Ingrid 't Hart

34 Chapter 2 2 Conclusions In conclusion, DSGb5 was synthesized by a chemoenzyma�c approach in which the oligosaccharide moiety of Gb5 was assembled chemically by a block coupling approach followed by enzyma�c sialyla�on using the mammalian sialyltransferases, ST3Gal1 and ST6GalNAc5 to install an α2,3- and α2,6-linked sialoside, respec�vely. Glycan microarray binding studies indicate that the oligosaccharide moiety of DSGb5 is not recognized by Siglec-7, and it is likely this ganglioside promotes tumorigenesis through other mechanisms such as increasing cell migra�on and invasion. Experimental Sec�on Chemical synthesis General procedures All chemicals were purchased from commercial sources. NMR spectra ( 1 H, 13 C, COSY, HSQC) were obtained on an Agilent 400-MR DD2 or Bruker 750 MHz. Chemical shi�s are reported in part per million (ppm) rela�ve to CDCl3 (7.26 ppm), TMS (0.00 ppm) or D2O (4.79 ppm). NMR data is presented as: chemical shi�, mul�plicity (where s = singlet, d = doublet, t = triplet, dd = doublet of doublets, m = mul�plet) and the coupling constant in Hertz (Hz). Mass spectra were obtained on a Shimadzu ESI LC-MS QP8000 or Kratos Analy�cal Maxima-CFR MALDI-TOF system (using 2,5-dihydroxybenzoic acid matrix). Reported HRMS data was obtained on an Agilent technologies 6560 Ion mobility Q-TOF. Semi-prepara�ve HPLC was performed on an Applied Biosystems 400 solvent delivery system and 757 Absorbance Detector (UV absorbance set on 214 nm) using HILIC column (XBridge® Amide 5 µm, 4.6 mm x 250 mm column, Waters). The mobile phase for analy�cal and semi-prepara�ve HPLC runs consisted of buffers A and B. For C18 columns buffer A is 0.1 % TFA in H 2 O and buffer B is 10 % A + 90 % CH 3 CN and a gradient was used. For HILIC column chromatography buffer A is 10 mM NH 4 COOH in H 2 O (pH = 4) and B is 10 % A + 90 % CH 3 CN at isocra�c condi�ons. Size exclusion chromatography was performed on Bio-Gel P-2 (45-90 µm) with water as the eluent. Column chromatography was performed on silica gel G60 (Silicycle 60 – 200 µm, 60 Å). TLC analysis was conducted on silica gel 60 F254 (EMD Chemicals Inc.) with detec�on by UV light (254 nm) and staining by 10 % H2SO4 in EtOH or p -anisaldehyde solu�on, followed by hea�ng for visualiza�on. Molecular sieves (4 Å) were flame-dried prior to use.