Isosteric and fluorescent DNA base pair formed by 4-amino-phthalimide and 2,4-diaminopyrimidine: melting, structure, and THz polar solvation dynamics

Abstract

An artificial base pair in the center of a duplex DNA oligomer, formed by 2,4-diaminopyrimidine and fluorescent 4-aminophthalimide C-nucleosides, is characterized spectroscopically, with a view towards its use in femtosecond solvation dynamics. Quantum-chemical calculations predict H-bonding energy equivalent to A:T. UV-vis absorption spectra provide insight into local melting at the 4-aminophthalimide modification site. Increase of temperature to nearly the melting temperature of the duplex leads to better hybridisation of the fluorescent nucleoside, contrary to native base pairs. This unusual observation is explained by the NMR solution structure of the duplex. Two conformations are adopted by the artificial pair due to backbone constraints, having either two or one interbase hydrogen bonds. In the latter, hydrogen bonding sites remain accessible for water solvation. The time-resolved dynamic Stokes' shift of 4-aminophthalimide fluorescence is consistent with that of a mixture of a slow and fast species. From the observations, the optimal linkage between 4-aminophthalimide and 2-deoxyribose for fitting into the duplex B-DNA structure is deduced.

An Isosteric and Fluorescent DNA Base Pair Consisting of 4‐aminophthalimide and 2,4‐diaminopyrimidine as C‐Nucleosides

“…4AP is approximately the size of a purine and should therefore be directly applicable as an isosteric DNA base surrogate . Theoretical calculations and NMR structures of dsDNA modified internally with C‐nucleoside 1 opposite 2,4‐aminopyrimidine (DAP) as C‐nucleoside 4 revealed that out of three possible H bonds between them, only two are actually formed . A more serious impediment to future use is the fact that a significant fraction of the duplexes forms only a single H bond between 4AP and DAP, and that the backbone structure adjacent to this artificial pair is distorted from native B‐DNA.…”

“…This is due to the syn conformation of 3 (as discussed below), which interferes with base pairing and inhibits proper stacking within the duplex. [21] Labels 01-26 for the nucleotides are given in Scheme 1a nd in the Supporting Information, together with further details.Inour previous work on the pairing properties of 1 and 4 in DNA1-4, [19] an additional imino proton signal was found that could only be assigned to ac entral A:Tb ase pair, thus indicating that two conformations coexist in that case.F or the current DNA2-4,w ef ind no evidence that asecond structure or conformation is hidden in the data. [17a] If this state is populated, the deactivation into the S 0 state occurs in aradiationless manner.…”

“…Except for DNA3-T,G.the T m values of DNA3-Y are all lower compared to the corresponding duplexes of DNA2-Y,w hich raises the suspicion that the dimethylamino group of 3 perturbs both the double-helical conformation and the base pairing,e specially in It is important to confirm the 4AP:DAP base pair with an NMR solution structure.T he assignment of the NOESY spectra of DNA2-4 was performed according to described methods. [21] Labels 01-26 for the nucleotides are given in Scheme 1a nd in the Supporting Information, together with further details.Inour previous work on the pairing properties of 1 and 4 in DNA1-4, [19] an additional imino proton signal was found that could only be assigned to ac entral A:Tb ase pair, thus indicating that two conformations coexist in that case.F or the current DNA2-4,w ef ind no evidence that asecond structure or conformation is hidden in the data. This is an important result since it shows that by changing the glycosidic connection of the 4AP from C6 (in 1)toC5(in 2), the second conformation is eliminated, as we had predicted.…”

“…[15a] Theoretical calculations and NMR structures of dsDNA modified internally with C-nucleoside 1 opposite 2,4-aminopyrimidine (DAP) as C-nucleoside 4 revealed that out of three possible Hbonds between them, only two are actually formed. [19] Amore serious impediment to future use is the fact that asignificant fraction of the duplexes forms only asingle Hbond between 4AP and DAP, and that the backbone structure adjacent to this artificial pair is distorted from native B-DNA. Obviously, 1 does not offer the correct angle between the glycosidic bond and the imide as a"base-pairing" interface.H ence the attachment of the 2'-deoxyribofuranoside was shifted from the 6-position (in 1)tothe 5-position (in 2)o ft he 4AP chromophore.W es how herein that the new geometry is properly adjusted, and that au nique fluorescent DNAbase pair is formed with DAPthat is well adapted to the overall B-DNAs tructure.T his allows application in primer extensions with different DNAp olymerases.F urthermore, the dimethylated derivative 3 shows increased steric hindrance at the amino substituent and thus serves as an important control for nucleoside syn conformation and its influence on fluorescence and base pairing.…”

Ein isosteres und fluoreszierendes DNA‐Basenpaar aus 4‐Aminophthalimid und 2,4‐Diaminopyrimidin als C‐Nucleoside

“…[18] 4AP weist ungefähr die Grçße eines Purins auf und sollte daher als isosteres DNA-Basensurrogat direkt anwendbar sein. [19] Die Tatsache, dass ein deutlicher Te il der Duplexe nur eine H-Brücke zwischen 4AP und DAPb ildet und dass die Rückgratstruktur neben diesem artifiziellen Paar von der nativen B-DNAa bweicht, ist ein noch ernsteres Hindernis fürd ie Zukunft. [19] Die Tatsache, dass ein deutlicher Te il der Duplexe nur eine H-Brücke zwischen 4AP und DAPb ildet und dass die Rückgratstruktur neben diesem artifiziellen Paar von der nativen B-DNAa bweicht, ist ein noch ernsteres Hindernis fürd ie Zukunft.…”