1. Carbohydrate Synthesis Part 1: Formation of the glycosidic linkage. “Essentials of Glycobiology” 3 June 2004 Michael VanNieuwenhze/Nathaniel Finney Dept. of Chemistry and Biochemistry UCSD msv@chem.ucsd.edu

2. Coverage Today we will discuss: Formation of glycosidic linkages. Chemical and chemoenzymatic synthesis of carbohydrates. Will not cover: Details of structure or nomenclature (see April 1 notes). Synthesis of monosaccharides. Glycoconjugates.

3. Relevant Primary Literature “Solid-Phase Oligosaccharide Synthesis and Combinatorial Carbohydrate Libraries.” Seeberger, et al. Chem. Rev. 2000, 100, 4349. “Stereocontrolled Glycosyl Transfer Reactions with Unprotected Glycosyl Donors.” Hanessian, et al. Chem. Rev. 2000, 100, 4443. “Synthesis of Complex Carbohydrates and Glycoconjugates: Enzyme-Based and Programmable One-Pot Strategies.” Wong, et al. Chem. Rev. 2000, 100, 4465.

4. Lecture Outline 1. Challenges in carbohydrate synthesis. 2. Glycosyl transfer: the glycosyl cation as a virtual (and sometimes real) intermediate. 3. “Protecting groups.” 4. Glycosyl cation precursors/equivalents. 5. Chemical glycosylation in solution.

5. Challenges in Carbohydrate Synthesis Let’s begin by assuming you are assigned the task of synthesizing D-Glc-  (1-4)-D-Glc. Quick reminders: check April 1 notes for nomenclature; remember that most chemists use chair or Mills structures for sugars.

6. Challenges in Carbohydrate Synthesis Immediate challenge: figure out how to assemble disaccharide from two monosaccharides. Chemists call this reverse thinking “retrosynthetic analysis” - working backwards from a desired target to precursors from which one might be able to make it.

7. Challenges in Carbohydrate Synthesis Thinking in the forward direction, 3 synthetic challenges become apparent: 1. Differentiation of numerous ~ equivalent hydroxyl (OH) groups in the two starting monosaccharides. 2. Control of “anomeric” configuration (  vs  ) in product.

8. Addressing the Challenges And the 3 rd synthetic challenge: 3. How do we choose which chemical bond to make in the forward direction? The combination of physical organic principles and the study of Nature tells us how to address this last issue: Use a “glycosyl cation” (or its equivalent) as an electrophile and one of the free hydroxyl groups of another sugar as a nucleophile.

9. Addressing the Challenges Glycosyl phosphates such as UDP-Glc act like a glycosyl cation.

10. Revisiting the Retrosynthesis Now that we have settled on using a glycosyl cation (or its equivalent): We have to worry about what we’re going to use as the cation equivalent or precursor, since you can’t put a cation in a bottle, as well as all those other hydroxyl groups (We’ll come back to the issue of anomeric stereochemistry later on.)

11. Revisiting the Retrosynthesis Restating it visually: So now we need to figure out what the “R” groups (called protecting groups) are, and what the “X” group in the glycosyl- X precursor to the glycosyl cation.

12. Working Forward Now Let’s start with the “protecting” (“R”) groups. An enormous number of different protecting groups have been used in carbohydrate chemistry. Indeed, much of the labor in chemical carbohydrate synthesis involves installing or removing protecting groups. Here are 6 of the more common protecting groups:

13. A Little More on the Protecting Group An important feature of these protecting groups is that they are removed under different conditions. This allows for the selective exposure of individual hydroxyl groups (provided they were selectively protected in the first place). For instance:

14. Now for the “X” Group Much like the protecting group, there are many variations on the identity of the anomeric X group. The common feature is that the X groups are stable enough that the glycosyl-X can be easily generated (and often isolated), but can be activated to either generate a glycosyl cation or act like a glycosyl cation. We’ll look at 4 of the more common variants on X:

15. How to Activate the “X” Groups: X = Br We’ll begin with X = Br. The following is the Koenigs-Knorr method, one of the oldest procedures for glycosylation. Pro: Anomeric halides are readily accessible. Reliable reaction driven by precipitation of AgBr. Con: Excess AgOTf ($). Hard to make  anomer. (See, however, “anchimeric assistance.”)

16. “X” Groups: X = Trichloroacetimidate X = trichloroacetimidate (TCA) is a more versatile alternative. Pro: Can prepare trichloroacetimidates in both  and  form.

17. X = Trichloroacetimidate, Part 2 Pro: Displacement usually S N 2-like, with inversion of configuration, allowing control of anomeric stereochemistry. Con: Choice of solvent and Lewis acid critical. Trichloroacetimidates are unstable and must be generated in situ.

18. Anomeric Sulfides, X = SPh Anomeric sulfides, X = SPh, are another alternative. Pro: They are easily prepared and can be isolated and stored. Con: Like Koenigs-Knorr, the  -anomer can be difficult to make (again, except for “anchimeric assistance, coming up). In addition, DMTST is highly reactive and difficult to handle.

19. Epoxides and the Glycal Method The final method we’ll look at starts with a “glycal,” from which a reactive epoxide intermediate can be generated. Pro: Very mild and efficient. Reaction can be modified to install NR group at C2. Glycals are readily available. Con:  -anomer general not accessible. DMDO is a nuisance to prepare and cannot be stored for any length of time.

20. “Anchimeric Assistance” The nature of the protecting group at C2 can dictate the stereochemical outcome of glycosylation reactions. When the protecting group at C2 does not interact with the glycosyl cation intermediate, the  -glycoside is the kinetic product of glycosylation.

21. Anchimeric Assistance 2 Protecting groups that can interact with the glycosyl cation protect the  -face, leading to  -glycosylation.

22. Other Glycosylation Protocols Worth Noting “Remote activation” of glycosyl donors (S. Hanessian). n-Pentenyl glycosides (B. Fraser-Reid). Glycal/catalytic sulfoxide (D. Y. Gin). Anomeric monophosphates (P. Seeberger).

23. Next Lecture 1. Iterative solution phase synthesis by Danishefsky’s glycal method. 2. Identity of glycosyl substituents alters the reactivity of glycosyl donors: Exploitation in Wong’s solution phase Optimer methodology. 3. Solid phase carbohydrate synthesis possesses many of the same advantages of solid phase peptide and oligonucleotide synthesis: Automated oligosaccharide synthesis. 4. Chemoenzymatic synthesis of oligosaccharides and glycoconjugates: Complementary to chemical methods; narrower is scope but more elegant and efficient in execution.