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Supporting information

Influence of the aromatic aglycon of galactoclusters on the binding of LecA: Case study with O-phenyl, S-phenyl, O-benzyl, S-benzyl, O-

biphenyl and O-naphthyl aglycons

Francesca Casoni,a Lucie Dupin,b Gérard Vergoten,c Albert Meyer,a Caroline Ligeour,a Thomas Géhin,b Olivier Vidal,c Eliane Souteyrand,b Jean-Jacques Vasseur,a Yann Chevolot,b François

Morvan*a

ContentsGeneral methods ..................................................................................................................................S3

Table S1. HPLC and MALDI-TOF MS of the glycoclusters G1-G24 ................................................S5

Table S2. IC50Lac and Kd values of glycoclusters G1-G24. .................................................................S6

Figure S1: HPLC profiles of purified glycoclusters G2-G24..............................................................S8

Figure S2: O-Biphenyl-, O-Naphthyl-, O-Phenyl- and S-Phenyl--D-galactoside in the carbohydrateS recognition domain (CRD) of LecA ............................................................................S9

Figure S3: O-Benzyl--D-galactoside (left) and S-Benzyl--D-galactoside (rigth) in the CRD of LecA.....................................................................................................................................................S9

Figure S4: Glycocluster G21 bound to LecA according to a parallel arrangement...........................S10

Figure S5: Glycocluster G21 bound to LecA according to a linear arrangement. ............................S11

Figure S6: Glycocluster G21 bound to LecA according to a Y arrangement....................................S12

Figure S7: Glycocluster G21 bound to LecA according to a T arrangement. ...................................S131H-NMR spectrum of 8 ......................................................................................................................S1413C-NMR spectrum of 8.....................................................................................................................S14

NMR HSQC Jmod 1H/13C spectrum of 8 ..........................................................................................S15

NMR NMR cosy 1H/1H spectrum of 8 ..............................................................................................S151H-NMR spectrum of 11....................................................................................................................S1613C-NMR spectrum of 11...................................................................................................................S16

NMR HSQC Jmod 1H/13C spectrum of 11 ........................................................................................S17

NMR cosy 1H/1H spectrum of 11.......................................................................................................S171H-NMR spectrum of 14....................................................................................................................S1813C-NMR spectrum of 14...................................................................................................................S18

Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry.This journal is © The Royal Society of Chemistry 2014

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NMR HSQC Jmod 1H/13C spectrum of 14 ........................................................................................S19

NMR cosy 1H/1H spectrum of 14.......................................................................................................S191H-NMR spectrum of 17....................................................................................................................S2013C-NMR spectrum of 17...................................................................................................................S20

NMR HSQC Jmod 1H/13C spectrum of 17 ........................................................................................S21

NMR cosy 1H/1H spectrum of 17.......................................................................................................S211H-NMR spectrum of 12....................................................................................................................S2213C-NMR spectrum of 12...................................................................................................................S22

NMR HSQC Jmod 1H/13C spectrum of 12 ........................................................................................S23

NMR cosy 1H/1H spectrum of 12.......................................................................................................S231H-NMR spectrum of 15....................................................................................................................S2413C-NMR spectrum of 15...................................................................................................................S24

NMR HSQC Jmod 1H/13C spectrum of 15 ........................................................................................S251H-NMR spectrum of 18....................................................................................................................S2613C-NMR spectrum of 18...................................................................................................................S26

NMR HSQC Jmod 1H/13C spectrum of 18 ........................................................................................S27

NMR cosy 1H/1H spectrum of 18.......................................................................................................S271H-NMR spectrum of 16....................................................................................................................S2813C-NMR spectrum of 16...................................................................................................................S28

NMR HSQC Jmod 1H/13C spectrum of 16 ........................................................................................S29

NMR cosy 1H/1H spectrum of 16.......................................................................................................S291H-NMR spectrum of 19....................................................................................................................S3013C-NMR spectrum of 19...................................................................................................................S30

NMR HSQC Jmod 1H/13C spectrum of 19 ........................................................................................S311H-NMR spectrum of 23....................................................................................................................S3213C-NMR spectrum of 23...................................................................................................................S32

NMR HSQC Jmod 1H/13C spectrum of 23 ........................................................................................S33

NMR cosy 1H/1H spectrum of 23.......................................................................................................S331H-NMR spectrum of 25....................................................................................................................S3413C-NMR spectrum of 25...................................................................................................................S34

NMR HSQC Jmod 1H/13C spectrum of 25 ........................................................................................S351H-NMR spectrum of 27....................................................................................................................S3613C-NMR spectrum of 27...................................................................................................................S36

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NMR HSQC Jmod 1H/13C spectrum of 27 ........................................................................................S37

NMR cosy 1H/1H spectrum of 27.......................................................................................................S371H-NMR spectrum of 29....................................................................................................................S3813C-NMR spectrum of 29...................................................................................................................S38

NMR HSQC Jmod 1H/13C spectrum of 29 ........................................................................................S391H-NMR spectrum of 24....................................................................................................................S4013C-NMR spectrum of 24...................................................................................................................S40

NMR HSQC Jmod 1H/13C spectrum of 24 ........................................................................................S41

NMR cosy 1H/1H spectrum of 24.......................................................................................................S411H-NMR spectrum of 26....................................................................................................................S4213C-NMR spectrum of 26...................................................................................................................S42

NMR HSQC Jmod 1H/13C spectrum of 26 ........................................................................................S43

NMR cosy 1H/1H spectrum of 26.......................................................................................................S431H-NMR spectrum of 28....................................................................................................................S4413C-NMR spectrum of 28...................................................................................................................S44

NMR HSQC Jmod 1H/13C spectrum of 28 ........................................................................................S451H-NMR spectrum of 33....................................................................................................................S4613C-NMR spectrum of 33...................................................................................................................S4631P-NMR spectrum of 33 ...................................................................................................................S47

General methodsAll reagents were commercial and used without further purification. Solvents were distilled over P2O5 (CH2Cl2) or CaH2 (CH3CN). All the reactions were performed under an argon atmosphere. Reactions under microwave activation were performed on Monowave 300 Anton Paar. The evolution of reactions was monitored by analytical thin-layer chromatography using silica gel 60 F254 precoated plates (E. Merck). TLC plates were inspected by UV light and developed by treatment with a mixture of 10% H2SO4 in EtOH/H2O (1:1 v/v) followed by heating. Purifications by column chromatography were performed using silica gel 60 (40-63 µm) with the indicated eluent. Optical rotations were measured using a Perkin Elmer 341 Polarimeter. Optical rotations were measured at 20 ± 2 °C in the stated solvent; []D values are given in deg.mL.g-1.dm-1. 1H NMR (600 MHz), 13C NMR (100 or 151 MHz), and 31P NMR (121 MHz) spectra were recorded in the stated solvent at 298 K using a 600 or 300 MHz Bruker Avance III Spectrometer. Shifts are referenced relative to deuterated solvent residual peaks. Analysis and assignments were made using NMR COSY and HSQC experiments. High resolution (HR-ESI-QToF) mass spectra were recorded using a Waters Synapt G2 S spectrometer.

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High performance liquid chromatography (HPLC) purifications were performed on a Waters Millipore instrument equipped with a reodyn injector a 600S controller and a Model 996 photodiode array detector. HPLC analyses were performed on a Dionex Ultimate 3000 instrument equipped with a reodyn injector and a photometer DAD 3000. For analyses, a reverse phase C18ec Nucleodur (3 μm) column (75 x 4.6 mm; Macherey-Nagel, Germany) was used at a flow rate of 1 mL min-1 using a linear gradient of acetonitrile in 0.05 M aqueous triethylammonium acetate (pH 7). For purifications, a reverse phase C18ec Nucleodur (7 μm) column (125 x 8 mm; Macherey-Nagel, Germany) was used at a flow rate of 2 mL min-1 using a linear gradient 4% to 48% of acetonitrile in 0.05 M aqueous triethylammonium acetate (pH 7) for 20 min . MALDI-TOF mass spectra were recorded on a Voyager mass spectrometer (Perspective Biosystems, Framingham, MA) equipped with a nitrogen laser. MALDI conditions were: accelerating voltage 24000V; guide wire 0.05% of the accelerating voltage; grid voltage 94% of the accelerating voltage; delay extraction time 500 ns. 1 μL of sample was mixed with 5μL of a saturated solution of matrix in acetonitrile/water (1:1, v/v) containing 10% of ammonium citrate. Then, 1 μL of the mixture was placed on a plate and dried at room temperature and pressure. Matrix: HPA: 3-Hydroxypicolinic Acid.

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Table S1. HPLC and MALDI-TOF MS of the glycoclusters G1-G24

Oligonucleotide glycoclusters

HPLC RT(min)a

MALDI-TOF MS in HPA[M-H]-

m/z Calcd; Found

nmolb

isolated

G1 See reference 1

G2 10.83 C277H372N72O150P20S47857.04; 7857.44

39.7

G3 10.90 C285H387N72O158P207968.99; 7969.62

23.2

G4 10.30 C285H388N72O154P20S48033.25; 8033.42

29.2

G5 10.20 C279H371N76O149P20S47921.09; 7921.24

40.9(71)

G6 10.65 C277H371N72O146P20S87921.31; 7921.37

46(83)

G7 10.60 C285H387N72O154P20S48033.25; 8033.49

36.0

G8 10.90 C285H388N72O150P20S88097.53; 8097.08

34.0

G9 9.40 C281H380N72O154P207848.88; 7848.31

35.8(91.6)

G10 11.17 C281H380N72O150P20S47913.15; 7913.06

41.3

G11 10.08 C291H396N76O157P208089.14; 8089.32

35.9

G12 11.02 C289H396N72O154P20S48089.36; 8089.06

32,5

G13 10.40 C281H379N72O150P20S47913.15; 7913.81

11.0

G14 10.72 C281H379N72O146P20S87977.42; 7977.86

48(79)

G15 10.35 C291H395N76O153P20S48153.41; 8153.84

45.0

G16 11.26 C289H395N72O150P20S88153.63; 8153.47

28.0

G17 10.97 C309H404N72O154P208209.37; 8209.59

16.5(92)

G18 11.82 C309H404N72O150P20S48273.64; 8273.51

3.7(76)

G19 12.22 C318H417N83O149P208404.65; 8404.78

19.2(79)

G20 12.28 C317H420N72O154P20S48449.85; 8449.86

11.0

G21 10.28 C301H400N72O154P208109.26; 8109.67

24.7(92)

G22 11.51 C301H400N72O150P20S48173.52; 8173.58

17.4

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G23 11.67 C310H413N83O149P208304.53; 8304.89

27.8(79)

G24 11.74 C309H416N72O154P20S48349.73; 8349.03

25.5

a Linear gradient from 4% to 48% of acetonitrile in 50 mM TEAAc buffer pH 7 over 20 min. bIn bracket starting amount in nmoles of crude tetraalkynylated mannoside oligonucleotide conjugate, if not specified 100 nmoles.

1. B. Gerland, A. Goudot, C. Ligeour, G. Pourceau, A. Meyer, S. Vidal, T. Gehin, O. Vidal, E. Souteyrand, J. J. Vasseur, Y. Chevolot and F. Morvan, Bioconjugate Chem., 2014, 25, 379-392.

Table S2. IC50Lac and Kd values of glycoclusters G1-G24.Oligonucleotide

glycoclusters IC50 Lac (mM) Kd (nM)aLength number

of atomsG1 103.1 39 27G2 36.2 63 21G3 122.7 28 24G4 17.7 76 24G5 45.1 51 21G6 38.2 55 21G7 63.9 49 24G8 19.7 70 24G9 8.1 99 22G10 5.7 170 22G11 9.1 85 25G12 8.4 125 25G13 12.0 85 22G14 18.5 71 22G15 13.3 76 25G16 18.1 75 25G17 160.3 20 27G18 91.6 36 21G19 68.9 46 30G20 81.6 43 30G21 178.3 14 25G22 160.2 20 25G23 68.0 48 28G24 129.4 31 28

aLength in atom between the C1 of galactose and O atom of mannose core.

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Figure S1: HPLC profiles of purified glycoclusters G2-G24. G1 was previously reported.1

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Figure S2: O-Biphenyl-, O-Naphthyl-, O-Phenyl- and S-Phenyl--D-galactoside in the carbohydrate recognition domain (CRD) of LecA

Figure S3: O-Benzyl--D-galactoside (left) and S-Benzyl--D-galactoside (rigth) in the CRD of LecA

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Figure S4: Glycocluster G21 bound to LecA according to a parallel arrangement.(Calcium ions are shown as green spheres).

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Figure S5: Glycocluster G21 bound to LecA according to a linear arrangement. (Calcium ions are shown as green spheres).

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Figure S6: Glycocluster G21 bound to LecA according to a Y arrangement. (Calcium ions are shown as green spheres).

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Figure S7: Glycocluster G21 bound to LecA according to a T arrangement. (Calcium ions are shown as green spheres).

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1H-NMR spectrum of 8

13C-NMR spectrum of 8

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NMR HSQC Jmod 1H/13C spectrum of 8

NMR NMR cosy 1H/1H spectrum of 8

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1H-NMR spectrum of 11

13C-NMR spectrum of 11

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NMR HSQC Jmod 1H/13C spectrum of 11

NMR cosy 1H/1H spectrum of 11

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1H-NMR spectrum of 14

13C-NMR spectrum of 14

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NMR HSQC Jmod 1H/13C spectrum of 14

NMR cosy 1H/1H spectrum of 14

S20

1H-NMR spectrum of 17

13C-NMR spectrum of 17

S21

NMR HSQC Jmod 1H/13C spectrum of 17

NMR cosy 1H/1H spectrum of 17

S22

1H-NMR spectrum of 12

13C-NMR spectrum of 12

S23

NMR HSQC Jmod 1H/13C spectrum of 12

NMR cosy 1H/1H spectrum of 12

S24

1H-NMR spectrum of 15

13C-NMR spectrum of 15

S25

NMR HSQC Jmod 1H/13C spectrum of 15

S26

1H-NMR spectrum of 18

13C-NMR spectrum of 18

S27

NMR HSQC Jmod 1H/13C spectrum of 18

NMR cosy 1H/1H spectrum of 18

S28

1H-NMR spectrum of 16

13C-NMR spectrum of 16

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NMR HSQC Jmod 1H/13C spectrum of 16

NMR cosy 1H/1H spectrum of 16

S30

1H-NMR spectrum of 19

13C-NMR spectrum of 19

S31

NMR HSQC Jmod 1H/13C spectrum of 19

S32

Benzyl-6-hydroxy-2-naphthoate (23):

1H-NMR spectrum of 23

13C-NMR spectrum of 23

S33

NMR HSQC Jmod 1H/13C spectrum of 23

NMR cosy 1H/1H spectrum of 23

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1H-NMR spectrum of 25

13C-NMR spectrum of 25

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NMR HSQC Jmod 1H/13C spectrum of 25

S36

1H-NMR spectrum of 27

13C-NMR spectrum of 27

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NMR HSQC Jmod 1H/13C spectrum of 27

NMR cosy 1H/1H spectrum of 27

S38

1H-NMR spectrum of 29

13C-NMR spectrum of 29

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NMR HSQC Jmod 1H/13C spectrum of 29

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1H-NMR spectrum of 24

13C-NMR spectrum of 24

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NMR HSQC Jmod 1H/13C spectrum of 24

NMR cosy 1H/1H spectrum of 24

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1H-NMR spectrum of 26

13C-NMR spectrum of 26

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NMR HSQC Jmod 1H/13C spectrum of 26

NMR cosy 1H/1H spectrum of 26

S44

1H-NMR spectrum of 28

13C-NMR spectrum of 28

S45

NMR HSQC Jmod 1H/13C spectrum of 28

S46

1H-NMR spectrum of 33

ppm (t1)1.02.03.04.05.06.07.0

4.1404.132

3.8773.7003.6813.478

2.7792.6092.5872.566

2.3732.3652.3571.941

1.678

1.1311.1201.1081.097

1.00

8.19

1.00

0.47

6.04

PN

OCN

O OOO

13C-NMR spectrum of 33

PN

OCN

O OOO

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31P-NMR spectrum of 33

PN

OCN

O OOO