Chemo-enzymatic synthesis of Neu 5 Gc-containing sialylated lactulose

Purpose: To synthesize novel sialylated lactuloses, namely, Neu5Gc-α2,3-lactulose and Neu5Gc-α2,6lactulose. Methods: ManNGc was chemically synthesized from commercially available N-acetylmannosaime (ManNAc), which was used as the donor substrate to synthesize α-(2→3) linkage and α-(2→6) linkage sialyllactulose from lactulose via sialyltransferases-catalyzed one-pot multienzyme (OPME) approach. The sialylated products were purified by silica gel flash chromatography column. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) were used to confirm the purity and characterize the structure of the new compounds. Results: Sialyllactulose with α-(2→3) linkage (Neu5Gc-α2,3-lactulose) and α-(2→6) linkage (Neu5Gcα2,6-lactulose) were efficiently synthesized by an efficient one-pot multienzyme sialylation approach from ManNGc, sodium pyruvate, CTP, and lactulose. The molecular weight of the two products, based on mass spectral data was 648 Da while NMR data indicated the formation of sialylated glycans. Conclusion: Novel sialylated oligosaccharides have been efficiently synthesized from lactulose using highly efficient OPME sialylation approaches. Further investigations are required to ascertain the probiotic activities for possible applications in pharmaceutical and food industries.


INTRODUCTION
Sialic acids are nine-carbon sugars and there are over fifty structurally distinct forms of sialic acids found in nature.Sialic acids have three basic forms, including N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), and deaminoneuraminc acid (KDN) [1][2][3].Sialylated glycoconjugates play important roles in physiological and pathological processes, such as cellular recognition, bacterial and viral infection, and tumor development, differentiation, etc [3,4].Currently, the intestinal microbiome is increasingly being recognized as an important determinant of health.Sialylated oligosaccharides are known to be one of the receptors for pathogens and they could inhibit pathogen adhere to the intestinal epithelia [5,6] and block the adhesion of Helicobacter pylori to intestinal cells [7].
The particular difficulty of the application of sialylated oligosaccharides is the lack of sufficient material for performing preclinical trials.Enzymatic approaches have approved to be one of the efficient way to produce many novel synthetic sialylated oligosaccharides which are most likely to have antimicrobial actions.

Lactulose (4-O-β-D-galactopyranosyl-D-fructose)
does not exist in nature.It is usually synthesized from lactose by isomerization and bioconversion [8][9][10].Lactulose is non-absorbable in the upper gastrointestinal tract but can be metabolized by colonic bacteria [12].It has been used to treat hepatic encephalopathy in patients with hyperammonemia [13], and also widely used in food industries because of its prebiotic activities [14].On the other hand, non-human sialic acid Nglycolylneuraminic acid (Neu5Gc) is broadly presented in tissues of animals other than human and has also been shown to be expressed on glycolipids and glycoproteins in human melanoma, colon, and breast cancers [15,16].It is interesting to get knowledge of whether Neu5Gc-containing lactuloses have any increased prebiotic activities.
In this paper, we report here the efficient chemoenzymatic synthesis of Neu5Gc-containing sialylated lactuloses with α-(2→3) linkage and α-(2→6) linkage, via one-pot multienzyme sialylation approach from chemically synthesized ManNGc as the donor substrate and commercially available lactulose as the acceptor substrate.The outcome of this work should provide useful information in the development of Neu5Gc containing sialylated oligosacchride.

Chemical synthesis of ManNGc (1) ManNH 2 . HCl
ManNAc (5 g) was dissolved in 20 mL of 2.5 M HCl.The mixture was reacted at 60 °C (waterbath) for 1 to 2 h.After removal of the solvent, the residue was dried under vacuum. (

(3) ManNGc
ManNGcAc (1 g) was dissolved in dry MeOH (20 mL), and NaOMe (200 mg) was then added to give pH about 9. The reaction mixture was stirred for overnight at rt. Resin (H + ) was added to the reaction mixture to neutralize the NaOMe.After filtration and concentration, the desired ManNGc was obtained (Yield: around 50 %).Reactions were allowed to proceed for 4.5 h at 37 °C.The reaction process was monitored by thin layer chromatography (TLC) and mass spectra.

Mass spectrometry
Purified samples (0.5 mg) were dissolved in 100 μL distilled water at room temperature.Mass Spectrum analyses were carried out on a TQD LC/MS system (Waters, USA), equipped with

ManNGc
As shown in Figure 1, ManNAc, ManNH 2 , ManNGc, and ManNGcAc were separated by the developing solvent (EtOAc:MeOH:H 2 O = 6:2:1) in thin layer chromatography (TLC).Silica gel flash chromatography column was used to purify the products in series of gradient eluting solvent.As shown in Figure 2, using lactulose as the sialyltransferase acceptor, α2-3-sialylated and α2-6-sialylated trisaccharides were readily obtained using an efficient OPME approach.The purified products were checked by TLC and Mass spectrum.Figure 3 showed the compounds were pure.From Figure 4, the molecular weight of Neu5Gcα2-3lactulose and Neu5Gcα2-6Lactulose based on MS data both showed about 648 Da (the mass of lactulose is 342 Da), these results confirmed that the obtained products are the desired sialylated lactuloses.

NMR data
As shown in Figure 5, the structures of sialyllactuloses were confirmed by 1 H and 13 C NMR spectra.The NMR data are as follows: Neu5Gcα2-3Lactulose: Yield, 85 %; white solid.

DISCUSSION
The sialic acid modification on cell surface glycoproteins and glycolipids plays a crucial role in many biological processes, including cell adhesion, antigen recognition and signal transduction [17].Studies have shown that sialylated glycans have a good prospect in food, especially the developing nervous system in infants [18].Chemical synthesis of sialylated glycans is very difficult, and the enzymatic synthesis provides an effective method for generating sialyl linkages [19].
In this paper, in order to obtain Neu5Gccontaining sialosides via enzymatic approaches, firstly ManNGc was synthezied from ManNAc.We found that most ManNGcAc (around 70 %) was deacetylated to ManNGc because of alkaline condition when the reaction of formation of ManNGcAc prolonged to 48 h.The two products were separated by silica gel flash