Chiral combinatorial preparation and biological evaluation of unique ceramides for inhibition of sphingomyelin synthase

Article abstract of DOI:10.1002/chir.23179

Enantiomers or diastereomers of chiral bioactive compounds often exhibit different biological and toxicological properties. Here, we report the efficient synthesis of four stereoisomers of sphingosine and derivatization of unique chiral ceramides through a combinatorial chemistry by solid-phase activated resin ester. In addition, to test the effectivity of stereochemistry of ceramide, we demonstrated a cell-based assay of sphingomyelin synthase inhibition in the presence ofchiral unique ceramides, which suggested that libraries of this sort will be a rich source of biologically active synthetic molecules.

Full text of DOI:10.1002/chir.23179

Received: 7 December 2019
DOI: 10.1002/chir.23179
Revised: 23 December 2019
Accepted: 26 December 2019
S P E C I A L I S S U E A R T I C L E
Chiral combinatorial preparation and biological evaluation
of unique ceramides for inhibition of sphingomyelin
synthase
Sajeer Koolath1
| Yuta Murai1,2
| Yoshiko Suga2 | Kenji Monde1,2
1Graduate School of Life Science,
Abstract
Hokkaido University, Sapporo, Japan
2Faculty of Advanced Life Science,
Hokkaido University, Sapporo, Japan
Enantiomers or diastereomers of chiral bioactive compounds often exhibit dif-
ferent biological and toxicological properties. Here, we report the efficient syn-
thesis of four stereoisomers of sphingosine and derivatization of unique chiral
ceramides through a combinatorial chemistry by solid-phase activated resin
ester. In addition, to test the effectivity of stereochemistry of ceramide, we
demonstrated a cell-based assay of sphingomyelin synthase inhibition in the
presence ofchiral unique ceramides, which suggested that libraries of this sort
will be a rich source of biologically active synthetic molecules.
Correspondence
Yuta Murai and Kenji Monde, Graduate
School of Life Science, Hokkaido
University, Kita 21 Nishi 11, Sapporo
001-0021, Japan.
Email: ymurai@sci.hokudai.ac.jp;
kmonde@sci.hokudai.ac.jp
Funding information
K E Y W O R D S
Hokkaido University; MEXT, Grant/
Award Numbers: 17K19188, 18K14350,
19H02836; MEXT, Grant/Award Number:
Doctoral program for Data Related
Innovation Exper
chiral ceramide, chiral combinatorial chemistry, functional resin, inhibitor, sphingomyelin
synthase
1 | INTRODUCTION
crystallography3 have often provided detailed structural
information on target biomolecules and novel chiral drug
Chirality is a property of matter found throughout biolog-
ical systems, from basic building blocks of biomolecules
such as amino acids, carbohydrates, and lipids. In the
field of new drug discovery and phramacotherapeutics,
information on the stereoisomerism of a bioactive com-
pound is very important because living systems are them-
selves chiral and each of the stereoisomers of chiral drugs
show very different effects in vivo. In fact, over 50% of
commercially available drugs are chiral substances,1 and
it is therefore also important to realize the safety and effi-
cacy of chiral drugs in order to avoid drug-induced inci-
dents such as the thalidomide disaster in Japan in the
late 1950s. Over the past several decades, protein
structure-based drug design in silico using structural
information from solution-state NMR2 and X-ray
candidates. In the case of membrane proteins, such as a
membrane receptor and enzyme, it is often more efficient
to design drug candidates from original substrates due to
difficulty in using NMR and X-ray methods.
In this study, we established new chiral inhibitors
based on the ceramide backbone for the membrane pro-
tein sphingomyelin synthase (SMS) and evaluated their
inhibitory efficacy. SMS catalyzes ceramide and phospha-
tidylcholine as substrates to produce sphingomyelin
(SM) and diacylglycerol.4 It has been shown that SMS
modulates the levels of SM and other sphingolipids
levels, thereby regulating membrane fluidity, ceramide-
dependent apoptosis, lipid metabolism, and signal trans-
duction.5 In addition, it was reported that mice with SMS
knockout or knockdown showed a decrease in plasma
inflammatory cytokines6 and showed resistance to the
development of high-fat diet-induced obesity,7 insulin
resistance,8 Alzheimer's disease,9 and tumorigenesis.10
This work is dedicated to the memory of Prof Koji Nakanishi,
outstanding leader in natural products chemistry and chemical biology.
Chirality. 2020;1–6.
wileyonlinelibrary.com/journal/chir
© 2020 Wiley Periodicals, Inc.
1

2
KOOLATH ET AL.
FIGURE 1 De novo pathway of
endogenous (D)-erythro sphingosine and
unnatural stereoisomers of sphingosine, (L)-
erythro, (D)-threo, and (L)-threo sphingosines
Therefore, SMS inhibition is
approach for these diseases.
Our strategy for creating SMS inhibitors is to design
the structure of SMS inhibitors from ceramide as an
a
novel therapeutic
original substrate. The most common ceramide in mam-
mals consists of the amino alcohol sphingosine as a back-
bone moiety, which has two chiral centers at the C-2 and
C-3 positions. Theoretically, there are four stereoisomers,
FIGURE 2 Efficient preparation of unique chiral
ceramides by active ester resin

KOOLATH ET AL.
3
(D-erythro: DE), (L-erythro: LE), (D-threo: DT), and (L-
threo: LT), in sphingosine, but only D-erythro type [(2S,
3R, 4E)-2-aminooctadec-4-ene-1,3-diol] has actually been
found in mammals (Figure 1). Nakanishi and Berova et al
determined their absolute configurations by sensitive and
reliable stereochemical analysis utilizing high-
performance liquid chromatography11 and circular
dichroism12 techniques. We have also established an
efficient methodology for those of sphingosine by using
the vibrational circular dichroism technique,13 which
measures the differential absorption of left versus right
circularly polarized infrared radiation. For investigating
the relationship between stereochemistry and biological
activity, the four isomers were synthesized in some
studies and their biological activities were evaluated.14
In addition to this, Inokuchi and Norman reported
TABLE 1 IC50 values are the means of each of the four unique chiral ceramides in separate determinations for SMS1 or SMS2
expressed in SMS1/2 double knockout mouse fibroblast cell lysate and were determined by using more than four concentrations of each
inhibitor
SMS1 (IC50) mM
SMS2 (IC50) mM
entry (# of R)
DE
20
3
LE
>100
15
LT
70
DT
>100
30
DE
7
LE
15
LT
20
0.3
80
9
DT
30
0.4
>100
4
1
2
7
>100
25
20
70
25
>100
>100
3.5
30
25
6
0.2
>100
10
3
1
>100
9
3
50
4
>100
50
>100
>100
20
4
5
5
20
6
3
4
6
30
3
100
30
70
30
8
60
7
60
15
40
90
5
40
20
>100
>100
3
7
70
8
>100
>100
0.8
100
50
20
>100
15
30
20
4
>100
>100
10
>100
>100
20
70
70
20
30
30
9
100
80
10
25
30
10
>100
10
20
10
6
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
90
>100
>100
50
10
7
10
40
6
60
20
2
>100
20
60
9
>100
30
>100
15
10
4
50
3
>100
5
40
20
15
15
7
70
4
7
50
20
2
5
25
20
1.5
1
1.5
2
3
1
15
30
5
7
15
30
1
50
7
20
9
10
15
7
4
5
40
8
15
10
3
3
5
9
6
20
3
5
15
8
20
15
15
10
30
20
3
25
10
2
7
4
7
30
30
5
1.5
4
4
5
8
10
3
5
6
50
20
2
50
50
30
10
2
15
15
5
9
7
20
20
7
7
10
15
2
4
10
50
30
16
>100
100
100
30
40
50
40
20
80
3
50
30
50
20
15
20
40
10
40
30
70
20
100
100
30
30
30
12

4
KOOLATH ET AL.
FIGURE 3 Heatmap
analysis of IC50 values of each of
the four unique chiral ceramides
for SMS1 and SMS2
that an inhibitor, (D)-threo-1-phenyl-2-decanoylamino-
3-morpholino-1-propanol (D-PDMP), of glucosylceramide
synthase suppressing GM3 in adipocytes showed a TNF-
α-induced defect in insulin-dependent tyrosine phosphor-
ylation of insulin receptor substrate-1.15 Notably, the ste-
reochemistry of active D-PDMP is different from that of
endogenous ceramide. Stereospecific activity is a com-
mon feature for sphingolipids, suggesting that their tar-
gets also have specific spatial configurations; therefore,
focusing on the stereochemistry of sphingolipids is a
much attractive research point for revealing the
structure-activity relationships of these pharmacologi-
cally active derivatives. However, a deeper understanding
of the biological role of ceramide stereochemistry in
human pathophysiology and the potential therapeutic
use of SMS has still been veiled. In this study, to address
these unclear points, we synthesized several unique
ceramides of all of four stereoisomers by solid-phase acti-
vated resin ester and evaluated the importance of their
stereochemistry efficacy towards lipid-metabolizing
enzymes.
selected ceramides among the 128 ceramides (see the
ESI).
Finally, to evaluate the inhibitory efficacy of the ste-
reochemistry of each of the chiral four unique ceramides,
we carried out cell-based assays for both of the isozymes
SMS1 and SMS218 in the presence of the 128 unique
ceramides. This assay uses a fluorescent (D)-erythro
C6-NBD (4-nitrobenzo-2-oxa-1,3-diazole)-Cer analog as a
substrate into SM moiety. As shown in Table 1, several
ceramides showed relatively moderate inhibitory activi-
ties (IC50 0.2-1 μM; Table 1) compared with those of our
previously reported natural compounds.19 Furthermore,
to determine the differences in the inhibitory activities of
the four stereoisomers, we represented the IC50 values as
a heatmap graphical representation in which individual
values contained in a matrix are displayed as colors.
Although the natural stereoisomer is (D)-erythro type, the
unnatural (L)-threo stereoisomer indicated that the ratio
of ceramides showing strong inhibitory activities towards
SMS1 and SMS2 is high (Figure 3). In addition to this,
there might be a possibility that (D)-erythro ceramide
analogue also become a substrate for SMS. Therefore,
(L)-threo sphingolipid is the promising scaffold for the
development of ceramide-based SMS inhibitors. In the
present study, we demonstrated that the chiral chemistry
of ceramide affected its inhibitory activity towards the
lipid-metabolizing enzymes.
2 | RESULTS AND DISCUSSION
First, we started to synthesize all four stereoisomers of
sphingosine according to established methods16 from (L)-
or (D)-serine with slight modification (Scheme S1). The
specific rotations of the stereoisomers were measured,
DE: [α]D −4.4, LE: +4.6, DT: −2.9, and LT: +2.2 (c 1.0;
CHCl3). Next, to prepare unique ceramides efficiently
through a combinatorial chemistry, we prepared several
activated esters coupling with 32 commercially available
carboxylic acids or acid chlorides and nitrophenol on
polystyrene as reported by Chang et al17 (Scheme S2).
Subsequently, each of the four chiral shingosines was
subjected to acylation with 32 kinds of activated ester
resin, followed by filtration to generate 128 unique
ceramides without starting material sphingosine and
byproducts (Figure 2). The products and their purity were
characterized by measuring electrospray ionization-mass
spectrometry (ESI-MS) and 1H-NMR of 40 randomly
3 | CONCLUSION
We successfully synthesized 128 unique chiral ceramides
through combinatorial chemistry by solid-phase activated
ester without starting material sphingosine and
byproducts. Additionally, we proved that these ceramides
have good inhibitory activities (IC50 = 0.2-1 μM) for SMSs
1 and 2. Furthermore, according to heatmap analysis of
IC50 values, we confirmed that most of the unnatural (L)-
threo stereoisomer ceramide derivatives showed strong
inhibitory activities towards SMS1 and SMS2, respec-
tively, compared with the inhibitory activities of other
stereoisomers. Therefore, to create competent bioactive

KOOLATH ET AL.
5
promotes clearance of amyloid-β by microglia. J Biol Chem.
2012;287:10977-10989.
compounds based on the sphingolipid moiety, it is impor-
tant to thoroughly consider the information on the ste-
reoisomerism of them.
6. Fan Y, Shi F, Liu J, et al. Selective reduction in the
sphingomyelin content of atherogenic lipoproteins inhibits
their retention in murine aortas and the subsequent develop-
ment of atherosclerosis. Arterioscler Thromb Vasc Biol. 2010;30:
2114-2120.
7. Mitsutake S, Zama K, Yokota H, et al. Dynamic modification of
sphingomyelin in lipid microdomains controls development of
obesity, fatty iver, and type 2 diabetes. J Biol Chem. 2011;
286(32):28544-28555.
8. Li Z, Zhang H, Liu J, et al. Reducing plasma membrane
sphingomyelin increases insulin sensitivity. Mol Cell Biol. 2011;
31(20):4205-4218.
9. Yuyama K, Mitsutake S, Igarashi Y. Pathological roles of
ceramide and its metabolites in metabolic syndrome
and Alzheimer's disease. Biochim Biophys Acta. 1841;2014:
793-798.
ACKNOWLEDGEMENTS
The authors are grateful to Dr. Shinji Nakaoka for help-
ing with the heatmap analysis. This work was supported
by a grant-in-aid for scientific research KAKENHI (grants
19H02836, 17K19188, and 18K14350) from the MEXT of
Japan, the MEXT Doctoral program for Data Related
Innovation Expert Hokkaido University (D-DRIVE-HU)
program, and the Photo-excitonix Project in Hokkaido
University. This work was inspired by JSPS Asian CORE
Program “Asian Chemical Biology Initiative” and JSPS
A3 Foresight Program. A. A. S. thanks the International
Graduate Program (IGP), Hokkaido University, for the
scholarship given.
10. Ohnishi T, Hashizume C, Taniguchi M, et al. Sphingomyelin
synthase 2 deficiency inhibits the induction of murine colitis-
associated colon cancer. FASEB J. 2017;31(9):3816-3830.
11. Kawamura A, Berova N, Dirsch V, et al. Picomole scale stereo-
chemical analysis of sphingosines and dihydrosphingosines.
Bioorg Med Chem. 1996;4(7):1035-1043.
ORCID
Sajeer Koolath https://orcid.org/0000-0002-3789-6547
Yuta Murai https://orcid.org/0000-0002-9115-6478
Kenji Monde https://orcid.org/0000-0002-1424-1054
12. a) Dirsch V, Frederico J, Zhao N, et al.
A two-step
chemical and circular dichroic method for assigning the
absolute configurations of sphingosines. Tetrahedron Lett.
1995;36:4959-4962. b) Jiang H, Huang X, Nakanishi K,
Berova N. Nanogram scale absolute configurational assignment
of ceramides by circular dichroism. Tetrahedron Lett. 1999;40:
7645-7649.
REFERENCES
1. Hutt AJ. The development of single-isomer molecules: why
and how. CNS Spectr. 2002;7(4 Suppl 1):14-22.
2. a) Zheng D, Huang Y, Moseley H, et al. Automated protein fold
determination using a minimal NMR constraint strategy. Pro-
tein Sci. 2003;12(6):1232-1246. b) Oezguen N, Adamian L,
Xu Y, Rajarathnam K, Braun W. Automated assignment and
3D structure calculations using combinations of 2D homonu-
clear and 3D heteronuclear NOESY spectra. J Biomol NMR.
2002;22(3):249-263. c) Bailey-Kellogg C, Widge A, Kelley JJ,
Berardi MJ, Bushweller JH, Donald BR. The NOESY Jigsaw:
automated protein secondary structure and main-chain assign-
ment from sparse, unassigned NMR data. J Comput Biol. 2000;
7(3-4):537-558. d) Pervushin K, Riek R, Wider G, Wutrich K.
Attenuated T2 relaxation by mutual cancellation of dipole–
dipole coupling and chemical shift anisotropy indicates an ave-
nue to NMR structures of very large biological macromolecules
in solution. Proc Natl Acad Sci U S A. 1997;94(23):12366-12371.
3. Erickson J, Neidhart DJ, VanDrie J, et al. Design, activity, and
2.8 Å crystal structure of a C2 symmetric inhibitor complexed
to HIV-1 protease. Science. 1990;249(4968):527-533.
4. a) Ullman MD, Radin NS. The enzymatic formation of
sphingomyelin from ceramide and lecithin in mouse liver.
J Biol Chem. 1974;249:1506-1512. b) Merrill AH, Jones DD. An
update of the enzymology and regulation of Sphingomyelin
metabolism. Biochim Biophys Acta, Lipids Lipid Metab. 1990;
1044:1-12.
5. a) Bienias K, Fiedorowicz A, Sadowska A, Prokopiuk S, Car H.
Regulation of sphingomyelin metabolism. Pharmacol Rep.
2016;68(3):570-581. b) Hannun YA. Functions of ceramide in
coordinating cellular responses to stress. Science. 1996;
274(5294):1855-1859. c) Yuyama K, Sun H, Mitsutake S,
Igarashi Y. Sphingolipid-modulated exosome secretion
13. Nakahashi A, Siddegowda AKC, Hammam MAS, Gowda SGB,
Murai Y, Monde K. Stereochemical study of sphingosine by
vibrational circular dichroism. Org Lett. 2016;18:2327-2330.
14. a) Bielawska A, Linardic CM, Hannun YA. Ceramide-
mediated biology. Determination of structural and stereospe-
cific
requirements
through
the
use
of
N-acyl-
phenylaminoalcohol analogs. J Biol Chem. 1992;267:18493-
18497. b) Bielawska A, Crane HM, Liotta D, Obeid LM,
Hannun YA. Selectivity of ceramide-mediated biology. Lack
of activity of erythro-dihydroceramide. J Biol Chem. 1993;
268(35):26226-26232. c) Chalfant CE, Szulc Z, Roddy P,
Bielawska A, Hannun YA. The structural requirements for
ceramide activation of serine-threonine protein phosphatases.
J Lipid Res. 2004;45(3):496-506.
15. Inokuchi J, Norman SR. Preparation of the active isomer of
1-phenyl-2-decanoylamino-3-morpholino-1-propanol, inhibitor
of murine glucocerebroside synthetase. J Lipid Res. 1987;28(5):
565-571.
16. a) Koskinen AMP, Koskinen PM. Synthetic studies towards
amino alcohols. Diastereocontrolled reduction of α'-chiral α,β-
enones. Tetrahedron Lett. 1993;34:6765-6768. b) Katsumura S,
Yamamoto N, Fukuda E, Iwama S. Highly diastereoselective
synthesis of D-threo- and D-erythro-sphingosine from glycidol.
Chem Lett. 1995;393-395. c) Saito S, Murai Y, Usuki S, et al.
Synthesis of nontoxic fluorous sphingolipids as molecular pro-
bes of exogenous metabolic studies for rapid enrichment by
fluorous solid phase extraction. Eur J Org Chem. 2017;1045-
1051.

6
KOOLATH ET AL.
17. Chang Y-T, Choi J, Ding S, et al. The synthesis and biological
characterization of a ceramide library. J Am Chem Soc. 2002;
124:1856-1857.
18. Zama K, Mitsutake S, Watanabe K, Okazaki T, Igarashi Y. A
sensitive cell-based method to screen for selective inhibitors of
SMS1 or SMS2 using HPLC and a fluorescent substrate. Chem
Phys Lipids. 2012;165(7):760-768.
19. a) Swamy MMM, Murai Y, Ohno Y, et al. Structure-inspired
design of a sphingolipid mimic sphingosine-1-phosphate recep-
tor agonist from a naturally occurring sphingomyelin synthase
inhibitor. Chem Commun. 2018;54(90):12758-12761. b)
Othman MA, Yuyama K, Murai Y, et al. Malabaricone C as
natural sphingomyelin synthase inhibitor against diet-induced
obesity and its lipid metabolism in mice. ACS Med Chem Lett.
2019;10:1154-1158.
SUPPORTING INFORMATION
Additional supporting information may be found online
in the Supporting Information section at the end of this
article.
How to cite this article: Koolath S, Murai Y,
Suga Y, Monde K. Chiral combinatorial
preparation and biological evaluation of unique
ceramides for inhibition of sphingomyelin
synthase. Chirality. 2020;1–6. https://doi.org/10.
1002/chir.23179