Download - ChemComm2016 ESI final · Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin** p a g e 6 | 105 Compound 13b was prepared in the same

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

[email protected]

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


p a g e 1 | 105

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


3.4. 1H- and 13C-NMR of compound 14b (400 MHz, CDCl3) _________________________________ 96




p a g e 2 | 105



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



p a g e 3 | 105

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.



p a g e 4 | 105

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



p a g e 5 | 105

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.



p a g e 6 | 105

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.



p a g e 7 | 105

(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:



p a g e 8 | 105

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),



p a g e 9 | 105

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.



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.



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



p a g e 12 | 105

(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%).


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.



p a g e 13 | 105

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).


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



p a g e 14 | 105

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.



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.



p a g e 16 | 105

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)



p a g e 17 | 105

(MI, OF) (OF) (MI, OF)

Tetraketides

(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)


Hexaketides

(MI, OF) (MI, OF)

(MI, OF)

Heptaketides

(MI) (MI)

Octaketides

(MI, OF) (MI, OF)

Nonaketides

(MI) (MI) (MI)

(MI) (MI)



p a g e 18 | 105

Undecaketides

(MI)

(MI

Dodecaketides

(MI) (MI) (MI)

(MI)

(MI) (MI) (MI)

(MI) (MI) (MI)



p a g e 19 | 105

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:


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.



p a g e 20 | 105

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.



p a g e 21 | 105

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.



p a g e 22 | 105

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


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


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.



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.



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


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


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.



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


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




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


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


+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.



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


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


39


+extracted ion




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


+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.



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


RT: 18.57


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




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


21.16

C)

D)


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]


+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).



p a g e 31 | 105

RT: 18.08


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


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


+extracted ion




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


+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


RT: 20.52


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




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


476.4101

477.4136

478.4205

72.8 min

0

2

4

4x10

Intens.

476 477 478 m/z

RT: 21.86


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


+extracted ion




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


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


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


+extracted ion




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


+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).



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


RT: 21.40


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


+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)).



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


RT: 17.86


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


+extracted ion




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


+extracted ion


A)



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


+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).



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


+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)



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)


+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).



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


NL: 2.90E9

m/z= 256.2245-256.2297


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


+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.



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



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


+extracted ion




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


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


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


+extracted ion




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


110.5

m/z

0

20

40

60

80

100

120

Rel

ativ

eA

bu

nd

ance

110.0965

RT: 15.26


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




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


RT: 15.48


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






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


RT: 15.84


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


+extracted ion




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



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


+extracted ion




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


EIC 338.3054 ± 0.0100

controlfeeding

RT: 17.90


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


+extracted ion




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


RT: 16.97


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


+extracted ion




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


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


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


+extracted ion




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


RT: 18.55


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



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


RT: 19.11


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


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


+extracted ion




p a g e 54 | 105

RT: 18.89


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


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






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


NH

C17H30N+

m/z [M+H]+: 248.2373

C17H29O+

m/z [M+H]+: 249.2213

RT: 20.57


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


+extracted ion




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


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


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


+extracted ion




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


+extracted ion




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


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


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


+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)).



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


+extracted ion


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


+extracted ion




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


+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).



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


+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.



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


RT: 19.04


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).



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


+extracted ion




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)


+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).



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


NL: 8.62E8

m/z= 297.2270-297.2300


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)


+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.



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


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


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


+extracted ion




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


+extracted ion


5 10 15 20 250

50000

100000

150000

200000

250000

300000

Inte

nsi

ty

13.83NL: 3.46E5

m/z= 323.2426-323.2458


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


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




A)



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


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


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


+extracted ion




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


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






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


+extracted ion


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


C13aH21O+

m/z [M+H]+: 193.1587

RT: 17.35


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


+extracted ion chromatogram (EIC) (Rt = 17.40 min) and (B)

fragmentation of putative intermediate 66 with putative fragment structural assignment are shown.



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



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


RT: 18.03


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


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


+extracted ion




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


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


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






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


+extracted ion




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


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


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


+extracted ion


A)



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


+extracted ion


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


+extracted ion




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


+extracted ion




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


+extracted ion




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)


+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).



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


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


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.



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


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



p a g e 82 | 105

RT: 13.08


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


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)


+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.



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


RT: 13.89


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


+extracted ion chromatogram (EIC)

(Rt = 13.92 min min) and (B) fragmentation of putative intermediate 79 with putative fragment structural assignmentare shown.



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



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


RT: 14.95


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


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)


+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).



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



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


RT: 14.52


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


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)


+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).



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


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


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)



p a g e 89 | 105

RT: 18.31


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


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)


+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).



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


+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).



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


+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).



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)


+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).



p a g e 93 | 105

3. Spectra

3.1. 1H- and 13C-NMR of compound 13a (400 MHz, CDCl3)



p a g e 94 | 105

3.2. 1H- and 13C-NMR of compound 13b (400 MHz, CDCl3)



p a g e 95 | 105




p a g e 96 | 105




p a g e 97 | 105




p a g e 98 | 105




p a g e 99 | 105




p a g e 100 | 105




p a g e 101 | 105

3.9. 1H- and 13C-NMR of compound 18 (400 MHz, CDCl3)

NH

O

O

OS S



p a g e 102 | 105

3.10. 1H- and 13C-NMR of compound 24 (400 MHz, D2O)

NH

O

O

OS S

K



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



p a g e 104 | 105

3.12. 1H- and 13C-NMR of compound 20