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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

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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 Table 6.

Please see accompanying Excel file.

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

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

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