JPS5829299B2 - how to use ester - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for extracting N-blocked amino acids from aqueous solutions of N-f', A-blocked amino acids, particularly fermentation broths.

As used herein, the term "N-blocked amino acids" includes N-blocked peptides.

Amino acids and N
- It is often necessary to isolate occluded amino acids.

In such isolations, for example, many naturally occurring N-blocked amino acids are relatively unstable in aqueous media and many fermentation and enzymatic reactions yield the desired product only in dilute solution. They are often complicated by the fact that they are produced, often requiring complex and expensive isolation techniques.

That is, for example, penicillin G [(3S・5R・6R)
-2,2-dimethyl-6-phenylacedoamitopenane-3-carboxylic acid] is in the free acid form (especially in aqueous systems)
It is unstable and requires special techniques to effectively isolate it from the fermentation broth.

Such techniques are, for example, rapid with selective and somewhat expensive purification steps to isolate penicillin G products (e.g. salts) of acceptable purity that are more stable than the free acid. Includes extraction.

Alternatively, penicillin G may be isolated by sulfoxidation and separation of the resulting penicillin G sulfoxide.

Cephalosporin C [(6R・7R)-3-acetoxymethyl-7(R-5-amino-5-carboxypentanamide) cef-3-emu-4-carboxylic acid] is characterized by its amphoteric structure and hydrophilic nature. Its separation from the fermentation broth is therefore equally difficult.

Deacetylcephalosporin C [(6R・7R)-7-
(R-5-amino-5-carboxypentanamide)-
Isolation of 3-hydroxymethylcephalosporin produced by fermentation such as 3-hydroxymethylcephalosporin (3-hydroxymethylceph-3-em-4-carboxylic acid) is difficult because in such compounds the hydroxyl group in the 3-position side chain is 4- Problems arise as well because of the pronounced tendency to form lactones by reacting with carboxy groups.

Examples of techniques used in the isolation of such N-blocked amino acids are the use of ion exchange resins and solvent extraction.

However, the former technique tends to be somewhat cumbersome and expensive to process dilute solutions on an industrial scale, and the latter technique suffers from the fact that the isolated acids often exhibit relatively low stability in organic solvents. The disadvantages are that solvent conditions and operating costs are high.

However, N-blocked amino acids can be N-blocked in a particularly simple manner by subjecting them to substantially simultaneous esterification and solvent extraction by reaction with diazoalkanes in the presence of a water-immiscible organic solvent. It has been found that effective separation of amino acids from aqueous solutions can be achieved.

Such a method will hereinafter be referred to as extractive esterification.

To the best of the inventor's knowledge, no esterification reaction using diazoalkanes in a two-phase aqueous organic solvent system has been previously reported.

It is perhaps surprising that such reactions proceed smoothly and efficiently.

This is because, given the number of examples, it is expected that other substances, such as water, present in competition with the carboxyl group of the N-blocked amino acid will be alkylated in the reaction with the diazoalkane.

However, such interference is generally minimal in the extractive esterification process of the present invention.

Thus, according to one aspect of the invention, esters of N-blocked amino acids in organic solvents are prepared by treating a solution of N-blocked amino acids with a diazoalkane in the presence of a water-immiscible organic solvent. consisting of producing a solution;
A method is provided for extracting an N-blocked amino acid from an aqueous solution thereof.

The diazoalkane esterifying agent used in this method has the general formula (wherein R1 represents a hydrogen atom or an organic group and R2
represents an organic group, or R1 and R2 together with the carbon atoms to which they are bonded form a cyclic organic group.

That is, a suitable diazoalkane is R1 and /
or R2 is the same or different and is a carbocyclic aryl group such as phenyl or naphthyl, a 5- or 6-membered heterocycle containing one or several atoms of 0, N and S (e.g. cheno-2-yl, 2-ylmata is pyricin-2-yl), aralkyl groups (e.g. monocyclic aryl groups and groups containing 1 to 6 carbon atoms in the alkyl moiety, such as benzyl), heterocyclic substituted alkyl groups (e.g. chenoyl), groups containing 1 to 6 carbon atoms in the alkyl moiety, such as -2-ylmethyl or flu-2-ylmethyl); groups containing 5 to 7 carbon atoms in the ring, such as cycloalkyl groups (such as cyclopentyl or cyclohexyl),
unsaturated analogs of said groups, such as carbocyclic or heterocyclic aralkenyl groups, lower (e.g. C2-6) alkenyl groups (e.g. vinyl or allyl) and cycloalkenyl groups (e.g. evacyclohexenyl); or a group containing 5 to 7 carbon atoms, such as cyclopentadienyl), or one or several halogen atoms, cyano, nitro, alkyl, alkylsulfonyl, or an alkoxy group (the last-mentioned group above). For example, methyl, ethyl, n-propyl, isopropyl, ethoxy,
1 to 6 carbon atoms, such as isopropyl or methylsulfonyl).

Alternatively, R1 and R2 together with the carbon atoms to which they are attached are cycloalkyl groups (e.g. groups containing 5 to 7 carbon atoms, such as cyclopentyl or cyclohexyl) or cycloalkenyl groups (e.g. cyclohexenyl or 5-7 like cyclopentagenyl
a C6-2o cycloaliphatic group such as a group containing 1 carbon atoms or 1 selected from oxygen, nitrogen and sulfur
A small number (with one 5
form a cyclic structure that is a heterocyclic group (eg, a monocyclic group such as pyranyl or piperidinyl) containing a membered or six-membered ring.

Particularly useful diazoalkanes of formula (3) are one or two carbocyclic or heterocyclic groups in which the resulting ester group R'R2CH- in R1 and R2 is bonded to the C-1 atom of the lower (e.g. 01-6) alkyl moiety. Aralkyl groups containing aryl groups, such as benzyl, ■-phenylethyl,
Diphenylmethyl, naphthylphenylmethyl, cy(cheno-2-yl)methyl, phenyl(cheno-2-yl)
Compounds that are methyl or substitutes of any of these groups, such as phenyl(0-)ruyl)methyl or (p-methoxyphenyl)phenylmethyl, are included.

This is because such ester groups can be easily decomposed in the next step of the reaction sequence.

However, this list is not complete.

The N-occluded amino acid to be extracted has the formula Q-CO
OH (where Q is an organic radical containing, for example, 1 to 50 carbon atoms and at least one blocked amino group) and is a mono-, di- or polycarboxylic acid; sell.

If a diazoalkane of the formula (1) is used, the ester obtained corresponds to the formula Q-COOCHRIR2, in which R1 and R2 have the meanings given above.

Examples of N-blocked amino acids that can be extractively esterified according to the invention are the N- Blocked derivatives, especially N-blocked peptides obtained from fermentation broths, plant and animal extracts, and complex aqueous reaction mixtures resulting from peptide synthesis, such as e.g. glutathione, and especially fermentation and other enzyme-catalyzed processes. natural or semi-synthetic penicillin or cephalosporin compounds obtained from and related degradation products such as benyronic acid or benicillonic acid.

The aqueous solution of the N-blocked amino acid to be extractively esterified may optionally be eg a lower alkanol (e.g. methanol), a ketone (e.g. acetone), in order to increase the solubility of the N-blocked amino acid in the aqueous medium.
, one or more water-miscible organic solvents such as esters (e.g. ethyl acetate), N-N-disubstituted amines (e.g. dimethylacetamide) and ethers (e.g. cyclic ethers such as dioxane). It may be contained in a proportion (for example, 30% v/v or less).

Alternatively, if the N-blocked amino acid exhibits particularly low solubility in water, the aqueous solution may contain solid undissolved N-
N- in contact with the occluded amino acid and in equilibrium
It may also take the form of a slurry consisting of a saturated aqueous solution of occluded amino acids.

References herein to aqueous solutions of N-blocked amino acids should therefore be construed to include such slurries and aqueous organic solutions.

Examples of penicillins and cephalosporins that can be extractively esterified according to the invention are compounds with the skeletal formula, where Z is 〉S or 〉S→O, and X is of the formula (a
) is a divalent group selected from

In the above formula, Y is methyl, substituted methyl, e.g. is an unsaturated group such as vinyl, and the 23- and 4-
A dotted line between the positions indicates that the compound is a cef-2em or cef-3-em compound, and R is an occluded It is an amino group or a protonated amino group (NH3+) and Ra is hydrogen or a lower (eg C1-4) alkyl, alkoxy (eg a methoxy group) or an alkylthio group.

Where R is a carboxyl acylamido group, the acyl moiety may be selected from a wide range of descriptions of such acyl groups in the penicillin and cephalosporin literature.

Specific acyl groups are illustrated below, but are not meant to be limited to these.

(i) 2nCO in RuCn - where Ru is aryl (carbocyclic or heterocyclic), cycloalkyl, substituted aryl, substituted cycloalkyl, cycloalkagenyl or a non-aromatic heterocyclic or mesoionic group; And n is 0 or an integer of 1-4.

Examples of this group include phenylacetyl, cheno-2-ylacetyl and cheno-3-ylacetyl, substituted or unsubstituted 3- and 4-inxazolylacetyl, pyridylacetyl, tetrazoylacetyl or cytononeacetyl groups. .

When n is other than 0, in particular when n is 1, the α-carbon atom of the acyl group can be, for example, a hydroxy group, an esterified hydroxy group (e.g. lower alkanoyloxy such as acetoxy), a blocked amino group (e.g. hydroxyimino groups, acyloxyimino groups (such as acetoxyimino, or halo-substituted lower plucyloxyimino such as mono- or di-chloroacetoxyimino) lower alkanoyloxyimino) or esterified oximino groups (e.g. lower alkoxyiminos such as methoxyimino or tertiary-7 toximino, cycloalkyloxyiminos such as cyclopentyloxyimino or aryloxyiminos such as phenoxyimino) may be substituted with groups such as.

Examples of α-substituted acyl groups of this type are 2-hydroxy-2-phenylacetyl, N-blocked 2-amino-
2-phenylacetyl and 2-(fluor-2-yl)-
Includes 2-hydroxyiminoacetyl.

(ii) CnH2n+I CO- where n is 0 or an integer from 1 to 7.

Alkyl groups may be straight-chain or branched and, for example, cyano, carboxy, alkoxycarbonyl,
The functional group may be substituted by a hydroxy group, a blocked amino group or a carboxycarbonyl group (-CO-COOH) or any group in which the functional group is blocked.

Examples of such groups are formyl, glutaroyl and N-
occluded (e.g. N-ethoxycarbonyl) R-5-
Includes amino-5-carboxypentanoyl.

and may additionally be benzyl.

RV and RW may be the same or different;
Each represents hydrogen, phenyl, benzyl, phenethyl or lower alkyl and Z is oxygen or sulfur.

Examples of this group include phenoxyacetyl or pyridylthioacetyl.

It should be understood that the skeletal formula ■ includes within its structure compounds not included in groups (a) and (b), such as 2-acetoxymethylpenicillin and 2-methyl and 2-methylene cephalosporins. .

Compounds of formula (nb) in which Y' is the residue of a nucleophile,
Compounds of formula (nb) in which Y' is acetoxy are combined with nucleophilic reagents such as pyridine or other tertiary amines as described in GB 912,541, as described in GB 1,012,943 sulfur-binding, nitrogen-binding or inorganic nucleophiles as described in GB 1059562; sulfur binding nucleophiles as described in GB 1030630; Nitrogen-bonding nucleophiles as described in British Patent Nos. 1082943 and 1082962 or sulfur-bonding nucleophiles as described in British Patent Nos. 1101423 and 1206305. It can be prepared by reacting with a reagent.

This description is given purely by way of illustration rather than by way of limitation.

Compounds of formula (nb) in which Y' is a hydroxy group may be prepared by the method described in GB 1,121,308.

Compounds in which Y is a carbamoyloxy group are described in Belgian Patent No. 764160.

When Y in formula (nb) is a methyl group, the compound may be prepared by the method described in British Patent No. 957,569.

As mentioned above, the amino acids to be separated using the method of the invention should be in the N-blocked form.

This is because this generally increases the solubility of the resulting ester in organic solvents and improves the efficiency of the extraction.

Thus, acids containing one or several free amine groups, such as simple amino acids or penicillins or cephalosporic acids containing an amino group in the 6- or 7-position side chain, can be extractively esterified. If necessary, such groups should be occluded before esterification by protonation or by substitution with an occluded functional group.

When protonation is used to occlude amino groups, the protonation is preferably carried out using a strong acid that has some lipid solubility in the resulting protonated amino acid.

Strong acids suitable for this purpose include, for example, aromatic sulfonic acids such as lower alkyl-substituted benzenesulfones (e.g. p-toluenesulfonic acid) and naphthalenesulfonic acid.

The N-blocking groups that can be used to protect amino groups by substitution are monovalent or divalent, and suitable groups include substituted groups such as acyl groups, lower alkanoyls such as acetyl, lower haloalkanoyls such as chloroacetyl. aryl lower alkanoyl such as phenylacetyl, aroyl such as benzoyl or phthaloyl, lower alkoxycarbonyl groups such as ethoxycarbonyl, imbutyloxycarbonyl or tert-butyloxycarbonyl, and 2,2,2 Substituted lower alkoxycarbonyl groups such as lower haloalkoxycarbonyl such as trichloroethoxycarbonyl, aryl lower alkoxycarbonyl groups such as benzyloxycarbonyl, sulfonyl groups such as lower furkylsulfonyl such as methanesulfonyl and pencenesulfonyl-elf Arylsulfonyls such as p-)luenesulfonyl, ylidene groups produced by reaction with aldehydes and ketones to form Schiff bases, such as benzaldehyde, salicylaldehyde or acetoacetate, and where the nitrogen atom is part of the dihydropyridine ring. (such protecting groups are obtained by the reaction of formaldehyde with a β-ketoester, such as e.g. acetoacetate) as described in Belgian Patent No. 771,694. ing.

Generally such N-blocking functional groups include, for example, acyl N-
This can be introduced by methods known per se, such as the reaction of an amino acid with an acyl halide when a blocking functional group is introduced.

The N-blocking functional group is preferably introduced at a pH above the isoelectric point of the amino acid, preferably in the range of 6 to 10, and the reaction temperature is advantageously relatively low to minimize acid decomposition. be.

Other groups present on the amino acid, such as hydroxyl or thiol groups, may likewise be occluded if desired prior to extractive esterification.

The water-immiscible solvent used in extractive esterification should be essentially inert to the reaction conditions and should be able to at least partially dissolve the esterified N-blocked amino acid. The nature of the solvent therefore depends on the properties of the N-blocked amino acids to be extracted.

In general, organic solvents that may be used are chlorinated hydrocarbons, such as methylene chloride, chloroform or chlorobenzene, aliphatic and aromatic esters, such as ethyl acetate, butyl acetate or ethyl benzoate, such as methyl ethyl ketone or methyl isobutyl. Included are water-immiscible ketones such as ketones, aliphatic and aromatic hydrocarbons such as benzene, and alcohols such as water-immiscible lower alkanols such as n-butanol.

Mixtures of the aforementioned solvents may also be used.

Extractive esterification is conveniently carried out by adding a solution of the diazoalkane in the selected organic solvent to an aqueous solution containing the N-blocked amino.

This is because in this way the esterification of the N-blocked amino acid and the extraction into the organic solvent are essentially paralleled at the same time, increasing the efficiency of the process.

Other methods of addition are possible, although the yield of isolated N-blocked amino acid ester may be lower.

Thus, for example, the aqueous solution is first treated with a water-immiscible organic solvent to effect at least partial extraction of the N-blocked amino acids, and then the diazoalkane is added to promote the desired esterification.

Extractive esterification of N-blocked amino acids can optionally be carried out in the presence of one or more further acids.

pKa less than that of N-occluded amino acids
The presence of mineral acids, such as sulfuric acid, orthophosphoric acid or perchloric acid, does not interfere with extraction esterification, but in many cases tends to primarily react with and esterify the weak acids present. , that is, it tends to react preferentially with N-occluded amino acids.

The amount of strong acid added to the reaction system is governed, at least in part, by the stability of the components of the reaction system under acidic conditions, as side reactions, including decomposition of diazoalkanes, tend to predominate at lower pH values. For example, the pH of the reaction solution should preferably not be lowered below about 1.5, and generally has a pH above about 1.7, e.g.
Preference is given to using solutions with H.

Extractive esterification may under certain circumstances be carried out in the presence of weaker acids than the N-blocked amino acids.

Thus, for example, penicillin V and N-blocked cephalosporin C can be selectively extractively esterified in the presence of the weak acids phenoxyacetic acid and acetic acid, respectively.

While not wishing to be bound by theory, the solubility of N-blocked amino acids in water-immiscible organic solvents is an important contributing factor in such extractive esterifications; It is believed that the esterification rate and selectivity of the method increases as the solubility of the N-blocked amino acid increases.

Since it is known that the stability of diazoalkanes is relatively low in acidic media, it is surprising that satisfactory extractive esterification can be carried out under acidic conditions using diazoalkane esterifying agents. .

The order of addition of the diazoalkane, organic solvent, and any strong acid added to the aqueous N-blocked amino acid generally depends on the nature of the amino acid substrate.

The mode of addition is thus not critical if the N-M-blocked amino acids are acid-stable; however, acid-sensitive substrates such as penicillin G or N-blocked deacetyl cephalosporin C are It is preferred to add a strong acid after the diazoalkane and the organic solvent when extracted, since under such conditions the esterification of the N-blocked amino acid with the diazoalkane and the subsequent conversion of the amino acid as an ester. The stabilization of the amino acids has been found to proceed extremely rapidly so that acid-induced degradation or other transformations of the amino acids occur to a minimum.

When using this mode of addition, the aqueous solution of acid-sensitive N-blocked amino acids maintains a pH 1, e.g., a neutral or basic pH, at which degradation of the N-blocked amino acids is minimized or prevented. This can be stabilized up to the point of extractive esterification.

Lowering the pH by acidifying the solution after addition of the diazoalkane and organic solvent allows rapid esterification of the N-blocked amino acid with the diazoalkane.

It is often preferred to use an excess of diazoalkane in the extractive esterification, the exact amount depending on the nature of the diazoalkane and the N-blocked amino acid.

Typically the amount of diazoalkane required is about 1.0 to 1.5 moles per equivalent of acid.

For example, if extraction of a dibasic acid, such as a cephalosporin diacid, is desired, it is convenient to use 2 to 3 moles of diazoalkane, such as about 2.1 moles per mole of dibasic acid.

The extractive esterification method according to the invention can be performed, for example, from 10 to +1
The evolution of nitrogen from the reaction system is carried out at a temperature in the range of 00°C, for example from 0 to 50°C, preferably at room temperature, and is a substantially quantitative indicator of the extent of decomposition of the diazoalkane, or by IR spectroscopy. The formation of ester bonds or the decomposition of the diazo group as evidenced by loss of IR and UV or visible light absorption can be monitored by spectroscopic techniques that follow.

After completion of the extractive esterification, the N-blocked amino acid ester can be isolated, for example, by using conventional techniques.

That is, the organic solvent can be separated from the aqueous solution, purified, for example by washing, and the solvent evaporated to produce the desired ester.

Alternatively, the organic solution may be subjected to further reaction without separating the N-blocked amino acid ester intermediate.

For example, penicillin esters so extracted can be converted directly to sulfoxides for use in ring expansion reactions.

As mentioned above, the extractive esterification method of the present invention is of particular value in isolating naturally produced amino acids and N-blocked amino acids, particularly penicillins and cephalosporins, from fermentation liquors.

Compounds that can be extractively esterified using the method of the invention include naturally occurring penicillins such as penicillin G, penicillin V and their hydroxylated and 6α-
Methoxylated analogs and naturally occurring cephalosporins such as cephalosporin C1 desacetylcephalosporin C1 desacetoxycephalosporin C1
3 carbamoyloxymethylcephalosporin and 7
Includes analogs of these compounds that are α-methoxylated, in which any free amino groups are first occluded.

This method is also advantageously used in the isolation of N-blocked amino acids obtained from fermentative reactions, e.g.
7R)-3-acetoxymethyl-7-(4-carboxybutanamido)cef-3-em-4-carboxylic acid is an example, which can be prepared by enzymatic oxidation of cephalosporin C. And because of their low solubility in water-immiscible organic solvents, they have hitherto been somewhat difficult to isolate, for example by solvent extraction.

Particularly important applications of the process of the invention include the extractive esterification of cephalosporins C and 3-hydroxymethylcephalosporins, especially desacetylcephalosporin C.

3-Hydroxymethyl cephalosporins are difficult to isolate using conventional techniques due to their pronounced tendency to form lactones, especially under acidic conditions.

In the case of desacetylcephalosporin C, the lactone produced is a stable compound which has almost no antibacterial activity and is of no value as a starting material in the production of semisynthetic cephalosporin antibiotics.

The carboxylate salt of 3-hydroxymethyl cephalosporin, however, is substantially resistant to such lactone formation, so that effective isolation according to the present invention is limited to aqueous 3-hydroxymethyl cephalosporin at basic pH. performing any pretreatment of the solution (e.g. N-blocking);
This can be accomplished by subsequently adding a diazoalkane and a water-immiscible organic solvent and a strong acid as described above to promote extractive esterification.

For example, in the case of extractive esterification of desacetylcephalosporin C, the necessary N-blocking treatment is carried out under basic conditions, e.g. with an excess of base (e.g. alkali metal hydroxide such as sodium hydroxide or potassium phosphate). This can be carried out by reaction with a suitable acylating agent (eg benzoyl chloride or an alkyl haloformate such as ethyl chloroformate) in the presence of a suitable buffer.

The by-products of the N-occlusion reaction are then removed by adding a solution of the diazoalkane (e.g. diphenyldiazomethane) in a suitable solvent, optionally after extraction, at a substantially neutral pH (e.g. in the pH range 5-8). and the solution is diluted with a strong acid (e.g. sulfuric acid or a mineral acid such as orthophosphoric acid) to a pH in the range of 2 to 4 (e.g. about pH 3,
5) Extract esterification is promoted by acidification.

Desirably, the starting aqueous desacetylcephalosporin C solution in such extraction is substantially free of cephalosporin C.

The ability to extract desacetylcephalosporin C effectively and economically would be extremely valuable.

This is because typical cephalosporin C fermentation broth obtained by fermentation of Cephalosporium acremonium contains significant amounts of desacetylcephalosporin C, the majority of which is produced by conventional cephalosporin C. This is because when C is isolated, it is usually lost.

The wastage of potentially valuable waste materials in the production of semisynthetic cephalosporins is clearly undesirable and if desacetylcephalosporin C of the fermentation broth
It will be seen that significant economic advantages arise if cephalosporin C is isolated in addition to cephalosporin C.

The extractive esterification method of the present invention can be used in multiple ways in treating cephalosporin fermentation broths depending on the desired product.

For example in this way, optionally after a pretreatment such as filtration, first an esterase which acts to deacetylate the cephalosporin C contained in the broth, advantageously a yeast organism of the genus Rhodotorula or a variant thereof, such as Rhodotorula rubra sp. Treating the broth with an esterifying agent produced by culturing the microorganism and subsequently subjecting the reaction product to an N-occlusion reaction (e.g. by treatment with benzoyl chloride or ethyl chloroformate under basic conditions) and extractive esterification. It is advantageous to produce a substantially pure N-blocked desacetyl cephalosporin C ester derivative.

Alternatively, cephalosporin C and desacetylcephalosporin C in broth are first subjected to an N-blocking reaction and extractive esterification to obtain N-blocked cephalosporin C and desacetylcephalosporin C ester derivatives. to obtain a mixture of

The N-blocked desacetyl cephalosporin C ester of the mixture is then treated with an acetylating agent (e.g., acetyl chloride) to acetylate and thereby remove the substantially pure N-blocked cephalosporin C ester derivative. A product is obtained.

The diazoalkanes used as esterification agents in the process of the invention can be prepared by combining suitable hydrazones with organic peracids, periodic acids, hypohalous acids or hypohalite salts, as described in Belgian Patent No. 802,112. They may be conveniently prepared by oxidation using esters, chromic acid, chlorine, bromine or positive halogen sources such as N-haloamides or imides.

Peracetic acid is a particularly preferred oxidizing agent for this purpose.

The oxidation is preferably carried out in the presence of a base or advantageously also an oxidation catalyst such as iodine.

Next, the present invention will be explained with reference to Examples, but the present invention is not limited thereto.

All temperatures are in °C.

Example 1 (6R・7R)-7-(R-5-benzoylamino-5
-carboxypentanamide)-3hydroxymethylcef-3-em-4-carboxylic acid bisdiphenylmethyl ester (6R-7R)-7-(R5
Benzoyl chloride (3,5rrLl, 30mM) and acetone (51.1. 'nl) mixture.

The mixture is stirred for 1.5 hours at room temperature while the pH is maintained at 85 by addition of 50% potassium phosphate.

The pH is adjusted to 5.0 with orthophosphoric acid and the solution is extracted with chloroform (100 rnl) to remove benzoic acid and benzoyl chloride.

Diphenyldiazomethane (51,26771M), dichloromethane (50%) and ethanol (10T111
) containing ethyl acetate (90rrLO) is added to the aqueous solution and the mixture is stirred for 45 minutes while the pH is adjusted to 2.0 with orthophosphoric acid.

After separation, the solvent was replaced with 5% sodium bicarbonate solution (lQOm7
) and water (1007rlO).

The solvent is removed under vacuum and the gum is dissolved in isopropanol (25TrLl) at 30°.

Petroleum ether (boiling point 30-400), QTL0, is added and the solution is cooled to -5°.

The product was washed with petroleum ether (15m) and dried under vacuum at room temperature to give the title compound (10,5y').
get.

Chloroform:acetone:acetic acid (80:2) was used as a developing agent.
Thin layer chromatography on silica gel GF'254 plates using 0:1) shows that the product is mainly the title compound with trace impurities.

Example 2 Diphenylmethyl (3S.5R.6R)-2.2-dimethyl-6-phuninoxyacetamidopenamyl 3-carboxylate 1-oxide in water (100 TILl) (3S
・5R・6R)-2,2-dimethyl-6-funinoxyacetamidopenamu-3-carboxylic acid potassium (7,8P
, 20mM: dichloromethane (75rrLl)
A solution of medium diphenyldiazomethane (41,20 mM) is added.

The mixture is stirred for 15 minutes at 10° C. while the pH is adjusted to 3.5 by addition of orthophosphoric acid.

The mixture was separated and the solvent layer was washed with water (lOOwLl), 5
% sodium bicarbonate solution (1007 rLl) and water (Loom/Li).

Peracetic acid (18.5 ml, 37% w/v) is added to the solution at 100 for 15 minutes and stirred further for 30 minutes.

The solvent is washed with water (100 rul), 5% sodium bicarbonate solution (100TL0) and water (100711O).

The solvent is removed under vacuum and the title product is crystallized from hot inproper tool (9,1).

IR and NMR data confirm the structure of the title compound.

Example 3 (6R・7R)-3-acetoxymethyl-7(R-5-
benzoylamino-5-carboxypentanamide)-
Cef-3-M-4-carboxylic acid bisdiphenylmethyl ester (6R・7.R)-3-acetoxymethyl-7-(R-5-benzoylamino 5-carboxypentanamide) Cef-3-M-4-carboxylic acid bisdiphenylmethyl ester in water (100T/Ll) -3-M-4-carboxylic acid (4,1', dichloromethane (
751 rLl), diphenyldiazomethane (41,
Add a solution of 20mM).

The mixture was stirred for 30 minutes while adjusting the pH to 2.0 with orthophosphoric acid.
Stir for 0 minutes at room temperature.

After separation, the solvent was diluted with 5% bicarbonate) IJum solution (100
7rLl) and water (100 mA).

After drying, the solvent is removed under vacuum and the gum is treated with petroleum ether (40-60°) to give the title compound (6,02).

IR and NMR data confirm the structure of the title compound.

Example 4 (6R・7R) -7-(R-5-carboxy-5-inbutyloxycarbonylaminopentanamide)-3-
Hydroxymethylceph-3M-4-carboxylic acid bisdiphenylmer x sterisobutyl chloroformate (50TLl, 375rI
(6R·7R)-7-(R-5-amino-5-carboxypentanamide)-3-hydroxymethylcef-3 at pH 7,8-8.0 and 5° for 1 h.
-Add to the solution of potassium em-4-carboxylate.

The mixture is stirred for a further 30 minutes while maintaining a temperature of 5° and a pH of 7.8-8.0.

The solution was diluted with chloroform (2507'/
Extract with L.

Dichloromethane (250ml), ethyl acetate (100ml)
ml) and a solution of diphenyldiazomethane (251,126 rILM) in ethanol (30 mz) is added to the aqueous solution.

The mixture was stirred for 30 minutes while adjusting the pH to 2.0 with orthophosphoric acid.
Stir at 15° for 0 minutes.

After separation, the solvent was replaced with 5% sodium bicarbonate solution (2 x 100
Wash with 1rLO and water (100 mA').

After drying, the solvent is removed under vacuum and the gum is treated with petroleum ether (40-600) to give the title compound (45, Fl).

IR and NMR data confirm the structure of the title compound.

Example 5 (6R・7R)-7-(R-5-benzenesulfonylamino-5-carboxypentanamide)3-hydroxymethylcef-3-em-4carboxylic acid bisdiphenyl methyl ester sodium bicarbonate (91) Water containing (
(6R/7R)-7-(R-5-7 Mino 5 in 120m0
-carboxypentanamide)-3-hydroxymethylcef-3-em-4-carboxylic acid potassium (4,IP,
10mM) in acetone (15rrLl).
) over a period of 30 minutes.

Reaction temperature (10°) and solution pH (7,8-8.0)
Hold for an additional 90 minutes.

The solution was diluted with chloroform (2xlOOm
Extract with A').

dichloromethane (80rIlO1 ethyl acetate (150r)
A solution of diphenyldiazomethane (6, Of, 30 mM) in IL and ethanol (207 rLl) is added to the aqueous solution and the mixture is stirred for 30 minutes while the pH is adjusted to 2.0 with orthophosphoric acid.

After separation, the solvent was replaced with 5% sodium bicarbonate solution (2 x 100
rrLl) and water (100m wash).

After drying, the title compound (5,1') is obtained by removing the solvent under vacuum and treating the gum with petroleum ether (40-600).

TLC and IR data confirm the structure of the title compound.

Example 6 Diphenylmethyl (6R・7R)-3-hydroxymethyl-7-(cheno-2-ylacetamido)cef-3-
emu 4-carboxylate (6
R-7R)-3-1:: A solution of potassium droxymethyl-7-(cheno-2-ylacetamido)cef-3-em-4-carboxylate (4,Of, 10 mM) in dichloromethane (75 mO and ethanol ( A solution of 101077 l of diphenyldiazomethane (2, C1) in 10 mO is added.

The mixture was adjusted to 2.0 with orthophosphoric acid.
Stir for 5 minutes.

After separation, the solvent is washed with water (100 rIl, 5% sodium bicarbonate solution (2 x 100 mO) and water (1,007 d).

After drying, the solvent is removed under vacuum and the gum is treated with petroleum ether (40-60°) to give the title compound (5,15f).

IR and NMR data confirm the structure of the title compound.

Example 7 (6R・7R)-3-acetoxymethyl-7(4-carboxybutanamide)77-3-emu 4-carboxylic acid bisdiphenylmethylnis7(6R・7R)-3-acetoxymethyl-7 -(4-Carboxybutanamide)cef-3-em 4-carboxylic acid (0,24P, 0.575 mM) was added to a slurry of diphenyldiazomethane (0,:
l', 1.5mM).

While adjusting the pH to 2.0 with orthophosphoric acid, the mixture was
Stir for 5 minutes.

The mixture is then stirred for a further 75 minutes at room temperature.

After separation, the solvent was dissolved in 5% sodium bicarbonate solution (2 x 25 r
Ill) and water (25rrLO).

After drying, the title compound (0,38P) is obtained by removing the solvent under vacuum and treating the gum with petroleum ether (40-600).

IR and NMR data confirm the structure of the title compound.

Example 8 (6R・7R) -7-(R-5-inbutyroxysulfonylamino-5-carboxypentanamide)-3-
Hydroxymethylceph-3-em-4-carboxylic acid bisphenyl (o-1) methyl ester (6R 7R) -7-(R5-
Inbutyl chloroformate (16 ml, 120771
M) is added over 1 hour at 10° and pH 7,8-8.0.

The mixture was further heated for 3 hours while maintaining the pH between 7.8 and 8.0.
Stir for 0 minutes.

The pH is adjusted to 5.0 with orthophosphoric acid and extracted with chloroform (2x10mO).

Dichloromethane (1207711), ethyl acetate (60
A solution of (0-)lyl)phenyldiazomethane (4,2P, 40mM) in a mixture of m0 and ethanol (20m0) is added to the aqueous solution.

The mixture was heated to
Stir for 5 minutes.

After separation, the solvent is washed with water (lQQm), 5% sodium bicarbonate solution and water (100 mQ).

After drying, the title compound (+6.i') is obtained by removing the solvent under vacuum and treating the gum with petroleum ether (40-600).

Example 9 (6R・7R) -7-CR-s-carboxy5-(3
・5-Jethoxycarbonyl-2,6 dimethyl-1,4
-dihydropyridin-1yl)pentanamide]-3-
Hydroxymethylcef-3-em-4-carboxylic acid bisdiphenylmethyl ester (6R・7R)-7-(R-5-amino-5carboxypentanamide)-3-hydroxymethylcef-3-em-4-carvone Potassium acid (purity 70%, 121.2
0TrLM) in a solution of 37% formaldehyde solution (
18,77711,249T71M) and ethyl acetoacetate (25.2 ml.

199rrLM) is added separately over 1 hour at 5°.

The pH of the solution is maintained at 7.0 by adding 25% potassium phosphate solution.

After stirring for an additional 30 minutes, the solution was dissolved in dichloromethane (20
0 ml).

Dichloromethane (150 ml) containing diphenyldiazomethane (101,52 yyiM) is added to the aqueous solution and the mixture is stirred for 45 minutes while the pH is adjusted to 2.0 with orthophosphoric acid.

After separation, the solvent layers were dissolved in water (200 rILl), 5% sodium bicarbonate solution (200 TlO and water (200 rILl)).
).

After drying over magnesium sulfate, the solution was dried under vacuum for 75 min.
A solution of the title compound is obtained by concentration to a volume of 7711.

Example 10 (6R・7S)-7-(R-5-benzyloxycarbonylamino-5-carboxypentanamide)-3-carbamoyloxymethyl-7methoxycefu-3-M-
4-Carboxylic acid bisdiphenyl methyl ester (pendyl chloroformate) (3,5 m7) in water (80 m0 containing sodium bicarbonate (61) (6R.7S)
)-7-(R-5-Amino-5-carboxypentanamide)-3-carbamoyloxymethyl-7-medoxycefu 3-M-4 carboxylic acid monoammonium salt (approximately 5% purity, 41). Heat at 10° for 1 hour.

The pH is maintained at 7.8-8 by adding IN (IJ) hydroxide.

The mixture is stirred for a further 90 minutes at 10° and pH 7,8-8.

The pH is adjusted to 5 with orthophosphoric acid and the solution is washed with chloroform (2 x 10011 Ll).

Ethyl acetate (50 ml) and ethanol (10 ml) were added to the aqueous phase.
A solution of m0 and methylene chloride or diphenyldiazomethane (50 ml, approximately 0.2M) is added.

The mixture was heated to 15° while adjusting the pH to 2 with orthophosphoric acid.
Stir for 45 minutes.

After separation, the organic phase was dissolved in 5% aqueous sodium bicarbonate (2 x 7
51rLl) and water (lOOTLl), dried Na25O4) and evaporated to a gum (2,9f).

Purification by column chromatography (silica gel, ethyl acetate and chloroform) followed by preparative thin layer chromatography (silica gel, chloroform and methanol) gives the title compound as a white foam (260 .mu.).

IR, NMR, UV and microanalytical data confirm the structure of the title compound.

Example 11 (6R・7S)-7-CR-s-carboxy5-(2・
2,2-Trichloroethoxycarbonylamino)-pentanamide io 3-carbamoyloxymethyl-7-methoxycef-3em-4-carboxylic acid hisdiphenylmethyl ester 2,2,2-trichloroethyl chloroformate (3r
fLl) in water (8?) containing sodium bicarbonate (6?).
(6R-78)-7-(R-5-amino-5
-carboxypentanamide)-3carbamoyloxymethyl-7-medoxycefu to a stirred solution of 3-em-4-carboxylic acid monoammonium salt (approximately 5% purity, 4 grams) at 100 °C over 1 hour.

The pH is maintained at 7.8-8 by adding 1N sodium hydroxide.

The mixture is stirred for a further 90 minutes at 10° and pH 7,8-8.

The pH is adjusted to 5 with orthophosphoric acid and the solution is washed with chloroform (2 x 1001N).

Ethyl acetate (50 mA'), ethanol (10
m0 and a solution of diphenyldiazomethane in methylene chloride (50ml ca. 0.2M) is added.

The mixture was heated to 15° while adjusting the pH to 2 with orthophosphoric acid.
Stir for 30 minutes.

After separation, the organic layer was dissolved in 5% aqueous sodium bicarbonate solution (2
A gum (2,8P) is obtained by washing with x80I710 and water (100 ml), drying (Na25O4) and evaporating.

Purification by column chromatography (silica gel, chloroform and ethyl acetate) followed by preparative thin layer chromatography (silica gel, chloroform and methanol) gives the title compound as a white foam (410 .mu.m).

IR, NMR, UV and microanalytical data confirm the structure of the title compound.

Example 12 (6R・7S) -7-(R-5-benzoylamino-5-carboxypentanamide)-3carbamoyloxymethyl-7-nodoxycef 3-M-4-carboxylic acid hisdiphenylmethyl ester water (100rIlO Monoammonium salt of (6R-78)-7-(R5-amino-5-carboxypentanamide)3-carbamoyloxymethyl-7-medoxycefu 3-M-4-carboxylic acid (purity about 8%, 51, approx. A solution of 0.85 mM) is adjusted to pH 8.3 with 50% potassium phosphate solution.

Benzoyl chloride (3 mA!) in acetone (5 ml).
, 26 mM) and the mixture is stirred at room temperature for 1.5 hours while the pH is kept at 8.3 by addition of 50% potassium phosphate.

The pH was adjusted to 5 with phosphoric acid and the solution was dissolved in chloroform (
Wash with 100TIL Shun.

Ethyl acetate (90 TLl), ethanol (10
A solution of diphenyldiazomethane in methylene chloride (50 ml, approximately 0.2 M) is added.

The mixture is stirred for 45 minutes while the pH is adjusted to 2 with orthophosphoric acid.

After separation, the organic layer was dissolved in 5% aqueous sodium bicarbonate (100
of water and water (100 TLl) and dry with N.
a25O4) and evaporate to a gum (2,6P).

Purification by preparative thin layer chromatography (silica gel, chloroform and ethyl acetate) gives the title compound as a white foam (554 .mu.m).

The structure of the title compound is confirmed by IR, NMR, UV and microanalysis.

Example 13 (6R・7R)-7-CR-5~carboxy5-(2
・2,2-Trichloroethoxycarbonylamino)pentanamide 3-hydroxymethylcef-3-em-
4-Carboxylic acid bisdiphenyl methyl ester 2,2,2-trichloroethyl chloroformate (5r
ILl, 37iM) in water (100rrLl) (6R-
7R) -7-(R-s-amino-5-carboxypentanamide)-3-hydroxymethylcef-3-em-
Add to a solution of potassium 4-carboxylate (20mM).

The pH of the solution is maintained at 75-8.0 for 30 minutes by using 10% w/v sodium hydroxide solution.

The solution was then dichloromethane (50 r
Extract with rL0.

A solution of dienyldiazomethane (8,5P, 44mM) in dichloromethane (150mO) was added to the aqueous layer and p
Adjust H to 3.5 by use of 10% v/v sulfuric acid for 15 min.

After separation the solvent is washed with water (3 x 150 ml).

The solvent is removed under vacuum to yield the title compound (20,5P).

IR and NMR data confirm the structure of the title compound.

Example 14 (6R・7R) -7-(R-5-carboxy5-(2
・2.2-)IJ dichlorotoxycarbonylamino)pentanamide io 3-hydroxymethylcef-3-em-4-carboxylic acid bisnaphthylphenyl methyl ester A solution of naphthylphenyldiazomethane (approximately 447 FLM) in dichloromethane (150 ml) was dissolved in dichloromethane. The title compound (
23,3y) is obtained.

IR and NMR data confirm the structure of the title compound.

Example 15 (6R・7R)-7-CR-5-carboxy5-(2・
2,2-Trichloroethoxycarbonylamino)pentanamide)-3-hydroxymethylcef-3-em-4
-Carboxylic acid bis(p-methoxyphenyl)phenylmethyl ester Example 13 except that a solution of (p-methoxyphenyl)phenylcyazomethane (approximately 44 mM) in dichloromethane (150 mO) is used instead of a solution of diphenyldiazomethane in dichloromethane. The title compound (6,=l) is obtained by repeating the method.

IR and NMR data confirm the structure of the title compound.

Example 16 (6R・7R)-7-[R-5-carboxy5-(2・
2.2- ) IJ chloroethoxycarbonylamino)pentanamide io-3-hydroxymethylcef-3-
Em-4-carboxylic acid bis-1-phenylethyl ester The procedure of Example 13 was repeated, except that a solution of 1-phenyldiazoethane (approximately 44 mM) in dichloromethane (150 mO) was used instead of a solution of difenino 1/diazomethane in dichloromethane. The title compound (18,
6P) is obtained.

IR and NMR data confirm the structure of the title compound.

Example 17 (6R・7R)-7-(R-5-carboxy5-ethoxycarbonylaminopentanamide)-3-hydroxymethylcef-3-em-4carboxylic acid bisdiphenyl methyl ester ethyl coo formate (5 ml , 52m
The title compound (15,75') is obtained by repeating the method of Example 13, except that M) is used instead of 2,2,2-trichloroethyl chloroformate.

IR and NMR data confirm the structure of the title compound.

Example 18 Diphenylmethyl (6R・7R)-3-acetoxymethyl-7-(cheno-2-ylacetamide) cef-3-
M-4-carboxylate water (in 5QmA (6R・
7R)-3-7-ttoxymethyl-7-(cheno-2-
To a solution of sodium (4,2P, 10mM) dichloromethane (30m7) containing dienyldiazomethane (2,IP, 11mM) is added.

Adjust the pH of the solution to 3.0 by using 10% V/V sulfuric acid for 15 min.

Separate the mixture and add the solvent layer to water (4 x 50TrLl)
Wash with

The title compound (6,9
6P) is obtained.

IR and NMR data confirm the structure of the title compound.

Example 19 Diphenylmethyl (5R.6R)-'2,2-dimethyl-6-phenoxyacetamidopenam 3-carboxylate (5,R-6R)-2-2-dimethyl-6-phenoxy in water (1 oomz) A solution of potassium acetamidopenam-3carposiate (1 (1, 25, 7mM) and phenoxyacetic acid (2), 13.1mM) was dichloromethane (1
Diphenyldiazomethane (5,2f, 277
71M) is added.

The pH is adjusted to 4.0 and the mixture is stirred for 10 minutes.

After separation, the solvent was dissolved in water (100 mA'), 5% w/v sodium bicarbonate solution (room) and water (1001 nA').
Wash with l).

The title compound (11,
4P).

Only 30% as impurity by IR and NMR data
Confirm the structure of the title compound containing the original phenoxyacetic acid.

Example 2O N-(2,2,2-trichloroethoxycarbonyl)glutamate bisdiphenylmethyl ester 2,2,2-trichloroethylchloroforme) (5,
5ml, 40mM) is added to a solution of DL glutamic acid (3,31', 20rrLM) in water (100TLl).

The pH of the solution is kept at 7.5-8.0 for 30 minutes by using 10% w/v sodium hydroxide solution.

The solution was then dichloromethane (2×1
00Tll).

A solution of diphenyldiazomethane (85i, 44rrLM) in dichloromethane (140 mO) is added to the aqueous layer and the pf is adjusted to 2.0 by use of 10% w/v sulfuric acid over 15 minutes.

After separation, the solvent is washed with water (3 x 150 rIl).

The title compound (14,
3IR and NMR data confirm the structure of the title compound.

Example 21 N-benzyloxycarbonylglycine bisdiphenylmethyl ester dichloromethane (diphenyldiazomethane (in 32 mO)
2, IP, 11 mM) in water (50 ml) with N
-benzyloxycarbonylglycine (2,12,10
(mM) solution.

The pH is adjusted to 3.5 using 10% v/v sulfuric acid for 15 minutes.

After separation, the solvent is washed with water (3 x 50 mO) and then removed under vacuum.

The product is then precipitated by addition of excess ether.

The title compound (3,4f) is obtained by filtering the precipitate and washing with ether (15m).

IR and NMR data confirm the structure of the title compound.

Example 22 N-(2.2.2-) IJ dichlorotoxycarbonyl) glutathione bisdiphenylmethylesarglutathione/(6,14P, 20rrLM) (6R-
7R)-7-(R-5-amino-5-carboxypentanamide)-3-hydroxymethylcef-3-em-4-
Example 13 except that potassium carboxylate was replaced with treatment.
By repeating the method, the title compound (11,:l
') get.

IR and NMR data confirm the structure of the title compound.

Example 23 DL-methionine diphenyl methyl ester p-toluenesulfonate DL-methionine (3,0P, 20771M) and p
-) luenesulfonic acid monohytray) (3.8 g, 2
0rrLM) is dissolved in water (50mM) at room temperature.

Di7 Engineering: /L/Diazomethane (4,85P, 25m
M) in dichloromethane (50 mA) is added and the mixture is stirred at pH 2 for 1 hour.

After separation, the solvent layer was dissolved in 5% sodium bicarbonate solution (50 m
0 and water (50 Tll).

The solvent is removed under reduced pressure and the product is crystallized from acetonitrile.

The title compound (5,4?) is obtained by filtration and washing with acetonitrile (15 ml).

Melting point and IR and NMR data confirm the structure of the title compound.

Example 24 Diphenylmethyl (6R・7R)-7-amino 3-acetoxymethylcef-3-em-4carboxylate hydrochloride 2.2.2-trichloroethylchloroforme) (4
, 2 ml, 31 mmol) to (6R-7R)7-(R
-5-amino-5-carboxypentanamide)-3-
Acetoxymethylcef-3-em-4-carboxylic acid (1
0 mmol) and (6R・7R)-7-(R-5-
An aqueous solution (100 mL) containing each kawaram salt of amino-5-carboxypentanamide)-3-hydroxymethylceph-3-em-4-carboxylic acid (10 mmol) is added.

The pH of the solution is maintained at 7.5-8.0 for 1 hour using 10% w/v sodium hydroxide solution.

The solution was then dichloromethane (501
Extract with rLO.

A solution of diphenyldiazomethane (8,5S', 4.4 mmol) in dichloromethane (150 ml) is added to the aqueous phase and the pH is adjusted to 3.5 using 10% v/v sulfuric acid for 30 minutes.

After separation, the solvent is washed with water (3×1507111) and stirred with anhydrous magnesium sulfate (IOS').

After filtering the solid, the solution is distilled under reduced pressure to obtain 50%
Concentrate to ml.

Acetyl chloride (4,3rnl, 60 mmol) and pyridine (4,8ml, 1160 mmol) are added to this solution with cooling to maintain a temperature of 5-10°.

After stirring for 30 minutes the solution is filtered and added to a slurry of phosphorus pentachloride (9,Or, 43 mmol) and pyridine (3.5ml 1143 mmol) in dichloromethane (40 rfLO).

Humidity is maintained at -5° during the addition. The mixture is stirred for 50 minutes to maintain a temperature of 0-5°.

After cooling to -10° methanol (4 Qm) is added and stirring is continued for a further 15 minutes.

Then water (40 ml) and di-isopropyl ether (
250 mA is added and the product is allowed to crystallize for 1 hour.

The product is filtered, washed with a mixture of methanol and diisopropyl ether (30 min. 0.100 m) and dried under reduced pressure to give the title compound (7,8P).

IR and NMR data confirmed the structure of the title compound.

The gist of the present invention and representative examples of embodiments are shown below.

1. characterized in that an ester solution of the N-blocked amino acid in an organic solvent is produced by treating an aqueous solution of the N-blocked amino acid with a diazoalkane in the presence of a water-immiscible organic solvent; A method for extracting N-blocked amino acids from an aqueous solution of N-blocked amino acids.

2. The above-mentioned diazoalkane in which the ester group introduced into the N-blocked amino acid is an aralkyl group consisting of a lower alkyl group having one or two carbocyclic or heterocyclic aryl substituents at the 1-position. The method described in Section 1.

3. The method according to item 2 above, wherein the aralkyl group is a diphenylmethyl group.

4. A method according to any preceding clause, wherein the N-occluded amino acid is derived from a fermentation broth.

5. The method of item 4 above, wherein the N-occluded amino acid is a naturally occurring penicillin or cephalosporin compound in which all free amino groups are occluded.

6. The method according to item 5 above, wherein the N-occluded amino acid is an N-occluded derivative of desacetylcephalosporin C.

7. N-occluded amino acids form cephalosporin C
and a N-blocked derivative of desacetylcephalosporin C.

8. The method according to items 1 to 4 above, wherein the N-blocked amino acid is an N-blocked derivative of glutathione.

9. N- in solution of diazoalkane in organic solvent
A method according to any preceding paragraph, wherein the occluded amino acid is added to an aqueous solution.

10. Decrease the pH of the neutral or basic solution of the N-blocked amino acid after addition of the diazoalkane and the water-immiscible organic solvent using a strong acid with a pKa less than the pKa of the N-blocked amino acid. A method according to any preceding clause, wherein the extractive esterification is carried out by:

11. The method of paragraph 1, above, substantially as herein described.

12. A method for extracting N-blocked amino acids from an aqueous solution of N-blocked amino acids substantially as described in any of the preceding Examples.

13. An N-blocked amino acid ester produced by the method described in any of Items 1 to 12 above.

Claims (1)

[Claims] 1. An organic solvent solution of an N-blocked amino acid ester is produced by treating an aqueous solution of an N-blocked amino acid with a diazoalkane in the presence of a water-immiscible organic solvent. , a method for extracting N-occluded amino acids.