Organic Syntheses, CV 7, 30
Submitted by H. P. Fahrni, U. Lienhard, and M. Neuenschwander
1.
Checked by David R. Bolin and Gabriel Saucy.
1. Procedure
A.
Benzyloxycarbonyl-L-alanyl-L-cysteine methyl ester. A
round-bottomed, three-necked, 100-mL flask is equipped with a
magnetic stirring bar,
10-mL dropping funnel,
thermometer, and
nitrogen bubbler (Note
1). The apparatus is flushed with dry
nitrogen and then charged with
446.5 mg (0.002 mol) of benzyloxycarbonyl-L-alanine in
10 mL of dry dichloromethane. The mixture is stirred until solution is complete and then cooled to 0°C. Within 20 min a solution of
525.5 mg (0.002 mol) of 1-(4-chlorophenyl)-3-(4'-methyl-1'-piperazinyl)-2-propyn-1-one (Note
2) in
5 mL of dry dichloromethane is added. Stirring is continued for 1 hr at 0°C and for an additional hour at room temperature (
t1). The mixture is cooled again to 0°C and a suspension of
343.3 mg (0.002 mol) of L-cysteine methyl ester hydrochloride is quickly added, followed by a solution of
202.3 mg (0.002 mol) of N-methylmorpholine in
5 mL of dry dichloromethane. While the
nitrogen atmosphere is maintained, the mixture is allowed to warm up and is stirred for 12 hr (
t2) at room temperature. The solvent is removed by rotary evaporation, and the residue is shaken intensively with
30 mL of ethyl acetate and 10 mL of water. The organic layer is extracted two times with
10-mL portions of aqueous 10% citric acid and once with
5 mL of 1 N sodium hydrogen carbonate. The organic phase is dried over
sodium sulfate. The solvent is removed by rotary evaporation to leave
647 mg (
95%) of the crude pale-yellow dipeptide. Recrystallization from
ethyl acetate provides
551 mg (
81%) of colorless crystals of
benzyloxycarbonyl-L-alanyl-L-cysteine methyl ester, mp
115–117°C;
[α]20D − 26.4° (CH
3OH,
c 1.29) (Note
3).
B.
Benzyloxycarbonyl-L-aspartyl-(tert-butyl ester)-L-phenylalanyl-L-valine methyl ester. A round-bottomed, three-necked, 100-mL flask is equipped with a 10-mL dropping funnel, thermometer, magnetic stirring bar, and a nitrogen bubbler. The flask is flushed with dry
nitrogen and then charged with a solution of
941.1 mg (0.002 mol) of benzyloxycarbonyl-L-aspartyl-(tert-butyl ester)-L-phenylalanine (Note
4) in
10 mL of dry dichloromethane. The flask is maintained under a dry
nitrogen atmosphere and cooled to 0°C with an
ice–salt bath. The mixture is stirred and a solution of
525.5 mg (0.002 mol) of 1-(4-chlorophenyl)-3-(4'-methyl-1'-piperazinyl)-2-propyn-1-one (Note
2) in
5 mL of dry dichloromethane is added during a period of 20 min. Stirring is continued for 1 hr at 0°C and for 5 hr at room temperature (
t1). The mixture is again cooled to 0°C and a suspension of
335.3 mg (0.002 mol) of L-valine methyl ester hydrochloride and
202.3 mg (0.002 mol) of N-methylmorpholine in
5 mL of dichloromethane is added. After 30 min the reaction mixture is allowed to warm up and is stirred overnight (18 hr;
t2) at room temperature. The solvent is removed by rotary evaporation and the residue is shaken intensively with
40 mL of ethyl acetate and 10 mL of water. The organic layer is extracted twice with
10-mL portions of aqueous 10% citric acid and once with
5 mL of 1 N sodium hydrogen carbonate.
The organic phase is dried over
sodium sulfate, and solvent is removed by rotary evaporation to leave
1132 mg (
97%) of the crude pale-yellow tripeptide. For further purification the crude product is dissolved in
ethyl acetate, treated with some activated
carbon, and filtered through Celite. Removal of the solvent and crystallization from
ethyl acetate/
ether/
petroleum ether (ca. 2 : 1 : 1) yields
993 mg (
85%) of colorless crystals of
benzyloxycarbonyl-L-aspartyl-(tert-butyl ester)-L-phenylalanyl-L-valine methyl ester; mp
119–120°C (Note
5).
2. Notes
2. This reagent is available from Fluka Chemical Corp.
3. The literature
2 value is
[α]20D − 26.5° (CH
3OH,
c 1.27). The reported
2 mp is
116.5–118°C.
4.
Benzyloxycarbonyl-L-aspartyl-(tert-butyl ester)-L-phenylalanine dicyclohexylamine salt was conveniently prepared by standard procedures.
3 The salt was dissolved in
ethyl acetate and extracted three times with aqueous
10% citric acid, and once with water. The organic phase was dried and solvent was removed to leave the dipeptide as an oil.
5. The product has the following physical properties: Specific rotation:
[α]20D − 36.5° (C
2H
5OH,
c 2); IR (KBr), cm
−1: 3285, 1732, 1691, 1640, 1531, 1367, 1229, 1158, 1050, 746, 701;
1H NMR (100 MHz, CDCl
3) δ: 0.81 (d, 3 H,
J = 7) 0.84 (d, 3 H,
J = 7), 1.41 (s, 9 H), 1.8–2.3 (m, 2 H), 2.62 (d of d, 1 H,
J = 17,
J' = 6), 2.86 (d of d, 1 H,
J = 17,
J' = 5), 3.08 (d, 1 H,
J = 8), 3.71 (s, 3 H), 4.3–4.8 (m, in total 3 H), 5.11 (s, 2 H), 5.78 (d, 1 H,
J = 8), 6.28 (d, 1 H,
J = 8), 7.02 (d, 1 H,
J = 8), 7.21 (s, 5 H), 7.57 (s, 5 H).
3. Discussion
The preparation of
Cbz-L-alanylcysteine methyl ester shows the advantage of using, as the amine component, an amino acid with an unprotected sulfhydryl moiety. No problems were encountered with the use of amino acid derivatives with unprotected hydroxyl or sulfhydryl groups as either the amine
6 or carboxyl component.
8 This procedure is based on the pronounced selective reactivity of the enol ester, which is generated by the addition of carboxylic acids to "push–pull acetylenes." Generally, the yields of peptides are good and a broad variety of solvents (e.g.,
dichloromethane, tetrahydrofuran, acetonitrile, dimethylformamide) may be used, depending on the solubility of the coupling components. It is also possible to change the solvent after the activation step or to isolate the activated components. Normally, however, this is neither necessary nor recommended. Purification of the reaction mixture is simple, since the
piperazine by-product is conveniently extracted with an acidic water phase.
The following peptides and further examples have been prepared
6 by the following procedure.
|
Peptide |
t1a |
t2b |
Yield (%)c |
|
Cbz-L-Ala-Gly-OMe |
2d |
12 |
91 |
Cbz-L-Ala-L-Val-OMe |
2d |
24 |
84 |
Cbz-L-Ala-L-Phe-OMe |
2d |
18 |
88 |
Cbz-Gly-L-Phe-Gly-OEt |
2 |
12 |
90 |
Cbz-L-Asp(O-t-Bu)-L-Phe-L-Val-OMe |
6 |
18 |
85 |
Cbz-L-Ile-L-Ile-OBzl |
18 |
24 |
75 |
Cbz-L-Ala-L-Ser-OMe |
2d |
72 |
90 |
Cbz-L-Ala-L-Tyr-OMe |
2d |
72 |
91 |
Cbz-L-Ala-L-Cys-OMe |
2d |
12 |
81 |
Cbz-L-Ala-L-Met-OMe |
2d |
15 |
85 |
Cbz-L-Ser-Gly-OEt |
2 |
24 |
81 |
|
a t1: time for activation of the carboxylic component (see procedures).
|
b t2: time for coupling (see procedures).
|
c Yield of pure recrystallized product.
|
d Stirring 1 hr at 0°C, then 1 hr at 20°C.
|
During our experiments no side reactions were detected. This is in contrast to peptide synthesis with isoxazolium salts,
9 10 where some side reactions, one leading to a diacyl amino compound, were observed.
11 In most cases, these side reactions are due to a secondary amino group in the reagent which is impossible in the case of push–pull acetylenes.
Compared with ynamines, which have also been applied to peptide synthesis,
12 push–pull acetylenes are much more selective. They do not show the side reactions observed with ynamines,
13 and the yields are not markedly influenced by the sequence of addition of compounds in the activation step or by excess of
acetylene reagent.
A crucial point in peptide synthesis is racemization of the activated amino acid. Three different tests were made to evaluate the degree of racemization. Using the Anderson test peptide
14 Cbz-Gly-Phe-Gly-OEt, no racemization could be detected when the peptide was prepared in
dichloromethane,
acetonitrile, or
tetrahydrofuran. This means racemization is below the detection limit of 1%.
Benzylleucylglycine ethyl ester is used in the very sensitive Young test.
15 In this test, designed to exaggerate racemization, we found 5% of racemate, when the solvent was
dichloromethane. In the more polar solvent,
dimethylformamide, this value rose to 12%. Therefore, racemization is in the same range as that observed for the racemization-resistant azide procedure. The coupling of
Cbz-L-aspartyl(O-t-Bu)-L-phenylalanine with
valine methyl ester is reported to be very sensitive to racemization.
16 The tripeptide was prepared as described above, and the crude product was hydrolyzed. Gas-layer chromatography (GLC) showed the presence of 2-3%
D-phenylalanine. Again, in contrast to the ynamine procedure,
17 racemization seems to be no problem when push–pull acetylenes are used.
So far, the only observable disadvantage of the reagent is the somewhat long reaction time for the coupling of the activated amino acids (or peptides) with the amine component. The increase in reaction time t2 could be a limiting factor, if longer peptide fragments are to be linked.
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