In solid-phase oligonucleotide synthesis, phosphoramidite monomers are iteratively and efficiently assembled on solid support. Using this principle, almost any small molecule can be covalently attached to oligonucleotides provided that the corresponding phosphoramidite is available and the small molecule is stable during oligonucleotide synthesis and deprotection. To develop a cathepsin B-cleavable dipeptide phosphoramidite for traceless release of payload after enzymatic cleavage, a self-immolative spacer was designed to link to the dipeptide on one side, and a small molecule on the other side via a phosphodiester linkage. p-Aminobenzyl alcohol is a typical self-immolative linker that has been used in ADCs such as Adcetris. However, we previously reported that the p-acetamidobenzyl(PAB)phosphodiester structure has poor stability during oligonucleotide synthesis/deprotection. We speculate that the acetamido group at the paraposition leads to degradation of the PAB-phosphodiester during oligonucleotide synthesis/deprotection. To evaluate this hypothesis, benzyl, PAB and N-methylated PAB (mPAB) phosphoramidites were synthesized and conjugated to DNA (Figure ). After oligonucleotide synthesis, the DNA conjugates were deprotected with concentrated aqueous ammonia at room temperature for two hours. As shown in Figures and, consistent with the previous report, DNA-PAB conjugate (ODN2) was completely degraded, and was even unstable to ultra-mild deprotection by 50 mM K2CO3 in methanol at room temperature for 4 hours (Figure ). However, DNA-benzyl (ODN1) and DNA-mPAB (ODN3) conjugates have better stability compared to ODN2, with 40.8% degradation for ODN1 and 56.2% degradation for ODN3 (Figures ). These results indicate that PAB-phosphodiester structure has poor stability during oligonucleotide synthesis/deprotection while N-methylation of PAB-phosphodiester improves the stability.