An Efficient Synthetic Method of N-Protected Dipeptide Acids Using Amino Acid Calcium Carboxylates in an Organic Solvent

Abstract

The syntheses of N-protected dipeptide acids using alkaline earth metal (Mg, Ca, and Ba) carboxylates of an amino acid in organic solvents were investigated. It was found that amino acid calcium carboxylates are the most effective among the carboxylates of the amino acids tested for coupling with active esters of Boc-Ala-OH in organic solvents. The coupling of Boc-Ala-ONp or Boc-Ala-ONSu with amino acid calcium carboxylates in DMF gave the desired N-protected dipeptide acids in high yields (92-100%).

Key words

N-protected peptide acid - amino acid calcium carboxylate - organic solvent - active ester method - synthesis

References and Notes

• 1 Bodansky M. Klausner SY. Ondetti AM. Peptide Synthesis   2nd ed.:  John Wiley & Sons; New York: 1976.  p.49 
• 2 Lansbury TP. Hendrix CJ. Coffman IA. Tetrahedron Lett.  1989,  30:  4915 
• 3 Chen ST. Wang KT. J. Chem. Soc., Chem. Commun.  1990,  1045 
• 4 The amino acids used were of l-configuration. The abbreviations used are those recommended by the IUPAC-IUB Joint Commission on Biochemical Nomenclature and Symbolism for Amino Acids and Peptides. See:Eur. J. Biochem.  1984,  138:  9 
• 8 Fox S. Büsching I. Barklage W. Strasdeit H. Inorg. Chem.  2007,  46:  818 
• For the values of specific rotation, see:
• 9a Boc-Ala-Gly-OH˙H₂O: [α]_D ^²7 -23.2˚ (MeOH, c = 2.0): Wolman Y. Schwarzberg M. Frankel M. Isr. J. Chem.  1970,  8:  53 
• 9b Boc-Ala-Pro-OH: [α]_D ^²8 -89.9˚ (EtOH, c = 1): McDermott JR. Benoiton NL. Can. J. Chem.  1973,  51:  2555 

5

Typical Procedure for the Preparation of Amino Acid Calcium Carboxylates; (H-Leu-O) ₂ Ca: To a solution of
H-Leu-OH (2.623 g, 20.0 mmol) dissolved in distilled H₂O (100 mL) was added a solution of Ca(OH)₂ (0.815 g, 11.0 mmol) dissolved in distilled H₂O (50 mL). After the mixture was stirred for 30 min at r.t., the H₂O was removed by eva-poration. The obtained solid was dried over SiO₂ for 1 d in vacuo. The solid was ground into a powder using a mortar and pestle. The powder, (H-Leu-O)₂Ca, was washed onto a filter funnel with THF and Et₂O, and then dried over SiO₂ in vacuo. Yield: 2.943 g (98%).

6

Typical Procedure for the Determination of HPLC Yield of N-Protected Dipeptide Acids; Boc-Ala-Leu-OH: To a solution of Boc-Ala-ONp (0.0931 g, 0.300 mmol) dissolved in DMF (4.0 mL) was added (H-Leu-O)₂Ca (0.0496 g, 0.165 mmol). The mixture was stirred at 25 ˚C for 24 h. To the mixture were then added MeCN (10 mL), H₂O (10 mL), and Boc-Val-OH (0.300 g, 1.38 mmol) as the internal standard reagent. The solution was applied to an HPLC column under the conditions described below. From the HPLC result, the yield of the product, Boc-Ala-Leu-OH, was determined using a calibration curve with a straight line plotted against three different concentration ratios of Boc-Ala-Leu-OH and Boc-Val-OH. Yield: 99%. t _R (Boc-Ala-Leu-OH) = 21.9 min, and t _R (Boc-Val-OH) = 16.4 min. HPLC conditions: column, Waters µ-Bondasphere (3.9 × 150 mm, C18, 5 µm, and 300 Å); solvent: 14-41% MeCN-H₂O-0.1% TFA; linear gradient, 60 min; flow rate: 1.0 mL/min; and detection, λ = 210 nm.

7

Typical Procedure for the Determination of Isolation Yield of N-Protected Dipeptide Acids; Boc-Ala-Ser(Bzl)-OH: To a solution of Boc-Ala-ONp (0.465g, 1.50 mmol) dissolved in DMF (5.0 mL) was added [H-Ser(Bzl)-O]₂Ca (0.354g, 0.825 mmol). After the mixture was stirred at 25 ˚C for 24 h, the solution was condensed in vacuo. The residue was then dissolved in EtOAc (ca. 100 mL). The organic layer was washed with 5% aq citric acid (1 × 20 mL, 3 × 10 mL) and H₂O (5 × 10 mL), and then dried over anhyd Na₂SO₄. After the removal of the solid, the filtrate was condensed in vacuo. The residue was recrystallized from EtOAc-hexane. Yield: 0.511 g (93%); mp 155.4-156.0 ˚C. MS (FAB): m/z = 337 [M + H]⁺ and 359 [M + Na]⁺. t _R = 23.3 min. HPLC conditions: column, Waters µ-Bondasphere (3.9 × 150 mm, C18, 5 µm, and 300 Å); solvent: 5.0-90.5% MeCN-H₂O-0.1% TFA; linear gradient, 60 min; flow rate, 1.0 mL/min; and detection, λ = 210 nm.

10

Alternative Synthetic Method of Boc-Ala-Leu-OH: Boc-Ala-OH (6.24 g, 33.0 mmol) in DMF (15 mL), ice-cooled, was coupled with TFA˙H-Leu-OPac (30.0 mmol) containing Et₃N (4.17 mL, 30.0 mmol) in DMF (15 mL) ice-chilled using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC˙HCl; 6.90 g, 36.0 mmol) and 1-hydroxybenzotriazole (HOBt; 6.08 g, 45.0 mmol). The solution was stirred for 6 h in an ice-bath and then for 18 h at r.t. After the treatment of the produced Boc-Ala-Leu-OPac (3.91 g, 9.30 mmol) with Zn powder (19.62 g, 300 mmol) in AcOH (30 mL), the desired product Boc-Ala-Leu-OH (1.13 g, 3.74 mmol) was obtained by the recrystalliza-tion from EtOAc-Et₂O-hexane. Boc-Ala-Leu-OH: mp 137.6-137.8 ˚C. MS (FAB): m/z = 303 [M + H]⁺, 325 [M + Na]⁺. t _R = 13.6 min. HPLC conditions: column, Waters
µ-Bondasphere (3.9 × 150 mm, C18, 5 µm, and 300 Å); solvent: 5-95% MeCN-H₂O-0.1% TFA; linear gradient, 30 min; flow rate: 1.0 mL/min; and detection, λ = 210 nm; [α]_D ^²² -40.7˚ (MeOH, c = 1.00).