chemoenzymatic synthesis of heparan sulfate mimetic

Electronic Supplementary Information (ESI)

Chemoenzymatic Synthesis of Heparan Sulfate

Mimetic Glycopolymers and Their Interactions with

the Receptor for Advanced Glycation End-Products

Jun Li,ad Chao Cai,*abc Lihao Wang,a Chendong Yang,a Hao Jiang,abc

Miaomiao Li,e Ding Xu,e Guoyun Li,abc Chunxia Li,abc and Guangli Yu*abc

a Key Laboratory of Marine Drugs, Ministry of Education, School of Medicine and Pharmacy,

Ocean University of China, Qingdao 266003, China.

b Laboratory for Marine Drugs and Bioproducts, Pilot National Laboratory for Marine Science

and Technology (Qingdao), Qingdao 266237, China.

c Shandong Provincial Key Laboratory of Glycoscience and Glycotechnology, School of

Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China.

d CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key

Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, South China

Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China.

e Department of Oral Biology, School of Dental Medicine, University at Buffalo, SUNY, Buffalo,

NY, 14214, USA.

E-mail addresses: [email protected] (C. Cai), [email protected] (G. Yu)

Table of Contents:

1. Materials and methods .................................................. 1

1.1 Materials .......................................................... 1

1.2 NMR spectroscopy analysis ........................................ 1

1.3 Determination of molecular weight .................................. 1

1.4 Degree of sulfation on HS analogues determined by ion chromatography 2

1.5 Zeta potential ...................................................... 2

1.6 Synthesis of the HS-mimetic glycopolymer ........................... 2

1.6.1 Synthesis of the glycomonomer ................................... 2

1.6.2 General procedure of the free radical polymerization ................ 3

1.6.3 General procedure of the enzymatic elongation of poly(GlcA-AAM) ... 4

1.6.4 Preparation of the HS-mimetic glycopolymers by chemical sulfation .. 4

1.7 Immobilization of RAGE on a CM5 Sensor Chip ....................... 5

1.8 SPR analysis of the RAGE interactions with HS-mimetic glycopolymers 5

1.9 Competition assay of HS-mimetic glycopolymers and Heparin in RAGE

using SPR ............................................................ 6

2. SDS-PAGE of PmHS2 and KfiA ............................................ 7

3. The specific refractive index increment (dn/dc) determination ................ 9

4. The size-exclusion chromatography (SEC) spectra ......................... 10

5. HRMS and NMR spectra .................................................. 13

6. Surface Plasmon Resonance measurements ............................... 19

7. Zeta potential analysis ................................................... 21

1

1. Materials and methods

1.1 Materials

4-Nitrophenyl β-D-glucuronide (GlcA-pNP), uridine 5′-diphosphoglucuronic acid

trisodium salt (UDP-GlcA), uridine 5′-diphospho-N-acetylglucosamine sodium

salt (UDP-GlcNAc), sulfur trioxide pyridine complex, sulfur trioxide

trimethylamine complex, and formamide were purchased from Sigma-Aldrich

Corporation (Saint Louis, Missouri, USA) and used without further purification.

Acryloyl chloride and Pd/C (10% Pd, contains 40-60% H2O) were purchased

from Aladdin Corporation (Shanghai, China). 2,2'-azobis-2-methyl-

propanimidamide, dihydrochloride (AAPD) was purchased from Energy

Chemical (Shanghai, China). Acrylamide and heparin sodium (Mw: 10-20 kDa)

from porcine intestinal mucosa were purchased from Sangon Biotech Co., Ltd.

(Shanghai, China). Heparosan synthase-2 (PmHS2) from Pasteurella

multocida and N-acetyl glucosaminyl transferase of E. coli K5 (KfiA) were

expressed and purified as previously described.1,2 The receptor for Advanced

Glycation End-products (RAGE, isoelectric point: 9.5; molecular weight: 23 kDa)

was expressed and supplied by Dr. Miaomiao Li and Dr. Ding Xu (University at

Buffalo, the State University of New York). Sephadex™ G10, 10×PBS-P buffer,

CM5 chip and amino coupling kit were purchased from GE healthcare (USA).

1.2 NMR spectroscopy analysis

For NMR analysis, the samples were freeze-dried with 500 μL D2O (99.9%)

thrice before final dissolution in 500 μL D2O (99.9%). The NMR experiments

were conducted on an Agilent DD2 500 MHz instrument (Agilent, USA) at room

temperature.

1.3 Determination of molecular weight

The purified samples (5 mg/mL) were dissolved in 0.1 M Na2SO4. Molecular

weights (Mw) were characterized by SEC-MALLS using high-performance

liquid chromatography (Agilent 1260, USA) system equipped with two OHpak

columns (SB-804 HQ, SB-803 HQ, Shodex, Japan). The signals were detected

by refractive index detector together with eighteen-angle laser scattering

(miniDAWN, Wyatt Technology, USA). The column temperature was set at

35 °C, and 0.1 M Na2SO4 in H2O was employed as an eluent at a flow rate of

2

0.6 mL/min. The refractive index increment (dn/dc) of the polymer solution was

determined as the slope of the dependence of the refractive index (n) as a

function of the concentration (c), which was measured by using a differential

refraction detector (Wyatt Optilab T-rEX).3

1.4 Degree of sulfation on HS analogues determined by ion

chromatography

Sulfur content (S%) was determined by ion chromatography as described by Li

with minor modification.4 Briefly, 20 μL of sample (5 mg/mL) was hydrolyzed in

ampoule with 1 M HCl at 110 °C for 8 h. The hydrolysate was dried under

vacuum before dissolved in deionised water (2 mL). Subsequently, sulfur

quantification was performed by using ion chromatography (CIC-100, Qingdao

ShengHan Chromatograph Technology Co., Ltd) equipped with a suppressed

conductivity detector. The column was a ShengHan SH-AC-1 operated at 35 °C.

The eluent was 3.6 mM Na2CO3 - 4.5 mM NaHCO3 aqueous solutions at a flow

rate of 1.5 mL/min.

The DSs (degree of sulfate substitution), which was the average number of O-

sulfate groups on each glycounit of HS-mimetic glycopolymers, was calculated

by the following formula: DSs = 896S%/(3200-102S%) for di-polymer OS; DSs

= 1072S%/(3200-102S%) for tri-polymer OS; DSs = 1275S%/(3200-102S%) for

tetra-polymer OS.

1.5 Zeta potential

The glycopolymers were dissolved (1 mg/mL) in NaCl 0.1 mM. The zeta

potential measurements were performed on a Malvern Zetasizer Nano ZS

(Malvern Instruments, UK).

1.6 Synthesis of the HS-mimetic glycopolymer

1.6.1 Synthesis of the glycomonomer

Scheme S1. Synthetic route to the glycomonomer.

According to the protocol from Miura5, the glycomonomer was prepared from

commercial GlcA-pNP with minor modifications. Briefly, the GlcA-pNP (1 equiv.)

3

was dissolved in a mixture of MeOH/H2O (v/v=1), and Pd/C was added to the

solution under H2. After hydrogenation, the Pd/C was removed by filtration. The

filtrate was cooled at -10 °C for 30 min, and then Na2CO3 (11 equiv.) and acrloyl

chloride (10 equiv.) were added to the reaction mixture. After stirring for 6 h, the

reaction mixture was evaporated under vacuum. The aqueous solution of the

crude product was purified on a Sephadex G10 column eluted with H2O.

Fractions were collected, and those containing the product, as determined by

UV absorbance at 254 nm, were combined and freeze-dried to afford a white

solid (1, yield: ~50%). 1H NMR (500 MHz, D2O) δ 7.48 (d, J = 8.2 Hz, 1H), 7.18

(d, J = 8.3 Hz, 1H), 6.45 (dd, J = 16.9, 10.3 Hz, 1H), 6.35 (d, J = 17.0 Hz, 1H),

5.89 (d, J = 10.1 Hz, 1H), 5.13 (d, J = 5.9 Hz, 1H), 3.92 (d, J = 8.6 Hz, 1H), 3.64

(s, 3H). 13C NMR (126 MHz, D2O) δ 175.33, 166.88, 154.16, 131.79, 130.21,

128.19, 123.65, 117.20, 116.48, 100.33, 76.16, 75.28, 72.71, 71.67. HRMS

(negative ion): calculated for C15H17NO8 [M-H]- = 338.0954, found: 338.1000.

1.6.2 General procedure of the free radical polymerization

Scheme S2. Synthesis of poly(GlcA-AAM).

Acrylamide (AAM) and the glycomonomer (compound 1) were copolymerized

in deionized water. The synthesis of the glycopolymer was conducted via free

radical polymerization using 2,2’-azobis(2-amidinopropane) dihydrochloride

(AAPD) as a radical initiator. After adding AAPD, nitrogen was bubbled through

the reaction mixture for 30 min, then heated at 60 °C. Following the end of the

reaction, the reaction mixture was cooled to room temperature, and then

dialyzed (molecular weight cut off [MWCO] = 1 kDa) against distilled water for

2 days. The dialysate was lyophilized to give a white solid (G1).

4

1.6.3 General procedure of the enzymatic elongation of poly(GlcA-AAM)

Scheme S3. Enzymatic elongation of poly(GlcA-AAM).

To introduce a GlcNAc residue, the reaction substrate was incubated with KfiA

(50 μg/mL) in a buffer containing Tris (25 mM, pH 7.2), MnCl2 (10 mM), and

UDP-GlcNAc (0.25 mM, ∼1.01 equiv., based on the number of oligosaccharide

units in the side chain) at room temperature overnight. To introduce a GlcA

residue, the reaction substrate was incubated with PmHS2 (50 μg/mL) in a

buffer containing Tris (25 mM, pH 7.2), MnCl2 (10 mM), and UDP-GlcA (0.25

mM, ∼1.01 equiv., based on the number of oligosaccharide units in the side

chain) at room temperature overnight.

The reaction mixture was heated in a boiling water bath for about 10 min until

the protein precipitated completely. The crude product was brought into the

supernatant by centrifugation at 12,000 rpm for 15 min, which was then dialyzed

(MWCO = 1 kDa) against distilled water for 2 days. Subsequently, NaCl was

added into the dialysate to a final concentration of 16% NaCl, and precipitation

was induced by adding four volumes of ethanol. The precipitate was centrifuged

at 12,000 rpm for 10 min to yield the precipitated product, which was dissolved

in water, dialyzed (MWCO = 1 kDa) against distilled water for 1 day, and

lyophilized to give a white solid (G2-4).

1.6.4 Preparation of the HS-mimetic glycopolymers by chemical sulfation

For 6-O-sulfation, the glycopolymer (G2-4) with varying side-chain lengths was

dissolved in formamide and a sulfur trioxide trimethylamine complex (∼5 equiv.

per free hydroxyl group was added, based on the number of oligosaccharide

5

units in the side chain). The reaction was carried out in a CEM microwave

reactor and heated at 60 °C for 1 h. For O-sulfation, the glycopolymer (G2-4)

with varying side-chain lengths was dissolved in formamide and a sulfur trioxide

trimethylamine complex (∼40 equiv. per free hydroxyl group was added, based

on the number of oligosaccharide units in the side chain). The reaction was

carried out in a CEM microwave reactor and heated at 60 °C for 2 h.

All the products were transferred into ethanol at room temperature before the

addition of 1% aqueous NaCl. After neutralizing by adding saturated sodium

bicarbonate solution, the reaction was dialyzed (MWCO = 3.5 kDa) against

distilled water for 2 days, and the dialysate was lyophilized to give a white

powder, which was dissolved in 16% aqueous NaCl. The addition of four

volumes of ethanol and centrifugation at 12,000 rpm for 10 min yielded the

precipitated product, which was then dissolved in water and dialyzed (MWCO

= 3.5 kDa) against distilled water for 2 days. The dialysate was lyophilized to

give a white solid.

1.7 Immobilization of RAGE on a CM5 Sensor Chip

To immobilize RAGE for surface plasmon resonance (SPR) analysis, a CM5

chip was first set in a Biacore T200. After washing the surface of the chip with

PBS-P running buffer, the sensor chip coated with carboxymethylated dextran

was activated with 0.4 M EDC/0.1 M NHS for 420 s at a flow rate of 10 μL/min.

Immediately after the activation, a RAGE solution (20 μg/mL) in sodium acetate

buffer (pH 5.0) was added onto the chip surface for 30 s at a flow rate of 10

μL/min. The unreacted, activated carboxylic acids on the chip surface were

blocked with ethanolamine.

1.8 SPR analysis of the RAGE interactions with HS-mimetic

glycopolymers

The baseline was allowed to stabilize for at least 2 h in PBS-P running buffer

before injecting test samples. Varying concentrations (6.25-200 nM) of HS-

mimetic glycopolymer were dissolved in the PBS-P running buffer and injected

for 120 s at 30 μL/min, followed by a 600 s dissociation phase. The sensor

surface was regenerated by 0.5 mM NaOH for 5 s. The response was monitored

as a function of time (sensorgram) at 25 °C and subtracted from the response

6

of the reference surface. Kinetic parameters were evaluated using the

BIAevaluation software 4.1.

1.9 Competition assay of HS-mimetic glycopolymers and Heparin in

RAGE using SPR

The baseline was allowed to stabilize for at least 2 h in PBS-P running buffer

before injecting test samples. Varying concentrations (6.25-200 nM) of HS-

mimetic glycopolymer were dissolved in PBS-P running buffer with 2.23 nM of

heparin and injected for 120 s at 30 μL/min, followed by a 600 s dissociation

phase. The sensor surface was regenerated by 1.25 mM NaOH for 9 s. The

response was monitored as a function of time (sensorgram) at 25 °C and

subtracted from the response of the reference surface. 2.23 nM of heparin in

PBS-P buffer was selected as a blank, which was detected with the same

program. Finally, signal of each sample was deducted from that of the blank

and kinetic parameters were evaluated using the BlAevaluation software 4.1.

7

2. SDS-PAGE of PmHS2 and KfiA

Figure S1. SDS-PAGE analysis of purified PmHS2. Affinity chromatography-

purified PmHS2 protein was resolved on a precasted 10% SDS-PAGE gel. The

gel was stained with Coomassie blue. Migration positions of molecular markers

(from Thermo Fisher Scientific) are indicated. The apparent mass was

approximately 72 kDa. Molecular mass standards (top to bottom: 170, 130, 95,

72, and 55 kDa)

8

Figure S2. SDS-PAGE analysis of purified KfiA. Affinity chromatography-

purified KfiA protein was resolved on a precasted 12% SDS-PAGE gel. The gel

was stained with Coomassie blue. Migration positions of molecular markers

(from Thermo Fisher Scientific) are indicated. The apparent mass was

approximately 30 kDa. Molecular mass standards (top to bottom: 170, 130, 95,

72, 55, 43, 34 and 26 kDa).

9

3. The specific refractive index increment (dn/dc) determination

Figure S3. The standard curve for the specific refractive index increment (dn/dc)

determination.

10

4. The size-exclusion chromatography (SEC) spectra

Entry 3, Table 1

Entry 4, Table 1

Entry 5, Table 1

Entry 6, Table 1

11

Entry 7, Table 1

Entry 8, Table 1

Entry 9, Table 1

Entry 10, Table 1

12

Figure S4. The size-exclusion chromatography spectra of G1 in table 1.

Figure S5. The size-exclusion chromatography (SEC) spectra of G2-4.

G2

G3

G4

Entry 11, Table 1

13

5. HRMS and NMR spectra

Figure S6. The HRMS of p-(N-Acrylamido)phenyl β-D-glucuronic acid.

Figure S7. Free radical polymerization of poly(acrylamide/GlcA) with different

monomer ratio in 50 mol% of AAPH.

14

Figure S8. 1H-NMR spectrum of G2-OS, G3-OS and G4-OS. S-convers

represents for sulfate ester conversion; S% represents for sulfur content; DSs

represents for degree of sulfate substitution.

12

Figure S9. 1H-NMR spectrum of p-(N-Acrylamido)phenyl β-D-glucuronic acid.

Figure S10. 13C-NMR spectrum of p-(N-Acrylamido)phenyl β-D-glucuronic acid.

13

Figure S11. 1H-NMR spectrum of Poly(acrylamide/GlcA), (acrylamide/GlcA=1).


14


Figure S14. 1H-NMR spectrum of G2.

15



16

Figure S17. 1H-NMR spectrum of G2-6S.


17


Figure S20. 1H-NMR spectrum of G2-OS.

18



19

6. Surface Plasmon Resonance measurements

Figure S23. SPR measurement of the direct binding between RAGE and G2-6S under different

concentrations of glycopolymer.



20



Figure S26. SPR measurement of the direct binding between RAGE and G4 (no sulfation) under

different concentrations of glycopolymer.

21

Figure S27. Competition assay of HS-mimetic glycopolymers and Heparin in RAGE using SPR. a)

G2-OS; b) G4-OS; c) G3-OS; ka: association rate constant; kd: dissociation rate constant; KD:

apparent equilibrium dissociation constant.

7. Zeta potential analysis

-6

-5

-4

-3

-2

-1

0

Ze

ta p

ote

nti

al

(mV

)

G 2 -6 S

G 2 -O S

G 3 -6 S

G 3 -O S

G 4 -6 S

G 4 -O S

Figure S28. Zeta potential of synthetic HS-mimetic glycopolymers in a 0.1 M solution of sodium

chloride measured at room temperature.

References

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2 Y. Xu, C. Cai, K. Chandarajoti, P. H. Hsieh, L. Li, T. Q Pham, E. M. Sparkenbaugh, J. Sheng, N. S.

Key, R. Pawlinski, E. N. Harris, R. J. Linhardt and J. Liu, Nat. Chem. Biol., 2014, 10, 248.

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chemoenzymatic synthesis of heparan sulfate mimetic

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