Diverted natural Lossen rearrangement for bioconjugation through in

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Diverted natural Lossen rearrangement for bioconjugation through in situ myrosinase-triggered isothiocyanate synthesis Cyrille Sabot, Jean Wilfried Fredy, Giuliano Cutolo, Benjamin Poret, Reine Nehmé, Marie HubertRoux, Pierrick Gandolfo, Helene Castel, Marie Schuler, Arnaud Tatibouët, and Pierre-Yves Renard Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00153 • Publication Date (Web): 01 Apr 2019 Downloaded from http://pubs.acs.org on April 5, 2019

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Bioconjugate Chemistry

Diverted natural Lossen-type rearrangement for bioconjugation through in situ myrosinase-triggered isothiocyanate synthesis† Jean Wilfried Fredy,‡ † Giuliano Cutolo,‡ ⸸ Benjamin Poret,$ Reine Nehmé,⸸ Marie Hubert-Roux,† Pierrick Gandolfo,$ Hélène Castel,$ Marie Schuler,⸸ Arnaud Tatibouët,*,⸸ Cyrille Sabot*,† and PierreYves Renard† †

Normandie Univ, CNRS, UNIROUEN, INSA Rouen, COBRA (UMR 6014), 76000 Rouen, France.

⸸ Institut

de Chimie Organique et Analytique - ICOA UMR 7311 CNRS Université d’Orléans - Rue de Chartres, BP6759, 45067 Orléans cedex 02, France.

$ Normandie

Univ, UNIROUEN, INSERM U1239, DC2N, 76000 Rouen, France, Institute for Research and Innovation in Biomedicine (IRIB), 76000 Rouen, France. ‡ These authors contributed equally to this work.

RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to) CORRESPONDING AUTHOR FOOTNOTE (Word Style “FA_Corresponding_Author_Footnote”). To whom correspondence should be addressed. For A.T.: e-mail: [email protected]. For C.S.: email: [email protected].

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Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT. The fluorescein isothiocyanate FITC is one of the most extensively used fluorescent probe for the labeling of biomolecules. The isothiocyanate function reacts with lysine residues of proteins to provide a chemically stable thiourea linkage without releasing any by-product. However, diversification of isothiocyanate-based reagents is still hampered by the lack of mild conditions to generate isothiocyanate chemical functions, as well as by their poor stability and limited solutions available to increase water solubility, restricting the use of isothiocyanate labeling to highly water soluble fluorophores. Inspired by plant biological processes, we report a safe and biocompatible myrosinase-assisted in situ formation of isothiocyanate conjugates from a highly water soluble and stable glucosinolate precursor. This method was applied for the fluorescence labeling of a plasmatic protein and fluorescence imaging of living cells.

INTRODUCTION Recent years have witnessed a dramatic increase in the use of modified biopolymers in current and emerging scientific research areas. Modified proteins are widely used in chemical biology for probing complex cellular processes in living systems, in medicinal chemistry for designing modern biotherapeutics,1 or in material sciences for creating new materials with advanced properties.2 Accordingly, it is of prime interest to continue efforts to develop original and effective labeling tools to mitigate existing limitations in bioconjugate chemistry.3 In contrast to genetic encoding strategies that require specific skills and equipment,4, 5 the chemical modification of native proteins has emerged as a practical and effective tool for rapid proteins conjugation.6 In this approach, the selective chemical incorporation of a specific probe, fluorophore, metal chelating agent or drug into a protein is achieved through chemoselective reactions which target specific amino acid residues such as cysteine, tyrosine, tryptophane, histidine, or lysine. 7-9 Lysine is one of the most abundant residues in native proteins, often exposed to the surface of proteins and thus readily accessible to the solvent. Its derivatization ensures a fast and effective tagging of a large number of biomolecules. In this context, stable chemical modification strategies include aza-electrocyclization,10,

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[1,5]-H sigmatropic rearrangement,12


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reductive amination,13, reaction.18,

19

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nucleophilic aromatic substitution (SNAr),15,

16

sulfonylation17 or acylation

Although very useful, these approaches involve, nevertheless, sensitive starting

derivatives, such as aldehydes that are prone to side reactions and oxidation, or N-hydroxysuccinimide (NHS) esters whose hydrolytic stability does not exceed a few hours under physiological pH conditions.20 Moreover, NHS esters suffer from poor selectivity, they also react with tyrosines, histidines, serines, and threonines,20, 21 and often require a slightly basic buffer for a rapid and efficient ligation. On the other hand, isothiocyanates readily undergo the nucleophilic addition of amino and sulfhydryl functions, at physiological pH, to provide corresponding thioureas and dithiocarbamates. However, the formation of dithiocarbamates being reversible, the reaction is in fine thermodynamically driven towards the formation of more stable thioureas. Although fluorescein isothiocyanate FITC developed more than 50 years ago22 is one of the most popular fluorescent bioconjugable probes created and routinely used, the diversification of the isothiocyanate-based bioconjugation is still hampered by the total absence of a general, safe, and biocompatible access to isothiocyanate derivatives, as well as their poor storage stability and a poor water solubility. This latter limits their use to highly water-soluble fluorophores, and post-synthetic water-solubilization strategies or commonly used water-solubilization functional groups such as sulfonates, phosphonates, carboxylates or sugar derivatives, which show limited compatibility either with isothiocynate derivatives or with synthetic routes to this highly electrophilic moiety.23, 24 Moreover, synthetic methods to prepare isothiocyanates involve highly toxic electrophilic reactants such as thiophosgene, triphosgene, or carbon disulfide,25 cytotoxic metal catalysts,26 basic conditions, or high temperatures.27 These constraints and poor functional group tolerance conditions have dramatically limited the structural diversity of isothiocyanate derivatives that are available for bioconjugation. The diversity of enzymatic reactions involved in plant biological processes are largely underexplored to generate highly reactive chemical species under mild, aqueous conditions compatible with proteins. The release of isothiocyanate derivatives from glucosinolate precursors is a well-established defense mechanism against any aggression, which takes place specifically in plant families of the Brassicales ACS Paragon Plus Environment

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order.28-34 This biochemical process is promoted by myrosinases, which specifically hydrolyzes the anomeric carbon-sulfur bond of glucosinolates to generate isothiocyanates through a Lossen-type rearrangement (Figure 1). HO HO HO

O

S

R

OH

N O3SO K

- Biocompatible - Chemically inert towards proteins - Water soluble

glucosinolate H 2O myrosinase D-glucose HS O3SO

R N K Lossen-type rearrangement

SO42-

S

C

N

R

- Highly electrophilic - Biologically reactive

Figure 1. Myrosinase-driven release of isothiocyanates for bioconjugation. In this communication, we questioned whether this unique mode of action which occurs in complex biological environments, could be diverted to the in situ preparation of tailor-made isothiocyanate conjugates from chemically inert towards proteins and water soluble precursors. It is noteworthy that rare examples of enzymatically-driven formation of bioconjugable chemical functions have been reported, most of them using horseradish peroxidase (HRP).35, 36 Based on structural analysis of natural glucosinolates composed of about 130 members, and artificial glucosinolate-like structures, it seems that modifications on the side chain R should not significantly affect the recognition process, and more importantly the hydrolytic ability of myrosinase.37-39 Accordingly, the introduction of (bio)conjugable handles or probes on the aglycon moiety should not interfere with the chemoenzymatic release of isothiocyanate. In order to minimize structural differences in the vicinity of the cleavage site, modifications were brought on the phenyl ring of the natural substrate, glucotropaeolin. Moreover, the formation of isothiocyanates from glucosinolate precursors was investigated for three different classes of function


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that are commonly used in the modification of biomolecules, i.e. a biorthogonal group (azide), an affinity label (biotine), and two fluorophores (sulforhodamine, sulfocyanine). RESULTS AND DISCUSSION To address this point, a series of four synthetic heterosides bearing either a bioorthogonal azide functional group, a biotin, a sulforhodamine, or a sulfocyanine 5.0 were prepared from 1-thio-β-Dglucose tetraacetate and ethyl 2-(4-hydroxyphenyl)acetate as key starting materials, as well as sinigrin 1a serving as a model substrate in the enzymatic reaction (Scheme 1).37, 40

HO HO HO

O OH

S

HO HO HO

R N

OEt a-c)

S

OH K

O3SO K (R = CHCH2, sinigrin 1a) (R = Ph, glucotropaeolin) O

O

O3SO

N

R

O

(analogues for bioconjugation) OAc

OAc

OH N

H

OH

O

AcO AcO

d)

OAc

Br

O

Br

S N

O

KO3SO

O

S3

O

O

HO HO

g)

S

OH N KO3SO

N3 h)

S

OH N KO3SO j)

i)

HN NH

O

NH N H

N H N 5

O

N H N

S SO3 3

R = NH2

R

O

O

O

N

OH

2

R =

N3

S

OAc

S2

OH HO HO

O

AcO AcO

O

HO

S1 f)

e)

SO3

2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


O

N 5

4

5 SO3

SO3K

Scheme 1. Preparation of synthetic glucosinolates 2-5. Conditions: a) 1,2-dibromoethane, K2CO3, MeCN, 78 h; b) Diisobutylaluminium hydride, CH2Cl2, -78 °C, 80 min.; c) hydroxylamine hydrochloride, Na2CO3, H2O/MeOH, 4 h, 82% (over three steps); d) sodium hypochlorite (12.5 % active chlorine), CH2Cl2, 20 min., then 2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranose, Et3N, 2 h, 100%; e) sodium azide, N,N-dimethylformamide, 50 °C, 4 h, 83%; f) potassium methoxide, MeOH, 5 h, 81%; g) triphenylphosphine, MeOH, 24 h; h) biotine N-hydroxysuccinimide ester, Et3N, N-methyl-2ACS Paragon Plus Environment

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pyrrolidone, 6 h, 82% (over steps g-h); i) sulforhodamine carboxylic acid, benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), N,N-diisopropylethylamine , N-methyl2-pyrrolidone, 10 h, 47% (over steps g-i); j) cyanine carboxylic acid, benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), N,N-diisopropylethylamine , N-methyl2-pyrrolidone, 18 h, 42% (over steps g-j). Compounds 3-5 were obtained from common key intermediate 2 which was first converted into the corresponding amine through the Staudinger reaction mediated by triphenylphosphine. Subsequent amidation reactions with biotin, sulforhodamine and cyanine carboxylic acids afforded 3, 4 and 5, respectively. The unsymmetrical red emitting sulforhodamine precursor bearing a carboxylic acid handle was prepared according to a recent methodology developed by our team.41, 42 The purity of each compound determined through RP-HPLC analyses was found to be equal to or above 98% (Supporting Information, S8-S10). With these compounds in hand, the myrosinase activity was monitored by capillary electrophoresis, by following the hydrolysis of the glucosinolate substrate. In fact, SO42produced during the hydrolysis/Lossen-type rearrangement cascade of glucosinolates by myrosinase was on-line detected for quantification.38, 39 Satisfyingly, by this method we observed that myrosinase was able to promote the formation of isothiocyanates from all synthetic glucosinolates 2-5, albeit with different kinetics (Supporting Information, S21). However, we have not been able to determine the Michaelis-Menten constants Km for the modified glucosinolates 3, 4 and 5. The Vmax was not reached for the bulkier fluorescent analogues and a sigmoid curve could be observed for the biotinylated glucosinolate conjugate 3, while for the glucosinolate 2, the Km could be determined and was found in good agreement with it small structure (Km = 1.06±0.39 mM ; Vmax = 0.12±0.01 mM min-1 for glucosinolate 2 versus Km = 0.63±0.1 mM, Vmax = 0.31±0.01 mM min-1 for the reference compound 1a). To complete this study, the time course of isothiocyanate formation was obtained by HPLC analysis for compounds 3-5 (Figure 2).


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Figure 2. Comparative kinetic studies of isothiocyanates release from glucosinolates 3 (blue), 4 (green), and 5 (red) in the presence of myrosinase, in PB pH 7.41. These results also showed that the bulkier the R-group is, the slower the formation of isothiocyanate is, which is consistent with previous studies.38,

39

Following these encouraging results, labeling of a

model protein (bovin serum albumin, BSA) with two red-emitting dyes, a sulforhodamine and a cyanine 5.0 respectively, was next investigated. Taking advantage of the high substrate specificity of myrosinase, a one-pot procedure was undertaken by mixing the enzyme with the corresponding substrate 4 or 5 (5 equiv. relative to BSA), together with BSA, which contains one free cysteine and ~30-35 accessible lysine residues. Solutions were incubated in 0.1 M phosphate buffer (PB), pH 7.4 at 37 °C for 24 h. The resulting sulforhodamine- or cyanine-modified protein BSA-4, and -5 was washed with an ultracentrifugal filter device with a 10 kDa cutoff. Subsequent characterization of bioconjugates was achieved by means of gel electrophoresis, mass spectrometry, UV-visible and fluorescence spectroscopy. Conjugation products were first analyzed by denaturing SDS-PAGE, followed by Coomassie blue staining and fluorescence detection (Figure 3). Both conjugates BSA-4 and -5 were conveniently tagged, as shown by the presence of a fluorescent band in the region of 66 kDa that corresponds to the molecular weight of the native protein. A comparative MALDI-TOF analysis of native BSA, BSA-4 and -5 was consistent with the attachment of 2.4 and 1.6 equiv. of fluorophore on the protein, respectively, corresponding to a satisfactory 48% (respectively 32%) labeling yield.43 Enlargement of peaks observed after labeling are associated with the heterogeneous distribution of the number of fluorophores per protein. ACS Paragon Plus Environment

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Figure 3. One-pot myrosinase-promoted labeling of BSA. A) Reaction of BSA with glucosinolate 4 or 5. B) SDS PAGE analysis of BSA-4 and -5 followed by Coomassie blue staining (left, B-1) and fluorescence detection (right, B-2). C) MALDI-TOF mass spectra of native BSA (tops), BSA-4 (bottom left), and BSA-5 (bottom right). With regards to photophysical properties, the absorption spectra of bioconjugates BSA-4 and -5 correspond to a linear combination of the absorption of BSA and dyes, which also supports the efficient labeling of the protein. For comparison purposes, the labeling was also performed with one of the most widely use fluorophore, FITC. The fluorescence spectrum of bioconjugates (BSA-4, BSA-5 and BSAFITC) matches well with that of dyes 4, 5 and FITC, but systematic lower quantum yields were observed with the dye-modified proteins, the most important loss being obtained with the FITC dye (73%, Table 1). This may be attributed to the formation of dye aggregates at the surface of the protein and/or quenching induced by interactions between fluorophores and aromatic ring systems of side chains of amino acids (e.g. tryptophan, tyrosine, phenylalanine).44, 45 The fluorophore to protein molar ratio (F/P) for BSA-4 and -5 was also estimated from the relative intensities of protein and dye absorption (Supporting Information, S18). F/P was found to be 2.5, 1.3, and 1.6 when labeling BSA with 5 equiv. of 4, 5 and FITC, respectively. These results are in rather good agreement with those


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obtained by mass spectrometry. Besides, the superior labeling ability observed with 4 stems presumably from (1) its higher water solubility than FITC due to the presence of two sulfonate functions, and (2) the superior chemical stability of rhodamine over cyanine dyes.

Cmpd a

F/P

λabs (nm) b

λem (nm)

ФF

4

N.A. b

568

588

0.70 c

5

N.A. b

648

665

0.40 d

FITC

N.A. b

493

518

0.62 e

BSA-4

2.5

568

587

0.25 c

BSA-5

1.3

650

669

0.16 d

BSA-FITC

1.6

500

521

0.17 e

All measurements were made at 25 °C, in 0.1 M PB pH 7.4. b Not applicable. c Relative method that used sulforhodamine 101 (SR101) as standard (ФF = 0.95 in EtOH) with excitation at 520 nm. d Relative method that used Nile Blue as standard (ФF = 0.27 in EtOH) with excitation at 610 nm. e Relative method that used Fluorescein as standard (ФF = 0.91 in 0.1 M NaOH) with excitation at 470 nm.

a

Table 1: Spectroscopic data of isothiocyanate-modified BSAs and precursors a

Isothiocyanate-based fluorophores released under myrosinase treatment, revealed suitable photophysical properties and good bioconjugation ability with an isolated protein. The small size of organic fluorophores making them very attractive for imaging proteins inside cells, we next decided to assess the biocompatibility of the tandem myrosinase/glucosinolate with biological systems by labeling live human embryonic kidney HEK-293 cells in culture through in situ myrosinase-mediated formation of the isothiocyanate dye 4. Here consistent with previous observations demonstrating interaction of myrosinase with plasma membrane,46, 47 we took advantage of this property to pre-incubate HEK-293 cells in culture medium with myrosinase to let some molecules (dye 4) interacting with plasma ACS Paragon Plus Environment

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membrane and rinsed once to roughly remove 4 in the supernatant. By reaction with the administered glucosinolate, we expect to allow labeling between 4 and the biological matrix containing inserted proteins by covalent modification. Thus, cells were first incubated for 15 min in the absence or the presence of myrosinase, rinsed with HBSS, and treated for 60 min with glucosinolate 4. Confocal microscopy analyses revealed a non-significant red fluorescence signal (background noise) in cells in control conditions (Figure 4A). In contrast, a myrosinase treatment led to a homogeneous fluorescent labeling in cells (Figure 4B), indicating the occurrence of a covalent labeling between dye 4 and the biological matrix in the absence of permeabilization. Therefore, cells have a fluorescence intensity two and about six times higher than the control conditions after 20 min and 60 min of treatment, respectively (Figure 5). Thereby, these results demonstrate that the myrosinase-glucosinolate system is specific and operational in live cells.


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Figure 4. Impact of myrosinase pretreatment on cell labeling of dye 4. Live HEK293 cells in culture were previously incubated with Hank's Balanced Salt Solution (HBSS) (A) or myrosinase (0.07 U, 15 min, 37 °C) (B) and then treated for 60 min with dye 4 (0.01 mM, 37 °C). After rising, cells were fixed with paraformaldehyde (4%, 10 min) and counterstaining with 4′,6-diamidino-2-phenylindole (DAPI) (1


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µg/mL, 5 min) to label nuclei. Cells were imaged through the membrane permeable PKH67 by confocal microscopy at 488 nm (excitation) and 502 nm (emission) (A1-B1), and 558 nm (excitation) and 605 nm (emission) for dye 4 (A2-B2), 405-461 nm for 4′,6-diamidino-2-phenylindole (DAPI) dye (A3-B3). A4-B4 microphotographs show the overlay and A5-B5 are digitally zoomed region corresponding to the white box of A4-B4 images.

Figure 5. Kinetic activity of myrosinase on the in cellulo cleavage of dye 4. Results were expressed as the ratio of fluorescence intensity obtained from HEK293 cells pretreated with myrosinase (0.07 U, 15 min, 37 °C) vs HBSS, and incubated from 0 to 60 min with dye 4 (0.01 mM, 37 °C). CONCLUSIONS This study reports a one-step labeling protocol of proteins from safe, chemically inert and water-soluble glucosinolate derivatives, which are substrates of myrosinase, a readily available enzyme. The strategy involves enzymatic activation of latent isothiocyanate-based conjugates, which can then readily react with proteins. Beyond practical concerns, the formation of a bioconjugable handle upon controllable enzymatic stimulus is a sought-after tool which may find valuable applications in various fields of biological chemistry and native life sciences. MATERIALS AND METHODS General. TLC were performed on Merck DC Kieselgel 60 F-254 aluminum sheets. The spots were visualized by illumination with a UV lamp (λ = 254 nm) and by charring with a 10% H2SO4 ethanolic solution or a solution of potassium permanganate. All chemicals were used as received from commercial sources without further purification unless otherwise stated. Peptide-synthesis-grade N,N-


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diisopropylethylamine (DIEA) and N-methyl-2-pyrrolidinone (NMP) were used. Solvents were dried by standard methods: MeCN was purified with a dry station immediately prior use, dichloromethane was distilled over P2O5; N,N-dimethylformamide was dried over molecular sieves, and triethylamine over potassium hydroxide. Molecular sieves were activated prior to use by heating for 4 h at 500 °C. All reactions were carried out under a dry argon atmosphere. Phosphate-buffered (PB, 100 mM phosphate, pH 7.4) and the aqueous mobile phases for HPLC were prepared with water purified with a Milli-Q system (purified to 18.2 MΩ cm). Thioglucosidase from Sinapis alba (white mustard) seed (myrosinase, EC 3.2.1.147, 25U, ≥100 units. g-1) was purchased from Sigma–Aldrich (Saint-Quentin Fallavier, France). Instruments and Methods. 1H and

13C

NMR spectra were recorded with a 300 or 400 MHz

spectrometers. Chemical shifts are expressed in parts per million (ppm) from the residual non-deuterated solvent signal. J values are expressed in Hz. For the 13C NMR in deuterated water, acetone or methanol was added as an internal standard. Analytical HPLC were performed with a Thermo Scientific Surveyor Plus instrument equipped with a photodiode array (PDA) detector. Semi-preparative HPLC were performed with a Thermo Scientific Spectra system liquid chromatography system (P4000) equipped with a UV/Visible 2000 detector. Gram-scale purification column chromatography on C-18 reverse phase was performed using a Reveleris® flash chromatography system. Centrifugations were carried out with a Microcentrifuge Micro Star 12. Melting points were determined in open capillary tubes using a Büchi 510 apparatus and are uncorrected. Optical rotation were measured at 20 °C using a C using a Perkin Elmer 341 polarimeter with a path length of 1 dm, values are given in degdm-1g-1mL-1 with concentrations reported in g100 mL-1. High-resolution mass spectra (HRMS) analyses were performed with an LCT Premier XE bench top orthogonal acceleration time-of-flight (oa-TOF) mass spectrometer (Waters Micromass) or a Maxis Bruker 4G by the “Federation de Recherche” ICOA/CBM (FR2708) platform equipped with an electrospray source and in the positive and negative modes (ESI+/–). Lowresolution mass spectra (LRMS) analyses were obtained with a Finnigan LCQ Advantage MAX (ion trap) apparatus equipped with an ESI source. MALDI-TOF-MS experiments were performed on an ACS Paragon Plus Environment

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Autoflex III time-of-flight mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a frequency-tripled Nd:YAG laser emitting at 355 nm. FlexControl (3.3) and FlexAnalysis (3.3) software package (Bruker Daltonics, Bremen, Germany) were used for data acquisition and processing. Spectra were acquired in the positive-ion linear mode at 100 Hz laser shot frequency. The acceleration voltage was set to 20 kV and the extraction delay time used was set to 250 ns in MS mode. Samples were prepared using the dried droplet method: 1 µL of sinapinic acid as matrix (10 mg mL-1 in acetonitrile/H2O 0.1%TFA) and 1 µL of protein solution were mixed and 1 µL of the mixture was then deposited on the target plate. The recorded mass spectra were the result of 600-2000 individual spectra averaged, depending of the samples. The laser fluence was set slightly higher than matrix desorption threshold (~25-50% of maximum laser fluence). External and internal calibrations were carried out using the doubly and singly charged ions of BSA (respectively m/z 33215.5 and 66431). IR spectra were measured with a Perkin Elmer Spectrum 100 FTIR-spectrometer. UV/Visible spectra were obtained with a Varian Cary 50 scan spectrophotometer by using a rectangular quartz cell (Varian, standard cell, open Top, 10 × 10 mm, chamber volume: 3.5 mL). Fluorescence spectroscopic studies (emission/excitation spectra) were performed with a Varian Cary Eclipse spectrophotometer by using a semi-micro quartz fluorescence cell (Hellma, 104F-QS, light path: 10 × 4 mm, chamber volume: 1400 μL). The emission spectra were recorded under the same conditions after excitation at the corresponding wavelength (excitation and emission filters: auto, excitation and emission slit = 5 nm). The quantum yields were measured at 25 °C by a relative method with: sulforhodamine 101 (SR101) as a standard (ΦF = 95% in EtOH, λEx = 520 nm) for rhodamine derivatives; Nile Blue as standard (ΦF = 27% in EtOH, λEx = 610 nm) for cyanine derivatives; Fluorescein as standard (ΦF = 91% in 0.1 M NaOH, λEx = 470 nm) for FITC derivatives. Equation (1) was used to determine the relative fluorescence quantum yield: ΦF(x) = (AS/AX)(FX/FS)(nX/nS)2ΦF(s) (1)


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where A is the absorbance (in the range 0.01–0.1 arbitrary unit, a.u.), F is the area under the emission curve, n is the refractive index of the solvent (at 25 °C) used in the measurements, and the subscripts S and X represent standard and unknown, respectively. The following refractive index values were used: 1.362 for EtOH, and 1.337 for PB pH 7.41 0.1 M, 1.33 for NaOH 0.1 M. HPLC Separations. Several chromatographic systems were used for the analytical experiments and purification steps. System A (analytical): Injection volume: 8 L; column temperature: 18-20 °C; Hypersil GOLD C18 column, (particule size: 5 μm; 2.1 × 100 mm) with CH3CN and 0.1% aqueous trifluoroacetic acid (aq.TFA, 0.1%, pH 2.2) as eluents [0% CH3CN (5 min), followed by a linear gradient from 0 to 100% (40 min) of CH3CN] at a flow rate of 0.25 mL/min. UV/Vis detection was achieved with the “Max Plot” mode (i.e., chromatogram at the absorbance maximum for each compound, 220–700 nm). System B: Semi-preparative RP-HPLC (Varian Kromasil C18 column, 10 μm, 21.2 × 250 mm) with CH3CN and 0.1% aq.TFA as eluents [0% CH3CN (10 min), followed by a linear gradient from 0 to 100% (200 min) of CH3CN] at a flow rate of 15.0 mL/min. Wavelength detections were chosen according to the compound (260 nm for biotine derivative 3, 550 nm for rhodamine derivative 4, and 590 nm for cyanine 5 derivative 5). Electrophoresis. SDS-PAGE was performed using 10 % of acrylamide. Fisher BioReagents™ EZRun™ Prestained Rec Protein Ladder was used as references for molecular weight of the protein. Coomassie Brilliant Blue was used to stain the gel. Fluorescence of the gel was registered using Typhoon™ FLA 9500 biomolecular imager. For the rhodamine derivative the excitation wavelength was 532 nm and for the cyanine derivative it was 635 nm. Capillary Electrophoresis. Procedure followed protocols earlier developed in our laboratory.38,

39

Substrates are used at large excess relative to the enzyme (about 100 times). For this reason, Myrosinase was used at 0.05 U.mL-1 for all assays. The myrosinase activity was determined by following the hydrolysis of the glucosinolate substrate, the SO42- produced was detected by the capacitively-coupled contactless conductivity detector (C4D) and quantified. The volume of the reaction mixture was set


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down to 7 µL, instead of the 100 µL used previously, and was carried out in a micro-vial of the CE instrument autosampler. The nonlinear curve fitting program PRISM® 5.04 (GraphPad, San Diego, California, USA) was used to determine Km and Vmax according to the following equation: Vi 

Vmax  [ S ] K m  [S ]

where Vi is the reaction rate, Km is the Michaelis Menten constant, Vmax is the maximum reaction velocity and [S] is the substrate (glucosinolate) concentration. Cell lines culture and transfections. Human embryonic kidney HEK-293 (ATCC®, CRL1573™) cell line was generously given by Dr Prézeau (IGF laboratory, Montpellier, France) and was routinely maintained according to the instructions from ATCC®. More precisely, cells were cultured with Dulbecco's Modified Eagle Medium (DMEM) media supplemented with 1% sodium pyruvate (ThermoFisher Scientific, Montigny-Le-Bretonneux, France) and 10% fetal bovine serum (FBS, Lonza, Levallois-Perret, France). For cytochemistry experiments, membranes of HEK-293 cells were platted at 60,000 cells/well into transparent, 24-well plates (Corning, Bagneaux-sur-Loing, France), previously coated with human fibronectin (25 µg/mL, overnight, 4 °C). Cells were labeled with the lipid dye PKH67 (Sigma-Aldrich, Saint-Ouen, France) and incubated overnight with medium. The day of experiments, cells were rinsed and incubated with myrosinase or HBSS (0.07 U, 15 min, 37 °C). They were gently rinsed with HBSS before treatment with glucosinase-sulforhodamine 4 (0.1 mM or 0.01 mM during 0, 10, 20, 30 or 60 min at 37°C). Therefore, cells were rinsed with PBS before fixation with paraformaldehyde (4%, 10 min) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (1 µg/mL, 5 min) to label nuclei. Cells were imaged by confocal microscopy (Leica TCS SP8 confocal laser scanning microscope). Synthesis, characterization and bioconjugation experiments Azido-Glucosinolate 2


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2-(4-(2-bromoethoxy)phenyl)acetaldehyde oxime S1. 1,2-Dibromoethane (19 mL, 0.22 mol, 10 equiv.) was added to a solution of ethyl 2-(4-hydroxyphenyl)acetate (4 g, 0.022 mol, 1 equiv.) and potassium carbonate (5.5 g, 0.040 mol, 1.8 equiv.) in anhydrous acetonitrile (70 mL). The reaction mixture was heated 18 h at 70 °C, then 9 supplementaries equivalents of 1,2-dibromoethane (17 mL, 0.20 mol) were added in 5 portions over a period of 60 h. The reaction mixture was then cooled down to room temperature and the solvent was evaporated under reduced pressure. The residue was taken up with ethyl acetate and washed once with water, then twice with NaOH 1M. The organic phase was dried over MgSO4, filtered and the solvent evaporated under reduced pressure to give a yellowish solid. The solid was dissolved in anhydrous CH2Cl2 (220 mL, 0.1 M) at -78 °C, then a solution of diisobutylaluminium hydride 1.2M in toluene (20 mL, 0.024 mol, 1.1 equiv.) was added dropwise. The solution was stirred at -78 °C for 80 min and then was quenched by addition of a saturated aqueous solution of sodium potassium tartrate, and then stirred for 20 min. The aqueous phase was then extracted twice with CH2Cl2, then the combined organic phases were washed twice with 1 M HCl aqueous solution, dried over MgSO4 and the solvent was evaporated under reduced pressure to give the crude aldehyde as a yellow oil. Finally, the aldehyde was dissolved in a mixture of H2O/MeOH (1/2) (20 mL, 0.75 M) to which hydroxylamine hydrochloride (1.16 g, 0.017 mol, 1.1 equiv.) and solid potassium carbonate (940 mg, 0.006 mol, 0.45 equiv.) were added. The reaction mixture was stirred at room temperature for 4 h and then the solvent was evaporated under reduced pressure. The crude residue was then taken up with ethyl acetate and washed twice with water then once with brine; after drying over MgSO4, the solvent was evaporated under reduced pressure to give the desired product as a mixture of E/Z isomers as a pale yellow solid (3.22 g, 82% over 3 steps). Rf = 0.6 (Petroleum ether (PE)/Ethylacetate(EA) : 70/30). 1H NMR (250 MHz, CDCl3) δ 8.41 (bs, 1H, NOH), 7.16 (d, J = 8.7 Hz, 2H, CHAr), 6.91 - 6.83 (m, 3H, CHAr, HC=N), 4.28 (t, J = 6.3 Hz, 2H, CH2O), 3.68 (d, J = 5.4 Hz, 2H, CH2CN), 3.63 (t, J = 6.3 Hz, 2H, CH2Br). 13C NMR (100 MHz, CDCl3) δ 157.1 (C=N), 151.5 (Cq Ar), 151.1 (Cq Ar), 130.1 (CH Ar), 115.2 (CH Ar), 68.1 (CH2O), 35.2 (CH2Br), 29.2 (CH2CN). IR (neat) ν =


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3201, 2866, 1608, 1511, 1242, 813 cm-1. HRMS (ESI+): m/z calculated for C10H12BrNO2 [M+H]+: 258.0124, found 258.0125. (Z)-S-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-[4-(2-bromoethoxy)phenyl]-2-acetothio hydroximate S2. A sodium hypochlorite solution (12.5 % active chlorine) (33.8 mL, 69.7 mmol, 3 equiv.) was added to a vigorously stirred solution of crude oxime S1 (6.0 g, 23.2 mmol, 1 equiv.) in anhydrous CH2Cl2 (115 mL), the colour of the solution changed from yellow to blue before returning to yellow. The solution was then stirred for 20 min at room temperature. After separation, the organic phase was slowly added to a solution of 2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranose (4.23 g, 11.6 mmol, 0.5 equiv.) in 62 mL of anhydrous CH2Cl2 at -10 °C under argon atmosphere, then triethylamine (9.7 mL, 69.7 mmol, 3 equiv.) was added dropwise and the solution was allowed to warm up to room temperature. After stirring for 2 h at room temperature, the reaction was quenched by addition of water and the aqueous phase was extracted twice with CH2Cl2. The combined organic layers were then washed twice with a 0.5 M aqueous HCl solution, dried over MgSO4, filtered and the solvent was evaporated under reduced pressure. The crude product was finally crushed in warm MeOH and filtered, 𝟐𝟎

to give a yellow solid (7.4 g, 100%). Rf = 0.56 (PE/EA : 4/6). m.p. = 125 °C; [α] 𝑫 = +19.1 (c = 0.25 in MeOH). 1H NMR (250 MHz, CDCl3) δ 8.66 (s, 1H, NOH), 7.16 (d, J = 8.5 Hz, 2H, CHAr), 6.89 (d, J = 8.5 Hz, 2H, CHAr), 5.11 - 4.90 (m, 3H, H2, H3, H4), 4.81 (d, J = 9.7 Hz, 1H, H1), 4.30 (t, J = 6.2 Hz, 2H, CH2O), 4.15 (dd, J = 12.3 Hz, J = 6.2 Hz, 1H, H6a), 4.04 (dd, J = 12.3 Hz, J = 2.1 Hz, 1H, H6b), 3.88 (s, 2H, CH2CN), 3.65 (t, J = 6.2 Hz, 2H, CH2Br), 3.59 - 3.49 (m, 1H, H5), 2.07, 2.01, 1.97 (3 × s, 12H, CH3 of Ac);

13C

NMR (100 MHz, CDCl3) δ 170.6, 170.3, 169.4, 169.2 (C=O), 157.6 (Cq Ar),

151.7 (C=N), 129.4 (CH Ar), 128.5 (Cq Ar), 115.4 (CH Ar), 79.5 (C-1), 75.9 (C-5), 73.8 (CH), 70.2 (CH), 68.2 (CH), 68.1 (CH2O), 62.4 (C-6), 38.2 (CH2CN), 29.2 (CH2Br), 20.9, 20.7, 20.68 (CH3 of Ac). IR (neat) ν = 3164, 1748, 1601, 1514, 1243, 1053, 983 cm-1. HRMS (ESI+): m/z calculated for C24H30NO11S [M+H]+: 620.0795, found 620.0801.


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(Z)-S-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-[4-(2-azidoethoxy)phenyl]-2-acetothio hydroximate N,O- sulfate potassium salt S3. Sodium azide (690 mg, 10.6 mmol, 3 equiv.) was added to a solution of thiohydroximate S2 (2.2 g, 3.5 mmol, 1 equiv.) in anhydrous N,N-dimethylformamide (12 mL). The mixture was heated at 50 °C for 4 h. The solvent was then evaporated under reduced pressure and the residue was taken up with ethyl acetate and washed three times with brine. The organic layer was dried over MgSO4, filtered and the solvent evaporated under reduced pressure to give the desired product as a yellow solid. The crude azidothiohydroximate (2 g, 3.4 mmol) was dissolved in anhydrous CH2Cl2 (49 mL), sulfur trioxide-pyridine complex (2.7 g, 17 mmol, 5 equiv.) was added and the suspension was heated at reflux for 24 h. The reaction was then cooled down to 0 °C and quenched by addition of a 0.5 M aqueous KHCO3 solution (2.4 g, 23.8 mmol, 10 equiv.) and left under stirring for 45 minutes at room temperature. The solvent was then evaporated under reduced pressure and the residue was purified using silica gel column chromatography (ethyl acetate/methanol: 9/1) to give the 𝟐𝟎

sulfated compound S3 as a white resin (1.96 g, 83%). Rf = 0.3 (EA/MeOH : 9/1); [α] 𝑫 = -5.78 (c = 0.83 in MeOH); 1H NMR (400 MHz, CD3OD) δ 7.33 (d, J = 8.6 Hz, 2H, CHAr), 6.97 (d, J = 8.7 Hz, 2H, CHAr), 5.13 (t, J = 9.2 Hz, 1H, H3), 5.05 (d, J = 10.2 Hz, 1H, H1), 5.01 – 4.77 (m, 1H, H4), 4.87 (t, J = 10.1 Hz, J = 9.6 Hz, 1H, H2), 4.22 - 4.11 (m, 3H, CH2O H6a), 4.07 - 3.94 (m, 3H, CH2CN, H6b), 3.78 3.72 (m, 1H, H5), 3.60 (t, 3J = 4.8 Hz, 1H, CH2N3), 2.06, 2.00, 1.94, 1.92 (4 × s, 12H, CH3 of Ac). 13C NMR (100 MHz, CD3OD) δ 172.2, 171.4, 171.1, 170.8 (C=O), 159.2 (Cq Ar), 158.3 (C=N), 130.5 (CH Ar), 129.3 (Cq Ar), 116.1 (CH Ar), 80.7 (C-1), 76.7 (C-5), 75.0 (C-3), 71.2 (C-2), 69.3 (C-4), 68.4 (CH2 O), 63.2 (C-6), 51.3 (CH2N3), 38.7 (CH2CN), 20.7, 20.53, 20.5 (CH3 of Ac). IR (neat) ν = 3160, 2111, 1745, 1511, 1368, 1216, 794 cm-1. HRMS (ESI-): m/z calculated for C24H29N4O14S2 [M]- = 661.1127, found 661.1125. (Z)-S-(β-D-glucopyranosyl)-[4-(2-azidoethoxy)phenyl]-2-acetothiohydroximate

N,O-sulfate

potassium salt 2. Potassium methoxide (76 mg, 1.08 mmol, 0.4 equiv.) was added to a solution of the acetylated compound S3 (1.9 g, 2.7 mmol, 1 equiv.) in anhydrous methanol (39 mL). The reaction


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mixture was stirred at room temperature for 5 h then the solvent was evaporated under reduced pressure. The crude product was finally purified on C-18 reverse phase column chromatography (H2O/MeOH : 𝟐𝟎

100/0 to 0/100) to give the azido glucosinolate 2 as a white resin (1.17 g, 81%). [α] 𝑫 = +10.0 (c = 0.34 in MeOH); 1H NMR (400 MHz, D2O) δ 7.34 (d, J = 8.5 Hz, 2H, CHAr), 6.93 (d, J = 8.6 Hz, 2H, CHAr), 4.57 - 4.54 (m, 1H, H1), 4.21 - 4.14 (m, 3H, CH2O, CH2CN), 4.02 (d, J = 15.8 Hz, 1H, CH2CN), 3.86 (bd, J = 12.5 Hz, 1H, H6a), 3.66 (dd, J = 12.5 Hz, J = 5.3 Hz, 1H, H6b), 3.58 (t, J = 4.8 Hz, 2H, CH2N3), 3.28 - 3.14 (m, 2H, H4, H5), 3.22 - 3.11 (m, 2H, H2, H3); 13C NMR (100 MHz, D2O, internal methanol) δ 161.9 (Cq Ar), 159.5 (C=N), 131.0 (CH Ar), 130.2 (Cq Ar), 116.5 (CH Ar), 83.3 (C-1), 82.6 (CH), 79.8 (CH), 74.6 (CH), 71.6 (CH), 68.9 (CH2O), 63.2 (C-6), 51.8 (CH2N3), 39.4 (CH2CN); IR (neat) ν = 3480, 2110, 1745, 1513, 1216, 794 cm-1. HRMS (ESI-): m/z calculated for C16H21N4O10S2 [M]- = 493.0704, found 493.0705. Glucosinolate-biotine 3. To a solution of azido-glucosinolate 2 (10 mg, 0.019 mmol, 1 equiv.) dissolved in MeOH (100 µL), triphenylphophine (5.4 mg, 0.021 mmol, 1.1 equiv.) was added. The reaction mixture was stirred for 24 h at room temperature, and then quenched with water (1 mL). A precipitate was formed corresponding to the by-product triphenylphosphine oxide. Then, the mixture was centrifuged 4 times (10000 rpm for 15 min) and the supernatants were collected and freeze-dried to obtain the glucosinolate-NH2 as a white solid (10.5 mg) The crude was used without any further purification. To a solution of glucosinolate-NH2 (10.5 mg, 0.019 mmol, 1 equiv) and biotine Nhydroxysuccinimide ester,48 (8.2 mg, 0.023 mmol, 1.2 equiv) in 200 µL of N-methyl-2-pyrrolidone was added triethylamine (4 µL, 0.030 mmol, 1.5 equiv). The reaction mixture was stirred at room temperature for 6 h. The completion of the reaction was confirmed by HPLC (System A). The reaction mixture was diluted in H2O (1 mL) and the crude product purified by preparative HPLC (System B) to afford the desired product 3 as a white solid (11 mg, 0.015 mmol, 82% over 2 steps). 1H NMR (300 MHz, D2O) δ 7.29 (d, J = 8.7 Hz, 2H), 6.98 (d, J = 8.7 Hz, 2H), 4.71 (dd, J = 5.2, 4.3 Hz, 2H), 4.42 (dd, J = 7.9, 4.4 Hz, 1H), 4.18 – 3.97 (m, 5H), 3.58 (ddd, J = 14.2, 7.8, 3.6 Hz, 4H), 3.43 – 3.19 (m, 4H),


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3.01 (ddd, J = 8.5, 6.3, 4.6 Hz, 1H), 2.81 (dd, J = 13.1, 4.9 Hz, 1H), 2.65 (d, J = 13.0 Hz, 1H), 2.22 (t, J = 6.8 Hz, 2H), 1.66 – 1.17 (m, 6H).

13C

NMR (75 MHz, D2O) δ 177.0, 165.1, 162.5, 157.5, 129.3,

127.6, 115.2, 81.3, 79.8, 76.9, 71.8, 68.7, 66.7, 61.7, 60.3, 60.1, 55.2, 39.7, 38.8, 37.3, 35.4, 27.5, 27.4, 25.1. HRMS (ESI): m/z calc [M]- (C26H37N4O12S3) 693.1570; found: 693.1558 IR (neat) ν = 3301, 2938, 1669, 1510, 1239, 1051, 796 cm-1 HPLC (System A): tR = 17.5 min, purity > 99% Glucosinolate-sulforhodamine 4. To a solution of glucosinolate-NH2 (10.5 mg, 0.019 mmol, 1 equiv) and sulforhodamine carboxylic acid37 (12.5 mg, 0.019 mmol, 1 equiv) in 200 µL of N-methyl-2pyrrolidone, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (11.2 mg, 0.021 mmol, 1.1 equiv) and N,N-diisopropylethylamine (10 µL, 0.059 mmol, 3 equiv) were added. The reaction mixture was stirred at room temperature for 10 h. The completion of the reaction was confirmed by HPLC. The reaction mixture was diluted with H2O (1 mL) and the crude mixture purified by preparative HPLC (System B) to afford the desired compound 4 as a purple solid (10 mg, 0.0085 mmol, 47% over 2 steps). 1H NMR (300 MHz, DMSO) δ 8.24 (d, J = 1.6 Hz, 1H), 7.72 (dd, J = 7.9, 1.6 Hz, 1H), 7.26 – 7.07 (m, 4H), 6.95 – 6.74 (m, 5H), 6.62 (s, 1H), 4.35 (dd, J = 22.3, 8.9 Hz, 1H), 4.12 – 3.87 (m, 4H), 3.80 – 3.72 (m, 2H), 3.58 – 3.44 (m, 5H), 3.38 (d, J = 3.6 Hz, 4H), 3.25 (s, 2H), 3.19 – 3.08 (m, 2H), 2.97 (dd, J = 16.6, 9.8 Hz, 6H), 2.63 (s, 2H), 2.10 (d, J = 6.9 Hz, 3H), 1.98 (s, 2H), 1.84 (s, 2H), 1.55 (dd, J = 13.5, 7.0 Hz, 5H), 1.33 (s, 3H).

13C

NMR (101 MHz, DMSO) δ 172.4, 157.8,

157.3, 156.6, 151.7, 151.1, 148.8, 146.6, 129.4, 129.4, 129.3, 129.1, 129.0, 128.5, 125.6, 125.3, 123.0, 114.6, 114.5, 113.4, 104.1, 81.4, 81.2, 78.0, 72.8, 69.9, 66.3, 61.0, 50.4, 49.9, 42.5, 40.2, 39.9, 39.7, 39.5, 39.3, 39.1, 38.9, 38.2, 37.0, 35.2, 28.0, 26.9, 26.1, 24.9, 20.1, 19.4, 19.2. HRMS (ESI): m/z calc [M-H]- (C47H53N4O18S4) 1089.2243; found: 1089.2238. IR (neat) ν = 3278, 1603, 1513, 1314, 1054, 955, 791, 568 cm-1 HPLC (System A): tR = 25.7 min, purity > 99%. The peak is broad probably due to the interaction of the sulfonate groups with the residual silanol groups present in the stationary phase. Glucosinolate-cyanine 5. To a solution of glucosinolate-NH2 (10.5 mg, 0.019 mmol, 1.3 equiv) and cyanine carboxylic acid49 (9 mg, 0.013 mmol, 1 equiv) in 200 µL of N-methyl-2-pyrrolidone, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (7.2 mg, 0.014 mmol, ACS Paragon Plus Environment

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1.1 equiv) and diisopropylethylamine (10 µL, 0.059 mmol, 3 equiv) were added. The reaction mixture was stirred at room temperature for 18 h. The completion of the reaction was confirmed by HPLC. The reaction mixture was diluted with H2O (1 mL) and the crude product purified by preparative HPLC (System B) to afford the desired compound 5 as a blue solid (9 mg, 0.0076 mmol, 42% over 2 steps). 1H NMR (300 MHz, DMSO) δ 8.36 (t, J = 13.0 Hz, 2H), 8.07 (t, J = 5.3 Hz, 1H), 7.81 (s, 2H), 7.64 (d, J = 8.1 Hz, 2H), 7.33 (dd, J = 8.3, 2.3 Hz, 2H), 7.19 (dd, J = 16.5, 8.5 Hz, 3H), 6.90 (t, J = 9.2 Hz, 2H), 6.59 (t, J = 12.3 Hz, 1H), 6.31 (d, J = 13.7 Hz, 2H), 4.34 (t, J = 11.1 Hz, 1H), 3.40 (dd, J = 10.5, 5.8 Hz, 4H), 3.20 – 2.91 (m, 5H), 2.09 (t, J = 6.7 Hz, 2H), 1.68 (s, 15H), 1.59 – 1.49 (m, 3H), 1.42 – 1.17 (m, 7H). 13C NMR (75 MHz, DMSO) δ 173.0, 172.7, 172.7, 172.4, 157.4, 155.3, 154.4, 154.3, 145.1, 145.0, 142.1, 141.6, 140.7, 140.6, 129.2, 128.6, 126.2, 120.0, 114.6, 110.3, 110.1, 103.5, 103.3, 81.4, 81.2, 78.1, 72.9, 69.9, 66.3, 61.1, 49.0, 48.9, 35.05, 27.14, 27.0, 25.8, 24.9, 12.2. HRMS (ESI): m/z calc [MH]2- (C47H53N4O18S4) 552.24; found: 552.23. IR (neat) ν = 3402, 2972, 1492, 1460, 1381, 1171, 1154, 1102, 1015, 926 cm-1. HPLC (System A): tR = 21.98 min, purity > 99%. Enzymatic hydrolysis of glucosinolates 3-5 To a solution of the corresponding glucosinolate (1 mg) in PB (1 mL, 0.1 M, pH 7.41) was added 20 L (0.28 U) of a solution of myrosinase (14000 U.L-1 in Milli-Q-water). The mixture was incubated at 37 °C for 24 h. The completion of the hydrolysis was monitored with HPLC. Each compound was isolated by RP-HPLC (system B), and analyzed with analytical HPLC (System A) and HRMS. ITC-3. tR = 27.2 min. HRMS (ESI): m/z calc [M-H]- (C20H25N4O3S2) 433.1368; found: 433.1366 ITC-4. tR = 28.3 min. HRMS (ESI): m/z calc [M]- (C41H41N4O9S3) 829.2036; found 829.2029. ITC-5. tR = 27.1 min. HRMS (ESI): m/z calc [M-]- (C43H43N4O8S3) 845.2713; found 845.2701 Fluorescent labeling of bovine serum albumin (BSA) Prior to the labeling of BSA, the following solutions were prepared: - Solution A: 1 mL stock solution of sulforhodamine 4 at 10-3 M in Milli-Q- water; - Solution B: 1 mL stock solution of cyanine 5 at 10-3 M in Milli-Q- water; - Solution C: 1 mL stock solution of FITC at 10-3 M in a solution of Milli-Q- water/DMSO (20:1); ACS Paragon Plus Environment

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- Solution D: 50 µL stock solution of myrosinase (14000 U.L-1 in Milli-Q- water). Fluorescent probe solutions 3-5 were stored at -25 °C, and the myrosinase solution was stored at 4 °C. To a solution of BSA (1 mg, 15 µmol) in PB (1.0 mL, 0.1 M, pH 7.41) was added the solution A, B or C (80 µL, 5 equiv.) and 5 µL of solution D (0.07 U). The mixture was incubated at 37 °C for 24 h. Then, the excess of sulforhodamine ITC-4, cyanine ITC-5 or FITC was removed by filtration through 10 kDa molecular weight cutoff Corning® Spin-X® UF 500 µL Concentrators. The protein was washed 4 times (800 µL) with Milli-Q water (10000 rpm, 15 min). The labeled proteins were stored at -25 °C before analyses. ASSOCIATED CONTENT Supporting Information. 1H,

13C

NMR data for all new compounds, spectroscopic data for

compounds 4, 5, BSA-4, BSA-5 and BSA-FITC (PDF). ACKNOWLEDGMENTS This work was partially supported by the Centre National de la Recherche Scientifique (CNRS), INSA Rouen, Normandie Rouen University, Inserm, the Region Centre Val de Loire, the University of Orléans and the Labex SynOrg (ANR-11-LABX-0029). We also thank Laurence Menu-Bouaouiche (Université de Rouen-Normandie) and Julien Roussel for technical assistance in the gel electrophoresis experiments and Albert Marcual (CNRS) for HRMS analyses. REFERENCES (1) Akkapeddi, P., Azizi, S.-A., Freedy, A. M., Cal, P. M. S. D., Gois, P. M. P., and Bernardes, G. J. L. (2016) Construction of homogeneous antibody–drug conjugates using site-selective protein chemistry. Chem. Sci. 7, 29542963. (2) Witus, L. S., and Francis, M. B. (2011) Using Synthetically Modified Proteins to Make New Materials. Acc. Chem. Res. 44, 774783. (3)

Hermanson, G. T. (2013) Bioconjugate Techniques, 3rd ed., Elsevier, Waltham, MA.

(4) Lang, K., Davis, L., Torres-Kolbus, J., Chou, C., Deiters, A., and Chin, J. W. (2012) Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction. Nat. Chem. 4, 298304.


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(5) Nguyen, T.-A., Cigler, M., and Lang, K. (2018) Expanding the genetic code to study protein– protein interactions. Angew. Chem. Int. Ed. 130, 14548–14559. (6) Tamura, T., and Hamachi, I. (2019) Chemistry for covalent modification of endogenous/native proteins: from test tubes to complex biological systems. J. Am. Chem. Soc. 141, 2782−2799. (7) Koniev, O., and Wagner, A. (2015) Developments and recent advancements in the field of endogenous amino acid selective bond forming reactions for bioconjugation. Chem. Soc. Rev. 44, 54955551. (8) Renault, K., Fredy, J. W., Renard, P.-Y., and Sabot, C. (2018) Covalent Modification of Biomolecules through Maleimide-based Labeling Strategies. Bioconjugate Chem. 29, 2497–2513. (9) Sakamoto, S., and Hamachi, I. (2019) Recent progress in chemical modification of proteins. Anal Sci. 35, 527. (10) Tanaka, K., Kamatani, M., Mori, H., Fujii, S., Ikeda, K., Hisada, M., Itagaki Y., and Katsumura, S. (1998) Synthesis of a new phospholipase A2 inhibitor of an aldehyde terpenoid and its possible inhibitory mechanism. Tetrahedron Lett. 39, 11851188. (11) Tanaka, K., Masuyama, T., Hasegawa, K., Tahara, T., Mizuma, H., Wada, Y., Watanabe, Y., and Fukase, K. (2008) A Submicrogram‐Scale Protocol for Biomolecule‐Based PET Imaging by Rapid 6π‐Azaelectrocyclization: Visualization of Sialic Acid Dependent Circulatory Residence of Glycoproteins. Angew. Chem. Int. Ed. 47, 102105. (12) Tung, C. L., Wong, C. T. T., Fung, E. Y. M., and Li, X. (2016) Traceless and Chemoselective Amine Bioconjugation via Phthalimidine Formation in Native Protein Modification. Org. Lett. 18, 26002603. (13) Jentoft, N., and Dearborn, D. G. (1979) Labeling of proteins by reductive methylation using sodium cyanoborohydride. J. Chem. Biol. 254, 43594365. (14) McFarland, J. M., and Francis, M. B. (2005) Reductive alkylation of proteins using iridium catalyzed transfer hydrogenation. J. Am. Chem. Soc. 127, 13490–13491. (15) Gimenez, D., Mooney, C. A., Dose, A., Sandford, G., Coxon, C. R., and Cobb, S. L. (2017) Reductive Alkylation of Proteins Using Iridium Catalyzed Transfer Hydrogenation. Org. Biomol. Chem. 15, 4086–4095. (16) Banks, P. R., and Paquette, D. M. (1995) Comparison of Three Common Amine Reactive Fluorescent Probes Used for Conjugation to Biomolecules by Capillary Zone Electrophoresis. Bioconjugate Chem. 6, 447–458. (17) Weber, G. (1952) Polarization of the fluorescence of macromolecules. Biochem. J. 51, 155–167. (18) Lomant, A. J., and Fairbanks, G. (1976) Chemical probes of extended biological structures: Synthesis and properties of the cleavable protein cross-linking reagent [35S]dithiobis(succinimidyl propionate). J. Mol. Biol. 104, 243261. (19) Diethelm, S., Schafroth M. A., and Carreira, E. M. (2014) Amine-Selective Bioconjugation Using Arene Diazonium Salts. Org. Lett. 16, 39083911. (20) Kalkhof, S., and Sinz, A. (2008) Chances and pitfalls of chemical cross-linking with aminereactive N-hydroxysuccinimide esters. Anal. Bioanal. Chem. 392, 305312. ACS Paragon Plus Environment

24

Page 25 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(21) Abello, N., Kerstjens, H. A. M., Postma D. S., and Bischoff, R. (2007) Selective acylation of primary amines in peptides and proteins. J. Proteome Res. 6, 47704776. (22) Jorbágy, A., and Király, K. (1966) Chemical characterization of fluorescein isothiocyanateprotein conjugates. Biochim. Biophys. Acta 124, 166175. (23) Niu, S. L., Ulrich, G., Ziessel, R., Kiss, A., Renard, P.-Y., and Romieu, A. (2009) Water-soluble BODIPY derivatives. Org. Lett. 11, 20492052. (24) Roubinet, B., Chevalier, A., Renard, P.-Y., and Romieu, A. (2015) Synthetic Route to 3(Heteroaryl)-7-Hydroxycoumarins Designed for Biosensing Applications. Eur. J. Org. Chem. 166182 (25) Mandapati, U. Pinapati S., and Rudraraju, R. (2017) Copper promoted desulfurization towards the synthesis of isothiocyanates. Tetrahedron Lett. 58, 125128. (26) Liao, Y.-Y., Deng, J.-C., Ke, Y.-P., Zhong, X.-L., Xu, L., Tang, R.-Y., and Zheng, W. (2017) Isothiocyanation of amines using the Langlois reagent. Chem. Commun. 53, 60736076. (27) Li, Z.-Y., Ma, H.-Z., Han, C., Xi, H.-T., Meng, Q., Chen, X., and Sun, X.-Q. (2013) Synthesis of Isothiocyanates by Reaction of Amines with Phenyl Chlorothionoformate via One-Pot or Two-Step Process. Synthesis 45, 16671674. (28) Bednarek, P., and Osbourn, A. (2009) Plant-Microbe Interactions: Chemical Diversity in Plant Defense. Science, 324, 746–748. (29) Agerbirk, N., and Olsen, C. E. (2012) Glucosinolate structures in evolution. Phytochemistry, 77, 16–45. (30) Jeschke, V., Gershenzon, J., and Vassão, D. G. (2015) The Formation, Structure and Activity of Phytochemicals (Ed.: R. Jetter), Springer International Publishing, Cham, pp. 163–194. (31) Halkier, B. A., and Gershenzon, J. (2006) Biology and biochemistry of glucosinolates. Annu. Rev. Plant Biol. 57, 303333. (32) Traka, M., and Mithen, R. (2009) Glucosinolates, isothiocyanates and human health. Phytochem Rev. 8, 269282. (33) Montaut, S., Zhang, W.-D., Nuzillard, J.-M., De Nicola, G. R., and Rollin, P. (2015) Glucosinolate Diversity in Bretschneidera sinensis of Chinese Origin. J. Nat. Prod. 78, 20012006. (34) Hanschen, F. S., Lamy, E., Schreiner, M., and Rohn, S. (2014) Reactivity and Stability of Glucosinolates and Their Breakdown Products in Foods. Angew. Chem. Int. Ed. 53, 1143011450. (35) Zhang, H., Trout, W. S., Liu, S., Andrade, G. A., Hudson, D. A., Scinto, S. L., Dicker, K. T., Li, Y., Lazouski, N., Rosenthal, J., et al. (2016) Rapid Bioorthogonal Chemistry Turn-on through Enzymatic or Long Wavelength Photocatalytic Activation of Tetrazine Ligation. J. Am. Chem. Soc. 138, 59785983. (36) Sato, S., Nakamura, K., and Nakamura, H. (2017) Horseradish-Peroxidase-Catalyzed Tyrosine Click Reaction. ChemBioChem 18, 475478. (37) Cerniauskaite, D., Rousseau, J., Sackus, A., Rollin, P., and Tatibouët, A. (2011) Glucosinolate Synthesis: a Hydroxamic Acid Approach. Eur. J. Org. Chem. 2011, 22932300.


25


Page 26 of 27

(38) Nehmé, R., Nehmé, H., Roux, G., Cerniauskaite, D., Morin, P., Rollin, P., and Tatibouët, A. (2014) Contactless conductivity detection for screening myrosinase substrates by capillary electrophoresis. Anal. Chim. Acta 807, 153158. (39) Cutolo, G., Reise, F., Schuler, M., Nehmé, R., Despras, G., Brekalo, J., Morin, P., Renard, P.-Y., Lindhorst, T. K., and Tatibouët, A. (2018) Bifunctional mannoside–glucosinolate glycoconjugates as enzymatically triggered isothiocyanates and FimH ligands. Org. Biomol. Chem. 16, 49004913. (40) Vo, Q. V., Trenerry, C., Rochfort, S., Wadeson, J., Leyton, C., and Hughes, A. B. (2013) Synthesis and anti-inflammatory activity of aromatic glucosinolates. Bioorg. Med. Chem. 21, 5945– 5954. (41) Chevalier, A., Renard, P.-Y., and Romieu, A. (2014) Straightforward Access to Water‐Soluble Unsymmetrical Sulfoxanthene Dyes: Application to the Preparation of Far‐Red Fluorescent Dyes with Large Stokes’ Shifts. Chem. Eur. J. 20, 83308337. (42) Chevalier, A., Renault, K., Boschetti, F., Renard, P.-Y., and Romieu, A. (2015) Rapid Synthesis of Unsymmetrical Sulforhodamines Through Nucleophilic Amination of a Monobrominated Sulfoxanthene Dye. Eur. J. Org. Chem. 2015, 152165. (43) calculated from the following equation: (F/P)exp(F/P)theo (44) Kobayashi H., and Choyke, P. L. (2011) Target-Cancer-Cell-Specific Activatable Fluorescence Imaging Probes: Rational Design and in Vivo Applications. Acc. Chem. Res 44, 8390. (45) Jouanno, L.-A., Chevalier, A., Sekkat, N., Perzo, N., Castel, H., Romieu, A., Lange, N., Sabot, C., and Renard, P.-Y. (2014) Kondrat’eva Ligation: Diels–Alder-Based Irreversible Reaction for Bioconjugation. J. Org. Chem. 79, 1035310366. (46) Folkertsma R. T., Rouppe van der Voort J. N., de Groot K. E., van Zandvoort P. M., Schots A., Gommers F. J., Helder J., and Bakker J. (1996) Gene pool similarities of potato cyst nematode populations assessed by AFLP analysis. Mol Plant Microbe Interact. 9, 4754. (47) Luthy, B., Matile, P. (1984) The mustard oil bomb; Rectified analysis of the subcellular organization of the myrosinase system. Biochem Physiol Pflanz 179, 512. (48) Hollas, M. A., Webb, S. J., Flitsch S. L., and Fielding, A. J. (2017) A Bifunctional Spin Label for Ligand Recognition on Surfaces. Angew. Chem. Int. Ed. 56, 94499453. (49) Park, J. W., Kim, Y., Lee, K.-J., and Kim, D. J. (2012) Novel Cyanine Dyes with Vinylsulfone Group for Labeling Biomolecules. Bioconjugate Chem. 23, 350362.


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Diverted natural Lossen rearrangement for bioconjugation through in

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