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S1 A glyco-gold nanoparticle based high-throughput activity assay for α-2,8-polysialyltransferase from Neisseria meningitidis Ching-Ching Yu, a Li-De Huang, a David H. Kwan, b Warren W. Wakarchuk, c Stephen G. Withers,* b Chun-Cheng Lin* a Department of Chemistry, National Tsing Hua University, 101, Sec. 2, Kuang Fu Rd. Hsinchu, 30013, Taiwan Centre for High-throughput Biology (CHiBi) and Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada. § Department of Chemistry and Biology, Ryerson University, 350 Victoria St. Toronto, Ontario M5B 2K3 Canada. *To whom correspondence should be addressed E-mail: [email protected]; [email protected] Supporting Information Electronic Supplementary Material (ESI) for Chemical Communications This journal is © The Royal Society of Chemistry 2013
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Transcript of α-2,8-polysialyltransferase from · 2013-09-09 · S1 A glyco-gold nanoparticle based...

  • S1

    A glyco-gold nanoparticle based high-throughput activity assay for

    α-2,8-polysialyltransferase from Neisseria meningitidis

    Ching-Ching Yu,a Li-De Huang,a David H. Kwan,b Warren W. Wakarchuk,c Stephen G. Withers,*b Chun-Cheng Lin*a

    †Department of Chemistry, National Tsing Hua University, 101, Sec. 2, Kuang Fu Rd.

    Hsinchu, 30013, Taiwan ‡Centre for High-throughput Biology (CHiBi) and Department of Chemistry,

    University of British Columbia, 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada.

    §Department of Chemistry and Biology, Ryerson University, 350 Victoria St. Toronto, Ontario M5B 2K3 Canada.

    *To whom correspondence should be addressed

    E-mail: [email protected]; [email protected]

    Supporting Information

    Electronic Supplementary Material (ESI) for Chemical CommunicationsThis journal is © The Royal Society of Chemistry 2013

    mailto:[email protected]

  • S2

    Table of Contents General procedures………………………………………………….………………..S3

    Chemical synthesis experimental section and NMR data...........................................S4

    Protein cloning and overexpression.............................................................................S9

    Overexpression and purification of the truncated α-2,8-polysialyltransferase from

    Neisseria meningitidis (PST).......................................................................................S9

    Overexpression and purification of the bifunctional α-2,3/2,8-sialyltransferase from

    Campylobacter jejuni (Cst-IIΔ32).............................................................................S10

    Overexpression and purification of the α-2,3-sialyltransferase from Campylobacter

    jejuni (Cst-I)...............................................................................................................S10

    Overexpression and purification of the endo-sialidase double mutant from

    bacteriophage K1F (EndoNF-DM)............................................................................S10

    Synthesis of citrate-stabilized gold nanoparticles (AuNPs).......................................S12

    Fabrication of oligosaccharide conjugated gold nanoparticles..................................S12

    E. coli. cell lysate preparation....................................................................................S12

    Enzymatic polysialylation catalyzed by α-2,8-polysialyltransferase and detection on

    oligosaccharide-assembled gold nanoparticles..........................................................S13

    Figure S1. The stability test for the oligosaccharide functionalized gold

    nanoparticles.......................................................................................S14

    Figure S2. Control experiment of EndoNF-DM-mediated assembly of AuNPs for

    PST activity assay....................................................................................S14

    Figure S3. Optimal concentration of EndoNF-DM for PST activity

    assay…………………………………………………………..............S15

    Figure S4. Time course analysis of PST activity with GD3 functionalized gold

    nanoparticle assay...................................................................................S15

    Scheme S1. Synthesis of LacPEG6C10SH...................................................................S4

    S c h e m e S 2 . S y n t h e s i s o f G M 3 P E G 6 C 1 0 S H a n d

    G D 3 P E G 6 C 1 0 S H . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S 7 1H spectra for compounds 1, 2, 3, 5, and 6.........................................................S17-S21

    Electronic Supplementary Material (ESI) for Chemical CommunicationsThis journal is © The Royal Society of Chemistry 2013

  • S3

    General procedure: Chemical synthesis:

    Solvents and chemicals were of analytical grade and were purchased from the Sigma‐Aldrich company. N,N‐Dimethylformamide and triethylamine were dried and stored over 4Å molecular sieves. Pyridine was dried over potassium hydroxide. All other organic solvents were distilled prior to use in reactions. All reactions were carried out in oven-dried glassware under an atmosphere of argon unless indicated otherwise. All solvents were dried and distilled by standard techniques. Analytical techniques:

    Analytical thin‐layer chromatography (TLC) was performed on Merck Silica Gel 60F254 glass plates. TLC plates were visualized using UV light (254 or 365 nm) and/or by immersion in either 10% ammonium molybdate in 2 M H2SO4, 5% (v/v) H2SO4 in ethanol, 0.3% (w/w) p-anisaldehyde and 0.7 M H2SO4 in ethanol or 0.2% (w/v) ninhydrin in ethanol followed by heating. Silica gel flash column chromatography was conducted on Silicagel E60 (Merck, 40-63 µm). Reversed‐phase silica gel chromatography was performed using Waters Sep‐Pak® tC18 cartridges with various sizes. Cartridges were preconditioned by washing with 5 bed volumes of methanol followed by 5 bed volumes of 50% methanol in deionized water and then 5 bed volumes of deionized water (For water/methanol gradient elution – see experiments for specific information). Chromatography on Bio-gel P-2 (1.5 x 50 cm) was conducted in deionized water at a flow rate of 10 mL∙hour-1 unless otherwise noted. Alternative chromatographic techniques are described in detail in the experimental section. 1H and 13C Nuclear Magnetic Resonance spectra were recorded at the National Tsing-Hua University NMR facility, using Bruker AV-400 MHz and DMX-600 MHz. All spectra were recorded using an internal lock (deuterium) and are referenced to a residual solvent peak. Chemical shifts are expressed in ppm using residual CDCl3 (7.24 ppm), CD3OD (3.31 ppm) or D2O (4.67 ppm at 298 k) as internal standard. 1H and 13C chemical shifts are quoted in parts per million (ppm) downfield of tetramethylsilane, and chemical shifts (δ) are rounded to the nearest 0.1 ppm unless increased precision was required to distinguish resonances. Low-resolution and high-resolution mass spectra were recorded under ESI-TOF Mass spectra conditions. Transmission electron microscopy spectra were performed on a JEOL microscope (Model JEOL JEM-2100) that operated at 200 kV.

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  • S4

    Chemical synthesis experimental section and NMR data:

    OO O

    HO

    HOOH

    OH

    HOO

    ON3NH

    O

    O

    O2N

    H2NO

    NH2 BocHNO

    NH 10

    4 5

    6

    7 9

    2

    a, b

    c-e

    f-i

    j-l

    8

    HO

    OH

    OHOH

    OO

    OAc

    AcO

    AcO

    AcO OOAc

    AcOOAc

    OO

    2

    OO

    OH

    OH

    HO

    HO OOH

    HOOH

    OHN

    HNO

    OHN

    O2 2 O10

    N3

    2 2

    OBr

    ONH 102

    OSAc

    SH

    Scheme S1. Synthesis of LacPEG6C10SH. Reagents and conditions: (a) 4, Boc2O, DCM; (b) EDCI, HOBt, TEA, 11-Bromoundecanoic acid, DCM, 72% for two steps; (c) KSAc, DMF; (d) TFA, DCM; (e) NPC, TEA, DCM, 46% for three steps; (f) Ac2O, Pyr.; (g) H2NNH2-HOAc, DMF, 71% for two steps; (h) CCl3CN, Cs2CO3, DCM; (i) TMSOTf, 8, 4Å MS, DCM, 65% for two steps; (j) Pd/C, H2, MeOH; (k) 6, TEA, DCM; (l) NaOMe, MeOH, 42% for three steps.

    As shown in Scheme S1, the linker 6 was synthesized from diamine 4 in which one of the amino groups was selectively protected with Boc anhydride and then conjugated with 11-bromoundecanoic acid to give compound 5. Displacement of the bromide in 5 using KSAc was followed by removal of the Boc group and then reaction with 4-nitrophenyl chloroformate (NPC) to yield carbamate spacer 6 in a total 46% yield for five steps. The synthesis of LacPEG6C10SH 2 started from 7. Glycosylation of triethylene glycol spacer 8 with lactosyl imidate1 afforded β-lactoside 9.2 Hydrogenation of the azide in 9 yielded an amine derivative, which was reacted with spacer 6. The resulting product was hydrolyzed under basic conditions to yield the desired product 2 in a total yield of 27% over four steps.

    BocHNO

    OHN

    O

    Br

    Tert-butyl [2-(2-{2-[(11-bromoundecanoyl)amino]ethoxy}ethoxy)ethyl] carba- mate (5). To a solution of 2,2’-(ethylenedioxy)-bis-ethylamine (4.80 g, 33 mmol) in dry CH2Cl2 (20.0 mL) was added a solution of di-tert-butyldicarbonate (2.40 g, 11

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  • S5

    mmol) in CH2Cl2 (20.0 mL) dropwise at 0 oC over 3 h under inert atmosphere. After additional stirring for 2 h at room temperature, the reaction solution was washed with water and brine, dried over anhydrous MgSO4, and concentrated to yield yellow oil {2-[2-(2-Amino-ethoxy)-ethoxy]ethyl}-carbamic acid t-butyl ester. The obtained t-butyl ester and 11-bromo-undecanoic acid (2.32 g, 8.7 mmol) were dissolved in 60 mL. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI; 3.13 g, 16.4 mmol), hydroxybenzotriazole (HOBt; 1.47 g, 10.9 mmol), and triethylamine (1.5 mL, 10.8 mmol) were subsequently added. The reaction mixture was concentrated under reduced pressure after being stirred for 12 h. The crude product was re-dissolved in ethyl acetate and washed with a 1 N HCl aqueous solution, saturated NaHCO3 solution, and water. The organic layer was dried over MgSO4, concentrated, and subjected to a flash silica gel column chromatography to give 5 (3.92 g, 7.9 mmol, 72% yield). Rf = 0.17 (2:1 EtOAc–hexane); 1H NMR (400 MHz, CDCl3) δ 6.41 (br s, 1H), 5.14 (br s, 1H), 3.36 (br s, 4H), 3.26-3.32 (m, 4H), 3.11-3.21 (m, 4H), 2.99-3.09 (m, 2H), 1.94 (t, J = 7.5 Hz, 2H), 1.54-1.64 (m, 2H), 1.31-1.42 (m, 2H), 1.11-1.26 (m, 12H), 1.03 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 172.8, 155.4, 78.4, 69.6, 69.5 (x2), 69.3, 39.7, 38.5, 35.9, 33.4, 32.2, 28.8 (x2), 28.7, 28.7, 28.1, 27.8, 27.5, 25.2; HRMS (ESI) m/z calculated for C22H43N2O5BrNa 517.2253 [M+Na]+, found 517.2256.

    NH

    OO

    HN

    O

    O

    O

    O2N

    SAc

    4-nitrophenyl N-{9-[11-(acetylthio)decanoyl]-12-oxo-3,6-dioxa-9-azadecanoyl} carbamate (6). KSAc (1.40 g, 12.3 mmol) and 5 (3.00 g, 6.1 mmol) were dissolved in DMF (40 mL) and the mixture was stirred at rt for overnight. After the completion of the reaction (checked by TLC), the solution was evaporated in vacuo and then diluted with EtOAc and the resulting organic layer was washed with water and brine. The organic layer was dried over MgSO4 and concentrated. The resulting thioacetate product was directly subjected to the next reaction. The obtained crude product was dissolved in 20% TFA in 70 mL CH2Cl2 and the mixture was stirred for 2 h. The reaction mixture was concentrated in vacuo and the residue was used without further purification. The above product was dissolved in CH2Cl2 (60 mL) and the mixture was cooled to 0 °C. After 10 min, 4-nitrophenylchloroformate (1.22 g, 6.1 mmol) and triethylamine were added sequentially. After being stirred for 30 min at 0 °C, the solution was washed by water, saturated NaHCO3 solution, 10% aqueous HCl, brine, and dried with MgSO4. The mixture was concentrated and subjected to a short pad of silica gel to give product 6 in 46% (1.55 g, 2.8 mmol) for three steps. Rf = 0.27 (3:1 EtOAc–hexane); 1H (400 MHz, CDCl3) δ 8.18 (d, J = 9.0, 2H), 7.26 (d, J = 9.0, 2H),

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  • S6

    6.00 (t, J = 5.1, 1H), 5.95 (t, J = 5.4, 1H), 3.55-3.65 (m, 6H), 3.48-3.55 (m, 2H), 3.37-3.48 (m, 4H), 2.79 (t, J = 7.4, 2H), 2.26 (s, 3H), 2.10 (t, J = 7.6, 2H), 1.44-1.60 (m, 4H), 1.13-1.34 (m, 12H); 13C (100 MHz, CDCl3) δ 196.0, 173.3, 158.2, 155.9, 153.2, 144.6, 125.0, 121.9, 70.2, 70.1, 69.9, 69.5, 41.0, 39.0, 36.6, 30.5, 29.3, 29.2, 29.2, 29.0, 28.9, 28.6, 25.6; HRMS (ESI) m/z calculated for C26H41N3O8SNa 578.2512 [M+Na]+, found 578.2520.

    OO

    OH

    OH

    HO

    HO OOH

    HOOH

    OHN

    HNO

    OHN

    O2 2 OSH10

    N-{19-[(O-β-D-galactopyranosyl-(1→4)-O-β-D-glucopyranosyl)oxy]-10-oxo-3,6,14, 17-tetraoxa-9,11-diazanonadec-1-yl}-11-mercapto-undecamide (2). Lactoside 9 (1.00 g, 1.3 mmol) was dissolved in MeOH (10 mL). The solution was stirred with a catalytic amount of 10% palladium on carbon (200 mg) under hydrogen atmosphere for 6 h. The solid was filtered off, washed with methanol, and the filtrate was concentrated under reduced pressure to give corresponding amine. To a solution of the above product in CH2Cl2 (50 mL), triethylamine (0.36 mL, 2.6 mmol), and compound 6 (720 mg, 1.3 mmol) were added. The reaction mixture was stirred at room temperature for 30 min, and then washed by water, saturated NaHCO3 solution, 10% aqueous HCl, and brine. The organic layer was concentrated and the resulting residue was subjected to deacetylation directly without further purification. The crude product was dissolved in MeOH/H2O (1/1, 10 mL). NaOH aqueous solution (1 N, 1 mL) was added and the solution was stirring for 30 min. The reaction mixture was neutralized by addition of 1 N HCl aqueous solution, concentrated in vacuo, and purified by column chromatography on silica gel to give desired pure product 2 in 42% (0.45 g,

    0.53 mmol). Rf = 0.60 (1:5 MeOH–CH2Cl2); ][ 33Dα +1.32 (c = 0.5, water); 1H NMR

    (400 MHz, D2O) δ 4.37 (d, J = 7.9 Hz, 1H), 4.31 (d, J = 7.7 Hz, 1H), 3.89-3.99 (m, 1H), 3.84 (br d, J = 11.6 Hz, 1H), 3.79 (br d, J = 2.4 Hz, 1H), 3.33-3.75 (m, 26H), 3.09-3.33 (m, 7H), 2.56 (t, J = 7.2 Hz, 2H), 2.10 (t, J = 7.3 Hz, 2H), 1.35-1.6 (m, 4H), 1.1-1.35 (m, 12H); 13C NMR (100 MHz, CD3OD) δ 173.6, 158.3, 101.2, 100.4, 76.7, 73.6, 73.0, 72.6, 71.1, 70.8, 69.2, 68.2, 68.1, 67.9 (x4), 67.7, 67.4, 66.9, 66.8, 59.3, 58.3, 37.7 (x2), 37.2 (x2), 34.2, 28.0 (x2), 27.9, 27.8, 27.5 (x2), 26.9, 24.1; HRMS (ESI) m/z calculated for C36H69N3O17SNa 870.4245 [M+Na]+, found 870.4259.

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  • S7

    OO

    OH

    OH

    HO

    OO

    -OOC

    HO

    OOH

    HOOH

    AcHNHO OH

    HO

    O

    2 3

    1

    a

    b

    OO

    OH

    OH

    HO

    OO

    -OOC

    HO

    OOH

    HOOH

    AcHNO OH

    HO

    O

    O

    -OOC

    HOAcHN

    HO OH

    HO

    OHN

    HN

    OHN

    O2 2 OSH10

    OHN

    HN

    OHN

    O2 2 OSH10

    Scheme S2. Synthesis of GM3PEG6C10SH and GD3PEG6C10SH. Reagents and conditions: (a) HEPES 50 mM, pH 7.5, MgCl2 20 mM, CMP-sialic acid, Cst-I, alkaline phosphatase, 37 oC, 85% (b) HEPES 50 mM, pH 7.5, MgCl2 20 mM, CMP-sialic acid, Cst-II, alkaline phosphatase, 37 oC, 35%.

    The LacPEG6C10SH 8 was then sialylated using α-2,3-sialyltransferase Cst-I and bifunctional α-2,3/2,8-sialyltransferase Cst-II, respectively, to afford ganglioside analogs GM3PEG6C10SH 3 and GD3PEG6C10SH 1 (Scheme S2). Compound 3 was obtained (in 85% isolated yield) by use of 1.5 equiv. of CMP-sialic acid with the mono-specific sialyltransferase Cst-I. Compound 1 was obtained as the major product along with mono-(9), tri-(GT3), and oligo-sialyl lactosides, by use of the dual specificity Cst-II and 5 equiv. of CMP-sialic acid. A combination of gel filtration chromatography on BioGel P-2 gel and C18 reverse phase silica column allowed purification of GD3PEG6C10SH 1 in 35% isolated yield.

    OO

    OH

    OH

    HO

    OO

    -OOC

    HO

    OOH

    HOOH

    AcHNHO OH

    HO

    OHN

    HNO

    OHN

    O2 2 OSH10

    N-{19-[(O-(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonic acid)-(2 → 3)-O-β-D-galactopyranosyl-(1 → 4)-O-β-D-glucopyranosyl)oxy]-10- oxo-3,6,14,17-tetraoxa-9,11-diazanonadec-1-yl}-11-mercapto-undecamide (3). A buffered (50 mM HEPES, pH 7.5) solution (12 mL) of 2 (50 mg, 59 µmol) containing manganese chloride (20 mM) was incubated at 37 oC with CMP-N-acetyl neuraminic acid (75 mg, 118 µmol) in the presence of Cst-I (20 mg/mL, 50 µL) and alkaline phosphatase (1 units/µL, 10 µL) for 12 h. The reaction was quenched by addition of an equal volume of EtOH when TLC analysis (EtOAc:MeOH:H2O:HOAc=4:2:1:0.5) indicated completion of reaction. The reaction solution was centrifuged (10,000 x g, 10 min) to remove proteins and insoluble precipitates. The supernatant was filtered (0.45-µm filter; Millipore), concentrated, and passed through a BioGel P-2 gel

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  • S8

    (packed in BioRad column #737-1576 1.5 cm x 50 cm) with water (10 mL/h) to obtain the desired product. Product-containing fractions were identified by TLC analysis, and were pooled together then lyophilized. The mixture of oligomers obtained was purified by C18 reverse phase silica column (Sep-Pak Vac tC18 cartridge 20cc/5g 37-55µm, Waters) and compound 3 was eluted out by 40% → 70% MeOH. Product-containing fractions were confirmed by TLC analysis and were pooled together followed by lyophilization. Rf = 0.25 (EtOAc:MeOH:H2O:HOAc=

    4:2:1:0.5); ][ 33Dα -0.62 (c = 0.5, water); 1H NMR (600 MHz, D2O) δ 4.42 (d, J = 8.3

    Hz, 1H), 4.41 (d, J = 8.2 Hz, 1H), 4.01 (dd, J = 3.1 and J = 9.9 Hz, 1H), 3.96 (dt, J = 11.5 Hz, J = 4.2 Hz and J = 4.3 Hz, 1H), 3.90-3.84 (m, 2H), 3.81-3.69 (m, 5H), 3.68-3.44 (m, 27H), 3.28 (t, J = 5.4 Hz, 2H), 3.24 (dd, J = 8.6 Hz, 1H), 3.21 (t, J = 5.5 Hz, 4H), 2.65 (dd, J = 12.5 and J = 4.7 Hz, 1H), 2.43 (t, J = 7.1 Hz, 2H), 2.14 (t, J = 7.3 Hz, 2H), 1.93 (s, 3H), 1.70 (t, J = 12.2 Hz, 1H), 1.49 (m, 4H), 1.27 (m, 2H), 1.18 (m, 10H); 13C NMR (150 MHz, D2O) δ 176.80, 174.82, 173.57, 160.16, 102.50, 101.95, 99.57, 78.12, 75.31, 74.96, 74.60, 74.12, 72.71, 72.62, 71.52, 69.65, 69.50, 69.45, 69.36, 69.33, 69.24, 69.18, 68.77, 68.53, 68.10, 67.92, 67.29, 39.41, 39.25, 38.71, 35.60, 33.00, 28.56, 28.35, 28.16, 28.10, 27.47, 25.23, 23.72, 21.85; HRMS (ESI) m/z calculated for C47H86N4O25S 1137.5224 [M-H]-, found: 1137.5222.

    OO

    OH

    OH

    HO

    OO

    -OOC

    HO

    OOH

    HOOH

    AcHNO OH

    HO

    OHN

    HNO

    OHN

    O2 2 OSH10

    O

    -OOC

    HOAcHN

    HO OH

    HO

    N-{19-[(O-(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonic acid)-(2 → 8)-O-(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulo -pyranosylonic acid)-(2 → 8)-O-(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto -2-nonulopyranosylonic acid)-(2 → 3)-O-β-D-galactopyranosyl-(1 → 4)-β-D-gluco -pyranosyl)oxy]-10-oxo-3,6,14,17-tetraoxa-9,11-diazanonadec-1-yl}-11-mercapto-undecamide (1). A buffered (50 mM HEPES, pH 7.5) solution (24 mL) of 2 (100 mg, 118 µmol) containing manganese chloride (20 mM) was incubated at 37 oC with CMP-N-acetyl neuraminic acid (375 mg, 590 µmol) in the presence of Cst-II (10 mg/mL, 50 µL) and alkaline phosphatase (1 units/µL, 50 µL) for 12 h. The reaction was quenched by addition of same amount of EtOH when TLC analysis (EtOAc:MeOH:H2O:HOAc=4:2:1:0.5) indicated completion of reaction. The reaction solution was centrifuged (10,000 x g, 10 min) to remove proteins and insoluble precipitates. The supernatant was filtered (0.45-µm filter; Millipore), concentrated and passed through a BioGel P-2 gel (packed in BioRad column #737-1576 1.5 cm x

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  • S9

    50 cm) with water (10 mL/h) to obtain the desired product. Product-containing fractions were identified by TLC analysis and were pooled together followed by lyophilization. The mixture of oligomers obtained was purified by C18 reverse phase silica column (Sep-Pak Vac tC18 cartridge 20cc/5g 37-55µm, Waters) and compound 1 was eluted by 20% → 45% MeOH. Product-containing fractions were confirmed by TLC analysis and were pooled together followed by lyophilization. Rf = 0.15

    (EtOAc:MeOH:H2O:HOAc=4:2:1:0.5); ][ 33Dα -0.84 (c = 0.5, water); 1H NMR (600

    MHz, D2O) δ 4.39 (d, J = 7.9 Hz, 1H), 4.38 (d, J = 8.0 Hz, 1H ), 4.04 (dd, J = 3.7 and J = 12.1 Hz, 1H), 4.02-3.99 (m, 1H), 3.98-3.96 (m, 2H), 3.89-3.82 (m, 2H), 3.79-3.66 (m, 7H), 3.65-3.41 (m, 29H), 3.25 ( t, J = 5.3 Hz, 2H), 3.21 (dd, J = 9.1 Hz and J = 8.1 Hz, 1H), 3.18 (t, J = 5.4 Hz, 4H), 2.64 (dd, J = 12.4 and J = 4.6 Hz, 1H), 2.54 (dd, J = 12.3 and J = 4.4 Hz, 1H), 2.41 (t, J = 7.2 Hz, 2H), 2.12 (t, J = 7.3 Hz, 2H), 1.93 (s, 3H), 1.91 (s, 3H), 1.61 (t, J = 12.2 Hz, 2H), 1.46 (m, 4H), 1.24 (m, 2H), 1.15 (m, 10H); 13C NMR (150 MHz, D2O) δ 177.25, 174.86, 173.36, 173.21, 160.28, 102.58, 102.01, 100.38, 100.07, 78.06, 77.95, 75.33, 75.08, 74.70, 74.11, 73.88, 72.71, 72.53, 71.59, 69.67, 69.55, 69.49, 69.40, 69.34, 69.29, 69.23, 69.16, 68.77, 68.58, 68.35, 68.03, 67.80, 67.33, 62.46, 61.44, 60.95, 59.90, 59.33, 52.16, 51.64, 40.38, 39.57, 39.31, 38.75, 35.62, 32.85, 28.38, 28.17, 27.98, 27.93, 27.32, 25.19, 23.66, 22.19, 21.90; HRMS (ESI) m/z calculated for C58H103N5O33S: 1428.6178 [M-H]-, found: 1428.6173. Protein cloning and overexpression: Overexpression and purification of the truncated α-2,8-polysialyltransferase from Neisseria meningitidis (PST). The amplification of pstNM from chromosomal DNA of N. meningitidis 992B and subsequent cloning into pCWmalE-thrombin were reported by Wakarchuk et al.3 The construct used in this study expresses a full length PSTNM fused to the C-terminus of maltose binding protein (MalE) and Escherichia coli BL21 (DE3) was used as the expression host. The recombinant E. coli strain BL21 (DE3) carrying the PSTNM-encoding plasmid was grown in LB broth containing ampicillin (100 µg/mL) at 37 °C for 2 h. Gene expression was induced with 0.5 mM isopropyl-thio-α-D-galactopyranoside and grown at 20 °C for 16-20 h. The cells overexpressing MalE-PSTNM were resuspended in 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, and lysed by French Press in the presence of a protease inhibitor mixture pellet (Roche Applied Science) and DNaseI (20 µg/mL). The lysate was centrifuged at 27000 x g for 30 min at 4 °C to remove cell debris. The supernatant was applied to amylose resin (New England Biolabs) equilibrated in 20 mM Tris-HCl (pH 7.5 with 500 mM NaCl). MalE-PSTNM was eluted with 10 mM maltose in 20

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  • S10

    mM Tris-HCl (pH 7.5 with 500 mM NaCl) and the fractions were analyzed by SDS-PAGE. Fractions with significant amounts of MalE-PSTNM were pooled. If the crude cell lysate was used without further purification, extreme care was taken to keep ratio of E. coli cell weight to re-suspension buffer volume constant (1 g cell / 20 mL buffer) to ensure reproducible amount of PST present in the lysate. Overexpression and purification of the bifunctional α-2,3/2,8-sialyltransferase from Campylobacter jejuni (Cst-IIΔ32). Cloning of the truncated version of Cst-II (32 residue C-terminal deletion) from Campylobacter jejuni strain OH4384 in pET-41a vector (Novagen) with an N-terminal hexahistidine tag was done as previously reported.4 Cst-II was expressed in Escherichia coli BL21 (DE3). The recombinant E. coli strain BL21 (DE3) carrying the Cst-II-encoding plasmid was grown in LB broth containing ampicillin (100 µg/mL) at 37 °C for 2 h. Gene expression was induced with 0.5 mM isopropyl-thio-α-D-galactopyranoside and grown at 20 °C for 20 h. The cells overexpressing Cst-II were resuspended in 20 mM Tris-HCl (pH 7.5 with 500 mM NaCl) and lysed by French Press in the presence of a protease inhibitor mixture pellet (Roche Applied Science) and DNaseI (20 µg/mL). The lysate was centrifuged at 27000 x g for 30 min at 4 °C to remove cell debris. The supernatant of a cell lysate was chromatographed on a 1-mL HisTrap FF column (GE Healthcare) with a linear gradient of 0-100% using the following buffers: A, Tris-HCl 25 mM, NaCl 500 mM, imidazole 10 mM, pH 7.5; B, Tris-HCl 25 mM, NaCl 500 mM, imidazole 500 mM, pH 7.5, and the fractions were analyzed by SDS-PAGE. Fractions with significant amounts of Cst-II were pooled. Overexpression and purification of the α-2,3-sialyltransferase from Campylobacter jejuni (Cst-I). The Cst-I from Campylobacter jejuni strain OH4384 was cloned in pET-28a vector (Novagen) with an N-terminal hexahistidine tag and and subsequently transformed into Escherichia coli BL21 (DE3). The overexpression and purification were the same as Cst-II purification mentioned above except using kanamycin as antibiotics. Overexpression and purification of the endo-sialidase double mutant from bacteriophage K1F (EndoNF-DM). Endosialidase from bacteriophage K1F (EndoNF), carrying an E581A mutation was cloned from a vector, pCW-MalE-EndoNF(E581A), which encoded EndoNF E581A mutant as C-terminal fusion to maltose-binding protein (linked by a thrombin cleavage site).5 The DNA encoding amino acid residues 246-1065 of EndoNF E581A, along with the upstream thrombin cleavage site was PCR amplified using the primers GCG TCG ACA GCG

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    GGG GTG GCG GAC ATA TCT TTA ACC and GCC GTA GCC TCG AGT TAC TTC TGT TCA AGA GCA GAA AGT C. The PCR product was digested with SalI and XhoI restriction enzymes and ligated to pET-15b-GFP which had been cut with XhoI. The resulting plasmid, pET15-GFP(L17)EndoNF(E581A) in which the gene had been inserted in the correct orientation encoded the EndoNF E581A mutant as an C-terminal fusion linked to GFP by a 17-amino acid linker sequence including a thrombin-cleavage site. A second mutation was required to completely abolish hydrolytic activity of EndoNF,6 and pET15-GFP(L17)EndoNF(E581A) was used as a template for site-directed mutagenesis using primers TAT ACC TTA TCA CTG CTG GCA CTC GTG GTG AT and ATC ACC ACG AGT GCC AGC AGT GAT AAG GTA TA to introduce the mutation R596A. The resulting plasmid pET15-GFP(L17)EndoNF(E581A+R596A) encoded the GFP-fused endosialidase double mutant, which was used in other relative work, was only used as parent plasmid of endosialidase double mutant in this study.

    The endosialidase double mutant was also cloned into pET-15b without a GFP fusion partner. The DNA encoding amino acid residues 246-1065 of EndoNF E581A+R596A was excised from pET15-GFP(L17)EndoNF(E581A+R596A) by digestion with NdeI and XhoI restriction enzymes, and the fragment was ligated to pET-15b cut with the same enzymes. The resulting plasmid pET15- EndoNF-(E581A+R596A) encoded endosialidase double mutant with an N-terminal hexa-histidine tag in the absence of any other fusion partner.

    This clone was expressed in Escherichia coli BL21 (DE3) by the same method. The recombinant E. coli strain BL21 (DE3) carrying either the plasmid encoding the GFP-fused EndoNF double mutant, or the plasmid encoding the EndoNF without a fusion partner was grown in LB broth containing ampicillin (100 µg/mL) at 37 °C for 2 h. Gene expression was induced with 0.5 mM isopropyl-thio-α-D-galactopyranoside and grown at 20 °C for 20 h. The cells overexpressing protein were resuspended in 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, and lysed by French Press in the presence of a protease inhibitor mixture pellet (Roche Applied Science) and DNaseI (20 µg/mL). The lysate was centrifuged at 27000 x g for 30 min at 4 °C to remove cell debris. The supernatant of a cell lysate was chromatographed on a 1-mL HisTrap FF column (GE Healthcare) with a linear gradient of 0-100% using the following buffers: A, Tris-HCl 25 mM, NaCl 500 mM, imidazole 10 mM, pH 7.5; B, Tris-HCl 25 mM, NaCl 500 mM, imidazole 500 mM, pH 7.5, and the fractions were analyzed by SDS-PAGE. Fractions with significant amounts of protein were pooled. Synthesis of citrate-stabilized gold nanoparticles (AuNPs). Citrate-stabilized gold nanoparticles of 13 nm were prepared according to the Frens method.7 All glassware

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    were cleaned with piranha solution (volume ratio 3:1, H2SO4/H2O2), and rinsed thoroughly with Millipore-Q water prior to use. 190 mL of aqueous solution of HAuCl4 (72 µmol) was brought to a rolling boil with vigorous stirring. Rapid addition of 10 mL of 1% sodium citrate solution (340 µmol) resulted in a color change from pale yellow to deep blue within 30 sec. Boiling was continued for another 15 min after the final color changed to burgundy and then gradually cooled to room temperature. The resulting solution of colloidal particles was stored at 4 oC for future functionalization. Fabrication of oligosaccharide conjugated gold nanoparticles. Five milligrams of the thiolated oligosaccharides were added to 200 mL of citrate-stabilized AuNPs, in which the solution basicity was pre-adjusted to pH 11 by 0.5 N NaOH, and the solution was left to self-assemble for 20 h. Excess oligosaccharide was removed via centrifugation. The UV-visible spectrum of the AuNP clearly showed characteristic plasmon absorption bands at 520 nm. The red-shift of the band to 525 nm after addition of oligosaccharide indicated the successful replacement of citrate molecules to oligosaccharide on surface of AuNPs. The sizes of gold nanoparticles were verified through transmission electron microscope analysis with an average size of 13 ± 1.2 nm (100 particles sampled). Spherical nanoparticles are the most abundant (80%) component of the mixture, followed by around 10% of triangular prisms and 10% of other shapes including cubes and rods. Transmission electron microscopy was performed to characterize the size and dispersion of the AuNPs. A drop of the AuNP solution (1 µL) was placed on a carbon-coated 200-mesh copper grid. The grid was left to dry at room temperature for several hours, and then subsequently dried under vacuum overnight and analyzed. Mean particle size and standard deviation were determined by measuring at least 100 particles. E. coli cell lysate preparation. E. coli strain BL21 (DE3) bearing pTXB1 (NEB) was grown in LB broth containing ampicillin (100 µg/mL) at 37 °C for 2 h. Gene expression was induced with 0.5 mM isopropyl-thio-α-D-galactopyranoside and grown at 20 °C for 16-20 h. The cells were resuspended in 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, and lysed by ultrasonication in the presence of a protease inhibitor mixture pellet (Roche Applied Science) and DNaseI (20 µg/mL). The lysate was centrifuged at 27000 x g for 30 min at 4 °C to remove cell debris. The resulting supernatant was collected and used as cell lysate without PST. Enzymatic polysialylation catalyzed by α-2,8-polysialyltransferase and detection on oligosaccharide-assembled gold nanoparticles. The oligosaccharide-AuNPs (5

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    nM, calculated the concentration of the AuNPs in solution using the molar extinction coefficient 2×108 M−1 cm−1 at 520 nm) were incubated either in 1.5 mL micro centrifugation tube or regular 96-well micro-titer plates with CMP-sialic acid (1 mM) and PST (10 µL of desired concentration for purified PST, 10% cell lysate diluted with reaction buffer used in both EndoNF-DM optimal concentration experiment and time course PST assay), and the enzymatic reaction was allowed to proceed at 37 oC for 1 h in 50 mM Na HEPES pH 7.5 with 20 mM MgCl2, adjusted the final volume to 100 µL. Individual experiment contained negative controls: negative controls of oligosaccharide-AuNP incubation with CMP-sialic acid only (no enzyme) and of oligosaccharide-AuNP incubation with enzyme only.

    Subsequently, the EndoNF DM was added to the reaction solution directly with final protein concentration at 0.03-300 µg/mL, adjusted the solution to 110 µL and reacted at room temperature for 30 min. After centrifugation (1000 x g) for 3 min at room temperature, the supernatant was transferred to regular 96-well microplates followed by measuring the absorbance at 525 nm using ELISA plate reader (SpectraMax M2e microplate reader, Molecular Devices).

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    Figure S1. The stability test for the oligosaccharide functionalized gold nanoparticles (A) The UV-visible absorption spectra of gold nanoparticle functionalized with sodium citrate, lactose, GM3 and GD3, respectively, before and after addition of sodium chloride (0.1 M) to examine the resistance of high ionic strength environment; (B) Photographs of visual color change of the functionalized gold nanoparticles after addition of sodium chloride (0.1 M) for 2 h; (C) Photographs of visual color change of the glyco-gold nanoparticles dispersed in HEPES 50 mM, MgCl2 20 mM, pH 7.5 for 24 h; (D) Photographs of visual color change of the glyco-gold nanoparticles dispersed in Tris 20 mM, NaCl 500 mM, pH 7.5, containing 50% PST crude lysate for 24 h.

    Figure S2. Control experiment of EndoNF-DM-mediated assembly of AuNPs for PST activity assay. In the control experiments, either CMP-sialic acid or PST was not included in the [email protected] solution during the incubation step, followed by the

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    addition of EndoNF-DM; a) control: no CMP-sialic acid and PST; b) no CMP-sialic acid; c) no PST.

    Figure S3. Optimal concentration of EndoNF-DM for PST activity assay. (A) The absorption spectra of GD3-functionalized gold nanoparticle solutions incubated for 30 minutes with fixed concentrations of PST and CMP-sialic acid, followed by addition of different amounts of EndoNF-DM (from 0 to 270 µg/mL); the inset shows the correlation plot of the A525nm versus the EndoNF-DM concentration. (B) Photographs of visual color fading of the AuNPs (5 nM in HEPES 50 mM, pH 7.5, MgCl2 20 mM buffer solution) containing different amounts of EndoNF-DM (from 0 to 270 µg/mL).

    Figure S4. Time course analysis of PSA formation on GD3 functionalized gold nanoparticle assay. The analysis of successive reaction time points shows the gradual

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    decrease of the absorbance of gold nanoparticles, indicating the formation of polysialic acid products. References: 1. Filice, M.; Palomo, J. M.; Bonomi, P.; Bavaro, T.; Fernandez-Lafuente, R.; Guisan,

    J. M.; Terreni, M. Tetrahedron 2008, 64, 9286. 2. Kato, H.; Uzawa, H.; Nagatsuka, T.; Kondo, S.; Sato, K.; Ohsawa, I.;

    Kanamori-Kataoka, M.; Takei, Y.; Ota, S.; Furuno, M.; Dohi, H.; Nishida, Y.; Seto, Y. Carbohydr. Res. 2011, 346, 1820.

    3. Willis, L. M.; Gilbert, M.; Karwaski, M.-F.; Blanchard, M.-C.; Wakarchuk, W. W. Glycobiology 2008, 18, 177.

    4. Chiu, C. P. C.; Watts, A. G.; Lairson, L. L.; Gilbert, M.; Lim, D.; Wakarchuk, W. W.; Withers, S. G.; Strynadka, N. C. J. Nat. Struct. Mol. Biol. 2004, 11, 163-170.

    5. Morley, T. J.; Willis, L. M.; Whitfield, C.; Wakarchuk, W. W.; Withers, S. G. J. Biol. Chem. 2009, 284, 17404.

    6. Stummeyer, K.; Dickmanns, A.; Mühlenhoff, M.; Gerardy-Schahn, R.; Ficner, R. Nat. Struct. Mol. Biol. 2005, 12, 90.

    7. Frens, G. Nat. Phys. Sci. 1973, 241, 20.

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