Partially phosphonated polyethylenimine-coated nanoparticles as convenient support for enzyme immobilization in bioprocessing

Sensors and Actuators B 192 (2014) 269–274

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Partially phosphonated polyethylenimine-coated nanoparticles as convenient support for enzyme immobilization in bioprocessing Clément Monteil a , Nathalie Bar a , Richard Retoux b , Joël Henry c,d , Benoît Bernay d , Didier Villemin a,∗ a

UMR CNRS 6507 LCMT, Université de Caen-ENSICAEN, 6 boulevard du Maréchal Juin, 14050 Caen, France UMR CNRS 6508 CRISMAT, Université de Caen-ENSICAEN, 6 boulevard du Maréchal Juin, 14050 Caen, France c CNRS INEE – FRE3484 BioMEA, Biologie des Mollusques marins et des Ecosystèmes Associés – Université de Caen Basse Normandie, 14032 Caen, France d PROTEOGEN Platform, SF ICORE 4206 – Université de Caen Basse Normandie, CS 14032 Cedex 5 Caen, France b

a r t i c l e

i n f o

Article history: Received 24 July 2013 Received in revised form 10 September 2013 Accepted 23 September 2013 Available online 18 October 2013 Keywords: Trypsin Phosphonated polyethylenimine Magnetic nanoparticles Enzyme immobilization

a b s t r a c t Partially phosphonated polyethylenimine (PEIP) has been developed as an easily functionalisable coating agent for iron oxide nanoparticles. Trypsin immobilization takes special advantage of the properties of this new material. Numerous enzymes can be loaded on the polymer by a covalent bounding with numerous amino groups. The PEIP contributes to the high stability of the material, through a strong covalent P O Fe bond. Resistance to hydrolysis and to temperature increasing ensure to obtain a highly recyclable magnetic nanomaterial designed for proteomic analysis. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Trypsin (EC 3.4.21.4) is an essential key in mass spectroscopydriven proteomics [1–3]. Its ability for specifically sequence C-terminal peptide bonds of arginine and lysine residues in a protein makes it a very useful tool for protein identification and analysis [4,5]. However, the conventional in-solution digestion for proteomic is limited by two principal factors [6]. Trypsin has a low thermostability at 37 ◦ C, and undergoes a rapid autolysis at basic pH. Many processes have been developed to overcome these two major drawbacks [7,8]. Among them, the covalent immobilization of trypsin on a magnetic support is widely studied, and the benefits of this methodology are multiple [9–11]. First, the restriction of degrees of freedom enhances stability of enzyme and permit reduction of autolysis. In addition, the magnetic support leads to easy enzymes recovery with an electromagnetic field and the so grafted enzyme can be reused; it clearly helps the purification of products and elimination of trypsin mass-fingerprints. Magnetic microbeads for enzyme support have been the subject of many investigations [12–19]. In these cases, in addition

∗ Corresponding author. Tel.: +33 2 31 45 28 40. E-mail addresses: [email protected] (C. Monteil), [email protected] (D. Villemin). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.09.096

to a low specific surface area, the micro or millimetric scale of support can highly hamper the approach of the substrate [20,21]. Even if the affinity of trypsin for substrate is more or less preserved, proteolysis speeds can be extremely decreased. Considering this point of view, we try to focus on a material at the nanometric scale, with a size similar to an enzyme. The support would only slightly hinder the substrate docking conducting to better speed. We have chosen to work on magnetic maghemite nanoparticles (NP) suitable for biofunctionalization. A poly (aminomethylenephosphonic) acid has been previously synthetized in our laboratory as a sorbent for metal ions in wastewaters [22,23]. Then, an only partially phosphonated version (PEIP) has been used for coating nanoparticles (NP-PEIP) [24]. The nanometric size and high number of free primary amine on PEIP permit the grafting of biomolecules and offers a well-adapted material for enzyme immobilization. The choice of the support has been carefully selected: compared to commonly used supports, like chitosan, alginate or silica, the stability is better toward acidic or basic hydrolysis and temperature increasing [25–28]. P O Fe covalent bonds between maghemite and PEIP achieve a strong stability of the material. The consequence is that NP-PEIPs properties are not deteriorated; and a large number of reuses can be obtained with a major stability toward increasing of temperature.

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2. Materials and methods 2.1. Materials Hyperbranched polyethylenimine with a molecular weight of 25,000 Da was purchased from BASF. Dialysis tubing was obtained from Roth. Phosphorous acid, formaldehyde, hydrochloric acid, nitric acid, aqueous ammonia solution were purchased from VWR; ferrous, ferric and nitrous chloride from Alfa. Glutaraldehyde 50% in water, sodium cyanoborohydride, trypsin from pancreas porcine (EC 3.4.21.4) and albumin from bovine serum were bought from Sigma–Aldrich. Unless otherwise noted, all reagent-grade chemicals were used as received. Millipore water was used in the preparation of all aqueous solutions. 2.2. Preparation of polyethylenimine with 5% of amine phosphonated-PEIP A mixture of PEI (0.186 mmol, 4.64 g), phosphorous acid (5.40 mmol, 0.443 g, 0.05 equiv. of the critical amount of PEI monomers MW = 43) in 10 mL of water was vigorously stirred, then irradiated at 150 W in a microwave oven (Prolabo Synthewave) for one minute. Then, pH was adjusted to 4 with hydrochloric acid 6 mol L−1 . A formaldehyde aqueous solution (35%, 10.8 mmol, 0.1 equiv.) was added and the mixture was irradiated for 5 min. The excess of formaldehyde was eliminated in vacuo. The polymer was dialyzed in water with a nitrocellulose membrane, yielding therefore 85% of PEIP (8.25 g). 2.3. Synthesis of maghemite nanoparticles (NPs) and coating on PEIP First, maghemite nanoparticles were synthesized as previously described [24]. Ferrofluid was obtained with an iron concentration of 1.24 mol L−1 . Micrographs from TEM show a system where nanoparticles have a nearly spherical structure. According to parameters fit of the magnetization curve and crystalline size calculated from Debye Scherer formula, medium diameter of nanoparticles is estimated to 7 nm and polydispersity to 0.32. 0.5 mL of diluted precursor acid ferrofluid with a ferric concentration of 10−2 mol L−1 was slowly added to 2 mL of a solution of 50 g L−1 PEIP in water adjusted to pH 2 with concentrated nitric acid, under vigorous stirring. After 5 min, the mixture was basified by adding sodium hydroxide until a precipitation was observed. Supernatant containing excess of PEI was removed and the precipitate redispersed in a diluted nitric acid solution. In a second time, the solution was concentrated by evaporating water and acetone was added leading to the flocculation of PEIP-coated nanoparticles (NP-PEIP). 2.4. Trypsin immobilization Glutaraldehyde was first mixed with active charcoal and filtered. This operation was repeated until the absorbance peak at 235 nm on UV-spectroscopy had disappeared. 2 mL of a solution of filtered glutaraldehyde (25%) were mixed to 2 mL of an aqueous solution of previously synthetized NP-PEIP ([Fe] = 10−2 mol L−1 ) and 10 mL of acetate buffer (0.1 mol L−1 , pH 4.8), and the mixture was stirred during 2 h. Excess of glutaraldehyde was eliminated by ultracentrifugation (20,000 rpm, 30 min.). After removing of supernatant, the precipitate was redispersed in 25 mL of acetate buffer. Rinse cycles were effected two times. 10 mg of trypsin were introduced by small portions to the mixture, followed by addition of 100 mg of sodium cyanoborohydride previously dispersed in 5 mL of acetate buffer. After 5 h of incubation, immobilized trypsin was obtained by decantation of the mixture on magnetic plates.

Supernatant was removed, and particles redispersed in water. Operation was repeated until no free trypsin was detected in wash water by spectrophotometry. The amount of immobilized trypsin was calculated by the difference between the introduced protein amount and that found in the supernatants. The UV absorption value of the supernatant solutions was measured at 280 nm and compared to the UV absorption value of the trypsin solution before immobilization. 2.5. Determination of activity Concerning the unmodified trypsin, the substrate solution consists in 4.2 mmol of N␣-Benzoyl-l-arginine 4-nitroanilide (BApNA) dissolved in 150 mL of Tris–HCl buffer (pH = 7.8, 0.1 mol L−1 , containing 20% of DMSO) at 25 ◦ C. Free enzyme solution was prepared by dissolving 5 mg of trypsin in 3 mL of a 2 mol L−1 HCl solution. At the start of the run, 0.1 mL of enzyme solution was then added, and the mixture maintained at 25 ◦ C. Activity of immobilized enzyme was assayed by the introduction of 10 mg of NP-PEIP-T in 150 mL of the Tris–HCl buffer containing 4.2 mmol L−1 of BAPNA, under stirring at 25 ◦ C. Stability tests are started after 30 min of incubation at the required temperature. The released p-nitroaniline was estimated by spectrophotometry against a blank after 15 min at 405 nm using a molar extinction coefficient value of 8800 L mol−1 cm−1 . This measure was repeated three times and the mean and standard deviation were calculated. The enzymatic activity was defined as the amount of enzyme able to hydrolyze one mmol of BApNA per minute under the established conditions. 2.6. Mass spectrometry A solution of bovine serum albumin (BSA) at 5 g L−1 was first prepared by dissolving BSA in Tris-buffer (20% DMSO, 0.1 mol L−1 , pH 7). The in-solution digestion was performed by adding free trypsin into 10 mL of the protein solution at a substrate-toenzyme ratio of 50:1. Evaluation of supported trypsin consisted in adding 2 mL of NP-PEIP-T dispersed in the same Tris-buffer ([Fe] = 5 × 10−3 mol L−1 ) into 10 mL of protein solution. Both mixtures were incubated at 37 ◦ C for 12 h. After digestion, 1 ␮L of nitric acid was added into the solutions to stop the reaction, and in the second case magnetic particles were separated by using a permanent magnet. MS experiments were carried out on an AB Sciex 5800 proteomics analyzer equipped with TOF TOF ion optics and an OptiBeamTM on-axis laser irradiation with 1000 Hz repetition rate. The system was calibrated immediately before analysis with a mixture of Angiotensin I, Angiotensin II, Neurotensin, ACTH clip (1–17), ACTH clip (18–39) and mass precision was better than 50 ppm. For MS analysis, a 1 ␮L volume of the peptide solution was mixed with 10 ␮L volumes of solutions of 5 g L−1 ␣-cyano-4-hydroxycinnamic acid matrix prepared in a diluent solution of 50% ACN with 0.1% trifluoroacetic acid. The mixture was spotted on a stainless steel Opti-TOFTM 384 targets; the droplet was allowed to evaporate before introducing the target into the mass spectrometer. A laser intensity of 3200 was typically employed for ionizing. MS spectra were acquired in the positive reflector mode by summarizing 1000 single spectra (5 × 200) in the mass range from 800 to 4000 Da. 3. Results and discussion 3.1. Immobilization of trypsin on coated nanoparticles 3.1.1. Synthesis of PEIP-coated nanoparticles (NP-PEIP) Whole reaction pathway of the trypsin immobilization is resumed in Scheme 1. The first part concerns the preparation

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3.1.2. Immobilization of trypsin Trypsin was covalently bound on NP-PEIP according to Scheme 1 with insertion of glutaraldehyde as spacer arm between enzyme and nanomaterial. This immobilization strategy is a well-known used method, however we devote some attention to the purification of the glutaraldehyde from impurities. Previous studies highlight the inhibition of enzymes activity due to the presence of polymeric residues from this compound [31]. A simple filtration on charcoal permits to recover pure glutaraldehyde. Trypsin was covalently bound to NP-PEIP by condensation of the amino group of lysine of trypsin with aldehyde function of glutaraldehyde. This step is followed by addition of sodium cyanoborohydride to reduce iminiums in irreversible C N bonds. It corresponds to our main objective: the use of PEIP for bioprocessing where a high number of nanocomposite reuses is expected. The amount of immobilized trypsin was evaluated by testing the supernatant after incubation of trypsin by spectrophotometry. Experiment was repeated ten times, and the quantity of enzyme immobilized was assessed about 26.41 mg per mg of maghemite, corresponding to an average of 5 molecules of trypsin per NP-PEIP. Characterization was first performed by FTIR. Free trypsin shows bands at 1639 cm−1 and 1530 cm−1 , indicating the vibrations corresponding to amide I and amide II bands of protein molecules (Fig. 1a). The frequencies of characteristic peaks in NP-PEIP-T added to Fe O P bands confirm the grafting of trypsin onto support surface. Then, NP-PEIP-T structure was investigated by TEM microscopy. To improve the contrast between the material and the grid, and consequently the observation of coated nanoparticles, we used a carbon grid perforated with holes. NP-PEIP-T which protrude into the hole of the carbon film avoid the overlapping problem. As illustrated in Fig. 1b pattern of maghemite core is easily discernible due to its cristallinity. Specific feature of enzyme is recognizable, and it appears that trypsin is bonded to PEIP. The bottom picture shows multiple units of trypsin surrounding the NP-PEIP support, corroborating the fact that several enzymes seem grafted on one NP-PEIP structure. Scheme 1. General scheme of trypsin immobilization on NP-PEIP. Coating takes place at acidic pH, for maintaining NP dispersed. Glutaraldehyde first reacts on remaining primary amines of PEIP, then with trypsin. The sodium cyanoborohydride reduces iminium groups into irreversible C N bonds.

of the partially phosphonated polyethylenimine. An exhaustive description of the PEIP synthesis is presented in a previous published work [22,24]. The phosphonation step is derived from the Moedritzer and Irani method [29]. Despite the hyperbranched character of the PEI it will be considered as a succession of monomers with a medium molecular weight of 43 g mol−1 corresponding to (CH2 CH2 NH)n . Only five percent of amino groups were converted into aminomethylphosphonate. The choice of a low modified PEIP corresponds to an objective to preserve a maximum of the primary amines in order to successfully immobilize a large amount of enzyme. The analysis of NP-PEIP on 31 P NMR in D2 O shows presence of phosphorus signals at ı = 6.50 and 6.93 ppm characteristic of PEIP. The ferrofluid, synthetized according the Massart protocol [30], must be diluted before grafting on the PEIP solution and the addition is carefully done slowly to avoid coating of clusters. The phosphonate groups of PEIP are condensed on hydroxyls of the maghemite surface. The so formed Fe O P is observed in FTIR (Fig. 1a) as broad bands at 1033 and 1076 cm−1 , and the strong band at 574 cm−1 characterizes of Fe O vibration correlated to the magnetic core. As previously reported, thermogravimetric analysis reveals that one PEIP is on average grafted on one NP [24].

3.2. Activity and reaction kinetics of the immobilized trypsin The catalytic activity of NP-PEIP-T was assayed by the conventional hydrolysis of N␣ -Benzoyl-dl-arginine p-nitroanilide (BApNA) method under standard conditions and was compared to the native enzyme [32]. Trypsin-catalyzed hydrolysis of chromogenic substrate BApNA as illustrated in Fig. 2a results in the formation of N-benzoylarginine and p-nitroaniline which can be quantified by spectrophotometry at 410 nm. The enzymatic activity was evaluated in terms of the classical Michaelis–Menten kinetics, according to the following equation: V = Vmax [S]/(Km + [S]) where Km is the Michaelis constant representing the affinity of enzyme for a substrate and Vmax is the maximum rate attained at infinite concentration of substrate. Km , Vmax are graphically obtained by a Lineweaver–Burk plot representation [33,34]. It appears that bound trypsin presents a Km of 7.56 mmol L−1 close to the free trypsin one measured in the same experimental conditions (2.89 mmol L−1 ). The affinity of the bound enzyme for the substrate is preserved and is not affected by the high amount of enzymes bounded to NP-PEIP. In others studies on nanometric supports, both Km have also the same order of magnitude [35,36]. kcat are calculated from the relation kcat = Vmax /[E] and are determined to 3.06 s−1 and 0.96 s−1 respectively for free and bound trypsin. As expected, a lower kinetic is obtained for immobilized one. However, speed values are of the same order of magnitude. The gain of speed obtained compared to microspheres or membrane systems are substantial, and the difference could increase again with proteolysis of macromolecules like proteins.

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Fig. 1. (a) FTIR spectra of NP-PEIP, free and immobilized trypsin on NP-PEIP. This last spectrum showed the presence of both amide bands specific to trypsin, and Fe O P covalent bond resulting from grafting of PEIP on maghemite. (b) TEM images of NP-PEIP-T. Trypsin linked to the support is particularly observable in the upper picture. The lower picture showed the numerous trypsins surrounding the NP-PEIP structure.

To evaluate residual activity after lot of reuses, the immobilized trypsin was reutilized more than 21 times in exactly same conditions. Experiments were carried a long-time period (5 months), confirming a high level of reusability. At every time, the bound enzyme was easily recovered by magnetically induced precipitation. As indicated on Fig. 2b it presents a mean activity equal to superior to 90% of the initial specific activity. Trypsin in solution is usually stored at low temperature in an acidic medium. In solution at pH 8, trypsin undergoes fast autolysis and loses almost all its activity after few hours. Positive effects on stability are obtained by chemical modification, adsorption or

covalent modification [37]. Generally, High storage stabilities have been reported for immobilized enzymes with a magnetic support [38,39]. The covalently bound trypsin NP-PEIP was conserved at room temperature at neutral pH during few months without important loss of activity (Fig. 2b) corroborating the fact that coating is important for immobilized enzyme performance providing higher stability. One way to reduce time of incubation is heating the proteolysis reaction. Each native enzyme has an optimum temperature for digestion of proteins but they can be irremediably denatured by

Fig. 2. (Solid and dashed lines correspond to the supported trypsin and the free trypsin respectively) (a) reaction of the hydrolysis of BApNA by trypsin. The p-nitroaniline formation can be observed by spectrophotometry at 410 nm. (b) Conservation of the activity of the enzyme at pH = 7. By contrast with trypsin, NP-PEIP-T presents a stable activity over five months. (c) Residual activity at high temperature after 30 min of incubation. T50 are determined at 45 ◦ C and 78 ◦ C for free and bound trypsin respectively. (d) Residual activity at prolonged period at 50 ◦ C. Even after 3 h, no significant loss of activity is detected on NP-PEIP-T.

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Fig. 4. MALDI-TOF peptide mass fingerprint for BSA digested by NP-PEIP-T. The dotted arrow indicates were the most frequent autolytic peptides of trypsin should appear.

Fig. 3. Comparison of MS profiles for (a) soluble and (b) bound trypsin. Multiple peaks characteristic of autolysis are detected in the first case. Even at high concentration, NP-PEIP-T does not present autolysis reaction.

heating. In this perspective many systems for stabilizing enzyme at higher temperature have been developed and incubation times are reduced from hours to few minutes [40,41]. Tests conducted on NP-PEIP-T lead to the same result: the polymeric support greatly protects trypsin not only against autolysis, but also against denaturation due to heat increase. To estimate the thermostability of NP-PEIP-T, we have determined the T50 constant, defined as the temperature at which 50% of the activity is retained upon 30 min of incubation. The persistent activity of NP-PEIP-T is defined by a T50 estimated to 75 ◦ C. Its stability, represented in Fig. 2c and d, is about 30 ◦ C higher than the trypsin (T50 = 45 ◦ C, to compare with previously determined to 41 ◦ C). We then investigated the resistance of immobilized trypsin on a longer time. A stability test at 50 ◦ C during more than 5 h does not report any noticeable drop of enzymatic activity after consecutive five hours of incubation. Catalytic activity of free trypsin is given as comparison. To resume, the covalent link of trypsin on NPPEIP resulted in an increase of thermostability and preservation of catalytic activity of trypsin after anchoring. In comparison, the trypsin immobilized on magnetic nanoparticles coated by modified chitosan has led to comparable T50 values (61 ◦ C). The slight difference could be attributed to the coating by PEIP, most resistant than chitosan regardless to increasing of temperatures [6,41] (Fig. 3). 3.3. Application in bioprocesses: test on MALDI mass-analysis 3.3.1. Detection of autolysis In addition to thermal stability, the application of trypsin in proteomics is limited by a rapid autolysis under common working conditions [42]. The presence of autolysis residues peaks in a protein sample digest can represent a drawback, as it makes the interpretation of the MALDI–TOF peptide mass fingerprint more

difficult, a phenomenon amplified when working at low sample concentration. This has a negative impact on the sample identification by database search. Work with a catalytic amount of enzyme during hydrolysis of protein is required to avoid too pollution on spectra but it lengthens the digestion time. To determine abundance of autolysis products of immobilized enzyme, we performed control digest of blank gel slabs of trypsin at high concentration (50 mg of NP-PEIP-T in 50 mL of 0.05 mol L−1 tris-buffer at 37 ◦ C overnight). MALDI-TOF spectra were acquired and compared with the autolytic peptide pattern of free trypsin (2 × 10−6 mol L−1 in 0.05 mol L−1 tris-buffer at 37 ◦ C overnight). None of the autolytic trypsin fragments were observed for the supported trypsin (Fig. 3b). Peaks detected come from the matrix and always appear on the spectra in normal conditions. 3.3.2. Performance of NP-PEIP-T on proteins The next step of our study was to determine ability of trypsin to sequence proteins in a catalytic concentration. Consequently we based our work on hydrolysis of a standard protein, the bovine serum albumin (BSA). A comparison of the peptide mass spectrum of BSA digested by NP-PEIP-T (Fig. 4) with that obtained using soluble trypsin (not shown) revealed no alteration in the cleavage performance. This result was confirmed by the peptide and sequence coverage obtained in MALDI-TOF for the experiments. Moreover, as demonstrated previously whatever the quantities of immobilized enzyme used, trypsin does not undergo any trace of autolysis. 4. Conclusions Trypsin has successfully been immobilized on a new magnetic nanometric support designed for biofunctionalization. The strong stability of the NP-PEIP system coupled to an irreversible covalent grafting of the enzyme ensures a high number of reuse without altering catalytic properties, and the opportunity of working under harder condition. Immobilized trypsin has a high efficiency in protein digestion Absence of characteristic autolytic peaks of trypsin in MALDI–TOF MS of a digested standard protein as BSA allows fast in-solution digestion of proteins and their identification by mass spectrometry. References [1] R. Aebersold, A mass spectrometric journey into protein and proteome research, J. Am. Soc. Mass Spectrom. 14 (2003) 685–695. [2] T. Stosová, M. Sebela, P. Rehulka, O. Sedo, J. Havlis, Z. Zdráhal, Evaluation of the possible proteomic application of trypsin from Streptomyces griseus, Anal. Biochem. 376 (2008) 94–102.

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Biographies Clément Monteil graduated from the University of Aix-Marseille in 2009, receiving a MS degree in organic chemistry. Currently he is pursuing a PhD degree at the University of Caen, working on elaboration of a hybrid organic–inorganic platform and its application to catalysis and depollution. Nathalie Bar received her PhD in 1990 in the area of enzymatic synthesis of organosilicon compounds from the University of Bordeaux 1, under the supervision of Prof. B. de Jeso. She was a postdoctoral fellow in the University of Wuppertal by Prof. M. P. Schneider group. Currently, she is lecturer in the University of Caen (UCBN) since 1997. Her research interests are focussing on new methodologies of synthesis including enzymatic synthesis, supported catalysis and on column reaction. Richard Retoux was born in 1963, Material Science Engineer, he received his PhD degree in Material Science studying superconducting oxide materials at Caen University in 1990. Accredited to supervise research in 2005 he is specialist in Transmission Electron Microscopy studies with a view to characterize, from macro to nano scale, relationships between structures and properties in complex materials. He is involved in research fields from solid state chemistry as oxide materials to hybrid, polymers, composites or soft materials. He has authored over 100 scientific publications. Joël Henry was born in 1963 in Paris. He graduated from the University of Paris VIIJussieu and from the University of Western Brittany (Brest). He received the PhD degree of Marine Biology at the University of Caen (UCBN) in 1993. His research interests involve regulation of reproduction mechanisms in the cuttlefish Sepia officinalis. He has authored over 30 international scientific publications. Benoît Bernay was born in 1979 in Evreux, France. He graduated from the Faculty of Biology Science at University of Caen in 2005, receiving the PhD degree in biology and biochemistry. His research interests involve mass spectrometry and proteomics. Didier Villemin obtained his PhD and his doctorate Es Sciences (1978) from the University P. and M. Curie (UPMC) of Paris. After a two-year postdoctoral stay with Sir D.H.R. Barton at ISCN, Dr. Villemin jointed the Chemistry School of Engineering of Paris (ENSCP) as lecturer. He moved as full Professor in 1985 to the School of Engineering of Caen (ISMRa, then ENSICAEN). Dr. D. Villemin and his coworkers has authored over more 280 papers in chemistry. His research interests are focusing on the development of new methodologies in organic synthesis based on catalysis and applications of chemoinformatics. He is known for his pioneer work in green chemistry in particular, for alkenes and alkynes metathesis (1972–1982), solventless reactions (1983) and microwave activation (1989). More recently, he had developed synthesis of phosphonates and their applications, for immobilization of catalysts, as anti-corrosion agents, as metal extractants and for precursor of MOF.