table s1 analytical characteristics for all sterols shown ... lipolytica, erg accumulation under...

Table S1 Analytical Characteristics for all sterols shown in the biosynthesis figure S1

Code IUPAC name Trivial name CAS number Chemical formula

MR

[g/mol] MR

(TMS) [g/mol]

RRT (cholestane)

Base peak (m/z)

Origin Reference

1*

(6E,10E,14E,18E)-2,6,10,15,19,23-Hexamethyl-tetracosa-2,6,10,14,18,22-

hexaene Squalene 7683-64-9 C30H50 410.7 (-) 0.95 69 R 2

2*

2,2-Dimethyl-3-((3E,7E,11E,15E)-3,7,12,16,20-pentamethylhenicosa-

3,7,11,15,19-pentaenyl)-oxirane Squalene epoxide 7200-26-2 C30H50O 426.7 (-) 1.05 81 R 2

3* 4,4,14-Trimethylcholesta-8,24(28)-dien-3β-

ol Lanosterol 79-63-0 C30H50O 426.7 498.9 1.44 393 R 1,2

4*

4,4-Dimethylcholesta-8,14,24-trien-3β-ol FF-MAS 64284-64-6 C29H44O 410.7 482.9 1.48 397 AF 1

5* 4,4-Dimethylcholesta-8,24-dien-3β-ol T-MAS 7448-02-4 C29H48O 412.7 484.9 1.47 379 AF 1

6 4-Methylcholesta-8,24-dien-3β-ol 4-Methylzymosterol C28H46O 398.7 470.9

7*

Cholesta-8,24-dien-3β-ol Zymosterol 128-33-6 C27H44O 384.7 456.9 1.31 351 R 1,2

8*

Ergosta-8,24(28)-dien-3β-ol Fecosterol 516-86-9 C28H46O 398.7 470.9 1.37 365 CG ERG2 (G)

9*

Ergosta-7,24(28)-dien-3β-ol Episterol 474-68-0 C28H46O 398.7 470.9 1.41 343 AF 1

10*

Ergosta-5,7,24(28)-trien-3β-ol 5-Dehydroepisterol 23582-83-4 C28H44O 396.7 468.9 1.39 363 SC ERG5 (I) 1

11*

Ergosta-5,7,22,24(28)-tetraen-3β-ol Dehydroergosterol 29560-24-5 C28H42O 394.7 466.8 1.36 361 CG ERG5 (I) 1

12*

Ergosta-5,7,22-trien-3β-ol Ergosterol 57-67-4 C28H44O 396.7 468.9 1.33 363 R 1

13*

4-Methylergosta-8,24(28)-dien-3β-ol 4-Methylfecosterol C29H48O 412.7 484.9 1.45 379 AF

14 Ergosta-8,22,24(28)-trien-3β‑ol C28H44O 396.7 468.9

15*

Ergosta-7,22-dien-3β‑ol 5-Dihydroergosterol/

Stellasterol 2465-11-4 C28H46O 398.7 470.9 1.35 343 R 1

16*

Ergosta-7,22,24(28)-trien-3β-ol C28H44O 396.7 468.9 1.37 343 AF 1

17*

Ergosta‑8,22‑dien‑3β‑ol 6673-68-3 C28H46O 398.7 470.9 1.32 365 SC ERG2 (G) 1

18*

Ergosta-8-en-3β-ol 30365-65-2 C28H48O 400.7 472.9 1.38 472 CG ERG2 (G) 1

19*

Ergosta-7-en-3β-ol 516-78-9 C28H48O 400.7 472.9 1.42 472 AF 1

20*

Ergosta-5,7-dien-3β-ol 516-79-0 C28H46O 398.7 470.9 1.39 365 YL ERG5 (I) 1

21*

Ergosta-5,8,24(28)-trien-3β‑ol C28H44O 396.7 468.9 1.36 363 AF

22*

Ergosta-5,8,22,24(28)-tetraen-3β-ol C28H42O 394.7 466.9 1.35 466 YL ERF2 (G)

23*

Ergosta-5,8,22-trien-3β-ol Lichesterol 50657-31-3 C28H44O 396.7 468.9 1.30 363 CG ERG2 (G) 1

24*

Ergosta-5,8-dien-3β‑ol C28H46O 398.7 470.9 1.36 339 R 3

25*

4,4,14-Trimethylergosta-8,24(28)-dien-3β-ol Eburicol 6890-88-6 C31H52O 440.7 512.9 1.50 407 CK ERG11 (C) 4

26*

4,14-Dimethylergosta-8,24(28)-dien-3β-ol Obtusifoliol C30H50O 426.7 498.9 1.43 393 AF ERG11 (C)

27*

14-Methylergosta-8,24(28)-dien-3β-ol 14-Methylfecosterol 33886-74-7 C29H48O 412.7 484.9 1.38 379 AF ERG11 (C) 1

28

4,4,14-Trimethylergosta-8-en-3β-ol C31H54O 442.7 514.9

Nature Protocols: doi:10.1038/nprot.2017.005

29 4,4,14-Trimethylergosta-8,22-dien-3β-ol C31H52O 440.7 512.9

30*

4,4,14-Trimethylergosta-7,22-dien-3β-ol C31H52O 440.7 512.9 1.48 407 CK ERG11 (C)

31 4,4,14-Trimethylergosta-8,22,24(28)-trien-

3β-ol C31H50O 438.7 510.9

32*

14-Methylergosta-8,24(28)-dien-3β,6α-diol 120523-06-0 C29H48O2 428.7 573.1 1.43 377 CK ERG11 (C) 1,4

33* 14-Methylergosta-8,22,24(28)-trien-3β,6α-

diol C29H46O2 426.7 571.1 1.39 123 CK ERG11 (C) 4

34*

14-Methylergosta-8,22-dien-3β,6α-diol C29H48O2 428.7 573.1 1.36 253 CK ERG11 (C) 4

35 14-Methylergosta-8-en-3β,6α-diol C29H50O2 430.7 575.1

36*

14-Methylergosta-7,24(28)-dien-3β,6α-diol C29H48O2 428.7 573.1 1.49 377 CK ERG11 (C) 4

37 14-Methylergosta-7,22,24(28)-trien-3β,6α-

diol C29H46O2 426.7 571.1

38 14-Methylergosta-7,22-dien-3β,6α-diol C29H48O2 428.7 573.1

39 14-Methylergosta-7-en-3β,6α-diol C29H50O2 430.7 575.1

40*

Cholesta-7,24-dien‑3β-ol 651-54-7 C27H44O 384.7 456.8 1.34 343 CG ERG6 (F) 1

41*

Cholesta-5,7,24‑trien‑3β-ol 7-Dehydrodesmosterol 1715-86-2 C27H42O 382.7 454.8 1.32 349 YL ERG6 (F) 1

42*

Cholesta-5,7,22,24‑tetraen‑3β-ol C27H40O 380.6 452.8 1.35 347 YL ERG6 (F) 2

43 Cholesta-8,22,24‑trien‑3β-ol C27H42O 382.6 454.8

44 Cholesta-7,22,24‑trien‑3β-ol C27H42O 382.6 454.8

45*

Cholesta-5-en-3ß-ol Cholesterol 57-88-5 C27H46O 386.6 458.6 1.25

(RT 14.35 min) 368 R 2

46*

Cholestane 481-20-9 C27H48 372.7 (-) 1.00

(RT 11.55 min) 217 R 2

AF Aspergillus fumigatus, CK Candida krusei, CG Candida glabrata, R synthetic or isolated reference material, RT retention time; RRT relative retention time, SC Saccharomyces cerevisiae, YL

Yarrowia lipolytica, ERG accumulation under specified enzyme inhibition (Enzyme code). * sterol can be found in spectral library. Sterol without * are only listed because of their appearance in the

biosynthesis figure 1, no analytical data is available.

1 Müller, C. et al. A convenient cellular assay for the identification of the molecular target of ergosterol biosynthesis inhibitors and quantification of their effects

on total ergosterol biosynthesis. Steroids 78, 483-493 (2013). 2 Giera, M., Müller, C. & Bracher, F. Analysis and experimental inhibition of distal cholesterol biosynthesis. Chromatographia 78, 343-58 (2014). 3 Giera, M. & Bracher, F. First total synthesis of ergosta-5,8-dien-3β-ol. Sci. Pharm. 76, 599-604 (2008). 4 Keller, P. et al. An antifungal benzimidazole derivative inhibits ergosterol biosynthesis and reveals novel sterols. Antimicrob. Agents Chemother. 59, 6296-6307

(2015).


Table S2 Mass spectra of ergosterol biosynthesis intermediates. For all sterols, the free

alcohols are depicted, without stereo-chemical assignments

IUPAC name (6E,10E,14E,18E)-2,6,10,15,19,23-Hexamethyl-tetracosa-

2,6,10,14,18,22-hexaene

trival name Squalene

code 1 RRT 0.95

CAS number 7683-64-9 MR [g/mol] 410

chemical formula C30H50 MR (TMS)

[g/mol] (-)


IUPAC name 2,2-Dimethyl-3-((3E,7E,11E,15E)-3,7,12,16,20-pentamethyl-

henicosa-3,7,11,15,19-pentaenyl)-oxirane

trival name Squalene epoxide

code 2 RRT 1.05


chemical formula C30H50O MR (TMS)

[g/mol] (-)


IUPAC name 4,4,14-Trimethylcholesta-8,24(28)-dien-3β-ol

trival name Lanosterol

code 3 RRT 1.44



[g/mol] 498


IUPAC name 4,4-Dimethylcholesta-8,14,24(28)-trien-3β-ol

trival name FF-MAS

code 4 RRT 1.48



[g/mol] 482


IUPAC name 4,4-Dimethylcholesta-8,24-dien-3β-ol

trival name T-MAS

code 5 RRT 1.47



[g/mol] 484


IUPAC name Cholesta-8,24-dien-3β-ol

trival name Zymosterol

code 7 RRT 1.31



[g/mol] 456


IUPAC name Ergosta-8,24(28)-dien-3β-ol

trival name Fecosterol

code 8 RRT 1.37



[g/mol] 470


IUPAC name Ergosta-7,24(28)-dien-3β-ol

trival name Episterol

code 9 RRT 1.41



[g/mol] 470


IUPAC name Ergosta-5,7,24(28)-trien-3β-ol

trival name 5-Dehydroepisterol

code 10 RRT 1.39



[g/mol] 468


IUPAC name Ergosta-5,7,22,24(28)-tetraen-3β-ol

trival name Dehydroergosterol

code 11 RRT 1.36



[g/mol] 466


IUPAC name Ergosta-5,7,22-trien-3β-ol

trival name Ergosterol

code 12 RRT 1.33



[g/mol] 468


IUPAC name 4-Methylergosta-8,24(28)-dien-3β-ol

trival name 4-Methylfecosterol

code 13 RRT 1.45

CAS number (-) MR [g/mol] 412


[g/mol] 484



trival name (-)

code 14 RRT 1.33



[g/mol] 468


IUPAC name Ergosta-7,22-dien-3β-ol

trival name 5-Dihydroergosterol/Stellasterol

code 15 RRT 1.35



[g/mol] 470



trival name (-)

code 16 RRT 1.37



[g/mol] 468



trival name (-)

code 17 RRT 1.32



[g/mol] 470


IUPAC name Ergosta-8-en-3β-ol

trival name (-)

code 18 RRT 1.38



[g/mol] 472


IUPAC name Ergosta-7-en-3β-ol

trival name (-)

code 19 RRT 1.42



[g/mol] 472



trival name (-)

code 20 RRT 1.39



[g/mol] 470



trival name (-)

code 21 RRT 1.36



[g/mol] 468


IUPAC Name Ergosta-5,8,22,24(28)-tetraen-3β-ol

trival name (-)

code 22 RRT 1.35



[g/mol] 466


IUPAC name Ergosta-5,8,22-trien-3β-ol

trival name Lichesterol

code 23 RRT 1.30



[g/mol] 468



trival name (-)

code 24 RRT 1.36



[g/mol] 470


IUPAC name 4,4,14-Trimethylergosta-8,24(28)-dien-3β-ol

trival name Eburicol

code 25 RRT 1.50



[g/mol] 512


IUPAC name 4,14-Dimethylergosta-8,24(28)-dien-3β-ol

trival name Obtusifoliol

code 26 RRT 1.43



[g/mol] 498


IUPAC name 14-Methylergosta-8,24(28)-dien-3β-ol

trival name 14-Methylfecosterol

code 27 RRT 1.38



[g/mol] 484


IUPAC name 4,4,14-Trimethylergosta-7,22-dien-3β-ol

trival name (-)

code 30 RRT 1.48



[g/mol] 512


IUPAC name 14-Methylergosta-8,24(28)-dien-3β,6α-diol

trival name (-)

code 32 RRT 1.43


chemical formula C29H48O2 MR (TMS)

[g/mol] 572


IUPAC name 14-Methylergosta-8,22,24(28)-trien-3β,6α-diol

trival name (-)

code 33 RRT 1.39



[g/mol] 570


IUPAC name 14-Methylergosta-8,22-dien-3β,6α-diol

trival name (-)

code 34 RRT 1.36



[g/mol] 572


IUPAC name 14-Methylergosta-7,24(28)-dien-3β,6α-diol

trival name (-)

code 36 RRT 1.49



[g/mol] 572


IUPAC name Cholesta-7,24-dien-3β-ol

trival name (-)

code 40 RRT 1.34



[g/mol] 456


IUPAC name Cholesta-5,7,24-trien-3β-ol

trival name 7-Dehydrodesmosterol

code 41 RRT 1.32



[g/mol] 454


IUPAC name Cholesta-5,7,22,24-tetraen-3β-ol

trival name (-)

code 42 RRT 1.35



[g/mol] 452


IUPAC name Cholesta-5-en-3ß-ol

trival name Cholesterol

code 45 RRT 1.25



[g/mol] 458


IUPAC name Cholestane

trival name Cholestane

code 46 RRT 1.00


chemical formula C27H48 MR (TMS)

[g/mol] (-)


Table S3 – semi-quantitative results for the incubation of A. fumigatus and S. cerevisiae with different inhibitors

Sterol % of total area

3 9 10 12 20 22 23 25 sum

(I) 0.4 1.0 0.7 93.1 1.5 1.1 1.3 0.90 100

SD 0.06 0.0 0.06 0.15 0.0 0.06 0.1 0.0

(II) 2.5 0.7 0.3 73.4 2.2 1.2 1.0 18.6 100

SD 0.7 0.4 0.07 7.5 1.0 0.7 0.3 8.1

t-test * ns * * ns ns ns *

Fold Difference (II / I)

6.3 0.7 0.4 0.8 1.5 1.1 0.8 20.6

Table S3A Semi-quantitative evaluation of an enzyme inhibition in distal ergosterol biosynthesis (n=3 for control; n=5 for Posaconazole treatment) relative sterol composition of

total area(%), (I) Aspergillus fumigatus without treatment, (II) Aspergillus fumigatus under treatment with Posaconazole (0.5 µg/ml), marker sterols for an inhibition of the

sterol C14-demethylase in red (compare table 1 and figure 1&3), SD standard deviation, t-test – two-tailed Mann Whitney test, * p < 0.05; ** p < 0.01; ns not significant

Sterol % of total area 3 10 12 20 sum

(I) 9.3 0.0 78.7 12.0 100

SD 1.3 0.0 1.2 0.9

(II) 6.6 9.9 56.9 26.6 100

SD 1.7 0.4 5.7 4.0

t-test ns - p=0.07 p=0.08

Fold Difference (II / I) 0.7 - 0.7 2.2

Table S3B Semi-quantitative evaluation of an enzyme inhibition in distal ergosterol biosynthesis (n=3 for control; n=3 for 22-desaturase inhibitor treatment) relative sterol

composition of total area(%), (I) Saccharomyces cerevisiae without treatment, (II) Saccharomyces cerevisiae under treatment with a desaturase inhibitor (cmpd 4 in ref. 13) (0.5

µg/ml), marker sterols for an inhibition of the sterol C22-desaturase in red (compare table 1 and figure 1&3), SD standard deviation, t-test – two-tailed Mann Whitney test, * p

< 0.05; ** p < 0.01; ns not significant

Sterol % of total area 3 10 11 12 20 sum

(I) 9.3 0.0 0.0 78.7 12.0 100

SD 1.3 0.0 0.0 1.2 0.9

(II) 9.6 12.0 5.1 45.4 28.0 100

SD 0.6 1.7 0.4 5.4 3.6

t-test ns - - p=0.1 p=0.07

Fold Difference (II / I) 1.0 - - 0.6 2.3

Table S3C Semi-quantitative evaluation of an enzyme inhibition in distal ergosterol biosynthesis (n=3 for control; n=3 for 22-desaturase inhibitor treatment) relative sterol

composition of total area(%), (I) Saccharomyces cerevisiae without treatment, (II) Saccharomyces cerevisiae under treatment with a desaturase inhibitor (cmpd 4 in ref. 13)(1.0

µg/ml), marker sterols for an inhibition of the sterol C22-desaturase in red (compare table 1 and figure 1&3), SD standard deviation, t-test – two-tailed Mann Whitney test, * p

< 0.05; ** p < 0.01; ns not significant


table s1 analytical characteristics for all sterols shown ... lipolytica, erg accumulation under...

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