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Supplementary Material
Proteome-wide Mapping of Cholesterol-Interacting Proteins in Mammalian Cells
Jonathan J. Hulce, Armand B. Cognetta, Micah J. Niphakis, Sarah E. Tully,
Benjamin F. Cravatt*
Nature Methods: doi:10.1038/nmeth.2368
Supplementary Table 1.
Please see accompanying Excel file.
Supplementary Table 2.
Please see accompanying Excel file.
Supplementary Table 3.
Please see accompanying Excel file.
Supplementary Table 4.
Please see accompanying Excel file.
Nature Methods: doi:10.1038/nmeth.2368
Supplementary Table 5
Protein name Protein
symbol
Confidence
group
Functional relationship to
cholesterol References
caveolin-1
CAV1 Group I
scaffolding protein of
caveolae
1
tetraspanin-27
CD82 Group I
tetraspanin functions in
cholesterol-rich
microdomains
2
SREBP cleavage
activating protein SCAP Group I
major sterol sensor and
controller of SREBP
3
3-hydroxy-
methylglutaryl CoA
reductase
HMGCR Group I
early cholesterol and
mevalonate biosynthetic
enzyme with sterol sensing
domain
4
progesterone
receptor, membrane
component 1/2
PGRMC1/
2 Group I
membrane components of
progesterone receptors
5
24-
dehydrocholesterol
reductase DHCR24 Group I
catalyzes cholesterol-
producing step of
biosynthesis
6
protein ARV1
ARV1 Group I
involved in transport of
sterols through ER
7
C4-methylsterol SC4MOL Group I cholesterol biosynthetic 8
Nature Methods: doi:10.1038/nmeth.2368
oxidase enzyme; C4-demethylase
sterol-O-Acyl
transferase 1
SOAT1 Group II
transfers fatty acyl groups to
cholesterol hydroxyl to
generate cholesterol esters
9
lanosterol-14-
demethylase CYP51A1 Group II
cholesterol biosynthetic
enzyme; C14-demethylase
10
translocator protein;
peripheral
benzodiazepine
receptor TSPO Group III
translocator protein of
cholesterol from OMM to
IMM
11
NAD(P)-dependent
steroid
dehydrogenase-like NSDHL Group III
cholesterol biosynthetic
enzyme;C4-decarboxylase
12
7-dehydrocholesterol
reductase
DHCR7 Group III
cholesterol biosynthetic
enzyme; 7-8 alkene
reductase
13
cholestenol delta-
isomerase
EBP Group III
cholesterol biosynthetic
enzyme; 8-9 alkene
isomerase
14
Niemann-Pick C1
protein NPC1 Group III
lysosomal cholesterol
trafficking protein
15
Supplementary Table 5. Representative known cholesterol-interacting proteins identified
in Groups I-III. Proteins that have been shown previously to interact with cholesterol/sterols
identified in Groups I-III are presented along with literature references describing these
Nature Methods: doi:10.1038/nmeth.2368
interactions. References in this table: 1) Schroeder F, Huang H, McIntosh AL, Atshaves BP,
Martin GG, Kier AB. Caveolin, sterol carrier protein-2, membrane cholesterol-rich microdomains
and intracellular cholesterol trafficking. Subcell Biochem 51, 279-318 (2010); 2) Charrin S,
Manié S, Thiele C, Billard M, Gerlier D, Boucheix C, Rubinstein E. A physical and functional link
between cholesterol and tetraspanins. Eur J Immunol 33 (2003); 3) Motamed M, Zhang Y,
Wang ML, Seemann J, Kwon HJ, Goldstein JL, Brown MS. Identification of luminal Loop 1 of
Scap protein as the sterol sensor that maintains cholesterol homeostasis. J Biol Chem 286,
18002-18012 (2011); 4) Miao H, Jiang W, Ge L, Li B, Song B. Tetra-glutamic acid residues
adjacent to Lys248 in HMG-CoA reductase are critical for the ubiquitination mediated by gp78
and UBE2G2. Biochim Biophys Sin (Shanghai) 42, 303-310 (2012); 5) Liu L, Wang J, Zhao L,
Nilsen J, McClure K, Wong K, Brinton RD. Progesterone increases rat neural progenitor cell
cycle gene expression and proliferation via extracellularly regulated kinase and progesterone
receptor membrane components 1 and 2. Endocrinology 150, 3186-3196 (2009); 6) Mirza R,
Hayasaka S, Takagishi Y, Kambe F, Ohmori S, Maki K, Yamamoto M, Murakami K, Kaji T,
Zadworny D, Murata Y, Seo H. DHCR24 gene knockout mice demonstrate lethal dermopathy
with differentiation and maturation defects in the epidermis. J Invest Dermatol 123, 638-647
(2006); 7) Tong F, Billheimer J, Shechtman CF, Liu Y, Crooke R, Graham M, Cohen DE, Sturley
SL, Rader DJ. Decreased expression of ARV1 results in cholesterol retention in the
endoplasmic reticulum and abnormal bile acid metabolism. J Biol Chem 285, 33632-33641
(2010); 8) He M, Kratz LE, Michel JJ, Vallejo AN, Ferris L, Kelley RI, Hoover JJ, Jukic D, Gibson
KM, Wolfe LA, Ramachandran D, Zwick ME, Vockley J. Mutations in the human SC4MOL gene
encoding a methyl sterol oxidase cause psoriasiform dermatitis, microcephaly, and
developmental delay. J Clin Invest 121, 976-984 (2011); 9) Wollmer MA, Streffer JR, Tsolaki M,
Grimaldi LM, Lütjohann D, Thal D, von Bergmann K, Nitsch RM, Hock C, Papassotiropoulos A.
Genetic association of acyl-coenzyme A: cholesterol acyltransferase with cerebrospinal fluid
cholesterol levels, brain amyloid load, and risk for Alzheimer's disease. Mol Psychiatry 8, 635-
Nature Methods: doi:10.1038/nmeth.2368
638 (2003); 10) Korosec T, Acimovic J, Seliskar M, Kocjan D, Tacer KF, Rozman D, Urleb U.
Novel cholesterol biosynthesis inhibitors targeting human lanosterol 14alpha-demethylase
(CYP51). Bioorg Med Chem 16, 209-221 (2008); 11) Falchi AM, Battetta B, Sanna F, Piludu M,
Sogos V, Serra M, Melis M, Putzolu M, Diaz G. Intracellular cholesterol changes induced by
translocator protein (18 kDa) TSPO/PBR ligands. Neuropharmacology 53, 318-329 (2007); 12)
McLarren KW, et al. Hypomorphic temperature-sensitive alleles of NSDHL cause CK syndrome.
Am J Hum Genet 87, 905-914 (2010); 13) Nowaczyk MJ, Irons MB, Smith-Lemli-Opitz
syndrome: Phenotype, natural history, and epidemiology. Am J Med Genet C Semin Med Genet
160C, 250-262 (2012); 14) Steijlen PM, van Geel M, Vreeburg M, Marcus-Soekarman D,
Spaapen LJ, Castelijns FC, Willemsen M, van Steensel MA. Novel EBP gene mutations in
Conradi-Hünermann-Happle syndrome. Br J Dermatol 157, 1225-1229 (2007); 15) Infante RE,
Radhakrishnan A, Abi-Mosleh L, Kinch LN, Wang ML, Grishin NV, Goldstein JL, Brown MS.
Purified NPC1 protein: II. Localization of sterol binding to a 240-amino acid soluble luminal loop.
J Biol Chem 283, 1064-1075 (2008).
Nature Methods: doi:10.1038/nmeth.2368
Supplementary Fig. 1
Supplementary Fig. 1. Competition of trans probe with various competitors. Competition
of the trans-sterol probe labeling profile with a panel of competitor lipids at 10x (100 µM); 1:
cholesterol; 2: estradiol; 3: testosterone; 4: ganaxolone; 5: hyodeoxycholic acid; 6: 7--hydroxy
cholesterol; 7: cholesteryl acetate; 8: C17-monoalkylglycerol ether; 9: di-C15-diacylglycerol.
Nature Methods: doi:10.1038/nmeth.2368
Supplementary Fig. 2
Supplementary Fig. 2. Comparison of proteome-labeling profiles for sterol probes by
SILAC ratio histograms. Heavy/light (20 µM trans / 20 µM epi or cis) SILAC ratio histograms
for trans/epi (white bars) and trans/cis (black bars) comparisons, with mean (), and standard
deviation () for each comparison noted.
Nature Methods: doi:10.1038/nmeth.2368
Supplementary Fig. 3
Supplementary Fig. 3. Comparison of cholesterol competition profiles for cis- and trans-
sterol probes. (a) Correlation plot of the SILAC ratio obtained from two independent
experiments: cholesterol competition for the cis probe (heavy: 10 µM cis probe + 100 µM
cholesterol, light: 10 µM cis probe) and cholesterol competition for the trans probe (as described
above); y-axis and x-axis, respectively. Their linear correlation and the resultant confidence cut-
offs (2.0 for cis and 1.5 for trans) are shown with black and red lines respectively. (b) Venn
diagram of proteins competed by cholesterol (ratios above confidence cut-offs in part a) for cis-
versus trans-sterol probes. This analysis was limited to proteins found in both cis and trans
probe cholesterol-competition data sets performed in duplicate and quadruplicate, respectively.
Nature Methods: doi:10.1038/nmeth.2368
Supplementary Fig. 4
Supplementary Fig. 4. Histogram of group I gene expression profiles. Histogram of gene
gene expression data from Supplementary Table 4, including only Group I proteins. The
means () and standard deviations () of each distribution are noted, with '30 min' in black bars
and '12 h' in white.
Nature Methods: doi:10.1038/nmeth.2368
Supplementary Fig. 5
Supplementary Fig. 5. Comparison of protein abundances in unenriched (cell
membranes) versus trans-sterol probe-enriched membrane proteome data sets. Spectral
counts of trans probe targets identified in unenriched membrane fractions were compared to the
their spectral counts in trans-sterol probe enrichments. A positive correlation was not found (r2 =
0.12) between a protein's abundance in unenriched membrane proteomes versus trans probe-
enriched samples. This analysis was limited to proteins that showed 10+ mean spectral counts
across quadruplicate runs in either mode of analysis, and to those with known or predicted
transmembrane domains.
Nature Methods: doi:10.1038/nmeth.2368
Supplementary Fig. 6
Supplementary Fig. 6. Experimental validation of novel protein-cholesterol interactions.
Candidate cholesterol-binding proteins were over-expressed by transient transfection of their
corresponding cDNAs under a CMV promoter in HeLa cells and compared to a sample
Nature Methods: doi:10.1038/nmeth.2368
transfected with a distinct protein as a control (Ctrl). For each recombinantly expressed Group I
protein, three profiles are presented: top) fluorescence-scan of trans-sterol probe labeling
with/without 10X cholesterol competition; middle) western blot with commercial antibody specific
to the protein; and bottom) anti-actin western blot loading control. Representative MS1 traces
for each protein from our chemoproteomic experiments that led to their assignment as Group I
proteins are shown. See Supplementary Fig. 7 for full blots and gels of cropped images shown
in this Supplementary Figure.
Nature Methods: doi:10.1038/nmeth.2368
Supplementary Fig. 7
Supplementary Fig. 7. Full-length blots and gels found in Supplementary Fig. 6.
Recombinantly expressed proteins of interest are noted in each blot and gel with blue dashes.
Each lane designated as control ('Ctrl') is from the same gel or blot as the Group I protein of
interest, scanned at the same intensity or exposure, and represents a transfection with a distinct
protein or with an empty vector (in the case of PGRMC1).
Nature Methods: doi:10.1038/nmeth.2368
Supplementary Note. Synthesis of chemical probes.
Materials
Hyodeoxycholic acid (98%) was obtained from Alfa Aesar. Undecanolide (98%) was obtained from
Sigma-Aldrich. All 1H and 13C NMR spectra were obtained on a Bruker 500 MHz or a Bruker 600 MHz
Cryo Probe. Coupling constants (J) are reported in hertz, and key characteristic peaks were used to
verify C3 and C5 stereochemistries along with crystal structures. The C3 proton was found to present as
a sharp multiplet ('sm') with a chemical shift >4.0 ppm when in an equatorial orientation, as in the epi
probe. This proton is found to be a broad multiplet ('bm') with a chemical shift <4.0 ppm when in an axial
position, as in the cis and trans probes. Finally, the largely irresolvable alkyl proton region (gen. between
0.8 and 2.0 ppm) is designated as many multiplets ('mm') in each spectrum. HRMS service was
performed by the TSRI Center for Mass Spectrometry. X-ray diffraction structures were obtained by the
Small Molecule X-ray Facility at University of California, San Diego. Three-dimensional crystal structures
of each intermediate were deposited in the Cambridge Crystallographic Database Centre (CCDC) under
the accession numbers CCDC 917972-917974 (trans, epi, cis).
Nature Methods: doi:10.1038/nmeth.2368
Synthesis of sterol probes
(4R)-4-((3R,5R,10R,13R,17R)-3-hydroxy-10,13-dimethyl-6-oxohexadecahydro-1H-
cyclopenta[]phenanthren-17-yl)pentanoic acid (1): To a stirred solution of hyodeoxycholic acid (10.0
g, 25.5 mmol, 1 equiv) in glacial acetic acid (135 ml) was added an aqueous solution of potassium
chromate (4.95 g, 1 equiv, in 12 ml) dropwise at room temperature. The resulting solution was stirred at
room temperature overnight, before being diluted slowly with sat. aq. NaHCO3 (100 ml) and water (200
ml) on ice. The resulting suspension was stirred to room temperature for 1 h before being extracted with
CH2Cl2 (3x). The combined organic extracts were dried over Na2SO4 and concentrated under reduced
pressure. The residue was purified by SiO2 flash chromatography (5% MeOH/ CH2Cl2) to provide the title
compound (6.18 g, 62%). The title compound was recrystallized from aqueous MeOH by vapor diffusion
to obtain a diffraction-quality crystal, and its structure was determined by X-ray diffraction (Fig 1b,
purple). 1H NMR (600 MHz, CDCl3) 3.64 (bm, 1H), 2.39 (m 1H), 2.27 (m, 1H), 2.18 (m, 1H), 2.12 (m,
1H), 2.04 (m, 1H), 1.0-1.91 (mm, 23H), 0.94 (d, J = 6 Hz, 3H), 0.84 (s, 3H), 0.65 (s, 3H); 13C NMR (150
MHz, CDCl3) 214.7, 179.4, 71.1, 60.2, 57.7, 56.6, 44.0, 43.8, 40.8, 40.5, 39.5, 37.9, 36.1, 35.8, 35.5,
Nature Methods: doi:10.1038/nmeth.2368
31.5, 31.0, 28.9, 24.8, 24.0, 21.7, 19.1, 12.8, 0.9; HRMS (ESI-TOF+) m/z calc’d for C24H39O4 [M+H]+:
391.2843, found 391.2846.
(4R)-methyl 4-((3R,5S,10R,13R,17R)-3-hydroxy-10,13-dimethyl-6-oxohexadecahydro-1H-
cyclopenta[]phenanthren-17-yl)pentanoate (2): Intermediate 1 (1.0 g, 2.56 mmol, 1 equiv) was
dissolved in anhydrous 2 N HCl in MeOH (10 ml), and the resulting solution was allowed to equilibrate
overnight. At this time, the solution was neutralized carefully with sat. aq. NaHCO3 (20 ml) and the mixed
diastereomers were extracted with CH2Cl2 (3x) . The combined organic extracts were dried over Na2SO4
and concentrated in vacuo. The title compound was obtained (537 mg, 52%) as a single diastereomer
(>95% by 1H NMR) after two recrystallizations from aqueous MeOH. 1H NMR (600 MHz, CDCl3) 4.16
(sm, 1H), 3.66 (s, 3H), 2.71 (m, 1H) 2.30 (m, 1H), 2.28 (m, 1H), 2.21 (m, 1H), 2.00 (m, 2H), 1.87 (m, 1H),
1.79 (m, 2H), 1.70 (m, 2H), 1.0-1.69 (mm, 17H), 0.92 (d, J = 6 Hz, 3H), 0.72 (s, 3H), 0.65 (s, 3H); 13C
NMR (150 MHz, CDCl3) 213.6, 175.5, 66.3, 57.6, 56.7, 54.6, 52.5, 52.4, 48.7, 44.6, 42.2, 40.1, 38.8,
35.7, 32.5, 31.9, 31.8, 28.8, 28.6, 28.5, 24.8, 21.9, 19.1, 13.2, 12.9; HRMS (ESI-TOF+) m/z calc’d for
C25H40O4 [M+H]+: 405.2999, found 405.2998.
(4R)-4-((3R,5S,10R,13R,17R)-3-hydroxy-10,13-dimethyl-6-oxohexadecahydro-1H-
cyclopenta[]phenanthren-17-yl)pentanoic acid (3): The title compound was prepared using the base
hydrolysis procedure described in the preparation of 6. Intermediate 2 (76.1 mg, 0.171 mmol,1 equiv)
yielded 3 (63 mg, 94%), which was used without further purification. A diffraction-quality crystal was
obtained by recrystallization of the crude material from CH2Cl2 and pentane by vapor diffusion and the
structure of the title compound was determined by X-ray diffraction (Fig 1b, cyan). 1H NMR (600 MHz,
D3OD/CDCl3) 4.03 (sm, 1H), 2.74 (m, 1H), 2.20 (m, 2H), 2.07 (m, 2H), 1.88 (m, 1H), 1.78 (m, 2H), 1.0-
1.67(mm, 20H), 0.96 (d, J = 6 Hz, 3H), 0.72 (s, 3H), 0.70 (s, 3H); 13C NMR (150 MHz, D3OD/CDCl3)
215.1, 175.0, 65.5, 57.4, 56.9, 54.5, 52.3, 47.1, 42.1, 40.3, 38.9, 36.6, 35.8, 33.3, 32.3, 28.5, 28.2, 27.9,
24.4, 21.6, 18.3, 12.1, 11.9; HRMS (ESI-TOF+) m/z calc’d for C24H38O4 [M+H]+: 391.2843, found
391.3843.
Nature Methods: doi:10.1038/nmeth.2368
(4R)-methyl 4-((3R,5S,10R,13R,17R)-10,13-dimethyl-3-((methylsulfonyl)oxy)-6-oxohexadecahydro-
1H-cyclopenta[]phenanthren-17-yl)pentanoate (4): To a stirred solution of 2 (537 mg, 1.33 mmol, 1
equiv) in CH2Cl2 (7 ml) at 0 oC was added Hunig's base (515 l, 3 equiv), catalytic DMAP and mesyl
chloride (160 l,1.5 equiv; dropwise). The resulting solution was warmed to room temperature and stirred
for 4 h before dilution with CH2Cl2 (10 ml) and subsequently quenched by vigorous stirring in water (10
ml). The phases from the quenched reaction were separated, and the organic phase was washed with
sat. aq. NaHCO3, dried over Na2SO4 and concentrated in vacuo. The title compound (640 mg, 99%) was
obtained following purification by SiO2 flash chromatography (40% EtOAc/hexanes). 1H NMR (600 MHz,
CDCl3) 5.04 (sm, 1H), 3.66 (s, 3H), 2.99 (s, 3H), 2.63 (m, 1H), 2.38 (m, 1H), 2.31 (m, 1H), 2.25 (m, 1H),
1.0-2.04 (mm, 22H), 0.93 (d, J = 6 Hz, 3H), 0.74 (s, 3H), 0.66 (s, 3H); 13C NMR (150 MHz, CDCl3) 211.2,
174.7, 78.8, 56.7, 55.7, 53.5, 51.9, 51.5, 46.6, 43.0, 41.1, 38.5, 37.9, 37.2, 35.3, 31.7, 31.0, 30.9, 27.9,
26.2, 25.7, 23.3, 22.2, 18.2, 12.5, 12.0; HRMS (ESI-TOF+) m/z calc’d for C26H42O6S [M+Na]+: 505.2594,
found 505.2597.
(4R)-methyl 4-((3S,5S,10R,13R,17R)-3-acetoxy-10,13-dimethyl-6-oxohexadecahydro-1H-
cyclopenta[]phenanthren-17-yl)pentanoate (5): To a stirred solution of 4 (640 mg, 1.33 mmol, 1
equiv) in toluene (2.8 ml) was added a pre-mixed solution of DBU (640 l, 3 equiv) and glacial acetic acid
(504 l, 6 equiv) in toluene (1 ml) dropwise. The resulting solution was heated with stirring at 80 oC for 6
h. The solution was then cooled to room temperature, diluted with EtOAc (10 ml), and washed first with
aq. 1 N HCl, water, and then with sat. aq. NaHCO3. The resulting organic phase was dried (Na2SO4) and
concentrated in vacuo. The remaining residue was purified by SiO2 flash chromatography (20%
EtOAc/hexanes) to provide the title compound (503 mg, 85%). 1H NMR (600 MHz, CDCl3) 4.67 (bm,
1H), 3.66 (s, 1H), 2.3 (m, 4H), 2.04 (m, 1H), 2.02 (s, 3H), 1.0-2.0 (mm, 23H), 0.93 (d, J = 6 Hz, 3H), 0.76
(s, 3H), 0.66 (s, 3H); 13C NMR (150 MHz, CDCl3) 210.3, 174.7, 170.6, 72.8, 56.6, 56.5, 55.7, 53.8, 51.5,
46.6, 43.2, 41.0, 39.4, 37.8, 35.3, 34.8, 31.0, 30.9, 26.8, 26.1, 26.0, 24.9, 24.8, 23.9, 18.2, 13.0, 12.0;
HRMS (ESI-TOF+) m/z calc’d for C27H42O5 [M+H]+: 447.3105, found 447.3108.
Nature Methods: doi:10.1038/nmeth.2368
(4R)-4-((3S,5S,10R,13R,17R)-3-hydroxy-10,13-dimethyl-6-oxohexadecahydro-1H-
cyclopenta[]phenanthren-17-yl)pentanoic acid (6): To a solution of 5 (503 mg, 1.12 mmol, 1 equiv)
in THF (2.8 ml) was added 2 N aqueous LiOH (2.8 ml). The resulting mixture was stirred vigorously
overnight and then neutralized by the addition of 1 N aq. HCl (12 ml) on an ice bath. The product was
extracted with CH2Cl2 (3x), dried over Na2SO4, concentrated under reduced pressure. The remaining
residue was recrystallized once from aqueous MeOH to provide the title compound (250 mg, 57%). The
title compound was recrystallized from aqueous EtOH by vapor diffusion to obtain a diffraction-quality
crystal, and its structure was determined by X-ray diffraction (Fig 1b, green). 1H NMR (600 MHz, CDCl3)
3.54 (bm, 1H), 2.37 (m, 1H). 2.29 (m, 1H), 2.22 (m, 2H), 2.03 (m, 1H), 1.96 (m, 1H), 1.76-1.92 (mm, 6H),
1.62 (m, 1H), 1.54 (m, 1H), 1.0-1.52 (mm, 14H), 0.94 (d, J = 6 Hz, 3H), 0.75 (s, 3H), 0.67 (s, 3H); 13C
NMR (150 MHz, CDCl3) 212.0, 177.0, 70.3, 56.8, 56.7, 55.9, 53.9, 49.5, 46.7, 43.1, 41.4, 39.5, 38.0,
36.7, 35.3, 31.2, 31.0, 30.7, 29.2, 24.8, 21.7, 18.2, 13.1, 12.0; HRMS (ESI-TOF+) m/z calc’d for C24H38O4
[M+H]+: 391.2843, found 391.2848.
(4R)-hex-5-yn-1-yl 4-((3S,5S,10R,13R,17R)-3-hydroxy-10,13-dimethyl-
1,2,3,4,5,7,8,9,10,11,12,13,14,15,16,17-hexadecahydrospiro[cyclopenta[]phenanthrene-6,3'-
diazirin]-17-yl)pentanoate (trans probe) (7): A round bottom flask containing 6 (100 mg, 0.256 mmol,
1equiv) was cooled to 0 oC under N2, 7 N NH3 in MeOH (2.5 ml) was added slowly, and the resulting
solution was stirred at 0 oC for 3 h. At this time, an anhydrous methanolic solution of hydroxylamine-O-
sulfonic acid (41 mg, 1.4 eq, in 0.3 ml) was added dropwise at 0 oC. The resulting solution was allowed to
stir to room temperature overnight, and became increasingly turbid. The following day, the mixture was
evaporated to dryness in the reaction vessel under a stream of dry N2, and the resulting residue was then
resuspended in anhydrous MeOH. The mixture was filtered, and the filter cake washed with additional
dry MeOH. The total filtrate was then concentrated under reduced pressure, and re-dissolved in dry
methanol (2.5 ml) in an amber flask. The solution was cooled to 0 oC, and Hunig's base was added (0.1
ml), followed by iodine in small portions, until a dark brown color persisted in the solution for more than 30
minutes, indicating total oxidation of the previously formed diaziridine. The solution was then diluted with
EtOAc, and washed successively with 1 N aq. HCl and then sat. aq. Na2S2O3 until the organic phase was
Nature Methods: doi:10.1038/nmeth.2368
clarified (2x). The organic phase was then dried (Na2SO4) and concentrated in vacuo in an amber flask to
yield the crude diazirine acid, which was immediately esterified without further purification. The crude
residue was dissolved in CH2Cl2 (2 ml), cooled to 0 oC, and 5-hexyn-ol (75 mg, 3eq), a catalytic amount of
DMAP, followed by DCC (69 mg, 1.3 eq) were added. The resulting solution was stirred to room
temperature overnight and then filtered and concentrated under reduced pressure. The trans probe was
then obtained (41 mg, 33% over three steps) by SiO2 flash chromatography (92.5:7.5:0.5,
CH2Cl2:EtOAc:MeOH). 1H NMR (600 MHz, CDCl3) 4.08 (t, J = 6 Hz, 2H), 3.47 (bm, 1H), 2.34 (m, 1H),
2.21 (m, 3H) 2.00 (m, 1H), 1.96 (sm, 1H), 0.75-1.75 (mm, 24H), 1.1 (s, 3H), 0.92 (d, J = 6 Hz, 3H), 0.79
(m, 2H), 0.69 (s, 3H), 0.42 (m, 2H); 13C NMR (150 MHz, CDCl3) 174.3, 83.9, 71.0, 68.7, 63.7, 56.0, 55.8,
53.6, 45.2, 43.1, 39.9, 37.5, 37.4, 36.3, 33.8, 33.1, 31.5, 31.4, 31.3, 29.2, 27.8, 27.7, 25.0, 24.9, 23.9,
21.2, 18.2, 18.1, 13.0, 12.1; HRMS (ESI-TOF+) m/z calc’d for C30H46N2O3 [M+H]+: 483.3581, found
483.3575.
Nature Methods: doi:10.1038/nmeth.2368
(4R)-hex-5-yn-1-yl 4-((3S,5R,10R,13R,17R)-3-hydroxy-10,13-dimethyl-
1,2,3,4,5,7,8,9,10,11,12,13,14,15,16,17-hexadecahydrospiro[cyclopenta[]phenanthrene-6,3'-
diazirin]-17-yl)pentanoate (cis probe) (8): In the same manner as described above for the preparation
of the trans probe, the cis probe (47.2 mg, 26% over three steps) was obtained from keto-acid 1 (150 mg,
0.384 mmol). 1H NMR (600 MHz, CDCl3) 4.08 (t, J = 6 Hz, 2H), 3.48 (bm, 1H), 2.33 (m, 1H), 2.24 (m,
2H), 2.21 (m, 1H), 2.02 (m, 1H), 1.95 (sm, 1H), 1.92 (m, 1H), 0.8-1.9 (mm, 25H), 1.16 (s, 3H), 0.92 (d, J =
6 Hz, 3H), 0.67 (s, 3H), 0.20 (m, 2H); 13C NMR (150 MHz, CDCl3) 175.2, 84.7, 71.3, 69.6, 64.6, 57.0,
56.7, 49.7, 43.6, 40.7, 40.6, 37.2, 36.1, 35.4, 34.8, 34.1, 33.8, 32.0, 31.8, 30.6, 28.9, 28.6, 25.8, 24.7,
24.0, 21.4, 18.9, 18.8, 12.9, 0.85; HRMS (ESI-TOF+) m/z calc’d for C30H46N2O3 [M+H]+: 483.3581, found
483.3588.
Nature Methods: doi:10.1038/nmeth.2368
(4R)-hex-5-yn-1-yl 4-((3R,5S,10R,13R,17R)-3-hydroxy-10,13-dimethyl-
1,2,3,4,5,7,8,9,10,11,12,13,14,15,16,17-hexadecahydrospiro[cyclopenta[]phenanthrene-6,3'-
diazirin]-17-yl)pentanoate (epi probe) (9): In the same manner as described above for the preparation
of the trans probe, the epi probe (17.2 mg, 23% over three steps) was prepared from keto-acid 3 (63 mg,
0.156 mmol). 1H NMR (600 MHz, CDCl3) 4.08 (t, J = 6 Hz, 2H), 3.95 (sm, 1H), 2.33 (m, 1H), 2.21 (m,
3H), 2.00 (m, 1H), 1.95 (sm, 1H), 0.8-1.9 (mm, 26H), 1.11 (s, 3H), 0.92 (d, J = 6 Hz, 3H), 0.69 (s, 3H),
0.57 (m, 1H), 0.39 (m, 1H); 13C NMR (150 MHz, CDCl3) 174.3, 83.9, 68.7, 65.2, 63.7, 56.0, 55.7, 53.9,
43.1, 39.7, 39.5, 38.2, 37.4, 35.2, 33.8, 31.5, 31.2, 30.9, 30.6, 29.2, 28.0, 27.7, 27.4, 24.9, 23.8, 20.8,
18.2, 18.1, 12.1, 12.0; HRMS (ESI-TOF+) m/z calc’d for C30H46N2O3 [M+H]+: 483.3581, found 483.3568.
Nature Methods: doi:10.1038/nmeth.2368
Synthesis of PEA-DA probe
Methyl 11-hydroxyundecanoate (10). Undecanolide (1.0 g, 5.4 mmol, 1.0 equiv) was added to 5%
H2SO4 in MeOH (20 mL). The reaction mixture was refluxed for 2 h and subsequently cooled to room
temperature. The product was extracted with Et2O (100 mL, 3x) and combined organic layers were
washed successively with H2O (100 mL, 2x), sat. NaHCO3 (100 mL, 2x) and brine (100 mL, 1x). The
Nature Methods: doi:10.1038/nmeth.2368
organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to provide the
crude methyl ester product (1.15 g, 98%), which was used in subsequent steps without further
purification: 1H NMR (500 MHz, CDCl3) 3.62 (s, 3H), 3.58 (t, J = 6.67 Hz, 2H), 2.26 (t, J = 7.55 Hz, 2H),
1.99 (bs, 1H), 1.60 – 1.48 (m, 4H), 1.33 – 1.21 (m, 12H); 13C NMR (150 MHz, CDCl3) 174.34, 62.79,
51.37, 33.99, 32.63, 29.40, 29.29, 29.24, 29.11, 29.00, 25.63, 24.82; HRMS (ESI-TOF+) m/z calc’d for
C12H24O3 [M+H]+: 217.1798, found 217.1795.
Methyl 11-hydroxy-16-(trimethylsilyl)hexadec-15-ynoate (11). Swern Oxidation: A solution of oxalyl
chloride (0.72 mL, 8.3 mmol, 2.0 equiv) in dry CH2Cl2 (50 mL) was cooled to –78 ºC. DMSO (1.18 mL,
16.6 mL, 4.0 equiv) was added dropwise and the reaction mixture was stirred for 15 min. Methyl 11-
hydroxyundecanoate (900 mg, 4.16 mmol, 1.0 equiv) was then added and the reaction was stirred for
another 15 min. Triethylamine (2.3 mL, 16.6 mmol, 4.0 equiv) was then added to the reaction mixture.
After 15 min, the reaction mixture was warmed to 0 ºC and stirred at that temperature for 30 min. The
reaction mixture was then passed over a silica plug with EtOAc:Hex (1:2) and the eluent concentrated
under reduced pressure. The remaining residue (822 g, 92%) was used immediately without further
purification.
Preparation of Grignard reagent: To a flame dried, two-necked round-bottomed flask fitted with a reflux
condenser containing magnesium turnings (418 mg, 17.2 mmol, 1.5 equiv) and anhydrous THF (20 mL)
was added iodine (~10 mg). The mixture was stirred at room temperature until the solution had become
clear. A few drops of a solution of (5-chloropent-1-yn-1-yl)trimethylsilane (2.0 g, 11.4 mmol, 1.0 equiv) in
THF (5 mL) was added to the reaction mixture and the reaction was subsequently warmed to reflux with
stirring. The remaining portion of the (5-chloropent-1-yn-1-yl)trimethylsilane solution was then added
dropwise. When the addition was complete, the reaction was refluxed for another 2 h and subsequently
cooled to room temperature. The concentration of Grignard reagent was determined to be 0.5 M.
Grignard reaction: To a stirring solution of methyl 11-oxoundecanoate (772 mg, 3.60 mmol, 1.0 equiv) in
anhydrous THF (40 mL) at –78 ºC was added (5-(trimethylsilyl)pent-4-yn-1-yl)magnesium chloride (7.92
mL, 3.96 mmol, 1.1 equiv, 0.5 M in THF) dropwise. The reaction was warmed to 0 ºC and stirred for 30
min before being quenched by the addition of sat. NH4Cl (50 mL). The product was extracted with EtOAc
Nature Methods: doi:10.1038/nmeth.2368
(100 mL, 3x) and the combined organic layers were dried of Na2SO4 and concentrated under reduced
pressure. The remaining residue was purified by SiO2 flash chromatography (10% EtOAc/hexanes, 1%
MeOH) providing the title compound as a colorless oil (1.02 g, 80%): 1H NMR (500 MHz, CDCl3) 3.64 (s,
3H), 3.63 – 3.57 (m, 1H), 2.27 (t, J = 7.55 Hz, 2H), 2.23 (t, J = 6.45 Hz, 2H), 1.68 – 1.36 (m, 8H), 1.32 –
1.22 (m, 12H), 0.12 (s, 9H); 13C NMR (125 MHz, CDCl3) 174.25, 107.28, 84.66, 71.29, 51.36, 37.47,
36.36, 34.04, 29.58, 29.46, 29.30, 29.15, 29.05, 25.56, 24.88, 24.65, 19.77, 0.11; HRMS (ESI-TOF+) m/z
calc’d for C20H38O3Si [M+H]+: 355.2663, found 355.2654.
Methyl 11-oxo-16-(trimethylsilyl)hexadec-15-ynoate. A solution of oxalyl chloride (0.27 mL, 3.12 mmol,
2.0 equiv) in dry CH2Cl2 (20 mL) was cooled to –78 ºC. DMSO (0.44 mL, 6.25 mL, 4.0 equiv) was added
dropwise and the reaction mixture was stirred for 15 min. Methyl 11-hydroxy-16-(trimethylsilyl)hexadec-
15-ynoate (554 mg, 1.56 mmol, 1.0 equiv) was then added and the reaction was stirred for another 15
min. Triethylamine (0.87 mL, 6.25 mmol, 4.0 equiv) was then added to the reaction mixture. After 15
min, the reaction mixture was warmed to 0 ºC and stirred at that temperature for 30 min. The reaction
mixture was then passed over a silica plug with EtOAc:Hex (1:2) and the eluent concentrated under
reduced pressure. The remaining residue was purified by SiO2 flash chromatography (5-10 %
EtOAc/hexanes) to provide the title compound (308 mg, 56%) as a white solid: 1H NMR (500 MHz, CDCl3)
3.65 (s, 3H), 2.52 (t, J = 7.24 Hz, 2H), 2.39 (t, J = 7.47 Hz, 2H), 2.29 (t, J = 7.57 Hz, 2H), 2.24 (t, J =
6.88 Hz, 2H), 1.76 (p, J = 7.07 Hz, 2H), 1.65 – 1.52 (m, 4H), 1.32 – 1.22 (m, 10H), 0.13 (s, 9H); 13C NMR
(150 MHz, CDCl3) 210.73, 174.27, 106.37, 85.28, 51.42, 42.97, 41.05, 34.06, 29.30, 29.22, 29.18,
29.16, 29.07, 24.90, 23.85, 22.36, 19.15, 0.10; HRMS (ESI-TOF+) m/z calc’d for C20H36O3Si [M+H]+:
353.2506, found 353.2504.
11-Oxohexadec-15-ynoic acid (12). To a stirring solution of methyl 11-oxo-16-(trimethylsilyl)hexadec-
15-ynoate (270 mg, 0.77 mmol, 1.0 equiv) in 1:1 MeOH:H2O (5 mL) was added NaOH (153 mg, 3.83
mmol, 5.0 equiv). The reaction mixture was stirred for 24 h at room temperature. The reaction mixture
was poured into a separatory funnel containing 50 mL of Et2O and 50 mL of 1 M HCl. The product was
extracted with Et2O (50 mL, 3x) and the combined organic layers were dried over Na2SO4 and
Nature Methods: doi:10.1038/nmeth.2368
concentrated under reduced pressure. The remaining residue was purified by SiO2 flash chromatography
(25% EtOAc/hexanes, 1% HCO2H) to provide the title compound as a white solid (198 mg, 97 %): 1H
NMR (500 MHz, CDCl3) 2.63 (t, J = 7.21 Hz, 2H), 2.48 (t, J = 7.46 Hz, 2H), 2.42 (t, J = 7.51 Hz, 2H),
2.31 (tdd, J = 0.91, 2.62, 6.84 Hz, 2H), 2.04 (td, J = 0.93, 2.65 Hz, 1H), 1.87 (p, J = 7.12 Hz, 2H), 1.76 –
1.60 (m, 4H), 1.47 – 1.30 (m, 10H); 13C NMR (125 MHz, CDCl3) 211.12, 179.70, 84.05, 69.37, 43.38,
41.42, 34.30, 29.70, 29.60, 29.58, 29.54, 29.40, 25.05, 24.24, 22.65, 18.18; HRMS (ESI-TOF–) m/z calc’d
for C16H26O3 [M–H]–: 265.1809, found 265.1812.
10-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)decanoic acid (13). To a sealed tube containing a stir bar and
11-oxohexadec-15-ynoic acid (172 mg, 0.65 mmol, 1.0 equiv) was added 7 N NH3 in MeOH (5.0 mL).
The reaction mixture was cooled to 0 ºC on an ice bath and stirred for 3 h. Hydroxylamine-O-sulfonic acid
(84 mg, 0.74 mmol, 1.15 equiv) was dissolved in MeOH (2 mL) and added to the reaction mixture
dropwise. The reaction mixture was allowed to warm to room temperature overnight after which the
solvent was evaporated under a stream of N2. To the remaining residue was added Et2O (5.0 mL)
resulting in a suspension of insoluble salts which were filtered away. The residue from the concentrated
filtrate was dissolved in anhydrous CH2Cl2 (10 mL) and pyridine (1.5 mL). To this mixture was added
PCC (280 mg, 1.30 mmol, 2.0 equiv). After being stirred for 2 h at room temperature, the reaction mixture
was passed through a pad of silica with 50% EtOAc/hexanes (1% HCO2H) and concentrated under
reduced pressure. The remaining residue was further purified by SiO2 flash chromatography (15%
EtOAc/hexanes, 1% HCO2H) to provide the title compound as a white solid (62 mg, 34 %): 1H NMR (500
MHz, CDCl3) 2.34 (t, J = 7.51 Hz, 2H), 2.15 (td, J = 2.65, 6.96 Hz, 2H), 1.94 (t, J = 2.62 Hz, 1H), 1.62 (p,
J = 7.47 Hz, 2H), 1.53 – 1.45 (m, 2H), 1.39 – 1.17 (m, 14H), 1.08 (p, J = 7.09 Hz, 2H); 13C NMR (125 MHz,
CDCl3) 179.69, 83.19, 68.59, 33.73, 32.58, 31.57, 29.02, 28.95, 28.87, 28.72, 28.16, 24.36, 23.53,
22.48, 17.69; HRMS (ESI-TOF–) m/z calc’d for C16H26N2O2 [M–H]–: 277.1921, found 277.1923.
N-(2-hydroxyethyl)-10-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)decanamide (14, PEA-DA). To a stirring
solution of 10-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)decanoic acid (25 mg, 0.09 mmol, 1.0 equiv) and N-
hydroxysuccinimide (16 mg, 0.13 mmol, 1.5 equiv) in CH2Cl2 (2.0 mL) was added EDCI (25 mg, 0.13
Nature Methods: doi:10.1038/nmeth.2368
mmol, 1.5 equiv). The reaction mixture was stirred until the starting material had disappeared as judged
by TLC (~ 4 h), at which point 5 drops of ethanolamine was added. After stirring for another 60 min, the
reaction mixture was poured into a separatory funnel containing brine (20 mL) and the product was
extracted with CH2Cl2 (20 mL, 3x). The combined organic layers were dried over Na2SO4, concentrated
under reduced pressure and purified by SiO2 flash chromatography (2% MeOH/EtOAc) to provide the title
compound as a white solid (25 mg, 87%): 1H NMR (500 MHz, CDCl3) 6.01 – 5.86 (m, 1H), 3.72 (t, J =
4.83 Hz, 2H), 3.42 (q, J = 5.25 Hz, 2H), 2.19 (t, J = 7.48 Hz, 2H), 2.16 (td, J = 2.65, 6.96 Hz, 3H), 1.94 (t,
J = 2.65 Hz, 1H), 1.62 (p, J = 7.73 Hz, 2H), 1.50 – 1.46 (m, 2H), 1.38 – 1.19 (m, 14H), 1.06 (p, J = 6.96
Hz, 2H); 13C NMR (150 MHz, CDCl3) 174.51, 83.45, 68.86, 62.68, 42.47, 36.63, 32.79, 31.80, 29.27,
29.23, 29.22, 29.19, 29.10, 28.45, 25.64, 23.77, 22.72, 17.93; HRMS (ESI-TOF+) m/z calc’d for
C18H31N3O2 [M+H]+: 322.2489, found 322.2488
Nature Methods: doi:10.1038/nmeth.2368
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