New Glycosidase Substrates for Droplet-Based Microfluidic Screening

Article pubs.acs.org/ac

New Glycosidase Substrates for Droplet-Based Microfluidic Screening Majdi Najah,†,‡ Estelle Mayot,†,‡ I Putu Mahendra-Wijaya,†,‡ Andrew D. Griffiths,*,†,§ Sylvain Ladame,†,∥ and Antoine Drevelle*,†,‡ †

Institut de Science et d’Ingénierie Supramoléculaires (ISIS), Université de Strasbourg, CNRS UMR 7006, 8 allée Gaspard Monge, 67083 Strasbourg Cedex, France ‡ Ets J. Soufflet, division Biotechnologies-OSIRIS, quai Sarrail, 10400 Nogent-sur-Seine, France § ́ Ecole Supérieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI ParisTech), 10 rue Vauquelin, 75231 Paris Cedex, France ∥ Department of Bioengineering, Imperial College London, South Kensington Campus, London SW72AZ, United Kingdom S Supporting Information *

ABSTRACT: Droplet-based microfluidics is a powerful technique allowing ultra-high-throughput screening of large libraries of enzymes or microorganisms for the selection of the most efficient variants. Most applications in droplet microfluidic screening systems use fluorogenic substrates to measure enzymatic activities with fluorescence readout. It is important, however, that there is little or no fluorophore exchange between droplets, a condition not met with most commonly employed substrates. Here we report the synthesis of fluorogenic substrates for glycosidases based on a sulfonated 7-hydroxycoumarin scaffold. We found that the presence of the sulfonate group effectively prevents leakage of the coumarin from droplets, no exchange of the sulfonated coumarins being detected over 24 h at 30 °C. The fluorescence properties of these substrates were characterized over a wide pH range, and their specificity was studied on a panel of relevant glycosidases (cellulases and xylanases) in microtiter plates. Finally, the β-D-cellobioside-6,8-difluoro-7-hydroxycoumarin-4-methanesulfonate substrate was used to assay cellobiohydrolase activity on model bacterial strains (Escherichia coli and Bacillus subtilis) in a dropletbased microfluidic format. These new substrates can be used to assay glycosidase activities in a wide pH range (4−11) and with incubation times of up to 24 h in droplet-based microfluidic systems.

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sorting (FACS)4−6 have been described for directed evolution of a number of enzymes, including β-glucosidase.7 This method relies on analysis of single cells compartmentalized in water-inoil-in-water emulsions.6 It inspired the development of dropletbased microfluidic systems for ultra-high-throughput screening of enzymes and microorganisms, which allows the production, manipulation, and sorting of highly monodisperse picoliter-size microreactors at high frequencies (kilohertz).8 This has allowed the directed evolution of horseradish peroxidase displayed on the surface of Saccharomyces cerevisiae9 and the evolution of sulfatase expressed in Escherichia coli.10 Droplet-based microfluidic systems have also been used to sort mammalian cells,11 viruses,12 or even genes expressed in vitro.13,14 For screening in droplet-based microfluidic systems, fluorogenic substrates based on a fluorescent leaving group are typically used to measure enzymatic activities.13,15 For instance, coumarin-based substrates have been widely used for

espite recent progress in opening up vast new fossil fuel reserves, environmental concerns linked to the production of greenhouse gases and side effects of hydraulic fracturing make sustainable production of commodities such as biofuels and other chemicals an attractive proposition. For a sustainable bioprocess, it is preferable to reserve starch for the food/feed industry and to instead use abundant renewable materials rich in cellulose and/or hemicellulose as feedstocks. All conversion technologies of these feedstocks proceed via hydrolysis of the biological polymers into simple sugars (mainly glucose and/or xylose), a sustainable bioprocess that requires efficient glycosidases (EC 3.2.1), most notably cellulases and xylanases.1 As a result of this, improving the efficacy of known glycosidases or finding new glycosidases, especially enzymes involved in the last steps of degradation releasing monosaccharides, which can be converted into commodities by fermentation process in a “sugars” biorefinery,2 has become a major challenge for the bioconversion industry. In this regard, an efficient and powerful high-throughput screening system is needed to select suitable enzymes. Screening methods based on in vitro compartmentalization3 (IVC) combined with fluorescence-activated cell © 2013 American Chemical Society

Received: July 23, 2013 Accepted: September 6, 2013 Published: September 6, 2013 9807

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Xylobiose and xylopolyose were acetylated using conventional procedures.34 The polyacetylated saccharides were then brominated as described by Camponovo et al.35 and used without further purification. General Procedure for the Synthesis of Sodium Saccharide-6,8-difluoro-7-hydroxycoumarin-4-methanesulfonate Derivatives 7−9. To a solution of coumarin 4, 5, or 6 (0.4 mmol) in anhydrous DMF (10 mL) kept under nitrogen and protected from light was added 5 equiv of silver carbonate. A solution of aceto-bromo-saccharide (5 equiv, except 3 equiv for aceto-bromo-xylobiose and for aceto-bromo-xylopolyose) in anhydrous DMF (10 mL) was then slowly added, and the reaction mixture was stirred at room temperature until the reaction had reached completion as indicated by LC/MS analysis (16−40 h). The mixture was then filtered through a pad of Celite, and the filtrate was collected and the solvent evaporated under reduced pressure. General Procedure for Acetyl Deprotection. To a solution of aceto-saccharide-coumarin (0.05 mmol) in anhydrous methanol (6 mL) kept under nitrogen at 0 °C was slowly added a solution of sodium methanoate 1% (w/w) in methanol (200 μL). The reaction was monitored by LC/MS and the mixture stirred at 0 °C until the reaction had reached completion (1−4 h). The reaction mixture was then neutralized by addition of Amberlite IR120 acid exchange resin, and the resin was filtered off and the filtrate concentrated under reduced pressure. The coumarin-saccharide conjugate was then purified by reverse phase chromatography. Sodium β-D-cellobioside-6,8-difluoro-7-hydroxycoumarin-4methanesulfonate 7a: yield 30%; 1H NMR (D2O) δ 7.55 (d, 1H), 6.60 (s, 1H), 5.17 (d, 1H), 4.45 (d, 1H), 4.38 (s, 2H), 3.9−3.2 (m, 12H). Sodium β-D-glucoside-6,8-difluoro-7hydroxycoumarin-4-methanesulfonate 7b: yield 54%; 1H NMR (D2O) δ 7.52 (d, 1H), 6.54 (s, 1H), 5.11 (d, 1H), 4.32 (s, 2H), 3.78 (m, 1H), 3.63 (m, 1H), 3.6−3.4 (m, 4H). Sodium β-D-xyloside-6,8-difluoro-7-hydroxycoumarin-4-methanesulfonate 7c: yield 62%; 1H NMR (D2O) δ 7.64 (d, 1H), 6.66 (s, 1H), 5.14 (d, 1H), 4.43 (s, 2H), 4.02 (m, 1H), 3.5−3.7 (m, 3H), 3.35 (m, 1H). Sodium β-D-xylobioside-6,8-difluoro-7hydroxycoumarin-4-methanesulfonate 7d: yield 40%; 1H NMR (D2O) δ 7.56 (d, 1H), 6.58 (s, 1H), 5.11 (d, 1H), 4.40 (d, 1H), 4.36 (s, 2H), 4.10 (m, 1H), 3.91 (m, 1H), 3.82 (m, 1H), 3.7− 3.5 (m, 3H), 3.40 (m, 2H), 3.21 (m, 2H). Sodium β-Dglucoside-5,6-benzo-7-hydroxycoumarin-4-methanesulfonate 8a: purification, HPLC eluent of 95:5 water/acetonitrile; yield 33%; 1H NMR (D2O) δ 8.24 (d, 1H), 8.18 (d, 1H), 7.53 (m, 2H), 6.80 (s, 1H), 6.24 (s, 1H), 5.25 (d, 1H), 4.49 (m, 2H), 4.0−3.5 (m, 6H). Sodium β-D-xyloside-5,6-benzo-7-hydroxycoumarin-4-methanesulfonate 8b: purification, HPLC eluent of 90:10 water/acetonitrile; yield 35%; 1H NMR (D2O) δ 8.09 (d, 1H), 7.99 (d, 1H), 7.46 (m, 1H), 7.38 (m, 1H), 6.51 (s, 1H), 6.16 (s, 1H), 4.98 (m, 1H), 4.37 (m, 2H), 4.01 (m, 1H), 3.8−3.4 (m, 4H). Sodium β-D-glucoside-7-hydroxy-8-methylcoumarin-4-methanesulfonate 9a: yield 34%; 1H NMR (D2O) δ 7.53 (d, 1H), 7.06 (d, 1H), 6.26 (s, 1H), 5.15 (d, 1H), 4.24 (m, 2H), 3.90 (m, 1H), 3.73 (m, 1H), 3.6−3.4 (m, 4H), 2.13 (s, 3H). Sodium β-D-galactoside-7-hydroxy-8-methylcoumarin4-methanesulfonate 9b: yield 36%; 1H NMR (D2O) δ 7.51 (d, 1H), 7.07 (d, 1H), 6.24 (s, 1H), 5.09 (d, 1H), 4.0−3.6 (m, 8H), 2.13 (s, 3H). Spectroscopic Studies. Buffer solutions at pH 3−8 were prepared by mixing 0.1 M citric acid and 0.2 M disodium phosphate solutions in different ratios (McIlvaine’s buffer36).

more than 30 years in cellulase and xylanase assays and to measure exoglycosidase (β-glucosidase or β-xylosidase) activity in microtiter plates.13,16−18 Unfortunately, these substrates are not suitable for droplet-based microfluidic systems because of the fast exchange of the coumarin product between droplets in emulsions produced with mineral oil16,19 or fluorinated oil.16,20 Exchange between droplets can occur directly through the carrier oil16,21,22 or by micellar transport.23−25 It can be reduced by using fluorinated carrier oils,26 in which nonfluorinated compounds are highly insoluble,27,28 or by using additives such as bovine serum albumin (BSA) in the aqueous phase.24,29 An alternative strategy is to modify chemically the coumarin with a sulfonate group to increase its water solubility, which results in a reduction in the rate of exchange between droplets.20 This strategy was used to develop substrates based on a sodium 7aminocoumarin-4-methanesulfonate scaffold to assay amidases in droplet-based microfluidic systems.20 Alternative fluorogenic substrates for glycosidases have been described previously30,31 and used recently in heterogeneous enzyme assays in a dropletbased microfluidic format.31,32 Unfortunately, this cellobiohydrolase substrate is based on resorufin, an orange-red fluorophore, which is prone to leakage in fluorinated emulsions13 and which is fluorescent only under neutral or alkaline conditions, whereas fungal glycosidases are efficient at acidic pH. Here we describe the development of fluorogenic assays for four glycosidases, β-glucosidase (EC 3.2.1.21), β-xylosidase (EC 3.2.1.37), cellobiohydrolase (EC 3.2.1.91), and endo-1,4β-D-xylanase (EC 3.2.1.8), based on the enzymatically catalyzed release of a sulfonated coumarin fluorophore. Three different sodium 7-hydroxycoumarin-4-methanesulfonate derivatives were synthesized by adapting a procedure previously reported by Gee et al.33 and present different spectroscopic properties and pH sensitivities. A set of nine different glycosidase fluorogenic substrates were synthesized by covalent attachment of these three coumarins to various oligosaccharides. Among them, four were tested and validated in microtiter plate assays against a panel of relevant fungal and bacterial glycosidases. One substrate was used in a droplet-based microfluidic assay to detect cellobiohydrolase activity in droplets containing two model bacterial strains.

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EXPERIMENTAL SECTION Synthesis. Reagents and Characterization. All reagents were used as received. Starting materials and reagents came from the following suppliers: Manchester Organics (1), ABCR (3), Wako (xylobiose and xylopolyose), Orgentis Chemicals (aceto-xylose), and Sigma-Aldrich (2, aceto-bromo-glucose, aceto-bromo-galactose, and all the other reagents). NMR spectra were recorded on a Bruker Advance-400 instrument and calibrated using residual undeuterated solvent as an internal reference. Splitting patterns are designated as s (singlet), d (doublet), dd (double doublet), q (quartet), and m (multiplet). LC/MS analyses were conducted on an Accela HPLC system with a Surveyor MSQ Plus, LCQ Fleet three-dimensional ion trap mass spectrometer (Thermo Scientific). The reverse phase HPLC purifications were performed either on Interchim reverse phase prepacked columns or with a Biotage Isolera One system with SNAP KP-C18-HS cartridges. Coumarins 4−6 were synthesized by adapting previously reported procedures.33 The detailed experimental conditions are described in the Supporting Information. 9808

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(Figure S1A of the Supporting Information). Syringes were connected to the microfluidic device using 0.6/25 mm Neolus needles (Terumo Corp.) and PTFE tubing with an internal diameter of 0.56 mm and an external diameter of 1.07 mm (Fisher Bioblock Scientific). Liquids were pumped into the microfluidic device using standard-pressure infusion-only PHD 22/2000 syringe pumps (Harvard Apparatus Inc.). Each aqueous solution (i.e., cell suspension or dye solutions) was infused at a rate of 100 μL/h into the microfluidic device. The oil phase was composed of EA surfactant (Raindance Technologies), a PFPE-PEG-PFPE amphiphilic block copolymer,38 at 2.5% (w/w) in Novec7500 oil (3M). It was infused at a rate of 400 μL/h to produce 37 pL droplets. The resulting emulsion, containing two different droplets populations, was collected in a glass capillary with an internal diameter of 5 mm connected to the device outlet by 10 cm length of Intramedicpoly(ethylene terephthalate) PE20 tubing (Becton, Dickinson and Company) for off-chip incubation.39 For reinjection, a syringe was connected via a PE20 tubing to the other end of the capillary. The emulsion was reinjected at a rate of 30 μL/h into the reinjection and analysis device (Figure S1B of the Supporting Information), and droplets were spaced with surfactant-free Novec7500 fluorinated oil, infused at a rate of 75 μL/h, to perform single-droplet analysis. Coumarin Retention. Solutions of coumarin 4 at 1 and 10 μM were prepared in buffer at pH 5, 7, and 9 (see above). After collection, a fraction of the emulsion was immediately reinjected into the analysis device (Figure S1B of the Supporting Information). The droplet fluorescence was monitored by exciting the coumarin at 375 nm and detecting the emitted light at 452 nm using the optical setup detailed in Figure S2 of the Supporting Information to obtain the fluorescence data at time zero. The capillary was incubated at 30 °C for 24 h, and then the rest of the emulsion was reinjected and analyzed again under the same conditions to obtain the fluorescence measurements at 24 h. Enzyme Kinetic Measurement. Bulk enzymatic assays were performed in a 384-well microtiter plate by adding enzymes to the different substrates. Twenty-five microliters of the enzyme at various concentrations was added to 25 μL of the different substrates at the same concentration (0.25 mM) in McIlvaine’s buffer36 (pH 4.5 or 7). All enzymes were supplied by Megazymes: β-D-glucosidase was from Aspergillus niger (EBGLUC), cellobiohydrolase I from Trichoderma sp. (E-CBHI), endo-β-glucanase from A. niger (E-CELAN), endo-β-glucanase from Bacillus amyloliquifaciens (E-CELBA), transglucosidase from A. niger (E-TRNGL), endo-1−4-β-xylanase from A. niger (E-XYAN4), β-D-xylanase from Bacillus stearothermophillus (EXYNBS), exo-1,4-β-D-xylosidase from Bacillus pumilus (EBXSEBP), and exo-1,4-β-D-xylosidase from Selenomonas ruminantium (E-BXSRB). All enzymes were tested at pH 4.5 and 60 °C except the two exo-1,4-β-D-xylosidases, which were tested at pH 7.0 and 37 °C. Fluorescence was monitored over 30 min using a spectrofluorophotometer (SpectraMax M5, Molecular Devices) with the following setup: λex and λem values of 350 and 470 nm for measurements at pH 4.5 and λex and λem values of 370 and 470 nm for measurements at pH 7, respectively. Initial rates (vi) were determined by fitting the slope over the first 5 min (when vi > 1000 RFU/min) or over the first 30 min (when vi < 1000 RFU/min). All kinetics experiments were performed in duplicate. Cell Preparation. The two strains used in this study were E. coli C41(DE3) (hereafter called Ec) and Bacillus subtilis

Buffer solutions at pH 9−11 were prepared by mixing 0.1 M sodium hydrogen carbonate and 0.1 M sodium carbonate solutions in different ratios (carbonate buffer). Coumarin solutions were prepared by dissolving coumarins 4 and 6 at 10 μM and coumarin 5 at 100 μM in each buffer. Excitation and emission spectra were recorded on a SpectraMax M5 spectrofluorophotometer (Molecular Devices). For each coumarin, the maximal emission wavelength was determined by recording their emission spectra between 400 and 700 nm with excitation at 370 nm. The maximal excitation wavelengths of the basic and acidic forms of each coumarin were then determined by recording the excitation spectra between 300 and 450 nm of each coumarin at every pH, fixing the emission wavelength at 470, 510, and 490 nm for derivatives 4−6, respectively. Microfluidic Systems. All microfluidic experiments were performed with dual-dropmaker or reinjection and analysis devices (Figure S1 of the Supporting Information). Devices were fabricated using soft lithography37as previously described.8 SU-8 2015 was used to prepare 30 μm deep molds. After the PDMS device had been sealed with a microscope slide, the surface of the channels was modified after being flushed with 1H,1H,2H,2H-perfluorooctyltrichlorosilane (Sigma-Aldrich) in 1% (v/v) fluorinated oil Novec7500 (3M), then with pure Novec7500 (3M), and finally nitrogen. Optical Setup, Data Acquisition, and Control System for Droplet Analysis. The optical setup (Figure S2 of the Supporting Information) consisted of a TI-U inverted microscope (Nikon SAS) mounted on a vibration-dampening platform (Clean Top II, TMC). The microscope was equipped with two epifluorescence stages: the higher one dedicated to epifluorescence illumination (an Intensilight illuminator was connected to the higher-stage back port through a fluorescence illuminator unit) and the lower one dedicated to fluorescence quantification in droplets [the lower stage back port was connected to the fluorescence detection system (FDS) described below]. A high-speed camera (Eosens MC1310, Mikrotron GmbH) was connected on the left-side port, and a color camera (DS-Fi1-12 bits, Nikon) was connected to the side port of an eyepiece tube base unit. The FDS was composed of three integrated lasers (a LightHUB combining a 20 mW, 375 nm Luxx diode laser, a 80 mW, 488 nm Luxx diode laser, and a 150 mW, 561 nm Coblot Jive DPSS laser, supplied by Omicron-Laserage Laserprodukte GmbH) and four photomultiplier tubes (PMT, H10723-20 from Hamamatsu Photonics KK). The combined laser beams from the LightHUB were focused by the microscope objective (CFI Plan Fluor 20×, Nikon SAS) on a channel within the microfluidic device. Dichroic filters were used to separate the incoming laser beams from the emitted fluorescence and to split the fluorescence to four PMTs. Bandpass filters were used to assign spectra to the four PMTs, allowing detection of fluorescence at 452, 514, 579, and 641 nm. A more detailed description of FDS is given in Figure S2 of the Supporting Information. Data acquisition (DAQ) and control were performed with a PCI-7831R Multifunction Intelligent DAQ card (National Instruments Corp.) executing a program written in LabView 8.2 (National Instruments Corp.). The data acquisition rate for the system was 200 kHz. Device Operation. Droplet production for coumarin retention experiments and enzymatic assays on cells in the droplet were conducted using the dual-dropmaker device 9809

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NCIM2724 (hereafter called Bs). Cell suspensions for emulsification were prepared following the same procedure. First, the strain was grown on tryptic soy agar (TSA) plates overnight at 30 °C to obtain isolated colonies. Then single colonies were used to inoculate 10 mL of tryptic soy both (TSB) medium for overnight incubation at 30 °C while the sample was being shaken at 200 rpm. Ten microliters of the growing culture was diluted in 10 mL of fresh TSB medium and grown under the same conditions until midlog phase (OD600 = 0.5−0.6). The cultures were harvested by centrifugation at 3000g for 5 min at 4 °C. The supernatant was removed and the cell pellet resuspended in fresh TSB medium containing sulforhodamine 101 (Sigma-Aldrich) (2 μM for Bs and 10 μM for Ec) and cellobiase substrate (compound 7a) at 0.25 mM. The OD600 values of cell suspensions were adjusted to achieve, on average, either 0.04 or 1 cell/droplet after emulsification. Analysis of Fluorescence Data. All data from droplet experiments were analyzed using Spotfire from TIBCO software.

Coumarin-sugar conjugates were then synthesized via a Koenigs−Knorr reaction40 between the hydroxy-coumarins and a range of polyacetylated saccharides brominated on their anomeric position. As a result of this covalent attachment, the fluorescence of the coumarin is almost completely quenched while the saccharide-free hydroxy-coumarin is strongly fluorescent. Both monosaccharides (i.e., xylose, glucose, and galactose) and polysaccharides (i.e., cellobiose, xylobiose, and xylopolyose) were coupled to the three hydroxy-coumarins to obtain a small set of β-glycosyl-coumarins. 40,41 After purification by reverse phase chromatography column, nine glycosyl-coumarins were obtained and tested as fluorogenic substrates suitable for monitoring enzymatic activity both in microtiter plates and in microfluidic devices. Spectroscopic Properties of Glycosidase Substrates. The spectroscopic properties of dyes 4−6 were determined at various pH values (Figure 2) to characterize the fluorescence

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RESULTS AND DISCUSSION Synthesis of Glycosidase Substrates. Three different coumarins were synthesized by adapting a protocol previously described by Gee et al.33 (Figure 1). The synthetic route

Figure 2. Fluorescence properties of coumarins 4−6 as a function of pH. Each coumarin was dissolved in a solution at a defined pH, at a concentration of 10 μM (4 and 6) or 100 μM (5). The dyes were excited at the maximal excitation wavelength of (a) the protonated form or (b) the deprotonated form. The fluorescence values were measured at the maximal emission wavelength of each coumarin.

properties of the coumarins in their basic form (i.e., when the hydroxyl group is deprotonated) and in their acidic form (i.e., when the hydroxyl group is protonated). These two forms have two different maximal excitation wavelengths, so both of them were used to excite the dyes and to measure the emitted fluorescence. From this spectroscopic study, estimated pKa values of 4.5, 6.5, and 8.0 were found for coumarins 4−6, respectively. It is noteworthy that for all three coumarins tested the fluorescence intensity was always higher at high pH values (i.e., when the coumarin is under its basic/anionic form). The maximal excitation and emission wavelengths were different for the three dyes in their protonated form and deprotonated form (Figure S3 of the Supporting Information). Because of their unique spectroscopic specificities, these three coumarins are compatible with a wide range of enzymatic assays. For example, coumarin 4 is likely to be better suited for experiments performed at pH ≥6, the presence of the electron-withdrawing fluorine atoms lowering the pKa value of the neighboring hydroxyl group. In contrast, coumarin 6, which remains strongly fluorescent at acidic pH, is best suited for assessing the activity of enzymes working at lower pH values (3−6). Finally, coumarin 5 offers the advantage of having red-shifted excitation and emission wavelengths (compared to those of coumarins 4 and 6) because of an extended aromatic surface and could therefore be excited with a different laser (e.g., a 405 nm laser diode).

Figure 1. Synthetic scheme and structures of sulfonated coumarins and their corresponding β-glycosyl conjugates.

consists of a Pechmann condensation between a resorcinol derivative and ethyl 4-chloroacetoacetate, followed by a sulfonation with sodium sulfite. The sulfonate moiety was selected because it carries a permanent negative charge that allows the coumarin to remain confined inside the water-in-oil droplets for multiple hours by strongly increasing the hydrophilicity of the fluorophore.20 Coumarins with different spectroscopic properties were obtained by introducing chemical modifications on their aromatic core (e.g., -Me, -F, and -Ar). 9810

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Coumarin 4 Retention. As a model system, enzymatic substrates based on coumarin 4 were used for droplet-based microfluidic experiments using the microfluidic station described in Figure S2 of the Supporting Information (laser at 375 nm, PMT at 452/45 nm). For the assay to be conducted in water-in-oil emulsions, it is vital that the free coumarin released upon action of the enzyme remains confined inside the droplet microreactor. To evaluate the potential exchange of 4 between droplets, two populations of droplets containing 1 and 10 μM compound 4 were produced. The fluorescence intensity of each drop was measured immediately after the production and after incubation at 30 °C for 24 h. The study was performed at pH values of 5, 7, and 9 to check if the pH had an influence on the retention. The results are presented in Figure 3. In all the experiments, the ratio of the mean fluorescence of

represent less than 2% of the total population. This comes from rare droplet fusion events that lead to drops with a concentration of 4 between 1 and 10 μM. The pH has no effect on the retention of coumarin 4; however, the lower fluorescence intensity at pH 5 was confirmed. Enzymatic Assays. Various sugars were grafted on the three coumarins leading to nine different fluorogenic substrates for glycosidases (compounds 7a−e, 8a, 8b, 9a, and 9b in Figure 1). Glycosyl conjugates made from coumarin 4 (i.e., derivatives 7a−e) were selected for further biological screening because of the stronger brightness and compatibility with a wider pH range [pH 4−11 (Figure 2)]. The four substrates 7a−d were assayed with fungal and bacterial glycosidases. All four conjugates function as substrates for both fungal and bacterial enzymes. As expected, cellulolytic enzymes (β-glucosidase, β-glucanase, and cellobiohydrolase) are strongly active on glucose-based substrates (7a and 7b) and hemicellulolytic enzymes are active on xylose-based substrates (7c and 7d). Exoglycosidases are active on monosaccharideand disaccharide-based substrates, whereas endoglycosidases (cellobiohydrolase, endo-β-glucanase, and endo-1−4-β-xylanase) are active mainly on disaccharide-based substrates (7a and 7d). These four substrates are, therefore, stable, specific, and well adapted to assay glycosidases for analytical or screening purposes. Some unexpected activity can be observed in the case of endo-β-glucanase and β-glucosidase from A. niger on xylosebased (7c) and xylobiose-based (7d) substrates, respectively. This may be due to secondary activities in these products, which are not fully purified enzymes, or the ability of these enzymes to easily accommodate the small coumarin leaving group in their active site. Moreover, the xylose/xylobiose-based substrates (7c and 7d) are less stable at 60 °C (the temperature used for cellulase assays) than glucose/cellobiose-based substrates (7a and 7b). This means that 7c and 7d are easier to hydrolyze under these conditions. At 37 °C, all substrates are stable. Cellobiase Assays on Bacterial Cells in Droplets. To assay cellobiase activity produced by bacteria grown in droplets, we performed experiments on cells using compound 7a. Two model strains, Ec (no cellobiase activity) and Bs (cellobiase producer), were compartmentalized in droplets at a 1:1 ratio using a dual-dropmaker (Figure S1A of the Supporting Information) with, on average, either 0.04 cell/droplet or 1

Figure 3. Coumarin 4 retention as a function of pH. Retention of coumarin 4 was assayed at three different pH values (5, 7, and 9). The distribution of the blue fluorescence of droplets containing 1 or 10 μM coumarin 4 was analyzed before (0 h) and after incubation (24 h) at 30 °C. For each sample, 10000 droplets were analyzed. Data were split into 500 bins.

the droplets containing 1 and 10 μM 4 was almost unchanged before and after incubation: the variation of the fluorescence ratio between time zero and 24 h was ≤5.6%, and this slight variation is likely to be due solely to minor modifications of the manual focus in the z-axis between each measurement. This indicates that there was no detectable exchange of coumarin 4 between droplets over 24 h. This contrasts with a half-life of 2.1 s for exchange of the nonsulfonated 7-amino-4-methylcoumarin analogue.20 In every case, the number of drops in each population represents 50 ± 4.6% of the total number of droplets. The droplets with intermediate fluorescence intensity Table 1. Enzyme Kinetic Measurements with Substrates 7a−da

7a with cellobiose enzymes

buffer

β-glucosidase from A. niger cellobiohydrolase from Trichoderma sp. endo-β-glucanase from A. niger endo-β-glucanase from B. amyloliquifaciens transglucosidase from A. niger endo-1−4-β-xylanase from A. niger β-D-xylanase from B. stearothermophillus exo-1,4-β-D-xylosidase from B. pumilus exo-1,4-β-D-xylosidase from S. ruminantium McIlvaine’s buffer at pH 4.5 and 60 °C McIlvaine’s buffer at pH 7 and 37 °C

235.3 1211.9 103.1 2447.0