Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Synthesis of a peptide nucleic acid oligomer of a Janus heterocycle Hasan, Syed 2013

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
24-ubc_2014_spring_hasan_syed.pdf [ 19.48MB ]
Metadata
JSON: 24-1.0166851.json
JSON-LD: 24-1.0166851-ld.json
RDF/XML (Pretty): 24-1.0166851-rdf.xml
RDF/JSON: 24-1.0166851-rdf.json
Turtle: 24-1.0166851-turtle.txt
N-Triples: 24-1.0166851-rdf-ntriples.txt
Original Record: 24-1.0166851-source.json
Full Text
24-1.0166851-fulltext.txt
Citation
24-1.0166851.ris

Full Text

 SYNTHESIS OF A PEPTIDE NUCLEIC ACID OLIGOMER OF A JANUS HETEROCYCLE    by  Syed Hasan  B.Sc., McMaster University, 2005      A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF    MASTER OF SCIENCE   in   The Faculty of Graduate and Postdoctoral Studies  (Chemistry)     THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   December 2013   ? Syed Hasan, 2013   ii Abstract This thesis describes the oligomerization of the Janus AT heterocycle on a peptide nucleic acid scaffold, a molecule that is capable of concurrently hydrogen bonding with adenine and thymine. An oligonucleotide incorporating this moiety should form sequence specific Watson-Crick based triplexes with DNA by strand invasion. Initially, the synthesis of diamino Janus AT peptide nucleic acid monomer 1 was attempted, but the monomer was too insoluble for solid phase synthesis. To curtail this issue, attempts were made to synthesize a more hydrophobic surrogate of 1 wherein the exocyclic amines were masked (2, 3 and 4). The synthesis of an undecamer was undertaken, wherein 4 was alternated with a 6-methyluracil spacer. Finally, a dodecamer of 6MU was synthesized to examine its biophysical properties in the context of base pairing with DNA.   HNN NNNH2NH2OOONNHRHOOHNNNNH2SOOONNHRHOOHNN NNClClOOONNHRHOOHNN NNNH2SOOONNHRHOOPhHNNOOONNHRHOOPhOO1 2 6-Methyluracil PNA Monomer 3 4  iii Preface All of the experiments described within this thesis were conducted by the author, Syed Hasan. The author and Dr. David Perrin conceived the protocols for the experiments described herein, except for previously reported/published procedures, which are referenced in the appropriate location. Computational and ongoing biophysical experiments were performed by Dr. Eric Largy. The analysis of data was performed by the author in consultation with Dr. Perrin and Dr. Largy.    iv Table of Contents Abstract??????????????????????????????..... ii!Preface???????????????????????????????.. iii!Table of Contents??????????????????????????..... iv!List of Tables????????????????????????????? vi!List of Figures???????????????????????????..? vii!List of Equations???????????????????????????.. ix!List of Schemes????????????????????????????. x!List of Abbreviations?????????????????????????.. xii!Acknowledgements??????????????????????????.. xv!Chapter 1: Introduction????????????????????????..  1 1.1  Introduction to DNA Structure????????????????. 1   1.1.1 Importance of DNA??????????????????. 1  1.1.2 Primary, Secondary and Tertiary Structure?????????. 2 1.2  Recognition of DNA????????????????????. 4   1.2.1 Overview of DNA recognition?????????????? 4  1.2.2 Minor Groove Recognition???????????????.. 5  1.2.3 Major Groove Recognition???????????????.. 7 1.3  Janus Bases???????????????????????? 8  1.3.1 Introduction and History of Janus Heterocycles???????.. 8   1.3.2 Supramolecular Chemistry of Janus Heterocycles??????. 10  1.3.3 Biological Chemistry of Janus Heterocycles????????.. 12 1.4  Goals of This Thesis???????????????????? 14   1.4.1 Overall Goals????????????????????. 14   1.4.2 Peptide Nucleic Acid Scaffold?????????????? 15 Chapter 2: Results and Discussion???????????????????? 19 2.1 Syntheses of PNA Backbones and Glycine Precursor??????? 20 2.2 Synthesis of Janus AT 1??????????????????.. 23 2.3 Synthesis Towards Janus AT 2????????????????.26   2.3.1 Janus AT 2 via Allylamine???????????????. 28  v   2.3.2 Janus AT 2 via Ethanolamine??????????????. 29 2.4 Synthesis Towards Janus AT 3???????????????? 32 2.5 Synthesis of Janus AT 4 and its Solid Phase Synthesis??????.. 40   2.5.1 Synthesis of the Monomers 4 and 47???????????. 41   2.5.2 Stability Tests on Monomers??????????????. 44   2.5.3 Solid Phase Oligomerization of Janus AT?????????. 46 2.6 Synthesis of 6-Methyluracil 5 and its Solid Phase Synthesis????. 49   2.6.1 Synthesis of 6-Methyluracil Monomer??????????.. 50   2.6.2 Solid Phase Oligomerization of 6-Methyluracil??????? 51 Chapter 3: Conclusion????????????????????????? 53 Chapter 4: Experimental???????????????????????? 54 4.1 General Experimental Procedure??????????????? 54 4.2 Chemical Methods????????????????????. 55 4.3 Protocol for Solid Phase Synthesis?????????????? 126 Bibliography ????????????????????????????.. 131!!   vi List of Tables Table 2.1. Bases screened for the deprotonation of 37?????????????. 38 Table 2.2. Screening of carbonylation agents ????????????????. 39 Table 2.3. Chemical stability tests for 4 and 47???????????????? 45    vii List of Figures Figure 1.1: Double helical DNA features??????????????????? 2 Figure 1.2: Watson-Crick hydrogen-bonding?????????????????.. 2 Figure 1.3: Major and minor grooves of dsDNA????????????????. 4 Figure 1.4: Example of hydrogen bonding??????????????????.. 5 Figure 1.5: Distamycin A?????????????????????????. 6 Figure 1.6: Polyamides halting transcription in dsDNA?????????????.. 6 Figure 1.7: Schematic view of a triple helix?????????????????? 7 Figure 1.8: Theoretical model of DNA recognition using Janus a heterocycle????. 9 Figure 1.9: Janus CU heterocycle?????????????????????.. 9 Figure 1.10: Supramolecular Janus heterocyclic architectures??????????.. 10 Figure 1.11: Three congeners of Janus AT?????????????????? 11 Figure 1.12: PNA oligomer of Janus wedge?????????????????.. 12 Figure 1.13: Janus GC on a silyl ribosyl scaffold???????????????.. 13 Figure 1.14: Olgiomer of hypothetical Janus bases??????????????.. 14 Figure 1.15: Peptide nucleic acids?????????????????????. 16 Figure 1.16: Solid phase synthesis of peptide nucleic acids???????????.. 17 Figure 1.17: Downloading the PS-MBHA resin???????????????? 18 Figure 2.1: Janus and 6-methyluracil PNA monomers?????????????.. 20 Figure 2.2: Generalized synthesis of PNA monomers?????????????.. 20 Figure 2.3: The synthesized PNA backbones????????????????? 21 Figure 2.4: Diamino Janus AT 1?????????????????????? 24 Figure 2.5: Mass spectrum from the ozonolysis of 26?????????????.. 29  viii Figure 2.6: Protonation disrupts the H-bonding of the adenine face????????. 32 Figure 2.7: UV-vis spectra of 4??????????????????????.. 43 Figure 2.8: Structure of 6-methyluracil PNA monomer 5????????????. 48 Figure 2.9: UV-vis spectrum of the 6-MU oligomer??????????????. 51 Figure 2.10: Crude HPLC chromatogram of 6MU 12mer????????????. 52        ix List of Equations Equation 2.1: The solid phase sequence??????????????????? 47 Equation 2.2: Modified sequence to avoid J-to-J couplings???????????.. 48 Equation 2.3: Re-modified sequence????????????????????. 49 Equation 2.4: Sequence synthesized for 6-methyluracil????????????? 51       x List of Schemes Scheme 2.1: Synthesis of esters 12 and 13?????????????????? 22 Scheme 2.2: Synthesis of Boc backbones 6, 7 and 8??????????????. 22 Scheme 2.3: Synthesis of the Fmoc backbone 9???????????????? 23 Scheme 2.4: Synthesis of tert-butylglycine 15????????????????.. 23 Scheme 2.5: Synthesis of 18???????????????????????.. 24 Scheme 2.6: Synthesis of diamino Janus AT 20???????????????? 25 Scheme 2.7: Synthesis of 1???????????????????????? 26 Scheme 2.8: Overall scheme for synthesis of 2????????????????. 27 Scheme 2.9: Synthesis of 22 from 16???????????????????? 27 Scheme 2.10: Synthesis of allyl Janus heterocycle 26?????????????.. 28 Scheme 2.11: Synthesis of 31 from 22???????????????????.. 30 Scheme 2.12: Failed attempts at oxidation of the pendant alcohol of 31??????.. 31 Scheme 2.13: Overall scheme for synthesis of dichloro 3????????????. 33 Scheme 2.14: Synthesis of 34??????????????????????? 34 Scheme 2.15: Synthesis of 37??????????????????????? 35 Scheme 2.16: Small scale reactions of 37??????????????????.. 35 Scheme 2.17: Smalls scale synthesis of PNA monomer 3????????????. 36 Scheme 2.18: Putative pathways during ring closure?????????????? 37 Scheme 2.19: Synthesis of 47??????????????????????? 41 Scheme 2.20: Synthesis of 41 and 43???????????????????? 42 Scheme 2.21: Synthesis of amide 46???????????????????? 43 Scheme 2.22: Synthesis of PNA monomers 4 and 47?????????????. 44  xi Scheme 2.23. Synthesis of Rapoport?s capping reagent 50???????????... 47 Scheme 2.24: Synthesis of 6-methyluracil monomer 5?????????????. 50     xii List of Abbreviations Abbreviation Meaning ? Chemical shift 6MU 6-methyluracil A Adenine or Acceptor Ac2O Acetic anhydride AcOH Acetic Acid AEEA Aminoethoxyethoxyacetic acid Boc Di-tert-butyl dicarbonate C Cytosine d Doublet D Donor or Deuterium DCM Dichloromethane DFT Density Functional Theory DIPEA Diisopropylethylamine DMF Dimethylformamide DMSO Dimethylsulfoxide DNA Deoxyribonucleic acid dsDNA Double stranded deoxyribonucleic acid EDC N-ethyl N?-(3-Dimethylaminopropyl) carbodiimide ESI Electrospray Ionization EtOAc Ethyl Acetate Fmoc Fluorenylmethyloxycarbonyl G Guanine  xiii H-bond Hydrogen bond HBTU O-Benzotriazole-N,N,N?,N?-tetramethyl-uronium-hexafluorophosphate HMDS Hexamethyldisilazide HPLC High Performance Liquid Chromatography Hz Hertz ID Inner Diameter J or J Coupling Constant or Janus m Multiplet m/z Mass to charge ratio MALDI Matrix Assisted Laser Desorption Ionization MS Mass Spectrometry NMP N-methylpyrrolidinone NMR Nuclear Magnetic Resonance OD Outer Diameter PNA Peptide Nucleic Acid ppm Parts per million PS-MBHA Polystyrene Methyl Benzhydrylamine Rf Retention Factor rt Room temperature s Singlet ssDNA Single stranded deoxyribonucleic acid T Thymine t Triplet TBDMS Tert-butyldimethylsilyl chloride  xiv TFA Trifluoroacetic acid TFO Triplex forming oligonucleotide TFMSA Trifluoromethanesulfonic acid THF Tetrahydrofuran TLC Thin layer chromatography UV-vis Ultraviolet visible   A note on the usage of ?base?: The word base is used in three contexts within this thesis: (1) denoting the opposite of acid, i.e. alkali; (2) as a contraction of the word ?nucleobase?, in the context of nucleic acids chemistry; (3) as synonym for Janus heterocycles, because they are designed to be nucleobase analogs.     xv Acknowledgements I would like to express my gratitude to my supervisor Dr. David Perrin for his guidance. Without his support, this thesis would never have been possible.  I would also like to thank members of the Perrin group. I would like to especially mention Ms. Tahereh Azadbakht, Mr. Wenbo Liu, Dr. Eric Largy and Mr. Shenal Cooray.  Finally, I would like to thank Professors Michael Wolf and Glenn Sammis for taking their time to serve as members of my examination committee.  1 Chapter 1 - Introduction 1.1 Introduction to DNA Structure 1.1.1 Importance of DNA A detailed understanding of the interactions of small molecules with deoxyribonucleic acids is of tremendous importance due to the role that DNA plays in determining both the physical traits, as well as the disease susceptibility of organisms. 1 Thus, a DNA-binding agent that could recognize an arbitrary sequence of DNA with high sequence specificity would find use as a modulator of gene expression and perhaps even as an antiretroviral agent against cancers and HIV. However, accomplishing such a lofty goal is far from simple, and the development of a code for the sequence specific recognition of longer sequences of DNA remains an unsolved problem. 2  Due to the ample availability of hydrogen bonds and negative charges in double stranded DNA, a strategy employing non-covalent forces is very fruitful for the development of a sequence-specific binder (Figure 1.1). Accordingly, the design and development of synthetic DNA binding agents falls within the domain of ?supramolecular? chemistry.1 To rationally design a DNA binder, an understanding of the structure of the nucleobases as well as the macrostructures of double stranded DNA is necessary.   2  Figure 1.1: DNA features an abundance of non-covalent binding regions. Figure used without permission. 3  1.1.2 Primary, Secondary and Tertiary DNA Structure The primary structure of DNA is defined by the nucleobase sequence of the strands. On the other hand, the secondary structure of DNA consists of a two-stranded dimer containing two complementary base pairing schemes. These Watson-Crick base-pairing motifs are adenine-thymine (A-T) and guanine-cytosine (G-C) and are mandated for the survival of all life (Figure 1.2). The individual strands of the DNA duplex are assembled through numerous hydrogen bonds (Section 1.2.1). 4   Figure 1.2: Watson-Crick hydrogen-bonding; Adenine associates with Thymine; Guanine associates with Cytosine. NNNNNONONNONNNNNNNOHHHHHHHHAdenine ThymineGuanine CytosineRRRR 3 In addition to the Watson-Crick base pairing motifs, several alternative mechanisms of hydrogen bonding can manifest in double stranded DNA, as well. Examples of these include Hoogsteen and reverse Hoogsteen pairings, wobble pairings, and i-motifs. 5-7 However, these modes of hydrogen bonding are not discussed in this introduction.  Despite the sound understanding of secondary structures, the design of medicinally employable sequence-specific DNA binding agents is non-trivial. 8 Perhaps this is due to the sheer size and structural intricacies that prevail in dsDNA, as well as the constraint on physical properties required for a successful drug (for instance, Lipinski?s rules). The challenge of designing a sequence specific DNA binding agent is further complicated by the tertiary structure of nucleic acid oligomers.  Tertiary structures of dsDNA are the numerous conformational isomers defining the double helix, of which the most common are the A-form, B-form and Z-form. 4 The adopted conformation of strands depends upon their base sequence, the inclusion of modified bases, the degree of supercoiling, and the composition of the solution, among other factors. The two strands forming the double helix make one complete turn on their axis every 10 base pairs. The hydrophobic nucleobases are concealed from the aqueous solution in the core of the helix, whereas the phosphate backbones face into solution. Due to an offset in the spacing between adjacent strands (there is asymmetry in the phosphodiester backbones), the structure adopts two differently sized binding pockets termed the major and minor grooves (Figure 1.3). Generally, the width and depth of the major groove is greater than that of the minor groove, although the exact dimensions are  4 sequence-dependent. The major groove?s larger size permits access to its nucleobases to both macromolecules such as nucleic acids, as well as small molecules. 4   Figure 1.3: The major and minor grooves of dsDNA; the sugar-phosphate backbone is purple and base pairs are red. Figure is reproduced without permission. 9  1.2 Recognition of DNA 1.2.1 Overview of DNA Recognition As alluded to in Section 1.1, the recognition specificity of nucleic acids can be attributed to non-covalent forces of which hydrogen bonding is arguably the most important. Hydrogen bonding is defined as the non-covalent, directional and attractive force between a hydrogen donor and an acceptor. The hydrogen atom must be covalently bonded to an electronegative atom and the acceptor is usually an electron rich moiety (Figure 1.4). The strength of this interaction is dependent on temperature, pressure and phase; for instance, in the gas phase, the strength of the Watson-Crick bonds between the A-T and G-C base pairs is 17 and 20 kcal/mol, respectively. 10, 11  5   Figure 1.4: Hydrogen bonding between N-ethylacetamide and N-methylacetamide.  Hydrogen bonding has been exploited for the design of binding agents in attempts to sequence-specifically recognize double and single-stranded DNA. Broadly, this work has given rise to two themes of small molecule DNA targeting agents: (i) minor groove binding agents, and (ii) small molecules that bind in the major groove.  1.2.2 Minor Groove Binding The minor groove possesses a unique landscape of charge, hydrophobic ?walls? (due to presence of H atoms on the DNA backbone), and a sequence-dependent groove width. Based on these factors, the most successful synthetic paradigm for dsDNA recognition in the minor groove was developed in Peter Dervan?s lab. 12 This strategy employs moieties derived from the polyamide antibiotic distamycin A, whose planar geometry and aromaticity allows its interaction with dsDNA through H-bonding, van der Waals forces and ?-? stacking. Attachment of pendant cationic residues on the polyamides permits favorable electrostatic interaction with the phosphate backbones, thereby increasing binding affinity (Figure 1.5). 2, 12  6  Figure 1.5: Distamycin A has been modified into a minor groove binding code.  The sequence recognition code consists of four heterocycles obtained by altering the core structure of distamycin. To this end, hydroxypyrrole-, pyrrole-, and imidazole-modified derivatives of distamycin were designed to interact with canonical DNA bases pairs. 2 Oligomers of these amides possess appreciable binding affinities (high association constants), and can inhibit helicase, nuclease and polymerase activity (Figure 1.6). 12   Figure 1.6: Two polyamides (pink and orange) can occupy the dsDNA minor groove, halting transcription. Figure reproduced without permission. 1  Although these polyamides can theoretically recognize any DNA sequence, there remain limitations. 13 Firstly, due to the microstructures of DNA (Section 1.1.2), not all sequences bind equally well, and recognition is thus context-sensitive. Secondly, many polyamides have been found that possess poor nuclear uptake for unknown reasons. Lastly, N-terminal residues have more spatial freedom than internal residues, which sometimes reduces the specificity of the polyamide by altering its conformation.  7 1.2.3 Major Groove Binding Sequence-specific binding by a single-stranded oligonucleotide into the major groove of dsDNA leads to the formation of triple helices. 14 Although such ?triplexes? have weaker binding affinities than the two strands of a duplex, they possess the potential for biological regulation. Triplex formation is guided by the formation of two Hoogsteen hydrogen bonds per base pair between the TFO and the DNA duplex. 15  Because Hoogsteen bonds are only limited to purines, triplex formation is only effective at binding tracts of guanines or adenines, with lengths of typically 10-30 nucleotides. Triplexes form in an antiparallel fashion and usually a TFO?s guanine base forms Hoogsteen bonds with a Watson-Crick guanine and a TFO?s adenine base with a Watson-Crick adenine. Pyrimidines (in particular C) require acidic pH to form triplexes, and even then, do so with relatively poor affinity. The landmark study pertaining to TFOs (in 1987) demonstrated that a 15mer oligonucleotide could form a triple helix with gigabyte-sized DNA, propelled by the formation of 30 hydrogen bonds (Figure 1.7). 14-16   NNNNRN HHNNOO RNNONNNRHHHNNNHHHO RNNO NHHNNRHGNNNHHNNRGCTAA 8  Figure 1.7: Top: Hoogsteen bonds (red denotes Watson-Crick pairing and bold blue denotes Hoogsteen). Bottom: Schematic view of a triplex. Used without permission. 14  Although triplex forming oligonucleotides appear promising for the sequence specific recognition of dsDNA, they also possess several drawbacks: (1) the requirement for low pH for binding with pyrimidines (C has a pKa of 4.3); 15 (2) the need Mg2+ to stabilize the numerous phosphate groups; and, finally, (3) poor in-cell stability (nucleases). 1, 16  1.3 Janus Heterocycles 1.3.1 Introduction and History of Janus Heterocycles An underexploited strategy for the recognition of dsDNA is a system containing two synthons, able to target each pair of DNA bases with absolute sequence specificity. For the design of such synthons supramolecular chemistry provides a rich inspiration of model compounds. A heterocycle can be envisaged that features two covalently linked faces with each one capable of forming Watson-Crick-like hydrogen bonds with one nucleotide. Such heterocycles are termed Janus bases, after the two faced Roman god. The insertion of a suitable oligomer of this heterocycle into an intact duplex should result in a stable triplex-like structure, as proposed by Lehn. The great enthalpic gain conferred by the simultaneous formation of multiple hydrogen bonds should provide high binding affinities, and the ability to form Watson-Crick bonds with both DNA nucleobases  9 simultaneously rather than just through the major groove (as outlined in Section 1.2.3) should enhance duplex stability (Figure 1.8). 17   Figure 1.8: Janus heterocycles (triangle) can interact with both DNA base pairs concurrently. R = rest of DNA oligonucleotide.  The first-reported Janus molecule formed a structurally regular mesoscopic supramolecular assembly. Although this molecule was not self-complementary, it could intercept a cytosine-uracil mismatch by forming a triad structure (Figure 1.9). A non-canonical base pair was deliberately chosen as the target in order to disfavor self-association; it was hypothesized that self-association could have led to the undesired formation of a multimeric cyclical rosette of the Janus heterocycle. 18, 19   Figure 1.9: The Janus CU heterocycle; R = phenyl derivatives. NNNNNONONHHHRRDADADATNNNNNONONHHHRRDADADATN NNROHNH HR2HNNOOHNNNOPhHHWC U 10 Since this thought-provoking report of the wedge, the Janus heterocycles? field has been broadly divided into two sections: (i) the design of supramolecular architectures (Section 1.3.2), and (ii) the use of Janus bases for biological goals (Section 1.3.3).  1.3.2 Supramolecular Chemistry of Janus Heterocycles Although supramolecular architectures are not directly related to DNA recognition, the field provides a gallery from which inspiration for the design of novel Janus heterocycles can be gleaned. The self-assembly of Janus heterocycles has been extensively studied, commencing with the first Janus base described in Section 1.3.1. Substitution with various side chains permits controlled, self-directed formation of ?-stacked nanotubes, rosettes of tunable pore sizes, and linear or crinkled ribbons (Figure 1.10). 18, 19    Figure 1.10: Architectures of: a) two-component hexameric rosette, b) helically coiled nanotubes, c) one-component ribbon.  Figure used without permission. 17, 18  The first self-complementary Janus GC heterocycle was reported by Mascal and coworkers and featured an AAD-DDA hydrogen-bonding motif. 20 This heterocycle was designed to permit the creation of a hexagonal supramolecular rosette propelled by the spontaneous formation of 18 hydrogen bonds. Unfortunately, NMR studies of the  11 hydrogen bonding in this GC heterocycle were difficult due to poor solubility in numerous organic solvents. 21 To avert such solubility issues, non-polar substituents (crown ether or alkyl) as well as larger ?-systems have been incorporated into variants of the Janus GC heterocycle. Fenniri et al. have synthesized a series of sterically hindered congeners that self-assemble into tubular or multimeric rosettes with tunable pore diameters. Pendant chiral auxiliaries lead to the formation of enantiopure helices for which numerous applications have been claimed. 22, 23  Despite exhaustive reports pertaining to modifications of the Janus GC motif, the first synthesis of a self-complementary Janus AT base was not reported until more recently. 24 One AT heterocycle, possessing diaminopurine- and thymine-like faces, crystallized in an extended ribbon array but two other congeners could not be crystallized without the addition of formic acid that incorporated into the crystal structure (Figure 1.11).   Figure 1.11: The three congeners of Janus AT (R = Butyl and heptyl). Bottom shows the heterocycle that crystallized as a ribbon. Figure reproduced without permission. 24   12 1.3.3 Biological Chemistry of Janus Heterocycles Even though at their conception Janus heterocycles were envisioned as wedges for the formation of a new kind of triplex with dsDNA, to date such a strategy has yet to be fully exploited. McLaughlin reported the oligomerization of the Lehn?s Janus wedge, and it was determined that the PNA octamer strand-invaded a duplex DNA containing eight CT mismatches (Figure 1.12). 25 It is noteworthy that this triplex is solely associated through Watson-Crick interactions and lacks any Hoogsteen hydrogen bonds. Indeed, biophysical experiments indicated a greater magnitude of stabilization than a conventional triple helix. Although this work was groundbreaking, the Janus wedge that was employed could only target mismatches, not canonical base pairs.   Figure 1.12: PNA octamer of the Janus wedge. Figure reproduced without permission. 25  Attempts towards binding ?matched? base pairs were commenced in Brodbelt?s lab with the synthesis of a ribosyl Janus GC base (Figure 1.13). 26 The implications of this heterocycle in the context of supramolecular chemistry are obvious but curiously the authors made no mention of the possibilities of RNA recognition. A phosphoramidite of  13 this heterocycle would have facilely permitted access to an oligomer that should form wedged triplexes with duplex poly(C)-poly(G).   Figure 1.13: Janus GC on a silyl ribose scaffold. Figure used without permission. 26  The Fenniri group also reported efforts towards ?-glycoside-functionalized Janus GC heterocycles. 27 Although a route towards a deoxyribose phorphoramidite of Janus GC was proposed, the authors did not report a synthesis of the amidite or of the oligomer. Instead, the target was intended for obtaining cyclic rosettes of pre-defined lengths. Later, He and coworkers published the synthesis of a Janus GC heterocycle on a ribosyl scaffold, which was screened by an in vitro replication inhibition assay. However, DNA hybridization properties were not examined in this work. 28  Reports pertaining to the scaffolding of Janus AT on biological supports are even sparser. Tor?s group reported the synthesis of a phosphoramidite of a self-complementary Janus AT congener that was incorporated into the central position of a 17mer and the hydrogen bonding between the Janus heterocycle and DNA bases was probed. It was reported that the stability of the hydrogen bond between the Janus base and either A or T exhibited  14 similar stability to that of canonical DNA base pairs. 29 Unfortunately, this work did not attempt to probe binding with dsDNA with tracts containing multiple Janus heterocycles.  1.4 Goals of This Thesis 1.4.1 Overall Goals To achieve the hallmark goal of sequence specific DNA recognition of canonical base pairs, we hypothesize that an oligomer of a Janus wedge can be employed. 17 Such an oligomer should overcome two of the greatest limitations of Hoogsteen-based TFOs (namely the inability to recognize pyrimidines, and high susceptibility to nucleases within the cell). Sequence-specific DNA recognition guided by wedging would proceed via the formation of stable Watson-Crick triplexes as outlined in Figure 1.14.   Figure 1.14: Two suitably oligomerized Janus synthons (green and blue) can recognize duplex DNA by formation of H-bonds (red) with canonical base pairs.  It might be argued that the formation of multimeric rosettes such as (Janus AT)6 (analogous to Figure 1.13) would compete with the formation of triplexes such as  15 (DNA)2-(Janus AT) as outlined in Figure 1.14 due to the enthalpic cost associated with formation of triplexes (the breakage and reformation of Watson-Crick bonds required for triplex formation is not needed in rosette formation). However, we propose that the seemingly lower enthalpic cost of rosette formation is in fact countered by the large price in entropy associated with what could be a sixth order process. In comparison, DNA triplex formation might proceed by only second order kinetics. To further encourage triplex formation, longer oligomers of the Janus wedge could be employed. However, such a synthesis might be troublesome due to low solubility of both Janus monomers and oligomers. Regardless, we propose to test this hypothesis by synthesizing an octamer of the Janus AT heterocycle.  1.4.2 Peptide Nucleic Acid Scaffold For this dissertation, the Janus AT heterocycle was scaffolded upon a peptide nucleic acid backbone, in order to discourage nuclease degradation. PNAs, designed in 1991 as synthetic triplex forming oligonucleotides in P.E. Nielsen?s laboratory, are analogs of DNA and their structure is derived by replacing the DNA phosphate backbone with acyclic 2-aminoethylglycine units. The nucleobases are attached to this 2-aminoethylglycine backbone via methylene carbonyl linkers (Figure 1.15). Due to structural dissimilarities with DNA, peptide nucleic acids are nuclease and protease resistant in cells, and this partly prompted us to choose this scaffold for our oligomerization. 30   16  Figure 1.15: Peptide nucleic acids (top) are synthetic analogs of DNA (bottom).  The discovery of PNAs was guided by the desire to obtain a water-soluble and electronically neutral mimic of single stranded DNA, and the chosen structure was found by computer screening to be homomorphous to the DNA backbone. Despite the apparently drastic deviation from deoxyribonucleotides, a PNA oligomer strictly obeys Watson-Crick hydrogen bonding rules. Moreover, it was found that PNA oligomers possess a greater specificity and stronger binding affinity for ssDNA in both ?antiparallel? and ?parallel? fashions, where the 5? is defined as equivalent to the N-atom of DNA. This is attributed to the lack of electrostatic repulsion between the strands, and therefore a DNA/PNA mismatch is more destabilizing than an analogous DNA/DNA mismatch. Also due to the lack of electrostatic repulsions, the binding strength of PNA duplexes and triplexes are not affected as much by the composition of the solution. 30  A PNA oligomer is iteratively synthesized by solid phase peptide synthetic protocols using either Boc or Fmoc chemistry (as an example, Boc chemistry is shown in Figure 1.16). The polystyrene 4-methyl benzhydrylamine resin is utilized due to its facile cleavage conditions, as well as its TFA stability as compared to other solid supports. 30 OOPOOOPOOOPOTGCOOO-O-O-ONOAHNONOHNOCNOGOHNNOH2NOTOCHONH2OH 17   Figure 1.16: The solid phase synthesis of peptide nucleic acids. Shaded circle represents the PS-MBHA resin. X = nucleobase (A, T, C, G or modified heterocycle)  The loading of the commercially obtained PS-MBHA resin must be reduced (this action is termed ?downloading?) prior to solid phase synthesis of the PNA. This is accomplished by coupling it with a controlled amount of the Fmoc-AEEA-OH spacer, followed by capping (Figure 1.17). This spacer is commonly employed for PNA synthesis because of its ideal length as well as its high aqueous solubility due to the incorporated ethylene NHONNHOXOtBuOHN ONNH3+OXCF3COO-HNONHN OXONOXNHOtBuO1. Deprotect Boc 2. Neutralization 3. Couple next monomer 4. Capping Cleavage from Resin PNA Oligomer  NNHOtBuOHOOOX 18 glycol unit. The downloading of the resin prevents interchain aggregation as well as entangling for longer sequences, which would lower the yield of the synthesized PNA.     Figure 1.17: Downloading of the PS-MBHA resin.  With the downloaded resin in hand, any conjured PNA sequence can be synthesized. First, the resin-terminal protecting group is deprotected (usually with TFA in DCM) and the resulting ammonium salt is neutralized. The resulting free base is coupled with an excess of the desired PNA monomer in a polar aprotic solvent such as NMP or DMF. Once the coupling is deemed complete by the Kaiser test, the unreacted amines are capped with an acetyl group to prevent truncates during future couplings. The sequence is then cleaved from the solid phase and purified by HPLC or gel electrophoresis, and can then be used for biophysical or cell studies. HNNHFmocNHOOO OOHNOO?MBHA Resin Resin with AEEA linker  19 Chapter 2 ? Results and Discussion The present chapter is divided into five sections. The synthesis of some key precursors and several PNA backbones is described in Section 2.1 and the synthesis towards diamino Janus AT (1) is described in Section 2.2. It was determined that 1 was inadequately soluble in most solvents, which impeded its purification. To ameliorate this, we endeavored on the synthesis of 2 (Section 2.3) by two pathways, but failures in late stage oxidations precluded the formation of the desired heterocycle. Regardless, it was determined from molecular modeling calculations that 2 possessed an undesirably high pKa, which would render it uncooperative in biophysical experiments. Thus, the synthesis of redesigned Janus AT 3 (Section 2.4) was undertaken, whereupon it was found that the penultimate ring-closure step was unsuccessful upon scale up. Finally, monomer 4 (Section 2.5) was successfully synthesized and employed for the synthesis of an undecamer that incorporated four Janus residues.  Due to poor Janus-to-Janus couplings encountered during the solid phase synthesis of the aforementioned undecamer of Janus AT 4, 6-methyluracil was incorporated in the sequence as a spacer. This thymine analog had been only briefly alluded to in the literature and, therefore, a foray into the biophysical properties of 6-methyluracil (5; Section 2.6) was undertaken, for which a homo(dodecamer) was synthesized (Figure 2.1).     20    Figure 2.1: Structures of Janus PNA monomers (1, 2, 3, and 4) and 6-methyluracil PNA monomer (5); R = protecting group (details in the relevant sections).  2.1 Syntheses of PNA Backbones and Glycine Precursor The solid phase synthesis of peptide nucleic acids employs monomers bearing the Boc or Fmoc protecting group. These monomers are crafted via solution phase coupling between the secondary amine of a ?PNA backbone? and a nucleobase precursor possessing a carboxymethyl tail (Figure 2.2). 31   Figure 2.2: Synthesis of a PNA monomer in solution. R = H or alkyl; R? = Fmoc or Boc HNN NNNH2NH2OOONNHRHOOHNNNNH2SOOONNHRHOOHNNOOONNHRHOOPhOOA ATTHNN NNClClOOONNHRHOOHNN NNNH2SOOONNHRHOOPhAAT THNNHR'ORONNHR'OROONucleobaseO OHNucleobaseCoupling Agent1 2 5 3 4  21 Because it was initially unknown whether Fmoc- or Boc-based chemistry would be ultimately employed, several PNA backbones were synthesized, viz. methyl/Boc (6), propyl/Boc (7), carboxylate/Boc (8) and t-Butyl/Fmoc (9)  (Figure 2.3). Generally, Fmoc chemistry is promoted in the literature since it lends itself well to automation and mandates shorter reaction times, but Boc monomers afford cleaner reactions and better coupling yields during manual synthesis. 32   Figure 2.3: PNA backbones that were synthesized ? N-Boc protected 6, 7, and 8 and N-Fmoc protected 9  Acetic acid was ?-brominated in a Hell-Volhard-Zelinskii reaction, furnishing bromoacetic acid 10. Seeking to alkylate at the ?-position of the carbonyl group, the incubation of 10 with ethylene diamine was attempted, but contrary to several reports in the literature, no reaction was observed. 33-35 An alternative route was sought wherein the acidity of the carboxylic acid was mitigated and, thus, bromoacetic acid was first converted to the methyl (11) and propyl (12) ester analogs via Fischer esterification (Scheme 2.1). The purpose for synthesizing these esters and the two PNA backbones derived therefrom will be elucidated in detail Section 2.5.1. OHNNHOO OOHNNHOO OLiOHNNHOO OOHNNHOO O6 7 8 9  22  Scheme 2.1: Synthesis of esters 11 and 12. A) PBr3, Br2 then water (95%) B) H2SO4, excess ROH (average 71%)  Ethylene diamine was protected with one equivalent of Boc anhydride in high dilution to furnish 13 as a pasty oil. This intermediate was then alkylated with either ester 11 or 12 to afford the desired PNA backbones 6 and 7 after chromatographic purification. 36 A small amount of 6 was saponified in aqueous methanol and stored as the more stable lithium carboxylate salt 8 (Scheme 2.2).   Scheme 2.2: Synthesis of Boc backbones 6, 7 and 8. A) Boc2O, DCM, 18hrs B) DIPEA, 11/12, DCM (average 64%) C) LiOH (1.1 equiv.), H2O: MeOH (quantitative yield)  In addition to amines 6, 7 and 8, the synthesis of the Fmoc-protected 9 was also undertaken (Scheme 2.3). 37 For this, excess ethylene diamine was alkylated with bromo OHOOHOBrOOBrOOBrA)B)NH2H2NNo ReactionH2NNH2H2NHNOONHHNOOOROBrOROB)A)NHHN OOOLiOC)13 11/12 In 6/11: R = Me In 7/12: R = Pr 8 10 11 12 6/7  23 tert-butylacetate and the resulting intermediate 14 was acylated with Fmoc-NHS. The product 9 was precipitated as the HCl salt, and it was found to be stable for at least one year upon storage at -20?C, unlike 6 and 7, which possess more limited half lives.     Scheme 2.3: Scheme for the synthesis of the Fmoc backbone 9. A) DCM, room temperature, overnight (uncalc. yield) B) DCM, DIPEA, Fmoc-OSu, overnight (27%)  The precursor to PNA monomers possesses a carboxymethyl tail (Figure 2.2), which is introduced into the heterocycle via tert-butylglycine. The glycinate was synthesized in one step by the reaction of tert-butyl bromoacetate with liquid ammonia, after which vacuum distillation was performed to obtain pure 15 as an oil (Scheme 2.4). This intermediate was utilized for the synthesis of heterocycles 1, 3, and 4.   Scheme 2.4: The synthesis of tert-butylglycine 15. A) NH3 (g), Et2O, -78?C, overnight   2.2 Synthesis of Diamino Janus AT Because a similar, high-yielding synthesis had been previously reported in our group, the NH2H2NBrOOtBu+A)NHH2NHNNHOOB)OtBuOOtBuOOA)OBrOOH2N14 9 15  24 diamino Janus AT 1 was initially identified for synthesis (Figure 2.4). 38   Figure 2.4: The initial target is the diamino Janus AT on an Fmoc PNA backbone.  We commenced the synthesis from the thymine face, for which urethane was first condensed with 2-cyanoacetic acid in the presence of phosphorus oxychloride to provide intermediate 16. This material was dissolved in DMF and subsequently treated with potassium carbonate followed by carbon disulfide to garner potassium salt 17. This was dissolved in water and acetonitrile and treated with methyl iodide, which upon reflux supplied 18 as a yellow solid (Scheme 2.5).    Scheme 2.5: The synthesis of intermediate 18. A) POCl3, N,N-DMF, toluene, reflux (76%) B) K2CO3, CS2, DMF, room temperature (94%) C) MeI, MeCN:H2O, reflux (78%)  The ring closure of 18 with tert-butylglycine acetate was unsuccessful with both triethylamine and potassium carbonate as bases, leading to undesired hydrolysis of the thiomethyl group upon workup. Instead, it was found that the reaction of 18 with the free NONHNOONNNH2NH2OtBuO NHOOEtO NHO OCNSSEtOONHONEtOONHONKS SKB) C)EtOONH2HOONA)1 18 17 16  25 amine of 15 in ethanol led to the desired product 19 in good yield. Analogous to the heptyl analog reported earlier, purified 19 was subjected to free guanidine base in ethanol, which precipitated the Janus heterocycle 20 as a white solid (Scheme 2.6). 38    Scheme 2.6: Synthesis of diamino Janus AT 20. A) EtOH, reflux (74%) B) NaOEt (1.1 equiv.), guanidine hydrochloride (1.1 equiv.), 19 (1.0 equiv.), EtOH, reflux (25%)  Unfortunately, intermediate 20 was found to possess low solubility in most solvents including acetone, EtOAc, sulfolane, ethylene carbonate, DMSO, dimethylacetamide, DMF and NMP, of which the latter two are commonly employed for PNA solid phase synthesis. This result was perhaps expected because compound 20 possesses the ability for self-association via formation of numerous hydrogen bonds. In striving to improve the solubility, the masking of the exocyclic amines as phthalimide groups was attempted. However, it was found that compound 20 was refractory to acylation despite employing several bases or heating.  Unsuccessful at masking the exocyclic amines, we set out to elaborate the Janus heterocycle into PNA monomer (1 or 1?) anyway, hoping that the introduction of increased organic character might impart favorable solubility properties. For this, the ester in 20 was converted to carboxylic acid 21 by reaction with TFA. The acid was OA)OH2NOONHNOOCNSEtO NHO OCNSSOONHNOO NNH2N NH2B)+19 18 15 20  26 activated with EDC in DMSO (the reaction failed in DMF) and subsequently coupled with 7 or 9. A new TLC spot formed, but it could not unfortunately be purified by silica chromatography, C18 SepPak chromatography, filtration or recrystallization (Scheme 2.7). Endeavoring to improve the solubility of the Janus heterocycle, we decided to embark upon an alternative synthesis.    Scheme 2.7: Purification of 1 was confounded due to low solubility. 1: R = tBu; R? = Fmoc. 1?: R = Me; R? = Boc A) TFA/DCM, 5 hours, rt B) 21, EDC-HCl, DMSO then 7 or 9, overnight at rt  2.3 Synthesis Towards Janus AT 2 In view of the poor solubility of diamino Janus AT (1; Section 2.2), a second target was identified to conceal the exocyclic amines (2; Figure 2.1), as the synthesis of a similar heterocycle had been previously reported. 38 The synthesis relied upon the condensation of an amine with carbamate 22, followed by ring closure and ammonolysis. The resulting intermediate could, after several steps, be converted to a monomer amenable for solid phase synthesis (Scheme 2.8). Ammonolysis after solid phase synthesis could nucleophilically cleave the sulfone, thereby revealing the fully elaborated diaminopyridine (adenine) face of the Janus heterocycle.  OONHNOO NNH2N NH2B)A)OHONHNOO NNH2N NH2NONHNOO NNH2N NH2NHR'OORNHFmocHNtBuOONHBocHNMeOO20 21 1 or 1? 7 or 9  27   Scheme 2.8: Synthesis of 2; R denotes a handle that can be unmasked into COOH; R? denotes Fmoc or Boc.  Intermediate 22 was synthesized in one step from 16 (synthesis previously disclosed) by treatment with triethylorthoacetate and acetic anhydride, which produced 22 as a pale white solid (Scheme 2.9).   Scheme 2.9: The synthesis of 22. A) POCl3, DMF, toluene (76%) B) triethyl orthoacetate, Ac2O, reflux (62%).  Initial attempts (not delineated here) involved the reaction of tert-butylglycine 15 with carbamate 22 but were unsuccessful, a result that we attributed to undesired over-reactivity of the t-butyl group (for instance, the acidic ?-position might be deprotonated under the basic conditions employed during the reaction carbon disulfide, Scheme 2.8). HNNNNH2SOOONNHR'HOOPhOOEtO NHO OCNOHNNCNOOHNNOO SSNH2HNNOO SHNNH2HNNOO SNNH2PhR-NH2CS2NH4OHBnBrNaOH[O]CoupleR RR RO NH2OHOOCNEtO NHO ONEtO NHO ONOEtA)B)22 2 22 16  28 Thus, we opted to use a less reactive amine than 15 that could be oxidized to the carboxylic acid late-stage. To this end, schemes with allylamine (Section 2.3.1) or ethanolamine (Section 2.3.2) were envisaged, wherein the alcoholic or olefinic handle would be oxidized or ozonolyzed in the final step, respectively.  2.3.1 Janus AT 2 via Allylamine The reaction of carbamate 22 and allylamine produced intermediate 23. Gratifyingly, this intermediate proceeded to 24 upon treatment with CS2 and KOtBu. The product was heated in excess ammonia, which caused the displacement of the intracyclic sulfur atom and furnished 25. The adenine face was then rendered aromatic by treatment with benzyl bromide under basic conditions to form 26 as a yellow solid (Scheme 2.10).     Scheme 2.10: Synthesis of 26. A) Allylamine, EtOH, Et3N, reflux (94%) B) THF, CS2 (1 equiv.), KOtBu (33%) C) NH4OH (excess), 100?C D) NaOH, BnBr, MeOH:H2O (42%)  EtO NHO ONOEtHNNCNOOHNNOOSSNH2HNNOONSHNH2HNNOONSNH2A)B)D)C)22 23 24 25 26  29 Unfortunately, the ozonolysis of olefin 26 was not successful. Upon treatment of the starting material with O3 in aqueous H2O2, the major product from the complex TLC of the reaction was putatively assigned to a structure wherein both the pyridine and the thioether underwent oxidations to the N-oxide and sulfone, respectively (Figure 2.5). Oxidation of the thioether was welcomed, since we had planned to perform this reaction later on anyway (Scheme 2.8, compound 2). The N-oxidation was not too surprising either given the strongly oxidizing conditions that heterocycle 26 was subjected to during ozonolysis. For instance, the oxidation of aminopyridine to the corresponding N-oxide can be affected with oxidants as mild as m-chloroperoxybenzoic acid. 39 In light of this failure, we focused on a different approach to the desired product, as outlined in the following section.   Figure 2.5: Based on this mass spectrum from the ozonolysis of 26, the base peak of m/z 429.4 was putatively assigned to the [M+Na]+ adduct of the boxed structure.  2.3.2 Janus AT via Ethanolamine For the installation of an ethanolamine side chain onto the Janus heterocycle, the alcohol must first be protected to prevent side reactions. This was accomplished with tert-butyldimethylsilyl chloride and the resulting intermediate (27) was reacted with HNNN+OOSNH2PhOHOO-OOHNNN+OOSNH2PhO-OO[O] 30 previously synthesized 22 (see Section 2.3.1). With the thymine face formed, 22 was transformed into 30 over two steps by reacting with carbon disulfide to form 29. The reaction of this intermediate with ammonia furnished the desired 30, with concomitant removal of the TBDMS group. 40  Next, the adenine face of the heterocycle was rendered aromatic by alkylating with benzyl bromide thereby generating 31 (Scheme 2.11).    Scheme 2.11: Synthesis of 31. A) TBDMSCl, imidazole, DMF (81%) B) 27, Et3N, EtOH, reflux (63%) C) THF, CS2 (1 equiv.), KOtBu (49%) D) NH4OH (excess), 100?C, pressure tube (yield uncalculated) E) NaOH, BnBr, MeOH:H2O (30%)  EtO NHO ONOEtHNNCNOOOTBDMSHNNOOOTBDMSSSNH2HNNOOOHNSHNH2HNNOOOHNSNH2B)C)D)E)OHH2NOH2NSiA)31 22 27 28 29 30  31 Two conditions for oxidation were identified for the conversion of 31 to aldehyde 31? ? pyridinium chlorochromate and Swern. We deliberately investigated weaker oxidizing agents to prevent an over-oxidation similar to the one encountered during the ozonolysis of 26 (Section 2.3.1; Figure 2.5). With aldehyde 31? (Scheme 2.12) in hand, we could screen for oxidants to convert it to the carboxylic acid. A similar two-step oxidation strategy has been previously employed to shield the nucleobases of fully elaborated PNAs from overly oxidizing conditions. 41 In accord with this logic, both oxidizing agents were first validated by a successful test-oxidation of benzyl alcohol. However, attempts to oxidize 31 were unsuccessful under either condition. Upon incubation of the starting material the oxidizing agents, neither the NMR nor the infrared spectra of the newly formed spot indicated the presence of an aldehyde or its hydrate (Scheme 2.12).     Scheme 2.12: Failed attempts at oxidation of the pendant alcohol of 31.  Unable to affect this transformation, we decided to circumvent the poor oxidative inclination of 31 by screening for stronger oxidizing agents. However, it was discovered at this time in our lab through theoretical chemistry 40 that the adenine face of diamino-2 possessed a pKa of 6.79, which would induce protonation of an appreciable amount of the heterocycle at the neutral pH employed for biophysical experiments. This protonation HNNOOOHNSNH2HNNOOONSNH2HPCCor(COCl)2DMSO31 31?  32 would preclude the formation of the DAD H-bonding pattern required for the Janus heterocycle to effectively hydrogen bond with thymine (Figure 2.6). Because this protonation is inherent to 2, this synthetic scheme was not further examined.   Figure 2.6: Protonation disrupts the H-bonding of the adenine face. R = carbocycle. 40  2.4 Synthesis of Janus AT 3 We sought to synthesize a pyrimidine-derived Janus heterocycle, which, due to the presence of the additional nitrogen, should suppress the pKa of the adenine face by increasing electron deficiency. To incorporate a pyrimidine heterocycle and concurrently mask the exocyclic amines for solubility reasons, we proposed 3 as a target (Scheme 2.13; forays into masking the exocyclic amine functionality as 2-octylthiopyridine and 2-benzylthiopyridine analogs were both unsuccessful due to low yields. Masking as a phthalimide was attempted, but the molecule unfortunately possessed undesirable over-reactivity (phthalimide was too electrophilic). As described previously, the exocyclic amines of 1 were refractory to acylation with phthaloyl dichloride).  The general strategy is outlined in Scheme 2.13 and commences with the carboxylation of trichloropyrimidine. The amide, derived from the carboxylic acid, would be subjected to nucleophilic substitution with a glycine derivative to elaborate the adenine face. Ring HNNOORNNH2NH2HNNOORN+NH2NH2HpH 7 33 closure would complete the thymine face and coupling with the PNA backbone would afford a PNA monomer. After completion of solid phase synthesis, the two exocyclic chlorine atoms could be nucleophilically removed via SNAr substitution with methanolic ammonia to yield the desired diamino Janus AT moiety. 42    Scheme 2.13: Synthesis of dichloro Janus AT. X = the leaving group.  To obtain barbituric acid, first diethyl malonate was condensed with commerically available urea, which resulted in the formation of compound 32 (the scheme is shown on the following page) as a white solid. The barbituric acid was then chlorinated with refluxing POCl3 under basic conditions to supply 33 after a column chromatographic purification. The yellow oil was then deprotonated with freshly-generated lithium diisopropylamide in anhydrous THF, followed by a quench with gaseous dry carbon dioxide, which garnered intermediate 34 as a white solid (Scheme 2.14).   N NClCl ClN NClCl ClNH2ON NClCl NHNH2ON NClCl NNHO OLDACO2(COCl2)NH4OHOtBuOH2NOX XN NClCl ClOHOO OtBu O OtBu39 33 34 36 15 37  34   Scheme 2.14: Synthesis of carboxylic acid 34. A) NaOEt, EtOH, reflux, 8 hours (67%) B) POCl3, Et3N, reflux (64%) C) LDA, CO2(g), THF, -78?C (65%) D) N,N-DMF, POCl3, dimethylaniline, reflux (55%) E) Oxone, N,N-DMF, rt (not purified)  The identity of the 34 was doubly confirmed via another pathway. Barbituric acid 32 was first subjected to Vilsmeier-Haack conditions to provide aldehyde 35, which proceeded to a carboxylic acid upon treatment with Oxone? in DMF, as evidenced by TLC staining with bromocresol green. Upon comparison of this compound with the carboxylic acid generated from 33, it was found that both materials were identical!  Next, 34 was converted to the acyl chloride in situ, which was quickly quenched with concentrated NH4OH (yield decreases upon increased incubation time due to aromatic substitutions), affording 36 after purification. Finally, the SNAr substitution of amide 36 with tert-butylglycine 15 was undertaken, and supplied 37 in high yields. With 37 in hand, reactions were performed to screen the most favorable conditions for ring closure (Scheme 2.15). N NClCl ClN NClCl ClOHOC)H2N NH2OEtO OEtO ON NOHHO OHA)B)N NClCl ClE)D)O H34 35 32 33  35   Scheme 2.15: Synthesis of intermediate 37. A) Oxalyl chloride, N,N-DMF, DCM then conc. NH4OH (66%) B) Et3N, 15.HOAc, DCM (93%)  It was discovered that the incubation of 37 with triphosgene at room temperature led exclusively to nitrile 38, a result that was rationalized in several ways (Scheme 2.16 and Scheme 2.18 Path A). First, nitrile formation (37 to 38) involves favorable sterics as compared to ring closure (37 to 39), which demands a planar geometry. Secondly, the mechanism for nitrile formation expels a molecule of CO2 from triphosgene, thereby propelling the reaction on entropic and enthalpic grounds, while concurrently extending the conjugation of the ? system present within the heterocycle. Fortunately, it was found that deprotonation of 37 with 60% NaH followed by the addition of methyl chloroformate formed 39, which could be purified by preparatory TLC.     Scheme 2.16: Intermediate 37 can produce nitrile 38 or desired material 39.  N NClCl ClOHON NClCl ClNH2ON NClCl NHNH2OOtBuOA) B)N NClCl NNHON NClCl NHNH2ON NClCl NHTriphosgeneOtBuONOtBuOO OtBuO60% NaHMeOCOCl34 36 37 38 39 37  36 Next, the tert-butyl ester of 39 was cleaved with 50% TFA to furnish 40, which was subjected to EDC-mediated coupling with Fmoc-PNA-backbone 9. Preparatory TLC purification yielded a few milligrams of 3 (Scheme 2.17). The small-scale synthesis precluded structural proof in the form of NMRs; instead, the reactions were characterized by ESI-MS and UV-vis spectroscopy.    Scheme 2.17: Synthesis of PNA monomer 3 was successful on the small scale.  Armed with a successful small-scale synthesis of PNA monomer 3, the reaction scheme delineated above was scaled up to 1.5 mmol. At this scale, however, a side reaction was detected which served to greatly diminish the yield of 3. In the reaction conditions employed (p-dioxane, NaH, methyl chloroformate), one of the exocyclic chlorine atoms was found to be nucleophilically cleaved by the methoxide anion (X-) produced from ring closure to convert the product 39 to 39? (Scheme 2.18, Path B). Worse, the side product coincidentally co-eluted with the desired product on silica, which impeded the large-scale purification of the product. Although the exact regioselectivity of the SNAr reaction (i.e. 39 to 39?) was not probed, there is literature precedence to suggest that cleavage of the 2-position predominates rather than the 4-position. 43  N NClCl NNHOO OtBuON NClCl NNHOO OHON NClCl NNHOO NONHFmocOOtBuTFANHNHFmocOtBuO39 40 3 9  37 At this point the possibility of the ammonolysis of one exocyclic chlorine atom of 37 (conversion of 37 to 37?; Path C in Scheme 2.18) was considered, which may have avoided the side reaction encountered in Path B by altering the electronics of the heterocycle. However, we could not predict the solubility properties of 37? or the likelihood of 37? to undergo a similar SNAr nucleophilic substitution as 37. Furthermore, under ammonolysis conditions, 37? could de-methylate rather than de-methoxylate. In fact, a reaction involving the incubation of 37 with ammonia led to the formation of 37? as well as diamino 37 as determined by MS and thus this route was not further explored.   Scheme 2.18: Putative mechanisms of the side reactions during ring closure. X refers to leaving group on a carbonylation reagent. N NClCl NHNH2OO OtBuXXON NClCl NHNHOO OtBuXON NClCl NNHOO OtBuOX-XXON NClCl NHNOO OtBuXO:BHN NXCl NNHOO OtBuON NClCl NHCO OtBuNN NNH2Cl NHNH2OO OtBuN NNH2Cl NNHOO OtBuOX XO1 eq. NH339? 39 38 Path A Path B Path C 37? 37  38 Instead, we chose to screen conditions to cleanly effect the desired transformation from 37 to 39. Clearly, the N-anion rather than the O-anion is required for successful ring closure and, therefore, several bases were screened. The chosen base must be strong enough to deprotonate the amide and also needs to be non-nucleophilic to prevent decomposition of the carbonylation reagent. It was found that 60% sodium hydride produced the highest yields of the product as outlined in Table 2.1.  Entry Base pKa Result A None Not applicable 38 B K2CO3 10 38 C 60% NaH 35 39 + 39? D 60% NaH with tBuOH 19 or 35 Intractable mass E 60% NaH (excess) 35 Intractable mass F 95% NaH 35 39 + 39? G LDA 36 Intractable mass H KOtBu 19 Intractable mass I KHMDS 26 Intractable mass Table 2.1: Bases screened for the deprotonation of amide 37, with methyl chloroformate as carbonylation reagent; all reactions performed in p-dioxane.  N NClCl NHNH2OO OtBuN NClCl NHNHOO OtBuXON NClCl NNHOO OtBuO1. Base2. X2CON NClCl NHNH-OO OtBuOXX 39 Unable to suppress the formation of 39? with choice of base, we attempted to change solvents (among THF, DMF and dioxane, the latter was optimal), concentration (0.125M), reaction time (8 hours is optimal), and temperature (below 0?C no reaction occurs and at temperatures greater than 20?C the yield is not improved). All of these reactions were performed with NaH and methyl chloroformate, and unfortunately none of these conditions was successful in preventing the formation of 39?. Finally, a host of carbonylation agents were screened (Table 2.2).    Entry Carbonylation agent pKa of X Result X A 2-chloroethyl chloroformate 14.3 44 39 + 39?  B Methyl chloroformate 15.5 39 + 39?  C Phenyl chloroformate 10.0 39 + 39?  D Carbonyl diimidazole 7.1 39 + 39?  E Triphosgene -7.0 (HCl) 38 (trace 39) - F Phosgene in toluene -7.0 (HCl) 38 (trace 39) - G Boc anhydride 18.0 (tBuOH) N-Boc - N NXCl NNHOO OtBuON NClCl NNHOO OtBuON NNH2Cl NHNH2OO OtBuXXOClOOONN37 39 39?  40 Adduct H Disuccinimidyl dicarbonate 6.0 (OSu) 45 Intractable mass - I Chlorocarbonyl isocyanate 3.7 46 39 + 39?  J Phenyl isocyanate 4.9 39 + 39?  Table 2.2: Carbonylation agent screening; 60% NaH was used in each case. Desired product = 39, nitrile = 38, and 39? is desired product with cleavage of exocyclic Cl.  Somewhat unexpectedly, it was found that the phenoxide anion generated from phenyl chloroformate effectuated an SNAr displacement (Entry C, Table 2.2), and even more surprisingly imidazole-adducts from carbonyl diimidazole were observed (Entry D). On the other hand, phosgene and triphosgene exclusively formed nitrile 38 (Entries E and F), which has been rationalized previously. When Boc anhydride was employed, the N-Boc intermediate was observed (Entry G) that failed to ring-close probably on electronic grounds (pKa of tBuOH is 18). The use of chlorocarbonyl isocyanate (Entry I) was attempted, whose conjugate acid has a boiling point of 23.5?C and a pKa of 3.7. 46 However, isocyanate substitution adducts were detectable under these conditions, which is not too surprising given the basic conditions employed. The failure met in obtaining a clean scaled-up ring closure of 38 prompted us to abandon this scheme.  2.5 Synthesis of Janus AT 4 and its Solid Phase Synthesis Having met with failure in masking the exocyclic amines as chlorides, we decided to embark on the synthesis of Janus heterocycle 47, the synthesis of which was reported NC OHN 41 recently in our group. Within this heterocycle, one exocyclic amine is masked as a sulfone. After incorporation of the monomer 47 into an oligomer, ammonolysis would furnish the desired diamino Janus AT motif (Scheme 2.19).    Scheme 2.19: Synthesis of 47. R = tBu or Pr or Me; R? = Fmoc or Boc (see text).  2.5.1 Synthesis of the Monomers 4 and 47 For the synthesis of monomers 4 and 47, we commenced from intermediate 19 whose synthesis was previously described (Section 2.2). To elaborate the adenine-face of 19, however, urea 41 is required. Thus, the reaction of thiourea and benzyl bromide was performed under acidic conditions to afford benzylthiourea as a white solid in high yield (Scheme 2.20).40 Next, 19 was reacted with 41 under basic conditions in refluxing ethanol to generate 42 in moderate yield. Finally, the TFA-mediated deprotection of the tert-butyl ester was undertaken, leading to carboxylic acid 43 (Scheme 2.20).  HNN SMeCNOOOOtBuHNN NOOOOtBuNSNH2HNN NOOONNSBnNH2NHR'HNN NOOONNSNH2NHR'OOHNSNH2Et3N, EtOH1. TFA2. Couple1. [O]2. TFAPhPhOHOORO 42   Scheme 2.20: Synthesis of 43 and 41. A) Thiourea, HCl, EtOH then BnBr, MeOH (90%) B) 15, EtOH, 100?C, 20 hrs (74%) C) EtOH, 41, Et3N (30%) D) TFA/DCM, 5 hours, (quant.)  With 43 in hand, solution phase coupling with carboxylate backbone 8 was attempted. It was found that in addition to the expected product 4, a significant amount of double coupling was observed that manifested as 44. To improve the yield, the coupling of 43 with methyl ester backbone 6 was carried out, which averted the formation of the double-coupled side product, as expected. However, yet another side reaction prevailed that yielded an uncharacterized impurity that coincidentally co-eluted with the desired product 45. Therefore, the propyl PNA backbone (7), which possessed a slightly higher Rf, was used for coupling, and provided 46 in good yield after purification (Scheme 2.21). O NHO OS SNHNN SCNOOOOtBuHNN NOOOOtBuNSNH2PhHNN NOOOOHNSNH2PhB)C)D)H2NSNH2A)-Cl+H2NSNH241 18 19 43 42  43   Scheme 2.21: Synthesis of amide 46. A) EDC, HOBt DIPEA, DMF (81% for 46) B) EDC, HOBt, DIPEA, DMF Or pivaloyl chloride, N-ethylmorpholine  Ester 46 was saponified with sodium hydroxide in 1:1 propanol and water, and the product was isolated as carboxylic acid 4 after extraction (Scheme 2.22). The UV-vis spectrum of 4 in DMSO is depicted in Figure 2.7, and compares well with that of the phosphoramidite precursor reported previously, with which 4 shares a ?-system. 40    Figure 2.7: The UV-vis spectra of carbocyclic Janus AT and 4. Extinction coefficient not calculated.  HNN NOOOOHNSNH2PhHNN NOOONNSNH2NHNN NOOONNSNH2NHBocOOROHNO NHBocRPhB)A)OOLiO NHBocPhBocHNLiOHNO NHBoc0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 250 350 450 550 650 750 Absorbance (arbitrary units) Wavelength (nm) UV-vis Spectrum of 4 and Carbocyclic  Janus AT Phosphoramidite Precursor PNA Monomer 4 Phosphoramidite Precursor !44 43 8 6: R = Me 7: R = Pr 45: R = Me 46: R = Pr HNN NNOO SNH2PhONHOONHBocHNN NNOO SNH2PhHOHO 44 Oxidation of a small portion of 4 was carried out in the presence of excess mCPBA to produce sulfone 47 after workup and chromatography (Scheme 2.22). Interestingly, this oxidation was unsuccessful with H2O2 and formic acid, one of the literature-reported methods, which we rationalize based on the increased electronegativity present in 4. 38 Regardless, the purification of 47 was not facile due to pervasive streaking on TLC (which is expected because of the free carboxylic acid) and, therefore, the oxidation reaction was not scaled up.    Scheme 2.22. Final steps towards synthesis of PNA monomers 4 and 47; A) NaOH, H2O/n-propanol (87%) B) MeOH, mCPBA  2.5.2 Stability Tests on Monomers With both sulfone 47 and thioether 4 in hand, solubility and chemical stability tests were conducted on these monomers. Gratifyingly, both monomers demonstrated appreciable solubility (at least 0.1 M) in DMF as well as NMP. This high solubility resolved the problem encountered with diamino Janus AT 1 (Section 2.2), which could not be purified because of its low solubility. Tests for chemical stability were undertaken at conditions chosen to mimic those encountered during solid phase PNA synthesis, at a concentration of 0.05 mol/L (Table 2.3). From these results, it is clear that with respect to thioether 4 there is great freedom in the choice for solid phase conditions. However, sulfone 47 was unstable in all bases (Table 2.3 Entries C to F) as well as the capping solution (Table 2.3, HNN NOOONNSNH2NHBocOOHNN NOOONNSNH2NHBocHOOA)HNN NOOONNSNH2NHBocHOOPhPh PhB)OO46 4 47  45 Entries G and H). It was found by Mr. Wenbo Liu that this pyridine-mediated decomposition occurred concomitantly with the formation of a Zincke salt, wherein the base displaces the sulfone moiety. This problem is acceptable in the case of a phosphoramidite, because DNA oligonucleotides are synthesized quicker than their PNA counterparts; PNA couplings are 30 minutes each, vs 2 minutes for DNA couplings.      4  47 Entry Condition Reason Estimated t? of 3 Estimated t? of 47 A DMF Solid phase solvent Indefinite Indefinite B NMP Solid phase solvent Indefinite Indefinite C NEM Coupling base Indefinite 20 minutes D DIPEA Coupling base Indefinite 1 hour E DBU Coupling base Indefinite 1 hour F Et3N Coupling base Indefinite 1.5 hours G Pyridine Capping solution Indefinite 2 hours H Lutidine Capping solution Indefinite 3 hours Table 2.3: Results from chemical stability tests for 4 and 47. Abbreviations: NEM = N-ethylmorpholine, DBU = Diazabicyclo[5.4.0]undecane, Et3N = triethylamine  HNN NOOONNSNH2NHBocHOOPh HNN NOOONNSNH2NHBocHOOPhOO 46 It is worth noting that the early Janus residues of a PNA sequence would be exposed to adverse conditions repeatedly. For example, the first residue in a homo-octamer of 47 would cumulatively face 16 hours of total exposure to triethylamine (during the coupling of residues 1 through 8) and 14 hours to pyridine (during capping of residue 1 to 7). In light of the data from Table 2.3, these numbers implicate that the first Janus residues would be appreciably degraded towards the end of the solid phase synthesis.  2.5.3 Solid Phase Oligomerization of Janus AT Commercially purchased PS-MBHA resin was downloaded to a final loading of 0.20 mmol/gram. Next, the Fmoc residue was deprotected and coupling was performed iteratively with HBTU in 0.05M NMP with excess DIPEA and 5 equivalents of the monomer for 120 minutes. 31, 32, 47 After the addition of each monomer to the solid phase, the Kaiser test was performed and the unreacted functional groups were capped. Once the synthesis was complete the sequence was cleaved from the resin using standard TFMSA/TFA conditions. 31  In order to avoid exposing sulfone 47 to pyridine (or lutidine) to which it appeared to be unstable, we chose to synthesize Rapoport?s reagent for capping the resin. 47 First, methyl sulfonate 48 was synthesized from sulfonic anhydride via methanolysis. Meanwhile, imidazole was treated with benzyl chloroformate, yielding intermediate 49 after purification, which was subsequently treated with aforementioned sulfonate 48 to precipitate 50 as a white solid (Scheme 2.23).  47  Scheme 2.23. Synthesis of capping reagent 50; A) 0?C, MeOH B) Imidazole, toluene, benzyl chloroformate, 12 hours (89%) C) 48, DCM, 0?C (69%)  Unfortunately, false positive Kaiser tests were too readily obtained on test sequences with this reagent, possibly due to removal of the Cbz group upon extended heating of the beads. Therefore, the capping solution was switched to the more conventional 50% acetic anhydride and pyridine in DMF. Unfortunately, this capping solution prevented the usage of sulfone monomer 47 for the solid phase synthesis and thus we opted to oligomerize thioether 4, and perform oxidation and ammonolysis of the resulting oligomer. Based on the hypothesis (Section 1.4.1), the synthesis of the sequence outlined in Equation 2.1 was proposed (J refers to monomer 4; K refers to lysine for solubility purposes).  N? - K K K - J J J J J J J J - C?   11 10 9  8 7 6 5 4 3 2 1   Equation 2.1: The first sequence that was identified for synthesis.  To our delight, the coupling of the first Janus residue proceeded uneventfully. However, the second unit?s coupling was deemed incomplete by Kaiser test despite increased coupling time (to 2.5 hours) and double couplings. This is unsurprising in light of results concerning the carbocyclic Janus AT phosphoramidite, which exhibited poor Janus-to-NHNNNOOPhN N+OOPhCF3SO2-A)S OOOF3C S CF3OOS OOOF3CB)C)50 49 48  48 Janus couplings, as well, and the synthesized sequence could only incorporate one Janus moiety. 40 We rationalize this poor reactivity on steric grounds, and it is presumed that the benzyl group inhibits the arrival of the incoming Janus monomer.  In light of failure, the target sequence was amended to avoid J-to-J couplings. The sequence was modified by alternating 4 with 6-methyluracil 5 (Section 2.6, Figure 2.8). This nucleotide was chosen because of its simplified solution phase synthesis due to the absence of exocyclic amines, which obviates the need for protecting groups. We had hoped the inclusion of 5 would permit high yielding couplings between Janus heterocycle 4 and the 6MU, by reducing steric demand.   Figure 2.8: Structure of 6-methyluracil used as a spacer in the sequence.  In accord with this logic, the sequence was modified (U refers to 5), as outlined in Equation 2.2.  N? - K K K J - U J U J U J U - C?   11 10 9 8  7 6 5 4 3 2 1   Equation 2.2: The modified sequence avoided J-to-J couplings. J = 4, U = 5  HNNOONOHOONHBoc5  49 The synthesis of this sequence (Equation 2.2) was undertaken in a similar fashion to that previously described (Equation 2.1). However, a poor coupling efficiency was observed for the sixth sequence (J), which was again attributed to steric-like factors. Therefore, the sequence was modified again to include two 6MU residues at the sixth position, thereby avoiding the problematic J-U-J-U-J motif, as outlined in Equation 2.3.  N? - K K K - U J U J U U U J U J U - C?       11 10 9 8 7 6 5 4 3 2 1   Equation 2.3: The sequence that was ultimately synthesized. J = 4, U = 5  Gratifyingly, this synthesis proceeded smoothly as evidenced by Kaiser tests. Nevertheless, the resin was capped after each coupling and three lysine residues bearing 2-chlorobenzyloxycarbonyl groups at N? were appended to the N-terminus. The lysines impart increased solubility and provide a handle through which gel electrophoresis of the oligonucleotide may be performed. Oxidation and ammonolysis of the resin were undertaken by subjecting the beads to mCPBA, followed treatment with excess ammonia in methanol for several hours. Standard TFMSA cleavage and precipitation with diethyl ether furnished the crude PNA as a white pellet. This was desalted and analyzed by HPLC and MALDI mass spectrometry. Currently, purification and biophysical experiments of the oligomer are underway.  2.6 Synthesis of 6-Methyluracil 5 and its Solid Phase Synthesis The 6-methyluracil monomer 5 was designed as a spacer for the Janus AT sequence (Section 2.5), to relieve steric demand during solid phase couplings. Curiously, we  50 discovered that the biophysical properties of a poly(6MU) oligomer had never been reported and therefore set out to synthesize a PNA dodecamer of 6MU.  2.6.1 Synthesis of 6-Methyluracil Monomer The synthesis of the PNA monomer is outlined in Scheme 2.24. The alkylation of commerically available 6-methyluracil with bromo tert-butylacetate was undertaken first and the resulting product 51 was subjected to TFA to produce carboxylic acid 52. 48 This acid was coupled with the propyl PNA backbone 7 (synthesis described in Section 2.1) using EDC and HOBt in DMF to afford intermediate 53, which was saponified with sodium hydroxide, garnering acid 5 as a white solid.     Scheme 2.24: Synthesis of 5. A) DMF, K2CO3, bromo t-butylacetate, 12 hours (36%) B) TFA, DCM, 6 hours (98%) C) HOBt, EDC, DMF, 7, 0?C (95%) D) MeOH, H2O, NaOH, 2 hours (88%) HNNHOOHNNOOOtBuOHNNOOOHOHNNOONOOONHBocHNNOONOHOONHBocB)D)C)A)5 51 52 53  51 2.6.2 Solid Phase Oligomerization of 6-Methyluracil The solid phase synthesis for 6-methyluracil was undertaken with the aim of synthesizing the sequence outlined in Equation 2.4.  N? - U U U U U U U U U U U U - C?   12 11 10 9 8 7 6 5 4 3 2 1   Equation 2.4: Sequence synthesized for 6-methyluracil. U = 5  Synthesis was performed on a 10 ?mol scale on the MBHA resin with a loading of 0.20 mmol/gram. The coupling times were kept to 30 minutes and double couplings were unnecessary but the resin was nevertheless capped with acetic anhydride. We had anticipated high aqueous solubility of the oligomer because the lack of purine residues; therefore, the use of terminal lysines was avoided. Cleavage from the resin was performed with TFMSA. The supernatant was dropped in ether to precipitate (6MU)12 as a pellet, which was desalted. The UV-vis spectrum of the oligomer exhibited a ?max of 263 nm that correlates with that of thymine (Figure 2.9). 49   Figure 2.9: UV-vis spectrum of the 6-MU oligomer; theoretical ?max of T is 264 nm. 49  0 0.05 0.1 0.15 0.2 220 240 260 280 300 320 Absorbance (au) Wavelength (nm) UV-vis Spectrum of 6-Methyluracil Dodecamer  52 Analysis by reversed phase HPLC and MALDI confirmed the presence of the desired oligomer. Purification and biophysical studies are currently underway (Figure 2.10).   Figure 2.10: Crude HPLC chromatogram of 6MU 12mer.   53 Chapter 3 ? Conclusion The synthesis of diamino Janus AT PNA monomer 1 was attempted. Upon discovering poor solubility of this heterocycle, two synthetic avenues were pursued to mask the exocyclic amine as 2 or 3, such that it could be deprotected at will via ammonolysis. Unfortunately, both routes failed at the penultimate step of oxidation and ring closure, respectively.  Finally, the peptide nucleic acid monomer 4 was synthesized and subsequently manually oligomerized on the solid phase. Due to poor Janus-to-Janus coupling efficiencies, 6-methyluracil had to be alternated with 4 within the sequence. On this note, a homo-oligomer of 6-methyluracil was also synthesized to examine its biophysical properties, which had not been reported previously.  Currently, biophysical studies such as circular dichroism and UV melting pertaining to the synthesized oligonucleotides are underway. This work complements the studies with the deoxyribose Janus AT because the PNA oligomer incorporates more than one Janus heterocycle. Thus, we would like to probe whether oligonucleotides can strand invade canonical DNA or PNA duplexes when they incorporate multiple Janus residues.  Finally, in the quest for a universal strategy for sequence specific DNA recognition, the goal remains to elaborate this work for a GC heterocycle, as well. It is anticipated that with both motifs in hand, any known sequence of DNA can be recognized, which may lead to potential anti-gene strategies based on strand invasion.  54 Chapter 4 ? Experimental 4.1 General Experimental Procedure All reagents were obtained from Sigma Aldrich, Acros or Alfa-Aesar, and used as received. DMSO, NMP, DMF and ethanol (absolute) were dried overnight over triply changed, oven-baked (at 170?C) 3? molecular sieves. DCM, MeOH, pyridine, triethylamine, DIPEA and THF were distilled over ninhydrin, Na, CaH2 or MgSO4 prior to use. 50  Thin layer chromatography was performed on Merck silica gel 60 F254 plates. The plates were developed with anisaldehyde, ninhydrin, bromocresol green or KMnO4 after being visualized by a UV lamp at 260 nm. Silica gel chromatography was performed with SiliCycle 40-63 ?m silica (column dimensions are indicated in Section 4.2). All reactions were performed under Ar (introduced by a rubber balloon) in flame-dried flasks equipped with Teflon stir bars and capped with rubber septa. Extraction volumes are listed in Section 4.2 for EACH extraction (i.e. ?5x, 50ml? implies 250 ml total solvent volume).  All nuclear magnetic resonance spectra were recorded on a Bruker AV-300 spectrometer, calibrated with internal residual protonated solvent peaks. Deuterated NMR solvents were purchased from Cambridge Isotope Labs or Sigma Aldrich. Electrospray ionization mass spectra under the positive or negative ion mode were obtained at the UBC Mass Spectrometry Facility; the solvents are indicated below. The diluted sample (10 ?M) was injected into the ion source by a syringe pump at a flow rate of 0.01 ml/min. High-resolution mass spectra were submitted to the UBC Mass Spectrometry Facility.  55 4.2 Chemical Methods Note: Compounds in this section are in the order they appear in Chapter 2.  Synthesis of 2-Bromoacetic Acid (10)  Procedure adapted from Workhoven et al. 51  To a round bottom flask containing acetic acid (8.0 g, 7.6 ml, 133.2 mmol) was slowly added phosphorus tribromide (12.6 ml, 35.9 g, 132.8 mmol) and the solution was cooled to 0?C. Liquid bromine (15.6 ml, 48.4 g, 302.9 mmol) was added to this solution over 25 minutes upon which evolution of gaseous HBr was observed. The solution was stirred at room temperature for 15 minutes, heated to 75?C for 2.5 hours, re-cooled to 0?C and finally quenched with water (20 ml, over 15 minutes). Excess bromine was reduced by addition of sodium thiosulfate (1M aqueous solution) and the resulting slurry was extracted with diethyl ether (8x, 75 ml), dried over MgSO4 and the solvent was evaporated to yield 10 as a very pale yellow solid (17.6 g, 95%) that was used without further purification. ESI-MS (MeOH): m/z 137.1 (100%), 139.0 (90%) [M-H]- 1H-NMR (DMSO-d6): ? 4.01 (s, 2H) TLC: Rf = 0.55 in 100% EtOAc OHOBr 56     DMSO H2O  57 Synthesis of Methyl 2-Bromoacetate (11) and n-Propyl 2-Bromoacetate (12)    To a round bottom flask charged with bromoacetic acid 10 (10.0 g, 72.0 mmol) was added either methanol (45.0 ml) or n-propanol (45.0 ml), followed by concentrated sulfuric acid (4.0 ml). The solution was stirred at reflux for 3 hours, after which the solvent was removed. The remaining starting material was basified with saturated sodium bicarbonate (100 ml) to a final pH of 7.5 and the solution was then washed with diethyl ether (2x, 100 ml). The combined organic extracts were washed with saturated NaHCO3 (1x, 75 ml) and dried with MgSO4. Removal of the solvent furnished 11 or 12 as colorless lachrymatory oils (11: 8.2 g, 74% 12: 8.9 g, 68%). ESI-MS (MeOH): These materials cannot be ionized under ESI conditions. 1H-NMR (CDCl3) for 11: ? 3.79 (s, 2H), 3.75 (s, 3H) 1H-NMR (CDCl3) for 12: ? 4.11 (t, J = 6.7 Hz, 1H), 1.67 (sextet, J = 7.1 Hz, 1H), 0.94 (t, J = 7.4 Hz, 1H)  OOBrOOBr1H-NMR for 11 OOBrCHCl3 Et2O Et2O H2O  58   Synthesis of alkyl 2-((2-((tert-butoxycarbonyl)amino)ethyl)amino)acetate, alkyl denotes methyl (6) or n-propyl (7)  Procedure adapted from Kofoed et al. 52  To a round bottom flask were added ethylene diamine (11.2 ml, 10.1 g, 167.7 mmol) and DCM (50.0 ml, 3.6 mol/L). Meanwhile, an addition funnel was charged with Boc anhydride (6.1 g, 27.9 mmol) dissolved in DCM (400.0 ml, 0.07 mol/L) and this solution was added to the ethylene diamine solution over 180 minutes with rapid stirring. The reaction mixture was stirred overnight, following which the solvent was removed by evaporation. The resulting solution was diluted with sodium carbonate (2M, 250 ml) and washed with DCM (2x, 150 ml). The organic extracts were dried with MgSO4 and evaporated to afford 13 as a clear colorless paste. NHHN OOORO1H NMR for 12 OOBrCHCl3  59 ESI-MS (MeOH): m/z 161.4 (80%) [M+H]+, 105.2 (100%) [M-tBu+H]+ TLC: Rf 0.15 in 40% MeOH/EtOAc.    To a solution of intermediate 13 (4.1 g, 25.3 mmol) in DCM (52.0 ml, 0.49 mol/L) was added DIPEA (8.7 ml, 6.5 g, 49.9 mmol) and the resulting solution was cooled to 0?C. A solution of bromo methylacetate 11 (3.9 g, 25.2 mmol) or bromo propylacetate 12 (4.5 g, 25.2 mmol) in DCM (10.0 ml, 2.5 mol/L) was added dropwise over 50 minutes to the solution of 13. The reaction was stirred overnight, evaporated to dryness and then diluted with water and extracted with DCM (5x, 50 ml). The organic fractions were dried with Na2SO4 and the white solid resulting after evaporation was purified by column chromatography (5 cm OD x 20 cm, prepared in 10% EtOAc/DCM, loaded with DCM, eluted with 10% EtOAc/DCM ! 75% EtOAc/DCM). The products 6 or 7 eluted as clear yellow oils (6: 3.9 g, 66%; 7: 4.1 g, 62%). ESI-MS (MeOH) for 6: m/z 233.4 (60%) [M+H]+, 177.4 (100%) [M-tBu+H]+ ESI-MS (MeOH) for 7: m/z 261.5 (85%) [M+H]+, 205.4 (100%) [M-tBu+H]+ 1H-NMR (CDCl3) for 6: ? 5.05 (s, 1H), 3.78 (s, 3H), 3.44 (s, 2H), 3.22 (q, J = 5.4 Hz, 2H), 2.75 (t, J = 9.4 Hz, 2H), 1.95 (s, 1H), 1.48 (s, 9H) 1H-NMR (CD2Cl2) for 7: ? 4.09 (t, J = 9.5 Hz, 2H), 3.16 (q, J = 5.8 Hz, 2H), 2.71 (t, J = 5.8 Hz, 2H), 1.66 (sextet, J = 7.1 Hz, 2H), 0.94 (t, J = 7.4 Hz, 3H)  60 13C-NMR (DMSO-d6)   for 7: ? 172.3, 155.9, 78.2, 66.0, 50.2, 48.8, 40.1, 28.1, 21.9, 10.1 TLC for 6: Rf = 0.50 in 20% MeOH/EtOAc TLC for 7: Rf = 0.50 in 8% MeOH/EtOAc  ESI-MS for 6  ESI-MS for 7:       For 6 CHCl3 EtOAc EtOAc EtOAc NHHN OOOO 61       Synthesis of lithium 2-((2-((tert-butoxycarbonyl)amino)ethyl)amino)acetate (8)  To ester 6 (0.4 g, 1.7 mmol) was added MeOH and water (4.0 ml each, 0.20 mol/L final concentration) to yield a clear solution. Lithium hydroxide (44.4 mg, 1.85 mmol) was NHHN OOOLiOFor 7 DCM NHHN OOOO 62 added with sonication and the reaction was stirred for 2 hours at room temperature. The solvent was removed by rotary evaporation to yield the product 8 as a whitish yellow solid (quantitative yield). ESI-MS (H2O): m/z 225.5 (100%) [M+H]+ 1H-NMR (D2O): ? 3.31-2.82 (broad, 4H), 2.52 (s, 2H), 1.25 (s, 9H) TLC: Rf = 0.50 in 100% MeOH    H2O in D2O  63 Synthesis of tert-butyl 2-((2-((Fmoc)amino)ethyl)amino acetate (9)  Procedure adapted from Milic et al. 53  A mixture of ethylene diamine (6.0 ml, 5.4 g, 89.9 mmol) dissolved in DCM (11.0 ml, 8.2 mol/L) was cooled to 0?C in an ice bath. Over 60 minutes, a solution of bromo tert-butylacetate (1.0 ml, 1.3 g, 6.9 mmol) in DCM (11.0 ml, 0.62 mol/L) was added. The solution was stirred overnight at room temperature. After 20 hours, the solution was washed with water (3x, 60 ml) and the aqueous extracts were washed with DCM (2x, 50 ml). The organic extracts were dried with MgSO4, and the solvent was removed by rotary evaporation to yield intermediate 13 as light yellow murky oil (yield uncalculated). ESI-MS (DCM): m/z 119.1 (100%) [M-tBu+H]+, 175.5 (50%) [M+H]+ TLC: Rf = 0.1 in 10% MeOH/EtOAc.   To a solution of 13 (0.6 g, 3.2 mmol) in DCM (11.0 ml, 0.29 mol/L) was added DIPEA (0.6 ml, 0.4 g, 3.2 mmol). Meanwhile, an addition funnel was charged with Fmoc OSU (1.0 gram, 3.0 mmol) and DCM (7.0 ml, 0.42 mol/L) and this solution was added over 40 HNNHOOOtBuO 64 minutes to the solution of 13 followed by overnight stirring. The reaction mixture was washed with 1M HCl (5x, 50 ml) and brine and then subjected to rotary evaporation to a volume of 7 ml. Cooling the organic layer to -20?C overnight caused precipitation of 9 as a white solid, which was filtered and washed with cold DCM (0.35 g, 27%). More of the product was detected in the filtrate, which could be re-purified if desired. ESI-MS (MeOH): m/z 397.4 (100%) [M+H]+, 341.4 (70%) [M-tBu+H]+ 1H-NMR (CD2Cl2): ? 9.93 (s, 1H), 7.77 (d, J = 7.4 Hz, 2H), 7.68 (d, J = 6.9 Hz, 2H), 7.44-7.28 (m, 4H), 6.84 (s, 1H), 4.33-4.24 (m, 3H), 3.76 (s, 2H), 3.73-3.64 (m, 2H), 3.27-3.24 (m, 2H), 1.56 (s, 9H) 13C-NMR (DMSO-d6): ? 166.0, 156.7, 144.3, 141.2, 128.1, 127.5, 125.7, 120.6, 83.3, 66.2, 55.4, 47.8, 47.2, 37.2, 28.1 TLC: Rf = 0.15 in 75% EtOAc/hexanes   65    Synthesis of tert-butyl 2-aminoacetate (15)  Procedure adapted from Siebum et al. 54  OOH2NDCM Acet. H2O  66 Commercially purchased tert-butyl 2-bromoacetate (10.0 ml, 13.4 g, 68.6 mmol) was dissolved in diethyl ether (10.0 mL, 6.85 mol/L) in a pressure tube, and the reaction was cooled to -78?C. Ammonia gas (approximately 15 ml) was condensed by bubbling it through the solution and the reaction was stirred overnight, slowly warming to room temperature. The next day, the reaction was re-cooled and opened to the atmosphere and the mixture was diluted with diethyl ether (30 ml), washed with water (30 ml, 2x) and then dried with MgSO4. The solvent was removed in vacuo and purified via vacuum distillation at 1 mmHg to furnish 42 as a colorless oil (5.9 g, 66%). ESI-MS (MeOH): m/z 132.2 (80%) [M+H]+ 1H-NMR (CDCl3): ? 3.40 (s, 2H), 1.48 (s, 9H) TLC: Rf = 0.15 in 100% EtOAc     Acetone CHCl3  67 Synthesis of ethyl (2-cyanoacetyl)carbamate (16)  Procedure adapted from Bach et al. 55  To a round bottom flask containing cyanoacetic acid (2.1 g, 25 mmol) and urethane (2.45 g, 25 mmol) were added toluene (5.0 ml, 5.0 mol/L), phosphorus oxychloride (1.3 ml, 2.1 g, 13 mmol) and DMF (1.0 ml, 0.9 g, 13.0 mmol). The reaction was heated under reflux (100?C) for 2 hours and then cooled to rt. Excess POCl3 was destroyed by addition of water and the peach solid that formed was filtered to afford pure 16 (3.0 g, 76%). ESI-MS (MeOH): m/z 155.2 (95%) [M-H]-, 109.0 (100%) [M-EtOH-H]- 1H-NMR (CDCl3): ? 7.63 (s, 1H), 4.29 (q, J = 7.1 Hz, 2H), 4.06 (s, 2H), 1.35 (t, J = 7.1 Hz, 4H) TLC: Rf = 0.50 in 50% EtOAc/hexanes  EtOONHON 68   Synthesis of potassium 2-cyano-3-((ethoxycarbonyl)amino)-3-oxopropenebisthiolate (17)  Procedure adapted from Asadi et al. 56  Cyanoacetylurethane (1.6 g, 10.0 mmol) and freshly ground K2CO3 (1.4 g, 10.0 mmol) in anhydrous DMF (25.0 mL, 0.40 mol/L) were stirred at room temperature for two hours. Carbon disulfide (1.3 ml, 1.6 g, 21.5 mmol) was then added to the mixture dropwise, and stirring was continued overnight to yield a yellow-orange suspension. Absolute ethanol (100 mL) was added, upon which a precipitate formed that was filtered and washed with EtOONHONKS SKCHCl3 H2O  69 diethyl ether (150 mL) and dried on the vacuum to afford 17 as a yellow solid (2.9 g, 94%). ESI-MS (MeCN+H2O): m/z 309.2 (100%) [M+H]+, 233.2 (25%) [M-2K+3H]+ 1H-NMR (DMSO-d6): ? 3.97 (q, J = 7.0 Hz, 2H), 1.15 (t, J = 7.0 Hz, 3H)     DMSO H2O  70 Synthesis of diethyl (2-cyano-3,3-bis(methylthio)acryloyl)carbamate (18)  Procedure adapted from Asadi et al. 56  Compound 17 (2.9 g, 9.4 mmol) was dissolved in 37.8 ml of H2O and 71.4 ml of MeCN (0.30 mol/L), fitted with a reflux condenser and stirred at room temperature for 30 minutes. Methyl iodide (1.2 mL, 4.6 mmol) dissolved in acetonitrile (2.9 ml, 1.61 mol/L) was then added slowly and the reaction was stirred for 30 minutes, followed by refluxing for 3 hrs. The mixture was cooled to room temperature, concentrated in vacuo to remove the acetonitrile, transferred to a separatory funnel and then extracted with EtOAc (3x, 75ml). The organic phases were washed with brine (60 ml) and dried over Na2SO4. Evaporation of this solution revealed an orange oil, which solidified into a dark yellow/brown solid upon standing at room temperature under vacuo. It can be further purified by column chromatography (1.25? OD by 6.5?, prepared in 15% EtOAc/hexanes, loaded with EtOAc, eluted with 30% EtAOc/hexanes ! 60% EtOAc/hexanes) (6.5 g, 78%). ESI-MS (MeOH): m/z 283.2 (100%) [M+Na]+ 1H-NMR (CDCl3): ? 8.01 (s, 1H), 4.27 (q, J = 6.4 Hz, 2H), 3.92 (s, 3H), 3.7 (s, 3H), 1.33 (t, J = 6.6 Hz, 3H) TLC: Rf = 0.45 in 50% EtOAc/hexanes EtO NHO OCNSS 71      Synthesis of tert-butyl 2-(5-cyano-6-(methylthio)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetate (19)  OONHNOOCNSCHCl3 H2O Acet.  72 A solution of 18 (0.48 g, 1.8 mmol) in absolute ethanol (14.0 mL, 0.13 mol/L) was stirred at 25?C in a round bottom flask and a solution of 15 (0.25 ml, 0.24 g, 1.8 mmol) was added dropwise over 15 minutes (Note: The melting point for 15 is 20?C; gentle warming is required). The flask was fitted with a condenser and the reaction heated at reflux for 20 hours, after which the mixture was cooled to room temperature and the ethanol removed in vacuo. The crude material was purified by column chromatography (2.5 cm OD by 18 cm, prepared in DCM, loaded with DCM, eluted with DCM ! 8% EtOAc/DCM) to yield 19 as a tan yellow solid (0.4 g, 74%). ESI-MS (DCM): m/z 296.3 (100%) [M-H]- 1H-NMR (Acetone-d6): ? 4.91 (s, 2H), 2.94 (s, 3H), 1.51 (s, 9H) 13C-NMR (DMSO-d6): ? 166.9, 166.3, 159.3, 149.6, 114.4, 94.1, 83.1, 48.4, 28.0, 19.7 TLC: Rf = 0.60 in 60% EtOAc/hexanes    73       Acetone  74 Synthesis of tert-butyl 2-(5,7-diamino-2,4-dioxo-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)acetate (20)  Procedure adapted from Asadi et al. 56  Sodium metal (39.9 mg, 1.7 mmol) was added to a solution of ethanol (1.5 ml) at 0?C under argon. Upon complete dissolution, guanidinium hydrochloride (0.15 g, 1.6 mmol) was added and the mixture was stirred at 40?C for 20 minutes. The resulting heterogeneous solution was filtered (through Pasteur pipette with glass wool) into a solution of 19 (0.4 g, 1.3 mmol) dissolved in absolute ethanol (5 mL). The reaction was fitted with a reflux condenser and heated to 110?C for 24 hours, after which it was cooled to room temperature. The resulting precipitate was filtered and washed with EtOH (125 ml), EtOAc (125 ml), H2O/EtOH (10 ml each) and finally EtOH (30 ml). Pure 20 was obtained as a white solid (90.0 mg, 25%). ESI-MS (DMSO): m/z 307.3 (100%) [M-H]- 1H-NMR (DMSO-d6): ? 11.24 (s, 1H), 8.00 (s, 1H), 7.51 (s, 1H), 7.17 (s, 2H), 4.61 (s, 2H), 1.47 (s, 9H) TLC: Rf = 0.10 in 100% EtOAc  OONHNOO NNH2N NH2 75    DMSO H2O  76 Synthesis of 2-(5,7-diamino-2,4-dioxo-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)acetic acid (21)  A solution of 20 (0.10 g, 0.30mol) in ice-cold trifluoroacetic acid (1.4 ml, 0.21 mol/L) was stirred overnight, slowly warming to room temperature. The excess TFA was removed in vacuo revealing a dense orange paste from which the product was precipitated by addition of diethyl ether (5 ml). Upon re-evaporation of the solvent and drying on the pump at 60?C, the product was obtained as an off-white powder (quantitative yield). ESI-MS (DMSO): m/z 251.3 (100%) [M-H]- 1H-NMR (DMSO-d6): ? 11.31 (s, 1H), 8.12 (s, 1H), 7.60 (s, 1H), 7.10 (s, 2H), 4.66 (s, 2H) TLC: Rf = 0.33 in 50% MeOH/EtOAc OHONHNOO NNH2N NH2 77    Integrates for 0.25; unknown impurity Water in TFA DMSO TFA or COOH?  78 Synthesis of (E)-ethyl (2-cyano-3-ethoxybut-2-enoyl)carbamate (22)  Procedure adapted from Asadi et al. 56  To a round-bottom flask containing carbamate 16 (3.1 g, 20.0 mmol) was added triethyl orthoacetate (3.7 ml, 3.3 g, 20.2 mmol) and acetic anhydride (8.0 ml, 8.7 g, 84.8 mmol). The reaction was heated to 110?C for one hour and then cooled to 0?C, which resulted in the formation of crystals. These were filtered and washed with hexanes and cold diethyl ether to afford 22 as white needles (2.8 g, 62%). ESI-MS (MeOH): m/z 249.4 (100%) [M+Na]+, 475.4 (75%) [Mx2+Na]+ 1H-NMR (CDCl3): ? 8.99 (s, 1H), 4.34 (q, J = 7.0 Hz, 2H), 4.21 (q, J = 7.1 Hz, 2H), 2.46 (s, 3H), 1.50 (t, J = 7.0 Hz, 3H), 1.27 (t, J = 7.1 Hz, 3H) TLC: Rf = 0.10 in 75% EtOAc/hexanes   EtO NHO ONOEt 79   Synthesis of 1-allyl-6-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (23)  Procedure adapted from Asadi et al. 56 To a round-bottom flask containing carbamate 22 (1.0 gram, 4.4 mmol) dissolved in absolute ethanol (15.0 ml, 0.30 mol/L) was added allylamine (0.33 ml, 0.3 g, 4.4 mmol) dropwise, followed by anhydrous triethylamine (1.0 ml, 0.7 g, 7.2 mmol). Once the evolution of gas had subsided, the reaction mixture was fitted with a reflux condenser and heated to 100?C. After two hours, the round bottom flask was cooled to room temperature and the excess solvent was removed by rotary evaporation. Column chromatography purification of this solid was performed (1.75? OD by 6.75?, prepared in hexanes, loaded HNNCNOOCHCl3 H2O  80 with 50% EtOAc/hexanes, eluted with 70% EtOAc/hexanes ! 100% EtOAc), which furnished pure 23 as a white solid (1.6 g, 94%). ESI-MS (MeOH): m/z 190.3 (100%) [M-H]- 1H-NMR (DMSO-d6): ? 11.97 (s, 1H), 5.93-5.82 (m, 1H), 5.20-5.13 (m, 2H), 4.50-4.49 (m, 2H), 2.47 (s, 3H). TLC: Rf = 0.66 in 100% EtOAc        DMSO  H2O  81 Synthesis of Allyl-5-amino-7-thioxo-1H-thiopyrano[4,3d]pyrimidine-2,4(3H,7H)dione (24)  Procedure adapted from Asadi et al. 56  To a solution of 23 (0.8 g, 3.9 mmol) in THF (48.0 ml, 0.08 mol/L) was added potassium tert-butoxide (1.6 g, 13.8 mmol). The resulting solution was stirred for 30 minutes and then carbon disulfide (0.6 ml, 0.8 g, 9.9 mmol) was added. After four hours, the reaction was evaporated to dryness and the reaction was diluted with water (15.0 ml). The hydroxide that formed was acidified with concentrated HCl to pH 5. The resulting reddish precipitate was filtered off and the filtrate was extracted with EtOAc (4x, 75 ml) and dried over Na2SO4. The solid was combined with the organic extracts and chromatographed (1.75? OD by 5?, prepared in 50% EtOAc/hexanes, loaded with EtOAc, eluted with 60% EtOAc/hexanes ! 80% EtOAc/hexanes), affording 24 as an orange solid (0.35 g, 33%). ESI-MS (MeOH): m/z 266.3 (100%) [M-H]- 1H-NMR (DMSO-d6): ? 11.68 (s, 1H), 10.53 (s, 1H), 9.83 (s, 1H), 6.61 (s, 1H), 5.86-5.80 (m, 1H), 5.19-5.16 (m, 2H), 4.58 (broad s, 2H) TLC: Rf = 0.4 in 20% Et2O/DCM.  HNNOOSSNH2 82     DMSO  H2O  83 Synthesis of 1-allyl-5-amino-7-mercaptopyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (25)  Procedure adapted from Asadi et al. 56  To a pressure-tube containing 24 (0.2 g, 0.7 mmol) was added concentrated ammonium hydroxide (10.0 ml), giving a turbid orange solution. The tube was sealed and heated to 95?C, upon which the solution turned clear and brown. The following day, the pressure tube was cooled and the solvent was removed to reveal 25 as a dark yellow solid (uncalculated yield). The intermediate was used without purification. ESI-MS (MeOH): m/z 249.3 (100%) [M-H]- TLC: Rf = 0.3 in 50% EtOAc/DCM      HNNOONSHNH2 84 Synthesis of Allyl-5-amino-7-(benzylthio)pyrido[4,3-d]pyrimidine-2,4(1H,3H)dione (26)  Procedure adapted from Asadi et al. 56  To a round bottom flask containing the starting material 25 (0.2 g, 0.7 mmol) was added sodium hydroxide (0.1 g, 2.5 mmol). The reagents were dissolved in hot water (95?C, 2.1 ml, 0.33 mol/L) and the mixture was stirred for 10 minutes, after which benzyl bromide (0.13 ml, 0.19 g, 1.1 mmol) was added dropwise. MeOH (6.0 ml) was then added and the reaction was stirred for 2 hours. Upon completion, the reaction mixture was evaporated and water was added (50 ml), followed by acidification to pH 3 with concentrated HCl. The aqueous layer was repeatedly extracted with EtOAc (5x, 100 ml) and then dried over Na2SO4. The crude material was purified by chromatography (2 cm OD by 18 cm, prepared in hexanes, loaded with EtOAc, eluted with 40% EtOAc/hexanes ! 60% EtOAc/hexanes) to furnish 26 as a white solid (0.1 g, 42%). ESI-HRMS (MeOH): m/z 341.1072 (100%) [M+H]+ 1H-NMR (DMSO-d6): ? 11.41 (s, 1H), 8.20 (s, 1H), 7.50 (s, 1H), 7.41 (d, J = 6.9 Hz, 2H), 7.30-7.21 (m, 3H), 6.17 (s, 1H), 5.82-5.73 (m, 1H), 5.06 (m, 2H), 4.49 (m, 2H), 4.38 (s, 2H) 13C-NMR (DMSO-d6): ? 163.7, 159.2, 150.3, 149.3, 138.5, 132.3, 129.4, 128.8, 127.5, 116.8, 94.6, 90.7, 44.8, 33.4 HNNOONSNH2 85 TLC: Rf = 0.50 in 50% EtOAc/hexanes       H2O  86   Synthesis of 2-((tert-butyldimethylsilyl)oxy)ethanamine (27)  To a solution of ethanolamine (0.4 g, 6.5 mmol) in DCM (7.0 ml, 0.93 mol/L) was added tert-butyldimethylsilyl chloride (1.1 g, mmol) and imidazole (0.9 g, 7.3 mmol), upon which the solution turned opaque. The reaction was complete in three hours and was then diluted with water (15 ml) and extracted with DCM (3x, 50 ml). The organic extracts were dried with MgSO4 and evaporated to dryness to reveal the product as a yellow oil, which was used without further purification (0.9 g, 81%). ESI-MS (DCM): 176.4 (100%) [M+H]+  OH2NSi 87 1H-NMR (CDCl3): ? 3.65 (t, J = 5.2 Hz, 2H), 2.81 (t, J = 5.2 Hz, 2H), 0.88 (s, 9H), 0.05 (s, 6H) TLC: Rf = 0.25 in 10% MeOH/EtOAc       Synthesis of 1-(2-((tert-butyldimethylsilyl)oxy)ethyl)-6-methyl-2,4-dioxo-1,2,3,4-tetra hydropyrimidine-5-carbonitrile (28)  HNNCNOOOTBDMS?  88 Procedure adapted from Asadi et al. 56  To a round bottom flask charged with 22 (0.2 g, 0.9 mmol) was added ethanol (3.0 ml, 0.29 mol/L) and 27 (0.12 g, 0.68 mmol). The flask was equipped with a condenser and then triethylamine (0.2 ml, 0.15 g, 1.4 mmol) was added upon which the mixture turned reddish brown. The reaction was then brought to reflux for 2.5 hours and then evaporated to dryness. The crude material was purified by column chromatography (2 cm OD by 15 cm, prepared in 40% EtOAc/hexanes, loaded with 40% EtOAc/hexanes, eluted with 40% EtOAc/hexanes ! 50% EtOAc/hexanes) to afford the product as a white solid (0.13 g, 63%). ESI-MS (MeOH): m/z 310.4 (100%) [M+H]+, 308.3 (100%) [M-H]- 1H-NMR (CDCl3): ? 8.62 (s, 1H), 4.10 (t, J = 4.9 Hz, 3H), 3.91 (t, J = 4.8 Hz, 2H), 2.73 (s, 3H), 0.89 (s, 9H), 0.05 (s, 6H) TLC: Rf = 0.85 in 100% EtOAc.    89   Synthesis of 5-amino-1-(2-((tert-butyldimethylsilyl)oxy)ethyl)-7-thioxo-1H-thiopyrano-[4,3-d]pyrimidine-2,4(3H,7H)-dione (29)  Procedure adapted from Asadi et al. 56  To a round bottom flask charged with 28 (0.13 mg, 0.4 mmol) and THF (5.2 ml, 0.08 mol/L) was added potassium tert-butoxide (0.16 g, 1.4 mmol) and the resulting mixture was stirred for 15 minutes. Then, carbon disulfide (64 ?L, 80.7 mg, 1.1 mmol) was added at once, followed by stirring at room temperature for 1.5 hours. The reaction mixture was then evaporated to reveal an orange powder, which was dissolved in water (10 ml) and HNNOOOTBDMSSSNH2CHCl3 H2O  90 acidified to pH 5 with concentrated HCl. The precipitate was filtered, washed with water and then dried in vacuo to afford 29 as an orange/yellow solid (75 mg, 49%). ESI-MS (MeOH): m/z 384.2 (100%) [M-H]-, 270.2 (30%) [M-TBDMS-H]- 1H-NMR (Acetone-d6): ? 10.81 (s, 1H), 10.35 (s, 1H), 8.88 (s, 1H), 7.05 (s, 1H), 4.23 (t, J = 5.8 Hz, 2H), 3.94 (d, J = 5.9 Hz, 2H), 0.88 (s, 9H), 0.06 (s, 6H). TLC: Rf = 0.45 in 50% EtOAc/hexanes; 29 co-elutes with 28 in this solvent system.    91   Synthesis of 5-amino-(2-hydroxyethyl)-7-mercaptopyrido[4,3-d]pyrimidine-2,4 (1H,3H)-dione (30)  Procedure adapted from Asadi et al. 56  To a pressure tube with 29 (0.7 g, 1.8 mmol) was added concentrated NH4OH (35.0 ml). The tube was sealed and heated to 110?C. The following day, the mixture was cooled to room temperature and excess water was removed by rotary evaporation. The solid HNNOOOHNSHNH2Acet. H2O  92 material was filtered, washed with water and EtOAc and dried to afford 30, which was used without purification (0.40 g, 87% assuming complete conversion). ESI-MS (MeOH): m/z 255.3 (100%) [M+H]+ TLC: Rf = 0.40 in 40% MeOH/EtOAc.   Synthesis of 5-amino-7-(benzylthio)-1-(2-hydroxyethyl)pyrido[4,3-d]pyrimidine-2,4 (1H,3H)-dione (31)  HNNOOOHNSNH2 93 Procedure adapted from Asadi et al. 56  Distilled water (2.0 ml, 0.51 mol/L) was added to a solution of starting material 30 (0.26 g, 1.0 mmol) and sodium hydroxide (0.05 g, 1.2 mmol), and the solution was stirred for ten minutes. Benzyl bromide (0.12 ml, 0.17 g, 1.0 mmol) was then added to the solution dropwise followed by MeOH (6.0 ml). After two hours, the reaction was deemed complete by TLC analysis and the mixture was evaporated to dryness. This resulting white powder was diluted with water (50 ml) and acidified from pH 10 to pH 5.5 with concentrated HCl. The aqueous solution was then extracted with EtOAc (5x, 105 ml) and the combined organic extracts were dried with Na2SO4 and concentrated to afford a white solid. This was purified by chromatography (2 cm OD by 19 cm, prepared in 40% EtOAc/hexanes, loaded with THF, eluted with 40% EtOAc/hexanes ! 10% MeOH/EtOAc) to afford pure 31 as a white solid (0.1 g, 30%). ESI-HRMS  (MeOH): m/z 345.1021 (100%) [M+H]+ 1H-NMR (Acetone-d6): ? 8.42 (s, 1H, NH), 7.46 (d, J = 7.5 Hz, 2H, phenyl), 7.33-7.22 (m, 5H, phenyl + NH2), 6.46 (s, 1H, CH), 4.43 (s, 2H, SCH2), 4.09 (t, J = 6.0 Hz, 3H, CH2 + OH), 3.76 (t, J = 5.7 Hz, 2H, CH2). TLC: Rf = 0.25 in 80% EtOAc/hexanes    94    Synthesis of pyrimidine-2,4,6-triol (32)  Procedure adapted from Dickey et al. 57  To a round-bottom flask with freshly cut Na (2.5 g, 106.5 mmol) was added absolute ethanol (100.0 ml, 1.1 mol/L). Once the emission of hydrogen gas had subsided, diethyl malonate (15.2 ml, 16.0 g, 100.0 mmol) and urea (6.0 g, 100.1 mmol) were added sequentially and the reaction was stirred at reflux overnight. The following day, the mixture was cooled to room temperature and the precipitated solid was filtered. It was dissolved in water (65 ml), acidified to pH 2 with concentrated HCl, and re-filtered to afford a white solid, which was washed with water (100 ml) and ethanol (150 ml). After drying in vacuo, 32 was obtained as a white solid (8.6 g, 67%). ESI-MS (DMSO): m/z 127.1 (100%) [M-H]- 1H-NMR (DMSO-d6): ? 10.00 (s, 2H), 3.55 (s, 2H) (NMR signals from tautomer of 32). N NOHHO OH 95       Synthesis of 2,4,6-trichloropyrimidine (33)  Procedure adapted from Langerman et al. 58  To a round bottom flask equipped with a condenser were added 32 (6.0 g, 46.8 mmol) and triethylamine (2.3 ml, 1.6 g, 16.1 mmol) followed by POCl3 (35.0 ml, 57.6 g, 375.5 mmol, 1.33 mol/L), and the solution was brought to reflux overnight. The next day the solution was cooled and excess POCl3 was quenched by the addition of icy water (110 ml), after which it was neutralized with 2M NaOH(aq). The aqueous layer was washed with DCM (5x, 170 ml) and the organic extracts were dried with MgSO4. The crude was N NClCl ClDMSO H2O  96 purified by chromatography (2.125? OD by 3.875?, prepared in hexanes, loaded with DCM, eluted with 20% DCM/hexanes ! 50% DCM/hexanes) to afford 33 as a dense yellowish-orange oil (5.5 g, 64%). ESI-MS: Product does not ionize in ESI or APCI conditions. 1H-NMR (CDCl3): ? 5.21 (s, 1H). 13C-NMR (CDCl3): ? 163.1 (C4 and C6), 159.7 (C2),  120.9 (C5) TLC: Rf = 0.75 in 50% hexanes in DCM.       Acetone CHCl3 Acetone H2O  97 Synthesis of 2,4,6-trichloropyrimidine-5-carbaldehyde (35)  Procedure adapted from Borzsonyi et al. 59  To a dry round bottom flask charged with cooled anhydrous DMF (10.0 ml, 9.5 g, 129.7 mmol) was slowly added POCl3 (50.0 ml, 82.3 g, 536.4 mmol). The solution was stirred at 45?C for 15 minutes to yield a clear gold solution. Barbituric acid 32 (8.3 g, 64.8 mmol) was added portionwise over 15 minutes and the reaction was stirred for 1 hour. The solution was heated to 100?C for 4 hours and excess POCl3 was then removed in vacuo (80?C), affording an orange oil to which was added ice (230 ml). The precipitated aldehyde was filtered and purified by column chromatography (2? OD by 6?, prepared and loaded in DCM, eluted with DCM ! 10% EtOAc/DCM) to afford 35 as a bright yellow solid (7.5 g, 55%). ESI-MS: Does not ionize under ESI; converted to p-methoxybenzylamine derivative for MS purposes. MeOH: m/z 411.3 (100%) [M-H]- 1H-NMR (DMSO-d6): ? 10.48 (s, 1H) TLC: Rf = 0.6 in DCM N NClCl ClO H 98     Synthesis of 2,4,6-trichloropyrimidine-5-carboxylic acid (34)   N NClCl ClOHON NClNHNHO HOHO 99 Method A was adapted from Seto et al. 60 Method B was adapted from Travis et al. 61  Method A (from 33): To an RB flask at -78?C containing THF (11.4 ml, 0.81 mol/L vs. diisopropylamine) was added diisopropylamine (1.3 ml, 0.9 g, 9.3 mmol) and n-butyllithium (6.3 ml, 9.2 mmol, 1.46 mol/L against diphenylacetic acid). The resulting solution was stirred at -78?C for 1hr after which a solution of 33 (1.4 g, 7.6 mmol) in THF (6.0 ml, 1.27 mol/L) was added dropwise. The resulting black solution was stirred for 6 hours and then quenched with CO2 (g) vapors. The reaction mixture was stirred at  -78?C for 15 minutes and then at room temperature for 45 minutes. Water (20 ml) was added and the solution was acidified with 1M HCl to pH 2. The aqueous layer was extracted with EtOAc (3x, 75 ml) and the organic extracts were dried (MgSO4) and chromatographed (1.75? OD by 5.5?, prepared in DCM, loaded with 5% MeOH/DCM, eluted with 14% MeOH/DCM) to afford 34 as a tan brown solid (1.1 g, 65%). ESI-MS (50% MeOH/DCM): m/z 225.2 (100%) 227.2 (95%) 229.2 (33%) [M-H]- 13C-NMR (CDCl3): ? 162.7 (COOH), 159.0 (C4 and C6), 158.5 (C2), 127.9 (C5) 1H-NMR: No peaks were observed in DMSO or chloroform TLC: Rf = 0.2 in 15% MeOH/DCM   100    Method B (from 35): To an Eppendorf with 35 (5 mg, 0.02 mmol) was added DMF (0.7 ml, 0.03 mol/L) and Oxone? (12.1 mg, 0.02 mmol). The solution was stirred at rt, upon which the yellow color subsided and TLC analysis indicated conversion to a more polar spot. Mass-spectrometric analysis confirmed the spot to be 34. Acetone  101 Synthesis of 2,4,6-trichloropyrimidine-5-carboxamide (36)  Procedure adapted from Balsamo et al. 62  To an RB flask with 34 (0.35 g, 1.5 mmol) was added DCM (12.8 ml, 0.12 mol/L) and oxalyl chloride (2.7 ml, 4.0 g, 31.5 mmol). To this, anhydrous DMF (0.25 ml, 0.24 mg, 3.2 mmol) was added, upon which evolution of smoke was observed. The reaction was stirred for 3 hours and then cooled to -5?C. It was quenched with concentrated NH4OH (4.8 ml) and quickly extracted with EtOAc (5x, 100 ml). The combined organic extracts were washed with brine (1x, 100 ml) and dried over MgSO4 and evaporated to afford a yellow solid. The crude material was purified by column chromatography (3 cm OD by 15.5 cm, prepared in 35% EtOAc/hexanes, loaded with 50% EtOAc/hexanes, eluted with 35% EtOAc/hexanes ! 50% EtOAc/hexanes) to supply 36 as a white solid (0.2 g, 66%). ESI-MS: 36 does not ionize; it was derivatized with cyclohexylamine in MeOH. Shows m/z 287.3 (100%) 289.3 (65%) [M-H]- 1H-NMR (DMSO-d6): ? 7.65 (broad s, 1H), 7.52 (broad s, 1H). 13C-NMR (DMSO-d6): ?162.4, 159.7, 158.4, 129.9 TLC: Rf = 0.45 in 50% EtOAc/hexanes   N NClCl ClO NH2 102     N NClHN ClO NH2H2O EtOAc  103   Synthesis of tert-butyl 2-((5-carbamoyl-2,6-dichloropyrimidin-4-yl)amino)acetate (37)   Procedure adapted from Johnson et al. 63  The starting material 36 (27 mg, 0.12 mmol) was solubilized in DCM (1.0 ml, 0.12 mol/L), following which the acetate salt of 15 (27.4 mg, 0.14 mmol) was added. The solution was cooled to -78?C and triethylamine (38 ?L, 27.6 mg, 0.27 mmol) was added. After 1.5 hours, the solution was warmed to -30?C and after another hour to rt. After stirring for yet another hour, the reaction was quenched with water (3.0 ml, solution is pH 6). The two layers were separated and the aqueous layer was washed with DCM (4x, 25 ml). The combined organic extracts were washed once with brine and dried with MgSO4. The 45 mg of crude white material was purified by column chromatography (1 cm ID by N NClCl NHO NH2OtBuOAcetone  104 7 cm, prepared in 70% EtOAc/hexanes, loaded with 100% EtOAc, eluted with 80% EtOAc/hexanes) to afford 37 as an off white solid (36 mg, 93%). ESI-MS (MeOH): m/z 319.3 (100%) 321.3 (64%) [M-H]- 1H-NMR (acetone-d6): ? 8.03 (s, 1H), 7.63 (s, 1H), 7.47 (s, 1H), 4.17 (d, J = 7.6 Hz, 2H), 1.49 (s, 9H). 13C-NMR (acetone-d6): ? 168.5, 164.4, 161.4, 157.7, 154.6, 112.5, 81.4, 44.1, 28.2 TLC: Rf = 0.45 in 80% EtOAc/hexanes      Acetone H2O  105   Synthesis of 2-benzylisothiouronium chloride (41)  To a solution of thiourea (3.0 g, 39.4 mmol) in ethanol (25.0 ml, 1.58 mol/L) was added concentrated HCl (7.5 ml). The reaction was stirred for 45 minutes and then solvent was evaporated to afford a wet residue. This was taken up in MeOH (25.0 ml, 1.58 mol/L) and benzyl bromide (5.5 ml, 7.9 g, 46.2 mmol) was added. After three hours, the reaction was evaporated to dryness, affording a solid. This crude material was recrystallized in methanol and filtered to furnish 41 as a white solid (7.1 g, 90%). ESI-MS (MeOH): 167.3 (100%) [M-HCl+H]+ 1H-NMR (DMSO-d6): ? 9.30 (broad s, 2H), 9.22 (broad s, 2H), 7.51-7.24 (m, 5H), 4.49 (s, 2H) TLC: Rf = 0.35 in 10% MeOH/EtOAc  -Cl+H2NSNH2 106       DMSO H2O Et2O Et2O  107 Synthesis of tert-butyl 2-(5-amino-7-(benzylthio)-2,4-dioxo-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)acetate (42)  To a round bottom flask charged with 19 (0.4 g, 1.4 mmol) was added anhydrous ethanol (6.0 ml, 0.23 mol/L) and triethylamine (0.2 ml, 0.16 g, 1.6 mmol), followed by benzylthiourea 41 (0.3 g, 1.6 mmol). The reaction was stirred under reflux for 5 hours and then cooled to room temperature, upon which an orange solid precipitated. The volatiles were removed in vacuo and the crude material was purified by column chromatography (1.0? OD by 7.5?, prepared in 20% EtOAc/hexanes, loaded with 10% acetone/EtOAc, eluted with 30% EtOAc/hexanes ! 50% EtOAc/hexanes) to afford 42 as an off-white solid (0.17 g, 30%). ESI-MS (MeOH): m/z 438.3 (15%) [M+Na]+, 416.3 (100%) [M+H]+, 360.3 (45%) [M-tBu+H]+ 1H-NMR (DMSO-d6): ? 11.76 (s, 1H), 8.44-8.17 (m, 2H), 7.48-7.11 (m, 5H), 4.62 (s, 2H), 4.33 (s, 2H), 1.39 (s, 9H) TLC: Rf = 0.40 in 40% EtOAc/hexanes  HNN NOOOOtBuNSNH2Ph 108     DMSO H2O EtOAc EA EA  109 Synthesis of 2-(5-amino-7-(benzylthio)-2,4-dioxo-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)acetic acid (43)  To a round bottom flask containing the starting material 42 (77.0 mg, 0.19 mmol) was added DCM (2.0 ml, 0.09 mol/L) followed by TFA (0.2 ml, 5.22 mmol). The reaction was stirred for 5 hours and the volatiles were removed by rotary evaporation to afford a brown paste. Cold diethyl ether (3 ml) was added to cause the precipitation of a light brown solid. The supernatant was removed to reveal 43, which was used without further purification (quantitative yield). ESI-MS (MeOH): m/z 358.0611 (100%) [M-H]+ 1H-NMR (Acetone-d6): ? 11.77 (s, 1H), 8.43-8.12 (m, 2H), 7.45-7.10 (m, 5H), 4.68 (s, 2H), 4.27 (s, 2H) TLC: Rf = 0.05 in 100% EtOAc   HNN NOOOOHNSNH2Ph 110   Synthesis of propyl 2-(2-(5-amino-7-(benzylthio)-2,4-dioxo-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)-N-(2-((tert-butoxycarbonyl)amino)ethyl)acetamido)acetate (46)  Procedure adapted from Bezer et al. 64  To a round bottom flask with carboxylic acid 43 (0.3 g, 0.72 mmol) was added HOBt-H2O (0.1 g, 0.81 mmol based on HOBt) and anhydrous DMF (10.5 ml, 0.07 mol/L). The flask was cooled to 0?C and EDC-HCl (0.15 g, 0.79 mmol) was added. The reaction was stirred for 10 minutes and then it was warmed up to room temperature for 2.5 hours. The flask was re-cooled to 0?C and PNA backbone 7 (0.2 g, 0.81 mmol) dissolved in DMF (1.0 ml, 0.81 mol/L) was added, after which the reaction mixture was warmed to room temperature overnight. Excess DMF was removed by rotary evaporation and the orange paste was purified by column chromatography (3 cm OD by 16.5 cm, prepared in 60% HNN NOOONNSNH2PhOO NHBocEtOAc + H2O in TFA  111 EtOAc/hexanes, loaded with 50% EtOAc/hexanes + 0.5% AcOH, eluted with 50% EtOAc/hexanes + 0.5% AcOH ! 80% EtOAc/hexanes + 0.5% HOAc) to afford a mixture of 43 and 46 (yellow oil). The combined fractions were diluted with NaHCO3 to pH 8.5 and extracted with EtOAc (75 ml, 3x). The organic extracts were dried over Na2SO4 and evaporated to afford 46 as a pale yellow solid (0.35 g, 81%). The aqueous portion could be acidified and extracted with EtOAc to regenerate 43. ESI-MS (MeOH): m/z 624.2 (100%) [M+Na]+ 1H-NMR (DMSO-d6): ? 11.71 (s, 1H), 8.34 (s, 1H), 8.26 (s, 1H), 7.40 (t, J = 5.9 Hz, 2H), 7.34-7.25 (m, 3H), 4.96 (s, 2H), 4.39 (s, 2H), 4.03 (s, 2H), 3.99 (t, J = 6.6 Hz, 2H), 3.52-3.43 (m, 2H), 3.23-3.13 (m, 2H), 1.57 (t, J = 7.1 Hz, 2H), 1.37 (s, 9H), 0.85 (t, J = 6.2 Hz, 3H) TLC: Rf = 0.66 in 100% EtOAc   112   Synthesis of 2-(2-(5-amino-7-(benzylthio)-2,4-dioxo-3,4-dihydropyrimido[4,5-d] pyrimidin(2H)-yl)-N-(2-((tert-butoxycarbonyl)amino)ethyl)acetamido)acetic acid (4)   To a round-bottomed flask containing starting material 44 (0.35 g, 0.6 mmol) was added water (6.0 ml) and MeOH (7.0 ml, final concentration 0.044 mol/L). Sodium hydroxide (58 mg, 1.5 mmol) was added and the reaction stirred for 3 hours. The volatiles were removed by rotary evaporation and the residue taken up in NaHCO3 (120 ml, pH 8.5). The aqueous layer was extracted with ethyl acetate (2x, 75 ml; these washes discarded) and the aqueous portion was acidified to pH 2 with concentrated HCl, upon which a white solid precipitated. The aqueous layer was extracted with ethyl acetate (5x, 80 ml), HNN NOOONNSNH2PhOHO NHBocDMSO H2O Acet. Unknown impur. integrates for 0.2  113 and the combined organic extracts were dried with Na2SO4 and evaporated to afford 4 as a white solid (0.28 g, 87%). ESI-MS (MeOH): m/z 582.3 (100%) [M+Na]+ 1H-NMR (DMSO-d6): ? 11.69 (s, 1H), 8.29 (s, 1H), 8.21 (s, 1H), 7.42-7.27 (m, 5H), 4.95 (s, 2H), 4.38 (s, 2H), 3.94 (s, 2H), 3.22 (m, 4H), 1.37 (s, 9H). 13C-NMR (DMSO-d6): ? 174.5, 171.0, 167.2, 163.1, 162.0, 157.9, 156.2, 150.5, 137.7, 129.4, 128.9, 127.6, 87.1, 78.5, 49.6, 48.1, 47.6, 42.3, 34.8, 28.6 TLC: Rf = 0.30 in 40% MeOH/EtOAc    Acet. H2O in DMSO (baseline) EtOAc?  114    Synthesis of methyl trifluoromethanesulfonate (48)  Procedure adapted from Booth et al. 65  In a dry round bottom flask was added triflic anhydride (3.4 ml, 5.6 g, 20.0 mmol). After cooling the flask to 0?C, anhydrous methanol (0.8 ml, 19.8 mmol) was added dropwise and the reaction was stirred for 15 minutes. The rubber septum was replaced with a distillation condenser and methyl sulfonate 48 was distilled under Ar a temperature of 100?C as a clear light yellow liquid (yield uncalculated). A pig adapter is employed because methanol (boiling point 65?C) and triflic anhydride (82?C) distill first. S OOOF3CImpurity?  115 ESI-MS: Product does not ionize under ESI-MS conditions. 1H-NMR (CDCl3): ? 4.21 (s, 3H). 19F-NMR (CDCl3): ? -74.23 (s, 3H), -76.14 (s 1H)       Synthesis of benzyl 1H-imidazole-1-carboxylate (49)   NN OOPhCHCl3 Trifluoromethyl sulfonic acid?  116 Procedure adapted from Komiyama et al. 66  To a flask at 0?C with imidazole (11.2 g, 164.5 mmol) and toluene (80.0 ml, 2.05 mol/L) was added benzyl chloroformate (14.6 ml, 17.4 g, 102.3 mmol) and the mixture was warmed to room temperature overnight. The next day, the mixture was filtered to afford a yellow filtrate, which was evaporated to dryness. Purification by chromatography (5 cm OD by 20 cm, prepared, loaded and eluted with 50% EtOAc/hexanes) furnished 49 as a clear colorless oil (9.2 g for half of the crude, 89% yield). 1H-NMR (Acetone-d6): ? 8.22 (s, 1H), 7.52 (broad s, 6H), 7.11 (s, 1H), 5.47 (s, 2H) TLC: Rf = 0.40 in 50% EtOAc/hexanes   Synthesis of 1-((benzyloxy)carbonyl)-3-methyl-1H-imidazol-3-ium trifluoromethane-sulfinate (50)  Procedure adapted from Komiyama et al. 66  N N+OOPhCF3SO2-Acetone and EtOAc EtOAc  117 To a solution of 49 (4.7 g, 23.2 mmol) in DCM (20.0 ml, 1.16 mol/L) at 0?C was added 48 (produced from 3.36 ml of triflic anhydride). The reaction was stirred for 30 minutes at rt, after which cold diethyl ether (15 ml) was added. The precipitate was filtered and washed with ether (120 ml) to afford 50 as a white solid (6.3 g, 69%). ESI-MS (MeCN): Does not exhibit molecular ion peak under ESI conditions. 1H-NMR (DMSO-d6): ? 9.84 (s, 1H), 8.13 (s, 1H), 7.81 (s, 1H), 7.48-7.21 (m, 5H), 5.51 (s, 2H), 3.87 (s, 3H). 19F-NMR (CDCl3): -78.16 (s, 1H) 13C-NMR (DMSO-d6): ? 146.3, 139.1, 134.0, 129.5, 129.2, 129.1, 125.1, 120.4, 71.9, 36.9.    DMSO H2O DCM  118    Synthesis of tert-butyl 2-(6-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetate (51)  Procedure adapted from Boens et al. 67  HNNOOOtBuO 119 To a round bottom flask containing commerically available 6-methyluracil (3.1 g, 24.6 mmol) and DMF (80 ml, 0.31 mol/L) was added freshly ground potassium carbonate (3.4 g, 24.6 mmol). Commercially purchased bromo tert-butyl acetate (3.6 ml, 4.76 g, 24.6 mmol) was added dropwise over 15 minutes and the heterogeneous reaction mixture was stirred overnight at room temperature. The following day, the mixture was diluted with 5% aqueous LiCl (200 ml) and extracted with EtOAc (15x, 150 ml). The combined organic extracts were dried over MgSO4 and concentrated to dryness. Column purification of the crude material (1.5? OD by 6.75?, prepared in 50% EtOAc/hexanes, dry loaded, eluted with 60% EtOAc/hexanes ! 100% EtOAc) afforded 51 as a white solid (2.1 g, 36%). ESI-MS (MeOH): 239.4 (100%) [M-H]- 1H-NMR (DMSO-d6): ? 11.29 (s, 1H), 5.53 (s, 1H), 4.48 (s, 2H), 2.11 (s, 3H), 1.41 (s, 9H). TLC: Rf = 0.45 in 100% EtOAc  120     DMSO H2O  121 Synthesis of 2-(6-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetic acid (52)  To a round bottom flask charged with 51 (2.0 g, 8.3 mmol) was added DCM (18.0 ml, 0.46 mol/L) followed by trifluoroacetic acid (11.2 ml, 146.3 mmol). The reaction was stirred for 6 hours at room temperature and the volatiles were then removed on a rotary evaporator equipped with a water aspirator to afford an orange paste. Addition of diethyl ether (20 ml) caused precipitation of a solid that was filtered, washed with diethyl ether and dried in vacuo to afford 52 as a white solid (1.5 g, 98%). ESI-MS (MeOH): m/z 183.3 (100%) [M+H]+ 1H-NMR (DMSO-d6): ? 11.27 (s, 1H), 5.53 (s, 1H), 4.49 (s, 2H), 2.09 (s, 3H). TLC: Rf = 0.20 in 100% EtOAc    HNNOOOHO 122   Synthesis of propyl 2-(N-(2-((tert-butoxycarbonyl)amino)ethyl)-2-(6-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)acetate (53)  Procedure adapted from Bezer et al. 64  To a round bottom flask containing 52 (0.5 g, 2.7 mmol) was added HOBt-H2O (0.4 g, 3.0 mmoles based on anhydrous HOBt), followed by dry DMF (39.0 ml, 0.07 mol/L vs. starting material). The reaction mixture was cooled to 0?C and EDC-HCl (0.6 g, 3.0 mmol) was added, followed by stirring for 10 minutes. The reaction mixture was warmed to room temperature and stirred for 2.5 hours then re-cooled to 0?C. A solution of propyl PNA backbone 7 (0.8 g, 3.0 mmol) in DMF (22 ml, 0.14 mol/L) was then added followed HNNOONOOONHBocH2O DMSO  123 by stirring for 15 minutes. The reaction mixture was allowed to warm up to room temperature overnight. The following day, evaporation at 45?C under a 1 mmHg vacuum revealed a yellow solution, which was purified by chromatography (1.5? ID by 8.5?, prepared in 0.6% AcOH/EtOAc, loaded with EtOAc, eluted with 0.6% AcOH/EtOAc ! 2% MeOH/EtOAc / 0.5% AcOH) to afford 53 as a yellow oil (1.1 g, 95%). ESI-MS (MeOH): m/z 425.5 (100%) [M-H]- 1H-NMR (CDCl3): ? 10.13 (s, 1H), 5.79 (s, 1H), 5.49 (s, 1H), 4.71 (s, 2H), 4.14 (s, 1H), 3.99 (t, J = 6.8 Hz, 3H), 3.44 (m, 4H), 2.08 (s, 3H), 1.57 (sextet, J = 6.9 Hz, 2H), 1.34 (s, 9H), 0.83 (t, J = 7.4 Hz, 3H) 13C-NMR (CDCl3): ? 169.7, 167.3, 163.2, 156.1, 154.4, 152.0, 102.0, 79.7, 67.1, 48.7, 44.6, 38.6, 28.3, 21.8, 19.8, 10.2 TLC: Rf = 0.20 in 100% EtOAc    124       CHCl3 CDCl3 ? Ethyl acetate EtOAc Ethyl acetate  125 Synthesis of 2-(N-(2-((tert-butoxycarbonyl)amino)ethyl)-2-(6-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)acetic acid (5)  To a round bottom flask charged with 53 (1.2 g, 2.6 mmol) was added water (28.0 ml) and MeOH (32.0 ml, final concentration is 0.045 mol/L). Sodium hydroxide (0.3 g, 6.8 mmol) was added and the reaction was stirred at room temperature for 2 hours. The MeOH was removed in vacuo and the aqueous layer was diluted with saturated NaHCO3 (120 ml). The aqueous extract was washed with EtOAc (2x, 75 ml), and these washes were discarded. The aqueous portion was acidified to pH 2 with concentrated HCl and extracted with EtOAc (2x, 100 ml). The combined organic layers were dried over Na2SO4 and evaporated to afford 5 as a white solid (0.9 g, 88%). ESI-MS (MeOH): m/z 407.1618 (100%) [M+Na]- 1H-NMR (DMSO-d6): ? 11.19 (s, 1H), 6.90 (s, 1H), 5.51 (s, 1H), 4.79 (s, 2H), 3.96 (s, 2H), 3.32-3.03 (m, 4H), 2.08 (s, 3H), 1.37 (s, 9H) 13C-NMR (DMSO-d6): ? 170.8, 167.7, 163.0, 156.2, 155.0, 152.1, 101.3, 78.5, 49.4, 48.0, 44.5, 38.4, 28.6, 19.4 TLC: Rf = 0.45 in 50% MeOH/EtOAc  HNNOONOHOONHBoc 126      4.3 Protocol for Solid Phase Synthesis Downloading the Resin (Procedure adapted from Nielson et al. 68) A 10 ml centrifuge column was charged with PS-MBHA resin HCl (100-200 Mesh, advertised at 0.67 mmol/g) and gently shaken overnight with DCM (8 ml). The solvent was drained and the resin was washed with 5% DIPEA/DCM (1 min) and DCM (1 min).   127 Falcon tube A: Fmoc-AEEA-OH (Panagene, 77 mg, 0.20 mmol), NMP (2.5 ml, 0.080 mol/L) and diisopropylethylamine (72 ?L, 0.40 mmol). Falcon tube B: HBTU (77 mg, 0.20 mmol) and NMP (1.4 ml, 0.15 mol/L).  The contents of A and B were mixed with shaking (2 min), and added to the resin, after which NMP (2 ml) was added. The column was shaken for 5 hours, and then the resin was washed with DCM (1x, 1 min), DMF (1x, 1 min), DCM (1x, 1 min), 5% DIPEA/DCM (1x 1 min) and DCM (1x, 1 min). Kaiser test proved positive.  To the resin was added Ac2O/DMF/pyridine (2/48/50, 6.0 ml) and the reaction was shaken gently overnight. The next day the beads were washed with DCM (300 ml) and the Kaiser test proved negative. The centrifuge column was dried for 2.5 hours.  Determining Loading (performed in duplicate) A Falcon tube containing the resin (50.0 mg) and 2% DBU/DMF (20 ml) was shaken for 1 hour. The contents were quantitatively transferred to a 100.00 ml volumetric flask and diluted with acetonitrile. From this solution, 2.0 ml of the supernatant was removed and diluted to 10.00 ml. The absorbance was recorded at 294 nm and 304 nm and the loading was 0.2036 mmol/gram as determined by standard methods. 69  Deprotection of Resin (Removal of Terminal Protecting Group) (References 66, 68) Terminal Fmoc: The downloaded resin (49.2 mg, 10 ?mol) was swollen for two hours in DCM (1.5 ml). The resin was washed with 20% piperidine/DMF (4x, 1 min, 2.0 ml)  128 followed by DMF (4x, 2 ml, 2 min), DCM (1x, 2 min), 5% DIPEA/DMF (4x, 1 min) and DCM (1 min). The Kaiser test proved positive. Terminal Boc: The dried resin was washed with TFA/DCM/m-cresol (4x, 50/45/5, 2 ml, 3 min), DCM (100 ml) and DMF (2x, 30 sec). Then it was washed with 5% DIPEA/DCM (2x, 1 min, 2 ml), DMF (30 sec) and DCM (10 sec). Kaiser test proved positive.  Coupling of Monomers In an Eppendorf tube were added the PNA monomer (For 5: 19.0 mg, 49.4 ?mol; for 4: 27.5 mg, 49.2 ?mol) and HBTU (For 4 and 5: 19.0 mg, 50.1 mmol). NMP (1.00 ml, 0.05 mol/L; concentration increased to 0.1 mol/L for difficult couplings) and diisopropylethylamine (17.4 ?L, 100 ?mol) were added, and the tube was gently shaken for 1.5 minutes at rt.  The pre-activated monomer was transferred to the resin and the column was shaken (for 5: 30 min; for 4: 2 hours). The solvent was removed and the beads were washed with DMF (2 min) and DCM (2 min). The Kaiser test was performed alongside the crude MBHA resin in order to avoid false positives.  Capping The resin was treated with Ac2O/DMF/pyridine (30/20/50, 2 ml) and gently shaken for two hours. The solvent was removed and the beads were washed with DCM (200 ml). The Kaiser test proved negative.   129 Oxidation and Ammonolysis (only applicable to Janus AT sequence) For oxidation, 25 mg (5 ?mol) of the resin was thoroughly dried and then suspended in DCM (1.0 ml). Then, mCPBA (5 equivalents per Janus heterocycle, 22 mg, 77% effective, 0.1 mmol) was dissolved and the resin was shaken for 6 hours at room temperature. The solvent was then drained and the resin was washed with DCM (2 min), DMF (2 min) and then DCM (2 min), and then dried in vacuo. The beads were transferred to an Eppendorf tube, sealed, re-suspended in 7N ammonia in methanol (1.5 ml) and incubated at 50?C for 4 hours. After this time, the excess solvent was removed by centrifugation, transferred to a centrifuge column, and then washed with DCM (200 ml). The crude oligomer was cleaved from the solid support as described below.  Removal from Solid Support The resin was cooled to 4?C and treated with TFA (0.66 ml, 8.6 mmol) and m-cresol (67 ?L, 0.64 mmol), followed by the dropwise addition of TFMSA (100 ?L, 1.13 mmol). The resin was suspended for 30 minutes and then shaken vertically for 95 minutes at room temperature, after which the supernatant was filtered and the beads were washed with neat TFA (0.3 ml). The isolated black liquid was cooled to 0?C and treated with cold diethyl ether (8.5 ml), which precipitated the crude PNA as a fine white residue. The solution was divided into 10 Eppendorfs and centrifuged at 10,000g at 4?C for 15 minutes. The supernatant was discarded and fresh ether (1.0 ml) was added to each Eppendorf. After performing the centrifugation three times in total, the white PNA pellets were dried at 35?C under vacuo. The pellets were desalted on a Sephadex G45 spin  130 column and purified by HPLC, and MALDI characterization was performed using a sinapinic acid matrix.  131 Bibliography 1. Hannon, M. J., Chemical Society Reviews 2007, 36 (2), 280-95. 2. Neidle, S., Nat Prod Rep 2001, 18 (3), 291-309. 3. Wheeler, R. DNA orbit Animated. http://commons.wikimedia.org/wiki/File:DNA_orbit_animated.gif (accessed Jul 18, 2013). 4. Voet, D.; Voet, J. G., Biochemistry. John Wiley & Sons: 2010. 5. Phan, A. T.; Mergny, J. L., Nucleic Acids Research 2002, 30 (21), 4618-25. 6. Crick, F. H. C., Journal of Molecular Biology 1966, 19 (2), 548-55. 7. Sun, J.-S.; Helene, C., Current Opinion in Structural Biology 1993, 3 (3), 345-56. 8. Tse, W. C.; Boger, D. L., Chemistry & Biology 2004, 11 (12), 1607-17. 9. Horvath, M. P. Grooves of DNA. http://courses.biology.utah.edu/horvath/biol.3525/1_DNA/Fig2/ (accessed July 18, 2013). 10. Arunan, E.; Desiraju, R.; Klein, R. A.; Sadlej, J.; Scheiner, S.; Alkorta, I.; Clary, D. C.; Crabtree, R. H.; Dannenberg, J. J.; Hobza, P.; Kjaergaard, H. G.; Legon, A. C.; Mennucci, B.; Nesbitt, D. J., Pure and Applied Chemistry 2011, 83 (8), 1. 11. Sponer, J.; Leszczynski, J.; Hobza, P., The Journal of Physical Chemistry 1996, 100 (5), 1965-74. 12. Baraldi, P. G.; Tabrizi, M. A.; Preti, D.; Fruttarolo, F.; Avitable, B.; Bovero, A.; Pavani, G.; Nunez Carretero, M. D. C.; Romagnoli, R., In DNA minor-groove binders. Design, synthesis, and biological evaluation of ligands structurally related to CC-1065, distamycin, and anthramycin. Pure and applied chemistry: Research Triangle Park, NC, 2003; 75, p 8. 13. Dervan, P. B., Bioorganic and Medicinal Chemistry 2001, 9 (9), 2215-35. 14. Knauert, M. P.; Glazer, P. M., Human Molecular Genetics 2001, 10 (20), 2243-51. 15. Thuong, N. T.; H?l?ne, C., Angewandte Chemie International Edition in English 1993, 32 (5), 666-90. 16. Guntaka, R. V.; Varma, B. R.; Weber, K. T., Int J Biochem Cell Biol 2003, 35 (1), 22-31. 17. Lehn, J.-M.; Mascal, M.; DeCian, A.; Fischer, J., Journal of the Chemical Society, Perkin Transactions 2 1992,  (4), 461-7. 18. Kimizuka, N.; Fujikawa, S.; Kuwahara, H.; Kunitake, T.; Marsh, A.; Lehn, J.-M., Journal of the Chemical Society, Chemical Communications 1995,  (20), 2103-4. 19. Branda, N.; Kurz, G.; Lehn, J.-M., Chemical Communications 1996,  (21), 2443-4. 20. Mascal, M.; Hext, N. M.; Warmuth, R.; Arnall-Culliford, J. R.; Moore, M. H.; Turkenburg, J. P., Journal of Organic Chemistry 1999, 64 (23), 8479-84. 21. Mascal, M.; Farmer, S. C.; Arnall-Culliford, J. R., Journal of Organic Chemistry 2006, 71 (21), 8146-50. 22. Moralez, J. G.; Raez, J.; Yamazaki, T.; Motkuri, R. K.; Kovalenko, A.; Fenniri, H., Journal of the American Chemical Society 2005, 127 (23), 8307-9.  132 23. Fenniri, H.; Mathivanan, P.; Vidale, K. L.; Sherman, D. M.; Hallenga, K.; Wood, K. V.; Stowell, J. G., Journal of the American Chemical Society 2001, 123 (16), 3854-5. 24. Asadi, A.; Patrick, B. O.; Perrin, D. M., Journal of Organic Chemistry 2007, 72 (2), 466-75. 25. Chen, H.; Meena; McLaughlin, L. W., Journal of the American Chemical Society 2008, 130 (40), 13190-1. 26. Sessler, J. L.; Jayawickramarajah, J.; Sathiosatham, M.; Sherman, C. L.; Brodbelt, J. S., Org Lett 2003, 5 (15), 2627-30. 27. Beingessner, R. L.; Diaz, J. A.; Hemraz, U. D.; Fenniri, H., Tetrahedron Letters 2011, 52 (6), 661-4. 28. Pan, M.-Y.; Hang, W.; Zhao, X.-J.; Zhao, H.; Deng, P.-C.; Xing, Z.-H.; Qing, Y.; He, Y., Organic & Biomolecular Chemistry 2011, 9 (16), 5692-702. 29. Shin, D.; Tor, Y., Journal of the American Chemical Society 2011, 133 (18), 6926-9. 30. Nielson, P. E.; Egholm, M., Peptide Nucleic Acids: Protocols and Applications. Horizon Scientific Press: Wymondham, England, 1999. 31. Nielson, P. E.; Egholm, M., Peptide Nucleic Acids: Protocols and Applications. Horizon Scientific Press: Wymondham, England, 1999. 32. Vearing, C. J.; Fecondo, J. V., Letters in Peptide Science 2002, 9 (4-5), 211-9. 33. Bialy, L.; D?az-Moch?n, J. J.; Specker, E.; Keinicke, L.; Bradley, M., Tetrahedron 2005, 61 (34), 8295-305. 34. Pl?ger, T. A.; von Kiedrowski, G., Helvetica Chimica Acta 2011, 94 (11), 1952-80. 35. P?tureau, B. M.; Hudson, R. H. E.; Damha, M. J., Bioconjugate Chemistry 2007, 18 (2), 421-30. 36. Riva, E.; Comi, D.; Borrelli, S.; Colombo, F.; Danieli, B.; Borlak, J.; Evensen, L.; Lorens, J. B.; Fontana, G.; Gia, O. M.; Via, L. D.; Passarella, D., Bioorganic & Medicinal Chemistry 2010, 18 (24), 8660-8. 37. Milic, D.; Prato, M., European Journal of Organic Chemistry 2010, 2010 (3), 476-83. 38. Asadi, A.; Patrick, B. O.; Perrin, D. M., Journal of Organic Chemistry 2007, 72 (2), 466-75. 39. Jung, M.-H.; El-Gamal, M. I.; Abdel-Maksoud, M. S.; Sim, T.; Yoo, K. H.; Oh, C.-H., Bioorganic & Medicinal Chemistry Letters 2012, 22 (13), 4362-7. 40. Largy, E.; Liu, W.; Hasan, S.; Perrin, D., Chembiochem 2013, In Press. 41. Hollenstein, M.; Leumann, C. J., The Journal of Organic Chemistry 2005, 70 (8), 3205-17. 42. Seto, S.; Okada, K.; Kiyota, K.; Isogai, S.; Iwago, M.; Shinozaki, T.; Kitamura, Y.; Kohno, Y.; Murakami, K., Journal of Medicinal Chemistry 2010, 53 (13), 5012-24. 43. Li, C.; Rosenau, A., Tetrahedron Letters 2009, 50 (43), 5888-93. 44. Carruthers, W., Cycloaddition reactions in organic synthesis. Pergamon Press: 1990. 45. Adalsteinsson, O.; Lamotte, A.; Baddour, R. F.; Colton, C. K.; Pollak, A.; Whitesides, G. M., Journal of Molecular Catalysis 1979, 6 (3), 199-225.  133 46. Roberts, J. M.; Veres, P. R.; Cochran, A. K.; Warneke, C.; Burling, I. R.; Yokelson, R. J.; Lerner, B.; Gilman, J. B.; Kuster, W. C.; Fall, R.; de Gouw, J., Proceedings of the National Academy of Sciences 2011, 108 (22), 8966-71. 47. Komiyama, M.; Aiba, Y.; Ishizuka, T.; Sumaoka, J., Nature Protocols 2008, 3 (4), 646-54. 48. Hudson, R. H. E.; Wojciechowski, F., Canadian Journal of Chemistry 2005, 83 (10), 1731-40. 49. Wilson, W., Journal of Chemical Society 1948, 1157-61. 50. Armarego, W. L. F.; Chai, C. L. L., Purification of Laboratory Chemicals. Butterworth-Heinemann: 2003. 51. Werkhoven, T. M.; van Nispen, R.; Lugtenburg, J., European Journal of Organic Chemistry 1999, 1999 (11), 2909-14. 52. Kofoed, T.; Hansen, H. F.; ?rum, H.; Koch, T., Journal of Peptide Science 2001, 7 (8), 402-12. 53. Milic, D.; Prato, M., European Journal of Organic Chemistry 2010, 2010 (3), 476-83. 54. Siebum, Arjan H. G.; Woo, Wei S.; Lugtenburg, J., European Journal of Organic Chemistry 2003, 2003 (23), 4664-78. 55. Bach, A.; Stuhr-Hansen, N.; Thorsen, T. S.; Bork, N.; Moreira, I. S.; Frydenvang, K.; Padrah, S.; Christensen, S. B.; Madsen, K. L.; Weinstein, H.; Gether, U.; Stromgaard, K., Organic & Biomolecular Chemistry 2010, 8 (19), 4281-8. 56. Asadi, A.; Patrick, B. O.; Perrin, D. M., Journal of Organic Chemistry 2007, 72 (2), 466-75. 57. Dickey, J. B.; Gray, A. R., Barbituric Acid. In Organic Syntheses, John Wiley & Sons, Inc.: 2003. 58. Langerman, M.-J.; Banks, C. K., Journal of the American Chemical Society 1951, 73 (7), 3011-2. 59. Borzsonyi, G.; Beingessner, R. L.; Yamazaki, T.; Cho, J.-Y.; Myles, A. J.; Malac, M.; Egerton, R.; Kawasaki, M.; Ishizuka, K.; Kovalenko, A.; Fenniri, H., Journal of the American Chemical Society 2010, 132 (43), 15136-9. 60. Seto, S.; Tanioka, A.; Ikeda, M.; Izawa, S., Bioorganic & Medicinal Chemistry Letters 2005, 15 (5), 1485-8. 61. Travis, B. R.; Sivakumar, M.; Hollist, G. O.; Borhan, B., Organic Letters 2003, 5 (7), 1031-4. 62. Balsamo, A.; Crotti, P.; Lapucci, A.; Macchia, B.; Macchia, F.; Cuttica, A.; Passerini, N., Journal of Medicinal Chemistry 1981, 24 (5), 525-32. 63. Johnson, R. S.; Yamazaki, T.; Kovalenko, A.; Fenniri, H., Journal of the American Chemical Society 2007, 129 (17), 5735-43. 64. Bezer, S.; Rapireddy, S.; Skorik, Y. A.; Ly, D. H.; Achim, C., Inorganic Chemistry 2011, 50 (23), 11929-37. 65. Booth, B. L.; Haszeldine, R. N.; Laali, K., Journal of the Chemical Society, Perkin Transactions 1 1980,  (0), 2887-93. 66. Komiyama, M.; Aiba, Y.; Ishizuka, T.; Sumaoka, J., Nature Protocols 2008, 3 (4), 646-54. 67. Boens, B.; Teste, K.; Hadj-Bouazza, A.; Ismaili, J.; Zerrouki, R., Nucleosides, Nucleotides and Nucleic Acids 2012, 31 (3), 197-205.  134 68. Nielson, P. E.; Egholm, M., Peptide Nucleic Acids: Protocols and Applications. Horizon Scientific Press: Wymondham, England, 1999. 69. EMD Millipore Novabiochem Catalog for 2010/2011.   

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.24.1-0166851/manifest

Comment

Related Items