1 SUPPORTING INFORMATION Noncovalent ... AWS
SUPPORTING INFORMATION
Noncovalent PEGylation via Lectin–Glycopolymer Interactions Paweł M. Antonik,a,b Ahmed M. Eissa,c,d,e Adam R. Round,f Neil R. Cameron,c,d and Peter B. Crowleya,* a
School of Chemistry, National University of Ireland Galway, University Road, Galway,
Ireland. b
c
Teagasc Food Research Centre, Ashtown, Dublin 15, Ireland.
Department of Chemistry, Durham University, Science Laboratories, South Road, Durham
DH1 3LE, United Kingdom. d
Present addresses: School of Engineering, University of Warwick, Coventry, CV4 7AL,
United Kingdom; Department of Materials Science and Engineering, Monash University, Clayton 3800, Victoria, Australia. e
Department of Polymers, Chemical Industries Research Division, National Research Centre
(NRC), El-Bohoos Street, Dokki, Cairo, Egypt. f
European Molecular Biology Laboratory Grenoble Outstation, 71 Avenue des Martyrs,
38042 Grenoble Cedex 9, France and Faculty of Natural Sciences, Keele University, Keele, Staffordshire, ST5 5BG, UK.
*Correspondence to: [email protected], +353 91 49 24 80
1
Scheme S1 Fuc-PEG synthesis Figure S1
1
H-NMR spectra of PEG precursors
Figure S2
1
H-NMR spectra of fucose precursor and Fuc-PEG
Figure S3 ATR-FTIR spectra of precursors and Fuc-PEG Figure S4 SEC of RSL bound to Fuc-PEG or Me-fuc Figure S5 Comparison of HSQC and TROSY-HSQC spectra of RSL bound to Fuc-PEG Figure S6 TROSY-HSQC spectra of RSL bound to Me-fuc or Fuc-PEG Table S1 Line widths of 1HN resonances of RSL bound to Me-fuc or Fuc-PEG Figure S7 TROSY-HSQC spectra of RSL in the presence of PEG 2000 Figure S8 ITC of RSL binding to Fuc-PEG
2
Scheme S1. Synthesis of fucose-capped PEG (Fuc-PEG) via click chemistry.
3
Figure S1. 1H-NMR spectra of (A) PEG and (B) Alk-PEG. Data collection was performed on a 400 MHz Varian Mercury spectrometer. Chemical shifts are relative to tetramethylsilane (TMS). Peak x denotes CDCl3.
4
Figure S2. 1H-NMR spectra of (A) 2’-azidoethyl α-L-fucose and (B) Fuc-PEG. Data collection was performed as per Figure S1. Peak x denotes D2O.
5
wavenumber (cm-1)
Figure S3. ATR-FTIR spectra of (A) PEG (B) 2’-azidoethyl α-L-fucose and (C) Fuc-PEG. Spectra were recorded on a Perkin Elmer 1600 series FTIR spectrometer fitted with a Diamond ATR crystal unit.
6
Figure S4. SEC of RSL in the presence of 12 equivalents of Fuc-PEG (red) or Me-fuc (blue). The elution buffer was 20 mM potassium phosphate, 50 mM NaCl, pH 6.0.
7
Figure S5. Two dimensional 1H-15N HSQC watergate (top) and TROSY-HSQC (bottom) spectra of 0.5 mM RSL bound to Fuc-PEG. Sample conditions were 20 mM potassium phosphate, 50 mM NaCl, pH 6.0 at 303 K.
8
Figure S6. The overlaid 1H−15N TROSY-HSQC spectra of 0.5 mM RSL bound to Me-fuc (navy contours) or Fuc-PEG (red).
9
Table S1. Line widths of 1HN resonances extracted from the TROSY-HSQC spectra of RSL bound to Me-fuc or Fuc-PEG.* 1
Residue
V3 Q4 T5 A7 T8 S9 W10 G11 T12 V13 S15 I16 R17 Y19 T20 A21 N22 N23 G24 K25 T27 E28 W31 Y37 T38 G39 F41 N42 E43
HN line-width (Hz)
Me-fuc
Fuc-PEG
16.0 21.6 22.3 22.0 19.6 18.2 19.5 17.8 20.3 20.0 22.2 25.2 18.7 23.6 24.7 22.8 18.7 17.2 16.9 19.0 21.7 17.9 18.8 20.6 16.4 22.8 19.3 19.4 19.3
26.1 34.3 34.8 31.4 29.1 29.5 38.2 31.4 22.3 27.8 32.2 25.9 27.5 29.5 30.0 23.3 29.5 23.3 26.3 22.9 24.4 28.4 32.3 28.1 28.3 30.7 25.8 27.9 32.0
1
Residue
G45 D46 N47 V48 S52 W53 V55 S57 A58 I59 R62 Y64 A65 G68 T70 T71 T72 E73 C75 W76 N79 T82 K83 G84 A85 T87 A88 T89 N90
Average line-width (Hz)
HN line-width (Hz)
Me-fuc
Fuc-PEG
17.3 22.1 20.6 20.9 17.7 20.2 20.7 24.3 19.8 18.6 21.0 21.1 20.2 16.5 20.0 21.1 20.1 19.8 19.7 21.8 20.5 21.4 15.6 21.2 17.7 13.1 19.3 18.1 16.7
25.9 32.9 25.4 30.0 24.6 30.9 28.2 31.1 23.7 31.0 27.4 33.4 34.1 38.4 25.0 26.6 30.2 30.8 33.5 30.9 25.2 23.5 21.1 30.5 25.9 35.0 30.8 21.4 20.9
Me-fuc = 19.8 (±2.3) Fuc-PEG = 28.6 (±4.1)
*Line-widths were measured in CCPNmr for non-overlapping cross-peaks (~65 % of total). Six resonances with line widths ≥ 40 Hz were considered outliers and were excluded from the analysis.
10
Figure S7. Spectral region of overlaid 1H-15N TROSY-HSQC spectra of sugar-free RSL (black contours) and RSL in the presence of 6 (magenta) or 12 (cyan) equivalents of PEG 2000.
11
Figure S8. Isothermal titration calorimetry of RSL (24 µM) binding to Fuc-PEG (1.5 mM) in 0.1 M Tris-HCl pH 7.5 at 25° C. The fit parameters were Kd = 1.3 ±0.3 µM, n = 2.5 ±0.2, ∆G = 33.7 ±0.5 kJ/mol, -∆H = 38.1 ±0.8 kJ/mol and T∆S = -4.4 ±1.1 kJ/mol.
12
Noncovalent PEGylation via Lectin–Glycopolymer Interactions Paweł M. Antonik,a,b Ahmed M. Eissa,c,d,e Adam R. Round,f Neil R. Cameron,c,d and Peter B. Crowleya,* a
School of Chemistry, National University of Ireland Galway, University Road, Galway,
Ireland. b
c
Teagasc Food Research Centre, Ashtown, Dublin 15, Ireland.
Department of Chemistry, Durham University, Science Laboratories, South Road, Durham
DH1 3LE, United Kingdom. d
Present addresses: School of Engineering, University of Warwick, Coventry, CV4 7AL,
United Kingdom; Department of Materials Science and Engineering, Monash University, Clayton 3800, Victoria, Australia. e
Department of Polymers, Chemical Industries Research Division, National Research Centre
(NRC), El-Bohoos Street, Dokki, Cairo, Egypt. f
European Molecular Biology Laboratory Grenoble Outstation, 71 Avenue des Martyrs,
38042 Grenoble Cedex 9, France and Faculty of Natural Sciences, Keele University, Keele, Staffordshire, ST5 5BG, UK.
*Correspondence to: [email protected], +353 91 49 24 80
1
Scheme S1 Fuc-PEG synthesis Figure S1
1
H-NMR spectra of PEG precursors
Figure S2
1
H-NMR spectra of fucose precursor and Fuc-PEG
Figure S3 ATR-FTIR spectra of precursors and Fuc-PEG Figure S4 SEC of RSL bound to Fuc-PEG or Me-fuc Figure S5 Comparison of HSQC and TROSY-HSQC spectra of RSL bound to Fuc-PEG Figure S6 TROSY-HSQC spectra of RSL bound to Me-fuc or Fuc-PEG Table S1 Line widths of 1HN resonances of RSL bound to Me-fuc or Fuc-PEG Figure S7 TROSY-HSQC spectra of RSL in the presence of PEG 2000 Figure S8 ITC of RSL binding to Fuc-PEG
2
Scheme S1. Synthesis of fucose-capped PEG (Fuc-PEG) via click chemistry.
3
Figure S1. 1H-NMR spectra of (A) PEG and (B) Alk-PEG. Data collection was performed on a 400 MHz Varian Mercury spectrometer. Chemical shifts are relative to tetramethylsilane (TMS). Peak x denotes CDCl3.
4
Figure S2. 1H-NMR spectra of (A) 2’-azidoethyl α-L-fucose and (B) Fuc-PEG. Data collection was performed as per Figure S1. Peak x denotes D2O.
5
wavenumber (cm-1)
Figure S3. ATR-FTIR spectra of (A) PEG (B) 2’-azidoethyl α-L-fucose and (C) Fuc-PEG. Spectra were recorded on a Perkin Elmer 1600 series FTIR spectrometer fitted with a Diamond ATR crystal unit.
6
Figure S4. SEC of RSL in the presence of 12 equivalents of Fuc-PEG (red) or Me-fuc (blue). The elution buffer was 20 mM potassium phosphate, 50 mM NaCl, pH 6.0.
7
Figure S5. Two dimensional 1H-15N HSQC watergate (top) and TROSY-HSQC (bottom) spectra of 0.5 mM RSL bound to Fuc-PEG. Sample conditions were 20 mM potassium phosphate, 50 mM NaCl, pH 6.0 at 303 K.
8
Figure S6. The overlaid 1H−15N TROSY-HSQC spectra of 0.5 mM RSL bound to Me-fuc (navy contours) or Fuc-PEG (red).
9
Table S1. Line widths of 1HN resonances extracted from the TROSY-HSQC spectra of RSL bound to Me-fuc or Fuc-PEG.* 1
Residue
V3 Q4 T5 A7 T8 S9 W10 G11 T12 V13 S15 I16 R17 Y19 T20 A21 N22 N23 G24 K25 T27 E28 W31 Y37 T38 G39 F41 N42 E43
HN line-width (Hz)
Me-fuc
Fuc-PEG
16.0 21.6 22.3 22.0 19.6 18.2 19.5 17.8 20.3 20.0 22.2 25.2 18.7 23.6 24.7 22.8 18.7 17.2 16.9 19.0 21.7 17.9 18.8 20.6 16.4 22.8 19.3 19.4 19.3
26.1 34.3 34.8 31.4 29.1 29.5 38.2 31.4 22.3 27.8 32.2 25.9 27.5 29.5 30.0 23.3 29.5 23.3 26.3 22.9 24.4 28.4 32.3 28.1 28.3 30.7 25.8 27.9 32.0
1
Residue
G45 D46 N47 V48 S52 W53 V55 S57 A58 I59 R62 Y64 A65 G68 T70 T71 T72 E73 C75 W76 N79 T82 K83 G84 A85 T87 A88 T89 N90
Average line-width (Hz)
HN line-width (Hz)
Me-fuc
Fuc-PEG
17.3 22.1 20.6 20.9 17.7 20.2 20.7 24.3 19.8 18.6 21.0 21.1 20.2 16.5 20.0 21.1 20.1 19.8 19.7 21.8 20.5 21.4 15.6 21.2 17.7 13.1 19.3 18.1 16.7
25.9 32.9 25.4 30.0 24.6 30.9 28.2 31.1 23.7 31.0 27.4 33.4 34.1 38.4 25.0 26.6 30.2 30.8 33.5 30.9 25.2 23.5 21.1 30.5 25.9 35.0 30.8 21.4 20.9
Me-fuc = 19.8 (±2.3) Fuc-PEG = 28.6 (±4.1)
*Line-widths were measured in CCPNmr for non-overlapping cross-peaks (~65 % of total). Six resonances with line widths ≥ 40 Hz were considered outliers and were excluded from the analysis.
10
Figure S7. Spectral region of overlaid 1H-15N TROSY-HSQC spectra of sugar-free RSL (black contours) and RSL in the presence of 6 (magenta) or 12 (cyan) equivalents of PEG 2000.
11
Figure S8. Isothermal titration calorimetry of RSL (24 µM) binding to Fuc-PEG (1.5 mM) in 0.1 M Tris-HCl pH 7.5 at 25° C. The fit parameters were Kd = 1.3 ±0.3 µM, n = 2.5 ±0.2, ∆G = 33.7 ±0.5 kJ/mol, -∆H = 38.1 ±0.8 kJ/mol and T∆S = -4.4 ±1.1 kJ/mol.
12
