The Seeberger Lab

1.

New Methods for the Solution and Solid-Phase Synthesis of Oligosaccharides - Development of an Automated Oligosaccharide Synthesizer

In order to allow for the synthesis of oligosaccharides to reach a level of sophistication already common to peptide and oligonucleotide assembly, ready access to differentially protected building blocks that are highly efficient and completely selective in coupling reactions will be needed. The first building block will be connected via a linker to a polymer support and repetitive coupling cycles will facilitate the stepwise growth of the desired sequence from the reducing end. A novel cap-tag feature will prevent the accumulation of internal deletion sequences and marks side products for rapid removal.

We have introduced straightforward methods for the preparation of glycosyl phosphate and glycosyl dithiophosphate donors from glycals. These glycosylating agents serve to create most glycosidic linkages in excellent yield. Even traditionally difficult to prepare linkages such as b-mannosides and glucosamines were installed in reasonable selectivity using glycosyl phosphates. New one-pot procedures for the preparation of oligosaccharides not relying on electronic effects but rather on the different reactivities of a- and b-glycosyl phosphates allowed for the minimization of protecting group manipulations. Glycosyl phosphates have proven efficient for the preparation of C-aryl and C-alkyl glycosides.

Plante, O.J.; Palmacci, E.R.; Andrade, R.B.; Seeberger, P.H. Oligosaccharide Synthesis Using Glycosyl Phosphate and Dithiophosphate Triesters as Glycosylating Agents., J. Am. Chem. Soc. 2001, 123, 9545-9554.

The differential protection of functional groups of similar reactivity is a major challenge for the synthesis of oligosaccharides when highly branched structures necessitate several selectively removable blocking groups. We developed halogenated benzyl ethers as protective groups that may be removed in the presence of other common modes of protection by Pd-catalyzed amination followed by brief exposure to Lewis acids or protic acids (collaboration with Prof. S. Buchwald, MIT). The 2-methyl-azido benzoate (Abz) and p-chlorophenylcarbonate groups provide additional degrees of orthogonality required for the synthesis of complex structures in solution and on solid support.

Plante, O.J.; Buchwald, S.L.; Seeberger, P.H. Halobenzyl Ethers as Protecting Groups for Organic Synthesis., J. Am. Chem. Soc. 2000, 122, 7148-7149.

The chemical nature of the linker used to attach the first monosaccharide to a solid support determines the scope of reaction conditions that may be used during oligosaccharide assembly. Our octenediol linker is cleaved by olefin cross-metathesis or ozonolysis has proven versatile and provided the basis for solid-phase synthesis protocols and automated oligosaccharide assembly. Different cleavage protocols provide access to oligosaccharides for conjugation to surfaces for the creation of carbohydrate chips and glycoconjugates.

Andrade, R.B.; Plante, O.J.; Melean, L.G.; Seeberger, P.H. Solid-Phase Oligosaccharide Synthesis: Preparation of Complex Structures Using a Novel Linker and Different Glycosylating Agents, Organic Letters 1999, 1, 1811-1814.

We departed from the traditional goal of oligosaccharide total synthesis striving for maximum convergency, and followed a linear synthesis approach based on monosaccharide building blocks. Using this method similar to that practiced for peptides and oligonucleotides we assembled several complex structures.

1.4.1 Synthesis of High Mannose Structures of HIV gp120

We completed the synthesis of a series of highly branched mannosides found on gp120 of HIV. Two different trisaccharides, a hexa-, and a nonasaccharide were prepared in conjugatable form. These structures were used to investigate the interaction of cyanovirin-N, a highly potent topical anti-HIV agent, with gp120. In collaboration with Barry O'Keefe (NCI) and Angela Gronenborn (NIH) isothermal calorimetry and high-field NMR were used to establish the minimal binding sequence and to map the binding site on the protein.

Ratner, D.M.; Plante, O.J.; Seeberger, P.H.; A Linear Synthesis of Branched High-Mannose Oligosaccharides from the HIV-1 Viral Surface Envelope Glycoprotein gp120; Eur. J. Org. Chem. 2002, 826-833.

1.4.2 Synthesis of Oligosaccharide Antigens Involved in Cancer and Bacterial Infections

Cell surface carbohydrates act as biological markers for various tumors and are involved in bacterial and parasitic infections. Specific carbohydrate structures are found on particular cell populations and may be used to induce a specific immune response. These complex structures require reliable methodologies for their assembly. The Lewis antigens, a class of glycosphingolipids, is essential for cellular adhesion and recognition. In addition to their role in normal cellular adhesion processes such as the inflammatory response they have been implicated in many types of cancer and bacterial infections. We developed new synthetic routes for the modular assembly of the Lewis antigens as demonstrated on the example of H-type II that lend themselves to automation (see 1.5). Other tumor-associated antigens including Gb3 have also been prepared. The oligosaccharides obtained from these syntheses are currently being attached to surfaces to enable rapid screening of carbohydrate-protein interactions.

Love, K.R.; Andrade, R.B.; Seeberger, P.H.; Linear Synthesis of a Protected H-type II Pentasaccharide Using Glycosyl Phosphate Building Blocks; J. Org. Chem. 2001, 66, 8165-8176.

On the basis of the glycosyl phosphate building blocks (see 1.1), the new protecting groups (see 1.2) and linkers (see 1.3) we adapted a peptide synthesizer to automated assembly of oligosaccharides on solid support. A temperature controlled reaction vessel was designed and synthetic cycles for oligosaccharide assembly were developed. The first automated solid-phase oligosaccharide synthesizer was used to prepare a branched dodecasaccharide phytoalexin elicitor.
Branching on solid support was achieved by employing orthogonal protecting groups to assemble the Leishmania tetrasaccharide antigen (see 2.2). The automated synthesis allowed for the preparation of this antigen in less than nine hours compared to more than one week when performed manually.
Although automated solid-phase synthesis greatly simplifies the assembly process by eliminating the time consuming purification of intermediates, this very feature often renders the final purification challenging. Particularly, the removal of sequences deficient by just one unit stemming from incomplete couplings at any stage of the synthesis can be very difficult. We developed two novel capping-and-tagging (cap-tag) methods to aid in the purification of oligosaccharides assembled by automated solid-phase synthesis.

Plante, O.J.; Palmacci, E.R.; Seeberger, P.H. Automated Solid-Phase Synthesis of Oligosaccharides, Science 2001, 291, 1523-1527.