Sterol synthesis. Preparation and characterization of fluorinated and deuterated analogs of oxygenated derivatives of cholesterol

Chemistry and Physics of Lipids

99 (1999) 33–71

Sterol synthesis. Preparation and characterization of ﬂuorinated

and deuterated analogs of oxygenated derivatives of cholesterol

Shengrong Li, Jihai Pang, William K. Wilson *, George J. Schroepfer, Jr. ¹

Departments of Biochemistry and Cell Biology and of Chemistry, Rice Uni6ersity, 6100 Main St., Houston, TX 77005-1892, USA

Received 20 November 1998; received in revised form 5 January 1999; accepted 5 January 1999

Abstract

Oxygenated sterols, including both autoxidation products and sterol metabolites, have many important biological

activities. Identiﬁcation and quantitation of oxysterols by chromatographic and spectroscopic methods is greatly

facilitated by the availability of authentic standards, and deuterated and ﬂuorinated analogs are valuable as internal

standards for quantitation. We describe the preparation, puriﬁcation and characterization of 43 oxygenated sterols,

including the 4b-hydroxy, 7a-hydroxy, 7b-hydroxy, 7-keto, and 19-hydroxy derivatives of cholesterol and their

analogs with 25,26,26,26,27,27,27-heptaﬂuoro (F₇) and 26,26,26,27,27,27-hexadeuterio (d₆) substitution. The 7a-hy-

droxy, 7b-hydroxy, and 7-keto derivatives of (25R)-cholest-5-ene-3b,26-diol (1d) and their 16,16-dideuterio analogs

were also prepared. These d₂-26-hydroxysterols and [16,16-²H₂]-(25R)-cholest-5-ene-3b,26-diol (1e) were synthesized

from [16,16-²H₂]-(25R)-cholest-5-ene-3b,26-diol diacetate (2e), which can be prepared from diosgenin. The highly

speciﬁc deuterium incorporation at C-16 in 1e and 2e should be useful in mass spectral analysis of 26-hydroxycholes-

terol samples by isotope dilution methods. The D⁵-3b,7a,26- and D⁵-3b,7b,26-triols were regioselectively oxidized/iso-

merized to the corresponding D⁴-3-ketosteroids with cholesterol oxidase. Also described are

5,6a-epoxy-5a-cholestan-3b-ol, its 5b,6b-isomer, cholestane-3b,5a,6b-triol, their F₇and d₆derivatives, and d₃-25-hy-

droxycholesterol, which was prepared from 3b-acetoxy-27-norcholest-5-en-25-one (30). The 43 oxysterols and most

synthetic intermediates were isolated in high purity and characterized by chromatographic and spectroscopic methods,

Abbre6iations: COSYDEC, f₁-decoupled ¹H–¹H correlation spectroscopy; COSY-DQF, ¹H–¹H correlation spectroscopy with

double-quantum ﬁltering; GC, gas chromatography; HMBC, heteronuclear multiple bond correlation; HPLC, high performance

liquid chromatography; HSQC, heteronuclear single-quantum coherence; IR, infrared (spectroscopy); m.p., melting point; MPLC,

medium pressure liquid chromatography; MS, mass spectrometry or mass spectrum; NMR, nuclear magnetic resonance (spec-

troscopy); NOE, nuclear Overhauser enhancement; SC, side chain; TBDMS, tert-butyldimethylsilyl; TES, N-tris(hydrox-

ymethyl)methyl-2-aminoethanesulfonic acid, sodium salt; TLC, thin-layer chromatography; TMS, trimethylsilyl.

* Corresponding author. Tel.: +1-713-527-4865; fax: +1-713-285-5154.

E-mail address: billw@bioc.rice.edu (W.K. Wilson)

¹Deceased 11 December 1998.

PII: S0009-3084(99)00005-5

34

S. Li et al. / Chemistry and Physics of Lipids 99 (1999) 33–71

including mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Detailed mass spectral assign-

1

ments are presented, and H NMR stereochemical assignments are derived for the C-19 protons of 19-hydroxysterols

Keywords: Oxygenated sterols; Deuterium-labeled 26-hydroxycholesterol; Alumina–silver nitrate chromatography;

NMR; Mass spectrometry

1. Introduction

Science, Gibbstown, NJ). Components of the

plates were visualized after spraying with 5% am-

monium molybdate in 10% sulfuric acid followed

by heating. TLC solvent systems were: SS-1, ethyl

acetate–hexane 1:9; SS-2, ethyl acetate–hexane

15:85; SS-3, ethyl acetate–hexane 2:8; SS-4, ethyl

acetate–hexane 3:7; SS-5, ethyl acetate–hexane

1:1; SS-6, ethyl acetate–hexane 6:4; SS-7,

methanol–chloroform 1:9. MPLC was done on

glass columns; unless speciﬁed otherwise, columns

were dry-packed with silica gel (230–400 mesh;

EM Science), and fraction volumes were 20 ml.

Low-resolution and high-resolution mass spectra

were recorded after direct-inlet sample introduc-

tion on a VG ZAB-HF reverse-geometry double-

sector instrument at 70 eV with an

electron-impact ion source (200°C). Mass spectral

data are given as m/z (relative abundance, sug-

gested assignment or molecular formula). Relative

abundances (for m/z]50) are from low-resolu-

tion spectra, and exact masses are from high-reso-

lution data. Exact masses are reported as the

average of ꢀﬁve scans; standard deviations were

typically 1–1.5 mmu (millimass units) for ions of

]10% relative abundance. Ions attributable to

loss of CH₃, side chain (SC), CH₃COOH, H₂O,

HBr, Br; (CH₃)₃C(CH₃)₂SiOH, C₄H₉(of TBDMS

ethers); CH₂O, CH₂OH, and CH₂O–2 (of 19-hy-

droxysterols), or combinations thereof are marked

by an asterisk (*). Ions showing exact masses

within 93.0 mmu of the indicated or implied

assignments are marked by †. Bromine-containing

ions are marked by §; only ions containing ⁷⁹Br

are listed. NMR spectra were acquired as de-

scribed previously (Swaminathan et al., 1993;

Wilson et al., 1996) on a Bruker AMX500 (5–15

Oxysterols constitute a class of lipids of high

interest in biology and medicine. For bioanalyti-

cal studies based upon GC-MS, we required la-

beled analogs of major oxygenated sterols.

Described herein are syntheses of deuterium- and

ﬂuorine-substituted analogs of a number of oxys-

terols of high current biomedical interest. Also

presented are detailed spectral (MS, H and ¹³C

1

NMR, and, in most cases, IR) characterization of

the synthetic sterols. Although deuterium-labeled

analogs of some of the oxysterols have been pre-

pared previously (Bjo¨rkhem and Kallner, 1976;

Javitt et al., 1981, 1982; Bjo¨rkhem, 1986; Breuer

and Bjo¨rkhem, 1990; Shoda et al., 1993a; Breuer,

1995; Dzeletovic et al., 1995; Krut et al., 1997),

little or no characterization of the sterols (or

mixtures of same) were presented. Among new

synthetic oxysterol analogs described herein are

the 25,26,26,26,27,27,27-heptaﬂuoro analogs of a

number of oxysterols, (25R)-[16,16-²H₂]cholest-5-

ene-3b,26-diol, and the corresponding d₂analogs

of

(25R)-3b,26-dihydroxycholest-5-en-7-one,

(25R)-cholest-5-ene-3b,7a,26-triol, and (25R)-

cholest-5-ene-3b,7b,26-triol.

2. Experimental procedures and results

2.1. Materials and methods

Melting points (m.p.) were measured with a

Thomas–Hoover apparatus in sealed, evacuated

capillary tubes. IR spectra were obtained from

KBr pellets on a Mattson Galaxy 6020 Fourier-

transform infrared spectrometer. TLC was carried

out on aluminum-backed, silica gel 60 plates (EM

1

mM CDCl₃solution at 25°C for H; 10–120 mM

CDCl₃solution at 22°C for ¹³C; CHCl₃solution

S. Li et al. / Chemistry and Physics of Lipids 99 (1999) 33–71

35

at 25°C for ²H, unless speciﬁed otherwise) or

AC250 spectrometer (CDCl₃solution at ꢀ22°C

for ¹⁹F). NMR chemical shifts were referenced to

(CH₃)₄Si (¹H), CDCl₃at l_C77.0 (¹³C), CDCl₃at

l_D7.26 (²H), and the downﬁeld line of the

3:3:1:0.1 isotope pattern for CFCl₃(¹⁹F). Chemi-

cal shifts were corrected for strong coupling in 1D

spectra by analogy with the analysis of AB spin

systems and in COSYDEC spectra by adjustments

based on spectral simulations with NMRSIM

(Bruker Instruments, Billerica, MA). Signal as-

signments were made from HSQC, COSYDEC,

COSY-DQF, and 1D spectra as described previ-

ously (Wilson et al., 1996). PCMODEL 7.0 (Ser-

ena Software; Bloomington, IN) was used for

modeling of sterol structures by molecular me-

(]99% D, Isotech; Miamisburg, OH) were pur-

chased and used without further puriﬁcation.

25,26,26,26,27,27,27-Heptaﬂuorocholest-5-en-3b-

ol (1b; F₇-cholesterol) (Swaminathan et al., 1993),

(25R)-bis(3b,26 -tert - butyldimethylsilyloxy)cho-

lest - 5 - en -16-one (9) (Kim et al., 1989), (25R)-

cholest-5-ene-3b,26-diol (1d) (Kim et al., 1989),

and its diacetate 2d (Siddiqui et al., 1992) were

prepared as described previously and character-

ized as follows: 9, m.p. 80–81°C, lit. 81.5–82.5°C

(Kim et al., 1989); 1d, m.p. 176–177°C, lit. 177–

178°C (Kim et al., 1989), single component by

TLC (R_f0.65, SS-5); 2d, m.p. 126.5–127°C, lit.

130.5–131.5°C (Siddiqui et al., 1992), single com-

ponent by TLC (R_f0.60, SS-2).

1

chanics and for predicting vicinal H–¹H NMR

2.2. 25,26,26,26,27,27,27-Heptaﬂuorocholest-5-

en-3i-ol acetate (2b), cholest-5-en-3i-ol acetate

(2a), and [26,26,26,27,27,27-²H₆]cholest-5-en-

3i-ol acetate (2c)

coupling constants. The purity of sterol samples

was judged by TLC and ¹H NMR (500 MHz

spectrum; methyl region and l_H2.5–7.0 region).

Stated purities are exclusive of traces of solvent

and (in the case of 18a, 18b, 18c, 19a, 19b, 19c,

and 21b) non-steroidal long chain alkyl contami-

nants arising from NMR sample preparation

techniques.

To a solution of F₇-cholesterol (1b; 100 mg,

0.20 mmol) in pyridine (1 ml) was added acetic

anhydride (40 mg, 0.40 mmol). The reaction mix-

ture was heated to reﬂux for 1 h, allowed to cool,

and poured into ice-water (200 ml). The resulting

precipitate was collected by ﬁltration, washed

with water, and dried in vacuo. Filtration through

silica gel in a Pasteur pipette (elution with ethyl

acetate–hexane 2:98) gave 2b as a white solid (96

mg, 89% yield, 98% purity): m.p., 135–136°C (lit.

137–139°C (Carroll et al., 1998)); single compo-

Butyl acetate, N-bromoacetamide, m-chloro-

perbenzoic acid (]85% purity), hydrazine hy-

drate (85%), hydrazine hydrochloride, lead

tetraacetate, lithium aluminum hydride, methyl

iodide-d₃(96% d₃, 4% d₂by MS), potassium

permanganate, pyridinium chlorochromate, L-Se-

lectride (lithium tri-sec-butyl borohydride), sele-

nium dioxide, and tetrabutylammonium ﬂuoride

were purchased from Aldrich Chemical Co. (Mil-

waukee, WI). TES buffer, cholesterol oxidase

from Streptomyces sp., and catalase were pur-

chased from Sigma Chemical Co. (St. Louis,

MO). Alumina–silver nitrate for MPLC was pre-

pared as described previously (Pascal et al., 1980)

from neutral aluminum oxide (ICN Biomedicals;

Costa Mesa, CA). CDCl₃for NMR analysis was

ﬁltered through furnace-dried basic alumina prior

to use. [26,26,26,27,27,27-²H₆]cholest-5-en-3b-ol

(1c; Medical Isotopes, Inc.; Pelham, NH; ]99%

purity by ¹H NMR; 93% d₆, 7% d₅by MS),

3b-acetoxy-27-norcholest-5-en-25-one (30; South-

eastern Biochemicals, Augusta, GA; ꢀ95% pu-

rity by ¹H NMR), lithium aluminum deuteride

1

nent by TLC (R_f0.50, SS-1); 98% purity by H

NMR

(contains

1.5%

(23E)-3b-acetoxy-

25,26,26,26,27,27,27-heptaﬂuorocholesta-5,23-di-

ene); high-resolution MS, calcd. for C₂₇H₃₇F₇

(M−60), 494.2783, found 494.2777; MS, 494*^†

(100), 479*^†(16), 386^†(7, C₁₉H₂₅F₇), 373^†(19,

C₁₈H₂₄F₇), 331^†(5, C₁₅H₁₈F₇), 283^†(4, M−

CH₃COOHꢀC₆H₆F₇), 255*^†(9), 239^†(4, SC), 213^†

(10, C₁₆H₂₁), 147^†(31, C₁₁H₁₅), 105^†(27, C₈H₉);

IR, 2947, 2905, 2874, 2852, 1736, 1467, 1440,

1377, 1346, 1313, 1278, 1246, 1222, 1134, 1037

cm^{−1; 1}H NMR, Table 1; ¹³C NMR, Table 2.

Similar acetylation of 1a (2.5 g) gave 2a (2.5 g,

90% yield): m.p. 114–115°C; single component by

TLC (R_f0.60, SS-1); MS, 368* (100, M−60),

36

S. Li et al. / Chemistry and Physics of Lipids 99 (1999) 33–71

Table 1

¹H NMR chemical shifts of F₇-cholesterol acetate (2b), 26-hydroxy derivatives (11, 15, 16d, 18d, 19d), synthetic precursors (26a, 27a,

28b) to 19-hydroxysterols (29b), and 27-norketone 30^a,b

26-Hydroxy derivatives

16a-OH D¹⁶

7-Keto

Acetate

16d

7a-OAc

Acetate

18d

7b-OAc

Acetate

19d

6b-OH

6b,19-O 19-OH

19-OH 25-Keto

3b-OAc TBDMS Acetate

3b-OAc 3b-OAc

3b-OAc 3b-OH 3b-OAc

2b

11

15

26a

27a

28b

29b

30

H-1a

1.133

1.859^†

1.858^†

1.587

4.604

2.327^†

2.308^†

5.375

1.541

1.971

1.452

0.953

1.511^†

1.455^†

1.178

2.005

1.012^†

1.597

1.084^†

1.819

1.255

1.101

0.684

1.020

–

1.049

1.800

1.716

1.531

3.487

2.170

2.261

5.315

1.593

1.952

1.465

0.975

1.507^†

1.424^†

1.283

1.979

1.386

1.529^†

1.620^†

–

1.141

1.858^†

1.865^†

1.590

4.609

2.339^†

2.321^†

5.397

1.621^†

2.010

1.662^†

1.037

1.609^†

1.537^†

1.405

1.776

1.329

2.050

1.836

(5.286)

–

1.265

1.966

1.985

1.683

4.716

2.548

2.468

5.703

–

1.203

1.879^†

1.605

4.675

2.356^†

5.585

–

4.963

1.594

1.370^†

1.535^†

1.478^†

1.192

2.004

1.342^†

1.430^†

1.074

1.828

1.263

1.137

0.669

1.013

–

1.133

1.869

1.899

1.581

4.602

2.339^†

2.319^†

5.240

5.034

–

1.691

1.131

1.556^†

1.483^†

1.156

2.015

1.150

1.445

1.285^†

1.807

1.237^†

1.055

0.693

1.084

–

1.763^†

1.584^†

1.962

1.635^†

5.475

2.193

2.505

4.181

2.243

1.667

1.755

1.577

1.432

1.301^†

1.201^†

1.992

1.165^†

1.574

1.064^†

1.838

1.257

1.126

0.678

1.320

–

1.993

1.695

1.648

1.512

5.197

2.320

2.268

4.055

1.517

1.993

1.609

1.662

1.142

1.396

1.232

1.993

1.220

1.503

1.076

1.836

1.243

1.124

0.693

3.919

3.743

1.366

0.894

1.326^†

0.988

1.326^†

1.13

1.144

1.954

1.871

1.494

4.645

2.421

2.267

5.777

1.544

2.031

1.820

0.924

1.547^†

1.625^†

1.170

2.040

0.922

1.571

1.091

1.810

1.256

1.093

0.736

3.835

3.621

1.420^†

0.937

1.413^†

1.089

1.431

1.628

1.967^†

2.043^†

–

1.093

1.935

1.853

1.405

3.578

2.386

2.192

5.752

1.533

2.033

1.836

0.906

1.131

1.856^†

1.583

4.602

2.322^†

2.305^†

5.372

1.534

1.964

1.45

H-1b

H-2a

H-2b

H-3a

H-4a

H-4b

H-6(a)

H-7a

H-7b

–

H-8b

2.232

1.529^†

1.564^†

1.54

H-9a

0.945

H-11a

H-11b

H-12a

H-12b

H-14a

H-15a

H-15b

H-16a

H-16b

H-17a

H-18

1.549^†1.500^†

1.633^†1.446^†

1.142

2.032

1.343^†

2.405

1.245^†

1.894

1.272^†

1.082

0.683

1.209

–

1.379

0.922

1.355^†

1.037

1.22

1.161

2.042

0.912

1.569

1.093

1.806

1.258

1.087

0.739

3.825

3.609

1.161

1.997

0.992

1.575

1.066

1.818

1.235

1.106

0.673

1.016

–

1.022

0.697

0.992

–

0.780

1.058

–

H-19R

H-19S

H-20

1.418^†

0.940

1.417^†

1.093

1.434^†

1.633

1.971^†

2.044^†

–

1.539

0.941

1.592

1.147

1.256^†

1.420^†

1.350^†

1.075^†

1.586

3.355

3.436

2.081

0.991

1.470*

1.338^†*

1.232^†*

1.33*

1.354^†

1.118

1.761^†

3.838

3.935

0.911

2.034^d

1.385

0.926

1.345^†

1.027

1.225^†

1.34

1.363^†

0.913

1.332^†

1.018

1.218^†

1.326^†

1.304^†

1.123^†

1.766

3.842

3.939

0.914

2.021^d

1.368

0.906

1.332^†

0.994

1.331^†

1.14

1.422^†1.393

H-21

0.938

0.932

H-22R

H-22S

H-23R

H-23S

H-24R

H-24S

H-25

H-26

H-27

Other

1.412^†1.342

1.090^†1.039

1.432

1.627

1.966

2.042

–

1.442

1.652

2.361

2.406

–

2.131

–

1.33

1.311^†

1.127^†

1.769

3.846

3.938

0.915

2.052^d

1.303^†

1.125^†

1.769

3.840

3.945

0.919

2.056^d

1.10

1.13

1.10

1.13

1.516

0.861

–

0.866

2.034

1.512

0.859

–

0.863

2.030

–

0.864

c

2.032

2.031

2.030

^aData obtained at 500 MHz in 5–15 mM CDCl₃solution at 25°C and referenced to Si(CH₃)₄.

^bChemical shifts given to two (three) decimal places are generally accurate to 90.01 (90.001) ppm except that values marked

by † are accurate to about 90.003 ppm. R and S denote pro-R and pro-S hydrogens. An asterisk indicates that stereochemical

assignments may be interchanged.

^cTMDMS signals: l_H0.892 (s), 0.889 (s), 0.058 (s), 0.036 (s).

^dAdditional acetate signals: l_H2.051 (15); 2.057 (16d); 2.043, 2.057 (18d); 2.030, 2.056 (19d).

S. Li et al. / Chemistry and Physics of Lipids 99 (1999) 33–71

37

Table 2

¹³C NMR chemical shifts of F₇-cholesterol acetate (2b), 26-hydroxy derivatives (11, 15, 16d, 18d, 19d), synthetic precursors (26a, 27a,

28b) to 19-hydroxysterols (29b), and 27-norketone 30^a,b

26-Hydroxy derivatives

16a-OH D¹⁶

7-keto

Acetate

16d

7a-OAc

Acetate

18d

7b-OAc

Acetate

19d

6b-OH

3b-OAc 3b-OAc

26a 27a

6b,19-O

19-OH

3b-OAc 3b-OH 3b-OAc

28b 29b 30

19-OH

25-keto

3b-OAc TBDMS Acetate

2b

11

15

C-1

C-2

C-3

C-4

C-5

C-6

C-7

C-8

36.96

37.25

36.92

35.97

27.30

72.16

37.69

163.85

126.65

201.92

45.35

49.72

38.26

21.11

38.59

43.07

49.88

26.24

28.53

54.68

11.93

17.21

35.60

18.76

35.94

23.20

33.63

32.41

69.55

16.72

21.24

170.27

20.97

171.31

–

36.48

27.47

73.12

37.80

146.47

120.82

68.24

35.72

43.00

37.24

20.63

38.99

42.23

49.14

24.02

28.13

55.83

11.43

18.13

35.64

18.66

36.00

23.25

33.72

32.49

69.57

16.78

21.34

170.41

20.97

171.29

21.34

170.79

36.48

27.57

73.18

37.48

144.17

122.21

75.49

36.42

48.02

36.45

20.99

39.27

42.79

55.41

25.09

28.35

55.30

11.74

18.95

35.57

18.65

35.92

23.17

33.63

32.41

69.56

16.72

21.35

170.34

20.98

171.31

21.63

171.09

35.08

32.80

33.07

33.32

36.94

27.73

73.93

38.08

139.63

122.56

31.83

31.81

49.94

36.55

20.98

39.65

42.33

56.59

24.21

28.20

55.86

11.82

19.29

35.53

18.46

35.99

18.09

32.03

72.55

42.77

141.51

120.98

31.83

31.19

49.99

36.57

20.73

39.85

44.12

53.65

27.74

73.92

38.12

139.87

122.54

31.59

30.53

50.65

36.80

20.75

35.02

46.89

57.28

26.32

72.13

38.39

86.71

75.72

34.54

30.54

47.38

40.31

21.27

39.62

42.66

55.68

24.03

28.17

56.06

12.16

18.01

35.74

18.64

36.10

23.78

39.47

27.99

22.54

22.80

21.36

170.48

–

23.24

69.99

41.31

74.54

82.30

26.86

33.25

48.64

45.81

22.64

39.70

43.13

54.32

23.45

28.25

55.97

12.38

67.48

35.71

18.58

36.09

23.75

39.47

27.99

22.55

22.80

21.31

170.36

–

28.05

73.36

38.16

134.48

128.26

31.16

33.32

50.21

41.55

21.69

39.91

42.53

57.46

24.00

28.19

55.79

12.17

62.68

35.49

18.45

35.97

18.06

29.37

91.81

121.10

21.37

170.53

–

31.91

71.32

42.25

135.50

127.33

31.17

33.37

50.29

41.47

21.73

39.96

42.54

57.55

24.01

28.20

55.80

12.18

62.69

35.48

18.45

35.97

18.04

29.37

91.81

121.08

–

27.73

73.94

38.08

139.62

122.59

31.84

31.81

49.94

36.55

20.97

39.64

42.28

56.61

24.23

28.18

55.75

11.81

19.28

35.61

18.57

35.40

20.38

44.25

209.43

29.87

–

C-9

C-10

C-11

C-12

C-13

C-14

C-15

C-16

C-17

C-18

C-19

C-20

C-21

C-22

C-23

C-24

C-25

C-26

C-27

Other

36.86

30.95

c

66.96

13.40

19.41

33.92

18.88

35.92

24.03

33.42

35.75

68.55

16.62

−4.61

18.26

25.93

−5.33

18.36

25.96

160.52

16.16

19.21

32.17

21.87

36.65

24.73

33.50

32.46

69.47

16.86

21.41

170.52

20.96

171.28

–

29.38

c

121.11

21.43

170.54

–

21.44

170.53

–

^aChemical shifts (90.03 ppm) obtained at 125 MHz at 22°C in 20–120 mM CDCl₃solution and referenced to the CDCl₃signal

at 77.0 ppm.

^bCoupling patterns for F₇sterols 2b, 28b, and 29b: C-23, broad (W_1/29 Hz); C-24, d, 20 Hz; C-25, d of quintet, 202, 32 Hz; C-26

and C-27, qd, 286, 28 Hz.

^cNot determined because of insufﬁcient sensitivity due to splittings from coupling to ²H or ¹⁹F.

(10, C₁₈H₂₅D₆), 213^†(8, C₁₆H₂₁), 211^†(2,

Similar acetylation of 1c (500 mg) gave 2c (510

mg, 92% yield, 98% purity): m.p., 111–113°C;

C₁₅H₁₉D₆), 147^†(21, C₁₁H₁₅), 105^†(19, C₈H₉); IR,

2964, 2937, 2904, 2862, 2827, 2216, 2121, 2065,

1730, 1465, 1440, 1369, 1249, 1199, 1134, 1118,

1039 cm⁻¹; NMR, l_H5.375 (m), 4.603 (m), 2.32

(m), 2.31 (m), 2.033 (s, 3H), 1.018 (d, 0.6 Hz, 3H),

single component by TLC (R_f0.60, SS-1); high-

resolution MS, calcd. for C₂₇H₃₈D₆(M−60),

374.3820, found 374.3796; MS, 374*^†(100), 359*^†

(12), 283 (1), 266^†(5, C₁₉H₂₆D₆), 255*^†(9), 253^†

38

S. Li et al. / Chemistry and Physics of Lipids 99 (1999) 33–71

0.914 (d, 6.6 Hz, 3H), 0.676 (d, 0.5 Hz, 3H), l_C

170.54, 139.62, 122.63, 73.97, 56.65, 56.09, 49.99,

42.28, 39.69, 39.36, 38.09, 36.96, 36.56, 36.16,

35.78, 31.87, 31.82, 28.21, 27.74, 27.51, 24.26,

23.80, 21.44, 21.00, 19.29, 18.69, 11.83 (no signals

observed for C-26, C-27).

19.9, 6.6 Hz), −76.74 and −76.95 (A₃B₃portion

of A₃B₃X system (Swaminathan et al., 1993)).

Similar treatment of 2a (0.43 g) gave 3a as a

white solid (0.16 g, 40% yield; \99% purity): m.p.,

176–177°C (lit. 176–177°C (Rosenheim and Star-

ling, 1937), 175–175.5°C (Breuer, 1995)); single

component by TLC (R_f0.57, SS-5); MS, 402 (46,

M⁺), 387* (39), 384* (100), 369* (30), 366* (23),

358 (84), 345 (30), 342 (12), 327 (14), 287 (13), 275

(18), 271* (29), 261 (17), 253 (9), 247 (33), 229 (49),

124 (51), 105 (56); ¹H and ¹³C NMR data identical

(90.001 ppm for ¹H, 90.03 ppm for ¹³C) to those

of 3c except for the occurrence of signals at l_H

1.517 (nonet, 6.6 Hz), 0.867 (d, 6.6 Hz, 3H), 0.862

(d, 6.6 Hz, 3H), l_C39.48 (replacing 39.35), 27.99

(replacing 27.50), 22.80, 22.54.

Similar treatment of 2c (52 mg) gave 3c as a

white solid (20 mg, 41% yield; ]99% purity): m.p.,

174–175°C; single component by TLC (R_f0.57,

SS-5); high-resolution MS, calcd. for C₂₇H₄₀D₆O₂,

408.3874, found 408.3877; MS, 408^†(22, M⁺),

393*^†(16), 390*^†(100), 375*^†(18), 364^†(40, M−

C₂H₄O), 351^†(12, M−C₃H₅O), 348 (15), 333^†(8,

M−C₃H₇O₂), 293^†(5, M−C₆H₁₁O₂), 281^†(13,

M−C₇H₁₁O₂), 271*^†(22), 267^†(24, M−

C₈H₁₃O₂), 253^†(32, M−C₉H₁₅O₂and M−

2H₂OꢀSC), 229^†(55, C₁₆H₂₁O), 124^†(73, C₈H₁₂O),

105^†(32, C₈H₉); IR, 3416, 3386, 2937, 2904, 2866,

2820, 2204, 1456, 1381, 1363, 1070, 1094, 964

cm⁻¹; NMR, l_H5.681 (dd, 4.6, 1.8 Hz), 4.139 (dd,

3.5, 1.2 Hz), 3.562 (dt, 11.8, 4.2 Hz), 2.087 (m),

2.014 (dt, 12.7, 3.6 Hz), 1.908 (dt, 13.3, 3.5 Hz),

1.649 (ddtd, 12.6, 4.8, 3.5, 1.3 Hz), 1.182 (d, 0.7 Hz,

3H), 0.914 (d, 6.7 Hz, 3H), 0.680 (d, 0.5 Hz, 3H),

l_D0.83 (s), l_C142.72, 128.78, 77.25, 72.45, 56.88,

56.06, 50.15, 42.28, 39.65, 39.35, 36.89, 36.16,

35.97, 35.76, 32.06, 31.78, 28.20, 27.50, 25.36,

24.23, 23.79, 21.03, 20.51, 18.68, 11.84 (no signals

observed for C-26, C-27).

2.3. 25,26,26,26,27,27,27-Heptaﬂuorocholest-

5-ene-3i,4i-diol (3b), cholest-5-ene-3i,4i-diol

(3a), and [26,26,26,27,27,27-²H₆]cholest-5-

ene-3i,4i-diol (3c)

To a solution of acetate 2b (80 mg, 0.14 mmol)

in toluene (20 ml) were added selenium dioxide

(15.4 mg, 0.14 mmol) and potassium phosphate

monobasic (19 mg, 0.14 mmol). The reaction mix-

ture was reﬂuxed for 4 h, followed by removal of

the inorganic salts by ﬁltration. The ﬁltrate was

dried over anhydrous sodium sulfate and evapo-

rated to a light yellow solid that was subjected to

MPLC (1000×10 mm i.d. column; elution with

ethyl acetate–hexane 5:95). The crude acetate

product (30 mg, ꢀ90% purity) was dissolved in

methanol (20 ml), sodium carbonate (10 mg, 0.1

mmol) was added, and the resulting solution was

stirred at room temperature overnight. TLC anal-

ysis showed a major product at R_f0.53 (SS-5).

After evaporation of methanol, the resulting

residue was dissolved in dichloromethane (100 ml),

washed with brine (10 ml), dried over anhydrous

sodium sulfate, and evaporated to a white solid.

MPLC (1000×10 mm i.d. column; elution with

ethyl acetate–hexane 15:85) followed by evapora-

tion of fractions 49–56 furnished 3b as a white

solid (26 mg, 34% yield; 98% purity): m.p., 178.5–

179.5°C; single component by TLC (R_f0.53, SS-5);

high-resolution MS, calcd. for C₂₇H₃₉O₂F₇,

528.2838, found 528.2834; MS, 528^†(24, M⁺),

513*^†(17), 510*^†(63), 495*^†(15), 484^†(41, M−

C₂H₄O), 471^†(15, M−C₃H₅O), 468^†(9, M−

C₂H₄O₂), 453^†(9, M−C₃H₇O₂), 413^†(8,

M−C₆H₁₁O₂), 401^†(11, M−C₇H₁₁O₂), 387^†(10,

M−C₈H₁₃O₂), 373^†(17, M−C₉H₁₅O₂), 271*^†

(12), 253*^†(5), 239^†(13, SC), 229^†(24, C₁₆H₂₁O),

124^†(40, C₈H₁₂O), 105^†(55, C₈H₉), 55 (100, C₄H₇);

IR, 3418, 2942, 2899, 2872, 1468, 1318, 1223, 1159,

2.4. 5,6h-Epoxy-25,26,26,26,27,27,27-hepta-

ﬂuoro-5h-cholestan-3i-ol acetate (4b), 5,6h-

epoxy-5h-cholestan-3i-ol acetate (4a), and

[26,26,26,27,27,27-²H₆]5,6h-epoxy-5h-cholestan-

3i-ol acetate (4c)

1

1074, 966 cm⁻¹; H NMR, Table 3; ¹³C NMR,

Table 4; ¹⁹F NMR, l_F−184.16 (dd of septet, 21.2,

To a solution of acetate 2b (70 mg, 0.13 mmol)

Table 3

¹H NMR chemical shifts of sterols oxygenated in rings A or B^a,b

4b-OH

3b-OH

3b

a-Epox

3b-OAc

4b

a-Epox

3b-OH

5b

b-Epox

3b-OAc

6b

b-Epox

3b-OH

7b

5a,6b-OH

3b-OH

8b

7-Keto

3b-OAc

16b

7-Keto

3b-OH

17b

7a-OAc

3b-OAc

18b

7b-OAc

3b-OAc

19b

7a-OH

3b-OH

20b

7b-OH

3b-OH

21b

H-1a

1.076

1.831

1.651

1.909

3.563

4.140

–

5.681

1.57

2.090

1.55

0.894

1.472^†

1.447^†

1.164

2.010

0.998^†

1.599

1.096^†

1.821

1.265

1.096

0.688

1.184

1.421^†

0.940

1.416^†

1.094

1.436^†

1.631

1.971^†

2.044^†

–

1.423

1.694

1.995

1.637

4.949

1.328

2.158

2.892

1.498

1.908

1.368

1.284^†

1.377

1.243^†

1.125

1.942

0.969^†

1.576

1.015^†

1.804

1.220

1.059

0.616

1.074

1.381^†

0.912

1.399^†

1.069^†

1.416^†

1.624

1.963^†

2.034^†

2.013

–

1.367

1.690

1.921

1.606

3.914

1.295

2.073

2.902

1.489

1.914

1.376

1.257^†

1.382

1.257^†

1.119

1.944

0.961^†

1.577

1.020^†

1.802

1.223

1.056

0.618

1.061

1.385^†

0.912

1.397^†

1.069

1.418^†

1.623

1.964^†

2.035^†

–

1.320

1.963

1.830

1.497

4.768

1.437

2.113

3.076

1.208

2.074

1.477

0.626

1.372^†

1.404^†

1.075

1.952

0.888

1.598

1.076

1.801

1.245

1.064

0.645

1.004

1.404^†

0.916

1.400^†

1.086^†

1.423

1.618

1.963^†

2.038^†

2.029

–

1.259

1.967

1.811

1.411

3.700

1.430

2.041

3.061

1.201

2.077

1.489

0.609

1.378^†

1.408^†

1.064

1.954

0.877

1.594

1.081

1.797

1.246

1.058

0.647

0.996

1.405^†

0.916

1.401^†

1.085^†

1.424

1.617

1.962^†

2.038^†

–

1.54

1.265

1.967^†

1.987^†

1.685

4.717

2.551

2.469

5.707

–

1.211

1.955^†

1.945^†

1.624

3.680

2.511

2.401

5.696

–

1.204

1.880^†

1.606

4.676^†

2.358^†

5.585

–

1.134

1.870

1.901

1.583

4.603^†

2.343^†

2.319^†

5.239

5.038

–

1.696

1.135

1.566^†

1.487^†

1.171

2.014

1.166

1.462

1.299^†

1.802

1.238^†

1.066

0.701

1.086

1.405^†

0.939

1.411^†

1.086^†

1.429^†

1.631

1.971^†

2.045^†

2.023

2.030

1.121

1.868^†

1.858^†

1.524

3.591

2.349

2.289

5.609

–

1.059

1.854^†

1.856^†

1.520

3.553

2.338

2.258

5.294

3.845

–

1.399

1.038

1.543^†

1.468^†

1.154

2.017

1.164

1.832^†

1.450

1.872^†

1.307

1.087

0.703

1.054

1.429^†

0.949

1.433^†

1.106

1.438^†

1.637

1.976^†

2.050^†

–

H-1b

1.422

1.867

1.52

H-2a

H-2b

H-3a

4.098

1.614

2.088

3.545

1.642^†

1.593^†

1.732

1.242

1.400^†

1.337^†

1.168

1.994

1.094^†

1.589

1.092^†

1.813

1.253

1.102

0.687

1.184

1.412^†

0.930

1.41

H-4a

H-4b

H-6

H-7a

/

H-7b

–

4.966

1.600

1.374

1.545^†

1.485^†

1.206

2.004

1.353

1.449

1.092

1.822

1.265

1.150

0.677

1.015

1.422^†

0.951

1.424^†

1.091

1.439^†

1.642

1.974^†

2.045^†

2.038

2.046

3.852

1.477^†

1.224

1.549^†

1.494^†

1.183

2.001

1.449^†

1.734

1.157

1.886

1.290

1.170

0.694

0.998

1.437^†

0.953

1.420^†

1.114

1.436^†

1.632

1.974^†

2.048^†

–

H-8b

2.235

1.54

1.59

2.243

1.506^†

1.59

H-9a

H-11a

H-11b

H-12a

H-12b

H-14a

H-15a

H-15b

H-16a

H-16b

H-17a

H-18

1.56

1.57

1.156

2.030

1.356^†

2.424

1.264^†

1.889

1.270^†

1.092

0.690

1.212

1.419^†

0.948

1.437^†

1.106

1.431^†

1.636

1.975^†

2.046^†

2.052

–

1.143

2.030

1.348

2.427

1.254^†

1.887

1.272^†

1.088

0.691

1.202

1.423^†

0.948

1.437^†

1.107

1.435^†

1.635

1.976^†

2.047^†

–

(

H-19

H-20

H-21

H-22R

H-22S

H-23R

H-23S

H-24R

H-24S

Acetate

1.086

1.430^†

1.627^†

1.966^†

2.042^†

–

^aSee footnote a of Table 1.

^bSee footnote b of Table 1.

Table 4

¹³C NMR chemical shifts of 25,26,26,26,27,27,27-heptaﬂuoro derivatives of sterols oxygenated in rings A or B^a,b,c

4b-OH

3b-OH

3b

a-Epox

3b-OAc

4b

a-Epox

3b-OH

5b

b-Epox

3b-OAc

6b

b-Epox

3b-OH

7b

5a,6b-OH

3b-OH

8b

7-Keto

3b-OAc

16b

7-Keto

3b-OH

17b

7a-OAc

3b-OAc

18b

7b-OAc

3b-OAc

19b

7a-OH

3b-OH

20b

7b-OH

3b-OH

21b

C-1

C-2

C-3

C-4

C-5

C-6

C-7

C-8

36.88

25.34

72.45

77.23

142.71

128.70

32.02

31.77

50.10

35.96

20.49

39.61

42.33

56.81

24.18

28.18

55.80

11.82

21.01

35.50

18.46

35.98

18.06

29.38

c

32.09

27.17

71.34

36.07

65.17

59.11

28.68

29.81

42.35

34.95

20.53

39.29

42.34

56.67

23.97

28.05

55.62

11.81

15.81

35.52

18.38

35.93

18.16

29.34

91.80

121.09

21.31

170.23

32.36

31.04

68.68

39.82

65.68

59.24

28.75

29.85

42.48

34.82

20.59

39.34

42.35

56.76

23.97

28.05

55.62

11.82

15.89

35.51

18.38

35.94

18.14

29.35

91.81

121.08

–

36.65

27.16

71.29

37.95

62.50

63.53

32.39

29.69

50.90

34.98

21.87

39.69

42.28

56.06

24.10

28.10

55.86

11.71

17.00

35.43

18.42

35.92

18.00

29.36

c

37.21

31.00

69.39

42.18

62.92

63.67

32.55

29.74

51.25

34.82

21.94

39.75

42.30

56.12

24.11

28.10

55.88

11.72

17.03

35.42

18.42

35.93

18.00

29.36

91.81

121.09

–

32.34

30.82

67.61

40.72

76.07

76.04

34.47

30.17

45.79

38.27

21.13

39.87

42.76

55.85

24.07

28.18

55.94

12.13

16.86

35.51

18.43

35.96

18.09

29.38

c

35.96

27.30

72.15

37.71

163.90

126.65

201.83

45.33

49.70

38.28

21.10

38.58

43.11

49.86

26.21

28.54

54.53

11.93

17.22

35.48

18.60

35.96

18.12

29.35

c

36.30

31.11

70.43

41.78

165.30

126.00

202.26

45.32

49.82

38.25

21.15

38.61

43.09

49.86

26.22

28.53

54.53

11.92

17.26

35.46

18.59

35.97

18.10

29.34

91.79

121.09

–

36.47

27.45

73.10

37.79

146.48

120.78

68.19

35.69

42.96

37.23

20.61

38.95

42.28

49.11

23.99

28.12

55.66

11.43

18.14

35.52

18.47

35.94

18.15

29.33

c

36.48

27.57

73.17

37.48

144.19

122.18

75.44

36.42

48.00

36.45

20.98

39.25

42.83

55.36

25.04

28.34

55.15

11.73

18.94

35.45

18.49

35.94

18.14

29.34

91.79

121.09

21.34^d

170.36^d

36.97

31.31

71.27

41.97

146.25

123.79

65.29

37.45

42.19

37.36

20.65

39.11

42.15

49.37

24.21

28.25

55.58

11.60

18.22

35.52

18.49

35.99

18.00

29.40

c

36.93

31.55

71.40

41.71

143.51

125.46

73.30

40.88

48.20

36.43

21.06

39.52

42.96

55.90

26.34

28.55

55.21

11.82

19.16

35.50

18.55

36.02

18.10

29.40

c

/

C-9

C-10

C-11

C-12

C-13

C-14

C-15

C-16

C-17

C-18

C-19

C-20

C-21

C-22

C-23

C-24

C-25

C-26

C-27

Acetate

(

121.10

–

121.09

21.30

170.56

c

121.10

21.36^d

170.45^d

121.10

–

121.13

–

21.25

170.29

–

^aSee footnote a of Table 2.

^bSee footnote b of Table 2.

^cSee footnote c of Table 2.

^dAdditional acetate signals: l_C21.36, 170.81 (18b), 21.62, 171.10 (19b).

S. Li et al. / Chemistry and Physics of Lipids 99 (1999) 33–71

41

in dichloromethane (15 ml) were added m-

2.5. 5,6h-Epoxy-25,26,26,26,27,27,27-hepta-

ﬂuoro-5h-cholestan-3i-ol (5b), 5,6h-epoxy-5h-

chloroperbenzoic acid (26 mg, 85% purity, 0.13

mmol) and sodium bicarbonate (17 mg, 0.20

mmol). The reaction mixture was stirred at room

temperature for 36 h, followed by dilution with

dichloromethane (20 ml), washing with 10%

aqueous sodium hydroxide solution (2×10 ml),

water (10 ml), and brine (10 ml), and drying over

anhydrous sodium sulfate. Evaporation gave a

white solid that was puriﬁed by MPLC on alu-

mina–AgNO₃(1000×10 mm i.d. column; elution

with ethyl acetate–hexane 3:97). Evaporation of

fractions 60–65 gave 4b (40 mg, 56% yield, 98%

purity, containing 0.2% 6b): m.p., 154.5–155.5°C;

single component by TLC (R_f0.24, SS-1); high-

resolution MS, calcd. for C₂₉H₄₁O₃F₇, 570.2944,

found 570.2926; MS, Table 5; IR, 2955, 2874,

2852, 1738, 1470, 1381, 1311, 1244, 1223, 1157,

cholestan-3i-ol (5a), and [26,26,26,27,27,27-²H₆]

5,6h-epoxy-5h-cholestan-3i-ol (5c)

To a solution of 4b (30 mg, 0.053 mmol) in

methanol (10 ml) was added sodium carbonate

(10 mg, 0.1 mmol), and the reaction mixture was

stirred at room temperature overnight. TLC anal-

ysis showed the disappearance of 4b and the

formation of a polar compound at R_f0.32 (SS-4).

The solvent was evaporated to a residue that was

dissolved in dichloromethane, washed with brine

(10 ml), and dried over anhydrous sodium sulfate.

Evaporation gave a white solid that was ﬁltered

through silica gel (30×5 mm i.d. column; elution

with ethyl acetate–hexane 15:85). Evaporation

gave 5b as a white solid (25 mg, 90% yield, 98%

purity): m.p., 169–170°C; single component by

TLC (R_f0.32, SS-4); high-resolution MS, calcd.

for C₂₇H₃₉O₂F₇, 528.2838, found 528.2827; MS,

Table 5; IR, 3478, 2938, 2870, 1468, 1314, 1221,

1

1132, 1042, 939 cm⁻¹; H and ¹³C NMR, Tables

3 and 4.

Similar treatment of 2a (200 mg) gave 4a (146

mg, 70% yield, ꢀ98% purity, containing 0.2%

6a): m.p., 98–100°C (lit. 97–98°C (Kudo et al.,

1989); 101°C (Fieser and Fieser, 1959)); single

component by TLC (R_f0.31, SS-1); MS, Table 5;

¹H and ¹³C NMR data identical (90.001 ppm for

¹H, 90.05 ppm for ¹³C) to those of 4c except for

the occurrence of signals at l_H1.510 (nonet, 6.6

Hz), 0.862 (d, 6.6 Hz, 3H), 0.857 (d, 6.6 Hz, 3H),

l_C39.45 (replacing 39.32), 27.97 (replacing 27.48),

22.80, 22.53.

Similar treatment of 2c (25 mg) gave 4c (20 mg,

77% yield, ꢀ99% purity, containing 0.5% 6c):

m.p., 94–96°C; single component by TLC (R_f

0.31, SS-1); high-resolution MS, calcd. for

C₂₉H₄₂D₆O₃, 450.3980, found 450.3981; MS,

Table 5; IR, 2943, 2868, 2212, 1736, 1467, 1377,

1240, 1028 cm⁻¹; NMR, l_H(50 mM solution)

4.949, (tt, 11.5, 4.9 Hz), 2.888 (d, 4.3 Hz), 2.157

(dd, 12.7, 11.6 Hz), 2.013 (s, 3H), 1.904 (ddd,

15.6, 11.9, 4.5 Hz), 1.494 (dd, 15.5, 9.8 Hz), 1.324

(ddd, 12.7, 5.0, 2.2 Hz), 1.073 (d, 0.5 Hz, 3H),

0.886 (d, 6.6 Hz, 3H), 0.607 (s, 3H); l_C170.21,

71.37, 65.16, 59.14, 56.72, 55.79, 42.38, 42.28,

39.32, 39.32, 36.09, 36.09, 35.74, 34.95, 32.08,

29.81, 28.72, 28.05, 27.48, 27.18, 24.01, 23.80,

21.33, 20.54, 18.60, 15.82, 11.82 (no signals ob-

served for C-26, C-27).

1

1161, 1061, 966 cm⁻¹; H and ¹³C NMR, Tables

3 and 4; ¹⁹F NMR, l_Fca. −184 (dd of septet,

21.1, 19.9, 6.7 Hz), −76.74 and −76.94 (A₃B₃

portion of A₃B₃X system (Swaminathan et al.,

1993)).

Similar treatment of 4a (50 mg) gave 5a as a

white solid (41 mg, 91% yield, 99% purity): m.p.,

140–141.5°C (lit. 142.5°C (Fieser and Fieser,

1959)); single component by TLC (R_f0.35, SS-4);

MS, Table 5; ¹H and ¹³C NMR data identical

(90.004 ppm for ¹H, 90.06 ppm for ¹³C) to

those of 5c except for the occurrence of signals at

l_H1.510 (nonet, 6.6 Hz), 0.862 (d, 6.6 Hz, 3H),

0.857 (d, 6.6 Hz, 3H), l_C27.97 (replacing 27.49),

22.80, 22.53.

Similar treatment of 4c (15 mg) gave 5c as a

white solid (12 mg, 88% yield, 99% purity): m.p.,

139–140°C; single component by TLC (R_f0.35,

SS-4); high-resolution MS, calcd. for C₂₇H₄₀D₆O₂,

408.3874, found 408.3888; MS, Table 5; IR, 3450,

2934, 2866, 2214, 1466, 1443, 1371, 1337, 1099,

1063, 1042 cm⁻¹; NMR, l_H(50 mM solution)

3.908 (tt, 11.3, 4.8 Hz), 2.899 (d, 4.3 Hz), 2.071

(dd, 12.7, 11.3 Hz), 1.910 (ddd, 15.6, 7.5, 4.5 Hz),

1.689 (ddd, 13.3, 4.2, 2.9 Hz), 1.603 (dddd, 13.5,

12.7, 11.3, 4.3 Hz), 1.484 (dd, 15.4, 9.8 Hz), 1.290

Table 5

Comparison of mass spectral data for a and b epoxides 5a and 7a, their 25,26,26,26,27,27,27-heptaﬂuoro (F₇) derivatives 5b and 7b, their 26,26,26,27,27,27-hexadeu-

terio (d₆) derivatives 5c and 7c, and acetate derivatives of the six epoxides^a

3b-Hydroxy (R=H)

3b-Acetates (R=Ac)

F₇-a

5b

F₇-b

7b

a

5a

b

7a

d₆-a

5c

d₆-b

7c

F₇-a

4b

F₇-b

6b

a

4a

b

6a

d₆-a

4c

d₆-b

6c

M+

M−Me

M−H₂O

528 (65)

513 (1)

510 (49)

495 (18)

–

(56)

(6)

(48)

(20)

–

(21)

(16)

(17)

(5)

(2)

(9)

(5)

(15)

(4)

(6)

(2)

(6)

(4)

(17)

(4)

(11)

(6)

(16)

(45)

(100)

402 (85)

387 (1)

384 (72)

369 (34)

–

(67)

408 (61)

393 (1)

390 (89)

375 (34)

–

(73)

(9)

(95)

(37)

–

570 (5)

555 (1)

552 (3)

537 (2)

510 (100)

495 (25)

492 (43)

482 (15)

481 (7)

477 (15)

466 (9)

401 (4)

387 (5)

373 (12)

333 (4)

319 (7)

331 (4)

271 (7)

253 (12)

239 (6)

230 (3)

229 (8)

211 (13)

120 (26)

107 (33)

55 (53)

(5)

(1)

(2)

(1)

(100)

(30)

(34)

(42)

(29)

(8)

(4)

(6)

(5)

(14)

(3)

(4)

(6)

444 (10)

429 (2)

426 (7)

411 (3)

384 (100)

369 (26)

366 (46)

356 (13)

355 (5)

351 (16)

340 (13)

275 (5)

261 (5)

247 (9)

207 (3)

193 (5)

331 (8)

271 (13)

(8)

(4)

(3)

(1)

(100)

(21)

(19)

(32)

(14)

(7)

(5)

(4)

(13)

(1)

450 (4)

435 (0)

432 (4)

417 (2)

390 (100)

375 (20)

372 (37)

362 (6)

361 (14)

357 (12)

346 (10)

281 (6)

267 (5)

253 (25)

213 (7)

199 (4)

331 (2)

271 (11)

(4)

(1)

(100)

(20)

(18)

(36)

(23)

(5)

(3)

(7)

(4)

(20)

(6)

(7)

(2)

(8)

(20)

(100)

(36)

–

(27)

(24)

(20)

(9)

(1)

(9)

(11)

(29)

(2)

/

M−H₂OꢀMe

M−AcOH

M−AcOHꢀMe

M−ROHꢀH₂O

M−ROHꢀCO

M−ROHꢀCHO

M−ROHꢀH₂OꢀMe 477 (10)

M−ROHꢀC₂H₄O

Ion B₁

Ion B₂

Ion B₃

Ion C₁

Ion C₂

M−SC

M−SCꢀROH

M−SCꢀROHꢀH₂O 253 (5)

SC

C₁₆H₂₂

C₁₆H₂₁

–

492 (14)

482 (4)

481 (5)

366 (17)

356 (6)

355 (5)

351 (17)

340 (4)

275 (12)

261 (25)

247 (24)

207 (5)

193 (14)

289 (10)

271 (22)

372 (27)

362 (12)

361 (12)

357 (11)

346 (3)

281 (7)

267 (10)

253 (20)

213 (9)

199 (11)

289 (4)

271 (15)

(35)

(44)

(56)

(8)

466 (3)

401 (8)

387 (7)

373 (15)

333 (5)

319 (9)

289 (3)

271 (8)

(2)

(13)

(10)

(27)

(11)

(10)

(13)

(27)

(7)

(3)

(

(29)

(24)

(11)

(13)

(10)

253 (11)

253 (20)

253 (25)

b

239 (16)

230 (3)

(5)

(2)

(7)

(10)

(8)

O

230 (7)

(5)

(25)

(24)

(18)

(50)

(34)

230 (6)

(12)

(24)

(13)

(25)

(72)

(100)

230 (3)

(4)

230 (4)

(4)

(12)

(7)

(12)

(25)

229 (13)

211 (22)

120 (40)

107 (49)

55 (100)

229 (19)

211 (20)

120 (65)

107 (71)

55 (100)

229 (19)

211 (14)

120 (30)

107 (60)

55 (100)

229 (13)

211 (16)

120 (30)

107 (32)

55 (45)

(10)

(15)

(19)

(39)

(49)

229 (11)

211 (15)

120 (28)

107 (26)

55 (22)

C₁₆H₁₉

C₉H₁₂

C₈H₁₁

C₄H₇

(24)

(30)

^aMass spectral data are given in the form m/z (relative abundance of a isomer) (relative abundance of b isomer). Ions B₁, B₂, B₃, C₁, and C₂are deﬁned in Fig.

^bExact masses of ions at m/z 113 and 119 did not correspond to C₈H₁₇or C₈H₁₁D₆.

7.

S. Li et al. / Chemistry and Physics of Lipids 99 (1999) 33–71

43

(ddd, 12.8, 4.9, 2.3 Hz), 1.059 (d, 0.6 Hz, 3H),

0.886 (d, 6.6 Hz, 3H), 0.609 (d, 0.5 Hz, 3H), l_C

(25°C) 68.72, 65.68, 59.29, 56.84, 55.85, 42.54,

42.32, 39.86, 39.40, 39.34, 36.13, 35.74, 34.84,

32.39, 31.08, 29.88, 28.81, 28.06, 27.49, 24.03,

23.82, 20.63, 18.62, 15.91, 11.85 (no signals ob-

served for C-26, C-27).

(replacing 39.32), 27.98 (replacing 27.49), 22.79,

22.53.

Similar treatment of 2c (25 mg) gave 6c (22 mg,

85% yield, \99% purity): m.p., 108–109°C; sin-

gle component by TLC (R_f0.31, SS-1); high-reso-

lution MS, calcd. for C₂₉H₄₂D₆O₃, 450.3980,

found 450.4003; MS, Table 5; IR, 2932, 2866,

2214, 1730, 1468, 1366, 1248, 1041 cm⁻¹; NMR,

l_H(50 mM solution) 4.767 (ddt, 11.9, 10.3, 4.8

Hz), 3.073 (br dd, 2.7, 0.8 Hz), 2.112 (ddd, ꢀ13,

ꢀ12, 0.5 Hz), 2.073 (dtd, 14.4, 3.3, 0.5 Hz), 2.028

(s, 3H), 1.575 (dddd, 12.3, 9.9, 7.3, 3.0 Hz), 1.435

(ddd, 13.1, 5.1, 2.2 Hz), 1.202 (ddd, 14.6, 10.9, 1.1

Hz), 1.002 (s, 3H), 0.889 (d, 6.6 Hz, 3H), 0.637 (s,

3H); l_C71.31, 63.57, 62.49, 56.13, 56.13, 50.94,

42.23, 39.73, 39.32, 37.97, 36.64, 36.09, 35.70,

34.98, 32.42, 29.69, 28.12, 27.49, 27.17, 24.14,

23.76, 21.89, 18.64, 17.01, 11.73 (no signals ob-

served for C-26, C-27).

2.6. 5,6i-Epoxy-25,26,26,26,27,27,27-hepta-

ﬂuoro-5i-cholestan-3i-ol acetate (6b), 5,6i-

epoxy-5i-cholestan-3i-ol acetate (6a), and

[26,26,26,27,27,27-²H₆]5,6i-epoxy-5i-choles-

tan-3i-ol acetate (6c)

Copper sulfate pentahydrate (0.25 g) and potas-

sium permanganate (0.50 g) were ground together

to a ﬁne powder, to which water (40 ml) was

added. This paste was transferred to a 15 ml ﬂask

containing a solution of 2b (80 mg, 0.144 mmol)

in dichloromethane (10 ml), followed by addition

of tert-butanol (0.1 ml). The reaction mixture was

heated to reﬂux for 10 min and stirred at room

temperature overnight. TLC analysis showed the

absence of 2b and the formation of polar mate-

rial. The reaction mixture was ﬁltered through a

pad of silica gel (230–400 mesh), followed by

washing with dichloromethane (50 ml). The

ﬁltrate was dried over anhydrous sodium sulfate

and evaporated to a white solid, which was sub-

jected to MPLC on alumina–AgNO₃(1000×10

mm i.d. column; elution with ethyl acetate–hex-

ane 3:97). Evaporation of fractions 25–40 gave 6b

(45 mg, 55% yield, ꢀ97% purity): m.p., 136–

137°C; single component by TLC (R_f0.24, SS-1);

high-resolution MS, calcd. for C₂₉H₄₁O₃F₇,

570.2944, found 570.2928; MS, Table 5; IR, 2949,

2870, 1738, 1470, 1379, 1312, 1226, 1155, 1032,

966, 939 cm⁻¹; ¹H and ¹³C NMR, Tables 3 and 4.

Similar treatment of 2a (200 mg) gave 6a (165

mg, 80% yield, ꢀ99% purity): m.p., 109.5–

110.5°C (lit. 110°C (Marchon and Ramasseul,

1989), 112–113°C (Kudo et al., 1989), 113°C

(Fieser and Fieser, 1959)); single component by

2.7. 5,6i-Epoxy-25,26,26,26,27,27,27-hepta-

ﬂuoro-5i-cholestan-3i-ol (7b), 5,6i-epoxy-5i-

cholestan-3i-ol (7a), and [26,26,26,27,27,27-²H₆]

5,6i-epoxy-5i-cholestan-3i-ol (7c)

As described above for 4b, saponiﬁcation of 6b

(40 mg, 0.070 mmol) in methanol (10 ml) and

sodium carbonate (10 mg, 0.1 mmol) gave after

workup 7b as a white solid (34 mg, 92% yield,

98% purity): m.p., 138.5–140°C; single compo-

nent by TLC (R_f0.32, SS-4); high-resolution MS,

calcd. for C₂₇H₃₉O₂F₇, 528.2838, found 528.2841;

MS, Table 5; IR, 3439, 3366, 2944, 2870, 1468,

1

1314, 1221, 1159, 1065, 938 cm⁻¹; H and ¹³C

NMR, Tables 3 and 4; ¹⁹F NMR, l_F−184.18

(dd of septet, 21.2, 19.8, 6.6 Hz), −76.73 and

−76.95 (A₃B₃portion of A₃B₃X system (Swami-

nathan et al., 1993)).

Similar treatment of 6a (100 mg) gave 7a (85

mg, 94% yield, \99% purity): m.p., 129–130°C

(lit. 131°C (Baxter and Spring, 1943), 132°C

(Fieser and Fieser, 1959)); single component by

1

TLC (R_f0.35, SS-4); MS, Table 5; H and ¹³C

1

TLC (R_f0.31, SS-1); MS, Table 5; H and ¹³C

NMR data identical (90.003 ppm for ¹H, 90.04

ppm for ¹³C) to those of 7c except for the occur-

rence of signals at l_H1.510 (nonet, 6.6 Hz), 0.862

(d, 6.6 Hz, 3H), 0.858 (d, 6.6 Hz, 3H), l_C39.46

NMR data identical (90.001 ppm for ¹H, 90.05

ppm for ¹³C) to those of 6c except for the occur-

rence of signals at l_H1.511 (nonet, 6.6 Hz), 0.862

(d, 6.6 Hz, 3H), 0.858 (d, 6.6 Hz, 3H), l_C39.45

44

S. Li et al. / Chemistry and Physics of Lipids 99 (1999) 33–71

(replacing 39.34), 27.98 (replacing 27.50), 22.77,

22.53.

calcd. for C₂₇H₄₁O₃F₇, 546.2944, found 546.2940;

MS, 546^†(2, M⁺), 528*^†(49), 510*^†(100), 495*^†

(20), 492*^†(27), 474^†(10, M−C₄H₈O), 457^†(10,

M−C₄H₉O), 429^†(6, M−C₆H₁₃O), 401^†(5,

C₂₀H₂₈F₇), 373^†(12, C₁₈H₂₄F₇), 319^†(5,

C₁₄H₁₈F₇), 271*^†(6), 253*^†(4), 247^†(13,

C₁₆H₂₃O₂), 244^†(25, C₁₇H₂₄O), 239^†(11, SC), 229^†

(18, C₁₆H₂₁O), 211^†(13, C₁₆H₁₉), 107^†(32, C₈H₁₁);

IR, 3434, 3399, 2946, 2916, 2870, 1468, 1445,

Similar treatment of 6c (18 mg) gave 7c (14 mg,

86% yield, 99% purity): m.p., 130–131°C; single

component by TLC (R_f0.35, SS-4); high-resolu-

tion MS, calcd. for C₂₇H₄₀D₆O₂, 408.3874, found

408.3874; MS, Table 5; IR, 3420, 2936, 2866,

2216, 1468, 1447, 1375, 1057 cm⁻¹; NMR, l_H(50

mM solution) 3.695 (dddd, 11.8, 10.3, 4.8, 4.4

Hz), 3.057 (br dd, 2.9, 1.0 Hz), 2.075 (dddd, 14.3,

3.7, 2.7, 0.7 Hz), 2.038 (br dd, 13.0, 11.8 Hz),

1.479 (qd, 11.0, 4.0 Hz), 1.426 (ddd, 13.1, 4.8, 2.3

Hz), 1.196 (ddd, 14.6, 11.0, 1.2 Hz), 0.993 (s, 3H),

0.889 (d, 6.6 Hz, 3H), 0.639 (d, 0.4 Hz, 3H); l_C

69.42, 63.71, 62.92, 56.22, 56.20, 51.32, 42.27,

42.22, 39.82, 39.34, 37.21, 36.12, 35.71, 34.84,

32.60, 31.04, 29.76, 28.13, 27.50, 24.17, 23.79,

21.98, 18.66, 17.03, 11.74 (no signals observed for

C-26, C-27).

1

1379, 1316, 1221, 1159, 1046 cm⁻¹; H and ¹³C

NMR, Tables 3 and 4; ¹⁹F NMR, l_F−184.15

(dd of septet, 21.2, 19.9, 6.6 Hz), −76.74 and

−76.94 (A₃B₃portion of A₃B₃X system (Swami-

nathan et al., 1993)).

Similar treatment of 1a (1.0 g) gave 8a as a

white solid (0.75 g, 69% yield, \99% purity):

m.p., 233.5–235°C (lit. 236–238°C (Baxter and

Spring, 1943)); single component by TLC (R_f

0.10, SS-6); MS, 420 (4, M⁺), 402* (100), 388

(10), 384* (88), 369* (30), 366* (11), 348 (10), 331

(17), 303 (9), 271* (13), 262 (25), 253* (5), 247

(34), 244 (28), 229 (29), 211 (17), 193 (4), 107 (39);

¹H and ¹³C NMR data identical (90.003 ppm for

¹H, 90.04 ppm for ¹³C) to those of 8c except for

the occurrence of signals at l_H1.515 (nonet, 6.6

Hz), 0.865 (d, 6.6 Hz, 3H), 0.861 (d, 6.6 Hz, 3H),

l_C39.47 (replacing 39.34), 27.99 (replacing 27.50),

22.81, 22.55.

Similar treatment of 1c (20 mg) gave 8c as a

white solid (17 mg, 78% yield, 99% purity): m.p.,

235–236°C; single component by TLC (R_f0.10,

SS-6); high-resolution MS, calcd. for C₂₇H₄₂D₆O₃,

426.3980, found 426.3980; MS, 426^†(3, M⁺),

408*^†(100), 390*^†(83), 375*^†(24), 372*^†(16),

354^†(10, M−C₄H₈O), 337^†(20, M−C₄H₉O),

309^†(7, M−C₆H₁₃O₂), 289*^†(9), 281^†(4,

C₂₀H₂₉D₆), 271*^†(13), 268^†(17, M−C₈H₁₄O₃),

253^†(14, C₁₈H₂₅D₆and C₁₉H₂₅), 247^†(31,

C₁₆H₂₃O₂), 244^†(26, C₁₇H₂₄O), 229^†(26,

C₁₆H₂₁O), 211^†(18, C₁₆H₁₉), 199^†(9, C₁₄H₁₉D₆),

107^†(36, C₈H₁₁); IR, 3583, 3562, 3543, 3420,

3400, 3273, 2937, 2866, 2214, 1647, 1456, 1375,

1292, 1085, 1043, 1014 cm⁻¹; NMR, l_H4.098 (tt,

11.0, 5.4 Hz), 3.542 (t, 2.9 Hz), 2.086 (dd, 12.9,

11.1 Hz), 1.999 (dddd, 12.6, 3.8, 3.1, 0.4 Hz),

ꢀ1.632 (ddd, 14.1, 11.4, 3.4 Hz), 1.616 (ddd,

12.8, 5.2, 1.9 Hz), ꢀ1.594 (ddd, 14.2, 5.1, 2.7

Hz), 1.184 (d, 0.4 Hz, 3H), 0.904 (d, 6.6 Hz, 3H),

2.8. 25,26,26,26,27,27,27-Heptaﬂuoro-5h-

cholestane-3i,5,6i-triol (8b), 5h-cholestane-

3i,5,6i-triol (8a), and [26,26,26,27,27,27-

²H₆]5h-cholestane-3i,5,6i-triol (8c)

A suspension of 1b (40 mg, 0.078 mmol) in 95%

formic acid (2 ml) was heated with stirring to

75°C for 5 min; an oil layer formed on the sur-

face. The mixture was cooled to 25°C and 30%

hydrogen peroxide (0.5 ml) was added dropwise.

The reaction was stirred for 6 h, followed by

addition of boiling water (50 ml). The white pre-

cipitate that formed was collected by ﬁltration,

dried in vacuo, and dissolved in methanol (5 ml).

Sodium hydroxide (10 mg) was added, and the

resulting solution was stirred at room temperature

for 0.5 h. TLC analysis (SS-7) showed a predomi-

nant product at R_f0.21. The methanol was evapo-

rated, and the resulting residue was dissolved in

dichloromethane (10 ml), followed by washing

with brine (10 ml) and drying over anhydrous

sodium sulfate. Evaporation gave a white solid

that was puriﬁed by MPLC (1000×10 mm i.d.

column; elution with methanol–chloroform 5.95).

Evaporation of fractions 24–29 gave 8b as a white

solid (32 mg, 75% yield, ꢀ98% purity): m.p.,

260–261°C; single component by TLC (R_f0.09,

SS-6 and R_f0.21, SS-7); high-resolution MS,

S. Li et al. / Chemistry and Physics of Lipids 99 (1999) 33–71

45

0.679 (d, 0.5 Hz, 3H), l_D0.84 (s), l_C76.08, 76.05,

67.62, 56.20, 55.90, 45.87, 42.72, 40.70, 39.91,

39.34, 38.27, 36.13, 35.78, 34.51, 32.35, 30.83,

30.17, 28.20, 27.50, 24.12, 23.83, 21.15, 18.64,

16.89, 12.14 (no signals observed for C-26, C-27).

Fractions 50–60 gave 11 as a white solid (0.50

g, 20% yield, ]99% purity): m.p.: 100–101°C;

single component by TLC (R_f0.47, SS-1); high-

resolution MS, calcd. for C₃₉H₇₃DO₃Si₂,

647.5239, found 647.5240; MS (\99% d₁), 647^†

(1, M⁺), 632*^†(4), 629*^†(4), 590*^†(73), 572*^†

(40), 500*^†(10), 498*^†(11, M−H₂OꢀTBDMSO),

458*^†(65), 440*^†(6), 366*^†(10, 498−TBDM-

SOH), 330 (10), 256^†(16, C₁₉H₂₆D), 254^†(18,

C₁₉H₂₄D), 213^†(13, C₁₆H₁₉D), 159^†(30, C₁₂H₁₅),

115^†(12, C₆H₁₅Si), 105^†(27, C₈H₇D), 75^†(100,

C₂H₇OSi); IR, 3622, 2949, 2931, 2885, 1471, 1383,

2.9. [16h-²H]-(25R)-Bis(3i,26-tert-butyldi-

methylsilyloxy)cholest-5-en-16i-ol (10) and

[16i-²H]-(25R)-bis(3i,26-tert-butyldi-

methylsilyloxy)cholest-5-en-16h-ol (11)

To a solution of 16-ketosterol 9 (2.50 g, 3.9

mmol) in anhydrous diethyl ether (200 ml) at 0°C

was added lithium aluminum deuteride (0.16 g,

3.8 mmol) under nitrogen. The reaction mixture

was stirred at room temperature overnight, fol-

lowed by addition of ﬁve drops of water. The

resulting slurry was ﬁltered and washed with di-

ethyl ether (50 ml). The ﬁltrate was evaporated to

a residue that was subjected to MPLC (500 mm×

25 mm i.d. column; elution with ethyl acetate–

hexane 4:96). Fractions 30–44 gave 10 as a white

solid (1.80 g, 72% yield, 99% purity, \99% d₁by

¹H NMR): m.p., 121.5–122.5°C (lit. m.p. for

protio analog, 123–124°C (Kim et al., 1989));

single component by TLC (R_f0.57, SS-1); high-

resolution MS, calcd. for C₃₉H₇₃DO₃Si₂,

647.5239, found 647.5228; MS (\99% d₁), 647^†

(1, M⁺), 632*^†(3), 629*^†(2), 590*^†(68), 572*^†

(20), 500*^†(5), 498*^†(3, M−H₂OꢀTBDMSO),

458*^†(45), 440*^†(4), 366*^†(6, 498−TBDM-

SOH), 330 (8), 256^†(9, C₁₉H₂₆D), 254^†(12,

C₁₉H₂₄D), 213^†(8, C₁₆H₁₉D), 159^†(20, C₁₂H₁₅),

115^†(8, C₆H₁₅Si), 105^†(20, C₈H₇D), 75^†(100,

C₂H₇OSi); IR, 3628, 2957, 2930, 2883, 2856, 1469,

1249, 1085, 839, 773 cm⁻^{1 1}H and ¹³C NMR

;

(Tables 1 and 2) showed absence of signals for

C-16 and H-16b (B0.01H).

2.10. [16h-²H]-(25R)-Bis(3i,26-tert-butyldi-

methylsilyloxy)cholest-5-en-16i-ol

methanesulfonate ester (12)

To a solution of 10 (1.71 g, 2.64 mmol) in

pyridine (50 ml) at 0°C was added methanesul-

fonyl chloride (1.4 ml, 18.1 mmol) dropwise under

nitrogen. The reaction mixture was stirred at

room temperature for 24 h, poured into ice-water

(200 ml), and extracted with dichloromethane

(3×100 ml). The combined extracts were washed

with cold 5% sulfuric acid (3×100 ml) and brine

(30 ml), dried over anhydrous sodium sulfate, and

evaporated to give 12 as a white solid (2.0 g, 104%

yield): TLC (R_f0.45, SS-1).

2.11. [16,16-²H₂]-(25R)-Bis(3i,26-tert-butyldi-

methylsilyloxy)cholest-5-ene (13) and [16-²H]-

(25R)-bis(3i,26-tert-butyldimethylsilyloxy)

cholesta-5,16-diene (14)

1384, 1251, 1076, 837, 775 cm⁻^{1 1}H and ¹³C

;

NMR identical (90.002 ppm for ¹H, 90.02 ppm

for ¹³C) to those of the undeuterated sterol²(Kim

et al., 1989) except for small upﬁeld shifts for ring

D protons, H-20, C-15 and C-17 (0.11–0.13 ppm)

and C-16 (0.5 ppm). These ¹³C signals appeared

as a weak multiplets, and the residual H-16a

signal at l_H4.35 integrated for 0.004 H.

A solution of crude 12 (1.9 g, 2.6 mmol) in

diethyl ether (20 ml) was added dropwise to a

solution of lithium aluminum deuteride (94 mg,

2.2 mmol) in diethyl ether (100 ml) at 0°C. The

reaction mixture was stirred at room temperature

for 5 h and quenched with water (ﬁve drops). The

inorganic precipitate was removed by ﬁltration

and thoroughly washed with diethyl ether. Evapo-

ration gave a white powder (1.6 g): single compo-

nent by TLC (R_f0.85, ethyl acetate–hexane 5:95);

²NMR data were compared with recent spectra of un-

deuterated 25- and 26-hydroxysterols in dilute solution; these

unpublished data differ slightly from those in the cited refer-

ences.

1

9:2 mixture of 13 and 14 by H NMR. Owing to

46

S. Li et al. / Chemistry and Physics of Lipids 99 (1999) 33–71

the hydrophobicity of the TBDMS groups, the

mixture was not retained on alumina–AgNO₃

(elution with hexane), and the sample was con-

verted to the diacetate derivative for puriﬁcation

as described below.

Fractions 80–86 gave 15 as a white solid (0.20

g, 16% yield from 12, ꢀ98% purity): m.p., 126–

127°C; single component by TLC (R_f0.62, SS-2);

high-resolution MS, calcd. for C₃₀H₄₄DO₄,

470.3381 (M−15), found 470.3392; MS (ꢀ99%

d₁), 470*^†(2), 425*^†(72), 410*^†(46), 365*^†(2),

358^†(3, C₂₅H₂₆O₂), 350*^†(4), 314*^†(4), 304^†(4,

M−C₁₁H₁₇O₂), 282^†(10, C₂₁H₂₈D), 266^†(3,

C₂₀H₂₄D), 254*^†(72), 236^†(21, C₁₆H₂₆DO and

C₁₈H₁₈D), 221 (37), 212^†(10, C₁₆H₁₈D), 180^†(79,

C₁₄H₁₀D), 165^†(86, C₁₂H₁₉D), 57^†(100, C₄H₉);

IR, 2960, 2939, 2924, 2910, 2852, 1732, 1465, 1448,

1438, 1369, 1246, 1035 cm⁻¹; ¹H NMR (50 mM in

CDCl₃), l_H5.397 (br dq, 5.2, 1.3 Hz), 5.29 (m,

0.01H), 4.608 (m), 3.935 (dd, 10.7, 5.9 Hz), 3.837

(dd, 10.7, 6.9 Hz), 2.34 (m), 2.32 (m), 2.050 (s,

3H), 2.034 (s, 3H), 1.058 (d, 0.5 Hz, 3H), 0.991 (d,

6.9 Hz, 3H), 0.911 (d, 6.8 Hz, 3H), 0.781 (d, 0.4

Hz, 3H); ¹³C NMR, Table 2.

2.12. [16,16-²H₂]-(25R)-Cholest-5-ene-3i,26-diol

diacetate (2e) and [16-²H]-(25R)-cholesta-

5,16-diene-3i,26-diol diacetate (15)

Tetrabutylammonium ﬂuoride (15 ml, 1.0 M

solution in tetrahydrofuran) was added to the 9:2

mixture of 13 and 14 in tetrahydrofuran (100 ml).

The reaction mixture was stirred for 24 h and

analyzed by TLC, which showed disappearance of

13 and 14 and the formation of polar material

with the same mobility as 1d (R_f0.62, SS-5).

Extraction with ethyl acetate (2×150 ml) fol-

lowed by washing with water (100 ml) and brine

(50 ml), drying over anhydrous sodium sulfate,

and evaporation to dryness furnished a white solid

(1.0 g): single component by TLC (R_f0.6, SS-5).

To a solution of the crude diol in pyridine (50 ml)

was added acetic anhydride (5 ml). The reaction

mixture was stirred overnight at room tempera-

ture, poured into ice-water (200 ml), and extracted

with dichloromethane (3×100 ml). The combined

organic extracts were washed with cold 5% sulfuric

acid (3×100 ml) and brine (30 ml) and dried over

anhydrous sodium sulfate. Evaporation gave a

white solid that was subjected to MPLC (500×25

mm i.d. column, elution with ethyl acetate–hexane

4:96). Evaporation of fractions 42–60 gave 2e as a

white solid (0.90 g, 70% yield from 12, ]99%

purity): m.p., 126–127°C; single component by

TLC (R_f0.62, SS-2); high-resolution MS, calcd.

for C₂₉H₄₄D₂O₂, 428.3623 (M−60), found

428.3625; MS (ꢀ99% d₂), 428*^†(100), 413*^†(10),

368*^†(25), 307^†(14, M−C₁₁H₁₇O₂), 257*^†(13),

255*^†(8), 213^†(15, C₁₆H₂₁), 158^†(35, C₁₂H₁₄),

145^†(33, C₁₁H₁₃), 105^†(24, C₈H₉); IR, 2945, 2910,

2872, 2850, 2831, 1736, 1466, 1377, 1365, 1238,

2.13. [16,16-²H₂]-(25R)-Cholest-5-ene-3i,26-diol

(1e)

To a solution of 2e (300 mg, 0.61 mmol) in

methanol (50 ml) was added sodium carbonate

(100 mg, 1.0 mmol). The reaction mixture was

stirred at room temperature overnight, concen-

trated in vacuo, and extracted with ethyl acetate

(100 ml). The organic layer was washed with water

(2×50 ml) and dried over sodium sulfate. Evapo-

ration gave a white solid that was subjected to

MPLC (500×10 mm i.d. column; elution with

ethyl acetate–hexane 2:8). Evaporation of frac-

tions 34–51 gave 1e as a white solid (220 mg, 89%

yield, ]99% purity): m.p., 173–174°C; single

component by TLC (R_f0.60, SS-5); high-resolu-

tion MS, calcd. for C₂₇H₄₄D₂O₂, 404.3623, found

404.3626; MS (96% d₂, 2% d₁, 2% d₀),³404 (100,

M⁺), 389*^†(30), 386*^†(55), 371*^†(28), 347^†(4,

M−C₃H₅O), 344^†(7, M−C₃H₈O), 319^†(35,

M−C₅H₉O), 293^†(66, M−C₇H₁₁O), 275*^†(22),

1033 cm^{−1 1}H and ¹³C NMR data essentially

;

³Mass spectra of diacetates 2e and 16e showed 100% d₂, but

lower values (90–98% d₂)were sometimes observed for diols 1e

and 17e. Because acetate hydrolysis should not affect deu-

terium at C-16, the lower values were attributed to dehydro-

genation (M−2), which was observed in low and variable

abundance for the undeuterated diols 1d and 17d.

identical to those of the undeuterated sterol²(Sid-

diqui et al., 1992) except for the deuterium isotope

effects and the absence of signals and couplings

2

for C-16, H-16a, and H-16b; H NMR (90.003

ppm), 1.819 (s), 1.237 (s).

S. Li et al. / Chemistry and Physics of Lipids 99 (1999) 33–71

47

272^†(9, C₁₉H₂₄D₂O), 265^†(13, C₁₈H₂₉D₂O), 257*^†

(26), 231^†(18, C₁₆H₂₃O), 228^†(10, C₁₇H₂₄), 213^†

(34, C₁₆H₂₁), 199^†(9, C₁₅H₁₅D₂), 159^†(32,

C₁₂H₁₅), 145^†(45, C₁₁H₁₃), 119^†(37, C₉H₁₁); IR,

3329, 3319, 2960, 2933, 2897, 2877, 2850, 1464,

(Morand and Van Tongerloo, 1973)); single com-

ponent by TLC (R_f0.21, SS-1); MS, 442 (0.6,

M⁺), 382* (100), 367* (11), 340 (3), 297* (3),

269* (20), 242 (5), 229 (9), 227 (9), 174 (73), 161

1

(33); H NMR data identical (90.001 ppm) to

1437, 1375, 1357, 1057, 1022 cm⁻¹

;

¹H NMR

those of 16c except for the occurrence of signals

at l_H1.517 (nonet, 6.6 Hz), 0.866 (d, 6.6 Hz, 3H),

0.862 (d, 6.6 Hz, 3H).

data essentially identical to those of the undeuter-

ated sterol²(Kim et al., 1989) except for deu-

terium isotope effects and the absence of signals

Similar treatment of 2c (170 mg) gave 16c as a

white solid (110 mg, 63% yield; 98% purity): m.p.,

156–157°C; single component by TLC (R_f0.21,

SS-1); high-resolution MS, calcd. for C₂₉H₄₀D₆O₃,

448.3824, found 448.3836; MS, 448^†(1, M⁺),

388*^†(100), 373*^†(5), 346^†(2, M−C₅H₁₀O₂), 297

(2), 269*^†(14), 253 (1, C₁₈H₂₅D₆), 242^†(3,

C₁₇H₂₂O), 229^†(6, C₁₆H₂₁O), 227^†(6, C₁₆H₁₉O),

174^†(59, C₁₂H₁₄O), 161^†(26, C₁₁H₁₃O); IR, 2939,

2906, 2885, 2868, 2214, 1734, 1703, 1467, 1442,

1381, 1255, 1037 cm⁻¹; NMR, l_H(50 mM solu-

tion) 5.702 (d, 1.7 Hz), 4.715 (tdd, 11.5, 5.1, 4.3

Hz), 2.547 (ddd, 14.1, 5.1, 2.3 Hz), 2.467 (ddd,

13.9, 11.6, 2.0 Hz), 2.40 (m), 2.231 (dd, 12.0, 10.8

Hz), 2.051 (s, 3H), 1.683 (distorted dddd, 14.2,

12.5, 11.8, 3.6), 1.209 (d, 0.6 Hz, 3H), 0.922 (d,

6.6 Hz, 3H), 0.682 (s, 3H), l_C201.94, 170.26,

163.82, 126.69, 72.19, 54.75, 49.93, 49.79, 45.40,

43.09, 39.32, 38.64, 38.29, 37.73, 36.16, 35.98,

35.70, 28.52, 27.48, 27.33, 26.28, 23.80, 21.24,

21.15, 18.84, 17.24, 11.95 (no signals observed for

C-26, C-27).

2

and couplings for H-16a, and H-16b; H NMR

(90.003 ppm), 1.815 (s), 1.244 (s).

2.14. 3i-Acetoxy-25,26,26,26,27,27,27-hepta-

ﬂuorocholest-5-en-7-one (16b), 3i-acetoxy-

cholest-5-en-7-one (16a), [26,26,26,27,27,27-

²H₆]3i-acetoxycholest-5-en-7-one (16c),

(25R)-3i,26-diacetoxycholest-5-en-7-one (16d),

and [16,16-²H₂]-(25R)-3i,26-diacetoxy-

cholest-5-en-7-one (16e)

To a solution of 2b (170 mg, 0.31 mmol) in

benzene (100 ml; dried over sodium) were added

pyridinium chlorochromate (1.6 g, 7.4 mmol) and

molecular sieve (0.1 g; type 3A) in one portion.

The reaction mixture was reﬂuxed under nitrogen

for 24 h, ﬁltered through a pad of silica gel

(230–400 mesh) with thorough elution using ethyl

acetate–hexane 1:1. The ﬁltrate was dried over

anhydrous sodium sulfate and evaporated to a

white solid that was subjected to MPLC (1000×

10 mm i.d. column; elution with ethyl acetate–

hexane 3:97 (1000 ml) and ethyl acetate–hexane

6:94). Evaporation of fractions 52–60 gave 16b as

a white solid (113 mg, 65% yield, ]99% purity):

single component by TLC (R_f0.27, SS-1); high-

resolution MS, calcd. for C₂₇H₃₅OF₇(M−60),

508.2576, found 508.2561; MS, 568^†(0.1, M⁺),

508*^†(100), 493*^†(6), 489*^†(4), 466^†(2, M−

C₅H₁₀O₂), 297^†(3, M−CH₃COOHꢀC₆H₆F₇),

269*^†(10), 242^†(3, C₁₇H₂₂O), 239^†(2, SC), 229^†

(4, C₁₆H₂₁O), 227^†(5, C₁₆H₁₉O), 174^†(42,

C₁₂H₁₄O), 161^†(30, C₁₁H₁₃O); IR, 2945, 2874,

2862, 1734, 1670, 1464, 1377, 1238, 1157, 1039

Similar treatment of 2d (300 mg) gave 16d as a

white solid (212 mg, 69% yield; \99% purity):

m.p., 119.5–120.5°C (lit. 121–122°C (Noll et al.,

1973); single component by TLC (R_f0.21, SS-2);

high-resolution MS, calcd. for C₃₁H₄₈O₅,

500.3502, found 500.3523; MS, 500^†(1, M⁺),

440*^†(100), 425*^†(3), 380*^†(3), 365*^†(1), 269*^†

(15), 242^†(3, C₁₇H₂₂O), 229^†(6, C₁₆H₂₁O), 228^†

(5, C₁₆H₂₀O), 227^†(6, C₁₆H₁₉O), 213^†(3,

C₁₅H₁₇O), 187^†(16, C₁₃H₁₅O), 174^†(45, C₁₂H₁₄O),

161^†(23, C₁₁H₁₃O); IR, 2949, 2874, 1736, 1676,

1

1643, 1462, 1371, 1238, 1030 cm⁻¹; H and ¹³C

NMR, Tables 1 and 2.

1

cm⁻¹; H and ¹³C NMR, Tables 3 and 4.

Similar treatment of 2e (500 mg) gave, after

MPLC (1000×10 mm i.d. column, elution with

ethyl acetate–hexane 6:94) and evaporation of

fractions 67–79, 16e as a white solid (360 mg,

70% yield, 99% purity): mp, 117–118.5°C; single

Similar treatment of 2a (450 mg) gave 16a as a

white solid (300 mg, 65% yield, ]99% purity):

m.p., 161–162°C (lit. 161–163°C (Dauben et al.,

1969), 162–163°C (Kudo et al., 1989), 163–164°C

48

S. Li et al. / Chemistry and Physics of Lipids 99 (1999) 33–71

component by TLC (Rf 0.21, SS-2); high-resolu-

tion MS, calcd. for C₃₁H₄₆D₂O₅, 502.3627, found

502.3626; MS (ꢀ99% d₂), 502^†(2, M⁺), 442*^†

(100), 427*^†(3), 382*^†(3), 367*^†(2), 271*^†(17),

242^†(6, C₁₇H₂₂O), 229^†(6, C₁₆H₂₁O), 228^†(8,

C₁₆H₂₀O), 227^†(8, C₁₆H₁₉O), 213^†(3,

C₁₅H₁₃D₂O), 174^†(62, C₁₂H₁₄O), 161^†(31,

C₁₁H₁₃O); IR, 2951, 2881, 1736, 1678, 1464,

165.5–166.5°C; single component by TLC (R_f

0.49, SS-6); high-resolution MS, calcd. for

C₂₇H₃₈D₆O₂, 406.3718, found 406.3736; MS, 406^†

(100, M⁺), 391*^†(3), 388*^†(45), 373*^†(17),

347^†(2, M−C₃H₇O), 287*^†(10), 269*^†(7), 245^†

(7, C₁₆H₂₁O₂), 205^†(8, C₁₃H₁₇O₂), 174^†(44,

C₁₂H₁₄O), 161^†(39, C₁₁H₁₃O); IR, 3435, 2936,

2866, 2214, 1665, 1462, 1439, 1379, 1230, 1057

cm⁻¹; NMR, l_H(50 mM solution) 3.674 (tdd,

11.2, 4.8, 4.1 Hz), 2.507 (ddd, 13.9, 4.7, 2.3 Hz),

2.400 (ddd, 13.8, 11.4, 2.0 Hz), 2.40 (m), 2.239

(dd, 12.4, 10.7 Hz), 2.034 (distorted dt, 12.7, 3.5

Hz), 1.198 (d, 0.6 Hz, 3H), 0.921 (d, 6.7 Hz,

3H), 0.683 (d, 0.4 Hz, 3H), l_C(25°C) 202.35,

165.15, 126.07, 70.48, 54.77, 49.94, 49.90, 45.39,

43.08, 41.79, 39.32, 38.68, 38.26, 36.32, 36.16,

35.69, 31.15, 28.52, 27.48, 26.30, 23.80, 21.20,

18.85, 17.29, 11.95 (no signals observed for C-26,

C-27).

1

1377, 1238, 1031 cm⁻¹; H and ¹³C NMR data

essentially identical to those of 16d except for

deuterium isotope effects and the absence of sig-

nals and couplings for C-16, H-16a, and H-16b.

2.15. 3i-Hydroxy-25,26,26,26,27,27,27-hepta-

ﬂuorocholest-5-en-7-one (17b), 3i-hydroxy-

cholest-5-en-7-one (17a), [26,26,26,27,27,27-

²H₆]3i-hydroxycholest-5-en-7-one (17c),

(25R)-3i,26-dihydroxycholest-5-en-7-one (17d),

and [16,16-²H₂]-(25R)-3i,26-dihydroxy-

cholest-5-en-7-one (17e)

Similar treatment of 16d (60 mg) gave 17d as

a white solid (45 mg, 90% yield): m.p., 173–

174°C; single component by TLC (R_f0.30, SS-5);

high-resolution MS, calcd. for C₂₇H₄₄O₃,

416.3290, found 416.3284; MS, 416^†(100, M⁺),

401*^†(3), 398*^†(20), 383*^†(11), 380*^†(5), 365*^†

(2), 359 (3), 358^†(3, C₂₄H₃₈O₃), 287*^†(17), 269*^†

(9), 260^†(4, C₁₇H₂₄O₂), 247^†(7, C₁₆H₂₃O₂), 245^†

(8, C₁₆H₂₁O₂), 227^†(6, C₁₆H₁₉O), 213^†(5,

C₁₅H₁₇O), 209^†(6, C₁₄H₂₅O and C₁₆H₁₇), 205^†

(16, C₁₃H₁₇O₂), 192^†(35, C₁₂H₁₆O₂), 187^†(24,

C₁₃H₁₅O); IR, 3365, 2935, 2866, 1670, 1460,

As described above for 4b, saponiﬁcation of

16b (50 mg, 0.088 mmol) in methanol (10 ml)

and sodium carbonate (20 mg) gave after

workup 17b as a white solid (41 mg, 89% yield,

98% purity): m.p., 164–165°C; single component

by TLC (R_f0.48, SS-6); high-resolution MS,

calcd. for C₂₇H₃₇O₂F₇, 526.2682, found 526.2674;

526^†(100, M⁺), 511* (3), 508*^†(25), 493*^†(21),

467^†(2, M−C₃H₇O), 315^†(2, M−C₆H₆F₇),

287*^†(5), 269*^†(4), 245^†(3, C₁₆H₂₁O₂), 239^†(6,

SC), 205^†(4, C₁₃H₁₇O₂), 174^†(7, C₁₂H₁₄O), 161^†

(13, C₁₁H₁₃O); IR, 3522, 2939, 2864, 1664, 1464,

1373, 1356, 1181, 1076, 1045, 1024 cm^{−1 1}H

;

NMR, l_H5.693 (d, 1.7 Hz), 3.678 (tq, 11.3, 4.4

Hz), 3.504 (dt, 10.6, 5.2 Hz), 3.422 (ddd, 10.7,

6.5, 4.5 Hz), 1.199 (d, 0.6 Hz), 0.925 (d, 6.6 Hz),

0.914 (d, 6.7 Hz), 0.683 (d, 0.4 Hz).

1

1311, 1219, 1159.3, 1058, 943, 719 cm⁻¹; H and

¹³C NMR, Tables 3 and 4.

Similar treatment of 16a (100 mg) gave 17a as

a white solid (85 mg, 94% yield, ]99% purity):

m.p., 171–172°C (lit. 171–172°C (Parish and

Scott, 1983)); single component by TLC (R_f0.49,

SS-6); MS, 400 (100, M⁺), 385* (5), 382* (10),

367* (14), 341 (2), 287* (12), 269* (4), 245 (7),

174 (12), 161 (26); ¹H NMR data identical

(90.004 ppm) to those of 17c except for the

occurrence of signals at l_H1.517 (nonet, 6.6

Hz), 0.866 (d, 6.6 Hz, 3H), 0.861 (d, 6.6 Hz,

3H).

Similar treatment of 16e (50 mg) gave, after

MPLC (300×10 mm i.d. column, elution with

ethyl acetate–hexane 25:75) and evaporation of

fractions 25–39, 17e as a white solid (38 mg,

91% yield, ꢀ99% purity): m.p., 172–173.5°C;

single component by TLC (R_f0.30, SS-5); high-

resolution MS, calcd. for C₂₇H₄₂D₂O₃, 418.3416,

found 418.3416; MS (ꢀ99% d₂),³418^†(100,

M⁺), 400*^†(8), 385*^†(5), 289*^†(15), 271*^†(4),

260^†(6, C₁₇H₂₄O₂), 247^†(6, C₁₆H₂₃O₂), 245^†(9,

C₁₆H₂₁O₂), 227^†(4, C₁₆H₁₉O), 213^†(4,

C₁₅H₁₃D₂O), 205^†(11, C₁₃H₁₇O₂), 192^†(33,

Similar treatment of 16c (20 mg) gave 17c as a

white solid (16 mg, 88% yield, 99% purity): m.p.,

S. Li et al. / Chemistry and Physics of Lipids 99 (1999) 33–71

49

C₁₂H₁₂D₂O₂), 187^†(14, C₁₃H₁₅O), 135 (20); IR,

3365, 2964, 2933, 2877, 2864, 1668, 1635, 1465,

yield, ]98% purity): m.p., 121–122°C (lit. 122–

123°C (Nickon and Mendelson, 1965)); single

component by TLC (R_f0.66, SS-2); MS, Table 6;

¹H and ¹³C NMR data identical (90.001 ppm

for ¹H, 90.01 ppm for ¹³C) to those of 18c

except for the occurrence of signals at l_H1.518

(nonet, 6.6 Hz), 0.869 (d, 6.6 Hz, 3H), 0.864 (d,

6.6 Hz, 3H), l_C39.45 (replacing 39.31), 27.99

(replacing 27.50), 22.81, 22.53.

1359, 1292, 1184, 1076, 1058, 1026 cm⁻^{1 1}H

;

NMR data essentially identical to those of 17d

except for deuterium isotope effects and the ab-

sence of signals and couplings for H-16a, and

H-16b.

2.16. 25,26,26,26,27,27,27-Heptaﬂuorocholest-

5-ene-3i,7h-diol diacetate (18b), cholest-5-

ene-3i,7h-diol diacetate (18a), and [26,26,26,27,

27,27-²H₆]cholest-5-ene-3i,7h-diol diacetate (18c)

and their 7i epimers 19b, 19a, and 19c

Fractions 46–72 gave 19a as a white solid (180

mg, 61% yield, ]98% purity): m.p., 106–108°C

(lit. 106°C (Nickon and Mendelson, 1965)); sin-

gle component by TLC (R_f0.68, SS-2); MS,

Table 6; ¹H and ¹³C NMR data identical (9

1

To a solution of 16b (100 mg, 0.19 mmol) in

anhydrous diethyl ether (30 ml) was added

lithium aluminum hydride (29 mg, 0.76 mmol) in

one portion. The reaction mixture was stirred

under nitrogen at room temperature overnight.

Water (two drops) was added to decompose the

excess lithium aluminum hydride, and the inor-

ganic precipitate was removed by ﬁltration. The

ﬁltrate was evaporated to a white solid that was

dissolved in pyridine (2 ml) and acetic anhydride

(0.3 ml). The reaction mixture was heated to

100°C for 1 h, cooled to room temperature, and

evaporated to a residue that was subjected to

MPLC on alumina–AgNO₃(1000×10 mm i.d.

column; elution with ethyl acetate–hexane 5:95

(1000 ml) and ethyl acetate–hexane 7:93). Evap-

oration of fractions 33–45 furnished 18b as a

white solid (20 mg, 19% yield, ]98% purity):

m.p., 98–100°C; single component by TLC (R_f

0.61, SS-2); high-resolution MS, calcd. for

C₂₉H₃₉O₂F₇(M−60), 552.2838, found 552.2838;

MS, Table 6; IR, 2957, 2895, 2854, 1726, 1442,

0.002 ppm for H, 90.01 ppm for ¹³C) to those

of 19c except for the occurrence of signals at l_H

1.515 (nonet, 6.6 Hz), 0.865 (d, 6.6 Hz, 3H),

0.860 (d, 6.6 Hz, 3H), l_C39.43 (replacing 39.30),

27.96 (replacing 27.47), 22.79, 22.53.

Similar treatment of 16c (85 mg) gave 18c

(fractions 30–40) as a colorless oil (20 mg, 21%

yield, ]98% purity): single component by TLC

(R_f0.66, SS-2); high-resolution MS, calcd. for

C₂₉H₄₀D₆O₂(M−60), 432.3874, found 432.3879;

MS, Table 6; IR, 2941, 2895, 2870, 2721, 2677,

2214, 1730, 1442, 1369, 1238, 1221, 1031, 1014,

cm⁻¹; NMR, l_H(50 mM solution) 5.585 (dt,

5.2, 1.3 Hz), 4.962 (br t, ꢀ4.6 Hz), 4.674 (m),

2.36 (m, 2H), 2.040 (s, 3H), 2.036 (s, 3H), 2.008

(br dt, 12.6, 4.0, 3.0 Hz), 1.013 (s, 3H), 0.925 (d,

6.6 Hz, 3H), 0.669 (s, 3H), l_C(25°C) 170.83,

170.45, 146.46, 120.82, 73.13, 68.27, 55.90, 49.13,

43.00, 42.22, 39.31, 38.98, 37.79, 37.23, 36.46,

36.14, 35.75, 35.70, 28.12, 27.50, 27.46, 24.03,

23.85, 21.36, 21.36, 20.63, 18.70, 18.14, 11.43 (no

signals observed for C-26, C-27).

Fractions 70–85 gave 19c as a colorless oil (65

mg, 70% yield, ]98% purity): single component

by TLC (R_f0.66, SS-2); high-resolution MS,

calcd. for C₂₉H₄₀D₆O₂(M−60), 432.3874, found

432.3864; MS, Table 6; IR, 2945, 2872, 2854,

2214, 1736, 1456, 1373, 1240, 1030, 1020, 949

cm⁻¹; NMR, l_H(50 mM solution) 5.240 (m),

5.034 (br dt, 8.6, 2.0 Hz), 4.601 (distorted dddd,

11.5, 10.6, 6.3, 4.3 Hz), 2.339 (distorted ddd,

13.2, 6.0, 2.0 Hz), 2.319 (distorted ddt, 13.2,

10.5, 2.0 Hz), 2.028 (s, 3H), 2.019 (s, 3H), 1.692

(ddd, 11.9, 10.8, 8.7 Hz), 1.085 (d, 0.4 Hz, 3H),

1

1238, 1157, 1031, 1014, 935 cm⁻¹; H and ¹³C

NMR, Tables 3 and 4.

Fractions 90–113 gave 19b as a white solid (65

mg, 60% yield, ]98% purity): m.p., 59–62°C;

single component by TLC (R_f0.63, SS-2); high-

resolution MS, calcd. for C₂₉H₃₉O₂F₇, 552.2838

(M−60), found 552.2834, MS, Table 6; IR,

2947, 2872, 1736, 1467, 1440, 1373, 1311, 1240,

1

1159, 1022, 949 cm⁻¹; H and ¹³C NMR, Tables

3 and 4.

Similar treatment of 16a (270 mg) gave 18a

(fractions 12–20) as a white solid (63 mg, 21%

Table 6

Comparison of mass spectral data for 7a and 7b hydroxycholesterol (20a and 21a), their 25,26,26,26,27,27,27-heptaﬂuoro (F₇) derivatives 20b and 21b, their

26,26,26,27,27,27-hexadeuterio (d₆) derivatives 20c and 21c, and diacetate derivatives of the six diols^a

3b,7-Diols (R=H)

3b,7-Diacetates (R=CH₃CO)

F₇-7a

20b

F₇-7b

21b

7a

20a

7b

21a

d₆-7a

20c

d₆-7b

21c

F₇-7a

18b

F₇-7b

19b

7a

18a

7b

19a

d₆-7a

18c

d₆-7b

19c

M⁺

528 (3)

510 (100)

495 (3)

(2)

(40)

(4)

402 (1)

384 (29)

369 (4)

(0.3)

(9)

(2)

408 (3)

390 (67)

375 (3)

(5)

(75)

(7)

612 (−)

552 (13)

537 (−)

510 (79)

492 (100)

477 (9)

451 (1)

387 (1)

373 (20)

333 (3)

319 (3)

313 (1)

253 (10)

239 (6)

211 (9)

143 (47)

119 (45)

105 (26)

57 (16)

(−)

(11)

(−)

(74)

(100)

(9)

(1)

(14)

(2)

486 (−)

426 (6)

(−)

(10)

(0.4)

(36)

(100)

(17)

(2)

(1)

(30)

(2)

492 (−)

432 (1)

(−)

(9)

M−ROH

M−ROHꢀMe

M−AcOHꢀC₂H₂O

M−2ROH

M−2ROHꢀMe

M−2ROHꢀC₃H₅

Ion B₂

Ion B₃

Ion C₁

Ion C₂

/

411 (0.2)

384 (61)

366 (100)

351 (11)

325 (1)

261 (1)

247 (20)

207 (2)

417 (0.5)

390 (67)

372 (100)

357 (11)

331 (1)

267 (1)

253 (39)

213 (5)

(0.3)

(55)

(100)

(10)

(1)

(21)

(4)

492 (53)

477 (8)

451 (2)

387 (1)

373 (12)

333 (1)

319 (3)

271 (1)

253 (4)

239 (31)

211 (11)

143 (38)

119 (34)

105 (38)

57 (46)

(100)

(12)

(4)

(1)

(19)

(2)

(1)

(5)

(18)

(9)

366 (100)

351 (10)

325 (2)

261 (1)

247 (18)

207 (1)

193 (3)

271 (2)

(63)

(8)

(2)

(1)

(11)

(1)

(3)

(2)

(8)

372 (100)

357 (11)

331 (2)

267 (1)

253 (24)

213 (4)

199 (6)

271 (2)

(100)

(18)

(6)

(2)

(26)

(6)

(8)

(4)

(26)

193 (2)

313 (0.5)

(4)

(0.5)

(20)

199 (10)

313 (1)

(9)

(1)

(21)

M−SCꢀROH

M−SCꢀ2ROH

SC

(0.5)

(9)

(8)

253 (7)

253 (24)

253 (12)

253 (39)

b

C₁₆H₁₉

211 (5)

143 (27)

119 (21)

105 (14)

57 (91)

(5)

211 (8)

143 (46)

119 (31)

105 (27)

57 (7)

(10)

(52)

(40)

(38)

(10)

(9)

211 (9)

143 (52)

119 (31)

105 (30)

57 (36)

(14)

(59)

(40)

(38)

(41)

211 (16)

143 (52)

119 (31)

105 (27)

57 (9)

(9)

)

C₁₁H₁₁

(45)

(46)

(32)

(19)

(46)

(22)

(30)

(100)

(41)

(36)

(25)

(28)

(38)

(20)

(7)

C₉H₁₁

C₈H₉

C₄H₉

c

^aMass spectral data are given in the form m/z (relative abundance of 7a isomer) (relative abundance of 7b isomer). Ions B₂, B₃, C₁, and C₂are deﬁned in Fig. 7.

^bExact masses of ions at m/z 113 and 119 did not correspond to C₈H₁₇or C₈H₁₁D₆.

c

The high relative abundances for m/z 57 for sterols lacking ﬂuorine or deuterium suggested partial origination from the side-chain terminus; deuterated sterols

did show m/z 63 ions of moderate abundance corresponding to C₄H₃D₆, but m/z 183 of the ﬂuorinated sterols corresponded mainly to C₁₄H₁₅(although C₄F₇H₂ions

were observed in low abundance).

S. Li et al. / Chemistry and Physics of Lipids 99 (1999) 33–71

51

0.914 (d, 6.6, 3H), 0.694 (s), l_C(25°C) 171.11,

170.35, 144.17, 122.23, 75.52, 73.20, 55.42, 55.33,

48.04, 42.78, 39.30, 39.28, 37.49, 36.49, 36.46,

36.43, 36.09, 35.65, 28.34, 27.58, 27.47, 25.11,

23.75, 21.63, 21.35, 21.00, 18.96, 18.72, 11.74.

NMR data essentially identical to those of 18d

except for deuterium isotope effects and the ab-

sence of signals and couplings for C-16, H-16a,

and H-16b.

Similar treatment of 16d (120 mg) gave (after

MPLC on alumina–AgNO₃) 18d (110 mg, 84%

yield, ꢀ98% purity) as a colorless oil: single

component by TLC (R_f0.15, SS-1); high-resolu-

tion MS, calcd. for C₃₁H₄₈O₄(M−60),

484.3553, found 484.3536; MS 484*^†(3), 442*^†

(20), 424*^†(100), 409*^†(9), 364*^†(4), 349*^†(3),

305^†(31, C₂₀H₃₃O₂), 281^†(2, C₂₁H₂₉), 279^†(2,

C₂₁H₂₇), 277^†(2, C₁₈H₂₉O₂), 269^†(1, C₁₉H₂₅O),

253^†(22, C₁₉H₂₅), 251^†(13, C₁₉H₂₃), 159^†(29,

C₁₂H₁₅), 157^†(39, C₁₂H₁₃), 143^†(63, C₁₁H₁₁); IR

(CHCl₃solution), 2944, 2870, 1734, 1469, 1373,

2.17. [16,16-²H₂]-(25R)-Cholest-5-ene-3i,7h,26-

triol triacetate (18e) and (25R)-cholest-5-ene-

3i,7h,26-triol triacetate (18d)

L

-Selectride (2.5 ml, 1 M solution) was added

dropwise to a solution of 16e (120 mg, 0.24

mmol) in anhydrous tetrahydrofuran (30 ml) at

–78°C. The reaction mixture was stirred at

−78°C for 5 h, warmed to room temperature,

and quenched with water (2.5 ml). To this solu-

tion was added 6 M sodium hydroxide (2.5 ml)

and 30% hydrogen peroxide (2.5 ml). The reac-

tion mixture was stirred at room temperature for

1 h and extracted with chloroform–methanol

(10:1). Evaporation of the combined extracts

gave a white solid that was dissolved in pyridine

(5 ml) and acetic anhydride (0.5 ml) and heated

at 100°C for 1 h under nitrogen. After cooling

to room temperature, the mixture was poured

into ice-water (50 ml) and extracted with

dichloromethane (3×30 ml). The combined ex-

tracts were washed with cold 5% sulfuric acid

(3×30 ml) and brine (30 ml) and dried over

anhydrous sodium sulfate. Evaporation gave a

yellow oil that was subjected to MPLC on alu-

mina–AgNO₃(1000×10 mm i.d. column, elu-

;

1242, 1035, 935 cm^{−1 1}H and ¹³C NMR, Ta-

bles 1 and 2.

2.18. [16,16-²H₂]-(25R)-Cholest-5-ene-3i,7i,26-

triol triacetate (19e) and (25R)-cholest-5-ene-3i,

7i,26-triol triacetate (19d)

Reduction of (16e) (120 mg) with lithium alu-

minum hydride (34 mg) as described for the

preparation of 18b, followed by acetylation and

MPLC on alumina–AgNO₃(1000×10 mm i.d.

column; elution with ethyl acetate–hexane 8:92)

and evaporation of fractions 55–74, gave 19e

(95 mg, 73% yield, ꢀ97% purity) as a colorless

oil: single component by TLC (R_f0.15, SS-1);

high-resolution MS, calcd. for C₃₁H₄₆D₂O₄

(M−60), 486.3678, found 486.3674; MS 486*^†

(30), 444*^†(100), 426*^†(38), 411*^†(9), 384*^†

(15), 366*^†(3), 351*^†(3), 307^†(6, C₂₀H₃₁D₂O₂),

271^†(10, C₁₉H₂₃D₂O), 255^†(16, C₁₉H₂₃D₂), 253^†

(8, C₁₉H₂₁D₂), 226^†(5, C₁₇H₁₈D₂), 213^†(12,

C₁₆H₁₇D₂), 212^†(12, C₁₆H₂₀), 211^†(11, C₁₆H₁₉),

159^†(63, C₁₂H₁₅), 146^†(38, C₁₁H₁₄); IR (CHCl₃

solution), 2941, 2877, 2854, 1726, 1456, 1375,

tion

with

ethyl

acetate–hexane

8:92).

Evaporation of fractions 20–40 gave 18e as a

colorless oil (115 mg, 88% yield, ꢀ98% purity

excluding non-steroidal material): single compo-

nent by TLC (R_f0.15, SS-1); high-resolution

MS, calcd. for C₃₁H₄₆D₂O₄(M−60), 486.3678,

found 486.3673; MS 486*^†(32), 444*^†(100),

426*^†(35), 411*^†(12), 384^†(17, C₂₇H₄₀D₂O),

367^†(4, C₂₇H₃₉D₂), 366*^†(3), 351*^†(4), 307^†(7,

C₂₀H₃₁D₂O₂), 283^†(4, C₂₁H₂₇D₂), 271^†(11,

C₁₉H₂₃D₂O), 255^†(17, C₁₉H₂₃D₂), 253^†(8,

C₁₉H₂₁D₂), 239^†(5, C₁₈H₁₉D₂), 226^†(6,

C₁₇H₁₈D₂), 213^†(13, C₁₆H₁₇D₂), 211^†(13,

C₁₆H₁₉), 159^†(58, C₁₂H₁₅), 146^†(35, C₁₁H₁₄); IR

(CHCl₃solution), 2939, 2895, 2872, 1726, 1458,

1

1249, 1033 cm⁻¹; H and ¹³C NMR data essen-

tially identical to those of 19d except for deu-

terium isotope effects and the absence of signals

and couplings for C-16, H-16a, and H-16b.

Similar treatment of 16d (120 mg) gave 19d

(50 mg, 38% yield, ꢀ97% purity) as a colorless

oil: single component by TLC (R_f0.15, SS-1);

high-resolution MS, calcd. for C₃₁H₄₈O₄,

1

1438, 1373, 1246, 1112, 1033 cm⁻¹; H and ¹³C

52

S. Li et al. / Chemistry and Physics of Lipids 99 (1999) 33–71

484.3553 (M−60), found 484.3542; MS, 484*^†

(8), 442*^†(53), 424*^†(100), 409*^†(11), 364*^†(7),

349*^†(3), 305^†(22, C₂₀H₃₃O₂), 269^†(2, C₁₉H₂₅O),

253^†(27, C₁₉H₂₅), 251^†(13, C₁₉H₂₃), 225^†(5,

C₁₇H₂₁), 211^†(15, C₁₆H₁₉), 199^†(12, C₁₅H₁₉), 159^†

(35, C₁₂H₁₅), 157^†(30, C₁₂H₁₃), 143^†(46, C₁₁H₁₁);

IR (CHCl₃solution), 2945, 2872, 1734, 1471,

ppm for ¹H, 90.07 ppm for ¹³C) to those of

20c except for the occurrence of signals at l_H

1.517 (nonet, 6.6 Hz), 0.867 (d, 6.6 Hz, 3H),

0.863 (d, 6.6 Hz, 3H), l_C39.49 (replacing

39.34), 27.99 (replacing 27.49), 22.79, 22.55.

Similar treatment of 18c (16 mg) gave 20c as

a white solid (12 mg, 90% yield, ]99% purity):

m.p., 185–186°C; single component by TLC (R_f

0.36, SS-6); high-resolution MS, calcd. for

C₂₇H₄₀D₆O₂, 408.3874, found 408.3874; MS,

Table 6; IR, 3367, 2933, 2864, 2214, 1458, 1375,

1336, 1259, 1055, 1012, 950 cm⁻¹; NMR, l_H

(50 mM solution) 5.601 (br dd, 5.3, 1.9 Hz),

3.852 (ddd, 5.3, 3.6, 1.7 Hz), 3.580 (tdd, 11.1,

5.1, 4.2 Hz), 2.343 (ddd, 13.1, 5.1, 2.2 Hz),

2.283 (ddt, 13.1, 11.2, 1.9 Hz), 2.003 (br ddd,

12.7, 4.2, 2.9 Hz), 1.705 (dddd, 12.3, 9.7, 6.9,

2.9 Hz), 0.994 (d, 0.6 Hz, 3H), 0.926 (d, 6.6 Hz,

3H), 0.685 (s, 3H), l_C146.21, 123.77, 71.21,

65.32, 55.79, 49.36, 42.19, 42.09, 41.97, 39.34,

39.12, 37.45, 37.34, 36.97, 36.13, 35.74, 31.28,

28.24, 27.49, 24.25, 23.67, 20.66, 18.70, 18.22,

11.60 (no signals observed for C-26, C-27).

1

1456, 1373, 1240, 1034 cm⁻¹; H and ¹³C NMR,

Tables 1 and 2.

2.19. 25,26,26,26,27,27,27-Heptaﬂuorocholest-

5-ene-3i,7h-diol (20b), cholest-5-ene-3i,7h-diol

(20a), [26,26,26,27,27,27-²H₆]cholest-5-ene-

3i,7h-diol (20c), (25R)-cholest-5-ene-3i,7h,26-

triol (20d), and [16,16-²H₂]-(25R)-cholest-5-ene-

3i,7h,26-triol (20e)

To a solution of 18b (20 mg, 0.033 mmol) in

methanol (10 ml) was added sodium hydroxide

(20 mg, 0.5 mmol), and the reaction mixture

was stirred at room temperature for a week.

TLC analysis showed the disappearance of 18b

and the formation of polar material at R_f0.32

(SS-6). The reaction mixture was evaporated to

a residue that was dissolved in dichloromethane,

washed with brine (10 ml), and dried over anhy-

drous sodium sulfate. Evaporation gave a white

solid that was further puriﬁed by chromatogra-

phy (200×10 mm i.d. gravity column; 230–400

mesh silica gel; elution with ethyl acetate–hex-

ane 3:7). Evaporation of fractions 6–14 gave

20b as a white solid (15 mg, 87% yield, ꢀ97%

purity): m.p., 212–214°C; single component by

TLC (R_f0.32, SS-6); high-resolution MS, calcd.

for C₂₇H₃₉O₂F₇, 528.2838, found 528.2830; MS,

Table 6; IR, 3483, 3393, 2935, 2858, 1467, 1311,

Similar treatment of 18d (100 mg) gave com-

pound 20d (65 mg, 85% yield, ꢀ99% purity) as

a white solid: m.p., 203–204°C; single compo-

nent by TLC (R_f0.60, SS-7); high-resolution

MS, calcd. for C₂₇H₄₆O₃, 418.3447, found

418.3434; MS, 418^†(5, M⁺), 400*^†(100), 382*^†

(8), 367^†(4, C₂₆H₃₉O), 341^†(1, C₂₄H₃₇O), 271*^†

(1), 253*^†(2), 225^†(6, C₁₇H₂₁), 211^†(2, C₁₆H₁₉),

153^†(17, C₁₂H₉), 119^†(6, C₉H₁₁); IR, 3324,

2965, 2949, 2926, 2905, 2874, 2857, 1458, 1441,

1373, 1364, 1057, 1042, 1013 cm⁻^{1 1}H NMR,

;

l_H5.607 (dd, 5.3, 1.7 Hz), 3.853 (m), 3.591 (m),

3.506 (br dd, 10.2, 5.9 Hz), 3.422 (br dd, 10.2,

6.6 Hz), 2.348 (ddd, 13.2, 5.0, 2.0 Hz), 2.286

(ddt, 13.2, 11.2, 2.0 Hz), 0.997 (d, 0.6 Hz, 3H),

0.930 (d, 6.7 Hz, 3H), 0.916 (d, 6.7 Hz, 3H),

0.687 (d, 0.4 Hz, 3H).

1244, 1221, 1161, 1055 cm⁻¹

;

¹H and ¹³C

NMR, Tables 3 and 4; ¹⁹F NMR, l_F−184.14

(dd of septet, 21.1, 19.9, 6.7 Hz), −76.75 and

−76.92 (A₃B₃portion of A₃B₃X system (Swami-

nathan et al., 1993)).

Similar treatment of 18e (100 mg) gave, after

MPLC (300×10 mm i.d. column, elution with

methanol–chloroform 3:97) and evaporation of

fractions 19–29, 20e as a white solid (69 mg,

90% yield, ]99% purity): m.p., 198–199°C; sin-

gle component by TLC (R_f0.60, SS-7); high-res-

olution MS, calcd. for C₂₇H₄₄D₂O₃, 420.3572,

Similar treatment of 18a (50 mg) gave 20a as

a white solid (38 mg, 92% yield, ꢀ99% purity):

m.p., 186–188°C (lit. 183–186°C (Kumar et al.,

1987), 183–184°C (Kudo et al., 1989), 183–

184°C (Lythgoe and Trippett, 1959)); single

component by TLC (R_f0.36, SS-6); MS, Table

6; ¹H and ¹³C NMR data identical (90.011

S. Li et al. / Chemistry and Physics of Lipids 99 (1999) 33–71

53

found 420.3583; MS, 420^†(2, M⁺), 402*^†(77),

384*^†(100), 369*^†(16), 366*^†(7), 343^†(4, M−

C₃H₉O₂), 273*^†(4), 265^†(27, C₁₈H₂₉D₂O), 255*^†

(19), 227^†(5, C₁₇H₁₉D₂), 211^†(14, C₁₆H₁₅D₂),

158^†(31, C₁₂H₁₀D₂), 137 (60); IR, 3314, 2964,

2951, 2928, 2901, 2876, 2854, 1458, 1438, 1376,

Table 6; IR, 3331, 2935, 2903, 2864, 2850, 2214,

1464, 1375, 1354, 1302, 1136, 1055, 1014, 947

cm⁻¹; NMR, l_H(50 mM solution) 5.289 (td,

2.0, 0.5 Hz), 3.843 (dt, 7.9, 2.3 Hz), 3.544 (tdd,

11.3, 5.0, 4.1 Hz), 2.331 (ddd, 13.2, 4.9, 2.2 Hz),

2.253 (ddt, 13.3, 11.4, 2.1 Hz), 2.021 (br ddd,

12.7, 4.0, 2.9 Hz), 1.804 (dddd, 12.3, 9.7, 7.2,

2.6 Hz), 1.050 (d, 0.5 Hz, 3H), 0.922 (d, 6.6 Hz,

3H), 0.694 (d, 0.5 Hz, 3H), l_C143.43, 125.40,

73.31, 71.36, 55.91, 55.40, 48.20, 42.88, 41.68,

40.83, 39.51, 39.33, 36.89, 36.39, 36.17, 35.70,

31.51, 28.52, 27.49, 26.35, 23.79, 21.04, 19.13,

18.74, 11.79 (no signals observed for C-26, C-

27).

1363, 1246, 1051, 1039, 1010 cm⁻^{1 1}H NMR

;

data essentially identical to those of 20d except

for deuterium isotope effects and the absence of

signals and couplings for H-16a and H-16b.

2.20. 25,26,26,26,27,27,27-Heptaﬂuorocholest-

5-ene-3i,7i-diol (21b), cholest-5-ene-3i,7i-diol

(21a), [26,26,26,27,27,27-²H₆]cholest-5-ene-

3i,7i-diol (21c), (25R)-cholest-5-ene-3i,7i,26-

triol (21d), and [16,16-²H₂]-(25R)-cholest-5-ene-

3i,7i,26-triol (21e)

Similar treatment of 19d (50 mg) gave com-

pound 21d (30 mg, 78% yield, ꢀ99% purity) as

a white solid: m.p., 198–199°C; single compo-

nent by TLC (R_f0.60, SS-7); high-resolution

MS, calcd. for C₂₇H₄₄O₂(M−18), 400.3341,

found 400.3342; MS, 418^†(7, M⁺), 400*^†(100),

382*^†(51), 367*^†(13), 341^†(5, C₂₄H₃₇O), 271*^†

(3), 263^†(10, C₁₈H₃₁O), 253*^†(10), 225^†(3,

C₁₇H₂₁), 211^†(10, C₁₆H₁₉), 157^†(28, C₁₂H₁₃),

143^†(48, C₁₁H₁₁), 119^†(35, C₉H₁₁); IR, 3306,

2964, 2926, 2886, 1456, 1437, 1417, 1398, 1375,

Saponiﬁcation of 19b (60 mg) as described for

18b gave 21b as a white solid (48 mg, 93%

yield, ]98% purity): m.p., 176–177°C; single

component by TLC (R_f0.36, SS-6); high-resolu-

tion MS, calcd. for C₂₇H₃₉O₂F₇, 528.2838, found

528.2822; MS, Table 6; IR, 3536, 3362, 2969,

2942, 1468, 1314, 1223, 1157, 1057, 937 cm⁻¹

;

¹H and ¹³C NMR, Tables 3 and 4; ¹⁹F NMR,

l_F−184.18 (dd of septet, 21.2, 19.9, 6.7 Hz),

−76.73 and −76.94 (A₃B₃portion of A₃B₃X

system (Swaminathan et al., 1993)).

1356, 1064, 1041, 1020, 976 cm⁻¹; H NMR, l_H

1

5.294 (t, 2.0 Hz), 3.846 (ddt, 8.0, 6.0, 2.3 Hz),

3.552 (m), 3.504 (br dt, ꢀ10.8, ꢀ6 Hz), 3.427

(br dt, ꢀ10.4, ꢀ6 Hz), 2.337 (ddd, 13.3, 5.0,

2.2 Hz), 2.257 (ddt, 13.3, 11.1, 2.1 Hz), 1.052 (s,

3H), 0.926 (d, 6.6 Hz, 3H), 0.917 (d, 6.7 Hz,

3H), 0.696 (d, 0.4 Hz, 3H).

Similar treatment of 19a (100 mg) gave 21a as

a white solid (72 mg, 87% yield, ]99% purity):

m.p., 177–178°C (lit. 174–178°C (Kumar et al.,

1987), 180.5–181.0°C (Kudo et al., 1989), 172–

175°C (Smith and Price, 1967), 176–182°C

(Ruzicka et al., 1944)); single component by

Similar treatment of 19e (80 mg) gave com-

pound 21e (58 mg, 94% yield, 99% purity) as a

white solid: m.p., 195–196°C, single component

by TLC (R_f0.60, SS-7); high-resolution MS,

calcd. for C₂₇H₄₄D₂O₃, 420.3572, found

420.3549; MS, 420^†(4, M⁺), 402*^†(100), 384*^†

(31), 369*^†(14), 366*^†(4), 343^†(6, M−

C₃H₉O₂), 273*^†(6), 265^†(9, C₁₈H₂₉D₂O), 255^†

(11), 227^†(3, C₁₇H₁₉D₂), 211^†(8, C₁₆H₁₅D₂),

158^†(14, C₁₂H₁₀D₂), 137 (21); IR, 3313, 3225,

2964, 2928, 2885, 2864, 1460, 1373, 1168, 1138,

1

TLC (R_f0.38, SS-6); MS, Table 6; H and ¹³C

NMR data identical (90.007 ppm for ¹H, 9

0.02 ppm for ¹³C) to those of 21c except for the

occurrence of signals at l_H1.519 (nonet, 6.6

Hz), 0.869 (d, 6.6 Hz, 3H), 0.864 (d, 6.6 Hz,

3H), l_C39.46 (replacing 39.33), 27.99 (replacing

27.49), 22.80, 22.54.

Similar treatment of 19c (60 mg) gave 21c as

a white solid (45 mg, 90% yield, \99% purity):

m.p., 176–178°C; single component by TLC (R_f

0.38, SS-6); high-resolution MS, calcd. for

C₂₇H₄₀D₆O₂, 408.3874, found 408.3897; MS,

1

1047, 1014, 980 cm⁻¹; H NMR data essentially

identical to those of 21d except for deuterium

isotope effects and the absence of signals and

couplings for H-16a and H-16b.

54

S. Li et al. / Chemistry and Physics of Lipids 99 (1999) 33–71

†

2.21. (25R)-26-Hydroxycholest-4-en-3-one (22d),

[16,16-²H₂]-(25R)-26-hydroxycholest-4-en-3-one

(22e), 3:1 mixture of (25R)-26-hydroxycholest-

4-ene-3,7-dione (23d) and (25R)-26-hydroxy-

cholest-5-ene-3,7-dione, (25R)-7h,26-dihydroxy-

cholest-4-en-3-one (24d), [16,16-²H₂]-(25R)-

7h,26-dihydroxycholest-4-en-3-one (24e), (25R)-

7i,26-dihydroxycholest-4-en-3-one (25d), and

[16,16-²H₂]-(25R)-7i,26-dihydroxy-

(5), 277 (26, M−C₈H₁₁O), 271*^†(12), 263^†(5,

C₁₈H₃₁O), 253*^†(3), 229^†(54, C₁₆H₂₁O and

C₁₇H₂₅), 211^†(8, C₁₆H₁₉), 149^†(12, C₁₁H₁₇), 147^†

(25, C₁₁H₁₅), 124^†(100, C₈H₁₂O); IR, 3431, 2931,

2870, 2856, 1660, 1612, 1465, 1458, 1448, 1433,

1377, 1361, 1230, 1043 cm⁻¹; H NMR, l_H5.724

(m), ꢀ3.503 (br dd, ꢀ10.5, 6 Hz), ꢀ3.426 (br

dd, ꢀ10.5, 6 Hz), 1.181 (d, 0.6 Hz, 3H), 0.915 (d,

6.7 Hz, 6H), 0.709 (d, 0.6 Hz, 3H).

1

cholest-4-en-3-one (25e)

Similar treatment of 1e (23 mg) gave 22e (19

mg, 83% yield, ]99% purity) as a white solid:

m.p., 134–135°C; single component by TLC (R_f

0.85, SS-7); high-resolution MS, calcd. for

C₂₇H₄₂D₂O₂, 402.3467, found 402.3486; MS, 402^†

A solution of 20e (30 mg) in butyl acetate (6

ml) was added to a TES buffer solution (6 ml, 50

mM, pH 7.5) containing cholesterol oxidase from

Streptomyces sp. (50 units) and catalase (40 000

units). The two-phase mixture (Lee and Biell-

mann, 1988) was stirred in a vial at ca. 22°C with

a magnetic stirrer and occasionally shaken vigor-

ously by hand. After 30 h, TLC analysis indicated

complete disappearance of 20e and formation of a

less polar product (R_f0.70, SS-7). The reaction

mixture was extracted with 10% methanol–chlo-

roform (2×20 ml), and the organic extracts were

washed with water and brine and dried over anhy-

drous sodium sulfate. Evaporation gave a white

solid that was subjected to MPLC (300×10 mm

i.d. column; elution with methanol–chloroform

3:97). Evaporation of fractions 36–48 gave 24e as

a white solid (25 mg, 86% yield, 99% purity):

m.p., 173–174°C; single component by TLC (R_f

0.70, SS-7); high-resolution MS, calcd. for

C₂₇H₄₂D₂O₃, 418.3416, found 418.3415; MS 418^†

(11, M⁺), 400*^†(22), 390*^†(7), 385*^†(6), 362^†

(22, M−C₃H₄O), 271*^†(8), 265^†(4, C₁₈H₂₉D₂O),

227^†(6, C₁₆H₁₅D₂O), 215^†(4, C₁₅H₁₅D₂O), 187^†

(6, C₁₃H₁₁D₂O), 174^†(12, C₁₂H₁₀D₂O); IR, 3338,

2962, 2930, 2885, 2862, 1664, 1618, 1458, 1375,

(70, M⁺), 387*^†(8), 384*^†(8), 369*^†(4), 360^†(20,

†

M−C₂H₂O), 345^†(6, M−C₃H₅O), 317

(5,

C₂₂H₃₃D₂O), 301 (3), 279^†(27, M−C₈H₁₁O),

273*^†(11), 265^†(5, C₁₈H₂₉D₂O), 255*^†(3), 229^†

(16, C₁₆H₂₁O), 211^†(10, C₁₆H₁₉), 149^†(25, C₁₁H₁₇

and C₁₀H₁₃O), 147^†(10, C₁₁H₁₅), 124^†(100,

C₈H₁₂O); IR, 3433, 2931, 2862, 1664, 1616, 1465,

1458, 1437, 1377, 1338, 1236, 1039 cm⁻¹; H and

1

¹³C NMR data essentially identical to those of

22d except for deuterium isotope effects and the

absence of signals and couplings for C-16, H-16a,

and H-16b.

Similar treatment of 17d (40 mg; 48 h reaction

time) gave mainly recovered starting material, but

a 3:1 mixture of 23d and (25R)-26-hydroxy-

cholest-5-ene-3,7-dione was isolated as a pale yel-

low solid (4 mg, 9% yield): two components by

TLC (R_f0.80 and 0.83, SS-7); high-resolution MS,

calcd. for C₂₇H₄₄O₃, 414.3134, found 414.3138;

MS, 414^†(100, M⁺), 399*^†(5), 396*^†(11), 386^†

(3, M−CO), 381*^†(3), 291^†(7, C₁₉H₃₁O₂), 285*^†

(34), 245^†(18, C₁₆H₂₁O₂), 203^†(20, C₁₆H₁₁), 190^†

(47, C₁₂H₁₄O₂), 124^†(16, C₈H₁₂O₂); ¹H NMR

(23d), l_H5.709 (dt, 2.5, 0.8 Hz), 3.503 (dd, 10.5,

5.9 Hz), 3.461 (ddd, 15.1, 2.5, 1.0, H-6b), 3.426

(dd, 10.5, 6.5 Hz), 3.051 (dd, 15.0, 0.7 Hz, H-6a),

1.419 (d, 0.7 Hz, 3H), 0.925 (d, 6.7 Hz, 3H), 0.915

1043, 1031, 1007 cm⁻¹; H NMR data essentially

identical to those of 24d except for deuterium

isotope effects and the absence of signals and

couplings for H-16a and H-16b.

Similar treatment of 1d (25 mg) gave 22d (22

mg, 83% yield, 98% purity (contains 1% 1d)) as a

white solid: m.p., 132–134°C; single component

by TLC (R_f0.85, SS-7); high-resolution MS,

calcd. for C₂₇H₄₄O₂, 400.3341, found 400.3326;

MS, 400^†(80, M⁺), 385*^†(8), 382*^†(6), 367*^†(3),

358^†(22, M−C₂H₂O), 343^†(7, M−C₃H₅O), 315^†

1

(d, 6.7 Hz, 3H), 0.709 (d, 0.5 Hz, 3H); H NMR

(D⁵isomer of 23d), l_H5.676 (dt, 2.4, 0.7 Hz),

3.505 (dd, 10.5, 5.9 Hz), 3.426 (dd, 10.5, 6.5 Hz),

3.424 (ddd, 17.5, 2.4, 0.6, H-4b), 3.076 (ddd, 17.5,

2.2, 0.6 Hz, H-4a), 2.573 (ddd, 15.9, 13.2, 5.8 Hz),

ꢀ2.426 (m, H-2a), 2.41 (m, H-15a), 2.309 (dd,

11.9, 10.8 Hz, H-8b), 2.142 (ddd, 13.8, 5.8, 4.0

S. Li et al. / Chemistry and Physics of Lipids 99 (1999) 33–71

55

Hz, H-1b), 1.362 (d, 0.4 Hz, 3H), 0.934 (d, 6.7 Hz,

3H), 0.917 (d, 6.7 Hz, 3H), 0.717 (d, 0.5 Hz, 3H);

partial ¹³C NMR (D⁵isomer of 23d; data from

HSQC spectrum), 54.75 (C-17), 47.4 (C-4), 45.3

(C-8), 37.0 (C-2), 35.0 (C-1), 26.2 (C-15), 18.7

(C-21), 17.2 (C-19), 12.0 (C-18). Similar reaction

of 17e also gave mainly recovered starting mate-

rial, with a small amount of a 3:1 mixture of the

deuterated analogs of 23d and its D⁵isomer.

Similar treatment of 20d (60 mg) gave 24d (54

mg, 90% yield, 99% purity) as a white solid: m.p.,

175–176°C (lit. 168–169°C (Kim et al., 1997));

single component by TLC (R_f0.70, SS-7); high-

resolution MS, calcd. for C₂₇H₄₄O₃, 416.3290,

found 416.3271; MS, 416^†(13, M⁺), 401*^†(1),

398*^†(24), 383*^†(3), 380*^†(1), 360^†(18,

C₂₄H₄₀O₂), 269*^†(7), 263^†(3, C₁₈H₃₁O), 245^†(2,

C₁₆H₂₁O₂) 145^†(8, C₁₁H₁₃), 124^†(100, C₈H₁₂O),

109^†(11, C₇H₉O); IR, 3350, 2932, 2886, 2860,

1665, 1464, 1375, 1276, 1188, 1049, 1005, 949

289*^†(2), 271*^†(21), 265^†(10, C₁₈H₂₉D₂O), 257^†

(3, C₁₈H₂₅O), 238^†(5, C₁₆H₂₆D₂O), 228^†(8,

C₁₄H₂₇O₂), 213 (5), 152^†(17, C₉H₁₂O₂), 124^†(100,

C₈H₁₂O); IR, 3445, 2935, 2854, 1647, 1637, 1608,

1456, 1419, 1373, 1338, 1278, 1232, 1195, 1060,

1

1043, 945 cm⁻¹; H NMR data essentially identi-

cal to those of 25d except for deuterium isotope

effects and the absence of signals and couplings

for H-16a and H-16b.

2.22. 5-Bromo-6i,19-epoxy-5h-cholestan-3i-ol

acetate (27a), 5-bromo-6i,19-epoxy-25,26,

26,26,27,27,27-heptaﬂuoro-5h-cholestan-3i-ol

acetate (27b), and [26,26,26,27,27,27-²H₆]5-

bromo-6i,19-epoxy-5h-cholestan-3i-ol acetate

(27c)

To a solution of acetate 2a (2.15 g, 5.0 mmol)

in dioxane (30 ml) were added water (2.5 ml) and

perchloric acid (70%, ﬁve drops), followed by

cooling to 0°C and addition of N-bromo-

acetamide (0.70 g, 5.1 mmol) in one portion. The

reaction mixture was stirred at 0°C in the dark for

1 h, warmed to room temperature, and stirred for

another 12 h in the dark. Crude 5-bromo-5a-

cholestane-3b,6b-diol 3-acetate (26a) was precipi-

tated by addition of water (300 ml). The white

precipitate (2.2 g) was collected by ﬁltration,

washed with water, dried in vacuo, and used

without further puriﬁcation. Similar reaction of

2a (1.0 g) gave crude 26a showing by TLC (SS-4)

1

cm⁻¹; H NMR, l_H5.805 (m), 3.969 (m), 3.501

(dd, 10.5, 5.9 Hz), 3.426 (dd, 10.5, 6.5 Hz), 1.193

(d, 0.7 Hz, 3H), 0.922 (d, 6.6 Hz, 3H), 0.915 (d,

6.7 Hz, 3H), 0.717 (d, 0.5 Hz, 3H).

Similar treatment of 21d (28 mg) gave 25d (26

mg, 94% yield, ]99% purity) as a white solid:

m.p., 158–159°C; single component by TLC (R_f

0.75, SS-7); high-resolution MS, calcd. for

C₂₇H₄₄O₃, 416.3290, found 416.3297; MS, 416^†(7,

M⁺), 398*^†(100), 388*^†(1), 383*^†(7), 380*^†(9),

365*^†(4), 360^†(3, C₂₄H₄₀O₂), 287*^†(1), 269*^†

(34), 263^†(19, C₁₈H₃₁O), 255^†(6, C₁₈H₂₃O), 245^†

(6, C₁₆H₂₁O₂), 227^†(15, C₁₆H₁₉O and C₁₇H₂₃), 213

(11), 175^†(34, C₁₂H₁₅O), 161^†(36, C₁₁H₁₃O and

C₁₂H₁₇), 136^†(54, C₉H₁₂O), 95^†(70, C₇H₁₁O and

C₆H₇O); IR, 3418, 2938, 2866, 1655, 1465, 1373,

1

and H NMR a 6:3:1 mixture of 26a (R_f0.65),

6b-bromo-5a-cholestane-3b,5-diol 3-acetate (R_f

0.77), and unreacted 2a (R_f0.95). Trituration with

ethyl acetate (6 ml) followed by recrystallization

from hexane–acetone (95:5, ꢀ20 ml) furnished

an analytical sample of 26a (0.20 g, 99% purity,

containing 1% 6b-bromo-5a-cholestane-3b,5-diol

3-acetate): m.p.; 167–168°C (lit. 168–169°C

(Kalvoda et al., 1963)); MS, 524*^§(0.8, M⁺),

506*^§(0.5), 491*^§(0.6), 444* (31), 429* (8), 426*

(13), 411* (2), 385* (68), 384* (100), 369* (36),

366* (45), 356 (14), 351* (10), 331* (26), 271*

(17), 253* (15), 247 (12), 244 (12), 229 (23), 211

1

1232, 1039, 947 cm⁻¹; H NMR, l_H5.761 (m),

3.501 (dd, 10.5, 6.5 Hz), 3.460 (ddd, 11.2, 9.2, 5.2

Hz), 3.428 (dd, 10.5, 5.9 Hz), 1.210 (d, 0.6 Hz,

3H), 0.929 (d, 6.7 Hz, 3H), 0.916 (d, 6.7 Hz, 3H),

0.734 (d, 0.6 Hz, 3H).

Similar treatment of 21e (29 mg) gave 25e (25

mg, 85% yield, ꢀ98% purity) as a white solid:

m.p., 156–157°C; single component by TLC (R_f

0.75, SS-7); high-resolution MS, calcd. for

C₂₇H₄₂D₂O₃, 418.3416, found 418.3426; MS, 418^†

(52, M⁺), 400*^†(70), 390*^†(5), 385*^†(8), 382*^†

(5), 362^†(14, M−C₃H₄O), 356^†(9, C₂H₆O₂),

1

(19), 107 (40); H and ¹³C NMR, Tables 1 and 2.

To a suspension of calcium carbonate (4.0 g)

and lead tetraacetate (15.0 g, dried overnight in a

desiccator over NaOH) in cyclohexane (300 ml) at

56

S. Li et al. / Chemistry and Physics of Lipids 99 (1999) 33–71

27.99) and the absence of signals corresponding to

H-26, H-27, C-26, and C-27.

80°C were added crude 26a (2.0 g) and iodine (2.0

g) in one portion. The reaction mixture was

stirred vigorously under reﬂux with irradiation

from a 500 watt halogen utility lamp. After about

1.5 h, when the deep red solution had become

colorless, the reaction mixture was cooled to

room temperature and the solid material was

removed by ﬁltration. The ﬁltrate was washed

with saturated sodium thiosulfate and brine and

evaporated to a pale yellow oil, which was sub-

jected to MPLC (300×25 mm i.d. column; elu-

tion with ethyl acetate–hexane 4:96). Evaporation

of fractions 18–32 gave a clean 3:1 mixture of 27a

and epoxide 4a (1.4 g, 53% crude yield). Further

puriﬁcation of a portion (ꢀ10 mg) of this mix-

ture on MPLC (1000×10 mm i.d. column; elu-

tion with ethyl acetate–hexane 4:96), followed by

evaporation of fractions 19–23 gave an analytical

sample of 27a (8 mg, 95% purity): m.p., 143–

144°C (lit. 143–144°C (Kalvoda et al., 1963));

single component by TLC (R_f0.32, SS-1); MS,

522*^§(0.4, M−H₂O), 462*^§(0.3), 443* (16), 383*

(100), 365* (7), 353 (18), 289 (8), 247 (1), 253* (1),

2.23. 25,26,26,26,27,27,27-Heptaﬂuorocholest-

5-ene-3i,19-diol 3-acetate (28b), cholest-

5-ene-3i,19-diol 3-acetate (28a), and [26,26,26,

27,27,27-²H₆]cholest-5-ene-3i,19-diol 3-acetate

(28c)

Zinc dust (1.5 g) was added to a solution of

crude 27b (250 mg) in absolute ethanol (50 ml),

and the reaction mixture was heated at reﬂux for

6 h. TLC analysis (SS-3) showed the disappear-

ance of 27b and formation of polar material at R_f

0.36. Unreacted zinc dust was removed by ﬁltra-

tion, and the ﬁltrate was evaporated to an oil,

which was subjected to MPLC (1000×10 mm i.d.

column; elution with ethyl acetate–hexane 6:94).

Evaporation of fractions 34–40 gave 28b as a

white solid (120 mg, 55% yield, ꢀ98% purity):

m.p., 134–135°C; single component by TLC (R_f

0.36, SS-3); high-resolution MS, calcd. for

C₂₇H₃₇OF₇(M−60), 510.2734, found 510.2733;

MS, 570^†(0.1, M⁺), 552*^†(0.3), 510*^†(19), 492*^†

(10), 480* (67), 479*^†(100), 478*^†(25), 375^†(15,

C₁₈H₂₆F₇), 373^†(22, C₁₈H₂₄F₇), 333^†(8, M−

C₁₄H₂₁O₃), 319^†(8, C₁₄H₁₈F₇), 291^†(5, C₁₂H₁₄F₇),

265^†(6, C₁₀H₁₂F₇), 253*^†(4), 241^†(8, C₁₈H₂₅),

239^†(8, SC), 227^†(5, C₁₇H₂₃), 213^†(7, C₁₆H₂₁),

211^†(7, C₁₆H₁₉), 145^†(51, C₁₁H₁₃); IR, 3601,

2943, 2893, 1732, 1709, 1474, 1441, 1381, 1317,

1

243 (13), 229 (18), 211 (11), 143 (17), 121 (23); H

and ¹³C NMR, Tables 1 and 2.

Similar treatment of 2b (425 mg) gave crude

27b (300 mg, 60% yield), which was used without

further puriﬁcation.

Similar treatment of 2c (140 mg) gave crude 27c

(90 mg, 53% yield). Further puriﬁcation of a

portion (20 mg) of this mixture on MPLC

(1000×10 mm i.d. column; elution with ethyl

acetate–hexane 4:96), followed by evaporation of

fractions 28–31 gave an analytical sample of 27c

(14 mg, ꢀ90% purity (containing 5% 4c)): m.p.,

142–143°C; single component by TLC (R_f0.32,

1

1238, 1159, 1034 cm⁻¹; H and ¹³C NMR, Tables

1 and 2.

Similar treatment of 27a (1.0 g) gave 28a as a

white solid (0.63 g, 74% yield, 99% purity): m.p.,

120–121°C (lit. 119–120°C (Kalvoda et al.,

1963)); single component by TLC (R_f0.38, SS-3);

MS, 426 (0.5, M−18), 384* (19), 366* (14), 354*

(68), 353* (100), 352* (19), 253* (5), 249 (6), 247

(14), 241 (12), 227 (8), 213 (7), 211 (7), 207 (3),

145 (45); ¹H and ¹³C NMR data identical (9

SS-6);

high-resolution

MS,

calcd.

for

C₂₉H₄₃D₆O⁷₄⁹Br (M−18), 528.3085, found

528.3069; MS, 528*^†§(0.3), 468*^†§(0.3), 449*^†

(13), 389*^†(100), 371*^†(7), 359^†(18, C₂₆H₃₅D₆),

289^†(7, C₁₈H₂₅O₃), 253^†(3, C₁₈H₂₅D₆and

C₁₉H₂₅), 243^†(12, C₁₇H₂₃O), 229^†(20, C₁₆H₂₁O),

211^†(9, C₁₆H₁₉), 143^†(14, C₁₁H₁₁); IR, 2940,

1

0.002 ppm for H, 90.04 ppm for ¹³C) to those

of 28c except for the occurrence of signals at l_H

1.514 (nonet, 6.6 Hz), 0.865 (d, 6.6 Hz, 3H), 0.860

(d, 6.6 Hz, 3H), l_C39.47 (replacing 39.35), 27.98

(replacing 27.50), 22.80, 22.54. Early fractions

from the puriﬁcation of 28a contained the a-epox-

2866, 2212, 1736, 1445, 1371, 1263, 1036 cm⁻¹

;

¹H and ¹³C NMR data identical (90.002 ppm for

¹H, 90.04 ppm for ¹³C) to those of 27a except

for l_C39.32 (replacing 39.47) and 27.48 (replacing

S. Li et al. / Chemistry and Physics of Lipids 99 (1999) 33–71

57

1

ide acetate 4a, which showed H and ¹³C NMR

data essentially identical to those in Tables 1 and

2.

189°C; single component by TLC (R_f0.30, SS-6);

high-resolution MS, calcd. for C₂₇H₃₉O₂F₇,

528.2838, found 528.2829; MS, 528^†(0.4, M⁺),

510*^†(25), 498*^†(15), 497*^†(13), 496*^†(5),

492*^†(4), 480*^†(100), 479*^†(41), 478*^†(4), 465^†

(3, M−C₂H₇O₂), 437^†(3, M−C₄H₁₁O₂), 373^†

(22, M−C₉H₁₅O₂), 319^†(4, M−C₁₃H₂₁O₂), 265^†

(3, C₁₀H₁₂F₇), 253*^†(1), 241^†(9, C₁₈H₂₅), 239^†

(11, SC), 211^†(5, C₁₆H₁₉), 145^†(40, C₁₁H₁₃), 131

(27, C₁₀H₁₁); IR, 3405, 3293, 2944, 2907, 2872,

Similar treatment of 27c (75 mg) gave 28c as a

white solid (39 mg, 52% yield, \99% purity):

m.p., 118–119°C; single component by TLC (R_f

0.38, SS-3); high-resolution MS, calcd. for

C₂₇H₃₈D₆O (M−60), 390.3769, found 390.3781;

MS, 432*^†(0.4), 390*^†(11), 372*^†(10), 360*

(55), 359*^†(100), 358*^†(18), 255^†(2, C₁₈H₂₇D₆),

253^†(10, C₁₈H₂₅D₆and C₁₉H₂₅), 241^†(7,

C₁₈H₂₅), 227^†(4, C₁₇H₂₃), 213^†(5, C₁₅H₂₁D₆),

211^†(6, C₁₆H₁₉), 145^†(33, C₁₁H₁₃); IR, 3491,

2940, 2868, 2214, 1768, 1703, 1467, 1448, 1381,

1265, 1038 cm⁻¹; NMR, l_H(50 mM solution)

5.772 (dt, 5.2, 2.1 Hz), 4.644 (tt, 11.5, 4.8 Hz),

3.828 (dd, 11.4, 0.6 Hz), 3.623 (br d, 11.4 Hz),

2.417 (ddd, 13.1, 4.9, 2.3 Hz), 2.267 (ddddd,

12.9, 11.5, 3.3, 2.7, 1.9 Hz), 2.029 (s, 3H), 1.957

(ddd, 14.0, 4.1, 3.2 Hz), 1.618 (br qd, 13.2, 4.0

Hz), 1.494 (dddd, 14.2, 12.9, 11.5, 4.1 Hz), 1.258

(dddd, ꢀ13, 11.5, 9.5, 3.0 Hz), 0.911 (d, 6.5 Hz,

3H), 0.728 (d, 0.4 Hz, 3H), l_C(25°C) 170.49,

134.50, 128.30, 73.39, 62.70, 57.55, 56.08, 50.29,

42.50, 41.58, 39.98, 39.35, 38.20, 36.16, 35.76,

33.35, 33.07, 31.22, 28.21, 28.09, 27.50, 24.06,

23.80, 21.75, 21.37, 18.68, 12.19 (no signals ob-

served for C-26, C-27).

2851, 1474, 1445, 1314, 1223, 1159, 1038 cm⁻¹

;

¹H and ¹³C NMR, Tables 1 and 2; ¹⁹F NMR, l_F

−184.16 (dd of septet, 21.3, 19.9, 6.6 Hz), −

76.73 and −76.95 (A₃B₃portion of A₃B₃X sys-

tem (Swaminathan et al., 1993)).

Similar treatment of 28a (200 mg) gave 29a as

a white solid (158 mg, 87% yield, 99% purity):

m.p., 160–162°C (lit. 162–164°C (Kalvoda et al.,

1963)); single component by TLC (R_f0.34, SS-6);

MS, 402 (0.8, M⁺), 384* (30), 372* (23), 371*

(18), 370* (8), 366* (7), 354* (100), 353* (42),

352* (6), 339 (5), 311 (2), 253* (2), 247 (22), 241

(18), 211 (4), 145 (30), 131 (19); ¹H and ¹³C

NMR data identical (90.006 ppm for ¹H, 9

0.04 ppm for ¹³C) to those of 29c except for the

occurrence of signals at l_H1.514 (nonet, 6.6 Hz),

0.865 (d, 6.6 Hz, 3H), 0.860 (d, 6.6 Hz, 3H), l_C

39.47 (replacing 39.35), 27.99 (replacing 27.49),

22.80, 22.54.

2.24. 25,26,26,26,27,27,27-Heptaﬂuorocholest-

5-ene-3i,19-diol (29b), cholest-5-ene-3i,19-diol

(29a), and [26,26,26,27,27,27-²H₆]cholest-5-

ene-3i,19-diol (29c)

Similar treatment of 28c (30 mg) gave 29c as a

white solid (23 mg, 85% yield, 99% purity): m.p.,

159–161°C; single component by TLC (R_f0.26,

SS-5);

high-resolution

MS,

calcd.

for

C₂₇H₄₀D₆O₂, 408.3874, found 408.3897; MS, 408^†

(0.8, M⁺), 390*^†(21), 378*^†(44), 377*^†(19),

376*^†(20), 372*^†(8), 360*^†(100), 359*^†(46),

358*^†(18), 345^†(5, M−C₂H₇O₂), 317^†(1, M−

C₄H₁₁O₂), 259*^†(6), 253^†(22, M−C₉H₁₅O₂and

C₁₉H₂₅), 241^†(22, C₁₈H₂₅), 211^†(7, C₁₆H₁₉), 199^†

(23, M−C₁₃H₂₁O₂), 145^†(52, C₁₁H₁₃), 131^†(37,

C₁₀H₁₁); IR, 3402, 2934, 2866, 2213, 1467, 1442,

1377, 1333, 1051 cm⁻¹; NMR, l_H(50 mM solu-

tion) 5.744 (dt, 5.3, 2.0 Hz), 3.817 (dd, 11.4, 0.7

Hz), 3.607 (d, 11.4 Hz), 3.570 (tt, 11.2, 4.7 Hz),

2.379 (ddd, 13.0, 4.7, 2.4 Hz), 2.189 (ddddd,

13.1, 11.3, 3.3, 2.6, 2.0 Hz), 2.046 (br dt, ꢀ12.9,

3.6 Hz), 2.029 (dtd, 17.9, 5.3, 2.6 Hz), 1.937

To a solution of 28b (100 mg, 0.175 mmol) in

methanol (20 ml) was added sodium carbonate

(100 mg, 1.0 mmol), the reaction mixture was

stirred at room temperature overnight. TLC

analysis (SS-6) showed the disappearance of 28b

and the formation of polar material at R_f0.30.

The solvent was evaporated to a residue that was

dissolved in dichloromethane, washed with brine

(10 ml), and dried over anhydrous sodium sul-

fate. Evaporation gave a white solid that was

subjected to MPLC (200×10 mm i.d. column,

elution with ethyl acetate–hexane 2:8). Evapora-

tion of fractions 31–47 gave 29b as a white solid

(78 mg, 84% yield, 98% purity): m.p., 188–

58

S. Li et al. / Chemistry and Physics of Lipids 99 (1999) 33–71

(ddd, 13.8, 4.0, 3.0 Hz), 1.625 (br qd, 13.2, 3.9 Hz),

1.403 (dddd, 14.2, 12.9, 11.3, 4.1 Hz), 1.260 (dddd,

13.3, 11.5, 9.5, 3.1 Hz), 0.911 (d, 6.6 Hz, 3H), 0.730

(d, 0.5 Hz, 3H), l_C(25°C) 135.52, 127.37, 71.34,

62.71, 57.64, 56.10, 50.37, 42.51, 42.30, 41.50,

40.02, 39.35, 36.16, 35.75, 33.39, 33.32, 31.95,

31.23, 28.21, 27.49, 24.06, 23.80, 21.79, 18.68, 12.20

(no signals observed for C-26, C-27).

2.26. Stereochemical NMR assignments of the

C-19 protons of 27a–c, 28a–c, and 29a–c

Stereochemical assignments of the dias-

tereotopic C-19 protons of 6b,19-ether 27a and

19-hydroxycholesteryl acetate (28a) were deter-

mined from 1D NOE difference experiments on

non-degassed 50–100 mM samples (1 s weak irra-

diation at a single frequency; 90° pulse; 2.7 s

acquisition time). For 27a, irradiation of H-8b (l_H

1.61) led to enhancement of the upﬁeld H-19 signal

(l_H3.745), and irradiation of H-4b (l_H2.27)

resulted in enhanced signals at l_H3.922 (dd, 8.3,

1.3 Hz) and 4.06 (d, 4.5 Hz, 3-OH)⁵. These obser-

vations demonstrate that the downﬁeld C-19 pro-

ton of 27a is positioned over ring A (pro-R). For

28a, irradiation of the downﬁeld C-19 proton (l_H

3.83) led to enhancements of H-2b (strong), H-4b

(strong), and H-8b (weak), whereas irradiation of

the upﬁeld C-19 proton (d_H3.62) gave enhance-

ments for H-1b (weak), H-4b (weak), and H-11b

(very strong). Also, the downﬁeld H-19 signal was

enhanced by irradiation of H-2b (strong), H-4b

(strong), and H-11b (weak), whereas the upﬁeld

H-19 signal was enhanced by irradiation of H-1b

(weak), H-4b (weak), and H-11b (strong). These

observations are compatible only with a predomi-

nant conformation having oxygen over ring B, with

the downﬁeld C-19 proton positioned over ring A

(pro-R). Weaker NOEs correspond to a minor

conformer with oxygen positioned between C-1 and

C-11. The major and minor conformer are pre-

dicted by molecular mechanics (MMX and MM3

force ﬁelds) to be similar in energy but ca. 1.5

kcal/mol lower in energy than the rotamer with

oxygen over ring A. Based on these results, stereo-

chemical assignments for the C-19 protons of

27b–c, 28b–c, and 29a–c were made by chemical

shift comparisons.

2.25. [26,26,26-²H₃]-(25RS)-cholest-5-ene-3i,

25-diol (1g and 1h)

A solution of CD₃I (160 mg, 1 mmol) in diethyl

ether (2 ml) was added dropwise, followed by

stirring at room temperature for 2 h. After removal

of excess magnesium turnings, the reaction mixture

was poured into water, extracted with diethyl ether

(20 ml), and washed with brine (10 ml). The organic

extracts were dried over anhydrous sodium sulfate

and evaporated to a residue that was puriﬁed by

MPLC (elution with ethyl acetate–hexane 15:85) to

give a 1:1 mixture of C-25 epimers 1g and 1h as a

white solid (85 mg, 95% yield, ꢀ96% purity): m.p.:

178–180°C, single component by TLC (R_f0.20,

ethyl acetate–hexane 1:3); high-resolution MS,

calcd. for C₂₇H₄₃D₃O₂, 405.3686, found 405.3681;

MS (98% d₃, 2% d₂), 405^†(34, M⁺), 390* (8), 387*^†

(96), 372*^†(46), 369* (27), 354* (28), 302 (13), 300^†

(18, C₂₁H₃₂O), 299^†(14, C₂₁H₃₁O), 276^†(37,

C₂₀H₃₀D₃), 273*^†(20), 271^†(70, M−SCꢀ2), 255*^†

(26, C₁₉H₂₇), 253^†(16, M−H₂OꢀSCꢀ2), 231^†(16,

C₁₆H₂₃O), 213^†(38, C₂₁H₁₆), 145^†(55, C₁₁H₁₃), 114^†

(9, SC–H₂O), 62^†(100, C₃H₄D₃O); IR, 3250, 2935,

2862, 2220, 1469, 1375, 1230, 1195, 1136, 1055, 956,

920 cm⁻¹; NMR data matched chemical shifts

reported²(Wilson et al., 1994) for the undeuterated

sterol 1f to 90.002 ppm for ¹H (except H-3a) and

90.02 ppm for ¹³C except for deuterium isotope

effects at the end of the side chain⁴.

⁴The ¹³C NMR spectrum showed two terminal methyl

singlets of roughly equal but diminished intensity, and the ¹H

NMR spectrum showed two singlets of equal intensity at l_H

1.208 and 1.210 (1.214 and 1.215 for the undeuterated sterol).

The HSQC spectrum showed correlations between the

downﬁeld proton and the downﬁeld carbon and between the

upﬁeld proton and upﬁeld carbon, but assignments of the

signals to individual C-25 epimers could not be established.

⁵Approximately one-third of the ¹H NMR oxysterol spectra

showed a ca. 4–5 Hz coupling of H-3a to the OH proton. This

coupling, which we have sometimes observed (without com-

ment) among a variety of other 3b-hydroxysterols in CDCl₃

solution, is routinely present in dimethylsulfoxide-d₆spectra

and is attributable to slow exchange of the hydroxyl proton.

Similar couplings were also frequently observed for hydroxyl

at other positions.

S. Li et al. / Chemistry and Physics of Lipids 99 (1999) 33–71

59

2.27. Stereochemical NMR assignments of the side

chain methylene protons

HMBC, and NOE difference spectra readily fur-

nished complete ¹H and ¹³C NMR assignments for

the major component of the 3:1 mixture of 3,7-

dione isomers except for the ene–dione system.

Assignments for 25-norketone 30 were based on

previously described conformational analyses of

sterol side chains (Swaminathan et al., 1993; Wilson

et al., 1994). The downﬁeld C-22 proton (l_H1.342;

dddd, 13.3, 11.4, 5.7, 2.9 Hz) was assigned as pro-R

based on its small coupling (2.9 Hz) to H-20 as well

as on chemical shift comparisons with other sterols

described herein. Matching coupling constants of

the downﬁeld C-23 proton (l_H1.652; ddddd, 13.5,

11.2, 8.8, 6.4, 4.7 Hz) with those of H-22R indicated

a 11.3 Hz mutual coupling. Thus, the two protons

are anti in the predominant extended conforma-

tion, and the downﬁeld C-23 proton is pro-S. These

coupling assignments were conﬁrmed in the dou-

ble-quantum ﬁltered COSY spectrum, which also

showed active couplings from H-23R to the upﬁeld

and downﬁeld C-24 protons of 8.6 and 6.2 Hz,

respectively. Based on molecular modeling results

showing the extended side chain conformation to

be favored over the other C23–C24 rotamers by 0.7

kcal/mol, this establishes the upﬁeld C-24 proton

as pro-R. The similarity of the downﬁeld shifts for

H-23R and H-23S (0.30, 0.31 ppm) and for H-24R

and H-24S (1.26, 1.28 ppm) relative to correspond-

ing protons of cholesteryl acetate (Wilson et al.,

1996) suggests that, in the overall distribution of

side chain conformers, the C-25 carbonyl and

methyl groups are each symmetrically disposed

about the plane bisecting the side chain (in its

extended conformation). The effect of the hep-

taﬂuoro substitution in F₇-cholesterol also pro-

duced almost identical downﬁeld shifts for

diastereotopic methylene side-chain protons, which

have been assigned previously (Swaminathan et al.,

1993; Wilson et al., 1994). Chemical shift compari-

sons with the NMR data for F₇-cholesterol pro-

vided the corresponding assignments for the F₇

sterols described herein. Stereochemical assign-

ments for the side-chain protons of 26-hydroxys-

terols will be described elsewhere.

1

Comparison of observed H and ¹³C NMR chem-

ical shifts with those predicted for the D⁴- and

D⁵-3,7-dione isomers (based on substituent incre-

ments for 7-keto and 26-hydroxy groups) suggested

the major component to be the D⁴isomer: predicted

for C-2 of D⁴, D⁵, l_C33.4, 37.1; observed for major,

minor components, l_C33.6, 37.0; predicted for C-8,

l_C49.1, 45.3; observed, l_C49.3, 45.3. An HMBC

correlation of the non-conjugated carbonyl (l_C

206.5) to H-8 (l_H2.53) provided a more deﬁnitive

assignment of the major component as the D⁴

isomer. Irradiation of the major and minor H-19

signals in NOE difference experiments led to stere-

ochemical assignments for the allylic methylene

protons (H-6 or H-4). The partial NMR data for

the minor component (notably ¹H and ¹³C data for

protonated carbons in rings A and B) were in

reasonable agreement with predicted values for

1

the D⁵isomer and with H NMR data reported

for cholest-5-ene-3,7-dione (Schabdach et al.,

1998).

2.29. Deuterium isotope effects on NMR chemical

shifts

Relative to their undeuterated analogs, the

16,16-dideuterio sterols showed upﬁeld shifts for

C-15 (0.21 ppm), C-17 (0.15 ppm), C-20 (0.05

ppm), H-14a (0.003 ppm), H-15a (0.015 ppm),

H-15b (0.015 ppm), H-17a (0.014 ppm), and H-18

(0.002 ppm). Similar upﬁeld shifts were observed

for 11: C-15 (0.13 ppm), C-17 (0.08 ppm), C-20

(0.05 ppm), H-15a (0.008 ppm), H-15b (0.008

ppm), H-17a (0.004 ppm), and H-18 (0.002 ppm).

Shieldings of other nuclei were essentially un-

1

changed (50.002 ppm for H and 50.02 ppm

for ¹³C). The 26,26,26,27,27,27-hexadeuterios-

terols

showed

similar

upﬁeld

shifts

for C-24 (0.13 ppm), C-25 (0.49 ppm), and

H-25, (0.038 ppm). The d₃-25-hydroxysterol

preparation (1f, 1g) showed upﬁeld shifts for C-24

(0.06 ppm), C-25 (0.11 ppm), C-27 (0.06 ppm;

diminished intensity), and H-27 (0.005 ppm)

2.28. NMR structure determination of

Z⁴-3,7-dione 23d and its Z⁵isomer

Inspection of ¹H, ¹³C, COSYDEC, HSQC,

60

S. Li et al. / Chemistry and Physics of Lipids 99 (1999) 33–71

3. Discussion

therein). Thus, potassium permanganate oxida-

tion of 2a–c in the presence of copper sulfate

(Syamala et al., 1992) followed by MPLC puriﬁ-

cation on alumina–AgNO₃afforded the b-epox-

ide acetates 6a–c in 55–85% yield, and

saponiﬁcation gave the free sterols 7a–c in 86–

94% yield. This route represents a useful alter-

native to the classical synthesis of b-epoxide 7a

via the bromohydrin 26a (Fried and Sabo,

1957), especially for small-scale syntheses. Triols

8a–c were prepared in 69–78% yield by oxida-

tion of the D⁵free sterols 1a–c with hydrogen

peroxide in formic acid, followed by saponiﬁca-

tion of any formate esters formed during the

reaction (Fieser and Rajugopalan, 1949).

3.1. Chemical syntheses

We have prepared 43 oxygenated sterols with

a diversity of functionality. Functional groups in

rings A and B include D⁵-3b,4b-dihydroxy (3),

D⁵-3b,7a-dihydroxy (20), D⁵-3b,7b-dihydroxy

(21), D⁵-3b,19-dihydroxy (29), 3b,5a,6b-trihy-

droxy (8), 3b-hydroxy-5a,6a-epoxy (5), 3b-hy-

droxy-5b,6b-epoxy (7), D⁵-3b-hydroxy-7-keto

(17), and D⁴-3-keto derivatives of 7,26-oxy-

genated sterols (22–25). Substituents on the par-

ent C₈H₁₇side chain (a) include 25,26,

26,26,27,27,27-heptaﬂuoro (b), 26,26,26,27,27,27-

hexadeuterio (c), (25R)-26-hydroxy (d), [16,16-

²H₂]-(25R)-26-hydroxy (e), and 25-hydroxy with

deuteration at one of the terminal methyls (g,

h). The deuterated and ﬂuorinated sterols were

prepared for use as internal standards to facili-

tate the quantitation of oxysterols by GC-MS.

The ﬂuorinated sterols are also of interest be-

cause of their anticipated blockage of oxidative

metabolism at the side-chain terminus.

As shown in Fig. 2, [16,16-²H₂]-(25R)-cholest-

5-ene-3b,26-diol diacetate (2e) was prepared in

53% yield from (25R)-bis(3b,26-tert-butyldi-

methylsilyloxy)cholest-5-en-16-one (9), which has

been synthesized from diosgenin in 37% yield

(Kim et al., 1989). The synthesis of 2e from

diosgenin requires three chromatographic sepa-

rations but can readily be carried out on a gram

scale. The free sterol 1e was obtained in 89%

yield by saponiﬁcation of acetate 2e. Deuterium

was introduced speciﬁcally at C-16 by two re-

ductions with LiAlD₄, ﬁrst on 16-ketosterol 9,

and then on mesylate 12. Use of LiAlD₄of ]

99% isotopic purity led to high levels of deu-

terium incorporation at C-16. Mass spectral

analysis showed ꢀ99% d₂for 2e, and ¹H and

¹³C NMR spectra indicated the virtual absence

of 16-protio material. ²H NMR demonstrated

the absence of deuterium at other positions. The

LiAlD₄reductions produced minor byproducts

14 (characterized as diacetate 15) and 11, which

were separated from the main products and

identiﬁed by NMR. The present synthesis of

(25R)-26-hydroxycholesterol, with a high incor-

poration of the isotopic label at a single carbon

atom, has clear advantages over previous syn-

theses of the deuterated sterol (Shoda et al.,

1993b; Ni et al., 1994; D’Ambra et al., 1997;

and Ref. therein). Moreover, the label at C-16 is

at a relatively inert position with respect to

known sterol metabolism and to mass spectral

fragmentation.

The parent oxygenated sterols (a) described

herein and their 25,26,26,26,27,27,27-heptaﬂuoro

(b)

and

26,26,26,27,27,27-hexadeuterio

(c)

derivatives were synthesized from cholesterol

(1a), F₇-cholesterol (1b), d₆-cholesterol (1c), or

their acetate derivatives 2a–c by modiﬁcations

of previously described approaches. As shown in

Fig. 1, the 4b-hydroxysterols 3a–c were pre-

pared in 34–41% yield by oxidation of the D⁵-

steryl acetates 2a–c with selenium dioxide

(Rosenheim and Starling, 1937) followed by sa-

poniﬁcation of the acetate. Oxidation of the D⁵-

steryl acetates 2a–c with m-chloroperbenzoic

acid gave predominantly the a-epoxide acetates

4a–c, which were puriﬁed (Kudo et al., 1989)

by alumina–AgNO₃chromatography prior to

saponiﬁcation to the free sterols 5a–c. The

epoxidation reactions proceeded in 56–77%

yield and the saponiﬁcations in 88–91% yield.

The usual preference for a-epoxidation (owing

to steric effects of the C-10 methyl group) can

be reversed by blocking the a-face of the D⁵

bond with a transition metal ion under speciﬁc

reaction conditions (Parish and Li, 1996 and ref.

S. Li et al. / Chemistry and Physics of Lipids 99 (1999) 33–71

61

Fig. 1. Synthesis of cholest-5-ene-3b,4b-diol (3a), 5,6a-epoxy-5a-cholestan-3b-ol (5a), 5,6b-epoxy-5b-cholestan-3b-ol (7a), 5a-

cholestane-3b,5,6b-triol (8a), and their 25,26,26,26,27,27,27-heptaﬂuoro and 26,26,26,27,27,27-hexadeuterio derivatives.

62

S. Li et al. / Chemistry and Physics of Lipids 99 (1999) 33–71

Fig. 2. Synthesis of deuterium-labeled (25R)-26-hydroxycholesterol (1e) and its diacetate derivative (2e).

The D⁵-7-ketosterols 17a–c were prepared as

21d) and [16,16-²H₂]-(25R)-26-hydroxy derivatives

(17e, 20e, and 21e) from (25R)-26-hydroxycholes-

terol diacetates 2d and 2e. The 7a-hydroxy sterols

20d and 20e were obtained by reduction of the

shown in Fig. 3 from the D⁵-steryl acetates 2a–c

in 55–61% overall yield by oxidation with pyri-

dinium chlorochromate in reﬂuxing benzene, fol-

lowed by saponiﬁcation (Parish et al., 1986;

Parish and Wei, 1987). Lithium aluminum hy-

dride reduction (Cheng et al., 1977) of the D⁵-7-

keto acetates 16a–c gave a 3:1 mixture of the 7b-

and 7a-hydroxysterols, which could be separated

as the diacetates (18a–c, 19a–c) by MPLC on

alumina–AgNO₃as reported previously by Kudo

et al. (1989). Saponiﬁcation of the diacetates gave

the 7a-hydroxysterols 20a–c (16–19% yield from

16a–c) and the 7b-hydroxysterols 21a–c (53–63%

yield from 16a–c). The 7-keto, 7a-hydroxy, and

7b-hydroxy sterols were also prepared similarly as

their (25R)-26-hydroxy derivatives (17d, 20d, and

7-ketosterols with L-Selectride, which gives almost

exclusively the 7a isomer (Kumar et al., 1987).

As shown in Fig. 4, (25R)-cholest-5-ene-3b,26-

diol (1d), its 7a- and 7b-hydroxy derivatives (20d,

21d), and their 16,16-dideuterio derivatives (1e,

20e, 21e) were regioselectively oxidized/isomerized

to the D⁴-3-ketosteroids (22d, 22e, 24d, 24e, 25d,

25e) on a 20–60 mg scale with cholesterol oxi-

dase. This enzyme can be regarded a useful

reagent for selectively oxidizing the 3b-hydroxyl

of oxygenated sterols. Reactions on a 10–100 mg

scale require attention to the limited solubility of

sterols in aqueous medium and the limited stabil-

Article Doi

Sterol synthesis. Preparation and characterization of fluorinated and deuterated analogs of oxygenated derivatives of cholesterol

DOI: 10.1016/S0009-3084(99)00005-5

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Authors:

Article abstract of DOI:10.1016/S0009-3084(99)00005-5

Full text of DOI:10.1016/S0009-3084(99)00005-5

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