功能化磁性纳米粒子在固定化酶研究中的应用

酶是高效的生物催化剂,在生物技术领域有广泛的应用。然而,不可再生催化的高成本和酶的有效成分分离回收,是实现大规模工业化应用需要解决的关键问题。磁性纳米粒子(magnetic nanoparticles,MNPs)具有优异的磁回收性质。通过设计和制备功能化MNPs作为固定化酶的多功能载体,是解决这一问题的有效途径之一,可为酶的工业化大规模应用提供条件。近年来,功能化磁性纳米粒子在酶的固定化领域基于载体性质、固定化方法和应用有广泛研究。文中重点介绍了近年来各种功能化磁性纳米载体,特别是Fe3O4纳米粒子,在固定化酶中的应用。根据功能化试剂的差异分类,实例讨论了不同功能化修饰的磁性纳米载体对酶的固定化,包括硅烷修饰的磁性纳米载体、有机聚合物修饰的磁性纳米载体、介孔材料修饰的磁性纳米载体以及金属-有机骨架材料(metal-organic framework,MOF)修饰的磁性纳米载体。同时,结合可持续工业催化的发展要求,对磁性复合载体固定化酶的发展前景进行了展望。     

Multi-point enzyme immobilization,surface chemistry,and novel platforms: a paradigm shift in biocatalyst design

Engineering enzymes with improved catalytic properties in non-natural environments have been concerned with their diverse industrial and biotechnological applications. Immobilization represents a promising but straightforward route, and immobilized biocatalysts often display higher activities and stabilities compared to free enzymes. Owing to their unique physicochemical characteristics, including the high-specific surface area, exceptional chemical, electrical, and mechanical properties, efficient enzyme loading, and multivalent functionalization, nano-based materials are postulated as suitable carriers for biomolecules or enzyme immobilization. Enzymes immobilized on nanomaterial-based supports are more robust, stable, and recoverable than their pristine counterparts, and are even used for continuous catalytic processes. Furthermore, the unique intrinsic properties of nanomaterials, particularly nanoparticles, also confer the immobilized enzymes to be used for their broader applications. Herein, an effort has been made to present novel potentialities of multi-point enzyme immobilization in the current biotechnological sector. Various nano-based platforms for enzyme/biomolecule immobilization are discussed in the second part of the review. In summary, recent developments in the use of nanomaterials as new carriers to construct robust nano-biocatalytic systems are reviewed, and future trends are pointed out in this article.     

Control of protein immobilization: coupling immobilization and site-directed mutagenesis to improve biocatalyst or biosensor performance

Mutagenesis and immobilization are usually considered to be unrelated techniques with potential applications to improve protein properties. However, there are several reports showing that the use of site-directed mutagenesis to improve enzyme properties directly, but also how enzymes are immobilized on a support, can be a powerful tool to improve the properties of immobilized biomolecules for use as biosensors or biocatalysts. Standard immobilizations are not fully random processes, but the protein orientation may be difficult to alter. Initially, most efforts using this idea were addressed towards controlling the orientation of the enzyme on the immobilization support, in many cases to facilitate electron transfer from the support to the enzyme in redox biosensors. Usually, Cys residues are used to directly immobilize the protein on a support that contains disulfide groups or that is made from gold. There are also some examples using His in the target areas of the protein and using supports modified with immobilized metal chelates and other tags (e.g., using immobilized antibodies). Furthermore, site-directed mutagenesis to control immobilization is useful for improving the activity, the stability and even the selectivity of the immobilized protein, for example, via site-directed rigidification of selected areas of the protein. Initially, only Cys and disulfide supports were employed, but other supports with higher potential to give multipoint covalent attachment are being employed (e.g., glyoxyl or epoxy-disulfide supports). The advances in support design and the deeper knowledge of the mechanisms of enzyme-support interactions have permitted exploration of the possibilities of the coupled use of site-directed mutagenesis and immobilization in a new way. This paper intends to review some of the advances and possibilities that these coupled strategies permit.     

Development of Silica Gel-Supported Modified Macroporous Chitosan Membranes for Enzyme Immobilization and Their Characterization Analyses

The present work was aimed at developing stability enhanced silica gel-supported macroporous chitosan membrane for immobilization of enzymes. The membrane was surface modified using various cross-linking agents for covalent immobilization of enzyme Bovine serum albumin. The results of FT-IR, UV–vis, and SEM analyses revealed the effect of cross-linking agents and confirmed the formation of modified membranes. The presence of silica gel as a support could provide a large surface area, and therefore, the enzyme could be immobilized only on the surface, and thus minimized the diffusion limitation problem. The resultant enzyme immobilized membranes were also characterized based on their activity retention, immobilization efficiency, and stability aspects. The immobilization process increased the activity of immobilized enzyme even higher than that of total (actual) activity of native enzyme. Thus, the obtained macroporous chitosan membranes in this study could act as a versatile host for various guest molecules.     

Formulation of organic and inorganic hydrogel matrices for immobilization of β‐glucosidase in microfluidic platform

The aim of this study was to formulate silica and alginate hydrogels for immobilization of β‐glucosidase. For this purpose, enzyme kinetics in hydrogels were determined, activity of immobilized enzymes was compared with that of free enzyme, and structures of silica and alginate hydrogels were characterized in terms of surface area and pore size. The addition of polyethylene oxide improved the mechanical strength of the silica gels and 68% of the initial activity of the enzyme was preserved after immobilizing into tetraethyl orthosilicate–polyethylene oxide matrix where the relative activity in alginate beads was 87%. The immobilized β‐glucosidase was loaded into glass–silicon–glass microreactors and catalysis of 4‐nitrophenyl β‐d ‐glucopyranoside was carried out at various retention times (5, 10, and 15 min) to compare the performance of silica and alginate hydrogels as immobilization matrices. The results indicated that alginate hydrogels exhibited slightly better properties than silica, which can be utilized for biocatalysis in microfluidic platforms.     

Porous zirconia: a new support material for enzyme immobilization

Four different proteases (trypsin, chymotrypsin, papain and pepsin) were covalently attached to the surface of a new type of porous zirconia, as well as a conventional porous silica, activated with 3-isothiocyanatopropyltriethoxy silane (NCS-silane). The immobilization efficiency onto the porous zirconia material was evaluated in terms of the amount of enzyme attached to the particles and from the biological activity remaining after the immobilization step. The results were compared with the corresponding experiments with a porous silica of similar surface area/g support material. In addition, the storage stability of the modified zirconia and silica biocatalysts were evaluated. These results indicated that specific immobilized enzyme biocatalysts can be achieved with this new zirconia support material which exhibits different properties to those observed with the more conventional silica-based materials. Moreover, the results with the enzyme-zirconia biocatalysts also indicate different characteristics when compared with data for the same enzymes immobilized under similar buffer conditions to organic support materials as previously described by various other investigators. The advantages of zirconia-based immobilized enzyme biocatalysts in terms of their density and chemical robustness are also described relative to other alternative support materials currently in use.     

Immobilization of adenosine deaminase onto agarose and casein

In the present study adenosine deaminase (ADA) was immobilized onto two different polymeric materials, agarose and casein. The factors affecting the amount of enzyme attachment onto the polymeric supports such as incubation time were investigated. The maximum amount of enzyme immobilized onto different polymeric supports occurred at incubation pH value 7.5 and ADA concentration 42 units/g and the incubation time needed for the maximum amount of enzyme attachment to the polymeric supports was found to be 8 h. Some phsicochemical properties of the free and immobilized ADA such as operational stability, optimum temperature and thermal stability, pH optimum and stability, storage stability, and the effect of gamma-radiation were studied. The operational stability of the free and immobilized enzyme showed that the enzyme immobilized by a cross-linking technique using gultaric dialdehyde showed poor durability and the relative activity decreased sharply due to the leakage after repeated washing, while the enzymes immobilized by covalent bonds to the carriers showed a slight decrease in most cases in the relative activity (around 20%) after being used 10 times. Storage for 4-6 months, showed that the free enzyme lost its activity, while the immobilized enzyme showed the opposite behavior. Subjecting the immobilized enzyme to a dose of gamma radiation of 0.5-10 Mrad showed complete loss in the activity of the free enzyme at a dose of 5 Mrad, while the immobilized enzymes showed relatively high resistance to gamma radiation up to a dose of 5 Mrad.     

Immobilization of enzymes on heterofunctional epoxy supports

Immobilization of enzymes and proteins on activated supports permits the simplification of the reactor design and may be used to improve some enzyme properties. In this sense, supports containing epoxy groups seem to be useful to generate very intense multipoint covalent attachment with different nucleophiles placed on the surface of enzyme molecules (e.g., amino, thiol, hydroxyl groups). However, the intermolecular reaction between epoxy groups and soluble enzymes is extremely slow. To solve this problem, we have designed "tailor-made" heterofunctional epoxy supports. Using these, immobilization of enzymes is performed via a two-step process: (i) an initial physical or chemical intermolecular interaction of the enzyme surface with the new functional groups introduced on the support surface and (ii) a subsequent intense intramolecular multipoint covalent reaction between the nucleophiles of the already immobilized enzyme and the epoxy groups of the supports. The first immobilization may involve different enzyme regions, which will be further rigidified by multipoint covalent attachment. The design of some heterofunctional epoxy supports and the performance of the immobilization protocols are described here. The whole protocol to have an immobilized and stabilized enzyme could take from 3 days to 1 week.     

Enzyme stabilization by nano/microsized hybrid materials

Immobilization is a key technology for successful realization of enzyme‐based industrial processes, particularly for production of green and sustainable energy or chemicals from biomass‐derived catalytic conversion. Different methods to immobilize enzymes are critically reviewed. In principle, enzymes are immobilized via three major routes (i) binding to a support, (ii) encapsulation or entrapment, or (iii) cross‐linking (carrier free). As a result, immobilizing enzymes on certain supports can enhance storage and operational stability. In addition, recent breakthroughs in nano and hybrid technology have made various materials more affordable hosts for enzyme immobilization. This review discusses different approaches to improve enzyme stability in various materials such as nanoparticles, nanofibers, mesoporous materials, sol–gel silica, and alginate‐based microspheres. The advantages of stabilized enzyme systems are from its simple separation and ease recovery for reuse, while maintaining activity and selectivity. This review also considers the latest studies conducted on different enzymes immobilized on various support materials with immense potential for biosensor, antibiotic production, food industry, biodiesel production, and bioremediation, because stabilized enzyme systems are expected to be environmental friendly, inexpensive, and easy to use for enzyme‐based industrial applications.     

Chemical modification of protein surfaces to improve their reversible enzyme immobilization on ionic exchangers

The enzyme penicillin G acylase (PGA) is not adsorbed at pH 7 on DEAE- or PEI-coated supports, neither is it adsorbed on carboxymethyl (CM)- or dextran sulfate (DS)-coated supports. The surface of the enzyme was chemically modified under controlled conditions: chemical amination of the protein surface of carboxylic groups (using soluble carbodiimide and ethylendiamine) and chemical succinylation (using succinic anhydride) of amino groups. The full chemical modification produced some negative effects on enzyme stability and activity, although partial modification (mainly succinylation) presented negligible effects on both enzyme features. The chemical amination of the protein surface permitted the immobilization of the enzyme on CM- and DS-coated support, while the chemical succinylation permitted the enzyme immobilization on DEAE- and PEI-coated supports. Immobilization was very strong on these supports, mainly in the polymeric ones, and dependent on the degree of modification, although the enzymes still can be desorbed after inactivation by incubation under drastic conditions. Moreover, the immobilization on ionic polymeric beds allowed a significant increase in enzyme stability against the inactivation and inhibitory effects of organic solvents, very likely by the promotion of a certain partition of the organic solvent out of the enzyme environment. These results suggest that the enrichment of the surface of proteins with ionic groups may be a good strategy to take advantage of the immobilization of industrial enzymes via ionic exchange on ionic polymeric beds.     

Overview on immobilization of enzymes on synthetic polymeric nanofibers fabricated by electrospinning

The arrangement and type of support has a significant impact on the efficiency of immobilized enzymes. 1-dimensional fibrous materials can be one of the most desirable supports for enzyme immobilization. This is due to their high surface area to volume ratio, internal porosity, ease of handling, and high mechanical stability, all of which allow a higher enzyme loading, release and finally lead to better catalytic efficiency. Fortunately, the enzymes can reside inside individual nanofibers to remain encapsulated and retain their three-dimensional structure. These properties can protect the enzyme's tolerance against harsh conditions such as pH variations and high temperature, and this can probably enhance the enzyme's stability. This review article will discuss the immobilization of enzymes on synthetic polymers, which are fabricated into nanofibers by electrospinning. This technique is rapidly gaining popularity as one of the most practical ways to fibricate polymer, metal oxide, and composite micro or nanofibers. As a result, there is interest in using nanofibers to immobilize enzymes. Furthermore, present research on electrospun nanofibers for enzyme immobilization is primarily limited to the lab scale and industrial scale is still challanging. The primary future research objectives of this paper is to investigate the use of electrospun nanofibers for enzyme immobilization, which includes increasing yield to transfer biological products into commercial applications.     

Optimizing Immobilization and Stabilization of Feruloyl Esterase from Humicola Insolens and its Application for the Feruloylation of Oligosaccharides

Feruloyl esterase (FAE)-catalyzed esterification reaction is as a potential route for the biosynthesis of feruloylated oligosaccharides as functional ingredients. Immobilization of FAE from Humicola insolens on metal chelate-epoxy supports was investigated. The study of effects of immobilization parameters using response surface methodology revealed the significance of enzyme/support ratio (3.25-29.25 mg/g support), immobilization time (14-38 h), buffer molarity (0.27-1.25 M) and pH (4.0-8.0). The interactions between enzyme-to-support ratio/buffer molarity and enzyme-to-support ratio/pH were found to be critical for the modulation of the immobilization activity yield and the retention of specific activity, respectively. Optimum conditions for FAE-immobilization on metal chelate Sepabeads® EC-EP R were identified to be 22.75 mg FAE/g support, pH of 5.0, 27.7 h and buffer molarity of 0.86 M. At these conditions, an activity yield of 82.4%, a specific activity retention of 143.4%, and an enzyme activity of 395.4 μmol/min. g support were achieved. Further incubation of the immobilized FAE at pH 10.0 improved its thermostability. Increasing the pore size of the epoxy support improved the retention of FAE hydrolytic activity and the esterifying efficiency of the immobilized biocatalyst. Optimally immobilized and stabilized FAE on metal chelate-epoxy support retained up to 92.9% of the free enzyme feruloylation efficiency to xylooligosaccharides..     

Immobilization of enzymes in porous supports: Effects of support-enzyme solution contacting

Proteins have been immobilized in porous support particles held in a fixed-bed reactor through which protein solution is continuously circulated. Changing the recirculation flow rate alters the observed immobilization kinetics and the maximum enzyme loading which can be achieved for glucose oxidase and glucoamylase on carbodiimide-treated activated carbon and for glucoamylase immobilized on CNBr-Sepharose 4B. Direct microscopic examination of FITC-labelled protein in sectioned Sepharose particles and indirect activity-loading studies with activated carbon-enzyme conjugates all indicate that immobilized enzyme is increasingly localized near the outer surface of the support particles at larger recirculation flow rates. Restricted diffusion of enzymes may be implicated in this phenomenon. These contacting effects may be significant considerations in the scaleup of processes for protein impregnation in porous supports, since apparent activity and stability of the final preparation depend on internal protein distribution.     

Immobilized Candida antarctica lipase B: Hydration, stripping off and application in ring opening polyester synthesis

This work reviews the stripping off, role of water molecules in activity, and flexibility of immobilized Candida antarctica lipase B (CALB). Employment of CALB in ring opening polyester synthesis emphasizing on a polylactide is discussed in detail. Execution of enzymes in place of inorganic catalysts is the most green alternative for sustainable and environment friendly synthesis of products on an industrial scale. Robust immobilization and consequently performance of enzyme is the essential objective of enzyme application in industry. Water bound to the surface of an enzyme (contact class of water molecules) is inevitable for enzyme performance; it controls enzyme dynamics via flexibility changes and has intensive influence on enzyme activity. The value of pH during immobilization of CALB plays a critical role in fixing the active conformation of an enzyme. Comprehensive selection of support and protocol can develop a robust immobilized enzyme thus enhancing its performance. Organic solvents with a log P value higher than four are more suitable for enzymatic catalysis as these solvents tend to strip away very little of the enzyme surface bound water molecules. Alternatively ionic liquid can work as a more promising reaction media. Covalent immobilization is an exclusively reliable technique to circumvent the leaching of enzymes and to enhance stability. Activated polystyrene nanoparticles can prove to be a practical and economical support for chemical immobilization of CALB. In order to reduce the E-factor for the synthesis of biodegradable polymers; enzymatic ring opening polyester synthesis (eROPS) of cyclic monomers is a more sensible route for polyester synthesis. Synergies obtained from ionic liquids and immobilized enzyme can be much effective eROPS.     

An approach for the stable immobilization of proteins

An approach is presented for the stable covalent immobilization of proteins with a high retention of biological activity. First, chemical modification studies were used to establish enzyme structural and functional properties relevant to the covalent immobilization of an enzyme to agarose based supports. Heparinase was used as a model enzyme in this set of studies. Amine modifications result in 75-100% activity loss, but the effect is moderated by a reduction in the degree of derivatization. N-hydroxysuccinimide, 1,1,1-trifluoroethanesulfonic acid, and epoxide activated agarose were utilized to determine the effect of amine reactive supports on immobilized enzyme activity retention. Cysteine modifications resulted in 25-50% loss in activity, but free cysteines were inaccessible to either immobilized bromoacetyl or p-chloromercuribenzoyl groups. Amine reactive coupling chemistries were therefore utilized for the covalent immobilization of heparinase. Second, to ensure maximal stability of the immobile protein-support linkage, the identification and subsequent elimination of the principal sources of protein detachment were systematically investigated. By using high-performance liquid chromatography (HPLC), electrophoresis, and radiolabeling techniques, the relative contributions of four potential detachment mechanisms-support degradation, proteolytic degradation, desorption of noncovalently bound protein, and bond solvolysis-were quantified. The mechanisms of lysozyme, bovine serum albumin, and heparinase leakage from N-hydroxysuccinimide or 1,1,1-trifluoroethanesulfonic acid activated agarose were elucidated. By use of stringent postimmobilization support wash procedures, noncovalently bound protein loss. An effective postimmobilization washing procedure is presented for the removal of adsorbed protein and the complete elimination of immobilized protein loss.     

Activity studies of immobilized subtilisin on functionalized pure cellulose-based membranes

The activity of immobilized subtilisin BPN' on pure cellulose-based membrane support was investigated using site-directed and random immobilization approaches. The catalytic activity of site-directed immobilized subtilisin on pure cellulose fiber-based materials was found to be 81% of that in homogeneous solution, while that of randomly immobilized subtilisin was 27%. Pure cellulose membrane supports provided large surface areas for high enzyme loading without diffusional limitations. The activity of immobilized subtilisin on pure cellulose support was more than twice that on a modified polyether sulfone (MPS) membrane, which was attributed to the higher hydrophilicity of cellulose. Immobilized subtilisin maintained its initial activity for 14 days at 4 degrees C and 7 days at 24 degrees C. The immobilized enzyme could resist higher temperature and operate over a wider range of pH without loss of activity. This study showed that pure cellulose fiber-based membranes are well suited for enzyme immobilization and biocatalysis.     

Novel polymeric microspheres: Synthesis,enzyme immobilization,antimutagenic activity,and antimicrobial evaluation against pathogenic microorganisms

New polymeric microspheres containing azomethine ( 1a ‐ 1c and 2a ‐ 2c ) were synthesized by condensation to compare the enzymatic properties of the enzyme glucose oxidase (GOx) and to investigate antimutagenic and antimicrobial activities. The polymeric microspheres were characterized by elemental analysis, infrared spectra (FT‐IR), proton nuclear magnetic resonance spectra, thermal gravimetric analysis, and scanning electron microscopy analysis. The catalytic activity of the glucose oxidase enzyme follows Michaelis‐Menten kinetics. Influence of temperature, reusability, and storage capacity of the free and immobilized glucose oxidase enzyme were investigated. It is determined that immobilized enzymes exhibit good storage stability and reusability. After immobilization of GOx in polymeric supports, the thermal stability of the enzyme increased and the maximum reaction rate (V_max) decreased. The activity of the immobilized enzymes was preserved even after 5 months. The antibacterial and antifungal activity of the polymeric microspheres were evaluated by well‐diffusion method against some selected pathogenic microorganisms. The antimutagenic properties of all compounds were also examined against sodium azide in human lymphocyte cells by micronuclei and sister chromatid exchange tests.     

Enzyme immobilization on electrospun polymer nanofibers: An overview

Enzyme immobilization has attracted continuous attention in the fields of fine chemistry, biomedicine, and biosensor. The performance of immobilized enzyme largely depends on the structure of supports. Nanostructured supports are believed to be able to retain the catalytic activity as well as ensure the immobilization efficiency of enzyme to a high extent. Electrospinning provides a simple and versatile method to fabricate nanofibrous supports. Compared with other nanostructured supports (e.g. mesoporous silica, nanoparticles), nanofibrous supports show many advantages for their high porosity and interconnectivity. This review mainly discusses the recent advances in using nanofibers as hosts for enzyme immobilization by two different methods, surface attachment and encapsulation. Surface attachment refers to physical adsorption or covalent attachment of enzymes on pristine or modified nanofibrous supports, and encapsulation means electrospinning a mixture of enzyme and polymer. We make a detailed comparison between these two immobilization approaches and highlight their distinct characteristics. The prospective applications of enzyme immobilized electrospun nanofibers in the development of biosensors, biofuel cells and biocatalysts are also discussed.     

Immobilization of lipases on hydrophobic supports: immobilization mechanism,advantages, problems,and solutions

Lipases are the most widely used enzymes in biocatalysis, and the most utilized method for enzyme immobilization is using hydrophobic supports at low ionic strength. This method allows the one step immobilization, purification, stabilization, and hyperactivation of lipases, and that is the main cause of their popularity. This review focuses on these lipase immobilization supports. First, the advantages of these supports for lipase immobilization will be presented and the likeliest immobilization mechanism (interfacial activation on the support surface) will be revised. Then, its main shortcoming will be discussed: enzyme desorption under certain conditions (such as high temperature, presence of cosolvents or detergent molecules). Methods to overcome this problem include physical or chemical crosslinking of the immobilized enzyme molecules or using heterofunctional supports. Thus, supports containing hydrophobic acyl chain plus epoxy, glutaraldehyde, ionic, vinylsulfone or glyoxyl groups have been designed. This prevents enzyme desorption and improved enzyme stability, but it may have some limitations, that will be discussed and some additional solutions will be proposed (e.g., chemical amination of the enzyme to have a full covalent enzyme-support reaction). These immobilized lipases may be subject to unfolding and refolding strategies to reactivate inactivated enzymes. Finally, these biocatalysts have been used in new strategies for enzyme coimmobilization, where the most stable enzyme could be reutilized after desorption of the least stable one after its inactivation.     

Site-specific immobilization of enzymes on magnetic nanoparticles and their use in organic synthesis

Magnetic nanoparticles (MNPs) are attractive materials that serve as a support for enzyme immobilization and facilitate separations by applying an external magnetic field; this could facilitate the recycling of enzymes and broaden their applications in organic synthesis. Herein, we report the methods for the immobilization of water-soluble and membrane-bound enzymes, and the activity difference between free and immobilized enzymes is discussed. Sialyltransferase (PmST1, from Pasteurella multocida ) and cytidine monophosphate (CMP)-sialic acid synthetase (CSS, from Neisseria meningitides ) were chosen as water-soluble enzymes and expressed using an intein expression system. The enzymes were site-specifically and covalently immobilized on PEGylated-N-terminal cysteine MNPs through native chemical ligation (NCL). Increasing the length of the PEG linker between the enzyme and the MNP surface increased the activity of the immobilized enzymes relative to the free parent enzymes. In addition, the use of a fluorescent acceptor tag for PmST1 affected enzyme kinetics. In contrast, sialyltransferase from Neisseria gonorrheae (NgST, a membrane-bound enzyme) was modified with a biotin-labeled cysteine at the C-terminus using NCL, and the enzyme was then assembled on streptavidin-functionalized MNPs. Using a streptavidin-biotin interaction, it was possible to immobilize NgST on a solid support under mild ligation conditions, which prevented the enzyme from high-temperature decomposition and provided an approximately 2-fold increase in activity compared to other immobilization methods on MNPs. Finally, the ganglioside GM3-derivative (sialyl-lactose derivative) was synthesized in a one-pot system by combining the use of immobilized PmST1 and CSS. The enzymes retained 50% activity after being reused ten times. Furthermore, the results obtained using the one-pot two-immobilized-enzyme system demonstrated that it can be applied to large-scale reactions with acceptable yields and purity. These features make enzyme-immobilized MNPs applicable to organic synthesis.