Diketopiperazines formation from amino acids

The stepwise polymerisation of activated amino acids leads to the formation of activated dimers, which very often cyclise to diketopiperazines and are thus removed from the chain elongation process (Orgel, 1989). 

Diazines other than diketopiperazines can also be prepared on insoluble supports (Table 15.31 see also Figure 3.13 [382]). Most strategies are based on intramolecular nucleophilic substitutions or acylations. Several examples of the solid-phase preparation of quinoxalinones have been reported. In most cases, the compounds have been prepared from support-bound 2-fluoronitrobenzenes according to the strategies outlined in Figure 15.18. Alternatively, a-amino acid esters bound to polystyrene as IV-benzyl derivatives can be N-arylated with 2-fluoronitrobenzene. Reduction of the resulting 2-nitroaniline leads to the formation of quinoxalinones [383]. 1,4-Diazines have been chemically modified by N- or C-alkylation on insoluble supports (Entries 9 and 10, Table 15.31). 

Among the six-membered ring heterocycles, diketopiperazines are most commonly prepared by cyclative cleavage. Indeed, diketopiperazine formation is often observed as an undesirable by-product during peptide synthesis20 and the facile nature of this cyclization makes it an obvious choice for library generation. In an early example from Pfizer,21 a set of 10 immobilized a-amino acids was reductively alkylated with 10 aldehydes, followed by acylation with 10 a-amino acids and cyclization (Fig. 6). By... 

Trityl-based resins are highly acid-labile. The steric hindrance of the linker prevents diketopiperazine formation and the resins are recommended for Pro and Gly C-terminal peptides. Extremely mild acidolysis conditions enable the cleavage of protected peptide segments from the resin. These resins are commercially available as their chloride or alcohol precursors. The trityl chloride resin is extremely moisture-sensitive, so reagents and glassware should be carefully dried before use to avoid hydrolysis into the alcohol form. It is necessary to activate the trityl alcohol precursor and it is highly recommended to reactivate the chloride just before use see Note 4). After activation, attachment of the first residue occurs by reaction with the Fmoc amino acid derivative in the presence of a base. This reaction does not involve an activated species, so it is free from epimerization. Special precautions should be taken for Cys and His residues that are particularly sensitive to epimerization during activation (Table 2). 

Another main type of pyrolysis products for peptides includes the diketopiperazines (DKP) and their secondary fragmentation products. The formation of DKP from oligopeptides showed that the generation of DKPs always takes place from neighboring amino acids. The mechanism of DKP formation seems to be the following ... 

The ester was prepared from and amino acid and the alcohol using DCC/DMAP. It was developed to prevent diketopiperazine formation during the formation and deprotection at the dipeptide stage of the growing peptide. It is cleaved with TBAF at approximately half the rate of TMSE cleavage." ... 

Oxidative degradation has confirmed that the amino-acid units in echinulin (18) are derived from acids in the L-series. The total synthesis (Scheme 5) of (18) has been announced it uses a previously established method for producing an appropriately a-substituted indole and newly developed methods for the introduction of benzene ring substituents and the formation,with partial selectivity, of the desired c/s-substituted diketopiperazine ring. 

Fischer and Fourneau, in 1901, found that 2, 5-diketopiperazine, or glycine anhydride, as it is now best termed, was converted by boiling with concentrated hydrochloric acid into the hydrochloride of an amino acid of the formula C HgNjOs, from which they obtained the free acid by treatment with the calculated quantity of caustic soda, or by means of silver oxide. Its formation is represented by the equation... 

The further attempts to again prepare this substance did not succeed, since the exact conditions leading to its formation could not be repeated, but in 1906 Fischer and Abderhalden obtained the anhydride of this body by a new method which they had discovered for isolating such compounds when mixed with amino acids and higher polypeptides. This method depends upon the different behaviour of the esters of these compounds those of the simple mono-amino acids are easily volatile in vacuo and are therefore easily removed, whereas those of the dipeptides are converted by the action of ammonia into their anhydrides or diketopiperazines which crystallise readily and are therefore easily separated from the esters of the higher polypeptides. They thus obtained a methyl diketopiperazine. 

Peptide synthesis is quite regularly accompanied by undesired side reactions, for instance by the alkylation of side chains during deprotection. In addition to such general problems for which at least partial remedies have been found, also a special problem is encountered in solid phase syntheses the formation of deletion peptides and truncated sequences . Both incomplete acylation and incomplete deprotection result in peptide chains from which one or more amino acid residues are missing. A second kind of deletion, the loss of the C-terminal dipeptide sequence, is caused by diketopiperazine formation followed by acylation of the resulting hydroxymethyl polymer (Fig. 8). Premature chain termination (truncation) takes place if acetic acid or trifluoroacetic acid are not completely removed after deprotection and then co-activated in the following coupling step. 

Diketopiperazines are cyclic dipeptides synthesized from two (activated) v-amino acids. The cyclic peptides built may be further transformed by monooxygenation (formation of iV-oxides, C 2.6.2., cf. also D 23.2), reduction and modification of the side chains (Fig. 333). 

Diketopiperazine Formation during Attempted Incorporation of Third Amino Acid. The free iV -amino group of an anchored dipeptide is poised for acid- or base-catalyzed intramolecular attack upon the C-terminal carbonyl (Fig. 6). The formation and release of the six-membered-ring diketopiperazine from the resin is kinetically favorable for residues that can form cis-peptide bonds, for example, Gly, Pro, iV-methylamino acids, and D-amino acids in either of the dipeptide residues. A hydroxymethyl group remains on the resin. Diketopiperazine formation is also a problem when the third amino acid is added to a BAL-anchored (Table 5) sequence (105) or to an activated leaving group such as the 0-nitrobenzyloxy handle (22). However, for most syntheses this problem can be adequately handled. 

First reports on the cell-free assembly of ergopeptines came from Abe s group who demonstrated the formation of ergokryptine and ergotamine in extracts from C. purpurea from preformed d-lysergyl amino acids and diketopiperazines. The enzyme system was not characterised and thus the protein components and the mechanism of formation remained obscure (Abe et al., 1971 Abe, 1972 Ohashi et al, 1972). 

In a similar manner resonance affects greatly the rate of racemization by reducing the energy required for ionization. This point is discussed with reference to a-phenylamino acids and particularly with regard to amide resonance in acylamino acids, peptides, diketopiperazines and hydantoins. The literature on the racemization of proteins by alkali is also reviewed. The racemization which is observed during acylation of amino acids is also discussed, and it is pointed out that apart from oxazolone formation, mixed anhydrides may also occur as intermediates and be partly responsible for the racemization observed. 

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