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  • 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

    M.Tosin@warwick.ac.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**

    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

    3.3. 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

    Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**

    p a g e 2 | 105

    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

    Ina Wilkening,* Silvia Gazzola,* Elena Riva, James S. Parascandolo, Lijiang Song and Manuela Tosin**

    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.

  • 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 4 | 105

    1.2.Synthesis of probes 15a-b

    Route 1

    N H

    O

    R

    O

    O

    OO N H

    O

    R

    O

    O

    O

    N H

    O

    R

    O

    O

    OO

    O

    O

    N H

    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

    N H

    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**

    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 (C