Abstract:A study was conducted to determine the effect of amino acid sequence on the rate of formation of pyroglutamate (pyrrolidone carboxylate) residues from amino‐terminal glutaminyl residues in peptides. The extent of cyclization from H‐Gln‐X‐OCH2‐resin was determined by a quantitative reaction, and found to be quite pronounced with X being Asn, Leu and Gly. The peptide‐resin with Leu was used to examine the influence of various reagents on the rate of pyroglutamate formation.
“…The other peak is at m / z = 583, which corresponds to loss of NH 3 from [QPG 4 K+2H] 2+ . The m / z = 583 product is a pyroglutamate moiety that is produced by the cyclization of Gln. − Scheme illustrates a simple mechanism of pyroglutamate formation through nucleophilic attack by the N-terminal amino group on the carbonyl carbon of the Gln side chain. The same reaction can happen with glutamate at the N terminus but with a slower rate as shown in a previous study .…”
Diketopiperazine
(DKP) formation is an important degradation pathway
for peptides and proteins. It can occur during synthesis and storage
in either solution or the solid state. The kinetics of peptide cleavage
through DKP formation have been analyzed for the model peptides Xaa1-Pro2-Gly4-Lys7 [Xaa = Gln,
Glu, Lys, Ser, Phe, Trp, Tyr, Cha (β-cyclohexylalanine), Aib
(α-aminoisobutyric acid), Gly, and Val] at multiple elevated
temperatures in ethanol with ion mobility spectrometry–mass
spectrometry (IMS–MS). When Xaa is an amino acid with a charged
or polar side chain, degradation is relatively fast. When Xaa is an
amino acid with a nonpolar alkyl side chain, the peptide is relatively
stable. For these peptides, a bulky group on the α carbon speeds
up dissociation, but the kinetic effects vary in a complicated manner
for bulky groups on the β or γ carbon. Peptides where
Xaa has a nonpolar aromatic side chain show moderate dissociation
rates. The stability of these peptides is a result of multiple factors.
The reaction rate is enhanced by (1) the stabilization of the late
transition state through the interaction of an aromatic ring with
the nascent DKP ring or lowering the activation energy of nucleophilic
attack intermediate state through polar or charged residues and (2)
the preference of the cis proline bond favored by
the aromatic N-terminus. The number of unseen intermediates and transition
state thermodynamic values are derived for each peptide by modeling
the kinetics data. Most of the transition states are entropically
favored (ΔS
⧧ ∼ −5
to +31 J·mol–1·K–1),
and all are enthalpically disfavored (ΔH
⧧ ∼ 93 to 109 kJ·mol–1). The Gibbs free energy of activation is similar for all of the
peptides studied here (ΔG
⧧ ∼ 90–99 kJ·mol–1).
“…The other peak is at m / z = 583, which corresponds to loss of NH 3 from [QPG 4 K+2H] 2+ . The m / z = 583 product is a pyroglutamate moiety that is produced by the cyclization of Gln. − Scheme illustrates a simple mechanism of pyroglutamate formation through nucleophilic attack by the N-terminal amino group on the carbonyl carbon of the Gln side chain. The same reaction can happen with glutamate at the N terminus but with a slower rate as shown in a previous study .…”
Diketopiperazine
(DKP) formation is an important degradation pathway
for peptides and proteins. It can occur during synthesis and storage
in either solution or the solid state. The kinetics of peptide cleavage
through DKP formation have been analyzed for the model peptides Xaa1-Pro2-Gly4-Lys7 [Xaa = Gln,
Glu, Lys, Ser, Phe, Trp, Tyr, Cha (β-cyclohexylalanine), Aib
(α-aminoisobutyric acid), Gly, and Val] at multiple elevated
temperatures in ethanol with ion mobility spectrometry–mass
spectrometry (IMS–MS). When Xaa is an amino acid with a charged
or polar side chain, degradation is relatively fast. When Xaa is an
amino acid with a nonpolar alkyl side chain, the peptide is relatively
stable. For these peptides, a bulky group on the α carbon speeds
up dissociation, but the kinetic effects vary in a complicated manner
for bulky groups on the β or γ carbon. Peptides where
Xaa has a nonpolar aromatic side chain show moderate dissociation
rates. The stability of these peptides is a result of multiple factors.
The reaction rate is enhanced by (1) the stabilization of the late
transition state through the interaction of an aromatic ring with
the nascent DKP ring or lowering the activation energy of nucleophilic
attack intermediate state through polar or charged residues and (2)
the preference of the cis proline bond favored by
the aromatic N-terminus. The number of unseen intermediates and transition
state thermodynamic values are derived for each peptide by modeling
the kinetics data. Most of the transition states are entropically
favored (ΔS
⧧ ∼ −5
to +31 J·mol–1·K–1),
and all are enthalpically disfavored (ΔH
⧧ ∼ 93 to 109 kJ·mol–1). The Gibbs free energy of activation is similar for all of the
peptides studied here (ΔG
⧧ ∼ 90–99 kJ·mol–1).
“…The highest yields of DKP peptides are obtained with Glu(OtBu) at the N‐terminus (Figure ). A few examples have been described in the literature of the Glu→Pyr transition during peptide synthesis as the lactamization reaction is generally catalyzed by weak acidic media . However, Pyr formation at the expense of a preformed Cγ peptide bond is a novel reaction in peptide chemistry. Cyclic peptides (43) to (45) obtained as mentioned earlier with relatively high yields can be used for synthesis of derivatives (46) to (54) containing Ile or Trp as R1 substituents.…”
A series of linear peptides with the general formula H-Glu(R1)-Glu(R2)-OH was subjected to cyclization under standard conditions. Formation of respective 2,5-diketopiperazines was accompanied by transformation of the N-terminal Glu(R1) to pyroglutamic acid residue. Even in the case R1 is an amino acid residue attached to the N-terminal γ-carboxyl group, lactamization leads to its elimination. The observed reaction has not been reported so far in the literature. Correspondingly, an alternative route to Glu(R1)-Glu(R2)-containing 2,5-diketopiperazines was applied to improve the overall yields.
“…It is well known that both glutamic acid and glutamine residues are prone to cyclization when located at the N ‐terminus of peptide sequence25, 26. Moreover, glutamic acid can be easily transformed to pyroglutamic acid in water solution at elevated temperature27.…”
N-terminal modification of peptides by unnatural amino acids significantly affects their enzymatic stability, conformational properties and biological activity. Application of N-amidino-amino acids, positively charged under physiological conditions, can change peptide conformation and its affinity to the corresponding receptor. In this article, we describe synthesis of short peptides, containing a new building block-N-amidino-pyroglutamic acid. Although direct guanidinylation of pyroglutamic acid and oxidation of N-amidino-proline using RuO(4) did not produce positive results, N-amidino-Glp-Phe-OH was synthesized on Wang polymer by cyclization of alpha-guanidinoglutaric acid residue. In the course of synthesis, it was found that literature procedure of selective Boc deprotection using TMSOTf/TEA reagent is accompanied by concomitant side reaction of triethylamine alkylation by polymer linker fragment. It should be mentioned that independently from cyclization time and coupling agent (DIC or HCTU), the lactam formation was incomplete. Separation of the cyclic product from the linear precursor was achieved by HPLC in ammonium formate buffer at pH 6. HPLC analysis showed N-amidino-Glp-Phe-OH stability at acidic and physiological pH and fast ring opening in water solution at pH 9. The suggested method of N-amidino-Glp residue formation can be applied in the case of short peptide chains, whereas synthesis of longer ones will require fragment condensation approach.
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