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Synthetic Efforts toward Self-Complementary Quadruply Hydrogen Bonded Ureidopurines

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Synthetic Efforts toward Self-Complementary Quadruply Hydrogen Bonded Ureidopurines
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GIESSERT, RACHEL ( Author, Primary )
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2008

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Dimers ( jstor )
DNA ( jstor )
Hydrogen ( jstor )
Hydrogen bonds ( jstor )
Isocyanates ( jstor )
Molecules ( jstor )
Nucleobases ( jstor )
Polymers ( jstor )
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University of Florida
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University of Florida
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Copyright Rachel Giessert. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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12/31/2008
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SYNTHETIC EFFORTS TOWARD S ELF-COMPLEMENTARY QUADRUPLY HYDROGEN BONDED UREIDOPURINES By RACHEL GIESSERT A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Rachel Giessert

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This document is dedicate d to Thomas William Chornoby.

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ACKNOWLEDGMENTS I want to thank Petros for his love and support. I want to thank my parents for always encouraging me and loving me no matter what. I also want to thank the Castellano group: Alisha and Roslyn for laboratory help, Ron for constant guidance, and Andy for answering chemistry questions. To all my other friends and family, I give my gratitude for my support network. I want to thank the Alumni Fellowship for funding and The University of Florida for facilities. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF FIGURES..........................................................................................................vii ABSTRACT.........................................................................................................................x CHAPTER 1 INTRODUCTION........................................................................................................1 Hydrogen Bonds...........................................................................................................1 Hydrogen Bonding in Organic Crystal Structures........................................................1 Hydrogen Bonding in DNA..........................................................................................3 Applications of Nucleobases and Nucleobase Analogues............................................4 Guanine and Cytosine Dimers...............................................................................4 Guanosine Quadruplexes.......................................................................................6 Synthetic Nucleobases...........................................................................................7 DNA Nanotechnology...........................................................................................7 Quadruple Hydrogen Bonds.........................................................................................8 Secondary Interactions..........................................................................................8 Preorganization and Tautomerization..................................................................10 Self-Complementary Systems.............................................................................10 Complementary Systems.....................................................................................11 Applications of Quadruple Hydrogen Bonds in Functional Materials and Assemblies.......................................................................................................13 Supramolecular polymers.............................................................................13 Host-guest chemistry....................................................................................14 Self-assembled architectures........................................................................15 Functional materials.....................................................................................16 Beyond Quadruply Hydrogen Bonded Systems.........................................................17 2 DESIGN OF QUADRUPLE HYDROGEN BONDING UREIDOPURINES...........20 3 RESULTS AND DISCUSSION.................................................................................26 General Synthetic Considerations...............................................................................26 Strategy 1....................................................................................................................27 Strategy 2....................................................................................................................29 v

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Strategy 3....................................................................................................................33 Strategy 4....................................................................................................................34 4 CONCLUSION AND OUTLOOK.............................................................................36 5 EXPERIMENTAL......................................................................................................37 General........................................................................................................................37 2-Amino-6-chloro-purine (3a)....................................................................................37 2-Amino-6-chloro-9-benzyl-purine (3b)....................................................................38 4-n-Heptylphenyl isocyanate (8)................................................................................39 9-(2,3,5-Tri-O-acetyl--D-ribofuranosyl)-2-amino-6-chloro-purine (10)..................39 2,6-Diamino--D-ribofuranosylpurine (4a)................................................................40 2,6-Diamino-9-[(3’,5’-O-tetraisopropyldisiloxane-1,3-diyl)--D-ribofuranosyl]purine (4b)........................................................................................40 2,6-Diamino-9-[(3’,5’-O-tetraisopropyldisiloxane-1,3-diyl)-2’-O-methyl--D-ribofuranosyl]purine (4c)........................................................................................41 2-Phenylurea-6-amino-9-[(3’,5’-O-tetraisopropyldisiloxane-1,3-diyl)-2’-O-methyl--D-ribofuranosyl]purine (1a)....................................................................42 APPENDIX NMR SPECTRA..........................................................................................43 LIST OF REFERENCES...................................................................................................44 BIOGRAPHICAL SKETCH.............................................................................................48 vi

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LIST OF FIGURES Figure page 1-1 All molecules shown form two hydrogen bonds........................................................2 1-2 A dimer of melamine and cyanuric acid binds noncovalently via three hydrogen bonds, which forms a lattice in the solid state............................................................3 1-3 Watson-Crick base pairing of guanine and cytosine..................................................4 1-4 Nucleobase-substituted porphyrins that experience photoinduced energy transfer when brought close in space by rigid triple hydrogen bonds.....................................5 1-5 Hydrogen bond directed complexation of a CG base pair bound to The Zimmerman Groups’ receptor unit.............................................................................5 1-6 Guanosine quadruplex with a cation bound (for example K + )...................................6 1-7 Synthetic nucleobase pair of pyAAD and puDDA....................................................7 1-8 Heterodimers showing Jorgensen’s secondary interactions.......................................8 1-9 Representation of Jorgensen’s theory of secondary interactions...............................9 1-10 Meijer and coworkers’ ADAD self-complementary dimers....................................11 1-11 Examples of one conformation of one tautomer of a self-complementary DDAA dimer.........................................................................................................................11 1-12 Three complexes containing DAAD-ADDA hydrogen bonding.............................12 1-13 Hydrazide based quadruply hydrogen bonded heterodimer with an ADDADAAD pattern..........................................................................................................12 1-14 Representation of a supramolecular polymer...........................................................13 1-15 Corbin and Zimmerman’s three tautomeric forms of a heterocycle, which all form quadruply hydrogen bonded dimers................................................................14 1-16 A schematic representation of a barbiturate binding site.........................................14 1-17 Utilizing 2-ureido-4[1H]-pyrimidinone units on a surface to form a monolayer.....15 vii

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1-18 Poly(ethylene glycol) with terminal functional groups [2-(6-isocyanatohexylaminocarbonylamino)-6-methyl-4[1H]pyrimidinone quadruple hydrogen bonding units for use as a supramolecular electrolyte host in dye-sensitized solar cells.................................................................................................16 1-19 Corbin and Zimmerman’s heterodimer of DDAADD bis-ureido-naphthyridine complexed with AADDAA bis-naphthyrdinyl-urea................................................18 1-20 Bing Gong and coworkers’ heterodimer DADDAD-ADAADA of oligoamides....18 2-1 Heterocycles that form self-complementary and complementary quadruply hydrogen bonded units.............................................................................................21 2-2 A ureidopurine, based on guanosine, and the two conformers formed from different intramolecular hydrogen bonds.................................................................23 2-3 Basic purine system showing the four likely conformers in solution......................23 2-4 Ureidopurines synthesized by Roslyn LaMastus.....................................................24 2-5 X-ray crystal structure of 2-phenylurea-6-dimethylamino-9-benzylpurine.............25 2-6 If the NMe 2 of B in Figure 2-4 is replaced with NH 2 , aryl interactions stabilize an intramolecularly hydrogen bonded conformer leading to a dimer......................25 3-1 Scheme of the general design to synthesize a quadruply hydrogen bonded ureidopurine.............................................................................................................26 3-2 Synthetic route to form 3a starting from 5...............................................................27 3-3 Benzylation of 3a to form 3b...................................................................................28 3-4 Preparation of 8 from heptylphenyl acid chloride (6) using sodium azide via a Curtius rearrangement..............................................................................................28 3-5 Unsuccessful reaction of 3b and 8 to form 2a.........................................................29 3-6 The new starting material 4c....................................................................................29 3-7 Reaction route showing the conversion of 9 to 2,6-diaminopurine 4a....................30 3-8 Scheme of the reaction of 4a with TIPDSiCl 2 to protect the 3’ and 5’ hydroxyl groups to form 4b.....................................................................................................31 3-9 Alkylation of the unprotected 2’ hydroxyl group of 4b to form 4c.........................31 3-10 Synthesis of 1a from 4c............................................................................................32 3-11 Conformers of 1a.....................................................................................................33 viii

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3-12 Target compound of Strategy 3, 1b..........................................................................33 3-13 Reaction of 4c with CDI to form an active acyl intermediate 11.............................34 3-14 Scheme of the reaction of 4d with PhNCO to form the urea 1c..............................35 ix

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science SYNTHETIC EFFORTS TOWARD SELF-COMPLEMENTARY QUADRUPLY HYDRODEN BONDED UREIDOPURINES By Rachel Giessert December 2005 Chair: Ronald K. Castellano Major Department: Chemistry Quadruply hydrogen bonded dimers, both complementary and self-complementary, are useful for the construction of supramolecular polymers and functional -conjugated materials. Quadruple hydrogen bonding units derived specifically from nucleobases, pyrimidines and purines may also have the ability to intercalate DNA and to function in synthetic biopolymers. The design of and synthetic efforts toward the first self-complementary quadruply hydrogen bonded ureidopurines are presented. Four synthetic strategies are described that take into consideration solubility of the heterocycles, tautomerization possibilities, intramolecular hydrogen bonding potential, and reactivity. The most successful approach has resulted in 2-phenylurea-6-amino-9-[(3’,5’-O-tetraisopropyldisiloxane-1,3-diyl)-2’-O-methyl--D-ribofuranosyl]purine in three steps from commercially-available material. Although the target is easily accessed and soluble in organic solvents, steric interactions between the N(9) sugar and phenyl urea result in a monomer conformation that precludes formation of a quadruply hydrogen bonded dimer. x

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Exclusive intramolecular hydrogen bonding to N(1) has been characterized by 1 H NMR spectroscopy and molecular modeling. The synthetic advances made in this work have led to a new synthetic approach to the self-complementary ureidopurines that currently is in progress. xi

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CHAPTER 1 INTRODUCTION Hydrogen Bonds Noncovalent interactions, namely hydrogen bonds, have been of interest in supramolecular chemistry for many years. 1-5 Hydrogen bonds are theoretically well-understood and oft employed throughout chemical and biological research. Hydrogen bonds are arguably the most useful noncovalent interaction in supramolecular chemistry for both noncovalent and kinetic reasons. In supramolecular assemblies, hydrogen bonds allow control in assembly construction because of their intermediate strength and directional nature. Many architectures in biology take advantage of the fact that hydrogen bonds fall in an energy range between a van der Waals interaction and a covalent bond. This allows hydrogen bonds to form and break continuously at room (or physiological) temperature, and specificity to be realized in biological reactions quickly. 1-5 This thesis focuses on quadruple hydrogen bonding between bio-derived building blocks, purines, as the basis for new functional self-assembling units. Background information on hydrogen bonding is described to place the project in a general context. Hydrogen Bonding in Organic Crystal Structures Chemists have looked to both the crystal structures of small organic molecules and those of biomolecules to gain inspiration for molecular design. Doubly hydrogen bonded motifs are known for amides, 6 sulfamides, 7 carboxylic acids, 8 pyridone derivatives, 9 and 2-aminopyridine-carboxylic acid complexes 10 in the solid state. A few examples of 1

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2 molecules that can form self-complementary complexes employing two hydrogen bonds are shown below (Figure 1-1). Each of the monomers contains hydrogen bond donors (D) and acceptors (A). Figure 1-1. All molecules shown form two hydrogen bonds. (A) Linear (one-dimensional) chains that arise from secondary amides, 6 (B) carboxylic acid dimers; 8 (C) an acetylenic dipyridone that forms cyclic triplexes (A = hydrogen bond acceptor, D = hydrogen bond donor). 9 Triply hydrogen bonded systems have also shown aggregation properties in cyanuric acid-melamine 11 and cytosine-guanine systems. 12 The triply hydrogen bonded dimer of cyanuric acid-melamine shown in Figure 1-2 aggregates via complementary hydrogen bond donors (D) and hydrogen bond acceptors (A). The cytosine-guanine motif will be discussed further in the next section. Multiply hydrogen bonded systems offer additional strength and specificity over doubly hydrogen bonded assemblies. The strength of a multiply hydrogen bonded system arises from the cooperative nature of hydrogen bonds. 1 The reversible formation of hydrogen bonds in solution ensures their specificity. The remainder of this thesis will focus on multiply (3 or more) hydrogen bonded systems.

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3 Figure 1-2. A dimer of melamine and cyanuric acid binds noncovalently via three hydrogen bonds, which forms a lattice in the solid state. 11 Hydrogen bond patterns in the solid state are usually well defined chains or arrays. Functional groups have preferences for specific crystal structure hydrogen bond patterns that can be explained as empirical rules. 13 The rules are formed from studying the patterns that functional groups repeatedly show and are also based upon the stereoelectronic hydrogen bond preferences and selectivity. There are three general hydrogen bonding rules for the patterns that neutral organic compounds form. First, all donors and acceptors participate in hydrogen bonding. Second, a six-membered ring formed from an intramolecular hydrogen bond will form before an intermolecular hydrogen bond. Lastly, once all intramolecular hydrogen bonds form, the remaining donors and acceptors form intermolecular hydrogen bonds. 13 Hydrogen Bonding in DNA The hydrogen bonding typical of DNA, a natural macromolecule, has been extensively studied and inspired the design of many synthetic assemblies. 14 The selfassembly of a DNA duplex at first glance appears to violate traditional molecular recognition rules and electrostatics. Looking only at the backbone of DNA, a poly-anion binds to a poly-anion; the fact that the poly-phosphates bind seems to not adhere to

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4 Coloumb’s Law. 14 DNA duplex formation overcomes the binding of poly-phosphates from a combination of hydrophobic effects, stacking, and hydrogen bonding. The ability of DNA to form hydrogen-bonded pairs in water arises from the fact that they are well-shielded from the bulk solvent; they offer the important specificity of binding that underlies the formation of this macromolecule. Guanine (G) and cytosine (C) interact with three hydrogen bonds in a Watson-Crick base pair shown in Figure 1-3. Figure 1-3. Watson-Crick base pairing of guanine and cytosine. 15 Although Watson-Crick base pairs are standard, there exist 28 possible base pairing motifs involving at least two hydrogen bonds, in addition to, a triplex formed from three bases interacting (C + :G:C). A simple GC interaction has a strength of 10 3 M -1 in CDCl 3 , although poor solubility (especially G) has made them undesirable candidates for many synthetic systems. 15 Applications of Nucleobases and Nucleobase Analogues Guanine and Cytosine Dimers As far as standard nucleobase hydrogen bonding interactions are concerned, the adenine (A) and thymine (T) pair is the weakest with a K a = 50 M -1 in CDCl 3 and GC is the strongest base pair as mentioned above. 15 The Rich Group 16 studied the hydrogen bonding interactions between derivatives of adenine and uracil. Although there have been many nucleobase interactions studied, the next sections will only focus on two of them, the GC dimer and guanosine quadruplexes. Using cytidine and guanosine

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5 functionalized porphyrins, Sessler et al. studied the effect hydrogen bonding has on the photoinduced energy transfer from a donor porphyrin to an acceptor. 17 The hydrogen-bonded GC system that Sessler et al. used is abbreviated in Figure 1-4. 17 Figure 1-4. Nucleobase-substituted porphyrins that experience photoinduced energy transfer when brought close in space by rigid triple hydrogen bonds. 17 Sessler et al. have also produced self-assemblies using a trimeric species of cytidine-guanosine for potential use in construction of self-assembled dendrimers 18 and selective nucleotide membrane transportation using synthetic GC. 19 There have been some practical applications using the GC complex, although not many. DNA intercalation, binding the major groove, is also possible by using a receptor designed by The Zimmerman Group to specifically interact with the CG base pair as shown in Figure 1-5. 20 Figure 1-5. Hydrogen bond directed complexation of a CG base pair bound to The Zimmerman Groups’ receptor unit. 20

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6 Guanosine Quadruplexes In addition to the unique structural properties of the DNA duplex (and higher multiplexes), nucleobases are also involved in other interesting natural and synthetic structures. Guanosine quadruplexes, hydrogen-bonded macrocycles, have been known since 1962. 21 There was renewed attention to the macrocycle in the late 1980s because the idea emerged that when the guanosine quadruplex was formed in DNA it might have biological significance. 21 Today the interest continues with thousands of papers about the guanosine quadruplex. Guanosine can form quadruplexes having applications in cancer research and also nanotechnology. 21 Using analogues of guanosine can increase the number of hydrogen bonding units, which assist in the self-assembly of the quadruplex. 22 Guanosine quadruplex formation begins with cation templated self-assembly to form the hydrogen-bonded macrocycle. Through interactions ranging from stacking to hydrogen bonding, guanosine quadruplexes can display molecular recognition properties. 21 An example of a guanosine quadruplex is shown in Figure 1-6. 21 Figure 1-6. Guanosine quadruplex with a cation bound (for example K + ). 21

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7 Synthetic Nucleobases A growing field of synthetic biology uses synthetic nucleobases to move past simple biomimetic chemistry to reproducing biological behavior, i.e. replication, selection, and evolution. 14 It is now known that ribose is specific to nucleobases; no other sugars will successfully form the backbone. 14 Non-conventional base pairs, formed from synthetic nucleobases, as sequences have been successfully replicated via polymerase chain reactions. 23 Synthetic nucleobases hydrogen bond and form molecular recognition units that can be both enzymatic and nonenzymatic in function. 14 In Figure 1-7 a synthetic nucleobase pair is shown with py denoting a pyrimidine-type structure and pu denoting a purine-type structure and AAD describing the hydrogen bonding pattern. 14 As a result of these synthetic nucleobases’ ability to pair via hydrogen bonds and also form a small strand of synthetic DNA, there is now an Artificially Expanded Genetic Information System (AEGIS). AEGIS currently assists in monitoring HIV and hepatitis C patients’ viral load and more applications are possible in the future. 14 Natural nucleobases also pair with synthetic nucleobases to form everything from single-stranded DNA to a full double helix. 24 Figure 1-7. Synthetic nucleobase pair of pyAAD and puDDA. 14 DNA Nanotechnology Closely related to building structures directly from DNA is the relatively new field of structural DNA nanotechnology, which utilizes DNA motifs to produce three

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8 dimensional materials. 25 Since as early as 1980, DNA nanotechnology has focused on the properties of DNA, such as the affinity of the complementary base pairs, stacked backbone molecules, persistance length of 50 nm, and a discernable code sequence. 26-30 Conventional linear cellular DNA is not an appropriate vehicle to pursue structural DNA nanotechnology; the focus is on branched systems. These branched DNA systems are found in nature as part of the recombination process rather than for information storage. So far DNA catenanes, nanomechanical devices, and two-dimensional arrays have been successfully produced from this new field. 25 Quadruple Hydrogen Bonds Quadruple hydrogen bonding arrays have evolved from attempts to strengthen triply hydrogen bonded systems. Secondary Interactions Figure 1-8. Heterodimers showing Jorgensen’s secondary interactions. Complex (A) has a K a of 10 4 -10 5 M -1 in chloroform and (B) has a K a of 170 M -1 in chloroform. 31 Jorgensen’s theory of secondary interactions was first noticed with triply hydrogen bonded complexes. For two superficially similar heterodimeric complexes both containing the same types of hydrogen bonds, NH 2 O, NHN, and NH 2 O, the binding constants have a difference of four orders of magnitude (Figure 1-8). Since each additional hydrogen bond adds an order of magnitude in strength, from where does this

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9 drastic difference arise between two triply hydrogen bonded complexes? Jorgensen and Pranata considered for the first time the importance of secondary interactions on hydrogen bond strength. 31 In a triply hydrogen bonded system there are three different potential patterns, ADA-DAD (Figure 1-2 and Figure 1-8B), ADD-DAA (Figure 1-3, Figure 1-4, Figure 1-5, Figure 1-7, and Figure 1-8A), and AAA-DDD (A = Acceptor and D = Donor). Although the AAA-DDD is the strongest triple hydrogen bond, very few structures have been synthesized using this pattern. Figure 1-9. Representation of Jorgensen’s theory of secondary interactions 31 showing only positive primary and secondary hydrogen bond interactions for triply hydrogen bonded systems. The patterns are listed in increasing strength because the AAA-DDD pattern has four positive secondary interactions, while the ADA-DAD contains no favorable secondary interactions. Therefore, the large binding constant difference between complex A and B in Figure 1-8 arises from complex A having positive secondary interactions while complex B only has negative secondary interactions. 31 In more objective terms, The Jorgensen Group assigns each primary hydrogen bond an energy of -7.5 kcal/mol and each secondary interaction +/-2.5 kcal/mol. 32 Mathematically speaking, complex A in Figure 1-8 has a binding energy of -22.5 kcal/mol while compound B only has a binding energy of -12.5 kcal/mol, further explaining the large difference in association constants.

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10 Preorganization and Tautomerization The extent to which molecules are preorganized and tautomerize affects the stability and specificity of their hydrogen-bonded complexes. Cram has coined the term “preorganization” meaning that a complex is most stable when the host and guest are organized for binding and have very few solvent molecules bound. 33 Intramolecular hydrogen bonding can preorganize a compound so that quadruple hydrogen bonding will occur. Tautomerization can affect strength and specificity of a quadruply hydrogen bonding unit; if the desired tautomer is not favored energetically, the energy price can be too high to form the desired complex. Quadruple hydrogen bonding is affected by primary hydrogen bond strength and secondary hydrogen bonding interactions. 34 Structures that will best form quadruple hydrogen bonds are geometrically well defined, tautomerically stable, and have general synthetic approaches. 35 Self-Complementary Systems In the literature there are many examples of heteroaromatic molecules inspired by nature, such as pyrimidines and triazines, that form quadruple hydrogen bonds. 35 Although many examples employ heteroaromatic molecules for self-assembled systems, there are solubility problems and it is often difficult to control tautomerization. 34 The Meijer Group have synthesized diaminotriazines and diaminopyrimidines that form ADAD-DADA associations and examples are shown in Figure 1-10. 36 Jorgensen’s secondary interactions describe the ADAD-DADA form as the weakest quadruply hydrogen bonded system.

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11 Figure 1-10. Meijer and coworkers’ ADAD self-complementary dimers. Assembly (A) is an acylated diaminotriazine with K dimer = 530 M -1 in CDCl 3 and (B) is an acylated diaminopyrimidine with K dimer = 2 10 5 M -1 in CDCl 3 . 34 Next, the DDAA array was considered due to its increased binding strength; the appropriate molecule needed to follow a short, scalable synthetic route from inexpensive starting material. 34 There are compounds by both Corbin-Zimmerman 37 and The Meijer Group 38 of the DDAA form and examples are shown in Figure 1-11. Figure 1-11. Examples of one conformation of one tautomer of a self-complementary DDAA dimer. (A) A dimer by Corbin and Zimmerman with K dimer > 3 10 7 M -1 ; (B) a dimer by Meijer and coworkers of 2-ureido-4[1H]-pyrimidinone monomers with K dimer > 10 6 M -1 . 34 Complementary Systems The use of complementary quadruple hydrogen bonding units to form self-assembling arrays has not been explored extensively. 39 The ADDA hydrogen bonding

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12 array creates a system that is not self-complementary, but can form complementary associations or heterodimers. Corbin and Zimmerman 37 have synthesized three complexes containing the DAAD-ADDA motif shown in Figure 1-12. Complex C has a much lower association constant than A and B because it must break an intramolecular hydrogen bond and unfold to form the complementary system. Figure 1-12. Three complexes containing DAAD-ADDA hydrogen bonding. Association constants were measured in chloroform-d. (A) and (B) have K a > 3 x 10 7 M -1 while for (C) K a = 2000 M -1 . 34 Hydrazide-based quadruple hydrogen bonded heterodimers have been synthesized and one example containing the motif is shown in Figure 1-13. 35 These dimers uniquely employ non-natural molecular recognition units that have programmable strength and specificity, namely the lack of self-complementarity. 35 Figure 1-13. Hydrazide based quadruply hydrogen bonded heterodimer with an ADDADAAD pattern. 35

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13 Applications of Quadruple Hydrogen Bonds in Functional Materials and Assemblies Supramolecular polymers Applications of quadruply hydrogen bonding units in functional materials and assemblies range from supramolecular polymers to nanoscale devices. As with most applications of quadruple hydrogen bonding, supramolecular polymers (Figure 1-14) require a straightforward synthesis to create a functional system. 39 Figure 1-14. Representation of a supramolecular polymer. Self-complementary systems, or homodimers, are advantageous for supramolecular polymers because they allow precise control of stoichiometry. 39 Note that triply hydrogen bonded compounds do not have the ability to form homodimers. For quadruply hydrogen bonded systems that have well defined dimerization properties, the architecture can be tuned via chemical modification. A quadruple hydrogen bonding polymer can have specific features of association, branching, and crosslink distances, in addition to thermodynamically controllable formation. 38 Figure 1-15 shows a compound that forms a quadruply hydrogen bonded dimer in each of its tautomeric forms. Corbin and Zimmerman’s supramolecular polymers focus on producing predictable tautomerism and therefore forming the strongest dimer by

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14 paying the lowest energy price. If a compound does not have to form an energetically unfavored tautomer prior to complexation, then the association constant is higher. 37 Figure 1-15. Corbin and Zimmerman’s three tautomeric forms of a heterocycle, which all form quadruply hydrogen bonded dimers. 37 Host-guest chemistry Although the field of supramolecular polymers is growing rapidly and relies strongly on quadruple hydrogen bonding units, there are other important applications to consider. The three main areas are host-guest chemistry, self-assembled architectures, and functional electronic materials. Host-guest systems employing quadruple hydrogen bonds include ureas, 40 barbiturates (Figure 1-16), 41 and recognition, functionalized monolayers. 42 The Hamilton Group has produced a series of synthetic receptors with strong selectivity toward barbiturates from six hydrogen bonds. 41 Figure 1-16. A schematic representation of a barbiturate binding site. 41

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15 Functionalized monolayers on a surface that interact via hydrogen bonds can recognize biological molecules. 42 For example, a biotin functionalized monolayer can recognize the bacterial protein streptavidin. 42 Self-assembled architectures Self-assembled architectures include everything from helices to fibers. Many types of organic nanotubes have been formed utilizing quadruple hydrogen bonding interactions. 43 The Fuijiki Group have formed a hyperhelical supramolecular assembly from interactions such as quadruple hydrogen bonding and stacking of a phthalocyanine unit. The -conjugated nature of phthalocyanine allows for future work into electronic and optoelectronic devices. 44 Figure 1-17. Utilizing 2-ureido-4[1H]-pyrimidinone units on a surface to form a monolayer with quadruple hydrogen bonds that can be controlled via solvent or temperature. 46

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16 Some consider crystal engineering a subfield of supramolecular chemistry, making the use of quadruple hydrogen bonding units the logical next step for the construction of crystalline materials. Examples formed from diaminopyrimidines have potential for use in solar cells (vide infra) as a gas storage device, a sensor, or an optical switch. 45 Self-assembled monolayers employing self-complementary quadruple hydrogen bonds can be used to construct more complex supramolecular architectures on surfaces (Figure 1-17). 46 Functional materials Another interesting application of quadruple hydrogen bonding is in solar energy devices. More specifically, dye-sensitized solar cells are low-cost, relatively efficient (12%) energy devices that employ supramolecular electrolyte hosts (Figure 1-18). 47 The device uses the injection of electrons from the photo-excited state of the dye molecules to the conduction band of TiO 2 . In order for the solar cell device to run efficiently there must be both good contact between the electrolyte and the semiconductor nanoparticles and also good ionic conductivity. Both of these parameters are significantly improved by using the supramolecular electrolyte host shown in Figure 1-18. 47 Figure 1-18. Poly(ethylene glycol) with terminal functional groups [2-(6-isocyanatohexylaminocarbonylamino)-6-methyl-4[1H]pyrimidinone quadruple hydrogen bonding units for use as a supramolecular electrolyte host in dye-sensitized solar cells. 47 The following is a list of other functional materials using quadruple hydrogen bonding units.

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17 Photovoltaic devices: Organic photovoltaic devices utilizing a -conjugated polymer as an electron donor and a fullerene derivative as an electron acceptor have been synthesized. Rispens et al. synthesized [60]fullerene containing a 2-ureidopyrimidin-4-one unit as a model compound for further studies. 48 Semiconductors: Conjugated polymers can be employed as a platform for electronic semiconductor properties. The self-complementary quadruple hydrogen bonding units allow for direct self-assembly of -conjugated oligomers. 49 Sensors: Conjugated hydrogen bonding compounds can employ their photosensitive nature as a probe for recognizing a guest molecule. Utilizing fluorescence microscopy, the artificial receptor with a quadruple hydrogen bonding DAAD array binding a carbohydrate (ADDA) can be monitored. A drastic change in fluorescence properties is attributed to the receptor-carbohydrate complexation that induces charge transfer via multiple hydrogen bonds. 50 Monolayers: As mentioned previously, supramolecular monolayers can be formed on a surface. 46 In addition, entire hydrogen-bonded nanostructures can be grown on a monolayer. 51 Nanowires: Different spatial arrangements of guanine can form nanowires that have electronic properties. 52 Hydrogen-bonded aggregates, quadruple helices, and planar ribbons of guanine, show interesting properties for molecular electronics applications in the push for continued miniaturization of electronic circuits. 52 Molecular machines: In biology there are complex machines, biomotor molecules, that can serve as systems to mimic on a simpler scale. Badji et al. successfully made a molecular elevator. 53 Beyond Quadruply Hydrogen Bonded Systems There have been examples of linear six-hydrogen bonded systems by both Corbin and Zimmerman 34 and Gong and coworkers. 54 Corbin and Zimmerman used a DDAADD bis-ureido-naphthyridine complexed with AADDAA bis-naphthyrdinyl-urea to form a complementary six hydrogen bonded system. 34 Both compounds in the complex have to overcome energy barriers from intramolecular hydrogen bonds, but the K a (CDCl 3 ) is estimated to be about 10 6 M -1 . 34 Shown in Figure 1-19 is Corbin and Zimmerman’s heterodimer. 34

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18 Figure 1-19. Corbin and Zimmerman’s heterodimer of DDAADD bis-ureido-naphthyridine complexed with AADDAA bis-naphthyrdinyl-urea. 34 Gong and coworkers have shown another complementary system forming six hydrogen bonds using the DADDAD-ADAADA hydrogen bonding pattern. 54 The association constant is 10 9 M -1 and the complex is shown in Figure 1-20. 54 Figure 1-20. Bing Gong and coworkers’ heterodimer DADDAD-ADAADA of oligoamides. 54 The reason for research to increase the number of hydrogen bonds between dimers (complementary and self-complementary) is the greater increase in strength and specificity that they impart. For more than four hydrogen bonds, the strength of a multiply hydrogen bonded system gradually approaches that of a covalent bond but still retains reversibility. Recently, The Zimmerman Group have synthesized ureido-naphthyridine oligomers that have a self-complementary DDAADDAA hydrogen bonding array with eight hydrogen bonds. 55 The association constant was measured in

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19 DMSO-d 6 /CDCl 3 mixtures and was 4.5 10 5 M -1 , even in the presence of DMSO as a competitive solvent. 55

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CHAPTER 2 DESIGN OF QUADRUPLE HYDROGEN BONDING UREIDOPURINES Chapter 1 showed that quadruple hydrogen bonding units can be based on heteroaromatics. For example, Figure 1-15 shows an intramolecularly hydrogen bonded compound in all of its tautomeric forms that can form quadruply hydrogen bonded dimers. 37 As the complication level increases in a self-assembled structure, the need for a recognition unit containing four hydrogen bonds arises to ensure specificity. Figure 2-1 shows representative examples of quadruply hydrogen bonded systems, both self-complementary (AA, BB, and CC) and complementary (BD, ED, FG, HE, and AD). Many of the examples shown in Figure 2-1 use an intramolecularly hydrogen bonded conformer to form the quadruple hydrogen bonded complex. The Meijer Group’s self-complementary structure (AA) in Figure 2-1 uses an intramolecular hydrogen bonded conformer. 38 AA has been used in many applications described in Chapter 1 and is actually the most widely used quadruple hydrogen bonding unit. 38,39,46-49 The reason for this is its ease of synthesis 56 and strong dimerization constant (K dimer > 10 6 M -1 in CHCl 3 ). 38 Although Figure 2-1 shows a greater number of complexes that are complementary, self-complementary complexes remain the most well studied. The reason is that self-complementary units were the first quadruple hydrogen bonded systems discovered and are more useful for forming supramolecular assemblies (due to stoichiometric considerations), as described in Chapter 1. Corbin and Zimmerman 36 have focused on developing molecular subunits for supramolecular assembly that do not have unfavorable tautomers interfering with the quadruple hydrogen bonded systems. The 20

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21 self-complementary system (BB) shown in Figure 2-1 for Corbin and Zimmerman has a K dimer > 10 7 M -1 in CDCl 3 . 36 Figure 2-1. Heterocycles that form self-complementary and complementary quadruply hydrogen bonded units. Unless noted, the association constants are taken in CDCl 3 . Other complexes shown include work mainly from Lning 57 and coworkers (HE) and Z.-T. Li 35,58,59 and coworkers (FG and AD). Shown in Figure 2-1 is complementary complex HE that has K dimer = 2000 M -1 in CDCl 3 , a complementary complex FG that has a K dimer = 4.7 x 10 4 M -1 in CDCl 3 , and a self-complementary

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22 complex AD that has K dimer = 10 4 M -1 (7% DMSO-d 6 /CDCl 3 ). A complementary quadruply hydrogen bonded complex (i.e. AD) can be formed from the related self-complementary complex (i.e. AA + D) and is driven by the additional donor-acceptor interactions. 59 This strategy is used for building novel supramolecular structures. 59 Aromatic dimer CC is interesting because although monomer C is capable of forming two intramolecular hydrogen bonds, the unfolded conformer has more hydrogen bonding sites for intermolecular hydrogen bonds, and therefore is preferred at equilibrium. Nonetheless, breaking the two intramolecular hydrogen bonds comes at cost and the K a is only 95 M -1 in CDCl 3 . 60 In addition, complementary complex ED has to break intramolecular hydrogen bonds to form the DAAD-ADDA quadruply hydrogen bonded dimer, explaining its low association constant. 61 The Zimmerman Group have shown a structure that is preorganized forms a stronger dimer because the intramolecular hydrogen bond creates a preference for that conformer (BB and BD). 36 Although there is a range of association constants that vary depending upon the heterocycle used, both self-complementary and complementary complexes have strong interactions if the monomers are preorganized to form a quadruply hydrogen bonded complex. 13,33 Ureidopyrimidines, and similar structures, intramolecularly hydrogen bond with the advantage of forming a quadruply hydrogen bonded dimer of great strength. Recently, The Zimmerman Group synthesized the first ureidopurine that has G dimer = -11.6 kcal/mol and is shown in Figure 2-2. Although the ureidopurine chosen (Figure 2-2) only weakly self-associates, it forms a strong heterodimer useful for making supramolecular polymer blends. 62 The ribose unit adds more sites that can be functionalized. The

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23 compound shown on the right as a heterodimer in Figure 2-2 is the dominant assembly in chloroform-d and stabilizes the tautomer shown. 62 Figure 2-2. A ureidopurine, based on guanosine, and the two conformers formed from different intramolecular hydrogen bonds. The conformer on the right is shown captured in a heterodimeric complex. 62 For a generic ureidopurinyl structure there are four possible conformers that are possible in solution. Shown in Figure 2-3 are the extended or unfolded conformers (A and B) and the two intramolecularly hydrogen bonded conformers C and D (to N(3) and N(1), respectively). Depending upon the specific purinyl compound used, one or all of the conformers in Figure 2-3 could form quadruply hydrogen bonded complexes. If only one conformer forms a quadruply hydrogen bonded complex but is not energetically favored, then the complex will not form. Figure 2-3. Basic purine system showing the four likely conformers in solution. Conformers C and D feature intramolecular hydrogen bonding. Previous work in the Castellano group by Rosyln LaMastus on intramolecularly hydrogen bonded purines resulted in the first ureidopurines based on the 2-aminopurine platform (Figure 2-4). 63

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24 Figure 2-4. Ureidopurines synthesized by Roslyn LaMastus. (A) 2-Phenylurea-9-benzyl-purine; (B) 2-phenylurea-6-dimethylamino-9-benzyl-purine; (C) 2-phenylurea-6-chloro-9-benzyl-purine; (D) 2-(p-ethoxyphenyl)urea-6-chloro-9-benzyl-purine; (E) 2-(p-fluorophenyl)urea-6-dimethylamino-9-benzyl-purine. 63 The general synthetic procedure involves reaction of an aryl isocyanate with a 2-aminopurine derivative. The reactions are performed at room temperature in pyridine with no additional solvent or reagents present. Unlike the guanine analogues that The Zimmerman Group 61 have introduced, the ureidopurines shown in Figure 2-4 are tautomerically stable, making them desirable compounds. Compound B (Figure 2-4) could be studied by x-ray crystallography (Figure 2-5). The asymmetric unit is shown that displays intramolecular hydrogen bonding of the urea to N(3). This is the appropriate preorganization to form a quadruple hydrogen bonding system. If the dimethylamino group is replaced in the 6-position with an amino group (–NH 2 ), the monomer would have a DADA arrangement capable of forming a self-complementary dimer (Figure 2-6). The favorable interactions (edge-to-face) between the arylurea and 9

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25 benzyl group further ensure that this conformer is favored, allowing the desired assembly to form. Figure 2-5. X-ray crystal structure of 2-phenylurea-6-dimethylamino-9-benzylpurine. 63 Evidence for intramolecular hydrogen bonding also comes in solution from 1 H NMR spectroscopy that shows an N-H shift for the N(12)-H to 11.4 ppm (CDCl 3 ). Figure 2-6. If the NMe 2 of B in Figure 2-4 is replaced with NH 2 , aryl interactions stabilize an intramolecularly hydrogen bonded conformer leading to a dimer. The structures from left to right are the conformers with an intramolecular hydrogen bond to N(1), an intramolecular hydrogen bond to N(3), and the resulting dimer.

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CHAPTER 3 RESULTS AND DISCUSSION General Synthetic Considerations The general strategies to form a quadruply hydrogen bonded ureidopurine are outlined in Figure 3-1. As described previously, when the urea functionality intramolecularly hydrogen bonds to N(3) in 1, the molecule is preorganized to form a quadruply hydrogen bonded self-complementary dimer (11). All four strategies attempted have similar initial concerns. Figure 3-1. Scheme of the general design to synthesize a quadruply hydrogen bonded ureidopurine. 26

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27 The R 1 group determines the solubility of the purine reagent and final products in organic solvents. Initially chosen was a benzyl group (Strategies 1 & 4) or a protected sugar (Strategies 2 & 3) in this position; benzyl was inspired by our earlier work and analysis (e.g. Figure 2-5). The R 2 group is important for isocyanate reactivity, urea hydrogen bonding strength, and for interactions (favorable or unfavorable) with R 1 . Both aryl and alkyl ureas are considered in the strategies. Strategy 1 then more specifically attempts to simply displace the 6-chloro group of 2 (available from 3) with ammonia. Strategies 2 through 4 rely on the known, but not often used, differential nucleophilicities of the 2and 6-amino groups of 4 for reaction with an isocyanate directly. 64 Strategy 1 Using guanine (5) as a starting material and following a procedure similar to a patent preparation, 65 2-amino-6-chloro-purine (3a) was synthesized (Figure 3-2). The first step uses phosphorus oxychloride to convert the carbonyl of 5 to a chloro group and also a series of acid and base steps to convert the 2-imine back to an amino group. The yield is only moderate for 3a because of the steps required, however the route is preferred over buying the very expensive reagent. Figure 3-2. Synthetic route to form 3a starting from 5. Benzylation at N(9) was then pursued according to the general methodology of Camaioni et al. 66 Deprotonation at N(9) of 3a and subsequent reaction with benzyl

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28 bromide by an S N 2 displacement (Figure 3-3) gave 2-amino-6-chloro-9-benzyl-purine (3b). Figure 3-3. Benzylation of 3a to form 3b. The yield of 3b is low because the nitrogen at the 7 position also reacts, creating the need to separate the isomers. Once 3b was purified by column chromatography and characterized, an isocyanate was chosen to form the desired urea functionality. An aryl isocyanate was synthesized with a solubilizing para alkyl chain (Figure 3-4). An aryl isocyanate was chosen because it reacts more readily than an alkyl isocyanate with the 2-position amino group. 63 According to a preparation by Ronald Castellano, 67 4-n-heptylphenyl isocyanate (8) was prepared via a Curtius rearrangement (Figure 3-4). Figure 3-4. Preparation of 8 from heptylphenyl acid chloride (6) using sodium azide via a Curtius rearrangement. Starting with heptylphenyl acid chloride (6), sodium azide was used to form the acyl azide (7) by an S N 2 reaction, which, once heated, rearranged to form 8. The reaction resulted in reasonable yields, although it was not quantitative because of the reactivity of

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29 8, the product. With the aminopurine and isocyanate prepared, the next step was to form the ureidopurine. Urea formation was attempted by reacting 3b and 8 but the yield of the product, 2-(p-heptylphenyl)urea-6-chloro-9-benzyl-purine (2a), was too low for successful isolation (Figure 3-5). The reactivity of 8 to form a hydrolysis side product may be a factor in the unsuccessful nature of the reaction as was the poor solubility and low reactivity of 3b. Figure 3-5. Unsuccessful reaction of 3b and 8 to form 2a. Strategy 2 After the reaction of 3b and 8 did not result in an ureidopurine product, another starting material had to be chosen. Since 3b still had solubility problems that inhibited the reaction, protected 2,6-diamino--D-ribofuranosylpurine (4c) was chosen as the starting material shown in Figure 3-6 with generic protecting groups. Figure 3-6. The new starting material 4c. The change from the 9-benzyl to a ribose was an effort to improve solubility without affecting reactivity. Another benefit of the sugar is the possibility of using the

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30 product as a synthetic nucleobase. As mentioned in the general design section, the 2-amino group of 4 should be more reactive than the 6-amino group toward electrophiles. Starting with guanosine (not shown), the sugar was protected with acetate groups to form acylated guanosine 9 (Figure 3-7, following Matsuda’s procedure 68 ) to safeguard the sugar in the next step. Shown is the conversion of the 6-position carbonyl group of 9 to the 6-chloro derivative 10 using a procedure similar to Robins and Uznaski. 69 Then methanolic ammonia was used to replace the chloro group with an amino group and additionally deprotect the sugar (Figure 3-7) to form 2,6-diamino--D-ribofuranosylpurine (4a). 58 Figure 3-7. Reaction route showing the conversion of 9 to 2,6-diaminopurine 4a. When characterizing 4a, its absolute purity could not be confirmed. The 1 H NMR spectrum of 4a showed a few stray peaks even after purification, in addition the 13 C NMR displayed impurity peaks. The problem lay in the methanolic ammonia step. Perhaps the

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31 commercially available methanolic ammonia was not as reactive toward the 6-position chloro group as desired. Figure 3-8. Scheme of the reaction of 4a with TIPDSiCl 2 to protect the 3’ and 5’ hydroxyl groups to form 4b. The decision was made to purchase 4a commercially and continue with the synthesis to protect the sugar hydroxyl groups (2’, 3’, and 5’) on 4a following Beigelman et al.’s procedure (Figure 3-8). 70 The first step was protection of the 3’ and 5’ hydroxyl groups on the sugar using TIPDSiCl 2 . This protecting group was chosen because it is fairly stable and a very good solubilizing moiety. 70 When TIPDSiCl 2 was added to 4a slowly at low temperature the reaction proceeded with good yield (Figure 3-8). Since the protecting group was added dropwise by syringe, for smaller scale reactions the protecting group was not added at the same rate as larger scale reactions. This is one reason the yield of 2,6-diamino-9-[(3’,5’-O-tetraisopropyldisiloxane-1,3-diyl)--D-ribofuranosyl]purine (1b) varied greatly. Figure 3-9. Alkylation of the unprotected 2’ hydroxyl group of 4b to form 4c.

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32 Once 4b was synthesized, purified, and characterized it was then carried on in the preparation of 2,6-diamino-9-[(3’,5’-O-tetraisopropyldisiloxane-1,3-diyl)-2’-O-methyl--D-ribofuranosyl]purine (4c) by protecting the 2’ hydroxyl group (Figure 3-9) by alkylation. The alkylation reaction used methyl iodide as the alkylating agent and sodium hydride as the base. The reaction shown in Figure 3-9 proved to be troublesome, only working once in moderate yield. If the reaction time was increased or another equivalent of methyl iodide was added to force the reaction to completion, then only side products were formed. Other procedures were ultimately attempted to produce 4c (vide infra). Figure 3-10. Synthesis of 1a from 4c. The synthesis of 2-phenylurea-6-amino-9-[(3’,5’-O-tetraisopropyldisiloxane-1,3-diyl)-2’-O-methyl--D-ribofuranosyl]purine (1a) in high yield was realized at room temperature using pyridine as a base (Figure 3-10). Once characterized by 1 H NMR, 13 C NMR, and LRMS, 1a was determined to be at least 95% pure. 1 H NMR analysis immediately revealed a problem (confirmed by molecular modeling): 1a does not preorganize to allow quadruple hydrogen bonding to occur. The sugar and the phenyl ring appear to suffer steric repulsion when intramolecular hydrogen bonding occurs to N(3) and the intramolecular hydrogen bond shown in Figure 3-11(1a-A) does not form. Observed by 1 H NMR is exclusive formation of conformer 1a-B.

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33 Figure 3-11. Conformers of 1a. (A) Desired conformer allowing quadruple hydrogen bonding to occur. (B) Actual conformer formed where no dimerization is possible. Strategy 3 To reduce steric interactions between the 2-phenylurea and 9-sugar, a less sterically hindered urea was chosen, a propyl urea. Work then began on synthesizing 2-propylurea-6-amino-9-[(3’,5’-O-tetraisopropyldisiloxane-1,3-diyl)-2’-O-methyl--D-ribofuranosyl]purine (1b) shown in Figure 3-12. Figure 3-12. Target compound of Strategy 3, 1b. After synthesizing 4c again, the methylation reaction was realized to be highly problematic and alternate procedures were attempted. Different bases, i.e. NaHMDS, and different reaction conditions, i.e. lower temperature, were employed from ideas presented by Chow et al. 71 Many variations were used to react the aminopurine functionality with the propyl isocyanate. The procedure first attempted employed the same conditions as shown in Figure 3-10 using methylene chloride as a solvent, pyridine as a base, and room

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34 temperature conditions. This reaction did not succeed. Heating to reflux in methylene chloride was next tried but the temperature was not high enough to drive the reaction forward. Finally, pyridine was used as the solvent so that the reaction temperature could be increased, but this created modification issues with the isocyanate evaporating and loss of the sugar protecting group. Neither increasing the temperature nor increasing reaction times resulted in an isolable ureidopurine. After many attempts and different procedures, it was concluded that the alkyl isocyanate was simply not reactive enough with the aminopurine functionality of 4c. Work then branched to another strategy to use carbonyl diimidazole (CDI) to react with 4c to form activated 2-imidazolide-6-amino-9-[(3’,5’-O-tetraisopropyldisiloxane-1,3-diyl)-2’-O-methyl--D-ribofuranosyl]purine (11) (Figure 3-13). The next step would be reaction with propyl amine (at the activated acyl group) to form 1b. Figure 3-13. Reaction of 4c with CDI to form an active acyl intermediate 11. The concept for the procedure came from ideas in a paper by The Meijer Group 72 An excess of diaminopurine 4c was used to prevent disubstitution of the CDI. Unfortunately, 11 was not formed so the strategy shifted for a final time. Strategy 4 The ongoing strategy comes full circle back to the idea that the 9-benzyl and 2-phenylurea groups are sterically compatible. Shown in Figure 3-14 is the reaction

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35 proposed between 2,6-diamino-9-benzyl-purine (4d) and PhNCO to form 2-phenylurea-6-amino-9-benzyl-purine (1c). This work is currently in progress. Figure 3-14. Scheme of the reaction of 4d with PhNCO to form the urea 1c.

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CHAPTER 4 CONCLUSION AND OUTLOOK A ureidopurine, 2-phenylurea-6-amino-9-[(3’,5’-O-tetraisopropyldisiloxane-1,3-diyl)-2’-O-methyl--D-ribofuranosyl]purine, was successfully synthesized from this research. Unfortunately, steric interactions between the 9-ribose and the urea phenyl group shift the equilibrium exclusively to an intramolecular hydrogen bonded conformer that does not form a quadruply hydrogen bonded dimer. Nonetheless, significant progress has been made and insight gained in the deisgn of self-complementary ureidopurines. Alkyl ureidopurines are difficult to form due to the fact that alkyl isocyanates are unusually unreactive with 2-aminopurine derivatives. In addition, sugar protecting groups prove troublesome. Under harsh conditions, such as high reaction temperatures or introduction of base, the sugar can be deprotected or lost. Demonstrated in the synthesis of 1a was that the 2-amino group on 2,6-diaminopurine is a much better nucleophile than the 6-amino group, which is emphasized in Strategies 2. This final strategy also takes advantage of the fact that the 9-benzyl group aids in solubility and interacts appropriately with a phenyl urea functionality. In the future, this project will continue with the synthesis of ureidopurines employing strategies shown in this thesis. The ultimate goal is to produce a ureidopurine that forms a quadruply hydrogen bonded dimer. Quadruply hydrogen bonded heterodimers and homodimers have potential applications as supramolecular polymers and functional -conjugated materials. Specifically quadruply hydrogen bonding purinyl compounds also have the ability to intercalate DNA and to act as synthetic nucleobases. 36

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CHAPTER 5 EXPERIMENTAL General Unless otherwise noted, all reagents and solvents were purchased from Acros, Aldrich, and Fluka and not purified further. CH 2 Cl 2 and DMF were degassed in 20 L drums and passed through two sequential purification columns (activated alumina for CH 2 Cl 2 ; molecular sieves for DMF) under a positive argon atmosphere using the GlassContour solvent system (GlassContour, Inc.). Pyridine was distilled from calcium hydride onto 4 molecular sieves. From Berry and Associates 2,6-diamino--D-ribofuranosylpurine was purchased. Thin layer chromatography (TLC) was performed on commercially prepared DURASIL TLC silica gel plates on aluminum with visualization by UV light. Column chromatography was performed using Whatman silica gel (60) as a solid support. 1 H NMR (300 MHz) and 13 C NMR (75 MHz) were recorded on either a Varian Gemini 300 or a VXR 300 spectrometer. Chemical shifts () are given in parts per million (ppm) and referenced to either TMS or residual protonated solvent. Abbreviations used are s (singlet), d (doublet), t (triplet), q (quartet), quin (quintet), m (multiplet) and b (broad). MS spectra (LRMS) were performed by The University of Florida Mass Spectroscopy Services using Electrospray Ionization (ESI) as the ionization method and a Bruker Apex II FTICR mass spectrometer. 2-Amino-6-chloro-purine (3a) 65 Under argon, POCl 3 (25.0 mL, 248 mmol) was added dropwise from an addition funnel to the reaction flask at 0 C containing stirring DMF (60 mL). The red reaction 37

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38 was warmed to rt and stirred for 10 min. A suspension of guanine 5 (15.0 g, 99.3 mmol) in DMF (30 mL) was added to the reaction and the orange solution was heated to 100 C for 5 h. After cooling to rt a saturated aq NaHCO 3 solution (500 mL) was slowly added to the reaction and stirring was continued for 15 min. The orange solid product was collected and dissolved in HOAc solution (12% aq) and heated to 70 C for 3 h. The resulting solid was filtered, collected, and dissolved in NaOH (10% aq) and stirred at rt for 1.5 h. The reaction was then neutralized using conc. HCl and upon adjustment of the pH to 7, a solid product formed. The white product was filtered, collected and rinsed with water, then dissolved in NH 4 OH. To the stirring solution at rt, activated carbon was added to decolorize the solution. After the carbon and solvent were removed, the white product, 3a (6.78 g, 40%), was collected, rinsed with water, and dried under high vacuum overnight. 1 H NMR (DMSO-d 6 ): 12.84 (s, 1H), 8.08 (s, 1H), 6.77 (s, 2H). 2-Amino-6-chloro-9-benzyl-purine (3b) 66 To a suspension of 3a (0.295 g, 1.74 mmol) and K 2 CO 3 (0.481 g, 3.48 mmol) in DMF (10 mL), benzyl bromide was added (0.248 mL, 2.09 mmol). The reaction was stirred at rt for 4 h and monitored by TLC. Once the reaction was complete, the solvent was removed by evaporation and the residue was dissolved in CHCl 3 (200 mL). The insoluble solid (excess K 2 CO 3 ) was removed. The organic layer was rinsed with water (2 100 mL) and dried with sodium sulfate. Upon removal of the solvent, the resulting residue was purified by column chromatography (0.5% MeOH in CHCl 3 ) to give a white solid, 3b (0.129 g, 29%). 1 H NMR (CDCl 3 ): 7.75 (s, 1H), 7.36 (bd, 1H), 7.27 (s, 2H), 5.27 (s, 2H), 5.10 (s, 2H).

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39 4-n-Heptylphenyl isocyanate (8) 67 To acetone (10 mL) cooled in an ice bath, heptylphenyl acid chloride, 6 (0.200 g, 0.838 mmol), was added. Then a solution of NaN 3 (0.218 g, 3.35 mmol) in water (0.5 mL) was added to the stirring reaction. The reaction was stirred for 30 min at 3 C. Then the reaction was evaporated and the residue was dissolved in CH 2 Cl 2 (20 mL) and rinsed with water (2 10 mL), and then brine (20 mL), and dried over MgSO 4 . Gradually CH 2 Cl 2 was replaced with toluene through evaporation cycles. The reaction was heated to reflux for 2.5 h. After the solvent was removed, the colorless oil, 8, was afforded in 61% yield (0.154 g). 1 H NMR (CDCl 3 ): 7.10 (d, 2H, J = 8.2 Hz), 6.96 (d, 2H, J = 8.3 Hz), 2.54 (t, 2H, J = 7.4 Hz), 1.57.52 (m, 2H), 1.27.24 (m, 8H), 0.85 (t, 3H, J = 6.6 Hz). 9-(2,3,5-Tri-O-acetyl--D-ribofuranosyl)-2-amino-6-chloro-purine (10) 69 Acylated guanosine, 9 (10.0 g, 24.4 mmol), 68 was dried overnight under vacuum in the reaction flask and Et 4 NCl (8.28 g, 50.0 mmol) was dried overnight under vacuum at 85 C. Et 4 NCl, DMF (40 mL), and diethylaniline (4.0 mL, 25 mmol) were added sequentially to the reaction flask under argon at rt with stirring. POCl 3 (14.0 mL, 150 mmol) was added slowly to the reaction and then stirred for 10 min at rt, and for 10 min at 100 C with monitoring by TLC. The reaction was added to CHCl 3 (150 mL) and ice, then stirred for 15 min. The organic layer was rinsed with water (4 20 mL) and the aqueous layer was back-extracted with CHCl 3 (4 20 mL). The pH was adjusted to neutral by rinsing the organic layer with cold NaHCO 3 (5% aq) and was then dried over sodium sulfate. Gradually isopropanol (60 mL) was exchanged with CHCl 3 in a series of

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40 evaporations. The product was purified by column chromatography (5% EtOH in CHCl 3 ) to yield the yellow sticky solid, 10 (3.75 g, 36%). 1 H NMR (CDCl 3 ): 8.77 (s, 1H), 8.04 (s, 1H), 6.31 (d, 1H, J = 5.2 Hz), 5.88 (t, 1H, J = 6.6 Hz), 5.64 (t, 1H, J = 6.6 Hz), 4.40 (m, 2H), 3.21 (d, 3H, J = 5.7 Hz), 2.92 (d, 2H, J = 21.9 Hz), 2.11 (t, 6H, J = 10.3 Hz). 2,6-Diamino--D-ribofuranosylpurine (4a) 69 Directly into a pressure tube 10 (0.500 g, 1.17 mmol) was weighed and MeOH/NH 3 (10 mL) was added. The reaction was heated to 95 C and monitored by TLC. When complete, the solvent was removed and the orange residue was purified by column chromatography (EtOAc/acetone/EtOH/water 4:1:1:0.5) to yield 4a in quantitative yield (1.65 g). 1 H NMR (DMSO-d 6 ): 7.92 (s, 1H), 7.30 (s, 2H), 6.70 (s, 2H), 5.81 (s, 2H), 5.75 (d, 1H, J = 6.1 Hz), 4.46 (bt, 1H), 4.09 (q, 1H, J 1 = 3.3 Hz, J 2 = 4.9 Hz), 3.89 (bq, 1H), 3.63 (dd, 1H, J 1 = 3.1 Hz, J 2 =12.1 Hz), 3.51 (dd, 1H, J 1 = 3.8 Hz, J 2 =12.1 Hz), 3.36 (s, 3H), 2.89 (s, 1H), 2.73 (s, 1H), 1.76 (s, 4H). 2,6-Diamino-9-[(3’,5’-O-tetraisopropyldisiloxane-1,3-diyl)--D-ribofuranosyl]purine (4b) 70 Under vacuum, 4a (0.500 g, 1.78 mmol) was dried o(5 mL) and then pyridine (8 mL) were added to the reaction. The suspension was cooled to 0 C in an ice/NaCl/water bath with stirring. TIPDSiCl2 (0.700 g, mmol) was added dropwise over 30 min and the temperature was maintained at 0 C. The stirring reaction vernight. Under argon, DMF 2.12

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41 was then warmed to rt and monitored by TLC. When complete, the reaction was quenched with EtOH (1 mL). Once the solvent was removed, the residue was dissolvedin EtOAc (5 mL) and then partitioned with saturated aq NaHCO3 (5 mL). After the solvent was removed the residue was purified by column chromatography (EtOAc/hexanes/EtOH 2:1:1) to give 4b in quantitative yield (0.929 g). 1 H NMR (DMSO-d 6 ): 7.77 (s, 1H), 6.77 (s, 2H), 5.77 (s, 1H), 5.71 (d, 1H, J = 1.4 Hz), 5.58 (d, 2H, J = 4.8 Hz), 4.44 (q, 1H, J 1 = 5.1 Hz, J 2 = 8.1 Hz), 4.29 (t, 1H, J = 4.8 Hz), 4.10.90 (m, 3H), 3.44.33 (bd, 2H, J = 0.5 Hz), 1.06 (bd, 16H), 1.02 (d, 12H, J = 3.7 Hz). LRMS (ESI, [M + H] + ) calcd for C 22 H 41 N 6 O 5 Si 2 : 525; found: 525. 2,6-Diamino-9-[(3’,5’-O-tetraisopropyldisiloxane-1,3-diyl)-2’-O-methyl--D-ribofuranosyl]purine (4c) 70,71 To a solution of 4b (0.375 g, 0.710 mmol) in DMF (7.3 mL) under argon, MeI (0.133 g, 2.10 mmol) was added. The reaction was cooled to 0 C in an ice/NaCl/water bath with stirring. Once cooled, NaH (0.043 g, 1.80 mmol) was added to the reaction. The reaction was stirred for 40 min at 0 C. Once complete (TLC) the reaction was quenched with EtOH (1 mL). To the reaction, cold CH 2 Cl 2 (20 mL) was added. The organic layer was washed with saturated aq NH 4 Cl (20 mL), water (2 20 mL) and dried over sodium sulfate. Once the solvent was removed the yellow oil was recrystallized (EtOH/water 1:1) to yield a white product 4c in 31% yield (0.121 g). 1 H NMR (CDCl 3 ): 7.81 (s, 1H), 5.86 (s, 1H), 5.40 (s, 2H), 4.73 (s, 2H), 4.60 (q, 1H, J 1 = 5.0 Hz, J 2 = 9.2 Hz), 4.20 (d, 1H, J = 13.4 Hz), 4.10 (d, 1H, J = 9.6 Hz), 4.02 (d, 1H, J = 4.8 Hz), 3.98 (d, 1H, J = 3.2 Hz), 3.72 (d, 1H, J = 7.1 Hz), 3.67 (s,

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42 2H), 1.79 (s, 4H), 1.25 (q, 2H, J 1 = 5.5 Hz, J 2 = 8.4 Hz), 1.10.04 (m, 29H). LRMS (ESI, [M + H] + ) calcd for C 23 H 43 N 6 O 5 Si 2 : 539; found: 539. 2-Phenylurea-6-amino-9-[(3’,5’-O-tetraisopropyldisiloxane-1,3-diyl)-2’-O-methyl--D-ribofuranosyl]purine (1a) After 4c (0.050 g, 0.0090 mmol) was dried under vacuum for 1 h, it was dissolved in CH 2 Cl 2 (4 mL) under argon. To the stirring solution, pyridine (0.011 mL, 0.012 mmol) was added followed by phenyl isocyanate (0.013 mL, 0.012 mmol) at rt. The reaction was monitored by TLC and stirred at rt for 2 h. After the solvent was removed the residue was purified by column chromatography (5% MeOH in CH 2 Cl 2 ) to yield the product 1a in 90% yield (0.054 g). 1 H NMR (CDCl 3 ): 11.77 (s, 1H), 8.04 (s, 1H), 7.68 (d, 2H, J = 8.4 Hz), 7.35 (t, 2H, J = 8.2 Hz), 7.09 (t, 2H, J = 7.4 Hz), 6.06 (s, 1H), 4.49 (q, 1H, J 1 = 4.3 Hz, J 2 = 9.2 Hz), 4.30 (d, 1H, J = 13.1 Hz), 4.19 (d, 1H, J = 10.0 Hz), 4.03 (d, 1H, J = 13.1 Hz), 3.85 (d, 1H, J = 4.0 Hz), 1.55 (s, 2H), 1.24 (s, 2H), 1.10–0.99 (m, 29H). 13 C NMR (CDCl3): 159.10, 155.32, 151.58, 138.85, 137.04, 129.41, 123.99, 122.11, 120.44, 115.09, 88.23, 84.00, 81.57, 70.09, 60.37, 59.91, 30.16, 17.79, 13.44, 13.40. LRMS (ESI, [M + Na] + ) calcd for C 30 H 47 N 7 NaO 6 Si 2 : 680; found: 680.

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APPENDIX NMR SPECTRA 1 H NMR spectrum for 2-phenylurea-6-amino-9-[(3’,5’-O-tetraisopropyldisiloxane-1,3-diyl)-2’-O-methyl--D-ribofuranosyl]purine (1a) in CDCl 3 (ca. 3 mM). 43

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BIOGRAPHICAL SKETCH Rachel Giessert was born in North Tonawanda, New York, on October 15, 1980. Her family moved to Vero Beach, Florida, in 1982 and has resided there ever since. She graduated from Sebastian River High School with an International Baccalaureate diploma in June 1999, where her interest in chemistry began. Rachel received her Bachelor of Science degree in chemistry from the University of North Carolina at Chapel Hill (UNC-CH) in May 2003. While attending UNC-CH, she performed research with Dr. Dorothy Erie in the biochemistry division of the chemistry department. Her project focused on nucleobase binding sites of RNA polymerase. Rachel spent the summers of 2002 and 2003 working for Merck Research Laboratories in West Point, Pennsylvania, as a Medicinal Chemistry Intern. During this time, her work focused on synthetic organic chemistry techniques and modern methods of compound purification. Rachel began graduate school at the University of Florida in August 2003. After graduating with a Master of Science degree in organic chemistry in December 2005, Rachel intends to pursue a career in the pharmaceutical industry, as a medicinal chemist in a drug discovery department. 48