Synthesis and structure-activity relationships of cerebroside analogues as substrates of cerebroside sulphotransferase and discovery of a competitive inhibitor

Article abstract of DOI:10.1080/14756366.2020.1791841

Metachromatic leukodystrophy (MLD) is a rare genetic disease characterised by a dysfunction of the enzyme arylsulphatase A leading to the lysosomal accumulation of cerebroside sulphate (sulphatide) causing subsequent demyelination in patients. The enzyme galactosylceramide (cerebroside) sulphotransferase (CST) catalyses the transfer of a sulphate group from 3′-phosphoadenosine-5'-phosphosulphate (PAPS) to cerebrosides producing sulphatides. Substrate reduction therapy for arylsulphatase A by inhibition of CST was proposed as a promising therapeutic approach. To identify competitive CST inhibitors, we synthesised and investigated analogues of the substrate galactosylceramide with variations at the anomeric position, the acyl substituent and the carbohydrate moiety, and investigated their structure–activity relationships. While most of the compounds behaved as substrates, α-galactosylceramide 16 was identified as the first competitive CST inhibitor. Compound 16 can serve as a new lead structure for the development of drugs for the treatment of this devastating disease, MLD, for which small molecule therapeutics are currently not available.

Full text of DOI:10.1080/14756366.2020.1791841

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Synthesis and structure-activity relationships
of cerebroside analogues as substrates of
cerebroside sulphotransferase and discovery of a
competitive inhibitor
Wenjin Li , Joren Guillaume , Younis Baqi , Isabell Wachsmann , Volkmar
Gieselmann , Serge Van Calenbergh & Christa E. Müller
To cite this article: Wenjin Li , Joren Guillaume , Younis Baqi , Isabell Wachsmann , Volkmar
Gieselmann , Serge Van Calenbergh & Christa E. Müller (2020) Synthesis and structure-activity
relationships of cerebroside analogues as substrates of cerebroside sulphotransferase and
discovery of a competitive inhibitor, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1,
1503-1512, DOI: 10.1080/14756366.2020.1791841
To link to this article: https://doi.org/10.1080/14756366.2020.1791841
© 2020 The Author(s). Published by Informa
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 1503–1512
https://doi.org/10.1080/14756366.2020.1791841
RESEARCH PAPER
Synthesis and structure-activity relationships of cerebroside analogues as
substrates of cerebroside sulphotransferase and discovery of a
competitive inhibitor
a
b
c
, Isabell Wachsmannd, Volkmar Gieselmannd, Serge Van
ꢀ
ꢀ
Wenjin Li , Joren Guillaume , Younis Baqi
Calenberghb
and Christa E. Muller
a
€
aDepartment of Pharmaceutical & Medicinal Chemistry, PharmaCenter Bonn, Pharmaceutical Institute, University of Bonn, Bonn, Germany;
c
bLaboratory for Medicinal Chemistry, Gent, Belgium; Department of Chemistry, College of Science, Sultan Qaboos University, Muscat, Oman;
d
€
Institut fur Biochemie und Molekularbiologie, University of Bonn, Bonn, Germany
ABSTRACT
ARTICLE HISTORY
Received 27 March 2020
Revised 9 June 2020
Accepted 29 June 2020
Metachromatic leukodystrophy (MLD) is a rare genetic disease characterised by a dysfunction of the
enzyme arylsulphatase A leading to the lysosomal accumulation of cerebroside sulphate (sulphatide) caus-
ing subsequent demyelination in patients. The enzyme galactosylceramide (cerebroside) sulphotransferase
(CST) catalyses the transfer of a sulphate group from 30-phosphoadenosine-5’-phosphosulphate (PAPS) to
cerebrosides producing sulphatides. Substrate reduction therapy for arylsulphatase A by inhibition of CST
was proposed as a promising therapeutic approach. To identify competitive CST inhibitors, we synthesised
and investigated analogues of the substrate galactosylceramide with variations at the anomeric position,
the acyl substituent and the carbohydrate moiety, and investigated their structure–activity relationships.
While most of the compounds behaved as substrates, a-galactosylceramide 16 was identified as the first
competitive CST inhibitor. Compound 16 can serve as a new lead structure for the development of drugs
for the treatment of this devastating disease, MLD, for which small molecule therapeutics are currently
not available.
KEYWORDS
Capillary electrophoresis
(CE); galactosylceramide
sulphotransferase (CST);
enzyme assay;
metachromatic leukodystro-
phy (MLD); competitative
inhibitor; galactosyl
ceramide analogues
1. Introduction
We, therefore, aim at developing CST inhibitors to reduce the
biosynthesis of sulphatide to prevent sulphatide aggregation in
the central and peripheral nervous system7. Sulphation is a widely
observed biological reaction conserved from bacterium to human
that plays a key role in various biological processes8,9. Deficiencies
due to the lack of the ubiquitous sulphate donor PAPS are lethal
in humans8–10. A large group of enzymes called sulphotransferases
catalyses the transfer reaction of the sulphuryl group of PAPS to
the acceptor group of numerous biochemical and xenobiotic sub-
strates11. Structure-based sequence alignments based on X-ray
crystal structures indicate that the PAPS-binding site is con-
served8,9,12. Therefore, competitive inhibitors for the CST substrate
galactosylceramide are expected to have less side effects com-
pared to inhibitors competing with the co-substrate PAPS.
However, so far only few weakly potent, non-selective CST inhibi-
tors have been described (Figure 2)13.
A reaction mechanism between the enzyme, the co-substrate
PAPS, and the substrate galactosylceramide has been proposed
based on crystal structures14–16. A transition state mimetic might
efficiently inhibit the CST-catalysed reaction17. In the present
study, we synthesised and investigated a series of substrate ana-
logues with the aim to study their structure–activity relationships
(SARs) as substrates and to identify competitive inhibitors.
Metachromatic leukodystrophy (MLD) is a rare genetic disease
characterised by a dysfunction of the enzyme arylsulphatase A1.
This defect leads to the lysosomal accumulation of cerebroside
sulphate (sulphatide, 1) in various cells such as tubular kidney
cells, bile duct epithelia, some neurons, oligodendrocytes and
Schwann cells. In particular accumulation in the latter two results
in progressive demyelination finally causing lethal symptoms in
patients. Recently haematopoetic stem cell-based gene therapy
has been shown to be effective in patients in early preclinical
states of disease only2. Thus, there is an urgent need to develop
alternative strategies to treat MLD. One of these strategies is sub-
strate reduction therapy in which galactosylceramide (cerebroside)
sulphotransferase (CST; EC 2.8.2.11), the enzyme which synthesises
sulphatide, is inhibited. This would diminish the load of accumu-
lated sulphatide in the patient. Such a strategy has been shown
to be effective in Gaucher disease, another lysosomal sphingolipid
storage disorder3. Inhibition of galactosylceramide sulphotransfer-
ase has been proposed as a promising new therapeutic strategy
for the treatment of MLD1,4. CST catalyses the transfer of a sul-
phate group from the coenzyme 30-phosphoadenosine-50-phos-
phosulphate (PAPS, 2) to galactosylceramide (3) yielding
galactosylceramide sulphate (2) and adenosine-30,50-bisphosphate
(PAP, 4) (Figure 1)5–7
.
€
CONTACT Christa E. Muller
christa.mueller@uni-bonn.de
Department of Pharmaceutical & Medicinal Chemistry, PharmaCenter Bonn, Pharmaceutical Institute,
University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
ꢀ
These authors contributed equally to this work.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1504
W. LI ET AL.
Figure 1. Sulphatide synthesis by CST: galactosylceramide (cerebroside) is converted to sulphatide by CST in the presence of PAPS as sulphate donor.
Figure 2. Chemical structures of aromatic dyes known as CST inhibitors competing with the co-substrate PAPS.
performed on a Waters LCT Premier XE TOF equipped with an
electrospray ionisation interface and coupled to a Waters Alliance
HPLC system. Samples were infused in a CH3CN/HCO2H (1000:1)
2. Materials and methods
2.1. Commercial compounds
Psychosine was purchased from Sigma (Steinheim, Germany). mixture at 10 ml/min.
Galactosylceramide and glucosylceramide were obtained from
Matreya LLC (Pleasant Gap, PA); according to the supplier, cere-
2.2.1. General procedure for Staudinger reduction and acylation
reaction for compounds 24 and 25
broside consists of a mixture of saturated or unsaturated fatty
acid residues (C16:0, C18:0, C20:0, C22:0, C23:0, C24:0–C27:0,
C24:1–C27:1) or hydroxyacyl residues (C18:0(2–OH), C20:0(2–OH),
To a solution of azide 23 (400 mg, 0.42 mmol, 1 eq.) in 16 ml of
tetrahydrofuran (THF) at room temperature, a 1 M solution of
PMe3 in THF (6.3 ml, 6.3 mmol, 15 eq.) was added dropwise. After
stirring for 3 h, 2 ml of H2O were added and the reaction mixture
was allowed to stir overnight at room temperature. Then the solv-
ent was removed under reduced pressure and the residue was co-
evaporated with toluene to afford the crude amine. A mixture of
the crude amine, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC, 131 mg, 0.84 mmol, 2 eq.) and the appropriate fatty acid
(0.63 mmol, 1.5 eq.) in 10 ml of CH2Cl2 was stirred for 24 h at room
temperature. The reaction mixture was diluted with CH2Cl2,
washed with H2O (2 ꢂ 10 ml) and brine (1 ꢂ 10 ml), dried over
Na2SO4, filtered, and evaporated to dryness. Purification by col-
umn chromatography using 10% ethyl acetate in hexane gave the
desired amides in the indicated yield.
C22:0(2–OH),
C23:0(2–OH),
C24:0(2–OH),
C24:1(2–OH),
C25:0(2–OH)) and glucosylceramide are a mixture of glucosylcera-
mide with saturated or unsaturated fatty acid residues (C16:0,
C18:0, C20:0, C22:0, C23:0, C24:0, C24:1). 3-Phosphoadenosine-5-
phosphosulphate (PAPS) was purchased from Bellbrook Labs (No.
2059) in high purity. Other commercial sources of PAPS typically
contain significant amounts of PAP and are, therefore, not suitable
for the assay18. a-Galactosylceramide (KRN7000) was purchased
from Avanti Polar Lipids, (Alabaster, AL). b-KRN7000 was synthes-
ised and provided by the laboratory of S. van Calenbergh.
2.2. Chemistry
Precoated Macherey-Nagel SIL G/UV254 plates were used for TLC
and spots were examined under UV light at 254 nm and further
visualised by sulphuric acid-anisaldehyde spray or by spraying
2.2.1.1. [(1S,2S,3R)-1-[[[2,3-Bis-O-(phenylmethyl)-4,6-O-[(S)-phenyl-
methylene]-a-D-galactopyranosyl]oxy]methyl]-2,3-bis(phenylme-
thoxy)heptadecyl]-N-pentanamide (24). Yield: 74%. 1H NMR
(300 MHz, CDCl3): d 0.87 (t, J ¼ 7.2 Hz, terminal CH3), 0.88 (t,
J ¼ 6.7 Hz, terminal CH3), 1.18 ꢃ 1.35 (m, 26 H, CH2), 1.37–1.53 (m,
with
a
solution of
(NH4)6Mo7O24ꢁ4H2O
(25 g/L) and
(NH4)4Ce(SO4)4ꢁ2H2O (10 g/L) in H2SO4 (10%) followed by charring.
Column chromatography was performed on Biosolve silica gel
(32–63 mm, 60 Å). NMR spectra were obtained with a Varian
Mercury 300 Spectrometer. Chemical shifts are given in ppm (d) 2 H, CH2), 1.55–1.69 (m, 2 H, CH2), 1.85–1.92 (m, 2 H, CH2),
relative to the residual solvent signals, in the case of CDCl3: 3.51–3.57 (m, 1 H, H-4), 3.58 (br s, 1 H, H-4”), 3.74–3.82 (m, 2 H, Hb-
d ¼ 7.26 ppm for 1H and d ¼ 77.4 ppm for 13 C and in the case of 1 and H-3), 3.88–3.99 (m, 3 H, Hb-6”, Ha-1 and H-3”), 4.05–4.15 (m,
pyridine-d5: d ¼ 8.74, 7.58 and 7.22 ppm for 1H and d ¼ 149.9, 2 H, H-2” and Ha-6”), 4.18 (d, J ¼ 3.3 Hz, 1 H, H-5”), 4.24–4.34 (m,
135.5 and 123.5 ppm for 13C. Exact mass measurements were 1 H, H-2), 4.46–4.57 (m, 3 H, CH2Ph), 4.60–4.66 (m, 1 H, CH2Ph),

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1505
4.69–4.80 (m, 3 H, CH2Ph), 4.85 (d, J ¼ 11.6 Hz, 1 H, CH2Ph), 4.96 (d, (d, J ¼ 3.8 Hz, 1 H, H-1”), 6.24 (br s, 6 H, OH), 8.48 (d, J ¼ 8.8 Hz, 1 H,
NH). 13 C NMR (75 MHz, pyridine-d5) d 14.68, 23.34, 26.79, 26.91,
J ¼ 3.5 Hz, 1 H, H-1”), 5.46 (s, 1 H, H-8”), 5.76 (d, J ¼ 8.2 Hz, 1 H,
NHCO), 7.19–7.42 (m, 23 H, arom H), 7.48–7.55 (m, 2 H, arom H).
13 C NMR (75 MHz, CDCl3) d 13.82, 14.11, 22.37, 22.67, 25.82, 27.71,
29.36, 29.68, 29.71, 29.79, 30.27, 31.91, 36.41, 50.35, 62.91, 68.22,
69.41, 71.72, 71.90, 73.28, 73.79, 74.37, 75.69, 76.16, 77.20, 79.44,
79.92, 99.68, 101.00, 126.31, 127.54, 127.57, 127.60, 127.66, 127.71,
127.82, 127.88, 128.08, 128.31, 128.35, 128.43, 128.82, 137.82,
138.40, 138.52, 138.56, 138.66, 172.85.
30.02, 30.16, 30.21, 30.27, 30.33, 30.40, 30.42, 30.55, 30.76, 32.53,
34.75, 37.20, 51.85, 63.06, 69.08, 70.71, 71.39, 72.01, 72.89, 73.45,
77.13, 101.94, 173.62. HRMS (ESI) m/z: calculated for C43H86NO9
[M þ H]þ 760.6297; found 760.6267.
2.2.3. Synthesis of tert-butyl-N-[(1S,2S,3R)-1-[[[2,3-bis-O-(phenyl-
methyl)-4,6-O-[(S)-phenylmethylene]-a-D-galactopyranosyl]oxy]-
methyl]-2,3-bis(phenylmethoxy)heptadecyl]carbamate (26)
2.2.1.2. [(1S,2S,3R)-1-[[[2,3-Bis-O-(phenylmethyl)-4,6-O-[(S)-phenyl-
methylene]-a-D-galactopyranosyl]oxy]methyl]-2,3-bis(phenylme-
thoxy)heptadecyl]-N-non-adecanamide (25). Yield 84%. 1H NMR
(300 MHz, CDCl3): d 0.86–0.92 (m, 6 H, 2 ꢂ terminal CH3) 1.16–1.37
(m, 54 H, CH2), 1.37–1.55 (m, 2 H, CH2), 1.55–1.72 (m, 2 H, CH2),
1.81–1.97 (m, 2 H, CH2), 3.50–3.57 (m, 1 H, H-4), 3.59 (br s, 1 H, H-
4”), 3.74–3.82 (m, 2 H, Hb-1 and H-3), 3.88–3.98 (m, 3 H, Hb-6”, Ha-
1 and H-3”), 4.04–4.15 (m, 2 H, H-2” and Ha-6”), 4.18 (d, J ¼ 3.1 Hz,
1 H, H-5”), 4.25–4.35 (m, 1 H, H-2), 4.47–4.63 (m, 3 H, CH2Ph),
4.65–4.80 (m, 4 H, CH2Ph), 4.86 (d, J ¼ 11.7 Hz, 1 H, CH2Ph), 4.95 (d,
J ¼ 3.4 Hz, 1 H, H-1”), 5.46 (s, 1 H, H-8”), 5.77 (d, J ¼ 8.2 Hz, 1 H,
NHCO), 7.22–7.43 (m, 23 H, arom H), 7.48–7.55 (m, 2 H, arom H).
13C NMR (75 MHz, CDCl3) d 14.11, 22.67, 25.71, 25.83, 29.36, 29.45,
29.71, 29.80, 30.26, 31.91, 36.73, 50.33, 62.93, 68.19, 69.41, 71.72,
71.90, 73.29, 73.81, 74.36, 75.69, 76.15, 77.20, 79.49, 79.85, 99.64,
101.00, 126.31, 127.56, 127.68, 127.82, 127.88, 128.08, 128.29,
128.32, 128.35, 128.43, 128.84, 137.82, 138.40, 138.53,
138.64, 172.89.
To a solution of azide 23 (800 mg, 0.84 mmol) in THF (30 ml) at
room temperature, a 1 M solution of PMe3 in THF (12.6 ml,
12.6 mmol) was added dropwise. After stirring for 3 h at room
temperature, H2O (4 ml) was added and the reaction mixture was
allowed to stir overnight at room temperature. Then the solvent
was removed under reduced pressure and additional co-evapor-
ation with toluene to afford the crude amine. The latter was dis-
solved in CH2Cl2 (13 ml) and Et3N (3.3 ml) followed by the addition
of Boc2O (1.1 g, 5.03 mmol). The reaction mixture was stirred over-
night at room temperature, evaporated under reduced pressure
and purified by column chromatography (0%!20% EtOAc in hex-
anes) to yield 26 (703 mg, 81%) as a colourless oil. 1H NMR
(300 MHz, CDCl3): d 0.90 (t, J ¼ 6.6 Hz, terminal CH3), 1.21–1.34 (m,
20 H, CH2), 1.43 (br s, 9 H, tBu), 1.47–1.73 (m, 6 H, CH2), 3.52–3.58
(m, 1 H, H-4), 3.59 (br s, 1 H, H-5”), 3.72–3.81 (m, 2 H, Hb-1 and H-
3), 3.82–3.96 (m, 3 H, Hb-6”, Ha-1 and H-2), 3.96–4.09 (m, 2 H, H-2”
and H-3”), 4.09–4.13 (m, 1 H, Ha-6”), 4.15–4.21 (m, 1 H, H-4”),
4.42–4.56 (m, 2 H, CH2Ph), 4.56–4.68 (m, 2 H, CH2Ph), 4.73–4.88 (m,
5 H, CH2Ph and NHCO), 4.95 (d, J ¼ 3.1 Hz, 1 H, H-1”), 5.47 (s, 1 H,
H-8”), 7.18–7.44 (m, 23 H, arom H), 7.50–7.55 (m, 2 H, arom H). 13 C
NMR (75 MHz, CDCl3) d 14.11, 22.69, 25.83, 28.01, 28.40, 29.36,
29.65, 29.71, 31.92, 51.68, 62.82, 68.48, 69.41, 71.83, 73.61, 74.51,
75.60, 76.10, 77.20, 79.21, 79.43, 79.75, 99.42, 101.01, 126.31,
127.54, 127.57, 127.79, 127.88, 128.08, 128.26, 128.29, 128.32,
128.35, 128.82, 137.83, 138.47, 138.55, 138.64, 138.73, 155.34.
HRMS (ESI) m/z: calculated for C64H86NO10 [M þ H]þ 1028.6246;
found 1028.6260.
2.2.2. General procedure for the debenzylation reaction to prepare
12 and 17
A solution of the protected glycoside (0.06 mmol) in CHCl3 (3 ml)
and EtOH (9 ml) was hydrogenolysed under atmospheric pressure
in the presence of palladium black (35 mg). Upon reaction com-
pletion, the mixture was filtered through celite. The filter cake was
rinsed with CHCl3 and EtOH and the filtrate was evaporated to
dryness. After purification by column chromatography (10% !;
18% MeOH in CH2Cl2), the final compounds were obtained as
white powders in the indicated yield.
2.2.4. Synthesis of tert-butyl-N-[(1S,2S,3R)-1-[(a-D-galactopyrano-
syloxy)methyl]-2,3-dihydroxyheptadecyl]carbamate (27)
2.2.2.1. [(1S,2S,3R)-1-[(a-D-Galactopyranosyloxy)methyl]-2,3-dihy-
droxyheptadecyl]-N-pentanamide (12). Yield: 70%. 1H NMR
(300 MHz, pyridine-d5): d 0.79 (t, J ¼ 7.4 Hz, 3H, terminal CH3), 0.87
(t, J ¼ 6.7 Hz, 3H, terminal CH3), 1.16–1.51 (m, 24 H, CH2), 1.57–1.79
(m, 3 H, CH2), 1.80–1.99 (m, 2 H, CH2), 2.21–2.35 (m, 1 H, CH2), 2.39
(t, J ¼ 7.9 Hz, 2H, CH2), 4.27–4.33 (m, 2 H), 4.34–4.46 (m, 4 H), 4.51
(t, J ¼ 6.0 Hz, 1 H), 4.56 (d, J ¼ 2.7 Hz, 1 H), 4.62–4.71 (m, 2 H),
5.21–5.31 (m, 1 H, H-2), 5.57 (d, J ¼ 3.8 Hz, 1 H, H-1”), 6.39 (br s,
6 H, OH), 8.43 (d, J ¼ 8.5 Hz, 1 H, NH). 13 C NMR (75 MHz, pyridine-
d5) d 14.55, 14.84, 23.25, 23.48, 27.06, 28.93, 30.15, 30.46, 30.54,
A solution of 26 (703 mg, 0.68 mmol) in CHCl3 (6 ml) and EtOH
(18 ml) was hydrogenolysed under atmospheric pressure in the
presence of palladium black (50 mg). Upon reaction completion,
the mixture was filtered through celite. The filter cake was rinsed
with CHCl3 and EtOH and the filtrate was evaporated to dryness.
After purification by column chromatography (10% !18% MeOH
in DCM), compound 27 (242 mg, 61%) was obtained as a pale
1
yellowish solid. H NMR (300 MHz, pyridine-d5): d 0.88 (t, J ¼ 6.4 Hz,
30.69, 30.89, 32.67, 34.84, 36.99, 52.06, 63.16, 69.02, 70.85, 71.49, 3H, terminal CH3), 1.15–1.34 (m, 21 H, CH2), 1.37–1.46 (m, 1 H,
72.13, 73.03, 73.54, 77.14, 102.01, 173.90. HRMS (ESI) m/z: calcu-
CH2), 1.51 (s, 9 H, tBu), 1.60–1.75 (m, 1 H, CH2), 1.80–1.98 (m, 2 H,
CH2), 2.22–2.35 (m, 1 H, CH2), 4.24–4.35 (m, 3 H, Ha-1, H-3”, H-4),
4.38–4.53 (m, 4 H, CH2-6”, H-4” and H-3”), 4.57 (d, J ¼ 3.0 Hz, 1 H,
H-5”), 4.61–4.74 (m, 2 H, H-2” and Hb-1), 4.91–5.01 (m, 1 H, H-2),
5.56 (d, J ¼ 3.8 Hz, 1 H, H-1”), 6.41 (br s, 6 H, OH), 7.46 (d, J ¼ 9.1 Hz,
1 H, NH). 13 C NMR (75 MHz, pyridine-d5) d 14.86, 23.50, 27.03,
29.13, 30.17, 30.48, 30.54, 30.58, 30.68, 30.90, 32.68, 34.87, 53.09,
63.10, 68.94, 70.82, 71.47, 72.16, 72.94, 73.41, 77.24, 79.06, 101.81,
157.14. HRMS (ESI) m/z: calculated for C34H63N2O10
lated for C29H58NO9 [M þ H]þ 564.4106; found 564.4094.
2.2.2.2. [(1S,2S,3R)-1-[(a-D-Galactopyranosyloxy)methyl]-2,3-dihy-
droxyheptadecyl]-N-non-adecanamide (17). Yield 42%. 1H NMR
(300 MHz, pyridine-d5): d 0.88 (t, J ¼ 6.4 Hz, 6H, 2 ꢂ terminal CH3),
1.11–1.50 (m, 52 H, CH2), 1.60–1.75 (m, 1 H, CH2), 1.76–1.99 (m, 4 H,
CH2), 2.23–2.37 (m, 1 H, CH2), 2.46 (t, J ¼ 7.5 Hz, 2 H, CH2),
4.30–4.36 (m, 2 H), 4.38–4.48 (m, 4 H), 4.53 (t, J ¼ 6.1 Hz, 1 H), 4.56
(d, J ¼ 3.1 Hz, 1 H), 4.63–4.72 (m, 2 H), 5.23–5.33 (m, 1 H, H-2), 5.59 [M þ pyridine þ H]þ 659.4477; found 659.4462.

1506
W. LI ET AL.
2.2.5. Synthesis of [(1S,2S,3R)-1-[(a-D-galactopyranosyloxy)- (adjusted by phosphoric acid), electrokinetic injection (ꢃ10 kV,
30 s). The capillary was washed with 0.2 M NaOH for 2 min, and
running buffer for 2 min before each injection. Data collection and
corrected peak area analysis were performed with the 32 Karat
software obtained from Beckman Coulter (Fullerton, CA). Further
data analysis was carried out with Graph Pad Prism 4 (Graph Pad
Software, Inc. San Diego, CA) and Excel. The human CST enzyme
methyl]-2,3-dihydroxyheptadecyl]-N-tetracos-15-enamide (22)
The Boc-protected glycophytosphingosine 27 (150 mg, 0.26 mmol)
was dissolved in CH2Cl2 (20 ml) and 1 M HCl in 90% aqueous
AcOH solution (100 lL) was added at room temperature. After
35 min TLC showed incomplete conversion of the starting material
and another amount of the HCl solution (100 lL) was added. This
process was repeated till TLC (CH2Cl2/MeOH: 8/2) showed full con- was obtained by heterologous expression in CHO cells in analogy
to a described procedure19. The CE assay method has previously
version of the starting material. This required a total addition of
800 lL of the HCl solution. The solvents were removed under
been reported18.
reduced pressure and the residue was co-evaporated with MeOH
(3 ꢂ 5 ml). The resulting crude amine (used without further purifi-
2.3.1. Determination of kinetic parameters for CST
cation) was dissolved in a biphasic mixture of THF (2.5 ml) and
saturated aqueous NaOAc (2.5 ml). In a separate flask, nervonic
acid (114 mg, 0.31 mmol) was refluxed for 2 h in oxalyl chloride
(4 ml) and the crude formed acyl chloride, obtained after evapor-
ation of the solvent by a stream of nitrogen and subsequent dry-
ing on high-vacuum, was dissolved in THF (2.5 ml) and added
dropwise to the biphasic mixture. The reaction mixture was stirred
for 2 h at room temperature and TLC showed complete conversion
of the starting material. Next, the aqueous layer was extracted
with THF (3 ꢂ 15 ml) and the combined organic layer was dried
over Na2SO4, filtered and evaporated. The crude residue was puri-
fied by column chromatography (10% ! 16% MeOH in CH2Cl2)
furnishing final compound 22 (79 mg, 37%) as a pale solid. 1H
NMR (300 MHz, pyridine-d5): d 0.86 (dt, J ¼ 6.7, 5.4 Hz, 3H, terminal
CH3), 1.15–1.49 (m, 54 H, CH2), 1.55–1.74 (m, 1 H, CH2), 1.74–1.98
(m, 4 H, CH2), 2.07–2.17 (m, 4 H, CH2), 2.21–2.35 (m, 1 H, CH2), 2.43
(t, J ¼ 7.4 Hz, 2 H, CH2), 4.21–4.37 (m, 2 H, H-3, H-4), 4.38–4.48 (m,
4 H, Ha-1, H-3” and CH2-6”), 4.53 (t, J ¼ 6.3 Hz, 1 H, H-5”), 4.57 (d,
J ¼ 2.9 Hz, 1 H, H-4”), 4.63–4.73 (m, 2 H, H-2” and Hb-1), 4.96 (br s,
1 H, OH), 5.24–5.33 (m, 1 H, H-2), 5.49–5.55 (m, 2H, CH ¼ CH), 5.59
(d, J ¼ 3.7 Hz, 1 H, H-1”), 6.10 (br s, 1 H, OH), 6.21–6.79 (m, 3 H, 3 x
OH), 6.96 (br s, 1 H, OH), 8.48 (d, J ¼ 8.8 Hz, 1 H, NH). 13 C NMR
(75 MHz, pyridine-d5) d 14.77, 23.41, 26.89, 26.99, 28.02, 30.03,
30.11, 30.26, 30.31, 30.38, 30.41, 30.49, 30.60, 30.64, 30.86, 32.58,
32.61, 34.81, 37.28, 51.97, 63.14, 69.13, 70.79, 71.48, 72.09, 72.97,
73.52, 77.17, 102.01, 130.73, 173.77. HRMS (ESI) m/z: calculated for
C48H94NO9 [M þ H]þ 828.6923; found 828.6942.
For the determination of kinetic parameters (Km and Vmax), eight
different substrate concentrations were chosen. Negative controls
were performed in the presence of heat-inactivated enzyme
(10 min, 60 ꢄC). Each analysis was repeated three times in inde-
pendent experiments.
2.3.2. Investigation of CST inhibitors
For CST inhibitor characterisation, full concentration–inhibition
curves were determined by testing a suitable range of inhibitor
concentrations, to determine IC50 values. Ki values were calculated
according to the Cheng–Prusoff equation20. The substrate concen-
tration was 100 mM of galactosylceramide (3), and 466 mM of
KRN7000 (18), respectively, and the concentration of the cofactor
PAPS was 30 lM. Substrate conversion was strictly controlled to
be below 15%. Negative controls were performed in the presence
of heat-inactivated enzyme (10 min, 60 ꢄC). Each analysis was
repeated three times in independent experiments.
3. Results and discussion
3.1. Chemistry
A series of analogues of the natural CST substrate galactocerebro-
side with variations in the galactose moiety (a- and b-glycosides,
substitution of the sugar moiety), in the hydroxylated alkyl chain,
and in the fatty acid residue was designed and synthesised.
b-KRN700021 and a-glycosides 11, 13–16 and 18–21 were pre-
pared as previously described.22–27 The synthetic route to obtain
a-galactosylceramides 12, 17 and 22 is depicted in Scheme 1.
The required azidoglycoside 23 was obtained through Lewis
acid-catalysed glycosidation reaction as previously reported28.
Staudinger reduction and subsequent EDC-mediated acylation
with the appropriate fatty acid furnished 24 and 25. Final catalytic
hydrogenolysis yielded a-galactosylceramides 12 and 17. To avoid
saturation of the cis-double bond of 15-tetracosanoic acid, the
amino group generated after Staudinger reduction was Boc-pro-
tected prior to the removal of the benzyl groups to afford inter-
mediate 27. Subsequent removal of the Boc moiety with HCl in
acetic acid gave the corresponding psychosine derivative, which
was subjected to Schotten–Baumann acylation with 15-tetracosa-
noyl chloride to afford glycolipid 22.
2.3. Biological evaluation
The CST reaction was carried out in a total volume of 50 ml con-
taining 30-phosphoadenosine-50-phosphosulphate and galactosyl-
ceramide
(concentrations
of
30-phosphoadenosine-50-phos
phosulphate and galactosylceramides varied according to assay
type) in reaction buffer (10 mM HEPES, 16 mM MgCl2, 0.2% (v/v)
Triton X-100, pH 7.1). All lipids and Triton X-100 were dissolved in
chloroform/methanol (1:1), pipetted into reaction vials, and the
chloroform/methanol mixture was removed by drying before add-
ing reaction buffer. Reactions were initiated by the addition of
938 ng of human galactosylceramide sulphotransferase (CST), and
then incubated at 37 ꢄC for 30 min. All enzymatic reactions were
stopped by heating for 10 min at 60 ꢄC.
Analytical experiments were carried out by using a P/ACE MDQ
capillary electrophoresis (CE) system (Beckman Instruments,
Fullerton, CA) equipped with a DAD detection system. The capil-
lary temperature was kept constant at 15 ꢄC. The electrophoretic
separations were carried out by using fused-silica capillary of
60 cm total length (50 cm effective length) ꢂ 75.5 mm (id) ꢂ
363.7 mm (od) obtained from Optronis GmbH. The following condi-
tions were applied: kmax ¼ 260 nm, voltage ¼ ꢃ15 kV, running
3.2. Biological evaluation
The synthesised compounds were studied using a previously
developed capillary electrophoresis-based assay, in which the con-
version of the co-substrate PAPS, acting as a sulphate donor, to
adenosine-30,50-bisphosphate (PAP) was measured18. In addition to
buffer 75 mM phosphate buffer, 0.002% polybrene, pH 5.6 the natural substrate galactosylceramide (3), which contains a

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1507
Scheme 1. Reagents and conditions: (a) (i) PMe3, THF, then H2O; (ii) appropriate RCOOH, EDC, CH2Cl2; (b) H2, Pd black; (c) (i) PMe3, THF, then H2O; (ii) (Boc)2O, Et3N,
CH2Cl2; (d) (i) HCl, AcOH, H2O; (ii) nervonic acid, oxalyl chloride, reflux; the acyl chloride is added to crude amine in THF/aq. NaOAc.
b-galactosyl residue and is present in nerve tissues as a constitu- (42% compared to 100% for galactosylceramide) and an increased
ent of myelin, the corresponding b-glucosyl derivative glucocere-
broside (9) was investigated, which is mainly found in liver and
spleen. As another naturally occurring sphingolipid psychosine
(galactosyl-b-sphingosine, 8) was investigated, which is a cytotoxic
derivative of galactosylceramide (3) that lacks its fatty acid resi-
due. Moreover, 13 glycolipids, four of which (12, 17, 20 and 22)
are new compounds, not previously described in literature, were
investigated as artificial substrate analogues and/or competitive
inhibitors of CST (Table 1). Enzyme kinetic parameters
(Michaelis–Menten constant and maximal velocity) were deter-
mined for all (artificial) substrates for which significant conversion
of >20% as compared to the natural substrate (set at 100%)
was observed.
Km value of 550 mM (compared to 60 mM for galactosylceramide)
determined under the same conditions. Interestingly, the a-galac-
toside anomer of compound 10, KRN7000 (18) showed about the
same conversion rate and Km value indicating that for this series
of more polar, hydrated sphingosine-derived synthetic lipids, the
enzyme did not discriminate between a- and b-glycosidic config-
uration. Thus, we investigated further a-galactosyl-lipids (11–17,
19–22) derived from KRN7000 (18) mainly with modification of
the fatty acid residue. KRN7000 (18) had previously been found to
display immunostimulatory and antitumor activity in several
in vivo models, and was advanced to clinical trials29–31
.
Using the artificial CST substrate KRN7000 (18) as a lead struc-
ture, we modified the fatty acid amide moiety. Compound 21,
which lacks the fatty acid residue, was not well accepted as a sub-
strate in this series (19% conversion). We subsequently investigated
in a systematic manner the introduction of saturated fatty acid resi-
dues with increasing chain length. Compound 11 having a short
fatty acid residue, namely acetyl, was tolerated as a substrate (40%
conversion) confirming that a long fatty acid residue is not required
for interaction with the enzyme and acceptance as a substrate.
Probing the optimal length of the fatty acid residue led to the fol-
lowing result: C2 (acetyl, 11), C5 (12), C8 (13), C11 (14) and C19
3.3. Structure–activity relationships
3.3.1. Substrates
The natural substrates of CST in nerve tissues are b-galactosylcera-
mides (3) which are converted into 3-O-sulphogalactosylcerebro-
side that constitutes about 4% of total myelin lipids. They are
sphingolipids consisting of (i) a sphingosine residue, (ii) a b-galact-
ose, and (iii) a fatty acid residue attached via an amide linkage.
Natural galactosylceramide (3) may contain different saturated or (17) were about similarly good substrates (Table 1). Medium chain
unsaturated (hydroxy)fatty acid residues with chain lengths mostly
between C18 and C27, frequently C24. Galactosylceramide dis-
played a Km value of 60.3 mM determined in our recently devel-
oped capillary electrophoresis-based assay and was the best
substrate of all compounds investigated in the present study19.
The corresponding b-glucosylceramide (9) were shown to be
much weaker substrates with only 19% conversion compared to
that of 3 (set at 100%) determined under the same conditions.
Another natural sphingolipid that had previously been shown to
be a substrate of CST is psychosine (8) which is lacking the fatty
acid residue of galactosylceramides (3). Psychosine was still effi-
ciently sulphurylated by the enzyme showing an only moderately
reduced Km value of 103 mM in the same assay19. A synthetic gal-
actosylceramide analogue, b-KRN7000 (10), in which the double
bond of the sphingosine core structure is hydrated and, therefore,
fatty acid residues appeared to be less well tolerated than some
shorter chain analogs. In particular, palmitic acid (C16, 16) showed
a sulphurylation rate below 10%. Interestingly, further increase of
the chain length in compound 18 (C26) led to the best substrate in
this series of a-galactosides with 51% conversion. Aromatic substitu-
tion in fatty acid amide analogue 19 was tolerated (compare 19
with 13 and 14), while the C24-fatty acid containing a cis-double
bond was not a very good substrate. A bulky aromatic substitution
on the sugar moiety in 20 strongly reduced the conversion rate
(compare 20 with 18). Typical Michaelis–Menten curves are shown
in Figure 3 for selected substrates (10, 11, 13 and 18).
3.3.2. Inhibitors
Substrate analogues that were not or only poorly converted by CST
contains an additional hydroxy group, led to reduced conversion with a conversion rate below 20% as compared to galactosylceramide

1508
W. LI ET AL.
Table 1. Investigation of analogues of galactocerebroside as substrates of CSTa.
Percent conversiona
Vmax
a
a
a
Compound
Structure
at 100 mM
Km (mM)
(mmol/min/mg protein)
kcat/Km
b-Glycosides
3 Galactosylceramideb
100%c
60.318
0.073818
12318
8 Psychosine
n.d.
10318
0.10518
10218
9 Glucosylceramideb
19%
n.d.
n.d.
n.d.
10 b-KRN7000
42%
550
0.113
20.5
a-Glycosides
11
40%
30%
22%
32%
907
358
392
493
0.1080
0.0449
0.0584
0.0590
11.9
12.5
15.0
12.0
12
13
14
15
20%
n.d.
n.d.
n.d.
(continued)

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1509
Table 1. Continued.
Percent conversiona
Vmax
a
a
a
Compound
Structure
at 100 mM
Km (mM)
(mmol/min/mg protein)
kcat/Km
16
9%
n.d.
n.d.
n.d.
17
25%
51%
521
0.0519
10.0
18 KRN7000
438
751
0.0758
0.0834
17.3
11.1
19
32%
20
15%
n.d.
n.d.
n.d.
21
22
16%
19%
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
aStandard errors were typically below 30% of the reported mean values.
bR: saturated or unsaturated alkyl or hydroxyalkyl residue (C16–C27).
cConversion of physiological substrate was set at 100%.
Km and Vmax values are shown in bold.
might still be able to bind to the enzyme and thereby act as competi- concentration. Only derivative 16 displayed measurable enzyme inhib-
tive inhibitors. Therefore, we investigated compounds 9, 16, and ition at this concentration (Table 2).
10–22 for inhibition of CST activity using two different substrates, the
Subsequently, concentration-dependent inhibition was deter-
natural galactosylceramide (3) and the synthetic a-galactoside mined against both substrates, and nearly identical Ki values were
KRN7000 (18). Initial inhibitor screening was performed at 100 mM determined against both substrates, 127 mM and 159 mM,

1510
W. LI ET AL.
10
18
respectively (see Table 2 and Figure 4). To our knowledge, this is
the first reported substrate-competitive inhibitor of CST.
0.06
11
13
0.04
0.02
4. Conclusions
In conclusion, we synthesised and investigated selected analogues
of the natural CST substrate galactosylceramide (3), which were
related to the artificial substrate KRN7000 (18). Our aim was to
study structure–activity relationships for substrates of the enzyme,
and to identify competitive inhibitors. We obtained detailed SAR
information for the CST galactosylceramide binding site by analy-
sing artificial substrates. Most importantly, a novel, competitive
CST inhibitor (16) was identified. Compound 16 can serve as a
0.00
0
100
200
300
400
[Substrate], M
Figure 3. Enzyme kinetics of galactosylceramide sulphotransferase for selected
substrates. For Km and Vmax values see Table 1.
Table 2. CST inhibitory activity of galactocerebroside analogues
Ki SEM (mM) versus
galactosylceramide (% inhibition at
Ki SEM (mM) versus KRN7000 (%
inhibition at 100 mM)
Compound
Structure
100 mM)
b-Glycosides
9 Glucosylceramide
(9%)
(ꢃ5%)
R= saturated or unsaturated alkyl
or hydroxyalkyl residue
R ¼ saturated or unsaturated alkyl
or hydroxyalkyl residue
a-Glycosides
16
127 12
159 55
20
(4%)
(1%)
21
22
(4%)
(ꢃ30%)
(ꢃ7%)
(1%)

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1511
125
100
75
50
25
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Disclosure statement
The authors declare no conflict of interest, financial or otherwise.
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W. L. is grateful for support by the Deutscher Akademischer
Austauschdienst (DAAD, research assistantship stipend). Y. B. was
funded by Arab-German Young Academy of Sciences and
Humanities (AGYA). J. G. is a fellow of the Agency for Innovation
by Science and Technology (IWT) of Flanders.
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ORCID
Younis Baqi
http://orcid.org/0000-0002-9659-8419
Serge Van Calenbergh
http://orcid.org/0000-0002-4201-1264
http://orcid.org/0000-0002-0013-6624
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galactosylceramide sulphotransferase. Biochem J 2002;368:
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