Acetylation

Acetylation is a post-translational modification consisting of grafting an acetyl group (-COCH₃) onto a peptide, typically at the free N-terminal amine (α-acetylation) or a lysine side-chain amine (ε-acetylation). In mammals, it is catalyzed by various acetyltransferases (NAT, HAT) using acetyl-coenzyme A as acetyl donor, and can be reversed by deacetylases (HDAC, sirtuins).

Functionally, acetylation plays several critical roles in peptide biology. (1) N-terminal protection: by blocking the free amine, it makes the peptide resistant to aminopeptidases, including the formidable DPP-4. N-terminal acetylation extends plasma half-life by an order of magnitude. (2) Activity modulation: some native peptides are acetylated by the body to activate or regulate their function (α-MSH is an acetylated derivative of ACTH). (3) Chromatin regulation: histone tail acetylation regulates global gene expression.

In peptide research, N-terminal acetylation is a common synthetic strategy to stabilize vulnerable sequences. It is obtained at SPPS end by coupling acetic anhydride to the free α-amine of the last N-terminal residue, before resin cleavage. Extra cost is minimal and stability gain often spectacular.

Several RUO-commercialized peptides exploit this modification: acetylated TB-500 fragment (Ac-Leu-Lys-Lys-Thr-Glu-Thr-Gln), Ac-SDKP (serylaspartyllysylproline, hematopoietic regulation peptide), certain α-MSH analogs. The acetyl presence changes molecular mass by +42 Da compared to the free form, a signature easily verifiable by LC-MS during quality control.

A peptide COA must always specify if the product is acetylated (noted "Ac-" before the name) and provide the corresponding MS mass. Confusing free and acetylated forms is a classic error that can invalidate comparative pharmacology protocols.

Acetylation is a reversible post-translational modification catalysed by histone acetyltransferases (HAT) and removed by histone deacetylases (HDAC), regulating chromatin (acetylation of lysines 9, 14, 27 of H3 and lysines 5, 8, 12, 16 of H4) and protein activity (p53, tubulin, transcription factors). On a synthetic peptide, acetylation is chemically incorporated during SPPS by reaction with acetic anhydride (Ac2O) in presence of DIEA on resin before cleavage, or by coupling of a preactivated acetyl-amino acid (e.g., Ac-Gly-OH).

In peptide pharmacology, N-terminal acetylation primarily serves to: (1) block serum aminopeptidases that degrade native peptides from the amine end, significantly extending in vivo half-life; (2) mimic native post-translational state for certain endogenous peptides (N-acetylated thymosin β-4, Ac-SDKP); (3) mask the free amine to avoid parasitic interactions in biochemical assays; (4) facilitate membrane crossing by increasing lipophilicity. Acetylation increases molecular mass by 42.01 Da (addition of -COCH3 replacing an H), easily detectable by ESI-HRMS and used as reaction control.