Small‐Molecule Inhibitors of Dna Polymerase Function

SMALL‐MOLECULE INHIBITORS OF DNA POLYMERASE FUNCTION

DISSERTATION

zur Erlangung des Akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.)

vorgelegt von

Tobias Strittmatter aus Küssaberg‐Kadelburg

an der Universität Konstanz Mathematisch‐Naturwissenschaftliche Sektion Fachbereich Chemie

2014

Tag der mündlichen Prüfung: 05. Dezember 2014 Prüfungsvorsitz: Prof. Dr. Gerhard Müller 1. Referent: Prof. Dr. Andreas Marx 2. Referent: Prof. Dr. Thomas U. Mayer

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-274434

„Meinen Eltern”

Danksagung

Die vorliegende Arbeit entstand in der Zeit von November 2009 bis August 2014 in der Arbeitsgruppe von Prof. Dr. Andreas Marx am Lehrstuhl für Organische und Zelluläre Chemie am Fachbereich Chemie der Universität Konstanz.

Nach diesem Zeitraum geprägt von intensiver Forschungsarbeit liegt nun meine Dissertation vor und ein weiteres Kapitel meiner beruflichen Karriere kann nun erfolgreich abgeschlossen werden. Aus diesem Grund ist es jetzt auch an der Zeit, mich bei all denen zu bedanken, die mich während dieser spannenden Zeit begleitet, unterstützt und gefördert haben.

Meinem Doktorvater Prof. Dr. Andreas Marx danke ich ganz herzlich für die Überlassung der sehr interessanten und interdisziplinären Themenstellung, sowie für das in mich gesetzte Vertrauen, welches mir viel Raum für die selbstständige Bearbeitung und kreative Gestaltung des Themas erlaubte. Ihm, wie auch den weiteren Mitgliedern meines „Thesis Committee“, Prof. Dr. Thomas Mayer und Herrn Prof. Dr. Michael Berthold, danke ich zudem für die anregenden wissenschaftlichen Diskussionen und die geleisteten Hilfestellungen. Prof. Dr. Thomas Mayer danke ich außerdem für die Übernahme des Zweitgutachtens und Herrn Prof. Dr. Gerhard Müller für die Übernahme des Prüfungsvorsitzes.

Natürlich möchte ich mich auch bei allen früheren und jetzigen Freunden und Kollegen der Arbeitsgruppe Marx und der Graduiertenschule Chemische Biologie für die unvergessliche Zeit, die tolle Arbeitsatmosphäre und die Hilfsbereitschaft bedanken. Besonderer Dank gilt hier Dr. Norman Hardt, Magdalena Grzywa, Matthias Drum und Dr. Nina Blatter für die sehr gute, langjährige Zusammenarbeit und die tolle Laboratmosphäre. Zudem danke ich Dr. Karl‐ Heinz Jung für die interessanten wissenschaftlichen Fachgespräche. Weiterhin danke ich ganz herzlich meinen sehr talentierten Bachelorstudenten Annika Hantusch, Moritz Pott, Joos Aschenbrenner und Melina Hoffmann, sowie allen Mitarbeiterpraktikanten und wissenschaftlichen Hilfskräften für ihre Leistung, ihr Engagement und ihr Interesse.

Bei Dr. Thomas Huhn, Dr. Timo Immel und Malin Bein bedanke ich mich für die Durchführung der ersten Zytotoxizitätsmessungen. Bei Herrn Prof. Dr. Thomas Brunner und Anette Brockmann möchte ich mich besonders für die großartige Hilfe bei komplexen zellbiologischen Fragestellungen und die fruchtbare Zusammenarbeit bei der Durchführung der Zellassays bedanken.

Meinen Lektoren Dr. Norman Hardt, Karin Reichardt, Matthias Drum und Joachim Braun danke ich für die Durchsicht meiner schriftlichen Arbeit.

Meinen Freunden, meiner Familie und ganz besonders meinen Eltern danke ich für die liebevolle und bedingungslose Unterstützung in allen Lebenslagen.

Publikationen

Teile dieser Arbeit sind veröffentlicht in:

 T. Strittmatter, B. Bareth, T. A. Immel, T. Huhn, T. U. Mayer & A. Marx ”Small Molecule Inhibitors of Human DNA Polymerase λ” ACS Chem. Biol. 2011, 6 (4), 314 – 319

 T. Strittmatter, J. Aschenbrenner, N. Hardt & A. Marx ”Synthesis of 4′‐C‐alkylated‐5‐iodo‐2′‐deoxypyrimidine nucleosides” ARKIVOC 2013, (ii) Issue in Honor of Prof. Richard R. Schmidt, 46 – 59

 T. Strittmatter, A. Brockmann, M. Pott, A. Hantusch, T. Brunner & A. Marx ”Expanding the Scope of Human DNA Polymerase λ and β Inhibitors” ACS Chem. Biol. 2014, 9 (1), 282–290.

Weitere Publikationen:

 M. Catarinella, T. Grüner, T. Strittmatter, A. Marx & T. U. Mayer ”BTB‐1: A Small Molecule Inhibitor of the Mitotic Motor Protein Kif18A” Angew. Chem. Int. Ed. 2009, 48, 9072 – 9076 Angew. Chem. 2009, 121, 9236 – 9240

 B. Reichmann, M. Drexler, B. Weibert, N. Szesni, T. Strittmatter & H. Fischer ”Amino‐substituted Butatrienes: Unusual η1Ligands Formed by an Unusual Reaction” Organometallics 2011, 30 (5), 1215 – 1223

 O. B. Gutiérrez Acosta, N. Hardt, S. M. Hacker, T. Strittmatter, B. Schink & A. Marx ”TPP stimulates acetone activation by D. biacutus as monitored by a fluorogenic ATP analogue” ACS Chem. Biol. 2014, 9 (6), 1263 – 1266.

 A. Brockmann, T. Strittmatter, S. May, A. Marx & T. Brunner ”Structure‐function relationship of thiazolide‐induced apoptosis in colorectal tumor cells” ACS Chem. Biol. 2014, 9 (7), 1520 – 1527.

 J. Braun, M. M. Möckel, T. Strittmatter, A. Marx, U. Groth & T. U. Mayer ”Synthesis and Biological Evaluation of Optimized Inhibitors of the Mitotic Kinesin Kif18A” ACS Chem. Biol. (DOI: 10.1021/cb500789h), in press.

Table of contents

Table of Contents

Introduction...... 3 1) Chemical genetics...... 3 2) DNA polymerases ...... 5 2.1) General ...... 5 2.2) DNA polymerases and the polymerisation reaction...... 6 2.3) DNA polymerase λ and β ...... 8 2.4) Herpes virus DNA polymerase...... 11 3) DNA polymerases as drug targets ...... 12 3.1) General ...... 12 3.2) Screening methods for DNA polymerase inhibitors ...... 14

Concepts and objectives...... 20

Results and discussions ...... 22 4) Small‐molecule inhibitors of human pol λ and pol β...... 22 4.1) Introduction ...... 22 4.2) Biochemical evaluation of the 1st small‐molecule generation...... 24 4.2.1) Conclusion ...... 29 4.3) Establishment of a 2nd generation compound library of potential active molecules against pol λ and β...... 29 4.3.1) Design of the 2nd small‐molecule generation SM12, 29‐58 ...... 29 4.3.2) Synthesis of 2nd small‐molecule generation SM12, 29‐58 ...... 31 4.3.3) Conclusion ...... 34 4.4) Biochemical evaluation 2nd small‐molecule generation...... 35 4.4.1) Evaluation of the 2nd small‐molecule generation ...... 35 4.4.2) Side‐by‐side comparison of SM1 and SM49 with reported inhibitors...... 38 4.4.3) Discussion and structure‐activity relationships...... 39 4.4.4) Conclusion ...... 41 4.5) Cellular investigations of the 1st and 2nd small‐molecule generation ...... 42 4.5.1) Introduction...... 42 4.5.2) Cell viability measurements of potential rhodanine probes ...... 43 4.5.3) Co‐treatment experiments with genotoxic agents and probes SM1 or SM49...... 44 4.5.4) Conclusion ...... 47 5) Synthesis of 4’‐C‐alkylated‐5‐iodo‐2’‐deoxypyrimidine nucleosides as potential antiviral drugs and synthetic building blocks ...... 49 5.1) Introduction ...... 49 5.2) Synthesis of 4‐C‐modified carbohydrate building blocks ...... 52 5.3) Synthesis of 4’‐C‐alkylated‐pyrimidine nucleosides ...... 54 5.4) Synthesis of 4’‐C‐alkylated‐5‐iodo‐2’‐deoxypyrimidine nucleosides...... 54

1 Table of contents

5.5) Evaluation of the synthons ‐ exemplified by 3’,5’‐di‐O‐acetyl‐2’‐deoxy‐5‐iodo‐4’‐C‐ propyluridine N12c in a Sonagashira test‐reaction ...... 56 5.6) Discussion and Conclusion...... 57

Conclusion ...... 59

Zusammenfassung ...... 65

Materials and methods ...... 72 6) Chemistry (nucleosides)...... 72 6.1) General ...... 72 6.2) Synthesis of 4‐C‐modified carbohydrate building blocks ...... 73 6.3) Synthesis of 4`C‐alkylated‐pyrimidine nucleosides ...... 75 6.4) Synthesis of 4’‐C‐alkylated‐5‐iodo‐2’‐deoxypyrimidine nucleosides...... 80 6.5) Sonagashira test‐reaction...... 87 7) Chemistry (small‐molecules) ...... 90 7.1) General ...... 90 7.2) Synthesis of precursor aldehydes and acetophenon SM59‐84...... 90 7.3) Synthesis of small‐molecules SM1, 4, 10, 12, 16, 17, 21, 23, 27‐58...... 102 8) Molecular biological and biochemical methods ...... 123 8.1) Chemicals, reagents, inhibitors and solvents...... 123 8.2) Expression and purification of pol λ and pol β...... 123 8.2.1) Nucleotide‐ and amio acid sequences of pol β and pol λ ...... 124 8.3) SDS‐PAGE ...... 126 8.4) Nucleotides and oligonucleotides ...... 126 8.4.1) DNA oligonucletide purification ...... 126 8.4.2) 5’‐Radioactive labelling of DNA oligonucletides ...... 127 8.5) Radiometric primer extension...... 127 8.5.1) Pol λ PEX assay with variable small‐molecule concentrations...... 128 8.5.2) Pol λ PEX assay with variable dNTP concentrations ...... 128 8.5.3) Pol λ‐TdT assay with variable small‐molecule concentrations ...... 128 8.5.4) Pol β PEX assay with variable small‐molecule concentrations ...... 129 8.6) Polyacrylamide gel electrophoresis (PAGE)...... 129 8.6.1) Quantitative analysis of gel images ...... 129 9) Cell biological methods ...... 131 9.1) Cell cultivation...... 131 9.2) AllamarBlue assay...... 131 9.2) MTT assay ...... 132

References ...... 133

2 Introduction

Introduction

1) Chemical genetics

The illustrious quotation from the great chemist Marcelin Berthelot (*1827 ‐ †1907) “La chimie crée son objet. Cette faculté créatrice, semblable à celle de l'art lui‐même, la distingue essentiellement des sciences naturelles et historiques. Les derniers ont un objet donné d'avance et indépendent de la volonté et de l'action du savant.”1 expresses impressively the capability of a synthetic chemist to create and design molecules with novel molecular structures and, in consequence, novel properties and features.2,3 Because of the creative passion of synthetic chemists, chemistry has been assigned various enabling roles and several of its sister disciplines have grown “chemical” branches such as chemical genetics.2‐4

As result from the melting of organic chemistry with cell biology, Timothy J. Mitchison and Stuart L. Schreiber described for the first time the essential elements of the interdisciplinary chemical genetics approach at the end of the last century.4,5 The young discipline took up the cause of clarifying biological pathways, or rather the functions of genes and gene products, with the aid of small‐molecule probes (molecular weight < 900 g mol‐1)6.4,5,7‐11

Figure 1. Reverse chemical genetics ‐ gene or gene product (e.g. DNA polymerase) to phenotype. Figure was adapted from literature 9.

3 Introduction

In classical genetics, biological processes are elucidated in living cells by the induction of dysfunctions, and analysis of the resulting phenotypes of interest. This can be achieved by manipulation of the genetic information, antibodies, or RNA interference, for example.8‐11 Traditional forward genetics operates from phenotype to genotype and reverse genetics the other way round.8‐11 However, chemical genetics differs fundamentally from the classical methods and has several advantages:4,5,7‐11

 Small‐molecules probes show on cells a fast and strong effect.  The biological effect is due to cell metabolism mostly reversible. So, the dynamic analysis of protein regulation and functions are amenable.  The effect can be influenced by small‐molecule concentrations, and hence, different phenotype characteristics can be achieved.  The phenotype can be studied in the organism at any time of its physiological development stage. For example, gene knockouts cannot be examined in adult organisms, when they are lethal to the embryonic stage.  Small‐molecules can differentiate between proteins, coded on the same gene.  And for conserved targets, one and the same small‐molecule probe can be used in various organisms and systems.

As in classical genetics, chemical genetics can be subdivided into two approaches ‐ the “forward” and the “reverse” approach.4,5,7‐11 Within forward chemical genetics, phenotype inducing small‐molecules are screened on cells up to multicellular organisms. Identified compounds ‐ that induce a phenotype of interest ‐ are selected and the biological targets must be examined in later phases of the study.4,5,7‐11 On the other hand, a selected biological target is screened in the “reverse” approach (Figure 1). Afterwards, the specificity of the found probe is investigated, and lastly, phenotypes are examined in a cellular context.4,5,7‐11

In order to develop powerful molecular probes for dissecting biological processes, diverse small‐molecule libraries are created12‐14 and screened under great technical and scientific effort.4,7‐11 The compounds are natural products (from plants, animals or micro‐organisms) or from synthetic origin.4,8‐11 In both approaches, the effort and complexity of the screening‐ system can vary greatly depending on the type of bio‐assay and method to determine the phenotype.4,7‐11 Due to the fact that chemical genetics is closely related to pharmaceutical research, the discovered molecular tools not only might be of great value for basic science but also may open up novel avenues for the treatment of diseases.2,4,5,7‐13

4 Introduction

The main part of the work presented herein is based on a reverse chemical genetics approach. Therefore, DNA polymerases were selected as molecular targets to further elucidate their respective cellular functions (Figure 1).

2) DNA polymerases

2.1) General

The basic functions of the six “classical” DNA polymerases have been elucidated from catalytic properties, and observation of cell physiology. Pol α catalyses the initiation of chromosomal DNA replication at origins of replication and at Okazaki fragments on the lagging‐strand,27,28 pol β is involved in base excision repair (BER),29‐31 pol γ synthesizes mitochondrial DNA,32 pol δ has a role in lagging‐strand synthesis,33,34 pol ε participates in the synthesis of the leading‐strand of chromosomal DNA,35 and TdT facilitates antigen receptor diversity36.15,20

5 Introduction

In the course of these and other cellular processes or by environmental conditions, DNA mutations and damages occur.16 To maintain the genetic integrity of the genome, an elaborate set of sophisticated repair mechanisms have evolved. The set includes, amongst others, the “novel” specialized DNA polymerases (pol η, θ, κ, λ, μ, ν, ι, ζ, and REV1).15‐17,20,37 Features of some of these enzymes are known (Table 1), but to understand in depth the task of a particular enzyme stills await clarification in the majority of the cases. For that reason, there is a great demand for appropriate methods and reagents (e.g. small‐molecule probes) to dissect the cellular functions of DNA polymerases.

Table 1. Human DNA polymerasesa

DNA polymerase Gene Protein size (kDa) Family Main Function pol α (alpha) POLA1 166 B DNA replication priming pol β (beta)b POLB 38 X DNA repair pol γ (gamma) POLG1 140 A Mitochondrial DNA replication and repair pol δ (delta) POLD1 124 B DNA replication (lagging‐strand) pol ε (epsilon) POLE 262 B DNA replication (leading‐strand) TdT DNTT 58 X DNA repair, V(D)J recombination pol η (eta) POLH 78 Y Bypass synthesis (inserter) pol ι (iota) POLI 80 Y Bypass synthesis (inserter) pol κ (kappa) POLK 99 Y Bypass synthesis (inserter/extender) pol λ (lambda)b POLL 63 X DNA repair, V(D)J recombination pol μ (mu) POLM 55 X DNA repair, V(D)J recombination pol θ (theta) POLQ 290 A DNA repair pol ζ (zeta) POLZ 353 B Bypass synthesis (extender) REV 1 REV1 138 Y Bypass synthesis (inserter) pol ν (nu) POLN 100 A DNA repair a table was adapted from literature 15,17,20. b see also chapter 2.3)

2.2) DNA polymerases and the polymerisation reaction

All previously discovered DNA polymerases share a common, over several steps well‐ coordinated reaction mechanism (Figure 2) for the synthesis of the helical DNA polymer (composed of a sugar phosphate backbone, to which the four heterocyclic bases adenine (A), guanine (G), thymine (T), and cytosine (C) are attached38) (Figure 3).38‐42

6 Introduction

dNTP 2 conformational DNA binding E:DNA E:DNA :dNTP n n 3 change dNTP binding DNAn

1 E*:DNAn:dNTP

processive pyrophosphyl E 7 k 4 DNA synthesis pol transfer

8 E*:DNAn+1:PPi DNA pyrophosphate n+1 conformational release enzyme change E:DNA E:DNA :PP 5 dissociation n+1 n+1 i 6 PPi 2Pi hydrolyosis

Figure 2. Mechanism of DNA polymerase catalyzed nucleotide incorporation. After binding of a DNA primer template complex (DNAn), the DNA polymerase (E) binds an incoming dNTP that is afterwards tightly bound and arranged for the chemical step by a conformational change of the enzyme (E*). After bond formation and an other conformational change, pyrophosphate (PPi) is released to start another cycle of catalysis. Figure was adapted from literature 41‐43.

A B

synthesis 5`‐end NH2 N direction O N O N A incoming 5`‐end N dNTP DNA polymerase O N OH H O O HN O O -O P primer N O P O- 2 PO - H N G P 2 O P O O- O C N N O O- O- O C O O O- O N H NH O H O H N OH 3`‐end - 2+ O O O 3`‐end O H O Mg N C - N N H O PO2 H O G N O primer P O O G O N O Mg2+ O N - O O N H PO2 O- template O H O - H N - O HO O H N 5`‐end O P - O N - O O PO2 O O P N A O O N O incoming O T N H N dNTP O O 3`‐end PO - 2

Figure 3. Enzymatic DNA polymerization. (A) Schematic representation of template directed DNA synthesis catalyzed by a DNA polymerase. (B) Schematic representation of the corresponding trigonal bipyramidal transition state in the active site of a DNA polymerase (template not shown). Figure was adapted from literature 24,44.

DNA replication proceeds semiconservative. In doing so, DNA polymerases use one DNA parent strand as a template for synthesis of the exact complementary replica.18,20,45 During the synthesis, the single‐stranded template dictates to the enzyme according to

7 Introduction

Watson‐Crick38, in which sequence the four native 2´‐deoxynucleoside‐5`‐triphosphates (dNTPs) have to be connected to the 3`‐OH end of the hybridized primer. Thereby, the primer is always extended in the 5` to 3` direction (Figure 3A).20,42

From a chemical point of view, the addition of a dNTP to the primer is performed according to the mechanism of nucleophilic substitution (SN2). In the active site of the enzyme, the trigonal bipyramidal transition state is stabilized by two metal ions, e.g. magnesium (II) ions. For that reason, the reaction mechanism is also called the “two metal ion mechanism” 24 (Figure 3B). The SN2 reaction is accompanied by pyrophosphate release. Its subsequent hydrolysis favors DNA synthesis and prevents the reverse reaction. According to the latest findings, the rate limiting step of the reaction is a local reorganization step in the active site 46 of the enzyme. Nevertheless, the velocity of the polymerisation (kpol) of correct paired ‐1 ‐1 nucleotides achieves 1000 dNTP s , and the catalytic efficiency (Kd kpol ) is in the region of diffusion control (~107 M‐1 s‐1).41,42 Due to this facts, DNA polymerases belong to most powerful enzymes.47

2.3) DNA polymerase λ and β

Most of the work presented herein covers the “classical” pol β31,32,48 and the recently discovered “novel” pol λ49. Both nonreplicative human enzymes are members of the DNA polymerase X‐family (Table 1).15,17,20,22,37 The exonuclease‐deficient pol λ (64 kDa) contains all the structural features required for DNA binding, nucleotide binding and selection, and catalysis of DNA polymerization, which are conserved in pol β (39 kDa) ‐ the smallest known human DNA polymerase (Table 1). On this account, the primary sequence and the 3D‐ structure of the catalytic core of both DNA polymerase are highly homologous (Figure 4).49,50

Because of its ability to remove the 5`‐deoxyribose phosphate (dRP) generated after incision by an abasic (AP) endonuclease (dRP‐lyase activity) and its DNA synthesis specificity for short gaps, pol β is the prime DNA polymerase participating in BER (Figure 5).15,17,20,30,37,51 In addition, pol β is able to associate with other downstream enzymes of the BER pathway like DNA ligase I, AP endonuclease, and XRCC1‐DNA ligase III.30,37 Extensive studies show that pol β bypasses several DNA lesions via translesion synthesis (TLS), for example, AP sites52 and cisplatin adducts53.

8 Introduction

Figure 4. Family X DNA polymerase λ and β. (A) Schematic representation of pol β (red) and pol λ (green). Pol λ consists of a nuclear localization signal (NLS), a BRCA1‐C terminal (BRCT) domain (residues 36‐132), a proline‐ rich region (residues 133‐243), and a pol β‐like catalytic core region (residues 244‐575), with a helix‐hairpin‐ helix (HhH) and a DNA polymerase X motif.49 (B) Superimposition of the pol β‐like catalytic core region (residues 244‐575) of pol λ (green) and pol β (red). PDB IDs 2PFN and 2FMP (shown without DNA).

Pol λ, the other DNA polymerase of interest, is unique in possessing all the enzymatic activities which are individually present in the other X‐family members.20 Pol λ is capable of synthesizing DNA de novo as well as template‐dependent, and displays dRP‐lyase and TdT activity.54‐60 It is implicated that pol λ is involved in gap filling during nonhomologous end joining36,61,62, TLS52,63,64, and BER54,65,66.

Moreover, studies with chicken DT40 cells67, as well as mammalian fibroblasts68, showed that pol λ has a backup role for pol β in BER. Experiments with reactive oxygen species (ROS) indicate that pol λ protects cells from oxidative damage66,69, and there is also evidence that pol λ is required for cell cycle progression and is functionally connected to the S phase DNA damage response machinery in cancer cells.69

9 Introduction

Figure 5. Schematic representation of BER imbalance by targeting pol β. BER is a highly coordinated, multistep pathway, that removes a damaged DNA base and replaces it with the correct base. The genotoxic TMZ induce the formation of a base damage (e.g. 3‐methyladenine (3meA)), which is excised by DNA glycosylase (AAG) to produce an apurinic site (AP). Afterwards, an AP endonuclease (APE) incises the DNA backbone (5´ to the AP site) and generates a single‐strand break. Then, pol β removes the 5´‐dRP moiety through its intrinsic lyase activity and fills in the resulting gap. In the final step, the nick is sealed by DNA ligase (LIG) to finish base excision repair (BER). If BER is inhibited, or downstream steps of BER are limiting, then toxic intermediates accumulate and can lead to cell death. In this way, the dosage of the DNA‐damaging agent can be reduced. Figure was adapted from literature 16,70.

It is well known that aberrant levels of specialized DNA polymerases might cause genomic instability.15,20,71 A recent investigation of the expression patterns of specialized DNA polymerases in 68 different tumor samples revealed that in more than 45% of these tumors at least one specialized DNA polymerase was two‐fold‐enhanced expressed. Of particular interest was the fact that over 30% of all samples had either pol λ or β overexpressed.72 Consequently, the regulation of both DNA polymerases could be crucial in cancer treatment, since many chemotherapeutic regimes in use depend at least in part on the artificial induction of DNA damage. If effective, their utility is typically limited by the severity of side effects caused by the nonselective targeting of cancerous and healthy tissue in addition to the potential to induce mutagenic events that can actually accelerate disease

10 Introduction development.41,70,73 The clinical efficacy of anticancer drugs like cisplatin and monofunctional alkylating agents (e.g. temozolomid (TMZ)) is often reduced by cellular DNA repair mechanisms.15‐17,20,37,51,70 Consequently, both enzymes specialized for DNA repair are discussed as promising future drug targets, to reduce the dosage of DNA‐damaging agents while improving their activity via targeting of their respective repair pathways (Figure 5).15,17,20,37,51,70,74 To date, several inhibitors of DNA polymerase λ and β were developed and investigated (see also chapter 4.4.4).37,51,74‐79 However, one remaining challenge is still to find novel potent small molecule inhibitors that selectively inhibit one of these enzymes. In addition, a discriminating inhibitor could facilitate the targeting of one of these DNA polymerases over the other, to probe the enzymes’ respective cellular functions.

2.4) Herpes virus DNA polymerase

Viral infections are the leading cause of many critical illnesses. Without exception, all viruses are obligate, intracellular molecular parasites that replicate only inside the cell of a living organism.80,81 For the reproduction of the viral genome, viruses often encode an own DNA polymerase.20 Due to the importance of these enzymes for the amplification of the genetic code, viral DNA polymerases are established targets for current chemotherapies.41,81 These facts are also relevant for the family of the Herpes viruses (see also chapter 5.1), which reproduce their genetic code with their own DNA‐dependent DNA polymerase in the nucleus of a host cell. Among the nine representatives of the morphologically very similar Herpesviridae, herpes simplex virus‐1 (HSV‐1) is the most researched member.20 For that reason, the HSV‐1 DNA polymerase serves herein as model enzyme, which is illustrated briefly. After the HSV‐1 infection of mammalian cells, Keir et al. discovered in 1966 for the first time DNA polymerase activities that deviated from the features of host enzymes.82 Later, Weissbach et al. purified and characterized a viral DNA polymerase with a high molecular weight (180 kDa) from HSV‐1 infected human cells.83 It was found, that the enzyme favoured ‐ very likely due to the composition of the HSV genome (67% GC content)20 ‐ the synthesis of GC rich DNA.83 Today, the 3D‐structur of the HSV‐1 DNA polymerase is known. The enzyme is a heterodimer, composed of a catalytic subunit (UL30), whose structure is similar to B‐family 84‐86 DNA polymerase structures, and a processivity factor (UL42) that increases the fidelity. The catalytic subunit consists of six domains. The N‐terminal part contains a pre‐N‐terminal,

11 Introduction an N‐terminal, and an exonuclease domain, that ensures a high fidelity of DNA replication as well as an RNase H activity.20,87 The C‐terminal part adopts the usual right hand folding in palm, fingers and thump domains.85 Based on sequence alignments, the replicative HSV DNA 20 polymerase (UL30) belongs to the B‐family of DNA polymerases (see also chapter 2.1).

3) DNA polymerases as drug targets

3.1) General

As foreshadowed in previous chapters, DNA replication serves not only as a target for answering chemical genetic issues, but has been tried and tested for decades as prominent target for life‐saving medicines. Numerous pathological states, like cancer, autoimmune disease, and many bacterial and viral infections can be traced back to uncontrolled DNA metabolism.15‐17,20,37,51,70,79 For that reason, it is one of modern medicine's top priorities to combat those diseases with novel and innovative drugs. If one wants to specifically interfere in DNA metabolism, DNA polymerases of humans, animals, viruses, and bacteria are potential key drug targets, which are responsible for the correct synthesis and repair of the genetic code (see also chapter 2).15‐17,37,41,53,57,80 Due to the fact, that DNA polymerases share a similar 3D‐structure, and reaction mechanism to synthesise DNA, it is very challenging to address a drug molecule for a particular enzyme of interest (see also chapter 2.2). However, if the drug has a poor selectivity in its mode of action, the therapy is limited and inevitably associated with severe side effects. Current medicines employed to target the metabolism of DNA often induce DNA damage (see also Figure 5, and chapter 2.3),70 influence the dNTP pool, that is provided for DNA synthesis by the cell, or inhibit the enzymatic synthesis of DNA.41 For the last‐mentioned therapeutic strategy, which is relevant for this work, nucleoside analogs are a frequently used type of drug. Nucleosides, that are able to enter a cell (Figure 6, see also chapter 5.1), function as so called “prodrugs”, which have to be transformed by specific cellular nucleoside kinases to the actual enzyme substrate analogue; the nucleoside‐5`‐triphosphates.20,41 Nucleoside‐5`‐triphosphates derivatives that are not/hardly able to enter cells cause often complications. In order to be effective, they have to compete with the natural dNTP substrate pool for the active site of the DNA polymerase target.20,41 On this account, the respective nucleosides must be administered in high

12 Introduction concentrations to be effective, which can result in turn in selectivity and drug resistance problems.20,41 An additional disadvantage is, that nucleotide analogues and nucleoside‐ 5`‐triphosphates can also be utilized, modified, or degraded by a multiplicity of cellular pathways or other enzymes.13,15,17,20,37,42,70,74,79,81 Thus, in developing nucleoside analogues, it is necessary to investigate not only the optimisation of their interaction with DNA polymerases, but also their ability to be transformed to the 5`‐triphosphates without being degraded or showing serious side effects.20

Figure 6. Chemical structures of approved modified nucleoside analogues. (A) Current antiviral drugs (see also chapter 5.1). (B) Currant anticancer drugs. Figure was adapted from literature 20,41.

To address these problems and other issues, it is an ongoing concern to develop innovative therapeutic approaches and novel drugs.88‐91 In the following, some for this work relevant examples and perspectives are given. One current option to open up the way for novel drugs and nucleoside analogues is, for example, to study certain structural features of various known molecules. Afterwards, the interesting features are fused together in one novel drug‐like molecule, bearing all desired properties (see also chapter 5.1).

Other research approaches proceed towards the development of non nucleosidic small‐ molecule inhibitors, which act on the activity of a respective DNA polymerase. Thereby, the

13 Introduction small‐molecule could act directly on the active site of the enzyme, induce conformational changes in further protein domains (e.g. finger or thumb domain) to act indirectly on the enzymes` activity, or block protein‐protein interactions that are important for the processivity.12,13,20,41,92‐94 In general, small‐molecule DNA polymerase inhibitors have important advantages over substrate analogues. They are ideally suited to tune for a high target selectivity, they are capable to enter a cell and do not require intercellular activation.13,20,41

Many chemotherapeutic regimes in use depend at least in part on the artificial induction of DNA damage. The clinical efficacy of anticancer drugs is often reduced by cellular DNA repair mechanisms.15‐17,20,37,51,70,78,79 Consequently, specialized DNA polymerases are discussed as promising future drug targets, to reduce the dosage of DNA‐damaging agents while improving their activity via inhibition of their respective DNA repair pathways (see also chapter 4).17,37,51,70,78,79

3.2) Screening methods for DNA polymerase inhibitors

In the preceding chapters it was described in detail, that there is a great demand for novel DNA polymerases inhibitors in basic as well as in the applied sciences. The discovery process of novel agents against a chosen protein target usually involves high‐throughput screening (HTS) or high‐content screening (HCS) techniques, wherein large compound libraries are screened for the desired effect (see also chapter 1).95 Apart from emerging in silico based methods20,96‐101, one needs for such large‐scale screening campaigns automated, robust, reliable, and cheap in vitro assays.95 DNA polymerase inhibitors generally exert their effects through interference with the enzyme and/or cofactors, or the direct interaction with DNA.102 While there is no simple way to study DNA replication in vitro, the world‐renowned polymerase chain reaction (PCR) is an ideally suited enzyme assay, which involves a similar set of DNA replication transactions.103‐105 Classic PCR and the respective electrophoresis techniques104,105 have been applied previously for the in vitro discovery and characterisation of several inhibitors of thermostable DNA polymerases.103,106‐110 However, to screen thousands of molecules, the modern real‐time PCR is considerably more favourable, and hundreds of in vitro reactions could be screened in parallel.111,112 Real‐time PCR instruments are able to monitor the concentration of the arising dsDNA products in multi‐well formats by

14 Introduction measuring for example the emission of sequence‐specific fluorescent oligonucleotide probes or fluorogenic dsDNA binders like SYBR® Green I.111

Because the common mammalian, viral, and bacterial target DNA polymerases are thermolabile, these proteins are not applicable for modern PCR and other screening methods are required that can be performed at moderate temperatures.

The primer extension reaction (PEX) is an effective and useful way to explore thermolabile and thermostable DNA polymerases in the laboratory.43,44,75,76,113‐119 To study the DNA template dependent DNA polymerization function, a short radioactive or fluorescence labeled DNA primer strand gets annealed to a longer DNA template strand, and gets elongated by enzymatic dNMP incorporation (Figure 7A). Noteworthy, to analyze TdT enzyme activities the reaction is performed without the template strand and is named single‐ stranded PEX.59,60,120 However, after a defined period of time, the PEX reactions are quenched and quantitatively analyzed via polyacrylamide gel electrophoresis (PAGE) (Figure 7A).

Figure 7. Generally applicable radioactive PEX assays to characterize and screen small‐molecule inhibitors of thermolabile DNA polymerases (A) Principle of the radioactive PEX assay to analyze the effect of an inhibitor on the DNA template‐dependent polymerisation function of a respective DNA polymerase.75,76 (B) Assay scheme for inhibitor screening via the scintillation method. DNA polymerase polymerizes isotopic phosphate labelled dNMPs. The exact amount of formed radioactive DNA can be determined by scintillation measurements.

15 Introduction

Another common technique to analyse the PEX reaction mixtures is the scintillation method. Therefore, the reaction is performed with an unlabelled primer template‐complex and isotopic labelled dNTPs as enzyme substrates. After the reaction, the radioactivity of the novel synthesized DNA is quantitatively measured by scintillation (Figure 7B).120‐125

So far, both PEX methods were implemented for the screening and characterisation of inhibitors,75,76,100,120,124,126,127 but to screen huge compound libraries, PAGE analytics and accordingly the usage of radioactive reagents is not practicable. Therefore, novel automatable assay readouts and the respective tailor‐made reagents were evolved. As a first excellent example, Summerer et al. developed a Förster Resonance Energy Transfer (FRET)‐ based assay format that translates the proceeding DNA synthesis into a fluorescent signal in real‐time (Figure 8A).128,129 The fluorescence signal is generated by the DNA polymerase triggering opening of a molecular beacon, by extension of the primer strand.128,129 The resulting distance alteration is reported by FRET between two dyes introduced into the molecular beacon stem and enables the quantitative characterization of inhibitors.128,129 Recently, the elegant real‐time strategy was adopted from others130 and successfully utilised in a further developed fashion for screening campaigns of human DNA polymerases (Figure 8B).131‐133

Figure 8. Screening for inhibitors via real‐time FRET methods. (A) The template probe labelled with donor (grey) and acceptor (brown) has a hairpin extension in closed conformation before start of reaction. While extension proceeds, the DNA polymerase (blue) opens the stem and prevents re‐annealing by DNA duplex formation. The increase in the distance between the two labels is reported by restoration of donor emission. Figure was adapted from literature 128,129. (B) Strand displacement DNA synthesis assay. DNA polymerase incorporates dNTP thereby extending the primer strand and displacing the downstream reporter strand labelled with a 3`‐fluorophore donor, leading to an increased fluorescence signal. Figure was adapted from literature 131‐133.

16 Introduction

To access continuous FRET‐based PEX assays, some researchers focused on the enzymatic turnover of fluorescently labled dUTP substrates. For that reason, Cauchon et al. desined a primer‐template complex, that was labelled at the 5`‐template‐terminus with a donor fluorophore.134 Polymerisation mediated by incubation with a DNA polymerase, dNTPs, and an acceptor labeled dUTP juxtaposes the donor‐acceptor pairs, resulting in donor quenching (Figure 9A).134 On the other hand, Krebs et al. reported an FRET assay that quantifies the incorporation of complementary pairs of fluorescently labeled dUMP into the DNA product, and taking advantage, that the dye‐conjugated dNTP pairs in solution do not interact to produce a FRET signal (Figure 9B).135

Figure 9. Screening for inhibitors using fluorescently labled dUTP substrates. (A) The substrate is a short DNA/DNA primer/template. The template strand is labelled with a donor fluorophore (grey). Polymerization mediated by incubation with DNA polymerase (blue), dNTP, and an acceptor‐labelled dUTP (brown) juxtaposes the donor‐acceptor pairs, resulting in donor quenching. Figure was adapted from literature 134. (B) Dye‐ conjugated nucleotide pairs in solution do not interact to produce a FRET signal. DNA polymerase incorporates the dye‐nucleotides into the DNA. The close proximity of the two dyes in the polymer allows interaction between the dyes causing the generation of a FRET signal proportional to the amount of DNA produced in the sample. Figure was adapted from literature 135.

Other interesting examples amenable to automation are fluorometric PEX techniques that monitor the concentration of the arising dsDNA products.75,136‐138 The increase of the fluorescence signal caused by PicoGreen™136 or SYBR® Green I75,137,138 emitting upon binding to dsDNA was investigated as a fast readout for DNA polymerase activity (Figure 10). PEX resulted in high concentrations of dsDNA when the respective DNA polymerase was not inhibited.75,137,138 On the contrary, when the enzyme was inhibited, the primer was not extended and the fluorescence signal was low in relation to control reactions.75 Using that simple and economical method, diverse compound libraries were screened and potential

17 Introduction inhibitors of bacterial139 and human75 DNA polymerases were identified (Figure 10, see also chapter 4.1). Recently, Dallmann et al. extended the scope of this readout and established an in vitro assay for the parallel multiplicative target HTS against divergent bacterial replicases.140

Figure 10. Principle of the SYBR® Green I (stars) ‐ based HCS assay.75

Of note, a possible approach could also be the measurement of the arising pyrophosphates ions (PPi) that are produced by enzymatic dNTP consumtion (see also Figure 2). Reportedly,

PPi release of DNA polymerases was investigated in real‐time for instance by different colorimetric and/or enzyme coupled assays.141‐143 In the recent years, DNA arrayed ultra‐HTS formats for PEX reactions were developed. Interestingly, these systems are time and cost efficient, and require only minimal amounts of reagents. DNA arrays are based on the spatial separation of on a surfaces immobilized or covalently bound primers.20,53,119,144 The enzymes, templates, screening compounds, buffers, and natural as well as labelled (e.g. radioactive or fluorescent) dNTP substrates can also be applied with spatial separation to perform the PEX reactions.53,119,144 After the reaction, the surfaces are washed several times ‐ whereas the primers remain ‐ and can be analysed by phosphor imaging144 or fluorescents measurements53,119, (Figure 11). By employment of this time and cost efficient concepts, Boudsocq et al. could identify a variety of natural products that inhibit the BER enzyme pol β.53

18 Introduction

Figure 11. Principle of DNA arrayed HTS format ‐ using fluorescently labelled dNTP substrates.

As one can see in this overview, many automatable techniques to screen DNA polymerase inhibitors are established and are ready for their application to screen the chemical space. These methods have indeed the potential to discover novel interesting molecules that not only might be of great value for basic science but also may open up novel avenues for the treatment of diseases related to genome integrity.

19 Concepts and objectives

Concepts and objectives

The survival and development of each organism relies on the equal distribution of its genome during cell division. DNA polymerases are key enzymes to pass the exact genomic information down generations.15,17,20,22 In the last decades several novel DNA polymerases were discovered, and so, at least 15 different human DNA polymerases are known today.15,17,20,22 Features of some of these enzymes are known, but to understand in depth the task of a particular enzyme stills await clarification in the majority of the cases. For that reason, there is a great demand for appropriate methods and molecular tools to dissect the respective biological functions of DNA polymerases.

The aim of this work (chapter 4) deals with the discovery and development of tailored small‐ molecule probes in order to gain insights into the functions of human pol λ and β. In previous surveys several small‐molecule inhibitors of pol λ and few moderate inhibitors of pol β were discovered.75,76 Importantly, the rhodanine‐based compounds were the most active inhibitor class and some of them could even discriminate between the highly homologous pol λ and β.75,76 Due to this facts, the rhodanine‐based small‐molecules were seen as an appropriate starting point for the development of molecular probes to specifically investigate the biological functions of pol λ and β.75,76 For this purpose, systematic synthetic optimization should be undertaken in order to further expand the chemical diversity and to find novel and more potent small‐molecule inhibitors of pol λ and β. The most promising inhibitors of the first and second generation should be further investigated enzymatically and should be evaluated in comparison to reference inhibitors. Out of the generated in virto data extensive SAR should be established and discussed in detail. With the aim to further develop the molecular probes in a cellular context, the probes should be explored on different human cell lines. Afterwards, the suited molecular probes should be investigated in proof‐of‐concept studies to specifically target the pol λ and β in their respective biological pathways.

In addition, DNA polymerases serve not only as a target for molecular probes to dissect their biological functions, but have been tried and tested for decades as prominent target for life‐ saving medicines.15,17,20,22,37,41 In the last decades, 4’‐C‐modified nucleoside analogues aroused scientific interest, because a couple of derivatives of this interesting compound class showed antiviral activity145‐153, even against multi‐drug resistant pathogens.146,154 Because

20 Concepts and objectives there is a great demand for the development of drugs and consequently also for novel nucleoside analogues, novel potentially antiviral 4’‐C‐modified nucleoside analogues should be designed and developed in the second part of this work (chapter 5). Therefore, the molecular features of 4’‐C‐modified nucleosides and other pharmacologically interesting nucleoside analogues should be fused together into one inventive small‐molecule. Afterwards, the synthetic route for these innovative analogues and their versatile synthetic building blocks should be developed and performed.

21 Results and discussions

Results and discussions

4) Small‐molecule inhibitors of human pol λ and pol β

4.1) Introduction

DNA polymerases are key enzymes in DNA metabolism. They are involved in the chromosomal duplication, DNA recombination, and DNA repair.15,17,20,22,37,41,42 So far, 15 different DNA polymerases have been discovered in human cells.15,17,20,22 The functions of some of these 15 enzymes are known, but for the majority, e.g. repair enzymes, the exact roles still await clarification. The entire process of DNA metabolism takes minutes, and individual steps take place in seconds.39,41,42 For that reason, there is a great demand for appropriate tools and reagents to study the exact cellular functions of DNA polymerases. Given their fast mode of action, cell‐permeable small‐molecule inhibitors are ideally suited to interfere with the highly dynamic replication process (see also chapter 1). Additionally, these molecules might not only be of great benefit for basic research, but may also open up novel avenues for the therapy of diseases related to genome integrity.2,4,5,8‐10,15,17,41,79

In order to identify small‐molecule inhibitors of thermolabile DNA polymerases, Marx et al. developed a SYBR® Green I based assay for inhibitor high‐content screening (HCS) (Figure 10, see also chapter 3.2).75,137,138 The enhancement of the fluorescence signal (> 1000‐fold) caused by SYBR® Green I emitting upon binding to double‐stranded (ds) DNA155 was used as readout for the activity of human family X DNA polymerases.75 For the parallelized primer extension reactions (PEX) a 20‐nucleotide primer strand was annealed to a 90‐nucleotide template strand. PEX resulted in high concentrations of dsDNA when human DNA polymerase was not inhibited.75 On the contrary, when the enzyme was inhibited, the primer was not extended and the fluorescence signal was low in relation to the control reactions.75 Using that cheap and elegant method, a commercially available 9009‐member pharmacophore library was screened against the DNA repair enzyme pol λ at a compound concentration of about 65 μM. Small‐molecules were considered as “hits”, if the relative fluorescence was significantly lower than the positive controls.75

The discovered hits were additionally studied with the aid of a more sensitive method. Hence, a PEX assay with a radioactive labeled primer with the same sequence context and a

22 Results and discussions shorter 33 nucleotide template was utilized (Figure 12B).75,76 In this way, eleven highly active molecules against pol λ could be identified out of 159 initial hits. On the basis of the chemical structures, the eleven hits could be subdivided into three inhibitor classes: Class I are rhodanines (5‐arylidene‐2,4‐thiazolidinediones), class II are carbohydrazides, and class III contains a common 2,4‐pentadione substructure element (Figure 12A).76

Next, the three classes were analyzed towards inhibiting the DNA polymerase function of the homologous pol β in the radioactive PEX assay. The rhodanines, classified as an excellent scaffold for the development of biologically active molecules,156‐161 inhibited the polymerase function of pol λ and were able to discriminate between the two X‐family DNA polymerases.75 Small‐molecule SM1 was the most potent and discriminating inhibitor. In addition SM1 did not inhibit (IC50 > 100 μM) the DNA polymerases 9°N, Therminator, Pfu, and Dpo4.75 Importantly, chemical identity of SM1 could be confirmed via re‐synthesis, and the re‐synthesized SM1 showed the same activity and selectivity as the purchased SM1.76

Figure 12. (A) Chemical structures of the most potent hit of each pol λ inhibitor class. According the blue substructures, the classes were subdivided. SM1 was the most potent pol λ inhibitor and discriminates between pol λ and β. (B) Representative PAGE analysis of IC50 determination of pol λ for 1. Lane 1: Primer only; lane 2: DMSO control; lane 3‐8: same as lane 2 increasing concentrations of SM1 (2.5, 5.0, 7.5, 10.0, 20.0, 50.0 μM compound). (C) Dose‐response curves of re‐synthesized SM1, which inhibited dose‐dependently the polymerization function of pol λ (■) with an IC50 value of 5.9 ± 1.1 μM and pol β (▲) with an IC50 of 64.4 ± 75,76 1.0 μM. Averages of at least three independent experiments and standard deviations are shown.

To measure the exact IC50 values of the compounds against pol λ and β, a radioactive PEX assay in the presence of increasing concentrations of inhibitor or DMSO as a solvent control

23 Results and discussions was developed.75,76 It was found out, that SM1 inhibits dose‐dependently the polymerization function of pol λ with an IC50 value of 5.9 μM and pol β with an IC50 of 64.4 μM, and could hence discriminate between the two homologous family X DNA polymerases with a factor of about ten (Figure 12B,C).75,76 For that reason, an initial 56‐member small‐molecule library was designed.76 The bulk of the molecules of the 1st generation was from commercial and the lower proportion from synthetic origin (for details see chapter 8.1).76 During the course of evaluating the inhibitory potential of the compound library in vitro, further rhodanine‐based inhibitors (SM10, 16, 21, 23) were discovered whose properties are comparable with SM1 and there were early indications that some compounds (e.g. SM11, 12, 20) also showed activity against pol β.76 Finally, preliminary basic structure‐activity relationships (SAR) could be established out of the in vitro data.76

4.2) Biochemical evaluation of the 1st small‐molecule generation

On purpose to design a molecular scaffold for the creation of the 2nd small‐molecule generation, first, the pol λ and β in vitro PEX data of the 1st compound generation76 had to be reproduced and completed to the required triplicate determinations (see also chapter 4.1). Therefore, 26 representative compounds were selected and their inhibitory potential against pol λ was screened (Table 2, Figure 13).

24 Results and discussions

Table 2. SM1 and the 1st generation analogues in the pol λ and β PEX assaysa

Pol λ Pol β Pol λ Pol β No. R1 R2 R3 Z conversion [%], conversion [%], IC50 [µM] IC50 [µM] 20 µM compound 50 µM compound b,c SM1 A ‐NO2 4‐Me‐Ph‐ ‐S‐ 3 92 5.9 64.4 b SM2 ‐NO2 A 4‐Me‐Ph‐ ‐S‐ ‐ ‐ 10.0 45.5

b SM3 ‐NO2 A 4‐Cl‐Ph‐ ‐S‐ ‐ ‐ 8.1 42.9

SM4 A ‐NO2 ‐ ‐Cl 92 ‐ ‐ ‐

SM5 A ‐O‐CH2‐COOH ‐ ‐H 93 ‐ ‐ ‐ SM6 5‐(perfluorobenzylidene)‐2‐thioxothiazolidin‐4‐one 82 ‐ ‐ ‐ SM7 A ‐Cl ‐Et ‐O‐ 59 ‐ ‐ ‐

SM8 A ‐NO2 HO(CH2)2‐ ‐S‐ 94 ‐ ‐ ‐

SM9 ‐NO2 A 4‐CF3‐Py‐ ‐S‐ 12 ‐ ‐ ‐

SM10 A ‐NO2 4‐CF3‐Py‐ ‐S‐ 6 92 11.0 >100

SM11 ‐NO2 A ‐Cy ‐S‐ 3 11 ‐ ‐

SM12 A ‐NO2 ‐Cy ‐S‐ 3 7 ‐ ‐

SM13 A ‐NO2 4‐Cl‐Ph‐ ‐S‐ 13 ‐ ‐ ‐

SM14 A ‐NO2 4‐Br‐Ph‐ ‐S‐ 26 ‐ ‐ ‐

SM15 A ‐NO2 Py‐ ‐S‐ 82 ‐ ‐ ‐

SM16 A ‐Br 4‐F‐Ph‐ ‐CH2O‐ 3 89 8.3 80.0

SM17 A ‐NO2 4‐F‐Ph‐ ‐CH2O‐ 11 ‐ ‐ ‐

SM18 A ‐H 4‐F‐Ph‐ ‐CH2O‐ 15 ‐ ‐ ‐ SM19 B ‐OMe 3‐Br‐Ph‐ ‐COO‐ 11 ‐ ‐ ‐ SM20 A ‐OMe 3‐Br‐Ph‐ ‐COO‐ 3 11 ‐ ‐ SM21 A ‐H 2‐Cl‐Ph‐ ‐COO‐ 4 95 12.4 88.8 SM22 A ‐OMe 2,4‐Cl‐Ph‐ ‐COO‐ 39 ‐ ‐ ‐

SM23 A ‐NO2 4‐Me‐Ph‐ ‐O‐ 4 96 9.3 >100

SM24 B ‐Br 4‐F‐Ph‐ ‐CH2O‐ 23 ‐ ‐ ‐

SM25 ‐NO2 ‐NO2 4‐Cl‐Ph‐ ‐S‐ 99 ‐ ‐ ‐

SM26 ‐COH ‐NO2 4‐Me‐Ph‐ ‐S‐ 82 ‐ ‐ ‐

SM27 C ‐NO2 4‐Me‐Ph‐ ‐S‐ 98 ‐ ‐ ‐

SM28 D ‐NO2 4‐Me‐Ph‐ ‐S‐ 100 99 ‐ ‐ aaverages of three independent experiments are shown, b from literature 76, c from literature 75

As result, the initial data of ten inactive compounds (SM4‐8, 15 and 25‐28), eight compounds (SM9, 13, 14, 17‐19, 22 and 24) with a moderate activity (11‐50% conversion), and eight highly active compounds (SM1, 10‐12, 16, 20, 21, and 23) with conversion below 10% was confirmed (Figure 13A; Table 2). In addition, the exact IC50 values were re‐measured for the

25 Results and discussions four highly active and self‐synthesised compounds (SM10, 16, 21, and 23) (Figure 13B; Table 2). SM10, 16, 21, and 23 inhibited dose‐dependently the polymerization function of pol λ with IC50 values of 11.0, 8.3, 12.4, and 9.3 μM and are thus comparable active as lead SM1 (see also Figure 12).

Figure 13. Evaluation of a representative compound selection of the 1st compound generation towards pol λ (listed in Table 2). (A) Evaluation of the compounds at 20 μM in the pol λ PEX assay. Inactive compounds are shown in red. Compounds with moderate activity (11−50% conversion) are shown in yellow. Compounds with less than 10% conversion (green) were chosen to determine their exact IC50 value and were evaluated towards pol β, in order to find a pol λ selective probe. (B) Dose‐response curves of compounds SM10, 16, 21, and 23, which dose‐dependently inhibited the polymerization function of pol λ with IC50 values of 11.0 ± 1.1, 8.3 ± 1.0, 12.4 ± 1.1, and 9.3 ± 1.1 μM. In general averages of at least three independent experiments and standard deviations are shown.

As already mentioned, pol λ is suggested to back up base excision repair (BER) by pol β.67,68 With intend to develop molecules that selectively inhibit one of these enzymes over the

26 Results and discussions other to probe the enzymes’ roles in DNA repair, the initial data76 of the eight highly active compounds was also re‐proved in the pol β PEX assay. As expected, SM11, 12, 20 targeted also significantly pol β (Figure 14). SM12 was the most active molecule and turned out to be a very interesting starting point for the development of pol β inhibitors. Importantly, SM1,

10, 16, 21, and 23 showed at 50 µM no activity against pol β and the exact IC50 values for those compounds was ≥80 µM. And so, it was verified that SM10, 16, 21, and 23 are like SM1 potentially useful molecular probes to inhibit pol λ over pol β.

Figure 24. Evaluation of the 1st compound generation towards pol β (listed in Table 2). (A) Evaluation of the compounds at 50 μM in the pol β PEX assay. Compounds showing activity against pol β are shown in green. Interesting pol λ selective inhibitors, with high conversions in the pol β PEX assay, are shown in red and were chosen to determine their exact IC50 values. (B) Dose‐response curves of compounds SM16, and 21, which inhibited dose‐dependently the polymerization function of pol β with IC50 values of 80.0 ± 1.0, and 88.8 ± 1.0 μM. The exact IC50 value of SM10, and 23 was not determined (IC50 > 100 μM). In general averages of at least three independent experiments and standard deviations are shown.

Reportedly, the family X member pol λ has a TdT activity, and its involvement in recombination pathways was implicated.59,60 On purpose to test whether SM1 targets also the TdT activity of pol λ, the inhibitory potential of SM1 was studied using the radioactive assay of single‐stranded PEX analogical to what has been applied earlier (Figure 15A, B).120 Interestingly, it was found out that SM1 inhibits dose‐dependently also the TdT function of pol λ with an IC50 value of 4.5 μM (Figure 15C).

27 Results and discussions

Figure 15. Analysis of SM1 on TdT function of pol λ. (A) Principle of the single‐stranded radioactive pol λ PEX assay. (B) PAGE analysis showing the influence of SM1 on TdT function of pol λ. Lane 1: DMSO control; lane 2‐7: increasing concentrations of SM1 in DMSO (2.5, 5.0, 7.5, 10.0, 20.0, 50.0 μM compound). (C) Dose‐response curve. The conversion of primer depicted in A), lane 1 was set as 100%. Compound SM1 inhibited dose‐ dependently the TdT function of pol λ with an IC50 value of 4.5 ± 1.1 μM. Averages of at least three independent experiments and standard deviations are shown.

To investigate, if SM1 competes with natural dNTP substrates, PEX assays in presence of a given amount of 50 µM inhibitor and variable concentrations of dNTPs, and respective control experiments without SM1 were conducted (Figure 16A). The absence of reaction products even in the presence of increasing amounts of dNTP suggests that SM1 inhibits pol λ without directly competing for the same binding site.

Figure 16. PAGE analysis of the pol λ PEX assay with varied dNTP concentrations. (A) Principle of radioactive pol λ PEX assay with variable dNTP concentrations. (B) Lane 1: Primer only; lane 2: DMSO control (get to 100% conversion); lanes 3‐8: 50 μM SM1 and increasing concentrations of dNTPs (15, 30, 60, 120, 240, 480 μM). (C) Lane 1: Primer only; lanes 2‐7: DMSO control reactions (without inhibitor) and increasing concentrations of dNTPs (15, 30, 60, 120, 240, 480 μM).

28 Results and discussions

4.2.1) Conclusion

st In conclusion, the initial screening and IC50 data of selected compounds of the 1 generation were confirmed. Additionally, it was ascertained that SM1 acts also dose‐dependently on the

TdT function of pol λ with an IC50 value of 4.5 μM. Continuing experiments with constant SM1 and increasing dNTP concentrations suggested that SM1 inhibits pol λ without directly competing for the substrate binding site. Based on the reproduced initial data, a molecular scaffold could be introduced for the design of the 2nd small‐molecule generation.

4.3) Establishment of a 2nd generation compound library of potential active molecules against pol λ and β

4.3.1) Design of the 2nd small‐molecule generation SM12, 29‐58

During the course of evaluating the inhibitory potential of the initial compound library, further rhodanine‐based inhibitors were found whose properties are comparable with SM1. In addition, there were early indications that some compounds (e.g. SM11, 12, 20) also showed activity against pol β (Table 2, see also chapter 4.2).76

With intend to further expand the chemical diversity and to find novel and more potent small‐molecule inhibitors of pol β and λ, 30 (SM29‐58) analogues of SM1 and SM12 were designed and synthesized (Table 3). Molecule SM12 was re‐synthesized because of its highest activity against pol β in the initial screen. To design the novel compound series, the structures of the 26 small‐molecule entities (SM1, SM4‐28) of the 1st generation were analysed and subdivided into a molecular scaffold (Table 1, and 2). The scaffold consists of three variable parts, R1, R2, and R3, which are connected via an aromatic core and a variable Z‐linkage.

29 Results and discussions

Table 3. 2nd generation small‐molecules SM1, 12, 29‐58 and their biochemical evaluation.a

Pol λ Pol λ Pol β 1 2 3 Pol λ Pol β No. R R R Z conversion [%], conversion [%], a conversion [%], a a a IC50 [µM] a IC50 [µM] 20 µM compound 10 µM compound 50 µM compound b SM1 A ‐NO2 4‐Me‐Ph‐ ‐S‐ 3 8 5.9 92 64.4

b SM12 A ‐NO2 ‐Cy ‐S‐ 4 19 <10 32 <50

SM29 B ‐NO2 4‐Me‐Ph‐ ‐S‐ 4 26 <10 90 >50

SM30 E ‐NO2 4‐Me‐Ph‐ ‐S‐ 80 ‐ ‐ 88 ‐

SM31 F ‐NO2 4‐Me‐Ph‐ ‐S‐ 91 ‐ ‐ 93 ‐

SM32 G ‐NO2 4‐Me‐Ph‐ ‐S‐ 97 ‐ ‐ 98 ‐

SM33 H ‐NO2 4‐Me‐Ph‐ ‐S‐ 5 19 <10 38 <50

SM34 I ‐NO2 4‐Me‐Ph‐ ‐S‐ 46 ‐ ‐ 100 ‐

SM35 A ‐NO2 4‐Me‐Ph‐ ‐SO2‐ 23 ‐ <20 96 ‐ SM36 A ‐ ‐H ‐H 99 ‐ ‐ 98 ‐ SM37 A ‐H 4‐Me‐Ph‐ ‐S‐ 3 31 <10 37 <50 SM38 A ‐F 4‐Me‐Ph‐ ‐S‐ 2 14 <10 3 38.7 SM39 A ‐Cl 4‐Me‐Ph‐ ‐S‐ 2 9 5.7 82 >50 SM40 A ‐Br 4‐Me‐Ph‐ ‐S‐ 2 19 <10 87 >50

SM41 A ‐CF3 4‐Me‐Ph‐ ‐S‐ 2 14 <10 7 28.1 SM42 A ‐CN 4‐Me‐Ph‐ ‐S‐ 2 11 <10 93 ‐

SM43 A ‐NO2 4‐Et‐Ph‐ ‐S‐ 3 14 <10 20 <50

SM44 A ‐NO2 4‐F3C‐Ph‐ ‐S‐ 2 18 <10 90 >50

SM45 A ‐NO2 4‐F3CO‐Ph‐ ‐S‐ 18 ‐ <20 83 >50

SM46 A ‐NO2 3‐F,4‐Me‐Ph‐ ‐S‐ 3 14 <10 90 >50 2,3,5,6,‐F; SM47 A ‐NO2 ‐S‐ 3 18 <10 76 >50 4‐F3C‐Ph‐

SM48 A ‐NO2 3‐Me‐Ph‐ ‐S‐ 2 10 6.0 8 29.8

SM49 A ‐NO2 2‐Me‐Ph‐ ‐S‐ 3 6 3.9 5 18.2

SM50 A ‐NO2 2,5‐Me‐Ph‐ ‐S‐ 2 15 <10 31 <50

SM51 A ‐NO2 2‐Naphtyl‐ ‐S‐ 3 19 <10 67 >50

SM52 A ‐NO2 Cyclopentane‐ ‐S‐ 17 ‐ <20 90 >50 SM53 A ‐Br 3‐F,4‐Me‐Ph‐ ‐S‐ 2 5 4.0 84 >50 SM54 A ‐H 2‐Me‐Ph‐ ‐S‐ 4 21 <10 47 ~50 SM55 A ‐F 2‐Me‐Ph‐ ‐S‐ 3 14 <10 50 ~50

SM56 A ‐CF3 2‐Me‐Ph‐ ‐S‐ 3 15 <10 37 <50 (Z)‐5‐(2,4‐bis(p‐tolylthio)benzylidene)‐2‐ SM57 2 10 4.0 27 <50 thioxothiazolidin‐4‐one (Z)‐5‐((4‐bromo‐5‐(p‐tolylthio)thiophen‐2‐ SM58 4 17 <10 22 <50 yl)methylene)‐2‐thioxothiazolidin‐4‐one a averages of three independent experiments are shown, b from literature 76, c re‐synthesized

30 Results and discussions

To project the 2nd generation of small‐molecule inhibitors (Table 3), the compounds were further derivatized in all parts of the scaffold. The heterocyclic rhodanine, moiety A in R1, proved to be highly important for the inhibitory activity against pol λ (Table 2).76 However, it could be shown that moiety A can be replaced in some cases by the 2,4‐thiazolidinone moiety B. Motivated by this fact, the synthesis of heterocyclic molecules SM29‐34 ‐ bearing moiety B and the further well‐known pharmacophoric moieties E, F, G, H, and I ‐ were investigated. The thioether‐Z‐linkage proved not to be essential for high activity towards pol λ and could be replaced by ester‐, benzyl phenyl ether‐, or diphenyl ether‐Z‐linkages (Table 2). Thus, this modification site was not in the main focus, and so SM35 with the sulphone as Z‐ linkage, and the oversimplified structure SM36 were sampled only. Importantly, it could be shown that the variation of the substituents in R2 and R3 can influence the inhibitory activity against pol λ. On the other hand there was evidence that some of these changes in R2 and R3 forced the inhibitory activity against pol β. To further expand the compound series with modifications in part R2 and R3, analogues SM37‐56 were designed and synthesized. In the initial screen, SM2 also proved to be a pol λ inhibitor in the low micromolar range (IC50 = 76 10.0 μM). For that reason, this compound (apart from the nitro groups) was “combined” with SM1 to generate molecule SM57. Lastly, the inhibitory potential of the first compound with a heterocyclic aromatic core structure was explored and SM58 was created.

4.3.2) Synthesis of 2nd small‐molecule generation SM12, 29‐58

For the chemical synthesis of the small‐molecule entities SM12, 29‐58 the previously developed synthesis strategy was followed.76 The strategy can be subdivided into two parts (Figure 17, and 18).

In the first part, the precursor aldehydes SM59‐61 and 63‐84, and for compound SM33 a precursor acetophenone SM62 were built up. To synthesize the new precursors SM59‐83 in good to excellent yields, the nucleophilic aromatic substitution (SNAr) was applied under basic conditions in DMF to displace activated halides (F, Cl, or Br) at the aromatic core by aromatic or aliphatic thiols (Figure 17).

To synthesize the 3‐nitro‐4‐tosylbenzaldehyde precursor SM84 in a three step sequence, literature‐known synthesis strategies162‐164 were assigned starting from 3‐nitro‐4‐(p‐

31 Results and discussions tolylthio)benzaldehyde SM59. Therefore, SM59 was reduced with sodium borohydride

(NaBH4) in methanol to yield the (3‐nitro‐4‐(p‐tolylthio)phenyl)methanol intermediate in

98%. Next, the intermediate was refluxed together with H2O2 in acetic acid to give (3‐nitro‐4‐ tosylphenyl)methanol SM85 in 61% yield. In the last step, SM85 was oxidized with DMP165 to furnish precursor SM85 in 57% yield (Figure 17).

Figure 17. Part one: Synthesis of precursor aldehydes and acetopenone SM59‐84. Reagents and conditions: a) Corresponding thiol and benzaldehyde (or rather acetophenone), K2CO3, DMF, 80°C, up to 89%; b) Corresponding thiol and benzaldehyde, KOH, DMF, 0°C to r.t., up to 97%; c) NaBH4, methanol, r.t., 1 h, 98%; d) 30% H2O2, AcOH, reflux 61%; e) DMP, CH2Cl2, r.t., over night, 53%.

In the second part, the Knoevenagel condensations were performed by fusing the precursor molecules together with varying heterocycles (Figure 18). In doing so, an exocyclic double bond is generated, whereas in theory two diastereomeres E and Z can be formed. Reportedly, the Z‐configuration is thermodynamically more stable than the E‐configuration.166‐168 For that reason, SM12, 29‐33 and 35‐58 were synthesized under thermodynamic reaction control. The precursor aldehydes SM59‐61 and 63‐84 were refluxed with rhodanine, thioazolidine‐ 2,4‐dione, thiohydantoine, rhodanine‐3‐acetic acid, or pseudothiohydantoin in a solution of

32 Results and discussions anhydrous NaOAc in glacial acetic acid to obtain the isomeric pure compounds SM12, 29‐32, and 35‐58 in good to excellent yields (Figure 18).76,169

Figure 18. Part two: Synthesis of small‐molecules SM12, 29‐58. Reagents and conditions: a) Corresponding aldehyde, rhodanine (or rather thioazolidine‐2,4‐dione, thiohydantoine, rhodanine‐3‐acetic acid, pseudothiohydantoin), NaOAc (2.5 M in AcOH), reflux, over night, up to 98%; b) NH4OAc, toluene, reflux, over night, 78%; c) Diethyl 2,6‐dimethyl‐1,4‐dihydro‐3,5‐pyridinedicarboxylate (Hantzsch ester), activated silica gel 60, toluene, reflux for 24 h in the dark, 82%.

1H NMR spectra analysis of compounds SM12, 29‐30, 32, and 35‐58 gave only one signal for the 5‐methylidene proton in the range 7.50 to 7.80 ppm and for SM31 at 6.50 ppm at lower field values than those expected for the E isomers,170,171 which strongly indicates the formation of the more stable Z isomers. To attach rhodanine to the acetophenone precursor 172 SM62, the reaction was performed according to Unangst et al. in a mixture of NH4OAc and toluene heated under reflux to yield SM33 in 78% (Figure 18). 1H NMR spectra of SM33 shows also for the 5‐ethylidene methyl group one sharp signal implying Z‐configuration. The exclusive formation of the thermodynamically stable Z isomers of SM12, 29‐33, and 35‐58 is in agreement with various publications of related small‐molecules.160,167,169‐175 For the

33 Results and discussions synthesis of SM34, with the free rotatable pharmacophoric moiety I, hydrogenation of the exocyclic double bond of SM1 was investigated. Only a few procedures have been published to reduce the exocyclic double bond in 5‐benzylidene‐2‐thioxothiazolidin‐4‐ones. The first attempts to reduce the double bond of SM1 by using lithium borohydride (LiBH4) and pyridine in THF were unsuccessful even at elevated temperatures. Reportedly, Kikelj and Peterlin Mašič et al. 170,171,174 adopted the Hantzsch ester (diethyl 2,6‐dimethyl‐1,4‐dihydro‐ 3,5‐pyridinedicarboxylate) on silica gel 60 method.176 The chemoselective method lead to success and SM1 was reduced to the racemic SM34 in a very good yield (Figure 18).

4.3.3) Conclusion

By systematic synthetic optimization, the chemical diversity of rhodanine‐based small‐ molecules with potential interesting pharmacological features could be further expanded starting from cheap and commercially available building blocks. The scaffold oriented synthesis of the drug‐like molecules SM12, 29‐58 was subdivided into two parts.

In the first part, the precursor aldehydes SM59‐61 and 63‐83 and for compound SM33 a precursor acetophenone SM62 were built up using high‐yielding SNAr reactions under basic conditions. Thereby activated halides were displaced at the aromatic core of the scaffold by the corresponding thiols. For the synthesis of precursor SM84, an elegant literature‐known three step synthesis sequence163 was assigned starting from SM59. Therefore, SM59 was reduced with NaBH4 to yield the methanol intermediate in 98%. Afterwards the intermediate was refluxed together with H2O2 to give SM85 in 61% yield. In the last step, SM85 was oxidized with DMP to furnish precursor SM84 in 57% yield.

By performing the Knoevenagel condensation in the second part, several precursor molecules were fused together with varying heterocycles. To obtain the Z‐isomeres exclusively, SM12, 29‐33, and 35‐58 were built up under thermodynamic reaction control in good yields. For the synthesis of racemic SM34 the exocyclic double bond of SM1 was reduced by the Hantzsch ester on silica gel method176.

34 Results and discussions

4.4) Biochemical evaluation 2nd small‐molecule generation

4.4.1) Evaluation of the 2nd small‐molecule generation

Holding 2nd small‐molecule generation in hand, the novel entities were tested in the pol λ

PEX assay in comparison to inhibitor SM1 at 20 μM (Figure 19, Table 3). In doing so, four inactive compounds (SM30‐32, and 36), four compounds (SM34, 35, 45, and 52) with a moderate activity (11‐50% conversion), and 23 highly active compounds (SM12, 29, 33, 37‐ 44, 46‐51, and 53‐58) with conversion below 10% were found (Figure 19A; Table 3).

Next, the 23 highly potent small‐molecules were studied for their effect in pol λ PEX assay at

10 μM to get a further selection criterion. Interestingly, all tested compounds had an IC50 value of less than 10 μM against pol λ (Figure 19B; Table 3). The exact IC50 value was determined for the five most active compounds (SM39, 48, 49, 53, and 57) with a turnover of ≤10% in the PEX assay (Figure 19C; Table 3). SM39, 48, 49, 53, and 57 inhibited dose‐ dependently the polymerization function of pol λ with IC50 values of 5.7, 6.0, 3.9, 4.0, and

4.0 μM, respectively, and are thus equally or even more active than the reference compound

SM1 (IC50 = 5.9 μM). To test the effect of SM39, 48, 49, 53, and 57 on the TdT activity of pol λ, the radioactive assay of single‐stranded PEX was performed. All investigated molecules inhibited the TdT function in a low micromolar range comparable to SM1 (IC50 = 4.5 μM) (Figure 19D, see also Figure 15).

35 Results and discussions

Figure 19. Evaluation of the 2nd compound generation towards pol λ (listed in Table 3). (A) Evaluation of the compounds at 20 μM in the pol λ PEX assay. Inactive compounds are shown in red. Compounds with moderate activity (11−50% conversion) are shown in yellow. Compounds with less than 10% conversion (green) were chosen to be analyzed at 10 μM in the pol λ PEX assay. (B) Evaluation of the compounds at 10 μM in the pol λ PEX assay. Compounds with a conversion below 50% are shown in green. Compounds with less than 10% conversion (blue) were chosen to determine their exact IC50 value. (C) Dose‐response curves of synthesized compounds SM39, 48, 49, 53,and 57, which dose‐dependently inhibited the polymerization function of pol λ with IC50 values of 5.7 ± 1.0, 6.0 ± 1.0, 3.9 ± 1.1, 4.0 ± 1.1, and 4.0 ± 1.0 μM. In general averages of at least three independent experiments and standard deviations are shown. (D) PAGE analysis showing the influence of SM39, 48, 49, 53, and 57 on TdT function of pol λ. Lane 1, primer only; lane 2, DMSO control; lanes 3−7, compounds SM39, 48, 49, 53, and 57 (each 10 μM).

To test for selectivity and to identify inhibitors of pol β, the new small‐molecule series was screened at 50 μM in the pol β PEX assay (Figure 20, Table 3). In this manner, ten compounds (SM29, 39, 40, 42, 44, 46, 47, and 51‐53) were identified that selectively inhibited at low concentrations pol λ but not pol β. Compounds that were not (SM30‐32, and 36) or were

36 Results and discussions moderately (SM34, 35, 45, and 52) active against pol λ showed no effect against pol β. The re‐synthesized entity SM12 belongs to the ten small‐molecules (SM12, 33, 37, 43, 50, and 54‐ 58) showing a moderate activity (15‐50% conversion) against pol β. Interestingly, four highly active molecules (SM38, 41, 48, and 49) were identified with a conversion below 10%

(Figure 20A; Table 3). For those four small‐molecules, the exact IC50 values were measured. It was found that SM38, 41, 48, and 49 dose‐dependently inhibited the polymerization function of pol β with IC50 values of 38.7, 28.1, 29.8, and 18.2 μM (Figure 20B, Table 3).

Figure 20. Evaluation of the 2nd compound generation towards pol β (listed in Table 3). (A) Evaluation of the compounds at 50 μM in the pol β PEX assay. Compounds inactive against pol β and pol λ are shown in grey. Inactive compounds, with a high conversion, are shown in red. Compounds with moderate activity (15‐50% conversion) are shown in yellow. Interesting compounds with less than 10% conversion (green) were chosen to determine their exact IC50 values. (B) Dose‐response curves of synthesized compounds SM39, 41, 48, and 49 which inhibited dose‐dependently the polymerization function of pol β with IC50 values of 38.7 ± 1.0, 28.1 ± 1.0, 29.8 ± 1.0, and 18.2 ± 1.0 μM.

37 Results and discussions

4.4.2) Side‐by‐side comparison of SM1 and SM49 with reported inhibitors

In order to audit the developed pol λ and β PEX assays, and to draw comparisons between the discovered small‐molecule inhibitors and literature known inhibitors, several inhibitors of pol λ and β were acquired.

So far, the published inhibitors of pol λ are mainly based on natural products.37,75,76 The strongest known inhibitor of the polymerase function of pol λ was (‐)‐epigallocatechin gallate 177 (EGCG) (Figure 21C), isolated from green tea. The catechin was reported with an IC50 value 177 of 3.8 μM.

Figure 21. Side‐by‐side comparison of SM1 with EGCG177 in the pol λ PEX assay. (A) Potency of SM1 against pol λ compared with EGCG using the same reaction conditions (100 μM compound). (B) Dose‐response curves of EGCG, which inhibited dose‐dependently the polymerization function of pol λ with an IC50 value of 34.4 ± 1.1 μM. (B) Chemical structure of EGCG. Averages of at least three independent experiments and standard deviations are shown.

To evaluate the fully synthetic SM1 (IC50 = 5.9 μM), EGCG was explored in a side‐by‐side comparison using the same pol λ PEX conditions. It was found, that EGCG is significant less active than SM1 (Figure 21A) and inhibits dose‐dependently the polymerization function of pol λ with an IC50 value of 34.4 μM (Figure 21B). In consequence, the rhodanines are currently the strongest inhibitors for pol λ.

38 Results and discussions

Figure 22. Side‐by‐side comparison of SM49 with betulinic acid, oleanolic acid, and lithocholic acid in the pol β PEX assay. (A) Potency of compound SM49 against pol β compared with betulinic acid,178 oleanolic 179 180 acid, and lithocholic acid using the same reaction conditions (500, 250, and 125 μM compound). (B) Chemical structures of betulinic acid, oleanolic acid, and lithocholic acid. In general, averages of at least three independent experiments and standard deviations are shown.

For the comparison of strongest identified inhibitor of pol β SM49 (IC50 = 18.2 μM), a variety of reported pol β inhibitors (betulinic acid,178 oleanolic acid,179 and lithocholic acid180 (Figure 22B)) were investigated in a side‐by‐side comparison using the pol β PEX assay under the same conditions. It was found out, that the IC50 values of the naturally occurring 178,179 180 triterpenoids and bile acid were above 100 μM, and thus much less active than SM49

(Figure 22A). Anyway, the IC50 of SM49 is comparable to the best of previously reported pol β inhibitors.51,78,181

4.4.3) Discussion and structure‐activity relationships

At the beginning, the results of the previous work76, or more precisely the screening data of st 26 representative compounds of the 1 compound generation (SM1‐28) and IC50 data of selected compounds were reproduced and completed to the required triplicate determinations (Table 2, Figure 13, and 14). During the course of confirming the inhibitory potential of the initial compound library, other rhodanine‐based inhibitors were found that are highly active against pol λ. Furthermore, SM10, 16, 21, and 23 are like SM1 potentially useful molecular probes to inhibit selective pol λ over pol β to probe the enzymes’ roles in DNA repair. As expected, SM11, 12, 20 targeted also significantly pol β (Figure 13). SM12 was

39 Results and discussions most active and turned out to be a very interesting starting point for the development of novel pol β inhibitors. Based on the reproduced initial data, a molecular scaffold could be introduced for the design of a novel compound series (SM29‐58) (Table 2, and 3). The molecular scaffold consists of three variable parts R1, R2, and R3, which are connected via an aromatic core and a variable linkage Z. Based on the biochemical evaluation of the 2nd compound generation (SM12, 29‐ 58) in radioactive PEX assays, several further highly potent inhibitors of pol λ and β were discovered and the previous reported basic SAR was confirmed and significantly extended. The survey herein clearly demonstrates the remarkable importance of the heterocyclic warhead for the effectiveness of the small‐molecule inhibitors against both human DNA polymerases. It could be shown that the hydrogen atom in moiety A cannot be replaced. In line with the substitution of the hydrogen atom to an allyl group SM27 (moiety C) or a phenyl group SM28 (moiety D)76 also the replacement by an acetic acid group (moiety E) resulted in a completely inactive compound (SM30) against pol λ and β. Moreover, the endocyclic sulphur atom in moiety A proved to be important since the substitution with an NH group (moiety F) in compound SM31 resulted in a loss of activity. In contrast, modifications at the exocyclic double bond (moieties H, and I) were tolerated. In consequence of methylation of the double bond in SM33 (moiety H), the high potency against pol λ was conserved, but resulted in a compound with moderate activity against pol β. The hydrogenation of SM1 to SM34 was attended by the change of the hybridization state of the exo‐ and endocyclic carbon atoms (sp3 to sp2). Pol λ could be inhibited by SM34, but with a lower effectiveness than SM1. The replacement of the exocyclic sulphur of moiety A by an oxygen atom (moiety B) or an NH group (moiety G) resulted in indifferent results. Moiety G containing SM32 was totally inactive against both family X DNA polymerases. Interestingly, by substitution of moiety A with moiety B, SM29 was generated, showing the same inhibitory properties as lead compound SM1, but with a 2,4‐thiazolidinone warhead.

In the screening of the 1st compound generation, also SM2 proved to be a pol λ inhibitor in the low micromolar range (IC50 = 10.0 μM). For that reason, SM2 was “combined” with SM1 and so the very potent pol λ inhibitor SM57 was created (IC50 = 4.0 μM). SM57 was moderately active against pol β, too. As mentioned before, the thioether used as Z linkage proved not to be essential for high activities or selectivity and could be replaced by ester, benzyl phenyl ether, or diphenyl ether linkages (Table 2). So only the sulphone linkage was

40 Results and discussions installed in SM35 herein. By oxidation of the thioether to the sulphone Z linkage, the pol λ selective inhibitor SM35 with a lower activity than SM1 was generated.

Comparing SM37‐56, it is evident that the variation of the substituents in position R2 and R3 of the scaffold is also able to influence the activity against both family X DNA polymerases. 2 With the introduction of a variety of substituents in position R (like H, F, Cl, Br, CF3, CN, NO2), high inhibitory activity was maintained against pol λ without exception. Interestingly, variation of the R2 substituent to Cl (SM39), Br (SM40), and CN (SM42) led to compounds that are able to discriminate between pol λ and β. By analyzing SM37, 38, 41, 54‐56, it turned out that the activity increased considerably against pol β in the specified order H ≤ F < 3 CF3. Re‐proved by SM36, R belongs to the inhibitory core structure and therefore it cannot be waived. Looking at both compound series with variations only in R3, it was found that all tested compounds (except SM15, R3 = 2‐pyridine) were able to inhibit pol λ. It is noteworthy 3 that the R modifications 4‐F3C‐Py‐ or 4‐F3C‐Ph‐ to 4‐F3CO‐Ph‐ and cyclohexane to cyclopentane led to moderately active pol λ inhibitors. A particularly interesting discovery is that the alkylation pattern of the aromatic ring in R3 not only affects the activity against pol λ but also against pol β. A close look at SM1, 43, 48‐50, and 54‐56 reveals that the IC50 value against pol λ decreases in the same order (4‐Me‐Ph > 3‐Me‐Ph > 4‐Me‐Ph), as the activity against pol β is modulated (4‐Me‐Ph << 4‐Et‐Ph = 3‐Me‐Ph < 4‐Me‐Ph). Lastly, the first compound (SM58) with a heterocyclic aromatic core structure was analyzed. As result of the bioisosteric thiophene ring, the activity against pol λ was preserved, but the selectivity dropped.

4.4.4) Conclusion

Due to the biochemical evaluation of the novel entities, 23 highly active compounds against pol λ (SM12, 29, 33, 37‐44, 46‐50, 53‐58) with IC50 values less than 10 μM were discovered. Interestingly, ten of these small‐molecules (SM29, 39, 40, 42, 44, 46, 47, 51‐53) selectively inhibited pol λ in a low micromolar range but not pol β. The exact IC50 values were determined for the five most active compounds. Compounds SM39, 48, 49, 53, and 57 dose‐ dependently inhibited the polymerization function of pol λ with IC50 values of 5.7, 6.0, 3.9,

4.0, and 4.0 μM and are thus equally or even more active than lead compound SM1 (IC50 =

41 Results and discussions

75,76 5.9 μM) . Moreover, SM39, 48, 49, 53, and 57 act also as inhibitors of the TdT function of pol λ. In addition, 14 novel small‐molecules (SM12, 33, 37, 38, 41, 43, 48‐50, 54‐58) that target pol β were identified. The exact IC50 values were determined in turn for the four most active compounds. SM38, 41, 48, and 49 inhibited dose‐dependently the polymerization function of pol β with IC50 values of 38.7, 28.1, 29.8, and 18.2 μM. With the aid of the molecular scaffold and the novel PEX data, the initial reported basic SAR76 could be extended and discussed in depth.

In order to draw comparisons between the discovered small‐molecule pol λ and β inhibitors (SM1, 49) and literature known inhibitors, a variety of reported inhibitors were explored in side‐by‐side comparisons using the same PEX conditions. SM1, 49 were much more active than the on natural products based inhibitors. In consequence, the rhodanines are currently the strongest inhibitors for pol λ and comparable to the best of previously reported pol β inhibitors.37,51,78,177,181

4.5) Cellular investigations of the 1st and 2nd small‐molecule generation

4.5.1) Introduction

During the course of evaluating the compound libraries of the 1st and 2nd generation in vitro, several inhibitors of pol λ and or β with different very interesting features were discovered. In general, rhodanines are classified as nonmutagenic,182 and a long‐term study on the clinical effects of the rhodanine‐based Epalrestat demonstrated, that it is well tolerated by patients.183 Nevertheless, to further develop the novel rhodanine based molecular probes and to study the exact roles of pol λ and β in a cellular environment (see also chapter 1), the effect of the small‐molecules itself on human cell lines has to be investigated first. In the next step, the applicative probes can be utilized to target known cellular DNA polymerase pathways or rather the respective DNA polymerase. Given the facts that human pol β and λ are key enzymes of the BER pathway (Figure 5) and discussed as promising cellular targets, especially to overcome resistance of DNA damaging agents in the case of cancer treatment.15,17,20,37,51,70,74,78,79,100 Consequently, the induction of artificial DNA damage ‐ that

42 Results and discussions is repaired via the BER pathway ‐ would be ideally suited to study the novel probes for the first time in a cellular regime.

4.5.2) Cell viability measurements of potential rhodanine probes

Given that pol λ is discussed as future drug target and motivated by the promising in vitro data, the effect of novel rhodanine based small‐molecules (1st generation) on human cell lines was investigated. For this purpose, the highly potent and selective pol λ inhibitors SM1, 10, 16, 21, and 32 of synthetic origin were selected (Table 2).

Figure 23. Results of cell viability measurements on human cancer cell lines. Increasing concentrations of compounds SM1, 10, 16, 21, and 32 were used to estimate the EC50 values (red lines) on HeLa‐S3 and Hep‐G2 cells by the adopted AlamarBlue assay. Averages of four independent experiments and standard deviations are shown.

43 Results and discussions

The half‐maximal inhibitory concentration of the cell viability (EC50) of the rhodanines was determined using an adapted version184 of a previously described AlamarBlue assay185 and two human cancer cell lines, the cervix carcinoma cell line, HeLa‐S3, and the hepatocellular carcinoma cell line, Hep‐G2 (Figure 23). In both of these cancer types pol λ is overexpressed.72,186 As shown in Figure 23, viability of both cell lines was suppressed dose‐ dependently by each small‐molecule after 48 h incubation. SM10, with EC50 values of 7.9 and

6.1 μM against HeLa‐S3 and Hep‐G2 cells, was due to the 4‐(trifluoromethyl)pyridine substituent the most toxic compound. The other compounds affected the viability of these cell lines at concentrations 2 to 5‐fold higher than the IC50 values of pol λ (Table 2). These results suggest that SM1, 10, 16, 21, and 32 are suited small‐molecule probes for broader studies in a cellular context.

4.5.3) Co‐treatment experiments with genotoxic agents and probes SM1 or SM49

Many chemotherapeutic regimes in use depend at least in part on the artificial induction of DNA damage and the clinical efficacy of these therapies is often reduced by cellular DNA repair mechanisms.15,17,37,51,70,74,78,79,100 Hence, there is an urgent need for the development of novel approaches to enhance tumor cell cytotoxicity of chemotherapeutics for the treatment of cancers.37,51,70,74,78,79,100 Motivated by the facts, that the BER repair proteins pol

β and λ are discussed as promising future drug targets to overcome resistance of genotoxic agents15,17,37,51,70,74,78,79,100 and the novel rhodanines are suited small‐molecule probes for broader studies in a cellular context (Figure 23), co‐treatment experiments of DNA‐damaging agents with rhodanine probes were explored.

The anticancer regime was investigated with the approved monofunctional alkylating drug temozolomide (TMZ) and the model ROS inducer H2O2 on the human colon carcinoma cell line Caco‐2. This cell line is especially interesting since colorectal cancer is the second leading cause of all cancer‐related deaths in developed countries.187 In addition, pol β and λ were found to be overexpressed in colorectal cancer tissue.72 To find suitable conditions for this proof‐of‐concept study, first the previously described 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐ diphenyltetrazolium bromide (MTT) assay188,189 was adapted. Afterwards, the half‐maximal nd inhibitory concentration of the cell viability (EC50) of SM1, SM49 (2 generation), TMZ, and

44 Results and discussions

H2O2 was measured. As shown in Figure 24, cell viability was suppressed dose‐dependently by SM1 (EC50 = 58.1 μM) and SM49 (EC50 = 63.8 μM) after 96 h incubation and by H2O2 (EC50 =

540 μM) after 72 h incubation. Because of the low cellular response of the Caco‐2 cells to

TMZ, the exact EC50 value of TMZ could not be determined (EC50 > 1000 μM).

Figure 24. Determination of cell death of compounds SM1 and SM49, model ROS inducer H2O2 and monoalkylating drug TMZ on Caco‐2 cells by the MTT assay. Cells were incubated with compound SM1 and SM49 for 96 h with increasing concentrations (6.25, 12.5, 25, 50, 75, 100, 500 μM); with H2O2 for 72 h with increasing concentrations (62.5, 125, 250, 500, 625, 750, 1000 μM) and with TMZ for 72 h with increasing concentrations (62.5, 125, 250, 500, 750, 1000 μM). EC50 values are shown in red. Co‐treatment experiments were performed at concentrations that yielded responses below 30% cell death (green line). Averages of at least three independent experiments and standard deviations are shown.

Afterwards, co‐treatment experiments were assayed at concentrations that yielded responses below 30% cell death, to study whether SM1 or SM49 can sensitize Caco‐2 cells towards TMZ (Figure 25) or H2O2 (Figure 26).

45 Results and discussions

Figure 25. Compounds SM1 and SM49 sensitize colorectal cancer cells to the monoalkylating drug TMZ. Caco‐2 cells were preincubated with (A) 12.5 μM and (B) 25 μM SM1 or SM49. After 24 h, cells were treated with 500 μM TMZ for 72 h. Then, cell death induction was measured by the MTT assay. Averages of at least seven independent experiments and standard deviations are shown.

Figure 26. Compounds SM1 and SM49 sensitize colorectal cancer cells to the model ROS inducer H2O2. Caco‐2 cells were preincubated with (A) 12.5 μM and (B) 25 μM SM1 or SM49. After 24 h, cells were treated with 125 μM H2O2 for 72 h. Then, cell death induction was measured by the MTT assay. Averages of at least seven independent experiments and standard deviations are shown.

46 Results and discussions

The resultant MTT assay data shows, that SM1 and SM49 enhanced the sensitivity of human

Caco‐2 cells at 12.5 µM and 25 µM towards TMZ (Figure 25) and H2O2 (Figure 26). Indeed, the major pathway for oxidative and monoalkylated DNA damage recognition and repair is the BER.16,17,51,70,78,100 The additive or even synergistic effect of cell death induction in Caco‐2, caused by inhibitor SM1 and SM49 together with the ROS inducer H2O2 or monoalkylating drug TMZ, suggests that both small‐molecules act in a way that affects the BER pathway. To prove these facts in detail is particularly challenging, but these findings support the efforts to further develop the discovered pol λ and β inhibitors in vitro and in a cellular context.

4.5.4) Conclusion

With the aim to further develop the rodanine based probes in a cellular environment, first the effect of the small‐molecules itself on human cell lines was investigated. Cell viability of HeLa‐S3 and Hep‐G2 cancer cell lines was suppressed dose‐dependently by selected small‐ molecules of the first generation (SM1, 10, 16, 21, and 32). Except SM10 (with an 4‐(trifluoromethyl)pyridine substituent in R3), the rhodanines showed moderate toxicities on both human cell lines. In addition, the results suggested that SM1, 16, 21, and 32 are suited small‐molecule probes for broader studies in a cellular context. Next, the discovered compounds were explored in co‐treatment experiments on colorectal cancer cells (Caco‐2). In these proof‐of‐concept studies, it was assayed, whether the discovered compounds sensitize cancer cells towards the artificial induction of DNA‐damage. Therefore, human colorectal cancer cells (Caco‐2) were co‐treated with the discovered probes SM1 and SM49 and the approved monoalkylating drug TMZ or the model ROS inducer H2O2. Importantly, the tested small‐molecules SM1 and SM49 were pharmacologically active and sensitized Caco‐2 cells towards both genotoxic agents. Because chemotherapies depend frequently in part on the artificial induction of DNA damage, consideration of targeting DNA repair capacity is an ongoing concern in improving responses to treatments.15,17,20,37,51,70,74,78,79,100 Like other gene products involved in DNA damage repair, the regulation of pol β and λ, which are overexpressed in cancer tissue,72 could be fundamental in cancer treatment.16,17,51,70,78,100 The herein reported cellular studies support this notion, as SM1 and SM49 enhanced the sensitivity of human colorectal cancer cells

47 Results and discussions

towards the genotoxic TMZ and H2O2 considerably. The additive or even synergistic effects indicate that SM1 and SM49 have the potential to reduce the dosage of DNA damaging reagents, while improving their activity via inhibition of the BER pathway. For this reason, the discovered small‐molecules not only might be of great value for basic science but may also serve as lead structures for the development of novel avenues for the treatment of diseases related to genome integrity.

48 Results and discussions

5) Synthesis of 4’‐C‐alkylated‐5‐iodo‐2’‐deoxypyrimidine nucleosides as potential antiviral drugs and synthetic building blocks

5.1) Introduction

For years, chemically modified nucleoside analogues have been outstanding life‐saving medicines.13,20,37,41,190‐197 This pharmacologically manifold compound class, which contains structural features of the skeleton of natural nucleosides, is frequently used for cancer therapy and the treatment of viral infections.20,41,81,190‐196,198‐200 Along with human immunodeficiency virus (HIV) and hepatitis virus (HV); herpes simplex virus (HSV) and varicella‐zoster virus (VZV) are prominent pathogens. In addition to the approved drugs brivudine, acyclovir, and vidarabine, HSV and VZV are also medicated with the prototype of antiviral drugs 2’‐deoxy‐5‐iodouridine (also known as iodoxuridine) N1 since over 40 years (Figure 27).20,190,192,193,196,201

O H N O N O OH HO O O N N NH N Br HO NH2 brivudine acyclovir

N NH2 HO O N N N HO OH

vidarabine

H O N O N O NH2 HO O N HO O N I I HO HO N1 N2

Figure 27. Chemical structures of the approved drugs brivudine, acyclovir, vidarabine, 2’‐deoxy‐5‐iodouridine N1, and 2’‐deoxy‐5‐iodocytidine N2. Figure was adapted from literature 20.

Drug N1, launched for example as Herples®, Stoxil®, Iodoxene®, and Virodox®, targets the DNA replication of the viruses.193,196,201 Thereby, N1 acts as an antagonist of thymidine, its

49 Results and discussions natural nucleoside counterpart, and targets the thymidylate phosphorylase and the viral DNA polymerase ‐ the workhorse of the DNA replication.41,193,196,201 In general, 5‐substituted‐ 2´‐deoxycytidines are appreciably more selective, but equally or slightly less potent in their anti‐HSV activity than the corresponding 5‐substituted‐2´‐deoxyuridines.196,197,202,203 Thus, the antiviral spectrum of 2’‐deoxy‐5‐iodocytidine N2 (Figure 27), marketed as Cebeviran® or Cuterherpes®, is similar to N1 to which medicine N2 is turned over by enzymatic deamination.196,197,202,204 Besides 5‐halopyrimidine nucleosides, 4’‐C‐modified nucleosides aroused scientific interest, because a couple of derivatives of this interesting compound class showed antiviral activity.145,146,148‐153 In doing so, 4’‐C‐modified nucleosides functions as nucleoside reverse transcriptase inhibitors (NRTIs)145‐147,154,205 and showed even activity against multi‐drug resistant virus strains.151,154 The evolution of viral resistance boosts the urgent need for new effective drugs and therapies against viral infections.88,89 Because there is a great demand for the development of drugs2,12,13,88,90,91 and consequently also for novel nucleoside analogues, a synthetic route for 4´‐C‐alkylated‐5‐iodo‐2´‐deoxyuridines N3a‐c and 4´‐C‐alkylated‐5‐iodo‐2´‐deoxycytidines N4a‐c was designed and developed (Figure 28). The resulting novel entities N3a‐c and 4a‐c could be of great pharmaceutical interest, because they fuse the structural features of the marketed drug N1 or 2, and 4´‐C‐alkylated nucleosides in one small molecule. In addition, the intermediates on synthesis route to N3a‐c and 4a‐c, or the developed compounds itself could function as key building blocks in order to open up the way to several further 4´‐C‐modified‐5‐substituted nucleoside analogues of scientific and pharmaceutical interest.

H O N O N R O R NH2 HO O N HO O N I I HO HO N3a-c N4a-c

R:Me(a); Et (b); n-Pr (c)

Figure 28. Chemical structures of the designed 4’‐C‐alkylated‐5‐iodo‐2’‐deoxyuridines N3a‐c and 4’‐C‐alkylated‐5‐iodo‐2’‐deoxycytidines N4a‐c.

It is noteworthy to mention that 4´‐C‐modifications of nucleosides always comprise the generation of quaternary carbon centres including the obstacles associated with the

50 Results and discussions respective chemistry.206 Three main methodologies have been evolved for the synthesis of 4´‐C‐modified nucleosides (Figure 29). In the first methodology a 4´‐C‐branch is attached to 2´‐C‐deoxynucleosides;206,207 in the second methodology the asymmetric SAMP/RAMP‐ hydrazone α‐alkylation and diastereoselective nucleophilic 1,2‐addition with Grignard and organocerium reagents is capitalized;208,209 and in the third methodology suitable 4‐C‐ribose glycosyl donors164 are synthesized for the nucleoside formation using Vorbrüggen`s method210,211 (Figure 29, 30).206

methodology one

O O O HO Base Base R PGO O Base 2`-deoxynucleoside PGO PGO PGO

methodology two

O R OH R PGO R O OPG O Base OO PGO O OOO PGO PGO

methodology three

O O R R O PGO O Base D-glucose O O PGO PGO OAc O PGO OAc PGO OAc

Figure 29. Main methodologies for the synthesis of 4’‐C‐modified nucleosides analogues. In the first methodology a 4´‐C‐branch is attached to 2´‐C‐deoxynucleosides;206,207 the second methodology the asymmetric SAMP/RAMP‐hydrazone α‐alkylation and diastereoselective nucleophilic 1,2‐addition is capitalized;208,209 and in the third methodology flexible 4‐C‐ribose glycosyl donors are synthesized for the nucleoside formation using Vorbrüggen`s method208‐211. R = modification, PG = protection group. Figure was adapted from literature 206,212.

Over the past years Marx et al. designed and synthesized a series of 4´‐C‐modified nucleosides and nucleotides.147,164,213‐221 In the present work this profound knowledge was combined with literature known synthesis strategies for N1222 and 12a153 in order to open up the way towards N3a‐c. On the basis of the transformation of uridines or thymidines into the respective cytidine analogues,216,223,224 N3a‐c were planed to be converted into the 4’‐C‐alkylated‐5‐iodo‐2’‐deoxycytidines N4a‐c. To evaluate whether the novel 4’‐C‐alkylated‐ 5‐iodo‐2’‐deoxypyrimidines are utilizable as building blocks to get access to further modified

51 Results and discussions nucleosides, compound N12c with the biggest hydrophobic 4’‐C‐modification was investigated in a Sonagashira test reaction.

5.2) Synthesis of 4‐C‐modified carbohydrate building blocks

Suitable 4‐C‐modified glycosyl donors ‐ methodology three ‐ have been widely used as precursors for the synthesis of 4´‐C‐modified nucleosides and nucleotides, because they are producible in a multigram‐scale starting from D‐glucose or D‐allofuranose (Figure 29, 30).206,225 After benzylation and selective acid hydrolysis of the

5,6‐O‐isopropylidene group of D‐allofuranose, periodate cleavage yielded an aldehyde intermediate.206,225 Afterwards, the 4‐C‐hydroxymethyl branch was installed by using formaldehyde in an aldol reaction that was followed by a Cannizzaro reduction in one pot.225,226 The selective silylation with tert‐butyldiphenylsilyl chloride (TBDPSCl) leaded to the versatile key ribose building block N6 (Figure 30).227

Figure 30. Retrosynthetic scheme for key building block N6 starting from D‐allofuranose.

Recently, Rangam et al. reported a nine‐steps reaction sequences for 4`‐C‐methyl‐ and 4`‐C‐ethyl‐substituted deoxyuridines N5a‐b.164 According to these synthetic routes, nucleosides N5a‐b and the respective glycosyl donors N8a‐b were obtained.164 In addition, the synthesis of the novel 4`‐C‐propyl‐substituted deoxyuridines N5c and the corresponding novel glycosyl donor N8c were investigated (Figure 31).

52 Results and discussions

164 Figure 31. Synthesis of glycosyl donors N8a‐c. Reagents and Conditions: i) Ph3P, imidazole, I2, toluene, reflux; 164 164 ii) n‐Bu3SnH, AIBN, toluene, reflux, 96% over 2 steps; iii) DMP, CH2Cl2, r.t., 91%; iv) MePPh3Br, n‐BuLi, THF, 164 164 164 r.t., 97%; iv) EtPPh3Br, t‐BuOK, THF, r.t., 84%; vi) AcOH, Ac2O, H2SO4, r.t, 79% (N8a) , 90% (N8b) , 64% (N8c).

After conversion of N6 with triphenylphosphine (Ph3P) and iodine, to the 4‐C‐iodomethyl derivative, the iodo intermediate was subsequently reduced with tributyltin hydride 164 (n‐Bu3SnH) to the 4‐C‐methyl derivative N7a. Acetolysis of N7a led to the 4‐C‐methyl‐ bis‐ acetate N8a (Figure 31).164,206

In order to synthesize the 4‐C‐vinylated and 4‐C‐(Z)‐prop‐1‐enylated bis‐acetates N8b‐c, compound N6 was oxidised with Dess‐Martin periodinane (DMP)165 to yield the corresponding aldehyde.164 Afterwards, Wittig reaction was used for C‐C‐bond formation to yield the 4‐C‐vinylated N7b164 and 4‐C‐(Z)‐prop‐1‐enyl ribose analogs N7c. Wittig reaction afforded for N7b the introduction of the C1‐unit with small steric restraints. The reaction could be carried out with methyltriphenylphosphonium bromide (MePPh3Br) and n‐butyllithium (n‐BuLi) as base.164 In contrast, bulky alkoxides have previously been reported to be the bases of choice in Wittig reactions involving sterically encumbered substrates.218,228 Thus, the reaction was performed with potassium tert‐butoxide (t‐BuOK) and ethyltriphenylphosphonium bromide (EtPPh3Br) as C2‐synthon. In addition, these cis‐ selective conditions (JHC=CH for N7c = 11.8 Hz) had the positive side effect that the possible products in the resulting diastereomeric mixture were brought to a minimum. By protection group manipulations N7b and 7c were converted to the substituted ribosyl acetates N8b and 8c in good yields (Figure 31).164

53 Results and discussions

5.3) Synthesis of 4’‐C‐alkylated‐pyrimidine nucleosides

Holding the substituted 4‐C‐modified glycosyl donors N8a‐c in hand, the nucleobase uracil was fused with N8a‐c according to the Vorbrüggen glycosylation210,211 (Figure 32). The reaction with bis(trimethylsilyl)uracil, which is formed as an intermediate by silylation of uracil with bis(trimethylsilyl)acetamide (BSA), and trimethylsilyl triflate (TMSOTf) as catalyst gave stereoselectively the β‐configurated 4´‐C‐methyl164, 4´‐C‐(Z)‐vinyl164, and (Z)‐prop‐ 1‐enyl substituted nucleoside N9a‐c.

Figure 32. Synthesis of 2´‐deoxy‐4´‐C‐methyl‐, 2´‐deoxy‐4´‐C‐ethyl‐, and the new 2´‐deoxy‐4´‐C‐propyluridine N5a‐c. Reagents and conditions: i) Uracil, BSA, TMSOTf, MeCN, reflux, 77% (N9a)164, 74% (N9b)164, 71% (N9c); ii) 164 NaOMe, MeOH, r.t.; iii) PhOCSCl, DMAP, MeCN, r.t.; iv) n‐Bu3SnH, AIBN, toluene, reflux; 70% (N10a) , 72% 164 164 164 (N10b) , 83% (10c) over 3 steps; v) 10% Pd/C, H2, EtOH, r.t.; vi) TBAF, THF, r.t, 79% (N5a) 89% (N5b) , 65% (N5c) over 2 steps.

After deacetylation with sodium methoxide (NaOMe) and reaction with phenyl chlorothionoformate (PhOCSCl) in the presence of 4‐dimethylaminopyridine (DMAP) the thiocarbonate esters were obtained, which were successive reduced with tributyltin hydride

(n‐Bu3SnH) to the 2´‐deoxyuridine derivates N10a‐c. Catalytic hydrogenation with Pd/C followed by desilylation with tetrabutylammonium fluoride (TBAF) furnished the 2´‐deoxy‐ 4´‐C‐methyl‐, 2´‐deoxy‐4´‐C‐ethyluridines N5a‐b164 and the new 2´‐deoxy‐4´‐C‐propyluridine N5c (Figure 32).

5.4) Synthesis of 4’‐C‐alkylated‐5‐iodo‐2’‐deoxypyrimidine nucleosides

For the halogenation in 5‐position of the uridines, a literature known synthesis strategy for compound N1222 and 12a153 was followed. Therefore nucleosides N5a‐c were acetylated to yield compounds N11a‐c. Next, the diammonium cerium (IV) nitrate (CAN) mediated

54 Results and discussions iodination (N12a‐c), followed by deprotection furnished in good to excellent yields the desired 4´‐C‐methyl‐, 4´‐C‐ethyl‐ and 4´‐C‐propyl‐5‐iodo‐2´‐deoxyuridine analogues N3a‐c (Figure 33).

Figure 33. Synthesis of 4´‐C‐alkylated‐2´‐deoxy‐5‐iodouridine derivates N3a‐c. Reagents and conditions: i) Et3N, Ac2O, DMAP, MeCN, r.t., 63% (N11a), 74% (N11b), 93% (N11c); ii) I2, CAN, MeCN, reflux, 89% (N12a), 98% (N12b), 89% (N12c); iii) NaOMe, MeOH, r.t., 91% (N3a), 97% (N3b), 97% (N3c).

The strategy to synthesize the 4´‐C‐alkylated‐5‐iodo‐2´‐deoxycytidines N4a‐c was based on the tranformation of uridines or thymidines into the corresponding cytidine derivates (Figure 34).216,223,224 In the first trail to make N4a‐c accessible, the acetylated 5‐iodo‐2´‐ deoxyuridines N12a‐c were treated unsuccessfully with the 2,4,6‐ triisopropylbenzenesulfonyl chloride (TPSCl)‐Et3N‐DMAP system followed by aminolysis with ammonium hydroxide (NH4OH) in order to generate the exocyclic amino functions and to deacetylate the sugar moieties.

Figure 34. Synthesis of 4´‐C‐alkylated‐2´‐deoxy‐5‐iodocytidine analogues N4a‐c. Reagents and conditions: i) TBDMSCl, imidazole, DMF, r.t., 81% (N13a), 90% (N13b), 91% (N13c); ii) 1.) TPSCl, DMAP, Et3N, MeCN, r.t.; 2.) 28% NH4OH, 78% (N14a), 77% (N14b), 80% (N14c); iii) TBAF, THF, r.t., 92% (N4a), 89% (N4b), 81% (N4c).

Because no product could be isolated in the first trail, the protecting group strategy had to be changed and N3a‐c were silylated with TBDMSCl in the presence of imidazole to yield

55 Results and discussions compounds N13a‐c. After silylation, the conversion of N13a‐c into N14a‐c with the (TPSCl)‐

Et3N‐DMAP system followed by aminolysis with ammonium hydroxide (NH4OH) to install the exocyclic amino functions was successful. Finally, desilylation with TBAF yielded the 4´‐C‐ methyl‐, 4´‐C‐ethyl‐ and 4´‐C‐propyl‐ 5‐iodo‐2´‐deoxycytidine analogues N4a‐c (Figure 34).

5.5) Evaluation of the synthons ‐ exemplified by 3’,5’‐di‐O‐acetyl‐2’‐deoxy‐5‐ iodo‐4’‐C‐propyluridine N12c in a Sonagashira test‐reaction

Beside the usage of 5‐halopyrimidine nucleosides as antiviral drugs, they have been applied extensively as key building blocks for the synthesis of a wide range of biological probes and other modified nucleosides of great pharmaceutical interest.117,153,190,198,229‐236 To get access to further 5‐modified pyrimidines starting from 5‐halopyrimidines nucleosides and nucleotides, palladium catalysed cross‐coupling reactions emerged to popular reaction types.153,198,230‐233,235‐241

Figure 35. Evaluation of 3’,5’‐di‐O‐acetyl‐2’‐deoxy‐5‐iodo‐4’‐C‐propyluridine N12c in a Sonagashira test‐ reaction. Reagents and conditions: i) 0.1 eq Pd(PPh3)4, 0.2 eq CuI, NEt3, DMF, r.t., 57%; ii) NaOMe, MeOH, r.t., 97%.

56 Results and discussions

To evaluate whether the novel 5‐iodo‐2’‐deoxypyrimidine nucleosides with hydrophobic 4’‐ C‐modifications are utilizable as reactants in palladium catalysed coupling reactions, 3’,5’‐di‐ O‐acetyl‐2’‐deoxy‐5‐iodo‐4’‐C‐propyluridine N12c, was subjected to a Sonagashira test reaction (Figure 35). The coupling reaction between N12c and a representative terminal alkyne‐biotin conjuate N15242 was carried out in the presence of the catalyst tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), cuprous iodide (CuI) and dry NEt3 to yield N16c in 57%. In an additional step the ribose moiety was deacetylated to furnish N17c in 97% yield. It turned out that the 4’‐C‐alkylated‐5‐iodo‐2’‐deoxypyrimidine nucleosides are also valuable intermediates to open up the synthetic way to further 4’‐C‐modified nucleosides.

5.6) Discussion and Conclusion

In order to extend the chemical diversity of 4’‐C‐modified uridine and cytosine analogues with potential interesting pharmacological features, the well established methodology three

(Figure 29) was applied to build up of several known and novel 4’‐C‐alkylated uridines and cytosines. By using methodology three, 4‐C‐alkylated glycosyl donors N8a‐c or rather the 4’‐

C‐alkylated uridines N9a‐c were synthesized in multistep reaction sequences, starting from relative cheap and commercially available D‐allofuranose. Afterwards, analogues N9a‐c were successfully transformed according to standard procedures into the 2’‐deoxygenated analogues N5a‐c and those in turn into the corresponding 4’‐C‐alkylated‐5‐iodo‐2’‐ deoxyuridines 3a‐c. In the end an exocyclic amino group could be installed effectually to yield the 4’‐C‐alkylated‐5‐iodo‐2’‐deoxycytidines N4a‐c.

The novel nucleoside analogues N3a‐c and 4a‐c are 4’‐C‐alkylated derivatives of the approved antiviral drugs 2’‐deoxy‐ 5‐iodouridine N1 and 2’‐deoxy‐5‐iodocytidine N2. Due to the fact, that several derivatives of 4’‐C‐modified nucleosides also showed antiviral activity, the here reported molecules are generally of great interest because they fuse the structural features of the marketed drug N1 or 2, and 4´‐C‐alkylated nucleosides in one small molecule. Additionally, the reported molecules could serve as useful chemical building blocks for the development of further compounds of scientific and pharmaceutical interest. This fact could be verified by a Sonogashira test‐reaction. In doing so N12c was reacted with a

57 Results and discussions representative alkyne‐biotin conjuate N15 to yield the 4´‐C‐alkylated‐5‐alkynylated nucleoside N16c and so the herein reported molecules can also act as versatile synthetic tools to open up the synthetic way for further 4’‐C‐modified nucleosides.

58 Conclusion

Conclusion

The survival and development of each organism relies on the equal distribution of its genome during cell division. Errors in this process can lead to severe developmental defects, cancer, or even death.15‐17 DNA polymerases are key enzymes to pass the exact genomic information down generations (see also chapter 2). Over 50 years ago, Kornberg et al. discovered the first enzyme (DNA polymerase I), that catalyses the accurate replication of DNA.18,19 Since this and other pioneering discoveries, it was assumed for a long period of time, that only six “classical” DNA polymerases (pol α, β, γ, δ, ε, and TdT) are responsible for DNA replication and repair in mammalian cells.15,17,20,21 For that reasons, the discovery of several “novel” specialized DNA polymerases (pol η, θ, κ, λ, μ, ν, ι, ζ, and REV1) was a real sensation in the last decades (Table 1).15,17,20,22 So far, at least 15 different human DNA polymerases are known.15,17,20,22 In the course of errors in replication or by environmental conditions, DNA mutations and damages occur.16 To maintain the genetic integrity of the genome, an elaborate set of sophisticated repair mechanisms have evolved. The set includes, amongst others, the “novel” specialized DNA polymerases.15‐17,20,37 Features of some of these specialized enzymes are known, but to understand in depth the task of the majority the exact roles still await clarification. For that reason, there is a great demand for appropriate methods and reagents (e.g. the chemical genetics approach with its small‐molecule probes, see also chapter 1)4,5,7‐11 to dissect the cellular functions of DNA polymerases. The entire process of DNA metabolism takes minutes, and individual steps take place in seconds. Given their fast mode of action, cell‐permeable small‐molecules are ideally suited to interfere with the highly dynamic replication process.4,5,7‐11

In addition, DNA polymerases serve not only as a target for molecular probes to dissect their cellular functions, but have been tried and tested for decades as prominent target for life‐ saving medicines.15,17,20,22,37,41 Numerous pathological states, like cancer, autoimmune disease, and many bacterial and viral infections can be traced back to uncontrolled DNA metabolism.15,17,20,37,41,51,79 Today, it is one of modern medicine's top priorities to combat those diseases with novel and innovative drugs. For that reasons, these novel small‐molecule probes not only might be of great value for basic science but also may open up novel avenues for the treatment of diseases related to genome integrity.

59 Conclusion

The main part of this work deals with a reverse chemical genetics approach, in order to discover and develop molecular tools to dissect the functions of the homologous BER enzymes pol λ and β (chapter 4, see also chapter 2.3). In previous surveys several small‐ molecule inhibitors of human pol λ were identified and evaluated by applying SYBR® Green I based screening and radioactive primer extension (PEX) techniques (see also chapter 3.2).75,76 Some rhodanine‐based compounds, classified as a privileged drug scaffold,156‐161 were found to be the most active inhibitor class. The most potent inhibitor obtained was compound SM1, with an IC50 value of 5.9 μM. Interestingly, SM1 is up to ten 75,76 times less active against the highly homologous pol β (IC50 = 64.4 μM). Additionally, SM1 75 did not inhibit the DNA polymerases 9°N, Therminator, Pfu, and Dpo4 (IC50 > 100 μM). During the course of evaluating an initial compound library (1st compound generation) further rhodanine‐based inhibitors (SM10, 16, 21, 23) were found whose properties are comparable with SM1 and there were early indications that some compounds (e.g. SM11, 12, 20) also showed activity against pol β.76 Finally, preliminary basic structure‐activity relationships (SAR) could be established out of the in vitro data76, and thus, the rhodanine‐ based small‐molecules could be seen as an appropriate starting point for the development of molecular probes to specifically investigate the biological functions of pol λ and β.

On purpose to design a molecular scaffold for the creation of the 2nd small‐molecule st generation, first, the initial screening and IC50 data of the 1 compound generation were confirmed. Based on the reproduced in vitro data, a suitable molecular scaffold could be introduced for the design of the 2nd small‐molecule generation. Additionally, it was ascertained that SM1 acts also dose‐dependently on the TdT function of pol λ with an IC50 value of 4.5 μM. Continuing experiments with constant SM1 and increasing dNTP concentrations suggested that SM1 inhibits pol λ without directly competing for the substrate binding site.

In the next steps, the chemical diversity of rhodanine‐based small‐molecules with potential interesting pharmacological features could be further expanded by systematic synthetic optimization starting from cheap and commercially available building blocks. The scaffold oriented synthesis of the drug‐like molecules SM12, 29‐58 was subdivided into two parts. In the first part, the precursor aldehydes SM59‐61, 63‐83, and for compound SM33 a precursor

60 Conclusion

acetophenone SM62 were built up using high‐yielding SNAr reactions under basic conditions. Thereby activated halides were displaced at the aromatic core of the scaffold by the corresponding thiols. For the synthesis of precursor SM84, an elegant literature‐known three step synthesis sequence163 was assigned starting from SM59. Therefore, SM59 was reduced with NaBH4 to yield the methanol intermediate in 98%. Afterwards the intermediate was refluxed together with H2O2 to give SM85. In the last step, SM85 was oxidized with DMP to furnish precursor SM84. By performing the Knoevenagel condensation in the second part, several precursor molecules were fused together with varying heterocycles. To obtain the Z‐ isomeres exclusively, SM12, 29‐33, and 35‐58 were built up under thermodynamic reaction control in good yields. For the synthesis of racemic SM34 the exocyclic double bond of SM1 was reduced by the Hantzsch ester on silica gel method176.

4.0, and 4.0 μM and are thus equally or even more active than lead compound SM1 75,76 (IC50 = 5.9 μM) . Moreover, SM39, 48, 49, 53, and 57 act also as inhibitor of the TdT function of pol λ. In addition, 14 novel small‐molecules (SM12, 33, 37, 38, 41, 43, 48‐50, 54‐

58) that target pol β were identified. The exact IC50 values were determined in turn for the four most active compounds. SM38, 41, 48, and 49 inhibited dose‐dependently the polymerization function of pol β with IC50 values of 38.7, 28.1, 29.8, and 18.2 μM. With the aid of the molecular scaffold and the novel PEX data, the initial reported initial SAR76 could be extended and discussed in depth (chapter 4.4.5).

61 Conclusion

Next, the two representatives of the discovered compounds (SM1, SM49) were explored in co‐treatment experiments on colorectal cancer cells (Caco‐2). In these proof‐of‐concept studies, it was assayed, whether the discovered compounds sensitize cancer cells towards the artificial induction of DNA‐damage. Therefore, human Caco‐2 cells were co‐treated with the discovered probes (SM1, SM49) and the approved monoalkylating drug TMZ or the model ROS inducer H2O2. Importantly, SM1 and SM49 were pharmacologically active and sensitized Caco‐2 cells towards both genotoxic agents. Because chemotherapies depend frequently in part on the artificial induction of DNA damage, consideration of targeting DNA repair capacity is an ongoing concern in improving responses to treatments.15,17,20,37,51,74,78,79,100 Like other gene products involved in DNA damage repair, the regulation of pol β and λ, which are overexpressed in cancer tissue,72 could be fundamental in cancer treatment.15,17,37,51,53,78,100 The herein reported cellular studies support this notion, as SM1 and SM49 enhanced the sensitivity of human colorectal cancer cells towards the genotoxic TMZ and H2O2 considerably. The additive or even synergistic effects indicate that SM1 and SM49 have the potential to reduce the dosage of DNA damaging reagents, while improving their activity via inhibition of the BER pathway. For this reason, the discovered small‐molecules not only might be of great value for basic science but may also serve as lead structures for the development of novel avenues for the treatment of diseases related to genome integrity (see also chapter 3).

For years, chemically modified nucleoside analogues have been outstanding life‐saving medicines. This pharmacologically manifold compound class, which contains structural features of the skeleton of natural nucleosides, is frequently used for cancer therapy and the treatment of viral infections (see also chapter 3).20,41,81,190‐196,198‐200 Along with human

62 Conclusion immunodeficiency virus (HIV) and hepatitis virus (HV); herpes simplex virus (HSV) and varicella‐zoster virus (VZV) are prominent pathogens. In addition to the approved drugs brivudine, acyclovir, and vidarabine, HSV and VZV are also medicated with the prototype of antiviral drugs 2’‐deoxy‐5‐iodouridine (also known as iodoxuridine) N1 since over 40 years.20,190,192,193,196,201 N1 targets the DNA replication of the viruses. Thereby, N1 acts as an antagonist of thymidine, its natural nucleoside counterpart, and targets the thymidylate phosphorylase and the viral DNA polymerase ‐ the workhorse of the DNA replication.41,193,196,201 In general, 5‐substituted‐2´‐deoxycytidines are appreciably more selective, but equally or slightly less potent in their anti‐HSV activity than the corresponding 5‐substituted‐2´‐deoxyuridines.196,197,202,203 Thus, the antiviral spectrum of 2’‐deoxy‐5‐ iodocytidine N2 is similar to N1 to which medicine N2 is turned over by enzymatic deamination.196,197,202,204 Besides 5‐halopyrimidine nucleosides, 4’‐C‐modified nucleosides aroused scientific interest, because a couple of derivatives of this interesting compound class showed antiviral activity,145‐153 even against multi‐drug resistant virus strains.151,154 Because there is a great demand for the development of drugs2,12,13,88,90,91 and consequently also for novel nucleoside analogues, a synthetic route for the interesting 4´‐C‐alkylated‐5‐ iodo‐2´‐deoxyuridines N3a‐c and 4´‐C‐alkylated‐5‐iodo‐2´‐deoxycytidines N4a‐c was designed and developed in the second part of this work (chapter 5).

It is noteworthy to mention that 4´‐C‐modifications of nucleosides always comprise the generation of quaternary carbon centres including the obstacles associated with the respective chemistry.206 Three main methodologies have been evolved for the synthesis of 4´‐C‐modified nucleosides. In the first methodology a 4´‐C‐branch is attached to 2´‐C‐deoxynucleosides;206,207 in the second methodology the asymmetric SAMP/RAMP‐ hydrazone α‐alkylation and diastereoselective nucleophilic 1,2‐addition with Grignard and organocerium reagents is capitalized;208,209 and in the third methodology suitable 4‐C‐ribose glycosyl donors 164 are synthesized for the nucleoside formation using Vorbrüggen`s method210,211.206

In order to extend the chemical diversity of 4’‐C‐modified uridine and cytosine analogues with potential interesting pharmacological features, the well established methodology three was applied to build up several novel 4’‐C‐alkylated uridines and cytosines. By using methodology three, 4‐C‐alkylated glycosyl donors N8a‐c or rather the 4’‐C‐alkylated uridines

N9a‐c were synthesized in multistep reaction sequences, starting from relative cheap and

63 Conclusion

164,206 commercially available D‐allofuranose. Afterwards, analogues N9a‐c were successfully transformed according to standard procedures into the 2’‐deoxygenated analogues N5a‐c and those in turn into the corresponding 4’‐C‐alkylated‐5‐iodo‐2’‐deoxyuridines 3a‐c. In the end an exocyclic amino group could be installed effectually to yield the 4’‐C‐alkylated‐5‐iodo‐ 2’‐deoxycytidines N4a‐c.

64 Zusammenfassung

Zusammenfassung

Das Überleben und die Entwicklung aller Lebewesen hängen von der exakten Teilung des Erbguts während der Zellteilung ab. Fehler in diesem Prozess können zu schweren Entwicklungsstörungen, Krebs oder sogar zum Tode führen.15‐17 DNA‐Polymerasen sind Schlüsselenzyme, welche die exakte genomische Information über Generationen weiter geben (Kapitel 2). Vor über 50 Jahren, entdeckte Kornberg et al. das erste Enzym (DNA‐ Polymerase I), das in der Lage war die akkurate DNA‐Replikation zu katalysieren.18,19 Seit dieser und weiterer bahnbrechenden Entdeckungen wurde für einen sehr langen Zeitraum angenommen, dass nur sechs „klassische“ DNA‐Polymerasen (pol α, β, γ, δ, ε und TdT) in Säugetierzellen für die DNA‐Replikation verantwortlich sind.15,17,20,22 Infolgedessen waren die Entdeckungen von „neuen” spezialisierten DNA‐Polymerasen (pol η, θ, κ, λ, μ, ν, ι, ζ und REV1) eine echte Sensation in den letzten Jahrzehnten (Tabelle 1).15,17,20,22 Heute sind mindestens 15 menschliche DNA‐Polymerasen bekannt.15,17,20,22 Durch Fehler während der DNA‐Replikation oder durch externe Umwelteinflüsse können DNA‐Schädigungen oder Mutationen entstehen.16 Deshalb hat sich um die genetische Integrität des Genoms zu bewahren ein gut durchdachter Satz an komplexen Reparaturmechanismen entwickelt. Unter anderem beinhaltet das Reparaturset die „neuen“ spezialisierten DNA‐Polymerasen.15‐17,20,37 Einige Aufgaben dieser hochspezialisierten Enzyme sind bekannt, doch um die Funktionen der Mehrzahl tiefgründig zu verstehen, bedarf es noch weiterer Aufklärung. Aus diesem Grund gibt es großen Bedarf an nützlichen Methoden und innovativen Reagenzien (wie z.B. den chemisch genetischen Ansatz mit seinen niedermolekularen Sonden, siehe Kapitel 1)4,5,7‐ 11 um die exakten zellulären Funktionen der DNA‐Polymerasen zu klären. Der gesamte Prozess der DNA‐Replikation benötigt Minuten, jedoch laufen einzelne Schritte in Sekunden ab. Deshalb sind zellpermeable niedermolekulare Moleküle durch ihre schnelle Wirkungsweise sehr gut geeignet um in den sehr dynamischen Replikationsprozess einzugreifen.4,5,7‐11

Hinzukommend fungieren DNA‐Polymerasen nicht nur als Wirkziel um chemisch genetische Fragestellungen zu beantworten, sondern sind seit Jahrzehnten erprobte Ziele für lebensrettende Medikamente. Viele sehr ernstzunehmende Krankheiten, wie z.B. Krebs, Autoimmunerkrankungen und diverse bakterielle und virale Infektionen, können unter

65 Zusammenfassung anderem auf einen unkontrollierten DNA‐Metabolismus zurückgeführt werden.15,20,37,41,51,79 Heutzutage ist es eines der Hauptziele der modernen Medizin, diese Erkrankungen mit neuen und innovativen Arzneimitteln zu bekämpfen. Aus diesem Grunde könnten diese neuen niedermolekularen Wirkstoffsonden nicht nur großen Nutzen für die Grundlagenforschung, sondern auch neue Wege zur Behandlung von Krankheiten eröffnen.

Im haupsächlichen Focus der hier vorliegenden Arbeit steht ein reverser chemisch genetischer Forschungsansatz zur Erforschung der humanen pol λ and β. Der Ansatz verfolgt hierbei das Ziel, neue niedermolekulare Sonden zu entdecken und weiter zu entwickeln, die es ermöglichen, die Funktionen beider BER Enzyme pol λ and β der DNA‐Polymerasenfamilie X in ihrem biologischen Kontext zu untersuchen (Kapitel 4, siehe auch Kapitel 2.3). In diesem Werk vorangehenden Arbeiten wurden niedermolekulare Inhibitoren der menschlichen pol λ durch eine auf SYBR® Green I basierende Durchmusterung und durch die Anwendung von radioaktiven Primerverlängerungsreaktions (PEX)‐Techniken identifiziert (siehe auch 3.2).75,76 Die Verbindungsklasse der Rhodanine, die als privilegiertes Wirkstoffgerüst klassifiziert 156‐161 wurden , stellten sich als die aktivsten Inhibitoren heraus. Mit einem IC50 Wert von 75,76 5.9 μM war SM1 das aktivste Molekül. Interessanterweise war SM1 zehnfach weniger aktiv gegen die strukturell homologe pol β (IC50 = 64.4 μM) und wirkte nicht gegen die DNA‐ 75 Polymerasen 9°N, Therminator, Pfu und Dpo4 (IC50 > 100 μM). Durch die Evaluation einer ersten selbstgenerierten Verbindungsbibliothek (1st compound generation) wurden weitere Rhodaninanaloga (SM10, 16, 21, 23) entdeckt, deren Eigenschaften vergleichbar mit SM1 waren.76 Zudem gab es erste Anzeichen, dass manche Verbindungen (z.B. SM11, 12, 20) auch Aktivität gegen die pol β zeigten.76 Zuletzt konnten aus den in vitro Daten erste Struktur‐ Wirkbeziehungen (SAR) aufgestellt werden, welche untermauerten, dass die gefundenen Rhodanine als Ausgangspunkt für die Entwicklung von Sonden zur Untersuchung der biologischen Funktionen von den pol λ und β geeignet sind.76

Mit der Absicht ein molekulares Grundgerüst zur Kreierung einer zweiten Generation an niedermolekularen Verbindungen (2nd compound generation) zu designen, wurden zuerst die vorangehenden Durchmusterungs‐ und IC50‐Daten der ersten Molekülgeneration reproduziert. Basierend auf diesen in vitro Daten konnte ein geeignetes Grundgerüst für das Moleküldesign der zweiten Generation etabliert werden. Des weiteren wurde festgestellt, dass SM1 die TdT‐Funktion der pol λ dosisabgängig mit einem IC50 Wert von 4.5 μM inhibiert. Aus weiteren Experimente mit konstanter SM1 und steigender dNTP Konzentrationen konnte

66 Zusammenfassung geschlossen werden, dass SM1 die pol λ inhibiert ohne direkt mit den Substraten zu konkurrieren.

In den nächsten Schritten wurde die chemische Diversität der pharmakologisch interessanten niedermolekularen Rhodanine, ausgehend von billigen und kommerziell erhältlichen Synthesebausteinen, durch systematische synthetische Optimierung erweitert. Die am molekularen Designgerüst orientierte Synthese, der wirkstoffähnlichen Molekülen SM12, 29‐ 58 wurde dabei in zwei Teile unterteilt. Im ersten Teil wurden die Präkursoraldehyde SM59‐ 61, 63‐83 und für Verbindung SM33 das Präkursoracetophenon SM62 in hohen Ausbeuten mittels basischen SNAr Reaktionen aufgebaut. Dabei wurden am aromatischen Gerüstkern aktivierte Halogene durch die gewünschten Thiole ersetzt. Für die Synthese von Präkursor SM84, ausgehend von SM59, wurde eine elegante literaturbekannte Synthesesequenz163 angepasst. Dazu wurde SM59 mit NaBH4 in 98%‐iger Ausbeute zum Methanolintermediat reduziert. Danach wurde die Zwischenstufe zusammen mit H2O2 refuxiert und man erhielt SM85. Im letzten Schritt wurde SM85 mit DMP zum Präkursor SM84 oxidiert. Im zweiten Syntheseteil wurde die Präkursormoleküle mit variierenden Heterozyklen durch die Knoevenagelkondensation verbunden. Um exklusiv die Z‐Isomere zu erhalten, wurden SM12, 29‐33 und 35‐58 unter thermodynamischer Reaktionskontrolle in guten Ausbeuten hergestellt. Zur Synthese des Racemates von SM34 wurde die exozyclische Doppelbindung von SM1 mit der Hantzsch‐Ester auf Silikagel Methode176 reduziert.

Durch die biochemische Evaluation der neuen Substanzbibliothek, wurden 23 gegen die pol λ hochaktive Verbindungen (SM12, 29, 33, 37‐44, 46‐50, 53‐58) mit IC50 Werten kleiner als

10 μM entdeckt. Interessanterweise inhibierten zehn dieser Moleküle (SM29, 39, 40, 42, 44, 46, 47, 51‐53) im micromolaren Bereich selektiv die pol λ, jedoch nicht die pol β. Der exakte

IC50 Wert wurde für die fünf aktivsten Verbindungen bestimmt. SM39, 48, 49, 53 und 57 inhibierten dosisabhängig die Polymerisationsfunktion von pol λ mit IC50 Werten von 5.7, 6.0, 75,76 3.9, 4.0 und 4.0 μM und sind somit gleich aktiv, oder sogar aktiver als SM1 (IC50 = 5.9 μM) . Ferner fungieren SM39, 48, 49, 53 und 57 auch als Inhibitoren der TdT Funktion von pol λ. Des Weiteren zielten 14 der niedermolekularen Verbindungen (SM12, 33, 37, 38, 41, 43, 48‐ 50, 54‐58) auch auf die pol β ab und für die vier Aktivsten (SM38, 41, 48 und 49) wurde der

IC50 Wert ermittelt. SM38, 41, 48 und 49 inhibierten dosisabhängig die

Polymerisationsfunktion von pol β mit IC50 von 38.7, 28.1, 29.8 und 18.2 μM. Basierend auf

67 Zusammenfassung dem zuvor erstellten Grundgerüst zum Verbindungsdesign und den neuen PEX Data konnten die anfänglichen SAR76 bestätigt, erweitert und tiefgehend diskutiert werden (Kapitel 4.4.5).

Mit dem Ziel, Vergleiche zwischen den entdeckten niedermolekularen pol λ und β Inhibitoren (SM1, 49) und literaturbekannten Inhibitoren zu ziehen, wurde eine Vielzahl an publizierten Inhibitoren zugänglich gemacht und unter den gleichen PEX Bedingungen getestet. SM1 und 49 waren aktiver als die auf Naturstoffe basierenden Verbindungen. Dies hat zur Folge, dass die Rhodanine momentan die stärksten bekannten pol λ Inhibitoren und sicherlich mit den aktivsten literaturbekannten pol β Inhibitoren mithalten können.37,51,75,76,78,177,181

Mit der Absicht die Rhodaninesonden in einem zellulären Umfeld weiterzuentwickeln wurde zuerst der Effekt von einigen Rhodaninen der ersten Generation auf menschlichen Zelllinien studiert. Die Zellviabilität von Gebärmutterhalskrebs (HeLa‐S3) und Leberkrebs (Hep‐G2) Zelllinien wurden dosisabhängig unterdrückt durch Moleküle (SM1, 10, 16, 21 und 32). Außer SM10 zeigten alle Rhodanine eine moderate Toxizität auf beide humanen Zelllinien. Zusätzlich konnte aus den Ergebnissen geschlossen werden, dass SM1, 10, 16, 21 und 32 als niedermolekulare Sonden geeignet sind.

Anschließend wurden zwei der entdeckten Verbindungen (SM1, SM49) repräsentativ in Experimenten zur Mitbehandlung von Darmkrebszellen (Caco‐2) eingesetzt. Genauer wurde in diesen Studien untersucht, ob die neu entdeckten Verbindungen Krebszellen auf die Einführung von artifiziellen DNA‐Schäden sensibilisieren. Dafür wurden menschliche Caco‐2‐ Zellen mit SM1 und SM49 und dem zugelassenen monoalkylierenden Medikament TMZ oder der ROS induzierenden Modellverbindung H2O2 behandelt. Bedeutenderweise waren SM1 und SM49 pharmakologisch aktiv und sensibilisierten die Caco‐2‐Zellen auf beide genotoxische Reagenzien. Da Chemotherapien häufig auf die artifizielle Induktion von DNA‐ Schäden beruhen ist es ein andauerndes Vorhaben die DNA‐Reparaturmaschinerie zu bekämpfen, um die Wirkung der Medikamente zu erhöhen.15,17,20,37,51,53,78,79,100 Wie auch andere Genprodukte, die DNA‐Reparatur involviert sind, könnte die Regulierung der in Krebsgewebe überexprimierten pol β and λ72 auch fundamental wichtig in der Behandlung von Krebs sein.15,17,37,51,53,78,100 Die hier gezeigten zellulären Studien unterstützen diese Überlegungen, da SM1 und SM49 die Sensibilität von menschlichen Darmkrebszellen auf die genotoxischen Substanzen TMZ und H2O2 deutlich erhöhten. Durch die additiven oder sogar synergistischen Effekte wurde gezeigt, dass SM1 und SM49 das Potential hat, die Dosierung

68 Zusammenfassung von DNA‐schädigenden Agenzien zu reduzieren. Zudem liegt die Vermutung nahe, dass die Reperatur der DNA‐Schäden durch SM1 und SM49 inhibitirt wird und dadurch die Aktivität der Argentien erhöht wird. Aus diesem Grunde sind die hier entdeckten niedermolekularen Sonden nicht nur von großer Interesse für die Grundlagenforschung, sondern könnten auch als Ausgangspunkt für die Entwicklung neuer Ansätze zur Behandlung von Erkrankungen sein, die mit Störungen in der genomischen Integrität in Verbindung stehen (siehe auch Kapitel 3).

Seit vielen Jahren dienen modifizierte Nukleosid‐Analoga als sehr bedeutende, lebensrettende Medikamente. Die pharmakologisch sehr vielseitige Verbindungsklasse, welche die strukturellen Eigenschaften der natürlichen Nukleoside enthält wird sehr häufig zur Behandlung von Krebs und viralen Infektionen eingesetzt (siehe auch Kapitel 3).20,41,81,190‐ 196,198‐200 Zusammen mit HIV und HV, sind HSV und VZV weit verbreitete Phatogene. HSV und VZV werden neben den zugelassenen Medikamenten Brivudine, Acyclovir und Vidarabine seit über 40 Jahren mit dem Prototyp der antiviralen Wirkstoffe 2’‐Deoxy‐5‐iodouridin (Idoxuridine) N1 behandelt.20,190,192,193,196,201 N1 zielt auf die DNA‐Replikation der Vieren ab. Dabei fungiert N1 als Antagonist von dem natürlichen Gegenstück Thymidien und wirkt auf die Thymidylatphosphorylase und auf das Arbeitspferd der viralen DNA‐Replikation, die DNA‐Polymerase.41,193,196,201 Generell sind 5‐substituierte 2´‐Deoxycytidine gleich oder ein kleines bisschen weniger potent in ihrer HSV‐Aktivität, als die entsprechenden 5‐substituierte 2´‐Deoxyuridines.196,197,202,203 Desshalb ist das antivirale Spektrum von 2’‐Deoxy‐5‐iodocytidin N2 vergleichbar mit dem von N1, zu welchem das N2 durch enzymatische Deaminierung umgewandelt wird.196,197,202,204

Neben 5‐Halopyrimidinnukleosiden stieg das wissenschaftliche Interesse in den letzten Jahren an 4’‐C‐modifizierten Nukleosiden, da einige Verbindungen dieser Substanzklasse antivirale Aktivitäten145‐153 ‐ sogar gegen multiresistente Virusstämme ‐ zeigten.151,154 Weil es einen sehr großen Bedarf für die Entwicklung von neuen Medikamenten2,12,13,88,90,91 und damit auch für neue Nucleosid‐Analoga gibt, wurden im zweiten Teil dieser Arbeit Syntheserouten für weitere hochinteressante 4´‐C‐alkylierte 5‐Iodo‐2´‐deoxyuridine N3a‐c und 4´‐C‐alkylierte 5‐Iodo‐2´‐deoxycytidine N4a‐c designed und entwickelt (Kapitel 5).

In diesem Zusammenhang ist anzumerken, dass die Einführung von 4´‐C‐Modifikationen immer die Generierung eines quaternären Kohlenstoffzentrums mit sich bringt und somit die

69 Zusammenfassung

Hürden der damit verbundenen Chemie beinhalted.206 Zur Synthese von 4´‐C‐modifzierten Nukleosiden wurden im Wesentlichen drei wichtige Methoden entwickelt. In der ersten wird eine 4´‐C‐Verzweigung an das entsprechende 2´‐C‐Deoxynukleosides gehängt;206,207 die zweite Methode beinhaltet die assymetrische SAMP/RAMP‐Hydrazone α‐Alkylierung und die diastereoselektive, nukleophile 1,2‐Addition mit Griniard‐ und Organocerreagenzien208,209 und in Methode drei werden geeignete 4‐C‐modifizierte Ribosen als Glycosyldonoren generiert, die anschließend mittels der Vorbrüggen‐Methode210,211 in das Nucleosid überführt werden164.206

Mit dem Ziel die chemische Vielfalt der 4´‐C‐modifzierten Uridin‐ und Cytosin‐Analoga mit potentiell interessanten pharmakologischen Eigenschaften zu erweitern, wurde die etablierte Methode drei angewandt um eine Vielzahl von 4´‐C‐alkylierten Uridinen und Cytosinen von Grund auf aufzubauen. Mittels Methode drei wurden zuerst die 4‐C‐alkylierten Glycosyldonoren N8a‐c beziehungsweise die 4´‐C‐alkylierten Uridine N9a‐c in mehrstufigen Synthesesequenzen ausgehend von der kommerziell erhältlichen 164,206 D‐Allofuranose synthetisiert. Anschließend wurden N9a‐c erfolgreich mit Standardmethoden in die entsprechenden 2’‐doxygenierten Analoga N5a‐c und diese wiederum in die 4’‐C‐alkylierten 5‐Iodo‐2’‐deoxyuridine 3a‐c überführt. Schlussendlich konnte dann eine exozyklische Aminogruppe effektiv installiert und dadurch die 4’‐C‐alkylierten 5‐Iodo‐2’‐deoxycytidines N4a‐c erfolgreich erhalten werden.

Diese neuen Nukleosid‐Analoga N3a‐c und 4a‐c sind Derivative der zugelassenen antiviralen Medikamente N1 and N2. Basierend darauf, dass einige Analoga von 4’‐C‐modifizierten Nukleoside ebenfalls antivirale Aktivitäten zeigten sind die hier beschriebenen Moleküle von großem Interesse, da sie die strukturellen Eigenschaften der zugelassenen Medikamente N1 oder N2 und den 4´‐C‐alkylierten Nukleosiden in einem niedermolekularen Molekül vereinen. Darüber hinaus könnten die hier vorgestellten Moleküle auch als nützliche Synthesebausteine zur Synthese von weiteren Verbindungen von wissenschaftlichem oder pharmazeutischem Nutzen sein. Dieser Punkt wurde abschließend durch eine Sonogashira Testreaktion untersucht. Hierbei wurde N12c mit einem repräsentativen Alkin‐Biotin‐ Konjugat N15 umgesetzt, wobei das 4´‐C‐alkylierte‐5‐alkinyllierte Nukleosid N16c in guten Ausbeuten erhalten werden konnte. Damit konnte belegt werden, dass die hier beschriebenen Moleküle grundsätzlich auch als Synthesebausteine eingesetzt werden

70 Zusammenfassung können und durch sie daduch der Weg zu weiteren interessanten 4’‐C‐modifizierten Nukleosiden geöffnet werden kann.

71 Materials and methods

Materials and methods

6) Chemistry (nucleosides)

6.1) General

Chemicals, reagents and solvents

All solvents, reagents and fine chemicals are commercially available (Sigma‐Aldrich, Acros, Merck, Fluka, Roth, TCI, MCAT, ABCR, or Carbosynth) and used without further purification. Dess‐Martin periodinane (DMP) was prepared as described in literature 165. The petroleum ether (PE) that was used had a boiling point range of 35‐80°C. Triethylamine and MeCN used was dried by distillation from CaH2, degassed, and stored over 4 Å molecular sieves. All solvents were dried over molecular sieves and used directly without further purification.

Chemical reactions

All reactions were performed under exclusion of air and moisture in oven dried glassware (120°C). All temperatures quoted are uncorrected. The reported yields refer to the analytically pure substance and are not optimized.

Chromatography

Flash chromatography was performed on Merck silica gel 60 with a pressure of 0.2‐0.4 bar and solvent mixtures or gradients as stated in the corresponding procedures. Merck precoated aluminium plates (silica gel 60 F254) were used for thin layer chromatography (TLC). Compounds were detected by the extinction of the fluorescence under UV light at 254 nm and stained by moistening the TLC plates with the following solutions and moderate heating afterwards:

• 20 mL para‐Anisaldehyde, 20 mL conc. H2SO4, 4 mL AcOH in 360 mL ethanol.

• 5 g Ce(SO4)2, 12.5 g (NH4)6Mo7O24, 50 mL conc. H2SO4 in 450 mL H2O.

• 3 g KMnO4, 20 g K2CO3, 2.5 mL 10% NaOH solution in 400 mL H2O. • 4.5 g Ninhydrin in 600 mL ethanol.

72 Materials and methods

Instrumental and chemical analysis

1H, 13C and 19F nucleic magnetic resonance (NMR) spectra were recorded on Avance III 400 MHz spectrometer (Bruker) at room temperature. Spectra were processed with the software MestReNova 6.1.1 (MestRelab Research) and 1H and 13C chemical shifts are reported relative to the residual solvent peak. A BBFOplus probe with actively shielded z‐gradient was used with its inner (BB‐) coil tuned to 19F. Electron spray ionization mass spectrometry (ESI‐IT) spectra were measured on an Esquire 3000 plus (Bruker) in positive or negative mode, ‐1 samples were diluted to 1‐15 μg∙mL with MeCN or MeCN/H2O (1:1) and directly injected with a flow General rate of 5 μL∙min‐1. High resolution (HRMS) mass spectra were recorded on a Daltronics micrOTOF‐Q II ESI‐Qq‐TOF (Bruker) in positive or negative mode, samples ‐1 were diluted to 10‐200 μg∙mL with MeCN or MeCN/H2O (1:1) and processed by HPLC

(column: Chromolith FastGradient RP‐18e 50‐2 (Merck), linear gradient MeCN/H2O 2‐100%), before. The melting points are uncorrected and were determined on a Gallenkamp melting point apparatus. For the CHN‐analysis the elementar vario MICRO Cube was used.

Memorandum

Parts of the results presented in this section were subject of a thesis of the bachelor's degree program Life Science at the University of Konstanz. The thesis was successfully completed by B. Sc. Joos Aschenbrenner.

6.2) Synthesis of 4‐C‐modified carbohydrate building blocks

3‐O‐Benzyl‐5‐O‐tert‐butyldiphenylsilyl‐4‐C‐formyl‐1,2‐O‐isopropylidene‐α‐D‐ribofuranose‐ 2’‐Deoxy‐4’‐C‐methyluridine

O O TBDPSO O BnO O

The aldehyde intermediat was synthesized according to literature.164 White solid, yield 1 17.78 g, 91%. H NMR (400 MHz, CDCl3) δ 0.92 (s, 9H), 1.30 (s, 3H), 1.56 (s, 3H), 3.74 (d, J 11.5 Hz, 1H), 3.82 (d, J 11.5 Hz, 1H), 4.48 (d, J 4.4 Hz, 1H), 4.56 (d, J 12.2 Hz, 1H), 4.60 (dd, J

73 Materials and methods

4.2, 3.5 Hz, 1H), 4.69 (d, J 12.2 Hz, 1H), 5.81 (d, J 3.3 Hz, 1H), 7.19 – 7.39 (m, 11H), 7.47 – 7.57 13 (m, 4H), 9.85 (s, 1H). C NMR (101 MHz, CDCl3) δ 19.29, 26.21, 26.73, 26.83, 63.09, 72.79, 78.62, 79.12, 90.69, 104.98, 114.23, 127.85, 127.88, 127.92, 128.17, 128.60, 129.90, 129.96, + 132.57, 132.88, 135.57, 135.65, 137.06, 200.28. ESI‐MS: m/z [M+Na] calcd for C32H38O6Si: 569.2; found: 569.2.

3‐O‐Benzyl‐5‐(O‐tert‐butyldiphenylsilyl)‐4‐C‐(Z)‐prop‐1‐enyl‐1,2‐O‐isopropylidene‐α‐D‐ ribofuranose N7c

O TBDPSO O BnO O

The suspension of EtPPh3Br (23.43 g, 63.1 mmol) and t‐BuOK (10.78 g, 96.0 mmol) in THF (100 mL) was stirred at r.t. for 2 h. Then aldehyde intermediate164 (15.00 g, 27.4 mmol) in THF (20 mL) was added and stirring was continued for 17 h. The reaction mixture was quenched with aq sat. NaHCO3 solution (40 mL) and extracted with CH2Cl2 (3×60 mL). The combined organic layers were dried over MgSO4, concentrated and purified by silica gel column chromatography (EtOAc‐PE, 1:6) to give N7c. Yellow gum, yield 12.86 g, 84%, Rf 0.55 1 (EtOAc‐PE, 1:4). H NMR (400 MHz, CDCl3) δ 0.97 (s, 9H), 1.29 (s, 3H), 1.51 (s, 3H), 1.65 (dd, J 7.2, 1.7 Hz, 3H), 3.49 (d, J 11.7 Hz, 1H), 3.69 (d, J 11.7 Hz, 1H), 4.36 (d, J 4.6 Hz, 1H), 4.60 (dd, J 4.6, 3.9, 1H), 4.67 (d, J 12.3 Hz, 1H), 4.83 (d, J 12.3 Hz, 1H), 5.52 (dq, J 11.9, 7.2 Hz, 1H), 5.74 (d, J 3.9 Hz, 1H), 5.80 (dd, J 11.8, 1.7 Hz, 1H), 7.25‐7.42 (m, 11H), 7.60‐7.68 (m, 4H). 13C NMR

(101 MHz, CDCl3) δ 14.79, 19.48, 25.58, 26.48, 27.01, 64.58, 72.76, 78.37, 86.54, 100.23, 103.91, 113.36, 126.97, 127.76, 127.87, 128.03, 128.05, 128.63, 128.65, 129.81, 129.81, + 133.27, 133.94, 135.02, 135.75, 136.09, 138.21. ESI‐MS: m/z [M+Na] calcd for C34H42O5Si: 581.3; found: 581.1.

74 Materials and methods

1,2‐Di‐O‐acetyl‐3‐O‐benzyl‐5‐(O‐tert‐butyldiphenylsilyl)‐4‐C‐(Z)‐prop‐1‐enyl‐α,β‐D‐ ribofuranose N8c

O TBDPSO OAc BnO OAc

To a solution of compound N7c (12.80 g, 22.9 mmol) in a mixture of AcOH (208 mL) and Ac2O

(32.4 mL, 247.7 mmol) was added concd H2SO4 (200 μL) and the mixture was stirred for 24 h at r.t. After completion of the reaction, the mixture was concentrated and coevaporated with toluene (2 × 100 mL). The residue was diluted with CH2Cl2 (100 mL) and washed with aq sat.

NaHCO3 (25 mL) and demin. H2O (25 mL), dried over MgSO4, concentrated, and purified by silica gel column chromatography (EtOAc‐PE, 1:4) to give N8c. Yellow gum, yield 8.91 g, 64%, 1 Rf 0.68 (EtOAc‐PE, 1:3). H NMR (400 MHz, CDCl3) δ 1.03 (s, 9H), 1.74 (dd, J 3.8, 1.5 Hz, 3H), 1.84 (s, 3H), 2.05 (s, 3H), 3.59 (d, J 11.4 Hz, 1H), 3.74 (d, J 11.4 Hz, 1H), 4.52 (d, J 11.6 Hz, 1H), 4.62 (d, J 4.9 Hz, 1H), 4.67 (d, J 11.6 Hz, 1H), 5.36 (d, J 4.9 Hz, 1H), 5.49‐5.60 (m, 2H), 6.21 (s, 13 1H), 7.25‐7.42 (m, 11H), 7.60‐7.72 (m, 4H). C NMR (101 MHz, CDCl3) δ 14.45, 19.58, 20.98, 21.17, 27.05, 65.07, 73.55, 74.82, 76.69, 88.78, 98.24, 100.20, 126.35, 127.82, 127.86, 127.96, 128.01, 128.50, 128.60, 129.86, 129.97, 133.40, 135.01, 135.79, 135.84, 138.02, + 169.71, 170.20. ESI‐MS: m/z [M+Na] calcd for C35H42O7Si: 625.3; found: 625.1. HRMS: m/z + [M+Na] calcd for C35H42O7Si: 625.2592; found: 625.2565.

6.3) Synthesis of 4`C‐alkylated‐pyrimidine nucleosides

2’‐O‐Acetyl‐3’‐O‐benzyl‐5’‐(O‐tert‐butyldiphenylsilyl)‐4’‐C‐(Z)‐prop‐1‐enyluridine N9c

O H N O TBDPSO O N

BnO OAc

Compound N8c (7.63 g, 12.8 mmol) and uracil (2.87 g, 25.6 mmol) were solved in MeCN (40 mL) and N,O‐bis(trimethylsilyl)acetamide (18.4 mL, 76.8 mmol) was added. The mixture was refluxed for 1 h and after cooling to r.t. Me3SiOTf (3.0 mL, 16.64 mmol) was added. After

75 Materials and methods

refluxing again for 1 h the mixture was quenched with aq sat. NaHCO3 solution (10 mL), evaporated and extracted with CH2Cl2. The organic layer was dried over MgSO4, concentrated and purified by silica gel column chromatography (EtOAc‐PE, 3:7) to give N9c. White foam, 1 yield 5.72 g, 71%, Rf 0.13 (EtOAc‐PE, 1:3). H NMR (400 MHz, CDCl3) δ 1.07 (s, 9H), 1.69 (dd, J 7.1, 1.6 Hz, 3H), 2.07 (s, 3H), 3.66 (d, J 11.9 Hz, 1H), 3.90 (d, J 11.9 Hz, 1H), 4.44 (s, 1H), 4.44 (d, J 10.5 Hz, 1H), 4.64 (d, J 11.2 Hz, 1H), 5.23 (dd, J 8.1, 2.3 Hz, 1H), 5.33 (dd, J 6.1, 2.7 Hz, 1H), 5.48 (dd, J 11.9, 1.7 Hz, 1H), 5.57‐5.69 (m, 1H), 6.07 (d, J 2.7 Hz, 1H), 7.25‐7.46 (m, 11H), 13 7.53‐7.65 (m, 4H), 7.68 (d, J 8.2 Hz, 1H), 8.15 (s, 1H). C NMR (101 MHz, CDCl3) δ 14.05, 19.41, 20.74, 27.04, 64.13, 73.87, 74.19, 75.81, 77.21, 87.11, 87.98, 100.01, 102.63, 124.43, 127.72, 127.95, 128.01, 128.03, 128.48, 130.08, 130.12, 130.18, 132.13, 132.94, 135.34, + 135.62, 137.31, 139.77, 149.75, 162.42, 169.94. ESI‐MS: m/z [M+Na] calcd for C37H42N2O7Si: 677.3; found: 677.9.

3’‐O‐Benzyl‐5’‐(O‐tert‐butyldiphenylsilyl)‐2’‐deoxy‐4’‐C‐(Z)‐prop‐1‐enyl‐uridine N10c

O H N O TBDPSO O N

BnO

Compound N9c (5.88 g, 9.0 mmol) was solved in MeOH (100 mL) and NaOMe (0.73 g, 13.5 mmol) was added. The mixture was stirred at r.t. for 2 h. After completion of the reaction, the mixture was treated with aq concd tartaric acid (50 mL) and extracted with

CH2Cl2 (3×80 mL). The combined organic layers were dried over MgSO4, concentrated and purified by silica gel column chromatography (EtOAc‐PE, 4:1). The resulting compound was dissolved in MeCN (65 mL), DMAP (3.31 g, 27.0 mmol) and PhOCSCl (1.5 mL, 10.8 mmol) were added and the mixture was stirred at r.t. for 1 h. After completion of the reaction the mixture was concentrated, diluted in CH2Cl2 (60 mL), washed with aq 5% citric acid (30 mL) and demin. H2O (20 mL). The aqueous layer was extracted with CH2Cl2 (50 mL), the combined organic layers dried over MgSO4 and evaporated. To a solution of the residue in toluene were added n‐Bu3SnH (12.57 g, 43.2 mmol) and a catalytic amount of AIBN. The mixture was refluxed for 1 h. After completion of the reaction the solvent was removed

76 Materials and methods under reduced pressure and the residue was purified by silica gel column chromatography

(EtOAc‐PE, 3:7) to give N10c. White foam, yield 4.44 g, 83%, Rf 0.31 (EtOAc‐PE, 1:1). 1 H NMR (400 MHz, CDCl3) δ 1.07 (s, 9H), 1.73 (dd, J 7.2, 1.6 Hz, 3H), 2.12‐2.24 (m, 1H), 2.38‐ 2.47 (m, 1H), 3.73 (d, J 11.7 Hz, 1H), 3.94 (d, J 11.8 Hz, 1H), 4.46‐4.55 (m, 2H), 4.59 (d, J 11.7 Hz, 1H), 5.21 (dd, J 8.2, 2.1 Hz, 1H), 5.53 (dd, J 11.9, 1.6 Hz, 1H), 5.69 (dq, J 11.9, 7.1 Hz, 1H), 6.12 (dd, J 7.3, 3.0 Hz, 1H), 7.26‐7.46 (m, 11H), 7.51‐7.67 (m, 4H), 7.92 (d, J 8.2 Hz, 1H), 8.14 13 (s, 1H). C NMR (101 MHz, CDCl3) δ 14.30, 19.63, 27.26, 37.80, 64.50, 72.75, 75.66, 77.43, 83.20, 88.99, 100.21, 102.19, 124.68, 127.71, 128.15, 128.18, 128.21, 128.75, 130.25, 130.34, 131.65, 132.46, 133.20, 135.55, 135.79, 137.79, 140.43, 150.17, 162.96. ESI‐MS: m/z + [M+Na] calcd for C35H40N2O5Si: 619.3; found: 619.6.

2’‐deoxy‐4’‐C‐propyluridine N5c

To a solution of compound N10c (4.06 g, 7.1 mmol) in EtOH (50 mL) was added an equivalent weight amount of 10% Pd/C and the mixture was stirred at r.t. for 8 h under H2 atmosphere (balloon). After completion of the reaction the mixture was filtered through Celite on a sintered funnel and washed thoroughly. The solvent was removed and the residue was dissolved in THF (40 mL) and a 1 M solution of TBAF (9.1 mL, 9.1 mmol) was added. The mixture was stirred at r.t. for 16 h, concentrated and purified by silica gel column chromatography (EtOAc→MeOH‐EtOAc, 1:9) to give N5c. White foam, yield 1.25 g, 65%, 1 Rf 0.48 (MeOH‐EtOAc, 1:9). H NMR (400 MHz, MeOD) δ 0.92 (t, J 7.1 Hz, 3H), 1.31‐1.70 (m, 4H), 2.28‐2.34 (m, 2H), 3.55 (d, J 11.7 Hz, 1H), 3.63 (d, J 11.7 Hz, 1H), 4.40 (t, J 5.5 Hz, 1H), 5.65 (d, J 8.1 Hz, 1H), 6.16 (t, J 6.5 Hz, 1H), 8.03 (d, J 8.1 Hz, 1H). 13C NMR (101 MHz, MeOD) δ 15.41, 18.28, 35.00, 41.81, 65.55, 72.96, 85.76, 91.12, 102.50, 142.83, 152.43, 166.51. ESI‐ + + MS: m/z [M+Na] calcd for C12H18N2O5: 293.1; found: 293.3. HRMS: m/z [M+H] calcd for

C12H18N2O5: 271.1289; found: 271.1286.

77 Materials and methods

2’‐Deoxy‐4’‐C‐methyluridine N5a

O H N O HO O N

164 N5a was synthesized according to literature. White foam, yield 0.79 g, 79%, Rf 0.43 (MeOH‐EtOAc, 1:9). 1H NMR (400 MHz, MeOD) δ 8.05 (d, J 8.1 Hz, 1H), 6.18 (t, J 6.3 Hz, 1H), 5.67 (d, J 8.1 Hz, 1H), 4.37 (t, J 5.9 Hz, 1H), 3.60 (d, J 11.7 Hz, 1H), 3.55 (d, J 11.7 Hz, 1H), 2.48 – 2.23 (m, 2H), 1.17 (s, 3H); 13C NMR (101 MHz, MeOD) δ 166.32, 152.24, 142.67, 102.33, + 89.28, 85.35, 72.34, 67.44, 41.27, 17.89; ESI‐MS: m/z [M+Na] calcd for C10H14N2O5: 265.1, + found: 265.2; HRMS: m/z [M+H] calcd for C10H14N2O5: 243.0976; found: 243.0974.

2’‐Deoxy‐4’‐C‐ethyluridine N5b

H O N O HO O N

164 N5b was synthesized according to literature. White foam, yield 1.19 g, 89%, Rf 0.59 (MeOH‐EtOAc, 1:9). 1H NMR (400 MHz, MeOD) δ 8.05 (d, J 8.1 Hz, 1H), 6.19 (t, J 6.5 Hz, 1H), 5.68 (d, J 8.1 Hz, 1H), 4.44 (t, J 5.6 Hz, 1H), 3.66 (d, J 11.7 Hz, 1H), 3.58 (d, J 11.7 Hz, 1H), 2.34 (t, J 6.4 Hz, 2H), 1.74 (dq, J 15.2, 7.6 Hz, 1H), 1.61 (dq, J 14.8, 7.5 Hz, 1H), 0.97 (t, J 7.6 Hz, 3H); 13C NMR (101 MHz, MeOD) δ 166.33, 152.29, 142.66, 102.36, 91.09, 85.64, 72.78, 65.01, + 41.70, 25.00, 8.51; ESI‐MS: m/z [M+Na] calcd for C11H16N2O5: 279.1; found: 279.2; HRMS: + m/z [M+H] calcd for C11H16N2O5: 257.1132; found: 257.1129.

General synthetic procedure, exemplified by 3’,5’‐di‐O‐acetyl‐2’‐deoxy‐4’‐C‐methyluridine N11a O H N O AcO O N

AcO

78 Materials and methods

To a suspension of compound N5a (0.62 g, 2.56 mmol) in MeCN (14 mL) was added NEt3

(1.43 mL, 10.2 mmol), Ac2O (0.96 mL, 10.2 mmol) and a catalytic amount of DMAP. The mixture was stirred at r.t. for 20 h and then diluted with CH2Cl2 (40 mL) and washed with demin. H2O (3×30 mL). The organic layer was dried over MgSO4, concentrated and purified by silica gel column chromatography (EtOAc‐PE, 6:1).

1 N11a. White foam, yield 0.55 g, 63%, Rf 0.30 (EtOAc‐PE, 6:1). H NMR (400 MHz, CDCl3) δ 1.27 (s, 3H), 2.10 (s, 3H), 2.12 (s, 3H), 2.33 (dt, J 14.2, 7.1 Hz, 1H), 2.52 (ddd, J 14.3, 6.1, 3.6 Hz, 1H), 4.12 (d, J 11.9 Hz, 1H), 4.18 (d, J 11.9 Hz, 1H), 5.31 (dd, J 6.8, 3.6 Hz, 1H), 5.76 (d, J 7.4 Hz, 1H), 6.24 (t, J 6.7 Hz, 1H), 7.55 (d, J 8.2 Hz, 1H), 9.35 (s, 1H). 13C NMR (101 MHz,

CDCl3) δ 18.40, 20.92, 20.95, 38.71, 67.98, 73.82, 84.21, 85.08, 102.84, 139.02, 150.44, + 163.19, 170.21. ESI‐MS: m/z [M+Na] calcd for C14H18N2O7: 349.1; found: 349.3.

3’,5’‐Di‐O‐acetyl‐2’‐deoxy‐4’‐C‐ethyluridine N11b

O H N O AcO O N

AcO

1 White foam, yield 1.18 g, 74%, Rf 0.31 (EtOAc‐PE, 6:1). H NMR (400 MHz, CDCl3) δ 0.96 (t, J 7.5 Hz, 3H), 1.60 (dq, J 14.8, 7.4 Hz, 1H), 1.74 (dq, J 15.1, 7.6 Hz, 1H), 2.09 (s, 3H), 2.10 (s, 3H), 2.32 (dt, J 14.3, 7.1 Hz, 1H), 2.47 (ddd, J 14.3, 6.1, 3.5 Hz, 1H), 4.16 (s, 2H), 5.37 (dd, J 6.9, 3.5 Hz, 1H), 5.75 (d, J 8.2 Hz, 1H), 6.17 (t, J 6.6 Hz, 1H), 7.55 (d, J 8.2 Hz, 1H), 9.65 (s, 1H). 13 C NMR (101 MHz, CDCl3) δ 7.98, 20.94, 24.83, 38.92, 66.02, 74.01, 84.24, 86.80, 102.81, + 139.04, 150.52, 163.38, 170.07, 170.27. ESI‐MS: m/z [M+Na] calcd for C15H20N2O7: 363.1; found: 363.3.

3’,5’‐Di‐O‐acetyl‐2’‐deoxy‐4’‐C‐propyluridine N11c

O H N O AcO O N

AcO

79 Materials and methods

1 White foam, yield 1.21 g, 93%, Rf 0.67 (EtOAc‐PE, 4:1). H NMR (400 MHz, CDCl3) δ 0.93 (t, J 7.1 Hz, 3H), 1.27‐1.69 (m, 4H), 2.09 (s, 3H), 2.11 (s, 3H), 2.30 (dt, J 14.2, 7.1 Hz, 1H), 2.47 (ddd, J 14.3, 6.1, 3.5 Hz, 1H), 4.16 (s, 2H), 5.35 (dd, J 6.8, 3.5 Hz, 1H), 5.74 (dd, J 8.2, 2.0 Hz, 13 1H), 6.20 (dd, J 7.0, 6.5 Hz, 1H), 7.53 (d, J 8.2 Hz, 1H), 8.61 (s, 1H). C NMR (101 MHz, CDCl3) δ 14.87, 17.12, 21.07, 21.09, 34.36, 39.05, 66.49, 74.10, 84.37, 86.81, 102.88, 139.09, + 150.28, 162.82, 170.17, 170.32. ESI‐MS: m/z [M+Na] calcd for C16H22N2O7: 377.1; found: 377.3.

6.4) Synthesis of 4’‐C‐alkylated‐5‐iodo‐2’‐deoxypyrimidine nucleosides

General synthetic procedure, exemplified by 3’,5’‐di‐O‐acetyl‐2’‐deoxy‐5‐iodo‐4’‐C‐ methyluridine N12a

Compound N11a (0.46 g, 1.40 mmol), iodine (0.21 g, 0.84 mmol) and CAN (0.38 g, 0.70 mmol) were solved in MeCN (23 mL) and refluxed for 1 h. After completion of the reaction the solvent was removed under reduced pressure and the residue was partitioned between EtOAc (40 mL), aq sat. NaCl (20 mL) and aq 5% NaHSO4 (5 mL). The aqueous layer was extracted with EtOAc (2×40 mL) and the combined organic layers were washed first with aq 5% NaHSO4 (5 mL) and then with aq sat. NaCl (25 mL) and demin. H2O (2×15 mL), dried over MgSO4, concentrated and purified by silica gel column chromatography (EtOAc‐PE, 2:1). 1 N12a. White foam, yield 0.56 g, 89%, Rf 0.57 (EtOAc‐PE, 5:1). H NMR (400 MHz, CDCl3) δ 1.26 (s, 3H), 2.12 (s, 3H), 2.21 (s, 3H), 2.36 (dt, J 14.2, 7.0 Hz, 1H), 2.53 (ddd, J 14.3, 6.2, 3.8 Hz, 1H), 4.13 (d, J 12.1 Hz, 1H), 4.21 (d, J 12.0 Hz, 1H), 5.32 (dd, J 6.9, 3.8 Hz, 1H), 6.21 (t, J 13 6.6 Hz, 1H), 8.02 (s, 1H), 9.44 (s, 1H). C NMR (101 MHz, CDCl3) δ 18.42, 20.89, 21.32, 39.09, 67.90, 68.79, 73.51, 84.44, 85.39, 143.98, 150.13, 159.96, 170.21. ESI‐MS: m/z [M+Na]+ calcd for C14H17IN2O7: 475.0; found: 475.1.

80 Materials and methods

3’,5’‐Di‐O‐acetyl‐2’‐deoxy‐5‐iodo‐4’‐C‐ethyluridine N12b

1 White foam, yield 1.56 g, 98%, Rf 0.71 (EtOAc‐PE, 5:1). H NMR (400 MHz, CDCl3) δ 0.97 (t, J 7.5 Hz, 3H), 1.60 (dq, J 14.8, 7.4 Hz, 1H), 1.75 (dq, J 15.1, 7.6 Hz, 1H), 2.11 (s, 3H), 2.21 (s, 3H), 2.38 (dd, J 14.3, 7.1 Hz, 1H), 2.50 (ddd, J 14.4, 6.2, 3.8 Hz, 1H), 4.19 (d, J 12.2 Hz, 1H), 4.22 (d, J 12.2 Hz, 1H), 5.38 (dd, J 7.0, 3.8 Hz, 1H), 6.20 (t, J 6.6 Hz, 1H), 8.02 (s, 1H), 9.65 (s, 13 1H). C NMR (101 MHz, CDCl3) δ 8.01, 20.92, 21.36, 24.95, 39.27, 66.19, 68.86, 73.60, 84.43, + 87.10, 143.98, 150.20, 160.04, 170.08, 170.32. ESI‐MS: m/z [M+Na] calcd for C15H19IN2O7: 489.0; found: 489.1.

3’,5’‐Di‐O‐acetyl‐2’‐deoxy‐5‐iodo‐4’‐C‐propyluridine N12c

1 White foam, yield 1.53 g, 89%, Rf 0.63 (EtOAc‐PE, 3:1). H NMR (400 MHz, CDCl3) δ 0.92 (t, J 7.1 Hz, 3H), 1.26‐1.69 (m, 4H), 2.09 (s, 3H), 2.19 (s, 3H), 2.33 (dt, J 14.3, 7.0 Hz, 1H), 2.47 (ddd, J 14.4, 6.2, 3.8 Hz, 1H), 4.16 (d, J 12.4 Hz, 1H), 4.19 (d, J 12.4 Hz, 1H), 5.34 (dd, J 7.0, 3.8 13 Hz, 1H), 6.17 (t, J 6.6 Hz, 1H), 7.99 (s, 1H), 9.41 (s, 1H). C NMR (101 MHz, CDCl3) δ 14.83, 17.10, 21.01, 21.46, 34.42, 39.35, 66.61, 68.91, 73.69, 84.50, 87.06, 144.05, 150.22, 160.03, + 170.16, 170.38. ESI‐MS: m/z [M+Na] calcd for C16H21IN2O7: 503.0; found: 503.4.

General synthetic procedure, exemplified by 2’‐deoxy‐5‐iodo‐4’‐C‐methyluridine N3a

O H N O HO O N I HO

81 Materials and methods

Compound N12a (0.08 g, 0.17 mmol) was stirred with 0.1 M NaOMe/MeOH (8 mL) at r.t. for

1 h. After the reaction was completed, addition of 2 mL of demin. H2O was followed by neutralization (pH 6) with Amberlite IR‐120 (H+ form) ion‐exchange resin. The resin was filtered and washed with 50% aq MeOH (20 mL). The combined filtrate and washings were evaporated and purified by silica gel column chromatography (EtOAc). 1 N3a. White foam, yield 0.057g, 91%, Rf 0.35 (EtOAc). H NMR (400 MHz, MeOD) δ 1.16 (s, 3H), 2.30‐2.44 (m, 2H), 3.57 (d, J 11.7 Hz, 1H), 3.62 (d, J 11.7 Hz, 1H), 4.40 (t, J 6.2 Hz, 1H), 6.14 (t, J 6.0 Hz, 1H), 8.64 (s, 1H). 13C NMR (101 MHz, MeOD) δ 18.01, 41.55, 67.01, 67.82, + 71.82, 85.73, 89.51, 147.47, 152.02, 162.92. ESI‐MS: m/z [M+Na] calcd for C10H13IN2O5: + 391.0; found: 391.1. HRMS: m/z [M+H] calcd for C10H13IN2O5: 368.9942; found: 368.9933.

2’‐Deoxy‐5‐iodo‐4’‐C‐ethyluridine N3b

O H N O HO O N I HO

1 White foam, yield 0.093 g, 97%, Rf 0.43 (EtOAc). H NMR (400 MHz, MeOD) δ 0.97 (t, J 7.6 Hz, 3H), 1.58 (dq, J 14.8, 7.5 Hz, 1H), 1.72 (dq, J 15.1, 7.6 Hz, 1H), 2.30‐2.42 (m, 2H), 3.58 (d, J 11.6 Hz, 1H), 3.71 (d, J 11.6 Hz, 1H), 4.46 (t, J 5.9 Hz, 1H), 6.14 (t, J 6.2 Hz, 1H), 8.63 (s, 1H). 13C NMR (101 MHz, MeOD) δ 8.52, 25.15, 41.96, 64.68, 67.86, 72.23, 85.93, 91.30, 147.47, + 152.03, 162.91. ESI‐MS: m/z [M+Na] calcd for: C11H15IN2O5: 405.0; found: 405.1. HRMS: m/z + [M+H] calcd for C11H15IN2O5: 383.0098; found: 383.0086.

2’‐Deoxy‐5‐iodo‐4’‐C‐propyluridine N3c

O H N O HO O N I HO

1 White foam, yield 0.089 g, 97%, Rf 0.63 (EtOAc). H NMR (400 MHz, MeOD) δ 0.95 (t, J 7.0 Hz, 3H), 1.28‐1.69 (m, 4H), 2.29‐2.43 (m, 2H), 3.58 (d, J 11.6 Hz, 1H), 3.70 (d, J 11.6 Hz, 1H), 4.45 (t, J 5.9 Hz, 1H), 6.14 (t, J 6.2 Hz, 1H), 8.62 (s, 1H). 13C NMR (101 MHz, MeOD) δ 15.40,

82 Materials and methods

18.27, 35.13, 42.07, 65.26, 68.02, 72.43, 86.07, 91.33, 147.62, 152.18, 163.05. ESI‐MS: m/z + + [M+Na] calcd for C12H17IN2O5: 419.0; found: 419.2. HRMS: m/z [M+H] calcd for C12H17IN2O5: 397.0255; found: 397.0249.

General synthetic procedure, exemplified by 3’,5’‐di‐(O‐tert‐butyldimethylsilyl)‐2’‐deoxy‐5‐ iodo‐4’‐C‐methyluridine N13a

To a solution of N3a (0.463 g, 1.26 mmol) in DMF (3 mL) TBDMSCl (1.22 g, 8.1 mmol) and imidazole (0.81 g, 11.6 mmol) were added. The clear solution was stirred at r.t. for 60 h.

Demin. H2O (15 mL) was added, the aqueous layer was extracted with EtOAc (4×50 mL), dried over MgSO4, concentrated and purified by silica gel column chromatography (EtOAc‐ PE, 1:4). 1 N13a. White foam, yield 0.605 g, 81%, Rf 0.30 (EtOAc‐PE, 1:4). H NMR (400 MHz, CDCl3) δ 0.07 (s, 3H), 0.08 (s, 3H), 0.15 (s, 3H), 0.15 (s, 3H), 0.91 (s, 9H), 0.95 (s, 9H), 1.15 (s, 3H), 2.18 (ddd, J 13.4, 7.4, 6.2 Hz, 1H), 2.33 (ddd, J 13.2, 5.9, 3.1 Hz, 1H), 3.55 (d, J 10.9 Hz, 1H), 3.71 (d, J 10.9 Hz, 1H), 4.33 (dd, J 6.1, 3.1 Hz, 1H), 6.18 (dd, J 7.3, 6.0 Hz, 1H), 8.13 (s, 1H), 8.17 13 (s,1H). C NMR (101 MHz, CDCl3) δ ‐5.03, ‐4.94, ‐4.93, ‐4.53, 18.21, 18.50, 18.72, 25.87, 26.36, 42.63, 68.04, 68.24, 73.08, 84.94, 89.15, 144.77, 149.74, 159.82. ESI‐MS: m/z [M+Na]+ calcd for C22H41IN2O5Si2: 619.2; found: 619.0.

3’,5’‐Di‐(O‐tert‐butyldimethylsilyl)‐2’‐deoxy‐5‐iodo‐4’‐C‐ethyluridine N13b

1 White foam, yield 1.670 g, 90%), Rf 0.31 (EtOAc‐PE, 1:4). H NMR (400 MHz, CDCl3) δ 0.07 (s, 3H), 0.08 (s, 3H), 0.15 (s, 3H), 0.16 (s, 3H), 0.90 (s, 9H), 0.94 (t, J 7.5 Hz, 3H), 0.95 (s, 9H), 1.47 (dq, J 14.8, 7.5 Hz, 1H), 1.74 (dq, J 15.1, 7.6 Hz, 1H), 2.11‐2.21 (m, 1H), 2.31 (ddd, J 13.2, 5.9, 2.9 Hz, 1H), 3.57 (d, J 10.8 Hz, 1H), 3.74 (d, J 10.8 Hz, 1H), 4.40 (dd, J 6.2, 2.9 Hz, 1H), 6.16

83 Materials and methods

13 (dd, J 7.5, 6.0 Hz, 1H), 8.11 (s, 1H), 8.26 (s, 1H). C NMR (101 MHz, CDCl3) δ ‐5.06, ‐4.95, ‐ 4.92, ‐4.44, 8.41, 18.17, 18.68, 24.96, 25.88, 26.35, 42.69, 66.34, 68.14, 73.38, 84.98, 90.62, + 144.76, 149.79, 159.87. ESI‐MS: m/z [M+Na] calcd for C23H43IN2O5Si2: 633.2; found: 633.1.

3’,5’‐Di‐(O‐tert‐butyldimethylsilyl)‐2’‐deoxy‐5‐iodo‐4’‐C‐propyluridine N13c

O H N O TBDMSO O N I TBDMSO

1 White foam, yield 1.13g, 91%, Rf 0.39 (EtOAc‐PE, 1:4). H NMR (400 MHz, CDCl3) δ 0.07 (s, 3H), 0.08 (s, 3H), 0.15 (s, 3H), 0.15 (s, 3H), 0.90 (s, 9H), 0.91 (t, J 2.9 Hz, 3H) 0.94 (s, 9H), 1.23‐ 1.51 (m, 2H), 1.63 (dt, J 9.5, 5.8 Hz, 2H), 2.05‐2.23 (m, 1H), 2.31 (ddd, J 13.2, 5.9, 2.9 Hz, 1H), 3.56 (d, J 10.9 Hz, 1H), 3.74 (d, J 10.9 Hz, 1H), 4.38 (dd, J 6.2, 2.9 Hz, 1H), 6.16 (dd, J 7.5, 6.0 13 Hz, 1H), 8.11 (s, 1H), 8.59 (s, 1H). C NMR (101 MHz, CDCl3) δ ‐5.06, ‐4.95, ‐4.93, ‐4.45, 14.95, 17.24, 18.17, 18.68, 25.79, 25.87, 26.35, 34.75, 42.68, 66.72, 68.20, 73.42, 84.99,

90.53, 144.75, 149.93, 160.05. ESI‐MS: m/z [M+Na]+ calcd for C24H45IN2O5Si2: 647.2; found: 647.0.

General synthetic procedure, exemplified by 3’,5’‐di‐(O‐tert‐butyldimethylsilyl)‐2’‐deoxy‐5‐ iodo‐4’‐C‐methylcytidine N14a

O N NH2 TBDMSO O N I TBDMSO

The solution of DMAP (0.113 g, 0.93 mmol), TPSCl (0.282 g, 0.86 mmol) and compound 13a

(0.191 g, 0.32 mmol) in MeCN (9 mL) was treated with freshly distilled Et3N (0.65 mL, 4.67 mmol). After the yellow mixture was stirred for 50 h at room temperature, a 28% aq solution of NH4OH (14 mL) was added and stirring was maintained for 3 h. MeCN was removed under vacuum and the aqueous layer was extracted with EtOAc (4×50 mL). The organic layer was dried over MgSO4, concentrated and purified by silica gel column chromatography (EtOAc‐ PE, 4:1).

84 Materials and methods

1 N14a. White foam, yield 0.148 g, 78%, Rf 0.17 (EtOAc‐PE, 3:1). H NMR (400 MHz, CDCl3) δ 0.04 (s, 3H), 0.06 (s, 3H), 0.12 (s, 3H), 0.13 (s, 3H), 0.89 (s, 9H), 0.93 (s, 9H), 1.15 (s, 3H), 2.12 (dt, J 13.2, 6.5 Hz, 1H), 2.49 (ddd, J 13.4, 6.1, 4.0 Hz, 1H), 3.54 (d, J 10.8 Hz, 1H), 3.68 (d, J 10.8 Hz, 1H), 4.29 (dd, J 6.3, 4.0 Hz, 1H), 5.55 (s, 1H), 6.12 (t, J 6.3 Hz, 1H), 8.10 (s, 1H), 8.63 13 (s, 1H). C NMR (101 MHz, CDCl3) δ ‐5.06, ‐5.01, ‐4.98, ‐4.45, 18.16, 18.32, 18.66, 25.87, 26.32, 42.84, 55.91, 67.83, 72.50, 85.78, 88.82, 146.79, 154.88, 163.84. ESI‐MS: m/z [M+Na]+ calcd for C22H42IN3O4Si2: 618.2; found: 618.2.

3’,5’‐Di‐(O‐tert‐butyldimethylsilyl)‐2’‐deoxy‐5‐iodo‐4’‐C‐ethylcytidine N14b

O N NH2 TBDMSO O N I TBDMSO

1 White foam, yield 0.150 g, 77%, Rf 0.18 (EtOAc‐PE, 3:1). H NMR (400 MHz, CDCl3) δ 0.04 (s, 3H), 0.06 (s, 3H), 0.12 (s, 3H), 0.12 (s, 3H), 0.88 (s, 9H), 0.92 (s, 9H), 0.93 (t, J 7.72 Hz, 3H), 1.48 (dq, J 14.8, 7.4 Hz, 1H), 1.73 (dq, J 15.1, 7.6 Hz, 1H), 2.09 (dt, J 13.3, 6.6 Hz, 1H), 2.46 (ddd, J 13.4, 6.1, 3.8 Hz, 1H), 3.54 (d, J 10.8 Hz, 1H), 3.71 (d, J 10.8 Hz, 1H), 4.37 (dd, J 6.5, 3.8 Hz, 1H), 5.56 (s, 1H), 6.10 (t, J 6.4 Hz, 1H), 8.07 (s, 1H), 8.63 (s, 1H). 13C NMR (101 MHz,

CDCl3) δ ‐5.09, ‐5.01, ‐4.37, 8.31, 18.11, 18.61, 24.63, 25.87, 26.30, 42.96, 56.11, 65.83, + 72.87, 85.78, 90.28, 146.73, 154.91, 163.89. ESI‐MS: m/z [M+Na] calcd for C23H44IN3O4Si2: 632.2; found: 632.2.

3’,5’‐Di‐(O‐tert‐butyldimethylsilyl)‐2’‐deoxy‐5‐iodo‐4’‐C‐propylcytidine N14c

O N NH2 TBDMSO O N I TBDMSO

1 White foam, yield 0.160 g, 80%, Rf 0.21 (EtOAc‐PE, 3:1). H NMR (400 MHz, CDCl3) δ 0.04 (s, 3H), 0.06 (s, 3H), 0.11 (s, 3H), 0.12 (s, 3H), 0.88 (s, 9H), 0.90‐0.95 (m, 12H), 1.21‐1.50 (m, 3H), 1.57‐1.67 (m, 1H), 2.01‐2.13 (m, 1H), 2.45 (ddd, J 13.4, 6.0, 3.7 Hz, 1H), 3.54 (d, J 10.8 Hz, 1H), 3.71 (d, J 10.8 Hz, 1H), 4.35 (dd, J 6.5, 3.7 Hz, 1H), 5.56 (s, 1H), 6.10 (t, J 6.4 Hz, 1H), 8.06

85 Materials and methods

13 (s, 1H), 8.84 (s, 1H). C NMR (101 MHz, CDCl3) δ ‐5.11, ‐5.02, ‐5.01, ‐4.39, 14.95, 17.15, 18.11, 18.60, 25.86, 26.29, 34.42, 42.97, 56.20, 66.23, 72.59, 85.79, 90.21, 146.68, 154.93, + 163.94. ESI‐MS: m/z [M+Na] calcd for C24H46IN3O4Si2: 646.2; found: 646.1.

General synthetic procedure, exemplified by 2’‐deoxy‐5‐iodo‐4’‐C‐methylcytidine N4a

O N NH2 HO O N I HO

Compound N14a (0.148 g, 0.25 mmol) was dissolved in THF (10 mL), and a 1 M solution of TBAF (1.0 mL, 1.0 mmol) was added. The mixture was stirred at r.t. for 16 h, concentrated and purified by silica gel column chromatography (EtOAc→MeOH‐EtOAc, 1:9). 1 N4a. White foam, yield 0.84 g, 92%, Rf 0.15 (MeOH‐EtOAc, 1:10). H NMR (400 MHz, MeOD) δ 1.20 (s, 3H), 2.29 (ddd, J 13.7, 6.8, 4.9 Hz, 1H), 2.50 (dt, J 13.2, 6.5 Hz, 1H), 3.61 (d, J 11.7 Hz, 1H), 3.66 (d, J 11.7 Hz, 1H), 4.39 (t, J 6.6 Hz, 1H), 6.11 (dd, J 6.5, 5.0 Hz, 1H), 8.69 (s, 1H). 13C NMR (101 MHz, MeOD) δ 17.93, 41.93, 56.46, 66.75, 71.29, 86.54, 89.42, 149.40, 157.37, + + 165.88. ESI‐MS: m/z [M+Na] calcd for C10H14IN3O4: 390.0; found: 390.0. HRMS: m/z [M+H] calcd for C10H14IN3O4: 368.0102; found: 368.0095.

2’‐Deoxy‐5‐iodo‐4’‐C‐ethylcytidine N4b

O N NH2 HO O N I HO

1 White foam, yield 0.85 g, 89%), Rf 0.16 (MeOH‐EtOAc, 1:10). H NMR (400 MHz, MeOD) δ 1.01 (t, J 7.6 Hz, 3H), 1.62 (dq, J 14.8, 7.5 Hz, 1H), 1.77 (dq, J 15.2, 7.6 Hz, 1H), 2.28 (ddd, J 13.7, 6.8, 5.4 Hz, 1H), 2.49 (ddd, J 13.7, 6.8, 5.4 Hz, 1H), 3.60 (d, J 11.6 Hz, 1H), 3.76 (d, J 11.6 Hz, 1H), 4.32‐4.63 (m, 1H), 6.12 (dd, J 6.3, 5.6 Hz, 1H), 8.67 (s, 1H). 13C NMR (101 MHz, MeOD) δ 8.48, 24.97, 42.41, 56.53, 64.38, 71.81, 86.83, 91.26, 149.41, 157.37, 165.86. ESI‐ + + MS: m/z [M+Na] calcd for C11H16IN3O4: 404.0; found: 404.0. HRMS: m/z [M+H] calcd for

C11H16IN3O4: 382.0258; found: 382.0249.

86 Materials and methods

2’‐Deoxy‐5‐iodo‐4’‐C‐propylcytidine N4c

O N NH2 HO O N I HO

1 White foam, yield 0.80 g, 81%, Rf 0.17 (MeOH‐EtOAc, 1:10). H NMR (400 MHz, MeOD) δ 0.99 (t, J 7.1 Hz, 3H), 1.40‐1.61 (m, 3H), 1.61‐1.73 (m, 1H), 2.28 (ddd, J 13.6, 6.7, 5.5 Hz, 1H), 2.49 (dt, J 13.6, 6.2 Hz, 1H), 3.60 (d, J 11.6 Hz, 1H), 3.75 (d, J 11.6 Hz, 1H), 4.46 (t, J 6.2 Hz, 1H), 6.12 (dd, J 6.4, 5.6 Hz, 1H), 8.67 (s, 1H). 13C NMR (101 MHz, MeOD) δ 15.26, 18.09, 34.81, 42.38, 56.55, 64.83, 71.84, 86.82, 91.13, 149.41, 157.36, 165.84. ESI‐MS: m/z [M+Na]+ + calcd for C12H18IN3O4: 418.0; found: 418.0. HRMS: m/z [M+H] calcd for C12H18IN3O4: 396.0415; found: 396.0402.

6.5) Sonagashira test‐reaction

Biotin‐Alkyne Conjuate N15

O O H HN H NH N N H S H

N15 was synthesized according to literature 242.

3’,5’‐Di‐O‐(acetyl)‐γ‐[N‐(biotin‐6‐amino‐hexanoyl)]‐5‐(aminopropargyl)‐2‘‐desoxy‐4‘‐C‐ propyluridine N16c

Compound N12c (250 mg, 0.52 mmol), Biotin‐Alkyne Conjuate N15 (226 mg, 0.57 mmol) and

CuI (20 mg, 0.10 mmol) were solved in DMF (10 mL) under N2 atmosphere. To the mixture was added Pd(PPh3)4 (60 mg, 0.05 mmol) and dry NEt3 (144 μL, 1.04 mmol). The mixture was

87 Materials and methods stirred at r.t. for 16 h, concentrated and purified by silica gel column chromatography

(CH2Cl2→MeOH‐CH2Cl2, 1:9). The product was crystallized from methanol‐diethyl ether to 1 give N16c (223 mg, 57%) as a white solid. Rf 0.41 (MeOH – CH2Cl2, 1:9). H NMR (400 MHz, DMSO‐d6) δ 11.66 (s, 1H), 8.26 (t, J 5.5 Hz, 1H), 7.91 (s, 1H), 7.71 (t, J 5.5 Hz, 1H), 6.40 (s, 1H), 6.34 (s, 1H), 6.07 (t, J 6.5 Hz, 1H), 5.35 (dd, J 7.1 Hz, 4.8 Hz, 1H), 4.27–4.33 (m, 1H), 4.18 (d, J 11.8 Hz, 1H), 4.07–4.15 (m, 2H), 4.05 (d, J 5.4 Hz, 2H), 3.05‐3.14 (m, 1H), 2.99 (dd, J 12.8 Hz, 6.7 Hz, 2H), 2.82 (dd, J 12.4 Hz, 5.1 Hz, 1H), 2.53‐2.65 (m, 2H), 2.34–2.44 (m, 1H), 1.99– 2.15 (m, 10H), 1.14–1.69 (m, 16H), 0.89 (t, J 7.2 Hz, 3H).13C NMR (101 MHz, DMSO‐d6) δ 171.74, 169.91, 169.68, 162.67, 161.47, 149.32, 143.26, 98.33, 90.00, 85.89, 83.88, 73.92, 73.23, 65.14, 61.01, 59.16, 55.39, 38.24, 36.72, 35.18, 35.02, 33.30, 28.95, 28.18, 28.00, + 26.11, 25.29, 24.83, 20.63, 20.56, 16.33, 14.54. ESI‐MS: m/z [M+Na] calcd for C35H50N6O10S: 769.3; found: 770.4.

γ‐[N‐(biotin‐6‐amino‐hexanoyl)]‐5‐(aminopropargyl)‐2‘‐desoxy‐4‘‐C‐propyluridine N17c

Compound N16c (60 mg, 0.08 mmol) was stirred with 0.1 M NaOMe/MeOH (3 mL) at r.t. for

1 h. After the reaction was completed, addition of 1 mL of demin. H2O was followed by neutralization (pH 6) with Amberlite IR‐120 (H+ form) ion‐exchange resin. The resin was filtered and washed with 50% aq MeOH (20 mL). The combined filtrate and washings were concentrated in vacuo, coevaporated with an ethanol‐EtOAc‐toluene mixture (1:1:2, 2 x 10 mL) and crystallized from methanol‐diethyl ether to give N17c (40 mg, 75%) as a white 1 solid. Rf 0.07 (MeOH–CH2Cl2, 1:9). H NMR (400 MHz, DMSO) δ 11.57 (s, 1H), 8.27 (t, J 5.4 Hz, 1H), 8.22 (s, 1H), 7.72 (t, J 5.6 Hz, 1H), 6.40 (s, 1H), 6.34 (s, 1H), 6.04 (t, J 6.5 Hz, 1H), 5.12 (br s, 2H), 4.30 (dd, J 7.6 Hz, 4.8 Hz, 1H), 4.23‐4.27 (m, 1H), 4.12 (dd, J 7.6 Hz, 4.5 Hz, 1H), 4.06 (d, J 5.4 Hz, 2H), 3.49 (d, J 11.5 Hz, 1H), 3,42 (d, J 11.6 Hz, 1H), 3.04–3.14 (m, 1H), 3.00 (q, J 6.4 Hz, 2H), 2.82 (dd, J 12.4 Hz, 5.1 Hz, 1H), 2.57 (d, J 12.4 Hz, 1H), 2.25 (dt, J 13.1, 6.5, 1H), 2.21 – 2.12 (m, 1H), 2.06 (m, 4H), 1.15–1.66 (m, 16H), 0.87 (t, J 7.2 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 171.78, 162.69, 161.68, 149.45, 143.75, 97.83, 89.60, 83.85, 74.36, 70.74,

88 Materials and methods

63.61, 61.02, 59.18, 55.39, 40.57, 38.26, 35.19, 35.02, 33.60, 28.95, 28.50, 28.19, 28.00, + 26.12, 25.30, 24.83, 16.67, 14.90. ESI‐MS: m/z [M+Na] calcd for C31H46N6O8S: 685.3; found: + 686.1. HRMS: m/z [M+H] calcd for C31H46N6O8S: 663.3171; found: 663.3155.

89 Materials and methods

7) Chemistry (small‐molecules)

7.1) General

For general experimental details see chapter 6.1.

Memorandum

7.2) Synthesis of precursor aldehydes and acetophenon SM59‐84

3‐Nitro‐4‐(p‐tolylthio)benzaldehyde SM59

A solution of 4‐methylbenzenethiol SM86 (1 g, 5.4 mmol) and 4‐chloro‐3‐nitrobenzaldehyde

(0.58 g, 5.4 mmol), NaHCO3 (0.45 g, 5.4 mmol), 3.33 ml water and 6.66 mL ethanol was heated at reflux for 2 h. After being allowed to cool to room temperature, the precipitate was filtered out, washed with H2O and dried under vacuum. The crude product was re‐crystallized from AcOH to yield yellow crystalline SM59 in 92% (1.29 g (4.7 mmol). 1 M.p. 142°C. Rf 0.4 (PE–EtOAc, 5:1 ). H NMR (400 MHz, DMSO‐d6) δ 9.95 (s, 1H), 8.67 (d, J 1.8, 1H), 7.77 (dd, J 8.5, 1.8, 1H), 7.47‐ 7.43 (m, 2H), 7.31 (d, J 7.9, 2H), 6.98 (d, J 8.5, 1H), 2.44 (s, 3H). 13C NMR (101 MHz, DMSO‐d6) δ 189.26, 148.20, 144.87, 141.52, 136.07, 133.21, 131.90, 131.51, 128.93, 128.04, 126.26, 21.66. CHN analysis: calcd: C 61.52, H 4.06, N 5.12; found: C 61.47, H 4.09, N 5.24.

General procedure for the synthesis of precursor SM60‐75

To a stirred solution of the corresponding thiophenol (1 eq) in anhydrous DMF (~3 mL per

1 mmol thiol) K2CO3 (2 eq) was added, followed by the corresponding para‐haloginated‐ benzaldehyde or acetophenone (1 eq.). The reaction mixture was then heated to 80°C until

90 Materials and methods the reaction was completed. The reaction mixture was allowed to cool to room temperature and partitioned between 20 mL ether and 10 mL H2O. The phases were separated and the aqueous layer was washed with ether (3×25 mL). The combined organic layers were washed with 10 mL of H2O and 10 mL brine, dried over MgSO4 and concentrated in vacuo. The crude product was purified by silica gel column chromatography using n‐hexane/EtOAc mixtures or by recrystallization from isopropyl alcohol to yield the pure product.

3‐Nitro‐4‐(o‐tolylthio)benzaldehyde SM60

Reaction batch: 2‐Methylbenzenethiol SM87 (1.0 g, 8.1 mmol), 4‐chloro‐3‐ nitrobenzaldehyde (1.49 g, 8.1 mmol). Golden solid in 79% (1.74 g, 6.4 mmol) yield. 1 M.p. 91°C. Rf = 0.51 (PE–EtOAc, 4:1). H NMR (400 MHz, CDCl3) δ 9.97 (s, 1H), 8.73 (d, J 1.7 Hz, 1H), 7.79 (dd, J 8.5, 1.8 Hz, 1H), 7.59 (d, J 7.6 Hz, 1H), 7.48 (td, J 7.5, 1.0 Hz, 1H), 7.43 (d, J 7.0 Hz, 1H), 7.34 (td, J 7.5, 0.9 Hz, 1H), 6.84 (d, J 8.5 Hz, 1H), 2.36 (s, 3H). 13C NMR (101 MHz,

CDCl3) δ 189.17, 146.81, 144.95, 143.47, 137.23, 133.15, 132.09, 131.83, 131.53, 128.76, 128.22, 128.13, 128.10, 20.63. CHN analysis: calcd: C 61.52, H 4.06, N 5.12; found: C 61.47, H 4.01, N 5.28.

3‐Nitro‐4‐(m‐tolylthio)benzaldehyde SM61

Reaction batch: 3‐Methylbenzenethiol SM88 (373 mg, 3.0 mmol), 4‐chloro‐3‐ nitrobenzaldehyde (557 mg, 3.0 mmol). Yellow solid in 85% (693 mg, 2.5 mmol). M.p. 97°C. 1 Rf = 0.48 (PE–EtOAc, 4:1). H NMR (400 MHz, CDCl3) δ 9.97 (s, 1H), 8.70 (d, J 1.7 Hz, 1H), 7.80 (dd, J 8.5, 1.8 Hz, 1H), 7.46 – 7.38 (m, 3H), 7.38 – 7.34 (m, 1H), 7.01 (d, J 8.5 Hz, 1H), 2.42 (s, 13 3H). C NMR (101 MHz, CDCl3) δ 188.89, 147.53, 144.48, 140.45, 136.17, 132.83, 132.72, 131.57, 131.41, 130.13, 129.13, 128.68, 127.65, 21.14. CHN analysis: calcd: C 61.52, H 4.06, N 5.12; found: C 61.58, H 4.02, N 5.15.

91 Materials and methods

1‐(3‐Nitro‐4‐(p‐tolylthio)phenyl)ethanone SM62

Reaction batch: 4‐Methylbenzenethiol SM86 (372 mg, 3.0 mmol), 1‐(4‐chloro‐3‐nitrophenyl) ethanone (598 mg, 3.0 mmol). Yellow solid in 70% (598 mg, 2.1 mmol). M.p. 106°C. Rf 0.44 1 (PE–EtOAc, 2:1). H NMR (400 MHz, CDCl3) δ 8.77 (d, J 1.9 Hz, 1H), 7.87 (dd, J 8.6, 2.0 Hz, 1H), 7.49 – 7.44 (m, 2H), 7.32 (d, J 7.9 Hz, 2H), 6.93 (d, J 8.6 Hz, 1H), 2.60 (s, 3H), 2.45 (s, 3H). 13C

NMR (101 MHz, CDCl3) δ 195.25, 146.43, 144.45, 141.26, 136.03, 133.75, 131.95, 131.35, 128.43, 126.41, 126.06, 26.60, 21.58. CHN analysis: calcd: C 62.70, H 4.56, N 4.87; found: C 62.62, H 4.70, N 5.25.

3‐Fluoro‐4‐(p‐tolylthio)benzaldehyde SM63

Reaction batch: 4‐Methylbenzenethiol SM86 (497 mg, 4.0 mmol), 3,4‐difluorobenzaldehyde

(568 mg, 4.0 mmol). Light yellow solid in 78% (768 mg, 3.1 mmol) yield. M.p. 52°C. Rf 0.64 1 (PE–EtOAc, 6:1). H NMR (400 MHz, CDCl3) δ 9.87 (d, J 1.8 Hz, 1H), 7.52 (dd, J 9.6, 1.5 Hz, 1H), 7.46 – 7.42 (m, 3H), 7.27 (d, J 6.6 Hz, 2H), 6.90 (t, J 7.6 Hz, 1H), 2.42 (s, 3H). 13C NMR (101

MHz, CDCl3) δ 190.22 (d, J 2.0 Hz), 158.86 (d, J 247.5 Hz) 140.26, 136.03 (d, J 17.1 Hz), 135.28, 135.21, 130.97, 128.31 (d, J 2.4 Hz), 126.34 (d, J 3.0 Hz), 125.66 (d, J 1.6 Hz), 114.90 19 (d, J 21.9 Hz), 21.47. F NMR (376 MHz, CDCl3) δ ‐111.07. CHN analysis: calcd: C 68.27, H 4.50; found: C 68.33, H 4.68.

5‐Formyl‐2‐(p‐tolylthio)benzonitrile SM64

92 Materials and methods

Reaction batch: 4‐Methylbenzenethiol SM86 (497 mg, 4.0 mmol), 2‐fluoro‐5‐ formylbenzonitrile (597 mg, 3.0 mmol). Light yellow solid in 75% (748 mg, 3.0 mmol) yield. 1 M.p. 131°C. Rf 0.53 (PE–EtOAc, 4:1). H NMR (400 MHz, CDCl3) δ 9.89 (s, 1H), 8.06 (d, J 1.7 Hz, 1H), 7.77 (dd, J 8.4, 1.8 Hz, 1H), 7.48 – 7.45 (m, 2H), 7.31 (d, J 8.0 Hz, 2H), 6.94 (d, J 8.4 13 Hz, 1H), 2.44 (s, 3H). C NMR (101 MHz, CDCl3) δ 189.22, 153.08, 141.25, 135.66, 134.96, 133.28, 132.61, 131.33, 126.87, 124.80, 115.83, 110.42, 21.54. CHN analysis: calcd: C 71.12, H 4.38, N 5.53; found: C 71.08, H 4.59, N 5.58.

4‐((3‐Fluoro‐4‐methylphenyl)thio)‐3‐nitrobenzaldehyde SM65

Reaction batch: 3‐Fluoro‐4‐methylbenzenethiol SM89 (250 mg, 1.76 mmol), 4‐chloro‐3‐nitro‐ benzaldehyde (326 mg, 1.76 mmol). Yellow solid in 83% (424 mg, 1.5 mmol) yield. 1 M.p. 167°C. Rf 0.64 (PE–EtOAc, 3:1). H NMR (400 MHz, CDCl3) δ 9.98 (s, 1H), 8.70 (d, J 1.7 Hz, 1H), 7.82 (dd, J 8.5, 1.8 Hz, 1H), 7.46 – 7.42 (m, 1H), 7.42 – 7.38 (m, 1H), 7.17 (t, J 8.8 Hz, 13 1H), 6.99 (d, J 8.5 Hz, 1H), 2.34 (d, J 1.8 Hz, 3H). C NMR (101 MHz, CDCl3) δ 189.15, 163.03 (d, J 251.3 Hz), 147.65, 144.77, 139.33 (d, J 6.0 Hz), 135.50 (d, J 8.7 Hz), 133.27, 132.02, 128.74, 128.03 (d, J 18.3 Hz), 127.97, 124.53 (d, J 3.7 Hz), 117.51 (d, J 23.2 Hz), 14.73 (d, J 3.4 19 Hz). F NMR (376 MHz, CDCl3) δ ‐112.72. CHN analysis: calcd: C 57.72, H 3.46, N 4.81; found: C 57.57, H 3.48, N 4.94.

3‐Bromo‐4‐((3‐fluoro‐4‐methylphenyl)thio)benzaldehyde SM66

Reaction batch: 3‐Fluoro‐4‐methylbenzenethiol SM89 (250 mg, 1.76 mmol), 3‐bromo‐4‐ fluorobenzaldehyde (357 mg, 1.76 mmol). White solid in 65% (369 mg, 1.1 mmol) yield. 1 M.p. 77°C. Rf 0.46 (PE–EtOAc, 10:1). H NMR (400 MHz, CDCl3) δ 9.85 (s, 1H), 8.00 (d, J 1.6 Hz, 1H), 7.57 (dd, J 8.2, 1.7 Hz, 1H), 7.43 (dd, J 7.1, 1.6 Hz, 1H), 7.41 – 7.36 (m, 1H), 7.13 (t, J 13 8.8 Hz, 1H), 6.73 (d, J 8.2 Hz, 1H), 2.32 (d, J 1.8 Hz, 3H). C NMR (101 MHz, CDCl3) δ 189.82,

93 Materials and methods

162.56 (d, J 250.4 Hz), 149.31, 139.03 (d, J 5.9 Hz), 135.15 (d, J 8.6 Hz), 134.32, 133.62, 128.39, 127.50 (d, J 18.1 Hz), 126.51, 124.66 (d, J 3.9 Hz), 120.30, 117.07 (d, J 23.1 Hz), 14.55 19 (d, J 3.5 Hz). F NMR (376 MHz, CDCl3) δ ‐113.83. CHN analysis: calcd: C 51.71, H 3.10; found: C 51.50, H 3.25.

3‐Nitro‐4‐((2,3,5,6‐tetrafluoro‐4‐(trifluoromethyl)phenyl)thio)benzaldehyde SM67

Reaction batch: 2,3,5,6‐Tetrafluoro‐4‐(trifluoromethyl)benzenethiol SM90 (750 mg, 3.0 mmol), 4‐chloro‐3‐nitro‐benzaldehyde (557 mg, 3.0 mmol). Yellow solid in 85% (1.01 g, 2.5 1 mmol) yield. M.p. 136°C. Rf 0.58 (PE–EtOAc, 4:1). H NMR (400 MHz, CDCl3) δ 10.05 (s, 1H), 8.80 (d, J 1.7 Hz, 1H), 8.00 (dd, J 8.4, 1.8 Hz, 1H), 7.04 (d, J 8.4 Hz, 1H). 13C NMR (101 MHz,

CDCl3) δ 188.64, 149.14 – 146.36 (m), 146.22 – 143.28 (m), 145.84, 140.05, 134.83, 133.21, 128.16, 128.04, 121.96 – 121.79 (m), 119.20 – 119.03 (m), 115.63 – 114.98 (m). 19F NMR

(376 MHz, CDCl3) δ ‐56.44 (t, J 22.0 Hz, 3F), ‐127.96 – ‐128.17 (m, 2F), ‐136.59 – ‐136.97 (m, 2F). CHN analysis: calcd: C 42.12, H 1.01, N 3.51; found: C 42.00, H 1.48, N 3.75.

3‐Bromo‐4‐(p‐tolylthio)benzaldehyde SM68

S Br

Reaction batch: 4‐Methylbenzenethiol SM86 (500 mg, 4.0 mmol); 3‐bromo‐4‐ fluorobenzaldehyde (818 mg, 4.0 mmol). Light yellow crystals in 83% (1.03 g, 3.3 mmol) 1 yield. M.p. 92°C. Rf 0.70 (PE–EtOAc, 1:1). H NMR (400 MHz, CDCl3) δ 9.83 (s, 1H), 7.99 (d, J 1.6 Hz, 1H), 7.54 (dd, J 8.3, 1.6 Hz, 1H), 7.49 – 7.44 (m, 2H), 7.30 (d, J 7.9 Hz, 2H), 6.74 (d, J 13 8.3 Hz, 1H), 2.44 (s, 3H). C NMR (101 MHz, CDCl3) δ 190.02, 149.90, 140.76, 135.87, 134.33, 133.74, 131.19, 128.44, 126.69, 126.48, 120.43, 21.55. CHN analysis: calcd: C 54.74, H 3.61; found: C 54.72, H. 3.74

94 Materials and methods

4‐(p‐Tolylthio)benzaldehyde SM69

Reaction batch: 4‐Methylbenzenethiol SM86 (500 mg, 4.0 mmol); 4‐fluorobenzaldehyde

(496 mg, 4.0 mmol). Light yellow crystals in 89% (814 mg, 3.6 mmol) yield. M.p. 73°C. Rf 0.93 1 (PE–EtOAc, 1:1). H NMR (400 MHz, CDCl3) δ 9.90 (s, 1H), 7.73 – 7.68 (m, 2H), 7.46 – 7.42 (m, 13 2H), 7.28 – 7.23 (m, 2H), 7.22 – 7.18 (m, 2H), 2.42 (s, 3H). C NMR (101 MHz, CDCl3) δ 191.33, 148.32, 139.86, 134.99, 133.63, 130.80, 130.23, 127.42, 126.74, 21.46. CHN analysis: calcd: C 73.65, H 5.30; found: C 73.61, H 5.35.

3‐Chloro‐4‐(p‐tolylthio)benzaldehyde SM70

Reaction batch: 4‐Methylbenzenethiol SM86 (509 mg, 4.1 mmol); 3‐chloro‐4‐ fluorobenzaldehyde (648 mg, 4.1 mmol). White solid in 61% (651 mg, 2.5 mmol) yield. 1 M.p. 72°C. Rf 0.57 (PE–EtOAc, 5:1). H NMR (400 MHz, CDCl3) δ 9.85 (s, 1H), 7.82 (d, J 1.7 Hz, 1H), 7.49 (dd, J 8.3, 1.7 Hz, 1H), 7.48 – 7.44 (m, 2H), 7.32 – 7.28 (m, 2H), 6.77 (d, J 8.2 Hz, 13 1H), 2.43 (s, 3H). C NMR (101 MHz, CDCl3) δ 190.13, 147.91, 140.70, 135.87, 134.21, 131.15, 131.03, 130.20, 127.97, 126.69, 125.93, 21.52. CHN analysis: calcd: C 64.00, H 4.22; found: C 63.57, H 4.17.

4‐(p‐Tolylthio)‐3‐(trifluoromethyl)benzaldehyde SM71

Reaction batch: 4‐Methylbenzenethiol SM86 (503 mg, 4.0 mmol); 4‐fluoro‐3‐ (trifluoromethyl) benzaldehyde (776 mg, 4.0 mmol). Yellowish solid in 59% (701 mg, 1 2.4 mmol) yield. M.p. 58°C. Rf 0.47 (PE–EtOAc, 5:1). H NMR (400 MHz, CDCl3) δ 9.93 (s, 1H), 8.11 (d, J 1.5 Hz, 1H), 7.72 (dd, J 8.3, 1.5 Hz, 1H), 7.49 – 7.42 (m, 2H), 7.29 (d, J 7.9 Hz, 2H),

95 Materials and methods

13 7.01 (d, J 8.3 Hz, 1H), 2.43 (s, 3H). C NMR (101 MHz, CDCl3) δ 190.11, 148.15, 140.78, 135.82, 132.91, 131.94, 131.18, 128.72, 128.30 (q, J 5.6 Hz), 127.43 (q, J 32.0 Hz), 126.23 (q, J 19 1.7 Hz), 126.11 (q, J 274.1 Hz), 21.51. F NMR (376 MHz, CDCl3) δ ‐62.15. CHN analysis: calcd: C 60.80, H 3.74; found: C 60.86, H 3.90.

3‐Fluoro‐4‐(o‐tolylthio)benzaldehyde SM77

Reaction batch: 2‐Methylbenzenethiol SM87 (87 mg, 0.7 mmol); 3,4‐difluorobenzaldehyde 1 (99 mg, 0.7 mmol). Colorless oil in 86% (151 mg, 0.6 mmol) yield. Rf 0.56 (PE–EtOAc, 6:1). H

NMR (400 MHz, CDCl3) δ 9.87 (d, J 1.9 Hz, 1H), 7.55 – 7.54 (m, 1H), 7.54 – 7.51 (m, 1H), 7.43 (dd, J 8.1, 1.6 Hz, 1H), 7.41 – 7.36 (m, 2H), 7.30 – 7.24 (m, 1H), 6.76 – 6.69 (m, 1H), 2.39 (s, 13 3H). C NMR (101 MHz, CDCl3) δ 190.16 (d, J 2.1 Hz), 159.01 (d, J 247.5 Hz), 142.95, 136.59, 135.15 (d, J 5.8 Hz), 134.92 (d, J 17.3 Hz), 131.45, 130.49, 128.16 (d, J 1.4 Hz), 127.67 (d, J 2.5 19 Hz), 127.59, 126.47 (d, J 3.0 Hz), 114.88 (d, J 21.7 Hz), 20.69. F NMR (376 MHz, CDCl3) δ ‐ 111.10. CHN analysis: calcd: C 68.27, H 4.50; found: C 68.63, H 4.53.

4‐(o‐Tolylthio)‐3‐(trifluoromethyl)benzaldehyde SM73

Reaction batch: 2‐Methylbenzenethiol SM87 (62 mg, 0.5 mmol); 4‐fluoro‐3‐(trifluoromethyl) benzaldehyde (96 mg, 0.5 mmol). Colorless oil in 80% (124 mg, 0.4 mmol) yield. Rf 0.51 1 (PE–EtOAc, 6:1). H NMR (400 MHz, CDCl3) δ 9.83 (s, 1H), 8.02 (d, J 1.6 Hz, 1H), 7.61 (dd, J 8.3, 1.4 Hz, 1H), 7.48 (dd, J 7.6, 1.1 Hz, 1H), 7.36 – 7.31 (m, 1H), 7.31 – 7.27 (m, 1H), 7.24 – 13 7.17 (m, 1H), 6.76 (d, J 8.3 Hz, 1H), 2.24 (s, 3H). C NMR (101 MHz, CDCl3) δ 190.05, 147.03, 143.21, 137.19, 132.92, 132.09, 131.69, 130.96, 128.75 (q, J 1.6 Hz), 128.35 (q, J 5.6 Hz), 19 127.99, 127.74, 127.54 (q, J 31.9 Hz), 123.53 (q, J 274.3 Hz), 20.66. F NMR (376 MHz, CDCl3) δ ‐62.43. CHN analysis: calcd: C 60.80, H 3.74; found: C 60.95, H 4.07.

96 Materials and methods

2,4‐Bis(p‐tolylthio)benzaldehyde SM74

Reaction batch: 4‐Methylbenzenethiol SM86 (2eq, 1.03 g, 8.3 mmol); 2,4‐ difluorobenzaldehyde (1eq, 595 mg, 4.1 mmol); K2CO3 (4eq, 2.29 g, 16.6 mmol). Yellow solid 1 in 78% (1.12 g, 3.2 mmol) yield. M.p. 74°C. Rf 0.45 (PE–EtOAc, 10:1). H NMR (400 MHz,

CDCl3) δ 10.20 (s, 1H), 7.64 (d, J 8.2 Hz, 1H), 7.28 – 7.23 (m, 4H), 7.14 – 7.09 (m, 4H), 6.93 (dd, J 8.1, 1.8 Hz, 1H), 6.58 (d, J 1.8 Hz, 1H), 2.39 (s, 3H), 2.39 (s, 3H). 13C NMR (101 MHz,

CDCl3) δ 190.40, 148.19, 144.17, 139.65, 139.18, 135.04, 134.58, 132.75, 130.58, 130.57, 129.87, 127.92, 126.64, 125.24, 122.88, 21.51, 21.49. CHN analysis: calcd: C 71.96, H 5.18; found: C 72.01, H 5.16.

4‐Bromo‐5‐(p‐tolylthio)thiophene‐2‐carbaldehyde SM75

Reaction batch: 4‐Methylbenzenethiol SM86 (186 mg, 1.5 mmol); 4,5‐dibromothiophene‐2‐ carbaldehyde (405 mg, 1.5 mmol). White solid in 87% (407 mg, 1.3 mmol) yield. M.p. 106°C. 1 Rf 0.33 (PE–EtOAc, 8:1). H NMR (400 MHz, CDCl3) δ 9.68 (s, 1H), 7.60 (s, 1H), 7.50 – 7.45 (m, 13 2H), 7.28 – 7.24 (m, 2H), 2.42 (s, 3H). C NMR (101 MHz, CDCl3) δ 180.75, 149.60, 141.41, 140.69, 139.03, 133.94, 131.02, 127.89, 110.67, 21.47. CHN analysis: calcd: C 46.01, H 2.90; found: C 46.02, H 3.01.

General procedure for the synthesis of precursor SM76‐83

A mixture of the corresponding thiol (1 eq), KOH (1 eq) and methanol (~3 mL per 1 mmol thiol) was stirred at room temperature until the KOH was dissolved. After evaporation of the

97 Materials and methods solvent under reduced pressure, the thiolate was dissolved in anhydrous DMF (~3 mL per 1 mmol thiol). The corresponding para‐haloginated‐benzaldehyde (1eq) was added to the stirred mixture in small portions at 0°C. The reaction mixture was allowed to warm to room temperature and was stirred till TLC indicated completion. The reaction mixture was partitioned between ether 20 mL and 10 mL H2O. The aqueous layer was washed with ether

(3×25 mL) and the combined organic layers were washed with 10 mL of H2O and 10 mL brine, dried over MgSO4 and concentrated in vacuo. The crude product was purified by silica gel column chromatography using n‐hexane/EtOAc mixtures or by recrystallization from isopropyl alcohol to give the pure product.

4‐(Cyclohexylthio)‐3‐nitrobenzaldehyde SM76

Reaction batch: Cyclohexanethiol SM91 (630 mg, 5.4 mmol); 4‐chlor‐3‐nitrobenzaldehyde

(1.0 g, 5.4 mmol). Orange solid in 35% (504 mg, 1.9 mmol) yield. M.p. 94°C. Rf 0.67 1 (PE–EtOAc, 1:1). H NMR (400 MHz, CDCl3) δ 9.99 (s, 1H), 8.62 (d, J 1.8 Hz, 1H), 8.01 (dd, J 8.4, 1.8 Hz, 1H), 7.60 (d, J 8.5 Hz, 1H), 3.53 – 3.26 (m, 1H), 2.16 – 2.07 (m, 2H), 1.90 – 1.84 13 (m, 2H), 1.77 – 1.66 (m, 1H), 1.63 – 1.22 (m, 5H). C NMR (101 MHz, CDCl3) δ 189.26, 146.31, 145.33, 132.48, 131.75, 128.39, 127.69, 44.30, 32.44, 26.05, 25.65. CHN analysis: calcd: C 58.85, H 5.70, N 5.28; found: C 58.99, H 5.62, N 5.40.

4‐(Naphthalene‐2‐ylthio)‐3‐nitrobenzaldehyde SM77

Reaction batch: Naphthalene‐2‐thiol SM92 (481 mg, 3.0 mmol), 4‐chlor‐3‐nitrobenzaldehyde

(557 mg, 3.0 mmol). Yellow solid in 33% (296 mg, 1.0 mmol) yield. M.p. 124°C. Rf 0.70 1 (PE–EtOAc, 1:1). H NMR (400 MHz, CDCl3) δ 9.97 (s, 1H), 8.72 (d, J 1.8 Hz, 1H), 8.20 (s, 1H), 7.98 (d, J 8.5 Hz, 1H), 7.96 – 7.87 (m, 2H), 7.76 (dd, J 8.5, 1.8 Hz, 1H), 7.62 (pd, J 7.0, 1.5 Hz, 13 2H), 7.53 (dd, J 8.5, 1.8 Hz, 1H), 7.03 (d, J 8.5 Hz, 1H). C NMR (101 MHz, CDCl3) δ 189.17,

98 Materials and methods

147.46, 136.52, 134.16, 133.97, 133.30, 131.99, 131.37, 130.57, 129.19, 128.29, 128.22, 128.14, 127.93, 127.45, 126.92. CHN analysis: calcd: C 66.01, H 3.58, N 4.53; found: C 65.85, H 3.74, N 4.73.

4‐(3,5‐Dimethylphenylthio)‐3‐nitrobenzaldehyde SM78

Reaction batch: 3,5‐Dimethylbenzenethiol SM93 (300 mg, 2.2 mmol), 4‐chlor‐3‐ nitrobenzaldehyde (403 mg, 2.2 mmol). Golden yellow needles in 49% (308 mg, 1.1 mmol) 1 yield. M.p. 112°C. Rf 0.69 (PE–EtOAc, 1:1). H NMR (400 MHz, CDCl3) δ 9.97 (s, 1H), 8.69 (d, J 1.8 Hz, 1H), 7.81 (dd, J 8.5, 1.8 Hz, 1H), 7.21 (s, 2H), 7.17 (s, 1H), 7.03 (d, J 8.5 Hz, 1H), 2.37 (s, 13 6H). C NMR (101 MHz, CDCl3) δ 189.13, 140.39, 133.38, 132.96, 132.55, 131.70, 129.04, 129.00, 128.97, 127.87, 125.20, 21.22. CHN analysis: calcd: C 62.70, H 4.56, N 4.87; found: C 62.69, H 4.70, N 5.15.

4‐(4‐Ethylphenylthio)‐3‐nitrobenzaldehyde SM79

Reaction batch: 4‐Ethylbenzenethiol SM94 (300 mg, 2.2 mmol), 4‐chlor‐3‐nitrobenzaldehyde

(403 mg, 2.2 mmol). Orange crystals in 73% (457 mg, 1.6 mmol) yield. M.p. 93°C. Rf 0.80 1 (PE–EtOAc, 1:1). H NMR (400 MHz, CDCl3) δ 9.97 (s, 1H), 8.70 (d, J 1.7 Hz, 1H), 7.80 (dd, J 8.5, 1.8 Hz, 1H), 7.52 – 7.48 (m, 2H), 7.38 – 7.34 (m, 2H), 7.01 (d, J 8.5 Hz, 1H), 2.75 (q, J 7.6 13 Hz, 2H), 1.31 (t, J 7.6 Hz, 3H). C NMR (101 MHz, CDCl3) δ 189.22, 148.18, 147.62, 136.08, 135.39, 133.11, 131.85, 130.25, 128.89, 128.02, 126.34, 28.89, 15.37. CHN analysis: calcd: C 62.70, H 4.56, N 4.87; found: C 62.74, H 4.77, N 4.91.

99 Materials and methods

3‐Nitro‐4‐(4‐(trifluoromethyl)phenylthio)benzaldehyde SM80

Reaction batch: 4‐(Trifluoromethyl)benzenethiol SM95 (600 mg, 3.4 mmol), 4‐chlor‐3‐ nitrobenzaldehyde (631 mg, 3.4 mmol). Yellow solid in 62% (687 mg, 2.1 mmol) yield. 1 M.p. 114°C. Rf 0.74 (PE–EtOAc, 1:1). H NMR (400 MHz, CDCl3) δ 10.00 (s, 1H), 8.72 (d, J 1.7 Hz, 1H), 7.86 (dd, J 8.5, 1.8 Hz, 1H), 7.79 (d, J 8.4 Hz, 2H), 7.74 (d, J 8.4 Hz, 2H), 7.00 (d, J 8.4 13 Hz, 1H). C NMR (101 MHz, CDCl3) δ 188.97, 145.53, 145.28, 136.18, 134.85, 133.76, 133.02, 19 132.69, 132.35, 129.02, 127.83, 127.44 (q, J 3.6 Hz), 125.04. F‐NMR (376 MHz, CDCl3) δ ‐ 62.97. CHN analysis: calcd: C 51.38, H 2.46, N 4.28; found: C 51.08, H 2.69, N 4.53.

4‐(Cyclopentylthio)‐3‐nitrobenzaldehyde SM81

Reaction batch: Cyclopentanethiol SM96 (300 mg, 2.9 mmol), 4‐chlor‐3‐nitrobenzaldehyde

(546 mg, 2.9 mmol). Orange crystals in 47% (349 mg, 1.4 mmol) yield. M.p. 76°C. Rf 0.79 1 (PE–EtOAc, 1:1). H NMR (400 MHz, CDCl3) δ 9.99 (s, 1H), 8.65 (d, J 1.8 Hz, 1H), 8.01 (dd, J 8.4, 1.8 Hz, 1H), 7.65 (d, J 8.5 Hz, 1H), 3.83 – 3.66 (m, 1H), 2.36 – 2.15 (m, 2H), 1.89 – 1.78 13 (m, 2H), 1.79 – 1.65 (m, 4H). C NMR (101 MHz, CDCl3) δ 189.27, 147.26, 145.77, 132.41, 131.85, 128.25, 128.10, 44.03, 33.28, 25.41. CHN analysis: calcd: C 57.35, H 5.21, N 5.57; found: C 57.18, H 5.23, N 5.74.

3‐Nitro‐4‐((4‐(trifluoromethoxy)phenyl)thio)benzaldehyde SM82

Reaction batch: 4‐(Trifluoromethoxy)benzenethiol SM97 (311 mg, 1.6 mmol), 4‐chlor‐3‐ nitrobenzaldehyde (297 mg, 1.6 mmol). Yellow solid in 77% (430 mg, 1.3 mmol) yield.

100 Materials and methods

1 M.p. 96°C. Rf 0.56 (PE–EtOAc, 5:1). H NMR (400 MHz, CDCl3) δ 9.99 (s, 1H), 8.71 (d, J 1.7 Hz, 1H), 7.85 (dd, J 8.5, 1.7 Hz, 1H), 7.71 – 7.59 (m, 2H), 7.38 (d, J 8.5 Hz, 2H), 6.99 (d, J 8.5 Hz, 13 1H). C NMR (101 MHz, CDCl3) δ 189.04, 151.21, 151.20, 146.50, 144.98, 137.81, 133.53, 19 132.27, 128.74, 128.29, 127.88, 122.70, 120.47 (q, J 259.0 Hz). F‐NMR (376 MHz, CDCl3) δ ‐ 57.69. CHN analysis: calcd: C 48.98, H 2.35, N 4.08; found: C 48.56, H 2.72, N 4.18.

4‐(o‐Tolylthio)benzaldehyde SM83

O S

Reaction batch: 2‐Methylbenzenethiol SM87 (1.61g, 13.0 mmol), 4‐fluorobenzaldehyde

(1.61 g, 13.0 mmol). White solid in 97% (2.91 g, 12.6 mmol) yield. M.p. 44°C. Rf 0.70 1 (PE–EtOAc, 4:1). H NMR (400 MHz, CDCl3) δ 9.79 (s, 1H), 7.62 – 7.57 (m, 2H), 7.44 (d, J 7.5 Hz, 1H), 7.32 – 7.23 (m, 2H), 7.20 – 7.13 (m, 1H), 7.04 – 6.99 (m, 2H), 2.27 (s, 3H). 13C NMR

(101 MHz, CDCl3) δ 191.30, 147.34, 142.66, 136.42, 133.59, 131.35, 130.32, 130.20, 129.89, 127.42, 126.43, 20.84. CHN analysis: calcd: C 73.65, H 5.30; found: C 73.64, H 5.29.

Synthesis of 3‐nitro‐4‐tosylbenzaldehyde SM84

NaBH4 (416 mg, 11.0 mmol) was added portion wise to a suspension of 33 (3.0 g, 11.0 mmol) in methanol (25 mL). The reaction mixture was stirred at room temperature for 1 h. H2O (30 mL) was added, and the reaction mixture was extracted with EtOAc (4×30 mL). The organic layers were combined, washed with brine (20 mL), dried over MgSO4 and concentrated under reduced pressure to give the (3‐nitro‐4‐(p‐tolylthio)phenyl)methanol intermediate as a yellow solid in 98% (2.94 g, 10.8 mmol) yield. M.p. 96°C. Rf 0.42 1 (PE–EtOAc, 1:1). H NMR (400 MHz, CDCl3) δ 8.22 (d, J 1.4 Hz, 1H), 7.47 – 7.43 (m, 2H), 7.33 (dd, J 8.4, 1.7 Hz, 1H), 7.28 (d, J 8.0 Hz, 2H), 6.85 (d, J 8.4 Hz, 1H), 4.70 (s, 2H), 2.43 (s, 3H). Refluxing of (3‐nitro‐4‐(p‐tolylthio)phenyl)methanol (1.5 g, 5.5 mmol) in 20 mL AcOH containing 2.73 mL H2O2 (30%) furnished the (3‐nitro‐4‐tosylphenyl)methanol intermediate

101 Materials and methods

SM85. The solvent was removed in vacuo and the residue was recrystallized from AcOH to yield the light yellowish product SM85 in 61% (1.02 g, 3.3 mmol) yield. M.p. 131°C. Rf 0.21 1 (PE–EtOAc, 1:1). H NMR (400 MHz, CDCl3) δ 8.31 – 8.27 (m, 1H), 7.88 – 7.81 (m, 2H), 7.75 – 7.69 (m, 2H), 7.34 (d, J 8.1 Hz, 2H), 4.84 (s, 2H), 2.43 (s, 3H).

To a solution of SM85 (1.0 g, 3.3 mmol) in CH2Cl2 (40 mL), DMP (5.29 g, 12.4 mmol) was added portion wise and the mixture was stirred at room temperature over night. After completion of the reaction, the mixture was quenched with 100 mL of a 1.3 M NaOH solution. The organic layer was separated and the aqueous layer was extracted with ether

(3×20 mL). The combined organic layers were dried over MgSO4, concentrated and purified by silica gel column chromatography (n‐hexane/EtOAc mixtures) to give the white crystals of 1 SM84 in 53% (0.63 g, 1.7 mmol) yield. M.p. 150°C. Rf 0.50 (PE–EtOAc, 1:1). H NMR (400

MHz, CDCl3) δ 10.11 (s, 1H), 8.51 (d, J 8.0 Hz, 1H), 8.22 (dd, J 8.1, 1.5 Hz, 1H), 8.16 (d, J 1.5 13 Hz, 1H), 7.94 – 7.82 (m, 2H), 7.38 (d, J 8.1 Hz, 2H), 2.45 (s, 3H). C NMR (101 MHz, CDCl3) δ 188.41, 149.10, 145.92, 140.21, 139.46, 136.58, 132.75, 132.67, 130.13, 128.84, 124.85, 21.89. CHN analysis: calcd: C 55.08, H 3.63, N 4.59; found: C 54.68, H 3.91, N 4.83.

7.3) Synthesis of small‐molecules SM1, 4, 10, 12, 16, 17, 21, 23, 27‐58

Memorandum

Small‐molecules SM1, 4, 10, 16, 17, 21, 23, 27, 28 were synthetisized during my privious diploma thesis76 and are listed herin for completeness.

General procedure for the synthesis of small‐molecule SM1, 4, 10, 12, 16, 17, 21, 23, 27‐32, 35‐58

The condensation products SM1, 4, 10, 12, 16, 17, 21, 23, 27‐32, 35‐58 were synthesized in a millimol range as described in literature. This was performed by refluxing 1 eq of the corresponding aldehyde, 1 eq rhodanine (or rather N‐allyl rhodanine, thioazolidine‐2,4‐ dione, thiohydantoine, rhodanine‐3‐acetic acid, 3‐phenyl‐2‐thioxothiazolidin‐4‐one, pseudothiohydantoin) together with (12 mL per 1 mmol aldehyde) an anhydrous NaOAc solution (2.5 M in AcOH) over night (thermodynamic reaction control). Afterwards the mixture was allowed to cool to room temperature and the products were isolated by

102 Materials and methods

filtration. The yellowish products were washed thoroughly with toluene, H2O, ethanol and ether and purified via recrystallization or column chromatography.

(Z)‐5‐(3‐Nitro‐4‐(p‐tolylthio)benzylidene)‐2‐thioxothiazolidin‐4‐one SM1

Reaction Batch: 3‐Nitro‐4‐(p‐tolylthio)benzaldehyde SM59 (0.273 g, 1 mmol); rhodanine

(0.133 g, 1 mmol). Yellow solid 96% (0.374 g, 0.96 mmol) yield. M.p. 264°C. Rf 0.33 (PE–EtOAc, 3:2). 1H NMR (400 MHz, DMSO‐d6) δ 13.83 (bs, 1H), 8.39 (d, J 2.0 Hz, 1H), 7.61 (dd, J 8.7, 2.1 Hz, 1H), 7.57 (s, 1H), 7.44 (d, J 8.1 Hz, 2H), 7.31 (d, J 7.9 Hz, 2H), 6.83 (d, J 8.6 Hz, 1H), 2.30 (s, 3H). 13C NMR (101 MHz, DMSO‐d6) δ 182.99, 144.56, 140.82, 140.77, 135.62, 134.24, 134.24, 131.25, 130.41, 130.41, 128.63, 128.36, 127.60, 125.72, 20.91. ESI‐MS: m/z ‐ ‐ [M‐H] calcd for C17H12N2O3S3: 387.0; found: 387.0. HRMS: m/z [M‐H] calcd for C17H12N2O3S3: 386.9937; found: 386.9922.76

(Z)‐5‐(4‐Chloro‐3‐nitrobenzylidene)‐2‐thioxothiazolidin‐4‐one SM4

Reaction Batch: 4‐Chloro‐3‐nitrobenzaldehyde (0.186 g, 1 mmol); rhodanine (0.133 g, 1 mmol). Orange solid in 96% (0.374 g, 0.96 mmol) yield. M.p. 255°C. Rf 0.42 (PE–EtOAc, 1:1). 1H NMR (400 MHz, DMSO‐d6) δ 14.00 (bs, 1H), 8.31 (d, J 2.1 Hz, 1H), 7.91 (d, J 8.5 Hz, 1H), 7.83 (dd, J 8.5, 2.1 Hz, 1H), 7.69 (s, 1H). 13C NMR (101 MHz, DMSO‐d6) δ 195.05, 169.27, 147.94, 134.12, 133.46, 132.68, 129.17, 127.67, 127.13, 126.39. ESI‐MS: m/z [M‐H]‐ calcd for ‐ C10H5ClN2O3S2: 298.9; found: 298.9. HRMS: m/z [M‐H] calcd for C10H5ClN2O3S2: 298.9357; found: 298.9350.76

103 Materials and methods

(Z)‐5‐(3‐Nitro‐4‐(5‐(trifluoromethyl)pyridin‐2‐ylthio)benzylidene)‐2‐thioxothiazolidin‐4‐one SM10

Reaction Batch: 3‐Nitro‐4‐((5‐(trifluoromethyl)pyridin‐2‐yl)thio)benzaldehyde (0.456 g, 1.39 mmol); rhodanine (0.185 g, 1.39 mmol). Yellow solid in 62% (0.381 g, 0.86 mmol) yield. 1 M.p. 255°C. Rf 0.42 (PE–EtOAc, 1:1). H NMR (400 MHz, DMSO‐d6) δ 14.00 (bs, 1H), 8.90 (s, 1H), 8.43 (s, 1H), 8.23 (d, J 6.2 Hz, 1H), 7.77 (m, 4H), 7.74 (s, 1H). 13C NMR (101 MHz, DMSO‐ d6) δ 195.00, 169.30, 160.41, 149.41, 147.05, 135.38, 135.30, 135.07, 135.02, 133.85, 133.72, 129.83, 128.93, 128.06, 127.04, 125.38. 19F NMR (376 MHz, DMSO‐d6): δ ‐60.87. ESI‐MS: m/z ‐ ‐ [M‐H] calcd for C16H8F3N3O3S3: 442.0; found: 441.9. HRMS: m/z [M‐H] calcd for 76 C16H8F3N3O3S3: 441.9607; found: 441.9597.

(Z)‐5‐(4‐(Cyclohexylthio)‐3‐nitrobenzylidene)‐2‐thioxothiazolidin‐4‐one SM12

Reaction batch: 4‐(Cyclohexylthio)‐3‐nitrobenzaldehyde SM91 (200 mg, 0.75 mmol), rhodanine (100 mg, 0.75 mmol). Orange solid in 45% (128 mg, 0.34 mmol) yield. M.p. 237°C. 1 Rf 0.49 (PE–EtOAc, 1:1). H NMR (400 MHz, DMSO‐d6) δ 13.95 (bs, 1H), 8.37 (d, J 1.5 Hz, 1H), 7.87 – 7.83 (m, 2H), 7.70 (s, 1H), 3.65 – 3.58 (m, 1H), 2.06 – 1.99 (m, 2H), 1.79 – 1.58 (m, 2H), 1.51 – 1.17 (m, 6H). 13C NMR (101 MHz, DMSO‐d6) δ 146.45, 137.59, 133.94, 129.98, 128.95, ‐ 127.44, 43.08, 31.97, 25.30, 25.10. ESI‐MS: m/z [M‐H] calcd for C16H16N2O3S3: 379.0; found: ‐ 379.0. HRMS: m/z [M‐H] calcd for C16H16N2O3S3: 379.0239; found: 379.0229.

104 Materials and methods

(Z)‐5‐(3‐Bromo‐4‐(4‐fluorobenzyloxy)benzylidene)‐2‐thioxothiazolidin‐4‐one SM16

Reaction Batch: 3‐Bromo‐4‐((4‐fluorobenzyl)oxy)benzaldehyde (0.309 g, 1 mmol); rhodanine

(0.133 g, 1 mmol). Yellow solid 90% (0.381 g, 0.90 mmol) yield. M.p. 206°C. Rf 0.26 (PE–EtOAc, 5:2). 1H NMR (400 MHz, DMSO‐d6) δ 13.83 (bs, 1H), 7.89 (d, J 2.2 Hz, 1H), 7.60 (s, 1H), 7.59‐7.51 (m, 3H), 7.37 (d, J 8.8 Hz, 1H), 7.29‐7.22 (m, 2H), 5.29 (s, 2H). 13C NMR (400 MHz, DMSO‐d6) δ 195.27, 156.06, 135.43, 132.25, 131.01, 130.12, 129.82, 129.73, 127.27, 124.16, 115.51, 115.29, 114.62, 112.08, 69.71. 19F NMR (376 MHz, DMSO‐d6) δ ‐114.00. ESI‐ ‐ 79 81 79 MS: m/z [M‐H] calcd for C17H11BrFNO2S2: 421.9 ( Br), 423.9 ( Br); found: 421.9 ( Br), 423.9 81 ‐ 79 81 ( Br). HRMS: m/z [M‐H] calcd for C17H11BrFNO2S2: 421.9326 ( Br), 423.9305 ( Br); found: 421.9316 (79Br), 423.9294 (81Br).76

(Z)‐5‐(4‐(4‐Fluorobenzyloxy)‐3‐nitrobenzylidene)‐2‐thioxothiazolidin‐4‐one SM17

Reaction Batch: 4‐((4‐Fluorobenzyl)oxy)‐3‐nitrobenzaldehyde (0.275 g, 1 mmol); rhodanine

(0.133 g, 1 mmol). Yellow solid in 89%: (0.347 g, 0.89 mmol) yield. M.p. 228°C. Rf 0.23 (PE–EtOAc, 2:1). 1H NMR (400 MHz, DMSO‐d6) δ 13.90 (bs, 1H), 8.18 (d, J 2.2 Hz, 1H), 7.85 (dd, J 8.9, 2.3 Hz, 1H), 7.66 (s, 1H), 7.61 (d, J 9.0 Hz, 1H), 7.55‐7.49 (m, 2H), 7.30‐7.22 (m, 2H), 5.38 (s, 2H). 13C NMR (101 MHz, DMSO‐d6) δ 195.17, 169.30, 163.20, 160.77, 152.03, 139.84, 135.52, 131.75, 131.72, 129.93, 129.84, 129.19, 127.17, 125.78, 125.70, 116.49, 115.57, 115.36, 70.25. 19F NMR (376 MHz, DMSO‐d6) δ ‐113.72. ESI‐MS: m/z [M‐H]‐ calcd for ‐ C17H11FN2O4S2: 389.0; found: 389.0. HRMS: m/z [M‐H] calcd for C17H11FN2O4S2: 389.0071; found: 389.0063.76

105 Materials and methods

(Z)‐4‐((4‐Oxo‐2‐thioxothiazolidin‐5‐ylidene)methyl)phenyl‐2‐chlorobenzoat SM21

Reaction Batch: 4‐Formylphenyl 2‐chlorobenzoate (0.261 g, 1 mmol); rhodanine (0.133 g, 1 mmol). Yellow solid in 84% (0.315 g, 0.84 mmol) yield. M.p. 246°C. Rf 0.26(PE–EtOAc, 5:2). 1H NMR (400 MHz, DMSO‐d6) δ 13.89 (bs, 1H), 8.41‐7.97 (m, 1H), 7.73 (d, J 8.7 Hz, 2H), 7.69 (s, 1H), 7.70‐7.66 (m, 2H), 7.61‐7.53 (m, 1H), 7.51 (d, J 8.7 Hz, 2H). 13C NMR (101 MHz, DMSO‐d6) δ 195.76, 169.69, 163.12, 151.66, 134.23, 132.70, 131.99, 131.96, 131.15, 130.48, ‐ 128.47, 127.61, 126.00, 123.01. ESI‐MS: m/z [M‐H] calcd for C17H10ClNO3S2: 374.0; found: ‐ 76 374.0. HRMS: m/z [M‐H] calcd for C17H10ClNO3S2: 373.9718; found: 373.9702.

(Z)‐5‐(3‐Nitro‐4‐(p‐tolyloxy)benzylidene)‐2‐thioxothiazolidin‐4‐one SM23

Reaction Batch: 3‐Nitro‐4‐(p‐tolyloxy)benzaldehyde (0.257 g, 1 mmol); rhodanine (0.133 g, 1 mmol). Orange crystals in 60% (0.225 g, 0.60 mmol) yield. M.p. 246°C. Rf 0.21 (PE–EtOAc, 5:2). 1H NMR (400 MHz, DMSO‐d6) δ 13.92 (bs, 1H), 8.33 (d, J 2.3 Hz, 1H), 7.80 (dd, J 8.9, 2.3 Hz, 1H), 7.69 (s, 1H), 7.29 (d, J 8.2 Hz, 3H), 7.15‐7.05 (m, 3H), 2.33 (s, 3H). 13C NMR (101 MHz, DMSO‐d6) δ 195.26, 169.39, 152.11, 151.44, 140.55, 135.46, 134.92, 130.92, 128.75, 127.98, ‐ 127.94, 126.84, 119.92, 119.76, 20.37. ESI‐MS: m/z [M‐H] calcd for C17H12N2O4S2: 371.0; ‐ 76 found: 371.0. HRMS: m/z [M‐H] calcd for C17H12N2O4S2: 371.0166; found: 371.0148.

106 Materials and methods

(Z)‐3‐Allyl‐5‐(3‐nitro‐4‐(p‐tolylthio)benzylidene)‐2‐thioxothiazolidin‐4‐one SM27

Reaction Batch: 3‐Nitro‐4‐(p‐tolylthio)benzaldehyde SM59 (0.119 g, 0.5 mmol); N‐allyl rhodanine (0.087 g, 0.5 mmol). Yellow solid in 63% (0.103 g, 0.96 mmol) yield. M.p. 243°C. 1 Rf 0.65 (PE–EtOAc, 1:1). H NMR (400 MHz, DMSO‐d6) δ 8.58 (d, J 2.0 Hz, 1H), 7.90 (s, 1H), 7.77 (dd, J 8.7, 2.1 Hz, 1H), 7.58‐7.53 (m, 2H), 7.42 (m, 2H), 6.96 (d, J 8.6 Hz, 1H), 5.89‐5.78 (m, 1H), 5.21‐5.10 (m, 2H), 4.66‐4.62 (m, 2H), 2.41 (s, 3H). 13C NMR (101 MHz, DMSO‐d6) δ 192.54, 166.51, 144.58, 141.30, 140.87, 135.71, 134.31, 131.32, 130.18, 130.13, 128.72, 128.10, 125.67, 124.16, 120.81, 117.90, 46.18, 20.96. CHN analysis: calcd: C 56.05, H 3.76, N 6.54; found: C 56.25, H 3.87, N 6.57.76

(Z)‐5‐(3‐Nitro‐4‐(p‐tolylthio)benzylidene)‐3‐phenyl‐2‐thioxothiazolidin‐4‐one SM28

Reaction batch: 3‐Nitro‐4‐(p‐tolylthio)benzaldehyde SM59 (273 mg, 1.0 mmol), 3‐phenyl‐2‐ thioxothiazolidin‐4‐one (209 mg, 1.0 mmol). Yellow solid in 56% (262 mg, 0.6 mmol). 1 M.p. 251°C. Rf 0.30 (PE–EtOAc, 5:1). H NMR (400 MHz, DMSO‐d6) δ 8.60 (d, J 2.0 Hz, 1H), 7.90 (s, 1H), 7.82 (dd, J 8.7, 2.0 Hz, 1H), 7.61 – 7.51 (m, 5H), 7.46 – 7.39 (m, 4H), 6.99 (d, J 8.6 Hz, 1H), 2.42 (s, 3H). 13C NMR (101 MHz, DMSO‐d6) δ 193.32, 166.80, 144.57, 141.18, 140.87, 135.73, 135.64, 135.09, 134.26, 131.35, 131.31, 130.39, 129.55, 129.36, 128.72, 127.97, 125.63, 125.12, 20.95. No response in ESI and HRMS. CHN analysis: calcd: C 59.46, H 3.47, N 6.03; found: C 59.41, H 3.58, N 6.20.76

107 Materials and methods

(Z)‐5‐(3‐Nitro‐4‐(p‐tolylthio)benzylidene)thiazolidine‐2,4‐dione SM29

Reaction batch: 3‐Nitro‐4‐(p‐tolylthio)benzaldehyde SM59 (273 mg, 1.0 mmol), thioazolidine‐2,4‐dione (117 mg, 1.0 mmol). Orange solid in 51% (189 mg, 0.5 mmol) yield. 1 M.p. 291°C (decomposition). Rf 0.24 (PE–EtOAc, 1:2). H NMR (400 MHz, DMSO‐d6) δ 8.42 (d, J 1.6 Hz, 1H), 7.70 (dd, J 8.6, 1.7 Hz, 1H), 7.55 – 7.51 (m, 2H), 7.54 (s, 1H), 7.52 (d, J 8.8 Hz, 2H), 7.39 (d, J 8.1 Hz, 2H), 6.89 (d, J 8.6 Hz, 1H), 2.40 (s, 3H). 13C NMR (101 MHz, DMSO‐d6) δ 209.67, 174.69, 170.87, 144.58, 140.62, 138.82, 135.62, 134.16, 132.15, 131.21, 128.36, ‐ 126.09, 126.03, 123.94, 20.95. ESI‐MS: m/z [M‐H] calcd for C17H12N2O4S2: 371.0; found: ‐ 370.8. HRMS: m/z [M‐H] calcd for C17H12N2O4S2: 371.0155; found: 371.0149.

(Z)‐2‐(5‐(3‐Nitro‐4‐(p‐tolylthio)benzylidene)‐4‐oxo‐2‐thioxothiazolidin‐3‐yl)acetic acid SM30

Reaction batch: 3‐Nitro‐4‐(p‐tolylthio)benzaldehyde SM59 (273 mg, 1.0 mmol), rhodanine‐3‐ acetic acid (191 mg, 1.0 mmol). Yellow solid in 83% (371 mg, 0.8 mmol) yield. M.p. 284°C. 1 Rf 0.45 (CH2Cl2–MeOH, 1:0.1). H NMR (400 MHz, DMSO‐d6) δ 13.46 (bs, 1H), 8.59 (d, J 1.9 Hz, 1H), 7.95 (s, 1H), 7.78 (dd, J 8.7, 1.9 Hz, 1H), 7.55 (d, J 8.0 Hz, 2H), 7.42 (d, J 8.0 Hz, 2H), 6.97 (d, J 8.6 Hz, 1H), 4.74 (s, 2H), 2.41 (s, 3H). 13C NMR (101 MHz, DMSO‐d6) δ 192.60, 167.23, 166.22, 144.53, 141.56, 140.87, 135.70, 134.26, 131.30, 131.02, 130.10, 128.69, + 128.33, 125.62, 123.51, 45.08, 20.95. ESI‐MS: m/z [M+Na] calcd for C19H14N2O5S3: 469.0; ‐ found: 469.4. HRMS: m/z [M‐H] calcd for C19H14N2O5S3: 444.9981; found: 444.9982.

108 Materials and methods

(Z)‐5‐(3‐Nitro‐4‐(p‐tolylthio)benzylidene)‐2‐thioxoimidazolidin‐4‐one SM31

Reaction batch: 3‐Nitro‐4‐(p‐tolylthio)benzaldehyde SM59 (273 mg, 1.0 mmol), thiohydantoine (116 mg, 1.0 mmol). Orange solid in 20% (65 mg, 0.2 mmol) yield. 1 M.p. 253°C. Rf 0.64 (PE–EtOAc, 2:3). H NMR (400 MHz, DMSO‐d6) δ 12.45 (bs, 1H), 12.24 (bs, 1H), 8.46 (d, J 1.8 Hz, 1H), 7.81 (dd, J 8.6, 1.9 Hz, 1H), 7.59 – 7.47 (m, 2H), 7.39 (d, J 8.0 Hz, 2H), 6.77 (d, J 8.6 Hz, 1H), 6.50 (s, 1H), 2.40 (s, 3H). 13C NMR (101 MHz, DMSO‐d6) δ 179.53, 165.46, 144.96, 140.57, 138.16, 135.56, 134.64, 131.12, 130.14, 129.32, 127.90, ‐ 126.45, 126.16, 108.05, 20.88. ESI‐MS: m/z [M‐H] calcd for C17H13N3O3S2: 370.0; found: ‐ 369.8. HRMS: m/z [M‐H] calcd for C17H13N3O3S2: 370.0315; found: 370.0322.

(Z)‐2‐Imino‐5‐(3‐nitro‐4‐(p‐tolylthio)benzylidene)thiazolidin‐4‐one SM32

Reaction batch: 3‐Nitro‐4‐(p‐tolylthio)benzaldehyde SM59 (273 mg, 1.0 mmol), pseudothiohydantoin (116 mg, 1.0 mmol). Yellow solid in 89% (331 mg, 0.9 mmol). 1 M.p. 296°C. Rf 0.33 (PE–EtOAc, 1:2). H NMR (400 MHz, DMSO‐d6) δ 9.55 (bs, 1H), 9.27 (bs, 1H), 8.41 (d, J 2.0 Hz, 1H), 7.74 (dd, J 8.7, 2.0 Hz, 1H), 7.59 (s, 1H), 7.56 – 7.50 (m, 2H), 7.40 (d, J 7.9 Hz, 2H), 6.91 (d, J 8.6 Hz, 1H), 2.40 (s, 3H). 13C NMR (101 MHz, DMSO‐d6) δ 180.04, 175.06, 144.60, 140.70, 139.49, 135.61, 134.76, 131.69, 131.40, 131.25, 128.50, 126.04, ‐ 126.02, 125.42, 20.95. ESI‐MS: m/z [M‐H] calcd for C17H13N3O3S2: 370.0; found: 370.1. ‐ HRMS: m/z [M‐H] calcd for C17H13N3O3S2: 370.0315; found: 370.0323.

109 Materials and methods

(Z)‐5‐(3‐Nitro‐4‐tosylbenzylidene)‐2‐thioxothiazolidin‐4‐one SM35

Reaction batch: 3‐Nitro‐4‐tosylbenzaldehyde SM84 (305 mg, 1.0 mmol), rhodanine (133 mg,

1.0 mmol). Yellow solid in 69% (290 mg, 0.7 mmol) yield. M.p. 270°C. Rf= 0.30 (PE–EtOAc, 1:6). 1H NMR (400 MHz, DMSO‐d6) δ 13.89 (bs, 1H), 8.43 (d, J 8.3 Hz, 1H), 8.24 (d, J 1.7 Hz, 1H), 8.01 (dd, J 8.5, 1.7 Hz, 1H), 7.89 – 7.83 (m, 2H), 7.68 (s, 1H), 7.50 (d, J 8.0 Hz, 2H), 2.41 (s, 3H). 13C NMR (101 MHz, DMSO‐d6) δ 195.11, 169.43, 148.00, 145.52, 139.98, 136.66, 132.87, 132.66, 132.30, 132.04, 130.21, 127.98, 126.49, 126.28, 21.17. ESI‐MS: m/z [M+Na]+ calcd ‐ for C17H12N2O5S3: 443.0; found: 442.5. HRMS: m/z [M‐H] calcd for C17H12N2O5S3: 418.9825; found: 418.9815.

(Z)‐5‐Benzylidene‐2‐thioxothiazolidin‐4‐on SM36

Reaction batch: Benzaldehyde (101 μL, 1 mmol), rhodanine (133 mg, 1 mmol). Orange solid 1 in 73% (161 mg, 0.73 mmol) yield. M.p. 203°C. Rf 0.64 (PE–EtOAc, 1:1). H NMR (400 MHz, DMSO‐d6) δ 13.84 (bs, 1H), 7.65 (s, 1H), 7.63 – 7.48 (m, 5H). 13C NMR (101 MHz, DMSO‐d6) δ 195.76, 169.45, 132.97, 131.58, 131.25, 130.72, 130.45, 129.44, 128.21, 125.58. No response ‐ in ESI‐MS. HRMS: m/z [M‐H] calcd for C18H14N2O3S3: 219.9885; found: 219.9895.

(Z)‐2‐Thioxo‐5‐(4‐(p‐tolylthio)benzylidene)thiazolidin‐4‐one SM37 O

NH S S S

Reaction batch: 4‐(p‐Tolylthio)benzaldehyde SM69 (300 mg, 1.3 mmol), rhodanine (175 mg,

1.3 mmol). Yellow solid 66% (295 mg, 0.9 mmol) yield. M.p. 197°C. Rf 0.90 (PE–EtOAc, 1:1).

110 Materials and methods

1H NMR (400 MHz, DMSO‐d6) δ 13.83 (bs, 1H), 7.55 (s, 1H), 7.52 – 7.48 (m, 2H), 7.44 – 7.40 (m, 2H), 7.32 – 7.29 (m, 2H), 7.24 – 7.19 (m, 2H), 2.35 (s, 3H). 13C NMR (101 MHz, DMSO‐d6) δ 195.79, 170.15, 141.65, 139.16, 134.02, 131.24, 130.75, 130.57, 130.45, 127.60, 127.42, ‐ 125.30, 20.81. ESI‐MS: m/z [M‐H] calcd for C17H13NOS3: 342.0; found: 342.0. HRMS: m/z [M‐ ‐ H] calcd for C17H13NOS3: 342.0076; found: 342.0070.

(Z)‐5‐(3‐Fluoro‐4‐(p‐tolylthio)benzylidene)‐2‐thioxothiazolidin‐4‐one SM38

Reaction batch: 3‐Fluoro‐4‐(p‐tolylthio)benzaldehyde SM63 (246 mg, 1.0 mmol); rhodanine

(133 mg, 1.0 mmol). Yellow solid in 77% (280 mg; 0.8 mmol) yield. M.p. 182°C. Rf 0.31 (PE– EtOAc, 3:1). 1H NMR (400 MHz, DMSO‐d6) δ 13.86 (bs, 1H), 7.58 (s, 1H), 7.53 (dd, J 11.0, 1.5 Hz, 1H), 7.44 – 7.40 (m, 2H), 7.34 – 7.29 (m, 3H), 7.03 (t, J 8.1 Hz, 1H), 2.35 (s, 3H). 13C NMR (101 MHz, DMSO‐d6) δ 195.37, 169.54, 158.50 (d, J 244.1 Hz), 139.28, 133.70, 132.90 (d, J 7.9 Hz), 130.80, 129.96 (d, J 2.2 Hz), 129.46, 128.00 (d, J 17.4 Hz), 126.74, 126.50 (d, J 2.9 Hz), 125.99 (d, J 1.1 Hz), 117.34 (d, J 22.8 Hz), 20.75. 19F NMR (376 MHz, DMSO‐d6) δ ‐110.97. ‐ ‐ ESI‐MS: m/z [M‐H] calcd for C17H12FNOS3: 360.0; found: 359.9. HRMS: m/z [M‐H] calcd for

C17H12FNOS3: 359.9981; found: 360.0021.

(Z)‐5‐(3‐Chloro‐4‐(p‐tolylthio)benzylidene)‐2‐thioxothiazolidin‐4‐one SM39

Reaction batch: 3‐Chloro‐4‐(p‐tolylthio)benzaldehyde SM70 (262 mg, 1.0 mmol); rhodanine

(133 mg, 1.0 mmol). Yellow solid in 81% (311 mg, 0.8 mmol) yield. M.p. 197°C. Rf 0.37 (PE–EtOAc, 1:1). 1H NMR (400 MHz, DMSO‐d6) δ 13.86 (bs, 1H), 7.77 – 7.75 (m, 1H), 7.57 (s, 1H), 7.51 – 7.47 (m, 2H), 7.42 – 7.35 (m, 3H), 6.80 (d, J 8.4 Hz, 1H), 2.40 (s, 3H). 13C NMR (101 MHz, DMSO‐d6) δ 195.24, 169.36, 141.00, 140.12, 135.07, 131.49, 131.08, 130.17, 129.46,

111 Materials and methods

‐ 128.62, 127.45, 126.20, 126.15, 125.38, 20.84. ESI‐MS: m/z [M‐H] calcd for C17H12ClNOS3: ‐ 376.0; found: 375.8. HRMS: m/z [M‐H] calcd for C17H12ClNOS3: 375.9686; found: 375.9688.

(Z)‐5‐(3‐Bromo‐4‐(p‐tolylthio)benzylidene)‐2‐thioxothiazolidin‐4‐one SM40

Reaction batch: 3‐Bromo‐4‐(p‐tolylthio)benzaldehyde SM68 (305 mg, 1.0 mmol), rhodanine (133 mg, 1.0 mmol). Yellow solid in 71% (295 mg, 0.7 mmol) yield. Visual nature. M.p. 213°C. 1 Rf 0.88 (PE–EtOAc, 1:1). H NMR (400 MHz, DMSO‐d6) δ 13.86 (bs, 1H), 7.88 (d, J 1.8 Hz, 1H), 7.53 (s, 1H), 7.50 – 7.46 (m, 2H), 7.40 (dd, J 8.6, 1.9 Hz, 1H), 7.37 (d, J 8.0 Hz, 2H), 6.72 (d, J 8.4 Hz, 1H), 2.38 (s, 3H). 13C NMR (101 MHz, DMSO‐d6) δ 195.59, 170.05, 143.00, 140.21, 135.17, 134.72, 134.71, 131.68, 131.14, 129.07, 127.22, 126.54, 125.91, 120.15, 20.90. ESI‐ ‐ 79 81 79 MS: m/z [M‐H] calcd for C17H12BrNOS3: 419.9 ( Br), 421.9 ( Br); found: 419.9 ( Br), 421.9 81 ‐ 79 81 ( Br). HRMS: m/z [M‐H] calcd for C17H12BrNOS3: 419.9181 ( Br), 421.9160 ( Br); found: 419.9171 (79Br), 421.9153 (81Br).

(Z)‐2‐Thioxo‐5‐(4‐(p‐tolylthio)‐3‐(trifluoromethyl)benzylidene)thiazolidin‐4‐one SM41

Reaction batch: 4‐(p‐Tolylthio)‐3‐(trifluoromethyl)benzaldehyde SM71 (297 mg, 1.0 mmol); rhodanine (133 mg, 1.0 mmol). Yellow solid in 61% (253 mg, 0.6 mmol) yield. M.p. 235°C. 1 Rf 0.29 (PE–EtOAc, 3:1). H NMR (400 MHz, DMSO‐d6) δ 13.86 (bs, 1H), 8.02 (d, J 1.6 Hz, 1H), 7.68 (s, 1H), 7.65 (dd, J 8.5, 1.5 Hz, 1H), 7.50 – 7.44 (m, 2H), 7.37 – 7.33 (m, 2H), 7.08 (d, J 8.5 Hz, 1H), 2.38 (s, 3H). 13C NMR (101 MHz, DMSO‐d6) δ 195.14, 169.32, 140.63, 140.11, 134.90, 133.09, 131.12, 131.04, 130.59, 129.84, 129.44, 129.36, 126.75, 126.53, 126.22, 125.92, 124.79, 122.06, 20.85. 19F NMR (376 MHz, DMSO‐d6) δ ‐60.58. ESI‐MS: m/z [M‐H]‐ calcd for

112 Materials and methods

‐ C18H12F3NOS3: 410.0; found: 409.8. HRMS: m/z [M‐H] calcd for C18H12F3NOS3: 409.9949; found: 409.9982.

(Z)‐5‐((4‐Oxo‐2‐thioxothiazolidin‐5‐ylidene)methyl)‐2‐(p‐tolylthio)benzonitrile SM42

Reaction batch: 5‐Formyl‐2‐(p‐tolylthio)benzonitrile SM64 (253 mg, 1.0 mmol); rhodanine

(133 mg, 1.0 mmol). Yellow solid in 83% (322 mg, 0.8 mmol) yield. M.p. 247°C. Rf 0.24 (PE–EtOAc, 3:1). 1H NMR (400 MHz, DMSO‐d6) δ 13.88 (bs, 1H), 8.11 (d, J 2.0 Hz, 1H), 7.68 (dd, J 8.8, 2.1 Hz, 1H), 7.58 (s, 1H), 7.53 – 7.47 (m, 2H), 7.40 – 7.38 (m, 2H), 7.03 (d, J 8.6 Hz, 1H), 2.38 (s, 3H). 13C NMR (101 MHz, DMSO‐d6) δ 195.27, 169.54, 145.20, 140.26, 136.15, 134.64, 134.43, 133.74, 131.10, 131.02, 128.42, 128.05, 125.07, 115.97, 110.39, 20.82. ESI‐ ‐ ‐ MS: m/z [M‐H] calcd for C18H12N2OS3: 367.0; found: 366.9. HRMS: m/z [M‐H] calcd for

C18H12N2OS3: 367.0028; found: 367.0027.

(Z)‐5‐(4‐((4‐Ethylphenyl)thio)‐3‐nitrobenzylidene)‐2‐thioxothiazolidin‐4‐one SM43

Reaction batch: 4‐((4‐Ethylphenyl)thio)‐3‐nitrobenzaldehyde SM79 (287 mg, 1.0 mmol), rhodanine (133 mg, 1.0 mmol). Yellow solid in 80% (322 mg, 0.8 mmol) yield. M.p. 254°C. 1 Rf 0.83 (PE–EtOAc, 1:1). H NMR (400 MHz, DMSO‐d6) δ 13.90 (bs, 1H), 8.48 (s, 1H), 7.76 – 7.69 (m, 1H), 7.68 (s, 1H), 7.56 (d, J 7.9 Hz, 2H), 7.44 (d, J 8.0 Hz, 2H), 6.95 (dd, J 8.6, 0.9 Hz, 1H), 2.71 (q, J 7.6 Hz, 2H), 1.24 (t, J 7.6 Hz, 3H). 13C NMR (101 MHz, DMSO‐d6) δ 195.10, 169.41, 146.83, 144.54, 140.88, 135.71, 134.28, 130.38, 130.09, 128.64, 128.51, 127.71, ‐ 127.43, 125.97, 27.96, 15.20. ESI‐MS: m/z [M‐H] calcd for C18H14N2O3S3: 401.0; found: 401.0. ‐ HRMS: m/z [M‐H] calcd for C18H14N2O3S3: 401.0083; found: 401.0071.

113 Materials and methods

(Z)‐5‐(3‐Nitro‐4‐((4‐(trifluoromethyl)phenyl)thio)benzylidene)‐2‐thioxothiazolidin‐4‐one SM44

Reaction batch: 3‐Nitro‐4‐((4‐(trifluoromethyl)phenyl)thio)benzaldehyde SM80 (300 mg, 0.9 mmol), rhodanine (122 mg, 0.9 mmol). Yellow solid in 80% (339 mg, 0.7 mmol) yield. 1 M.p. 268°C. Rf 0.73 (PE–EtOAc, 1:1): H NMR (400 MHz, DMSO‐d6) δ 13.93 (bs, 1H), 8.46 (d, J 1.8 Hz, 1H), 7.91 (d, J 8.4 Hz, 2H), 7.86 (d, J 8.3 Hz, 2H), 7.73 (dd, J 8.6, 2.0 Hz, 1H), 7.56 (s, 1H), 7.08 (d, J 8.6 Hz, 1H). 13C NMR (101 MHz, DMSO‐d6) δ 196.88, 145.60, 137.18, 135.76, 135.57, 134.39, 132.00, 130.91 – 130.57 (m), 130.41, 130.09, 129.83, 127.23, 127.13 (q), 126.48 – 125.77 (m), 125.25, 122.55. 19F NMR (376 MHz, DMSO‐d6) δ ‐61.36. ESI‐MS: m/z ‐ ‐ [M‐H] calcd for C17H9F3N2O3S3: 441.0; found: 441.0. HRMS: m/z [M‐H] calcd for

C17H9F3N2O3S3: 440.9644; found: 440.9669.

(Z)‐5‐(3‐Nitro‐4‐((4‐(trifluoromethoxy)phenyl)thio)benzylidene)‐2‐thioxothiazolidin‐4‐one SM45

Reaction batch: 3‐Nitro‐4‐((4‐(trifluoromethoxy)phenyl)thio)benzaldehyde SM82 (275 mg, 0.8 mmol), rhodanine (107 mg, 0.8 mmol). Yellow solid in 65% (236 mg, 0.5 mmol) yield. 1 M.p. 281°C. Rf 0.32 (PE–EtOAc, 1:4). H NMR (400 MHz, DMSO‐d6) δ 13.91 (bs, 1H), 8.49 (d, J 1.9 Hz, 1H), 7.84 – 7.78 (m, 2H), 7.73 (dd, J 8.6, 1.9 Hz, 1H), 7.66 (s, 1H), 7.57 (d, J 8.1 Hz, 2H), 7.00 (d, J 8.6 Hz, 1H). 13C NMR (101 MHz, DMSO‐d6) δ 195.28, 169.93, 149.82, 144.86, 139.23, 137.67, 134.35, 130.90, 129.01, 128.80, 128.12, 127.94, 127.58, 122.70, 121.22, 121.49 – 118.27 (m). 19F NMR (376 MHz, DMSO) δ ‐56.67. ESI‐MS: m/z [M‐H]‐ calcd for ‐ C17H9F3N2O4S3: 357.0; found: 356.8. HRMS: m/z [M‐H] calcd for C17H9F3N2O4S3: 456.9593; found: 456.9619.

114 Materials and methods

(Z)‐5‐(4‐((3‐Fluoro‐4‐methylphenyl)thio)‐3‐nitrobenzylidene)‐2‐thioxothiazolidin‐4‐one SM46

Reaction batch: 4‐((3‐Fluoro‐4‐methylphenyl)thio)‐3‐nitrobenzaldehyde SM65 (291 mg, 1.0 mmol), rhodanine (133 mg, 1.0 mmol). Yellow solid in 77% (312 mg, 0.8 mmol) yield. 1 M.p. 262°C. Rf 0.61 (PE–EtOAc, 1:4). H NMR (400 MHz, DMSO‐d6) δ 13.92 (bs, 1H), 8.50 (d, J 1.9 Hz, 1H), 7.73 (dd, J 8.7, 2.0 Hz, 1H), 7.70 (s, 1H), 7.64 (dd, J 7.3, 1.8 Hz, 1H), 7.54 (ddd, J 7.4, 4.9, 2.3 Hz, 1H), 7.42 – 7.34 (m, 1H), 6.98 (d, J 8.6 Hz, 1H), 2.29 (d, J 0.8 Hz, 3H). 13C NMR (101 MHz, DMSO‐d6) δ 195.15, 169.62, 162.11 (d, J 248.3 Hz), 144.44, 140.49, 139.09 (d, J 5.9 Hz), 135.53 (d, J 8.8 Hz), 134.30, 130.49, 128.72, 128.25, 127.62, 127.16 (d, J 18.2 Hz), 124.48 (d, J 3.3 Hz), 117.27 (d, J 23.1 Hz), 14.03 (d, J 3.1 Hz). 19F NMR (376 MHz, DMSO‐d6) δ ‐ ‐ ‐114.06. ESI‐MS: m/z [M‐H] calcd for C17H11FN2O3S3: 405.0; found: 404.8. HRMS: m/z [M‐H] calcd for C17H11FN2O3S3: 404.9832; found: 404.9853.

(Z)‐5‐(3‐Nitro‐4‐((2,3,5,6‐tetrafluoro‐4‐(trifluoromethyl)phenyl)thio) benzylidene)‐2‐thioxo‐ thiazolidin‐4‐one SM47

Reaction batch: 3‐Nitro‐4‐((2,3,5,6‐tetrafluoro‐4‐(trifluoromethyl)phenyl)thio)benzaldehyde SM67 (399 mg, 1.0 mmol), rhodanine (133 mg, 1.0 mmol). Yellow solid in 80% (401 mg, 0.8 1 mmol) yield. M.p. 296°C (decomposition). Rf 0.29 (PE–EtOAc, 1:4). H NMR (400 MHz, DMSO‐ d6) δ 13.99 (bs, 1H), 8.58 (d, J 1.9 Hz, 1H), 7.79 (s, 1H), 7.76 (dd, J 8.6, 2.0 Hz, 1H), 7.42 (d, J 8.5 Hz, 1H). 13C NMR (101 MHz, DMSO‐d6) δ 194.81, 169.26, 149.04 – 145.91 (m), 145.85 – 145.56 (m),145.05, 135.24, 133.98, 132.20, 129.07, 128.38, 128.12, 127.77, 122.40 – 121.23 (m), 119.34 – 119.19 (m), 114.93 (t, J 21.2 Hz). 19F NMR (376 MHz, DMSO‐d6) δ ‐55.78 (t, J 21.5 Hz, 3F), ‐129.95 – ‐130.48 (m, 2F), ‐139.46 – ‐139.89 (m, 2F). ESI‐MS: m/z [M‐H]‐ calcd

115 Materials and methods

‐ for C17H5F7N2O3S3: 512.9; found: 512.7. HRMS: m/z [M‐H] calcd for C17H5F7N2O3S3: 512.9267; found: 512.9271.

(Z)‐5‐(3‐Nitro‐4‐(m‐tolylthio)benzylidene)‐2‐thioxothiazolidin‐4‐one SM48

Reaction batch: 3‐Nitro‐4‐(m‐tolylthio)benzaldehyde SM61 (273 mg, 1.0 mmol); rhodanine

(133 mg, 1.0 mmol). Yellow solid in 81% (301 mg, 0.8 mmol) yield. M.p. 247°C. Rf 0.30 (PE–EtOAc, 3:1). 1H NMR (400 MHz, DMSO‐d6) δ 13.89 (bs, 1H), 8.48 (s, 1H), 7.75 – 7.69 (m, 1H), 7.67 (s, 1H), 7.53 – 7.40 (m, 4H), 6.98 (d, J 8.5 Hz, 1H), 2.37 (s, 3H).13C NMR (101 MHz, DMSO‐d6) 195.02, 169.30, 144.58, 140.56, 140.28, 135.93, 134.28, 132.65, 131.45, 130.43, ‐ 129.01, 128.81, 128.52, 127.67, 127.37, 20.80. ESI‐MS: m/z [M‐H] calcd for C17H12N2O3S3: ‐ 387.0; found: 386.8. HRMS: m/z [M‐H] calcd for C17H12N2O3S3: 386.9926; found: 386.9926.

(Z)‐5‐(3‐Nitro‐4‐(o‐tolylthio)benzylidene)‐2‐thioxothiazolidin‐4‐one SM49

Reaction batch: 3‐Nitro‐4‐(o‐tolylthio)benzaldehyde SM60 (273 mg, 1.0 mmol), rhodanine

(133 mg, 1.0 mmol). Golden solid in 73% (284 mg, 0.7 mmol) yield. M.p. 245°C. Rf 0.30 (PE–EtOAc, 1:4). 1H NMR (400 MHz, DMSO‐d6) δ 13.90 (bs, 1H), 8.53 – 8.59 (m, 1H), 7.74 – 7.69 (m, 1H), 7.68 (s, 1H), 7.63 (d, J 7.5 Hz, 1H), 7.58 – 7.50 (m, 2H), 7.40 (td, J 7.4, 2.1 Hz, 1H), 6.79 (d, J 8.5 Hz, 1H), 2.30 (s, 3H). 13C NMR (101 MHz, DMSO‐d6) δ 194.97, 169.25, 144.70, 142.74, 139.53, 136.79, 134.42, 131.62, 131.34, 130.36, 128.50, 128.26, 128.02, ‐ 127.93, 127.89, 127.33, 20.00. ESI‐MS: m/z [M‐H] calcd for C17H12N2O3S3: 387.0; found: ‐ 386.8. HRMS: m/z [M‐H] calcd for C17H12N2O3S3: 386.9926; found: 386.9929.

116 Materials and methods

(Z)‐5‐(4‐((3,5‐Dimethylphenyl)thio)‐3‐nitrobenzylidene)‐2‐thioxothiazolidin‐4‐one SM50

Reaction batch: 4‐((3,5‐Dimethylphenyl)thio)‐3‐nitrobenzaldehyde SM78 (300 mg, 1.0 mmol), rhodanine (133 mg, 1.0 mmol). Yellow solid in 55% (230 mg, 0.6 mmol) yield. 1 M.p. 266°C. Rf 0.39 (PE–EtOAc, 1:1). H NMR (400 MHz, DMSO‐d6) δ 13.94 (bs, 1H), 8.49 (d, J 2.0 Hz, 1H), 7.72 (dd, J 8.7, 2.0 Hz, 1H), 7.68 (s, 1H), 7.29 (s, 2H), 7.25 (s, 1H), 7.00 (d, J 8.6 Hz, 1H), 2.34 (s, 6H). 13C NMR (101 MHz, DMSO‐d6) δ 195.15, 169.50, 144.60, 140.63, 140.03, 134.28, 133.01, 132.26, 130.42, 128.88, 128.72, 128.46, 127.65, 127.49, 20.70. ESI‐MS: m/z ‐ ‐ [M‐H] calcd for C18H14N2O3S3: 401.0; found: 401.0. HRMS: m/z [M‐H] calcd for C18H14N2O3S3: 401.0083; found: 401.0111.

(Z)‐5‐(4‐(Naphthalen‐2‐ylthio)‐3‐nitrobenzylidene)‐2‐thioxothiazolidin‐4‐one SM51

Reaction batch: 4‐(Naphthalen‐2‐ylthio)‐3‐nitrobenzaldehyde SM77 (240 mg, 0.8 mmol), rhodanine (103 mg, 0.8 mmol). Yellow solid in 74% (245 mg, 0.6 mmol) yield. M.p. 251°C. 1 Rf 0.70 (PE–EtOAc, 1:1). H NMR (400 MHz, DMSO‐d6) δ 13.88 (bs, 1H), 8.46 (d, J 1.9 Hz, 1H), 8.37 (d, J 1.4 Hz, 1H), 8.10 (d, J 8.6 Hz, 1H), 8.07 – 8.01 (m, 2H), 7.72 – 7.58 (m, 4H), 7.50 (s, 1H), 7.00 (d, J 8.6 Hz, 1H). 13C NMR (101 MHz, DMSO‐d6) δ 199.21, 174.16, 144.92, 135.62, 134.30, 133.59, 133.23, 131.12, 130.22, 129.17, 128.07, 127.89, 127.84, 127.16, 127.00, ‐ ‐ 126.94. ESI‐MS: m/z [M‐H] calcd for C20H12N2O3S3: 423.0; found: 423.0. HRMS: m/z [M‐H] calcd for C20H12N2O3S3: 422.9926; found: 422.9919.

117 Materials and methods

(Z)‐5‐(4‐(Cyclopentylthio)‐3‐nitrobenzylidene)‐2‐thioxothiazolidin‐4‐one SM52

Reaction batch: 4‐(Cyclopentylthio)‐3‐nitrobenzaldehyde SM81 (200 mg, 0.8 mmol), rhodanine (106 mg, 0.8 mmol). Yellow solid in 74% (216 mg, 0.6 mmol) yield. M.p. 257°C. 1 Rf 0.65 (PE–EtOAc, 1:1). H NMR (400 MHz, DMSO‐d6) δ 13.93 (bs, 1H), 8.40 (s, 1H), 7.83 (s, 2H), 7.72 (s, 1H), 4.06 – 3.70 (m, 1H), 2.38 – 2.13 (m, 2H), 1.79 – 1.34 (m, 6H). 13C NMR (101 MHz, DMSO‐d6) δ 194.91, 169.29, 145.52, 140.18, 134.10, 129.53, 128.97, 128.78, 127.47, ‐ 126.95, 43.04, 32.64, 24.79. ESI‐MS: m/z [M‐H] calcd for C15H14N2O3S3: 365.0; found: 365.0. ‐ HRMS: m/z [M‐H] calcd for C15H14N2O3S3: 365.0083; found: 365.0115.

(Z)‐5‐(3‐Bromo‐4‐((3‐fluoro‐4‐methylphenyl)thio)benzylidene)‐2‐thioxothiazolidin‐4‐one SM53

Reaction batch: 3‐Bromo‐4‐((3‐fluoro‐4‐methylphenyl)thio)benzaldehyde SM66 (241 mg, 0.7 mmol); rhodanine (98 mg, 0.7 mmol). Yellow solid in 98% (334 mg, 0.7 mmol) yield. 1 M.p. 257°C. Rf 0.37 (PE–EtOAc, 3:1). H NMR (400 MHz, DMSO‐d6) δ 13.85 (bs, 1H), 7.90 (d, J 1.9 Hz, 1H), 7.58 (dd, J 7.5, 2.0 Hz, 1H), 7.56 (s, 1H), 7.47 (ddd, J 7.6, 4.9, 2.4 Hz, 1H), 7.43 (dd, J 8.6, 1.9 Hz, 1H), 7.38 – 7.30 (m, 1H), 6.77 (d, J 8.4 Hz, 1H), 2.28 (d, J 1.5 Hz, 3H). 13C NMR (101 MHz, DMSO‐d6) δ 195.15, 169.26, 161.85 (d, J 247.7 Hz), 142.89, 138.65 (d, J 5.9 Hz), 135.05 (d, J 8.7 Hz), 134.78, 131.59, 129.44, 129.13, 127.25, 127.05 (d, J 18.1 Hz), 125.99, 124.55 (d, J 3.6 Hz), 120.04, 117.16 (d, J 23.0 Hz), 14.06 (d, J 3.1 Hz). 19F NMR (376 MHz, ‐ 79 81 DMSO‐d6) δ ‐114.73. ESI‐MS: m/z [M‐H] calcd for C17H11BrFNOS3: 437.9 ( Br), 439.9 ( Br); 79 81 ‐ 79 found: 437.7 ( Br), 439.7 ( Br). HRMS: m/z [M‐H] calcd for C17H11BrFNOS3: 437.9086 ( Br), 439.9066 (81Br); found: 437.9081 (79Br), 439.9065 (81Br).

118 Materials and methods

(Z)‐2‐Thioxo‐5‐(4‐(o‐tolylthio)benzylidene)thiazolidin‐4‐one SM54

Reaction batch: 4‐(o‐Tolylthio)benzaldehyde SM83 (228 mg, 1.0 mmol), rhodanine (133 mg,

1.0 mmol). Yellow solid in 90% (309 mg, 0.9 mmol) yield. M.p. 178°C. Rf 0.51 (PE–EtOAc, 4:1). 1H NMR (400 MHz, DMSO‐d6) δ 13.82 (bs, 1H), 7.57 (s, 1H), 7.54 – 7.46 (m, 3H), 7.45 – 7.39 (m, 2H), 7.34 – 7.27 (m, 1H), 7.18 – 7.13 (m, 2H), 2.31 (s, 3H). 13C NMR (101 MHz, DMSO‐d6) δ 195.47, 169.48, 141.29, 140.84, 135.22, 131.37, 131.24, 130.96, 130.30, 129.92, 129.88, ‐ 127.53, 127.37, 124.80, 20.22. ESI‐MS: m/z [M‐H] calcd for C17H13NOS3: 342.0; found: 342.0. ‐ HRMS: m/z [M‐H] calcd for C17H13NOS3: 342.0076; found: 342.0103.

(Z)‐5‐(3‐Fluoro‐4‐(o‐tolylthio)benzylidene)‐2‐thioxothiazolidin‐4‐one SM55

Reaction batch: 3‐fluoro‐4‐(o‐tolylthio)benzaldehyde SM72 (150 mg, 0.6 mmol); rhodanine

(79.1 mg, 0.6 mmol). Yellow solid in 88% (192 mg; 0.53 mmol) yield. M.p. 161°C. Rf 0.63 (PE– EtOAc, 2:1). 1H NMR (400 MHz, DMSO‐d6) δ 13.80 (bs, 1H), 7.51 (dd, J 11.1, 1.6 Hz, 1H), 7.46 (s, 1H), 7.45 – 7.36 (m, 3H), 7.33 – 7.26 (m, 2H), 6.91 (t, J 8.1 Hz, 1H), 2.33 (s, 3H). 13C NMR (101 MHz, DMSO‐d6) δ 197.06, 158.90 (d, J 244.1 Hz), 140.73, 134.25, 133.72 (d, J 7.6 Hz), 131.18, 129.92, 129.86 (d, J 2.0 Hz), 129.63, 129.37 (d, J 2.9 Hz), 128.99, 128.68 (d, J 4.9 Hz), 127.55, 127.38 (d, J 5.5 Hz), 126.47 (d, J 2.7 Hz), 117.11 (d, J 22.7 Hz), 20.02. 19F NMR (376 ‐ MHz, DMSO‐d6) δ ‐110.93. ESI‐MS: m/z [M‐H] calcd for C17H12FNOS3: 360.0; found: 359.7. ‐ HRMS: m/z [M‐H] calcd for C17H12FNOS3: 359.9981; found: 359.9990.

119 Materials and methods

(Z)‐2‐Thioxo‐5‐(4‐(o‐tolylthio)‐3‐(trifluoromethyl)benzylidene)thiazolidin‐4‐one 56

Reaction batch: 4‐(o‐Tolylthio)‐3‐(trifluoromethyl)benzaldehyde SM73 (107 mg, 0.4 mmol); rhodanine (48 mg, 0.4 mmol). Yellow solid in 91% (131 mg; 0.3 mmol) yield. M.p. 217°C. Rf 0.60 (PE–EtOAc, 2:1). 1H NMR (400 MHz, DMSO‐d6) δ 13.89 (bs, 1H), 8.02 (d, J 1.2 Hz, 1H), 7.63 (s, 1H), 7.66 – 7.59 (m, 1H), 7.54 (d, J 7.5 Hz, 1H), 7.49 – 7.44 (m, 2H), 7.40 – 7.31 (m, 1H), 6.90 (d, J 8.5 Hz, 1H), 2.29 (s, 3H). 13C NMR (101 MHz, DMSO‐d6) δ 196.18, 142.32, 139.76, 136.61, 133.64, 132.03, 131.28, 131.14, 130.03 – 129.64 (m), 129.59, 129.22 – 128.94 (m), 128.28, 128.21 – 128.09 (m), 127.08, 126.78, 125.32, 122.59, 20.52. 19F NMR ‐ (376 MHz, DMSO‐d6) δ ‐60.89. ESI‐MS: m/z [M‐H] calcd for C18H12F3NOS3: 410.0; found: ‐ 409.7. HRMS: m/z [M‐H] calcd for C18H12F3NOS3: 409.9949; found: 409.9956.

(Z)‐5‐(2,4‐Bis(p‐tolylthio)benzylidene)‐2‐thioxothiazolidin‐4‐one SM57

Reaction batch: 2,4‐Bis(p‐tolylthio)benzaldehyde SM74 (355 mg, 1.0 mmol); rhodanine

(133 mg, 1.0 mmol). Yellow solid in 45% (210 mg, 0.5 mmol) yield. M.p. 209°C. Rf 0.51 (PE–EtOAc, 3:1). 1H NMR (400 MHz, DMSO‐d6) δ 13.84 (bs, 1H), 7.80 (s, 1H), 7.37 (d, J 8.3 Hz, 1H), 7.30 – 7.25 (m, 2H), 7.22 – 7.11 (m, 7H), 6.62 (d, J 2.0 Hz, 1H), 2.35 (s, 3H), 2.33 (s, 3H). 13C NMR (101 MHz, DMSO‐d6) δ 195.38, 169.10, 142.80, 140.76, 139.29, 138.65, 134.18, 132.97, 130.59, 130.51, 128.87, 128.50, 127.65, 127.06, 126.66, 126.58, 126.06, 124.96, 20.82, 20.75. No response in ESI and HRMS. CHN analysis: calcd: C 61.90, H 4.11, N 3.01; found: C 61.66, H 4.11, N 3.17.

120 Materials and methods

(Z)‐5‐((4‐Bromo‐5‐(p‐tolylthio)thiophen‐2‐yl)methylene)‐2‐thioxothiazolidin‐4‐one SM58

Reaction batch: 4‐bromo‐5‐(p‐tolylthio)thiophene‐2‐carbaldehyde SM75 (150 mg, 0.48 mmol); rhodanine (64 mg, 0.48 mmol). Yellow solid 83% (171 mg; 0.4 mmol) yield. 1 M.p. 209°C. Rf 0.57 (PE–EtOAc, 2:1). H NMR (400 MHz, DMSO‐d6) δ 13.86 (bs, 1H), 7.76 (s, 1H), 7.76 (s, 1H), 7.37 – 7.32 (m, 2H), 7.26 (d, J 8.1 Hz, 2H), 2.32 (s, 3H). 13C NMR (101 MHz, DMSO‐d6) δ 193.95, 168.98, 139.33, 138.92, 138.73, 136.99, 130.84, 130.61, 129.37, 125.23, ‐ 79 81 122.37, 116.88, 20.69. ESI‐MS: m/z [M‐H] calcd for C15H10BrNOS4: 425.9 ( Br), 427.9 ( Br); 79 81 ‐ 79 found: 425.6 ( Br), 427.6 ( Br). HRMS: m/z [M‐H] calcd for C17H11BrFNOS3: 425.8745 ( Br), 427.8724 (81Br); found: 425.8752 (79Br), 427.8727 (81Br).

Synthesis of (Z)‐5‐(1‐(3‐Nitro‐4‐(p‐tolylthio)phenyl)ethylidene)‐2‐thioxothiazolidin‐4‐one SM33

A mixture of 1‐(3‐nitro‐4‐(p‐tolylthio)phenyl)ethanone SM62 (191 mg, 0.67 mmol), rhodanine (144 mg, 1.08 mmol and NH4OAc (81 mg, 1.03 mmol) in toluene (5 mL) was stirred at reflux until the reaction was completed. The reaction mixture was cooled to room temperature and H2O (6 mL) was added. The precipitate was washed thoroughly with toluene, H2O, ethanol and ether to get the pure ocher SM33 in 78% (210 mg, 0.52 mmol) 1 yield. M.p. 228°C. Rf= 0.70 (PE–EtOAc, 1:1). H NMR (400 MHz, DMSO‐d6) δ 13.42 (bs, 1H), 8.34 (d, J 1.9 Hz, 1H), 7.59 (dd, J 8.5, 2.0 Hz, 1H), 7.56 – 7.52 (m, 2H), 7.40 (d, J 7.9 Hz, 2H), 6.76 (d, J 8.5 Hz, 1H), 2.40 (s, 3H), 2.21 (s, 3H). 13C NMR (101 MHz, DMSO‐d6) δ 194.37, 165.77, 143.77, 143.31, 140.60, 138.57, 135.79, 135.70, 133.89, 131.17, 126.99, 126.04, ‐ 125.61, 99.51, 26.77, 20.89. ESI‐MS: m/z [M‐H] calcd for C18H14N2O3S3: 401.0; found: 400.8. ‐ HRMS: m/z [M‐H] calcd for C18H14N2O3S3: 401.0083; found: 401.0077.

121 Materials and methods

Synthesis of 5‐(3‐Nitro‐4‐(p‐tolylthio)benzyl)‐2‐thioxothiazolidin‐4‐one SM34

A stirred suspension of SM1 (0.504 g, 1.30 mmol) in toluene (50 mL) was treated with diethyl 2,6‐dimethyl‐1,4‐dihydro‐3,5‐pyridinedicarboxylate (0.427 g, 1.69 mmol) and silica gel 60 (1.3 g, 1 g mmol‐1), previously activated by heating at 120°C for 5 h. The mixture was heated to 100°C for 24 h under N2 atmosphere and the exclusion of light. The reaction mixture was cooled and filtered. The filter cake was rinsed with EtOAc. The filtrates were combined, rinsed and concentrated in vacuo. The residue was dissolved in EtOAc (30 mL) and washed with aq HCl (1 M, 3×30 mL) and brine (30 mL). The organic phase was dried over MgSO4, concentrated in vacuo and the crude product was purified by silica gel column chromatography using n‐hexane/EtOAc mixtures to give compound SM34 as a yellow solid in 1 82% (0.413 g, 1.06 mmol) yield. M.p. 80°C. Rf= 0.30 (PE–EtOAc, 3:1). H NMR (400 MHz, DMSO‐d6) δ 13.18 (bs, 1H), 8.15 (d, J 1.8 Hz, 1H), 7.52 – 7.48 (m, 2H), 7.45 (dd, J 8.4, 1.9 Hz, 1H), 7.37 (d, J 7.9 Hz, 2H), 6.77 (d, J 8.4 Hz, 1H), 5.03 (dd, J 8.2, 5.4 Hz, 1H), 3.41 (dd, J 14.2, 5.4 Hz, 1H), 3.28 (dd, J 14.2, 5.4 Hz, 1H), 2.39 (s, 3H). 13C NMR (101 MHz, DMSO‐d6) δ 203.15, 177.82, 144.28, 140.33, 136.81, 135.48, 135.37, 134.93, 131.07, 127.99, 126.43, ‐ 126.25, 54.81, 35.14, 20.87. ESI‐MS: m/z [M‐H] calcd for C17H14N2O3S3: 389.0; found: 388.8. ‐ HRMS: m/z [M‐H] calcd for C17H14N2O3S3: 389.0083; found: 389.0090.

122 Materials and methods

8) Molecular biological and biochemical methods

8.1) Chemicals, reagents, inhibitors and solvents

All solvents, reagents and fine chemicals used in the molecularbiological and biochemical part are commercially available (Sigma‐Aldrich, Acros, Merck, Fluka, Roth, TCI, MCAT, Riedel de Haën or Amersham Pharmacia Biotech) and are qualified for molecular biologiy and biochemistry. Milli‐Q water was genereted with Easy pure UV/UF pure water system (Werner). Small‐molecule analogues SM1, 4, 10, 12, 16, 17, 21, 23, 27‐32, and 35‐58 are of synthetic origin, whereat SM1, 4, 10, 16, 17, 21, 23, 27, and 28 were synthesized in my privious diploma thesis76 (see chapter 7). SM2, 3, 5‐9, 11, 13‐15, 18‐20, 22, and 24‐26 were purchased from the suppliers Maybridge, Labotest OHG and Key Organics. (‐)‐ Epigallocatechin gallate (EGCG), betulinic acid, oleanolic acid and lithocholic acid were purchased from Sigma Aldrich to evaluate the discovered small‐molecule inhibitors and the

PEX assays. All compounds were stored in 10 or 5 µM DMSO stock solutions at ‐20°C.

8.2) Expression and purification of pol λ and pol β

Human recombinant pol λ and pol β were expressed and purified as described in literature55,243. Pol β‐wt ORF in pGDR11 plasmid and pol λ‐wt in pGDR11 plasmid in E.coli strain XL10 Gold was kindly provided by Bettina Bareth.75 The purity of the enzymes was >95% as controlled by SDS‐PAGE analysis (Figure 36). Enzyme concentrations were determined by the Bradford assay244 using standart protocols (Roth).

Figure 36. SDS‐PAGE analysis of the recombinant human DNA polymerases. Lane M = marker (kDa), lane 1 = pol λ and lane 2 = pol β.

123 Materials and methods

8.2.1) Nucleotide‐ and amio acid sequences of pol β and pol λ

Coding DNA sequence of 6xHis‐tagged pol β‐wt ORF in pGDR11:

ATG AGA GGA TCT CAC CAT CAC CAT CAC CAT ACG GAT CCG ATG AGC AAA CGT AAA GCG CCG CAG GAA ACC CTG AAC GGC GGC ATT ACC GAT ATG CTG ACC GAA CTG GCC AAC TTT GAA AAA AAC GTG AGC CAG GCG ATC CAT AAA TAT AAC GCG TAT CGT AAA GCG GCG AGC GTG ATT GCG AAA TAT CCG CAC AAA ATT AAA AGC GGT GCG GAA GCG AAA AAA CTG CCG GGC GTG GGC ACC AAA ATT GCG GAA AAA ATC GAT GAA TTT CTG GCC ACC GGC AAA CTG CGT AAA CTG GAA AAA ATT CGC CAG GAT GAT ACC AGC AGC AGC ATT AAC TTT CTG ACC CGT GTG AGC GGC ATT GGT CCG AGC GCG GCG CGT AAA TTT GTG GAT GAA GGC ATC AAA ACC CTG GAG GAT CTG CGT AAA AAC GAA GAT AAA CTG AAC CAT CAT CAG CGT ATT GGC CTG AAA TAT TTT GGC GAT TTC GAA AAA CGT ATT CCG CGT GAA GAA ATG CTG CAG ATG CAG GAT ATT GTG CTG AAC GAA GTG AAA AAA GTG GAT AGC GAA TAT ATT GCG ACC GTG TGC GGC AGC TTT CGT CGT GGC GCG GAA AGC AGC GGC GAT ATG GAT GTG CTG CTG ACC CAT CCG AGC TTT ACC AGC GAA AGC ACC AAA CAG CCG AAA CTG CTG CAT CAG GTG GTG GAA CAG CTG CAG AAA GTG CAT TTT ATT ACC GAT ACC CTG AGC AAA GGC GAA ACC AAA TTT ATG GGC GTG TGC CAG CTG CCG AGC AAA AAC GAT GAA AAA GAA TAT CCG CAT CGC CGT ATT GAT ATT CGT CTG ATC CCG AAA GAT CAG TAT TAT TGC GGC GTG CTG TAT TTT ACC GGC AGC GAT ATC TTC AAC AAA AAC ATG CGT GCG CAT GCG CTG GAA AAA GGC TTT ACC ATC AAC GAA TAC ACC ATT CGT CCG CTG GGC GTG ACC GGT GTT GCG GGT GAA CCG CTG CCG GTG GAT AGC GAA AAA GAT ATC TTC GAT TAC ATC CAG TGG AAA TAT CGT GAA CCG AAA GAT CGT AGC GAA TAA

Bold: Start‐ and Stopp codon Bold and underline: N‐terminal His‐Tag Underline: Coding DNA sequence of human pol β‐wt

Protein sequence of human pol β‐wt in Plasmid pGDR11:

MRGSHHHHHHTDPMSKRKAPQETLNGGITDMLTELANFEKNVSQAIHKYNAYRKAASVIAKYPHKIKSGAEAKKL PGVGTKIAEKIDEFLATGKLRKLEKIRQDDTSSSINFLTRVSGIGPSAARKFVDEGIKTLEDLRKNEDKLNHHQR IGLKYFGDFEKRIPREEMLQMQDIVLNEVKKVDSEYIATVCGSFRRGAESSGDMDVLLTHPSFTSESTKQPKLLH QVVEQLQKVHFITDTLSKGETKFMGVCQLPSKNDEKEYPHRRIDIRLIPKDQYYCGVLYFTGSDIFNKNMRAHAL EKGFTINEYTIRPLGVTGVAGEPLPVDSEKDIFDYIQWKYREPKDRSE

Bold: Sequence of human pol β Underline: N‐terminales His‐Tag

124 Materials and methods

Coding DNA sequence of 6xHis‐tagged pol λ‐wt ORF in pGDR11:

ATG AGA GGA TCT CAC CAT CAC CAT CAC CAT ACG GAT CCG CGT GGC ATT CTG AAA GCG TTC CCG AAA CGC CAG AAA ATT CAT GCG GAT GCC AGC AGC AAA GTG CTG GCC AAA ATT CCG CGT CGT GAA GAA GGC GAA GAA GCG GAA GAA TGG CTG TCT AGC CTG CGT GCG CAT GTG GTG CGT ACC GGC ATT GGT CGT GCG CGT GCG GAA CTG TTT GAA AAA CAA ATT GTG CAG CAT GGT GGC CAA CTG TGT CCG GCC CAG GGT CCG GGT GTG ACG CAT ATT GTG GTG GAT GAA GGC ATG GAT TAT GAA CGT GCG CTG CGT CTG CTG CGC CTG CCG CAA CTG CCG CCG GGT GCG CAG CTG GTT AAA AGC GCG TGG CTG TCT CTG TGC CTG CAG GAA CGT CGT CTG GTT GAC GTG GCG GGC TTC AGC ATT TTT ATT CCG AGC CGC TAT CTG GAT CAT CCG CAG CCG AGC AAA GCG GAA CAG GAT GCG AGC ATT CCG CCG GGC ACC CAT GAA GCG CTG CTG CAG ACC GCC CTG TCT CCG CCG CCG CCG CCG ACC CGT CCG GTT AGC CCG CCG CAG AAA GCG AAA GAA GCG CCG AAC ACC CAG GCG CAG CCG ATT AGC GAT GAT GAA GCG AGC GAT GGC GAA GAA ACC CAG GTG AGC GCG GCG GAT CTG GAA GCG CTG ATT AGC GGC CAT TAT CCG ACC AGC CTG GAA GGC GAT TGC GAA CCG AGT CCG GCA CCG GCG GTT CTG GAT AAA TGG GTG TGC GCC CAG CCG AGC AGC CAG AAA GCG ACC AAC CAT AAC CTG CAT ATC ACC GAA AAA CTG GAA GTT CTG GCC AAA GCG TAT AGC GTG CAG GGC GAT AAA TGG CGT GCG CTG GGC TAT GCG AAA GCG ATT AAC GCG CTG AAA TCT TTT CAT AAA CCG GTG ACC AGC TAT CAG GAA GCG TGC AGC ATT CCG GGC ATT GGC AAA CGT ATG GCG GAA AAA ATC ATC GAA ATT CTG GAA AGC GGC CAT CTG CGT AAA CTG GAT CAT ATT AGC GAA AGC GTG CCG GTG CTG GAA CTG TTT AGC AAC ATT TGG GGT GCG GGC ACC AAA ACC GCG CAG ATG TGG TAT CAG CAG GGC TTT CGT AGC CTG GAA GAT ATT CGT AGC CAG GCG AGC CTG ACC ACC CAG CAG GCG ATT GGC CTG AAA CAT TAC AGC GAT TTT CTG GAA CGT ATG CCG CGT GAA GAA GCG ACC GAA ATT GAA CAG ACC GTG CAG AAA GCG GCG CAG GCG TTT AAC AGC GGC CTG CTG TGC GTT GCG TGC GGC AGC TAT CGT CGT GGC AAA GCG ACC TGC GGT GAT GTG GAT GTG CTG ATT ACC CAC CCG GAT GGC CGT AGC CAT CGT GGC ATT TTT AGC CGT CTG CTG GAT AGC CTG CGT CAG GAA GGC TTT CTG ACC GAC GAT CTG GTG AGC CAG GAA GAA AAC GGC CAG CAG CAG AAA TAT CTG GGC GTG TGC CGT CTG CCG GGT CCG GGT CGT CGT CAT CGT CGT CTG GAT ATT ATT GTG GTG CCG TAT AGC GAA TTT GCG TGT GCG CTG CTG TAT TTC ACC GGC AGC GCG CAT TTT AAC CGT AGC ATG CGT GCG CTG GCC AAA ACC AAA GGC ATG AGC CTG AGC GAA CAT GCC CTG AGC ACC GCC GTG GTG CGT AAC ACC CAT GGC TGC AAA GTT GGT CCG GGC CGT GTT CTG CCG ACC CCG ACC GAA AAA GAT GTG TTT CGT CTG CTG GGT CTG CCG TAT CGT GAA CCG GCG GAA CGT GAT TGG TAA

Bold: Start‐ and Stopp codon Bold and underline: N‐terminal His‐Tag Underline: Coding DNA sequence of human pol λ‐wt

Protein sequence of human pol λ ‐wt in Plasmid pGDR11:

MRGSHHHHHHTDPRGILKAFPKRQKIHADASSKVLAKIPRREEGEEAEEWLSSLRAHVVRTGIGRARAELFEKQI VQHGGQLCPAQGPGVTHIVVDEGMDYERALRLLRLPQLPPGAQLVKSAWLSLCLQERRLVDVAGFSIFIPSRYLD HPQPSKAEQDASIPPGTHEALLQTALSPPPPPTRPVSPPQKAKEAPNTQAQPISDDEASDGEETQVSAADLEALI SGHYPTSLEGDCEPSPAPAVLDKWVCAQPSSQKATNHNLHITEKLEVLAKAYSVQGDKWRALGYAKAINALKSFH KPVTSYQEACSIPGIGKRMAEKIIEILESGHLRKLDHISESVPVLELFSNIWGAGTKTAQMWYQQGFRSLEDIRS QASLTTQQAIGLKHYSDFLERMPREEATEIEQTVQKAAQAFNSGLLCVACGSYRRGKATCGDVDVLITHPDGRSH RGIFSRLLDSLRQEGFLTDDLVSQEENGQQQKYLGVCRLPGPGRRHRRLDIIVVPYSEFACALLYFTGSAHFNRS MRALAKTKGMSLSEHALSTAVVRNTHGCKVGPGRVLPTPTEKDVFRLLGLPYREPAERDW

Bold: Sequence of human pol λ Underline: N‐terminales His‐Tag

125 Materials and methods

8.3) SDS‐PAGE

Denaturing sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) was performed to separate proteins. SDS‐PAGE was performed using a stacking along with a separating gel. First, a 12% separating gel was prepared by mixing 1.7 mL H2O, 1.25 mL SDS‐ PAGE separating gel buffer, 50 μL 10% SDS, and 2 mL 30% bis‐acrylamide with 50 μL 10% APS and 5 μL TEMED. The solution was applied to the gel chamber and covered with 2‐ propanole. After polymerization, 2‐propanole was removed and the stacking gel including the gel pockets was prepared (1.22 mL H2O, 0.5 mL SDS‐PAGE stacking gel buffer, 10 μL 10% SDS, 270 μL 30% bis‐acrylamide, 20 μL 10% APS, and 5 μL TEMED). Protein samples were mixed with one sixth of 6 SDS‐PAGE loading buffer and denaturated (5 min, 95°C). PageRuler unstained protein ladder (Fermentas) was used as a standard. Separation was achieved by applying 35 mA in 1x SDS‐PAGE electrophoresis buffer. Afterwards the gel was stained according to standard protocols using coomassie brilliant blue staining‐ and destaining solutions (Carl Roth) respectively.

8.4) Nucleotides and oligonucleotides dNTPs were purchased from Roche, [γ‐32P]ATP from Hartmann Analytics and oligonucleotides were purchased from Purimex and purified as described in chapter 8.4.1 Employed oligonucleotide sequences: Primer F20H, 5`‐d(CGT TGG TCC TGA AGG AGG AT); template F33A, 5`‐d(AAA TCA ACC TAT CCT CCT TCA GGA CCA ACG TAC).

8.4.1) DNA oligonucletide purification

DNA oligonucletides were purified via PAGE. First, a 12% gel was prepared by reacting

120 mL 25% acrylamide‐bisacrylamide in 8.3 M urea, 105 mL 8.3 M urea, and 25 mL 8.3 M urea in 10 TBE buffer with 1.8 mL 10% APS in H2O and 90 μL TEMED. Final gel thickness was 1.5 mm. DNA samples containing denaturing PAGE loading buffer (stop solution) were separated by applying up to 100 W and 3000 V, respectively, in 1x TBE buffer at up to 45°C. Afterwards DNA was visualized by UV light absorption (shadowing) and excised with a scalpel. Gel pieces were scrambled by pushing them trough a syringe. DNA was eluted by

126 Materials and methods

adding H2O and incubating the mixture at 55°C overnight. The mixture was filtered using a syringe with a pad of silanised glass‐fibres wool. Finally, DNA was purified by ethanol precipitation. For DNA precipitation 0.1 volumes of 3 M NaOAc/HOAc (pH 5.3) followed by 2.5 volumes of 100% ethanol were added to the DNA sample. After incubation for >30 min at 20°C centrifugation was carried out at 4°C and 20,000×g for 30 min. The supernatant was discarded and 500 μL pre‐cooled 70% ethanol were added to wash the DNA pellet followed by another centrifugation step for 10 min. After removing the supernatant, the pellet was dried in vacuo (Speed‐Vac) and resolved in H2O. If the starting volume of the DNA sample was less than 1 mL volumes were adjusted. To determine DNA concentrations, 1.5 to 2.0 μL sample were applied to the Nanodrop pedestals (Nanodrop ND1000). DNA was measured at 260 nm wavelength and the concentrations were calculated using the Lambert‐Beer law. The molar extinction coefficient for single stranded DNA resulted from the formula ‐1 ‐1 ε[mM cm ] = 15.2(A) + 12.01(G) + 8.4(T) + 7.05(C) at which A, G, C and T are the numbers of the respective nucleotide in the sequence.

8.4.2) 5’‐Radioactive labelling of DNA oligonucletides

DNA oligonucleotide primers were radioactively labelled at the 5’ terminus by a 32P containing phosphate group using T4 polynucleotide kinase (PNK) (from Fermentas) which transfers the γ phosphate group from [γ‐32P]ATP (Hartmann Analytics) to the 5’ hydroxyl group. The reactions contained primer (0.4 μM), PNK forward reaction buffer (1), [γ‐32P]ATP (0.8 μCi μL‐1) and T4 PNK (0.4 U μL‐1) in a final volume of 50 μL and were incubated for 1 h at 37°C. The reaction was stopped by denaturing the T4 PNK for 2 min at 95°C and buffers and excess [γ‐32P]ATP were removed by gel filtration (MicroSpin Sephadex

G‐25). Addition of unlabelled primer (20 μL, 10 μM) led to a final concentration of 3 μM of diluted radioactive labelled primer.

8.5) Radiometric primer extension

Enzyme activity, small molecule evaluation and IC50 measurements were done using a radiometric primer extension (PEX) assay with a radiometric product analysis. All primer

127 Materials and methods template complexes were annealed by heating a mixture of primer and template in the respective reaction buffer to 95°C for 5 min and subsequent cooling to room temperature prior to applications in all PEX assays.

8.5.1) Pol λ PEX assay with variable small‐molecule concentrations

Pol λ reactions were made in a final volume of 20 μL containing: 50 mM Tris‐HCl pH 7.5,

1.5 mM MgCl2, 5% (v/v) glycerol, 1 mM DTT (dithiothreitol), 250 nM purified recombinant ‐1 pol λ, 150 nM radioactively labled primer F20H, 225 nM template F33A, 0.1 mg mL BSA (bovine serum albumin) (from Thermo Scientific), 1 μL compound solution (variable concentrations in DMSO) and accordingly 1 μL DMSO for the solvent control. Reactions and positive controls were started by addition of 5 μL dNTP solution (15 μM final). After 30 min incubation at 37°C, reactions and controls were subsequently quenched using 45 μL PAGE loading solution (formamide 80% (v/v), EDTA 20 mM, bromophenol blue 0.05% (w/v), xylene cyanol 0.05% (w/v)) per well. Then the reaction mixture was denaturated for 5 min at 95°C and analyzed by 12% PAGE containing 8 M urea. Visualization was performed using phosphorimaging.

8.5.2) Pol λ PEX assay with variable dNTP concentrations

Assay was carried out as described above (pol λ PEX assay with variable small‐molecule concentrations). Except that the inhibitor concentration was 50 μM and accordingly 0 μM for the solvent control. Positive control was started by addition of 5 μL dNTP solution (15 μM final). Reactions were started by addition of variable concentrations of 5 μL dNTP solution

(15, 30, 60, 120, 240, 480 μM final).

8.5.3) Pol λ‐TdT assay with variable small‐molecule concentrations

Assay was carried out as described above (pol λ PEX assay with variable small‐molecule concentrations) with the exception that no template was used and the reaction was incubated for 120 min.

128 Materials and methods

8.5.4) Pol β PEX assay with variable small‐molecule concentrations

Pol β reactions were made in a final volume of 20 μL containing: 50 mM Tris‐HCl (pH 7.9),

20 mM KCl, 5% (v/v) glycerol, 1 mM DTT, 2 mM MnCl2, 250 nM purified recombinant pol β, ‐1 150 nM radioactively labled primer F20H, 225 nM template F33A, 0.1 mg mL BSA, 1 μL compound solution (variable concentrations in DMSO) and accordingly 1 μL DMSO for the solvent control. Reactions and positive controls were started by addition of 5 μL dNTP solution (15 μM final). After 30 min incubation at 37°C, reactions and controls were subsequently quenched using 45 μL PAGE loading solution (formamide 80% (v/v), EDTA

20 mM, bromophenol blue 0.05% (w/v), xylene cyanol 0.05% (w/v)) per well. Then the reaction mixture was denaturated for 5 min at 95°C and analyzed by 12% PAGE containing

8 M urea. Visualization was performed using phosphorimaging.

8.6) Polyacrylamide gel electrophoresis (PAGE)

Denaturing polyacrylamide gels (12%) were prepared by polymerization of a solution of urea

(8.3 M) and bisacrylamide/acrylamide (12%) in TBE buffer using ammonium peroxodisulfate (APS, 0.08%) and N,N,N’,N’‐tetramethylethylenediamine (TEMED, 0.04%). Immediately after addition of APS and TEMED the solution was filled in a sequencing gel chamber and left for polymerization for at least 45 min. After addition of TBE buffer (1) to the electrophoresis unit, gels were prewarmed by electrophoresis at 100 W for 30 min and samples were added and separated during electrophoresis (100 W) for approx. 1.5 h. The gel was transfered to whatman filter paper, dried (80°C, in vacuo, using a gel dryer model 583, BioRad) and exposed to a phosphor screen.

8.6.1) Quantitative analysis of gel images

After visualization of the gel, the image was analyzed with the software QuantityOne from BioRad. Every band of a lane was marked and quantified using the software. The generated percental values of every band of a lane were weighted with a factor (factor equates to the number of incorporated dNTPs) and summated. DMSO was used as solvent control and the activity of the enzyme in its presence was set to 100% conversion. Afterwards the

129 Materials and methods conversion of the reaction in presence of the compound (in their respective concentration) was calculated. The resulted conversions (in %) of independently conducted experiments were used to fit the data using the non‐linear regression calculation of Prism 4 (GraphPad). ‐1 Y=Bottom + (Top‐ Bottom) (1+10^((LogIC50‐X) HillSlope)) .

130 Materials and methods

9) Cell biological methods

9.1) Cell cultivation

HeLa‐S3 and Hep‐G2

Human cervix carcinoma cell line HeLa‐S3 and human liver carcinoma cell line Hep‐G2 were obtained from European Collection of Cell Cultures (ECACC) and were cultured as described previously.184

HeLa‐S3 and Hep‐G2 cells were cultivated at 37°C in humidified 5% CO2 atmosphere using Dulbecco’s DMEMmedia (Invitrogen) containing 10% fetal calf serum, 1% penicillin and 1% streptomycin. Cells were split every three days. Both cell lines were tested for mycoplasma infections using a mycoplasma detection kit (Roche Applied Science).

Caco‐2

The human colon cancer cell line Caco‐2 (ATCC HTB‐37) was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and was cultured as described previously.245 Cells were grown in Iscove’s Modified Dulbecco’s Media supplied with 10% fetal calf serum, ‐1 50 mg mL gentamicin at 37°C and 5% CO2. Cells were split every three days.

9.2) AllamarBlue assay

To measure the cell viability we adapted the previously described AlamarBlue assay184,185 AlamarBlue (BioSource Europe), the dark blue coloured sodium salt of resazurin (7‐hydroxy‐ 3H‐phenoxazin‐3‐one‐10‐oxide) was used to measure growth and viability of cells which are capable of reducing it to the fluorescent, pink colored resorufin (7‐hydroxy‐3H‐phenoxazin‐ 3‐one). Cells were seeded in 96‐well plates (4,000 HeLa‐S3 cells per well or 8,000 Hep‐G2 cells per well) and allowed to attach for 24 h. Small‐molecules to be tested were dissolved in a suitable amount of DMSO. Different concentrations were prepared by serial dilution with medium to give final concentrations with a maximum DMSO content of 1%. The cells were then incubated for 48 h with 100 μL each of above dilution series. Alamar Blue (10 μL) was added and the cells were incubated for another hour. After excitation at 530 nm, fluorescence at 590 nm was measured using a FL600 Fluorescence Microplate Reader

131 Materials and methods

(Bio‐TEK). Cell viability is expressed in percent with respect to a control containing only pure medium and 1% DMSO incubated under identical conditions. All experiments were repeated for a minimum of three times with each experiment done in four replicates. The resulting curves were fitted using the software Prism 4 (GraphPad).

9.2) MTT assay

To measure the cell viability we adapted the previously described 3‐(4,5‐dimethylthiazol‐2‐ yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay.188,189 2,000 cells per well were seeded in a 96‐well plate format and allowed to recover overnight. On the next day, different concentrations of small‐molecules to be tested or DMSO controls were prepared by serial dilution with medium to give final concentrations with a maximum DMSO content of 1%. The dilutions of the compounds were added and the cells were pre‐incubated for 24 h. The following day, different concentrations of DNA damaging agents were tested (H2O2, TMZ) or controls (DMSO for TMZ) were prepared by serial dilution with medium to give final concentrations with a maximum DMSO content of 1% (DMSO of small‐molecules dilution series included). Different dilutions of the agents were added and the cells were incubated for additional 72 h. Afterwards the medium was replaced with fresh culture medium containing 0.5 mg mL‐1 MTT. The cells were incubated another 2.5 h. Then, the MTT medium was discarded again and replaced by DMSO to dissolve the formazan. Absorbance was measured at 565 nm using a microplate reader (TECAN Sunrise Remote). Cell viability (or cell death respectively) was expressed as a percentage of according controls. The resulting diagrams and curves were generated using the software Prism 4 (GraphPad).

132 References

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