Stereochemistry of benzylic carbon substitution coupled with ring modification of 2-nitrobenzyl groups as key determinants for fast-cleaving reversible terminators.

Next-generation sequencing (NGS) technologies have facilitated important biomedical discoveries, yet high error rates and slow cycle times warrant further improvements in the chemistry.1a Such technologies that employ the cyclic reversible termination (CRT) method1a,b typically utilize 3′-O-blocked reversible terminators.2a–c Recently, we described a novel 3′-OH-unblocked reversible terminator based on 2-nitrobenzyl-modified 5- hydroxymethyl-2′- deoxyuridine(HOMedU) 5′-triphosphate.3 Our study revealed that the proximity of the 2-nitrobenzyl group to the nucleobaseand the size of the alkyl group attached to its α-methylene carbon are important structural features that confer the unique properties of single-base termination, efficient incorporation, and high nucleotide selectivity (i.e., high fidelity) to these 3′-OH-unblocked nucleotides.3 These properties have the potential to improve accuracy and read-lengths in the CRT method. As HOMedU is a naturally found hypermodified nucleoside,4a we set out to identify other such examples. 5- Hydroxymethyl-2′- deoxycytidine(HOMedC) is found naturally in the genomes of T-even bacteriophages4a,b and mammals.5 Pyrrolopyrimidine (7-deazapurine) is also found naturally in nucleoside antibiotics6 and tRNAs.7 Thus, various analogues of 2-nitrobenzyl-modified 7-deaza-7- hydroxymethyl-2′- deoxyadenosine(C7-HOMedA),8 HOMedC, 7-deaza-7- hydroxymethyl-2′- deoxyguanosine(C7-HOMedG),9 and HOMedU were synthesized with the goal of developing a complete set of reversible terminators (Figure 1).