Carbohydrate Research 368 (2013) 52–56

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Carbohydrate Research

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A straightforward access to TMG-chitooligomycins and theirevaluation as b-N-acetylhexosaminidase inhibitors

0008-6215/$ - see front matter � 2012 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.carres.2012.12.007

⇑ Corresponding author. Tel.: +33 476 037 666; fax: +33 476 547 203.E-mail address: sami.halila@cermav.cnrs.fr (S. Halila).

Sami Halila a,⇑, Eric Samain a, Constantin E. Vorgias b, Sylvie Armand a

a Centre de Recherches sur les Macromolécules Végétales CERMAV-CNRS (Affiliated with Université Joseph Fourier, and Member of the Institut de Chimie Moléculaire de Grenoble),BP 53, 38041 Grenoble cedex 9, Franceb National and Kapodistrian University of Athens, Faculty of Biology, Dept. of Biochemistry-Mol. Biology, Panepistimiopolis-Zographou, 157 84 Athen, Greece

a r t i c l e i n f o

Article history:Received 28 September 2012Received in revised form 29 November 2012Accepted 7 December 2012Available online 19 December 2012

Dedicated to Dr. Hugues Driguez on theoccasion of his retirement

Keywords:ChitooligosaccharidesHexosaminidaseInhibitorsBiotechnologyN-Quaternization

a b s t r a c t

A chemo-biotechnological approach is reported for the synthesis of TMG-chitooligomycins, CO-n (NMe3).Their abilities to inhibit b-N-acetylhexosaminidases (HexNAcases), from Aspergillus oryzae (AoHex, fungi),Canavalia ensiformis (CeHex, plant) HexNAcases and a chitobiase from Serratia marcescens (SmCHB, bac-teria) were studied and compared with their precursors CO-n (N). CO-n (NMe3) were revealed as potentinhibitors for AoHex and SmCHB with a proved chain length effect while CO-n (N) was a highly selectiveinhibitor of SmCHB. This route can be considered as the privileged way to produce easily and in largescale a wide range of size-defined chitooligosaccharide-based inhibitors to fine-tune the structure–activ-ity relationships for inhibition of HexNAcases from various origins.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

TMG-chitotriomycin (CO-IV (NMe3))1 (Fig. 1), isolated from theculture filtrate of actinomycete Streptomyces anulatus NBRC13369strain, was recently discovered as a potent and selective inhibitorof b-N-acetyl-hexosaminidase (HexNAcase, EC 3.2.1.52) from fungi,bacteria, and insects compared to mammalian and plant sources(http://www.cazy.org/). Therefore, this natural active oligosaccha-ride was depicted as an attractive and potentially highly selectivebioproduct for the development of a new class of natural bacteri-cides, fungicides, and insecticides without adverse effect on humanand animal health or on the environment. At first, the atypicalstructure was proposed as a unique N,N,N-trimethyl-D-glucosami-nyl (TMG) residue a-(1 ? 4)-linked at the non-reducing end of atri-NI,NII,NIII-acetylchitotriose. Afterward, Yang et al.2 have pro-posed a revised structure, determined by comparison of the 1HNMR spectra with synthetic compounds. They revealed that theTMG residue is rather linked in a b-(1 ? 4) manner. They obtainedthe TMG-chitotriomycin by a multi-step synthesis sequence (28steps) with 3% overall yield, starting from a 2-azido-2-deoxy-glu-copyranosyl donor. Originally, CO-IV (NMe3) was isolated fromStreptomyces anulatus NBRC13369 strain culture in concentration

up to 46.6 mg/L.1 Thus, both previous approaches are limited bythe tedious and time-consuming work and featured by very lowoverall yields. In general, the preparation of well-defined chitooli-gosaccharides or their derivatives starting from chitin hydrolysis orfrom chemical synthesis is particularly laborious. For almost20 years now, we have developed a process for the production ofa large range of recombinant oligosaccharides in Escherichia coli,3

including chitooligosaccharides (CO-n, n = number of saccharideunits in roman numeral) and their N-deacetylated derivatives(CO-n (N)) at the non-reducing end unit with high yields.4–6 Thisprocess, termed as ‘cell factory’, is based on the metabolicallyengineered E. coli cells, where the overproduction of recombinantproteins NodC (chitooligosaccharide synthase) and NodB (chitool-igosaccharide N-deacetylase) enabled the high intracellular con-centrations of CO-n (N) with n = II,4 IV,5 or V6 saccharidic units.This technology in our hands allows to suggest CO-n (N) as excel-lent intermediates for the preparation of TMG-chitotriomycin CO-IV (NMe3) and analogues CO-n (NMe3) with n = II or V, also termedas chitooligomycins (Fig. 1). Moreover, previous works have shownthat CO-n (N) are competitive inhibitors of a chitobiase from Serra-tia marcescens,7 and a comparative study on inhibition propertiesbetween CO-n (N) and CO-n (NMe3) has therefore to be envisioned.

In this communication, we demonstrate the versatile chemo-bio-technological approach for the preparation of TMG-chitooligomycinsCO-n (NMe3) starting from ‘recombinant’ chitooligosaccharides CO-n

OHOHO

N

OHO

OHO AcNH OH

OH

n-2

n = II; CO-II (NMe3)n = IV; CO-IV (NMe3) or TMG-Chitotriomycinn = V; CO-V (NMe3)

n = II; CO-II (N)n = IV; CO-IV (N)n = V; CO-V (N)

TMG

OOHO

AcNH

OH

OHOHO

NH2

OHO

OHO AcNH OH

OH

n-2

OOHO

AcNH

OH

MeI, NaOH 5 MNMP

Figure 1. Mono-N-deacetylated chitooligosaccharides (CO-n (N)) and their N-methyl quaternized analogues (CO-n (NMe3)).

S. Halila et al. / Carbohydrate Research 368 (2013) 52–56 53

(N) with n = II, IV, or V. We focus our interest on the influence ofthe saccharidic chain length and of N-methyl quaternization oninhibition properties against HexNacases from various sourcesbut belonging to GH20 family. During the course of our work, Usu-ki et al.8 have published the production of TMG-chitooligomycinsby Streptomyces cell culture that afforded, after 14 days, CO-II(NMe3) and CO-III (NMe3) with maximum concentration of around8 nmol mL�1. At the same time, Yang et al.9 developed 20-step syn-thesis for CO-II (NMe3) with 13% or 7% overall yield starting fromthe glycosyl donor or acceptor, respectively while CO-V (NMe3)was achieved by a convergent 36-step synthesis with less than4% overall yield.

2. Results and discussion

2.1. Preparation of CO-n (NMe3)

Recombinant CO-n (N) with n = II, IV, or V were first obtained byhigh cell density culture of appropriate Escherichia coli strains aspreviously described, with production yields reaching up to4 g L�1.4–6 The TMG-chitotriomycin or CO-IV (NMe3) was synthe-sized from CO-IV (N) by reaction at room-temperature with methyl

OO

H

O

O

O

NH

O RHO

HO

OHβ

R = -(GlcNAc) n-

OO

H

O

O

O

OHO

HOHO

NO

H

R

Michaelis Complex

-1 +1

R'

R' = Ac

OO

H

O

O

O

NH

O RHO

HO

OHβ

R = -(GlcNAc)n-

-1 +1

H

-1

HO

HOHO

Mic

skew-boatconformation

4C1 chairconformation

A

B C

He

CO-n (NMe3)CO-n (N)

Figure 2. (A) Catalytic mechanism of GH20 HexNAcases relying on Michaelis compleglucosaminide ring at the non-reducing end of (B) CO-n (N) and (C) CO-n (NMe3). (D) St

iodide (30 equiv) in the presence of sodium hydroxide (4 equiv)using N-methyl-2-pyrrolidone (NMP) as solvent. TMG-chitotrio-mycin was easily isolated from the reaction mixture by precipita-tion in acetone followed by centrifugation with 85% yield. Thesame procedure was repeated for CO-II (N) and CO-V (N) to giveCO-II (NMe3) and CO-V (NMe3) with 47% and 78% yields, respec-tively. All compounds displayed in 1H NMR spectra a doublet(1H) with a weak coupling constant (J = 4 Hz) around 5.35 ppmand a singlet around 3.15 ppm (9H) characteristic of the anomericproton of TMG residue and of the trimethylammonium group,respectively.1,2 Moreover under these specific conditions, thesimultaneous occurrence of O-methylation was prevented as dem-onstrated in mass spectra even if we observe a slight additional O-methylation for CO-II (NMe3) (See Supplementary data). CO-I(NMe3) or TMG was not synthesized since Usuki et al.1 as well asYang et al.9 have already shown that this monosaccharide wasnot an inhibitor.

2.2. Inhibition properties

Aspergillus oryzae (AoHex, fungi), Canavalia ensiformis (CeHex,plant) HexNAcases and S. marcescens (SmCHB, bacteria) chitobiase

O

O

Oxazolinium ion intermediate

O

NH

HOHO

OH

O

OO

HO

RO

O

H

O

O

O

NH

O HO

ROHO

OHβ

hydrolyzed product

OO

H

O

O

R = -(GlcNAc)n-

+1

O

O

N

R

mimics of thehaelis Complex

D

OO

H

O

O

-1 +1

mimic of the bicyclicoxazoline intermediate

O

N

HOHO

OH

S

NAG-thiazoline

xNAcasesGH20

x and oxazolinium ion intermediate10 with the expected conformations for theructure of the NAG-thiazoline, an inhibitor that mimics the oxazoline intermediate.

Table 1IC50 (lM) and Ki (lM) values of chitooligosaccharide-based inhibitors against various HexNAcases

HexNAcases NAG-thiazoline n CO-n (NMe3) CO-n (N)

Ki IC50 Ki IC50 Ki

SmCHB II n.d.b 0.04 ± 0.03 n.d. 167 ± 12c

>200a IV n.d. 0.12 ± 0.01 n.d. 50 ± 7c

V n.d. 0.13 ± 0.01 n.d. 14.1 ± 1.5c

AoHex II 56.7 ± 4.2 100 ± 12 >3000d n.d.76.5 ± 7.8 IV 2.3 ± 0.08 1.00 ± 0.07 >250e n.d.

V 0.97 ± 0.08 1.20 ± 0.01 >250f n.d.

CeHex II No inhibition0.71 ± 0.06 IV

V

a <28% inhibition at 200 lM.b n.d. for not determined.c Reference 7.d <29% inhibition at 3 mM.e <20% inhibition at 250 lM.f <40% inhibition at 250 lM.

54 S. Halila et al. / Carbohydrate Research 368 (2013) 52–56

all belonging to the family 20 of glycoside-hydrolases (GH20), wereused to investigate the inhibitory activity of CO-n (N) and CO-n(NMe3). Both chitooligosaccharide-based inhibitors will be com-pared with NAG-thiazoline (Fig. 2D),10 a potent transition-state-mimicking inhibitor (Fig. 2A) of GH20 and GH84 HexNAcases.11

As shown in Table 1, obtained by the inhibition graphs, the resultsof this study have demonstrated potent and selective competitiveinhibition with Ki ranging from 200 to 0.04 lM, except for CeHexwhere no inhibition was observed. Significant differences wereshown depending on HexNAcase origins, chitin chain length, andN-methyl quaternization (Table 1). For a better comprehension ofthis work, it is worthy to note that Yang et al.12 have resolvedthe crystal structure of a b-N-acetyl-D-hexosaminidase (OfHex 1)in complex with TMG-chitotriomycin and have shown that TMGis well located at the �1 subsite in the active pocket. Since for Hex-NAcases belonging to the same family, the active site is highly con-served, we can assume that for all studied HexNAcases the TMGresidue binds the �1 subsite while the GlcNAc units bind to the po-sitive subsites (Fig. 2C). Moreover, TMG alone did not display inhi-bition against various HexNAcases1,8,9 emphasizing the major roleof chitin chain in the binding affinity with the enzymes.

2.2.1. SmCHB inhibition assaysConsidering SmCHB, CO-IV (NMe3) exhibited a Ki value of

0.12 lM. This value was in accordance with the one recently re-ported by Yang et al.13 (Ki = 0077 lM). The addition of a fourth Glc-NAc unit on the reducing end to afford CO-V (NMe3) gave similarinhibition constant (Ki = 0.13 lM) indicating a minor contributionof the subsite +4 in substrate-binding. However with CO-II(NMe3), the inhibition constant was roughly threefold decreasedto give a sub-micromolar inhibition (Ki = 0.04 lM). This studyshowed that the increase of the saccharide chain length providedunfavorable contributions to SmCHB binding and the Ki values re-mained about constant at least beyond the chitintriose or +3 sub-site. The studies led by Yang et al.9 on a bacterial HexNAcase fromStreptomyces plicatus with synthetic CO-n (NMe3) (n = III, IV, or V)showed no effect of the number of saccharide units on inhibitoryproperties. While Usuki et al.8 have described a better affinity ofCO-III (NMe3) for HexNAcase from Streptomyces coelicolor thanCO-II (NMe3). These works emphasize that the binding affinitywith the chitin chain is very dependent on the enzyme origin.Our data obtained with CO-n (NMe3) have to be compared to thestudy engaged by Drouillard et al.7 who have reported that the ser-ies of well-defined chitooligosaccharides selectively N-deacety-lated at the non-reducing end, CO-n (N) with n = II-V, wascompetitive inhibitor of SmCHB and that the efficiency was im-

proved with chitin chain length. For this reason, SmCHB was con-sidered as an exo-chitooligosaccharide N-acetylglucosaminidase.In comparison to CO-n (N), the significant difference in chainlength effect of CO-n (NMe3) that we found, could result fromthe ring conformation of the TMG unit linked to the chitin chain,compared to the GlcNH2 unit. In fact, based on NMR data, Yanget al.2 have first reported that TMG unit exists in a twist-boat con-formation like in the Michaelis complex (Fig. 2C) while GlcNH2 unitis known to be in a 4C1 chair form (Fig. 2B).These glucosaminederivatives located at the non-reducing end of the oligosaccharidemay drive the orientation of the GlcNAc units and certainly affectthe binding affinity with adjacent subsites. In any case, it is note-worthy that CO-n (NMe3) were better inhibitors than CO-n (N).For example, CO-II (NMe3) was 4.4.103 times more potent thanCO-II (N). Surprisingly, NAG-thiazoline displayed a weak inhibitionactivity (28% inhibition at 200 lM) that underlines the importanceof a GlcNAc unit, at the +1 subsite for the stabilization of the inhib-itor-SmCHB complex. As reported by Tews et al.,14 SmCHB that re-moves a single GlcNAc unit from the non-reducing end of a smallchitin oligomer, exhibits a rich domain of aromatic residues atthe +1 subsite that strongly stabilize the substrate through hydro-phobic interactions with the GlcNAc sugar.

2.2.2. AoHex inhibition assaysConcerning the AoHex, we have observed the same behavior in

inhibitory properties with CO-n (NMe3) as those reported by Usukiet al.8 The Ki value for CO-IV (NMe3) was around 1 lM and theaddition of another GlcNAc unit changed nothing. However, withCO-II (NMe3) the Ki value increased sharply to reach 100 lM high-lighting on the basis of this study that AoHex has an extended sub-strate-binding pocket with at least 4 contributive subsites. Theseexperimental results were quite surprising since di-N-acetyl-chito-biose was described as the most effective substrate for AoHex com-pared to longer N-acetyl-chitooligomers.15 Secondly, we havetested the series of CO-n (N) and have revealed a weak inhibitionwith at best 40% inhibition at 250 lM with CO-V (N) and a relativedecrease in inhibition with the reduction of chitin chain length.This weak inhibition gives Supplementary data arguing that theN-methyl quaternization is an essential parameter for the selectiveinhibition since it ensures the pyranose ring distortion toward askew-boat conformation, thus correctly mimicking the Michaeliscomplex.8,9,12 Another reason could come from the topology ofthe active site that would preferentially accomodate bulky substi-tutes, such as methyl groups, on the quaternized amine. In spite ofthe recent structural elucidation of AoHex,16 this hypothesis can-not be assessed. Finally, as expected, NAG-thiazoline that exhibited

S. Halila et al. / Carbohydrate Research 368 (2013) 52–56 55

a Ki value of 76.5 lM toward AoHex remained a weaker inhibitorthan CO-n (NMe3) with n > 2.

2.2.3. CeHex inhibition assaysNo inhibition of activity was noted on CeHex up to a concentra-

tion of 1 mM for series of CO-n (N) and 150 lM for series of CO-n(NMe3). NAG-thiazoline inhibited CeHex with a Ki value of0.71 lM that was in accordance with the literature.10 The lack ofinhibition with both chitooligosaccharide families suggested thatthe chitin chain has a negative impact in the positioning and stabil-ization of the complex inhibitor-enzyme.

3. Conclusion

In conclusion, we have developed a straightforward chemo-bio-technological approach for the rapid production of CO-n (NMe3)compared to previous works.1,2 The series of CO-n (NMe3) was po-tent and selective inhibitor of GH20 HexNAcases from fungi (Ao-Hex) or bacteria (SmCHB) origin on the contrary to GH20HexNAcases from plant (CeHex) and are much better than NAG-thiazoline. The sensitivity to CO-n (NMe3) inhibitors for AoHexand SmCHB was in this order: CO-II (NMe3) < CO-IV (NMe3) = CO-V (NMe3) and CO-II (NMe3) > CO-IV (NMe3) = CO-V (NMe3), respec-tively. The CO-n (N) were highly selective inhibitors of SmCHB witha chitin chain length effect opposite to CO-n (NMe3). In contrast tomonosaccharide-based inhibitors, non-hydrolyzable oligosaccha-ride-based inhibitors have the advantage to combine mimics ofthe transition-states, with interactions along many subsites otherthan those close to the point of enzymatic cleavage. These charac-teristics contribute to binding affinity and selectivity allowing thediscrimination between glycoside-hydrolases belonging to the samefamily. The versatility of our approach allows us to design a setof N-quaternized derivatives that will take advantage of structuraldifferences in the substrate-binding pocket of HexNAcases. This willpermit us to finely tune the structural parameters of future biopes-ticides or drugs. To design potent and selective inhibitors, crystallo-graphic studies providing insight into the molecular basis ofsubstrate recognition by SmCHB or AoHex will be performed.

4. Experimental

4.1. General methods

All reagents, methyl iodide, sodium hydroxide, and 1-methyl-2-pyrrolidone were used as received from Sigma–Aldrich Co. Biosyn-thesis and purification of CO-n (N) were performed as previouslydescribed.4 Visualization of the TLC plates was performed by char-ring with sulfuric acid solution (H2SO4/MeOH/H2O 3:45:45). NMRspectra were recorded on Bruker Avance 400 at 298 K. Chemicalshift values are reported in ppm with the solvent resonance asthe internal standard (CD3OD 3.31 ppm, HDO 4.79 ppm, for 1HNMR). MALDI-TOF measurements were performed on a BrukerDaltonics Autoflex apparatus and ESI mass spectrum was recordedon a ZQ Waters micromass spectrometer. High-resolution massspectra were recorded in positive mode on a ZabSpec TOF (Micro-mass, UK) tandem hybrid mass spectrometer with EBETOFgeometry.

4.2. General procedure for the N-trimethylation of CO-n (N)with n = II, IV, or V

To a solution of CO-n (N) (17 mM) dissolved in 1-methyl-2-pyr-rolidone (NMP) were successively added 30 equiv of methyl iodideand 4 equiv of aqueous sodium hydroxide solution 5 M. After 8 h ofreaction under stirring at room-temperature in a sealed tube, the

reaction mixture was diluted with acetone (10 volumes) and theprecipitate was washed and isolated twice by centrifugation inacetone (9600 g; 6 min; 4 �C). The solid was dissolved in waterand then freeze-dried to give CO-n (NMe3) with n = II, IV, and Vwith 47%, 85%, or 78% yield, respectively. High-resolution massspectrometry and 1H NMR spectral data of the isolated productsare presented below.

4.2.1. (2-Trimethylammonio-2-deoxy-b-D-glucosaminyl)-(1 ? 4)-N-acetyl-D-glucosamine (CO-II (NMe3))

White amorphous solid; (D2O, 400 MHz): d 5.43 (bs, 1H, H-1II),5.20 (bs, 0.93H, H-1aI), 4.00–3.50 (m, 12H, H-2I–II, H-3I–II, H-4I–II,H-5I–II, H-6I–II), 3.33 (s, 9H, NCH3), 2.07 (s, 3H, COCH3); ESI-MS:m/z 425.19 [M]+; HR-ESI-MS Calcd for [C17H33N2O10]+: 425.2118.Found: 425.2135.

4.2.2. (2-Trimethylammonio-2-deoxy-b-D-glucosaminyl)-(1 ? 4III)-NI,NII,NIII-triacetylchitotriose (CO-IV (NMe3))

White amorphous solid; (CD3OD, 400 MHz): d 5.30 (d, J = 4.0 Hz,1H, H-1IV), 5.01 (d, J = 1.8 Hz, 1H, H-1aI), 4.51–4.42 (m, 2H, H-1II–III),3.90–3.33 (m, 24H, H-2I–IV, H-3I–IV, H-4I–IV, H-5I–IV, H-6I–IV), 3.26(s, 9H, NCH3), 1.96 (s, 3H, COCH3), 1.93(s, 3H, COCH3), 1.91 (s, 3H,COCH3); MALDI-TOF: m/z 831.33 [M]+; HR-ESI-MS Calcd for[C33H59N4O20]+: 831.3715. Found: 831.3723.

4.2.3. (2-Trimethylammonio-2-deoxy-b-D-glucosaminyl)-(1 ? 4IV)-NI,NII,NIII,NIV-tetraacetyl-chitotetraose (CO-V (NMe3))

White amorphous solid; (CD3OD, 400 MHz): d 5.42 (d, J = 4.0 Hz,1H, H-1V), 5.20 (bs, 0.92H, H-1aI), 4.63–4.58 (m, 3H, H-1II–IV), 4.06–3.49 (m, 30H, H-2I–V, H-3I–V, H-4I–V, H-5I–V, H-6I–V), 2.08 (s, 3H,COCH3), 2.07 (s, 9H, 2 x COCH3), 2.05 (s, 3H, COCH3); MALDI-TOF:m/z 1034.46 [M]+; HR-ESI-MS Calcd for [C41H72N5O25]+:1034.4525. Found: 1034.4516.

4.3. Enzymatic assays

b-N-Acetyl-D-hexosaminidases CeHex from Canavalia ensiformisand AoHex from Aspergillus oryzae were purchased from Sigma–Al-drich. Chitobiase (SmCHB) from Serratia marcescens was preparedaccording to the method described by C.E. Vorgias (University ofAthens, Greece).17 Initially, concentrations of inhibitors causing50% inhibition of enzyme activity (IC50 values) were determined,followed by further determination of inhibition constants (Ki val-ues) and inhibition modes for those showing IC50 values of lessthan 200 lM. The amount of HexNAcases was adjusted so thatthe increase in absorbance at 415 nm of the control reaction with-out the inhibitor was around 0.5 after 1 h. Typically, an appropriateamount of SmCHB (5 lL, 4.6 lg/mL) in 50 mM phosphate buffer(pH 7.8) or AoHex (5 lL, 0.1 U/mL) in 100 mM sodium acetate buf-fer (pH 5.5) or CeHex (5 lL, 1 U/mL) in 100 mM acetate buffer (pH5.0) containing 100 mM NaCl and 0.01% (w/v) bovine serum albu-min, was added to a pre-incubated solution (115 lL, 37 �C) con-taining 0.5 mM pNP-b-D-GlcNAc and varying concentrations ofinhibitors from 0.1 to 600 lM. The mixture was incubated for 1 hat 37 �C, then terminated by adding 1 M Na2CO3 (120 lL) and theabsorbance at 415 nm was measured. To determine the Ki valueof chitooligosaccharide-based inhibitors for HexNAcases, a seriesof initial reaction rates were measured at five concentrations ofsubstrate, pNP-b-D-GlcNAc, and a range of inhibitor concentrations(typically bracketing the Ki value ultimately determined such that0.2Ki < [inhibitor] < 5Ki). These data were fit to a competitive inhi-bition model using nonlinear regression analysis, as performed bythe GraFit (GraFit version 7.0.1; Erithacus Software Ltd, London,UK), to provide the Ki value. Values are means of triplicate determi-nations. Lineweaver�Burke plots of each data set validated the useof a competitive inhibition model.

56 S. Halila et al. / Carbohydrate Research 368 (2013) 52–56

Acknowledgments

We acknowledge the financial support from the CNRS. Wethank D. Vocadlo (Simon-Fraser University, Canada) for the kindgift of NAG-thiazoline, S. Cottaz and H. Driguez (CERMAV-CNRS,France) for their fruitful discussions on interpretation of inhibi-tion data, ICMG mass spectrometry platform for analyses andD. Lesur (Université Picardie Jules-Verne, France) for ESI-HRMSexperiments.

Supplementary data

Supplementary data (1H NMR and MS spectra and Lineweaver–Burk plots of inhibitors) associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.carres.2012.12.007.

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