Electronic Supplementary Material (ESI) for Green Chemistry. This journal is © The Royal Society of Chemistry 2021 Supplementary Material Gram-scale production of sugar nucleotides and their derivatives Shuang Li[a]#, Shuaishuai Wang[b]#, Yaqian Wang[a], Jingyao Qu[c], Xian-wei Liu[a], Peng George Wang[d], and Junqiang Fang*[a] [a] Junqiang Fang, Shuang Li, Yaqian Wang, Xian-wei Liu National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Glycochemistry and Glycobiology, Shandong University, Qingdao, Shandong 266237, People’s Republic of China *Email: [email protected] [b] Shuaishuai Wang Department of Chemistry, George State University, Atlanta, GA, 30302-4098, US [c] Jingyao Qu State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong 266237, People’s Republic of China [d] Peng George Wang School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong 518055, People’s Republic of China # These authors contributed equally to this paper. I. Supplementary Figures..................................................................................................................................1 Figure S1. Effects of GlcNAc substrate concentration on conversion rate of UDP-GlcNAc......................1 Figure S2. Effect of buffer sytem on enzymatic conversion rate of UDP-GlcNAc.....................................1 Figure S3. Evaluation of recovery and recyclability of enzymes for UDP-GlcNAc...................................1 Table S1. Enzymes used in this work ..........................................................................................................2 Table S2. Reaction system for substrate concentration optimization. .........................................................2 Table S3. Initial data of AAS.......................................................................................................................3 Table S4. Initial data of ICP-MS .................................................................................................................3 II. Experientials information ............................................................................................................................4 Materials.......................................................................................................................................................4 Expression and purification of enzymes ......................................................................................................4 Optimization of reaction system concentration............................................................................................4 Optimization of buffer system .....................................................................................................................5 Evaluation of recovery and recyclability of enzymes for UDP-GlcNAc.....................................................5 Precipitation efficiency of metal ion............................................................................................................5 XPS of formed precipitate............................................................................................................................5 13P ssNMR of formed precipitate.................................................................................................................6 AAS and ICP-MS of UDP-GlcNAc.............................................................................................................6 Synthesis of uridine 5′-diphosphate glucuronic acid (UDP-GlcA)..............................................................6 Synthesis of uridine 5′- diphosphate galacturonic acid (UDP-GalA) ..........................................................7 Synthesis of uridine 5′- diphosphate N-acetyl glucosamine (UDP-GlcNAc) and its derivatives................8 Synthesis of uridine 5′- diphosphate N-acetyl galactosamine (UDP-GalNAc) and its derivatives .............9 Synthesis of uridine 5′- diphosphate galactose (UDP-Gal)........................................................................10 Synthesis of guanosine 5′- diphosphate fucose (GDP-Fuc).......................................................................11 Synthesis of guanosine 5′- diphosphate mannose (GDP-Man)..................................................................11 Synthesis of cytidine 5’-monophosphate N-acetyl neuraminic acid (CMP-Neu5Ac) ...............................12 Enzymatic synthesis of compound 2: Gal1-4Gal1-4Glc-PEG-N3 ......................................................13 Enzymatic synthesis of compound 3: GalNAc1-3Gal1-4Gal1-4Glc-PEG-N3 .................................13 Enzymatic synthesis of compound 4: GalNTFA1-3Gal1-4Gal1-4Glc-PEG-N3...............................14 Enzymatic synthesis of compound 5: Gal1-3GalNAc1-3Gal1-4Gal1-4Glc-PEG-N3 ....................15 Enzymatic synthesis of compound 6: Gal1-3GalNTFA1-3Gal1-4Galβ1-4Glc-PEG-N3 .................15 Enzymatic synthesis of compound 7: Fucα1-2Galβ1-3GalNAcβ1-3Galα1-4Galβ1-4Glcβ-N3.................16 Enzymatic synthesis of compound 8: Fucα1-2Galβ1-3GalNTFAβ1-3Galα1-4Galβ1-4Glcβ-N3..............17 III. ESI and NMR spectrum............................................................................................................................18 -Q ESI-MS profile of UDP-GlcA ..........................................................................................................18 1H NMR Spectrum of UDP-GlcA .........................................................................................................18 13C NMR Spectrum of UDP-GlcA ........................................................................................................19 -Q ESI-MS profile of UDP-GalA ..........................................................................................................20 1H NMR Spectrum of UDP-GalA .........................................................................................................20 13C NMR Spectrum of UDP-GalA ........................................................................................................21 -Q model ESI-MS profile of UDP-GlcNAc...........................................................................................22 1H NMR Spectrum of UDP-GlcNAc.....................................................................................................22 13C NMR Spectrum of UDP-GlcNAc....................................................................................................23 -Q model ESI-MS profile of UDP-GlcNTFA........................................................................................24 1H NMR Spectrum of UDP-GlcNTFA..................................................................................................24 13C NMR Spectrum of UDP-GlcNTFA.................................................................................................25 -Q model ESI-MS profile of UDP-GlcNAz...........................................................................................26 1H NMR Spectrum of UDP-GlcNAz.....................................................................................................26 13C NMR Spectrum of UDP-GlcNAz....................................................................................................27 -Q model ESI-MS profile of UDP-GalNAc...........................................................................................28 1H NMR Spectrum of UDP-GalNAc.....................................................................................................28 13C NMR Spectrum of UDP-GalNAc....................................................................................................29 -Q model ESI-MS profile of UDP-GalNTFA........................................................................................30 1H NMR Spectrum of UDP-GalNTFA..................................................................................................30 13C NMR Spectrum of UDP-GalNTFA.................................................................................................31 -Q model ESI-MS profile of UDP-GalNAz...........................................................................................32 1H NMR Spectrum of UDP-GalNAz.....................................................................................................32 13C NMR Spectrum of UDP-GalNAz....................................................................................................33 -Q model ESI-MS profile of UDP-Gal ..................................................................................................34 1H NMR Spectrum of UDP-Gal ............................................................................................................34 13C NMR Spectrum of UDP-Gal ...........................................................................................................35 -Q model ESI-MS profile of GDP-Fuc ..................................................................................................36 1H NMR Spectrum of GDP-Fuc ............................................................................................................36 13C NMR Spectrum of GDP-Fuc...........................................................................................................37 -Q model ESI-MS profile of GDP-Man.................................................................................................38 1H NMR Spectrum of GDP-Man...........................................................................................................38