Supporting Information
Second-generation probes for biosynthetic intermediate capture:towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
Department of Chemistry
University of Warwick
Library Road, Coventry CV4 7AL, UK
Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2016
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
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Table of Content
1. Synthesis of novel chemical probes______________________________________________ 3
1.1. General methods _______________________________________________________________ 3
1.2. Synthesis of probes 15a-b ________________________________________________________ 4
1.2.1. Methyl 2-(2-(3-acetamidopropyl)-4-phenyl-1,3-dioxolan-2-yl)acetate (13a) _____________________ 5
1.2.2. Methyl 2-(2-(3-d3-acetamidopropyl)-4-phenyl-1,3-dioxolan-2-yl)acetate (13b) ___________________ 5
1.2.3. Acetoxymethyl 2-(2-(3-acetamidopropyl)-4-phenyl-1,3-dioxolan-2-yl)acetate (14a) _______________ 6
1.2.4. Acetoxymethyl 2-(2-(d3-acetamidopropyl)-4-phenyl-1,3-dioxolan-2-yl)acetate (14b) ______________ 7
1.2.5. Methyl 2-[2-(3-acetamidopropyl)-1,3-dithiolan-2-yl]acetate (16a) _____________________________ 8
1.2.6. Acetoxymethyl 2-[2-(3-acetamidopropyl)-1,3-dithiolan-2-yl]acetate (17a)_______________________ 9
1.2.7. Acetoxymethyl 6-acetamido-3-oxohexanoate (15a)_________________________________________ 9
1.2.8. Acetoxymethyl 6-d3-acetamido-3-oxohexanoate (15b) _____________________________________ 10
1.3. Synthesis of N-(4,6-dioxoheptyl)decanamide (20) ____________________________________ 10
1.3.9. Methyl-2-(2-(3-decanamidopropyl)-1,3-dithiolan-2-yl)acetate (18) ___________________________ 11
1.3.10. Potassium 2-(2-(3-decanamidopropyl)-1,3-dithiolan-2-yl)acetate (26) _________________________ 12
1.3.11. Acetoxymethyl-2-(2-(3-decanamidopropyl)-1,3-dithiolan-2-yl)acetate (19) _____________________ 12
1.3.12. Acetoxymethyl-6-decanamido-3-oxohexanoate (20) _______________________________________ 13
1.3.13. Synthesis of methyl 6-(10-azidodecanamido)-3-oxohexanoate (4) ____________________________ 13
2. Growth of S. lasaliensis ACP12 (S970A) and mass spectrometry analysis_______________ 14
2.1. Summary of captured intermediates for S. lasaliensis ACP12(S970A) ____________________ 16
2.2. Intermediates captured by methyl 6-decanamido-3-oxohexanoate (3) ___________________ 19
2.3. Intermediate capture by N-(4,6-dioxoheptyl)decanamide (20)__________________________ 42
2.4. Intermediate capture by methyl 6-(10-azidodecanamido)-3-oxohexanoate (4)_____________ 65
2.5. Staudinger-phosphite derivatisation of azido intermediates ___________________________ 80
3. Spectra ___________________________________________________________________ 93
3.1. 1H- and 13C-NMR of compound 13a (400 MHz, CDCl3) _________________________________ 93
3.2. 1H- and 13C-NMR of compound 13b (400 MHz, CDCl3) _________________________________ 94
3.3. 1H- and 13C-NMR of compound 14a (400 MHz, CDCl3) _________________________________ 95
3.4. 1H- and 13C-NMR of compound 14b (400 MHz, CDCl3) _________________________________ 96
3.5. 1H- and 13C-NMR of compound 16a (400 MHz, CDCl3) _________________________________ 97
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
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3.6. 1H- and 13C-NMR of compound 17a (400 MHz, CDCl3) _________________________________ 98
3.7. 1H- and 13C-NMR of compound 15a (400 MHz, CDCl3) _________________________________ 99
3.8. 1H- and 13C-NMR of compound 15b (400 MHz, CDCl3) ________________________________ 100
3.9. 1H- and 13C-NMR of compound 18 (400 MHz, CDCl3) _________________________________ 101
3.10. 1H- and 13C-NMR of compound 24 (400 MHz, D2O) __________________________________ 102
3.11. 1H- and 13C-NMR of compound 19 (400 MHz, CDCl3) _________________________________ 103
3.12 1H- and 13C-NMR of compound 20 _______________________________________________ 104
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
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1. Synthesis of novel chemical probes
1.1.General methods
Solvents and reagents: Unless specified otherwise, chemicals were purchased from Sigma Aldrich, Fisher
Scientific, Carbosynth or Alfa Aesar and were used without further purification. Anhydrous
dichloromethane and tetrahydrofurane were purchased from VWR International (AR grade) and dried using
solvent towers. Anhydrous ethyl acetate and methanol were purchased from Fisher Scientific or Sigma
Aldrich. Reagent grade dichloromethane, ethyl acetate, methanol, acetonitrile, cyclohexane and chloroform
were purchased from Fisher Scientific.
Chromatography: Analytical thin-layer chromatography (TLC) was performed on aluminium sheets
precoated with silica gel 60 (F254, Merck) and visualized under ultra-violet light (short and long-wave) and
using potassium permanganate (KMnO4) or vanillin stains. Silica gel was purchased from Sigma Aldrich
(Tech Grade, pore size 60 Å, 230-400 mesh).
NMR Spectroscopy: 1H and 13C NMR spectra were recorded in d4-MeOD, CDCl3 or D2O on the following
Bruker Avance instruments: DPX-400 400 MHz, DRX-500 500 MHz, AV III-500 HD 500 MHz or AV-600
600 MHz.
LC-HRMS: High-resolution mass spectra (HRMS) of newly made compounds were obtained using
electrospray ionization (ESI) on a MaXis UHR-TOF (Bruker Daltonics) or on Bruker MaXis (ESI-HR-MS).
HPLC: Compounds were purified by semipreparative HPLC on a Phenomenex synergi™ Polar RP 80 Å (250 x
10.0 mm, 4 µm) column. The mobile phase consisted of a gradient of water and acetonitrile (HPLC grade,
containing 0.1% (v/v) trifluoroacetic acid) at a flow rate of 2.5 mL/min, with UV detection at 210, 254 and
280 nm.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
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1.2.Synthesis of probes 15a-b
Route 1
NH
O
R
O
O
OONH
O
R
O
O
O
NH
O
R
O
O
OO
O
O
NH
O
R
O
O
O
O
O
OH
OH
chlorotrimethylsilane
CH2Cl2, 40 °C, 18 h
(CH3)3SiOK, 3 Å
THF, r. t., 18 h
THF, r. t., 3 h
NH
O
R
O
O
OO
K O
O
Br
H2 (g)Pd(OAc)2, Pd(CF3COO)2
EtOAc, r. t., 48 h
90%
98% over 2 steps
40%R = CH3 or CD3
1a-b
13a-b
22a-b
14a-b
15a-b
Route 2
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
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1.2.1. Methyl 2-(2-(3-acetamidopropyl)-4-phenyl-1,3-dioxolan-2-yl)acetate (13a)
To a solution of the methyl ester 1a1 (1.40 g, 7.00 mmol) in dry dichloromethane (40 mL), 1-phenyl-1,2-
ethanediol (1.92 g, 13.9 mmol) and chlorotrimethylsilane (3.51 mL, 27.9 mmol) were added dropwise under
argon atmosphere. The reaction mixture was heated under reflux at 40 °C for 18 h.2 The solvent was
removed in vacuo, the resulting residue was dissolved in EtOAc (40 mL) and washed with 5% (w/v) NaHCO3
(20 mL). The organic layer was dried over MgSO4 and concentrated affording 13a as a yellow oil (2.01 g,
90%; Rf = 0.60 in 9:1 CH2Cl2:MeOH), which was used directly in the next step. A sample of this crude
material was purified by semipreparative HPLC for accurate NMR characterisation (Rt = 23 min, with a
gradient elution from 0 to 100% MeCN over 30 minutes). A double set of signals was observed for two
major isomers present in 1:1 ratio. 1H-NMR (400 MHz, CDCl3) δ = 1.68-1.79 (4H, m, CH2CH2CH2), 1.95 and
1.97 (each 3H, s, CH3CONH), 1.92-2.04 (4H, m, CH2CO2), 2.80 and 2.80 (each 2H, d, J = 15.0 Hz, CH2CO), 3.27-
3.33 (4H, m, CH2NH), 3.68-3.73 (2H, m, CH2O), 3.71 (6H, s, OCH3), 4.31-4.37 (2H, m, CH2O), 5.05 (1H, dd, J =
6.0, 9.3 Hz, CH), 5.15 (1H, dd, J = 6.5, 8.4 Hz, CH), 5.95 and 5.98 (each 1H, br s, NH), 7.29-7.37 (10H, m, ArH);
13C-NMR (125 MHz, CDCl3) δ = 23.1 (CH3CO), 23.1 (CH3CO), 23.5 (CH2CH2CH2), 23.6 (CH2CH2CH2), 35.1
(CH2CO2), 35.2 (CH2CO2), 39.4 (CH2NH), 39.5 (CH2NH), 42.4 (CH2CO), 43.1 (CH2CO), 51.8 (CH3O), 51.8 (CH3O),
71.9 (CH2O), 71.9 (CH2O), 78.0 (CHO), 78.9 (CHO), 109.8 (CO2), 109.9 (CO2), 126.1 (CH arom), 126.3 (CH
arom), 128.2 (CH arom), 128.3 (CH arom), 128.5 (CH arom), 128.6 (CH arom), 137.5 (C arom), 137.7 (C
arom), 169.7 (CONH), 169.8 (CONH), 170.3 (CO2Me), 170.3 (CO2Me); m/z (HR-ESI-MS): found [M+Na]+
344.1470, C17H23NO5Na+ requires 344.1468.
1.2.2. Methyl 2-(2-(3-d3-acetamidopropyl)-4-phenyl-1,3-dioxolan-2-yl)acetate (13b):
1(a) Tosin, M.; Betancor, L.; Stephens, E.; Li, W. M. A.; Spencer, J. B.; Leadlay, P. F. ChemBioChem, 2010, 11, 539-546;
(b) Tosin, M.; Demdychuk, Y.; Parascandolo, J. S.; Blasco Per, C.; Leeper, F. J.; Leadlay, P. F. Chem. Commun., 2011, 47,
3460-3462.
2Chan, T. H.; Brook, M. A.; Chaly, T. Synthesis, 1983, 3, 203-205.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
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Compound 13b was prepared in the same way as 13a from methyl 6-(d3-acetamido)-3-oxohexanoate 1b.1b
1H-NMR (400 MHz, CDCl3) δ = 1.66-1.80 (4H, m, CH2CH2CH2), 1.90-2.05 (4H, m, CH2), 2.80 and 2.81 (each 2H,
d, J = 15.0 Hz, CH2CO), 3.27-3.34 (4H, m, CH2NH), 3.71 (6H, s, OCH3), 3.71 (2H, t, J =8.5 Hz, CH2O), 4.32-4.38
(2H, m, CH2O), 5.06 (1H, dd, J = 9.2, 5.9 Hz, CH), 5.16 (1H, dd, J = 8.5, 6.5 Hz, CH), 5.90 and 5.94 (each 1H, br
s, NH), 7.28-7.39 (10H, m, ArH); 13C-NMR (125 MHz, CDCl3) δ = 23.1 (CD3), 23.1 (CD3), 23.5 (CH2CH2CH2),
23.6 (CH2CH2CH2), 35.1 (CH2CO2), 35.2 (CH2CO2), 39.4 (CH2NH), 39.5 (CH2NH), 42.4 (CH2CO), 43.2 (CH2CO),
51.8 (CH3O), 51.8 (CH3O), 71.9 (CH2O), 72.0 (CH2O), 78.0 (CHO), 79.0 (CHO), 109.8 (CO2), 109.9 (CO2), 126.1
(CH arom), 126.3 (CH arom), 128.3 (CH arom), 128.4 (CH arom), 128.5 (CH arom), 128.6 (CH arom), 137.5 (C
arom), 137.7 (C arom), 169.8 (CONH), 169.8 (CONH), 170.4 (COMe), 170.4 (COMe); m/z (HR-ESI-MS): found
[M+Na]+ 347.1651, C17H20D3NO5Na requires 347.1657.
1.2.3. Acetoxymethyl 2-(2-(3-acetamidopropyl)-4-phenyl-1,3-dioxolan-2-yl)acetate (14a)
To a solution of 13a (2.00 g, 6.2 mmol) in dry THF (40 mL) containing 3 Å molecular sieves, potassium
trimethylsilanolate (3.19 g, 24.9 mmol) was added. The reaction mixture was stirred under argon
atmosphere and at room temperature for 3 h.3 The reaction was then filtered and the solvent removed in
vacuo. The residue was partitioned between EtOAc (20 mL) and water (20 mL), the aqueous layer was
lyophilised affording crude potassium 2-(2-(3-acetamidopropyl)-4-phenyl-1,3-dioxolan-2-yl)acetate (22a,
2.14 g, 100%), which was carried forward to the next step without further purification.
For this intermediate a double set of signals was observed for two major isomers present in 1:1 ratio; 1H-
NMR (600 MHz, D2O) δ = 1.65-1.78 (4H, m, CH2CH2CH2), 1.93-2.03 (4H, m, CH2), 1.94 and 1.95 (each 3H, s,
CH3CONH), 2.48-2.64 (4H, m, CH2CO), 3.22 (4H, t, J = 6.7 Hz, CH2NH), 3.78-3.86 (2H, m, CH2O), 4.34-4.45
(2H, m, CH2O), 5.15 (1H, dd, J = 6.2, 8.9 Hz, CH), 5.24-5.31 (1H, m, CH), 7.36-7.53 (10H, m, ArH); 13C-NMR
(125 MHz, D2O) δ = 22.6 (CH3), 22.6 (CH3), 23.6 (CH2CH2CH2), 23.6 (CH2CH2CH2), 36.2 (CH2CO2), 36.4
3Barrett, A. G. M.; Peña, M.; Willardsen, J. A. J. Org. Chem., 1996, 61, 1082–1100.
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(CH2CO2), 40.1 (CH2CO), 40.1 (CH2CO), 46.4 (CH2NH), 46.9 (CH2NH), 71.9 (CH2O), 71.9 (CH2O), 78.5 (CH), 79.3
(CH), 111.7 (CO2), 111.8 (CO2), 127.1 (CH arom), 127.6 (CH arom), 128.9 (CH arom), 129.4 (CH arom), 129.5
(CH arom), 129.6 (CH arom), 138.0 (C arom), 138.3 (C arom), 176.3 (CONH), 176.4 (CONH), 180.3 (COO-),
180.3 (COO-); m/z (HR-ESI-MS): found [M+Na]+ 330.1311, C16H21NO5Na requires 330.1312).
To a solution of crude potassium 2-(2-(3-acetamidopropyl)-4-phenyl-1,3-dioxolan-2-yl)acetate (22a, 2.10 g,
6.1 mmol) in dry THF (40 mL), bromomethyl acetate (650 µl, 6.7 mmol) was added and the reaction mixture
was stirred for 18 h at room temperature. The solvent was removed in vacuo to give 14a as a yellow oil
(2.27 g, 98%), used for the next step without further purification, Rf = 0.25 (EtOAc). A sample of this crude
material was purified by semipreparative HPLC for accurate NMR characterisation (Rt = 37 min, with a
gradient elution from 0 to 50% MeCN over 30 min, then 50 to 100% MeCN over 15 min). A double set of
signals was observed for two major isomers present in 1:1 ratio. 1H-NMR (400 MHz, CDCl3) δ = 1.65-1.79
(4H, m, CH2CH2CH2), 1.95 and 1.97 (each 3H, s, CH3CONH), 1.92-2.04 (4H, m, CH2), 2.04 and 2.05 (each 3H, s,
CH3COO) 2.83 and 2.83 (each 2H, d, J = 13.0 Hz, CH2CO), 3.26-3.33 (4H, m, CH2NH), 3.70 (2H, t, J = 8.3 Hz,
CH2O), 4.29-4.37 (2H, m, CH2O), 5.04 (1H, dd, J = 9.4, 5.9 Hz, CH), 5.15 (1H, dd, J = 8.4, 6.7 Hz, CH), 5.73-
5.78 (4H, m, OCH2O), 6.00 and 6.03 (each 1H, br s, NH), 7.28-7.38 (10H, m, ArH); 13C-NMR (125 MHz, CDCl3)
δ = 20.6 (CH3), 20.6 (CH3), 23.1 (CH3), 23.1 (CH3), 23.4 (CH2CH2CH2), 23.5 (CH2CH2CH2), 35.2 (CH2CO2), 35.3
(CH2CO2), 39.3 (CH2NH), 39.4 (CH2NH), 42.4 (CH2CO), 43.1 (CH2CO), 71.9 (CH2O), 72.0 (CH2O), 78.1 (CHO),
79.1 (CHO), 79.1 (OCH2O), 79.1 (OCH2O), 109.6 (CO2), 109.7 (CO2), 126.1 (CH arom), 126.2 (CH arom), 128.3
(CH arom), 128.4 (CH arom), 128.6 (CH arom), 128.6 (CH arom), 137.2 (C arom), 137.6 (C arom), 167.9 (C
arom), 167.9 (C arom), 169.6 (CONH), 169.7 (CONH), 170.3 (CO), 170.3 (CO); m/z (HR-ESI-MS): found
[M+Na]+ 402.1521, C19H25NO7Na requires 402.1523.
1.2.4. Acetoxymethyl 2-(2-(d3-acetamidopropyl)-4-phenyl-1,3-dioxolan-2-yl)acetate (14b)
Compound 14b was prepared in the same way as 14a from 13b as starting material, through potassium 2-
(2-(3-d3-acetamidopropyl)-4-phenyl-1,3-dioxolan-2-yl)acetate (22b) as intermediate:
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
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Intermediate characterisation of 22b: 1H-NMR (400 MHz, MeOD) δ = 1.65-1.82 (4H, m, CH2CH2CH2), 1.95-
2.08 (4H, m, CH2), 2.48-2.64 (4H, m, CH2CO), 3.18-3.25 (4H, m, CH2NH), 3.54-3.65 (2H, m, CH2O), 4.31 (1H,
dd, J = 6.0, 8.2 Hz, CH2O), 4.36 (1H, dd, J = 6.5, 7.7 Hz, CH2O), 5.03 (1H, dd, J = 7.0, 11.9 Hz, CH), 5.23 (1H,
dd, J = 6.5, 8.4 Hz, CH), 7.36-7.53 (10H, m, ArH); 13C-NMR (125 MHz, D2O) δ = 24.4 (CH2CH2CH2), 24.4
(CH2CH2CH2), 24.9 (CD3), 24.9 (CD3), 36.4 (CH2CH), 36.7 (CH2CH), 40.9 (CH2NH), 40.9 (CH2NH), 47.4 (CH2CO2),
48.0 (CH2CO2), 73.0 (CH2O), 73.2 (CH2O), 79.1 (CH), 80.1 (CH), 112.3 (CO2), 112.5 (CO2), 127.6 (CH arom),
127.7 (CH arom), 129.2 (CH arom), 129.4 (CH arom), 129.6 (CH arom), 129.7 (CH arom), 140.0 (C arom),
140.3 (C arom), 171.8 (CONH), 171.8 (CONH), 176.4 (COO-), 176.4 (COO-); m/z (HR-ESI-MS): found [M+Na]+
333.1487, C16H18D3NO5Na+ requires 333.1500.
For 14b: 1H-NMR (500 MHz, CDCl3) δ = 1.65-1.80 (4H, m, CH2CH2CH2), 1.92-2.04 (4H, m, CH2), 2.05 and 2.06
(each 3H, s, CH3COO), 2.83 and 2.84 (each 2H, d, J = 16.0 Hz, CH2CO), 3.28-3.35 (4H, m, CH2NH), 3.70 (2H, t,
J = 8.3 Hz, CH2O), 4.32-4.36 (2H, m, CH2O), 5.04 (1H, dd, J = 6.6, 8.4 Hz, CH), 5.15 (1H, dd, J = 6.0, 9.3 Hz,
CH), 5.73-5.78 (4H, m, OCH2O), 6.08 and 6.13 (each 1H, br s, NH), 7.29-7.39 (10H, m, ArH); 13C-NMR (125
MHz, CDCl3) δ = 20.6 (CH3), 20.6 (CH3), 23.0 (CD3), 23.0 (CD3), 23.4 (CH2CH2CH2), 23.5 (CH2CH2CH2), 35.2
(CH2CO2), 35.3 (CH2CO2), 39.4 (CH2CO), 39.4 (CH2CO), 42.4 (CH2NH), 43.1 (CH2NH), 71.9 (CH2O), 72.0 (CH2O),
78.1 (CH2CH), 79.1 (CH2CH), 79.1 (OCH2O), 79.1 (OCH2O), 109.6 (CO2), 109.7 (CO2), 126.1 (CH arom), 126.3
(CH arom), 128.3 (CH arom), 128.4 (CH arom), 128.6 (CH arom), 128.6 (CH arom), 137.3 (C arom), 137.6 (C
arom), 167.9 (CO), 167.9 (CO), 169.7 (CONH), 169.8 (CONH), 170.5 (CO), 170.5 (CO); m/z (HR-ESI-MS):
found [M+Na]+ 405.1701, C19H22D3NO7Na+ requires 405.1682.
1.2.5. Methyl 2-[2-(3-acetamidopropyl)-1,3-dithiolan-2-yl]acetate (16a)
To a solution of 1a (968 mg, 4.8 mmol) in dry CH2Cl2 (30 mL), 1,2-ethanedithiol (2.02 mL, 24.1 mmol) was
added. The reaction mixture was cooled to 0°C before dropwise addition of BF3·Et2O (2.97 mL, 24.1 mmol)
then the reaction was heated under reflux at 40 °C for 18 h. The reaction was quenched with saturated
NaHCO3 solution, the organic layer was dried over MgSO4 and concentrated in vacuo. The crude product
was purified by flash chromatography (9:1 DCM:MeOH, Rf = 0.59) to give 16a (1.20 g, 90%). A sample of this
crude material was purified by semipreparative HPLC for accurate NMR characterisation (Rt = 24.5 min,
with a gradient elution 0 to 40% over 15 min, then 40 to 50 over 30 min, then 50 to 100% MeCN over 5
min). 1H-NMR (400 MHz, CDCl3) δ = 1.62-1.80 (2H, m, CH2CH2CH2), 2.00 (3H, s, CH3CONH), 2.11-2.16 (2H, m,
CH2CS2), 3.05 (2H, s, CH2), 3.20-3.24 (2H, m, CH2NH), 3.30 (4H, s, SCH2CH2S), 3.70 (3H, s, OCH3), 5.90 (1H, br
s, NH); 13C-NMR (125 MHz, CDCl3) δ = 23.0 (CH3), 26.8 (CH2CH2CH2), 39.3 (CH2), 39.5 (CH2), 39.8 (SCH2CH2S),
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
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48.2 (CH2CO), 51.7 (CH3O), 66.6 (CS2), 170.4 (CONH), 170.4 (COMe); m/z (HR-ESI-MS): found [M+Na]+
300.0701, C11H19NO3S2Na+ requires 300.0699.
1.2.6. Acetoxymethyl 2-[2-(3-acetamidopropyl)-1,3-dithiolan-2-yl]acetate (17a)
Compound 17a was prepared in the same way as 14a from 16a as starting material, proceeding through the
intermediate potassium 2-[2-(3-acetamidopropyl)-1,3-dithiolan-2-yl]acetate (23a):
23a: 1H-NMR (700 MHz, MeOD) δ = 1.72-1.78 (2H, m, CH2CH2CH2), 1.92 (3H, s, CH3CONH), 2.16-2.19 (2H, m,
CH2), 2.89 (2H, s, CH2CO), 3.16 (2H, t, J = 7.0, CH2NH), 3.21-3.26 (4H, m, SCH2CH2S); 13C-NMR (176 MHz,
MeOD) δ 24.4 (CH3), 24.4 (CH2), 40.1 (CH2), 40.2 (CH2), 40.8 (CH2), 41.5 (CH2), 53.0 (CH2CO), 69.4 (CS2), 173.3
(CONH), 178.3 (COO-); m/z (HR-ESI-MS): found [M+H]+ 286.0542, C10H17NO3S2Na+ requires 286.0542.
A sample of 17a was purified by semipreparative HPLC for accurate NMR characterisation (Rt = 19.5 min,
with a gradient elution 0 to 65% MeCN over 15 min, then 65 to 70 MeCN over 15 min, then 70 to 100%
MeCN over 5 min). 1H-NMR (400 MHz, CDCl3) δ = 1.69-1.77 (2H, m, CH2CH2CH2), 1.98 (3H, s, CH3CONH),
2.08-2.14 (2H, m, CH2CS2), 2.11 (3H, s, CH3COO), 3.06 (2H, s, CH2CO2R), 3.26-3.31 (2H, m, CH2NH), 3.28 (4H,
s, SCH2CH2S), 5.72 (2H, s, CH2), 6.01 (1H, br s, NH); 13C-NMR (125 MHz, CDCl3) δ = 20.7 (CH3), 23.1 (CH3),
26.8 (CH2CH2CH2), 39.3 (CH2), 39.3 (CH2), 39.3 (CH2), 48.1 (CH2CO), 66.2 (CS2), 79.1 (OCH2O), 168.5 (CONH),
169.7 (CO), 170.3 (CO); m/z (HR-ESI-MS): found [M+Na]+ 358.0753, C13H21NO5S2Na+ requires 358.0753.
1.2.7. Acetoxymethyl 6-acetamido-3-oxohexanoate (15a)
Method A
To a solution of 14a (250 mg, 0.7 mmol) in dry EtOAc (20 mL) under argon atmosphere, Pd(OAc)2 (200 mg,
0.9 mmol) and Pd(CF3COO)2 (100 mg, 0.3 mmol) were added 4. H2(g) (1 bar) was then flushed through and
4Conway, S. J.; Thuring, J. W.; Andreu, S.; Kvinlaug, B. T.; Roderick, H. L.; Bootman, M. D.; Holmes, A. B. Aust. J.
Chem., 2006, 59, 887–893.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
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p a g e 10 | 105
the mixture was stirred at room temperature for 48 h. The reaction mixture was filtered through Celite and
the solvent removed in vacuo; following HPLC purification (see conditions below), 15a was obtained as a
yellow oil (68 mg, 40%).
Method B
To a solution of 17a (250 mg, 0.7 mmol) dissolved in a mixture of CH3CN (20 ml) and water (2 ml)
[Bis(trifluoroacetoxy)iodo]benzene (864 mg, 2.1 mmol) was added. The reaction was stirred at room
temperature for 20 minutes and then quenched with a 1:1:1 water/NaHCO3/Na2SO3 mixture (20 ml in
total). After an extraction with EtOAc (20 mL), the organic phase was dried over MgSO4 and the solvent
evaporated under reduced pressure. The crude residue was then purified by semipreparative HPLC (Rt = 24
min, with a gradient elution from 0 to 50% MeCN over 30 min, then 50 to 100% MeCN over 15 min)
affording 15a as a yellow oil (125 mg, 78%). 1H-NMR (500 MHz, (CD3)2SO) δ = 1.57 (2H, app quin, J = 7.1 Hz,
CH2CH2CH2), 1.77 (3H, s, CH3CONH), 2.07 (3H, s, CH3COO), 2.52 (2H, t, J =7.3 Hz, CH2), 2.97 (2H, m, CH2NH),
3.68 (2H, s, COCH2CO), 5.68 (2H, s, OCH2O), 7.80 (1H, br s, NH); 13C-NMR (150 MHz, (CD3)2SO) δ = 20.4 (CH3),
22.6 (CH3), 23.0 (CH2CH2CH2), 37.6 (CH2), 39.5 (CH2), 48.4 (CH2), 79.1 (OCH2O), 166.3 (CONH), 169.1 (CO),
169.2 (CO), 202.7 (CO); m/z (HR-ESI-MS): found [M+Na]+ 282.0942, C11H17NO6Na+ requires 282.0948.
1.2.8. Acetoxymethyl 6-d3-acetamido-3-oxohexanoate (15b)
Compound 15b was prepared in the same way as 15a (see above) from 14b as starting material.
1H-NMR (500 MHz, (CD3)2SO) δ = 1.57 (2H, app quin, J = 7.1 Hz, CH2CH2CH2), 2.08 (3H, s, CH3COO), 2.52 (2H,
t, J = 7.3 Hz, CH2), 2.97 (2H, app q, J = 6.6, CH2NH), 3.68 (2H, s, COCH2CO), 5.68 (2H, s, OCH2O), 7.80 (1H, br
s, NH); 13C-NMR (150 MHz, (CD3)2SO) δ = 20.4 (CH3), 22.6 (CH3), 23.0 (CH2CH2CH2), 37.6 (CH2), 39.6 (CH2),
48.4 (COCH2), 79.1 (OCH2O), 166.3 (CONH), 169.1 (CO), 169.2 (CO), 202.7 (CO); m/z (HR-ESI-MS): found
[M+Na]+ 285.1132, C11H14D3NO6Na+ requires 285.1136.
1.3.Synthesis of N-(4,6-dioxoheptyl)decanamide (20)
Compound 3 was prepared as reported previously.5
5 E. Riva, I. Wilkening, Ina, S. Gazzola, W. M. A. Li, L Smith, P. F. Leadlay, M. Tosin, Angew. Chem. Int. Ed., 2014, 53,11944—11949.
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p a g e 11 | 105
C9H19 Cl
O
H2NOH
O
, Et3N
MeOH, 0 oC to r. t., 16 h
NH
OHC9H19
O
O O
OO
THF, r. t., 16 hEDC*HCl, Meldrum's acid, DMAP
25
65 oC, 16 h
MeOHNH
O
C9H19
O
O
O
3
24
85%
1.
2.
30%
1.3.9. Methyl-2-(2-(3-decanamidopropyl)-1,3-dithiolan-2-yl)acetate (18)
Compound 3 (800 mg, 2.55 mmol) and 1,2-ethanthiol (1.07 mL, 12.8 mmol) were dissolved in
dichloromethane (16 mL) under argon atmosphere. The solution was cooled to 0 °C before BF3·OEt2
(1.57 mL, 12.8 mmol) was slowly added. The reaction was stirred under reflux overnight. Then, a saturated
solution of NaHCO3 was added at 0 °C up to pH 9 and the mixture was extracted with CH2Cl2 (3x10 mL). The
crude product was purified by column chromatography (cyclohexane: EtOAc, 7:3 to 1:1)) and 18 was
obtained as a white powder (600 mg, 61%).
1H-NMR (400 MHz, CDCl3): δ = 0.87 (3H, t, J = 6.5 Hz, CH3), 1.27 (12H, br s, (CH2)6), 1.58 (2H, m,
CH2CH2CONH), 1.75 (2H, m, NHCH2CH2), 2.14 (2H, m, CH2CS2), 2.14 (2H, m, CH2CONH), 3.04 (2H, s, CH2COO),
3.29 (2H, m, NHCH2), 3.30 (4H, s, SCH2CH2S), 3.70 (3H, s, OCH3), 5.51 (1H, br s, NH); 13C-NMR (101 MHz,
CDCl3): δ = 14.1 (CH3), 22.6 (CH2CH3), 22.6 (CH2CH2CS2), 25.8 (CH2), 27.1(CH2), 29.3 (CH2), 29.3 (CH2), 29.4
(CH2), 29.4 (CH2), 36.9 (CH2CONH), 39.1 (CH2CS2), 39.4 (SCH2CH2S), 39.8 (NHCH2), 48.2 (CH2COO), 51.7
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(OCH3), 66.6 (CS2), 170.5 (C=O), 173.1 (C=O); m/z (HR-ESI-MS): found [M+H]+ 412.1945, C19H36NO3S2+
requires 412.1951.
1.3.10. Potassium 2-(2-(3-decanamidopropyl)-1,3-dithiolan-2-yl)acetate (26)
To a solution of compound 18 (600 mg, 1.54 mmol) in dry THF (10 mL) containing 3 Ȧ molecular sieves, was
added potassium trimethilsilanolate (789 mg, 6.16 mmol). The reaction was stirred under argon
atmosphere for 3 h at room temperature. Then the reaction was filtered under vacuum and concentrated.
The residue was dissolved in water (5 mL), EtOAc (5 mL) was added, and the two phases were separated.
The aqueous layer was freeze-dried affording 26 as a white powder (565 mg, 86%).
1H-NMR (400 MHz, D2O): δ = 0.79 (3H, br s, CH3), 1.22 (12H, br s, (CH2)6), 1.53 (2H, br s, CH2CH2CONH), 1.64
(2H, br s, NHCH2CH2), 1.84 (4H, s, CH2CS2), 2.01 (2H, m, CH2CO), 2.17 (2H, t, J = 7.2 Hz, CH2COOK), 3.13 (2H,
m, CH2NH), 3.22 (4H, m, SCH2CH2S); 13C-NMR (101 MHz, D2O): δ = 13.2 (CH3), 21.9 (CH2CH3), 25.3
(CH2CH2CS2), 26.1 (CH2), 28.2 (CH2), 28.4 (CH2), 28.5 (CH2), 28.7 (CH2), 31.1 (CH2CONH), 35.5 (CH2CONH),
38.2 (CH2CS2), 38.7 (SCH2CH2S), 39.4 (SCH2CH2S), 50.1 (CH2COO), 67.1 (CS2), 175.7 (C=O), 177.4 (C=O), 180.9
(C=O); m/z (ESI-MS): found [M-H]- 374.2, C18H32NO3S2- requires 374.2.
1.3.11. Acetoxymethyl-2-(2-(3-decanamidopropyl)-1,3-dithiolan-2-yl)acetate (19)
Compound 26 (545 mg, 1.32 mmol) was suspended in dry THF (9 mL) and bromoethylacetate (140 µL,
1.58 mmol) was added. The reaction was stirred at room temperature overnight and afterwards
concentrated under vacuum. The crude product was purified by column chromatography (EtOAc/CycloHex
6:4) to yield 19 as a white powder (220 mg, 40%).
1H-NMR (400 MHz, CDCl3): δ = 0.88 (3H, t, J = 6.4 Hz, CH3), 1.29 (12H, br s, (CH2)6), 1.27 (2H, m,
CH2CH2CONH), 1.56 (2H, m, NHCH2CH2), 2.12 (3H, s, COCH3), 2.13 (2H, m, CH2CS2), 2.13 (2H, m, CH2CONH),
3.08 (2H, s, CH2COCH3), 3.30 (2H, m, NHCH2), 3.31 (4H, s, SCH2CH2S), 5.55 (1H, br s, NH), 5.75 (1H, s,
OCH2O); 13C-NMR (101 MHz, CDCl3): δ = 14.1 (CH3), 20.7 (COCH3), 22.7 (CH2CH3), 22.7 (CH2CH2CS2), 25.8
(CH2), 27.1 (CH2), 29.3 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 36.9 (CH2CONH), 39.1 (CH2CS2), 39.4
(SCH2CH2S), 39.8 (NHCH2), 48.2 (CH2COO), 66.4 (CS2), 79.2 (OCH2O), 168.6 (C=O), 169.9 (C=O),173.2 (C=O);
m/z (HR-ESI-MS): found [M+Na]+ 470.2006, C21H37NNaO5S2+ requires 470.2005.
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1.3.12. Acetoxymethyl-6-decanamido-3-oxohexanoate (20)
Compound 19 (200 mg, 0.45 mmol) was dissolved in acetonitrile (12 mL) and water (1.12 mL) and
[bis(trifluoroacetoxy)iodo]benzene (576 mg, 1.34 mmol) was added. The reaction was stirred at room
temperature for 10 minutes, quenched with H2O/NaHCO3 sat./Na2S2O3 sat. solution (1:1:1, 12 mL). The
organic solvent was separated and removed under reduced pressure. The final mixture was extracted with
dichloromethane (3x5 mL), the organic layer was dried, filtered, concentrated and the crude was purified
by column chromatography (eluent 7:3 EtOAc/CycloHex). 20 was obtained as a white powder (80 mg, 40%).
The compound was further purified by semipreparative HPLC (Rt = 17.7 min, with a gradient elution from 30
to 70% MeCN over 10 min, 70% MeCN for 10 min, then 70 to 100% MeCN over 5 min).
1H-NMR (400 MHz, CDCl3): δ = 0.86 (3H, t, J = 6.1 Hz, CH3), 1.25 (12H, br s, (CH2)6), 1.59 (2H, m,
CH2CH2CONH), 1.80 (2H, m, CH2CH2CO), 2.12 (3H, s, COCH3), 2.14 (2H, m, CH2CONH), 2.60 (2H, t, J = 6.8 Hz,
CH2CO), 3.24 (2H, m, NHCH2), 3.50 (2H, s, COCH2CO), 5.73 (1H, br s, NH), 5.75 (1H, s, OCH2O); 13C-NMR
(101 MHz, CDCl3): δ = 14.1 (CH3), 20.5 (COCH3), 22.1 (CH2CH3), 23.9 (CH2), 25.8 (CH2), 29.3 (CH2), 29.3 (CH2),
29.4 (CH2), 31.8 (CH2), 31.8 (CH2), 38.5 (CH2CONH), 40.4 (NHCH2), 40.4 (CH2COCH2), 48.7 (COCH2CO), 79.5
(OCH2O), 166.1 (C=O), 169.7 (C=O), 173.4 (C=O), 200.8 (C=O); m/z (HR-ESI-MS): found [M+Na]+ 394.2202,
C19H33NNaO6+ requires 394.2200.
1.3.13. Synthesis of methyl 6-(10-azidodecanamido)-3-oxohexanoate (4)
Compound 4 was prepared as reported previously.5
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2. Growth of S. lasaliensis ACP12 (S970A) and mass spectrometry analysis
S. lasaliensis ACP12 (S970A)6 was grown in M79 medium (10 mL) for 3 days at 30 ºC. This seed culture was
used to inoculate MYM liquid cultures (10 mL, in duplicate copy). These were incubated at 30°C and shaken
at 180 rpm for a total of 5 days. After the first day of incubation, the 0.01 mmol of probes (3, 21, 8) were
added daily in 50 µL of MeOH. Control liquid cultures in absence of the probes were also prepared. After
incubation all the cultures were extracted twice with ethyl acetate (10 mL x 2). The extracts were
concentrated and the residues were redissolved in HPLC-grade methanol (1 mL) for mass spectrometry
analysis. The Staudinger reaction on organic extracts of feeding experiments with probe 8 was performed
as described preiviously.5
HPLC-HR-ESI-MS analyses of S. lasaliensis ACP12 (S970A) extracts were performed on:
1) a MaXis Impact UHR-ESI-TOF (Bruker Daltonics). Method 1: 10% B 0-2.7 min; 10-100% B 2.7-42.7 min;
100% B 42.7-62.7 min; 100-10% B 62.7-65.7 min; 10% B 65.7-77.7 min, using an Acquity UPLC HSS T3
column at a flow rate of 0.05 mL/min. Method 2: 5% B 0-5.3 min; 5-100% B 5.3-17.3 min; 100% B 17.3-22.3
min; 100-5% B 22.3-25.3 min; 5% B 25.3-35.3 min, using Agilent Eclipse C18 at a flow rate of 0.2 mL/min.
Spectra were recorded in positive ionisation mode, scanning from m/z 50 to 2000, with Capillary Voltage
set at 3500 V, Dry Heater set at 180°C and UV Lamp set at 210 nm. Selected ion search within 5 ppm was
performed, as well as high resolution fragmentation for the putative biosynthetic intermediates.
2) a Thermo Orbitrap Fusion (Q-OT-qIT, Thermo) instrument. Reversed phase chromatography was used to
separate the mixtures prior to MS analysis. Two columns were utilized: an Acclaim PepMap µ-precolumn
cartridge 300 µm i.d. x 5 mm 5 μm 100 Å and an Acclaim PepMap RSLC 75 µm x 15 cm 2 µm 100 Å (Thermo
Scientific). The columns were installed on an Ultimate 3000 RSLCnano system (Dionex). Mobile phase buffer
A was composed of 0.1% (v/v) aqueous formic acid and mobile phase B was composed of 100% acetonitrile
containing 0.1% (v/v) formic acid. Samples were loaded onto the µ-precolumn equilibrated in 2% aqueous
acetonitrile containing 0.1% (v/v) trifluoroacetic acid for 8 min at 10 µL min-1 after which compounds were
eluted onto the analytical column following a 75 min gradient for which the mobile phase B concentration
was increased from 50% B to 99.5% over 15 min, then maintained at 99.5% B for 35 minutes, then
decreased to 50% over 16 min, followed by a 9 min wash at 50% B. Eluting cations were converted to gas-
phase ions by electrospray ionization and analyzed. Survey scans of precursors from 150 to 1500 m/z were
performed at 60K resolution (at 200 m/z) with a 5 × 105 ion count target. Tandem MS was performed by
isolation at 0.7 Th with the quadrupole, HCD fragmentation with normalized collision energy of 30, and
rapid scan MS analysis in the ion trap. The MS2 ion count target was set to 104 and the maximum injection
time was 35 ms. A filter targeted inclusion mass list was used to select the precursor ions. The dynamic
6M. Tosin, L. Smith, P. F. Leadlay Angew. Chem. Int. Ed, 2011, 50, 11930-11933.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
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p a g e 15 | 105
exclusion duration was set to 45 s with a 10 ppm tolerance around the selected precursor and its isotopes.
Monoisotopic precursor selection was turned on. The instrument was run in top speed mode with 5 s
cycles, meaning the instrument would continuously perform MS2 events until the list of nonexcluded
precursors diminishes to zero or 5 s, whichever is shorter. Fusion runs were performed with Survey scans of
precursors from 150 to 1500 m/z 60K resolution (at 200 m/z) with a 1 × 106 ion count target. Tandem MS
was performed by isolation at 1.8 Th with the ion-trap, CAD fragmentation with normalized collision energy
of 32, and 15K resolution scan MS analysis in the Orbitrap. The data dependent top 20 precursors were
selected for MS2. MS2 ion count target was set to 4 × 106 and the max injection time was 50 ms. The
dynamic exclusion duration was set to 40 s with a 10 ppm tolerance around the selected precursor and its
isotopes.
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2.1.Summary of captured intermediates for S. lasaliensis ACP12(S970A)
Figure 1S: Overview of ester probes utilized for in vivo intermediate capture in S. lasaliensis ACP12 (S970A) togetherwith estimated in vivo deprotection yields (qualitative estimation by LC-MS).
Table 1S: Overview of intermediates captured and characterized from S. lasaliensis ACP12 (S970A) viaprobes 1a-b, 3, 4, 15a-b and 20 (detected on Maxis impact, MI, and Orbitrap Fusion, OF).
intermediate putative structure
probes
1 a-b[c]
R1= CH3/CD3
P= CH3
short chain/
Me
probe
3[b]
R1= (CH2)8CH3
P= CH3
deca chain/
Me
probe
4[c]
R1= (CH2)8N3
P= CH3
N3 deca/
Me
probes
15 a-b[d]
R1= CH3/CD3
P= CH2CO2CH3
short chain/
AM
probe
20[d]
R1= (CH2)8CH3
P= CH2CO2CH3
deca chain/
AM
Diketides(MI, OF) (MI, OF) (MI, OF)
(OF) (MI, OF) (MI, OF)
(OF) (MI, OF)
Triketides
(OF) (MI, OF) (OF)
(OF) (MI, OF)
(OF) (MI, OF)
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(MI, OF) (OF) (MI, OF)
Tetraketides
(MI, OF) (MI, OF)
(OF) (MI, OF) (MI, OF)
(OF) (MI, OF) (OF)
PentaketidesR1
O
NH
O O
(MI, OF) (MI, OF) (MI, OF)
(MI, OF) (OF) (OF)
(MII) (MI)
(OF) (MI, OF) (MI, OF)
Hexaketides
(MI, OF) (MI, OF)
(MI, OF)
Heptaketides
(MI) (MI)
Octaketides
(MI, OF) (MI, OF)
Nonaketides
(MI) (MI) (MI)
(MI) (MI)
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Undecaketides
(MI)
(MI
Dodecaketides
(MI) (MI) (MI)
(MI)
(MI) (MI) (MI)
(MI) (MI) (MI)
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2.2. Intermediates captured by methyl 6-decanamido-3-oxohexanoate (3)
5 10 15 20 25 30
Time (min)
0
200000000
400000000
600000000
800000000
0
500000000
1000000000
1500000000
2000000000
2500000000
3000000000
Inte
nsi
ty13.82
13.65
NL: 3.44E9
m/z= 314.2310-314.2342 F:
FTMS + p NSI Full ms
NL: 8.52E8
m/z= 256.2258-256.2284 F:
FTMS + p NSI Full ms
RT: 13.81
314 315 316
m/z
0
20
40
60
80
100
Rel
ativ
eA
bu
nd
ance
314.2328
315.2327
316.2358
RT: 13.65
256 257 258
m/z
0
20
40
60
80
100
Rel
ativ
eA
bu
nd
ance
256.2274
257.2293
258.2328
3 3I
A)
B)
C) D)
Figure 2S: (A) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in thepresence of 3 (final concentration 4 mM): [M+H]
+extracted ion chromatogram (EIC) for probe 3 (Rt = 13.82 min) and
(B) EIC for the decarboxylated probe 31 (Rt = 13.65 min) are shown. (C) The high resolution masses of probe 3 and (D)of the hydrolysed-decarboxylated probe 31 are shown.
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298.2384
299.2425
0.0
0.5
1.0
1.5
5x10
Intens.
298 299 300 301 m/z
5 10 15 20 25 30
Time (min)
0
5000000
10000000
Inte
nsi
ty
13.01 NL: 1.45E7
m/z= 298.2362-298.2392F: FTMS + p NSI Full ms
20.2
18.0 18.5 19.0 19.5 20.0 20.5 21.0 21.5 22.0 Time [min]
0
1
2
5x10
Intens.EIC 298.2377 ± 0.0100
controlfeeding
RT: 13.00Full ms2 [email protected]
120 140 160 180 200 220 240 260 280 300
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
144.1020
127.0755
155.1433
172.1697
C7H11O2+
calc: 127.0754
-NH3
A)
B)
C)
D)
20.2 min
300.2441 32
Figure 3S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 3 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 32 (Rt = 20.2 min). (C) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in the presence of 3(final concentration 4 mM): EIC (Rt = 13.01 min) and (D) fragmentation of 32 with putative fragment structuralassignment.
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5 10 15 20 25
Time (min)
0
50000
100000
150000
200000
250000In
ten
sity
12.79
NL: 2.97E5m/z= 300.2518-300.2548F: FTMS + p NSI Full ms
RT: 12.62-13.19
Full ms2 [email protected]
150 155 160 165 170 175 180
m/z
0
20
40
60
80
100
120
140
160
180
200
220
Rel
ativ
eA
bu
nd
ance
155.1428
172.1694
A)
B) 33
Figure 4S: (A) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in thepresence of 3 (final concentration 4 mM): EIC (Rt = 12.79 min) and (B) fragmentation of putative intermediate 33 withputative fragment structural assignment.
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5 10 15 20 25 30
Time (min)
0
10000000
20000000
30000000
40000000
50000000
Inte
nsi
ty
14.75 NL: 5.04E7
m/z=282.2414-282.2442
F: FTMS + p NSI Full ms
m/z
O
NH
OH
C17H32NO2+
m/z[M+H]+: 282.2428
C7H11O+
m/z [M+H]+: 111.0804
RT: 14.74
Full ms2 [email protected]
110.0 110.5 111.0
m/z
0
20
40
60
80
100
Rel
ativ
eA
bu
nd
ance
110.0965
111.0805
RT: 14.74
Full ms2 [email protected]
120 140 160 180 200 220 240 260 280
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
155.1430172.1691 282.2428
RT: 14.75
Full ms [150.0000-1500.0000]
282.0 282.5 283.0 283.5 284.0 284.5
m/z
0
20
40
60
80
100
Rel
ativ
eA
bu
nd
ance
282.2422
283.2458
284.2490
A)
B)
C)
34
Figure 5S: (A) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in thein the presence of 3 (final concentration 4 mM): [M+H]
+extracted ion chromatogram (EIC), (B) high resolution mass
(Rt = 14.75 min) and (C) fragmentation of putative intermediate 34 with putative fragment structural assignment areshown.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 23 | 105
Figure 6S: (A) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in thepresence of 3 (final concentration 4 mM): EIC (Rt = 15.43 min) and (B) fragmentation of putative intermediate 35 withputative fragment structural assignment.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 24 | 105
5 10 15 20 25 30
Time (min)
0
200000
400000
600000In
ten
sity
15.88 NL: 7.58E5
m/z= 354.2985-354.3021
F: FTMS + p NSI Full ms
NH OH
C11H20NO+
m/z [M+H]+: 182.1539
O
C10H19O+
m/z [M]+: 155.1430
C11H18N+
m/z [M+H]+: 164.1434
-H2O
RT: 15.87
Full ms2 [email protected]
120 140 160 180 200 220 240
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
182.1539
164.1436
155.1438
A)
B)
36
Figure 7S: A) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in thepresence of 3 (final concentration 4 mM): EIC (Rt = 15.88 min) and (B) fragmentation of putative intermediate 36 withputative fragment structural assignment.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 25 | 105
5 10 15 20 25 30
Time (min)
0
10000
20000
30000
40000
50000
Inte
nsity
12.20
17.05
NL: 5.99E4
m/z=336.2880-336.2914
F: FTMS + p NSI Fullms
NH
C11H18N+
m/z [M+H]+: 164.1434
RT: 17.06
Full ms2 [email protected]
150 160 170 180 190 200 210 220 230 240
m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive
Abu
nd
an
ce
161.0954
164.1421
186.0313215.4193
A)
B)
37
Figure 8S: A) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in thepresence of 3 (final concentration 4 mM): EIC (Rt = 17.05 min) and (B) fragmentation of putative intermediate 37 withputative fragment structural assignment.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 26 | 105
338.3057
339.3083340.3108
59.3 min
0
2
4
4x10
Intens.
338.0 339.0 340.0 m/z
5 10 15 20 25 30
Time (min)
0
100000
200000
300000
400000
500000
Inte
nsi
ty
17.95 NL: 5.50E5m/z= 338.3037-338.3071F: FTMS + p NSI Full ms
59.3
30 35 40 45 50 55 60 65 70 Time [min]0
1
2
3
4
4x10
Intens.EIC 338.3054 ± 0.0100
controlfeeding
-NH3
RT: 17.97
Full ms2 [email protected]
120 140 160 180 200 220 240 260 280 300 320 340
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
166.1595
184.1689
167.1432
C11H19O+
m/z [M+H]+: 167.1430
A)
B)
C)
D)
38
Figure 9S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 3 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 38 (Rt = 59.3 min). (C) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in the presence of 3(final concentration 4 mM): EIC (Rt = 17.95min) and (D) fragmentation of 38 with putative fragment structuralassignment.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 27 | 105
O
NH
O OH
C23H42NO3+
m/z [M+H]+: 380.3159
380.3168
381.3183
382.3221
59.6 min
0.0
0.5
1.0
4x10
Intens.
380 381 382 m/z
59.6
30 35 40 45 50 55 60 65 70 Time [min]
0.00
0.25
0.50
0.75
1.00
4x10
Intens.EIC 380.3159 ± 0.0100
controlfeeding
A)
B)
C)
D)
RT: 17.14
Full ms2 [email protected]
150 200 250 300 350
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lati
veA
bu
nd
ance
208.1685
5 10 15 20 25 30
Time (min)
0
20000
40000
60000
Inte
nsi
ty
17.12
23.61
NL: 7.42E4
m/z= 380.3121-380.3197
F: FTMS + p NSI Full ms
39
Figure 10S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 3 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 39 (Rt = 59.6 min). (C) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in the presence of 3(final concentration 4 mM): EIC (Rt = 17.12 min) and (D) fragmentation of 39 with putative fragment structuralassignment.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 28 | 105
5 10 15 20 25 30
Time (min)
0
50000
100000
150000
200000
Inte
nsity
17.15NL: 2.24E5m/z= 382.3297-382.3335F: FTMS + p NSI Full ms
RT: 17.14
382.5 383.0 383.5 384.0 384.5
m/z
0
20
40
60
80
100
Rela
tive
Abundance
382.3314
383.3347
384.2735
RT: 17.14
Full ms2 [email protected]]
185 190 195 200 205 210 215 220 225
m/z
0
10
20
30
40
50
60
70
80
90
100
110
Rela
tive
Abu
nd
ance
210.1858
192.1750
193.1579
C13H21O+
m/z [M+H]+: 193.1587
-H2O
-NH3O
H3N
C13H24NO+
m/z [M+H]+: 210.1852
A)
B)
C)
40
Figure 11S: (A) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in thein the presence of 3 (final concentration 4 mM): [M+H]
+extracted ion chromatogram (EIC) (Rt = 17.15 min), (B) high
resolution mass and (C) fragmentation of putative intermediate 40 with putative fragment structural assignment areshown.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 29 | 105
5 10 15 20 25 30
Time (min)
0
20000
40000
60000
80000
Inte
nsity
18.58
NL: 9.83E4
m/z= 364.3192-364.3228
F: FTMS + p NSI Full ms
RT: 18.57
Full ms2 [email protected]
185 190 195 200 205 210 215 220
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Abu
nd
an
ce
192.1739
193.1589
C13H21O+
m/z [M+H]+: 193.1587
A)
B)
41
Figure 12S: A) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in thepresence of 3 (final concentration 4 mM): EIC (Rt = 18.58 min) and (B) fragmentation of putative intermediate 41 withputative fragment structural assignment.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 30 | 105
434.3637
435.3672
436.3706
63.2 min
0.00
0.25
0.50
6x10
Intens.
434 435 436 437 m/z
63.2
68.0
30 35 40 45 50 55 60 65 70 Time [min]
0.0
0.5
1.0
6x10
Intens.EIC 434.3629 ± 0.0100
controlfeeding
434.3629
435.3661
436.3690
68.0 min
0.0
0.5
6x10
Intens.
434.5 435.0 435.5 436.0 m/z
A)
B)
42
5 10 15 20 25
Time (min)
0
200000
400000
600000
800000
Inte
nsi
ty
19.00 NL: 8.02E5
m/z= 434.3607-434.3651
F: FTMS + p NSI Full ms
21.16
C)
D)
RT: 19.00Full ms2 [email protected]
150 200 250 300 350 400
m/z
0
20
40
60
80
100
Rel
ativ
eA
bu
nd
ance
262.2158
150 200 250 300 350 400
m/z
0
20
40
60
80
100
Rel
ativ
eA
bu
nd
ance
262.2154 RT: 21.16Full ms2 [email protected]
Figure 13S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 3 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 42 (Rt = 63.2 and 68.0 min).(C) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in the presence of3 (final concentration 4 mM): EIC (Rt = 19.00 and 21.16 min) and (D) fragmentation of 42 with putative fragmentstructural assignment. Double peaks may arise from isomerisation (currently under investigation).
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 31 | 105
RT: 18.08
Full ms2 [email protected]
200 210 220 230 240 250 260 270 280
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lati
veA
bu
nd
ance
264.2317
246.2206
62.6
66.6
30 35 40 45 50 55 60 65 70 Time [min]
0.0
0.5
1.0
1.5
2.0
4x10
Intens.EIC 436.3785 ± 0.0100
controlfeeding
5 10 15 20 25 30
Time (min)
0
20
40
60
80
100
Rel
ativ
eA
bu
nd
ance
18.08
18.31
NL: 2.84E5
m/z= 436.3763-436.3807
F: FTMS + p NSI Full ms
A)
B)
C)
D)
436.3756
437.3798438.3902
66.6 min
0
1
24x10
Intens.
436 437 438 m/z
436.3796
437.3838
61.8 min
0
1
4x10
Intens.
436 437 438 439 m/z
43
Figure 14S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 3 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 43 (Rt = 62.6 and 66.6 min).(C) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in the presence of3 (final concentration 4 mM): EIC (Rt = 18.08 and 18.31 min) and (D) fragmentation of 43 with putative fragmentstructural assignment. Double peaks may arise from isomerisation (currently under investigation).
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 32 | 105
A)
B)
62.4
30 35 40 45 50 55 60 65 70 Time [min]
0.0
0.5
1.0
1.5
2.0
4x10
Intens.
418.3685
419.3707420.3467
62.4 min
0.0
0.5
1.0
4x10
Intens.
418.0 419.0 420.0 m/z
EIC 418.3680 ± 0.0100
controlfeeding
44
Figure 15S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 3 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 44 (Rt = 62.4 min).
5 10 15 20 25 30
Time (min)
0
5000
10000
15000
20000
25000
30000
Inte
nsi
ty
20.46
NL: 1.47E4
m/z= 420.3815-420.3857
F: FTMS + p NSI Full ms
RT: 20.52
Full ms2 [email protected]
180 200 220 240 260 280 300 320
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
248.2373
A)
B)45
Figure 16S: A) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in thepresence of 3 (final concentration 4 mM): EIC (Rt = 20.46 min) and (B) fragmentation of putative intermediate 45 withputative fragment structural assignment.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 33 | 105
72.8
30 35 40 45 50 55 60 65 70 Time [min]0
2
4
6
4x10
Intens.
8 10 12 14 16 18 20 22 24 26 28 30
Time (min)
0
50000
100000
150000
Inte
nsi
ty
21.85
NL: 1.61E5
m/z= 476.4074-476.4122
F: FTMS + p NSI Full ms
476.4101
477.4136
478.4205
72.8 min
0
2
4
4x10
Intens.
476 477 478 m/z
RT: 21.86
Full ms2 [email protected]
200 220 240 260 280 300 320 340 360 380
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
304.2641
EIC 476.4098 ± 0.0100
controlfeeding
A)
B)
C)
D)
46
Figure 17S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 3 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 46 (Rt = 72.8 min). (C) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in the presence of 3(final concentration 4 mM): EIC (Rt = 21.85 min) and (D) fragmentation of 46 with putative fragment structuralassignment.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 34 | 105
65.1
30 35 40 45 50 55 60 65 70 Time [min]
0.0
0.2
0.4
0.6
0.8
4x10
Intens.
5 10 15 20 25 30
Time (min)
0
10000
20000
30000
40000
Inte
nsi
ty
19.59
NL: 1.81E4
m/z= 478.4231-478.4279
F: FTMS + p NSI Full ms
478.4265
479.4279
480.4240
65.1 min
0.00
0.25
0.50
4x10
Intens.
478 479 480 m/z
RT: 19.58
Full ms2 [email protected]
260 280 300 320 340 360 380
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
306.2817
NH
C20H36NO+
m/z [M+H]+: 306.2791
OH
EIC 478.4255 ± 0.0100
controlfeeding
A)
B)
C)
D)
47
Figure 18S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 3 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 47 (Rt = 65.1 min). (C) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in the presence of 3(final concentration 4 mM): EIC (Rt = 19.59 min) and (D) fragmentation of 47 with putative fragment structuralassignment.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 35 | 105
70.2 73.1
30 35 40 45 50 55 60 65 70 Time [min]
0
1
2
4x10
Intens.
548.4677
549.4708550.4758
70.2 min
0
1
2
4x10
Intens.
548 549 550 551 m/z
548.4675
549.4707
73.1 min
0
1
2
4x10
Intens.
548 549 550 551 m/z
550.4759
EIC 548.4673 ± 0.0100
controlfeeding
A)
B)
48
Figure 19S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 3 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 48 (Rt = 70.2 and 73.1 min).Double peaks may arise from intramolecular cyclisation or isomerisation (currently under investigation).
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 36 | 105
72.9
30 35 40 45 50 55 60 65 70 Time [min]
0
1
2
3
5x10
Intens.
604.4941
605.4974
606.5068
72.8 min
0
1
2
3
5x10
Intens.
604 605 606 607 608 m/z
5 10 15 20 25 30 35
Time (min)
0
100000
200000
300000
400000
500000
600000
Inte
nsi
ty
21.40 NL: 6.31E5
m/z= 604.4906-604.4966
F: FTMS + p NSI Full ms
RT: 21.40
Full ms2 [email protected]
150 200 250 300 350 400 450 500 550 600
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
282.2062
128.0706
151.1481 205.1951
246.1234
163.1485364.2861
300.2159
432.4531
O
NH
O OC16H28NO3
+
m/z [M]+: 282.2064
EIC 604.4936 ± 0.0100
controlfeeding
A)
B)
C)
D)
49
Figure 20S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 3 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 49 (Rt = 72.9 min). (C) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in the presence of 3(final concentration 4 mM): EIC (Rt = 21.40 min) and (D) fragmentation of 49 with putative fragment structuralassignment. A compound of m/z [M+H]= 586.4830 (C37H64NO4
+) resulting from the loss of water from 49 was also
detected at an identical retention time (and characterised by the same fragment highlighted in D)).
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 37 | 105
73.0
30 35 40 45 50 55 60 65 70 Time [min]
0
2
4
4x10
Intens.
662.5368
663.5399
664.5409
73.0 min
0
1
2
3
4x10
Intens.
662 663 664 665 m/z
Time (min)
5 10 15 20 25 300
10000
20000
30000
40000
Inte
nsi
ty
17.83
NL: 4.98E4
m/z= 684.5140-684.5208
F: FTMS + p NSI Full ms
RT: 17.86
Full ms2 [email protected]
150 200 250 300 350 400 450 500 550 600 650
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
282.2061
EIC 662.5354 ± 0.0100
controlfeeding
A)
B)
C)
D)
50
Figure 21S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 3 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 50 (Rt = 73.0 min). (C) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in the presence of 3(final concentration 4 mM): EIC (Rt = 17.83 min) and (D) fragmentation of 50 with putative fragment structuralassignment.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 38 | 105
72.5
30 35 40 45 50 55 60 65 70 Time [min]
0.00
0.25
0.50
0.75
1.00
4x10
Intens.
732.5777
733.5797
734.5914
72.5 min
0.0
0.5
1.0
1.54x10
Intens.
732 733 734 m/z
EIC 732.5773 ± 0.0100
controlfeeding
B)
52
Figure 22S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 3 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 52 (Rt = 72.5 min).
A)
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 39 | 105
377.2655
472.3009 826.5801
+MS2(826.5797), 44.8eV, 67.6 min
0.0
0.2
0.4
0.6
0.8
1.0
1.24x10
Intens.
300 400 500 600 700 800 m/z
67.1
67.7
30 35 40 45 50 55 60 65 70 Time [min]
0
2000
4000
6000
8000
Intens.
826.5806
827.5761
828.5900
67.1 min
0
2000
4000
Intens.
826 827 828 829 m/z
827.5844
826.5806
828.5845
67.7 min
0
2000
4000
6000
Intens.
826 827 828 829 m/z
EIC 826.5804 ± 0.0100
controlfeeding
A)
B)
C)
53
Figure 23S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 3 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC), (B) high resolution mass are shown for the putative intermediate 53 (Rt = 67.1 and 67.7 min) and(C) fragmentation of 53 with putative fragment structural assignment. Double peaks may arise from intramolecularcyclisation or isomerisation (currently under investigation).
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 40 | 105
70.2
70.6
30 35 40 45 50 55 60 65 70 Time [min]0
1
2
5x10
Intens.
810.5856
811.5889
812.5917
70.2 min
0.0
0.5
1.0
1.5
2.0
5x10
Intens.
810 811 812 813m/z
810.5754
811.5788
70.6 min
0.0
0.5
1.0
5x10
Intens.
810 811 812 813m/z
812.5914
EIC 810.5754 ± 0.0100
controlfeeding
B)
54
Figure 24S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 3 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 54 (Rt = 70.2 and 70.6 min).Double peaks may arise from intramolecular cyclisation or isomerisation (currently under investigation).
70.4
73.3
20 30 40 50 60 70 Time [min]
0
1
2
5x10
Intens.
808.5709
810.5777
809.5745
70.4 min
0.0
0.2
0.4
0.6
5x10
Intens.
809 810 m/z
808.5715
810.5788
809.5756
73.3 min
0
1
2
5x10
Intens.
809 810 m/z
EIC 808.5698 ± 0.0100
controlfeeding
A)
B)
55
A)
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 41 | 105
377.2671
454.2941
808.5719
+MS2(809.5757), 44.3 eV, 70.5 min
0
1
2
3
4
5
4x10
Intens.
300 400 500 600 700 800 m/z
377.2672
454.2936
808.5731
+MS2(809.5727), 44.3 eV, 73.4 min
0
2
4
6
4x10
Intens.
300 400 500 600 700 800 m/z
C)
Figure 25S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 3 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC), (B) high resolution mass are shown for the putative intermediate 55 (Rt = 70.5 and 73.4 min) and(C) fragmentation of 55 with putative fragment structural assignment. Double peaks may arise from intramolecularcyclisation or isomerisation (currently under investigation).
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 42 | 105
2.3. Intermediate capture by N-(4,6-dioxoheptyl)decanamide (20)
0 5 10 15 20 25
Time (min)
0
20
40
60
80
100
0
20
40
60
80
100
Rel
ativ
eA
bu
nd
ance
13.44
13.00
NL: 7.31E7
m/z= 372.2344-372.2418
FTMS + p NSI Full ms
NL: 2.90E9
m/z= 256.2245-256.2297
FTMS + p NSI Full ms
RT: 13.00
256.5 257.0 257.5 258.0
m/z
0
20
40
60
80
100
Rel
ativ
eA
bu
nd
ance
256.2271
257.2284
258.2318
RT: 13.44
372.5 373.0 373.5 374.0
m/z
0
20
40
60
80
100
Rel
ativ
eA
bu
nd
ance
372.2377
373.2408
374.2433
A)
B)
C) D)
2031
Figure 26S: (A) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in thein the presence of 20 (final concentration 4 mM): [M+H]
+extracted ion chromatogram (EIC) for probe 20 (Rt = 13.44
min) and (B) EIC for the decarboxylated probe 31 (Rt = 13.00 min) are shown. (C) The high resolution masses of probe20 and (D) of the hydrolysed- decarboxylated probe 31 are shown.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 43 | 105
4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
1000000
2000000
3000000
4000000
5000000
Inte
nsi
ty
12.99 NL: 5.87E6
m/z= 298.2362-298.2392
F: FTMS + p NSI Full ms
RT: 12.99Full ms2 [email protected]
120 140 160 180 200 220 240 260 280 300
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
144.1019
127.0754
155.1431
172.1697
H3N
O O
C7H14NO2+
m/z [M+H]+: 144.1019
C7H11O2+
calc: 127.0754
-NH3
A)
B)
C)
D)
EIC 298.2377 ± 0.0100
controlfeeding
298.2385
299.2420 300.2477
20.2 min
0
1
2
5x10
Intens.
298.0 299.0 300.0 m/z
20.2
5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5Time [min]0
1
2
5x10
Intens.
32
Figure 27S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 20 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 32 (Rt = 20.2 min). (C) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in the presence of 20(final concentration 4 mM): EIC (Rt = 12.99 min) and (D) fragmentation of 32 with putative fragment structuralassignment.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 44 | 105
48.4
30 35 40 45 50 55 60 65 Time [min]
0.00
0.25
0.50
0.75
6x10
Intens.
5 10 15 20 25 30
Time (min)
0
5000000
10000000
15000000
20000000
Inte
nsi
ty
12.88
NL: 2.46E7
m/z= 300.2518-300.2548
F: FTMS + p NSI Full ms
300.2530
301.2564302.2587
48.4 min
0.00
0.25
0.50
0.75
6x10
Intens.
300 301 302 303m/z
EIC 300.2533 ± 0.0100
controlfeeding
A)
B)
C)
D)
RT: 12.89
Full ms2 [email protected]
145 150 155 160 165 170 175
m/z
0
20
40
60
80
100
120
140
160
Rel
ativ
eA
bu
nd
ance
155.1431
172.1698
33
Figure 28S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 20 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 33 (Rt = 48.4 min). (C) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in the presence of 20(final concentration 4 mM): EIC (Rt = 12.88 min) and (D) fragmentation of 33 with putative fragment structuralassignment.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 45 | 105
15.8
5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5Time [min]
0
2
4
5x10
Intens.
5 10 15 20 25 30
Time (min)
0
10000000
20000000
30000000
40000000
50000000
60000000
Inte
nsi
ty
15.26NL: 6.18E7
m/z= 282.2414-282.2442
F: FTMS + p NSI Full ms
110.5
m/z
0
20
40
60
80
100
120
Rel
ativ
eA
bu
nd
ance
110.0965
RT: 15.26
Full ms2 [email protected]
120 140 160 180 200 220 240 260 280
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
155.1433 172.1700
282.2427
283.2468284.2506
15.8 min
0
1
2
3
4
5x10
Intens.
282 283 284 285 m/z
EIC 282.2414 ± 0.0100
controlfeeding
A)
B)
C)
D)
34
Figure 29S: A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 20 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 34 (Rt = 15.8 min). (C) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in the presence of 20(final concentration 4 mM): EIC (Rt = 15.26 min) and (D) fragmentation of 34 with putative fragment structuralassignment.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 46 | 105
RT: 15.52
352.0 352.5 353.0 353.5 354.0
m/z
0
20
40
60
80
100
Rel
ativ
eA
bu
nd
ance 352.2846
353.2875
5 10 15 20 25 30
Time (min)
0
200000
400000
600000
800000In
ten
sity
15.52 NL: 8.97E5
m/z= 352.2828-352.2864
F: FTMS + p NSI Full ms
RT: 15.48
Full ms2 [email protected]
150 200 250 300 350
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
180.1385
162.1283
-H2O
C11H16N+
m/z [M+H]+: 162.1277
A)
B)
C)
35
Figure 30S: (A) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in thein the presence of 20 (final concentration 4 mM): [M+H]
+extracted ion chromatogram (EIC) (Rt = 15.52 min), (B) high
resolution mass and (C) fragmentation of putative intermediate 35 with putative fragment structural assignment areshown.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 47 | 105
354.2996
355.3031
52.6min
0
1
2
4x10
Intens.
354.5 355.0 355.5 356.0 m/z
52.6
30 35 40 45 50 55 60 65 Time [min]
0
1
2
4x10
Intens.
5 10 15 20 25 30
Time (min)
0
100000
200000
300000
400000
Inte
nsi
ty
15.83NL: 4.78E5
m/z= 354.2985-354.3021
F: FTMS + p NSI Full ms
RT: 15.84
Full ms2 [email protected]
155 160 165 170 175 180 185 190
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
182.1539
164.1431
165.1272161.0959
155.1429182.9924168.0650
-H2O
C11H18N+
m/z [M+H]+: 164.1434
EIC 354.3003 ± 0.0100
controlfeeding
A)
B)
C)
D)
36
Figure 31S: A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 20 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 36 (Rt = 52.6 min). (C) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in the presence of 20(final concentration 4 mM): EIC (Rt = 15.83 min) and (D) fragmentation of 36 with putative fragment structuralassignment.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 48 | 105
52.6
30 35 40 45 50 55 60 65 Time [min]
0
2
4
6
4x10
Intens.
336.2890
337.2925
52.7 min
0
2
4
4x10
Intens.
336 337 338 m/z
10 15 20 25 30 35
Time (min)
0
50000
100000
150000
Inte
nsi
ty
16.94
NL: 1.86E5
m/z= 336.2880-336.2914
F: FTMS + p NSI Full ms
RT: 16.94Full ms2 [email protected]
155 160 165 170 175 180 185 190
m/z
0
10
20
30
40
50
60
70
80
90
100
110
Rel
ativ
eA
bu
nd
ance
164.1425
NH
C11H18N+
m/z [M+H]+: 164.1434
EIC 336.2897 ± 0.0100
controlfeeding
A)
B)
C)
D)
37
Figure 32S: A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 20 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 37 (Rt = 52.6 min). (C) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in the presence of 20(final concentration 4 mM): EIC (Rt = 16.94 min) and (D) fragmentation of 37 with putative fragment structuralassignment.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 49 | 105
58.3
30 35 40 45 50 55 60 65 Time [min]
0
2
4
4x10
Intens.
338.3047
339.3076
58.3 min
0
1
2
3
4
4x10
Intens.
338 339 340 m/z
5 10 15 20 25 30
Time (min)
0
200000
400000
600000
800000
Inte
nsi
ty
17.95NL: 9.77E5
m/z= 338.3037-338.3071
F: FTMS + p NSI Full ms
EIC 338.3054 ± 0.0100
controlfeeding
RT: 17.90
Full ms2 [email protected]
160 165 170 175 180 185 190
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
166.1586
167.1427
184.1688
C11H19O+
m/z [M+H]+: 167.1430
-NH3
A)
B)
C)
D)
38
Figure 33S: A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 20 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 38 (Rt = 58.3 min). (C) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in the presence of 20(final concentration 4 mM): EIC (Rt = 17.95 min) and (D) fragmentation of 38 with putative fragment structuralassignment.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 50 | 105
57.5
30 35 40 45 50 55 60 65 Time [min]
0.0
0.5
1.0
1.5
2.05x10
Intens.
380.3159
381.3193382.3171
57.5 min
0.0
0.5
1.0
1.5
5x10
Intens.
380.5 381.0 381.5 382.0 m/z
EIC 380.3159 ± 0.0100
controlfeeding
5 10 15 20 25 30
Time (min)
0
200000
400000
600000
800000
1000000
Inte
nsi
ty
17.05
NL: 1.10E6
m/z= 380.3121-380.3197
F: FTMS + p NSI Full ms
RT: 16.97
Full ms2 [email protected]
150 200 250 300 350
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
208.1699
190.1594
NH O
C13H22NO+
m/z [M+H]+: 208.1696
-H2O
C13H20N+
m/z [M+H]+: 190.1590
A)
B)
C)
D)
39
Figure 34S: A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 20 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 39 (Rt = 57.5 min). (C) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in the presence of 20(final concentration 4 mM): EIC (Rt = 17.05 min) and (D) fragmentation of 39 with putative fragment structuralassignment.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 51 | 105
24.5
5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 Time [min]0
1
2
3
4x10
Intens.
5 10 15 20 25 30
Time (min)
0
50000
100000
150000
Inte
nsi
ty
17.25
NL: 1.83E5
m/z= 382.3297-382.3335
F: FTMS + p NSI Full ms
382.3308
383.2771384.2739
24.5 min
0
1
2
4x10
Intens.
382.5 383.0 383.5 384.0 m/z
RT: 17.25
Full ms2 [email protected]]
170 180 190 200 210 220
m/z
0
10
20
30
40
50
60
70
80
90
100
110
Rel
ativ
eA
bu
nd
ance
210.1853
192.1743
193.1587
172.1692
C13H21O+
m/z [M+H]+: 193.1587
-H2O
-NH3
EIC 382.3316 ± 0.0100
controlfeeding
A)
B)
C)
D)
40
Figure 35S: A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 20 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 40 (Rt = 24.5 min). (C) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in the presence of 20(final concentration 4 mM): EIC (Rt = 17.25 min) and (D) fragmentation of 40 with putative fragment structuralassignment.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 52 | 105
5 10 15 20 25 30
Time (min)
0
10000
20000
30000
40000
50000
60000In
ten
sity
18.50
NL: 6.71E4
m/z= 364.3192-364.3228
F: FTMS + p NSI Full ms
RT: 18.55
Full ms2 [email protected]
120 140 160 180 200 220 240 260 280 300
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
192.1747
C13H21O+
m/z [M+H]+: 193.1587
193.1595
A)
B)
41
Figure 36S: A) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in thepresence of 20 (final concentration 4 mM): EIC (Rt = 18.50 min) and (B) fragmentation of putative intermediate 41with putative fragment structural assignment.
24.1
25.9
5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5Time [min]
0
1
2
3
6x10
Intens.
456.3458
457.3491458.3528
24.1 min
0
1
2
6x10
Intens.
456 457 458 m/z
456.3462
457.3497
458.3534
25.9 min
0
2
4
6
5x10
Intens.
456 457 458 m/z
EIC 456.3448 ± 0.0100
controlfeeding
A)
B)
42
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 53 | 105
5 10 15 20 25 30
Time (min)
0
200000
400000
600000
800000
Inte
nsi
ty
19.16NL: 8.26E5
m/z= 434.3607-434.3651
F: FTMS + p NSI Full ms
RT: 19.11
Full ms2 [email protected]
150 200 250 300 350 400
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
262.2154
RT: 21.12
Full ms2 [email protected]]
150 200 250 300 350 400
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lati
veA
bu
nd
ance
262.2156
C)
D)
21.12
Figure 37S: A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 20 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 42 (Rt = 24.1 and 25.9 min).(C) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in the presence of20 (final concentration 4 mM): EIC (Rt = 19.16 and 21.12 min) and (D) fragmentation of 42 with putative fragmentstructural assignment. Double peaks may arise from isomerisation (currently under investigation).
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 54 | 105
RT: 18.89
Full ms2 [email protected]
240 245 250 255 260 265 270
m/z
0
10
20
30
40
50
60
70
80
90
100
110
120
Rel
ativ
eA
bu
nd
ance
264.2307
246.2212
5 10 15 20 25 30 35
Time (min)
0
20
40
60
80
100
Rel
ativ
eA
bu
nd
ance
18.89NL: 1.77E5
m/z= 436.3763-436.3807
F: FTMS + p NSI Full ms
RT: 18.85
437 438 439
m/z
0
50
100
Rel
ativ
eA
bu
nd
ance
436.3786
437.3817438.3794
A)
B)
C)
43
Figure 38S: (A) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in thein the presence of 20 (final concentration 4 mM): [M+H]
+extracted ion chromatogram (EIC) (Rt = 18.89 min), (B) high
resolution mass and (C) fragmentation of putative intermediate 43 with putative fragment structural assignment areshown.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 55 | 105
46.5
30 35 40 45 50 55 60 65 Time [min]
0.0
0.5
1.0
1.5
2.0
5x10
Intens.
442.3650
443.3689444.3721
46.5 min
0
1
5x10
Intens.
442.0 442.5 443.0 443.5 444.0 m/z
A)
B)
EIC 442.3656 ± 0.0100
controlfeeding
C)
D)
5 10 15 20 25 30
Time (min)
0
10000
20000
30000
40000
50000
60000
Inte
nsi
ty
20.50 NL: 6.94E4
m/z= 420.3815-420.3857
F: FTMS + p NSI Full ms
NH
C17H30N+
m/z [M+H]+: 248.2373
C17H29O+
m/z [M+H]+: 249.2213
RT: 20.57
Full ms2 [email protected]]
248.0 248.2 248.4 248.6 248.8 249.0 249.2
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
248.2370
249.2213
45
Figure 39S: A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 20 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 45 (Rt = 46.5 min). (C) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in the presence of 20(final concentration 4 mM): EIC (Rt = 20.50 min) and (D) fragmentation of 45 with putative fragment structuralassignment.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 56 | 105
8 10 12 14 16 18 20 22 24 26 28 30
Time (min)
0
20000
40000
60000
80000
Inte
nsi
ty
21.93
NL: 9.06E4
m/z= 476.4074-476.4122
F: FTMS + p NSI Full ms
m/z
43.2
30 35 40 45 50 55 60 65 Time [min]
0
2
4
6
5x10
Intens.
498.3915
499.3952
43.3 min
0
2
4
5x10
Intens.
498 499 500 m/z
RT: 21.92
Full ms2 [email protected]
220 240 260 280 300 320 340
0
10
20
30
40
50
60
70
80
90
100
110
120
Rel
ativ
eA
bu
nd
ance
304.2628
A)
B)
C)
D)
EIC 476.4098 ± 0.0100
controlfeeding
46
Figure 40S: A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 20 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 46 (Rt = 43.2 min). (C) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in the presence of 20(final concentration 4 mM): EIC (Rt = 21.93 min) and (D) fragmentation of 46 with putative fragment structuralassignment.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 57 | 105
27.1
25 26 27 28 29 30 31 Time [min]
0
2000
4000
6000
Intens.
570.4495
571.4502 572.4952
27.1 min
0.0
0.5
1.0
4x10
Intens.
570 571 572 m/z
A)
B)
EIC 570.4493 ± 0.0100
controlfeeding
48
Figure 41S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 20 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 48 (Rt = 27.1 min).
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 58 | 105
23.0
5 10 15 20 25 30 Time [min]
0.0
0.5
1.0
1.5
5x10
Intens.
5 10 15 20 25 30
Time (min)
0
50000
100000
150000
Inte
nsi
ty
21.44NL: 1.93E5
m/z= 604.4906-604.4966
F: FTMS + p NSI Full ms
627.4730
628.4716
626.4746
23.0 min
0.0
0.5
1.0
1.55x10
Intens.
626 627 628 629 m/z
A)
B)
C)
D)
EIC 626.4755 ± 0.0100
controlfeeding
RT: 21.43
Full ms2 [email protected]
260 270 280 290 300 310 320 330 340
m/z
0
10
20
30
40
50
60
70
80
90
100
110
Rel
ativ
eA
bu
nd
ance
282.2065
49
Figure 42S: A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 20 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 49 (Rt = 23.0 min). (C) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in the presence of 20(final concentration 4 mM): EIC (Rt = 21.44 min) and (D) fragmentation of 49 with putative fragment structuralassignment. A compound of m/z [M+H]= 586.4830 (C37H64NO4
+) resulting from the loss of water from 49 was also
detected at an identical retention time (and characterised by the same fragment highlighted in D)).
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 59 | 105
71.2
30 35 40 45 50 55 60 65 70 Time [min]
0
1
2
4x10
Intens.
662.5355
663.5396
664.5448
71.2 min
0
1
2
4x10
Intens.
662 663 664 m/z
A)
B)
EIC 662.5354 ± 0.0100
controlfeeding
50
Figure 43S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 20 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 50 (Rt = 71.2 min).
28.9
5 10 15 20 25 30 Time [min]0
1
2
3
4
4x10
Intens.
671.5427
672.5437
670.5403
28.9 min
0
2
4x10
Intens.
671 672 673 m/z
A)
B)
EIC 670.5381 ± 0.0100
controlfeeding
56
Figure 44S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 20 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 56 (Rt = 28.9 min).
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 60 | 105
25.1
27.3
5 10 15 20 25 30 Time [min]
0
2
4
4x10
Intens.
756.5744
757.5780
758.5849
27.3 min
0
1
2
4x10
Intens.
756.5 757.0 757.5 758.0 758.5 m/z
A)
B)
EIC 756.5749 ± 0.0100
controlfeeding
57
Figure 45S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 20 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 57 (Rt = 27.3 min). Doublepeaks may arise from intramolecular cyclisation or isomerisation (currently under investigation).
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 61 | 105
826.5800
827.5832
828.5861
26.0 min
0
1
2
3
4
4x10
Intens.
827 828 m/z
5 10 15 20 25 30 Time [min]
0
1
2
3
4x10
Intens.
26.0
A)
B)
O O
O
OHNa
C21H38NaO4+
m/z [M+Na]+: 377.2662
377.2671
472.3063
826.5833
+MS2(826.5833), 44.8eV, 26.0 min
0.00
0.25
0.50
0.75
1.00
1.25
6x10
Intens.
100 200 300 400 500 600 700 800 m/z
D)
C)
EIC 826.5804 ± 0.0100
controlfeeding
53
Figure 46S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 20 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC), (B) high resolution mass are shown for the putative intermediate 53 (Rt = 26.0 min) and (C)fragmentation of 53 with putative fragment structural assignment.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 62 | 105
25.4
26.7
5 10 15 20 25 30 Time [min]
0.0
0.5
1.0
5x10
Intens.
828.5965
829.5997
830.6028
25.4 min
0.0
0.5
1.0
1.55x10
Intens.
828 829 830 m/z
A)
B)
EIC 828.5960 ± 0.0100
controlfeeding
5 10 15 20 25 30
Time (min)
0
20
40
60
80
100
Rela
tive
Ab
und
an
ce
20.74
19.65
NL: 1.42E5
m/z= 806.6060-806.6222
F: FTMS + p NSI Full ms
RT: 19.04
Full ms2 [email protected]
200 300 400 500 600 700 800
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
377.2661
828.5974
829.6004
830.6042
26.7 min
0.0
0.2
0.4
0.6
0.85x10
Intens.
829 830 m/z
C)
D)
58
Figure 47S: LC-HRMS analysis (MaXis Impact) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in theabsence (red) and in the presence (blue) of 20 (final concentration 4 mM). [M+H]
+extracted ion traces A) and high
resolution mass B) are shown for a putative intermediate 58 (Rt = 25.4 and 26.7 min). Double peaks may arise fromintramolecular cyclisation or isomerisation (currently under investigation).
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 63 | 105
262.1800
377.2666
456.3107
810.5868
MS2(810.5868), 44.3 eV, 27.9 min
0.0
0.5
1.0
1.5
2.0
5x10
Intens.
100 200 300 400 500 600 700 800 m/z
811.5906
812.5939
810.5872
28.0 min
0.0
0.5
1.0
6x10
Intens.
810 811 812 813 m/z
5 10 15 20 25 30 Time [min]
0.0
0.5
1.0
6x10
Intens.
28.0
A)
B)
C)
EIC 810.5854 ± 0.0100
controlfeeding
54
Figure 48S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 20 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC), (B) high resolution mass are shown for the putative intermediate 54 (Rt = 27.9 min) and (C)fragmentation of 54 with putative fragment structural assignment.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 64 | 105
26.9
28.6
5 10 15 20 25 30 Time [min]
0
2
4
6x10
Intens.
808.5718
809.5751
810.5784811.5822
28.6 min
0.0
0.5
1.0
1.5
6x10
Intens.
808 809 810 811 m/z
809.5754
810.5786
808.5725
811.5822
26.9 min
0
2
6x10
Intens.
808 809 810 811 m/z
O
NH
O
Na
HO
OH
O
OO
OH
C47H79NNaO8+
m/z [M+Na]+: 808.5698
A)
B)
EIC 808.5698 ± 0.0100
controlfeeding
55
377.2666
454.2949
808.5715
+MS2(808.5715), 44.3 eV, 28.5 min
0.00
0.25
0.50
0.75
1.00
1.25
6x10
Intens.
400 500 600 700 800 m/z
377.2666
454.2949
+MS2(808.5715), 44.3 eV, 26.9 min
0.00
0.25
0.50
0.75
1.00
1.25
6x10
Intens.
400 500 600 700 800 m/z
808.5728
O O
O
OH
Na
C21H38NaO4+
m/z [M+Na]+: 377.2662 O
NH
O
Na
HO
OC26H41NNaO4+
m/z [M+Na]+: 454.2928
O O
O
OH
Na
C21H38NaO4+
m/z [M+Na]+: 377.2662
O
NH
O
Na
HO
OC26H41NNaO4+
m/z [M+Na]+: 454.2928
C)
Figure 49S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 20 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC), (B) high resolution mass are shown for the putative intermediate 55 (Rt = 26.9 and 28.5min) and(C) fragmentation of 55 with putative fragment structural assignment. Double peaks may arise isomerisation(currently under investigation).
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 65 | 105
2.4. Intermediate capture by methyl 6-(10-azidodecanamido)-3-oxohexanoate (4)
5 10 15 20 25
Time (min)
0
200000000
400000000
600000000
800000000
Inte
nsi
ty
0
100000000
200000000
300000000
Inte
nsi
ty13.02
12.83
NL: 3.52E8
m/z= 355.2322-355.2358
FTMS + p NSI Full ms
NL: 8.62E8
m/z= 297.2270-297.2300
FTMS + p NSI Full ms
RT: 13.05
355 356 357
m/z
0
20
40
60
80
100
Rel
ativ
eA
bu
nd
ance
355.2341
356.2339
357.2374
RT: 12.84
297.0 297.5 298.0 298.5 299.0 299.5
m/z
0
20
40
60
80
100
Rel
ativ
eA
bu
nd
ance
297.2283
298.2284
299.2332
O
NH
O
O
OH
N3
C17H31N4O4+
m/z [M+H]+: 355.23404 59
A)
B)
C) D)
Figure 50S: (A) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in thein the presence of 4 (final concentration 4 mM): [M+H]
+extracted ion chromatogram (EIC) for probe 4 (Rt = 13.02 min)
and (B) EIC for the decarboxylated probe 59 (Rt = 12.83 min) are shown. (C) High resolution masses of probe 4 and (D)of hydrolysed- decarboxylated probe 59 are shown.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 66 | 105
5 10 15 20 25 30
Time (min)
0
1000000
2000000
3000000
4000000
5000000
6000000
Inte
nsi
ty
12.23 NL: 6.66E6
m/z= 339.2374-339.2408
F: FTMS + p NSI Full ms
339.2398
340.2428
40.7 min
0
1
2
3
4
5x10
Intens.
339 340 341 m/z
40.7
20 30 40 50 60 70 Time [min]
0
1
2
3
5x10
Intens.EIC 339.2391 ± 0.0100
controlfeeding
RT: 12.22
Full ms2 [email protected]
120 140 160 180 200 220 240 260 280 300 320 340
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
144.1018
127.0753
168.1382-NH3
C7H11O2+
calc: 127.0754
A)
B)
C)
D)
60
Figure 51S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 4 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 60 (Rt = 40.7 min). (C) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in the presence of 4(final concentration 4 mM): EIC (Rt = 12.23 min) and (D) fragmentation of 60 with putative fragment structuralassignment.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 67 | 105
O
NH
O OHH
N3
C17H33N4O3+
m/z [M+H]+: 341.2547
15.9
5 10 15 20 25 30 Time [min]
0.0
0.5
1.0
1.5
2.0
4x10
Intens.
341.2556
342.2555343.2357
15.9 min
0.0
0.5
1.0
1.5
2.04x10
Intens.
341 342 343 m/z
EIC 442.3656 ± 0.0100
controlfeeding
B)
61
Figure 52S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 4 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 61 (Rt = 15.9 min).
5 10 15 20 250
50000
100000
150000
200000
250000
300000
Inte
nsi
ty
13.83NL: 3.46E5
m/z= 323.2426-323.2458
F: FTMS + p NSI Full ms
RT: 13.83
323.5 324.0 324.5 325.0 325.5
m/z
0
20
40
60
80
100
Rel
ativ
eA
bu
nd
ance
323.2442
325.2122324.2471
C7H11O+
m/z [M+H]+: 111.0804
RT: 13.79
Full ms2 [email protected]]
120 130 140 150 160 170
m/z
0
10
20
30
40
50
60
70
80
90
100
110
Rel
ativ
eA
bu
nd
ance
111.0806
168.1375
RT: 13.79Full ms2 [email protected]]
110.1
m/z
0
20
40
60
80
100
Rel
ativ
eA
bu
nd
ance
110.0965
A)
B)
C)
62
Figure 53S: (A) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in thein the presence of 4 (final concentration 4 mM): [M+H]
+extracted ion chromatogram (EIC) (Rt = 13.83 min), (B) high
resolution mass and (C) fragmentation of putative intermediate 62 with putative fragment structural assignment areshown.
A)
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 68 | 105
O
NH
O OH
N3
C21H37N4O3+
m/z [M+H]+: 393.2860
5 10 15 20 25 30
Time (min)
0
50000
100000
150000
200000
Inte
nsi
ty
12.53NL: 2.01E5
m/z= 393.2840-393.2880
F: FTMS + p NSI Full ms
5 10 15 20 25 30 Time [min]
0
1000
2000
3000
4000
Intens.EIC 393.2860 ± 0.0100
controlfeeding
393.2842
394.2848
395.2859
26.1 min
0
2000
4000
Intens.
393 394 395 m/z
C11H16N+
m/z [M+H]+: 162.1277
RT: 12.47
Full ms2 [email protected]
160 165 170 175 180
m/z
0
10
20
30
40
50
60
70
80
90
100
110
120
130
Re
lati
veA
bu
nd
ance
180.1387
162.1274
168.1380
A)
B)
C)
D)
26.1
63
Figure 54S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 4 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 63 (Rt = 26.1 min). (C) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in the presence of 4(final concentration 4 mM): EIC (Rt = 12.53 min) and (D) fragmentation of 63 with putative fragment structuralassignment.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 69 | 105
5 10 15 20 25 300
100000
200000
300000
400000
500000
Inte
nsi
ty
16.83
NL: 5.57E5
m/z= 379.3049-379.3087
F: FTMS + p NSI Full ms
RT: 16.80
379 380 381 382
m/z
0
20
40
60
80
100
Rel
ativ
eA
bu
nd
ance
379.3061
380.3362
381.3032
NH
C11H20N+
m/z [M+H]+: 166.1590
C11H19O+
m/z [M+H]+: 167.1430
RT: 16.79Full ms2 [email protected]]
160 165 170 175 180 185
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
166.1593
167.1429
A)
B)
C)
64
Figure 55S: (A) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in thein the presence of 4 (final concentration 4 mM): [M+H]
+extracted ion chromatogram (EIC) (Rt = 16.83 min), (B) high
resolution mass and (C) fragmentation of putative intermediate 64 with putative fragment structural assignment areshown.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 70 | 105
21.5
5 10 15 20 25 30 Time [min]0
2000
4000
6000
Intens.EIC 423.3330 ± 0.0100
controlfeeding
423.3296
424.3370
425.3390
21.5 min
0
2000
4000
Intens.
423.0 424.0 425.0 m/z
A)
B)
65
Figure 56S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 4 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 65 (Rt = 21.5 min).
5 10 15 20 25 30
Time (min)
0
20000
40000
60000
80000
Inte
nsi
ty 17.40
NL: 9.84E4
m/z= 405.3204-405.3244
F: FTMS + p NSI Full ms
C13aH21O+
m/z [M+H]+: 193.1587
RT: 17.35
Full ms2 [email protected]]
191.5 192.0 192.5 193.0 193.5 194.0 194.5
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
192. 1743
193.1579
A)
B)
66
Figure 57S: (A) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in thein the presence of 4 (final concentration 4 mM): [M+H]
+extracted ion chromatogram (EIC) (Rt = 17.40 min) and (B)
fragmentation of putative intermediate 66 with putative fragment structural assignment are shown.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 71 | 105
498.3495
497.3463
499.3502
58.9 min
0
2
4
4x10
Intens.
497 498 499 m/z
58.9
63.4
20 30 40 50 60 70 Time [min]
0
1
2
3
4
4x10
Intens.EIC 497.3462 ± 0.0100
controlfeeding
498.3499
497.34663
499.3505
63.4 min
0
2
4
4x10
Intens.
497 498 499 m/z
A)
B)
67
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 72 | 105
5 10 15 20 25 30
Time (min)
0
1000000
2000000
3000000
4000000
5000000
Inte
nsi
ty
18.08NL: 5.60E6
m/z= 475.3619-475.3667
F: FTMS + p NSI Full ms
RT: 18.03
Full ms2 [email protected]
120 140 160 180 200 220 240 260 280
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
262.2161
244.2063
C17H26N+
m/z [M+H]+: 244.2060
-H2O
165 170
m/z
0
20
40
60
80
100
Rel
ativ
eA
bu
nd
ance
168.1378
RT: 19.70
Full ms2 [email protected]
120 140 160 180 200 220 240 260 280
m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive
Abu
nda
nce
262.2161
170
m/z
0
20
40
60
80
100
Rela
tive
Abu
nda
nce
168.1378
C)
D)
O
HN
C10H18NO+
m/z [M+H]+: 168.1383
O
HN
C10H18NO+
m/z [M+H]+: 168.1383
Figure 58S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 4 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 67 (Rt = 58.9 and 63.4 min).(C) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in the presence of4 (final concentration 4 mM): EIC (Rt = 18.08 and 19.70 min) and (D) fragmentation of 67 with putative fragmentstructural assignment. Double peaks may arise from isomerisation (currently under investigation).
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 73 | 105
5 10 15 20 25 30
Time (min)
0
100000
200000
300000
400000
Inte
nsi
ty
17.80 NL: 4.67E5
m/z= 477.3775-477.3823
F: FTMS + p NSI Full ms
RT: 17.75
478 479 480
m/z
0
20
40
60
80
100
Rel
ativ
eA
bu
nd
ance
477.3805
478.3822
479.2024
RT: 17.80
Full ms2 [email protected]
120 140 160 180 200 220 240 260 280
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
168.1382
264.2319
A)
B)
C)
68
Figure 59S: (A) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in thein the presence of 4 (final concentration 4 mM): [M+H]
+extracted ion chromatogram (EIC) (Rt = 17.80 min), (B) high
resolution mass and (C) fragmentation of putative intermediate 68 with putative fragment structural assignment areshown.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 74 | 105
481.3525
482.3570
25.7 min
0
1
2
4x10
Intens.
481 482 483 484 m/z
25.7
5 10 15 20 25 30 Time [min]
0
1
2
34x10
Intens.EIC 481.3513 ± 0.0100
controlfeeding
A)
B)
69
Figure 60S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 4 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 69 (Rt = 25.7 min).
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 75 | 105
25.0
5 10 15 20 25 30 Time [min]
0
1
2
34x10
Intens.
5 10 15 20 25
Time (min)
0
2000
4000
6000
8000
10000
Inte
nsi
ty
20.15NL: 1.19E4
m/z=461.3827-461.3873
F: FTMS + p NSI Full ms
461.3871
462.3944 463.2793
25.0 min
0.0
0.5
1.0
4x10
Intens.
461.5 462.0 462.5 463.0 m/z
EIC 461.3850 ± 0.0100
controlfeeding
NH
C17H30N+
m/z [M+H]+: 248.2373
RT: 20.15
Full ms2 [email protected]]
120 140 160 180 200 220 240 260 280
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
248.2378
B)
C)
D)
70
Figure 61S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 4 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 70 (Rt = 25.0 min). (C) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in the presence of 4(final concentration 4 mM): EIC (Rt = 20.15 min) and (D) fragmentation of 70 with putative fragment structuralassignment.
A)
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 76 | 105
21.5
5 10 15 20 25 30 Time [min]
0
2
4
4x10
Intens.
725.5192
726.5221
21.5 min
0
2
4
4x10
Intens.
725 726 727 m/z
EIC 725.5188 ± 0.0100
controlfeeding
A)
B)
71
Figure 62S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 4 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 71 (Rt = 21.5 min).
711.5408
712.5451
713.5418
20.6 min
0.0
0.5
1.0
1.54x10
Intens.
712 713 m/z
20.6
5 10 15 20 25 30 Time [min]
0.0
0.5
1.0
4x10
Intens.EIC 711.5395 ± 0.0100
controlfeeding
A)
B)
72
Figure 63S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 4 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 72 (Rt = 20.6 min).
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 77 | 105
20.0
5 10 15 20 25 30 Time [min]
0.0
0.2
0.4
0.6
0.8
5x10
Intens.
317.1842
377.2669
485.3028
+MS2(867.5854), 46.0eV, 20.0 min
0.00
0.25
0.50
0.75
1.00
1.25
5x10
Intens.
200 300 400 500 600 700 800 m/z
868.5857
869.5874
867.5819
20.0 min
0
2
4
6
4x10
Intens.
867 868 869 870 m/z
EIC 867.5818 ± 0.0100
controlfeeding
A)
B)
C)
8
73
Figure 64S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 4 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC), (B) high resolution mass are shown for the putative intermediate 73 (Rt = 20.0 min) and (C)fragmentation of 73 with putative fragment structural assignment.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 78 | 105
852.5895
853.5920
851.5868
21.1 min
0.0
0.5
1.0
1.5
5x10
Intens.
851 852 853 854 m/z
21.1
5 10 15 20 25 30 Time [min]
0
1
2
5x10
Intens.EIC 851.5868 ± 0.0100
controlfeeding
377.2666
469.3059
MS2(851.5896), 45.5eV, 21.1 min
0.0
0.5
1.0
1.5
2.0
2.5
5x10
Intens.
300 400 500 600 700 800 m/z
A)
B)
C)
74
Figure 65S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 4 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC), (B) high resolution mass are shown for the putative intermediate 74 (Rt = 21.1 min) and (C)fragmentation of 74 with putative fragment structural assignment.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 79 | 105
20.6
21.2
5 10 15 20 25 30 Time [min]
0
1
2
3
6x10
Intens.EIC 849.5712 ± 0.0100
controlfeeding
850.5737
851.5778
849.5705021.2 min
0
1
2
6x10
Intens.
849 850 851 m/z
850.5753
851.5783
849.571720.6 min
0
2
4
6
5x10
Intens.
850 851 m/z
A)
B)
75
377.2669
467.2908
MS2(849.5743), 45.5eV, 20.5 min
0
2
4
6
8
5x10
Intens.
100 200 300 400 500 600 700 800 m/z
377.2674
467.2899
MS2(849.5713), 45.5eV, 21.2 min
0.0
0.5
1.0
1.5
2.0
2.5
6x10
Intens.
100 200 300 400 500 600 700 800 m/z
C)
Figure 66S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 4 (final concentration 4 mM): [M+H]
+extracted ion
chromatogram (EIC), (B) high resolution mass are shown for the putative intermediate 75 (Rt = 20.6 and 21.2 min) and(C) fragmentation of 75 with putative fragment structural assignment. Double peaks may arise from isomerisation(currently under investigation).
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 80 | 105
2.5.Staudinger-phosphite derivatisation of azido intermediates
16.0
6 8 10 12 14 16 18 20 22 24 Time [min]
0.0
0.5
1.0
5x10
Intens.
2 4 6 8 10 12 14 16
Time (min)
0
200000
400000
600000
800000
Inte
nsi
ty
9.57 NL: 9.75E5
m/z= 421.2420-421.2504
F: FTMS + p NSI Full ms
421.2469
422.2508423.2535
16.0 min
0.0
0.5
1.0
5x10
Intens.
421 422 423 m/z
H3N
O O
C7H14NO2+
m/z [M+H]+: 144.1019
RT: 9.57Full ms2 [email protected]
150 200 250 300 350 400
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
278.1515
250.1568
144.1016
C11H25NO3P+
m/z [M]+: 250.1567
-CO
A)
B)
C)
D)
EIC 421.2462 ± 0.0100
controlfeeding
76
Figure 67S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 4 after treatment with trimethyl phosphite: [M+H]
+extracted
ion chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 76 (Rt = 16.0 min). (C)LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in the presence of 4after treatment with trimethyl phosphite (final concentration 4 mM): EIC (Rt = 9.57 min) and (D) fragmentation of 76with putative fragment structural assignment.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 81 | 105
5 10 15 20 25 30
Time (min)
0
20
40
60
80
100
Rel
ativ
eA
bu
nd
ance
10.44NL: 1.85E6m/z= 405.2472-405.2554F: FTMS + p NSI Full ms
RT: 10.42
405 406 407
m/z
0
50
100
Rel
ativ
eA
bu
nd
ance
405.2511
406.2545
407.2299
RT: 10.44
Full ms2 [email protected]
150 200 250 300 350 400
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
278.1515
250.1565
293.2213
C11H25NO3P+
m/z [M]+: 250.1567
-CO125 130
m/z
0
50
100
Rel
ativ
eA
bu
nd
ance
128.1065
110.0 110.2
m/z
0
20
40
60
80
100
Rel
ativ
eA
bu
nd
ance
110.0965
A)
B)
C)
77
Figure 68S: (A) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in thein the presence of 4 after treatment with trimethyl phosphite: [M+H]
+extracted ion chromatogram (EIC)
(Rt = 10.44 min min), (B) high resolution mass and (C) fragmentation of putative intermediate 77 with putativefragment structural assignment are shown.
19.2
12 14 16 18 20 22 24 26 28 Time [min]
0
2
4
4x10
Intens.19.2
462.3192463.3213
19.2 min
0
2
4
6
4x10
Intens.
461.5 462.0 462.5 463.0 m/z
461.3155
A)
B)
EIC 461.3139 ± 0.0100
controlfeeding
78
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 82 | 105
RT: 13.08
Full ms2 [email protected]
160 180 200 220 240 260 280
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
278.1512
166.1590
250.1561
184.1687
166 167
m/z
0
20
40
60
80
100
120
Rel
ativ
eA
bu
nd
ance 166.1590
167.1430
C11H25NO3P+
m/z [M]+: 250.1567
-CO
C11H19O+
m/z [M+H]+: 167.1430
-NH3
RT: 13.37
Full ms2 [email protected]]
160 180 200 220 240 260 280
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
278.1509
166.1588
250.1561
184.1691
166 167
m/z
0
20
40
60
80
100
120
Rel
ativ
eA
bu
nd
ance 166.1588
167.1427
C11H25NO3P+
m/z [M]+: 250.1567
-CO
C11H19O+
m/z [M+H]+: 167.1430
-NH3
D)
Figure 69S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 4 after treatment with trimethyl phosphite: [M+H]
+extracted
ion chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 78 (Rt = 19.2 min). (C)LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in the presence of 4after treatment with trimethyl phosphite (final concentration 4 mM): EIC (Rt = 13.09 and 13.37 min) and (D)fragmentation of 78 with putative fragment structural assignment.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 83 | 105
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Time (min)
0
20
40
60
80
100
Rel
ativ
eA
bu
nd
ance
13.92NL: 3.59E4
m/z= 487.3285-487.3305
F: FTMS + p NSI Full ms
RT: 13.89
Full ms2 [email protected]
120 140 160 180 200 220 240 260 280
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
278.1515
192.1739
A)
B)
79
Figure 70S: (A) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in thein the presence of 4 after treatment with trimethyl phosphite: [M+H]
+extracted ion chromatogram (EIC)
(Rt = 13.92 min min) and (B) fragmentation of putative intermediate 79 with putative fragment structural assignmentare shown.
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 84 | 105
14 16 18 20 22 24 26 28 30 Time [min]
0.00
0.25
0.50
0.75
6x10
Intens.
557.3712
558.3747
20.6 min
0
2
4
6
5x10
Intens.
557 558 559 m/z
557.3710
558.3744
22.2 min
0.0
0.5
1.0
6x10
Intens.
557 558 559 m/z
20.6
22.2
A)
B)
EIC 557.3714 ± 0.0100
controlfeeding
80
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 85 | 105
2 4 6 8 10 12 14 16 18 20 22 24 26 28
Time (min)
0
20
40
60
80
100
Rel
ativ
eA
bu
nd
ance
14.95
17.02
NL: 2.08E6
m/z= 557.3686-557.3742
F: FTMS + p NSI Full ms
RT: 14.95
Full ms2 [email protected]
120 140 160 180 200 220 240 260 280 300
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
278.1514
262.2162
250.1562
O
(MeO)2(O)PHN
C12H25NO4P+
m/z [M]+: 278.1516
C11H25NO3P+
m/z [M]+: 250.1567
-CO
RT: 16.98Full ms2 [email protected]
120 140 160 180 200 220 240 260 280 300
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
278.1516
262.2161
250.1564
NH O
C17H28NO+
m/z [M+H]+: 262.2165
C11H25NO3P+
m/z [M]+: 250.1567
-CO
C)
D)
Figure 71S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 4 after treatment with trimethyl phosphite: [M+H]
+extracted
ion chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 80 (Rt = 20.6 and 22.2min). (C) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in thepresence of 4 after treatment with trimethyl phosphite (final concentration 4 mM): EIC (Rt = 14.95 and 17.02 min) and(D) fragmentation of 80 with putative fragment structural assignment. Double peaks may arise from isomerisation(currently under investigation).
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 86 | 105
20.1
14 16 18 20 22 24 26 28 Time [min]
0.0
0.5
1.0
4x10
Intens.
559.3874
560.3855
20.1 min
0.0
0.5
1.0
4x10
Intens.
559.0 559.5 560.0 560.5 m/z
A)
B)
EIC 559.3855 ± 0.0100
controlfeeding
81
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 87 | 105
5 10 15 20 25 30
Time (min)
0
20000
40000
60000
80000
100000
Inte
nsi
ty
15.8714.50 NL: 1.07E5
m/z= 559.3843-559.3899
F: FTMS + p NSI Full ms
RT: 14.52
Full ms2 [email protected]
180 190 200 210 220 230 240 250 260 270 280 290
m/z
0
20
40
60
80
100
120
140
Rel
ativ
eA
bu
nd
ance
278.1514
RT: 15.87Full ms2 [email protected]
274 276 278 280 282 284 286
m/z
0
10
20
30
40
50
60
70
80
90
100
110
120
Rel
ativ
eA
bu
nd
ance
278.1528
O
(MeO)2(O)PHN
C12H25NO4P+
m/z [M]+: 278.1516
C)
D)
Figure 72S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 4 after treatment with trimethyl phosphite: [M+H]
+extracted
ion chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 81 (Rt = 20.1 min). (C)LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in the presence of 4after treatment with trimethyl phosphite (final concentration 4 mM): EIC (Rt = 14.50 and 15.87 min) and (D)fragmentation of 81 with putative fragment structural assignment. Double peaks may arise from isomerisation(currently under investigation).
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 88 | 105
23.5
12 14 16 18 20 22 24 26 28 30 Time [min]
0
1
2
4x10
Intens.
599.4187
600.4227
601.4216
23.5 min
0
1
2
4x10
Intens.
599.0 599.5 600.0 600.5 601.0 m/z
B)
EIC 599.4184 ± 0.0100
controlfeeding
82
Figure 73S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 4 treated with trimethyl phosphite: [M+H]
+extracted ion
chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate 82 (Rt = 23.5 min).
5 10 15 20 25 30
Time (min)
0
10000
20000
30000
40000
Inte
nsi
ty
18.60
18.26
NL: 4.02E4
m/z= 727.4985-727.5057
F: FTMS + p NSI Full ms
RT: 18.53
727.5 728.0 728.5 729.0
m/z
0
20
40
60
80
100
120
Rel
ativ
eA
bu
nd
ance 727.5012
728.5005
RT: 18.28
727.5 728.0 728.5 729.0
m/z
0
20
40
60
80
100
120
Rel
ativ
eA
bu
nd
ance 727.5028
728.5069
A)
B)
83
A)
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 89 | 105
RT: 18.31
Full ms2 [email protected]
150 200 250 300 350 400 450
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
278.1528
RT: 18.60
Full ms2 [email protected]
150 200 250 300 350 400 450
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
eA
bu
nd
ance
278.1529
C)
Figure 74S: (A) LC-HRMS analysis (Orbitrap Fusion) of the organic extracts of S. lasaliensis ACP12 (S970A) grown in thein the presence of 4 after treatment with trimethyl phosphite: [M+H]
+extracted ion chromatogram (EIC) (Rt = 18.26
and 18.60 min), (B) high resolution mass and (C) fragmentation of putative intermediate 83 with putative fragmentstructural assignment are shown. Double peaks may arise from intramolecular cyclisation or isomerisation (currentlyunder investigation).
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 90 | 105
21.8
12 14 16 18 20 22 24 26 28 30 Time [min]
0
2
4
4x10
Intens.
949.5888
950.5920
951.5951
21.8 min
0
2
4
4x10
Intens.
950 951 m/z
A)
B)
EIC 949.5889 ± 0.0100
controlfeeding
84
Figure 75S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 4 treated with trimethyl phosphite (final concentration 4mM): [M+H]
+extracted ion chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate
84 (Rt = 21.8 min).
22.421.5
12 14 16 18 20 22 24 26 28 30 Time [min]
0
1000
2000
3000
Intens.
951.6045
952.6081953.6107
21.5 min
0
1000
2000
3000
Intens.
952 953 m/z
951.6042
953.6168
952.6021
22.4 min
0
2000
4000
Intens.
952 953 m/z
A)
B)
EIC 951.6045 ± 0.0100
controlfeeding
85
Figure 76S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 4 treated with trimethyl phosphite (final concentration 4mM): [M+H]
+extracted ion chromatogram (EIC) and (B) high resolution mass are shown for the putative intermediate
85 (Rt = 21.5 and 22.4 min). Multiple peaks may arise from intramolecular cyclisation or isomerisation (currently underinvestigation).
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 91 | 105
O
NH
ONa O OH O O
O
OH
(MeO)2(O)PHN
C49H87N2NaO11P+
m/z [M+Na]+: 933.5940
22.9
12 14 16 18 20 22 24 26 28 30 Time [min]
0.0
0.5
1.0
5x10
Intens.
934.5971
935.5992
933.593722.9 min
0.0
0.5
1.0
5x10
Intens.
934 935 m/z
278.1509377.2660
579.3158
933.5949
MS2(933.5949), 48.0eV, 22.8 min
0.0
0.2
0.4
0.6
0.8
5x10
Intens.
100 200 300 400 500 600 700 800 900 m/z
O
(MeO)2(O)PHN
C12H25NO4P+
m/z [M]+: 278.1516
A)
B)
C)
EIC 933.5940 ± 0.0100
controlfeeding
86
Figure 77S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 4 treated with trimethyl phosphite (final concentration 4mM): [M+H]
+extracted ion chromatogram (EIC), (B) high resolution mass are shown for the putative intermediate 86
(Rt = 22.8 min) and (C) fragmentation of 86 with putative fragment structural assignment. Double peaks may arisefrom intramolecular cyclisation or isomerisation (currently under investigation).
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 92 | 105
23.0
22.6
12 14 16 18 20 22 24 26 28 30 Time [min]
0.0
0.5
1.0
1.5
6x10
Intens.
932.5809
933.5845
931.5775
22.6 min
0.0
0.5
1.0
6x10
Intens.
932 933 m/z
932.5802
933.5837
931.5772
23.0 min
0
1
2
6x10
Intens.
932 933 m/z
A)
B)
EIC 931.5783 ± 0.0100
controlfeeding
87
278.1512
377.2663
577.3010
931.5775
MS2(931.5775), 47.9eV, 22.6 min
0.0
0.2
0.4
0.6
0.8
1.0
1.2
6x10
Intens.
100 200 300 400 500 600 700 800 900 m/z
278.1509377.2660
577.3012
931.5777
MS2(931.5777), 47.9eV, 23.0 min
0.0
0.5
1.0
1.5
2.0
2.5
6x10
Intens.
200 300 400 500 600 700 800 900 m/z
C)
Figure 78S: (A) LC-HRMS analysis (MaXis Impact, method 2) of the organic extracts of S. lasaliensis ACP12 (S970A)grown in the absence (red) and in the presence (blue) of 4 treated with trimethyl phosphite (final concentration 4mM): [M+H]
+extracted ion chromatogram (EIC), (B) high resolution mass are shown for the putative intermediate 87
(Rt = 22.6 and 23.0 min) and (C) fragmentation of 87 with putative fragment structural assignment Double peaks mayarise from isomerisation (currently under investigation).
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 93 | 105
3. Spectra
3.1. 1H- and 13C-NMR of compound 13a (400 MHz, CDCl3)
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 94 | 105
3.2. 1H- and 13C-NMR of compound 13b (400 MHz, CDCl3)
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 95 | 105
3.3. 1H- and 13C-NMR of compound 14a (400 MHz, CDCl3)
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 96 | 105
3.4. 1H- and 13C-NMR of compound 14b (400 MHz, CDCl3)
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 97 | 105
3.5. 1H- and 13C-NMR of compound 16a (400 MHz, CDCl3)
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 98 | 105
3.6. 1H- and 13C-NMR of compound 17a (400 MHz, CDCl3)
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 99 | 105
3.7. 1H- and 13C-NMR of compound 15a (400 MHz, CDCl3)
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 100 | 105
3.8. 1H- and 13C-NMR of compound 15b (400 MHz, CDCl3)
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 101 | 105
3.9. 1H- and 13C-NMR of compound 18 (400 MHz, CDCl3)
NH
O
O
OS S
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 102 | 105
3.10. 1H- and 13C-NMR of compound 24 (400 MHz, D2O)
NH
O
O
OS S
K
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 103 | 105
3.11. 1H- and 13C-NMR of compound 19 (400 MHz, CDCl3)
NH
O
O
OS S
O
O
Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly
Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**
p a g e 104 | 105
3.12. 1H- and 13C-NMR of compound 20