DE102009045969B4 - Process and means for cleaving esters, amides and thioesters of formic acid - Google Patents

Abstract

Use of a formate dehydrogenase (E.C. 1.2.1.2) to cleave formyl protecting groups on hydroxyl groups, the formyl protecting group being converted to CO.

Description

The invention relates to a novel process and means for cleaving esters, amides and thioesters of formic acid.

The esterification with formic acid is used in organic synthesis primarily for the protection of alcohols, amides, amino acids, carbohydrates and steroids ( JH Babler, BJ Invergo, Tetrahedron Lett. 22, 1981, 621 , // HJ Ringold, B. Löken, G. Rosenkranz, F. Sondheimer, J. Am. Chem. Soc. 78, 1956, 816 , // M. Dymicky, Org. Prpe. Proced. Int. 14, 1982, 177 .). The state of the art for the preparation of formate-protected chiral alcohols, carboxylic acids, and amines based on classical chemical reactions ( TW Greene, PGM Wuts, Protective Groups in Organic Chemistry, 4th edition, Wiley & Sons 2006, ISBN 10: 0471697540, MB Smith, J. March, March's Advanced Organic Chemistry Reactions, Mechanisms and Structure, 6th edition, Wiley-Interscience, 2007, ISBN 10: 0-471-72091-7 ). By attaching a formyl group to the alcohol, the formoxy group (-O-CHO) and thus stable compounds are formed, which are modified in subsequent chemical processes to give the functional, protected unit. The cleavage of the ester group proceeds according to the prior art via chemical and biochemical methods, such as the hydrolysis of esters by strong acids or bases (A. Streitwieser, CH Heathcock, EM Kosower, Organic Chemistry, 2nd edition, VCH Verlag, Weinheim , 1994.), such as concentrated mineral acids, as well as potash and caustic soda. These methods are characterized by complete conversion, but lead to side reactions on unprotected functional units or partial to complete degradation in acid or base labile compounds. In the case of relatively high molecular weight compounds, such as polysiloxanes, the acid- or base-catalyzed ester hydrolysis leads to cleavages on the siloxane backbone, with formation of undesired by-products ( MA Brook, Silicon in Organic, Organometallic and Polymer Chemistry, John Wiley & Sons, Inc., Canada, 2000 .).
A preferred prior art method for cleaving esters is the use of hydrolases which cleave protecting groups such as formyl and acetyl groups under mild physiological conditions ( K. Drauz, H. Waldmann, Enzyme Catalysis in Organic Synthesis A Comprehensive Handbook, VCH-Verlag, Weinheim, 1995 ).

The hydrolysis of a formic acid ester of an alcohol R-OH with a hydrolase is illustrated by formula 1:

The mild conditions cause fewer side reactions. Disadvantageously, however, the liberated alcohol and the corresponding carboxylic acid must be worked up via subsequent purification steps. The hydrolase-catalyzed reaction is an equilibrium reaction which, as such, per se, is particularly disadvantageous in that it never leads to complete separation. In addition, the specificity of hydrolases for individual esters is low, d. H. a selective cleavage of different ester groups is not possible.

In US 2002/002250 A1 and US 2005/0124677 A1 For example, the peptide deformylase (PDF) is used together with a formate dehydrogenase (FDH, F-91666 Sigma chemicals) from Pseudomonas oxalaticus in an activity assay in which the peptide deformylase forms an N-formylated peptide (N-formyl-Met-Ala-Ser) of formate (HCOO ^- ) splits. The formed formate (HCOO ^- ) is oxidized by the FDH to CO ₂ .
Uotila L and Koivusalo M, Archives of Biochemistry and Biophysics 196: 33-45 (1979) describe the purification of formate dehydrogenase (FDH) from pea seeds.
Egorov et al. Biochemical and Biophysical Research Communications 104, pp 1-5 (1982) disclose that S-formyl-glutathione is a substrate of the methylotrophic bacterium Achromobacter parvolus and the methylotrophic yeast Candida methylica.

DE 103 13 972 A1 relates to a coupled enzymatic reaction system, which is characterized in that it is carried out in a homogeneous solvent mixture. In particular, the invention is directed to a reaction system comprising a cofactor-dependent enzymatic transformation of an organic compound, wherein the cofactor is regenerated enzymatically in the same system.

Donato H. et al. Journal of Biological Chemistry 282 (47), pp. 34159-34166 (2007) describe a formyl tetrahydrofolate dehydrogenase (EC 1.5.1.6). An N-carbonyl group is implemented.

The object of the invention is to specify an improved process for the cleavage of esters, amides and thioesters of formic acid or for elimination of formyl groups and means for carrying out the process.

According to the invention, the object is achieved by the use of a formate dehydrogenase (E.C. 1.2.1.2) for splitting off formyl protective groups onto hydroxyl groups having the features of claim 1. Further embodiments contain the features of claims 2 to 7.

Formate dehydrogenase (FDH) offers a novel possibility for efficient cleavage of the formyl protecting group from formyl-protected compounds. By using FDH, in particular, the ester is split into an alcohol and carbon dioxide. The gaseous carbon dioxide escapes during the reaction, so that a shift of the equilibrium irreversibly in favor of the products occurs. This leads to a quantitative conversion and thus to a significant increase in the space-time yield in comparison to the hydrolases used in the prior art.

The process is advantageously applicable to formyl-protected hydroxy groups of a variety of different molecules (various R) (alcohols, sugars, sterols, siloxanes). The formic acid ester (compound of the HCO-OR formula Formula 2 'type) is preferably added in a concentration range of 0.1 μmol / L to 1 mol / L. Furthermore, the method is also applicable to formyl protected thio and nitrogen compounds (n-formyl).

mit X = O und R ausgewählt aus substituierten oder unsubstituierten Kohlenwasserstoffresten, Silyl und Hydrocarbylsilyl (ein mit Kohlenwasserstoffresten substituiertes Silicium). Die Kohlenwasserstoffreste (bzw. Hydrocarbylreste) sind bevorzugt ausgewählt aus Alkyl, Alkenyl, Aryl, Zuckerresten und Steranen. Besonders bevorzugte R sind substituierte oder unsubstituierte Alkyl, Aryl, Alkylsilyl und Arylsilyl. Bevorzugte Alkyl- und Alkenylgruppen haben eine Kohlenstoffkette oder ein cyclisches Ringsystem mit 1 bis 32 C-Atomen, bevorzugt mindestens 5 und bevorzugt bis 16 C-Atomen. Bevorzugte Arylreste haben ein cyclisches Ringsystem mit 1 bis 32 C-Atomen und ggf. bis zu 10, bevorzugt bis zu 5 Heteroringatomen (wie N, O und S), bevorzugt mindestens 5 und bevorzugt bis 16 C-Atomen, wie z. B. Phenyl, Benzyl und Indolresten und
X ausgewählt aus O, S und N-R1, wobei R1 Wasserstoff ist oder ausgewählt ist wie R. Bevorzugte R1 sind Wasserstoff, Alkyl mit 1 bis 6 C-Atomen, vorzugsweise 1 bis 3 C-Atomen, oder Aryl mit 5 bis 7 C-Atomen.The esters are preferably compounds of the following formula

with X = O and R selected from substituted or unsubstituted hydrocarbon radicals, silyl and hydrocarbylsilyl (a hydrocarbon radical-substituted silicon). The hydrocarbon radicals (or hydrocarbyl radicals) are preferably selected from alkyl, alkenyl, aryl, sugar residues and sterols. Particularly preferred R are substituted or unsubstituted alkyl, aryl, alkylsilyl and arylsilyl. Preferred alkyl and alkenyl groups have a carbon chain or a cyclic ring system having 1 to 32 C atoms, preferably at least 5 and preferably up to 16 C atoms. Preferred aryl radicals have a cyclic ring system having 1 to 32 carbon atoms and optionally up to 10, preferably up to 5 hetero ring atoms (such as N, O and S), preferably at least 5 and preferably up to 16 C atoms, such. As phenyl, benzyl and indole radicals and
X is selected from O, S and N-R1, wherein R1 is hydrogen or is selected as R. Preferred R1 are hydrogen, alkyl having 1 to 6 carbon atoms, preferably 1 to 3 carbon atoms, or aryl having 5 to 7C -atoms.

wobei R wie oben ausgewählt ist.According to the invention, esters having a structure of the formula (2 '):

wherein R is selected as above.

In the process according to the invention, the following reaction is catalyzed by the FDH:

Surprisingly, it could be shown that in the presence of FDH, in particular from esters of formic acid (HCO-OR), the underlying alcohol is formed.

The reaction can be advantageously carried out under mild reaction conditions (neutral pH, room temperature, aqueous system), thereby preventing side reactions. Separation of the alcohol is advantageous for enzyme stability, but not necessary.

Advantageously (unlike the hydrolase-catalyzed ester cleavage) no acid is formed in the process of the invention which could chemically modify the product or damage the stability of the enzyme by denaturation.

In contrast to hydrolases, the FDH also has a very high selectivity. Thus, for example, in a sugar in which an OH group is protected by a formyl group and other OH groups by acetyl groups, the formyl group can be cleaved off selectively. If the selectivity of FDH does not play a role in the case where the substrate contains no other acyl groups, technical enzyme preparations can also be used.

The FDH is used natively (in solution, suspension or emulsion) or by the usual methods as immobilized catalyst.

The process according to the invention is preferably carried out in an aqueous medium or a mixture of water and an organic solvent (as cosolvent). As organic solvents, water-miscible solvents, preferably protic solvents, such as alcohols, preferably primary, secondary or tertiary alcohols having 1 to 10 carbon atoms, are used. The proportion of organic solvent in the mixture is preferably up to 50%, particularly preferably up to 20%. Cosolvent serves to improve the solubility of the organic substrates and products. A particularly preferred alcohol used as cosolvent is isopropanol. Surprisingly, it was found that the FDH in the aforementioned concentrations of alcohol, in particular isopropanol, up to 50%, are active. Significant enzyme deactivation by alcohols liberated in the reaction, such as methanol, ethanol and n-propanol, only occurs above an alcohol concentration of 2 mol / L and the alcohol tolerance increases with decreasing chain length of the alcohol.

The FDH, especially the FDH from Candida boidinii, shows advantageous process stability over 5 h at a reaction temperature of 10 ° C to 50 ° C and stability at 60 ° C for at least 3 hours. The optimal process conditions are achieved in the pH range 6-8 for all esters to counteract a base- and acid-catalyzed cleavage. The formate dehydrogenase is preferably added in the range 0.1-5% by volume. The process preferably proceeds without pressure.

The invention therefore relates to the use of a formate dehydrogenase (FDH) for the cleavage of esters of formic acid or for cleavage of formyl protective groups. The formyl protecting group in the substrate is bonded to a hydroxy group (carbinol). The substrate is preferably immobilized on a support.

The formate dehydrogenase used in the invention is a nicotinamide adenine dinucleotide (NAD ⁺ ) or nicotinamide adenine dinucleotide phosphate (NADP ⁺ ) -dependent formate dehydrogenase (EC No. 1.2.1.2):

The reaction is an oxidation in which the cleaved formyl group is oxidized to CO ₂ and the cofactor (in formula 4 NAD ⁺ or NADP ⁺ ) is reduced. The reaction is carried out in the presence of water, that water consumed in the reaction is not shown separately in formulas 3 'and 4.

Other (not NAD ⁺ - or NADP ⁺ -dependent) FDH belong to a group of iron-sulfur proteins that have different electron acceptors as cofactors.

The (NAD ⁺ -dependent) FDH preferably originates from bacteria or yeasts, preferably from the genus Escherichia, in particular Escherichia coli, Candida, in particular Candida boidinii, or Saccharomyces, in particular Saccharomyces cerevisiae.

The cofactor NAD ⁺ or NADP ⁺ consumed in the reaction is preferably regenerated in situ. By in situ is meant herein within the same reaction vessel, preferably in the reaction mixture.

For the regeneration of NADH to NAD ⁺ or NADPH to NADP ⁺ chemical processes, such as electrochemical and photochemical, as known from the prior art ( Nakamura et al., 1988, Electroenzymology coenzyme regeneration , Springer Verlag; Ruppert & Steckhan, 1988, J. Chem. Soc. Chem. Commun. 1150-1151 ; Fry et al., 1994, Tetrahedron Lett. 35, 5607-5610 ) be applied. In these methods, the cofactor is often nonspecifically regenerated, but the rates of catalysis are generally high.

According to the invention, the regeneration of the cofactor preferably takes place with an alcohol dehydrogenase (ADH). The ADH reduces ketones or aldehydes to the corresponding alcohol, converting NADH to NAD ⁺ or NADPH to NADP ⁺ .

The alcohol dehydrogenase is preferably of the genus Candida, in particular Candida boidinii (ADH CB). The ADH is also used natively (in solution, suspension or emulsion) or by the usual methods as immobilized catalyst. Both enzymes (FDH and ADH) are preferably immobilized on the same support.

The substrate used for the ADH is preferably a ketone or aldehyde having 1 to 15 carbon atoms, preferably acetone. Preferably, a ketone or aldehyde is used, which is reduced by the ADH to the alcohol used as Cosolvens. Thus, the preferred acetone is converted by the ADH to isopropanol. The substrate for the ADH is either initially charged or added continuously or semicontinuously.

The reaction apparatus used in the invention preferably contains a gas-permeable membrane to ensure the liberation of carbon dioxide, and preferably a stirrer.

The invention is particularly suitable for use in the field of polymer chemistry, in particular for the removal of formyl groups from polymers having functionalized units in the side chain or terminal formyl groups. The or part of the hydroxyl groups in the starting monomers are protected here by formyl groups. The protected monomers are then linked by known polymerization techniques to higher molecular weight compounds. After completion of the reaction and adjustment of a defined chain length, formate dehydrogenase dissolved in aqueous buffer is added to the reaction mixture.

The reaction system is preferably mixed intensively and separated to the outside through a gas-permeable membrane, so that CO ₂ can escape. A particularly preferred field of application is the preparation of chiral, demanding alcohols by FDH-catalyzed cleavage of formyl groups.

The invention also provides the use of a kit for the cleavage of formic acid esters, amides or thioesters or for the removal of formyl protective groups containing a formate dehydrogenase and an alcohol dehydrogenase, preferably immobilized on a common carrier.

EXAMPLES

The invention is explained in more detail below by exemplary embodiments, without limiting the invention to these:

In the examples, formate dehydrogenase EC (E. coli), alcohol dehydrogenase CB (Candida boidinii) and NAD ⁺ from Julich Chiral Solutions GmbH, Jülich, DE and formate dehydrogenase CB (Candida boidinii) from Sigma Aldrich Co., St. Louis, MO, USA used.

FDH-catalyzed cleavage of formic esters of different alcohols:

The cleavage of formic acid esters by FDH was tested on the formic acid esters of methanol, ethanol, n-propanol and phenol:

Of the formic acid ester in each case a 50 mmol / L stock solution is prepared in 20 vol.% Isopropanol.

The qualitative conversion of the formic acid ester is monitored by the formation of NADH at 340 nm in a UV / vis spectrophotometer according to the method described above. The reaction is carried out in vessels which are sealed with a gas-permeable membrane to ensure the release of CO ₂ . To a solution of 100 mmol / L potassium phosphate buffer at pH 7.5 (940 μL + 60 μL isopropanol) is added 250 μL of the formic acid ester (50 mmol / L diluted in isopropanol) and 100 mmol / L NAD ⁺ ( 250 μL dissolved in 100 mmol / L potassium phosphate buffer). After 10 minutes of stirring and homogeneous mixing of the reactants, the reaction is started by adding 50 μL of dilute FDH EC or FDH CB (in the range 1-5 U / mL). The decrease in the educt and the formation of the product is monitored by gas chromatography over 4 hours.

As a result, both FDH of C. boidinii (FDH CB) and E. coli (FDH EC) quantitatively cleaved all the formic acid esters tested to release CO ₂ and the corresponding alcohol. Both FDH convert esters with different R-OH with R = alkyl or aryl. In control experiments with NAD ⁺ but without enzyme) no ester cleavage was observed.

In 1 exemplified is the course of the reaction by GC for formic acid ethyl ester (AEE) as FDH substrate over a period of 4 hours. As can be seen from the chromatogram, the substrate peak decreases significantly and the ethanol peak increases significantly.

The biocatalytic oxidation by FDH proceeds quite fast. All tested formic acid esters were converted to 60% after only 2 hours. The reactions were almost quantitatively completed with 97.5% conversion after 4 hours. In none of the tested formulations was it possible to detect free formic acid ester or free formic acid after completion of the reaction (maximum 5 hours).

The reaction rate in 20 vol.% Isopropanol is in 2 for the different formic acid esters tested. For phenylformate (APhE), this was even two to four times higher than for the alkyl formates (AME, AEE, APE abbreviations, see Table 1).

The FDH-mediated ester cleavage follows the rules of Michaelis-Menten kinetics. The K _m values given in Table 1 were determined using Eadie-Hofstee diagrams.

In all experiments, no adverse effect of alcohols at concentrations less than 20% by volume could be observed. In addition to the cosolvent isopropanol, these include the released alcohols from the formic acid esters. Table 1 R ester K _m (mmol / L) methyl Formic acid methyl ester (AME) 2.9 ethyl Formic acid ethyl ester (AEE) 4.1 n-propyl Formic acid n-propyl ester (APE) 4.7 phenyl Formic acid phenyl ester (APhE) 1.7

Regeneration of the process solution

The regeneration of the cofactors NAD ⁺ and NADP ⁺ in the process solution was carried out by adding ADH to the process solution used in Example 1 with 20% by volume of isopropanol with addition of acetone as substrate for the ADH. The ADH activity and regeneration of the cofactor NAD ⁺ and NADP ⁺ was determined photometrically at 340 nm.

FDH-catalyzed cleavage of siloxanes:

The cleavage of formic acid ester by FDH was tested on siloxanes of the following formula:

The reaction was carried out with a siloxane content of 1-20 mmol in 20% by volume of isopropanol according to Example 1. As a comparative experiments, the hydrolases Lipase CS and Novozyme 735 ^{® were} used instead of the FDH.

With FDH, the cleavage of the formyl group proceeds within 4 hours with both n = 1 and polysiloxanes with n to 150 quantitatively:

The comparative experiments with the hydrolases Lipase CS and Novozyme ^735® each resulted in a conversion of less than 20% after 96 hours.

Used abbreviations:

• FDH = formate dehydrogenase
• ADH = alcohol dehydrogenase
• M = mol / L
• GC = gas chromatography

Claims (7)

Use of a formate dehydrogenase (EC 1.2.1.2) for the removal of formyl protective groups on hydroxyl groups, wherein the formyl protective group is converted to CO ₂ . Use after Claim 1 , characterized in that the formate dehydrogenase is used natively or as immobilized catalyst. Use after Claim 1 or 2 , characterized in that a formate dehydrogenase from bacteria or yeasts, preferably of the genus Escherichia, Candida or Saccharomyces, is used. Use according to one of Claims 1 to 3 , characterized in that the reaction is carried out in a mixture of water with an organic solvent. Use after Claim 4 , characterized in that the proportion of organic solvent is up to 50%. Use according to one of Claims 1 to 5 , characterized in that as cofactor NAD ⁺ or NADP ^{+ is} used, which is regenerated with an alcohol dehydrogenase. Use of a kit comprising a formate dehydrogenase (E.C. 1.2.1.2) and an alcohol dehydrogenase, preferably immobilized on a common carrier for the removal of formyl protective groups.

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