Main Part

1. Investigation of Metal Complex – Amino Acid Side Chain Interactions by Potentiometric Titration

This chapter deals with the application of potentiometric titrations as a screening method for interactions of metal complexes with additional substrates related to side chains of amino acids.ⁱⁱ The resulting data was analysed by a computer program (HyperQuad2000) to determine every equilibrium constant involved in the titration experiment.ⁱⁱⁱ

i Kruppa, M.; Frank, D.; Leffler-Schuster, H.; König, B. Inorg Chem. 2005, under revision.

ii All potentiometric titrations were performed by H. Leffler-Schuster.

iii Data analysis was carried out by D. Frank as reported in his Zulassungsarbeit.

1.1 Introduction

In 1975 Porath published a new type of chromatography which was first called "metal chelate chromatography", but later termed "immobilised metal (ion) affinity chromatography" (IMAC).¹ The technique uses the different affinity of proteins to metal complexes immobilised on a chromatographic support.²

To find suitable metal complexes for this purification method it is necessary to know how strong metal ions are bound to a chelate (problem of metal leaching³). This information can be obtained using potentiometric titration of desired metal and chelate.⁴ IDA and NTA complexes are well investigated in this respect.^5,6 But next to the binding between metal and immobilised chelate the interactions of substrate and metal complex are essential for the use in chelating purification methods. Weak binding will not generate a good separation. Very strong interactions will block all binding sites. Potentiometric titration can reveal equilibria between metal complexes and additional ligands.⁷ The binding of amino acids towards different metal complexes was investigated,⁸ but in most of the data the α-amine and Carboxylate amino acid functional group are used to chelate the metal complex.

Working with peptide or proteins, these binding processes are not relevant. C-terminal Carboxylate and N-terminal amine of one protein will not chelate a metal complex. The support of additional side chain functionalities is essential for a binding event. The histidine-tag strategy, in which several imidazole side chains and the N-terminal amino group reversibly coordinate to a metal complex, demonstrates this impressively. To understand which side chains of natural amino acids are suitable for metal coordination potentiometric titration may provide information.

We report here the use of potentiometric titration to screen interactions between several metal complexes (figure 76) and functional groups of amino acid side chains. In addition to the well investigated IMAC metal complexes 72-75 we focus our investigation on M(II) cyclen complexes 76-79.

H2O N

Figure 76. Structures of metal complexes 72-79 investigated in this study.

We represent typical amino acids side chain functional groups by butyl amine (Lsy), acetic acid (Glu, Asp), ethanol (Ser, Thr), ethane thiole (Cys), imidazole (His), N-ethylguanidine hydrogenchloride (Arg), phenol (Tyr) and disodium phenylphosphate (phosphorylated Tyr). The investigation of each metal complex – substrate combination consists of three experiments. In two initial potentiometric titrations, we examine the properties of substrate and metal complex separately. The obtained pK_s values for substrate and metal complex are then used to analyse the titration curve of a 1:1 mixture of substrate and metal complex.

1.2 Results and Discussion

First, the substances resembling the side chain functional groups of natural amino acids (with exception of amide and thioether) were titrated, to determine their pKa values for our experimental conditions. Table20 summarises the results and compares with literature reported values.

Table 20. Determination of pKa values for the different functional group

Compound Deprotonation

reaction(s) pKa

(this work)

pKa (Lit.)

butylamine But-NH3+

/ But-NH2 + H⁺ 11.3 10.6⁶ di-Sodium-

phenylphosphate ^a

RPO4H2 / RPO4H^-+ H⁺ 1.0 0.8⁹

RPO4H^- / RPO4

2--+ H⁺ 6.2 6.2¹⁰

acetic acid HAc / Ac^- + H⁺ 4.7 4.7⁶

ethanol EtOH / EtO^- + H^{+ b} 15.9⁶

ethanthiole EtSH / EtS^- + H⁺ 13.2 10.1 – 11.3¹¹ imidazole^a ImH2+

/ ImH + H⁺ 7.3 7.0⁶

ImH / Im^- + H^{+ b} 14.4⁶

N-ethylguanidine-hydrogenchloride RNH3+

/ RNH2 + H^{+ b} 13.4¹²

phenol PhOH / PhO^- + H⁺ 10.2 10.0 ⁶

a 1 eq HClO4 for each protonation step was added

b Determination of pKa value not possible using TEAOH

The potentiometric titration of the metal complexes 72-79 was used to determine the pKa

values of the deprotonation of coordinated water molecule(s). In cases of 74 and 75 an initial deprotonation of the third carbonic acid is taking place before the required NTA complex is formed.

Table 21. Potentiometric Titration of metal complexes investigated in this study

Metal complex Deprotonation reaction(s) pKa

Cu(II)-IDA (72) [Cu(ida)(H2O)3] / [Cu(ida)(OH)(H2O)2]^- + H⁺ 9.0 Ni(II)-IDA (73) [Ni(ida)(H2O)3] / [Ni(ida)(OH)(H2O)2]^- + H⁺ 9.8

Cu(II)-NTA

(74) [Cu(ntaH)(H2O)3] / [Cu(nta)(H2O)2]^- + H⁺ 2.1 [Cu(nta)(H2O)2]^- / [Cu(nta)(OH)(H2O)]^2- + H⁺ 9.9 [Cu(nta)(H2O)(OH)]^- / [Cu(nta)(OH)2]^3- + H⁺ 12.8 Ni(II)-NTA

(75) [Ni(ntaH)(H2O)3] / [Ni(nta)(H2O)2]^- + H⁺ 2.1 [Ni(nta)(H2O)2]^- / [Ni(nta)(OH)(H2O)]^2- + H⁺ 13.8 Cu(II)-Cyc (76) [Cu(cyc)(H2O)]²⁺ / [Cu(cyc)(OH)]⁺ + H⁺ 12.9 Ni(II)-Cyc (77) [Ni(cyc)(H2O)]²⁺ / [Ni(cyc)(OH)]⁺ + H⁺ 12.5 Zn(II)-Cyc (78) [Zn(cyc)(H2O)]²⁺ / [Zn(cyc)(OH)]⁺ + H⁺ 8.1 Cd(II)-Cyc (79) [Cd(cyc)(H2O)]²⁺ / [Cd(cyc)(OH)]⁺ + H⁺ 11.2

The potentiometric titrations to identify interactionsbetween a functional group and a metal complex were carried out at a constant 1:1 substrate to metal complex ratio. Variation of the ratio was not expected to yield extra information, and was not attempted.

M(II)-IDA Titration

As an already established system for poly-His-tag binder M(II)-IDA complexes were used to verify the screening method. Cu(II)- and Ni(II)-IDA complexes are known to have the highest affinity towards histidines.² Figure 77 shows the affinities of all tested substrates towards these metal complexes 72 and 73. As expected, among all tested compounds only imidazole showed significant binding affinity to the metal complexes.

Butylamine

Figure 77. Potentiometric screening results for Cu(II)-IDA 72 and Ni(II)-IDA 73 complexes.

All other experiments showing no binding event were analysed by two independent equilibria already known from the separate titration experiments. The pKa values fit very well to the data obtained and summarised in table 20 and table 21.

The equilibria shown in scheme 23 were used to analyse the potentiometric data. The initial deprotonation step (pK_a1 values can be found in table 21) is followed by a 1:1 complexation of imidazole and M(II)-IDA¹³ (pK_a2 (72) = 13.0, pK_a2 (73) = 12.9).

Scheme 23. Deprotonation and binding equilibria used to fit potentiometric titration data