The lower molecular weight peaks associated with the CSO-oxidized oligo are deletion mutants (-1, -2 and -3 dTs), which suggests the oxidation time of 3 minutes should have been increased slightly for an oligo of this length. (The splitting of the -DBCO peaks is 14 Da, indicating the formation of both the amide and N-methylamide linkers which results from the oligo being deprotected in AMA). During deprotection, the DBCO is cleaved off, leaving a hexamido linker present. We introduce the concept of positional diversity by placing the DNA attachment at either of two possible sites on the. What appears to have occurred during oxidation with iodine is the formation of an N-iodo amide, making the amide linkage unstable. Here we report the successful construction of a novel, stereochemically diverse DNA-Encoded Chemical Library (DECL) by utilizing 24 enantiomerically pure trifunctional 2, 6- di-substituted piperazines as central cores. It is clear that the DBCO moiety is being cleaved off when exposed to iodine-based oxidizers. Figure 3 shows the deconvolved electrospray MS data for the same sequence synthesized using standard 0.02 M Iodine versus 0.5 M CSO with the target mass being 20,511 Da. by Microsynth), the number of high-quality sequences retained for each. So, when the 63-mer was re-synthesized using 0.5 M CSO in acetonitrile (40-4632-xx) and a 3 minute oxidation time, the hoped-for improvement was dramatic. Total DNA was extracted from soil samples using the FastDNA SPIN Kit for soil (MP. With other nucleoside analogs with sensitivity to iodine, we have achieved good results using an alternative oxidizer, (1S)-(+)-(10-Camphorsulfonyl)-oxaziridine (CSO) (2).įigure 2: RP HPLC of Test 12-mer containing three DBCO-dT additions As shown in Figure 2b, the resulting degradation was quite dramatic. This exposure is equivalent to roughly 20 synthesis cycles. To test this hypothesis, CPG from the 12-mer synthesis (Figure 2a) was subjected to treatment with standard DNA synthesis oxidizer, 0.02 M Iodine, for 5 minutes at room temperature. However, when analyzed by RP HPLC, the resulting oligonucleotide looked very impure and mass spec analysis confirmed that the DBCO had been substantially cleaved off the linker.Ĭonceptually, this was unexpected since amide linkages are resistant to hydrolysis, which implied that DBCO-dT is sensitive to one or more of the synthesis reagents and that the repeated exposure during the synthesis of long oligos led to cleavage of the DBCO. Since the performance of this batch of DBCO-dT looked good, as a courtesy, we offered to synthesize the customer's longer sequence. DBCO-dT (1) CSO (2) Figure 1: Structures of DBCO-dT and CSO
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