Figure 4.

Cleavage and re‐ligation of the squaramide backbone. Note that oligonucleotides are colour‐coded in all graphs based on the text and box colours in the reaction Scheme in (A). A) The various products generated upon treatment of squaramide‐containing oligonucleotides with ethylenediamine. Conversion of the 3′‐squaramide‐ethylenediamine adduct to the free 3′‐amine is the rate‐determining step (r.d.s.) to regenerate the 5′‐amino and 3′‐amino oligonucleotides for re‐ligation. B) An example of squaramide cleavage (1:3 v/v EDA to oligonucleotide in water, 3 h at 55 °C) and re‐ligation (squarate ester in 15 mm sodium borate buffer, pH 8.5 containing 0.2 m NaCl). Each reaction was analysed by UPLC‐MS with the UPLC traces shown here. Relative amounts of the squaramide‐containing oligonucleotide were determined by integration of the traces, which were all normalised to the splint peak intensity (grey). C) Time‐dependent cleavage of squaramide‐containing oligonucleotides using 1:3 v/v of ethylenediamine to oligonucleotide in water at 55 °C. Both UPLC traces and mass spectrometry deconvolution of these peaks are shown to highlight that the removal of the 3′‐squaramide‐EDA adduct is rate limiting in obtaining the free amino oligonucleotides. UPLC peaks were integrated to determine relative conversion of the squaramide‐adduct oligonucleotide to the free amino oligonucleotides. Note that all traces are normalised to the splint peak intensity (grey). For mass spectrometry, the ratio of deconvoluted masses is shown to estimate squaramide‐adduct amounts. This assumes ionisation of the free amino oligonucleotide and squaramide‐ethylenediamine adduct is comparable and dominated by the negative charge of the oligonucleotide. m/z (3′‐squaramide‐EDA adduct)=3957 (expected), 3957 (found); m/z (5′‐squaramide‐EDA adduct)=6405 (expected), 6406 (found). The oligonucleotides used for (B) are listed in Supplementary Table 2 and for (C) in Supplementary Table 1.