On June 19, 2024, a collaborative team of Benjamin G. Davis from the University of Oxford and Ming Joo Koh from the National University of Singapore published a paper titled "Direct radical functionalization of native sugars" in Nature. They developed a biomimetic "cap and glycosylate" strategy that does not require metal catalysis and protecting groups, providing a simple method for directly functionalizing native sugars to form stable glycosides.
Native sugars and Carbohydrates play a key role in biological processes, but due to the difficulty in obtaining pure samples, scientists mostly study their functions through synthetic means. Non-enzymatic chemical glycosylation is the cornerstone of carbohydrate chemistry, but due to the site selectivity of hydroxyl groups, cumbersome protecting group strategies are required. Direct, efficient and selective conversion of native sugars into glycosides remains a major challenge.
Inspired by biological S-glycosylation, the authors speculated that a biomimetic approach could be used to preferentially activate and replace the anomeric hydroxyl group (hemiacetal) in native sugars to form a thioglycoside intermediate (Cap), which could then be subjected to stereo-controlled single-step desulfurization cross-coupling under appropriate conditions to achieve Glycosylation. After exploring a large number of substrates, the authors verified that the thioglycoside intermediate could undergo stereo-controlled desulfurization free radical cross-coupling reactions with a variety of electrophilic reagents after photoactivation, thereby obtaining a series of C-, S-, Se- and O-glycosylated compounds with high yields, high regio- and stereo-selectivity. Most importantly, this method can also be used for direct post-translational glycosylation of aqueous proteins, further demonstrating the high efficiency of the method and the excellent biocompatibility of this new type of Glycosyl Donor.
Fig. 1 Introduction to glycosylation of native sugars and design of the "cap and glycosylate" strategy. (Jiang, et al., 2024)
After proposing the above reaction design, the authors first selected D-glucose 1 as the template substrate to explore the optimal reaction conditions for regio-selective nucleophilic substitution. The results showed that when 2-chloro-1,3-dimethylimidazolinium chloride (DMC) was used as an activator, triethylamine as a base, C5F4N–SH as a nucleophile at 0 °C for 2 h, 2,3,5,6-tetrafluoropyridine-4-thioglycoside derivative 2 could be obtained with a theoretical yield of 85% and a selectivity of > 95:5 β:α. This derivative was reported for the first time and has extremely strong stability. Subsequently, the authors explored the glycosylation reaction performance of the above derivatives. The results showed that the desulfurization carbonyl coupling reaction of thioglycoside donor 2 was carried out at room temperature under blue LED irradiation using Hantzsch ester as a reducing agent, 1,4-diazabicyclo[2.2.2]octane (DABCO) and dimethyl sulfoxide (DMSO) as solvents, and the yield of unprotected C-alkyl glucoside 11 was 96% and more than 95% α selectivity. In addition, the authors also selected other thioglycoside donors (6 - 9) with lower redox activity for the reaction, but the conversion rate was poor, which reflects the importance of thioglycoside donor 2 in the cross-coupling reaction.
Next, the authors found in the experiment that even if D-glucose and D-maltose were used as substrates to synthesize thioglycoside donors 2 and 13 with opposite stereoselectivity, they would obtain products 11 and 15 with the same stereoselectivity after cross-coupling reaction, which aroused the authors' enthusiasm for exploring the reaction mechanism. Therefore, they added exogenous free radical scavenger TEMPO to the reaction system during the cross-coupling process, inhibiting the photoinduced transformation of 2 to 11 that should have occurred according to theory. The cross-coupling reaction mainly generated TEMPO-glycoside adduct 16, which proved that Glycoside free radical species were indeed generated during the cross-coupling reaction. The thioglycoside donor 2, Hantzsch ester and DBACO can form a ternary complex, which will cleave to generate glycosyl free radicals after absorbing visible light. From this, the authors deduced the reaction mechanism of the "cap and glycosylate" strategy. First, DMC selectively caps the more acidic anomeric hydroxyl group and forms an activated leaving group, which is nucleophilically attacked by C5F4N–SH under alkaline conditions to form a thioglycolate donor and a byproduct 1,3-dimethylimidazolidin-2-one (DMI). Subsequently, the resulting thioglycolate donor forms a ternary complex with Hantzsch ester and DABCO, which can absorb visible light and induce photoinduced electron transfer (PET), thereby obtaining a dihydropyridine radical 17 and a radical anion 18, which can desulfurize and cleave the thioglycolate donor to obtain a glycosyl radical and 2,3,5,6-tetrafluoropyridine-4-thiolate. Finally, the glycosyl radical is cross-coupled with an electrophilic reagent under the promotion of 17 to achieve a stereoselective coupling reaction, thereby obtaining the desired unprotected glycoside.
Afterwards, the authors explored the versatility of the strategy. They found that the strategy not only has good adaptability to common native sugars, rare sugars, non-native sugars and Oligosaccharides, and can prepare target C-glycoside products with high yields and good stereoselectivity, but also can be universally reacted with a variety of electrophilic coupling reagents to obtain glycoside compounds with high yields and good stereoselectivity.
Finally, the authors further explored the biocompatibility of the reaction. They turned their attention to glycoproteins, which are extremely important in the field of biology, so the authors tried to use this strategy to achieve protein glycosylation reactions. The results showed that regardless of the size and folding shape of the protein, the reaction can obtain the corresponding unprotected C-alkyl glycoprotein with good conversion rate and α-selectivity.
Fig. 2 Application of the "cap and glycosylate" strategy in protein glycosylation. (Jiang, et al., 2024)
In short, the authors reported a photoinduced unprotected group "cap and glycosylate" strategy by simulating the glycosylation process in nature. Through free radical cross-coupling, a series of glycoside compounds with diverse structures, high yield and good stereoselectivity were directly synthesized from native sugars. They extended the reaction to post-translational glycosylation of proteins, providing a new idea for the preparation of non-native glycoproteins. The Glycoproteins synthesized by this method have potential application value in the biomedical field and are expected to promote the research and application of glycosylated compounds in the fields of biochemistry, medicinal chemistry and materials science.
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