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Title:
ASYMMETRIC HYDRATION OF 4-HYDROXYSTYRENE DERIVATIVES EMPLOYING DECARBOXYLASES
Document Type and Number:
WIPO Patent Application WO/2013/186358
Kind Code:
A1
Abstract:
A promiscuous catalytic hydratase-activity of phenolic acid decarboxylases allows the asymmetric addition of H2O or of a non-natural nucleophile across the C=C bond of hydroxystyrene derivatives.

Inventors:
WUENSCH CHRISTIANE (AT)
GROSS JOHANNES (AT)
GLUECK SILVIA M (AT)
FABER KURT (AT)
Application Number:
PCT/EP2013/062372
Publication Date:
December 19, 2013
Filing Date:
June 14, 2013
Export Citation:
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Assignee:
ACIB GMBH (AT)
KARL FRANZENS UNI GRAZ (AT)
International Classes:
C12P7/22; A23L29/00; C12N9/88; C12P13/00
Other References:
POELARENDS, G.J. ET AL.: "Inactivation of Malonate Semialdehyde Decarboxylase by 3-Halopropiolates: Evidence for Hydratase Activity", BIOCHEMISTRY, vol. 44, no. 26, 5 July 2005 (2005-07-05), pages 9375 - 9381, XP002692732
WUENSCH, C. ET AL.: "Regioselective Enzymatic Carboxylation of Phenols and Hydroxystyrene Derivatives", ORGANIC LETTERS, vol. 14, no. 8, 3 April 2012 (2012-04-03), pages 1974 - 1977, XP009162441
MCINTIRE, W. ET AL.: "Stereochemistry of 1-(4'-hydroxyphenyl)ethanol produced by hydroxylation of 4-ethylphenol by p-cresol methylhydroxylase", BIOCHEMICAL JOURNAL, vol. 224, no. 2, December 1984 (1984-12-01), pages 617 - 621, XP002692733
WUENSCH, C. ET AL.: "Asymmetric Enzymatic Hydration of Hydroxystyrene Derivatives", ANGEWANDTE CHEMIE INTERNATIONAL EDITION, vol. 52, no. 8, 17 January 2013 (2013-01-17), pages 2293 - 2297, XP002692734
DATABASE CAPLUS [online] CHEMICAL ABSTRACTS SERVICE; 5 January 2007 (2007-01-05), "A microbial biocatalyst for preparing pharmaceutical compounds", XP002697945, Database accession no. 2007:416876
MADALINSKA, L. ET AL.: "Investigations on enzyme catalytic promiscuity: The first attempts at a hydrolytic enzyme-promoted conjugate addition of nucleophiles to alpha,beta-unsaturated sulfinyl acceptors", JOURNAL OF MOLECULAR CATALYSIS. B, ENZYMATIC, vol. 81, 2 May 2012 (2012-05-02), pages 25 - 30, XP028495604
WEINER, B. ET AL.: "Biocatalytic Enantioselective Synthesis of N-Substituted Aspartic Acids by Aspartate Ammonia Lyase", CHEMISTRY - A EUROPEAN JOURNAL, vol. 14, no. 32, 8 October 2008 (2008-10-08), pages 10094 - 10100, XP002713766
Attorney, Agent or Firm:
GASSNER, Birgitta et al. (Donau-City-Straße 1, Vienna, AT)
Download PDF:
Claims:
CSaims:

1 . A process for the stereoselective conversion of a substrate compound using a decarboxylase, wherein said stereoselective conversion is hydration or addition of a non-natural nucleophile.

2. The process according to claim 1 , wherein the decarboxylase is selected from the group consisting of phenolic acid decarboxylase (PAD), feruiic acid decarboxylase (FDC), and β-coumaric acid decarboxylase (PDC).

3. The process according to claim 1 or 2, wherein said stereoselective conversion is addition of a non-natural nucleophile.

4. The process according to claim 3, wherein the non-natural nucleophile is selected from the group consisting of hydroxylamine, 2-mercaptoethanol,

methoxylamine, sodium azide, hydrazine, ammonium acetate, methylamine, sodium formate, nitromethane, sodiumbicarbonate, ammonium cyanate, sodium phosphate, ammonium thiocyanate, sodium phosphite, ammonium bromide, sodium borate, ammonium iodide, sodium sulfide, sodium nitrite, sodium sulfite, and ammonia.

5. The process according to claim 1 or 2, wherein said stereoselective conversion is hydration.

6. The process according to any one of claims 1 to 5, wherein the substrate com ound is of formula I,

wherein

each R is independently from one another selected from the group consisting of hydroxy, halogen, Ci-3alkoxy, C -salkyl, Ci-3alkenyl, -NH2, and

n is 0, 1 , 2, 3 or 4.

7. The process according to any one of claims 1 to 5, wherein the substrate com ound is of formula la, wherein

each R1 is independently from one another selected from the group consisting of hydroxy, halogen, C -3alkoxy, Ci-3alkyl, Ci-3alkenyl, -NH2, and

n is 0, 1 , 2, 3 or 4.

8. A process for making a compound of formula II, comprising the step of

(II)

wherein

each R1 is independently from one another selected from the group consisting of hydroxy, halogen, d-3alkoxy, Ci-3alkyl, Ci-3alkenyl, -NH2, and

n is 0, 1 , 2, 3 or 4.

9. A process for making a compound of formula lla, comprising the step of

wherein

each R1 is independently from one another selected from the group consisting of hydroxy, halogen, Ci-3alkoxy, Ci-3alkyl, Ci-3alkenyl, -NH2, and

n is 0, 1 , 2, 3 or 4.

10. The process according to claim 8 or 9, wherein a base is added.

1 1 . The process according to claim 8 or 9, wherein the base is bicarbonate, or carbonate.

12. The process according to any one of claims 8 to 1 1 , wherein compound II or compound I la is obtained with at least 30%, preferably with at least 50%, more preferred with at least 70% conversion.

13. The process according to any one of claims 8 to 12, wherein compound II or compound I la is obtained with at least 25%, preferably with at least 40%, more preferred with at least 55% enantiomeric excess (e.e.).

14. A process for manufacturing a hyd rated compound, comprising the steps of: i) suspending host cells which overexpress the decarboxylase in a buffer;

ii) adding the substrate compound;

iii) adding a base; and

iv) obtaining the hyd rated compound.

Description:
ASYMMETRIC HYDRATION OF 4-HYDROXYSTYRENE DERIVATIVES EMPLOYING DECARBOXYLASES

FIELD OF THE INVENTION

The present invention relates to the regio- and stereoselective conversion of a substrate compound using a decarboxylase.

BACKGROUND OF THE INVENTION

The stereoselective addition of water across C=C double bonds is a highly valuable transformation of alkenes to non-racemic alcohols, which represents a major challenge in synthetic organic chemistry. Acid catalysed alkene hydration— following the rule of Markovnikov— usually proceeds with low to moderate regioselectivity and is often plagued by rearrangement, which yields regioisomeric product mixtures.

Despite the simplicity and the 100% atom economy of this transformation, no generally applicable protocol has been developed so far, with few examples, such as the production of ferf-butanol from -butene [1]. In a complementary fashion, base- catalysed 1 ,4-addition of water onto α,β-unsaturated (Michael) acceptors would be a complementary method to furnish non-racemic β-hydroxycarbonyl and -carboxyl compounds. However, due to the reversibility of 1 ,4-addition and the poor

nucleophilicity of water, this method is not well established in asymmetric synthesis [2]. In general, the equilibrium of alkene hydration is not far from unity, it is slightly favoured in 1 ,4-additions, and somewhat disfavoured on isolated C=C bonds[5].

Overall, only an astonishing small number of alkene-hydration protocols are reported in literature: (i) The stereoselective hydration of α,β-unsaturated carboxylic acids using a heterobimetallic chiral biopolymer (wool-Pd"-Co") catalyst furnished β- hydroxy carboxylic acids in high optical purities[3], and the asymmetric syn-hyd ration of α,β-unsaturated acyl imidazoles applying a DNA-based Cu" catalyst yielded β- hydroxy carbonyl compounds in moderate enantiomeric purities [4]. In order to compensate for the insufficient nucleophilicity of water, indirect methods based on various strong nucleophiles— alkoxides, AZ-silyloxycarbamates, oximes, silicon- and boron-reagents— have been employed, which require cumbersome reductive or oxidative follow-up chemistry to yield the desired β-hydroxy carbonyl or -carboxyl compounds [2].

In contrast, direct asymmetric addition of water across isolated or conjugated C=C bonds is an important process in biology [5]. The corresponding enzymes belong to the class of lyases and are termed 'hydro-lyases' or 'hydratases'. Mechanistically, these enzymes seem to fall into two categories, acting via (Lewis) acid catalysed 1 ,2- addition (v, vi) and (Michael-type) nucleophilic 1 ,4- or 1 ,6-addition involving quinone- methide enolates (i-iv) [6].

i) Butenedioate hydratases are key enzymes in the citric acid cycle and catalyse the anti-hydration of fumarate yielding (S)-malate, a process which is successfully operated in industrial scale (~2,000t/a) [7]. Unfortunately, malease, citraconase and mesaconate hydratase show a very narrow substrate spectrum [8].

ii) Enoyl-CoA hydratases (ECH) require the (ATP-energy-consuming) activation of their (monoacid) substrates via a thioester bond onto coenzyme A (CoA) [9,10,11].

ECHs, such as crotonase, are key enzymes in the β-oxidation pathway and catalyse the syn- hydration of c/s- or frans-2-enoyl-CoA to the corresponding (R)- or (S)-3- hydroxyacyl-CoA hydration products. A few processes on industrial scale have been established, such as the hydration of crotonobetain to L-carnitine and the conversion of 2-methylpropenoic acid (obtained via enzymatic dehydrogenation of i-butyric acid) to yield (R)- or (S)-3-hydroxy-/-butyric acid [7]. Due to the dependence on ATP, whole cells are employed.

iii) Hydroxycinnamoyl- CoA hydratase-lyase (HCHL) belongs to the crotonase superfamily and requires ATP-dependent substrate activation with CoA. This enzyme catalyses the two-step degradation of feruloyl CoA via stereospecific hydration of the C=C-bond, followed by retro-aldol C-C-cleavage to yield vanillin and acetaldehyde

[6,12,13,14].

iv) Michael hydratase alcohol dehydrogenase (MhyADH) is a bifunctional enzyme, which catalyses the 1 ,4-hydration of a range of α,β-unsaturated carbonyl compounds followed by oxidation of the β-hydroxy moiety to yield the corresponding β- oxo-aldehyde or -ketone in the presence of an oxidant; in the absence of an electron acceptor the hydration product could be identified [15,16]. Based on sequence analysis the enzyme is a member of the molybdopterin containing oxidoreductase family comprising molybdenum, iron and zinc as cofactor. In view of the narrow substrate tolerance of hydratases, MhyADH is exceptional due to its broad substrate spectrum. v) Acetylene hydratase (AH) is a rare tungsten-protein, which enables the Lewis-acid catalysed hydration of acetylene to furnish acetaldehyde [17,18,19,20]. AH is a member of the dimethyl sulfoxide reductase family carrying an iron-sulfur [4Fe-4S] cluster and two molybdopterin-guanosine-dinucleotide ligands coordinated to the metal. However, the high substrate specificity and oxygen-sensitivity severely limit the practical applicability of this enzyme.

vi) The 1 ,2-hydration of isolated C=C bonds occurs in fatty acids (oleate hydratase yields (R)-10-hydroxy-stearate from oleic acid) [21 ,22], in natural products (kievitone and phaseollidin hydratase [23], carotenoid 1 ,2-hydratase [24]) and in terpenoids (linalool dehydratase-isomerase) [25]. The latter enzyme catalyses the hydration of myrcene to yield linalool, which is subsequently isomerised to geraniol. However, all enzymes from this group are very substrate specific.

vii) Dehydratases [5,8]: a number of dehydration reactions play an important role in the metabolism of carbohydrates and amino acids (galactonate and glucarate dehydratase; serine and threonine dehydratases) and since the reaction is reversible it can run in both directions (elimination versus addition of water). Diol-dehydratases act via a radical mechanism mediated by B12 as cofactor [26,27]. DETAILED DESCRIPTION OF THE INVENTION

Herein the unprecedented stereoselective asymmetric conversion of

hydroxystyrene-type substrates employing various decarboxylases is described.

One aspect of the invention relates to such process wherein the decarboxylase is selected from the group consisting of phenolic acid decarboxylase (PAD), ferulic acid decarboxylase (FDC), and β-coumaric acid decarboxylase (PDC).

A further aspect of the invention relates to a process as described above, wherein said stereoselective conversion is hydration or addition of a non-natural nucleophile.

A further aspect of the invention relates to a process as described above, wherein the non-natural nucleophile is selected from the group consisting of

hydroxylamine, 2-mercaptoethanol, methoxylamine, sodium azide, hydrazine, ammonium acetate, methylamine, sodium formate, nitromethane, sodiumbicarbonate, ammonium cyanate, sodium phosphate, ammonium thiocyanate, sodium phosphite, ammonium bromide, sodium borate, ammonium iodide, sodium sulfide, sodium nitrite, sodium sulfite, and ammonia.

A further aspect of the invention relates to a process as described above, wherein the substrate compound is of formula I,

wherein

each R 1 is independently from one another selected from the group consisting of hydroxy, halogen, Ci- 3 aikoxy, C h alky!, Ci-3alkenyl, -NH2, and

n is 0, 1 , 2, 3 or 4.

A further aspect of the invention relates to a process as described above, wherein the substrate compound is of formula la, wherein

each R 1 is independently from one another selected from the group consisting of hydroxy, halogen, C - 3 alkoxy, C h alky!, C -3alkenyl, -NH2, and

n is 0, 1 , 2, 3 or 4.

A further aspect of the invention relates to a process for making a compound of formula I I, comprising the step of

(I) (II) wherein

each R 1 is independently from one another selected from the group consisting of hydroxy, halogen, Ci^alkoxy, C h alky!, Ci-salkenyl, -NH 2 , and

n is 0, 1 , 2, 3 or 4.

A further aspect of the invention relates to a process for making a compound of formula I la, comprising the step of

wherein each R 1 is independently from one another selected from the group consisting of hydroxy, halogen, Ci^alkoxy, C 1.3a Iky I, C -3alkenyl, -NH 2 , and

n is 0, 1 , 2, 3 or 4.

A further aspect of the invention relates to such process, wherein a base is added.

A further aspect of the invention relates to a process, wherein the base is bicarbonate, or carbonate.

A further aspect of the invention relates to a process, wherein compound II or compound I la is obtained with at least 30%, preferably with at least 50%, more preferred with at least 70% conversion.

A further aspect of the invention relates to such process, wherein compound II or compound I la is obtained with at least 25%, preferably with at least 40%, more preferred with at least 55% enantiomeric excess (e.e.).

A further aspect of the invention relates to a process for manufacturing a hyd rated compound, comprising the steps of:

i) suspending host cells which overexpress the decarboxylase in a buffer;

ii) adding the substrate compound;

iii) adding a base; and

iv) obtaining the hyd rated compound.

A further aspect of the invention relates to a compound obtained in a process as described above for use in pharmaceutical, cosmetic and/or food industry.

The promiscuous catalytic 'hydratase-activity' of these enzymes was discovered during studies on the regioselective β-carboxylation of p-vinylphenol [28], which expectedly gave p-coumaric acid, but unexpectedly also furnished (S)-1 -(p- hydroxyphenyl)-ethanol as side-product derived via enzymatic hydration of the C=C- bond.

Detailed literature studies revealed puzzling similarities between the (proposed) mechanism of p-coumaric acid decarboxylase from Lactobacillus plantarum [29] and that of hydroxycinnamoyl-CoA hydratase-lyase from Pseudomonas fluorescens

[5,6,12], both of which act via a quinone-methide enolate intermediate. The latter represents a good Michael-acceptor for the nucleophilic 1 ,6-addition of water, which provides a plausible explanation for the enzymatic hydratase-promiscuity of

decarboxylases. Scheme 1 : Asymmetric enzymatic hydration of hydroxystyrenes employing phenolic acid decarboxylases.

First results were obtained when the phenolic acid decarboxylases from

Lactobacillus plantarum (PAD_Lp) and from Bacillus amyloliquefaciens (PAD_Ba) overexpressed in E. coli were applied to p-vinylphenol (1a) in carbonate buffer (3M, pH 8.5) to yield (S)-4-(1 -hydroxyethyl)-phenol (1 b) with good conversions (PDC_Lp: c 82%, e.e. 43%; PDC_Ba: c 64%, e.e. 53%, Table 1 , entry 1 ). The corresponding carboxylation product (p-coumaric acid) was observed only as minor side product

(≤ 5%). In order to examine the potential of this method, further enzymes as well as a range of various substrates were investigated. Based on similarity search (30-80% identity) using available sequences in the NCBI genebank, the following enzymes were chosen: phenolic acid decarboxylases from Mycobacterium colombiense (PAD_Mc), Methylobacterium sp. (PAD_Msp), Pantoea sp. (PAD_Psp), Lactoccocuslactis

(PAD_LI), ferulic acid decarboxylase from Enterobacter sp. (FDC_Esp) and p- hydroxycinnamoyl CoA hydratase-lyase from Pseudomonas fluorescens (HCHL). All genes were synthesised at geneart AG (Regensburg) and subcloned in a common pET vector (pET 21 a or pET 28a). The obtained plasmids were transformed in a standard E. coli host [BL21 (DE3)] using IPTG-induction for overexpression. According to SDS-PAGE analysis, successful overexpression was obtained for all enzymes, which were employed as lyophilized whole-cell biocatalyst to a range of substrates. The absence of competing hydratase-activity of empty E. coli host cells was verified in separate blank experiments. To our delight, almost all decarboxylases were able to catalyse the hydration of hydroxystyrene-type substrates 1a-6a except PDC_Mc from Mycobacterium colombiense. The broad range of hydratase activities was even more remarkable since the 'real' hydratase, hydroxycinnamoyl-CoA hydratase-lyase (HCHL), which was selected for reason of comparison, did not show any activity at all on the hydroxystyrene derivatives described above. All active enzymes displayed similar levels of conversion with respect to a given substrate (c 73-82% for 1a, c 24-45% for 2a, c 63-76% for 3a, c 34-47% for 4a, c 3- 25% for 5a, c < 1 % for 6a). Overall, the data indicate that steric effects play a major role, because p-vinylphenol (1a) and the chloro-analog 3a were accepted best (c 63- 82%, entries 1 , 3) while the conversion continuously dropped by increasing the size and/or number of substituents. An additional methyl or methoxy group (2a, 4a), led to moderate conversions (c 24-47%, entries 2, 4), and more bulky substituents, like an ethoxy group (5a) resulted in a significant decrease of conversion (entry 5) indicating the limit of substrate tolerance. Compound 6a, carrying three substituents on the phenyl ring, was not accepted as substrate (c < 1 , entry 6). It seemed that electronic effects played only a minor rule.

In order to get deeper insight into the enzymatic hydration, the conversion of p- vinylphenol (1a) was monitored over time employing whole cells of PAD_l_p by taking data points for e.e. and c at 0.1 , 0.2, 0.5, 1 , 4, 6, 16 and 22 hours (Figure 1 ). The conversion reached a plateau after approximately 18 hours (c 90%) and carboxylation occurred only as minor side reaction (c < 2%). However, the enantiomeric excess of hydration product 1 b continuously dropped during the course of reaction from ~ 87% at the onset of the reaction to ~ 47% at 22h reaction time. It must be mentioned that a small amount of spontaneous (non-enzymatic) background reaction took place in the absence of biocatalysts (c ~ 6%). All conversion data in Table 1 are corrected for this value accordingly to accurately describe the enzyme-catalysed activity. An

approximate calculation of the stereoselectivity by excluding the non-enzymatic

(spontaneous) background hydration gives an a value of ca. 20.

In order to elucidate the correlation between the hydration and the concentration of bicarbonate, a set of experiments using various buffer systems were performed using 1a as test substrate and whole cells of PAD_Lp as biocatalyst (see fig. 2). These studies showed that an increasing concentration of bicarbonate goes in hand with an increasing formation of the hyd rated product 1 b reaching an optimum at 3M

bicarbonate. The corresponding carboxylation product (p-coumaric acid) was observed only as minor side product (< 5%). The enantiomeric excess of the hydration product (S)-4-(1 -hydroxyethyl)-phenol (1 b) remained constant (e.e. ~ 55%); in the absence of bicarbonate, however, 1 b was obtained in racemic form (e.e.≤ 1 %), which roughly correlates to the non-enzymatic (spontaneous) background reaction, which proved to be independent on the concentration of bicarbonate (c ~ 6%, blank). ln addition, a set of experiments using carbonate buffer (1 M) at different pH values (pH 6.5, 7.5, 8.5, 9.5, see fig. 3) was performed. At pH 6.5 and 7.5 the conversion of the hydration product 1 b was around 45% and dropped significantly by increasing the pH until only spontaneous reaction at pH 9.5 (c -8%) remained. The course of the enantiomeric excess of 1 b showed the same picture which was -34% e.e. at pH 6.5 and 7.5 and dropped to -23% e.e. at pH 8.5. Racemic 1 b was obtained at pH 9.5. The conversion of the carboxylation reaction increased from c -1 .5% at pH 6.5 to c -12% at pH 9.5. Furthermore, two further buffer systems were applied: Tris- buffer (pH 8.5, 100mM) and phosphate buffer (pH 8.5, 100mM) showed hydration of 13% and 28% (e.e. <4%), respectively, whereas the carboxylation reaction was completely eliminated due to the absence of a CO 2 source. Overall, a significant positive influence of bicarbonate on the hydration activity was proven.

Various bicarbonate sources were applied for the hydration reaction differing in the type of the cation (see fig. 4) employing 1a as substrate and PAD_Ba as biocatalyst. Best results were obtained applying KHCO 3 (63% conversion, 55% e.e.), the concurring carboxylation reaction was only 5%. The use of NaHCO 3 displayed similar results concerning conversion and enantiomeric excess (c 52%, e.e. 56%) and the carboxyiated product was obtained as minor side product (c 6%). Similar results (c 52%, e.e. 54%) were obtained with NH 4 HCO3, however, undesired carboxylation increased slightly (c 10%). The use of CSHCO3 resulted in a significant decrease of conversion and enantiomeric excess in the hydration (c 34%, e.e. 44%) and the ratio between hydration and carboxylation became unfavourable, which was even worse using carbonate-based ionic liquids (1 -butyl-3-methylimidazolium hydrogen carbonate solution -50% in methanol/water 2/3; 200 μΙ_).

The influence of the ratio of hydration versus carboxylation depending on the substrate concentration was investigated by using 2a and PaD_Ba. Fig. 5 shows that the hydration clearly dominates over the carboxylation at reduced substrate

concentrations, while the e.e. of 2b was not significantly affected. AC002P 2013-06-14

-9-

Table 1. Activities and stereoselectivities of the asymmetric enzymatic hydration of hydroxystyrenes 1a-6a.

Reaciion conditions: whole lyophilised cells containing the overexpressed enzyme (30mg), substrate (10mM), KHC0 3 (3M), Pi buffer (pH 5.5, 100mM), final pH ~ 8.5, 30°C, 120rpm, 24h; n.d. = not determined due to low conversion; a spontaneous (non-enzymatic) hydration in absence of biocatalyst; b absolute configuration not determined due to low e.e.

Apart from their carboxylation and promiscuous hydration activity, phenolic acid decarboxylases (PADs) are able to catalyze the stereoselective addition of non -natural nucleophiles (Nu) onto the C=C bond of styrene-type compounds. This novel biocatalytic strategy enables a very straightforward route towards interesting synthons for asymmetric synthesis such as thiols, amines etc.

Scheme 2: Stereoselective addition of non-natural nucleophiles onto the C=C bond of p-hydroxystyrene-type substrates.

First results were obtained when methoxylamine was used as non-natural nucleophile applying various recombinant phenolic and ferulic acid decarboxylases (PAD_Lp, PAD JJ, PAD_Ps and FDC_Es) as biocatalyst.

DESCRIPTION OF THE DRAWINGS

Figure 1 : Time study of the hydration of 1a employing PAD_Lp.; e.e. of 1 b. Figure 2: Hydration 1a employing PAD_l_p using various concentrations of bicarbonate; e.e. of 1 b.

Figure 3: Hydration of 1a employing PAD_Lp using a carbonate buffer (1 M) at different pH values; e.e. of 1 b.

Figure 4: Hydration of 1a employing PAD_Ba using various bicarbonate salts; e.e. of 1 b; IL = ionic liquid (1 -butyl-3-methylimidazolium hydrogen carbonate solution -50% in methanol/water 2/3; 200μΙ_).

Figure 5: Relative ratio of hydration versus carboxylation depending on the substrate concentration employing 2a and PaD_Ba; e.e. of 2b.

Fig. 6: Time study of the stereoselective addition of methoxylamine to the C=C bond of 1a employing FDC Es.; e.e. of 1c. Fig. 7: Stereoselective addition of methoxyiamine to the C=C bond of 1a employing FDC Es as biocatalyst at various concentrations of bicarbonate; e.e. of 1c.

Fig. 8: Stereoselective addition of methoxyiamine to the C=C bond of 1a employing FDC Es as biocatalyst at various pH values; e.e. of 1c.

The subject matter of the following definitions is considered embodiments of the present invention:

1 . A process for the stereoselective conversion of a substrate compound using a decarboxylase, wherein said stereoselective conversion is hydration or addition of a non-natural nucleophile.

2. The process according to claim 1 , wherein the decarboxylase is selected from the group consisting of phenolic acid decarboxylase (PAD), ferulic acid

decarboxylase (FDC), and β-coumaric acid decarboxylase (PDC).

3. The process according to claim 1 or 2, wherein said stereoselective conversion is addition of a non-natural nucleophile.

4. The process according to claim 3, wherein the non-natural nucleophile is selected from the group consisting of hydroxylamine, 2-mercaptoethanol,

methoxyiamine, sodium azide, hydrazine, ammonium acetate, methylamine, sodium formate, nitromethane, sodiumbicarbonate, ammonium cyanate, sodium phosphate, ammonium thiocyanate, sodium phosphite, ammonium bromide, sodium borate, ammonium iodide, sodium sulfide, sodium nitrite, sodium sulfite, and ammonia.

5. The process according to claim 1 or 2, wherein said stereoselective conversion is hydration.

6. The process according to any one of claims 1 to 5, wherein the substrate com ound is of formula I,

wherein

each R 1 is independently from one another selected from the group consisting of hydroxy, halogen, Ci -3 alkoxy, C -3 alkyl, Ci. 3 alkenyl, -NH 2 , and

n is 0, 1 , 2, 3 or 4.

7. The process according to any one of claims 1 to 5, wherein the substrate compound is of formula la, wherein

each R 1 is independently from one another selected from the group consisting of hydroxy, halogen, C . 3 alkoxy, C -3 alkyl, Ci -3 alkenyl, -NH 2 , and

n is 0, 1 , 2, 3 or 4.

8. A process for making a compound of formula II, comprising the step of

wherein

each R 1 is independently from one another selected from the group consisting of hydroxy, halogen, Ci-3alkoxy, Ci -3 alkyl, Ci-3alkenyl, -NH 2 , and

n is 0, 1 , 2, 3 or 4.

9. A process for making a compound of formula lla, comprising the step of

wherein

each R 1 is independently from one another selected from the group consisting of hydroxy, halogen, Ci -3 alkoxy, Ci. 3 alkyl, Ci -3 alkenyl, -NH 2 , and

n is 0, 1 , 2, 3 or 4.

10. The process according to claim 8 or 9, wherein a base is added.

1 1 . The process according to claim 8 or 9, wherein the base is bicarbonate, or carbonate.

12. The process according to any one of claims 8 to 1 1 , wherein compound I I or compound l la is obtained with at least 30%, preferably with at least 50%, more preferred with at least 70% conversion. 13. The process according to any one of claims 8 to 12, wherein compound II or compound I la is obtained with at least 25%, preferably with at least 40%, more preferred with at least 55% enantiomeric excess (e.e.).

14. A process for manufacturing a hyd rated compound, comprising the steps of:

i) suspending host cells which overexpress the decarboxylase in a buffer;

ii) adding the substrate compound;

iii) adding a base; and

iv) obtaining the hyd rated compound.

Examples

Materials and Methods

2-Methoxy-4-vinylphenol (2a), KHC0 3 , KH 2 P0 4 and Na 2 P0 4 * 2 H 2 0 were from Sigma Aldrich, p-vinylphenol (1a) was purchased from Alfa Aesar, petroleum ether (boiling range 60 - 95°C) was acquired from VWR.

TLCs were run on silica plates (Merck, silica gel 60, F 254 ); for column chromatography silica gel (Merck, silica gel 60) was used, compounds were detected using UV (254 nm); NMR experiments were acquired either on a Bruker Avance III 300 MHz spectrometer using a 5 mm BBO probe with z-axis gradients at 300 K. Chemical shifts (δ) are reported in ppm and coupling constants (J) are given in Hz. All GC-MS measurements were carried out on an Agilent 7890A GC system, equipped with an Agilent 5975C mass selective detector (electron impact, 70 eV) and a HP-5-MS column (30 m x 0.25 mm x 0.25 μηι) using helium as carrier gas at a flow of

0.55 mL/min. Following temperature program was used in all GC-MS measurements: initial temperature: 100 °C, hold for 0.5 min, 10°C/min, to 300 °C.

Phenolic acid decarboxylase from Lactobacillus plantarum (PAD_Lp, Gl:

300769086) and from Bacillus amyloliquefaciens (PAD_Ba, Gl: 308175189), subcloned in a pET 28a (+) vector, were provided by Byung-Gee Kim (Seoul National University, Korea). Phenolic acid decarboxylase from Mycobacterium colombiense (PAD_Mc), Methylobacterium sp. (PAD_Msp), Pantoea sp. (PAD_Psp),

Lactoccocuslactis (PAD_LI), ferulic acid decarboxylase from Enterobacter sp.

(FDC_Esp) and p-hydroxycinnamoyl CoA hydratase-lyase from Pseudomonas fluorescens (HCHL) were synthesized at Geneart AG (Germany, Regensburg). Their DNA sequences were allocated in the NCBI Genebank (Gl: 31 16017 for HCHL, Gl: 323462934 for FDC_Esp, Gl: 304396594 for PAD_Psp, Gl: 15673912 for PAD_LI, Gl: 342860341 for PAD_Mc, Gl: 168197631 for PAD_ sp).

Codon-optimised sequences of decarboxylases and HCHL (SEQ ID NO:1 ) HCHL (Gl: 31 16017)

MSTYEGRWKTVKVEIEDGIAFVILNRPEKRNAMSPTLNREMIDVLETLEQDPAAGVL V LTGAGEAWTAGMDLKEYFREVDAGPEILQEKIRREASQWQWKLLRMYAKPTIAMVN GWCFGGGFSPLVACDLAICADEATFGLSEINWGIPPGNLVSKAMADTVGHRQSLYYI MTGKTFGGQKAAEMGLVNESVPLAQLREVTIELARNLLEKNPWLRAAKHGFKRCRE LTWEQNEDYLYAKLDQSRLLDTEGGREQGMKQFLDDKSIKPGLQAYKR PAD Ba (Gl: 308175189) (SEQ ID NO:2)

MENFIGSHMIYTYENGWEYEIYIKNDHTIDYRIHSGMVGGRWVRDQEVNIVKLTEGVY KVSWTEPTGTDVSLNFMPNEKRMHGIIFFPKWVHEHPEITVCYQNDYIDVMKESREK YDTYPKYWPEFADITYLNNAGINNEALISEAPYEGMTDDIRAGKLK PAD_Lp (Gl: 300769086) (SEQ ID NO:3)

MTKTFKTLDDFLGTHFIYTYDNGWEYEWYAKNDHTVDYRIHGGMVAGRWVTDQKAD IVMLTEGIYKISWTEPTGTDVALDFMPNEKKLHGTIFFPKWVEEHPEITVTYQNEHIDL MEQSREKYATYPKLWPEFANITYMGDAGQNNEDVISEAPYKEMPNDI RNGKYFDQN YHRLNK

FDC_Esp (Gl: 323462934) (SEQ ID NO:4)

MNTFDKHDLSGFVGKHLVYTYDNGWNYEIYVKNDNTIDYRIHSGLVGNRWVKDQEA YIVRVGESIYKISWTEPTGTDVSLIVNLGDSLFHGTIFFPRWVMNNPEPTVCFQNDHIP LMNSYREAGPAYPTEVIDEFATITFVRDCGANNESVIACAASELPKNFPDNLK

PAD_Psp (Gl: 304396594) (SEQ ID NO:5)

MSTFDKHDLSGIVGKHLVYTYDNGWNYELYIKNASTIDYRIHSGMVGNRWVKNQHVY WRLAQDVYKVSWTEPTGTDVSLAVNLADKIFHGTIFFPRWVMNNPEKTVCFQNDHL EEMAKFRETGPAYPTEIIDEFATLTVIREVGENNESVIDCPPDQLPANWPANLQN

PAD_LI (Gl: 15673912) (SEQ ID NO:6)

MKTFKSLEDFVGTHFIYTYDNGWEYELYVKNDHTIDYRIHGGMVAGRWVKDQEVSLV MLTEGIYKITWTEPTGTDVALDFLPNEGKLHGMIFFPKWVEEHPEITVCFQNDFIDLMH ESREKYETYPKYLVPEFAKITYAAEAGKDNDDVVAQAPYKEMTNDIRNGKYFDQNYK MIKH

PAD_Mc (Gl: 342860341 ) (SEQ ID NO:7)

MTSVTNPIPPQDLSGIVGHRFIYTYANGWQYEMYVKNVTTIDYRIHSGHVGGRWVKG QQVNLVQLDDDSFKISWTEPTGTCVAVNVLPGKRRIHGVIFFPQWIRMHGEHTVCFQ NDHLDEMRAYRDRGPTYPIYEVPEFAYITLFEYVGTDDETVIDTGPEHLPQGWSNRT

N

PADJVisp (Gl: 168197631 ) (SEQ ID NO:8)

MSDLFASTRREEIAPFLGKHFIYTYENGWQYEMYIKNERTIDYRIHSGIVGGRWVRD Q VAHIVRLSDEWKISWDEPTGTTVSVAVNFEERRIHGVIFFPQWIAQDPKRTVCFQNE HLDRMRQYRDAGPTYPKLWDEFASVTFLEDCGIDNQEVIACAPADLPDGYAARQN

Cloning and overexpression of enzymes

Genes were synthesized at Geneart AG (Germany, Regensburg) and subcloned in a pET vector (pET21 a or pET28a). The obtained plasmid was then transformed into chemically competent Escherichia coli BL21 (DE3) cells and heterologous

overexpression was performed as follows:

For preculturing 500 ml_ LB medium [Trypton (10 g/L, Oxoid L0042), yeast extract (5 g/L, Oxoid L21 ), NaCI (5 g/L, Roth 9265.1 )] supplemented with the appropriate antibiotics [ampicillin (100 g/mL, Sigma Aldrich) for PAD_Mc, PAD_Msp, PAD_Psp, PAD_LI and FDC_Esp], kanamycin (50 pg/mL, Roth) for HCHL, PAD_Lp and PAD_Ba] were inoculated with 3 mL ONC (starter culture) and incubated at 37°C and 120 rpm until an ODeoo of 0.6-1 .0 was reached. Then IPTG [175 g/mL, 0.5 mM, Peqlab] was added for induction and the cells left overnight at 20°C and 120 rpm. The next day the cells were harvested by centrifugation (20 min, 8000 rpm, 4°C), washed with phosphate buffer (5 mL, 50 mM, pH 7.5) and centrifuged under the same conditions. The cell pellet thus obtained was resuspended in phosphate buffer (5 mL, 50 mM, pH 7.5), shock frozen in liquid nitrogen followed by lyophilization. Lyophiiized cells were stored at +4°C.

Example 1 : General procedure for the asymmetric enzymatic hydration of substrates 1a-6a

Enzymatic hydration reactions were performed in glass vials capped with septums. Lyophiiized whole cells (30 mg E. coli host cells containing the corresponding overexpressed enzyme) were suspended in phosphate buffer (1 mL, pH 5.5, 100 mM) and were rehyd rated for 30 min. The substrate (10 mM) was added, followed by addition of KHCO 3 (3 M, 300 mg). Thereafter the vials were immediately tightly closed and the mixture was shaken at 30°C and 120 rpm. After 24 h the mixture was centrifuged (13000 rpm, 15 min), an aliquot of 100 pL of each sample was diluted in 1 mL of H 2 O/MeCN (1 :1 ) supplemented with TFA (30 L). After incubation at room temperature for 5 min, the samples were again centrifuged ( 3000 rpm, 15 min) and analyzed on reverse-phase HPLC to determine the conversion.

For the determination of the enantiomeric excess, products were extracted after 24 h with EtOAc (2 x 500 μΐ_) and the combined organic phases were dried over Na 2 S0 . The organic solvent was removed, the residue was dissolved in n-heptane//- propanol (90/10) and analyzed on HPLC to determine the e.e.

Determination of absolute configuration

The absolute configuration of hydration products 2b-5b was determined via co- injection of the hydration product with independently synthesised reference material of known absolute configuration. The latter were obtained via oxidative kinetic resolution of rac-alcohols or by asymmetric bioreduction of the corresponding ketones using alcohol dehydrogenases of known stereopreference: (S)-selective ADH-A from

Rhodococcus ruber [1 ,2], (S)-selective ADH-005 (corresponds to ADH-T from Julich chiral solutions) and (R)-selective ADH from Lactobacillus brevis [3]. Additional proof was obtained by comparison of optical rotation values for 1 b and 2b with literature data.

-(1 -Hydroxyethyl)-2-methoxyphenol (2b)

: NADPH-Oxidase; Scheme 2: Enantioselective enzymatic oxidation using (S)-selective ADH-005.

An aliquot of ADH-005 (50 μΙ_, 77.5 U, corresponds to ADH-T, Julich chiral solutions) was added to a phosphate buffer solution (950 μΙ_, 50 mM, pH 7.5) containing rac-4-(1 -hydroxyethyl)-2-methoxyphenol (50 mM), cofactor NADP + (1 mM) and nicotinamide oxidase YncD (10 μΙ_) for cofactor-recycling [4,5]. The mixture was in- cubated at 30°C and 120 rpm. After 24 h products were extracted with EtOAc

(2x500 μΙ_). The combined organic phases were dried over Na2S0 4 , the organic solvent was removed and the residue was dissolved in a mixture of n-heptane//- propanol (90:10). The samples were analyzed on a chiral HPLC column to s

the enantiomeric excess.

Example 3: 4-(1 -Hydroxyethyl)phenol (1b), 4-(1 -hydroxyethyl)-2- chlorophenol (3b) and 4-(1 -hydroxyethyl)-2-methylphenol (4b)

Scheme 3: Asymmetric bioreduction of ketones using (S)-selective ADH-A [1 ,2]

An aliquot of ADH-A (50 μΙ_, 3 U) was added to a phosphate buffer solution (950 μΙ_, 50 mM, pH 7.5) containing the substrate (4-hydroxyacetophenone, 3-chloro-4- hydroxyacetophenone, or 3-methyl-4-hydroxyaceophenone, 50 mM), the cofactor NAD + (1 mM) and the co-substrate /-propanol (5 μΙ_). The mixture was incubated at 30°C and 120 rpm. After 24h products were extracted with EtOAc (2x500 μΙ_). The combined organic phases were dried over Na 2 SO 4 , the solvent was removed and the residue was dissolved in n-heptane//-propanol (90:10). The samples were analyzed on a chiral HPLC to determine the enantiomeric excess.

Example 4: Determination of optical rotation of (S)-4-(1 -hydroxy- ethyl)phenol (1 b) and (S)-4-(1 -hydroxyethyi)-2-methoxy-phenol (2b)

To determine the optical rotation value of 1 b and 2b the asymmetric hydration of 1a and 2a applying PAD from Bacillus amyloliquefaciens was scaled up 10 times. After 24h, hydration products were extracted with EtOAc (2 x 500μΙ_) and the combined organic phases were dried over Na 2 SO 4 . Afterwards the solvent was removed and the optical rotation was measured at 589 nm (Na-line) using a Perkin-Elmer polarimeter 341 .

1 b: [α]ο 20 -37 (c 0.125, EtOH). The absolute configuration of 1 b was determined to be (S) by comparison with literature values [a] D 20 -48 (c 4.2, EtOH) [6].

(2b: [a] D 20 -41 (c 0.23, CHCI 3 ). The absolute configuration of 2b was determined to be (S) by comparison with literature values [a] D 20 +41 (c 0.8, CHCI 3 ) for the (R)- enantiomer [7].

Example 5: Synthesis of substrates Hydroxystyrenes 3a-6a were synthesized according to the following general Wittig-reaction protocol [8]

2-Chloro-4-vinylphenol (3a), 2-methyl-4-vinylphenol (4a), 2-ethoxy-4-vinylphenol (5a) and 2,6-dimethoxy-4-vinylphenol (6a): Sodium bis(trimethylsilyl)amide (1 .05 g, 5.7 mmol, Sigma Aldrich) was added under cooling to a stirred solution of methyl triphenylphosphonium bromide (1 .85 g, 5.2 mmol, Sigma Aldrich) in freshly distilled THF (8 mL). Immediately afterwards a yellow colour change could be observed. After 1 .5 h of stirring the corresponding solid aldehyde (2.54mmol, Sigma Aldrich) was added to the ylid-solution and the stirring was continued for 4 h. The mixture was acidified using H 2 SO 4 (0.1 M, 5 mL) and extracted with CH2CI2. The combined organic phases were dried over Na 2 S0 4 , evaporated and purified by flash chromatography on silica.

2-Chloro-4-vinylphenol (3a): Eluent for flash chromatography: petroleum ether/EtOAc (2:1 ), isolated yield 80% (0.31 g, 2.03 mmol), TLC: R f = 0.63 (silica, petroleum ether/EtOAc 2:1 ); GC-MS: m/z 154; 1 H-NMR (MeCN-d 3 ): δ 5.07 (1 H, dd, J = 0.56 and 10.95); δ 5.58 (1 H, dd, J = 0.67 and 17.63); δ 6.54 (1 H, dd, J = 10.95 and 17.63); δ 6.84 (1 H, d, J = 8.40); δ 7.17 (1 H, dd, J = 1 .97 and 8.21 ); δ 7.35 (1 H, d, J = 2.03); 13 C-NMR (MeCN-d 3 ): δ 1 12.3, 1 16.6, 120.2, 126.0, 127.4, 131 .1 , 135.1 , 152.0.

2-Methyl-4-vinylphenol (4a): Eluent for flash chromatography: petroleum ether/EtOAc (5:1 ), isolated yield 63% (0.21 g, 1 .60mmol), TLC: R, = 0.48 (silica, petroleum ether/EtOAc 5:1 ); GC-MS: m/z 134; 1 H-NMR (MeCN-d 3 ): δ 2.07 (3H, s), δ 4.96 (1 H, dd, J = 0.79 and 10.93); δ 5.50 (1 H, dd, J = 0.87 and 17.64); δ 6.53 (1 H, dd, J = 10.94 and 17.65); δ 6.64 (1 H, d, J = 8.22); δ 6.76 (1 H, s); δ 7.02 (1 H, dd, J = 1 .66 and 8.21 ); δ 7.12 (1 H, s); 13 C-NMR (MeCN-d 3 ): δ 15.14, 1 10.3, 114.7, 124.4, 124.9, 128.7, 129.6, 136.6, 154.9.

2-Ethoxy-4-vinylphenol (5a): Eluent for flash chromatography: petroleum ether/EtOAc (10:1 ), isolated yield 16% (67.3mg, 0.41 mmol), TLC: R f = 0.39 (silica, petroleum ether/EtOAc 10:1 ); GC-MS: m/z 164 1 H-NMR (acetone-d 6 ): δ 1 .39 (3H, t, J = 6.98); δ 4.13 (2H, q, J = 6.98); δ 5.05 (1 H, dd, J = 0.78 and 10.91 ); δ 5.62 (1 H, dd, J = 0.89 and 17.60); δ 6.65 (1 H, dd, J = 10.91 and 17.61 ); δ 6.80 (1 H, d, J = 8.13); δ 6.91 (1 H, dd, J = 1.75 and 8.13), δ 7.09 (1 H, d, J = 1 .73), δ 7.65 (1 H, s); 13 C-NMR (acetone- de): δ 14.17, 64.12,109.93, 1 10.14, 1 14.82, 1 19.72, 129.69, 136.93, 146.72, 146.93. (acetone-d6 contained H2O δ H2O peak in NMR) 2,6-Dimethoxy-4-vinylphenol (6a): Eluent for flash chromatography: CH 2 CI 2 /MeOH (10:1 ), isolated yield 59% (0.27g, 1 .50mmol), TLC: R, = 0.94 (silica, CH 2 CI 2 /MeOH 10:1 ); GC-MS: m/z 180; Ή-NMR (CDC ): δ 3.93 (6H, s); δ 5.17 (1 H, dd, J = 0.61 and 10.83); δ 5.56 (1 H, s); δ 5.62 (1 H, dd, J = 0.70 and 17.50); δ 6.64 (1 H, dd, J = 10.78 and 17.47); δ 6.67 (2H, s); 13 C-NMR (CDCI 3 ): δ 56.24, 56.27, 103.0, 1 1 1 .8, 129.2, 134.8, 136.8, 147.1 . NMR data corresponded to literature [9].

Example 6: Synthesis of reference materials

Racemic reference material for alcohols 1 b, 3b and 4b was obtained via reduction of the corresponding ketones.

rac-4-(1 -Hydroxyethyl)phenol (1b), rac-4-(1 -hydroxyethyl)-2-chlorophenol (3b) and rac-4-(1 -hydroxyethyl)-2-methylphenol (4b) [10]: Ketone (6 mmol, Sigma Aldrich) was added to a stirred solution of CeC x 7H 2 0 (6 mmol, Sigma Aldrich) in methanol (20 ml_). Then sodium borohydride (6 mmol) was added in small portions over 3 min, the once clear solution turned milky-white and the stirring was continued for 20 min at room temperature. The mixture was quenched with a saturated (NH 4 ) 2 S0 4 -solution (50 ml_) and extracted with diethyl ether (3 x 30 ml_). The combined organic phases were washed with brine, dried over Na 2 S0 4 , evaporated and purified by flash

chromatography on silica.

1 b: Eluent for flash chromatography: petroleum ether/EtOAc (1 :2), isolated yield 79% (0.72g, 5.24mmol), TLC: R f = 0.38 (silica, petroleum ether/EtOAc 1 :2); GC-MS: m/z 138. Ή-NMR (MeCN-d 3 ): δ 1 .37 (3H, d, J = 6.43), δ 3.14 (1 H, d, J = 3.94); δ 4.70- 4.78 (1 H, m); δ 6.78 (2H, d, J = 8.54); δ 6.89 (1 H, s); δ 7.21 (2H, d, J = 8.41 ); 13 C-NMR (MeCN-ds): δ 24.83, 68.81 , 114.8, 126.7, 138.1 , 155.8.

3b: Eluent for flash chromatography: petroleum ether/EtOAc (1 :2), isolated yield 78% (0.82 g, 4.76 mmol), TLC: R, = 0.70 (silica, petroleum ether/EtOAc 1 :2); GC-MS: m/z 172. 1 H-NMR (MeCN-d 3 ): δ 1 .36 (3H, d, J = 6.44), δ 3.25 (1 H,s), δ 4.43 (1 H, q, J = 6.16 and 6.20); δ 6.93 (1 H, d, J = 8.34); δ 7.16 (1 H, dd, J = 1 .93 and 8.39; δ 7.34 (1 H, d, J = 2.01 ); 13 C-NMR (MeCN-ds): δ 24.74, 68.22, 116.3, 1 19.6, 125.3, 126.8, 140.0, 151 .1 .

4b: Eluent for flash chromatography: petroleum ether/EtOAc (1 :1 ), isolated yield

87% (0.70 mg, 4.58 mmol), TLC: R f = 0.51 (silica, petroleum ether/EtOAc 1 :1 ); GC- MS: m/z 152. 1 H-NMR (MeOH-d 4 ): δ 1.29 (3H, d, J = 0.47), δ 2.07 (1 H, s); δ 4.59 (1 H, q, J = 6.44); δ 6.58 (1 H, d, J = 8.17); δ 6.88 (1 H, dd, J = 8.17 and 2.07); δ 6.89 (1 H, s) (H 2 0 peak from solvent: δ 4.78, 2H, s); 13 C-NMR (MeOH-d 4 ): δ 16.45, 25.58, 70.84, 1 15.4, 125.1 , 125.4, 129.3, 138.3, 155.8.

4-(1 -Hydroxyethyl)-2-ethoxyphenol (5b): 3-ethoxy-4-hydroxybenzaldehyde (1.9 mmol, 310 mg, Sigma Aldrich) dissolved in anhydrous diethyl ether (2 ml_) was added under argon to a stirred solution of CS2CO3 (1.95 g, 6 mmol, Sigma Aldrich) in diethyl ether (4 ml_) at room temperature. After 30 min methyl magnesium bromide (1 ml_ of a 3M solution, 3 mmol, Sigma Aldrich) was slowly added under ice-cooling and stirring was continued for 2 h. Thereafter diethyl ether (1 ml_) and another portion of methyl magnesium bromide (1 ml_) were added and stirring was continued overnight. The reaction was quenched with a saturated NH 4 CI-solution, the pH was set to 4 with HCI (6 M) and products were extracted with diethyl ether (3 x 30ml_). The combined organic phases were dried over Na 2 S0 4 , the solvent was evaporated and products were purified by flash chromatography on silica using petroleum ether/EtOAc (1 :1 ) as eluent; isolated yield 6.4% (22 mg, 0.12 mmol), TLC: R f = 0.51 (silica, petroleum ether/EtOAc 1 :1 ); GC-MS: m/z 182. Ή-NMR (MeOH-d 4 ): δ 1 .40-1 .45 (6H, m), 5 4.10 (2H, q, J = 6.99); δ 4.74 (1 H, q, J = 6.42); δ 6.75-6.81 (2H, m); δ 6.96 (1 H, s) (H 2 0 peak from solvent: δ 4.89, 2H, s) ; 13 C-NMR (MeOH-d 4 ): δ 13.78, 24.16, 64.10, 69.39, 1 10.23, 1 14.56, 1 17.85, 137.72, 145.50, 146.54.

Example 7: Determination of conversion

All analyses were carried out on a Shimadzu HPLC system from equipped with a diode array detector (SPD-M20A) and a reversed-phase Phenomenex Luna column

C18 (2) 100A 250 * 4.600 mm 5 pm, column temperature 24°C. Conversions were determined by comparison with calibration curves for products and substrates prepared with authentic reference material.

The method for 1a/1 b, 2a/2b and 5a/5b was run over 12min with H 2 0/TFA (0.1 %) as the mobile phase at a flow rate of 1 mL/min and a MeCN/TFA (0.1 %) gradient (0-2 min 15%, 2-10 min 0-100%, 10-12 min 100%). The column temperature was 24°C and compounds were spectrophotometrically detected at 254 or 270 nm.

Retention times: 1a 10.83 min, 1 b 7.62 min, 2a 1 1 .01 min, 2b 7.90 min, 5a 1 1 .81 min and 5b 8.77 min.

The method for 3a/3b and 4a/4b was run over 17 min with H 2 0/TFA (0.1 %) as the mobile phase at a flow rate of 1 mL/min and a MeCN TFA (0.1 %) gradient (0-2 min 0%, 2-15 min 0-100%, 15-17 min 100%). The column temperature was 24°C and compounds were spectrophotometrically detected at 270 and 280 nm respectively. Retention times: 3a 15.22 min, 3b 1 1 .75 min 4a 15.1 1 min, 4b 1 1 .24 min. The method for 6a was run over 32min with H 2 O/TFA (0.1 %) as the mobile phase at a flow rate of 0.5 mL/min and an MeCN/TFA (0.1 %) gradient (0-2min 0%, 2- 30 min 0-100%, 30-32 min 100%). Compounds were spectrophotometncaily detected at 280 nm. Retention time: 6a 26.20 min.

Example 8: Determination of enantiomeric excess

All analyses were carried out on a normal-phase Shimadzu HPLC system. The absolute configurations were determined by co-injection with independently

synthesized reference material, which was obtained as described above.

The enantiomeric excess of 1 b-5b was determined using a Chiralcel OD-H column (25 cm x 0.46 cm). The method for 1b, 2b, 4b and 5b was run over 20 min with n-heptane//-propanol (90:10) as the mobile phase at a flow rate of 1 .0 mL/min. The column temperature was 25°C and compounds were spectrophotometncaily detected at 270 nm or 280 nm. Retention times: (R)-1 b 1 1 .40 min, (S)-1 b 12.40 min, (R)-2b 15.26 min, (S)-2b 16.26 min, (R)-4b 9.30 min, (S)-4b 10.79 min, rac-5b 1 1 .28 and 12.51 min, absolute configuration was not determined.

The method for 3b was run over 60min with n-heptane//-propanol (95:5) as the mobile phase at a flow rate of 0.5 mL/min. The column temperature was 25°C and compounds were spectrophotometncaily detected at 280nm. Retention times: (R)-3b 39.66 min, (S)-3b 43.17 min.

Example 9: Addition of non-natural nucleo hiles

1a 1c

1a was used as substrate in carbonate buffer (100 mM, pH 8.5) to yield non- racemic 4-(1 -(methoxyamino)ethyl)phenol (1c) with excellent conversions (see table 2). Furthermore, a set of blank experiments were carried out in order to verify that the observed addition of the non-natural nucleophile is truly enzyme catalysed and not only a spontaneous non-enzymatic background reaction. For the control experiments the standard reaction conditions were applied either in the absence of the biocatalyst or employing heat denatured enzyme. In both cases only traces of 1c were observed (<1 %). However, a small amount of the standard hydration product (sec-alcohol 1 b) has been detected (5% and 7%, respectively employing no or heat denatured biocatalyst, after 24 h) due to spontaneous (non-enzymatic) background reactions.

Table 2. Conversions and optical purity (expressed as e.e. of the product 1 c) of the stereoselective addition of methoxylamine applying various phenolic acid decarboxylases. enzyme conversion [%] e.e. of 1c [%]

FDC_Es 94 38

PAD_Lp 81 1 1

PAD_LI 80 rac

PAD_Ps 93 13

Example 10: Time study

The conversion of 1 a was monitored over time employing recombinant FDC Es - overexpressing whole cells by taking data points for e.e. and conversion at 0, 0.5, 1 , 4, 6, and 24 hours (Figure 6). The conversion steadily increased (-20% after 30 min) to almost 95% after 24 hours. The standard hydration product [sec-alcohol, (S)-4-(1 - hydroxyethyl)-phenol (1 b)] occurred only as minor side product (-6% conversion after 24 h). The optical purity of the corresponding non-racemic product 4-(1 -(methoxy- amino)ethyl)phenol (1 c) reached a plateau after approximately 6 h (e.e. -38%) and stayed constant for the residual reaction time (latest data point after 24 hours).

Example 11 : Bicarbonate concentration study

In order to elucidate the correlation between the stereoselective addition of non- natural nucleophiles and the concentration of bicarbonate, a set of experiments using various bicarbonate concentrations (20 mM - 200 mM) were performed. In the model reaction 1 a was applied as test substrate, methoxylamine as non-natural nucleophile and whole cells of FDC Es as biocatalyst (see fig. 7). These studies showed that an increasing concentration of bicarbonate goes hand in hand with a slight decrease in conversion of the corresponding product 1c (conv. = 94% at 20 mM bicarbonate; conv. = 88% at 200 mM bicarbonate). The corresponding sec-alcohol (standard hydration product 1 b) was observed only as side product (< 10%) which correlated to the time study. The enantiomeric excess of 1c remained constant (e.e. -34%) at different bicarbonate concentrations.

Example 12: pH study

In addition, a set of experiments using carbonate buffer (100 mM) at different pH values (pH 7.5 - 9.5) (see fig. 3) was performed. In the model reaction 1a was applied as test substrate, methoxylamine as non-natural nucleophile and whole cells of FDC Es as biocatalyst (see fig. 3). The pH window of the enzymatic addition reaction is rather broad since no considerable difference in conversion (~ 89%) as well as enantiomeric excess (-20%) of the corresponding product 1c has been detected. The corresponding sec-alcohol (standard hydration product 1 b) was observed only as side product (< 10%) which correlated to the studies described above.

Based on these results various non-natural nucleophiles will be evaluated. The candidates are summarized in Table 2.

Table 3: Non-natural nucleophile candidates [30]. non-natural nucleophiles non-natural nucleophiles hydroxylamine 2-mercaptoethanol

methoxylamine sodium azide

hydrazine ammonium acetate

methylamine sodium formate

nitromethane sodiumbicarbonate

ammonium cyanate sodium phosphate

ammonium thiocyanate sodium phosphite

ammonium bromide sodium borate

ammonium iodide sodium sulfide

sodium nitrite sodium sulfite

ammonia

Example 13: Addition of n on -natural nucleophiles to the C=C bond of styrene-type substrates

Lyophilized whole cells (30 mg E. coli host cells containing the corresponding overexpressed enzyme) were resuspended in bicarbonate buffer (900 μΙ_, pH 8.5, 100 mM) and rehyd rated for 30 min. Thereafter the non-natural nucleophile (100 mM) and the substrate 1a (10 mM, 12 μΙ_ of a 10% solution) were added and the reaction mixture was incubated at 30°C and 120 rpm. After ~-24h the reaction mixture was extracted with ethyl acetate (2 x 500 μ[_), the organic phases were combined and dried over Na 2 S0 4 . After that the solvent volume was reduced under a N 2 gas stream (from 1 ml_ to -300 μΙ_) and the conversion was measured on the GC-MS.

For the bicarbonate concentration study as well as the pH study the applied bicarbonate buffer was changed accordingly. In case of the time study, standard reaction conditions were applied but the reaction was stopped after different time intervals (0.5, 1 , 4, 6, and 24 hours). Work up was performed as mentioned above for all screening procedures.

Determination of conversion

All nucleophilic addition reactions were analysed via GC-MS. The

measurements were carried out on an Agilent 7890A GC system, equipped with an Agilent 5975C mass selective detector (electron impact, 70eV) and a HP-5-MS column (30 m x 0.25 mm x 0.25 m) using helium as carrier gas at a flow of 0.55 mL/min. The following temperature program was used: initial temperature: 100°C, hold for 0.5 min, 10°C/min to 300°C.

Determination of enantiomeric excess

Analyses of the enantiomeric excess of 4-(1 -(methoxyamino)ethyl)phenol (1 c) were carried out on a Shimadzu HPLC system equipped with a diode array detector (SPD-M20A and a Chiralpak AD column (25cm x 0.46cm). The method for was run over 22min with n-heptane/i-propanol (90:10) as the mobile phase at a flow rate of 1 .0 mL/min. The column temperature was 25°C and compounds were

spectrophotometrically detected at 280nm. Retention times: 1 st enantiomer: 9.25 min; 2 nd enantiomer: 9.99 min. References:

[I] H.-D. Hahn, G. Dambkes, N. Rupprich, H. Bahl, Butanols in: LJISmann's

Encyclopedia of Industrial Chemistry, Electronic version, Wiley-VCH, Weinheim, 2010.

[2] E. Hartmann, D. J. Vyas, M. Oesterreich, Chem. Commun. 201 1 , 47, 7917-1932.

[3] S.-Q. Wang, Z.-W. Wang, L.-C. Yang, J.-l. Dong, C.-Q. Chi, D.-N. Sui, Y.-Z. Wang, J.-G. Ren, M.-Y. Hung, Y.-Y. Jiang, J. Mol. Catal. A: Chem. 2007, 264, 60-65.

[4] A. J. Boersma, D. Coquiere, D. Geerdink, F. Rosati, B. L. Feringa, G. Roelfes, Nat. Chem. 2010, 2, 991 -995.

[5] J. Jin, U. Hanefeld, Chem. Commun. 201 1 , 47, 2502-2510.

[6] J. P. Bennett, L. Bertin, B. Moulton, I. J. S. Fairlamb, A. M. Brzozowski, N. J.

Walton, G. Grogan, Biochem. J. 2008, 414, 281 -289.

[7] A. Liese, K. Seelbach, A. Buchholz, J. Haberland, in: Industrial Biotransformations, ed. A. Liese, K. Seelbach, C. Wandrey, Wiley-VCH, Weinheim, 2006, 2nd ed, pp 465 and pp 488.

[8] M. Wubbolts in: Enzyme Catalysis in Organic Synthesis, ed. K Drauz, H.

Waldmann, Wiley-VCH, Weinheim, 2002, 2nd ed, pp 686-697.

[9] G. Agnihotri, H.-w. Liu, Bioorg. Med Chem. 2003, 11 , 9-20.

[10] B. J. Bahnson, V. E. Anderson, G. A. Petsko, Biochem. 2002, 41 , 2621 -2629.

[I I] A. F. Bell, Y. Feng, H. A. Hofstein, S. Parikh, J. Wu, M. J. Rudolph, C. Kisker, A. Whitty, P. J. Tonge, Chem. Biol. 2002, 9, 1247-1255.

[12] P. M Leonard, A. M Brzozowski, A. Lebedev, C. M. Marshall, D. J. Smith, C. S. Verma, N. J. Walton, G. Grogan, Acta Cryst. D 2006, 62, 1494-1501.

[13] P. M Leonard, C. M. Marshall, E. J. Dodson, N. J. Walton, G. Grogan, Acta Cryst. D 2004, 60, 2343-2345.

[14] A. Mitra, Y. Kitamura, M. J. Gasson, A. Narbad, A. J. Parr, J. Payne, M. J. C.

Rhodes, C. Sewter, N. J. Walton, Arch. Biochem. Biophys. 1999, 365, 10-16.

[15] J. Jin, P. C. Oskam, S. K. Karmee, A. J. J. Straathof, U. Hanefeld, Chem.

Commun. 2010, 46, 8588-8590.

[16] J. Jin, A. J. J. Straathof, M. W. H. Pinske, U. Hanefeld, Appl. Microbiol. Biotechnol. 2011 , 89, 1831 -1840.

[17] F. tenBrink, B. Schink, P. M. H. Kroneck, J. Bacteriol. 201 1 , 193, 1229-1236.

[18] R.-Z. Liao, F. Himo, ACS Catal. 2011 , 1 , 937-944.

[19] R.-Z. Liao, J.-G. Yu, F. Himo, Proc. Natl. Acad. Sci. USA 2010, 107, 22523-22527. [20] G. B. Seiffert, G. M. Ullmann, A. Messerschmidt, B. Schink, P. M. H. Kroneck, O.

EinsSe, Proc. Natl. Acad. Sci. USA 2007, 104, 3073-3077.

[21] L. E. Bevers, M. W. H. Pinkse, P. D. E. M. Verhaert, W. R. J. Hagen, J. Bacteriol. 2009, 191 , 5010-5012.

[22] A. Voikov, A. Liavonchanka, O. Kamneva, T. Fiedler, C. Goebel, B. Kreikemeyer, I. Feussner, J. Biol. Chem. 2010, 285, 10353-10361 .

[23] C. S. Turbek, D. A. Simth, C. L. Schardl, FEMS Microbiol. Lett. 1992, 94, 187-190.

[24] J. A. Maresca, J. E. Graham, A. D. Pryant, Photosynth. Res. 2008, 97, 121 -140.

[25] D. Brodkorb, M. Gottschall, R. Marmulla, F. Luddeke, J. Harder, J. Biol. Chem. 2010, 285, 30436-30442.

[26] D. M. Smith, B. T. Golding, L. Radom, J. Am. Chem. Soc. 1999, 121 , 5700-5704.

[27] T. Kamachi, T. Toraya, K. Yoshizawa, J. Am Chem. Soc. 2004, 126, 16207- 16216.

[28] C. Wuensch, S. M. Glueck, J. Gross, D. Koszelewski, M. Schober, K. Faber, Org. Lett. 2012, 14, 1974-1977.

[29] H. Rodriguez, I. Angulo, B. de las Rivas, N. Campillo, J. A. Paez, R. Munoz, J. M. Mancheno, Proteins 2010, 78, 1662-1676.

[30] B. Weiner, G. J. Poelarends, D. B. Janssen, B. L. Feringa, Chem. Eur. J. 2008,

14, 10094-10100.