2.3. Stepwise Target Guided Synthesis
2.3.1. Synthesis of Bidentate Receptors
2.3.2.3. Binding Affinities of a Cu(II)-NTA-Zn(II)-Porphyrin Complex
As the porphyrin moiety of 109 showed a strong fluorescence, which was expected to change during the binding process,69 first the spectroscopic characteristics of 109 were investigated and compared with 62. The UV/Vis-spectrum of 109 did not show any significant changes compared to 62 – the soret band was neither shifted nor was there a change of the extinction coefficient. However, the fluorescence spectrum of 109 showed a decrease of emission intensity by nearly 50 %, compared to 62. This was due to the Cu(II)-NTA moiety which quenched the emission of the Zn(II)-porphyrin moiety (fig. 29).
350 400 450 500 550 600 650 0,0
0,4 0,8 1,2
500 525 550 575 600 625 650 0,00
550 575 600 625 650 675 700 725 750
0
1) Binding affinities to single amino acids:
In a first series of experiments, the binding of single amino acids to 109 was investigated. The tested amino-acids were: H-His-OMe, Boc-His-OH, H-Lys-OH, H-Gly-OH, H-Ser-OH. Experiments were performed in hepes buffer pH 7.5 (c = 50 mmol/L). Results revealed that only H-His-OMe was bound by 109 with an association constant of Ka = 1.5 x 105 L/mol (1:1 stoichiometry).70 This was expected as Cu(II)-NTA complexes selectively bind terminal histidines, whereas Zn(II)-porphyrins show only a weak binding of amines by the zinc cation.71 During the titration of 109 (c = 2.7 µM) with H-His-OMe (c = 270 µM) the fluorescence increased and reached in the end almost the intensity of 62 (fig. 30).72 This showed that, after binding of a terminal histidine, the copper-NTA complex no longer quenched the fluorescence of the zinc porphyrin. Thus, the Zn(II)-porphyrin moiety also can be used to investigate binding-events of the Cu(II)-NTA moiety.
∆
Figure 30: Measured and calculated curve of titration of 109 with H-His-OMe in 50 mM hepes buffer, pH 7.5 (left). Job’s plot of titration showed a 1:1 stoichiometry (right).
2) Binding affinity to pentapeptide 63:
In order to check the influence of a hydrophobic peptide residue, connected to a N-terminal His, on the binding to 109, the binding affinity of the pentapeptide 63 to 109 was investigated. It was expected that 63 shows a higher binding affinity than H-His-OMe because of interactions between the non-polar amino acid sequence –Leu-Leu-Val-Phe-OH and the porphyrin.
The fluorescence titration of 109 with 63 in hepes buffer was performed under the same conditions like the titration of 109 with H-His-OMe. However, results revealed only an insignificant higher binding affinity of 2.1 x 105 L/mol (1:1 stoichiometry).
Therefore, the influence of the non-polar Leu-Leu-Val-Phe-OH moiety has to be considered rather low.
Maybe, a longer non-polar peptide residue with more aromatic side chain functionalities (π- π interactions with the porphyrin) would result in a significant stronger binding to 109. However, it has to be noted that an elongation of the non-polar peptide moiety is restricted due to decreasing solubility in aqueous solution.
3) Binding affinity to Proteins:
Porphyrin 109 was synthesized in order to achieve selective bindings to non-polar regions on a protein surface with a nearby exposed histidine. Proteins which fulfill these criteria are for example hen eggwhite lysozyme (HEL) or myoglobine (horse muscle myoglobine, Myo). Figure 31 shows the titration curve of 109 vs. HEL and figure 32 shows the titration of 109 with Myo. Results revealed in both cases a decrease of fluorescence, a 1:1 binding of the receptor to the protein and high binding affinities of 1.7 x 105 L/mol for HEL respectively 4.1 x105 L/mol for Myo.
Compared to HEL, the decrease of fluorescence of 109, after binding to Myo, was stronger, because Myo contains a paramagnetic FeII in its active center, which quenches the fluorescence more effectively.
∆
∆
∆∆ emission 109 vs. HEL log b = 5.24
0 50 100 150 200 250
0 1 2 3 4 5 6 7 8 9 10
eq (HEL)
∆∆∆∆ intensity [a.u.]
measured calculated
0 5 10 15 20 25 30 35 40 45
0,0 0,2 0,4 0,6 0,8 1,0 x (109)
x (109) * ∆∆∆∆ emission
Figure 31: Measured and calculated curve of titration of 109 with HEL in 50 mM hepes buffer, pH 7.5 (left). Job’s plot of titration showed a 1:1 stoichiometry (right).
∆
∆∆
∆ emission 109 vs. Myo log b = 5.61
0 60 120 180 240 300 360 420
0 3 6 9 12 15
eq (Myo)
∆∆∆∆ intensity [a.u.] measured calculated
0 10 20 30 40 50 60 70 80 90
0,0 0,2 0,4 0,6 0,8 1,0 x (109)
x (109) * ∆∆∆∆ emission
Figure 32: Measured and calculated curve of titration of 109 with Myo in 50 mM hepes buffer, pH 7.5 (left). Job’s plot of titration showed a 1:1 stoichiometry (right).
As both, HEL and Myo, have got several hydrophobic areas on their surface a non-specific binding of the porphyrin moiety was expected. An enhancement of binding affinity as well as specificity should be achieved by the additional binding site of the Cu(II)-NTA moiety. Figure 33 shows a 3D illustraion of HEL with depiction of the polarity on its surface. One can see the predominant hydrophobic areas. Only a few polar amino acid side chain residues (e.g. of Lys [basic, blue] or Asp [acidic, red]) increase the polarity. A comparison with 109 with matching scale shows that, after binding of the Cu(II)-NTA moiety to His 15, there are several non polar areas, where the porphyrin moiety can bind to.
Figure 33: Electrostatic representation of HEL. Hydrophobic patches are represented in white, acidic patches in red and basic patches in blue. Locations of amino acid residues of His 15 and nearby Asp 87, Lys 96 and Lys 97 are assigned. A depiction of 109 with matching scale is added in overlaid fashion.
In order to confirm that the additional binding site in 109 results in an enhancement of binding affinity, the titration experiments were repeated with 62. The measurements had to be performed in solutions saturated with Ar.73 Otherwise it was not possible to obtain constant values of fluorescence intensity.74 A possible explanation for this is the formation of singlet oxygen by 62.75 This was not observed for 109, as the copper complex quenches the energy of the excited zinc-poprphyrin.76
However, the binding constants of 62 to HEL or Myo did not differ significantly from values obtained for 109. The association constant (Ka) for 62 to HEL was 1.9 x 105 L/mol, for 62 to Myo Ka was 4.9 x 105 L/mol. A possible explanation for the slight increase of binding affinity of 62 to the proteins compared to 109 is the higher hydrophilicity of 109, due to the copper-NTA moiety. The final conclusion that can be drawn from this observation is that the additional Cu(II)-NTA binding site results in no enhancement of binding affinity towards the tested proteins.
His 15 Lys 96
Lys 97
Asp 87
Cu2+
H2O OH2 O O
N
O O O O O
N N
N N
HOOC
COOH
COOH
N N N
N H Zn2+ O
2.4. Summary and Conclusions
Novel receptor building blocks for different binding motifs have been synthesized.
Zn(II)-cyclen complexes for imide groups, bis-Zn(II)-cyclen complexes for phosphate groups, Zn(II) and Cu(II) nitrilo-triacetic acid complexes for N-terminal histidines, guanidines for carboxylic acids and a Zn(II)-porphyrin for non-polar regions on a protein surface. All compounds bear either an azide or an alkyne function and therefore can be connected in any combination using the Cu(I)-catalyzed cycloaddition.
A biological relevant pentapeptide, with a histidine and a carboxyl-group as possible binding motifs, has been used as target in a kinetically controlled target guided reaction. In order to identify a bidentate receptor with a high binding constant for the target, combinations of Zn(II)- and Cu(II)-NTA complexes and guanidines have been tested in this reaction. Hit-detection was performed in situ with MS-spectroscopy.
Results revealed one possible high affinity receptor out of 12 combinations.
Six bidentate receptors have been synthesized and their binding interactions with the peptide target have been investigated with NMR- MS- and fluorescence-spectroscopy. All methods confirmed binding interactions.
Results of NMR-investigations allowed a prediction of the structural relations of the formed complex between target and receptor. This prediction was confirmed by Spartan-calculations.
Further, the association constant of this complex was determined with fluorescence spectroscopy to be in the micro molar range. However, a definite determination, as well as a comparison of the association constants of these six receptors was not possible.
Fluorescent labelled N-terminal histidines, with a dansyl- or a fluorescein group have been synthesized. The binding interactions between the histidine-moiety and heavy metal cations as well as Cu(II)-NTA complexes has been investigated with fluorescence spectroscopy. Results revealed high association constants (> 1011 L2/mol2) of these indicators towards Cu2+ and Cu(II)-NTA complexes.
Therefore, these indicators can be used in very sensitive detection assays for Cu(II) and corresponding complexes.
Using the Cu(I)-catalyzed cycloaddition, several bidentate receptor molecules with different binding sites have been synthesized. Combinations of receptor building blocks were: bis-Zn(II)-cyclen-complexes with Zn(II)-NTA-complexes or guanidine compounds and further a Cu(II)-NTA- with a Zn(II)-porphyrin complex.
The binding interactions between the bis-Zn(II)-cyclen compounds and phosphorylated peptides were investigated with a fluorescence polarisation assay.
Results confirmed a slight increase of the binding affinity (factor 2) compared to a monodentate bis-Zn(II)-cyclen reference compound.
The binding interactions between the Zn(II)-porphyrin-Cu(II)-NTA complex and amino acids, a pentapeptide and two His-containing proteins (HEL and Myo) were investigated with fluorescence titrations. Results showed a specific binding of histidine as well as of peptides with an N-terminal His. Binding affinities were in the micromolar range. The binding affinities of the porphyrine receptor towards His-containing proteins revealed to be also in the micromolar range, but unspecific.
These results not only show the possibilities, but also emphasize the importance of the Cu(I)-catalyzed “click-reaction” for the “Target Guided Synthesis”-approach in the field of drug-discovery.
Further, this work shows that the Cu(I)-catalyzed “click-reaction” is a valuable tool to connect diverse kinds of building blocks in any way, which enables the easy synthesis of polydentate receptors with increased affinity for various target molecules.
2.5. Experimental Part
2.5.1. General Information
All reactions were performed under an inert atmosphere of N2 using standard Schlenk techniques if not otherwise stated.
2.5.1.1. Spectroscopy
Emission Spectroscopy
Fluorescence measurements were performed with UV-grade solvents (Baker or Merck) at 20 °C in 1 cm quartz cuvettes (Hellma) and recorded on a Varian ‘Cary Eclipse’ fluorescence spectrophotometer.
Absorption Spectroscopy
Varian Cary BIO 50 UV/VIS/NIR Spectrometer. Use of a 1 cm quartz cell (Hellma) and Uvasol solvents (Merck or Baker).
NMR Spectra
Bruker Avance 600 (1H: 600.1 MHz, 13C: 150.1 MHz, T = 300 K), Bruker Avance 400 (1H: 400.1 MHz, 13C: 100.6 MHz, T = 300 K), Bruker Avance 300 (1H: 300.1 MHz,
13C: 75.5 MHz, T = 300 K). The chemical shifts are reported in δ [ppm] relative to external standards (solvent residual peak). The spectra were analysed by first order, the coupling constants are given in Hertz [Hz]. Characterisation of the signals: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, bs = broad singlet, psq = pseudo quintet, dd = double doublet, dt = double triplet, ddd = double double doublet.
Integration is determined as the relative number of atoms. Assignment of signals in
13C-spectra was determined with DEPT-technique (pulse angle: 135 °) and given as (+) for CH3 or CH, (-) for CH2 and (Cquat) for quaternary C. Error of reported values:
chemical shift: 0.01 ppm for 1H-NMR, 0.1 ppm for 13C-NMR and 0.1 Hz for coupling constants. The solvent used is reported for each spectrum.
Mass Spectra
Varian CH-5 (EI), Finnigan MAT 95 (CI; FAB and FD), Finnigan MAT TSQ 7000 (ESI). Xenon serves as the ionisation gas for FAB.
IR Spectra
Recorded with a Bio-Rad FTS 2000 MX FT-IR and Bio-Rad FT-IR FTS 155.
2.5.1.2. Synthesis
Melting Points were determined on a Tottoli micro melting point apparatus and are uncorrected. TLC analyses were performed on silica gel 60 F-254 with a 0.2 mm layer thickness. Detection via UV light at 254 nm / 366 nm or through discolouration with ninhydrin in EtOH. For preparative column-chromatography, Merck Geduran SI 60 silica gel was used. Commercially available solvents of standard quality were used. If otherwise stated, purification and drying was done according to accepted general procedures.77 Elemental analyses were carried out by the Center for Chemical Analysis of the Faculty of Natural Sciences of the University Regensburg.
2.5.2. Synthesis of Building Blocks for Kinetically Controlled TGS