Fragmentation / deletion Δm loss of N-terminal dipeptide severity: high

Aib²-mediated DKP in GLP-1/tirzepatide-class peptides

Tirzepatide and GLP-1 analogs containing Aib at position 2 suffer from accelerated DKP formation after the second Fmoc deblock. The dialkyl Aib stabilizes a cis-amide, just like Pro does, and the dipeptide cyclizes off the resin. Documented as a major industrial-scale issue.

Affected residue(s): any (Aib at position 2)

Neighbour(s) that trigger it: Aib MeAA

Why it happens (mechanism)

Identical to ordinary DKP (see DKP entry) but Aib's two methyl groups force the Cα of the bis-α-substituted residue into a constrained geometry that favors cis-amide pre-organization. After Fmoc-removal of residue 2, the free α-amine is geometrically locked in for nucleophilic attack on the resin-tethering ester. The yield drop can exceed 30% in poorly-controlled batches.

When it strikes (triggers)

Aib (or any α,α-dialkyl AA, or N-Me-Xaa) at position 2 of an SPPS sequence. Wang/HMPA resins (good leaving group). Slow coupling of residue 3 (e.g., when residue 3 is hindered, like another Aib). Prolonged base exposure between deblock and coupling. Worse in microwave-assisted SPPS.

How to spot it (MS signature)

Yield loss + cyclo(Xxx-Aib) DKP visible by HPLC of the post-coupling-3 wash. Truncated peptide missing the first two residues.

How to prevent it

Use CTC resin for any peptide with Aib/MeAA at position 2 — bulky linker disfavors the cyclization. This is now standard for tirzepatide-class production.
Couple residue 3 immediately after Fmoc-removal of residue 2 — minimize the time the dipeptide-Aib sits exposed to base.
Pre-form the residue-3 active ester before beginning the Fmoc deblock of residue 2 (parallel preparation).
Use DIC + Oxyma for residue-3 coupling — works at room temperature, no microwave needed.
Avoid Wang or HMPA resin for these sequences; if using PAL/Rink (amide C-terminus), the issue is similar but slightly less pronounced.

If it already happened (salvage)

Once detached, the dipeptide is gone — the resin is now -2 residues short. No salvage. Re-load with proper protocol.

Source

Yi Yang, Side Reactions in Peptide Synthesis (Elsevier, 2016), Chapter 1, §1.10 (DKP background); literature: ACS Omega 2023, doi:10.1021/acsomega.2c05915 (tirzepatide DKP study).