EP0970235A1

EP0970235A1 - Process for the selective enzymatic hydroxylation of aldehydes and ketones

Info

Publication number: EP0970235A1
Application number: EP98914885A
Authority: EP
Inventors: Anna STÜTZ-DE RAADT; Herfried Griengl; Irene Kopper; Markus Klingler; Gerhart Braunegg
Original assignee: DSM Fine Chemicals Austria Nfg GmbH and Co KG
Current assignee: Patheon Austria GmbH and Co KG
Priority date: 1997-03-18
Filing date: 1998-03-13
Publication date: 2000-01-12
Also published as: WO1998041647A1; CA2284256A1; KR20000076361A; US6287829B1; AT406960B; ATA46897A; AU6921498A; JP2001515357A

Abstract

The invention concerns a process for the selective enzymatic hydroxylation of aldehydes and ketones using chiral anchor/protective groups.

Description

Process for the selective enzymatic hydroxylation of aldehydes and ketones

The subject invention relates to a process for the selective hydroxylation of aldehydes and ketones using enzymes or microorganisms.

Technological background

Hydroxylated aldehydes and ketones are of great industrial importance as synthetic intermediates. For example, DE 36 22 839 discloses the synthesis of cardioactive compounds using hydroxylated ketones.

WO 86/0761 1 discloses the synthesis of 4-hydroxycyclopent-2-en-1-one and 3-hydroxycyclopentanone. These compounds are used in the manufacture of prostaglandins.

Another area of application is the preparation of compounds for liquid crystal compositions (F. Hoffman-La Röche, CA 113: 58558, Jpn. Kokai Tokkyo Koho JP 02 25,451 [90 25,451]).

Linking a hydroxyl group to an unactivated carbon atom is a very difficult task if only "conventional" chemical methods are used for this. Looking at the more recent works with so-called gif systems (DHR Barton and W. Chavasiri; Tetrahedron., 1994, 50, 19; DHR Barton and RD Doller, Acc. Chem. Res., 1992, 25, 504) ab, there are no regioselective, purely chemical methods for the hydroxylation of unactivated carbon atoms. However, it was also not possible to make the hydroxyation enantioselective with these Gif systems. In addition, the yield is so low that these processes are out of the question for industrial implementation.

It is not only important to position the hydroxyl group regioselectively on a specific carbon atom relative to the carbonyl group. A new chirality center is usually formed during the hydroxyation. Particularly as an intermediate for pharmaceuticals, but also for the agricultural and cosmetic sectors, it is important that one of the two enantiomers or diastereomers is formed preferentially.

Chemical methods for enantioselective hydroxyiation are only available for the α carbon atom. These are preferably reactions via corresponding enolates, a certain enantioselectivity being achieved by suitable complexation with chiral auxiliaries. A disadvantage of using this enantiomeric However, enriched α-hydroxycarbonyl compounds are such that racemization easily occurs at the α-carbon atom

Enzymatic hydroxylation is therefore the only method for solving the task of enantioselective hydroxylation of aldehydes and ketones. Above all, numerous examples of the hydroxylation of steioids are known, where due to the rigid molecular structure, with suitable selection of the enzymes or microorganisms, both good regions as well as stereoselectivities were achieved. These reactions are used technically for the representation of pharmaceutical intermediates

However, there is no general method for the enzymatic hydroxylation of aldehydes and ketones. One reason for this is that the carbonyl group itself is reduced by the oxidoreductases present in all microorganisms, which often results in no more hydroxylation

With the help of the concept of reversible anchor / protective groups, it was possible to carry out regioselective hydroxylation with chemical yields of up to 70%

The concept is based on deπvatisation of the carbonyl group, whereby it precedes dei

Biotransformation is protected by the oxidoreductases

It is also possible to use this protective group to chemical and physical

To model the properties of the substrate and thus, for example, to reduce the alignment, which is disruptive for some processes, or to introduce a chromophore necessary for some chromatographic separations

Acetals, aminoacetals, mercaptals or aminals are particularly suitable as anchor / protective groups

After the enzymatic hydroxylation has been carried out, these protective / anchor groups are gently split off again

So far, only non-chiral protective / anchor groups have been used. Two good chemical yields have been achieved, but the enantiomeric excess is a maximum of 40%.

(Biohydroxylation as the key step in the synthesis of optically active 3-substituted

Cyclopentanones Derivatives, G Braunegg, A de Raadt et al, BIOTRANS ^'95 Universitv of

Warwick, Coventry, 5-8 September 1995, UK)

Due to the constantly increasing need for optically pure hydroxyhered oxo compounds, it is desirable to provide processes which are suitable for producing these compounds in high optical yield Surprisingly, it has now been found that both the chemical yield and the enantiomeric excess can be increased considerably when using chiral protective / anchor groups.

The invention therefore relates to a process for the preparation of compounds of the formula

m ^CH ~ ^{~ z} _? ⁾ _m ~ ^A '(H), where

wherein in formula II one of the radicals X, Y is hydrogen, n is an integer 0, 1, 2 or 3, 0, the compounds of

Formula I optionally contain a double bond in the cycle and m denotes one of the numbers 0 or 1, Z | u. Z ₂ independently of one another denote a Cj to C _x alkylene radical which can optionally be substituted by C 1 to C ₄ alkyl and / or unsaturated.

Ri, R independently of one another hydrogen or straight-chain or branched or cyclic Cι. ₄ -alkyl or Rj and R ₂ together with the cycle containing group A are a bicyclic compound of the structure bicyclo [a, b, c] heptane to decane (a, b, c = 0, 1, 2, 3 or 4) form, which can optionally be substituted by d- C ₄ alkyl and / or unsaturated, and

R, R- ₄ , can be hydrogen or a straight-chain, branched or cyclic Cj - alkyl,

characterized in that a compound of formula

where A, A ', Rj, R ₂ , R ₃ , Rj, ZZ ₂ , m and n have the above meaning, is protected with a suitable chiral anchor / protective group, the compound thus protected is hydroxylated enzymatically, regioselectively and stereoselectively, if appropriate the hydroxyl group is protected with a suitable compound and the

Anchor / protection group is split off.

A compound of the formulas (III) or (IV) can, for example, be one of the following,

Other suitable compounds of the formula III or IV are, for example

Bicyclo [2.2.1] heptan-2-one

(1R) and (1S) -l, 7,7, -trimethyl-bicyclo [2.2. 1] heptan-2-one

Trans-1 -decalon

2-decalon

(IS) and (IR) -l, 3,3-trimethyl-bicyclo [2.2.1] heptan-2-one

Bicyclo [3.3.0] octane-3,7-dione

Bicyclo [3.3.0] octan-3-one

Bicyclo [3.3.0] oct-7-en-2-one

Bicyclo [3.3.0] oct-6-en-2-one

Bicyclo [4.2.0] oct-2-en-7-one

Bicyclo [3.2.0] hept-2-en-7-one Bicyclo [3.2.0] hept-2-en-6-one

Chiral acetals, aminoacetals, mercaptals or aminals are particularly suitable as protecting / anchor groups. These can be cyclic or non-cyclic. These are preferably 1, 2- and 1, 3-diols, amino alcohols, dithiols and aminodiols, and also 1,2- and 1,3-diamines. These must have at least one chiral center. These compounds can be aliphatic, alicyclic or heterocyclic. It is also possible for these protective / anchor groups to carry further functional groups in addition to the groups which are required for fixation to the substrate molecule.

A preferred anchor / protecting group is a compound of the formulas

(V) or ( ^VI ), where R ' straight-chain or branched Cι-4-alkyl means, for example, the N-benzoyl derivatives of (R) -2-amino-l-propanol, (S) -2-amino-l-propanol, (R) -l-amino -2-propanol, (S) - l-amino-2-propanol, (R) -2-amino-l-butanol, (S) -2-amino-l-butanol, (R) - l-amino-2 -butanol or (S) - l -amino-2-butanol.

After the enzymatic hydroxylation has been carried out, these protective / anchor groups are gently split off again. The cleavage can be carried out using standard chemical methods. (T.W. Greene P.G.M. Wuts, "Protective Groups in Organic Synthesis". John Wiley & Sons, Inc. 1991). such as by acid catalyzed hydrolysis. Electrolysis or by using metal salts

The following species are preferred as microorganisms for the hydroxyation.

Asper i Uns ochraceus ATCC 18500. Bacillus egaterium CCM 2037, Bacillus megaleriuw DSM 32, Beauveria bassiana ATCC 7159, Calonectria decυra DSM 879, Chaetomium cochlioides DSM 831, Chaetomium globosum DSM 1962, Cornyespora cass cola DSMella245, 574 DSM 1906. Cunninghamella echinulala DSM 1905, Cunninghamella elegans DSM 1908, Diplodia gossypina ATCC 10936, Fusarium solani DSM 62416, Mortierella alpina ATCC 8979, Mucor plumheus CBS 1 10.16., Pseudonionas put / da ATCC 29607, Pellicularium rickia filament 10490, Polyporus ostreiformis CBS 36234, Staurophoma species DSM 858 and Strepto yces griseus ATCC 13273.

The microorganisms suitable for the method according to the invention are also known in part under various synonyms, as listed for example in SC Jong, JM Birmingham, G. Ma, "ATCC Names of Industrial Fungi", American Type Culture Collection, USA, 1994. However, it is possible in the same way to find other microorganisms via screening or to isolate them from natural sources such as soil samples, waste or waste water using enrichment and selection techniques

The enzyme preparations used according to the invention are not restricted with regard to purity and the like and can be used, for example, as a coarse enzyme solution

However, they can also consist of optionally immobilized cells which have the desired activity or consist of a homogenate of cells of the desired activity

The invention is in no way restricted by the form in which the enzyme is used. Within the scope of the invention it is also possible to use enzymes which differ from one another

Derive mutants, or use genetically modified microorganisms

Examples

Example 1: (/?) - 3-Benzyloxycvclopentanone

(3R) -4-benzoyl-3-methyl-l-oxa-4-azaspiro | ^~ 4 4 ^" ) nonane (Saaverda. J Org Che, 1985, 50 2379)

Cyclopentanone (1 80 g, 0.021 mol) and (R) -2-amino-l-propanol (1.07 g, 0 014 mol) were converted into a suspension of K ₂ CO ₃ (3 94 g, 0 029 mol) in CH ₂ C1 ₂ (10 ml) added and stirred for 48 hours at room temperature. Benzoyl chloride (2.01 g, 0.014 mol) was added to the reaction mixture and the mixture was stirred for a further 48 hours. The mixture was filtered and the filtrate successively with 100 ml of 5% aq. HC1, sat. aq Na ₂ CO ₃ and H ₂ 0 washed. The organic phase was dried over Na ₂ SO ₄ and evaporated (3R) -4-benzoyl-3-methyl-1-oxa-4-azaspiro [4.4] nonane was obtained as a light yellow solid after column chromatography and recrystallization from ethyl acetate / petroleum ether (yield 75%)

ee: 98% (HPLC, CHIRALCEL OD-H, n-heptane EPA, 7 3, p = 38 bar, flow rate = 0 5 ml / min)

Mp 65 5 - 67 5 c

[α] _D ²⁰ = -79 8 ° (c 2 1, CH ₂ C1 ₂ )

1H-NMR δ 0 96 (d, J _3Λ i _e = 6.5 Hz, 3H, Me), 1.64 - 1 98, 2 37 - 2 71 (2 xm, 6H & 2H, H6, 7, 8, 9), 3 60 (m, IH, H2), 4 00 (m, 2H, H2.3), 7 40 (s, 5H, benzoyl)

"C-NMR δ 20 1 (Me), 24 7, 24 8 (C7.8), 35 0, 36 5 (C6, 9), 54 1 (C3), 70 0 (C2), 105 0 (C5) , 126 2, 128 4, 128 5, 129 4, 138 2, 168 1 (benzoyl)

(3R. 5S. 77) -4-Benzoyl-3-methyl-l-oxa-4-azaspiro | ^" 4 4] nonan-7-ol

The Beauvena bassiana ATCC 7159 mushroom was placed in Petri dishes for 1 week on Medium E (15 g / 1 agar, 10 g / l glucose, 5 g / 1 peptone, 5 g / 1 malt extract, 2 g / 1 yeast extract, 2 g / 1 KH2PO4) 1 cm was used to inoculate the preculture (70 ml, medium E, 30 C, 120 rpm). After 72 hours, the main culture in the fermenter was inoculated with 10% of the volume of preculture (conditions in fermenter 1, 6 1 working volume, pH = 6.6-7, T = 28 C, ventilation l, 6 NL / min, 400 rpm, Medium E, 0.25 ml PPG 2000 as anti-foaming agent)

After 24 hours of main culture growth, 0 16 g (3 /?) - 4-benzoyl-3-methyl-l-oxa-4-azaspiro [4.4] nonane, dissolved in EtOH (solution 20% w / v), were added Substrate (0 64 g) was added after a further 12 hours

The fermentation was terminated as soon as no substrate could be detected during the hydroxylation process using GC (HP 5890, Series II, column HP 5, 25 m, FID) and DC (Merck 5642 001). The culture was extracted twice with ethyl acetate, the organic phase dried over Na2SO4, filtered and the filtrate rotated in. This was done by column chromatography (Merck Kieselgel 60, 70-230 medium grain size) hydroxy product, (3R, 5S, 7R) -4-benzoyl-3-methyl-l-oxa-4-azaspιro [4 4] nonan-7-ol, isolated. (0.72 g, 84%)

The diastereomer excess was determined using 1 1 1 (HPLC, CHIRALCEL OD-H - n-heptane IPA, 7 3, P = 37 bar, flow rate = 0 50 ml / min - before column chromatography), and on the other hand using 20 1 (HPLC, determined by column chromatography)

[α] _D 20 ₌ _ _{79 6} o _(c ! _{5 ^ C} H ₂ C1 ₂ )

M.p .: 106-108 ° C

NMR NMR δ 0 93 (d, J ₃ , _Me = 5 7 Hz, 3H, Me), 1 73 - 2 41 (m, 4H, H6, 8, 9, 9), 2 57 - 2 69 (m, 2H, H8, OH), 2 75, 2.94 (2 x dd, J ₆ -, ₇ = 5 7 Hz, J ₆ , ₆ > = 14 0 Hz, IH, ratio 20 1, H6), 3 63 (dd, J _2.3 = 5 1 Hz, J _2.2 = 1 1 3 Hz, IH, H2), 3 98 (m, 2H, H2, H3), 4 42 (br s, IH, H7), 7 37 ( s, 5H, benzoyl)

¹³ C-NMR δ 20 1 (Me), 34 7, 34 8 (C8, 9), 43 2 (C6), 54 0 (C3), 70 3 (C2), 72 7 (C7), 104 0 (C5 ), 126 2, 128 6, 129 6, 137 7, 168 2 (benzoyl)

(3R. 5S. 7 /?> 4-benzoyl-7-benzyloxy-3-methyl-l-oxa-4-azaspirof4 4] nonane

Before the protective group was split off, (3R, 55, 7Λ) -4-benzoyl-3-methyl-l-oxa-4-azaspiro [4 4] nonan-7-ol was derivatized (NaH, benzyl bromide, THF, DMF, room temperature) After column chromatography, (3R, 5S, 7 /?) - 4-benzoyl-7-benzyloκ \ -3-methyl-l-oxa-4-azaspiro [4 4] nonane was obtained as fine needles in a yield of 85%

de: 89% (NMR)

[α] _D 20 = .75 ₂ ° ( _c 3 0, CH ₂ C1 ₂ )

Mp: 85-86 ° C (CH ₂ C1 ₂ / Pet Ether)

Η-NMR δ (small quantity isomer printed in bold) 0 95 (d, J _{3 e} = 6 6 Hz, 3H, Me), 1 83 - 2 67 (m, 5H, H6,8,9), 2 77, 2.96 (2 x dd, J ₆ , ₇ = 6 7 Hz, J ₆ , ₆ = 14 4 Hz, IH, ratio 18 1, H6), 3 64 (m, IH, H2), 4 03 (m, 2H, H2, H3 ), 4 33 (br s, IH, H7), 4 54 (s, 2H, CH ₂ Ph), 7 34 (m, 10H, COPh, CH ₂ Ph)

¹³ C NMR δ 20 1 (Me), 31 7, 35 3 (C8, 9), 41 9 (C6), 54 0 (C3), 70 4 (C2), 70 9 (CH ₂ Ph), 79 2 (C7), 103 3 (C5), 126 3, 127 5, 127 7, 128 4, 128 4, 128 6, 129 7, 137 9, 138 9 (COPh, CH ₂ Ph), 168 2 (COPh)

(R) -3-benzyloxycyclopentanone

(3R, 5S, 7R) -4-benzoyl-7-benzyloxy-3-methyl-l-oxa-4-azaspiro [4 4] nonane (250 mg,

0.7 mmol) were dissolved in 5 ml CH.CN IR 120 [H ⁺ ] (2x with methanol and 1x with H ₂ 0 washed) was added to a pH of 5-6. The mixture was stirred at room temperature until no more educt was detectable. The reaction mixture was filtered and the filtrate was evaporated. After column chromatography, (R) -3-benzyloxycyclopentanone (81 mg, 0 4 mmol) obtained

ee: 84% (chiral GC, Macherey - Nagel, Lipodex E)

Yield: 61%

[α] _D 20 = _ _{43 0} ° ( _c i _0ι CH ₂ C1 ₂ )

F.p .: syrup

Η-NMR and ¹ C-NMR are identical to the literature values (TH Eberlein, FG West, R W. Tester, J. Org Chem., 1992, 57, 3479 - 3482)

Examples 2 to 7 were carried out in the manner described in Example 1, but using other aldehydes / ketones and anchor / protective groups

Example 2: (R) -3-Benzyloxycvclopentanone

(3R -4-Benzoyl-3-ethyl-1-oxa-4-azaspiro [4 4] nonane

ee: 90%. HPLC (CHIRALCEL OD-H, n-heptane IPA, 7 3rd P = 40 bar, flow rate = 0 50 mL / min)

Yield: 57%

[α] _D 20 ₌ .59 ₄ ^o _(c 3 9 CH ₂ C1 ₂ )

F.p .: waxy

NMR NMR. δ 0.61 (t, J = 7.3 Hz, 3H, CH ₂ CH ₃ ), 1.31 (m, 2H, CH ₂ CH ₃ ), 1 58 - 2 03, 2 3 1 - 2.72 (2 xm, 6H & 2H, H6 , 7, 8, 9), 3 68 - 3 99 (m, 3H, H2,3), 7 37 (s, 5H, benzoyl)

¹³ C NMR. δ 9.9 (CH ₂ CH ₃ ), 24 8, 24.8, 26.4 (C7, 8, CH ₂ CH ₃ ), 35 0, 36 3 (C6, 9), 59 6 (C3), 67.3 (C2), 104 9 (C5), 126.3, 128.4, 129.4, 138.2, 168 1 (benzoyl)

(3R.7R) -4-benzoyl-3-ethyl-l-oxa-4-azaspiroι ^r 4 4] nonan-7-ol

de: 7: 1 (75%) (after column chromatography)

Yield: 82%. Mp: Su up

¹ H-NMR δ (small amount isomer printed in bold) 0 60 (t, J = 7 4 Hz, 3H, CH ₂ CH, 1 1 1 - 1 43 (br m, 2H, CH ₂ CH ₃ ), 1 69 - 2 62 (m, 6H, H6, 8, 9, OH), 2 69, 2.90 (2 x dd, J _{6 7} = 5 7 Hz, J ₆ _.6 - = 13 7 Hz, IH, ratio 7 1, H6), 3 70 - 3 99 (m, 3H, H2,3), 4 41 (br s, IH, H7), 7 36 (s, 5H, benzoyl)

¹ C-NMR δ (small quantity isomer printed in bold) 9 9 (CH ₂ QL), 26.3, 26 4 (CH ₂ CH ₃ ) 33.6, 34 5, 43 1, 44.7 (C6, 8, 9), 59 4 (C3) , 67.5, 67 6 (C2), 72.7, 72 8 (C7), 103 9 (C5), 126 2, 128.4, 128 4, 128.5, 129 7, 137 7, 168 3 (benzoyl)

(3R, 7R) -4-benzoyl-7-benzyloxy-3-ethyl-l-oxa-4-azaspιror4 4] nonane

de: 7 1 (75%) (NMR)

Yield: 75%

F.p .: syrup

"H-NMR δ (small amount isomer shown in bold) 0 64 (t, J = 7 4 Hz, 3H, CH ₂ C), 1 20 - 1 43 (br m, 2H, CH ₂ CH ₃ ), 1 84 - 2 09 & 2 20 - 2 65 (2 xm, 5H, H6, 8, 9), 2 76, 2.98 (2 x dd, J ₆ - ₇ = 6 9 Hz, J _{6 6} = 14 1 Hz, IH, ratio 7 l , H6), 3 76 - 4 03 (m, 3H, H2,3) 4 33 (bi s, IH, H7), 4 55 (m, 2H, CH ₂ Ph) 7 22 - 7 47 (m, 10H, CH ₂ P_h, COPh)

^I3 C-NMR δ (small amount isomer shown in bold) 9 9 (CH ₂ CH ₃ ), 26 5 3 1 8, 34.0, 35 3 (C8, 9, CH ₂ CH ₃ ), 42 1, 43.0 (C6), 59 6 , 59.7 (C3), 67.8, 67 9 (C2), 71 0, 71.3 (CH ₂ Ph), 79.3 79 6 (C7), 103 5 (C5), 126 5, 127 6 127 8. 128 5, 128 7 , 129 7, 129 8, 138 2, 139 2 (CH ₂ Ph, COPh), 168 4 (COPh)

(R) -3-benzyloxycyclopentanone

ee: 76% (chiral GC, Macherey - Nagel, Lipodex E)

Yield: 77%>

F.p .: syrup

¹ H-NMR and ¹³ C-NMR are identical to the literature values (TH Eberlein, FG West, RW Tester, J Org Che, 1992 57, 3479 - 3482)

(2S) -4-Benzoyl-2-methyl-l-oxa-4-azaspιror4 4] nonane ee: 99%

Yield: 50%

[J _D 20 = _{+ 12} o 6 (c 2 2, CH ₂ C1 ₂ )

F.p.:Sιrup

'H NMR δ 1 27 (d, J _{2 Me} = 5 9 Hz, 3H, Me), 1 59 - 2 03 & 2 35 - 2 75 (2 xm, 6H, 2H, H6, 7, 8, 9) , 3 19 (dd, J ₂ , "= JV," = 9 5 Hz, IH, H3 *), 3 45 (dd, J ₂ , = 5 2 Hz, IH, H3), 4 04 (m, IH, H2), 7 33 - 7 51 (m, 5H, benzoyl)

^{, 3} C NMR δ 17 5 (Me), 24 5, 25 2 (C7, 8), 35 2, 36 1 (C6, 9), 55 6 (C3), 70 8 (C2), 105 0 (C5 ), 126 6, 128 4, 129 8, 137 9, 167 4 (benzoyl)

(2S 7R -4-Benzoyl-2-methyl-1-oxa-4-azaspiro [ ^" 4 4] nonan-7-ol

de: 1 1 1 (HPLC, CHIRALCEL OD-H - n-heptane IPA, 7 3, P = 38 bar, flow rate = 0 50 mL / min - after chromatography)

Yield: 77%

[αJ _D 20 _{== + 87 8} ° ( _C i _6ι CH ₂ C1 ₂ )

F.p .: syrup

'H-NMR δ (small quantity isomer printed in bold) 1 26 (d, J _{2 e} = 6 0 Hz, 3H, Me), 1 69 -2 37 & 2 53 - 2 69 (m, 5H, H6, 8, 9) , 2 76 2.93 (2 x dd, J ₆ „ ₇ = 5 9 Hz, J _{6 6} , = 13 9 Hz, IH, ratio 1 1 1, H6) 3 19 (dd, J ₂ -, - = J-, . = 9 6 Hz, IH, H3 ^S ), 3 45 (dd J _{2 3} = 5 3 Hz, IH, H3), 4 06 (m, IH, H2), 4 46 (m, IH, H7), 5 15 (br s, IH, OH), 7 42 (m, 5H, benzoyl)

¹³ C NMR δ 17 4 (Me), 34 2, 43 3 (C6, 8, 9), 55 3 (C3), 71 4 (C2), 73 3 (C7), 104 0 (C5), 126 6 , 128 4, 130 1, 137 3, 167 7 (benzoyl)

(2S, 7R) -4-benzoyl-7-benzyloxy-2-methyl-l-oxa-4-azaspιroι ^r 4 4] nonane

de: 18 1 (NMR)

Yield: 43%

[α] _D 20 _{= + 62} 7 ⁰ _{(c 2} 7, CH ₂ C1 ₂ )

F.p.:Sirup

'H-NMR δ (small quantity isomer printed in bold) 1 31 (d, J _{2 Me} = 5 9 Hz, 3H, Me), 1 84 -2 39 & 2 52 - 2 73 (m, 5H, H6, 8, 9) , 2 81, 2.96 (2 x dd, J _{6Ä 7} = 7 0 Hz, J _{6 6} * = 13 8 Hz, IH, Ratio 1 8 1, H6) 3 22 (dd, J _2,3 - = J, _fl = 9 6 Hz, 1 H, H3 ^ff ), 3 47 (dd, J _2,3 = 5 2 Hz, 1 H, H3), 4 06 (m, IH, H2), 4 38 (m, IH, H7), 4 55 (s, 2H, CH ₂ Ph), 7 21 - 7 57 (m, 10H, CH ₂ Ph, COPh )

"C NMR δ 17 6 (Me), 3 1 5, 34 9 (C8, 9), 42 3 (C6), 55 5 (C3), 70 9, 71 1 (C2, CH ₂ Ph), 79 8 (C7), 103 2 (C5), 126 8, 127 0, 127 5, 127 8, 128 4, 128 4, 128 8, 130 1, 137 6, 139 0 (CH ₂ P_h, COPh), 167.6 (COPh )

(7 ^) - 3-Benzyloxycyclopentanone

ee: 91% (chiral GC)

Yield: 82%

F.p .: syrup

Η-NMR and ¹ C-NMR are identical to the literature values (TH Eberlein, FG West, RW Testei, J Org Chem, 1992, 57, 3479 - 3482)

Example 4: (3S. 4-S ') - 4-hvdroxy-3-n.ethyl-cvclohexanone

(3R.5R / S, 7 / τ?) - 4-Benzoyl-3.7-dimethyl-l-oxa-4-azaspirof4 4] decane

de: SR 5S 4 4 1 (HPLC, CHIRALCEL OD-H - n-heptane IPA. 4 1, P = 34 bar, flow rate =

Purity of the starting materials (Aldrich) (/) - (+) - 3-methyl-cyclohexanone (98% ee) (/ <-) - (-) - 2-amino-l-propanol (98% ee)

Yield: 76%

[αI _D 20 ₌ _45 ₄ o ( _{cl 4ιCH2C} , ₂₎

Mp: 62 - 72 ° C

Η-NM R δ 0 82 - 1.16 (m, 6H, 2 x Me), 1 43 - 2 87 (m, 9H, H6, 7, 8, 9, 10), 3 60 (m, IH, H2), 4 01 (m, 2H, H2, 3), 7 38 (s, 5H, benzoyl)

¹³ C-NMR δ (small quantity isomer printed in bold) 19.9, 20 7, 20.8 22 4 (Me), 22 8 (C9), 28.2, 29 9 (C7), 30.1, 30.2 (C8), 33 3, 34.8 (C10) , 43 3 (C6), 54.2, 54 5 (C3), 69.3, 69 4 (C2), 97 4 (C5), 126 1, 128 5, 129 2, 138 6, 168 4 (benzoyl)

(3R. 5 R. IS. 8ΛV4-Benzoyl-3.7-dimethyl-l-oxa-4-azaspiror4 4] decan-8-o1 de: 95% HPLC (CH1RALCEL OD-H, n-heptane IPA, 7 3, P = 40 bar, flow rate = 0

Yield: 40%

[α] _D 20, =. 33 ₂ ° (c 0 5 _, CH ₂ C1 ₂ )

M.p .: 215-218 ° C

NMR NMR δ 0 98 & 1 03 (2 xd, J = 6 7 & 6 4 Hz, 6H, 2 x Me), 1 54 - 1 95 & 2 63 (m & br, 6H & 2H, H6, 7, 9, 10, OH), 3 31 (ddd, J ₈ , _9a χ = Js.vx = 10 8 Hz, J _{8) 9eq} = 4 2 Hz, IH, H8), 3 63 (m, IH, H2), 4 02 (m, 2H, H2, 3), 7 37 (s, 5H, benzoyl)

¹³ C-NMR δ 18 2, 20 6 (Me), 29 2 (C9), 31 8 (CIO), 36 7 (C7), 41 1 (C6), 54 6 (C3), 69 4 (C2), 74 8 (C8), 96 3 (C5), 126 1, 128 5, 129 3, 138 3, 168 5 (benzoyl)

(3S.4S) -4-hydroxy-3-methylcyclohexanone

en: 1 diastereoisomer

Yield: 81%

(α] _D 20 = + 22 0 ° (c 0 6, CH ₂ C1 ₂ )

F.p.:Sιrup

NMR NMR δ 1 05 (d, J = 6 1 Hz, 3H, Me), 1 68 - 2 59 (m, 8H, H2, 3, 5, 6, OH), 3 69 (ddd, J, 5. _x ~ J ₄ , ₃ a \ = 8 1 Hz, J 5-q ⁼ 3 7 Hz, 1 H, H4)

¹³ C-NMR δ 18 6 (Me), 32 4, 38 4, 39 5 (C2, 5, 6), 45 7 (C3), 72 8 (C4), 210 5 (Cl)

Example 5: 3-Acetoxy-cvclopentanecarboxaldehyde

(4R) -N-Benzoyl-2-cvclopentyl-4-methyloxazohdιn

de: 1 diastereoisomer (ΝMR)

Yield: 28%

[α] _D 20 = + ιo7 5 ° (c 1 1, CH ₂ C1 ₂ )

Mp: pale yellow 01 'H-NMR: δ 1. 13 - 1.85 (br m, 1 IH, H2 \ 3', 4 ', 5', Me), 2.29 (br s, IH, H l '), 3.54 - 4.03 ( m, 3H, H4, 5), 5.46 (d, J _{2, r} = 5.7 Hz, IH, H2), 7.38 (m, 5H, benzoyl).

^I C-NMR: δ 20.9 (Me), 25.3, 27.3, 28.4 (C2 \ 3 ', 4', 5 '), 44.1 (CT), 54.3 (C4), 71.9 (C5), 92.5 (C2), 126.6 , 128.5, 130.0, 137.4, 170.5 (benzoyl).

(47) -N-Benzoyl-2- (3'-hydroxycyclopentyl) -4-methyloxazolidine

Yield: 30%.

[αJ _D 20 = + ₁₀ 4.9 ° (c 3.9, CH ₂ C1 ₂ )

Mp: 83-85 ° C

'H-ΝMR: δ (small quantity isomer printed in bold) 1. 14 - 2.22 (4 x br m, 10H, H2 \ 4', 5 ', Me, OH), 2.64 (br s, IH, Hl'), 3.64 - 3.99 (m, 3H, H4, 5), 4.21, 4.33 (2 x br s, IH, ratio: 1: 3.5, H3 '), 5.42 (m, IH, H2), 7.43 (m, 5H, benzoyl).

¹³ C-ΝMR: δ 20.9, 21.1 (Me), 25.3, 25.4, 26.0 (C5 '), 35.0, 35.1, 35.7, 35.7, 37.1, 38.2, 40.1 (C2 \ 4'), 41.5, 41.9 (Cl ') , 54.3, 54.5 (C4), 71.6, 71.9, 72.0, (C5), 73.1, 73.3, 73.3, 73.4 (C3 '), 92.2, 92.4, 92.5 (C2), 126.4, 126.5, 126.6, 126.9, 127.0, 127.1 , 127.1, 128.4, 128.5, 128.9, 130.1, 137.1, 137.2, 171.2 (benzoyl).

(4R) -N-Benzoyl-2- (3 '-acetoxy-cyclopentyl) -4-methyloxazolidine

Yield: 83%.

F.p .: syrup.

Η-ΝMR: δ (small quantity isomer printed in bold) 1.29 (i.e. _{4th 4th ΝL} . = 6.4 Hz, 3H, Me), 1.54 - 2.26 (3 xm, s, 9H, H2 ', 4', 5 ', COCH ₃ ), 2.40, 2.66 (2 x br s, IH, ratio: 1: 3.5, H l '), 3.65 - 3.98 (m, 3H, H4, 5), 5.03, 5.24 (2 x br s, IH, H3' ). 5.42, 5.49 (2 xd, J _2. , - = 5. 1 & 5.7, ratio 3: 1, IH, H2), 7.42 (m, 5H, benzoyl).

^I C-NMR: δ (small quantity isomer printed in bold) 21.0, 21.4 (2 x Me), 24.9, 26.1, 26.1 (C5 '), 32.1, 32.2, 33.7, 35.2 (C2 \ 4'), 41.2, 41.6, 42.5 ( CL), 54.4 (C4), 72.0 (C5), 75.9, 76.7 (CT), 91.8, 92.0, 92.0 (C2), 126.6, 128.5, 130.1, 137.2, 170.8 (benzoyl).

Example 6: (R) -3-Benzyloxycyclopentanone

(2R) -4-B enzo yl-2-methyl-1-oxa-4-azaspiro f 4.41 nonane

ee: 98% Yield: 39%

l ^α l _D ^{2 (} > = - 123 5 ° (c 2 3, CH ₂ C1 ₂ )

F.p.:Sιrup

NMR NMR δ 1 27 (d, J _{2, Me} = 6 1 Hz, 3H, Me), 1 56 - 2 05 & 2 36 - 2 75 (2 xm, 6H, 2H, H6, 7, 8, 9) , 3 20 (dd, J _{2 #} = J ₃ , * = 9 5 Hz, IH, H3 ^# ), 3 45 (dd, J _{2 3} = 5 1 Hz, IH, H3), 4 03 (m, IH, H2), 7 33 - 7 51 (m, 5H, benzoyl)

(2R, 7R) -4-benzoyl-2-methyl-1-oxa-4-azaspιror4 4 nonan-7-ol

de: 1 6 (71%, NMR, after chromatography)

Yield: 28%

l ^α J _D ²⁰ = not determined due to low de

F.p.:Sιrup

NMR-NMR δ (small quantity isomer printed in bold) 1 23 (d, J ₂ = 5 9 Hz, 3H, Me), 1 68 -2 56 (m, 5H, H6, 8, 9), 2.73, 2 90 (2 x dd, J ^ ₇ = 6 3 Hz, J _{6 6} - = 14 0 Hz, l H, Veι haltnιs 6 1, H6)

3 17 (dd, J _{2 Ä} = J _{3 3 #} = 9 7 Hz, IH, H3 ^" ), 3 43 (dd, J _{2 3} = 5 3 Hz, IH, H3), 4 03 (m, IH, H2 )

4 46 (m, IH, H7), 5 04 (br s, I H, OH), 7 39 (m, 5H, benzoyl)

^I3 C-NMR δ (small quantity isomer shown in bold) 17 4 (Me), 33 8, 34 6, 43.2 44 5 (C6, 8, 9), 55 3 (C3), 71 4 (C2), 72 2, 73.3 ( C7), 103 5, 104.0 (C5), 126 6, 128 4, 130 2, 137 2, 167 8 (benzoyl)

(2R, 7 /?) - 4-Benzoyl-7-benzyloxy-2-methyl-l-oxa-4-azaspιro [4 4] nonane

de: 6 1 (71%, NMR)

Yield: 56%>

[α] -20 = _j _we g _s low de not determined

F.p.:Sιrup

1H-NMR δ ( _{small quantity isomer} printed in bold) 1 31 (d, J _{2 Ml} = 6 0 Hz, 3H, Me), 1 77 - 2 70 (m, 5H, H6, 8, 9), 2 81, 2.94 (2nd x dd, J _{6S 7} = 7 7 Hz, J _{6 6S} = 14 1 Hz, IH, ratio 6 1, H6) 3 22 (dd, J _{2 3} = J _{3 vl} = 9 5 Hz, IH, H3 *), 3 48 (dd, J _{2 3} = 5 2 Hz, IH, H3), 4 08 (m, IH, H2), 4 40 (m, IH, H7), 4 54 (s, 2H, CH ₂ Ph), 7 21 - 7 57 (m, 10H, CH ₂ Ph, COPh)

¹³ C-NMR δ 17 5 (Me), 32 1, 33 9 (CS, 9), 42 8 (C6), 55 3 (C3), 71 3, 71 3 (C2, CH ₂ Ph), 78 8 ( C7), 102 7 (C5), 126 7, 126 7, 127 4, 127 8, 128 4, 128 4, 130 1, 137 5, 139 0 (CH ₂ Ph, COPh), 167 5 (COPh)

(7 -3-Benzyloxycyclopentanone

ee: 71% (chiral GC, Macherey - Nagel, Lipodex E)

Yield: 77%

[α] _D 20 =.

F.p .: syrup

1H-NMR and ° C-NMR are identical to the literature values (T H Eberlein, F G West, R W Tester, J Org Chem, 1992, 57, 3479 - 3482)

Example 7: (R) -3-Benzyloλvcvclopentanone

(3Λ ^τ ) -4-benzoyl-3-methyl-l-oxa-4-azaspιro [4 4] nonane

ee: 98%

Yield: 89%

[α] _D 20 = + _{76 4} ^o ( _{C 2} 7, CH ₂ C1 ₂ )

M.p .: 65 5-67 5 ° C (EtOAc / Pet Ether) pale yellow solid

Η NMR δ 0 93 (m, 3H, Me), 1 57 - 2 03, 2 32 - 2 71 (2 xm, 6H & 2H, H6, 7, 8, 9), 3 57 (m, IH, H2 ), 3 98 (m, 2H, H2.3), 7 38 (s, 5H, benzoyl)

¹³ C-NMR δ 20 1 (Me), 24 7, 24 8 (C7.8), 35 0, 36 5 (C6, 9), 54 1 (C3), 70 0 (C2), 105 0 (C5) , 126 2, 128 4, 128 5, 129 4, 138 2, 168 0 (benzoyl)

(3S, 7R) -4-benzoyl-3-methyl-l-oxa-4-azaspιro [4 4 ^" | nonan-7-ol

de: 1 3 (50%), NMR, after chromatography

Yield: 35% , ^α ' _D ²⁰ = not determined due to low de

F.p .: syrup

¹ H-NMR δ ( _{low-quantity isomer} printed in bold) 0 94 (m, 3H, Me), 1 74 - 2 70 (m, 6H, H6, 8, 9, OH), 2.76, 3 01 (2 x dd, J _6V7 = 5 9 Hz, J _{6 6.} = 14 0 Hz, IH, ratio 3 1, H6), 3 63 (m, IH, H2), 4 02 (m, 2H, H2, H3), 4 47 (br s , IH, H7), 7 38 (s, 5H, benzoyl)

"C-NMR δ (small quantity isomer printed in bold) 20 1, 20.3 (Me), 33 8, 34 6, 34.7, 34.9 (C8, 9), 43.4, 45 0 (C6), 54 2 (C3), 70 5 ( C2), 73 0 (C7), 104 0, 104.2 (C5), 126 4, 128 7, 129 8, 138 1, 168 5 (benzoyl)

⁽³ R, 7 R) -4-Benzoyl-7-benzyloxy-3-methyl-1-oxa-4 azaspιrof4 4] nonane

de: 1 2 (33%, NMR)

Yield: 78%

ι ^α J _D ²⁰ = not determined due to low de.

F.p .: syrup

¹ H-NMR δ (small amount isomer printed in bold) 0 96 (m, 3H, Me), 1 79 - 2 66 (m, 5H, H6, 8, 9), 2.78, 2 96 (2 x dd, J _{6> 7} = 7 3 Hz, J _{6 6} = 14 4 Hz, IH, ratio 2 1, H6), 3 56 (m, IH, H2), 4 03 (m, 2H, H2, H3), 4 33 (br s, IH, H7) 4 54 (m, IH, CH ₂ Ph), 7 34 (m, 10H, benzoyl, benzyl)

¹ C-NMR δ (small quantity isomer printed in bold) 20 1 (Me), 31 5, 31.7, 33 9, 34.1 (C8, 9), 41.9, 43 0 (C6), 54.0, 54 1 (C3), 70 3, 70.4 (C2), 70.9, 71 1 (CH ₂ Ph), 79 1, 79.2 (C7), 103 0 (C5), 126 2, 126 2, 127 4, 127 8, 128 3, 128 5, 129 6, 129 7, 138 0, 138 8, 168 3 (benzoyl, benzyl)

(R) -3-benzyloxycyclopentanone

ee: 29%> (chiral GC, Macherey - Nagel, Lipodex E)

Yield: 74%

F.p .: syrup

1H-NMR and ¹³ C-NMR are identical to the literature values (TH Eberlein, FG West, RW Tester, J Org Chem, 1992, 57, 3479 - 3482)

Claims

Claims:

1. Process for the preparation of compounds of the formula

~ l ₇ r- A '(II), where

where in formula II one of the radicals X, Y is hydrogen, n is one of the integers 0, 1, 2 or 3, the compounds of

Formula I optionally contain a double bond in the cycle and m denotes one of the numbers 0 or 1,

Room and Z ₂ independently of one another denote a Ci to Cs alkylene radical which can optionally be substituted by Ci to C alkyl and / or unsaturated. R 1, R ₂ independently of one another denote hydrogen or straight-chain or branched or cyclic C 1-4 -alkyl or R _t and R ₂ together with the group A containing cycle form a bicyclic compound of the structure bicyclo [a, b, c] heptane to decane (a, b, c = 0, 1, 2, 3 or 4), which may be replaced by C

Cj-alkyl may be substituted and / or unsaturated, and

R ₃ , R ₊ Wassei substance or a straight-chain, branched or cyclic Ci - Cg-alkyl can be.

characterized in that a compound of formula

where A, A \ Ri, R ₂ , R ₃ , R-ι, Zi, Z ₂ , m and n have the meaning given above, is protected with a suitable chiral anchor / protective group which hydroxylates the compound protected in this way enzymatically, regioselectively and stereoselectively is, if necessary, the hydroxyl group is protected with a suitable compound and the

Anchor / protection group is split off

2. The method according to claim 1, characterized in that as a compound of the formula

(III) or (IV) one of the following compounds is used "

or Bιcyclo [2 2 1] heptan-2-one, (IR) and (lΛ ^' ) -l, 7,7, -tπmethyl-bicyclo [2 2 l] heptan-2-one Trans-1 -decalon, 2- Decalon, (IS) and (l /) - 1,3,3-trimethyl-bicyclo [2 2 l] heptan-2-one bicyclo [3 3 0] octane-3,7-dione, bιcyclo [3 3 0] octane 3-one, bicyclo [3 3.0] oct-7-en-2-one bicyclo [3 3 0] oct-6-en-2, bicyclo [4 2 0] oct-2-en-7-one, bicyclo [3.2.0] hept-2-en-7-one bicyclo [3.2 0] hept-2-en-6-one

3 The method according to claim 1, characterized in that as a compound of the formula

(III) or (IV) one of the following compounds is used

4. The method according to any one of claims 1 or 2, characterized in that a compound of the formula as an anchor / protective group

, (V) or (VI), where R 'is straight chain or branched Cι. ₄ alkyl means is used.

5. The method according to any one of claims 1 to 4, characterized in that the N-benzoyl derivative of one of the following compounds is used as anchor / protective group: R-2-amino-l-propanol, S-2-amino-l -propanol, Rl-amino-2-propanol, Sl-amino-2-propanol, R-2-amino-l-butanol, S-2-amino-l-butanol, R-l-amino-2-butanol or SI -amino-2-butanol

6. The method according to any one of claims 1-5, characterized in that the enzymatic hydroxyation is carried out with one of the following microorganisms: Aspergillus ochraceus ATCC 18500, Bacillus megaterium CCM 2037, Bacillus megaterium DSM 32, Beauveria bassiana ATCC 7159, Calonectria decora DSM 879 , Chaetomium cochlioides DSM 83 1, Chaetomium globosum DSM 1962, Comyespora casshcola DSM 62474, Corticum sasakii NPvRL 2705, Cunninghamella blakesleeana DSM 1906, Cunninghamella echinulala DSM 1905, Cunninghamella elegans DSM 1908 ATMinaina DSM 1908, Diplodia24ellaellayp 8979, Mucor plumbeus CBS 1 10. 16, Pseudomonas put / da ATCC 29607, Pellicularia filamentosa IFO 6298, Penicillium rastrickii ATCC 10490, Polyporus ostreiformis CBS 36234, S / aurophoma species DSM 858 and Streptomyces gnseus ATCC 13273.

7. The method according to any one of claims 1-6, characterized in that a microorganism of one of the species Beauveria bassiana ATCC 7159 or Cunninghamella blakesleeana DSM 1906 is used for the hydroxyation.