DD151304A1 - PROCESS FOR THE PREPARATION OF TYROSINE DERIVATIVES - Google Patents

Abstract

The invention relates to a process for the preparation of sulfuric acid esters of tyrosine, its derivatives and tyrosine-containing peptides. The preparation of the O-sulfate ester of the compounds mentioned by means of a mixed anhydride from a carboxylic acid and sulfuric acid in an organic solvent. The process allows to convert both amino- and carboxyl-free tyrosine derivatives, N-protected activated esters of tyrosine, and tyrosine-containing peptides of different chain lengths to sulfate esters. The products can be obtained after conversion to corresponding alkali metal salts in good yields and in high purity.

Description

22,1677 rl-

Process for the preparation of sulfuric acid esters of tyrosine, its derivatives and tyrosine-containing peptides

'Field of the invention

The invention relates to a process for the preparation of sulfuric acid esters of tyr'osine and its derivatives, which can be used according to methods known in peptide chemistry for the preparation of O-sulfated tyrosyl peptides, in particular for the production of O "sulfate esters of gastrointestinal hormones, and of tyrosine-containing peptides.

Characteristic of the known, technical solutions

The gastrointestinal hormones of the gastrin family gastrin II, cholecystokinin pancreozymin (CCK-PZ) and caerolein have a sulfated tyrosine residue in the C-terminal sequence

From the literature it is known that so far in the peptide chemistry for the preparation of tyrosine ~ 0-sulfuric acid "star two SuI-.

fatation reactions find application:

1. the reaction with concentrated sulfuric acid

2. the reaction with adducts of sulfur trioxide and tertiary amines.

Concentrated sulfuric acid reactions lead to low levels of tyrosine (DOGSON, Biochem. J. 71, 10 (1959)) and of tyrosine-containing peptides (DOS 1922185 and J. Amer. Chem. Soc., 9, 195 (197O)) Yields of corresponding sulfuric acid esters. Moreover, in this reaction, substantial nuclear sulfonation occurs on the tyrosine aromatic ring. Better yields can be achieved in the sulfation reaction with concentrated sulfuric acid, if according to HOI

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BERG and MlTOIA dicyclohexylcarbodiiniide, which probably forms a mixed anhydride sulfating reagent with the sulfuric acid (J.Amer, formerly Soc., 9JL, 1273 (1969)). However, due to the facile activation of carboxyl groups of the amino acids and peptides, this method can only be applied to carboxyl-protected amino acids and peptides or cyclopeptides (WIELATTD et al., USA Pat., 3,793,304). "Most commonly used for the 0-sulfation of tyrosyl peptides are adducts of tertiary amines with sulfur trioxide. Thus carbobenzoxy-tyrosine can be almost quantitatively converted without Kernsulfonierung in the T-yrosin-O-schwefelsäureester (WÜNSCH, Hoppe-Seyler's Z.Physiol.Chem ^f. ^ ~ SO 60 with the adduct of pyridine in organic solvents, 787 ( 1979)). The suicide

tion of tyrosine-containing peptides with these reagents leads to different yields, on the corresponding tyrosine-0-sulfuric acid esters, such as protected sequences of the CCK-PZ (ONDETTI et al., J. Amer et al., Soc (1970), PEME and co-workers DDR Pat 129782, NIEDRICH et al., DDR Pat., 128973), the gastrin (MORLEY, Proc, 9 Europ. Peptide Symp., Orsay, North-Holland Publ.Comp. Amsterdam 1968, p. 251) and of the caerulein (BERNARDI et al., Experientia 2%, 699 (1967)). ,

While sulfation with concentrated sulfuric acid must be regarded as unsatisfactory according to the literature, sufficient yields of tyrosine O-sulfate esters can be achieved with the SO 2 / tert-amine method, but only when very large excesses are applied to the respective sulfate reagent. For this reason, all other functional groups including the amino function and the terminal carboxyl group must be blocked by protective groups during the sulfation reaction. The subsequent, usually acidolytic deblocking of the peptides then leads in most cases to partial desulfation. Another disadvantage is the large quantities of inorganic salts resulting from the decomposition of the sulfation complex in an aqueous-alkaline solution, which permit only a loss-rich isolation of the sulfate esters

 It is also known that mixed anhydrides from carboxylic acids

.and sulfuric acid alcoholic Hydroxylfunittionen partially

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acylate and partially sulfated, the mixed anhydrides of the lower carboxylic acids with sulfuric acid but preferably sulfating them (van PESIiI, Recueil Trsv.chim Pays-Bas 40, 103 (1921)).

Object of the invention. ". '

The aim of the invention is the development of a process for the preparation of sulfuric acid esters of tyrosine and its derivatives, which are suitable for use as coupling components in 'peptide synthesis, and of tyrosine-containing peptides.

Explanation of the essence of the invention

The invention has for its object to develop a method that allows only a small excess of reagents, the selective sulfation of OH-free tyrosine derivatives and tyrosine-containing peptides, the given after sulfation given if a4s carboxyl or amiriokomponenten in the peptide synthesis v / can ground. In this way, while avoiding the disadvantages of known processes, the introduction of the sulfate ester group onto the tyrosine can take place in a targeted manner and, if appropriate, the incorporation into the desired peptide via peptide-synthetic knotting steps can be carried out clearly with high purity and yield.

It has now been found that mixed anhydrides of carboxylic acids and sulfuric acid exclusively sulphate phenolic hydroxyl groups. As the carboxylic acid can be advantageously used acetic acid. The object of the present work is thus a new process for the preparation of tyrosine O-sulfuric acid esters of general formula I.

R ₁ -TnH-CHR-CoI -NH-CH-CO-Fm-CHR-CoI-R

2 2 16 7 7 ⁴

In the R, the same or different known, optionally protected amino acid side chains

R ^ is a hydrogen atom or an amino protecting group known in peptide chemistry

R _{2 is} an OH group, an alkoxy group, an amino group. or a residue of an active ester component known in peptide chemistry

X is a metal ion, suitably an alkali metal ion, a cation of an organic base or a hydrogen ion

η and m are zero or arbitrary integers

which is characterized by reacting tyrosine, tyrosine derivatives or tyrosine-containing peptides with suitable salts of mixed anhydrides of carboxylic acids and sulfuric acid, preferably with the pyridinium salt of acetylsulfuric acid, which can be easily prepared from acetic anhydride, sulfuric acid and pyridine, at room temperature. It has surprisingly been found that with equimolar addition of reagent according to the invention, the sulfation reaction is quantitative. It proved to be advantageous to work up the resulting sulfate esters as alkali metal salts. Preferably, it was neutralized with potassium hydroxide or potassium bicarbonate. Purification after the sulfation reaction, two variants were developed, generally with 10 % excess of the corresponding base was worked:

Variant A: Addition of the calculated amount of alkali metal hydroxide in ethanol (E) or in water (W) with ice cooling

Variant B: Addition of the calculated amount of alkali metal bicarbonate to the reaction mixture with ice cooling and subsequent dropwise addition of water.

In both work-up variants, the reaction mixtures were then generally concentrated and the products were extracted with a suitable organic solvent. The results obtained are shown in Tables 1 and 2:

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Table 1: Sulfation of tyrosine and its derivatives with the pyridinium salt of acetyl sulfuric acid

tyrosine solution ' molar " Time On ar at · » yield medium Reagent/ (H) tungsva- umkri3t. substratum riante Prod. (%) _T Ac-Tyr-OH DCHA pyridine 1.7 24 A- (E) " ₇₂ b) Z-Tyr-OH DMP 10 A (E) ₇₅ a) Z ~ Tyr-OH CHCl ₃ 2 4 A (E) 80 ^a) Boc-Tyr-OSu EE 1.3 6 A (E) 86 ^a) H-Tyr-OMe HCl CHCl ₃ 1.1 4 A (W) 85 ^C) H-Tyr-OH DMP 2 f ^J 24 B ₇₉ a)

a) monoclalum salt; b) dipotassium salt; c) betaine

Table 2: Sulfation of tyrosine-containing peptides with the pyridinium salt of acetylsulfuric acid

Tyrosylpeptid

Boc-Tyr GIy-OH

Solution Molverh. Time recovery yield of reagent / (h) tan (%) substrate variant

Boc AlaTyr-

-Gy-OEt.

-Boc-Tyr Gly-Trp -MetAspPhe-NHg

EE 2 .5 * 5 A (E) EE / CHCI ₇ (1: 1) ³ 1 4 A (W) EE / DMP (5: 1) 2 .5 6 B -DMP 2 4 · B

74 81

83

78

a)

a)

a) monopotassium salt; EE = ethyl acetate; DMP = dimethylformamide

Table 1 shows that with the sulfation reaction according to the invention both carboxyl group-free and salt-protected, amino group-free tyrosine derivatives can be sulfated in good yields. Even unprotected tyrosine can be reacted intereesanterweise completely selective to tyrosine O-sulfate. ·

The sulfation of activated tyrosine esters is also surprisingly smooth, as shown by the example of the reaction of the tert -butyloxycarboriyl-tyro.sin-hadroxysuccinimide ester.

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As the results summarized in Table 2 show, sulfate esters of tyrosine-containing peptides can also be prepared in good yields with the sulfation reaction according to the invention without large excesses having to be applied to the sulfation reagent. These examples also show that the protection of certain functional groups, for example the terminal and side-chain carboxyl groups and the indole-M group of the tryptophan, can be dispensed with in the new sulfation method.

Thereafter, as advantages of the method according to the invention result:

1. A high selectivity of the reaction

2. The use of only small excesses of the sulfonation reagent

3. An uncomplicated work-up of the sulfated tyrosine derivatives and tyrosine-containing peptides as stable alkali metal salts

4 »The feasibility of the reaction in various types

Solvents 5 * The sulfatability of amino- or carboxyl-unprotected tyrosine derivatives or tyrosine-containing peptides and of their active esters containing the versatile, direct.

Enable use of sulfated compounds in peptide synthesis.

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Embodiments. ''

EXAMPLE 1 Preparation of Pyridinium Acetyl Sulfate To 80 ml of acetic anhydride is added, while cooling, first 6 ml (0.11 mol) of concentrated sulfuric acid and then, with vigorous stirring, 9 ml (0.11 mol) of pyridine so that the reaction temperature does not exceed .degree , Wake the Pyridinzugabe, is stirred for a further hour at room temperature. Then _(to be added to the reaction mixture to complete precipitation of the product, where I50 ml of ether. The Peststoff is filtered off, washed twice with 100 ml of ether and dried. The pyridinium salt of the Acetylschwefelsäure is highly hygroscopic and decomposes quickly .an the air. In the desiccator, it is Durable over KOH months.

Example 2: General sulfation method for tyrosine, tyrosine derivatives and tyrosine-containing peptides

0.1 mol of the compound to be sulfated are dissolved or suspended in about 100 ml of a suitable, dry, organic solvent. 1 bia 2 equivalents of pyridinium acetylsufate are then added to this reaction mixture at room temperature and, approx. Stirred for 4 hours. The course of the reaction is monitored by thin-layer chromatography. After complete sulfation, the reaction mixture is worked up under the following variants with ice-cooling:

Variant A: addition of the calculated amount of alkali metal hydroxide in ethanol (E) or in water (W)

Variant B: Addition of the calculated amount of alkali metal bicarbonate and subsequent dropwise addition of water

After neutralization, the reaction mixtures are concentrated and the sulfate esters obtained are extracted from the inorganic constituents with a suitable organic solvent. Following this general reaction procedure, the tyrosine O-sulfuric acid esters listed in Tables 1 and 2 were

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manufactured. Information on the solvents used, reagent excesses, reaction times and working-up variants can be found in the tables. The R.sub.i values were determined on Silufol Ferroplates of the company Kavalier (CSSR) in the following eluent systems:

System 1: n-butanol / 3% ammonia (3: 1)

System 2: Chloroform / methanol (2: 1)

System 3: Butanol / glacial acetic acid / water (4: 1: 1)

System 4: Ethanol / Water (7: 3)

Example 2.1.: Z-TyT (SO ₃ K) -OH. .. \ Yield: 75 % d. Th.

Fc 177-18O ⁰ C; [α] £ ° - 0.85 (c = 1, DMF); R _f 0.4 (2) C ₁₇ H ₁₆ NO ₈ SK (433.5)

Ber. C 47.10 H 3.73 N 3.24 S 7.40 K 9.02 Gef. 46.98 3.65 3.17 7.55 9.11

Example 2.2 .: H-TyT (SO ₃ K) -OH Yield: 79 % d. Th.

F. 173-175 ⁰ C; [Ά]! ⁰ 13.2 (c = 1, DMF); R _f 0.64 (4) C H ₁₀ NO ₆ SK (299.3)

Ber. C 36,12 H 3,37 N 4,68 S 10,72 K 13,07 Gef. ' 35.98 3.30 4.57 10.78 1.2,95

Example 2.3 .: H-TyT (SO ₃ H) -OCH ₃ Yield: 85% of theory. Th.

F. 238-241 ° C (dec.); \ Δψ - 9.0 ⁰ C (c = 1, water); R _f 0.35 (D.

0.24 (3) C ₁₀ H ₁₃ NO ₆ S (275.3)

Ber. C 43.63 H 4.77 N 5.09 S 11.65 '.

Gef. 43,54 4,86 5,19 11,54

'.-. i i ki. A ύ C i'l · .. · ti i. /

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Example 2.4. BoC-TyT (So ₃ K) -OSu Yield: 79 % d. Th.

P. 186 ° C; ^ cJg ⁰ - 35.8 ° (c = 1, DMP); R _f 0.35 '(D, 0.4 (2),

0.75 (4) C ₁₈ H ₂₁ N ₂ O ₁₀ SK (496.5)

Ber. C 43.54 H 4.28 Έ 5.65 S 6.46 K 7.88 Vessel 43.98 4.23 5.52 6.51 8.01

Example 2.5 »: BoC-TyT (SO ₃ K) -Gly-OH Yield: 74 % of theory . th

P. 163-167 ⁰ C; [ed C ₁₆ H ₂₁ O ₉ IT ₂ SK (456.5)

Ber. C 42.10 H 4.65 N6, H S 7.03 K 8.57 Gef, 41.95 4.79 6.08 7.13 8.47

Example 2.6 .: ₃₂

Yield: 81 % d. Th.

P. 195-198 ⁰ C jk *] ^{from 20 to} 3.8 ° (c = 1, DMP); R _f 0.67 (2) C ₁₈ H ₂₅ JT ₂ O ₉ SK (484.6)

Ber. C 44.61 H 5.21 N 5.78 S 6.62 K 8.07 Gef. 44.49 5.18 5.67 6.71 8.11

Example 2.7 .: BoC-Ala-TyT (SO ₃ K) -Gy-OC ₂ H ₅ Yield: 83 % of theory . Th.

P. 198 ⁰ C (dec.); ^ "] ²⁰ - 10.2 ° (c = 1, DMP); R _f 0.53 (2) C ₂₁ H ₃₀ N ₃ O ₁₀ SK (555.7)

Ber. C 45.39 H 5.46 N 7.57 S 5.77 K 7.04 Gef. 45.28 5.39 7.48 5.82 7.12

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Example 2.8 .: Boc-TyrCSO 4 -Gly-Trp-Met-Asp-Phe-NHg Yield: 78 % of theory . Th.

P. 255 ⁰ C (Zers.); [o $ f - 20.42 (c = 1, DMF); R _f 0.43 (D.

  0.25 (2)

C ₄₅ H ₅₅ I ₈ O ₁₄ S ₂ K (1035.3)

Ber. C 52.21 H 5.37 N 10.83 S 6.20 K 3.78 Vessel 52.64 5.69 10.91 5.92 3.38

Claims (4)

22 1 6 77 11 Inventor claim 1. A process for the preparation of tyrosine derivatives of the general formula I. R ₄ -F] JH-CHR-COj -NH-CH-CO- [MH-CHR-Co] -R, CH ₂ OSO ₃ X where R is the same or different from the known, optionally protected amino acid side chains R ^ is a hydrogen atom or an amino protecting group known in peptide chemistry Rp represents an OH group, an alkoxy group, an amino group or a residue of an active ester component known in peptide chemistry X is a metal ion, suitably an alkali metal ion, a cation of an organic base or a hydrogen peroxide, and
, η and m are zero or arbitrary integers mean, characterized in that an unsulfated tyrosine derivative according to the general. Formula II -nh-ch-co-Ttth-chr-coI-r, η ι L -i m ί II in which R, R ^, Rp, η and m have the abovementioned meaning, wherein in the case of R, equal to hydrogen corresponding ammonium salts are used, is sulfated with a salt of a mixed anhydride of a carboxylic acid and sulfuric acid and optionally corresponding to the meaning of X is worked up by neutralization or ion exchange. 22 1 6 77 12 2 # procedure after. Point 1 " _f characterized by the fact that salts of acetylsulfuric acid, preferably the salts of tertiary bases, which is prepared by one-pot reaction from acetic anhydride, concentrated sulfuric acid and the tertiary base, are expediently used as the sulfating reagent. 3. The method according to item 1j, characterized in that one uses the SuIfatierungsreagens, preferably the pyridinium salt of acetylsulfuric acid, equimolar or in a slight excess and the resulting sulfate ester after neutralization with a base as salts, preferably as stable alkali metal salts isolated. 4. The method according to item 1, characterized in that a tyrosine derivative of the general formula II, in the R U-terminal starting side chains of glycine, tryptophanes, methionines, aspartic acid and phenylalanines, R ^ Tert'rButyloxycarbonyl, Rp the amino group and n = 0 and m = 5, is reacted with the pyridinium salt of acetylsulfuric acid to give a compound of the general formula I in which R, R 1, R 2, n and m are as defined above and X is potassium.