Stereoselective synthesis and NMR characterization of C-24 Epimeric Pairs of 24-Alkyl oxysterols

Article abstract of DOI:10.1007/s11745-012-3739-1

Two pairs of C-24 epimeric (24R)-/(24S)-24-hydroxy-24-methyl-5α- cholestan-3β-yl acetates and (24R)-/(24S)-25-hydroxy-24-methyl-5α- cholestan-3β-yl acetates as well as some related 24-ethyl oxysterol analogs were stereoselectively synthesized directly from the respective parent 24-alkyl sterols by a remote O-insertion reaction with 2,6-dichloropyridine N-oxide (DCP) in the presence of a catalytic amount of (5,10,15,20- tetramesitylporphrinate) ruthenium(II) carbonyl complex [Ru(TMP)CO] and HBr. ¹H- and ¹³C-NMR signals serving to differentiate each of the two epimeric pairs were interpreted. The C-24 alkyl oxysterols epimeric at C-24 were found to be effectively characterized by the aromatic solvent-induced shift (ASIS) by C₅D₅N, particularly for the difference in the ¹³C resonances in the substituted cholestane side chain. A method for differentiating the ¹H and ¹³C signal assignment of the terminal 26-/27-CH₃ in the iso-octane side chain was also discussed on the basis of a combined use of the preferred conformational analysis and HMQC and HMBC techniques. The present method may be useful for determining the stereochemical configuration at C-24 of this type of 24-alkyl oxysterols.

Full text of DOI:10.1007/s11745-012-3739-1

Lipids (2013) 48:197–207
DOI 10.1007/s11745-012-3739-1
METHODS
Stereoselective Synthesis and NMR Characterization
of C-24 Epimeric Pairs of 24-Alkyl Oxysterols
•
Shoujiro Ogawa Hiroaki Kawamoto
•
•
Takashi Mitsuma Hiroki Fujimori
•
•
Tatsuya Higashi Takashi Iida
Received: 24 August 2012 / Accepted: 22 October 2012 / Published online: 30 November 2012
Ó AOCS 2012
Abstract Two pairs of C-24 epimeric (24R)-/(24S)-24-
hydroxy-24-methyl-5a-cholestan-3b-yl acetates and (24R)-/
(24S)-25-hydroxy-24-methyl-5a-cholestan-3b-yl acetates
as well as some related 24-ethyl oxysterol analogs were
stereoselectively synthesized directly from the respective
parent 24-alkyl sterols by a remote O-insertion reaction
with 2,6-dichloropyridine N-oxide (DCP) in the presence
of a catalytic amount of (5,10,15,20-tetramesitylpor-
phrinate) ruthenium(II) carbonyl complex [Ru(TMP)CO]
and HBr. 1H- and 13C-NMR signals serving to differentiate
each of the two epimeric pairs were interpreted. The C-24
alkyl oxysterols epimeric at C-24 were found to be effec-
tively characterized by the aromatic solvent-induced shift
Abbreviations
ASIS
LIS
Aromatic solvent-induced shift
Lanthanide-induced shift
NMR
Nuclear magnetic resonance
HMQC Heteronuclear multiple quantum coherence
HMBC Heteronuclear multiple bond correlation
DEPT
HR
Distortionlessenhancementby polarizationtransfer
High resolution
ESI
Electrospray ionization
MS
Mass spectrometry
(ASIS) by C5D5N, particularly for the difference in the 13
C
Introduction
resonances in the substituted cholestane side chain. A
1
method for differentiating the H and 13C signal assign-
Cholesterol is biosynthesized in the liver of mammals
including humans and then transformed into various ste-
roid hormones and bile acids. Phytosterols (e.g., cam-
pesterol, stigmasterol, and b-sitosterol) are cholesterol-like
naturally occurring steroids found in many plants [1].
They block the absorption of cholesterol in the small
intestines and can help lower the level of serum choles-
terol and reduce the risk of heart disease [2]. Ergosterol
(provitamin D2) and its analogs, which are components of
fungal cell membranes, do not belong to phytosterols, but
their chemical structures are also very similar to phytos-
terols. These steroids in biological materials are all clas-
sified into the category of so-called ‘‘sterols’’, because
their chemical structures vary only in the presence or
absence of an alkyl group and/or a double bond in the C27
parent cholestane structure.
ment of the terminal 26-/27-CH3 in the iso-octane side
chain was also discussed on the basis of a combined use of
the preferred conformational analysis and HMQC and
HMBC techniques. The present method may be useful for
determining the stereochemical configuration at C-24 of
this type of 24-alkyl oxysterols.
Keywords Alkylsterol Á Oxysterol Á 24-Hydroxylation Á
1
25-Hydroxylation Á (24R)-/(24S)-Epimer Á H NMR Á
13C NMR Á Aromatic solvent-induced shift by C5D5N
S. Ogawa Á T. Higashi
Faculty of Pharmaceutical Sciences, Tokyo University
of Science, Noda, Chiba 278-8510, Japan
H. Kawamoto Á T. Mitsuma Á H. Fujimori Á T. Iida (&)
Department of Chemistry, College of Humanities and Sciences,
Nihon University, Sakurajousui, Setagaya,
Tokyo 156-8550, Japan
Meanwhile, oxysterols are defined as the oxygenated
derivatives of sterols having additional hydroxy and/or oxo
groups. In human, oxysterols have a remarkably diverse
e-mail: takaiida@chs.nihon-u.ac.jp
123

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Lipids (2013) 48:197–207
Instruments
profile of biological activities, including effects on cho-
lesterol homeostasis, sphingolipid metabolism, platelet
aggregation, apoptosis, and protein prenylation [3–9]. It is
also suggested that 24-hydroxycholesterol, 25-hydroxy-
cholesterol, and 27-hydroxycholesterol in serum can be
used as a marker of Alzheimer’s disease, cerebrotendinous
xanthomatosis, Smith-Lemli-Opitz syndrome, and neuro-
degenerative diseases, respectively [10–15].
All melting points (mp) were determined on a micro hot-
stage apparatus and are uncorrected. NMR spectra were
recorded at 23 °C in either CDCl3, C6D6 or C5D5N by
using a JEOL ECA-500 instrument (500.2 MHz for 1H and
125.8 MHz for 13C). Chemical shifts were expressed in d
(ppm) scale, relative to internal Me4Si and coupling con-
stants (J) in Hz. The H and 13C resonance assignments
1
In addition, a considerable number of new oxysterols
have recently been identified, mainly from patients with a
hepatobiliary disease and marine organisms, which contain
additional alkyl and/or hydroxy groups at other positions of
the C8iso-octane (cholestane) side chain [10–15]. These
substituted oxysterols possess an added a chiral center and
present the possibility of the appearance in nature of two
epimers from the chiral carbon. Thus, the epimers of ox-
ysterols do exist, either individually or as a mixture of the
two epimers, in various organisms and the determination of
the stereochemistry at a chiral center of oxysterols has been
recognized to be important from both biogenetic and
phylogenetic viewpoints. Furthermore, oxysterols in bio-
logical fluids are of keen current interest in metabolic and
catabolic studies. Interest in oxysterols is also due to their
physiological function and bioactivity and use as important
biomarkers of various human diseases. However, these
naturally occurring and specific oxysterols are present only
in extremely low concentrations relative to the parent
sterols.
were made using a combination of two-dimensional (2D)
homonuclear (1H–1H) and heteronuclear (1H–13C) shift-
1
correlated techniques, which include H detected hetero-
nuclear multiple quantum correlation (HMQC; 1H–13C
coupling) and 1H detected heteronuclear multiple bond
1
correlation (HMBC; long-range H–13C coupling) experi-
ments. These 2D-NMR spectra were recorded using stan-
dard pulse sequences and parameters recommended by the
manufacturer. The 13C distortionless enhancement by
polarization transfer (DEPT; 135°, 90°, and 45°) spectra
were also measured to determine the exact 13C signal
multiplicity and to differentiate between CH3, CH2, CH,
and C based on their proton environments. High-resolution
mass spectra using an electrospray ionization (HR–ESI–
MS) were carried out using a JEOL AccuTOF JMS-
T100LC liquid chromatograph-mass spectrometer equip-
ped with an ESI source and coupled to an Agilent 1100
series binary pump (Agilent Technologies Inc., Santa
Clara, CA, USA) operated in the positive ion mode. The
HR-ESI–MS of compounds 2 and 4 were carried out in the
flow injection mode, using 10 mM ammonium formate (pH
6.5) and methanol mixture (1:99, v/v) as the mobile phase
at a flow rate of 200 lL/min. The ionization conditions
were as follows: needle voltage, 2 kV; ion guide peak
voltage, 2 kV; ion source temperature, 80 °C; desolvation
temperature, 250 °C; orifice voltage, 75 V; mass range,
m/z 50–1,000; nebulizing gas, N2 gas. Normal-phase TLC
was performed on pre-coated silica gel plates (0.25 mm
layer thickness; E. Merck, Darmstadt, Germany) using a
mixture of hexane–EtOAc (4:1, v/v) as the developing
solvent.
As part of a program in our laboratory to synthesize
novel and scarce oxysterols that have clinical utility as
biomarkers of diseases as well as authentic reference
compounds in chromatographic and spectroscopic analysis
[16], we report herein the chemical synthesis of 24- and
25-hydroxylated derivatives of 24-alkylsterols epimeric at
1
C-24 and some related compounds and their H and 13C
NMR characteristics.
Materials and Methods
Materials and Reagents
Stereoselective Synthesis of Compounds 2 and 4
Campesterol [(24R)-24-methyl-cholest-5-en-3b-ol], obtained
from Tama Biochemical Co. Ltd., (Tokyo, Japan), was treated
with acetic anhydride and pyridine, followed by catalytic
hydrogenation of the resulting acetate derivative with a PtO2
catalyst according to the general procedure to give campesta-
nyl acetate [(24R)-24-methyl-5a-cholestan-3b-yl acetate].
Ergostanyl acetate [(24S)-24-methyl-5a-cholestan-3b-yl ace-
tate] was from a collection in our laboratory. All other chem-
icals and solvents were of analytical reagent grade and
available from commercial sources.
To a magnetically stirred solution of campestanyl acetate
(30 mg, 68 lmol) in dry benzene (0.2 mL) and molecular
˚
sieves (50 mg, 4 A), freshly prepared 2,6-dichloropyridine
N-oxide (DCP) (38 mg, 230 lmol), (5,10,15,20-tetra-
mesitylporphyrinate) ruthenium(II) carbonyl complex
[Ru(TMP)CO] (0.3 mg, 0.33 lmol), and HBr (3 lL) were
successively added, and the mixture was stirred at 50 °C
for 48 h; the reaction was monitored by TLC. After cooling
at room temperature, the reaction product was extracted
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Lipids (2013) 48:197–207
199
with toluene, and the combined extract was washed with
water, dried with DrieriteÒ, and evaporated to dryness
under reduced pressure. The oily product, which consisted
of a mixture of two major components by TLC, was
chromatographed on a column of silica gel (10 g). Elution
with hexane–EtOAc (9:1–8:2, v/v) provided two-well
separated fractions.
acetate; (24S)-24-methyl-5a-cholestan-3b-yl acetate] and
campestanyl acetate [(24R)-24-methyl-5a-cholestan-3b-yl
acetate], which are derived from ergosterol and campes-
terol, respectively, by acetylation and subsequent hydro-
genation of the resulting acetate derivatives. When the
remote O-insertion reaction by Ru(TMP)CO/DCP/HBr was
subjected to ergostanyl acetate under mild conditions (see
Materials and Methods), two major products were isolated
after chromatographic purification. Based on the previous
findings [17, 18], the two products were identified as (24R)-
24-hydroxy-ergostanyl acetate [(24R)-24-hydroxy-24-
methyl-5a-cholestan-3b-yl acetate; 1] and (24S)-25-
hydroxy-ergostanyl acetate [(24S)-25-hydroxy-24-methyl-
5a-cholestan-3b-yl acetate; 3] without conclusive evidence.
In a similar manner, the O-insertion reaction of campestanyl
acetate with Ru(TMP)CO/DCP/HBr afforded exclusively
(24S)-24-hydroxy-campestanyl acetate [(24S)-24-hydroxy-
24-methyl-5a-cholestan-3b-yl acetate; 2] and (24R)-25-
hydroxy-campestanyl acetate [(24R)-25-hydroxy-24-methyl-
5a-cholestan-3b-yl acetate; 4].
The less polar fraction was identified as (24S)-24-
hydroxy-campestanyl acetate (2) which crystallized from
aqueous methanol as colorless needles; yield, 5.2 mg (17 %);
mp, 172–174 °C. High resolution LC–ESI–MS, m/z Calc. for
C30H53O3 [M ? H]?: 461.3995, Found: m/z 461.4002.
The more polar fraction was recrystallized from aqueous
methanol to give (24R)-25-hydroxy-campestanyl acetate
(4) as colorless needles; yield, 7.6 mg (24 %); mp,
159–160 °C. High resolution LC–ESI–MS, m/z Calc. for
C30H53O3 [M ? H]?: 461.3995, Found: m/z 461.4014.
Stereoselective Synthesis of Compounds 1 and 3
Ergostanyl acetate (500 mg, 1.1 mmol), subjected to the
remote-oxidation with Ru(TMP)CO/DCP/HBr and pro-
cessed as described for the preparation of 2 and 4, afforded
two major products. After chromatographic purification on
a column of silica gel, eluting with hexane–EtOAc
(9:1–8:2, v/v), gave two well-separated fractions. The less
polar component was determined as (24R)-24-hydroxy-
ergostanyl acetate (1) (100 mg, 19 %) and the more polar
component as (24S)-25-hydroxy-ergostanyl acetate (3)
(120 mg, 23 %), according to mp, LC–MS, and NMR
comparisons with the authentic samples [17, 18].
For the purpose of comparison, (24S)-24-hydroxy-stigm-
astanyl acetate [(24S)-24-ethyl-24-hydroxy-5a-cholestan-
3b-yl acetate; 5] and (24R)-25-hydroxy-stigmastanyl acetate
[(24R)-24-ethyl-25-hydroxy-5a-cholestan-3b-yl acetate; 6],
prepared in the previous papers [17, 18], were also examined.
The chemical structures of stereoisomeric 24-alkyl oxysterol
(1–6) examined in this study are depicted in Fig. 1. These
compounds differ from one another in the stereochemical
configuration at C-24, the site of hydroxy group (at C-24 or
C-25), and/or alkyl substituents (methyl or ethyl at C-24) in
the cholestane side chain attached at C-17.
1
The H- and 13C-NMR spectra of sterols and oxysterols
and their conjugates (e.g., sulfated derivatives) are con-
ventionally measured in CDCl3 or CD3OD as a solvent,
depending upon their solubility. However, our preliminary
work revealed that when (24S)-24-hydroxy-campestanyl
acetate (2) was measured in CDCl3, its 500 MHz 1H-NMR
spectrum was unsatisfactory because of the serious CH3
Results and Discussion
We have recently reported a new stereospecific remote-
hydroxylation of a variety of steroids with ethyl(trifluoro-
methyl)dioxirane [16], dimethyldioxirane [17] or
2,6-dichloropyridine N-oxide (DCP) catalyzed by (5,10,15,
20-tetramesitylporphrinate) ruthenium(II) carbonyl com-
plex [Ru(TMP)CO] and HBr [18]. The remote O-insertion
reaction proceeded smoothly, particularly at the hydrogen
atom of an unactivated methine carbon (R3C–H) in the
substrates, to give the corresponding hydroxylated deriva-
tives (R3C–OH) stereoselectively [19, 20]. Alternatively,
the stereochemical nature of a newly inserted hydroxy
group (e.g., R3C OH) of the oxygenated products was
completely retained to that of the hydrogen atom
(R3C H) in the starting substrates.
1
signal overlapping. To improve H and 13C signal resolu-
tion, the application of aromatic solvent-induced shift
(ASIS) and lanthanide-induced shift (LIS) techniques have
been well recognized in previous literatures [21, 22]. Of the
two techniques, the ASIS is available in view of the simple
procedure, short measuring time, and easy recovery of
1
samples, compared to the LIS. Figure 2 illustrates the H-
NMR spectra of the compound 2 measured in three variants
of the solvent, i.e., CDCl3, C6D6, and pyridine-d6 (C5D5N).
As can be seen it, the spectral patterns as well as the
1
chemical shifts of each H signal were significantly chan-
Our initial effort was directed toward stereoselective
synthesis of the two C-24 epimer pairs of 24-alkyl oxys-
terols (1 vs. 2; 3 vs. 4). The starting materials chosen for our
synthesis were ergostanyl acetate [tetrahydrobrassicasteryl
ged by the ASIS effect on the CH3 signals occurring at up-
field region of ca. 0.6–1.4 ppm. Particular noteworthy was
that although four CH3 signals (21-, 26-, 27-, and 28-CH3)
measured in CDCl3 and C6D6 solvents are partially
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Lipids (2013) 48:197–207
(a)Solvent: CDCl3
21-CH3
0.89 ppm (d)
27-CH3, 26-CH3
0.92 ppm (d)
28-CH3
1.07 ppm
19-CH3
18-CH3
(b)Solvent: C6D6
26-CH3
0.93 ppm (d)
19-CH3
28-CH3
1.01 ppm
18-CH3
21-CH3
0.87 ppm (d)
27-CH3
1.01 ppm (d)
(c) Solvent: C5D5N
26-CH3
1.09 ppm (d)
19-CH3
18-CH3
27-CH3
1.17 ppm (d)
28-CH3
1.33 ppm
21-CH3
1.02 ppm (d)
Fig. 1 Chemical structures of 24- and 25-hydroxylated oxysterols
(1–6) examined
1.4
1.2
1.0
0.8
0.6
overlapped, these signals are completely resolved from one
another in C5D5N. Therefore, the use of C5D5N seemed to
be a more suitable solvent for measuring this type of
24-alkyl oxysterols.
1
Fig. 2 A part of the H-NMR spectra of (24S)-24-hydroxy-campes-
tanyl acetate (2) measured in a CDCl3, b C6D6, and c C5D5N
analogy with the ASIS HDd- and CDd-values mentioned
above, the epimeric HDd- and CDd-values represent the
differences in the dH and dC values in two C-24 epimeric
pairs of 24-methyl oxysterols: i.e., 1 vs. 2; 3 vs. 4.
Table 1 compiles the 1H and 13C chemical shift data for
the compound 2 measured in CDCl3, C6D6, and C5D5N
solvents. The 13C signal assignments were made exclu-
sively by the use of the DEPT spectra. 2D heteronuclear
(1H–13C) shift-correlated HMQC and HMBC techniques
A large number of the 1H- and 13C-NMR data of steroids
as well as sterols have been reviewed by Kirk et al. [23],
Blunt and Stothers [24], and Goad and Akihisa [25, 26].
According to the previous observation by Rubinstein et al.
[27], eight pairs of epimeric 24-alkylsterols could be dis-
tinguished by 220 MHz 1H-NMR spectra. Similarly,
identification of six pairs of epimeric 24-alkylsterols was
attained at 25.16 MHz 13C NMR by Wright et al. [28].
As shown in Fig. 4, when a pair of C-24 epimeric (24R)-
24-hydroxy-ergostanyl acetate (1) and (24S)-24-hydroxy-
campestanyl acetate (2) were measured in C5D5N, both the
epimers exhibited essentially identical 1H-NMR spectra
1
were also used to further confirm the H and 13C correla-
tions, as shown in Fig. 3. The differences in the 1H and 13
C
chemical shift (dH and dC) values of the substrate measured
in CDCl3 vs. C5D5N (or CDCl3 vs. C6D6) were expressed
in terms of the ASIS HDd- and CDd-values, respectively;
the negative and positive values show the down-field and
up-field shifts, respectively, relative to the dH (or dC) val-
ues measured in CDCl3.
Based on the above findings, the dH and dC values and
signal multiplicities for all of the six 24-alkyl oxysterols
(1–6) measured in C5D5N are compiled in Table 2. In
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Lipids (2013) 48:197–207
201
Table 1 1H- and 13C-NMR chemical shifts for (24S)-24-hydroxy-campestanyl acetate (2) measured in different solvents
Carbon no Type CDCI3
13C
C6D6
13C
ASISHDd- andCDd-
values
C5D5N
ASISHDd- andCDd-
values
1H
1H
13C
1H
13C
1H
13C
1H
1
CH2
CH2
CH
CH2
CH
CH2
CH
CH
CH
C
36.74
27.46
36.90
27.94
-0.16
-0.48
0.28
36.84a
27.81
73.69
34.34
44.67
28.77
32.13
35.52
54.25
35.52
21.40
40.14
42.74
56.46
24.39
28.51
56.38
12.22
12.24
36.61
19.09
29.78
36.83a
73.57
37.37
18.02
17.61
23.85
170.30
21.31
-0.10
-0.35
0.08
2
3
73.77 4.68 (m)
34.01
73.49 4.89 (m)
34.50
-0.21
4.86 (m)
-0.18
4
-0.49
-0.02
-0.26
-0.26
-0.21
-0.11
-0.09
-0.30
-0.39
-0.24
-0.24
-0.33
-0.38
-0.46
-0.16
-0.09
-0.44
-0.33
-0.03
-0.19
-0.17
-0.08
-0.06
-0.08
-0.23
-0.20
-0.15
-0.08
-0.21
-0.30
-0.43
-0.16
-0.03
-0.48
5
44.64
44.66
6
28.58
28.84
7
31.96
32.22
8
35.44
35.65
9
54.19
54.30
10
35.44
35.53
11
CH2
CH
C
21.17
21.47
12
39.94
40.33
13
42.59
42.83
14
CH
CH2
CH2
CH
CH3
CH3
CH
CH3
CH2
CH2
C
56.38
56.62
15
24.18
24.51
16
28.21
28.59
17
55.95
56.41
18
12.06 0.65 (s)
12.21 0.82 (s)
36.13
12.22 0.65 (s)
12.30 0.70 (s)
36.57
0.00
0.12
0.64 (s)
0.76 (s)
0.01
0.06
19
20
21
18.74 0.89 (d, 6.9)
29.14
19.07 0.87 (d, 6.9) -0.33
0.02
1.02 (d, 6.9) -0.35
-0.64
-0.13
22
29.57
36.47
74.08
36.99
-0.43
-0.51
0.71
23
35.96
-0.87
24
74.79
1.22
25
CH
CH3
CH3
CH3
C
36.59
-0.40
-0.78
26
17.48 0.92 (d, 6.9)
16.94 0.92 (d, 6.9)
23.32 1.07 (s)
170.71
17.68 0.93 (d, 6.9) -0.20
17.16 1.01 (d, 6.3) -0.22
-0.01
-0.09
0.06
1.09 (d, 6.9) -0.54
1.17 (d, 6.9) -0.67
-0.17
-0.25
-0.26
27
28
23.58 1.01 (s)
169.70
-0.26
1.01
1.33 (s)
-0.53
0.41
OCOCH3
OCOCH3
CH3
21.48 2.02 (s)
21.06 1.77 (s)
0.42
0.25
2.07 (s)
0.17
-0.05
The difference in the chemical shifts between the substrate measured in two different solvents
a
Assignments along a vertical column may be interchanged
and were indistinguishable from each other. However, a
careful comparison of 1 and 2 revealed that small, but
distinct differences for characterizing each epimer are
detected in the 13C-NMR spectra. Thus, the 13C signals
arising from the C-23 and C-25 in 1 resonated at 37.11 and
36.79 ppm, respectively, while the corresponding signals
in 2 appeared at 36.83 and 37.37 ppm, respectively: the
epimeric CDd-values observed are 0.28 and -0.59 ppm.
The dC differences should therefore be utilized to charac-
terize each of the two epimers, except for the C-26 and
acetate (3) and (24R)-25-hydroxy-campestanyl acetate (4).
As shown in Fig. 5, there is no significant difference in
1
the H-NMR spectra of 3 and 4, but significant features to
characterize the individual epimers were observed on the
13C-NMR spectra. Those were the 13C signals arising
from the C-20–C-22 and C-28; the epimeric CDd-values
observed were 0.42–0.57 ppm. Of further importance was
that a more diagnostic, large difference is detected for the
C
epimeric Dd-value (0.85 ppm) for the chiral carbon itself
at C-24.
1
1
In the H-NMR spectrum of (24S)-24-hydroxy-stigm-
C-27 H and 13C resonances (see below).
A similar relationship was also observed between a
astanyl acetate (5), one triplet signal appearing at 1.07 ppm
and two doublet signals occurring at 1.13 and 1.15 ppm
pair of C-24 epimeric (24S)-25-hydroxy-ergostanyl
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Lipids (2013) 48:197–207
(a)(24S), 24-OH, 24-Me (2)
(24R), 25-OH, 24-Me (4)
(b)
1H
26-CH3
1H
28-CH3
27-CH3
19-CH3
19-CH3 18-CH3
26-CH3
27-CH3
18-CH3
28-CH3
21-CH3
21-CH3
13C
13C
ppm
ppm
26-CH3/ C-27
27-CH3/ C-26
26-CH3/ C-27
21-CH3/ C-22
28-CH3/ C-23
C-27
C-26
C-27
20.0
30.0
40.0
C-26
C-23
30.0
21-CH3/ C-22,
21-CH3/ C-20
27-CH3/ C-26
C-22
28-CH3/ C-23,
28-CH3/ C-25
C-22
C-8, C-10
C-20
C-1
C-20
C-23, C-1
C-25
C-12
C-13
C-5
26-CH3/ C-24,
27-CH3/ C-24
40.0
C-12
28-CH3/ C-24
21-CH3/ C-20
C-13
27-CH3/ C-25
C-5
C-24
26-CH3/ C-25
1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7
ppm
ppm
1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7
Fig. 3 A part of the HMBC spectra of a (24S)-24-hydroxy-campestanyl acetate (2) and b (24R)-25-hydroxy-campestanyl acetate (4)
were assigned to the 29-CH3 and the 26- and 27-CH3
signals, respectively. The remaining 18-, 19-, and 21-CH3
signals in 5 showed the same chemical shifts to those
observed in (24S)-24-hydroxy-campestanyl acetate (2),
probably because 2 and 5 differ only the alkyl substituent at
C-24. In addition, the 13C-NMR spectra of the two com-
pounds differed remarkably from each other, particularly in
the dC values of the C-23, C-24, C-25, and C-28 signals,
due to the a- and b-effects of a different length of the alkyl
substituents (see Table 2).
that the 24-OH in the (24R)-24-hydroxy compound (1) is
antiperiplanar with respect to the 27-CH3, the electron-
donating 26-CH3 which has a gauche arrangement may be
more effectively shifted to down-field (deshielding) by the
inductive effect of the electron-withdrawing 24-OH:
1
thereby 26-CH3 H signal (doublet) resonates at 1.17 ppm
and the 27-CH3 at 1.08 ppm. Meanwhile, differentiation
between the C-26/C-27 13C signals in 1 was basically made
on the gauche c-substituent effect. Thus, both the C-26/
C-27 are situated in the c-position with respect to the C-24
bearing the 24-OH. The conformational arrangement of the
24-OH vs. 26-CH3 is gauche, while that of the 24-OH vs.
27-CH3 is anti. These facts suggest that shift to up-field
(shielding) caused by gauche c-substituent is much more
Analogously, predominant a-, b- and c-shifts of the
neighboring 13C signals were also observed between (24R)-
25-hydroxy-stigmastanyl acetate (6) and (24R)-25-
hydroxy-campestanyl acetate (4).
There is considerable uncertainty in the literature [25,
1
pronounced for the C-26 than for the 27-CH3. Thus, the 13
C
26] on the H and 13C assignments of the terminal iso-
signal appearing at up-field of 17.51 ppm was assigned to
the C-26 and the other one occurring at down-field of
18.09 ppm to the C-27. The validity of the above tentative
1H and 13C assignments in 1 was further confirmed by
measuring the 2D HMQC and HMBC spectra (Fig. 3).
On the other hand, the reverse relationship is true for the
propyl methyl (26-/27-CH3) signals. The cholestrane side
1
chain without a substituent affords the 26-/27-CH3 H sig-
nals at 0.85–0.89 ppm as equivalent doublets, because both
the CH3 groups are chemically equivalent by the free
rotation of the r-bond between the C-24 and C-25. On the
other hand, the two 26-/27-CH3 signals in alkylsterol and
oxysterols having a substituent in the side chain render
nonequivalent and usually show different dH values. In
analogy with the 26-/27-CH3, the C-26/C-27 13C signals in
these compounds are also nonequivalent. In the case of the
compounds 1–6 examined in this study, the major reason
1
(24S)-hydroxy epimer (2): the 26-CH3 (1.09 ppm) H sig-
nal resonates at higher-field than the 27-CH3 (1.17 ppm).
Based on the 1H signal assignments, the corresponding
C-26/C-27 13C signals were directly correlated by the
HMQC and HMBC spectra. The result exhibited that the
13C signals occurring at 18.02 and 17.61 ppm in 2 were
assigned to the C-26 and C-27, respectively.
1
for the nonequivalence of the 26-/27-CH3 H signals arises
from a combined effect of the inductive effect and steric
interaction of the substituents (i.e., CH3 and OH groups),
whereas that of the C-26/C-27 13C signals principally
suffers non-bonded interaction so-called ‘‘gauche c-sub-
stituent effect’’ [24, 29, 30].
Meanwhile, if the 25-OH and 23-CH2- in (24S)-25-
hydroxy compound (3) has antiperiplanar conformation,
shift to up-field by the inductive effect of the 28-CH3 on
the gauche 26-CH3 seems to be slightly effective, com-
1
pared to that on the anti 27-CH3. The CH3 H signal (sin-
Figure 6 shows Newman’s projection formula for the
most preferred conformation of compounds 1–4. Assuming
glet) occurring at 1.39 ppm was therefore assigned to the
26-CH3 and the other one appearing at 1.41 ppm to the
123

Lipids (2013) 48:197–207
203
Table 2 1H- and 13C chemical shifts for compounds 1–6 measured in C5D5N
Carbon no
Type
1
2
Epimeric HDd- and CDd-valuesa
13C 1H
13C
1H
13C
1H
1
CH2
CH2
CH
CH2
CH
CH2
CH
CH
CH
C
36.84
36.84b
0.00
2
27.80
73.69
34.34
44.67
28.77
32.13
35.53
54.25
35.53
21.41
40.14
42.74
56.47
24.40
28.53
56.32
12.22
12.24
36.55
19.09
29.72
37.11
73.55
36.79
17.51
18.09
23.84
170.30
21.31
27.81
73.69
34.34
44.67
28.77
32.13
35.52
54.25
35.52
21.40
40.14
42.74
56.46
24.39
28.51
56.38
12.22
12.24
36.61
19.09
29.78
36.83b
73.57
37.37
18.02
17.61
23.85
170.30
21.31
-0.01
0.00
3
4.86 (m)
4.86 (m)
0.00
4
0.00
5
0.00
6
0.00
7
0.00
8
0.01
9
0.00
10
0.01
11
CH2
CH
C
0.01
12
0.00
13
0.00
14
CH
CH2
CH2
CH
CH3
CH3
CH
CH3
CH2
CH2
C
0.01
15
0.01
16
0.02
17
-0.06
0.00
18
0.65 (s)
0.76 (s)
0.64 (s)
0.76 (s)
0.01
0.00
19
0.00
20
-0.06
0.00
21
1.02 (d, 6.9)
1.02 (d, 6.9)
0.00
22
-0.06
0.28
23
24
-0.02
-0.58
-0.51
0.48
25
CH
CH3
CH3
CH3
C
26
1.17 (d, 6.9)
1.08 (d, 6.9)
1.32 (s)
1.09 (d, 6.9)
1.17 (d, 6.9)
1.33 (s)
0.08
-0.09
-0.01
27
28
-0.01
0.00
OCOCH3
OCOCH3
CH3
2.07 (s)
1H
2.07 (s)
1H
0.00
0.00
Carbon no
Type
3
4
Epimeric HDd- and CDd-values
13C 1H
13C
13C
1
CH2
CH2
CH
CH2
CH
CH2
CH
CH
CH
C
36.86
27.82
73.70
34.35
44.69
28.78
32.15
35.56
54.28
35.56
21.41
40.13
42.72
56.41
36.84
27.81
73.69
34.35
44.67
28.77
32.15
35.55
54.26
35.53
21.41
40.17
42.76
56.47
0.02
2
0.01
0.01
3
4.86 (m)
4.86 (m)
0.00
4
0.00
5
0.02
6
0.01
7
0.00
8
0.01
9
0.02
10
11
12
13
14
0.03
CH2
CH
C
0.00
-0.04
-0.04
-0.06
CH
123

204
Lipids (2013) 48:197–207
Table 2 continued
Carbon no
Type
3
4
Epimeric HDd- and CDd-values
13C
1H
13C
1H
13C
1H
15
CH2
CH2
CH
24.41
24.41
0.00
16
28.46
56.17
12.23
12.24
36.64
19.27
35.42
28.22
45.95
72.21
26.46
28.18
15.42
170.30
21.32
28.55
56.47
12.22
12.24
36.07
18.85
34.92
28.17
45.10
72.12
27.95
26.71
14.93
170.30
21.31
-0.09
-0.30
0.01
0.00
0.57
0.42
0.50
0.05
0.85
0.09
-1.49
1.47
0.49
0.00
0.01
17
18
CH3
CH3
CH
0.65 (s)
0.76 (s)
0.64 (s)
0.76 (s)
0.01
19
0.00
20
21
CH3
CH2
CH2
CH
1.02 (d, 6.9)
1.02 (d, 6.9)
0.00
22
23
24
25
C
26
CH3
CH3
CH3
C
1.39 (s)
1.41 (s)
-0.02
0.02
27
1.41 (s)
1.39 (s)
28
1.12 (d, 6.9)
1.12 (d, 6.9)
0.00
OCOCH3
OCOCH3
CH3
2.07 (s)
2.07 (s)
0.00
1H
Carbon no
Type
5
6
13C
1H
13C
1
CH2
CH2
CH
CH2
CH
CH2
CH
CH
CH
C
36.87
27.83
73.70
34.36
44.68
28.79
32.15
35.55
54.26
35.57
21.43
40.17
42.78
56.49
24.43
28.60
56.46
12.24
12.24
36.93
19.11
29.70
32.58
75.01
34.63
17.46
17.33
29.06
36.85
27.82
73.70
34.35
44.68
28.78
32.16
35.56
54.27
35.56
21.43
40.18
42.77
56.51
24.43
28.60
56.33
12.24
12.24
36.64
19.00
36.39
27.48
52.47
72.74
28.11
28.00
24.20
2
3
4.86 (m)
4.86 (m)
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
CH2
CH
C
CH
CH2
CH2
CH
CH3
CH3
CH
CH3
CH2
CH2
C
0.64 (s)
0.76 (s)
0.65 (s)
0.76 (s)
1.03 (d, 6.3)
1.05 (d, 6.3)
CH
CH3
CH3
CH2
1.13 (d, 5.2)
1.15 (d, 5.2)
1.42 (s)
1.42 (s)
123

Lipids (2013) 48:197–207
205
Table 2 continued
Carbon no
Type
5
6
13C
1H
13C
1H
29
CH3
C
8.35
1.07 (t, 7.4)
14.51
1.12 (t, 7.2)
2.07 (s)
OCOCH3
OCOCH3
170.30
21.33
170.30
21.31
CH3
2.07 (s)
a
1
The difference in the H or 13C chemical shifts between an epimeric pair of the substrates
b
Assignments along a vertical column may be interchanged
(a) (24R), 24-OH, 24-Me (1)
(24S), 25-OH, 24-Me (3)
(a)
18-CH3
19-CH3
19-CH3
26-CH3
1.39 ppm
27-CH3
1.08 ppm (d)
27-CH3
1.41 ppm
18-CH3
26-CH3
1.17 ppm (d)
28-CH3
1.32 ppm
28-CH3
1.12 ppm (d)
21-CH3
1.02 ppm (d)
21-CH3
1.02 ppm (d)
(b) (24S),24-OH, 24-Me(2)
(b)(24R), 25-OH, 24-Me (4)
19-CH3
18-CH3
19-CH3
18-CH3
26-CH3
1.09 ppm (d)
28-CH3
1.33 ppm
27-CH3
26-CH3
27-CH3
1.17 ppm (d)
1.39 ppm
1.41 ppm
21-CH3
1.02 ppm (d)
28-CH3
1.12 ppm (d)
21-CH3
1.02 ppm (d)
1.4
1.2
1.0
0.8
0.6
1.4
1.2
1.0
0.8
0.6
C-28
(c) (24R), 24-OH, 24-Me (1)
(c)(24S), 25-OH, 24-Me (3)
15.42 ppm
C-20
36.55 ppm
C-25
36.79 ppm
C-8, C-10
C-20
36.64 ppm
C-21
19.27 ppm
C-24
45.95 ppm
C-1
36.84 ppm
C-23
37.11 ppm
C-7
C-4
C-22
35.42 ppm
C-21
18.85 ppm
C-28
14.93 ppm
(d)(24S), 24-OH, 24-Me (2)
(d) (24R), 25-OH, 24-Me (4)
C-20
36.61 ppm
C-24
45.10 ppm
C-20
36.07 ppm
C-8, C-10
C-25
37.37 ppm
C-23
C-1
36.83 ppm
36.84 ppm
C-22
C-7
C-4
34
34.92 ppm
50
40
30
20
38
36
32
Fig. 5 A part of the 1H-NMR spectra of a (24S)-25-hydroxy-
ergostanyl acetate (3) and b (24R)-25-hydroxy-campestanyl acetate
(4) and their 13C-NMR DEPT spectra [c and d] measured in C5D5N
Fig. 4 A part of the 1H-NMR spectra of a (24R)-24-hydroxy-
ergostanyl acetate (1) and b (24S)-24-hydroxy-campestanyl acetate
(2) and their 13C NMR DEPT spectra [c and d] measured in C5D5N
123

206
Lipids (2013) 48:197–207
In conclusion, the ASIS by C5D5N was recommended as
a suitable solvent to determine the (24R)-/(24S)-configu-
ration of epimeric 24-alkyl oxysterols. Differentiation
between two epimeric pairs of the 24- and 25-hydroxylated
derivatives of 24-alkylsterols was particularly achieved by
the 13C-NMR measurement in C5D5N. Studies of the nat-
ural occurrence of 24-alkyl oxysterols and their biological
and physiological importance as well as their use as bio-
markers of human diseases are now progressing in our
laboratory. Availability of the synthetic 24-alkyl oxysterols
as authentic reference compounds may also be useful for
their identification and analysis in biological materials. A
further detailed study is now progressing in our laboratory,
and the result will be reported at a later date.
Acknowledgments This work was supported in part by a Grant-in-
Aid for Scientific Research from the Ministry of Education, Culture,
Sports, Sciences and Technology of Japan (to T.I., 24550107) for
2012–2014 and for the Strategic Research Base Development Pro-
gram for Private Universities subsidized MEXT 2009 (S0901022) for
2009–2013.
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