PT97847B - PROCESS FOR THE PREPARATION OF BETA-HYDROXY-ALPHA-AMINOACIDES - Google Patents

Abstract

This invention relates to a process for the preparation of β-hydroxy-α-amino acids which are obtained by the condensation of aldehydes with glycine catalysed by serine hydroxymethyl transferase. The predominant product with the majority of aldehydes is the L-erythro diastereoisomer. For example, the methyl ester of succinic semialdehyde is condensed with glycine in the process to provide the monomethyl ester of L-erythro- α-amino-β-hydroxy adipic acid.

Description

DESCRIPTION

GIVES

INVENTION PATENT

No. 97.84?

APPLICANT: THE UNIVERSITY of notre dame du.lac., North american, industrial, in Soíith Bend india na 46556 - UNITED STATES NORTH AMERICA

EPIGRAPH: PROCESS FOR THE PREPARATION OF B-HYDROXY-AMINO ACIDS

INVENTORS:

MARVIN JOSEPH MILLER

Claim of the right of priority under Article 4? of the Paris Convention of 20 March 1883.

UNITED STATES OF NORTH AMERICA, ON JUNE 4, 1990, UNDER NO.07 / 532,845

THE UNIVERS1TY OF NOTRE DAME DU LAC

PROCESS FOR THE PREPARATION OF B-HYDROXY-a-AMINO ACIDS ®tôRXA_DiS ^ mVA h ^ SU / BO

The present invention relates to a process for the preparation of B-Hydroxy '' - amino acids which are obtained by condensing aldehydes with glycine catalyzed by hydroxymethyltransfsrsss of sarins. The predominant product with most aldehydes is the L-erythro diastereomer »For example? succinic semisldehyde methyl ester is condensed with glycine in the process to provide monomethyl ester of L ~ e r i t r o acid - · «- a mi η o - β - · h i tí r o x i a d ί p i c o =, ζ:

Λ '\

This invention relates to a process for the preparation of B-hydroxy-α-amino acids »In particular? it concerns an enzymatic process for the preparation of β-hydroMÍ-a-amino acids that are substantially in the L-erythro form.

Do B-hydroxy-camcino acids have many uses? including -the use as intermediate compounds in the preparation of β-lactam antibiotics. To see? for example? Mattingly? P »G„ j 1981? 46? 1557 and Milier? M »J»? et al »7026» Are several B-hydroxy-a-amino acid methods known? Yet? disadvantages salts. Do these requirements require chiral auxiliary agents? or production? of the treo (or sin) isomers »

η x 1 λ e r? H »J» »J» Org „Chem J »Am = Chem« Soc «1980? 10 chemicals from preparation of majority has one or absence of generality?

include the disabled? mainEnzymatic processes have some obvious advantages over chemical processes »For example? are carried out in aqueous systems under mild conditions? often being stereoselective »Is the enzyme used in the process of the invention known? In general? as an a1do1ase. One of these aldolases is serine hydroxymethyltransfrase (HMTS)? L »Adv» Enzymol. Report Mol Areas »Biol» 1982? 53? 83 and Schirch? L »§ Sross? T »Biol» Chem »1968» 245? 56-51 »The natural biological roles of aldolases involve the transfer of units of a carbon atom to or from serine and - retroaldol dissociation from β-hydroxy-a-amino acids such as threoniria and 1 p-tr eonin to give rise to an aldehyde and glycine »

Do aldolases exist in olantas? bacteria and animals.

for example? corn sprouts? mung bean sprouts ¹ rabbit liver »Does the process of this invention comprise the use of HMTS to prepare? under mild conditions?

prepare ?

precursors (5-h id rax i -amino acids provides in many isles L-erythro pus

of antibiotics ^ -lactam, cases ο β — hydroxy amino acid in. shape is the desired form of the precursor to propose the process by providing the correct diastereomeric form of the antibiotic

-lactate.

The invention provides an enzymatic process for the preparation of β-hydroxy-a-amino acids which comprises incubation in an aqueous medium at a pH value between about 7 s and about Q and at a temperature between about 3 ° C and about 55 ° C glycine and an aliphatic aldehyde, eg acetaldehyde or butanal, or an aromatic aldehyde, eg. 2-furfural, with CHMTS5 serine hydroxymethyltransferase. For example, a succinic semialdehyde ester is converted in the process to β-hydro> acid monoester; i — cí — aminoadí pico „ _; ..

A process presented here for the (5-hydroxy-P-amino acids represented by the formula 1

it comprises mixing in an aqueous medium at a pH of between about 5, 5 and about 9 of glycine and an RCHO aldehyde in the presence of serine hydroxymethyltransferase and 5'— pyridoxal phosphate. Is the process carried out at a temperature between about 3 €? ^C 'C and about qs and preferably & about ί * 7 ** Ό.ϊ

The relative proportions of glycine and RCHO aldehyde may vary%, however, the highest product yields appear to be obtained when using equimolar amounts. THE

The amount of enzyme used depends on the degree of purification of the enzyme and the effect that any impurities present may have on the enzyme activity.

The pH of the reaction medium is buffered at a value between 5.5 s and about 9 and, with most substrates, preferably at a pH value between 7.0 and about, 8.8. Phosphate buffers are suitable to obtain the desired pH range.

As with other enzymatic reactions, cofactors can influence the specificity of the substrate. enzyme as well as a. enzyme efficiency in. catalysis of. reaction of a given substrate. An essential cofactor for serine hydroxymethyltransferase in the process is pyridoxal 5'-phosphate. Factors other than FPL that can be added have a beneficial effect on the yield of the product obtained with some substrates. · For example, tetrahydrophallic acid enhances the activity of the enzyme in. conversion of the pyruvaldehyde substrate. Also, sodium metavanadate serves as a cofactor for converting the same aldehyde.

The aforementioned formula shows the β-hydroxy-α-amino acids provided by the process in the form of internal salt. Is this salt form the one expected with the pH value of the process? however, the non-ionic form represented by the formula.

OH

I h ₂ n-ch-ch-r

I

COOH can also exist under other conditions.

aldehyde, RCHO, used in the process, can be an alkyl-, alkyl- or alkynylaldehyde of the straight chain or

branched or an aromatic or heterocyclic aldehyde. The aldehyde may have substituent groups;, for example, alkoxy such as methoxy or ethoxy, esterified carboxy such as C ₁ -C ₄ alkyl esters of the carboxy group; cyan? hydroxy? acylated hydroxy such as formyloxy, acetoxy, propionoxy, or butyloxy, halogen such as fluorine or chlorine; haloalkyl such as trifluoromethyl or chloromethyl; or the aliphatic aldehyde may contain oxo groups eg RCHO may be a ketoaldehyde such as 4 — oxopentanal or pyruvaldehyde and the like. The aromatic aldehyde or heterocyclic aldehyde can likewise be substituted on the aromatic or heterocyclic ring or any one thereof. alkyl or alkenyl portion. Examples of alkyl-, alkenyl- and alkynylaldehydes RCHO that can be used in the process acetaldehyde, propionaldehyde, buficraldehyde, valeraldehyde, succinic semiadehyde methyl ester, succinic semiladehyde benzyl ester, 4-hydroxy-valeraldside, semiaidide methyl pyridate, pyridine hydroxide, pyridyl hydroxide, pyridine hydroxide, pyridine hydroxide, pyridine oxide. , O-formyl “4-hydroxiva1era1dehyde, 3 - f 1uorova1era1dehyde, 5-c1orovaleraldehyde, 2-clearpropionaldside, propargylaldehyde, and alkenes of alkenes represented by the formulas

0

HC-CH = CH-R ', HC-CH ₂ -CH ₂ -CH = CH-R' have R 'is hydrogen or C.-C' alkyl such as acrolein, crotonaldehyde and 4-pentenal.

Examples of aromatic and heterocyclic aldehydes RCHO for use in the benzaldehyde, tolualdehyde, anhydrous anhydride, aldehyde veratrium, acetaldehyde, -3 — phenyl propional, dehydric, 2-phenylpropionaldehyde, furfural, 2 process ~ <2 ~ furyl) acetaldehyde,

3-furylacrolein, 3-phenylacrolein, 2-thiophenoaldehyde, 3- <2-tie3- <methoxyphenylJacrolein, and aromatic aldehydes nilIscrolsius, analogues.

X

In the process of the invention, R d of formula 1 is preferably C. <-C. alkenyl, C ^-C,, alkynyl, or C-C, alkyl substituted with esterified carboxy, C ^-C ^ alkanoxyloxy, or a Cj-C _A alkyl group substituted with arylalkoxy. As used herein, the term C'-C .. alkenyl refers to unsaturated hyrocarbon chains of linear or branched structure such as ethylenyl, propenyl, butenyl, alchemy rstsrs if groups

C .-- C, pentenium and nexenium; ethynyl, propynylOj, butynyl, pentynyl and hexynyl which may have branched structure, alkyl substituted with esterified carboxy refers to 'ί' -'ώ straight chain or branched alkyl groups substituted with an esterified carboxy group in which the ester group έ C, -C, alkyl, phenyl, benzyl, substituted benzyl such as p-methoxybenzyl, methyl benzyl, p-nitrobenzyl, diphenylmethyl; conventional carboxy protectors.

or other groups are examples of such ethoxycarbonylmethyl, 2-methyloxycarbonylethyl, 3-Ct-butyloxycarbonyl> propyl, 3 ~ <benzyloxycarbonyl> butyl groups, • C 4-methoxy and benzyloxycarbonylhexyl, and similar alkyl groups substituted with substituted carboxy groups. The term C ^-C ^, alkyl substituted with Cj-C ^ alkoxy refers to methoxymethyl, 2-ethoxyethyl, 4-t-butyloxybutyl, 3-isopropoxymethyl, 2-ethoxyethyl, 4-1-butoxyoxy, 3- isopropoxypentyl, 2-ethoxyhexyl are other analogous groups;

alkyl substituted with fluorine or chlorine refers to groups such as 2-fluoroethyl, 2-chloroethyl, 4-chlorobutyl, 5-chloropentyl, chloromethyl, fluoromethyl, 3-chloro-4-methylpentyl and other analogous groups; Cj-C & cyano-substituted alkyl refer to cyanomethyl, 2-cyanoethyl, 4-cyanobutyl, 3-cyanobutyl, 5-cyano-hexyl and other similar groups? C --- C, hydroxy substituted alkyl refers to hydroxymethyl, 2-hydroxymethyl, 4-hydroxybutyl, 3-hydroxypropyl, 3-hydroxybexyl, and other analogous groups? ^c i ^{c is} alkyl substituted with C C-C ^ alkanoyloxy refers to groups such as 2-acetoxyethyl, 2-formyloxyethyl, 3-acetoxypropyl, 4-propionoxybutyl and other analogous groups? and C, - C ..

<3

arylalkoxy substituted alkyl refers to benzyloxymethyl _: diphenyl 1 -methyl, 4-benzyloxybutyl, 2- (4-methoxy benzyloxyethyl, 3-diphenylmethoxypropyl, and other similar groups.

Especially preferred aldehydes for use in the process are represented by RCHO where R is an alkenoaldehyde

HC-CH2CH2 "CH = CH-R as defined above, or C, -C" alkyl substituted with a esterified carboxy group or I ^λ γ a Cj-C ^ alloanoyloxy group "Examples of such 2-Cmethoxycarbonyl) ethyl groups, 2-benzyloxycarbonyl1) ethyl, 3-ethoxycarbonyl) ~ propyl, 2-C formyloxy) ethyl and 2-acetoxyethyl »

The aldehydes, RCHO, used in the process are all known compounds available on the market or that can be prepared by conventional methods:

Preferred process of the invention comprises the use of the aldehyde RCHO in which R is 2— (carboxy esterified) ethyl, 2-cyanoethyl, 2-C carboxyl esterified) ethynyl, 2-carboxy: L esterified) vinyl, esterified 3-carboxy) propyl, 2-formyloxystyl, 2-acetoxymethyl, 3-formyloxypropyl, or 3-acetoxypropyl, where the ester fraction of the esterified carboxy group is C C-C ^alkyl, eg methyl, ethyl or t-butyl§ phenyl, benzyl, diphenylmethyl, trityl or substituted benzyl e.g. 4— methoxyhenzyl or 4 — nitrobenzyl »An especially preferred aldehyde for use in the invention is a succinic semialdehyde ester» Another especially preferred aldehyde is a qlutarxco ssmxaldehyde ester »The following diagram illustrates the reaction taking place in the process of this invention» you

RCHQ + WH ^ CH ^ COOH> R-CH (OH) -CH C NH ^. ’> COO where R is such a coma ago has been defined.

As described above, serine hydroxymethyltransferase can be obtained at. from a variety of sources »Behind these sources are rabbit liver (LaVerne Schirch and Merle Mason, J» Biol »Chem», Vol, 237, N2 8, August 1962), corn sprouts (Masuda, T ,, et al », Agric» Biol »Chem» 19665®, 2763) and “mung” bean sprouts (Rao, D »N = e Rao, NA., Plant Physiol»

QQO i.D -ί 1

X is 5 / / X X J.? tt

Table 1 below shows one. list of representative aldehyde substrates that were converted in the process to--hydroxy-cyclic amino acids with HMTS from two sources »The conversion of the substrates in Table 1 was carried out by incubating the substrate and glycine with HMTS under the process conditions» A series of process conditions used were common to all substrates and are specified in the following paragraphs. Exceptions, where they exist, are indicated in each case »

The incubations were carried out in phosphate buffer 1® mM, pH 7.3 containing 5'-pyridoxal phosphate at a concentration of about 8® μΜ »All solutions were prepared from distilled and deionized water to obtain the best results» Conversions were carried out at 37 ° C in sealed polypropylene flasks suitable for 50® μΐ microcentrifuges in a water bath, with constant temperature, using streaks of light except when they were removed from the water bath to remove the parts. all quotas for analysis »With all conversions, a blank incubation was carried out parallel to the HMTS incubation» The total volume of the incubation mixture was typically 2®® μΐ »The conditions in the blank incubation were identical to those of the mixture incubation »à © da. enzyme"

QtlfiDRQ 1

Su bs a rts a I from C RCHQ) to. HMTS

Aldehyde CRCHO) R = _. 1 th.

Rabbit liver

Kebentos de Mi 1 ho

1/

-ch ₃ 4 · -ch ₂ ch ₂ ch ₃ + ~ ch ₂ ch ₃ ND -ch ₂ ch ₂ co ₂ ch ₃ + -CH ₂ CH ₂ CO ₂ (CH ₃ ) ₂ + -co ₂ h - -ch ₂ ch ₂ ch ₂ co ₂ ch ₃ + -C (O) CH ₃ ² + -ch ₂ ch ₂ ch ₂ ocho + c ₆ h ₅ + -ch ₂ och ₂ c ₆ h _s ND 2-furyl + 2- (2-furyl) ethyl +

+

ND +

+

ND +

+

ND +

+ conversions

- conversion not detectable? MD = not determined.

HMTS VQ_ was present with rabbit liver and HffTS tetrahydrofolate was present with corn.

aliquots of the β-hydroxy-a-amino acid sample analysis by removing the incubation mixture from time to time and by CLEP separation and fluorescence detection of isoindoles derived from o-phthalaldehyde from all primary amine compounds present in the mixture. The experimental procedure is described by Janes, BN? Gilligan, JP, J, Chroni. 1985, 266, 4711 Jones, B, W. et al., J. Liq. Chrom »19814,565, and Simons, Jr =, S. S, et al., J. Am. Chem. Soe. 1976, 98 CLEP analysis of both the incubation mix and the ^!! blank ”allowed the determination of which or which piooCs) in the chromatogram could be attributed to enzymatic products.

incubation with aldehyde in a cofactor solution ε after Alternatively, the enzyme solution of the invention is ramified by mixing with a buffered solution and mixed

It is also possible to add the aldehyde enzyme solution to glycine in the presence of the buffer solution carried out by adding the glycine containing FLP to another buffered solution of the enzyme, that of glycine containing FLP and a. the aldehyde is then added.

to a solution containing FLP.

of

It is not necessary for the aldehyde, RCHO, to be dissolved to give its conversion. Aldehydes that are only partially soluble in the aqueous also serve as a substrate for the enzyme.

be in the middle of completing it. Reactions to the aphiphatic acid process i = aldehyde function are the diastereomer L-er However, when the sub is attached to the aromatic carbonyl) the product of the invention in which the aldehyde, RCHO, is an e. the carbon attached to the carbonyl group of the endure, CH.-,) preferably provides itro of the B-hydroxy-a-amino acid or ester, the aldehyde layer is aromatic Ci.e. ç? carbon of the aldehyde function is part of a system is obtained in both threo and erythro forms in

approximately equal amounts »For example» benzaldehyde

It also makes the threus isomer like B-phenylserine erythro.

Examples of p-hydroxy-α-amino acids obtained in the process of the invention | 3-phenylserine _s 13-C 2 — furi 1) serine, β-hydroxy-antinoadipic acid, B-hydroxy-α-aminobexanoic acid, β -hydroxy-a-amino-S — formyloxyhexanoic .. β — hydroxy-a-amino-Γ -Qxovaleric acid, β-hydroxy-a-am.ino-T-phenylbutyric acid, β-hydroxy-a-sminoheptanoic acid » β-hydroxy-a-amino-rC2-furyl> -psntanoic, and analogous amino acids »

In a preferred embodiment of the process ..

a succinic semialdehyde ester such as a C.-C „alkyl ester e.g. the methyl ester or the isopropyl ester is incubated with glycine and HMTS in phosphate buffer in the presence of FLP in order to give rise to the β-hydroxy-α-aminoadipic acid semiester in the form of the L-erythro isomer »In another embodiment preferred part of the process.

a glutaric semialdehyde ester, eg methyl ester, is incubated with HMTS to provide the β-hydroxy-s-aminopimelic acid semiester »

The two embodiments of the aforementioned invention are illustrated by the following reaction scheme »

II

RiOC- (CH ₂ ) _cho + H ₂ NCH ₂ C00H

II

OH NH ₂

RiOC- (CH ₂ ) _n -CH-CH-COOH where R ₄ is C alkyl, phenyl, benzyl '. diphenylmethyl.

4-methoxybenzyl or 4-nitrobenzyl and n is 2 or 3 ..

«,

This invention further provides Ο 'amino acid, β-hydroxy-s-aminoadipic acid, its mono- and di-R ^ salts and esters represented by the formula RjOOC-CH ₇ ~ CH ₇ "CH (OH) CHiNH ^) COORj', where R R is as defined above and R ^ 'is hydrogen or R », This amino acid is obtained in the process in the form L-erythro. The salts of your diacid or monoester can be formed by conventional methods »These salts include alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium salt» and ammonium salts and amine salts such as those formed with benzylamine, dibenzylamine; cyclohexyl amine, dicyclobexylamine, C »-C., primary alkylamines

J.

and secondary ones such as methylamine, ethylamine, diethylamine, di-Cn-butylamine, ethanolamine, diethanolamine, dipropanolamine, and analogous salts »These salts are useful in the isolation and purification of the diacid or a stable monoester and amino acid for storage» provide qq flavors Also provided are the acid addition salts formed with the amino acid and its esters »Such salts are formed with acids that are stronger than the acidic carboxylic acid groups of the amino acid,» Examples of such acids are hydrochloric, hydrobromic acids, phosphoric and sulfuric, and sulfonic acids such as toiuenesulfonic acid, naphthalenesulfonic acid, methanesulfonic acid and n-butanesulfonic acid '

Its esters R ^ where R ^ has the same siqn for the reaction schemes »monomethyl, monoethyl, and di-t-butyl, benzyl esters refer to the mono- and di-esters mentioned above Examples of such esters are its lyric, dimethyl, diethyl, t-butyl, 4 — methoxybenzyl and dibenzyl

The j3-hydroxy-α-amino acids provided in this invention are useful intermediates for β-lactam compounds. Products are converted by the indirect method by methods

known in β-lactam compounds which are '0-3' i-ir £ Ο * Γ3ι_άΟ LH, - .;

known antibiotics. ' For example, o are converted to hydroxamate derivatives to form β-Iactam 3-amino-4 compounds

R-’n.i d ro κ i-s-aminacid and they are cyclized • -replaced »as shown in the following reaction scheme

OH

I

H ₃ N_CH — CH — R ₂ I co ~

0 * r ₁ onh ₂

OH

I

H ₂ N— CH — CH— RI

C —MH - · 0R ₂

where R has the same meanings as defined above for formula 1 and R. _t is an alkyl, alkanoyl or aralkyl group »The formation of the hydroxamate and the cyclization of the hydroxamate to give rise to the compound β — lactam are carried cable by the ficcfcs method. Chem »Res» 1986, 19, 49;

. Tetrahedron Lett., 1987, 28, 6257, u = Tetrahedron Lett ', 1987, 28, 1861,

described by i ’ Li 11 and r, M, J Rajendra, B » Miller, M « and Ko1asa, T » and Miller, M,

can

Alternatively »β-hydroxy-a-amino acids can be converted to substituted N-methyl) azetidinones as described by Miller, K„ u », U.S. Patent, NS 4,595,532» In addition, and in addition, the process amino acids can. converted to N-Cphosphonomethyl) USA, NS

azstxdinonas 4 _v 820. 8 to 5 ..

: as by

The following preparations and examples further describe the process of the invention, and should in no way be construed as constituting a limitation.

Preparation of Serine Hydroxymethyltransferase from Corn Shoots

All the procedures described below were performed

4’'C

Corn sprouts with washed days were

13,800 xg for 40 µm _? pH 7.8 ₃ containing mM. The solution was homogenized in a Waring mixer in 100 mM potassium monohydrogen phosphate containing disodium ethylenediaminetetraacetate (1 mKj), 1 mH dithiotrsitol and pyridoxal 5'-phosphate 25 μΜ. The mixture contained approximately 960 g of sprouts per liter. The homogenate was filtered through a cheesecloth and the filtrate was subjected to a precipitation process with ammonium sulfate. The protein that precipitated with ammonium sulphate after centrifugation at minutes was dissolved in 5'-pyridoxal phosphate phosphate buffer 25 μΜ and EDTA subjected to concentrated dialysis to 5-15 mg protein / ml in the same buffer solution without the presence of EDTA . The test for the presence of protein aldols activity consisted of a system, coupled between the retroaldol catalyzed by L-allo-threine aldolase with the reduction NADH C reduced form of nicotinamide and adenine-dependent acetaldehyde-dinucleotide resulting by dehydrogenation of yeast alcohol (CftDH). Specific activity has the mean value of / 8 ml / units of protein. Lima unit of enzymatic activity is defined as the amount of enzyme needed to cause the variation of the optical density value in a unit Ca 340 nm) for minutes at temperature

• 5 * · * »iJ environment in the presence of ADH, L-allo-threonine 1.2® mM ₅ FLP 125 μΜ _? and NADH 120 μΜ in phosphate buffer 100 ml% pH 7 _Ρ 5 =

AHMTS when not used once prepared was kept either in a freeze-dried form or frozen with 10% glycerol.

In the examples that follow _? and unless otherwise specified, the buffer system consisted of 10 mM potassium phosphate _s pH 7 _P 3 _P and contained pyridoxal 5'-phosphate (FLP) at a concentration of about 30 μΜ. All solutions were prepared in distilled and deionized water.

The enzyme incubations were carried out at 37 ° C in sealed polypropylene bottles suitable for 500 μ microcentrifuge machines! _? in a constant temperature water bath.

examples can t> s vary from time to time indicated in the CLEP analyzes from time to time due to slight variations in factors such as the column _? the balance. buffer solutions, etc.

The HMTS used in the following examples was obtained h (above) 'from Tíqsoo ob coeino _? by L. hem

Example 1 f irBunine repair

An. glycine solution (34 mM) was prepared in the phosphate buffer containing FLP _? and freshly distilled acetaldehyde was added in sufficient quantity for its. cough concentration also 34 mM. When added to the enzyme solution, the concentration of each substrate is 17 ml

X

X. 'b A solution of HMTS (Omg) in 20® μΐ of standard phosphate buffer was placed in a 2 ml glass flask. An equal volume of the substrate solution was added and the flask was sealed with rubber and copper wire to minimize ds loss

COi »Uffl SSpuD acetaldehyde»

This flask is an enzyme seduction ^! 'blank were incubated at 3, - ^f, U «

Aliquots (ζ10 μΐ) were removed using a 5®μ1 microlit.ro syringe at the end of the following incubation times 1 _? ® _? 2, ®, 3 _S 0 »4, ® and 2®, 5 hours and analyzed by means of CL.EP» chromatogram revealed the highest peak for the L-erythro isomer at the retention time value of 21.9 minutes and a minor peak for the threo isomer »The identity of the produced amino acids was confirmed by co-injection with authentic commercially available threonine isomers»

Example 2

A c i d a-siBi η o-15-hydro x icapróic

A substrate solution was prepared by dissolving glycine (25.5 mg, ®, 34® mmol) and n-butanal (25.3 mg, 0.351 mmol) in 10 ml of the standard phosphate buffer, providing concentrations of 34 mM and 35 mM of two substrates-, respectively »mg) in i®® μΐ of ® polypropylene, 5 ml

A solution of HMítí (®, the buffer was placed in a bottle of other ί®® μ.1 buffer solution without enzyme were placed in another bottle, and 1®® μ.1 of the substrate solution „solution was added to each bottle v- ·

'_ * ι.ύ V

Aliquots were removed from the blank 'ensim solutions' at the end of the following incubation times 1.0

2b and 21 hours, which were analyzed using CLEP.

Two product peaks were observed in the chromatogram, a large one at 25 = -, 7 minutes and a small one at 24.3 minutes. The peaks corresponded to those of the authentic racemic product »The authentic material was prepared racemically by an aldol reaction on a copper glycine complex.» S »Akabari et al», Archives od Biochefflistry and Biophysics, 195 ?, 83, 1, and J. P »

Greenstein and M. Winítz, Chemistry of Amino Acids · (Chemistry. Amino Acids), John Wiley & Sons, New York, 1961, Vol »3. 2249-2250» <3 OS

PP

Example cc-amino-B-hydroxydipic acid monomethyl ester

Glycine <13.3

IlfQ ,, mmol) and methyl ester of

Xu- * without i to 1 of the suc c ict (20 mg, 0.172 imo 1)

5.0 ml of buffer solution to provide concentrations of each 35 mM and 34 mM, respectively. The aldehyde used in this experiment was free of DMSO, although previous incubations with this aldehyde still contained a. impurity (residue resulting from its synthesis by ozon61i.se of CH ₇ = CH ~ CH _n —CH ^ -CDD — CH _? followed by processing with dimethylsulfoxide).

were dissolved in

To a solution of HMTS (0.1 mq) in 100 μ1 of buffer solution was added 100 μΐ of the substrate mixture. Were aliquots removed at the end of the following incubation periods? 0 »5, 1„ 0, .X n ‘-5 * Sf; 4 hours.

higher product peak CL-erit.ro) was observed at u <n t value

minutes reaching its maximum at two o'clock

During the initial two hours, a small new peak emerged in the CLEP analysis with a retention time of approximately 8 minutes. During a 24-hour period, this peak increased in intensity while the peaks corresponding to the starting glycine and the intermediate product methyl ester of α-amino-hydroxydipic acid decreased »After 24 hours» the ratio between the new peak and the glycine ds and the ester was 38s47s15 «The structure of the compound corresponding to the new peak was determined to be L-erythro-a-amino- | 5-hydroKiadipic acid CR = CH ^ -CH ^ -CQ ^ H) by an independent synthesis and co-injection of the authentic product with that produced enzymatically. The chemical synthesis of the authentic material was carried out by condensing the aldol of the methyl ester of succinic semialdehyde with a boron enolate from a chiral oxazine (Reno, D »S» s Lot, B »T«? Miller »ΙΊ» J « Tetrahsdron Lett,., 1990 * 51 «827)» followed by deprotection and hydrolysis to give the described compound »

The formation of this original amino acid during enzymatic synthesis takes place by hydrolysis of the inonomethyl ester of the amino acid Initially produced »Since the product has an additional ionized carboxylic acid group it does not constitute a substrate for the enzymatically reversible aldol condensation» Thus »the new amino acid product (L-erifcro-a-amino-fl-hydroxyacidic acid) accumulates» As a dicarboxylic acid amino acid product, it can be purified from the reaction mixture by means of CSreensteirs ion exchange chromatography » J «p« ₅ Winitz »M», “Cbemistry of tbe Amino Acids ' ¹ ' (Chemistry of Amino Acids) Vol. 2» pp 1452 -4646 ₅ Uliley »New York»

M = V ,. 1961) «

Example 4

mono-isopropyl ester of s-~ amino acid-3-hydroxydiphy-O

Slicin (26.1 mg ₅ 0 _- 348 mmol) was dissolved in t® _s ® ml of buffer solution and 34 mg of isopropyl succinic isopropyl ester (containing DMSO as a contaminant from its preparation) was added. The presence of DMSO in. incubation has no harmful effects on the enzyme or the reaction »The concentration of aldehyde in buffer ₅ after taking into account a. presence of DMSO, was 18 mmolar. The initial concentrations of glycine and aldehyde in the incubation mixture were 17.4 mM and 9 mM respectively.

The substrate mixture (100 μΐ) was added to a solution of HMTS Í® ₉ 1 mg) in Í0S μΐ of buffer solution and to the blank preparation ”„

Aliquot parts were collected for incubation periods: ® _? 5 ₅ ? ₅ @ ₅ 2 _? 0 _? 4 ^: im of the following

17,? fl rf -.- u n hours and examined by means of CLEP »The main product peak (L-erythro) appeared at a T_ value of 23 minutes, after k

hours and grew to a maximum in the space of 2-4 hours »

Example 5 uranino-β-hydroxyipimelic acid ester □ qlutaric semialdehyde methyl ester used in this incubation contained DMSO in a proportion of about 1% by weight »DMSO is a by-product of the preparation of aldehyde by ion osonolysis -methoxycyclopsentene that includes DMSO in the etrahedron processing? (Cline? D, L »0 =?

Leti ipse,

v ¹ '

Russel, C »

1399) = The substrate solution was prepared by dissolving glycine (25.6 mg? 0.341 mmol) ε d-the aldehyde / DMSO mixture (& 6? 7 mg) in 10? 0 ml of buffer solution to provide glycine concentrations. and 34 aldehyde ?! and approximately 25 mM »

The enzyme solution consisted of 0.1 dissolved HMTS. in 40 u], of buffer solution instead of ui. The substrate solution (40 μΐ> was added to the enzyme and blank solutions and aliquots 0.5? 1.0?

incubated in the usual way. Were parts (10 μΐ) collected at the end of the following 1? 5? 2? 0? 3.0, 4.0 s 17, õ hours, After 0.5 hours a peak produced by the enzyme at 23.7 minutes corresponding to the desired product appeared in the CLEP and a yield of about 202 was intergrated »A second smaller peak induced by the enzyme appeared with a retention time of 22? 3 minutes? and may correspond to a very small amount of the isometric isthmus.

t-xsinplo &

Ac i d o <x ~ am i n o — fi h i d r ox i — Γοχ ον a 1 é r i c ο

Glycine (25.5 mg - 0.0340 mmol) and pyruvaldehyde (61 µl of a 402 weight solution, 0.327 mmol of pxruvaldehyde) were dissolved in 10 ml of buffer solution? pH 7? 2? to provide concentrations of 34 mM (glycine) and 32.7 mM (pxruvaldehyde) in the stack solution. Sodium metavanadate (1.5 mg pure decomposed at 902 Aldrich's) was added. After 30 minutes had it dissolved? providing a concentration of 1.11 mM, to the enzyme solution Co? í mg ds HMTS sm 80 μΐ) 80μ1 of the substrate solution were added, Aliquots part 1010 μΐ)

- ί ί (· 'were removed at the end of the following incubation periods! 9,5 _,; 9, 5, noraí

The analysis of amino acids by means of CLEp indicated a retention time of 8.5 minutes for the new amino acid »To confirm that it was the desired amino acid, authentic racemic material was prepared by the copper glycanate procedure» Thus »4,23 g of copper (II) glycyanate (20 mmol) were dissolved in 10 ml of 9M aqueous KOH solution „Pyruvaldehyde (16.2 ml of a 40% by weight seduction in water, corresponding to 7.0 g , 9.71 mmol of aldehyde) ,, After 5.25 hours, resultante ,, ΟΗ 3M (40 ml) was added to the resulting brown solution and the solvents were evaporated to provide a brown oil »The oil was passed through a Dowex-50 column (water-eluted form to provide the amino acid without purification „

Example 7

Alpha-6-formi1ox i-6-formi1ox i haxanoic aldehyde acid in this incubation also retained a significant amount of DMSO from its preparation by means of dihydropyran ozonolysis followed by processing with DMSO »A 10, ml of solution buffer, O-formyl 4-hydroxybuthanal (39.5 mg, about 9.20 mmol, taking into account mg, 0.340 ml) was added »

The = ubstrate solution (40 pl) was added to the enzyme solution 0.1 mg of HMTS in 49 μΐ) and to the blank solution »The concentrations of the two subtracts in the incubation mixture were 17 mM (glycine) and 10 mri (aldehyde). Aliquot parts (10 μΐ) mM (aldehyde)

's $ II \ were collected- at the end of the following incubation periods' ί. ο..0!} 7.3 and 24 hufsSi

The amino acid analysis of the first aliquot revealed two new amino acids »A small peak appeared at 15.6 minutes, just after the glycine peak» The other was a large and accentuated peak, at 22.5 minutes »at three hours, the peak at 22.5 minutes corresponding to the expected amino acid reached a maximum at about 10% of the glycine peak and then decreased slowly, while the peak at 15.6 minutes slightly increased in intensity »The amino acid that elutes at 15.6- minutes is the product of slow hydrolysis of the ester format in the reaction medium »The structure corresponds to« -amino — β — hydroxy — 6 ~ hydroxyhexanoic acid «

Example 8 β — Fenilseri na

The buffer solution used contained FLP at a concentration of 160 μΜ instead of 83 μΜ »Benzaldehyde was distilled immediately before use» The buffer solution was deoxygenated by bubbling nitrogen in it before adding the substrates »This was done to minimize the possibility of benzaldehyde being oxidized while in solution »

C25.4 mg, solution (133 μΐ) mg of enzyme) »second period

Glycyr 3.2.38 mmol) and benzaldehyde (35 μΐ, '3.344 mmol 1' were dissolved in 13.3 ml of buffer solution).

Aliquot parts for s, 3, o, 25 and />, & hours.

μΐ of the HMTtí solution (3 ~ sm collected at the end of dt

Two orifice oicos were visible in the chromatograms.

the largest at 24.8 minutes and the smallest at minute

X. χ ίζ-V · 'product mix from this experiment was compared with authentic, racemic β-phenyl · serine, available on the market. The peaks of the two mixtures exactly matched. In addition, when compared by co-injection with authentic B-phenylserine treo, the first (smaller) pica was intensified in relation to final tare, the pairs of

These results indicate that the enzyme produces both enantiomers of the aromatic amino acids. although it favors the isomers I enter.

Example 9

U-Amino-R.-hydroxy-p-Cz-furyl) propionic acid

2-furfural used in this example was prepared by means of two distillations on carbonate, sodium with a. second distillation under a nitrogen atmosphere. The distilled aldehyde was stored, before use, dried, under a nitrogen atmosphere and protected from light.

ft substrate solution was prepared by dissolving glycine <14, £ J mg, 0.186 mmol) and furaldehyde (15.5 μΐ, 0.187 mmol) in buffer solution (10.0 ml). When 36 © μΐ of this solution were * added to the enzyme solution (0.2 mg of HMTS in 40 μΐ), the concentration of each substrate became 77 mM.

Aliquots (10 μΐ) were collected at the end of the following incubation periods ·; 1.0, 2.0, 4.0, 5.0, 18, 24 o'clock Tim, analysis by means of dos

CL.C.P largest peak that revealed the production of two new amino acids. The one corresponding to an isomer had a retention period of 21-22 minutes, while the smallest peak (<SO5 of the largest peak) corresponded to the other isomer (threus) and had a retention time of 19-2 minutes.

à X LfU

imino - $ - hydroxy-5- C 2-fu 1) va 1 etheric

The fifteen minute solution of the glycine sulfate (25.5 mg »0.34 © mmol) in i®, © .ml of buffer solution» was prepared by dissolving and the aldehyde (46.3 mg, 0.373 mmol) aldehyde (a liquid) took about

The CIO® ul substrate solution added enzyme solution (0.1 mg HMTS in 100 μΐ incubated at 3z ' ^: ' C = aliquots were incubated for the following incubation periods 0.55, 1.0, 2, solution buffer) and Ihidas at the end of 0, 4.25, 8.0 and 21 showed a peak conversion hours »After 0.5 hours the analysis by CLEP with a retention time of 21.3 minutes, irsdic in about 3Z of glycine in the product aldol »xemp.

X — amino — ji — hydroxy and j — enoic acid is obtained by following the incubation procedures described in the previous examples with propargyl aldehyde »

ç. xemp a o

S — amino — β — hydroxyhex — 5 — enoic acid is obtained with allyl aldehyde under the incubation conditions described in the previous examples »

b.XSiii lo 13

Obtains

ν · · 'α-affli no-β-hydroxy-pen t-4 ~ only with acrolein following the procedures and under the previous examples condition

SC r * i ffiS

It produces a-amino acid -j the conditions of the previous examples • hydroxycid fl-ci δ ^: ™ ait ο γ ΰΛ 1 and í * -LC í ~ 't ~ · Anopropionaldehyde.

Claims (2)

CLAIMED iâ. Process for the preparation of a hydroxy-cyclic amino formula in which R is substituted chlorine? aroma, or benzyloxy? «-C, alkenyl; C .-? ~ C, alkynyl? cyan, bxdroxx, Ί “4 ienxlo? or yurilu? '1 **! «. · J Ί ^ 4 alkanoyloxy, phenyl. alkyl 1 u u r o .. furyl? Characterized at about 3 ° C between about where R is hydroxystyrene because it comprises the mixture? at a temperature between and about 55 '-' C? in an aqueous solution with a pH of 5? 5 and about 9? of an aldehyde of formula RCHD? the meanings defined above? and glycine with serine ansferase in the presence of pyridoxal 5'-phosphate termed by R substituted alkyl or subalkyl Process according to Claim 1, comprising C, - C, alkenyl, C, C, alkynyl, or C, C, x ^1; z & o uido with esterified carboxy? C. — C »alkanoyloxy, X * r substituted with arylalkoxy» 2 / .X <3ã, t e r i z a d o p r Processes in accordance with Claim 1, character or C,; _{It is also used as a} sterified carbox, or L-, -C, alkanoyloxy .aa. Process according to Claim ί, characterized in that the aldehyde RLHO is IJ u R »OC-ÍCí-U> _CHO, i Ζ. ΐ I where R »is alkyl, benzyl, diphenylmethyl or 4-nitrobenzyls and n is 2 or 3» ethyl, me tox i oen zi02 r. termed by the succinic » Process according to the aldehyde RCHD ester Wxivxnaication 4, semialdehyde caracmethyl 2 - Process according to the claimed claim that the aldehyde RCHO is qlutaric ester. mM t.x 13.co from semi a1dexdu 7. A process according to Claim, characterized in that R is alkyl substituted with carboxy, alkanoyloxy, phenyl, furyl or benzyloxy. Cctr'S.C ~ sterile, - Process according to Claim 1, characterized in that the α-amino-fS-hydroxy acid is the L-erythro diastereomer »