DE1770720A1 - Peptide derivatives - Google Patents

Description

Peptide derivatives

This invention relates to a novel method of manufacture of N-thiocarboxy amino acid anhydrides.

The controlled, stepwise synthesis of polypeptides, in particular of heteropeptides, is a problem that evolves in technology has posed for a long time. Such products are useful as intermediates in protein synthesis. Some of them are therapeutic effective. They are also useful in the study and analysis of proteins, particularly studies based on them are turned off to gain insight into the mode of action of enzymes, hormones and other proteins with important punctures in the body to win.

The controlled, stepwise synthesis of heteropeptides and proteins can be carried out by adding an amino acid or a Peptide, for example a tetrapeptide, in an aqueous medium

1098ÖV / 181Ö

under controlled conditions τοη concentration, temperature » Time and hydrogen ion concentration is reacted with an N-thiocarboxyamino acid anhydride to form an intermediate thiocarbamate, which is then usually by lowering the hydrogen ion consistency to pH 3 bis 5 is dethiocarboxylated to form the desired product. Bas method can be used in sequential reactions without isolation of intermediates to generate heteropeptides using exceptionally high molecular weights are carried out. on this method is referred to below under the designation TCA method.

Generally speaking, the method comprises reacting the selected amino acid thioanhydride with the amino group of a second Amino acid or a peptide under such conditions that the only one in a significant concentration in the course of the implementation of « essential, amino group is that which should take part in the reaction. The exact conditions under which a special Amino acid thioanhydride is an amino acid or a peptide reacts depend on the chemical and physical properties of the reactants. The main factor is the relative basicity of the reactants and intermediates. The reaction is usually carried out in such a way that the thloanhydride to an aqueous Medium containing the amino acid orter the peptide at pH 7 to 10, usually from Ö to 10, at low temperature, see B. 0 to 25 ° 0, contains, given and the coupling realization untor

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" ² " BATH ORIGINAL

Completion of a thiocarbamate occurring as an intermediate product by taking two or three hours for a period of time that normally does not exceed 30 minutes can be stirred quickly. The intermediate product is then reduced by lowering it dethiocarboxylated the pH to about 3 to 5 to form the desired product.

This invention provides a novel and useful method for preparing τοη useful for syntheses H-thioarboxyaminoaur anhydrides ready. The method according to the invention to manufacture of N-thiocarboxy amino acid anhydrides generally includes cyclization an alkylthionourethane derivative of an amino acid amide in the presence of a strong acid in an inert, polar solvent,

The method under consideration can be expressed by the equation R-CH-COHH ₂ RCfH-C.

H +

HH -C = O

are shown, in which: R denotes an aromatic acid radical, typically hydrogen or lower alkyl _f lower aralkyl, lower aminoalkyl, lower alkoxyalkyl, lower hydroxyalkyl, lower mercaptoalkyl, lower (lower alkyl mercapto) alkyl, lower guanidyl alkyl lower indolyl and lower indolyl lower alkyl

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R ^{1 is} lower alkyl or aralkyl. The term lower alkyl embraces alkyl groups containing up to 8 carbon atoms.

The CyclisleruTigsreaktlon is in the presence of a strong acid, preferably one of the inorganic mineral acids, such as hydrochloric acid, carried out. It is also carried out in an inert, polar, organic solvent, the term "Inert" means that the solvent with the thionourethane derivative, the thiocarboxy anhydride or the strong acid is not reacted. The Hitra lower alkanes, especially nitromethane, are suitable polar solvents; however, it is also possible to use other solvents, in particular esters, ethers or halogenated hydrocarbons, which contain, for example, up to 8 carbon atoms.

If the reaction is carried out with oil, the thionourethane derivative is used dissolved or dispersed in the polar solvent, and the strong acid is introduced by, for example, bubbling gaseous hydrogen chloride through the solution. Moderate reaction temperatures of the order of magnitude from about 0 to about 40 ° C. are suitable} However, slightly higher or lower temperatures can also be used applied throw. In general, it is most convenient to run the reaction at or around room temperature. Atmospheric pressure or higher pressures can be used. The formation of the product thiocarboxy anhydride is caused by the occurrence a precipitate of the ammonium alale of the acid used

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shown, and implementation continues "until there is no further precipitation forms more or until the ultrared spectra indicate that the implementation is complete, which is typical is reached in about 0.5 to 10 hours.

The thiocarboxy anhydride is obtained by extracting with an inert organic solvent such as methylene chloride or ethyl acetate, washing with water, drying the organic phase and removing the solvent. The purity of the product is determined by recrystallization from a suitable solvent such as n -Hexane-ethyl acetate; improved. If necessary, the anhydride can be cleaved into its optical isomers by methods well known in the art.

Bas thionourethane derivative can expediently either from the Staom amino acid amide by reaction with a xanthate diester or from the parent amino acid itself. Implementation with a xanthate dieater and subsequent reaction with ammonia and a mild one Dehydrating agents are produced.

The synthesis of thionourethane from the amino acid amide is carried out by the implementation

BOHCOHH ₀ + R ¹ OO-SR ¹ > ROHOOlIH ₀ + R ¹ SH

^l 2 ^l 2

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illustrates in which R and R have the meanings described above and the two R groups are identical or different. The reaction takes place easily at moderate temperatures of the order of about 30 to about 80 ° C, although somewhat higher or lower temperatures can also be used. If desired, solvents can be used to ensure homogeneity and to promote mobility of the reaction mixture. The distance the by-product mercaptan as it forms is desirable; for this reason the short chain xanthate esters, such as methyl xanthate, are preferred. The history The reaction can conveniently be followed by a nitrogen carrier stream through the reaction mixture and from there through an aqueous one Lead acetate solution is passed. The presence of the mercaptan by-product in the gas stream is determined by the continued precipitation of a yellow to orange lead captids displayed, the formation of which stops, as soon as the main reaction is complete. The thlourethane can be sufficiently purified for the subsequent reaction by conventional methods by, for example, in one Inert, organic solvent is dissolved and washed with aqueous sodium chloride, the organic layer is separated and the Solvents are removed.

The production of thionourethane from the parent amino acid is made through the reaction scheme

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11334. 7th

S RGHCOOH + R ¹ OC-SR ¹ f RCHCOOH + R ¹ SH

σ. * s

OR ¹
(1) HH, / (2) mild dehydration aitt *! ** '

RCHCOHHj

J »S
k ¹

in which R and R ^{1 have} the meanings given above and the two R ¹ groups can be identical or different. It is desirable to use a strong base such as an alkali metal hydroxide in water, alcohol, or a water-alcohol solution in the first stage of this synthesis. In other respects the translation conditions are similar to those given above for the reaction of the amino acid amide with the xanthate ester.

The aralnoic acid thionourethane is isolated by converting the product into an aqueous, alkaline solution and washed with an inert, organic solvent, such as ether, by then the aqueous layer is acidified and back extracted with an inert organic solvent such as ethyl acetate. the organic layer is retained and washed with aqueous sodium chloride washed, cann separated and evaporated to dryness.

The conversion of the Araiuosäure-thionourethans in the corresponding

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BATH ORIGINAL

11334 β

Amide is done in such a way that it is successively combined with ammonia and a mild dohydrate agent · The ammonia converts the acid into the ammonium salt and the dehydrating agent removes 1 pint of water to form the amide. In the typical case, the amino acid thionourethane is in an inert, Organic solvents such as ether are dissolved and dry ammonia gas is introduced into bubbles to precipitate the ammonia salt. Excess ammonia is then removed and the theoretical amount of dehydrating agent added. The preferred Dehydrating agents are the dialkylcarbodiimides, such as dioyclohexylcarbodiimide. The reaction mixture is stirred until the reaction is complete, typically about 5 to 20 times For hours. With the preferred dehydrating agents proceeds the reaction smoothly at room temperature, so higher temperatures are not required. The work-up of the amino acid amide thionourethane obtained as a product for use in the manufacture of a thiocarboxy anhyaride is carried out as described above.

Starting substances which are suitable for use in the inventive Examples of suitable methods are the following amino acids in the right-handed or left-handed form or racemic mixtures as well as their amides or thionourethanes:

Alanine
Leucine
Cysteine
Phenylalanine

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Glutamic acid - as the α-amide, laoglutaiain

Aspartic acid - ala the α-Ajnid, - Isoaeparagin

Cystine

Arginine

Glycine

Proline

Serine

Isoleucine

Valine

tyrosine

Threonine

and similar as well as substituted derivatives thereof such as those Compounds that have lower alkyl, aryl, aralkyl, Carry alkaryl or halogen groups.

If the amino acid is a simple amino acid, such as leucine or alanine, the synthesis is in a simple manner as indicated above. If the amino acid contains other potentially reactive groups, these are preferably protected during the synthesis and during possible subsequent peptide syntheses. The blocking group should be resistant to cleavage by the strong acid used in the cyclization reaction. For example, amine groups are easily blocked with im-benzyl groups. Thiocarboxylic acid anhydrides of dioarboxyl-aminoslinron are best .hergoatellt via the α-amides. Analogous methods for treating other reactive groups are known to those skilled in the art.

As discussed earlier, any of the subsequent

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8 sharing listed or similar compounds are used ι Dimethylxanthate, diethylxanthate, di-n-propylxanthate, di-n-butylxanthate, di-n-hexylxanthate, dibenzylxanthate, di-8-phenylethylxanthate, etc. Unsymmetrical xanthates can, if appropriate, also be used.

The following examples serve to illustrate the invention, but are not intended to limit it.

Example 1 Leucinamide-Nethylthionoureth & n

A solution of 2.6 g (20 millimoles; "M moles") of L-leucine amide in 5 al of methanol is placed in a 50 ml mash neck flask, the one with a mechanical stirrer, a nitrogen inlet valve, Thermometer and a reflux condenser sprinkler ("Reflux condenser scrubbing apparatus "). A total of 2.17 ml (21 millimoles) dimethylxanthate are pipetted into the mixture. A stream of dry nitrogen gas is passed through the mixture and the exiting gas is bubbled through aqueous lead acetate. A yellow to orange colored precipitate of blel mercaptide is immediately observed.

The mixture is stirred for 3 1/2 hours at room temperature and then heated to 50 ° C for 30 minutes. At this point the lead acetate test is practically negative. The oil obtained would be seen

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taken up in 10 ml of ethyl acetate, twice with 7 ml of an aqueous, Washed 10bigen sodium chloride solution, then dried over sodium sulfate and evaporated to dryness. The product obtained is dissolved in 18 ml of 2B alcohol and allowed to evaporate slowly. Man this gives 3.5 g of L-Ieucinamidemethylthionourethane, which slowly crystallizes.

Following the same procedure, the corresponding methylthionourethanes are prepared with alanine anide or phenylalanine amide. Dimethylxanthate is obtained in a similar manner by replacing the dimethylxanthate by diethylxanthate, di-n-butylxanthate or dibenzylxanthate the corresponding ethyl-, η-butyl- and benzylthiouourethane.

Example 2

Leuc in N-thiocarboxy anhydride

Inagosamt 0.45 g (2.2 millimoles) of the product of Example 1 will be dispersed in 5 ml of hitromethane, and dry hydrogen chloride gas is bubbled through for one hour at tree temperature. The corrosion mixture immediately becomes cloudy. After an hour a 0.2 ml portion removed and evaporated to dryness in vacuo. the Solids are taken up in about 0.4 ml of methylene chloride filtered through a plug of glass wool and subjected to the ultra-red analysis, which revealed the presence of leucine-H-thiocarboxy-anhydride indicates.

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The rest of the reaction mixture is left for an additional hour Treated with hydrogen chloride and then in 5 al ethyl acetate and 5 ml of water added. The aqueous layer is discarded and the organic layer washed with an additional 5 ml of water. The organic layer is again isolated and dried over sodium sulfate. It is then evaporated to dryness, and the The residue is recrystallized from 2 ecm of cyclohexane. You get C, 17 g of leucine ^ -N-thiocarboxy anhydride.

Any of the other thionourethanes described in Example 1 can be used under similar conditions to prepare the corresponding thioarboxy anhydrides.

Beia-plel 3 leucln-methylthionourethaa

A total of 52.5 g of leucine are added to 54.2 ml of 5% ethanolic potassium hydroxide aiifgenome and 10 ml of fiber. 43.9 g of diethyl ether in 40 ml of Kothanoi are added to the solution. The two-phase Syetea is heated to about 50 ° C. in order to form a single-phase Syatem with the development of Ke thy liner cap tan. The solution is held at 50 ° C. for about 90 minutes and concentrated to remove most of the ethanol. The residue is diluted with 100 ral Weecor and washed twice with 100 ml portions of ethyl acetate in order to remove any non-replaced Xanthate. The aqueous layer is added to 200 ml of Ethylacetate-S, the 34 ic] konr.on-

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trated hydrochloric acid included, given, and additional 50 ml of water are added. This treatment converts the potassium salt of H-thiocarbalcoxy-leucine into the desired acid, which is extracted into the organic layer. The aqueous layer is washed with additional ethyl acetate and the combined Organic layers are made twice with 40 ml portions a saturated solution of sodium chloride, washed over anhydrous dried sodium sulfate and filtered. The piltrate is concentrated under reduced pressure to a viscous oil, which on standing crystallized. Ijeucine-methyl-thionourethane is obtained.

Similarly, alanine and phenylalanine are made with dimethylxanthate with formation of the corresponding methylthionourethane and Leucine, alanine and phenylalanine with diethyl xanthate, di-n-butyl xanthate and dibenzylxanthate with the formation of ethyl, η-butyl and Benzylthionourethane implemented.

Example 4
Leucine amide methylthionourethane

The product of Example 3 (2.05 g, 10.0 millimoles) is in 15 ml dry ether is dissolved in a 50 ml single-necked flask, and dry ammonia gas is bubbled through it for about 5 minutes an oil forms, which settles out of the solution. The mixture is used to remove ether and excess ammonia in vacuo concentrated and with 10 ml of acetonitrile and 2.06 g (10 millimoles)

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11334 β

Dioyclohexylcarbodiiraid added. The mixture is stirred overnight at room temperature, extracted and old ether. The ether extract is dried over sodium sulfate, filtered and concentrated. Kan receives Leucinanld-methyl-thlonourethane. The product is cyclized under the conditions of Example 2. Leucin-N-thiocarboxy anhydride is obtained.

Similarly, the remaining thionourethenes of the examples 3 converted into the amidthionourethane and bu the corresponding Thioarboxy anhydrides celiated.

Example 5 Alanyl-leucyl-phenylalanine

Äicaes example illustrates the use of the Thiοcarboxyanhydride for the controlled production τοη polypeptides · To a mixture of 3 millimoles of phenylalanine in 30 ml aqueous Kaliruaborat buffer at pH 10 is added with rapid stirring 3 _f 2 Millinol solid H-Thlocarboxy-leucln anhydride, while the temperature is kept between 0 and 40 ° C. The pH is then adjusted to 3 with concentrated sulfuric acid at 0 ° C while a nitrogen atom is bubbled through the mixture. The product leucyl-phenylalanine is not isolated.

The pH-value of the mixture is adjusted to 10.0 aingeotelJt by adding kcnr.öat, riertön ksliiuahydroxids, and 3 _f 4 milliwol alanine-V-thlo-

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BATH ORIGINAL

¹¹ " ⁴ AS

oarboxy anhydride are poured out with stirring while the Temperature is maintained at about 25 ° G. The reaction is continued for 20 minutes and by adjusting the pH using Sulfuric acid on 3 causes the sethiocarboxylation. The desired product is chromatographed on a Si3f ± ceg «l table isolated. Similarly, others are described above Thiocarboxy anhydrides for controlled, stepwise synthesis Clay polypeptides used.

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Claims (1)

1. Process for the preparation of an amino acid B-thiocarboxy anhydride, characterized in that one is an amino acid amide thionourethane cyclized in the presence of a strong acid in an inert, polar., organic solvent. 2. Method of making an amino acid IT thiocarboxy anhydride the formula RAW- in which R dm-. Hßst eiiifsr AmiT7.or * iiure means öDäurch ^ e denotes, <lfiD ma ^ an Arainoßiä'.ires.mid-thionourethan der formula P. - Hit α m s OR in WOIu ¹ KIr J ?. ¹ Wi. «Arigalky :. or A: mlk; r: i with liio cu β carbons clays Ti σ ^ c .tatet, in Ocfvonwar ·; an otarken S? iura and cyclized in © tnom i.norti'r, polar, organic solvent, _{1 cm} 109884/18 10 BATH ORIGINAL 3. Ancestors according to Anspruoii 2. _t üacLnrch geksnnseiclmet that the acid rips a mineral acid. 4. The method according to Anspruoii 3 »üödurch characterized in that Mineral acid is Ohlorwasoeretoffsäitre. 5. The method according to claim 2 _r characterized »that the acid is Ohlorwassoratoffsätire _r the solvent is hitromethane and E ^{1 is} methyl. 6 »Procedure Kum producing an Ansinosöure-li-thiocarboxy-anhydride, as a result, there is no need for that! »to an amino acid amide with a xanthate oiler to form an amino acid amide thiottouretlmne converts and iiieses AminfsaäTiraiMaid-thionourethan in the presence of a strong acid and in an inert, polar, organic solvent cyclisiort. 7. Verebren mm Harstellon elvea amino acid-H-thiocarboxy-en- M animal? O'i "iel Z «DII - V in ;; e:.! chor H data Rast eic or Arainogäui © means, datlxirch marked, düsa nmvi oia Aiainoetlureartild der! Porr: el 109884/1810 / ftf R - CHCONH ₂ with a xanthate diester of the formula SR ¹ OC- SR ¹ in which each R ^{1 is} lower alkyl or aralkyl with up to θ Konlenetoffatomen, with formation of an amino acid eaniidthionourethans of the formula R - OHCONH ₂ C = S and this amino acid amld-thionourethane in the presence a strong acid and cyclized in an inert, polar, organic solvent. 8, a process for the production of an amino acid-N-thiooarboxy-anhydrideSf characterized »that one uses an amino acid a xanthate diester to form an amino acid thionourethano, then this amino acid thionourethane with ammonia and a mild dehydrating agent Formation of an amino acid amld-thlonourethane converts and this - ¹⁸ - 109884/1810 ORIGINAL Amino acid amide thionourethane in the presence of a strong acid and cyclized in an inert, x> oleren, organic solvent. 9. A method for producing an amino acid thiocarboxylic acid anhydride the formula 0
R-CH- <T Bra - in which R denotes the remainder of an amino acid, characterized in that, that you can get an amino acid formula R - CHCOOH S with an Aentbatdiestar the? Orroel RO-C = SR, in which each 'R ¹ means low-Vyl or AralVT-1 with up to 8 carbon atomsj with formation of an Amiirashure-thionourethane of the? Orme? _ R - CHCOOH ! ^S OR ¹ 109804/1 ^ 11334 Φ0 implemented, then this amino acid tiiionourethBn with Ammonia and a mild dehydrating agent implemented and this amino acid urothlonourethane in the presence of a strong acid and in an inert, polar, organic solvent oyoli eiort. 10. Aadnosäureamid-thionourethane of the formula HCBCOlIH ₂ in which R is the bed of an amino acid and R is lower alkyl or Äralky.T with up to 8 carbon atoms. 11 · Compound according to claim 10, characterized in »that R Hydrogen, lower alkyl, halo-aralkyl, lower aminoalkyl, Lower oxyalkyl, lower hydroxyalkyl, lower aptoalkyl, Lower- (niedri & nlkyliaercaptο) -elkyl, lower guanidylalkyl, Liiedrigindclylal> yl or 'iicdrigimldasolylalkyl means. 12. Connection according to claim 10, characterized in that H Means hydrogen. 13 · Compound according to claim 10, characterized in that R Means isobutyl. - t ° - 109884/1810 U. A compound according to claim 10, characterized in that dees E Benzyl means 15. A compound according to claim 10, characterized in ',; -. daa «H means p-hydroxybenisyl. 16. A compound according to claim 10, characterized in that K Isopropyl means - 21 - 109884/1810