Synthesis of the glycoprotein having N-linked complex type oligosaccharide

 Couple Fmoc-Thr-(OtBu)-OH (3 equiv.) to the HMPB-PEGA resin using MSNT (3.0 equiv.) and N-methylimidazole (2.75 equiv.) in CH₂Cl₂ (concentration of Fmoc-Thr(OtBu)-OH was adjusted to 250 mM).

 Add 20% piperidine in DMF and react for 20 min to deprotect the Fmoc group.

 Couple the amino acid by use of Fmoc-amino acid derivatives (5.0 equiv.) according to the target amino acid sequence, diisopropylcarbodiimide (DIC, 5.0 equiv.) and 1-hydroxybenzotriazole (HOBt, 5.0 equiv.) in DMF (concentration of Fmoc-amino acid was adjusted to 0.4 M) and react for 1.0 h. Deprotect the Fmoc group by the treatment with 20% piperidine in DMF for 20 min after each Fmoc-amino acid coupling.

 Add the Fmoc-Asn(oligosaccharide)-OH 1 (2.0 equiv.) to the peptide-resin with DEPBT (3.0 equiv.) and DIPEA (2.0 equiv.) in DMF (concentration of 1 was adjusted to 30 mM), followed by deprotection of the Fmoc group by the treatment with 20% piperidine in DMF for 20 min.

 Repeat the same manner as step 3 until the last amino acid residue but adjust the concentration of the Fmoc-amino acid to 40 mM.

 Add the solution of acetic acid : trifluoroethanol (1 : 1, e.g. 2.0 mL for 2 micro mol scale glycopeptide synthesis) and repeat this twice in order to obtain side chain protected glycopeptide 2.

 Concentrate the solution containing 2 in vacuo.

 Dissolve the residue in DMF and concentrate in vacuo for three times.

 Dissolve the residue containing the glycopeptide 2 in DMF (the concentration was adjusted to 5 mM).

 Add 4 Å molecular sieves (10 mg for 2 micro mol scale glycopeptide synthesis) and benzylthiol (30 equiv.) to the DMF solution and incubate for 1 h at −20˚C.

 Add PyBOP (5 equiv.) and DIPEA (5 equiv.) to this mixture and stirr the mixture at −20˚C for 4 h.

 Filter the solution and add ethyl ether (e.g. 6.0 mL for 2 micro mol scale glycopeptide synthesis) to give a precipitation of glycopeptide-alpha-thioester 3. Collect the precipitate by a centrifugation.

 Add a solution containing 95% TFA, 2.5% triisopropyl silane (TIPS), and 2.5% H₂O to the precipitate to remove side-chain protecting groups. After 2 h, concentrate the solution in vacuo.

 Purifiy the residue by reverse phase-HPLC to obtain sialylglycopeptide-alpha-benzylthioester 4 (3.0 mg, 43%).

¹H-NMR (400 MHz, 295K in D₂O, HOD = δ 4.81) δ 7.54-7.35 (m, 15H, Ph,), 5.38 (d, 2H, J = 11.8 Hz, PhCH₂), 5.28 (d, 2H, J = 11.8 Hz, PhCH₂), 5.10 (s, 1H, Man4-H-1), 5.01 (d, 1H, J = 9.6 Hz, GlcNAc1-H-1), 4.92 (s, 1H, Man4’-H-1), 4.77 (s, 1H, Man3-H-1), 4.70 (dd, 1H, Pyr-αH), 4.25 (bs, 1H, Man3-H-2), 2.91 (bdd, 1H, Asn-βCH₂), 2.75 (bdd, 1H, Asn-βCH₂), 2.65 (bdd, 2H, NeuAc7, 7’-H_3eq), 1.84 (m, 3H, Ile-β CH₂, NeuAc7, 7’-H-3_ax), 1.23, 1.15, 1.13 (each d, each 3H, Thr-γCH₃), 1.97, 1.93 (each d, each 3H, Val-γCH₃), 1.86 (d, 3H, Ile-γCH₃), 1.82 (dd, 3H, Ile-γCH₃) ; ESI-MS m/z calcd for [M+H]⁺ 3490.4, found 3490.2 (deconvoluted).

 Dissolve the peptide segment-2 (5, 2 mM) and segment-3 (6, 2 mM) in 0.1 M phosphate buffer (pH 7.5) solution containing 6 M Gn-HCl, 1% thiophenol (v/v) and 1% benzylmercaptan (v/v).

 Adjust the pH of the reaction solution to 4.0 by using 0.2 M methoxyamine-HCl to convert the N-terminal Thz-group to Cys residue.

 Purify the reaction mixture by reverse phase-HPLC to obtain polypeptide segment-4 (7) (ESI; m/z calcd for [M+H]⁺ 7854.1, found 7855.1 (deconvoluted)).

 Dissolve the peptide segment-4 (7, 2 mM) and segment-1 (sialylglycopeptide-alpha-benzylthioester 4, 2 mM) in 0.1 M phosphate buffer (pH 7.6) solution containing 6 M Gn-HCl, 1% thiophenol (v/v) and 1% benzylmercaptan (v/v) and incubate for 17 h at 37˚C.

 Add the sialylglycopeptide-alpha-benzylthioester 4 (ca. 0.5–0.6 equiv. to the used segment-4) to the reaction mixture, and incubate this mixture for 17 h at 37˚C.

 Dilute the mixture by adding five times volume of 0.1M Tris/HCl buffer (pH 8.0) and then bubble the air for 1min to perform folding reaction. Incubate the reaction mixture for 24 h at room temperature.

 Purify the mixture by reverse phase-HPLC to afford the desired product (ESI; m/z calcd for [M+7H]⁷⁺ 1617.4, [M+8H]⁸⁺ 1415.3, [M+9H]⁹⁺ 1258.2, [M+10H]¹⁰⁺ 1132.4, [M+11H]¹¹⁺ 1029.6, [M+12H]¹²⁺ 943.9, found 1617.5, 1415.5, 1258.4, 1132.7, 1029.9, 944.2).

 Add the 50 mM NaOH and leave for 10 min to remove the benzyl ester groups.

 Purify the mixture by reverse phase-HPLC to afford glycosylated MCP-3 9 ESI; m/z calcd for [M+7H]⁷⁺ 1591.6, [M+8H]⁸⁺ 1392.8, [M+9H]⁹⁺ 1238.1, [M+10H]¹⁰⁺ 1114.4, [M+11H]¹¹⁺ 1013.2, [M+12H]¹²⁺ 928.9, found 1591.8, 1393.1, 1238.5, 1114.7, 1013.5, 929.1).