Bifunctional mannoside-glucosinolate glycoconjugates as enzymatically triggered isothiocyanates and FimH ligands

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Reise, M. Schuler, R. Nehme, G. Despras, J. Brekalo, P. Morin, P. Renard, T. K. Lindhorst and A. tatibouet,

Org. Biomol. Chem., 2018, DOI: 10.1039/C8OB01128A.

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Bifunctional mannoside-glucosinolate glycoconjugates as

enzymatically triggered isothiocyanates and FimH ligands

G. Cutolo,^aF. Reise,^bM. Schuler,^aR. Nehmé,^aG. Despras,^bJ. Brekalo,^a,bP. Morin,^aP.-Y. Renard,^c

T. K. Lindhorst^band A. Tatibouët*^a

Received 00th January 20xx,

Accepted 00th January 20xx

Glucosinolates are sulfur-containing secondary metabolites found in plants of the Brassicale order. They are precursors of

isothiocyanate species, resulting from C-S hydrolysis catalysed by the thioglucohydrolase myrosinase. We describe the

synthesis of bifunctional glucosinolate-mannoside glycoconjugates combining both the structural features of a substrate of

myrosinase and a ligand of the lectin FimH. We show that these glycoconjugates serve as enzyme substrates and that

myrosinase can indeed hydrolyze the glucosinolate moiety with affinities (K_M, V_max) comparable to the natural substrates

glucomoringin and sinigrin. This enzymatic hydrolysis of the thioglycosidic bond led to the efficient formation of an

isothiocyanate which was assessed by the formation of the corresponding dithiocarbamates derivatives. Finally, we show

that our synthetic bifunctional glycoconjugates also serve as FimH ligands where the glucosinolate moiety does not hamper

the interaction with the lectin. Our findings set the stage for an original bioconjugation tool, allowing for myrosinase-

triggered specific labelling of lectins using glucosinolate glycoconjugates as non-toxic, water soluble isothiocyanate

precursors.

DOI: 10.1039/x0xx00000x

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leads to the hydrolysis of the C-S anomeric bond of glucosinolates,

forming D-glucose and a transient thiohydroxamic species which in

turn undergoes a Lossen rearrangement liberating sulfate and an

isothiocyanate (ITC) (2) (Figure 2A). This unique enzymatic reaction

Introduction

The myrosinase-glucosinolate (MG) reaction is a unique biochemical

transformation in Nature, able to produce a reactive isothiocyanate,

which is a well-known bioconjugation tool. Glucosinolates (Figure 2A,

1) as substrates of myrosinase have a long chemical and biochemical

history. As early as the identification of the first glucosinolate

sinalbine (Figure 2A, 1a) in 1831 by P. J. Robiquet and F. Boutron-

Charlard, the presence of a sulfur atom in the structure was assessed.

The glucosinolate was identified as the preactive molecule, which

could be further transformed into mustard oil.^[1]This transformation,

known as the "mustard oil bomb", is due to the activity of

myrosinase.^[2]The first synthesis of the glucosinolate glucotropaeolin

(1b) by Ettlinger led to the structural assignment of these secondary

metabolites.^[3]They are specific biomarkers of the Brassicale order in

which more than 140 glucosinolates were found associated with

thioglucoside glucohydrolases named myrosinases (EC 3.2.1.147).^[4]

induced numerous studies on the enzymatic mechanism, as well as

on the biological (antifungal, antibacterial, antiviral, insecticidal,

repellent to animals) and even therapeutic activities

(chemopreventive effects) of these secondary metabolites^[6]which

are mainly attributed to the isothiocyanate products. Thus

glucosinolate extraction, analysis and synthesis have attracted

numerous research efforts,^[7]whereas little work has been devoted

to evaluate myrosinase activity with various natural and artificial

glucosinolates.^[8]Indeed, as strong electrophiles, the ITCs are well

known ligation agents, but are toxic, not easy to prepare and to store.

Using the natural myrosinase-glucosinolate (MG) reaction would

allow for the in-situ preparation of ITCs from a stable, non-toxic and

water soluble GL precursor (Figure 1).

Glucosinolates and myrosinase root their relationship in

a

mechanism of defense of Brassicale plants against any aggression

from bacteria to mammals.^[5]The enzymatic action of myrosinase

Figure 1 The Myrosinase-Glucosinolate (MG) tandem as a new tool to release reactive ITC?

^a.Institut de Chimie Organique et Analytique - ICOA UMR 7311 CNRS Université

d’Orléans - Rue de Chartres, BP6759, 45067 Orléans cedex 02, France. Fax:

(+33)2-3841-7281. E-mail: arnaud.tatibouet@univ-orleans.fr . http://www.icoa.fr

^b.Otto Diels Institute of Organic Chemistry, Christiana Albertina University of Kiel

Otto-Hahn-Platz 3-4, D-24118 Kiel, Germany.

Thus, the aim of this work is the study of the MG reaction as a

potential new bioconjugational tool for specific labelling^[9]

through the use of synthetic glucosinolate glycoconjugates,

designed to interact with a specific biological target. This initial

study targets the bacterial lectin FimH, which has a well-defined

^c.Normandie Université, Université de Rouen; INSA Rouen; CNRS, 76000 Rouen,

COBRA UMR 6014 & FR 3038 IRCOF, 1, Rue Tesnières, 76821 Mont-Saint-Aignan

cedex, France.

specificity for -D

-manno pyranosides.^[10]

Electronic Supplementary Information (ESI) available: see

DOI: 10.1039/x0xx00000x

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Results and Discussion

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DOI: 10.1039/C8OB01128A

other hand. Indeed, it is known that an aromatic aglycone

moiety considerably adds to the affinity of FimH mannoside

Our approach/strategy is to combine two structural features in

one molecule in order to design/prepare glycoconjugates which

can act at the same time as a substrate for the enzyme

myrosinase and as a ligand for a specific biological target, such

as lectin FimH in this present study. The design of the target

glycoconjugates was based on our knowledge of myrosinase

substrate specificity and the chemistry of glucosinolates on one

hand and of synthetic ligands of the bacterial lectin FimH on the

ligands^[10]due to

- stacking interactions at the entrance of the

lectin’s carbohydrate recognition domain.^[11]Recently, B. Ernst

and co-workers^[12a]and Janetka et al.^[12b]have shown that

biphenyl

-D-mannopyranosides show especially high affinity

for FimH with K_dvalues in the nanomolar range in specific

assays.^[12c]

Figure 2 A: Generic glucosinolate structures and some classical examples (1a

-

1d). Myrosinase catalyzes the hydrolysis of glucosinolate thioglycosidic bond, generating

a transient sulfated thiohydroxamic acid, which decomposes into sulfate and a reactive isothiocyanate ( 2). B: Targeted glucosinolate glycoconjugates (3-6), comprising



-D-mannopyranosyl moieties as FimH ligands.

Accordingly, glucosinolate glycoconjugates

3-6 (Figure 2B) achieved using various conditions starting from a nitroalkane, a

comprising high-affinity binding motifs for FimH were first nitrovinyl, an aldoxime or using the most recent approach

designed to explore the possibility of triggering isothiocyanate starting from lactones.^[7e,13]We chose to follow previous

formation with myrosinase. The 4'-O-mannopyranosyl sinalbine methods from our laboratory applying the Kulkarni conditions

analogue 3, where the O-sulfated thiohydroximate part is on nitrovinyl derivatives. First, the phenyl mannoside derivative

separated from the

constituted a first model substrate, and the biphenyl analogues reported by us.^[14]To prepare the required biphenyl nitrovinyl

, and were envisaged as advanced glucosinolate compounds 14 16, a three-step sequence was employed

glycoconjugates. The O-glycosides and were directly inspired (Scheme 1). Formation of the O-glycoside intermediates bearing

by literature^[11]while the S-glycosidic mannoside

was a biphenyl moiety was achieved according to methods

considered to prevent hydrolysis of the O-glycosidic bond by an developed in the literature.^[12]Thus, glycosylation of the per-

-mannosidase.^[12d]Hence, the replacement of the anomeric acetylated mannose with p-bromophenol gave the

oxygen atom by a sulfur atom might lead to a more glycosidase- bromophenyl mannoside in 68% yield. Then, a Suzuki-Miyaura

resistant ligand, without modifying much the ligand-protein reaction of the bromophenyl mannopyranoside and para- or

interaction and could consequently lead to improved meta-formylphenylboronic acid, respectively, gave the biphenyl

bioavailability. Glucosinolate was designed in order to mannoside intermediates 11 and 13 in 84% and 80% respective

evaluate the influence of the spatial orientation of the -D- yields. In the next step, Henry condensation afforded the

-D-mannopyranoside by a p-benzyl moiety

3 (Fig. 1) was prepared following the procedures previously

4

,

5

6

-

3

4

5



9

6



mannopyranoside part in relation to the biphenyl aglycone nitrovinyl derivatives 14 and 16, respectively, again in very good

moiety on FimH binding. The key step in glucosinolate synthesis yields. Synthesis of the S-glycosidic biphenyl analogue was

is the formation of the thiohydroximate function. This can be inspired by the efficient protocol developed by S. Messaoudi

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and coworkers applying palladium catalysis. ^[15]Tetra-O-acetyl- condensation was applied to obtain the nitrovinyl_Vd_iee_wri_Av_rta_ict_li_ev_Oe_nl1_in5_e

1-thio- -D-mannopyranoside ( ) was used as a 80/20

/β- in quantitative yield. In order to evaluat^De^Ot^Ih^:e¹⁰i^.1n⁰f³lu⁹e^/Cn⁸c^Oe^Bo⁰¹f¹t²h⁸e^A

anomeric mixture to furnish the p-phenyl- glucosinolate moiety on FimH binding, the unprotected

thiomannopyranoside 10 after a short optimisation process in intermediate aldehydes 17 and 18 were also prepared. Thus,

87% as a 80/20 /β-anomeric mixture. This ratio suggests that aldehydes 11 and 13 were deacetylated under Zemplén



8



no anomerisation occurs during the coupling process. In the conditions using potassium methoxide in methanol to lead to

next step, the Suzuki-Myaura cross-coupling reaction with an the desired compounds in reasonable yields after purification

excess of 4-formylphenylboronic acid afforded the by reverse phase chromatography (50% and 79% respectively).

biphenylaldehyde 12 in 88% yield. Then again Henry

Scheme 1 Synthesis of the nitrovinyl precursors 14 to 16

.

The three biphenyl nitrovinyl mannoside precursors 14-16 were mannopyranosides on the other hand might be the result of the

then used in the following key step towards the target Kulkarni conditions. Indeed, the Lewis acid activation of the

glucosinolates. For the formation of the thiohydroximate nitrovinyl moiety with titanium chloride could induce, through

function the Kulkarni protocol was applied to prepare the the conjugated system, a glycosidic bond cleavage. This could

hydroximoyl chloride with titanium chloride and triethylsilane explain the lower yields observed with the p-substituted S- and

(Scheme 2).^[16]Then, under basic conditions a transient nitrile O-glycosidic bond for the thiohydroximate 19 and 20. The m-

oxide was formed and trapped with -thio-D-glucopyranose to nitrovinyl derivative 16 resulted in the thiohydroximate 21 in a

form the thiohydroximate function in acceptable yields in most better 70% yield. Sulfation of the thiohydroximate moiety was

cases. A modest yield of 53% was observed with the para- next achieved using pyridine sulfur trioxide complex in DMF.

substituted biphenyl derivative 14 while with the meta- Again, with the O-glycosidic bond the sulfation was very

substituted biphenyl derivative 16 the yield was better (70%). efficient, producing the sulfated glucosinolates 22 and 24 in

The preparation of the S-mannopyranoside 20 turned out to be very good yield (80% and 86%) while a lower yield of 26% was

more difficult. The yields were much lower when conditions observed for 23. The synthesis of the glucosinolates 4, 5, and 6

developed for the O-mannopyranoside were applied (26% was finalized by transesterification using a catalytic amount of

yield). Decreasing the temperature to -10°C during the Kulkarni potassium methoxide and purification through C18 reverse

reaction allowed some improvement and isolation of the phase chromatography to remove traces of sulfate salts. There

thiohydroximate 20 in 48% yield. The different yields observed again the O-glycosidic bonds were more stable under these

between m- and p-substitution on one hand and O- an S- conditions and afforded

4

and

6

efficiently with yields close to

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80%. While with the S-mannoside 23, some degradation good yields (80%, 84%, 50% and 64% respectively)_Vfo_iewllo_Arw_tici_ln_eg_Ont_lh_ine_e

occurred during the deacetylation reaction and hence the final same deprotection process as previ^Do^Ou^Is^:l¹y^0.10d³e⁹s^/c^Cr⁸ib^Oe^Bd⁰¹¹²f⁸o^Ar

glucosinolate

5

was obtained in a much lower 40% yield. The aldehydes 17 and 18

.

non-sulfated thiohydroximates 25-28 were also prepared in

Scheme 2 Synthesis of glucosinolates from the corresponding nitrovinyl derivatives.

Myrosinase activity

Table 1. Screening of myrosinase* substrates using capillary electrophoresis

(for structures of 1a-1d see Figure 2).

Once the synthesis of the glucosinolate analogues completed, the

next step was to determine whether myrosinase could still

hydrolyze those artificial GLs or not. To evaluate the enzyme

activity, we followed a procedure previously developed in our

laboratory.^[8]

Entry

K_M(mM) ^[a]

V_max

V_max/K_M

Glucosinolates

(mM.min^-1) (min^-1)

The myrosinase hydrolysis of both natural and synthetic

glucosinolates could be followed by CE/C4D (capillary

electrophoresis/contactless capacitively coupled conductivity

detector) in less than 10 min using 7 µL reaction mixture. This

approach to enzyme analysis by CE is very simple, economic,

fast and robust. Two steps are required: 1) incubation of the

reaction mixture in a micro vial; 2) electrophoretic analysis in

the CE capillary to detect the released sulfate product (SO₄^2-).

Using this technique, the K_Mfor the enzymatic reaction with

sinigrin 1c was determined as 0.63 mM, (Table 1, entry 1). This

value is in the same range as the one we previously reported, as

well as the value obtained using the conventional photometric

approach to enzyme activity determination. This K_Mvalue

served as a reference for comparison with the different

0.63±0.10

0.11±0.01^[b]

1b (glucotropaeolin) 0.12±0.01^[b]

1a (sinalbin)

0.07±0.01^[b]

1d (glucomoringin) 2.60±0.19^[b]

0.31±0.01

0.49

-

0.09

0.18

0.35

0.20

1

1c (sinigrin)

-

2

3

4

5

6

7

8

3

4

5

6

1.31±0.26

3.33±0.79

1.19±0.28

1.78±0.23

0.12±0.01

0.59±0.04

0.42±0.02

0.36±0.01

*

myrosinase : thioglucosidase from Sinapis alba (white mustard) seed

^[a] K_M= Michaelis Menten constant (mM); [b] data from our previous work ^[8]

.

Natural glucosinolates with small aromatic side chains such as

benzyl (glucotropaeolin) 1b or p-hydroxybenzyl (sinalbin) 1a

(Table 1, entries 2 and 3) showed higher affinities than the

bulkier natural glucomoringin 1d which incorporate in its

structure a L-rhamnopyranoside moiety.^[14]The results obtained

glucosinolate derivatives 3, 4, 5, and 6. V_maxare also reported

(myrosinase was used at 0.05 U. mL⁻¹for all enzymatic assays).

with the small library of glucosinolates

3-6 (Table 1) were

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consistent with our previous data.^[8]The K_Mvalue determined

for these four glucosinolates were 1.31, 3.33, 1.19, and 1.78

mM, respectively (Table 1, entries 5-8) which show that these

artificial glucosinolates are still good substrates of myrosinase.

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DOI: 10.1039/C8OB01128A

The analogue

glucomoringin 1d. In terms of catalytic efficiency, glucosinolates

and seemed to be hydrolysed with similar rates although

with lower overall efficiency than the natural substrate sinigrin

1c. The thiomannopyranoside (entry 7) turned out to be

3 (entry 5) showed a similar K_Mvalue as

3

,

4

6

5

slightly better substrate of myrosinase than the three O-

glycosides, having a global efficiency closer to sinigrin. Finally, it

can be concluded from these experiments that the biphenyl

moiety do not dramatically influence the myrosinase activity

when compared to the natural glucosinolate glucomoringin 1d

and the S-glycoside analogue

5 is also well recognized by

myrosinase. Our project relies on the myrosinase ability to

convert the glucosinolate moiety into a reactive isothiocyanate

function. We have thus optimized a process to prove the

formation of the isothiocyanate product by trapping it with a

thiol to form a dithiocarbamate derivative, which is easier to

detect analytically, especially by NMR. The reactivity of

myrosinase on glucosinolates could be assessed using a simple

biphasic system or direct reaction in water.

Myrosinase was diluted in a phosphate buffer at pH 7, and the

same volume of dichloromethane containing an excess of

ethanethiol and triethylamine was added. The glucosinolate

analogues were then added and reacted with myrosinase at

37°C until hydrolysis was completed. The formed isothiocyanate

was subsequently trapped in the dichloromethane phase by

Scheme 3: Hydrolysis of the glucosinolates

3, 4, and 6 by myrosinase and

subsequent trapping reaction with mercaptans.

ethanethiol. This protocol was applied to glucosinolates

, the resulting dithiocarbamates 29 and 30 were isolated in

64% and 81% yields respectively (conditions A, Scheme 3).

To evaluate the reactivity of the isothiocyanate in the buffer

solution only, we tested the MG reaction without CH₂Cl₂and

Et₃N. Surprisingly, the condensation with the isothiocyanate

3 and

The interaction with ten mannose selective lectins were

explored: six lectins from vegetal (ConA, LcH, PSA, VFA, GNA,

HHA), two bacterial lectins (BC2L-A, FimH) and two recombinant

human lectins (Langerin and DC SIGN) and a further nine other

lectins chosen for their various specificity toward carbohydrate

4

structures.^[18a]The three artificial glucosinolates

3, 4, 6 tested,

was not observed. The glucosinolate analogues

also tested with benzylmercaptan and the corresponding

dithiocarbamates 31 32, and 33 were isolated in reasonable to

3, 4 and 6 were

showed good inhibition levels of the interaction with mannose

selective lectins with a stronger effect on ConA, PSA, BC2LA and

FimH, while no or very little inhibition effect was detected with

the other set of lectins. On the contrary, glucomoringin 1d

showed no or little inhibition of the interaction with various

carbohydrate selective lectins except for the specific

L-rhamnose lectin CorM. Finally, this glycoprofile allowed us to

assess that these novel glycoconjugates retain good affinities

with lectins, the glucosinolate moiety having a small impact on

the recognition pattern, and that the side chain mainly drives

the interaction with lectins.

,

good yields (47%, 50%, and 80%, respectively). These

complementary results confirm that the synthetic

glucosinolates prepared here, are indeed substrates of

myrosinase from Sinapis alba. The observed affinities as well as

reactivities are comparable to the hydrolysis of the natural

substrates of myrosinase. Moreover, the MG reaction produced

the corresponding isothiocyanates, as proven by trapping with

benzylmercaptan at neutral pH. Further work is ongoing in the

lab on the study of the kinetics of the hydrolysis and the

reactivity of the ITC towards nucleophiles.

Impact of glucosinolate moieties on the interaction with

lectins

Having the glucosinolate mannosides 3-6 in hands, we decided

to explore their ability to inhibit the interaction with various

lectins using the “glycoprofile” technique.^[17]Four

glucosinolates 3, 4 and 6 and natural glucomoringin 1d were

explored for their interaction with 19 lectins using GLYcoDIAG

company technology.^[18]

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GlycoPROFILE of glucosinolate 3

100

80

60

40

20

0

2mM

1mM

0,5mM

Lectins

Figure 3 : Example of a glycoprofile for Glucosinolate 3 : inhibition of the interaction using three different concentrations of glucosinolate (2mM, 1mM and 0.5 mM)

with 19 lectins using Concanavalin A (ConA), Lens Culinaris Agglutinin (LcH), Pisum Sativum Agglutinin (PSA), Vicia Faba Agglutinin (VFA) ; Galanthus Nivalis

Agglutinin (GLN/GNA), Hippeastrum Hybrid Agglutinin (HHA), Burkholderia cenocepacia lectin A (BC2LA), E. coli Type I fimbrial lectin (FimH), DC-SIGN extracellular

domain (DC-SIGN), Langerin extracellular domain (Langerin), Artocarpus intergrifolia Agglutinin (AIA), Peanut Agglutinin (PNA), Griffonia Simplicifolia Lectin II (GSLII),

Wheat Germ Agglutinin (WGA), Aleuria Aurantia Lectin (AAL), Ulex Europeus Agglutinin (UEA-I), Maackia amurensis Agglutinin (MAA), Elderberry Lectin (SNA),

Coregonus lavaretus marenae (CorM). Each experiment has been set in triplicate.

Furthermore, we studied glucosinolates 3-6, for their potential

Table 2. Inhibition of type 1 fimbriae-mediated E. coli adhesion to mannan-

to inhibit the adhesion of live type 1 fimbriated E. coli bacteria

to mannan, which is mediated by the lectin FimH. To gain a

more detailed understanding of the interaction of particular

moieties of our synthetic ligands with FimH, the deacetylated

desulfoglucosinolate 19 and 21 were also tested. Hence, the

coated microtiter plates under static conditions.^[a]

IC₅₀(SD)^[b]

(mol/L)

Entry

compounds

RIP (SD)^[c]

p-NPMan

187 (10.3)

144.9 (5.9)

81.4 (15.3)

myrosinase substrates

25 28 (Scheme 2) and 17 and 18 (Scheme 1) were compared in

microplate-based adhesion inhibition assays, where methyl

D-mannopyranoside (MeMan) and p-nitrophenyl

3-6, the desulfoglucosinolate analogues

-

1

2

3

4

34.75 (1.05)

-

20.55 (3.15) 267.0 (11.0)

-D-

glucosino-

lates

mannopyranoside (pNPMan) were tested on the same

microtiter plate. We used fluorescent E. coli bacteria according

to a published assay^[19]where fluorescent read-out can be

correlated with bacterial adhesion. In the employed adhesion

inhibition assay, the mannan-coated microplate surface

competes with the tested inhibitors for binding to the bacteria.

Serial dilutions of inhibitors were applied to obtain sigmoidal

inhibition curves from which IC₅₀values can be deduced,

reflecting the inhibitor concentration, which causes 50%

inhibition of bacterial binding to the polysaccharide mannan.

However, as the absolute IC₅₀values obtained in independent

assays are typically found to differ significantly, the inhibitory

potency of synthetic inhibitors is related to standard inhibitors

(MeMan, pNPMan), which are tested in parallel in the same

experiment on the same plate. Thus relative inhibitory

potencies (RIP values) are obtained, which are suited to

compare individual compounds even when they were not

assayed in the same experiment. The determined IC₅₀and RIP

values of the tested compounds are collected in Table 2. All

tested inhibitors exceeded the inhibitory potency of MeMan

(Table 2) and were thus more potent inhibitors of type 1

fimbriae-mediated bacterial adhesion to the mannan-coated

surface employed in our assay.

3

5

36.0 (19.0)

41.4 (12.0)

82.8 (0.1)

169.7 (38.4)

260.0 (2.75)

60.8 (0.7)

4

6

5

25

26

27

28

17

18

desulfo-

glucosino-

sinolates

6

31.85 (3.85) 218.5 (10.5)

7

23.0 (3.0)

33.0 (15.0)

23.4 (16.0)

6.5 (1.0)

174.8 (5.4)

511.0 (22.0)

441.0 (20.8)

776.0 (1.14)

8

9

aldehydes

10

[a]

IC₅₀and RIP (relative inhibitory potency, based on methyl

-D-

mannopyranoside (MeMan) with IP = 1) values are averaged from

mean values from three independent tests; [b] Note that IC₅₀values

can vary significantly in independent experiments as live bacteria are

investigated. Therefore, IC₅₀values cannot be correlated with RIP

values throughout the table. However, all RIP values can be compared

to one another as they are all referenced to the same standard

compound (MeMan) tested in the same experiment; [c] RIP = IC₅₀

(MeMan)/IC₅₀(compound) as tested in the same individual

experiment. SD: standard deviation; pNPMan: p-nitrophenyl--D-

mannopyranoside.

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docking studies with the program Glide^[20]implemented in the

As expected, the biphenyl mannoside structures (

18

Table 2) were more potent ligands than the Schrödinger software package. For that pu^Dr^Op^I:o¹s⁰e^.1,⁰g³l⁹u^/c^Co⁸s^Oin^Bo⁰¹la¹²t⁸e^As

phenylglucosinolate and the corresponding desulfo- and , desulfoglucosinolates 26 and 28, and the aldehydes 17

glucosinolate 25 with RIP values at least 4 times higher than and 18 were chosen. As the FimH protein occurs with the

4-6, 26-28, 17-

View Article Online

,

3

4

6

3

or 25. Interestingly, the aldehyde derivatives 17 and 18 showed tyrosine gate flanking the entrance of the carbohydrate binding

especially high RIP values, 18 surpassing the inhibitory potency domain (Tyr 48 and Tyr 137) in a “closed” conformation as well

of MeMan by almost 780 times. This corresponded to an IC₅₀as in a more “open” form, we used both the open^[21]and the

value of 6.5



mol in our experiment. In addition, all tested closed gate crystal structures^[22]of FimH for docking. Docking

biphenyl mannosides showed better inhibition than pNPMan, was carried out in extra precision mode and keeping the

which equally provides an aromatic aglycon to interact with the receptor in a fixed conformation. Moreover, flexibility was

bacterial lectin’s binding site and thus can be regarded as a allowed for the input ligands, meaning that several conformers

suitable reference compound for comparison. Overall, analysis per ligand (five at most) were generated.

of the results obtained with the desulfoglucosinolates 25

and the glucosinolates revealed some reduction of the listed in Table 3 (for comprehensive results, see Supporting

corresponding RIP values in comparison to the tested Information). More negative docking scores correlate with

-28 Results, showing the highest docking score for each ligand, are

3-6

aldehydes.

higher affinity.

Molecular modeling/docking

In order to rationalize the differences observed for the RIP

values of the synthetic myrosinase substrates and to obtain

some insight in the molecular details of binding, we performed

Table 3. Docking scores^[a]and binding energies^[b]in open and closed gate conformation of FimH for selected glycoside ligands.

Docking

score

Open gate

-8.514

-10.082

-11.677

-9.85

-11.101

-8.781

Docking

score

Closed gate

-8.212

-9.81

-10.971

-9.067

-8.88

-8.557

-9.785

Binding energy

(kJ.mol^-1)

Open gate

-53.44

Binding energy

(kJ.mol^-1)

Closed gate

-53.634

Entry

Ligand

1

2

3

4

5

6

7

MeMan

4

6

26

28

17

18

-87.681

-86.179

-64.511

-89.092

-70.932

-73.13

-85.935

-91.79

-72.885

-92.167

-74.287

-77.781

-8.847

[a] Calculated with the Glide program; [b] calculated via MM-GBSA method from the docking output

For a better correlation with the experimental results, docking results in the flanking of the biphenyl linker by the tyrosine

outputs were re-scored through a MM-GBSA^[23]calculation in residues 48 and 137 (Tyr gate) while the glucoside portion binds

which the two important tyrosine residues (Tyr 48 and 137) via an H-bond network to a lectin subsite located above the Tyr

were set flexible. The binding energies corresponding to the gate. A comparable situation is observed for the p-substituted

score values are listed in Table 3. Hence, the expected affinity derivative

ranking is found for the glucosinolates ( ) and their desulfo the aforementioned subsite. However, a π-π stacking of the

counterparts (26 28) with FimH in the open gate conformation. aromatic linker with the tyrosine gate may favor FimH binding

This is slightly different in the closed gate conformation, where of over . As shown by the values of binding energies (Table

and 28 bind with very similar affinity. However, the calculated 3), the mode of interaction varies when FimH is used in the

4 but with no binding of the glucoside residue into

4, 6

,

4

6

binding energy values for the aldehyde derivatives 17 and 18 do closed gate conformation (Fig. 5). Here, the benefit of the m-

not correlate with the respective RIPs in both open and closed substitution can be seen by first comparing the complexes with

gate conformation of FimH. This finding might be rationalized the glucosinolates. Ligand

6 binds stronger than 4 because its

with the possibility of a strong non-specific interaction of the sulfate group is positioned so as to allow an ionic interaction

aromatic aldehyde moiety with a peripheral area of the with the side chain of Arg 98. As observed in the open gate

carbohydrate recognition domain.^[24]The structures of the conformation, the binding of the desulfoglucosinolate

ligand-FimH complexes resulting from the docking confirm the derivatives with the closed gate FimH is clearly influenced by

expected recognition mode of FimH: a tight interaction with the the substitution pattern on the biphenyl linker. While the two

biphenylmannoside residue while the side chain of the compounds 26 and 28 exhibit the same number of H-bonds with

glucosinolate protrudes on the outside of the lectin (Fig. 4). the protein residues, the orientation of their respective

Additionally, our docking poses hint on the influence of the aglycone part is quite different. The biphenyl moiety of 26

,

position of the glucosinolate substituent on the biphenyl which is lifted to the right, lays above a hydrophilic domain

moiety. In the open gate conformation, the m-substitution

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ARTICLE

Journal Name

whereas the same aromatic part on 28 is set in a much favorable

situation, close to the phenyl ring of tyrosine 48.

View Article Online

DOI: 10.1039/C8OB01128A

Figure 4 : Complexes of the open gate crystal structure of FimH with ligands

4

,

6

,

26 and 28, respectively; the docking results from the MM-GBSA calculation. Top

row: partial charge coloured Connolly descriptions ^[25](negative partial charges coloured in red, positive in blue). Bottom row: highlights of the interactions of the

ligands with some residues of the lectin; the dotted yellow lines represent the H-bonds, the dotted blue lines represent the π-π stacking.

Figure 5. Complexes of the closed gate crystal structure of FimH with ligands

row: partial charge coloured Connolly descriptions. Bottom row: highlights of the interactions of the ligands with some resid ues of the lectin; the dotted yellow lines

represent the H-bonds, the dotted blue lines represent the π-π stacking; in the case of ligand , the green highlight represents the ionic interaction between the

sulfate and the arginine 98.

4, 6, 26 and 28, respectively; the docking results from the MM-GBSA calculation. Top

6

Thus, myrosinase-glucosinolate reaction proved to be a

valuable biochemical system able to produce isothiocyanates in

vitro which can be further reacted with nucleophiles. The next

step is to use it as a labelling tool that could be extended to

other biologically relevant ligand/drug. In doing so, the MG

system could be used as a new bioconjugation device for in vitro

chemical ligation. Further work is currently ongoing in our group

in this perspective, in terms of both synthetic design and

biological application.

Conclusions

Our study showed that combining within the same molecule a

FimH ligand and a substrate of myrosinase preserves the

recognition of the two moieties by both proteins, without

significant loss of activity of any of the two. Myrosinase was still

able to hydrolyze the glucosinolate moiety with fairly good and

comparable activities (K_M, V_max) to the natural substrate

glucomoringin. The feasibility of the hydrolysis and the Lossen

rearrangement leading to an isothiocyanate, which could be

trapped with simple thiols, was also verified. The RIP values

Experimental

determined in adhesion inhibition studies with E. coli proved General methods: flash silica column chromatography was

that the glucosinolate moiety of our synthetic mannosides is not performed on silica gel 60N (spherical, neutral, 40-63µm,

detrimental to the interaction with FimH. Moreover, molecular Merck) or using a Reveleris® flash chromatography system. The

modelling gave us some hint on the possible mode of reactions were monitored by thin layer chromatography (TLC)

interaction with FimH, in favour of m-substitution for the on silica gel 60F254 precoated aluminium plates. Compounds

biphenylmannoside while the docking results for the aldehyde were visualised under UV light and by charring with a 10% H₂SO₄

derivatives do not fit with the experimental data. In this regard, ethanolic solution, a solution of potassium permanganate.

further practical and theoretical investigations will be carried Solvents were dried by standard methods: THF was purified

out in order to understand the binding details of these ligands. with

a dry station GT S100 immediately prior use,

dichloromethane was distilled over P₂O₅; dried methanol from

8 | J. Name., 2012, 00, 1-3

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3

ACROS ORGANICS, N,N-dimethylformamide and dioxane were 5.08 (0.30H, dd, J_3-410.0 Hz, ³J_3-23.4 Hz, H_3β), 4.98 (0.25H, bs,

View Article Online

2

3

DOI: 10.1039/C8OB01128A

dried over molecular sieves; pyridine and triethylamine were H_1β), 4.59 - 4.50 (1H, m, H_5α), 4.32 (1.4H, dd, J _6a-6b12.2 Hz, J _6a-

dried over potassium hydroxide. Molecular sieves were ₅5.6 Hz, H_6aα, H_6aβ), 4.21 (0.30H, dd, ²J _6a-6b12.2 Hz, ³J _6a-52.6 Hz,

activated prior to use by heating for 4h at 500°C. All other H_6bβ), 4.15 (1H, dd, ²J _6a-6b12.2 Hz, ³J _6a-52.4 Hz, H_6bα), 3.78 - 3.71

commercial solvents and reagents were used without further (0.30H, m, H_5β), 2.22 (0.8H, s, CH_{3 Ac β}), 2.16 (3H, s, CH_{3 Ac α}), 2.10

purification. All reactions were carried out under dry argon (0.91H, s, CH_{3 Ac β}), 2.08 (3H, s, CH_{3 Ac α}), 2.04, 2.03, (7.1H, s x 3,

atmosphere. Melting points were determined in open capillary CH_{3 Ac α,}_), 1.99 (0.83H, s, CH_{3 Ac β}); δ_C(100 MHz, CDCl₃) 191.8

tubes using a Büchi 510 apparatus and are uncorrected. Optical (HC=O), 170.6 (C=O _{Ac β}), 170.5 (C=O _{Ac α}), 170.1 (C=O _{Ac β}), 170

rotation were measured at 20°C using a Perkin Elmer 341 (C=O _{Ac β}), 169.9 (C=O _{Ac α}), 169.8 (C=O _{Ac α}), 169.7 (C=O _{Ac α}),

polarimeter with a path length of 1 dm, values are given in deg 169.6 (C=O _{Ac β}), 145.98 (Cq _Ar_), 145.95 (Cq _{Ar α}), 139.4 (Cq _Ar_),

1

dm^-1g^-1mL^-1with concentrations reported in g.100 mL^-1. H 139.37 (Cq _{Ar α}), 135.5 (Cq _{Ar α}), 133.9 (Cq _), 133.3 (Cq _{Ar α}),

Ar

NMR and ¹³C NMR were recorded with Bruker Avance II 400 or 132.2 (CH _), 132.1 (CH _{Ar α}), 130.7 (CH _), 130.3 (CH _{Ar α}),

Ar

Bruker DPX 250 spectrometers. Assignments were based on 128.0 (CH _{Ar α}), 127.9 (CH _{Ar β}), 127.54 (CH _{Ar α}), 126.9 (CH _),

Ar

DEPT 135 sequence, homo- and heteronuclear correlations. 85.5 (C-1 _α), 85.4 (C-1 _β), 76.6 (C-5 _β), 71.8 (C-3 _β), 70.9 (C-2 _α),

Chemical shifts were reported in parts per million (ppm) using 70.6 (C-2 _β), 69.7 (C-5 _α), 69.3 (C-4 _αor C-3 _α), 66.3 (C-3 _αor C-4

tetramethylsilane as the internal standard. For the ¹³C NMR in _α),65.8 (C-4 _β), 62.8 (C-6 _β), 62.4 (C-6 _α), 20.9 (CH_{3Ac α}), 20.8 (CH_3Ac

deuterated water, acetone was used as internal standard. _), 20.7, 20.63 (2 x CH_{3Ac α}), 20.6, 20.5 (CH_{3Ac α}); IR (neat)



(cm^-

Coupling constants (J) are reported and expressed in Hertz (Hz), ¹) = 1736 (C=O), 1698 (HC=O), 1604, 1515, 1483 (C=C _Ar), 1212

splitting patterns are designated as b (broad), s (singlet), d (C-O), 812,751 (C_sp2-H _Ar); ESI⁺HRMS [M+Na]⁺m/z calcd.

(doublet), dd (doublet of doublet), q (quartet), dt (doublet of 567.1295 for C₂₇H₂₉NaO₁₀S, found 567.1298.

triplet), ddd (doublet of doublet of doublet), m (multiplet).

High-resolution mass spectra (HRMS) were performed on a [4'-(2,3,4,6-Tetra-O-acetyl-α-

Maxis Bruker 4G by the “Federation de Recherche” ICOA/CBM 4-yl]carboxaldehyde 11: general procedure 1 was followed

(FR2708) platform in the electrospray ionisation (ESI) mode. The with tetraacetylated 4-bromophenyl-α-D-mannoside (1.6

D-mannopyranosyloxy)biphenyl-

9

infrared spectra of compounds were recorded on a Thermo g),^[14]4-formylphenylboronic acid (1.43 g), caesium carbonate

Scientific Nicolet iS10. The following solvents have been (3.11 g), and tetrakis(triphenylphosphine)palladium (356 mg) in

abbreviated: ethyl acetate (EA), petroleum ether (PE), dioxane/water (84/16) (58 mL). The desired product 11 was

tetrahydrofuran (THF), diethyl ether (Et₂O) and N,N- obtained as a yellow foam (1.42 g, 84%).

[ ]²⁰

dimethylformamide (DMF), methanol (MeOH). Thioglucosidase R_f= 0.46 (PE/EA : 5/5);

α

+79.6 (c 0.69 in MeOH); δ_H(400

D

from Sinapis alba (white mustard) seed (myrosinase, EC MHz, CDCl₃) 10.05 (1H, s, HC=O), 7.94 (2H, d, ³J 7.9 Hz, H_Ar), 7.71

3.2.1.147, 25U, ≥100 units. g⁻¹) was purchased from Sigma– (2H, d, ³J 7.9 Hz, H_Ar), 7.6 (2H, d, ³J 8.4 Hz, H_Ar), 7.20 (2H, d, ³J 8.4

Aldrich (Saint-Quentin Fallavier, France). Experimental Hz, H_Ar), 5.61 - 5.57 (2H, m, H₁, H₃), 5.49 - 5.46 (1H, m, H₂), 5.40

3

procedures and analytical data for compounds

6

,

13

,

16

,

21

,

24

,

(1H, t, ³J_4-3J_4-510.2 Hz, H₄), 4.30 (1H, dd, ³J_6a-6b12.2 Hz, ³J_6a-55.2

Hz, H_6a), 4.15 - 4.07 (2H, m, H_6b, H₅), 2.22, 2.07, 2.05, 2.04 (12H,

s x 4, CH_{3 Ac}); δ_C(100 MHz, CDCl₃) 192 (HC=O), 170.6, 170.1,

26 28 and 17 18 are given in the supporting information.

-

General procedure 1: tetraacetylated (4-bromophenyl)-α-D- 170.07, 169.8 ( 4 x C=O _Ac), 156.1 (Cq _Ar) , 146.4 (Cq _Ar), 135.2 (Cq

mannopyranoside 9-10 (1 equiv.), 4- or 3-formylphenyl boronic _Ar), 134.7 (Cq _Ar), 130.5 (CH _Ar), 128.8 (CH _Ar), 127.4 (CH _Ar), 117.1

acid (3 equiv.), caesium carbonate (3 equiv.), and (CH _Ar), 95.9 (C-1), 69.5 (C-2 or C-5), 69.49 (C-5 or C-2), 69.0 (C-

tetrakis(triphenylphosphine)palladium (10 mol%) were heated 3), 66.1 (C-4), 62.2 (C-6), 21.0, 20.85, 20.83, 20.82 (4 x CH_{3 Ac});

at 80°C in dioxane/water (84/16, 0.055 M) for 1 h, under argon IR (neat) ν (cm^-1) = 1742 (C=O _Ac), 1698 (C=O ald), 1602, 1523,

atmosphere. The solvent was removed under reduced pressure 1495 (C=C _Ar), 1212 (C-O), 847, 818 (C_sp2-H _Ar); ESI⁺HRMS [M+H]⁺

and the crude was purified by silica gel column chromatography m/z calcd. 529.1704 for C₂₇H₂₉O₁₁, found 529.1708.

(PE/EA: 100/0 to 40/60).

General procedure 2: ammonium acetate (1.1 equiv.) was

added to a solution of mannoside 11-13 (1 equiv.) in

[4'‐(2,3,4,6-Tetra-O-acetyl-α-D-mannopyranosylsulfanyl)

biphenyl‐4‐yl]carboxaldehyde 12: general procedure 1 was

followed with tetraacetylated (4-bromophenyl)-1-thio-α-D-

mannopyranoside 10 (3 g, 5.79 mmol), 4-formylphenyl boronic

acid (2.6 g), caesium carbonate (3.11 g), and

nitromethane (0.08 M). The reaction mixture was heated for 2

to 24h. The solvent was removed under reduced pressure and

the crude was purified by silica gel column chromatography

(PE/EA : 100/0 to 40/60) (compound 16). For compounds 14 and

15, the residue obtained after evaporation was taken up with

ethyl acetate and washed 2 times with saturated aqueous

NH₄Cl, dried over MgSO₄, filtered and the solvent evaporated

under reduced pressure to give the desired product which was

used in the next step without any further purification.

tetrakis(triphenylphosphine)palladium

(650

mg)

in

dioxane/water 84/16 (105 mL). The desired product 12 was

obtained as a mixture of α/β anomers (80/20) as a yellow foam

(2.7 g, 88%).

R_f= 0.45 (PE/EA : 5/5); δ_H(400 MHz, CDCl₃) 10.05 (1.2H, s, CHO),

3

7.95 (2.4H, d, J 7.9 Hz, CH_Ar), 7.71 (2.4H, bd, J 7.9 Hz, CH_Ar),

7.61 - 7.53 (4.8H, m, CH_Ar), 5.69 (0.30H, d, ³J_2,33.4 Hz, H₂_), 5.56

(1H, bs, H ), 5.51 (1H, s, H ), 5.45 - 5.26 (2.4H, m, H , H , H ),

General procedure 3, thiohydroximate formation: titanium

tetrachloride (2.2 equiv.) was added dropwise to a stirred



4

1α

2α

3α

4α

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solution of triethylsilane (2.1 equiv.) and biphenylnitrovinyl and triethylsilane (1.31 mL) in anhydrous dichloromethane (65

View Article Online

DOI: 10.1039/C8OB01128A

derivative 14-16 (1 equiv.) in anhydrous dichloromethane (0.06 mL). For the second step, were used 2,3,4,6-tetra-O-acetyl-1-

M) under argon atmosphere. The mixture was stirred at room thio--D-glucopyranose (818 mg, 0.9 equiv.) and triethylamine

temperature overnight. The reaction mixture was quenched by (1.64 mL) and 65 mL of anhydrous dichloromethane. The

addition of water, then the aqueous phase was extracted 2 desired product 20 was obtained after purification (PE/EA:

times with dichloromethane. The combined organic phases 100/0 to 20/80) as a mixture of α/β anomers (75/25) as a yellow

were dried over MgSO₄and the solvent evaporated under foam (1 g, 48%).

reduced pressure. The residue was taken up with anhydrous R_f=0.25 (PE/EA : 5/5); δ_H(400 MHz, CDCl₃) 8.25-8.20 (m, 1H,

dichloromethane (0.06 M) and 2,3,4,6-tetra-O-acetyl-1-thio-

D-glucopyranose (1.2 equiv.) and triethylamine (3 equiv.) were _{Ar α+β}), 5.68 (0.2H, d, J 3.2 Hz, H_2β), 5.55-5.50 (2H, m, H_{1M α}



-

NOH), 7.60-7.52 (7.2H, m, CH _{Ar α+β}), 7.34 (2.4 H, d, ³J 7.8 Hz, CH

3

,

2-3

sequentially added and the reaction mixture was stirred for 3h H_{2M α}), 5.39-5.26 (2.3H, m, H_{3M α}, H_{4M α}, H ), 5.12-4.95 (3.7H, m,



4

at rt. The solvent was evaporated under reduced pressure and H_2G, H_3G, H_4G,H_3β,H_1β), 4.87 (1H, d, ³J_1-210.0 Hz, H_1G), 4.59-4.53

the crude residue was purified by silica gel column (1H, m, H_{5M α}), 4.32 (1.2H, dd, ²J_6a-6b12.2 Hz, ³J_6a-55.5 Hz, H_6aMα

chromatography (PE/EA: 100/0 to 50/50) to give the desired H_{6aM β}), 4.21-4.01 (3.3H, m, H_{6bM α}, H_6aG), 3.98 (2H, bs, CH₂C=N),

,

product.

3.76-3.70 (0.2H, m, H_{5M β}), 3.60-3.53 (1H, m, H_5G), 2.22 (0.6H, s,

CH_{3 M β}), 2.16 (3H, s, CH₃), 2.10 (0.6H, s, CH_{3 M β}), 2.08, 2.07, 2.05

(9H, s x 3, CH₃), 2.04 (0.6H, s, CH_{3 M β}), 2.02, 2.00 (6H, 2s, CH₃),

1.99 (0.6H, s, CH_{3 M β}), 1.97, 1.96 (6H, 2s, CH₃); δ_C(100 MHz,

CDCl₃) 170.7 (C=O_), 170.6, 170.5, 170.2 (C=O_), 170.2, 170.1

(C=O_), 170.0, 169.9, 169.8, 169.6 (C=O_), 169.2, 169.1 (C=O),

151.0 (C=N), 140.4 (Cq _Ar_), 140.3 (Cq _Ar), 139.3 (Cq _Ar), 135.4 (Cq

_Ar), 132.5 (CH _Ar_), 132.4 (CH _Ar), 131.9 (Cq _Ar), 128.8, 127.8,

(Z)-S-(2,3,4,6-Tetra-O-acetyl--D-glucopyranosyl) [4'-(2,3,4,6-

tetra-O-acetyl-α-D-mannopyranosyloxy)biphenyl-4-yl]aceto

thiohydroximate 19: general procedure 2 was followed from

mannoside 11 (1.75 g), ammonium acetate (280 mg) in

nitromethane (47 mL) for 2h at reflux. The desired product 14

was obtained after work up as a yellowish foam (1.86 g, 99%).

General procedure 3 was then followed from biphenyl nitrovinyl

derivative 14 (300 mg), titanium tetrachloride (130

triethylsilane (180 L) in anhydrous dichloromethane (9 mL).

For the second step, were used mL of anhydrous

dichloromethane, 2,3,4,6-tetra-O-acetyl-1-thio- -D-gluco

pyranose (232 mg) and triethylamine (220 L). The desired

127.6 (CH _Ar_), 127.5 (CH ), 85.8 (C-1_M), 85.7 (C-1_), 79.6 (C-

Ar

L.) and

1_G), 76.5 (C-5_), 75.8 (C-5_G), 73.8 (C-2_Gor C-3_Gor C-4_G), 71.9 (C-

3_), 71.0 (C-1 or C-2_M), 70.7 (C-2 _), 70.1 (C-2_Gor C-3_Gor C-4_G),

69.7 (C-5_M), 69.5 (C-3_Mor C-4_M), 68.1 (C-2_Gor C-3_Gor C-4_G), 65.8

(C-4 _), 66.4 (C-3_Mor C-4_M), 62.8 (C-6 _), 62.5 (C-6_Gor C-6_M), 62.3

(C-6_Mor C-6_G), 38.5 (CH₂C=N), 21.0, 20.8, 20.7, 20.65, 20.62 (CH₃



9





product 19 was obtained after purification as a yellowish foam

(260 mg, 53%).

R_f= 0.60 (PE/EA : 4/6);

_Ac); IR (neat)



(cm^-1) = 1743 (C=O), 1662 (C=O), 1484 (C=C _Ar),

1217 (C-O), 912 (C_sp2-H _Ar); ESI⁺HRMS [M+H]⁺m/z calcd.

[ ]²⁰

+36.5 (c 0.94 in MeOH); δ_H(400

α

D

936.2413 for C₄₂H₅₀NO₂₀S, found 936.2414.

MHz, CDCl₃) 8.67 (s, 1H, NOH), 7.58 - 7.50 (4H, m, H_Ar), 7.33 (2H,

d, ³J 7.6 Hz, H_Ar), 7.17 (2H, d, ³J 8.0 Hz, H_Ar), 5.64 - 5.55 (2H, m,

General Procedure 4, sulfation: sulfur trioxide-pyridine

complex (5 equiv.) was added to a solution of thiohydroximate

(1 equiv.) in anhydrous DMF (0.4 M). The suspension was

heated at 50°C overnight. It was then cooled at 0°C, quenched

by addition of a 0.5M aqueous KHCO₃solution (10 equiv.) and

then stirred for 30 minutes at room temperature. The solvent

was evaporated under reduced pressure and the residue was

purified by silica gel column chromatography (EA/MeOH 9/1).

H_3M, H_1M), 5.49-5.46 (1H, m, H_2M), 5.40 (1H, t, ³J _4-5J _4-310.0 Hz,

3

H_4M), 5.13-4.95 (m, 3H, H_2G, H_3G, H_4G), 4.88 (1H, d, ³J_1-210.0 Hz,

3

H_1G), 4.30 (1H, dd, J_6a-6b12.2 Hz, J_6a-54.6 Hz, H_6aM), 4.17-3.97

(6H, m, H_6bM, H_5M,H_6aG, H_6bG, CH₂C=N ), 3.61-3.52 (1H, m, H_5G),

2.22, 2.08, 2.07, 2.05, 2.01, 1.97 (s x 6 , 24H, CH_{3 Ac}); δ_C(100

MHz, CDCl₃) 170.7, 170.6, 170.3, 170.2, 170.1, 169.9, 169.4,

169.2 (C=O), 155.3 (Cq _Aror C=N), 151.1 (C=N or Cq _Ar), 139.6 (Cq

_Ar), 135.5 (Cq _Ar), 134.7 (Cq _Ar), 128.6 (CH _Ar), 128.3 (CH _Ar), 127.4

(CH _Ar), 117.0 (CH _Ar), 96.0 (C-1_M), 79.6 (C-1_G), 75.8 (C-5_G), 73.8, (Z)-S-(2,3,4,6-Tetra-O-acetyl-

-D-glucopyranosyl) [4'-(2,3,4,6-

70.1 (C-2_Gor C-3_Gor C-4_G), 69.5 (C-2_M), 69.3 (C-5_M), 69.0 (C-3_M), tetra-O-acetyl-α- -mannopyranosyloxy)biphenyl-4-yl]aceto

D

68.1 (C-2_Gor C-3_Gor C-4_G), 66.1 (C-4_M), 62.2, 62.3 (C-6_M, C-6_G), thiohydroximate N,O-sulfate potassium salt 22 : general

38.5 (CH₂C=N), 20.9, 20.8, 20.79, 20.78, 20.66, 20.62 (6 x CH₃); procedure 4 was followed from thiohydroximate 17 (440 mg)

IR (neat)

1220, 1186, 1127 (C-O), 847, 818 (C_sp2-H _Ar); ESI⁺HRMS [M+H]⁺DMF (6 mL). The desired compound 22 was obtained as a

m/z calcd. 920.2641 for C₄₂H₅₀NO₂₀S, found 920.2636. yellowish oil (400 mg, 80%).

[ ]²⁰

+34.6 (c 1.53 in MeOH); δ_H(400



(cm^-1) = 1742 (C=O), 1632 (C=C), 1602, 1496 (C=C _Ar), and sulfur trioxide-pyridine complex (380 mg) in anhydrous

R_f= 0.37 (EA/MeOH : 9/1);

α

D

(Z)-S-(2,3,4,6-Tetra-O-acetyl-

tetra-O-acetyl-α- -mannopyranosylsulfanyl)biphenyl-4-

yl]acetothiohydroximate 20 general procedure

-D

-glucopyranosyl) [4'-(2,3,4,6- MHz, CDCl₃) 7.65-7.58 (4H, m, CH_Ar), 7.47 (2H, d, ³J 7.9 Hz, CH_Ar),

D

7.22 (2H, d, ³J 8.4 Hz, CH_Ar), 5.67 (s, 1H, H_1M), 5.53-5.46 (2H, m,

3

:

2

was H_3M, H_2M), 5.33 (1H, t, ³J _4-3J _4-59.8 Hz, H_4M), 5.20-5.09 (2H, m,

followed from mannoside 12 (2.6 g), ammonium acetate (420 H_1G, H_2G), 4.99 (1H, t, ³J _4-3J_4-59.7 Hz, H_4G), 4.88 (1H, t, ³J_3-2J_3-4

3

mg) in nitromethane (70 mL) for 2h at reflux. The desired 9.6 Hz, H_3G), 4.24 (1H, dd, ²J_6a-6b11.9 Hz, ³J_6a-55.3 Hz, H_6aM), 4.17-

product 15 was obtained after work up as a yellowish foam (2.9 4.05 (5H, m, H_5M, H_6aG, H_6bM, CH₂C=N), 3.91 (1H, d, J_6b-6a10.8

2

g, 100%). General procedure 3 was then followed from biphenyl Hz, H_6bG), 3.77-3.71 (1H, m, H_5G), 2.19, 2.06, 2.03, 2.01, 1.98,

nitrovinyl derivative 15 (2.3 g), titanium tetrachloride (946

L.) 1.96, 1.93, 1.91 (24H, 8 s, CH_{3 Ac}); δ_C(100 MHz, CDCl₃) 172.22,

10 | J. Name., 2012, 00, 1-3

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172.2, 171.6, 171.5, 171.48, 171.4, 171.1, 170.8 (C=O _Ac), 157.8 (1H, m, H_5G); δ_C(100 MHz, D₂O, internal acetone) _V1_ie6_w3_A.0_rtic(_lC_eq_On_A_li_r_n)_e,

(C=N or Cq _Ar), 156.5 (Cq _Aror C=N), 140.7 (Cq _Ar), 136.8 (Cq _Ar), 155.7 (C=N), 139.4 (Cq _Ar), 135.0 (Cq _Ar), 1^D3^O4^I.^:5¹⁰(C^.1q⁰³_A⁹_r^/)^C, 1^8O29^B0.2¹¹(²C⁸H^A

135.9 (Cq _Ar), 130.0 (CH _Ar), 129.2 (CH _Ar), 128.2 (CH _Ar), 118.2 (CH _Ar), 128.6 (CH _Ar), 127.7 (CH _Ar), 117.9 (CH _Ar), 98.7 (C-1_M), 82.1

_Ar), 97.1 (C-1_M), 80.8 (C-1_G), 76.7 (C-5_G), 75.0 (C-3_G), 71.3 (C-2_G), (C-1_G), 80.4 (C-5_G), 77.6 (C-2_Gor C-3_G), 73.9 (C-5_M), 72.5 (C-3_Gor

70.63, 70.59, 70.5 (C-5_M, C-3_M, C-2_M), 69.3 (C-4_G), 67.1 (C-4_M), C-2_G), 71.1 (C-3_M), 70.6 (C-2_M), 69.3 (C-4_G), 67.1 (C-4_M), 61.2

63.4 (C-6_Mor C-6_G), 63.1 (C-6_Gor C-6_M), 39.2 (CH₂CN), 20.64, (CH₂C=N), 60.9 (C-6_G), 38.6 (C-6_M); IR (neat)

20.62, 20.60, 20.53, 20.49 (CH_{3 Ac}); IR (neat)

(cm^-1) = 1742 H), 1607, 1497 (C=C), 1231, 1056 (C-OH); ESI^-HRMS [M-K]^-m/z



(cm^-1) = 3384 (O-



(C=O), 1666 (C=C), 1603, 1497 (C=C _Ar), 1230, 1127 (C-O), 847, calcd. 662.1219 for C₂₆H₃₂NO₁₅S₂, found 662.1224.

818 (C_sp2-H Ar); ESI^-HRMS [M-K]^-m/z calcd. 998.2064 for

C₄₂H₄₈NO₂₃S₂, found 998.2069.

(Z)-S-(-D-Glucopyranosyl) [4'-(α-D-mannopyranosylsulfanyl)

biphenyl-4-yl]acetothiohydroximate N,O-sulfate potassium

(Z)-S-(2,3,4,6-Tetra-O-acetyl-

tetra-O-acetyl-α-



-

D

-glucopyranosyl) [4'-(2,3,4,6- salt 5: general procedure 5 was followed from acetylated

D

-mannopyranosylsulfanyl)biphenyl-4-yl]

compound 23 (280 mg, 0.27 mmol, 1 equiv.) to give product 5

acetothiohydroximate N,O-sulfate potassium salt 23: general as a white resin (76 mg, 40%).

[ ]²⁰

procedure 4 was followed from thiohydroximate 20 (950 mg)

α

+126.6 (c 1.23 in MeOH); δ_H(400 MHz, CD₃OD) 7.66-7.55

D

and sulfur trioxide-pyridine complex (811 mg) in anhydrous (6H, m, CH _Ar), 7.50 (2H, d, ³J 8.2 Hz, CH _Ar), 5.48 (1H, d, ³J_1-21.5

DMF (15 mL). After 24h at 50°C, sulfur trioxide-pyridine complex Hz, H_1M), 4.56 (1H, d, J 9.5 Hz, H_1G), 4.30 (1H, d, J 16.1 Hz,

3

2

1-2

(811 mg, 5.1 mmol, 5 equiv.) was added again, and the reaction CH₂C=N), 4.13-4.03 (3H, m, H_2M, H_4M,CH₂C=N), 3.89-3.68 (5H,

mixture was heated for an additional 24h. The desired m, H_3M, H_5M, H_6aG, H_6aM, H_6bM), 3.66-3.58 (1H, m, H_6bG), 3.27-3.21

compound 23 was finally obtained as a yellow foam (290 mg, (2H, m, H_3G, H_5G), 3.19-3.13 (2H, m, H_2G,H_4G); δ_C(100 MHz,

26%).

CD₃OD) 159.1 (C=N), 139.8, 139.0, 135.5, 133.5 (Cq _Ar), 131.9,

+122.1 (c 0.13 in MeOH); δ_H128.6, 127.0, 126.9 (CH _Ar), 89.0 (C-1_M), 81.5 (C-1_G), 80.8 (C-5_G

[ ]²⁰

R_f= 0.35 (EA/MeOH : 9/1);

α

D

(250 MHz, CD₃OD) 7.70-7.57 (6H, m, CH_Ar), 7.49 (2H, d, ³J 8.2 Hz, or C-3_G), 78.0 (C-2_Gor C-4_G), 74.3 (C-4_M), 72.8 (C-4_Gor C-2_G), 72.3

CH_Ar), 5.70-5.57 (1H, m, H_1M), 5.54-5.48 (1H, m, H_2M), 5.32-5.27 (C-2_M), 71.8 (C-5_Mor C-3_M), 69.8 (C-3_Gor C-5_G), 67.3 (C-3_Mor C-

(1H, m, H_3M), 5.20-5.09 (2H, m, H_1G, H_2G), 5.05-4.83 (3H, m, H_3G

H_4G,H_5M), 4.62-4.49 (1H, m, H_4M), 4.37-4.20 (1H, m, H_6aM), 4.18- (neat)

4.04 (4H, m, H_6’M, H_6aG, CH₂CN), 3.91 (1H, dd, ²J_6a-6b12.3 Hz, ³J_6-(C_sp2-H _Ar); ESI^-HRMS [M-K]^-m/z calcd. 678.0990 for

,

5_M), 61.4 (C-6_Gor C-6_M), 61.2 (C-6_Mor C-6_G), 37.9 (CH₂C=N); IR



(cm^-1) = 3380 (O-H), 1484 (C=C _Ar), 1276 (C-O), 1057

2.4 Hz, H_6aG), 3.83-3.66 (1H, m, H_5G), 2.15, 2.08, 2.02, 2.00, C₂₆H₃₂NO₁₄S₃, found 678.0996.

5

1.99, 1.98, 1.93, 1.91 (24H, 8s, CH_{3 Ac}); δ_C(62.5 MHz, CD₃OD)

172.3, 172.2, 171.5, 171.47, 171.1, 170.8 (C=O), 157.6 (C=N), General Procedure 6: 0.67 U of myrosinase from Sinapis alba

141.8 (Cq _Ar), 140.3 (Cq _Ar), 136.7 (Cq _Ar), 133.9 (CH _Ar), 132.7 (Cq (white mustard) seed (10 U/mL) solution was added to a

_Ar), 130.1 (CH _Ar), 128.8 (CH _Ar), 128.4 (CH _Ar), 86.6 (C-1_M), 80.9 solution of glucosinolate (0.1 mmol,

(C-1_G), 76.7, 75.0, 71.9, 71.3, 71.0, 71.8, 69.3, 67.5, 63.6, 63.1, benzylmercaptan (0.3 mmol, 3 equiv.), in a mixture of

39.2 (CH₂CN), 20.67, 20.61, 20.5, 20.45 (CH₃); IR (neat)

(cm^-1) water/phosphate buffer pH = 7 (2/1) (0.029 M). The mixture

1

equiv.) and



= 1742 (C=O), 1666, 1603 (C=O), 1497, 1434, 1367 (C=C _Ar), 1230 was stirred at 37°C for 24h, then purified using Reveleris®

(C-O), 1035, 1001, 979 (C_sp2-H _Ar); ESI^-HRMS [M-K]^-m/z calcd. column chromatography on C-18 reverse phase (H₂O/MeOH :

1014.1836 for C₄₂H₄₈NO₂₂S₃, found 1014.1858.

100/0 to 0/100).

S-Benzyl-N-(4-(α-

31: general procedure 6 was followed from glucosinolate

D

-mannopyranosyloxy)benzyl)dithiocarbamate

General procedure 5: potassium methoxide (0.4 equiv.) was

added to a solution of acetylated compound (1 equiv.) in

anhydrous methanol (0.15 M). The reaction mixture was stirred

at room temperature during 6h. The solvent was then

evaporated under reduced pressure and the crude product was

purified using Reveleris® column chromatography on C-18

reverse phase (H₂O/MeOH : 100/0 to 0/100).

3

(30

mg, 0.0480 mmol, 1 equiv.) to give product 31 as a solid (10.2

mg, 47%).

[ ]²⁰

α

+72.6 (c 0.74 in MeOH); δ_H(400 MHz, CD₃OD) 7.39-7.04

D

(9H, m, CH _Ar), 5.46 (1H, s, H₁), 4.84 (2H, s, CH₂N) 4.54 (2H, s,

CH₂S), 3.99 (1H, s, H₂), 3.89 (1H, dd, ³J_3-49.4 Hz, ³J_3-23.4 Hz, H₃),

3.78-3.68 (3H, m, H_6a, H_6b, H₄), 3.61-3.55 (1H, m, H₅); δ_H(100

(Z)-S-(



-

D

-Glucopyranosyl)

[4'-(α-D-mannopyranosyloxy) MHz, CD₃OD) 197.6 (C=S), 155.9 (Cq _Ar), 137.3 (Cq _Ar), 131.1 (Cq

biphenyl-4-yl]acetothiohydroximate N,O-sulfate potassium _Ar), 129.0 (CH _Ar), 128.7 (CH _Ar), 128.1 (CH _Ar), 126.8 (CH _Ar), 116.4

salt 4: Ggeneral procedure 5 was followed with acetylated (CH _Ar), 98.8 (C-1), 73.9 (C-5), 71.0 (C-3), 70.6 (C-2), 66.9 (C-4),

compound 22 (400 mg, 0.38 mmol, 1 equiv.) to give product

4

61.3 (C-6), 49.4 (CH₂N), 38.81 (CH₂S); ESI⁺HRMS [M+H]⁺m/z

as a white resin (207 mg, 78 %).

calcd. 452.1196 for C₂₁H₂₅NO₆S₂, found 452.1195.

[ ]²⁰

α

+55.7 (c 0.98 in MeOH); δ_H(400 MHz, D₂O) 7.43 (4H, d, ³J

D

8.3 Hz

CH_Ar), 5.59 (1H, s, H_1M), 4.77-4.70 (1H, m, H_1G), 4.18 (1H, bs, dithiocarbamate 32: general procedure 6 was followed from

, D-mannopyranosyloxyphenyl)benzyl)

CH_Ar), 7.36 (2H, d, ³J 8.0 Hz, CH_Ar), 7.10 (2H, d, ³J 8.5 Hz, S-Benzyl-N-(4-(4-(α-

3

H_2M), 4.13-4.04 (3H, m, H_6M, H_3M), 3.82 (1H, t, J_4-3J_4-59.8 Hz, glucosinolate

4 (16 mg, 0.0228 mmol, 1 equiv.) to give product

H_4M), 3.78-3.65 (3H, m, CH₂C=N, H_5M), 3.65-3.54 (2H, m, H_6G), 32 as a solid (6 mg, 50 %).

3.48-3.41 (1H, m, H_4G), 3.41-3.32 (2H, m, H_2G, H_3G), 3.24-3.18

J. Name., 2013, 00, 1-3 | 11

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Journal Name

[ ]²⁰

α

+66.9 (c 0.35 in MeOH); δ_H(400 MHz, CD₃OD) 7.63-7.16 bacterial pellet resulting after centrifugation and d_Ve_ic_ewan_Art_ta_ict_leio_On_nlio_nef

D

(13H, m, CH_Ar), 5.52 (1H, s, H₁), 4.94 (2H, s, CH₂N), 4.56 (2H, s, media was washed twice with PBS (2 mL)^Da^On^Id^{: 1}s⁰u^.1s⁰p³e⁹n^/Cd⁸e^Od^Bi⁰n¹¹P²B^8AS

CH₂S), 4.03 (1H, s, H₂), 3.92 (1H, dd, ³J_3-49.4 Hz, ³J_3-23.4 Hz, H₃), buffer afterwards. The bacterial suspension was adjusted to

3.80-3.70 (3H, m, H₆, H₄), 3.66-3.60 (1H, m, H₅); δ_C(100 MHz, OD₆₀₀= 0.4 with PBS.

CD₃OD) 199.2 (C=S), 157.5 (Cq _Ar), 141.1 (Cq _Ar), 138.7 (Cq _Ar), The inhibition assay was performed according to the

137.3 (Cq _Ar), 136.2 (Cq _Ar), 130.1 (CH _Ar), 129.5 (CH _Ar), 129.0 (CH literature.^[19]Black microtiter plates (Nunc, MaxiSorp) were

_Ar), 128.3 (CH _Ar), 127.7 (CH _Ar), 118.1 (CH _Ar), 100.2 (C-1), 75.4 incubated overnight with mannan from Saccharomyces

(C-5), 72.4 (C-3), 72.0 (C-2), 68.4 (C-4), 62.7 (C-6), 51.0 (CH₂N), cerevisiae (1.2 mg/mL carbonate buffer, 120 μL/well) at 37°C at

40.3 (CH₂S); ESI⁺HRMS [M+H]⁺m/z calcd. 528.1509 for 100 rpm. After washing three times with PBST microtiter plates

C₂₇H₂₉NO₆S₂, found 528.1508.

have been blocked with PVA (poly vinyl alcohol) by adding a

solution of 1 % PVA in PBS (120 μL/well) and incubation at room

temperature, 3 h, 100 rpm. Afterwards plates were washed

S-Benzyl-N-(3-(4-(α-D-mannopyranosyloxyphenyl)benzyl)

dithiocarbamate 33: general procedure 6 was followed from with PBST twice and PBS once. Finally, a serial dilution of the

glucosinolate (30 mg, 0.0427 mmol, 1 equiv.) to give the particular inhibitor was prepared (50 μL/well) and the bacterial

6

desired product as a solid 33 (18 mg, 80%).

suspension was added (50 μL/well). After incubation for one

[ ]²⁰

α

+75.3 (c 1.5 in MeOH); δ_H(400 MHz, CD₃OD) 7.58-7.15 hour at 37°C and 100 rpm, microtiter plates were washed three

D

(13H, m, CH _Ar), 5.53 (1H, s, H₁), 4.97 (2H, s, CH₂N), 4.56 (2H, s, times with PBS and filled with PBS (100 μL/well) for terminal

CH₂S), 4.03 (1H, s, H₂), 3.93 (1H, dd, ³J_3-49.4 Hz, ³J_3-23.3 Hz, H₃), fluorescence intensity read out (excitation wavelength 485 nm,

3.81-3.70 (3H, m, H₆, H₄), 3.66-3.60 (1H, m, H₅); δ_C(100 MHz, emission wavelength 535 nm).

CD₃OD) 199.3 (C=S), 157.5 (Cq _Ar), 142.2 (Cq _Ar), 139.2 (Cq _Ar),

138.7(Cq _Ar), 136.4 (Cq _Ar), 130.5 (CH _Ar), 130.1 (CH _Ar), 129.5 (CH

Molecular modeling

Molecular modeling was performed using the Schrödinger

software package implementing the Maestro interface.^[27]The

ligands were built using Maestro then minimized using

MacroModel,^[28]with the OPLS3 force field in implicit water

(GB/SA continuum solvation model). The minimized structures

were prepared for docking using LigPrep.^[29]The docking studies

were performed on the open gate (PDB code: 1klf),^[21]or on the

closed gate (PDB code: 1uwf) crystal structure of FimH.^[22]

Receptor grids suitable for docking were built with the Glide

docking software,^[20]by defining a outer box of 20 Å centered

on the ligand from the crystal structure. Each grid was

generated using the OPLS3 force field and including aromatic

protons as H-bond donors. Extra precision (XP) docking was

carried out with Glide, setting the ligand flexible and including

aromatic protons as H-bond donors. At most five poses per

ligand were written out, discarding poses as duplicate if both

RMS deviation was less than 0.5 Å and the maximum atomic

displacement was less than 1.3 Å. For re-scoring, the docking

outputs were processed in a MM-GBSA (molecular mechanics-

generalized born surface area) calculation,^[23]giving the free

energy of binding in kJ.mol^-1. The MM-GBSA calculation was

performed using the VGSB solvation model and the OPLS3 force

field. Hierarchical sampling was carried out, allowing flexibility

of the two important tyrosine residues (Tyr 48 and 137) of the

_Ar), 129.1 (CH _Ar), 128.2 (CH _Ar), 127.5 (CH _Ar), 127.2 (CH _Ar), 126.7

(CH _Ar), 118.1 (CH _Ar), 100.2 (C-1), 75.4 (C-5), 72.4 (C-3), 72.0 (C-

2), 68.4 (C-4), 62.7 (C-6), 51.3 (CH₂N), 40.2 (CH₂S); ESI⁺HRMS

[M+H]⁺m/z calcd. 528.1509 for C₂₇H₂₉NO₆S₂, found 528.1510

Capillary Electrophoresis procedure followed protocols earlier

developed in our laboratory.^[8]Substrates are used at large

excess relative to the enzyme (about 100 times). For this reason,

Myrosinase was used at 0.05 U.mL^-1for all assays. The

myrosinase activity was determined by following the hydrolysis

of the glucosinolate substrate, the SO₄^2-produced was detected

by the C⁴D and quantified. The volume of the reaction mixture

was set down to 7 L, instead of the 100 µL used previously, and

was done in a mico-vial of the CE instrument autosampler.

The nonlinear curve fitting program PRISM® 5.04 (GraphPad,

San Diego, California, USA) was used to determine K_Mand V_max

according to the following equation:

V_max[S]

V_i

K_m[S]

where V_iis the reaction rate, K_Mis the Michaelis Menten

constant, V_maxis the maximum reaction velocity and [S] is the

substrate (glucosinolate) concentration.

Adhesion inhibition tests:

Bacterial inhibition studies: buffers: PBS buffer: PBS tablets receptor. The extensive docking and MM-GBSA results are listed

were obtained from GIBCO containing phosphate (as sodium in Tables S2-S5 (see Supporting Information).

phosphates), 10 mM, potassium chloride (KCl), 2.68 mM,

sodium chloride (NaCl), 140 mM, pH = 7.45; PBST buffer: PBS

buffer + 0.05% v/v Tween®20; carbonate buffer solution (pH

Conflicts of interest

9.6): sodium carbonate (10.6 g) and sodium hydrogen

carbonate (8.40 g) were dissolved in bidest. Water (1.0 L), pH

values were adjusted by using 0.1 M HCl or 0.1 M NaOH.

There are no conflicts to declare.

Acknowledgements

This work has been partly supported by the Université Orléans,

the Centre National de la Recherche Scientifique (CNRS), the

Bacterial culture: The bacterial E. coli strain PKL1162^[26]was

cultured from a frozen stock in LB media (+ampicillin 100 mg/mL

and chloramphenicol 50 mg/mL) overnight at 37°C. The

12 | J. Name., 2012, 00, 1-3

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16 a) G. Kumaran, G. H. Kulkarni, Tetrahedron Lett. 1994 , 35 ,

Labex SynOrg (ANR-11-LABX-0029). G. C. thanks the Labex

SynOrg (ANR-11-LABX-0029) for the doctoral fellowship. T.K. L.

thanks the International Center at Christiana Albertina

University of Kiel for funding this French-German collaboration.

5517-5518; b) G. Kumaran, G. H. Kulkarni, Tetr^Va^ieh^wed^Arr^to^icln^{e O}Lⁿe^ltⁱⁿt^e.

DOI: 10.1039/C8OB01128A

1994, 35, 9099-9100.

17 Landemarre, L.; Duverger, E. Lectin Glycoprofiling of

Recombinant Therapeutic Interleukin-7. In Glycosylation

Engineering of Biopharmaceuticals: Methods and Protocols;

Beck, A., Ed.; Humana Press: Totowa, NJ, 2013; pp 221–226.

18 a) see supporting information for results for compounds 4 and 6;

Notes and references

b) http://www.glycodiag.com/; c) L. Landemarre; P. Cancellieri;

E. Duverger Glycoconjugate Journal 2013, 30, 195-203.

19 M. Hartmann, A. K. Horst, P. Klemm, T. K. Lindhorst, Chem.

Commun. 2010, 46, 330-332.

1

2

P. J. Robiquet, F. Boutron, J. Pharm. Chim., 1831, 279-298.

a) A. M. Bones, J. T. Rossiter, Physiol. Plant. 1996, 97, 194-208;

b) A. Ratzka, H. Vogel, D. J. Kliebenstein, T. Mitchell-Olds, J.

Kroymann, Proc. Natl. Acad. Sci. USA 2002, 99, 11223-11228.

a) M. G. Ettlinger, A. J. Lundeen, J. Am. Chem. Soc. 1956, 78,

4172-4173; b) M. G. Ettlinger, A. J. Lundeen, J. Am. Chem. Soc.

1957, 79, 1764-1765.

a) J. W. Fahey, A. T. Zalcmann, P. Talalay, Phytochemistry

2001, 56, 5-51; b) N. Agerbirk, C. E. Olsen, Phytochemistry

2012, 77, 16-45.

20 Schrödinger Release 2016-1: Glide, version 7.0, Schrödinger,

LLC, New York, NY, 2016.

3

4

21 C. S. Hung, J. Bouckaert, D. Hung, J. Pinkner, C. Widberg, A.

DeFusco, C. G. Auguste, R. Strouse, S. Langermann, G.

Waksman, S. J. Hultgren, , Mol. Microbiol. 2002, 44, 903-15.

22 J Bouckaert, J. Berglund, M. Schembri, E. De Genst, L. Cools,

M. Wuhrer, C.-S. Hung, J. Pinkner, R. Slättegård, A. Zavialov,

D. Choudhury, S. Langermann, S. J. Hultgren, L. Wyns, P.

Klemm, S. Oscarson, S. D. Knight, H. De Greve, Mol. Microbiol.

2005, 55, 441-455.

23 Schrödinger Release 2016-1: Prime, Schrödinger, LLC, New

York, NY, 2016.

24 C. Grabosch, M. Hartmann, J. Schmidt-Lassen, T. K. Lindhorst,

ChemBioChem 2011, 12, 1066-1074.

25 a) M. L. Connolly, Science 1983, 221, 709-713; b) M. L.

Connolly, J. Appl. Crystallogr. 1983, 16, 548-558.

26 (a) A. Reisner, J. A. J. Haagensen, M.A. Schembri, E.L. Zechner,

S. Molin, Mol. Microbiol. 2003, 48, p33-946; (b) The GFP-

tagged strain PKL1162 was constructed by introduction of the

plasmid pPKL174 into strain SAR18. The plasmid pPKL174

contains the fim gene cluster, which is required for type 1

fimbriae assembly and expression. The chromosome of strain

SAR18 contains the GFP gene, controlled by a constitutive

promoter.

27 Schrödinger Release 2016-1: Maestro, version 10.5,

Schrödinger, LLC, New York, NY, 2016.

28 Schrödinger Release 2016-1: Maestro, version 11.1,

Schrödinger, LLC, New York, NY, 2016.

5

6

A. Mithöfer, W. Boland, Annual Review of Plant Biology 2012

63, 431-450.

,

a) W. P. Burmeister, S. Cottaz, H. Driguez, R. Iori, S. Palmieri,

B. Henrissat, Structure 1997, 5, 663-676; b) P. Bednarek, M.

Pislewska-Bednarek, A. Svatos, B. Schneider, J. Doubsky, M.

Mansurova, M. Humphry, C. Consonni, R. Panstruga, A.

Sanchez-Vallet, Science 2009, 323, 101-106; c) M. Stahl, V.

Dufour, C. Baysse, Microbiology 2015, 161, 229-243; d) Y.

Zhang, Mol. Nutr. Food Res. 2010, 54, 127-135.

7

a) M. S. C. Pedras, Q. H. To, G. Schatte, Chem. Commun. 2016

52, 2505-2508; b) Q. V. Vo, C. Trenerry, S. Rochfort, J.

Wadeson, C. Leyton, A. B. Hughes, Bioorg. Med. Chem. 2014

,

22, 856–864; c) E. Elhalem, R. Recio, S. Werner, F. Lieder, J. M.

Calderón-Montaño, M. López-Lázaro, I. Fernández, N. Khiar,

Eur. J. Med. Chem. 2014, 87, 552-563; d) Q. V. Vo, C. Trenerry,

S. Rochfort, J. Wadeson, C. Leyton, A. B. Hughes, Bioorg. Med.

Chem. 2013, 21, 5945-5954; e) P. Rollin, A. Tatibouët, CR

Chimie 2011, 14, 194-210.

8

9

R. Nehmé, H. Nehmé, G. Roux, D. Cerniauskaite, P. Morin, P.

Rollin, A. Tatibouët, Anal. Chim. Acta, 2014, 807, 153–158.

a) O. Planas, T. Gallavardin, S. Nonell, S. Chem. Commun.

2015, 51, 5586-5589; b) Hermanson, G. T. Bioconjugate

29 Schrödinger Release 2016-1: LigPrep, Schrödinger, LLC, New

York, NY, 2016.

Techniques, 3rd ed.; Academic Press: Waltham, MA, 2013

.

10 M. Hartmann, Th. K. Lindhorst, Eur. J. Org. Chem. 2011, 3583-

3609

11 a) M. Scharenberg, O. Schwardt, S. Rabbani, B. Ernst, J. Med.

Chem. 2012, 55, 9810-9816; b) L. Pang, S. Kleeb, L. Lemme, S.

Rabbani, M. Scharenberg, A. Zalewski, F. Schädler, O.

Schwardt, B. Ernst, ChemMedChem 2012, 7, 1404-1422.

12 a) T. Klein, D. Abgottspon, M. Wittwer, S. Rabbani, J. Herold,

X. Jiang, S. Kleeb, C. Lüthi, M. Scharenberg, J. Bezençon, E.

Gubler, L. Pang, M. Smiesko, B. Cutting, O. Schwardt, B. Ernst,

J. Med. Chem. 2010, 53, 8627-8641; b) Z. Han, J. S. Pinkner, B.

Ford, R. Obermann, W. Nolan, S. A. Wildman, D. Hobbs, T.

Ellenberger, C. K. Cusumano, S. J. Hultgren, J. W. Janetka, J.

Med. Chem. 2010, 53, 4779-4792; c) S.Kleeb, L. Pang, K.

Mayer, D. Eris, A. Sigl, R. C. Preston, P. Zihlmann, T. Sharpe, R.

P. Jakob, D. Abgottspon, A. S. Hutter, M. Scharenberg, X. Jang,

G. Navarra, S. Rabbani, M. Smiesko, N. Ludin, J. Bezencon, O.

Schward, T. Maier, B. Ernst, J. Med. Chem. 2015, 58, 2221-

2239; d) L. Mydock-McGrane, Z. Cusumano, Z. Han, J. Binkley,

M. Kostakioti, T. Hannan, J. Pinkner, R. Klein, V. Kalas, J.

Crowley, N. P. Rath, S. J. Hultgren, J. W. Janetka J. Med. Chem.

2016, 59, 9390−9408.

13 D. Cerniauskaite, J. Rousseau, A. Sackus, P. Rollin, A.

Tatibouet, Eur. J. Org. Chem. 2011, 2293-2300.

14 D. Gueyrard, R. Iori, A. Tatibouët, P. Rollin, P. Eur. J. Org.

Chem. 2010, 3657-3664.

15 E. Brachet, J.-D. Brion, S. Messaoudi, M. Alami, Adv. Synth.

Catal. 2013, 355, 477-490.

J. Name., 2013, 00, 1-3 | 13

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DOI: 10.1039/C8OB01128A

Bifunctional mannoside-glucosinolate glycoconjugates as enzymatically triggered isothiocyanates and FimH

ligands.

Giuliano Cutolo, Franziska Reise, Marie Schuler, Reine Nehmé, Guillaume Despras, Jasna Brekalo, Philippe

Morin, Pierre-Yves Renard, Thisbe K. Lindhorst, Arnaud Tatibouët*

The synthesis of glucosinolate-mannoside glycoconjugates combining both the structural features of a

myrosinase substrate and a FimH ligand are described.

Article Doi

Bifunctional mannoside-glucosinolate glycoconjugates as enzymatically triggered isothiocyanates and FimH ligands

DOI: 10.1039/C8OB01128A

Source and publish data:

Authors:

Article abstract of DOI:10.1039/C8OB01128A

Full text of DOI:10.1039/C8OB01128A

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