Supporting Information for: New Insights into Nisin’s Antibacterial Mechanism Revealed by Binding Studies with Synthetic Lipid II Analogues
Peter ‘t Hart[a], Sabine F. Oppedijk[b], Eefjan Breukink[b] and Nathaniel I. Martin*[a]
[a] Department of Medicinal Chemistry & Chemical Biology Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands. [b] Department of Biochemistry of Membranes Bijvoet Center for Biomolecular Research, Utrecht University Padualaan 8, 3584 CH Utrecht, The Netherlands
[email protected]
Table of Contents Page
S2
General Procedures
S3-‐4
Synthesis of compound 5, 7, 8 and 10
S5
TLC of used lipids
S6
HPLC traces for compounds 6, 7 and 8
S7-‐10
NMR spectra for compounds 5, 7, and 8
S11-‐15 Isothermal titration calorimetry S16-‐19 Dye leakage experiments S20-‐23 Pore stability experiments S24
References
1
General Procedures Reagents, solvents and solutions. All reagents employed were of American Chemical Society (ACS) grade or finer and were used without further purification unless otherwise stated. All reactions and fractions from column chromatography were monitored by thin layer chromatography (TLC) using plates with a UV fluorescent indicator (normal SiO2, Merck 60 F254). One or more of the following methods were used for visualization: UV absorption by fluorescence quenching; iodine staining; phosphomolybdic acid:ceric sulfate:sulfuric acid:H2O (10 g:1.25 g:12 mL:238 mL) staining; and ninhydrin staining. Flash chromatography was performed using Merck type 60, 230-‐400 mesh silica gel. Compounds 2, 6, 8, and 11 were prepared as previously described.1–4 Instrumentation for Compound Characterization. 1H NMR spectra were recorded at 400 or 500 MHz with chemical shifts reported in parts per million (ppm) downfield relative to tetramethylsilane (TMS). 1H NMR data are reported in the following order: multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; qn, quintet and m, multiplet), number of protons and coupling constant (J) in Hertz (Hz). When appropriate, the multiplicity is preceded by br, indicating that the signal was broad. 13C NMR spectra were recorded at 100 or 125 MHz with chemical shifts reported relative to CDCl3 (δ 77.0 ppm). 31P NMR spectra were recorded at 162 MHz with chemical shifts reported relative to PPh3 (δ -‐6 ppm) which was used as external reference. High-‐resolution mass spectrometry (HRMS) analysis was performed using an ESI-‐TOF instrument. All literature compounds had NMR spectra and mass spectra consistent with the assigned structures.
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Synthesis of compounds 5, 7, 8 and 10 Synthesis of (2R,3S,4R,5R,6R)-‐5-‐acetamido-‐2-‐(acetoxymethyl)-‐4-‐(((R)-‐1-‐amino-‐1-‐oxopropan-‐2-‐ yl)oxy)-‐6-‐((bis(benzyloxy)phosphoryl)oxy)tetrahydro-‐2H-‐pyran-‐3-‐yl acetate (5) Compound 3 (303.1 mg, 0.38 mmol) was dissolved in DCM (10 ml) and DBU (112 µl, 0.75 mmol) was added dropwise. The mixture was stirred for 2 h before it was diluted with DCM (10 ml) and extracted with 1M HCl (20 ml). The organic layer was dried over Na2SO4, filtered and evaporated to dryness. The compound was further purified by column chromatography (5% MeOH in DCM à 5% MeOH in DCM + 1% AcOH). After dissolving the free carboxylic acid in DMF (4 ml), BOP (417.2 mg, 0.94 mmol), NH4Cl (207.5 mg, 3.88 mmol) and DIPEA (660 µl, 3.80 mmol) were added and the mixture was stirred for 24 h. After removal of the solvents under vacuum, the residue was dissolved in EtOAc (20 ml) and extracted with 1M KHSO4 (10 ml), sat NaHCO3 (10 ml), and brine (10 ml). The organic layer was dried over Na2SO4, filtered and evaporated to dryness. The compound was further purified by column chromatography (CHCl3/Acetone 2:1à1:2). Fractions containing product were pooled and the product was obtained as an off white solid. Yield: 121.2 mg (0.190 mmol, 51%). HRMS ESI-‐TOF m/z calcd for C29H37N2O12P ([M+Na]+): 659.1976, found: 659.1975. 1H NMR (400 MHz, CDCl3) δ 7.41 – 7.30 (m, 10H), 6.39 (s, 1H), 6.00 (d, J = 9.5 Hz, 1H), 5.56 (dd, J = 5.8, 3.2 Hz, 1H), 5.32 (s, 1H), 5.14 – 4.99 (m, 5H), 4.40 – 4.32 (m, 1H), 4.10 (dd, J = 13.0, 4.6 Hz, 1H), 3.91 (t, J = 2.4 Hz, 1H), 3.90 – 3.82 (m, 2H), 3.43 (t, 1H), 2.07 (s, 3H), 1.99 (s, 3H), 1.76 (s, 3H), 1.30 (d, J = 6.8 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 174.7, 171.0, 170.7, 169.2, 129.3 (2x), 129.0 (2x), 128.3 (2x), 97.2, 79.1, 78.0, 70.4, 70.3, 70.2, 68.8, 61.6, 52.9 (2x), 23.2, 21.0, 20.9, 18.82. 31P NMR (162 MHz, CDCl3) δ -‐2.36. Synthesis of Undecaprenol-‐MurNAc-‐amide (7) Synthesis done as described by Blasczcak et al. but starting from compound 5 (22.5 mg, 35.4 µmol).2 Compound 7 was purified using a Dr. Maisch Reprospher 100 C8-‐Aqua column (250 x 20 mm, 10 µm) eluting with a gradient of 25% to 100 % buffer B over 1 h with a flow of 12 ml/min. Product eluted at 43.0 min. Buffer A: 10 mM NH4HCO3, Buffer B: MeCN. Yield: 7.1 mg (5.91 µmol, 17%). HRMS ESI-‐TOF m/z calcd for C66H110N2O13P2 ([M-‐H]-‐): 1199.7405, Found 1199.7447. 1H NMR (500 MHz, CD3OD/CDCl3 (1:1)) δ 5.50 (d, 1H), 5.41 (t, 1H), 5.14 (s, 11H), 4.48 (s, 2H), 4.18 (q, 1H) 4.13 (d, 1H) 3.99 (t, J = 10.8 Hz, 2H), 3.72 – 3.54 (m, 3H), 3.32 (t, 1H), 2.14-‐1.92 (m, 43H), 1.74 (s, 3H), 1.69 (s, 21H), 1.62 (s, 3H), 1.60 (s, 9 H), 1.44 (d, J = 6.6 Hz, 3H). 13C NMR (125 MHz, CD3OD/CDCl3 (1:1)) δ 147.2, 146.7, 143.9, 117.1, 103.0, 100.5, 95.9, 92.9, 85.5, 84.5, 75.4, 62.1, 54.5, 48.7, 46.3, 45.4, 45.3, 44.4, 41.0, 37.9. 31P NMR (162 MHz, CDCl3) δ -‐9.55, -‐11.80.
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Synthesis of undecaprenol-‐pyrophosphate (10) Undecaprenol (11.3 mg, 14.8 µmol) was dissolved in dry DCM (2 ml) and CBr4 (14.7 mg, 44.4 µmol) was added, followed by PPh3 (9.7 mg, 37 µmol). After 20 min TLC indicated full consumption of the starting material. The reaction mixture was evaporated to dryness and applied to a short silica column eluting with 5% EtOAc in hexanes + 1% NEt3. Fractions containing product were pooled evaporated and immediately dissolved in dry chloroform (0.5 ml), tris(tetra-‐n-‐butylammonium) hydrogen pyrophosphate (53.7 mg, 59.5 µmol) was added and the mixture was stirred for 16 h under an argon atmosphere. The reaction mixture was diluted with (iPrOH/25% NH4OH/H2O 7:2:1) until a homogenous solution was obtained. The solution was applied to a silica column eluting with iPrOH/25% NH4OH/H2O (7:2:1). Fractions containing product were pooled and concentrated to approx. 0.5 ml. After dilution with 25 mM NH4HCO3 (10 ml) Dowex 50wx8 (NH4+ form) was added and the mixture was shaken for 1h. The Dowex was removed by filtration and the aqueous solution concentrated to 5 ml. Chloroform (5 ml) was added and after thorough shaking a suspension was obtained. Addition of small amounts of MeOH allowed the mixture to separate. The aqueous solution was treated this way once more and the organic layers were combined. After drying over Na2SO4 the product was kept in the organic solution (MeOH/CHCl3) as rapid degradation of the dried compound was observed. The yield was determined by measuring the total amount of free phosphate by the method of Rouser et al.5 (5.64 mg, 5.89 µmol, 39.8%) HRMS ESI-‐TOF m/z calcd for C55H92O7P2 ([M-‐H]-‐): 925.6240, Found 925.6227. Product was identical to reference material obtained from the Institute of Biochemistry and Biophysics of the Polish Academy of Sciences.
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SI figure 1: TLC of all used phospholipids TLC eluent: CHCl3/MeOH/H2O/NH4OH (88:48:10:1) Lane 1: Lipid II (2)
Rf: 0.11
Lane 2: Lipid I (6)
Rf: 0.13
Lane 3: Compound 7
Rf: 0.48
Lane 4: C55-‐PP (10)
Rf: 0.37
Lane 5: C55-‐P (11)
Rf: 0.46
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HPLC data for compounds 7 Compounds 7 was analysed on a Dr. Maisch Reprospher 100 C8 Aqua column (250 mm x 4.6 mm, 5 µm) eluting with a linear gradient of 25 % buffer B to 100 % buffer B over 36 min. Buffer A: 10 mM NH4HCO3, Buffer B: MeCN.
Arbitrary Units
SI figure 2: HPLC trace of compound 7 900000 800000 700000 600000 500000 400000 300000 200000 100000 0 -‐100000 0
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NMR spectra for compounds 5 and 7 SI figure 3: 1H-‐NMR spectrum for compound 5 (CDCl3)
SI figure 4: 13C-‐APT spectrum for compound 5 (CDCl3)
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SI figure 5: 31P-‐NMR spectrum for compound 5 (CDCl3)
SI figure 6: 1H-‐NMR spectrum for compound 7 (CDCl3/CD3OD 1:1)
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SI figure 7: COSY spectrum for compound 7 (CDCl3/CD3OD 1:1)
SI figure 8: HSQC spectrum for compound 7 (CDCl3/CD3OD 1:1)
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SI figure 9: 31P-‐NMR spectrum for compound 7 (CDCl3/CD3OD 1:1)
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Isothermal Titration Calorimetry Large unilamellar vesicles were prepared containing 10 mM DOPC and 0.1 mM of the lipid of interest by suspending the dried lipid films in 50 mM Tris and 100 mM NaCl (pH 7.0). The solution was then extruded through 0.2 µm pore filters ten times. For C55-‐PP the vesicles were prepared using 10 mM DOPC and 0.5 mM C55-‐PP. The nisin solution used was freshly prepared before every experiment in the same buffer system used for preparing the vesicles. All binding experiments were performed using a MicroCal-‐iTC200 microcalorimeter (Malvern). Each binding experiment consisted of 25 separate 1.5 µL injections (following an initial injection of 0.5 µl) delivered into the sample cell which contained a 200 uL volume of nisin at a concentration of 20 µM (or 50 µM for binding measurements with C55-‐PP). The intervening time between each injection was 180 seconds and all measurements were performed at 25°C with the reference power set at “2”. Feedback mode/Gain was set at “low” to obtain a better signal to noise ratio. For the titration with compound 8 a modified protocol was employed wherein compound 8 was dissolved at 800 µM and nisin at 80 µM. The binding measurements with compound 8 were performed by administering 19 separate injections of 2 µL (following an initial injection of 0.5 µl) at 180s intervals into the sample cell containing the nisin solution. All ITC binding experiments were conducted in triplicate and corrected by subtraction of a “blank” titration of the corresponding syringe solution into buffer. The binding data obtained was analysed using the Origin 7.0 software supplied with the instrument.
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SI figure 10: Isotherms for titration of vesicles containing Lipid II (1) into nisin
SI figure 11: Isotherms for titration of vesicles containing Lipid I (6) into nisin
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SI figure 12: Isotherms for titration of vesicles containing compound 7 into nisin
SI figure 13: Isotherms for titration of vesicles containing C55-‐PP (10) into nisin
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SI figure 14: Isotherms for titration of vesicles containing C55-‐P (11) into nisin
SI figure 15: Isotherms for titration of vesicles containing C55-‐OH into nisin
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SI figure 16: Isotherms for titration of vesicles containing compound 8 into nisin
SI figure 17: Isotherms for titration of empty DOPC vesicles into nisin
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Dye leakage experiments Dye leakage assays were conducted as previously described4 with all experiments performed in triplicate. In short, carboxyfluorescein loaded large unilamellar vesicles were prepared from 10 mM DOPC containing 0.2% of the lipid of interest. These vesicles were used at a concentration of 25 µM in the cuvette and fluorescence was measured for 200s. At approximately 40s nisin was added at a concentration of 10 nM and after 140s triton-‐x 100 was added at a final concentration of 0.1%. The baseline signal was determined as the average of the first 40 sec (A0) and the maximum signal as the average of the last 40 sec (Amax). The value at 120 sec (Ameasured) was used to calculate the percentage of dye leakage using the formula: % 𝑙𝑒𝑎𝑘𝑎𝑔𝑒 =
!!"#$%&"' !!! !!"# !!!
×100%.
SI figure 18: Dye leakage experiments using Lipid II containing vesicles
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SI figure 19: Dye leakage experiments using Lipid I (6) containing vesicles
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SI figure 20: Dye leakage experiments using compound 7 containing vesicles
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SI figure 21: Dye leakage experiments using C55-‐PP (10) containing vesicles
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Pore stability experiments Pore stability experiments were performed as previously described.6 For these experiments nisin was premixed with “empty” DOPC vesicles containing the lipid of interest. The cuvette was filled with CF loaded lipid II vesicles and after 40 s the premixed vesicles added. At t=140s triton-‐x 100 was added to obtain the 100% fluorescence signal. Each measurement was performed in duplicate. For these experiments the fluorescent signal obtained when using nisin alone (no empty vesicles added) was considered to be 100%. SI figure 22: Pore stability measurement using Lipid II containing vesicles
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SI figure 23: Pore stability measurement using Lipid I (6) containing vesicles
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SI figure 24: Pore stability measurement using compound 7 containing vesicles
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SI figure 25: Pore stability measurement using nisin only
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M. S. VanNieuwenhze, S. C. Mauldin, M. Zia-‐Ebrahimi, J. A. Aikins, L. C. Blaszczak, J. Am. Chem. Soc. 2001, 123, 6983–6988.
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L.-‐Y. Huang, S.-‐H. Huang, Y.-‐C. Chang, W.-‐C. Cheng, T.-‐J. R. Cheng, C.-‐H. Wong, Angew. Chem. Int. Ed. 2014, 402, 8060–8065.
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G. Rouser, S. Fleischer, A. Yamamoto, Lipids 1970, 5, 494–496.
6.
E. Breukink, H. E. van Heusden, P. J. Vollmerhaus, E. Swiezewska, L. Brunner, S. Walker, A. J. R. Heck, B. de Kruijff, J. Biol. Chem. 2003, 278, 19898–19903.
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