4.1 The Isovaleric Acid Mixed Anhydride Method [1]
6 · H3NCH2CO-OC2H5
N1C2H5)3
A solution ofbenzyloxycarbonyl-L-Ieucine (2.65 g, 10 mmol) and triethylamine (1.01 g= 1.4 ml, 10 mmol) in a mixture of toluene (12.5 ml) and chloroform (12.5 ml) is cooled to - 5 °C. Isovaleryl chloride (1.205 g = 1.27 ml, 10 mmol) is added and the mixture allowed to stand at - 5 °C for one and a half hours for the formation of the mixed anhydride. At that time a solution of glycine ethyl ester hydrochloride (1.4 g, 10 mmol) and triethylamine (1.4 ml, 10 mmol) in chloroform (25 ml), cooled to - 5 °C, is added and the reaction mixture is set aside in a refrigerator at about 5- 10 0C. Next day the solution is washed with equal volumes of water, 0.5 M KHC0 3 solution, and water again. It is dried over MgS04 , filtered from the drying agent and concentrated in vacuo to about 15 ml. On dilution with hexane (75 ml) the product separates in crystalline form. It is collected on a filter, washed with hexane and dried in air. The crude dipeptide derivative is dissolved in boiling 95% ethanol on a steam bath and diluted with warm distilled water until turbid. On cooling, crystals separate. After several hours at room temperature the product is collected on a filter, washed with a small volume of 50% ethanol, dried in air and finally in
vacuo over PzOs. The protected dipeptide ester weighs 2.45 g (70%) [2J and melts at 105-106°C; [exJ54 - 25.6° (c 5, ethanol).
1. Vaughan JR, Jr, Osato RL (1951) J Amer Chern Soc 73: 5553 2. The same method used in the coupling of benzyloxycarbonyl-L-proline to L-leucyl-glycine
ethyl ester yielded benzyloxycarbonyl-L-prolyl-L-Ieucyl-glycine ethyl ester with m.p. 145-146 0c. A single recrystallization from aqueous ethanol afforded the protected tripeptide derivative in analytically pure form. (Ressler C, du Vigneaud V (1954) J Amer Chern Soc 76: 3107)
Benzyloxycarbonyl-ex-methylalanine (2.37 g, 10 mmol) and triethylamine (1.01 g= 1.40 ml, 10 mmol) are added to dry toluene (5 ml). The solution is cooled to - 5°C and treated with trimethylacetyl chloride (pivaloyl chloride, 1.21 g = 1.23 ml, 10 mmol). The mixture is stirred at - 5 °C for two hours then at room temperature for one hour. The precipitated triethylammonium chloride is removed by filtration and the filtrate evaporated to dryness in vacuo. The residue, the mixed anhydride (3.17 g, 99%), melts at 81-83 DC; its i.r. spectrum shows carbonyl bands at 1805 and 1736 em - 1, characteristic for anhydrides of carboxylic acids. Satisfactory values are obtained for C, Hand N on elemental analysis.
The anhydride (3.21 g, 10 mmol) and ex-methylalanine methyl ester (1.29 g, 11 mmol) are added to dry toluene (50 ml), the solution is heated at 60°C for three hours and allowed to stand at room temperature overnight. It is diluted
with ethyl acetate (50 ml), washed with 0.5 N KHC0 3 (50 ml), water (50 ml), 0.5 N HCI (50 ml) and water (50 ml), dried over anhydrous Na2S04 and evaporated to dryness in vacuo. Crystallization of the residue from etherhexane yields 2.95 g (88%) of the dipeptide derivative melting at 107-109°C. Recrystallization from the same solvents can raise the m. p. to 109-111 dc.
[QJ N
7N-S02~CH3 (CH2)4
o-CH20-CO-NH-tH-CO-NH-CH2-CO-OC2 HS
C2SH33N307S (5196)
N~-Benzyloxycarbonyl-N'-tosyl-Iysine [3J (4.34 g, 10 mmol) is dissolved in chloroform (20 ml) by the addition of pyridine (0.79 g = 0.80 ml, 10 mmol). The solution is cooled to - 3°C and treated with trimethylacetyl chloride (pivaloyl chloride, 1.21 g = 1.23 ml, 10 mmol). After about 10 minutes at 0 to - 3°C the mixture is cooled to -10°C and glycine ethyl ester [4J (1.03 g, 10 mmol) is added. The reaction mixture is allowed to warm up and is kept at room temperature for about 30 min. The solvent is removed in vacuo, the residue shaken with a mixture of water (20 ml) and ethyl acetate (30 ml), the crystalline product collected on a filter and washed with ethyl acetate (20 ml). The first crop thus obtained weighs 3.85 g. A second crop (0.56 g) is secured by washing the combined filtrates and washes with 0.5 N KHC0 3 (20 ml), water (20 ml), 0.5 N HCI (20 ml), water (20 ml) drying the solution over anhydrous Na2S04 ,
and evaporation in vacuo. The two crops are combined and recrystallized from water-saturated ethyl acetate by dilution with hexane. The purified protected dipeptide ester (4.2 g, 81 %) melts at 155-156 DC; [ex]52 - 5 ° (c 2, chloroform).
1. Leplawy MT, Jones DS, Kenner GW, Sheppard RC (1960) Tetrahedron, 11: 39 2. Zaoral, M (1959) Angew Chern 71: 743; Collect Czechoslov Chern Commun 27: 1273 (1962) 3. Hofmann K, Thompson TA, Yajima H, Schwartz ET, Inouye H (1960) J Am Chern Soc 82:
3715 4. Prepared from glycine ethyl ester hydrochloride. A solution of the latter in water is covered
with ether and cooled with ice water during the addition of a 50% solution of K2C03• The ether extract is dried and the ether is evaporated under moderately reduced pressure (to avoid too heavy losses of the volatile ethyl glycinate).
A one molar solution [3J ofbenzyloxycarbonyl-L-Ieucine in toluene (500 ml) is placed in a 3 liter round bottom flask protected from moisture by a cotton containing drying tube and diluted with chloroform (500 ml). The solution is stirred and neutralized with triethylamine [4J (50.6 g = 70 ml, 500 mmol). The mixture is cooled to about -15°C and ethyl chlorocarbonate (about 97% pure, 50 ml = 56 g, 505 mmol) is added in 3 portions. Within a few minutes a mass of crystals separates. About 20 minutes later a precooled (ca-lO°C) solution of glycine ethyl ester hydrochloride (73 g, 525 mmol) and triethylamine (73 ml = 53 g, 525 mmol) in chloroform (1 liter) is added and stirring is continued at a bath temperature of -10 to O°c. When all the solid material dissolves the mixture is heated to 50°C (C0 2 evolution) for about half an hour and then cooled to room temperature. The solution is washed with N HCI, H 20, 0.5 M KHC0 3 , again with H 20, dried over MgS04 , filtered and evaporated in vacuo to about 0.7 liter. On dilution with hexane (3 liters) the product separates in crystalline form. It is collected on a filter, washed with hexane (0.7 liter) and dried in air [5J. The protected dipeptide ester, 149 g (85%), m.p. 103-104 °C, [cxJ64 - 26.8° (c 5, ethanol) is sufficiently pure for most practical purposes.
1. Boissonnas RA (1951) Helv Chirn Acta 34: 874; Vaughan JR, Jr (1951) J Am Chern Soc 73: 3547; Wieland T, Bernhard H (1951) J Liebigs Ann Chern 572: 190; Vaughan JR, Jr, Osato RL (1952) J Am Chern Soc 74: 676
2. Bodanszky M, unpublished 3. A one rnl aliquot of a solution ofbenzyloxycarbonyl-L-Ieucine in toluene is diluted with a few
rnl of ethanol and titrated with 0.1 N NaOH in the presence of phenolphthaleine.
IsobutyIcarbonic Acid Mixed Anhydrides [IJ 93
4. Triethylamine is satisfactory in the activation and coupling of most amino acids protected by urethane type amine blocking groups. When conservation of chiral purity appears as a problem, for instance in connection with the activation and coupling of peptides, triethylamine should be replaced by N-methylmorpholine (Anderson OW, Zimmerman IE, Callahan FM (1966) I Am Chern Soc 88: 1338).
5. Drying in air should be carried out in a hood and the danger of fire (hexane vapors) should not be forgotten.
A solution of glycine ethyl ester hydrochloride (1.40 g, 10 mmol) in dimethylformamide (20 ml) is prepared by gentle warming. The solution is cooled to room temperature and treated with triethylamine [3] (1.01 g= 1.40 ml, 10 mmo!).
A solution of benzyloxycarbonyl-glycyl-L-phenylalanine (3.56 g, 10 mmol) in dry tetrahydrofurane (50 ml) is cooled to -15°C and neutralized with Nmethylmorpholine (1.01 g-1.10 ml, 10 mmol). Isobutyl chlorocarbonate [4J (1.37 g= 1.32 ml, 10 mmol) is added, followed, about one minute later, by the solution of glycine ethyl ester described above. About 2 ml dimethylformamide is used for rinsing. The reaction mixture is allowed to warm up to room temperature. The hydrochlorides of N -methylmorpholine and triethylamine are removed by filtration and washed with tetrahydrofurane. The combined filtrate and washings are concentrated in vacuo to about 25 ml and diluted with water (50 ml) [5]. The precipitate is collected on a filter and washed with water, 0.5 N KHC0 3 , again with water. The dry product weighs 4.0 g (91 %) and melts at 118-120°C [6].
1. Vaughan JR Jr, Osato RL (1951) J Amer Chern Soc 73: 3547; 74: 676 (1952) 2. Anderson GW, Zimmerman JE, Callahan FM (1967) J Amer Chern Soc 89: 5012 3. Triethylamine can be replaced with the equivalent amount of N-methylmorpholine (1.01 g
= 1.12 ml) 4. Commercially available as isobutyl chloroformate. Slightly better yields were reported (cf. ref.
1) with sec-butyl chlorocarbonate and, in recent years, with isopropyl chlorocarbonate. 5. Alternatively, the solvent is removed in vacuo, the residue dissolved in a mixture of ethyl
acetate (150 ml) and water (50 ml) and the organic phase is washed with 0.5 N KHC03
(50 ml), 0.5 N HCI (50 ml), water (50 ml), dried over anhydrous Na2S04 and evaporated to dryness in vacuo.
6. Fractional crystallization from 200 ml abs. ethanol yields no racemate (cf. Anderson GW, Callahan FM (1958) J Amer Chern Soc 80: 2092) and the recovered material (3.92 g, 84%) melts at 120-121 DC.
4.5 Coupling with the Aid of o-Phenylene Phosphorochloridite [1]
The protected pentapeptide derivative N-benzyloxycarbonyl-S-benzyl-Lcysteinyl-L-tyrosyl-L-isoleucyl-L-glutaminyl-L-asparagine [2,3J (8.7 g, 10 mmol) and the partially blocked tetrapeptide amide S-benzyl-L-cysteinyl-Lpropyl-L-Ieucyl-glycinamide [2J (5.5 g, 11.5 mmol) are dissolved in dimethylformamide (50 ml) and the solution is cooled in an ice-water bath. Triethylamine (3.75 g = 5.2 mI, 37 mmol) is added with stirring followed by o-phenylene phosphorochloridite (3.75 g, 21.5 mmol). The mixture is allowed to warm
Coupling with the Aid of o-Phenylene Phosphorochloride [1] 95
up to room temperature and stirred overnight. The reaction mixture is diluted with ice water under vigorous stirring. Acetic acid (about 1 ml) is added to adjust the reaction of the suspension to slight acidity (ca pH 6). The precipitate is collected on a filter, thoroughly washed with water and dried in air. The crude product (12.2 g, m.p. 223-224 °C dec.) is suspended in methanol (300 ml) and the suspension is stirred at room temperature for about an hour. The insoluble material is filtered, washed with methanol, dried in air and finally in vacuo over phosphorus pent oxide. The protected nonapeptide derivative (with the sequence of oxytocin) weighs 9.6 g (72 %) and melts at 235-236 °C dec. [4J.
1. Anderson GW, Blodinger J, Young RW, Welcher AD (1952) J Amer Chern Soc 74: 5304; Anderson GW, Young RW (1952) ibid. 74: 5307; Anderson GW, Blodinger J, Welcher AD (1952) ibid. 74: 5390
2. Bodanszky M, du Vigneaud V (1959) J Amer Chern Soc 81: 2504 3. Activation of the carboxyl group of an asparagine residue is usually complicated by the
formation of cyanoalanine derivatives. Yet, in the present example the expected product was obtained in fair yield and thus the dehydration reaction does not seem to interfere with the process to an unacceptable degree.
4. On deprotection by reduction with sodium in liquid ammonia, followed by air-oxidation of the resulting disulfhydro intermediate to oxytocin 200 units of biologically active material were obtained from 1 mg of the protected nona peptide. This compares favorably with the results of several syntheses of oxytocin. When, however, instead of condensation of segments the stepwise strategy was applied (Bodanszky M, du Vigneaud V (1959) J Amer Chern Soc 81: 5688) the same protected nonapeptide was obtained with a higher melting point (245-248° dec.) and it yielded more biological activity on reduction and cyclization.
