Artificially expanded genetic information system: a new base pair with an alternative hydrogen bonding pattern

Abstract

To support efforts to develop a 'synthetic biology' based on an artificially expanded genetic information system (AEGIS), we have developed a route to two components of a non-standard nucleobase pair, the pyrimidine analog 6-amino-5-nitro-3-(1'-beta-D-2'-deoxyribofuranosyl)-2(1H)-pyridone (dZ) and its Watson-Crick complement, the purine analog 2-amino-8-(1'-beta-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one (dP). These implement the pyDDA:puAAD hydrogen bonding pattern (where 'py' indicates a pyrimidine analog and 'pu' indicates a purine analog, while A and D indicate the hydrogen bonding patterns of acceptor and donor groups presented to the complementary nucleobases, from the major to the minor groove). Also described is the synthesis of the triphosphates and protected phosphoramidites of these two nucleosides. We also describe the use of the protected phosphoramidites to synthesize DNA oligonucleotides containing these AEGIS components, verify the absence of epimerization of dZ in those oligonucleotides, and report some hybridization properties of the dZ:dP nucleobase pair, which is rather strong, and the ability of each to effectively discriminate against mismatches in short duplex DNA.

Figure 1

The AEGIS that follows closely the geometry and hydrogen bonding architecture of natural nucleotides (1,2). The various hydrogen bonding patterns are named pu or py, depending on whether they are presented on a large (purine-like) or small (pyrimidine-like) heterocyclic ring system, with hydrogen bond donor (D) and acceptor (A) groups listed starting in the major groove and ending in the minor groove. Unshared pairs of electrons (or ‘electron density’) presented to the minor groove are shown by the shaded lobes. The pyDDA:puAAD hydrogen bonding pattern, the subject of this paper, is at the bottom right.

Figure 2

Conditions: (a) N, N-Diisobutylformamide dimethyl acetal, MeOH, rt; (b) (MeO)₂TrCl, Py, Et₃N, DMAP, 0°C to rt.

Figure 3

Conditions: (a) DEAD, Ph₃P, dioxane/DMF (4/1, v/v), 0°C to rt, 92%; (b) N, N-Diisobutyl formamide dimethyl acetal, MeOH, rt; (c) i) BzCl, DMAP, Py, Et₃N, 0°C to rt; ii) H₂O, NH₄OH (29%), 0°C, 73% (over two steps); (d) Pd(OAc)₂, Ph₃As, Et₃N, DMF, 55°C; (e) NaBH(OAc)₃, AcOH, CH₃CN, 0°C, 98%; (f) Bu₄NF, AcOH, THF, 0°C, 90%; (g) BzCl, DMAP, Py, Et₃N, rt, 85%; (h) 1 M NaOH / H₂O, 1,4-dioxane, rt, 90%; (i) (MeO)₂TrCl, Py, Et₃N, rt, 80%; (j) iPr₂N(OC₂H₄CN)PCl, DIPEA, CH₂Cl₂, 0°C to rt, 71%.

Figure 4

Conditions: (a) (MeO)₂TrCl, Py, Et₃N, rt, 80%; (b) Ac₂O, Py, rt, 94%; (c) HCl, MeOH, CH₂Cl₂, 0°C, 90%; (d) i) 2-Chloro-4H-1,3,2-benzodioxaphosphorin-4-one, Py, dioxane, rt; ii) tributylammonium pyrophosphate, n-Bu₃N, DMF, rt; iii) I₂, H₂O, Py, rt; (e) i) DBU, CH₃CN, rt; ii) H₂O, NH₄OH, rt, 10% (over two steps).

Figure 5

Conditions: (a) Dibutylformamide dimethyl acetal, MeOH, 40°C, β isomer 53%, α isomer 37%; (b) (MeO)₂TrCl, Py, Et₃N, rt, 75%; (c) iPr₂N(OC₂H₄CN)PCl, DIPEA, CH₂Cl₂, 0°C to rt, 76% ; (d) Ac₂O, Py, rt, 93%; (e) HCl, MeOH, CH₂Cl₂, 0°C, 99%; (f) i) 2-Chloro-4H-1,3,2-benzodioxaphosphorin-4-one, Py, dioxane, rt; ii) tributylammonium pyrophosphate, n-Bu₃N, DMF, rt; iii) I₂, H₂O, Py, rt; iv) H₂O, NH₄OH, 45°C, 20% (over two steps).