Robust transition metal markers for labelling of peptides via solid phase synthesis methods

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1 Robust transition metal markers for labelling of peptides via solid phase synthesis methods Dissertation for the degree of Doktor der aturwissenschaften in the Fakultät für Chemie at the Ruhr-Universität Bochum presented by Dave Richard van Staveren Bochum, June 2001

2 This work was carried out between July 1999 and May 2001 at the Max-Planck-Institut für Strahlenchemie Mülheim an der Ruhr, Germany Submitted on: May 16 th 2001 Examination: June 27 th 2001 Referent: Prof. Dr. K. Wieghardt Korreferent: Prof. Dr. W. S. Sheldrick Prüfer: Prof. Dr. W. Sander

3 Acknowledgements I would like to acknowledge everybody who showed interest in my work and supported me during this Ph. D. period. I am especially indebted to: Prof. Dr. Karl Wieghardt, for the opportunity to work in his group and the access to chemicals and equipment. I really appreciate that I was allowed to complete my work after my advisor joined another university. Prof. Dr. ils Metzler-olte, for the freedom he gave me during my Ph. D. research, his constant encouragement, many helpful comments and the skills in MR spectroscopy he taught me. Dr. Thomas Weyhermüller and Heike Schucht for the numerous high-quality X-ray crystal structure determinations. I appreciate that I was given the opportunity to have a close look at the process of X-ray data collection and the subsequent elucidation and refinement of the structure. Dr. Eberhard Bothe, Petra Höfer and Helmut Schmidt for their technical assistance with the electrochemical and spectro-electrochemical measurements. I am grateful to Dr. E. Bothe for his assistance with the low temperature electrochemical measurements and his helpful discussions. Dr. Michael Bühl, for performing the Density Functional Theory calculations and the pleasant cooperation. The accuracy of the results impressed me to a large extent. Dr. Eckard Bill, Frank Reikowski and Bernd Mienert for their help with the EPR and Mössbauer spectroscopic data acquisition and interpretation.

4 Jörg Bitter and Kerstin Sand for running many MR spectra, and their patience and willingness to cooperate. Manuela Trinoga for the numerous skillfully performed HPLC purifications and the nice conversations. Herr Selbach for his assistance with the solid phase peptide syntheses. I also would like to thank Thomas Happ and Ulrich Hoffmanns for their preliminary investigations concerning the solid phase peptide synthesis. Andy Göbels for his assistance with the circular dichroism spectroscopic measurements. Prof. Dr. Phalguni Chaudhuri for his interest in the molybdenum chemistry and his helpful discussions. I also would like to thank Dr. Craig Grapperhaus and Dr. Bas de Bruin for the nice conversations and the many helpful suggestions and comments along the way. I also would like to acknowledge Tapan Paine for his helpful comments concerning the organic syntheses. Furthermore, I also would like to thank Udo Beckmann, Ricardo Garcia, Dr. Diran Herebian, Dr. Shuji Kimura for the conversations and helpful comments. Weiterhin möchte ich mich ganz herzlich bei Silke Klein bedanken. bwohl Du keinen wissenschaftlichen Beitrag leistetest, war Deine Unterstützung in jeglicher Hinsicht doch von unschätzbarem Wert. hne Dich, liebe Silke, hätte diese Doktorarbeit auf keinen Fall diesen Umfang und diese Qualität erreicht. Eveneens wil mijn moeder bedanken. Zonder jouw steun al die jaren tijdens mijn schooltijd, studie en mijn promotieonderzoek had dit proefschrift nooit tot stand kunnen komen. Echt heel erg bedankt voor alles, en gelukkig kunnen we ons op een aanzienlijk stressvrijere toekomst verheugen.

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6 Table of contents Chapter 1 Introduction General introduction Metal ions in biological systems Bio-organometallic chemistry Application of organometallic complexes in immuno-assays bjectives and outline of this thesis 12 Chapter 2 Peptides General aspects Peptide synthesis in solution Solid phase peptide synthesis Biological properties of enkephalin 20 Chapter 3 The marker Mo(η-Cp-CH)(η-allyl)(C) Reasons for selecting molybdenum carbonyl complexes as markers Properties of Mo(η-Cp)(η-allyl)(C) 2 and synthesis of the marker Coupling of the marker with amino acids and peptides X-ray crystal structure of the phenylalanine derivative H MR spectroscopic investigations Infrared spectroscopy Electrochemistry and air-sensitivity 35 i

7 Table of contents Chapter 4 Fluxional processes in complexes of the type 37 Mo(His)(η-2-R-allyl)(C) Introduction Synthesis of the complexes Solid state structures Behaviour in solution Results from Density Functional Theory calculations Electrochemical investigations Electronic and infrared spectroscopic investigations EPR spectroscopic investigations Density Functional Theory calculations on the oxidised complexes Concluding remarks 78 Chapter 5 Markers based on the complex Mo(His)(η-allyl)(C) General introduction Synthesis of the enantiomeric markers and diastereomeric 81 bioconjugates 5.3 X-ray crystallography MR spectra of the phenylalanine and dipeptide derivatives Solid phase synthesis MR spectra of the [Leu]-enkephalin bioconjugates Circular dichroism spectroscopy Electrochemistry and infrared spectroscopy Concluding remarks 97 Chapter 6 Spectroscopic properties and reactivity of the complexes 98 Mo(bpa)(C) 3 and Mo(benzyl-bpa)(C) General introduction Synthesis X-ray crystallography MR and electronic spectroscopy Electrochemistry 109 ii

8 Table of contents 6.6 Spectro-electrochemistry EPR spectroscopy Concluding remarks 116 Chapter 7 The Mo(bpa)(C) 3 unit as a marker General introduction Synthesis in solution Solid phase synthesis Infrared spectroscopic and electrochemical investigations Concluding remarks on the labelling of [Leu]-enkephalin and future outlook 125 Chapter 8 Ferrocene and cobaltocenium conjugates of amino acids and 127 dipeptides, a hydrogen bonding study 8.1 Introduction Synthesis X-ray crystallography Investigation of the conformation in solution Mössbauer spectroscopy and electrochemistry Concluding remarks 146 Chapter 9 Summary 148 Experimental Section 155 Crystallographic data 192 References 200 Curriculum Vitae 212 iii

9 Abbreviations Å Ar B br CD cm CT CV d dd δ E Q E E p,a E p,c EI EPR esd ESI Et ε FAB G g hr HM angström aryl group magnetic field broad circular dichroism centimeter charge transfer cyclic voltammetry doublet double doublet chemical shift, isomer shift quadrupole splitting potential anodic potential cathodic potential electron impact electron paramagnetic resonance estimated standard deviation electro-spray interface ethyl molar extinction coefficient fast atom bombardment gauss g-value hour highest occupied molecular orbital iv

10 Abbreviations HPLC Hz I IR J K LUM λ m M Me min M m/z MR ν TTLE-cell ppb ppm q RT S s SM σ T t tert UV Vis vs. w high performance liquid chromatography hertz nuclear spin infrared coupling constant kelvin lowest unoccupied molecular orbital wavelength multiplet, meter, milli-, medium (intensity) molar, megamethyl minute molecular orbital mass per charge product nuclear magnetic resonance stretching vibration optically transparent thin layer electrochemical cell parts per billion parts per million quartet room temperature spin singlet, second, strong (intensity) single occupied molecular orbital standard deviation tesla, temperature triplet tertiary ultra violet Visible versus weak (intensity) v

11 Abbreviations Abbreviations for chemicals and solvents Ala b-bpa benzyl-bpa bpa Boc 2-ClTrt Cys DCM dipea DMF DMS EtAc EtH Fmoc Gly HBTU His HBt Leu Lys MeC MeH MST PEA Phe tacn TBTU TFA THF TIS Tyr alanine -benzyl-,-di(2-picolyl)amine -benzyl-,-di(2-picolyl)amine di(2-picolyl)amine tert-butyloxycarbonyl 2-chloro-trityl cysteine dichloromethane di-isopropyl-ethylamine dimethylformamide dimethylsulfoxide ethylacetate ethanol Fluorenyl-9-methoxycarbonyl Glycine -(benzotriazole-1-yl)-,,, -tetramethylurionium hexafluorophosphate histidine 1-hydroxy-1H-benzotriazole leucine lysine acetonitrile methanol 1-(mesitylene-2-sulphonyl)-3-nitro-1H-1,2,4-triazole S-1-phenyl-ethylamine phenylalanine 1,4,7-triazacyclononane -(benzotriazole-1-yl)-,,, -tetramethylurionium tetrafluoroborate trifluoro-acetic acid tetrahydrofuran tri-isopropylsilane tyrosine vi

12 1 Introduction 1.1 General introduction During the second half of the 20 th century the well-defined borders between the classical disciplines of chemistry, which are organic chemistry, inorganic chemistry and biochemistry, have started to disappear. Each of these fields has profited to a large extent from achievements made in the other areas of chemistry. owadays a fairly good share of reactions in organic chemistry employ (transition) metals, either as elements or in the form of compounds [1]. Also the use of biochemical reagents in the form of immobilised enzymes has become of great importance for organic chemistry, in particular for stereospecific reactions [2]. The biochemists on their turn have benefited immensely from discoveries made by their organic colleagues. For example, Merrifield s invention of solid phase peptide synthesis [3] provided (bio)chemists a method by which small peptides (up to 40 amino acids) with essentially any desired primary structure can be prepared [4-6]. In the last few decades the areas of biochemistry and inorganic chemistry have become much more related as well, because it has been discovered that a variety of metal ions is indispensable for living cells. 1

13 Chapter Metal ions in biological systems Until about 60 years ago, only some (earth-)alkaline metal ions, such as a +, K +, Ca 2+ and Mg 2+, were considered essential for life. The earth-alkaline metal ion Ca 2+ plays a number of important roles in organisms [7-9]. For example bones consist of an organic phase, mainly collagen, and an inorganic phase, of which hydroxyapatite with chemical formula Ca 5 (P 4 ) 3 H is the main constituent. Furthermore, Ca 2+ ions act as messengers in eukaryotic cells in a manner similar to the action of camp. Transient increases of the Ca 2+ concentration in the cytosol trigger various cellular responses, like muscle contraction, release of neurotransmitters and the breakdown of glycogen to glucose-6-phosphate. Moreover, Ca 2+ serves as a regulator of the citric acid cycle, by activating the enzymes pyruvate dehydrogenase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase. The alkaline metal ions a + and K + are also important for cells [7-9]. The plasmamembrane enzyme [a + -K + ]-ATPase pumps two K + ions out of and three a + ions into the cell with the concomitant hydrolysis of intracellular ATP. This enzyme therefore regulates the concentration of electrolytes in the cell, which enables animal cells, all of which lack a cell wall, to control their water content osmotically. Furthermore, the electrochemical gradient that is generated by the enzyme is important for the signal transmission by nerve cells. The earth-alkaline metal ion Mg 2+, the fourth most abundant cation in the human body after a +, K + and Ca 2+, is indispensable for life as well [7-9]. For example, stabilisation of the helical structure of DA is accomplished by Mg 2+ ions in cooperation with a + ions, by neutralising the negatively charged phosphate backbone. In fact, enzymes that mediate reactions with nucleic acids or nucleotides, e.g. ATP, usually require Mg 2+ for activity [7-9]. At the present time, it is more or less general knowledge that apart from these four (earth-)alkaline metal ions, also numerous transition metal ions are necessary for living organisms [9-11]. The roles of the transition metal ions are diverse and include involvement in catalytic reactions, transporting tasks as well as electron transfer processes. Moreover, the metal ion can also have a structural role, for example in the case of a zinc-finger in proteins. This structural motif consists of a Zn 2+ ion, which is in an [S 4 ], [S 3 ] or [ 2 S 2 ] coordination environment, contributed by cysteinato sulfur atoms and histidine ε atoms. These zinc 2

14 Introduction fingers constitute important secondary structural elements that are found predominantly in DA-binding proteins [12, 13]. As a result of this zinc-finger, the protein possesses a looplike structure, which enables it to bind efficiently and selectively to the DA s major groove at specific nucleobase sequences. In this way, the protein regulates vital processes, such as the expression or inhibition of genes. When the active site of an enzyme contains a metal ion that is coordinated by functional groups from the protein, it is called a metallo-enzyme. These kinds of enzymes utilise the versatile chemistry of the transition metal ions. A metallo-enzyme with a transporting function is for example hemoglobin, which delivers dioxygen from the lungs to the tissues. In its deoxy form, the active site of hemoglobin consists of a low-spin Fe 2+ ion that is coordinated by a planar tetradentate heme ligand and by a histidine ε atom, with the sixth coordination site available for dioxygen. The binding of dioxygen is accompanied by the transfer of an electron from the Fe-ion to the dioxygen molecule, resulting in a high-spin Fe 3+ ion and a superoxide ligand. Upon release of dioxygen the reverse process takes place, yielding again deoxy hemoglobin. Catalytic reactions of metallo-enzymes can be divided in two classes, i.e. reactions that occur without a change of the metal ion s valency and those that involve a change of the oxidation state of the metal ion. An example of the former is the Zn 2+ containing enzyme carbonic anhydrase, which catalyses the reaction of H 2 and C 2 to HC - 3 and a proton. The Zn 2+ ion in human carbonic anhydrase I and II is coordinated by two histidine ε atoms, one histidine δ atom and a water molecule or hydroxide ion, as revealed by protein X-ray crystallography [14, 15]. The role of the metal ion is to serve as a Lewis acid and, thus, to decrease the pk a of the coordinated water molecule, in this way activating a nucleophilic attack of the Zn-H moiety on the C 2 carbon atom. A catalytic reaction involving a change of the metal ion s oxidation state takes for example place in Cu-Zn Superoxide Dismutase (Cu-Zn SD). The active site of this enzyme consists of a Zn 2+ and a Cu 2+ ion, which are bridged by a deprotonated imidazole from a histidine, with the δ coordinated to Zn 2+ and the ε to the Cu 2+ ion. Furthermore, the Cu 2+ ion is ligated by two additional histidine- ε atoms and one histidine δ atom, whereas the Zn 2+ is further coordinated by two histidine δ atoms and a carboxylate oxygen atom from an aspartate. This 3

15 Chapter 1 enzyme catalyses the reaction of two superoxide-ions with two protons to yield one molecule of dioxygen and one molecule of hydrogen peroxide. The produced hydrogen peroxide, which is also a harmful substance for living cells, is subsequently transformed by the enzyme catalase. It has been revealed that the catalytic mechanism of Cu-Zn SD consists of two steps: the so-called ping-pong mechanism. The Cu(II) state of the enzyme undergoes a redoxreaction with an - 2 molecule, yielding a Cu(I) state and a molecule of dioxygen. In the second step, this Cu(I) state reacts with a second - 2 molecule and two protons, resulting in formation of a H 2 2 molecule and regeneration of the Cu(II) state. The metallo-enzymes mentioned thus far have transition metal ions that are merely coordinated by classical coordination chemical donor atoms, such as, and S atoms. However, also metallo-enzymes with organometallic ligands are known to be present in nature, one example thereof being the [Fe-i]-hydrogenase, which contains a Fe(C) 2 (C) fragment. The active site of the reduced (active) and the two-electron oxidised (inactive) forms of this enzyme, isolated from the bacterium D. Gigas, is depicted in Scheme 1.1, as revealed by protein X-ray crystallography [16, 17]. In the reduced form, the nickel ion is coordinated by two terminal cysteinato sulfur atoms and by two cysteinato sulfur atoms that bridge to the Fe(C) 2 (C) moiety. In the two-electron oxidised form, an extra bridge in the form of an hydroxo or oxo ligand is present. Scheme 1.1 Representation of the active site of [i-fe]-hydrogenase from D.Gigas as revealed by protein X-ray crystallography [16, 17]: left: active (reduced) form; right: inactive (oxidised) form. Cys S S Cys Cys i Cys S S Fe C C C Cys S S Cys Cys i S S (H) Cys Fe C C C 4

16 Introduction Apart from the active reduced form (the i-b form) and the inactive two-electron oxidised form (the i-a form), also two one-electron oxidised forms are known to exist, as concluded from EPR and infrared spectroscopic investigations [18-24]. This enzyme is capable to transform dihydrogen efficiently in two protons and two electrons. In the case of D. Gigas these electrons are used to fulfil the function of the enzyme, namely the reduction of sulfate. Recently a review on the bio-organometallic chemistry of the metal containing hydrogenases ([Fe only], [ife] and [ifese]) appeared in the literature [25]. 1.3 Bio-organometallic chemistry The era of organometallic chemistry started with the discovery of ferrocene by Kealy and Pauson in 1951 [26]. Since then, research in this area has developed rapidly, which has lead to characterisation of a large variety of structurally exotic and novel compounds. In the early years of organometallic chemistry, there was a common belief among scientists that these compounds had no biological relevance. It was therefore a huge surprise that metal carbon bonds were actually found to be present in nature as well, the first example being the X-ray crystal structure of vitamin B 12 [27], which possesses a Co-CH 3 bond. n the border between organometallic chemistry and biochemistry a new field emerged in the past few decades, which is called bio-organometallic chemistry. To most chemists, the name bio-organometallic chemistry still implies a contradiction, because organometallic complexes are thought of to be very reactive compounds, only being stable in the absence of water and dioxygen. However, this generalisation is not justified, since many organometallic complexes display high stability in aerobic aqueous media, e.g. ferrocene or the cobaltocenium cation. The field of bio-organometallic chemistry can be divided in five classes of compounds, based upon structural features and properties. The first class consists of organometallic compounds that are encountered in nature, like vitamin B 12 and the enzyme [Fe-i]-hydrogenase. To the second class belong complexes that contain in addition to a classical organometallic ligand, such as a Cp-ring or a carbonyl ligand, also a biologically relevant molecule, like an amino acid, peptide or nucleic acid, coordinated to the metal ion. 5

17 Chapter 1 The third class is comprised of compounds that resemble structural features of biomolecules, e.g. amino acids, but at the same time also possessing an organometallic fragment. Among the fourth class of compounds, organometallic complexes are classified that do not contain a single biomolecule, but exhibit biological activity, for example against tumors. The fifth class of compounds constitutes organometallic complexes that are bound in a covalent way to biomolecules. Because the structure of the active site of the [Fe-i]-hydrogenase, which is a class one compound, has already been depicted and discussed above, only examples of each of the other four remaining classes will be presented at this stage. The synthetic bio-organometallic chemistry started more or less with the use of amino acids as ligands. Each of the naturally occurring amino acids contains two potential donor atoms, namely the carboxylate oxygen atom and the nitrogen atom of the primary amine or in the case of proline the secondary amine. Several of the amino acids contain, in addition to these two potential ligating atoms, side-chains with another potential donor atom. The nature of these additional donor atoms varies largely and constitutes carboxylates (Asp, Glu), amino groups (Lys), aromatic -donors (His), thiolate and thioether sulfur atoms (Cys and Met, respectively) as well as aliphatic and aromatic alkoxides (Ser and Tyr, respectively). In the past two decades a large number of class two compounds has been reported by the groups of Sheldrick and Beck, and recently an overview of these appeared in the literature [28]. Two class two compounds showing different ways via which amino acids can bind to metal ions are depicted in Scheme 1.2 [29]. The Ru 2+ ion in the complex on the left of Scheme 1.2 is coordinated by a η 5 -pentamethyl cyclopentadienyl ligand and by the carboxylate oxygen atom, amine nitrogen atom and thioether sulfur atom of a deprotonated methionine. The Ru 2+ ion in the sandwich compound displayed on the right of Scheme 1.2 on the other hand is ligated in a η 5 -fashion by a pentamethyl cyclopentadienyl ligand and in a η 6 -manner by the phenyl ring of a phenylalanine. 6

18 Introduction Scheme 1.2 Molecular structure of two Ru(II) complexes illustrating different binding possibilities of amino acids (from ref. [29]) + S Ru H 2 Ru H 2 C 2 H In Scheme 1.3 another example of a class two compound is shown, with a modified nucleobase serving as a didentate ligand. In this complex, the Mo 4+ ion is coordinated by two η 5 -cyclopentadienyl ligands and the -3 and exocyclic -atom of a -1 methylated thymine [30]. In addition to the presence of a methyl group on -1, the coordinated thymine differs from the free nucleobase in another way, i.e. the exocyclic H 2 group is now deprotonated, making it an amido ligand. Scheme 1.3 Molecular structure of a Mo(IV) complex with a modified nucleobase as a chelating ligand (from ref. [30]) Mo H + The class three compounds consist of substances that structurally resemble biomolecules, for example amino acids, while at the same time containing an organometallic fragment. To this class belong the unnatural amino acids ferrocenylalanine and cymantrenylalanine, the molecular structures of which are depicted in Scheme 1.4. The difference between these two complexes and the Ru-phenylalanine sandwich compound displayed in Scheme 1.2 is evident. The ruthenium compound constitutes an unnatural amino acid as well, but it contains the amino acid phenylalanine. Ferrocenylalanine and cymantrenylalanine on the other hand cannot be dissected into a naturally occurring amino acid and a metal fragment. 7

19 Chapter 1 Both ferrocenylalanine and cymantrenylalanine are quite stable compounds, and can be introduced into a peptide, even by solid phase peptide synthesis methods [31-34]. However, these unnatural amino acids have a drawback, because their synthesis yields a racemic mixture of the D and L forms that can be separated into both enantiomers, but this is a very timeconsuming process [35-37]. Cymantrenylalanine can be modified before or after the peptide synthesis by photochemical substitution of a carbonyl ligand by a trisubstituted phosphane ligand [38, 39]. Because tri-alkyl and tri-aryl phosphanes with various substituents can be introduced, the bulkiness of this unnatural amino acid and the electron-availability of the Mn + ion can be fine-tuned this way. Scheme 1.4 Molecular structures of cymantrenylalanine (left) and ferrocenylalanine (right) Mn C C C C 2 H H 2 C 2 H Fe H 2 To the class four compounds belong for example several metallocenes, like titanocene dichloride, vanadocene dichloride and a cationic molybdenocene dichloride derivative, whose molecular structures are depicted in Scheme 1.5. Although these compounds contain no biomolecule at all, they are classified under the field of bio-organometallic chemistry because of their anti-tumor activity against a variety of tumors, as discovered by Köpf and Köpf-Maier [40-42]. f these three anti-tumor agents, titanocene dichloride displays the highest activity, albeit less than several bis-β-diketonato titanium compounds, some of which already entered clinical trials [43, 44]. Scheme 1.5 Molecular structures of three anti-tumor active organometallic complexes 2+ Ti Cl Cl V Cl Cl Mo Cl Cl 8

20 Introduction The fifth class consists of organometallic complexes that are bound to a biomolecule or biologically relevant molecule in a covalent way and these compounds outnumber the combined number of class 1-4 complexes. In the last decades several strategies have been developed to tether organometallic complexes to amino acids or peptides and two examples are shown in Scheme 1.6. n the left the complex Co(Cp)(C) 2 is covalently bound to β- alanine via an amide bond [45]. The formation of an amide linkage from an organometallic acid, in this case Co(Cp-CH)(C) 2, and an amine, in this case β-alanine, constitutes a very attractive method because standard peptide synthesis methods (see Chapter 2) can be applied. n the left of Scheme 1.6, the complex Cr(η-benzene)(C) 3 is covalently bound to a Bocprotected leucine derivative via an alkyne linkage [46]. This alkyne bond is formed from Cr(η-C 6 ClH 5 )(C) 3 and the alkyne modified Boc-leucine via a Pd-catalysed Sonagoshira reaction, employing CuI and Pd(PPh 3 ) 2 Cl 2 under exclusion of dioxygen. Scheme 1.6 Two examples of class five bio-organometallic compounds (from ref. [45, 46]) C Co C H CH C Cr C C H H 1.4 Application of organometallic complexes in immuno-assays Quantitative analytical methods with antigens and antibodies are nowadays part of many clinical, pharmaceutical and basic scientific investigations [47-51]. In these so-called immuno-assays, either the antigen (also called hapten) or antibody is labelled with a substance that displays spectroscopic properties that are characteristic for this compound. In this way the concentration of the analyte, for example peptides or hormones, can be determined accurately and quickly, also at very low concentrations. Even when the analyte could be determined feasibly by other methods, like chromatography, immuno-assays are often preferred due to their speed and simplicity. 9

21 Chapter 1 In the past, labelling of the antigens or antibodies was mainly performed by introduction of radioactive isotopes, e.g. 3 H and 125 I [52-56]. However, these radiological immuno-assays have a number of disadvantages, such as the consequent need for protection against radiation and the generation of radioactive waste. Another drawback is the short lifetime of some labelling reagents, which requires that the markers need to be synthesised constantly. These shortcomings of radioactive labels stimulated the investigation and development of nonradioactive markers, or cold markers. In the late 1970 s, Cais and co-workers reported the first examples of immuno-assays with haptens bound to organometallic complexes [57, 58], with detection and quantification of the analyte taking place by Atomic Absorbtion Spectroscopy (AAS). A few years later it was demonstrated that this method is as accurate as radio-immunological assays for several clinical determinations [59]. Apart from AAS, also other quantitative analytic methods applicable to metal complexes have been used for detection, which include luminescence and electrochemistry. For the former detection method, several complexes with the lanthanide ions Sm 3+, Eu 3+ and Tb 3+ bound to the haptens have been used [60-65]. The quantification of the analyte occurred in these cases by time-resolved luminescence measurements. For electrochemical detection, ferrocene labelled compounds have been widely studied. Morphine covalently attached to the ferrocene moiety was the first biologically relevant molecule subjected to electrochemical investigations [66, 67]. It was demonstrated that binding of the conjugate to the corresponding receptor resulted in a diminished accessibility of the Fc/Fc + transition due to the fact that the protein matrix of the receptor surrounds the ferrocene. Unfortunately, this effect alone did not lend itself to quantification of the analyte at concentrations that are easily and accurately determined by AAS [68]. However, this problem can be overcome if unlabelled hapten is added, together with the use of oxidases as signal amplifiers. In that case the unlabelled hapten will compete with the labelled hapten for binding to receptor sites because its affinity is at least as high as that of the labelled hapten. Subsequently, the liberated labelled hapten will serve as a substrate for the oxidases, and therefore the measured current depends on the amount of liberated labelled hapten. This somewhat cumbersome method has been applied successfully for the quantification of thyroxine [69, 70], lidocaine [71] and human choriongonadotropine [72, 73], which demonstrates its potential usability. 10

22 Introduction After these first reports of quantification by electrochemical methods, amino acids and proteins have been labelled with the ferrocene moiety, with detection taking place by HPLC- ECD (High Performance Liquid Chromatography-ElectroChemical Detection) [74-76]. The limit of detection was in these cases in the picomolar range. The ferrocene derivative shown in Scheme 1.7 has been used for determination of alkaline phosphatase [77]. Hydrolysis of the phosphate moiety lowers the redox potential by 210 mv, a difference that allows quick and reliable determination of the enzyme by electrochemical methods. Scheme 1.7 Application of a ferrocene derivative for the determination of alkaline phosphatase (from ref. [77]) Fe H H P H + H 2 alkaline phosphatase E 1/2 = +390 mv vs Fc/Fc + Fe H H + H 3 P 4 E 1/2 = +180 mv vs Fc/Fc + In the last 15 years, Jaouen and co-workers developed a new immuno-assay method based upon metal carbonyl complexes as markers, which is called Carbonyl Metal Immuno-Assay (CMIA) [78-103]. After the pioneering work by Cotton and Kraihanzel in the early sixties [104], it has been well known that metal bound carbonyls display intense and characteristic bands in the cm -1 region of the infrared spectrum. Biomolecules show almost no absorption in this range, in this way not obscuring the detection of the C stretching vibrations of the metal-bound carbonyls. Jaouen and co-workers have shown that this method is applicable to the quantification of various hormones, drugs and proteins, and recently they published examples of simultaneous determination and quantification of several steroids [105, 106]. 11

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