nm. The compounds
were solubilized in acetonitrile at approximately 0.01 M,
and for each measurement, 10 scans were performed and
averaged. ECD spectra of the solvent under the same
experimental conditions were subtracted. The molar
ellipticity was calculated by correcting the OD for the
molar concentration of the compound.
2.9 | Predicted electronic circular
dichroism
MacroModel and Jaguar subroutine implemented in
Schrodinger Suite31 were used for conformational search
and ECD spectrum prediction, respectively. Conforma-
tional analyses of (R)- and (S)-1 and 2 were performed
first with Merck molecular force field (MMFF), in a
mixed torsional/low-mode sampling mode. 10 k maxi-
mum number of steps per molecule and 100 steps per
rotatable bond were set. Redundant conformers within a
maximum atom deviation cut-off of 0.5 Å were elimi-
nated. All the conformers found within 10 kcal/mol in
energy compared with the most stable ones were further
optimized at DFT level. Only the optimized conformers,
which differed from the most stable ones within 2 kcal/
mol in relative free energy, were considered for ECD
spectrum calculation. ECD spectrum simulation by time-
dependent density functional theory (TD-DFT) was car-
ried out using the B3LYP-D3 exchange correlation func-
tional at the 6-31G** basis set level. The Tamm–Dancoff
approximation for the singlet excited state was used, and
10 number of excited states were considered. The polariz-
able continuum model (PCM) was used to consider the
solvent effects of acetonitrile. Velocity representations
were used to simulate the ECD spectra. The calculated
ECD spectra were the result of a weighted average of the
calculated ECD spectra of each conformer based on
Boltzmann distribution.32
2.10 | 3D homology modeling
The primary sequences for PKCαC1B and PKCδC1B were
downloaded in FASTA format from UniProt (ID: P17252
and ID: Q05655, respectively), and they were aligned by
multiple sequence alignment. The two C1B domains pos-
sess a 62% identity. The available crystallographic
6 LINCIANO ET AL.
structure of PKCδC1B (PDB ID: 1PTR) was used as suit-
able template for homology modeling. The 3D model of
PKCαC1B was built using Prime in Schrodinger Suite.
The cognate ligand P13A present in the crystallographic
structure of PKCδC1B was retained in the homology
model. The modeled 3D structure of PKCαC1B was
further optimized using molecular dynamics
(MD) simulations. Because the C1B domain of PKCs is
associated with the plasmatic membrane, the modeled
PKCαC1B was further refined by partially inserting the
protein within a phospholipidic bilayer. The orientation
of PKCδC1B in the membrane available in the Orienta-
tions of Proteins in Membranes (OPM) Database was
used as template. The modeled PKCαC1B and the mem-
brane were solvated in a SPC water orthorhombic box
40  10  10 Å in size. System charge was neutralized
using Na+ ions, and 0.15 M NaCl was added. OPLS3
force field was used to model the system. The MD system
was relaxed using the default Desmond protocol.33,34 The
10 ns MD simulations were then carried out with the
equilibrated systems at a temperature of 300 K and at a
constant pressure of 1 atm, under the NPT (normal pres-
sure and normal temperature) ensemble with a time step
of 2 fs. To check the convergence of MD simulations, we
investigated the protein Cα and ligand root-mean-square
deviation (RMSD) plots for each trajectory. Clearly, the
relatively flat plots within last 4 ns indicate that the com-
plex systems have reached a steady state. The Desmond
Simulation Interaction Diagram35 was used to analyze
the interaction between the protein and the ligand. Des-
mond trajectory clustering tool was used to group similar
structures from the last 4 ns of the MD simulation.36 The
representative structure of the most abundant cluster was
used for docking calculation.
2.11 | Docking calculation
The prepared homology model of PKCαC1B was used for
docking calculation. It was prepared by using the Protein
Preparation Wizard utility of the Schrödinger Suite.31
The receptor grid was generated around the residues lin-
ing the binding site of P13A. The Glide software was used
as an engine for the docking calculations, which were
performed with default settings of the Standard Precision
(SP) protocol.37 The docking protocol was validated by
redocking the P13A into its parent receptor, providing an
RMSD value lower than 1.2 Å. The new compounds
investigated were first designed in ChemDraw, imported
into the Maestro software,38 and prepared using the
LigPrep utility available within the Schrodinger Suite.39
All potential states of ionization and tautomerism at a
physiological pH of 7.4 ± 0.5 were generated. The
prepared compounds were subjected to 2000 minimiza-
tion steps with MacroModel using the OPLS3e force field.
The ligands thus prepared were finally docked to the pro-
tein using the default settings of the SP protocol.37 The
docking poses with the best score were further subjected
to an additional InducedFit docking setting the side
chains of the amino acid within a 5 Å radius from the
ligand flexible. The resulting ligand–protein complexes
were visually inspected.
2.12 | Production of recombinant hPKCα
and binding assay
The expression and production of recombinant hPKCα
and the affinity of the homochiral compounds 1 and 2 to
the C1 domains of PKCα were performed using the proto-
col previously reported.23,27,40,41
2.13 | Cell culture and treatments
Human neuroblastoma SH-SY5Y cells (CRL-2266, ATCC,
Manassas, VA, USA) were cultured in Eagle's minimum
essential medium (MEM) supplemented with 10% fetal
calf serum (FCS), 100 units/ml penicillin, 100 μg/ml
streptomycin, 1% non-essential amino acids, 1 mM
sodium pyruvate. Cells were maintained at 37
C in an
atmosphere of 5% CO2 and 95% air in 100% relative
humidity and grown to 80% confluence in Petri dishes.
For preliminary studies, cells were exposed to vehicle
(dimethyl sulfoxide [DMSO]) or increasing concentra-
tions (from 1 to 300 μM) of 1 and 2 racemates for differ-
ent times (from 15 to 120 min) to find the best conditions
for PKCα activation. In the experiments, the DAG ana-
logue phorbol-12-myristate-13-acetate (PMA, 100 nM for
15 min) was used as a positive control of PKCα activation
(not shown). For further studies, cells were exposed to
vehicle, 25 μM 1 (racemate or enantiomers, for 15 min)
or 1 μM 2 (racemate or enantiomers, for 60 min). All
drug treatments were carried out in cells falling within
23–28 passages in normal cell culture medium and stan-
dard conditions.
2.14 | Sample preparation, SDS-PAGE,
and Western blotting analysis
After treatments, the cells were washed twice with ice-
cold phosphate-buffered saline, harvested, and homoge-
nized in ice-cold lysis buffer (20 mM Tris (pH 7.4), 2 mM
EDTA, 0.5 mM EGTA, 50 mM mercaptoethanol,
0.32 mM sucrose, protease and phosphatase inhibitors
LINCIANO ET AL.
according to manufacturer's instructions) by using a Tef-
lon/glass homogenizer. Total cell homogenates were then
subjected to fractioning by ultracentrifugation to obtain
cytosol, membranes and cytoskeleton accordingly to a
published method.17 Protein concentrations in cellular
fractions were determined using Bradford's method. Sam-
ples for sodium dodecyl sulfate polyacrylamide gel elec-
trophoresis (SDS-PAGE) and Western blotting were
prepared by diluting the homogenates from each fraction
in SDS loading solution, boiled for 5 min, and separated
on 12% SDS-PAGE, subsequently transferred to nitrocel-
lulose membranes, and processed following standard pro-
cedures. Briefly, membranes were washed in 0.1% Tween
20 in Tris-buffered saline (TTBS), blocked for 1 h at room
temperature (RT) with 5% nonfat milk powder in TTBS
(milk–TTBS), and incubated overnight at 4
C with the
primary antibody in milk–TTBS. The primary antibody
dilutions were 1:500 for anti-PKCα and 1:1000 for anti-
α-tubulin mouse antibodies, respectively. Membranes
were then washed and incubated at RT for 1 h with a
horseradish peroxidase-conjugated secondary antibody
diluted at 1:3000 in milk–TTBS. After washing with
TTBS, the immunoreactive bands were visualized with
enhanced chemiluminescence. The α-tubulin was used to
normalize the data. Statistical analysis of Western blot-
ting data was performed on the densitometric values
obtained using the NIH Image software. The data were
subjected to one-way analysis of variance (ANOVA),
followed by Dunnett's multiple comparisons test. Differ-
ences were considered statistically significant when p-
values were ≤0.05.
3 | R ES U L T S A N D D I S C U S S I O N
Compounds 1 and 2 have been synthesized as racemate
according to the procedure previously reported.18 To
obtain homochiral 1 and 2 in suitable amount (approxi-
mately 50–60 mg of each homochiral compound) for
chemical and chiroptical characterization, configura-
tional assignment, and biological evaluation, an
enantioselective HPLC approach was applied.
A screening for resolving the synthesized compounds
1 and 2 was performed using chiral selectors based on
polysaccharides, consisting of amylose- and cellulose-
derived CSPs. Basing on our previous experience,42,43
Chiralpack® IA (amylose-based CSP, 0.46 cm dia-
meter  25 cm length, 5 μm) and Chiralpak® IC (cellu-
lose-based CSP, 0.46 cm diameter  25 cm length, 5 μm)
columns were used, and HPLC screening was performed
under two possible chiral separation modes, namely, nor-
mal and polar organic phase. The solvents used for the
screening have been selected from an environmental
7
perspective using n-heptane, greener than the commonly
used n-hexane, and alcohols.44 Accordingly, the mobile
phases used were a mixture of n-heptane and 2-propanol
(IPA) or ethanol, or pure alcohols (methanol or ethanol).
Several types of mobile phase compositions were investi-
gated by changing percentage of the mixture compo-
nents. The main results of the screening protocol
obtained for each compound, expressed in term of reten-
tion factor (K1 and K2), separation factor (α), and resolu-
tion factor (Rs), are reported in Table SI-1.
To transfer the analytical methods to semipreparative
scale, chromatographic conditions characterized by both
good enantioresolution and short retention times have
been considered, resulting in: n-heptane/ethanol 90/10
(v/v) for both rac-1 (tr1 = 15.1 min, tr2 = 18.3 min,
α = 1.27, Rs = 3.34) and rac-2 (tr1 = 14.8 min,
tr2 = 16.6 min, α = 1.15, Rs = 2.64) on Chiralpack® IA
and Chiralpack® IC, respectively. The analytical chro-
matographic UV traces (λ = 254 nm) for racemate 1 and
2 are reported (black line) in Figure 3.
According to the preliminary screening results
described, suitable separation of rac-1 was obtained on
the Chiralpack® IA column (1  25 cm, 5 μm) with the
n-heptane/ethanol mixture (90/10, v/v) at flow rate
4 ml/min. 150 mg of rac-1 (10 mg/ml, injection volume
1 ml) were processed in 15 cycles, properly fractionating
the eluate according to the cut-points reported in
Figure SI-1A. The collected fractions were analyzed for
optical rotations and enantiomeric purity. In this way,
62 mg of the first eluted enantiomer {[α]20405 = +37.7

(c = 0.5 in methanol) and e.e. = 99.9%} and 59 mg of the
second eluted one {[α]20405 = 35.2
(c = 0.5 in metha-
nol) and e.e. = 95.1%} were successfully isolated
(Figure 3A), together with an intermediate fraction of
21 mg as a mixture of the two enantiomers. Applying the
same elution conditions, 180 mg of rac-2 (10 mg/ml,
injection volume 1 ml) were processed in 18 cycles on the
Chiralpack® IC column (1  25 cm, 5 μm) and fraction-
ated as stated in Figure SI-1B, affording 76 mg of the first
eluted enantiomer {[α]20405 = +37.5
(c = 0.4 in metha-
nol) and e.e. = 98.8%}, 66 mg of the second eluted one
{[α]20405 = 37.4
(c = 0.4 in methanol) and e.e.
= 98.3%} (Figure 3B) and 35 mg of an almost racemic
fraction. To sum up, the semipreparative enantioselective
HPLC protocol applied afforded homochiral 1 and 2 with
e.e. higher than 98% and in amount suitable for the next
steps, including configurational study and biological
investigations.
The next step of the work consisted in AC assign-
ment, because it is pivotal to get a better understanding
of the role of chirality on the interaction with the biologi-
cal target. Among the different possible approaches,
NMR technique was herein exploited. It was considered a
8 LINCIANO ET AL.
F I G U R E 3 (A) Analytical separation of rac-1 on Chiralpak® IA, eluent: n-heptane/IPA 90:10, flow rate 1 ml/min. (B) Analytical
separation of rac-2 on Chiralpak® IC, eluent: n-heptane/EtOH 90:10, flow rate 1 ml/min
suitable strategy for our purposes, because compounds
under investigations are characterized by the presence of
a primary alcohol function, easily to derivatize with chi-
ral derivatizing agents (CDA) such as (R)- and (S)-met-
hoxy-α-trifluoromethyl-α-phenylacetic acid (MTPA, also
known as Mosher's reagent).45,46 Only few examples deal-
ing with the use of MPTA for the configurational study of
primary alcohols have been reported.47–51 Homochiral
(+)-1 and ()-1 were therefore esterified with (R)- and
(S)-MTPA, thus obtaining the corresponding diastereo-
meric derivatives 3–4 [starting from (+)-1] and 5–6
[starting from ()-1]. Briefly, the appropriate MTPA was
converted in the corresponding chloride by reaction with
an excess of thionyl chloride using DMF as catalyst. The
prepared MTPAcyl chloride was not isolated, and it was
directly reacted with the homochiral (+)-1 and ()-1.
The esterification was easily carried out in dic-
hloromethane at room temperature for 72 h using
4-dimethylaminopyridine (DMAP) as catalyst. DMAP
was supported on polymer (PS-DMAP) to simplify the
reaction workup procedures (Scheme 1). It is worth to
point that after esterification with Mosher's acid, due to
the change in the priority of the substituents around the
chiral center of the esters 3–6, a change in the absolute
configuration with the respect of the parent alcohols
1 and 2 is observed. Thus, (R)- absolute configuration at
C3 in compounds 1 and 2 will become (S)- in the
corresponding Mosher's ester and vice versa.
The 1H NMR spectra of the synthesized esters were
acquired in CDCl3, and the ΔδSR between the protons of
the diastereoisomers for each couple 3–4 and 5–6 were
calculated (Table 1).
The comparison of the NMR spectra of the two dia-
steroisomers allows the evaluation of the shielding/
deshielding effect due to the presence of the CDA groups
on the chemical shift values of L1 and L2 substituents of
the unknown stereocenter. The chemical shift variation
contains the necessary information for configurational
assignment because they indicate the relative position.
However, because we are dealing with primary alcohols,
two main issues should be taken into account in inter-
preting these results: (i) The distance between groups
L1/L2 of the substrate and the aryl ring of the auxiliary
reagent is greater than that in secondary alcohols,
because the asymmetric center is at the β-position, and
(ii) the presence of an extra C–C bond between the chiral
center and the auxiliary reagent reduces the conforma-
tional preference by increasing the rotational freedom.
As a result, the ΔδSR values for primary alcohols would
be expected to be smaller and less significant than those
usually obtained for secondary alcohols. For the final
determination of the absolute configurations, the confor-
mation of (R)- and (S)-MTPA esters reported in literature
is used,46 in which C in α position, the carbonyl, and the
CF3 groups are situated in the same plane (Figure 4) of
the two substituents with respect to the anisotropic group
(in our case, the benzyl group).
The analysis of the NMR spectra of the diastereomeric
couple of the MTPA esters of homochiral (+)-1 com-
pound (3 and 4) revealed a quite small but significant dif-
ference in the chemical shifts. Focusing on the ΔδSR
difference in chemical shift, the protons of
4-benzyloxyphenyl group resulted uniformly shifted
toward higher chemical shift. In details, the aromatic
protons H5–H50 close to the chiral center of 3 are slightly
shielded (δ 7.11 ppm) compared with the corresponding
protons of 4 (δ 7.12 ppm), with a calculated ΔδSR of
0.01 ppm. Similarly, the same behavior resulted for the
aromatic protons H6–H60 (δ 6.89 and δ 6.91 ppm for
3 and 4, respectively) with a calculated ΔδSR of
0.02 ppm, and for the methylene protons H8 (δ 5.06
and δ 5.07 ppm for 3 and 4, respectively) with a
LINCIANO ET AL. 9

S C H E M E 1 Reagents and
conditions: (a) (R)- or (S)-MTPA, SOCl2
(excess), DMF (cat.), 0
C to r.t, 1 h;
(b) (R)- or (S)-MTPAcyl chloride
(1 equiv.), PS-DMAP (cat.), dry
dichloromethane, N2, 72 h, r.t

T A B L E 1 Differences in chemical shifts observed in the 1H NMR spectra of the diastereoisomeric couples 3–4 and 5–6 at room
temperature

3 (δS) 4 (δR) 5 (δS) 6 (δR)

Protons (+)-1-(S)-MTPA (+)-1-(R)-MTPA Δδ SR
(3–4) ()-1-(S)-MTPA ()-1-(R)-MTPA ΔδSR (5–6)
H5–H50 /H6–H60 7.11/6.89 7.12/6.91 0.01/0.02 7.03/6.82 7.02/6.81 0.01/0.01
H 8
5.06 5.07 0.01 4.98 4.97 +0.01
H1/H10 4.31/4.21 4.30/4.17 +0.01/+0.04 4.21/4.08 4.22/4.12 0.01/0.04
CH3 1.17 1.16 +0.01 1.07 1.08 0.01

calculated ΔδSR of 0.01 ppm (Table 1). So, this group
should correspond to the L1 substituent at the chiral cen-
ter. Accordingly, the protons corresponding to L2 substit-
uent can be considered the methylene pivalate, because it
has the opposite behavior with a ΔδSR > 0. The dia-
stereotopic protons H1/H10 are slightly deshielded (δ 4.31
and 4.21 ppm) compared with the corresponding protons
of 4 (δ 4.30 and 4.17 ppm), with a calculated ΔδSR of
+0.01 and +0.04 ppm. The same positive difference
(+0.01 ppm) was observed for the protons of the t-butyl
group between diastereomer 3 and 4 (Table 1). So, based
on the observed ΔδSR, the (R) AC was assigned to com-
pound (+)-1 (Figure 5).
As expected, for 5 and 6, an opposite behavior with
respect to the diastereoisomeric couple 3 and 4 (Table 1)
has been observed (Figure SI-4).
10 LINCIANO ET AL.

F I G U R E 4 (A,B) The model used to determine the AC of analysed compounds. (C) The substituent with positive difference in proton
chemical shifts between (S)- and (R)- MTPA derivatives was placed on the right side of MTPA plane, while the substituent with negative
difference on the left side

F I G U R E 5 Attribution of the AC to
(+)-1 according to the model proposed
in Figure 4

To confirm the AC, the experimental ECD registered
for homochiral (+)-1 and ()-1 were registered and
compared with the calculated ECD spectra for com-
pounds (R)-1 and (S)-1. As reported in Figure 6A, the
ECD spectra of (+)-1 in acetonitrile showed a profile and
a Cotton's effect comparable with the predicted ECD
spectra for (R)-1, thus confirming as the AC assigned by
NMR. Accordingly, the ECD spectra of ()-1 showed a
comparable profile to the predicted ECD spectra for
(S)-1.
Therefore, combining NMR and ECD studies, the
absolute configuration (R) was undoubtedly assigned to
(+)-1 and (S) to ()-1.
To assign the absolute configuration to (+)-2 and
()-2 compound, (R)-1 and (S)-1 were used as reference
compounds. Experimental ECD spectra of (+)-2 and
LINCIANO ET AL.
FIGURE 6 ECD spectra for homochiral compounds 1 (A) and 2 (B) and superimposition with the corresponding predicted ones
()-2 acquired in acetonitrile showed profile and Cot-
ton's effect comparable with those of (+)-(R)-1 and
()-(S)-1 (Figure 6B). Moreover, ECD spectra predicted
in the same solvent match well with experimental ECD
spectra. Taken together, these results allowed to assign
the (R)- and (S)-configuration to (+)-2 and ()-2,
respectively.
The binding affinity of rac-1, (R)-1, (S)-1, rac-2, (R)-
2 and (S)-2 with PKCα was determined by assessing the
capability of the ligands to compete for binding of
[20-3H] phorbol-12,13-dibutylate (PDBU) to recombinant
PKC.52 Compound (R)-1 resulted about two-fold more
affine than the corresponding enantiomer (S)-1 with a Ki
of 0.98 ± 0.04 and 2.08 ± 0.02 μM, respectively, whereas
no significant differences in the binding affinity were
observed between (R)-2 and (S)-2 (Ki of 2.37 ± 0.22
and Ki of 1.51 ± 0.16 μM, respectively). Unexpectedly,
compounds herein studied showed a slight or null
enantiopreference toward the target protein.
11
To justify and explain the binding results from a
molecular point of view, the docking studies were per-
formed. At present, no crystallographic structure for the
human PKCαC1B domain is available; therefore, the 3D
structure of the target was built by homology modeling.
The PKCδC1B domain was used as template because its
x-ray crystallographic structure is resolved (PDB ID:
1PTR; resolution = 1.9 Å),53 and it shares with
PKCαC1B a 62% identity in primary sequence
(Figure 7A). Homology model for PKCαC1B was gener-
ated with Prime. The backbone alignment of the
modeled PKCαC1B with the template PKCδC1B showed
a high degree of homology with a RMSD = 0.765 Å
(Figure 7B). Furthermore, the predicted model has
secondary structure (β-sheet and loops) similar to the
template. The cognate ligand P13A present in crystallo-
graphic structure of PKCδC1B was retained in the
homology model (Figure 7C). Before proceeding to
docking calculation, the PKCαC1B model was refine
12 LINCIANO ET AL.

F I G U R E 7 Homology model for PKCαC1B. (A) Alignment of the primary amino acid sequences of PKCα (UNIPROT: P17252) and
PKCδ (PDB ID: 1PTR) C1B domain. (B) Superimposition of the crystallographic structure of PKCδC1B (PDB ID: 1PTR, in green line and
cartoon) and the 3D structure of PKCαC1B (in blue lines and cartoon) predicted by homology modeling and after relaxation through 10 ns
MD. (C) Focus on the binding mode of P13A at the PKCαC1B binding site. The protein is represented in blue cartoon; important interacting
residues are in line representation with blue carbons, and P13A is stick representation in blue carbons. Model atoms except for carbons are
color-coded: oxygen (red) and nitrogen (blue). H-bonds are represented as yellow dotted lines

and subjected to 10-ns MD simulation using Des-
mond.33,34 To mimic the real physiological environ-
ment, the PKCαC1B domain was partially inserted into
a phospholipidic membrane using the orientation of
PKCδC1B in the membrane available in the OPM
Database as template (Figure SI-2). The stability of the
PKCαC1B model after MD was measured by its devia-
tion from the initial structure in terms of RMSD. The
RMSD values of the backbone atoms and ligand atoms
in the entire MD simulation trajectory are reported in
the Supporting Information. The model PKCαC1B
structure reached a stable state after 5 ns where the
RMSD of the protein backbone atoms and of the ligand
atoms converged to 1.2–1.5 and 0.5–0.9 Å, respectively.
The root-mean-square fluctuations (RMSF) of PKCαC1B
generated during the MD simulation were calculated to
characterize the mobility of individual residues
(Figure SI-3). The Cα and side chains of all the residues
showed low RMSF in the range 0.4–1.2 and 0.7–1.5 Å,
respectively. Only the residues from 147 to
151, corresponding to the truncate C-terminal loop, had
high peaks in the RMSF plot indicating large fluctua-
tions of those residues. The Ramachandran plot
(Figure SI-2) of the optimized PKCαC1B structure
showed that all the residues were placed in allowed
regions, with the 92% of them located in the favored
zone. This indicates that the optimized PKCαC1B struc-
ture should be stable and reliable for use in elucidating
of PKCαC1B-ligand binding modes through molecular
docking. The MD calculation were performed in dupli-
cate, and the results converged.
Homochiral compounds 1 and 2 were docked against
PKCαC1B. (S)-1/(R)-1 and (S)-2/(R)-2 enantiomers
were analyzed, and the predicted binding mode is
reported in Figure 8. According to our calculations, both
the couple of enantiomers feature the same overall posi-
tioning within the PKCαC1B binding site and establish
the same key interactions observed in the binding mode
of the cognate ligand P13A. In particular, the primary
alcoholic function is always positioned within a protein
LINCIANO ET AL. 13

cavity and establish a network of H-bond with the back-
bone of Thr113 and Leu121. Conversely, the two main
hydrophobic moieties of the ligands, namely, the ben-
zyloxyphenyl group and the t-butyl (in compound 1) or
the benzyl esters (in compound 2) are located over the
hydrophobic surface of the protein edged by Tyr123,
Pro112, and Thr108, whereas the carbonyl ester accepts
an additional H-bond from Gly124.
The two enantiomers of both 1 and 2 establish the
same kind of interactions with the key amino acidic resi-
dues of the C1 domain and assume a comparable overall
active conformation on the surface of the binding pocket.
No significant differences in the binding mode of the
enantiomeric couple of both compounds 1 and 2
were observed, in accordance with the absence of
enantiopreference experimentally observed.
Lastly, the effect induced by homochiral and racemic
1 and 2 upon binding to PKCα was evaluated in SH-SY5Y
cells. The translocation of the protein from cytosol
(soluble fraction) to other cellular compartments
(membrane, cytoskeleton), as an index of the PKCα
activation, was measured. SH-SY5Y cells were initially
treated with increasing concentrations (1–300 μM) of
racemic 1 or 2, and the translocation of PKCα was
quantified by Western blotting analysis at different times
(15–120 min). As reported in Figure 9, the exposure of

F I G U R E 8 Predicted binding poses for compound (R)-1 and (S)-1 (A, in pale cyan and deep teal stick carbons, respectively) and (R)-2
and (S)-2 (B, in pink and deep purple stick carbons, respectively) within the binding site of PKCαC1B built by homology modeling. The
main amino acid residues of the pocket involved in the binding with the ligands are reported in blue line. The heteroatoms are color-coded:
oxygen in red and nitrogen in blue. The H-bonding interactions are represented in yellow dotted lines

F I G U R E 9 Effects of racemic and

enantiomeric 1 on PKCα translocation.
Densitometric analysis of PKCα/α-
tubulin in the soluble (A) and
cytoskeletal (B) fractions of human SH-
SY5Y cells after exposure to vehicle
(control; Ctrl) and racemic and
homochiral 1 at 1 μM for 60 min.
Results are expressed as mean
percentages ± SEM. with respect to
control (100%). *p < 0.05, **p < 0.01,
Dunnett's multiple comparisons test,
n = 3–6
14
SH-SY5Y cells to rac-1 at 1 μM for 60 min was able to
trigger PKCα translocation as testified by the reduction of
PKCα level in the soluble fraction and the corresponding
increment of the protein level in the cytoskeletal fraction,
both in a statistically significant manner. Based on these
results, homochirals 1 were investigated in the same
conditions. (R)-1 was able to produce the same effect of
the corresponding racemate in SH-SY5Y cells, whereas
(S)-1 has a not significant effect. These results are in
accordance with the binding affinities, being (R)-1 the
most affine enantiomer. Conversely, for compounds
rac-2, (R)-2, and (S)-2, no effect on translocation was
observed up to 25 μM (data not shown).
4 | C ON C L U S I ON
In conclusion, we shed light on the role of chirality of
the previously identified 2-aryl-3-hydroxypropyl esters
1–2 in the binding at the C1 domain of PKCα and the
consequent effect induced on the protein. The key
steps of the work consisted in the preparation of
homochiral 1 and 2, in the AC assignment and in the
study of the role of chirality in the interaction with C1
domain of PKCα.
Racemic 1 and 2 have been resolved via
enantioselective semipreparative HPLC starting from the
corresponding racemic mixture. The previously reported
enantioselective HPLC screening was revised and
extended by considering amylose- and cellulose-derived
CSP column and solvents with a lower environmental
impact. The developed method used ecofriendly solvents
as eluent and was suitable for obtaining a quick access to
enantiomeric 1 and 2 with high enantiomeric excess and
amounts sufficient for biological assays. The AC was
assigned to (+)- and ()-1 combining NMR experiments
with chiroptical studies. The derivatization of the primary
alcoholic function of 1 into the corresponding diastereo-
meric ester of Mosher's acid and the interpretation of the
change in the proton chemical shifts allowed to assign the
(R)-configuration to (+)-1 and the (S)-configuration to
()-1. Further confirmation of the AC derived from the
match between the experimental and predicted ECD
spectra. These results proved that MTPA esters are a
suitable strategy for the determination of the AC of
chiral primary alcohols. Using (R)-1 and (S)-1 as reference
compounds, the AC was then assigned to (+)-2 and
()-2.
Regarding the binding affinity to PKCα C1 domain,
no significative differences in the Ki of the enantiopure
compounds were observed, thus suggesting that chirality
does play a relevant role in protein affinity. Furthermore,
to get more insight on the interaction between
LINCIANO ET AL.
enantiomeric 1 and 2 and PKCα, a new homology model
for the human PKCαC1B domain was postulated.
Molecular modeling studies contributed to rationalize the
experimental binding data.
ACKNOWLEDGMENT
PL gratefully acknowledge the Department of Drug
Science of the University of Pavia for Fondo Ricerca
Giovani.
DA TA AVAI LA BI LI TY S T ATE ME NT
no data are available
ORCID
Daniela Rossi https://orcid.org/0000-0002-2458-3728
RE FER EN CES
1. Reyland ME. Protein Kinase C Isoforms: Multi-Functional
Regulators of Cell Life and Death. Front Biosci (Landmark Ed).
2009;14:2386-2399. https://doi.org/10.2741/3385
2. Talman V, Pascale A, Jäntti M, Amadio M, Tuominen RK.
Protein Kinase C Activation as a Potential Therapeutic Strategy
in Alzheimer's Disease: Is There a Role for Embryonic Lethal
Abnormal Vision-like Proteins? Basic Clin Pharmacol Toxicol.
2016;119(2):149-160. https://doi.org/10.1111/bcpt.12581
3. Steinberg SF. Structural Basis of Protein Kinase C Isoform
Function. Physiol Rev. 2008;88(4):1341-1378. https://doi.org/10.
1111/bcpt.12581
4. Kolczynska K, Loza-Valdes A, Hawro I, Sumara G.
Diacylglycerol-Evoked Activation of PKC and PKD Isoforms in
Regulation of Glucose and Lipid Metabolism: A Review. Lipids
Health Dis. 2020;19(1):113 https://doi.org/10.1186/s12944-020-
01286-8
5. Hurley JH, Newton AC, Parker PJ, Blumberg PM, Nishizuka Y.
Taxonomy and Function of C1 Protein Kinase C Homology
Domains. Protein Sci. 1997;6(2):477-480. https://doi.org/10.
1002/pro.5560060228
6. Tahara E, Kadara H, Lacroix L, Lotan D, Lotan R. Activation
of Protein Kinase C by Phorbol 12-Myristate 13-Acetate Sup-
presses the Growth of Lung Cancer Cells through KLF6 Induc-
tion. Cancer Biol Ther. 2009;8(9):801-807. https://doi.org/10.
4161/cbt.8.9.8186
7. Dempsey EC, Newton AC, Mochly-Rosen D, et al.
Protein Kinase C Isozymes and the Regulation of Diverse Cell
Responses. Am J Physiol Cell Mol Physiol. 2000;279(3):L429-
L438. https://doi.org/10.1152/ajplung.2000.279.3.L429
8. Battaini F, Pascale A, Paoletti R, Govoni S, Battaini F. The Role
of Anchoring Protein Rack1 in Pkc Activation in the Ageing
Rat Brain. Trends Neurosci. 1997;20(9):410-415. https://doi.org/
10.1016/S0166-2236(97)01084-9
9. Singh RM, Cummings E, Pantos C, Singh J. Protein Kinase C
and Cardiac Dysfunction: A Review. Heart Fail Rev. 2017;22(6):
843-859. https://doi.org/10.1007/s10741-017-9634-3
10. Sadeghi MM, Salama MF, Hannun YA. Protein Kinase C as a
Therapeutic Target in Non-Small Cell Lung Cancer. Interna-
tional Journal of Molecular Sciences. 2021;22:(11):5527-5540.
https://doi.org/10.3390/ijms22115527
LINCIANO ET AL.
11. Sun M-K, Alkon DL. The “Memory Kinases”: Roles of PKC
Isoforms in Signal Processing and Memory Formation. Prog
Mol Biol Transl Sci. 2014;122:31-59. https://doi.org/10.1016/
B978-0-12-420170-5.00002-7
12. Lucchi L, Pascale A, Battaini F, Govoni S, Trabucchi M. Cogni-
tion Stimulating Drugs Modulate Protein Kinase C Activity in
Cerebral Cortex and Hippocampus of Adult Rats. Life Sci. 1993;
53(24):1821-1832. https://doi.org/10.1016/0024-3205(93)
90490-T
13. Lucke-Wold BP, Turner RC, Logsdon AF, et al. Common
Mechanisms of Alzheimer's Disease and Ischemic Stroke: The
Role of Protein Kinase C in the Progression of Age-Related
Neurodegeneration. J Alzheimers Dis. 2014;43(3):711-724.
https://doi.org/10.3233/JAD-141422
14. Battaini F, Pascale A. Protein Kinase C Signal Transduction
Regulation in Physiological and Pathological Aging. Ann N Y
Acad Sci. 2005;1057(1):177-192. https://doi.org/10.1196/annals.
1356.011
15. Racchi M, Mazzucchelli M, Pascale A, Sironi M, Govoni S. Role
of Protein Kinase Cα in the Regulated Secretion of the Amyloid
Precursor Protein. Mol Psychiatry. 2003;8(2):209-216. https://
doi.org/10.1038/sj.mp.4001204
16. Etcheberrigaray R, Tan M, Dewachter I, et al. Therapeutic
Effects of PKC Activators in Alzheimer's Disease Transgenic
Mice. Proc Natl Acad Sci. 2004;101(30):11141-11146. https://
doi.org/10.1073/pnas.0403921101
17. Talman V, Amadio M, Osera C, et al. The C1 Domain-Targeted
Isophthalate Derivative HMI-1b11 Promotes Neurite Out-
growth and GAP-43 Expression through PKCα Activation in
SH-SY5Y Cells. Pharmacol Res. 2013;73:44-54. https://doi.org/
10.1016/j.phrs.2013.04.008
18. Sajan MP, Hansen BC, Higgs MG, et al. Atypical PKC, PKCλ/ι,
Activates β-Secretase and Increases Aβ1–40/42 and Phospho-
Tau in Mouse Brain and Isolated Neuronal Cells, and May
Link Hyperinsulinemia and Other APKC Activators to Devel-
opment of Pathological and Memory Abnormalities in
Alzheimer's. Neurobiol Aging. 2018;61:225-237. https://doi.org/
10.1016/j.neurobiolaging.2017.09.001
19. Rui M, Nasti R, Bignardi E, Della Volpe S, Rossino G, Rossi D,
Collina S. PKC in Regenerative Therapy: New Insights for Old
Targets. Pharmaceuticals. 2017;10:(4):46-60. https://doi.org/10.
3390/ph10020046
20. Thomason HA, Cooper NH, Ansell DM, et al. Direct Evidence
That PKCα Positively Regulates Wound Re-Epithelialization:
Correlation with Changes in Desmosomal Adhesiveness.
J Pathol. 2012;227(3):346-356. https://doi.org/10.1002/path.
4016
21. Cooper NH, Balachandra JP, Hardman MJ. Global Gene
Expression Analysis in PKCα / Mouse Skin Reveals Struc-
tural Changes in the Dermis and Defective Wound Granulation
Tissue. J Invest Dermatol. 2015;135(12):3173-3182. https://doi.
org/10.1038/jid.2015.338
22. Wallis S, Lloyd S, Wise I, Ireland G, Fleming TP, Garrod D.
The α Isoform of Protein Kinase C Is Involved in Signaling the
Response of Desmosomes to Wounding in Cultured Epithelial
Cells. Mol Biol Cell. 2000;11(3):1077-1092. https://doi.org/10.
1091/mbc.11.3.1077
23. Rossi D, Talman V, Gennäs GBA, et al. Beyond the Affinity
for Protein Kinase C: Exploring 2-Phenyl-3-Hydroxypropyl
15
Pivalate Analogues as C1 Domain-Targeting Ligands.
Fortschr Med. 2015;6(4):547-554. https://doi.org/10.1039/
C4MD00564C
24. Castagna M, Takai Y, Kaibuchi K, Sano K, Kikkawa U,
Nishizuka Y. Direct Activation of Calcium-Activated,
Phospholipid-Dependent Protein Kinase by Tumor-Promoting
Phorbol Esters. J Biol Chem. 1982;257(13):7847-7851. https://
doi.org/10.1016/S0021-9258(18)34459-4
25. Kortmansky J, Schwartz GK. Bryostatin-1: A Novel PKC
Inhibitor in Clinical Development. Cancer Invest. 2003;21(6):
924-936. https://doi.org/10.1081/CNV-120025095
26. Kang J-H, Siddiqui MA, Sigano DM, et al. Conformationally
Constrained Analogues of Diacylglycerol. 24. Asymmetric
Synthesis of a Chiral (R)-DAG-Lactone Template as a
Versatile Precursor for Highly Functionalized DAG-Lactones.
Org Lett. 2004;6(14):2413-2416. https://doi.org/10.1021/
ol0492041
27. Af Gennäs GB, Talman V, Aitio O, et al. Design, Synthesis, and
Biological Activity of Isophthalic Acid Derivatives Targeted to
the C1 Domain of Protein Kinase C. J Med Chem. 2009;52(13):
3969-3981. https://doi.org/10.1021/jm900229p
28. Lee J, Lee J-H, Kim SY, et al. 2-Benzyl and 2-Phenyl-
3-Hydroxypropyl Pivalates as Protein Kinase C Ligands. Bioorg
Med Chem. 2006;14(6):2022-2031. https://doi.org/10.1016/j.
bmc.2005.10.051
29. Das J, Rahman GM. C1 Domains: Structure and Ligand-
Binding Properties. Chem Rev. 2014;114(24):12108-12131.
https://doi.org/10.1021/cr300481j
30. Rossi D, Tarantino M, Rossino G, Rui M, Juza M, Collina S.
Approaches for Multi-Gram Scale Isolation of Enantiomers
for Drug Discovery. Expert Opin Drug Discovery. 2017;12(12):
1253-1269. https://doi.org/10.1080/17460441.2017.1383981
31. Schrödinger Suite 2014–3: Protein Preparation Wizard; Epik,
Schrödinger. NY [USA]: LLC New York; 2014.
32. Cavalloro V, Marrubini G, Stabile R, Rossi D, Linciano P,
Gheza G, Assini S, Martino E, Collina S. Microwave-Assisted
Extraction and HPLC-UV-CD Determination of (S)-usnic Acid
in Cladonia foliacea. Molecules. 2021;26:(2):455-470. https://
doi.org/10.3390/molecules26020455
33. Raval A, Piana S, Eastwood MP, Dror RO, Shaw DE.
Refinement of Protein Structure Homology Models via Long,
All-Atom Molecular Dynamics Simulations. Proteins Struct
Funct Bioinforma. 2012;80(8):2071-2079. https://doi.org/10.
1002/prot.24098
34. Shaw DE. Schrödinger Release 2018–4: Desmond Molecular
Dynamics System. Maestro-Desmond Interoperability Tools. New
York, NY: Schrödinger; 2021.
35. Bowers KJ, Sacerdoti FD, Salmon JK, Shan Y, Shaw DE,
Chow E, Xu H, Dror RO, Eastwood MP, Gregersen BA,
Klepeis JL, Kolossvary I, Moraes MA. Molecular Dynamics---
Scalable Algorithms for Molecular Dynamics Simulations on
Commodity Clusters. In Proceedings of the 2006 ACM/IEEE
conference on Supercomputing – SC'06; ACM Press: New York,
New York, USA, 2006; p 84
36. Shaw Research DE. Schrödinger Release 2014–2: Desmond
Molecular Dynamics System. New York, NY: Schrödinger, LLC;
2014.
37. Schrodinger Release 2014–3: Glide (Version6.8). New York, NY
[USA]: Schrodinger LLC; 2014.
16
38. Schrodinger Release 2014–3: Maestro. Schrodinger LLC, New
York, NY [USA]; 2014.
39. Schrodinger Release 2014–3: LigPrep. New York, NY [USA]:
Schrodinger LLC; 2014.
40. Sandler C, Ekokoski E, Lindstedt KA, et al. Chemically Modi-
fied Tetracycline (CMT)-3 Inhibits Histamine Release and
Cytokine Production in Mast Cells: Possible Involvement of
Protein Kinase C. Inflamm Res. 2005;54(7):304-312. https://doi.
org/10.1007/s00011-005-1358-5
41. Tammela P, Ekokoski E, García-Horsman A, et al. Screening of
Natural Compounds and Their Derivatives as Potential Protein
Kinase C Inhibitors. Drug Dev Res. 2004;63(2):76-87. https://
doi.org/10.1002/ddr.10399
42. Rossi D, Nasti R, Collina S, et al. The Role of Chirality in a
Set of Key Intermediates of Pharmaceutical Interest, 3-Aryl-
Substituted-γ-Butyrolactones, Evidenced by Chiral HPLC Sep-
aration and by Chiroptical Spectroscopies. J Pharm Biomed
Anal. 2017;144:41-51. https://doi.org/10.1016/j.jpba.2017.
01.007
43. Gaggeri R, Rossi D, Daglia M, et al. An Eco-Friendly
Enantioselective Access to (R)-Naringenin as Inhibitor of
Proinflammatory Cytokine Release. Chem Biodivers. 2013;
10(8):1531-1538. https://doi.org/10.1002/cbdv.201200227
44. Byrne FP, Jin S, Paggiola G, et al. Tools and Techniques for
Solvent Selection: Green Solvent Selection Guides. Sustain
Chem Process. 2016;4(1):7 https://doi.org/10.1186/s40508-016-
0051-z
45. Wenzel TJ, Chisholm CD. Assignment of Absolute Configura-
tion Using Chiral Reagents and NMR Spectroscopy. Chirality.
2011;23(3):190-214. https://doi.org/10.1002/chir.20889
46. Seco JM, Quiñoa E, Riguera R. The Assignment of Absolute
Configuration by NMR. Chem Rev. 2004;104(1):17-117. https://
doi.org/10.1021/cr000665j
47. Tsuda M, Toriyabe Y, Endo T, Kobayashi J. Application of
Modified Mosher's Method for Primary Alcohols with a Methyl
Group at C2 Position. Chem Pharm Bull. 2003;51(4):448-451.
https://doi.org/10.1248/cpb.51.448
48. Galman JL, Hailes HC. Application of a Modified Mosher's
Method for the Determination of Enantiomeric Ratio and
LINCIANO ET AL.
Absolute Configuration at C-3 of Chiral 1,3-Dihydroxy
Ketones. Tetrahedron Asymmetry. 2009;20(15):1828-1831.
https://doi.org/10.1016/j.tetasy.2009.07.023
49. Fukui H, Fukushi Y, Tahara S. NMR Determination of the
Absolute Configuration of β-Chiral Primary Alcohols. Tetrahe-
dron Lett. 2005;46(30):5089-5093. https://doi.org/10.1016/j.
tetlet.2005.05.028
50. Rustoy EM, Baldessari A, Monsalve LN. A Conformational
Model for MTPA Esters of Chiral N -(2-Hydroxyalkyl)Acrylam-
ides. Adv Chemother. 2014;2014:1-6. https://doi.org/10.1155/
2014/736417
51. Xu S, Oda A, Kamada H, Negishi E. Highly Enantioselective
Synthesis of -, , and -Chiral 1-Alkanols via Zr-Catalyzed
Asymmetric Carboalumination of Alkenes (ZACA)-Cu- or Pd-
Catalyzed Cross-Coupling. Proc Natl Acad Sci. 2014;111(23):
8368-8373. https://doi.org/10.1073/pnas.1401187111
52. Lewin NE, Blumberg PM. [3H]Phorbol 12,13-Dibutyrate Bind-
ing Assay for Protein Kinase C and Related Proteins. In: Protein
Kinase C Protocols. New Jersey: Humana Press:129-156.
53. Zhang G, Kazanietz MG, Blumberg PM, Hurley JH. Crystal
Structure of the Cys2 Activator-Binding Domain of Protein
Kinase Cδ in Complex with Phorbol Ester. Cell. 1995;81(6):
917-924. https://doi.org/10.1016/0092-8674(95)90011-X
SU PP O R TI N G I N F O RMA TI O N
Additional supporting information may be found in the
online version of the article at the publisher's website.
How to cite this article: Linciano P, Nasti R,
Listro R, et al. Chiral 2-phenyl-3-hydroxypropyl
esters as PKC-alpha modulators: HPLC
enantioseparation, NMR absolute configuration
assignment, and molecular docking studies.
Chirality. 2021;1-16. doi:10.1002/chir.23406