Process Engineering and Glycosyltransferase Improvement for Short Route Chemoenzymatic Total Synthesis of GM1 Gangliosides

Abstract

Large-scale synthesis of GM1, an important ganglioside in mammalian cells especially those in the nervous system, is needed to explore its therapeutic potential. Biocatalytic production is a promising platform for such a purpose. We report herein the development of process engineering and glycosyltransferase improvement strategies to advance chemoenzymatic total synthesis of GM1. Firstly, a new short route was developed for chemical synthesis of lactosylsphingosine from the commercially available Garner's aldehyde. Secondly, two glycosyltransferases including Campylobacter jejuni β1-4GalNAcT (CjCgtA) and β1-3-galactosyltransferase (CjCgtB) were improved on their soluble expression in E. coli and enzyme stability by fusing with an N-terminal maltose binding protein (MBP). Thirdly, the process for enzymatic synthesis of GM1 sphingosines from lactosylsphingosine was engineered by developing a multistep onepot multienzyme (MSOPME) strategy without isolating intermediate glycosphingosines and by adding a detergent, sodium cholate, to the later enzymatic glycosylation steps. Installation of a desired fatty acyl chain to GM1 glycosphingosines led to the formation of target GM1 gangliosides. The combination of glycosyltransferase improvement with chemical and enzymatic process engineering represents a significant advance in obtaining GM1 gangliosides containing different sialic acid forms by total chemoenzymatic synthesis in a short route and with high efficiency.

Enabling Chemoenzymatic Strategies and Enzymes for Synthesizing Sialyl Glycans and Sialyl Glycoconjugates

“…10.1002/chem.202300005202329e202300005 . A user-friendly highly efficient chemoenzymatic strategy showcased for the synthesis of complex gangliosides on gram scales by the one-pot enzymatic glycosylation of chemically synthesized lactosyl sphingosine followed by chemical acylation, with highlights of short-route chemical synthesis, glycosylation process engineering, and biocatalyst improvement.…”

“…The second approach developed by our collaborators Peng G. Wang and Jun Yin’s groups started from an inexpensive N -Boc l -serine methyl ester to form a β-ketophosphonate intermediate which reacted with an alkyl aldehyde in the presence of potassium carbonate in acetonitrile and water to form exclusively the desired E -olefin derivative in the sphingosine acceptor (Acceptor 2, Figure b) via the Horner–Wadsworth–Emmons reaction . The third approach used ( S )-Garner’s aldehyde and 1-pentadecyne as starting materials to form the sphingosine Acceptor 1 (Figure c) in a short four-step route with two column purification steps . Chemical glycosylation of Acceptor 1 or Acceptor 2 with a per- O -benzoylated trichloroacetimidate lactosyl donor followed by deprotection formed the desired LacβSph (Figure d) ready for enzymatic glycosylation.…”

“…The ganglio-series GSLs obtained included prioritized cancer antigens GM3, fucosyl GM1, GD3, and GD2; GM1 (Figure e), which is a therapeutic candidate for Huntington’s and Pakinson’s diseases; and 9- N -acetyl analogs of 9- O -acetylated b-series ganglio-series gangliosides including 9NAc-GD3, 9NAc-GD2, 9NAc-GD1b, and 9NAc-GT1b (Figure f) . The sphingosine hydrophobic tail in the glycosyl sphingosine product facilitated the product purification from OPME reactions in less than 30 min with a single C18 cartridge, which was much simpler compared to the multiple column purification processes needed for purifying glycosphingolipid glycans containing a free reducing end or a propyl azide aglycone. , Initial synthesis involved purifying individual intermediate glycosyl sphingosines, which was necessary when they were all desired targets but was not needed when a more complex GSL such as GM1 was the target . The use of the OPME sialylation system allowed the introduction of different sialic acid structures to the products as demonstrated for the synthesis of GM3 glycosphingosines containing various sialic acid forms .…”

“… 40 The third approach used ( S )-Garner’s aldehyde and 1-pentadecyne as starting materials to form the sphingosine Acceptor 1 ( Figure 6 c) in a short four-step route with two column purification steps. 4 Chemical glycosylation of Acceptor 1 or Acceptor 2 with a per- O -benzoylated trichloroacetimidate lactosyl donor followed by deprotection formed the desired LacβSph ( Figure 6 d) ready for enzymatic glycosylation.…”

“…Complex glycosyl sphingosines, including those in the neolacto series (e.g., Lc 3 βSph, nLc 4 βSph, and Galα3nLc 4 βSph) 38 and the ganglio series, were readily produced from LacβSph using sequential OPME reactions. The ganglio-series GSLs obtained included prioritized cancer antigens GM3, fucosyl GM1, GD3, and GD2; 39 GM1 ( Figure 6 e), which is a therapeutic candidate for Huntington’s and Pakinson’s diseases; 4 and 9- N -acetyl analogs of 9- O -acetylated b-series ganglio-series gangliosides including 9NAc-GD3, 9NAc-GD2, 9NAc-GD1b, and 9NAc-GT1b ( Figure 6 f). 41 The sphingosine hydrophobic tail in the glycosyl sphingosine product facilitated the product purification from OPME reactions in less than 30 min with a single C18 cartridge, which was much simpler compared to the multiple column purification processes needed for purifying glycosphingolipid glycans containing a free reducing end 35 or a propyl azide aglycone.…”