Several conditions have been used in the coupling reaction of stepwise SPPS at elevated temperature (SPPS-ET), but we have elected the following as our first choice: 2.5-fold molar excess of 0.04-0.08 M Boc or Fmoc-amino acid derivative, equimolar amount of DIC/HOBt (1:1) or TBTU/DIPEA (1:3), 25% DMSO/toluene, 60 degrees C, conventional heating. In this study, aimed to further examine enantiomerization under such condition and study the applicability of our protocols to microwave-SPPS, peptides containing L-Ser, L-His, L-Cys and/or L-Met were manually synthesized traditionally, at 60 degrees C using conventional heating and at 60 degrees C using microwave heating. Detailed assessment of all crude peptides (in their intact and/or fully hydrolyzed forms) revealed that, except for the microwave-assisted coupling of L-Cys, all other reactions occurred with low levels of amino acid enantiomerization (<2%). Therefore, herein we (i) provide new evidences that our protocols for SPPS at 60 degrees C using conventional heating are suitable for routine use, (ii) demonstrate their appropriateness for microwave-assisted SPPS by Boc and Fmoc chemistries, (iii) disclose advantages and limitations of the three synthetic approaches employed. Thus, this study complements our past research on SPPS-ET and suggests alternative conditions for microwave-assisted SPPS.

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Highly electrophilic α-dicarbonyls such as diacetyl, methylglyoxal, 3-deoxyglucosone, and4,5-dioxovaleric acid have been characterized as secondary catabolites that can aggregate proteins and form DNA nucleobase adducts in several human maladies, including Alzheimer's disease, rheumatoid arthritis, diabetes, sepsis, renal failure, and respiratory distress syndrome. In vitro, diacetyl and methylglyoxal have also been shown to rapidly add up the peroxynitrite anion (k2 ~ 10(4)-10(5) M(-1) s(-1)), a potent biological nucleophile, oxidant and nitrosating agent, followed by carbon chain cleavage to carboxylic acids via acetyl radical intermediate that can modify amino acids. In this study, we used the amino acid derivatives Ac-Lys-OMe and Z-Lys-OMe and synthesized the tetrapeptides H-KALA-OH, Ac-KALA-OH, and H-K(Boc)ALA-OH to reveal the preferential Lys amino group targeted by acyl radical generated by the α-dicarbonyl/peroxynitrite system. The pH profiles of the reactions are bell-shaped, peaking at approximately 7.5; hence, they are close to the pKa values of ONOOH and of the catalytic H2PO4(-) anion. RP-HPLC and ESI-MS analyses of reaction products confirmed (α)N- and (ϵ)N-acetylation of Lys by diacetyl as well as acetylation and formylation by methylglyoxal, with preference for the α-amino group. These data suggest the possibility of radical acylation of proteins in epigenetic processes, where enzymatic acetylation of these biomolecules is a well-documented event, recently reported to be as critical to the cell cycle as phosphorylation. Also noteworthy is the observed formylation of L-Lys containing peptides by methylglyoxal never reported to occur in amino acid residues of peptides and proteins.

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