Methanol esterification of Asp/Glu/C-term

MeOH + TFA + free carboxyl → methyl ester. +14 Da. Common artifact when peptide is concentrated from MeOH/TFA HPLC fractions.

Why it happens (mechanism)

Acid-catalyzed Fischer esterification: -COOH + MeOH → -COOMe + H₂O. Slow at RT but accelerates dramatically during rotary evaporation (heat + acid + concentrated MeOH).

When it strikes (triggers)

MeOH-based HPLC mobile phase + TFA buffer. Concentration by rotavap of the collected fractions. Asp- or Glu-containing peptides hold methyl ester for hours.

How to spot it (MS signature)

+14.02 Da. Multiple Asp/Glu = multiple esterifications possible. Check by prolonged 0.1 M NaHCO₃ wash (mild base hydrolyzes methyl ester slowly).

How to prevent it

• Switch to MeCN/H₂O/TFA HPLC (acetonitrile doesn't esterify). Caveat: trace AcOH from MeCN can acetylate.
• Lyophilize directly from HPLC fractions; avoid rotavap concentration.
• If MeOH must be used, use a higher-pH buffer (5 mM ammonium acetate) instead of TFA for that purification.

If it already happened (salvage)

• Mild aqueous base (pH 9-10, RT, 2-4 h) hydrolyzes methyl ester back to acid. Monitor by HPLC.

Source

Yi Yang, Side Reactions in Peptide Synthesis (Elsevier, 2016), Chapter 14, §14.3; 8.1.2.