Protecting amino

The following short descriptions of the steps involved in the synthesis of a tripeptide will demonstrate the complexity of the problem amino acid units. In the later parts of this section we shall describe actual syntheses of well defined oligopeptides by linear elongation reactions and of less well defined polypeptides by fragment condensation. 

Step 1 The Boc protected amino acid is anchored to the resin Nucleophilic substitution of the benzylic chloride by the carboxylate anion gives an ester... 

An O acylisourea is formed by addition of the Z protected amino acid to N N dicyclo hexylcarbodiimide as shown in Figure 27 13 This O acylisourea is attacked by p nitrophenol... 

Anhydride Formation. The carboxyl group ia A/-protected amino acids is converted iato the symmetrical anhydride on treatment with the carbodiimide (84). 

Protected Amino Acids. Various types of protected amino acids for peptide synthesis are available commercially (243). 

Polypeptide Synthesis and Analysis. Sihca or controUed-pore glass supports treated with (chloromethyl)phenylethyltrimethoxysilane [68128-25-6] or its derivatives are replacing chloromethylated styrene—divinylbenzene (Merrifield resin) as supports in polypeptide synthesis. The sdylated support reacts with the triethyl ammonium salt of a protected amino acid. Once the initial amino acid residue has been coupled to the support, a variety of peptide synthesis methods can be used (34). At the completion of synthesis, the anchored peptide is separated from the support with hydrogen bromide in acetic acid (see Protein engineering Proteins). 

Amino Acids. Chloroformates play a most important role for the protection of the amino group of amino acids (qv) during peptide synthesis (32). The protective carbamate formed by the reaction of benzyl chloroformate and amino acid (33) can be cleaved by hydrogenolysis to free the amine after the carboxyl group has reacted further. The selectivity of the amino groups toward chloroformates results in amino-protected amino acids with the other reactive groups unprotected (34,35). Methods for the preparation of protected amino acids on an industrial scale have been developed (36,37). A wide variety of chloroformates have been used that give various carbamates that are stable or cleaved under different conditions. 

Nitro groups have been reduced to amino groups, whilst amino groups in the 3- and 6-positions of pyrido[2,3-f ]pyrazines and in the 5-position of the [3,4-f ] isomers have been hydrolyzed to the corresponding hydroxy derivatives with alkali. Protected amino groups have been liberated by hydrolysis or reduction in deazapteridine syntheses. 

RCO2H = Ph, 2,4,6-Me3C6H2—, N-protected amino acids R = Me, Et, PhCH2, -Bu... 

Fluorenylmethyl esters of A -protected amino acids were prepared using the DCC/ DMAP method (50-89% yield) or by imidazole-catalyzed transesterification of... 

The carboxamidomethyl ester was prepared for use in peptide synthesis. It is formed from the cesium salt of an A-protected amino acid and a-chloroacetamide (60-85% yield). It is cleaved with 0.5 M NaOH or NaHCOa in DMF/H2O. It is stable to the conditions required to remove BOC, Cbz, Fmoc, and r-butyl esters. It cannot be selectively cleaved in the presence of a benzyl ester of aspartic acid. ... 

The Dppe group was developed for carboxyl protection in peptide synthesis. It is formed from an N-protected amino acid and the alcohol (DCC, DMAP, 3-12 h, 0°, It). It is most efficiently cleaved by quatemization with Mel followed by treatment with fluoride ion or K2CO3. The ester is stable to HBr/AcOH, BF3 Et20, and CF3CO2H. ... 

Phenyl esters can be prepared from A-protected amino acids (PhOH, DCC, CH2CI2, -20° 20°, 12 h, 86% yield PhOH, BOP, Et3N, CH2CI2, 25°, 2 h. 

The / -(methylmercapto)phenyl ester has been prepared from an /-protected amino acid and 4-tH3SC6H40H (DCC, CH2CI2, 0°, 1 h 20°, 12 h, 60-70% yield). The p-(methylmercapto)phenyl ester serves as an unactivated ester that is activated on oxidation to the sulfone (H2O2, AcOH, 20°, 12 h, 60-80% yield) which then serves as an activated ester in peptide synthesis. ... 

This derivative is prepared from an A-protected amino acid and the anthrylmethyl alcohol in the presence of DCC/hydroxybenzotriazole. It can also be prepared from 2-(bromomethyl)-9,10-anthraquinone (Cs2C03). It is stable to moderately acidic conditions (e.g., CF3COOH, 20°, 1 h HBr/HOAc, / 2 = 65 h HCl/ CH2CI2, 20°, 1 h). Cleavage is effected by reduction of the quinone to the hy-droquinone i in the latter, electron release from the —OH group of the hydroqui-none results in facile cleavage of the methylene-carboxylate bond. The related 2-phenyl-2-(9,10-dioxo)anthrylmethyl ester has also been prepared, but is cleaved by electrolysis (—0.9 V, DMF, 0.1 M LiC104, 80% yield). ... 

The Tcrom ester is prepared from the cesium salt of an N-protected amino acid by reaction with 2-(trifluoromethyl)-6-chromylmethyl bromide (DMF, 25°, 4 h, 53-89% yield). Cleavage of the Tcrom group is effected by brief treatment with n-propylamine (2 min, 25°, 96% yield). It is stable to HCl/dioxane, used to cleave a BOC group. ... 

Sulfobenzyl esters were prepared (cesium salt or dicyclohexylammonium salt, Na03SC6H4CH2Br, DMF, 37-95% yield) from A -protected amino acids. They are cleaved by hydrogenolysis (H2/Pd), or hydrolysis (NaOH, dioxane/water). Treatment with ammonia-or hydrazine results in formation of the amide or hydrazide. The ester is stable to 2 M HBr/AcOH and to CF3SO3H in CF3CO2H. The relative rates of hydrolysis and hydrazinolysis for different esters are as follows ... 

Carbamates can be used as protective groups for ammo acids to minimize race-mization in peptide synthesis. Raccmi/ation occurs during the base-catalyzed coupling reaction of an W-protected, catboxyl-uc ivated amino acid, and takes place in the intermediate oxazolone that foro S readily from an N-acyl protected amino... 

This method is suitable for the preparation of BOC, Fmoc, Adoc, and Bpoc protected amino acids. The acid chloride is a stable, storable solid. ... 

This vinylogous amide has been prepared in 70% yield to protect amino acid esters. It is cleaved by treatment with either aqueous bromine or nitrous acid (90% yield). ... 

This group, which is more stable than the 2-hitrobenzenesulfenamide, has been developed to protect amino acids. It is readily introduced with the sulfenyl chloride (52-74% yield) and is cleaved with triphenylphosphine or 2-thiopyridine N-oxide. It is stable to CF3COOH but can be cleaved with 0.1 M HCl. ... 

To fonn a peptide bond between two suitably protected amino acids, the free carboxyl group of one of them must be activated so that it is a reactive acylating agent. The most ffflniliar- acylating agents are acyl chlorides, and they were once extensively used to couple fflnino acids. Certain drawbacks to this approach, however, led chemists to seek alternative methods. 

In the second major method of peptide synthesis the carboxyl group is activated by converting it to an active ester, usually a p-nitrophenyl ester. Recall from Section 20.12 that esters react with ammonia and amines to give fflnides. p-Nitrophenyl esters are much more reactive than methyl and ethyl esters in these reactions because p-nitrophenoxide is a better (less basic) leaving group than methoxide and ethoxide. Simply allowing the active ester and a C-protected amino acid to stand in a suitable solvent is sufficient to bring about peptide bond formation by nucleophilic acyl substitution. 

B enzy loxy c arbony 1 -protected amino acid... 

Section 27.17 Peptide bond fonnation between a protected amino acid having a free carboxyl group and a protected amino acid having a free amino group can be accomplished with the aid of A, A/ -dicyclohexylcaibodiimide (DCCI). 

What protected amino acid would you anchor to the solid support in the first step of a synthesis of oxytocin (see Figure 27.8) by the Merrifield method ... 

Polymer-supported esters are widely used in solid-phase peptide synthesis, and extensive information on this specialized protection is reported annually. Some activated esters that have been used as macrolide precursors and some that have been used in peptide synthesis are also described in this chapter the many activated esters that are used in peptide synthesis are discussed elsewhere. A useful list, with references, of many protected amino acids (e.g., -NH2, COOH, and side-chain-protected compounds) has been compiled/ Some general methods for the preparation of esters are provided at the beginning of this chapter conditions that are unique to a protective group are described with that group/ Some esters that have been used as protective groups are included in Reactivity Chart 6. 

Phenyl esters can be prepared from A-protected amino acids (PhOH, DCC, CH2CI2, -20° 20°, 12 h, 86% yield PhOH, BOP, Et.N, CH2CI2, 25°, 2 h, 73-97% yield ). Phenyl esters are readily cleaved under basic conditions (H2O2, H2O, DMF, pH 10.5, 20°, 15 min). ... 

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