Expeditious chemoenzymatic synthesis of homogeneous N-glycoproteins carrying defined oligosaccharide ligands

Abstract

An efficient chemoenzymatic method for the construction of homogeneous N-glycoproteins was described that explores the transglycosylation activity of the endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) with synthetic sugar oxazolines as the donor substrates. First, an array of large oligosaccharide oxazolines were synthesized and evaluated as substrates for the Endo-A-catalyzed transglycosylation by use of ribonuclease B as a model system. The experimental results showed that Endo-A could tolerate modifications at the outer mannose residues of the Man3GlcNAc-oxazoline core, thus allowing introduction of large oligosaccharide ligands into a protein and meanwhile preserving the natural, core N-pentasaccharide (Man3GlcNAc2) structure in the resulting glycoprotein upon transglycosylation. In addition to ligands for galectins and mannose-binding lectins, azido functionality could be readily introduced at the N-pentasaccharide (Man3GlcNAc2) core by use of azido-containing Man3GlcNAc oxazoline as the donor substrate. The introduction of azido functionality permits further site-specific modifications of the resulting glycoproteins, as demonstrated by the successful attachment of two copies of alphaGal epitopes to ribonuclease B. This study reveals a broad substrate specificity of Endo-A for transglycosylation, and the chemoenzymatic method described here points to a new avenue for quick access to various homogeneous N-glycoproteins for structure-activity relationship studies and for biomedical applications.

Figure 1

A chemoenzymatic strategy for N-glycoprotein synthesis

Figure 2

Synthetic oligosaccharide oxazolines

Figure 3

The ESI mass spectra of the synthetic glycoproteins. A, glycoprotein 36; B, glycoprotein 37; and C, glycoprotein 38. Peaks are labeled according to the corresponding charge states.

Figure 4

SDA-PAGE and ESI-MS analysis of the αGal-incorporated glycoprotein product (42) generated by “Click” chemistry. A, Coomassie blue-stained SDS-PAGE gel (Lane M is protein marker with sizes on the left; Lane 1 is the starting material, glycoprotein 38, and lane 2 is the purified product, glycoprotein 42); B, The ESI mass spectrum of glycoprotein 42. Charge states are labeled.

Figure 5

SPR sensorgrams of the binding between respective synthetic glycoproteins and immobilized lectin or human serum antibody. A, binding to immobilized ConA; B, binding to immobilized PNA; and C, binding to immobilized whole IgG-type antibodies from human serum. For comparison, respective glycoprotein or ribonuclease A was injected onto the sensor chip surface at a concentration of 1.0 µM.

Scheme 1

Synthesis of oligosaccharide oxazoline 3 Reagents and conditions: (a) BSP, TTBP, Tf₂O, CH₂Cl₂, 67%; (b) TFA, CH₂Cl₂, 85%; (c) TMSOTf, CH₂Cl₂, 79%; (d) AcSH, 86%; (e) (i) Pd(OH)₂-C, H₂, MeOH; (ii) Ac₂O, pyridine, 90% (2 steps); (f) TMSBr, BF₃·OEt₂, 2,4,6-collidine, CH₂Cl₂, 67%; (g) MeONa, MeOH, quant.

Scheme 2

Synthesis of sugar oxazoline 4 Reagents and conditions: (a) DDQ, CH₂Cl₂, H₂O, 80%; (b) Cu(OTf)₂, BH₃·THF, THF, 73%; (c) TMSOTf, CH₂Cl₂, 96%; (d) AcCl, CH₂Cl₂, MeOH, 90%; (e) TMSOTf, CH₂Cl₂, 65%; (f) (i) MeONa, CH₂Cl₂, MeOH; (ii) Ac₂O, pyridine; (iii) AcSH, pyridine, CHCl₃, 79% (3 steps); (g) (i) Pd(OH)₂-C, H₂, MeOH; (ii) Ac₂O, pyridine, 77% (2 steps); (h) TMSBr, BF₃·OEt₂, 2,4,6-collidine, CH₂ClCH₂Cl, 35%; (i) MeONa, MeOH, quant.

Scheme 3

Synthesis of sugar oxazoline 5 Reagents and conditions: (a) AcSH, pyridine, CHCl₃, 85%, (b) AcCl, CH₂Cl₂, MeOH, 72%; (c) (i) TsCl, pyridine; (ii) NaN₃, DMF, 84% (2 steps); (d) MeONa, MeOH, 85%; (e) Pd(OH)₂-C, H₂, MeOH; (f) TfN₃, K₂CO₃, CuSO₄, CH₂Cl₂, MeOH, H₂O; (g) Ac₂O, pyridine, 61% (3 steps); (h) TMSBr, BF₃·OEt₂, 2,4,6-collidine, CH₂Cl₂, 52%; (i) MeONa, MeOH, quant.

Scheme 4

enzymatic transglycosylation Reagents and conditions: (a) Endo-A, phosphate buffer (50 mM, pH 6.5), 82%; (b) Endo-A, phosphate buffer (50 mM, pH 6.5), 96%; (c) Endo-A, phosphate buffer (50 mM, pH 6.5), 71%; (d) Endo-A, phosphate buffer (50 mM, pH 6.5), 38%; (e) Endo-A, phosphate buffer (50 mM, pH 6.5), 89%;

Scheme 5

Synthesis of alkyne-functionalized αGal epitope Reagents and conditions: (a) NaHCO₃, MeOH, MeCN, H₂O, 79%.

Scheme 6

Catalytic 1,3-dipolar cycloaddition to introduce αGal epitope Reagents and conditions: (a) Tris buffer (0.1 M, pH 8.0), CuSO₄, l-ascorbic acid, bathophenanthrolinedisulfonic acid, 87%.