First-principles study of radiation-induced radicals in solid-state amino acids and sugars: confrontation of density-functional calculations with experimental results

In this work, we present an extensive computational study of several radiationinduced radicals of biomolecules. In particular, two specific types of molecular systems will be highlighted: amino acids and sugars. Both systems are abundantly present in the natural world and are vital to the existence of life in all its forms. Amino acids are the building blocks of polypeptides and proteins, which are involved in nearly all biochemical processes. Sugars (or carbohydrates) also play a key role, not merely as sweeteners but rather as essential components in the biological energy storage and transport systems of animals and as chief structural material in plants. As can be expected, the radical adducts of these compounds have an equal importance in biochemistry. These radicals can arise in chemical reactions or can be induced as the result of radiation damage. Such species are normally very shortliving in gas phase or solution. In crystals on the other hand, the radicals become “trapped” inside the amino-acid or carbohydrate lattice and their reactivity will be sharply reduced. The solid state therefore offers the opportunity to extensively study the nature and structure of the (radiation-)induced organic radicals using various experimental techniques, of which Electron Paramagnetic Resonance spectroscopy (EPR) can be favoured as it can access an abundance of structural information about the radical. However, this technique does not provide the information as such, instead it has to be deduced from the EPR spectroscopic parameters, an analysis that is often complex and open to ambiguity. In addition, the radiation chemistry of sugars and especially amino acids in the solid state is an elaborate field of study and requires a profound understanding of the different physical and chemical processes taking place inside the crystal. This area of interest has received considerable attention in view of interesting applications in EPR dosimetry. Within this respect, we refer to the success of the alanine dosimeter for reference- and routine dosimetry in radiation therapy, biological research and industrial high-dose irradiation facilities. However, it was only after the publication of a detailed EPR study on this amino acid that an enhanced understanding of its radiation chemistry was established. Three radical species were in this way identified as contributing significantly to the observed composite spectrum and hence also to the overall dosimetric characteristics of the alanine system. As a result of the often-cumbersome analysis of the EPR parameters and the complexity of the associated radiation chemistry, the experimentalist is faced with a delicate task to propose appropriate models for the paramagnetic species present in the crystals. Over the last few years, it has become increasingly popular to rely on ab-initio molecular modeling techniques for this purpose. This success is in part due to the spectacular expansion of recent computer capabilities but is not in the least a result of the ongoing development of theoretical models and numerical algorithms in the field of quantum chemistry. Especially since the introduction of Density Functional Theory (DFT), a sharp quantitative as well as qualitative increase of theoretical calculations has been witnessed. The effectiveness of DFT can be largely attributed to a better incorporation of electron correlation as compared to more conventional ab-initio methods (such as e.g. Hartree Fock). Furthermore, this DFT algorithm does not require considerably more computer time as compared to conventional HF calculations but, in contrast, is significantly faster in comparison with other high-level correlation calculations (e.g. post HF), which renders it a very cost-effective method. Not only can these types of ab-initio calculations identify and verify proposed radical structures with the aid of optimization routines, predictions can also be made founded on entirely theoretical grounds. In addition, these methods offer the possibility to reproduce EPR quantities based on first principles. Evidently this presents a powerful tool to the experimentalist for the interpretation and analysis of EPR spectra. By now comparing measured and predicted spectroscopic parameters with each other, the true identity of an experimentally observed paramagnetic species can be linked directly to the structural characteristics of a theoretical model proposed for the specified radical. In this work, we will specifically make use of the link with experiment to characterize the radiation-induced radicals of amino acids and sugars from a theoretical point of view. A general computational strategy is reported, which outlines a basic procedure for the theoretical treatment and simulation of radicals in a solid state. This strategy is composed of four main steps. In an initial step, one or more radical models are proposed that might be consistent with the experimental EPR data of an observed paramagnetic species. The structures of these radical models are subsequently optimized within a well-defined model space, in either a DFT or semi-empirical framework. A third step concerns the determination of EPR parameters for the optimized structures, adopting an ab-initio level of theory. The results of these EPR calculations can also be sensitive to the used model space. In the final step, a conclusive analysis between calculated and measured EPR parameters is then possible. Applied on amino-acid and sugar systems, the drafted procedure will enable us to formulate specific conclusions with regard to the nature and identity of the radiation induced radicals, on the condition that an appropriate approximation is made for the solid-state environment of the radical. The extent of the model space during the optimization and EPR calculations is therefore of particular importance. In this work, it is examined what effect the size of the model space and the applied level of theory have on the calculated structural and spectroscopic properties of a simulated radical. This is achieved by introducing several model space approaches – classified from “single molecule”, over “cluster” to “periodic” – which incorporate an increasing amount of intermolecular interactions between the radical and its crystalline environment. Eventually, it is argued that the model space indeed plays a considerable role for the determination of a radical geometry and its associated EPR parameters. This aspect must therefore be carefully considered when initiating a computational study of radicals in the solid state. This work is organized in two main sections. The first section contains chapters 2 to 4 and outlines the conceptual framework of this thesis. In chapter 2, a concise overview is presented of some general principles in molecular modeling that are relevant to this work. The subsequent chapter deals with the basic concepts and theory of EPR spectroscopy. In the fourth chapter, we will introduce a general computational strategy that will be followed in the applications-section to determine EPR parameters on theoretical grounds. In the second, applied section (chapters 5 to 10), several investigations are made of radiation-induced radicals in solid-state systems. Chapters 5 and 6 deal with the amino-acid systems, alanine and glycine, respectively. After a general introduction into the applications and occurrence of radicals in sugar crystals (chapter 7), a report is given on the radicals in β-D-fructose (chapter 8), α-D-glucose (chapter 9) and α-L-sorbose (chapter 10). In the final chapter, some general conclusions are formulated

LC-MS characterization and cell-binding properties of chelate modified somatropin

Somatropin, a recombinant protein containing 191 amino acids, is derived from the endogenous human growth hormone, somatotropin. This protein is clinically used in children and adults with inadequate endogenous growth hormone to stimulate a normal bone and muscle growth. In addition, somatropin is recently being investigated for the diagnosis and radiotherapy of certain hormonal cancers. In some of these cancers, over-expression of the human growth hormone receptor (hGHR) is described. The modification of the protein with a chelating agent like NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) allows the inclusion of metals coupled to the protein. The NOTA unit is selectively introduced on a lysine side chain. As site-specific labelling is necessary to avoid active region interactions (1-16, 41-68, 103-119 and 167-175), characterization of the chelate-modified somatropin is indispensable. Therefore, we have applied an enzymatic digestion procedure using trypsin, chymotrypsin and a combination of both enzymes. The resulting peptides were then monitored using HPLC-MSn, allowing the investigation of the exact amino acid modifications. The use of a mixture of trypsin and chymotrypsin gave an enhanced information efficiency. Moreover, the intact protein, without enzymatic degradation, was analysed on a protein HPLC column using UV detection for quantification and ESI-MS/MS for characterization. Based upon the HPLC-MSn results of the digested somatropin, the chelating molecule is mainly bound to a specific lysine amino acid that is located away from the receptor binding site. Therefore, the cell-binding functionality of the characterized NOTA-somatropin is measured, using a HepG2 cell line

Radiation-induced radical formation in solid state sugars: a review of recent EMR and DFT results

Carbohydrates are important constituents of several biological systems, including DNA, and elucidating their radiation chemistry is therefore of general importance. In particular, sugar radicals play a crucial role in radiation-induced single and double strand breaks in DNA, which can lead to mutations and, finally, cancer. Certain sugars such as sucrose (table sugar) are also promising dosimetric materials. An advanced knowledge of the radiation-induced processes in carbohydrates may therefore provide better insight into the DNA radiation chemistry and aid in establishing reliable sugar dosimetry protocols. The first step in acquiring such knowledge is identification of the radical structures. Electron Magnetic Resonance (EMR) experiments on irradiated sugar single crystals allow a very detailed characterisation of the radicals via the g-tensor and the hyperfine interactions between the unpaired electron spin and the nuclear spins in the lattice. Single crystals also offer the advantage of mimicking to some extent the compact structure of chromosomal DNA. Numerous EMR studies on single crystals of sugars and sugar derivatives have been performed the last decades, but radical identification by EMR experiments alone is often ambiguous and sometimes not feasible. The last few years, highly accurate Density Functional Theory (DFT) calculations on extended organic solid state systems have become possible. These provide a powerful tool to help clarify and interpret experimental results and enable unambiguous structural identifications that were not possible before. In this talk, an overview will be given of recently identified radiation-induced radicals in single crystals of sugars (e.g. sucrose,1,2,3 fructose4) and sugar derivatives (e.g. glucose 1-phosphate5,6). The results pertain to primary as well as intermediate and stable species and the identifications are mainly based on the agreement, both in principal values and directions, between experimentally determined and DFT calculated proton hyperfine tensors. Common structural features are highlighted and possible commonly operative formation mechanisms are discussed

Modeling Radiation-Damage Processes in Organic Solids via DFT Calculations of EMR Parameters

High-energy radiation induces radicals in organic materials. When created in biological macromolecules such as DNA, these can cause harm to living organisms. This detrimental effect is also exploited for the sterilisation of e.g. foodstuffs, and radiation-induced radicals are used for dosimetry purposes. Knowledge of the structure of the radicals and their formation mechanisms is therefore of fundamental importance. In particular, radiation-induced radicals in solid sugars are studied (i) as model systems to gain insight into the precise role of the deoxyribose unit in the radiation chemistry of DNA and (ii) because of their potential as (emergency) dosimeters. X-irradiation typically gives rise to a variety of primary radicals in these systems, which then transform into stable radicals or diamagnetic species via one or more radical reactions. A prerequisite for unraveling the formation mechanisms is the identification of the different intermediate (semistable) radicals. Experimentally, solid-state sugar radicals can be characterised in detail by electron magnetic resonance (EMR) experiments. These allow determination of EMR parameters which describe the interaction of the unpaired-electron spin with its lattice environment, e.g. with (nearby) nuclear spins. Theoretical calculations of EMR parameters with DFT codes are increasingly being used to help clarify, interpret and explain experimental results. Recently we have managed, in a combined experimental and theoretical approach, to identify the structure of the major radiation-induced stable radicals in solid sucrose [1,2,3] (see Figure). We currently are investigating their formation mechanism, also via both EMR experiments and DFT modeling. A summary of the results obtained so far are presented

Assessment of atomic charge models for gas-phase computations on polypeptides

The concept of the atomic charge is extensively used to model the electrostatic properties of proteins. Atomic charges are not only the basis for the electrostatic energy term in biomolecular force fields but are also derived from quantum mechanical computations on protein fragments to get more insight into their electronic structure. Unfortunately there are many atomic charge schemes which lead to significantly different results, and it is not trivial to determine which scheme is most suitable for biomolecular studies. Therefore, we present an extensive methodological benchmark using a selection of atomic charge schemes [Mulliken, natural, restrained electrostatic potential, Hirshfeld-I, electronegativity equalization method (EEM), and split-charge equilibration (SQE)] applied to two sets of penta-alanine conformers. Our analysis clearly shows that Hirshfeld-I charges offer the best compromise between transferability (robustness with respect to conformational changes) and the ability to reproduce electrostatic properties of the penta-alanine. The benchmark also considers two charge equilibration models (EEM and SQE), which both clearly fail to describe the locally charged moieties in the zwitterionic form of penta-alanine. This issue is analyzed in detail because charge equilibration models are computationally much more attractive than the Hirshfeld-I scheme. Based on the latter analysis, a straightforward extension of the SQE model is proposed, SQE+Q0, that is suitable to describe biological systems bearing many locally charged functional groups