Pyroglutamates, formation

Enzymes can also undergo other side reactions under conditions that divert a chemically reactive intermediate from its usual catalytic function. Again, glutamine synthetase is an excellent example (see figure above), because its side reactions include acyl-phosphate reduction by borohydride, pyroglutamate formation, and the formation of y-glutamyl hydroxamate in the presence of hydroxylamine and arsenate. 

The absolute purity of a biological substance is hard - if at all possible - to determine. Regular and sometimes only subtle protein modifications such as glycosyla-tion, alternative disulphide bond formation, deamidation, oxidation, phosphorylation, acetylation, sulfation, sulfoxidation, y-carboxylation, and pyroglutamate formation lead to protein variants that may have more or less different characteristics. Also, truncated protein variants might be generated by the presence of cryptic or alternative start sites of transcription, by premature stop of the peptide chain... 

Glutaminyl cyclase (QC), EC 2.3.2.5, an acyltransferase responsible for N-terminal pyroglutamate formation from glutaminyl precursors in peptides and proteins. The first QC was isolated from the latex of Car-ica papaya in 1963. Later, it was established that glutaminyl cyclases occur in both animal and plant sources. They are abundant in mammalian neuroendocrine tissues, such as hypothalamus and pituitary, and are highly conserved from yeast to human. From the crystal structure of human glutaminyl cyclase it follows that a single zinc ion in the active site is coordinated... 

Mass spectrometry, in addition to RP-HPLC, serves as a powerful technique for assessing disulfide bond formation. Additionally, tandem mass spectrometry (MS-MS) can in some instances be used to define the pairing of cysteines, like the non-conserved disulfide bond in CCL28 that finks C30 to C80 (Thomas et al., 2015), or confirm the locations of posttranslational modifications, like pyroglutamate formation in CCL2 as shown in Figs. 4 and 5, respectively. 

Pyroglutamate formation. Occurs in acidic aqueous media if side-chain not protected... 

FIGURE 4 Pyroglutamate formation occurs when a glutamine at the NHa-terminus reacts with amine groups. 

Pyroglutamate formation should not strictly be categorized as a degradation event, since this amino acid residue imparts aminopeptidase resistance to the pro-tein and the presence of glutamine or pyroglutamic acid in the N-terminal position of many proteins is a naturally occurring source of heterogeneity. 

L-Glutamic acid does not racemize in neutral solution, even at 100°C. Deviation of pH from neutral to greater than 8.5 results in thermal racemization with loss of taste characteristics. Racemization in neutral solution occurs at 190 °C after formation of the lactam, 5-oxo-L-proline, pyroglutamic acid [98-79-3]. 

Patients have metabolic acidosis caused by excessive formation of 5-oxoproline (pyroglutamic acid Fig. 40-6, reaction 2). This occurs because the diminution of intracellular glutathione relieves the feedback inhibition on the y-glutamylcysteine synthetase pathway (reaction 1), thereby augmenting the concentration of y-glutamylcys-teine and the subsequent conversion of this dipeptide to cysteine and 5-oxoproline in the cyclotransferase pathway (reaction 4). 

An angiotensin-converting enzyme inhibitor 64 was prepared in several steps from L-pyroglutamic acid. The formation of the bicyclic system was achieved with methylene glutaric anhydride (89TL3621). 

When solid-phase peptide synthesis was initially being developed, the question of whether or not a separate neutralization step is necessary was considered. Since it was known from the work of others that the chloride ion promotes racemization during the coupling step in classical peptide synthesis, and since we were deprotecting the Boc group with HC1, it seemed advisable to neutralize the hydrochloride by treatment with TEA and to remove chloride by filtration and washing. This short, additional step was simple and convenient and became the standard protocol. Subsequently, we became aware of three other reasons why neutralization was desirable (1) to avoid weak acid catalysis of piperazine-2,5-dione formation, 49 (2) to avoid acid-catalyzed formation of pyroglutamic acid (5-oxopyr-rolidine-2-carboxylic acid), 50 and (3) to avoid amidine formation between DCC and pro-tonated peptide-resin. The latter does not occur with the free amine. 

Scheme 1 Formation of Pyroglutamic Acid from Glutamine, Glutamic Acid, and Related Derivatives...

Base-catalyzed pyroglutamic acid formation has been reported for Fmoc-Glu(OBzl)-peptide in piperidine deprotection steps. By the use of short deprotection times (1 min) with 50% piperidine/DMF this side reaction was no longer detectable. 39 ... 

The key step is the construction of a bicyclo[4.2.1] system. To this end, pyrrolidine derivative 95 having fif-substituents is synthesized from (+)-pyroglutamic acid and subjected to enyne metathesis using Ig to result in formation of 96 in 84% yield. From 96, synthesis of (+)-anatoxin-a is straightforward and successfully achieved. 

A novel reaction of pyroglutamate (6) and an isocyanate promoted by NaH in THF leads to functionalized hydantoins (7) in good yields. The reaction involves the ring closure of intermediate (8) by a nucleophilic attack on the carbonyl of the ester function followed by expulsion of an alkoxide anion resulting in the formation of the bicyclic intermediate (9). The alkoxide anion in turn can open this bicyclic intermediate with formation of anions (10) and (11) leading to the final racemic hydantoins (7) (Scheme 3).8... 

Other degradation mechanisms. Additional degradation reactions include N-terminal degradation to form pyroglutamic acid formation (Fig. 137) (193) and N-terminal degradation diketopiperazine formation (Fig. 138) (194). 

Oxidation of proline and arginine residues leads also to formation of glutamate semialdehyde and, upon its further oxidation, to pyroglutamic acid (A9). Oxidized proline produces also 2-pyrrolidone (K6) (Fig. 5). 

To avoid the intramolecular cyclization of N-terminal Gin peptide leading to the formation of N-terminal pyroglutamic acid peptide, it is recommended to cleave the peptide from the support before removing the N-Fmoc terminal protection. The DMF/piperidine treatment has to be performed in solution followed by concentration of the mixture. The target peptide is then precipitated by MTBE and filtered. To minimize epimerization during N-protected cysteine coupling, some coupling methods are recommended ... 

Glutamic acid was replaced by tyrosine in position one of a-conotoxin GI to prevent pyroglutamic acid formation at the N-terminal. This strategy also introduced an amino acid detectable at = 280 nm by UV. 

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