Systematic chemoenzymatic synthesis of O-sulfated sialyl Lewis x antigens

Abstract

O-Sulfated sialyl Lewis x antigens play important roles in nature. However, due to their structural complexity, they are not readily accessible by either chemical or enzymatic synthetic processes. Taking advantage of a bacterial sialyltransferase mutant that can catalyze the transfer of different sialic acid forms from the corresponding sugar nucleotide donors to Lewis x antigens, which are fucosylated glycans, as well as an efficient one-pot multienzyme (OPME) sialylation system, O-sulfated sialyl Lewis x antigens containing different sialic acid forms and O-sulfation at different locations were systematically synthesized by chemoenzymatic methods.

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Introduction

O-Sulfated sialyl Lewis x structures play important roles in immune regulation, inflammation, and cancer metastasis.¹ For example, 6-O-sulfo-sialyl Lewis x [6-O-sulfo-sLe^x (1), Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAc6SβOR] with an O-sulfate group at the carbon-6 of the N-acetylglucosamine (GlcNAc) residue (Fig. 1) is a well known ligand for L-selectin, a C-type (Ca²⁺-dependent) carbohydrate-binding protein (lectin) expressed broadly in most leukocytes in the blood.^1,2 The interaction of 6-O-sulfo-sLe^x (1) and L-selectin plays a critical role in lymphocyte homing to the peripheral lymph nodes² and in chronic inflammation.³ It has also been shown that human sialic acid-binding immunoglobulin-like lectin⁴ Siglec-9 binds strongly^5,6 to 6-O-sulfo-sLe^x, but the biological importance of this interaction is less well understood.

Fig. 1 Structures of O-sulfated sialyl Lewis x including 6-O-sulfo-sLe^x (1), 6′-O-sulfo-sLe^x (2), and 6′, 6-di-O-sulfo-sLe^x (3).

On the other hand, 6′-O-sulfo-sialyl Lewis x [6′-O-sulfo-sLe^x (2), Neu5Acα2-3Gal6Sβ1-4(Fucα1-3)GlcNAcβOR] with an O-sulfate group at the carbon-6 of the galactose (Gal) residue (Fig. 1),⁷ in addition to 6′-O-sulfo-sialyl-N-acetyllactosamine (6′-O-sulfo-sLacNAc, Neu5Acα2-3Gal6Sβ1-4GlcNAcβOR),⁸ was shown by glycan microarray studies to be a preferred glycan ligand for Siglec-8 and for its paralog mouse Siglec-F.⁹ Siglec-8 is expressed in human allergic inflammatory cells including eosinophils, mast cells, and basophils.^5,10 Reducing the number of eosinophils, such as by soluble 6′-O-sulfo-sLe^x synthetic polymer induced apoptosis,¹¹ has been suggested as an approach for asthma therapies.¹² Furthermore, 6′-O-sulfo-sLe^x (2), in addition to 6′-O-sulfo-sLacNAc and 6′-O-sulfo-sialyl-lacto-N-neotetraose (6′-O-sulfo-sLNnT, Neu5Acα2-3Gal6Sβ1-4GlcNAcβ1-3Galβ1-4GlcβOR), was shown to bind to langerin,¹³ a C-type (Ca²⁺-dependent) lectin specific to Langerhans cells (immature antigen-presenting specific T cell immunity initiating dendritic cells of epidermis and mucosal tissues).¹⁴

Scheme 1 Sequential OPME synthesis of 6-O-sulfo-Le^xβProN₃ (8) from 6-O-sulfo-GlcNAcβProN₃ (7) using an OPME β1-4-galactosyl activation and transfer system for the formation of 6-O-sulfo-LacNAcβProN₃ (4) followed by an OPME α1-3-fucosyl activation and transfer system for the formation of 6-O-sulfo-Le^xβProN₃ (8). Enzymes and abbreviations: SpGalK, Streptococcus pneumoniae TIGR4 galactokinase;⁴⁴ BLUSP, Bifidobacterium longum UDP-sugar pyrophosphorylase;⁴⁵ PmPpA, Pasteurella multocida inorganic pyrophosphorylase;⁴³ Hp4GalT, Helicobacter pylori β1-4-galactosyltransferase;⁴³ BfFKP, Bacteroides fragilis bifunctional L-fucokinase/GDP-fucose pyrophosphorylase;⁴² and Hp3FT, Helicobacter pylori α1-3-fucosyltransferase.^39,41

Although less efficient than Neu5Acα2-8Neu5Acα2-3LacNAc, both 6-O-sulfo-sLe^x (1) and 6′-O-sulfo-sLe^x (2) bound moderately to human Siglec-7.⁵ Both are present in glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), an L-selectin ligand,¹⁵ with 6′-O-sulfo-sLe^x (2) as the major sulfated form.^16–18 Gal-6-O-sulfotransferase and GlcNAc-6-O-sulfotransferase have been found to synergistically produce L-selectin ligands. This indicates either the potential synergistic involvement of both 6-O-sulfo-sLe^x (1) and 6′-O-sulfo-sLe^x (2) or the involvement of 6′,6-di-O-sulfo-sLe^x (3) (Fig. 1) with O-sulfate groups at both the Gal and GlcNAc residues of sLe^x in L-selectin-binding.¹⁹ Human Siglec-7 and -8 have also been shown to bind more strongly to 6′,6-di-O-sulfo-sLe^x (3) than their mono-O-sulfated derivatives (1 and 2), while mouse Siglec-F has been shown to bind with similar strength to 6′,6-di-O-sulfo-sLe^x (3) and 6′-O-sulfo-sLe^x (2).⁶

The biological importance of O-sulfated sLe^x structures makes them attractive synthetic targets. However, the structures of these compounds are relatively complex and include synthetically challenging α2-3-linked sialic acid, which suffers from low stereoselectivity and a high 2,3-elimination rate in chemical synthesis,^20–22 as well as the acid labile O-sulfate group.^23,24 Chemically^20,25,26 or chemoenzymatically²⁷ synthesized Neu5Acα2-3Gal building blocks have been used as effective synthons for constructing more complex sialosides including sLe^x and 6-O-sulfo-sLe^x (1).²⁰ Several examples of the chemical^22,28 or chemoenzymatic²⁹ synthesis of 6-O-sulfo-sLe^x (1) as well as the chemical synthesis of 6′-O-sulfo-sLe^x (2)^22,30,31 and 6′,6-di-O-sulfo-sLe^x (3)³² have been reported. All these examples are, however, limited to compounds with the most abundant sialic acid form, N-acetylneuraminic acid (Neu5Ac). Despite the presence of more than 50 different sialic acid forms identified in nature,^33,34O-sulfated sLe^x containing a sialic acid form other than Neu5Ac has not been synthesized.

We report here the development of efficient chemoenzymatic methods for the systematic synthesis of O-sulfated sLe^x containing different sialic acid forms. The methods are demonstrated for representative examples of 6′-O-sulfo-sLe^x (1), 6-O-sulfo-sLe^x (2) and/or 6′,6-di-O-sulfo-sLe^x (3) containing the most abundant Neu5Ac form and N-glycolylneuraminic acid (Neu5Gc), a sialic acid form commonly found in mammals other than humans, but which can be incorporated into the human glycome from dietary sources.³⁵

One efficient approach for the synthesis of O-sulfated sLe^x with different sialic acid forms would be by direct sialylation of O-sulfated Le^x using one-pot multienzyme (OPME) sialylation systems³⁶ containing an α2-3-sialyltransferase and a CMP-sialic acid synthetase (CSS),³⁷ with or without a sialic acid aldolase.³⁸ Such an approach has been successfully demonstrated for direct sialylation of non-sulfated Le^x for the synthesis of sLe^x containing a diverse array of naturally occurring and non-natural sialic acid forms, using OPME systems containing a recombinant viral α2-3-sialyltransferase vST3Gal-I³⁹ or a bacterial multifunctional sialyltransferase mutant, Pasteurella multocida α2-3-sialyltransferase 1 (PmST1) M144D.⁴⁰ The latter, with a high expression level (98 mg L^-1 culture, >1000-fold higher than that of vST3Gal-I) and high promiscuity in tolerating different modifications on the sialic acid in the substrates, is a superior choice for the synthesis.⁴⁰ However, it is not clear whether O-sulfated Le^x structures could be used with PmST1 M144D as suitable acceptors in the OPME sialylation process to produce the desired O-sulfated sLe^x with different sialic acid forms.

Results and discussion

Synthesis of O-sulfated disaccharides and O-sulfated Le^x

In order to obtain O-sulfated Le^x as potential acceptor substrates for PmST1 M144D, enzyme-catalyzed α1-3-fucosylation of the corresponding O-sulfated disaccharides was tested as a potential strategy. A one-pot three-enzyme (OP3E) α1-3-fucosylation system (Scheme 1)^39,41 containing Bacteroides fragilis bifunctional L-fucokinase/GDP-fucose pyrophosphorylase (BfFKP),⁴²Pasteurella multocida inorganic pyrophosphorylase (PmPpA),⁴³ and Helicobacter pylori α1-3-fucosyltransferase (Hp1-3FTΔ66 or Hp3FT) was used for this purpose. The O-sulfated disaccharides tested were 6-O-sulfo-LacNAcβProN₃ (4) (Scheme 1), 6′-O-sulfo-LacNAcβProN₃ (5), and 6,6′-di-O-sulfo-LacNAcβProN₃ (6) (Fig. 2). LacNAcβProN₃ (ref. 43) without any O-sulfate groups was used as a positive control.

Fig. 2 Structures of chemically synthesized 6′-O-sulfo-LacNAcβProN₃ (5) and 6,6′-di-O-sulfo-LacNAcβProN₃ (6).

6-O-Sulfo-LacNAcβProN₃ (4) was synthesized from 6-O-sulfo-GlcNAcβProN₃ (7)⁴³ using an improved OPME galactosyl activation and transfer system (Scheme 1) containing Streptococcus pneumoniae TIGR4 galactokinase (SpGalK),⁴⁴Bifidobacterium longum UDP-sugar pyrophosphorylase (BLUSP),⁴⁵ PmPpA, and a Helicobacter pylori β1-4-galactosyltransferase (Hp1-4GalT or Hp4GalT).⁴³ The EcGalK, BLUSP, and PmPpA allowed in situ formation of the donor substrate of Hp4GalT, uridine 5′-diphosphate-galactose (UDP-Gal), from monosaccharide galactose (Gal).⁴⁵ It was previously shown that Hp4GalT, but not Neisseria meningitidis β1-4-galactosyltransferase (NmLgtB), was able to use 6-O-sulfated GlcNAc and derivatives as acceptor substrates for the synthesis of β1-4-linked galactosides.⁴³ The activity of Hp4GalT in synthesizing 6-O-sulfo-LacNAcβProN₃ (4) was confirmed again here using the improved OPME approach.^45,46 An excellent 89% yield was obtained, comparing favourably to the previous Hp4GalT-dependent OPME β1-4-galactosylation approach (70% yield) which used Escherichia coli K-12 glucose-1-P uridylyltransferase (EcGalU), Escherichia coli UDP-galactose-4-epimerase (EcGalE), and PmPpA to produce UDP-Gal in situ from glucose-1-phosphate.⁴³ 6′-O-Sulfo-LacNAcβProN₃ (5) and 6,6′-di-O-sulfo-LacNAcβProN₃ (6) (Fig. 2) were chemically synthesized (see ESI†).

Scheme 2 Chemical synthesis of 6′-O-sulfo-Le^xβProNH₂ (9) and 6,6′-di-O-sulfo-Le^xβProNH₂ (10). Reagents and conditions: (a) N-iodosuccinimide (NIS), TMSOTf, MS 4 Å, CH₂Cl₂, −40 °C, 30 min; (b) N-iodosuccinimide (NIS), TMSOTf, MS 4 Å, CH₂Cl₂–Et₂O (1 : 1), −18 °C, 45 min; (c) H₂N(CH₂)₂NH₂, n-BuOH, 90 °C, 8 h; (d) pyridine, Ac₂O, r.t., 10 h; (e) HF pyridine, 0 °C to r.t., overnight; (f) SO₃ pyridine, pyridine, 0 °C to r.t.; (g) 0.1 M NaOMe, MeOH, r.t., 3 h; (h) Pd(OH)₂/C, H₂, CH₃OH, 48 h.

Among the three O-sulfated disaccharides tested, only 6-O-sulfo-LacNAcβProN₃ (4) was a suitable acceptor for Hp3FT to produce the desired 6-O-sulfo-Le^xβProN₃ (8). In contrast, 6′-O-sulfo-LacNAcβProN₃ (5) and 6,6′-di-O-sulfo-LacNAcβProN₃ (6) were not used efficiently by Hp3FT for the synthesis of the corresponding O-sulfated Le^x derivatives. With the positive outcome in small scale reactions for fucosylation of 6-O-sulfo-LacNAcβProN₃ (4), the preparative-scale synthesis of 6-O-sulfo-Le^xβProN₃ (8) was carried out using the OP3E α1-3-fucosyl activation and transfer system (Scheme 1). A yield of 70% was obtained. The combined sequential OPME β1-4-galactosylation and OPME α1-3-fucosylation (Scheme 1) was an effective approach for obtaining 6-O-sulfo-Le^xβProN₃ (8) from a simple monosaccharide derivative 6-O-sulfo-GlcNAcβProN₃ (7) in an overall yield of 62%.

As Hp3FT was not able to use 6′-O-sulfo-LacNAcβProN₃ (5) or 6,6′-di-O-sulfo-LacNAcβProN₃ (6) efficiently as acceptors for fucosylation to obtain the desired Le^x trisaccharides, the target trisaccharides 6′-O-sulfo-Le^xβProNH₂ (9) and 6,6′-di-O-sulfo-Le^xβProNH₂ (10) were chemically synthesized (Scheme 2) from monosaccharide synthons 11, 12,²⁷13, and 14.²⁷ Notable features of the synthetic strategy include: (a) application of an efficient general protection strategy⁴⁷ for the synthesis of the two trisaccharides (i.e. similar protecting groups were used in the syntheses and the same reagents were used for their removal); (b) use of similar thioglycoside derivatives as glycosyl donors in all glycosylations; (c) high regio- and stereoselectivity in product formation; (d) one step removal of benzyl ethers and reduction of the azido group using 20% Pd(OH)₂/C (Pearlman's catalyst) and H₂.⁴⁸ More specifically, for the synthesis of 9 and 10, two N-phthalimide glucosamine derivatives 11 and 12 selectively protected at C6 with benzyl and tert-butyldiphenylsilyl ether (TBDPS), respectively, were coupled stereoselectively with thioglycoside donor 13, which was selectively protected with TBDPS at C6, in the presence of N-iodosuccinimide (NIS) and trimethylsilyl trifluoromethanesulfonate (TMSOTf)⁴⁹ in dichloromethane. Disaccharide derivatives 15 and 16 were obtained in 72% and 78% yields, respectively. The bulky N-phthalimido protecting group in acceptors 11 and 12 provides steric hindrance to the neighboring C-3 hydroxyl group and decreases the reactivity of the C-3 hydroxyl group. Therefore, glycosylation occurs regioselectively at the C-4 hydroxyl group.²⁷ Initial attempts to glycosylate acceptors 15 and 16 in dichloromethane with 1.2 equivalents of thiophenyl fucoside 14 produced trisaccharides in alpha and beta mixtures. In contrast, stereospecific formation of trisaccharides was achieved when a mixed solvent of diethylether and dichloromethane (1 : 1)^50,51 was employed. The reaction of acceptors 15 and 16 with 1.2 equivalents of fucosyl donor 14 produced compounds 17 and 18 in 68% and 65% yields, respectively. Compounds 17 and 18 were then subjected to a series of synthetic transformations: (a) conversion of the N-phthaloyl group to an acetamido group by removing the phthaloyl group using ethylenediamine, followed by N- and O-acetylation using acetic anhydride and pyridine; (b) HF-pyridine-mediated selective removal of the TBDPS group;⁵² (c) O-sulfation of the primary hydroxyl group by SO₃ pyridine complex;^52,53 (d) deacetylation by NaOMe in MeOH;⁵⁴ and (e) hydrogenation using Pd(OH)₂/C and H₂ (ref. 55) to obtain the desired 6′-O-sulfo-Le^xβProNH₂ (9) and 6,6′-di-O-sulfo-Le^xβProNH₂ (10).

Enzymatic synthesis of O-sulfated sLe^x

With chemoenzymatically synthesized 6-O-sulfo-Le^xβProN₃ (8) as well as chemically synthesized 6′-O-sulfo-Le^xβProNH₂ (9) and 6,6′-di-O-sulfo-Le^xβProNH₂ (10) in hand, a one-pot two-enzyme (OP2E) sialylation system (Scheme 3) was used to test the tolerance of PmST1 M144D⁴⁰ for using these O-sulfated Le^x compounds as potential acceptor substrates. PmST1 M144D was previously engineered by protein crystal structure-assisted design. It has 20-fold reduced CMP-sialic acid (donor) hydrolysis activity and significantly (5588-fold) decreased α2-3-sialidase activity compared to the wild-type enzyme. It was used efficiently in a one-pot three-enzyme (OP3E) sialylation system for the synthesis of non-sulfated sLe^x tetrasaccharides containing diverse sialic acid forms from Le^x.⁴⁰ To our delight, PmST1 M144D also tolerated O-sulfated Le^x containing O-sulfate at C-6, C-6′, or both. In addition to N-acetylneuraminic acid (Neu5Ac), N-acetylneuraminic acid (Neu5Gc) was also successfully introduced to compounds 8–10. O-Sulfated sLe^x tetrasaccharides 6-O-sulfo-Neu5Acα2-3Le^xβProN₃ (1a, 80 mg, 85%), 6-O-sulfo-Neu5Gcα2-3Le^xβProN₃ (1b, 22 mg, 47%), 6′-O-sulfo-Neu5Acα2-3Le^xβProNH₂ (2a, 75 mg, 82%), 6′-O-sulfo-Neu5Acα2-3Le^xβProNH₂ (2b, 45 mg, 60%), 6,6′-di-O-sulfo-Neu5Acα2-3Le^xβProNH₂ (3a, 42 mg, 64%), and 6,6′-di-O-sulfo-Neu5Gcα2-3Le^xβProNH₂ (3b, 40 mg, 38%) were successfully obtained using this highly efficient one-pot two-enzyme system containing Neisseria meningitidis CMP-sialic acid (NmCSS)³⁷ and PmST1 M144D⁴⁰ from the corresponding acceptors 8–10 and Neu5Ac or Neu5Gc, respectively. In general, Neu5Gc was used less efficiently by the OPME sialylation system, leading to lower yields for 1b–3b (38–60%) compared to their Neu5Ac-counterparts 1a–3a (64–85%). O-Sulfated sLe^x glycans with a propyl amine aglycone (compounds 2a, 3a, 2b and 3b) were found to be more challenging for column purification compared to the ones with a propyl azide aglycone (compounds 1a and 1b). When a desired sialic acid is readily available such as in the case presented here, a one-pot two-enzyme (OP2E) system is sufficient. When only the 6-carbon precursors of the desired sialic acid forms are available, the one-pot three-enzyme (OP3E) sialylation system including an aldolase in addition to NmCSS and PmST1 M144D⁴⁰ should be used.

Scheme 3 PmST1 M144D-mediated one-pot two-enzyme (OP2E) sialylation of O-sulfo analogues of Lewis^x. Yields obtained for O-sulfated sLe^x tetrasaccharides: 1a, 85%; 1b, 47%; 2a, 82%; 2b, 60%; 3a, 64%; 3b, 38%. Enzymes and abbreviations: NmCSS, Neisseria meningitidis CMP-sialic acid;³⁷ PmST1 M144D, Pasteurella multocida α2-3-sialyltransferase 1 (PmST1) M144D mutant.⁴⁰

Conclusions

In conclusion, we have successfully developed an efficient chemoenzymatic method for the systematic synthesis of synthetically challenging O-sulfated sLe^x (1a–3a and 1b–3b) containing different sialic acid forms (Neu5Ac or Neu5Gc) by direct sialylation of the corresponding O-sulfated Le^x structures 8–10 using an efficient one-pot two-enzyme (OP2E) system containing NmCSS and PmST1 M144D. The method can be extended to the synthesis of O-sulfated sLe^x structures containing other sialic acid forms. We have also shown here that a relatively complex trisaccharide 6-O-sulfo-Le^xβProN₃ (8) can be efficiently produced from a simple monosaccharide derivative 6-O-sulfo-GlcNAcβProN₃ (7) by a sequential OPME β1-4-galactosylation and OPME α1-3-fucosylation process. PmST1 M144D has been demonstrated to be a powerful catalyst not only for synthesizing non-sulfated sLe^x structures as shown previously,⁴⁰ but also for producing biologically important but difficult-to-obtain O-sulfated sLe^x.

Acknowledgements

This work was supported by NIH grants R01GM094523 (to X. C.) and R21AI097354 (to A. Y. L.) as well as NSF grant CHE-1300449 (to X. C.). M. M. M. acknowledges UC-Davis and USDE for a GAANN Fellowship (P200A120187). A Bruker Avance-800 NMR spectrometer was funded by NSF grant DBIO-722538.

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Footnotes

† Electronic supplementary information (ESI) available: Materials, experimental details of the synthesis, analytical data of 4–10, 1a–3a, and 1b–3b, and NMR spectra of synthesized compounds. See DOI: 10.1039/c5sc04104j

‡ Current address: Children's National Medical Center, Washington DC, 20010, USA.

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