chem. commun., 2013,49, 7237-7239 suppinfo

8/13/2019 Chem. Commun., 2013,49, 7237-7239 SuppInfo

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Supporting Information

Fluorescence-Activated Cell Sorting and Directed Evolutionof -N-Acetylgalactosaminidases Using a Quenched Activity-

Based Probe (qABP)

Kamaladasan Kalidasan,aYing Su,bXiaoyuan Wu,bShao Q. Yao*band MaheshUttamchandani*a,b,c

aDefence Medical and Environmental Research Institute, DSO National Laboratories, 27 MedicalDrive, Singapore 117510, SingaporebDepartment of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore117543, SingaporecDepartment of Biological Science, National University of Singapore, 14 Science Drive 4,Singapore 117543, Singapore.

Joint first authors*Address correspondence to: [email protected], [email protected]

Contents

Page S2-S7: Probe synthesis and characterization of intermediates.

Page S7-S8: General procedures.

Page S9-S11: Results and discussion

Page S12-S20: 1H and 13C NMR

ectronic Supplementary Material (ESI) for Chemical Communicationsis journal is The Royal Society of Chemistry 2013


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1. Chemistry.

Scheme S1.Detailed synthesis of fluorescently quenched Probe A.

(2S,4R,5R,6R)-6-(acetoxymethyl)-3-azidotetrahydro-2H-pyran-2,4,5-triyl

triacetate (2). Commercial galactosamine (0.54g, 2.5mmol) was dissolved in dry

MeOH, followed by addition of K2CO3 (0.485g, 3.5mmol), CuSO45H2O (6.25mg,0.025mmol) and 1H-imidazole-1-sulfonyl azide (0.625g, 3mmol). The above mixturewas stirred at r.t. for 18 h andconcentrated in vacuo. The residue was redissolved in5ml of Ac2O with 2ml pyridine and stirred at r.t. for another 3 h, after which thesolvent was removed in vacuoand H2O was added to stop the reaction. The residuewas extracted with ethyl acetate and separated by flash chromatography(EA/Hex=1/2) to give2(0.65g, 70%). 1H-NMR (500 MHz, CDCl3) 6.26 (d,J= 3.15Hz, 0.35H), 5.51 (d, J= 8.15 Hz, 0.65H), 5.42 (d, J= 3.15 Hz, 0.35H), 5.32 (d, J=3.15 Hz, 0.65H), 5.25 (dd, J1= 11.05 Hz, J2= 3.45 Hz, 0.35H), 4.87 (dd, J1= 10.7Hz, J2 = 3.15 Hz, 0.65H), 4.25-4.22 (m, 0.4H), 4.11-4.02 (m, 2H), 4.01-3.97 (m,0.6H), 3.88 (dd, J1= 10.73 Hz, J2= 3.78 Hz, 0.35H), 4.75 (dd, J1= 10.73 Hz, J2=

8.85 Hz, 0.65H), 2.14 (s, 1.8H), 2.11 (s, 1.2H), 2.10 (s, 3H), 2.01 (s, 1.2H), 2.00 (s,1.8H), 1.97 (s, 1.2H), 1.97 (s, 1.8H). 13C-NMR (125 MHz, CD3CCl3) 170.1, 169.7,



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169.6, 169.4, 168.3, 92.6, 90.2, 71.5, 71.1, 68.5, 66.7, 66.1, 60.9, 60.8, 59.5, 56.7,20.6, 20.6, 20.410, 20.3, 20.3.

(2R,3R,4R,5R)-2-(acetoxymethyl)-5-azido-6-hydroxytetrahydro-2H-pyran-3,4-

diyl diacetate (3).2(0.49g, 1.3mmol) was dissolved in 2ml of dry DMF, followed byaddition of hydrazine acetate (0.32g, 3.25mmol). The above mixture was stirred at r.t.for 0.5h and concentrated in vacuo. The residue was redissolved in 10ml of ethylacetate and washed with H2O, brine, and dried on Na2SO4. The crude product was

purified by flash column (EA/Hex=5/1) to obtain 3 (0.31 g, 60%). 1H-NMR (500MHz, CDCl3) 5.44 (d,J= 3.15 Hz, 0.6H), 5.40 (d,J= 3.8 Hz, 0.85H), 5.37 (d, J=

3.8 Hz, 0.27H), 5.32 (d, J= 3.15 Hz, 0.36H), 4.81 (dd, J1= 10.7 Hz, J2= 3.15 Hz,0.37H), 4.69 (d,J=8.2, 0.38H), 4.45 (t,J= 6.62, 0.63H), 4.12-4.05 (m, 2H), 3.90 (t,J= 6.3, 0.47H), 3.72 (dd,J1= 11.025,J2= 3.48, 0.64H), 3.64 (dd,J1= 10.725,J2= 7.6,0.4H), 2.13 (s, 3H), 2.04 (s, 3H), 2.04 (s, 3H). 13C-NMR (125 MHz, CDCl3) 170.6,170.6, 170.1, 169.9, 96.3, 92.3, 71.1, 70.8, 68.3, 67.7, 66.4, 61.9, 61.7, 61.5, 58.0,20.6, 20.5, 20.5.

(2R,3R,4R,5R,6S)-2-(acetoxymethyl)-5-azido-6-(2,2,2-trichloro-1-

iminoethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate (4). 3 (0.31g, 0.92mmol) wasdissolved in 5ml dry DCM on ice bath, followed by addition of DBU (135l,

0.92mmol), and stirred at 0oC for 10 min. Subsequently, 2,2,2-trichloroacetonitrile(0.93ml, 9.2mmol) was added dropwise, and the mixture was stirred for another 2 h.The solvent was remove in vacuo and the resulting mixture waspurified by flashchromatography to obtain 4(0.32g, 71%). 1H-NMR (500 MHz, CDCl3) 8.78 (s,1H),6.46 (d,J= 3.15 Hz, 1H), 5.49 (d, J= 1.90 Hz, 1H), 5.33 (dd,J1= 3.88 Hz,J2= 3.15Hz, 1H), 4.37 (t,J= 6.625 Hz, 1H), 4.10 (dd, J1= 11.35 Hz,J2= 6.95 Hz, 1H), 4.03-3.98 (m, 2H), 2.12 (s, 3H), 2.02 (s, 3H), 1.95 (s, 3H). 13C-NMR (125 MHz, CDCl3) 170.0, 169.7, 169.5, 160.4, 94.3, 90.4, 68.9, 68.5, 66.7, 61.0, 56.9, 20.4, 20.4, 20.3.

(2R,3R,4R,5R,6R)-2-(acetoxymethyl)-6-(4-(2-((2-(allyloxy)-2-

oxoethyl)amino)-1-hydroxy-2-oxoethyl)phenoxy)-5-azidotetrahydro-2H-pyran-3,4-diyl diacetate (5). Flame-driedschlenk flask was filled with 200mg of oven-dried 4



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MS and 20ml of dry DCM. 4 (0.26 g, 0.55 mmol) was added and the mixture wascooled on ice bath, followed by addition ofallyl 2-(2-hydroxy-2-(4-hydroxyphenyl)acetamido)acetate (0.13g, 0.5mmol).The mixture was further stirred at-20oCfor 30 min, followed by dropwise addition ofTMSOTf (110 l, 0.62mmol) overa period of 20 min. The reaction was continued on ice-salt bath for another 2h.Finally, the solvent was removed in vacuoand resulting mixture waspurified by flashchromatography to give5(0.165 g, 53%). 1H-NMR (500 MHz, CDCl3) 7.45 (d,J=8.2 Hz, 2H), 7.12 (d, J= 8.5 Hz, 2H), 5.94-5.88 (m, 1H), 5.76 (d, J= 2.75 Hz, 1H),5.51 (s, 1H), 5.33-5.20 (m, 2H), 5.05 (s, 1H), 4.61 (d,J= 4.35 Hz, 1H), 4.40 (m, 1H),4.23 -3.93 (m, 6H), 2.15 (s, 3H), 2.05 (s, 3H), 1.91 (s, 3H). 13C-NMR (125 MHz,CDCl3) 174.6, 170.7, 170.5, 170.0, 169.4, 156.0, 135.1, 131.9, 128.3, 117.3, 116.7,116.3, 114.7, 99.8, 96.9, 73.5, 71.0, 70.7, 68.3, 67.5, 66.6, 65.4, 61.5, 60.8, 60.1, 57.4,40.3, 19.3, 19.1.

(2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(4-(2-((2-(allyloxy)-2-

oxoethyl)amino)-1-hydroxy-2-oxoethyl)phenoxy)tetrahydro-2H-pyran-3,4-diyl

diacetate (6). 5 (0.165 g, 0.31 mmol) was dissolved inAcSH and the reaction wasstirred at r.t. for 12 h. Uponsolventremoval, the residue was redissolved in ethylacetate and purified by flash chromatography to give 6 (80 mg, 47%). 1H-NMR (500MHz, MeOD) 7.44 (d,J=8.5 Hz, 2H), 7.11 (d, J=8.2 Hz, 2H), 5.95-5.87 (m, 1H),5.61 (d,J=3.4 Hz, 1H, form), 5.50 (d, J=5.5 Hz, 1H), 5.38 (dd, J1=11.55 Hz, J2

=3.15 Hz, 1H), 5.33-5.20 (m, 2H), 5.04 (d, J=1.5 Hz, 1H), 4.61 (d,J=5.65 Hz, 2H),4.37 (t,J=6.375 Hz, 1H ), 4.15-3.99 (m, 5H), 2.16 (s, 3H), 1.99 (s, 3H), 1.95 (s, 3H),1.90 (d,J= 3.4 Hz, 3H). 13C-NMR (125 MHz, CDCl3) 174.6, 172.4, 171.5, 170.7,170.517, 169.4, 156.3, 134.9, 131.9, 128.3, 117.3, 116.7, 96.6, 73.5, 68.1, 67.3, 65.4,61.6, 60.1, 40.4, 29.899, 21.2, 21.1, 19.6, 19.3.

(2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(4-(2-((2-(allyloxy)-2-

oxoethyl)amino)-1-(((4-nitrophenoxy)carbonyl)oxy)-2-oxoethyl)phenoxy)tetrahydro-

2H-pyran-3,4-diyl diacetate (7).6 (80 mg, 0.14 mmol) was dissolved in dry DCM,followed by slow addition of pyridine (1ml, 0.87mmol) and p-nitrochloroformate(60mg, 0.29 mmol). The reaction was stirred at r.t. for 6 h. Upon solvent removal, theresidue was redissolved in ethyl acetate and purified by flash chromatography togive7(22.0 mg, 22%). The unstable product was used immediately in the next step.



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(2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(4-(1-(4-((E)-(4-

(dimethylamino)phenyl)diazenyl)phenyl)-1,7,10,13-tetraoxo-8,14-dioxa-2,6,11-

triazaheptadec-16-en-9-yl)phenoxy)tetrahydro-2H-pyran-3,4-diyl diacetate (8).7(22.0 mg, 0.029 mmol) was dissolved in dry THF, followed by addition ofthe Dabcylderivative (15 mg, 0.035mmol) and DIEA (50 l, 0.26mmol). The reaction wasstirred at r.t. for 3 h. Upon solvent removal, the residue was redissolved in DCM and

purified by flash chromatography to give8 (33 mg, 90%). 1H-NMR (500 MHz,MeOD) 7.93 (d,J= 8.20Hz, 2H), 7.86-7.81 (m, 4H), 7.49 (d,J= 8.80Hz, 2H), 7.14(d,J= 8.85Hz, 2H), 6.81 (d,J= 8.85, 2H), 5.96 (s, 1H), 5.92-5.84 (m, 1H), 5.63 (d,J= 3.8Hz, 1H, form), 5.49 (d,J= 3.15Hz, 1H), 5.38 (dd, J1=11.35 Hz,J2=3.15 Hz,1H), 5.30-5.18 (m, 2H), 4.59 (d,J= 5.70Hz, 2H), 4.33 (t,J= 5.70Hz, 1H), 4.12-4.02(m, 4H), 3.45 (t,J= 6.95Hz, 2H), 3.33-3.32 (m, 1H), 3.25 (dd,J1=12.60 Hz,J2=6.3Hz, 2H), 3.09 (s, 6H), 2.16 (s, 3H), 2.00 (s, 3H), 1.97 (s, 3H), 1.88 (s, 3H).13C-NMR(125 MHz, CDCl3) 173.7, 172.4, 172.0, 172.0, 171.8, 170.5, 169.5, 158.2, 157.2,156.3, 154.5, 144.7, 135.6, 133.2, 131.9, 130.4, 130.3, 130.2, 129.2, 126.4, 122.9,118.6, 118.1, 118.1, 112.6, 97.8, 76.4, 69.4, 68.7, 68.5, 66.7, 63.0, 41.8, 40.3, 39.4,38.3, 30.5, 22.4, 20.6, 20.5. LC-MS (IT-TOF) calcd for [M+H]+: 946.383, Found946.352.

9-(4-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-

(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-1-(4-((E)-(4-

(dimethylamino)phenyl)diazenyl)phenyl)-1,7,10-trioxo-8-oxa-2,6,11-triazatridecan-

13-oic acid (9).8 (33.0 mg, 0.035 mmol) was dissolved in dry THF, followed by

addition ofPd(PPh3)4 (0.1eq) and PhSiH3(2eq) under Ar protection. The reaction wasstirred at r.t. for 2 h. Upon solvent removal, the residue was redissolved in DCM andpurified by flash chromatography to give9 (25 mg, 90%). 1H-NMR (500 MHz,CD3OD/CDCl3=10/1) 7.89 (d,J= 8.10Hz, 2H), 7.80 (d,J= 9.05Hz, 2H), 7.76 (d, J= 7.40Hz, 2H), 7.43 (d,J= 7.95Hz, 2H), 7.06 (d,J= 7.95Hz, 2H), 6.75 (d,J= 8.90,2H), 5.92 (s, 1H), 5.57 (d, J= 3.8 Hz, 1H), 5.44 (d, J= 4.65Hz, 1H), 5.34 (d, J=11.4Hz, 1H), 4.61 (d, J= 13.85Hz, 1H), 4.27 (s, 1H), 4.04-3.99 (m, 2H), 3.85-3.70(m, 2H), 3.41-3.21 (m, 4H), 3.05 (s, 6H), 2.13 (s, 3H), 1.97 (s, 3H), 1.92 (s, 3H), 1.82(s, 3H).13C-NMR (125 MHz, MeOD/CDCl3=10/1) 173.5, 171.9, 171.9, 171.7,169.4, 158.0, 157.2, 156.2, 154.4, 144.6, 135.4, 132.2, 130.1, 129.2, 126.3, 122.9,118.0, 112.51, 97.7, 69.3, 68.79, 68.6, 68.4, 62.8, 40.4, 26.3, 22.5, 20.6, 20.6, 20.5.

LC-MS (IT-TOF) calcd for [M+H]+: 906.352, Found 906.323.



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(4)5-((9-(4-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-

(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-1-(4-((E)-(4-

(dimethylamino)phenyl)diazenyl)phenyl)-1,7,10,13-tetraoxo-8-oxa-2,6,11,14-

tetraazaheptadecan-17-yl)carbamoyl)-2-(6-hydroxy-3-oxo-3,10-dihydroanthracen-9-

yl)benzoic acid (10). 9(25.0 mg, 0.028 mmol) was dissolved in 2ml DMF, followed

by addition ofthe fluorescent derivative (14mg, 0.03mmol) and HATU (10.5 mg,0.028mmol). The reaction was stirred at r.t. overnight. The crude product was purifiedby flash chromatography to give10 (22 mg, 61%). 1H-NMR (500 MHz, CD3OD) 8.15-8.05 (m, 2H), 7.88 (t, J= 8.2Hz, 2H), 7.81-7.74 (m, 4H), 7.46-7.41 (m, 2H),7.10 (t, J= 7.55Hz, 2H), 6.78 (t, J= 9.45Hz, 2H), 6.67-6.51 (m, 7H), 5.75 (d, J=3.80Hz, 1H), 5.60 (d,J= 2.55Hz, 1H), 5.45 (s, 1H), 5.35 (d,J= 8.80Hz, 1H), 4.60 (d,

J= 12.0 Hz, 1H), 4.29 (s, 1H), 4.08-3.68 (m, 6H), 3.34-3.18 (m, 10H), 3.07 (s, 6H),2.14 (s, 3H), 1.97 (s, 3H), 1.94 (s, 3H), 1.84 (s, 3H). 13C-NMR (125 MHz, CD3OD) 173.8, 172.9, 172.8, 172.0, 172.0, 171.8, 171.5, 171.4, 171.0, 169.6, 168.4, 168.2,258.2, 157.8, 175.8, 156.3, 154.6, 144.7, 137.6, 135.6, 131.1, 130.5, 130.4, 130.0,129.2, 126.4, 122.982, 118.2, 118.1, 112.6, 103.7, 97.7, 69.4, 68.7, 68.5, 62.9, 55.8,

40.3, 38.4, 37.9, 30.5, 22.4, 20.6, 20.5, 20.5. LC-MS (ESI) calcd for [M+H]+

:1320.47, Found: 1320.65; calcd for [M+2H]2+/2: 660.74, Found: 660.65.

5-((9-(4-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-

(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-1-(4-((E)-(4-

(dimethylamino)phenyl)diazenyl)phenyl)-1,7,10,13-tetraoxo-8-oxa-2,6,11,14-

tetraazaheptadecan-17-yl)carbamoyl)-2-(6-hydroxy-3-oxo-3,10-dihydroanthracen-9-

yl)benzoic acid (Probe A).10(10.2 mg, 0.008 mmol) was dissolved in 2ml CH3OH,followed by addition of NaOCH3(2.5 mg, 0.046mmol). The reaction was stirred at r.t.for 1h. The crude product was purified on semi-preparative HPLC to give Probe A(2.5 mg, 28%). 1H-NMR (500 MHz, DMSO/H2O = 3/4) 8.24-8.22 (m, 1H), 7.97-7.93 (m, 2H), 7.75-7.54 (m, 6H), 7.33-7.27 (m, 2H), 7.04-7.01 (m, 2H), 6.74-6.46 (m,7H), 5.65-5.58 (m, 1H), 5.34 (d, J = 2.00Hz, 1H), 4.13-4.15 (m, 1H), 3.86-3.44 (m,

6H), 3.28-3.02 (m, 10H), 2.96 (s, 6H), 1.83 (s, 3H). Chemical Formula: C61H63N9O17,HR-MS for [M+Na]+calcd 1216.4234, found 1216.4234.



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2. Biology and Screening.

2.1Cloning and Generation of Random-Mutagenesis Library.

Strains reported in Liu et al., were sourced from ATCC.1 Specifically theElizabethkingia miricola (ATCC Strain no: 33958) strain was acquired. GenomicDNA was extracted using the bacterial DNA extraction kits (Qiagen). There wassome confusion as to the actual source of the enzymes reported by Clausen et al., andwhich exact strains they came from. This was because there were discrepancies in thesequences deposited by the authors in NCBI, against their actual reported primersequences and proteins in the supporting information. After going through thesedetails carefully, we realized that the authors had wrongly reported the strain fromwhich they isolated the NAG enzyme, which should have been Elizabethkingeamiricola instead of Elizabethkingea meningosepticum. This was confirmed by ourown sequencing results which confirmed the former strain. We thus cloned out therelevant galactosidase sequence using Gateway Cloning Technology by following

the manufacturers instructions (Invitrogen). Briefly, the NAG gene was amplified byPCR with designed primers (Table S1), then was ligated into TOPO S/D vector andtransformed into Top10 cells. After verification by colony PCR and insert DNAsequencing, LR reaction was performed with pDEST17 vector and then the finalconstruct was transformed into NovaBlue(DE3) competent cells (NovaGen). Theconstruct was subjected to random mutagenesis using a GeneMorph II EZcloneDomain mutagenesis kit (Stratagene). 10ng of template plasmid DNA per reactionwas used to establish the mutant library, with the expected mutation frequency of 9-16 mutations/kb. The mutant plasmid library was transformed into NovaBlue(DE3)competent cells by electroporation to get the high transformation efficiency estimatedat 1108cfu/ng. Then we randomly sequenced 20 colonies from the electroporated

mutant library, each provided diverse sequences confirming the vast diversitygenerated.

2.2Hypotonic Loading prior to Flow Cytometric Screening.

1 ml of induced cell culture was spun down and re-suspended in 25l ofhypotonic buffer (50% LB in autoclaved water) and warmed at 37 oC for 5 min.200M of probes were prepared and warmed at 37oC for 5 min. 25l of the pre-warmed probe was added to pre-warmed cells to a final probe concentration of100M. The cell suspension was re-incubated 37oC for a further 1-5 min. A 10-folddilution with ice-chilled 100% LB produced a final volume of 500l with final probeconcentration of 10M and 92.5% LB. The tubes were kept on ice at 4 oC for 30 min

before FACS analysis.

2.3Protein Expression and Purification.

Proteins were successfully purified with Ni-NTA agarose using standard affinitypurification procedures (Qiagen Ni-NTA handbook). Briefly, 200l of Ni-NTAagarose bead suspension (Qiagen) was pipetted into a fritted column and washed. 10ml of the lysate was added and incubated with the Ni-NTA beads for 2 h at 4 oC. Thecolumn was gently mixed during this incubation. The flow through was then drained.Repeated washing was then performed to remove background proteins. Elution was

performed in PBS buffer containing 200mM imidazole. Typical protein purificationgels obtained are as shown in Fig. 2A of the maintext.



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2.4Kinetic Analysis.

The enzyme activity assays were carried out in 50l reactions, containing 1PBS(pH7.4) with either 2nM and 20nM of the purified NAG protein or approximately

10g of crude NAG-overexpressing cell lysates. Coumarin AMethylumbelliferyl2-acetamido-2-deoxy--D-galactopyranoside; purchased from Carbosynth with Cat#EM04782) or Probe A working stocks in DMSO were added to varying finalconcentrations. The mixtures were monitored at 360nm/420nmand 490nm/520nmat 37Con a BioTek Microplate reader, in a time-dependent manner (Fig. S1). Forcomparison of the activity of NAG protein and the mutant, assays were performed intriplicate with Coumarin A, over 8 different probe concentrations over 5M and200M, along with a 10nM of total enzyme. The initial enzyme kinetics data and plotwas monitored by Gen5 software at 360nm/420nm. These were plotted on a Michealis-Menten plot, as shown in Fig. 4A of the maintext, using the Graphpad software. Non-linear regression was used to derive the KMand kcatvalues for both the wildtype and

variant NAG enzyme, as shown in Fig. 4B of the main text.

2.5Protein Labeling and In-Gel Fluorescence Scanning.

After enzyme activity assay, the samples were separated by SDS-PAGE, and thegels were scanned on the Typhoon fluorescence gel scanner for detection offluorescently labeled protein bands under the FITC channel (Fig. S2).



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3. Results and Discussion.

Table S1. Primer sequences used/derived in the current study.NAG Forward Primer CACCCCTAAAAAGGTAAGAATAGC

NAG Reverse Primer GGGTTAGTAGTCGTCATTTATTGCForward Sequencing Primer (T7-F) TAATACGACTCACTATAGGG

Reverse Sequencing Primer (T7-R) TATGCTAGTTATTGCTCAG

Coumarin A

Fig. S1 Continuous microplate monitoring of enzymatic activity. (A) Structure ofcommercial Coumarin A fluorogenic substrate. Time-dependent protein activity of

NAGusing (B) Coumarin A substrate and (C) Probe A. Left panels are assaysconducted with purified wildtype NAG protein and right panels are assays withinduced NAG cell lysates. Red curves depict assays run enzyme, blue curves depict

blanks with probe only. The activity was monitored at 360nm/420nm and 490nm/520nm,respectively, using 20 nM of enzyme/approximately 10g of cell lysate and 20 M of

probe, buffered in PBS (pH 7.4).

0

20

40

60

0 15 30

RFU(

100)

Time (min)

0

20

40

60

0 15 30

RFU

(100)

Time (min)

0

50

100

150

0 15 30

RFU

(100)

Time (min)

A

B

0

20

40

60

0 15 30

RFU

(100)

Time (min)

C



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Fig. S2 In-gel fluorescence scanning of NAG-overexpressingbacterial cell lysatesupon labeling with Probe A. MW marker; Lane 1: Probe A only; Lane 2:

NovaBlue(DE3) cell lysates labeled with Probe A; Lane 3: NAG-overexpressingNovaBlue(DE3) cell lysates labeled with Probe A. (Left - Fluorescence image, Right

- Coomassie Blue stained image of the same gel). Results indicated that manyproteins, including NAG, in the NAG-overexpressing lysate was labeled by the probe,and the labeling was NAG activity-dependent (as no labeling was observed in lane 2).

Table S2.Mutations in identified NAG variant

Fig. S3 Sequence information for identified variant. The chromatograms indicate the

bi-directional reads with the highlighted yellow bands indicating each of the non-synonymous mutations identified.

Reference

Position

Nucleotide

Change

Amino Acid

Change

Mutation Location from active site

311 A>T H104L Yes Within 10 angstroms

528 A>T T176T Silent

1041 T>A S347R Yes Within 20 angstroms

Lanes: MW 1 2 3 1 2 3



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0.3

55

3

0.63

3

5

0.3

60

3

0.6

41

7

0.4

95

0

0.667

2

0.4

10

8

2.1

04

9

0.9

12

2

0.380

0

0.5

89

2

1.95

6

0

1.1

81

8

2.6

04

6

1.3

16

3

1.703

6

2.6

87

9

In

teg

ra

l

7.26

0

6

6.269

7

6.2

63

4

5.5

23

3

5.5

07

0

5.4

26

3

5.4

20

0

5.3

25

4

5.31

9

1

5.273

7

5.266

2

5.2

58

6

5.2

51

0

5.2

44

7

4.8

85

4

4.8

79

1

4.86

4

0

4.85

7

7

4.250

0

4.2

36

1

4.2

22

3

4.1

10

1

4.0

97

5

4.0

88

6

4.0

74

8

4.06

4

7

4.05

2

1

4.045

8

4.0

42

0

4.0

31

9

4.0

29

4

4.0

24

3

4.0

11

7

4.00

1

6

3.99

6

6

3.989

0

3.9

84

0

3.9

70

1

3.9

02

0

3.8

94

5

3.8

80

6

3.8

73

0

3.79

7

4

3.77

9

8

3.776

0

3.7

58

3

2.1

42

1

2.1

16

9

2.1

06

8

2.0

14

7

2.0

03

4

1.97

6

9

1.973

1

(ppm )

0. 01. 02. 03. 04. 05. 06. 07. 08. 09. 0

* * * C u r r e n t D a t a P a r a m e t e r s * * *

N A M E : s u y 1 1 1 0

E X P N O : 3P R O C N O : 1

* * * A c q u i s i t io n P a r a m e t e r s * * *

N S : 8

O 1 : 3 0 8 8 .5 1 Hz

P U L P R O G : z g 3 0

S F O 1 : 5 00 .1 3 3 0 8 8 5 M H z

S O L V E N T : C D C l3

S W : 2 0 .6 5 5 7 pp m

* * * P r o c e s s i n g P a r a m e t e r s * * *

L B : 0 .3 0 H z

S F : 5 0 0 .1 3 0 0 1 2 7 M Hz

S W _ p : 1 03 3 0.5 78 51 24

1 H A M X 5 0 0

s u y 1 1 1 0 3

A c N 3

17

0

.1

218

16

9.

7574

16

9.

64

81

16

9

.4

149

16

8.

38

74

92

.678

9

90

.27

40

77

.251

5

76

.99

64

76

.7487

71

.545

5

71

.10

82

68

.579

5

66

.73

58

66

.1018

60

.949

6

60

.84

03

59

.572

3

56

.70

11

20

.6942

20

.657

8

20

.41

00

20

.3663

20

.344

4

(ppm)

-100102030405060708090100110120130140150160170180190200210220

*** Cur rent Data Parameters ***

N AM E : suy1110

EXPN O : 4

PR O C N O : 1

*** Acquisit ion Parameters ***

BF1 : 12 5.7 5778 90 M H z

L OC NU C : 2H

N S : 84

O 1 : 1 3204 .57 Hz

PU L PRO G : zg pg30

SFO 1 : 12 5.7 7099 36 M Hz

SOLV EN T : C DC l3

SW : 2 38.76 75 p pm

*** Process ing Parameters ***

L B : 1 .00 H z

PH C 0 : 2 17.2 36 deg ree

PH C 1 : 42.1 30 d eg ree

13C AMX500

suy1110 4

Ac N3



13/20

S13

0

.6

4

4

6

0

.9

1

1

3

0

.2

9

5

6

0

.3

8

6

6

0

.4

0

0

0

0

.4

0

9

9

0

.6

8

3

1

2

.1

5

0

2

0

.5

0

4

4

0

.6

7

9

4

0

.4

1

2

6

3

.0

2

6

6

5

.9

1

5

1

In

teg

ra

l

7

.2

6

0

6

5

.4

4

7

7

5

.4

4

1

4

5

.4

0

7

4

5

.3

9

9

8

5

.3

9

2

2

5

.3

7

7

1

5

.3

6

9

5

5

.3

2

9

2

5

.3

2

2

9

4

.8

2

3

6

4

.8

1

7

3

4

.8

0

2

2

4

.7

9

5

9

4

.7

0

0

1

4

.6

8

3

7

4

.4

6

5

6

4

.4

5

3

0

4

.4

3

9

1

4

.1

2

6

5

4

.1

1

2

6

4

.0

9

8

7

4

.0

8

9

9

4

.0

7

9

8

4

.0

6

5

9

4

.0

5

7

1

3

.9

1

8

4

3

.9

0

5

8

3

.8

9

3

2

3

.7

3

8

2

3

.7

3

0

6

3

.7

1

5

5

3

.7

0

9

2

3

.6

6

3

8

3

.6

4

8

6

3

.6

4

2

3

3

.6

2

7

2

2

.1

4

7

1

2

.1

3

4

5

2

.0

4

7

5

2

.0

4

3

7

2

.0

3

9

9

(pp m )

0. 01. 02. 03. 04. 05. 06. 07. 08. 09. 0

* * * C u r r e n t D a t a P a r a m e t e rs * * *

N A M E : s u y 1 1 1 5

E X P N O : 1

P R O C N O : 1

* * * A c q u i s i t i o n P a r a m e t e r s * * *

L O C N U C : 2 H

N S : 8

N U C L E U S : o ff

O 1 : 3 0 8 8 .5 1 Hz

P U L P R O G : z g 3 0

S F O 1 : 5 00 .1 3 3 0 8 8 5 M H z


S W : 2 0 .6 5 5 7 p p m

T D : 3 2 7 6 8

T E : 3 0 0 .0 K


L B : 0 .3 0 H z

S F : 5 0 0 .1 3 0 0 1 2 7 M Hz

* * * 1 D N M R P l o t P a r a m e t e rs * * *


1 H A M X 5 0 0

s u y 1 1 1 5 1

s u g a r A O A c t o O H

0

.6

4

4

6

0

.9

1

1

3

0

.2

9

5

6

0

.3

8

6

6

0

.4

0

0

0

0

.4

0

9

9

0

.6

8

3

1

2

.1

5

0

2

0

.5

0

4

4

0

.6

7

9

4

0

.4

1

2

6

In

teg

ra

l

5

.4

4

1

4

5

.4

0

7

4

5

.3

9

9

8

5

.3

9

2

2

5

.3

7

7

1

5

.3

6

9

5

5

.3

2

9

2

5

.3

2

2

9

4

.8

2

3

6

4

.8

1

7

3

4

.8

0

2

2

4

.7

9

5

9

4

.7

0

0

1

4

.6

8

3

7

4

.4

6

5

6

4

.4

5

3

0

4

.4

3

9

1

4

.1

2

6

5

4

.1

1

2

6

4

.0

9

8

7

4

.0

8

9

9

4

.0

7

9

8

4

.0

6

5

9

4

.0

5

7

1

3

.9

1

8

4

3

.9

0

5

8

3

.8

9

3

2

3

.7

3

8

2

3

.7

3

0

6

3

.7

1

5

5

3

.7

0

9

2

3

.6

6

3

8

3

.6

4

8

6

3

.6

4

2

3

3

.6

2

7

2

(p p m )

3 .63. 84. 04. 24 .44. 64. 85. 05. 25 .45. 6

1

7

0

.6

5

38

1

7

0

.6

2

46

1

7

0

.1

5

09

1

6

9

.9

6

15

9

6

.3

7

3

6

9

2

.3

0

7

2

7

7

.2

5

8

8

7

7

.0

0

3

7

7

6

.7

4

8

7

7

1

.1

8

8

4

7

0

.8

4

5

9

6

8

.3

0

9

9

6

7

.7

0

5

1

6

6

.4

8

8

1

6

1

.9

7

7

2

6

1

.7

5

1

3

6

1

.5

3

9

9

5

8

.0

3

4

7

2

0

.6

2

1

3

2

0

.5

9

2

2

2

0

.5

4

8

5

(p p m )

02 04 06 08 01 001 2 01 401 6 01 8 0


N A M E : s u y 1 1 1 5

E X P N O : 2

P R O C N O : 1


L O C N U C : 2 H

N S : 8 1


O 1 : 1 3 2 0 4 .5 7 H z

P U L P R O G : z g p g 3 0

S F O 1 : 1 2 5 .7 7 0 9 9 3 6 M H z


S W : 2 3 8 .7 6 7 5 p p m

T D : 6 5 5 3 6

T E : 3 0 0 .0 K


L B : 1 .0 0 H z

S F : 1 2 5 .7 5 7 7 9 5 2 M H z

* * * 1 D N M R P l o t P a r a m e t er s * * *


1 3 C A M X 5 0 0

s u y 1 1 1 5 2

s u g a r A O A c t o O H



14/20

S14

0

.9

9

7

9

1

.0

0

0

0

1

.0

6

1

2

1

.0

6

0

0

1

.0

3

9

8

1

.0

9

0

2

2

.0

7

6

8

3

.0

9

6

8

3

.0

9

7

6

2

.9

8

6

6

In

teg

ra

l

8

.7

8

4

9

7

.2

6

0

6

6

.4

7

0

2

6

.4

6

3

9

5

.4

9

3

1

5

.4

8

9

3

5

.3

4

4

3

5

.3

3

8

0

5

.3

2

1

6

5

.3

1

5

3

4

.3

8

8

7

4

.3

7

6

1

4

.3

6

2

2

4

.1

2

0

2

4

.1

0

6

3

4

.0

9

7

5

4

.0

8

3

6

4

.0

3

3

2

4

.0

1

9

3

4

.0

1

0

5

4

.0

0

2

9

3

.9

9

6

6

3

.9

8

7

8

3

.9

8

1

5

2

.1

2

1

9

2

.0

2

7

4

1

.9

5

6

8

(p p m )

0. 01 .02. 03. 04. 05. 06. 07 .08. 09. 0


N A M E : s u y 1 1 2 9

E X P N O : 1

P R O C N O : 1


L O C N U C : 2 H

N S : 8


O 1 : 3 0 8 8 . 5 1 H z

P U L P R O G : z g 3 0

S F O 1 : 5 0 0 .1 3 3 0 8 8 5 M H z


S W : 2 0 . 6 5 5 7 pp m

T D : 3 2 7 6 8

T E : 3 0 0 .0 K


L B : 0 . 3 0 H z

S F : 5 0 0 .1 3 0 0 1 3 4 M H z

* * * 1 D N M R P l o t P a r a m e t er s * * *


1 H A M X 5 0 0

s u y 1 1 2 9 1

A p r o b e N 3 C N C C l 3

1

7

0

.0

7

7

9

1

6

9

.7

5

7

3

1

6

9

.5

0

2

2

1

6

0

.4

5

1

3

9

4

.3

3

3

0

9

0

.4

7

8

0

7

7

.2

5

8

7

7

7

.0

0

3

6

7

6

.7

4

8

5

6

8

.9

7

2

9

6

8

.5

1

3

8

6

6

.7

9

4

0

6

1

.0

5

1

6

5

6

.8

9

7

7

2

0

.4

3

9

0

2

0

.4

0

2

6

2

0

.3

8

8

0

(ppm )

0204 06 08010 012 014 016 018 0


N A M E : s u y 1 1 2 9

E X P N O : 2

P R O C N O : 1


L O C N U C : 2 H

N S : 4 2


O 1 : 1 3 2 0 4 .5 7 H z

P U L P R O G : z g p g 3 0

S FO 1 : 1 2 5 . 7 7 0 9 9 3 6 M H z


S W : 2 3 8 .7 6 7 5 p p m

T D : 6 5 5 3 6

T E : 3 0 0 .2 K


L B : 1 .0 0 H z

S F : 1 2 5 . 7 5 7 8 0 5 3 M H z

* * * 1 D N M R P l o t P a r a m e t e rs * * *


1 3 C A M X 5 0 0

s u y 1 1 2 9 2

A p r o b e N 3 C N C C l 3



15/20

S15

2

.1

21

3

2

.115

8

1

.000

0

0

.791

2

1

.209

4

2

.131

9

1

.042

7

2

.30

5

8

0

.791

4

0

.790

1

2

.146

9

1

.883

4

1

.250

1

3

.2

47

5

2

.3

30

5

2

.21

5

9

2

.34

9

2

In

teg

ral

7.

4

67

4

7.

4

5

1

0

7

.1

35

4

7.

1

18

4

7

.0

8

5

9

7

.0

69

0

5.

9

4

0

0

5

.9

30

4

5.

9

19

4

5

.9

07

9

5.

8

96

0

5.

8

8

5

0

5

.7

62

3

5.

7

56

8

5.

5

1

4

9

5

.3

30

4

5.

2

96

0

5

.2

27

8

5.

2

06

7

5

.0

54

2

4.

8

05

0

4

.6

23

2

4.

6

14

5

4.

4

00

6

4

.2

31

6

4.

2

18

8

4.

1

9

2

2

4

.1

69

3

4.

1

59

2

4

.1

48

2

4.

1

37

2

4.

1

1

3

0

4

.1

00

6

4.

0

85

5

4.

0

7

4

9

4

.0

67

6

4.

0

32

4

4

.0

09

0

3.

9

74

2

3.

9

6

0

9

3

.9

38

5

3.

9

33

0

3

.3

07

3

2.

1

53

6

2.

0

57

9

2

.0

35

0

2.

0

25

8

2.

0

1

0

7

1

.9

09

9

1.

9

04

0

(ppm)

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5

*** Current Data Parameters ***

N AM E : suy 0117

Aprobe after phenol copule alpha

E XP NO : 1

P RO C NO : 1

*** Acquisition Parameters ***

B F1 : 5 00 .2300000 M Hz

LO C NUC : 2H

N S : 2

O 1 : 2751 .27 Hz

P ULP RO G : zg

S FO 1 : 500 .2327513 M Hz

S OLV EN T : M eOD

S W : 15 .0080 ppm

*** Processing Parameters ***

LB : 0 .10 Hz

P HC0 : 307 .249 d egree

P HC1 : -9 .562 d egree

174

.6

114

170.

7718

1

70.

57

50

170

.0

650

1

69.

48

94

156

.0

906

1

35.

13

63

135

.1

145

131

.9

233

128

.3

459

117

.3

951

1

16.

73

94

116

.6

811

116.

3897

1

16.

35

33

114

.7

868

99.

8652

9

7.

016

4

96.

97

27

7

3.

5048

73.

453

8

71.

06

40

7

0.

794

4

68.

3609

6

7.

617

8

67.

50

85

6

6.

648

7

65.

43

20

61.

5340

6

1.

344

6

60.

84

91

60.

1861

5

7.

410

2

48.

20

08

48.

0332

4

7.

858

4

47.

69

08

4

7.

523

2

47.

34

83

47.

18

08

4

0.

375

7

1

9.

348

6

19.

33

40

1

9.

195

6

(ppm)

2030405060708090100110120130140150160170

*** Cur rent Data Parame ters ***

NA ME : su y01 17

Aprobe af t er phenol coup

EXPN O : 3

PR OC N O : 1

*** Acquis it ion Parameters ***BF1 : 1 25 .782 93 40 M H z

LO CN U C : 2H

NS : 3 00

O1 : 125 77.84 Hz

PU LPR O G : zgp g30

SFO 1 : 1 25 .795 51 18 M Hz

SOL V EN T : C DC l3

SW : 238 .72 10 p p m


LB : 1 .00 H z

PH C 0 : 18 0.2 73 d egree

PH C 1 : 3 5.2 43 d e gree



16/20

S16

2

.0

00

0

1

.9

71

1

1

.0

12

8

0

.9

73

9

0

.9

6

5

8

0

.9

9

8

1

2

.3

4

8

0

1

.0

11

8

2

.4

27

7

0

.9

87

9

1

.1

3

5

1

2

.1

2

5

6

1

.7

8

3

4

3

.0

2

9

0

3

.1

43

8

3

.3

19

2

2

.8

95

6

In

te

g

ral

7

.4

57

9

7

.4

40

9

7

.1

26

5

7

.1

10

1

5

.9

52

7

5

.9

4

1

5

5

.9

31

1

5

.9

20

4

5

.9

07

2

5

.8

96

8

5

.8

8

6

1

5

.8

74

8

5

.6

19

4

5

.6

1

2

6

5

.5

07

8

5

.5

02

6

5

.3

99

2

5

.3

92

9

5

.3

7

6

1

5

.3

69

8

5

.3

32

0

5

.3

29

2

5

.2

97

4

5

.2

94

7

5

.2

25

8

5

.2

05

0

5

.0

5

0

2

5

.0

47

2

4

.7

97

3

4

.6

2

3

7

4

.6

12

4

4

.3

8

8

0

4

.3

74

9

4

.3

62

5

4

.1

52

1

4

.1

40

9

4

.1

2

9

6

4

.1

18

4

4

.1

04

1

4

.0

89

9

4

.0

75

6

4

.0

41

0

4

.0

30

9

4

.0

17

2

4

.0

1

0

9

4

.0

05

1

3

.9

99

6

3

.3

10

0

2

.1

6

3

1

1

.9

89

7

1

.9

57

4

1

.9

06

6

1

.8

9

9

8

(ppm)

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5


N AM E : su y011 7

E XPNO : 6

P ROC N O : 1


B F1 : 500 .230 0000 M H z

LO CN UC : 2H

N S : 2

O1 : 225 9.37 Hz

P ULPRO G : zg

S FO1 : 500 .232 2594 M Hz

S OL V EN T : M e OD

S W : 8 .9886 ppm


LB : 0.10 Hz

P HC0 : 618 .859 degre e

P HC1 : -9.723 d egre e

1H

suy0117 6

AcS H u p M eO D

174.

6989

172.

43

29

171

.5951

170.

77

18

170

.7426

170.

5167

169.

48

94

169

.4749

156.

38

21

134.

9542

134.

93

23

131

.9524

128.

30

22

128

.2730

117.

3951

116.

71

75

116

.6957

96.

6448

96.

60

11

73.

5266

73.

4756

68.

1569

67.

3773

67.

28

99

65.

4101

61.

6943

60.

17

88

48.

22

27

48.

0551

47.

8875

47.

71

26

47.

5451

47.

3775

47.

20

26

40.

4704

19.

6036

19.

3850

19.

35

59

19.

2757

(ppm)

0102030405060708090100110120130140150160170180190


N AM E : su y01 17

E XPN O : 7

P ROC N O : 1*** Acquisit ion Parameters ***

B F1 : 125.782 9340 M Hz

LO C NUC : 2 H

N S : 320

O1 : 125 77.84 H z

P ULPRO G : zgpg 30

S FO 1 : 125.795 5118 M Hz

S OLV EN T : M e OD

S W : 238 .7210 p p m


LB : 1.00 Hz

P HC0 : 167 .558 de gree

P HC1 : 3 1.832 de gree

13 C

suy0117 7

AcS H u p M eO D



17/20

S17

1

.897

5

4

.490

3

2

.128

6

1

.888

1

2

.156

6

0

.7

79

6

1

.000

0

0

.865

0

0

.970

2

0

.997

5

2

.253

6

0

.900

2

2

.357

9

0

.807

5

1

.999

8

2

.129

6

2

.125

1

0

.695

4

1

.9

49

2

6

.4

21

2

3

.1

27

2

2

.4

72

9

2

.507

3

2

.853

4

Inte

gral

7.

94

17

7

.9

253

7.

861

0

7

.8

421

7.

8333

7.

816

9

7

.5

042

7.

486

6

7.

15

63

7

.1

386

6.

824

7

6

.8

070

5.

968

6

5.

92

33

5

.9

119

5.

901

8

5.

89

05

5

.8

779

5.

866

5

5

.8

564

5.

845

1

5.

63

58

5

.6

282

5.

494

6

5

.4

883

5.

4038

5.

397

5

5

.3

811

5.

374

8

5.

30

05

5

.2

664

5.

207

2

5

.1

845

4.

878

1

4

.6

575

4.

649

9

4.

63

48

4

.6

272

4.

610

8

4

.5

970

4.

585

6

4.

34

48

4

.3

335

4.

322

1

4

.1

217

4.

1103

4.

099

0

4

.0

889

4.

070

0

4.

05

74

4

.0

322

4.

015

8

3

.4

724

3.

459

8

3.

44

59

3

.3

350

3.

332

5

3.

32

87

3

.2

707

3.

258

1

3

.2

455

3.

232

9

3.

09

04

2

.1

688

2.

002

4

1

.9

696

1.

8864

1.

883

9

(ppm)

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5


NA M E : suy0 120

EXP N O : 1

PR O C N O : 1*** Acquisit ion Parameters ***

BF1 : 50 0.13 000 00 M H z

L OCN U C : 2H

NS : 43

O 1 : 3 088 .51 Hz

PU LPRO G : zg30

SFO1 : 50 0.13 308 85 M Hz

SOLV EN T : M eOD

SW : 2 0.65 57 ppm


L B : 0 .30 Hz

PH C 0 : 3 50.4 49 degre e

PH C 1 : -2.7 45 d egre e

1H AV500

suy0120 1

NHAc dabcy l

173.

7454

1

72.

47

01

172

.0

839

1

72.

05

47

171

.82

88

170.

5

098

1

69.

58

43

158

.2

087

1

57.

22

49

156

.3

358

154.

5796

144.

7562

1

35.

69

07

133

.2

130

131.

9158

1

30.

42

19

130

.3

490

1

30.

25

43

129

.2

486

126.

4211

1

22.

99

61

118

.6

892

1

18.

1572

118.

11

35

1

12.

61

88

97.

84

73

9

7.

8109

7

6.

451

6

69.

44

11

6

8.

7561

68.

588

5

66.

73

75

6

3.

006

3

4

9.

510

1

49.

34

25

49.

1676

4

9.

000

0

48.

89

07

48.

8324

4

8.

657

5

48.

48

99

4

1.

865

6

40.

38

63

39.

4462

3

8.

309

4

30.

52

65

2

2.

4666

20.

652

1

20.

53

55

(ppm)

0102030405060708090100110120130140150160170180190


N A M E : suy 012 0

E XP NO : 2

P RO CN O : 1

*** Acquis i tion Parameters ***

B F1 : 12 5.7 577 890 M Hz

L O CN U C : 2H

N S : 440

O 1 : 1 320 4.57 H z

P ULPR O G : zg pg3 0

S FO1 : 12 5.7 709 936 M Hz

S OL VEN T : M e OD

S W : 2 38.7 675 p p m


L B : 1.00 H z

P HC0 : 3 25.536 de gree

P HC1 : 37 .276 d egree

13C AV500

suy0120 2

NHAc dabcy l



18/20

S18

2.

000

0

3.

962

4

2.

011

2

1

.6

505

2.

089

8

0.

848

3

0

.8

308

1

.0

737

1

.3

035

1

.1

079

1.

3903

2.

206

8

1.

699

7

1

.9

766

1

.0

633

1.

6137

2

.5

139

3.

0183

3.

3118

2.

9068

Inte

gral

7.

9994

7

.9

832

7.

906

9

7

.8

888

7.

8633

7.

848

5

7

.5

394

7.

523

4

7.

1734

7

.1

575

6.

8629

6

.8

451

6

.0

257

5.

677

6

5

.6

724

5.

547

0

5.

5377

5

.4

538

5.

431

0

5.

4271

4

.9

153

4.

720

5

4.

7156

4

.6

977

4.

692

8

4.

368

6

4

.1

653

4.

154

1

4.

1425

4

.1

318

4.

107

7

4

.0

929

4.

076

4

4.

0731

3

.9

845

3.

983

7

3

.9502

3.

9

497

3.

881

4

3

.8

490

3.

848

2

3.

8474

3

.8

274

3.

814

2

3

.8

010

3.

517

9

3.

508

6

3

.4

450

3.

409

3

3.

4063

3

.4

030

3.

400

0

3

.3

103

3.

151

7

2.

2336

2

.0

693

2.

025

7

1

.9170

(ppm)

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5


NAM E : suy0 117

EXPNO : 10

PRO C N O : 1


BF1 : 50 0.23 0000 0 M Hz

LOC N UC : 2 H

NS : 10 9

O1 : 22 59.3 7 Hz

PUL PRO G : zg

SFO1 : 50 0.23 2259 4 M Hz

SOL V EN T : M eOD

S W : 8.988 6 ppm


LB : 0.1 0 Hz

PHC 0 : 76 6.27 1 de gree

PHC 1 : 16.86 0 d egree

1H

suy0117 10

af t er deal ly l 40mg M eOD/CDCl3=10/1

173

.5893

1

71

.96

45

171

.9

427

1

71

.7387

169

.46

55

1

58

.01

20

157

.2

324

156

.2342

1

54

.42

73

144

.6

496

1

35

.40

38

132

.2

271

130

.1142

1

29

.22

53

126

.3

619

1

22

.9230

118

.0

560

112

.5

405

97.

7210

7

9.

324

0

79.

06

17

78.

7994

69.

3496

6

8.

759

4

68.

61

37

68.

4680

6

2.

857

8

4

9.

510

0

49.

33

51

49.

1676

4

9.

000

0

48.

82

51

48.

6576

4

8.

482

7

40.

40

26

26.

37

72

22.

5448

2

0.

686

9

20.

65

78

2

0.

5922

(ppm)

0102030405060708090100110120130140150160170180190


NAM E : suy 011 7E XP NO : 1 1

P RO CN O : 1


B F1 : 125.7 829 340 M H z

LO CN U C : 2H

NS : 5 441

O1 : 1 257 7.84 H z

P ULPR OG : zg pg3 0

S FO1 : 1 25.7 955 118 M Hz

S OL VEN T : M e OD

S W : 2 38.7 210 p pm

*** Processing Parameters ***

LB : 1.00 H z

P HC0 : 1 35.060 deg ree

P HC1 : 81 .169 d eg ree

13 C

suy0117 10

af t er deal ly l 40mg MeOD/CDCl3=10/1



19/20

S19

0.

454

3

2.

124

3

2.

4193

2.

8929

2.

4472

0.

7543

2.

8698

2.

448

7

2.

699

6

2.

486

4

2.

648

1

1.

648

8

0.

843

3

0

.4629

0.

7446

1.

0000

0.

9963

1.

4602

1.

3535

1

.1

784

2.

830

8

2.

516

9

3

.1

902

2

.2

033

4

.2

039

2

.8

612

6

.9

275

3.

2043

3.

9295

3.

1667

3.

7424

Integ

ral

7.

8978

7.

881

4

7

.8

650

7.

812

1

7.

7995

7

.7

944

7.

781

8

7

.7

629

7.

749

0

7.

7452

7

.6

545

7.

461

6

7.

4465

7

.4

250

7.

411

2

7

.1

212

7.

106

1

7.

0922

6

.8

022

6.

783

3

6

.7

644

6.

6749

6.

669

8

6

.6

661

6.

633

3

6.

6156

6

.5

299

6.

512

3

5.

8151

5.

811

3

5

.7

609

5.

753

3

5.

6033

5

.5

982

5.

457

0

5.

3700

5

.3

637

5.

346

1

4

.8

330

4.

6212

4

.5

972

4.

293

4

4.

084

1

4.

0614

4

.0

500

4.

023

6

4.

0084

3

.8

521

3.

835

7

3

.8

181

3.

802

9

3.

7639

3

.7

298

3.

713

4

3

.7

008

3.

6870

3.

347

8

3

.3

163

3.

312

5

3.

3100

3

.3

062

3.

192

8

3

.1

839

3.

075

5

2.

1451

2

.1

363

1.

976

1

1.

9724

1

.9

434

1.

845

0

1

.8

400

(ppm)

1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0

*** Cur rent Data Parame ters ***

N AM E : suy0 208

E XP NO : 1

P RO CN O : 1


B F1 : 500 .13 0000 0 M H z

LO C N UC : 2 H

N S : 2 4

O 1 : 30 88.5 1 Hz

P ULPR O G : zg30

S FO 1 : 500 .13 3088 5 M Hz

S OLVEN T : M eOD

S W : 20 .655 7 ppm


LB : 0.3 0 H z

P HC 0 : 26 4.98 0 d egre e

P HC 1 : -0.95 5 d egre e

1H AMX500

suy0208 1

A p rob e F l lin ke r c ou pl

1

73.

84

74

172

.9

074

172.

8709

1

72.

09

12

172

.0

547

1

71.

84

34

171

.5

228

171.

4207

1

71.

02

72

169

.6

208

1

68.

4329

1

68.

23

61

158

.2

451

157.

8371

1

57.

77

88

156

.3

577

1

54.

62

33

144

.74

89

137.

6

656

1

35.

61

78

131

.1

798

130.

5604

1

30.

42

19

130

.2

179

1

30.

07

94

129

.2

487

126

.4

212

1

22.

9815

118

.2

083

118.

1791

112

.6

261

1

03.

70

64

9

7.

7963

6

9.

4484

68.

763

4

68.

59

58

6

2.

991

8

55.

83

56

49.

51

74

49.

3425

4

9.

174

9

49.

00

00

4

8.

8324

48.

664

8

48.

48

99

4

0.

371

7

38.

45

52

37.

9159

3

0.

511

9

22.

45

94

2

0.

637

5

20.

593

8

20.

51

36

(ppm)

0102030405060708090100110120130140150160170180190


NA M E : suy02 08

EXP N O : 2

PR O C NO : 1

*** Acquisit ion Parameters ***BF1 : 12 5.75 7789 0 M Hz

L OCN U C : 2 H

NS : 115 99

O 1 : 132 04.5 7 Hz

PU LPRO G : zgpg 30

SFO1 : 12 5.77 0993 6 M Hz

SOLV EN T : M eOD

SW : 238 .767 5 p pm


L B : 1.00 H z

PH C 0 : 2 03.5 49 d egree

PH C 1 : 32.9 17 d egree

13C AMX500

suy0208 2

A p rob e F l lin ke r c ou pl



20/20

S20

5. References.

1. Q. P. Liu, G. Sulzenbacher, H. Yuan, E. P. Bennett, G. Pietz, K. Saunders, J. Spence,E. Nudelman, S. B. Levery, T. White, J. M. Neveu, W. S. Lane, Y. Bourne, M. L.Olsson, B. Henrissat and H. Clausen,Nat. Biotechnol., 2007, 25, 454-4640.


chem. commun., 2013,49, 7237-7239 suppinfo

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