Clay S. Bennett

Chemoenzymatic approaches to glycoprotein synthesis

The construction of homogeneous glycoproteins presents a formidable challenge to the synthetic chemist. Over the past few years there has been an explosion in the number of methods developed to address this problem. These methods include the development of novel ligation technologies for the synthesis of the protein backbone, as well chemical and enzymatic approaches for introducing complex glycans into the peptide backbone. This tutorial review discusses the application of these techniques to the synthesis of peptides and proteins possessing well defined glycans.

Chemoenzymatic elaboration of monosaccharides using engineered cytochrome P450BM3 demethylases

Polysaccharides comprise an extremely important class of biopolymers that play critical roles in a wide range of biological processes, but the synthesis of these compounds is challenging because of their complex structures. We have developed a chemoenzymatic method for regioselective deprotection of monosaccharide substrates using engineered Bacillus megaterium cytochrome P450 (P450BM3) demethylases that provides a highly efficient means to access valuable intermediates, which can be converted to a wide range of substituted monosaccharides and polysaccharides. Demethylases displaying high levels of regioselectivity toward a number of protected monosaccharides were identified using a combination of protein and substrate engineering, suggesting that this approach ultimately could be used in the synthesis of a wide range of substituted mono- and polysaccharides for studies in chemistry, biology, and medicine.

Advances in glycoprotein synthesis

The development of chemical and enzymatic methods for the synthesis of homogeneous glycoproteins is a fascinating challenge at the interface between chemistry and biology. Discussed here are the currently available methods for preparation of homogeneous glycoproteins. These methods include (1) glycopeptide ligation; (2) glycoprotein remodeling; and (3) in vivo suppressor tRNA technology.

A reagent-controlled SN2-glycosylation for the direct synthesis of β-linked 2-deoxy-sugars.

The efficient and stereoselective construction of glycosidic linkages remains one of the most formidable challenges in organic chemistry. This is especially true in cases such as β-linked deoxy-sugars, where the outcome of the reaction cannot be controlled using the stereochemical information intrinsic to the glycosyl donor. Here we show that p-toluenesulfonic anhydride activates 2-deoxy-sugar hemiacetals in situ as electrophilic species, which react stereoselectively with nucleophilic acceptors to produce β-anomers exclusively. NMR studies confirm that, under these conditions, the hemiacetal is quantitatively converted into an α-glycosyl tosylate, which is presumably the reactive species in the reaction. This approach demonstrates that use of promoters that activate hemiacetals as well-defined intermediates can be used to permit stereoselective glycosylation through an SN2-pathway.

Sugar-Assisted Glycopeptide Ligation with Complex Oligosaccharides: Scope and Limitations

We have previously shown sugar-assisted ligation (SAL) to be a useful method for the convergent construction of glycopeptides. However to date SAL has only been carried out on systems where the thiol auxiliary is attached to a monosaccharide. For SAL to be truly applicable to the construction of fully elaborated glycopeptides and glycoproteins, it must be possible to carry out the reaction when the thiol auxiliary is attached to more elaborate sugars, as these are frequently what are observed in nature. Here we examine the effects of glycosylation at C-3, C-4, and C-6 of the C-2 auxiliary-containing glycan. Model glycopeptides where synthesized chemoenzymatically and reacted with peptide thioesters used in our previous work. These studies reveal that SAL is sensitive to extended glycosylation on the auxiliary-containing sugar. While it is possible to carry out SAL with extended glycosylation at C-4 and C-6, the presence of glycosylation at C-3 prevents the ligation from occurring. Additionally, with glycosylation at C-4 the ligation efficiency is affected by the identity of the N-terminal AA, while the nature of the C-terminal residue of the peptide thioester does not appear to affect ligation efficiency. These studies provide useful guidelines in deciding when it is appropriate to use SAL in the synthesis of complex glycopeptides and glycoproteins and how to choose ligation junctions for optimal yield.

Reagent controlled β-specific dehydrative glycosylation reactions with 2-deoxy-sugars.

N-Sulfonyl imidazoles activate 2-deoxy-sugar hemiacetals for glycosylation presumably by converting them into glycosyl sulfonates in situ. By matching the leaving group ability of the sulfonate with the reactivity of the donor, it is possible to obtain β-specific glycosylation reactions. The reaction serves as proof of the principle that, by choosing promoters that can modulate the reactivity of active intermediates, it is possible to place glycosylation reactions entirely under reagent control.

Cyclopropenium Cation Promoted Dehydrative Glycosylations Using 2-Deoxy- and 2,6-Dideoxy-Sugar Donors

Dehydrative glycosylation reactions using 2-deoxy- and 2,6-dideoxy-sugar donors promoted by a combination of 3,3-dichloro-1,2-diphenylcyclopropene and tetrabutylammonium iodide (TBAI) are described. The reactions are α-selective and proceed under mild conditions at room temperature without the need for special dehydrating agents. The reaction is shown to be effective with a number of glycosyl acceptors, including those possessing acid and base sensitive functionality.

Selective synthesis of 1,2-cis-α-glycosides without directing groups. Application to iterative oligosaccharide synthesis.

A method for the highly selective synthesis of 1,2-cis-α-linked glycosides that does not require the use of the specialized protecting group patterns normally employed to control diastereoselectivity is described. Thioglycoside acceptors can be used, permitting iterative oligosaccharide synthesis. The approach eliminates the need for lengthy syntheses of monosaccharides possessing highly specialized and unconventional protecting group patterns.

Design, synthesis, and biological evaluation of diminutive forms of (+)-spongistatin 1: lessons learned.

The design, synthesis, and biological evaluation of two diminutive forms of (+)-spongistatin 1, in conjunction with the development of a potentially general design strategy to simplify highly flexible macrocyclic molecules while maintaining biological activity, have been achieved. Examination of the solution conformations of (+)-spongistatin 1 revealed a common conformational preference along the western perimeter comprising the ABEF rings. Exploiting the hypothesis that the small-molecule recognition/binding domains are likely to comprise the conformationally less mobile portions of a ligand led to the design of analogues, incorporating tethers (blue) in place of the CD and the ABCD components of the (+)-spongistatin 1 macrolide, such that the conformation of the retained (+)-spongistatin 1 skeleton would mimic the assigned solution conformations of the natural product. The observed nanomolar cytotoxicity and microtubule destabilizing activity of the ABEF analogue provide support for both the assigned solution conformation of (+)-spongistatin 1 and the validity of the design strategy.

Principles of modern solid-phase oligosaccharide synthesis.

The past few decades have seen a renewed interest in the chemical synthesis of oligosaccharides, owing to increased recognition of their biological importance. Of the different approaches to oligosaccharides, solid-phase synthesis has emerged as a particularly attractive option, due in large part to its potential for automation. This perspective describes principles to consider when planning solid-phase oligosaccharide synthesis (SPOS), including recent achievements and areas where future work is necessary.