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Synthesis, Structure, and Photophysical Properties of Donor-Acceptor Purines

Permanent Link: http://ufdc.ufl.edu/UFE0021319/00001

Material Information

Title: Synthesis, Structure, and Photophysical Properties of Donor-Acceptor Purines
Physical Description: 1 online resource (147 p.)
Language: english
Creator: Butler, Roslyn Susan
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: acceptor, donor, fluorescence, molecular, nucleobase, optoelectronics, probes, purines, recognition, supramolecular
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Donor-pi-acceptor (D-A) molecules have photophysical, electronic, and optoelectronic properties that make them suitable as sensors, wires, and fluorophores. Here the syntheses and properties of the first D-A systems based on purines are reported. Results show that simple chemical modifications to the heterocycle significantly improve its inherent optical properties (even beyond 2-aminopurine); the resulting fluorophores are candidates as both biological probes and optoelectronic device components. The molecular recognition functionality of purines, multiple nitrogen atoms capable of hydrogen bonding and an extended aromatic surface for pi-stacking, can potentially be exploited to control molecular association and ordering. C(2) and C(6) donor substituted purines are brominated at C(8) and then transformed via a palladium(0)-catalyzed cyanation reaction to their corresponding nitriles. Conversion of the nitriles to methyl esters comes by treatment with acidic methanol. The C(8) acceptor groups dramatically increase the fluorescence quantum yields of the purines (versus a hydrogen in the C(8) position) in organic solvents to in many cases near unity (representing, for one derivative, a quantum yield enhancement of > 2500% in methylene chloride). Absorption (lambda max 310?365 nm), emission (lambda max 360?465 nm), and fluorescence lifetime data (8 ns ? fluorescence lifetime ? 0.5 ns) were collected for 12 D-A purines in four organic solvents and even water (for the 2-aminopurine analogues). Four of the purines could be studied in the solid state by X-ray crystallography. Theoretical calculations (DFT and ZINDO/CI) determined the absorption spectra, HOMO (?6.5 to ?5.5 eV) and LUMO (?2.5 to ?1.5 eV) energies, and the ground and excited state dipoles. The calculated orbital energies are in good agreement with solution-phase cyclic voltammetry data and the calculated excited state dipoles agree well with results from a Lippert-Mataga treatment of the solvent-dependent emission spectra. One D-A purine was successfully tested as the emissive layer in an organic light emitting diode (OLED). Amide bonds can be formed at the purine C(8) position (via an intermediate carboxylic acid) toward use of the molecules as heterocyclic amino acids. The amides show high quantum yields (quantum yield > 0.85) in all organic solvents and well-defined conformations in the solid state.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Roslyn Susan Butler.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Castellano, Ronald K.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021319:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021319/00001

Material Information

Title: Synthesis, Structure, and Photophysical Properties of Donor-Acceptor Purines
Physical Description: 1 online resource (147 p.)
Language: english
Creator: Butler, Roslyn Susan
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: acceptor, donor, fluorescence, molecular, nucleobase, optoelectronics, probes, purines, recognition, supramolecular
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Donor-pi-acceptor (D-A) molecules have photophysical, electronic, and optoelectronic properties that make them suitable as sensors, wires, and fluorophores. Here the syntheses and properties of the first D-A systems based on purines are reported. Results show that simple chemical modifications to the heterocycle significantly improve its inherent optical properties (even beyond 2-aminopurine); the resulting fluorophores are candidates as both biological probes and optoelectronic device components. The molecular recognition functionality of purines, multiple nitrogen atoms capable of hydrogen bonding and an extended aromatic surface for pi-stacking, can potentially be exploited to control molecular association and ordering. C(2) and C(6) donor substituted purines are brominated at C(8) and then transformed via a palladium(0)-catalyzed cyanation reaction to their corresponding nitriles. Conversion of the nitriles to methyl esters comes by treatment with acidic methanol. The C(8) acceptor groups dramatically increase the fluorescence quantum yields of the purines (versus a hydrogen in the C(8) position) in organic solvents to in many cases near unity (representing, for one derivative, a quantum yield enhancement of > 2500% in methylene chloride). Absorption (lambda max 310?365 nm), emission (lambda max 360?465 nm), and fluorescence lifetime data (8 ns ? fluorescence lifetime ? 0.5 ns) were collected for 12 D-A purines in four organic solvents and even water (for the 2-aminopurine analogues). Four of the purines could be studied in the solid state by X-ray crystallography. Theoretical calculations (DFT and ZINDO/CI) determined the absorption spectra, HOMO (?6.5 to ?5.5 eV) and LUMO (?2.5 to ?1.5 eV) energies, and the ground and excited state dipoles. The calculated orbital energies are in good agreement with solution-phase cyclic voltammetry data and the calculated excited state dipoles agree well with results from a Lippert-Mataga treatment of the solvent-dependent emission spectra. One D-A purine was successfully tested as the emissive layer in an organic light emitting diode (OLED). Amide bonds can be formed at the purine C(8) position (via an intermediate carboxylic acid) toward use of the molecules as heterocyclic amino acids. The amides show high quantum yields (quantum yield > 0.85) in all organic solvents and well-defined conformations in the solid state.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Roslyn Susan Butler.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Castellano, Ronald K.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021319:00001


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1 SYNTHESIS, STRUCTURE, AND P HOTOPHYSICAL PROPERTIES OF DONOR-ACCEPTOR PURINES By ROSLYN SUSANNE BUTLER A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Roslyn Susanne Butler

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3 For Mom, Carolyn Adwell

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4 ACKNOWLEDGMENTS I would like to thank my mom, Carolyn Ad well, for her unwavering love, support, and encouragement. I would also like to thank my dad, Jewell Butler, who has always let me know how proud he is of me and has always encourag ed me in all of my pursuits. When I was young, they both instilled in me the im portance of a good educat ion. Without that I would not be where I am today. I would like to thank my entire fam ily, they have always loyally helped me in any way possible and for that I am extremely grateful Special thanks go to Dr. Andrew Lampkins who has become an important person in my life. He has been a wonderful colleague and friend. I would also like to thank Dixie, Dexter, and Cali Butler (my three pugs) who have continuously been by my side and have given me unconditional love. I do not know what I would have done without them. I cannot leave out little Newman, the chihuahua; I cannot wait to add him to my pack of dogs. I would like to thank my advisor, Prof. Ron Castellano, who has scien tifically guided and motivated me to achieve more than I ever though t I could. He is a wonderful scientist, person, and friend. I am very proud to say I come from his research group, and I am excited about the future of his research. I would also like to thank Dr Robert Holman, my research advisor at Western Kentucky University, and Dr. Spiro Alexandr atos, my REU advisor at Univer sity of Tennessee. They both sparked my interest in organic chemistry and in spired me to further my scientific pursuits. I would like to thank former and present memb ers of the Castellano group. I would also like to thank collaborators, Prof. John Reynolds, Dr. Khalil Abboud, Prof. Kirk Schanze, Dr. Katsu Ogawa, Dr. Garry Cunningham, and Aubrey Dyer, for their scientific expertise and assistance with my work.

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5 Finally, I would like to thank my committee me mbers: Prof. Ken Wagener, Prof. Nicole Horenstein, Prof. Lisa McElwee-White, and Prof Joanna Long for their scientific advice and wisdom.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES................................................................................................................ .......10 ABSTRACT....................................................................................................................... ............15 CHAPTER 1 INTRODUCTION TO DONOR-ACCEPTOR SYSTEMS AND DONOR-ACCEPTOR PURINES........................................................................................................................ ........17 Conjugated Systems............................................................................................................. ...17 Donor-Acceptor Systems.............................................................................................19 Supramolecular Control of Donor-Acceptor Systems.................................................22 Purines as Conjugated Materials............................................................................................24 Nucleobases in Synthetic Functional Architectures........................................................27 Design of Donor-Acceptor Purines Based on 2-Aminopurine....................................29 2 SYNTHESIS AND STRUCTURE OF NO VEL DONOR-ACCEPTOR PURINES.............31 Introduction................................................................................................................... ..........31 Chemical Manipulation of the Puri ne C(2) and C(6) Positions..............................................32 Addition of Acceptor Groups to C(8).....................................................................................34 Formation of the C(8)-CN Purines..................................................................................34 Formation of the C(8)-COOMe Purines..........................................................................35 Solid State Structures......................................................................................................... .....36 Molecular Level Structure...............................................................................................36 Crystal Packing of 2.7b, 2.12b, and 2.12c.......................................................................38 Original Synthetic Strategies..................................................................................................41 Experimental Section........................................................................................................... ...43 Synthesis of Compounds.................................................................................................43 X-ray Crystallography.....................................................................................................54 3 PHOTOPHYSICAL AND ELECTRONIC PR OPERTIES OF DONOR-ACCEPTOR PURINES........................................................................................................................ ........56 UV/vis data for C(8)-H, C(8)-CN, and C( 8)-COOMe Purines in Methylene Chloride.........57 Steady State Fluorescence of C(8)-H, C(8)-C N, and C(8)-COOMe Purines in Methylene Chloride....................................................................................................................... ........61 Quantum Yield Measurements in Methylene Chloride...................................................62 Stokes Shifts of Purine Com pounds in Methylene Chloride...........................................65 Fluorescence Lifetime Data for C(8)-CN and -COOMe in CH2Cl2.......................................66

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7 Solvatochromism Studies for C(8)-H, -CN, and -COOMe Purines.......................................67 Solvent Influence on Absorption and Emission Spectra.................................................68 A Lippert-Mataga Analysis of the C( 8)-H, -CN, and -COOMe Purines.........................75 The Chemical and Photochemical Stabil ity of C(8)-H, C(8)-CN, and -COOMe Purines........................................................................................................................ ..78 Electronic Properties of the D-A Purines...............................................................................81 Theoretical Calculations..................................................................................................81 Cyclic Voltammetry Studies............................................................................................84 Device Measurements for D-A Purine 2.7b....................................................................85 Experimental Section........................................................................................................... ...87 4 USING THE DONOR-ACCEPTOR PURI NE UNIT AS A FUNCTIONAL SYNTHETIC BUILDING BLOCK.......................................................................................91 Introduction................................................................................................................... ..........91 A Complementary Thrust: -Purinyl Amino Acids................................................................91 Amide Functionality at the C(8) Position...............................................................................95 Crystal Structure of the C(8)-Amide Purine 4.7.....................................................................98 Molecular Level Structure...............................................................................................98 Crystal Packing for Compound 4.7.................................................................................99 Photophysical Data for the C(8)-Amide Derivatives............................................................100 C(8)-Amide Photostability............................................................................................102 Theoretical Data............................................................................................................103 Amide Functionalization and Boc protection of the Primary Amine Donors......................105 Amide Formation at C(2) of the Purine Core................................................................105 Boc Protection of the C(2) and C(6) Primary Amines..................................................106 Urea Formation on the C(2) Amine: Building Blocks for Self-assembled Structures.........107 Critical Crystal Structure of Ureidopurine 4-16...................................................................109 Conclusion and Future Directions........................................................................................110 Experimental Section........................................................................................................... .113 Synthesis of Compounds...............................................................................................113 X-ray Crystallography...................................................................................................123 APPENDIX A 1H NMR SPECTRA OF SELECTED PURINES.................................................................125 B X-RAY CRYSTAL ST RUCTURE DATA..........................................................................138 LIST OF REFERENCES.............................................................................................................140 BIOGRAPHICAL SKETCH.......................................................................................................147

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8 LIST OF TABLES Table page 2-1 Selected Bond Lengths for 2.7b 2.12b 2.12c and 2-AP.................................................37 3-1 Absorption and Emission Propert ies for C(8)-H Purines in CH2Cl2.................................61 3-2 Absorption and Emission Propert ies for C(8)-CN Purines in CH2Cl2...............................61 3-3 Absorption and Emission Propert ies for C(8)-COOMe Purines in CH2Cl2......................61 3-4 Absorption and Emission Properties fo r C(8)-H Purines in 1,4-Dioxane.........................72 3-5 Absorption and Emission Properties fo r C(8)-H Purines in Acetonitrile..........................72 3-6 Absorption and Emission of C(8)-H Purines in Methanol.................................................73 3-7 Absorption and Emission of C( 8)-CN Purines in 1,4-Dioxane.........................................73 3-8 Absorption and Emission of C( 8)-CN Purines in Acetonitrile..........................................73 3-9 Absorption and Emission of C( 8)-CN Purines in Methanol..............................................74 3-10 Absoption and Emission of C(8)-COOMe in 1,4-Dioxane...............................................74 3-11 Absorption and Emission of C(8 )-COOMe Purines in Acetonitrile..................................74 3-12 Absorption and Emission of C( 8)-COOMe Purines in Methanol......................................75 3-13 Lippert-Mataga Data for Selected Purines.........................................................................76 3-14 Selected Bond Lengths for 2.7b and 2-AP from the crystal, and their 9-methyl derivatives from computation............................................................................................82 3-15 Electronic Structure Data for D-A Purines........................................................................83 3-16 Amino and Dimethylamino Group Wagging Angles ( in degrees) Calculated from the Optimized Geometries of the D-A Purines..................................................................90 4-1 Photophysical data for compounds 4.3 and 4.5 in 1,4-dioxane.......................................100 4-2 Photophysical data for compounds 4.3 and 4.5 in methylene chloride...........................101 4-3 Photophysical data for compounds 4.3 and 4.5 in acetonitrile........................................101 4-4 Photophysical data for compounds 4.3 and 4.5 in methanol...........................................102

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9 4-5 Electronic Structure Data for the D-A Pu rine Amide Derivatives Compared to the Corresponding Nitriles and Esters...................................................................................103 B-1 Crystal Structure Parameters............................................................................................138

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10 LIST OF FIGURES Figure page 1-1 Jablonski diagram showing radiative and nonradiative electronic transitions...................18 1-2 Traditional building blocks for some D-A systems and the resulting zwitterionic resonance generally used to descri be their electronic structure.........................................20 1-3 Selective hydrogen bonding be tween natural nucleobases................................................22 1-4 Aggregates (2:1) formed through th e Watson-Crick and Hoogsteen hydrogen bonding sites.................................................................................................................. ....23 1-5 Quadruple hydrogen bonding in the urei dopyrimidinone supramolecular polymers of Meijer and Sijbesma..........................................................................................................24 1-6 Conventional numbering of the purine core......................................................................24 1-7 Common fluorescent nucleoba se analogue 2-aminopurine...............................................25 1-8 Fluorescent nucleobase analogues.....................................................................................26 1-9 Ribbons formed by the self-assembly of guanosine derivatives........................................27 1-10 G-Quartet formed by the self-assembly of guanosine derivatives in the presence of a metal cation................................................................................................................... .....28 1-11 G-Pentamer formed from the self-assem bly of an isomer of guanosine in the presence of a metal cation..................................................................................................28 1-12 Hydrogen-bonded network formed from the guanine nucleobase.....................................29 1-13 Donor-acceptor purines based on 2-aminopurine..............................................................30 1-14 Zwitterionic resonance stru ctures of D-A purines.............................................................30 2-1 Synthesis of key synthetic inte rmediates 2-amino-6-chloropurine 2.4 and 2-amino-6chloro-9-benzylpurine 2.5 ..................................................................................................31 2-2 C(2) and C(6) derivatives from 2-amino-6-chloro-9-benzylpurine...................................33 2-3 C(2) dimethylaminopurines formed fro m derivatives of 2-amino-6-chloro-9benzylpurine ( 2.5 )..............................................................................................................33 2-4 Functionalization of the C(8) position of the purine to form the D-A purines..................35 2-5 Single molecules from the crys tal structure and their numbering.....................................37

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11 2-6 Noncovalent intermolecula r interactions present in the crystal structure of 2.7b .............39 2-7 Crystal packing of 2.7b along its three crystallographic axes...........................................39 2-8 Noncovalent intermolecula r interactions present in the crystal structure of 2.12b ...........39 2-9 Crystal packing of 2.12b along its three crys tallographic axes.........................................40 2-10 Noncovalent intermolecula r interactions present in the crystal structure of 2.12c ............40 2-11 Crystal packing of 2.12c along its three crys tallographic axes..........................................40 2-12 Preparation of starting material 2.17 used in an early synt hetic approach to D-A purines........................................................................................................................ ........41 2-13 Deprotonation attempts of the C(8) position of purines....................................................42 2-14 Successful C-C bond formation by C(8) deprotonation using LDA..................................42 2-15 Attempted lithium-halogen exchange at the C(8) position of the purine followed by electrophile addition.......................................................................................................... .42 3-1 Donor-acceptor purines targ eted for photophysical studies...............................................56 3-2 Absorption spectra for compound 2.7b upon dilution in methylene chloride...................59 3-3 Absorption intensity at 334 nm vs. concentration for compound 2.7b ..............................59 3-4 Absorption spectra for C(8)-H ( 2.7 ), C(8)-CN ( 2.7b ), and C(8)-COOMe ( 2.7c ) containing the same C(2) and C(6) substituents................................................................60 3-5 Emission spectra of C(8)-CN purines in methylene chloride............................................65 3-6 Emission spectra of C(8)-COOMe compounds in methylene chloride.............................66 3-7 Absorption data for 40 M solutions of compound 2.7b in different solvents.................68 3-8 Emission spectra for 2.10b in solvents of varying polarities.............................................69 3-9 Emission spectra for 2.10c in solvents of varying polarites..............................................69 3-10 Emission spectra of 2.6b in solvents of varying polarities................................................70 3-11 Emission spectra for 2.6c in solvents of varying polarities...............................................71 3-12 Lippert-Mataga plot for 2.6 ................................................................................................77 3-13 Lippert-Mataga plot for 2.12b ...........................................................................................77

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12 3-14 Lippert-Mataga plot for 2.12c ............................................................................................78 3-15 Changes observed in the absorption spectrum of a 60 M solution of 2.14b in methylene chloride after exposure to 254 nm ultraviolet light at varying time intervals...................................................................................................................... ........79 3-16 Changes observed in the absorption spectrum of a 60 M solution of 2.14c in methylene chloride after exposure to 254 nm ultraviolet light at varying time intervals...................................................................................................................... ........79 3-17 Changes observed in the absorption spectrum of a 60 M solution of 2.14c in 1,4dioxane after exposure to 254 nm ultravio let light at varying time intervals....................80 3-18 Changes observed in the absorption spectrum of a 60 M solution of 2.6c in water after exposure to 254 nm ultraviolet light at varying time intervals..................................80 3-19. Orbital density plots (Molden v. 4.6) fo r D-A purines calculated from the B3LYP/631++G** optimized geometries.........................................................................................84 3-20 Organic light emitting diode (OLED) for 2.7b ..................................................................86 3-21 Measurements taken with the OLED device.....................................................................86 3-22 Schematic model of the OLED device using 2.7b as the emissive layer..........................86 4-1 Novel -purinyl amino acids..............................................................................................92 4-2 Homopurinyl oligopeptide from -purinyl amino acids....................................................93 4-3 Computationally derive d torsion angle preferences for a dipurinyl peptide constructed from deprotected monomer 2-7c ....................................................................93 4-4 Oligopeptide curvature w ith potential molecular r ecognition and supramolecular capabilities................................................................................................................... ......93 4-5 Design for a hetero-o ligopeptide containing -purinyl amino acids linked with conventional -amino acids...............................................................................................94 4-6 Folded structure predic ted (by computation) for -purinyl amino acids linked with Gly-DPro-Gly.....................................................................................................................94 4-7 C(8) amide formation via an acid chloride........................................................................96 4-8 Unsuccessful coupling reaction to synthesi ze a purine dimer via the acid chloride..........96 4-9 C(8) amide formation using the DCC/DMAP conditions..................................................97 4-10 Coupling reaction to form a purinyl dimer using DCC/DMAP.........................................98

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13 4-11 Single molecule from the crystal structure of 4.7 ..............................................................98 4-12 Dominant noncovalent interactions found in the crystal structure of 4.7 ........................100 4-13 The crystal packing of 4.7 along its crystallographic axes..............................................100 4-14 Changes observed in the absorption spectrum of a 60 M solution of 4.3 in methylene chloride after exposure to 254 nm ultraviolet light at varying time intervals...................................................................................................................... ......102 4-15 Orbital density plots for D-A purinyl amide derivatives calculated from the B3LYP/6-31++G** optimized geometries (using Molden v. 4.6)..................................105 4-16 Amide formation on the C(2) amine via an acid chloride...............................................106 4-17 Boc protection of primary amines on the purine core......................................................107 4-18 General design of self-complementary quadruply hydrogen bonded dimers based on purines........................................................................................................................ ......108 4-19 Urea formation using the C(2) primary amine.................................................................108 4-20 Intramolecular hydrogen bonding of 4-16 in the solid state (ellipsoids drawn at the 50% probability level). The side view of a CPK representation shows the extent of contact achievable for the two phenyl ri ngs in the N(3) H-bonded conformer...............110 4-21 Design of donor-acceptor purines to be prepared for future photophysical studies........111 4-22 Potential supramolecular structure of an C(8)-amide adenine analogue.........................111 4-23 Potential supramolecular structure of C(8)-amide 2-aminopurine derivatives................112 A-1 1H NMR of 2.7 in CHCl3.................................................................................................125 A-2 1H NMR of 2.10 in CHCl3...............................................................................................125 A-3 1H NMR of 2.12 in DMSOd6..........................................................................................126 A-4 1H NMR of 2.14 in DMSOd6..........................................................................................126 A-5 1H NMR of 2.7a in CHCl3...............................................................................................127 A-6 1H NMR of 2.8a in CHCl3...............................................................................................127 A-7 1H NMR of 2.10a in CHCl3.............................................................................................128 A-8 1H NMR of 2.12a in CHCl3.............................................................................................128 A-9 1H NMR of 2.14a in DMSOd6........................................................................................129

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14 A-10 1H NMR of 2.6b in DMSOd6.........................................................................................129 A-11 1H NMR of 2.7b in DMSOd6.........................................................................................130 A-12 1H NMR of 2.8 in DMSOd6............................................................................................130 A-13 1H NMR of 2.10b in CHCl3.............................................................................................131 A-14 1H NMR of 2.12b in DMSO-d6......................................................................................131 A-15 1H NMR of 2.14b in CHCl3.............................................................................................132 A-16 1H NMR of 2.6c in CHCl3...............................................................................................132 A-17 1H NMR of 2.7c in CHCl3...............................................................................................133 A-18 1H NMR of 2.8 in CHCl3.................................................................................................133 A-19 1H NMR of 2.10c in CHCl3.............................................................................................134 A-20 1H NMR of 2.12c in DMSOd6........................................................................................134 A-21 1H NMR of 2.14c in DMSOd6........................................................................................135 A-22 1H NMR of 4.3 in DMSOd6............................................................................................135 A-23 1H NMR of 4.5 in CHCl3.................................................................................................136 A-24 1H NMR of 4.6 in CHCl3.................................................................................................136 A-25 1H NMR of 4.7 in DMSOd6............................................................................................137 A-26 1H NMR of 4.8 in DMSOd6............................................................................................137

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15 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy SYNTHESIS, STRUCTURE, AND P HOTOPHYSICAL PROPERTIES OF DONOR-ACCEPTOR PURINES By Roslyn Susanne Butler August 2007 Chair: Ronald Castellano Major: Chemistry Donor-acceptor (D-A) molecules have photophys ical, electronic, and optoelectronic properties that make them suitabl e as sensors, wires, and fluor ophores. Here the syntheses and properties of the first D-A systems based on purin es are reported. Resu lts show that simple chemical modifications to the heterocycle signi ficantly improve its inhe rent optical properties (even beyond 2-aminopurine); the resulting fluorophor es are candidates as both biological probes and optoelectronic device components. The mo lecular recognition functionality of purines, multiple nitrogen atoms capable of hydrogen bonding and an extended aromatic surface for stacking, can potentially be e xploited to control molecular association and ordering. C(2) and C(6) donor substituted purines are brom inated at C(8) and then transformed via a palladium(0)-catalyzed cyanation reaction to th eir corresponding nitriles. Conversion of the nitriles to methyl esters comes by treatment w ith acidic methanol. The C(8) acceptor groups dramatically increase the fl uorescence quantum yields (F) of the purines (versus a hydrogen in the C(8) position) in organic solvents to in many cases near unity (representing, for one derivative, a F enhancement of > 2500% in methylene chloride). Absorption (max 310 nm), emission (max 360 nm), and fluorescence lifetime data (8 ns F 0.5 ns) were

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16 collected for 12 D-A purines in four organic so lvents and even water (for the 2-aminopurine analogues). Four of the purines could be studi ed in the solid state by X-ray crystallography. Theoretical calculations (DFT and ZINDO/CI) determined the absorption spectra, HOMO (.5 to .5 eV) and LUMO (.5 to .5 eV) energies, and the ground and excited state dipoles. The calculated orbital energies are in good agreemen t with solution-phase cyclic voltammetry data and the calculated excite d state dipoles agree well with resu lts from a Lippert-Mataga treatment of the solvent-dependent emission spectra. On e D-A purine was successfully tested as the emissive layer in an organic light emitting diode (OLED). Amide bonds can be formed at the purine C(8) position (via an in termediate carboxylic acid) towa rd use of the molecules as heterocyclic amino acids. The amides show high quantum yields (F > 0.85) in all organic solvents and well-defined confor mations in the solid state.

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17 CHAPTER 1 INTRODUCTION TO DONOR-ACCEPTOR SYSTEMS AND DONOR-ACCEPTOR PURINES Conjugated Systems All molecules undergo an electronic process as they absorb energy (photons at different wavelengths of light). This proc ess is expressed by equation 1.1, where is the energy absorbed, h is Plancks constant, c is the speed of light, and is the wavelength of light.1 = hc / The absorbed energy ( ) increases the energy of a conjugated system by promoting an electron from the molecules highest occupied molecular orbital (HOMO) to its lowest unoccupied molecular orbital (L UMO) resulting in molecula r excitation. For conjugated molecules as conjugation length is increased the energy gap between the HOMO and LUMO is decreased enabling longer wave lengths (lower energy photons) to promote electrons to the LUMO. If this gap is lowere d enough, absorption enters the visible range (400 nm) and can be detected by the human eye as color. If th ese electronic transitions are promoted by photons between 200 nm and 800 nm, the electronic absorp tion transitions that occur can be monitored using ultraviolet and visible (UV/vis) spectroscopy. A UV/vi s spectrometer determines the absorption using the Beer-Lambert law (equation 1.2).1 A = log I0/ I = lc (1.2) The absorption is A I0 is the intensity of light entering the sample, I is the intensity of the light exiting the sample, l is the pathlength of the sample cell, and c is the concentration (mol/L) of the sample. The molar extinction coefficient ( is the experimentally determined efficiency of absorption that is unique for each molecule. As conjugated systems become more complex, the

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18 number of electronic transitions that can occur upon their interaction with light increases and UV/vis spectra can become quite complicated as multiple absorption bands begin to overlap.1 S00 1 2S1S2T1 Absorption Internal Conversion Intersystem Crossing Fluorescence Phosphorescence A A F P Figure 1-1. Jablonski diagram showing radiative and nonradia tive electronic transitions. The release of absorbed energy can take pl ace through nonradiative (heat generating) or radiative (light emitting) processes as shown in the simplified Jablonski diagram in figure 1-1.2 Nonradiative processes include relaxation, in tersystem crossing, and internal conversion. Radiative processes include fluorescence (e mission from the excited singlet state, S1) and phosphorescence (emission from the excited triplet state, T1) and are almost always initiated from the lowest excited state (S1 and T1).2 The term luminescence is used for light emitting processes when the source of the emission is undetermined or contains both fluorescence and phosphorescence. Time resolved data can determ ine the source of a molecules luminescence through emission rates which are ty pically between 1 and 10 ns fo r fluorescence and on the order of milliseconds to minutes for phosphorescence. A molecules luminescence is typically reported as an emission spectrum that shows the wavelength of light the molecule emits

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19 (emission maximum) and its intensity. Once the ener gy is released, the molecule is structurally unchanged from the original species.2 Absorption and emission spectra provide additi onal quantitative information related to a molecules structure and to its potential appl ications. The Stokes shift, for example, is determined as the difference between the absorption and emission maximum for a given molecule in a given environment. Large variatio ns in Stokes shift due to solvent polarity can indicate that a fluorophore will be a useful biological probe. Th e quantum yield (of fluorescence or luminescence) is a defining parameter of a lu minophore and is defined as the total number of photons emitted relative to the number of photons absorbed (highest value, F = 1); this value gives insight into how a conjugated system will pe rform in an electronic device or as a probe. Implications of large Stokes shifts and quantum yields in the contex t of fluorophore design will be discussed more thoroughly in Chapter 3.3 Fluorescence spectroscopy is a powerful rese arch technique for probing the dynamics of biomolecules (e.g. conformational changes upon bi nding). Proteins and DNA are often studied by analyzing the changes in their fluorescence spectra under different conditions. Fluorescence spectroscopy also identifies organic molecules suitable for use in optoelectronic devices, and helps characterize optoelectronic devices such as organic light emitting diodes.2 Donor-Acceptor Systems Conjugated systems can be functionalized with electron donor and electron acceptor groups to create donor-acceptor (D-A or D-A) systems (Figure 1-2).4 D-A systems can consist of small molecules, oligomers, or polym ers. The oligomers and polymers can be either linear or star-shaped.4 The electron donor and electron accep tor create a push-pu ll delocalization

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20 mediated by the -spacer, the flow of -electrons is often represented by simple valence bond theory and a zwitterionic resonance form (Figure 1-2).4 D = Donor A = Acceptor D A D A D A NR2 OR O NO2 CHO CN S * * * n n n n n n Figure 1-2. Traditional bu ilding blocks for some D-A systems and the resulting zwitterionic resonance generally used to descri be their electronic structure. The magnitude of this push-pull effect is re lated to the strength (e .g. the ionization energy of an amine substituent) and placement of the donor and acceptor groups, to the nature of the conjugated system to which they are introduced, and to the envir onment in which the system is placed. This molecular design can result in mol ecules with outstanding el ectrical, optical, and optoelectronic properties. The nonlinear optic al (NLO) properties associated with donoracceptor systems have emerged among the most important for optical data storage, data processing, and data transfer.5 Additional applications for D-A molecules incl ude use in electronic devices such as fieldeffect transistors,6 organic light emitting diodes,7 and solar cells.8 Field-effect transistors are a type of transistor that relies on an electric field to control the conductivity of a channel in a

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21 semiconductor material. Field-effect transi stors in complementary symmetry metal oxide semiconductors are the basis for modern digital in tegrated circuits that are responsible for the function of microprocessors. Organic light em itting diodes are semiconduc tors that emit light from an electroluminescent layer consisting of a film of organic compounds (discussed more thoroughly in Chapter 3). This technology is used for creating displays that can have manufacturing and energy requireme nt advantages over plasma and liquid crystal displays. Solar cells or photovoltaic cells are devices that convert light energy into electrical energy. A critical and challenging issue for creat ing electronic devices from organic D-A molecules is the control of molecular organiza tion when incorporating these systems into a device.8 A noncentrosymmetric ordering of D-A mol ecules is required for organic materials used for second order nonlinear optics and in optoelectronic devi ces. This is why many devices fabricated from materials based on D-A molecules with excellent photophysic al properties fail to perform well or at all. A larg e number of D-A molecules form -stacked centrosymmetric (antiparallel) dimers due to electrostatic inte rmolecular interactions arising from their large ground state dipole moments. The centrosymm etric face-to-face stacked dimer generally quenches fluorescence by causing tw o exciton states to arise wher e only transition to the higher energy state is allowed; this can be observed experimentally as a blue-shifted absorption band (when compared to absorption for the single molecu le). Rapid internal conversion to the lower energy exciton state decreases the transition probab ility for a radiative pr ocess to the ground state and the fluorescence is quenched. The use of additional noncovalent interactions to control supramolecular structure could be adva ntageous for constructing devices.

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22 Supramolecular Control of Donor-Acceptor Systems Organizational hierarchy and se lf-assembly through molecular recognition are the focuses of supramolecular chemistry, so it is only fitting that incorp orating aspects of molecular recognition into a D-A molecule could improve control over its orientation in a device, thereby improving the performance of the device.9 Experimental time scale disintegration and reassembly of reversible supr amolecular interactions also a llows optimization of noncovalent interactions between monomers re sulting in annealing and self-hea ling of structural defects. Two of the most common noncovalent intermolecu lar interactions employed in supramolecular chemistry are and hydrogen bonding interactions.8 Logically, -interactions are going to occur in D-A systems, and due to their highly speci fic and directional nature hydrogen bonds are ideal for creating controlled supramolecular assemblies.8 Nature elegantly demonstrates how -stacking and hydrogen bondi ng can create highly ordered structures with phenomenal function.10 Self-assembly of the nucleic acids, to form double(or multiple-) stranded DNA and RNA, is a quintessential example. At the heart of nucleic acid assembly and function are th e pyrimidine and purine nucleobases. N N N N N N N O O R X H H H N N N N O N NN N O H H H H H R R R T : X = Me U : X = H A G CAB Figure 1-3. Selective hydrogen bonding between natural nucleobases. A) Two-point hydrogen bonding between adenine (A) and either t hymine (T) or uracil (U). B) Triple hydrogen bonding between guanine (G) and cytosine (C).

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23 The pyrimidines thymine (T), cytosine (C), and uracil (U) selectively base pair through multiple hydrogen bonds with the purine nucleobases aden ine (A) and guanine (G). Supramolecular structures of nucleic acids are further stabilized by -stacking and hydroph obic interactions.11 The purines base pair through their WatsonCrick hydrogen bonding site (Figure 1-3A,B); further assembly via the Hoogsteen site is possible and leads to the formation of 2:1 aggregates (Figure 1-4). Access to such well-defined patt erns has made the pyrimidine and purine cores attractive building blocks for creating highl y controlled supramolecular structures.11 N N N N N N N O O R X H H H N N N N O N NN N O H H H H H R R R A G C N N O O H R N HN N O H H H C T TA B Figure 1-4. Aggregates (2:1) formed thr ough the Watson-Crick a nd Hoogsteen hydrogen bonding sites. A) adenine (A) and thymine(T). B) guanine (G) and cytosine (C). Meijer and Sijbesma have utilized the pyr imidine core to creat e a urea-functionalized pyrimidine (ureidopyrimidinone) uni t capable of self-dimeriza tion through four hydrogen bonds which results in the formation of a supramolecular polymer (Figure 1-5).12-14 Such polymers offer the promise of merging the desirable ma terials properties of conventional covalent polymers with the self-healing behavior typical of reversibly-formed assemblies.15-18 Pyrimidines have been used in a number of ot her interesting supramol ecular systems; however, from here on the focus will shift to supramol ecular structures created by compounds containing the purine core.11

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24 N N O H N N O N N O R H N N O H H H H N N O H N N O N N O R H N N O H H H H n Figure 1-5. Quadruple hydrogen bonding in the ureidopyrimidinone supramolecular polymers of Meijer and Sijbesma.16 Purines as Conjugated Materials Purines as heterocyclic aromatics have plen ty of synthetic and supramolecular potential.11 The planar extended aroma tic core can engage in -interactions. The four nitrogen atoms offer opportunity for hydrogen bonding and metal binding inte ractions while the nitrogens in the 7 and 9 positions can be functionalized (See Figure 1-6 for numbering). Carbons in the 2, 6, and 8 positions can be functionalized by addition of substituents through standard reactions that involve cationic, anionic, or ra dical intermediates. The conjuga tion of the system offers the opportunity to use it for photophys ical and electronic applicati ons. Sources of purine core precursors would not necessarily have to rely on traditional petroleum feed stocks if a method could be devised to extract and purify pur ine compounds from a biological source. N N N H N 12 6 3 4 5 7 9 8 Figure 1-6. Conventional numbering of the purine core. The modification of purine nucleobases in orde r to enhance their op tical properties for biological applications has a ri ch history. Natural nucleobases are essentially non-fluorescent (F = 0.0001) making the study of nucleic acids by fluorescence spectroscopy basically

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25 impossible without modification.19 2-Aminopurine (2-AP) is a common fluorescent nucleobase analogue of adenine (Figure 17A) that in water has a high fluorescence quantum yield (F = 0.68) and can establish stable Watson-Crick interactions with thymine (Figure 1-7B).19,20 The red-shifted absorption (305 nm ) of 2-AP also allows it to be sele ctively excited in the presence of the natural nucleobases.20 In order to study DNA using fluor escence spectroscopy, 2-AP or other fluorescent nucleobase analogues are incorporat ed into a DNA strand in place of their corresponding nucleobase, requiri ng that they be amenable to sugar functionalization.2 N N N N NH2 N N N N NH2 R R N N N N N R NN O O R H H H 2-AP Adenine 2-Aminopurine TAB Figure 1-7. Common fluorescen t nucleobase analogue 2-aminopur ine. A) Adenine analogue, 2aminopurine (2-AP). B) Watson-Crick base pairing between 2-AP and thymine. 2-AP has been used to study local interac tions between nucleobases in DNA, electron transfer reactions throug h oligonucleotide strands,21,22 and DNA damage due to ultraviolet radiation.23 Recently, Turro and coworkers used 2-AP as a fluorescent base in a molecular beacon that displays an enhanced se nsitivity for detecting complementary DNA.24 When 2-AP is incorporated into oligonucleotide s, its fluorescence is dramatically quenched up to 100-fold due to -stacking interactions.19 The quenching can take plac e to a lesser degree if the -stacking of the oligonucleotide is less effici ent or if the stacking is interr upted by some other interaction.25-27 2-APs sensitivity to its environment can lead to observations about the dynamics of the system

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26 that it is incorporated into; however, the reduc tion of fluorescence requires high concentrations of the 2-AP labeled oligonucleotide in order for the system to be studied effectively.28 Many researchers have prepared fluorescent nucleobase analogues through modification of natural nucleobases. Systems have been formed where the conjugation of a natural nucleobase is extended through formation of an etheno derivative,20,29,30 or by incorporating an additional aromatic ring in between the imidazole and pyr imidine portion of the purine core (Figure 18A).31 Systems have been formed by adding weak electron acceptor functionality to the C(8) position of natural purine nucleobases (Figure 1-8B ). Firth and coworkers created 8-alkynylated guanosine (Figure 1-8B) and adenine (not shown).32 Tor and coworkers found increased luminescence by incorporating a furan moiety onto the C(8) position of natural purine nucleosides.33 Some researchers, like Kool have ev en shown that the base structure can be altered completely, preservi ng base stacking but introduci ng additional functionality (luminescence, enhanced metal binding, etc.) (Figure 1-8C).34-38 All of these fluorescent analogues have exhibited increased fluorescen ce when compared to that of the natural nucleobases. HN N N N O H2N N N N N NH2 R R O R N N N N R N H N N R N N NH2 N N N N R N N N N R ABC Figure 1-8. Fluorescent nucleobase analogues. A) Formed by exte nded conjugation. B) Formed by addition of acceptor group. C) Formed by us ing a complete alteration of base structure.

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27 Nucleobases in Synthetic Functional Architectures Remarkable examples of complex purine s upramolecular structure have been achieved with guanine and guanosine derivatives, driven by hydrogen bonding. Ribbons, for example, may form with (Figure 1-9A) or without (Figure 19B) a net dipole 39and the preference can be controlled by solvent interactions and/or by the nature of the R group.39 Three-terminal devices are found to act as a field effect transistor by using polar ribbons (Figure 1-9A) to connect the drain and source terminals.40 N N N N O N N N N N O N N N N N O N N N N N O N H H H N N N N O N H H H H H H H H H N N NN O N N N N N O N NH N NN O N H H H H H H H H H H H R R R R R R R R A B Figure 1-9. Ribbons formed by the self-assemb ly of guanosine derivatives. A) Ribbon that contains a net dipole. B) Ribbon that has no net dipole. In the presence of sodium or potassium cations the guanine unit can form a self-assembled quartet structure known as the G-quartet driven by hydrogen-bondi ng interactions between four guanine derivatives (Figure 1-10).41 The quartets are further st abilized by the cations that coordinate with the carbonyl oxygen of each guanine in the quartet.42 The G-quartets can then stack with one another to form colu mnar nanostructures, the G-quadruplex.43

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28 N N N N O N N N N N O N N N N N O N N N N N O N H H H H H H H H H H H H M R R R R N N N N O N H H H R M Figure 1-10. G-Quartet formed by the self-assembly of guanosine de rivatives in the presence of a metal cation. Through simple switching of the carbonyl and primary amine of guanosine, a G-pentamer is formed instead (Figure 1-11).44 The guanine, which has a hydr ogen atom in place of the R group, can lead to a hydrogen bonded network of G-quartets without the need for a cation template (Figure 1-12).45 Addition of an oxo group to the C(8) position of a guanosine can create a self-assembled helical structure.39 All of these examples show how simple chemical changes to the purine core can result in predic table and controllable supramolecular structures with unique functions. N N N N N O H H H R N N N N N O H H H R N N N N N O H H H R N N N N N O H H H R N N N N N O H H H R N N N N N O H H H R M M Figure 1-11. G-Pentamer formed from the self -assembly of an isomer of guanosine in the presence of a metal cation.

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29 N N N N O N N N N N O N N N N N O N N N N N O N H H H H H H H H H H H H H H H H N N N N O N N N N N O N N N N N O N N N N N O N H H H H H H H H H H H H H H H H N N N N O N N N N N O N N N N N O N N N N N O N H H H H H H H H H H H H H H H H N N N N O N N N N N O N N N N N O N N N N N O N H H H H H H H H H H H H H H H H NH N N H N O NH2 Figure 1-12. Hydrogen-bonded network fo rmed from the guanine nucleobase. Design of Donor-Acceptor Purines Based on 2-Aminopurine We hoped to extend the traditional donor-acceptor concept to 2-aminopurine in order to study the fundamental changes that would result, increase the luminescence of the system, and potentially exploit the multiple noncovalent interactions availabl e to the purine core in the context of optoelectronic materials. The design of our D-A purines utilizes the purine core as the -linker and features a relatively strong electron-accepting substituent in the C(8) position (Figure 1-13). Donor groups placed in the C(2) and C(6) positions of the purine core create the desired push-pull delocalization, and both donors can communicate with the acceptor in this fashion (Figure 114A,B).

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30 N N N N D D Bn A D = Dono r 1 2 3 4 5 6 8 7 9 A =Accepto r H2N (Me)2N BnO H CN CO2Me Figure 1-13. Donor-acceptor purines based on 2-aminopurine. N N N N D D Bn A N N N N D D Bn A N N N N D D Bn A N N N N D D Bn A A B Figure 1-14. Zwitterionic resonance structures of D-A purines. A) Resonance through the C(2) position. B) Resonance through the C(6) position. Photophysical investigation of D-A purines prepared in this thesis reveal the highest reported quantum yield values for simple purines with intrinsic values approaching unity. Furthmore, many of the compounds exhibit high se nsitivity to their environment making them suitable for function as biological probes or nuc leobase surrogates prov ided modification to impart water solubility. Examination of so lid state crystalline structures of the D-A purines shows how slight changes to the donor a nd acceptor groups can ch ange their packing preferences. These results are encouraging that the supramolecula r structure of such purines can be tailored for use in optoelectronic devices.

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31 CHAPTER 2 SYNTHESIS AND STRUCTURE OF NOVEL DONOR-ACCEPTOR PURINES Introduction Synthesis of the D-A purines starts with the biological compound guanine 2.1 a cheap readily available starting material costing le ss than one dollar per gr am. Using a four-step protocol (Figure 2-1) adapted from an established patented procedure46 guanine is transformed into 2-amino-6-chloropurine ( 2.4 ) in 56% yield. 2-Amino-6-chloropurine ( 2.4 ) is otherwise a commercially available but very expensive prec ursor costing approximately $68 per gram. Due to the need for large quantities of 2.4 it was more cost effective to make this molecule in-house for approximately $7 per gram (c ost of material s, not labor). HN N O N H N H2N 1) POCl3/ DMF 2) NaHCO3N N Cl N H N N N acetic acid N N Cl N H N HN 1) NaOH 2) HCl 3) NaOH O N N Cl N H N H2N 56% from 2.1 N N Cl N N H2N Bn 59% K2CO3, BnBr DMF 2.1 2.4 2.5 2.2 2.37 9 Figure 2-1. Synthesis of key synthetic intermediates 2-amino-6-chloropurine 2.4 and 2-amino-6chloro-9-benzylpurine 2.5 In order to add tautomeric stability to the imidazole portion of the purine core, key intermediate 2.4 is benzylated47 to afford 2-amino-6chloro-9-benzylpurine ( 2.5 ) (Figure 2-1) and 2-amino-6-chloro-7-benzylpurine47 (not shown). Even though both benzylated regioisomers are formed, the desired N(9) isom er is synthesized in higher yiel d and is easily separated from the N(7) isomer by flash chroma tography or by recrystallization. The benzyl group was chosen as the N(9) alkylating group over simple alkyl groups for two reasons. Simple alkyl groups

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32 remain in the plane of the purine ring allowing extended -stacking to occur making the species very insoluble; the benzyl group, not being planar to the purine core, is able to frustrate longrange -stacking and allow for more efficient solvation.48 Second, as a robust protecting group the benzyl substituent is capable of withst anding conditions needed for functional group transformations at other positions on the purine. The benzyl group can also be removed from the purine core using Pd/C, H2, and concentrated HCl.47 Chemical Manipulation of the Pu rine C(2) and C(6) Positions Versatile intermediate 2.5 can be selectively modified to cr eate a variety of C(2) and C(6) substituted purines (Figure 2-2). The C(6)-chl oro substituent can be removed using catalytic hydrogenation to give the pare nt 2-aminopurine derivative 2.6 ,49 while nucleophilic aromatic substitution reactions like amination50 and alcoholysis can displace the chlorine and provide compounds 2.7 and protected guanosine derivative 2.8 ,51 respectively. Transformation of the C(2) primary amine is achieved using a chloro-dediazotization52 procedure to produce 2,6dichloro-9-benzylpurine 2.9 All the products formed in figure 2-2 are stable solids that can be handled easily and formed on the gram scale. These compounds do not require stringent purification and can generally be used in subsequent reactions di rectly after removing the solvent and drying under vacuum. The C(2) and C(6) positions of purine compounds 2.6 and 2.9 can be transformed further into C(2) dimethylaminopurine deriva tives (Figure 2-3A,B). Compound 2.9 can be heated with dimethylamine50 in ethanol at 100 C to give 2.10 (Figure 2-3A). The C(6) chlorine of 2.9 can also be selectively displaced with a primary amine using methanolic ammonia at 60 C to produce 2.11 .50 The C(2) chlorine of compound 2.11 is then transformed to a dimethylamino function50 using dimethylamine and ethanol to form 2.12 (Figure 2-3A). 2-Amino-9-

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33 benzylpurine ( 2.6 ) is placed under the same ch loro-dediazotization conditions52 used to form compound 2.9 and offers 2-chloro-9-benzylpurine ( 2.13 ) which can then be heated with dimethylamine and ethanol at 100 C to produce 2-dimethylamino-9-benzylpurine ( 2.14 ) (Figure 2-3B). N N N N H2N Cl Bn N N N N H2N N Bn N N N N H2N Bn N N N N H2N OBn Bn N N N N Cl Cl Bn Pd/C,H2EtOH H2O BnOH, K2CO3, DABCO TMSCl isoamylnitrite CH2Cl292% 86% 95% 63% NH(Me)2, EtOH 2.7 2.6 2.5 2.9 2.86 2 Figure 2-2. C(2) and C(6) derivatives from 2-amino6-chloro-9-benzylpurine. N N N N N N Bn 2.10 76% N N N N Cl NH2 Bn N N N N N NH2 Bn NH3/MeOH 64%99% 2.12 2.11 N N N N H2N Bn 2.6 N N N N Cl Bn N N N N N Bn NH(Me)2EtOH TMSCl isoamylnitrite CH2Cl22.132.14 34% 86% N N N N Cl Cl Bn 2.9 NH(Me)2EtOH NH(Me)2EtOHB A Figure 2-3. C(2) dimethylaminopurines formed from derivatives of 2-amino-6-chloro-9benzylpurine ( 2.5 ).

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34 Addition of Acceptor Groups to C(8) Using Br2 under ambient light, the C( 8) position of compounds 2.1 2.6 is selectively brominated53 in 70-90% in almost all cases (Figure 2-4), which provides a chemical handle for its further functionalization. Alt hough also often used for purines, bromination of C(8) with NBS in THF at room temperature only rendered th e unreacted starting pur ine and succinimide. Formation of the C(8)-CN Purines Several methods for creating purinecarbonitrile s from precursor halopurines have been used and suffer from various disadvantages includ ing low yields and the need for highly reactive precursor halo or pseudohalopurines such as iodopurines or sulfonylpurines. These methods include the treatment of the precu rsor purine with copper(I) cyanide,54 displacement reactions involving potassium cyanide or sodium cyanide,55 reactions with tetraeth ylammonium cyanide in the presence of trimethylamine,56 and Pd-catalyzed couplings with potassium cyanide57 and tributyltin cyanide.58 We identified the Pd-catalyzed cross coupling method developed by Gundersen and coworkers as the most promisi ng for our systems. In their work, C(8) purinecarbonitriles were prepared in 0-81% yield using Pd(PPh3)4, Zn(CN)2,and NMP at 90 C.59 Conversion of the bromides, compounds 2.6a 2.8a 2.10a 2.12a 2.14a to the nitriles using a slightly modified Gundersen method cr eated the first series of donor-acceptor (D-A) purines, compounds 2.6b 2.8b 2.10b 2.12b 2.14b (Figure 2-4). The primary amine functionality and nitrogen rich na ture of the purine core presented many challenges for the metal catalyzed cyanation reaction. T horough degassing and drying of the solvent was necessary due to the sensitivity of the Pd(0) metal to oxygen. The Pd(0) metal also easily complexes to the nitrogen donor atoms of the purine requiring the use of larg e amounts of the catalyst (20 40%) which is significantly more than the 0.07 mmol catalyst loading used in Gundersens method. After many inconsistent results fr om these reactions a literature investigation revealed that Pd

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35 ((0) and (II))-catalyzed reactions are sensit ive to cyanide ions;60 in particular, when in excess they can render the catalyst inactive61 by forming species such as [Pd0(CN)n]n-.62 With this knowledge, consistent results were obtained by a dding the catalyst to the purine and heating for approximately 30 min before slowly adding Zn(CN)2/solvent mixture by syringe (over 5 hours). 2.6 R1 = NH2, R2 = H 2.7 R1 = NH2, R2 = N(Me)2 2.8 R1 = NH2, R2 = OBn 2.10 R1 = N(Me)2, R2 = N(Me)2 2.12 R1 = N(Me)2, R2 = NH2 2.14 R1 = N(Me)2, R2 = H N N N N R1 R2 Bn N N N N R1 R2 Bn N N N N R1 R2 Bn N N N N R1 R2 Bn 2.6a R1 = NH2, R2 = H 2.7a R1 = NH2, R2 = N(Me)22.8a R1 = NH2, R2 = OBn 2.10a R1 = N(Me)2, R2 = N(Me)2 2.12a R1 =N(Me)2, R2 = NH2 2.14a R1 = N(Me)2, R2 = H Br CN O O Br2, CH2Cl2Zn(CN)2Pd[PPh3]4NMP 1. NH3/MeOH 2. HCl 24% 78% 77% 89% 68% 80% 80% 91% 42% 93% 77% 39% 60% 86% 82% 64% 75% 71% 2.6b R1 = R1 = NH2, R2 = H 2.7b R1 =NH2, R2 = N(Me)22.8b R1 =NH2, R2 = OBn 2.10b R1 = N(Me)2, R2 = N(Me)2 2.12b R1 = N(Me)2, R2 = NH2 2.14b R1 = N(Me)2, R2 = H 2.6c R1 = R1 = NH2, R2 = H 2.7c R1 = NH2, R2 = N(Me)2 2.8c R1 = NH2, R2 = OBn 2.10c R1 = N(Me)2, R2 = N(Me)2 2.12c R1 = N(Me)2, R2 = NH2 2.14c R1 = N(Me)2, R2 = H Figure 2-4. Functionalization of the C(8) position of the purine to form the D-A purines. Formation of the C(8)-COOMe Purines The second series of D-A purines the C(8) methyl esters ( 2.6c 2.8c 2.10c 2.12c 2.14c ) were subsequently synthesized by formation of the methyl imidate intermediate using known methodology that involves treating the nitrile derivatives with methanolic ammonia (Figure 24).63 The methyl imidate was then readily hydrolyzed to give the C(8) methylester.63 The C(8) methylester compounds are more soluble in acet onitrile and methanol th an the C(8) cyano;

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36 accordingly, thin layer chromatography (silica, 5% MeOH/CH2Cl2) indicates that they are a more polar species. Solid State Structures The structures of compounds 2.7b 2.12b and 2.12c as determined in the solid state by Xray crystallography, perfomed by Dr. Khalil Abb oud, are shown in Figure 2-5. Single crystals were grown by slow evaporation of solutions of each compound in a methanol/methylene chloride mixture. Crystals for the C(8) purinecarbonitriles 2.7b and 2.12b formed overnight while those for the C(8) purine methylester 2.12c formed upon slow evaporation over one week. Molecular Level Structure The X-ray structure of 2.7b (Figure 2-5A) reveals the dimethylamine substituent, defined by atoms N(10), C(14), and C(15), to be planar at N(10) and with the purine core. The sum of the N(10) bond angles (e.g., C(14)-N(10 )-C(15)) is 359.72, consistent with sp2 hybridization at nitrogen. The dimethylamine substituents on 2.12b (Figure 2-5B) and 2.12c (Figure 2-5C) show nearly identical geometry where the sum of the N(11) bond angles are 359.95 and 359.74, respectively. In the case of 2.12c the crystal structure also shows that the nonhydrogen atoms defining the C(8) methyl ester s ubstituent, C(14), O(15), O(16), a nd C(17) deviate from the mean plane defined by the purine core by no more than 0.087 The N(9) benzyl substituent geometry can be defined by torsion angles defined by a-b-c-d) and defined by b-c-d-e) for 2.7b 2.12b and 2.12c (Figure 2-5D). The values are = 81.55 and = 104.60 for 2.7b = 90.93 and = 108.54 for 2.12b and = 103.10 and = 16.64 for 2.12c In all cases the benzyl group extends nearly perpendicu lar to the plane of the purine core ( ); this is also consistent with its preferred geometry by computation. Angle is sensitive to the intermolecular interactions involving the phenyl group in the solid state.

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37 Figure 2-5. Single molecules from the crys tal structure and thei r numbering. A) Compound 2.7b B) Compound 2.12b. C) Compound 2.12c Selected bond lengths derived from the cr ystal structure for the D-A purines and 2aminopurine (2-AP),64 one of just four 2-aminopurine stru ctures available (not including those presented here),65,66 are shown in table 2-1. Upon examination, the bond lengths between 2.7b 2.12b 2.12c and 2-AP deviate by little more than 0.015 Table 2-1. Selected Bond Lengths for 2.7b 2.12b 2.12c and 2-AP. Bond 2.7b 2.12b 2.12c 2-AP N(1)-C(6) 1.3411(15) 1.3292(19) 1.3407(16) 1.332(3) N(1)-C(2) 1.3536(15) 1.3654(19) 1.3635(16) 1.365(2) C(2)-N(3) 1.3484(15) 1.3577(18) 1.3524(16) 1.343(2) C(2)-N(11) 1.3563(15) 1.3506(19) 1.3590(17) 1.353(3) N(3)-C(4) 1.3446(14) 1.3323(18) 1.3402(16) 1.329(3) C(4)-N(9) 1.3749(14) 1.3779(17) 1.3710(16) 1.368(3) C(4)-C(5) 1.3952(16) 1.390(2) 1.3890(17) 1.400(2) C(5)-C(6) 1.4301(16) 1.415(2) 1.4177(18) 1.381(3) C(5)-N(7) 1.3810(14) 1.3791(18) 1.3778(16) C(6)-N(10) 1.3450(15) 1.342(2) 1.3339(17) N(7)-C(8) 1.3129(16) 1.3192(19) 1.3212(16) 1.318(2) C(8)-N(9) 1.3834(14) 1.3779(19) 1.3910(15) 1.360(3) C(8)-C(12) 1.4308(15) 1.438(2) C(8)-C(14) 1.4895(18) Bond length alternation seen in the X-ray structure of a donor-ac ceptor molecule has been found to be a good indication that there is charge-tra nsfer from the donor to the acceptor in the ground state.67 This is nicely illustrated for the cyanoethynylethenes67,68 where bond lengths can deviate by 0.2 or greater between the donor a nd non-donor substituted derivatives.68 The analysis shows that there may only be a very small amount of charge transfer in the ground state of the D-

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38 A purines reported here, a result that is confirmed by electroni c structure calculations (vide infra). Important to note, however, is that comp arison of 2-AP to the D-A purines reported here is somewhat skewed due to the tautomeric instab ility that sees 13% of the N(7) tautomer found in the 2-AP crystal structure.64 Crystal Packing of 2.7b, 2.12b, and 2.12c Examination of the crystal packing of all th ree structures reveals many of the expected noncovalent intermolecular inte ractions. All three purines form dimers through hydrogen bonding (Figures 2-6A, 2-8,A and 2-10A) s howing typical hydrogen bonding distances69 and all three exhibit antiparallel dipolar -stacking (Figures 2-6B, 28B, 2.10B) which is a common motif for D-A molecules.67,68,70-73 Purine 2.7b forms a two-point hydrogen bonded dimer via the N(3) face (N(3)N(11) = 3.11 )(Figure 2-6A). Purine 2.12b, which is the structural isomer of 2.7b forms a similar dimer with hydrogen bonding taking place this time from the Hoogsteen edge (N(7)N(10) = 3.00 ) (Figure 2-8A). The C(8) methylester purine derivative 2.12c also forms a dimer but does so through four hydrogen bonds (O(15)N(10) = 2.87 and N(7)N(10) = 3.03 ) (Figure 2-10A). Due to strong dipolar inte ractions, nitriles 2.7b and 2.12b approach absolute antip arallel alignment of neighboring purines and short -stacking distances of 3.32 for 2.7b and 3.38 for 2.12b (Figures 2-6b and 2-8b). Methylester derivative 2.12c shows antiparallel dipolar stacking of 3.30 but the molecule is slipped with respect to its neighbor such that the methylester portion of one mo lecule stacks with the purine core of the next (Figure 2-10B). The noncovale nt intermolecular interactions revealed from these crystal structures suggest how the molecula r recognition features of the nucleobases might be useful in controlling their supram olecular and solid state architectures.

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39 Figure 2-6. Noncovalent interm olecular interactions present in the crystal structure of 2.7b A) Hydrogen bonding interactions. B) -stacking and dipolar interactions. Figure 2-7. Crystal packing of 2.7b along its three crystall ographic axes. A) Axis a B) Axis b. C) Axis c Figure 2-8. Noncovalent interm olecular interactions present in the crystal structure of 2.12b A) Hydrogen bonding interactions. B) -stacking and dipolar interactions.

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40 Figure 2-9. Crystal packing of 2.12b along its three crystall ographic axes. A) Axis a B) Axis b. C) Axis c Figure 2-10. Noncovalent interm olecular interactions present in the crystal structure of 2.12c A) Hydrogen bonding interactions. B) -stacking and dipolar interactions. Figure 2-11. Crystal packing of 2.12c along its three crystall ographic axes. A) Axis a B) Axis b. C) Axis c

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41 Original Synthe tic Strategies During the early stages of this research guanosine ( 2.15 ) was selected as the purine with which to initiate the D-A purine synthesis (Fi gure 2-12). Protection for the N(9) sugar hydroxy groups was necessary in order to transform the C(6) carbonyl to a chlorine; this was done by treatment with acetic anhydride using a literature procedure74 (Figure 2-12). Subsequent conversion to the 6-chloro derivative was successful to give 2.1775 (Figure 2-12), however decomposition of the sugar moiety led to a low product yield of 46%. The low yield along with the lability of the N(9) sugar functionality encour aged a reexamination of the synthetic approach. HN N N N H2N O O HO OH OH Ac2O TEA DMAP acetonitrile HN N N N H2N O O A cO OAc OAc N N N N H2N O Cl A cO OAc OAc POCl3DEA 98% 46% 2.15 2.16 2.17 Figure 2-12. Preparatio n of starting material 2.17 used in an early synthetic approach to D-A purines. The first synthetic strategy entertaine d for carbon-carbon bond formation on the C(8) position was deprotonation of C(8) using a st rong bulky base, LTMP or LDA, followed by the addition of an electrophile (car bon dioxide, methylchloroformat e, or benzylchloroformate) (Figure 2-13). This stategy was inspired by previous direct C( 8) lithiation of the purine core done in the lab of Miyasaka.76 Some of the problems encount ered with Miyasakas route included low yields of the desired product due to side product formation. Our deprotonation reactions were attempted many times and included varying the substituents on the purine, the base, equivalents of base used (1 5 equiv), temperature of the reaction ( 50 C), and the electrophile. The C(2) amine groups were functiona lized with the Boc protecting group to see if

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42 the free amine was complicating the reaction. Se veral of these reactions proved unsuccessful (Figure 2-13). N N N N R1 R2 Bn N N N N R1 R2 Bn O R4 O 1. LTMP or LDA THF Cl O O R3 2. R2 = Cl R2 = N(Me)2 R2 = Cl R1 = NH2R1 = NH2R1 = N(Boc)22.5 2.6 2.18 R4 = Me, Bn, or H R3 = Me or Bn CO2or Figure 2-13. Deprotonation attempts of the C(8) position of purines. One attempt, using 2-amino-9-benzyl purine ( 2.6 ), three equivalents of LDA, and one equivalent of methylchlorofomate at C su ccessfully formed the desired product 2.6c in 5% yield. This reaction, while encouraging, was still not su fficiently high yielding to permit continued investigation of depr otonation strategies. N N N N H2N Bn N N N N H2N Bn O Me O 1.LD A ,THF Cl O O Me 2. 2.6 2.6c 5% Figure 2-14. Successful C-C bond forma tion by C(8) deprotonation using LDA. Lithium-halogen procedures have been used to modify the C(8) position of C(8) bromopurines77and were also attempted. Reacting molecules like 2.7a with 1.2 6 equivalents of n -butyllithium and 2 10 equivalents of an electrophile ga ve no desired C(8) carbon-carbon bond formation and a complicated pr oduct mixture (Figure 2-15). N N N N H2N Bn N N N N H2N Bn O Me O 1. n -buLi, THF Cl O O Me 2. N Br N 2.7a Figure 2-15. Attempted lithium-halogen exchange at the C(8) position of the purine followed by electrophile addition.

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43 After this series of unsucce ssful C(8) carbon-carbon bond formation reactions, a change of synthetic direction was necessary to effectively construct the desi red C(8) modified products. It was at this time that the Pd(0)-mediated cyana tion reactions discussed earlier were conceived. Experimental Section General. Reagents and solvents were purchased from Aldrich, Fluka, or Acros and used without further purification unless ot herwise specified. THF, ether, CH2Cl2, and DMF were degassed in 20 L drums and passed through two sequential purification columns (activated alumina; molecular sieves for DMF) under a pos itive argon atmosphere. Flash chromatography (FC) was performed on Purasil SiO2-60, 230 400 mesh from Whatman. Thin layer chromatography (TLC) was performed on SiO2-60 F254 aluminum plates from EMD Chemicals with visualization by UV light or staining (e.g. KMnO4). Melting points (m.p.) were determined on a Mel-temp electrothermal melting point apparatus and are uncorrected. 1H (300 MHz) and 13C NMR (75 MHz) spectra were recorded on Varian Mercury 300, Gemini 300, and VXR 300 spectrometers. Chemical shifts ( ) are given in parts per million (ppm) relative to residual protonated solvent (CHCl3: H 7.26 ppm, C 77.00 ppm). Abbreviations used are s (singlet), d (doublet), t (triplet), q (quartet), quin (quintet ), b (broad), and m (mu ltiplet). High resolution mass spectra (HRMS) were obtained by the Univer sity of Florida Mass Spectrometry Services using a Finnigan MAT95Q Hybrid Sector spectrometer. Compound 2.1 was purchased from Aldrich. Purines 2.246, 2.346, 2.446, 2.547, 2.649, and 2.851 were obtained as described in the literature with some modification. Synthesis of Compounds General Method A ( C(8) bromination of purines; c onversion of C(8)-H to C(8)-Br ). To a solution (6 mM) of starting material in CH2Cl2 was added bromine (33 equiv.) dropwise and the mixture was stirred at room temperature for 1 h to 24 h. The reaction was monitored by TLC to

PAGE 44

44 determine when the reaction was complete. Th e reaction mixture was poured into 10% aqueous Na2S2O3. The organic layer was separated and washed with brine, dried over MgSO4, and concentrated under reduced pr essure. The residue was pur ified by silica gel column chromatography (1% MeOH/CH2Cl2) to give the C(8) brominated product. General Method B ( coupling of bromopurines with zinc cyanide; conversion of a to b ). To a solution (0.16 M) of C(8) bromopurine in NMP was added tetrakis(triphenylphosphine) palladium(0) (20 mol %), which was purified by washing with benzene and methanol. After stirring at 90 C for 20 min a mixture of zinc cyanide (2 eq uiv.) suspended in NMP (0.90 mM) was added dropwise to the reaction (10 drops ever y 30 min for 4 h). The reaction was stirred at 90 C overnight and then cooled and treated with aqueous ammonia (2 N, 15 mL). The mixture was extracted with ethyl acetate (3 x 100 mL). Th e combined organic extracts were washed with 2 N NH4OH and brine, dried over MgSO4, and concentrated under reduced pressure. The residue was purified by silica gel co lumn chromatography (0.5% MeOH/CH2Cl2). General Method C ( methanolysis of C(8) cyanopurin es; conversion of b to c ). A solution of methanolic ammonia (7 N) and C(8) cyanopurin e was stirred at room temperature overnight. The residue, after evaporati on, was stirred in methanol wh ile 1 N aqueous HCl was added dropwise. The solution was stirred for 2 h at ro om temperature, neutralized with Dowex 1 (OH) resin, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (1% MeOH/CH2Cl2). N N N N H2N N

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45 2-Amino-6-dimethylamino-9-benzylpurine (2.7). Dimethylamine (4.0 mL, 33% in abs ethanol) was added to 2.5 (1.0 g, 3.9 mmol) in abs. ethanol (40 mL) and the solution was heated to 60 C for 20 h. The reaction mixture was evapor ated to dryness and tr iturated with deionized H2O. The white crystals (0.95 g, 92%) were then collected by vacuum f iltration and dried under high vacuum: m.p. 170 173 C. 1H NMR (300 MHz, CDCl3) 3.46 (s, 6H), 4.61 (s, 2H), 5.21 (s, 2H), 7.27 (m, 5H), 7.42 (s, 1H). 13C NMR (75 MHz, CDCl3) 38.22, 46.39, 114.98, 127.49, 127.93, 128.82, 135.87, 136.36, 152.91, 155.39, 159.40. HRMS calcd for C14H17N5 [M+H]+: 269.1509, found: 269.1516. N N N N N N 2,6-Bis(dimethylamino)-9-benzylpurine (2.10). Dimethylamine (50 mL) was added to 2.9 (2.5 g, 9.0 mmol) and the solution was heated to 100 C for 20 h and then concentrated under reduced pressure. The crude solid was purifie d by silica gel column chromatography (20% EtOAc/hexanes) to afford a white solid (1.98 g, 76 %): m.p. 98 C. 1H NMR (300 MHz, CDCl3) 3.17 (s, 6H), 3.46 (s, 6H), 5.22 (s 2H), 7.30 (m, 5H), 7.40 (s, 1H). 13C NMR (75 MHz, DMSOd6) 37.55, 38.19, 46.34, 113.55, 128.22, 128.44, 129.21, 137.16, 138.27, 153.40, 154.84, 159.38. HRMS calcd for C16H21N6 [M+H]+: 297.1828, found: 297.1820. N N N N N NH2 2-Dimethylamino-6-amino9-benzylpurine (2.12). Dimethylamine (10 mL, 33% in abs ethanol) was added to 2.11 (0.30 g, 1.2 mmol) in a sealed tube. The reaction was stirred at 100

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46 C for 20 h, and then concentrat ed under reduced pressure. The crude solid was purified by silica gel column chromatography (1% MeOH/CH2Cl2) to afford a white solid (0.31 g, 99%): m.p. 168 171 C. 1H NMR (300 MHz, DMSOd6) 3.07 (s, 6H), 5.19 (s, 2H), 6.73 (s, 2H), 7.34 (m, 5H), 7.82 (s, 1H). 13C NMR (75 MHz, DMSOd6) 37.00, 45.64, 112.39, 127.50, 127.77, 128.48, 137.35, 137.56, 151.60, 155.54, 159.48. HRMS calcd for C14H17N6 [M+H]+: 269.1509, found: 269.1515. N N N N N 2-Dimethylamino-9-benzylpurine (2.14). Dimethylamine (4.0 mL, 33% in abs ethanol) was added to 2.13 (0.42 g, 1.7 mmol) and the solution was heated to 100 C for 20 h and then concentrated under reduced pre ssure. The crude solid was purified by silica gel column chromatography (20% EtOAc/hexanes) to affo rd an off-white solid (0.37 g, 86%): m.p. 104 C. 1H NMR (300 MHz, DMSOd6) 3.15 (s, 6H), 5.30 (s, 2H), 7.34 (m, 5H), 8.22 (s, 1H), 8.68 (s, 1H). 13C NMR (75 MHz, DMSOd6) 37.12, 45.71, 126.07, 127.74, 127.86, 128.63, 136.90, 142.81, 148.51, 152.85, 159.28. HRMS calcd for C14H15N5 [M+H]+: 254.1400, found: 254.1412. N N N N H2N Br N 2-Amino-6-dimethylamino-8-br omo-9-benzylpurine (2.7a). Compound 2.7 (0.50 g, 1.8 mmol) was reacted under the conditions of general method A to yield a white solid (0.52 g, 81%): m.p. 168 169 C. 1H NMR (300 MHz, CDCl3) 3.42 (s, 6H), 4.69 (s, 2H), 5.24 (s, 2H), 7.28 (m, 5H). 13C NMR (75 MHz, CDCl3) 38.27, 46.69, 115.54, 121.10, 127.40, 127.77,

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47 128.63, 135.87, 154.13, 154.21, 159.19. HRMS calcd for C14H15BrN6 [M+H]+: 347.0620, found: 347.0618. N N N N H2N O Br 2-Amino-6-benzyloxy-8-bromo-9-benzylpurine (2.8a). Compound 2.8 (0.38 g, 0.93 mmol) was reacted under the conditions of general method A to yield a white solid (0.44 g, 77%): m.p. 164 166 C. 1H NMR (300 MHz, CDCl3) 7.50 (m, 2H), 7.30 (m, 8H), 5.53 (s, 2H), 5.26 (s, 2H), 4.90 (s, 2H). 13C NMR (75 MHz, CDCl3) 47.11, 68.25, 115.85, 125.56, 127.50, 128.03, 128.07, 128.39, 128.75, 135.37, 136.20, 155.29, 159.17, 159.85. HRMS calcd for C19H17BrN6O [M+H]+: 410.0616, found: 410.0627. N N N N N N Br 2,6-Bis(dimethylamino)-8-bro mo-9-benzylpurine (2.10a). Compound 2.10 (1.5 g, 5.1 mmol) was reacted under the conditions of general method A to yield a white solid (1.3 g, 70 %): m.p. 130 C. 1H NMR (300 MHz, DMSOd6) 3.08 (s, 6H), 3.33 (bs, 6H), 5.21 (s, 2H), 7.31 (m, 5H). 13C NMR (75 MHz, DMSOd6) 36.74, 37.55, 46.06, 113.20, 120.29, 127.30, 127.63, 128.61, 136.43, 152.99, 153.93, 158.43. HRMS calcd for C16H20BrN6 [M+H]+: 375.0933, found: 375.0943.

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48 N N N N N NH2 Br 2-Dimethylamino-6-amino-8-br omo-9-benzylpurine (2.12a). Compound 2.12 (0.59 g, 2.2 mmol) was reacted under the conditions of general method A to yield a white solid (0.52 g, 68%): m.p. 195 197 C. 1H NMR (300 MHz, CDCl3) 3.15 (s, 6H), 5.21 (bs, 2H), 5.23 (s, 2H), 7.29 (m, 5H). 13C NMR (75 MHz, CDCl3) 37.33, 46.11, 46.90, 113.31, 122.36, 127.90, 128.06, 128.61, 136.03, 153.87, 159.77. HRMS calcd for C14H16BrN6 [M+H]+: 347.0614, found: 347.0626. N N N N N Br 2-Dimethylamino-8-bromo9-benzylpurine (2.14a). Compound 2.14 (0.40 g, 1.6 mmol) was reacted under the cond itions of general method A to yield a white solid (0.42 g, 80%): m.p. 128-129 C. 1H NMR (300 MHz, CDCl3) 3.16 (s, 6H), 5.30 (s, 2H), 7.31 (m, 5H), 8.67 (s, 1H). 13C NMR (75 MHz, CDCl3) 37.07, 46.11, 126.18, 127.41, 127.83, 128.52, 128.74, 135.88, 147.68, 153.71, 159.06. HRMS calcd for C14H14BrN5 [M+H]+: 332.0506, found: 332.0517. N N N N H2N CN 2-Amino-8-cyano-9-benzylpurine (2.6b). Compound 2.6a (0.10 g, 0.33 mmol) was reacted under the conditi ons of general method B to yield a white solid (0.065 g, 80%): m.p. 139 142 C. 1H NMR (300 MHz, DMSOd6) 5.40 (s, 2H), 7.26 (m, 7H), 8.88 (s, 1H). 13C

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49 NMR (75 MHz, DMSOd6) 46.62, 112.17, 124.40, 126.58, 127.87, 128.87, 128.90, 129.67, 136.11. HRMS calcd for C13H10N6 [M+]: 250.0967, found: 250.0973. N N N N H2N CN N 2-Amino-6-dimethylamino-8-cyan o-9-benzylpurine (2.7b). Compound 2.7a (0.20 g, 0.58 mmol) was reacted under the conditions of general method B to yield a white solid (0.15 g, 91%): m.p. 175 176 C. 1H NMR (300 MHz, DMSOd6) 3.40 (bs, 6H), 5.33 (s, 2H), 6.44 (s, 2H), 7.33 (m, 5H). 13C NMR (75 MHz, DMSOd6) 47.56, 112.08, 114.91, 117.72, 126.90, 127.90, 128.78, 135.82, 152.77, 154.86, 161.50. HRMS calcd for C15H16N7 [M+H]+: 294.1467, found: 294.1479. N N N N H2N CN O 2-Amino-6-benzyloxy-8-cyano-9-benzylpurine (2.8b). Compound 2.8a (0.30 g, 0.73 mmol) was reacted under the conditions of general method B to yield a white solid (0.14 g, 54%): m.p. 246 248 C. 1H NMR (300 MHz, DMSOd6) 5.37 (s, 2H), 5.52 (s, 2H), 7.14, (s, 2H) 7.38 (m, 10H). 13C NMR (75 MHz, DMSOd6) 46.22, 67.61, 111.58, 114.62, 121.17, 127.01, 128.07, 128.25, 128.42, 128.64, 128.88, 135.48, 135.89, 153.69, 161.10, 161.79. HRMS calcd for C20H17N6O [M+H]+: 357.1464, found: 357.1432.

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50 N N N N N N CN 2,6-Bis(dimethylamino)-8-cyano-9-benzylpurine (2.10b). Compound 2.10a (0.20 g, 0.53 mmol) was reacted under the conditions of general method B to yield a white solid (0.16 g, 93 %): m.p. 174 C. 1H NMR (300 MHz, CDCl3) 3.20 (s, 6H), 3.43 (bs, 6H), 5.32 (s, 2H), 7.30 (m, 3H), 7.45 (m, 2H). 13C NMR (75 MHz, DMSOd6) 35.11, 36.78, 46.03, 109.15, 112.23, 114.39, 117.79, 127.62, 128.09, 128.85, 135.95, 152.62, 154.22, 159.94. HRMS calcd for C17H19N7 [M+H]+: 322.1780, found: 322.1783. N N N N N NH2 CN 2-Dimethylamino-6-amino-8-cyan o-9-benzylpurine (2.12b). Compound 2.12a (0.10 g, 0.29 mmol) was reacted under the conditions of general method B to yield a white solid (85 mg, 77 %): m.p. 205 209 C. 1H NMR (300 MHz, DMSOd6) 3.12 (s, 6H), 5.33 (s, 2H), 7.34 (m, 7H). 13C NMR (75 MHz, DMSOd6) 36.90, 46.02, 112.25, 113.92, 118.50, 127.67, 128.10, 135.86, 136.00, 151.49, 156.28, 161.02. HRMS calcd for C15H16N7 [M+H]+: 294.1467, found: 294.1492. N N N N N CN 2-Dimethylamino-8-cyano-9benzyl purine (2.14b). Compound 2.14a (0.20 g, 0.60 mmol) was reacted under the conditions of general method B to yield a white solid (0.065 g, 39 %): m.p. 111 C. 1H NMR (300 MHz, CDCl3) 3.30 (s, 6H), 5.41 (s, 2H), 7.35 (m, 5H)

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51 8.83 (s, 1H). 13C NMR (75 MHz, CDCl3) 29.93, 38.01, 47.19, 111.51, 126.04, 128.76, 128.98, 129. 27, 134.78, 134.99, 151.78. HRMS calcd for C15H14N6 [M+H]+: 279.1353, found: 279.1355. N N N N H2N O O 2-Amino-8-methylester-9benzylpurine (2.6c) Compound 2.6b (0.10 g, 0.40 mmol) was reacted under the conditi ons of general method C to yield a white solid (0.068 g, 60%): m.p. 226 228 C. 1H NMR (300 MHz, CDCl3) 3.97 (s, 3H), 5.27 (s, 2H ), 5.74 (s, 2H), 7.29 (m, 5H), 8.84 (s, 1H). 13C NMR (75 MHz, CDCl3) 47.05, 53.22, 127.18, 127.92, 128.13, 128.87, 136.71, 151.75, 153.26, 159.86, 161.43. HRMS calcd for C14H13N5O2 [M+H]+: 283.1069, found: 283.1086. N N N N H2N N O O 2-Amino-6-dimethylamino-8-methyl ester-9-benzylpurine (2.7c). Compound 2.7b (0.062 g, 0.21 mmol) was reacted under the general method C to yield a white solid (0.054 g, 86%): m.p. 175 180 C. 1H NMR (300 MHz, CDCl3) 3.51 (bs, 6H) 3.91 (s, 3H) 4.77 (s, 2H) 5.70 (s, 2H) 7.26 (m, 5H). 13C NMR (75 MHz, DMSOd6) 37.00, 46.12, 52.02, 114.23, 126.49, 127.12, 128.41, 132.89, 137.89, 137.60, 154.38, 155.46, 159.28, 161.02. HRMS calcd for C16H19N6O2 [M+H]+: 327.1569, found: 327.1583.

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52 N N N N H2N O O O 2-Amino-6-benzyloxy-8-methyleste r-9-benzylpurine (2.8c). Compound 2.8b (0.037 g, 0.10 mmol) was reacted under the conditions of general method C to yield a white solid (0.038 g, 95%): m.p. 180 185 C. 1H NMR (300 MHz, CDCl3) 3.90 (s, 3H), 5.03 (s, 2H), 5.53 (s, 2H), 5.70 (s, 2H), 7.31 (m, 10H). 13C NMR (75 MHz, CDCl3) 47.20, 52.64, 68.46, 115.51, 127.52, 127.69, 128.18, 128.37, 128.53, 128.65, 135.90, 136.74, 137.13, 155.26, 159.70, 160.53, 162.56. HRMS calcd for C21H19N5O3 [M+H]+: 390.1566, found: 390.1544. N N N N N N O O 2,6-Bis(dimethylamino)-8 -methylester-9-benzyl purine (2.10c). Compound 2.10b (0.10 g, mmol) was reacted under the conditions of general method C to yield a white solid ( 0.070 g, 64 %): m.p. 147 C. 1H NMR (300 MHz, CDCl3) 3.20 (s, 6H), 3.50 (bs, 6H), 3.90 (s, 3H), 5.70 (s, 2H), 7.26 (m, 5H). 13C NMR (75 MHz, DMSOd6) 36.77, 46.27, 52.05, 113.63, 127.24, 127.29, 128.40, 132.91, 137.64, 154.21, 154.77, 159.35, 159.57. HRMS calcd for C18H23N6O2 [M+H]+: 355.1882, found: 355.1881.

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53 N N N N N NH2 O O 2-Dimethylamino-6-amino-8-methyl ester-9-benzylpurine (2.12c). Compound 2.12b (0.10 g, 0.34 mmol) was reacted under th e conditions of general method C to yield a white solid (0.067 g, 60%): m.p. 250 252 C. 1H NMR (300 MHz, DMSOd6) 3.10 (s, 6H), 3.80 (s, 3H), 5.55 (s, 2H), 7.26 (m, 7H). 13C NMR (75 MHz, DMSOd6) 36.91, 46.25, 51.94, 113.12, 127.30, 128.39, 133.75, 137.67, 153.04, 156.83, 159.39, 160.58. HRMS calcd For C16H18N6O2 [M+]: 326.1491, found: 326.1488. N N N N N O O 2-Dimethylamino-8-methylest er-9-benzyl purine (2.14c). Compound 2.14b (0.043 g, 0.16 mmol) was reacted under the conditions of general method C to yield a white solid (0.034 g, 71 %): m.p. 167 C. 1H NMR (300 MHz, DMSOd6) 3.21 (s, 6H), 3.87 (s, 3H), 5.64 (s, 2H), 7.28 (m, 5H), 8.92 (s, 1H). 13C NMR (75 MHz, DMSOd6) 37.86, 47.05, 53.33, 125.56, 128.07, 128.23, 129.24, 137.79, 139.27, 152.69, 154.34, 160.07, 160.83. HRMS calcd for C16H17N5O2 [M+H]+: 312.1455, found: 312.1468. N N N N Cl O O N O O

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54 2-Di-tertbutoxycarbonlyamine-6-chloro-9-benzylpurine (2.18) Compound 2.5 (0.10 g, 0.39 mmol), (Boc)2O (0.25 g, 1.2 mmol), and DMAP (0.004 g, 0.04 mmol) were dissolved in dry THF (2 mL) and stirred at rt overnight. The solvent was rem oved under reduced pressure and the product was purified by flash ch romatography (10% EtOAC/hexa nes) to yield a white solid (0.074 g, 42%): 1H NMR (300 MHz, DMSOd6) 1.31 (s, 18H) 5.53 (s, 2H) 7.32 (m, 5H) 8.94 (s, 1H).13C NMR (75 MHz, DMSOd6) 27.3, 47.1, 83.3, 127.4, 128.1, 128.8, 129.4, 135.9, 149.0, 149.2, 150.0, 150.9, 152.5. HRMS calculated for C22H26N5O4Na [M+Na]+: 482.1566, found: 482.1573. X-ray Crystallography General. Data were collected at 173 K on a Siemens SMART PLATFORM equipped with a CCD area detector and a grap hite monochromator utilizing MoK radiation ( = 0.71073 ). Cell parameters were refined using up to 8192 reflections. A full sphere of data (1850 frames) was collected using the -scan method (0.3 frame width). The first 50 frames were remeasured at the end of data collection to mon itor instrument and crys tal stability (maximum correction on I was < 1 %). Absorption corrections by integration were applied based on measured indexed crystal faces. The structures were solved by the Direct Methods in SHELXTL6 and refined using fullmatrix least squares. The non-H atoms were tr eated anisotropically, wh ereas the hydrogen atoms were calculated in ideal positions and were riding on their respective carbon atoms. Compound 2.7b The methyl H atoms on C(14) and C(15) are disordered and each set was refined in two parts and in a riding model with their site occupation f actors fixed at 50%. A total of 208 parameters were refined in the fina l cycle of refinement using 4656 reflections with I > 2 ( I ) to yield R1 and w R2 of 3.83% and 10.27%, respectively. Refinement was done using F2.

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55 Compound 2.12b. A total of 207 parameters were refine d in the final cycle of refinement using 3304 reflections with I > 2 ( I ) to yield R1 and w R2 of 4.75% and 12.0%, respectively. Refinement was done using F2. Compound 2.12c The two protons on N(10) were obtained from a Difference Fourier map and refined without any constraints. A tota l of 227 parameters were refined in the final cycle of refinement using 2572 reflections with I > 2 ( I ) to yield R1 and w R2 of 3.92% and 9.82%, respectively. Refinement was done using F2.

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56 CHAPTER 3 PHOTOPHYSICAL AND ELECTRONIC PROPER TIES OF DONOR-ACCEPTOR PURINES Before molecules are considered for optoelect ronic devices or used as biological probes it is critical to determine their pho tophysical and electronic properties, stability, and behavior in the solid state and in solution.78 Knowing the solvent-induced ab sorption and emission changes for a fluorophore is relevant to its potential use fo r reporting structural changes and intermolecular interactions within biol ogical microenvironments.79,80 For materials applications, the ability to tailor optical properties in the context of controlled molecular (a nd supramolecular) architectures is important. This chapter describes the UV/vi s, fluorescence, and solvatochromism properties of the D-A purines (Figure 3-1). Cyclic volta mmetry and computational data are also provided that correlates well with photophysic al data. Finally, the first usage of a purine in a light emitting device is reported. N N N N Bn N N N N N N Bn N NH2 N N N N Bn H2N N N N N N Bn N N N N N Bn H2N N N N N Bn H2N O X X X X X X b X = CN c X = COOMe 2.6b,c2.7b,c 2.8b,c 2.10b,c2.12b,c2.14b,c Figure 3-1. Donor-acceptor purines targeted for photophysical studies. Interest in the photophysical pr operties of substituted purin es and 2-aminopurine (2-AP) derivatives spans over six decades. Studies of 2AP derivatives have been traditionally resigned to aqueous solutions81 (vide supra) due to their poor solubil ity in organic solvents and their more

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57 typical application in aqueous (b iological) environments. Limite d attention has been given to purines as fluorophores that might be used in or processed from organic solution. Photophysical measurements in organic solvents have been performed (although not comprehensively so) for 2AP19 and 2-AP derivatives such as 2-dimethylaminopurine82 and the ethenopurines.83 Tors recently prepared C(8)-furano substituted puri nes represent new compounds for such studies (Figure 1-8B).33 The N(9) benzyl group of the purines in Figure 3-1 makes their photophysical measurement in organic solvents possible wher efrom they can potentially be processed for materials applications. The photoph ysical studies discussed in this chapter show that addition of acceptor groups to the purine C(8) position appears as a general way to produce the first nucleobase derivatives that have near unity quantum yields acro ss a range of organic solvents.38,83-85 Upon removal of the benzyl (or sim ilar) protecting group, the compounds should be quite suitable for applications in water. To this end, preliminar y photophysical data for compounds 2.6b and 2.6c (with the benzyl group in place) in aqueous solution shows that they indeed maintain (and even enhance) th eir exceptional optical properties. UV/vis data for C(8)-H, C(8)-CN, and C( 8)-COOMe Purines in Methylene Chloride UV/vis measurements were performed for each C(8)-H, -CN, and -COOMe purine (Tables 3-1 3-3) at 5, 10, 20, 40, and 60 M in methylene chloride. A representative series of absorption spectra are shown for nitrile 2.7b in figure 3-2. Examination of the UV/vis spectra reveals two significant low-energy bands w hose maximum absorption wavelengths are concentration independent. By analogy to 2-AP81 and further confirmed by computation (vide infra), the lowest energy (emitting) band is assigned to the 1( La) transition, and the higher energy non-emitting band to the 1( Lb) transition.86

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58 Mason,81 Drobnik87,88, and Smagowicz82 concluded early on from analysis of the absorption bands of substituted and unsubsti tuted purines that the long-wavelength 1( La) absorption band is due principally to a longi tudinally (along the long axis of the purine) polarized transition. Substituents on the C(2) posi tion of the purine core, positioned at an angle 30 from this axis, therefore significantly affect its energy (and max), as do substituents on C(8). Substituents in the C(6) position, situated tr ansverse to the long axis, contribute less.81 For example, adenine (C(6)-amino) and 6-dimet hylaminopurine show a modest difference in max values for their 1( La) absorption (260 versus 275 nm in water, respectively), while the difference between 2-aminopurine and 2-dimethyl aminopurine is much larger (305 versus 332 nm in water, respectively).81 C(6) substituents do, however, more significantly affect the higher energy 1( Lb) absorption band. These general trends are apparent in the long-wavelength absorption data shown for the C(8)-H purines used in this work (Table 3-1). Purines 2.6 and 2.14 for example, bearing donors only in the C(2) position, show the most red-shifted 1( La) absorption bands. As in Masons work,81 the stronger C(2)-dimethylamino donor of 2.14 extends max to ~ 330 nm. Data from figure 3-2 could be plotted to confirm that the optical density varies linearly with concentration (Figure 3-3). This experiment repeated for each of the purines discussed in this work, provides some assurance that the mo lecules are not significan tly aggregating under the solvent and concentration conditions chose n. Self-association could occur by hydrogen bonding89 or dipolar -stacking67,71 for these nucleobases. Extinction coefficients, presented as log could then be calculated by multiplying the slope of the line from the absorption vs. concentration plot by 106. Only the absorption data for the lowest energy emitting 1( La) transition are shown in tables 3-1 3-12.

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59 Figure 3-2. Absorpti on spectra for compound 2.7b upon dilution in methylene chloride. Figure 3-3. Absorption intensity at 3 34 nm vs. concentration for compound 2.7b

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60 Figure 3-4. Absorption spectra for C(8)-H ( 2.7 ), C(8)-CN ( 2.7b ), and C(8)-COOMe ( 2.7c ) containing the same C(2) and C(6) substituents. Figure 3-4 shows graphically the respons e of the absorption maxima of the 1( Lb) and 1( La) bands upon installing a -CN or -COOMe acceptor group in the C(8) position of 2.7 ; the 1( Lb) is red-shifted by about 20 nm while the 1( La) is shifted by a larger 44 nm. These changes are consistent with an increase in co njugation length. Inte restingly, the absorption maxima are comparable between the C(8)-CN and -COOMe derivatives 2.7b and 2.7c a trend that exists for nearly all of the purines studied (where max(CN/COOMe) ~ 6 nm). The bathochromic shifts observed upon C(8)-acceptor s ubstitution are universal for the D-A purines prepared, and the largest ch ange is ~51 nm (for the 2.10 series). Even the parent 2.6 series shows a red-shift of ~26 nm upon CN/COOMe in troduction, a potentially useful improvement for selective excitation over 2-AP for biological applications. Finally, th e extinction coefficient maxima are uniformly high for the C(8)-H, -C N, and -COOMe purines and comparable to literature values (Tables 3-1 3-12).81,90

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61 Table 3-1. Absorption and Emission Pr operties for C(8)-H Purines in CH2Cl2.a Purine max, abs/nm log /M-1 cm-1 max, b em/nm max, em-abs/nm F c 2.6 304 3.9 357 53 0.20 2.7 286 4.1 351 65 0.013 2.8 281 4.0 360 79 0.033 2.10 297 4.1 392 95 0.033 2.12 299 3.9 360 61 0.12 2.14 330 3.8 393 d 63 0.76 aAll measurements performed at room temperature. bAll experiments were performed using optical densities 0.1 at the excitation wavelength ( ex = 290 nm). cFluorescence quantum yields are relative to the quantum yield of quinine sulfate in 0.5 M H2SO4 (F = 0.546). dMeasurement performed at the excitation wavelength ( ex = 320 nm). Table 3-2. Absorption and Emission Pr operties for C(8)-CN Purines in CH2Cl2.a Purine max, abs/nm log /M-1 cm-1 max, em/nm max, b em-abs/nm F c F/ns 2.6b 326 3.2 371 45 0.20 0.5.2 2.7b 324 4.2 375 51 0.30 1.6.3 2.8b 311 4.3 355 44 0.81 3.1.2 2.10b 348 4.4 388 40 0.20 7.6.1 2.12b 336 4.1 387 51 0.97 3.2.3 2.14b 361 4.2 429 68 0.90 4.2.2 aAll measurements performed at room temperature. bAll experiments were performed using optical densities 0.1 at the excitation wavelength ( ex = 320 nm). cFluorescence quantum yields are relative to the quantum yield of quinine sulfate in 0.5 M H2SO4 (F = 0.546). Table 3-3. Absorption and Emission Pr operties for C(8)-COOMe Purines in CH2Cl2.a Purine max, abs/nm log /M-1 cm-1 max, b em/nm max, em-abs/nm F c F/ns 2.6c 328 4.1 379 51 0.42 1.8.2 2.7c 330 4.2 393 63 1 3.1.3 2.8b 315 4.3 371 56 0.98 2.5.1 2.10c 348 4.3 409 61 0.90 2.7.3 2.12c 338 4.1 409 71 1 3.3.1 2.14c 362 4.1 433 71 0.81 3.4.2 aAll measurements performed at room temperature. bAll experiments were performed using optical densities 0.1 at the excitation wavelength ( ex = 320 nm). cFluorescence quantum yields are relative to the quantum yield of quinine sulfate in 0.5 M H2SO4 (F = 0.546). Steady State Fluorescence of C(8)-H, C(8 )-CN, and C(8)-COOMe Purines in Methylene Chloride Two different excitation wavele ngths were chosen based on the 1( La) absorption transition to examine the fluorescence of the purin e derivatives. The C(8)-H derivatives, with

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62 the exception of 2.14 were excited at 290 nm, while the C(8)-CN, and -COOMe derivatives, and compound 2.14, were excited at 320 nm. The shape of the excitation spectrum for compound 2.7c is essentially the same as its 1( La) absorption band; therefore, excitation at any point of the absorption band leads to the efficient conv ersion into the low-lying emitting state (the fluorescence emission is independent of the exci tation wavelength used as stated by Kashas rule).2 Emission spectra were recorded for C(8)-H -CN, and -COOMe purines in methylene chloride (Tables 3-1 3-3 and Figures 3-4 and 3-5). The emission data revealed significant spectral and quantum yield (F) differences as a result of C(8) substitution. The emission bands are generally red-shifted by ~ 20 nm upon additi on of the C(8)-CN acceptor group, expected due to the increased conjugation length of the molecu le. They are red-shifted an additional 5 nm upon the nitriles transformation to the met hyl ester. This is illustrated by the 2.6 series of compounds ( 2.6 2.6b and 2.6c ) as the emission maximum increases from 357 nm to 371 nm with C(8) cyano addition and from 371 nm to 379 nm when transformed to the methylester. Exceptions to this trend are seen with the 2.8 and 2.10 series of purines as the emission maxima decrease 4 and 5 nm, respectively, upon C(8)-CN a ddition. This result is most likely due to a small amount of water or methanol contamina tion present in the C(8)-H compounds. General solvatochromism studies from the literature reve al that small amounts (< 1%) of polar protic solvents can cause significant increases in em ission maxima due to sp ecific solvent effects.2 This is also consistent with solvent studies presented in this work. Quantum Yield Measurements in Methylene Chloride Quantum yields (F), the efficiency of absorbed photons that are emitted, were calculated (equation 3-1) from the absorbance ( A ) at the excitation wavelengt h, the area under the emission

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63 curve ( F ), and the solvent refractive index () of solutions of purine compounds (x) and standards (s) prepared using dried and degassed solvents. F(x) = ( As/ Ax)( Fx/ Fs)(x/ s)2F(s) (3-1) During the early stages of quantum yield de termination, many inconsistent results were obtained. The purity and quality of both the so lvent and compound were found to be extremely important. In addition to being pure, the solvents must also be dried a nd degassed to prevent any deviations in absorption maxima from water an d to prevent quenching of fluorescence resulting from molecular oxygen. Measurements from a standard with a known quantum yield are necessary for determination of new quantum yields The standard must be excited at the same wavelength as the unknown compound, so it must absorb in the same range as the unknown. After the evaluation of several standards, in cluding anthracene, 2,6-di phenylanthracene, and quinine sulfate, the latter 0.5 M H2SO4 (F = 0.546)3 appeared to give the most consistent results. In order to verify the literature quantum yield value of quinine su lfate, a second standard was measured; typically anthracene in ethanol (F = 0.27)3, and the quantum yield of each standard was calculated using the literature value of the other standard. This was done every time quantum yields for the purine compounds we re measured. The quantum yield of the standards and purines had to be m easured on the same day to circum vent day-to-day variations in the fluorometer laser. The quantum yields of the C(8)-H purines were found to vary with ch anges in substitution. The quantum yield of the parent 2-aminopurine derivative 2.6 in methylene chloride (F = 0.20) is somewhat higher than literature values for 2-aminopurine derivatives in nonprotic solvents. For 2-amino-9-ethylpurine, a quantum yi eld of 0.085 in chloroform is reported,19 and quantum yields of 0.10, 0.045, 0.12, and 0.08 are found82 for the same compound in ethyl ether, ethyl

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64 acetate, DMF, and acetonitrile, respectivel y. The 2-dimethylaminopurine derivative 2.14 was found to have a comparable quantum yield (F = 0.76) to literature values measured for 2dimethylaminopurine in sim ilarly nonprotic ether (F = 0.71), ethyl acetate (F = 0.64), and DMF (F = 0.72).82 The addition of C(8)-CN in general led to dr amatic quantum yield in creases. Compounds 2.7b (F = 0.013 0.30), 2.8b (F = 0.033 0.81), and 2.10b (F = 0.033 0.20) showed quantum yields that increased an order of ma gnitude upon addition of the C(8) cyano group and for compounds 2.12b and 2.14b quantum yields increased to 0.90. Once the C(8) cyano groups were transformed to methylesters the qua ntum yield increased again with nearly all compounds approaching unity (Table 3-3). Tor a nd Greco have recently found that they could transform the virtually non-emi ssive nucleosides guanosine and ad enosine into highly emissive variants by adding a furan to the C( 8) position (C(8)-furano guanosine (F = 0.69); C(8)-furano adenosine (F = 0.57)).33 Donor substituents on the C(2) and C(6) positions of the pur ine contribute considerably, but not equivalently, to the obser ved emission properties as seen from the increase in quantum yield of 2-dimethylaminopurine vs. 2-aminopurine (v ide supra). Comparison of isomeric species 2.7b and 2.12b (Figure 3-5) also nicely shows that intr oduction of the strongest donor to C(2), in this case N(CH3)2, has the most significant effect on the emission maximum. Compound 2.7b has a quantum yield of 0.30, however upon switching the C(2) and C(6) substituents the quantum yield becomes near unity for compound 2.12b (F = 0.97). This finding is consistent with the transition dipole moment being positioned along th e purines longitudinal axis, such that C(2) substituents (oriented only 30 from this axis) more significantly affect the energy of the

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65 emitting state. Transversely-oriented C(2) subst ituents are found to have a smaller effect on the emission maxima (Figure 3-4). The C(2) and C(6) donor substituents seem to affect the quantum yields of the C(8)-CN purines differently than the C(8)-COOMe purin es (Figures 3-5 and 3-6). This phenomenon could be a result of the cyano gr oup being aligned with the long ax is of the purine, allowing C(2) donor groups to influence most strongly the emission properties. The C(8)-COOMe acceptor group, however, features an off-axis geometry whic h allows it to borrow from both the C(2) and C(6) donors. Figure 3-5. Emission spectra of C(8 )-CN purines in methylene chloride. Stokes Shifts of Purine Comp ounds in Methylene Chloride The Stokes shift is the energy loss that o ccurs between excitation and emission of a fluorophore.2 Stokes shift values ( are calculated by taking the difference of the emission maximum and the absorption maximum. These en ergy losses are observe d for all fluorescent molecules in solution.2 Large Stokes shifts (35 nm) generally indicate a significant

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66 reordering of the surrounding solvent molecules upon excitation and accompany the existence of a charge separated excited state.2 The Stokes shifts are typically smallest for the C(8)-CN purines, from 40 nm, and largest for the C(8)-H purines, 53 nm. The C(8)-COOMe purines fall in between with a Stokes shif t range of 51 nm. The ground state and excited state dipoles are aligned more for the C(8)-CN purines leading to less solvent reorganization upon excitation and consequently a smaller Stokes shift. These numbers however, show that there is no significant increase upon addition of ac ceptor groups to C(8) in methylene chloride (Tables 3-1-3). Figure 3-6. Emission spectra of C(8)-C OOMe compounds in methylene chloride. Fluorescence Lifetime Data for C(8)-CN and -COOMe in CH2Cl2 Fluorescence lifetime data (time-resolved fluorescence) was performed using the time domain method (pulse fluorometry). In this meth od the sample is excited with a pulse of light that is preferably much s horter than the decay time () of the sample. The excitation pulse is first sent to the sample; the time dependent intensit y is then measured. Next, the decay time is

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67 calculated from the slope of a plot of log I ( t ) versus t or from the time at which the intensity ( I ) decreases to 1/ e of the intensity at t = 0.2 Time resolved emission data was collected for the C(8)-CN and -COOMe D-A purines in methylene chloride. Excitation was performe d for all compounds at 337 nm (all measured compounds absorbed at the wavelength) using a n itrogen laser with a pulse width of ~800 ps. This is the highest energy wavelength that can be used for this laser without passing the beam through a frequency doubler which decreases the intensity of the beam and makes the data collection more difficult and time consuming. The lifetime measuremen ts reveal that the F values correspond to the quantum yields, and the best emitters have F values of ~ 3 ns. All decays could be fit, with 2 values close to 1, to a single exp onential, and are consistent with fluorescence emission from the low-lying S1 state. Literature values show 2-dimethyladenosine with F values of 0.71 and 1.27 ns in acet onitrile and dioxane, respectively.91 Also found in the literature are lifetime values in water for 2-aminopurine (F = 11.8 ns) and 2-amino-9methylpurine (F = 11.1 ns).90 These values are difficult to compare with the D-A purines presented in this work, because of solvent and substitution, but they do fall into a similar range. Solvatochromism Studies for C( 8)-H, -CN, and -COOMe Purines Solvatochromism describes the influence of a medium on UV/vis and fluorescence spectra. The medium can be a solvent, solid, micelle, organized molecular film, even a vacuum.92 Here we discuss the effects of solv ent polarity on the ab sorption and emission properties of the C(8)-H, -CN, and -COOMe purines The solvents used for this study include 1,4-dioxane, methylene chloride, ace tonitrile, and methanol whose re lative polarities increase in the order in which they are listed.93 The same general emission trend observed in methylene chloride, where the C(8)-CN and C(8)-COOMe DA purines show higher quantum yields than

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68 the C(8)-H purines, are observed with 1-4 dioxane, acetonitrile, and methanol. Compounds 2.6b and 2.6c were also measured in water. Solvent Influence on Absorption and Emission Spectra Absorption and emission spectra we re obtained for C(8)-H purines 2.1.6 and for the C(8)-CN ( 2.1b.6b ) and -COOMe ( 2.1c.6c ) purines in 1,4-dioxane, methylene chloride, acetonitrile, and methanol (Tables 3-1 3-12). The absorption maximum (max) for each purine appears to have similar values regardless of th e polarity of the solvent used; for example, the 1( La) absorption maximum for 2.7b changes by only 8 nm across the polarity range of solvents (Figure 3-7). Figure 3-7. Absorption data for 40 M solutions of compound 2.7b in different solvents. The insensitivity of the absorption maxima to solvent indicates that the ground state and the excited state are solvated equally in all the solv ents. Again, absorption vs. concentration plots of each compound in all of the solvents verify th at their optical densities vary linearly with concentration. Linearity is found even in dioxane a solvent known to prom ote the association of

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69 polar -systems by dipolar -stacking as seen for the merocyanine dyes developed by Wrthner and coworkers.70 Figure 3-8. Emission spectra for 2.10b in solvents of varying polarities. Figure 3-9. Emission spectra for 2.10c in solvents of varying polarites.

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70 This result is consistent with the lower ground state dipole moment s calculated for the purines (~ 2 D) versus the merocyanine derivatives (12 D).71 The log() determined for each of the compounds also remains high despite the solvent used. The expected bathochromic shifts of the em ission peaks occur with increasing solvent polarity due to greater interaction of the excited state dipole with the solvent; this results in a reduction in energy of the exci ted state (Figures 3-8-11).94 Accordingly, there is an increase of the Stokes shift for each purine as solvent pola rity is increased indicat ing a greater degree of solvent reorganization.2 Figure 3-10. Emission spectra of 2.6b in solvents of varying polarities. Large bathochromic shifts are generally good fo r (biological) applications that involve microenvironment sensing/reporting. These change s make possible the fluorometric detection of small structural changes in biomolecules. The DA purines presented here are more sensitive to solvent effects than the unsubstituted C(8)-H purines reported in this work and in the literature. The parent 2.6 is found to share a solvent sensitivity similar to that of 2-AP reported in the

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71 literature. Swagowicz reported Stokes shifts fo r 2-AP of 51 nm in ethyl ether and 72 nm in water;82 a 21 nm range between the lowest and highe st polarity solvents studied. Similarly, a 20 nm range was found for compound 2.6 in going from 1,4-dioxane to water. Stokes shift ranges observed for compounds 2.6b and 2.6c are 25 nm and 40 nm, respectively, showing a slight increase in the C(8)-CN derivative and a signi ficant increase for the C(8)-COOMe derivative. Figure 3-11. Emission spectra for 2.6c in solvent of varying polarities. The quantum yield values for the major ity of the purine co mpounds decrease with increasing solvent polarity; for example, the quantum yield of 2.10b dramatically decreases from 0.96 in 1,4-dioxane to 0.032 in methanol (Figure 3-8). Luminescence quenching in polar solvents is commonly observed for chromophores. Although the nature of this quenching is not generally understood by any one e xplanation, it is linked to the energy gap law that describes the rates of non-radiative deactivations.92 The energy of the more polar excited state of the molecule is lowered through more efficient solvation as a resu lt of the more polar solv ent. This causes the radiationless transitions to become faster and/ or the radiative transiti ons to become slower making the effective transition energy diminish with increasing solvent polarity. Protic solvents

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72 also have the ability to quench luminescence by protonating some of the charge separated excited state molecules. Evidence of this has been seen by means of isotope exch ange experiments with naphthylamine.92 The C(8) methyl ester family of co mpounds shows only moderate decreases in luminescence, by comparison to C(8)-H and C( 8)-CN derivatives, as solvent polarity is increased. The quantum yield values for 2.10c are unity in 1,4-dioxane and 0.66 in methanol (Figure 3-9). Table 3-4. Absorption and Emission Propert ies for C(8)-H Purines in 1,4-Dioxane.a Purine max, abs/nm log /M-1 cm-1 max, b em/nm max, em-abs/nm F c 2.6 308 3.9 358 50 0.053 2.7 287 4.2 369 82 0.0077 2.8 283 4.1 372 89 0.012 2.10 293 4.1 367 74 0.41 2.12 293 4.0 355 62 0.085 2.14 327 3.9 391d 64 0.63 aAll measurements performed at room temperature. bAll experiments were performed using optical densities 0.1 at the excitation wavelength ( ex = 290 nm). cFluorescence quantum yields are relative to the quantum yield of quinine sulfate in 0.5 M H2SO4 (F = 0.546). dMeasurement performed at the excitation wavelength ( ex = 320 nm). A few of the purine compounds ( 2.6 2.6a b and 2.12 ) exhibit an overall enhanced luminescence as solvent polarity is increased, and for the parent series 2.6 this is dramatic (Figures 3-10 and 3-11); for example, th e quantum yield of the C(8)-CN derivative 2.6b increases from 0.34 in 1,4-dioxane to 0.88 in water. Table 3-5. Absorption and Emission Propert ies for C(8)-H Purines in Acetonitrile.a Purine max, abs/nm log /M-1 cm-1 max, b em/nm max, em-abs/nm F c 2.6 305 4.0 361 56 0.18 2.7 285 4.2 360 75 0.026 2.8 281 4.0 361 80 0.030 2.10 295 4.1 364 69 0.06 2.12 294 4.0 363 69 0.17 2.14 328 3.8 404d 76 0.58 aAll measurements performed at room temperature. bAll experiments were performed using optical densities 0.1 at the excitation wavelength ( ex = 290 nm). cFluorescence quantum yields are relative to the quantum yield of quinine sulfate in 0.5 M H2SO4 (F = 0.546). dMeasurement performed at the excitation wavelength ( ex = 320 nm).

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73 Table 3-6. Absorption and Emission of C(8)-H Purines in Methanol.a Purine max, abs/nm log /M-1 cm-1 max, b em/nm max, em-abs/nm F c 2.6 2.6e 310 303 3.8 ----368 373 58 70 0.47 0.91 2.7 286 4.2 368 82 0.0006 2.8 284 4.0 370 86 0.015 2.10 295 4.2 375 80 0.018 2.12 295 4.0 368 73 0.25 2.14 330 3.8 405d 75 0.46 aAll measurements performed at room temperature. bAll experiments were performed using optical densities 0.1 at the excitation wavelength ( ex = 290 nm). cFluorescence quantum yields are relative to the quantum yield of quinine sulfate in 0.5 M H2SO4 (F = 0.546). dMeasurement performed at the excitation wavelength ( ex = 320 nm). eMeasurements taken in water. Table 3-7. Absorption and Emission of C(8)-CN Purines in 1,4-Dioxane.a Purine max, abs/nm log /M-1 cm-1 max, b em/nm max, em-abs/nm F c 2.6b 331 4.2 381 50 0.34 2.7b 334 4.4 373 39 0.78 2.8b 313 4.3 362 49 0.73 2.10b 345 4.3 383 38 0.96 2.12b 339 4.2 382 43 0.92 2.14b 355 4.4 424 69 0.91 aAll measurements performed at room temperature. bAll experiments were performed using optical densities 0.1 at the excitation wavelength ( ex = 320 nm). cFluorescence quantum yields are relative to the quantum yield of quinine sulfate in 0.5 M H2SO4 (F = 0.546). Table 3-8. Absorption and Emission of C(8)-CN Purines in Acetonitrile.a Purine max, abs/nm log /M-1 cm-1 max, b em/nm max, em-abs/nm F c 2.6b 330 3.2 381 51 0.31 2.7b 326 4.3 378 52 0.075 2.8b 313 4.3 366 53 0.65 2.10b 344 4.3 394 50 0.26 2.12b 338 4.3 396 58 0.55 2.14b 357 3.9 440 83 0.53 aAll measurements performed at room temperature. bAll experiments were performed using optical densities 0.1 at the excitation wavelength ( ex = 320 nm). cFluorescence quantum yields are relative to the quantum yield of quinine sulfate in 0.5 M H2SO4 (F = 0.546). There are some literature examples of amplified quantum yields and this phenomenon is believed to result from an n state (nonluminescent) crossing to a state (luminescent) in a

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74 more polar solvent medium.92 This increase in quantum yield with increasing solvent polarity is seen with 2-AP in the literature.82 Table 3-9. Absorption and Emission of C(8)-CN Purines in Methanol.a Purine max, abs/nm log /M-1 cm-1 max, b em/nm max, em-abs/nm F c 2.6b 2.6bd 333 331 3.2 2.8 398 406 65 75 0.64 0.88 2.7b 327 4.2 389 62 0.014 2.8b 318 4.1 382 64 0.14 2.10b 342 4.4 403 61 0.032 2.12b 338 4.2 410 72 0.80 2.14b 358 4.0 456 98 0.14 aAll measurements performed at room temperature. bAll experiments were performed using optical densities 0.1 at the excitation wavelength ( ex = 320 nm). cFluorescence quantum yields are relative to the quantum yield of quinine sulfate in 0.5 M H2SO4 (F = 0.546).d Measurements taken in water. Table 3-10. Absoption and Emission of C(8)-COOMe in 1,4-Dioxane.a Purine max, abs/nm log /M-1 cm-1 max, b em/nm max, em-abs/nm F c 2.6c 331 4.2 386 55 0.64 2.7c 334 4.3 391 57 1 2.8c 313 4.3 376 63 0.97 2.10c 344 4.3 402 58 1 2.12c 340 4.4 399 59 1 2.14c 358 4.2 426 68 0.83 aAll measurements performed at room temperature. bAll experiments were performed using optical densities 0.1 at the excitation wavelength ( ex = 320 nm). cFluorescence quantum yields are relative to the quantum yield of quinine sulfate in 0.5 M H2SO4 (F = 0.546). Table 3-11. Absorption and Emission of C(8)-COOMe Purines in Acetonitrile.a Purine max, abs/nm log /M-1 cm-1 max, b em/nm max, em-abs/nm F c 2.6c 330 4.3 388 58 0.55 2.7c 332 4.4 399 67 0.83 2.8c 316 4.2 382 66 0.87 2.10c 347 4.3 415 68 0.86 2.12c 342 4.1 414 72 0.75 2.14c 359 4.1 444 85 0.60 aAll measurements performed at room temperature. bAll experiments were performed using optical densities 0.1 at the excitation wavelength ( ex = 320 nm). cFluorescence quantum yields are relative to the quantum yield of quinine sulfate in 0.5 M H2SO4 (F = 0.546).

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75 Table 3-12. Absorption and Emission of C(8)-COOMe Purines in Methanol.a Purine max, abs/nm log /M-1 cm-1 max, b em/nm max, em-abs/nm F c 2.6c 2.6c d 335 325 4.1 4.1 412 420 77 95 0.87 0.96 2.7c 331 4.2 420 89 0.39 2.8c 321 4.3 405 84 0.92 2.10c 347 4.2 432 85 0.66 2.12c 345 4.1 436 91 0.76 2.14c 362 4.1 466 104 0.17 aAll measurements performed at room temperature. bAll experiments were performed using optical densities 0.1 at the excitation wavelength ( ex = 320 nm). cFluorescence quantum yields are relative to the quantum yield of quinine sulfate in 0.5 M H2SO4 (F = 0.546). d Measurements taken in water. A Lippert-Mataga Analysis of the C(8)-H, -CN, and -COOMe Purines The Lippert-Mataga equation (E quation 3-2) was developed to approximate the energy difference between the ground state (represe nted by the ground state dipole moment, g)95 and the excited state (represented by the excited state dipole moment, e) of a molecule using the orientation polarizability ( f ) (Equation 3-3) of the solvent, a term derived from the solvents refractive index ( n ) and dielectric constant (). A F = 2(e-g)2 f / hca3 + C (3-2) f = [( 1)/(2 + 1)] [( n2 1)/(2 n2 +1)] (3-3) For the other terms in the equation that are not already defined h (= 6.626 x 10 34 Js) is Planks constant, c (= 2.998 x 108 m/s) is the speed of light, a represents the radi us of the cavity in which the fluorophore resides (the Onsager radius), and A F is the Stokes shift (in cm 1). This equation describes phenomena related to general solvent effects in which the molecular species is considered to be a dipole in a conti nuous medium of uniform dielectric constant and does not account for specific solvent-molecule interactions like hydrogen bonding or solutesolute interactions.2

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76 A fluorophores solvent sensitivity can be esti mated by a Lippert-Mataga (Lippert) plot. Here A F is plotted against f for several different bulk solvents or for mixtures of solvents. A linear plot indicates the dominance of general solvent effects. The slope ( k ) of the line can then be used to calculate eg (Equation 3-4).95 eg = khca3 (3-4) A larger k indicates more sensitivity of the mo lecule to its solvent environment. Lippert plots constructed for the purines show in general modest linearity, as given by the coefficient of determination, R2, calculated from a least squares analysis. Graphical examples for the best fitting purines in each class are provid ed as Figures 3-12 through 3-14. Specific solvent effects, including hydrogen bonding96 and preferential solv ation, may contribute to nonlinearity; the response of the purines to these effects is difficult to predict.2 The correlation coefficients do not generally improve if the methanol data is omitted, although this has been observed for other D-A fluorophores where hydrogen bonding by solvent has been implicated.80 Worth noting also is that on average larger slopes ( k ) are found for the C(8)-CN and C(8)-COOMe D-A compounds, demonstrating their hi gher sensitivity to solven t polarity than the C(8)-H derivatives. This higher sensit ivity is likely due to an enha nced charge separation for the acceptor-modified purines.2 Table 3-13. Lippert-Mataga Da ta for Selected Purines. Purine slope ( k ) cm-1 R2 eg, D g, D (theory) e, De, D (theory) 2.6 1978 0.99 4.10 3.85 7.95 5.02 2.12b 5190 0.75 8.12 5.66 13.78 11.04 2.12c 4417 0.69 7.49 3.16 10.65 9.77 Estimating eg from the Lippert plots is error prone in the cases of modest correlation values, nonetheless this has been done for the examples shown. Using Equation 3-4 and an estimated value for the Onsager radius for each purine (taken as 3.5 for 2.6 and 4.0 for both

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77 2.12b and 2.12c ; the latter two are similar in structure to PRODAN that has an accepted radius of 4.2 ),97 eg could be calculated. From this value and the theoretically-determined g (vide infra) a value emerges for the excited state dipole, e. The magnitude of e correlates very well with values from computation. Figure 3-12. Lippert-Mataga plot for 2.6 Figure 3-13. Lippert-Mataga plot for 2.12b

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78 Figure 3-14. Lippert-Mataga plot for 2.12c The Chemical and Photochemical Stability of C(8)-H, C(8)-CN, and -COOMe Purines The C(8)-H, -CN, and -COOMe purines s howed no decomposition by TLC analysis, 1H and 13C NMR, and HRMS after months of being stor ed neat in vials with exposure to air and ambient light. After days in solutions of 1,4-dioxane, methylene chloride, acetonitrile, and methanol with exposure to air and ambient lig ht the purines showed no significant loss of absorption (< 1%). Evaluation of an emitting compounds absorption loss is a method sometimes used to evaluate the molecules photostability.98 When the D-A purines were placed in solution and exposed to 254 nm light from a hand-held UV lamp for varying time intervals, changes were observed in the abso rption spectra. As shown for 2.14b and 2.14c (Figures 3-15 and 3-16), complete deterioration of the 1( La) absorption band occurs after ~ four hours in methylene chloride and a new blue-shifted absorption band (max = 309 nm) emerges. The 1( Lb) absorption band is shifted to th e red during the experiments. Th e structure of the new species that forms, possibly from direct reac tion with oxygen, has not been determined.

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79 Figure 3-15. Changes observed in the absorption spectrum of a 60 M solution of 2.14b in methylene chloride after exposure to 254 nm ultraviolet light at varying time intervals. Figure 3-16. Changes observed in the absorption spectrum of a 60 M solution of 2.14c in methylene chloride after exposure to 254 nm ultraviolet light at varying time intervals.

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80 Figure 3-17. Changes observed in the absorption spectrum of a 60 M solution of 2.14c in 1,4dioxane after exposure to 254 nm ultravio let light at varying time intervals. Figure 3-18. Changes observed in the absorption spectrum of a 60 M solution of 2.6c in water after exposure to 254 nm ultraviolet light at varying time intervals.

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81 The rate of decomposition is somewhat slowed in 1,4-dioxane as shown for 2.14c (Figure 3-17); in this case no significant new absorption bands are observed. Finally, the decomposition rate appears most suppressed in water as shown for 2.6c (Figure 3-18) that loses very little of its absorption intensity after 300 min of exposure. This last result bodes well for the use of the modified purines in aqueous-b ased sensing applications. Electronic Properties of the D-A Purines Theoretical Calculations Electronic structure calculations have beco me essential to chromophore design and study, particularly for establishing their potential use in optoelectronic applications. Both semiempirical and high-level DFT calculations have been used to shed additional light on the electronic nature of the D-A purines and th eir absorption properties. The ground state geometries, dipoles, and orbita l energies have been obtained for 9-methyl-2-aminopurine ( 2.6Me ), 2.6 2.14b and 2.6 2.14c from DFT calculations at the B3LYP/6-31++G** level (as implemented in Gaussian 03).99 Other levels of theory not supplemented by diffuse functions provided similar, but somewhat less accura te values (B3LYP/6-31G**, B3LYP/6-311G**, and B3LYP/aug-cc-pVDZ). For the D-A purines, the benz yl groups have been abbreviated to methyl (Me) substituents to save computational time, a nd frequency calculations have been performed in all cases to assign the optimized geometries as energy minima or transi tion states (all except 2.12b -Me are minima, vide infra). To demonstrat e the accuracy of the calculations, Table 3-14 compares the bond lengths for 2-AP and 2-7b (crystal structure data fr om Table 2-1) with their calculated 9-methyl analogues. The values are the same within ~ 0.02 In most cases, the optimized geometries do show slight pyramid alization of the C(2) and C(6) amino (or dimethylamino) nitrogens. These deviations have been analyzed in terms of the wagging angle, in the Experimental section.

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82 Table 3-14. Selected Bond Lengths for 2.7b and 2-AP from the crystal, and their 9-methyl derivatives from computation. Bond 2.7b 2.7b-Me 2-AP 2.6-Me N1-C6 1.3411(15) 1.346 1.332(3) 1.335 N1-C2 1.3536(15) 1.350 1.365(2) 1.358 C2-N3 1.3484(15) 1.344 1.343(2) 1.347 C2-N11 1.3563(15) 1.369 1.353(3) 1.371 N3-C4 1.3446(14) 1.335 1.329(3) 1.331 C4-N9 1.3749(14) 1.372 1.368(3) 1.374 C4-C5 1.3952(16) 1.410 1.400(2) 1.412 C5-C6 1.4301(16) 1.435 1.381(3) 1.395 C5-N7 1.3810(14) 1.374 C6-N10 1.3450(15) 1.358 N7-C8 1.3129(16) 1.321 1.318(2) 1.308 C8-N9 1.3834(14) 1.390 1.360(3) 1.390 C8-C12 1.4308(15) 1.421 Summarized in table 3-15 are the dipole and orbital energy data obtained for the D-A purines from the DFT calculations. Complement ary orbital density pl ots of the HOMO and LUMO levels could be generated using Molden v. 4.6100 to show graphica lly the electronic nature of the ground state (Figure 3-19). The or bital features are quite similar within and between the nitriles and methyl esters. The HO MO consists of delocal ized p orbitals on the purine plane, with the greatest density in the N( 9)-C(4)-C(5)-C(6) region consistent with what has been observed for parent 2-AP.101 Localized pz orbitals are seen on the exocyclic donor heteroatoms and the acceptor functionality, and ther e is more substantial ch arge separation in the HOMO for the nitriles (consistent also with thei r greater ground state di pole moments; average for nitriles = 5.73 D, esters = 3.96 D, 2.6-Me = 3.85 D). The LUMO is also of character but now largely concentrated on the ac ceptor substituent, th e cyano group or methyl ester. Generally the density on the C(2) donor group diminishes most substantially in the LUMO; the C(6) donor orbital coefficient is only slightly affected. The energies of the HOMO and LUMO levels scale with the substituents and substitution pa tterns in reasonable ways. For example, 2.6b bearing one donor group, has the lowest HOMO and LUMO energies. Purine 2.10b with two

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83 dimethylamino substituents, has the highest. In general the HOMO/LUMO values for the esters are ~ 0.3 eV higher than the nitr iles; the calculated HOMO-LUMO ga ps are slightly lower (~ 0.1 eV) for the esters. The results confirm that the cyano groups are stronger electron acceptors, although the optical properties al so clearly depend on the geometry/structure of the electron accepting group. Table 3-15. Electronic Struct ure Data for D-A Purines.a Purineb g HOMO calcd, CVc (eV) LUMO calcd, CVc (eV) E calcd, CVc (eV) E opticald (eV) max La calcd (nm, [eV]), fe max La CH2Cl2 (nm) 2.6b-Me 5.45 .52 .27 4.25 3.5 328, 0.57 326 2.7b-Me 5.00 .88, .48 .68, .28 4.20, 3.20 3.4 340, 0.60 334 2.8b-Me 6.60 .29 .87 4.42 3.6 327, 0.59 311 2.10b-Me 5.52 .64 .34 .60 .11 4.04 3.23 3.3 344, 0.62 348 2.12b-Me f 5.66 .75 .59 .68 .56 4.07 3.03 3.4 339, 0.61 336 2.14b-Me 6.13 .08 .14 3.94 3.1 333, 0.59 361 2.6c-Me 5.19 .24 .04 4.20 3.4 331, 0.58 328 2.7c-Me 2.43 .62 .55 4.07 3.3 349, 0.58 322 2.8c-Me 4.74 .03 .69 4.34 3.5 333, 0.59 315 2.10c-Me 2.81 .40 .48 3.92 3.2 354, 0.61 348 2.12c-Me 3.16 .53 .56 3.97 3.2 348, 0.61 338 2.14c-Me 5.42 .82 .94 3.88 3.1 338, 0.61 362 a See the Experimental section for computational details. b All benzyl groups have been replaced by methyl groups for the calculations. c Cyclic voltammetry data; for details, see the Experimental section. d Calculated as the end-absorption of the lowest energy transition in the UV-vis spectrum. e All correspond to one-electron HOMO (S0) LUMO (S1) transitions. f One imaginary frequency was identified for th is structure indicating a transition state. The frequency is associated with minor inversion of the C(2) amino group based on analysis of the coordinate files. ZINDO/S CI semi-empirical calculations102 using the optimized DFT ground state geometries reveal that the lowest energy transitio n is associated with promotion of an electron from the HOMO to the LUMO in all cases. Give n the large calculated oscillator strength ( f ) for this transition, it is an allowed process for which the trend in energy gap (327 nm; 3.79 3.50 eV) correlates well with the trend in max in CH2Cl2 (311 nm; 3.99.43 eV). Based on

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84 the plots in Figure 3-19, the tran sition mainly involves transfer of charge from the amino group on C(2) (and the pyrimidine ri ng) to the C(8) acceptor group. Figure 3-19. Orbital density plot s (Molden v. 4.6) for D-A purines calculated from the B3LYP/631++G** optimized geometries. Cyclic Voltammetry Studies The difference between the first oxidation potential and the first reduction potential, determined by cyclic voltammetry, can be rega rded as a measure for the HOMO-LUMO gap.

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85 Electrochemical studies have been performed, in collaboration with Aubrey Dyer (in the group of Prof. John Reynolds), for purines 2.7b 2.10b and 2.12b Measurements (at least three independent reductive and oxidative scans) we re performed using 10 mM solutions of the purines in CH2Cl2 with 0.2 M n -Bu4NClO4 as the supporting electrol yte; redox potentials were determined versus the ferricinium/ferrocene couple. The average HOMO and LUMO values, determined from the oxidation and reduction onset s (Table 3-15), respectively, are shown in Table 3-15. The most electron rich purine, 2.10b shows the highest HOMO and LUMO values. The HOMO values in general agree within 0.4 eV with the computa tional results and the calculated HOMO-LUMO gaps agre e within 0.2 eV of the optical band gaps determined from the solution absorption spectra. Given the latt er, the DFT calculations appear to largely overestimate the LUMO energies (> 0.5 eV). Nonetheless, the experiments show that the theoretically-determined HOMO energies and the op tical band gap are sufficient to determine the LUMO energies for the D-A purines within ~ 0.2 eV. Device Measurements for D-A Purine 2.7b D-A molecules are often targets for organic light emitting diode (OLED) research due to their ability to fluoresce efficiently, transport charge, and to undergo charge transfer to an electrode.78 Since the donor-acceptor compounds presente d in this work fluoresce efficiently in solvent, their capability of performing in a device was explored with D-A purine 2.7b in collaboration with Aubrey Dyer (in the group of Prof. John Reynolds) (Figure 3-20A). The result of this study revealed 2.7b to be a blue emitter (Figure 3-20B). One device was fabricated by spin-coating a dispersion of PEDOT:PSS (the oxidized form of poly(ethylenedioxythiophene) and poly(styrene sulfonate)) in water onto ITO (i ndium-doped tin oxide) deposited on glass. This PEDOT:PSS layer (~ 15 nm) acts to smooth out the ITO layer and aids in hole injection.

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86 Figure 3-20. Organic light emitting diode (OLED) for 2.7b A) The device used to test the electroluminescence. B) Electroluminescence of 2.7b seen in the device. Figure 3-21. Measurements taken with the O LED device. A) Electroluminescence spectra of 2.7b B) Graph showing the turn-on voltage of the OLED device. Figure 3-22. Schematic model of the OLED device using 2.7b as the emissive layer. The emissive layer, 2.7b was then spin-cast from a solution of chloroform onto the PEDOT:PSS layer. A thin layer of calcium (~ 10 nm) depos ited on top of the emi ssive layer acts as the

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87 cathode and finally aluminum was deposited (~ 150 nm) onto the calcium to encapsulate the device from oxygen and water. The electroluminescence spectrum shows two peaks of interest, max ~ 450 nm and max ~ 650 nm (Figure 3-21A). The higher energy peak is red-shifted approximately 60 nm when compared to the most bathochromic shift (Tab le 3-9) found in solution for this compound. Bathochromic shifts are genera lly observed when comparing photophysical measurements from solution to measurements in the solid state where -stacking interactions are more substantial.103 The turn-on voltage for 2.7b appears to be approximately 9 V (Figure 3-21B). This turn-on voltage appears to be slightly higher than turn-on voltages for optimized systems reported in the literature which are generally below 5 V.104,105 Though these results are promising, the electroluminescence for this device is very weak. One reason for th e poor efficiency may be that some crystallization of the compound has take n place upon substrate deposition. It is believed that vapor depositing the purine onto the substrat e could give more intense electroluminescence by forming a thinner emissive layer th at is less likely to crystallize. It is also possible that use of a carrier polymer (PMMA or PS) with the purin es could produce better films. Further experiments in this area are currently underway. Experimental Section UV-Visible Data Absorption spectra were measured for 5, 10, 20, 40, and 60 M solutions of the purines on a Cary 100 Bio UVVisible spectrophotometer (thermostatted at 25 C) using dried and degasse d methylene chloride (CH2Cl2). The absorption intensity at max was then plotted against th e concentration to confirm, by lin earity, that the compounds followed Beers law. Once confirmed, molar extinction coefficients () were determined from the linear plot for each compound (where A = bc ).

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88 Fluorescence Lifetime Measurements Fluorescence lifetime measurements were performed on a Photon Technology International, Inc. Time Master Fluorescence Lifetime system. Excitation was performed via a nitrogen laser at 337 nm (pulse width approximately 800 ps), and a quartz fiber optic was used to conve y light to the sample chamber. Samples for fluorescence lifetime measurements of th e C(8)-CN and C(8)-COOMe compounds were prepared by making ~ 5 M solutions of each compound in dried and degassed methylene chloride. Each compound was excited at 337 nm and the monochromator was set to the corresponding em(max) for each compound. After the measurement of each compound, a scattering agent (Ludox AM-30 colloidal silica, 30% wt. % suspension in water) was measured under the same parameters as the compound in orde r to find the instrument response time. The instrument response time along with the data collect ed for each sample was placed into the Time Master software to generate the fluorescence lifetime data. Fluorescence Quantum Yield Measurements Fluorescence quantum yield measurements were performed by collecting co rrelating absorption a nd steady-state emission spectra on a Perkin-Elmer Lambda 25 dua l beam absorption spectrometer and a SPEX Fluoromax spectrophotometer, respectively. Th e unknowns and standards were prepared with dried and degassed solvent at or below a 10 M concentration and absorbance below an intensity of 0.1. F(x) = ( As/ Ax)(Fx/Fs)(x/ s)2F(s) F is the fluorescence quantum yield, A is the absorbance at the excitation wavelength, F is the area under the emission curve, and is the refractive index of the so lvent used. Subscripts s and x refer to the standard and unknown, respectively. Quantum yi elds were calculated using quinine sulfate in 0.5 M H2SO4 (F = 0.546) as the standard.3

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89 Computation Starting geometries were obtained from semi-empirical calculations using the AM1 method as implemented in Hype rChem v. 7.5 for Windows (HyperCube, Inc., Gainesville, FL). The ground state geometries, di poles, and orbital energi es were then obtained from DFT calculations at the B3LYP/6-31++G** level as implemented in Gaussian 03.99 The DFT calculations were performed on the SG I Altix system Cobalt through available computational time (Prof. A. Roitberg, UF) at the National Center for Supercomputing Applications (NCSA) at the University of Illin ois, Urbana-Champaign. Frequency calculations were performed at the same computational leve l. The resultant geometries were used for ZINDO/S CI calculations102 using HyperChem v.7.5 where the highest 20 occupied and the lowest 20 unoccupied orbitals were considered for determination of the electronic transition energies, polarizations, and oscillator strengths. The currently accepted values for the overlap weighting factors of 1.267 (s-s ) and 0.585 (p-p) were used. In many cases the amino groups of the optimized purine structures show slight pyramidalization. These deviations can be pa rameterized in terms of the wagging angle, defined as the angle C(2 or 6)Ncentroid between R substituents (H or CH3) (Table 3-16). An alternative way of expressing th e same distortion is through th e N(1)-C(2)-N(11)-R and N(3)C(2)-N(11)-R torsion angles (that deviate from 0). For 2-AP-Me, these values are .8 and 16.4, respectively (compared with .8 and 23.1 from MP2/6-31G** calculations on 2-AP in the literature).106 In general, such slight pyramidalizat ion represents a small stability gain for the molecule in the gas phase (< 1 kcal mol-1).106 Cyclic Voltammetry Electrochemical measurements were performed using an EG&G PAR model 273A potentiostat/galvano stat in a three-electrode cell configuration consisting of a silver wire pseudo reference electrode calibrated with a ferrocene/ferricinium couple, platinum

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90 button as the working electrode, a nd a platinum flag as the counter electrode (scan rate = 75 mV s-1). For the studies, 10 mM solutions of the purines in CH2Cl2 containing 0.2 M n -Bu4NClO4 were prepared. All measurements were perfor med in a glove box and re peated in at least triplicate. Table 3-16. Amino and Dimet hylamino Group Wagging Angles ( in degrees) Calculated from the Optimized Geometries of the D-A Purines. Purine C(2)-NH2 C(2)-N(CH3)2 C(6)-NH2 C(6)-N(CH3)2 2.6-Me 27.4 2.6b-Me 16.8 2.7b-Me 26.2 (5.3)a 10.5 (17.0)a 2.8b-Me 21.9 2.10b-Me 15.6 11.2 2.12b-Meb 0.4 (2.3)a 0.4 (8.3)a 2.14b-Me 12.6 2.6c-Me 21.7 2.7c-Me 28.7 9.8 2.8c-Me 25.1 2.10c-Me 16.6 11.2 2.12c-Me 16.0 (5.2)a 2.1 (4.4)a 2.14c-Me 14.0 a Value from X-ray analysis (N(9)-benzyl). b One imaginary frequency was identified for this structure indicating a transition state. The frequency is associat ed with minor inversion of the C(2) amino group based on analysis of the coordinate files.

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91 CHAPTER 4 USING THE DONOR-ACCEPTOR PURINE UNIT AS A FUNCTIONAL SYNTHETIC BUILDING BLOCK Introduction The purine core is an excellent heterocycl ic building block for the construction of functional materials capable of molecular rec ognition, sensing, and displaying supramolecular behavior. The nitrogen-rich lining of the co re gives ample opportunity for hydrogen bonding or metal binding, and the polarizable -surface allows for efficient -stacking interactions. This Chapter identifies general ways, through synthe tic chemistry, that th e donor-acceptor (D-A) purines can be further functionaliz ed for incorporation into nonc ovalent supramolecular systems, or covalent oligomeric and/or pol ymeric architectures. Looking at the structures of the purines, there are several atomic starting points: C(2), C(6), C( 8), N(7), and N(9). The most attractive way to build off of C(8) while potentially pres erving the cores photophys ical properties is to employ the acid precursor functionality (CN or CO2Me); discussed here are ways to make the first amide bonds from this position. A second strategy worth explori ng is to use the NH2 group on C(2) or C(6) as a nucleophile ; early progress in this directi on is presented and some of its advantages and disadvantages disc ussed. Finally, presented in the Future Work section are ways that N(7) and N(9) coul d potentially be promising functionali zation points, particularly toward the incorporation of D-A purines into DNA, RNA, or PNA backbones. A Complementary Thrust: -Purinyl Amino Acids Numerous nitrogen rich hete rocycles have been employed in synthetic oligopeptides.107-109 The incorporation of heterocycles directly within the peptides mainchain is a powerful design to realize information-rich molecules capable of specifically recogni zing biological targets.110 It did not escape us that the C(8)-CN and -COOMe f unctionality of the D-A purines, together with

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92 C(2) amino functionality, affords novel -purinyl amino acids that can serve as scaffolds from which unique chemical architectures can be built. N N N N H2N R1 Bn OH O R1 = H, NH2, N(Me)2, OBn Figure 4-1. Novel -purinyl amino acids. Homoand heteropurinyl oligopep tides could be formed through direct covalent linkage of the -purinyl amino acids; the homopurinyl olig opeptide made from stringing identical molecules of -purinyl amino acids togeth er in a head-to-tail fashi on is shown in Figure 4-2. Simple molecular mechanics calculations with deprotected oligomers fu rther illustrate the concept and some of its attract ive features (Figures 4-3 and 4-4). The energy-minimized structure (MacroModel, Amber* fo rce field) shows a near planar dipurinyl peptide arrangement defined by torsion angles labeled and ( = 0 or 180) (Figure 4-3). Reduced torsional degrees of freedom, enforced by favorable electrostatic inte ractions between NH and C=O groups, maintain a curvature along the polar ed ge of the molecule as the length of the oligopeptide is extended. This curvature is ex pected to result in oli gopeptides suitable for binding grooved biomolecular surfaces or formi ng predictable three-di mensional structures. Computation shows that a thre e unit purinyl homopeptide fits into the minor groove of DNA and that an 18 unit purinyl homopeptide coul d form a covalent macrocycle with a 4.5 nm diameter that is lined with multiple binding site s. Given the interesting photophysical properties of the D-A monomers, it is expected that thes e architectures would present advantages in detection through increased conjugation length an d/or have unique optical properties.

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93 N N N N H2N R Bn N N N N R Bn N N N N HN R Bn N H O O n Figure 4-2. Homopurinyl oligopeptide from -purinyl amino acids. Figure 4-3. Computationally de rived torsion angle preferen ces for a dipurinyl peptide constructed from deprotected monomer 2-7c Figure 4-4. Oligopeptide curvat ure with potential molecular recognition and supramolecular capabilities. A) 2:1 Minor groove bind ing of DNA by a three unit homopurinyl peptide. B) 18 unit homopurinyl peptid e forming a covalent macrocycle. Hetero-oligopeptide structures are also con ceivable and one design might incorporate both -purinyl amino acids and conventional -amino acids into a peptidic main chain. The different noncovalent interactions unique to the amino acid components are expected to work cooperatively to form three-dimensional structur es capable of folding and unfolding in response to environmental stimulia new cl ass of foldamers could be born.111 There is much work being

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94 done to form synthetic, self-folding oli gomers capable of biomolecule-like function.111 An example of one hetero-o ligopeptide that might serve in this capacity is shown in Figure 4-5, where -purine amino acids are linked by an -amino acid tripeptide with the sequence Gly-DPro-Gly. N N N N N H N H N N H N N N N H2N R O O O O R R R O O n H Figure 4-5. Design for a hete ro-oligopeptide containing -purinyl amino acids linked with conventional -amino acids. The computationally-determined three-dimensional folded structure of such an oligomer is shown in Figure 4-6 as it comes from a Monte Carlo molecular dynamics simulation (MacroModel, Amber* force field, GB/SA water solvation treatment). The result shows a helical secondary struct ure as a result of the -stacking preferences of the purines and the -turns enforced by hydrogen bonding within the Gly-DPro-Gly sequences. Substitution on N(9) and C(6) of the purine core could in troduce other functiona lity and control sol ubility/aggregation. Finally, and importantly, the N-terminal -purinyl amino acid in such oligopeptide architectures could serve as a fluorescent reporter of secondary structure and conformation. Figure 4-6. Folded structure pr edicted (by computation) for -purinyl amino acids linked with Gly-DPro-Gly.

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95 Amide Functionality at the C(8) Position Using the C(8)-CN and -CO2Me derivatives as amide precursors appeared as a promising synthetic strategy to both elabor ate the core and work toward -purinyl amino acid structures. Conversion of the methyl esters to carboxylic acid inte rmediates (C(8)-CO2H) was attempted first, in hopes that the acid could be used conventionally in amide bond forming reactions (by direct coupling reactions or initia l conversion of the acid to an activated ester or acid chloride). Carboxylic acid functionality on the C(8) positio n of the purine is reported only once in the literature, however, and is reported to be very unstable.63 For the initial studies, to eliminate the possi bility of side reacti ons at C(2) or C(6), compound 2.10c containing a dimethylamino group at bot h positions was used as the starting methyl ester. The C(8) carboxylate 4.1 was formed initially from 2.10c by heating it to 100 C in a sealed tube with 10% aqueous sodium hydroxi de and methanol (Figur e 4-7). It was then discovered that a synthetic step in the seque nce could be saved by r eacting the C(8) cyano derivative 2-10b under the same conditions to form 4.1 directly. When 4.1 was isolated as its sodium salt it proved stable and could be le ft on the benchtop under air and ambient light indefinitely (consistent with the liter ature observation for a related compound).63 Once acidified, 4.1 rapidly decarboxylated to yield the C(8)-H compound 2.10 (monitored by TLC and 1H NMR). The reactivity of the carboxylic acid of 4.1 required that it be used immediately after work-up and that it be kept cold thoughout th is process. With care, the free acid of 4.1 was then heated with thionyl chloride to form the C(8) acid chloride 4.2 Compound 4.2 was quickly placed in THF with Et3N and butylamine (a reactive primary amine that would produce a soluble amide product); a complex mixture of products wa s obtained from the reaction that included a

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96 23% yield of the desired amide 4.3 (Figure 4-7). This shows that even with the lability of the acid, some was converted to the acid chlo ride and further to the desired product 4.3 N N N N N N Bn O O N N N N N N Bn O O N N N N N N Bn Cl O N N N N N N Bn O HN NaOH EtOH 100 C 1. HCl 2. SOCl2Et3N BuNH2THF 23%f r om 2.10c 83% 2.10c4.1 4.3 4.2 N N N N N N Bn CN 2.10b quant. 80 C1h r.t., overnight Na NaOH EtOH 100 C Figure 4-7. C(8) amide form ation via an acid chloride. Successful, albeit low-yielding formation of the C(8) amide 4.3 inspired attempt at the formation of a purinyl dimer by using the acid chloride 4.2 and a purine bearing a C(2) primary amine. This reaction resulted in a complex mixt ure of inseparable produ cts (Figure 4-8). The low yield of 4.3 and the complex product mixture of the reaction in figure 4-8 are most likely a result of slow reaction of the relatively weak ly nucleophilic C(2) amino group coupled with apparently rapid d ecarboxylation of the 4.1 carboxylic acid (or decomposition of 4.2 ) under the reaction conditions. N N N N N N Bn Cl O 4.2 N N N N H2N O Bn Br 2.8a + Bn N N N N N N Bn O N N N N HN O Bn Br Bn DIPEA THF r.t., overnight Figure 4-8. Unsuccessful coupling reaction to synthesize a purine dimer via the acid chloride.

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97 The use of conventional peptide coupli ng conditions direc tly from the acid 4.1 (e.g., DCC/DMAP) were next attempted; this approach eliminates the activation step and was found to improve the product yield dramatically. By this strategy, C(8) amide derivatives 4.3 and 4.5 4.7 were formed (Figure 4-9). Compound 4.3 was formed to compare product yields between amide formation via the acid chloride and DCC/DMAP coupling. This showed that product yields almost doubled (23 42%) by using DCC/DMAP coupling. Products 4.5 and 4.6 were formed using the methyl ester protected amino acids of glycine and 5-aminovaleric acid. The methylesters of these compounds can potentially be deprotected and further coupled to form oligomeric species. The formation of 4.7 shows that C(8) coupling with aliphatic amines can be performed in the presence a free pr imary amine on the C(2) position ( 4.4 to 4.7 ) since the latter is considerably less nucleophilic. N N N N R1 R2 Bn CN N N N N R1 R2 Bn OH O N N N N R1 R2 Bn O HN R3 2. HCl 0 C DCC DMAP primary amine 0 C, overnight2.10b R1 = N(Me)2, R2 = N(Me)22.12b R1 = N(Me)2, R2 = NH24.1 R1 = N(Me)2, R2 = N(Me)24.4 R1 = N(Me)2, R2 = NH2R1 = N(Me)2, R2 = N(Me)2, R3 = (CH2)3CH3R1 = N(Me)2, R2 = N(Me)2, R3 = CH2CO2CH3R1 = N(Me)2, R2 = N(Me)2, R3 = (CH2)4CO2CH3R1 = N(Me)2, R2 = NH2, R3 = CH2CO2CH342% from 2.10b82% from 2.10b45% from 2.10b51% from 2.12b 4.3 4.5 4.6 4.7 1.NaOH EtOH 100 C Figure 4-9. C(8) amide formati on using the DCC/DMAP conditions. Extension of the DCC/DMAP conditions to purin yl dimer formation resulted in none of the desired product, however, and instead a large percentage of C(8)-H derivative 2.10 was

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98 recovered from the reaction (Figur e 4-10). This suggests that th e acid or in situ generated activated ester of 4.1 decomposes prior to nucleophilic at tack by the N(2) primary amine of 2.8a Of note, very few C(8)-amido purines ha ve been reported in the literature.112,113 The C(8)-amido purines reported in this thesis appear to be the first formed through conventional amide chemistry using acid precursors. N N N N N N Bn OH O + N N N N H2N O Bn Bn Br N N N N N N Bn 4.12.8a DCC, DMAP CH2Cl276% 2.10 0 C, overnight Figure 4-10. Coupling reaction to form a purinyl dimer using DCC/DMAP. Crystal Structure of the C(8)-Amide Purine 4.7 Molecular Level Structure Single crystals suitable for X-ray diffraction, perfomed by Dr. Khalil Abboud, could be grown by slow evaporation of a meth anol/methylene chloride solution of 4.7 over one week. The X-ray structure of 4.7 (Figure 4-10a) reveals the dimethyl amino substituent, defined by atoms N(11), C(12), and C(13), to be plan ar at N(11) and with the purine core. The sum of the N(11) bond angles is 359.91, consistent with sp2 hybridization at nitroge n. Nearly identical geometries were observed in the crystal structures of 2.7b 2.12b and 2.10c Figure 4-11. Single molecule from the crystal structure of 4.7

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99 The N(9) benzyl substituent geometry can again be defined by torsion angles defined by a-b-c-d) = 88.79 and defined by b-c-d-e) = 76.96 for 4-7 (Figure 4-10b). The values show that as in 2.7b 2.12b and 2.12c the benzyl group extends nearly perpendicular to the plane of the purine core ( ), while angle remains sensitive to the inte rmolecular interactions involving the phenyl group. The crystal structure also co nfirms the expected positioning of the amide substituent with respect to the purine core; namely, planar an d with the NH group on the same side as N(7) (N(16)N(7) = 2.74 ). This pref erence is predicted by co mputation (vide supra) and speaks to the ability to control local conformation through favorable intramolecular electrostatic (e.g., hydrogen-bonding) interactions in these he terocyclic amide systems. Crystal Packing for Compound 4.7 The crystal packing for compound 4.7 reveals extended -stacking where the C(8) amide moiety of one molecule is extended over th e pyrimidine ring of a neighboring purine core (Figure 4-11a). The distance between the least squares planes defined by N(1), N(3), N(7), and N(9) is equal to 3.35 Intermolecular hydr ogen bonding is also observed between the amide carbonyl oxygen O(15) and the N( 10) hydrogen of an adjacent purine (N(15)N(10) = 2.97 (Figure 4-11b). That simple dipolar -stacking is not observed, but extended intermolecularly H-bonded chains are, reemphasizes how simple changes in functionality can influence supramolecular ordering. Particularly intere sting from the standpoint of optoelectronic applications (e.g., second-order nonlinear optics) is how intermolecular hydrogen bonding confers polar ordering to the molecules of 4.7 within one-dimensional ro ws (Figure 4-11b). The distance between these one-dimensional rows is 3.35 (benzyl groups pointing toward each other) and 3.52 (benzyl groups pointing away from each other).

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100 Figure 4-12. Dominant noncovalent interact ions found in the crystal structure of 4.7 A) stacking. B) Interand in tramolecular hydrogen bonding. Figure 4-13. The crystal packing of 4.7 along its crystallographic axes. A) Axis a B) Axis b C) Axis c Photophysical Data for the C(8)-Amide Derivatives Absorption and fluorescence emission meas urements were taken for C(8) amide compounds 4.3 and 4.5 Many of the photophysical trends re ported in Chapter 3, seen with the nitriles and esters, are also observed with these compounds. Table 4-1. Photophysical data for compounds 4.3 and 4.5 in 1,4-dioxane.a Purine max, abs/nm log /M-1 cm-1 max,b em/nm max, em-abs/nm F c 2.10b 345 4.3 392 38 0.96 2.10c 344 4.3 392 58 1 4.3 332 4.2 392 60 1 4.5 336 4.3 397 61 1 aAll measurements performed at room temperature. bAll experiments were performed using optical densities 0.1 at the excitation wavelength ( ex = 360 nm). cFluorescence quantum yields are relative to the quantum yield of quinine sulfate in 0.5 M H2SO4 ( F = 0.546).

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101 Photophysical comparisons for compounds 4.3 and 4.5 are made using compounds 2.10b and 2.10c the C(8)-CN and -COOMe purines, respectively, that have the same C(2) and C(6) donors as 4.3 and 4.5 (Tables 4-1-4). Table 4-2. Photophysical data for compounds 4.3 and 4.5 in methylene chloride.a Purine max, abs/nm log /M-1 cm-1 max, em/nm max,b em-abs/nm F c F/ns 2.10b 348 4.4 388 40 0.20 7.6.1 2.10c 348 4.3 409 61 0.90 2.7.3 4.3 338 4.2 402 64 0.87 1.9.3 4.5 343 4.3 407 64 0.91 1.6.4 aAll measurements performed at room temperature. bAll experiments were performed using optical densities 0.1 at the excitation wavelength ( ex = 360 nm). cFluorescence quantum yields are relative to the quantum yield of quinine sulfate in 0.5 M H2SO4 ( F = 0.546). The absorption maxima appear to be weak ly solvent dependent, vary linearly with concentration, and the molar extinction co efficients remain high. The emitting 1( La) absorption band is found to be blue-shifted for 4.3 and 4.5 when it is compared to 2.10b and 2.10c Table 4-3. Photophysical data for compounds 4.3 and 4.5 in acetonitrile.a Purine max, abs/nm log /M-1 cm-1 max,b em/nm max, em-abs/nm F c 2.10b 344 4.3 394 50 0.26 2.10c 347 4.3 415 68 0.86 4.3 333 4.2 405 72 1 4.5 336 4.3 409 73 1 aAll measurements performed at room temperature. bAll experiments were performed using optical densities 0.1 at the excitation wavelength ( ex = 360 nm). cFluorescence quantum yields are relative to the quantum yield of quinine sulfate in 0.5 M H2SO4 ( F = 0.546). This could be due to electron donation from the amide nitrogen to the am ide carbonyl interfering with conjugation between C(8) and the am ide carbonyl. The emission maximum for compound 4.3 is red shifted from 392 nm to 424 nm in so lvents of increasing pol arity, 1,4-dioxane and methanol, respectively. These values are compar able to the values observed for methyl ester compound 2.10c

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102 Table 4-4. Photophysical data for compounds 4.3 and 4.5 in methanol.a Purine max, abs/nm log /M-1 cm-1 max,b em/nm max, em-abs/nm F c 2.10b 342 4.4 403 61 0.032 2.10c 347 4.2 432 85 0.66 4.3 335 4.2 424 89 0.89 4.5 340 4.2 429 89 0.90 aAll measurements performed at room temperature. bAll experiments were performed using optical densities 0.1 at the excitation wavelength ( ex = 360 nm). cFluorescence quantum yields are relative to the quantum yield of quinine sulfate in 0.5 M H2SO4 ( F = 0.546). Stokes shifts also increase with increasing solvent polarity and the range between Stokes shifts for the lowest and highest polarity solvents are 29 nm for 4.3 and 28 nm for 4.5 ; these values show that the solvent sensitiv ity for these compounds is similar to 2.10c (27 nm Stokes shift). Of greater interest, the qua ntum yield values remain close to unity in spite of increases in solvent polarity, and are even higher for 4.3 (F = 0.89) and 4.5 (F = 0.90) than 2.10c (F = 0.66) in methanol. C(8)-Amide Photostability Figure 4-14. Changes observed in the absorption spectrum of a 60 M solution of 4.3 in methylene chloride after exposure to 254 nm ultraviolet light at varying time intervals.

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103 When a 60 M solution of 4.3 in methylene chloride was ex posed to 254 nm light from a hand-held UV lamp for varying time intervals, th e absorption spectra exhibited more dramatic changes (Figure 4-14) than were observ ed under similar conditions for compounds 2.14b and 2.14c (Figures 3-15 and 3-16). The 1( La) absorption band at 334 nm is completely deteriorated within 5 min of exposure and a ne w band appears at 306 nm. After 10 min this new band begins to lose absorption intensity. The 1( Lb) absorption band progressively blue shifts and loses intensity over the 240 min of UV exposure. Theoretical Data Again, both semiempirical and high-level DFT (B3LYP/6-31++G**) calculations have been used to shed light on the electronic na ture of the C(8) amide derivatives and their absorption properties. Table 4-5. Electronic Structure Data for the DA Purine Amide Derivatives Compared to the Corresponding Nitriles and Esters.a Purineb g HOMO calcd (eV) LUMO calcd (eV) E calcd (eV) E opticalc (eV) max La calcd (nm, [eV]), f d max La CH2Cl2 (nm) 2-APCONHMee 0.78 .16 .84 4.32 N.D.f 326, 0.53 N.D.f 2.6c-Me 5.19 .24 .04 4.20 3.4 331, 0.58 328 2.6b-Me 5.45 .52 .27 4.25 3.5 328, 0.57 326 4.3-Me 3.36 .36 .25 4.11 3.3 344, 0.57 338 2.10c-Me 2.81 .40 .48 3.92 3.2 354, 0.61 348 2.10b-Me 5.52 .64 .60 4.04 3.3 344, 0.62 348 4.7-Me 2.88 .47 .33 4.14 N.D.f 339, 0.57 N.D.f 2.12c-Me 3.16 .53 .56 3.97 3.2 348, 0.61 338 2.12b-Me 5.66 .75 .68 4.07 3.4 339, 0.61 336 a See the Experimental (Chapter 2)section for computational details. b All benzyl groups have been replaced by methyl groups for the calculations. c Calculated as the end-absorption of the lowest energy transition in the UVvis spectrum. d All correspond to one-electron HOMO (S0) LUMO (S1) transitions. e One imaginary frequency was identified for this structure indicating a transition stat e. The frequency is associated with rotation around the amide methyl group based on analysis of the coordinate files.f Not determined.

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104 Ground state geometries, dipoles, and orbi tal energies have been obtained for 4.3-Me 4.7-Me and the parent 2-AP derivative bearing a CONH Me group in the C(8) position (Me on N(9)), 2AP-CONHMe (structure not shown). In all cases, th e amide torsion angle used for the starting structures was that found in the Figure 4-11 X-ra y structure. Summarized in Table 4-5 are the dipole and orbital energy data obtained from the DFT calculations. Complementary orbital density plots of the HOMO and LU MO levels (Molden v. 4.6) to s how graphically the electronic nature of the ground state are shown in Figure 4-15. The HOMO consists of delocalized p orbitals on the purine plane, with the greatest density in the N9-C4-C5-C6 region consistent with the nitriles and esters. Localized pz orbitals are seen on the exocyclic donor heteroatoms and the accep tor functionality; the degree of charge separation in the HOMO is similar to that of the esters. Interestingly, some orbital density is noted on the amide nitrogen atom that is likely re lated to interaction with N(7). The LUMO is again concentrated on the acceptor portion, C(8) and the amide. The density on the C(2) donor group diminishes quite dramatically in the LU MO; the C(6) donor orbital coefficient is only slightly affected. In general the amides have s lightly higher HOMO energi es than the esters, but more significantly increased (~0.2 eV) LUMO en ergies (resulting in overall larger HOMOLUMO gaps). The LUMO energy may be aff ected by the intramolecular H-bonding interaction between the amide and N(7). ZINDO/S CI semi-empir ical calculations, as before, reveal that the lowest energy transition is associated with promotion of an electron from the HOMO to the LUMO in all cases. Although the experimental data is limited, the energy of the long wavelength transition correlates well with max in CH2Cl2. Based on the plots in Figure 4-15, the transition mainly involves transf er of charge from the amino group on C(2) (and the pyrimidine ring) to the C(8) acceptor group.

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105 Figure 4-15. Orbital density plots for D-A pur inyl amide derivatives calculated from the B3LYP/6-31++G** optimized geometries (using Molden v. 4.6). Amide Functionalization and Boc prot ection of the Primary Amine Donors Amide Formation at C(2) of the Purine Core Discussed in the introduction, also worth ex ploring is functionalization of the NH2 group on C(2) or C(6); we focused initially on acyla tion at this position due to its relevance to generating oligopeptide architec tures. A simple acylation reaction was explored by adding benzoyl chloride to compound 2.7 (Figure 4-16). The C(2) am ine was found to quickly react with this relatively active aci d chloride to produce amide 4.8 in reasonable yield. This result is consistent with any number of similar reactions from the literature that show acylation of 2aminopurine derivatives with acid chlorides114,115 and acetic anhydride.116

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106 N N N N Bn H2N N N N N N Bn N H N O TEA THF 70% Cl O + 2.74.8 Figure 4-16. Amide formation on the C(2) amine via an acid chloride. Milder coupling reagents were explored for C(2) amide formation to obviate HCl generation associated with the use of acid chlorides that could lead to epimerization of -amino acid sequences that might later be introduce d. Reactions using DCC/DMAP in methylene chloride or EDCI/HOBt/DMAP in DMF proved unsuccessful for coupling compound 2.7 with an aliphatic carboxylic acid, butyric acid. An alternative peptide coupling condition, HOAt/EDCI/amine base in DMF, has been found to successfully couple deactivated amines and would be worth trying in the future.117 If no mild coupling conditi ons are successful for C(2) amide formation, an acid chloride can be genera ted from the Fmoc-protected glycine to react with the C(2) amine. Once the amide on C(2) is formed, milder reagents can be used to form subsequent peptide bonds.117 Boc Protection of the C(2) and C(6) Primary Amines Along the same lines, employment of the purines described thus far as amino acid building blocks requires some investigation of their protection and deprotection chemistry. This was initially investigated using th e conventional Boc protecting group. The C(2) and C(6) primary amines on the C(8)-H (not shown) and the C(8)-B r purines could be successfully protected with Boc groups under mild conditions (Figure 4-17). The reaction conditions do require four equivalents of (Boc)2O (and a catalytic amount of DMAP in THF) since the reagent reacts twice with each primary amine (a surprising result give n steric considerations). Similar observations have been reported for purines118 and confirmed here by MS and 1H NMR (absence of amide

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107 NH peak). That the reactions shown in figure 4-17 work, while the ones shown in figures 4-8 and 4-10 fail, further implicates the relatively unstable C(8) carboxylic acid function as the source of the previous difficulties. Unfortunately, the (Boc)2O reaction (same reaction conditions) could not be ex tended to the D-A purines 2.7b and 2.7c no desired product was obtained and the D-A purines appeared to decompose. N N N N Bn H2N R1 + O O O O N N N N Bn N R1 O Br O O O O Br DMAP THF 2.6a R1 = H 2.7a R1 = N(Me)22.8a R1 = OBn 4.9 R1 = H 4.10 R1 = N(Me)24.11 R1 = OBn 48% 64% 77% N N N N Bn N NH2 + O O O O N N N N Bn N N O Br Br DMAP THF O O O O 56% 2.12a4.12A B Figure 4-17. Boc protection of primary amines on the purine core. A) On the C(2) amine. B) On the C(6) amine. Urea Formation on the C(2) Amine: Buildin g Blocks for Self-assembled Structures Early in this thesis, reactiv ity at the C(2) amino group was evaluated through reaction with isocyanate electrophiles in work aimed at e xpanding the hydrogen-bondi ng abilities of the purinyl building blocks. The general design is shown in Figure 4-18. Formation of an intramolecular hydrogen bond between the urea h ydrogen and purine N(3) exposes a hydrogen bond donor-acceptor-donor-acceptor face capable of dimer formation by four hydrogen bonds. Since its conception, self-complementary qua druply hydrogen bonded complexes from ureafunctionalized 2,6-diaminopurines have been ex plored and published by the Castellano Group.119

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108 N N N N N Ph N N O Ph H H H N N N N N Ph N N O Ph H H H H H Figure 4-18. General design of self-complem entary quadruply hydrogen bonded dimers based on purines. To initiate this work, the author explored the general reactivity of 2.5 2.6 and 2.7 toward isocyanates (Figure 4-19). E ach purine bears only one reacti ve amino group (unlike what is required for dimerization as shown in Figure 4-18) to simp lify the studies. N N N N Bn H2N + N N N N Bn N H DMAP THF CO N N H O N N N N Bn H2N + N N N N Bn N H DMAP THF CO N N H O N N N N Bn H2N + N N N N Bn N H DMAP THF CO N N H O Cl Cl R R = Me, F R 4.13 R = Me 4.14 R = F 93% 98% 2.5 2.6 53% N R R = H, Me, EtO, F, Br 2.7 N R 4.16 R = H 4.17 R = Me 4.18 R = EtO 4.19 R = F 4.20 R = Br 94% 95% 75% 76% 42% 4.15A B C Figure 4-19. Urea formation using the C(2) primary amine.

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109 The purinyl urea compounds were formed smoo thly upon treating 2-aminopurine derivatives with aryl isocyanate derivatives in pyridine/DMA P. Most of the reactions gave reasonable to good product yields. Urea compounds formed from compound 2.5 can in theory be treated with methanolic ammonia (that c onverts C(6)-Cl to C(6)-NH2) to produce self-complementary hydrogen bonded dimers (Figure 4-18), wh ile the ureas formed from compounds 2.6 and 2.7 served as model compounds for 1H NMR chemical shift comparis on. The solubility of ureas 4.13 4.20 did, however, prove to be quite low in solv ents that were critical for examination of their hydrogen bonding properties, li ke chloroform. Alkyl ureas were envisioned as an easy alternative to increase solubi lity, however, no reaction was f ound to occur between the purine C(2) amine and alkyl isocyanates under the reac tion conditions shown in figure 4-19. Efforts in the Castellano Group have since circumvented these reactivity issu es through elevated temperatures and direct de protonation of the amine. Critical Crystal Structure of Ureidopurine 4-16 Single crystals of 4.16 suitable for X-ray analysis, perfomed by Dr. Khalil Abboud, could be grown by slow evaporation (two weeks) from pyridine. The structure of the compound as determined in the solid state by X-ray crystallography is shown in figure 4-20.119 It critically reveals that intramolecular hydrogen bonding betw een the urea hydrogen on N(12) with N(3) (N(12)N(3) = 2.74 ) is accessible through a pl anar arrangement (despite the bulky N(9) benzyl substituent). There is also a near edge-to-face relationship between the aromatic substituents with the angle betw een the least squares planes on the aromatic rings equal to 86.3 and the closest carbon-carbon dist ance equal to 3.67 The dimethylamino substituent, defined by atoms N(19), C(20), and C(21), is largely planar but slightly twisted (~ 9) around the C(6)N(19) bond. Consistent with the solid-state data for 4-16 the chemical shift of its urea Hb proton in CDCl3 (~ 5 mM) is significantly deshield ed to 11.4 ppm (relative to TMS); Ha appears at ~ 7.2

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110 ppm. This conformation is identical to the one adopted by C(6)-amino (rather than dimethylamino) derivatives upon dimer form ation in solution and the solid state. Figure 4-20. Intramolecular hydrogen bonding of 4-16 in the solid state (ellipsoids drawn at the 50% probability level). The side view of a CPK representation shows the extent of contact achievable for the two phenyl ri ngs in the N(3) H-bonded conformer. Conclusion and Future Directions In conclusion, donor-acceptor (D-A) purines have been prepared, many of which have unity or near unity quantum yields in organic solution. Many of these compounds, especially the C(8)-COOMe purines, expressed more sensitiv ity to their solvent environment than 2aminopurine and other nucleobase analogues used for biological studies. Future work will include making and studying the photophysical properties of N(9) sugar functionalized derivatives of 2.6b and 2.6c along with new D-A compounds (Fi gure 4-21) that are water soluble and have the ability to base pair with natural nucleobases. X-ray crystal structures revealed that simple changes in the functionality of C(2), C(6), and C(8) dramatically changed the -stacking and hydrogen bonding of th e purines in the solid state. Future work in this area will include making lu minescent purine derivatives and evaluating their supramolecular properties. Some exampl es are shown in figures 4-21 and 4-22a,b.

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111 N N N N N H R X N N N N N H R X HN N N N N N H R X N N N N N N R NN O O R H H X N N N O H H R H N N N N N R X H H O H N N N O H H R N N N N N R X H H O H X = CN X = COOMe R = SugarA B C Figure 4-21. Design of donor-accep tor purines to be prepared for future photophysical studies. N N N N N R H O H N N N N N R H O H N N H H H H N N N N N R H O H N H H N N N N N R H O H N H H N N N N N R H O H N H H N N N N N R H O H N H H N N N N N R H O H N H H Figure 4-22. Potential supramolecular stru cture of an C(8)-amide adenine analogue.

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112 N N N N N R H O R N H H N N N N N R H O R N H H N N N N N R H O R N H H N N N N N R H O R N R H N N N N N R H O H N H H N N N N N R H O H N H H N N N N N R H O H N H H N N N N N R H O H N H H A B Figure 4-23. Potential supramolecular structur e of C(8)-amide 2-aminopurine derivatives. Figure 4-22 shows a C(8)-amide functionalized adenine forming a seven-membered ring directed by hydrogen bonding interactions. Figu re 4-23a,b shows C(8)-amide functionalized 2aminopurine forming two different ribbon structures directed by the N(9) functionality. When N(9) is functionalized, hydrogen bonding can poten tially direct the formation of the structure presented in figure 4-23a, and then a much di fferent ribbon (Figure 4-23 b) can be envisioned when N(9) is hydrogen. Compound 2.7b was found to emit light when in corporated (using a spin casting technique) into an OLED device. Work will continue with device exploration using the D-A purines as the emissive layer. The performan ce of the C(8)-COOMe and C(8)-amide derivatives will be investigated. Vapor deposited films of th e D-A compounds will also be tested in devices. Successful C(8)-amide functionalization of the D-A purines opens the potential for future work involving the development of homoand hete ro-oligopeptides discusse d at the beginning of

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113 this chapter. Synthetic stra tegies will be continued on progression toward C(2)-amide functionalization and oligopeptide formation. Experimental Section Synthesis of Compounds General Method A (Boc protection of C(2) or C(6) primary amine) (Boc)2O (4 equiv), DMAP (0.1 equiv), and a purine containing a primary amine were dissolved in dry THF (50 mL). The solution was stirred overnight, concentrated under reduced pressure, and the desired product was obtained by flash chro matography (40% EtOH/hexanes). N N N N N N HN O 2,6-Bis(dimethylamino)-8-butylam ide-9-benzyl purine (4.3). Compound 2.10b (0.10 g, 0.31 mmol) was heated overnight at 100 C w ith EtOH (25 mL) and 10% aqueous NaOH (25 mL). The ethanol was then removed under reduce d pressure and the solu tion, once cooled to 0 C, was acidified to a pH of 2. The solution was then extracted with cold ethyl acetate (100 mL x 5). The combined organic layers were drie d with magnesium sulfate and concentrated under reduced pressure with the soluti on kept below 20 C at all times. The white solid that resulted was dried under high vacuum for 15 min. The solid was then dissolved in cold, dry CH2Cl2 and added to butylamine (0.013 g, 0.18 mmol) in dry CH2Cl2 (150 mL) at 0 C. DCC (0.040 g, 0.19 mmol) and DMAP (0.024 g, 0.19 mmol) were then a dded and the reaction was stirred at room temperature overnight. The reaction was filte red, the solvent was removed under reduced pressure, and the crude solid was purified by co lumn chromatography (40%, EtOAc/hexanes) to yield a white solid (0.052 g, 42% from 2.10b ): m.p. 112 C. 1H NMR (300 MHz, DMSO-

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114 d6) 0.88 (t, 3H, J = 7.2 Hz), 1.27 (m, 2H), 1.47 (m, 2H ), 3.11 (s, 6H), 3.38 (q, 2H, J = 7.2), 3.45 (bs, 6H), 5.65 (s, 2H), 7.29 (m, 5H), 8.46 (t, 1H, J = 7.2 Hz). 13C NMR (75 MHz, DMSOd6) 13.67, 19.56, 31.29, 36.78, 38.18, 45.88, 111.98, 127.22, 127.66, 128.25, 136.44. 138.06, 149.96, 154.20, 154.49, 159.00, 159.15. HRMS calcd for C21H30N7O [M+H]+: 396.2512, found: 396.2506. N N N N N N HN O O O 2,6-Bis(dimethylamino)-8-carbonyl-amino-a cetic acid methyl ester-9-benzylpurine (4.5). Compound 2.10b (0.10 g, 0.31 mmol) was heated overni ght at 100 C with EtOH (25 mL) and 10% aqueous NaOH (25 mL). The ethanol was then removed under reduced pressure and the solution once cooled to 0 C wa s acidified to a pH of 2. The solution was then extracted with cold ethyl acetate (100 mL x 5). The combined organic layers were dried with magnesium sulfate and concentrated under redu ced pressure with the solution ke pt below 20 C at all times. The white solid that resulted was dried under high vacuum for 15 min. The solid was then dissolved in cold, dry CH2Cl2 and added to glycine methyles ter HCl (0.037 g, 0.29 mmol) in dry CH2Cl2 (150 mL) at 0 C. DCC (0.067 g, 0.32 mmol) and DMAP (0.08 g, 0.7 mmol) were then added and the reaction was stirre d at room temperature overnight The reaction was filtered, the solvent was removed under reduced pressure, and the crude solid wa s purified by column chromatography (40%, EtOAc/hexanes) to yield a white solid (0.10 g, 82% from 2.10b ): m.p. 133 C. 1H NMR (300 MHz, CDCl3) 3.30 (s, 6H), 3.46 (bs, 6H ), 3.78 (s, 3H), 4.19 (d, 2H, J = 5.6 Hz), 5.75 (s, 2H), 7.23 (m, 3H), 7.46 (m, 2H), 7.75 (t, 1H, J = 5.6 Hz). 13C NMR (75 MHz, CDCl3) 37.30, 38.27, 40.83, 46.72, 52.38, 113.27, 127.30, 128.23, 128.40, 135.40,

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115 138.00, 155.13, 159.55, 170.23. HRMS calcd for C20H26N7O3 [M+H]+: 412.2097, found: 412.2088. N N N N N N HN O O O 2,6-Bis(dimethylamino)-8-pentanoic amide methyl ester-9-benzyl purine (4.6). Compound 2.10b (0.10 g, 0.31 mmol) was heated overnight at 100 C with EtOH (25 mL) and 10% aqueous NaOH (25 mL). The ethanol was then removed under redu ced pressure and the solution once cooled to 0 C wa s acidified to a pH of 2. The solution was then extracted with cold ethyl acetate (100 mL x 5). The combined organic layers were dried with magnesium sulfate and concentrated under redu ced pressure with the solution ke pt below 20 C at all times. The white solid that resulted was dried under high vacuum for 15 min. The solid was then dissolved in cold, dry CH2Cl2 and added to methyl 5-aminova lurate (0.049 g, 0.29 mmol) in dry CH2Cl2 (150 mL) at 0 C. DCC (0.067 g, 0.32 mmol) and DMAP (0.08 g, 0.65 mmol) were then added and the reaction was stirre d at room temperature overnight The reaction was filtered, the solvent was removed under reduced pressure, and the crude solid wa s purified by column chromatography (40 %, EtOAc/hexanes) to yield a white solid (0.063g, 45% from 2.10b ): m.p. 85 C. 1H NMR (300 MHz, CDCl3) 1.70 (m, 4H), 2.37 (t, 2H, J = 6.9 Hz), 3.21 (s, 6H), 3.41 (q, 2H, J = 6.6 Hz), 3.47 (s, 6H), 3.68 (s, 3H), 5.81 (s, 2H), 7.24 (m, 3H), 7.37 (t, 1H, J = 6.3 Hz), 7.51 (m, 2H). 13C NMR (75 MHz, DMSOd6) 21.78, 28.53, 32.85, 36.75, 38.02, 45.87, 51.11, 111.98, 127.16, 127.65, 128.20, 136.34, 138.00, 154.18, 154.47, 159.04, 159.13, 173.20. HRMS calcd for C23H31N7O3 [M+H]+: 454.2561, found: 454.2597.

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116 N N N N N NH2 O HN O O [(6-Amino-9-benzyl-2-dimethylamino-9 H -purine-8-carbonyl)-amino]-acetic acid methyl ester (4.7). Compound 2.12b (0.10 g, 0.34 mmol) was heated overnight at 100 C with EtOH (25 mL) and 10% aqueous Na OH (25 mL). The ethanol was then removed under reduced pressure and the solution once cooled to 0 C wa s acidified to a pH of 2. The cold acidified solution was then extracted with cold ethyl acet ate (100 mL x 5). The combined organic layers were dried with magnesium sulfate and concentr ated under reduced pres sure with the solution kept below 20 C at all times. The white solid that resulted was dried under high vacuum for 15 min. The solid was then dissolved in cold, dry CH2Cl2 and added to glycine methylester HCl (0.040 g, 0.32 mmol) in dry CH2Cl2 (150 mL) at 0 C. DCC (0.079 g, 0.38 mmol) and DMAP (0.09 g, 0.77 mmol) were then added and the reaction was stirred at room temperature overnight. The reaction was filtered, the solvent was rem oved under reduced pressure, and the crude solid was purified by column chromatography (40%, Et OAc/hexanes) to yield a white solid (0.066 g, 51% from 2.12b ): m.p. 170 C. 1H NMR (300 MHz, DMSOd6) 3.11 (s, 6H), 3.65 (s, 3H), 4.04 (d, 2H, J = 6.3 Hz), 5.62 (s, 2H), 6.97 (s, 2H), 7.29 (m, 5H), 8.55 (t, 1H, J = 6.3 Hz). 13C NMR (75 MHz, DMSOd6) 36.91, 40.63, 46.04, 51.80, 111.79, 127.27, 127.74, 128.28, 136.77, 137.80, 153.23, 156.32, 159.40, 160.19, 160.19, 169.93. HRMS calcd for C18H21N7O3 [M+H]+: 384.1779, found: 384.1788.

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117 N N N N N H N O N-(9-Benzyl-6-dimethylamino-purin-2-yl)-benzamide (4.8). Benzoyl chloride (0.12 g, 0.82 mmol) was added dropwise to a solution of compound 2.7 (0.20 g, 0.75 mmol) and triethylamine (0.75 g, 0.75 mmol) in dry THF (100 mL). The reacti on was stirred at rt for 2 h, was concentrated under reduced pressure, and purified by flash chro matography (40% EtOAC/ Hexanes) to yield an off-white fo amy solid (0.19 g, 70%): m.p. 55 C. 1H NMR (300 MHz, DMSOd6) 3.35 (s, 6H), 5.33 (s, 2H), 7.23 (m, 5H), 7.47 (m, 3H), 7.90 (d, 2H), 8.16 (s, 1H), 10.40 (s, 1H). 13C NMR (75 MHz, DMSOd6) 37.59, 45.86, 116.57, 127.44, 127.61, 127.91, 128.10, 128.59, 131.38, 135.06, 137.16, 139.23, 151.39, 152.36, 154.26, 165.76. C21H20N6O [M+H]+: 373.1771, found: 373.1801. N N N N O O N O O Br 2-Di-tert-butoxycarbonylamine-8-bromo-9-benzylpurine (4.9). Compound 2.6a (0.21 g, 0.66 mmol) was reacted under the conditions of general method A to yield a white solid (0.16 g, 48%). 1H NMR (300 MHz, DMSOd6) 1.31 (s, 18H), 5.50 (s, 2H), 7.20 (m, 2H), 7.32 (m, 3H), 9.19 (s, 1H). 13C NMR (75 MHz, DMSOd6) 28.0, 47.6, 83.6, 127.6, 128.8, 129.5, 132.9, 135.9, 136.9, 148.7, 151.0, 152.8, 153.8. HRMS calculated for C22H27BrN5O4 [M+H]+: 504.1241, found: 504.1241.

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118 N N N N N O O N O O Br 2 -Di-tert-butoxycarbonlyamine-6-dimethyl amino-8-bromo-9-benzylpurine (4.10) Compound 2.7a (0.20 g, 0.58 mmol) was reacted under the conditions of general method A to yield a white solid (0.35 g, 64%). 1H NMR (300 MHz, CHCl3) 1.41 (s, 18H), 3.45 (bs, 6H), 5.35 (s, 2H), 7.28 (m, 5H). 13C NMR (75 MHz, CHCl3) 28.1, 38.6, 47.5, 82.8, 119.1, 125.1, 127.9, 128.2, 128.9, 135.7, 151.5, 152.7, 153.3, 154.4. HRMS calculated for C24H32BrN6O4 [M+H]+: 548.1692, found: 548.1701. N N N N O O O N O O Br 2-Di-tert-butoxycarbonylamine-6-ben zyloxy-8-bromo-9-benzylpurine (4.11) Compound 2.8a (0.38 g, 0.93 mmol) was reacted under the conditions of general method A to yield a white solid (0.44 g, 77%). 1H NMR (300 MHz, DMSOd6) 1.32 (s, 18H), 5.44 (s, 2H), 5.60 (s, 2H), 7.38 (m, 10H). 13C NMR (75 MHz, DMSOd6) 27.1, 27.3, 47.2, 68.4, 82.9, 119.2, 126.9, 128.0, 128.4, 128.5, 128.8, 131.7, 135.3, 135.7, 150.1, 153.8, 159.0. HRMS calculated for C28H32BrN5O5 [M+H]+: 610.1660, found: 610.1660.

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119 N N N N N Br N O O O O 2-Di-tert-butoxycarbonlyamine-6-dimethyl amino-8-bromo-9-benzylpurine (4.12) Compound 2.12a (0.20 g, 0.58 mmol) was reacted under the conditions of general method A to yield a white solid (0.31 g, 56%). 1H NMR (300 MHz, DMSOd6) 1.38 (s, 18H), 3.13 (s, 6H), 5.32 (s, 2H), 7.33 (m, 5H). 13C NMR (75 MHz, DMSOd6) 27.9, 47.3, 83.8, 120.4, 127.9, 128.0, 128.6, 129.0, 129.5, 136.5, 148.9, 150.6, 156.3, 159.2. HRMS calculated for C24H32BrN6O4 [M+H]+: 547.1663, found: 547.1701. N N N N N H N H O Cl 1-(9-Benzyl-6-chloro-purin-2-yl)-3p -tolyl-urea (4.13) Compound 2.5 (0.10 g, 0.39 mmol) and p -tolylisocyanate (0.077 g, 0.58 mmol) were disso lved in dry pyridine and stirred at rt. The reaction was monitored by TLC until comple te (no longer than 24 h). The pyridine was removed under reduced pressure and the crude pr oduct was purified by recrystallization in EtOH to yield a white solid (0 .14 g, 93%): m.p. 204 C. 1H NMR (300 MHz, DMSOd6) 2.25 (s, 3H), 5.55 (s, 2H), 7.09 (m, 2H), 7.29 (m, 7H), 8.62 (s, 1H), 10.34 (s, 1H), 10.68 (s, 1H). 13C NMR (75 MHz, DMSOd6) 20.3, 46.9, 119.4, 126.4, 127.0, 127.9, 128.3, 128.8, 129.2, 132.0, 135.7, 135.8, 146.1, 149.8, 150.8, 152.8. HRMS calcd for C19H16N6O [M+H]+: 393.1225, found: 393.1261.

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120 N N N N N H N H O F Cl 1-(9-Benzyl-6-chloro-purin-2-yl )-3-(4-fluoro-phenyl)-urea (4.14). Compound 2.5 (0.10 g, 0.39 mmol) and 4-fluorophenylisoc yanate (0.42 g, 3.1 mmol) were dissolved in dry pyridine (7 mL) and stirred at rt. The reaction was monitored by TLC until complete (no longer than 24 h). The pyridine was removed under reduced pre ssure and the crude product was purified from recrystallization in EtOH to yiel d a white solid (0.150 g, 98%). 1H NMR (300 MHz, DMSOd6) 5.54 (s, 1H), 7.16 (m, 2H), 7.35 (m, 7H), 8.60 (s, 1H), 10.34 (s, 1H ), 10.69 (s, 1H). 13C NMR (75 MHz, DMSOd6) 46.9, 115.2, 115.5, 121.2, 126.5, 127.1, 127.9, 128.8, 134.6, 135.8, 146.2, 149.8, 150.9, 152.0, 152.8. HRMS calcd for C19H15N6O [M+H]+: 397.0980, found: 397.0992. N N N N N H N H O 1-(9-Benzyl-9H-purin-2-yl)-3-phenyl-urea (4.15). Compound 2.6 (0.10 g, 0.44 mmol) and phenylisocyanate (0.85 g, 7.1 mmol) were dissolved in dry pyridine (7 mL ) and stirred at rt. The reaction was monitored by TLC until complete (no longer than 24 h). The pyridine was removed under reduced pressure and the crude product was purified from recrystallization in EtOH to yield a white solid (0.081 g, 53%): m.p. 260 C. 1H NMR (300 MHz, DMSOd6) 5.51 (s, 2H), 7.04 (m, 3H), 7.33 (m, 7H), 7.47 (m, 2H), 8.57 (s, 1H), 9.04 (s, 1H), 10.13 (s, 1H), 11.36 (s, 1H). 13C NMR (75 MHz, DMSOd6) 46.2, 119.3, 122.9, 127.2, 127.9, 128.3, 128.8, 129.4, 136.2, 138.5, 146.0, 148.7, 151.4, 151.7, 153.6. HRMS calcd for C19H17N6O [M+H]+: 345.1464, found: 345.1471.

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121 N N N N N H N H O N 1-(9-Benzyl-6-dimethylamino-pur in-2-yl)-3-phenyl-urea (4.16) Compound 2.7 (0.10 g, 0.37 mmol) and phenylisocyanate (0 .65 mL, 6.0 mmol) were dissolv ed in dry pyridine (7 mL) and stirred at rt. The reaction was monitored by TLC until complete (no longer than 24 h). The pyridine was removed under reduced pressure and the crude product was purified from recrystallization in EtOH to yield a white solid (0.137 g, 94%): m.p. 234 C. 1H NMR (300 MHz, DMSOd6) 3.62 (bs, 6H), 5.40 (s, 2H), 7.28 (m 10H), 8.13 (s, 1H), 9.41 (s, 1H), 11.45 (s, 1H). 13C NMR (75 MHz, DMSOd6) 46.1, 60.0, 115.46, 119.17, 122.7, 126.8, 126.9, 127.6, 128.7, 128.8, 136.8, 138.7, 138.9, 151.8, 153.0, 154.1. HRMS calculated for C21H22N7O [M+H]+: 388.1886, found: 388.1921. N N N N N H N H O N 1-(9-Benzyl-6-dimethyl amino-purin-2-yl)-3p -tolyl-urea (4.17) Compound 2.7 (0.10 g, 0.37 mmol) and p -tolylisocyanate (0.10 g, 0.75 mmol) we re dissolved in dry pyridine and stirred at rt. The reaction was monitored by TLC until complete (no longer than 24 h). The pyridine was removed under reduced pressure and the crude product was purified from recrystallization in EtOH to yield a wh ite solid (0.14 g, 95%): m.p. 204 C. 1H NMR (300 MHz, DMSOd6) 2.25 (s, 3H), 3.42 (bs, 6H), 5.41 (s, 2H), 7.07 (m, 2H), 7.27 (m, 7H), 8.14 (s, 1H), 9.34 (s, 1H), 11.38 (s, 1H). 13C NMR (75 MHz, DMSOd6) 21.0, 46.9, 116.2, 119.9,

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122 127.6, 128.4, 129.4, 129.9, 132.3, 136.8, 137.5, 139.6, 151.0, 152.5, 153.7, 154.8. HRMS calcd for C22H24N7O [M+H]+: 402.2042, found: 402.2061. N N N N N H N H O O N 1-(9-Benzyl-6-dimethylamino-purin-2 -yl)-3-(4-ethoxy-phenyl)-urea (4.18). Compound 2.7 (0.10 g, 0.37 mmol) and 4-ethoxyphenylisocyanate (0.091 g, 0.56 mmol) were dissolved in dry pyridine and stirred at rt. The reaction was monitored by TLC until complete (no longer than 24 h). The pyridine was removed under reduce d pressure and the crude product was purified from recrystallization in EtOH to yield a white solid (0.12 g, 75%). 1H NMR (300 MHz, CHCl3) 1.38 (t, 3H, J = 6.8 Hz), 3.48 (bs, 6H), 3.98 (q, 2H, J = 6.8 Hz), 5.27 (s, 2H), 6.82 (m, 2H), 7.24 (m, 8H), 7.59 (s, 1H), 11.18 (s, 1H). 13C NMR (75 MHz, CHCl3) 15.1, 47.4, 63.9, 115.1, 121.9, 127.6, 128.6, 129.3. HRMS calcd for C23H26N7O2 [M+H]+: 432.2142, found: 432.2164. N N N N N H N H O F N 1-(9-Benzyl-6-dimethylamino-purin-2 -yl)-3-(4-fluoro-phenyl)-urea (4.19) Compound 2.7 (0.10 g, 0.37 mmol) and 4-fluoro phenylisocyanate (0.10 g, 0.75 mmol ) were dissolved in dry pyridine (7 mL) and stirred at rt. The reacti on was monitored by TLC until complete (no longer than 24 h). The pyridine was removed under reduced pressure and the crude product was purified from recrystalli zation in EtOH to yield a white solid (0.064 g, 42%). 1H NMR (300 MHz, DMSOd6) 3.45 (bs, 6H), 5.42 (s, 2H), 7.14 (m, 2H), 7.33 (m, 7H), 8.14 (s, 1H), 9.45 (s,

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123 1H), 11.46 (s, 1H). 13C NMR (75 MHz, DMSOd6) 46.1, 115.1, 115.42, 120.9, 121.0, 126.8, 127.6, 128.7, 135.0, 136.8, 138.9, 150.2, 151.9, 152.9, 154.1. HRMS calcd for C21H21FN7O [M+H]+: 406.1792, found: 406.1789. N N N N N H N H O Br N 1-(9-Benzyl-6-dimethylamino-purin-2 -yl)-3-(4-bromo-phenyl)-urea (4.20). Compound 2.7 (0.10 g, 0.37 mmol) and 4-bromophenylisocyanate (0.11 g, 0.56 mmol) were dissolved in dry pyridine and stirred at rt. The reaction was monitored by TLC until complete (no longer than 24 h). The pyridine was removed under reduced pre ssure and the crude product was purified from recrystallization in EtOH to yiel d a white solid (0.13 g, 76%). 1H NMR (300 MHz, DMSOd6) 3.42 (bs, 6H), 5.43 (s, 2H), 7.30 (m, 9H), 7.15 (s, 1H), 9.53 (s, 1H), 11.54 (s, 1H). 13C NMR (75 MHz, DMSOd6) 46.1, 114.1, 115.5, 121.1, 126.7, 127.6, 128.7, 131.5, 136.8, 138.03, 139.0, 150.2, 151.7, 152.8, 154.1. HRMS calcd for C22H24N7O [M+H]+: 466.0985, found: 466.0978. X-ray Crystallography General. Data were collected at 173 K on a Siemens SMART PLATFORM equipped with a CCD area detector and a grap hite monochromator utilizing MoK radiation ( = 0.71073 ). Cell parameters were refined using up to 8192 reflections. A full sphere of data (1850 frames) was collected using the -scan method (0.3 frame width). The first 50 frames were remeasured at the end of data collection to mon itor instrument and crys tal stability (maximum correction on I was < 1 %). Absorption corre ctions by integration were applied based on measured indexed crystal faces. The struct ure was solved by the Direct Methods in SHELXTL6, and refined using full-matrix least squares. The non-H atoms were treated anisotropically,

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124 whereas the hydrogen atoms were calculated in ideal positions and were riding on their respective carbon atoms. Compound 4.7 The NH2 protons were obtained from a Difference Fourier map and refined without any constraints. A total of 268 parameters were refined in the final cycle of refinement using 3472 reflections with I > 2 ( I ) to yield R1 and w R2 of 4.42% and 11.01%, respectively. Refinement was done using F2. Compound 4.16 A total of 272 parameters were refi ned in the final cycle of refinement using 3194 reflections with I > 2 ( I ) to yield R1 and w R2 of 3.74% and 9.66%, respectively. Refinement was done using F2.

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125 APPENDIX A 1H NMR SPECTRA OF SELECTED PURINES Figure A-1. 1H NMR of 2.7 in CHCl3. Figure A-2. 1H NMR of 2.10 in CHCl3.

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126 Figure A-3. 1H NMR of 2.12 in DMSOd6. Figure A-4. 1H NMR of 2.14 in DMSOd6.

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127 Figure A-5. 1H NMR of 2.7a in CHCl3. Figure A-6. 1H NMR of 2.8a in CHCl3.

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128 Figure A-7. 1H NMR of 2.10a in CHCl3. Figure A-8. 1H NMR of 2.12a in CHCl3.

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129 Figure A-9. 1H NMR of 2.14a in DMSOd6. Figure A-10. 1H NMR of 2.6b in DMSOd6.

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130 Figure A-11. 1H NMR of 2.7b in DMSOd6. Figure A-12. 1H NMR of 2.8 in DMSOd6.

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131 Figure A-13. 1H NMR of 2.10b in CHCl3. Figure A-14. 1H NMR of 2.12b in DMSO-d6.

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132 Figure A-15. 1H NMR of 2.14b in CHCl3. Figure A-16. 1H NMR of 2.6c in CHCl3.

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133 Figure A-17. 1H NMR of 2.7c in CHCl3. Figure A-18. 1H NMR of 2.8 in CHCl3.

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134 Figure A-19. 1H NMR of 2.10c in CHCl3. Figure A-20. 1H NMR of 2.12c in DMSOd6.

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135 Figure A-21. 1H NMR of 2.14c in DMSOd6. Figure A-22. 1H NMR of 4.3 in DMSOd6.

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136 Figure A-23. 1H NMR of 4.5 in CHCl3. Figure A-24. 1H NMR of 4.6 in CHCl3.

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137 Figure A-25. 1H NMR of 4.7 in DMSOd6. Figure A-26. 1H NMR of 4.8 in DMSOd6.

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138 APPENDIX B X-RAY CRYSTAL STRUCTURE DATA Table B-1. Crystal Structure Parameters. Parameters 2.7b 2.12b 2.12c 4.7 4.16 Empirical formula C15H15N7 C15H15N7 C16H18N6O2 C18H21N7O3 C21H21N7O Formula weight 293.34 293.34 326.36 383.42 387.45 Temperature K 173(2) 173(2) 173(2) 173(2) 173(2) Wavelength 0.71073 0.71073 0.71073 0.71073 0.71073 Crystal system Triclinic Monoclinic Triclinic Monoclinic Monoclinic Space group P-1 P21/n P-1 P21/n P2(1)/n Unit cell dimensions a=8.5576(6) =79.739(1) b=9.1964(7) =74.878(1) c=10.2811(7) =65.164(1) a=10.0863(8) =0 b=8.6914(7) =98.137(2) c=16.9812(14) =90 A=6.4306(13) =84.052(4) b=10.050(2) =84.765(3) c=11.980(2) =88.383(4) a=10.6415(8) =90 b=13.5541(10) = 107.01(1) c=13.0430(9) =90 a=9.0629(6) =90 b=19.8629(1 =106.862(1) c=11.3182(7) =90 Volume 3 706.68(9) 1473.7(2) 766.7(3) 1799.0(2) 1949.9(2) Z 2 4 2 4 4 Density (calculated) Mg/m 3 1.379 1.322 1.414 1.416 1.320 Absorption coefficient mm -1 0.090 0.087 0.099 0.101 0.087 F(000) 308 616 344 808 816 Crystal size mm 3 0.19 x 0.18 x 0.15 0.21 x 0.19 x 0.15 0.13 x 0.09 x 0.07 0.32 x 0.08 x 0.06 0.19 x 0.18 x 0.17 Theta range for data collection 2.06 to 27.49 2.22 to 27.50 1.72 to 27.50 2.18 to 27.49. 2.05 to 27.49 Index ranges -11 h 9, 11 k 8, 13 l 12 -13 h 13, -10 k 11, 10 l 21 -8 h 8, 7 k 13, 13 l 15 -13 h 13, 15 k 17, 16 l 16 -11 h 10, 25 k 20, 14 l 14 Reflections collected 4656 9095 5277 12006 12603

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139 Table B-1. Continued Independent reflections 3099 [R(int) = 0.0258] 3304 [R(int) = 0.0615] 3445 [R(int) = 0.0248] 4125 [R(int) = 0.0418] 4406 [R(int) = 0.0337] Completeness to theta = 27.49 95.4 % 97.6 % 97.7 % 99.7 % 98.5 % Absorption correction Integration Integration Integr ation Integration Integration Max. and min. transmission 0.9894 and 0.9819 0.9895 and 0.9803 0.9931 and 0.9873 0.9941 and 0.9756 0.9943 and 0.9800 Refinement method Full-matrix least-squares on F 2 Full-matrix least-squares on F 2 Full-matrix least-squares on F 2 Full-matrix least-squares on F 2 Full-matrix least-squares on F 2 Data / restraints / parameters 3099 / 0 / 208 3304 / 0 / 207 3445 / 0 / 227 4125 / 0 / 268 4406 / 0 / 272 Goodness-offit on F 2 1.032 1.048 1.029 1.064 1.063 Final R indices [I>2sigma(I)] R1 = 0.0383, wR2 = 0.1027 [2624] R1 = 0.0475, wR2 = 0.1200 [2770] R1 = 0.0392, wR2 = 0.0982 [2572] R1 = 0.0442, wR2 = 0.1101 [3472] R1 = 0.0374, wR2 = 0.0966 [3194] R indices (all data) R1 = 0.0450, wR2 = 0.1065 R1 = 0.0569, wR2 = 0.1269 R1 = 0.0550, wR2 = 0.1046 R1 = 0.0545, wR2 = 0.1162 R1 = 0.0572, wR2 = 0.1038 Largest diff. peak and hole e. -3 0.229 and 0.266 0.275 and 0.228 0.200 and 0.228 0.304 and 0.206 0.185 and 0.176

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147 BIOGRAPHICAL SKETCH Roslyn Susanne Butler was born in 19 78 in Elizabethtown, KY. After growing up in South Central Kentucky, she graduated in 2001 with a B.S. in chemistry from Western Kentucky University, where she worked in th e lab of Prof. Robert Holman. During her undergraduate education she also briefly worked in the lab of Prof. Spiro Alexandratos at the University of Tennessee, and as a park ranger at Mammoth Cave National Park. After working on her M.S. in organic chemistry for a year at Western Kentucky Univers ity, she then moved to Gainesville, FL in 2002 to pursue her Ph.D. in or ganic chemistry at the University of Florida under the guidance of Prof. Ronald K. Castella no. In August 2007 she will join the teaching faculty at Marian High School in Mishaw aka, IN to teach chemistry and biology.