Synthesis of High-Mannose 1-Thio Glycans and Their Conjugation to Protein

Transcription

1 Electronic Supplementary Material (ESI) for Organic. This journal is The Royal Society of Chemistry 2014 Supplementary Data for Synthesis of High-Mannose 1-Thio Glycans and Their Conjugation to Protein Justin J. Bailey and David R. Bundle Alberta Glycomics Centre and the Department of Chemistry, University of Alberta Edmonton, Alberta, T6G 2G2, Canada TABLE OF CONTENTS LC-UV-MS analysis 1H NMR spectra S2-S13 S14-S35 S1

2 Quantifying the degree of conjugation of compound 37: N-succinimidyl iodoacetate linker addition to ubiquitin. Figure S1. Total Ion Current (TIC) chromatogram and extracted-ion chromatograms (EICs) representing the differentially conjugated ubiquitin species of 37. L=linkers, +40 signifies a mass increase of 40 MW, corresponding to an internal crosslink formed through reaction of the iodoacetyl moiety with a neighbouring amino acid functionality. S2

3 Response Units Response Units Table S1. Relative abundance of conjugate species of 37 as derived from peak integration from the PeakFit analysis of the UV chromatogram and identified by EIC correlation. Peak Identity Area Peak Identity Area 1 4L L L L L L L L L L L L L L L L L L L L L L L L L L L L L 0.27 Total Area: Table S2. Summary of linker distribution of conjugate Area % Area 4 Linkers % 5 Linkers % 6 Linkers % 7 Linkers % Total: % Average # of linkers: 5.3 Note: L = linkers; +40 signifies a mass increase of 40 MW, corresponding to an internal crosslink formed through reaction of the iodoacetyl moiety with a neighbouring amino acid functionality Acquisition time (min) Figure S2. UV chromatogram (@210 nm) obtained from LC-UV-MS (above) and simulated chromatogram obtained by use of PeakFit software (below) for ubiquitin conjugate Acquisition time (min) S3

4 Quantifying the degree of conjugation of compound 38: N-succinimidyl 4-pentynoate linker addition to ubiquitin. Figure S3. Total Ion Current (TIC) chromatogram and extracted-ion chromatograms (EICs) representing the differentially conjugated ubiquitin species of 38. L = linkers, +16 signifies a mass increase of 16 MW corresponding to one oxidation. S4

5 Response Units Response Units Table S3. Relative abundance of conjugate species of 38 as derived from peak integration from the PeakFit analysis of the UV chromatogram and identified by EIC correlation. Peak Identity Area 1 6L L L L L L L L 0.75 Total Area: Note: L = linkers; +16 denotes a mass increase of 16 MW corresponding to one oxidation. Table S4. Summary of linker distribution of conjugate Area % Area 5 Linkers % 6 Linkers % 7 Linkers % Total: % Average # of linkers: Acquisition time (min) Acquisition time (min) Figure S4. UV chromatogram (@210 nm) obtained from LC-UV-MS (above) and simulated chromatogram obtained by use of PeakFit software (below) for ubiquitin conjugate 38. The PEG contamination observed in the TIC chromatogram does not interfere with protein quantification by UV peak integration as it does not absorb UV (@210 nm) S5

6 Quantifying the degree of conjugation of compound 39: glycoconjugation of 1 (Man 4 ) to 37. Figure S5. Total Ion Current (TIC) chromatogram and extracted-ion chromatograms (EICs) representing the differentially conjugated ubiquitin species of 39. L = linkers, T = tetrasaccharides, +40 signifies a mass increase of 40 MW corresponding to an internal crosslink formed through reaction of the iodoacetyl moiety with a neighbouring amino acid functionality. S6

7 Response Units Response Units Table S5. Relative abundance of conjugate species of 39 as derived from peak integration from the PeakFit analysis of the UV chromatogram and identified by EIC correlation. Peak Identity Area Peak Identity Area 1 4L/4T L/6T L/5T L/6T L/4T L/7T L/5T L/4T L/6T L/5T L/6T L/5T L/4T L/6T L/5T L/5T L4T L/5T L/5T L/5T L/5T L/5T L/4T L/6T L/6T L/6T L/4T L/6T L/6T Total Area: Table S6. Summary of Man 4 distribution of conjugate Area % Area 4 Man Man Man Man Total: Average # of Man 4 : 5.3 Note: L = linkers; T = tetrasaccharides; +40 signifies a mass increase of 40 MW, corresponding to an internal crosslink formed through reaction of the iodoacetyl moiety with a neighbouring amino acid functionality Acquisition time (min) Acquisition time (min) Figure S6. UV chromatogram (@210 nm) obtained from LC-UV-MS (above) and simulated chromatogram obtained by use of PeakFit software (below) for ubiquitin conjugate 39. S7

8 Quantifying the degree of conjugation of compound 40: glycoconjugation of 2 (Man 5 ) to 37. Figure S7. Total Ion Current (TIC) chromatogram and extracted-ion chromatograms (EICs) representing the differentially conjugated ubiquitin species of 40. L = linkers, P = pentasaccharides, +40 signifies a mass increase of 40 MW corresponding to an internal crosslink formed through reaction of the iodoacetyl moiety with a neighbouring amino acid functionality. S8

9 Response Units Response Units Table S7. Relative abundance of conjugate species of 40 as derived from peak integration from the PeakFit analysis of the UV chromatogram and identified by EIC correlation. Peak Identity Area Peak Identity Area 1 4L/4P L/5P L/5P L/6P L/5P L/5P L/5P L/5P L/6P L/4P L/6P L/5P L/7P L/5P L/4P L/6P L/5P L/4P L/4P L/5P L/4P L4P L/6P L/5P L/5P L/4P L/5P L/4P L/5P L/4P L/4P Total Area: Table S8. Summary of Man 5 distribution of conjugate Area % Area 4 Man Man Man Man Total: Average # of Man 5 : 5.3 Note: L = linkers; P = Man 5 ; +40 signifies a mass increase of 40 MW, corresponding to an internal crosslink formed through reaction of the iodoacetyl moiety with a neighbouring amino acid functionality Acquisition time (min) Acquisition time (min) Figure S8. UV chromatogram (@210 nm) obtained from LC-UV-MS (above) and simulated chromatogram obtained by use of PeakFit software (below) for ubiquitin conjugate 40. S9

10 Quantifying the degree of conjugation of compound 41: glycoconjugation of 33 (Man 4 ) to 38. Figure S9. Total Ion Current (TIC) chromatogram and extracted-ion chromatograms (EICs) representing the differentially conjugated ubiquitin species of 41. L = linkers, T = tetrasaccharides. S10

11 Response Units (%) Response Units (%) Table S9. Relative abundance of conjugate species of 41 as derived from peak integration from the PeakFit analysis of the UV chromatogram and identified by EIC correlation. Peak Identity Area Peak Identity Area 1 6L/5T L/4T L/6T L/2T L/7T L/4T L/5T L/2T L/4T L/3T L/5T L/3T L/5T L/3T L/5T L/2T L/6T L/2T L/6T L/2T L/4T 0.33 Total Area: L/4T 0.41 Table S10. Summary of Man 4 distribution of conjugate Area % Area 2 Man Man Man Man Man Man Total: Average # of Man 4 : 4.9 Note: L = linkers; T = Man Acquisition Time (min) Acquisition Time (min) Figure S10. UV chromatogram (@210 nm) obtained from LC-UV-MS (above) and simulated chromatogram obtained by use of PeakFit software (below) for ubiquitin conjugate 41. S11

12 Quantifying the degree of conjugation of compound 42: glycoconjugation of 34 (Man 5 ) to 38. Figure S11. Total Ion Current (TIC) chromatogram and extracted-ion chromatograms (EICs) representing the differentially conjugated ubiquitin species of 42. L = linkers, P = pentasaccharides. Oxidized products shared similar retention times to their non-oxidized counter parts are were ignored for simplicity. S12

13 Response Units Response Units (%) Table S11. Relative abundance of conjugate species of 42 as derived from peak integration from the PeakFit analysis of the UV chromatogram and identified by EIC correlation. Peak Identity Area Peak Identity Area 1 6L/6P L/4P L/7P L/4P L/5P L/5P L/6P L/3P L/5P L/3P L/6P L/2P L/4P L/2P L/4P L/3P L/5P L/2P L/4P L/3P L/4P L/1P L/5P 0.46 Total Area: Table S12. Summary of Man 5 distribution of conjugate Area % Area 1 Man Man Man Man Man Man Man Average # of Man 5 : 4.7 Note: L = linkers; P = Man Acquisition Time (min) Acquisition Time (min) Figure S12. UV chromatogram (@210 nm) obtained from LC-UV-MS (above) and simulated chromatogram obtained by use of PeakFit software (below) for ubiquitin conjugate S13

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