N-Boc-amino acid-thioester

Scheme 13.16 Enzymatic kinetic resolution of N-Boc-amino acid thioesters coupled with base-catalyzed racemization.

Enzymatic Kinetic Resolution of N-Boc-Amino Add-Thioesters Coupled with Base-catalyzed Racemization Recently, a new method leading to the preparation of a number of aryl-glycines of the L-configuration has been published. The method is based on the hydrolysis of N-Boc-amino acid thioesters 15 catalyzed by an industrial preparation of the protease subtilisin (Scheme 13.16) [43]. 

Because ofthe presence ofalarge amount of water appears to considerably narrow the field of the previously illustrated resolution system, experiments were started with immobilized enzymes in organic solvents, in which the water concentration was considerably reduced. This proved to be quite an effective strategy, permitting to carry out a nicely working DKR on a representative array of aliphatic N-Boc-amino acid thioesters [62], which could be resolved in high yield and with excellent optical purity. Moreover, the choice of an immobilized form of the enzyme (Alcalase -CLEA ) permitted recovery and reuse of the catalyst for several consecutive batches [63]. The solvent of choice proved to be tert-butanol, which was able to dissolve the hydrophobic substrates, the organic base, and the strictly necessary amount of water (Table 8.6). 

Table 8.6 Results obtained with the hydrolysis of N-Boc-amino acid thioesters under DKR conditions.

Peptide thioester 84 was prepared from 81 (0.3 g of resin). All amino acids were protected with N-terminal Boc groups. The side-chain protections were as follows Arg(Tos), Asp(OCy), Cys[Bzl(4-Me)], Glu(OCy), His(Tos), Lys[Z(2-Cl)], Ser(Bzl), and Thr(Bzl). Each synthesis cycle consisted of (1) a 25-min deprotection with 55% TFA/CH2C12 and (2) coupling with Boc amino acid (4 equiv) and BOP or HBTU (4 equiv) in the presence of DIPEA (6 equiv) in DMF for 1 h. All couplings were monitored by the... 

DKR of Thioesters The acidity of the H in the a-position of a thioester is higher than the acidity of the same positioned H in esters, amides, or acids. This fact and the ability of subtUisin Carslberg to hydrolyze a-amino acid thioesters have been the keys to the successful DKR of several N-Boc-a-amino thioesters rac-118 developed by Servi et al. Reactions were carried out in a biphasic system of TBME/-water, at 37°C and pH = 8.0, with trioctylamine as the base to remove the acidic a-H of the substrates (Scheme 57.34). pH was kept constant at 8.0 throughout each process, which was stopped when all the starting substrate had been consumed. Finally, the L-Af-Boc-a-amino acids 119 were easily... 

Camarero et al. [108] used the hydrazine safety-catch linker to prepare peptide thioesters. After assembling the peptide using standard Fmoc protocols, the fully protected peptide resin was activated by mild oxidation with N-bromosuc-cinimide (NB S) in the presence of pyridine, forming a reactive acyl diazene that was then deaved with an a-amino add S-alkyl thioester such as H-AA-SEt, where AA is Gly or Ala. After TFA deprotection, peptide thioesters were obtained in good yields. Although the oxidation step did produce racemization, and other sensitive amino acids such as Tyr(tBu) and Trp(Boc) were not affected, Met and Cys presented some problems. Met was completely oxidized, and a reductive cleavage was required. For Cys, the Cys(Trt) derivative should be avoided and use of Cys(Npys) or Cys(S-StBu) is recommended instead. 

To a cold 0°C solution of 7V-Fmoc- or Boc-protected a-amino acid (10 mmol) and benzyl mercaptan (20 mmol, 2.48 g) in dichloromethane (30 mL) are successively added 4-(dimethylamino)pyridine (DMAP) (1 mmol, 122 mg) and 1,3-dicyclohexylcarbodiimide (DCC) (10.5 mmol, 2.17 g). After the reaction mixture is stirred overnight and the solvent removed in vacuo, ethyl acetate (100 mL) and a 1 N potassium hydrogen sulfate solution are added. The organic layer is washed with 1 N potassium hydrogen sulfate (2 X 60 mL) and saturated NaCl solution (60 mL), dried over magnesium sulfate, and filtered. The solvent is removed in vacuo and the 5-benzyl thioester is recovered as a white solid after precipitation with ether-hexane (1 1, v/v). 

In our laboratory we use two different SPPS-based approaches to synthesize cyclic disulfide-rich peptides. Most commonly we use the intramolecular native chemical ligation (NCL) reaction between a thioester group at the C-terminus and a cysteine residue at the N-terminus [16, 26-29]. For this approach the coupling of the thioester linker requires Boc-SPPS protection chemistry as the thioester is unstable in the basic conditions used for Fmoc chemistry. The first amino acid coupled after the thioester linker could be any residue however, Gly, Cys, and His are most favorable, and Leu, Thr, Val, He, and Pro are the least favorable amino acids, as reflected in their ligation rates with the N-terminal cysteine [26]. The second SPPS approach used in our laboratory to synthesize the cyclic disulfide-rich peptides involves an Fmoc-based method, in... 

C-terminal peptide tiiioesters are used extensively in synthetic protein chemistry, especially for native chemical ligation (NCL) and other chemoselective reactions, which has inspired a search for robust synthetic strategies. Initially, peptide thioesters were mainly prepared using solid-phase peptide synthesis with amino acids N -protected with Boc (Boc-SPPS) [1-3], see Chapter 4. However, this technique requires specialized equipment for handling of hydrofluoric acid (HP) for release of the peptide from the resin, and it is therefore currently not used in many laboratories. Furthermore, the HP treatment is incompatible with many post-translational modiflcations such as glycosylations or phosphorylations [4]. Boc-SPPS is described thoroughly in Chapter 4. 

---