Substrate analog studies of the ω-regiospecificity of Mycobacterium tuberculosis cholesterol metabolizing cytochrome P450 enzymes CYP124A1, CYP125A1 and CYP142A1

Accepted Manuscript

Substrate analog studies of the ω-regiospecificity of Mycobacterium tubercu‐

losis cholesterol metabolizing cytochrome P450 enzymes CYP124A1,

CYP125A1 and CYP142A1

Jonathan B. Johnston, Arti A. Singh, Anaelle A. Clary, Chiung-Kuan Chen,

Patricia Y. Hayes, Sharon Chow, James J. De Voss, Paul R. Ortiz de Montellano

PII:

S0968-0896(12)00362-8

DOI:

http://dx.doi.org/10.1016/j.bmc.2012.05.003

BMC 10030

Reference:

To appear in:

Bioorganic & Medicinal Chemistry

Received Date:

Revised Date:

Accepted Date:

31 March 2012

26 April 2012

2 May 2012

Please cite this article as: Johnston, J.B., Singh, A.A., Clary, A.A., Chen, C-K., Hayes, P.Y., Chow, S., De Voss,

J.J., Ortiz de Montellano, P.R., Substrate analog studies of the ω-regiospecificity of Mycobacterium tuberculosis

cholesterol metabolizing cytochrome P450 enzymes CYP124A1, CYP125A1 and CYP142A1, Bioorganic &

Medicinal Chemistry (2012), doi: http://dx.doi.org/10.1016/j.bmc.2012.05.003

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Substrate analog studies of the -

regiospecificity of Mycobacterium

tuberculosis cholesterol metabolizing

cytochrome P450 enzymes CYP124A1,

CYP125A1 and CYP142A1

Jonathan B. Johnston, Arti A. Singh, Anaelle A. Clary, Chiung-Kuan Chen, Patricia Y. Hayes, Sharon Chow,

James J. De Voss, Paul R. Ortiz de Montellano

Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com

Substrate analog studies of the -regiospecificity of Mycobacterium tuberculosis

cholesterol metabolizing cytochrome P450 enzymes CYP124A1, CYP125A1 and

CYP142A1

Jonathan B. Johnston^a, Arti A. Singh^b, Anaelle A. Clary^c, Chiung-Kuan Chen^d, Patricia Y. Hayes^b, Sharon

Chow^b, James J. De Voss^b, ^Paul R. Ortiz de Montellano^a

^aDepartment of Pharmaceutical Chemistry, University of California San Francisco, San Francisco 94158-2517 U.S.A.

^bSchool of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, 4072, Australia

^cBioengineering Department, Polytech Nice-Sophia, Antipolis Cedex 06903, France

^dDepartment of Molecular Biology & Biochemistry, University of California Irvine, Irvine 92697, U.S.A.

ARTICLE INFO

ABSTRACT

Article history:

Received

Revised

Accepted

Available online

We report the synthesis and evaluation of a series of cholesterol side-chain analogs as

mechanistic probes of three important Mycobacterium tuberculosis cytochrome P450 enzymes

that selectively oxidize the -position of the methyl-branched cholesterol side-chain. To probe

the structural requirements for the thermodynamically disfavored -regiospecificity we

compared the binding of these substrate analogs to each P450, determined the turnover rates, and

characterized the enzymatic products. The results are discussed in the context of the structure-

activity relationships of the enzymes and how their active sites enforce -oxidation.

Keywords:

Mycobacterium tuberculosis

cytochrome P450

substrate analog

steroid

-oxidation

———

 Corresponding author. Tel.: 415-476-2903; fax: 415-502-4728; e-mail: ortiz@cgl.ucsf.edu

Abbreviations: CYP – cytochrome P450; BSTFA – N,O-bis(trimethylsilyl)trifluoroacetamide; DIBAL-H – diisobutylaluminum hydride; MCD –

methyl -cyclodextrin; MCE – mammalian cell entry; MDR-TB – multi-drug resistant tuberculosis; Mtb – Mycobacterium tuberculosis; P450 –

cytochrome P450; PCC – pyridinium chlorochromate; TB – tuberculosis; TBS – tert-butyldimethylsilyl; XDR-TB – extremely drug resistant

tuberculosis.

1. Introduction

Mycobacterium tuberculosis (Mtb) is the pathogenic bacterium

that causes pulmonary tubercular infection (TB). Deaths due to

active TB infection, or arising from complications from HIV/AIDS

in conjunction with TB, continue to claim millions of lives per

year.¹In addition, 10% of individuals harboring latent infections

(approximately 2 billion people) experience activation to infectious

TB each year.¹The emergence of multi-drug resistant (MDR-TB)

and extremely drug resistant (XDR-TB) Mtb strains has rendered

the frontline antibiotics less effective and complicates efforts to halt

the spread of TB infection, particularly in developing countries.

New drugs and new drug targeting strategies are urgently needed to

slow the spread of tuberculosis infection.

fatty acids and isoprenoids but CYP125A1 does not.^11,20

CYP142A1 is more distantly related to CYP125A1 than

CYP124A1. Thermodynamic considerations suggest that the

terminal portion of the cholesterol (1) side-chain would be most

easily oxidized at the tertiary 25-position to give 25-

hydroxycholesterol,

a known potent modulator of cellular

processes. However, this product is not detectably formed. Instead,

only products resulting from the thermodynamically disfavored ω-

oxidation of the primary methyl group are observed.¹⁷

We report here the synthesis and evaluation of a series of

cholesterol (1) analogs bearing side-chain and ring modifications as

substrates of CYP125A1, CYP142A1 and CYP124A1. The results

provide a better understanding of the strategies used by each

enzyme to oxidize its substrates with high regiospecificity. They

also provide much-needed insight for the design of substrate-

analogs as mechanism-based inhibitors of a critical energy-

harvesting pathway in M. tuberculosis.

Like nearly all bacteria studied to date, M. tuberculosis is not

able to synthesize steroids de novo.^2-4Nevertheless, Mtb has

evolved a remarkable ability to thrive and persist within the human

macrophage, and cholesterol (1) is essential for its entry into the

macrophage.⁵Bacterial survival in the macrophage relies on the

uptake of lipids, including cholesterol (1), from the human host

cells during infection and their subsequent breakdown to derive

energy and carbon equivalents for building biomolecules such as

lipids.^2,6,7Cholesterol (1) is an important source of carbon and

energy during both chronic and latent phases of infection.

2. Results

2.1. Synthesis of cholesterol side-chain analogs 2-10

The steps necessary to utilize cholesterol (1) can be divided into

roughly four phases: (a) uptake of cholesterol (1) into the cell, (b)

its oxidation to cholest-4-en-3-one (1k), (c) side-chain degradation,

and (d) steroid ring degradation.²Cholesterol (1) (cholest-5-en-3-

ol) is transported into the cell by the MCE (mammalian cell entry)

family of proteins,⁸and is then converted to cholest-4-en-3-one

(1k) by a cholesterol oxidase.⁹An essential P450 activity is

required to activate the side-chain for entry into -oxidation, and

finally, the steroid ring system is degraded. The structures of

cholesterol (1) and cholest-4-en-3-one (1k) are shown in Figure 1.

The structures of cholesterol (1), the chemically synthesized

cholesterol side-chain analogs (2–10) and the other, commercially

available steroids (11–13) used in this study are shown in Figure 2.

Our initial strategy for the synthesis of the desired analogs 2–10

began with the TBS ether of commercially available 5-pregnen-3-

ol-20-one (14).^21-23Despite reports of similar steroids undergoing

Wittig condensation,^24-28all reaction conditions tested were

unsuccessful. An approach involving vinyl Grignard addition to C-

20 followed by oxidative rearrangement of the resulting tertiary

alcohol was thus adopted (Supplementary data).^23,29It was

envisioned that aldehyde (20E)-15 would provide easy access to

our desired sterols 2-10 via Wittig condensation and subsequent

Figure 1: Cholesterol (1) and cholest-4-en-3-one (1k).

Three Mtb P450 isoforms, CYP125A1, CYP142A1 and

CYP124A1, catalyze steroid side-chain oxidation.^10-15CYP125A1

is the primary enzyme responsible for this in vivo activity though

CYP142A1 provides functional redundancy in some Mtb

strains.^2,11,16In vivo, CYP142A1 but not CYP124A1 fully restores

the growth phenotype for the CYP125A1 strain.⁹The cholest-4-

en-3-one (1k) side-chain is oxidized at the ω-position in three

consecutive steps, first to the alcohol, then to the aldehyde and

finally to the carboxylic acid.^2,10,11,17The newly formed carboxylic

acid undergoes transesterification with coenzyme-A (CoA) in an

ATP-dependent step that primes the acyl-CoA chain for -

oxidation. Three cycles of -oxidation produces one acetyl-CoA

and two propionyl-CoA equivalents.

The critical function of these P450 enzymes for Mtb infection

and survival makes them potential drug targets.^2,18These particular

bacterial P450 isoforms show low sequence identity with human

cholesterol (1) side-chain oxidizing P450 enzymes. CYP125A1 is

distributed widely in various actinomycete genomes and parallels in

gene organization and functional assignment led to our initial

understanding of the M. tuberculosis CYP125A1 isoform.¹⁹All

three P450 enzymes catalyze the ω-oxidation of the steroid side-

chain.¹⁷Although CYP124A1 and CYP125A1 exhibit high

sequence identity and are closely related, CYP124A1 also oxidizes

side-chain functionalization.

respectively, in excellent yields. Alkene 21 was also subjected to

catalytic hydrogenation prior to TBS-deprotection, which afforded

a diastereomeric mixture of (25S)- and (25R)-5 contaminated with a

small amount of saturated 22 (~20%). While this strategy (Schemes

1 & 2) provided the desired cholesterol analogs 4–6, the production

and challenging chromatographic separation of the diastereomeric

mixtures obtained was unattractive. A more efficient, alternative

route for the synthesis of sterols 2, 3 and 7–10 was thus developed

(Scheme 3).

Figure 2: The sterols used in this study.

Horner-Wittig addition of diethyl 2-(methoxy(methyl)amino)-2-

oxoethylphosphonate³⁰(16) to a mixture of (20E)-15 and (20Z)-15

proceeded in near quantitative yield (Scheme 1), with exclusive

formation of the 23(24)E alkene³⁰affording only two

chromatographically separable, isomeric ,,γ,δ-unsaturated

Scheme 1. Synthesis of ketone (20R)-19 from TBS-protected 5-pregnen-3-ol-

20-one (14) via a mixture of isomeric aldehydes (20E)-15 and (20Z)-15 (~3:1).

Reagents and conditions: a) Phosphonate ester 16, NaH, THF, 0 °C, 99%, ~3:1

(20E,23E)-17: (20Z,23E)-17. b) H₂, PtO₂, 1,4-dioxane/AcOH (50:1), 88%, ~2:1

(20S)-18:(20R)-18. c) H₂, PtO₂, 1,4-dioxane/AcOH (50:1), 81%, ~95:5 (20R)-

18:(20S)-18. d) Methylmagnesium bromide, THF, −78 °C, 83%.

Weinreb amides, (20E,23E)-17

and (20Z,23E)-17. Catalytic

hydrogenation of various 20(22)E alkenes^26-28,31and conjugated

20(22),23(24) dienes^32-34generally, but not always,³⁵occurs

stereoselectively to provide the natural 20R stereochemistry of

cholesterol (1). In this case, hydrogenation of both (20E,23E)-17

and (20Z,23E)-17 separately over PtO₂

28,36

proceeded in good yield

(Scheme 1), with no observed reduction of the 5(6) double bond.

Unfortunately, reduction of the major isomer (20E,23E)-17

afforded an inseparable mixture of (20S)-18 and (20R)-18, with the

undesired 20S stereochemistry predominating (20S:R ~2:1).

Hydrogenation of (20Z,23E)-17 afforded the desired (20R)-18

almost exclusively (20S:R ~95:5), with the stereochemistry at C-20

Again starting with 14, (17Z)-23 was available in two steps

(Supplementary data)²²and a BF₃.Et₂O-catalyzed ene condensation

with paraformaldehyde cleanly provided the homoallylic alcohol

(20S)-24 in 74% yield (Scheme 3).^37,38Catalytic hydrogenation of

(20S)-24 (PtO₂, EtOAc) afforded alcohol 25 stereoselectively, with

the angular C-18 methyl group directing hydrogen addition from

the less hindered -face of the molecule.³⁹Oxidation (PCC,

CH₂Cl₂) of 25 provided aldehyde 26 as a key intermediate, which

permitted simple construction of 2, 3, and 7–10.

1

assigned based on comparison of the H NMR spectra of 18 with

data reported for similar compounds.²⁸

Reaction of methylmagnesium bromide with (20R)-18 afforded

the methyl ketone (20R)-19 (Scheme 1), and subsequent

condensation with either methyl or ethyltriphenylphosphonium

halides provided alkenes 20 (89% yield) and 21 (48%, 25E:Z ~1:1),

respectively (Scheme 2). Acid-catalyzed hydrolysis of the silyl

moiety of 20 and 21 afforded the desired sterols 4 and 5,

Scheme 2: Synthesis of sterols 4–6 from ketone (20R)-19. Reagents and

conditions: a) Methyltriphenylphosphonium iodide, n-BuLi, THF, −78 °C, 89%.

b) Dowex 50W-X8(H) resin, CH₂Cl₂/MeOH (1:1), quantitative. c)

Ethyltriphenylphosphonium bromide, n-BuLi, THF, −78 °C, 48%. d) Dowex

50W-X8(H) resin, CH₂Cl₂/MeOH (1:1), 96%. e) H₂, PtO₂, 1,4-dioxane/AcOH

(50:1). f) HCl, CH₂Cl₂/MeOH (1:1), quantitative over 2 steps, ~4:1 6:22.

Wittig condensation of 26 with n-butyltriphenylphosphonium

bromide gave 27 (87% yield; Scheme 3) as predominantly one

isomer, tentatively assigned as (22E)-27 based on comparison of

¹³C NMR spectral data with that reported for similar compounds,

and in accord with literature precedent.^40-42Catalytic hydrogenation

of 27 (PtO₂, 1,4-dioxane/AcOH) then provided 28 and acid-

catalyzed removal of the TBS group yielded 27-norcholesterol (2)

in near-quantitative yield.

Scheme 4. Synthesis of sterols 3 and 10 from aldehyde 26. Reagents and

conditions: a) Stabilized ylide 29, CH₂Cl₂, reflux, 93%. b) H₂, PtO₂, 1,4-

dioxane/AcOH (50:1), 97%. c) LiAlH₄, Et₂O, 98%. d) CBr₄, PPh₃, imidazole,

CH₃CN/Et₂O (1:3), 95%. e) KSAc, acetone, 95%. f) Dowex 50W-X8(H) resin,

CH₂Cl₂/MeOH (1:1), 92%. g) i) NaOMe, THF/MeOH (1:1), 0 °C. ii) MeI. iii)

HCl (2:1), 68% over 3 steps.

Wittig

addition

of

stabilized

ylide

ethyl

2-

Reaction of bromide 33 with potassium thioacetate provided 34

(95% yield; Scheme 4) and sodium methoxide-mediated thioester

hydrolysis, followed by alkylation of the thiolate generated in situ

with methyl iodide, and TBS-deprotection afforded the desired 25-

thia-27-norcholesterol (3) in 68% yield (over the three steps).

Alternatively, reaction of 33 with potassium cyanide afforded

nitrile 35 in 98% yield (Scheme 5), which then underwent

diisobutylaluminum hydride (DIBAL-H)-mediated reduction to

aldehyde 36 (76% yield). Subsequent Wittig condensation with

methyltriphenylphosphonium bromide afforded the terminal alkene

37, with cleavage of the TBS moiety providing sterol 7 (93%

yield).

(triphenylphosphoranylidene)acetate⁴³(29) to aldehyde 26 gave the

,-unsaturated ester (22E)-30 in 93% yield (Scheme 4), with the

coupling constants for H-22 (δ_H6.81 ppm, dd) and H-23 (5.71 ppm,

d) revealing the 22E stereochemistry (J = 15.6 Hz) of the major

isomer (>85%). Catalytic hydrogenation of 30, LiAlH₄-mediated

reduction of ester 31, and subsequent bromination of alcohol 32

afforded the primary bromide 33 in excellent yield. Bromide 33

was an important intermediate in the synthesis of sterols 3 and 7–9

(Schemes 4 & 5) and also yielded 10 via acid-catalyzed cleavage of

Addition of methylmagnesium bromide to aldehyde 36 afforded

alcohol 38 in 90% yield (Scheme 5). Direct conversion of 38 to

either 39 or 40 (PPh₃and CCl₄or CBr₄) was unsuccessful. Thus, 38

was converted to the corresponding mesylate, which was reacted

without purification with LiCl or LiBr to afford chloride 39 and

bromide 40, respectively, in quantitative yields. Finally, acid-

catalyzed removal of the TBS ether afforded the desired

halogenated norcholesterols 8 and 9 in high yields.

the TBS ether (Scheme 4).

Scheme 3: Synthesis of 27-norcholesterol (2) from compound 14, via aldehyde

26. Reagents and conditions: a) (CHO)_n, BF₃.Et₂O, CH₂Cl₂, 74%. b) H₂, PtO₂,

EtOAc, 86%. c) PCC, NaOAc, CH₂Cl₂, 87%. d) n-Butyltriphenylphosphonium

bromide, n-BuLi, THF, −78 °C, 87%. e) H₂, PtO₂, 1,4-dioxane/AcOH (50:1),

96%. f) Dowex 50W-X8(H) resin, CH₂Cl₂/MeOH (1:1), 97%.

Scheme 5. Synthesis of sterols 7–9 from bromide 33, via aldehyde 36. Reagents

and conditions: a) KCN, DMF, 80 °C, 98%. b) DIBAL-H, Et₂O, −78 °C, 76%.

c) Methyltriphenylphosphonium bromide, n-BuLi, THF, −78 °C, quantitative. d)

Dowex 50W-X8(H) resin, CH₂Cl₂/MeOH (1:1), 93%. e) Methylmagnesium

bromide, THF, −78 °C, 90%. f) MsCl, Et₃N, CH₂Cl₂. g) LiCl, THF, reflux,

quantitative over 2 steps. h) LiBr, THF, reflux, quantitative over 2 steps. i)

Dowex 50W-X8(H) resin, CH₂Cl₂/MeOH (1:1), 89%. j) Dowex 50W-X8(H)

resin, CH₂Cl₂/MeOH (1:1), 88%.

the coordinated water molecule by a heteroatom of the ligand, often

the lone pair of electrons on a nitrogen atom of an imidazole or

triazole ring.

Table 1 lists the observed characteristic spin shifts for each

cytochrome P450-substrate complex. Most of the compounds tested

(1–13 and 1k), with some exceptions, bound in the active sites as

Type-I ligands. Interestingly 25-thia-27-norcholesterol (3),

cholesta-5,25-dienol (4), and 24-bromochol-5-enol (10) bind in the

CYP125A1 active site as reverse-Type I ligands. A significant

fraction of the resting ferric CYP125A1 enzyme is in the high-spin

state,^10,14,15,46thus, the observed Type-Ia binding spectra for

compounds 3, 4, and 10 with CYP125A1 is consistent with

stabilization of the low-spin, water-bound form of the enzyme upon

ligand binding. The observation of Type-I/Type-Ia binding spectra

for nearly all of the compounds indicated that the main structural

determinant of binding resides in the steroid nucleus rather than the

side chain and suggested that they may also be substrates for the

P450 enzymes.

2.2. Spectroscopic characterization of P450-substrate analog

complexes

We probed the specific interaction of substrate analogs 2–13

with the active site heme of each cytochrome P450 enzyme using

UV-visible spectrophotometry. Specific ligand binding in the P450

active site usually perturbs the spin-state of the heme iron from the

low-spin, 6-coordinate water-bound form to the high-spin, 5-

coordinate form (a Type-I spectrum).⁴⁴A broad peak centered

between 385–390 nm and a trough at 418–422 nm characterizes a

Type-I difference binding spectrum.⁴⁵A Type-Ia ligand produces a

mirror image of the Type-I spectrum and indicates a shift of

equilibrium from the high-spin, 5-coordinate form to the low-spin,

6-coordinate form of the heme iron. Type-II difference spectra, on

the other hand, typically have a peak between 422–435 nm and an

absorbance minimum at 390–405 nm that indicates replacement of

Table 1. The characteristic binding shifts detected for each substrate with each P450 enzyme, and the side-chain of the primary

oxidations products.

#

Compound name

Characteristic binding type

Side-chain of primary

oxidation product

CYP124A1

CYP125A1

CYP142A1

1

2

Cholesterol

I

OH

R

27-Norcholesterol

I

OH

R

3

4

25-Thia-27-norcholesterol

Cholesta-5,25-dienol

I

IA

I

ND^a

OH

R

5

6

26-Methylcholesta-5,25(26)-dienol

26-Methylcholesterol

I/IA^b

I

OH

R

OH

7

8

9

27-Norcholesta-5,25-dienol

25-Chloro-27-norcholesterol

25-Bromo-27-norcholesterol

I

O

R

OH

R

Cl

Br

OH

R

10

11

24-Bromochol-5-enol

Desmosterol

I

IA

I

ND^a

H

OH

R

12

Coprostanol

I

13

1k

Lanosterol

I

H

OH

R

Cholest-4-en-3-one

^aND = not detected. ^bTwo distinct binding modes were observed: Type I at low substrate concentrations (0.05–0.20 M) and Type Ia at higher concentrations.

2.3. Substrate analog affinities for each P450 enzyme

(data for ketones 1k–13k not shown). We were unable to detect

enzymatic oxidation of 3 and 3k beyond the non-enzymatic

sulfoxide formation. Although both CYP125A1 and CYP142A1

catalyzed the oxidation of lanosterol (13) and its ketone form 13k,

CYP124A1 did not, suggesting differences in the ability of the

active sites to accommodate the extra methyl groups on the A and

D rings of lanosterol (13) and 13k. We were also unable to detect

the oxidation of 26-methylcholesterol (6) and ketone 6k by

CYP124A1 or the oxidation of 26-methylcholesta-5,25(26)-dienol

(5) and 5k by CYP142A1.

Steroid stock solutions typically require solubilization by

cyclodextrins, such as methyl -cyclodextrin (MCD), or other

compatible detergents in order to measure binding and observe

catalytic activity.¹¹We attempted to determine the dissociation

constants (K_d) across the panel of compounds with each P450

isoform by monitoring the concentration-dependence of the low-

spin/high-spin equilibrium. In most cases we were unable to

accurately determine K_dvalues because the signals exhibited

complex titration behavior (Supplementary data). The reversible

and erratic signals likely result from partitioning of the steroid

between three phases: the enzyme active site, the medium, and

bound with the detergent (MCD).⁴⁷In many cases the high-spin

absorbance rapidly increased in amplitude in a concentration

dependent manner but then quickly decayed over time. The

absorbance at ~650 nm increased in amplitude and then reversed

with time, paralleling the reversible Soret absorbance and providing

further support for rapid and reversible binding in the P450 active

site. We observed in several other cases that a substrate analog

produced different binding spectra at different concentration

ranges: for example, the titration of CYP124A1 with 26-

2.6. Catalytic activity comparison

Table 2 lists the apparent turnover numbers (k_cat^app) as well as

the relative turnover numbers (k_rel) for each compound 1–13 with

each enzyme (the oxidation of ketones 1k–13k parallaled these

results, data not shown). As already noted, we were unable to detect

oxidation of 25-thia-27-norcholesterol (3) and 24-bromochol-5-enol

(10) by the three enzymes. The rest of the tested compounds were

substrates. Substrates with methyl branching (2, 3, 7, 10) exhibited

lower turnover numbers than substrates without methyl branching

at the 25-position (4–6, 8, 9), in contrast to what we found

previously with CYP124A1.²⁰Our data with all these substrate

analogs parallels our earlier finding that CYP125A1 is generally the

best catalyst. However, and interestingly, CYP142A1 exhibits the

highest turnover numbers with lanosterol (13), suggesting that its

active site is more tolerant of structural modifications to the A and

D rings.

methylcholesta-5,25(26)-dienol (5) showed

a distinct Type-I

binding spectrum at low concentrations (0.05–0.2 M), but a

reverse-Type-I binding spectrum at higher concentrations.

Generally, each of the enzymes exhibited a large amplitude Type-I

binding spectrum at sub-stoichiometric ligand:enzyme ratios.

We also examined the absolute spectra of the P450-steroid

complexes and this data supports our conclusion that each

compound binds as a Type I ligand within the active site of each

enzyme. For several of the more stable P450-steroid complexes we

were able to measure equilibrium dissociation constants

(supplemental information) and they were generally high-affinity

interactions that were on the order of the binding parameters for the

We observed exclusively the formation of ω-oxidation products,

and found evidence for secondary oxidation at the same site to

produce aldehyde and carboxylic acid products. Two examples of

this are cholesta-5,25-dienol (4) and 26-methylcholesta-5,25(26)-

dienol (5). We have previously found that the three enzymes

oxidized the side-chain of cholesterol (1) and cholest-4-en-3-one

(1k) in a similar manner to form alcohol, aldehyde and carboxylic

acid products.¹¹CYP124A1 also catalyzes such sequential

oxidation at the same site in non-steroidal substrates such as

geranylgeraniol.²⁰

app

substrate, i.e. the K_dvalues of the analogs were similar to the

apparent values of the natural substrates cholesterol (1) and cholest-

4-en-3-one (1k).^9-13

2.4. Conversion of sterols to their respective ketone forms

Previous studies have established that cholest-4-en-3-one (1k) is

the better, and likely physiological, substrate for all three

enzymes.^2,10,11,15We tested the compounds with sterol (1–13) and

ketosteroid (2k-13k) ring cores. The alcohols were converted

enzymatically to the corresponding ketones using cholesterol

oxidase and the conversion was confirmed by GC-MS analysis.

Having demonstrated specific binding of the substrate analogs

within the active sites of all three enzymes, we next tested whether

each would be turned over by the different enzymes.

2.7. 27-Norcholesta-5,25-diene (7) is not a mechanism-based

inhibitor

Like most of the steroids examined, the unsaturated 7 binds

specifically and with high relative affinity in the active sites of the

P450 enzymes. Catalytic assays demonstrated that compound 7 is a

substrate for all three P450 isoforms, in each case forming the

terminal epoxide. However, in a competition between epoxidation

of the terminal double bond in analog 4 and allylic, terminal

hydroxylation, only terminal hydroxylation was observed. A similar

selectivity preference was observed in the oxidation of desmosterol

(11) and lanosterol (13). We were not able to detect inhibition by

these olefinic substrates beyond the competitive inhibition that

occurs during the pre-incubation experimental setup, which is

corrected for in the calculations.

2.5. Monooxygenation products formed by Mtb CYP124A1,

CYP125A1, and CYP142A1

Sterols 1–13 and ketones 1k–13k were evaluated as substrates

for the P450 enzymes. Nearly all of the compounds were substrates

for the three enzymes, with the exception of 25-thia-27-

norcholesterol (3), 24-bromochol-5-enol (10), and ketones 3k and

10k. Nevertheless, compounds 3 and 10 were both found to bind

specifically in the active site of each enzyme (Table 1). Table 2

lists the apparent turnover number (k_cat^app) for compounds 1–13

3. Discussion

3.1. Nor series of steroid side-chain analogs

Cytochrome P450 enzymes oxidize a wide variety of substrates,

often with very precise regio- and stereoselectivity.⁴⁴Enzymatic

oxidation of unactivated hydrocarbons is the most energetically

demanding cytochrome P450-catalyzed reaction.⁴⁸The primary

intermediate in the catalytic cycle is the fleeting oxoferryl species

(Fe=O^+, Compound I)⁴⁹that abstracts a hydrogen atom from the

substrate to produce a substrate-centered radical and Fe^IV-OH. The

reaction products are formed by recombination of the substrate

radical with the hydroxy radical equivalent. Based on

thermodynamic principles the oxidized product distribution should

reflect the ease with which C-H bonds undergo homolytic bond

cleavage, i.e., the primary C-H bonds are stronger than those at

secondary and tertiary positions and are disfavored sites of

reaction.^17,48Many cytochrome P450 enzymes, however, have

evolved to catalyze oxidation at these difficult primary positions,

inlcuding the ω-oxidation of various steroid side-chains by

CYP124A1, CYP125A1, and CYP142A1. It appears in these cases

that ω-oxidation is facilitated by preventing more reactive positions

of the substrate from encountering the reactive oxoferryl species.¹⁷

Exclusive ω-oxidation of steroid side-chains is observed for the

three P450 isoforms. All three P450 isoforms tolerate a variety of

substrate structural modifications while retaining their precise ω-

regiospecificity. Modifications to either the steroid rings (vide

infra, Section 3.3) or the steroid side-chain were tolerated, as

judged from the k_relvalues (Table 2). It appears that additions to

the side-chain are more readily tolerated than deletions: for

example, the relative turnover numbers of 27-norcholesterol (2) and

27-norcholesta-5,25-dienol (7) show that they are poorer substrates

than cholesterol (1). All three enzymes produced only products

resulting from ω-oxidation with none of the ω-1 or ω-2 oxidation

products observed previously in the oxidation of unbranched fatty

acids by CYP124A1.²⁰CYP124A1 thus appears to bear some

resemblance to the ―letterbox‖ active site of CYP125A1,^10,14in that

tight binding of the sterol nucleus defines the distance the sterol

side-chain can extend towards the heme. Indeed, strong support for

this model is provided by the inability of these enzymes to oxidize

24-bromochol-5-enol (10) despite its specific binding in the active

site. Based on previous studies, it is likely that the truncated side-

chain is too short for the terminal atom to be accessible to the iron-

based oxidizing species, presumably because the steroid nucleus

prevents the side-chain from moving closer to the heme iron,

whereas the phytanic acid CYP124A1 co-crystal structure²⁰shows

how lipids of varying chain length are able to bypass this steric

block and reach the heme for ω-oxidation. Thus, the steroid nucleus

appears to influence the binding interactions with the substrate of

CYP124A1 much like the CYP125A1 ―letterbox‖ active site and it

appears that the binding interactions of the steroid rings also control

substrate mobility in CYP124A1. Indeed, the turnover numbers of

CYP124A1 with methyl branched fatty acids and isoprenoid

alcohols (15-methylpalmitic acid, phytanic acid, farnesol,

geranylgeraniol: k_cat= 7.6  1.5, 9.9  2.7, 15.5  2.8, 9.6  3.1

min^-1, respectively)²⁰are similar to those for methyl branched

CYP124A1 binds and oxidizes a wider variety of substrates than

CYP125A1 and CYP142A1, including fatty acids and alcohols,

isoprenoid alcohols and diphosphates, and steroids.^11,20However,

CYP124A1 is a less efficient catalyst than CYP142A1 and

CYP125, at least for cholesterol (1).¹¹CYP124A1 catalyzes ω-

oxidation of methyl branched substrates and a co-crystal structure

of CYP124A1 with phytanic acid suggests a mechanism by which

the active site enforces unfavorable regioselectivity.^11,20A small

hydrophobic cavity located near the heme group binds one of the

terminal methyl groups such that the other is positioned near the

heme iron for oxidation. Substrates lacking a terminal methyl

branch bind in the active site with the end of the lipid chain bound

in the small hydrophobic pocket above the heme, preventing

oxidation of the methyl group. Oxidation occurs non-selectively at

the ω-1 and ω-2 positions due to minor conformations in which the

methyl terminus is not sequestered in the cavity.^11,17CYP124A1

catalyzes the ω-oxidation of lipids of varying lengths, which

suggests that the observed strict ω-regiospecificity is controlled by

constraints near the heme, whereas the rest of the active site has

some tolerance for structural variation.^11,20Indeed, the co-crystal

structure with phytanic acid revealed several large cavities (200 Å³

and 300 Å³) along the length of the substrate, while the terminal

portion is tightly bound by a hydrophobic complementary surface.

steroidal substrates [cholesterol (1), cholest-4-en-3-one (1k): k_cat

=

1.5  0.2, 11.7  1.3 min^-1].¹¹The turnover numbers of the

unbranched analogs [27-norcholesterol (2), cholesta-5,25-dienol

(4), 25-thia-27-norcholesterol (3), and 24-bromochol-5-enol (10):

k_cat= 0.7  0.1, 0.03  0.005 min^-1, ND, and ND, respectively) and

the lack of detectable ω-1 and ω-2 oxidation products from these

substrates supports this model. The turnover of unbranched palmitic

acid (K_cat= 0.07  0.003 min^-1) was greatly diminished relative to

branched substrates, importantly with the thermodynamically

favored ω/ω-1/ ω-2 product ratios.²⁰

In contrast, CYP125A1 for which only the ω-hydroxylation of

steroid side-chains has been observed, has a ―letterbox shaped‖

active site.^8,12,14The crystal structures of sterols in complex with

CYP125A1 suggest that many lipophilic interactions with the

steroid ring nucleus clamp the substrate and position the side-chain

at the correct distance for ω-oxidation.

The CYP142A1 crystal structure¹²reveals a hybrid of the

CYP124A1 and CYP125A1 active sites, with a ―letterbox‖ portion

of the active site, consistent with our observation that CYP142A1

does not oxidize fatty acids or methyl branched lipids. However,

the CYP142A1 co-crystal structure uncovered a small pocket near

the heme iron that may function similarly to that in CYP124A1 by

binding one methyl group and thereby placing the other methyl

terminus near the heme iron for oxidation.

3.2. Methyl branching side-chain analogs

Structural modifications in the methyl branched side-chain were

generally well tolerated among the enzymes, much better so than

the removal of carbon atoms as observed in the nor series. Similar

to earlier findings with CYP124A1, methyl branching here led to an

increase in turnover number. However, the effects of the additional

branch are less clear when comparing the turnover numbers of

steroids. 27-Norcholesterol (2) shows slightly higher activity than

most of the branched analogs, indicating that the effects of methyl

branching in steroids are much more subtle than those in simple

fatty acids with CYP124A1. In all of the steroid products we only

detected ω-oxidation of the side-chain. Interestingly the terminal

olefin of 27-norcholesta-5,25-dienol (7) was oxidized to the oxirane

ring, as confirmed by the mass spectrum and relative retention

times, as well as the appearance of the diol following acid treatment

(Supplementary data). The epoxide was detected with each enzyme,

a prerequisite in some cases for mechanism-based inhibition by

olefins of cytochrome P450 enzymes (vide infra, Section 3.4).⁴⁴

branched terminus. Furthermore, CYP125A1 and CYP142A1

tolerate modifications of the A and B ring system better than

CYP124A1. This information provides a starting point for the

development of specific inhibitors of these enzymes.

3.3. Steroid ring analogs

4. Materials and Methods

CYP125A1 and CYP142A1 accommodate modifications of the

steroid ring structure, as indicated by their apparent k_catvalues with

lanosterol (13). Lanosterol (13) has three additional methyl groups,

two at the 4’ position and one at the 14’ position at the juncture of

rings C and D (Figure 2). CYP125A1 and CYP142A1 show k_rel

values for lanosterol (13) of 37 and 132, respectively, when

compared to cholesterol (1) (Table 2). These high activities

indicate that the two enzymes readily adjust to the presence of the

additional methyl groups. As expected, the only product observed

was the terminal alcohol resulting from ω-hydroxylation. In

contrast, CYP124A1 did not detectably oxidize lanosterol (13) and

thus, despite its ability to bind and oxidize linear hydrocarbon

substrates, cannot cope with expansion of the sterol core.

Coprostanol (12), which has a saturated A/B ring system, was

turned over efficiently by all three enzymes, as shown by the k_rel

values. Comparison of the turnover numbers of coprostanol (12),

cholesterol (1) and cholest-4-en-3-one (1k) suggests that three

features of the steroid A/B ring system contribute to the catalytic

efficiency: (a) the level of oxidation at the 3 position, and both (b)

the presence and (c) position of a double bond in the A/B ring

system. The absolute k_catvalues parallel our earlier finding that

CYP125A1 is the best steroid oxidant, followed by CYP142A1 and

finally CYP124A1 (Table 2). Under our incubation conditions, two

of the substrates, cholesta-5,25-dienol (4) and 26-methylcholesta-

5,25(26)-dienol (5), with unsaturated, branched side-chains, were

found to be oxidized to the respective carboxylic acids (Table 1).

However, it is likely that the other analogs would undergo similar

secondary oxidation in extended incubations.

4.1. General

All materials and reagents were of the highest commercial grade

available. All chemicals, unless noted otherwise, were obtained

from Sigma-Aldrich (St. Louis, MO or Castle Hill, NSW,

Australia). DNA restriction enzymes were obtained from New

England Biolabs. Cholesterol (1), cholest-4-en-3-one (1k),

desmosterol (11), coprostanol (12), and lanosterol (13) were

obtained from Sigma-Aldrich. All sterol stocks were solubilized in

9% (w/v) methyl--cyclodextrin (MCD) at between 4–10 mM,

and diluted further as necessary. 26-Hydroxycholesterol and 26-

cholestanoic acid were obtained from Avanti Polar Lipids

(Alabaster, AL) and Research Plus, Inc (Barnegat, NJ). The

cloning, over-expression, purification and general characterization

of CYP124A1, CYP125A1, and CYP142A1 have been reported

previously.^10-15,20,46Ferredoxin (Spinacia oleracea), ferredoxin-

NADP⁺reductase (S. oleracea), catalase (bovine), glucose-6-

phosphate dehydrogense, and cholesterol oxidase (Streptomyces

sp.) were obtained from Sigma-Aldrich.

Anhydrous solvents were dried according to established

procedures and were distilled under vacuum or N₂immediately

prior to use. THF and Et₂O were distilled off sodium-benzophenone

ketyl. CH₂Cl₂, pyridine, and BF₃.Et₂O were dried over and distilled

from CaH₂. Moisture or air sensitive experiments were conducted

in oven-dried glassware under a N₂or Ar atmosphere. Flash column

chromatography was performed on Silica gel 60 (0.04–0.06 mm,

230–400 mesh, Scharlau or Merck). TLC was performed on

Kieselgel 60 F254 plates (Merck, aluminum backed). Compounds

were visualized by treatment with PMA (5% phosphomolybdic acid

in EtOH), KMnO₄(1.0 g KMnO₄, 5.0 g Na₂CO₃and 0.5 g NaOH

dissolved in 100 mL H₂O), or vanillin (6.0 g vanillin and 2.5 mL

3.4. Mechanism-based inhibition: 27-norcholesta-5,25-dienol (7)

concentrated H₂SO₄dissolved in 250 mL EtOH) dips. H and ¹³C

1

27-Norcholesta-5,25-dienol (7) undergoes olefin epoxidation.

Olefin epoxidation is known to cause inactivation of some P450

enzymes,⁴⁴either through heme or protein alkylation, but time-

dependent inhibition was not observed in extended incubations of

up to 3 hours. The epoxidation of compound 7 therefore does not

appear to be associated with inactivation of any of the three

enzymes.

NMR spectra were recorded on either a Varian Inova 400, Bruker

AV400 or Bruker AV500 spectrometer using CDCl₃(99.8 atom %

D) or C₆D₆(99.96 atom % D) as solvent. H and ¹³C signals are

1

recorded in part per million (ppm) on the δ scale, with the residual

solvent peaks (CDCl₃: δ_H7.24 and δ_C77.0; C₆D₆: δ_H7.18 and δ_C

128.0) as internal references. GC-MS analyses (positive ion EI

mode) were performed on a Shimadzu GC-MS QP5000 or QP2010-

plus GC-MS, operating at 70 eV, connected to a Shimadzu GC-17A

or GC-2010 gas chromatograph fitted with a DB-5 column (30 m,

internal diameter 0.25 mm, J&W Scientific), or on an Agilent 6850

gas chromatograph fitted with an HP-5MS column (30 m, internal

diameter 0.25 mm, Agilent), coupled to an Agilent 5973 network

mass selective detector operating at 70 eV. Standard GC-MS

programs: Shimadzu GC-MS, DB-5 column: split mode; column

flow 1.0 mL min^-1; total flow 102.2 mL min^-1; injector 250 °C;

detector 250 °C; oven 200 °C (1.0 min equilibration) held for 1.0

min, ramp 20 °C min^-1to 300 °C and held for 44.0 min (total

program time 50.0 min). Agilent GC-MS, HP-5MS column: split

mode; column flow 1.0 mL min^-1; total flow 10.0 mL min^-1; injector

250 °C; detector 230 °C; oven 70 °C (1.0 min equilibration) held

for 1.0 min, ramp 10 °C min^-1to 300 °C and held for 20.0 min

3.5. Conclusions and outlook

In summary, we have investigated a series of synthetic

cholesterol (1) side-chain analogs (2-10) and commercially

available ring analogs (11-13) as substrates for CYP124A1,

CYP125A1, and CYP142A1. Our results indicate that there are

differences in the tolerance of the three enzyme active sites to

modifications of the side-chain and steroid core, but, most

importantly, the enzymes do tolerate substantial changes. Thus

each P450 active site tolerates a range of structural modifications to

the steroid side chain and ring nucleus. The minimal sidechain

must extend to at least C25, but the sidechain can accept additional

carbon units at various positions extending from C25 to the methyl

(total program time 44.0 min). Melting points were measured on a

Buchi Dr Tottoli apparatus and are uncorrected. High resolution

mass spectra were recorded on a Bruker micrOTOFQ spectrometer

(positive ion ESI mode) or at the UC Berkeley Mass Spectrometry

Facility using EI-MS. Elemental microanalyses were performed by

the Microanalytical Service, School of Chemistry and Molecular

Biosciences, at the University of Queensland. Optical rotations

were measured at the sodium D line (589 nm) at ambient

temperature using a 1 mL quartz cell with a 10 cm path length,

using a Jasco P-2000 polarimeter.

(20Z,23E)-N-methoxy-N-methyl-24-(3-(tert-

butyldimethylsilyloxy)-chola-5,20(22),23-triene)carboxamide

1

[(20Z,23E)-17]: Mp. 161-163 °C. [‡]²_D³−20.6 (c 0.28, CHCl₃). H

NMR (400 MHz, CDCl₃): δ 0.04 (s, 6H, ROSi(CH₃)₂C(CH₃)₃), 0.63

(s, 3H, H₃-18), 0.82-1.08 (m, 16H, incl. 0.87 [s, 9H,

ROSi(CH₃)₂C(CH₃)₃], 0.96 [s, 3H, H₃-19]), 1.10-1.81 (m, 12H),

1.82-2.05 (m, 5H, incl. 1.87 (s, 3H, H₃-21]), 2.10-2.30 (m, 2H),

3.23 (s, 3H, RCON(CH₃)(OCH₃)), 3.47 (m, 1H, H-3), 3.68 (s, 3H,

RCON(CH₃)(OCH₃)), 5.30 (m, 1H, H-6), 6.22 (d, 1H, J = 11.8 Hz,

H-22), 6.32 (d, 1H, J = 14.8 Hz, H-24), 7.69 (dd, 1H, J = 14.8, 11.8

Hz, H-23). ¹³C NMR (100 MHz, CDCl₃):

δ –4.6 (2C,

ROSi(CH₃)₂C(CH₃)₃), 13.9 (C-18), 18.2 (ROSi(CH₃)₂C(CH₃)₃),

19.4 (C-19), 20.7, 24.1, 24.7, 24.9, 25.9 (3C, ROSi(CH₃)₂C(CH₃)₃),

31.97, 32.03 (2C), 32.4, 36.7 (C-10), 37.3, 37.6, 42.8, 46.4 (C-13),

50.2, 51.5, 55.8, 61.7, 72.5 (C-3), 116.2, 121.0 (C-6), 127.5, 140.2,

141.6 (C-5), 149.4 (C-20), 168.0 (C-25). Anal.: Calc. for

C₃₃H₅₅NO₃Si: C, 73.14; H, 10.23; N, 2.58. Found: C, 73.18; H,

10.28; N, 2.48. HRMS (ESI): Calc. for C₃₃H₅₆NO₃Si ([M+H]^+.):

542.4029. Observed: 542.4033.

4.2. Sterol synthesis

4.2.1. (20E,23E)-N-Methoxy-N-methyl-24-(3-(tert-

butyldimethylsilyloxy)-chola-5,20(22),23-triene)carboxamide

[(20E,23E)-17] and (20Z,23E)-N-methoxy-N-methyl-24-(3-(tert-

butyldimethylsilyloxy)-chola-5,20(22),23-triene)carboxamide

[(20Z,23E)-17].

NaH (60% dispersion in mineral oil, 190 mg, 4.75 mmol) was

added

portion-wise

to

a

solution

of

diethyl

2-

(methoxy(methyl)amino)-2-oxoethylphosphonate³⁰(16) (1.030 g,

4.31 mmol) in anhydrous THF (15 mL) stirring under a N₂

atmosphere at 0 °C. The mixture was stirred for a further 10 min

following the completion of effervescence of H₂, then a solution of

isomeric aldehydes (20E)-15 and (20Z)-15 (~75%:25%, 912 mg,

2.00 mmol) in anhydrous THF (10 mL) was added dropwise. The

reaction mixture was allowed to warm to room temperature and

was stirred for 16 h. Saturated aqueous NH₄Cl solution (10 mL)

was added to quench the reaction and the aqueous and organic

layers were separated. The aqueous layer was extracted with

CH₂Cl₂(3 x 20 mL). The combined organic extract was washed

with brine (20 mL), dried over anhydrous MgSO₄, filtered, and

concentrated in vacuo. The residue was purified by flash column

chromatography (silica gel, 100% CH₂Cl₂to 2% EtOAc in CH₂Cl₂)

to afford the major unsaturated Weinreb amide (20E,23E)-17 (810

mg, 1.49 mmol, 75%) and the minor isomer (20Z,23E)-17 (261 mg,

0.48 mmol, 24%; total 99% yield) as white solids.

4.2.2. (20S)-N-Methoxy-N-methyl-24-(3-(tert-

butyldimethylsilyloxy)-chol-5-ene)carboxamide [(20S)-18].

A suspension of PtO₂(40 mg, 0.18 mmol) in a mixture of 1,4-

dioxane and AcOH (50:1, 20 mL) was degassed-purged twice with

N₂(g), then twice with H₂(g). Triene (20E,22E)-17 (800 mg, 1.48

mmol) was added as a solution in a mixture of 1,4-dioxane and

AcOH (50:1, 5 mL) and the reaction mixture was stirred under a H₂

atmosphere at room temperature for 5 h. The mixture was then

filtered through a pad of Celite^TMand silica gel, which was washed

thoroughly with CH₂Cl₂, and the filtrate was concentrated in vacuo.

The residue was purified by flash column chromatography (silica

gel, 15% to 20% EtOAc in petroleum spirit 40-60) to afford (20S)-

18 (264 mg, 0.48 mmol, 33%), and a mixture comprised of

approximately 45% (20S)-18 and 55% (20R)-18 (444 mg, 0.81

mmol, 55%; total 88% yield, ~2:1 S:R) as white solids.

(20S)-N-Methoxy-N-methyl-24-(3-(tert-butyldimethylsilyloxy)-

chol-5-ene)carboxamide [(20S)-18]: Mp. 82-84 °C. [‡]²_D⁴−26.2 (c

0.14, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.03 (s, 6H,

ROSi(CH₃)₂C(CH₃)₃), 0.65 (s, 3H, H₃-18), 0.78-1.32 (m, 24H, incl.

0.82 [d, 3H, J = 6.6 Hz, H₃-21], 0.87 [s, 9H, ROSi(CH₃)₂C(CH₃)₃],

0.97 [s, 3H, H₃-19]), 1.35-1.62 (m, 8H), 1.62-1.85 (m, 4H), 1.88-

2.00 (m, 2H), 2.08-2.42 (m, 4H), 3.16 (s, 3H, RCON(CH₃)(OCH₃)),

3.45 (m, 1H, H-3), 3.66 (s, 3H, RCON(CH₃)(OCH₃)), 5.29 (m, 1H,

(20E,23E)-N-Methoxy-N-methyl-24-(3-(tert-

butyldimethylsilyloxy)-chola-5,20(22),23-triene)carboxamide

1

[(20E,23E)-17]: Mp. 175-177°C. [‡]²_D⁴−7.2 (c 0.77, CHCl₃). H

NMR (400 MHz, CDCl₃): δ 0.03 (s, 6H, ROSi(CH₃)₂C(CH₃)₃), 0.55

(s, 3H, H₃-18), 0.82-1.30 (m, 16H, incl. 0.86 [s, 9H,

ROSi(CH₃)₂C(CH₃)₃], 0.97 [s, 3H, H₃-19]), 1.33-1.61 (m, 6H),

1.62-2.04 (m, 10H, incl. 1.90 (d, 3H, J = 0.8 Hz, H₃-21]), 2.10-2.30

(m, 3H), 3.23 (s, 3H, RCON(CH₃)(OCH₃)), 3.46 (m, 1H, H-3), 3.69

(s, 3H, RCON(CH₃)(OCH₃)), 5.29 (m, 1H, H-6), 6.09 (d, 1H, J =

11.6 Hz, H-22), 6.36 (d, 1H, J = 14.9 Hz, H-24), 7.68 (dd, 1H, J =

14.9, 11.6 Hz, H-23). ¹³C NMR (100 MHz, CDCl₃): δ –4.6 (2C,

ROSi(CH₃)₂C(CH₃)₃), 13.0 (C-18), 18.2 (ROSi(CH₃)₂C(CH₃)₃),

19.2, 19.4 (C-19), 21.0, 24.4, 24.8, 25.9 (3C, ROSi(CH₃)₂C(CH₃)₃),

31.8, 32.0, 32.2, 32.5, 36.6 (C-10), 37.4, 38.6, 42.8, 44.8 (C-13),

50.3, 56.5, 59.7, 61.7, 72.6 (C-3), 116.6, 120.9 (C-6), 124.2, 139.8,

141.6 (C-5), 149.0 (C-20), 168.0 (C-25). Anal.: Calc. for

C₃₃H₅₅NO₃Si: C, 73.14; H, 10.23; N, 2.58. Found: C, 73.16; H,

10.19; N, 2.48. HRMS (ESI): Calc. for C₃₃H₅₆NO₃Si ([M+H]^+.):

542.4029. Observed: 542.4029.

H-6). ¹³C NMR (100 MHz, CDCl₃):

δ

–4.6 (2C,

ROSi(CH₃)₂C(CH₃)₃), 12.1 (C-18), 18.3 (ROSi(CH₃)₂C(CH₃)₃),

18.7, 19.4 (C-19), 21.1, 21.4, 24.2, 25.9 (3C, ROSi(CH₃)₂C(CH₃)₃),

28.0, 31.9 (2C), 32.1, 32.4, 35.0, 35.1, 36.6 (C-10), 37.4, 39.7, 42.4

(C-13), 42.8, 50.2, 55.5, 56.8, 61.2, 72.6 (C-3), 121.2 (C-6), 141.5

(C-5), 178.3 (C-25). GC-MS (EI): m/z (%) 545 (0.1, M^+.), 530 (2,

M^+.-CH₃), 490 (10), 489 (37), 488 (100, M^+.-tBu), 470 (2), 458 (3),

253 (1), 207 (5), 159 (4), 158 (3), 147 (3), 133 (3), 131 (3), 130 (1),

119 (3), 116 (1), 115 (1), 105 (4), 103 (1), 95 (4), 88 (1), 81 (7), 75

(19), 60 (1), 57 (2), 55 (5). Anal.: Calc. for C₃₃H₅₉NO₃Si: C, 72.60;

H, 10.89; N, 2.57. Found: C, 72.92; H, 10.83; N, 2.53. HRMS

(ESI): Calc. for C₃₃H₅₉NNaO₃Si ([M+Na]^+.): 568.4162. Observed:

568.4143.

4.2.3. (20R)-N-Methoxy-N-methyl-24-(3-(tert-

ROSi(CH₃)₂C(CH₃)₃), 11.8 (C-18), 18.3 (ROSi(CH₃)₂C(CH₃)₃),

18.6, 19.4 (C-19), 20.4, 21.0, 24.3, 25.9 (3C, ROSi(CH₃)₂C(CH₃)₃),

28.2, 29.9, 31.90, 31.92, 32.1, 35.4, 35.6, 36.6 (C-10), 37.4, 39.7,

42.3 (C-13), 42.8, 44.3, 50.2, 55.8, 56.8, 72.6 (C-3), 121.1 (C-6),

141.6 (C-5), 209.4 (C-25). GC-MS (EI): m/z (%) 500 (0.3, M^+.),

485 (3, M^+.-CH₃), 444 (34), 443 (94, M^+.-tBu and/or M^+.-C₃H₅O),

429 (1), 425 (1), 415 (1), 387 (1), 385 (1 M^+.-TBS), 369 (1, M^+.-

OTBS), 367 (3), 351 (21), 341 (1), 329 (2), 309 (6), 295 (3), 253

(11), 241 (8), 227 (6), 213 (11), 199 (9), 185 (10), 173 (13), 161

(18), 159 (30), 145 (48), 133 (20), 131 (14), 119 (24), 115 (7), 113

(9), 105 (25), 95 (40), 85 (4), 81 (37), 75 (100), 71 (6), 67 (16), 58

(9), 57 (8), 55 (22), 43 (40). Anal.: Calc. for C₃₂H₅₆O₂Si: C, 76.73;

H, 11.27. Found: C, 76.58; H, 11.28. HRMS (ESI): Calc. for

C₃₂H₅₆NaO₂Si ([M+Na]^+.): 523.3947. Observed: 523.3947.

butyldimethylsilyloxy)-chol-5-ene)carboxamide [(20R)-18].

A mixture of triene (20Z,22E)-17 (240 mg, 0.44 mmol) and PtO₂

(12 mg, 0.05 mmol) in 1,4-dioxane and AcOH (50:1, 5 mL) was

degassed-purged twice with N₂(g), then twice with H₂(g). The

reaction mixture was stirred under a H₂atmosphere at room

temperature for 20 h and then was filtered through a pad of Celite^TM

and silica gel, which was washed thoroughly with CH₂Cl₂. The

filtrate was concentrated in vacuo and the residue was purified by

flash column chromatography (silica gel, 80% CH₂Cl₂in petroleum

spirit 40-60) to afford (20R)-18 (195 mg, 0.36 mmol, 81%; ~95%

20R, 5% 20S) as a white solid. Mp. 122-124 °C. [‡]²_D⁴−13.4 (c

0.37, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.03 (s, 6H,

ROSi(CH₃)₂C(CH₃)₃), 0.65 (s, 3H, H₃-18), 0.80-1.30 (m, 24H, incl.

0.82 [d, ~0.14H, J = 6.6 Hz, H₃-21 of 20S-isomer], 0.86 [s, 9H,

ROSi(CH₃)₂C(CH₃)₃], 0.92 [d, ~2.86H, J = 6.6 Hz, H₃-21 of 20R-

isomer], 0.97 [s, 3H, H₃-19]), 1.32-1.60 (m, 8H), 1.62-1.87 (m,

4H), 1.88-2.02 (m, 2H), 2.10-2.42 (m, 4H), 3.15 (s, 3H,

RCON(CH₃)(OCH₃)), 3.45 (m, 1H, H-3), 3.65 (s, 3H,

RCON(CH₃)(OCH₃)), 5.28 (m, 1H, H-6). ¹³C NMR (100 MHz,

CDCl₃): δ –4.6 (2C, ROSi(CH₃)₂C(CH₃)₃), 11.8 (C-18), 18.3

(ROSi(CH₃)₂C(CH₃)₃), 18.6, 19.4 (C-19), 21.0, 21.2, 24.3, 25.9

(3C, ROSi(CH₃)₂C(CH₃)₃), 28.2, 29.7, 31.90, 31.92, 32.1, 32.4,

35.6, 36.7, 36.6 (C-10), 37.4, 39.8, 42.3 (C-13), 42.8, 50.2, 55.8,

56.8, 61.2, 72.6 (C-3), 121.1 (C-6), 141.6 (C-5), 178.3 (C-25). GC-

MS (EI): m/z (%) 545 (0.1, M^+.), 530 (2, M^+.-CH₃), 490 (11), 489

(37), 488 (100, M^+.-tBu), 470 (2), 458 (4), 253 (1), 207 (5), 159 (4),

158 (3), 147 (3), 133 (3), 131 (3), 130 (1), 119 (3), 116 (1), 115 (1),

105 (4), 103 (1), 95 (4), 88 (1), 81 (7), 75 (19), 60 (1), 57 (2), 55

(5). Anal.: Calc. for C₃₃H₅₉NO₃Si: C, 72.60; H, 10.89; N, 2.57.

Found: C, 72.67; H, 10.78; N, 2.55. HRMS (ESI): Calc. for

C₃₃H₅₉NNaO₃Si ([M+Na]^+.): 568.4162. Observed: 568.4147.

4.2.5. (20R)-3-(tert-Butyldimethylsilyloxy)cholesta-5,25-diene

(20).

A solution of n-BuLi (1.1 M in hexanes, 0.27 mL, 297 mol)

was added dropwise to a solution of methyltriphenylphosphonium

iodide (180 mg, 450 mol) in anhydrous THF (3 mL) stirring under

a N₂atmosphere at –78 C. The resulting orange solution was

stirred at –78 C for 40 min, then allowed to warm to room

temperature and stirred for a further 10 min. The reaction mixture

was re-cooled to –78 C and a solution of ketone (20R)-19 (34 mg,

68 mol) in anhydrous THF (1 mL) was added dropwise. The

orange reaction mixture was allowed to warm to room temperature

and stirred for a further 20 h. Saturated aqueous NH₄Cl solution (10

mL) was added to quench the reaction and the mixture was

extracted with Et₂O (20 mL) and CH₂Cl₂(3 x 20 mL). The

combined organic extract was washed with brine (20 mL), dried

over anhydrous MgSO₄, filtered, and concentrated in vacuo. The

crude product was purified by flash column chromatography (silica

gel, 25% CH₂Cl₂in petroleum spirit 40-60) to afford diene 20 (30

mg, 60 mol, 89%; ~95% 20R, 5% 20S) as a white solid. Mp. 125-

127 °C. [α]²_D³−15.6 (c 0.09, CHCl₃). ¹H NMR (400 MHz, CDCl₃):

δ 0.04 (s, 6H, ROSi(CH₃)₂C(CH₃)₃), 0.66 (s, 3H, H₃-18), 0.80-1.61

(m, 33H, incl. 0.81 [d, ~0.15H, J = 6.6 Hz, H₃-21 of 20S-isomer],

0.87 [s, 9H, ROSi(CH₃)₂C(CH₃)₃], 0.91 [d, ~2.85H, J = 6.6 Hz, H₃-

21 of 20R-isomer], 0.97 [s, 3H, H₃-19]), 1.65-1.74 (m, 4H, incl.

1.69 [s, 3H, H₃-27]), 1.75-1.85 (m, 2H), 1.87-2.03 (m, 4H), 2.10-

2.18 (m, 1H), 2.20-2.30 (m, 1H), 3.46 (m, 1H, H-3), 4.62-4.68 (m,

2H, H₂-26), 5.29 (m, 1H, H-6). ¹³C NMR (100 MHz, CDCl₃) δ –4.6

4.2.4. (20R)-3-(tert-Butyldimethylsilyloxy)-25-oxo-27-

norcholest-5-ene [(20R)-19].

Methylmagnesium bromide (3.0 M in Et₂O, 0.4 mL, 0.60 mmol)

was added dropwise to a solution of Weinreb amide (20R)-18 (180

mg, 0.33 mmol) in anhydrous THF (4 mL) stirring under a N₂

atmosphere at –78 C. The reaction mixture was stirred at –78 C

for 1 h, then allowed to warm to room temperature and stirred for a

further 24 h. Saturated aqueous NH₄Cl solution (10 mL) was added

to quench the reaction and the mixture was extracted with Et₂O (20

mL) and CH₂Cl₂(3 x 20 mL). The combined organic extract was

washed with brine (20 mL), dried over anhydrous MgSO₄, filtered,

and concentrated in vacuo. The crude product was purified by flash

column chromatography (silica gel, 50% to 60% CH₂Cl₂in

petroleum spirit 40-60) to afford methyl ketone (20R)-19 (137 mg,

0.27 mmol, 83%; ~95% 20R, 5% 20S) as a white solid. Mp. 135-

137°C, lit.⁵⁰mp. 120-122 °C. [‡]_D²⁴−12.9 (c 0.38, CHCl₃).

Synthesis of (20R)-19 via a different synthetic route has been

(2C,

ROSi(CH₃)₂C(CH₃)₃),

11.8

(C-18),

18.3

(ROSi(CH₃)₂C(CH₃)₃), 18.7 (C-21), 19.4 (C-19), 21.1, 22.4 (C-27),

24.1, 24.3, 25.9 (3C, ROSi(CH₃)₂C(CH₃)₃), 28.2, 31.91, 31.94,

32.1, 35.6, 35.7, 36.6 (C-10), 37.4, 38.3, 42.3 (C-13), 42.8, 50.2,

56.1, 56.8, 72.6 (C-3), 109.5 (C-26), 121.2 (C-6), 141.6 (C-5),

146.4 (C-25). GC-MS (EI): m/z (%) 498 (0.3, M^+.), 483 (2, M^+.-

CH₃), 443 (11, M^+.-C₄H₇), 442 (36), 441 (100, M^+.-tBu), 429 (1),

423 (3), 415 (1), 387 (1), 385 (2), 371 (1), 367 (2, M^+.-OTBS), 365

(11), 341 (1), 329 (2), 281 (2), 255 (4), 245 (3), 235 (4), 207 (4),

159 (10), 147 (8), 133 (6), 131 (5), 121 (7), 115 (3), 111 (9), 109

(12), 95 (16), 83 (3), 81 (16), 75 (77), 69 (15), 57 (4), 55 (13), 41

(8). Anal.: Calc. for C₃₃H₅₈OSi: C, 79.45; H, 11.72. Found: C,

79.13; H, 11.71.

1

reported,^50,51and the H and ¹³C NMR spectral data obtained here

are consistent with the limited data available in the literature.^{50 1}H

NMR (400 MHz, CDCl₃): δ 0.03 (s, 6H, ROSi(CH₃)₂C(CH₃)₃), 0.65

(s, 3H, H₃-18), 0.80-1.27 (m, 24H, incl. 0.81 [d, ~0.16H, J = 6.6

Hz, H₃-21 of 20S-isomer], 0.87 [s, 9H, ROSi(CH₃)₂C(CH₃)₃], 0.91

[d, ~2.84H, J = 6.6 Hz, H₃-21 of 20R-isomer], 0.97 [s, 3H, H₃-19]),

1.27-1.85 (m, 12H), 1.88-2.02 (m, 2H), 2.08-2.18 (m, 4H, incl. 2.11

[s, 3H, H₃-26]), 2.20-2.44 (m, 3H), 3.46 (m, 1H, H-3), 5.29 (m, 1H,

4.2.6. (20R)-Cholesta-5,25-dien-3-ol (4).

H-6). ¹³C NMR (100 MHz, CDCl₃):

δ

–4.6 (2C,

Dowex 50W-X8(H) standard grade resin (cat.) was added to a

solution of TBS ether 20 (22 mg, 44.1 mol) in a mixture of

CH₂Cl₂(1 mL) and MeOH (1 mL). The mixture was stirred at room

temperature for 66 h, and then the solvent was evaporated. The

residue was purified by flash column chromatography (silica gel,

5% to 17% EtOAc in petroleum spirit 40-60) to afford sterol 4 (17

mg, 44.1 mol, quantitative) as a white solid. Mp. 118-120°C, lit.⁵²

mp. 120.5-121.5°C. [‡]_D²⁴−17.4 (c 0.09, CHCl₃), lit.⁵²[α]²_D⁵−40.2

(c unspecified, CHCl₃). Synthesis of 4 has reportedly been

accomplished via different synthetic routes,^52,53however no spectral

26), 121.2 (C-6), 136.2 (C-25), 136.4 (C-25), 141.6 (C-5). GC-MS

(EI) m/z (%) More volatile isomer: 512 (0.2, M^+.), 497 (1, M^+.-

CH₃), 457 (7, M^+.-C₄H₇), 456 (25), 455 (64, M^+.-tBu), 437 (2), 381

(1, M^+.-OTBS), 379 (9), 359 (1), 331 (1), 309 (1), 281 (1), 255 (2),

235 (3), 207 (5), 159 (10), 131 (5), 125 (4), 115 (3), 105 (12), 97

(4), 95 (19), 83 (10), 81 (20), 75 (100), 69 (26), 57 (9), 55 (25), 41

(20). Less volatile isomer: 512 (0.1, M^+.), 497 (1, M^+.-CH₃), 457

(7, M^+.-C₄H₇), 456 (24), 455 (62, M^+.-tBu), 437 (2), 381 (1, M^+.-

OTBS), 379 (8), 359 (1), 331 (2), 309 (1), 281 (2), 255 (3), 235 (3),

207 (6), 159 (10), 131 (5), 125 (4), 115 (3), 105 (12), 97 (5), 95

(20), 83 (11), 81 (19), 75 (100), 69 (27), 57 (9), 55 (25), 41 (20).

HRMS (ESI): Calc. for C₃₄H₆₀NaOSi ([M+Na]^+.): 535.4311.

Observed: 535.4294.

1

data has been reported previously. H NMR (400 MHz, CDCl₃): δ

0.66 (s, 3H, H₃-18), 0.79-1.66 (m, 26H, incl. 0.91 [d, 3H, J = 6.5

Hz, H₃-21], 0.99 [s, 3H, H₃-19]), 1.69 (s, 3H, H₃-27), 1.74-1.88 (m,

3H), 1.88-2.07 (m, 3H), 2.25 (m, 2H), 3.50 (m, 1H, H-3), 4.65 (dd,

2H, J = 8.9, 0.8 Hz, H₂-26), 5.33 (m, 1H, H-6). ¹³C NMR (100

MHz, CDCl₃): δ 11.8 (C-18), 18.7 (C-21), 19.4 (C-19), 21.1, 22.4

(C-27), 24.1, 24.3, 28.2, 31.7, 31.9 (2C), 35.6, 35.7 (C-20), 36.5

(C-10), 37.2, 38.3, 39.8, 42.3, 42.3 (C-13), 50.1, 56.1, 56.8, 71.8

(C-3), 109.5 (C-26), 121.7 (C-6), 140.8 (C-5), 146.4 (C-25). GC-

MS (EI): m/z (%) Free alcohol: 384 (64, M^+.), 369 (16, M^+.-CH₃),

367 (5, M^+.-OH), 366 (15, M^+.-H₂O), 351 (24), 327 (5), 299 (30),

271 (46), 255 (21), 243 (10), 229 (16), 213 (34), 199 (20), 187 (14),

173 (20), 159 (41), 145 (52), 131 (35), 119 (43), 111 (29), 105 (77),

95 (69), 83 (16), 81 (78), 69 (63), 55 (100), 41 (54). TMS ether:

456 (66, M^+.), 441 (13, M^+.-CH₃), 367 (20, M^+.-OTMS), 366 (67),

351 (33), 343 (8), 327 (44), 310 (2), 299 (2), 281 (4), 271 (3), 255

(9), 245 (10), 227 (3), 213 (10), 199 (5), 187 (4), 173 (13), 159

(17), 145 (24), 129 (93), 121 (26), 111 (14), 105 (28), 95 (32), 91

(30), 89 (1), 83 (7), 81 (31), 75 (68), 73 (100, TMS^+.), 69 (63), 55

(60), 41 (36). HRMS (ESI): Calc. for C₂₇H₄₄NaO ([M+Na]^+.):

407.3290. Observed: 407.3275. HRMS (EI): TMS ether: Calc. for

C₃₀H₅₂OSi (M^+.): 456.3787. Observed: 456.3792.

4.2.8. (20R)-26-Methylcholesta-5,25(26)-dien-3-ol (5).

Dowex 50W-X8(H) standard grade resin (cat.) was added to a

solution of TBS ether 21 (8 mg, 15.6 mol) in a mixture of CH₂Cl₂

(1 mL) and MeOH (1 mL). The mixture was stirred at room

temperature for 44 h, and then the solvent was evaporated. The

residue was purified by flash column chromatography (silica gel,

5% to 9% EtOAc in petroleum spirit 40-60) to afford sterol 5 (6

mg, 15.1 mol, 96%; ~50% 25E, 50% 25Z) as a white solid. [‡]_D²⁴

1

−13.2 (c 0.10, CHCl₃). H NMR (400 MHz, CDCl₃) (mixture of E

and Z isomers): δ 0.66 (2 overlapping s, 3H, H₃-18), 0.78-1.70 (m,

32H, incl. 0.89 & 0.91 [2 overlapping d, 3H, J = 2.9 Hz, H₃-21],

0.99 [s, 3H, H₃-19]), 1.74-2.05 (m, 6H), 2.24 (m, 2H), 3.50 (m, 1H,

H-3), 5.17 (m, 1H, H-26), 5.33 (m, 1H, H-6). ¹³C NMR (100 MHz,

CDCl₃) (mixture of E and Z isomers): δ 11.8 (C-18), 13.2, 13.3,

15.6, 18.69, 18.73, 19.4 (C-19), 21.1, 23.4, 24.2, 24.3, 24.4, 28.2,

29.7, 31.7, 31.8, 31.9, 35.6 (C-20), 35.7 (C-20), 35.8, 36.5 (C-10),

37.2, 39.8, 40.1, 42.3 (2C, incl. C-13), 50.1, 56.06, 56.10, 56.8,

71.8 (C-3), 118.0 (C-26), 118.6 (C-26), 121.7 (C-6), 136.2 (C-25),

136.5 (C-25), 140.8 (C-5). GC-MS (EI): m/z (%) More volatile

isomer: Free alcohol: 398 (57, M^+.), 383 (16, M^+.-CH₃), 381 (5,

M^+.-OH), 380 (15, M^+.-H₂O), 365 (23), 341 (20), 313 (12), 299

(28), 281 (59), 271 (33), 255 (19), 231 (13), 213 (27), 199 (18), 185

(21), 173 (18), 161 (32), 145 (36), 133 (39), 125 (14), 119 (36), 105

(62), 93 (55), 83 (30), 81 (71), 69 (75), 55 (100), 41 (70). TMS

ether: 470 (77, M^+.), 455 (15, M^+.-CH₃), 400 (3), 381 (17, M^+.-

OTMS), 380 (61), 365 (16), 345 (3), 341 (58), 325 (2), 295 (3), 271

(11), 255 (14), 245 (6), 217 (11), 207 (6), 185 (5), 173 (6), 159

(16), 145 (24), 129 (100), 125 (5), 119 (21), 105 (28), 97 (6), 95

(34), 89 (2), 83 (13), 73 (97, TMS^+.), 69 (51), 55 (57), 41 (13). Less

volatile isomer: Free alcohol: 398 (47, M^+.), 383 (15, M^+.-CH₃),

381 (6, M^+.-OH), 380 (16, M^+.-H₂O), 365 (20), 341 (20), 313 (11),

299 (25), 281 (24), 271 (39), 255 (20), 231 (13), 213 (29), 199 (20),

185 (18), 173 (15), 161 (25), 145 (34), 133 (29), 125 (15), 119 (38),

105 (43), 93 (45), 83 (24), 81 (56), 69 (78), 55 (100), 41 (73). TMS

ether: 470 (73, M^+.), 455 (13, M^+.-CH₃), 381 (10, M^+.-OTMS), 380

(48), 373 (2), 365 (23), 345 (1), 341 (38), 329 (3), 313 (2), 281 (4),

271 (3), 259 (8), 243 (4), 215 (5), 207 (11), 185 (4), 173 (6), 159

(14), 145 (14), 129 (100), 125 (6), 121 (13), 107 (26), 97 (6), 95

(29), 89 (2), 83 (18), 73 (93, TMS^+.), 69 (47), 55 (60), 41 (49).

HRMS (ESI): Calc. for C₂₈H₄₆NaO ([M+Na]^+.): 421.3446.

Observed: 421.3434. HRMS (EI): TMS ether: Calc. for C₃₁H₅₄OSi

(M^+.): 470.3944. Observed: More volatile isomer: 470.3952, less

volatile isomer: 470.3949.

4.2.7. (20R)-3-(tert-Butyldimethylsilyloxy)-26-methylcholesta-

5,25(26)-diene (21).

A solution of n-BuLi (0.9 M in hexanes, 0.44 mL, 396 mol)

was added dropwise to a solution of ethyltriphenylphosphonium

bromide (99%, 368 mg, 981 mol) in anhydrous THF (4 mL)

stirring under a N₂atmosphere at –78 C. The resulting orange

solution was stirred at –78 C for 30 min, then allowed to warm to

room temperature and stirred for a further 30 min. The reaction

mixture was re-cooled to –78 C and a solution of ketone (20R)-19

(41 mg, 82 mol) in anhydrous THF (1 mL) was added dropwise.

The orange reaction mixture was allowed to warm to room

temperature and stirred for a further 40 h. The solvent was then

evaporated and the residue was purified by flash column

chromatography (silica gel, 100% petroleum spirit 40-60 to 9%

EtOAc in petroleum spirit 40-60) to afford diene 21 (20 mg, 39

1

mol, 48%; ~50% 25E, 50% 25Z) as a white solid. H NMR (400

MHz, CDCl₃) (mixture of E and Z isomers): δ 0.04 (s, 6H,

ROSi(CH₃)₂C(CH₃)₃), 0.65 & 0.66 (2 overlapping s, 3H, H₃-18),

0.78-2.06 (m, 46H, incl. 0.87 [s, 9H, ROSi(CH₃)₂C(CH₃)₃], 0.98 [s,

3H, H₃-19]), 2.15 (m, 1H), 2.25 (m, 1H), 3.46 (m, 1H, H-3), 5.17

(m, 1H, H-26), 5.30 (m, 1H, H-6). ¹³C NMR (100 MHz, CDCl₃)

(mixture of E and Z isomers): δ –4.6 (2C, ROSi(CH₃)₂C(CH₃)₃),

11.8 (C-18), 13.2, 13.3, 15.6, 18.3 (ROSi(CH₃)₂C(CH₃)₃), 18.69,

18.73, 19.4 (C-19), 21.1, 23.4, 24.2, 24.3, 24.4, 25.9 (3C,

ROSi(CH₃)₂C(CH₃)₃), 28.2, 31.8, 31.91, 31.94, 32.1, 35.63 (C-20),

35.64, 35.7 (C-20), 35.8, 36.6 (C-10), 37.4, 39.8, 40.1, 42.3 (C-13),

42.8, 50.2, 56.07, 56.10, 56.8, 72.6 (C-3), 117.9 (C-26), 118.6 (C-

4.2.9. (20R)-26-Methylcholest-5-en-3-ol (6) and (20R)-26-

Methylcholestan-3-ol (22).

4.2.10. (20S)-3-(tert-Butyldimethylsilyloxy)-20-

hydroxymethylpregna-5,16(17)-diene (24).^37,38

A mixture of 21 (9 mg, 17.5 mol) and PtO₂(3 mg, 13.2 mol)

in 1,4-dioxane and AcOH (50:1, 1 mL) was evacuated and purged

twice with N₂(g), then twice with H₂(g). The reaction mixture was

stirred under a H₂atmosphere at room temperature for 16 h, and

then filtered through a pad of Celite^TMand silica gel, which was

washed thoroughly with EtOAc. The filtrate was concentrated in

vacuo and the residue was dissolved in CH₂Cl₂(1 mL) and MeOH

(1 mL). Concentrated aqueous HCl solution (32%, 20 L) was

added and the solution was stirred at room temperature for 1 h. The

solvent was evaporated under reduced pressure and the residue was

purified by flash column chromatography (silica gel, 5% to 9%

EtOAc in petroleum spirit 40-60) to afford a mixture comprised of

approximately 80% 6 and 20% saturated analog 22 (7 mg, 17.5

mol, quantitative over 2 steps, mixture of diastereomers at C-25)

as a white solid. [‡]²_D⁴−11.9 (c 0.10, CHCl₃). No spectral data is

A solution of freshly distilled BF₃.Et₂O in anhydrous CH₂Cl₂

(1% v/v, 0.18 mL, 15 mol) was added dropwise to a solution of

alkene (17Z)-23 (~92% [5% (17E)-23, 3% 20(22) alkene, 3-(tert-

butyldimethylsilyloxy)pregna-5,20(21)-diene], 58 mg, 0.14 mmol)

and paraformaldehyde (5 mg) in anhydrous CH₂Cl₂(20 mL)

stirring under a N₂atmosphere at room temperature. The reaction

mixture was stirred for 5 min, and then saturated aqueous NaHCO₃

solution (10 mL) was added to quench the reaction. The mixture

was extracted with CH₂Cl₂(6 x 10 mL) and the combined organic

extract was washed with brine (10 mL), dried over anhydrous

MgSO₄, filtered, and concentrated in vacuo. The crude product was

purified by flash column chromatography (silica gel, 1% to 50%

Et₂O in petroleum spirit 40-60) to afford 24 (46 mg, 0.10 mmol,

74%) as a pale yellow solid. Mp. 144-146 °C. [‡]²_D⁵–32.3 (c 0.79,

CHCl₃). Synthesis of 24 has reportedly been achieved by this

method previously,^37,38but no experimental procedure or spectral

1

available for 6 or 22 in the literature. H NMR (400 MHz, CDCl₃)

(mixture of diastereomers at C-25; contains ~20% saturated steroid

analog 22): δ 0.63 (s, ~0.7H, H₃-18 of saturated compound 22),

0.66 (s, ~2.3H, H₃-18 of 6), 0.74-1.72 (m, 36H, incl. 0.89 [d,

~2.5H, J = 6.6 Hz, H₃-21 of 6], 0.99 [s, ~2.6H, H₃-19 of 6]), 1.73-

1.87 (m, ~3.5H), 1.90-2.04 (m, 2H), 2.15-2.32 (m, 2H), 3.44-3.62

(m, 1H, H-3), 5.33 (m, ~0.8H, H-6 of 6). ¹³C NMR (100 MHz,

CDCl₃) (mixture of diastereomers at C-25; contains ~20% saturated

steroid analog 22* [tentative assignments]): δ 11.38, 11.44, 11.8,

12.1*, 12.3*, 18.65*, 18.69, 18.74, 19.2, 19.3, 19.4, 21.1, 21.3*,

23.5, 24.2*, 24.3, 28.2, 28.7, 29.4, 29.65, 29.68*, 31.5*, 31.7, 31.9,

32.1*, 34.45, 34.46, 35.46*, 35.51*, 35.75, 35.81, 36.2, 36.3, 36.5,

37.0*, 37.1, 37.18*, 37.25, 38.2*, 39.8, 40.0*, 42.3, 42.6*, 44.9*,

50.1, 54.4*, 56.1, 56.2, 56.27*, 56.30*, 56.5*, 56.8, 71.4* (C-3),

71.8 (C-3), 121.7 (C-6), 140.8 (C-5). GC-MS (EI): m/z (%) (20R)-

26-Methylcholest-5-en-3-ol (6): Free alcohol: 400 (53, M^+.), 385

(26, M^+.-CH₃), 383 (9, M^+.-OH), 382 (34, M^+.-H₂O), 367 (24), 341

(14), 315 (44), 289 (38), 281 (22), 273 (20), 255 (28), 231 (22), 213

(39), 199 (17), 185 (16), 173 (19), 159 (31), 145 (51), 131 (37), 119

(45), 105 (67), 91 (62), 85 (13), 81 (56), 71 (25), 67 (39), 57 (100),

43 (65). TMS ether: 472 (59, M^+.), 457 (10, M^+.-CH₃), 415 (1), 383

(30, M^+.-OTMS), 382 (97), 373 (1), 367 (45), 345 (5), 343 (94),

315 (5), 289 (8), 255 (4), 233 (4), 213 (9), 187 (3), 173 (13), 159

(21), 145 (22), 129 (100), 127 (1), 119 (24), 105 (26), 99 (2), 91

(19), 89 (3), 85 (10), 81 (27), 75 (63), 73 (85, TMS^+.), 71 (15), 57

(88), 55 (40), 43 (40), 41 (30). (20R)-26-Methylcholestan-3-ol

(22): Free alcohol: 402 (36, M^+.), 387 (27, M^+.-CH₃), 385 (12, M^+.-

OH), 384 (8, M^+.-H₂O), 369 (14), 315 (11), 276 (18), 257 (12), 233

(73), 215 (79), 193 (16), 165 (29), 147 (59), 133 (32), 121 (37), 107

(66), 95 (72), 85 (14), 81 (73), 71 (24), 67 (56), 57 (100), 43 (67).

TMS ether: 474 (65, M^+.), 459 (73, M^+.-CH₃), 417 (25), 389 (5),

385 (5, M^+.-OTMS), 384 (24), 375 (2), 369 (32), 347 (1), 345 (6),

329 (4), 306 (18), 305 (17), 276 (8), 261 (3), 230 (6), 215 (51), 207

(12), 183 (6), 175 (6), 159 (7), 147 (14), 129 (16), 127 (2), 119

(15), 105 (19), 99 (1), 95 (19), 89 (1), 85 (11), 81 (25), 75 (100), 73

(44, TMS^+.), 71 (18), 69 (29), 57 (56), 55 (31), 43 (30), 41 (29).

HRMS (ESI): (20R)-26-Methylcholest-5-en-3-ol (6): Calc. for

C₂₈H₄₈NaO ([M+Na]^+.): 423.3603. Observed: 423.3593. (20R)-26-

Methylcholestan-3-ol (22): Calc. for C₂₈H₅₀NaO ([M+Na]^+.):

425.3759. Observed: 425.3742. HRMS (EI): (20R)-26-

Methylcholest-5-en-3-ol (6): TMS ether: Calc. for C₃₁H₅₆OSi

(M^+.): 472.4100. Observed: 472.4109. (20R)-26-Methylcholestan-

3-ol (22): TMS ether: Calc. for C₃₁H₅₈OSi (M^+.): 474.4257.

Observed: 474.4258.

1

data were reported. H NMR (500 MHz, CDCl₃): δ 0.04 (s, 6H,

ROSi(CH₃)₂C(CH₃)₃), 0.80 (s, 3H, H₃-18), 0.87 (s, 9H,

ROSi(CH₃)₂C(CH₃)₃), 1.00-1.03 (m, 6H, incl. 1.02 [s, 3H, H₃-19],

1.02 [d, 3H, J = 6.9 Hz, H₃-21]), 1.19-1.44 (m, 4H), 1.47-1.74 (m,

6H), 1.78 (m, 2H), 1.87 (m, 1H), 1.99 (m, 1H), 2.09 (m, 1H), 2.16

(m, 1H), 2.26 (m, 1H), 2.38 (m, 1H, H-20), 3.47 (m, 1H, H-3), 3.55

(m, 1H, H₂-22), 5.31 (m, 1H, H-6), 5.42 (m, 1H, H-16). ¹³C NMR

(125 MHz, CDCl₃): δ –4.6 (2C, ROSi(CH₃)₂C(CH₃)₃), 16.2 (C-18),

18.1 (C-21), 18.2 (ROSi(CH₃)₂C(CH₃)₃), 19.3 (C-19), 20.7, 25.9

(3C, ROSi(CH₃)₂C(CH₃)₃), 30.5, 31.2, 31.6, 32.0, 34.9, 35.3 (C-

20), 36.8 (C-10), 37.3, 42.8, 47.0 (C-13), 50.8, 57.4, 66.5 (C-22),

72.6 (C-3), 120.9 (C-6), 123.0 (C-16), 141.8 (C-5), 157.6 (C-17).

GC-MS (EI): m/z (%) 443 (0.02, M^+.-1), 429 (0.1, M^+.-CH₃), 427

(0.1, M^+.-OH), 387 (19, M^+.-tBu), 385 (1), 370 (1), 357 (7), 330 (1),

312 (1), 296 (1), 282 (1), 253 (5), 239 (1), 225 (1), 211 (2), 199 (2),

185 (3), 173 (5), 159 (10), 145 (8), 133 (8), 131 (6), 121 (9), 115

(3), 105 (17), 93 (15), 91 (19), 79 (15), 75 (100), 67 (14), 59 (11),

57 (10), 55 (20), 41 (41). Anal.: Calc. for C₂₈H₄₈O₂Si: C, 75.61; H,

10.88. Found: C, 75.57; H, 10.86. HRMS (ESI): Calc. for

C₂₈H₄₈NaO₂Si ([M+Na]^+.): 467.3321. Observed: 467.3316.

4.2.11. (20S)-3-(tert-Butyldimethylsilyloxy)-20-

hydroxymethylpregn-5-ene (25).

A mixture of diene 24 (125 mg, 0.28 mmol) and PtO₂(19 mg,

0.08 mmol) in EtOAc (20 mL) was degassed-purged twice with N₂

(g), then twice with H₂(g). The reaction mixture was stirred under

a H₂atmosphere at room temperature for 4 h, and then was filtered

through a pad of Celite^TMand silica gel, which was washed

thoroughly with additional EtOAc. The filtrate was concentrated in

vacuo and the residue was purified by flash column

chromatography (silica gel, 2% to 20% Et₂O in petroleum spirit 40-

60) to afford 25 (108 mg, 0.24 mmol, 86%) as a white solid. Mp.

151-152 °C, lit.²¹mp. 153.5-155.5 °C. [α]_D²⁵–33.2 (c 0.43, CHCl₃).

Synthesis of 25 via a different synthetic method has been

reported,²¹while the complete NMR spectral data obtained here is

consistent with the limited data available for 25 in the literature (¹H

NMR),²¹and with data reported for a deuterium-labeled analog (¹H,

¹³C).^{54 1}H NMR (500 MHz, CDCl₃):

δ

0.04 (s, 6H,

ROSi(CH₃)₂C(CH₃)₃), 0.68 (s, 3H, H₃-18), 0.72-1.36 (m, 22H, incl.

0.86 [s, 9H, ROSi(CH₃)₂C(CH₃)₃], 0.98 [s, 3H, H₃-19], 1.03 [d, 3H,

J = 6.6 Hz, H₃-21], 1.37-1.74 (m, 9H), 1.75-1.86 (m, 2H), 1.96 (m,

2H), 2.14 (m, 1H), 2.19-2.35 (m, 1H), 3.35 (dd, 1H, J = 10.5, 6.9

Hz, H-22), 3.45 (m, 1H, H-3), 3.61 (dd, 1H, J = 10.5, 3.3 Hz, H-

22), 5.29 (m, 1H, H-6). ¹³C NMR (125 MHz, CDCl₃): δ –4.6 (2C,

ROSi(CH₃)₂C(CH₃)₃), 11.9 (C-18), 16.7 (C-21), 18.3

(ROSi(CH₃)₂C(CH₃)₃), 19.4 (C-19), 21.0, 24.4, 25.9 (3C,

ROSi(CH₃)₂C(CH₃)₃), 27.7, 31.9, 31.9, 32.1, 36.6 (C-10), 37.4,

38.7 (C-20), 39.6, 42.4 (C-13), 42.8, 50.2, 52.4, 56.5, 68.0 (C-22),

72.6 (C-3), 121.1 (C-6), 141.6 (C-5). GC-MS (EI): m/z (%) 436 (1),

431 (1, M^+.-CH₃), 403 (1), 340 (12), 389 (12, M^+.-tBu), 388 (8), 387

(3), 371 (1), 353 (1), 313 (1), 298 (1), 283 (1), 255 (1), 240 (1), 213

(2), 199 (2), 187 (3), 173 (3), 159 (12), 145 (6), 133 (6), 131 (5),

121 (6), 115 (2), 105 (13), 91 (15), 81 (19), 75 (100), 67 (15), 59

(10), 57 (13), 55 (32), 41 (50). Anal.: Calc. for C₂₈H₅₀O₂Si: C,

75.27; H, 11.28. Found: C, 74.99; H, 11.50. HRMS (ESI): Calc. for

C₂₈H₅₀NaO₂Si ([M+Na]^+.): 469.3478. Observed: 469.3465.

NMR comparison.⁴⁰Mp. 122-124 °C. [‡]_D²⁴–20.3 (c 0.28, CHCl₃).

¹H NMR (500 MHz, CDCl₃): δ 0.04 (s, 6H, ROSi(CH₃)₂C(CH₃)₃),

0.70 (s, 3H, H₃-18), 0.79-1.27 (m, 25H, incl. 0.88 [s, 9H,

ROSi(CH₃)₂C(CH₃)₃], 0.89 [obscured t, 3H, J = 7.3 Hz, H₃-26],

0.94 [d, 3H, J = 6.6 Hz, H₃-21]), 0.99 [s, 3H, H₃-19]), 1.27-1.59 (m,

8H), 1.59-1.75 (m, 2H), 1.79 (td, 1H, J = 13.2, 3.4 Hz), 1.86-2.10

(m, 4H), 2.15 (m, 1H), 2.25 (m, 1H), 2.41 (m, 1H, H-20), 3.46 (m,

1H, H-3), 5.06-5.26 (m, 2H, H-22 & H-23), 5.30 (m, 1H, H-6). ¹³

C

NMR (125 MHz, CDCl₃): δ –4.6 (2C, ROSi(CH₃)₂C(CH₃)₃), 12.1

(C-18), 13.9 (C-26), 18.2 (ROSi(CH₃)₂C(CH₃)₃), 19.4 (C-19), 20.8

(C-21), 21.1, 23.0, 24.3, 25.8, 25.9 (3C, ROSi(CH₃)₂C(CH₃)₃),

28.1, 29.7, 31.9, 32.1, 34.2 (C-20), 36.6 (C-10), 37.4, 39.7, 42.2 (C-

13), 42.8, 50.3, 56.1, 56.9, 72.6 (C-3), 121.1 (C-6), 126.4 (C-23),

136.7 (C-22), 141.5 (C-5). GC-MS (EI): m/z (%) 484 (0.1, M^+.),

469 (1, M^+.-CH₃), 428 (17), 427 (46, M^+.-tBu), 409 (1), 353 (1, M^+.-

OTBS), 351 (6), 329 (3), 281 (2), 255 (4), 231 (2), 207 (9), 159 (7),

145 (6), 133 (7), 131 (5), 119 (6), 115 (3), 105 (10), 97 (62), 75

(100), 69 (10), 67 (11), 57 (6), 55 (71), 43 (5), 41 (14). Anal.: Calc.

for C₃₂H₅₆OSi: C, 79.27; H, 11.64. Found: C, 79.12; H, 11.28.

4.2.12. (20S)-3-(tert-Butyldimethylsilyloxy)-20-

oxomethylpregn-5-ene (26).

PCC (98%, 703 mg, 3.20 mmol) was added to a solution of

alcohol 25 (1.091 g, 2.44 mmol) and NaOAc (73 mg, 0.89 mmol) in

anhydrous CH₂Cl₂(20 mL) stirring under a N₂atmosphere at 0 °C.

The reaction mixture was allowed to warm to room temperature

and stirred for 42 h. The solvent was evaporated and the residue

was purified by flash column chromatography (silica gel, 100%

petroleum spirit 40-60 to 2% EtOAc in petroleum spirit 40-60) to

afford aldehyde 26 (947 mg, 2.13 mmol, 87%) as a white solid. Mp.

130-132 °C, lit.⁵⁵mp. 130-131 °C. [‡]_D²⁶–24.6 (c 0.26, CHCl₃),

lit.⁵⁵[‡]²_D⁵–42.4 (c 1.00, CHCl₃). Synthesis of 26 via a different

procedure has been reported,⁵⁵and the full ¹H and ¹³C NMR

spectral data obtained here (Supplementary data) is consistent with

the data reported previously. GC-MS (EI): m/z (%) 444 (0.1, M^+.),

429 (1, M^+.-CH₃), 388 (16), 387 (48, M^+.-tBu and/or M^+.-C₃H₅O),

359 (8), 341 (2), 329 (5, M^+.-TBS), 313 (1, M^+.-OTBS), 311 (2),

295 (5), 253 (8), 239 (3), 225 (2), 213 (5), 199 (5), 185 (5), 159

(16), 145 (12), 133 (12), 131 (9), 119 (12), 115 (5), 107 (20), 93

(21), 81 (23), 75 (100), 67 (15), 58 (5), 57 (10), 55 (18), 41 (19).

HRMS (ESI): Calc. for C₂₈H₄₈NaO₂Si ([M+Na]^+.): 467.3321.

Observed: 467.3318.

4.2.14. (20R)-3-(tert-Butyldimethylsilyloxy)-27-norcholest-5-

ene (28).

A mixture of diene 27 (91 mg, 0.19 mmol) and PtO₂(17 mg, 70

mol) in 1,4-dioxane (6 mL) and AcOH (0.12 mL) was degassed-

purged twice with N₂(g), then twice with H₂(g). The reaction

mixture was stirred under a H₂atmosphere at room temperature for

18 h, and then filtered through a pad of Celite^TMand silica gel,

which was washed thoroughly with EtOAc. The filtrate was

concentrated in vacuo to afford 28 (88 mg, 0.18 mmol, 96%) as a

1

white solid. Mp. 115-117 °C. [‡]²_D⁶–14.4 (c 0.59, CHCl₃). H

NMR (500 MHz, CDCl₃): δ 0.04 (s, 6H, ROSi(CH₃)₂C(CH₃)₃), 0.65

(s, 3H, H₃-18), 0.79-0.94 (m, 18H, incl. 0.88 [s, 9H,

ROSi(CH₃)₂C(CH₃)₃]), 0.94-1.59 (m, 22H, incl. 0.98 [s, 3H, H₃-

19]), 1.59-1.74 (m, 1H), 1.74-1.86 (m, 2H), 1.86-2.10 (m, 2H), 2.15

(m, 1H), 2.25 (m, 1H, H-20), 3.46 (m, 1H, H-3), 5.29 (m, 1H, H-6).

¹³C NMR (125 MHz, CDCl₃): δ –4.6 (2C, ROSi(CH₃)₂C(CH₃)₃),

11.8 (C-18), 14.1 (C-26), 18.2 (ROSi(CH₃)₂C(CH₃)₃), 18.7 (C-21),

19.4 (C-19), 21.1, 22.7, 24.3, 25.7, 25.9 (3C, ROSi(CH₃)₂C(CH₃)₃),

28.2, 31.90, 31.93, 32.1, 32.4, 35.7 (C-20), 35.9, 36.6 (C-10), 37.4,

39.8, 42.3 (C-13), 42.8, 50.2, 56.1, 56.8, 72.6 (C-3), 121.2 (C-6),

141.5 (C-5). GC-MS (EI): m/z (%) 486 (0.1, M^+.), 471 (1, M^+.-

CH₃), 430 (25), 429 (65, M^+.-tBu), 411 (2), 355 (2, M^+.-OTBS), 353

(10), 339 (1), 289 (1), 275 (1), 255 (1), 233 (4), 207 (4), 159 (10),

145 (8), 133 (6), 131 (5), 119 (7), 115 (3), 105 (11), 99 (1), 95 (18),

75 (100), 71 (3), 69 (14), 67 (10), 57 (31), 55 (19), 43 (17), 41 (17).

Anal.: Calc. for C₃₂H₅₈OSi: C, 78.94; H, 12.01. Found: C, 79.21; H,

12.00.

4.2.13. (20R)-3-(tert-Butyldimethylsilyloxy)-27-norcholesta-

5,22-diene (27).

A solution of n-BuLi (0.9 M in hexanes, 1.0 mL, 0.90 mmol)

was added dropwise to a solution of n-butyltriphenylphosphonium

bromide (607 mg, 1.52 mmol) in anhydrous THF (4 mL) stirring

under a N₂atmosphere at –78 C. The resulting orange solution was

stirred at –78 C for 30 min, then allowed to warm to room

temperature and stirred for a further 30 min. The reaction mixture

was re-cooled to –78 C and a solution of aldehyde 26 (114 mg,

0.26 mmol) in anhydrous THF (3 mL) was added dropwise. The

orange reaction mixture was allowed to warm to room temperature

and stirred for a further 46 h. The solvent was then evaporated in

vacuo and the residue was purified by flash column

chromatography (silica gel, 100% petroleum spirit 40-60 to 2%

EtOAc in petroleum spirit 40-60) to afford diene 27 (106 mg, 0.22

mmol, 87%) as a white solid. ¹³C NMR and GC-MS analysis

suggested that this consists of predominantly one isomer,

tentatively assigned as having 22E stereochemistry based on ¹³C

4.2.15. (20R)-27-Norcholest-5-en-3-ol (2).

Dowex 50W-X8(H) standard grade resin (cat.) was added to a

solution of TBS ether 28 (54 mg, 111 mol) in a mixture of CH₂Cl₂

(2 mL) and MeOH (2 mL). The mixture was stirred at room

temperature for 42 h, and then the solvent was evaporated. The

residue was purified by flash column chromatography (silica gel,

5% to 9% EtOAc in petroleum spirit 40-60) to afford sterol 2 (40

mg, 107 mol, 97%) as a white solid. Mp. 118-120 °C, lit.⁵⁶mp.

(recrystallized from MeOH) 131-134 °C. [‡]²_D⁴–14.5 (c 0.42,

CHCl₃). The synthesis of 2 via a different synthetic route has been

1

reported previously,⁵⁶and the full H NMR spectral data obtained

here is consistent with the limited data available in the literature.

4.2.17. Ethyl (20R)-3-(tert-butyldimethylsilyloxy)chol-5-en-24-

oate (31).

1

¹³C NMR data has not been reported previously. H NMR (400

MHz, CDCl₃): δ 0.65 (s, 3H, H₃-18), 0.73-1.72 (m, 32H, incl. 0.88

[apparent t, 3H, J = 7.5 Hz, H₃-26], 0.98 [s, 3H, H₃-19]), 1.72-1.88

(m, 3H), 1.97 (m, 2H), 2.25 (m, 2H), 3.50 (m, 1H, H-3), 5.33 (m,

1H, H-6). ¹³C NMR (100 MHz, CDCl₃): δ 11.8 (C-18), 14.1 (C-26),

18.7 (C-21), 19.4 (C-19), 21.1, 22.7, 24.3, 25.7, 28.2, 31.6, 31.9

(2C), 32.4, 35.7 (C-20), 35.9, 36.5 (C-10), 37.2, 39.8, 42.3 (2C,

incl. C-13), 50.1, 56.1, 56.8, 71.8 (C-3), 121.7 (C-6), 140.8 (C-5).

GC-MS (EI): m/z (%) Free alcohol: 372 (85, M^+.), 357 (31, M^+.-

CH₃), 355 (8, M^+.-OH), 354 (43, M^+.-H₂O), 339 (39), 312 (8), 287

(66), 273 (29), 261 (60), 255 (35), 231 (31), 213 (54), 199 (21), 173

(25), 159 (45), 145 (63), 133 (47), 119 (53), 105 (88), 91 (79), 81

(75), 67 (60), 57 (100), 43 (83). TMS ether: 444 (48, M^+.), 429 (7,

M^+.-CH₃), 355 (20, M^+.-OTMS), 354 (78), 345 (1), 315 (90), 287

(3), 255 (14), 233 (14), 213 (9), 199 (5), 185 (5), 173 (9), 159 (17),

145 (24), 129 (100), 105 (27), 99 (1), 91 (24), 89 (1), 73 (93,

TMS^+.), 71 (4), 57 (51), 55 (37), 43 (39), 41 (28). Anal.: Calc. for

C₂₆H₄₄O: C, 83.80; H, 11.90. Found: C, 83.43; H, 11.70. HRMS

(ESI): Calc. for C₂₆H₄₄NaO ([M+Na]^+.): 395.3290. Observed:

395.3284. HRMS (EI): TMS ether: Calc. for C₂₉H₅₂OSi (M^+.):

444.3787. Observed: 444.3781.

A mixture of ,-unsaturated ester 30 (224 mg, 0.44 mmol) and

PtO₂(38 mg, 0.17 mmol) in 1,4-dioxane (6 mL) and AcOH (0.12

mL) was degassed-purged twice with N₂(g), then twice with H₂

(g). The reaction mixture was stirred under a H₂atmosphere at

room temperature for 16 h, and then filtered through a pad of

Celite^TMand silica gel, which was washed thoroughly with EtOAc.

The filtrate was concentrated in vacuo to afford 31 (218 mg, 0.42

mmol, 97%) as a white solid. Mp. 90-92 °C. [α]²_D⁶–18.5 (c 0.59,

CHCl₃). ¹H NMR (400 MHz, CDCl₃):

δ 0.02 (s, 6H,

ROSi(CH₃)₂C(CH₃)₃), 0.64 (s, 3H, H₃-18), 0.72-2.03 (m, 39H, incl.

0.86 [s, 9H, ROSi(CH₃)₂C(CH₃)₃], 0.89 [d, 3H, J = 6.5 Hz, H₃-21]),

0.96 [s, 3H, H₃-19], 1.22 [t, 3H, J = 7.1 Hz, RCO₂CH₂CH₃]), 2.08-

2.38 (m, 4H), 3.44 (m, 1H, H-3), 4.08 (q, 2H, J = 7.1 Hz,

RCO₂CH₂CH₃), 5.28 (m, 1H, H-6). ¹³C NMR (100 MHz, CDCl₃): δ

–4.6 (2C, ROSi(CH₃)₂C(CH₃)₃), 11.8 (C-18), 14.2 (RCO₂CH₂CH₃),

18.2 (ROSi(CH₃)₂C(CH₃)₃), 18.3 (C-21), 19.4 (C-19), 21.0, 24.2,

25.9 (3C, ROSi(CH₃)₂C(CH₃)₃), 28.1, 31.0, 31.3, 31.9 (2C, incl. C-

20), 32.1, 35.3, 36.5 (C-10), 37.3, 39.7, 42.3 (C-13), 42.8, 50.1,

55.7, 56.7, 60.1 (RCO₂CH₂CH₃), 72.6 (C-3), 121.1 (C-6), 141.5 (C-

5), 174.2 (C-24). GC-MS (EI): m/z (%) 516 (0.2, M^+.), 501 (2, M^+.-

CH₃), 460 (28), 459 (84, M^+.-tBu), 441 (3), 413 (2), 385 (1), 383

(3), 339 (9), 321 (7), 295 (3), 281 (1), 253 (2), 227 (3), 213 (7), 199

(7), 175 (12), 159 (21), 145 (14), 133 (14), 131 (11), 129 (8), 121

(15), 115 (5), 105 (20), 101 (11), 93 (21), 88 (11), 87 (1), 81 (26),

75 (100), 73 (35), 67 (16), 57 (9), 55 (25). Anal.: Calc. for

C₃₂H₅₆O₃Si: C, 74.36; H, 10.92. Found: C, 74.22; H, 11.18. HRMS

(ESI): Calc. for C₃₂H₅₆NaO₃Si ([M+Na]^+.): 539.3896. Observed:

539.3899.

4.2.16. Ethyl (20R,22E)-3-(tert-butyldimethylsilyloxy)chola-

5,22-dien-24-oate (30).

A solution of aldehyde 26 (240 mg, 0.54 mmol) and ethyl 2-

(triphenylphosphoranylidene)acetate⁴³(29) (660 mg, 1.89 mmol) in

anhydrous CH₂Cl₂(20 mL) was heated at reflux under a N₂

atmosphere for 72 h. The reaction mixture was cooled to room

temperature and concentrated in vacuo, and the residue was

purified by flash column chromatography (silica gel, 100%

petroleum spirit 40-60 to 2% EtOAc in petroleum spirit 40-60) to

afford ,-unsaturated ester 30 (259 mg, 0.50 mmol, 93%, >85%

22E-isomer) as a white solid. Mp. 86-88 °C. [α]²_D⁶–19.0 (c 0.69,

CHCl₃). ¹H NMR (400 MHz, CDCl₃) (mixture of E and Z isomers):

δ 0.04 (s, 6H, ROSi(CH₃)₂C(CH₃)₃), 0.69 (s, 3H, H₃-18), 0.74-1.11

(m, 20H, incl. 0.87 [s, 9H, ROSi(CH₃)₂C(CH₃)₃], 0.98 [s, 3H, H₃-

19], 1.07 [d, 3H, J = 6.6 Hz, H₃-21]), 1.14-1.31 (m, 6H, incl. 1.26

[t, 3H, J = 7.2 Hz, RCO₂CH₂CH₃]), 1.34-1.87 (m, 8H), 1.95 (m,

2H), 2.14 (m, 1H), 2.24 (m, 2H), 3.45 (m, 1H, H-3), 4.06-4.21 (m,

2H, RCO₂CH₂CH₃, incl. 4.15 [q, J = 7.1 Hz] for major E-isomer),

5.29 (m, 1H, H-6), 5.67-5.77 (m, 1H, H-23, incl. 5.71 [d, J = 15.6

Hz] for E-isomer), 6.75-6.97 (m, 1H, H-22, incl. 6.81 [dd, J = 15.6,

8.9 Hz] for E-isomer). ¹³C NMR (100 MHz, CDCl₃): δ –4.6 (2C,

ROSi(CH₃)₂C(CH₃)₃), 12.1 (C-18), 14.3 (RCO₂CH₂CH₃), 18.2

(ROSi(CH₃)₂C(CH₃)₃), 19.2, 19.4, 21.0, 24.3, 25.9 (3C,

ROSi(CH₃)₂C(CH₃)₃), 28.1, 31.8, 31.9 (C-20), 32.1, 36.6 (C-10),

37.4, 39.6, 39.7, 42.7 (C-13), 42.8, 50.2, 54.9, 56.6, 60.1

(RCO₂CH₂CH₃), 72.6 (C-3), 118.9 (C-23), 121.0 (C-6), 141.5 (C-

5), 154.7 (C-22 of E-isomer), 155.5 (C-22 of Z-isomer), 166.9 (C-

24 of Z-isomer), 167.1 (C-24 of E-isomer). GC-MS (EI): m/z (%)

514 (0.2, M^+.), 499 (2, M^+.-CH₃), 458 (32), 457 (95, M^+.-tBu), 439

(2), 411 (2), 383 (3), 357 (4), 329 (3), 281 (3), 255 (7), 227 (3), 213

(5), 199 (6), 185 (5), 175 (14), 159 (19), 147 (15), 133 (14), 131

(11), 127 (2), 119 (14), 115 (6), 105 (22), 99 (8), 93 (23), 81 (26),

75 (100), 73 (36), 67 (15), 57 (8), 55 (21). Anal.: Calc. for

C₃₂H₅₄O₃Si: C, 74.65; H, 10.57. Found: C, 75.03; H, 10.25. HRMS

(ESI): Calc. for C₃₂H₅₄NaO₃Si ([M+Na]^+.): 537.3740. Observed:

537.3722.

4.2.18. (20R)-3-(tert-Butyldimethylsilyloxy)chol-5-en-24-ol

(32).

LiAlH₄(95%, 24 mg, 0.60 mmol) was added portion-wise to a

solution of ester 31 (200 mg, 0.39 mmol) in anhydrous THF (5 mL)

stirring under a N₂atmosphere at 0 C. The reaction mixture was

allowed to warm to room temperature and stirred for 44 h. Sodium

sulfate decahydrate (330 mg) was added to quench the reaction and

the mixture was stirred for 10 min and then filtered through a pad

of Celite^TMand silica gel, which was washed thoroughly with

EtOAc. The filtrate was concentrated in vacuo to afford alcohol 32

(180 mg, 0.38 mmol, 98%) as a white powder. Mp. 156-158 °C,

lit.⁵⁷mp. 164-165 °C for ent-32. [α]_D²⁶–11.2 (c 0.64, CHCl₃), lit.⁵⁷

[‡]²_D⁴+28.4 (c 1.00, CHCl₃) for ent-32. The full ¹H and ¹³C spectral

data obtained here (Supplementary data) matches that reported for

ent-32.⁵⁷GC-MS (EI): m/z (%) 474 (0.04, M^+.), 473 (0.2, M^+.-1),

459 (1, M^+.-CH₃), 457 (0.2, M^+.-OH), 419 (13), 417 (39, M^+.-tBu),

399 (2), 387 (4), 343 (1), 341 (3), 325 (3), 283 (1), 269 (1), 255 (3),

243 (2), 229 (3), 215 (6), 199 (4), 175 (7), 159 (16), 145 (12), 133

(12), 131 (9), 121 (13), 115 (5), 105 (19), 95 (30), 87 (1), 81 (29),

75 (100), 69 (31), 67 (20), 59 (6), 57 (13), 55 (24), 45 (4), 41 (25).

Anal.: Calc. for C₃₀H₅₄O₂Si: C, 75.88; H, 11.46. Found: C, 75.69;

H, 11.62. HRMS (ESI): Calc. for C₃₀H₅₄NaO₂Si ([M+Na]^+.):

497.3791. Observed: 497.3783.

4.2.19. (20R)-3-(tert-Butyldimethylsilyloxy)-24-bromochol-5-

ene (33).

CBr₄(99%, 198 mg, 0.59 mmol) was added portion-wise to a

solution of alcohol 32 (140 mg, 0.29 mmol), PPh₃(99%, 168 mg,

0.63 mmol) and imidazole (102 mg, 1.50 mmol) in a mixture of

anhydrous Et₂O (3 mL) and CH₃CN (1 mL) stirring under a N₂

atmosphere at 0°C. The reaction mixture was allowed to warm to

room temperature and stirred for 2.5 h, and then was re-cooled to

0°C. MeOH (0.2 mL) was added to quench the reaction and the

solvent was evaporated. The residue was purified by flash column

chromatography (silica gel, 100% petroleum spirit 40-60 to 2%

Et₂O in petroleum spirit 40-60) to afford bromide 33 (151 mg, 0.28

mmol, 95%) as a white solid. Mp. 108-110 °C. [α]²_D⁶–10.0 (c 0.16,

([M+Na]^+.): 445.2082/447.2062. Observed: 445.2065/447.2070.

HRMS (EI): Calc. for C₂₄H₃₉BrO (M^+.): 422.2184/424.2164.

Observed: 422.2186/424.2177.

4.2.21. (20R)-3-(tert-Butyldimethylsilyloxy)-24-

(acetylthio)chol-5-ene (34).

A solution of bromide 33 (52 mg, 97 mol) and potassium

thioacetate (98%, 32 mg, 275 mol) in freshly distilled acetone (5

mL) was stirred under a N₂atmosphere at room temperature for 23

h. The solvent was then evaporated and the residue was purified by

flash column chromatography (silica gel, 1% to 2% EtOAc in

petroleum spirit 40-60) to afford thioacetate 34 (49 mg, 92 mol,

95%) as a white solid. Mp. 90-92 °C. [α]²_D⁶–6.7 (c 0.50, CHCl₃).

¹H NMR (400 MHz, C₆D₆): δ 0.14 (d, 6H, J = 2.4 Hz,

ROSi(CH₃)₂C(CH₃)₃), 0.64 (s, 3H, H₃-18), 0.71-1.88 (m, 36H, incl.

0.91 [d, 3H, J = 6.6 Hz, H₃-21], 0.99 [s, 3H, H₃-19], 1.05 [s, 9H,

ROSi(CH₃)₂C(CH₃)₃]), 1.90-2.03 (m, 5H, incl. 1.94 [s, 3H,

RSCOCH₃), 2.41 (m, 1H), 2.52 (m, 1H), 2.83 (m, 2H, H₂-24), 3.65

(m, 1H, H-3), 5.41 (m, 1H, H-6). ¹³C NMR (100 MHz, C₆D₆): δ –

CHCl₃). ¹H NMR (400 MHz, CDCl₃):

δ 0.03 (s, 6H,

ROSi(CH₃)₂C(CH₃)₃), 0.66 (s, 3H, H₃-18), 0.74-2.05 (m, 38H, incl.

0.88 [s, 9H, ROSi(CH₃)₂C(CH₃)₃], 0.91 [d, 3H, J = 6.6 Hz, H₃-21]),

0.98 [s, 3H, H₃-19]), 2.14 (m, 1H), 2.24 (m, 1H), 3.35 (m, 2H, H₂-

24), 3.46 (m, 1H, H-3), 5.29 (m, 1H, H-6). ¹³C NMR (100 MHz,

CDCl₃): δ –4.6 (2C, ROSi(CH₃)₂C(CH₃)₃), 11.8 (C-18), 18.2

(ROSi(CH₃)₂C(CH₃)₃), 18.7 (C-21), 19.4 (C-19), 21.0, 24.2, 25.9

(3C, ROSi(CH₃)₂C(CH₃)₃), 28.2, 29.6, 31.89 (C-20), 31.90, 32.1,

34.45, 34.50, 35.2, 36.6 (C-10), 37.4, 39.8, 42.3 (C-13), 42.8, 50.2,

55.8, 56.8, 72.6 (C-3), 121.1 (C-6), 141.5 (C-5). GC-MS (EI): m/z

(%) 536/538 (0.2/0.1, M^+.), 521/523 (1/1, M^+.-CH₃), 479/481

(33/38, M^+.-tBu), 405/407 (7/3, M^+.-OTBS), 401 (7), 325 (8), 283

(3), 255 (4), 227 (3), 213 (5), 199 (4), 161 (8), 159 (20), 151 (3),

149 (6), 145 (14), 131 (9), 123 (5), 121 (10), 115 (5), 107/109

(15/14), 95 (30), 93 (18), 81 (23), 79 (13), 75 (100), 69 (28), 57

(10), 55 (26), 41 (22). Anal.: Calc. for C₃₀H₅₃BrOSi: C, 67.01; H,

9.93. Found: C, 66.61; H, 9.68. HRMS (ESI): Calc. for

C₃₀H₅₃BrNaOSi ([M+Na]^+.): 559.2947/561.2926. Observed:

559.2943/561.2940.

4.3

(2C,

ROSi(CH₃)₂C(CH₃)₃),

12.0

(C-18),

18.3

(ROSi(CH₃)₂C(CH₃)₃), 18.8 (C-21), 19.5 (C-19), 21.4, 24.5, 26.1

(3C, ROSi(CH₃)₂C(CH₃)₃), 26.7, 28.4, 29.8, 30.2 (RSCOCH₃), 32.2

(C-20), 32.4, 32.7, 35.4, 35.7, 36.8 (C-10), 37.7, 40.1, 42.5 (C-13),

43.5, 50.6, 56.2, 56.9, 72.9 (C-3), 121.5 (C-6), 141.5 (C-5), 194.3

(C=O). GC-MS (EI): m/z (%) 532 (0.1, M^+.), 517 (2, M^+.-CH₃), 476

(35), 475 (96, M^+.-tBu), 457 (3, M^+.-SAc), 433 (1), 417 (1, M^+.-

TBS), 401 (5, M^+.-OTBS), 400 (2), 399 (5), 383 (1), 357 (2), 329

(2), 255 (3), 213 (5), 161 (9), 159 (13), 145 (11), 131 (8), 119 (32),

117 (13), 115 (6), 105 (17), 103 (8), 95 (20), 89 (2), 81 (18), 76 (8),

75 (100), 69 (16), 57 (7), 55 (15), 43 (98). HRMS (ESI): Calc. for

C₃₂H₅₆NaO₂SSi ([M+Na]^+.): 555.3668. Observed: 555.3668.

4.2.20. (20R)-24-bromochol-5-en-3-ol (10).

Dowex 50W-X8(H) standard grade resin (cat.) was added to a

solution of TBS ether 33 (11 mg, 20.5 mol) in a mixture of

CH₂Cl₂(1 mL) and MeOH (1 mL). The mixture was stirred at room

temperature for 90 h, and then the solvent was evaporated. The

residue was purified by flash column chromatography (silica gel,

5% to 9% EtOAc in n-hexane) to afford sterol 10 (8 mg, 18.9 mol,

92%) as a white solid. The synthesis of 10 via a different synthetic

4.2.22. (20R)-25-Thia-27-norcholest-5-en-3-ol (3).

A solution of sodium methoxide in anhydrous MeOH (0.46 M,

0.50 mL, 228 mol) was added to a solution of thioacetate 34 (28

mg, 53 mol) in a mixture of anhydrous MeOH (1 mL) and THF (1

mL) stirring under an Ar atmosphere at 0 °C. The mixture was

allowed to warm to room temperature and stirred for 30 min. MeI

(99%, 20 L, 318 mol) was added and the reaction mixture was

stirred at room temperature for 2 h, and then concentrated under a

stream of N₂in order to evaporate excess MeI. The residue was re-

dissolved in anhydrous THF (1 mL), and MeOH (0.5 mL) and

concentrated aqueous HCl solution (32%, 10 L) were added. The

reaction mixture was stirred for 30 min, and then was concentrated

under a stream of N₂. The residue was purified by flash column

chromatography (silica gel, 5% to 17% EtOAc in petroleum spirit

40-60) to afford sterol 3 (14 mg, 36 mol, 68% over 3 steps) as a

pale yellow solid. Mp. 113-115 °C, lit.⁵⁹mp. (recrystallized from

hexane) 119-120 °C. [‡]_D²³–13.1 (c 0.40, C₆D₆). Synthesis of 3 via

a different synthetic route has been reported previously and only

1

route has been reported previously,⁵⁸and the full H NMR spectral

data obtained here is consistent with the very limited data available

in the literature. ¹³C NMR data has not been reported previously. ¹H

NMR (400 MHz, CDCl₃): δ 0.66 (s, 3H, H₃-18), 0.73-2.34 (m, 35H,

incl. 0.91 [d, 3H, J = 6.4 Hz, H₃-21]), 0.98 [s, 3H, H₃-19]), 3.36 (m,

2H, H₂-24), 3.51 (m, 1H, H-3), 5.33 (m, 1H, H-6). ¹³C NMR (100

MHz, CDCl₃): δ 11.8 (C-18), 18.7 (C-21), 19.4 (C-19), 21.1, 24.2,

28.2, 29.6, 31.6, 31.9, 34.5, 34.6, 35.1, 36.5, 36.6, 37.2, 39.7, 42.2,

42.3, 50.1, 55.8, 56.7, 71.8 (C-3), 121.7 (C-6), 140.7 (C-5). GC-MS

(EI): m/z (%) Free alcohol: 422/424 (52/53, M^+.), 409 (19), 407

(27), 405 (10), 404/406 (33/34, M^+.-H₂O), 389/391 (33/33),

362/364 (7/9), 337/339 (49/46), 311/313 (42/40), 283 (17), 273

(32), 255 (46), 231 (37), 213 (57), 145 (80), 133 (51), 121 (50), 121

(61), 109 (43), 107 (98), 105 (100), 95 (94), 93 (80), 91 (89), 79/81

(81/86, Br^+.), 69 (82), 55 (81), 41 (75). TMS ether: 494/496 (21/23,

M^+.), 479/481 (6/6, M^+.-CH₃), 429 (1), 405/407 (11/10, M^+.-

OTMS), 404/406 (32/33), 389/391 (19/15), 365/367 (54/40), 337

(1), 311 (5), 285 (11), 255 (7), 229 (2), 207 (7), 185 (7), 159 (16),

149/151 (6/5), 145 (20), 129 (83), 121 (15), 119 (22), 109 (13), 107

(22), 105 (27), 93/95 (21/28), 79/81 (22/22, Br^+.), 73 (100, TMS^+.),

69 (44), 55 (44), 41 (33). HRMS (ESI): Calc. for C₂₆H₄₄NaO

1

partial H NMR data obtained in CDCl₃has been reported.^{59 1}H

NMR (500 MHz, C₆D₆): δ 0.67 (s, 3H, H₃-18), 0.76-1.86 (m, 28H,

incl. 0.96 [d, 3H, J = 6.6 Hz, H₃-21], 0.98 [s, 3H, H₃-19]), 1.87 (s,

3H, H₃-26), 1.99 (m, 2H), 2.31 (m, 4H), 3.42 (m, 1H, H-3), 5.39

(m, 1H, H-6). ¹³C NMR (125 MHz, C₆D₆): δ 12.0 (C-18), 15.4 (C-

26), 18.9 (C-21), 19.5 (C-19), 21.4, 24.5, 26.1, 28.5, 32.1, 32.2 (C-

20), 32.3, 35.1, 35.4, 35.8, 36.8 (C-10), 37.7, 40.1, 42.5 (C-13),

42.9, 50.5, 56.3, 56.9, 71.6 (C-3), 121.6 (C-6), 141.3 (C-5). GC-MS

(EI): m/z (%) Free alcohol: 390 (65, M^+.), 375 (39, M^+.-CH₃), 373

(24, M^+.-OH), 372 (82, M^+.-H₂O), 357 (42), 330 (10), 305 (37), 279

(33), 273 (28), 255 (52), 213 (77), 199 (25), 185 (21), 173 (26), 161

(55), 145 (74), 131 (47), 117 (100), 105 (91), 95 (83), 81 (86), 69

(75), 61 (60), 55 (73), 41 (67). TMS ether: 462 (35, M^+.), 447 (15,

M^+.-CH₃), 405 (4), 373 (15), 372 (75), 357 (21), 345 (3), 333 (64),

305 (5), 281 (8), 255 (9), 251 (9), 237 (4), 227 (4), 215 (5), 207

(24), 199 (9), 185 (6), 175 (6), 161 (13), 145 (20), 129 (69), 121

(18), 117 (15), 105 (8), 91 (24), 89 (1), 81 (20), 75 (79), 73 (100,

TMS^+.), 69 (26), 61 (20), 55 (23), 47 (12), 45 (13). HRMS (EI):

TMS ether: Calc. for C₂₈H₅₀OSiS (M^+.): 462.3352. Observed:

462.3349.

CH₃), 430 (30), 429 (89, M^+.-tBu), 415 (1), 411 (3), 401 (11), 387

(1), 371 (1, M^+.-TBS), 355 (2, M^+.-OTBS), 353 (3), 337 (5), 295

(3), 281 (4), 267 (2), 255 (11), 241 (6), 227 (8), 213 (11), 199 (10),

185 (9), 173 (12), 159 (30), 145 (20), 133 (20), 131 (17), 121 (19),

115 (6), 105 (28), 99 (5), 95 (34), 81 (44), 75 (100), 71 (3), 67 (19),

57 (11), 55 (29), 43 (11), 41 (13).

A solution of n-BuLi (1.1 M in hexanes, 0.15 mL, 165 mol)

was added dropwise to a solution of methyltriphenylphosphonium

bromide (95 mg, 266 mol) in anhydrous THF (2 mL) stirring

under a N₂atmosphere at –78 C. The resulting orange solution was

stirred at –78 C for 40 min, then allowed to warm to room

temperature and stirred for a further 10 min. The reaction mixture

was re-cooled to –78 C and a solution of aldehyde 36 (25 mg, 51

mol) in anhydrous THF (1 mL) was added dropwise. The orange

reaction mixture was allowed to warm to room temperature and

stirred for a further 12 h. The solvent was then evaporated and the

residue was filtered through a pad of silica gel that was washed

thoroughly with additional EtOAc. The filtrate was concentrated in

vacuo to afford diene 37 (25 mg, 51 mol, quantitative) as a white

4.2.23. (20R)-3-(tert-Butyldimethylsilyloxy)-24-cyanochol-5-

ene (35).

Potassium cyanide (62 mg, 952 mol) was added to a solution of

bromide 34 (140 mg, 260 mol) in anhydrous DMF (4 mL) and the

reaction mixture was stirred under a N₂atmosphere at 80°C for 2 h.

The reaction mixture was then cooled to room temperature and

saturated aqueous Na₂CO₃solution (10 mL) was added to quench

the reaction. The mixture was extracted with EtOAc (3 x 10 mL)

and the combined organic extract was washed with saturated

aqueous Na₂CO₃solution (10 mL) and brine (10 mL), dried over

anhydrous MgSO₄, filtered, and concentrated in vacuo. The crude

product was purified by flash column chromatography (silica gel,

5% EtOAc in petroleum spirit 40-60) to afford nitrile 35 (123 mg,

solid. ¹H NMR (400 MHz, CDCl₃):

δ

0.04 (s, 6H,

ROSi(CH₃)₂C(CH₃)₃), 0.65 (s, 3H, H₃-18), 0.73-1.87 (m, 36H, incl.

0.87 [s, 9H, ROSi(CH₃)₂C(CH₃)₃], 0.90 [d, 3H, J = 6.5 Hz, H₃-21],

0.98 [s, 3H, H₃-19]), 1.90-2.07 (m, 4H), 2.10-2.18 (m, 1H), 2.20-

2.31 (m, 1H), 3.46 (m, 1H, H-3), 4.87-5.02 (m, 2H, H₂-26), 5.29

(m, 1H, H-6), 5.79 (m, 1H, H-25). ¹³C NMR (100 MHz, CDCl₃): δ

–4.6

(2C,

ROSi(CH₃)₂C(CH₃)₃),

11.8

(C-18),

18.3

(ROSi(CH₃)₂C(CH₃)₃), 18.7 (C-21), 19.4 (C-19), 21.1, 24.3, 25.3,

25.4, 25.9 (3C, ROSi(CH₃)₂C(CH₃)₃), 28.2, 31.92 (C-20), 31.94,

32.1, 34.2, 35.5, 35.6, 36.6 (C-10), 37.4, 39.8, 42.3 (C-13), 42.8,

50.2, 56.1, 56.8, 72.6 (C-3), 114.1 (C-26), 121.2 (C-6), 139.3 (C-

25), 141.6 (C-5). GC-MS (EI): m/z (%) 484 (0.2, M^+.), 469 (2, M^+.-

CH₃), 429 (8), 428 (36), 427 (100, M^+.-tBu), 409 (4), 353 (2, M^+.-

OTBS), 351 (11), 281 (2), 255 (3), 235 (3), 207 (4), 159 (12), 145

(8), 135 (7), 131 (6), 121 (7), 115 (3), 105 (11), 97 (6), 95 (19), 81

(19), 75 (79), 69 (7), 67 (10), 57 (4), 55 (21), 41 (7). HRMS (ESI):

Calc. for C₃₂H₅₆NaOSi ([M+Na]^+.): 507.3998. Observed: 507.3984.

1

254 mol, 98%) as a white solid. The full H and ¹³C spectral data

obtained here (Supplementary data) matches that reported for ent-

35.⁵⁷

4.2.24. (20R)-3-(tert-Butyldimethylsilyloxy)-27-norcholesta-

5,25-diene (37).

DIBAL-H (1.0 M in hexanes, 185 L, 185 mol) was added

dropwise to a solution of nitrile 35 (68 mg, 141 mol) in anhydrous

Et₂O (4 mL) stirring under a N₂atmosphere at –78 °C. The reaction

mixture was stirred at –78 °C for 1 h, and then allowed to warm to

room temperature and stirred for a further 4 h. Aqueous oxalic acid

solution (5%, 10 mL) was added to quench the reaction and the

mixture was stirred until clear phase separation was observed. The

organic and aqueous layers were separated and the aqueous solution

was extracted with EtOAc (3 x 10 mL). The combined organic

extract was washed with saturated aqueous Na₂CO₃solution (10

mL) and brine (10 mL), dried over anhydrous MgSO₄, filtered, and

concentrated in vacuo to afford the aldehyde (20R)-3-(tert-

butyldimethylsilyloxy)-24-oxomethylchol-5-ene (36) (52 mg, 107

mol, 76%) as a white solid that was used without further

purification. ¹H NMR (400 MHz, CDCl₃): δ 0.03 (s, 6H,

ROSi(CH₃)₂C(CH₃)₃), 0.65 (s, 3H, H₃-18), 0.73-1.85 (m, 36H, incl.

0.87 [s, 9H, ROSi(CH₃)₂C(CH₃)₃], 0.92 [d, 3H, J = 6.6 Hz, H₃-21]),

0.97 [s, 3H, H₃-19]), 1.89-2.01 (m, 2H), 2.10-2.19 (m, 1H), 2.19-

2.29 (m, 1H), 2.37 (m, 2H, H₂-24), 3.46 (m, 1H, H-3), 5.29 (m, 1H,

H-6), 9.74 (t, 1H, J = 1.8 Hz, H-25). ¹³C NMR (100 MHz, CDCl₃):

4.2.25. (20R)-27-Norcholesta-5,25-dien-3-ol (7).

Dowex 50W-X8(H) standard grade resin (cat.) was added to a

solution of TBS ether 37 (7 mg, 14.4 mol) in a mixture of CH₂Cl₂

(1 mL) and MeOH (1 mL). The reaction mixture was stirred at

room temperature for 90 h, and then the solvent was evaporated.

The residue was purified by flash column chromatography (silica

gel, 5% to 9% EtOAc in n-hexane) to afford sterol 7 (5 mg, 13.5

1

mol, 93%) as a white solid. H NMR (400 MHz, CDCl₃): δ 0.66

(s, 3H, H₃-18), 0.72-2.36 (m, 34H, incl. 0.90 [d, 3H, J = 6.4 Hz, H₃-

21], 0.99 [s, 3H, H₃-19]), 3.51 (m, 1H, H-3), 4.86-5.02 (m, 2H, H₂-

26), 5.33 (m, 1H, H-6), 5.78 (m, 1H, H-25). ¹³C NMR (100 MHz,

CDCl₃): δ 11.8 (C-18), 18.7 (C-21), 19.4 (C-19), 21.1, 24.3, 25.4,

28.2, 31.6, 31.9, 34.2, 35.4, 35.6, 36.5, 36.6, 37.2, 39.8, 42.25,

42.31, 50.1, 56.0, 56.8, 71.8 (C-3), 114.1 (C-26), 121.7 (C-6),

139.3 (C-25), 140.7 (C-5). GC-MS (EI): m/z (%) Free alcohol: 370

(63, M^+.), 355 (28, M^+.-CH₃), 353 (10, M^+.-OH), 352 (33, M^+.-H₂O),

337 (31), 300 (10), 285 (48), 273 (17), 271 (45), 255 (27), 231 (30),

213 (43), 199 (17), 185 (15), 173 (24), 159 (43), 145 (57), 133 (43),

119 (46), 107 (72), 105 (73), 97 (24), 95 (77), 81 (85), 69 (31), 67

(54), 55 (100), 41 (43). TMS ether: 442 (52, M^+.), 427 (13, M^+.-

CH₃), 400 (2), 353 (15, M^+.-OTMS), 352 (48), 337 (32), 313 (28),

284 (6), 255 (7), 207 (10), 159 (11), 145 (15), 129 (76), 119 (17),

δ

–4.6 (2C, ROSi(CH₃)₂C(CH₃)₃), 11.8 (C-18), 18.3

(ROSi(CH₃)₂C(CH₃)₃), 18.6 (C-21), 18.7, 19.4 (C-19), 21.0, 24.2,

25.9 (3C, ROSi(CH₃)₂C(CH₃)₃), 28.2, 31.9 (2C, incl. C-20), 32.1,

35.5, 35.6, 36.6 (C-10), 37.4, 39.8, 42.3 (C-13), 42.8, 44.3, 50.2,

55.8, 56.8, 72.6 (C-3), 121.1 (C-6), 141.6 (C-5), 202.9 (C-25). GC-

MS (EI): m/z (%) 486 (0.2, M^+.), 485 (0.3, M^+.-1), 471 (3, M^+.-

107 (20), 105 (20), 97 (14), 91 (30), 89 (1), 75 (64), 73 (100,

TMS^+.), 69 (18), 55 (94), 41 (37). HRMS (ESI): Calc. for

C₂₆H₄₂NaO ([M+Na]^+.): 393.3133. Observed: 393.3122. HRMS

(EI): TMS ether: Calc. for C₂₉H₅₀OSi (M^+.): 442.3631. Observed:

442.3630.

then cooled to room temperature and filtered through a pad of silica

gel, which was washed thoroughly with EtOAc. The filtrate was

concentrated under reduced pressure to afford chloride 39 (26 mg,

50 mol, quantitative, 1:1 mixture of diastereomers at C-25) as a

white solid. ¹H NMR (400 MHz, CDCl₃): δ 0.04 (s, 6H,

ROSi(CH₃)₂C(CH₃)₃), 0.65 (s, 3H, H₃-18), 0.73-1.87 (m, 41H, incl.

0.87 [s, 9H, ROSi(CH₃)₂C(CH₃)₃], 0.91 [d, 3H, J = 6.5 Hz, H₃-21],

0.98 [s, 3H, H₃-19], 1.48 [br d, 3H, J = 6.5 Hz, H₃-26]), 1.90-2.04

(m, 2H), 2.10-2.18 (m, 1H), 2.20-2.31 (m, 1H), 3.46 (m, 1H, H-3),

4.00 (m, 1H, H-25), 5.29 (m, 1H, H-6). ¹³C NMR (100 MHz,

4.2.26. (20R)-3-(tert-Butyldimethylsilyloxy)-27-norcholest-5-

en-25-ol (38).

CDCl₃) (mixture of diastereomers at C-25):

δ –4.6 (2C,

ROSi(CH₃)₂C(CH₃)₃), 11.8 (C-18), 18.3 (ROSi(CH₃)₂C(CH₃)₃),

18.6 (C-21), 18.7 (C-21), 19.4 (C-19), 21.1, 23.1, 23.2, 24.3, 25.3,

25.5, 25.9 (3C, ROSi(CH₃)₂C(CH₃)₃), 28.2, 31.91 (C-20), 31.92,

32.1, 35.3, 35.4, 35.6, 35.7, 36.6 (C-10), 37.4, 39.8, 40.8, 40.9, 42.3

(C-13), 42.8, 50.2, 55.97, 55.99, 56.8, 58.97 (C-25), 59.01 (C-25),

72.6 (C-3), 121.1 (C-6), 141.6 (C-5). GC-MS (EI): m/z (%) 520/522

(0.4/0.1, M^+.), 505/507 (2/1, M^+.-CH₃), 464/466 (30/11), 463/465

(85/35, M^+.-tBu), 445 (2), 429 (13), 427 (21), 415 (1), 389/391 (3/1,

M^+.-OTBS), 387 (5), 353 (32), 311 (3), 297 (2), 283 (4), 267 (4),

255 (13), 241 (7), 227 (7), 213 (12), 199 (8), 187 (8), 173 (14), 159

(34), 145 (23), 133/135 (23/13), 131 (13), 119 (15), 115 (6), 107

(20), 105 (23), 95/97 (38/13), 93 (22), 91 (16), 81 (35), 79 (14), 77

(7), 75 (100), 69 (17), 65 (17), 65 (1), 63 (1), 57 (15), 55 (36).

Methylmagnesium bromide (3.0 M in Et₂O, 20 L, 60 mol)

was added dropwise to a solution of aldehyde 36 (15 mg, 31 mol)

(see preparation of 37) in anhydrous THF (3 mL) stirring under a

N₂atmosphere at –78 C. The reaction mixture was stirred at –78

C for 1 h, then allowed to warm to room temperature and stirred

for 12 h. Saturated aqueous NH₄Cl solution (10 mL) was added to

quench the reaction and the mixture was extracted with Et₂O (3 x

10 mL). The combined organic extract was washed with brine (10

mL), dried over anhydrous MgSO₄, filtered, and concentrated in

vacuo. The crude product was purified by flash column

chromatography (silica gel, 20% Et₂O in petroleum spirit 40-60) to

afford alcohol 38 (14 mg, 27 mol, 90%, 1:1 mixture of

diastereomers at C-25) as a white solid. Synthesis of 38 via a

different synthetic route has been reported previously,⁵⁰and the full

¹H and ¹³C NMR spectral data obtained here is consistent with the

limited data available in the literature. ¹H NMR (400 MHz, CDCl₃):

δ 0.03 (s, 6H, ROSi(CH₃)₂C(CH₃)₃), 0.65 (s, 3H, H₃-18), 0.73-1.86

(m, 41H, incl. 0.87 [s, 9H, ROSi(CH₃)₂C(CH₃)₃], 0.90 [d, 3H, J =

6.5 Hz, H₃-21], 0.98 [s, 3H, H₃-19], 1.17 [d, 3H, J = 6.2 Hz, H₃-

26]), 1.89-2.04 (m, 2H), 2.10-2.18 (m, 1H), 2.19-2.35 (m, 1H), 3.46

(m, 1H, H-3), 3.78 (m, 1H, H-25), 5.29 (m, 1H, H-6). ¹³C NMR

(100 MHz, CDCl₃) (mixture of diastereomers at C-25): δ –4.6 (2C,

ROSi(CH₃)₂C(CH₃)₃), 11.8 (C-18), 18.3 (ROSi(CH₃)₂C(CH₃)₃),

18.7 (C-21), 19.4 (C-19), 21.1, 22.16, 22.24, 23.5, 23.6, 24.3, 25.9

(3C, ROSi(CH₃)₂C(CH₃)₃), 28.2, 31.90 (C-20), 31.93, 32.1, 35.5

(br), 35.69, 35.70, 35.88, 35.94, 36.6 (C-10), 37.4, 39.8, 42.3 (C-

13), 42.8, 50.2, 56.00, 56.01, 56.8, 68.2 (C-25), 68.3 (C-25), 72.6

(C-3), 121.1 (C-6), 141.6 (C-5). GC-MS (EI): m/z (%) 489 (2), 487

(2, M^+.-CH₃), 476 (2), 459 (1), 446 (22), 445 (65, M^+.-tBu), 443 (3),

429 (4), 428 (7), 427 (20), 415 (1), 401 (2), 387 (2, M^+.-OTBS),

371 (1), 369 (7), 353 (16), 341 (2), 329 (3), 311 (3), 297 (2), 281

(6), 255 (9), 241 (5), 227 (5), 213 (9), 203 (6), 187 (6), 173 (11),

159 (25), 147 (21), 133 (14), 131 (10), 121 (17), 115 (5), 105 (21),

95 (35), 87 (2), 81 (35), 75 (100), 73 (38), 69 (16), 67 (14), 59 (4),

57 (14), 55 (30), 45 (15).

4.2.28. (20R)-25-Chloro-27-norcholest-5-en-3-ol (8).

Dowex 50W-X8(H) standard grade resin (cat.) was added to a

solution of TBS ether 39 (13 mg, 24.9 mol) in a mixture of

CH₂Cl₂(1 mL) and MeOH (1 mL). The mixture was stirred at room

temperature for 90 h, and then the solvent was evaporated. The

residue was purified by flash column chromatography (silica gel,

5% to 9% EtOAc in n-hexane) to afford sterol 8 (9 mg, 22.1 mol,

89%, 1:1 mixture of diastereomers at C-25) as a white solid.

Synthesis of 8 via a different synthetic route has been reported

previously,⁶⁰but no NMR spectral data was reported. ¹H NMR (400

MHz, CDCl₃) (mixture of diastereomers at C-25): δ 0.66 (s, 3H, H₃-

18), 0.73-2.38 (m, 37H, incl. 0.91 [d, 3H, J = 6.8 Hz, H₃-21]), 0.99

[s, 3H, H₃-19], 1.48 [br d, 3H, J = 6.4 Hz, H₃-26]), 3.51 (m, 1H, H-

3), 4.00 (m, 1H, H-25), 5.33 (m, 1H, H-6). ¹³C NMR (100 MHz,

CDCl₃) (mixture of diastereomers at C-25): δ 11.9 (C-18), 18.6 (C-

21), 18.7 (C-21), 19.4 (C-19), 21.1, 23.16, 23.21, 24.3, 25.3, 25.5,

28.2, 31.6, 31.9 (2C, incl. C-20), 35.3, 35.4, 35.6, 35.7, 36.5 (C-

10), 37.2, 39.8, 40.8, 40.9, 42.25, 42.33, 50.1, 55.97, 55.99, 56.7

(br), 59.00, 59.03, 71.8 (C-3), 121.7 (C-6), 140.7 (C-5). GC-MS

(EI): m/z (%) Free alcohol: 406/408 (73/26, M^+.), 393 (9), 391 (27),

389 (12), 388/390 (40/15, M^+.-H₂O), 373/375 (39/14), 346 (8), 321

(58), 295 (58), 273 (30), 255 (43), 231 (34), 213 (64), 199 (22), 161

(47), 159 (46), 147 (40), 145 (63), 133 (45), 123 (22), 121 (41), 119

(48), 109 (37), 107 (76), 105 (72), 95 (73), 93 (58), 91 (60), 81

(76), 79 (54), 67 (47), 55 (100), 41 (36). TMS ether: 478/480 (16/6,

M^+.), 463/465 (7/3, M^+.-CH₃), 443 (1, M^+.-Cl), 442 (3, M^+.-HCl),

415 (1), 401 (1), 389/391 (14/6, M^+.-OTMS), 388 (54), 373 (26),

349 (63), 345 (1), 313 (5), 267 (10), 228 (5), 207 (14), 185 (7), 159

(26), 133/135 (12/8), 129 (67), 95 (37), 91/93 (31/21), 89 (1), 77/79

(12/7), 75 (57), 73 (100, TMS^+.), 63/65 (5/2), 55 (75), 41 (31).

4.2.27. (20R)-3-(tert-Butyldimethylsilyloxy)-25-chloro-27-

norcholest-5-ene (39).

Triethylamine (70 L, 502 mol) and methanesulfonyl chloride

(99%, 9 L, 115 mol) were added sequentially to a solution of

alcohol 38 (25 mg, 50 mol) in anhydrous CH₂Cl₂(2 mL) stirring

under a N₂atmosphere at 0 C. The reaction mixture was stirred at

0 C for 1 h and then allowed to warm to room temperature. H₂O (5

mL) was added to quench the reaction and the mixture was

extracted with EtOAc (3 x 10 mL). The combined organic extract

was washed with brine (10 mL), dried over anhydrous MgSO₄,

filtered, and concentrated in vacuo. The crude mesylate was

dissolved in anhydrous THF (3 mL) and LiCl (30 mg, 708 mol)

was added. The reaction mixture was heated at reflux for 1 h, and

HRMS

(ESI):

Calc.

for

C₂₆H₄₃ClNaO

([M+Na]^+.):

429.2900/431.2871. Observed: 429.2878/431.2872. HRMS (EI):

TMS ether: Calc. for C₂₉H₅₁ClOSi (M^+.): 478.3397/480.3368.

Observed: 478.3397/480.3416.

37.2, 39.8, 41.6, 41.7, 42.27, 42.34, 50.1, 52.0, 52.1, 55.97, 55.99,

56.7 (br), 71.8 (C-3), 121.7 (C-6), 140.7 (C-5). GC-MS (EI): m/z

(%) Free alcohol: 450/452 (30/29, M^+.), 437 (10), 435 (14), 433 (6),

432/434 (18/18, M^+.-H₂O), 417/419 (14/15), 392 (4), 365/367

(25/25), 339/341 (23/22), 311 (7), 285 (7), 273 (27), 271 (21), 255

(35), 231 (30), 213 (46), 199 (16), 179 (6), 177 (9), 159/161

(43/42), 151 (7), 149 (25), 147 (39), 145 (61), 131 (39), 123 (21),

121 (41), 119 (50), 109 (50), 107 (75), 105 (78), 95 (72), 93 (60),

91 (67), 81 (78), 79 (61), 67 (49), 55 (100), 41 (47). TMS ether:

522/524 (28/26, M^+.), 507/509 (11/9, M^+.-CH₃), 488 (5), 460 (2),

449 (2), 433/435 (21/15, M^+.-OTMS), 432/434 (35/39), 415 (2),

393/395 (46/49), 373 (3), 367 (10), 345 (2), 341 (13), 314 (9),

281/283 (16/14), 253/255 (13/13), 229 (8), 207 (54), 177/179 (5/5),

165 (19), 149/151 (6/5), 135/137 (10/11), 129 (100), 121/123 (5/4),

107 (28), 91 (31), 89 (1), 79/81 (3/3, Br^+.), 75 (61), 73 (94, TMS^+.),

55 (81), 40 (36). HRMS (ESI): Calc. for C₂₆H₄₃BrNaO ([M+Na]^+.):

473.2395/475.2375. Observed: 473.2376/475.2386. HRMS (EI):

Calc. for C₂₆H₄₂BrO (M^+.): 450.2497/452.2477. Observed:

450.2499/452.2498.

4.2.29. (20R)-3-(tert-Butyldimethylsilyloxy)-25-bromo-27-

norcholest-5-ene (40).

Triethylamine (40 L, 287 mol) and methanesulfonyl chloride

(99%, 5 L, 64 mol) were added sequentially to a solution of

alcohol 38 (14 mg, 28 mol) in anhydrous CH₂Cl₂(1 mL) stirring

under a N₂atmosphere at 0 C. The reaction mixture was stirred at

0 C for 1 h and then allowed to warm to room temperature. H₂O (5

mL) was added to quench the reaction and the mixture was

extracted with EtOAc (3 x 10 mL). The combined organic extract

was washed with brine (10 mL), dried over anhydrous MgSO₄,

filtered, and concentrated in vacuo. The crude mesylate was

dissolved in anhydrous THF (3 mL) and LiBr (30 mg, 345 (mol)

was added. The reaction mixture was heated at reflux for 1 h, and

then cooled to room temperature and filtered through a pad of silica

gel, which was washed thoroughly with EtOAc. The filtrate was

concentrated under reduced pressure to afford bromide 40 (16 mg,

28 mol, quantitative, 1:1 mixture of diastereomers at C-25) as a

white solid. ¹H NMR (500 MHz, CDCl₃): δ 0.03 (s, 6H,

ROSi(CH₃)₂C(CH₃)₃), 0.65 (s, 3H, H₃-18), 0.73-1.86 (m, 41H, incl.

0.87 [s, 9H, ROSi(CH₃)₂C(CH₃)₃], 0.91 [d, 3H, J = 6.5 Hz, H₃-21],

0.98 [s, 3H, H₃-19], 1.68 & 1.69 [2 overlapping d, 3H, J = 6.6 Hz,

H₃-26]), 1.90-2.01 (m, 2H), 2.11-2.17 (m, 1H), 2.20-2.28 (m, 1H),

3.46 (m, 1H, H-3), 4.11 (m, 1H, H-25), 5.29 (m, 1H, H-6). ¹³C

NMR (125 MHz, CDCl₃) (mixture of diastereomers at C-25): δ –

4.3. UV-vis spectrophotometry

Absolute and difference spectra were recorded using 1 cm path

length quartz cuvettes on

a dual-beam Cary UV-visible

spectrophotometer as described previously.¹¹Equilibrium

dissociation constants were obtained by measuring the

concentration-dependent spin-shifts and were fitted as described

elsewhere.¹¹

4.6

(2C,

ROSi(CH₃)₂C(CH₃)₃),

11.8

(C-18),

18.3

(ROSi(CH₃)₂C(CH₃)₃), 18.6 (C-21), 18.7 (C-21), 19.4 (C-19), 21.0,

24.27, 24.30, 25.9 (3C, ROSi(CH₃)₂C(CH₃)₃), 26.4, 26.6, 28.2,

31.89 (C-20), 31.92, 32.1, 35.2, 35.3, 35.5 (br), 35.6, 35.7, 36.6 (C-

10), 37.4, 39.8, 41.5, 41.7, 42.3 (C-13), 42.8, 50.2, 52.0 (C-25),

52.1 (C-25), 55.96, 55.98, 72.6 (C-3), 121.1 (C-6), 141.6 (C-5).

GC-MS (EI): m/z (%) 564/566 (0.2/0.2, M^+.), 549/551 (1/1, M^+.-

CH₃), 508/510 (17/17), 507/509 (48/51, M^+.-tBu), 433/435 (2/2,

M^+.-OTBS), 429 (22), 427 (33), 353 (36), 311 (4), 283 (3), 271 (2),

255 (11), 241 (6), 227 (5), 213 (10), 199 (7), 187 (7), 177/179 (3/4),

173 (12), 159 (30), 151 (4), 149 (8), 145 (20), 137 (5), 135 (12),

133 (21), 131 (12), 123 (10), 121 (15), 115 (6), 107/109 (18/19),

105 (21), 95 (34), 81 (32), 79 (13), 75 (100), 69 (18), 57 (15), 55

(32).

4.4. Conversion of sterols 2–13 to ketones 2k–13k

Sterols 2–13 were converted to the respective ketones 2k–13k

(cholest-4-en-3-one forms for 2k–10k) by incubation with 0.005

U/L units of cholesterol oxidase and 5 g catalase in 50 mM

potassium phosphate (pH 7.5, 0.5 mL) for 3 h at 25 °C. For GC-MS

analysis, the reaction mixtures were quenched by the addition of 1

N HCl (0.5 mL), then were rapidly mixed and extracted with

hexanes (2 x 5 mL). The combined organic extracts were dried over

anhydrous MgSO₄(~0.5 g), filtered and then dried under a gentle

stream of N₂. In order to derivatize any remaining sterols as their

trimethylsilyl (TMS) ethers, the residues were suspended in 50 L

hexanes and then were treated with 70 L BSTFA and incubated at

37 °C for 2 h prior to GC-MS analysis. For HPLC assays, the

reaction mixtures were quenched by the addition of acetonitrile

containing 0.2% (v/v) formic acid (0.5 mL). The organic phases

were analyzed directly without derivatization. The steroids for

further enzymatic assays were mixed with the appropriate reagents

in situ and were then processed for either HPLC and/or GC-MS

analyses as described above.

4.2.30. (20R)-25-Bromo-27-norcholest-5-en-3-ol (9).

Dowex 50W-X8(H) standard grade resin (cat.) was added to a

solution of TBS ether 40 (10 mg, 17.7 mol) in a mixture of

CH₂Cl₂(1 mL) and MeOH (1 mL). The mixture was stirred at room

temperature for 90 h, and then the solvent was evaporated. The

residue was purified by flash column chromatography (silica gel,

5% to 9% EtOAc in n-hexane) to afford sterol 9 (7 mg, 15.5 mol,

88%, 1:1 mixture of diastereomers at C-25) as a white solid.

Synthesis of 9 via a different synthetic route has been achieved

1

previously,⁶¹and the full H NMR spectral data obtained here is

consistent with the limited data reported. ¹³C NMR data has not

4.5. Enzymatic evaluation of compounds

1

been reported previously. H NMR (400 MHz, CDCl₃) (mixture of

Cytochrome P450 activity assays and product characterization

for each of the sterol analogs with the three enzymes (CYP124A1,

CYP125A1, or CYP142A1) were conducted by incubating 250 nM

P450 with substrate (50 M), 5 g catalase, 50 g/mL ferredoxin,

diastereomers at C-25): δ 0.66 (s, 3H, H₃-18), 0.73-2.38 (m, 37H,

incl. 0.91 [d, 3H, J = 6.4 Hz, H₃-21]), 0.99 [s, 3H, H₃-19], 1.68 &

1.69 [2 overlapping d, 3H, J = 6.8 Hz, H₃-26]), 3.50 (m, 1H, H-3),

4.12 (m, 1H, H-25), 5.33 (m, 1H, H-6). ¹³C NMR (100 MHz,

CDCl₃) (mixture of diastereomers at C-25): δ 11.9 (C-18), 18.6 (C-

21), 18.7 (C-21), 19.4 (C-19), 21.1, 24.27, 24.32, 26.4, 26.6, 28.2,

31.6, 31.9 (2C, incl. C-20), 35.2, 35.3, 35.6, 35.7, 36.5 (C-10),

0.16 U/mL ferredoxin-NADP⁺reductase,

1 mM glucose-6-

phosphate, 2.4 U/mL glucose-6-phosphate dehydrogenase, 5 mM

MgCl₂, and 800 M NADP⁺in 50 mM potassium phosphate (pH

7.5, 0.5 mL) at 25 °C for 1 h. The reaction mixtures were quenched

and the product mixtures derivatised as TMS ethers and analyzed

by GC-MS as described above. Control reactions omitted either the

cytochrome P450 enzyme or the NADPH. For assays conducted

using the ketone derivatives of the sterols, HPLC analysis was also

performed using diode array detection at 240 nm.

4.6. Time-dependent inhibition

27-Norcholesta-5,25-dienol (7) was tested with each of the P450

enzymes as a time-dependent inhibitor by pre-incubating 50 M

compound 7 with 5 M P450 in 50 mM potassium phosphate (pH

7.5, 0.5 mL) containing ferredoxin, ferredoxin-NADP⁺reductase,

catalase, and the NADPH-regenerating system, as described for

catalytic assays. The control pre-incubation reactions were identical

but instead contained only the MCD buffer used to dissolve the

steroids. Over time, portions of the pre-incubated enzyme/inhibitor

or enzyme/control reactions were diluted twenty-fold into fresh

buffer (0.5 mL) containing 50 M cholest-4-en-3-one (1k), spinach

ferredoxin, ferredoxin-NADP⁺reductase and 1 mM NADPH. These

reactions were allowed to proceed for 10 min at 25 °C before being

terminated and analyzed for residual catalytic activity by HPLC.

Residual activity percentages for each time point were calculated

by taking the ratio of enzyme activity towards cholest-4-en-3-one

(1k) remaining for the enzyme pre-incubated with compound 7

versus the control reaction and multiplying by 100.

Table 2. Apparent turnover numbers (k_cat^app) for CYP124A1, CYP125A1 and CYP142A1 with cholesterol side-chain and ring

analogs are listed in the first three columns. The second set of three columns lists the relative turnover numbers (k_rel), normalized to

cholesterol (1).

Apparent turnover number k_cat^app(min^-1)

Relative turnover number (k_rel)

#

Compound name

CYP124A1

CYP125A1

2 8  3^a

CYP142A1

CYP124A1

1 00

CYP125A1

1 00

CYP142A1

1 00

1 .5  0. 2^a

0 .7  0. 1

ND^b

1 6. 7  1 .3^a

1 .9  0. 4

ND^b

1

2

Cholesterol

27-Norcholesterol

4 8. 6

NA^c

1 8. 0

NA^c

1 1. 7

NA^c

5 .1  1. 6

ND^b

3

25-Thia-27-norcholesterol

Cholesta-5,25-dienol

26-Methylcholesta-5,25(26)-dienol

26-Methylcholesterol

27-Norcholesta-5,25-dienol

25-Chloro-27-norcholesterol

25-Bromo-27-norcholesterol

24-Bromochol-5-enol

Desmosterol

3

2 6. 2

2 2. 4

NA^c

3 7. 0

2 9. 9

6 8. 1

8 .0

6 .7

0 .4  0. 03

0 .3  0. 05

ND^b

1 0. 7  1 .4

8 .1  2. 1

1 9. 0  2 .0

2 .2  0. 7

7 .2 9  1 .5

1 8. 22  2 .3

ND^b

1 .1  0. 2

ND^b

5

NA^c

6

5 8. 7

6 .7

9 .8  1. 6

1 .1  0. 3

8 .9 7  1 .4

1 7. 94  1 .7

ND^b

7

1 .9

0 .0 3  0 .00 5

0 .4 5  0 .03 3

1 .0 1  0 .5

ND^b

8

2 9. 8

6 7. 3

NA^c

2 6. 0

6 5. 1

NA^c

5 3. 7

1 07 .4

NA^c

9

10

11

12

13

9 1. 2

6 8. 4

NA^c

4 7. 5

5 0. 9

3 7. 0

4 1. 4

4 3. 1

1 32 .3

1 .3 7  0 .3

1 .0 3  0 .3

ND^b

1 3. 29  2 .2

1 4. 24  2 .1

1 0. 37  1 .6

6 .9 2  1 .1

7 .2  1. 8

2 2. 09  3 .1

Coprostanol

Lanosterol

^aValues from Johnston et al., J. Biol. Chem. 2010, 285, 36352. ^bND = not detected. ^cNA = not applicable.

8.

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Mohn, W. W.; van der Geize, R.; Stewart, G. R.; Okamoto,

Acknowledgments

JBJ was supported by a Heiser Postdoctoral Fellowship for

Research in Leprosy and Tuberculosis. AAS was supported by an

Australian Postgraduate Award (PhD scholarship) and the 2009

Francine Kroesen Travel Fellowship (School of Chemistry and

Molecular Biosciences, The University of Queensland). The

authors thank Hugues Ouellet for samples of CYP125A1 and

CYP142A1. This research was supported by National Institutes of

Health grant AI074824 (PROM).

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Article Doi

Substrate analog studies of the ω-regiospecificity of Mycobacterium tuberculosis cholesterol metabolizing cytochrome P450 enzymes CYP124A1, CYP125A1 and CYP142A1

DOI: 10.1016/j.bmc.2012.05.003

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