Amino groups active-esters methods

The Active Esters Method. Selected amino-protecting groups and conditions required for their deprotection in organic solvents are shown in Table III. Active N-hydroxysuccinimide esters of f-butyloxycarbonyl-L-... 

Then another N-protected amino acid is coupled to the free amino group of the polymer-bound substrate using the dicyclohexylcarbodiimide activation or the active ester method. The N-deblocking and coupling steps are repeated until the desired sequence is formed. Finally the resin-peptide bond is split by a suitable acid cleavage reaction with HBr—AcOH, trifluoroacetic acid or HF. This results in a simultaneous N-deblocking and deprotection of most of the side-chain functionalities. 

The reaction of 42 with 43 was attempted with dicyciohexylcarbodiimide (DCC) or water-soluble carbodiimide in solvent systems. All these reactions seemed to be unsuccessful because of the low solubility and low reactivity of 43. Among the methods of activation of a carboxy group, only the activated ester method of p-nitrophenyl ester was successful. In the acid chloride method a side reaction with the amino group of adenine was reported56. ... 

An important feature of polymer synthesis by the active ester method is that displacement of the polymer-bound activating 0eaving) groups can be readily monitored by IR qiectroscopy of the polymer. Tl% IR spectrum generally shows the disappearance of the phenyl ester carbonyl at about 1760 cm, and the appearance of a new carbonyl absorption at 1620-80 cm (amide) and/or 1720-30 cm (ester), together with other characteristic absorptions due to A and A. In some cases, a relatively weak polystyrene band at ca. 1607 cm is also observed. A typical illustration is provided by Fig. IS, showing the amino-lysis of the activated polymer with 6-tert.-butoxycarbonylaminohexylamine, followed by reaction with dimethylamine (see the first entry in Table 7). 

With the dicyclohexylcarbodiimide (DCQ reagent racemization is more pronounced in polar solvents such as DMF than in CHjCl2, for example. An efficient method for reduction of racemization in coupling with DCC is to use additives such as N-hydroxysuccinimide or l-hydroxybenzotriazole. A possible explanation for this effect of nucleophilic additives is that they compete with the amino component for the acyl group to form active esters, which in turn reaa without racemization. There are some other condensation agents (e.g. 2-ethyl-7-hydroxybenz[d]isoxazolium and l-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline) that have been found not to lead to significant racemization. They have, however, not been widely tested in peptide synthesis. 

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. 

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. 

The alternative method for making activated esters is base-catalyzed transesterification. Fmoc-amino acids are esterified in excellent yields by reaction with pentafluorophenyl trifluoroacetate at 40°C in the presence of pyridine (Figure 7.13). A mixed anhydride is formed initially, and the anhydride is then attacked by the pentafluorophenoxy anion that is generated by the pyridine. Succinimido, chlorophe-nyl, and nitrophenyl esters were made by this method when it was introduced decades ago. A unique variant of this approach is the use of mixed carbonates that contain an isopropenyl group [Cf C CfyO-COjR]. These react with hydroxy compounds in the presence of triethylamine or 4-dimethylaminopyridine (see Section 4.19) to give the esters and acetone.30 35... 

If a free amino group forms a portion of the protein that is essential for activity (e.g., the antigen-combining site for antibody), biotinylation with the succinimide ester will lower or destroy the activity of the protein, and other methods of labeling should be tried. Biotin hydrazide has been used to modify the carbohydrate moieties of antibodies (10,11). Other alternatives are the thiol-reactive biotin maleimide (12) or biotin iodoacetamide (13). 

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