Synthesis of Globo H hexasaccharide

Globo H (GH; Fucα1→2Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glc) is a member of the globo series glycosphingolipids. It was first found and characterized in

21 human teratocarcinoma cells and breast cancer MCF-7 cells in 1983,^19,20,22 and was subsequently found overexpressed in many types of human cancer cells including breast, prostate, ovary, pancreas, brain, endometrium, gastric, colon and lung cancers.^21,23,24 Because of its biological importance as a tumor marker and potential anticancer vaccine, Globo H has been target for scientists. Since the difficulty of isolating Globo H from natural sources, total synthetic approach was the viable way to get homogenous Globo H. It was first synthesized in 1995 by Danishesky and co-workers using glycal assembly strategy,^93,94 Schmidt et al. reported the synthesis of Globo H using trichloroacetimidate methodology.⁹⁵ A two directional glycosylation strategy was published by Boons and co-workers.⁹⁶ The synthesis of Globo H was prepared via a one-pot strategy by our group using computer program OptiMer.^53,97 Many other chemical methods have also been explored, such as linear synthesis,⁹⁸ automated solid phase synthesis⁹⁹ by Seeberger and co-workers, multi-component one-pot synthesis and chemoenzymatic synthesis approach.¹⁰⁰

In 1995, Danishesky and co-workers discovered the first total synthesis of globo H using glycal assembly approach (Scheme 1.1).⁹⁴ The glycal assembly approach uses the glycan building blocks, possessing three hydroxyl groups and an olefinic handle, that server to build complex carbohydrates. A retrosynthetic divided into A3 and A9.

A3 was synthesized from acceptor A2 and donor A1 using Mukaiyama Nicolaou condition to get 54 % yiels of A3. A9 was synthesized from acceptor A7 and donor A5 with methyl triflate. It was feasible to synthesize from A9 to A10 via iodosulfonimidation and function group rearrangement. The [3+3] glycosylation strategy was perform with methyl triflate to get 10: 1 mixture of α;β. Treatment of A11 with 3,3-dimethyldioxirane followed by coupling of the epoxide with lipid A12 under zinc chloride, after acetylation, to get A13. After general deprotection, the

22 Globo H ceramide was get. In the following years, they disclosure a second-generation synthetic strategy, which gives easily access to significant quantities of materials.¹⁰¹

23 Another effective synthesis of Globo H was reported by our group using programmable one-pot approach (Scheme 1.2).¹⁰² This approach uses a computer program called ‘‘OptiMer’’ to list the appropriate tolylthioglycoside building blocks from a database for the one-pot synthesis. With this strategy, oligosaccharides are rapidly assembled in minutes or hours without intermediate workup and purification.

To perform a significant reactivity difference between the glycosides and control of the stereoselectivity of each glycosylation, the approach for the synthesis of protected-Globo H A17 involved the use of two one-pot reactions.⁵³ The first one-pot reaction is to build the two β-linkages to form the building block A15. The other one-pot reaction is to build the two α-linkages using three building blocks A14 (RRV

= 7.2 x 104), A15 (RRV = 6) and A16 (RRV = 0) and the yield is 62 %. The reactivities are mainly regulated by electron-donating groups (benzyl ether and 2,2,2-trichloroethylcarbamate) and electron-withdrawing groups (benzoyl, p-nitrobenzoyl and o-chlorobenzyl ethers).

With further refinement, [1+2+3] one-pot strategy improve the yield of the synthesis globo H from 62% to 83% and the challenging Gal α(1→4) linkage is formed (Scheme 1.3).⁹⁷ Therefore, I followed this efficient one-pot synthetic approach with an attempt to synthesize the Globo H and its analogues described later in chapter II. The synthetic glycan analogues can be used to develop the carbohydrate based vaccine and glycan microarray.^23,103

24 Pyridine; c) NaOMe; d) H2, Pd/C, HCOOH, 45% over four steps.

25 Scheme 1.3. One-Pot synthesis of Globo H using (1 + 2 +3) strategy.⁹⁷ (i) NIS, TfOH, -40°C. (ii) NIS, TfOH, -30°C, 75% from 3. (iii) a) Zn, AcOH; b) Ac2O, Pyridine; c) NaOMe; d) H2, Pd-black, HCOOH, 70% over four steps.

In 2007, Seeberger and co-workers reported automated solid-phase assembly the protected TACAs Gb3 and Globo H (Scheme 1.4).⁹⁹ Six building blocks A23, A24, A25, A28, A30 and A31 are prepared for the Globo H. It is assembled using fluorenylmethoxycarbonyl (Fmoc) as a temporary protecting group that is stable under acidic glycosylation conditions and is easily removed by a weak base.

Installation of α-galactosidic linkage of protected Gb3 A27 was achieved by β-anomer A25 and A24 with better α-selectivity. Tetrasaccharide A29 wasd assembled by using building block A27 and A28. A30 and A31 were sequentially to assemble the Globo

26 H A32. After cleavage from the solid support by olefin cross-metathesis and HPLC purification, product Globo H was obtained with 30% overall yield.

FmocO O piperidine (20% in 2 mL of DMF), repeated twice for 5 min each; (c) building block (5 equiv.), TMSOTf (0.5 equiv.), DCM, -30 ^oC, repeated once for 1 h each; (d) Grubbs’ catalyst (first generation), ethylene atmosphere, CHCl3, r.t., overnight; (e) building block (5 equiv.), TMSOTf (5 equiv.), Et2O, DCM, -50 ^oC, repeated once for 3 h each; (f) building block (3.3 equiv.), TMSOTf (3.3 equiv.), DCM, -15 ^oC, repeated twice for 25 min each; (g) building block (5 equiv.), TMSOTf (0.5 equiv.), DCM, -10 ^oC, repeated twice for 25 min each, 30% overall yield.

However, chemical synthesis methods often require multiple protection and deprotection steps, resulting in relatively low yields. An alternative efficient strategy was based on the use of enzymes coupled with effective sugar nucleotide regeneration (Figure 1.13).^54,104 Using this method developed by our lab, the GH and GH truncated forms were easily prepared using glycosyltransferases (LgtC, LgtD, Futc) and cofactor regeneration systems (UDP-Gal, UDP-GalNAc, GDP-Fuc). Gb3-allyl A34

27 was synthesized from synthesis of Lactose-allyl A33, α1,4-galactosyltransferase (LgtC) and the UDP-Gal regeneration system including UDP-sugar pyrophosphorylase (AtUSP), galactokinase (GalK), pyruvate kinase (PK) and inorganic pyrophosphatase (PPA). Next, the step from Gb3-allyl A34 to Gb4-allyl A35 was catalyzed by galactosamine, β1,3-N-acetylgalactosaminyltransferase (LgtD) and the UDP-GalNAc regeneration system including N-acetylhexosamine kinase (NahK), N-acetyl glucosamine-1-phosphate uridyltransferase (GlmU), pyruvate kinase (PK) and inorganic pyrophosphatase (PPA). Synthesis of Gb5-allyl A36 was achieved from Gb4-allyl A35 by using LgtD and the UDP-Gal regeneration system.

The final step of enzymatic transformation was employed from Gb5-allyl A36 by using α-1,2-fucosyltransferase (FutC) and GDP-Fuc regeneration system to obtain GH-allyl A37. The chemoenzymatic approach provides multigrams of GH with two or three purification steps and is useful for drug discovery research and clinical development.