Ingrid 't Hart

61 Synthesis of globo-series glycolipids 3 With protected Gb5-Sph 12 in hand (Scheme 1B), appropriate deprotec�on condi�ons were explored. Conven�onal benzyl ether cleavage by hydrogena�on could not be used for compound 12 , since the sphingosine double bond will be reduced. Therefore, the benzyl ethers need to be cleaved by Birch reduc�on to keep the unsaturated sphingosine intact. 43 As a model substrate, Gb3-Sph 9 was converted into compound 16 . The benzoyl ester of compound 9 was removed under Zemplén condi�ons ( cat. NaOMe in DCM/ MeOH) and the crude intermediate was treated with HF·pyridine to cleave the silane and provide compound 16 (Scheme 2). Birch reduc�on on compound 16 proved unsuccessful, even when different addi�on orders of reagents were used or when heated to refluxing temperature (-30 °C). A possible explana�on for the failure of the Birch reduc�on could be lack of the solubility of the substrate. Scheme 2. Deprotec�ons on compound 9 provided trisaccharide 16 . Birch reduc�on did not provide the desired deprotected Gb3Sph. Reagents and condi�ons: (a) i. NaOMe, DCM/MeOH, ii. HF·pyridine, pyridine; (b) Na, NH 3 , THF/t-BuOH, -78 °C to -30 °C. To obtain the desired globo-series glycosphingolipids we moved on to a chemoenzyma�c strategy, which we expected to overcome some of the difficul�es encountered in the chemical synthesis of Gb5-Sph. Chemoenzyma�c synthesis of Gb3-Cer, Gb4-Cer and Gb5-Cer. Enzyma�c synthesis is usually performed by bacterial enzymes, since these are generally easier to express than mammalian enzymes and have broad substrate specifici�es. The bacterial enzymes that have been used in the synthesis of globo-series glycan moie�es are: LgtC (Gb3), LgtD (Gb4, Gb5), CgtB (Gb5), FutC (Globo-H), JT-FAJ-16 (MSGb5), pmST1 (MSGb5), CST-I (MSGb5) and Psp2,6ST (DSGb5). 30,44,45,46 Two mammalian enzymes have also successfully been used for the synthesis of MSGb5 (ST3Gal1) and DSGb5 (ST6GalNAc5) by our group. 28 Only one enzyme has been reported to synthesize Gb4 from Gb3; the bacterial enzyme LgtD from Haemophilus influenzae . For the second enzyma�c extension, two enzymes can be used: CgtB and LgtD. LgtD is a bifunc�onal enzyme and it is able to catalyze both β1,3 GalNAc addi�on on Gb3 to form Gb4 and β1,3 Gal addi�on on Gb4 to form Gb5, however the efficiency of the la�er step is lower. 45,46,47 CgtB has been used on a variety of substrates, including the oligosaccharide moiety of Gb4. 48,24 O R 1 O OR 2 BnO BnO O O OBn BnO OBn O O OBn BnO BnO O C 13 H 27 OR 3 N 3 b 9: R 1 :R 2 = Si( t -Bu) 2 , R 3 Bz 16 , R 1 = R 2 = R 3 = H, 58% a O HO OH HO HO O O OH HO OH O O OH HO HO O C 13 H 27 OH N 3 =