Faculty of Health and Life Sciences

Master Thesis

Τοwards a Synthetic Tryptophan Aminotransferase

Author: Kleomenis Tsimpos Supervisor: Dr Suppan Kathiravan & Dr Jesper G. Wiklander Examiner: Prof. Ian Nicholls Date: 30 August 2017 Level:Master of Science Course code: 5KE02E

Abstract

The synthesis and evaluation of a molecularly imprinted polymer has been undertaken using an oxazine-based tryptophanamide transition state analogue (TSA) as template. An efficient route to the synthesis of oxazine-based TSAs for the reaction of pyridoxamine and indole-3-pyruvic acid has been established, with yields of up to 80%. NMR titration studies were performed to examine the interactions between the functional monomer, methacrylic acid and the template. Complexation of the template by functional monomer in the presence of crosslinker showed an apparent KD of 0.63-0.79 ± 0.04 M (293 K, acetonitrile-d3) based upon the chemical shift of the template amide protons. TSA-imprinted and non-imprinted reference polymers were synthesized by free radical polymerization in acetonitrile. Polymer monoliths were ground and fractionated into a 25-63 μm size range. Polymer-ligand recognition studies were conducted using the polymers as HPLC stationary phases. An imprinting factor (IF) of 2.93 was observed for the TSA, indicating the selectivity of the imprinted sites for the template. Studies using the D- and L-enantiomers of the phenylalaninamide analogue of the template showed enantioselectivity in the case of the imprinted polymer, α = 1.10, though not in the case of the non-imprinted reference polymer (1.00). Using UV-spectroscopy based polymer-ligand binding studies, a maximum -1 theoretical capacity (Bmax) of 0.059 ± 0.004 mmol·g was observed for the imprinted polymer. Conclusively, an imprinted polymer with binding sites selective for the TSA was successfully prepared and shall subsequently be studied with respect to its capacity to catalyse the transamination reaction between pyridoxamine and indole-3-pyruvic acid to yield pyridoxal and tryptophan.

Keywords aminotransferase; biomimetic; catalysis; catalyst; molecular imprinting; molecularly imprinted polymer; transition state analogue.

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Acknowledgments

First of all, I would like to thank professor Ian Nicholls for his great support and help for the past two years. He gave me the opportunity to be a part of his research group, which has given me more academic experience. I also want to thank him for helpful discussions about the project.

I would further like to thank my two supervisors Dr Jesper G. Wiklander and Dr Suppan Kathiravan. Their doors were always open whenever I had questions about my writing, or had troubles with my experimental work. They taught me so many things about my project and I gained so much experience working with them.

This study was funded by the Linnaeus University and the financial support is most gratefully acknowledged.

I would also like to thank Dr Gustaf Olsson. He was always there for me when I had theoretical questions about my project, and he also supported me psychologically when I needed it.

Of course, I want to thank Dr Panagiota Georgoulia. She contributed to the experimental work of my project. Also she has been an amazing friend for the past two years. She was there when I felt down and supported me. Panagiota thank you for your patience.

Lastly, I want to thank all my friends that they were around all those years supporting me and making this journey pleasant with amazing memories. I will not mention names. They know. Also so many thanks to my Father, mother and sister that they have always been there for me.

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Contents

1 Introduction ______1 1.1 Amino acid biosynthesis ______1 1.2 Artificial transaminases ______3 1.3 Molecular imprinting ______4 1.3.1 Molecular imprinting - applications ______5 1.3.2 Molecularly imprinted polymers in synthesis and catalysis ______6 1.4 Objectives of the thesis ______9

2 Materials & Methods ______10 2.1 General procedures ______10 2.2 Synthesis of transition state analogues ______10 2.2.1 L-2-(3-Hydroxy-5-hydroxymethyl-2-methyl-pyridin-4-yl-methyl)amino-N- phenylalaninamide (1a) ______10 2.2.2 L-2-(5-Hydroxymethyl-8-methyl-3.4-dihydropyrido[4.3-e]-1.3-oxazin-3- yl)phenylalaninamide (1b) ______11 2.2.3 D-2-(3-Hydroxy-5-hydroxymethyl-2-methyl-pyridin-4-yl-methyl)amino-N- phenylalaninamide (2a) ______12 2.2.4 D-2-(5-Hydroxymethyl-8-methyl-3.4-dihydropyrido[4.3-e]-1.3-oxazin-3- yl)phenylalaninamide (2b) ______12 2.2.5 L-2‐{[(2‐hydroxyphenyl)methyl]amino}‐3‐(1H‐indol‐3‐yl)propanamide (3a) ______13 2.2.6 L-2-({[2-hydroxy-6-(hydroxymethyl)-3-methylphenyl]methyl}amino)-3-(1H- indol-3-yl)propanamide (4a) ______13 2.2.7 L-2-[5-(hydroxymethyl)-8-methyl-2H,3H,4H-pyrido[4,3-e][1,3]oxazin-3-yl]- 3-(1H-indole-3-yl)propanamide (L-TSA) (4b) ______14 2.3 Polymer synthesis ______15 2.4 1H NMR-titrations ______15 2.5 Polymer recognition ______15 2.6 Polymer-ligand rebinding studies ______15

3 Results and Discussion ______16 3.1 Evaluation and synthesis of transition state analogues ______16 3.2 Polymer synthesis and analysis ______17 3.3 1H NMR-titrations, functional monomers interactions ______18 3.4 Polymer-ligand recognition studies ______20 3.5 Polymer-ligand rebinding studies ______22

4 Conclusion and Future ______23

References ______25

Appendices ______I

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1 Introduction

1.1 Amino acid biosynthesis

The α-amino acids are responsible for important functions in living systems. They are fuels and key intermediates in metabolism and they also constitute the building blocks of proteins and therefore are essential to virtually every biological process; from enzyme catalysis and cell-signalling to storage, transport and defense.1,2 Some 240 can be found in nature, though only 20 are encoded by the genetic code.3 Therefore these 20 amino acids are very important for the human body to grow and function properly and they are divided into two groups: the “essential” and the “non-essensial” amino acids. The nine essential amino acids cannot be produced by the body and they must be obtained from the diet. On the other hand, the non-essential amino acids can be produced by cells from metabolic intermediates in the human body. Accordingly, the importance of amino acid synthesis in biology has been of long standing interest, and approaches for the synthesis of amino acids found in biology or those of use in the synthesis of fine chemicals for other purposes, most often for use in agricultural, materials or medical contexts, has motivated the development of new approaches to their synthesis.

Figure 1. General structure of an amino acid.

Amino acids are both amines and acids. They are organic molecules containing one amino (-NH2) group, one carboxyl (-COOH) and one side-chain (-R group), figure 1. In biochemistry, amino acids having both the (-NH2) group and the (-COOH) group connected to the α-carbon (alpha carbon) are known as α-amino acids.2

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Transamination is one of the most important biological reactions and occurs when the amino group from an α-amino acid is transferred to an α-keto acid to form a new amino acid.4 Biological transamination is catalyzed by enzymes, called transaminases or aminotransferases, using pyridoxal 5΄-phosphate (PLP) or pyridoxamine 5΄-phosphate (PMP) as coenzymes.5,6 The coenzymes can act without the presence of an enzyme, but studies have shown that these reactions are much slower.7 PLP and PMP are also responsible for the catalysis of other reactions such as decarboxylation, aldol condensation and racemization.8 The unique properties of PLP and PMP in nature, have led to them being the subject of intense research and many efforts have been made to synthesize enzyme mimics using these cofactors.9 A complete cycle of the biological transamination reaction includes two half transaminations, figure 2. First PMP reacts with an α-keto acid to form a Schiff base. In the second step the Schiff base tautomerizes and cleaves to yield PLP and the amino acid. The reaction is reversed, using a second different α-amino acid to regenerate PMP forming a new α-keto acid thus completing the catalytic cycle.4,7,10,11 Developing enzyme mimics to imitate the biological transamination for the synthesis of α-amino acids is very important in the biochemisty field and offers potential in the synthesis of non-natural amino acids.4

Figure 2. A complete biological transamination of α-keto acids. In the first half-transamination, PMP transfers its NH2 group to α-keto acid 1 forming a Schiff base, which after hydrolysis yields α-amino acid 2 and PLP. In the second half-transamination a different α-amino acid 3 acts as the amino donor and reacts with PLP to regenerate PMP, forming a new α-keto acid 4, thus completing the cycle. [Adapted from Shi et al.]4

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1.2 Artificial transaminases

Natural enzymes (also known as biological catalysts) are most often macromolecular systems, e.g. proteins or oligonucleotides with particular structures, that help chemical reactions to proceed rapidly in living organisms.12 Enzymes found in nature accelerate the rate of chemical reactions and play an important role in the human body. They are responsible for various tasks in our body such as digestion (breaking down food), respiration, and responsible for destroying toxins. In the case of proteins with catalytic function, generally referred to as enzymes, they possess binding sites, often referred to as the active sites, that can accommodate specific substrates and speed up their reaction by lowering the reaction’s activation energy.1,13 One important aspect of naturally occurring enzymes that can be imitated by artificial enzymes, or enzyme mimics, is their selectivity. The selectivity can be divided into four categories: substrate selectivity, reaction selectivity, stereoselectivity and regioselectivity.14,15 Consequently, the unique properties and great importance of natural enzymes motivate researchers to develop enzyme mimics to test our capacity to mimic biology, and to explore possible routes to non-biological amino acids. Notable examples include cyclodextrin-based catalysts, molecularly imprinted polymers (MIPs) and catalytical antibodies.16 The first examples were presented by Breslow and co-workers, who developed a selective transaminase mimic using pyridoxamine connected to β-cyclodextrin,7 figure 3. The impressive results showed substrate specificity and a 200-fold rate enhancement for the reaction of indolepyruvic acid and pyridoxamine to yield tryptophan.7,17 Moreover, optically active amino acids were formed, due to the chirality of β- cyclodextrin. This serves as an excellent example of an artificial cyclodextrin-based transaminase that imitates the catalytical functions of the natural enzymes.

Figure 3. Structure of native cyclodextrin, with the three most common kinds (α, β, γ) (I). [Adapted from Murakami et al.].18 First artificial transaminase as presented by Breslow et al.,7 where pyridoxamine as the coenzyme is connected to β-cyclodextrin as a binding site (II).

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1.3 Molecular imprinting

Molecular imprinting is a technique for the synthesis of polymers which have specific molecular recognition abilities for a given molecule.19 Functional and cross-linking monomers are co-polymerized in the presence of a template (ligand) in an appropriate solvent (porogen). The interactions between the template and functional monomers in the pre-polymerization mixture can be either covalent or non-covalent (hydrogen bonds, ionic bonds, van der Waals forces), thus there are two ways that molecular imprinting can be approached. The self-assembly approach involves non-covalent interactions between the imprint molecule and the monomers. These self-assembly complexes are formed spontaneously in the solution (porogen) and polymerization then takes place. In this case an excess of functional monomers relative to the number of moles of the template is needed in the polymerization matrix. Hence the non-covalent protocol leads to a heterogeneous distribution of binding sites in the final imprinted polymer.20,21 The pre-organized approach involves strong and reversible covalent bonds between the template and the functional monomers before polymerization. In contrast to the self- assembly technique, the pre-organized strategy allows us to control the stoichiometry for the template-monomer complex, thus the binding groups are fixed in space and homogeneous distribution of binding sites affinities is also achieved.21,22 The combination of the two mentioned techniques is called semicovalent imprinting. Whitcombe et al. used covalent linkages in template-monomer complexes, and non- covalent interactions for rebinding of the target molecule to the MIP.23 Although this strategy combines the advantages of both the self-assembly and the pre-organized protocols, there are many drawbacks arising mostly from the covalent approach. Nowadays the non-covalent method to prepare molecularly imprinted polymers is preferred for the following reasons. The polymerization protocol and removal of the template are much easier using non-covalent interactions, and moreover reversible interactions with substrates are fast.22 The polymerization is then initiated by UV light or heat.19 After removal of the template molecule by chemical cleavage or solvent extraction, the polymer matrix contains cavities or recognition sites with a complementary shape and chemical functionality to those of the template,24 figure 4. The imprinted sites can therefore recognise and rebind the template structure. Over recent decades many different methods have been introduced for the preparation of molecularly imprinted polymers, such as bulk polymerization (MIP monoliths), suspension polymerization (MIP spherical particles), precipitation polymerization (MIP microspheres) and seed polymerization (monodisperse MIP beads). The molecular imprinting technique and its applications are being developed rapidly. It is a simple, fast and inexpensive method for producing synthetic recognition materials and it has attracted interest in many fields of chemistry. Consequently, the molecular imprinting technique with artificial binding sites and the special memory to recognise

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the template molecule, can be used as an alternative method for the development of a synthetic transaminase.

Figure 4. Schematic representation of the molecular imprinting process. Functional and cross- linking monomers are co-polymerized in the presence of template. The interactions between the template and functional monomers in the pre-polymerization mixture can be either covalent or non-covalent (or a mixture of both). In the last step the template is removed creating cavities with a complementary shape and chemical functionality to those of the template. [Permission to reproduce this schematic diagram has been granted by Gustaf Olsson].

1.3.1 Molecular imprinting - applications

MIPs can be used in many potential applications.25 Historically, one of the most explored fields of MIP applications has been chiral chromatographic separations.26 Imprinted polymers are used as stationary phases in liquid chromatography (LC) for the separation of racemic mixtures.27 Thus selective removal of small amounts of a non- pure optical compound can be achieved. Since most chiral drugs on the market are provided as racemic mixtures this application could allow us to obtain optically pure drugs in the future.26,28 MIPs for clinical drug separation applications have not been achieved yet because there are concerns regarding the safety between the imprinted polymers and the biological tissues.29 The imprinted polymers are usually prepared in organic solvents to promote electrostatic interactions and this may be harmful to the cells. Hydrophilic molecular imprinted polymers can be an alternative and more friendly approach in drug delivery applications. One of the main drawbacks of MIPs regarding the racemic separation is the excessive peak broadening and tailing. That limits the commercial applications of imprinted polymers as stationary phases in liquid chromatography for chiral separations.29 Antibody mimics are another interesting and promising application of MIPs for the determination of drugs in pseudo-immunoassays, as first presented by Vlatakis et al. for detection of theophylline in serum in 1993.30 Impressively, their imprinted materials showed strong binding comparable to that of antibodies. In 1995 a similar work in this field was reported by Andersson et al. using morphine and enkephalin polymers to

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mimic the binding activity of opiod receptors.31 The results were again impressive and the imprinted polymers showed high binding affinity and consequently had a capacity to mimic the binding site of the receptors. Moreover, in comparison with natural antibodies, MIPs can be prepared fast and at low cost, they do not involve the use of animals (synthetic products), and they are also stable in aggressive conditions.28,32 Unfortunately, the applications of this technology are limited and the progress is small since 1993 because it involves the use and the handling of radioactive materials. For this reason, alternative methods have been developed using fluorescent analogues of analytes which are called “fluoro-molecularly imprinted immunoassays”.33 Molecularly imprinted polymers can also be used as recognition elements in biosensors.20,34 A biosensor is a device that recognizes a target molecule (analyte) in a complex sample. The recognition element is connected to a transducer. When the recognition element specifically binds to the analyte the transducer converts the chemical signal into another signal that can be quantified. In many cases natural receptors are unstable and not suitable for the analysis, hence MIPs with high affinity can be used to replace these molecules as first reported by Mosbach et al.35 Solid phase extraction is a well-known method for sample preparation (clean-up and pre-concentration) from complex mixtures.21,36 Nowadays this application is used efficiently in combination with imprinted materials. The imprinted polymer is used as a stationary phase in an SPE cartridge. A mixture of analytes is then added and passed through the stationary phase where the separation of undesired compounds takes place.

1.3.2 Molecularly imprinted polymers in synthesis and catalysis

Over recent years, the potential to use MIPs as tools to guide synthesis and catalysis has been explored using an increasing range of systems.37 Imprinted materials are of particular interest in these applications due to their resistance to aggressive conditions such as high temperature and organic solvents, which do not normally support biological function. The first report of the use of a MIP in synthesis was presented by Damen and Neckers.38 They synthesized a series of styrene-divinylbenzene copolymers that were able to recognize the template and lead a 2+2 photochemical cycloaddition reaction to a specific stereochemical outcome. Importantly, this stereochemical preference was not observed when non-imprinted reference polymers were used. Catalysis using molecularly imprinted polymers as enzyme mimics, that imitate the efficient catalytic activities of natural enzymes, have been considered as possible alternatives, in part due to their stability, but also due to the (often) low cost preparation of MIPs and even the possibility of using them to drive reactions not found in nature. The principles underlying MIP-based catalysis follow those of enzyme-based catalysis, and the development of catalytic antibodies,39 where stabilization of the transition state of a reaction is the basis for the rate enhancement. For MIPs, or catalytic antibodies, this requires the use of a transition state analogue (TSA) as template or hapten in the case of catalytic antibodies. The site produced through the molecular imprinting process forms

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favourable interactions with the reaction components to stabilize the transition state so as to lower the activation energy, hence enhancing the rate of the reaction, figure 5.

Figure 5. Schematic illustration of a hypothetical energetic profile for an enzyme catalyzed reaction. Catalysis is achieved through the catalysts ability to stabilize the transition state TS#, lowering the activation energy requirement of the reaction. [Adapted from Alexander et al.]40

An important issue for MIP-based catalysts is that they must have lower product selectivity than affinity for intermediates of the reaction so as to allow turnover. Product inhibition and low catalytic turnover yields are problems often encountered in non- entropically-driven reactions, e.g. hydrolytic. The first example of MIP catalysis was presented by Robinson and Mosbach in 1989.41 They investigated the hydrolysis reaction for p-nitrophenyl acetate. p-Nitrophenol methylphosphonate was used as a template in the synthesis of the imprinted materials, as it resembles the transition state for the hydrolysis of p-nitrophenyl acetate. They found that the MIP was 60% more active, enhancing the rate of the ester hydrolysis than the non-imprinted reference polymer and also the template could be used as an inhibitor, proving that the hydrolysis was due to binding cavities in the imprinted polymer. Later, Mosbach at al. prepared a MIP artificial enzyme to catalyze the Diels-Alder reaction between tetrachlorothiophene dioxide and maleic anhydride.42 A 200-fold rate enhancement was observed for this reaction. This is an example of carbon-carbon bond formation, a significant challenge in organic chemistry. Another important study in this field was conducted by Svenson et al. in 2004.9 Svenson’s work was the major inspiration for the present study, forming the basics and motivating the development of molecularly imprinted polymer transaminase mimic using a transition state analogue a tryptophanamide oxazine-based compound as template. Their study described a MIP-based synthetic transaminase prepared using a transition state analogue for the reaction of phenylpyruvic acid and

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pyridoxamine to form phenylalanine and pyridoxal, figure 6. Polymer-ligand recognition studies showed that the imprinted polymers contained cavities selective for the transition state analogue in comparison with the reference polymer. The important goal of this study was to evaluate the influence of the polymers on the transamination reaction of phenylpyruvic acid and pyridoxamine to form phenylalanine and pyridoxal. The MIP-based synthetic transaminase catalyzed the transamination reaction with a 15-fold rate enhancement, exhibited enantioselectivity and functioned in aqueous media. The general importance of α-amino acids, both naturally occurring and prepared using synthetic strategies, makes developing new methods for their efficient synthesis of interest. From a sustainability perspective, methods capable of catalysing reactions in an atom-economical fashion are of particular interest. Accordingly, exploring the further development of MIP-based transaminases is desirable.

Figure 6. α-Amino acid synthesis by transamination: Phenylpyruvic acid reacts with pyridoxamine to yield pyridoxal and phenylalanine (a). General structure of proposed transition state (b), and structure of transition state analogue (c).

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1.4 Objectives of the thesis

The ultimate goal of this study is to explore the scope of MIP-based catalysts for pyridoxamine dependent transamination reactions. In the present study, the development of candidate polymer systems is to be undertaken. Three key objectives are necessary to achieve this goal:

- Establish a synthetic route to transition-state analogues for the transamination reaction of indole-3-pyruvic acid and pyridoxamine

- Synthesize and characterize polymers capable of the selective recognition of the transition state analogues

- Examine the catalytic behaviour of the polymer with respect to reaction kinetics, substrate selectivity and influence of reaction conditions.

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2 Materials & Methods

2.1 General procedures

NMR spectra were recorded with a Varian (500 MHz) spectrometer. The data is reported as follows: chemical shift (δ) in ppm, multiplicity (d=doublet, dd=double doublet, dt=double triplet, m=multiplet, s=singlet, t-triplet). Infrared spectra were recorded on an Agilent Cary 630 FTIR. Melting points were uncorrected. Thin layer chromatography was performed using silica gel plates with fluorescence indicator (254 nm), purchased from Sigma-Aldrich. HPLC tests were performed with a Merck-Hitachi system equipped with a L-7100 pump and L-7455 diode array detector. UV spectra were recorded on a Shimadzu UV-1800 spectrophotometer. Mass spectra were obtained on Agilent 1200 series mass spectrometer. Elemental analysis was performed by Eurofins Biopharma Product Testing (Uppsala, Sweden). The reagents and solvents including dichloromethane (Carlo Erba), acetonitrile (Fisher Chemical), L-phenylalaninamide (AK Scientific), pyridoxal hydrochloride (Sigma-Aldrich), sodium borohydride (Sigma-Aldrich), formaldehyde solution (37 wt. % in H2O Sigma-Aldrich), triethylamine (Chemtronica), D-phenylalaninamide hydrochloride (Combi-Blocks), L-tryptophanamide hydrochloride (Bachem), salicylaldehyde (Riedel-de Haën) were used as received. Methacrylic acid (MAA) (TCI) and ethylene glycol dimethacrylate (EGDMA) (Sigma-Aldrich) were purified by standard procedures. 2,2´-Azobisisobutyronitrile (AIBN) (Aldrich) was recrystallized from methanol. Methanol (MeOH) (Sigma-Aldrich) was purified by distillation from iodine and magnesium.43

2.2 Synthesis of transition state analogues

2.2.1 L-2-(3-Hydroxy-5-hydroxymethyl-2-methyl-pyridin-4-yl-methyl)amino-N- phenylalaninamide (1a)

L-phenylalaninamide (1.64 g, 10 mmol) and pyridoxal hydrochloride (1 eq, 10 mmol, 2.04 g) were added to a solution of dry methanol (90 ml). The reaction mixture was stirred at 0 oC for 45 min and then it was further stirred for additional 3 h at room

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temperature. The progress of the reaction was monitored by TLC. After completion of the reaction as indicated by TLC the mixture was cooled on ice, and sodium borohydride (50 mmol, 5 eq, 1.90 g) was added in portions. The solution was then warmed to room temperature and the reaction was allowed to continue for 1 hour. The resulting yellow solution was concentrated on rotary evaporator and was purified by flash chromatography on silica gel using dichloromethane/methanol (ratio: 95:5) as solvent for elution. Yield 65%. Pale yellow solid, Mp 172-175 oC; FTIR ν: 1665, 2913, -1 1 3161, 3375 cm ; H NMR (DMSO-d6), δ 7.73 (s, 1H), 7.57 (s, 1H), 7.22 (dd, J = 28.2, 12.3 Hz, 7H), 4.59 (s, 1H), 4.31 (d, J = 14.0 Hz, 3H), 3.87 (d, J = 14.4 Hz, 1H), 3.70 (d, J = 15.3 Hz, 2H), 3.28 (s, 2H), 3.15 (d, J = 6.7 Hz, 1H), 2.86 (d, J = 13.2 Hz, 1H), 2.72 13 (d, J = 7.5Hz, 1H); C NMR (DMSO-d6), 19.1, 40.3, 44.8, 59.4, 61.9, 126.7, 128.3, 128.5, 128.7, 129.5, 129.8, 133.1, 138.2, 138.5, 145.6, 152.5, 174,5 ppm. LCMS: calculated molecular weight for C17H21N3O3 315.15, found 315.10

2.2.2 L-2-(5-Hydroxymethyl-8-methyl-3.4-dihydropyrido[4.3-e]-1.3-oxazin-3- yl)phenylalaninamide (1b)

To a solution of 1a (0.4 g, 1.26 mmol) in dry methanol (30 ml) formaldehyde solution (2.52 mmol, 2 eq, 206 μl) was added and the reaction mixture was stirred for 30 min at room temperature. The mixture was then refluxed under inert atmosphere for 4 hours. The reaction was monitored by TLC. The resulting yellow suspension was concentrated in vacuo. The crude product was recrystallized from methanol affording the desired product. Yield 60%. Pale yellow solid, Mp 50-53 oC; FTIR ν: 1665, 2868, 3190, 3288 -1 1 cm ; H NMR (CDCl3), δ 7.88 (s, 1H), 7.35 – 7.04 (m, 7H), 5.03 (d, J = 10.1 Hz, 1H), 4.84 (d, J = 9.9 Hz, 1H), 4.49 (s, 2H), 4.27 (d, J = 16.9 Hz, 1H), 4.12 (d, J = 17.6 Hz, 1H), 3.83 – 3.50 (m, 2H), 3.21 (d, J = 6.8 Hz, 1H), 3.06 (d, J = 14.5 Hz, 1H), 1.26 (d, J 13 =9.9 Hz, 2H), 0.83 (s, 1H); C NMR (CDCl3), 18.5, 36.1, 45.5, 60.7, 66.2, 80.4, 126.4, 127, 128.1, 128.9, 131.4, 137.8, 139.2, 146.2, 148.8, 173.7 ppm. LCMS: calculated molecular weight for C18H21N3O3 327.15 found 327.15.

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2.2.3 D-2-(3-Hydroxy-5-hydroxymethyl-2-methyl-pyridin-4-yl-methyl)amino-N- phenylalaninamide (2a)

Triethylamine (1.3 eq, 6.47 mmol, 902 μl) was added to a stirred solution of D- phenylalaninamide hydrochloride (4.98 mmol, 1 g) in dry methanol (50 ml). The reaction mixture was stirred for 45 min at room temperature. Pyridoxal hydrochloride (1.3 eq, 6.47 mmol, 1.31 g) was then added and the solution was stirred at 0 oC for 45 min and then it was further stirred for an additional 3 h at room temperature. The progress of the reaction was monitored by TLC. The suspension was then cooled on ice and excess of sodium borohydride (5 eq, 0.94 g, 24.9 mmol) was added in portions. The solution was then warmed to room temperature and the reaction was allowed to continue for 1 hour. The resulting yellow solution was concentrated on rotary evaporator. The product was purified by flash chromatography on silica gel using dichloromethane/methanol (ratio: 95:5) as solvent for elution. Yield 66%. Pale yellow o solid, Mp 175-178 C. LCMS: calculated molecular weight for C17H21N3O3 315.15, found 315.05. The product had identical NMR and IR spectral properties as 1a.

2.2.4 D-2-(5-Hydroxymethyl-8-methyl-3.4-dihydropyrido[4.3-e]-1.3-oxazin-3- yl)phenylalaninamide (2b)

2b was synthesized in a similar manner as the 1b using D-phenylalaninamide hydrochloride. Yield 66%. Pale yellow solid, Mp 55-58 oC. LCMS: calculated molecular weight for C18H21N3O3 327.15, found 327.06. The product had identical NMR and IR spectral properties as the 1b.

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2.2.5 L-2‐{[(2‐hydroxyphenyl)methyl]amino}‐3‐(1H‐indol‐3‐yl)propanamide (3a)

A solution of triethylamine (1.3 eq, 10.84 mmol, 1.50 mL) was added to a stirred solution of L-tryptophanamide hydrochloride (8.343 mmol, 2 g) in dry methanol (90 mL). The reaction mixture was stirred for 45 min at room temperature. Salicylaldehyde (2 eq, 16.6 mmol, 1.77 ml) was then added and the solution was stirred at 0 oC for 45 min and then it was further stirred for an additional 1 h at room temperature. The reaction was monitored by TLC. After completion of the reaction the mixture was cooled on ice, and sodium borohydride (5 eq, 41.7 mmol, 1.57 g) was added in portions. The solution was then warmed to room temperature and the reaction was allowed to continue for 18 hours. The resulting solution was concentrated on rotary evaporator and the resulting residue was purified by flash chromatography on silica gel using dichloromethane/methanol (ratio: 95:5) as solvent for elution. Pale yellow oil. Yield -1 1 65%, FTIR ν: 1588, 1659, 2848, 2918, 3195 cm ; H NMR (Acetonitrile-d3), δ 9.71 (s, 1H), 7.61 (d, J = 7.7 Hz, 1H), 7.43 (d, J = 7.9 Hz, 1H), 7.14 (d, J = 9.3 Hz, 5H), 7.05 (d, J = 6.7 Hz, 2H), 7.00 (d, J = 6.9 Hz, 1H), 6.86 (d, J = 7.7 Hz, 2H), 6.76 (d, J = 7.7 Hz, 2H), 6.49 (s, 2H), 4.70 (d, J = 16.4 Hz, 2H), 3.97 (d, J = 13.4 Hz, 5H), 3.24 (dd, J = 13 14.3, 4.8 Hz, 2H), 3.14 – 3.05 (m, 2H); C NMR (Acetonitrile-d3), 28.5, 49.1, 60.8, 109.1, 111.5, 115.6, 117.5, 118.9, 119.5, 121.7, 124.2, 127.2, 128.1, 128.4, 129.6, 136.6, 156.9, 174.6 ppm. LCMS: calculated molecular weight for C18H19N3O2 309.14, found 309.05

2.2.6 L-2-({[2-hydroxy-6-(hydroxymethyl)-3-methylphenyl]methyl}amino)-3-(1H- indol-3-yl)propanamide (4a)

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Triethylamine (1.3 eq, 378 μl) was added to a stirred solution of L-tryptophanamide hydrochloride (2.085 mmol, 500 mg) in dry MeOH (23 ml). The reaction mixture was stirred for 45 min. Pyridoxal hydrochloride (1.3 eq, 550 mg) was then added and the reaction was stirred on ice for 45 min and then was further stirred for additional 1 hour to room temperature. The progress of the reaction was monitored by TLC. After completion of the reaction as determined by TLC the mixture was cooled on ice, and sodium borohydride (5 eq, 0.39 g) was added in portions. The solution was then warmed to room temperature and the reaction was allowed to continue for 1 hour. The resulting yellow solution was concentrated on rotary evaporator. The crude product was purified flash chromatography on silica gel using dichloromethane/methanol (ratio: 95:5) as solvent system for elution. Pale yellow solid. Yield 83%; Mp 144-147 oC; -1 1 FTIR ν: 1665, 2831, 2915, 3181, 3274 cm ; H NMR (DMSO-d6), δ 10.86 (s, 1H), 7.74 (s, 1H), 7.65 – 7.50 (m, 2H), 7.32 (d, J= 8.0 Hz, 1H), 7.18 (s, 1H), 7.12 (s, 1H), 7.04 (t, J = 7.5 Hz, 1H), 6.94 (dd, J = 15.8, 8.5Hz, 1H), 4.34 (s, 2H), 3.91 (d, J = 15.1 Hz, 1H), 3.77 (d, J = 15.2 Hz, 1H), 3.37 (t, J =6.4 Hz, 1H), 3.16 (s, 2H), 3.04 (dd, J = 14.1, 6.0 Hz, 1H), 2.89 (dd, J = 14.4, 7.2 Hz, 1H), 2.48 (s, 2H), 2.18 (s, 2H); 13C NMR (DMSO- d6), 19.1, 29.3, 40.3, 59.4, 61.1, 110.2, 111.7, 118.5, 118.8, 121.2, 124.1, 127.7, 128.4, 133.1, 136.5, 138.4, 145.7, 152.6, 175.1 ppm. LCMS: calculated molecular weight for C19H22N4O3 354.16, found 354.13.

2.2.7 L-2-[5-(hydroxymethyl)-8-methyl-2H,3H,4H-pyrido[4,3-e][1,3]oxazin-3-yl]-3- (1H-indole-3-yl)propanamide (L-TSA) (4b)

4a (0.4 g, 1.13 mmol) was dissolved in dry methanol (30 ml). Formaldehyde solution (2 eq, 183 μl) was added and the reaction mixture was stirred for 30 min to room temperature. The mixture was then refluxed under inert atmosphere for 4 hours. The reaction was monitored by TLC. The resulting yellow suspension was concentrated on rotary evaporator. The residue was recrystallized from methanol affording the desired product. Pale yellow solid. Yield 80%, Mp 85-88 oC; FTIR ν: 1668, 2845, 2904, 3184, -1 1 3292 cm ; H NMR (DMSO-d6), δ 7.86 (d, J = 4.9 Hz, 1H), 7.49 (s, 1H), 7.42 (d, J = 7.4 Hz, 1H), 7.29 (d, J = 7.9 Hz, 1H), 7.01 (dt, J = 29.7, 6.9 Hz, 1H), 4.55 (s, 1H), 4.37 (dd, J =27.3, 11.6 Hz, 1H), 4.07 (d, J = 14.4 Hz, 1H), 3.95 (d, J = 14.2 Hz, 1H), 3.81 (s, 1H), 3.74 (s, 1H), 3.22 (s, 1H), 3.18 (s, 4H), 3.09 (d, J = 14.9 Hz, 1H), 3.05 – 2.92 (m, 13 1H); C NMR (DMSO-d6), 19.15, 47.5, 54.3, 59.3, 61.4, 89.9, 105.2, 111.5, 117.8, 119, 121.2, 126.8, 128.1, 131.1, 134.1, 136.4, 138.4, 145.6, 152.1, 174.8 ppm. LCMS: calculated molecular weight for C20H22N4O3 366.16, found 366.15.

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2.3 Polymer synthesis

Imprinted (MIP) polymers with L-TSA P(L), were prepared according to the following procedure:9 The template (4b) (0.61 mmol), MAA (3.06 mmol), EGDMA (12.22 mmol), porogen (acetonitrile, 4.0 mL), and the initiator AIBN (0.27 mmol) were mixed into a glass tube. The mixture was then cooled on ice and sparged with nitrogen for 5 min. After that time the tube was placed in a water bath at 65oC for 24 h. The polymers were manually ground, and sieved to afford 25-63 μm particles. Sedimentation from acetone (6 × 40 mL) removed the fine particles. The polymer particles were packed in a stainless steel chromatography column and then washed with different solvents to remove the template as described elsewhere.44 The polymers were collected from the column and air dried. The non-imprinted (NIP) reference polymers P(B) were synthesized as described above but without using the template. P(L): Found: C, 58.8; H, 7.12; N, 0.48%. P(B): Found: C, 58.5; H, 7.17; N, 0.31%.

2.4 1H NMR-titrations

A solution of 4b (0.022 M) and EGDMA (1.86 M) in acetonitrile-d3 was titrated with a solution, ranging from 1-120 μL, containing MAA (5.895 M), 4b (0.022 M) and EGDMA (1.86 M) in acetonitrile-d3. GraphPad Prism software was used for the calculations of the apparent dissociation constants (app Kdiss) by non-linear regression using a one-site model.

2.5 Polymer recognition

A mixture of chloroform/acetonitrile (85/15, v/v) was used to suspend the washed polymer. The polymer was then slurry packed into a stainless steel chromatography column (100 x 4.6 mm) using acetone as solvent with a pump (Haskel Engineering) at 300 bar. Pure methanol was used to wash the columns until a stable baseline was achieved. The packed columns were used for recognition analyses with MeOH/0.1 M sodium acetate (aq) (1:1 v/v, pH 7) as buffer for isocratic elution at a flow rate of 0.5 mL/min. Analytes were dissolved (1 mM) in the mobile phase (except for indole-3- pyruvic acid which was dissolved in MeOH) and injected (10 μl) in triplicate. A non- interacting solvent (acetone) was used as void marker.

2.6 Polymer-ligand rebinding studies

Binding to both P(L) and P(B) was studied. In a typical experiment, 4b in concentrations ranging from 0.05-3.0 mM in acetonitrile was mixed with 5 mg of polymer in a total volume of 1 mL in acetonitrile. Each experiment was conducted in triplicate. The samples were incubated on a rocking table for 24 hours. The next day the tubes were centrifuged (5000g, 5 min). Thereafter, UV spectra were recorded for the supernatants using a Shimadzu UV-1800 spectrophotometer covering a spectral range from 200 to 350 nm. The instrument was calibrated with acetonitrile. Control samples were also prepared identically in the absence of polymer and were performed in duplicate.

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3 Results and Discussion

3.1 Evaluation and synthesis of transition state analogues

In the present work the TSA 4b was chosen in order for us to study the reaction between pyridoxamine and indole-3-pyruvic acid to yield pyridoxal and tryptophan, figure 7(b). The choice was based on a previous report in which TSA 1b was used by Svenson et al. to study the reaction between pyridoxamine and phenylpyruvic acid to yield pyridoxal and phenylalanine.9 Iskander et al., performed studies on transition state analogues as inhibitors for GABA-aminotransferase and they proposed that oxazine-based compounds are suitable as TSAs.45 Moreover, to avoid lactonization the amide was preferred from the carboxylic acid.

Figure 7. General structure of proposed transition state (a), and structure of the transition state analogue (b).

All the transition state analogues were synthesized in two steps, figure 8. In the first step the imine between the amide and the chosen aldehyde was formed by condensation and the reduction to amine was then achieved using NaBH4 as reducing reagent. In the second step the TSAs (oxazine-based compounds) were produced by Mannich cyclization using formaldehyde solution and recrystallization of the crude products then afforded the pure oxazine compounds. In the present study a different approach was developed for the synthesis of the transition state analogues. The known previous procedure in the literature reported 56% yield for the TSAs using paraformaldehyde. In our new approach the maintenance of the temperature at 80oC and the use of formaldehyde solution instead of paraformaldehyde without purification on silica, afforded TSAs with good to very good yields ranging from 60% to 80%. Furthermore, it was observed that the temperature has a huge impact on the yield in the second step. Reflux at lower temperature than 80oC afforded compounds in lower yields, while only side products were observed when the temperature was increased above 80oC. In cases

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where the amide was used as its hydrochloride form, triethylamine was added to the solution before the condensation with the aldehyde to remove the HCl, optimizing the reaction conditions. The TSAs 1b, 2b and 4b were successfully synthesized in good yields. The attempt to prepare the oxazine-based compound of the {[(2- hydroxyphenyl)methyl]amino}-3-(1H-indol-3-yl)propanamide 3a was unsuccessful with problems to isolate the final product giving low yield, 10-15 %.

o o Figure 8. L-TSA synthesis: (a) methanol, 45 min 0 C, 1-3 h r.t.; (b) methanol, NaBH4, 0 C; (c) methanol, formaldehyde solution, reflux, 4h.

3.2 Polymer synthesis and analysis

The imprinted and reference polymers were prepared by bulk polymerization. This method is the most popular and it does not require special operator skills and complicated instrumentation. The resulting polymer monolith is manually ground and sieved to afford particles with different size and shape. In the last step repeated sedimentation is applied to remove the fine particles (<1 μm). Particles with size 25 μm are usually used in chromatographic studies.46 In the present study the imprinted polymer P(L) was synthesized using 4b as template, EGDMA as cross-linker and MAA as functional monomer in a ratio of 1:20:5 (template:EGDMA:MAA). The polymerization was initiated by placing the polymerization mixture in a water bath at 65 oC for 24h using acetonitrile as the porogen and AIBN as the initiator. Particles (25-63 μm) were obtained after manual grinding, sieving and repeated sedimentation from acetone. The template was removed

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with washing steps according to a published protocol.44 The reference polymer P(B) was synthesized identically but in the absence of the template. FT-IR studies showed that the two polymers have identical functionalities. Moreover, elemental analysis was conducted for the two polymers and showed comparable values. The different values for nitrogen between the two polymers can be explained by the fact that some TSA residues are trapped inside P(L). SEM images were obtained to observe the surface morphology of the MIP and NIP polymers (25-63 μm particles), figure 9.

Figure 9. SEM images for particles (25-63 μm) by bulk polymerization. (a), (b), and (c) images correspond to P(L) at 60 000, 32 000 and 1 600 times magnification, respectively. (d), (e), and (f) images refer to P(B) at the same magnifications.

3.3 1H NMR-titrations, functional monomers interactions

1H NMR titrations experiments were performed to investigate the interactions between MAA and the template prior to polymerization. Interactions between the template and the functional monomer(s) can result in chemical shifts changes that can be used to calculate the apparent dissociation constants (Kd) of the complexes observed. Preferably, the interaction of a crosslinking agent with a template should be small to reduce the non-specific binding in the target polymer. In the current work, EGDMA is present during titrations thus it contributes in the complexation. Consequently in the current report a series of titrations were conducted exploring the strength of the interactions between MAA and the L-TSA 4b (in the presence of EGDMA) and the

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chemical shift changes were used to calculate the apparent dissociation constants for the observed interactions. MAA was added to a fixed concentration of L-TSA in acetonitrile to mimic the conditions prior to polymerization and the results are shown in figure 10. It is observed that the two non-equivalent amide protons have significant changes in chemical shifts upon subsequent additions of MAA. Apparent dissociation constants (Kd) of these protons were calculated to be Kd(b) = 0.63 ± 0.021 M and Kd(a) = 0.79 ± 0.039 M respectively. These values indicate the interactions (hydrogen bonds) between MAA and the amide functional group of the template.

Figure 10. Titration of L-TSA with MAA (EGDMA present).

Moreover, figure 11 represents the significant change in chemical shifts of protons, amide-NHa (6.645 ppm) and amide-NHb (6.208 ppm). Apparent dissociation constants of the protons on the pyridine ring, Pyr-CH3 and Pyr-CH, were determined to be Kd(CH3) = 0.27 ± 0.030 M and Kd(CH) = 0.26 ± 0.031 M, indicating interactions between MAA and the pyridine nitrogen. The total changes in chemical shifts for amide-NHb, amide-NHa, pyr-CH and pyr-CH3 were 1.305, 0.516, 0.302 and 0.153 ppm, respectively. In summary the NMR results indicate that the functional monomer interacts with the TSA in at least two different positions (both with the amide and with the pyridine) making the template-functional monomer complex suitable for the polymerization process

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Figure 11. 1H NMR spectra showing the change in chemical shifts of the two non-equivalent amide protons shifts upon subsequent additions of MAA.

3.4 Polymer-ligand recognition studies

Polymer-ligand recognition studies were performed to examine the selectivity of the imprinted sites for the template. P(L) and P(B) were packed into stainless steel chromatography columns and used as stationary phases in HPLC chromatography. A polar eluent (methanol/0.1 M sodium acetate (aq); 1:1 v/v; pH=7) was used as a mobile phase since the L-TSA failed to elute from the column when a non-polar buffer system (chloroform/acetic acid 96:4 v/v) was used. The retention times for a series of analytes were determined and used to calculate the corresponding capacity and imprinting factors, k´ and IF, Table 1. The capacity factor for L-TSA in P(L) was 9.45 while in P(B) a lower capacity factor of 3.22 was observed. Consequently, the imprinting factor for L-TSA is 2.93, indicating a strong evidence for the selectivity of P(L) for the template.

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Table 1. Capacity and imprinting factors

Analyte

Pyridoxamine Pyridoxal L-TSA Indole-3- L-Trp 1b 2b HCl HCl pyruvic acid

a k´P(L) 1.68 0.04 9.45 9.17 0.25 3.10 2.83 k´P(B) 0.91 0.00 3.22 8.77 0.12 1.35 1.33

IF 1.84b - 2.93 1.05 2.08 2.29 2.12 a k´= (tR-t0)/t0 where tR is the retention time for the analyte, and t0 is the retention time for a non- interacting solvent (acetone). The standard deviation was ± 0.07 or lower for all samples. b IF= k´P(L) /k´P(B)

Furthermore, polymer selectivity was examined for pyridoxamine, pyridoxal, indole- 3-pyruvic acid and tryptophan. Pyridoxamine was the only substrate to show a significant retention time on P(L) with an imprinting factor 1.84. On the other hand indole-3-pyruvic acid also displayed strong binding to both P(L) and P(B) mostly due to the similar structure to the template and also to the strong electrostatic interactions. In this case no selectivity was observed for this substrate in P(L) with a low imprinting factor of 1.05. L-tryptophan and pyridoxal were poorly retained. Since the template that was used in this study is a chiral compound, the use of the opposite enantiomer is essential and should provide an excellent way to establish the selectivity. Difficulties in the synthetic route combined with limited available time did not allow us to complete the synthesis of the D-TSA. Thus 1b and 2b were used as alternative controls to support the selectivity of the imprinted sites for L-TSA. The capacity factors of P(L) for 1b and 2b are 3.10 and 2.83 respectively. The difference between the two enantiomers in P(L) is not significant but some enantioselectivity was still observed (α = IF1b/IF2b = 1.1). This value is in agreement with the assumption that the imprinted cavities are more selective for the L-form since these enantiomers have a similar structure with the L-TSA. The interaction between those enantiomers and P(B) is the same. Furthermore, the imprinting factors of P(L) for 1b and 2b are 2.29 and 2.12 respectively. Therefore the analysis of the enantiomers 1b and 2b serves as an alternative and convincing evidence that the MIP contains cavities selective for the L- TSA. It is important to note that differences in surface areas make comparison of binding to imprinted and non-imprinted polymers difficult. BET-surface area data were not available at the time of writing. However, the observed enantioselectivity demonstrated for P(L) provides evidence for the presence of template-selective recognition sites in this polymer and the synthesis of the D-TSA in the future will establish the selectivity of the imprintes sites.

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3.5 Polymer-ligand rebinding studies

The rebinding of L-TSA to the MIP and the reference polymer NIP was examined using UV spectroscopy. It is a simple and straightforward technique that provides repeatable and accurate measurements. It is known that the solvent plays a major role in determining the binding process. A non-polar solvent stabilizes the electrostatic interactions, decreasing the non-specific binding and resulting in better specific binding for the target molecule. A quite polar solvent, acetonitrile, was used in this case to overcome the solubility issues of the L-TSA. Moreover, equilibrium binding analyses are usually performed in the same solvent as in the polymerization process. UV spectra were recorded at wavelengths ranging from 200 to 350 nm and all absorbance values were taken at 280 nm, which is the optimum absorbance wavelength for tryptophan. In order to determine the TSA concentration in solution, control samples of known concentration were used to construct a standard curve, figure 12(a). The samples with L-TSA concentrations 0.5, 1, 1.5 and 3 mM were diluted to keep their absorbance within the linear UV detection range. Consequently the binding (mmol·g-1) was determined for both polymers and for each L-TSA concentration. The specific binding is defined as the difference between the binding to the MIP and the binding to the reference polymer. Figure 12(b) shows the saturation curve of the total specific binding of L-TSA in acetonitrile. Bmax corresponds to the maximum theoretical amount of TSA that can bind to the polymer at saturation. Significant error bars at 3 mM concentration was observed due to the multiple dilutions needed for this sample. It has to be mentioned that the L-TSA also binds to the non-imprinted polymer due to non-specific interactions with the carboxyl groups of the functional monomer. Consequently these results confirm that the MIP has higher affinity for L-TSA than the NIP due to the imprinted sites. Furthermore, the MIP has a good capacity for the template, with a theoretical maximum specific binding of 0.059 ± 0.004 mmol·g-1 even though acetonitrile was the only option of the polymerization process.

Figure 12. (a) Standard curve from duplicated control samples. (b) Specific binding of L-TSA in acetonitrile.

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4 Conclusion and Future

Developing enzyme mimics to imitate the biological transamination for the synthesis of α-amino acids is a significant challenge in biochemistry and offers potential in the synthesis of non-natural amino acids. Molecularly imprinted polymers with artificial binding sites that resemble the active sites of the natural enzymes can be used as an alternative method for the development of a synthetic transaminase. In this study, we attempted to produce a synthetic aminotransferase as a catalyst for the transamination reaction between pyridoxamine and indole-3-pyruvic acid to yield pyridoxal and tryptophan, using the molecular imprinting technique. We used the TSA 4b as template to produce molecularly imprinted polymers and we suggest that their imprinted sites resemble the proposed transition state for the transamination reaction of tryptophan. The present work was based on a previous report in which a molecularly imprinted polymer-based synthetic transaminase successfully catalyzed the reaction between pyridoxamine and phenylpyruvic acid to yield pyridoxal and phenylalanine.9 The first step of this study was to synthesize the transition state analogue 4b. Here, a new approach is introduced for the TSA synthesis, achieving better yields for the oxazine-based compounds in comparison with the previously known procedure. The use of formaldehyde solution without any purification by flash column chromatography affords TSAs in good yields. Furthermore, two more transition state analogues (1b and 2b) were synthesized using this new procedure. Hence new oxazine-based compounds can be produced using our novel approach for future applications. The second step of this study was to prepare imprinted polymers using as template the transition state analogue 4b and to examine if they can catalyze the transamination reaction of tryptophan. 1H NMR studies were conducted to investigate the interactions between the functional monomer MAA and the template prior to polymerization. The results from 1H NMR titrations indicate interactions between MAA and the L-TSA, making the template-functional monomer complex suitable for the polymerization process. MAA and EGDMA were co-polymerized in the presence of 4b in acetonitrile. Control polymers were synthesized identically but in the absence of the template. BET- surface area analysis was not performed due to technical issues. However, its implementation in the future will provide more information about the surface morphology of the polymers. The combination of the polymer-ligand recognition and polymer-ligand rebinding studies provided evidence that the imprinted cavities are selective for the template. Furthermore, the TSAs 1b and 2b were used as an alternative control to support the selectivity of the imprinted sites for the L-TSA since the synthesis of the enantiomer D-TSA was not completed. Nevertheless some enantioselectivity was still observed (α = 1.1) between 1b and 2b in P(L), showing that the imprinted cavities are more selective for the L-form and overall that they are selective for the template. Limited available time did not allow us to investigate the catalytic properties of this imprinted polymer for the transamination reaction of tryptophan. In conclusion an imprinted polymer using as template a TSA with binding sites that resemble the proposed transition state for the transamination reaction of tryptophan, was

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successfully synthesized. There are two important on-going goals for this work. Firstly the synthesis of the enantiomer D-TSA is necessary and it will provide an excellent way to establish the selectivity of the imprinted sites for the L-TSA in the imprinted polymer. Secondly the main goal for this study is to examine if the polymer can catalyze the transamination reaction between pyridoxamine and indole-3-pyruvic acid to yield pyridoxal and tryptophan. If this polymer can be applied to catalyze the reaction, then it may be further applicable to the synthesis of non-natural amino acids as an artificial aminotransferase.

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Appendices

Appendix I. 1H NMR spectra showing the change in chemical shifts of the pyridine C-H proton.

1 Appendix II. H NMR spectrum of 1a in DMSO-d6

I

13 Appendix III. C NMR spectrum of 1a in DMSO-d6

1 Appendix IV. H NMR spectrum of 1b in CDCl3

II

13 Appendix V. C NMR spectrum of 1b in CDCl3

1 Appendix VI. H NMR spectrum of 4a in DMSO-d6

III

13 Appendix VII. C NMR spectrum of 4a in DMSO-d6

1 Appendix VIII. H NMR spectrum of 4b in DMSO-d6

IV

13 Appendix IX. C NMR spectrum of 4b in DMSO-d6

1 Appendix X. H NMR spectrum of 3a in acetonitrile-d3

V

13 Appendix XI. C NMR spectrum of 3a in acetonitrile-d3

Appendix XII. Mass spectrum (LCMS) of 1a

VI

Appendix XIII. Mass spectrum (LCMS) of 1b

Appendix XIV. Mass spectrum (LCMS) of 4a

Appendix XV. Mass spectrum (LCMS) of 3a

VII

Appendix XVI. Mass spectrum (LCMS) of 4b

Appendix XVII. FT-IR spectrum of the imprinted polymer

VIII

Appendix XVIII. FT-IR spectrum of the non-imprinted polymer

Linnæus University Sweden

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