Reaction SMILES: [CH3:1][C:2]([NH:4][C@H:5]1[C@H:10]([O:11][C@@H:12]2[C@@H:17]([OH:18])[C@H:16]([O:19][C@H:20]3[C@H:25]([OH:26])[C@@H:24]([OH:27])[CH:23]([OH:28])[O:22][C@@H:21]3[CH2:29][OH:30])[O:15][C@H:14]([CH2:31][OH:32])[C@@H:13]2[OH:33])[O:9][C@H:8]([CH2:34][OH:35])[C@@H:7]([O:36][C@@H:37]2[O:42][C@H:41]([CH2:43][OH:44])[C@H:40]([OH:45])[C@H:39]([OH:46])[C@H:38]2[OH:47])[C@@H:6]1[OH:48])=[O:3].[OH:49][C:50]([C:52]1([O:63][C@@H:62]([C@@H:64]([C@@H:66]([CH2:68][OH:69])[OH:67])[OH:65])[C@H:57]([NH:58][C:59]([CH3:61])=[O:60])[C@@H:55]([OH:56])[CH2:54]1)O)=[O:51].[Mg+2].[Cl-].[Cl-].[NH4+].[OH-]>C(O)C(N)(CO)CO.Cl.C(O)C.O.C(O)CC>[OH:51][C:50]([C@@:52]1([O:63][C@@H:62]([C@@H:64]([C@@H:66]([CH2:68][OH:69])[OH:67])[OH:65])[C@H:57]([NH:58][C:59]([CH3:61])=[O:60])[C@@H:55]([OH:56])[CH2:54]1)[O:46][C@H:39]1[C@@H:40]([OH:45])[C@@H:41]([CH2:43][OH:44])[O:42][C@@H:37]([O:36][C@@H:7]2[C@@H:8]([CH2:34][OH:35])[O:9][C@@H:10]([O:11][C@H:12]3[C@@H:13]([OH:33])[C@@H:14]([CH2:31][OH:32])[O:15][C@@H:16]([O:19][C@@H:20]4[C@@H:21]([CH2:29][OH:30])[O:22][CH:23]([OH:28])[C@H:24]([OH:27])[C@H:25]4[OH:26])[C@@H:17]3[OH:18])[C@H:5]([NH:4][C:2]([CH3:1])=[O:3])[C@H:6]2[OH:48])[C@@H:38]1[OH:47])=[O:49]
Reactant: CC(=O)N[C@H]1[C@H](O[C@H]2[C@@H](O)[C@@H](CO)O[C@@H](O[C@H]3[C@H](O)[C@@H](O)C(O)O[C@@H]3CO)[C@@H]2O)O[C@H](CO)[C@@H](O[C@@H]2O[C@H](CO)[C@H](O)[C@H](O)[C@H]2O)[C@@H]1O
Reactant: CC(=O)N[C@H]1[C@H]([C@H](O)[C@H](O)CO)OC(O)(C(=O)O)C[C@@H]1O
Agent: Cl
Agent: NC(CO)(CO)CO
Agent: [Cl-]
Solvent: CCO
Solvent: CCCO
Solvent: O
Product: CC(=O)N[C@H]1[C@H](O[C@H]2[C@@H](O)[C@@H](CO)O[C@@H](O[C@H]3[C@H](O)[C@@H](O)C(O)O[C@@H]3CO)[C@@H]2O)O[C@H](CO)[C@@H](O[C@@H]2O[C@H](CO)[C@H](O)[C@H](O[C@]3(C(=O)O)C[C@H](O)[C@@H](NC(C)=O)[C@H]([C@H](O)[C@H](O)CO)O3)[C@H]2O)[C@@H]1O
Reaction time: 48 h
Yield: 98.7 %

Procedure:
One-Pot Two-Enzyme Preparative-Scale Synthesis of Pentasaccharide 3′″-sLNnT Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc. To prepare the pentasaccharide, a reaction mixture in Tris-HCl buffer (100 mM, pH 8.5) in a total volume of 8 mL containing LNnT (100 mg, 0.14 mmol), Neu5Ac (65 mg, 0.21 mmol), CTP (119 mg, 0.21 mmol), MgCl2 (20 mM), NmCSS (2.0 mg), and PmST1 M144D (1.5 mg) was incubated in a shaker at 37° C. for 48 hrs. The reaction was monitored by TLC (n-PrOH:H2O:NH4OH=4:2:1 by volume and detected by p-anisaldehyde sugar stain) and mass spectrometry. When an optimal yield was achieved, the reaction mixture was added with the same volume (8 mL) of ethanol and incubated at 4° C. for 30 min. The precipitates were removed by centrifugation and the supernatant was concentrated and purified by a Bio-Gel P-2 gel column (water was used as an eluent). Further purification was achieved by silica gel chromatography (EtOAc:MeOH:H2O=5:3:2 by volume) to produce Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc (138 mg, 98%). 1H NMR (800 MHz, D2O) δ 5.16 (d, J=3.2 Hz, 0.4H), 4.65 (d, J=8.0 Hz, 0.4H), 4.64 (d, J=8.0 Hz, 0.6H), 4.60 (d, J=8.0 Hz, 0.6H), 4.50 (d, J=8.0 Hz, 1H), 4.38 (d, J=8.0 Hz, 1H), 4.10 (d, J=3.2 Hz, 1H), 4.06 (dd, J=3.2 and 9.6 Hz, 1H), 3.91-3.21 (m, 29H), 2.70 (dd, J=4.8 and 12.8 Hz, 1H), 1.97 (s, 6H), 1.74 (t, J=12.0 Hz, 1H). 13C NMR (200 MHz, D2O) β-isomer: δ 174.86, 174.77, 173.76, 102.78, 102.69, 102.38, 99.65, 95, 59, 81.90, 78.08, 77.77, 75.29, 75.00, 74.73, 74.37, 74.17, 73.59, 72.70, 71.96, 71.60, 71.22, 70.93, 69.81, 69.21, 68.20, 67.88, 67.28, 62.37, 60.87, 59.85, 59.72, 59.61, 56.03, 51.58, 39.92, 22.02, 21.89. HRMS (ESI) m/z calculated for C37H61N2O29 (M−H) 997.3360, found 997.3364.