JPS5848524B2 - Method for removing thiol protecting group - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION In general, in organic synthesis reactions, it is often necessary to protect and eliminate thiol groups, and protection and elimination are particularly important in peptide synthesis reactions.

The most widely used methods today for protecting a thiol group and removing the protecting group include, for example, protecting a thiol group with a benzyl group and then removing it with sodium metal in liquid ammonia, or protecting a thiol group with a p-methoxybenzyl group. The thiol group can be removed by boiling trifluoroacetic acid or anhydrous hydrogen fluoride, but the conditions for removing the protecting group are quite harsh, so the thiol group is removed while other functional groups and protecting groups remain. In some cases, it was not possible to selectively remove just the protecting group.

On the other hand, since t-butyl groups and adamantyl groups can be introduced into thiol groups very easily, various attempts have been made to use them as protective groups for thiol groups.
Since its removal is extremely difficult, no effective method for removing the protecting group is known yet.

In view of the above circumstances, the present inventors researched a method for protecting and removing thiol groups protected with p-methoxybenzyl, adamantyl or t-butyl groups, and found that these protecting groups can be removed under very mild conditions. In other words, we obtained an unexpected new finding that when treated with a mercury salt of carboxylic acid, it can be removed very easily and in good yield.

The present invention was completed based on this new knowledge, and p-
This method for removing the above-mentioned protecting group is characterized by treating a compound having a thiol group protected with a methoxybenzyl, adamantyl or t-butyl group with a mercury salt of a carboxylic acid.

In the method of the invention, a compound having a thiol group protected with a p-methoxybenzyl, adamantyl or t-butyl group is treated with a mercury salt of a carboxylic acid.

Examples of compounds having a thiol group include amino acids (e.g. cysteine, homocysteeine), peptides (
Examples include glutathione) and other organic compounds (eg cysteamine).

The protecting group removal method of the present invention can be advantageously applied to the synthesis of compounds having disulfide bonds (eg, cystine, lipoic acid, oxytocin, passopressin, somatostatin, insulin, calcitonin, trypsin, inhibitors, ribonuclease, lysozyme), and the like.

Examples of mercury salts of carboxylic acids that can be used in the method of the present invention include formic acid, acetic acid, propionic acid, plastic acid, pentanoic acid, dichloroacetic acid, trichloroacetic acid, trifluoroacetic acid, pentafluoropropionic acid, and heptafluoroplastic acid. Examples include mercury salts of carboxylic acids having 1 to 5 carbon atoms, and mercury salts of acetic acid and trifluoroacetic acid are particularly preferred.

These mercury salts are, for example, 1
~4 equivalents may be used.

In the method of the present invention, instead of directly adding such mercury salt itself to the reaction system, a raw material that can contain such mercury salt in the reaction system may be added to the reaction system.

For example, mercury oxide may be dissolved in the acid described above to form a mercury salt of the acid, which may be used in the present method.

The method of the present invention can usually be carried out in the presence of a solvent.

Examples of the solvent include carboxylic acids having 1 to 5 carbon atoms (e.g., formic acid, acetic acid, propionic acid, butyric acid,
Pentanoic acid, dichloroacetic acid, trichloroacetic acid, trifluoroacetic acid, pentafluoropropionic acid, heptafluoropropionic acid), or a mixture of these and water may also be used, and other water, alcohols (e.g., methanol, ethanol, propanol,
butanol, or a mixture of these with water), tetrahydrofuran, dioxane, dimethylformamide, chloroform, acetonitrile, or a mixture of these with water, etc., which dissolves the target product as easily as possible and is suitable for the desired reaction. It is preferable to use

The process of the invention may be carried out at any suitable temperature, for example in the range of about -20<0>C to 100<0>C, preferably about 00<0>C to about 80<0>C.

In the method of the present invention, the treatment time varies depending on the type and amount of the thiol compound, solvent, and mercury salt, but for example, about 30 seconds to 12 hours is often sufficient.

By the method of the present invention as described above, protective groups such as p-methoxybenzyl, adamantyl, or t-butyl groups of thiol groups can be removed, but the thiol compound may be a mercury thiolate, and it can be freed by a conventional method. of thiol compounds can be obtained.

For example, mercapto thiolate can be prepared as it is or precipitated, the solvent is distilled off under reduced pressure, it is dissolved or suspended in water or a water-containing solvent, or a solvent (e.g., formic acid, acetic acid, dimethylformamide, tetrahydrofuran), and mercapto
Treatment with hydrogen sulfide, mercaptoethanol, thioglycolic acid, sodium sulfide, ammonium sulfide) can lead to free thiols.

The method of the present invention has the following advantages.

(1) Protecting group removal conditions are very mild.

(2) Protecting groups can be removed with good yield.

(3) Operation and post-processing are simple.

(4) The protecting group for the thiol group can be selectively removed while leaving other functional groups and protecting groups intact.

Next, an example of a method for producing a thiol compound having a protecting group will be given as a reference example, and then an example of a method for removing the protecting group will be given as an example.

In this specification, when using abbreviations regarding amino acids, peptides, protecting groups, etc., they shall follow the IUPACIUB regulations or the abbreviations commonly used in the field.

For amino acids, if the L-form is possible, the L-form is indicated unless it is explicitly stated that the D-form is present.

Reference example 1 Adm? -Adamantyl-L-cysteine (H-Cys10H
) Production of 12.1 g of L-cysteine is dissolved in 120,771 l of trifluoroacetic acid, 15.22 g of I-adamantanol is added, and the mixture is stirred at room temperature for 12 hours.

Trifluoroacetic acid was distilled off under reduced pressure, and the remaining oil was diluted with water.
Dissolve in 0011L and cool on ice.

When the pH is adjusted to 6.0 with concentrated ammonia water, crystals precipitate.

This is collected by filtration, washed with water, ethanol, and ether, and then recrystallized from water.

Yield 23. Of, melting point 227. 0-2 2 8. 0
°C (decomposition), (b) -145 °C = 0.51, glacial acetic acid).

Elemental analysis Calculated value as C13H202N5: C,
60.90;H, 8.65;N, 5.4 6
;S, 12.51.

Analysis value: C, 5 9.5 1; H, 8.1 6
;N, 5.41;S1 12.33.

Reference example 2 tBu S-t-butyl-L-cysteine (H-CysOH)
It was produced from L-cysteine and t-butanol in the same manner as in the production reference example.

Melting point: 244.0-245.0°C, (2) Temperature: -15.4°
(C=0.57, glacial acetic acid) Elemental analysis C7H1502N
Calculated value as S: C, 45.13; H, 8.65; N
7.5 2; S, 1 7.22 Analysis value: C, 45
．． 1 1; H18.32; N, 7.47; S, ■7
．． 77.

Reference Example 3 t-7"}xycarbonyl-S-t-7"Thyl-L-cysteine dicyclohexylamine salt H-Cys-OH obtained in Reference Example 2 was converted to t-butyl-S-4,6-dimethylpyrimidine by a conventional method. t-Butoxycarbonylation with -2-ylthiocarbonate, which was crystallized as a salt of dicyclohexylamine.

Yield 90%, melting point 181.0-182.5°C, (b) f
i5+7. 4° (C=0.83, methanol).

Elemental analysis C Calculated value as 24H4604N2s: C
, 62.93; H, 10.10; N16.10;
S, 6.99 Analysis value: C, 62.92; H, 10.3
6; N, 5.95; S, 7.0 5.

t-7''}xycarbonyl-S-7 damantyl L-cysteine dicyclohexylamine salt?Boa-Cys
-OH-DCHA 9.29 and HONB 3.61 prepared in dioxane using dicyclohexylcarbodiimide 4.11 as a condensing agent) were added and stirred at room temperature for 12 hours.

Chloroform was distilled off under reduced pressure and the remaining oil was diluted with acetic acid.
Dissolve to ~150 ml, wash with 4% sodium hydrogen carbonate and saturated citric acid water, and then with water, and then ethyl acetate is distilled off.

When water is added, it forms crystals and is filtered out.

Yield 5.9'f! , melting point 7 3.0 -7 5.0°C,
P 17.9° (C=0.56, dimethylformamide).

Elemental analysis C Calculated value 2C as 15H2805N2S
, 5 1.70; H18.10; N18.04;
S, 9, 20 Analysis value: C, 5 1.7 0; H,
8.1 2 ; N, 8.0 5 ; S19.1 4
.

Boc-Cys-Gly-OMe5. 2 3?
was dissolved in 30 rnl of trifluoroacetic acid and diluted at room temperature for 15 min.
Stir for a minute.

Trifluoroacetic acid is distilled off under reduced pressure and dried.

Dissolve the residue in 30 TIL of dimethylformamide,
After cooling on ice, add triethylamine 7 and OTLl, and further add Z -G 1y -ONB 5. 8 Add P to 48
Stir the time.

Dimethylformamide was distilled off under reduced pressure, and the residue was dissolved in ethyl acetate, washed with 4% sodium bicarbonate, IN-hydrochloric acid and water, and dried under reduced pressure.

If petroleum ether is added to the residue and left to stand, crystals will precipitate and should be collected by filtration.

Yield 6. IP and melting point cannot be measured because it dissolves at room temperature.

@26-1 1.4° (C=0.46, dimethylformamide).

Elemental analysis Calculated value as C 20H290 6N3S:
C, 5 4.6 5; H16.6 5; N, 9.
5 6; S, 7. 3 0 Analysis value C, 5 5
．． 1 1 ; H, 6.6 0 ; N, 9.48; S,
6,55.

PMB Z-Gly-Cys-Gly-OMe was also produced in the same manner as above.

Melting point 90.0-95.0°C, @27-23.1° (C=
0.55, dimethylformamide) elemental analysis C24H
Calculated value as 2907N3S: C, 5 7.2 0;
H, 5.8 0; N, 8.3 4; S, 6.
37 Analysis value: C15 7.6 0; H, 5.8
1; N, 823; S, 6.36.

Drop *Example I Recovery of cysteine from S-protected cysteine.

(1) Dissolve S-protected cysteine (1mM) in 10ml of 1-refluoroacetic acid, add 0.2ml of anisono, cool to 0°C, and add 319my(1mM) of Hg(CH3COO)2 to it. and stir at 0°C for 15 minutes.

The 1 fluoroacetic acid was distilled off under reduced pressure for 5 minutes, and the residue was
Add rrll A and wash once with ether.

Blow H2S gas into this liquid for 30 minutes, and the resulting HgS
4. Repeat the procedure of filtering and concentrating the filtrate three times, and analyze the residue using an amino acid analyzer.

The results are summarized in 4.

Under these conditions, the S-benzyl group is not cleaved at all.

Further, even if the above reaction is carried out using Ag, Cu, Zn, Ni, or Pb acetate instead of Hg (CHsCOO)2, no cleavage of the protecting group occurs.

(2) S-protected cysteine (imM-) to 101rLl
of 80% formic acid or 80% acetic acid, 0. 2 T
Add anisole of Ll** and Hg(CH3COO)23 at room temperature.
Add 19my (1mM) and stir for 1 hour.

The results of amino acid analysis performed simultaneously with (1) are shown in the table.

(3) Dissolve S-protected cysteine (1mM) in 20rnl of 80% acetic acid, add 0.2ml of anisole, and add H
g (CF3COO)2512■ (1.2mM) and stirred at room temperature for 1 hour.

What are the results of amino acid analysis using the same procedure as in (1)? 1 y- Cys -G ly -OMe 3.
0 2? was dissolved in 45 ml of cold trifluoroacetic acid,
Add 1.5 nl of anisole and cool Hg (CH3C
OO ) 21. Add 9 1 9 and stir for 15 minutes.

Trifluoroacetic acid is distilled off under reduced pressure, ether is added, and the resulting precipitate is collected by filtration.

Yield 4. I P elemental analysis C raH2oC)sN3
Calculated value as sF3Hg: C, 31,06; H, 2.9
0; N, 6.04; S14.6 1; Hg, 28
．． 8 2; F18.1 9o analysis value 2C, 3 1.
02; H; 2.87; N, 6, 18; S,
4.6 0 :Hg 128.68 :F18.1 8
.

The above mercaptide 2.0'fI was dissolved in 307d dimethylformamide, H2S gas was passed through it for 1 hour and 20 minutes, the resulting HgS was filtered off through Celite, and the filtrate was dried under reduced pressure.

Since crystals remain, add water, filter, and recrystallize from methanol and water.

Yield 1.1f (100%), melting point 130-132℃, ■
Back -5.7° (C=0.57, dimethylformamide)
.

Elemental analysis Calculated value as C16H206N3S: C, 5
0.1 2 ; H, 5.5 2 ; Nl O. 9
6; S, 8, 36.

Analysis value: C, 49.61; H, 5.18; N, 1
0.97; S18.44o Elemental analysis Calculated value: C, 50.12; H, 5.52;
N, 1 0.9 6; S, 8.3 6: Analysis value: C, 50.13; H, 5.6 1; N, 1 0
．． 77; S, 8.27.

PMB 3) 80% of Z-Gly-Cys-cly-OMe
The yield of Z-GlyCys-Gly-OMe crystals by treatment with Hg(CF3COO)2 in acetic acid is shown in a table below.

PMB 1 Z -G ly-Cys -G ly -OMe 1
Hg(CF3C) was dissolved in 20rnl of 80% acetic acid.
OO) 2512mg (1.2mM) or 8
Add 537%/(2.0 mM) and incubate at room temperature.
Stir for 0 minutes.

Add 40 ml of 80% acetic acid to the reaction solution and add 20 ml of H2S gas.
HgS was filtered off, and the filtrate was dried under reduced pressure.

Water was added to the residue, and the resulting crystals were collected by filtration and recrystallized from ethyl acetate.

This reaction was carried out using Hg (
CC 1 3COO )21. When carried out with 2 equivalents, the yield was 74.9%.

Example 3 Production of bis-t-7"toxycarbonyl-L-cystine PMB BOC -Cys -OH 3.4 1? to 80
Dissolve in 200 TIL of methanol containing % Hg (CF
Add 25.12f (3COO) and stir at room temperature for 2 hours.

100 ml of 80% aqueous methanol is added to the reaction solution, and H2S gas is blown in for 30 minutes.

The generated H2S is filtered off, and the filtrate is concentrated under reduced pressure to about half its volume.

Add 421 sodium carbonate to this and further concentrate under reduced pressure.

After methanol was distilled off, 100,771 liters of water was added,
Oxidation is carried out by blowing air for 40 hours.

Acidify by adding citric acid and extract the resulting oil twice with 150 mlJ of ethyl acetate.

Ethyl acetate was distilled off under reduced pressure, and petroleum ether was added to the residue to form crystals, which were collected by filtration.

Recrystallize from ethyl acetate.

Yield 1.85P, melting point 1 4 9.0-1 5 0.0
°C (decomposition), CA',0-1 3 6.4° (C=1
．． 84, methanol) Elemental analysis Calculated value as C16H2808N2S2:
C, 43.62; H, 6.48; N16.36
;S, 14.55 Analysis value: C, 43.72;H, 6
．． 34; N, 6.24; S1 14.25.

Example 4 Production of oxytocin (1) Boc-Cys(PMB)-Tyr-I1e
-Gln-Asn-Cys (PMB) -P r
Production of o-Leu-Gly-resin Boc-101y-resin (Gly content 2.2 mmol
) using Shimadzu's automatic peptide synthesizer APS-800
(1) Dichloromethane washing (2 minutes x 3 times) (2) 50%
Trifluoroacetic acid monodichloromethane (10 minutes x 2 times) (3) Dichloromethane washing (2 minutes x 3 times) (4) Ethanol washing (2 minutes x 3 times) (5) Chloroform washing (2 minutes x 3 times) (6 ) 10% triethylamine-chloroform (2 minutes and 10 minutes, once each) (7) Chloroform washing (2 minutes x 3 times) (8) BOC-amino acid symmetric anhydride 6.6 mmol (60 minutes) or BOC −
Amino acid p-nitrophenyl ester 6.6 mmol (
12 hours, BOC-Asn, Boc-G In,
Boc-Tyr) to perform the condensation reaction.

(9) Dichloromethane washing (2 minutes x 3 times) (10) Acetylation of unreacted amino groups (4.5% acetic anhydride monodichloromethane) (1 liter) Dichloromethane washing (2 minutes x 3 times) Repeat the above steps in sequence .

After completion of all reactions, wash with acetic acid, dimethylformamide, and methanol, and dry.

Yield 1 3. 1 5? (2) Boc-Cys(
PMB) -Tyr-I1e-GinAsn-Cys
(PMB) 1Pro-Leu-Gly-NH2
Resin 12.1' of Preparation (1) was suspended in 70 TL of methanol containing 15.5% ammonia and stirred at room temperature for 46 hours.

Filter off the resin and add 20ml of dimethylformamide. Wash twice, combine the filtrate and washing liquid, and dry under reduced pressure.

Add 5 Qml of ether, filter the powder, and dry.

2.86SF. This is heated methanol, aqueous ethanol,
Purify by repeating reprecipitation with dimethylformamide and ethanol.

Yield: 1.01 This powder was further mixed with 2.8×4. Develop on OcrfL silica gel column (developing solvent: chloroform: methanol: water: pyridine: acetic acid, 1085-150-225:
63:84).

Collect fractions of the target substance and dry them.

Yield 768my, 21-39.1° (C=0.47, dimethylformamide) Elemental analysis C66H98016N
Calculated value as 12S2CH3COOH, H20: C, 5
5.52; H, 6.92; N, 1 1.78; S1
4.49, actual value: C155.24; H, 7,00;
N, 1 1.92; S, 443 (3) 125 rny of powder obtained in oxytocin production (2) and 0.2 anisole
21rLl was dissolved in 1m7 of trifluoroacetic acid, to which was added 127my of Hg (CH3COO)2 and left at room temperature for 30 minutes.

Add 30 TL of ether and collect the resulting precipitate by filtration (1
73rn9).

Dissolve this in 50% acetic acid water IA', add 0.31 rrLl of 2-mercaptoethanol, and filter off the precipitate.

The filtrate is dried under reduced pressure.

Dissolve this in 1 ml of 50% acetic acid water and use Sephadex G.
-15 column (2.2 x 92.5 CIIL) and elute with 50% acetic acid.

149-18877I. 1 of the eluted fractions are collected and lyophilized (85 ■).

This was dissolved in 150 ml of 0.1% acetic acid water, and adjusted to pH 7. with N ammonia water. Blow air as 3, 4.5
After an hour, adjust the pH to 3.5 and freeze-dry (76
).

Expand this to the same power ram as above and 154-188TL
1 portions are collected and lyophilized to yield 50 μl of oxytocin.

@Sweet -24.6° (C=0.47, N monoacetic acid water) Amino acid analysis Asp 1.03, Glu 1.13
, Pro 1.06, Guy 1.00, Cys
1.97, I1e 1. 0 3, Leu 1.
03, Tyr O. 90.

Thin layer chromatography: Rf (n-butanol: acetic acid:
Water, 3:1:1)-0.44Rf (n-butanol diacetate:acetic acid:water, BOC゜Ala゜G1
y゜CyS-LyS-ASn-Phe・Boc
PMBPhe-Trp-L
ys-Thr-Phe-Thr-Ser-Cys・Ot
Bu220rrigt(0.1mM
) in 20 rul of acetic acid and water (8:2) mixture, and
g(CF,COO)2171■(0.4mM
) and stir at room temperature for 12 hours.

Hydrogen sulfide gas is blown into this reaction solution for 1 hour.

The resulting black precipitate is separated using Celite, and the P solution is concentrated under reduced pressure.

The residue is collected as a powder by adding ether (containing a small amount of β-mercaptoethanol) and dried.

Yield 193■.

Dissolve this product in 500ml of dimethylformamide,
0% triethylamine-dimethylformua ※※Mito-0
．． Add 28 TILe, then add 30 my of 1,2-jodoethane, and stir for 30 minutes at room temperature.

The solvent is removed in vacuo and the residue is triturated with ether, drained and dried.

Yield 158■. Add this to trifluoroacetic acid and water (9:1)
Dissolve in 3771 liters of the mixed solution and stir at room temperature for 30 minutes.

Trifluoroacetic acid is distilled off under reduced pressure at 30° C. or below for 10 minutes.

Add ether and collect the resulting precipitate. This is dissolved in a small amount of water and passed through an Amberlite IRA410 (acetic acid type) column (2.0 x 7.0 cm) to perform ion exchange.

Collect 60rnl of the eluate and transfer it directly to Sephadex L.
Pour into a column of H-20 (5.5 x 3 5.0α).

It is developed using 0.1 N acetic acid as a developing solvent, and the main eluted fraction (560 ml/64,0771 l) is collected and freeze-dried.

Yield: 20.4 Hirodo: 31.2° (C=0.13,
1% acetic acid) Thin layer chromatography: Rf = 0.6
0 (n-butanol:pyridine:acetic acid:water-30:
20:6:24, Avicel) Rf=0.40 (n-butanol:ethyl acetate:
water = 121:1:1, silica gel), amino acid ratio of acid decomposition product (6N hydrochloric acid, 110°C, 24 hours) Lys, 1.9 7(2); Asp, 1.0
1 (1): Thr, 1.9 5 (2): Set
, 0.92(1), Guy, 1.0 0(1) ;Al
a.1. 0 2 (1); Half Gys, 1.
5 8 (2): Phe, 3,0 1 (3), average recovery rate 82% Somatostatin can also be synthesized by the route shown in Table 2 using compound (191).

Claims (1)

[Claims] 1. A method for removing the above-mentioned protecting group, which comprises treating a compound having a thiol group protected with a p-methoxybenzyl, adamantyl or t-butyl group with a mercury salt of a carboxylic acid.