WO2008145620A1 - Process for the deprotection of aryl group protected amines employing oxidases - Google Patents

Abstract

In organic synthesis, amine functionalities are often protected by a protecting group. The invention relates to a process for the deprotection of a protected amine compound, wherein the protect ed amine compound which comprises a protecting group attached to the N-atom of the amine, is contacted with an oxidative enzyme and an oxidising agent that may optionally be formed in situ. The invention also relates to the use of oxidative enzymes in a process for deprotection of a protected amine compound, in particular protected secondary or tertiary amine compounds.

Description

PROCESS FOR THE DEPROTECTION OF ARYL GROUP PROTECTED AMINES EMPLOYING OXIDASES

The invention relates to a process for the deprotection of protected amines and the use of oxidative enzymes in a process for the deprotection of protected 5 amines.

Protecting groups, in particular the para-methoxyphenyl (PMP) group, are increasingly used as a nitrogen protecting group for amines, in particular secondary and tertiary amines. In particular in proline-catalyzed asymmetric Mannich reactions, the PMP protecting group appeared to be a crucial element for reaching high 10 enantioselectivities, as has been described in for example M. M. B. Marques, Angew. Chem. 2006, 118, 356-360. Until recently, industrial application of the PMP-protecting group has been thwarted by a lack of cheap and practical deprotection methods. In most cases, eerie ammonium nitrate (CAN) has been used for deprotection, which is expensive and highly toxic. In for example Janey et al, J. Org. Chem. 2006, 71, 15 390-392, phenyl iodoacetate has been described as an alternative for CAN. Another chemical alternative for the deprotection has been disclosed by Verkade et al, Tetrahedron Lett.2006, 47, 8109-8113, comprising the use of electrophilic halogen- containing oxidants for PMP-deprotection. Another alternative for deprotection has been disclosed by De Lamo Marin et al, J. Org. Chem. 2005, 70, 10592-10595, 20 wherein electrochemical deprotection has been proposed. However, known approaches do not provide viable alternatives for industrial application.

Thus, the present invention aims at providing a further alternative for the deprotection of protected amine compounds.

Surprisingly, it has been found that oxidative enzymes may be used 25 to effect deprotection of protected amine compounds.

Therefore, the invention relates to a process for the deprotection of protected amines, wherein the protected amine compound is contacted with an oxidative enzyme and an oxidising agent.

It is an advantage of the process according to the invention that the

30 deprotection may be carried out under mild conditions. Mild conditions are for example ambient temperature, or temperatures not much higher than 40°C, or aqueous solutions, or conditions which allow reactions in the absence of strong acids or strong bases. It is an advantage of the process according to the invention that when protected amine compounds having one or more chiral centres at the α- and/or β carbon atom of 35 the amine are deprotected, the deprotection may be carried out under conditions mild enough not to cause racemisation or epimerization of the chiral centre(s) . It also is an advantage of the process according to the invention that no stoichiometric amounts of relatively expensive oxidants such as CAN, phenyl iodoacetate and halogen containing oxidants need to be used. In particular, the para-methoxybenzyl (PMB) group is increasingly used as a nitrogen protecting group for amines. PMB deprotection from amines can usually only be done by hydrogenation using a Pd containing catalyst (see also T.W. Greene and P. G. M. Wuts in "Protective groups in organic synthesis", 3^rd edition, Wiley, 1999 page 581 ). These conditions also cause deprotection of N-benzyl protected amines so that the PMB group can not be selectively deprotected from an amine in the presence of an N-benzyl protected amine in the same molecule. Thus, it is an advantage of the process according to the invention that it allows deprotection of N- PMB protected amines leaving N-benzyl protection that may be present in the same molecule or in another molecule present during the deprotection fo the PMB-protected amine intact.

According to the invention, the amino group of the protected amine compound is preferably protected by an optionally substituted aryl or arylmethyl group, most preferably by an optionally substituted C₆ - C₁₄ aryl or arylmethyl group. Suitable examples of such optionally substituted aryl or arylmethyl groups include optionally substituted phenyl, naphthyl and benzyl groups. Additionally, it is preferred that such aryl or arylmethyl groups are activated by electron-donating groups, wherein the aryl or arylmethyl group is preferably substituted at positions that maximise the activating effect of the electron-donating substituent. Such substitution patterns are well known to the person skilled in the art and include for example the ortho- and para-positions of a phenyl ring. Consequently, it is preferred according to the invention that the amino group of the protected amine compound is protected with a phenyl or benzyl group that is substituted with an electron-donating substituent, wherein the phenyl or benzyl group is substituted at an ortho-position and/or the para-position. Obviously, the phenyl or benzyl group may be substituted with more than one electron-donating substituents, wherein the electron-donating substituent may be the same or different. In case of more than one substituent the phenyl or benzyl group may be substituted with two substituents wherein one substituent is at the para-position and the other at the ortho- position. An electron-donating group is herein defined as a group that releases electrons into a reaction center and as such stabilizes electron deficient carbocatjons. Preferably, the electron-donating group has a Hammett σ_p-constant of less than -0.14. Examples for suitable electron-donating groups are methyl (σ_p = -0.14), t-butyl (σ_p = - 0.15) and methoxy (σ_p = -0.28). The values for σ_p are taken from J. March, Advanced Organic Chemistry, 4^th Ed., page 280 (Table 9.4), 1992. Preferred electron-donating groups include linear or branched Ci - C₆ alkyl groups, cyclic C₃ - C₆ alkyl groups, linear or branched Ci - C₆ alkoxy groups, cyclic C₃ - C₆ alkoxy groups, linear or branched Ci - C₆ alkylthio groups, cyclic C₃ - C₆ alkylthio groups and OH. More preferred electron-donating groups are linear or branched Ci - C₆ alkyl groups, cyclic C₃ - C₆ alkyl groups, linear or branched Ci - C₆ alkoxy groups and cyclic C₃ - C₆ alkoxy groups. Even more preferred electron-donating groups are linear or branched Ci - C₆ alkoxy groups and cyclic C₃ - C₆ alkoxy groups. Yet even more preferred electron-donating groups are linear or branched Ci - C₆ alkoxy groups, even yet more preferably linear or branched C₁ - C₄ alkoxy groups. Most preferably, the electron- donating group is methoxy. If the phenyl or benzyl group has more than one substituent, their combined electron-donating capability is preferably equivalent to a Hammett σ_p-constant of less than -0.14. It is well known to the person skilled in the art which combinations of substituents provide such an electron-donating capability.

Preferred protecting groups are ortho-methoxyphenyl, para- methoxyphenyl (PMP), ortho-hydroxyphenyl, para-hydroxyphenyl and para- methoxybenzyl. The most preferred protecting group is the p-methoxyphenyl-group.

The protected amine may be derived from the protecting of any N-atoms for which protection is known. Typically, the N-atom of a secondary or tertiary amine group is protected, and may now be deprotected by the process according to the invention. Conversion of protected imines may result in protected amines, that may also be deprotected by a process according to the invention.

In the framework of this invention deprotection is defined as removing the protecting group from the N-atom.

In Scheme I an example of a typical deprotection reaction has been shown. With [O] is meant the oxidant. - A -

Scheme 1

The process according to the invention may be suitably applied to effect deprotection of any protected amine compound. Preferred compounds to be deprotected by the process according to the invention are for example compounds according to formula 1.

Formula 1 wherein PG means Protecting Group,

R⁴ = H, or a (substituted) alkyl, (substituted) cycloalkyl, (substituted) alkenyl, (substituted) alkynyl or (substituted) aryl group

R¹, R² and R³ are each independently H or (substituted) alkyl, (substituted) cycloalkyl, (substituted) alkenyl, (substituted) alkynyl or (substituted) aryl groups or carboxylic acid groups or salts thereof, carboxylic esters, carboxylic thioesters or carboxamides. provided that at least two out of R¹, R² and R³ are not H and provided that not more than two groups chosen from R¹, R² and R³ are carboxylic acid groups or salts thereof, carboxylic esters, carboxylic thioesters or carboxamides.

R⁴ can also be a protecting group, which is the same or different from the PG group. In case R⁴ is the same PG then the removal of that group may take place simultaneously with the removal of the PG-group. Upon deprotection, a compound according to formula I is obtained, but with the one or two protecting groups replaced by H-atoms.

In case the protected amine contains an unprotected OH-group at the β-position, such as shown in formula 2, submission to PG removal by an oxidative enzyme of the present invention may result in occurrence of side reactions due to (partial) cleavage of the C-C bond of the 1 ,2-amino-alcohol moiety.

Formula 2

In formula 2, R² and R⁴ are as defined above in formula 1 and R⁵ is H or a (substituted) alkyl, (substituted) cycloalkyl, (substituted) alkenyl, (substituted) alkynyl or (substituted) aryl group. These side reactions may be partially or fully avoided by first protecting the OH-group at the β-position, then performing the amine deprotection with an oxidative enzyme of the present invention, and finally removing the OH-protecting group. For instance, it is envisaged that the OH-group can be protected with a benzyl group, using benzylbromide and sodium hydride, followed by amine deprotection with a laccase enzyme - with limited or no side reactions due to cleavage of the C-C bond of the 1 ,2-amino-alcohol moiety - and finally removal of the benzyl group using hydrogenation with hydrogen and Pd/C under acidic conditions, giving the desired deprotected 1 ,2-amino-alcohol in high yield. The OH-group can, for example, also be protected with a tert-butyl-diphenylsilyl group, using tert-butyl-diphenylsilylchloride in the presence of imidazole, followed by amine deprotection with a laccase enzyme - with limited or no side reactions due to cleavage of the C-C bond of the 1 ,2-amino- alcohol moiety - and finally removal of the tert-butyl-diphenylsilyl group by HF/pyridine, giving the desired deprotected 1 ,2-amino-alcohol in high yield.

Preferably, ,the method according to the invention is carried out with a protected amine compound not comprising an OH-gropu at the β-position.

The cycloalkyl groups in protected amines according to formula 1 or 2 may optionally have one or more unsaturated, exocyclic or endocyclic, carbon carbon bonds.

If substituted, the alkyl, cycloalkyl, alkenyl, alkynyl, and aryl groups that may be present in protected amines according to formula 1 or 2 may be substituted with optionally protected, functional groups comprising one or more heteroatoms such as for example oxygen, sulphur and nitrogen. Additionally, the alkyl, alkenyl and alkynyl groups may be interrupted by one or more heteroatoms such as oxygen, sulphur and nitrogen, whereas the cycloalkyl and aryl groups may comprise within their ring system one or more heteroatoms such as oxygen, sulphur and nitrogen.

The alkyl, alkenyl, and alkynyl groups that may be present in R¹, R², R³ , R⁴ and R⁵ each independently typically comprise 1-30 C atoms. Preferably, at least one of R¹, R², R³' R⁴ and R⁵ comprises 1-6 C atoms, more preferably, all R¹, R², R³, R⁴ and R⁵ comprise between 1-6 C atoms if they are an alkyl, alkenyl or alkynyl group.

The cycloalkyl or aryl group that may be present in R¹, R², R³ ,R⁴ and R⁵ typically comprises 1-30 C atoms. Preferably, at least one of R¹, R², R³ ,R⁴ and R⁵ comprises 3-6 C atoms, more preferably, all R¹, R², R³ R⁴ and R⁵ comprise between 3- 6 C atoms if they are a cycloalkyl or aryl group.

Any compound obtained by the process according to the invention may subsequently be isolated by known techniques, or may be further converted without being isolated. In the framework of this invention, an oxidative enzyme is defined as an enzyme which catalyses an oxidation reaction of a substrate, in this case the substrate being the compound to be deprotected, in the presence of an oxidant.

Since the process according to the invention involves the transfer of electrons, the enzyme used in the process according to the invention must be able to catalyse oxidations, more preferably, it is able to catalyse one electron oxidations.

An enzyme suitable for the process according to the invention typically comprises a protein part and one or more metal atoms, whereby the metal atom or atoms must be able to exist in at least two different oxidation states.

All enzymes known capable of transferring electrons may in principle be suitable to be used in the process according to the invention. Now that it has been found that an oxidative enzyme may be used to effect deprotection of protected amine groups, a person skilled in the art will be able to identify suitable enzymes for the process according to the invention, and suitable oxidants to use with the enzymes. The experiments as described in the experimental and examples section of this text, and the assay described therein may be suitable used for the screening of enzymes.

Preferably, the enzyme used in the process according to the invention is an oxidative enzyme chosen from enzyme class (E. C.) 1.10.3 or an oxidative enzyme of the enzyme class E. C. 1.1 1.

Most preferably, laccases (E. C. 1.10.3.2) are used in the process according to the invention. Even more preferably, laccases are used for the removal of an optionally substituted aryl or optionally substituted arylmethyl protecting group. Most preferably, laccases are used for the removal of a PMP-protecting group.

Laccases are multi-copper oxidases found in several trees and fungi, catalyzing the oxidation of various types of substances with concomitant reduction of oxygen to water, avoiding the formation of the hazardous hydrogen peroxide. Laccases are commercially available from e.g. Jϋlich Fine Chemicals and Sigma-Aldrich.

Suitable laccases that are commercially available are disclosed in the Examples section.

A mixture of enzymes may also be used in the process according to the invention.

The enzyme or enzymes used in the process according to the invention may be used in any form known, for example a such, as part of whole cells, or in immobilized form, for example as beads, etc.

The process according to the invention is carried out in the presence of an oxidising agent, also sometimes referred to as oxidant. In principle, any oxidising agent that is capable of oxidizing the protected amine may be used in the process according to the invention.

Each oxidative enzyme needs a suitable oxidant. Suitable oxidants for a particular enzyme are known to the skilled person, and can be found e.g. through the Enzyme Classification system.

For oxidative enzymes from the class E. C. 1.10.3, oxygen is a suitable oxidant. Typically, if oxygen is the oxidant used in the process according to the invention, the oxygen used in the process according to the invention is the oxygen dissolved in the reaction mixture. When no care is taken to exclude air from the reaction mixture, there will always be some oxygen present in the reaction mixture. However, it is also possible to create a higher concentration of oxygen in the reaction mixture, by contacting the reaction mixture with a gas containing oxygen in a higher concentration than air, or even with pure oxygen, for example by bubbling an oxygen containing gas or pure oxygen through the reaction mixture. For enzymes chosen from the class E. C. 1.1 1 , peroxides, for example

H₂O₂ may be suitably added as the oxidant.

Since the exact reaction mechanism is not known, it is also unknown exactly which species are involved in the deprotection process according to the invention. Thus, the process according to the invention also relates to a process for the deprotection of protected amine compounds according to the invention wherein the oxidant is formed in situ.

The preparation of protected amines is known to a person skilled in the art. The protected amine compounds can, for instance, be prepared through reductive amination of the corresponding ketones with p-anisidine as, for example, described by A. Pelter, R. Rosser, S. Mills, J. Chem. Soc, Perkin Trans. 1 1984, 4,

717-720. Alternatively, the protected amine compounds can be prepared via asymmetric Mannich reactions as, for instance, described by B. List et al, Angew.

Chem. Int. Ed, 2005, 44, 7424, B. List, Ace. Chem. Res. 2004, 37, 548-557, CF. Barbas III et al, Adv. Synth. Catal. 2004, 346, 1131-1 140 and CF. Barbas III et al,

J. Org. Chem. 2003, 68, 9624-9634.

Typically, at least some water is present in the process according to the invention. However, since enzymes are known that are active in an organic solvent, it may also be possible to carry out the process according to the invention in a reaction mixture not comprising any water.

Preferably, the process is carried out in a solution that comprises water. More preferably, at least one equivalent of water is present relative to the amine compound to be deprotected.

In a typical embodiment, the process according to the invention is carried out in a mixture of solvents, typically a mixture of water and one or more other solvents, also referred to as co-solvents. Any solvent may be used as a co-solvent.

Thus, the co-solvent may have a miscibility with water that ranges from immiscible to poorly miscible to well miscible. Accordingly, the process according to the invention may be carried out as a one phase process or as a two or more phase process. It is known that enzymes may be deactivated by organic solvents.

Thus, the process according to the invention is preferably carried out in the presence of water and a co-solvent that results in no or little deactivation during reaction.

Preferably, the process according to the invention is carried out in the presence of water and a solvent that together with water forms a homogeneous mixture. More preferably, the process is carried out in a mixture of water and a polar organic solvent, such as for example dimethylsulfoxide, methanol, acetonitrile, sulfolane, N,N-dimethylformamide, N-methyl-pyrrolidinone. The polar organic solvent may be protic or aprotic.

The amount of co-solvent may vary between wide ranges. Depending on the protected amine compound to be deprotected in the process according to the invention, it may be advantageous to increase the amount of protected amine compound that may be dissolved by increasing the amount of co-solvent. However, for solvents that cause deactivation of the enzyme used, it is preferred to use as little as possible. Therefore, the preferred amount of co-solvent depends on the protected amine compound used and the enzyme used. Typically, the amount of co-solvent used is more than 5 vol%, and typically the amount of co-solvent used is less than 50 vol%, relative to the total volume of the reaction mixture.

A person skilled in the art will be able to identify the optimum reaction conditions for each reaction with a given enzyme. Another factor that has influence on the process according to the invention is the pH, specifically the activity of the enzyme is pH dependent. The optimum pH for a process according to the invention can be determined by routine experimentation by a person skilled in the art. The pH may vary between wide limits, for example between 0 and 1 1. Typically, the optimum pH is lower than 9, preferably lower than 7, more preferably, lower than 6. Typically, the optimum pH is higher than 2, more preferably, higher than 2.5.

In the examples it has been demonstated that deprotection of Λ/-PMP protected benzylic amines involving laccases, is most effective at a pH below 4.

At too low pH values, enzymes may be deactivated. The process according to the invention wherein laccase is used as the oxidative enzyme, is therefore typically carried out at a pH higher than 3. To keep the pH at a desired value, a buffer may be used. In principle, any known buffer may be used. Preferably, the buffer is inert with respect to the compound to be deprotected and the (free) amine obtained after deprotection has taken place, the enzyme and the oxidant used. Thus, in this context inert is defined as not reactive towards the reactant and desired product under the conditions under which the process according to the invention is carried out.

Known buffer systems suitable for use in enzymatic reactions are phosphate buffers, acetate buffers and citric acid buffers. Depending on the desired pH and the type of enzyme used, a skilled person can select the most suitable buffer system.

The pressure at which the process according to the invention may be carried out is not critical and may vary between wide limits, for example 0.01 bar and 100 bar.

Typically, the process according to the invention is carried out under atmospheric pressure. Th e temperature at which the process according to the invention may be carried out is not critical, as long as it is not so high that the enzyme is deactivated. Typically, the process according to the invention is carried out at a temperature of 0°C or higher, preferably at a temperature of 10°C or higher, more preferably at a temperature of 15 °C or higher, even more preferably at a temperature of 20°C or higher, and most preferably at a temperature of 25°C or higher.

The process according to the invention is typically carried out at a temperature of 80°C or lower, preferably 70°C or lower, more preferably 60°C or lower, even more preferably 50°C or lower, and most preferably 40°C or lower. The amount of enzyme relative to the protected amine compound is not critical, and will be different for each enzyme. Typically, the amount of enzyme added is based on a compromise between enzyme cost and reaction time. Many factors influence the activity of an enzyme, for example temperature, pH, type of solvent etc, thus, and hence, the amount added will also be influenced by those factors. A person skilled I the art will be able to determine for each enzyme which amounts are effective amounts, i.e. which amounts result in the desired conversion under the chosen reaction conditions.

The suitable amount of oxidant relative to the protected amine compound can easily be determined by a person skilled in the art. For instance, if oxygen is used as the oxidant typically a large excess of oxidant is used for instance by contacting the reaction mixture with air or another gas containing oxygen. If hydrogen peroxide is used as the oxidant, the amount of oxidant is typically higher than 0.8 equivalent and preferably higher than 0.9 equivalent relative to the protected amine compound since full or almost full deprotection of the protected amine compound is usually desirable. If hydrogen peroxide is used as the oxidant, the amount of oxidant is also typically lower than 3 equivalent and preferably lower than 1.5 equivalent relative to the protected amine since a too high concentration of hydrogen peroxide may deactivate the enzyme.

In an embodiment, the process according to the invention is carried out in the presence of a mediator. A mediator is defined as an organic molecule acting as electron shuttle between the enzyme and the substrate. The use of a mediator may result in an improved conversion and/or selectivity relative to the same process according to the invention carried out in the absence of a mediator. Use of a mediator may also result in expanding the substrate scope of a given enzyme. In some cases, the enzymatic activity of a certain enzyme towards a certain protected amine compound only becomes easily detectable in the presence of a mediator.

It is assumed that mechanistically, the mediator is oxidized by the enzyme (e.g. laccase) and then converts the substrate into the oxidized form. The reduced mediator can subsequently be reoxidized by the enzyme so that both the enzyme and the mediator can be used in a catalytic fashion using e.g. oxygen as the stoichiometric oxidant (thereby producing water as the by-product).

In the examples it is shown that the presence of a mediator in the process according to the invention results in a higher conversion in a shorter time than when the same reaction is carried out in the absence of a mediator. Moreover, it is shown that mediators often result in widening the substrate scope of a particular enzyme. For the process according to the invention, the substrates are the protected amine compounds.

All known mediators may be used in the process according to the invention. Examples of mediators can be found in literature on enzymes. Suitable mediators for the process according to the invention are for example compounds comprising an N-hydroxy functional group, and other mediators known for laccases.

For laccases, suitable mediators have been described by D.

Rochefort et al., Green. Chem., 2004, 6, 14-24. Although the paper deals with the use of laccases in the paper pulp industry, the mediators described are also suitable for the process according to the invention.

Some examples of mediators for laccases are 2,2'- azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), violuric acid (VLA), 1 ,5- tetramethylpiperidine-N-oxide (TEMPO), hydroxybenzatriazole (HOBT), and syringaldehyde, as depicted in Figure 1 , and N-hydroxyacetanilide (NHA).

If a mediator is used, the suitable amount of mediator may vary between wide limits, for example 0.001 - 10 molar equivalents relative to the protected amine compound. However, the mediator typically only needs relatively small quantities to be effective. Therefore, when a mediator is used, the process of the invention is preferably carried out in the presence of 0.01-0.2 molar equivalents of mediator relative to the protected amine compound.

The invention also relates to a process for the deprotection of protected amines resulting from all possible combinations of claim 1 with one or more preferred embodiments, preferred protected amines, preferred reaction conditions, including preferred enzymes, oxidising agents or mediators, hitherto not explicitly disclosed as a combination. The invention also relates to the use of an oxidative enzyme for the deprotection of a protected amine compound as claimed in Claim 15, also in all possible combinations of one or more preferred features for the enzyme, the protected amine compound, the oxidising agent and any other applicable preferred feartures as disclosed herein.

The resulting deprotected amines can be used as a pharmaceutical, nutraceutical, food or feed ingedrient or agrochemical or as an intermediate in the process thereto. For instance, the resulting deprotected amines can be converted to heterocyclic compounds.

EXAMPLES General

Reagents and solvents were obtained at commercial quality and used without further purification. IR spectra were recorded on a Thermo Mattson IR300 equipped with a Harrick split pea ATR unit. ¹H NMR and ¹³C NMR spectra were recorded at 300 (75) or 400 (100) MHz. HRMS (ESI) spectra were recorded on a JEOL AccuTOF. Commercially available laccase T from Trametes versicolor was purchased from Jϋlich Fine Chemicals in two batches as a light brown lyophilized powder. The activity of the first batch (used in the experiments as shown in Table 1 , entries 1-32) was 24 U/mg (using syringaldazin). The activity of the second batch (used in the experiments as shown in Table 1 , entries 33-60, Tables 2 and 3) was 1.2 U/mg (using syringaldazin). Laccase AB from Agaricus bisporus was commercially available from Jϋlich Fine Chemicals as a brown lyophilized powder (activity 7.9 U/mg (using catechol)). Amine 12 is commercially available. Amines 10 and 13 were prepared through reductive amination of the corresponding ketones as described in A. Pelter, R. Rosser, S. Mills, J. Chem. Soc, Perkin Trans. 1 1984, 4, 717-20. Amines 1 and 14 were prepared according to known procedures as described in Y. Hayashi, W. Tsuboi, I. Ashimine, T. Urushima, M. Shoji, K. Sakai, Angew. Chem. Int. Ed. 2003, 42, 3677- 3680. Amine 15 was prepared by non-stereoselective reduction of the ketone function of Mannich adduct 15a using NaBH₄. Recorded spectra of 1 (see: W. Notz, F. Tanaka, S.-l. Watanabe, N. S. Chowdari, J. M. Turner, R. Thayumanavan, C. F. Barbas, J. Org. Chem. 2003, 68, 9624-9634, 10, (see: T. Itoh, K. Nagata, M. Miyazaki, H. Ishikawa, A. Kurihara, A. Ohsawa, Tetrahedron 2004, 60, 6649-6655) and 12-14 (see: W. Notz, F. Tanaka, S.-l. Watanabe, N. S. Chowdari, J. M. Turner, R. Thayumanavan, C. F. Barbas, J. Org. Chem. 2003, 68, 9624-9634 and T. Itoh, K. Nagata, M. Miyazaki, H. Ishikawa, A. Kurihara, A. Ohsawa, Tetrahedron 2004, 60, 6649-6655) were in accordance with those reported in the literature. The procedures for the preparation of amines 15, 16 and 16a are described below.

(2S,3S)-3-((4-Methoxyphenyl)(methyl)amino)-2-methyl-3-(4-nitrophenyl)propan-1-ol (15)

To a solution of 14 (3.0 g, 9.5 mmol) in MeCN (50 ml_), was added K₂CO3 (2.6 g, 19 mmol) and MeI (6.7 g, 47 mmol). The resulting suspension was stirred for 16 h at 40 ⁰C and subsequently filtered off. The filtrate was concentrated in vacuo and the resulting residue was taken up in CH₂Cb (50 ml_). The organic solution was washed with water (50 ml.) and brine (50 ml_), dried (MgSO₄) and concentrated in vacuo. The crude product was purified through flash chromatography (30% EtOAc/n- heptane) giving product 15 as a red syrup in quantitative yield. ¹H-NMR (CDCI₃, 300 MHz) δ 1.20 (d, J = 6.6 Hz, 3H), 1.89 (bs, 1 H), 2.58 (s, 3H), 3.38 (dd, J = 4.9, 10.6 Hz, 1 H), 3.58 (dd, J = 3.7, 10.7 Hz, 1 H), 3.75 (s, 3H), 6.80 (m, 4H), 7.26 (d, J = 8.1 Hz, 2H), 8.11 (d, J = 8.4 Hz, 2 H); ¹³C-NMR (CDCI₃, 75 MHz) δ 14.8, 34.1 , 35.7, 55.6, 67.7, 114.5, 118.7, 123.3, 128.8, 144.7, 145.1 , 147.0, 153.4; IR v (cm^"1): 3387, 2933, 1509, 1344, 1243, 1034, 811 ; HRMS (ESI+) calcd for Ci₈H₂₃N₂O₄ (M⁺) 331.1658, found 331.1670.

(S)-4-(3,4-Dimethoxyphenyl)-4-(4-methoxyphenylamino)-2-butanone (16a).

To a stirred solution of 3,4-dimethoxybenzaldehyde (10.1 g, 60.9 mmol) and p-anisidine (7.43g, 60.3 mmol) in DMSO/acetone (255ml, 1 :4) was added L-proline (1.47g, 12.75 mmol). The resulting suspension was stirred at room temperature for 21 h, quenched with saturated NH₄CI_(aq) (55 mL) and extracted with EtOAc (4 x 50 ml). The combined organic fractions were dried (Na₂SO₄) and concentrated. Precipitation occurred and the formed precipitate was filtered off, giving product 16a as an off-white solid (8.0 g, 39%). ¹H-NMR (CDCI₃, 400 MHz) δ 2.1 1 (s, 3H), 2.89 (d, J = 6.4 Hz, 2H), 3.70 (s, 3H), 3.85 (s, 3H), 4.69 (t, J = 6.8 Hz, 1 H), 6.52 (d, J = 8.8 Hz, 2H), 6.70 (d, J = 8.8 Hz, 2H), 6.81 (d, J = 8.8 Hz, 1 H), 6.89 (d, J = 6.0 Hz, 2H); ¹³C-NMR (CDCI₃, 75 MHz) δ 30.8, 51.4, 55.2, 55.7, 55.9, 109.5, 11 1.3, 11 1.7, 115.4, 118.2, 135.3, 141.1 , 148.1 , 149.2, 152.4; IR v (cm^"1): 3480, 1703, 1512, 1256, 1233, 1140, 1025, 813; HRMS (ESI+) calcd for Ci₉H₂₃NO₄ (M⁺) 329.1627, found 329.1617.

(4S)-4-(3,4-Dimethoxyphenyl)-4-(4-methoxyphenylamino)-2-butanol (16).

To a cooled stirred solution of 16a (1.08 g, 3.29 mmol) in MeOH (50 mL) was carefully added NaBH₄ (1.46 g, 38.6 mmol). The resulting suspension was stirred for 1 h and acidified with aqueous 5 M HCI to pH 1 and extracted with CH₂CI₂ (3 x 20 mL). 5 M NaOH was added until pH 11 was reached and the solution was extracted with EtOAc (4 x 30 mL). The combined organic fractions were dried (Na₂SO₄) and concentrated to give product 16 as an off-white solid (1.06 g, 97%, dr 2:1 ). ¹H- NMR (CDCI₃, 400 MHz) δ 1.25 and 1.23 (d, J = 6.2 Hz, 3H), 1.88 (m, 2H), 3.70 and 3.69 (s, 3H), 3.85 (m, 6H), 4.03 (m, 1 H), 4.50 and 4.39 (dd, J = 8.8; 7.4 Hz, 1 H), 6.55 (m, 2H), 6.68 (m, 2H), 6.83 (m, 3H); ¹³C-NMR (CDCI₃, 75 MHz) δ 23.8, 24.4, 46.8, 55.7, 55.9, 56.2, 65.4, 68.0, 11 1.2, 11407, 1 15.1 , 1 16.6, 118.2, 136.4, 140.9, 147.4, 148.7, 151.7, 152.1 ; IR v (cm^"1): 3494, 3203, 1511 , 1234, 1 138, 1019, 812; HRMS (ESI+) calcd for Ci₉H₂₅NNaO₄ (M+Na⁺) 354.1654, found 354.1667. 2-Methoxy-N-(4-phenylbutan-2-yl)aniline (22)

To a solution of acetic acid (1.91 ml_, 33.3 mmol), 4-phenylbutan-2- one (5.00 ml_, 33.3 mmol) and o-anisidine (3.76 ml_, 33.3 mmol) in 250 mL dichloromethane was added sodium triacetoxyborohydride (9.89 g, 46.6 mmol). The mixture was stirred for 7 days and quenched with 250 mL of a saturated aqueous solution of NaHCOs. After layer separation, the organic layer was dried (Na₂SO₄) and concentrated in vacuo. The residue was purified by flash column chromatography (ethylacetate/n-heptane 1/20). The combined fractions were concentrated in vacuo and the residue was taken up in 50 mL n-heptane and extracted with 4 x 100 mL 1 M aqueous hydrochloric acid. The combined aqueous layers were brought to pH 12 using a 5 M aqueous potassium hydroxide solution and extracted with 3 x 100 mL dichloromethane. The combined organic layers were dried (Na₂SO₄) and concentrated in vacuo, yielding the desired product 22 as a colorless oil (2.2 g, 8.6 mmol, 26% yield). ¹H-NMR (CDCI₃, 300 MHz) δ 1.24 (d, J = 6.0 Hz, 3H), 1.74-1.98 (m, 2H), 2.74 (t, J = 7.8 Hz, 2H), 3.40-3.60 (m, 1 H), 3.85 (s, 3H), 4.00-4.20 (m, 1 H), 6.51-6.87 (m, 4H), 7.19-7.31 (m, 5H). ¹³C-NMR (CDCI₃, 75 MHz) δ 21.2, 32.8, 39.1 , 47.9, 55.8, 109.9, 110.5, 116.1 , 121.6, 126.1 , 128.7, 128.8, 137.8, 142.4, 147.1.

2-(4-Phenylbutan-2-ylamino)phenol hydrochloric acid (23)

To a solution of acetic acid (1.91 mL, 33.3 mmol), 4-phenylbutan-2- one (5.00 mL, 33.3 mmol) and 2-aminophenol (3.64 g, 33.3 mmol) in 250 mL dichloromethane was added sodium triacetoxyborohydride (9.89 g, 46.6 mmol). The mixture was stirred for 3 days and quenched with 250 mL of a 1 M aqueous solution of hydrochloric acid. The resulting white precipitate was filtered off and redissolved in 25 mL methanol. Diisopropylether (300 mL) was added and the resulting white solid was filtered off and dried. Yield of 23: 7.1 g (25.6 mmol, 77% yield). ¹H-NMR (CD₃OD, 300 MHz) δ 1.41 (d, J = 6.3 Hz, 3H), 1.40-2.22 (m, 2H), 2.65-3.38 (m, 2H), 3.60-3.80 (m, 1 H), 6.99-7.41 (m, 9H). ¹³C-NfVtR (CD₃OD, 75 MHz) δ 17.5, 33.3, 36.6, 59.7, 118,7, 122.0, 123,1, 126.9, 128,2, 130.1, 130,4, 133.1, 142,5, 153.2.

4-(4-Phenylbutan-2-ylamino)phenol (24)

To a solution of acetic acid (1.91 ml_, 33.3 mmol), 4-phenylbutan-2- one (5.00 ml_, 33.3 mmol, and 2-aminophenol (3.64 g, 33.3 mmol) in 250 ml_ dichloromethane was added sodium triacetoxyborohydride (9.89 g, 46.6 mmol). The mixture was stirred for 3 days and quenched with 250 ml. of a 1 M aqueous solution of hydrochloric acid. The solution was concentrated in vacuo until full removal of the dichloromethane. The resulting aqueous phase was then washed with 3 x 250 ml. n- heptane and subsequently made slightly alkaline (pH 8) by the addition of an aqueous 5 M solution of potassium hydroxide. The resulting aqueous solution was extracted with 250 ml. dichloromethane. The combined organic phase was dried (Na₂SO₄) and concentrated in vacuo. The crude residue was recrystallized twice from diisopropyl ether to obtain the desired compound 24 as a white solid (2.5 g, 10 mmol, 31% yield). The resulting spectral data were in accordance with values reported in the literature (T. Suwa, E. Sugiyama, I. Shibata, A. Baba, Synthesis, 2000, 6, 789-800).

(2S.3SV3-(4-MethoxybenzylaminoV2-methyl-3-(4-nitrophenvnpropan-1-ol (25)

To a solution of compound 19. HCI (0.419 g, 1.698 mmol) in 100 ml_ acetonitrile was added p-methoxybenzylchloride (0.254 ml_, 1.868 mmol), LiI (0.023 g, 0.170 mmol) and NaHCO₃ (0.713 g, 8.49 mmol). The mixture was heated to reflux, stirred over night and poured out into a mixture of 200 ml. dichloromethane and 200 ml. saturated aqueous NaHCO₃ solution. The mixture was concentrated in vacuo until full removal of the acetonitrile and dichloromethane. The resulting aqueous solution was extracted with 200 ml. dichloromethane. The organic phase was dried with Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography, which gave product 25 as a colorless oil (0.309 g, 0.935 mmol). ¹H-NMR (CDCI₃, 300 MHz) δ 0.79 (d, J = 7.2 Hz, 3H), 2.09-2.18 (m, 1 H), 3.47-3.68 (m, 2 x 2H), 3.80 (s, 3H), 4.05 (d, J = 3.9 Hz, 1 H), 6.83-6.88 (m, 2H), 7.11-7.16 (m, 2H), 7.48-7.52 (m, 2H), 8.22-8.27 (m, 2H). ¹³C-NMR (CDCI₃, 75 MHz) δ 12.8, 39.8, 51.5, 55.7, 66.4, 66.9, 1 14.4, 123.9, 129.1 , 131.3, 147.7, 148.3.

EXAMPLE 1

Deprotection of PMP-protected amine 1 using laccase T and Laccase AB in the absence of a mediator.

An HPLC assay to screen laccases by monitoring the conversion of starting material, i.e. the Λ/-PMP protected amine into the desired free amine, was developed. As a benchmark substrate, PMP-protected 1 ,3-amino alcohol 1 was used. First, 4 mg of laccase T (from Trametes versicolor) was added to 1 mL of a solution of 1 (1.0 mg) in an acetonitrile/aqueous buffer (pH 5.0) mixture. Addition of the laccase immediately altered the color of the reaction mixture from colorless to purple, indicating enzymatic activity. Subjection of the crude reaction mixture to the HPLC assay clearly demonstrated the formation of the free amine 3 and benzoquinone (4, Table 1 ). After this first result, the influence of various reaction conditions was studied by carrying out experiments in which co-solvent and pH were varied. Two commercially available laccases were employed without modification, in lyophilized form, namely laccase T and laccase AB (from Agaricus bisporus).

rt=room temperature (about 20°C) Table 1a. Screening of reaction parameters entry laccase solvent vol% buffer PH t (h) conversion to 3 (%)^a

1 T MeCN 60 24 32

2 T MeCN 70 5 24 46

3 T MeCN 80 24 46

4 T MeCN 90 24 73

5 T THF 60 24 27

6 T THF 70 24 47

7 T THF 80 24 79

8 T THF 90 5 24 51

9 T EtOAc 60 24 O

10 T EtOAc 70 24 12

11 T EtOAc 80 24 21

12 T EtOAc 90 _{Il C C}NN

O O O O O O O O O O O O O O^iiiiiiiiii _iiii 24 58

13 T toluene 60 24 1 1

14 T toluene 70 24 23

15 T toluene 80 24 44

16 T toluene 90 24 44

17 AB MeCN 80 1 18 5

18 AB MeCN 80 18 2

19 AB MeCN 80 3 18 4

20 AB MeCN 80 4 18 24

21 AB MeCN 80 5 18 25

22 AB MeCN 80 6 18 20

23 AB MeCN 80 7 18 15

24 AB MeCN 80 8.5 18 13

25 T MeCN 80 1 18 O

26 T MeCN 80 18 8

27 T MeCN 80 3 18 82

28 T MeCN 80 4 18 74

29 T MeCN 80 5 18 61

30 T MeCN 80 6 18 37 Table 1 b. Screening of reaction parameters entry laccase solvent vol% buffer PH t (h) conversion to

3(%)^a

31 T MeCN 80 7 18 23

32 T MeCN 80 8.5 18 0

33 T MeCN 60 3 4 42

34 T MeCN 70 3 4 68

35 T MeCN 80 3 4 77

36 T MeCN 90 3 4 79

37 T MeCN 60 3 20 53

38 T MeCN 70 3 20 76

39 T MeCN 80 3 20 80

40 T MeCN 90 3 20 85

41 T THF 60 3 4 19

42 T THF 70 3 4 48

43 T THF 80 3 4 58

44 T THF 90 3 4 70

45 T THF 60 3 20 31

46 T THF 70 3 20 72

47 T THF 80 3 20 75

48 T THF 90 3 20 82

49 T DMSO 60 3 18 84

50 T DMSO 70 3 18 85

51 T DMSO 80 3 18 89

52 T DMSO 90 3 18 91

53 T MeOH 60 3 18 75

54 T MeOH 70 3 18 78

55 T MeOH 80 3 18 82

56 T MeOH 90 3 18 87

57 T MTBE 60 3 18 47

58 T MTBE 70 3 18 64

59 T MTBE 80 3 18 72

60 T MTBE 90 3 18 87 Conditions: laccase T or AB (4 mg) was added to a solution of 1 (1 mg) in the buffer/solvent mixture (1 ml_), rt. ^a Conversions were determined using HPLC (Inertsil ODS-3 column) on crude samples taken from the reaction mixture.

Thus, a series of experiments was carried out in a phosphate buffer (50 mM, pH 5)/co-solvent mixture, thereby varying the co-solvent. The conversions of substrate 1 into the free amine 3, as determined by HPLC, are depicted in Table 1a. (entries 1-16). These results show that the laccase-mediated PMP deprotection proceeds faster in homogeneous systems (e.g. MeCN, THF, MeOH, DMSO), but that the use of a biphasic system (e.g. EtOAc, toluene, MTBE) is also applicable. Additionally, it must be noted that higher amounts of co-solvent, although rendering the substrate more soluble, significantly decrease the overall reaction rate through enzyme deactivation. Evaluation of the pH dependence of the reaction in a buffer mixture/MeCN (4:1 ) using both laccases T and AB (entries 17-32). clearly showed that the conversions increased with lower pH values, albeit that too acidic conditions (pH < 3) led to enzyme deactivation.

A series of industrially viable solvents was evaluated in varying ratios with the buffer solution (entries 33-60). No real solvent limitations were identified in these experiments, although the use of acetonitrile and DMSO gave slightly better results. In addition, there was no clear difference between mono- and biphasic systems both giving high conversions to the desired product.

Subjection of protected amino alcohol 1 to laccase AB gave a maximum conversion of 25% (phosphate buffer pH 5/MeCN 4:1 , entry 21 ).

EXAMPLE Il Deprotection of PMP-protected amines 1 and 10 using laccase T and Laccase AB in the presence of a mediator.

The influence of various mediators on the process according to the invention was studied.

The reaction of experiment I (entry 21 ), was repeated in the presence of 1 equivalent ABTS (based on the protected amine compound). The conversion to 3 increased to 84% (instead of 25% in entry 1-21 ), 18 h, rt, 1 equiv of ABTS).

For each mediator, a blank reaction without enzyme was also conducted. In all cases there was no conversion of the protected amine substrate, except for TEMPO, which showed a percentage of conversions equivalent to the mol% of TEMPO added (e.g. 10mol% TEMPO added resulted in 10% conversion). In addition to increasing the conversion, it was also found that the substrate scope could be extended by using mediators.. Introduction of any of the mediators 5-9 led to high conversions to the corresponding amine 11 (Table 2).

Table 2. Influence of mediators. entry mediator conversion (18 h) conversion (48 h)

1 5 43 87

2 6 50 88

3 7 53 80

4 8 30 75

5 9 71 83

EXAMPLE Deprotection of various protected amine compounds and isolation of the deprotected amines

Procedure for the laccase-mediated PMP-deprotection of 1.

To a solution of 0.92 mmol of a PMP-protected amine (in case of 1 , 250 mg) in THF (40 ml.) and phosphate buffer (160 ml_, pH 3, 50 mM) was added laccase T (120 mg) and if desired ABTS (10 mol %). The resulting suspension was stirred for 20 h at room temperature, acidified to pH 1 with 5 N HCI and filtrated over Celite. The filtrate was washed with CH₂CI₂ (3 x 100 ml_). The resulting aqueous phase was subsequently brought to pH 10.5 via addition of 5 N NaOH and extracted with EtOAc (4 x 75 ml_). The combined organic fractions were dried (Na₂SO₄) and brought to pH 1 via addition of HCI/EtOAc, and concentrated under reduced pressure to afford the deprotected amine as the HCI-salt (when 1 was used, the salt was 2. HCI (124 mg, 67%) which was a white solid.

This procedure was conducted for substrate 1 as well as substrates 10, 13-16 with laccases T and AB (Table 3). It may be noted that subjection of PMP- protected benzylamine 12 to these enzymes did not produce benzylamine (17) itself. Instead, formation of benzaldehyde was observed, suggesting oxidation at the benzylic position, followed by hydrolysis of the resulting imine. In all other cases, however, the corresponding free amines 11 , 18-21 were isolated after workup in reasonable to good yields and excellent purity.

Table 3. Preparative deprotections using laccase T and AB.⁵ entry W-PMP amine laccase product (yield)^c

PMP^ χ\ N Ph H₂N^^Ph H

1

2*

3 13 T 18 (31%)

4* AB 18 (56%)

PMP

HNT NH₂

HO^^Ξ^^Ph HO^^r^^Ph

Me Me

5 1 T 2 (67%)

6* 1 AB 2 (71%)

11 16 T 21 (34%)

12* 16 AB 21 (41%)

^PMP NH₂ HN

Ph^^^ Ηe

Ph^^^Me

13* 10 T 11 (30%)

14* 10 AB 11 (47%) a Conditions: PMP-protected amine (0.92 mol), laccase (120 mg), THF/buffer mixture (1 :4, pH 3, 200 ml_), rt. ^b Reaction conducted in the presence of ABTS (10 mol%). ^c Products were isolated as HCI-salts. (2S,3S)-3-(4-Methoxyphenylamino)-2-methyl-3-phenylpropan-1 -ol hydrochloride (2)

OR [α]_D ²⁰ -24.3 (c 0.18, MeOH); ¹H-NMR (400 MHz, D₂O) δ 1.12 (d, J = 6.9 Hz, 3H), 2.36 (m, 1 H), 3.37 (dd, J = 5.4, 11.4 Hz, 1 H), 3.42 (dd, J = 5.4,1 1.4 Hz, 1 H), 4.33 (d, J = 8.2 Hz, 1 H), 7.50 (m, 5H); ¹³C-NMR (75 MHz, D₂O) δ 12.3, 37.9, 57.8, 62.6, 126.9, 128.7, 128.7, 134.6; IR v (cm^"1): 3330, 3112, 2975, 2871 , 1489, 1473, 1026, 706, 558; HRMS (ESI⁺) calcd for Ci₀H₁₆NO 166.1232, found, 166.1248; Anal. Calcd. for Ci₀H₁₆CINO: C, 59.55; H, 8.00, N, 6.94. Found: C, 58.05; H, 7.87; N, 6.84

4-Phenylbutan-2-amine hydrochloride (1 1 )

Spectral data were in accordance with those reported in the literature, (see: M. Kitamura, S. Chiba, K. Narasaka, Bull. Chem. Soc. Jpn. 2003, 76, 1063- 1070).

1-Phenylethanamine hydrochloride (18)

¹H-NMR (400 MHz, D₂O) δ 1.65 (d, J = 6.9 Hz, 3H), 4.55 (q, J = 6.2 Hz, 1 H), 7.49 (m, 5H); ¹³C-NMR (I OO MHz, D₂O) δ 19.5, 51.2, 126.7, 129.4, 129.4, 137.9; IR v (cm^"1): 2972, 2879, 1609, 1513, 1084, 1032, 763, 696, 537; HRMS (ESI+) calcd for C₈Hi₂N (M⁺) 122.0970, found 122.0981.

(2S,3S)-3-Amino-2-methyl-3-(4-nitrophenyl)propan-1 -ol hydrochloride (19)

¹H-NMR (H₂O, 300 MHz), δ 1.12 (d, J = 6.8 Hz, 3H), 2.41 (m, 1 H), 3.42 (d, J = 5.2 Hz, 1 H), 4.53 (d, J = 7.7 Hz, 2H), 7.69 (d, J = 8.5 Hz, 1 H), 8.37 (d, J ■ 8.5Hz, 1H); ¹³C-NMR (H₂O, 75 MHz), δ 12.1,38.0, 57.1,62.4, 123.7, 128.1, 142.0, 147.5; IR v (crrT¹): 3671, 2985, 2900, 1406, 1393, 1250, 1066, 1055, 891, 669; HRMS (ESI⁺) calcd for C₁₀H₁₅N₂O₃211.1083, found 211.1086; Anal. Calcd. for C₁₁H₁₅CIN₂O₃: C, 48.69; H, 6.13; N, 11.36. Found: C, 48.72; H, 6.12; N, 11.14.

(2S,3S)-2-Methyl-3-(methylamino)-3-(4-nitrophenyl)propan-1-ol hydrochloride (20)

¹H-NMR (400 MHz, D₂O) δ 1.04 (d, J = 6.8 Hz, 3H), 2.55 (m, 1 H), 2.59(s, 3H), 3.37 (dd, J=7.1, 11.4Hz, 1H), 3.45 (dd, J = 4.9, 11.5Hz, 1H), 4.44 (d, J = 6.5 Hz, 1 H), 7.71 (d, J = 8.6 Hz, 2H), 8.39 (d, J = 8.4 Hz, 2H); ¹³C-NMR (75 MHz, D₂O) 612.4,31.3,36.8,62.3,66.1, 123.9, 129.3, 138.6, 147.8; IR v (cm^"1): 3369, 2965, 1606, 1521, 1461, 1348, 1035,857, 701; HRMS (ESI+) calcd for C₁₁H₁₇N₁₂O₂

225.1239, found 225.1251; Anal. Calcd for C₁₁H₁₈CINO: C, 50.68; H, 6.57; N, 10.74. Found: C, 49.97; H, 6.65, N, 10.34.

(S)-4-Amino-4-(3,4-dimethoxyphenyl)-2-butanol hydrochloride (21 )

¹H-NMR(CD₃OD, 400 MHz) δ 1.19 and 1.22 (d, J = 6.2 Hz, 3H), 2.05 (m, 2H), 3.68 and 3.81 (m, 1 H), 4.48 (m, 1 H), 4.86 (s, 6H), 7.45 (m, 3H)

¹³C-NMR (CD₃OD, 75 MHz) δ 23.5, 24.5, 54.0, 55.6, 64.9, 78.7, 79.1, 79.5, 127.7, 128.1, 129.6, 130.1; IR v (cm^"1): 3190, 2943, 2908, 1524,939,696; HRMS (ESI+) calcd for C₁₂H₂₀NO₃226.1443, found 226.1432. EXAMPLE IV:

Deprotection of 2-methoxy-N-(4-phenylbutan-2-yl)aniline (22)

Compound 22 (0.200 g, 0.783 mmol) was dissolved in 20 ml. acetonitrile/80 mL aqueous phosphate buffer 100 mM pH 3. To the resulting solution was added 100 mg laccase T. The mixture was stirred for 5.5 hours, acidified with 5 M aqueous hydrochloric acid to pH 1.0 and subsequently washed with 3 x 75 ml. dichloromethane. The aqueous layer was brought to pH 1 1 with 5 M aqueous potassium hydroxide and extracted with 3 x 75 ml. ethyl acetate (while maintaining the pH at 11 by the addition of extra potassium hydroxide solution). The combined organic layers were dried, acidified with 2 ml. of a saturated solution of hydrochloric acid in ethylacetate and concentrated. The residue was purified with flash column chromatography (mixtures of dichloromethane/methanol). The combined organic fractions were acidified with HCI in ethylacetate and concentrated to yield product 11. HCI as an off-white solid (0.051 g, 0.28 mmol, 35 % yield).

EXAMPLE V:

Deprotection of 2-(4-phenylbutan-2-ylamino)phenol hydrochloric acid (23)

To a solution of compound 23 (0.218 g, 0.783 mmol) in 20 mL acetonitrile/80 mL aqueous phosphate buffer 100 mM pH 3 was added ABTS (0.043 mg, 0.078 mmol) and 100 mg laccase T. The mixture was stirred for 18 hours, acidified with 5 M aqueous hydrochloric acid to pH 1.0 and subsequently washed with 3 x 100 mL dichloromethane. The aqueous layer was brought to pH 11 with 5 M aqueous potassium hydroxide and extracted with 3 x 150 mL ethylacetate (while maintaining the pH at 11 by the addition of extra potassium hydroxide solution). The combined organic layers were dried, acidified with 2 mL saturated solution of hydrochloric acid in ethylacetate and concentrated in vacuo. Product 11. HCI was obtained as an off-white solid (0.043 g, 0.232 mmol, 30% yield).

EXAMPLE VI:

Deprotection of 4-(4-phenylbutan-2-ylamino)phenol (24)

laccase T

24 11. HCI

Compound 24 (0.189 g, 0.783 mmol) was dissolved in 20 mL acetonitrile/80 mL aqueous phosphate buffer 100 mM pH 3. To the resulting mixture was added 100 mg laccase T. The mixture was stirred for 19.5 hours, acidified with 5 M aqueous hydrochloric acid to pH 1.0 and subsequently washed with 3 x 75 mL dichloromethane. The aqueous layer was brought to pH 1 1 with 5 M aqueous potassium hydroxide and extracted with 3 x 75 mL ethylacetate (while maintaining the pH 11 by the addition of extra potassium hydroxide solution). The combined organic phase was dried (Na₂SO₄), acidified with 2 mL of a saturated solution of hydrochloric acid in ethylacetate and concentrated in vacuo. Product 11.HCI was obtained as an off-white solid (0.134 g, 0.722 mmol, 92 % yield).

EXAMPLE VII:

Deprotection of (2S,3S)-3-(4-methoxybenzylamino)-2-methyl-3-(4-nitrophenyl)propan-

1 -QI (25)

laccase T/ABTS

25 19.HCI To a solution of 50 mg (0.15 mmol) PMB-protected amine 25 in 50 ml. aqueous phosphate buffer (100 mM pH 3) was added 8.3 mg (0.015 mmol) ABTS followed by 50 mg laccase T. The mixture was stirred and after 20 hours, fresh laccase T (50 mg) and ABTS (8.3 mg, 0.015 mmol) were added. After an additional 24 hours of stirring, the reaction mixture was brought to pH 1 by the addition of 5 M hydrochloric acid and washed with 3 x 75 ml. dichloromethane. Subsequently, the resulting aqueous phase was brought to pH 1 1 with a 5 M aqueous solution of potassium hydroxide and extracted with 3 x 75 ml. ethylacetate (while maintaining the pH at 11 by the addition of extra potassium hydroxide solution). The combined organic phase was dried (Na₂SO₄), acidified with hydrochloric acid in ethylacetate and concentrated. The resulting residue was a mixture of the starting material and the desired deprotected amine as their HCI salts. The corrected yield for the product is 0.028 g (0.133 mmol, 75%).

EXAMPLE NX:

Deprotection using immobilised laccase T

CLEA laccase T

14 19.HCI

To a solution of compound 14 (0.316 g, 1.0 mmol) in 40 mL tetrahydrofuran/160 mL 100 mM aqueous phosphate buffer (pH = 3) was added 200 mg CLEA laccase T (obtained from CLEA Technologies, Delft, The Netherlands). The resulting suspension was stirred for 28 hours and washed with dichloromethane (2 x 200 ml_). The aqueous layer was brought to pH 11 with 5 M aqueous potassium hydroxide and extracted with 3 x 200 ml. ethylacetate (while maintaining the pH at 1 1 by the addition of extra potassium hydroxide solution). The combined organic layers were dried with Na₂SO₄, acidified with hydrochloric acid in ethyl acetate and concentrated in vacuo giving 19. HCI (0.19 g, 0.78 mmol, 78% yield).

EXAMPLE IX:

Deprotection using Horse Radish Peroxidase

14 19

A 3.5 wt% solution of H₂O₂ was prepared by diluting 1 ml. of an aqueous 35 wt% H₂O₂ solution with 9 ml. aqueous phosphate buffer (100 mM, pH 3). To a solution of 10 mg (0.032 mmol) of compound 14 in 2 ml. acetonitrile/8 ml. 100 mM aqueous phosphate buffer (pH = 3) was added 56 μl_ of the prepared H₂O₂ solution. To the resulting solution, 0.4 mg Horse Radish Peroxidase (obtained from Fluka,168 u/mg) was added. After 30 minutes, the reaction mixture was analyzed by HPLC, which showed full conversion of the starting material to the free amine 19. HPLC was conducted on an lnertsil ODS 3 column (150 mm length, 4.6 mm internal diameter) at a flow of 1.0 mL/min and using a UV detector at 210 and 254 nm. The eluent consisted of mixtures of solvent A (aqueous 10 mM phosphate buffer pH 3.0) and solvent B (acetonitrile). The applied gradient was as follows: t = 0-2 min: 0% B; t = 2-6 min: 0^50% B; t = 6-10 min: 50% B; t = 10-12 min: 50^70% B; t = 12-14 min: 70% B; t = 14 - 16.5 min: 70^0% B; t = 16.5-19 min: 0% B. Retention times: compound 19: 8.2 min; benzoquinone 9.2 min, compound 14: 15.3 min.

Claims

A process for the deprotection of a protected amine compound, wherein the protected amine compound which comprises a protecting group attached to the N-atom of the amine, is contacted with an oxidative enzyme and an oxidising agent that may optionally be formed in situ and wherein the protected amine compound to be deprotected is a compound according to formula 1

Formula 1 wherein PG means Protecting Group,

R⁴ = H, or a (substituted) alkyl, (substituted) cycloalkyl, (substituted) alkenyl, (substituted) alkynyl or (substituted) aryl group

R¹, R² and R³ are each independently H or (substituted) alkyl, (substituted) cycloalkyl, (substituted) alkenyl, (substituted) alkynyl or (substituted) aryl groups or carboxylic acid groups or salts thereof, carboxylic esters, carboxylic thioesters or carboxamides provided that at least two out of R¹, R² and R³ are not H and provided that not more than two groups chosen from R¹, R² and R³ are carboxylic acid groups or salts thereof, carboxylic esters, carboxylic thioesters or carboxamides. and wherein the protecting group is an optionally substituted aryl group or an optionally substituted arylmethyl group and wherein the enzyme is an oxidative enzyme of the enzyme class E. C. 1.10.3 or an oxidative enzyme of the enzyme class E. C. 1.11.

2. Process according to claim 1 , wherein the optionally substituted aryl or arylmethyl group is an optionally substituted C₆ - Ci₄ aryl or C₆ - Ci₄ arylmethyl group comprising one or more electron-donating groups.

3. Process according to any one of claims 1-2, wherein the protecting group is chosen from the group of ortho-methoxyphenyl, para-methoxyphenyl (PMP), ortho-hydroxyphenyl, and para-hydroxyphenyl.

4. Process according to any one of claims 1-3, wherein the protecting group is a p-methoxyphenyl-group.

5. A process according to any one of claims 1-4 wherein the enzyme is of the enzyme class E. C. 1.10.3.2.

6. Process according to any one of claims 1-5, wherein the process is carried out in a solution comprising water.

7. Process according to any one of Claims 1-6, wherein the process is carried out at a pH lower than 7.

8. Process according to any one of Claims 1-7, wherein the process is carried out at a pH lower than 5.

9. Process according to any one of Claims 1-8, wherein the process is carried out in the presence of a mediator.

10. Process according to Claim 9, wherein the process is carried out in presence of a substrate according to Formula 1 , wherein R¹, R² and R³ are all non-aryl groups.

11. Use of oxidative enzymes in a process for deprotection of a protected secondary or tertiary amine compound.

12. Use according to claim 11 , wherein the enzyme is a laccase.

13. Use according to claim 1 1 or 12, wherein the amine compound is protected with a PMP-group.