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Fungal peroxygenases (UPOs) have emerged as oxyfunctionalization catalysts of tremendous interest in recent years. However, their widespread use in the field of biocatalysis is still hampered by their challenging heterologous production, substantially limiting the panel of accessible enzymes for investigation and enzyme engineering. Building upon previous work on UPO production in yeast, we have developed a combined promoter and signal peptide shuffling system for episomal high throughput UPO production in the industrially relevant, methylotrophic yeast Pichia pastoris. Eleven endogenous and orthologous promoters were shuffled with a diverse set of 17 signal peptides. Three previously described UPOs were selected as first test set, leading to the identification of beneficial promoter/signal peptide combinations for protein production. We applied the system then successfully to produce two novel UPOs: MfeUPO from Myceliophthora fergusii and MhiUPO from Myceliophthora hinnulea. To demonstrate the feasibility of the developed system to other enzyme classes, it was applied for the industrially relevant lipase CalB and the laccase Mrl2. In total, approximately 3200 transformants of eight diverse enzymes were screened and the best promoter/signal peptide combinations studied at various cofeeding, derepression, and induction conditions. High volumetric production titers were achieved by subsequent creation of stable integration lines and harnessing orthologous promoters from Hansenula polymorpha. In most cases promising yields were also achieved without the addition of methanol under derepressed conditions. To foster the use of the episomal high throughput promoter/signal peptide Pichia pastoris system, we made all plasmids available through Addgene.

Unspecific peroxygenases (UPOs) enable oxyfunctionalizations of a broad substrate range with unparalleled activities. Tailoring these enzymes for chemo- and regioselective transformations represents a grand challenge due to the difficulties in their heterologous productions. Herein, we performed protein engineering in Saccharomyces cerevisiae using the MthUPO from Myceliophthora thermophila. More than 5300 transformants were screened. This protein engineering led to a significant reshaping of the active site as elucidated by computational modelling. The reshaping was responsible for the increased oxyfunctionalization activity, with improved kcat/Km values of up to 16.5-fold for the model substrate 5-nitro-1,3-benzodioxole. Moreover, variants were identified with high chemo- and regioselectivities in the oxyfunctionalization of aromatic and benzylic carbons, respectively. The benzylic hydroxylation was demonstrated to perform with enantioselectivities of up to 95% ee. The proposed evolutionary protocol and rationalization of the enhanced activities and selectivities acquired by MthUPO variants represent a step forward toward the use and implementation of UPOs in biocatalytic synthetic pathways of industrial interest M.J.W., A.K., and N.H. thank the Bundesministerium für Bildung und Forschung (“Biotechnologie 2020+ Strukturvorhaben: Leibniz Research Cluster”, 031A360B) for generous funding. P.P. thanks the Landesgraduiertenförderung Sachsen- Anhalt for a Ph.D. scholarship. M.G.-B. thanks the Generalitat de Catalunya AGAUR for a Beatriu de Pinós H2020 MSCACofund 2018-BP-00204 project and the Spanish MICINN (Ministerio de Ciencia e Innovación) for PID2019-111300GAI00 project, and J.S. thanks the Spanish MIU (Ministerio de Universidades) for a predoctoral FPU fellowship FPU18/ 02380. The computer resources at MinoTauro and the Barcelona Supercomputing Center BSC-RES are acknowledged (RES-QSB-2019-3-0009 and RES-QSB-2020-2-0016)

Fungal unspecific peroxygenases (UPOs) represent an enzyme class catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and rely on hydrogen peroxide as the oxygen source. However, their heterologous production in a fast-growing organism suitable for high throughput screening has only succeeded once—enabled by an intensive directed evolution campaign. We developed and applied a modular Golden Gate-based secretion system, allowing the first production of four active UPOs in yeast, their one-step purification and application in an enantioselective conversion on a preparative scale. The Golden Gate setup was designed to be universally applicable and consists of the three module types: i) signal peptides for secretion, ii) UPO genes, and iii) protein tags for purification and split-GFP detection. The modular episomal system is suitable for use in Saccharomyces cerevisiae and was transferred to episomal and chromosomally integrated expression cassettes in Pichia pastoris. Shake flask productions in Pichia pastoris yielded up to 24 mg/L secreted UPO enzyme, which was employed for the preparative scale conversion of a phenethylamine derivative reaching 98.6 % ee. Our results demonstrate a rapid, modular yeast secretion workflow of UPOs yielding preparative scale enantioselective biotransformations., Püllmann et al developed a modular Golden Gate-based secretion system, which enabled production and one-step purification of active fungal unspecific peroxygenases (UPOs) in yeast. Their system was applied to an enantioselective conversion on a preparative scale and may be used in the future for other genes of interest that are suitable for production in yeast.

A bioinspired emulsification strategy resulted in substantially increased enzymatic activities in the degradation of synthetic polyisoprene with the latex clearing protein LcpK30, even with cis : trans ratios of 56 : 27.

The oxidation of alkanes into valuable chemical products is a vital reaction in organic synthesis. This reaction, however, is challenging due to the inertness of C-H bonds. Transition metal catalysts for C-H functionalization are frequently explored. Despite chemical alternatives, nature has also evolved powerful oxidative enzymes (e.g. methane monooxygenases, cytochromeP450 oxygenases, peroxygenases) that are capable of transforming C-H bonds under very mild conditions, with only the use of molecular oxygen or hydrogen peroxide as electron acceptors. Although the progress of alkane oxidation has been reviewed extensively, little attention has been paid to small alkane oxidation. The latter holds great potential for the manufacture of chemicals. In this contribution, we therefore will give a concise overview of the most relevant enzyme classes capable of small alkanes (

Fungal peroxygenases (UPOs) have emerged as oxyfunctionalization catalysts of tremendous interest in recent years. However, their widespread use in the field of biocatalysis is still hampered by their challenging heterologous production, substantially limiting the panel of accessible enzymes for investigation and enzyme engineering. Building upon previous work on UPO production in yeast, we have developed a combined promoter and signal peptide shuffling system for episomal high throughput UPO production in the industrially relevant, methylotrophic yeast

A high throughput GC-MS approach was developed, permitting the simultaneous analysis of up to three substrates and six products quantitatively from one reaction mixture. This screening approach was applied to site-saturation libraries of the novel unspecific peroxygenaseMthUPO. Using this setup enabled substantial insights from a small mutant library. Enzyme variants were identified exhibiting selective alkene epoxidation and substantially shifted regioselectivities to 2- and 1-octanol formations. Computational modelling rationalised the observed selectivity changes M. J. W, A. K., and N. H. thank the Bundesministerium für Bildung und Forschung (“Biotechnologie 2020+ Strukturvorhaben: Leibniz Research Cluster”, 031A360B) for generous funding. P. P. thanks the Landesgraduiertenförderung Sachsen-Anhalt for a PhD scholarship. The authors thank Eugen Schell for fruitful discussions. M. G. B. thanks the Generalitat de Catalunya AGAUR for a Beatriu de Pinós H2020 MSCA-Cofund 2018-BP-00204 project, the Spanish MICINN (Ministerio de Ciencia e Innovación) for PID2019-111300GA-I00 project, and J. S. thanks the Spanish MIU (Ministerio de Universidades) for a predoctoral FPU fellowship FPU18/02380. The computer resources at MinoTauro and the Barcelona Supercomputing Center BSC-RES are acknowledged (RES-QSB-2019-3-262 0009 and RES-QSB-2020-2-0016)

Fungal Peroxygenases (UPOs) have emerged as oxyfunctionalization catalysts of tremendous interest in recent years. However, their widespread use in the field of biocatalysis is still hampered by their challenging heterologous production, substantially limiting the panel of accessible enzymes for investigation and enzyme engineering. Building upon previous work on UPO production in yeast, we have developed a combined promoter and -signal peptide shuffling system for episomal high throughput UPO production in the industrially relevant, methylotrophic yeast Pichia pastoris. 11 endogenous and orthologous promoters were shuffled with a diverse set of 17 signal peptides. Three previously described UPOs were selected as first test set, leading to the identification of beneficial promoter/signal peptide combinations for protein production. We applied the system then successfully to produce two novel UPOs: MfeUPO from Myceliophthora fergusii and MhiUPO from Myceliophthora hinnulea. To demonstrate the feasibility of the developed system to other enzyme classes, it was applied for the industrially relevant lipase CalB and the laccase Mrl2. In total, approximately 3200 transformants of eight diverse enzymes were screened and the best promoter/signal peptide combinations studied at various co-feeding, derepression and induction conditions. High volumetric production titers were achieved by subsequent creation of stable integration lines and harnessing orthologous promoters from Hansenula polymorpha. In most cases promising yields were also achieved without the addition of methanol under derepressed conditions. To foster the use of the episomal high throughput promoter/signal peptide Pichia pastoris system, we made all plasmids available through Addgene.

Fungal unspecific peroxygenases (UPOs) are biocatalysts of outstanding interest. Providing access to novel UPOs using a modular secretion system was the central goal of this work. UPOs represent an enzyme class, catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and solely rely on hydrogen peroxide as the oxygen source. Fungal peroxygenases are widespread throughout the fungal kingdom and hence a huge variety of UPO gene sequences is available. However, the heterologous production of UPOs in a fast-growing organism suitable for high throughput screening has only succeeded once—enabled by an intensive directed evolution campaign. Here, we developed and applied a modular Golden Gate-based secretion system, allowing the first yeast production of four active UPOs, their one-step purification and application in an enantioselective conversion on a preparative scale. The Golden Gate setup was designed to be broadly applicable and consists of the three module types: i) a signal peptide panel guiding secretion, ii) UPO genes, and iii) protein tags for purification and split-GFP detection. We show that optimal signal peptides could be selected for successful UPO secretion by combinatorial testing of 17 signal peptides for each UPO gene. The modular episomal system is suitable for use in Saccharomyces cerevisiae and was transferred to episomal and chromosomally integrated expression cassettes in Pichia pastoris. Shake flask productions in Pichia pastoris yielded up to 24 mg/L secreted UPO enzyme, which was employed for the preparative scale conversion of a phenethylamine derivative reaching 98.6 % ee. Our results demonstrate a rapid workflow from putative UPO gene to preparative scale enantioselective biotransformations.

Electrochemical in vitro reduction of P450 enzymes is a promising alternative to in vivo applications. Previously we presented three engineered P450 BM3 variants for aniline hydroxylation, equipped with a carbon nanotube binding-peptide (CNT-tag) for self-assembly on CNT electrodes. Compared to wildtype P450 BM3 the NADPH-dependent activity was enhanced, but the coupling efficiency remained low. For P450 BM3 Verma, Schwaneberg and Roccatano (2014, Biopolymers 101, 197–209) calculated putative electron transfer pathways (eTPs) by MD simulations. We hypothesised that knockouts of these transfer pathways would alter the coupling efficiency of the system. The results revealed no improved system for the electrically-driven P450s. For the NADPH-driven P450s, however, the most active eTP-mutant showed a 13-fold increased activity and a 32-fold elevated coupling efficiency using NADPH as reducing equivalent. This suggests an alternative principle of electron transport for the reduction by NADPH and an electrode, respectively. The work presents moreover a tool to improve the coupling and activity of P450s with non-natural substrates.

Fungal peroxygenases represent an exciting new enzyme class for stereo - selective hydroxylation reactions. They are capable of the oxyfunctionalisation of a large, diverse scope of substrates including alkanes and steroids as well as the heteroatoms sulfur and nitrogen. The outstanding activities and stabilities as well as their reliance on hydrogen peroxide as co-substrate renders it a highly interesting biocatalyst.

Directed evolution requires the screening of enzyme libraries in biological matrices. Available assays are mostly substrate or enzyme specific. Chromatographic techniques like LC and GC overcome this limitation, but require long analysis times. The herein developed multiple injections in a single experimental run (MISER) using GC coupled to MS allows the injection of samples every 33 s resulting in 96-well microtiter plate analysis within 50 min. This technique is implementable in any GC-MS system with autosampling. Since the GC-MS is far less prone to ion suppression than LCMS, no chromatographic separation is required. This allows the utilisation of an internal standards and the detection of main and side-product. To prove the feasibility of the system in enzyme screening, two libraries were assessed: i) YfeX library in an E. coli whole cell system for the carbene-transfer reaction on indole revealing the novel axial ligand tryptophan, ii) a library of 616 chimeras of fungal unspecific peroxygenase (UPO) in S. cerevisiae supernatant for hydroxylation of tetralin resulting in novel constructs. The data quality and representation are automatically assessed by a new R-script.

The structure of a P450 ω-hydroxylase bound to its fatty acid product was determined, which revealed a narrow substrate tunnel that leads to the heme. The introduction of an arginine side chain in proximity to the carboxyl group of the fatty acid led to a reduced KM value for dodecanoic acid, which suggests the importance of an anchoring point in the active site. An increase in the flexibility of the substrate recognition region was also engineered, which resulted in a threefold improved product formation.

The native redox partners of many novel cytochrome P450 monooxygenases (CYPs) are unknown. Therefore, they are combined with non-native redox partners to obtain catalytically active systems. Understanding the CYP-redox partner interactions is the basis of successful protein engineering. Six redox partner interaction sites (RPISs) were identified by systematic literature, sequence, and structure analyses. All six RPISs are proposed to contribute to class II CYP-redox partner interaction interface, whereas four and five contribute to the interaction interface in class I prokaryotic and mitochondrial CYPs, respectively. The significance of the identified RPISs was tested by designing seven variants of CYP153 A (class I) as a fusion protein with its non-native redox partner CYP102 A1 reductase (class II) and measuring electron coupling efficiencies with a precision of 1–3 %. The best variant K166Q had an improved electron coupling efficiency of 64 % as compared to 53 % for the wild type.

The Wittig-type carbonyl olefination reaction has no biocatalytic equivalent. To build complex molecular scaffolds, however, C−C bond-forming reactions are pivotal for biobased economy and synthetic biology. The heme-containing E. coli protein YfeX was found to catalyze carbonyl olefination by reaction of benzaldehyde with ethyl diazoacetate under aerobic conditions in the absence of a triphenylphosphine oxophile. The reaction was performed in whole cells and showed a product formation of 440 mg L−1 in 1 h. It was, moreover, shown that the reaction could be performed under Wittig-analogue conditions in the presence of triphenylphosphine or triphenylarsine.

Three different reductases have been fused to CYP153 monooxygenase from Marinobacter aquaeolei. The most promising candidate has been analysed in terms of its linker part, which connects the reductase with the haem domain through sequence alignment of the corresponding reductase family CYP116B. To improve the artificial fusion construct, the linker length has been varied, thereby only altering the non-conserved middle part of the linker. This way seven artificial fusion constructs have been engineered, which varied in linker length between 11 and 32 amino acids (“natural” is 16). These variations showed a substantial impact on the fusion construct. The best mutant, extended by two amino acids, showed an improved activity (67 %), higher stability (67 % more active haem domain after 2 h) and a coupling efficiency of 94 % (55 % higher than before). Presented in this paper is an approach to find and optimise artificial fusion constructs for P450 monooxygenases.

The screening effort of large protein variant libraries renders the probability of coincidental discovering a new enzyme with non-natural activity to almost zero - the so-called numbers problem. Insights into the origin of life, evolution and enzymatic promiscuity, combined with the inspiration of methods from organic chemistry, offer solutions for this problem. With the newly discovered enzymes synthetic micro production units shall be established in a Leibniz Research Cluster where engineering and biotechnology are combined.

The functionalization of C-H bonds with non-precious metal catalysts is an important research area for the development of efficient and sustainable processes. Herein, we describe the development of iron porphyrin catalyzed reactions of diazoacetonitrile with N-heterocycles yielding important precursors of tryptamines, along with experimental mechanistic studies and proof-of-concept studies of an enzymatic process with YfeX enzyme. By using readily available FeTPPCl, we achieved the highly efficient C-H functionalization of indole and indazole heterocycles. These transformations feature mild reaction conditions, excellent yields with broad functional group tolerance, can be conducted on gram scale, and thus provide a unique streamlined access to tryptamines.

Site-directed methods for the generation of genetic diversity are essential tools in the field of directed enzyme evolution. The Golden Gate cloning technique has been proven to be an efficient tool for a variety of cloning setups. The utilization of restriction enzymes which cut outside of their recognition domain allows the assembly of multiple gene fragments obtained by PCR amplification without altering the open reading frame of the reconstituted gene. We have developed a protocol, termed Golden Mutagenesis that allows the rapid, straightforward, reliable and inexpensive construction of mutagenesis libraries. One to five amino acid positions within a coding sequence could be altered simultaneously using a protocol which can be performed within one day. To facilitate the implementation of this technique, a software library and web application for automated primer design and for the graphical evaluation of the randomization success based on the sequencing results was developed. This allows facile primer design and application of Golden Mutagenesis also for laboratories, which are not specialized in molecular biology.

A readily available galactose oxidase (GOase) variant was used to develop a whole cell screening assay. This endpoint detection system was applied in a proof-of-concept approach by screening a focussed mutant library. This led to the discovery of the thus far most active P450 Marinobacter aquaeolei mutant catalysing the terminal hydroxylation of fatty acids.

The facile synthesis of rifamycin S from rifamycin B, a member of the ansamycin family of antibiotics, via the oxidation of rifamycin B was developed. Currently on an industrial scale, this oxidation is performed using harsh pH conditions and chlorinated solvents. With the development of a suitable buffer/methanol system, a similar yield and space-time-yield in comparison to the current process can be obtained renouncing chlorinated solvents. Employment of methanol as a reaction medium in this process is crucial for attaining high yields under mild reaction conditions. With this method a space-time-yield of 189 g L–1 h–1 of rifamycin S was achieved in one step.

Background Galactose oxidase (GOase) catalyses the highly selective oxidation of terminal galactosides on a wide range of natural glycoconjugates and has found wide applications in biotechnology - particularly in biocatalysis. GOase is copper dependent and uses oxygen to oxidise the C6-primary alcohol of galactose and produces hydrogen peroxide. The enzyme activity can be conveniently assessed by a colorimetric assay. Objectives The objective of the present study was to develop an assay system, which is independent of the hydrogen peroxide formation to identify possible fluorinated GOase inhibitors. In case that the inhibitor bears a primary or secondary alcohol, it could also be oxidised by the enzyme. In such case, the colorimetric assay is not able to distinguish between substrate and inhibitor, since oxidation of both molecules would result in the formation of hydrogen peroxide. Methods D-galactose (D-Gal) was immobilised onto a gold surface functionalised by selfassembled monolayers (SAMs,). A GOase solution was then added to the surface in a droplet for a certain period of time and thereafter washed away. The activity of GOase on the immobilised D-Gal can then be quantified by MALDI-ToF MS. Results For inhibition studies, GOase was incubated together with 62.5 mM of deoxy-fluorinated monosaccharides on the D-Gal displaying platform. Five deoxy-fluorinated D-Gal showed a g50% inhibition of its activity. The array system has been moreover utilised to determine the apparent IC50 value of 3-F-Gal 15 as a proof of principle. Conclusion The developed array platform allows the fast identification of GOase substrates and inhibitors from a library of deoxy-fluorinated sugars using MALDI-ToF MS as a label-free readout method. In addition, the enzymatic reaction enables for the in situ activation of sugar-coated surfaces to bioorthogonal aldehydes, which can be utilised for subsequent chemical modifications.

Electrochemical in vitro reduction of P450 enzymes is a promising alternative to in vivo applications. Previously we presented three engineered P450

CD73 is a dimeric ecto-5'-nucleotidase that is expressed on the exterior side of the plasma membrane. CD73 has important regulatory functions in the extracellular metabolism of certain nucleoside monophosphates, in particular adenosine monophosphate, and has been linked to a number of pathological conditions such as cancer and myocardial ischaemia. Here, we present the crystal structure of a soluble form of human soluble CD73 (sCD73) at 2.2 Å resolution, a truncated form of CD73 that retains ecto-5'-nucleotidase activity. With this structure we obtained insight into the dimerisation of CD73, active site architecture, and a sense of secondary modifications of the protein. The crystal structure reveals a conserved loop that is directly involved in the dimer-dimer interaction showing that the two subunits of the dimer are not linked by disulfide bridges. Using biophotonic microarray imaging we were able to confirm glycosylation of the enzyme and show that the enzyme is decorated with a variety of oligosaccharide structures. The crystal structure of sCD73 will aid the design of inhibitors or activator molecules for the treatment of several diseases and prove useful in explaining the possible roles of single nucleotide polymorphisms in physiology and disease.

O-mannosyl glycans are known to play an important role in regulating the function of α-dystroglycan (α-DG), as defective glycosylation is associated with various phenotypes of congenital muscular dystrophy. Despite the well-established biological significance of these glycans, questions regarding their precise molecular function remain unanswered. Further biological investigation will require synthetic methods for the generation of pure samples of homogeneous glycopeptides with diverse sequences. Here we describe the first total syntheses of glycopeptides containing the tetrasaccharide NeuNAcα2-3Galβ1-4GlcNAcβ1-2Manα, which is reported to be the most abundant O-mannosyl glycan on α-DG. Our approach is based on biomimetic stepwise assembly from the reducing end and also gives access to the naturally occurring mono-, di-, and trisaccharide substructures. In addition to the total synthesis, we have developed a "one-pot" enzymatic cascade leading to the rapid synthesis of the target tetrasaccharide. Finally, solid-phase synthesis of the desired glycopeptides directly on a gold microarray platform is described.

The enzymatic glycosylation of microarrays is a relatively young field in glycoscience. Platforms developed from other array technologies (e.g., proteins and nucleic acids) were successfully adopted in several proof-of-principle studies as a high-throughput tool for the generation of more complex carbohydrate structures using carbohydrate-processing enzymes. These arrays and the developed on-chip enzymatic glycosylation methodologies are reviewed in this article.

The synthesis of a number of aminoethyl glycosides of cell-surface carbohydrates, which are important intermediates for glycoarray synthesis, is described. A set of protocols was developed which provide these intermediates, in a short number of steps, from commercially available starting materials.

Lectin binding has been studied using the particle plasmon light-scattering properties of gold nanoparticles printed into an array format. Performance of the kinetic assay is evaluated from a detailed analysis of the binding of concanavalin A (ConA) and wheat germ agglutinin (WGA) to their target monosaccharides indicating affinity constants in the order of KD ∼10 nM for the lectin-monosaccharide interaction. The detection limits for the lectins following a 200 s injection time were determined as 10 ng/mL or 0.23 nM and 100 ng/mL or 0.93 nM, respectively. Subsequently, a nine-lectin screen was performed on the porcine and human fibrinogen glycoproteins. The observed spectra of lectin-protein specific binding rates result in characteristic patterns that evidently correlate with the structure of the glycans and allow one to distinguish between glycosylation of the porcine and human fibrinogens. The array technology has the potential to perform a multilectin screen of large numbers of proteins providing information on protein glycosylation and their microheterogeneity.

High-throughput microarray technology has been combined with ultrasensitive and high-resolution tritium autoradiography to create a new platform for the quantitative detection of glycosyltransferase activity on glycan arrays. In addition, we show full compatibility with the use of fluorescently labeled lectins to help with the stereochemical assignment of newly formed glycoside linkages.

This review gives an overview of enzymatic reactions that have been conducted on substrates attached to solid surfaces. Such biochemical reactions have become more important with the drive to miniaturisation and automation in chemistry, biology and medicine. Technical aspects such as choice of solid surface and analytical methods are discussed and examples of enzyme reactions that have been successful on these surfaces are provided.

Glycans functionalised with hydrophobic trityl groups were synthesised and adsorbed onto polystyrene and glass slides in an array format. The adsorbed glycans could be analysed directly on these minimally conducting surfaces by MALDI-TOF mass spectrometry analysis after aluminium tape was attached to the underside of the slides. Furthermore, the trityl group appeared to act as an internal matrix and no additional matrix was necessary for the MS analysis. Thus, trityl groups can be used as simple hydrophobic, noncovalently linked anchors for ligands on surfaces and at the same time facilitate the in situ mass spectrometric analysis of such ligands.

We report a highly efficient and selective method for the coupling of peptides and glycoconjugates bearing N-terminal cysteines to activated surfaces. This chemoselective method generates stable amide linkages without using any thiol additives.

There is a wide range of immobilisation reactions to tether substrates to a variety of surfaces for array-based analysis. Most of these immobilisation strategies are specific for a particular surface and require an additional linker to be attached to the substrate or the surface. Furthermore, the analysis of functionalised surfaces is often restricted to certain analytical techniques and therefore, different immobilisation strategies for different surfaces are desirable. Here we have tested an S-tritylated linker for non-covalent or covalent immobilisation of mannosides to polystyrene or gold surfaces. S-Tritylated mannosides with varying linkers were readily synthesised and used to add to biorepulsive maleimide-terminated preformed SAMs after in situ deprotection of the S-trityl group. In addition, S-tritylated mannosides themselves formed stable glycoarrays on polystyrene microtiter plates. The glycoarrays were successfully analysed by MALDI-ToF mass spectrometry, SPR spectroscopy, and interrogated with GFP-transfected Escherichia coli cells. This work has shown that a dual purpose linker can be used on multiple surfaces to form arrays allowing for different testing as well as analytical approaches.