HATU: A Versatile Compound Enhancing Efficiency in Peptide Synthesis and siRNA Conjugation

HATU, 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, is a commonly used condensation reagent and is widely used in the synthesis of amide bonds. HATU promotes the formation of amide bonds by activating the carboxyl group.

Figure 1. HATU

Application of HATU as a peptide coupling reagent

HATU is the N-guanidinium salt of HOAt (1-hydroxy-1H-1,2,3-triazolo[4,5-b]pyridine) and has recently been used for the macrocyclization of complex molecules. For example, Danishefsky reported the total synthesis of himastatin, which contains two identical subunits connected by a biaryl group. In this synthesis, a double macrocyclization of both ends of the linear precursor was achieved using HATU/HOAt/DIEA.

Application of HATU in organic synthesis

In organic synthesis, the application of HATU as a peptide coupling reagent is under investigation. Ehrlich studied the cyclization of all-L tetrapeptides or all-L pentapeptides and pointed out that care needs to be taken to avoid side reactions such as ring dimerization or significant isomerization of the C-terminal residue during the reaction. HOAt-based reagents such as HATU, HAPyU, and TAPipU are more efficient than TBTU or BOP. In particular, alternation of the α-configuration of the linear precursor ensures efficient cyclization. Marchelli successfully optimized the coupling conditions of PNA analogs in solution and solid phase using HATU/trimethylpyridine.

Toxicity information of HATU

When HATU was administered to rats via the oral route at a dose of 2000 mg/kg, limited toxicity was observed, but no mortality was observed. HATU was non-irritating to the skin of rabbits, but slightly irritating to the eyes of rabbits. Studies conducted in guinea pigs have shown that HATU causes contact allergies. In an in vivo micronucleus assay on male mouse bone marrow polychromatic erythrocytes, no HATU-induced micronuclei were observed.

Sensitization potential and mutagenicity testing of HATU as a peptide coupling agent

In the mutagenicity test, the results of the sensitization potential of HATU as a peptide coupling agent and the Salmonella-E. coli/mammalian microsome reverse mutation test are presented. HATU was negative in the mutagenicity test, but was not detected in the local lymph node assay (LLNA), while it was positive in the guinea pig maximal test.

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