Peptide bonds using acid anhydrides

The original procedure for the trifluoroacetylation of amino acids used trifluoroacetic anhydride [Acetic acid, trifluoro-, anhydride].4 This reagent, although inexpensive and readily available, has certain disadvantages it is a highly reactive compound and thus has caused undesired reactions such as the cleavage of amide or peptide bonds,5 unsymmetrical anhydrides are formed between the newly formed A-trifluoroacetylamino acids and the by-product trifluoroacetic acid, and excess trifluoroacetic anhydride has caused racemization of asymmetric centers. 

For efficient peptide bond formation acid halides azides 2,b l and Leuchs anhydridesb l were employed as the first activated species in peptide synthesis. Since then, besides considerable improvements to the azide (see Section 3.1) as well as the V-carboxyanhydride 7 procedure d (sgg Section 3.4.3), the methods have evolved over decades along a few basic principles as outlined in Scheme 2. The symmetrical the mixed carboxylic acid 4,b 5s] and the carbonic acid anhydrides were developed and remain useful despite the... 

There is no obvious advantage of purifying mixed anhydrides of /V-alkoxycar-bonylamino acids before reacting them with an /V-nuclcophilc to form a peptide bond. However, there are a few reports of success with the reaction of a purified mixed anhydride after the reaction with the anhydride in the presence of tertiary-amine salt had failed. It would seem that the prudent course of action is to try the reaction using both the crude and purified anhydrides in case one approach is better.18-19... 

Peptide bond formation. The process requires that the derivative (42), a carboxylic acid, should be caused to acylate the free base liberated from the hydrochloride (43). Activation of the carboxyl group is effected by conversion into a type of acid anhydride the mixed carbonic anhydride (44) is used here, and is prepared by reaction of the acid (42) with ethyl chloroformate. 

Limiting essential amino acids covalently attached to proteins by using activated amino acid derivatives can improve the nutritional quality and change the functional properties of proteins. The best chemical methods for incorporating amino acids into water-soluble proteins involve using car-bodiimides, N-hydroxysuccinimide esters of acylated amino acids, or N-carboxy-a-amino acid anhydrides. The last two methods can give up to 75% incorporation of the amount of amino acid derivative used. With the anhydride method, as many as 50 residues of methionine have been linked to the 12 lysine residues of casein. The newly formed peptide and isopeptide bonds are hydrolyzed readily by intestinal aminopeptidase, making the added amino acids and the lysine from the protein available nutritionally. 

When the second amino acid (valine) is added to the protected, activated alanine, the nucleophilic amino group of valine attacks the activated carbonyl of alanine, displacing the anhydride and forming a peptide bond. (Some procedures use an ester of the new amino acid to avoid competing reactions from its carboxylate group.)... 

In the case of the stepwise synthesis of peptide fragments starting from an amino acid without C-terminal protection, peptide bond forming methods are limited to the mixed anhydride method,the active ester method, and the azide methodJ l Although such peptide fragments can be directly used for the segment condensation reaction, the purification of the products after each coupling reaction is not so easy. 

Racemization of activated amino acids under mildly basic conditions has been studied in some detail for Boc-protected amino acidsj but recent reports confirm that the problem is not limited to Boc chemistry. Fmoc-Cys(Trt)-OH has been shown to racemize in TBTU-based couplings using excess DIPEA under certain conditions. In this instance the problem was avoided by using the preformed symmetrical anhydride for peptide bond formation. In a more recent publication the racemization of Fmoc-Ser(tBu)-OH under similar conditions using NMM as base, was reduced by using a hindered collidine base.P l... 

Acid anhydrides in the presence of basic catalysts can be used instead of acyl chlorides in acylation reactions of thiols.Mixed anhydrides of N-protected amino acids and ethyl carbonate yield the corresponding S-f-butyl thiocarboxylic esters, which are useful reagents for peptide syntheses (equation 22). Acylation of thiols with ketenes (equation 23) is a method of long standing. In many cases the yields are nearly quantitative. Functionalities such as acetamino groups or carbon-carbon double bonds in the thiol are not attacked under the mild reaction conditions and optically active thiol esters are obtained without racemization. 3 ... 

The oxidation-reduction method, developed initially by Mukaiyama et al. [133] and related to the previously described organophosphorus methods, has permitted a variety of important solid-phase applications. The mechanism of the activation is complex and involves the oxidation of the triaryl/ alkyl-phosphine to the oxide as well as reduction of the disulfide to the mercapto derivative. However, different active species, such as 81 (Fig. 11), the 2-pyridyl thioester, or even the symmetrical anhydride, have been postulated to form. For the intermediate 81, the peptide bond formation may proceed through a (cyclic transition state. The method has been used for conventional stepwise synthesis [134], acylation of the first protected amino acid to a hydroxymethyl resin, and to achieve segment condensation on a solid support in the opposite direction (N C) [135,136]. Lastly, it has been used for efficient grafting of a polyethylene glycol (molecular weight 2000) derivative to an aminomethyl resin to prepare PEG-PS resins [137]. 

---