2.4.2. Non-RAHB Cases

Deuterium isotope effects on 15N chemical shifts have been studied in the mono anion of 2,3-dipyrrol-2-ylquinoxaline and its 6,7-dintro derivative (see Figure 15E) [56]. The effects 1.13 ppm and 0.88 ppm (signs have been changed from the original publication) are rather large and indicate a strong hydrogen bond.

Deuterium isotope effects on 15N and 1H chemical shifts have been used to judge whether certain salt bridges, which are observed in the solid also exist in solution. An example is protein G, B1 domain. The isotope effect demonstrated that two salt bridges found in the X-ray structure did not exist in solution [77]. This approach was also used in Barnase [78]. Both types of isotope effects have also been treated theoretically [79].

In ubiquitin the one-bond deuterium isotope effects of hydrogen bonded NH of the back-bone is correlated to the back bone angles and the angle between the acceptor oxygen and the NH bond (Figure 21) [80].

**Figure 21.** Angles and distances for an inter-chain hydrogen bond. Taken from [80] with permission from Springer.

In DNA and RNA through hydrogen bond isotope effects on chemical shifts can be seen from H-3 to C-2 between adenine and thymine respectively uracil. The isotope effects on chemical shifts are found to be sensitive to the N1-N3 distance suggesting that the isotope effect is sensitive to hydrogen bond strength (see Figure 22) [81,82].

**Figure 22.** Example of hydrogen bonding in an adenine:thymine base pair.

Very long range isotope effects due to deuteriation at NH have been observed in N-substituted 3-(cycloamino)thioproionamides [83]. These effects were ascribed to electric field effects (Figure 23). The use of nuclei with a large chemical shift range like 19F makes this kind of effect very useful even for weaker hydrogen bonds.

**Figure 23.** Example of electric field isotope effect through space in N-substituted 3-(cycloamino)- thioproionamides.
