Marc Fouet

Sébastien Cargou

Rémi Courson

Marie-Charline Blatché

Armelle Montrose

Karine Reybier

Anne Marie Gué

C Blatche

A M Gue

Multilevel (3D) microfluidic technology for an innovative magnetic cell separation and couting platform

Keywords: Magnetic separation, Cell tagging, Planar coils, Multilayer (3D)

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Magnetophoresis is broadly used in medical and biological applications (from cellular to genomic scale) and has been frequently implemented in lab on chip. However magnetic handling tools are mostly external magnets limiting the range of functions that can be integrated: the magnetic field is consequently permanent and non-local and coupling several functions in the same chip remains quite impossible. In particular, magnetic labelling has to be performed out of the chip prior to sorting, reducing therefore the added value of the device. Here, we demonstrate a new concept of device which by combining 3D fluid engineering and localized magnetic actuation enables the full integration of a cell tagging and magnetic separation device.

MICROFABRICATION AND EXPERIMENTAL

The lab on chip microfabrication was done by using a novel negative epoxy based dry film DF-1050 (EMS) whose low price is a major advantage compared to well-known epoxy photoresists: it is at least ten times cheaper than SU-8 and TMMF [START_REF] Courson | Low cost integration of multilevel lab-on-achip using a new generation of dry film photoresists[END_REF]. Microchips were fabricated following the general scheme proposed by Fulcrand et al. [START_REF] Fulcrand | Development of a flexible microfluidic system integrating magnetic micro-actuators for trapping biological species[END_REF] and depicted in Figure 1.

RESULTS AND DISCUSSION

The magnetic labelling of monocytes was performed on chip. Solutions of cells and beads were introduced simultaneously in the micromixing module whose principle was based on baker's transformation applied to microfluidics. This principle was firstly proposed by the theoretical work of P. Carriere [START_REF] Carriere | On a three-dimensional implementation of the baker's transformation[END_REF]. Contrary to standard approaches which are more suited to molecular mixing than to particle mixing, this solution turned to be well suited to cellular labelling with an average labelling yield of 1-2 microbead/cell as shown in Figure 2. The separation buffer was introduced in the lower level channel while the sample was flowing in the upper one. Nine coils located before and on the separation stage enabled the tagged cells to be efficiently at-tracted across the superposed microchannels. Monocytes could be trapped on the micro-coil (Fig. 3 b), and the separation efficiency of magnetic beads was studied at different flow rates (Fig. 4).

Interestingly we demonstrated an abrupt transition from a "trapping mode" to a "continuous separation mode" depending on flow. The thermal safety of the device has also been checked using rhodamine B to ensure Joule heating (at a 100mA intensity) will not harm the monocytes.

CONCLUSION

While our magnetofluidic devices still undergo characterization tests, we endeavor to achieve even higher integration in implementing an electrical impedance measurement based detection system. Tagging, separating and counting different monocytes populations would have direct applications in detecting early stages of infectious diseases, and would give a good insight of new available tools for immunology.

Figure 1 :

 1 Figure 1: 3D fabrication process using DF dry film photoresists and lamination process.

Figure 2 :

 2 Figure 2: a) Comsol FEM analysis of the 3D mixer device for mixing an aqueous solution of fluorescein with water. b) Top view (optical microscope) of a mixer unit (3 loops) right after micro fabrication. c) Left: trajectory of a monocyte tagged with 4 magnetic beads inside a mixer unit. Right: examples of tagged monocytes obtained after 10 units (30 loops) with 1 and 2 magnetic beads. Downstream the magnetic actuation was driven by planar coils, which allowed a high flexibility in the design and a strong magnetic field. The separation principle is given in Figure 3 a).

Figure 3 :

 3 Figure 3: a) Scheme of the magnetic separation stage. Sample (monocytes and beads previously mixed) and buffer are injected in superposed channels. The coils attract magnetic beads and tagged monocytes and separate them from the upper channel. b) THP1 Monocytes trapped on a planar micro coil. If the flow rate is low, beads and monocytes can be trapped on a coil instead of flowing and performing a continuous separation.

Figure 4 :

 4 Figure 4: Graph of separation efficiency of 5 µm diameter magnetic beads. Trapping occurs at low flow rates, and those tests were carried out on short periods of time so the coils are never saturated. Proper separation with a satisfying efficiency can be achieved between 1.5 and 2 µL/min (about 3 mm/s for the average velocity of the flow according the the microchannel cross section).

ACKNOWLEDGEMENTS

This study was supported by the French National Research Agency (ANR) in the frame of the PIA -Nanobiotechnology project Digidiag and by the Defense Agency (DGA). It was also partly supported by the French Renatech Network


A Girard

N Coulon

C Cardinaud

T Mohammed-Brahim

F Geneste

Effect of doping on the modification of polycrystalline silicon by spontaneous reduction of diazonium salts

Keywords: Polycrystalline silicon, diazonium salt, spontaneous grafting, doping, crystallinity, functionalization

) used as references is investigated by spontaneous reduction of diazonium salts. The effectiveness of the grafting process on all polySi surfaces is shown by AFM and XPS analyses. The effect of substrate doping on the efficiency of the electrografting process is compared by using the thicknesses of the deposited organic films. For a better accuracy, two methods are used to estimate the thicknesses: XPS and the coupling of a O 2 plasma etching with AFM measurement.

Structural characteristics of the poly-Si films were investigated by Scanning Electron Microscopy and X-ray diffraction to find a correlation between the structure of the material and its reactivity.

Different parameters that could have an impact on the efficiency of the grafting procedure are discussed. The observed differences between differently doped silicon surfaces is rather limited, this is in agreement with the radical character of the reacting species.

Introduction

Silicon is the most widely used material in semiconductor devices because of the low cost of its raw material, the relatively simple processing, and a useful temperature range. Polycrystalline silicon, also called PolySi, is composed of multiple small silicon crystals. It is a material of choice in microelectronics for the fabrication of devices needing deposition of doped silicon films. For 1 example, it is widely used in the fabrication of field effect transistors [1][2][3][4][5], solar panels [6] and diodes [7,8]. Advantageously, the polySi deposition step is manufactured by Low Pressure Chemical Vapor Deposition (LPCVD), leading to deposited layers with good homogeneity, high stability and well-controlled doping concentrations. The process can also be performed at relatively low temperature allowing the deposition on many substrates like even glass.

The formation of organic thin films on silicon constitutes a major issue since it has been used in many electronic devices such as transistors, memories and diodes [9]. Among all the methods that have been developed to modify silicon surfaces [10][11][12][13], those involving radical reactions have the advantage to form a chemical bond directly on Si. Among them, the cathodic reduction of arenediazonium salts that has been widely used to functionalize surfaces [11,14,15] is particularly effective to prepare phenyl layers on H-terminated planar Si surfaces [16]. This efficient and fast grafting process leads to a strong covalent bond, and monolayers or thin films can be obtained in well-controlled conditions [17,18]. Interestingly, the modification procedure can also be performed spontaneously, simplifying the process and making it readily accessible [19]. A mechanism has been proposed for the attachment of the aryl radical on hydrogenated Si surfaces (Scheme 1) [16]. The first step involves an electron transfer from the surface at open-circuit potential to the diazonium salt generating, upon loss of N 2 , an aryl radical. Elimination of a proton leads to silicon radical that reacts with aryl radical to give C-Si bond. The presence of silicon radicals has been confirmed through screening of various reagents known to react rapidly with molecular silicon radicals [20].

The reactivity of polySi substrates to spontaneous grafting of diazonium salts has been reported in literature [19,21]. The electroless deposition can be performed both in the dark and under illumination. A better reactivity has been observed for polySi (N2+) than for Si (N2+) or Si (P2+) whatever the time exposure to the solution. It has been attributed to the roughness of the substrate giving rise to higher density of surface states, increasing the probability to ensure the spontaneous electron transfer leading to the reduction of the diazonium salt.

In view of the importance of polySi in the electronic devices fabrication, we investigated the effect of the nature and level of doping in polycrystalline silicon on the spontaneous reduction of diazonium salts. The effectiveness of the grafting process was evaluated by XPS and AFM. The different substrates were analyzed by X-ray diffraction and SEM in order to find a correlation between the structure of the material and the reactivity of the surface toward the modification procedure.

Experimental section

Chemicals

Reagents 4-carboxymethyl-benzenediazonium salts were prepared according to literature and stored under argon in a freezer [22]. All solvents were HPLC grade. Ultrapure water (18.2 Mω, Millipore Simplicity) was used for the grafting procedure.

Preparation of the modified surfaces

All manipulations were carried out under argon on a Schlenk line with standard airless techniques.

A 2% hydrofluoric acid solution was cannulated into a reactor cell containing the surfaces under argon to remove the native Si oxide. After 2 min of immersion, the HF solution was eliminated. A solution containing 5 mg of 4-carboxymethyl-benzenediazonium salts in 50 mL of anhydrous deoxygenated acetonitrile was cannulated into the reactor cell. After 1h, the surfaces were washed in six successive baths of acetonitrile of 10 min each. It is worth noting that the grafted layer was still observed in XPS when the samples were sonicated 3 times in acetonitrile for 5 min. Before analyses, the samples were stored under argon in a fridge.

Substrates

In this work, three types of materials composed of silicon were studied.

As a reference, we add a silicon monocristal wafer, oriented 100, which is a microelectronic grade material (high purity) with well-known properties. This phosphorous-doped (1-10 ohm/cm) n-type Si has a thickness of 280 μm. It was purchased from BT Electronics.

The materials composed with silicon are deposited in the IETR laboratory on glass substrates.

First, on this Corning Eagle glass substrates, a 250 nm buffer oxide layer was deposited by atmospheric pressure chemical vapor deposition (APCVD) technique at the temperature of 420°C

with SiH 4 -N 2 -O 2 gas mixture. This oxide layer acts as a diffusion barrier in order to avoid the possible contamination of the layers by impurities in the substrate.

Low Pressure Chemical Vapor Deposition (LPCVD)

The silicon layers (300 nm) were amorphously deposited at 550°C with a pressure of 90 Pa using a hot wall Low Pressure Chemical Vapor Deposition (LPCVD) reactor. Phosphorous (10 ppm or 1000 ppm PH 3 diluted in hydrogen) and boron (1000 ppm B 2 H 6 diluted in hydrogen) in situ doped silicon films are deposited with the same conditions using a mixture of silane as precursor gas and phosphine or diborane as a doping gas, respectively. Doping concentrations, measured by secondary ion mass spectroscopy (SIMS), are fixed at 3 × 10 18 and 2 × 10 20 atom/cm 3 . After depositing each layer, a solid phase crystallization (SPC) process for 12h at 600°C under vacuum was carried out to obtain polysilicon.

All the conditions are summarized in Table 1.

Instrumentation

AFM

The Atomic Force Microscopy (AFM) images were obtained in air, operating in tapping mode, using a Veeco AFM diCaliber High Value Scanning Probe Microscope with a standard antimony doped silicon tip which is a cantilever with a resonant frequency of 300 kHz. All the images were recorded with a 512 points per line resolution at a scan rate of 1Hz. Tapping mode was used to determine all the changes at the surface of our substrates caused by the grafting modification.

SEM

The SEM micrographs were obtained with a Jeol 6301F (9 kV) microscope. A scanning electron microscopy may be used not only to observe a surface image but also to provide a fine electron beam for charging an isolator surface simultaneously [23,24].

X-Ray Diffraction (XRD) X-Ray (Cu Kα) diffraction (Bruker D8 Advance) was used in the -2  configuration to evaluate :

(i) the evolution of crystallization as a function of the doping (ii) the existence of preferred grain orientations in the films XPS X-Ray photoelectron spectroscopy (XPS) has been carried out with a Kratos Axis Ultra HSA spectrometer using a monochromatic Al Kα excitation (1486.6 eV), with magnetic confinement charge neutralization, which is very effective in minimizing both uniform charging and differential charging on this large-band-gap semiconductor. The hybrid lens magnification mode was used with the slot aperture resulting in an analyzed area of 700 per 300 μm 2 . Charge stabilization was achieved by using the Kratos Axis device. The pass energy was set at 20 eV for the detailed scans.

In these conditions, the energy resolution gives a full width at half maximum (FWHM) of the Ag 3d5/2 peak of about 0.55 eV. Wide scans were recorded with a step size of 0.5 eV and a pass energy of 80 eV, for narrow scans the corresponding parameters were 0.1 and 20 eV, respectively.

The sampling depth of approximately 15 nm in normal XPS analysis provides information on the nature of the chemical bonds of samples through the determination of core-level binding energies.

To assess compositional variation with depth, angle-resolved XPS data were collected at nominal photoelectron take-off angles of 30° and 60°, where take-off angle is defined as the angle between the surface and the axis of the analyzer lens system. Using published electron mean free paths equations from Seah and Dench [25], the sampling depth (three times the mean free path) should decrease from ~ 11 to ~ 5 nm as the take-off angle decreases from 60° to 30°. The data were collected at room temperature, and the operating pressure in the analysis chamber was always below 10 -9 Torr. Peak positions were referenced to the average reported data in the literature of aliphatic carbon (C-C/C-H 1s peak) at 285.0 eV [START_REF] Beamson | High resolution XPS of organic polymers-the science ESCA science database[END_REF][START_REF] Fulghum | [END_REF][28].

Data treatment and peak fitting procedures were performed using Casa XPS software (version 2.3.16) and Kratos data base of relative sensitivity factors. Deconvolution of spectra into Gaussian-Lorentzian peaks (G/L=30), was also done using Casa XPS software, with peak area and width as free fitting parameters. A Shirley function was used to model the background. Peak assignments were made based on comparing the binding energies with reports in the literature for similar molecular species (Table 2). shows the influence of the doping level on the grain size. Thus, heavily N-type poly-Si clearly presents smaller grains and more grain boundaries than the weakly N-type poly-Si. The nature of the doping also influences the structure of the surface. As highlighted in Fig. 1 a) and c), N-type poly-Si surfaces contain less grain boundaries than P-type poly-Si for similar doping concentrations. The presence of defects also seems to show that the P-type poly-Si is less well crystallized after the Secco treatment.

X-Ray diffraction

The crystallinity and preferred orientation (texture) of the deposited polysilicon films were studied by X-ray diffraction (XRD). Fig. 2 shows the X-ray diffraction profiles for undoped, N and P-doped films. The features for these LPCVD films are similar. The existence of preferred grain orientations were analyzed using the intensities of the (111), ( 220), (311), (331), ( 422) and ( 511) peaks (Table 3). These intensities were corrected to take into account the effect of the film thickness. For a randomly oriented polysilicon film, the X-ray integrated intensity of the (111) reflection is the strongest. For 300 nm-thick P type film, the other intensities for (220), ( 311), (400), and (

reflections are lower by a factor of 0.25, 0.19, 0.18, and 0.06, respectively (Table 3).

Modification of the PolySi surfaces

PolySi surfaces were first treated with a 2% HF solution to achieve hydride termination. They were then immersed in a solution of 4-carboxymethyl-benzenediazonium salts in acetonitrile.

Spontaneous reduction of the diazonium salt occurred, leading to surfaces functionalized by carboxylic acids (Scheme 2). Even if a C1s peak was observed in the spectra of polySi, probably due to pollution by oil vapor pumping LPCVD system or atmospheric contamination, grafting obviously leads to an increase of the C1s peak centered at 285.0 eV. The presence of an O1s peak at 532.4 eV in the polySi spectra is due to the reoxidation of the substrate before XPS analysis. We also noticed that photoelectron peaks characteristic of the semiconductor substrates (Si2s, Si2p) are attenuated but still visible after the organic layer has been grafted. This result indicates either the formation of a homogeneous layer onto the substrate the thickness of which is thinner than the depth analyzed by the XPS technique, or the heterogeneous distribution of the organic matter onto the surface.

The presence of terminal grafted carboxylic functions was evidenced with XPS by analyzing C1s

and Si2p core level. The high-resolution C1s spectra of the polycrystalline silicon and the modified sample are shown in Fig. 4. All data shown were obtained on N-type polycrystalline silicon; however, other dopings were measured and showed similar results. C1s spectra of the polycrystalline silicon (Fig. 4a) can be decomposed into three features: one located at 284.8 eV assigned to C-C bonds, one at 286.7 eV assigned to C-O bonds and the last at 288.7 eV assigned to C=O bonds. With respect to the functionalized surface, the C1s peak at 285.2 characterized by C-C bonds increases significantly as a phenyl layer was grafted on the surface (Fig. 4b). The intensity of the peak around 287 eV, which is obviously attributed to the C-O bonds increased after grafting.

Besides, we still observe the presence of the C=O peak (288.1 eV) and another one at 289.6 eV.

This can be attributed to the functional group -COOH [10,16]. Interestingly, a new carbon peak appears at 283.3 eV in the spectrum of the modified sample that corresponds to a carbide silicon.

This small C1s signal is, however, really important as it was interpreted for many times in the literature as an evidence of the covalent metal-carbon bond between the metal surface and phenyl group grafted through the diazonium route [11,28]. The peak found at around 283 eV was reported for the first time to be a direct proof for the existence of a covalent bond between an aryl group and the iron substrate [32]. The presence of Si-C bonds is also highlighted on the Si2p spectra with a component at 100.6 eV in the deconvolution (Fig. 4).

heavily N-type polycristalline Si heavily N-type polycristalline Si with grafted layer Angle-resolved XPS was performed to probe the interfacial species formed between the diazonium salt and the polysilicon surface. The analyzed depth (d) decreases following the relation [START_REF] Briggs | Practical Surface Analysis by Auger and X-Ray Photoelectron Spectroscopy[END_REF]:

d = 3 λ cos a
where λ is the photoelectron mean free path for the analysed material, a is the photoelectron emission angle with respect to the detector axis; it is equal to the angle of sample rotation. Angular XPS analysis allows extinction of the signal from the deeper layers and hence, enhancement of the surface sensitivity. By this way, for thin-film deposits, depth resolution of different XPS detected bonds can be attained in order to permit a study of interfacial regions. The takeoff angles (TOAs) were 0, 30 and 60° relative to the surface normal.

Table 4 gives informations on elemental composition versus the TOAs. The silicon substrate is still visible even at 60°, an indication that the organic overlayer is thinner than the sampling depth of the technique. We observe an increase of the carbon content and a decrease of the silicon concentration (in the metallic and oxide forms). The oxygen concentration, however, is not affected by the angle, because this element belongs to both the inorganic substrate

as oxide forms and the organic overlayer in the form of COOH groups and superficial adventitious contamination.

The table 5 reports the concentrations (at%) of different types of bonds obtained after C1s

decomposition region in function of the analysis angle. From the results presented, it would appear that the carboxylic acid group is not exactly at the outermost surface of the organic layer because the relative intensity of the peak at 285.2 eV slightly decreases from 0 to 60°. Nevertheless, the carbide/COOH ratio (calculated from the results presented in Table 5) decreases versus the analysis angle, indicating that the COOH groups exist in all the thickness of the layer. This is in agreement with the formation of multilayers due to the growth of polyphenylene chains as it is well-known for grafting involving aryl radicals [43].

In addition to identifying elements present, XPS allows one to perform quantitative analysis.

Considering that a homogeneous layer is formed, the attenuation of the Si 2p photoelectron signal through the top layer yields insights into the molecular films thickness (z). This thickness (Table 6) could be estimated by the standard overlayer model [START_REF] Briggs | Practical Surface Analysis by Auger and X-Ray Photoelectron Spectroscopy[END_REF]:

  s s I I exp (-z/ λ s cos a) =  s I exp (-z/ λ s sin θ)
Where I s is the intensity of the attenuated Si 2p signal,  s I is the Si 2p signal coming from the Si sample without any overlayer, λ s (nm) is the photoelectron mean free path of the substrate-specific photoelectrons in the analysed material, a is the photoelectron emission angle with respect to the detector axis and θ is the analysis takeoff angle relative to the surface.

The electron attenuation length for Si was obtained from Henry de Villeneuve et al. [16], where the λ s value was equal to 4 ± 0.5 nm. In the case of a polycrystalline silicon layer, the thickness of the grafted layer is about 4 nm against 2.6 nm and 3.3 nm for monocrystalline silicon oriented (100) and (111) respectively. It seems that the texture favors grafting as well as an (111) orientation.

By AFM

These results were corroborated by Atomic Force Microscopy (AFM). Contact mode AFM images of all surfaces have been performed before and after modification (8 m  8 m), as exemplified in Fig. 5 with weakly N-type polySi. Since surface roughness is a current factor to the presence of a molecular layer on the surface, the RMS roughness of unmodified and modified polySi was also performed by measurements on 8 by 8 m images, in tapping mode AFM (Table 7).

The N-type polySi surface is flat and homogeneous showing topographical variations in the range 2 -6 nm. Disperse nodules of approximately 0.1 -0.2 m in diameter were observed. After grafting, the roughness changes appreciably giving rise to topographical variations of 5 to 7 nm, showing the effectiveness of the grafting process. The appearance of the modified surfaces is homogeneous with the presence of nodules with diameters between 0.05 and 0.36 m, in a larger amount than in the initial polySi surface. They can be attributed to the covering of the defects of polySi surfaces by a thin organic film after grafting. Whereas the roughness of Si (100) surfaces also increased after grafting, the behavior of the heavily P-type polySi was different. Indeed, since its rugosity is high, the presence of the electrografting film probably smoothed the surface. The thicknesses of the grafting layers estimated by XPS were confirmed with a plasma etching followed by AFM analysis [44]. The method consists in mechanically masking the grafted surfaces and then exposing them to an oxygen plasma to etch the organic layer. The thickness of the resulting step (Fig. 6) corresponding to the transition between the grafting layer and the cleaned substrate was measured by AFM. Results are given in Table 8. The thickness measurement of the step was difficult to achieve on heavily P-type polySi and Si(111) due to the high roughness of the modified surfaces.

Discussion

XPS and AFM analyses were performed to investigate the effect of doping on the spontaneous grafting of diazonium salts on different silicon substrates. The increase of the C1s and O1s components in XPS associated with an attenuation of the Si peak evidenced the effectiveness of the grafting process on all the studied surfaces. The covalent immobilization of the aryl compound deduced from the stability of the organic layer to sonication in acetonitrile was confirmed by the presence of a peak corresponding to the C-Si bond. AFM images corroborated XPS analyses and highlighted the homogeneity of the organic film deposited on the surfaces. To evaluate the effect of doping on the grafting method, the surface thicknesses were estimated by XPS and by a method combining a O 2 plasma with AFM step measurements. Results obtained by the two different methods were similar and allowed to conclude on the influence of the surface on the grafting process: weakly N-type polySi > heavily P-type polySi > heavily N-type polySi > Silicon (111) > Silicon (100). From these results, it is obvious that the method is less efficient on Si(100) and Si(111) than on polySi substrates, whatever the nature and amount of doping. This result is in accordance with those previously presented by Charlier et al. [21]. The authors have proposed that the higher roughness of the polySi surface, allowing a higher density of surface states, would explain its higher reactivity. However, our results do not exactly support this hypothesis since the more reactive surface is N-type polySi that is 3.4 times less rough than the heavily P-type polySi.

A relationship between the surface electron work function and the crystal structure has already been evocated for electrografting of diazonium salts on polycrystalline gold substrate [45]. Since the work function is the minimum energy required to release electrons from the reactive surface, it is linked to the capacity to lose electrons of the different polySi substrates. Thus, the crystallinity of the polySi should influence the spontaneous reduction of the diazonium salt and so the effectiveness of the grafting process. X-ray diffraction allowed us to get insight into the crystallinity and preferred orientation of the deposited polysilicon films. None of the orientations of polySi seems to favor a grafting process in the order of reactivity revealed by thickness measurements: weakly Ntype polySi > heavily P-type polySi > heavily N-type polySi. Since the integrated intensities of the (422) reflection in the different polySi substrates follow the opposite order, this orientation seems to be unfavorable to spontaneous grafting. By contrast, (220) and (511) orientations that predominate in the weakly N-type polySi would facilitate surface modification. However, it is difficult to conclude on the preferred orientation for spontaneous grafting on silicon from these results due to the combination of several orientations in polycrystalline Si. Pure material should be investigated to achieve a clear relationship between crystal structure of silicon and the effectiveness of spontaneous grafting of diazonium salts. Thus, the reactivity of Si(111) seems to be higher than Si(100), according to these experiments.

If the doping level plays a role in the resistivity of the material and so in the reactivity of the surface towards electrografting [46], it is not obvious for spontaneous reduction of diazonium salts. Indeed, the thickness of the grafted organic layer is higher for the weakly N-type polySi substrate than for the less resistant heavily N-type polySi surface. However, the nature and level of doping can influence other factors than resistivity. First, it affects the grain size of the polySi surface. Indeed, weakly N-type polySi has higher grain size than the heavily doped surfaces. Thus defects associated with grain boundaries could decrease the reactivity of the substrate, as if the grafting process preferentially occurred on the bulk surface. Second, the influence of doping on the Fermi level of polySi materials is well-known [47]. Thus, it has been shown that the Fermi level is significantly higher for weakly N-type polySi than for the heavily N-type polySi, which has also a higher Fermi level than the heavily P-type polySi. Furthermore, the spontaneous reduction of the diazonium salt depends on the difference between the Fermi level of the doped semi-conductor and the LUMO of the diazonium salt. An increase of the Fermi level should lead to a more efficient reduction step and so to higher kinetics for the modification process. Indeed, a good correlation was found between the reactivity of the weakly and heavily N-type polySi substrates and their Fermi levels. However, for the heavily N and P-type polySi, the Fermi levels are closer and the crystal orientations play probably a more important role on spontaneous grafting.

Conclusion

Whereas covalent grafting by reduction of diazonium salts is now a common method to modify silicon substrates, this work focused on the modification of polycrystalline silicon by spontaneous reduction of diazonium salts and highlighted the effect of doping on the functionalization procedure. XPS and AFM analyses revealed that the covalent grafting process was very efficient on all studied polySi substrates, even more than on monosilicon Si(100) and Si(111) taken as references. Comparison of the thickness of the formed deposited organic layer showed that the effectiveness of the modification procedure followed the order: weakly N-type polySi > heavily Ptype polySi > heavily N-type polySi. Whereas the resistivity does not influence the reactivity of the surface as for electrografting processes, other parameters such as the orientations of the crystal structure and the amount of grain boundaries can participate to the results. A good correlation between the measured surface thicknesses and the evolution of the Fermi levels with the doping level of the semi-conductor was also highlighted, underlining the role of the reducing power of the substrate for the effectiveness of the grafting procedure.

1 Figure 1 :

 11 Figure 1: Scanning electron micrographs of the surface of 300 nm-thick polysilicon films

Fig 2 :

 2 Fig 2: X-ray diffraction profiles for undoped, N and P-doped polysilicon films

Scheme 2 8 3. 3 Figure 3 :

 833 Scheme 2

Figure 4 :

 4 Figure 4: Deconvolution of C1s and Si2p core level spectra obtained with AlK excitation

Figure 5 :

 5 Figure 5: A top view representation of N-type polySi before (a) and after ( b) grafting.

Figure 6 :

 6 Figure 6: AFM images of a step created by O2 plasma etching on weakly N-type polySi and step measurement of the grafted surface.

Table 1 :

 1 LPCVD parameters

	Layer types	Diluted PH3	Diluted B2H6	Pure SiH4	T°C	Postbake°C
	Weakly N-type polycristalline Si	17	-	50	550	600
	Heavily N-type polycristalline Si	40	-	50	550	600
	Heavily P-type polycristalline Si	-	40	50	550	600

Table 2 :

 2 XPS reference table and peak fitting parameters used in this study

		Si-Si	99 -99.6
	Si (Si2p)	Si-C SiOx	101 102.7	[39] [40] [41]
		SiO2	103.5 -103.7
		C=O	531.0 -532.2
		Si-O-Si	531.4 -531.7
	O (O1s)	O-H	530.8 -532.0	[40] [32] [33] [37]
		SiO2	532.3
		C-O-C	533.5

  The I(220)/I(111) and I(311) /I(111) intensity ratios were calculated and normalized to their values in a randomly oriented powder. A normalized ratio of one would indicate that the grains are randomly oriented. Values lower than one indicate a (111) preferred orientation.

Table 3 :

 3 Intensities of the Si peaks

			(111)	(220)	(311)	(400)	(331)	(422)	(511)
	Weakly N-type	I	16.6	3.1	1.8	-	1.5	1.1	1.1
	polycristalline Si	I/I(111)	1	0.18	0.11	-	0.09	0.06	0.06
	Heavily N-type	I	1 9 . 2	2 . 9	2	-	1.6	1.5	0.9
	polycristalline Si	I/I(111)	1	0,15	0,1		0.08	0.07	0.05
	Heavily P-type	I	9.4	2.4	1.8	-	1.4	1.2	1.1
	polycristalline Si	I/I(111)	1	0.25	0.19	-	0.15	0.13	0.12
	Polysilicon	I I/I(111)	11.9 1	2.1 0.18	1.6 0.13	-	1.6 0.13	1.3 0.11	-

Table 4 :

 4 Analysis results of the grafted layer on heavily P type Poly Si by angular XPS (at%) depending of TOAs

	TOAs (°)	O (%)	C (%)	Si (%)
	0	21	34.3	44.7
	30	21.2	35	43.8
	60	20.9	51.1	37

Table 5 :

 5 Peak fitting results for C1s region in function of the analysis angle

	TOAs (°)	C-Si (%)	C-C/C-H (%)	CH2-COOH (%)	C-O (%)	C=O (%)	C-Si/COOH ratio
	0	1.4	82.5	8.2	5.5	2.4	0.17
	30	1.2	86.0	7.4	3.8	1.6	0.16
	60	1	87.7	7.3	2.9	1.1	0.14

Table 6 :

 6 Thickness (nm) of the grafted layer deduced from the attenuation of the signal

	Thickness of grafted diazonium layer (nm)

Table 7 :

 7 RMS roughness of samples estimated by AFM

	Layer types
	weakly N-type polySi
	heavily N-type polySi
	heavily P-type polySi
	Silicon (111)
	Silicon (100)

Table 8 :

 8 Thickness (nm) of the grafted layer deduced from plasma etching coupled with AFM

		Thickness of grafted diazonium layer (nm) a
	weakly N-type polySi	4.5 ± 0.3
	heavily N-type polySi	3.7 ± 0.4
	heavily P-type polySi	-
	Silicon (111)	-
	Silicon (100)	1.9 ± 0.2

a estimated from ten line measurements


Alizé Pennec

Richard Daniellou

Pascal Loyer

Caroline Nugier-Chauvin
email: caroline.nugier@ensc-rennes.fr

Vincent Ferrières

Araf51 with improved transglycosylation activities: One

Keywords: Furanosides, Transglycosylation, Random mutagenesis, Furanosidase 1

Introduction

One of the current needs in chemical research is the development of greener and more eco-friendly reactions. The glycosylation is an example thereof, being one of the most important and difficult reactions for biomolecules synthesis. Indeed, it still remains a challenge in chemistry requiring multiple reaction steps and the use of various protecting groups. In Nature there are multiple enzymes as glycosyltransferases, polysaccharide lyases and glycosidases able to assemble or cleave easily the glycosidic bond with high regioselectivity. 1 Nowadays, biotechnological approaches are recognized as powerful tools for the synthesis of glycoconjugates and improving biocatalyst efficiency seems to be one of the most promising approaches. [2][3][4][5][6][7] Moreover it is noteworthy that the chemical synthesis of arabinofuranosides with a high degree of stereocontrol and without any pyranosidic forms, suffers from a lack of generality. For instance the synthesis of alkyl arabinofuranosides is generally obtained from the key intermediates per-O-protected furanoses. 8-10 However, biocatalyzed furanosylation has been recently developed and its versatility was further extended to the preparation of non-natural furanosyl-containing conjugates (for a review, see reference 11 ). Indeed, glycosidases are good candidates in comparison to other enzymes, thanks to their stability, ease of production and low-cost of substrates which can be bio-sources. A recent example of directed evolution was applied to the βglycosidase of Thermus thermophilus in order to increase its ability to synthesize oligopyranosides vs. its hydrolytic activity. [12][13][14] To achieve these results, Koné et al. developed a high-throughput digital imaging screening methodology to detect transglycosidase mutants in E. coli cells. 14 Herein, we adapted and completed this kind of screening to improve arabinofuranosyl transglycosylation with the aim to apply it to a wide range of aliphatic alcohols (Scheme 1). As a model reaction, we chose to synthesize alkyl α-L-arabinofuranosides which are simple molecules with diverse properties and potential uses. Depending on the nature of the alkyl chain, they find applications as immunostimulating agents or antiparasitic drugs. [15][16][17] More generally, the alkyl pyranosidic counterparts act as chemical building blocks for further derivatization and industrial preparation of alkyl polyglycosides. 18,19 Scheme 1. Enzymatic synthesis of alkyl α-L-arabinofuranosides.

In this context, we performed random mutagenesis on the α-L-arabinofuranosidase (Araf51) from Clostridium thermocellum in an attempt to improve the transglycosylation activities for the synthesis of alkyl α-L-arabinofuranosides. For the selection of suitable variants, a screening in two steps has been developed. The initial one, directly on plates, allowed colony selection of mutants with good hydrolytic activity on an Araf-containing chromogenic substrate. Indeed in order to maintain the physical quality of the agar gels and cells viability, the presence of alcohols as acceptors was excluded for this first screening step. The second one assessed the transglycosylation activities towards the convenient alcohol acceptor of the previous selected enzymes by 96-well spectrophotometry. In this approach, one of the main objectives was to obtain mutants with high selectivity towards nucleophilic alcohols: one specific mutant for one specific acceptor.

2.

Results and discussion

2.1.

Identification of mutants with improved transglycosylation abilities.

The thermostable arabinofuranosyl hydrolase, Araf51, naturally removes α-(1 2), α-(1 3) and α-

(1 5)-linked L-arabinofuranosyl units from arabinans and arabinoxylans. To the best of our knowledge, this enzyme belongs to the only arabinofuranosidase family (GH51) showing the ability to catalyze transglycosylation reactions. In addition, Araf51 has been described several times for its substrate versatility as it recognizes arabinofuranosides as well as galactofuranosides. 17,[20][21][22][23] Preliminary experiments showed the ability of the Araf51 wild type (wt) to transglycosylate pNP-Araf to small aliphatic alcohol in order to obtain alkyl α-L-arabinofuranosides. 24 However, the activity strongly decreased when acceptors contain more than 3 carbons in their chain. With the aim to improve the transglycosylation activity towards aliphatic alcohols, mutants were thus created by random mutagenesis and selected from a simple screening procedure.

Firstly, a library of mutated Araf51 genes has been generated by error prone PCR and cloned into pCR® 2.1-TOPO® vector. After cell transformations, E. coli BL21 DE3 clones were spread on LB plates for the screening. This latter consisted in a first selection of positive colonies overexpressing an active Araf51. During this step, the colonies were platted on LB media containing IPTG and 1.5 mM of 5-bromo-indolyl α-L-arabinofuranosyl. 25 This molecule is hydrolyzed by the Araf51 releasing a blue air-oxidized di-indolyl compound. In this way, the colonies staining in blue expressed enzymes successfully cloned which still recognize the arabinofuranosyl moiety as donor. Ideally, mutants that tend to favor transglycosylation were supposed to perform slow aglycon release in the absence of a suitable acceptor and therefore display a pale blue staining. Nevertheless, a low expression level of some mutated enzymes could also result in a pale coloration. Also, the selection of all the blue colonies was performed and the transglycosylation properties of the resulting enzymes assayed.

This first step allowed us to select more than 120 colonies for further expression of the mutated enzymes. The corresponding cell free extracts were heated at 70 °C to remove most of the E. coli derived proteins and also confirmed the thermostability of the selected mutants. In the second step of the screening procedure, each enzyme was tested in the presence of pNP-Araf and an alcohol in order to evaluate their transglycosylation ability (T) over hydrolysis potential (H) (Scheme 2). UVmeasurements were performed during the initial time where the deglycosylation of the glycosylenzyme intermediate is the rate-limiting step. If the transglycosylation is favored, the initial speed rate in these conditions should be higher than the one during the hydrolysis. The initial reaction rates were determined thanks to the release of p-nitrophenol (405 nm). For each cell free extract, two types of reaction were performed, in presence and absence of alcohol acceptor. The alcohol volume was up to 25% (v/v) to limit the presence of water, and so to favor as much as possible the target transglycosylation. DMSO was added to the reaction mixture as an optimized solubility factor for aliphatic alcohol. Reactions were performed at pH 8 to ensure maintaining the phenolate form of the leaving group and thus proper UV-visible monitoring. A range of linear alcohols were tested from methanol to n-hexanol as well as i-propanol, allylic alcohol and solketal.

Scheme 2. Araf51-catalyzed transglycosylation with pNP-Araf as donor and alcohols as acceptors.

To easily reveal interesting enzymes, the initial speed rates obtained (Vi) were normalized with the one corresponding to the wild type. Then, a ratio R was calculated correlating directly to the increase of activity in presence of the acceptor (Equation 1).

R= H+T (Equation 1)

with Vi H+T corresponds to the initial rate in presence of alcohol, and Vi H to the initial rate without alcohol.

For each alcohol tested, around twenty mutants showed positive activation with R value above 1. In order to detect these mutants with potentially better transglycosylation than the wild type, analytic time course reactions were performed and conversions (Table 1) determined by NMR spectroscopy:

the protons corresponding to the pNP group are up-field shifted when released, and the appearance of a new anomeric proton from the product was clearly observed (see supplementary materials). On this basis, we focused our attention on five mutants. Then preparative reactions were performed, and the transglycosylation products were isolated by column chromatography (Table 1). The alkyl resulting α-L-arabinofuranosides were characterized by 1 H and 13 C NMR analysis. These data were in accordance with previously recorded results for compounds 1-5, 9 [26][27][28] and new furanosides 6-8 were fully described in the experimental part. i-propanol, solketal, n-butanol, n-hexanol) to excellent (methanol, ethanol) conversions in transglycosylated derivatives were generally obtained using the wild type biocatalyst but long reaction times were required (up to 2 hours). Secondly, it is important to note that no autocondensation product has been observed, probably due to the high concentration of alcohol acceptor that favored the desired transglycosylation. Subsequently, the mutation procedure of Araf51 wt enabled the isolation of 5 mutants with significant improved transglycosylation activities for n-propanol, i-propanol, n-butanol, n-pentanol, and n-hexanol (entries 3, 4, 7, 8, 9, respectively). In comparison to the native enzyme, the engineered biocatalysts were all more promising in term of reaction time and conversion in tranglycosylation, with improvement from + 13% for M20 (n-propanol, entry 3) to + 154% for M57 (n-hexanol, entry 9). As an example, time course evolution between the wild type biocatalyst and the M20 mutant was given in figure 1. Unfortunately, the proposed protocol did not afford improved biocatalyst for solketal and allyl alcohol. Nevertheless, under the transglycosylation conditions performed with the selected mutants, hydrolysis of the resulted arabinofuranosides was not observed, even over 120 minutes and after total consumption of the substrate. Finally, no other transglycosylation by-products (autocondensation) were observed, so that the targeted α-L-arabinofuranosides were specifically formed, confirming the diastereoselectivity of the mutants according to the glycosidic bond, whatever the nature of the biocatalyst. In order to evaluate the selectivity towards the aliphatic acceptor thanks to the mutated biocatalysts, transglycosylation reactions were performed with other structurally close alcohols (Table 2). It appeared that the mutants showed significantly better transglycosylation ability for the alcohol for which they were selected than for other acceptors. For instance, M20 mutant was particularly efficient and selective for n-propanol.

Kinetic characterization and mutation identification of the selected enzymes

Finally, the kinetic parameters of the different mutants were determined under hydrolysis conditions of pNP-Araf at 25 °C and pH 8 (50 mM Tris HCl buffer). Whereas Araf 51 is a thermophilic enzyme with T opt around 82 °C, the parameters were determined at 25 °C for practical reasons, as previously reported. 29 The data presented in table 3 compares the values obtained with the Araf51 wt to the ones calculated for the selected enzymes. Most of the new biocatalysts showed lower K m by comparison with the wild type, especially for M20 and M22. Furthermore, k cat values for the mutants were also lower than that for the wild type Araf51. On a structural point of view, the crystal structure of the wild type enzyme has been previously solved (PDB file: 2C7F) and showed that two essential catalytic residues are required for the hydrolysis reaction were assigned as the acid/base residue Glu173 and the nucleophile Glu292. Moreover, structural information was also obtained from the co-crystallised of the inactive mutant E173A form of Araf51 with natural arabinotriose and arabinoxylobiose. 29 These studies suggest little specificity for the exact orientation of the aglycone in the +1 subsite due to flexible conformation adopted by Trp178 which stacks against the aglycone sugar unit. The sequence analysis of the mutants from this study revealed from one to six mutations (Table 4, see also supplementary data). It clearly appeared that the mutations were neither within the active site nor close to it. Nevertheless, concerning the biocatalyzed synthesis of the target alkyl arabinofuranosides, we expected that the alkyl chains could fit the entry of the common GH51 (β/α) 8 barrel domain located at the extremity of the -1 subsite. Despite the location of the mutated sites, far from the active site, these mutations seemed to significantly affect the kinetics properties with regards to substrate affinity and transglycosylation reaction activity.

Conclusion

Thanks to the coupling of random mutagenesis method conducted on the arabinofuranosyl hydrolase Araf51 and an efficient screening, we have obtained five mutants able to catalyze the transfer of an arabinofuranosyl entity to various aliphatic alcohols. In comparison to the wild type enzyme, these new biocatalysts improved the transglycosylation conversions up to 96%. Additionally, the procedure used in the study yielded new biocatalysts with increased transglycosylation abilities and selectivity closely depending on the nature of the acceptor for which they were screened. This strategy was efficient with the aim of synthesizing valuable chemical building blocks such as alkyl α-Larabinofuranosides. Additional benefits mutations in these rationally designed enzymes appeared to be distant from the active site. Further directed evolution experiments targeted on these observed mutations are now required in order to study the properties and catalytic abilities of the generated enzymes and to accurately understand the involvement of each modified site.

Experimental

Expression and purification of Araf51 and mutant derivatives

Plasmid pET28a (+) (Novagen) containing Araf51 wild type from Clostridium thermocellum 29 and kanamycin resistance genes was provided by Prof G. Davies, University of York, U. K. Plasmid pCR®2.1-TOPO® (3.9 kb) from Invitrogen contains encoding mutated enzymes genes as well as ampicillin and kanamycin resistance genes. These plasmids were under the control of T7 promoter.

The enzymes were produced in Escherichia coli BL21 DE3 cells (Invitrogen) cultured in LB (Lysogeny broth) plates containing 0.1 mM of the corresponding selective agent at 37 °C. 10 millitres of LB medium supplemented with antibiotic were inoculated with one single positive colony E. coli BL21 (DE3) and incubated overnight at 37 °C, 250 rpm. That was subsequently used to prepare 1% inoculum (200 mL LB + antibiotic, 250 rpm, 37 °C). Cells were grown to mid-exponential phase [Absorbance, A 550 : 0.7] at which point i-propyl -D-thiogalactopyraoside (IPTG) was added to a final concentration of 1.0 mM and the cultures were incubated for 14 h at 37 °C. After centrifugation (20 min, 4000 rpm, 4 °C), the cells were resuspended in 50 mM Tris HCl buffer pH 8 and sonicated (3 × 10 s) for another centrifugation (30 min, 20000 rpm, 4 °C), the supernatant was heated at 70 °C for 15 min to remove a major amount of proteins and centrifuged again at 20000 rpm for 30 min. Protein concentrations were determined by the Bradford method. 30 The purity of His-tagged Araf51 was confirmed by SDS-PAGE after Ni-NTA agarose affinity chromatography (Novagen, USA).

Determination of Araf51 kinetic parameters

The kinetic parameters K m and k cat of Araf51 were quantified in triplicate by incubation of the enzyme (10 µL of 1 mg/mL) with different concentrations of p-nitrophenyl -L-arabinofuranoside (6 to 0.02 mM) in 50 mM Tris HCl buffer pH 8 at 25 °C. The release of p-nitrophenol was measured at 405 nm during 5 min (Microplates Spectrophotometer PowerWave XS/XS2, BioTek). The kinetic parameters were determined by calculations using GOSA software with repeatability.

Random mutagenesis

Screening of mutants

The blue colonies were selected and cultivated in 5 mL LB media containing 0.1 mM kanamycin LB media. The enzymes were purified as previously described and analyzed for their transglycosylation activities. As previously, protein concentration was estimated by Bradford method. Enzyme assays were performed to compare transglycosylation activities (presence of the alcohol acceptor) to hydrolytic activities (absence of the alcohol). Mutants and Araf51 wt enzymes were incubated in pH 8

Tris HCl 50 mM buffer with 20 mM pNP-Araf, 20% (v/v) DMSO, with or without 25% (v/v) alcohol for a final volume of 140 µL at 50 °C. The final concentration in enzyme necessarily reached 0.017 mg/mL, thus the collected volume having to be adapted to each attempt following the determination of the initial concentration by the Bradford method. The release of p-nitrophenol was measured at 405 nm during 5 min (Microplate Spectrophotometer Powerwase XS/XS2, Biotek) and data evaluated with Gen5 Data Analysis Software (Biotek). The initial activities of the enzyme and mutated enzymes were determined using the monitoring of UV curves of the enzymatic assays. This enabled to compare the slope between Araf51 wt and the one of the mutants with or without the presence of alcohol acceptors, and highlighted the mutants of interest. Indeed, the mutated enzymes presenting a higher slope than the one of the Araf51 wt, in presence of alcohol, showed higher reaction activations, meaning that transglycosylation was preferred.

Analytical scale of transglycosylation reactions, NMR kinetics

Enzymatic reactions were run from 20 mM pNP-Araf (4.3 mg) and 200 µL of alcohol acceptor incubated in pH 8 Tris HCl 50 mM buffer with 160 µL of DMSO. The enzyme preparation was added (a final concentration of 0.017 mg/mL is required) to the solution, and finally completed to a final volume of 800 µL and maintained at 50 °C during 3 h. Aliquots (100 µL) of the enzymatic reaction mixture were withdrawn at several times and directly freezed with liquid nitrogen. After complete lyophilization, samples were solubilized in 500 µL of CD 3 OD to enable the analysis by NMR.

Transglycosylation activities using pNP-Araf as glycosyl donor were determined by 1 H NMR. By monitoring the decrease of the pNP-Araf signal (aromatic protons =8.21 ppm and/or anomeric proton =5.66 ppm) and the release of p-nitrophenol (aromatic protons =8.12 ppm) corresponding to both the hydrolysis and the transglycosylation of the donor, the residual starting material can easily be quantified. The transglycosylation products were visualized by the emergence of the anomeric proton signal of the furanoside and/or the signal of the alkyl group protons. By reporting the relation between the proton signals, the resulting conversions were determined.

General procedure for the synthesis of alkyl arabinofuranosides in a preparative scale

Experiments were performed in 50 mM Tris HCl buffer, pH 8 at 50 °C in the presence of pNP-Araf (30 mg, 0.11 mM), 1.4 mL of alcohol and 1.12 mL of DMSO in a total reaction volume of 5.6 mL.

Araf51 wild type or mutated was added to a final concentration of 0.017 mg/mL and the reaction was performed at 50 °C during the optimal duration determined at the analytical scale experiment. Then the mixture was concentrated under reduced pressure and the reaction products were separated by column chromatography on silica gel (AcOEt/AcOH/H 2 O, 50:1:1) affording the desired product.

Purification and analysis of synthetic alkyl arabinofuranosides

Figure 1 .

 1 Figure 1. Time course of transglycosylation into propyl α-L-arabinofuranoside (black) from pNP-Araf (grey) by a) Araf51 wt and b) M20.

  Random mutagenesis was performed by GeneMorph II Random Mutagenesis kit (Stratagene) using mutagenic PCR. The open reading frame encoding Araf51 was amplified using the primers: forward T7 Promoter TACGACTCACTATAGGGGAA and reverse T7 Promoter GCTAGTTATTGCTCAGCGGT (Strategy 1); forward T7 Promoter TACGACTCACTATAGGGGAA and reverse T7 Promoter GTGAGTCGTATTAATTTCGCGGT (Strategy 2).For low mutation number (mutation frequency 0 -4.5 mutations / kb), 500 ng of the initial target DNA were mixed with 250 ng of each primer, 1 µ L of 40 mM dNTP mix (final concentration of 800 µ M each), 5 µL of 10 × Mutazyme II reaction buffer and 1 µL of Mutazyme II DNA polymerase (2.5 U/µL) completed to 50 µ L with H 2 O. The reaction was thermocycled as follows: one hot start cycle (95 °C, 2 min) then 10 cycles: first the denaturing step (95 °C, 30 s), the hybridation step (60 °C, 30 s) and the elongation step performed for 1min/kb (72 °C, 7 min or 10 min); and finally one cycle at 72 °C for 10 min. Only for the second strategy, mutagenesis PCR products were directly cloned into a plasmid vector using the TOPO TA Cloning® Invitrogen protocol. The fresh PCR product (2 µ L) was mixed with the different reagents provided in the TOPO TA Cloning® Invitrogen kit: 1 µ L salt solution, 1 µL pCR®2.1-TOPO® vector and H 2 O was added up to a final volume of 6 µ L. The reaction was incubated for 5 min at room temperature (22-23 °C).

Table 1 .

 1 Conversions and yields by transglycosylation using Araf51 wt and selected mutants for different alcohol acceptors.

	Entry Acceptor	Araf51 wt		Mutant			Improvement c
			Conversion a	Time	Conversion a	Time	Yield b
			(%)	(min)	(%)	(min)	(%)
	1	MeOH	95	60	-	-	-
	2	EtOH	90	60	-	-	-
	3	n-PrOH	85	120	96 (M20)	60	60	+ 13%
	4	i-PrOH	30	120	38 (M22)	60	30	+ 27%
	5	Solketal	38	120	-	-	-
	6	AllOH	76	120	-	-	-
	7	n-BuOH	42	50	92 (M12)	20	68	+ 119%
	8	n-PentOH 72	120	96 (M60)	120	80	+ 33%

a Conversion in transglycosylated product; b Isolated yield; c Was obtained as 100x(Conversion mutant -Conversion wt)/Conversion wt. Data from Table 1 firstly showed that moderate (

Table 2 .

 2 Selectivity of the mutated enzymes (% of transglycosylation conversion).

	Entry	Mutant	EtOH	n-PrOH	i-PrOH	n-BuOH	n-PentOH n-HexOH
	1	M20	20	96	4	10
	2	M22		26	38	25
	3	M12		19		92	35
	4	M60				65	96	79
	5	M57					65	94

Table 3 .

 3 Determination of Araf51 wt and selected mutants kinetic parameters for the hydrolysis reaction of pNP-Araf (pH 8, 50 mM of Tris HCl buffer, at 25 °C).

	Enzyme	K m (mM)	k cat (s -1 )	k cat /K m
	Araf51 wt	0.34 ± 0.08	143 ± 5	420
	M20	0.07 ± 0.02	133 ± 6.4	1900
	M22	0.07 ± 0.01	20.9 ± 1.2	298
	M12	0.26 ± 0.10	93.8 ± 12	360
	M60	0.17 ± 0.06	118 ± 8.7	694
	M57	0.16 ± 0.04	73.6 ± 4.7	460

Table 4 .

 4 Identified mutations.

	Enzyme	Mutations
	M20	R51G, V105L, T220S, S268T, A348S
	M22	P392S
	M12	K115T

  The desired furanoside 8 was obtained according to the general procedure for transglycosylation by incubation of solketal in the presence of the Araf51 wt. After chromatographic purification, it was isolated in 36% yield (10.5 mg). 1 H NMR (CD 3 OD): 4.92 (d, 1H, J 1,2 = 1.6 Hz, H-1), 4.31-4.27 (m, 1H, CH 2 CHCH 2 ), 4.05 (ddd, 1H, J = 2.4 Hz, J = 6.8 Hz, J = 8.4 Hz, O-1CH 2 CHCH 2 O), 3.99 (dd, 1H, J 2,3 = 3.6 Hz, H-2), 3.92-3.90 (m, 1H, H-4), 3.84 (dd, 1H, J 3,4 = 6.0 Hz, H-3), 3.78-3.74 (m, 2H, O-1CH 2 CH-CH 2, O-1CH 2 ), 3.71 (dd, 1H, J 4,5a = 5.2 Hz, J 5a,5b = 12 Hz, H-5a), 3.64 (dd, 1H, J 4,5b = 5.6 Hz, H-5b), 3.50-3.47 (m, 1H, O-1CH 2 ), 1.38 (s, 3H, CH 3 ), 1.32 (s, 3H, CH 3 ).13 C NMR(CD 3 OD): 109.7 (C-1), 85.7 (C-4), 83.1 (C-2), 78.8 (C-3), 75.9 (CH), 69.1 (O-1CH 2 CH), 67.6 (O-1CH 2 CHCH 2 ), 63.0 (C-5), 27.0 (CH 3 ), 25.6 (CH 3 ). HRMS (ESI + ): calcd for C 11 H 20 O 7 Na [M+Na] + m/z 287.1107; found, m/z 287.0985.

	time-of-flight analyzer, or a Q-tof 2 (Waters) equipped with electrospray ionization source, two NMR (CD 3 OD): 109.4 (C-1), 85.1 (C-4), 83.6 (C-2), 78.7 (C-3), 68.8 (OCH 2 CH 2 ), 63.0 (C-$ # ! 2! # 3 4 -!2 ! , ! " % 9 4 , !9 4 ) 6! 1 ! , " ) 4 "H ! ! ;! = !"
	quadrupole-orthogonal accelerators and reflection time-of-flight analyzer. 5), 32.8 (OCH 2 CH 2 ), 30.7 (CH 2 CH 2 CH 2 CH 3 ), 26.9 (CH 2 CH 2 CH 3 ), 23.7 (CH 2 CH 3 ), 14.4 ! &05 &050 & -((&% (((&
	(CH 3 ). HRMS (ESI + ): calcd for C 11 H 22 O 5 Na [M+Na] + m/z 257.1365; found, m/z 257.1915. ' 6 7 8 4 " ### $0&5 $0$' (& >F ? 6 F >F # > GF7 >F I!7 -2 8 7 ; 22 : * !
	4.8.	>	n-Pentyl α-L-arabinofuranoside (6) . 7 -4 ! ;! = !" '	!"	-9 7 : # 0&5& 0&5% 4 , ! " -
	n-Pentyl α-L-arabinofuranoside 6 was obtained according to the described general procedure 4.10. 3-O--L-arabinofuranosyl-O-isopropylidene-sn-glycerol (8) '&5 ' $ ( D ! 9 ) 9 : !" # EJ ! 4
	for transglycosylation by incubation of n-pentanol in the presence of the Araf51, and was / ; <!" " ) $ % '5 '%$ $ %% (& % (&('
	isolated in 66% yield (16.1 mg) after purification by column chromatography. 1 H NMR 5 ! !*! ! ;! = !" & ' (( 0& 0&/ (( D ! 9 ) 9 : EJ ! 4 $
	(CD 3 OD): 4.84 (d, 1H, J 1,2 = 2.0 Hz, H-1), 3.94 (dd, 1H, J 2,3 = 4.0 Hz, H-2), 3.92-3.89 (m, % 8 9 3 ' ( /$% /'. % ! .0/ .$$
	1H, H-4), 3.82 (dd, 1H, J 3,4 = 6.4 Hz, H-3), 3.73 (dd, 1H, J 4,5 = 3.2 Hz, J 5a,5b = 12.0 Hz, H-& -!1 -# 3 4 & ' 05.$ 05/0 (0 D ;! ) 9 : ! 9 ! EJ !
	5a), 3.71 (dt, 1H, J CH2,CH2 = 10.0 Hz, J CH2,CH2 = 6.4 Hz, OCH 2 CH 2 ), 3.62 (dd, 1H, J 4,5b = 5.2 ;! = !" : * ! > !*! 3 -? $ 4 . / % %.'0 %.''
	Hz, H-5b), 3.41 (dt, 1H, J CH2,CH2 = 9.6 Hz, J CH2,CH2 = 6.8 Hz, OCH 2 CH 2 ), 1.61-1.57 (m, 2H, ! ) " 3 @ ! ! A " (& $ ($ ! ! ) > ? ! ;! = !" : * ! > 0 *
	OCH 2 CH 2 ), 1.36-1.34 (m, 4H, CH 2 CH 2 CH 3 ), 0.92 (t, 3H, J CH2,CH3 = 7.2 Hz, CH 3 ). 13 C NMR $& 22 $& $ / " :K . L.0.550
	(CD 3 OD): 109.4 (C-1), 85.1 (C-4), 83.6 (C-2), 78.7 (C-3), 68.8 (OCH 2 CH 2 ), 63.0 (C-5), ( 3!1! ! 7B 4 1<1!7 2 1 -!< A 3 3! ! (' #! , -! # % / /'
	30.4 (OCH 2 CH 2 ), 29.5 (CH 2 CH 2 CH 3 ), 23.5 (CH 2 CH 3 ), 14.4 (CH 3 ). HRMS (ESI + ): m/z calcd ) * ( 0 % (. ; ?"7 ! -8 ! ) $ #! 0 % 0('
	for C 10 H 20 O 5 Na [M+Na] + m/z 243.1208 ; found, m/z 243.1676. 0 ;! * A ! 4 CC 3 3! ! (/ 1 6 6 " : 7 3 7 E	"	!	$	&
	#+ (/ 05	+! 0/&55 0/&%/
	4.9.		n-Hexyl α-L-arabinofuranoside (7) $ 6 D ; 3 ! #D ! (5 -3 7 A A !" 6 -! 4	&	3! ! %( %'' %'5	) *
	n-Hexyl α-L-arabinofuranoside 7 was obtained according to the described general procedure 0/ $$ (% 3 9 4 -: 3 7! * 4 J-1 -! 4 > !" 4
	Thin layer chromatography (TLC) analysis were conducted on E. Merck 60 F 254 Silica Gel non ' -! * ! B 4 !*! #! 1 -!1 " A ! & % 0 0/
	for transglycosylation by incubating n-hexanol in the presence of the Araf51 wt. It was
	activated plates and compounds were visualized by UV (254 nm), or a 5% solution of H 2 SO 4 solution + ( '. ( .. in EtOH containing orcinol, followed by heating. For column chromatography, Si 60 (40-63 µm) isolated in 71% yield (18.5 mg) after purification by column chromatography. 1 H NMR ! -! *! E # ! ) * ; ;! = !" ! * ! B ; $ 0& # C ( ($5 ('$
	(CD 3 OD): 4.84 (d, 1H, J 1,2 = 2.0 Hz, H-1), 3.94 (dd, 1H, J 2,3 = 4.0 Hz, H-2), 3.92-3.90 (m, Silica Gel was used. NMR spectra were recorded on a Bruker spectrometer ARX (400 MHz for 1 H and . !*! ) 4 ! : * ! > ;! = !" $
	100 MHz for 13 C). Chemicals shifts are expressed in units (ppm). Chemical shifts are calculated in 1H, H-4), 3.82 (dd, 1H, J 3,4 = 6.4 Hz, H-3), 3.74 (dd, 1H, J 4,5 = 3.2 Hz, J 5a,5b = 12.0 Hz, H-$%& $%' % , '$ '$'
	Hertz and pattern abbreviations are as follows: s (singlet), d (doublet), t (triplet), q (quartet), m 5a), 3.71 (dt, 1H, J CH2,CH2 = 6.8 Hz, J CH2,CH2 = 9.6 Hz, OCH 2 CH 2 ), 3.62 (dd, 1H, J 4,5b = 5.6 ( ) " * + , ! " -... ./$ / >F ? ; 22 -2 8 7 > GF7 >F ! : * ! >
	(multiplet), dd (double doublet)...The HRMS were measured with a MicrO-Tof-Q 2 (Bruker) equipped Hz, H-5b), 3.41 (dt, 1H, J CH2,CH2 = 9.6 Hz, J CH2,CH2 = 6.4 Hz, OCH 2 CH 2 ), 1.60-1.57 (m, 2H, 0 1 # .%/ /&% 6 F >F # ;! = !" ' + (&%( ( &(
	with electrospray and APCI ionization sources, two quadrupole-orthogonal accelerators and reflection OCH 2 CH 2 ), 1.35-1.32 (m, 6H, CH 2 CH 2 CH 2 CH 3 ), 0.91 (t, 3H, J CH2,CH3 = 6.8 Hz, CH 3 ). 13 C 5 > 9 "7 , 6 %( 0(5 0$'

Acknowledgments

We are grateful to the Région Bretagne for a grant to A. P. We also thank Prof. G. J. Davies for providing the wild type and Jean-Paul Guégan (ENSCR) for recording NMR spectra.