Org. Synth. 1979, 59, 159
DOI: 10.15227/orgsyn.059.0159
REMOVAL OF Nα-BENZYLOXYCARBONYL GROUPS FROM SULFUR-CONTAINING PEPTIDES BY CATALYTIC HYDROGENATION IN LIQUID AMMONIA: O-tert-BUTYL-L-SERYL-S-tert-BUTYL-L-CYSTEINE tert-BUTYL ESTER
Submitted by Arthur M. Felix, Manuel H. Jimenz, and Johannes Meienhofer
1,2.
Checked by László Révész and G. Büchi.
1. Procedure
Caution! All operations described in these procedures must be carried out in a well-ventilated hood, since ammonia is highly toxic, hydrogen is extremely flammable, and palladium black is pyrophoric.
A.
L-Methionine. A dry,
1-l., three-necked, round-bottomed flask is equipped with a dry
ice reflux condenser (Note 1), a
gas-inlet tube, and a
magnetic stirring bar as illustrated in
Figure 1. The reaction vessel is immersed in an
acetone–dry ice bath, and a total of
300 ml. of ammonia (Note 2) is passed through a
drying tower containing
potassium hydroxide pellets and collected in the flask. The bath is removed to permit the reaction to proceed at the boiling point of
ammonia (−33°), and a gentle stream of dry
nitrogen (Note 2) is bubbled into the flask. A solution of
0.708 g. (0.80250 mole) of N-benzyloxycarbonyl-L-methionine (Note 3) in
10 ml. of N,N-dimethylacetamide (Note 4),
1.02 g. (1.40 ml., 0.0101 mole) of triethylamine (Note 5), and
1.25 g. of freshly prepared palladium black (Note 6) are added. The
nitrogen stream is discontinued and replaced by a stream of
hydrogen (Note 2) that has been passed through a concentrated sulfuric acid scrubber. The mixture is stirred under reflux for 5.5 hours to effect hydrogenolysis
(Note 1). The
hydrogen stream is discontinued, a flow of
nitrogen is resumed, and the dry ice is removed from the reflux condenser, permitting rapid evaporation of
ammonia (Note 7). The flask is attached to a
rotary evaporator (Note 8), and the mixture is evaporated to dryness under reduced pressure. The residue is dissolved in water and filtered through a
sintered funnel of medium porosity to remove the catalyst. The filtrate is evaporated to dryness, and the residue (
354 mg.,
95%) is crystallized from water–ethanol. The white crystalline product, after drying under reduced pressure at 25°, weighs
272–305 mg. (
73–82%), m.p.
280–282° (dec.)
(Note 9),
[α]25D +23.1° (
c = 1, aqueous 5
N hydrochloric acid)
(Note 10).
Figure 1.
B.
O-tert-Butyl-L-seryl-S-tert-butyl-L-cysteine tert-butyl ester. A dry, 1-l., three-necked, round-bottomed flask is equipped with a dry ice reflux condenser
(Note 1), a gas-inlet tube, and a magnetic stirring bar, as illustrated in
Figure 1. The reaction vessel is immersed in an acetone–dry
ice bath, and a total of
300 ml. of ammonia (Note 2) is passed through a drying tower containing
potassium hydroxide pellets and then collected in the flask. The bath is removed, and a gentle stream of
nitrogen (Note 2) is bubbled into the flask. A solution of
200 mg. (0.392 mole) of Nα-benzyloxycarbonyl-O-tert-butyl-L-seryl-S-tert-butyl-L-cysteine tert-butyl ester (Note 11) in
4 ml. of N,N-dimethylacetamide (Note 4),
0.160 g. (0.220 ml., 0.00158 mole) of triethylamine (Note 5), and
200 mg. of palladium black freshly prepared from
333 mg. (0.00188 mole) of palladium(II) chloride (Note 6) are added. The
nitrogen stream is discontinued and replaced by a stream of
hydrogen (Note 2) that has been passed through a concentrated sulfuric acid scrubber. The mixture is stirred and hydrogenated at reflux temperature for 6 hours
(Note 1). The
hydrogen stream is discontinued, a stream of
nitrogen is again passed into the flask, and the dry ice is removed from the reflux condenser to permit rapid evaporation of
ammonia (Note 7). The flask is attached to a rotary evaporator
(Note 8) and evaporated to dryness under reduced pressure. The residue is dissolved in
50 ml. of methanol (Note 12), and the suspension is filtered through a 5 × 25-mm. bed of Celite
(Note 13) to remove the catalyst. The Celite bed is washed thoroughly with three
20-ml. portions of methanol. The filtrate is evaporated to dryness, and the residue is recrystallized from
petroleum ether (b.p.
60–90°). The white crystalline product, after drying under reduced pressure at 25°, weighs
121–127 mg. (
82–86%), m.p.
71–73° (Note 14),
[α]25D −5.8° (
c = 1,
methanol)
(Note 15).
2. Notes
1.
The condenser is filled with crushed dry ice (no solvent). More dry ice is added periodically as necessary throughout Parts A and B.
2.
Anhydrous
ammonia, prepurified nitrogen, and prepurified hydrogen were all purchased from Matheson Gas Products.
3.
N-Benzyloxycarbonyl-L-methionine was obtained from Bachem, Inc., 3132 Kashiwa Street, Torrance, California 90505.
4.
Spectrophotometric-grade N,N-dimethylacetamide was purchased from Aldrich Chemical Company, Inc., and stored over molecular sieves.
5.
Sequanal-grade triethylamine, obtained from Pierce Chemical Company, Rockford, Illinois, was distilled under
nitrogen from ninhydrin, which was also purchased from Pierce Chemical Company.
6.
The catalyst is prepared
4,5 with
palladium(II) chloride and 97–100% formic acid, which were purchased from Engelhard Industries Division (Engelhard Minerals and Chemicals Corporation) and MC and B Manufacturing Chemists, respectively. A 10.4-ml. aliquot containing
2.08 g. (0.0117 mole) of palladium(II) chloride from a stock solution of
palladium(II) chloride in
2 N hydrochloric acid (10 g. per 50 ml.) is added to 104 ml. of boiling water in a
600-ml. beaker. A
0.51-g. (0.42 ml., 0.011 mole) portion of formic acid and
33 ml. of aqueous 10% potassium hydroxide are added to the boiling solution. The pH of the resulting slightly alkaline solution (pH ~ 8) is adjusted to 6–7 by adding
formic acid, after which the mixture is allowed to boil for an additional 5 minutes. The catalyst is isolated by careful suction filtration.
Caution! The palladium catalyst is pyrophoric and must always be kept wet with water or methanol to prevent contact with air.To minimize the danger in handling
palladium black, the submitters recommend that filtration, washing, and transfer of the catalyst be performed with a "syringe filter."
6 This device was fashioned from a 10-ml. Plastipak syringe, purchased from Becton-Dickinson and Company, Rutherford, New Jersey, by cutting off the tip at the end of the cylindrical barrel and forcing a tight-fitting, porous disk of polypropylene into its place. The use of this "syringe filter" permits the removal of most of the solvent and the safe transfer of the catalyst to the flask with little danger of ignition or moisture absorption.
The catalyst is washed thoroughly with 100 ml. of water and with 200 ml. of absolute methanol to remove all traces of water, after which it is transferred to the flask under nitrogen with a minimal amount of absolute methanol. To be effective the catalyst must be pyrophoric, and extreme care must be taken during this operation to prevent ignition of the methanol or ammonia. The catalyst must not be allowed to become dry or to collect on the wall of the flask above the surface of the liquid ammonia.
7.
Evaporation of the
ammonia generally requires several hours. Toward the end of the evaporation, it is advantageous to immerse the flask in an acetone bath, taking care to avoid bumping.
8.
N,N-Dimethylacetamide remains in the flask and is removed by rotary evaporation under reduced pressure with a
water bath kept at a temperature lower than 35°. The submitters recommend that the evaporation be carried out directly in the same three-necked flask by stoppering the two side arms and adjusting the angle of the rotary evaporator.
9.
The melting point is corrected [lit.,
7 m.p.
280–281° (dec.)].
10.
The literature
8 reports
[α]25D +23.2° (
c = 1, 5
N hydrochloric acid). The product was analyzed by the submitters. Analysis calculated for C
5H
11NO
2S: C, 40.25; H, 7.43; N, 9.39; S, 21.49. Found: C, 40.14; H, 7.42; N, 9.50; S, 21.52. The product was homogeneous according to TLC on precoated silica gel G plates purchased from Analtech, Inc., Newark, Delaware, and developed with the following two solvent systems (solvents, volume ratios of solvents in the same order):
1-butanol–
acetic acid–
ethyl acetate–water, 1:1:1:1,
Rf 0.49;
1-butanol-
acetic acid-
pyridine-water, 15:3:10:12,
Rf 0.51.
11.
The protected dipetide was prepared by the procedure described in the following paragraph using
N-benzyloxycarbonyl-O-tert-butyl-L-serine purchased from Chemical Dynamics Corporation (P. O. Box 395, South Plainfield, New Jersey 07080),
tetrahydrofuran distilled from
lithium aluminum hydride [
Caution! For a warning regarding this method for purifying tetrahydrofuran, see
Org. Synth., Coll. Vol. 5, 976 (1973)], and
N-methylmorpholine distilled from
ninhydrin.
S-tert-Butyl-L-cysteine tert-butyl ester9 was prepared as follows: To a suspension of
10 g. (0.082 mole) of L-cysteine in 75 ml. of dry dioxane in a
200-ml. pressure bottle cooled in an ice bath are added
10.0 ml. of concentrated sulfuric acid and
56 g. (95 ml., 1.0 mole) of isobutylene. The pressure bottle is stoppered and shaken at room temperature for 18 hours. The mixture is cooled to 0°, the pH is adjusted to 10 by adding
160 ml. of aqueous 2 N sodium hydroxide, and the product is extracted with three
100-ml. portions of diethyl ether. The ethereal solution is washed with three
80-ml. portions of aqueous 5% sodium hydrogen carbonate and three 80-ml. portions of water, dried over anhydrous
magnesium sulfate, and concentrated to a volume of
ca. 200 ml. The concentrate is stirred and cooled at 0° as
90.8 ml. (0.0908 mole) of 1 M hydrogen chloride in ether is added. The resulting mixture is stirred for several minutes, the precipitated hydrochloride is filtered, and the filter cake is washed with
ether. The white crystalline product weighs
18.1 g. (
81%) and is used without further purification. The submitters caution that the hydrochloride sublimes under reduced pressure. Recrystallization from
chloroform–petroleum ether affords an analytical sample, double m.p.
187° and
219–222°,
[α]25D +5.85° (
c = 1,
methanol). Analysis calculated for C
11H
23NO
2S·HCI: C, 48.96; H,8.96; N, 5.19; S, 11.88; Cl, 13.14. Found: C, 49.07; H, 9.25; N, 5.13; S, 12.06; Cl, 13.07. The free base is obtained by dissolving the
hydrochloride in aqueous
10% sodium carbonate, extracting the mixture with
ether, and evaporating the ethereal solution under reduced pressure.
A dry, three-necked, round-bottomed flask is equipped with a mechanical stirrer, a rubber septum, and a 200-ml., pressure-equalizing dropping funnel mounted with a T-shaped gas-inlet connected to both a nitrogen source and a bubbler serving as a gas exit. The flask is charged with a solution of 14.9 g. (0.0506 mole) of Nα-benzyloxycarbonyl-O-tert-butyl-L-serine in 100 ml. of tetrahydrofuran and purged with nitrogen. The solution is stirred and cooled at −15° as 5.12 g. (5.67 ml., 0.0506 mole) of N-methylmorpholine and 6.91 g. (6.61 ml., 0.0506 mole) of isobutyl chloroformate are added rapidly through the septum with syringes. One minute after the addition is completed, a precooled (−20°) solution of 11.8 g. (0.0506 mole) of S-tert-butyl-L-cysteine tert-butyl ester in 100 ml. of tetrahydrofuran is added dropwise at −15°. The contents of the flask are stirred for 1 hour at −15° and for 3 hours at room temperature. The mixture is then evaporated to dryness under reduced pressure and dissolved in 150 ml. of ethyl acetate. The solution is washed with three 50-ml. portions of each of the following: 5% sodium hydrogen carbonate in water, water, 1 M citric acid in water, and water. The ethyl acetate solution is then dried over anhydrous magnesium sulfate, the solvent is evaporated, and the remaining solid is recrystallized from ethyl acetate–petroleum ether (b.p. 35–60°). The white crystalline product, after drying under reduced pressure at 25°, weighs 25.5 g. (98.6%), m.p. 94.5–95°, [α]25D −2.47° (c = 1, methanol). TLC of the product on plates precoated with silica gel G and purchased from Analtech, Inc., Newark, Delaware, each showed a single spot when developed with the following three solvent systems (solvents, volume ratio of solvents in the same order): 1-butanol–acetic acid–ethyl acetate–water, 1:1:1:1, Rf 0.90; 1-butanol–acetic acid–water, 4:1:1, Rf 0.81; 1-butanol–acetic acid–pyridine–water, 15:3:10:12, Rf 0.81. Analysis calculated for C26H42N2O6S: C, 61.15; H, 8.29; N, 5,49; S, 6.28. Found: C, 61.15; H, 8.52; N, 5.42; S, 6.44.
12.
Other solvents including
N,N-dimethylformamide and water may also be used to dissolve the products.
13.
In some cases the catalyst was not entirely removed, and the filtrate contained trace amounts of
palladium. In those instances the submitters evaporated the filtrate to a small volume and repeated the filtration using a large bed of Celite.
14.
The melting point was taken on a
Reichert hot stage microscope. This instrument is available from William J. Hacker and Company, Inc., P.O. Box 646, West Caldwell, New Jersey 07006.
15.
The recrystallized product was analyzed by the submitters. Analysis calculated for C
18H
36N
2O
4S: C, 57.41; H, 9.63; N, 7.43; S, 8.51. Found: C, 57.60; H, 9.66; N, 7.37; S, 8.25. TLC (
(Note 10)) run by the submitters showed a single spot for the product in each of three following solvent systems (solvents, volume ratio of solvents in the same order):
chloroform–
methanol–
acetic acid, 85:10:5,
Rf 1-butanol–
acetic acid–water, 4:1:1,
Rf 0.58;
1-butanol–
acetic acid–
pyridine–water, 15:3:10:12,
Rf 0.71.
3. Discussion
The protection of the amino terminus of a peptide with the benzyloxycarbonyl group combined with protection of the carboxyl terminus and all side-chain functions with
tert-butyl-derived groups
10 enables totally selective liberation of the terminal amino function by catalytic hydrogenolysis. This combination of protecting groups is currently considered "ideal" for peptide synthesis,
11 except for the rather serious limitation that catalyst poisoning has prevented its application to the preparation of peptides that contain
cysteine,
methionine, or other residues bearing divalent sulfur groups. The submitters have recently discovered
12 that catalyst poisoning is greatly diminished when liquid
ammonia is used as solvent for palladium-catalyzed hydrogenation. This solvent enables quantitative cleavage of
Nα-behzyloxycarbonyl groups on many protected peptides bearing
S-protected cysteine residues. The method has been used successfully in syntheses of oxytocin
13 and somatostatin.
3The present procedures illustrate this method with the regeneration of
L-
methionine and the preparation of the
tert-butyl ester of O-tert-butyl-L-seryl-S-tert-butyl-L-cysteine3 from their respective
Nα-benzyloxycarbonyl derivatives. No other procedures for the preparation of this protected dipeptide have been reported. These preparations and other peptide syntheses have served to establish the complete stability of the
S-methyl substituent and various protecting groups including the
tert-butyl ester (OBu
t),
tert-butyl ether (Bu
t),
N-tert-butyloxycarbonyl (Boc),
S-tert-butyl (Bu
t),
S-benzyl (Bzl), and
S-acetamidomethyl (Acm) groups.
14 Table I summarizes the results of hydrogenations carried out with sulfur-containing peptides having chain lengths varying from 6 to 13 amino acid residues, demonstrating the stability of various protecting groups to this procedure for hydrogenolysis. In each case the N
α-benzyloxycarbonyl (Z) group was removed quantitatively and the
tert-butyl based protecting groups were unaffected. As a result of these findings, sulfur-containing amino acids may now be used in peptide synthesis by the "ideal" combination of aminoterminal benzyloxycarbonyl protection with
tert-butyl-type blocking groups on all other functions.
TABLE I
REMOVAL OF Nα-BENZYLOXYCARBONYL GROUPS FROM SULFUR-CONTAINING PEPTIDES
|
|
Peptide a
|
Amount (g.) (mmole.)
|
Dimethylacetamide (ml.)
|
Ammonia (ml.)
|
Triethylamine (mmole.)
|
Palladium(II) Chloride (g.)
|
Yieldb (%)
|
|
|
|
3.05 (2.54)
|
10.0
|
300
|
40
|
2.08
|
98
|
|
|
0.418 (0.272)
|
3.0
|
35
|
4.0
|
0.227
|
81
|
|
|
0.263 (0.160)
|
3.5
|
30
|
4.0
|
0.133
|
91
|
|
|
0.305 (0.170)
|
3.0
|
60
|
4.7
|
0.532
|
100
|
|
|
0.368 (0.182)
|
3.0
|
70
|
4.7
|
0.596
|
89
|
|
|
0.281 (0.129)
|
2.0
|
40
|
10.0
|
0.596
|
86
|
|
|
0.178 (0.080)
|
1.3
|
40
|
10.0
|
0.380
|
85
|
|
a Protecting group abbreviations are as follows: N-benzyloxycarbonyl (Z), N-tert-butyloxycarbonyl (Boc), tert-butyl ether (But), and S-tert-butyl (But).
|
b The reaction time was 5.0–6.0 hours in all entries.
|
Since the use of N,N-dimethylacetamide and triethylamine improved the rate and extent of cleavage of the N-benzyloxycarbonyl group in several difficult cases, these additives have been incorporated into the submitters' standard procedure and are included in the present procedures. Deprotection with this method has been carried out with as much as 25 g. of the protected peptide.
This preparation is referenced from:
Appendix
Chemical Abstracts Nomenclature (Collective Index Number);
(Registry Number)
petroleum ether
palladium black
Nα-benzyloxycarbonyl-O-tert-butyl-L-serine
sulfuric acid (7664-93-9)
hydrogen chloride,
hydrochloric acid,
hydrochloride (7647-01-0)
acetic acid (64-19-7)
ammonia (7664-41-7)
ethyl acetate (141-78-6)
methanol (67-56-1)
ether,
diethyl ether (60-29-7)
hydrogen (1333-74-0)
sodium hydroxide (1310-73-2)
citric acid (77-92-9)
chloroform (67-66-3)
sodium hydrogen carbonate (144-55-8)
sodium carbonate (497-19-8)
formic acid (64-18-6)
nitrogen (7727-37-9)
1-butanol (71-36-3)
pyridine (110-86-1)
potassium hydroxide (1310-58-3)
palladium (7440-05-3)
palladium(II) chloride (7647-10-1)
magnesium sulfate (7487-88-9)
dioxane (123-91-1)
isobutylene (9003-27-4)
Methionine,
L-Methionine (63-68-3)
Tetrahydrofuran (109-99-9)
lithium aluminum hydride (16853-85-3)
N,N-dimethylformamide (68-12-2)
benzyloxycarbonyl (Z)
triethylamine (121-44-8)
S-tert-Butyl (1605-73-8)
N,N-dimethylacetamide,
Dimethylacetamide (127-19-5)
cysteine,
L-cysteine (52-90-4)
ninhydrin (938-24-9)
isobutyl chloroformate (543-27-1)
N-methylmorpholine (109-02-4)
butyloxycarbonyl (Boc), tert-butyl ether
O-tert-Butyl-L-seryl-S-tert-butyl-L-cysteine tert-butyl ester,
tert-butyl ester of O-tert-butyl-L-seryl-S-tert-butyl-L-cysteine (67942-95-4)
N-benzyloxycarbonyl-L-methionine
Nα-benzyloxycarbonyl-O-tert-butyl-L-seryl-S-tert-butyl-L-cysteine tert-butyl ester
N-benzyloxycarbonyl-O-tert-butyl-L-serine
S-tert-Butyl-L-cysteine tert-butyl ester (45157-84-4)
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