Due to the increased stability, when compared to other dipyrrinato-metal complexes, a large number of modifications to the BODIPY core have been achieved. The group that has received the most attention has been the aromatic group at the 8-position, which is an easily modified unit that can have a large effect on the fluorescent properties of the BODIPY. BODIPYs can also be synthesised with groups that allow in vitro chemical modifications to occur (e.g. metal ion complexation) promoting their use as fluorescent probes for biological systems. They can be used as sensors for redox active molecules,37, 60-62
pH probes,39 metal ions63-71 and probes for conjugation to biologically active substrates.72-74 In the examples presented in Figure 1.10, fluorescence is turned ‗ON‘ or ‗OFF‘ by photoinduced electron transfer (PeT) which is controlled by changes in the oxidation potentials of the 8-aryl groups when they interact with certain chemical species.
NH2
NH2
Nitric oxide probe
N pH probe N OH OH Pb2+ Pb2+probe B O O Monosaccaride probe
Figure 1. 10: Meso-modified BODIPY probes
2.4.1 Photoinduced electron transfer (PeT)
The primary factor exploited in BODIPYs to modify their fluorescence is photoinduced electron transfer. This involves the transfer of excited electrons between non-planar parts of a fluorescent molecule.75 This transfer of excited electrons has a significant effect on the fluorescence intensity of photoactive molecules. By calculating the orbital energy levels of the two non-planar parts of a molecule, it can be predicted whether a certain change will alter the fluorescence characteristics.76-78
Fluorescence LUMO HOMO meso-subst. BODIPY core Fluorescent N N R B F F
Figure 1. 11: BODIPY where no PeT can occur
For BODIPYs, the majority of substituents attached to the 8-position are aromatic groups. When this electron transfer occurs it prevents the loss of energy by fluorescence processes. It is worth noting that, during the design of new BODIPY probes that exploit this effect, that the fluorescence intensity of the BODIPY is still reduced by the effects of molecular motion. This means that the rotation would still have to be taken into account before synthesis is carried out to ensure that the target molecule‘s change in fluorescence intensity by PeT is large enough for it to be a suitable probe.
PeT can occur by either a reductive or oxidative process. If the substituent attached to the BODIPY core is donating electrons to the BODIPY core in the excited state, i.e. reducing it, then this is reductive PeT or a-PeT (―a‖ for acceptor). (Fig. 1.12)
LUMO HOMO meso-subst. BODIPY core Fluorescence reduced N N R B F F a-PeT e-
Figure 1. 12: Reductive PeT (a-PeT)
Alternatively, if the excited BODIPY core can donate electrons to the substituents LUMO then oxidative PeT, or d-PeT (―d‖ for donor), is occurring (Fig. 1.13).
LUMO HOMO meso-subst. BODIPY core Fluorescence reduced N N R B F F e- d-PeT
Figure 1. 13: Oxidative PeT (d-PeT)
The likelihood of electron transfer can be predicted by calculating the change in free energy (ΔGPeT). This can be calculated from the Rehm-Weller equation:79
C E A A E D D E GPeT 1/2( / ) 1/2( / ) 00
Where E1/2(D+/D) is the ground state oxidation potential of the electron donor, E1/2(A/A-) is the ground state reduction potential of the electron acceptor, ΔE00 is the excitation energy, and C is an electrostatic interaction term. The primary problem with using the approach of orbital energy level calculations is that the energy levels are calculated for the isolated meso-substituents. By attaching them to the BODIPY core, it is reasonable to assume that their energy levels are altered somewhat, giving this method a reasonably large margin for error. However, this approach is useful to give a reasonable prediction of the effect that certain substituents or structural changes will have on the fluorescence of this molecule.
A prime example of the use of this equation to predict PeT in BODIPY systems has been used for the development of a nitric oxide probe. For this probe, a diamine was converted
in vitro to a benzotriazole by reaction with nitric oxide.40 NH2 NH2 N N B F F HN N N N B F F N NaNO2/HCl
Fluorescence quantum yield = 0.012 Fluorescence quantum yield = 0.496
Scheme 1. 8: Mechanism of BODIPY nitric oxide probe
The HOMO energy levels of various potential meso-substituents were calculated including the diamine and the benzotriazole. The low fluorescence of the diamine was attributed to reductive PeT. This effect is quenched upon conversion to the benzotriazole which causes a large increase in fluorescence. This reductive PeT effect has also been exploited for zinc(II) and NO2+ sensors.80, 81 In these cases, coordination to the metal or nitration makes the reduction potential of the meso-substituent more negative, which quenches PeT and causes the BODIPYs to become fluorescent.
This ON/OFF switching has also been investigated with regard to oxidative PeT. It has been discovered that placing strongly electron withdrawing groups on the meso- substituent tends to lower the LUMO of the aromatic unit. If lowered to an appropriate level, this allows the meso-substituent to accept electrons from the excited state of the BODIPY core. This effect was investigated by attaching nitro groups to the meso- substituent and by attaching acetyl groups to the 2,6-positions of the BODIPY core. While the nitro groups lower the LUMO of the aromatic substituent, the acetyl groups lower the energy level of the BODIPY orbital containing the excited electron. This brings both orbitals to a similar energy level, promoting oxidative PeT.81 ON/OFF switching of PeT has also been observed for the phosphorylated forms of 8-(4-hydroxyphenyl) BODIPY. Phosphorylation caused PeT to switch off by lowering the HOMO energy level of the aryl substituents.82