Synthesis and enzymatic post-modification of chiral polymers

Smart materials, which respond to external stimuli, are well known for a long time. Typically these materials undergo an (ir)reversible property change upon exposure to an external stimulus. In the majority of cases the stimulus is a change in temperature, pH or is light induced. A relatively new concept that was recently developed involves smart materials that produce a change in response to the action of an enzyme, so called Enzyme Responsive Materials (ERMs). The response that the ERM produces can be any of a wide range of changes such as a change in fluorescence, the release of a drug or the generation of some form of physical aggregation such as the formation of micelles. Enzymes do not only exhibit regio-selectivity but they generally exhibit extremely high stereo-selectivity as well, i.e. the enzyme exclusively acts on one enantiomeric form of a molecule whilst leaving the other enantiomer untouched. Our approach to ERM synthesis is based on this principle, where the extent of the response that the ERM produces to the enzyme is encoded within the chirality of the material. By using different ratios of the enantiomers (building blocks) within the ERM, the extent of the response can be programmed-in without causing any changes in the general physical properties of the material. With this principle in mind, a number of acetophenone-derivatives with different functional groups in the para-position of the phenyl ring were synthesized (Chapter 2). In an attempt to reduce these prochiral ketones to their enantiomeric forms by employing two commercially available alcohol dehydrogenases (ADHs), namely (R)-producing Lactobacillus (ADH-LB) and (S)-producing Thermoanaerobacter sp. (ADH-T) as the catalysts for the reductions, a significant difference in the substrate acceptance by these enzymes was observed despite claims concerning their broad substrate spectrum. The examination of the various factors such as the solubility of the substrate in the reaction medium, the size of the substrate and the electronic character of the substituent at the para-position of the acetophenone derivatives did not offer any trend to explain the difference in the substrate acceptance by ADH-LB and ADH-T. Instead, we realized that non-polar para-substituents were favored over polar or ionizable para-substituents in the reductions. Mapping of the electronic charge distribution in the molecules revealed a correlation between the location of the highest electron density on the molecule and the success of the reaction. The substrates with the highest electron density on the carbonyl group could be reduced almost quantitatively to their enantiomeric forms. However, if the highest electron density is located on a different functional group of the substrate, then the substrate could not be reduced. ADH-catalyzed reduction of acetophenone-derivatives resulted in the synthesis of two types of enantiopure building blocks having an enzyme sensitive unit (phenyl ethanol): one with a polymerizable group, ((R)- and (S)-1-(4-vinylphenyl)ethanol), and the other one with a clickable moiety, ((R)- and (S)-1-(4-ethynylphenyl)ethanol). In chapter 3, homo and copolymers were synthesized from enantiopure (R)- and (S)-1-(4-vinylphenyl)ethanol and styrene by RAFT (co)polymerization. Well-defined polymers with low polydispersities were obtained. Kinetic investigations confirmed that the enantiopure monomers and styrene have similar reactivity ratios resulting in random copolymers. In chapter 4, block copolymers comprising two blocks with pendant hydroxy groups of opposite chirality were synthesized in which the length of starting block, (R)-block, was kept constant at 7,200 g/mol. The length of the second block, (S)-block, was varied from 2,100 to 6,400 g/mol. The optical rotation decreased from 29.8° to 1.5° with increasing (S)- to (R)- block length ratio. Noteworthy is that the blocky character of these chiral polymers would only manifest itself in the optical rotation since it is reasonable to claim that the chemical and physical properties of the individual chiral (S)- and (R)-blocks are identical. In chapter 5, linear and star-shaped poly(n-butyl acrylate)s (BAs) were prepared via ATRP of n-butyl acrylate by employing different ATRP initiators. Consecutive click reactions with (R)-1-(4-ethynylphenyl) ethanol after the chain end functionalization with an azide were not quantitative, probably due to loss of some of the end groups. In addition, chiral dendrimers were prepared from different mixtures of (R)- and (S)-1-(4-ethynylphenyl)ethanol, and the matching azide-functional bisMPA dendrimers using click chemistry. The specific optical rotation of the dendrimers increased linearly with increasing percentage of (R) end-groups in the dendrimer, which indicates that (R)- and (S)-building blocks had been incorporated into the dendrimer in agreement with the enantiomeric feed ratio in the click reaction. The molar rotation values of the dendrimers were found to be directly proportional to the number of (R)-building blocks clicked to the periphery, implying that each stereogenic group at the periphery behaves like an isolated molecule and does not induce any additional chiral substructure. Prior to the exposure of the synthesized chiral polymers to Candida Antarctica Lipase B to study the response of these macromolecules to an enantioselective enzyme, model reactions were performed to investigate the optimal reaction conditions. Since the chiral homopolymers synthesized in this study were not soluble in common hydrophobic solvents like hexane or toluene, in which CALB generally shows optimum activity, different organic solvent systems, both polar as well as mixtures of polar and apolar solvents, were employed for the CALB-mediated esterification of phenylethanol units with vinylacetate. Toluene/THF (2/1 v/v) was found to be the most appropriate solvent mixture for the post-functionalization of (R)-polymer. It was shown that the lipase-catalyzed polymer analogous esterification of the chiral hydroxy groups was strongly (R)-selective, in agreement with the preferred lipase enantioselectivity. However, even after extended reaction times, esterification on the (R)-polymers was limited to around 50 %, which was much lower than the results obtained with small model compounds. Increasing the molecular weight of (R)-polymers from 5,400 g/mol to 16,200 g/mol resulted in a decrease of the esterification yield (55 % to 42 %). This suggests that steric factors play a role in the esterification, although it cannot be ruled out that a decrease of polymer solubility in toluene/THF (2/1) with increasing molecular weight also contributes to this result. When (R)-1-(4-vinylphenyl)ethanol-styrene copolymers with different chirality were exposed to CALB, an increase in the extent of esterification (from 21 to 53 %) was observed whilst the composition ratio of (R)- 1-(4-vinylphenyl)ethanol/styrene was increased from 0.25 to 4. However, further increase in the enzyme-sensitive monomer concentration in the backbone did not further improve the extent of esterification (the maximum reached in these conditions with homopolymer was 55 %). This might suggest that not only steric effects play a role but that possibly also the local environment of the hydroxy groups is important for the extent of the reaction. CALB-catalyzed esterification of the chiral block copolymers was stereoselective and only one of the present blocks was esterified, thereby converting these chiral block copolymers into block copolymers with chemically and physically distinguishable blocks. CALB-catalyzed esterification of these (R)-phenyl ethanol groups at the chain end of polyBAs was successful and principally validated the proposed strategy of enantioselective enzymatic esterification of globular multifunctional chiral materials. The extremely high selectivity of CALB towards the R enantiomer of the 1-phenyl-ethanol moiety was retained also on the periphery of dendrimers. Furthermore, the chirality of the dendrimer directly correlates to a chemical reaction yield using an enantioselective catalyst. In the last chapter, vinyl methacrylate was used as the acyl donor in the CALB-catalyzed post-polymerization modification of selected (R)-chiral polymers as a comparison to vinyl acetate. These modifications provided pendant methacrylic double bonds which were utilized in thiol-ene reactions, viz. the Michael addition of poly(ethylene glycol) methyl ether thiol (PEG-SH), for further modification. The preliminary results showed that vinyl methacrylate could be used successfully as the acyl donor in the CALB-catalyzed esterifications. However, the extent of the esterification of pendant (R)-OHs with vinyl methacrylate was lower (35 %) compared to that with vinyl acetate (55%) performed under the same reaction conditions. In conclusion, the proof of principle described in this dissertation can be employed to program reactivity into otherwise indistinguishable molecules. It can be stated that chirality can be used as a means of encoding macromolecules that and a key characteristic of enzymes, i.e. enantioselectivity, can be utilized to read-out this code by correlating it to a chemical reaction

Lipases in polymer chemistry

Lipases are highly active in the polymerization of a range of monomers. Both ring-opening polymerization of cyclic monomers such as lactones and carbonates as well as polycondensation reactions have been investigated in great detail. Moreover, in combination with other (chemical) polymerization techniques, lipase-catalyzed polymerization has been employed to synthesize a variety of polymer materials. Major advantages of enzymatic catalysts are the often-observed excellent regio-, chemo- and enantioselectivity that allows for the direct preparation of functional materials. In particular, the application of techniques such as Dynamic Kinetic Resolution (DKR) in the lipase-catalyzed polymerization of racemic monomers is a new development in enzymatic polymerization. This paper reviews selected examples of the application of lipases in polymer chemistry covering the synthesis of linear polymers, chemoenzymatic polymerization and applications of enantioselective techniques for the synthesis and modification of polymers

Enantioselective enzymatic modification of chiral block copolymers

Block copolymers comprising blocks with pendant hydroxy groups of opposite chirality are synthesized by RAFT polymn. of (R)- and (S)-1-(4-vinylphenyl)ethanol (1R and 1S) as monomers. Initially, poly(styrene) macro-RAFT agents are chain-extended with both enantiomeric monomers to obtain poly(styrene-b-1R) and poly(styrene-b-1S) with controlled mol. wt. and low polydispersities. Enantioselective esterification with vinyl acetate by Candida Antarctica Lipase B (CALB) is only possible on the 1R-contg. block copolymer. This concept is extended to a series of chiral block copolymers poly(1R-b-1S) which, apart from their optical rotation, all behave like very similar homopolymers. By enzymic enantioselective esterification on the 1R-block, block copolymer structures were obtained

Investigation of asymmetric alcohol dehydrogenase (ADH) reduction of acetophenone derivatives: effect of charge density

In an effort to study the effect of substituent groups of the substrate on the alcohol dehydrogenase (ADH) reductions of aryl-alkyl ketones, several derivatives of acetophenone have been evaluated against ADHs from Lactobacillus brevis (LB) and Thermoanaerobacter sp. (T). Interestingly, ketones with non-demanding (neutral) para-substituents were reduced to secondary alcohols by these enzymes in enantiomerically pure form whereas those with demanding (ionizable) substituents could not be reduced. The effect of substrate size, their solubility in the reaction medium, electron donating and withdrawing properties of the ligand and also the electronic charge density distribution on the substrate molecules have been studied and discussed in detail. From the results, it is observed that the electronic charge distribution in the substrate molecules is influencing the orientation of the substrate in the active site of the enzyme and hence the ability to reduce the substrat

Efficiency of olopatadine hydrochloride 0.1% in the treatment of vernal keratoconjunctivitis and goblet cell density

Purpose: The aim of this study was to investigate the effect of topical 0.1% olopatadine hydrochloride on goblet cell density, clinical signs, and symptoms of patients with vernal keratoconjunctivitis

Synthesis of enantiopure homo and copolymers by raft polymerization and investigation of their enantioselective lipase-catalyzed esterification

Homo and copolymers were synthesized from enantionpure (R)- and (S)-1-(4-vinylphenyl)ethanol by reversible addition-fragmentation chain transfer polymerization. The polymerization conditions were optimized resulting in dioxane as the preferred reaction solvent. First-order polymerization kinetics and well-defined enantiopure homopolymers with low dispersities were obtained. In agreement with their enantiomeric composition, the (R) and (S)-polymers gave opposite optical rotation of light. Polymer analogous esterification of the chiral hydroxy groups catalyzed by enantioselective Candida antarctica Lipase B was strongly (R)-selective. Esterification on the homopolymer and copolymers could be achieved to a maximum of around 50 %. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 201

Lipases in polymer chemistry

Lipases are highly active in the polymerization of a range of monomers. Both ring-opening polymerization of cyclic monomers such as lactones and carbonates as well as polycondensation reactions have been investigated in great detail. Moreover, in combination with other (chemical) polymerization techniques, lipase-catalyzed polymerization has been employed to synthesize a variety of polymer materials. Major advantages of enzymatic catalysts are the often-observed excellent regio-, chemo- and enantioselectivity that allows for the direct preparation of functional materials. In particular, the application of techniques such as Dynamic Kinetic Resolution (DKR) in the lipase-catalyzed polymerization of racemic monomers is a new development in enzymatic polymerization. This paper reviews selected examples of the application of lipases in polymer chemistry covering the synthesis of linear polymers, chemoenzymatic polymerization and applications of enantioselective techniques for the synthesis and modification of polymers

Encoded dendrimers with defined chiral composition via 'click' reaction of enantiopure building blocks

Dendrimers with end-groups of defined chiral composition have been prepared from alkyne functional enantio-pure building blocks obtained by selective enzymatic (ADH) ketone reductions using click chemistry. Optical rotation and enantioselective enzymatic modification is in agreement with the chiral composition of the dendrimers and permits unique molecular-level encoding of stereoisomeric dendritic libraries

Human ocular onchocerciasis: further evidence on the zoonotic role of Onchocerca lupi

BACKGROUND: Among ocular vector-borne pathogens, Onchocerca volvulus, the agent of the so-called "river blindness", affects about 37 million people globally. Other Onchocerca spp. have been sporadically reported as zoonotic agents. Cases of canine onchocerciasis caused by Onchocerca lupi are on the rise in the United States and Europe. Its zoonotic role has been suspected but only recently ascertained in a single case from Turkey. The present study provides further evidence on the occurrence of O. lupi infesting human eyes in two patients from Turkey (case 1) and Tunisia (case 2). The importance of obtaining a correct sample collection and preparation of nematodes infesting human eyes is highlighted. METHODS: In both cases the parasites were identified with morpho-anatomical characters at the gross examination, histological analysis and anatomical description and also molecularly in case 1. RESULTS: The nematode from the first case was obviously O. lupi based on their morphology at the gross examination, histological analysis and anatomical description. In the second case, although the diagnostic cuticular characters were not completely developed, other features were congruent with the identification of O. lupi. Furthermore, the morphological identification was also molecularly confirmed in the Turkish case. CONCLUSIONS: The results of this study suggest that O. lupi infestation is not an occasional finding but it should be considered in the differential diagnosis of other zoonotic helminths causing eye infestation in humans (e.g., D. immitis and Dirofilaria repens). Both cases came from areas where no cases of canine onchocerciasis were previously reported in the literature, suggesting that an in depth appraisal of the infestation in canine populations is necessary. Physicians and ophthalmologists are advised on how to preserve nematode samples recovered surgically, to allow a definitive, correct etiological diagnosis