Human fertilization starts with spermatozoa binding to the oocyte coating which is termed the zona pellucida, the resulting fertilized egg is known as a zygote [41]. Glycans on the zona pellucida have been implicated in sperm binding, however, the structures of the glycans have remained enigmatic then. Until 2011, Pang et al. [25] reported that sLeX is the most abundant terminal sequence on the glycans of human zona pellucida glycoproteins involving in sperm-egg binding, which implied that sLeX sequence represents the main carbohydrate ligand for sperm-egg binding in humans. Recently, Wang et al. [26] found that the sLeX on zona pellucida could bind to a protein, chromosome 1 open reading frame 56 (C1orf56), on human spermatozoa.

Embryo development relies on the adhesion of trophoblast cells to the maternal uterine and the diversion of maternal blood to the placenta. The process of trophoblast adhesion is integrin dependent [42, 43]. Therefore, it is likely that trophoblast adhesion to the endometrium in implantation and placentation may follow a similar way that selectins and their carbohydrate-based ligands follow. Liu et al. [44] reported that the sLeX/L-selectin adhesion system at the maternal and embryonic interface regulates the adhesion of the embryo to the maternal uterine epithelium. They set up an in vitro implantation model using a human trophoblast cell line (JAR) and human uterine epithelial cell line (RL95-2); they found that sLeX was expressed on JAR, after being transfected with fucosyltransferase VII which is responsible for synthesizing sLeX, the synthesis of sLeX in JAR was increased, and simultaneously the percent adhesion of JAR to RL95-2 monolayer was markedly increased [43, 44]. In 2016 sLeX containing N-glycans was identified in human trophoblasts, which supported previous research and implicated the importance of sLeX in embryo development [12].

However, due to the sample limitation and ethical issues, it is not easy to perform research using a human embryo. Therefore, animal models were employed. The first comprehensive glycomic analysis on zebrafish embryos showed that oligomannose-type glycans and complex N-glycans with galactosyl sLeX antennae were major N-glycans at all developmental stages of the whole zebrafish embryonic samples [45, 46], and this suggested the importance of galactosyl sLeX in the embryo development.

sLeX had been identified in around 10% of resting human memory T lymphocytes via anti-sLeX antibodies [47, 48], and this indicates its importance in immunity.

In mammals, lymphocytes circulation occurs in lymphatic and vascular areas, and this allows lymphocytes exposed maximally to invading pathogens. Lymphocytes left the vascular area via lymph nodes, then passed the lymphoid organs, and finally returned to the vascular system [48–50]. This circulation path of lymphocytes is proposed to rely on glycans displayed on specialized endothelial cells, termed HEVs [51].

Lymphocyte homing is regulated via adhesive interactions between lymphocytes and HEVs, it particularly binds to the tetrasaccharide 6-sulfo-sLeX on HEVs. It has been reported found that GlcNAc6STs can control lymphocyte homing via the ligand 6-sulfo-sLeX synthesis on HEVs, and this ligand is on either mucin type branched core 2 O-glycan or extended core 1 O-glycan of endothelial sialomucin CD34 [31, 49]. The function of this carbohydrate structure in lymphocyte homing has been demonstrated mainly through researches using several mouse models with deficiencies in related glycosyltransferases [52], detailedly, studies employing β galactoside α2-3 sialyltransferases 4 and 6 double-deficient mice uncovered the coordinated involvement of these two sialyltransferases in the synthesis of functional oligosaccharides that mediate lymphocyte homing to HEVs [53]; studies using both α1-3 fucosyltransferase 4 and 7 deficiency mice model exposed that the fucosylation of 6-sulfo-sLeX in peripheral lymph nodes HEVs is vital for the interaction with L-selectin [54], compared to wild-type mice, lymphocyte homing to peripheral lymph nodes was significantly influenced in these two fucosyltransferase double-knockout (DKO) mice by more than 80% [52, 54]; studies using GlcNAc6STs 1 and 2 DKO mice illustrated more than 70% reduction in lymphocyte homing, which suggested the importance of GlcNAc-6-O-sulfation in L-selectin ligand synthesis [31, 55].

Asthma is a chronic inflammatory disease that results in severe leukocyte infiltration in the lungs. To achieve the infiltration, the binding of sLeX on the leukocytes to the E- and P-selectins on the endothelial surface at the inflammation area is required [56]. These two selectins probably function similarly in regulating the infiltration; sLeX capped O-glycans on P-selectin glycoprotein ligand 1 (PSGL-1) at the leukocyte surface binds to the two selectins and thus makes leukocytes roll following the blood flow direction [57–60], without the binding of PSGL-1 to the two selectins, leukocytes could not initiate rolling on the endothelial surface [27, 61, 62]. Overcoming the hydrodynamic force of the blood flow is an essential aim of selectin-sLeX binding for leukocyte rolling, Zhang et al. [63] identified the molecular determinants within sLeX that contributes to the binding using single-molecule dynamic force spectroscopy; two determinants in sLeX are required for the selectin binding, which is the Fuc and the terminal Neu5Ac.

sLeX is a well known cancer-associated carbohydrate structure [48, 64, 65]. It plays a vital role in tumor cell metastasis. Increased levels of sLeX positively correlated with metastasis [65]. For instance, mouse melanoma B16-F1 cells were transfected by α1-3 fucosyltransferase 3 to express sLeX, researchers found that transfected cells with high sLeX expression level became highly metastatic compared to the wild-type B16-F1 cells or the cells with low sLeX expression level. In addition, sLeX overexpression on B16-F1 cells resulted in apoptosis in lung tissues, indicating that these cells were eliminated by natural killer cells [66].

It also plays an importance role in cancer cell invasion. For instance, Gomes et al. [67] found that the expression of β galactoside α2-3 sialyltransferase 4 in MKN45 gastric cancer cells resulted in sLeX expression and subsequently caused an increased invasive phenotype in vitro and in the in vivo chicken chorioallantoic membrane (CAM) model via c-Met activation, however, it is not clear whether the sLeX is on glycoprotein or glycolipid.

Glycosylation has emerged as a cancer hallmark; some of the biomarkers used in oncology are cancer-associated glycans, and sLeX is one of them [68]. Serum sLeX has been proposed as a marker for the detection of breast cancer [69, 70]. An increase of sLeX level on ceruloplasmin in pancreatic adenocarcinoma patients was identified via N-glycan analysis on ceruloplasmin. After being immunoprecipitated with anti-ceruloplasmin antibody and analyzed by western blot, sLeX/ceruloplasmin ratio in sera from pancreatic adenocarcinoma patients tend to be higher than that from healthy controls and chronic pancreatitis patients [71]. Tang et al. [72] reported that sLeX could be used as a biomarker for pancreatic cancer in combination with sialyl Lewis A (sLeA), and this could differentiate 109 pancreatic cancers from 91 benign pancreatic diseases with 79% accuracy (74% sensitivity and 78% specificity), which was noticeably better than employing sLeA alone.

sLeX plays an important role in virus attaching. For instance, coronaviruses are able to result in human respiratory tract infections and outbreaks of deadly worldwide pneumonia [73]. Middle East respiratory syndrome coronavirus (MERS-CoV) targets the epithelial cells of the respiratory tract in humans. Proteins or glycolipids with sLeX on the surface of human airway epithelial cells can be used as an attaching receptor by MERS-CoV, thereby increasing infection efficiency. Removing cell surface Neu5Ac by neuraminidase inhibited MERS-CoV from entering human airway cells [73, 74].

Additionally, sLeX is essential for angiogenesis. The function of sLeX in angiogenesis is demonstrated by the observation that the emergence of tube-like networks of endothelial cells caused by the co-culture with cancer cells could be prohibited by sLeX antibodies [24, 75]. Once sLeX biosynthesis was blocked, the ability of hepatocarcinoma cells to promote angiogenesis was hindered [24, 76].

For the chemical synthesis of sLeX tetrasaccharide, the reported synthetic routes can be classified into four strategies according to the sequence of installing monosaccharide/disaccharide building blocks (Figure 3). For a designated strategy, the orthogonal protecting groups and glycosyl donor types (with different leaving groups) can be varied to pursue high reactivity and stereoselectivity.

In 1991, Nicolaou et al. [77] reported the chemical total synthesis of sLeX based on the Strategy 1 (Figure 4). The protected lactosamine 4 was synthesized from GlcNAc acceptor 3 and Gal fluoride donor 2. After releasing the 3-OH group by a two-step protocol of allyl removal (double bond migration and acidolysis), a highly efficient α-fucosylation was achieved using Fuc fluoride donor 5. Next, the sialic acid glycosyl donor 7 bearing an equatorial 3-PhS group was used to facilitate the α-sialylation. The 3-PhS served as an auxiliary and controlled the α-selectivity via neighboring group participation. Finally, the PhS group was removed via radical process, and the sLeX 1 was obtained after global deprotection. In the following years, this BC + D + A strategy (Figure 3) was adopted respectively by Hasegawa et al. [78, 79], Jain et al. [80, 81], Vig et al. [82], Ellervik and Magnusson [83], Herzner and Kunz [84], Filser et al. [85], and Lu et al. [86] to finish the syntheses of sLeX tetrasaccharide and related conjugates, using different sialic acid glycosyl donors and Fuc donors.

In 1992, Danishefsky et al. [87–89] reported the synthesis of 1 based on the Strategy 2 (Figure 5). The disaccharide 11 was first prepared via the α-fucosylation at the more reactive 3-OH of the glycal acceptor 10. Subsequently, without protecting group manipulation, the less reactive 4-OH was involved in the β-galactosylation using donor 12. After removing the three benzoyl groups on the Gal ring of the trisaccharide 13, the sialyl chloride donor 14 reacted selectively with the most reactive 3-OH followed by acetylation to give the tetrasaccharide 15 in good overall yield. Serving as the surrogate of the glucosamine, the glycal in 15 was subjected to the iodinium mediated two-step transformation (iodoamination and aziridine formation/ring-opening) to install the 2-amino group, and product 16 was deprotected globally to give 1. This CD + B + A (Figure 3) Strategy was adopted by Sprengard et al. [90], Kretzschmar and Stahl [91], Misra et al. [92], and Dekany et al. [93] in their synthetic works, while the glucosamine building blocks were used directly instead of the glycal in the first glycosylation step.

In 1998, to further improve the efficiency of the synthetic route, Baba et al. [94] reported the synthesis based on Strategy 3 (Figure 6), in which the tetrasaccharide was assembled in a convergent AB + CD manner (Figure 3). The disaccharide 19, which was prepared from 17 and 18 in high yield, was subjected to the regioselective benzylidene opening and glycosylation with disaccharide donor 20. The tetrasaccharide product 21 was then transformed to 1 after global deprotection. In this route, the two key glycosylation reactions were facilitated by dimethyl(thiomethyl)sulfonium triflate (DMTST) mediated activation of methyl thioglycosides, which was developed by the same group. This strategy was also adopted by Gege et al. [95] and Akçay et al. [96] respectively to achieve sLeX derivatives.

In 2003, Pazynina et al. [97] reported the Strategy 4, in which the tetrasaccharide 27 was assembled in a C + AB + D manner (Figure 3, Figure 7). The linear trisaccharide 24 was prepared from disaccharide donor 22 and acceptor 23, and the chloroacetyl (ClAc) group was selectively removed by ethylenediamine to release 3-OH. Subsequently, α-fucosylation with donor 25 gave product 26, and global deprotection gave 27 in good yield. This work also demonstrated that the hindered trisaccharide can serve as a good acceptor in the α-fucosylation.

In 2012, leveraging Strategy 4, Esposito et al. [98] and Kröck et al. [99] developed the solid phase synthesis of sLeX derivative 35 [98, 99]. As shown in Figure 8, resin 28 with a cleavable linker installed, was used as the support of the whole synthesis. The first GlcNAc building block was first installed by reaction with donor 29, and the resin-bound acceptor 30 was obtained after 9-fluorenylmethyloxycarbonyl (Fmoc) removal. Then, a Lewis acid catalyzed glycosylation using disaccharide donor 31 gave linear trisaccharide, and the new acceptor 32 was obtained after levulinoyl (Lev) deprotection. Finally, N-iodosuccinimide (NIS) mediated α-fucosylation using donor 33 gave resin-bound tetrasaccharide 34, which was transformed into 35 via radical based reduction of trichloroacetamide and global deprotection. This solid phase synthesis, comprised of 6 on-resin steps, 1 linker cleavage step, and 2 in-solution deprotection steps, gave 35 in overall 15% yield with minimized purification efforts.

Compared with chemical synthesis that needs great efforts in protecting group installation and removal, enzymatic synthesis (Figure 9) uses unprotected sugar compounds as building blocks and gives the product highly selective glycosylations. The first attempt at the enzymatic synthesis of sLeX was reported by Palcic et al. in 1989 [100]. In this work, the α-sialylation of disaccharide N-acetyllactosamine (LacNAc) was catalyzed by procine submaxillary α2-3 sialyltransferase, while the α-fucosylation was catalyzed by an α3/4 fucosyltransferase isolated from the milk of a human Le^a+b- donor. A similar synthesis using enzymes obtained from different sources was reported by de Vries et al. [101]. In 1991, Dumas et al. [102] reported that a recombinant α3/4 fucosyltransferase could catalyze the α-fucosylation of a series of disaccharide acceptors and the 3’-sialyl LacNAc. A chemically synthesized trisaccharide with a reducing end block was also suitable for this transformation [103, 104]. This strategy was also applied to the synthesis of sLeX analogs bearing N-modifications [105].

In 1992, Ichikawa et al. [106] reported the enzymatic total synthesis of sLeX from monosaccharides. In this work, uridine diphosphogalactose (UDP)-Gal, cytidine 5’-monophospho (CMP)-Neu5Ac, and guanosine diphosphate (GDP)-Fuc were used as donors respectively in the three enzymatic glycosylation steps. Using this process, a sLeX analog containing ¹³C labelled Gal was synthesized. The same process was also applied to the synthesis of sLeX containing glycopeptides [107] and further extended to the solid phase oligosaccharide synthesis [108]. Considering the limited availability of the glycosyl donors that hampered the scale-up synthesis, Ichikawa et al. [106] developed two multiple enzyme systems to generate the UDP-Gal and CMP-Neu5Ac from glucose-1-phosphate (Glc-1-P) and Neu5Ac respectively. In another work reported by Hayashi et al. [109], the UDP-Gal was generated from UDP-Glc by UDP-Gal epimerase (UDPGE) catalyzed epimerization. The linear trisaccharide was synthesized via enzymatic β-galactosylation and chemical α-sialylation. By this process, a series of sLeX analogues with N-modification on the glucosamine unit was obtained.

In 2011, a new multiple enzyme system for the α-sialylation reaction was developed by Sugiarto et al. [110]. In this system, the enzyme Neisseria meningitidis CMP-sialic acid synthetase (NmCSS) catalyzed the synthesis of CMP-Neu5Ac from Neu5Ac and cytidine triphosphate (CTP), while the maltose binding protein-viral α2-3 sialyltransferase I (N-terminal 30 amino acids truncated)-His6 tag fusion (MBP-Δ30vST3Gal-I-His6) catalyzed the α-sialylation. This system was used by the same group in the synthesis of sulfated sLeX analogues [29]. In 2019, Tasnima et al. [111] reported a new system to facilitate the gram-scale synthesis of sLeX. In this new progress, both UDP-Gal and GDP-Fuc were synthesized enzymatically from Gal and Fuc respectively. The CMP-Neu5Ac was synthesized from N-acetylmannosamine (ManNAc) by two enzymatic reactions catalyzed by Pasteurella multocida sialic acid aldolase (PmNanA) and NmCSS and used in the α-sialylation, as they demonstrated before [112]. More importantly, the mannosamine bearing modification on the 6-OH and 2-NH₂ sites were well adopted by these enzymes, and a series of sLeX analogues were synthesized in good yields.

Sialic acids, a series of nine carbon acidic monosaccharides, usually exist as the terminal sugars on glycoproteins at the cell surface [121, 122]. Neu5Ac is a member of the sialic acid family, and it is the most common sialic acid in humans [21, 123].

Usually, glycans containing Neu5Ac require extra considerations. Because sialic acid residues are highly labile compared to other glycosidic bonds, they are easy to lose during ionization in mass spectrometric analysis [124]. The presence of sialic acid on glycoconjugates offers other analytical difficulties. For instance, the negative charge on the monosaccharide results in quantitative difficulties. Additionally, the presence of sialyl linkage isomers increases the difficulty of analysis of sialylated glycans [124, 125].

There are seveal linkages for Neu5Ac; α2-3, α2-6, α2-8 and α2-9 [6, 21, 126]. In nature, Neu5Acs are α2-3 or α2-6 linked to Gal and N-acetylgalactosamine (GalNAc), α2-6 linked to GlcNAc, and α2-8 or α2-9 linked to the second Neu5Ac residue [21, 127]. In most cases, distinguishing α2-3 and α2-6 linked Neu5Ac is adequate.

Gal β1-4(Fucα1-3)GlcNAc is termed Lewis X.

Fuc is a 6-deoxy hexose in the L-configuration discovered in a great variety of different organisms. In mammals, there are 13 fucosyltransferases responsible for transferring Fuc from GDP-Fuc to glycoconjugates [148]. In humans, there are six α1-3 fucosyltransferases [20]. For a more detailed description of Fuc please read these review papers [148–150]. A research of 3,299 mammalian oligosaccharides showed that Fuc was found in approximately 7.2% of the oligosaccharides studied and thus was the second commonest component [151]. There are seveal linkages for Fuc; α1-2, α1-3, α1-4 and α1-6. In nature, Fucs are α1-2 linked to Gal, α1-3, α1-4, and α1-6 linked to GlcNAc, α1-2 and α1-4 linked to Fuc [148, 152]. In this case, determining α1-3 linked Fuc is required.

Gal has a different configuration of the hydroxyl group at the C4 position from glucose [153]. It exists either as a free sugar or bound to other monosaccharide units in various linkages in glycoproteins and glycolipids [154, 155]. For a more detailed description of Gal please read these review papers [154, 156, 157]. By far 19 distinct galactosyltransferase enzymes have been characterized in mammals, as a result of which there are four linkages for Gal; α1-3, α1-4, β1-3, and β1-4 linked [158]. Gals that are linked to GalNAc via α1-3 linkage can be observed in the O-glycan core 8 structure [21]. Gals that are linked to Gal via α1-4 linkage can be observed in glycosphingolipid Gb4 structure [159]. Gals that are linked to GlcNAc via β1-3 and β1-4 linkages can be observed in type 1 and type 2 LacNAc respectively [160, 161].