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Copper(II) amino acids complexes compose 5–10% of complexed copper in human blood plasma, competing against the Cu(II)-albumin-bound fraction 1. Cu(II) salts with the deprotonated amino acids histidine (L-His), threonine (L- Thr), asparagine (L-Asn), glutamine (L-Gln), and cysteine (L-Cys) were found to be the most prevalent copper(II) amino acid compounds in human blood serum 1. Due to their small size, the electrically neutral Cu(II)–amino acid complexes can diffuse easily through the biological membranes and participate in copper transport between blood and tissues. The study presented on this poster summarizes results of the conformational analysis of l-cysteinato-l- asparaginato-copper(II) in gas phase. Both cysteine (L-Cys) and asparagine (L-Asn) can donate electrons to copper and hence form coordinative bonds via their different hetero atoms in the main amino acid chain as well as in their side chain, namely sulfur in Cys and nitrogen or oxygen of the amido group in Asn. For this study only the so called glycinato- like mode was chosen, i.e. an approximately planar coordination to copper via amino nitrogen and one carbonyl oxygen from the amino acid main chain (see figure). Other binding modes are still a topic of ongoing research. Starting with this restriction of two five membered chelate rings, three possible torsion angles in both ligands are left to modify and give therefore the number of [2·(3·3·2)^2]=648 input structures. From these, 171 minima were obtained by optimizing the initial structures via DFT/B3LYP calculations with the Gaussian09 program package. The LAN double ζ basis set augmented by polarization and diffuse functions for O, N and C and an effective core potentials for Cu and S were employed. Reference 1. P. Z. Neumann, A. Sass-Kortsak, The State of Copper in Human Serum: Evidence for an Amino Acid- bound Fraction, J Clin Invest 46 (1967) 646 − 658.

L-threonine (L-Thr) is one of two essential α- amino acids with a hydroxyl group in the side chain. It is also one of two essential α-amino acids with a chiral side chain (the second one is L-isoleucine). Its diastereomer, L-allo- threonine (L-aThr), is rarely present in nature. In L-Thr, Cα and Cβ are L- and D- chiral, respectively. In L-aThr, both of these atoms are L-chiral. Bis(L- threoninato)copper(II), Cu(L-Thr)2, is part of the exchangeable pool of copper(II) in human blood plasma, whereas the bis(L-allo- threoninato)copper(II), Cu(L-aThr)2, does not regularly appear in the living organisms. One L-Thr, as well as L-aThr, can bind to Cu(II) via amino nitrogen (Nam), carboxylato oxygen (O), and hydroxy oxygen (Oh) atoms, in three coordination modes, as presented in Figure 1: 1. a glycine-like (via Nam and O atoms) – marked with G 2. an amino-hydroxy (via Nam and Oh atoms) – marked with No 3. a carboxy-hydroxy (via O and Oh atoms) – marked with Oo, yielding six possible coordination modes in Cu(L-Thr)2 and in Cu(L-aThr)2. Fig.1. Three coordination modes of binding of L-Thr to Cu(II) Information on the actual coordination modes to Cu(II) in Cu(L-Thr)2 and Cu(L-aThr)2 complex in aqueous solution are available from the experimental data, such as visible absorption and circular dichroism measurements. According to them, at physiological pH value, 7.4, both L-Thr and L- aThr ligands coordinate to Cu(II) in a glycine- like coordination mode. At higher pH values increasing up to ~ 9.4, coordination via Nam and Oh atoms to Cu(II) was observed in Cu(L- Thr)2, whereas only one Nam coordinated in Cu(L-aThr)2.1, 2 To examine the relative stabilities of these coordination modes and possible conformations of both systems in the gas phase, quantum chemical B3LYP/LanL2DZ{;D95v+ (d)}; conformational analyses were performed.3 They resulted in 196 and 267 gas-phase Cu(L- Thr)2 and Cu(L-aThr)2 conformers, respectively.3 The glycine-like mode is calculated as the most stable for both complexes.3 Intramolecular hydrogen bonds, as the most dominant weak molecular interaction in both copper compounds, generally create an important effect on molecular stability and relative energy of the two systems. For example, the position of the hydroxyl group in the physiological Cu(L-Thr)2 complex causes the described restriction of its conformational space, compared to Cu(L-aThr)2. To model the solvent effects, calculations with the polarizable continuum model (PCM) were performed.3 As initial structures, the five lowest-energy gas-phase conformers for each of the coordination modes were optimized by using B3LYP/LanL2DZ{;D95v+(d)}; in the aqueous medium using PCM.3 As a result, we found generally that the most stable gas-phase conformers are also the most stable ones in aqueous solution.3 The versatility of gas-phase and aqueous Cu(L- Thr)2 and Cu(L-aThr)2 minima obtained will be presented. As a conclusion, our data may explain why Cu(L-aThr)2 is not present in the body. Acknowledgement: M.M. is grateful for the financial support of “Scholarship of the Scholarship Foundation of the Republic of Austria for Postdocs” given via Österreichische Austauschdienst. This work has been supported by the Croatian Science Foundation under the Project No. IP-2014-09-3500. Computational resources were provided by the Graz University of Technology and by the Isabella cluster (isabella.srce.hr) at the Zagreb University Computing Centre. References: [1] P. Grenouillet, R. P. Martin, A. Rossi, M. Ptak, Biochim. Biophys. Acta, 322, 185 (1973). [2] V. Noethig-Laslo, N. Paulić, Monatsh. Chem. Chem. Mon., 128, 1101 (1997). [3] M. Marković, M. Ramek, C. Loher, J. Sabolović, Inorg. Chem., 55, 7694 (2016).

By now experimental studies for physiological ternary (L-histidinato)(Lthreoninato) copper(II) complex in solutions did not report any firm structural information [1, 2]. To complement experimental findings we performed conformational analyses for the ternary complex in the gas phase and in an implicitly modeled aqueous medium using the density functional theory (DFT) functional B3LYP [3]. 1728 initial structures, which cover the conformational space of the complex in three copper(II) coordination modes with trans- and cis-configuration, were used for geometry optimizations. The effects of intermolecular interactions on the complex properties are examined. Different energy landscapes in the gas phase and aqueous solution are obtained with much more possible aqueous conformers than vacuum conformers. Less strained structures are detected in aqueous solution than in the gas phase. While the trans conformers in glycine-like coordination are the most stable in vacuum, conformers in all coordination modes are predicted within 15 kJ mol-1 in aqueous solution. The diversity of lowest-energy aqueous geometries with or without the intra- and inter-residual hydrogen bonds, and comparisons with the previously studied parent binary complexes bis(L-histidinato)copper(II) [4] and bis(L-threoninato)copper(II) [5] reveal a pronounced conformational flexibility of the title complex in aqueous solution. The flexibility explains previous experimental findings [1, 2] on the lack of inter-residual strain and the abundance of the ternary over the parent complexes determined in aqueous solutions at physiological pH values. Acknowledgment: This work has been supported by the Croatian Science Foundation under the project IP-2014-09-3500. [1] H. C. Freeman, R.-P. Martin, J. Biol. Chem. 24, 4823 (1969). [2] O. Yamauchi, T. Sakurai, A. Nakahara, J. Am. Chem. Soc. 101, 4164 (1979). [3] M. Ramek, M. Marković, C. Loher, J. Sabolović, Polyhedron (submitted). [4] M. Marković, M. Ramek, J. Sabolović, Eur. J. Inorg. Chem. 198 (2014). [5] M. Marković, M. Ramek, C. Loher, J. Sabolović, Inorg. Chem. 55, 7694 (2016).