Synthesis of dinuclear metal complexes based on tyrosine

The general approach for the preparation of the symmetrically substituted dinuclear building blocks is shown in Scheme 2.5.

Scheme 2.5 General scheme for the preparation of the dinuclear building blocks 14 and 16 by a MANNICH reaction of Boc-Tyr-OMe and the synthesized tridentate ligand precursors 4 and 8.

Accordingly, the aforementioned ligand precursors, paraformaldehyde and Boc-Tyr-OMe were used in a MANNICH-reaction. The highest reaction yields were obtained using a slightly modified literature protocol.^[58] In contrast to the published procedure, in which all components were simultaneously added, it turned out to be beneficial to first generate the Schiff base before adding the amino acid. Hence, paraformaldehyde and the ligand precursor were suspended in ethanol/water (1:4) and stirred at 60 °C for 90 min. MANNICH

reactions are known to have individual pH optima, which are dependent on the amines used and the CH-acidic compounds.^[59] Due to resonance stabilization, the phenolic side chain of tyrosine almost exclusively exists in the enol form.^[60] As reported by MINAKAWA, the optimum conditions for the electrophilic substitution request slightly acidic conditions (pH 5.5 – 6.5) to efficiently perform the MANNICH reaction with tyrosine derivatives.^[59] However, this was restricted by the Boc protecting group, which might be cleaved by applying acidic conditions in combination with elevated temperatures for 36 Hence, Boc-Tyr-OMe was dissolved in

ethanol/water (1:4) and 2.5 equivalents of the preformed Schiff-base cocktail were added.

The pH was adjusted to approximately 6.5 – 7.0 by the addition of 1 M HCl (aq.) and the reaction mixture was stirred at 95 °C for 36 h.

It has been observed that minor amounts of the undesired mono-substituted byproduct were present after the reaction workup. Due to similarly low retardation factors of both compounds, an effective chromatographic separation and purification method could not be applied.

Therefore, aliquots were taken from the reaction mixture during the synthesis and analyzed on the presence of mono-substituted products by means of ESI mass spectrometry. In case of detecting the byproduct, further addition of the ligand precursor or an extension of the reaction time was applied. After the reaction has gone to completion, the crude product was extracted with chloroform and purified by RP-flash-column chromatography with water/ethanol (4:1) as eluting system.

In order to use the artificial amino acid in SPPS, a change of the N-terminal Boc protecting groups to Fmoc was necessary and, in addition, the C-terminal methyl ester had to be cleaved off. In order to generate the free carboxylic acid, the compounds were dissolved in methanol and an aqueous solution of sodium carbonate (1 M) was added in excess and refluxed until complete deprotection was observed by TLC. For the deprotection of the Boc group, trifluoroacetic acid was added to the residues and the mixture was agitated for 2 h.

The volatile components were removed in a nitrogen stream and the crude products were precipitated upon the addition of ice-cold diethyl ether. The completely unprotected amino acid was dissolved in water and sodium bicarbonate (3 eq) was added. The N-terminus was Fmoc protected by the addition of Fmoc-succinimide (2.2 eq) as a solution of para-dioxane and the reaction mixture was subsequently stirred at room temperature overnight.^[61] After the extraction of the crude product from the aqueous solution with ethyl acetate, the final products were purified by the previously described RP-flash-column chromatographic method to obtain the building blocks suitable for SPPS.

In a similar approach, the mono-substituted analogues (Figure 2.2) were synthesized in order to compare their hydrolysis rates with the di-substituted building blocks as described in section 2.3. The MANNICH conditions were slightly changed to exclude the formation of the di-substituted products. This was achieved by decreasing the amount of the ligand precursor to 0.95 equivalents with regard to the tyrosine concentration. This proceeding prevented on the one hand the formation of the di-substituted product, which was nearly inseparable from the mono-substituted compound and, on the other hand, it facilitated the purification due to different retardation factors with respect to the remaining Boc-Tyr-OMe. An exchange of the Boc protecting group for Fmoc and the removal of the C-terminal methyl ester (OMe) was not

performed due to the fact that these building blocks should not be used in the SPPS approach.

Figure 2.2 Synthesized mononuclear BMIA (17) and BPA (18) building blocks for comparative studies in kinetic experiments with their dinuclear analogues and DNA-model substrate.

As mentioned at the beginning, the synthesis of the unsymmetrically substituted building block was a big challenge due to the additionally required protecting group at the tert-butyl-(pyridin-2-ylmethyl)glycine (11) ligand precursor. The protecting group had to be fully orthogonal to the Fmoc group exclusively allowing for the use of acid labile protecting groups. Furthermore, it had to tolerate the applied conditions of the MANNICH reaction with regard to the acidic pH and longtime reflux. Thus, the Boc/OMe protected tyrosine used in the previous reactions was unsuitable due to the subsequent acidic deprotection of the Boc group. This would also cause a loss of the tert-butyl group of the ligand which could not be reintroduced in a region-selective manner at this position. Hence, Fmoc-Tyr-Bn (19) was used because this residue circumvents the need of Boc deprotection and, in addition, the benzyl group can be selectively deprotected by hydrogenation using palladium on charcoal.

In order to generate the unsymmetrically modified building block, the BPA ligand 8 was first attached to tyrosine in the manner described above using 0.95 equivalents of the BPA compound (Scheme 2.6). The Schiff base was generated by the addition paraformaldehyde in EtOH/water (1:4) at 65 °C for 2 h. The pH of the reaction mixture was adjusted to approximately 6.5 because the ligand shows enhanced basicity and tends to deprotect the Fmoc group of tyrosine upon addition. Fmoc-Tyr-Bn (19) was added and the reaction mixture was refluxed for 36 h, whereby the mono-substituted building block 20 was generated in yields of 16%. Afterwards, the Schiff base cocktail of the reaction between ligand 11 and paraformaldehyde was added to the intermediate product in order to be attached to the remaining ortho-position of tyrosine. The reaction process was monitored by ESI-MS that revealed the formation of several byproducts. In addition, the second substitution is known to be less effective due to the deactivation of the phenol ring by the first substituent.^[62] The additional aromaticity as well as the increased steric demand of the Fmoc and benzyl

protecting groups compared to the previously used Boc and OMe groups might explain the high content of side products. The unsymmetrically substituted building block 21 was obtained in 2% overall yield as confirmed by RP-HPLC. Besides, HPLC purification was the only way to isolate the target product because the high content of byproducts as well as their low retardation factors made it impossible to apply column chromatography. This fact as well as the generally difficult synthesis of 21 enormously decreased its suitability even though the hydrolysis ability might be potentially higher. Thus, future experiments were limited to the use of the successfully synthesized symmetrically modified building blocks 14 and 16.

Scheme 2.6 Synthetic route for the preparation of the asymmetrically substituted building block 21 by a MANNICH

reaction of Fmoc-Tyr-OBn (19) with the ligand precursors 8 and 11.

2.3 Evaluation of the hydrolysis ability of the building blocks towards DNA