Ingrid 't Hart

62 Chapter 3 3 Although bacterial enzymes LgtD and CgtB have been used for the synthesis of globo- series oligosaccharides, they have not been used for glycolipid synthesis. To ensure that all used enzymes were ac�ve, control reac�ons were performed on oligosaccharide substrates. Our chemoenzyma�c strategy started with the chemical synthesis of Gb3-Sph 1a according to a reported procedure. 49 Gb3-Sph 1a was mixed with UDP-GalNAc, MgCl 2 and LgtD in a Tris buffer at pH 7.7 (Scheme 3). Although full conversion was seen of the free oligosaccharide, only 30 % of product 2a (Gb4-Sph) was formed (based on MALDI-TOF). Earlier reports had indicated that even small subs�tuents at the reducing end such as a benzyl ether or glycolipid can reduce enzyme ac�vity. 50,51,52 The use of a larger quan�ty of LgtD and UDP-GalNAc did not drive the reac�on further. Therefore, the Gb3-Sph/Gb4- Sph 1a / 2a mixture was purified by C4 SPE cartridges. The obtained glycolipid mixture was resubmi�ed to fresh sugar nucleo�de and enzyme, and in 5 runs all substrate was converted to Gb4-Sph 2a (22%). Gb4-Sph 2a was mixed with UDP-Gal, MgCl 2 and CgtB in Tris buffer at pH 7.7. A�er 4 h, a mixture was obtained of Gb4-Sph ( 2a ), Gb5-Sph ( 3a ) and Gb5-Sph with a spectrum of 1-5 galactose addi�ons (based on MALDI-TOF). Over-galactosyla�on has been decribed before for CgtB variants. 53,54 Conversion of 2a by LgtD did not provide full conversion to 3a , however, no over-galactosyla�on was observed. Therefore, LgtD was the be�er choice for the synthesis of Gb5-Sph 3a (88%, 3 runs). Also, a highly improved isolated yield of 3a over 2a was obtained by changing SPE purifica�ons from C18 to C4. Pure Gb3- Sph 1a , Gb4-Sph 2a and Gb5-Sph 3a were characterized by 1 and 2D NMR spectroscopy and HRMS spectrometry. Scheme 3. Enzyma�c synthesis of Gb4-Sph 2a and Gb5-Sph 3a star�ng from chemically synthesized Gb3-Sph 1a. Reagents and condi�ons: substrate (10 mM), UDP-sugar (10-11 mM), Tris buffer pH 7.7 (100 mM), MgCl 2 (20 mM) and LgtD at 37 °C. O HO OH O AcHN O HO OH HO OH O HO OH O HO O O OH HO OH O O OH HO OH NH 2 OH C 13 H 27 O O HO OH HO AcHN O HO OH O HO O O OH HO OH O O OH HO OH NH 2 OH C 13 H 27 O O HO OH HO HO O O OH HO OH O O OH HO OH NH 2 OH C 13 H 27 O 1a (Gb3-Sph) 2a (Gb4-Sph), 22% 3a (Gb5-Sph), 88% UDP-GalNAc LgtD UDP-Gal LgtD