Enzyme catalysis on solid surfaces

Review

Enzyme catalysis on solid surfaces Nicolas Laurent, Rose Haddoub and Sabine L. Flitsch Manchester Interdisciplinary Biocentre (MIB) and School of Chemistry, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK

Enzyme-catalysed reactions in which substrates are bound (immobilised) to solid surfaces are becoming increasingly important in biotechnology. There is a general drive for miniaturisation and automation in chemistry and biology, and immobilisation of the reaction intermediates and substrates, for example on microarrays or nanoparticles, helps to address technical challenges in this area. In bionanotechnology, enzyme catalysis can provide highly selective and biocompatible tools for the modification of surfaces on the nano-scale. Here, we review the range of enzyme-catalysed reactions that have been successfully performed on the solid phase and discuss their application in biotechnology. Introduction Enzymes are attractive catalysts for organic synthesis because they generally promote reactions in high regioand stereoselectivities under very mild reaction conditions. Biocatalysis (synthesis of compounds using enzymes) is now a widely used tool for chemical synthesis that is complementary to purely chemical methods. In the past, enzymes have generally been studied either in solution phase (Figure 1a) or with the enzymes immobilised on a solid support (Figure 1b). Enzyme-catalysed reactions in which substrates are bound (immobilised) to solid surfaces (Figure 1c) are becoming increasingly important in biotechnology. With the drive for miniaturisation and automation in chemistry and biology, enzyme-catalysed reactions with substrates bound to a solid surface help to address technical challenges in this area. In bionanotechnology, enzyme catalysis can provide highly selective and biocompatible tools for the modification of surfaces on the nano-scale. When considering enzymatic transformations at a solid–liquid interface, one has to be aware of some complications that are likely to occur [1]: rates and yields of solid-phase reactions are often lower than solution-phase reactions because of accessibility problems (the enzyme might have difficulty in accessing the substrate), and such reactions might require a large excess of enzyme. Accessibility of the immobilised substrate is an important issue, and the nature of the linker between substrate and support and the support itself is crucial and differs between enzymes. Indeed, most of the solid supports commonly used in solid-phase chemistry, such as polystyrene and polyacrylamide, are not suitable for enzymatic reactions because of their low swelling properties in aqueous media. Porous polymers, such as TentaGelTM (polyethylene glycol grafted onto polystyrene), controlled pore glass (CPG) and Corresponding author: Flitsch, S.L. ([email protected]).

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PEGA (polyethylene glycol grafted onto polyacrylamide), enable high loading of the substrate but at the same time can limit accessibility to large molecules, in particular enzymes with molecular weights above 50 KDa [2–4]. Other important types of support, such as planar surfaces (e.g. glass slides), have mainly been used for the construction of chips for high throughput screening. An enzyme acting on a substrate bound to such a surface will not experience the same limitations as in the case of a resinbound substrate. However, in some cases a proper choice of linker and substrate density is necessary to achieve a high yield of transformation. A major issue for reactions on a solid phase is analysis of reaction products. A popular method that can also be used for high-throughput applications is fluorescence spectroscopy [5,6], but this method relies on labelling of reaction components, which is not always easy. For label-free analysis, mass spectrometry (MS) [particularly matrixassisted laser desorption ionization-time of flight (MALDI-TOF) MS] [7], quartz crystal microbalance (QCM) [8] or confocal Raman microscopy [4] have become increasingly useful and can often be performed directly on the surface. Some fundamental studies have shown that enzymecatalysed reactions on solid-supported substrates can have very different thermodynamics, kinetics and chemical selectivity to equivalent reactions in solution. These studies have recently been reviewed elsewhere [1,9]. The aim of the present review is to highlight important examples of the applications of enzymatic transformations on solid supports. There are now several diverse chemical reactions that have been shown to work on immobilised substrates, and some representative examples are summarised in Table 1. These include the synthesis of natural biopolymers: peptides, oligonucleotides and oligosaccharides. Enzymes have been used on the solid phase for kinetic resolutions and solid-phase enzyme screening. More recently, examples of C-C bond formation and synthetic biopolymer fabrication have been described on solid supports. Carbohydrate synthesis In contrast to proteomics and genomics, understanding of the molecular interactions of carbohydrates in living systems (so-called ‘glycomics’) has been hampered by the complexity of carbohydrate structures and the lack of high-throughput methods for their synthesis and analysis. Carbohydrate oligomers (glycans) isolated from natural sources are often a mixture of complex isoforms refered to as glycoforms. Chemical synthesis of naturally occurring carbohydrate structures or their analogues involves

0167-7799/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2008.03.003 Available online 20 April 2008

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Figure 1. Different types of enzymatic reactions. Enzyme catalysis has commonly been studied in solution phase (a). For practical reasons, enzymes can be immobilised on a polymeric support, which is also well documented (b). The present review discusses enzyme-catalysed reaction in which the substrate is attached to solid phase (c).

numerous protection and deprotection steps, often leads to variable glycosylation yields and, furthermore, faces issues of regio- and/or stereoselectivity, requiring tedious purifications. Given these problems with synthesis, there has been a lot of interest in combining solid phase synthesis, which has been so successful for peptides and nucleic acids, with enzymatic methods, which are highly selective and clean. Following the pioneering work of Schuster et al. [10], enzymatic approaches combined with solid-supported synthesis have gained increasing interest for the synthesis of complex glycans and glycopeptides, and several successful examples of this approach are shown in Figure 2. Such enzymatic glycosylation reactions have been performed on various surfaces to produce glyco-structures with varying degrees of complexity. Blixt and Norberg [11] have described the chemoenzymatic synthesis of oligosaccharides that were attached to a solid support by a cleavable linker that allows, after enzymatic elongation, the recovery of the reducing glycan structure without any remnant of

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the linker that was used for attachment to solid support. Fazio et al. [12] have used microtiter plates to attach oligosaccharides through a 1,3-dipolar cycloaddition between azide-functionalised saccharides and alkyne-terminated hydrocarbon chains that were non-covalently bound to the microtiter plate. Using this method, the sialyl Lex structure, an important tetrasaccharide structural motif that is found on many protein and cell surfaces, was successfully synthesised by enzymatic fucosylation of its trisaccharide precursor directly on the surface. Mrksich et al. have developed a platform in which enzyme substrates are immobilised as self-assembled monolayers (SAMs) of alkanethiols on gold surfaces and in which enzyme activity and protein binding can be monitored using either MALDI-TOF MS [7], fluorescence or surface plasmon resonance (SPR) [13]. Using this technology, galactosylation of an immobilised N-acetyl-glucosamine (GlcNAc) by a bovine b-1,4-galactosyltransferase was investigated [14]. The substrate was immobilised onto the SAM-coated gold surface at different surface densities. Interestingly, the substrate density was found to influence the yield of enzymatic galactosylation, and maximum yield was reached when the density of the enzyme substrate was 70%. Higher densities led to dramatically decreased yields, probably due to steric hindrance on the surface. Carbohydrate arrays (glycochips) are becoming increasingly important tools for studying carbohydrate–protein interactions. In such arrays, enzymatic reaction plays an important role both for defining enzyme specificity in terms of acceptor substrate and glycosidic linkage formed, as well as for the synthesis of oligosaccharides on the chip. An enzymatic approach was used by Park et al. [15] for the construction of carbohydrate chips on glass slides. Starting from a GlcNAc that was immobilised on the glass surface via an alkane linker, a series of three sequential enzymatic reactions were performed to ultimately yield an immobilised sialyl Lex structure. Each enzymatic transformation was monitored by a specific lectin that would only bind to

Table 1. Typical examples of enzymes acting on solid-supported substrates Enzyme b-1,4-Galactosyltransferase

Substrate Glycopeptides Oligosaccharides Sugars

Support Aminopropyl silica Sepharose Glass Gold Glycopeptides Aminopropyl silica a-2,3-Sialyltransferase Glass Oligosaccharides Oligosaccharides Glass a-1,3-Fucosyltransferase Microtiter plates Gold Polypeptide N-acetylgalactosaminyl transferase Peptides Sugars/glycopeptides PEGA Glycosynthase Peptides PEGA, PEGA1900 Protease Peptides PEGA Protease Peptides PEGA Thioesterase Glycopeptides PEGA1900 Protease Phenylacetamides PEGA Penicillin G amidase 3-phenylbutyric vinyl esters PEGA Lipase Oligonucleotides Gold nanoparticles DNA polymerase Magnetic beads Peptides Gold Kinases 4-hydroxyphenyl valerate Gold Cutinase Peptides Mica Serine V8 protease Oligonucleotides Gold DNase Hydroquinone Polystyrene Soybean peroxidase

Application Refs Glycopeptide synthesis [10] Carbohydrate synthesis [11] Microarray fabrication [15,16] Microarray fabrication [7] Glycopeptide synthesis [10] Microarray fabrication [15,16] Microarray fabrication [15,16] Microarray fabrication [12] Microarray fabrication [17] Glycopeptide/carbohydrate synthesis [24] Peptide synthesis [25,26] Design of antibiotics [30] Design of antibiotics [32,34] Cleavage from the support [42] Cleavage from the support [39,40] Kinetic resolution [66] Oligonucleotide synthesis [50] cDNA library synthesis [52] Enzyme activity screening [56,57] Enzyme activity screening [60] Biopolymer fabrication [68] Biopolymer fabrication [69] Biopolymer fabrication [73]

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Figure 2. Some typical examples of enzymatic glycosylations on solid support. (a) Synthesis of glycopeptides and enzymatic cleavage from the support [10]. (b) Enzymatic elongation of oligosaccharides and chemical release to yield the free reducing sugar [11]. (c) Enzymatic glycosylation of an immobilised N-acetyl glucosaminide on gold surface [14]. (d) Synthesis of glycopeptide arrays containing peptide fragments found in mucins on gold sufaces by enzymatic glycosylation [17].

the reaction product and not to the starting material. Detection of the sialyl LeX was then performed by incubating with anti-sialyl Lex antibody followed by a secondary fluorescent anti-antibody. More recently, the same authors described an elegant approach of combining hydrazidelinked glycans with epoxide-coated glass slides for the immobilisation of various glycans and for the screening of galactosyltransferase activity [16]. Glycosyltransferases that are involved in mucin-type glycopeptide biosynthesis have recently been used to glycosylate peptide arrays on gold surfaces [17]. The enzymatic glycosylation reactions were monitored in a label-free manner by MALDI-TOF MS, and a quantitative glycosylation could be demonstrated for several peptide substrates. Mucins often 330

present an aberrant glycosylation in tumour cells [18], and antibodies specific for the carbohydrate epitopes of mucins are widely used in diagnosis. The synthesis of these tumour-associated antigens has been investigated by several groups [19–21], but for practical applications, an efficient and straightforward preparation of these glycopeptides motifs is required. The chemo-enzymatic approach overcame some of the previous limitations associated with chemical methods and allowed the preparation of glycopeptide libraries on a single chip. Such arrays might find applications in screening antibodies in patient serum samples and might facilitate early detection of cancer. Furthermore, the screening of glycosyltransferase inhibitors as potential anti-cancer drugs can be achieved in an

Review automated and high-throughput manner. Using the same platform, the specificity of the bovine b1,4-GalT against a panel of immobilised mono- and disaccharides was monitored in an array format [22]. This proof-of-principle experiment showed that glycosyltransferase specificities can be easily monitored by a label-free MS technique and that libraries of glycosides can be generated by using a chemo-enzymatic approach. Although glycosyltransferases are naturally involved in the biosynthesis of complex glycans, their use in synthetic applications [19] is limited by their generally narrow substrate specificity. As an alternative, glycosidases have been used. Indeed, glycosidases normally catalyse the hydrolysis of glycosidic bonds, but they can also create sugar linkages by transglycosylation from chemically accessible substrates, such as p-nitrophenyl glycosides, albeit in moderate yields due to the reversibility of the reaction. Withers et al. have developed glycosynthases [23], mutant forms of glycosidases that lack the nucleophilic carboxylic acid residue responsible for hydrolysis in the active site. These novel enzymes are therefore unable to hydrolyse glycosidic bonds but can take advantage of the enzyme’s binding specificity to carry out the glycosylation reaction. Using this approach, sugars and glycopeptides linked to PEGA resin have been glycosylated with glycosynthase and a glycosyl fluoride donor [24]. Peptide synthesis Proteases are normally responsible for the hydrolysis of peptide bonds, but they have also been found to catalyse bond formation between amino acids when the substrate was immobilised on a PEGA solid support [25]. This remarkable shift in the equilibrium between synthesis and hydrolysis is believed to arise from the transferral of hydrophobic substrates from aqueous solution into the hydrophobic domain of the PEGA resin [26]. Due to their large substrate-binding sites, which typically encompass seven to eight amino acid residues, proteases can also bind two peptides at the same time and facilitate bond formation. This enzymatic segment condensation between peptide fragments can be used for the synthesis of longchain peptides and even small proteins in an efficient manner, thereby surpassing chemical methods limited to small-size peptides. Using a complex of sodium dodecylsulfate with subtilisin (SDS–subtilisin), three successive segment condensation steps of tripeptides on an aminosilochrom-immobilised tetrapeptide were achieved. Interestingly, the yield of the second and the third enzymatic couplings were significantly higher than that of the first coupling reaction, although no explanation was proposed for this result [27]. This finding – that on solid supports proteases catalyse peptide formation rather than hydrolysis – has been exploited recently for the screening of the activity of chymotrypsin and thermolysin [28]. A library of amino acids was linked to PEGA resin and incubated with the enzyme in the presence of Fmoc-protected amino acids as acyl donors in a 96 well plate format. The resulting dipeptides formed by enzyme-catalysed coupling were then detected and quantified by fluorescence of the Fmoc moiety. Hence, specificity of the thermolysin for large hydrophobic amino acids, such as phenylalanine or

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isoleucine, was clearly highlighted. This method provided an easy way to evaluate substrate specificity without the need for labelling amino acid partners and would be a valuable tool for better understanding the biological role of proteases. Proteases are also important targets for drug discovery. Many protease inhibitors are peptide-like molecules in which the scissible bond has been altered. Therefore, the enzyme binding site retains its affinity for these analogues while being unable to hydrolyse the modified bond. The ability of proteases to catalyse peptide cyclisation has been successfully used to identify promising macrocyclic antibiotic leads [29]. To this end, a panel of chemically synthesised peptides on PEGA resins were treated with a range of peptidases, including trypsin, chymotrypsin, pepsin and thermolysin. Those peptides that were found to cyclise were then used as macrocyclic motifs for the design of inhibitors of the enzymes [30]. Two of the molecules synthesised as mimics of the cyclic peptides were evaluated as competitive inhibitors of chymotrypsin with an inhibition constant in the low micromolar range. Non-ribosomal peptide synthetases are another class of enzymes involved in the biosynthesis of macrocyclic peptides. Contrary to the ribosomal pathway, which is gene encoded, the non-ribosomal pathway involves large multifunctional enzymes, the peptide synthetases, and leads to highly complex macromolecules with a broad range of structural diversity. These enzymes are composed of several domains that build the peptide covalently attached to the enzyme structure through a thioether linkage by sequential assembly until the product is finally released [31]. The last domain, called the thioesterase (TE) domain, promotes the macrocyclisation and release of the nascent peptide from the complex. Isolated TE domains have been used to catalyse the macrocyclisation and release of peptides from solid supports. Kohli et al. [32] screened a range of TE domains and found that the tyrocidine TE domain was a versatile catalyst for the formation of lactams and lactones. By using this enzyme domain, over 300 linear peptide substrates anchored on PEGA resin could be cyclised and released from the support. These cyclic peptides, analogues of the tyrocidine A antibiotic, were subsequently screened for enhanced activity. Several analogues were found to have enhanced antibiotic activity compared to tyrocidine A, and analogues with positively charged D-amino acids in place of the D-phenylalanine 4 of tyrocidine A showed the greatest increase in selectivity for bacterial membranes over eukaryotic cell membranes. However, the TE domain was found to suffer from a short lifetime, showing a decrease in catalytic activity within a few minutes. Furthermore, a significant proportion of the products were lost by direct peptide hydrolysis before cyclisation could occur. More recently, these problems have been addressed with the use of non-ionic detergents that were able to help maintain the proper folding of the enzyme and thus extend the catalytic activity for up to an hour. A sixfold enhancement in the rate of cyclisation reaction without an increase in the rate of hydrolysis also resulted from the addition of detergents. Taken together, these effects allowed increased net yields of the cyclised products by factors of 150 to 300 [33]. TE domains were also 331

Review used by Wu et al. [34] for the cyclisation of linear peptides. They were able to generate the potent peptide antibiotic gramicidin S, a unique peptide that contains the uncommon amino acids D-phenylalanine and ornithine. Moreover, seven analogues of this antibiotic were also generated by TE-catalysed cyclisation of an array of linear peptides bound to TentaGelTM resins, therefore demonstrating that this enzyme is also capable of cyclising substrates containing varied amino acid sequences. This approach would, therefore, be very valuable for the discovery of more potent gramicidin analogues to overcome potential antibiotic resistance problems. Enzyme-cleavable linkers In traditional solid-phase synthesis, a crucial step is the release of the molecule from the support after completion of the synthesis. This is a critical step because treatment of the anchored molecule often requires relatively harsh conditions [35]. For example, cleavage of a peptide from resin beads is typically performed with a 95% trifluoroacetic acid solution and needs careful control of the reaction conditions to avoid potential side reactions and degradation of the desired molecule. The linker also dictates the type of chemistry applicable: acid-sensitive linkers restrict the use of acidic reagents during synthesis steps. However, acid-sensitive compounds such as carbohydrates need to be anchored with special linkers that can be cleaved under mild conditions. As an alternative to chemical treatment, several enzymatic strategies for the release of molecules from solid supports under mild conditions have been developed. Endo- and exo-linkers used to attach molecules on a solid support can be distinguished depending on whether cleavage is achieved by endo- or exoenzymes, respectively (Figure 3) [36]. Endo-linkers contain a site for enzyme-catalysed hydrolysis to which the target molecule is directly linked in a linear arrangement. Examples for endo-linker cleavers are proteases (trypsin and chymotrypsin) and phosphodiesterases. Peptides [37] and oligosaccharides [38] synthesised on solid supports were successfully cleaved from endo-linkers. However, enzyme-mediated cleavage releases the target molecule, which in most cases is still attached by the functional group recognised by the enzyme. For instance, chymotrypsincatalysed hydrolysis of a peptide often requires an aromatic amino acid that will stay on the C-terminus of the liberated peptide. Conversely, exo-linkers rely on the recognition of a specific group (phenylacetamide [39,40] or ester [41]) by the enzyme, which upon cleavage undergoes an

Figure 3. Exo- (left) and endo- (right) linkers. Cleavage of an exo-linker (R1) leads to an intramolecular reaction that ultimately liberates the target molecule (R3), whereas direct cleavage of an endo-linker often releases the target (R3) tagged with the group providing the site for enzyme-catalysed hydrolysis (R1). Reproduced, with permission, from Ref. [36].

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intramolecular rearrangement to release the untagged molecule. Penicillin G amidase (PGA) and lipases have been used to cleave exo-linkers and release alcohol- [41,42], amine- [42] or carboxylic-acid- [43] containing molecules. Interestingly, the so-called Wang linker (hydroxymethyl-phenoxyacetic acid, HMPA), which is widely used in solid-phase peptide synthesis, was found to be susceptible to chymotrypsin-catalysed hydrolysis, and a broad range of amino acids were tolerated [42]. This result was applied in the enzymatic release of peptides and glycopeptides which were synthesised on solid support, and was an alternative to the use of strong acidic conditions potentially detrimental for the glycosidic bond. Niederhafner et al. [43] also employed enzymatic cleavage for the release of protected peptides from the human immunoglobulin G1 hinge region synthesised on polyethylene glycol (PEG) support, and in this case cleavage occurred between a terminal AlaLeu sequence, rather than at the linker position. Substrates anchored on positively charged PEGA polymers via an exo-linker could be successfully cleaved by penicillin G amidase (PGA), a hydrolysase specific for phenylacetamide and phenylhydrazide groups. Upon selective cleavage of a phenylacetic hydrazide moiety of the exo-linker, the highly reactive hydrazine group generated could undergo intramolecular nucleophilic attack leading to the release of the target molecule [44]. The efficiency of this cleavage was improved by introduction of permanent positive charges inside the polymer, thereby strengthening the favourable electrostatic interaction with the negatively charged PGA, resulting in an optimal enzyme diffusion into the resin [45]. We used a similar approach with a linker, in which hydrolysis of a phenylacetamide moiety by PGA generated an unstable hemiaminal that fragmented in aqueous solution and thus released the target molecule [39]. Similarly, Sauerbrei et al. released the desired compound via a lipase-catalysed cleavage of an acyl group [41]. More recently, the same group developed a novel exo-linker that was based on the intramolecular cyclisation of an intermediate, which was generated after enzymatic reaction by PGA released the target molecule [40]. Oligonucleotide and cDNA synthesis The synthesis of defined short sequences of nucleic acids is extremely useful in molecular biology applications. Typically, oligonucleotides are single-stranded DNA of around 15 to 20 bases, although automated synthesizers allow the synthesis of oligonucleotides up to 200 bases. They are widely used as primers for DNA sequencing and amplification or as probes for detecting cDNA (complementary DNA) or RNA via hybridisation. Furthermore, oligonucleotides have clinical applications in the so-called ‘antisense’ therapy against cancer or HIV [46,47]. To deliver DNA intracellularly and to overcome low efficiency or toxicity issues, conjugation of nucleotides with non-viral transfection agents, such as polymers, dendrimers, liposomes or nanoparticles, was investigated [48]. Oligonucleotidemodified gold nanoparticles are an example of such a system for intracellular gene regulation and control of protein expression [49]. These conjugates are traditionally prepared by coating the nanoparticle surface with a presynthesised oligonucleotide containing a thiol linker that

Review reacts with the gold surface. An enzymatic extension of gold nanoparticle-bound oligonucleotides by DNA polymerase was achieved by Nicewarner Pena et al. [50]. The effect of linker length and surface coverage was studied to optimize the enzymatic reaction and achieve high efficiency in hybridisation experiments. In nature, cDNA is synthesised from an mRNA (messenger RNA) template by the enzyme reverse transcriptase. cDNA is used to clone eukaryotic genes in prokaryotes and obtain the corresponding proteins [51]. cDNA libraries have been constructed on solid-phase supports with mRNAs isolated and captured by a biotinylated oligodTprimer bound to streptavidin-coated magnetic beads [52]. First-strand cDNA synthesis was then performed on the solid support primed by the same primer that was used to capture the mRNA. After the subsequent steps, such as second-strand synthesis with RNase H and DNA-polymerase, a blunting step catalysed by T4-DNA-polymerase, EcoRI adaptor ligation and kinasing of the cDNA, the liberation of the cDNA products from the beads was achieved by cleavage with the restriction enzyme NotI. According to claims by the author, this solid-phase approach would allow for the construction of a cDNA library, from the RNA isolation to transformation of competent bacteria, in a single day. Solid-phase enzyme screening Enzymatic reactions that are carried out on solid supports allow for miniaturisation of screening platforms for biocatalyst activities [53] and several examples have already been discussed above. For example, glycosyltransferase, protease and thioesterase activities have been successfully assayed on immobilised substrates. Seibel et al. [54] have used a carbohydrate microarray on a microtiter plate to identify new acceptor specificities of the non-Leloir glycosyltransferase R (GTFR) from Streptococcus oralis. Contrary to other glycosyltransferases that used sugarnucleotide donors, the non-Leloir enzymes are able to transfer a sugar from an oligosaccharide to another saccharide acceptor by trans-glycosylation. GFTR uses sucrose as a glucose donor and was capable of transferring it to maltose but also, and rather unexpectedly, to alcoholterminated long aliphatic chains. This finding was applied for the enzymatic synthesis of various glycosides and glycosyl amino acids. Another microarray approach was recently used by Blixt et al. [55] for the screening of sialyltransferase specificities. First, a biotinylated cytidine monophospho-N-acetylneuraminic acid (CMP-NeuNAc), the activated sugar nucleotide used by the sialyltransferase, was synthesised by a chemo-enzymatic method. The activated sugar nucleotide was then used as a donor substrate for the tested mammalian sialyltransferases, which transferred biotinylated sialic acid onto the glycan acceptors immobilised on a glass slide. The resulting biotinylated glycans were then detected by fluorescence via a fluorescein-streptavidin conjugate that would bind selectively to the biotinylated glycans formed by the enzyme. Thus, each sialyltransferase could be profiled in terms of acceptor specificity in a convenient and high-throughput manner, and new acceptor specificities were discovered by this method. These findings will now be useful in the

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chemo-enzymatic synthesis of glycan libraries, as well as in the understanding of the biological role of sialyltransferases and the diseases associated with them. Serine protease substrate specificity was assessed by means of an array of fluorogenic substrates that, upon enzymatic cleavage, unmasked a coumarin residue, allowing fluorescence read-out [5]. This peptide chip built on a glass slide contained 800 spots in an area of less than 1.7 cm2 and was used to obtain a proteolytic ‘fingerprint’ of the serine protease thrombin with a minimal amount of enzyme and substrates. Using peptide chips that were built from alkanethiolate SAMs carrying peptide motifs on gold surfaces, Mrksich’s group have profiled kinase activities by analysing the phosphorylated substrates with MALDI-TOF MS [56], surface plasmon resonance, fluorescence and phosphorimaging [57]. Kinases are involved in many signalling pathways and are currently a target of choice for the development of therapeutics for the treatment of neurodegenerative diseases, diabetes or cancers [58]. Understanding their mechanism of action and substrate specificities will open the way toward the discovery of potent and selective inhibitors that can further be used in drug design. As a proof-of-principle, the peptide array was assayed against the c-Src tyrosine kinase, and the substrate specificity of the enzyme was confirmed by all the above-mentioned analytical techniques. Furthermore, the same array was used to determine the inhibition constant of three known inhibitors of the kinase. This high-throughput screening would prove useful for the screening of new kinase inhibitors as potential drugs for the treatment of numerous diseases in which kinases play a key role. The discovery of 48 potential substrates of MPK3 (mitogenactivated protein kinase 3) and 39 of MPK6 was recently achieved by using an array of 1690 Arabidopsis thaliana protein substrates and radioactive ATP [59]. The MPKs are involved in a wide range of cellular signalling cascades, and the discovery of new substrates of these enzymes will give scientists important insights into their biological functions. This study showed that the MPKs are potentially involved in the phosphorylation of transcription factors, transcription regulators, histones, receptors, enzymes, etc., thereby indicating the broad range and complexity of MPK-mediated regulation. New methods for detecting enzymes’ action on anchored substrates have recently emerged. In particular, electrochemical measurements allow not only a sensitive detection of enzymatic transformation but also open the way to the design of electroactive interfaces that can either respond to or modify enzymes’ action. The activity of cutinase, a serine esterase that hydrolyses an acyl group from its substrate, could be monitored on gold surfaces via the enzymatic conversion of a redox-inactive molecule to a redox-active moiety that is linked to an electrode, and this enabled real time analysis via cyclic voltammetry (Figure 4a) [60]. In another study, faradaic impedance spectroscopy was used to detect enzymatic ligation, polymerisation or restriction of oligonucleotides immobilised on a gold electrode [61]. QCM is another method of choice for analysing enzymatic reactions on surfaces. QCM is a very sensitive mass-measuring device for aqueous solutions and is able to detect mass increases or decreases 333

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Figure 4. Enzymatic surface modifications. (a) Cutinase-catalysed cleavage of 4-hydroxyphenyl valerate on a gold surface generates an electroactive molecule that can be detected by electrochemical measurement. Reproduced, with permission, from Ref. [61]. (b) Soybean peroxidase-catalysed oligomerisation of resin-bound hydroquinone. Radical coupling of an apocynin monomer onto the supported hydroquinone leads to the formation of a C-C bond (highlighted in red) and therefore enables the formation of ortho-methoxyphenol oligomers Reproduced, with permission, from Ref. [73].

at the nanogram level. Therefore, all steps of enzyme catalysis, such as enzyme binding, catalytic activity and enzyme release, could be monitored. Nishino et al. [8,62] reported the use of QCM to study glucoamylase-catalysed enzymatic hydrolysis of glucan that was immobilised on the quartz, and they were even able to obtain kinetic parameters for the enzyme. Kinetic resolution Kinetic resolution is a standard and well-established method of obtaining enantiopure molecules from racemic mixture [63]. The principle of kinetic resolution relies on the difference of reactivity of the two enantiomers in a chemical transformation: the more-reactive enantiomer is consumed during the reaction, thereby creating an excess of the less-reactive one. However, the principal drawback of traditional kinetic resolution is that the maximum yield of enantiopure compound is 50%, and the desired product must be separated from the reaction mixture. Therefore, other chemical methods have been introduced to overcome these limitations, the most important one being dynamic kinetic resolution [64]. By combining the advantages of the high specificity of enzymatic transformations and the 334

solid-supported methodology, a few examples of enzymatic kinetic resolution performed on solid-supported substrates have been reported. Ulijn et al. showed that the thermolysin-catalysed amide-bond formation on solid support enabled the isolation of both L,L or L,D diastereoisomers of dipeptides and pure L-amino acids from racemic mixtures [65]. Indeed, when a racemic mixture of Fmoc-protected phenylalanine was used as the reagent, only the L,L diastereoisomer was formed on the beads by the thermolysin-mediated coupling. This approach might be particularly useful for non-natural L-amino acids that are not readily available because the resolution of enantiomers from racemic mixtures and the coupling is achieved in one step. The kinetic resolution of L,L and L,D dipeptides on a solid phase was also investigated by using the hydrolysis reaction catalysed by thermolysin. Because this hydrolysis reaction was highly selective for L-amino acids, only the terminal L-amino acid was released by enzymatic hydrolysis of a mixture of L,D and L dipeptides that was immobilised on the support, leaving only the L,D dipeptide on the solid support [65]. Lipases, another important class of hydrolases that are frequently used in biocatalysis, have been used to catalyse

Review kinetic resolution of chiral carboxylic acids via a ‘capture and release’ strategy [66]. Cyclohexane 1,3-dione (CHD) was first loaded onto PEGA resin, where it then reacted with a racemic mixture of vinyl esters of 3-phenylbutyric acid in the presence of Chromobacterium viscosum lipase. Once the favored enantiomer of the ester was captured on the resin by enzymatic esterification, treatment of the beads with sodium hydroxide allowed the release and recovery of the corresponding acid. Surprisingly, the reaction afforded the (R)-acid as the predominant isomer, with enantiomeric excess ranging from 59% to 99%, despite this enzyme’s commonly found selectivity for S-esters. Nanda et al. also reported the use of a lipase from porcine pancreas to separate enantiomers of carboxylic acids [67]. In this experiment, a racemic mixture of trans-substituted cyclopropane carboxylic acids was coupled to resin beads. The lipase was then applied to cleave the (R)-acid, which was obtained after separation from the reaction mixture with enantiomeric excess (ee) ranging from 80% to 94%. A chemical treatment of the resin after enzymatic reaction also afforded the (S)-acid enantiomer with similar ee. Surface modification Solid surfaces that are functionalised with grafted polymers have found a wide range of applications in nanotechnologies as biosensors or biocompatible materials. Enzymes can be directed to specific locations on the surface when they are immobilised on scanning probe microscopes to yield surface patterns with nanometer precision. For example, Staphyloccocal serine V8 protease that had been immobilised to an atomic force microscopy (AFM) tip was used to cleave glutamic- or aspartic-acid-containing peptides on a mica surface to create a square shape of 5 mm2 [68]. Dip-pen lithography was able to deliver DNase to an oligonucleotide self-assembled monolayer on gold [69], and in another approach, trypsin was successfully used to create grooves and channels in a film of BSA (bovine serum albumin) via a so-called nano fountain pen [70]. An enzymatic surface-initiated polymerisation (SIP) has been developed for the synthesis of biocompatible polymers of poly(3-hydroxybutyrate) as an alternative to traditional SIP, which involves radical, cationic, anionic or metal alkoxide-based chemical reactions [71]. Polymer films of polyhydroxyalkanoates (PHAs) are generated by a variety of microorganisms [72] and have been used for the development of biodegradable and biocompatible polymers. PHA-synthase, the enzyme involved in the biosynthesis of PHAs, was used to modify a surface of silicon with poly(3-hydroxybutyrate) polymer. Initiation of the polymerisation was achieved under physiological conditions and in a highly controlled manner. Analysis by AFM showed that the surface was coated with a film 200 nm in thickness. By employing several PHAsynthases, it is possible to produce tailored biomaterials that are useful in a wide range of applications, such as drug delivery, tissue engineering or surgical sutures. Enzymatic polymerisation could also be applied for the generation of oligomers of ortho-methoxyphenols on solidsupported substrates by the enzyme soybean peroxidase (Figure 4b) [73]. In this study, the peroxidaze initiated the polymerisation by oxidation of apocynin (acetovanillone) in

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solution. The oxidised apocynin radical then underwent radical transfer with a polymer-bound hydroquinone to yield a carbon-centered radical, followed by radical coupling with another apocynin monomer resulting in the formation of a diphenolic resin-bound molecule and, ultimately, higher order oligomers on the support. This result represented the first example of enzymatic carbon– carbon bond formation on solid support and expanded the repertoire of enzymatic catalysis available in combinatorial library synthesis. Enzymatic immobilisation of proteins onto a b-caseincoated surface has recently been described as an alternative route for the design and preparation of novel functional biomaterials or protein arrays [74]. The strategy took advantage of the ability of microbial transglutaminase (MTG) to catalyse cross-linking reactions. MTG catalyses an acyl transfer reaction between the g-carboxyamide of the glutamine residue and a variety of primary amines, including the e-amino group of lysine, with an associated loss of ammonia. Recombinant enhanced green fluorescent protein (EGFP) and glutathione S-transferase (GST), which were tagged with a glutamine peptide at the C-termini, were immobilised onto a b-casein-coated surface that provided the e-amino acceptor group. However, this immobilisation technique, which enabled functionalisation of biomaterials with high efficiency and great precision under smooth conditions, required the introduction of a special tag for the cross-linking reaction and furthermore, as in the case of EGFP, an additional linker that would provide the necessary flexibility to overcome steric hindrance. An additional complication arose from the fact that the C-terminal lysine residue of EGFP was recognised by the MTG enzyme and therefore had to be substituted to an arginine by site-directed mutagenesis. Similarly, one of the five glutamine residues in the wildtype GST was also recognised by the MTG and had to be mutated to an alanine before successful protein immobilisation. Although the sequence changes in these proteins did not cause any loss of native function, they nevertheless constituted additional undesirable steps in the protocol. Conclusion A wide range of biochemical reactions can now be performed on substrates that are linked to a polymer support, which includes hydrolysis and formation of the major biopolymers – nucleic acids, peptides and carbohydrates, but also more general reactions such as C-C bond formations and transesterifications. These examples have demonstrated proof-of-principle and have also established the type of solid support that is compatible with biocatalysis – in particular, polyethylene-glycol-coated surfaces have been very successful, either as a copolymer such as in PEGA (Table 1) or as coats for self-assembled monolayers on gold surfaces and nanoparticles. Several direct analysis methods for monitoring these enzymatic reactions have also been developed and have demonstrated that these enzymatic reactions can be achieved with very high yields. Most recently, biocatalysis on solid support has been used in some very elegant applications for developing enzyme-responsive surfaces and microarrays. With the groundwork now established, we should see many more 335

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