[ CAS No. 133-89-1 ] {[proInfo.proName]}

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Quality Control of [ 133-89-1 ]

COA NMR CNMR HPLC LC-MS

Related Doc. of [ 133-89-1 ]

SDS Specification

Alternatived Products of [ 133-89-1 ]

Product Details of [ 133-89-1 ]

CAS No. :	133-89-1	MDL No. :
Formula :	C₁₅H₂₄N₂O₁₇P₂	Boiling Point :	-
Linear Structure Formula :	-	InChI Key :	HSCJRCZFDFQWRP-JZMIEXBBSA-N
M.W :	566.30	Pubchem ID :	8629
Synonyms :		Chemical Name :	UDP-a-D-Glucose

Safety of [ 133-89-1 ]

Signal Word:	Warning	Class:
Precautionary Statements:	P280-P305+P351+P338	UN#:
Hazard Statements:	H302-H312-H315-H319	Packing Group:
GHS Pictogram:

Application In Synthesis of [ 133-89-1 ]

* All experimental methods are cited from the reference, please refer to the original source for details. We do not guarantee the accuracy of the content in the reference.

Upstream synthesis route of [ 133-89-1 ]
Downstream synthetic route of [ 133-89-1 ]

[ 133-89-1 ] Synthesis Path-Upstream 1~14

1
[ 133-89-1 ]
[ 58-97-9 ]
[ 58-98-0 ]

Reference： [1] Biochemical and Biophysical Research Communications, 2018, vol. 495, # 1, p. 401 - 407

2
[ 133-89-1 ]
[ 2196-14-7 ]
[ 578-74-5 ]

Reference： [1] Nature Communications, 2017, vol. 8, # 1,

3
[ 133-89-1 ]
[ 60-82-2 ]
[ 4192-90-9 ]
[ 60-81-1 ]

Reference： [1] Organic Letters, 2014, vol. 16, # 18, p. 4874 - 4877

4
[ 133-89-1 ]
[ 60-82-2 ]
[ 58-98-0 ]
[ 60-81-1 ]

Reference： [1] Green Chemistry, 2014, vol. 16, # 9, p. 4417 - 4425

5
[ 133-89-1 ]
[ 117-39-5 ]
[ 491-50-9 ]

Reference： [1] Organic Letters, 2014, vol. 16, # 18, p. 4874 - 4877

6
[ 133-89-1 ]
[ 64849-39-4 ]
[ 57817-89-7 ]

Reference： [1] Patent: WO2014/193888, 2014, A1, . Location in patent: Page/Page column 56
[2] Patent: WO2016/43926, 2016, A1, . Location in patent: Page/Page column 56
[3] Patent: WO2016/120486, 2016, A1, . Location in patent: Paragraph 00196

7
[ 133-89-1 ]
[ 135-19-3 ]
[ 6044-30-0 ]

Yield	Reaction Conditions	Operation in experiment
89%	With Aloearborescensglycosyltransferase*GT₃ In dimethyl sulfoxide at 30℃; for 12 h; Enzymatic reaction	General procedure: Generally 30-50 μmol of aglycon was solved in 0.5 mL DMSO and diluted with buffer solution (50 mM Tris HCl, pH 7.4, 25 mL total volume). UDP-Glc (60100 μmol) was added along with 50 mL of crude enzyme of AaGT3, extracted from 3 g in wet induced E. coli cells with pET28a-AaGT3. The reactions were performed at 30 °C for up to 12 h, followed by adding 5 × 100 mL of ethyl acetate to extract 5 times. The organic phase was evaporated to dryness under reduced pressure, and the residue was dissolved in 1.5 mL of methanol and purified by reverse-phase semi-preparative HPLC. The obtained products were analyzed by MS and NMR.

Reference： [1] Tetrahedron Letters, 2017, vol. 58, # 22, p. 2118 - 2121

8
[ 133-89-1 ]
[ 121-33-5 ]
[ 494-08-6 ]

Reference： [1] Phytochemistry, 2009, vol. 70, # 4, p. 473 - 482

9
[ 133-89-1 ]
[ 20675-96-1 ]
[ 118-34-3 ]

Reference： [1] Phytochemistry, 2014, vol. 102, p. 55 - 63

10
[ 133-89-1 ]
[ 20675-96-1 ]
[ 118-34-3 ]

Reference： [1] Journal of Biological Chemistry, 2015, vol. 290, # 30, p. 18770 - 18781

11
[ 133-89-1 ]
[ 2035-15-6 ]
[ 6807-83-6 ]

Reference： [1] Organic Letters, 2014, vol. 16, # 18, p. 4874 - 4877

12
[ 58-98-0 ]
[ 60-81-1 ]
[ 133-89-1 ]
[ 60-82-2 ]

Reference： [1] Green Chemistry, 2014, vol. 16, # 9, p. 4417 - 4425

13
[ 133-89-1 ]
[ 57817-89-7 ]
[ 58543-16-1 ]

Reference： [1] Patent: WO2014/193888, 2014, A1, . Location in patent: Page/Page column 9; 46-47; 52-53
[2] Patent: WO2016/43926, 2016, A1, . Location in patent: Page/Page column 47

14
[ 133-89-1 ]
[ 58543-16-1 ]

Reference： [1] Patent: WO2014/193888, 2014, A1,
[2] Patent: WO2016/43926, 2016, A1,

[ 133-89-1 ] Synthesis Path-Downstream 1~88

1
[ 133-89-1 ]
[ 123-31-9 ]
[ 497-76-7 ]

Reference： [1],1957,vol. 13,p. 514
Chem.Abstr.,1958,p. 8248
[2]Bioorganic and Medicinal Chemistry,2002,vol. 10,p. 1731 - 1741

2
[ 3387-36-8 ]
[ 57-50-1 ]
[ 133-89-1 ]

Reference： [1]Tetrahedron,1996,vol. 52,p. 2395 - 2404

3
[ 133-89-1 ]
[ 22427-39-0 ]
6-O-cellobiosyl-20-O-glucosylprotopanaxatriol [ No CAS ]
6-O-glucosyl-20-O-cellobiosylprotopanaxatriol [ No CAS ]

Reference： [1]Journal of Organic Chemistry,2001,vol. 66,p. 262 - 269

4
[ 133-89-1 ]
[ 22427-39-0 ]
6-O-lactosyl-20-O-glucosylprotopanaxatriol [ No CAS ]
C₄₈H₈₂O₁₉ [ No CAS ]
6,20-di-O-lactosylprotopanaxatriol [ No CAS ]

Reference： [1]Journal of Organic Chemistry,2001,vol. 66,p. 262 - 269
[2]Journal of Organic Chemistry,2001,vol. 66,p. 262 - 269

5
[ 133-89-1 ]
[ 22427-39-0 ]
6-O-lactosyl-20-O-glucosylprotopanaxatriol [ No CAS ]
6,20-di-O-lactosylprotopanaxatriol [ No CAS ]

Reference： [1]Journal of Organic Chemistry,2001,vol. 66,p. 262 - 269

7
[ 133-89-1 ]
[ 21293-29-8 ]
abscisic acid glucoside [ No CAS ]
[ 2228-72-0 ]
[ 14398-53-9 ]

Reference： [1]Tetrahedron Asymmetry,2005,vol. 16,p. 143 - 147

8
[ 133-89-1 ]
[ 117-39-5 ]
[ 482-35-9 ]
[ 6892-74-6 ]

Reference： [1]Phytochemistry,2006,vol. 67,p. 1598 - 1612

9
[ 63-37-6 ]
[ 133-89-1 ]

Reference： [1]Journal of Organic Chemistry,1990,vol. 55,p. 1834 - 1841
[2]Journal of Organic Chemistry,1990,vol. 55,p. 1834 - 1841

10
[ 133-89-1 ]
[ 501-36-0 ]
[ 38963-95-0 ]
[ 27208-80-6 ]

Reference： [1]Phytochemistry,2009,vol. 70,p. 473 - 482
[2]Organic Letters,2014,vol. 16,p. 4874 - 4877

11
[ 133-89-1 ]
[ 501-36-0 ]
[ 27208-80-6 ]

Reference： [1]Phytochemistry,2009,vol. 70,p. 473 - 482

12
[ 133-89-1 ]
[ 501-36-0 ]
[ 27208-80-6 ]
(E)-1-[3-β-D-(glucopyranosyloxy)-5-hydroxyphenyl]-2-(4-β-D-glucopyranosyloxyphenyl)ethene [ No CAS ]

Reference： [1]Phytochemistry,2009,vol. 70,p. 473 - 482

13
[ 133-89-1 ]
[ 33419-42-0 ]
[ 135970-29-5 ]

Reference： [1]Phytochemistry,2009,vol. 70,p. 473 - 482

14
[ 133-89-1 ]
[ 121-33-5 ]
[ 494-08-6 ]

Reference： [1]Phytochemistry,2009,vol. 70,p. 473 - 482

16
[ 133-89-1 ]
[ 117-39-5 ]
[ 482-35-9 ]

Reference： [1]Phytochemistry,2010,vol. 71,p. 1253 - 1263
[2]Journal of Agricultural and Food Chemistry,2018,vol. 66,p. 11701 - 11708
[3]Organic Letters,2019,vol. 21,p. 2241 - 2245

17
[ 133-89-1 ]
[ 529-44-2 ]
[ 15648-86-9 ]

Reference： [1]Phytochemistry,2010,vol. 71,p. 1253 - 1263
[2]Organic Letters,2019,vol. 21,p. 2241 - 2245

18
[ 133-89-1 ]
[ 65271-80-9 ]
[ 1295578-41-4 ]

Reference： [1]Organic Letters,2011,vol. 13,p. 2786 - 2788

19
[ 133-89-1 ]
[ 87-73-0 ]
[ 56-65-5 ]
thuringiensin [ No CAS ]

Reference： [1]Journal of Biological Chemistry,2010,vol. 285,p. 39191 - 39200

20
[ 133-89-1 ]
[ 529-44-2 ]
[ 15648-86-9 ]

Reference： [1]Glycoconjugate Journal,2012,vol. 29,p. 425 - 432

21
[ 133-89-1 ]
[ 6665-86-7 ]
[ 71802-05-6 ]

Yield	Reaction Conditions	Operation in experiment
	With oleandomycin glucosyltransferase; In dimethyl sulfoxide; at 37℃;pH 8.0;Enzymatic reaction;	General procedure: The OleDPSA-catalyzed reaction was carried out at 37 C in 100 mL of Tris-HCl buffer (100 mM, pH 8.0) containing quercetin (15.1 mg, 50 mumol), UDP-Glc (152.6 mg, 250 mumol), OleDPSA (9.8 mg) and 10% DMSO. The reaction was incubated overnight and quenched by adding an equal volume of ice-cold ethanol. The precipitates were removed by centrifugation, and the supernatant was concentrated. The crude product was purified via C-18 reverse phase chromatography (water: acetonitrile from 8:1 to 3:1) to give the product as light yellow solid

Reference： [1]Carbohydrate Research,2013,vol. 368,p. 73 - 77

22
[ 133-89-1 ]
[ 6665-86-7 ]
7-hydroxyflavone monoglucoside [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
	With recombinant glucosyltransferase 3 from Phytolacca americana; In aq. phosphate buffer; at 37℃; for 1h;pH 7.2;Enzymatic reaction;Kinetics;	General procedure: Glucosylation reactions were performed at 37 ?C for 60 min in0.5 mL of 50 mM potassium phosphate buffer (pH 7.2) supplementedwith 50 M hydroxyflavone, 200 M UDP-glucose, and5 M enzyme. The incubation was stopped by adding 1.5% trifluoroaceticacid, and the reaction mixture was analyzed by HPLC todetect the glucoside products. When the Km and kcat values weredetermined, the concentration of hydroxyflavone was varied as follows:For glucosylation of 6-hydroxyflavone by PaGT3, 50-150 M;for glucosylation of <strong>[6665-86-7]7-hydroxyflavone</strong> by PaGT3, 50-200 M; forglucosylation of 6-hydroxyflavone by PaGT2, 10-120 M. The averagevalues from more than three independent experiments wereused to construct Lineweaver-Burk plots.

Reference： [1]Journal of Molecular Catalysis B: Enzymatic,2013,vol. 90,p. 61 - 65

23
[ 133-89-1 ]
[ 501-36-0 ]
(E)-1-(3,5-bis-β-D-glucopyranosyloxyphenyl)-2-(4-hydroxyphenyl)ethene [ No CAS ]
[ 38963-95-0 ]
[ 27208-80-6 ]
(E)-1-[3-β-D-(glucopyranosyloxy)-5-hydroxyphenyl]-2-(4-β-D-glucopyranosyloxyphenyl)ethene [ No CAS ]

Reference： [1]Journal of Natural Products,2013,vol. 76,p. 279 - 286

24
[ 2280-44-6 ]
[ 1476-50-2 ]
[ 133-89-1 ]

Yield	Reaction Conditions	Operation in experiment
26%	With pyrophosphatase, inorganic from yeast; UTP-glucose-1-phosphate galactokinase from Streptococcus pneumoniae TIGR4; UTP-glucose-1-phosphate uridylyltransferase from Streptococcus pneumoniae TIGR4; adenosine 5'-triphosphate sodium salt; magnesium chloride; In aq. buffer; at 42℃; for 24.0h;pH 8;Enzymatic reaction;	General procedure: We chose 14 monosaccharides which could be used by GalK in previous report36 as substrates for one-pot three-enzyme system to synthesize various sugar nucleotides. The reactions were carried out in the system containing 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, various monosaccharides, 10 mM ATP, 10 mM UTP, 0.5 mg/mL SpGalK, 0.5 mg/mL SpGalU, and 0.1 U PPase. The reactions were incubated at 42 C for 24 h. The reactions were terminated by heating at 100 C for 5 min, followed by centrifugated at 13,400 rpm at room temperature for 30 min to remove protein precipitation. The products were determined by TLC, CE and MS. For CE analysis, 5 muL of each sample was diluted into 50 muL and subjected to CE analysis as described above.

Reference： [1]Carbohydrate Research,2013,vol. 373,p. 76 - 81

25
[ 133-89-1 ]
[ 3934-84-7 ]
1-O-vanilloyl-β-D-glucose [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
	With recombinant Quercus robur UGT84A13; In glycerol; at 30℃;pH 6.0;Enzymatic reaction;Kinetics;	General procedure: Standard UGT assays were performed in 0.1M MES, 10% glycerol, 0.01% -mercaptoethanol, 0.5mM EDTA (pH 6.0) containing 6mugml-1 of recombinant UGT84A13 protein, 4mM UDP-glucose, and 2mM of the hydroxybenzoic or hydroxycinnamic acid substrate. Reaction mixtures were incubated at 30C, and the reactions stopped after 1, 2, 3 and 4min by mixing aliquots of the assay mixture with equal volume of methanol (C6-C1 phenolic acids) or with 0.2 volume of trifluoroacetic acid (C6-C3 phenolic acids). Reaction products were analyzed by HPLC-PDA. Specific enzyme activities were calculated from the increase in the formation of reaction products and expressed as nmol of substrate glucosylated per second and per mg protein (nkatmg-1). Kinetic parameters were determined from enzyme assays under standard conditions (pH 6.0; 30C; 6mugml-1 recombinant UGT84A13). Initial reaction velocities were measured over a range of 0.2-3.0mM phenolic acid in the presence of 4mM UDP-glucose. Product formation was quantified after 1, 2 and 4min. KM and Vmax values were calculated from hyperbolic Michaelis-Menten saturation curves by non-linear fitting using the Enzyme Kinetics 1.3 module of Sigma Plot 10

Reference： [1]Phytochemistry,2014,vol. 99,p. 44 - 51

26
[ 133-89-1 ]
[ 60-82-2 ]
1-[3-(β-D-glucopyranosyl)-2,4,6-trihydroxyphenyl]-3-(4-hydroxyphenyl)propan-1-one [ No CAS ]

Reference： [1]Chemistry - A European Journal,2016,vol. 22,p. 5873 - 5877
[2]Advanced Synthesis and Catalysis,2013,vol. 355,p. 2757 - 2763
[3]Angewandte Chemie - International Edition,2015,vol. 54,p. 12678 - 12682
Angew. Chem.,2015,vol. 127,p. 12869 - 12873,5

27
[ 58-98-0 ]
[ 60-81-1 ]
[ 133-89-1 ]
[ 60-82-2 ]

Reference： [1]Green Chemistry,2014,vol. 16,p. 4417 - 4425

28
[ 133-89-1 ]
[ 60-82-2 ]
[ 58-98-0 ]
[ 60-81-1 ]

Reference： [1]Green Chemistry,2014,vol. 16,p. 4417 - 4425

29
[ 133-89-1 ]
[ 58543-16-1 ]

Reference： [1]Patent: WO2014/193888,2014,A1
[2]Patent: WO2016/43926,2016,A1

30
[ 133-89-1 ]
[ 57817-89-7 ]
[ 58543-16-1 ]

Yield	Reaction Conditions	Operation in experiment
	With UDP-glucosyltransferase UGT76G1; magnesium chloride; In aq. phosphate buffer; at 30℃;pH 7.2;Enzymatic reaction;Catalytic behavior; Kinetics;	The total volume of the reaction was 5.0 mL with the following composition: 50 mM sodium phosphate buffer pH 7.2, 3 mM MgCl2, 2.5 mM UDP-glucose, 0.5 mM Stevioside and 500 mu^ of UGT76G1 thawed lysate. The reactions were run at 30 C on an orbitary shaker at 135 rpm. For each sample, 460 muEpsilon of the reaction mixture was quenched with 40 mu, of 2N H2S04 and 420 mu, of methanol/water (6/4). The samples were immediately centrifuged and kept at 10 C before analysis by HPLC (CAD). HPLC indicated almost complete conversion of stevioside to rebaudioside A as seen in Figure 40
	With UGT76G1; magnesium chloride; In aq. phosphate buffer; at 30℃;	EXAMPLE 4Catalytic reaction with in-vivo produced UGT76G1The total volume of the reaction was 5.0 mL with the following composition: 50 mM sodium phosphate buffer pH 7.2, 3 mM MgCl2, 2.5 mM UDP-glucose, 0.5 mM Stevioside and 500 muL of UGT76G1 thawed lysate. The reactions were run at 30 C on an orbitary shaker at 135 rpm. For each sample, 460 muL of the reaction mixture was quenched with 40 muL of 2N H2SO4and 420 muL of methanol/water (6/4). The samples were immediately centrifuged and kept at 10 C before analysis by HPLC (CAD). HPLC indicated almost complete conversion of stevioside to rebaudioside A, as shown in FIG. 51.

Reference： [1]Patent: WO2014/193888,2014,A1 .Location in patent: Page/Page column 9; 46-47; 52-53
[2]Patent: WO2016/43926,2016,A1 .Location in patent: Page/Page column 47

31
[ 133-89-1 ]
[ 58543-16-1 ]
13-[(2-O-β-D-glucopyranosyI-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]-ent-kaur-16-en-19-oic acid [(6-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester] [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
	With UDP-glucosyltransferase S115N06 E1; magnesium chloride; In aq. phosphate buffer; at 30℃; for 24h;pH 7.2;Enzymatic reaction;Catalytic behavior;	430 mu, of a reaction mixture containing 0.5 mM Rebaudioside A, 3mM MgCl2, 50 mM phosphate buffer (pH7.2) and 2.5 mM UDP-glucose was added to a 1.5 mL sterile microtube. 52 of the enzyme expression medium was added and the resulting mixture was allowed to react at 30 C for 24 hours. 125 mu^ samples were taken after 2 hours, 16 hours and 24 hours and added to a 1 15 mu, of 60% methanol and 10 mu, of 2 N H2SO4. The quenched sample was centrifuged at 18,000 g for 2 minutes at RT. 200 mu, was transferred to an HPLC vial and analyzed

Reference： [1]Patent: WO2014/193888,2014,A1 .Location in patent: Page/Page column 61-62

32
[ 133-89-1 ]
[ 58543-16-1 ]
[ 63279-13-0 ]
13-[(2-O-β-D-glucopyranosyI-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]-ent-kaur-16-en-19-oic acid [(6-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester] [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
	With UDP-glucosyltransferase S115N05 A7; magnesium chloride; In aq. phosphate buffer; at 30℃; for 24h;pH 7.2;Enzymatic reaction;Catalytic behavior;	430 mu, of a reaction mixture containing 0.5 mM Rebaudioside A, 3mM MgCl2, 50 mM phosphate buffer (pH7.2) and 2.5 mM UDP-glucose was added to a 1.5 mL sterile microtube. 52 of the enzyme expression medium was added and the resulting mixture was allowed to react at 30 C for 24 hours. 125 mu^ samples were taken after 2 hours, 16 hours and 24 hours and added to a 1 15 mu, of 60% methanol and 10 mu, of 2 N H2SO4. The quenched sample was centrifuged at 18,000 g for 2 minutes at RT. 200 mu, was transferred to an HPLC vial and analyzed

Reference： [1]Patent: WO2014/193888,2014,A1 .Location in patent: Page/Page column 61-62

33
[ 133-89-1 ]
[ 58543-16-1 ]
[ 63279-13-0 ]

Yield	Reaction Conditions	Operation in experiment
	With UDP-glucosyltransferase UGTSL; magnesium chloride; In aq. phosphate buffer; at 30℃; for 45h;pH 7.2;Enzymatic reaction;Catalytic behavior; Kinetics;	Activity tests were performed at 5 mL scale with 1,000 mu of thawed lysate for the transformation of Rebaudioside A using 0.5 mM of substrate, 2.5 mM of UDP-Glucose and 3 mM MgCl2 in 50 mM Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed by HPLC as seen in Figures 48-52
	With UGTSL2; magnesium chloride; In aq. phosphate buffer; at 30℃; for 22h;	EXAMPLE 48 Directed evolution of UGTSL2 for the conversion of Rebaudioside A to Rebaudioside D (Round 2) Taking the native enzyme UGTSL2 (GI_460410132) as baseline, a list of 23 mutations was established containing different identified positive mutations for activity from the first round (EXAMPLE 45) and new mutations obtained by DNA2.0 ProteinGPSTMstrategy. This list of mutations was subsequently used to design 46 variant genes that contained each 3 different mutations. After codon-optimized for expression in E. coli the genes were synthesized, subcloned in the pET30a+ plasmid and used for transformation of E. coli BL21 (DE3) chemically competent cells. The obtained cells were grown in Petri-dishes on solid LB medium in the presence of Kanamycin. Suitable colonies were selected and allowed to grow in liquid LB medium in tubes. Glycerol was added to the suspension as cryoprotectant and 400 muL aliquots were stored at -20C and at -80C.These storage aliquots of E. coli BL21(DE3) containing the pET30a+_UGTSL2var plasmids were thawed and added to LBGKP medium (20 g/L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50 mM Phosphate buffer pH 7.00; 2.5 g/L glucose and 50 mg/L of Kanamycine). This culture was allowed to shake in a 96 microtiter plate at 30C for 8 h.3.95 mL of production medium containing 60 g/L of Overnight Express Instant TB medium (Novagen), 10 g/L of glycerol and 50 mg/L of Kanamycin was inoculated with 50 muL of above described culture. In a 48 deepwell plate the resulting culture was allowed to stir at 20C. The cultures gave significant growth and a good OD (600 nm) was obtained. After 44 h, the cells were harvested by centrifugation and frozen.Lysis was performed by addition of Bugbuster Master mix (Novagen) to the thawed cells and the lysates were recovered by centrifugation.In order to measure the activity of the variants for the transformation of Rebaudioside A to Rebaudioside D, 100 muL of fresh lysate was added to a solution of Rebaudioside A (final concentration 0.5 mM), MgCl2(final concentration 3 mM) and UDP-Glucose (final concentration 2.5 mM) in 50 mM phosphate buffer pH 7.2. The reaction was allowed to run at 30C and samples were taken after 2, 4, 6 and 22 h. to determine the initial rates after HPLC analysis (CAD detection) using the analytical method that was described above for the transformation of Rebaudioside A to Rebaudioside D.In parallel for the most active clones, 100 muL of fresh lysate was added to a solution of Rebaudioside D (final concentration 0.5 mM), MgCl2(final concentration 3 mM) and UDP-Glucose (final concentration 2.5 mM) in 50 mM phosphate buffer pH 7.2. The reaction was allowed to run at 30C and samples were taken after 2, 4, 6 and 22 h. to determine the initial rates for Rebaudioside D conversion after HPLC analysis (CAD detection).Apart from the new variants, both experiments were also performed with baseline clone, UGTSL2. The initial rates for the conversion of Rebaudioside A or Rebaudioside D for this baseline clone were defined as 100%.Activity of each clone was defined as normalized activity compared to baseline clone UGTSL2 whereas specificity of each clone was expressed as the ratio between the initial rates for the conversion of Rebaudioside A and Rebaudioside D.The normalized initial rate for the conversion of Rebaudioside A and the ratio between the initial rates for the conversion of Rebaudioside A and Rebaudioside D are depicted in the following table.n d 2dd0dd7ddd1675dd192ddd60d61*Mutations are noted as follows: reference gene-original amino acid-position-new amino acid: For example the mutation of an isoleucine at position 240 to a Leucine forUGTSL2 is noted asUGTSL2 (I240L). Nd means Not determined.Modeling of these results allowed to obtain a ranking of the effect of each mutation. The following mutations were determined as being beneficial for activity:N325S, G387E, A285V, I333L, V270I, Q27R, N278G, L393V, S258T, A341V, H247P and T392A.The following mutations were determined as being beneficial for an improved ratio between initial rate for the conversion of Rebaudioside A and Rebaudioside D:V270I, T392A, T329V, L276A, L393V, A341V and S255C.
	With EUGT11 enzyme (SEQ ID NO:16); In aq. buffer; at 30℃; for 24h;pH 8;Enzymatic reaction;	Chimeras were screened by adding 20 muIota_ purified UGT91 D2e-b, EUGT1 1 , or UGT91 D2e-b-EUGT11 chimeric enzymes (0.02 mg/ml_) to a total volume of 80 muIota_ reaction mixture comprising 100 mM Tris-HCI (pH 8.0), 5 mM MgCI2, 1 mM KCI, 300 muMu uridine diphosphate glucose (UDPG), and 100 muMu rubusoside or RebA. The reactions were incubated at 30C for 24 h, and levels of RebA, RebD, rubusoside, and 1 ,2-stevioside were measured by LC-MS. Not all of the chimeras purified were active in the above described assay (see Table 5 for enzymes having activity on rubusoside and/or RebA).

With uridine 5'-diphospho glycosyl transferase EUGT11, N-terminal HIS-tag, N-terminal GST-tag; In aq. buffer; at 37℃; for 48h;pH 7;Enzymatic reaction;Kinetics;

A series of experiments were performed to determine kinetic parameters of EUGT11 and 91D2e-b. For both enzymes, 100 muM RebA, 300 muM UDP-glucose, and 10 U/mL Alkaline Phosphatase (Fermentas/Thermo Fisher, Waltham, MA) were used in the reactions. For EUGT11, the reactions were performed at 37C using 100 mM Tris-HCl, pH 7, and 2% enzyme. For 91D2e-b, the reactions were performed at 30C using 20 mM Hepes-NaOH, pH 7.6, 20% (by volume) enzyme. The initial velocities (V0) were calculated in the linear range of a product versus time plot. To first investigate the linearity intervals, initial time-courses were done for each enzyme. EUGT11 was assayed at 37C for 48h at initial concentrations of 100 muM RebA and 300 muM UDP-glucose. UGT91D2e-b was assayed at 37C for 24h at initial concentrations of 200 muM RebA and 600 muM UDP-glucose. Based on these range-finding studies, it was determined that the initial 10 minutes in the case of EUGT11, and the initial 20 minutes in UGT91D2e-b would be in the linear range with respect to product formation, and therefore initial velocities of each reaction were calculated in those intervals. In the case of EUGT11, RebA concentrations assayed were 30 muM, 50 muM, 100 muM, 200 muM, 300 muM and 500 muM. Concentration of UDP-glucose was always three times the concentration of RebA and incubation was performed at 37C. By plotting the calculated V0 as a function of the substrate concentrations, Michaelis-Menten curves were generated. By plotting the reciprocal of V0 and the reciprocal of [S], a Lineweaver-Burk graphic was obtained, with y = 339.85x +1.8644; R2 = 0.9759.

Reference： [1]Patent: WO2014/193888,2014,A1 .Location in patent: Page/Page column 9-10; 47; 61-62; 67-69; 86; 91; 95-96
[2]Patent: WO2016/43926,2016,A1 .Location in patent: Page/Page column 136; 137; 138
[3]Patent: WO2016/120486,2016,A1 .Location in patent: Paragraph 0052; 00196
[4]Patent: EP3009508,2016,A1 .Location in patent: Paragraph 0054; 0360; 0361

34
[ 133-89-1 ]
[ 5858-17-3 ]
3,4-dichlorophenyl-1-thio-β-D-glucopyranoside [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
	With Pueraria lobata glycosyltransferase 7; at 35℃; for 16h;pH 9.0;Enzymatic reaction;	General procedure: The detecting enzyme reactions, conducted with 0.8 mM UDP-glucose, 0.4 mM aglycon and 300 mug of purified PlGT7 in a total volume of 100 muL (pH 9.0), were incubated at 35 C for 16 h and terminated with 200 muL ice-cold methanol. After centrifugation at 15,000 g for 30 min, the supernatant of each reaction was collected and analyzed by HPLC-UV/ESIMS as described above. For quantification, three parallel assays were performed routinely.

Reference： [1]Organic Letters,2014,vol. 16,p. 4874 - 4877
[2]Tetrahedron Letters,2016,vol. 57,p. 1518 - 1521
[3]Journal of the American Chemical Society,2020,vol. 142,p. 3506 - 3512

35
[ 133-89-1 ]
[ 2035-15-6 ]
[ 6807-83-6 ]

Reference： [1]Organic Letters,2014,vol. 16,p. 4874 - 4877

36
[ 133-89-1 ]
[ 60-82-2 ]
phloretin 4-O-β-D-glucoside [ No CAS ]
[ 4192-90-9 ]
[ 60-81-1 ]

Reference： [1]Organic Letters,2014,vol. 16,p. 4874 - 4877

37
[ 133-89-1 ]
[ 519-34-6 ]
[ 4773-96-0 ]

Reference： [1]Angewandte Chemie - International Edition,2015,vol. 54,p. 12678 - 12682
Angew. Chem.,2015,vol. 127,p. 12869 - 12873,5

38
[ 133-89-1 ]
[ 6681-15-8 ]
C₂₆H₃₀NO₉⁽¹⁺⁾*Cl^(1-) [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
	With Pueraria lobata glycosyltransferase 7; at 35.0℃; for 16.0h;pH 9.0;Enzymatic reaction;	General procedure: The detecting enzyme reactions, conducted with 0.8 mM UDP-glucose, 0.4 mM aglycon and 300 mug of purified PlGT7 in a total volume of 100 muL (pH 9.0), were incubated at 35 °C for 16 h and terminated with 200 muL ice-cold methanol. After centrifugation at 15,000 g for 30 min, the supernatant of each reaction was collected and analyzed by HPLC-UV/ESIMS as described above. For quantification, three parallel assays were performed routinely.

Reference： [1]Tetrahedron Letters,2016,vol. 57,p. 1518 - 1521

39
UDP [ No CAS ]
[ 120221-14-9 ]
[ 133-89-1 ]

Reference： [1]Angewandte Chemie - International Edition,2016,vol. 55,p. 12338 - 12342
Angew. Chem.,2016,vol. 128,p. 12526 - 12530,5

40
[ 4488-22-6 ]
[ 133-89-1 ]
C₂₆H₂₆N₂O₅ [ No CAS ]