Summary and conclusions

The described zinc-finger-based metal sensors derived from click-chemistry successfully demonstrated the use of CuAAC to introduce a fluorophore to a metal-coordination site of a peptide. The in-situ generated 1,2,3-triazole moiety was able to mimic the coordination properties of histidine and thereby maintained all characteristic structural features as confirmed by CD spectroscopy. The same method revealed the importance of the spacer-length that separates the fluorophore from the metal coordination site. With decreasing length, the metal binding ability of the peptide was inhibited and led to an inaccurate secondary structure formation as observed for Zf3COUM and Zf3ACEN. It can be assumed that the close proximity of the fluorophore to the metal coordinating N2 atom of the triazole moiety negatively influenced the metal coordination. The influence might either derive from shielding effects of the fluorophore on the metal binding site or due to additional functional groups of the fluorophores, which are also able to perform metal coordination such as the carboxyl oxygen of coumarin. This could have established a competing binding mode, which led to inaccurate peptide folding. To evaluate these assumptions, future experiments could target the precise impact of the spacer length. It remains to be emphasized that a spacer of similar length as the one used for Zf3DNS might also be beneficial for other fluorophores.

The hypothesis of a competing binding site needs to be evaluated for example by nuclear magnetic resonance spectroscopy to exactly prove if metal binding proceeds via the N2 or N3 nitrogen atom of the triazole. Such experiments were performed for the present systems but due to the enormous abundance of signals it was not possible to draw solid conclusions.

The use of a reduced model system could remedy this deficiency.

The aforementioned coordination mismatches for the metal complexes of Zf3COUM and Zf3ACEN were further confirmed by UV-vis titration experiments, which were performed to investigate the molar ratio of metal to peptide binding and to determine the dissociation constants for the zinc finger complexes with Co(II) and Zn(II). Whereas the spectra of Zf3COUM and Zf3ACEN showed no d-d transition upon Co(II) addition, the latter was obtained for Zf3DNS and used to determine the Kd for the ZF-Co^II complex to be 7.16 ± 0.4 ^M. In a reverse titration with Zn(II), the dissociation constant for the ZF-Zn^II complex was determined to be 150.2 ± 14.4 pM. These values are in overall good agreement with published dissociation constants for other zinc finger mutants found in the literature.^[69]

Fluorescence titration experiments for Zf3DNS with different divalent metal ions were performed to test its ability to function as fluorescent metal sensor. The findings clearly demonstrated that the peptide shows an increase in fluorescence intensity exclusively upon Zn(II) addition. This switch-on mechanism resulted in a 40% increased fluorescence at saturating Zn(II) levels with regard to the initial metal-free value. In contrast to that, all other examined divalent metal ions showed moderately decreased fluorescence intensities within an 8% range with regard to metal-free conditions (Figure 5.16).

These findings imply that the metal sensing ability of Zf3DNS relies on the PET mechanism described in section 5.3. This assumption is based on two facts. Firstly, the presence of a spacer that connects the metal coordination site and the fluorophore is a major indication for a PET mechanism. This condition was met with the ethylamine moiety that connects the dansyl group and the triazole. The absence of delocalized electrons within the spacer makes an ICT mechanism rather unlikely due to an insufficient signal transmission from the metal binding site to the fluorophore. Secondly, the metal addition led to a change in fluorescence intensity under retention of the absorbance wavelength. The still observed hypsochromic or bathochromic shifts may be attributed to peptide folding and therefore, to local changes in the environment of the fluorophore upon changing from the unfolded state to the folded state where the latter is partially shielded. An ICT mechanism as mentioned in the beginning can be virtually excluded due to the observation of minor spectral shifts within 7 nm. Due to the findings for multiple other ICT-based metal sensors, the expected spectral shift should be significantly more pronounced.^[185] In addition, the turn-on fluorescence sensing mechanism exclusively observed for Zn(II) is in very good agreement with other PET based metal

Figure 5.16 Fluorescence spectra of the dansyl modified zinc finger (Zf3DNS) saturated with different divalent metal ions (1.6 eq), as indicated. The peptide was used in a concentration of 25 M in HEPES buffer (20 mM, 500 M TCEP, pH 7.0). The initial fluorescence was determined by the addition of EDTA (50 M) to complex residual metals.

sensors.^[202] The fluorescence decrease observed for the other examined metals is rather due to the aforementioned folding event than due to a simultaneously occurring turn-off effect. Despite the fact, that only a few examples are known, which combine a turn-on and turn-off mechanism, the energy diagrams shown in Figure 5.6 clearly prohibit such a mechanism for solely PET based metal sensors.^[203] Hence, metal sensors that comprise a simultaneous on-off mechanism are usually composed of two different binding sites that also show different metal selectivity and fluorescence response upon complex formation.

The resolution of the Zf3DNS sensor to detect Zn(II) is rather limited to the relatively low brightness of the fluorophore than to the metal binding ability of the peptide, which was found to be in the upper nanomolar range. Compared to other fluorophores, the azido-N-dansylethylamine fluorophore has a comparably low molar extinction coefficient of 3400 M^-1 cm^-1.^[204] The abolition of the PET quenching upon Zn(II) addition increased its fluorescence intensity by 40%. In contrast to other fluorescence metal sensors this is a rather small increase, which might be attributed to the relatively long spacer. Thus, the efficiency of the PET could be reduced as indicated at the beginning. These characteristics leave room for improvements concerning the attachment of brighter fluorophores with variable spacer-lengths. However, the reported zinc-finger-based peptidyl metal sensor was found to be fully operable and able to distinguish between Zn(II) and other divalent metal ions the zinc finger is able to bind.

The successful synthesis of a zinc-finger-based peptidyl metal sensor suggests that histidines, which are part of a metal binding site in peptides, proteins or other molecules can be exchanged for a propargylglycine residue to enable metal coordination by a click-derived triazole moiety. The applied CuAAC allows the direct modification of the metal binding site with conjugated fluorophores in a mild, fast and orthogonal reaction. This could lead to new insights in processes, which involve metal coordination in biomolecules due to the possibility of metal sensing in an aqueous environment. A potential field of application is for example the pathogenesis of Alzheimer’s disease. In current studies, the involvement of histidine residues in Zn(II) and Cu(II) complexation is under investigation because this process is expected to cause the formation of highly neurotoxic amyloid- fibrils and plaques.^[205] An exchange of the eligible histidines for the herein described system, could help to spread further light on the progression of this disease. The modification of triazoles with other functional molecules instead of fluorophores was demonstrated by RAO.In her approach, a second metal binding site derived from a 1,4,7-triazacyclononane (TACN) modified triazole was in-situ generated in the zinc finger sequence in close proximity to the native metal binding site.^[206] The thus generated dinuclear Zn(II) complex was intended to be used as sequence specific artificial nuclease. It is important to note, that even with such a complex

system, the secondary structure of the zinc finger mutant was successfully established as confirmed by CD spectroscopy.

In summary, tailor-made post synthetic modifications of metal binding peptides or proteins, either synthesized by the means of solid phase peptide synthesis or recombinantly expressed, are easily accessible by CuAAC. 1,2,3-Triazoles are excellent surrogates for histidines, which can easily be generated from propargylglycine that is furthermore genetically encoded for the use in direct ribosomal synthesis.^[207] In combination with the highly orthogonal click reaction, this would even allow the modification of larger proteins.