Isotope-Labeling Studies Support the Electrophilic Compound i Iron Active Species, FeO3+, for the Carbon-Carbon Bond Cleavage Reaction of the Cholesterol Side-Chain Cleavage Enzyme, Cytochrome P450 11A1

Article abstract of DOI:10.1021/jacs.6b04437

The enzyme cytochrome P450 11A1 cleaves the C20-C22 carbon-carbon bond of cholesterol to form pregnenolone, the first 21-carbon precursor of all steroid hormones. Various reaction mechanisms are possible for the carbon-carbon bond cleavage step of P450 11A1, and most current proposals involve the oxoferryl active species, Compound I (FeO³⁺). Compound I can either (i) abstract an O-H hydrogen atom or (ii) be attacked by a nucleophilic hydroxy group of its substrate, 20R,22R-dihydroxycholesterol. The mechanism of this carbon-carbon bond cleavage step was tested using ¹⁸O-labeled molecular oxygen and purified P450 11A1. P450 11A1 was incubated with 20R,22R-dihydroxycholesterol in the presence of molecular oxygen (¹⁸O₂), and coupled assays were used to trap the labile ¹⁸O atoms in the enzymatic products (i.e., isocaproaldehyde and pregnenolone). The resulting products were derivatized and the ¹⁸O content was analyzed by high-resolution mass spectrometry. P450 11A1 showed no incorporation of an ¹⁸O atom into either of its carbon-carbon bond cleavage products, pregnenolone and isocaproaldehyde. The positive control experiments established retention of the carbonyl oxygens in the enzymatic products during the trapping and derivatization processes. These results reveal a mechanism involving an electrophilic Compound I species that reacts with nucleophilic hydroxy groups in the 20R,22R-dihydroxycholesterol intermediate of the P450 11A1 reaction to produce the key steroid pregnenolone.

Full text of DOI:10.1021/jacs.6b04437

Subscriber access provided by Northern Illinois University
Article
Isotope-Labeling Studies Support an Electrophilic Compound I Iron Active
Species (FeO3+) for the Carbon-Carbon Bond Cleavage Reaction of the
Cholesterol Side Chain Cleavage Enzyme, Cytochrome P450 11A1 (P450SCC)
Francis K Yoshimoto, I-Ji Jung, Sandeep Goyal, F. Peter Guengerich, and Eric Gonzalez
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b04437 • Publication Date (Web): 29 Aug 2016
Downloaded from http://pubs.acs.org on August 29, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of the American Chemical Society is published by the American Chemical
Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 60
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
Isotope-Labeling Studies Support an Electrophilic Compound I Iron Active
Species (FeO3+) for the Carbon-Carbon Bond Cleavage Reaction of the
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Cholesterol Side Chain Cleavage Enzyme, Cytochrome P450 11A1 (P450SCC
)
Francis K. Yoshimoto,1 I-Ji Jung,2 Sandeep Goyal,3 Eric Gonzalez, and F. Peter
Guengerich*
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville,
Tennessee 37232-0146, United States
Address correspondence to:
Prof. F. Peter Guengerich
Department of Biochemistry
Vanderbilt University School of Medicine
638B Robinson Research Building
2200 Pierce Avenue
Nashville, Tennessee 37232-0146, United States
Telephone: 1 (615) 322-2261
FAX:
1 (615) 343-0704
E-mail: f.guengerich@vanderbilt.edu
1
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 60
1
2
3
4
5
1Current address: Department of Chemistry, The University of Texas at San Antonio,
San Antonio, Texas
6
7
8
9
2Current address: Department of Chemistry, Columbia University, New York, New
York
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3Current address: Department of Chemistry and Biochemistry, University of
Arizona, Tucson, Arizona
*Author to whom correspondence should be addressed
Running title: Mechanism of P450 11A1 diol cleavage
2
ACS Paragon Plus Environment

Page 3 of 60
Journal of the American Chemical Society
1
2
3
4
ABSTRACT: The enzyme cytochrome P450 11A1 cleaves the C20,C22 carbon-carbon
5
6
7
8
bond of cholesterol to form pregnenolone, the first 21-carbon precursor of all
steroid hormones. Various reaction mechanisms are possible for the carbon-carbon
bond cleavage step of P450 11A1 and most current proposals involve the oxoferryl
active species, Compound I (FeO3+). Compound I can either (i) abstract an O-H
hydrogen atom or (ii) be attacked by a nucleophilic hydroxy group of its substrate,
20R,22R-dihydroxycholesterol. The mechanism of this carbon-carbon bond cleavage
step was tested using 18O-labeled molecular oxygen and purified P450 11A1. P450
11A1 was incubated with 20R,22R-dihydroxycholesterol in the presence of
molecular oxygen (18O2), and coupled assays were used to trap the labile 18O atoms
in the enzymatic products (i.e., isocaproaldehyde and pregnenolone). The resulting
products were derivatized and the 18O content was analyzed by high-resolution
mass spectrometry. P450 11A1 showed no incorporation of an 18O-atom into either
of its carbon-carbon bond cleavage products, pregnenolone and isocaproaldehyde.
The positive control experiments established retention of the carbonyl oxygens in
the enzymatic products during the trapping and derivatization processes. These
results reveal a mechanism involving an electrophilic Compound I species that
reacts with nucleophilic hydroxy groups in the 20R,22R-dihydroxycholesterol
intermediate of the P450 11A1 reaction to produce the key steroid pregnenolone.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 60
1
2
3
4
INTRODUCTION
5
6
7
8
9
Steroid hormone (e.g. progesterone, mineralocorticoids, glucocorticoids, androgens,
estrogens) biosynthesis begins with the conversion of cholesterol (Scheme 1, 1) to
pregnenolone (Scheme 1, 4), which is catalyzed by cytochrome P450 (P450) 11A1.
This reaction proceeds in three steps (Scheme 1). The first two steps involve
hydroxylation reactions at the C22- and C20- positions of cholesterol, and the third
step is a carbon-carbon (C-C) bond cleavage process. Alternatively, the 20R,22R-
dihydroxycholesterol intermediate (Scheme 1, 3) has been proposed to arise from
cholesterol through a C20,C22-epoxide intermediate, which in turn is hydrolyzed by
water to form the 1,2-diol moiety.1 Another early proposal was that a C20-
hydroperoxide is formed and then is converted to 20R,22R-dihydroxycholesterol.2
However, the C20- and C22- hydroxy groups of 20R,22R-dihydroxycholesterol
(Scheme 1, 3) both come from molecular oxygen, which was shown from an
incubation of cholesterol with adrenal microsomes in the presence of a mixture of
16O2 and 18O2.3 Therefore, the sequential hydroxylation mechanism catalyzed by
Compound I (FeO3+) to form 20R,22R-dihydroxycholesterol (Scheme 1, 1 to 3) is
favored through the 18O-labeling studies. Moreover, through electron paramagnetic
resonance and electron nuclear double resonance (EPR and ENDOR) studies,
Compound I has directly been shown to be the active iron species in both the
hydroxylation and C-C bond cleavage steps.4,5 (The role in the C-C cleavage is
supported by a 50% yield of the product pregnenolone from 20R,22R-
dihydroxycholesterol.5) Despite knowledge that Compound I is the active iron
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
ACS Paragon Plus Environment

Page 5 of 60
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
intermediate in the C20-C22 bond cleavage step (Scheme 1, 3 to 4 and 5), it is still
not clear how Compound I cleaves the side chain of cholesterol.6 Several possible
Compound I mechanisms for this C20-C22 lyase step are viable, and we have
addressed the issue with 18O-labeling studies.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 1. The three steps catalyzed by P450 11A1, which convert cholesterol
to pregnenolone, the first steroid precursor found in steroid hormone
biosynthesis.
Four mechanistic proposals can potentially explain the carbon-carbon bond
cleavage step of P450 11A1 using Compound I as the active iron species (Scheme
2).6-10 The four mechanisms can be divided into two types of reactivity: (i) one
involves the nucleophilic attack of the hydroxy group of the substrate (Scheme 2A, 3)
on the Compound I species of P450 11A1 (Scheme 2A),11 and (ii) the second type
5
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 60
1
2
3
4
5
6
7
8
9
involves the O-H hydrogen atom abstraction of the substrate (Scheme 2B, 3) by
Compound I (Scheme 2B).9 In the latter case, where an O-H hydrogen atom
abstraction occurs, an oxygen atom (e.g. 18O) from molecular oxygen (e.g. 18O2)
would be incorporated into one of the products (Scheme 2B, 4-L or 5-L) after the
oxygen rebound step. On the other hand, in the mechanisms involving nucleophilic
attack of the substrate on Compound I (i.e., “electrophilic Compound I”) no oxygen
atom from molecular oxygen is incorporated into either of the products,
pregnenolone or isocaproaldehyde (Scheme 2A, 4 and 5). Assuming that the
dehydration step (Scheme 2B) is not stereospecific and that the resulting product
isotopologues will be in an equivalent mixture (1:1 of 5:5-L or 4:4-L), we used 18O-
labeling experiments to determine whether P450 11A1 undergoes an electrophilic
Compound I mechanism (Scheme 2A) or an O-H abstraction mechanism (Scheme
2B) in the C-C bond cleavage step to form pregnenolone and isocaproaldehyde from
20R,22R-dihydroxycholesterol (Scheme 2, 3).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
ACS Paragon Plus Environment

Page 7 of 60
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 2. Four Compound I mechanisms grouped into two categories (A and
B) depicting the C-C bond cleavage step of P450 11A1. (A) involves an
electrophilic Compound I species while (B) shows an O-H hydrogen atom
abstraction by Compound I. (A) shows no incorporation of an oxygen into the
products (5 and 4) from molecular oxygen (*O2) and (B) indicates oxygen
incorporation from molecular oxygen into products 5-L and 4-L. *O: 18O.
EXPERIMENTAL SECTION
7
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 60
1
2
3
4
5
6
7
Chemical
Synthesis–General.
The
synthesis
of
20R,22R-
8
9
dihydroxycholesterol was accomplished following a modified route from a
previously reported procedure.12 As done previously, compounds were visualized
using either ceric ammonium molybdate stain (CAM, Hanessian’s stain: ammonium
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
molybdate (4.8% w/v), ceric ammonium molybdate (0.2% w/v), and H
2
SO (6%
4
v/v) in H2O)13 or a UV lamp (254 nm) on TLC plates with fluorescent indicator
(F254). D2O (99.9% enriched) was purchased from Sigma-Aldrich. Bruker NMR
spectrometers (600 MHz or 400 MHz) were used to acquire NMR spectra of
synthesized compounds in the Vanderbilt facility. The proton and carbon chemical
shifts of CDCl3 were referenced to
δ 7.26 ppm and 77.16 ppm, respectively, and
solvent impurities in the spectra of the purified compounds were identified from
previously reported chemical shifts.14 The D2O chemical shift in the proton NMR
spectrum was referenced to
δ 4.79 ppm. Bruker TopSpin 3.2 software was used to
process the NMR spectra. Other experimental details regarding the synthesis of
20R,22R-dihydroxy-[21,21,21-2H3]-cholesterol and 4-methylpentan-1-ol picolinoyl
ester (e.g. NMR spectra and Rf values of synthetic intermediates) are reported in the
Supporting Information. tert-Butyldimethylsilyl (TBDMS) chloride was purchased
from Oakwood Chemical (Columbia, SC). Silica gel (SiliaFlash® F60, for purification
of compounds) was purchased from Silicycle (Quebec, Canada). The ethylpyridine
acetate standard (compound 8d-0) was synthesized according to our previous
study.15
8
ACS Paragon Plus Environment

Page 9 of 60
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
Synthesis of Pregnenolone-3-OTBDMS Ether (Compound 6).
Pregnenolone (4, 5.0 g, 16 mmol), tert-butyldimethylsilyl chloride (6.6 g, 44 mmol,
2.8 mol eq), and imidazole (1.0 g, 15 mmol, 0.9 mol eq) were dissolved in CH3CN
(100 mL). The reaction was stirred for 17 h at room temperature. The reaction
mixture was diluted with H2O (200 mL) and extracted with ethyl acetate (300 mL).
The organic extracts were combined and concentrated. The crude material was
purified via flash column chromatography (100% hexanes to 20% ethyl acetate in
hexanes, v-v) to afford pregnenolone-3-OTBDMS ether 6 (5.7 g, 13 mmol, 84%) as a
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
white solid. 1H NMR (600 MHz, CDCl
) δ 5.32 (broad s, 1H), 3.51-3.45 (m, 1H), 2.53
3
(apparent t, J = 9.0 Hz, 1H), 2.31-2.23 (m, 1H), 2.21-2.14 (m, 2H), 2.12 (s, 3H), 2.07-
2.03 (m, 1H), 2.02-1.96 (m, 1H), 1.84-1.79 (m, 1H), 1.75-1.59 (m, 4H), 1.59-1.52 (m,
3H), 1.52-1.40 (m, 3H), 1.28-1.19 (m, 1H), 1.18-1.11 (m, 1H), 1.06 (td, J1 = 13.1 Hz,
J2 = 3.0 Hz, 1H), 1.00 (s, 3H), 0.99-0.94 (m, 1H), 0.89 (broad s, 9H), 0.62 (s, 3H), 0.06
(s, 6H).
Synthesis of Dithiane (Compound 7). n-Butyl lithium (3.68 mL of a 2.5 M
solution in hexanes, 9.2 mmol, 2.0 mol eq) was added dropwise to dithiane (1.11 g,
9.2 mmol, 2.0 mol eq) in THF (15 mL) at -30 °C under an atmosphere of argon. The
mixture was stirred for 10 min at the same temperature, and the reaction was
cooled to -78 °C. The reaction mixture was stirred for 2 h, and the temperature was
gradually warmed to -20 °C. Pregnenolone-3-OTBDMS ether 6 (2.0 g, 4.6 mmol, 1.0
mol eq) in THF (15 mL) was added at -78 °C and the reaction was stirred for 1 h. The
reaction was quenched with the addition of H2O (100 mL) and stirred for an
9
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 10 of 60
1
2
3
4
5
6
7
8
9
additional 10 min at room temperature. The reaction mixture was diluted with ethyl
acetate (200 mL) and the organic layer was extracted and concentrated under
reduced pressure. The crude material was purified by flash column chromatography
(300 mL increments of: 100% hexanes to 10% ethyl acetate in hexanes (v-v) to 20%
ethyl acetate/hexanes (v-v)) to afford dithiane 7 (550 mg, 0.64 mmol, 14%) as a
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
white solid. Rf 0.50 (hexanes:ethyl acetate, 4:1, v-v). 1H NMR (600 MHz, CDCl
) δ 5.24
3
(m, 1H), 4.07 (broad s), 3.45-3.37 (m, 1H), 2.91-2.85 (m, 1H), 2.85-2.73 (m, 2H),
2.21-2.15 (m, 1H), 2.12-2.06 (m, 1H), 2.06-1.99 (m, 1H), 1.94-1.87 (m, 1H), 1.86-1.70
(m, 3H), 1.69-1.61 (m, 1H), 1.61-1.53 (m, 1H), 1.52-1.39 (m, 4H), 1.36 (s, 3H), 1.27-
1.15 (m, 1H), 1.15-1.06 (m, 1H), 1.02-0.96 (m, 1H), 0.93 (s, 3H), 0.82 (broad s, 5H),
13
0.80 (broad s, 7H), -0.02 (s, 6H); C NMR (150 MHz, CDCl
3) δ 141.3, 120.9, 76.6,
72.4, 61.2, 56.7, 55.0, 50.0, 42.9, 42.7, 40.1, 37.3, 36.5, 32.0, 31.7, 31.4, 31.3, 30.8,
26.0, 25.9, 24.1, 23.7, 21.6, 20.9, 19.4, 18.1, 13.3, -4.6.
Synthesis of Aldehyde (compound 8). A modified procedure previously
reported16 was used. HgCl2 (345 mg, 1.27 mmol, 2.0 mol eq) and HgO (139 mg, 0.64
mmol, 1.0 mol eq) were added to a solution of dithiane 7 (550 mg, 0.64 mmol, 1.0
mol eq) in CH3CN (10 mL) and H2O (0.1 mL). The reaction mixture was heated under
reflux for 2.5 h. The reaction mixture was filtered through a short pad of silica gel
with ethyl acetate (1 L). The filtrate was concentrated and the crude material was
purified via flash column chromatography to afford the aldehyde 8 (90 mg, 0.20
mmol, 31%) as a white solid. 1H NMR (600 MHz, CDCl
) δ 9.56 (s, 1H), 5.31-5.30 (m,
3
1H), 3.50-3.45 (m, 1H), 2.29-2.23 (m, 1H), 2.18-2.11 (m, 2H), 1.99-1.93 (m, 1H),
10
ACS Paragon Plus Environment

Page 11 of 60
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
1.82-1.75 (m, 2H), 1.74-1.68 (m, 2H), 1.68-1.59 (m, 2H), 1.57-1.41 (m, 7H), 1.35 (s,
3H), 1.33-1.22 (m, 9H), 1.22-1.08 (m, 2H), 1.07-1.01 (m, 2H), 1.00 (s, 3H), 0.98-0.91
(m, 2H), 0.88 (s, 9H), 0.81 (d, J = 6.9 Hz, 3H), 0.79 (s, 3H), 0.05 (s, 6H).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis of Grignard Adduct (Compound 9). Isopentylmagnesium
bromide (21.7 mL, 2.5 M solution in diethyl ether, 43 mmol, 20 mol eq) was added
to a solution of aldehyde 8 (1.0 g, 2.2 mmol, 1.0 mol eq) in THF (100 mL) at -78 °C.
The reaction mixture was stirred for 1.5 h. The reaction was diluted with H2O (100
mL) at -78 °C and the reaction mixture was left to warm to room temperature. The
reaction mixture was extracted with CH2Cl2 (2 × 250 mL). The combined organic
extracts were concentrated and purified by flash column chromatography (300 mL
increments of: 100% hexanes to 10% ethyl acetate in hexanes (v-v) to 30% ethyl
acetate in hexanes (v-v)) to yield the diol 9 (220 mg, 0.41 mmol, 19%) as a white
1
solid. H NMR (600 MHz, CDCl
3
) δ 5.32-5.31 (m, 1H), 3.51-3.46 (m, 1H), 3.41-3.37
(m, 1H), 2.29-2.24 (m, 1H), 2.21 (d, J = 3.2 Hz, 1H), 2.17 (ddd, J1 = 13.2 Hz, J2 = 5.1
Hz, J3 = 2.3 Hz, 1H), 2.12 (dt, J1 = 12.5 Hz, J2 = 3.6 Hz, 1H), 2.00-1.95 (m, 1H), 1.85-
1.79 (m, 2H), 1.75-1.69 (m, 1H), 1.68-1.61 (m, 1H), 1.58-1.40 (m, 11H), 1.26-1.22 (m,
2H), 1.22 (s, 3H), 1.21-1.18 (m, 1H), 1.17-1.11 (m, 1H), 1.06-1.03 (m, 1H), 1.02-0.96
(m, 1H), 1.00 (s, 3H), 0.94-0.92 (m, 1H), 0.90 (s, 3H), 0.88 (broad s, 12H), 0.06 (s, 6H).
Synthesis of 20R,22R-Dihydroxycholesterol (Compound 3). To a stirring
solution of diol 9 (220 mg, 0.41 mmol) in CH3OH:CH2Cl2 (40 mL, 1:1, v-v) was added
conc HCl (12 M, 0.02 mL). The reaction was stirred for 15 min and the mixture was
11
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 12 of 60
1
2
3
4
5
6
7
8
9
concentrated under reduced pressure. The resulting crude material was purified by
flash column chromatography (100% hexanes to 50% hexanes/ethyl acetate, v-v) to
yield 20R,22R-dihydroxycholesterol (3, 50 mg, 0.12 mmol, 29%) as a white solid. 1H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
NMR (600 MHz, CDCl ) δ 5.34-5.33 (m, 1H), 3.55-3.46 (m, 1H), 3.39-3.35 (m, 1H),
3
2.33-2.26 (m, 2H), 2.25-2.19 (m, 1H), 2.12 (dt, J1 = 12.7 Hz, J2 = 3.5 Hz, 1H), 1.99-1.94
(m, 1H), 1.94-1.92 (m, 1H), 1.86-1.79 (m, 3H), 1.67-1.59 (m, 2H), 1.58-1.38 (m, 10H),
1.20 (s, 3H, C-21), 1.19-1.10 (m, 3H), 1.10-1.03 (m, 1H), 1.00 (s, 3H, C-19), 0.90 (d, J =
7 Hz, 6H), 0.89 (s, 3H) (1H NMR characterization matched the previously reported
synthesis12); 13C NMR (150 MHz, CDCl
) δ 140.9, 121.7, 77.5, 76.5, 71.8, 56.8, 54.9,
3
50.2, 43.3, 42.4, 40.3, 37.4, 36.6, 36.5, 31.9, 31.7, 31.4, 29.3, 28.2, 24.1, 23.1, 22.5,
22.1, 20.5, 19.5, 13.7.
Synthesis of [21,21,21-2H3]-Pregnenonlone-3-O-tert-butyldimethylsilyl
Ether (Compound 6a). NaH (0.89 g, 35 mmol, 3.1 mol eq) was added as a solid to a
solution of pregnenolone-3-O-tert-butyldimethylsilyl ether 6 (4.9 g, 11 mmol, 1.0
mol eq) in THF (200 mL) at 0 °C. The reaction flask was evacuated and backfilled
with argon. D2O (5 mL) was added and the reaction was allowed to stir for 8 days at
room temperature. The reaction mixture was concentrated and purified by flash
column chromatography (100% hexanes to 10% ethyl acetate in hexanes (v-v) to
30% ethyl acetate in hexanes (v-v)) to afford [21,21,21-2H3]-pregnenonlone-3-O-
tert-butyldimethylsilyl ether (6a, 4.0 g, 9.2 mmol, 84%) as a white solid. The proton
NMR showed a small peak corresponding to the 21-methyl protons (δ 2.12), which
integrated to 0.31 while the 18-methyl protons (δ 0.63) integrated to 3.00 and the
12
ACS Paragon Plus Environment

Page 13 of 60
Journal of the American Chemical Society
1
2
3
4
17-proton (δ 2.53) integrated to 0.96. 1H NMR (600 MHz, CDCl
) δ 5.32-5.31 (m,
3
5
6
7
8
9
1H), 3.52-3.44 (m, 1H), 2.53 (apparent t, J = 8.8 Hz, 1H), 2.31-2.21 (m, 1H), 2.21-2.13
(m, 2H), 2.12 (s, 0.3H), 2.11-2.01 (m, 1H), 2.07-1.96 (m, 2H), 1.82 (dt, J1 = 13.2 Hz, J2 =
3.4 Hz, 1H), 1.76-1.40 (m, 8H), 1.29-1.02 (m, 3H), 1.00 (s, 3H), 0.99-0.93 (m, 1H), 0.89
(s, 9H), 0.63 (s, 3H), 0.06 (s, 6H).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis of Isocaproaldehyde (5) via Swern Oxidation. Dimethyl
sulfoxide (20.2 mL, 284 mmol, 2.9 mol eq) was added to oxalyl chloride (12.6 mL,
147 mmol, 1.5 mol eq) in CH2Cl2 (500 mL) at -78 °C. After 10 min, 4-methylpentan-
1-ol (12.3 mL, 10 g, 97.9 mmol) was added. Triethylamine (41 mL, 294 mmol, 3.0
mol eq) was added with the use of an addition funnel. After 1 h, the reaction was
diluted with H2O (100 mL). The reaction mixture was washed with HCl (1 M, 3 ×
300 mL) and the organic layer was dried with anhydrous MgSO4 and concentrated
to yield isocaproaldehyde (5) as a clear oil (9 g, 90 mmol, 92%). The purity of the
1
crude material was determined by H NMR and used for subsequent experiments
(i.e., gem-diol/aldehyde equilibrium in D2O and yeast alcohol dehydrogenase
1
reduction rates). H NMR (600 MHz, CDCl
3)
δ9.77 (t, J = 2.0 Hz, 1H), 2.42 (td, J1 =
7.4 Hz, J2 = 1.9 Hz, 2H), 1.62-1.54 (m, 1H), 1.52 (apparent q, J = 7.4 Hz, 2H), 0.90 (d. J
= 6.6 Hz, 6H); 13C NMR (150 MHz, CDCl
) δ203.1 (C1), 42.1 (C2), 30.9 (C3, -CH2-),
3
27.8 (C4, -CH-), 22.5 (C5 and C6, -CH3).
Determination
of
the
gem-Diol/aldehyde
Equilibrium
of
1
Isocaproaldehyde. An H NMR spectrum of a solution of isocaproaldehyde in
13
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 14 of 60
1
2
3
4
5
6
7
8
9
potassium phosphate buffer (D2O) was acquired. The following buffer solution was
prepared: 100 mM potassium phosphate in H2O (pH = 7.8, 1 mL) was lyophilized
and D2O (1 mL) was added (pD 7.4).17-19 A solution (0.07 mL) of isocaproaldehyde
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
(2.7 mg/1 mL of D2O) was added to buffer A (0.63 mL) and the H NMR spectrum
was acquired on a 600 MHz NMR spectrometer.
Enzyme Incubations. 18O2 gas (99% 18O), yeast alcohol dehydrogenase (369
units/mg, one unit converted 1
25 °C, CAS: 9031-72-5), glucose 6-phosphate dehydrogenase (Type XV, 210
units/mg, one unit oxidized 1 mol of glucose 6-phosphate to 6-phospho-D-
ꢀ
mol of ethanol to acetaldehyde min-1 at pH 8.8 and
ꢀ
gluconate min-1 in the presence of NADP+ at pH 7.4 at 25 °C, CAS: 9001-40-5), and
NADH were purchased from Sigma-Aldrich. A 310 mM aqueous solution of (2-
hydroxypropyl)-β-cyclodextrin (β-cyclodextrin) was made for solubilization of
substrates in enzymatic incubations.20 An OLIS/Aminco DW2 spectrophotometer
(On-Line Instrument Systems, Bogart, GA) was used to measure P450
concentrations.21
Human Cytochrome P450 11A1 Expression. A codon-optimized P450
11A1 plasmid was purchased from Genewiz (South Plainfield, NJ) (nucleotide
sequence provided in Supporting Information). The P450 11A1 plasmid (pCWori+
vector22) was co-expressed with another plasmid containing the Escherichia coli
molecular chaperone GroEL/ES in E. coli JM109 cells in Luria-Bertani (LB) media
(100 mL) containing ampicillin (100
ꢀg/mL) and kanamycin (50
ꢀg/mL). This pre-
14
ACS Paragon Plus Environment

Page 15 of 60
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
culture was incubated overnight at 37 °C and 220 rpm). The scaled-up expression of
P450 11A1 (i.e., bulk culture) was grown in 4.5 L (6 × 750 mL/2.8 L flask) of Terrific
Broth (TB) media supplemented with the same concentrations of ampicillin and
kanamycin as the pre-culture, along with bactopeptone (2 g/L), trace elements (250
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ꢀ
L/L of culture),23 NaCl (1 mM), thiamine (1 mM), glycerol (4 mL/L), and the pre-
culture (dilution of 1:100, v/v). The bulk cultures were incubated at 37 °C and 225
rpm until the OD600 reached 0.7-0.75 (~6 h). P450 expression was induced by the
addition of isopropyl
β
-D-1-thiogalactopyranoside (IPTG) (1.0 mM),
δ-
aminolevulinic acid (1.0 mM), and arabinose (1.0 mM). The induced cultures were
incubated for 42 h at 28 °C and 190 rpm in a Multifors incubator. The cells were
harvested by centrifugation (3,500 × g for 20 min).
Cytochrome P450 11A1 Purification. All steps were carried out at 4 °C. The
cell pellets from bulk cultures (6 L) were resuspended in 300 mL of TES buffer (100
mM Tris-acetate (pH 7.5) containing 0.5 M sucrose and 0.5 mM EDTA) for each
pellet derived from 1 L of bulk culture. Lysozyme (2 mg/L of bulk culture) was
added and the suspension was incubated on ice for 60 minutes and then centrifuged
at 3,500 × g for 20 min. The spheroplasts were resuspended in sonication buffer (50
mL of 100 mM potassium phosphate (pH 7.5) containing 16% glycerol (v/v), 9 mM
magnesium acetate, 100
ꢀM dithiothreitol (DTT)), 1.0 mM phenylmethylsulfonyl
fluoride (PMSF), and two protease inhibitor tablets (Roche) for each 1 L of bulk
culture. After sonication at 0 °C, the contents were centrifuged at 12,000 × g for 15
min. The recovered supernatant was centrifuged at 140,000 × g for 60 min. The
15
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 16 of 60
1
2
3
4
5
6
pellet was collected, homogenized, and stirred in solubilization buffer (600 mL of
100 mM potassium phosphate buffer (pH 7.5) containing 20% glycerol (v/v), 0.1
7
8
9
mM EDTA, 10 mM
β-mercaptoethanol, 0.5 M KCl, and 1% (w/v) sodium 3-[(3-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) overnight at 4
°C. The resulting solution was centrifuged at 140,000 × g for 60 min, and the
supernatant was loaded onto a Ni-NTA (nitriloacetic acid) column (1.5 × 8 cm),
which had been previously equilibrated with 100 mM potassium phosphate buffer
(pH 7.5) containing glycerol (20%, v/v), 10 mM β-mercaptoethanol, 0.5 M KCl (0.5
M), and sodium CHAPS (1% w/v). The column was washed with the same
equilibration buffer containing 20 mM imidazole and 2.8 mM pentaethylene glycol
monooctyl ether (C8E5) detergent. The concentration of the imidazole was raised to
200 mM in the same buffer to elute P450 11A1. The eluted P450 11A1 fractions
(based on A418) were combined and dialyzed against 2 L of dialysis buffer (100 mM
potassium phosphate (pH 7.5) containing 20% glycerol (v/v)) 3 × for 24 h. The
concentration of P450 11A1 was measured using ferrous vs. ferrous-CO difference
spectra,21 and a typical yield of the protein was 135 nmol. The dialyzed protein was
divided into 150 ꢀL aliquots and stored at -20 °C until use for incubations.
Adrenodoxin (Adx) Expression. E. coli DH5
α cells were transformed with
the plasmid pBA1159 for bovine Adx expression.24-27 A pre-culture was prepared by
inoculating a single colony into 10 mL of LB media containing 100
ꢀg/mL of
ampicillin and grown at 37 °C with gyrorotary shaking at 220 rpm. The pre-culture
(5 mL) was then added to bulk culture medium (500 mL of TB media containing
16
ACS Paragon Plus Environment

Page 17 of 60
Journal of the American Chemical Society
1
2
3
4
ampicillin (100 ꢀg/mL), thiamine (340 ꢀ
g/mL), and trace elements23 (0.025% v/v).
5
6
7
8
9
The cultures were incubated at 37 °C with shaking at 220 rpm for 4 h. IPTG (1 mM)
was added to induce Adx expression. The induced cultures were incubated for 30 °C
for 24 h. The cells were pelleted by centrifugation (3000 × g) for 5 min, and the
pelleted cells were sonicated in 10 mM potassium phosphate buffer (pH 7.4). The
sonicated material was centrifuged at 100,000 × g for 30 min to remove cell debris,
and the supernatant was used for the purification step.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Adrenodoxin (Adx) Purification. The supernatant (vide supra) was loaded
onto a DEAE-Sepharose column (2.5 × 20 cm) that had been equilibrated with 50
mM potassium phosphate buffer (pH 7.4) containing 0.1 mM EDTA and washed with
1 L of the equilibration buffer. The bound Adx was eluted with the same buffer
containing NaCl (170 mM then 300 mM). The fractions that absorbed at 414 nm
from the 170 mM pool were highly purified, based on sodium dodecyl sulfate-
polyacrylamide electrophresis (15%, w/v, gel). The fractions from the 170 mM pool,
(pooled based on A414 readings) were dialyzed twice against 10 mM potassium
phosphate buffer (pH 7.4) containing 0.1 mM EDTA and applied to a DEAE-
Sepharose column (2.5 × 10 cm). The column was washed with the dialysis buffer
and eluted with the same buffer containing NaCl (500 mM). Fractions containing
adrenodoxin were combined based on A414 measurements. The impure fractions
from the DEAE steps were purified further with gel filtration chromatography on a
Sephadex G-75 (2.5 × 100 cm) using 10 mM potassium phosphate buffer (pH 7.4)
17
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 18 of 60
1
2
3
4
containing 0.1 mM EDTA (≤ 20 mL per run). The total yield (from 6 L) was 25.7
5
ꢀmol of Adx, which was stored in 1 mL aliquots at -70 °C.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Adrenodoxin Reductase (ADR) Expression. E. coli DH5
α cells were
transformed with the plasmid containing bovine ADR28 in a pCWori+ expression
vector22,26,27 with a (His)6 tag added to the C-terminus of the cDNA to facilitate
protein purification. LB media (10 mL containing 100
ꢀg/mL ampicillin) was
inoculated with a single colony and incubated overnight at 37 °C with gyrorotary
shaking at 220 rpm. The overnight culture (5 mL) was used to inoculate 500 mL of
TB media containing ampicillin (100
ꢀg/mL), thiamine (340 ꢀg/mL), and trace
elements (0.025%, v/v).23 The cultures were incubated at 37 °C and 220 rpm for 4
h. ADR expression was induced with the addition of 1.0 mM IPTG. following
induction, the induced cultures were incubated at 30 °C and 220 rpm for another 24
h.
ADR Purification. The cell cultures were harvested by centrifugation (3,000
× g) for 10 min. The cell pellet was sonicated in potassium phosphate buffer (100
mM, pH 7.4) containing EDTA (0.1 mM), glycerol (20% v/v), and protease inhibitor
tablets (Roche). The sonicated solution was centrifuged (100,000 × g) for 30 min to
remove the cell debris. The supernatant was loaded on a Ni-NTA column, which was
then washed with the sonication buffer containing 20 mM imidazole. ADR was
eluted with the sonication buffer containing 200 mM imidazole. The eluted ADR was
dialyzed against 40 volumes of 100 mM potassium phosphate buffer (pH 7.4)
18
ACS Paragon Plus Environment

Page 19 of 60
Journal of the American Chemical Society
1
2
3
4
5
6
containing glycerol (20% v/v) and 0.1 mM EDTA three times to remove the
imidazole and then stored at -80 °C.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Cytochrome P450 17A1. Human P450 17A1 was expressed and purified as
described elsewhere.15
Cytochrome b5 and NADPH-P450 Reductase. Recombinant human
cytochrome b529 and NADPH P450 rat reductase30 were expressed in E. coli and
purified as described previously.
P450 11A1 Coupled Assay with Yeast Alcohol Dehydrogenase
(Isocaproaldehyde Analysis). In preliminary assays, we measured a kcat of 4.5 s-1
and a Km (isocaproaldehyde) of 32 µM for the reduction of isocaproaldehyde by
yeast alcohol dehydrogenase (utilizing NADH oxidation at 340 nm). P450 11A1 (450
pmol), Adx (168 pmol), ADR (708 pmol), yeast alcohol dehydrogenase (5 mg) and L-
α-1,2-dilauroyl-sn-glycero-3-phosphocholine (50 µg) were added to a Thunberg
tube containing 800
the addition of either cholesterol (52 nmol) or 20R,22R-dihydroxycholesterol (142
nmol), which was dissolved in a solution of -cyclodextrin (310 mM). The cap of the
Thunberg tube contained the NADPH-regenerating system31 (100
L of the
following mixture: 8.3 mol of glucose 6-phosphate, 4 g of glucose 6-phosphate
dehydrogenase, 333 nmol of NADP+) plus NADH (100
L of a 100 mM solution). The
ꢀL of 100 mM potassium phosphate buffer (pH 7.4) followed by
β
ꢀ
ꢀ
ꢀ
ꢀ
Thunberg tube was purged with argon on a gas train by alternate evacuation with
19
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 20 of 60
1
2
3
4
5
6
7
8
9
reduced pressure and backfilling with argon (repeated 10×). Before injection of 18O2
gas, the tube was evacuated with reduced pressure for 3 minutes. After the 18O2 gas
was injected, the contents of the tube and cap were mixed and the incubation was
shaken at 50 rpm and 37 °C. After 15 min, the tube was placed on ice and CH2Cl2 (5
mL) was added. The organic layer was extracted and taken to dryness under a
stream of N2.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Derivatization with Picolinoyl Chloride. A solution of picolinoyl chloride in
THF (200 ꢀL of a 5 mg/mL solution) was added to the dried incubation extracts
from the P450 11A1-yeast alcohol dehydrogenase-18O2 reaction (vide supra). The
solution was mixed using a vortex device for 5 seconds and dried under a stream of
N2. The samples were dissolved in CH3CN (75 ꢀL) for LC-MS analysis.
P450 11A1 Coupled Assay with P450 17A1 (Pregnenolone Analysis).
P450 11A1 (900 pmol), Adx (840 pmol), ADR (900 pmol), P450 17A1 (5.6 nmol),
cytochrome b5 (11 nmol), NADPH P450 reductase (6.2 nmol), and L-α-1,2-dilauroyl-
sn-glycero-3-phosphocholine (50 µg) were added to a Thunberg tube containing 2
mL of 50 mM potassium phosphate buffer (pH 7.4). Cholesterol (1, 160 nmol in 1.5
ꢀ
mol
β
-cyclodextrin) or 20R,22R-dihydroxy-[21,21,21-2H3]-cholesterol (3a, 190
mol -cyclodextrin) were added to the Thunberg tube. The cap
nmol in 1.5
ꢀ
β
contained the NADPH-regenerating system.31 The Thunberg tube was purged with
argon on a gas train by evacuation with reduced pressure and backfilling with argon
(repeated 10×). Before injection of 18O2 gas, the tube was evacuated with reduced
20
ACS Paragon Plus Environment

Page 21 of 60
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
pressure for 3 minutes. After the 18O2 gas was injected, the contents of the tube and
cap were mixed and the incubation was shaken at 50 rpm at 37 °C. After 60 min, the
tube was placed on ice and CH2Cl2 (5 mL) was added. HCl (3 M, 1 mL, chilled to 0 °C)
was added and the solution was mixed with a vortex device and centrifuged at
1,500 × g for 1 minute. The organic layer was extracted and dried with anhydrous
MgSO4 (~50 mg). The MgSO4 was filtered using a Pasteur pipet with a cotton plug.
Diazo reagent (vide infra) was added to the CH2Cl2 extract of the reaction mixture
and the resulting solution was dried under a stream of nitrogen. The samples were
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
dissolved in CH3CN (75 ꢀL) and analyzed by LC-MS.
Derivatization with Diazo Reagent. The diazo reagent was generated by
the addition of NaOH (2 mL of a 30% w/v solution in H2O) to the nitrosourea
precursor in diethyl ether (~5 mg in 2 mL) in a borosilicate screw cap vial.15,32 The
two layers were mixed with a vortex device and the diethyl ether layer was
extracted and dried with anhydrous MgSO4 (~50 mg). The organic layer was filtered
through a Pasteur pipet with a cotton plug and was used immediately to treat the
incubation extracts prepared above. *Caution: Nitrosourea and diazo compounds are
carcinogenic and explosive. Extra care was used when handling the compounds.
Double-glove protection and proper personal protective equipment (safety glasses and
a laboratory coat) are required. No ground glass joints were used when handling these
compounds. A solution of acetic acid (50 mL) chilled on ice was set aside to rinse all of
the glassware to react any remaining diazo compound generated. All of the
experiments were performed in a fume hood.
21
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 22 of 60
1
2
3
4
5
6
7
Derivatization of P450 11A1/P450 17A1 Incubation Extracts with
8
9
Picolinoyl Chloride. The CH3CN solution remaining after the LC-MS analyses was
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
added to a solution of picolinoyl chloride in THF (200
The solution was mixed using a vortex device for 5 seconds and dried under a
stream of N2. The samples were dissolved in CH3CN (75 L) for LC-MS analysis.
ꢀL of a 5 mg/mL solution).
ꢀ
These samples were used to analyze the deuterium content of 17-
hydroxypregnenolone picolinoyl esters (Supporting Information).
LC-MS Analysis–General. An Acquity UPLC system was used for the LC-MS
analysis of compounds from the enzymatic incubations. Thermo XCalibur Qual
Browser 2.2 software (ThermoFisher Scientific) was used to process LC-MS data. An
LTQ Orbitrap XL mass spectrometer was used to detect derivatized products in the
ESI positive mode with a 100,000 resolution setting. LTQ Tune Plus software was
used to calibrate and tune the instrument. The mass window scanned was m/z 50-
800.
Calibration of the Mass Spectrometer (LTQ Orbitrap XL). The mass
spectrometer was calibrated before each sequence using Pierce® LTQ ESI Positive
Ion Calibration Solution (ThermoFisher) containing a mixture of caffeine, MRFA (the
peptide methionine-arginine-phenylalanine-alanine), and Ultramark 1621 in CH3CN,
CH3CO2H, and CH3OH by direct infusion (10 ꢀL/min). The mass spectrometer was
first tuned for MRFA (m/z 524) and then the tube lens voltage was adjusted to 145 V
22
ACS Paragon Plus Environment

Page 23 of 60
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
to fragment caffeine (m/z 195 to 138). The tune conditions were as follows: Sheath
Gas Flow Rate: 19, Aux Gas Flow Rate: 3, Sweep Gas Flow Rate: 0, Spray Voltage:
4.00 kV, Capillary Temperature: 300 °C, Capillary Voltage: 4.00 V, Tube Lens: 145.00
V. The exact masses used to calibrate the instrument were (m/z) 138.066190,
195.087652, 524.264964, 1121.997024, 1221.990636, 1321.984249, 1421.977862,
1521.971475, 1621.965088, and 1721.958701.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Mass Spectrometer Tune Conditions. The following tune settings were
used to detect the picolinoyl ester of the derivatized isocaproaldehyde product
(compound 15) in the APCI positive mode with the following settings (acquired
after tuning with the synthetic standard, Supporting Information): Vaporizer
Temperature: 450 °C, Sheath Gas Flow Rate: 50, Aux Gas Flow Rate: 5, Sweep Gas
Flow Rate: 5, Discharge Current (ꢀA): 5.00, Capillary Temperature: 275 °C, Capillary
Voltage: 12.00 V, Tube Lens: 50.00 V.
The tune settings used to detect pregnenolone in the APCI positive mode
were as follow: Vaporizer Temperature: 450 °C, Sheath Gas Flow Rate: 50, Aux Gas
Flow Rate: 5, Sweep Gas Flow Rate: 5, Discharge Current: 5.00
Temperature: 275 °C, Capillary Voltage: 43.00 V, Tube Lens: 55.00 V.
ꢀA, Capillary
The pyridine acetate of the derivatized acetic acid product (compound 18)
was detected in ESI positive mode with the following tune settings (acquired after
tuning with the synthetic standard): Sheath Gas Flow Rate: 15, Aux Gas Flow Rate: 5,
23
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 24 of 60
1
2
3
4
5
6
Sweep Gas Flow Rate: 0, Spray Voltage: 4.00 kV, Capillary Temperature: 300 °C,
Capillary Voltage: 16.00 V, Tube Lens: 30.00 V.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The tune settings for detecting picolinoylated pregnenolone and mono-
picolinoylated-20,22-dihydroxycholesterol (Supporting Information) using ESI
positive mode were as follows: Sheath Gas Flow Rate: 8, Aux Gas Flow Rate: 0,
Sweep Gas Flow Rate: 0, Spray Voltage: 5.00 kV, Capillary Temperature: 275 °C,
Capillary Voltage: 24.00 V, Tube Lens: 70.00 V.
Liquid Chromatography (LC) Methods–General. A Phenomenex Kinetex
octylsilane reversed-phase column (2.6 ꢀm, C8, 100 Å, 100 mm × 2.1 mm) was used
to separate all of the analytes. For every LC method, the column temperature was
held at 40 °C. In order to maintain consistent retention times in each run in the
sequence, the column was equilibrated with the initial gradient conditions for at
least 10 min until the delta value (i.e. maximum and minimum difference in
pressure) was <30 psi. Two to three sequential injections of the standards were
made until the retention times remained stable. LC-MS spectra were acquired in full
scan mode: m/z 100-1000 (for yeast alcohol dehydrogenase coupled assays), m/z
50-700 or 50-800 (for P450 17A1 coupled assays). ICIS peak algorithm detection
was used. Peaks in LC-MS data were acquired in profile mode.
LC Method to Detect Picolinoyl Ester of Isocaproaldehyde Derivative
(Compound 15) (Method A). The LC method used to detect the derivatized
24
ACS Paragon Plus Environment

Page 25 of 60
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
isocaproaldehyde product (compound 15) and pregnenolone were run with
solvents A (10 mM ammonium acetate, H2O:CH3CN, 95:5, v/v) and B (10 mM
ammonium acetate, CH3CN) with the following gradient: 95% A from 0-1.0 min,
95% A to 100% B from 1.0-4.0 min, 100% B from 4.0-5.0 min, 100% B to 95% A
from 5.0-5.5 min, 95% A (all v/v) from 5.5-8.0 min with a flow rate of 0.3 mL/min.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
LC Method to Detect Pyridine Acetate Derivatives (compound 18)
(Method B). The LC method to detect the derivatized acetic acid product
(compound 18) and picolinoylated pregnenolone was run with solvents A (10 mM
ammonium formate, H2O) and B (10 mM ammonium formate, CH3CN:H2O, 95:5, v/v)
with the following gradient: 100% A from 0-1.0 min, 100% A to 100% B from 1.0-
4.0 min, 100% B from 4.0-5.2 min, 100% B to 100% A from 5.2-5.3 min, 100% A (all
v/v) from 5.3-8.0 min with a flow rate of 0.3 mL/min.
RESULTS
Overall Strategy. In order to determine if an oxygen atom from molecular
oxygen is incorporated into either of the C-C bond cleavage products of P450 11A1
(Scheme 2,
4
and 5), incubations were performed using 20R,22R-
dihydroxycholesterol in the presence of purified P450 11A1 and 18O2. Because the
carbonyl oxygens in pregnenolone and isocaproaldehyde are readily exchangeable
with water, it was necessary to convert the enzymatic products (Scheme 2, 4 and 5)
25
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 26 of 60
1
2
3
4
5
6
7
8
9
to different chemical entities so that the oxygens would be incapable of exchanging
with water. The derivatized C-C bond cleavage products (4 and 5) would be
detected by LC-MS for analysis of 18O-content.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis of Substrates: 20R,22R-dihydroxycholesterol (3) and Its
Deuterated Isotopologue, 20R,22R-dihydroxy-[21,21,21-2H3]-cholesterol (3a).
The synthesis of 20R,22R-dihydroxycholesterol (3) commenced with pregnenolone
as the starting material (Scheme 1). The 3-hydroxy group was protected as a TBDMS
ether (Scheme 3, 7). One-carbon homologation of pregnenolone TBDMS ether (7)
with a 1,3-dithiane anion nucleophile followed. The resulting dithiane (8) was
converted to the aldehyde (9) with mercury-promoted hydrolysis. Grignard
addition onto the aldehyde with isopentylmagnesium bromide yielded the 1,2-diol
(10). The C3-oxygen was deprotected by removal of the TBDMS group in CH3OH
1
with HCl to furnish 20R,22R-dihydroxycholesterol (3). The H NMR spectrum
matched a previously reported spectrum.33
Additionally, 20R,22R-dihydroxy-[21,21,21-2H3]-cholesterol (3a) was
obtained by synthesizing [21,21,21-2H3]-pregnenolone-3-tert-butyldimethylsilyl
ether (6a) and performing the same steps to access 20R,22R-dihydroxy-[21,21,21-
2H3]-cholesterol from the deuterated silyl ether (Scheme 3). Pregnenolone-3-
OTBDMS (6) was deuterated on the C21-position by reacting with NaH and D2O in
THF at room temperature for eight days (Scheme 3, 6 to 6a). These reaction
conditions resulted in the regioselective deuteration of the C21 position over the
26
ACS Paragon Plus Environment

Page 27 of 60
Journal of the American Chemical Society
1
2
3
4
5
6
C17 position through the formation of the kinetic enolate (see Supporting
Information for 1H NMR spectrum).
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 3. Synthesis of 20R,22R-dihydroxycholesterol (3) and 20R,22R-
dihydroxy-[21,21,21-2H3]-cholesterol (3a) substrates for 18O2-incubation with
P450 11A1.
Gem-Diol/Aldehyde Equilibrium of Isocaproaldehyde (5) in D2O. The
equilibrium between the aldehyde and gem-diol forms of isocaproaldehyde in D2O
was measured by proton NMR. Similar to the case of 19-oxoandrostenedione,13 the
gem-diol and aldehyde were in a 0.9:1.0 ratio in D2O (Figure 1). The fact that
isocaproaldehyde is hydrated in water was important in the context of our 18O2
experiments. The purpose of this study is to distinguish between the two types of
mechanisms presented in Scheme 2 (i.e. electrophilic Compound I and O-H
27
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 28 of 60
1
2
3
4
5
6
7
8
9
abstraction). In other words, in order to determine whether or not P450 11A1
incorporates an 18O atom into isocaproaldehyde from molecular oxygen (18O2) (i.e. if
Scheme 2Ba supported), the aldehyde oxygen must be trapped at a state where it
cannot exchange with H2O.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
Figure 1. H NMR spectrum of isocaproaldehyde (5) in potassium phosphate buffer
in D2O (pD 7.4). The aldehyde (
δ 9.68) and the gem-dihydroxymethine (δ 5.01,
triplet, J = 5.4 Hz) protons are shown (600 MHz).
Design of Coupled Assays to Avoid Exchange of Carbonyl Oxygen Atoms
of Isocaproaldehyde and Pregnenolone with H2O. The carbonyl oxygens in
steroids can be exchanged readily with H2O.13 As shown in the proton NMR
28
ACS Paragon Plus Environment

Page 29 of 60
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
spectrum of isocaproaldehyde (one of the lyase products of P450 11A1) in D2O, the
aldehyde exists in equilibrium in the gem-diol form (Figure 1). Moreover, exchange
kinetics of ketones and aldehydes with water have also been determined
experimentally to occur on a time scale from seconds to minutes (i.e. less than one
hour)34,35 while the incubations in this study were run for 15 to 60 minutes in this
study. Therefore, coupled enzyme assays were necessary to trap the carbonyl
oxygens of the reaction products of P450 11A1, pregnenolone and isocaproaldehyde
(Scheme 4, 4 and 5), so that no exchange would occur with the water medium. Two
types of coupled assays (yeast alcohol dehydrogenase and P450 17A1) were
performed depending on the product (i.e. isocaproaldehyde or pregnenolone)
analyzed.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
29
ACS Paragon Plus Environment