Chemical Models of Zinc-dependent Enzymes: Methyltransferases and Class II Aldolases

Inauguraldissertation der Philosophisch-naturwissenschaftlichen Fakultät der Universität Bern

vorgelegt von Miguel A. S. Machuqueiro aus Portugal

Leiter der Arbeit: Prof. Dr. Jean-Louis Reymond Departement für Chemie und Biochemie der Universität Bern

– i –

Chemical Models of Zinc-dependent Enzymes: Methyltransferases and Class II Aldolases

Inauguraldissertation der Philosophisch-naturwissenschaftlichen Fakultät der Universität Bern

vorgelegt von Miguel A. S. Machuqueiro aus Portugal

Leiter der Arbeit: Prof. Dr. Jean-Louis Reymond Departement für Chemie und Biochemie der Universität Bern

Von der Philosophisch-naturwissenschaftlichen Facultät angenommen.

Bern, 24 April 2003 Der Dekan: Prof. Dr. G. Jäger

– ii –

To all members of my family… …past, present and future.

– iii –

Table of Contents

ACKNOWLEDGEMENTS ...... VII

ABSTRACT...... VIII

1. INTRODUCTION...... 1

1.1. The Importance of Zinc in Life ...... 1

1.2. Methyltransferases ...... 2 1.2.1. The Ada protein...... 2 1.2.2. Other Methyltransferases...... 6 1.2.2.1. Cobalamin-Independent Methionine Synthase...... 6 1.2.2.2. Cobalamin-Dependent Methionine Synthase ...... 7

1.3. Aldolases...... 8 1.3.1. Class I aldolases ...... 8 1.3.2. Class II aldolases ...... 9

1.4. The Aldol Reaction ...... 12 1.4.1. Direct Aldol Reaction...... 15 1.4.2. Catalytic Antibodies ...... 15

1.5. Organic Chemistry in H2O ...... 16 1.5.1. Direct Aldol in H2O...... 16 1.5.2. Prebiotic Chemistry ...... 17 1.5.2.1. Amino Acids Synthesis ...... 17 1.5.2.2. Sugar Synthesis ...... 17

1.6. Zinc Complexes...... 19 1.6.1. The Chelating Effect...... 20 1.6.2. NxSy Environment ...... 22 1.6.3. Zinc Complexation with Aminoacids...... 23

1.7. Chemical Models and Catalysts ...... 24 1.7.1. Methyltransferase Models ...... 24 1.7.2. Class II Aldolase Models...... 27 1.7.3. Small Molecules as Catalysts of the Aldol Reaction...... 29 1.7.3.1. Activation of the Acceptor ...... 29 1.7.3.2. Activation of the Donor...... 34 1.7.3.3. Activation of Both the Donor and the Acceptor...... 36 1.7.3.4. Proline-like Catalysts...... 38

– iv – 2. RESULTS AND DISCUSSION...... 40

2.1. Methyl Transfer to Sulfur...... 40 2.1.1. Our Project...... 40 2.1.2. MEPAH vs. Hexane-1-thiol ...... 41 2.1.2.1. The Non-chelating Alkyl Thiol: Hexane-1-thiol...... 41 2.1.2.2. The Chelating Alkyl Thiol: MEPAH...... 42 2.1.2.3. Kinetic study...... 46 2.1.2.4. Discussion...... 48 2.1.3. The Dinucleotide Model...... 49 2.1.4. The NxSy Ligands ...... 52 2.1.4.1. The NS2 Ligands...... 52 2.1.4.2. The S4 Ligand ...... 58 2.1.4.3. The N2S2 Ligand...... 60 2.1.4.4. Another NS2 Ligand ...... 62 2.1.4.5. Other NxSy Ligands ...... 62

2.2. Class II Aldolase System ...... 66 2.2.1. The N5 and N3O Ligands...... 66 2.2.1.1. The N5-NiCl2 Complex...... 67 2.2.1.2. The N5-CoCl2 Complex...... 68 2.2.1.3. The N3O-ZnCl2 Complex ...... 69 2.2.1.4. The N3O-CoCl2 Complex ...... 70 2.2.1.5. Direct Aldol Catalytic Test...... 71 2.2.2. The N3O Chiral Ligand...... 72 2.2.3. Zinc-Amino Acid Complexes...... 74 2.2.3.1. Synthesis...... 74 2.2.3.2. Catalysis of the reaction between aceton and p-nitrobenzaldehyde...... 77 2.2.3.3. Catalysis of other aldol systems ...... 83

2.3. Prebiotic Sugar Synthesis ...... 88 2.3.1. The Idea ...... 88 2.3.2. The Reaction of Hydroxy-acetaldehyde...... 89 2.3.3. Final Remarks and Outlook...... 97

3. EXPERIMENTAL PART...... 98

3.1. General Remarks...... 98 3.1.1. List of Abbreviations...... 98 3.1.2. Chemicals and Solvents...... 99 3.1.3. Materials and Methods ...... 99

3.2. Methyltransferase System...... 101 3.2.1. Chelating vs. Non-Chelating ...... 101 3.2.1.1. Compound Characterization...... 101 3.2.1.2. Reactivity Studies...... 105 3.2.1.3. Kinetic experiments...... 105 3.2.2. The Dinucleotide Model...... 106 3.2.2.1. Compound Characterization...... 106 3.2.2.2. Dinucleotide Reactivity Study...... 111 3.2.3. The NS2 Ligand ...... 111

– v – 3.2.3.1. Compound Characterization...... 111 3.2.3.2. Complexation Procedures...... 124 3.2.3.2. Reactivity Procedure ...... 125 3.2.4. The S4 Ligand ...... 125 3.2.4.1. Compound Characterization...... 125 3.2.5. The N2S2 Ligand...... 133 3.2.5.1. Compound Characterization...... 133 3.2.5.2. Complexation Studies...... 136 3.2.6. Other Ligands ...... 137 3.2.6.1. Compound Characterization...... 137

3.3. Class II Aldolase System ...... 146 3.3.1. The N5 and N3O Ligands...... 146 2+ 2+ 2+ 3.3.1.1. N5 complexation with Co , Cu and Ni , and direct aldol catalytic studies..146 2+ 2+ 2+ 3.3.1.2. N3O complexation with Co , Cu and Ni , and direct aldol catalytic studies146 3.3.1.3. Direct Aldol Catalytic Test:...... 147 3.3.1.4. X-Ray Section ...... 147 3.3.2. Chiral N3O Ligand...... 163 3.3.2.1. Compound Characterization...... 163 3.3.3. Zinc-Amino Acid Complexes...... 168 3.3.3.1. Synthesis...... 168 3.3.3.2. Compound Characterization...... 169 3.3.3.3. Catalytic Studies...... 173 3.3.3.4. Chiral HPLC measurements ...... 174 3.3.3.5. Kinetic Study...... 175

3.4. Prebiotic Sugar Synthesis ...... 176

3.5. Relevant Spectra...... 177

4. REFERENCES...... 184

5. PUBLICATIONS ...... 190

6. CURRICULUM VITAE...... 198

– vi – Acknowledgements

My first words have to go to Dr. T. Darbre for all the help, support and motivation throughout the thesis. Thank you Tamis for letting me work in such an interesting project and for sharing with me all those magnificent ideas and suggestions. My thanks go also to Prof. J.-L. Reymond and Prof. R. Keese for taking me in their groups and for all the advices and teachings. I would like to thank the members of the Analytical Research Service responsible for the NMR spectra measurements (group of Prof. P. Bigler) and the MS analysis (group of Dr S. Schürch and the group of Prof. Dr. Schaller). The x-ray analyses were performed by Prof. H. Stoeckli-Evans at the Small Molecule Crystallography Service (BENEFRI) in Neuchatel, and by Prof. C. Kratky in the University of Graz, Austria. I am also grateful the “Materialausgabe” team, U. Ruch, S. Lädrach, S. Thomi and R. Flückiger, who have always been there to solve every logistic problem with a smile. My particular gratitude goes to the secretary Frau R. Rohner who always dealt with my bureaucratic problems very efficiently. My particular gratitude goes to all the people with whom I worked the last four years, comprising members of the past group of Professor Keese, until the present people working in the Reymond group. Special thanks to: Lucas Gartenman for his friendship throughout the years and all the valuable scientific discussions; the French lobby, Manu, Caro, Nibe, David, Antony, Estelle and Johann for all the jokes and the nice environment in the lab; Francis Djojo for keeping me laughing for two years in a row; Christian Dubs for his friendship and for letting me share my own bench with him; Ruben Fernandez for showing me that there are nice Spanish around (I am crazy about you); José Freitas, Catarina Batista and Bruno Livramento for their friendship, support and practice of the Lusitanian language. A very warm thank you to Prof. Dr J.-L. Reymond, Prof. Dr. Thomas Ward and Prof. Dr. Silvio Decurtins for the reading and refereeing of my thesis and to the Swiss National Science Foundation for financial support of my work (Project 21-54130.98). Finally, there are no words to thank you Ana. You were always there… believing.

– vii – Abstract

The present work addresses two different topics in bioorganic chemistry, the methyl transfer to sulfur and the direct asymmetrical aldol reaction. Despite the differences between these two systems, living cells tend to rely on zinc for efficiently carry out both the jobs. In the first part of the thesis, a new model for the methyl transfer from trimethyl phosphate to a zinc thiolates in the absence of strong base is presented. The chelating thiol N- (2-mercaptoethyl)picolylamine (MEPAH) was methylated by trimethyl phosphate in the presence of zinc salts whereas the reaction did not take place in the absence of zinc. The pre- formed complex (MEPA)2Zn was methylated faster than the MEPAH in the presence of zinc ions. When non-chelating hexanethiol was the methyl acceptor, a slower reaction took place in the presence of pyridine which was independent of zinc. Kinetic studies of the methyl transfer from trimethyl phosphate to (MEPA)2Zn gave a second-order rate constant of 5.0´10-5 M-1s-1. The results obtained suggest that the methyl transfer to MEPA-Zn involves a zinc-bound thiolate. A methylated dinucleotide was synthesized for better mimicking the methylated DNA phosphate backbone. Other ligands were synthesized in order to increase the chelation effect and the number of sulfur atoms around zinc achieving better models of the Ada protein. In the second part of this work, is reported a series of zinc-amino acid complexes as catalysts of several different direct asymmetric aldol reactions. The zinc-proline complex catalyzed the direct aldol addition of p-nitrobenzaldehyde and acetone in aqueous medium, giving quantitative yields and enantiomeric excesses up to 56% with 5 mol% of the catalyst at room temperature. Other zinc-amino acid complexes catalyzed direct aldol reactions in water with similar enantioselectivities. The ability of many different zinc-amino-acid complexes to catalyze the direct aldol reaction in aqueous media led to a third part of this thesis, the synthesis of tetroses and hexoses under prebiotic environment. The multiple aqueous aldol condensation of hydroxyacetaldehyde in the presence of zinc complexes with proline, lysine and arginine produced the entire tetrose and hexose repertoire under mild conditions.

– viii – Chapter 1: Introduction

1. Introduction

1.1. The Importance of Zinc in Life

Zinc has been known to be an essential trace element in life for more than a century1. There are 2-3 g of zinc in adult humans, making it one of the most prevalent “trace” elements. However, no specific biological role for zinc was established until 1940, when it was shown to be required for the catalytic activity of carbonic anhydrase2. Now, many hundreds of enzymes are known to contain zinc, and high-resolution crystal structures and detailed mechanistic studies are generating significant information about the catalytic role of zinc in these enzymes. In proteins, zinc seems to play two distinct roles: structural, by stabilizing the holoprotein structure due to a large free energy decrease associated with the binding of zinc to the protein; and functional, with a direct involvement in catalysis3,4. A structural role for zinc in transcription factors was first proposed in 1983 for the protein transcription factor IIIA (TFIIIA)5. Subsequent analysis revealed the presence of small zinc-based domains (termed “zinc fingers”) in TFIIIA and in a wide variety of other proteins involved in gene regulation6. Over the past two decades, more than 10 classes of such zinc- based domains have been discovered and biochemically characterized7. In biological terms, zinc has two properties that make it well suited to its role as a structural element in nucleic acid-binding or other gene regulatory proteins. First, its lack of redox activity may be crucial for such a role. The use of redox-active metal ions such as copper and iron could easily lead to the promotion of radical reactions that result in nucleic acid damage8. Second, the relative facile ligand exchange reactions of zinc appear to be at least partially responsible for the ease of uptake and release of this metal ion9. Beyond the questions of zinc homeostasis, the possibility exists that zinc levels are used in vivo in an information-carrying role10. The largest group of zinc enzymes is the zinc-containing hydrolases. The active site of some phosphatases (for example: alkaline phosphatase and phospholipase C) contain three zinc ions, whereas a group of peptide bond cleaving enzymes (for example: aminopeptidases, b-lactamases and CPG2) contain binuclear zinc complexes at their active centers, a hydroxide group that bridges the two zinc atoms may be the nucleophile in these enzymes.

– 1 – Chapter 1: Introduction

Matthews and Golding11 showed that zinc can also activate sulfur ligands as nucleophiles to accept alkyl groups from appropriate donors.

1.2. Methyltransferases

Zinc-mediated nucleophilic attack of a thiolate on alkyl groups has been proposed as the catalytic mechanism for the Escherichia coli repair protein Ada12, cobalamin-dependent13 and cobalamin-independent methionine synthase14. While the general mechanism of zinc- catalyzed sulfur alkylation has been explored, the detailed catalytic mechanism remains to be elucidated for these metalloproteins.

1.2.1. The Ada protein

In most organisms, there are proteins capable of repairing DNA lesions created by non-enzymatic alkylating agents. These lesions can have toxic, mutagenic and carcinogenic consequences15. Escherichia coli uses, for example, two O6-meG-DNA methyltransferases to repair O6-meG-DNA adducts: a constitutive Ogt protein and an inducible Ada protein16 (Figure 1 and 2).

Figure 1. N-Ada 10 (DNA methyl- phosphotriester reppair domain, residues 1 - 92) (Zn was enlarged (5x) for a better visual effect17.

– 2 – Chapter 1: Introduction

39 kD Ada

HS 69 321 N C SH

H3N+ CO2-

N-terminal C-terminal domain domain N-Ada10 MeP Repair O6-MeG Repair 1 92 179 354

N-Ada20 C-Ada19

Figure 2. Schematics of the Ada protein, it’s constituent domains and a summary of Ada’s DNA repair function16,17.

Each exponentially growing cell contains only 2-4 molecules of Ada protein and 10- fold more Ogt protein. During active growth the prime defense against endogenous DNA methylation is, therefore, the Ogt protein. The Ada protein is induced by as much as 1000- fold upon exposure of cells to DNA-methylating agents and confers increased protection against mutation induction18. Ada is a 39 kD protein divided by two domains, the N-terminal domain with 20 kD and the C-terminal domain with 19 kD of weight. Based on the aminoacid sequence of the products and the nucleotide sequence of the ada gene, the major cleavage site was found to be between Lys178 and Gln179 of Ada protein19. The two domains of the protein exhibit distinct methyltransferase activities, but neither promoted transcription of the ada gene20. It’s important to notice that in vivo cleavage of Ada has not been detected and that observed in vitro cleavage is apparently due to the OmpT gene product, an outer membrane protease21. The primary injuries repaired by Ada protein are O6-methylguanine, O4- methylthymine (Figure 3), and the Sp diastereomer of methyl phosphotriesters (Figure 4).

– 3 – Chapter 1: Introduction

OMe OMe

N N N

N N O N H2N R R a) b)

Figure 3. Schematic drawing of: a) O6-methylguanine; b) O4-methylthymine.

B1 B1 HOCH2 HOCH O 2O

H3CO O O O

P P B2 B2 OCH OCH O 2O H3CO 2O

R OH S OH Figure 4. Schematic drawing of the R and S stereoisomers of methyl phosphotriesters produced by alkylation of DNA phosphate residues22.

Ada removes the offending methyl groups by direct, stoichiometric, and irreversible transfer to specific cysteine residues in the protein. Base methylation is repaired in the C- terminal portion of the Ada by Cys321 and it’s not know if there is a direct influence of zinc in this process. On the other hand, the phosphate damage is repaired by methyl transfer to Cys69, one of four cysteine residues bound to a zinc ion in the N-terminus of Ada12. DNA methyl phosphotriesters are formed by modification of phosphate residues of the DNA backbone. The reactions with an alkylating agent causes covalent linkage of a methyl group to either of the oxygen atoms not involved in the 5’-3’ phosphodiester bond. A pair of stereoisomers, in the R and S configuration (Figure 4), is generated. In addition to acting as a DNA methyltransferase, Ada regulates this adaptive response and controls expression of four inducible genes, ada, alkA, alkB and aidB (Table 1).

– 4 – Chapter 1: Introduction

Table 1. Some known inducible genes whose expression is controlled by Ada protein23,24.

Gene Encoded Protein Known Functions Repairs O6-alkylG, O4-alkylT, and alkyl-P lesions.

ada Ada Protein Regulates the transcription of genes, which encode for DNA alkylation repair proteins.

alkB AlkB Protein Membrane protein with unknown function. Repairs 3-methylA, 3-methylG and O2-methyl-pyridine AlkA Protein alkA (DNA glycosylase) lesions.

Confers resistance to MNNG and possibly have a role in aidB AidB Protein MNNG detoxification.

Methylation of Cys69 reveals in Ada a sequence-specific DNA binding activity that recognizes a promoter element – the “Ada box” – present in ada regulon genes18. Thus, methylation of Cys69 in Ada is the triggering mechanism for a genetic switch that regulates resistance to methylating agents in E. coli. It’s also known that the specific DNA affinity of the protein is increased three orders of magnitude (103) by transfer of a methyl group to Cys69. The mechanism by which the adaptive response shuts off has remained unknown. Since the methylation of Cys69 is apparently irreversible, it has been postulated that methylated Ada is simply diluted out during cell growth subsequent to removal of alkylated DNA damage. An alternative hypothesis is that the Ada protein can be inactivated in vivo by proteolytic cleavage at is Lys178-Gln179 bond19. However, there are no evidences in vivo of proteases capable of cleaving the Ada protein in that specifically bond. More recently, it was found that physiologically relevant higher concentrations of unmethylated Ada are able to inhibit the activation of ada transcriptor by methylated Ada, both in vitro and in vivo. It was also identified the C-terminal 10-20 % of Ada as the most important region of the protein that plays a role in this negative regulation24. Many proteins are known to coordinate zinc through four Cys residues. However, except for Ada, the metal has invariably been shown merely to stabilize the folded structure of the protein25. Ada differs even from the numerous transcription factors that contain structural

– 5 – Chapter 1: Introduction

26 (Cys)4- ligated zinc in that Ada’s sequence-specific DNA binding activity is potenciated only after methylation of one of its Cys ligands (Cys69). Ada is unusual in that the zinc serves to activate the nucleophilicity of Cys69 by stabilizing the thiolate form at neutral pH (Figure 5). The activation of Cys69 by Ada may be viewed as analogous to the activation of H2O by carbonic anhydrase because the metal assists deprotonation of the protonucleophile, presenting a metal-bound anion for reaction with an electrophile27.

Cys42-S S-Cys38 Zn S-Cys69 Cys72-S Figure 5. A model for metalloactivated repair of a DNA methylphosphotriester by Ada. residue H3C O Cys69, present either as a metal-bound

thiolate or a transiently free thiolate, attacks P RO OR the methyl carbon of a DNA O methylphosphotriester.

1.2.2. Other Methyltransferases

1.2.2.1. Cobalamin-Independent Methionine Synthase

Cobalamin-independent methionine synthase from E. Coli catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine14 (Figure 6).

CH3-H4folate + homocysteine ® H4folate + methionine

Figure 6. Reaction catalyzed by cobalamin-independent methionine synthase.

The nucleophilic displacement of the methyl group from a tertiary amine only occurs through a direct attack of a zinc coordinated (activated) homocysteine.

– 6 – Chapter 1: Introduction

1.2.2.2. Cobalamin-Dependent Methionine Synthase

Cobalamin-dependent methionine synthase catalyzes the transfer of a methyl group from methylcobalamin to homocysteine13. This reaction is a part of catalytic cycle (Figure 7).

+ CH3-H4folate + cob(I)alamin + H ® H4folate + methylcob(III)alamin

methylcob(III)alamin + homocysteine ® cob(I)alamin + methionine + H+

Figure 7. Two half reactions involved in the catalytic cycle of cobalamin-dependent methionine synthase

The mechanism of homocysteine activation seems to be very similar to the on from cobalamin-independent methionine synthase.

– 7 – Chapter 1: Introduction

1.3. Aldolases

Aldolases are a specific group of lyases able to catalyze the stereospecific addition of a ketone donor to an aldehyde acceptor28. Over 30 aldolases have been identified to date. They are present in all animals and in most microorganisms, and catalyze central steps of the carbohydrate metabolism (Figure 8). There are two distinct classes of aldolases according to their mechanism.

Figure 8. Reversible cleavage of fructose-1,6-bisphosphate.

1.3.1. Class I aldolases

Class I aldolases are found in animal and higher plant tissue, and have no requirement for a divalent metal cofactor. These aldolases bind fructose-1,6-bisphosphate (FBP) in its linear form as a protonated Schiff base. A tyrosine side chain in its phenolate form then abstracts a proton from the 4-hydroxy group of the substrate. This initiates an aldol cleavage reaction between the substrate's number-3 and number-4 carbons to yield one molecule of glyceraldehyde-3-phosphate (G3P) and an enamine intermediate. The reaction is catalyzed because the electron pair that formed the cleaved bond is readily delocalized by the protonated Schiff base to form the enamine. The G3P is released by the enzyme and proceeds along the glycolytic pathway. The enamine is protonated by the tyrosine side chain and the resulting imminium cation is hydrolyzed to yield the reaction's second product, dihydroxyacetone phosphate (DHAP) (Figure 9).

– 8 – Chapter 1: Introduction

Figure 9. Type I aldolase mechanism (taken from Ref. 38).

1.3.2. Class II aldolases

Class II aldolase is found in primitive cells such as yeasts and bacteria, and require a metal co-factor. Most of these aldolases use a divalent zinc cation to stabilize the enolate intermediate of the aldol reaction. A typical active site of the enzyme comprises three histidines and a glutamic acid all coordinated to the zinc cation (Figure 10)29.

– 9 – Chapter 1: Introduction

Figure 10. Active site of Fuc A. The zinc cation was enlarged (2.5x) for better visual effect.

Dreyer and Schultz29 proposed that upon binding of the substrate (DHAP) the carboxylate of Glu 73 rotates away providing the necessary space and deprotonating the carbon in the alpha position to the carbonyl to form the enolate. This enolate, stabilized by the zinc coordination, acts as a nucleophile and attacks the L-lactaldehyde. The protonation of the newly formed alkoxide is provided by the Tyr 113’. This tyrosine gets immediately protonated through Glu 73. Upon release of L-fuculose-1-phosphate, Glu 73 takes it’s initial position of coordination and the enzyme is ready for another catalytic cycle (Figure 11).

– 10 – Chapter 1: Introduction

His 155 Tyr 113' DHAP OH HN HO O N H O N Zn2+ Glu 73 O His 94 O N N O P O O NH L- fuculose- 1- phosphate

His 92 His 155 Tyr 113'

HN OH HO Tyr 113' O N H H O N Zn2+ OH Glu 73 His 94 - O N H OH O - N H O O NH Zn2+ O H P O - O O His 92

O O P L- Lactaldehyde O O O Tyr 113'

O HO OH

H OH O Zn2+

-O

O O P O O

Figure 11. Catalytic cycle of class II fuculose-1-phosphate aldolase.

– 11 – Chapter 1: Introduction

1.4. The Aldol Reaction

The aldol reaction is crucial for C–C bond formation in organic synthesis. Therefore, there has been a big development of methodologies for this type of reactions. In the aldol reaction, the a carbon of one aldehyde or ketone molecule adds to the carbonyl carbon of another30. The base most often used is OH–, though stronger bases, e.g., aluminum t-butoxyde, are sometimes employed. Hydroxide ion is not a strong enough base to convert substantially all of an aldehyde or ketone molecule to the corresponding enolate ion (Figure 12), i.e., the equilibrium lies well to the left for both aldehydes and ketones. Nevertheless, enough enolate ion is present for the reaction to proceed (Figure 13).

CH R OH C R C R

O O O

Figure 12. Keto-enolate equilibrium.

R' R'' C R O C R HO C R R' R' O O R'' O R'' O

Figure 13. Mechanism of the aldol addition.

The product is a b-hydroxy aldehyde (called an aldol) or ketone, which in some cases is dehydrated during the course of the reaction (aldol condensation). Even if the dehydration is not spontaneous, it can usually be done easily, since the new double bond is in conjugation with the C=O bond; so that this is a method of preparing a,b-unsaturated aldehydes and ketones as well as b-hydroxy aldehydes and ketones. The entire reaction is an equilibrium (including the dehydration step), and a,b-unsaturated and b-hydroxy aldehydes and ketones can be cleaved by treatment with OH– (the retro-aldol reaction). The scope of the aldol reaction may be discussed under five categories:

– 12 – Chapter 1: Introduction

1. Reaction between two molecules of the same aldehyde. The equilibrium lies far to the right, and the reaction is quite feasible. Of course, the aldehyde must possess an a- hydrogen. 2. Reactions between two molecules of the same ketone. In this case, the equilibrium lies well to the left, and the reaction is feasible only if the equilibrium can be shifted. 3. Reactions between two different aldehydes. In the most general case, this will produce a mixture of four products (eight, if the olefins are counted). However, if one aldehyde does not have an a-hydrogen, only two aldols are possible, and in many cases the crossed product is the main one. The crossed aldol reaction is often called the Claisen-Schmidt reaction. 4. Reaction between two different ketones. This is seldom attempted (except with the use of preformed enolates, see below), but similar considerations apply. 5. Reaction between an aldehyde and a ketone. This is usually feasible, especially when the aldehyde has no a-hydrogen, since there is no competition from ketone condensing with itself. This is also called the Claisen-Schmidt reaction. Even when the aldehyde has an a- hydrogen, it is the a-carbon of the ketone that adds to the carbonyl of the aldehyde, not the other way around. The reaction can be prepared made regioselective by preparing an enol derivative of the ketone separately and then adding this to the aldehyde (or ketone), which assures that the coupling takes place on the desired side of an unsymmetrical ketone. A number of these preformed enolates have been used, the most common of which is the silyl enol ether of the ketone (Figure 14). This can be combined with an

aldehyde or ketone, with TiCl4 (the Mukayama reagent), with various other catalysts, and even in aqueous solution with no catalyst at all. The large number of catalysts reported testify to the importance of this method30.

R2 1 3 4 3 R R R 1. TiCl4 R R2 R1 R4 2. H2O OSiMe3 O O OH Figure 14. Regioselective aldol reaction with the Mukayama reagent.

The aldol reaction can also be performed with acid catalysts, in which case dehydration usually follows. Here there is initial protonation of the carbonyl group, which attacks the a-carbon of the enol form of the other molecule (Figure 15).

– 13 – Chapter 1: Introduction

C R R' R'' R' R'' H H OH HO C R O OH R' R'' OH

HO C R (if a-H present) R' C R R' R'' O R'' O

Figure 15. Acid-catalyzed aldol reaction.

The aldol reaction creates two new chiral centers, and, in the most general case, there are four stereoisomers of the aldol product (Figure 16).

OH O OH O OH O OH O

R' R R' R R' R R' R

Syn (or erythro) (±) pair Anti (or threo) (±) pair

Figure 16. The stereoisomers obtained from an aldol reaction.

In general, most methodologies available for the asymmetric aldol reaction fall in one of the following categories31: i) The chiral auxiliary-assisted aldol reaction based on the use of stoichiometric quantities of the chiral appendage; ii) Chiral Lewis acid-catalyzed Mukaiyama-type and chiral Lewis base-catalyzed aldol reactions; iii) Heterobimetallic bifunctional Lewis acid/Brønsted base-catalyzed direct aldol reactions; iv) Aldol reactions catalyzed by aldolase enzymes and catalytic antibodies.

– 14 – Chapter 1: Introduction

A very important characteristic of the latter two methodologies is the employment of unmodified carbonyl compounds as aldol donor substrates (direct aldol reaction), whereas the two first methodologies require pre-activation of the substrates involved (e.g., the silyl enol ethers described above).

1.4.1. Direct Aldol Reaction

From all existing methods, the direct aldol reaction is the most attractive approach since it does not require the isolation of preformed enolates32, hence keeping the reaction highly atom economic33. An exciting challenge to enhance the efficiency of the aldol reaction is to find a compound that will catalyze the direct aldol addition without prior stoichiometric formation of the nucleophile and to do so asymmetrically34. Few examples exist of direct catalytic asymmetric aldol reactions, among them recent work by Shibasaki35 et al., List36 et al. and Trost34 et al. Detailed information on this examples can be found at sections 1.7.2. and 1.7.3.

1.4.2. Catalytic Antibodies

In recent years catalytic-antibody technology has provided a method of developing new protein catalysts that catalyze a variety of reactions37. In the past several years monoclonal antibodies elicited against a number of haptens designed to resemble the transition states of specific reactions have been shown to be capable of catalyzing those reactions with remarkable rate accelerations38. Catalytic antibodies prepared using diketone haptens and reactive immunization, have provided aldolase antibodies with broad scope and excellent enantioselectivities in a wide range of direct asymmetrical aldol reaction. Key to the function of these catalysts is an active site lysine residue with a highly perturbed pKa that is essential to the enamine mechanism of these antibody aldolases and their natural counterparts, the class I aldolase enzymes39. Aldolase catalytic antibodies developed recently have the ability to match the efficiency of the natural aldolases while accepting a more diverse range of substrates.

– 15 – Chapter 1: Introduction

1.5. Organic Chemistry in H2O

Organic synthesis in water has always been a major goal for most synthetic chemists. To run reactions in aqueous media have the following advantages compared with reactions under anhydrous conditions40:

i) It is not necessary to dry solvents and substrates for the reactions in aqueous media. This means that aqueous solutions of substrates or hydrated substrates can be directly used without further drying; ii) From the viewpoint of the recent environmental conscientiousness, it is desirable to use water instead of organic solvents as a reaction solvent, since water is a safe, harmless, and environmentally benign solvent. Therefore, development of organic reactions in water will contribute to the progress of green chemistry41; iii) Water has unique physical and chemical properties such as high dielectric constant and high cohesive energy density compared with most organic solvents. This unique nature of water is also essential for most enzymatic reactions in living systems.

Many enzymes catalyze desired reactions with high efficiency and excellent stereoselectivity under mild conditions in water, and this effectiveness is often regarded as a goal for chemists. Although many researchers have developed synthetic mimics of active sites of enzymes to realize enzymatic activity, little attention has been given to the medium, water, which plays major roles in the reactions. By utilizing the unique nature of water, it should be possible to develop reaction systems, which cannot be attained in dry solvents. In fact, water can have highly beneficial effects (mainly hydrophobic effect and hydrogen bonding) on the reactions42.

1.5.1. Direct Aldol in H2O

Catalytic aqueous aldol reactions are well documented. Almost all involve metal- based Lewis acid activation of the acceptor and very frequently use silyl enol ethers as aldol donors43.

– 16 – Chapter 1: Introduction

Aqueous catalysis via an enamine nucleophile, in analogy to the class I aldolases, is known to be difficult to achieve due to the known rapid hydrolysis of enamines in water44. Both enzymes and catalytic antibodies do not seem to have this problem. Recently, nornicotine was shown to catalyze aldol reactions in water via, under the authors suggestion, an enamine nucleophile45. However, no enantioselectivity could be observed. The enantioselective methods utilizing Lewis acids rely on the catalysis of metal complexes bearing chiral ligands. So far, all complexes of this type exhibit very high water sensitivity and, consequently, all test reactions have been carried out under anhydrous conditions in organic solvents. Chiral Lewis acid complexes that efficiently catalyze aldol reactions in aqueous medium have not been reported, although such catalyst would have important advantages over the rest40.

1.5.2. Prebiotic Chemistry

The complexity and diversity of contemporary life forms appears at first sight to provide few clues towards the possible origins of life. Closer inspection reveals extraordinary similarities in all organisms at the most basic of biochemical levels46.

1.5.2.1. Amino Acids Synthesis

Some of the first experiments in prebiotic chemistry were carried out by Miller and Urey in the early 1950s47. The authors passed an electric discharge through a gaseous mixture of methane, ammonia, hydrogen and water vapor and were able to isolate many organic compounds including the amino acids glycine, alanine and aspartic acid. Other similar experiments have since been carried out and many other amino acids have been isolated48.

1.5.2.2. Sugar Synthesis

The prebiotic formation of sugars has been a subject of several studies and debate. Pioneer work from Butlerow49 called attention to the prebiotic importance of formaldehyde in what has become know as the “formose reaction”. The author showed that a complex mixture

– 17 – Chapter 1: Introduction of sugars was formed from formaldehyde via an array of aldol condensations. The “formose reaction” can be initiated by an alkaline earth catalyst, such as calcium hydroxide, and yields a mixture of triose, tetrose, pentose, hexose and heptose sugars in aldose and ketose form, branched and straight chain in structure (Figure 17).

Ribulose Ribose Xylulose Arabinose Lyxose Xylose

CH2OH CHO CHO CH2OH HCHO CHO HCHO HCHO C=O CHOH HCHO CHOH C=O Ca(OH)2 CH2OH CHOH CHOH CH2OH CH2OH CH2OH CH2OH

Fructose Glucose Sorbose Mannose Retro-aldol + others + others

Figure 17. The “formose reaction”.

Eschenmoser and coworkers50 showed that the aldolisation of glycoaldehyde phosphate gives a less complex mixture of sugars due to the lack of aldose/ketose equilibria. The authors reported that the aldolisation of glycoaldehyde phosphate in aqueous NaOH solution, at room temperature, after seven days, gave a mixture of tetrose and hexose derivatives in a 1:10 ratio (aprox.), with racemic allose triphosphate being the major hexose formed with up to 50% of the mixture of sugar phosphates. This diastereoselectivity was attributed to chosen by nature and derived from a minimization of steric interactions in the transition state during formation. To date, there are no alternatives to the rather harsh conditions needed for the aldol reaction to take place, which do not seem to be compatible with prebiotic environment.

– 18 – Chapter 1: Introduction

1.6. Zinc Complexes

It is known since a long time ago that a neutral molecule with an electron pair (Lewis base) can donate these electrons to a metal ion or other electron acceptor (Lewis acid). We can define a ligand as any molecule or ion that has at least one electron pair that can be donated. Ligands may also be called Lewis bases; in the terms used in organic chemistry, they are nucleophiles. Metal ions or molecules such as BF3 with incomplete valence electron shells are Lewis acids or electrophiles51. The d10 configuration of Zn2+ indicates that zinc complexes are not subject to ligand field stabilization effects and so coordination number and geometry is only dictated by ligand size and charge. In enzymes, zinc shows a strong preference for tetrahedral coordination, which enhances both the Lewis acidity of a zinc center and the Brønsted acidity of a coordinated water molecule. Only CuII is a better Lewis acid3. The divalent zinc ion is exceptionally stable with respect to oxidation and reduction and so it does not participate in redox reactions, in contrast to Mn, Fe and Cu52. The flexibility in coordination geometry makes ligand exchange more facile than for Ni or Mg and enhances the ability of zinc to effect a catalytic cycle52. Zinc is an element of borderline hardness, so that nitrogen, oxygen and sulfur ligands can all be accommodated, in contrast to Mg and Ca which favor binding to oxygen. Which also explains why zinc binds strongly to many proteins52. In addition, anions such as HO–, RO– and RS– retain nucleophilic character when coordinated to zinc. The importance of studying synthetic analogues of zinc enzymes resides with the fact that such species are more amenable to structural, spectroscopic and mechanistic studies than the enzymes themselves. Accurate synthetic analogues of zinc enzymes are not, however, trivial to obtain. A simple illustration of this statement is provided by the fact that whereas pseudo-tetrahedral coordination geometry is prevalent in zinc enzymes (Figure 18), higher coordination numbers are common for simple zinc complexes in both the solid state and solution53. Furthermore, degradation pathways leading to polymerization are inhibited for the enzyme by virtue of the fact that the active sites are located in its interior. To circumvent such problems, considerable attention must be given to ligand design in order to procure synthetic analogues that mimic well the enzyme active sites.

– 19 – Chapter 1: Introduction

Figure 18. Structures of biological Zn(II) sites: structural sites in alcohol dehydrogenase (A) and a zinc finger (B); catalytic sites in carbonic anhydrase (C), carboxypeptidase A (D), alcohol dehydrogenase (E), aminopeptidase (F) and leucine aminopeptidase (G).

The high propensity of thiolate ligands to yield sulfur-bridged polymeric metal species is well-documented54. The strategies to avoid this problem rely either on the design of the ligand, or on the source of the zinc metal ion. Many ligands have been designed and synthesized with different bulky groups on the a-position to the thiol in order to avoid sulfur-bridging55. A very important disadvantage of this strategy it the decrease of nucleophilicity of the zinc thiolate when comparing a primary to a tertiary thiolate. An interesting approach, it to present the zinc metal ion already coordinated to a strong chelating ligand preventing the zinc coordination to more then one thiolate71. This will also decrease the ability of zinc to serve as a template in bringing together both reactants.

1.6.1. The Chelating Effect

The term “chelate effect” refers to the enhanced stability of a complex system containing chelate rings as compared to the stability of the system that is as similar as possible but contains none or fewer rings51. As an example, consider the following:

– 20 – Chapter 1: Introduction

2+ 2+ Ni (aq) + 6NH3(aq) = [Ni(NH3)6] (aq) log b = 8.61 2+ 2+ Ni (aq) + 3en(aq) = [Nien3] (aq) log b = 18.28

b = overall formation (stability) constant; en = ethylene diamine (NH2CH2CH2NH2);

2+ 10 The system [Nien3] in which three chelate rings are formed is nearly 10 times as stable as that in which no such ring is formed. Although the effect is not always so pronounced, such a chelate effect is a very general one. To understand this effect, we must invoke the thermodynamic relationships:

DG° = -RT ln b DG° = DH° -TDS°

Thus b increases as DGº becomes more negative. A more negative DGº can result from making DHº more negative or from making DSº more positive. In the example cited, the enthalpies make a slight favorable contribution, but the main source of the chelate effect is still to be found in the entropies. An example, which illustrates the existence of a chelate effect despite an unfavorable enthalpy term, is the following:

2+ 2+ [Ni en2(H2O)2] (aq) + tren(aq) = [Ni tren(H2O)2] (aq) + 2 en (aq) log b = 8.61

tren = N(CH2CH2NH2)3

For this reaction we have DHº = +13.0, –TDSº = –23.7 and DGº = –10.7 (all in kJ.mol–1). The positive enthalpy change can be attributed both to greater steric strain resulting from the presence of three fused chelate rings in Ni–tren, and to the inherently weaker M–N bond when N is a tertiary rather than a primary nitrogen atom. Nevertheless, the greater number of chelate rings (3 vs. 2) leads to greater stability, owing to an entropy effect that is only partially cancelled by the unfavorable enthalpy change. Probably the main cause of the large entropy increase in the cases we have been considering is the net increase in the number of unbound molecules-ligands. Another more

– 21 – Chapter 1: Introduction pictorial way to look at the problem is to visualize a chelate ligand with one end attached to the metal ion. The other end cannot then get very far away, and the probability of it, too, becoming attached to the metal atom is greater than if this other end were instead another independent molecule, which would have access to a much larger volume of a solution. The latter view provides an explanation for the decreasing magnitude of the chelate effect with increasing ring size. Off course, when the ring, that must be formed, becomes sufficiently large (seven membered or more), it becomes more probable that the other end of the chelate molecule will contact another metal ion than that it will come around to the first one and complete the ring.

1.6.2. NxSy Environment

In the case of NxSy environments, one needs multidentate N,S ligands which ideally should fulfill the following conditions56:

i) They should contain the correct number of N and S donors; ii) They should encapsulate the metal in such a way that oligomerizations via sulfur bridging are prevented; iii) They should leave room for a labile ligand which may be replaced by a reacting substrate; iv) They should provide the right charge and electronic environment to the metal to tune it for its catalytic function;

One of the most simplest and widely used methods of forming C–S bonds involves nucleophilic attack of a thiolate on a suitable C-centered electrophile such as an alkyl halide (Figure 19). Coordinated thiolate ligands behave as nucleophiles in exactly the same manner, and the method has been extensively used for the preparation of thioethers and their metal complexes. The use of a metal ion to control the reactivity of ambidentate ligands is also of great importance57. A typical example is seen in the methylation of bis(2- aminoethylthiolato)nickel(II) with methyl iodide to yield the bis(methylthioethylamine) nickel(II) cation (Figure 20).

– 22 – Chapter 1: Introduction

+ X RS X RS

Figure 19. The nucleophilic displacement of a leaving group by a thiolate is one of the common methods for the formation of C–S bonds.

H2N S H2N SMe MeI Ni Ni

H2N S H2N SMe

Figure 20. The alkylation of an aminothiolate can occur very selectively at the sulfur atom.

1.6.3. Zinc Complexation with Aminoacids

The renaissance of classical coordination chemistry 30-40 years ago, which was triggered by the advent of all modern physical methods, included the elucidation of the structural diversity of metal-aminoacid complexes. Nevertheless, today there are undoubtedly more metalloprotein crystal structures than metal-aminoacid or metal-peptide complex structures58. The reason for this is the reluctance of the latter to form crystals. There are just a few structure determinations of zinc-peptide complexes and eleven 59 60 2–61 62 binary zinc-aminoacid complexes, namely Zn(Gly)2 , Zn(Leu)2 , [Zn(Cys)2] , Zn(Met)2 , 63 64 65 66 58 58 Zn(His)2 , Zn(Ser)2 , Zn(Asp)(H2O)2 , Zn(Glu)(H2O) , Zn(Ile)2(H2O)2 , Zn(Phe)2 and 67 Zn(Pro)2 . Almost all the zinc-aminoacid complexes with the carboxylate-O and amino-N coordination (with the exception of Zn(Ile)2(H2O)2) are polymers held together by bridging carboxylate ligands. A common characteristic of all zinc-aminoacid complexes seems to be their low solubility in organic solvents. This might explain the difficulty to get suitable crystals for structural determination. Moreover, it can condition severely their use as catalysts for organic synthesis. To my knowledge, these binary zinc-aminoacid complexes were never studied for that purpose.

– 23 – Chapter 1: Introduction

1.7. Chemical Models and Catalysts

1.7.1. Methyltransferase Models

2- S. Lippard and J. Wilker reported a functional model for the [Zn(S-Cys)4] site of E. coli’s Ada that repairs the DNA alkyl-P lesions68,69. In this model, they used a system where a thiolate of [(CH3)4N]2[Zn(SC6H5)4] accepts a methyl group from (CH3O)3PO (TMP) (Figure 21). It’s a 2nd order reaction, 1st order with respect to each reagent, with a rate constant of (1.6 ± 0.3) ´ 10-2 M-1s-1 at 24.5 (± 0.6) °C. Ada repairs DNA with a me-P lesion in aqueous buffer at 4 °C with a second order rate constant of 2.8 ´ 102 M-1s-1. The authors attribute the significant difference in the rate constants to the usual ability of enzymes to provide optimal substrate binding, orientation and transition state stabilization. Solvent differences (H2O vs. DMSO) and the energetics of TMP rather than a DNA me-P are additional factors that contribute to kinetic differences between the protein and the model chemistry. This study also showed that metal bound thiolates have inferior nucleophilicity (and, therefore, rate constant) than the free thiolate. The thiol form does not react at all with the methylated substrates. Combining these results with those with Ada protein, these authors proposed a mechanism for methyl-P repair in the protein, where a transiently dissociated Cys69 ligand is the nucleophile responsible for accepting the methyl group from the DNA methyl-P lesion. With the zinc bound state preventing the protonation and the deactivation of the cysteine thiolate nucleophile.

2 2 CH3 O O S S S P P O CH DMSO-d6 S Zn S + H3C O 3 S Zn O O CH3 + O O S S CH 3 CH3

Figure 21. Reactional scheme of the model proposed by Lippard and Wilker68.

– 24 – Chapter 1: Introduction

Hammes and Carrano70 recently reported that methylation of the coordinated thiolate in pseudotetrahedral zinc complexes of the form [(L3S)ZnX] by a variety of alkylating agents appears to occur via a nondissociative route (Figure 22). In their case, the resulting thioether can remain coordinated to the metal center as it does in zinc dependent alkyl transfer enzymes.

Figure 22. Hammes and Carrano’s non- dissociative alkylation model70.

Vahrenkamp71 and coworkers reported a series of tripodal tris(pyrazolyl)borate complexes [TpRR’]ZnSR (Figure 23). All four complexes (1 and 2 in Figure 23) reacted with methyl iodide in a 1:1 ratio in chloroform at room temperature. The authors showed that the methylations occur at the zinc bound thiolates. Grapperhaus72 and coworkers reported dimeric dithiolate complexes with zinc and cadmium that mimic the active site of zinc-dependent methylation proteins. In their work, upon methylation of the thiolates with methyl iodide, the resulting thioethers dissociates from the metal, which suggests that a restrictive environment in the Ada protein is required to enforce thioether binding (Figure 24).

– 25 – Chapter 1: Introduction

Figure 23. Vahrenkamp’s tripodal model71.

Figure 24. Grapperhaus model with zinc72.

Bridgewater and Parkin73 also introduced the thiolate complex [TmPh]ZnSPh (Figure 25) as a model for the Ada protein, in which the [TMPh] ligand mimics the three cysteine residues that remain bound to zinc during the course of the alkylation reaction. The phenylthiolate ligand also possesses nucleophilic character and is alkylated by methyl iodide to give PhSMe and [TmPh]ZnI.

– 26 – Chapter 1: Introduction

Figure 25. Parkin’s model for Ada protein73.

1.7.2. Class II Aldolase Models

Up to date, there are not many models for class II aldolases and most of the ones existing are mainly structural or functional without stereoselectivity. Vahrenkamp74 and coworkers reported a modified trispyrazolyl borate zinc complex with three nitrogens and a water molecule coordinated with a zinc cation, mimicking the active site of the enzyme (Figure 26). Several crystal structures of the model complex bound to several aldolase inhibitors were obtained, but no testing for catalysis was performed.

– 27 – Chapter 1: Introduction

Figure 26. Vahrenkamp’s structural model74.

Recently, Kimura75 and coworkers reported also a structural model with four nitrogens and a carbonyl group coordinated to a zinc cation (Figure 27). These authors showed the first quantitative assessment of enolate formation promoted by a proximate zinc cation near neutral pH in aqueous solution. Nevertheless, no attempts to test for catalytic activity were performed.

Figure 27. Kimura’s structural mimic of the class II aldolase75.

– 28 – Chapter 1: Introduction

1.7.3. Small Molecules as Catalysts of the Aldol Reaction

The search for simple catalysts that mimic the selectivity of biochemical methods and yet offer more general substrate acceptance has been the subject of intense research in recent years76. Several methods have been reported over the years having in common one of the following strategies38:

1) Activation of the acceptor (aldehyde); 2) Activation of the donor (ketone); 3) Activation of both the donor and the acceptor; 4) Proline-like catalysts;

1.7.3.1. Activation of the Acceptor

The catalytic activation of the acceptor aldehyde toward the addition of a silyl enol ether, commonly referred to as the Mukaiyama reaction, has been a particularly successful method of performing aldol reactions. The catalyst is usually a Lewis acid. The catalyst coordinates to the aldehyde (acceptor) creating an asymmetric environment, which is then attacked by an enolate species (donor) from the less/hindered face to produce the aldol adduct (Figure 28). A successful catalyst must coordinate strongly enough with the oxygen atom of the aldehyde to form a tight transition state for effective transfer of chirality and at the same time not form such a strong bond with the resulting alkoxide that turnover does not occur38. A second common problem that must be overcome is the generation of M+,77 that may also catalyze the reaction through an achiral pathway and lead to decreased enantioselectivities.

Figure 28. The Mukaiyama reaction38.

– 29 – Chapter 1: Introduction

The first successful catalytic asymmetric Mukaiyama reactions were achieved with tin(II) complexes78. The catalysts were prepared in situ from the addition of tin(II) triflate to a chiral diamine derived from L-Proline (Figure 29).

Figure 29. Chiral diamines used in the preparation of Sn(II) complexes38.

The reaction between aldehydes and ketene silyl acetals prepared from either esters or thioesters is highly enantioselective with ee values usually greater than 98% (Figure 30).

Figure 30. Tin(II)-catalyzed asymmetric Mukaiyama-aldol reaction38.

– 30 – Chapter 1: Introduction

Considerable attention has been paid to titanium(IV) catalysts77. The most successful ones are the ones derived from a titanium(IV) source and an (R)- or (S)-BINOL-type ligand (Figure 31).

Figure 31. BINOL-titanium(IV) catalysts38.

The simple BINOL complexes are efficient catalysts for the aldol reaction of aldehydes and ketene silyl acetals of thioesters (Figure 32)79, giving good to excellent yields and excellent enantioselectivities (up to 98% ee).

Figure 32. BINOL-titanium- (IV)-catalyzed aldol

reaction38.

– 31 – Chapter 1: Introduction

Further improvement was made with the Carreira catalyst80, which is prepared in situ by combining the tridentate ligand derived from BINOL, Ti(OiPr)4 and salicylic acid in the presence of trimethylsilyl chloride and trimethyl amine (Figure 33). The reaction of ketene silyl acetals derived from esters affords b-hydroxy ester products in high yields and up to 98% ee.

Figure 33. Top: preparation of the Carreira catalyst. Bottom: Reactions performed with the catalyst38.

Bis(oxazolinyl)-copper(II) complexes have been shown to be effective chiral Lewis acid catalysts for the Mukaiyama aldol reaction of ketene silyl acetals (Figure 34)81,82. Acceptor aldehydes with a-benzyloxyaldehydes and pyruvate esters are very good substrates and afford the corresponding aldol adducts in very high yield (85–100%) with excellent enantiofacial selectivity (92–99% ee).

– 32 – Chapter 1: Introduction

Chiral boron catalysts, such as chiral oxaazoborolidinones83 and chiral (acyloxy)boranes84, have been used to catalyze the asymmetrical aldol condensation of aldehydes with ketene silyl acetals and silyl enol ethers with excellent enantioselectivity (Figure 35).

Figure 34. Copper-catalyzed asymmetric aldol reaction38.

Figure 35. Oxaazoborolidinone- and acyloxyborane-catalyzed asymmetric aldol reaction38.

The first example of a catalytic asymmetric aldol reaction was the gold(I)-catalyzed reaction of an a-isocyanoacetate and an aldehyde85. The reaction is both diastereo- and

– 33 – Chapter 1: Introduction enantioselective and gives trans-oxazolines with excellent enantioselectivities. These oxazolines can by converted in the corresponding syn-b-hydroxy-a-amino acids by hydrolysis (Figure 36).

Figure 36. Gold(I)-catalyzed reaction of a-isocyanocarboxylates or -amides with aldehydes38.

1.7.3.2. Activation of the Donor

The catalytic activation of the donor rather than the acceptor provides an alternative to the asymmetric Mukaiyama aldol reaction. Numerous approaches have been developed that incorporate a variety of catalysts from organometallic reagents to phosphoramide Lewis bases. The rhodium-catalyzed asymmetric aldol reaction is rather recent86 and consists of a rhodium(I) complex coordinated with the trans-chelating chiral diphosphane TRAP. This catalyst promotes the enantioselective condensation of a-cyanopropionates with aldehydes (Figure 37). Simple aliphatic aldehydes afford products with modest to good enantioselectivities (up to 86% ee), while glyoxaldehyde results in the highest level of selectivity (91% ee).

– 34 – Chapter 1: Introduction

Figure 37. Rhodium(I)–TRAP-catalyzed aldol reaction38.

Palladium catalysts have shown an unsurpassed degree of flexibility in the reactions they catalyze. A few examples exist that catalyze the asymmetric aldol condensation. A palladium(II) complex with chiral PCP-type tridentate ligands has been shown to catalyze the asymmetric aldol condensation between methyl isocyanoacetate and aldehydes (Figure 38)87. Similar to the gold(I) catalyst, the reaction produces predominantly the trans- oxazoline; however, in contrast the cis isomer is formed in higher ee values (up to 74%).

Figure 38. PCP-PdII-catalyzed asymmetric aldol reaction38.

In contrast with the organometallic reagents described above, phosphoramides act as Lewis bases to catalyze the aldol reaction by coordinating temporarily to the electrophilic silicon atom of trichlorosilyl enolates, thus generating a strongly activated donor which reacts with a variety of aldehydes. Chiral phosphoramides have been used successfully to afford

– 35 – Chapter 1: Introduction aldol adducts in excellent yields with a high degree of enantioselectivity (up to 97% ee) (Figure 39). The diphenyl-substituted catalyst was found to give the optimal results88.

Figure 39. Phosphoramide-catalyzed asymmetric aldol reaction38.

1.7.3.3. Activation of Both the Donor and the Acceptor

While catalytic activation of aldehyde acceptors by chiral Lewis acids has achieved great success in the asymmetric aldol condensation, pre-conversion of the donor to a more reactive species such as enol silyl ether, enol methyl ether, or ketene silyl acetal is a necessity. Some catalysts have been developed where both Lewis acidic and Brønsted basic sites participate in the reaction, which catalyze the reaction of unmodified ketones with aldehydes. The work of Shibasaki in recent years brought two different catalysts for asymmetric aldol condensations89. The multifunctional catalyst LLB catalyzes the direct transformation of aldehydes and ketones to aldol adducts. The catalyst encorporates a central lanthanum atom, which serves as a Lewis acid and a lithium binaphthoxide moiety, which serve as a Brønsted base (Figure 40). The reaction affords aldol adducts in excellent yields (87-90%), high enantiomeric excess (70- 95% ee), but not very high diastereoselectivity (1:2 to 1:5 syn:anti).

– 36 – Chapter 1: Introduction

A similar catalyst derived from linked BINOL and two equivalents of diethyl zinc showed promising results (Figure 40). The reaction gave very high yields (up to 95%), high diastereoselectivity (up to 97%) and excellent enantioselectivity (up to 99%).

Figure 40. Shibasaki’s catalysts for direct aldol condensation38.

Recently, Trost also reported a series of catalysts capable of asymmetric aldol reaction90. The catalyst was prepared by reacting the free ligand with two equivalents of diethyl zinc (Figure 41). The catalyst showed to be quite versatile and in an example90b case the authors reported for the reaction of hydroxyacetophenone with cyclohexanecarboxaldehyde, excellent yields (up to 97%), very high diastereoselectivity (up to 30:1 syn:anti) and very high enantioselectivity (up to 93%).

Figure 41. Trost’s catalyst for direct aldol condensation38.

– 37 – Chapter 1: Introduction

1.7.3.4. Proline-like Catalysts

The original studies of the utility of proline as asymmetric catalyst were performed by Hajos and Parrish91 and by Eder and coworkers92, while studying the Robinson annulation reaction (Figure 42). They found that the best enantioselectivities were achieved with proline.

Figure 42. Robinson annulation reactions catalyzed by proline91.

Agami93 and List94 showed the ability of proline to catalyze intramolecular aldol cyclodehydration reactions (Figure 43). The ideal reaction conditions for high enantioselectivities involve polar solvents, and the carboxylate functionality is crucial for asymmetrical induction.

R R = Ph (R) 47% ee R = Me (R) 42% ee O R O (L)-Proline 10 mol% R = n-C5H11 (R) 20% ee DMF, 5d R = i-Pr (R) 8% ee O R = t-Bu (R) 0% ee

Figure 43. Cyclodehydration reactions catalyzed by proline94.

Barbas III and coworkers36 showed recently that a variety of amino acids and proline derivatives catalyze the asymmetric aldol reaction of acetone with 4-nitrobenzaldehyde (Figure 44). Proline and 5,5-dimethyl thiazolidinium-4-carboxylate (DMTC) showed to be the most efficient catalysts, giving both high yields and high enantioselectivities.

– 38 – Chapter 1: Introduction

Figure 44. Proline and DMTC as catalysts of the direct asymmetric aldol reaction36.

The authors suggest that the reactions proceed via an enamine mechanism (Figure 45) and a highly organized metal-free Zimmerman-Taxler-type transition state95.

Figure 45. The enamine mechanism36.

– 39 – Chapter 2: Results and Discussion

2. Results and Discussion

2.1. Methyl Transfer to Sulfur

2.1.1. Our Project

2– The Ada protein uses an active site with the structure [Zn(S-cysteine)4] to repair methylphosphotriesters present in DNA alkylation products21. It has been proposed that zinc serves to activate Cys69 leading to a nucleophilic attack on the offending methyl groups12. Despite the several studies on this subject (see chapter 2.7.1.), there is no clear explanation of the mechanism for this action. All the models proposed until now lack important features that might be essential for a good mimic of the protein. These features are: the need for the use of primary alkyl thiols (to mimic the cysteine residue); the use of chelating ligands and determine their importance (the active site of the protein is a chelating environment); and the use of a sulfur-rich environment around zinc, assessing its importance (comparison between

ZnCys4 and ZnCys2His2 active sites in the different methyltransferases). We address this problem on a stepwise way. The first model studied was between two primary alkyl thiols, a chelating and a non-chelating. The non-chelating ligand used was the commercially available hexane-1-thiol and MEPAH was used as a chelating ligand. The system studied consisted in trimethyl phosphate (TMP) as methyl donor, mimicking the methylated DNA phosphate backbone, and a methyl acceptor in the form of thiol, naked thiolate and zinc coordinated thiolate. The study of reactivity towards TMP by the different ligands and complexes under different conditions should give some insight on the influence of chelation on zinc bound thiolate nucleophilicity. The model of the DNA’s phosphate backbone was also improved by replacing TMP with a methylated dinucleotide in the reactivity studies. The dinucleotide dTp(Me)dTAc was synthesized and used for this purpose. To address the importance of the sulfur-rich environment around zinc, we synthesized a variety of ligands possessing different binding motifs for zinc like NS2, N2S2, NS3 and S4.

– 40 – Chapter 2: Results and Discussion

2.1.2. MEPAH vs. Hexane-1-thiol

2.1.2.1. The Non-chelating Alkyl Thiol: Hexane-1-thiol

The influence of zinc on the methyl transfer from TMP to hexane-1-thiol was studied under different conditions (Figure 46).

O O Zn+2 SH + P SCH3 + P H3CO OCH3 H3CO OH OCH3 1 OCH3 TMP DMP Figure 46. Reaction scheme of methyl transfer to a hexane-1-thiol.

In this system, only the sodium thiolate (Table 2, Entry 7) reacts with TMP at room temperature. The presence of a base is essential for methyl transfer to take place and pyridine showed to be better than dimethyl aniline (DMA) (Table 2, Figure 47). This might be due to steric hindrance of DMP (the two bases have similar pKa’s). It is very clear that Zn+2 salts had no effect on the reaction suggesting that zinc activation of the thiol (if existent) is not able to compete with the reaction of the naked thiolate formed with the base.

Table 2. Methyl transfer from TMP to hexane-1-thiol in refluxing methanol for about 18 h.

Entry Base Zinc Temperature Yield [%] RT 0 1 - - RF 6 RT 0 2 Py - RF 42 RT 0 3 Py Zn(NO ) .6H O 3 2 2 RF 44 RT 0 4 Py ZnCl 2 RF 40 5 DMA - RF 20

6 DMA ZnCl2 RF 24

7a NaOH - RT 100

a) Performed at Room Temperature.

– 41 – Chapter 2: Results and Discussion

Methyl transfer to Hexane-1-thiol

100 90 80 70

] 60 % [

d

l 50 e

i RT

Y 40 RF 30 20 10 0 RF 1 2 3 RT 4 5 6 7

Figure 47. Graphical representation of the results in Table 2.

The reaction between pyridine and TMP with the formation of DMP and N- methylpyridinium has been described96. Under our reaction conditions, the reaction is slow and it can only account for a small fraction of methyl transfer from TMP. The methyl group from N-methylpyridinium was not transferred to hexane-1-thiol.

2.1.2.2. The Chelating Alkyl Thiol: MEPAH

The chelating alkyl thiol MEPAH was studied in the same system as before including some pre-formed complexes with zinc. It should be noted that the pyridine is incorporated in the molecule and that complexation with zinc was achieved.

Both MEPAH (2) and the complex [MEPAZnCl]n (6) were synthesized according to a 56 literature procedure . The synthesis of the complex (MEPA)2Zn (5) has also been reported 56 before starting from Zn[N(SiMe3)2]2. A simplification of this procedure was obtained when 2 was reacted with the commercially available diethyl zinc in toluene, giving 5 in good yield (Figure 48). The 1H-, 13C-NMR spectroscopy, m.p. and IR of the complex were identical to the one reported.

– 42 – Chapter 2: Results and Discussion

N S SH Et Zn H H 2 N N N Zn N Toluene S NH 82% 2 5

Figure 48. Synthesis of (MEPA)2Zn complex.

The reaction of MEPAH, (MEPA)2Zn and [MEPAZnCl]n with TMP to give DMP and Me-MEPA (3) was studied under different conditions (Figure 49).

O SH +2 H + P Zn N MeO OMe SMe OMe H N N 2 N 3 + N S O H O Zn N N + P P MeO OMe MeO OH S NH OMe OMe 5

- n N Cl

N S- O Zn+2 Zn+2 -S + P MeO OMe OMe Cl- N N 6 C16H22N4Cl2S2Zn2 536.16

Figure 49. Reaction scheme of methyl transfer from TMP to MEPAH, MEPA2Zn and

[MEPAZnCl]n.

The reaction does not take place at RT or in the absence of Zn+2 (Table 3, Figure 50). Refluxing the ligand with TMP yielded only 10% of DMP what can be considered as the hydrolysis product since we are measuring the DMP formed in the reaction. The reaction was run with three different zinc salts: ZnCl2, Zn(NO3)2.H2O and Zn(CH3COO)2.6H2O at room temperature and under reflux. In the presence of Zn+2, the reaction is complete under reflux in

– 43 – Chapter 2: Results and Discussion the time frames studied and that at room temperature only zinc acetate is able to catalyze the reaction. This might be explained by the better solubility of the zinc acetate complex compared with zinc chloride and zinc nitrate.

Table 3. Methyl transfer from TMP to MEPAH in methanol for about 18 h (RT) or 22 h (RF).

Entry Zinc Temperature Yield [%] RT 0 1 - RF 10 RT 0 a) 2 ZnCl2 RF 96 RT 8 a) 3 Zn(NO3)2.6H2O RF 100 RT 24 4 Zn(CH3COO)2.2H20 RF 100 a b RT 0 ) 5 [MEPAZnCl]n ) RF 35 a)

b RT 36 6 (MEPA)2Zn ) RF 100

A ratio of 2/2/1 for MEPAH/TMP/ zinc for entries 2-4 and 1/1 Zn complex/TMP for entries 5,6. a) The zinc complex formed was not completely soluble. b) The zinc complex was preformed and used in the pure form.

The reaction with the preformed complex (MEPA)2Zn gave a reasonable methylation at RT (36%). This reaction was also performed in chloroform with similar results; after 18 hours at RT, 34% of DMP was formed. Me-MEPA (3) was also obtained when MEPAH was treated with NaOH and TMP in 1 MeOH at RT for 26 h. In this case, traces (~1%) of Me2-MEPA (4) were also detected by H- NMR spectroscopy. Compound 4 was also independently prepared by methylation of MEPA- thiolate with MeI in toluene at RT for 2.5 h (34% Me2-MEPA and 66% Me-MEPA were formed) (Figure 51).

– 44 – Chapter 2: Results and Discussion

Methyl Transfer to MEPAH

100 90 80 70

] 60 % [

d

l 50

e RT i

Y 40 RF 30 20 10 0 1 2 3 4 5 6

Figure 51. Graphical representation of the results in Table 3.

NaH, MeI SH S S H H N Toluene N + N N N N 50% 2 3 4 2 : 1

Figure 51. Methyl iodide methylation of MEPAH.

The reaction with polymeric complex [MEPAZnCl]n (6) at reflux MeOH gave a lower yield then the reaction with ZnCl2/MEPAH or with the complex (MEPA)2Zn .The polymeric material is quite insoluble and seems to have a different composition that the complex formed 56 in situ with ZnCl2. Vahrenkamp solved the X-ray structure of 6 and showed that the thiolate in MEPAH is coordinated to two zinc atoms by a sulfur bridge, which might explain the lower reactivity.

– 45 – Chapter 2: Results and Discussion

2.1.2.3. Kinetic study

A kinetic analysis was undertaken to better understand methyl transfer from TMP to 1 (MEPA)2Zn. The reaction was followed by H-NMR in d4-MeOH (for details see experimental section) and showed to be first order with respect to TMP (Figure 52). By maintaining its concentration constant and varying [(MEPA)2Zn], data was obtained for a plot of pseudo-first-order constant k’ vs. (MEPA)2Zn concentration (Figure 53).

Pseudo First Order Fitting

-3,7 0 20000 40000 60000 80000 100000 120000

-3,8

-3,9 ]

P ln[TMP]

M -4

T Linear (ln[TMP]) [ n l

-4,1

-4,2

-4,3 Time (s)

Figure 52. The natural log of TMP concentration plotted against time. The linear fitting gives

us the pseudo-first-order rate constant (k’). ([TMP]=24 mM; [MEPA2Zn]=119 mM)

A second-order rate constant was obtained with the value 5´10–5 M–1 s–1. Lippard and Wilker68 obtained a rate constant of 1.6´10–2 M–1 s–1 for the methyl transfer from TMP to

[(PhS)4Zn][CH3)4N] in DMSO that was attributed to the reaction of a dissociated thiolate. This reaction is aprox. 300 times faster than our system, which suggests that our thiolate is bound to zinc.

– 46 – Chapter 2: Results and Discussion

Carrano70 reported a rate constant of 1.6´10–4 M–1 s–1 for the methyl transfer from methyl iodide to a thiophenolate coordinated to zinc bound to a scorpionate ligand in chloroform. This value is aprox. 30 times faster then our system and can be accounted for the difference in reactivity between MeI and TMP. Rate constants for the methyl transfer from TMP to thiophenolates (Li+, Na+, K+, + –4 –1 –1 97 Me4N ) in methanol (1.7–2´10 M s ) have also been reported . These values show that there is no effect from changing the anion and it is clear that the reaction is slower in methanol than in DMSO.

k' vs. [MEPA2Zn]

8.00E-06

7.00E-06 y = 4.97E-05x

6.00E-06

5.00E-06 ) 1 - s

( 4.00E-06

' k 3.00E-06

2.00E-06

1.00E-06

0.00E+00 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14

[MEPA2Zn] M

Figure 53. The pseudo-first-order rate constant plotted against the concentration of

MEPA2Zn.

– 47 – Chapter 2: Results and Discussion

2.1.2.4. Discussion

The methyl transfer from TMP to non-chelating alkyl thiols only took place in the presence of base and the effect of zinc could not be observed under the reported conditions. Considering that the thiol is not a good nucleophile, the thiol/thiolate equilibrium established in the presence of base should determine the reaction rate. When the reaction was conducted with a chelating thiol, the reaction rate was faster then the non-chelating case and, in the presence of Zn+2, it was complete after 18h reflux in methanol. The observed reactivity enhancement with chelation is very important in the biological context in analogy with Ada protein, which provides a sulfur rich environment around Zn+2 where four cysteines coordinate to the metal in a rigid chelating way. The fact that a slow second-order rate constant was obtained (5´10–5 M–1 s–1) is in agreement with what would be expected for the reaction of a zinc bound thiolate with TMP in a protic solvent.

The MEPA2Zn complex is a neutral compound and, therefore, should reduce the dissociation tendency (also indicated by sharp peaks in the 1H-NMR spectra of the complexes). The results of methyl transfer in chloroform, a solvent that does not favor the dissociation of the zinc thiolate to the same extent as methanol, were very similar to the ones with methanol also suggesting that methyl transfer is taking place through a zinc bound thiolate.

– 48 – Chapter 2: Results and Discussion

2.1.3. The Dinucleotide Model

The main reaction of interest is the methyl transfer from a methylated DNA phosphate backbone to Ada protein. To improve the model system, the synthesis of a methylated dinucleotide was performed (Figure 54). The compound was synthesized according to a literature procedure98.

O O O DMTO N DIAT O NH DMTO N NH NH HN N O N N 7 HO O + O 10 CH Cl 2 2 P N N O

P O O N O HO N NH Tetrazole

CH3CN O AcO 9

O O O O DMTO N HO N NH NH 1. I 2,6-Lutidine; THF; H O 2 2 O O CH3CN O O 2. CH COOH O 3 P P O O O O O O O O N N NH NH O 12 R, S O AcO AcO

Figure 54. Synthetic pathway of dTp(Me)dTAc (12).

Compounds 7 was obtained by protecting the commercially available tymidine with dimethoxytrityl chloride (DMTCl). Further protection with acetic anhydride and subsequent selective deprotection of the trityl group gave compound 9. The nucleoside phosphine 10 was prepared by reacting 7 with bis-diisopropylamino phosphine. The coupling with the protected

– 49 – Chapter 2: Results and Discussion nucleoside 9 gave the intermediate phosphine, which was directly oxidized with iodine followed by DMT deprotection, affording the dimer 12 in a diastereomeric mixture. The spectroscopic data of the dinucleotide 12 is in agreement with the literature98.

The methylated dinucleotide was tested as a methyl donor for (MEPA)2Zn in deuterated methanol at RT (Figure 55).

O O SMe HO N H NH N N O O O + P O O O H3C O N O O NH HO N NH O CD3OD AcO O + O O P O N S HO O O H N N Zn N NH S NH O AcO

Figure 55. Reaction between MEPA2Zn complex and the dinucleotide 12 in d4-methanol.

The formation of Me-MEPA was followed by 1H-NMR (Figure 56 left). At t = 0 h there are present only the peaks for the acetate and the two methyls in the thymidine of the dinucleotide. At 4 h there is already Me-MEPA (3) present in about 30%. The yield of the product increased to 66 % after 21 h of reactional time. It is worth mentioning that the acetate protecting group is hydrolyzed under the reaction conditions. A comparison between spectra of 31P-NMR (Figure 56 right) at t = 0 and t = 21 h shows a break down in the sharp peaks of the methylated dinucleotide with a small chemical shift change.

– 50 – Chapter 2: Results and Discussion

t = 0 h Ty-CH3 + Ty’-CH 3 Ac.

t = 0 h

t = 4 h

t = 21 h Me-MEPA (3) t = 21 h

Figure 56. Left: 1H-NMR spectra of reactional mixture at 0, 4 and 21 h. Right: 31P-NMR spectra of reactional mixture at 0 and 21 h.

– 51 – Chapter 2: Results and Discussion

2.1.4. The NxSy Ligands

A series of ligands were attempted in order to study the importance of the sulfur-rich environment around zinc in analogy to the Ada protein (Figure 57).

OH O O O HO O N N N N

SH SH SH SH SH SH SH SH SH SH

NS2 NS2 S4 NS3 SH

N N N N SH SH S SH HS N SH HS SH SH SH SH NS2 N2S2 NS3 S4

Figure 57. The different NxSy ligands envisioned.

From these ligands, going from NS2 to N2S2, NS3 and S4, there is an increase in complexity and sulfur content, which should give some insight on how primary thiols coordinate with zinc, and how this coordination affects their reactivity towards TMP.

2.1.4.1. The NS2 Ligands

The first strategy to achieve these ligands (Figure 58) hit a major setback in the synthesis of [1,2]dithiolan-4-one (Figure 59). The synthesis was started with bromination of acetone to give 1,3-dibromo-propan-2-one 13. Bromide displacement with potassium thioacetate afforded the dithioacetate 14.

– 52 – Chapter 2: Results and Discussion

O OH + R N R N Li S S SH SH Figure 58. First approach to obtain readily available thiol ligands. R= side chain to introduce more functionality, or to help solubilization of the future complex.

O O O O O Br2 CH3COSK AcOH/H O EtOH S S 2 O O Br Br HS SH S S 13 14

Figure 59. Synthesis of [1,2]dithiolan-4-one.

Attempts to hydrolyze compound 14 to form 1,3-dimercapto-propan-2-one were not successful. Acid and base catalyzed methanolysis always resulted in the loss of starting material and in the formation of polymer like crude. Iodine oxidation of this crude did not afford the desired [1,2]dithiolan-4-one. To solve this problem, alternative ketones were synthesized (Figure 60).

O O

Si O O Si O O 15 17

Figure 60. Alternative ketones for the pyridyl alkylation.

Ketone 15 was obtained from the protection of 1,3-dihydroxy-propan-2-one with t- butyldimethylsilyl chloride. The reaction with lithiated pyridine gave the alkylated compound 16 in very low yield (12%) (Figure 61). The causes for this low yield might lie on the high steric hindrance to access this ketone’s carbonyl. An alternative to this route was based on a different ketone (17) and better yields were obtained for the alkylation (Figure 62).

– 53 – Chapter 2: Results and Discussion

O OH N Li N O O Si Si THF Si O O Si 15 16

Figure 61. Alkylation with ketone 15 to give compound 16.

O OH OH N Li N HCl N THF O O O O THF quant. OH OH 77% 18 19

DMSO TsCl Py 56%

NaHS OH OH OH N N + N MeOH or DMF OTs OTs quant. SH SH 22 S S 21 20

Figure 62. Synthetic pathway for the formation of dithiol 22.

The alkylated product 18 was deprotected under acidic conditions giving the triol 19, which was dried very well for the subsequent tosylation. This step was very water sensitive and the best yield obtained was only 56%. The ditosylate 20 was the starting point for different attempts to get the dithiol 22. The reaction with thiourea is well known to convert bromides and chlorides into thiols99. In our case, the reaction led to the disappearance of the ditosylate and no desired product was formed. Another way to get the dithiol would be through a dithioacetate100, but in our case, the product was obtained in very low yield (10%). Tetraethylammonium sulfide101 reaction led to almost total starting material recovered, which suggests that there is not enough “naked” hydrogen sulfide (HS–) available for the nucleophilic displacement of the tosylate. The reaction with NaHS showed to be the most promising one to obtain dithiol 22. Its versatility to work under different conditions turned out to be a good advantage. In this specific case, the reaction of ditosylate 20 with NaHS, at room temperature, gave

– 54 – Chapter 2: Results and Discussion quantitatively a mixture of dithiol 22 and the disulfide 21. The ratio of dithiol/disulfide was increased by changing the solvent in the solution. The reaction run in MeOH/H2O (1:1) gave, in the best case, 55% of dithiol. When only MeOH was used, the relative amount of dithiol was increased to 77%, and when DMF was used, over 95% of dithiol was present in the mixture. One of the possible explanations for these findings might come from the degassing process of the different solvents, which, although it was the same method for all, gave rise to different amounts of oxygen in the reaction vessel. An interesting characteristic of this dithiol 22 was that it oxidizes slowly with air to give the disulfide 21. This conversion was also obtained by reacting the dithiol with DMSO at 70 ºC for 18 hours. A modification on this ligand was also obtained by methylating the alcohol group of compound 18 (Figure 63).

OH O O N NaH, MeI N HCl N THF THF O O O O OH OH 25 18 24 TsCl Py

O CH3COSK O N N EtOH:DMF (2:1) OTs OTs 26 SAc SAc 27

NaOH EtOH NaHS DMF

O O O N + N N

SH SH 28 S S 29 S 30

Figure 63. Synthetic pathway to obtain the methylated dithiol

The methylation of compound 18 with methyl iodide afforded compound 24 that was converted to the methylated ditosylate 26 following the same procedure used to convert 18 in

– 55 – Chapter 2: Results and Discussion

20. It was of some surprise that when reacting 26 with NaHS/DMF, instead of obtaining the corresponding dithiol 28, only the sulfide 30 could be observed. The structure of compound 30 was put forward taking into consideration several factors: (1) this compound was completely inert to oxidation with Ph3PO and was stable in an open flask, suggesting that could not be the dithiol 28; (2) the 13C-NMR of this sulfide showed a methylene carbon at 35.90 ppm, which could not be assigned to the disulfide 29 (> 50 ppm expected); (3) both ESI-MS and HR-ESI-MS showed 30 to be the compound obtained in the reaction. The compounds 28 and 29 were, indeed synthesized but through the dithioacetate 27 and with quite low yield (16%). Compounds 21, 29, 30 and a hypothetical sulfide 21b were simulated with Spartan® in geometry optimization using the PM3 method (Figure 64).

21 29 DH = 8.382 DH = 14.898 kcal/mol kcal/mol

21b 30 DH = 1.769 DH = 10.103

kcal/mol kcal/mol

Figure 64. Spartan simulation of 21, 21b, 29 and 30 (images rendered with POV-Ray®).

– 56 – Chapter 2: Results and Discussion

The simulations performed gave enthalpy values (DH) for the compounds. The differences between these values are not significant enough to discriminate between the compounds in terms of stability. Therefore, from these study it is not possible to understand that, under the same reaction conditions, the ditosylate 20 gives the disulfide/dithiol (21/22) mixture and the ditosylate 26 gives the sulfide 30. There are examples in literature where it is possible to form thiethanes (like compound 30) out of dichlorides102 and dibromides103 using NaHS and KHS in ethanol or DMSO. On the other hand, a very similar dichloride gives the dithiol/disulfide mixture when reacted with NaHS in ethanol104 or methanol105. These facts show that it is very difficult to predict the outcome of this reaction.

Complexation

Several attempts to complex ligands 22 and 28 were performed using different strategies. Reactions with ZnBr2, Zn(NO3)2, ZnCl2, Zn(CH3COO)2 and Et2Zn were tried and all led to polymeric material. The formed complexes were not soluble in any common solvent

(H2O, DMSO, CH3CN, MeOH, EtOH and CH2Cl2). A zinc complex with [2,2']bipyridinyl was prepared (see Exp. Section for details) and added to ligand 22. After 24 h, an insoluble polymeric material was formed leaving in solution only [2,2']bipyridinyl. This result suggests that the dithiol replaced the [2,2']bipyridinyl around zinc and the polymeric dithiol-zinc complex was formed.

Reactivity

A reactivity test was done with ligand 22 in the presence of zinc. Zinc acetate was added to a mixture of dithiol 22 and TMP in refluxing d4-methanol and the solution was stirred for 20 hours. 1H-NMR analyze of the mixture showed that no methyl transfer took place. An intriguing observation was the fact that no precipitation of the complex occurred. TMP is preventing the formation of polymeric complexes, probably by a direct coordination to zinc replacing the sulfur-bridging thiolates. To answer this question, several attempts were made to get single crystals suitable for X-ray crystallography, but without success.

– 57 – Chapter 2: Results and Discussion

2.1.4.2. The S4 Ligand

Using a similar strategy as for the NS2 ligands, we envisioned a new series of ligands with four thiolates available for zinc coordination. The four-thiolate ligand would be a very good model for the Cys4Zn active site for Ada protein. The ligands were synthesized as follows:

O OH OMe Br N Li Br N NaH, MeI Br N O O CH Cl THF O O 2 2 O O quant. 32 52% 31 O 17 n-BuLi

CH2Cl2 O O 40%

HO OMe OMe MeO OMe N MeO N N HCl NaH, MeI O O O O O O THF OH OH OH OH THF O O quant. quant. 35 34 HCl THF 33 TsCl Py quant. 77%

OMe OMe HO OMe TsCl HO N MeO N N Py OH OH OH OH OTs OTs OTs OTs OTs OTs OTs OTs 68% 36 39 38 NaHS DMF NaHS DMF 74%

OMe OMe HO OMe HO N MeO N N +

S S S SH SH S S S 37 40 41

Figure 65. Synthetic pathway to obtain tetrathiolate ligands.

The synthesis started with a lithium pyridine alkylation of the ketone 17, but now 2,6- dibromopyridine was used to generate the lithiated compound. When the reaction with THF

– 58 – Chapter 2: Results and Discussion as solvent using the conditions developed for the alkylation of 2-bromopyridine, a complex mixture of products was obtained with the desired compound 31 in very low yield (< 15%). Although butyllithium reacts with dichloromethane in THF at –100 ºC106, there is evidence that vinyllithiums are stable at –78 ºC in dichloromethane for several hours107. Peterson and Mitchell108 found out that 2,6-dibromopyridine undergoes remarkably clean lithium bromine exchange at –78 ºC and that dilithiation is much less favorable in dichloromethane then in either THF or Et2O.

By using CH2Cl2 as a solvent for the butyllithium reaction, compound 31 was obtained in good yield (52%). The methyl iodide alkylation gave 32 in quantitative yield. A second alkylation with ketone 17 gave compound 33 with smaller yield (40%). The synthesis of the tetratosylate 36 followed the procedure used to synthesize 26 from 18. When 36 was treated with NaHS in DMF, the bis-sulfide 37 was obtained as the main product (74%). This compound showed the same characteristics as compound 30. A synthetic variation was accomplished when the two compounds described previously (21 and 30) were comprised in only one molecule. The tetratosylate 39 was treated with NaHS/DMF under the same conditions to give a mixture of compounds where 40 and 41 were present. These compounds were detected by 1H-NMR spectroscopy and 40 was identified be HR-ESI-MS. The purification of these compounds was not performed. The synthesis of the tetrathioacetate (Figure 66) was tried but failed, leading to a complex mixture of products. Careful TLC analysis suggested that the harsh conditions (refluxing ethanol) used for the tosyl displacement to take place, are incompatible with this type of neopentyl thioacetates. Hence, before all substitutions occur, hydrolysis takes place leading to the observed mixture of compounds.

OMe CH3COSK OMe MeO N MeO N EtOH:DMF 2:1 OTs OTs OTs OTs SAc SAc SAc SAc

Figure 66. The attempted potassium thioacetate reaction.

Complexation with the bis-sulfide 37 and diethyl zinc was attempted, but only insoluble polymeric material was obtained.

– 59 – Chapter 2: Results and Discussion

2.1.4.3. The N2S2 Ligand

A different approach was undertaken with this ligand. It was shown by Darbre and 109 coworkers that ligands with N5 and N3O motifs formed soluble monomeric complexes with zinc salts. Inspired in this N3O ligand, a new synthetic pathway was conceived (Figure 67).

1. Na2CO3 Thiourea, EtOH 2. NaH, Br SH N 42 N N 3. NaHCO , H O Br OH HBr 3 2 Et2O:DMF 53%

N KOH Thiourea, S OH N 1. N TsCl EtOH N S SH S Cl DMF 2. NaHCO3, 79% N N H2O 44 quant. 45 47

Figure 67. Synthesis of the N2S2 ligand 47.

The commercially available 2-bromomethyl-pyridine hydrobromic acid was converted to pyridin-2-yl-methanethiol in the reaction with thiourea and, the direct coupling with 42 afforded compound 44. The next step turned out to be a very interesting one by accident. The original conditions were for a tosylation in KOH and THF129 but, by mistake, DMF was used. Instead of the tosylate, compound 45 was isolated from the crude. One possible route for the production of this chloride is through the Vilsmeier-Haack reaction110 (Figure 68). We propose that compound 45 could have been synthesized through a variation to the Vilsmeier-Haack reaction where DMF is the catalyst and TsCl is the source of chloride (Figure 69). From the synthetic point of view, it does not change much because the chloride is a good enough leaving group for the thiourea reaction, therefore, the ligand 47 was easily obtained.

– 60 – Chapter 2: Results and Discussion

O O O O Cl P O Cl P + N Cl P O Cl Cl N Cl Cl H Cl N Cl H H

NMe NMe NMe 2 2 2 O Cl2PO2 rearrangement Cl P O Cl N Cl Cl N + HO H H H O H NMe2 Vilsmeier-Reagent

Figure 68. The Vilsmeier-Haack reaction.

Cl O O O S OTs OTs N N N N O Cl Cl R O Cl S O O N Cl Cl R R

O O O R Cl + N N N Cl

Figure 69. Proposed mechanism for the production of 45 through a variation of the Vilsmeier-Haack mechanism.

Complexation with ligand 47 and zinc acetate was performed and some colorless crystals were obtained. Unfortunately, the crystals were too small and it was not possible to obtain a X-ray structure.

– 61 – Chapter 2: Results and Discussion

2.1.4.4. Another NS2 Ligand

A very simple NS2 ligand was had been reported in literature as a building block of more complex ligands99, 111 and, also as direct ligand for oxorhenium(V) complexes112. Such ligand provided information about the reactivity (or lack thereof) of sulfur-bridging zinc thiolates (Figure 70).

N Br Br 43 1. Thiourea, EtOH N 2. NaHCO3, H2O SH SH 58 N OTs OTs 57

Figure 70. Synthetic pathways for the synthesis of ligand 58.

The ligand 58 was synthesized from both the dibromide 43 and the ditosylate 57 in high yields via the thiourea reaction. Complexation of this ligand with ZnCl2 afforded insoluble material, probably due to sulfur-bridging polymerization. The reactivity of the dithiol 58 towards trimethyl phosphate in the presence of zinc acetate was tested. After refluxing for 20 h, 7% of dimethyl phosphate was formed, but no methyl sulfide was detected, suggesting that no methyl transfer took place and hydrolysis accounts for dimethyl phosphate production. The fact that no methyl transfer took place, indicates that sulfur-bridging zinc thiolates are less reactive (nucleophilic) than regular zinc thiolates.

2.1.4.5. Other NxSy Ligands

In this section, it will be described the effort to obtain different ligands with many different motifs and characteristics.

– 62 – Chapter 2: Results and Discussion

The NS3 Ligand

An alternative to the butyllithium alkylation came from the dimethyl malonate reaction. Better yields were obtained with this approach (Figure 71).

dimethyl malonate N K CO N 2 3 O O N AcO O O HO Br DMF 12% O OAc O OAc Imidazole 42 49 DMF O AcOAc 52 TBDMSCl O Et3N 57% DMAP MeOH LiAlH4 THF Py quant. dimethyl malonate N LiAlH N K2CO3 O 4 N O O Br Si O THF Si DMF quant. HO 73% O 50 O OH 51 OH TsCl Py 53

N N HS TsO SH OTs SH OTs

Figure 71. Synthetic pathways for the synthesis of a NS3 ligand.

The synthesis was carried on from two different pathways. A more direct one was achieved with the dimethyl malonate alkylation of compound 42 to give a mixture of compounds where 49 was afforded in only 12% yield. Reduction with LiAlH4 gave 53 in a straightforward way. In order to improve the yield of the alkylation, the alcohol function of 42 was protected with t-butyldimethylsilyl (TBDMS) chloride affording 50, which was alkylated in 73% yield to give compound 51. Surprisingly, LiAlH4 reduction also deprotected the alcohol function from the TBDMS group, affording directly 53. The acetylation/deacetylation steps were performed in order to get very pure triol for the tosylation reaction. Several tosylation attempts were performed but no product was obtained. The starting material was always obtained in a mixture with PTSA, suggesting that, if the tosylation does occur, hydrolysis takes place yielding the triol 53 back.

– 63 – Chapter 2: Results and Discussion

Another NS3 Ligand

A different NS3 ligand was envisioned (Figure 72). This approach was attempted in order to provide an extra “arm” to the NS2 ligand previously reported. The function of this “arm” is to close the previously open coordination position on zinc. By doing this, it is possible to avoid sulfur-bridging to a point where no more polymeric material is formed.

Br OH HO OMe OMe Br N N PCC CH2Cl2 O O n-BuLi, 32 O O Br O CH2Cl2 55 54 Br

HCl THF:H2O 1:1

HO OMe TsCl HO OMe N N

OTs OTs Py OH OH 56

Br Br

Figure 72. Synthetic pathway for the synthesis of another NS3 ligand.

The aldehyde 54 was obtained by PCC oxidation from the commercially available 6- bromo-hexan-1-ol, and alkylated with bromide 32 to afford compound 55. The deprotection step afforded quantitatively the triol 56. The tosylation step did not work and the starting material was not recovered.

– 64 – Chapter 2: Results and Discussion

Another S4 Ligand

Another S4 ligand was designed alongside with the NS3 ligand. The goal was the synthesis of a ligand with four thiol groups in two arms that would have enough freedom to coordinate to zinc in a tetrahedral fashion.

N N N Br Br 43 AcO OAc HS SH dimethyl OAc OAc SH SH malonate DMF 61 K2CO3 AcOAc Et3N DMAP MeOH

TsCl N LiAlH O O 4 N N O O THF Py O O O O HO OH TsO OTs 59 OH OH OTs OTs 60

Figure 73. Synthetic pathway to obtain another S4 ligand.

The synthesis of tetraol 60 followed the same lines as the one already described for ligand 53. The problem with the tosylation step was also encountered in this case. Both mono- and di-tosylation products were obtained, but the tetratosyl was never formed or was not stable enough. Several alternatives were tried without success, namely: different conditions of tosylation including KOH/THF and Et3N/CH2Cl2; the mesylation reaction with MsCl/Py afforded the starting material back; the tetrabromination with HBr led to decomposition of the product and no starting material was recovered; thionyl chloride reaction with SOCl2/Py also afforded the starting material back.

– 65 – Chapter 2: Results and Discussion

2.2. Class II Aldolase System

2.2.1. The N5 and N3O Ligands

Darbre and coworkers109 reported an efficient synthesis of two ligands containing nitrogen as binding sites (Figure 74). These ligands were coordinated with different zinc salts, the complexes characterized and their activity, as catalysts of the aldol reaction, was studied in the reaction of benzaldehyde and 2-hydroxyacetophenone (Figure 75). The authors reported that the zinc complexes catalyzed the aldol reaction in yields up to 62% and some diastereoselectivity (up to 3:1 syn/anti ratio).

N N NH NH NH OH

N N N

N5 N3O Figure 74. The ligands used by Darbre and coworkers109.

O O OH O Catalyst (7.5 mol%) H + THF or MeOH OH OH

Figure 75. Direct aldol reaction of benzaldehyde with 2-hydroxyacetophenone.

These ligands were used in the complexation with different metals affording new MII complexes (M= Ni+2, Co+2, Cu+2) (Figure 76).

– 66 – Chapter 2: Results and Discussion

N N MX2 NH NH NH NH +2 MeOH M

N N N N 2X-

+2 +2 +2 - - M = Ni , Co and Cu X = Cl and CH3COO

N N MX2 NH OH HN O +2 H MeOH M N N 2X-

+2 +2 +2 - - M = Ni , Co and Cu X = Cl and CH3COO

Figure 76. Complexation of N5 and N3O ligands with different metal salts.

Some complexes gave good crystals for X-ray crystallography characterization. The first crystal structure obtained was for the complex N3O-ZnCl2, which was synthesized by 109 Christian Dubs . Single crystals were also obtained for the complexes N5-NiCl2, N5-CoCl2 and N3O-CoCl2 (details in experimental section).

2.2.1.1. The N5-NiCl2 Complex

109 This complex resembles very much the N5-ZnCl2 complex already reported (Figure 77).

– 67 – Chapter 2: Results and Discussion

+ tm Figure 77. X-ray structure of the cationic fragment N5-NiCl (Image rendered with Pov-Ray tracer).

The ZnII ion is coordinated to the five nitrogen atoms of the ligand and to a chloride anion, producing a pseudo-C2 symmetry. The zinc atoms and the pyridine rings of the side chain are almost co-planar. In the molecular packing, there is a chloride nearby which acts as a counterion.

2.2.1.2. The N5-CoCl2 Complex

This cobalt complex turned out to be very intriguing because in the crystal packing there were CoII and CoIII in a 1:1 ratio. The CoIII might have been generated in solution but the possibility of the cobalt chloride being already oxidized before use should not be disregarded. The cationic fragment of the complex show a CoIII ion coordinated with the five nitrogen atoms of the ligand and to a chloride anion (Figure 78). The cobalt atom and the pyridine rings of the side chain are in the same plane but slightly distorted. Also present in the 2- packing, there is a chloride anion right next to the cationic unit, a cobalt atom of anion CoCl4 which occupies a twofold axes and a water molecule that is located always next to an inversion center.

– 68 – Chapter 2: Results and Discussion

2+ Figure 78. X-ray structure of the cationic fragment N5-CoCl (Image rendered with Pov- Raytm tracer).

2.2.1.3. The N3O-ZnCl2 Complex

The N3O-ZnCl2 complex gave very small crystals that, still, were suitable for X-ray analysis (Figure 79). The ZnII ion is coordinated to three nitrogens from the ligand and two chloride anions. Surprisingly, the alcohol function of the ligand is not coordinated to the metal. The two chlorides balance the charge, originating a neutral complex. In Figure 76, the drawing suggested a coordination between zinc and the alcohol in the ligand, which, now, we know that is not correct.

– 69 – Chapter 2: Results and Discussion

Figure 79. X-ray structure of complex N3O- tm ZnCl2 (Image rendered with Pov-Ray tracer).

2.2.1.4. The N3O-CoCl2 Complex

In this complex, there was also a mixture of CoII and CoIII in a 1:1 ratio. III II The complex N3O-CoCl2 has the generic formula: [Co (N3O)2][Co Cl4]Cl. The cobalt(III) ion is coordinated to two ligand molecules (Figure 80), one involving all four + + donor atoms (N3O - H ) and the second one involving only two donor atoms (N3O + H ), the non-coordinated pyridine ring is protonated and the OH group unchanged. In addition, there 2- are a [CoCl4] complex anion and a chloride anion in the asymmetric unit.

– 70 – Chapter 2: Results and Discussion

3+ Figure 80. X-ray structure of the cationic fragment (N3O)2-Co (Image rendered with Pov- Raytm tracer).

2.2.1.5. Direct Aldol Catalytic Test

II II II The complexes with N5 and N3O ligands (with Ni , Co and Cu in both chloride and acetate salts) were tested for catalytic activity in the reaction of benzaldehyde with 2- hydroxyacetophenone. In all cases, no conversion was observed under our reactional conditions (for details, see experimental section).

– 71 – Chapter 2: Results and Discussion

2.2.2. The N3O Chiral Ligand

109 The N3O-zinc complexes were reported to catalyze the reaction of benzaldehyde with 2-hydroxyacetophenone (Figure 75). The product was obtained in good yields but without any enantioselectivity and only a poor diastereoselectivity. In order to increase selectivity, a new N3O ligand was envisioned (Figure 81). This ligand features the same core as the previous one, but a new chiral part was introduced originating a chiral complex upon coordination with zinc. There are examples in literature34, 35 where chiral ligands coordinated with zinc gave excellent diastereoselectivities and enantioselectivities. * 109 The synthesis of the new N3O ligand was different from the original one because of the impossibility of doing a direct coupling to the chiral unit (menthyl group). The commercially available 2-bromo-6-methyl-pyridine was brominated with NBS to afford compounds 64 and 65 in a 4:1 ratio. From these compounds only 64 was used further. The tosylamine 66 was synthesized according to the literature113 from picolamine using a two- phase system. The bromide 64 was coupled with the tosylamine 66 also in a two phase system using (n-Bu)4NBr as a phase transfer catalyst to yield 67 in 72% yield. Tosyl deprotection with H2SO4 gave 68 in 92% yield. Menthane carbaldehyde 69 was obtained from (–)- menthone through a Wittig type reaction with (Ph)3PClCH2OCH3. Many difficulties arose in the last step, the n-BuLi alkylation of the aldehyde 69 with 68. The aldehyde showed low stability, leading to slow decomposition even at –18 ºC. Attempts to alkylate 68 with the ketone 17, that worked well previously, led to decomposition of the bromide without formation of the desired product. The possibility that butyllithium was forming an LDA-type base with the secondary amine of 68, led us to the attempt of direct alkylation of the tosylamine 67. This reaction led to the recovery of the starting material and no alkylation took place. Compounds 70 and 71 were synthesized in an attempt to solve the alkylation problem (Figure 82). Compound 70 was obtained from the MeI alkylation of 68 and compound 70 was obtained from the coupling of the tosylamine 66 with the commercially available pyridin-2- yl-methanol. All alkylation attempts with the aldehyde 69 led to starting material degradation. A close observation of 1H-NMR spectra of the several crudes revealed that the ratio between the methylene and the pyridine protons changed from 4:7 to aprox. 2:7, suggesting that the decomposition of starting material is occurring at the methylene sites.

– 72 – Chapter 2: Results and Discussion

NBS AIBN + Br Benzene Br Br N Br N Br N 47% Br 13% 64 65 + KOH TsCl (n-Bu) NBr NaOH 4 Ts NH NHTs N 2 Et2O CH2Cl2 Br N N H O N 2 H2O 89% 66 72% N H2SO4 92% 67 n-BuLi

Ph3PCl(CH2OCH3) H + N O Br N O THF 59% 69 N

68

N NH OH

* N N3O ligand

Figure 81. Synthetic pathway for the synthesis of the N3O chiral ligand.

– 73 – Chapter 2: Results and Discussion

N O Br N Br N

N N 70 71

Figure 82. Alternative reactants for the alkylation.

2.2.3. Zinc-Amino Acid Complexes

The direct asymmetric aldol reactions catalysts with higher enantiomeric excesses available in literature34, 35 consist in chiral zinc complexes difficult to obtain and rather complex to understand. This characteristic turns them mostly into intellectual curiosities but not into useful catalysts for the day-to-day synthetic necessities. Proline36, on the other hand, strikes us by its simplicity and usefulness. This aminoacid can catalyze direct asymmetric aldol reactions with both high yields and high e.e.’s36. It is important to note that none of these catalysts is efficient in aqueous media reducing in a great deal their usefulness A complex between zinc and optically active L-proline was prepared and tested for catalytic activity. In addition, other amino acids were tested whether, upon coordination with zinc, they catalyze a direct aldol reaction and what influence will have the solvent on the reaction.

2.2.3.1. Synthesis

Several zinc-amino acid complexes were synthesized in order to study their ability to catalyze an asymmetrical direct aldol reaction.

The zinc-L-proline complex (L-Pro2Zn) was synthesized in two different ways, with – Zn(CH3COO )2/Et3N and with Et2Zn (Figure 83).

– 74 – Chapter 2: Results and Discussion

O - Zn(CH3COO )2 Et N - 3 O O MeOH 81% NH - 2+ O Zn or N+ HN 2 H2 - Et2Zn O 72 DMSO 85% O

Figure 83. Synthesis of L-Pro2Zn.

Both reactions gave the same product (the ESI-MS looks identical) in similar yields. The 1H-NMR from the complex shows both a chemical shift deviation and a flattening of the peaks, upon coordination (Figure 84).

1 Figure 84. H-NMR of L-Proline and (L-Pro)2Zn.

– 75 – Chapter 2: Results and Discussion

L-Pro2Zn was obtained as a white powder very characteristic for its insolubility in common organic solvents (MeOH, EtOH, DMSO, Et2O, CH3CN, CH2Cl2 and Toluene). The complex was very soluble in water, thus opening a new window of advantages in dealing with such catalyst.

NH2 NH

NH2 N HN NH O O O O O

HO O- O- O- O- + + + H3N H3N H3N H3N+

L-Lysine L-Arginine L-Histidine L-Glutamic Acid

O O O O

HO HS O- O- O- O- + + + + H3N H3N H3N H3N

L-Serine L-Cysteine L-tert-Leucine L-Isoleucine

Figure 85. The different amino acid used in zinc complexation.

Table 4. Yields of the zinc-amino acid complexes.

Complex Yield (%)

(L-Pro)2Zn 81

(D-Pro)2Zn 75

(L-Lys)2Zn 38

(L-Arg)2Zn 81

(L-His)2Zn 33

(L-Glu)2Zn 60

(L-Ser)2Zn 38

(L-Cys)2Zn 33

(L-Ile)2Zn 23

(L-t-Leu)2Zn 95

– 76 – Chapter 2: Results and Discussion

Other amino acids were also coordinated with zinc acetate in the presence of Et3N to afford a variety of (L-a.a.)2Zn complexes (Figure 85). The yields of the complex formation reactions were not optimized (Table 4).

2.2.3.2. Catalysis of the reaction between aceton and p-nitrobenzaldehyde

The first system studied was the reaction of acetone with p-nitrobenzaldehyde (PNB) (Figure 86). The reaction was performed in water for a better solubility of the catalyst.

O O OH O [(L)-a.a.]2Zn + H 5 mol%

H2O NO2 (S)-1 NO2

Figure 86. Reaction of acetone with p-nitrobenzaldehyde in aqueous medium.

A solubility study of the system showed that the catalyst needed at least 10% water content in order to avoid precipitation. On the other hand, if the amount of water exceeds 66%, the PNB starts to precipitate. Taking these findings in consideration, the first catalysis studies were performed at 33% H2O (Table 5). A few other molecules were tested for catalysis in order to assess better the system (Table 6).

The complexes L-Pro2Zn, L-Lys2Zn and L-Arg2Zn showed to be efficient catalysts of the aldol reaction giving reasonable enantiomeric excesses. On the other hand, L-Cys2Zn, L-

Glu2Zn, L-His2Zn and L-Ser2Zn gave very small amounts of product after 24 h. Surprisingly, after 96 h, L-Glu2Zn complex gave a very high yield of the product (98%). When the reaction was run with the complexes L-Ile2Zn and L-t-Leu2Zn, significant amounts of aldol product were obtained and there was a significant increase in the e.e.’s, suggesting that steric hindrance promoted by the bulky side group might be playing a role.

– 77 – Chapter 2: Results and Discussion

Table 5. Conversion yields and enantiomeric excesses (e.e.) for the different catalysts.

L-a.a.a in Entry Time (h) Yield e.e. (S) Zn complex

1 Pro 24 100% 32%

2 Lys 24 100% 24%

3 Arg 24 100% 54%

4 Cys 24 3% - 5 Cys 96 13% 35%

6 Glu 24 < 1% - 7 Glu 96 98% 32%

8 His 24 4% - 9 His 96 45% 27%

10 Ser 24 5% - 11 Ser 96 24% 20%

12 Ile 24 10% - 13 Ile 96 36% 45%

14 t-Leu 24 15% - 15 t-Leu 96 74% 51%

a) L-amino acid in the Zn-complex

When L-proline was used, the reaction yielded a very small amount of product in 21% e.e. of the R enantiomer. The fact that the R enantiomer was observed in excess is in agreement with the reported results36. These findings suggest that the two reactions occur with two different mechanisms and that L-Pro2Zn does not dissociate under the reaction conditions. The low yield of conversion in the presence of proline alone is also an indication that the enamine-mechanism does not operate well in aqueous medium. When DMSO substituted H2O in this reaction, 57% of product was obtained in 77% e.e. of the R enantiomer.

– 78 – Chapter 2: Results and Discussion

Table 6. Conversion yields and enantiomeric excesses (e.e.) for other catalysts.

Entry Catalyst Time (h) Yield e.e.

a) 1 Et3N 24 100% rac.

2 Zn(CH3COO)2 (5%) 24 0% -

3 Zn(CH3COO)2 (5%) 96 6% -

4 L-Proline (10%) 24 6% 21% (R)

5 L-Lysine (5%) 24 74% 6% (R)

a) Et3N in 1 mol eq.; rac. = racemic mixture

General base catalysis was present when the reaction was run with Et3N and L-lysine, producing racemic mixtures of the product. The higher e.e. values observed with different amino acids, however, require catalysis by a chiral Lewis acid. Zinc acetate itself was not able to catalyze the reaction and even after 96 h only 6% of the product could be observed. Examination of the pH rate profile for the reaction (Table 7) showed that with decreasing pH there was decreased yield and increased e.e. (S-enantiomer).

Table 7. pH rate profile for the aldol reaction of acetone with p-nitrobenzaldehyde for 24 h

with 33 vol % of H2O and using L-Pro2Zn as the catalyst.

pH rate profile for the aldol reaction pH Yield e.e. (S) 100 6 18% 44% 80

7 33% 41% 60 % 8 96% 31% 40 Yield e.e. (S) 20 9 100% 32% 0

9 8 7 6 pH

Figure 87. Chart representation of the pH profile results.

– 79 – Chapter 2: Results and Discussion

The effect of water/acetone ratio on the reaction rate and on the enantiomeric excess of the product was also investigated (Table 8). The lower reaction rates with decreasing percentage of water reflect, at least in part, the decreasing solubility of the complexes in the reaction medium. The (L-Pro)2Zn complex becomes insoluble in 2% water/acetone yielding poor conversion and the R-enantiomer in excess. These results indicate that, at low amount water, the reaction is catalyzed by proline alone, formed from the slow dissociation from zinc.

Table 8. The effect of water in the yield and e.e. of the (L-Pro)2Zn catalyzed aldol reaction of acetone with p-nitrobenzaldehyde.

Entry % H2O Time (h) Yield e.e. enantiomer 1 66 24 100% 56% S

2 50 24 100% 44% S

3 33 24 100% 32% S

4 20 20 56% 26% S

5 10 20 6% - - 6 10 96 100% 25% S

7 7 20 5% - - 8 7 96 81% 26% S

9 2 20 2.5% - - 10 2 96 15% 17% R

11 0 20 3% - - 12 0 96 8% 56% R

Looking at Table 8, it is clear that the e.e. of the aldol product increases with an increase in the amount of water. The same does not happen when the (L-Arg)2Zn complex was used. In this case, the aldol reaction with 66% water gave the product in 64% yield and in 36% e.e. of the S enantiomer, which is smaller than when 33% water was used (see table 5).

The reaction was also carried out in (33%) H2O/THF with only 5 mol equivalents of acetone to give 63% yield and 50% e.e. (S enantiomer). This result shows that the reaction

– 80 – Chapter 2: Results and Discussion does not need the large excess of acetone in order to proceed. Nevertheless, because it is an equilibrium, the rate of conversion slows down when less equivalents of acetone are used. Several attempts were made to increase the enantioselectivity of the reaction by changing the conditions. It has been shown that temperature can have an effect on the enantiomeric excess of an 114 aldol product . This was tested and the reaction was run with 33% H2O/acetone for 22 h at 0 ºC, generating the product in 37% yield and in 28% e.e. of the S enantiomer. The reaction was slowed down with the decrease of the temperature but that had no effect on the e.e. of the product. Recently, Barbas III and coworkers115 showed that, with the use of sodium dodecyl sulfate (SDS, 0.1 equivalents), the solubility of the reactants was improved and the uncatalyzed reaction was completely suppressed. With this in mind, the reaction of acetone with PNB was run at 66% H2O/acetone and at 100% H2O (with 5 mol. equiv. acetone) giving the aldol product in 100% yield (e.e. = 17% S) and 17% (racemic), respectively. If, in the first case the reaction was a clear solution, in the second one, PNB precipitated leading to smaller conversion. The enantioselectivity was also not improved with the use of SDS. Kobayashi and coworkers114 showed that several different water-stable Lewis acids could be used in several carbon-carbon bond-forming reactions in aqueous media. Because II II 3 Cu is a better Lewis-acid then Zn , a (L-Pro)2Cu complex was synthesized following the procedure for (L-Pro)2Zn. When the reaction was run with 5% (L-Pro)2Cu for 48 h the following results were obtained:

Table 9. Results for the aldol reaction catalyzed by 5% (L-Pro)2Cu for 48 h.

Entry % H2O Yield e.e.

1a) 0 traces -

2 a) 33 traces -

3 66 11% rac.

a) The complex was not completely solubilized.

If in the first two entries, the lack of reactivity can be explained by the insolubility of the complex under the reaction conditions, it is also true that with 66% of H2O there is a big

– 81 – Chapter 2: Results and Discussion decrease in reaction rate when going from ZnII to CuII. Being CuII a better Lewis-acid, it was expected exactly the opposite. The fact that the product is completely racemic might be just a result of the uncatalyzed reaction. It is noteworthy the fact that in all tests of the reaction between acetone and PNB, no side products like the a,b-unsaturated ketone, derived from aldol condensation, were detected by 1H-NMR spectroscopy. This system was studied kinetically by 1H-NMR spectroscopy (for details see

Experimental Part). The reaction of PNB with d6-Acetone in 33% D2O with 5% (L-Pro)2Zn was run for 10 h at 23 °C, after which, 14% of the product was observed. A pseudo first-order rate constant was determined to be 4.2´10-6 s-1 (2.5´10-4 min-1) (Figure 88). The fact that this reaction is approximately one order of magnitude slower than the reaction with non- deuterated acetone, shows that there is a primary isotopic effect.

These results indicate that the rate-limiting step, in the (L-Pro)2Zn catalyzed reaction, has to be the C-H bond cleavage for the enolate formation.

L-Pro2Zn Catalysis

0,02

0 0 5000 10000 15000 20000 25000 30000 35000 40000 -0,02

-0,04 y = -4,2104E-06x + 3,6719E-03

] 2

B R = 9,9930E-01

N -0,06 P [

g -0,08 o L -0,1

-0,12

-0,14

-0,16 Time (s)

Figure 88. The natural log of PNB concentration plotted against time.

– 82 – Chapter 2: Results and Discussion

2.2.3.3. Catalysis of other aldol systems

The complex (L-Pro)2Zn was used in catalysis in many other different aldol systems with success. The reaction of acetone with benzaldehyde is an interesting system to assess the efficiency of the catalyst with less activated aldehydes (Figure 89).

O O OH O [(L)-Pro]2Zn + H 5 mol%

50% H2O

Figure 89. Aldol reaction between acetone and benzaldehyde catalyzed by (L-Pro)2Zn.

The reaction was run in 50% H2O/acetone with 5% catalyst and the product (4- hydroxy-4-phenyl-butan-2-one) was obtained in 49% yield. No e.e. was measured for this compound. This result clearly shows that with less activated aldehydes the reaction goes slower. Another aldol reaction studied was between hydroxyacetone and PNB (Figure 90).

O O O OH [(L)-Pro]2Zn + H 10 mol%

H2O OH OH NO2 NO2

Figure 90. Aldol reaction between hydroxyacetone and PNB catalyzed by (L-Pro)2Zn.

The reaction was run with 10% catalyst and with (33%) and without water (Table 10). This was possible because the catalyst was soluble in the hydroxyacetone.

– 83 – Chapter 2: Results and Discussion

Table 10. Results for the aldol reaction between PNB and hydroxyacetone catalyzed by 10%

(L-Pro)2Zn for 3 h.

Entry % H2O Yield d.e.

1 0 80% 12% (anti) 2 33 100% 19% (anti)

The aldol addition of hydroxyacetone to an activated aldehyde (PNB) went rather fast, yielding a mixture of diastereoisomers with very small diastereomeric excess. No e.e.’s were measured for this product. It is noteworthy that this reaction was regioselective, preferring the formation of 3,4- dihydroxy-4-(4-nitro-phenyl)-butan-2-one (Figure 90) to the 1,4-dihydroxy-4-(4-nitro- phenyl)-butan-2-one (Figure 91).

O OH

Figure 91. Structure of 1,4-dihydroxy-4- (4-nitro-phenyl)-butan-2-one. OH NO2

Hydroxyacetone was also reacted with naphthalene-2-carbaldehyde to yield also a mixture of diastereoisomers (Figure 92).

O O O OH Catalyst + H 20 mol% DMSO OH OH

Figure 92. Aldol reaction between hydroxyacetone and Naphtaldehyde in DMSO or without solvent.

– 84 – Chapter 2: Results and Discussion

The reaction was performed in DMSO/hydroxyacetone mixture or in neat hydroxyacetone and always in the absence of water. Proline and the complexes (L-Pro)2Zn and (D-Pro)2Zn were the catalysts used.

Table 11. Results for the aldol reaction between naphtaldehyde and hydroxyacetone catalyzed by 20% catalyst for 20 h.

Entry Catalyst Solvent Yielda d.e. (anti)

1 L-proline 80% DMSO 22% 28%

2 (L-Pro)2Zn 80% DMSO 40% 18%

3 (L-Pro)2Zn Neat 42% 20%

4 (D-Pro)2Zn Neat 50% 16% a) Isolated yields.

Barbas III and coworkers36 reported that L-proline catalyzes this reaction with 50% diastereomeric excess of the anti compound. Reproducing this experiment (Table 11), only 28% d.r. was obtained. Nevertheless, it allowed an unambiguous assignment of the anti- configuration. The results for the zinc catalysts show that the reaction goes faster but less diastereoselective. Interestingly, the main species was, in all cases, the anti-compound, contrasting with the syn-compound obtained via aldolase catalytic antibodies76. Running the reaction under neat hydroxyacetone gave better solubility of the catalyst, but had no effect on the rate of the reaction or on its stereoselectivity. The results also show no distinction between the L- and D-proline-zinc complex.

Two other ketones were tested for reactivity with both L-proline and (L-Pro)2Zn catalysts. Cyclohexanone was used to assess the efficiency of the catalyst with more rigid ketones (Figure 93) and butan-2-one was used to study possible regiospecificity (Figure 94).

The reactions were performed in 33% H2O/ketone for 24 or 96 h (Table 12). Once again, L-proline showed not to be an efficient catalyst in aqueous media, on the 1 other hand, (L-Pro)2Zn catalyzed both reactions with significant yields. H-NMR spectroscopy analysis of the butan-2-one aldol product indicates that 4-hydroxy-3-methyl-4- (4-nitro-phenyl)-butan-2-one (Figure 94) was the only product formed in a diastereomeric

– 85 – Chapter 2: Results and Discussion mixture. 1-Hydroxy-1-(4-nitro-phenyl)-pentan-3-one (Figure 95) was not present, suggesting that the enolate is preferentially formed on the C3 over the C1 of butan-2-one.

O O OH Catalyst O 5 or 10 mol% + H H2O NO 2 NO2

Figure 93. Aldol reaction between cyclohexanone and PNB in aqueous medium.

O O OH Catalyst O 5 or 10 mol% + H H2O NO 2 NO2

Figure 94. Aldol reaction between butan-2-one and PNB in aqueous medium.

Table 12. Results for the aldol reaction between PNB and cyclohexanone or butan-2-one in

33% H2O media.

Entry Ketone Catalyst Time (h) Yield

1 Cyclohexanone L-proline 24 4% 2 Cyclohexanone L-proline 96 9%

3 Cyclohexanone (L-Pro)2Zn 24 11%

4 Cyclohexanone (L-Pro)2Zn 96 23%

5 Butan-2-one L-proline 24 0% 6 Butan-2-one L-proline 96 1%

7 Butan-2-one (L-Pro)2Zn 24 17%

8 Butan-2-one (L-Pro)2Zn 96 45%

– 86 – Chapter 2: Results and Discussion

Barbas III and coworkers36 had shown that L-proline catalyzes this reaction in DMSO to give 1-hydroxy-1-(4-nitro-phenyl)-pentan-3-one and they suggest that steric hindrance is responsible for the observed selectivity. With the zinc catalyst, the reaction does not go through an enamine-type mechanism. To explain the observed results it is needed to go into detailed analysis of the ketone coordination to the zinc-proline complex.

O HO

NO2 Figure 95. Structure of 1-hydroxy-1-(4- nitro-phenyl)-pentan-3-one.

Through 1H-NMR spectroscopy analysis, it was also possible to observe that the butan-2-one and the cyclohexanone aldol products were present in 30% and 12% diastereomeric excesses, respectively. Unfortunately, the diastereoisomers were not assigned.

– 87 – Chapter 2: Results and Discussion

2.3. Prebiotic Sugar Synthesis

2.3.1. The Idea

The prebiotic formation of sugars has been a subject of several studies and speculation. Although no prevalent theory exist at the moment, it is possible that an asymmetrical aldol reaction in aqueous media might have played an important role.

We have previously shown that (L-Pro)2Zn catalyzes asymmetrically different aldol reactions in water. These findings, transform this catalyst into an interesting candidate to the source of chirality needed for asymmetric sugar synthesis. The simplicity of the catalyst is also in accordance with prerequisites needed for the job. ZnII is an abundant transition metal making its availability, in prebiotic era, not a problem. Miller and Urey47, 48 already showed a possible route for the formation of different amino acids in an early stage of prebiotic era, strengthening the idea that proline was available.

To model the prebiotic formation of sugars, a simple CnH2nOn molecule (n = 1 or 2) was needed. A simple way to approach this system would be with the use of formaldehyde (n = 1) or hydroxy-acetaldehyde (n = 2). We decided to use hydroxy-acetaldehyde because, in principle, we avoid the formation of a large variety of sugars (n = 3, 5 and 7). This way, only tetroses (n = 4) and hexoses (n = 6) can be formed. Another point in favor of using hydroxy- acetaldehyde is that to form it from two molecules of formaldehyde, no asymmetric induction is required; therefore, no asymmetric catalyst is needed. The work performed by Eschenmoser and coworkers50 gave an important starting point. These authors performed the reaction of hydroxy-acetaldehyde phosphate in aqueous NaOH solution and later, deprotected the sugars from the phosphate residue. The characterization of these sugars turned out to be very useful in our work, specially the 1H- NMR chemical shifts of many hexoses.

– 88 – Chapter 2: Results and Discussion

2.3.2. The Reaction of Hydroxy-acetaldehyde

The aldol reaction of hydroxy-acetaldehyde was performed with three different catalysts (Figure 96): (L-Pro)2Zn, (L-Lys)2Zn and (L-Arg)2Zn. The reaction of (L-Pro)2Zn was also performed at 40 ºC to determine the influence of the temperature in the sugar formation.

O OH O Catalyst OH O HO

H O + 2 HO OH OH RT, 6 days OH OH OH

Figure 96. The aldomerization of hydroxy-acetaldehyde to give tetroses and hexoses.

The reaction run for 6 days, after which, the solvent was removed affording quantitatively product plus catalyst. The mixture was analyzed by 1H-NMR spectroscopy (Table 13, Figure 97). The yields were determined by the integration of the corresponding 1 anomeric protons in the H-NMR spectrum recorded in D2O at 300 MHz.

Table 13. The yields of hexoses obtained from aldomerization of hydroxy-acetaldehyde in the presence of Zn-amino acid complexes.

Yields (%) a) Entry Hexose (L-Pro)2Zn (L-Pro)2Zn* (L-Lys)2Zn (L-Arg)2Zn 1 a-mannose 0 0 1.7 / 2.0 7.9 /9.0 2 b-mannose trace trace 7.9 / 9.1 b) 15.3 / 17.4 b) 3 a-glucose 24.0 / 37.2 11.1 / 14.0 c) 16.1 / 18.5 17.1 / 19.4 4 b-glucose 3.3 / 5.1 Trace 3.4 / 3.9 trace 5 a-galactose 0 d) c) c) c) 6 b-galactose 19.5 / 30.2 49.3 / 61.6 29.3 / 33.8 9.9 / 11.3 7 a-allose 0 0 Trace 7.5 / 8.5 8 b-allose trace Trace b) b) 9 a-altrose 0 0 0 0

– 89 – Chapter 2: Results and Discussion

10 b-altrose 0 0 6.1 / 7.0 6.5 / 7.4 11 a-gulose 0 0 0 0 12 b-gulose trace trace b) b) 13 a-idose 6.3 / 9.8 4.1 / 5.1 7.9 / 9.1 4.3 / 4.9 14 b-idose 6.6 / 10.2 4.1 / 5.1 5.5 / 6.3 5.3 / 6.0 15 a-talose 5.0 / 7.8 3.3 / 4.1 5.0 / 5.8 9.7 / 11.0 16 b-talose trace 7.9 / 9.9 3.9 / 4.5 4.3 / 4.9 a) The yields refer to: total sugar content (tetrose and hexose) / total hexose content; b) the value given is the sum of b-mannose, b-allose and b-gulose; c) the value given is the sum of a-glucose and a-galactose; d) determined by GC-MS; * at 40 ºC;

Hexose Ratio Production

70,0

60,0

50,0

s 40,0 d l e

i (L-Pro)2Zn

Y 30,0 (L-Pro)2Zn* (L-Lys)2Zn 20,0 (L-Arg)2Zn

10,0 Catalyst

0,0 (L-Arg)2Zn e (L-Lys)2Zn s e (L-Pro)2Zn* no os se e n l to s e a gu c co os se e (L-Pro)2Zn m b- la lu ct lo s e a- a g a al ro os se e + -g - al - lt tr lo s e e a b g a -a al u o s e s + - a - g id o os se llo b b a- a- -id l o -a se b -ta al b o a -t + uc b e gl s a- no an m Hexoses b-

Figure 97. Graphical representation of the hexoses yields from the different catalysts (in the x-axis a stands for a, and b stands for b).

– 90 – Chapter 2: Results and Discussion

The peaks were identified with spectra recorded for reference sugars and from literature (Table 14).

Table 14. Proton chemical shifts of the anomeric protons from all tetroses and hexoses.

Sugar d (ppm) J (Hz) a-erythrose a) 5.27 4.7 b-erythrose a) 5.25 3.4 a-threose a) 5.24 1.2 b-threose a) 5.40 4.1 a-mannose 5.07 1.47 b-mannose 4.78 0.75 a-glucose 5.15 3.60 b-glucose 4.56 7.89 a-galactose 5.15 3.30 b-galactose 4.47 7.74 a-allose 5.04 3.66 b-allose 4.78 8.07 a-altrose 4.88 3.30 b-altrose 5.01 1.47 a-gulose b) 5.10 n.d. b-gulose b) 4.79 8.4 a-idose b) 4.89 n.d. b-idose b) 4.98 n.d. a-talose 5.17 1.86 b-talose 4.70 1.11

a) Extracted from reference 116; b) Extracted from reference 50. For the other sugars, see Experimental Part.

Some sugars were very difficult to distinguish from each other, like in the case of b- mannose, b-allose and b-gulose which have an anomeric position rather close (4.78-4.79 ppm), a-glucose and a-galactose have also the same problem (5.15 ppm). If, in the case of the first, the discrimination between the sugars is not very relevant due to a quite small relative

– 91 – Chapter 2: Results and Discussion abundance, in the case of a-glucose and a-galactose it becomes important to know which of the sugars are present and in which ratio (see below). It is noteworthy that only the pyranose forms of the hexoses were present. The peaks for furanoses could not be observed. A close observation over Figure 97, shows that there was a clear preference, by all catalysts, for the formation of glucose and galactose over the other sugars. (L-Pro)2Zn, for instance, gave 27.3% glucose, 19.5% galactose, 12.9% idose, 5.0% talose, traces of mannose, allose and gulose, and no altrose. The hexoses that were not produced with (L-Pro)2Zn, could be observed with the other catalysts. The identification of the tetroses by 1H-NMR spectroscopy was quite difficult in the non-protected sugars, mainly due to the very close anomeric position of three of them (5.24- 5.27 ppm). Nevertheless, tetrose:hexose ratios were obtained by 1H-NMR spectroscopy integration (Table 15).

Table 15. Tetrose:hexose ratios for the sugar mixtures obtained with different Zn-complexes as catalysts (determined by 1H-NMR).

Catalyst Tetrose:Hexose ratio

(L-Pro)2Zn 1 : 1.8

(L-Pro)2Zn* 1 : 3.8

(L-Lys)2Zn 1 : 6.6

(L-Arg)2Zn 1 : 7.2

In order to solve some of these problems and get a better insight on the system, the sugar mixture from (L-Pro)2Zn was acetylated quantitatively with acetic anhydride and pyridine (for details, please see Experimental Part). With this protection, the identification of the tetroses became less complicated (Table 16). The acetylated crude was chromatographed into two fractions, one containing mainly the tetroses and a second one with a majority of hexoses (for details, see Experimental Part). In addition, the ratio between the different tetroses was different in the two chromatographed fractions. This difference in tetrose ratios, monitored by 1H-NMR spectroscopy and GC-MS, allowed an unambiguous assignment of the GC-MS peaks (Figure 98).

– 92 – Chapter 2: Results and Discussion

Table 16. Proton chemical shifts of the anomeric protons from all acetylated tetroses.

Sugar d (ppm) J (Hz) a-erythrose a) 6.34 4.6 b-erythrose a) 6.15 1.3 a-threose b) 6.13 0.3 b-threose b) 6.42 4.2

a) Extracted from reference 116; b) Extracted from reference 117;

b-threo a-threo

Table 17. Relative abundance tetroses produced by

the (L-Pro)2Zn catalyzed reaction.

b-eryth

Tetrose 1H-NMR (%) GC-MS (%)

a-eryth a-erythrose trace trace b-erythrose 29.3 19.0 a-threose 35.1 43.2 b-threose 35.6 37.9

Figure 98. GC-MS spectrogram of the tetrose region.

– 93 – Chapter 2: Results and Discussion

With the help of the peak assignment, it was possible to determine the relative abundance for all tetroses by both methods (Table 17). The GC-MS spectrogram was also integrated in both the tetrose and hexose regions to yield a 1:1.02 ratio, slightly different from the one obtained with 1H-NMR spectroscopy on the deprotected sugars (1:1.8). One problem that was not yet addressed was the question of distinction between a- glucose and a-galactose. Thus, commercially available a-glucose and a-galactose were acetylated following the same procedure used for the crude and independent GC-MS measurements were performed with co-injections of the crude and the sugars (Figure 99).

a-glu

a-gal

Figure 99. GC-MS spectrogram of the hexose region.

– 94 – Chapter 2: Results and Discussion

When the mixture was co-injected with pentaacetylated a-glucose, the peak at retention time 31.40 min was increased in about 20% in relation with the others, indicating that there was, in fact, this sugar in the mixture. On the other hand, when the co-injection was made with pentaacetylated a-galactose, a big new peak arose with a retention time between 31.30 and 31.35 min, suggesting that no relevant amount of a-galactose was present in the original mixture. The possible asymmetric induction from the catalyst was studied (Figure 100). Because the synthesis of hexoses in the system has to pass through one of the two available tetroses, there is a clear separation between pathways. From erythrose, four different sugars can be obtained, namely, allose, altrose, glucose and mannose. The other four sugars, talose, galactose, gulose and idose, are obtained from threose. In table 18, there is a direct comparison between the amount of tetroses and the total amount of hexoses directly derived from them.

– 95 – Chapter 2: Results and Discussion

CHO

OH hydroxy-acetaldehyde

CHO CHO

H OH HO H

H OH H OH

OH OH

D-erythrose D-threose

CHO CHO CHO CHO

H OH HO H HO H H OH

H OH H OH HO H HO H

H OH H OH HO H HO H

H OH H OH H OH H OH

CH2OH CH2OH CH2OH CH2OH D-allose D-altrose D-talose D-galactose

CHO CHO CHO CHO

H OH HO H H OH HO H

HO H HO H H OH H OH

H OH H OH HO H HO H

H OH H OH H OH H OH

CH2OH CH2OH CH2OH CH2OH D-glucose D-mannose D-gulose D-idose

Figure 100. Scheme of sugar synthesis (for simplicity only the D form of the sugars is shown).

– 96 – Chapter 2: Results and Discussion

Table 18. Correlation of relative abundance between tetroses and its directly derived hexoses

in the (L-Pro)2Zn catalyzed reaction.

Abundance Abundance Sugar (tetrose) (%) (derived hexoses) (%)

erythrose 29.3 49.9 threose 70.7 50.1

The excess of threose over erythrose in the sugar mixture apparently had no effect on the threose-derived hexoses. There was no difference between the sum of gulose, galactose, talose and idose and the sum of the other hexoses.

The acetylated crude from the (L-Pro)2Zn reaction was tested for optical activity and the [a]D = + 0.7 º was obtained at 21 ºC. As an example, the value reported for pentaacetylated a- glucose118 is + 100 º in methanol (c = 1 g/100 ml) at 28 ºC, and the value for pentaacetylated 119 b-glucose is + 3.8 º in CHCl3 (c = 1 g/100 ml) at 25 ºC. This difference in the values even between a- and b- forms of the sugars expose the difficulty in extracting information from values obtained for sugar mixtures. Nevertheless, the value obtained is encouraging taking in consideration the fact that the starting material was optically inactive.

2.3.3. Final Remarks and Outlook

With the system previously described, it was possible to produce the entire tetrose and hexose repertoire under mild conditions. As far as I know, this is the first system available that, in compliance with the prebiotic environment, provides a simple and effective route for the synthesis of sugars. A simple way to avoid the synthesis of tetroses and only form the hexoses is to use methoxy-acetaldehyde as the starting material. With this protection, the tetroses formed from the first condensation would not be able to close the five-membered ring and, therefore, would react further leading to the sole production of hexoses.

– 97 – Chapter 3: Experimental Part

3. Experimental Part

3.1. General Remarks

3.1.1. List of Abbreviations

Ac acetyl AcOH acetic acid AIBN a,a’-azoisobutyronitrile Approx. approximate Aq. aqueous DIAT diisopropylammonium tetrazolide DMAP dimethylaminopyridine DMF dimethylformamide DMSO dimethyl sulfoxide EI-MS electronic ionisation mass MS en ethane-1,2-diamine ESI-MS electron spray ionisation mass spectrometry

Et2O diethyl Ether

Et3N triethylamine EtOAc ethyl Acetate EtOH ethanol FAB-MS Fast atom bombardment mass spectrometry GC-MS Gas chromatography mass spectrometry h Hours Hex hexane HPLC high performance liquid chromatography HR-MS high resolution mass spectrometry IR infra-red LSI-MS liquid secondary ion mass spectrometry m.p. melting point MeI methyl iodide

– 98 – Chapter 3: Experimental Part

MeOH methanol MS mass spectrometry n-BuLi n-butyl lithium NMR nuclear magnetic resonance Ph phenyl ppm parts per million PTSA p-toluene sulfonic acid quant. quantitative RT room temperature RT room temperature TBDMS tertiobutyl-dimethylsilyl THF tetrahydrofurane TLC thin layer chromatography TMP trimethyl phosphate tren bis-N1-(2-amino-ethyl)- ethane-1,2-diamine

3.1.2. Chemicals and Solvents

All chemicals were bought in puriss or p.a. quality from Fluka, Sigma or Aldrich. All solvents used in reactions were bought in p.a. quality or distilled and dried prior to use. Solvents for extractions were distilled from technical quality.

3.1.3. Materials and Methods

Water or oxygen sensitive reactions were carried out under nitrogen or argon. All materials were dried before use.

· Thin Layer Chromatography (TLC): all the reactions were followed by TLC on Alugram SIL G/UV254 silica gel sheets (Macherey-Nagel) with detection by UV lamp at 254 nm and 365 nm, and/or coloration with revelators like ninhydrin solution (10% in EtOH),

– 99 – Chapter 3: Experimental Part phopsphomolybdic acid solution (10% in EtOH) and cerium solution (10.5 g Cer(IV)-sulfate, 21 g phopsphomolybdic acid, 60 mL conc. H2SO4 in 900 mL water) and heating. · Flash Chromatography: all cromatographies were performed in Silica Gel 60 from Fluka (0.04-0.063 nm; 230-400 mesh ASTM). All the solvents used were previously distilled. · High Pressure Liquid Chromatography (HPLC): Chiral HPLC measurements were performed using a Waters system consisting of a Waters 600 Controller, a Waters 600 Pump and a Waters 996 Photodiode Array Detector. The chiral columns used were the Chiralpak AS and OD from Dancel Chemical Industries, Ltd. · Melting Point (m.p.): Büchi 510, measured in an open glass capillary in a Tottili apparatus, without correction. · Optical Rotation: Polarimeter Perkin Elmer 241. The optical rotation [a] was determined at the given temperature and wavelength (Na-D-line: D = 589 nm) in a cell 10 cm long. · Kugelrohr Distillation: kugelrohr oven Büchi GKR-50. · Infrared Spectroscopy (IR): Perkin Elmer 1600 series FTIR. Spectra were measured as film between two NaCl cells (for the case of the oils) and as KBr pills (for the case of the solids). Frequencies n are given in cm-1 (br broad, s strong, m medium or w weak). · 1H-NMR Spectroscopy: Bruker AC-300 (300 MHz) and AM-500 (500 MHz).

Chemical shifts (d) are given in ppm referred to CHCl3. Coupling constants (J) are given in Hertz (Hz) and the multiplicities as follows: s (singlet), d (doublet), t (triplet), q (quartet) and m (multiplet). · 13C-NMR Spectroscopy: Bruker AC-300 (75 MHz). Chemical shifts (d) are given in ppm. Multiplicities were determined with DEPT (Distortion Enhancement by Polarization Transfers) techniques. · Mass Spectroscopy and GC-MS: were provided by the “Service of Mass Spectrometry” at the Department of Chemistry and Biochemistry, University of Bern.

The IUPAC-standard compound names were generated with Autonom® version 2.2 from Beilstein Informationssysteme GmbH.

– 100 – Chapter 3: Experimental Part

3.2. Methyltransferase System

3.2.1. Chelating vs. Non-Chelating

3.2.1.1. Compound Characterization

C7H16S S 132.26 1-Methylsulfanyl-hexane (Hexyl methyl sulfide) (1) Colorless oil

A solution of hexane-1-thiol (0.5 ml, 3.56 mmol), NaH (103 mg, 4.3 mmol) and methyl iodide (0.22 ml, 3.56 mmol) in THF (20 mL) was stirred at room temperature under

N2 for 0.5 h. Iced water (50 ml) was added to quench the reaction. Extraction with CH2Cl2 (2x

30 ml), drying with MgSO4 and evaporation under reduced pressure afforded the title compound in 50% yield (235 mg).

1 H-NMR (300 MHz, CDCl3): 0.83 (t, 3H, J= 6.63Hz), 1.19-1.37 (m, 6H), 1.47-1.58 (m, 2H), 2.02 (s, 3H), 2.42 (t, 2H, J= 6.99Hz). 13 C-NMR (75 MHz, CDCl3): 14.36 (q), 15.84 (q), 22.93 (t), 28.88 (t), 29.59 (t), 31.83 (t), 34.68 (t).

NH N C8H12N2S HS 168.26 2-[(Pyridin-2-ylmethyl)-amino]-ethanethiol (MEPAH) (2) Colorless oil

A solution of freshly distilled 2-picolamine (5.79 mg, 53.5 mmol) and ethylene sulfide (1.56 g, 26.0 mmol) in dry toluene (20 ml) was refluxed in a sealed flask for 16 h. The solvent was removed under reduced pressure and the crude was distilled (2 times) over a 5 cm

– 101 – Chapter 3: Experimental Part

Vigreux column yielding 43% (4.93 g) of the title compound as a colorless oil. The spectroscopic data is in agreement with the literature120. b.p. (0.15 mBar): 85-90 ºC 1 H-NMR (300 MHz, CDCl3): 1.91 (s, br, 2H), 2.71 (t, 2H, J= 6.26Hz), 2.89 (t, 2H, J= 6.44Hz), 3.94 (s, 2H), 7.18 (ddd, 1H, J1= 0.75Hz, J2= 4.97Hz, J3= 7.55Hz), 7.33 (d, 1H, J= 7.71Hz), 7.66 (ddd, 1H, J1= 1.83Hz, J2= 7.71Hz, J3= 7.71Hz), 8.57 (d, 1H, J= 4.77Hz). 13 C-NMR (75 MHz, CD3OD): 24.52 (t), 52.88 (t), 54.59 (t), 123.85 (d), 124.11 (d), 138.72 (d), 149.89 (d), 159.86 (s).

NH N C9H14N2S S 182.28 (2-Methylsulfanyl-ethyl)-pyridin-2-ylmethyl-amine (Me-MEPA) (3) Black oil

A solution of 2 (336 mg, 2 mmol), NaOH (80 mg, 2 mmol) and trimethyl phosphate

(308 mg, 2.2 mmol) in methanol (20 ml) was stirred at room temperature under N2 for 26 h. The solvent was removed under reduced pressure and the crude mixture was dried in the high vacuum overnight. Kugelröhr distillation afforded the title compound as a black oil (185 mg, 50%).

1 H-NMR (300 MHz, CDCl3): 2.08 (s, 3H), 2.40 (s, br, 1H), 2.69 (t, 2H, J= 6.44Hz), 2.87 (t, 2H, J= 6.44Hz), 3.94 (s, 2H), 7.15 (ddd, 1H, J1= 0.75Hz, J2= 5.52Hz, J3= 6.99Hz), 7.32 (d, 1H, J= 8.07Hz), 7.64 (ddd, 1H, J1= 1.83Hz, J2= 7.73Hz, J3= 7.73Hz), 8.55 (ddd, 1H, J1= 0.75Hz, J2= 0.92Hz, J3= 4.96Hz). 13 C-NMR (75 MHz, CDCl3): 15.22 (q), 34.11 (t), 47.47 (t), 54.58 (t), 121.93 (d), 122.00 (d), 136.45 (d), 149.28 (d), 159.12 (s). EI-MS m/z (%): 182 (M +) (1), 135 (35), 121 (100). HRMS m/z: (M+) calc: 182.087770; found: 182.087890.

– 102 – Chapter 3: Experimental Part

N N C10H16N2S S 196.31

Methyl-(2-methylsulfanyl-ethyl)-pyridin-2-ylmethyl-amine (Me2-MEPA) (4) Black oil.

A solution of 2 (336 mg, 2 mmol), NaH (48 mg, 2 mmol) and methyl iodide (284 mg,

2 mmol) in toluene (20 mL) was stirred at room temperature under N2 for 2.5 h. Iced water

(50 ml) was added to quench the reaction. Extraction with CH2Cl2 (2x 50 ml), drying with MgSO4 and evaporation under reduced pressure afforded a mixture (334 mg) of both 3 (34%) and 4 (66%). The residue was purified by column chromatography (elution with 2%

MeOH:CH2Cl2), the product containing fractions (Rf = 0.25) were combined and the solvent was removed to give the title compound in 34% yield (122 mg).

Rf (2% MeOH/CH2Cl2): 0.25 1 H-NMR (300 MHz, CDCl3): 2.10 (s, 3H), 2.31 (s, 3H), 2.69 (m, 4H), 3.71 (s, 2H), 7.16 (ddd, 1H, J1= 1.11Hz, J2= 4.96Hz, J3= 7.54Hz), 7.46 (d, 1H, J= 7.74Hz), 7.66 (ddd, 1H, J1= 1.83Hz, J2= 7.53Hz, J3= 7.53Hz), 8.55 (ddd, 1H, J1= 0.72Hz, J2= 1.74Hz, J3= 4.86Hz). 13 C-NMR (75 MHz, CDCl3): 15.76 (q), 31.90 (t), 42.39 (q), 56.88 (t), 63.70 (t), 122.00 (d), 123.07 (d), 136.42 (d), 149.10 (d), 159.24 (s). FAB-MS (matrix: DTT/DTE) m/z (%): 197 (M +) (100), 135 (25).

N S H N Zn N S NH C16H22N4S2Zn 399.88

MEPA2Zn Complex (5) Colorless crystals

A solution of diethyl zinc (1.82 mL, 2 mmol) in toluene was added dropwise to a solution of 2 (672 mg, 4 mmol) in freshly distilled toluene (15 mL) at 0 ºC under N2. After 1 h., the precipitate was filtered off, washed with toluene and dried under reduced pressure

– 103 – Chapter 3: Experimental Part yielding the title compound as a white powder (655 mg, 82%). Recrystallization from ethanol gave colorless crystals. The spectroscopic data of the complex is in accordance with the literature120. m.p.: 160 ºC 1 H-NMR (300 MHz, CD3OD): 2.57 (t, 4H, J= 4.77Hz), 2.70 (t, 4H, 4.77Hz), 4.08 (s, 4H), 7.43 (dd, 2H, J1= 6.33Hz, J2= 6.33Hz), 7.48 (d, 2H, J= 7.92Hz), 7.90 (ddd, 2H, J1= 1.47Hz, J2= 7.62Hz, J3= 7.62Hz), 8.64 (d, 2H, J= 4.77Hz). 13 C-NMR (75 MHz, d6-DMSO): 25.31 (t), 51.97 (t), 52.13 (t), 122.86 (d), 123.14 (d), 137.51 (d), 148.49 (d), 157.09 (s). FAB-MS (matrix: DTT/DTE) m/z (%): 399 (MH+) (10), 231 (70), 169 (100). IR (KBr pellets) n (cm-1): 3227.6 (s), 3064.3 (w), 3028.3 (m), 2999.0 (m), 2943.6 (s), 2920.0 (s), 2894.2 (m), 2859.3 (s), 2840.0 (m), 1606.7 (m), 1591.5 (s), 1565.1 (s), 1482.9 (s), 1455.9 (s), 1448.9 (s), 1442.1 (s), 1432.0 (s), 1292.2 (s), 1207.2 (m), 1172.2 (m), 1151.7 (s), 1104.9 (m), 1084.4 (s), 1056.9 (s), 1047.9 (s), 1028.3 (s), 993.5 (s), 966.9 (s), 947.0 (s), 936.2 (s), 836.2 (m), 821.4 (m), 774.9 (s), 733.8 (m), 665.4 (m), 633.3 (m), 608.0 (m).

- n N Cl

N S- Zn+2 Zn+2 -S

Cl- N N C16H22N4Cl2S2Zn2 536.16 (MEPAZnCl)n Complex (6) Pale yellow powder

A solution of ZnCl2 (136 mg, 1 mmol) in degassed MeOH (10 ml) was added to a solution of 2 (168 mg, 1 mmol) also in degassed MeOH (10 ml). After 1 h stirring, the precipitate formed was collected and dried under reduced pressure. The title compound was obtained in a polymeric form (32% yield) and showed similar characteristics and spectroscopic data as reported in literature120.

– 104 – Chapter 3: Experimental Part

1 H-NMR (300 MHz, d6-DMSO): 2.44-2.72 (m, br, 8H), 4.04 (s, br, 4H), 4.31 (s, br, 2H), 7.50 (s, br, 2H), 7.55 (d, 2H, J= 8.07Hz), 8.03 (dd, 2H, J1= 7.16Hz, J2= 7.16Hz), 8.68 (s, br, 2H). 13 C-NMR (75 MHz, d6-DMSO): 25.67 (t), 50.00 (t), 51.67 (t), 123.56 (d), 123.88 (d), 139.73 (d), 147.77 (d), 156.27 (s). EI-MS m/z (%): 373 (2), 321 (6), 231 (4), 169 (100).

3.2.1.2. Reactivity Studies

Methyl transfer from trimethyl phosphate to hexane-1-thiol: A solution of TMP (199 mg, 1.42 mmol), ZnCl2 or Zn(NO3)2.6H2O (1.42 mmol) and pyridine (0.57 ml, 7.1 mmol) in methanol (20 ml) was stirred until the solution was clear. Hexane-1-thiol (1 ml, 7.1 mmol) was added dropwise and the mixture stirred for 18 hours at the temperature indicated. The solvent was evaporated under vacuum and the residue was analyzed by 1H-NMR spectroscopy.

Methyl transfer from trimethyl phosphate to MEPAH (2): A solution of TMP (28 mg,

0.2 mmol), ZnX2 (0.1 mmol) and MEPAH (33.6 mg, 0.2 mmol) in methanol (5 ml) was stirred for 18 hours at room temperature or 22 hours at reflux. The crude was analyzed as above. In ZnX2, X stands for Cl, NO3 and CH3COO.

Methyl transfer from trimethyl phosphate to the preformed complexes (MEPA)2Zn (5) or (MEPAZnCl)n (6): A solution of trimethyl phosphate (33.6 mg, 0.2 mmol) and 5 or 6 (0.1 mmol) in 5 ml of MeOH stirred for 18 hours at room temperature or 22 hours at reflux and analyzed as above.

3.2.1.3. Kinetic experiments

st Reactions were run under pseudo - 1 order conditions with (MEPA)2Zn (5) (72, 95 and 119 mM) in excess (3, 4 and 5 mol equivalents) over TMP (24 mM). The reaction was 1 1 monitored by H-NMR spectroscopy in d4-MeOH at 24 ± 0.3 °C. Typical H-NMR parameters included 4 scans per spectrum, 40 s relaxation delay between scans and a total of

– 105 – Chapter 3: Experimental Part

64 scans. The total time of data collection varied between 18 and 32 h after which, we observed never less than 30% conversion. Pseudo-first order rate constants (k’) were obtained from the plot of the natural logarithm of TMP concentration against time:

-k’t [TMP]t = [TMP]0 e

The pseudo-first-order rate constants (k’) obtained are presented in table 19.

Table 19. Pseudo-first-order rate constant for the different runs performed.

-1 [(MEPA)2Zn] (mM) k’ (s ) 72 3.5´10-6 95 4.2´10-6 119 6.2´10-6

The plot of k’ vs. [MEPA2Zn] used in each run gave the final value of k (5.0 ± 0.6 ´ 10-5 M-1 s-1) as the slope of the line with a y-intercept at zero (Figure 53).

3.2.2. The Dinucleotide Model

3.2.2.1. Compound Characterization

O

O

NH

O O N O O H H H H C31H32N2O7 OH H 544.60 1-{5-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-tetrahydro-furan-2- yl}-5-methyl-1H-pyrimidine-2,4-dione (7) Brownish foam.

– 106 – Chapter 3: Experimental Part

A solution of thymidine (1 g, 4.1 mmol), 4,4’-dimethoxytrityl chloride (1.67 g, 4.9 mmol) in pyridine (20 ml) was stirred at RT for 168 h under N2 pressure. The mixture was poured into iced-H2O (50 ml) and the precipitate collected afforded quantitatively the title compound121.

1 H-NMR (300 MHz, CDCl3): 1.48 (s, 3H), 2.40 (m, 2H), 3.37 (dd, 1H, J1= 2.94Hz, J2= 10.47Hz), 3.48 (dd, 1H, J1= 2.66Hz, J2= 10.40Hz), 3.80 (s, 6H), 4.06 (d, 1H, J= 2.58Hz), 4.57 (s, br, 1H), 6.42 (t, 1H, J= 7.14Hz), 6.84 (d, 4H, J= 8.82Hz), 7.24-7.41 (m, 9H), 7.59 (s, 1H), 8.66 (s, br, 1H).

O

O

NH

O O N O O H H H H O H C33H34N2O8 O 586.64 Acetic acid 2-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-5-(5-methyl-2,4-dioxo- 3,4-dihydro-2H-pyrimidin-1-yl)-tetrahydro-furan-3-yl ester (8) Brittle yellow foam

Compound 7 (2.23 g, 4.1 mmol) was dissolved in pyridine (20 ml) and cooled down to 0 ºC. A catalytic amount (c.a. 5 mg) of DMAP was added to the mixture followed by the careful addition of acetic anhydride (3.85 ml, 41 mmol). The reaction stirred at RT for 16 h.

Quenching with H20 (150 ml) and extraction with CH2Cl2 (2x 100 ml) afforded quantitatively the title product (2.4 g)121.

1 H-NMR (300 MHz, CDCl3): 1.39 (s, 3H), 2.10 (s, 3H), 2.45 (m, 2H), 3.47 (m, 2H), 3.80 (s, 6H), 4.14 (d, 1H, J= 1.65Hz), 4.45 (d, 1H, J= 4.77Hz), 6.44 (t, 1H, J= 6.24Hz), 6.85 (d, 4H, J= 8.82Hz), 7.24-7.41 (m, 9H), 7.62 (s, 1H), 8.44 (s, br, 1H).

– 107 – Chapter 3: Experimental Part

O

NH

HO N O O H H H H O H C12H16N2O6 O 284.27 Acetic acid 2-hydroxymethyl-5-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)- tetrahydro-furan-3-yl ester (9) Colorless oil

A solution of 8 (2.4 g, 4.1 mmol) in 80% AcOH/H2O (50 ml) was stirred at RT for 16 h. Evaporation of the solvent under reduced pressure afforded colorless crude. The residue was purified by column chromatography (elution with EtOAc). The product containing fractions (Rf = 0.20) were combined and the solvent was removed to give the title compound as colorless oil in 70% yield (815 mg)121.

Rf (EtOAc): 0.20 1 H-NMR (300 MHz, CDCl3): 1.94 (s, 3H), 2.12 (s, 3H), 2.41 (m, 2H), 3.95 (m, 2H), 4.11 (dd, 1H, J1= 2.40Hz, J2= 4.79Hz), 5.36 (m, 1H), 6.25 (dd, 1H, J1= 6.42Hz, J2= 8.1Hz), 7.49 (s, 1H), 8.06 (s, br, 1H). FAB-MS (matrix: DTT/DTE) m/z (%): 303 (35), 285 (M+) (75), 127 (100).

O

O

NH

O O N O O H H H H O H P O N C38H48N3O8P 705.79 Diisopropyl-phosphoramidous acid 2-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]- 5-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)- tetrahydro-furan-3-yl ester methyl ester (10) Brittle yellow foam

– 108 – Chapter 3: Experimental Part

A solution of 7 (109 mg, 0.2 mmol) and DIAT (51 mg, 0.3 mmol) in CH2Cl2 (4 ml) was stirred at RT under N2 pressure. The freshly prepared phosphoramidite (i-Pr2N)2POMe (0.063 ml, 0.22 mmol) was added carefully and the mixture was stirred at RT for 24 h. The reaction mixture was poured into a sat. solution of NaHCO3 (20 ml), the organic layer was washed with brine (20 ml), dried with MgSO4 and evaporated under reduced pressure to afford a oil crude (150 mg). The crude was dissolved in hexane (5 ml) and taken to –70 ºC. The precipitate was collected by filtration and dried under reduced pressure to afford the title compound in 74% (100 mg)122.

1 H-NMR (300 MHz, CDCl3): 1.26 (m, 12H), 1.41 (s, 3H), 2.26-2.59 (m, 2H), 3.46-3.54 (m, 4H), 3.67 (d, 3H, J= 12.3Hz), 3.80 (s, 6H), 4.18 (dd, 1H, J1= 2.01Hz, J2= 9.93Hz), 4.67 (m, 1H), 6.41 (t, 1H, J1= 7.71Hz, J2= 13.59Hz), 6.84 (dd, 4H, J1= 1.65Hz, J2= 8.64Hz), 7.26- 7.42 (m, 9H), 7.64 (d, 1H, J= 6.78Hz), 7.93 (s, br, 1H). ESI-MS(–) m/z (%): 704 (M–) (100), 283 (25).

O

O

NH

O O N O O H H H H O O H P O O NH O N O O H H H H O H C44H49N4O15P O 904.86 DMT-dT(Me)pdT-Ac. (11) Brittle yellow foam

A solution of 10 (50 mg, 0.07 mmol), 9 (22.2 mg, 0.078 mmol) and tetrazole (14.9 mg, 0.21 mmol) in dry CH3CN (10 ml) was stirred under N2 pressure at RT for 20 h. The reaction mixture was treated with a solution of 1 M I2 in 2,6-lutidine, THF and H2O (2:2:1) until it turn into a persistent pale orange color. The reaction was quenched after 10 min, with the addition of sat. Na2S2O3 (10 ml). The mixture was poured into sat. NaHCO3 (50 ml),

– 109 – Chapter 3: Experimental Part

extracted with CH2Cl2 (2x 50 ml), washed with H2O (50 ml), dried with MgSO4 and evaporated under reduced pressure to afford the title compound in 50% (31 mg) over two steps. The spectroscopic data is in agreement with the literature122.

1 H-NMR (300 MHz, CDCl3): 1.40 (s, 3H), 1.92 (s, 3H), 2.11 (s, 3H), 2.42 (m, 4H), 3.37- 3.54 (m, 4H), 3.69-3.75 (m, 3H), 3.80 (s, 6H), 4.04-4.16 (m, 2H), 4.25-4.35 (m, 2H), 5.11- 5.35 (m, 2H), 6.25-6.36 (m, 1H), 6.42-6.47 (m, 1H), 6.84 (d, 4H, J= 8.64Hz), 7.26-7.42 (m, 9H), 7.53-7.62 (m, 2H).

O

NH

HO N O O H H H H O O H P O O NH O N O O H H H H O H C23H31N4O13P O 602.49 Acetic acid 2-{[2-hydroxymethyl-5-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)- tetrahydro-furan-3-yloxy]-methoxy-phosphoryloxymethyl}-5-(5-methyl-2,4-dioxo-3,4- dihydro-2H-pyrimidin-1-yl)-tetrahydro-furan-3-yl ester [dT(Me)pdT-Ac.] (12) Colorless foam

A solution of 11 (208 mg, 0.23 mmol) in 80% AcOH/H2O (5 ml) stirred at RT for 2 h. The solvent was removed under reduced pressure, and the crude was purified by column chromatography (elution with a gradient of 0-8% MeOH:CH2Cl2). The product fractions were combined and the solvent was removed to give the title compound as colorless foam in 85% yield (118 mg). The spectroscopic data is in agreement with the literature98.

1 H-NMR (300 MHz, CDCl3): 1.91 (s, 3H), 1.95 (s, 3H), 2.13 (s, 3H), 2.28-2.53 (m, 4H), 3.84 (d, 3H), 3.86-3.89 (m, 2H), 4.17-4.23 (m, 2H), 4.31-4.38 (m, 2H), 5.13-5.17 (m, 1H),

– 110 – Chapter 3: Experimental Part

5.27-5.33 (m, 1H), 6.14-6.21 (m, 1H), 6.31-6.36 (m, 1H), 7.41 (s, 1H), 7.46 (s, 1H), 8.7-9.3 (m, br, 2H). 13 C-NMR (75 MHz, CDCl3): 13.10 (q), 13.21 (q), 21.59 (q), 37.57 (t), 39.09 (t), 55.52 (q), 62.65 (t), 62.77 (t), 68.10 (t), 74.52 (d), 74.73 (d), 78.92 (d), 83.30 (d), 85.70 (d), 85.79 (d), 86.22 (d), 86.32 (d), 86.38 (d), 86.86 (d), 87.04 (d), 111.99 (s), 112.05 (s), 112.50 (s), 135.99 (d), 137.11 (d), 137.24 (d), 151.06 (s), 164.32 (s), 164.45 (s), 171.36 (s), 171.44 (s). 31 P-NMR (162 MHz, CD3OD): –0.49 (s), –0.26 (s). ESI-MS(–) m/z (%): 601 (M–) (100), 377 (35), 335 (40), 283 (70), 255 (35). FAB-MS (matrix: DTT/DTE) m/z (%): 603 (M+) (25), 379 (50), 303 (60), 225 (100), 193 (45), 127 (70).

3.2.2.2. Dinucleotide Reactivity Study

Methyl transfer from dT(Me)pdT-Ac to MEPA2Zn complex: A solution of dinucleotide

12 (72 mg, 0.012 mmol) and MEPA2Zn (4.6 mg, 0.012 mmol) in deuterated methanol (1 ml) was stirred for 21 hours at RT. The residue was analyzed by 1H- and 31P-NMR spectroscopy.

3.2.3. The NS2 Ligand

3.2.3.1. Compound Characterization

O

C3H4Br2O Br Br 215.87 1,3-Dibromo-propan-2-one (13) Colorless Oil

A mixture of acetone (50 ml), water (160 ml) and acetic acid (37.5 ml) was heated to 70 °C. Bromine (117 mmol, 65 ml) was added and the reaction mixture was stirred for 20 min. Cold water (80 ml) was added. The organic phase was distilled and the title compound was obtained as the main fraction with a boiling point of 87-88 °C. Note that this compound is extremely lacrimogen.

– 111 – Chapter 3: Experimental Part

1 H-NMR (300 MHz, CDCl3): 4.15 (s, 4H).

O

S S O O C7H10O3S2 206.27 Thioacetic acid S-(3-acetylsulfanyl-2-oxo-propyl) ester (14) Dark orange oil.

Potassium thioacetate (570 mg, 5 mmol) was dissolved in ethanol (50 ml) and cooled down to 0 °C. A solution of 13 (432 mg, 2 mmol) in ethanol (3 ml) was added dropwise to the mixture. The ice was removed and the reaction mixture was stirred at room temperature for about 2 h. Solvent was removed under reduced pressure and the crude redissolved in CH2Cl2

(50 ml). After washing 2 times with H2O (50 ml), CH2Cl2 was removed under vacuum to afford quantitatively the title compound in a pure form.

1 H-NMR (300 MHz, CDCl3): 2.31 (s, 6H); 3.81 (s, 4H). 13 C-NMR (75 MHz, CDCl3): 30.06 (q), 38.18 (t), 194.13 (s), 197.78. EI-MS m/z (%): 206 (M+) (4), 164 (60), 117 (75), 89 (65), 43 (100). HRMS m/z: calc. 206.007138, found 206.007080.

O

O O Si Si C15H34O3Si2 318.60 1,3-Bis-(tert-butyl-dimethyl-silanyloxy)-propan-2-one (15) Colorless oil

A solution of 1,3-dihydroxypropan-2-one (504 mg, 5.6 mmol) in DMF (4 ml) was cooled to 0 °C. Imidazole (950 mg, 14 mmol) and tert-butyldimethylsilyl chloride (2.11 g, 14 mmol) were added. The ice bath was removed and the mixture was allowed to stir 1 h at room

– 112 – Chapter 3: Experimental Part temperature. The reaction was quenched with the addition of iced water (50 ml) and the aqueous phase was extracted with diethyl ether (3x 50 ml). The organic phase was washed with brine, dried with MgSO4 and removed under reduced pressure. The residue was purified by column chromatography (elution with 4% EtOAc:Hexane). The product containing fractions (Rf = 0.44) were combined and the solvent was removed to give the title compound as a viscous oil in 90% yield. Spectroscopic data is in accordance with literature123.

Rf (4% EtOAc./Hex.): 0.44 1 H-NMR (300 MHz, CDCl3): 0.08 (s, 12H); 0.91 (s, 18H); 4.41 (s, 4H). 13 C-NMR (75 MHz, CDCl3): -5.56 (q), 18.32 (s), 25.75 (q), 67.90 (t).

OH N

O O Si Si C20H39NO3Si2 397.71 1,3-Bis-(tert-butyl-dimethyl-silanyloxy)-2-pyridin-2-yl-propan-2-ol (16) Viscous and colorless oil

A solution of 2-Bromo-pyridine (711 mg, 4.5 mmol) in dry THF (10 ml) were cooled down to –78 ºC under inert atmosphere. n-Butyllitium (2.81 ml, 4.5 mmol) was added carefully (temperature should not rise above –60 ºC to avoid decomposition) to the stirred suspension and was left to stir for 15 min. Compound 15 (477 mg, 1.5 mmol) in THF (5 ml) was added dropwise to the mixture. The reaction was stirred for 2 h below –65 ºC and was left to warm up to –40 ºC. The reaction mixture was quenched with H2O (10 ml) and the required product was extracted with diethyl ether (2x 20 ml), dried with MgSO4 and concentrated under reduced pressure (144 mg of crude). The residue was purified by column chromatography (elution with 4% EtOAc:Hexane) and the product containing fractions (Rf = 0.15) were combined and the solvent was removed to give the title compound (72.4 mg, 12%) as a viscous and colorless oil.

Rf (4% EtOAc./Hex.): 0.15

– 113 – Chapter 3: Experimental Part

1 H-NMR (300 MHz, CDCl3): -0.04 (s, 6H), -0.03 (s, 6H), 0.82 (s, 18H), 3.83 (d, 2H, J= 9.93Hz), 3.94 (d, 2H, J= 9.93Hz), 4.55 (s, 1H), 7.18 (m. 1H) 7.58-7.69 (m, 1H), 8.53 (m, 1H). 13 C-NMR (75 MHz, CDCl3): -5.53 (q), -5.52 (q), 18.25 (s), 25.82 (q), 66.95 (t), 77.05 (s), 121.75 (d), 122.10 (d), 135.86 (d), 147.62 (d), 161.22 (s). EI-MS m/z (%): 382 (M-15) (7), 340 (M-57) (100), 252 (60), 73 (80).

O

O O C6H10O3 130.14 2,2-Dimethyl-[1,3]dioxan-5-one (17) Highly volatile liquid

A mixture of 2-amino-2-(hydroxymethyl)-1,3-propandiol hydrochloride (69.3 g, 440 mmol), 2,2-dimethoxypropane (52.1 g, 500 mmol) and PTSA (3.5 g, 22 mmol) in dry DMF

(140 ml) was stirred at RT for 18h. After addition of Et3N (3.5 ml, 25 mmol) the solvent was removed under reduced pressure, the residue diluted with Et3N (56 ml, 400 mmol) and EtOAc (1000 ml). The ammonium salts were filtered off and the solvent evaporated under reduced pressure to afford a very viscous oil. Addition of a few drops of Et2O resulted into the precipitation of 5-amino-(2,2-dimethyl-1,3-dioxan-5-yl)methanol. This intermediate was dissolved, together with KH2PO4 (54.3 g, 440 mmol), in H2O (300 ml) and a solution of

NaIO4 (800 ml, 400 mmol) was added carefully at 0 ºC. The reaction was stirred for 5 h at

RT, extracted with Et2O (3x 200 ml), dried with MgSO4 and concentrated under reduced pressure to afford the title compound in 34 % yield (17.68 g) over two steps. The spectroscopic data is in agreement with the literature124.

1 H-NMR (300 MHz, CDCl3): 1.46 (s, 6H), 4.16 (s, 4H).

– 114 – Chapter 3: Experimental Part

OH N

O O C11H15NO3 209.25 2,2-Dimethyl-5-pyridin-2-yl-[1,3]dioxan-5-ol (18) Colorless oil

A solution of 2-Bromo-pyridine (122 mg, 0.77 mmol) in dry THF (5 ml) was cooled down to –78 ºC under inert atmosphere. n-Butyllitium (0.48 ml, 0.77 mmol) was carefully added at such rate that the temperature did not rise above –60 ºC. After stirring for 15 min., the ketone 17 (100 mg, 0.77 mmol) in THF (2 ml) was added to the mixture. The reaction was stirred for 2 h below –65 ºC and then was left to warm up to –40 ºC. The reaction mixture was quenched with H2O (15 ml) and the required product was extracted with diethyl ether (2x 20 ml), dried with MgSO4 and concentrated under reduced pressure. The residue was purified by column chromatography (elution with 50% EtOAc:Hexane) and the product containing fractions (Rf = 0.43) were combined and the solvent was removed to give the title compound (124 mg, 77%) as a colorless oil.

Rf (50% EtOAc./Hex.): 0.43 1 H-NMR (300 MHz, CDCl3): 1.57 (s, 3H), 1.61 (s, 3H), 3.80 (d, 2H, J= 11.76Hz), 4.27 (d, 2H, J= 11.76Hz), 4.59 (s, br, 1H), 7.24 (ddd. 1H, J1= 1.11Hz, J2= 4.77Hz, J3= 7.44Hz) 7.75 (ddd, 1H, J1= 1.83Hz, J2= 7.72Hz, J3= 7.72Hz), 7.86 (ddd, 1H, J1= 1.11Hz, J2= 1.11Hz, J3= 8.09Hz), 8.54 (ddd, 1H, J1= 1.11Hz, J2= 1.83Hz, J3= 4.77Hz). 13 C-NMR (75 MHz, CDCl3): 21.71 (q), 25.61 (q), 68.83 (t), 69.20 (s), 98.47 (s), 121.10 (d), 122.78 (d), 137.08 (d), 147.82 (d), 160.13 (s). ESI-MS(+) m/z (%): 210 (MH+) (80), 152 (100), 122 (90). HR-ESI-MS(+) m/z: (MH+) calc: 210.1130; found: 210.1142.

– 115 – Chapter 3: Experimental Part

OH N

C8H11NO3 OH OH 169.18 2-Pyridin-2-yl-propane-1,2,3-triol (19) Colorless oil

A mixture of 18 (41.6 mg, 0.2 mmol) in THF (2 ml) and HCl 1.2 M (2 ml) were stirred at RT for 4 h. The pH of the solution was taken to neutrality with the addition of

NaOH (2M). The neutral aqueous phase was washed with Et2O (2x 10 ml) and dried under reduced pressure. The crude was redissolved in 10% MeOH:CH2Cl2 in order to filter out the formed NaCl salts. Evaporation of the solvents under reduced pressure afforded the title compound in a pure form and in quantitative yield.

Rf (20% MeOH/CH2Cl2): 0.58 1 H-NMR (300 MHz, CD3OD): 3.82 (d, 2H, J= 11.22Hz), 3.91 (d, 2H, J= 11.40), 7.63 (ddd. 1H, J1= 1.11Hz, J2= 5.52Hz, J3= 7.72Hz) 7.97 (d, 1H, J=8.10Hz), 8.19 (ddd, 1H, J1= 1.83Hz, J2=7.71Hz, J3= 7.90Hz), 8.62 (ddd, 1H, J1= 0.72Hz, J2= 0.72Hz, J3= 5.51Hz). 13 C-NMR (75 MHz, CD3OD): 67.08 (t), 78.53 (s), 124.64 (d), 125.16 (d), 142.51 (d), 145.48 (d), 161.52 (s). ESI-MS(+) m/z (%): 170 (MH+) (100), 152 (70), HR-ESI-MS(+) m/z: (MH+) calc: 170.0817; found: 170.0811.

OH N

O O O O S S O O C22H23NO7S2 477.55 Toluene-4-sulfonic acid 2-hydroxy-3-toluene- 4-sulfonyloxy-2-pyridin-2-yl-propyl ester (20) Beige powder.

– 116 – Chapter 3: Experimental Part

A solution of 19 (265 mg, 1.57 mmol) in pyridine (10 ml) was cooled down to 0 ºC under N2 pressure. Tosyl chloride (744 mg, 3.90 mmol) was added in small portions to the stirring solution over 30 min. The mixture was left to warm slowly to RT. After 36 h of continuous stirring, the mixture was poured into 30 ml of iced H2O. The precipitate formed was collected by filtration and dried under high vacuum to afford the title compound in 56% yield (420 mg).

1 H-NMR (300 MHz, CD3OD): 2.45 (s, 6H), 4.16 (d, 2H, J= 9.93Hz), 4.27 (d, 2H, J= 9.93Hz), 4.60 (s, br, 1H), 7.24 (ddd. 1H, J1= 1.11Hz, J2= 4.79Hz, J3= 7.45Hz) 7.30 (dd, 4H, J1= 0.75Hz, J2= 8.45Hz), 7.54 (ddd, 1H, J1= 1.11Hz, J2= 1.11Hz, J3= 7.99Hz), 7.60 (dd, 4H, J1= 0.75Hz, J2= 8.45Hz), 7.73 (ddd, 1H, J1= 1.83Hz, J2= 7.71Hz, J3= 7.82Hz), 8.27 (ddd, 1H, J1= 1.11Hz, J2= 1.83Hz, J3= 4.78Hz). 13 C-NMR (75 MHz, CD3OD): 21.59 (q), 73.74 (t), 76.01 (s), 122.54 (d), 124.24 (d), 129.04 (d), 131.06 (d), 133.68 (s), 138.27 (d), 146.60 (s), 149.43 (d), 159.41 (s). FAB-MS (matrix: DTT/DTE) m/z (%): 478 (MH+) (100), 155 (50), 119 (65). HR-ESI-MS(+) m/z: (MH+) calc: 478.0994; found: 478.0997.

OH N C8H9NOS2 S S 199.29 4-Pyridin-2-yl-[1,2]dithiolan-4-ol (21) Pale yellow oil.

A solution of the ditosylate 20 (62 mg, 0.13 mmol) in degassed MeOH (20 ml) was cooled down to 0 ºC prior to the addition of NaHS (73 mg, 1.3 mmol) under Argon pressure.

The reaction mixture stirred for 18 h and degassed H2O (50 ml) was added. Extraction with

CH2Cl2 (2x 40 ml), drying with MgSO4 and evaporation of the solvent under reduced pressure afforded the title compound together with the reduced form 22 in 93% yield (24.2 mg). The ratio between the oxidized and reduced form depends on the amount of oxygen present in the reaction mixture. The title compound could be obtained from 22 (pure or in a mixture) by leaving the compound at open air or by oxidation with DMSO following a literature procedure125.

– 117 – Chapter 3: Experimental Part

1 H-NMR (300 MHz, CDCl3): 3.29 (d, 2H, J= 11.76Hz), 3.64 (d, 2H, J= 11.37), 4.74 (s, 1H), 7.27 (ddd. 1H, J1= 1.47Hz, J2= 4.76Hz, J3= 7.16Hz), 7.71-7.80 (m, 2H), 8.56 (d, 1H, J= 4.77Hz). 13 C-NMR (75 MHz, CDCl3): 52.65 (t), 86.39 (s), 120.29 (d), 122.94 (d), 137.23 (d), 148.19 (d), 159.96 (s). EI-MS m/z (%): 199 (M+) (25), 181 (100), 105 (50), 79 (80). ESI-MS(+) m/z (%): 200 (MH+) (10), 182 (55), 150 (100). HR-ESI-MS(+) m/z: (MH+) calc: 200.0203; found: 200.0200. IR (film) n (cm-1): 668.4 (m), 758.0 (s), 1038.9 (w), 1215.5 (s), 1436.3 (w). 1470.6 (w), 1591.1 (w), 2400.1 (w), 3018.8 (m).

OH N C8H11NOS2 SH SH 201.30 1,3-Dimercapto-2-pyridin-2-yl-propan-2-ol (22) Pale yellow oil.

A solution of the ditosylate 20 (95.4 mg, 0.2 mmol) in degassed DMF (5 ml) was cooled down to 0 ºC prior to the addition of NaHS (112 mg, 2 mmol) under Argon pressure.

The reaction mixture stirred for 18 h and degassed H2O (50 ml) was added. Extraction with

CH2Cl2 (2x 40 ml), drying with MgSO4 and evaporation of the solvent under reduced pressure afforded the title compound together with a very small amount (< 5%) of the oxidized form 21 in quantitative yield.

1 H-NMR (300 MHz, CDCl3): 3.35 (d, 2H, J= 10.29Hz), 3.94 (d, 2H, J= 10.29), 7.33 (ddd. 1H, J1= 1.11Hz, J2= 4.86Hz, J3= 7.44Hz), 7.75 (ddd, 1H, J1= 1.83Hz, J2=8.54Hz, J3= 8.54Hz), 7.87 (ddd, 1H, J1= 1.83Hz, J2=7.71Hz, J3= 7.71Hz), 8.41 (dd, 1H, J1= 1.11Hz, J2= 7.90Hz). 13 C-NMR (75 MHz, CDCl3): 44.94 (t), 92.45 (s), 120.30 (d), 122.85 (d), 137.23 (d), 148.51 (d), 159.48 (s). FAB-MS (matrix: DTT/DTE) m/z (%): 202 (MH+) (45), 200 (25), 119 (100).

– 118 – Chapter 3: Experimental Part

OH N

O S S O C12H15NO3S2 285.38 Thioacetic acid S-(3-acetylsulfanyl-2-hydroxy-2-pyridin-2-yl-propyl) ester (23) Pale yellow oil.

A solution of the ditosylate 20 (95.4 mg, 0.2 mmol) and potassium thioacetate (57.0 mg, 0.5 mmol) in EtOH (10 ml) was heated to reflux for 5 h. The cooled down mixture was poured into H2O (50 ml) and extracted with CH2Cl2 (2x 40 ml). The organic phase was washed with brine (50 ml) dried over MgSO4 and concentrated under reduced pressure. The residue was purified by column chromatography (elution with 20% EtOAc:Hexane), the product containing fractions (Rf = 0.21) were combined and the solvent was removed to give the title compound in a very low yield (10%).

Rf (20% EtOAc/Hex): 0.21 1 H-NMR (300 MHz, CDCl3): 2.24 (s, 6H), 3.86 (d, 2H, J= 13.95Hz), 4.08 (d, 2H, J= 13.95), 7.20 (ddd. 1H, J1= 1.11Hz, J2= 4.77Hz, J3= 7.35Hz), 7.38 (ddd. 1H, J1= 0.72Hz, J2= 0.72Hz, J3= 8.01Hz), 7.66 (ddd. 1H, J1= 1.83Hz, J2= 7.89Hz, J3= 7.89Hz), 8.58 (ddd. 1H, J1= 0.72Hz, J2= 0.92Hz, J3= 4.79Hz). 13 C-NMR (75 MHz, CDCl3): 30.21 (q), 35.79 (t), 84.36 (s), 120.66 (d), 122.71 (d), 135.94 (d), 148.83 (d), 169.36 (s), 193.81 (s). EI-MS m/z (%): 284 (M+) (15), 242 (35), 181 (75), 150 (50), 105 (55), 78 (50), 43 (100).

O N

O O C12H17NO3 223.27 2-(5-Methoxy-2,2-dimethyl-[1,3]dioxan-5-yl)-pyridine (24) Colorless oil

– 119 – Chapter 3: Experimental Part

To a solution of 18 (1.7 g, 8.1 mmol) in THF (50 ml), was added first NaH (583 mg, 24.3 mmol) and after MeI (1.48 ml, 24.3 mmol). The mixture stirred at RT for 18 h and then was quenched with H2O (100 ml). Extraction with CH2Cl2 (3x 100 ml), drying with MgSO4 and evaporation of solvents under reduced pressure, afforded the title compound quantitatively (1.8 g).

1 H-NMR (300 MHz, CDCl3): 1.53 (s, 3H), 1.55 (s, 3H), 3.31 (s, 3H), 4.01 (ddd, 2H, J1= 1.11Hz, J2= 1.11Hz, J3= 12.58Hz), 4.45 (ddd, 2H, J1= 1.11Hz, J2= 1.11Hz, J3= 12.59Hz), 7.22 (ddd. 1H, J1= 1.11Hz, J2= 4.79Hz, J3= 7.63Hz) 7.58 (ddd, 1H, J1= 1.11Hz, J2= 1.11Hz, J3= 8.08Hz), 7.73 (ddd, 1H, J1= 1.83Hz, J2= 7.35Hz, J3= 7.72Hz), 8.59 (ddd, 1H, J1= 1.11Hz, J2= 1.85Hz, J3= 4.88Hz). 13 C-NMR (75 MHz, CDCl3): 20.44 (q), 28.49 (q), 52.25 (q), 65.70 (t), 76.13 (s), 98.76 (s), 122.07 (d), 123.34 (d), 137.40 (d), 149.78 (d), 160.14 (s). ESI-MS(+) m/z (%): 224 (MH+) (40), 166 (100). HR-ESI-MS(+) m/z: (MH+) calc: 224.1286; found: 224.1290.

O N C9H13NO3 OH OH 183.21 2-Methoxy-2-pyridin-2-yl-propane-1,3-diol (25) Colorless oil

Same procedure as compound 19, starting from compound 24. The yield is also quantitative.

1 H-NMR (300 MHz, CD3OD): 3.40 (s, 3H), 3.95 (d, 2H, J= 12.12Hz), 4.06 (d, 2H, J= 11.76Hz), 7.58 (ddd. 1H, J1= 1.11Hz, J2= 5.33Hz, J3= 7.55Hz) 7.85 (d, 1H, J= 8.10Hz), 8.13 (ddd, 1H, J1= 1.47Hz, J2= 7.74Hz, J3= 7.82Hz), 8.61 (ddd, 1H, J1= 0.75Hz, J2= 1.83Hz, J3= 5.24Hz). 13 C-NMR (75 MHz, CD3OD): 51.54 (q), 63.48 (t), 83.64 (s), 124.99 (d), 125.05 (d), 141.58 (d), 146.65 (d), 160.39 (s).

– 120 – Chapter 3: Experimental Part

ESI-MS(+) m/z (%): 184 (MH+) (70), 152 (75) 122 (100). HR-ESI-MS(+) m/z: (MH+) calc: 184.0973; found: 184.0959.

O N

O O O O S S

O O C23H25NO7S2 491.57 Toluene-4-sulfonic acid 3-toluene-4-sulfonyloxy- 2-methoxy-2-pyridin-2-yl-propyl ester (26) Beige powder.

Same procedure as compound 20, starting from compound 25. The title compound was obtained in 78% yield.

1 H-NMR (300 MHz, CDCl3): 2.45 (s, 6H), 3.10 (s, 3H), 4.39 (d, 2H, J= 10.65Hz), 4.47 (d, 2H, J= 10.65Hz), 7.19 (ddd. 1H, J1= 1.11Hz, J2= 4.79Hz, J3= 7.53Hz) 7.31 (d, 4H, J= 8.07Hz), 7.40 (ddd, 1H, J1= 1.11Hz, J2= 1.11Hz, J3= 7.99Hz), 7.65 (ddd, 1H, J1= 1.83Hz, J2= 6.44Hz, J3= 7.36Hz), 7.66 (d, 4H, J= 8.10Hz),8.38 (ddd, 1H, J1= 1.11Hz, J2= 1.83Hz, J3= 4.78Hz). 13 C-NMR (75 MHz, CDCl3): 22.36 (q), 51.97 (q), 69.54 (t), 80.09 (s), 122.53 (d), 123.96 (d), 128.65 (d), 130.55 (d), 133.09 (s), 137.54 (d), 145.61 (s), 149.23 (d), 157.15 (s). FAB-MS (matrix: DTT/DTE) m/z (%): 492 (MH+) (100), 155 (15), 119 (20). HR-ESI-MS(+) m/z: (MH+) calc: 492.1150; found: 492.1162.

– 121 – Chapter 3: Experimental Part

O N

O S S O C13H17NO3S2 299.40 Thioacetic acid S-(3-acetylsulfanyl-2-methoxy-2-pyridin-2-yl-propyl) ester (27) Pale yellow oil.

A solution of 26 (100 mg, 0.2 mmol) and potassium thioacetate (91 mg, 0.8 mmol) in 2:1 EtOH/DMF (30 ml) was taken to reflux for 20 h. After cooling down, the crude was poured into H2O (50 ml), extracted with CH2Cl2 (2x 50 ml), dried with MgSO4 and evaporated under reduced pressure to afford a mixture of compounds. The residue was purified by column chromatography (elution with 10% EtOAc:Hexane) and the product containing fractions (Rf = 0.40) were combined and the solvent was removed to give the title compound in 16% yield (23 mg).

Rf (10% EtOAc/Hex): 0.40 1 H-NMR (300 MHz, CDCl3): 2.25 (s, 6H), 3.25 (s, 3H), 3.65 (d, 2H, J= 13.80Hz), 3.74 (d, 2H, J= 13.77), 7.20 (dd. 1H, J1= 4.77Hz, J2= 7.35Hz), 7.55 (d, 1H, J= 7.89Hz), 7.69 (dd, 1H, J1= 7.71Hz, J2=7.71Hz), 8.56 (d, 1H, J= 4.80Hz).

O N C9H13NOS2 SH SH 215.33 2-Methoxy-2-pyridin-2-yl-propane-1,3-dithiol (28) Pale yellow oil.

A solution of 27 (10 mg, 0.033 mmol) in degassed EtOH (0.5 ml) was cooled down to 0 ºC under argon pressure. An aqueous solution 2 M NaOH (0.05 ml) was added and the mixture was stirred at 0 ºC for 1 h. The pH of the solution was adjusted to 7 with the addition of 1 M HCl (c.a. 0.1 ml). The mixture was extracted with degassed CH2Cl2 (2x 10 ml), dried

– 122 – Chapter 3: Experimental Part

with MgSO4 and evaporated under reduced pressure to afford quantitatively the title compound with a small amount of the oxidized form 29 (< 10%)

1 H-NMR (300 MHz, CDCl3): 1.24 (t, 2H, J= 8.00Hz), 3.24-3.28 (m, 7H), 7.26 (dd. 1H, J1= 4.59Hz, J2= 7.53Hz), 7.64 (d, 1H, J= 7.92Hz), 7.76 (dd, 1H, J1= 7.89Hz, J2=7.89Hz), 8.59 (d, 1H, J= 4.80Hz). 13 C-NMR (75 MHz, CDCl3): 30.38 (t), 50.96 (q), 83.10 (s), 123.48 (d), 123.72 (d), 137.66 (d), 149.82 (d), 160.41 (s).

O N C9H11NOS2 S S 213.31 2-(4-Methoxy-[1,2]dithiolan-4-yl)-pyridine (29) Pale yellow oil.

The title compound was obtained following a slightly modified procedure described for the reduced form 28. In this case, the solvents were used without degassing leading to mainly the disulfide 29 (93%) and only traces of the dithiol 28 (7%).

1 H-NMR (300 MHz, CDCl3): 3.24 (s, 3H), 3.53 (d, 2H, J= 12.12Hz), 3.77 (d, 2H, J= 12.12), 7.26 (ddd. 1H, J1= 1.11Hz, J2= 4.86Hz, J3= 7.44Hz), 7.60 (ddd, 1H, J1= 1.11Hz, J2=1.01Hz, J3= 7.81Hz), 7.76 (ddd, 1H, J1= 1.86Hz, J2=7.72Hz, J3= 7.72Hz), 8.59 (ddd, 1H, J1= 0.75Hz, J2= 1.84Hz, J3= 4.78Hz). 13 C-NMR (75 MHz, CDCl3): 48.08 (t), 52.20 (q), 94.53 (s), 122.20 (d), 123.72 (d), 137.67 (d), 149.82 (d), 159.91 (s). EI-MS m/z (%): 213 (M+) (4), 181 (100), 105 (50), 78 (45). HR-ESI-MS(+) m/z: (M + Na+) calc: 236.0179; found: 236.0178.

– 123 – Chapter 3: Experimental Part

O N C9H11NOS S 181.25 2-(3-Methoxy-thietan-3-yl)-pyridine (30) Colorless oil

A solution of the ditosylate 26 (130 mg, 0.26 mmol) in degassed DMF (8 ml) was cooled down to 0 ºC prior to the addition of NaHS (150 mg, 2.6 mmol) under Argon pressure.

The reaction mixture stirred for 18 h and degassed H2O (30 ml) was added. Extraction with

CH2Cl2 (2x 30 ml), drying with MgSO4 and evaporation of the solvent under reduced pressure afforded the title compound in 82% yield (46 mg).

1 H-NMR (300 MHz, CDCl3): 3.01 (s, 3H), 3.66 (d, 2H, J= 10.29Hz), 3.73 (d, 2H, J= 9.57), 7.29 (ddd. 1H, J1= 1.11Hz, J2= 4.88Hz, J3= 7.63Hz), 7.54 (ddd, 1H, J1= 1.11Hz, J2=1.11Hz, J3= 7.99Hz), 7.78 (ddd, 1H, J1= 2.22Hz, J2=7.72Hz, J3= 7.72Hz), 8.69 (ddd, 1H, J1= 1.11Hz, J2= 1.83Hz, J3= 4.86Hz). 13 C-NMR (75 MHz, CDCl3): 35.90 (t), 50.42 (q), 83.27 (s), 120.51 (d), 122.99 (d), 137.05 (d), 148.70 (d), 160.29 (s). ESI-MS(+) m/z (%): 182 (MH+) (100), 150 (90), 117 (70). HR-ESI-MS(+) m/z: (MH+) calc: 182.0639; found: 182.0630. IR (film) n (cm-1): 651.7 (m), 728.9 (s), 906.8 (s), 2253.1 (w).

3.2.3.2. Complexation Procedures

Bipyridinyl zinc complex: To a solution of [2,2']bipyridinyl (78 mg, 0.5 mmol) in toluene (5 ml), diethyl zinc (0.45 ml, 0.5 mmol) was carefully added. The reddish mixture turned yellow after 1 h and a sample of the complex (0.3 mmol) was added to a solution of 22 in toluene (2 ml). Upon addition, a precipitate was formed and was filtered off. Both the precipitate and the solution were analyzed by 1H-NMR.

– 124 – Chapter 3: Experimental Part

3.2.3.2. Reactivity Procedure

Methyl transfer test: A solution of TMP (42 mg, 0.3 mmol), Zn(CH3COO)2 (11 mg, 0.05 mmol) and 22 (20.1 mg, 0.1 mmol) in degassed deuterated methanol (5 ml) was refluxed for 20 hours. A sample (0.6 ml) of the mixture was analyzed by 1H-NMR spectroscopy.

3.2.4. The S4 Ligand

3.2.4.1. Compound Characterization

OH Br N

O O C11H14BrNO3 288.14 5-(6-Bromo-pyridin-2-yl)-2,2-dimethyl-[1,3]dioxan-5-ol (31) Beige powder.

A solution of 2,6-dibromo-pyridine (474 mg, 2 mmol) in dry CH2Cl2 (5 ml) was cooled down to –78 ºC under inert atmosphere. n-Butyllitium (1.25 ml, 2 mmol) was carefully added. After stirring for 15 min., the ketone 17 (195 mg, 1.5 mmol) was added to the mixture. The reaction was stirred for 2 h below –70 ºC and then was left to warm up to –40 ºC. The reaction mixture was quenched with H2O (25 ml) and the required product was extracted with

CH2Cl2 (2x 30 ml), dried with MgSO4 and concentrated under reduced pressure. The residue was purified by column chromatography (elution with 20% EtOAc:Hexane), the product containing fractions (Rf = 0.41) were combined and the solvent was removed to give the title compound (226 mg, 52%) as a beige powder.

Rf (20% EtOAc/Hex): 0.41 1 H-NMR (300 MHz, CDCl3): 1.55 (s, 3H), 1.60 (s, 3H), 3.73 (d, 2H, J= 12.12Hz), 4.12 (s, 1H), 4.31 (d, 2H, J= 11.73), 7.42 (dd. 1H, J1= 1.08Hz, J2= 7.91Hz), 7.59 (d, 1H, J1= 7.73Hz, J2=7.73Hz), 7.82 (dd, 1H, J1= 1.08Hz, J2= 7.73Hz).

– 125 – Chapter 3: Experimental Part

13 C-NMR (75 MHz, CDCl3): 26.87 (q), 27.51 (q), 68.02 (t), 71.82 (t), 85.73 (s), 111.78 (s), 120.19 (d), 127.59 (d), 139.65 (d), 141.88 (s), 164.85 (s). EI-MS m/z (%): 272 (M+-15) (20), 274 (M+-15) (20), 199 (100), 201 (100), 157 (50), 159 (50).

O Br N

O O C12H16BrNO3 302.17 2-Bromo-6-(5-methoxy-2,2-dimethyl-[1,3]dioxan-5-yl)-pyridine (32) Colorless oil

To a solution of 31 (220 mg, 0.76 mmol) in THF (5 ml), was added first NaH (55 mg, 2.3 mmol) and after MeI (0.14 ml, 2.3 mmol). The mixture stirred at RT for 16 h and then was quenched with H2O (20 ml). Extraction with Et2O (3x 20 ml), drying with MgSO4 and evaporation of solvents under reduced pressure, afforded quantitatively the title compound (230 mg).

1 H-NMR (300 MHz, CDCl3): 1.52 (s, 3H), 1.56 (s, 3H), 3.34 (s, 3H), 3.97 (d, 2H, J= 12.66Hz), 4.42 (d, 2H, J= 12.48), 7.41 (dd. 1H, J1= 1.65Hz, J2= 7.17Hz) 7.53-7.62 (m, 2H). 13 C-NMR (75 MHz, CDCl3): 20.20 (q), 28.71 (q), 52.46 (q), 65.53 (t), 75.83 (s), 98.76 (s), 121.02 (d), 127.82 (d), 139.69 (d), 142.23 (s), 161.83 (s). ESI-MS(+) m/z (%): 324 (M+Na+) (25), 326 (M+Na+) (25), 302 (MH+) (10), 304 (MH+) (10), 244 (100), 246 (100). HR-ESI-MS(+) m/z: (MH+) calc: 302.0391; found: 302.0411.

HO O N

O O O O C18H27NO6 353.42 5-[6-(5-Methoxy-2,2-dimethyl-[1,3]dioxan-5-yl)- pyridin-2-yl]-2,2-dimethyl-[1,3]dioxan-5-ol (33) Colorless oil

– 126 – Chapter 3: Experimental Part

A solution of 32 (239 mg, 0.79 mmol) in dry CH2Cl2 (5 ml) was cooled down to –78 ºC under inert atmosphere. n-Butyllitium (0.5 ml, 0.79 mmol) was carefully added. After stirring for 15 min., the ketone 17 (103 mg, 0.79 mmol) was added to the mixture. The reaction was stirred for 2 h below –70 ºC and then was left to warm up to –40 ºC. The reaction mixture was quenched with H2O (25 ml) and the required product was extracted with

CH2Cl2 (2x 30 ml), dried with MgSO4 and concentrated under reduced pressure. The residue was purified by column chromatography (elution with 20% EtOAc:Hexane), the product containing fractions (Rf = 0.11) were combined and the solvent was removed to give the title compound (112 mg, 40%) as a beige powder.

Rf (20% EtOAc/Hex): 0.11 1 H-NMR (300 MHz, CDCl3): 1.53 (s, 3H), 1.55 (s, 3H), 1.57 (s, 3H), 1.60 (s, 3H), 3.34 (s, 3H), 3.78 (d, 2H, J= 12.06Hz), 4.02 (d, 2H, J= 11.49), 4.29 (d, 2H, J= 11.88Hz), 4.41 (d, 2H, J= 12.60), 7.50 (dd. 1H, J1= 2.64Hz, J2= 6.39Hz), 7.72-7.82 (m, 2H). 13 C-NMR (75 MHz, CDCl3): 19.32 (q), 26.13 (q), 27.91 (q), 30.91 (q), 51.74 (q), 65.03 (t), 68.86 (t), 75.21 (s), 97.98 (s), 98.41 (s), 119.95 (d), 120.31 (d), 137.84 (d). ESI-MS(+) m/z (%): 376 (M+Na+) (80), 354 (MH+) (100), 296 (40), 166 (60). HR-ESI-MS(+) m/z: (MH+) calc: 354.1916; found: 354.1909.

O O N

O O O O C19H29NO6 367.44 2,6-Bis-(5-methoxy-2,2-dimethyl-[1,3]dioxan-5-yl)-pyridine (34) Colorless oil

To a solution of 33 (150 mg, 0.42 mmol) in THF (5 ml), was added first NaH (51 mg, 2.1 mmol) and after MeI (0.13 ml, 2.1 mmol). The mixture stirred at RT for 16 h and then was quenched with H2O (20 ml). Extraction with Et2O (3x 20 ml), drying with MgSO4 and evaporation of solvents under reduced pressure, afforded quantitatively the title compound (156 mg).

– 127 – Chapter 3: Experimental Part

1 H-NMR (300 MHz, CDCl3): 1.53 (s, 6H), 1.58 (s, 6H), 3.38 (s, 6H), 4.01 (d, 4H, J= 12.69Hz), 4.44 (d, 4H, J= 12.66), 7.48 (d, 2H, J= 7.92Hz), 7.77 (t, 1H, J= 7.92Hz). 13 C-NMR (75 MHz, CDCl3): 19.70 (q), 28.95 (q), 52.52 (q), 65.78 (t), 76.10 (s), 98.61 (s), 120.77 (d), 138.32 (d), 159.45 (s). ESI-MS(+) m/z (%): 390 (M+Na+) (60), 368 (MH+) (100). HR-ESI-MS(+) m/z: (MH+) calc: 368.2073; found: 368.2072.

O O N C13H21NO6 OH OH OH OH 287.31 2-[6-(2-Hydroxy-1-hydroxymethyl-1-methoxy-ethyl)-pyridin-2-yl]- 2-methoxy-propane-1,3-diol (35) Colorless oil

A mixture of 34 (156 mg, 0.42 mmol) in THF (5 ml) and HCl 1.2 M (5 ml) were stirred at RT for 18 h. The pH of the solution was taken to neutrality with the addition of

NaOH (2M). The neutral aqueous phase was washed with Et2O (2x 20 ml) and dried under reduced pressure. The crude was dissolved in 10% MeOH:CH2Cl2 in order to filter out the formed NaCl salts. Evaporation of the solvents under reduced pressure afforded the title compound in a pure form and in quantitative yield.

1 H-NMR (300 MHz, CD3OD): 3.54 (s, 6H), 3.96 (d, 4H, J= 12.12Hz), 4.11 (d, 4H, J= 12.12), 8.13 (d. 2H, J= 8.10Hz), 8.60 (t, 1H, J= 8.10Hz). 13 C-NMR (75 MHz, CD3OD): 52.02 (q), 62.78 (t), 82.70 (s), 125.21 (d), 146.99 (d), 156.98 (s). ESI-MS(+) m/z (%): 288 (MH+) (100). HR-ESI-MS(+) m/z: (MH+) calc: 288.1451; found: 288.1447.

– 128 – Chapter 3: Experimental Part

O O N

O O O O O S O O S O O S O O S O

C41H45NO14S4 904.05 Toluene-4-sulfonic acid 3-toluene-4-sulfonyloxy-2-[6-(2-toluene-4-sulfonyloxy-1-toluene- 4-sulfonyloxymethyl-1-methoxy-ethyl)-pyridin-2-yl]-2-methoxy-propyl ester (36) Brownish foam.

A solution of 35 (120 mg, 0.42 mmol) in pyridine (5 ml) was cooled down to 0 ºC under N2 pressure. Tosyl chloride (480 mg, 2.52 mmol) was added in small portions to the stirring solution over 30 min. The mixture was left to warm slowly to RT. After 64 h of continuous stirring, the mixture was poured into 20 ml of iced H2O. The precipitate formed was collected by filtration and dried under high vacuum to afford the desired compound in a pure form (292 mg, 77%).

1 H-NMR (300 MHz, CDCl3): 2.45 (s, 12H), 3.02 (s, 6H), 4.32 (d, 4H, J= 10.68Hz), 4.40 (d, 4H, J= 10.65Hz), 7.30-7.38 (m, 10H), 7.65-7.73 (m, 9H). 13 C-NMR (75 MHz, CDCl3): 22.38 (q), 52.13 (q), 69.37 (t), 80.09 (s), 122.12 (d), 128.62 (d), 130.73 (d), 132.92 (s), 138.57 (d), 145.88 (s), 156.55 (s). ESI-MS(+) m/z (%): 904 (MH+) (100). HR-ESI-MS(+) m/z: (MH+) calc: 904.1801; found: 904.1767.

O O N C13H17NO2S2 S S 283.40 2,6-Bis-(3-methoxy-thietan-3-yl)-pyridine (37) Pale yellow oil.

– 129 – Chapter 3: Experimental Part

A solution of the tetratosylate 36 (50 mg, 0.055 mmol) in degassed DMF (3 ml) was cooled down to 0 ºC prior to the addition of NaHS (62 mg, 1.1 mmol) under Argon pressure.

The reaction mixture stirred for 18 h and degassed H2O (20 ml) was added. Extraction with

CH2Cl2 (2x 20 ml), drying with MgSO4 and evaporation of the solvent under reduced pressure afforded the title compound in good 74% yield (14 mg).

1 H-NMR (300 MHz, CDCl3): 3.06 (s, 6H), 3.66 (d, 4H, J= 10.29Hz), 3.86 (d, 4H, J= 9.93), 7.50 (d. 2H, J= 7.71Hz), 7.85 (t, 1H, J= 7.71Hz). 13 C-NMR (75 MHz, CDCl3): 36.49 (t), 51.23 (q), 84.33 (s), 120.31 (d), 138.79 (d), 159.67 (s). ESI-MS(+) m/z (%): 284 (MH +) (75), 252 (100). HR-ESI-MS(+) m/z: (MH+) calc: 284.0778; found: 284.0775.

HO O N C12H19NO6 OH OH OH OH 273.29 2-[6-(2-Hydroxy-1-hydroxymethyl-1-methoxy-ethyl)-pyridin-2-yl]- propane-1,2,3-triol (38) Colorless oil

A mixture of 33 (335 mg, 0.95 mmol) in THF (20 ml) and HCl 1.2 M (20 ml) were stirred at RT for 20 h. The pH of the solution was taken to neutrality with the addition of

NaOH (2M). The neutral aqueous phase was washed with Et2O (2x 50 ml) and dried under reduced pressure. The crude was dissolved in 10% MeOH:CH2Cl2 in order to filter out the formed NaCl salts. Evaporation of the solvents under reduced pressure afforded the title compound in a pure form and in quantitative yield.

1 H-NMR (300 MHz, CD3OD): 3.34 (s, 3H), 3.83 (d, 2H, J= 8.43Hz), 3.89 (d, 2H, J= 8.43Hz), 4.00 (d, 2H, J= 8.61Hz), 4.06 (d, 2H, J= 8.82Hz), 7.53 (dd, 1H, J1= 0.75Hz, J2= 5.96Hz) 7.62 (dd, 1H, J1= 0.75Hz, J2= 5.87Hz), 7.85 (dd, 1H, J1= 5.88Hz, J2= 5.97Hz. 13 C-NMR (75 MHz, CD3OD): 51.46 (q), 64.09 (t), 67.56 (t), 78.83 (s), 83.93 (s), 121.11 (d), 121.92 (d), 138.22 (d), 160.17 (s), 162.37 (s).

– 130 – Chapter 3: Experimental Part

ESI-MS(+) m/z (%): 274 (MH+) (100). HR-ESI-MS(+) m/z: (MH+) calc: 274.1290; found: 274.1278.

HO O N

O O O O O S O O S O O S O O S O

C40H43NO14S4 890.02 Toluene-4-sulfonic acid 2-hydroxy-3-toluene-4-sulfonyloxy-2-[6-(2-toluene-4-sulfonyl- oxy-1-toluene-4-sulfonyloxymethyl-1-methoxy-ethyl)-pyridin-2-yl]-propyl ester (39) Brownish foam.

A solution of 38 (205 mg, 0.75 mmol) in pyridine (10 ml) was cooled down to 0 ºC under N2 pressure. Tosyl chloride (1.4 g, 7.5 mmol) was added in small portions to the stirring solution over 30 min. The mixture was left to warm slowly to RT. After 48 h of continuous stirring, the mixture was poured into 50 ml of iced H2O. The precipitate formed was collected by filtration and dried under high vacuum to afford the desired compound in a pure form (454.4 mg, 68%).

1 H-NMR (300 MHz, CDCl3): 2.45 (s, 12H), 3.06 (s, 3H), 4.19 (d, 2H, J= 7.71Hz), 4.24 (d, 2H, J= 7.71Hz), 4.33 (d, 2H, J= 8.07Hz), 4.38 (d, 2H, J= 8.07Hz), 7.20 (d, 2H, J= 6.42), 7.30- 7.33 (m, 9H), 7.65-7.72 (m, 8H).

– 131 – Chapter 3: Experimental Part

HO O HO O N N C12H15NO2S3 C12H17NO2S3 S S S 301.44 SH SH S 303.45 4-[6-(3-Methoxy-thietan-3-yl)-pyridin- 2- 1,3-Dimercapto-2-[6-(3-methoxy-thietan- yl]-[1,2]dithiolan-4-ol (40) 3-yl)-pyridin-2-yl]-propan-2-ol (41) Pale yellow oil Pale yellow oil

A solution of the tetratosylate 39 (88.9 mg, 0.1 mmol) in DMF (5 ml) was cooled down to 0 ºC prior to the addition of NaHS (56 mg, 1 mmol) under Argon pressure. The reaction mixture stirred for 19 h and H2O (20 ml) was added. Extraction with CH2Cl2 (2x 20 ml), drying with MgSO4 and evaporation of the solvent under reduced pressure afforded a mixture of products. The residue was purified by column chromatography (elution with 25% EtOAc:Hexane), the product containing fractions (Rf = 0.40) were combined and the solvent was removed to give 40 (9.8 mg, 32%) as a pale yellow color. 1H-NMR shows the presence of another compound in small amount (< 10%). The chemical shifts suggest that it is 41 but no further characterization was done for this compound.

Compound 40:

1 H-NMR (300 MHz, CDCl3): 3.06 (s, 3H), 3.31 (d, 2H, J= 11.49Hz), 3.39 (d, 2H, J= 10.56), 3.75 (d, 2H, J= 11.67Hz), 3.98 (d, 2H, J= 9.03), 7.83 (d. 1H, J= 8.10Hz), 7.99 (d, 1H, J= 8.28Hz), 8.16 (t, 1H, J= 7.91Hz). 13 C-NMR (75 MHz, CDCl3): 36.42 (t), 51.29 (q), 53.03 (t), 83.85 (s), 87.20 (s), 118.76 (d), 120.60 (d), 140.14 (d), 159.75 (s), 161.65 (s). HR-ESI-MS(+) m/z: (MH+) calc: 302.0343; found: 302.0345.

– 132 – Chapter 3: Experimental Part

3.2.5. The N2S2 Ligand

3.2.5.1. Compound Characterization

N N OH Br Br Br

C7H8BrNO C7H7Br2N 202.05 264.95 (6-Bromomethyl-pyridin-2-yl)-methanol (42) 2,6-Bis-bromomethyl-pyridine (43) White crystals White crystals

A solution of 2,6-pyridinedimethanol (1 g, 7.2 mmol) in 48% HBr (15 ml, 89 mmol) was refluxed for 18 h. The excess of HBr was neutralized with 2 M NaOH and the aqueous phase was extracted with Et2O (2x 50 ml), dried with MgSO4 and concentrated under reduced pressure. The residue was purified by column chromatography (elution with 60% Et2O:Hex). The reaction gave quantitative yields and, by changing the original procedure (varying mol percent of HBr and reaction time), allowed changes in the 42 : 43 ratio according to our needs. The spectroscopic data is in according with the literature126.

Compound 42:

Rf (60% Et2O/Hex.): 0.10 1 H-NMR (300 MHz, CDCl3): 3.67 (t, 1H, J= 5.16Hz), 4.56 (s, 2H), 4.77 (d, 1H, J= 5.16Hz), 7.18 (d, 1H, J= 7.71Hz), 7.36 (d, 1H, J= 7.74Hz), 7.70 (dd, 1H, J1= 7.73Hz, J2= 7.73Hz).

Compound 43:

Rf (60% Et2O/Hex.): 0.50 1 H-NMR (300 MHz, CDCl3): 4.55 (s, 4H), 7.39 (d, 2H, J= 7.74Hz), 7.72 (dd, 1H, J1= 7.74Hz, J2= 7.74Hz).

– 133 – Chapter 3: Experimental Part

S N OH N C13H14N2OS 246.33 [6-(Pyridin-2-ylmethylsulfanylmethyl)-pyridin-2-yl]-methanol (44) Colorless oil

The HBr salt of 2-bromomethyl-pyridine (500 mg, 1.98 mmol) was added to a

Na2CO3 sat. aqueous solution (10 ml) and it was stirred for 10 min at 0 ºC. Extraction with

CH2Cl2 (2x 20 ml) and concentration under reduced pressure afforded a solution (3-5 ml). This solution was added to a stirring solution of thiourea (165 mg, 2.18 mmol) in EtOH (20 ml) and the mixture refluxed for 5 h. The solvent was removed under vacuum to afford the thiouronium salt as a white solid. This salt was refluxed in H2O (10 ml) with NaHCO3 (168 mg, 2 mmol) for 1 h under argon pressure. After cooling down, the pH of the solution was adjusted to 7 with 1 M HCl and the aqueous solution was extracted with Et2O (2x 20 ml). The ethereal phase was dried with MgSO4 and concentrated under reduced pressure until a final volume of 10 ml. NaH (48 mg, 2 mmol) was added to the previously formed thiol and, after 15 min, the bromide 42 (202 mg, 1 mmol) was also added. The reaction mixture was stirred under argon pressure for 24 h. Dry DMF (10 ml) was added to help solubilization and the reaction was stirred for 3 h more. The mixture was poured into H2O (50 ml), extracted with

CH2Cl2 (2x 50 ml), dried with MgSO4 and concentrated under reduced pressure to afford an oil mixture. The residue was purified by column chromatography (elution with 80% EtOAc:Hex) and the product containing fractions (Rf = 0.15) were combined and the solvent was removed to give the title compound in 53% yield (130 mg) over four steps.

Rf (80% EtOAc./Hex.): 0.15 1 H-NMR (300 MHz, CDCl3): 3.80 (s, 2H), 3.86 (s, 2H), 4.77 (s, 2H), 7.11 (d, 1H, J= 7.71Hz), 7.20 (ddd, 1H, J1= 1.10Hz, J2= 5.15Hz, J3= 7.35Hz), 7.28 (d, 1H, J= 8.10Hz), 7.43 (d, 1H, J= 8.10Hz), 7.63-7.72 (m, 2H), 8.59 (d, 1H, J= 4.05Hz). 13 C-NMR (75 MHz, CDCl3): 36.72 (t), 37.18 (t), 63.86 (t), 118.71 (d), 121.76 (d), 122.10 (d), 123.41 (d), 137.07 (d), 137.62 (d), 148.94 (d), 157.26 (s), 158.15 (s), 158.42 (s). FAB-MS (matrix: 3-NBA) m/z (%): 247 (MH+) (100).

– 134 – Chapter 3: Experimental Part

S N Cl N C13H13ClN2S 264.77 2-Chloromethyl-6-(pyridin-2-ylmethylsulfanylmethyl)-pyridine (45) Colorless oil

A solution of 44 (123 mg, 0.5 mmol) and KOH (42 mg, 0.75 mmol) in DMF (5 ml) was cooled down to 0 ºC under N2 pressure. Tosyl chloride (190 mg, 1 mmol) was added and the mixture stirred at RT for 2 h. The reaction was poured into 60 ml of iced H2O and the precipitate formed (PTSA) was filtered off. Extraction with CH2Cl2 (2x 50 ml), dried with

MgSO4 and concentrated under reduced pressure afforded the title compound in 79% yield (112 mg).

1 H-NMR (300 MHz, CDCl3): 3.81 (s, 2H), 3.85 (s, 2H), 4.66 (s, 2H), 7.17 (ddd, 1H, J1= 1.10Hz, J2= 4.97Hz, J3= 7.53Hz), 7.29-7.40 (m, 3H), 7.61-7.70 (m, 2H), 8.55 (ddd, 1H, J1= 1.10Hz, J2= 1.85Hz, J3= 4.95Hz).

- S Cl N S + N H2N C14H18N4S2Cl NH2 341.90 2-[6-(Pyridin-2-ylmethylsulfanylmethyl)-pyridin-2-ylmethyl]- isothiouronium chloride(46) White powder

A solution of 45 (65 mg, 0.25 mmol) and thiourea (20.5 mg, 0.27 mmol) in EtOH (3 ml) was refluxed for 5 h. The solvent was removed under vacuum to afford the thiouronium salt as a white solid in quantitative yield.

– 135 – Chapter 3: Experimental Part

1 H-NMR (300 MHz, CDCl3): 3.79 (s, 2H), 3.80 (s, 2H), 4.31 (s, 2H), 7.19 (ddd, 1H, J1= 1.11Hz, J2= 4.97Hz, J3= 7.54Hz), 7.23 (d, 1H, J= 7.71Hz), 7.35 (d, 1H, J= 7.71Hz), 7.38 (d, 1H, J= 8.07Hz), 7.69 (t, 1H, J= 7.71Hz), 7.70 (t, 1H, J= 7.71Hz), 8.55 (ddd, 1H, J1= 1.08Hz, J2= 1.75Hz, J3= 4.87Hz).

N S SH N

C13H14N2S2 262.39 [6-(Pyridin-2-ylmethylsulfanylmethyl)-pyridin-2-yl]-methanethiol (47) Pale yellow oil

A solution of 46 (86 mg, 0.25 mmol) and NaHCO3 (21 mg, 0.25 mmol) in H2O (3 ml) was refluxed for 1 h under argon pressure. After cooling down, the pH of the solution was adjusted to 7 with 1 M HCl and the aqueous solution was extracted with Et2O (2x 10 ml). The ethereal phase was dried with MgSO4 and concentrated under reduced pressure to afford quantitatively the title compound ready for the metal complexation.

1 H-NMR (300 MHz, CDCl3): 3.80 (s, 2H), 3.81 (d, 2H, J= 11.22Hz), 3.85 (s, 2H), 7.11-7.18 (m, 2H), 7.22-7.28 (m, 1H), 7.39 (dd, 1H, J1= 7.07Hz, J2= 7.07Hz), 7.55-7.65 (m, 2H), 8.54 (d, 1H, J= 4.77Hz).

3.2.5.2. Complexation Studies

Complexation with zinc acetate: A solution of 48 (60 mg, 0.23 mmol) and zinc acetate

(50 mg, 0.23 mmol) in 50% MeOH:CH2Cl2 (10 ml) was stirred at RT for 1 h. The reaction solution was stored at –18 ºC overnight but no crystals were formed. The solvent was removed under reduced pressure, the crude mixture was dissolved in EtOH and it was left standing in the bench for 3 months. Colorless crystals were formed and collected from the mother liquid.

– 136 – Chapter 3: Experimental Part

3.2.6. Other Ligands

3.2.6.1. Compound Characterization

N O O O O

O O O C16H19NO8 353.33 O 2-Methoxycarbonyl-3-[6-(2-methoxycarbonyl-acetoxymethyl)-pyridin-2-yl]- propionic acid methyl ester (49) Colorless oil

A solution of 42 (760 mg, 3.76 mmol), dimethyl malonate (993 mg, 7.52 mmol) and

K2CO3 (1466 mg, 9.40 mmol) in DMF (10 ml) was stirred at RT under argon atmosphere for 48 h. The crude was filtered, concentrated under reduced pressure and purified by column chromatography (elution with 50% EtOAc.:Hex.). The product containing fractions (Rf = 0.45) were combined and the solvent was removed to give the title compound in 12% yield (154 mg).

Rf (50% EtOAc./Hex.): 0.45 1 H-NMR (300 MHz, CDCl3): 3.41 (d, 2H, J= 7.53Hz), 3.51 (s, 2H), 3.74 (s, 6H), 3.77 (s, 3H), 4.13 (t, 1H, J= 7.53Hz), 5.24 (s, 2H), 7.14 (d, 1H J= 7.71Hz), 7.20 (d, 1H, J= 7.71Hz), 7.62 (t, 1H, J= 7.71Hz). 13 C-NMR (75 MHz, CDCl3): 36.02 (t), 41.21 (t), 50.46 (q), 52.58 (q), 67.42 (t), 119.35 (d), 122.61 (d), 137.18 (d), 154.55 (s), 157.28 (s), 166.08 (s), 166.82 (s), 169.59 (s).

N

Br O Si C13H22BrNOSi 316.31 2-Bromomethyl-6-(tert-butyl-dimethyl-silanyloxymethyl)-pyridine (50) Colorless oil

– 137 – Chapter 3: Experimental Part

A solution of 42 (720 mg, 3.56 mmol) in DMF (5 ml) was cooled to 0 °C. Imidazole (605 mg, 8.9 mmol) and tert-butyldimethylsilyl chloride (1.28 g, 8.5 mmol) were added. The ice bath was removed and the mixture was allowed to stir 20 h at room temperature. The reaction was quenched with the addition of iced water (50 ml) and the aqueous phase was extracted with diethyl ether (2x 50 ml). The organic phase was washed with brine, dried with

MgSO4 and evaporated under reduced pressure. The residue was purified by column chromatography (elution with 30% Et2O:Hex). The product containing fractions (Rf = 0.76) were combined and the solvent was removed to give the title compound as colorless oil in 57% yield (637 mg).

Rf (30% Et2O/Hex.): 0.76 1 H-NMR (300 MHz, CDCl3): 0.13 (s, 6H), 0.97 (s, 9H), 4.65 (s, 2H), 4.84 (s, 2H), 7.34 (d, 1H J= 7.71Hz), 7.48 (d, 1H, J= 7.74Hz), 7.75 (dd, 1H, J1= 7.71Hz, J2= 7.74Hz). 13 C-NMR (75 MHz, CDCl3): -4.68 (q), 19.05 (s), 26.60 (q), 47.13 (t), 66.43 (t), 120.14 (d), 121.67 (d), 138.64 (d), 155.95 (s), 161.96 (s).

N O O O Si C H NO Si O 18 29 5 O 367.52 2-[6-(tert-Butyl-dimethyl-silanyloxymethyl)-pyridin-2-ylmethyl]- malonic acid dimethyl ester (51) Colorless oil

A solution of 50 (630 mg, 2 mmol), dimethyl malonate (330 mg, 2.5 mmol) and

K2CO3 (495 mg, 3 mmol) in DMF (15 ml) was stirred at RT under argon atmosphere for 26 h. The crude was filtered, concentrated under reduced pressure and purified by column chromatography (elution with 30% Et2O:Hex). The product containing fractions (Rf = 0.5) were combined and the solvent was removed to give the title compound in 73% yield (536 mg).

– 138 – Chapter 3: Experimental Part

Rf (30% Et2O/Hex.): 0.50 1 H-NMR (300 MHz, CDCl3): 0.12 (s, 6H), 0.96 (s, 9H), 3.38 (d, 2H, J= 7.53Hz), 3.72 (s, 6H), 4.10 (t, 1H, J= 7.53Hz), 4.77 (s, 2H), 7.05 (d, 1H J= 7.53Hz), 7.34 (d, 1H, J= 7.53Hz), 7.61 (dd, 1H, J1= 7.71Hz, J2= 7.71Hz). 13 C-NMR (75 MHz, CDCl3): -4.69 (q), 19.03 (s), 26.59 (q), 36.89 (t), 51.57 (q), 53.26 (d), 66.68 (t), 118.63 (d), 122.16 (d), 137.69 (d), 156.50 (s), 160.90 (s). FAB-MS (matrix: DTT/DTE) m/z (%): 368 (MH+) (100), 310 (70).

N O O

O O O O C16H21NO6 323.35 Acetic acid 6-(3-acetoxy-2-acetoxymethyl-propyl)-pyridin-2-ylmethyl ester (52) Colorless oil

The three-step synthesis was carried out starting from compound 42 (400 mg, 1.98 mmol). The first step was previously described and gave compound 49 as a crude that was used without purification. This crude was dissolved in THF (20 ml) and cooled down to 0 ºC.

LiAlH4 (10 ml, 1M in THF) was added to the mixture and it was stirred at RT for 88 h. The reaction was quenched with the addition of H2O (40 ml) and neutralized with HCl 1M. The removal of H2O under reduced pressure and high vacuum afforded the tri-alcohol 53 ready for acetylation. The very well dried crude from the previous step was dissolved in pyridine (20 ml) and cooled down to 0 ºC. A catalytic amount (c.a. 5 mg) of dimethyl-pyridin-4-yl-amine (DMAP) was added to the mixture followed by the careful addition of acetic anhydride (3 ml). The reaction stirred at RT for 20 h. Quenching with H20 (50 ml) and extraction with

CH2Cl2 (2x 50 ml) afforded the title product (300 mg) in 47% yield over three steps.

1 H-NMR (300 MHz, CDCl3): 2.05 (s, 6H), 2.17 (s, 3H), 2.67 (m, 1H), 2.88 (d, 2H, J= 7.35Hz), 4.09 (m, 4H), 5.19 (s, 2H), 7.08 (d, 1H J= 7.71Hz), 7.20 (d, 1H, J= 7.74Hz), 7.62 (dd, 1H, J1= 7.71Hz, J2= 7.74Hz).

– 139 – Chapter 3: Experimental Part

13 C-NMR (75 MHz, CDCl3): 21.56 (q), 37.44 (t), 38.05 (d), 64.54 (t), 67.55 (t), 120.14 (d), 123.35 (d), 137.84 (d), 156.27 (s), 161.28 (s), 171.69 (s). EI-MS m/z (%): 323 (M+) (3), 280 (15), 264 (70), 250 (30), 220 (60), 204 (100).

N OH C10H15NO3 OH OH 197.23 2-(6-Hydroxymethyl-pyridin-2-ylmethyl)-propane-1,3-diol (53) Colorless oil. Very viscous and hygroscopic.

A solution of 52 (90 mg, 0.28 mmol) and triethylamine (2 drops, cat.) in MeOH (20 ml) was refluxed for 24 h. The cooled down mixture was evaporated under reduced pressure and high vacuum to give quantitatively the title compound (55 mg).

1 H-NMR (300 MHz, CD3OD): 2.07 (m, 1H), 2.83 (d, 2H, J= 7.17Hz), 3.56 (d, 4H, J= 5.52Hz), 4.67 (s, 2H), 7.21 (d, 1H J= 7.71Hz), 7.37 (d, 1H, J= 7.92Hz), 7.76 (dd, 1H, J1= 7.71Hz, J2= 7.74Hz). 13 C-NMR (75 MHz, CD3OD): 37.25 (t), 45.30 (d), 63.15 (t), 65.48 (t), 119.40 (d), 123.64 (d), 139.02 (d), 155.75 (s), 161.05 (s).

O C6H11BrO Br 179.06 6-Bromo-hexanal (54) Colorless oil

A solution of 6-bromo-hexan-1-ol (1060 mg, 5.86 mmol) and pyridinium chlorochromate (PCC, 1900 mg, 8.78 mmol) in CH2Cl2 (15 ml) was cooled down to 0 ºC and stirred for 3 h at this temperature. The reaction crude was concentrated under reduced pressure until a final volume of 6 ml. Et2O (20 ml) was added, the mixture was filtered with Celite and concentrated under reduced pressure. The residue was purified by column chromatography (elution with 5% Et2O:Hexane) and the product containing fractions (Rf =

– 140 – Chapter 3: Experimental Part

0.50) were combined and the solvent was removed to give the title compound (495 mg, 47%) as a colorless oil. Spectroscopic data is in accordance with the literature127.

Rf (5% Et2O/Hex.): 0.50 1 H-NMR (300 MHz, CDCl3): 1.46-1.52 (m, 2H), 1.62-1.70 (m, 2H), 1.84-1.94 (m, 2H), 2.48 (dt, 2H, J1= 1.47Hz, J2= 7.17Hz), 3.42 (t, 2H, J= 6.62Hz), 9.78 (d, 1H, J= 1.47Hz). 13 C-NMR (75 MHz, CDCl3): 21.17 (t), 27.64 (t), 32.38 (t), 32.59 (t), 43.65 (t), 202.14 (d).

HO O N

O O C18H28BrNO4 Br 402.33 6-Bromo-1-[6-(5-methoxy-2,2-dimethyl-[1,3]dioxan-5-yl)-pyridin-2-yl]-hexan-1-ol (55) Pale yellow oil.

A solution of 32 (92 mg, 0.3 mmol) in dry CH2Cl2 (5 ml) was cooled down to –78 ºC under inert atmosphere. n-Butyllitium (0.2 ml, 0.33 mmol) was carefully added. After stirring for 15 min., the aldehyde 54 (70 mg, 0.39 mmol) was added to the mixture. The reaction was stirred for 3 h below –70 ºC and then was left to warm up to –40 ºC. The reaction mixture was quenched with H2O (25 ml) and the required product was extracted with CH2Cl2 (2x 30 ml), dried with MgSO4 and concentrated under reduced pressure. The residue was purified by column chromatography (elution with 30% EtOAc:Hexane), the product containing fractions (Rf = 0.19) were combined and the solvent was removed to give the title compound (31 mg, 26%) as a pale yellow oil.

Rf (30% EtOAc/Hex.): 0.19 1 H-NMR (300 MHz, CDCl3): 1.49-1.54 (m, 4H), 1.53 (s, 3H), 1.55 (s, 3H), 1.61-1.69 (m, 1H), 1.86-1.90 (m, 3H), 3.31 (s, 3H), 3.41 (t, 2H, J= 6.78Hz), 4.02 (d, 2H, J=12.69Hz), 4.43 (d, 2H, J=12.69Hz), 4.75 (dd, 1H, J1= 3.96Hz, J2= 7.62Hz), 7.18 (d. 1H, J= 7.71Hz) 7.50 (d, 1H, J= 7.71Hz), 7.75 (dd, 1H, J1= 7.71Hz, J2= 7.71Hz). EI-MS m/z (%): 402 (M+) (1), 387 (1), 313 (40), 165 (100).

– 141 – Chapter 3: Experimental Part

HO O N

OH OH

C15H24BrNO4 Br 362.26 2-[6-(6-Bromo-1-hydroxy-hexyl)-pyridin-2-yl]-2-methoxy-propane-1,3-diol (56) Colorless oil

A mixture of 55 (28 mg, 0.07 mmol) in THF (1.5 ml) and HCl 1.2 M (1.5 ml) were stirred at RT for 18 h. The pH of the solution was taken to neutrality with the addition of

NaOH (2M). The neutral aqueous phase was washed with Et2O (2x 10 ml) and dried under reduced pressure. The crude was redissolved in 10% MeOH:CH2Cl2 in order to filter out the formed NaCl salts. Evaporation of the solvents under reduced pressure afforded the title compound in a pure form and in quantitative yield.

1 H-NMR (300 MHz, CD3OD): 1.43-1.47 (m, 4H), 1.74-1.86 (m, 4H), 3.28 (s, 3H), 3.43 (t, 2H, J= 6.78Hz), 3.93-4.09 (m, 4H), 4.69 (t, 1H, J= 5.16Hz), 7.38 (d. 1H, J= 7.71Hz) 7.45 (d, 1H, J= 7.71Hz), 7.80 (dd, 1H, J1= 7.71Hz, J2= 7.71Hz). 13 C-NMR (75 MHz, CD3OD): 24.46 (t), 28.07 (t), 32.68 (t), 33.76 (t), 38.34 (t), 51.71 (q), 64.99 (t), 65.04 (t), 72.12 (d), 98.08 (s), 119.34 (d), 120.09 (d), 137.74 (d), 158.07 (s), 161.33 (s).

N O O O O S S O O

C21H21NO6S2 447.52 Toluene-4-sulfonic acid 6-toluene-4-sulfonyloxymethyl-pyridin-2-ylmethyl ester (57) White powder.

A solution of 2,6-pyridinedimethanol (695 mg, 5 mmol) and KOH (806 mg, 14.4 mmol) in THF (20 ml) was taken to 0 ºC. Tosyl chloride (2.18 g, 14.4 mmol) was added

– 142 – Chapter 3: Experimental Part carefully and the mixture stirred at this temperature for 5 h and 14 h more at RT. The crude was filtered and concentrated under reduced pressure to afford a white solid. Crystalization with THF:Hexane afforded the title compound in 92% yield (2056 mg). The spectroscopic data is in agreement with the literature128.

1 H-NMR (300 MHz, CDCl3): 2.45 (s, 6H), 5.06 (s, 4H), 7.34 (m, 6H), 7.70 (t, 1H, J= 7.53Hz), 7.81 (d, 4H, J= 8.28Hz). 13 C-NMR (75 MHz, CDCl3): 21.64 (q), 71.27 (t), 121.36 (d), 128.05 (d), 129.91 (d), 132.76 (s), 137.85 (d), 145.13 (s), 153.54 (s).

N C7H9NS2 SH SH 171.28 (6-Mercaptomethyl-pyridin-2-yl)-methanethiol (58) Pale yellow oil.

The mixture of 43 (1 g, 3.8 mmol) and thiourea (575 mg, 7.6 mmol) in EtOH (15 ml) was heated to reflux for 5 h, after which the solution was evaporated to dryness under reduced pressure to give a white precipitate of solid thiouronium salt. This salt was treated with a solution of NaHCO3 (530 mg, 7.6 mmol) in H2O (25 ml) to give a clear solution and was refluxed for 45 min under N2 pressure. After cooling, the crude was extracted with CH2Cl2

(3x 30 ml), washed with brine (30 ml), dried with MgSO4 and evaporated under reduced pressure to afford the title compound in 96% yield (624 mg)99.

1 H-NMR (300 MHz, CDCl3): 2.05 (t, 2H, J= 7.71Hz), 3.84 (d, 4H, J= 7.71Hz), 7.23 (d, 2H, J= 7.74Hz), 7.65 (t, 1H, J= 7.74Hz).

– 143 – Chapter 3: Experimental Part

N O O O O

O O C17H21NO8 O O 367.36 3-[6-(2,2-Bis-methoxycarbonyl-ethyl)-pyridin-2-yl]-2-methoxycarbonyl- propionic acid methyl ester (59) Colorless oil

A mixture of 43 (3.67 g, 13.8 mmol), dimethyl malonate (7.286 g, 55.2 mmol) and potassium carbonate (10 g, 72.5 mmol) in DMF (40 ml) was stirred at RT for 48 h. The reaction mixture was filtered and the residue was thoroughly washed with CH2Cl2. The combined organic extract was concentrated under reduced pressure to afford a thick yellow oil (6.23 g), which was purified by column chromatography on silica gel (eluted with 40 % EtOAc:Hex) to give the title compound in 93% yield (5.43 g). The spectroscopic data is in agreement with the literature129.

1 H-NMR (300 MHz, CDCl3): 3.35 (d, 4H, J= 7.53Hz), 3.74 (s, 12H), 4.16 (t, 2H, J= 7.53Hz), 7.02 (d, 2H, J= 7.71Hz), 7.49 (t, 1H, J= 7.71Hz). 13 C-NMR (75 MHz, CDCl3): 36.15 (t), 50.28 (d), 52.51 (q), 121.17 (d), 136.70 (d), 156.81 (s), 169.73 (s). EI-MS m/z (%): 367 (M+) (25), 336 (35), 308 (75), 244 (100), 216 (50), 158 (55), 59 (90).

N

C13H21NO4 OH OH OH OH 255.31 3-[6-(3-Hydroxy-2-hydroxymethyl-propyl)-pyridin-2-yl]- 2-hydroxymethyl-propan-1-ol (60) Colorless oil. Very viscous and hygroscopic.

A solution of 61 (80 mg, 0.19 mmol) and triethylamine (2 drops, cat.) in MeOH (10 ml) was refluxed for 66 h. The cooled down mixture was evaporated under reduced pressure and high vacuum to give quantitatively the title compound (48 mg).

– 144 – Chapter 3: Experimental Part

1 H-NMR (300 MHz, CD3OD): 2.09 (m, 2H), 2.82 (d, 4H, J= 7.17Hz), 3.56 (d, 8H, J= 5.52Hz), 7.15 (d, 2H J= 7.71Hz), 7.65 (t, 1H, J= 7.71Hz). 13 C-NMR (75 MHz, CD3OD): 36.74 (t), 43.00 (d), 64.37 (t), 121.59 (d), 137.85 (d), 159.07 (s). ESI-MS(–) m/z (%): 254 (M–) (60), 138 (100).

N

O O O O O O O O C21H29NO8 423.46 Acetic acid 3-[6-(3-acetoxy-2-acetoxymethyl-propyl)-pyridin-2-yl]- 2-acetoxymethyl-propyl ester (61) Colorless oil

A solution of compound 59 (183.5 mg, 0.5 mmol) in THF (5 ml) was cooled down to

0 ºC. LiAlH4 (5 ml, 1M solution in THF) was added carefully and the mixture stirred at RT for 24 h. The reaction was quenched with the addition of H2O (20 ml) and neutralized with

HCl 1M. The removal of H2O under reduced pressure and high vacuum afforded the tetraalcohol ready for acetylation. The very well dried crude from the previous step was dissolved in pyridine (20 ml) and cooled down to 0 ºC. A catalytic amount (c.a. 5 mg) of dimethyl-pyridin-4-yl-amine (DMAP) was added to the mixture followed by the careful addition of acetic anhydride (10 ml). The reaction stirred at RT for 20 h. Quenching with H20 (50 ml) and extraction with

CH2Cl2 (2x 50 ml) afforded the title product (199 mg) in 94 % yield over two steps.

1 H-NMR (300 MHz, CDCl3): 2.06 (s, 12H), 2.65 (m, 2H), 2.84 (d, 4H, J= 7.35Hz), 4.02- 4.15 (m, 8H), 6.98 (d, 2H, J= 7.53Hz), 7.52 (t, 1H, J= 7.53Hz). 13 C-NMR (75 MHz, CDCl3): 20.82 (q), 36.79 (t), 37.40 (d), 63.92 (t), 123.74 (d), 136.78 (d), 158.66 (s), 170.91 (s). FAB-MS (matrix: DTT/DTE) m/z (%): 324 (MH+) (15), 391 (45), 167 (40), 149 (100), 113 (65).

– 145 – Chapter 3: Experimental Part

3.3. Class II Aldolase System

3.3.1. The N5 and N3O Ligands

2+ 2+ 2+ 3.3.1.1. N5 complexation with Co , Cu and Ni , and direct aldol catalytic studies

Pyridin-2-ylmethyl-(6-{[(pyridin-2-ylmethyl)-amino]-methyl}-pyridin-2-ylmethyl)- amine (62) (N5 Ligand): The compound was synthesized and kindly provided by Christian Dubs109.

Complex synthesis: A solution of 62 (150 mg, 0.47 mmol) and MX2 (0.47 mmol) in MeOH (10 ml) was refluxed for 4 h. After cooling down, the mixture was concentrated under reduced pressure until a volume of 2 ml and left at –18 ºC overnight. The precipitate formed was filtered out and re-crystallized with MeOH/Et2O affording the desired complexes in 41- 82% yields range. The crystals were used directly in the catalytic studies. M stands for Co2+, 2+ 2+ – – Cu and Ni , and X stands for Cl and CH3COO .

2+ 2+ 2+ 3.3.1.2. N3O complexation with Co , Cu and Ni , and direct aldol catalytic studies

(6-{[(Pyridin-2-ylmethyl)-amino]-methyl}-pyridin-2-yl)-methanol (63) (N3O Ligand): The compound was synthesized109 and kindly provided by Christian Dubs.

Complex synthesis: A solution of 63 (150 mg, 0.66 mmol) and MX2 (0.66 mmol) in MeOH (15 ml) was refluxed for 1 h. After cooling down, the mixture was concentrated under reduced pressure until a volume of 2 ml and left at –18 ºC over 3 nights. Precipitation was achieved with the addition of a few drops of Et2O. Filtration and re-crystallization with

MeOH/Et2O afforded the desired complexes in 40-80% yields range. The crystals were used directly in the catalytic studies. M stands for Co2+, Cu2+ and Ni2+, and X stands for Cl– and – CH3COO .

– 146 – Chapter 3: Experimental Part

3.3.1.3. Direct Aldol Catalytic Test:

A solution of 2-hydroxy-1-phenyl-ethanone (34 mg, 0.25 mmol), benzaldehyde (106 mg, 1 mmol) and metal catalyst (0.05 mmol) in MeOH (5 ml) was stirred at RT for 48 h. The solvent was removed under reduced pressure and a sample (5 mg) of the mixture was dissolved in CDCl3 (1 ml). The solution was passed through a small silica gel column (3 cm of silica in a Pasteur pipette) and used directly in 1H-NMR measurements.

3.3.1.4. X-Ray Section

Single-crystals from some complexes were obtained and characterized by X-ray crystallography. The structures from complexes N5-NiCl2, N5-CoCl2 and N3O-CoCl2 were determined by Prof. Helen Stoeckli-Evans at the BENEFRI crystallography service in

Neuchâtel. The complex N3O-ZnCl2 was achieved in the University of Graz by Prof. C. Kratky.

N5-NiCl2 complex

+ Figure 101. Cationic fragment [Ni(Ligand)Cl] in structure N5-NiCl2.

– 147 – Chapter 3: Experimental Part

Figure 102. Fragment of molecular packing in crystal compound N5-NiCl2 with 1D hydrogen bonding along crystallographic axes y.

Table 20. Crystal data table for N5-NiCl2. Methanol Solvate ______

Identification code N5-NiCl2

Crystal shape plate

Crystal colour blue

Crystal size 0.35 x 0.30 x 0.07 mm

Empirical formula C20 H25 Cl2 N5 Ni O

[(C19 H21 N5)NiCl]Cl . CH3OH

Formula weight 481.06

Crystal system Orthorhombic

Space group P 21/c

Unit cell dimensions a = 8.8320(8) A alpha = 90 deg. b = 9.1807(6) A beta = 95.035(11) deg. c = 26.635(2) A gamma = 90 deg.

Volume 2151.3(3) A^3

Cell refinement parameters Reflections 8000 (plus equivalents) Angle range 2.31 < theta < 25.92

Z 4

Density (calculated) 1.485 g/cm^3

Radiation used MoK\a

– 148 – Chapter 3: Experimental Part

Wavelength 0.71073 A

Linear absorption coefficient 1.172 mm^-1

Temperature 153(2) K ______

Table 21. Data Collection Details for N5-NiCl2. ______

Diffractometer STOE IPDS

Scan method phi oscillation

Number of Reflections measured 14378

Number of Independent reflections 4022

Number of observed reflections 2912

Criterion for recognizing I>2sigma(I)

R(int) = 0.0877

Theta range for data collection 2.31 to 25.92 deg.

Index ranges -10<=h<=10, -10<=k<=10, -32<=l<=32

Number of standards 0

Intensity variation 0 %

F(000) 1000 ______

Table 22. Refinement Details for N5-NiCl2. ______

Refinement method Full-matrix least-squares on F^2

Final R indices [I>2sigma(I)] R1 = 0.0399, wR2 = 0.0957

R indices (all data) R1 = 0.0577, wR2 = 0.1005

R1 [=SUM(||Fo|-|Fc||)/SUM|Fo|]

wR^2 {=[SUM(w(Fo^2-Fc^2)^2)/SUM(wFo^4)]^1/2}

H-locating and refining Method mixed

Number of reflections used 4022

Number of L.S. restraints 0

Number of refined Parameters 362

Goodness-of-fit on F^2 0.918 S {=[SUM w(Fo^2-Fc^2)^2]/(n-p)^1/2}, n= number of reflections, p= Parameters used. calc w=1/[\s^2^(Fo^2^)+(0.0585P)^2^] where P=(Fo^2^+2Fc^2^)/3

Maximum delta/sigma 0.001

Maximum e-density 0.699 e.A^-3

Minimun e-density -0.993 e.A^-3 ______

– 149 – Chapter 3: Experimental Part

Table 23. Computer Programs used for N5-NiCl2. ______

130 Data collection program EXPOSE

130 Cell refinement program CELL

130 Data reduction program INTEGRATE

131 Structure Solving Program SHELXS-97

132 Structure Refinement Program SHELXL-97

133 Pictures drawn with PLATON99

132 Tables made with SHELXL-97

______

Table 24. Atomic coordinates ( x 10^4) and equivalent isotropic displacement parameters

(A^2 x 10^3) for N5-NiCl2. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor.

______

x y z U(eq) ______

Ni(1) 10227(1) 3095(1) 1640(1) 16(1) Cl(1) 11474(1) 2342(1) 2434(1) 23(1) N(1) 8101(3) 2556(3) 1897(1) 19(1) N(2) 9837(3) 1050(3) 1273(1) 22(1) N(3) 9240(3) 3778(3) 969(1) 21(1) N(4) 10275(3) 5395(3) 1766(1) 21(1) N(5) 12357(3) 3616(3) 1380(1) 18(1) C(1) 7161(3) 3445(4) 2127(1) 22(1) C(2) 5731(3) 2986(4) 2242(1) 26(1) C(3) 5244(3) 1607(4) 2105(1) 25(1) C(4) 6213(3) 689(4) 1873(1) 24(1) C(5) 7648(3) 1201(4) 1783(1) 19(1) C(6) 8811(4) 219(4) 1576(1) 22(1) C(7) 9204(4) 1243(4) 745(1) 31(1) C(8) 8883(3) 2808(4) 606(1) 23(1) C(9) 8277(3) 3249(5) 131(1) 32(1) C(10) 8092(4) 4726(5) 44(1) 38(1) C(11) 8499(4) 5721(5) 423(1) 31(1) C(12) 9062(3) 5209(4) 886(1) 23(1) C(13) 9474(4) 6179(4) 1333(1) 27(1) C(14) 11898(3) 5812(4) 1846(1) 24(1) C(15) 12840(3) 4956(4) 1499(1) 19(1) C(16) 14170(3) 5527(4) 1332(1) 28(1) C(17) 15034(3) 4651(4) 1049(1) 30(1) C(18) 14561(4) 3256(4) 937(1) 29(1) C(19) 13210(3) 2771(4) 1104(1) 22(1) O(1) 5878(4) 8929(5) 726(1) 74(1) C(20) 5597(5) 8329(6) 248(2) 47(1) Cl(2) 2539(1) 8943(1) 1005(1) 39(1) ______

– 150 – Chapter 3: Experimental Part

Table 25. Bond lengths [A] and angles [deg] for N5-NiCl2. ______

Ni(1)-N(3) 2.019(2) Ni(1)-N(1) 2.112(2) Ni(1)-N(5) 2.116(2) Ni(1)-N(2) 2.132(3) Ni(1)-N(4) 2.138(3) Ni(1)-Cl(1) 2.3978(8) N(1)-C(5) 1.333(4) N(1)-C(1) 1.349(4) N(2)-C(7) 1.477(4) N(2)-C(6) 1.479(4) N(2)-H(2N) 0.86(4) N(3)-C(8) 1.332(4) N(3)-C(12) 1.339(4) N(4)-C(14) 1.481(4) N(4)-C(13) 1.486(4) N(4)-H(4N) 0.96(3) N(5)-C(15) 1.331(4) N(5)-C(19) 1.345(4) C(1)-C(2) 1.392(4) C(1)-H(1) 0.98(4) C(2)-C(3) 1.376(5) C(2)-H(2) 1.02(4) C(3)-C(4) 1.385(5) C(3)-H(3) 0.86(4) C(4)-C(5) 1.392(4) C(4)-H(4) 0.98(4) C(5)-C(6) 1.507(4) C(6)-H(6A) 0.86(3) C(6)-H(6B) 0.97(3) C(7)-C(8) 1.505(5) C(7)-H(7A) 0.90(4) C(7)-H(7B) 0.95(4) C(8)-C(9) 1.391(5) C(9)-C(10) 1.383(6) C(9)-H(9) 1.01(4) C(10)-C(11) 1.385(6) C(10)-H(10) 0.90(4) C(11)-C(12) 1.373(5) C(11)-H(11) 0.92(4) C(12)-C(13) 1.506(5) C(13)-H(13A) 0.91(4) C(13)-H(13B) 0.94(4) C(14)-C(15) 1.517(4) C(14)-H(14A) 1.00(3) C(14)-H(14B) 0.97(4) C(15)-C(16) 1.394(4) C(16)-C(17) 1.378(5) C(16)-H(16) 0.99(4) C(17)-C(18) 1.372(5) C(17)-H(17) 0.96(5) C(18)-C(19) 1.383(5) C(18)-H(18) 1.03(4) C(19)-H(19) 0.95(4) O(1)-C(20) 1.388(5) O(1)-H(1O) 0.82(6) C(20)-H(20A) 1.06(4) C(20)-H(20B) 0.98(5) C(20)-H(20C) 1.05(6)

N(3)-Ni(1)-N(1) 91.82(9) N(3)-Ni(1)-N(5) 88.09(9) N(1)-Ni(1)-N(5) 179.43(11) N(3)-Ni(1)-N(2) 80.03(11) N(1)-Ni(1)-N(2) 80.18(10) N(5)-Ni(1)-N(2) 99.25(10) N(3)-Ni(1)-N(4) 80.40(11) N(1)-Ni(1)-N(4) 100.77(10) N(5)-Ni(1)-N(4) 79.77(10) N(2)-Ni(1)-N(4) 160.42(10) N(3)-Ni(1)-Cl(1) 177.96(7) N(1)-Ni(1)-Cl(1) 90.00(7) N(5)-Ni(1)-Cl(1) 90.09(7)

– 151 – Chapter 3: Experimental Part

N(2)-Ni(1)-Cl(1) 101.19(8) N(4)-Ni(1)-Cl(1) 98.37(7) C(5)-N(1)-C(1) 118.9(3) C(5)-N(1)-Ni(1) 113.77(19) C(1)-N(1)-Ni(1) 127.2(2) C(7)-N(2)-C(6) 112.5(3) C(7)-N(2)-Ni(1) 111.4(2) C(6)-N(2)-Ni(1) 106.60(19) C(7)-N(2)-H(2N) 106(2) C(6)-N(2)-H(2N) 103(2) Ni(1)-N(2)-H(2N) 117(2) C(8)-N(3)-C(12) 121.3(3) C(8)-N(3)-Ni(1) 119.4(2)

C(12)-N(3)-Ni(1) 119.1(2) C(14)-N(4)-C(13) 112.0(3) C(14)-N(4)-Ni(1) 106.4(2) C(13)-N(4)-Ni(1) 110.8(2) C(14)-N(4)-H(4N) 104.9(19) C(13)-N(4)-H(4N) 111(2) Ni(1)-N(4)-H(4N) 112(2) C(15)-N(5)-C(19) 118.7(3) C(15)-N(5)-Ni(1) 113.98(19) C(19)-N(5)-Ni(1) 127.3(2) N(1)-C(1)-C(2) 121.6(3) N(1)-C(1)-H(1) 117.1(19) C(2)-C(1)-H(1) 121.2(19) C(3)-C(2)-C(1) 119.2(3) C(3)-C(2)-H(2) 124(2) C(1)-C(2)-H(2) 117(2) C(2)-C(3)-C(4) 119.2(3) C(2)-C(3)-H(3) 123(3) C(4)-C(3)-H(3) 117(3) C(3)-C(4)-C(5) 118.6(3) C(3)-C(4)-H(4) 120(2) C(5)-C(4)-H(4) 121(2) N(1)-C(5)-C(4) 122.4(3) N(1)-C(5)-C(6) 116.2(3) C(4)-C(5)-C(6) 121.4(3) N(2)-C(6)-C(5) 111.1(3) N(2)-C(6)-H(6A) 113(2) C(5)-C(6)-H(6A) 109(2) N(2)-C(6)-H(6B) 109.8(19) C(5)-C(6)-H(6B) 106.0(19) H(6A)-C(6)-H(6B) 108(3) N(2)-C(7)-C(8) 113.3(3) N(2)-C(7)-H(7A) 111(2) C(8)-C(7)-H(7A) 109(3) N(2)-C(7)-H(7B) 107(2) C(8)-C(7)-H(7B) 110(3) H(7A)-C(7)-H(7B) 106(4) N(3)-C(8)-C(9) 120.9(3) N(3)-C(8)-C(7) 115.6(3) C(9)-C(8)-C(7) 123.4(3) C(10)-C(9)-C(8) 117.9(3) C(10)-C(9)-H(9) 131(2) C(8)-C(9)-H(9) 112(2) C(9)-C(10)-C(11) 120.4(4) C(9)-C(10)-H(10) 120(3) C(11)-C(10)-H(10) 119(3) C(12)-C(11)-C(10) 118.6(4) C(12)-C(11)-H(11) 118(2) C(10)-C(11)-H(11) 124(2) N(3)-C(12)-C(11) 120.9(3) N(3)-C(12)-C(13) 115.6(3) C(11)-C(12)-C(13) 123.4(3) N(4)-C(13)-C(12) 113.1(3) N(4)-C(13)-H(13A) 108(2) C(12)-C(13)-H(13A) 108(2) N(4)-C(13)-H(13B) 113(2) C(12)-C(13)-H(13B) 108(2) H(13A)-C(13)-H(13B) 106(3) N(4)-C(14)-C(15) 110.8(3) N(4)-C(14)-H(14A) 104.2(16) C(15)-C(14)-H(14A) 110.0(17) N(4)-C(14)-H(14B) 115.9(18) C(15)-C(14)-H(14B) 111.7(18)

– 152 – Chapter 3: Experimental Part

H(14A)-C(14)-H(14B) 104(2) N(5)-C(15)-C(16) 122.2(3) N(5)-C(15)-C(14) 116.3(3) C(16)-C(15)-C(14) 121.5(3) C(17)-C(16)-C(15) 118.5(4) C(17)-C(16)-H(16) 124.3(18) C(15)-C(16)-H(16) 117.1(18) C(18)-C(17)-C(16) 119.4(3) C(18)-C(17)-H(17) 122(3) C(16)-C(17)-H(17) 118(3) C(17)-C(18)-C(19) 119.1(3) C(17)-C(18)-H(18) 122(2) C(19)-C(18)-H(18) 119(2) N(5)-C(19)-C(18) 122.0(3) N(5)-C(19)-H(19) 118(2) C(18)-C(19)-H(19) 119(2) C(20)-O(1)-H(1O) 113(4) O(1)-C(20)-H(20A) 109(2) O(1)-C(20)-H(20B) 114(3) H(20A)-C(20)-H(20B) 99(3) O(1)-C(20)-H(20C) 114(3) H(20A)-C(20)-H(20C) 104(4) H(20B)-C(20)-H(20C) 116(4) ______

Table 26. Torsion-angles for N5-NiCl2. ______

N(3)-Ni(1)-N(1)-C(5) 88.6(2) N(5)-Ni(1)-N(1)-C(5) 8(11) N(2)-Ni(1)-N(1)-C(5) 9.1(2) N(4)-Ni(1)-N(1)-C(5) 169.2(2) Cl(1)-Ni(1)-N(1)-C(5) -92.3(2) N(3)-Ni(1)-N(1)-C(1) -87.1(3) N(5)-Ni(1)-N(1)-C(1) -168(100) N(2)-Ni(1)-N(1)-C(1) -166.7(3) N(4)-Ni(1)-N(1)-C(1) -6.5(3) Cl(1)-Ni(1)-N(1)-C(1) 92.0(2) N(3)-Ni(1)-N(2)-C(7) 4.2(2) N(1)-Ni(1)-N(2)-C(7) 97.8(2) N(5)-Ni(1)-N(2)-C(7) -82.2(2) N(4)-Ni(1)-N(2)-C(7) 3.3(4) Cl(1)-Ni(1)-N(2)-C(7) -174.1(2) N(3)-Ni(1)-N(2)-C(6) -118.9(2) N(1)-Ni(1)-N(2)-C(6) -25.3(2) N(5)-Ni(1)-N(2)-C(6) 154.7(2) N(4)-Ni(1)-N(2)-C(6) -119.8(3) Cl(1)-Ni(1)-N(2)-C(6) 62.8(2) N(1)-Ni(1)-N(3)-C(8) -83.5(2) N(5)-Ni(1)-N(3)-C(8) 95.9(2) N(2)-Ni(1)-N(3)-C(8) -3.8(2) N(4)-Ni(1)-N(3)-C(8) 175.9(2) Cl(1)-Ni(1)-N(3)-C(8) 123(2) N(1)-Ni(1)-N(3)-C(12) 101.4(2) N(5)-Ni(1)-N(3)-C(12) -79.1(2) N(2)-Ni(1)-N(3)-C(12) -178.9(2) N(4)-Ni(1)-N(3)-C(12) 0.8(2) Cl(1)-Ni(1)-N(3)-C(12) -52(2) N(3)-Ni(1)-N(4)-C(14) -117.1(2) N(1)-Ni(1)-N(4)-C(14) 152.83(19) N(5)-Ni(1)-N(4)-C(14) -27.35(19) N(2)-Ni(1)-N(4)-C(14) -116.2(3) Cl(1)-Ni(1)-N(4)-C(14) 61.22(19) N(3)-Ni(1)-N(4)-C(13) 4.91(19) N(1)-Ni(1)-N(4)-C(13) -85.1(2) N(5)-Ni(1)-N(4)-C(13) 94.7(2) N(2)-Ni(1)-N(4)-C(13) 5.8(4) Cl(1)-Ni(1)-N(4)-C(13) -176.73(18) N(3)-Ni(1)-N(5)-C(15) 92.6(2) N(1)-Ni(1)-N(5)-C(15) 174(100) N(2)-Ni(1)-N(5)-C(15) 172.2(2) N(4)-Ni(1)-N(5)-C(15) 12.1(2)

– 153 – Chapter 3: Experimental Part

Cl(1)-Ni(1)-N(5)-C(15) -86.4(2) N(3)-Ni(1)-N(5)-C(19) -85.3(3) N(1)-Ni(1)-N(5)-C(19) -4(11) N(2)-Ni(1)-N(5)-C(19) -5.7(3) N(4)-Ni(1)-N(5)-C(19) -165.8(3) Cl(1)-Ni(1)-N(5)-C(19) 95.7(2) C(5)-N(1)-C(1)-C(2) -0.7(4) Ni(1)-N(1)-C(1)-C(2) 174.9(2) N(1)-C(1)-C(2)-C(3) -2.0(5) C(1)-C(2)-C(3)-C(4) 2.5(5) C(2)-C(3)-C(4)-C(5) -0.5(5) C(1)-N(1)-C(5)-C(4) 2.9(4) Ni(1)-N(1)-C(5)-C(4) -173.3(2) C(1)-N(1)-C(5)-C(6) -173.9(3) Ni(1)-N(1)-C(5)-C(6) 10.0(3) C(3)-C(4)-C(5)-N(1) -2.3(5) C(3)-C(4)-C(5)-C(6) 174.3(3) C(7)-N(2)-C(6)-C(5) -85.0(3) Ni(1)-N(2)-C(6)-C(5) 37.4(3) N(1)-C(5)-C(6)-N(2) -32.9(4) C(4)-C(5)-C(6)-N(2) 150.3(3) C(6)-N(2)-C(7)-C(8) 115.4(3) Ni(1)-N(2)-C(7)-C(8) -4.2(3) C(12)-N(3)-C(8)-C(9) -1.5(4) Ni(1)-N(3)-C(8)-C(9) -176.5(2) C(12)-N(3)-C(8)-C(7) 177.4(3) Ni(1)-N(3)-C(8)-C(7) 2.5(3) N(2)-C(7)-C(8)-N(3) 1.4(4) N(2)-C(7)-C(8)-C(9) -179.7(3) N(3)-C(8)-C(9)-C(10) 1.5(4) C(7)-C(8)-C(9)-C(10) -177.4(3) C(8)-C(9)-C(10)-C(11) -0.1(5) C(9)-C(10)-C(11)-C(12) -1.2(5) C(8)-N(3)-C(12)-C(11) 0.2(4) Ni(1)-N(3)-C(12)-C(11) 175.2(2) C(8)-N(3)-C(12)-C(13) 178.5(3) Ni(1)-N(3)-C(12)-C(13) -6.5(3) C(10)-C(11)-C(12)-N(3) 1.1(4) C(10)-C(11)-C(12)-C(13) -177.0(3) C(14)-N(4)-C(13)-C(12) 109.3(3) Ni(1)-N(4)-C(13)-C(12) -9.4(3) N(3)-C(12)-C(13)-N(4) 10.5(4) C(11)-C(12)-C(13)-N(4) -171.2(3) C(13)-N(4)-C(14)-C(15) -83.0(3) Ni(1)-N(4)-C(14)-C(15) 38.3(3) C(19)-N(5)-C(15)-C(16) 2.3(4) Ni(1)-N(5)-C(15)-C(16) -175.8(2) C(19)-N(5)-C(15)-C(14) -175.2(3) Ni(1)-N(5)-C(15)-C(14) 6.7(3) N(4)-C(14)-C(15)-N(5) -31.3(4) N(4)-C(14)-C(15)-C(16) 151.1(3) N(5)-C(15)-C(16)-C(17) -2.3(5) C(14)-C(15)-C(16)-C(17) 175.1(3) C(15)-C(16)-C(17)-C(18) 0.5(5) C(16)-C(17)-C(18)-C(19) 1.1(5) C(15)-N(5)-C(19)-C(18) -0.6(4) Ni(1)-N(5)-C(19)-C(18) 177.2(2) C(17)-C(18)-C(19)-N(5) -1.1(5) ______

– 154 – Chapter 3: Experimental Part

Table 27. Anisotropic displacement parameters (A^2 x 10^3) for N5-NiCl2. The anisotropic displacement factor exponent takes the form: -2 pi^2 [ h^2 a*^2 U11 + ... + 2 h k a* b* U12 ]

______

U11 U22 U33 U23 U13 U12 ______

Ni(1) 12(1) 14(1) 24(1) 0(1) 3(1) 0(1) Cl(1) 18(1) 25(1) 27(1) 5(1) 3(1) 0(1) N(1) 14(1) 18(2) 25(1) 2(1) 3(1) 2(1) N(2) 17(1) 17(2) 32(1) -2(1) 7(1) 2(1) N(3) 11(1) 22(2) 29(1) 1(1) 4(1) 0(1) N(4) 15(1) 19(2) 29(1) -2(1) 7(1) -1(1) N(5) 14(1) 19(2) 22(1) 3(1) 3(1) 2(1) C(1) 21(2) 19(2) 26(2) 1(1) 4(1) 3(1) C(2) 19(2) 29(2) 30(2) 5(1) 5(1) 8(1) C(3) 11(1) 31(2) 32(2) 8(1) 2(1) 0(1) C(4) 19(2) 24(2) 28(2) 3(1) 2(1) -4(1) C(5) 17(1) 17(2) 23(2) 3(1) 2(1) 0(1) C(6) 19(2) 15(2) 33(2) -2(1) 7(1) -2(1) C(7) 37(2) 26(2) 31(2) -6(2) 4(2) -7(2) C(8) 11(1) 32(2) 26(2) -3(1) 3(1) -4(1) C(9) 16(2) 51(3) 28(2) -2(2) 2(1) -6(2) C(10) 21(2) 58(3) 34(2) 17(2) 1(2) 4(2) C(11) 19(2) 32(3) 43(2) 13(2) 5(1) 5(1) C(12) 10(1) 21(2) 37(2) 5(1) 7(1) 3(1) C(13) 22(2) 15(2) 44(2) 4(2) 6(1) 4(1) C(14) 20(2) 19(2) 34(2) -5(1) 4(1) -7(1) C(15) 14(1) 19(2) 24(2) 3(1) -1(1) -1(1)

C(16) 15(2) 26(2) 42(2) 7(2) 0(1) -4(1) C(17) 15(2) 39(3) 36(2) 12(2) 7(1) 0(1) C(18) 20(2) 38(3) 29(2) 4(2) 6(1) 9(2) C(19) 21(2) 22(2) 23(2) 1(1) 4(1) 3(1) O(1) 31(2) 137(4) 55(2) -22(2) 3(2) -21(2) C(20) 32(2) 56(4) 51(2) -2(2) 6(2) 7(2) Cl(2) 37(1) 22(1) 63(1) -4(1) 22(1) 5(1) ______

Table 28. Hydrogen coordinates ( x 10^4) and isotropic displacement parameters (A^2 x

10^3) for N5-NiCl2.

______

x y z U(eq) ______

H(2N) 10600(40) 480(40) 1260(13) 30(10) H(4N) 9860(40) 5650(40) 2074(13) 24(9) H(1) 7540(40) 4410(40) 2221(12) 23(9) H(2) 5100(40) 3700(50) 2428(13) 38(10) H(3) 4410(50) 1240(50) 2193(15) 51(12) H(4) 5890(40) -310(40) 1775(12) 29(9) H(6A) 8340(30) -470(40) 1409(11) 15(8) H(6B) 9390(40) -200(40) 1867(12) 22(8) H(7A) 9830(50) 850(50) 529(14) 41(11) H(7B) 8290(50) 680(50) 703(15) 49(12) H(9) 8070(40) 2390(50) -104(14) 39(11) H(10) 7760(40) 5050(50) -266(15) 40(11) H(11) 8440(40) 6720(50) 378(12) 27(10) H(13A) 8600(40) 6560(40) 1437(12) 30(10) H(13B) 10030(40) 6980(40) 1226(12) 30(9) H(14A) 12200(30) 5540(30) 2206(11) 10(7) H(14B) 12110(30) 6840(40) 1834(11) 14(8)

– 155 – Chapter 3: Experimental Part

H(16) 14440(40) 6540(40) 1436(12) 25(9) H(17) 15890(50) 5090(60) 905(17) 66(14) H(18) 15150(40) 2590(40) 716(14) 36(10) H(19) 12930(40) 1780(40) 1055(12) 25(9) H(1O) 5110(70) 9010(70) 880(20) 100(20) H(20A) 4590(50) 8790(50) 71(15) 46(11) H(20B) 6310(50) 8650(50) 8(17) 60(13) H(20C) 5370(60) 7210(70) 250(20) 90(20) ______

Table 29. Hydrogen-bonds for N5-NiCl2 [A and deg.]. ______

D-H...A d(D-H) d(H...A) d(D...A) <(DHA)

N(2)-H(2N)...Cl(2)#1 0.86(4) 2.36(4) 3.201(3) 165(3) N(4)-H(4N)...Cl(1)#2 0.96(3) 2.41(4) 3.270(3) 150(3) O(1)-H(1O)...Cl(2) 0.82(6) 2.33(6) 3.105(3) 158(5) ______

Symmetry transformations used to generate equivalent atoms: #1 x+1,y-1,z #2 -x+2,y+1/2,-z+1/2

N5-CoCl2 complex

The intensity data of a dark-green crystal of N5-CoCl2, III II {[Co LCl]2[Co Cl4](Cl)2(H2O)} were collected at 153K on a Stoe Image Plate Diffraction System130 using MoKa graphite monochromated radiation, image plate distance 70mm, f oscillation scans 0 – 180°, step Df = 1.0°, 2q range 2.90– 48.4°, dmax -dmin = 12.45 - 0.81 Å. The structure was solved by direct methods and refined by least-squares techniques in the anisotropic approximation for all non hydrogen atoms by using the program SHELXS-97131 and SHELXL-97132. All hydrogen atoms were localized in difference Fourier map but placed in calculated position with Uiso = 1.2 Ueq of supporting atoms. Hydrogen atoms of solvate water molecule were calculated with Uiso = 1.5 Ueq (O1) and fixed position parameters in final refinement. 2- The cobalt atom of anion CoCl4 occupies a twofold axes and the water molecule is located next to an inversion center (Figure 103).

– 156 – Chapter 3: Experimental Part

Figure 103. Molecular structure and crystallographic numbering scheme (PLATON133 drawing).

133 Figure 104. Cationic fragment [Co(L)Cl]2+ (50% probability ellipsoids) (PLATON drawing).

– 157 – Chapter 3: Experimental Part

N3O-CoCl2 complex

III II The intensity data of a dark green-gray crystal of [Co L2][Co Cl4]Cl (N3O-CoCl2) were collected at 153K on a Stoe Image Plate Diffraction System130 using MoKa graphite monochromated radiation. Image plate distance 70mm, f oscillation scans 0 – 162.5°, step Df

= 1.3°, 2q range 1.93– 25.94, dmax -dmin = 12.45 - 0.81 Å. The structure was solved by direct methods and refined by least-squares techniques in the anisotropic approximation for all non- hydrogen atoms by using the programs SHELXS-97131 and SHELXL-97132. All hydrogen atoms were localized in difference Fourier map. In the final refinement cycle, the NH and OH hydrogen atoms were refined isotropically and the CH hydrogen were placed in calculated position with Uiso = 1.2 Ueq of supporting atoms as ‘riding’ model. III II In complex [Co L2][Co Cl4]Cl, the cobalt(III) ion is coordinated to two ligand molecules, one involving all four donor atoms (L-) and the second one involving only two donor atoms (L+), the non-coordinated pyridine ring is protonated and the OH group 2- unchanged. In addition, there are a [CoCl4] complex anion and a chloride anion in the asymmetric unit (Figure 105).

133 Figure 105. Molecular structure of N3O-CoCl2 (50% probability ellipsoids) (PLATON drawing).

– 158 – Chapter 3: Experimental Part

133 Figure 106. N3O-CoCl2 molecular packing (PLATON drawing).

– 159 – Chapter 3: Experimental Part

133 Figure 107. N3O-CoCl2 hydrogen bonding network (PLATON drawing).

N3O-ZnCl2 complex

109 The N3O-ZnCl2 complex was prepared and kindly provided by Christian Dubs . Attempts to crystallize the complex always resulted in very small crystals. Nevertheless, the X-ray structure determination was achieved (Figure 108, Table 30).

– 160 – Chapter 3: Experimental Part

Figure 108. ORTEP of refined structure of the N3O-ZnCl2 complex.

Table 30. List of important parameters of the data collection and the refinement.

Crystal data N3O-ZnCl2 complex

Molecular formula C13H15N3OCl2Zn Molecular weight 365.6

Crystal shape prisms Crystal dimensions [mm] 0.2 x 0.1 x 0.1 l [Å] 0.801 Radiation source beamline X13, DESY (Hamburg) Detector MarCCD (MarResearch) T [K] 100° Xtal-detector dist. [mm] 59 Images 40

– 161 – Chapter 3: Experimental Part

Df 5°

Space group P21/n a=7.651, b=13.793, c=13.743 Cell parameters [Å,°] b=98.37 V [Å3] 1434.8 Z 4 3 rc [g/cm ] 1.692 F(000) 744 µ [mm-1] 2.081

Resolution [Å] 20.0-0.90 (0.92-0.90) Measured reflections 16016 Unique reflections 2150 Completeness 99.2% (100%)

I/sI 70.8 (66.5)

Rsym 0.036 (0.059)

I>2sI 98.6% (97.5%)

NRefl 2041

NPar 183

NRestr 0

R1 0.0448 (0.0448 for all data) wR2 0.1156 S 1.128 dr -0.78/0.66

– 162 – Chapter 3: Experimental Part

3.3.2. Chiral N3O Ligand

3.3.2.1. Compound Characterization

Br Br N Br C6H5Br2N C6H4Br3N Br N 250.92 Br 329.82 2-Bromo-6-bromomethyl-pyridine (64) 2-Bromo-6-dibromomethyl-pyridine (65) White powder. White powder.

A solution of 2-bromo-6-methylpyridine (500 mg, 2.9 mmol), NBS (570 mg, 3.2 mmol) and AIBN (28 mg, 0.2 mmol) in benzene (40 ml) was refluxed for 6 h under Ar and irradiation (W lamp: 150 Watt). After cooling and separation of succinimide by filtration, the solvent was removed. The oily residue containing two compounds was purified by chromatography on silica gel. Elution with (30%) CH2Cl2/Hex afforded compound 65 (Rf= 0.37) in 13% yield (124 mg) and compound 64 in 47% yield (340 mg). The spectroscopic data is in agreement with the literature134.

Compound 64:

Rf (30% CH2Cl2/Hex.): 0.26 1 H-NMR (300 MHz, CDCl3): 4.51 (s, 2H), 7.43 (d, 2H, J= 7.56Hz), 7.57 (t, 1H, J= 7.56Hz). 13 C-NMR (75 MHz, CDCl3): 32.43 (t), 122.40 (d), 127.49 (d), 139.29 (d), 141.50 (s), 158.05 (s).

Compound 65:

Rf (30% CH2Cl2/Hex.): 0.37 1 H-NMR (300 MHz, CDCl3): 6.59 (s, 1H), 7.45 (d, 1H, J= 7.89Hz), 7.67 (dd, 1H, J1= 7.89Hz, J2= 7.71Hz), 7.83 (d, 1H, J= 7.71Hz). 13 C-NMR (75 MHz, CDCl3): 39.54 (d), 121.18 (d), 128.68 (d), 139.80 (d), 140.06 (s), 159.97 (s).

– 163 – Chapter 3: Experimental Part

O NH N S

O C13H14N2O2S 262.33 4-Methyl-N-pyridin-2-ylmethyl-benzenesulfonamide (66) White crystals

To a solution of picolamine (8 g, 74 mmol) and NaOH (4.4 g, 110 mmol) in H2O (20 ml), was added a solution of tosylchloride (14.12 g, 74 mmol) in Et2O (14 ml). The reaction stirred for 18 h and the ethereal phase was removed under reduced pressure. The pH of the aqueous phase was adjusted to neutrality with the addition of 1 M HCl solution. The formed white solid was collected by filtration to afford the title compound in 89% yield (17.36 g). The spectroscopic data is in agreement with the literature113.

1 H-NMR (300 MHz, CDCl3): 2.39 (s, 3H), 4.23 (d, 2H, J= 5.49), 5.90 (t, br, 1H), 7.14-7.19 (m, 2H), 7.23-7.26 (m, 2H), 7.61 (dd, 1H, J1= 7.25Hz, J2= 7.25Hz), 7.74 (d, 2H, J= 7.71Hz), 8.45 (d, 1H, J= 4.77Hz). 13 C-NMR (75 MHz, CDCl3): 22.17 (q), 48.05 (t), 122.65 (d), 123.29 (d), 127.91 (s), 130.80 (d), 137.52 (d), 144.04 (s), 149.57 (d), 155.47 (s).

O S O N Br N

N C19H18BrN3O2S 432.33 N-(6-Bromo-pyridin-2-ylmethyl)-4-methyl-N-pyridin- 2-ylmethyl-benzenesulfonamide (67) White powder

A solution of 66 (334 mg, 1.27 mmol) in CH2Cl2 (40 ml) was added to a solution of

(n-Bu)4NBr (409 mg, 1.27 mmol) and KOH (3.34 g, 60 mmol) in H2O (10 ml), and the two-

– 164 – Chapter 3: Experimental Part phase system was taken to reflux. The compound 64 (320 mg, 1.27 mmol) was added to the reaction and it refluxed for 16 h. After cooling, the organic phase was separated, dried with

MgSO4 and concentrated under reduced pressure to leave a brownish oil. The residue was purified by column chromatography (elution with 50% EtOAc:Hexane) and the product containing fractions (Rf = 0.52) were combined and the solvent was removed to give the title compound in 72% yield (392 mg).

Rf (50% EtOAc./Hex.): 0.52 1 H-NMR (300 MHz, CDCl3): 2.44 (s, 3H), 4.54 (s, 2H), 4.57 (s, 2H), 7.12 (dd, 1H, J1= 5.25Hz, J2= 7.08Hz), 7.24-7.30 (m, 4H), 7.35-7.42 (m, 2H), 7.58 (ddd, 1H, J1= 1.76Hz, J2= 7.64Hz, J3= 7.64Hz), 7.72 (d, 2H, J= 8.28Hz), 8.40 (d, 1H, J= 4.95Hz). 13 C-NMR (75 MHz, CDCl3): 21.53 (q), 53.25 (t), 54.08 (t), 121.30 (d), 122.60 (d), 123.08 (d), 126.62 (d), 127.40 (d), 129.70 (d), 136.20 (d), 136.88 (s), 138.69 (d), 141.19 (s), 143.63 (s), 148.64 (s), 156.03 (s), 157.98 (s).

NH Br N

N C12H12BrN3 278.15 (6-Bromo-pyridin-2-ylmethyl)-pyridin-2-ylmethyl-amine (68) Beige powder.

Compound 67 (200 mg, 0.46 mmol) was dissolved in 95% H2SO4 (2 ml) and heated to 120 ºC for 3 h. The darkened crude was poured into a solution of brine (10 ml) and 2 M

NaOH (10 ml), extracted with CH2Cl2 (3x 30 ml), dried with MgSO4 and evaporated under reduced pressure to give the title compound pure in 92% yield (111 mg)

1 H-NMR (300 MHz, CDCl3): 3.97 (s, 2H), 3.97 (s, 2H), 7.18 (dd, 1H, J1= 5.33Hz, J2= 6.98Hz), 7.33-7.38 (m, 3H), 7.52 (dd, 1H, J1= 7.62Hz, J2= 7.62Hz), 7.66 (ddd, 1H, J1= 1.83Hz, J2= 7.62Hz, J3= 7.62Hz), 8.57 (d, 1H, J= 4.77Hz). 13 C-NMR (75 MHz, CDCl3): 53.49 (t), 53.78 (t), 121.33 (d), 122.46 (d), 126.77 (d), 136.77 (d), 139.04 (d), 149.34 (d).

– 165 – Chapter 3: Experimental Part

O C11H20O 168.28

2-Isopropyl-5-methyl-cyclohexanecarbaldehyde (69) Colorless oil

This compound was synthesized following a literature procedure135. A suspension of methoxymethyl-chloro-triphenyl-l5-phosphane (4456 mg, 13 mmol) in freshly distilled THF (40 ml) was cooled down to 0 ºC before n-BuLi (11.4 ml, 18.2 mmol) was carefully added. The dark red solution stirred at 0 ºC for 30 min and (–)-menthone (2 g, 13 mmol) was added over 10 min. The mixture was left to warm slowly to RT and, after 18 h of continuous stirring, the reaction was quenched with the addition of HCl (5 ml). Concentration under reduced pressure resulted in precipitation of Ph3PO crystals, which were filtered off. The reaction crude was re-dissolved in CH2Cl2 (15 ml) with 36% HCl (2.2 ml) and the mixture stirred at RT for 4 h. The solvent was removed and H2O (100 ml) was added. Extraction with

Et2O (2x 100 ml), drying with MgSO4 and concentration under reduced pressure (no high vacuum) afforded a mixture of products. The mixture was purified by column chromatography (elution with 2% EtOAc:Hex) and the product containing fractions (Rf = 0.50) were combined and the solvent was removed to give the title compound (1286 mg, 59%) as a colorless oil. Spectroscopic data is in agreement with the literature135.

[a]D (in CH2Cl2 at 20 ºC): -24.7 º Rf (2% EtOAc/Hex.): 0.50 1 H-NMR (300 MHz, CDCl3): 0.80 (d, 3H, J= 6.99Hz), 0.93 (d, 6H, J= 6.99Hz), 1.01-1.09 (m, 2H), 1.19-1.24 (m, 2H), 1.51-1.58 (m, 1H), 1.65-1.75 (m, 4H), 2.19-2.28 (m, 1H), 9.50 (d, 1H, J= 4.77Hz). 13 C-NMR (75 MHz, CDCl3): 15.26 (q), 21.06 (q), 22.40 (q), 24.00 (t), 29.65 (d), 31.48 (d), 34.44 (t), 34.97 (t), 42.60 (d), 53.56 (d), 205.51 (d).

– 166 – Chapter 3: Experimental Part

N Br N

N C13H14BrN3 292.18 (6-Bromo-pyridin-2-ylmethyl)-methyl-pyridin-2-ylmethyl-amine (70) Colorless oil

A solution of 68 (95 mg, 0.36 mmol), Na2CO3 (717.5 mg, 6.8 mmol) and MeI (23 ml,

0.37 mmol) in CH3CN (10 ml) stirred under argon at RT for 3 days. The Na2CO3 was removed by paper filtration and the solvent was evaporated under reduced pressure. The resulting oil stirred for 1 h in Na2CO3 saturated aqueous solution. An extraction with CH2Cl2 afforded a mixture of compounds. The residue was purified by column chromatography (elution with 50% EtOAc:Hexane) and the product containing fractions (Rf = 0.25) were combined and the solvent was removed to give the title compound in 22% yield.

Rf (50% EtOAc./Hex.): 0.25 1 H-NMR (300 MHz, CDCl3): 2.41 (s, 3H), 3.85 (s, br, 4H), 7.38-7.41 (m, 2H), 7.53-7.58 (m, 5H). 13 C-NMR (75 MHz, CDCl3): 43.09 (q), 62.94 (t), 122.95 (d), 127.52 (d), 129.48 (d), 131.61 (d), 139.50 (d), 139.67 (d), 142.14 (s).

O Br N

N C12H11BrN2O 279.14 2-Bromo-6-(pyridin-2-ylmethoxymethyl)-pyridine (71) White powder

A mixture of pyridin-2-yl-methanol (131 mg, 1.2 mmol) and NaH (31 mg, 1.3 mmol) in dry THF (8 ml) refluxed for 15 min. The compound 64 (251 mg, 1 mmol) was added to the mixture and it was refluxed for 1 h. The reaction was quenched with the addition of H2O (20

– 167 – Chapter 3: Experimental Part

ml), extracted with CH2Cl2 (2x 20 ml), dried with MgSO4 and evaporated under reduced pressure to afford a mixture of compounds. The residue was purified by column chromatography (elution with 20% EtOAc:Hexane) and the product containing fractions (Rf = 0.10) were combined and the solvent was removed to give the title compound in 70% yield (195 mg).

Rf (20% EtOAc./Hex.): 0.10 1 H-NMR (300 MHz, CDCl3): 4.76 (s, 2H), 4.80 (s, 2H), 7.22-7.27 (m, 1H), 7.40 (d, 1H, J= 7.71Hz), 7.51-7.61 (m, 3H), 7.75 (dd, 1H, J1= 7.73Hz, J2= 7.73Hz), 8.59 (d, 1H, J= 4.59Hz). FAB-MS (matrix: DTT/DTE) m/z (%): 279-281 (MH+) (100), 230 (35).

3.3.3. Zinc-Amino Acid Complexes

3.3.3.1. Synthesis

Zn(a.a.)2 complex: The zinc-amino acid complexes were prepared by adding Et3N (0.6 ml, 4.34 mmol) to the amino acid (4.34 mmol) in MeOH (10 ml), followed, after 10 min, by zinc acetate (477 mg, 2.17 mmol). After stirring for 45 min., the white precipitate was collected by filtration (23-95% yields range). Complexes were characterized by 1H- and 13C- NMR and ESI-MS.

Cu(L-Pro)2 complex: The copper-proline complex was synthesized the same way as the zinc complexes. The complex was obtained in 20% yield and was characterized by ESI- MS.

Zn(L-Pro)2 complex (Alternative Method): L-proline (500 mg, 4.34 mmol) was dissolved in DMSO (20 ml) and diethyl zinc (1.97 ml, 2.17 mmol) was added to the solution. After stirring for 14 h, methanol (20 ml) was added to the milky suspension and the white precipitate was collected by filtration (542 mg, 85% yield). The complex showed identical 1H- NMR and ESI-MS to the one synthesized previously.

– 168 – Chapter 3: Experimental Part

3.3.3.2. Compound Characterization

O

O NH Zn2+ HN O C10H16N2O4Zn 293.63 O

L-Pro2Zn complex (72) White powder

1 H-NMR (300 MHz, D2O): 1.81 (m, br, 3H), 2.19-2.24 (m, br, 1H), 2.98 (m, br, 1H), 3.12- 3.18 (m, br, 1H), 3.85 (m, br, 1H). 13 C-NMR (75 MHz, CD3OD): 25.52 (t), 25.74 (t), 26.00 (t), 26.21 (t), 30.21 (t), 47.60 (t), 61.39 (d), 175.09 (s).

ESI-MS(+) m/z (%): 877, 879, 881, 883, 885 (L-Pro6Zn3, 16) (corresponding to the 3 zinc isotopes), 762, 764, 766, 768, 770 (L-Pro5Zn3, 10), 700, 702, 704, 706 (L-Pro5Zn2, 40), 585, 64 66 68 587, 589, 591 (L-Pro4Zn2, 55), 408 (L-Pro3 Zn, 70), 410 (L-Pro3 Zn, 40), 412 (L-Pro3 Zn, 64 66 68 25), 293 (L-Pro2 Zn, 100), 295 (L-Pro2 Zn, 60), 297 (L-Pro2 Zn, 40), 231 (L-Pro2, 40).

L-Lys2Zn complex (73) MW: 355.5 Brownish powder

64 66 68 ESI-MS(+) m/z (%): 355 (L-Lys2 Zn, 30), 357 (L-Lys2 Zn, 18), 359 (L-Lys2 Zn, 12),

293 (L-Lys2, 25), 147 (L-Lys, 100).

L-Arg2Zn complex (74) MW: 411.5 Brownish powder

– 169 – Chapter 3: Experimental Part

64 66 68 ESI-MS(+) m/z (%): 411 (L-Arg2 Zn, 18), 413 (L-Arg2 Zn, 10), 415 (L-Arg2 Zn, 6), 175 (L-Arg, 100).

L-Ile2Zn complex (75) MW: 325.5 White powder

64 ESI-MS(+) m/z (%): 518, 520, 522, 524 (L-Ile3Zn2, 25), 325 (L-Ile2 Zn, 32), 327 (L-Ile2 66 68 Zn, 18), 329 (L-Ile2 Zn, 12), 263 (L-Ile2, 60), 132 (L-Ile, 100).

L-t-Leu2Zn complex (76) MW: 325.5 White powder

64 66 68 ESI-MS(+) m/z (%): 456 (L-t-Leu3 Zn, 8), 458 (L-t-Leu3 Zn, 5), 460 (L-t-Leu3 Zn, 3), 64 66 68 325 (L-t-Leu2 Zn, 8), 327 (L-t-Leu2 Zn, 5), 329 (L-t-Leu2 Zn, 3), 263 (L-t-Leu2, 40), 132 (L-t-Leu, 100).

L-His2Zn complex (77) MW: 373.5 White powder

64 ESI-MS(+) m/z (%): 590, 592, 594, 596 (L-His3Zn2, 40), 373 (L-His2 Zn, 100), 375 (L-His2 66 68 Zn, 60), 377 (L-His2 Zn, 40), 311 (L-His2, 5), 156 (L-His, 90).

L-Ser2Zn complex (78) MW: 373.5 White powder

– 170 – Chapter 3: Experimental Part

64 ESI-MS(+) m/z (%): 440, 442, 444, 446 (L-Ser3Zn2, 35), 378 (L-Ser3 Zn, 35), 380 (L-Ser3 66 68 64 66 Zn, 22), 382 (L-Ser3 Zn, 15), 273 (L-Ser2 Zn, 100), 275 (L-Ser3 Zn, 60), 277 (L-Ser2 68Zn, 40).

L-Glu2Zn complex (79) MW: 357.5 White powder

64 66 68 ESI-MS(+) m/z (%): 357 (L-Glu2 Zn, 35), 359 (L-Glu2 Zn, 22), 361 (L-Glu2 Zn, 15), 322, 324, 326, 328 (?, 100).

L-Cys2Zn complex (80) MW: 305.5 White powder

64 ESI-MS(+) m/z (%): 467, 469, 471, 473 (L-Cys2Zn2, 30), 305 (L-Cys2 Zn, 30), 307 (L-Cys3 66 68 Zn, 20), 309 (L-Cys2 Zn, 12), 138 (?, 100).

O OH

C10H11NO4 NO2 209.20 4-Hydroxy-4-(4-nitro-phenyl)-butan-2-one (81)

1 H-NMR (300 MHz, CDCl3): (Please see section 3.5.)

– 171 – Chapter 3: Experimental Part

O OH

C10H12O2 164.20

4-Hydroxy-4-phenyl-butan-2-one (82) White solid

1 H-NMR (300 MHz, CDCl3): 2.20 (s, 3H), 2.84 (d, 1H, J= 3.66Hz), 2.88 (d, 1H, J= 8.46Hz), 3.27 (s, br, 1H), 5.16 (dd, 1H, J1= 3.69Hz, J2= 8.82Hz), 7.36-7.42 (m, 5H).

O OH

OH C10H11NO5 NO2 225.20 3,4-Dihydroxy-4-(4-nitro-phenyl)-butan-2-one (83) Pale yellow solid.

Syn diastereoisomer:

1 H-NMR (300 MHz, CDCl3): 2.37 (s, 3H), 4.42 (dd, 1H, J1= 2.58Hz, J2= 4.41Hz), 5.23 (dd, 1H, J1= 2.58Hz, J2= 7.91Hz), 7.62 (d, 2H, J= 8.82Hz), 8.25 (d, 2H, J= 8.46Hz).

Anti diastereoisomer:

1 H-NMR (300 MHz, CDCl3): 2.03 (s, 3H), 4.48 (dd, 1H, J1= 4.79Hz, J2= 4.79Hz), 5.10 (dd, 1H, J1= 4.35Hz, J2= 4.35Hz), 7.62 (d, 2H, J= 8.82Hz), 8.25 (d, 2H, J= 8.46Hz).

– 172 – Chapter 3: Experimental Part

O OH

C H O OH 14 14 3 230.26 3,4-Dihydroxy-4-naphthalen-2-yl-butan-2-one (84) White solid

Syn diastereoisomer:

1 H-NMR (300 MHz, CDCl3): 2.27 (s, 3H), 2.85 (d, 1H, J= 6.99Hz), 3.72 (d, 1H, J= 3.30Hz), 4.50 (dd, 1H, J1= 3.69Hz, J2= 4.41Hz), 5.19 (dd, 1H, J1= 3.30Hz, J2= 3.30Hz), 7.49-7.54 (m, 3H), 7.84-7.90 (m, 4H).

Anti diastereoisomer:

1 H-NMR (300 MHz, CDCl3): 1.97 (s, 3H), 2.94 (d, 1H, J= 3.66Hz), 3.70 (d, 1H, J= 3.30Hz), 4.59 (dd, 1H, J1= 4.41Hz, J2= 4.77Hz), 5.19 (dd, 1H, J1= 3.30Hz, J2= 3.30Hz), 7.49-7.54 (m, 3H), 7.84-7.90 (m, 4H).

3.3.3.3. Catalytic Studies

Reaction of acetone with p-nitrobenzaldehyde (PNB): A solution of PNB (15.1 mg,

0.1 mmol) and catalyst (5 mol %, 0.005 mmol) in acetone:H2O (1.5 ml) was stirred at RT for 24-96 h. A sample of the mixture (0.5 ml) was evaporated to dryness under reduced pressure.

The resulting solid was suspended in CDCl3 (0.7 ml) and filtered with cotton and MgSO4 to afford a solution of remaining PNB and the aldol product ready for 1H-NMR analysis. Reaction of acetone with benzaldehyde: A solution of freshly distilled benzaldehyde

(53 mg, 0.5 mmol) and (L-Pro)2Zn (7.3 mg, 0.025 mmol) in 50% acetone:H2O (2.5 ml) was stirred at RT for 48 h. The mixture was evaporated to dryness under reduced pressure. Under these conditions, both the starting materials and the solvent were evaporated. The yield (49%) was determined by subtracting the catalyst weight from the weight obtained for the solid mixture (87.5 mg). A sample of the resulting mixture (5 mg) was suspended in CDCl3 (0.7

– 173 – Chapter 3: Experimental Part

ml) and filtered with cotton and MgSO4 to afford a solution of pure aldol product ready for 1H-NMR analysis. Reaction of hydroxyacetone with PNB: A solution of PNB (15.1 mg, 0.1 mmol) and

(L-Pro)2Zn (2.9 mg, 0.01 mmol) in 33% H2O/hydroxyacetone (1.5 ml) or just hydroxyacetone (1 ml) was stirred at RT for 3 h. The mixture was evaporated to dryness under reduced pressure. A sample of the resulting crude (c.a. 5 mg) was suspended in CDCl3 (0.7 ml) and 1 filtered with cotton and MgSO4 to afford a solution ready for H-NMR analysis. Compound 83 (syn and anti) was purified by flash chromatography (eluting with 20-50% gradient of EtOAc/Hex). Reaction of hydroxyacetone with Naphtaldehyde: A solution of naphtaldehyde (31.2 mg, 0.2 mmol) and catalyst (20%, 0.04 mmol) in 80% DMSO/hydroxyacetone (2.5 ml) or just hydroxyacetone (1 ml) was stirred at RT for 20 h. The mixture was evaporated to dryness under reduced pressure. Compound 84 (syn and anti) was purified by flash chromatography (eluting with 50% EtOAc/Hex). Reaction of cyclohexanone or butan-2-one with p-nitrobenzaldehyde (PNB): A solution of PNB (15.1 mg, 0.1 mmol) and L-proline (10 mol %, 0.01 mmol) or (L-Pro)2Zn (5 mol %, 0.005 mmol) in 33% H2O/ketone (1.5 ml) was stirred at RT for 24-96 h. A sample of the mixture (0.5 ml) was evaporated to dryness under reduced pressure. The resulting solid was suspended in CDCl3 (0.7 ml) and filtered with cotton and MgSO4 to afford a solution of remaining PNB and the aldol product ready for 1H-NMR analysis.

3.3.3.4. Chiral HPLC measurements

4-Hydroxy-4-(4-nitro-phenyl)-butan-2-one: The crude aldol product (1 mg) was dissolved in 5% EtOH:Hex (1 ml) with the help of an Eppendorf centrifuge. The sample was injected (20 mL) in the HPLC running isocratic conditions of 5% EtOH:Hex (freshly prepared, water-free solvents) at a constant flow rate of 1.0 mL/min. The R enantiomer had a retention time of 39.3 min, whereas the S enantiomer had a retention time of 43.2 min.

– 174 – Chapter 3: Experimental Part

3.3.3.5. Kinetic Study

Reactions were run under pseudo first-order conditions with deuterated acetone (0.5 ml) in excess over PNB (7.6 mg, 0.05 mmol). (L-Pro)2Zn (0.8 mg, 2.5 mmol) was used as a catalyst (5%) and the reaction was performed in D2O (0.25 ml). The reaction was monitored by 1H-NMR spectroscopy for 10 h at 23 ± 0.3 °C. Typical 1H-NMR parameters included 4 scans per spectrum, 40 s relaxation delay between scans and a total of 64 scans. The total time of data collection was 10 h after which, 14% conversion was observed. Pseudo-first order rate constant (k’) was obtained from the plot of the natural logarithm of PNB concentration against time (Figure 88):

-k’t -6 -1 -4 -1 [PNB]t = [PNB]0 e k’ = 4.2´10 s (2.5´10 min )

– 175 – Chapter 3: Experimental Part

3.4. Prebiotic Sugar Synthesis

Sugar synthesis: A solution of hydroxy-acetaldehyde (60 mg, 1 mmol) and Zn-amino acid complex (15%, 0.15 mmol) in H2O (5 ml) was stirred for 7 days at the desired temperature. The solvent was removed by lyophilization and crude mixture was analyzed by 1H-NMR.

Equilibration of the reference sugars: The commercially available sugars (5 mg) stirred in H2O (2 ml) for 24 h at RT. The solvent was removed under reduced pressure and the 1 solid mixture was dissolved in D2O (0.6 ml) for H-NMR spectroscopy measurements at 300 MHz.

Sugar crude acetylation: A solution of the sugar mixture prepared above (60 mg),

DMAP (10 mg, cat.) and acetic anhydride (2 ml) in pyridine (2 ml) was stirred at RT under N2 pressure for 24 h. The reaction was quenched with H2O (20 ml), extracted with CH2Cl2 (3x 30 ml) and evaporated under reduced pressure to afford the acetylated mixture (100 mg). The mixture was analyzed by 1H-NMR and GC-MS.

Separation of tetroses and hexoses from the sugar mixture: The mixture was purified by column chromatography (elution with 50% EtOAc:Hex), the first fractions (Rf = 0.77– 0.57) were combined and the solvent was removed to give mainly tetroses. The late fractions (Rf = 0.55–0.15) were combined and the solvent removed to give a mixture of tetroses and hexoses.

– 176 – Chapter 3: Experimental Part

3.5. Relevant Spectra

N S H N Zn N S NH C16H22N4S2Zn 399.88

1 Figure 109. H-NMR (CD3OD) of product 5.

OH N C8H9NOS2 S S 199.29

1 Figure 110. H-NMR (CDCl3) of product 21.

– 177 – Chapter 3: Experimental Part

O N C9H11NOS S 181.25

1 Figure 111. H-NMR (CDCl3) of product 30.

O O N C13H17NO2S2 S S 283.40

1 Figure 112. H-NMR (CDCl3) of product 37.

– 178 – Chapter 3: Experimental Part

N OH C10H15NO3 OH OH 197.23 Et3N

1 Figure 113. H-NMR (CD3OD) of product 53.

N

C13H21NO4 OH OH OH OH 255.31

1 Figure 114. H-NMR (CD3OD) of product 60.

– 179 – Chapter 3: Experimental Part

O OH

C10H11NO4 NO2 209.20

1 Figure 115. H-NMR (CDCl3) of the crude product 81 from the aldol reaction with 100% yield.

1 Figure 116. H-NMR (CDCl3) of the aldol product 81 together with p-nitro benzaldehyde. The NMR yield calculated is 45%

– 180 – Chapter 3: Experimental Part

1 Figure 117. H-NMR (D2O) of the complex L-Pro2Zn and L-Proline (box)

– 181 – Chapter 3: Experimental Part

1 Figure 118. H-NMR of the complex L-t-Leu2Zn.

1 Figure 119. H-NMR of the complex L-Ile2Zn.

– 182 – Chapter 3: Experimental Part

O

O NH Zn2+ HN O

O

Figure 120. ESI-MS of the complex L-Pro2Zn.

Figure 121. ESI-MS of the complex L-His2Zn

– 183 – Chapter 4: References

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125. Uneme, H., Mitsudera, H., Kamikado, T., Kono, Y., Manabe, Y., Numata, M. (1992) Biosci. Biotech. Biochem., 56, 2023. 126. Chen, D., Martell, A.E., McManus, D. (1995) Can. J. Chem., 73, 264. 127. Hon, Y.-S., Chang, F.-J., Lu, L., Lin, W.-C. (1998) Tetrahedron, 54, 5233. 128. Bradshaw, J.S., Huszthy, P., McDaniel, C.W., Zhu, C.Y., Dalley, N.K., Izatt, R.M., Lifson, S. (1990) J. Org. Chem., 55, 10, 3129. 129. Newkome, G.R., Kawato, T., Kohli, D.K., Puckett, W.E., Olivier, B.D., Chiari, G., Fronczek, F.R., Deutsch, W.A. (1981) J. Am. Chem. Soc., 103, 3423. 130. Stoe & Cie (2000) IPDS Software. Stoe & Cie GmbH, Darmstadt, Germany. 131. Sheldrick, G.M. (1990) Acta Crystallogr., A46, 467. 132. Sheldrick, G.M. (1999) "SHELXL-97", Universität Göttingen, Göttingen, Germany. 133. Spek, A.L. (1990) Acta Crystallogr., A46, C-34. 134. Graf, E. (1992) Synthesis, 519. 135. Spino, C., Beaulieu, C. (2000) Angew. Chem. Int. Ed., 39 (11), 1930.

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Chapter 5: Publications

5. Publications

Poster Sessions:

Machuqueiro, M., Darbre, T. (2001) "Models for Zn-dependent Methyltransferases", Spring Meeting SCS, Lausanne, Switzerland.

Machuqueiro, M., Darbre, T. (2001) "Methyl Transfer to Zinc Thiolates: A model for Zn - dependent methyltransferases”, 10th ICBIC, Florence, Italy.

Oral Presentations:

Machuqueiro, M., Darbre, T. (2001) "Methyl Transfer to Zinc Thiolates: A Model for Zn- dependent Methyltransferases”, Catalysis in Organic and Bioorganic Chemistry, Champéry, Switzerland.

Machuqueiro, M., Darbre, T. (2001) "Models for Zn-dependent Methyltransferases", Fall Meeting SCS, Zurich, Switzerland.

Machuqueiro, M., Darbre, T. (1999) “Models for Zn-dependent Methyltransferases: The Ada Protein Model”, Strain in Chemistry Meeting, Gregynog, U.K.

Abstracts:

Machuqueiro, M., Darbre, T. (2001) “Models for Zn-dependent methyl-transferases”, J. Inorg. Biochem., 86, 325.

Machuqueiro, M., Darbre, T. (2001) “Models for Zn-dependent methyl-transferases”, Chimia, 55, 128.

– 190 – Chapter 5: Publications

Journal Publications:

Machuqueiro, M., Darbre, T. (2003) “Prebiotic sugar synthesis: Synthesis of tetroses and hexoses from hydroxyacetaldehyde catalyzed by Zn-proline complex”, to be submitted.

Machuqueiro, M., Darbre, T. (2003) “Zn-Proline catalyzed direct Aldol reaction in aqueous medium”, Chem. Commun., 1090-1091.

Machuqueiro, M., Darbre, T. (2003) “Zinc mediated methyl transfer from trimethyl phosphate to chelating and non-chelating thiols. Model for Zn-dependent methyltransferases”, J. Inorg. Biochem., 94, 193.

– 191 – Chapter 5: Publications

– 192 – Chapter 5: Publications

– 193 – Chapter 5: Publications

– 194 – Chapter 5: Publications

– 195 – Chapter 5: Publications

– 196 – Chapter 5: Publications

– 197 – Chapter 6: Curriculum Vitae

6. Curriculum Vitae

Name Miguel Ângelo dos Santos Machuqueiro

Date and Place of Birth December the 27th, 1974, Pinhal Novo, Portugal

Marital Status Married

Nationality Portuguese

E-mail [email protected] 1979-1984: Primary school at Escola Primária da Lagoa da Palha, Pinhal Novo;

1984-1987: Preparatory school at Escola Preparatória de Pinhal Novo;

1987-1991: Secondary school at Escola Secundária de Pinhal Novo; Education 1991-1992: 12th grade at the Escola Secundária n° 1 de Setúbal;

1992-1998: Diploma in Biochemistry, University of Lisbon, July 1998, with a final grade of 15 (out of 20);

1999-2003: PhD work in the Department of Chemistry and Biochemistry of the University of Bern, under the supervision of Dr. Tamis Darbre;

– 198 –