The Total Synthesis of Hexavalent Glycodendrimers

THE TOTAL SYNTHESIS OF HEXAVALENT GLYCODENDRIMERS

USING A DIVERGENT PATHWAY FOR

ANTI-HIV THERAPY

A Thesis

Presented to the faculty of the Department of Chemistry

California State University, Sacramento

Submitted in partial satisfaction of the requirements for the degree of

MASTER OF SCIENCE

Chemistry

Dustin Andres Dimas

SUMMER 2018

Dustin Andres Dimas

THE TOTAL SYNTHESIS OF HEXAVALENT GLYCODENDRIMERS

USING A DIVERGENT PATHWAY FOR

ANTI-HIV THERAPY

A Thesis

Dustin Andres Dimas

Approved by:

______, Committee Chair Dr. Katherine McReynolds

______, Second Reader Dr. Roy Dixon

______, Third Reader Dr. Cynthia Kellen-Yuen

______Date

iii

Student: Dustin Andres Dimas

I certify that this student has met the requirements for format contained in the University format manual, and that this thesis is suitable for shelving in the Library and credit is to be awarded for the thesis.

______, Graduate Coordinator ______Dr. Susan Crawford Date

Department of Chemistry

Abstract

THE TOTAL SYNTHESIS OF HEXAVALENT GLYCODENDRIMERS

USING A DIVERGENT PATHWAY FOR

ANTI-HIV THERAPY

Dustin Andres Dimas

The Human Immunodeficiency Virus (HIV) has caused a worldwide epidemic.

Currently, an estimated 36.9 million people are infected with HIV. The current treatment for HIV is antiretroviral therapy (ART). Around 20.9 million people living with HIV have access to ART therapy. Right now, only 57% of the people infected are currently using ART. As of now, ART can only slow the progression of the virus but doesn’t cure the disease. Additionally, ART can have toxic side effects or become ineffective over time through viral resistance. This is why finding a way to prevent new HIV infections is very important.

A promising new class of anti-HIV molecules are dendrimers. All dendrimers have common characteristics: A core, linkers that increase the number of ends, and terminal functional groups where the chemistry can occur. For glycodendrimers, the functional groups can include amino, carboxyl or aminooxy moieties. In our research, the dendrimers terminate in aminooxy groups, which can then react chemoselectively with sugars to yield oxime-linked glycodendrimers. As dendrimers and glycodendrimers have

globular structures with multiple ends, they can exhibit the multivalent effect.

Multivalency refers to the simultaneous interactions of multiple binding sites on one entity to multiple receptor sites on another. With this unique ability, sulfated glycodendrimers have been shown to bind to HIV virions and block fusion, and therefore the infection, of host cells. Our research is focused on the synthesis of multivalent glycodendrimers as HIV entry inhibitors.

In the present study, three diverse pathways were used to synthesize three hexavalent and one octavalent core. After completing each of the pathways, cellobiose or the dimer of colominic acid was used to create the desired terminated glycodendrimers.

The purpose of the first pathway was to create a flexible hexavalent core. This pathway began by synthesizing the linkers for the hexavalent core. The reaction involved the addition of an aminooxy group on one end of the diols, either diethylene glycol

(DEG) or 1,3-propandiol. Next, a methanesulfonyl group was added to the remaining OH to complete the linker synthesis. The second step in the process was to create a trivalent core to accept the linkers. Unfortunately, the linker would not react with the core so none of the desired product was obtained.

The next pathway explored increasing the valency of the core by using an octavalent dendrimer core. This second pathway began by synthesizing a new linker for the octavalent core. The first linker reaction involved the addition of an ether group to the linker. The second linker was synthesized to add the aminooxy group to the core. To make the tetravalent core, the mesylated linker was added to the core S under SN2 conditions. Next, a three-step process was used to obtain the desired amine-terminated

octavalent core. Finally, the addition of the aminooxy linker was added under carbamate coupling. Unfortunately, it was not possible to achieve the desired octavalent aminooxy- terminated dendrimer core, so no glycodendrimers could be made by this route.

This study entailed the determination of two failed and one effective route in synthesizing dendrimer cores. For the successful hexavalent dendrimer core, a longer linker was synthesized to add an ether group with a yield of 99.8%. Additionally, through the synthesis of this linker it was determined that purification methods of dialysis and

FPLC were all that was needed to achieve the best yield and time for purification with

68.2%. Finally, for both the cellobiose and dimer of colominic acid glycodendrimers, the use of a smaller microwave cavity allowed for excellent yields of 77.8 and 79.2%, respectively. Further research needs to be conducted on this new core. The addition of more sugars, and further optimization of the yield of the hexavalent aminooxy terminated core is needed. Future research will be conducted to optimize the above steps, and finally to assess to the anti-HIV activity of the sulfated products.

______, Committee Chair Dr. Katherine McReynolds

______Date

vii

ACKNOWLEDGEMENTS

I would like to extend my sincere appreciation and gratitude to those who have made completion of this Thesis and my Master of Science degree possible. First and foremost, I would like to thank my advisor Dr. Katherine McReynolds, PhD. I would like to thank her for putting up with all my edits and shenanigans in lab. I also would like to thank her for seeing potential in me when I didn’t see it in myself. Without Dr.

McReynolds’ mentorship I would not have attempted a Master Program let alone pursued a PhD. I would like to thank my committee, Dr. Roy Dixon and Dr. Cynthia Kellen-

Yuen, for dealing with my last minute edits before the deadline. I would also like to thank them for their open door policy which allowed me to talk to either of them at any time about any issues I encountered along the way. I would also like to thank Dr. Jeff Mack for helping me become the best teacher I could be. He provided me with the opportunity to TA Chemistry 1A which helped me decide on a career pathway into teaching.

Next, I would like to thank my research group. I would like to extend my largest thanks to James Cerney. Lauren Wells, Juan Gonzalez, and Ugbad Farah, for helping me synthesize many compounds used throughout this entire project. Without their help, it would have taken me months longer to finish my research. I would also like to thank

Grace Paragas, and Cory Vierra for just being there for me and allowing me to bounce ideas off them about edits on PowerPoints and my seminar. Additionally, I would like to thank all the other undergraduates who made coming to lab a joy through conversations and joking. They made whole group feel like a family.

viii

Finally, I would like to thank my family and friends. I appreciate my parents’ understanding that I could not go home to San Jose for more than a few days while I worked in my lab. I would like to thank my girlfriend, Belinda Vue, for realizing that my lab work had to come before her most days and for helping me find a few distractions, like going to a movie or dinner, to take my mind off my research. I would like to thank my friends and family for visiting me when they knew I couldn’t get away to visit them.

Finally, I would like to thank my great-grandparents. While they are no longer alive to see what I’ve achieved, their value of education was passed own to me so that I could complete a higher education.

TABLE OF CONTENTS

Acknowledgements ...... viii

List of Figures ...... xii

List of Schemes ...... xviii

Chapter

1. BACKGROUND ...... 1

HIV Background ...... 1

HIV-1 Life Cycle ...... 2

Life Cycle of HIV-1 ...... 6

Available Treatments for HIV ...... 8

NNRTI and NRTIs ...... 11

INI Inhibitors ...... 13

Protease inhibitors ...... 14

CCR5 Inhibitor and Fusion Inhibitors ...... 15

Drawbacks of using ART ...... 18

Dendrimers ...... 24

Current Dendrimers ...... 26

The Multivalent effect ...... 27

Sulfated Sugars ...... 29

Oxime and Aminooxy Linkages ...... 31

Testing for Inhibition ...... 32

Original Proposed Project ...... 34

2. RESULTS AND DISCUSSION ...... 53

3. CONCLUSIONS AND FUTURE WORK ...... 137

4. EXPERIMENTAL ...... 138

Methods and Material ...... 138

Appendix A.1H NMR Spectra ...... 156

Appendix B.13C NMR Spectra ...... 176

Appendix C. IR Spectra ...... 196

Appendix D. Mass Spectra ...... 198

References ...... 205

LIST OF FIGURES Page

1. A mature HIV-1 virion ...... 2

2. CD4 and CCR5/CXCR4, and HIV envelope glycoproteins, gp120 and gp41 ... 3

3. HSPG on a host cell and the HIV will attachment site to the host cell ...... 4

4. HIV viral attachment to a CD4 positive host cell ...... 6

5. The life cycle of HIV-1 ...... 8

6. ART drugs effects on the life cycle of HIV ...... 10

7. NNRTI compounds used for ART treatment ...... 12

8. NRTI compounds used for ART treatment ...... 13

9. INI compounds used for ART treatment ...... 14

10. PI compounds used for ART treatment ...... 15

11. Maraviroc, a CCR5 antagonist compound ...... 16

12. Enfuvirtide, a fusion inhibitor compound ...... 18

13. The four categories that microbicides ...... 20

14. Desired properties of an ideal prophylaxis agents ...... 21

15. The structure of a PAMAM ...... 25

16. Divergently or convergently synthesized ...... 25

17. Peptide dendrimer general structure ...... 27

18. Multivalent binding example ...... 28

19. Heparin sulfate and dextran sulfate structures ...... 31

20. Example of an oxime reaction ...... 32 xii

21. The cores that will be incorporated into dendrimers ...... 34

22. Sugars that will be incorporated into glycodendrimers ...... 35

23. Synthesis of the mono-phthalimide of 1,3-propandiol (Compound 2A)...... 54

24. The proton NMR for Compound 2A in CDCl3 at 500 MHz ...... 55

25. The carbon NMR for Compound 2A in CDCl3 at 125 MHz ...... 56

26. The synthesis of the mono-succinimide of 1,3-propandiol ...... 57

27. The proton NMR for Compound 2B in D2O at 500 MHz ...... 58

28. The carbon NMR for Compound 2B in D2O with a methanol internal

standard at 125 MHz ...... 59

29. The synthesis of the mesylated form of the mono-phthalimide of

1,3-propandiol (Compound 3A) ...... 60

30. The proton NMR for Compound 3A in CDCl3 at 500 MHz ...... 61

31. The carbon NMR for Compound 3A in CDCl3 at 125 MHz ...... 62

32. The synthesis of the mesylated form of the mono-succinimide of

1,3-propandiol (Compound 3B) ...... 63

33. The proton NMR for Compound 3B in CDCl3 at 500 MHz ...... 64

34. The carbon NMR for Compound 3B in CDCl3 at 125 MHz ...... 65

35. Formation of the mono-phthalimide of DEG ...... 66

36. The proton NMR for Compound 5A in CDCl3 at 500 MHz ...... 67

37. The carbon NMR for Compound 5A in CDCl3 at 125 MHz ...... 68

38. Formation of the mono-succinimide of DEG ...... 69

39. The proton NMR for Compound 5B in D2O at 500 MHz ...... 70

xiii

40. The carbon NMR for Compound 5B in D2O with a methanol internal

standard at 125 MHz...... 71

41. The formation of the mesylated the mono-phthalimide of

DEG (Compound 6A) ...... 72

42. The proton NMR for Compound 6A in CDCl3 at 500 MHz ...... 73

43. The carbon NMR for Compound 6A in CDCl3 at 125 MHz ...... 74

44. The formation of the mesylated the mono-succinimide of

DEG (Compound 6B) ...... 75

45. The proton NMR for Compound 6B in CDCl3 at 500 MHz ...... 76

46. The carbon NMR for Compound 6B in CDCl3 at 125 MHz ...... 77

47. The Michael addition of Core N and acrylonitrile Compound 7 ...... 78

48. HR-ESI of Compound 7...... 79

49. The proton NMR for Compound 7 in D2O at 500 MHz ...... 80

50. The carbon NMR for Compound 7 in D2O with a methanol internal

standard at 125 MHz ...... 81

51. Reduction and protection of the nitrile core ...... 82

52. The deprotection of trivalent amine core (Compound 9) ...... 83

53. HR-ESI of Compound 9...... 83

54. The proton NMR for Compound 9 in D2O at 500 MHz ...... 85

55. The carbon NMR for Compound 9 in D2O with a methanol internal

standard at 125 MHz ...... 86

56. The SN2 of Compounds 3A or 3B to the trivalent core to make

xiv

hexavalent cores (Compound 10)...... 88

57. The SN2of Compounds 6A or 6B to the trivalent core to make

hexavalent cores (Compound 11)...... 89

58. The reduction protection of the amine linker (Compound 19) ...... 90

59. The synthesis of the mesylated form of the BOC-protected amine linker

(Compound 20)...... 91

60. The proton NMR for Compound 20 in D2O at 500 MHz ...... 92

61. The Carbon NMR for Compound 20 in CDCl3 at 125 MHz ...... 93

62. The short aminooxy linker (Compound 22) ...... 94

63. The proton NMR for Compound 22 in D2O at 500 MHz ...... 95

64. The Carbon NMR for Compound 22 in D2O at 125 MHz with a methanol

internal standard at 125 MHz ...... 96

65. The synthesis of the tetravalent BOC core (Compound 23) ...... 97

66. The proton NMR for Compound 23 in C6D6 at 500 MHz ...... 98

67. The Carbon NMR for Compound 23 in C6D6 at 125 MHz ...... 100

68. The deprotection of the tetravalent core (Compound 24)...... 101

69. HR-ESI of Compound 24...... 101

70. The proton NMR for Compound 24 in D2O at 500 MHz ...... 103

71. The carbon NMR for Compound 24 in CD3OD at 125 MHz ...... 104

72. The Michael addition of acrylonitrile to synthesis the octavalent

nitrile core (Compound 25) ...... 105

73. IR of Compound 25, with a nitrile peak at 2250 cm-1 ...... 106

74. The esterification synthesis of the octavalent methyl ester

core (Compound 26) ...... 108

75. IR of Compound 26, carbonyl stretch 1708 cm-1 and C-O stretch 1220

and 1358 cm-1 ...... 108

76. The synthesis of the octavalent amine core (Compound 28)...... 109

77. The proton NMR for Compound 28 in D2O at 500 MHz ...... 110

78. The carbon NMR for Compound 28 in D2O with a methanol internal

standard at 125 MHz ...... 111

79. The synthesis of the octavalent aminooxy terminated core (Compound 29) .. 113

80. The synthesis of the t-butyl linker (Compound 32) ...... 114

81. The formation of mono-succinimide of t-butyl linker (Compound 33) ...... 114

82. The proton NMR for Compound 33 in CD2Cl2 at 500 MHz ...... 115

83. The carbon NMR for Compound 33 in CD2Cl2 at 125 MHz ...... 117

84. The deprotection of mono-succinimide of carboxy linker (Compound 34). .. 118

85. HR-ESI of Compound 34...... 118

86. The proton NMR for Compound 34 in D2O at 500 MHz ...... 119

87. The carbon NMR for Compound 34 in D2O with a methanol internal

standard at 125 MHz ...... 121

88. The synthesis of amine terminated hexavalent core (Compound 38)...... 122

89. The synthesis of aminooxy terminated hexavalent core (Compound 39)...... 124

90. HR-MALDI-TOF of Compound 39 ...... 125

xvi

91. The proton NMR for Compound 39 in D2O at 500 MHz ...... 126

92. The carbon NMR for Compound 39 in D2O with a methanol internal

standard at 125 MHz...... 127

93. Oxime coupling with cellobiose and the hexavalent aminooxy terminated

core (Compound 40)...... 128

94. HR-MALDI-TOF of Compound 40 ...... 129

95. The proton NMR for Compound 40 in D2O at 500 MHz ...... 130

96. The carbon NMR for Compound 40 in D2O with a methanol internal

standard at 125 MHz ...... 131

97. Oxime coupling with colomnic acid and the hexavalent aminooxy terminated

core (Compound 41)...... 132

98. HR-MALDI-TOF of Compound 41...... 133

99. The proton NMR for Compound 41 in D2O at 500 MHz ...... 134

100. The carbon NMR for Compound 41 in D2O with a methanol internal

standard at 125 MHz ...... 135

xvii

LIST OF SCHEMES Page

1. The synthesis of the mesylated short linker (Compound 3A and B) for

Core N synthesis ...... 37

2. The synthesis of the mesylated long linker (Compound 6A and B) for

Core N synthesis ...... 38

3. Functionalization of triethanolamine to make trivalent amine-terminated

Compound 9 ...... 39

4. The addition of Compounds 3A or 3B to the trivalent core to make the

hexavalent core (Compound 10) ...... 40

5. The addition of Compounds 6A or 6B to the trivalent core to make the

hexavalent core (Compound 11) ...... 41

6. Short linker glycodendrimers (Compounds 14 and 15) ...... 42

7. Long-linker glycodendrimers (Compounds 16 and 17) ...... 43

8. The synthesis of the mesylated Boc protected amine linker and

(Compound 20) ...... 44

9. BOC-aminooxyacetic acid (Compound 22) ...... 45

10. The functionalization of Core S to make tetravalent core (Compound 24) ...... 45

11. The functionalization of Core S to make octavalent core (Compound 28) ...... 46

12. The synthesis of Core S to make aminooxy octavalent core (Compound 29) .. 47

13. Core S glycodendrimers (Compound 30)...... 47

14. Core S glycodendrimers (Compound 31) ...... 48

xviii

15. The synthesis of the mono-succinimide of carboxy linker and (Compound 35)

for hexavalent core synthesis ...... 49

16. The synthesis of the hexavalent core (Compound 39) ...... 50

17. The addition of the mono-succinimide carboxy linker and (Compound 35) to

hexavalent core, yielding aminooxy hexavalent core (Compound 40)...... 51

18. The synthesis of hexavalent core glycodendrimer (Compound 41 and 42) ...... 52

xix 1

Chapter 1:

HIV Background

Infection caused by the Human Immunodeficiency Virus (HIV) is a current worldwide epidemic, which leads to the development of acquired immunodeficiency syndrome (AIDS). Currently, an estimated 36.9 million people are infected with HIV-1.1

HIV comes in two strains, HIV-1 and HIV-2. The similarities between these strains are the basic gene arrangement, modes of transmission, intracellular replication and progression into AIDS. 2 Some of the differences between the two strains are the transmission rate, disease progression and the location where the virus is confined.2 HIV-

1 develops faster and is transmitted more frequently than HIV-2. 2 HIV-2 is limited to

West Africa, while HIV-1 occurs worldwide.2 A 2014 survey reported that 2 million people are infected with the HIV virus each year, with 1.2 million annual deaths.1

HIV is one of the more complex retroviruses. It’s virions contain single-stranded

RNA, a viral envelope, capsid and enzymes. Figure 1 shows a representation of HIV-1.

The capsid provides protection for the key virion parts. Within the capsid is the viral core, which consists of viral RNA, reverse transcriptase, protease, and integrase proteins.

Finally, on the outside of virus, the viral envelope and the glycoproteins, gp120 and gp41, cover the surface of the virus. 3

Figure 1: A mature HIV-1 virion contain single-stranded RNA, a viral envelope, capsid, glycoproteins and enzymes.3 (Courtesy: U.S. Department of Health and Human Services)

HIV-1 Life Cycle:

Attachment:

In order to synthesize drugs to combat HIV, there is a need to understand all components of the virus and its life cycle. The HIV virus attaches to the host cell primarily through CD4-positive helper T-cells, macrophages, and dendritic cells.4 CD4- positive cells are white blood cells that play an important role in the immune system. The

HIV virus must introduce its viral RNA into the host cell so that it can replicate. In order to do this, the HIV virus must attach to the host cell using glycoproteins 120 (gp120) and glycoprotein 41 (gp41). Gp120 is a peripheral membrane protein, while gp41is an integral membrane protein on the HIV envelope surface.5 To incorporate gp120 and gp41 into the viral envelope, gp160 must first be synthesized. Gp160 undergoes a number of

3 maturation steps, one of which is extensive glycosylation,5 which prevents an immediate immune response to HIV.6 The primary attachment groups for both HIV and the host cell are shown in Figure 2.

Surface Heparan Sulfate Proteoglycans (HSPGs):

While using the CD4 receptor pathway is the most common entry mechanism for

HIV, this versatile virus can enter the host cell by a different process through the use of heparan sulfate proteoglycans (HSPGs) (Figure 3).8 HSPGs are glycoproteins that contain one or more covalently attached heparan sulfate (HS) chain glycosaminoglycans

(GAGS).9 GAGS are polysaccharides that consist with the presence of amino sugars (N- acetylglucosamine or N-acetylgalactosamine) and other sugars in a polyanionic form to help the HSPG become more hydrophobic. 9 These transmembrane receptors are located on epithelial cells, endothelial cells, and macrophages, and are poorly expressed on T-

4 lymphocyte cells.8 The way cells manufacture HSPGs is by a multi-step process where a unique core protein and multiple heparin polysaccharide chains are converted into more complex chain structure.10 Due to the extensive expression of HSPGs on cells, many pathogens take advantage, adhere, and invade the host tissues.11 It has been proposed that

HIV can bind to HSPGs with high-affinity due to the basic residues of the V3 loop.8, 11

Additionally, HSPGs have shown they can hold HIV until a CD4 host cell can approach and become infected.8, 12

Figure 3: Comparison of HIV-1 binding to HSPG and CD4 cell. In both cases, electrostatic interactions between the V3 loop basic region and the sulfated regions found on the host cells allow viral infectivity.8 (Courtesy: American Society for Biochemistry and Molecular Biology).

Gp120 and gp41:

HIV can enter the host cell by many different mechanisms, but all of the mechanisms use a similar interface between the host cell and the virus, gp120 and gp41.

The most prevalent attachment mechanism for the virus to the host cell is through the

CD4 receptor mechanisms, which is shown in Figure 4. On the gp120 surface, there are two important protein loops: the V2 and V3 loops. What makes the V2 loop so interesting is the presence of sulfated tyrosine residues which interact with the V3 loop, allowing gp120 to be stabilized while not attached to the host cell.13 This stabilization of gp120 allows the virus to be transmitted around the body so that gp120 has a greater ability to infect host cells. The V2 loop stabilizes gp120 before attachment to the host cell, which allows the V3 loop to attach more readily to the host cell.13 When gp120 binds to CD4 on the host cell, the receptor ratchets the HIV virion closer to the host cell membrane, whereby the host cell chemokine co-receptors (CCR5 or CXCR4) can then bind to V3 loop.14-15 The binding between the viral gp120 and the host cell coreceptors is efficient in this process due to the multivalent character of gp120. Multivalency is the simultaneous attachment of multiple binding sites on one entity to multiple receptor sites on another.4

Once attached, a conformational change occurs in the CD4 receptor on the host cell so that another viral glycoprotein, gp41, can penetrate the host cell membrane and introduce the viral RNA. Gp 41 also undergoes a conformational change.16 Resulting in a pre- hairpin intermediate that binds to CCR5 or CXCR4 coreceptor before the hydrophobic region inserts into the target cell membrane.17 Next, the pre-hairpin intermediate collapses into a stable six-helix bundle that results in the fusion of the virus and host cell membranes.17 This allows for the entry of the viral genome and proteins into the host cell.

Figure 4: In the HIV viral attachment to a CD4 positive host cell, a conformational change occurs between CD4, gp120 and gp41, which will allow the virus to deposit the viral load into the host cell.7 (© 2018 Remedy Health Media, LLC).

Life Cycle of HIV-1:

Once the virus has fused to the host cell, the HIV life cycle begins (Figure 5).

First, the viral RNA is reverse transcribed to viral DNA, so it can be integrated into the host cell DNA. This is accomplished in two-steps. First, the viral RNA is processed by reverse transcriptase, which reverse transcribes the viral RNA into viral DNA. Next, viral integrase incorporates the viral DNA in to the host cell DNA. The newly incorporated viral DNA reprograms the human cell to make more HIV. Now replication can occur, where the host cell will transcribe the incorporated viral DNA to form new viral RNA.

During transcription, the two strands of DNA divide and form a new strand of viral RNA.

Then translation will occur using the viral RNA to build new viral proteins, which will go on to form the new HIV particle, which assembles in the cell. The protein building blocks are then cleaved into smaller pieces by both cellular and viral proteases. These proteins

7 form the structure of the new HIV particle, including each of the enzymes and proteins needed to repeat the viral reproductive process. Once this assembly has occurred, the new viral particle buds off of the human cell and is able to infect other cells.

Figure 5: The life cycle of HIV-1.18 (Courtesy: National Institute of Allergy and Infectious Diseases).

Available Treatments for HIV:

Since HIV can infect the host in multiple ways, the ability to fight the virus and slow down the transmission of HIV can help combat the spread of the virus. In the beginning, HIV was fought by using a mono-therapeutic approach, which involved the use of a single drug to treat HIV.19 Now the primary treatment for HIV is ART

(antiretroviral therapy), which slows the progression of the virus. ART treatment relies on targeting a specific enzyme or protein in the HIV virus, then inactivating the virus. As of

June 2015, 15.8 million people living with HIV had access to antiretroviral therapy.1

When patients are on ART drugs, they are given a combination of non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleoside reverse transcriptase inhibitors (NRTIs), protease inhibitors (PI), integrase inhibitors (INI) or fusion inhibitors (FI). ART has two main goals to achieve in order to combat HIV. The first goal is to suppress viral replication in the patient.20 The second goal is to bring down the viral loads in the body below detectable levels, which in turn will improve the life expectancy and quality of life of the patient.20 Figure 6 shows where the ART drugs affect the HIV life cycle.

Figure 6: Target sites of FDA-approved ART drugs on the life cycle of HIV.3 (Courtesy: U.S. Department of Health and Human Services).

While the ART method for patients consists of NNRTIs, NRTIs, PI, INI and FI, only a combination of two to three different drugs inhibitors are used for treatment.19

Currently, the recommended treatment for HIV patients consists of NRTIs, NNRTIs and a PI.19 The World Health Organization (WHO) suggestion for medication consists of:

Zidovudine™ (AZT)+ Lamivudin™ (3TC)+ Neverapine™ (NVP) or AZT + 3TC +

Efaverence™ (EFV) or Tenofovir disoproxil fumarate™ (TDF) + 3TC or

Emtricitabine™ (FTC) + EFV, and finally TDF + 3TC or FTC + NVP.21

Non-nucleoside Reverse Transcriptase Inhibitors (NNRTIs) and Nucleoside Reverse Transcriptase Inhibitors (NRTIs): After the virion has fused with the host cell, the virus can infect the host cell and start its life cycle. NNRTIs (Figure 7) cause distortion in the pocket of the protein that inhibits the DNA polymerization function, by binding to an allosteric site and inactivating the protein.22 NRTIs (Figure 8) inhibit reverse transcriptase by using structurally similar compounds to nucleosides and nucleotides.22 The NRTIs resemble a nucleoside or nucleotide to such a sufficient degree that they can bind to the active site.

However, they are different enough that the normal function of the substrate can’t be carried out, resulting in chain termination of the viral DNA double stand.

Figure 7: NNRTI compounds used for ART treatment.23

Figure 8: NRTI compounds used for ART treatment. 23

Integrase Inhibitors (INI):

After the HIV RNA has been converted to DNA, it must be integrated into the host cell DNA. INI are another class of drugs that inhibit the integration of viral DNA into the host DNA.22 The current INIs that are Food and Drug Administration (FDA) approved for patients are Raltegravir™ (RAL), Elvitegravir™ (EVG) and Dolutegravir™ (DTG)

(Figure 9).24 Inhibition of integrase prevents the integration of the HIV genome into the host cell genome by targeting the strand transfer reaction between the viral DNA stands to the nuclear DNA. Therefore, integration of the viral genomes cannot occur, preventing the propagation of the viral infection.24

Figure 9: INI compounds used for ART treatment.25

Protease Inhibitors:

After the host cell replication cycle has made new viral RNA, the host cell will start to translate the viral RNA into the proteins needed to infect new cells. Most antiviral drugs currently used in ART treatment only target steps in viral replication cycle. The

HIV protease enzyme plays an essential role in HIV replication and the formation of the infectious virus. HIV protease is responsible for cleaving newly synthesized viral proteins to generate the active protein components required for viral propagation. Since 1994, the incorporation of PIs into ART treatment has been shown to significantly decrease the amount of mature virions in the system and increase lifespan of patients.26 Once the viral

RNA has been used to translate the essential viral proteins, PIs block the protease enzyme and prevents a mature viruses being produced.22 While the HIV virus still replicates in the host cell, the presence of PI will result in immature virions unable to infect new cells. An example of PI that are used in the treatment of HIV are Ritonavir and Indinavir (Figure

10)27.

Figure 10: PI compounds that are used for ART treatment. 27

CCR5 Inhibitor and Fusion Inhibitors:

Most of ART drugs target reverse transcriptase, integrase and protease enzymes for HIV, but recently, research in drug development has made progress towards preventing viral entry into the cell.28 The purpose of these inhibitors is to stop the interaction of the viral surface receptors with specific host cell surface CD4 receptor and the chemokine coreceptor CCR5. The types of compounds that can accomplish this goal are CCR5 antagonist or fusion inhibitors. CCR5 antagonists will attach to the host cell, while in contrast, fusion inhibitors bind directly to HIV proteins.

CCR5 Inhibitors:

The CCR5 coreceptor is a promising target for entry inhibitor therapies, because this class of inhibitor bind directly to the coreceptor, instead of binding to gp120. An example of a CCR5 antagonist compound is Maraviroc™, an FDA-approved drug

(Figure 11).28 Studies have shown the importance of the chemokine coreceptors in HIV infection that confer relative resistance to HIV infection though mutations of alleles.28

Maraviroc works as an antagonist by binding to the CCR5 coreceptor on the host cell and blocking it from attachment. Maraviroc’s function is due to a mutation that occurs in the human population. This mutation is known as at CCR5Δ32.28 With this, the host cell is now a locked door, which prevents the virus from entering the host cell. Thus, the existence of the CCR5 deletion in the human population makes Maraviroc a feasible target to help prevent this viral infection. 28

Fusion Inhibitors:

The feature of a good inhibitor would be to prevent virial infection of the host cell by blocking one of the glycoproteins on the surface. FI can play a critical role in preventing the start of the HIV life cycle. For FI, this interaction can trigger a different conformational change in the viral glycoprotein that activate the fusion mechanisms in a synthetic manner.29 This synthetic method of inducing fusion with the FI instead of the host cell will prevent the virus from replicating within the host cell. Fusion inhibitors will inhibit HIV before infection, an example is Enfuvirtide™, (Figure 12) would provide greater ability in preventing the virus from attaching to the host cell. Enfuvirtide functions as a fusion inhibitor by binding to gp41 of the virus. Enfuvirtide™ is a 36 amino acid gp41 analog that mimics the pre-hairpin intermediate of the N-terminal region of gp41.30 Enfuvirtide™ works by engaging in a coiled-coil interaction with the pre-

16 hairpin loop regions on gp41 preventing viral capsid entry into the host cell.31 FIs provide useful insight for the design of improved drugs with anti-escape properties.29

Figure 12: Enfuvirtide™, a fusion inhibitor compound used to fight HIV. 20 (Reprinted from De Clercq, E.; et al. Int. J. Antimicrob. Agents 2009, 33 (4), 307-20. Copyright 2018, with permission from Elsevier)

Drawbacks of Using ART:

While ART treatment has been very effective in decreasing viral loads in patients, there are many drawbacks that cannot be ignored. ART can become toxic or ineffective over time.32 The main reason for the reduced effectiveness of ART are viral mutations.

Drug resistance can be attributed to random mutations during viral replication, which lacks proofreading during the replication cycle, or can also be due to improper antiretroviral (ARV) management and incorrect ARV drug selection.33 ART treatment

17 relies on inhibiting the activity of a specific enzyme or protein in the HIV virus, which then inactivates the virus. The problem with this type of treatment is that the HIV virus quickly mutates so that the ART drugs become ineffective in as little as one month.32

Additionally, ART drugs can build up to toxic levels in the body very quickly. A study in

Belarus by the Setkina group looked at 518 ART-naïve HIV-positive patients.34 The group found that 65% of the patients’ experienced one or more adverse drug reactions

(ADRs) related to the components of ART. The patient’s most common side effects were hematotoxic, hepatotoxic, and neurotoxic adverse reactions.34 While ART has helped millions of people with HIV to live longer, healthier lives, additional drug therapies are needed due to resistance and toxicity issues.

Prophylaxis:

While ART has been effective in prolonging a patient’s life once infected with

HIV, the ability to fight the virus before one becomes infected is the new frontier. A new type of treatment has been developed called prophylaxis. HIV prophylaxis comes in different varieties: vaccines, macromolecular HIV entry inhibitors, antiretroviral drugs, and nucleic acid-based therapeutics (Figure 13).35 While each type of prophylaxis has vastly different problems in development, they all require beneficial properties to be useful in fighting HIV such as complex steps for testing, effectiveness, ease of synthesis, and cost effectiveness (Figure 14).35

Figure 13: The four categories that microbicides: vaccines, macromolecular HIV entry inhibitors, antiretroviral drugs, and nucleic acid-based therapeutics. 35(Reprinted from Date, A.; et al. Biomaterials 2013, 34 (26), 6202-6228. Copyright 2018, with permission from Elsevier)

Within the scientific community the development of new, safe and effective prophylaxis to combat the virus is needed. The development of a vaccine to fight HIV infection is a challenge for researchers. In spite of the extensive research on HIV, a safe and effective vaccine has not yet been created due to the rapid replication and extreme variability of the virus.36 Past attempts at developing an effective vaccine were failures and results were sobering.37 An HIV vaccine created by Merck (MRKAd5 HIV-1 gag/pol/nef) showed an increase in HIV infection among the patients in the study,37 while another studying using RV144 vaccine demonstrated only 31% protection from HIV infection.35

While an HIV vaccine has been elusive, the use of anionic macromolecules have been found to inhibit HIV. Macromolecules help inhibit the virus by interaction with

20 glycoproteins. Many clinical trials have already been performed with macromolecules.35

During the trials, entry inhibitors showed great promise in animal models, however, in the human trials failed to show any significant protection.35 Unfortunately, one of the macromolecules used in a trial, cellulose sulfate, showed an increased risk of HIV acquisition due to destruction of vaginal epithelium.35

Finally, siRNAs can be therapeutic agents for a variety of indications, since they are a sequence of a specific gene, which at a very small concentration can be effective combating HIV. In the last few years, a considerable amount of research on siRNA constructs for prophylaxis has been done using systemic or local delivery system.35 siRNA target various genes on HIV that have shown potential to inhibit infection or suppression in the cells.35 While siRNA have therapeutic potential to combat the virus, there are several tasks that need to be worked out before they can be a beneficial therapy. siRNAs are hydrophilic and carry anionic charge. These characteristics hamper the cell’s ability to uptake siRNA effectively.35 Another drawback in using siRNAs is the need to release them in the cytoplasm of the cells in order to achieve silencing.35 Finally, the delivery mechanism for siRNA in to the host cell can be degraded by lysosomes.35

Microbicides:

In developing an effective microbicide, finding an appropriate drug delivery vehicle and delivery system for targeting the virus with a specific time frame and appropriate dose is important. Microbicides have the capability of contacting the virus before the host cell has been infected. Research is being conducted with a wide range of systems, such as, gels, capsules, creams, tablets and rings.38 One of the first topical

21 nanomicrobicides, SPL7013 (VivaGel®), has been shown to have broad spectrum antiviral activity against HIV and Herpes Simplex Virus (HSV). VivaGel® is a polyanionic compound, currently in phase III trials, composed of naphthalene dendrimer sulfonate groups and has been found to be non-cytotoxic and effective against HIV in vitro.39

Another new prevention strategy that uses ART compounds, pre-exposure prevention (PrEP), antiretroviral therapy and topical microbicide have gained momentum from interventions for those who are in risk.36 PrEP pharmaceutical products are applied topically to mucosal surfaces such as vaginal or rectal tissues. PrEP products are designed for application before sexual contact to inhibit the viral infection process.40 Since microbicides are a type of topical treatment, they can be given in higher concentrations to exposed surfaces without significant systemic exposure, thereby reducing the risk of toxicity in healthy but at-risk individuals.41 Recent developments in anti-microbicide design consist of formulating combinations of anti-retroviral agents for further protection against HIV.

While there are currently no microbicides approved, there are many promising candidates. Three of these contenders are nucleotide reverse transcriptase inhibitors

(NtRTI), Tenofovir™ (TFV), and vaginal gelatin material. These are currently in phase

III of clinical trials. The effective activity of Tenofovir™ is both in suppression of viral reproduction with a favorable safety profile and a long half-life. Making it a perfect drug to be formulated as an anti-microbial agent.42-43 Phase II of the Tenofovir™ trial showed a 39% reduction in HIV acquisition along with a 54% reduction in HIV infection rate among women.40 The ongoing Phase III trials will provide valuable information for

22 future developments of this microbicide.40 As the virus continues to evolve and resist drugs, new innovative approaches to combat HIV are still needed.

Dendrimers:

While ART has proven benefits for patients with HIV, the side effects from the drugs are just too serious to ignore. In spite of what ART treatment does for patients, it only reduces the viral load after infection. A new treatment with an inhibitor compound that could inhibit the viral load before infection is needed. Dendrimers are very interesting carriers for fusion inhibitors because they are globular, multivalent polymers that contain many more binding sites (Figure 15). 44 Dendrimers which are currently being evaluated as HIV entry inhibitors are classical dendrimers with anionic end groups, carbohydrate terminated dendrimers (glycodendrimers), and dendrimers based on inorganic or organic backbones.35

The synthesis of these compounds can be done in two ways, either divergently or convergently (Figure 16).45 In divergent synthesis, the molecule is built from the dendrimer core outward. In convergent synthesis, the molecule is built by creating the outer pieces then connecting them to the core in final step.

Figure 15: The structure of a poly(amidoamine) (PAMAM) generation 2 dendrimer, showing the core, and generation 0, 1 and 2 branches.44

Figure 16: The left hand side of the figure shows the divergent synthesis of a dendrimer where the linker is added to the core. The right hand side of the figure shows the convergent synthesis where the linkers are added to each other, and in the final step they are added to the core. 45(Reprinted from Boas, U.; et al. Journal of Materials Chemistry 2006, 16 (38), 3785-3798. Copyright 2018, with permission from Elsevier)

Current Dendrimers:

Dendrimers are attractive polymers for a new generation of antiviral drugs.

Generally, dendrimers can be synthesized to exhibit a defined designs, highly branched structures, a high density of functional terminal groups, and controlled molecular weights.46 The majority of dendrimers that have been studied for viral inhibition are peptide dendrimers.47 48 49 These peptide dendrimers are linear dendritic polymers, which contain peptide bonds. The types of viruses that these peptide dendrimers are bioactive against are HIV-1, Herpes Simplex Virus 1 (HSV-1), HSV-2 and Human

Cytomegalovirus (HCMV).47 48 49 Bon’s group synthesized peptide dendrimers that were able to inhibit a wild-type HIV-1 strain.47 The type of the dendrimer the group used was a

SB105-A10 (Figure 17) dendrimer, a tetravalent dendrimer with a lysine core.47

Luganini and coworkers were able to synthesize one peptide dendrimer, SB105, that was able inhibit either HSV-1, HSV-2 or HCMV.48-49 The peptide dendrimers that have been studied showed water solubility, biocompatibility, biodegradability and low toxicity when tested against each of the viruses. While peptide dendrimers are a very promising area for research in combating viruses, the main challenges are the time for synthesis and the cost of the starting materials for the synthesis.46

The Multivalent Effect:

While many of the dendrimers that have been studied are peptide dendrimers, with a low degree of branching, the ability to make a highly branched dendrimer will yield a greater multivalent effect. One major advantages of a multivalent system is the set number of defined binding sites that can be chemically controlled.50 The multivalent effect for dendrimers can be described as a tailor made delivery system with diverse biomaterial properties like surface morphology, geometric size, shape and porosity.51

These submicron sized polymer particles provide vastly increased in vivo responses as opposed to linear chains.51 It has been hypothesized that HIV treatment using a drug with multiple binding sites could require a much smaller concentration of the compound, and therefore it would be more effective in preventing HIV.4

Having multivalent polymers containing an increased number of binding sites will lead to multiple interactions between the virus and the polymer. This interaction is very

26 important because it leads to stable adhesion.50 Figure 18 illustrates the increased number of interactions between a virus and a host cell, when a multivalent drug is used to combat the virus. The figure shows a higher affinity for the multivalent binding sites of the cell surface, compared to monovalent drugs, which can only be effective in very high doses.50

As a result, the synthesis and investigation of multivalent scaffold architectures have enormous potential for competitively and effectively fighting viruses such as HIV-1.50

Figure 18: a) Shows a multivalent virus binding to the host cell, b) Illustrates a monovalent drug interacting with virus c) Shows a multivalent drug interaction with the virus and shielding the host cell from the viral adhesion.50(Reprinted from Fasting, C.; et al. Angewandte Chemie International Edition 2012, 51 (42), 10472-10498. Copyright 2018, with permission from Elsevier)

Using Carbohydrates to Help Prevent Infections:

Classically, drug development has rarely used carbohydrates, but research has shown regarded as promising targets to help fight infections. While carbohydrates have a high density of functional groups per unit, the stereochemical linkages at the anomeric carbon has been challenging synthetically.52 Carbohydrates are very suitable in the

27 blocking or inhibition of lectins for the prevention and treatment of microbial diseases as they are nontoxic and immunogenic.53 Due to the structural variety of carbohydrate compounds, they have the ability to exceed the effectiveness compare to proteins and nucleic acid drugs. Vaccines typically protect individuals by inducing humoral or cellular immunity against pathogens, allowing the body to remember the infectious body.54 In the

90’s Haemophilus influenzae type b was prevalent in developed countries until the introduction of a successful carbohydrate vaccine.55 Extensive use of the carbohydrates as vaccines has offered a useful way to protect the population by improving long-lasting immunity. This has been achieved by covalently coupling the carbohydrates to carrier proteins.55 While carbohydrates show a lot of promise as vaccines, there is still a glaring problem with using them, carbohydrates can break down in the digestive tract thought enzymatic digestion or cleavage of the glycosidic bond in the stomach.

Sulfated Sugars:

Classically, dendrimers have been shown to be excellent drug scaffolding molecules due to the ability to exhibit the multivalent effect in biological systems. On their own, they do not possess inhibitory activity and require the addition of a terminal moiety where chemistry can occur, in this study, a sulfated sugar.4 Rather than attacking the virus itself, glycodendrimers interfere with the adhesion events between HIV and the host cell coreceptors CCR5 and or CXCR4. Glycodendrimers are therefore a very promising treatment for preventing the adhesion of the HIV.4

While focusing on inhibiting the virus once it has entered the host cell, which most current drugs do, newer alternatives need to be studied. One way to interfere with

28 the host cell binding sites is by creating novel sulfated compounds. Research on two natural sulfated polysaccharides, heparin (HS) and dextran sulfate (DS) (Figure 19), has shown that the negatively charged sulfates have an inhibitory effect on the binding of

HIV to the host cell membrane.56 Adding sulfate groups to glycodendrimers can mimic the negative charges on the CCR5 and CXCR4 gp120 binding sites through electrostatic interactions. The HIV virus will therefore bind to the glycodendrimers instead of the host cell. While these naturally sulfated polysaccharides have shown the ability to inhibit HIV, they can also hinder blood clotting and lack an efficient administration method.56

Another sulfated sugar, sulfated colominic acid was much more potent to HIV activities than non-sulfated colominic acids.57 Sulfated colominic acids showed no effect to T-cells and no anti-coagulant activity compare to DS.57 These properties may be therapeutically advantageous if these compounds were considered for possible use to combat the virus.

It’s been hypothesized that sulfated sugar molecules known as glycodendrimers can inhibit the entry of HIV into the host cell.

Figure 19: Heparin sulfate and dextran sulfate structures.

Oxime and Aminooxy Linkages:

When developing new dendrimers as drug carriers, the types of linkages on the carrier needs to be determined to see if there is any benefit for drug synthesis. The terminal end linkages that will be investigated in this study will be the aminooxy and corresponding oxime linkages (Figure 20). The aminooxy functionality reacts chemoselectively with aldehyde/ketone groups at room temperature in range of different solvents, which results in the oxime linkage.58 Aminooxy linkages are chemoselective for the reducing end of sugars and are more stable than glycosidic bonds.59

Figure 20: Example of an oxime reaction. Shown, an aminooxy group reacts with an aldehyde moiety to form an oxime.

Oxime-based click chemistry is advantageous because it can be conducted using commercially available unprotected reducing sugars.60 This high chemoselectively pathway is well-suited for the synthesis of therapeutic agents because of various carbohydrates that can be used to make the glycodendrimers.61 This is very promising since it makes the final step in the synthesis to glycodendrimers facile, while it also occurs in high yields.

Testing for Inhibition:

In order to assess the anti-HIV activity for the glycodendrimers, two techniques will be used. An in-house enzyme-linked immunosorbent assay (ELISA) will be used to determine if any of the glycodendrimers possesses binding affinity towards gp120.56 The way the ELISA assay works is through an enzyme conjugated to an antibody that will react with a colorless substrate to give a colored product which can then be measured using a spectrophotometer.62 The resultant absorbance is inversely proportional to the amount of the glycodendrimer bound to gp120. A common protein used for ELISA is avidin-labeled microwell plates and a biotin-labeled gp120.63 Upon completion of the

ELISA analysis, the absorbance data will be graphed to give a dose-response curve.

From the does-response graph, an IC50 can be calculated for a glycodendrimer. An IC50 value gives the concentration of the glycodendrimer will inhibit 50% of a biological

63 process. Glycodendrimers having an IC50 values in the nanomolar range are desired.

Some advantages of the ELISA are the ability to screen large number of samples,

31 sensitivity, ease of performance and also the cost-effectiveness of the method.62 One major disadvantage of the ELISA assay is the high incidence of false positive results.62

After the ELISA assay has confirmed binding between the glycodendrimers and gp120, they will be sent for luciferase reporter gene assay to our collaborators at Duke

University. The luciferase assay was first developed by Shaw, and was later optimized at

Duke University. 64 The luciferase assay provides a measurement of HIV neutralization as a function of reduction of the Tat-regulated Luc reporter gene expression after a single round of infection.64 The tat gene is the HIV-1 gene that encodes for the regulatory Tat protein, which the virus uses in viral transcription of HIV double stranded DNA. The cells used in the assay are further engineered to contain reporter genes for firefly luciferase and Escherichia coli β-galactosidase under control of an HIV long-terminal repeat sequence (LTR).64 Luciferase activity is directly related to the number of infection events by the modified HIV virus. This type of neutralization assay has advantages over other similar neutralization assays, including the use of a clonal cell line (TZM-bl) and the utilization of altered HIV strains with the capability of only one round of infection. 64

Original Proposed Project:

The goal of this study is to develop a synthetic strategy for the synthesis of oxime-linked glycodendrimers that could exhibit HIV inhibition properties. All

32 dendrimers possess: a core, linkers, and terminal functional groups where chemistry can occur.65 For dendrimers, these functional groups can include amino, carboxyl or aminooxy moieties. In our research, the dendrimers terminate in aminooxy groups, which can then react chemoselectively with reducing sugars to yield oxime-linked glycodendrimers. With this unique ability, sulfated glycodendrimers have been shown to bind to HIV virions and block infection of host cells.56

In the present study, two different cores were synthesized based on triethanolamine (Core N), and a second from cystamine dihydrochloride (Core S)

(Figure 21). Core N will yield four unique hexavalent glycodendrimers, through two unique pathways. While one pathway for Core S that will yield two desired glycodendrimers.

Figure 21: The cores that will be incorporated into dendrimers.

After completing the synthesis of the dendrimers, the dimer of colominic acid or cellobiose (Figure 22) was added to form to the target glycodendrimers. Evidence has shown that multivalency plays a very important role in inhibiting viral loads.50 Thus, synthesizing both hexavalent and octavalent glycodendrimers should decease the viral load more efficiently.

Figure 22: Sugars that will be incorporated into glycodendrimers.

When deciding what sugars to add to a glycodendrimer, a researcher must take into consideration the types of sugars and how they will be attached to the dendrimers.

Reducing sugars come in two types, aldoses and ketoses. Aldose sugars have aldehydes functional groups, while ketose sugars have ketones. For this study, the dimer of colominic acid, a ketose sugar, and cellobiose, an aldose sugar, were used. It is important to note that cellobiose, a plant based sugar, cannot be digested in animals, thus making this sugar a good choice for incorporation into a potential drug molecule. The acetal linkage for cellobiose has a b 1à4 linkage. This bond between the sugars results in a major difference in digestibility in humans. Humans are unable to hydrolyze these bonds because of the lack of appropriate enzymes to break it down.66

The linkers for each of the cores was synthesized first before adding them to their respective cores. Schemes 1 and 2 shows the synthesis of the1,3 propandiol (short linker) and of the diethylene glycol (long linker), respectively. The reason for using either the long or short linker is to give the dendrimer either more or less flexibility. The use of different protecting groups on the aminooxy group should allow us to see if the less bulky succinimide or the more bulky phthalimide would yield the most purified product. This

34 means the long linker might show improved binding to gp120 compared to the short linker due to this flexibility difference.

Scheme 1: The synthesis of the mesylated short linker (Compound 3A and B) for Core N synthesis.

Scheme 2: The synthesis of the mesylated long linker (Compound 6A and B) for Core N synthesis.

After confirmation of each of the linkers was achieved (Compounds 3A or 3B,

6A or 6B), they were added to Core N. Before Compounds 3A, 3B, 6A, 6B could be added to Core N, the functionalization of Core N was conducted as shown in Scheme 3.

Schemes 4 and 5 shows addition of both linkers (Compounds 3A or 3B and 6A or 6B), which resulted in two different hexavalent cores.

Scheme 3: Functionalization of triethanolamine to make trivalent amine-terminated Compound 9.

Scheme 4: The addition of Compounds 3A or 3B to the trivalent core to make the hexavalent core (Compound 10).

Scheme 5: The addition of Compounds 6A or 6B to the trivalent core to make the hexavalent core (Compound 11).

After completion of the hexavalent dendrimers, the next step was to add the sugar to the core. Since Core N ends in an aminooxy group, the sugar coupling was attempted via oxime coupling in a microwave (Schemes 6 and 7). Since Core N has both short and long linkers, addition of two different sugars would yield four unique glycodendrimers

(Compounds 14, 15, 16 and 17).

Scheme 6: Short linker glycodendrimers (Compounds 14 and 15).

Scheme 7: Long-linker glycodendrimers (Compounds 16 and 17).

The linkers for the cores were synthesized first before adding them to the core.

Schemes 8 and 9 shows the synthesis of long linker and short linker that was added to

Core S. The addition of the of the long linker was performed to achieve the tetravalent amine-terminated core, then short linker was added to yield an octavalent aminooxy termination dendrimer. The reason for using Core S is to give an octavalent dendrimer which will increase multivalency for these studies. By growing the number of ends on the dendrimer, the binding to gp120 should be improved. Schemes 10 shows the addition of

Compound 20 to Core S, which resulted in a tetravalent core. After deprotection

(Scheme 11), a three-step synthesis was completed to achieve with the last step consisting of addition of 1,3-diaminopropane to yield an octavalent amine-terminated core. With the addition of Compound 22 (Scheme 12) giving the desired octavalent aminooxy terminated dendrimer Cores S.

Scheme 8: The synthesis of the mesylated Boc-protected amine linker and (Compound 20).

Scheme 9: BOC-aminooxyacetic acid (Compound 22).67

Scheme 10: The functionalization of Core S to make tetravalent core (Compound 24).

Scheme 11: The functionalization of Compound 24 to make octavalent core (Compound 28).

Scheme 12: The synthesis of the aminooxy octavalent core (Compound 29).

After completing of the octavalent dendrimers, the addition of the sugars was attempted. The completed synthesis of the Core S octavalent glycodendrimer is shown in Scheme 13 and 14, yielding two unique glycodendrimers (compounds 31 and 32).

Scheme 13: Core S glycodendrimers (Compound 30).

Scheme 14: Core S glycodendrimers (Compound 31).

Modification of the Original Proposed Work:

Due to problems in the synthesis proposed in the original work, three new schemes were added to complete two new desired glycodendrimers. Scheme 15 shows the synthesis of a mono-succinimide carboxy linker to add an ether group to the dendrimer (Compound 34). The synthesis of the amine-terminated hexavalent core was completed by a lab member (Scheme 16). After confirmation of the linker was achieved, the addition to the core was completed next (Scheme 17). Finally completing the synthesis of the hexavalent glycodendrimers as shown in Scheme 18, will yield two unique glycodendrimers. After the desired glycodendrimers are confirmed, they will be sulfated. An in-house competitive ELISA assay will be performed to determine the binding inhibition properties of the sulfated glycodendrimers against gp120 on HIV-1.56,

Scheme 15: The synthesis of the mono-succinimide of carboxy linker and (Compound 34) for hexavalent core synthesis.

Scheme 16: Synthesis of the amine-terminated hexavalent core used in the new glycodendrimer syntheses(Compound 38).

Scheme 17: The addition of the mono-succinimide carboxy linker and (Compound 34) to the hexavalent core, yielding the aminooxy-terminated hexavalent core (Compound 39).

Scheme 18: The synthesis of hexavalent core glycodendrimer (Compounds 40 and 41)

Chapter 2:

Results and Discussion

The primary goal of this study was to develop an efficient strategy for the synthesis of both hexavalent and octavalent oxime-linked glycodendrimers that could potentially exhibit HIV inhibition properties. First, for the hexavalent dendrimers, an ether group was placed either close to the core or to the exterior in two different linkers, one short, one long. This will help to understand the flexibility of the dendrimer core and how this can impact the binding of the virus to the host cell. Second, the octavalent dendrimer has greater multivalency compared to hexavalent core. This will answer the question of how many ends are needed to inhibit viral attachment. Finally, for the sugars, a dimer of colominic acid, a ketose sugar, and cellobiose, an aldose sugar will be attached to the cores and these glycosylated dendrimers will be tested to determine which sugar type will be the most effective for HIV inhibition. Upon structural confirmation, each glycodendrimer will be sulfated and evaluated by another group member in our lab for their HIV inhibition properties. Sulfated glycodendrimers created in this study may exhibit potent HIV inhibition properties, and may therefore be candidates for a new class of anti-HIV inhibitory molecules, microbicides.

Synthesis of Compounds 2A and 2B, protecting of 1,3-propandiol

The first step in the synthesis of the hexavalent dendrimer was to make the desired aminooxy linker. The first step in the alteration of 1,3-propandiol was the synthesis of the mono-phthalimide of 1,3-propandiol (Compound 2A, Figure 23). The

51 synthesis began via a Mitsunobu reaction with 1,3-propandiol, where a phthalimide group was added via a mono-substitution of the diol. Purification of the reaction was difficult.

The original flash column conditions of 1:1 chloroform: methanol was too polar and the product eluted with undesired compounds. After switching to a less polar mobile phase,

2:1 ethyl acetate: hexanes, a clean product was obtained.

Figure 23: Synthesis of the mono-phthalimide of 1,3-propandiol (Compound 2A).

Figure 24 shows the 1H NMR for Compound 2A. The proton NMR for

Compound 2A spectra yielded six distinct peaks and two triplet patterns at 4.35 and 3.90 ppm that integrate for two protons each (HB and HD). Another distinct peak was present at 1.98 ppm a pentet pattern that is equivalent to two protons (HC). A broad singlet at

2.80 ppm is equivalent for one proton (HA). Finally, two doublet patterns in the aromatic region, displaying an ortho-substitution pattern for an aromatic ring, at 7.80 and 7.73 ppm are equivalent to two protons each (HE and HF). The identification of HD as the most downfield triplet is due to the proximity to the phthalimide group and yields a larger coupling constant of J=5.50 Hz. To identify the protons, HE and HF the chemical shift of the phthalimide needs to be considered. Since HE is closer to the carbonyl, which is an

electron withdrawing group (EWG), this would deshield the CH more resulting in HE being more downfield compared to HF.

Figure 24: The proton NMR for Compound 2A in CDCl3 at 500 MHz.

Figures 25 show the 13C NMRs for Compound 2A. The carbon NMR spectra yielded seven distinct peaks. The addition of four new carbon signals from the phthalimide group at 163.83, 134.65, 128.83 and 123.67 ppm, confirmed the attachment of the group onto the linker (C6-C7). The relative location of C6 and C7 in the spectrum needs to be considered. Since C6 is closer to the carbonyl, which is an EWG, the carbon would be more deshielded, yielding a more downfield signal relative to C7. C5 is the shortest of the carbon signals in the aromatic region because there are three carbon atoms attached with no proton. C4 is the carbonyl signal and being a quaternary, also results in a small signal. To identify, which signals corresponds to C1-C3, looking at what is adjacent to them will help with the assignment. C2 is only next to carbons, and thus, is the most

upfield signal. C3 is next to the phthalimide group and therefore must be the most downfield carbon. Finally, C1 is the carbon signal in between C3 and C2.

Figure 25: The carbon NMR for Compound 2A in CDCl3 at 125 MHz.

While the synthesis of Compound 2A was achievable, the ability to obtain pure product was more desirable. Due to phthalimide being bulky and difficult to purify, a different protecting group was incorporated into the short linker. The synthesis of the mono-succinimide of 1,3-propandiol (Compound 2B, Figure 26) was accomplished. The succinimide group was chosen because it is less bulky. The reaction time was cut in half from 48 hours to about 24 hours and purification of the linker involved just a simple extraction.

Figure 26: The synthesis of the mono-succinimide of 1,3-propandiol.

The proton spectrum (Figure 27) spectrum for Compound 2B yielded six distinct peaks and two triplet patterns at 4.23 and 3.85 ppm that integrate for two protons each

(HC and HA). Another distinct peak at 1.90 ppm, a pentet pattern, is equivalent to two protons (HB). While for Compound 2B, the aromatic protons are missing which

Compound 2A had instead a new singlet at 2.70 ppm that is equivalent to four protons

(HE) showing the addition of the succinimide. The identification of HC as the most downfield triplet is due to the proximity to the succinimide group. This will cause the

CH2 protons to shift more downfield and also yields a larger coupling constant of J=5.40

Hz compared to HA having J=5.25 Hz.

Figure 27: The proton NMR for Compound 2B in D2O at 500 MHz.

The carbon NMR (Figures 28) for Compound 2B yielded seven distinct peaks.

The addition of two new carbon signals from the succinimide group at 175.97 and 25.26 ppm, confirmed the attachment of the succinimide group onto the linker, while the aromatic carbons are missing. C5 is the tallest of the carbon signals, since the two methylene carbons are equivalent. C4 is the carbonyl signal and being a quanternary carbon, will have smallest signal. To identify which signals, correspond to C1-C3 it is helpful to inspect what is adjacent to them. C2 is only next to other methylene carbons, thus making it an upfield signal. C3 is next to the succinimide group, and therefore must be the most downfield of the alkyl chain carbons. Finally, C1 is the carbon signal in between C3 and C2.

Figure 28: The carbon NMR for Compound 2B in D2O with a methanol internal standard at 125 MHz.

Synthesis of Compounds 3A and 3B the mesylated form of each linker

The second step in the synthesis of the short linker was the formation of the mesylated mono-phthalimide of 1,3-propandiol (Compound 3A, Figure 29). This reaction was performed because the hydroxy group (OH) on Compound 2A needed to be converted into a better leaving group for the subsequent reaction. Since tosylates are good leaving groups, tosyl chloride was used in this procedure to synthesize Compound 3A.

The addition of the tosyl group proved to be difficult. This could be due the bulkiness of both the tosyl and phthalimide groups. By switching to a methanesulfonyl group, a much smaller leaving group, the desired product could be achieved. Compound 3A was

57 relative easy to clean with a simple extraction, using 1 M HCl to remove the excess pyridine and unreacted methanesulfonyl chloride, into the aqueous layer.

Figure 29: The synthesis of the mesylated form of the mono-phthalimide of 1,3- propandiol (Compound 3A).

The proton NMR for Compound 3A (Figure 30) spectra yielded six distinct peaks with the addition of a singlet at 3.11 ppm that is the methyl group (HF) from methanesulfonyl group, confirming the addition to the linker. HC was identified as the most downfield triplet because of the proximity to the phthalimide group. This will cause the CH2 protons to shift downfield and also results in a larger coupling constant of J=5.80

Hz. HA was also moved more downfield as a result of the addition of a sulfonyl group, which deshielded the CH2 protons. The assignment of HD and HE in the phthalimide group also needs to be considered. Since HD is closer to the carbonyl group, which is an

EWG, the resultant signal would be deshielded moving the CH signal more downfield compared to HE.

Figure 30: The proton NMR for Compound 3A in CDCl3 at 500 MHz.

The carbon NMR for Compound 3A (Figure 31) yielded eight distinct peaks.

The addition of a peak at 28.14 ppm, confirmed the addition of the leaving group onto the linker. The carbon signals from the phthalimide group at 163.83, 134.65, 128.83 and

123.67 ppm, confirmed that the phthalimide group remained after the reaction (C4-C7).

The locations of C6 and C7 in the spectrum needs to be considered. Since C6 is closer to the carbonyl, which is an EWG, the carbonyl would deshield that carbon more yielding a more downfield signal compared to C7. C5 is the shortest of the aromatic carbon signals because there are three carbon atoms attached with no protons. C4 is the carbonyl signal and being a quaternary carbon, will have smallest signal. To identify which of the signals correspond to C1- C3, it is helpful to look at what functional groups are adjacent. C2 is

only next to methylene carbons and is therefore the most upfield signal. C3 is next to the phthalimide groups, therefore it must be the most downfield carbon. Finally, C1 is the carbon signal in between C3 and C2.

Figure 31: The carbon NMR for Compound 3A in CDCl3 at 125 MHz.

The second step in the synthesis was the formation of the mesylated mono- succinimide 1,3-propandiol (Compound 3B, Figure 31). This reaction was performed because the OH group on Compound 2B needed to be converted into a better leaving group for the subsequent reaction. Again, the tosylated version of this compound proved difficult to synthesize. By switching to a methanesulfonyl group, a much smaller leaving

60 group, the desired product could be achieved. Compound 3A was relatively easy to purify with a simple extraction.

Figure 32: The synthesis of the mesylated form of the mono-succinimide of 1,3- propandiol (Compound 3B).

The proton NMR for Compound 3B (Figure 33) spectra yielded six distinct peaks with the addition of a singlet at 3.11 ppm that is the methyl group (HF) form methanesulfonyl group, confirming the addition to the linker. The identification of HC as the most downfield triplet is because of the proximity to the succinimide group. This will cause the CH2 protons to move the more downfield and also yields a larger coupling constant of J=5.80 Hz compared to HA having J=5.25 Hz. HA has also moved more downfield compared to HA from Compound 2B, as a result of the addition of a sulfonyl group, which deshielded the CH2 protons.

Figure 33: The proton NMR for Compound 3B in CDCl3 at 500 MHz.

The carbon NMR for Compound 3B (Figure 34) yielded six distinct peaks. The addition of a peak at 28.28 ppm confirmed the addition of the leaving group onto the linker (C8). The positions of C4 and C5 also needs to be considered. C5 is the tallest of the carbon signals, since the two methylene carbons of the succinimide group are equivalent.

C4 is the carbonyl signal and is also one of smallest signals because it is a quaternary carbon. To identify which of the signals correspond to C1-C3, looking at what is adjacent to them will help in the assignment. C2 is only next to other carbons and is thus the most upfield signal. C3 is next to the succinimide group, and therefore must be the most downfield alkyl carbon. Finally, C1 is the carbon signal in between C3 and C2.

Figure 34: The carbon NMR for Compound 3B in CDCl3 at 125 MHz.

Synthesis of Compounds 5A and 5B protecting of DEG (long linker).

The first step in the alteration of long linker was the addition of the phthalimide group (Compound 5A, Figure 35). The synthesis began via a Mitsunobu reaction with long linker, where a phthalimide was added to via a mono-substitution of the diol. The purification was difficult. The original flash chromatography solvent of 1:1 chloroform: methanol was too polar and the product eluted with undesired compounds. After

63 switching to a less polar mobile phase, 2:1 ethyl acetate: hexanes, clean linker was obtained.

Figure 35: Formation of the mono-phthalimide of DEG

The proton NMR for Compound 5A (Figures 36) yielded six distinct peaks with four triplets at 4.35, 3.86, 3.73 and 3.52ppm that integrate for two protons each (HD, HC,

HB and HA). Finally, two doublets appear in the aromatic region at 7.57 and 7.58 ppm, displaying an ortho-substitution pattern for an aromatic ring, integrating for two protons each (HE and HF). The identification of HD as the most downfield triplet is due to the proximity to the phthalimide group. This causes the CH2 protons to move more downfield and gives rise to a coupling constant of J=6.25 Hz. To identify HA, HB and HC the coupling constants were used. The coupling constants for these signals are HA: J= 5.90

Hz, HB: J= 5.75 Hz, and HC: J= 6.30 Hz. Since HD was already identified as the most downfield triplet this makes it possible to identity the other two triplets. Since the signals at 4.35 and 3.86 ppm, have the same coupling constant they must be next to each other, thus 3.86 ppm is HC. The triplets at 3.73 and 3.52 ppm also have the same coupling constant. Since HB is next to an ether group it will be more upfield compared to HA which is next to an alcohol group. To identify the HE and HF protons in the spectrum the

phthalimide group signal needs to be considered. Since HE is closer to the carbonyl, which is an EWG, the resonance would deshield the CH moving, it more downfield compared to HF.

Figure 36: The proton NMR for Compound 5A in CDCl3 at 500 MHz.

The carbon NMR for Compound 5A (Figures 37) spectra yielded seven distinct peaks. The addition of four new carbon signals from the phthalimide group at 166.23,

135.75, 129.26 and 121.45 ppm (C5-C8), confirmed the attachment of the group onto the linker. The location of C7 and C8 in the spectra first needs to be considered. Since C7 is closer to the carbonyl, which is an EWG, the carbon would be deshielded more and would move further downfield compared to C8. C6 is the shortest of the carbon signal in the aromatic region because there are three carbon atoms attached and no proton. C5 is the carbonyl signal and is also one of smallest signals, denoting a quaternary carbon. To identify the difference between the carbon signals C1-C4, examining what is adjacent to

each carbon will help in the assignment. C4 is next to the phthalimide group, therefore it must be the most downfield alkyl carbon. Since C1 is next to an alcohol, making it the next most downfield signal. Finally, C3 should be more downfield compared to C2 since

C3 is closer to the phthalimide group, deshielding C3 more.

Figure 37: The carbon NMR for Compound 5A in CDCl3 at 125 MHz.

Because the phthalimide is bulky and difficult to purify, a different protecting group was also utilized for the long linker, similar to the approach used for the short linker. The synthesis of the mono-succinimide of DEG (Compound 5B, Figure 38) was next performed. The reaction time was again cut in half from 48 hours to about 24 hours and purification of the linker was just a simple extraction.

Figure 38: Formation of the mono-succinimide of DEG.

The proton NMR for Compound 5B (Figure 39) yielded six distinct peaks with four triplets at 4.27, 3.77, 3.71 and 3.59 ppm that integrate for two protons each (HD, HC,

HB and HA, respectively). For Compound 5B the aromatic protons are missing, and instead Compound 5A has a new singlet at 2.70 ppm equivalent for four protons (HE), showing the addition of the succinimide group. The identification of HD as the most downfield triplet is because of the proximity to the succinimide group. This will cause the

CH2 protons to move more downfield and also yields a coupling constant of J=6.25 Hz.

To identify HA, HB and HC, the coupling constants were used. The coupling constants are: HA: J= 5.90 Hz, HB: J= 5.75 Hz and HC: J= 6.30 Hz. Since HD was identified as the most downfield triplet, this makes it possible to identity each of the other triplets. Since the signals at 4.27 and 3.78 ppm, have the same coupling constant, they must be next to each other, thus the signal at 3.78 ppm is HC. The triplets at 3.71 and 3.59 ppm also have the same coupling constant. Since HB is next to an ether group it will be more upfield compared to HA which is next to an alcohol group.

Figure 39: The proton NMR for Compound 5B in CDCl3 at 500 MHz.

The carbon NMR for Compound 5B (Figure 40) yielded six distinct peaks. The addition of two new carbon signals from the succinimide group at 175.67 and 25.26 ppm confirmed the attachment of the group onto the linker, while the aromatic carbons are missing. C6 is the tallest of the carbon signals and represents the two equivalent succinimide group methylene carbons. C5 is the carbonyl signal. To identify, C1-C4, looking at what is adjacent to them will help in the assignment of these signals. C4 is next to the succinimide group and must be the most downfield alkyl signal. Since C1 is next to an alcohol, making it the next most downfield signal. Finally, C3 should be more downfield compared to C2 since C3 is next to the succinimide group, deshielding C3 more.

Figure 40: The carbon NMR for Compound 5B in D2O with a methanol internal standard at 125 MHz.

Synthesis of Compounds 6A and B mesylated form of each linker.

The second step in the synthesis was the formation of the mesylated mono- phthalimide DEG (Compound 6A, Figure 41). This reaction was performed because the hydroxyl group (OH) on Compound 5A needed to be converted into a better leaving group for the subsequent reaction. Compound 6A was again relatively easy to purify with a simple extraction.

Figure 41: The formation of the mesylated the mono-phthalimide of DEG (Compound 6A).

The proton NMR for Compound 6A (Figure 42) yielded seven distinct peaks with the addition of a singlet at 3.14 ppm, corresponding to the methyl group from the methanesulfonyl group, confirming the addition to the linker. The identification of HD as the most downfield triplet is due to the proximity to the phthalimide group. This will cause the CH2 protons to move more downfield, and yields a coupling constant of J=4.20

Hz. To identify HA, HB and HC, the coupling constants were used. The coupling constant for the other triplets are HA: J= 2.65 Hz, HB: J= 2.60 Hz and HC: J= 4.20 Hz. Since HD was already identified as the most downfield triplet, it is possible to identity each of the other triplets. Since the signals at 4.33 and 3.83 ppm have the similar coupling constant, they must be next to each other, thus 3.83 ppm is HC. The triplets at 4.32 and 3.77 ppm have the similar coupling constant. Since HB is next to an ether group it will be more upfield compared to HA which is next to a methanesulfonyl group. To identify the HE and

HF protons in the spectrum, the phthalimide group needs to be considered. Since HE is closer to the carbonyl, which is an EWG, and in conjugation with the aromatic ring, this would deshield the CH moving, it more downfield compared to HF. Another confirmation of the addition of methanesulfonyl group is the downfield movement of HA as a result of the proximity to the methanesulfonyl group which deshields the CH2 protons.

Figure 42: The proton NMR for Compound 6A in CDCl3 at 500 MHz.

The carbon NMR for Compound 6A (Figure 43) yielded nine distinct peaks, with the addition of a peak at 22.54 ppm again confirming the addition of the mesyl group onto the linker. The location of C6 and C7 in the spectrum needs to be considered.

Since C6 is conjugated to the carbonyl, which is an EWG, the signal would be deshielded more, moving the signal downfield compared to C7. C5 is the shortest carbon signal in the aromatic region because it is a quaternary carbon. To identify, which signals correspond to C1-C4, examining what is adjacent to them will help in the assignments. C4 is next to the succinimide group and is the most downfield alkyl signal. Since C1 is next to an alcohol it is the next most downfield signal. Finally, C3 should be more downfield compared to C2 since C3 is closer to the succinimide group, deshielding C3 more.

Figure 43: The carbon NMR for Compound 6A in CDCl3 at 125 MHz.

The second step in the alternative synthesis of the long linker was the formation of the mesylated mono-phthalimide DEG (Compound 6 B, Figure 44). Compound 6B was also relative easy to purify with a simple extraction, using 1 M HCl to move the excess pyridine and unreacted methanesulfonyl chloride into the aqueous layer.

Figure 44: The formation of the mesylated the mono-succinimide of DEG (Compound 6B).

The proton NMR for Compound 6B (Figure 45) yielded six distinct peaks with the addition of a singlet at 3.11 ppm, that is the methyl group (HF) from the methanesulfonyl group, confirming the addition to the linker. Additionally, four triplets at

4.37, 4.28, 3.84 and 3.75 ppm integrate for two protons each (HD, HC, HB and HA, respectively). For Compound 6B, compared to Compound 6A, the aromatic protons are missing. Additionally, Compound 6A has a new singlet at 2.72 ppm, equivalent for four protons (HE) showing the addition of the succinimide group. The identification HD as the most downfield triplet is because of the proximity to the succinimide group. This causes the CH2 protons to shift more downfield and yields a coupling constant of J=2.50 Hz. To identify HA, HB and HC, the coupling constants were used. The coupling constant for the other triplets are HA: J= 1.95 Hz, HB: J= 2.00 Hz and HC: J= 2.50 Hz. Since HD was already identified as the most downfield triplet, this makes it possible to identity each of the other triplets. Since the signals at 4.37 and 3.75 ppm have the equivalent coupling constants they must be next to each other, thus 3.75 ppm is HC. Similarly, the triplets at

4.28 and 3.84 ppm also have the equivalent coupling constants. Since Since HB is next to an ether group it will be more downfield compared to HA which is next to an

73 methanesulfonyl group. Another confirmation of the addition of the methanesulfonyl group is the downfield movement of HA which will is further deshielded.

Figure 45: The proton NMR for Compound 6B in CDCl3 at 500 MHz.

The carbon NMR for Compound 6B (Figure 46) yielded seven distinct peaks.

With the addition of a peak at 22.51 ppm (C9), this confirmed the addition of the methanesulfonyl group onto the linker. C6 is the tallest of the carbon signals and represents the two equivalent succinimide group methylenes carbons. C5 is the carbonyl signal and being a quaternary carbon, has a smaller signal. To identify C1-C4, looking at

what is adjacent to them will help in the assignment. C4 is next to the succinimide group and is the most downfield alkyl signal. Since C1 is next to an alcohol, making it the next most downfield signal. Finally, C3 should be more downfield compared to C2 since C3 is closer in proximity the to succinimide group, deshielding C3 more.

Figure 46: The carbon NMR for Compound 6B in CDCl3 at 125 MHz.

Synthesis of Compound 7 synthesis of the trivalent nitrile core.

After the alteration of both the short and long linker, Core N needed to be functionalized to accept the new linkers. The first step in the modification of Core N was the synthesis of the trivalent nitrile core (Compound 7, Figure 47). The synthesis began via a Michael addition with acrylonitrile and Core N. This step in the functionalization

75 allows the linker to be added to give the desired hexavalent dendrimer. Originally, this reaction was performed at room temperature and at a higher percent (40% (w/v)) of

NaOH overnight. After finding a new protocol as an undergraduate by heating the reaction to 45oC and using a lower concentration (10% (w/v)) of NaOH, the reaction was now complete in five hours. The yields between the two methods are comparable.

Figure 48 shows the mass spectrum for Compound 7 showing a mass of M/Z 309.19212

(M+H)+ with an error of 0.27 ppm.

Figure 47: The Michael addition of Core N and acrylonitrile Compound 7.68

Figure 48: HR-ESI of Compound 7.

The proton NMR for Compound 7 (Figure 49) yielded four distinct triplets at

3.94, 3.80, 3.63, 2.82 ppm that integrate for two protons each. To identity HG, HI, HJ, and HK, the coupling constants were used. The peaks at 3.94 and 3.63 ppm are coupling with J= 5.10 and 5.05 Hz, respectively, while 3.80 and 2.82 ppm are coupling with each other with J= 5.80 and 5.85 Hz, respectively. Since HG is next to a tertiary nitrogen the deshielding effect from this nitrogen will be less making the signal more upfield. Because the HG coupling constant is 5.85 Hz and the signal at 3.80 ppm has a coupling constant of

5.80 Hz that means that HI is the triplet at 3.80 ppm. Also, as HJ is next to oxygen on the

77 core it will be one of more downfield triplets. This is because the nitrile group is an

EWG. Since HJ has already been identified as the signal at 3.94 ppm HK must be the signal at 3.63 ppm.

Figure 49: The proton NMR for Compound 7 in D2O at 500 MHz.

The carbon NMR for Compound 7 (Figure 50) yielded five distinct peaks. The identity of C14 as the carbon on the nitrile group was made because there is a triple bond to a nitrogen this will cause C14 to be the most deshielded carbon signal, at 115.35 ppm.

Next, the most upfield carbon signal is C10. This is due to the limiting withdrawing effect from the tertiary nitrogen, thus being the carbon signal at 13.60 ppm. C13 will be the next

78 upfield carbon signal since the withdrawing effect of the nitrile group moves the carbon more downfield compared to C10. For C11 and C12 these are both next to an oxygen atom, but C12 will also feel a small effect from the nitrile group, causing the carbon signal to be a little bit more downfield compared to C11 since the nitrile is an EWG.

Figure 50: The carbon NMR for Compound 7 in D2O with a methanol internal standard at 125 MHz.

Synthesis of Compound 8 through reduction/protection

The next step in the functionalization of the core was a two-step, one-pot reaction, which reduced the nitrile to the primary amine and then BOC-protected the amine end groups (Figure 51, Compound 8). The reason for adding the BOC group to the amine terminated core was to prevent any potential side reactions. However, the addition of the BOC group prevents the acquisition of a clear proton NMR and instead gives rise to broadened signals without distinct splitting. Thus, the NMR cannot be used to show the proper proton count or peak splitting for Compound 8. Since the NMR was inconclusive the next reaction was performed to determine if the reaction worked.

Figure 51: Reduction and protection of the nitrile core.67

Synthesis of Compound 9 through deprotection.

The last step in functionalization of the trivalent core was the synthesis of the trivalent deprotected amine core (Compound 9, Figure 52). This is the last step in the functionalization to transform Core N to the trivalent dendrimer. Figure 53 shows the mass spectrum for Compound 9, giving a mass of M/Z 321.18597 [M+H]+ with an error of 0.14ppm.

Figure 52: The deprotection of trivalent amine core (Compound 9).

Figure 53: HR-ESI+ of Compound 9.

The proton NMR for Compound 9 (Figure 54) yielded five distinct peaks with four triplets at 3.84, 3.64, 3.53 and 3.07 ppm that integrate for two protons each. A pentet pattern at 1.95 ppm, equivalent to two protons, confirms the reduction of the nitrile. To

identify HG, HI, HJ, HK, and HL, the coupling constants were used. The peaks at 3.84 and

3.64 ppm are coupling to each other with J= 4.65 and 4.75 Hz, respectively, while the signals at 3.53 and 3.07 ppm are coupling to each other with J= 6.05 and 7.25 Hz, respectively. After characterizing the pentet, which has a coupling constant of J= 6.95 and 13.70 Hz, shows that the signals at 3.53 and 3.07 ppm are near each other. The two triplets at 3.53 and 3.07 ppm must be coupling with HK. The reason for the large coupling constant difference between HJ and HL is due to the exchange of a deuterium from the

NMR solvent for one of the hydrogen on to the primary amine. Since deuterium gives rise to different splitting multiplicities, this will cause HL to increase coupling constant away from 6.05 Hz to 7.25Hz. Since HL is next to the primary amine while HJ is next to oxygen, the oxygen will deshield the CH2 more causing HJ to be more downfield compared to HL, which is next to the primary amine and is the most upfield triplet. Since

HI is next to oxygen on the core it will be one of more downfield triplets. Since HJ has already been identified as the signal at 3.53 ppm, HI must be the signal at 3.84 ppm.

Finally, HG can be identified as the last triplet at 3.64 ppm.

Figure 54: The proton NMR for Compound 9 in D2O at 500 MHz.

The carbon NMR for Compound 9 (Figure 55) spectra yielded five distinct peaks. The only way to distinguish the carbon signals from each other is to look at what is adjacent to the carbon in question. Both C11 and C12 are next to oxygen, while C10 and

C14 are next to nitrogen. C13 is only adjacent to other methylene carbons, thus will be the most upfield carbon signal. C10 and C13 will be the next upfield carbon signals, since they are next to nitrogens. Since C13 is next to a primary amine and C10 is next to a tertiary amine, C10 will move more downfield compared to C13 since the tertiary amine has more of a withdrawing effect compared to the primary amine. For C11 and C12, these are both next to an oxygen atom, but C12 will feel more of an effect from the primary amine group, causing the carbon signal to be more upfield compared to C11.

Figure 55: The carbon NMR for Compound 9 in D2O with a methanol internal standard at 125 MHz.

Synthesis of Compounds 10 and 11 through hexavalent branching

Once the trivalent amine core and both linkers were synthesized, they were added together under SN2 conditions. This synthesis yielded two unique hexavalent dendrimer cores. The difference between the cores are the location of the ether groups, for

Compound 10, the ether groups closer to the interior of the core, while for Compound

11, the ether groups are both in the interior and exterior of the core. While many attempts

84 were made to synthesize Compounds 10 and 11 using either Compound 3A, 3B, 6A, and 6B (Figures 56 and 57), all of them failed to yield the desired compounds. First,

Compounds 3A and 6A, the phthalimide linkers, did not add to chosen core. It was reasoned that due to the bulkiness of phthalimide, the ability to add to the terminal amine could have been sterically hindered. Since each linker has to add to the amine group twice this bulkiness might prevent the double addition, only allowing for only a single addition of the linker.

While the phthalimide reaction was sterically hindered, and this prevented the reaction from going to completion, the succinimide reaction with core also had problems, but for different reasons. Whereas the succinimide is less bulky than phthalimide, in this experiment there was a side reaction that was out-competing the reaction with the core.

While trying to clean up the synthesis via either FPLC or HPLC, the only compound isolated was the starting material, Compound 9. The observed side reaction was the linker reacting with itself and not the core. When taking crude NMRs for each reaction, it appeared that the reaction was complete, but after purification, the 1H NMRs would show an incorrect number of peaks. As a consequence of being unable to synthesize either

Compounds 10 or 11, the reaction pathway to complete the synthesis the hexavalent glycodendrimers was abandoned.

Figure 56: The SN2 reactions of Compounds 3A or 3B with the trivalent core to make hexavalent cores (Compound 10).

Figure 57: The SN2 reactions of Compounds 6A or 6B with the trivalent core to make hexavalent cores (Compound 11).

Synthesis of Compounds 18 and 19 for the ether amine linker.

Beyond the hexavalent core synthesis, the octavalent core synthesis was also attempted. The first part of the synthesis of the linker was performed as an undergraduate

(Figure 58).68 The synthesis of Compound 18 places an ether in the linker to allow for greater flexibility for the core later on. For Compound 19, the reduction of the nitrile to the primary amine allowed the core to go from tetravalent to octavalent through branching. Converting the core to octavalent is hypothesized to be more effective in inhibiting viral binding through the increased valency.

Figure 58: The reduction protection of the amine linker (Compound 19).68

Synthesis of Compound 20, the mesylated form of the linker

The addition of a methanesulfonyl group onto the BOC-protected amine linker

(Compound 20, Figure 59), was the next step in the process. This reaction was performed because the OH group on Compound 19 needed to be converted into a better leaving group for the subsequent reaction. Since sulfonates are good leaving groups, tosyl chloride was first used in this procedure. The addition of the tosyl group proved to be difficult, giving low yields, around 30.3%. By switching to methanesulfonyl group, a much smaller leaving group, the yield increased to 50% for the linker Compound 20.

Additionally, while the tosylate linker required a flash column for purification the methanesufonyl-containing linker only required a simple extraction, which included four washes with 1 M HCl to remove the excess pyridine and unreacted methanesulfonyl chloride into the aqueous layer.

Figure 59: The synthesis of the mesylated form of the BOC-protected amine linker (Compound 20).

The proton NMR for Compound 20 (Figure 60) yielded six distinct peaks with the addition of a singlet at 3.06 ppm from the methyl on the methanesulfonyl group (HF), confirming its addition to the linker. HP is the pentet from Compound 20 and equates for two protons. There are four other triplets: HM, HN, HO and HQ. To assign each triplet, the coupling constants were used to determine which signals were coupling with each other.

The triplets at 4.35 and 3.69 have J= 2.05 and 1.95 Hz, respectively while the triplets at

3.56 and 3.22 ppm have J= 5.60 and 5.21 Hz, respectively. This means that the two most downfield triplets (HM and HN) are coupling with each other, while the two most upfield signals (HO and HQ) are also coupling with each other. Since HM is close to the methanesulfonyl group and will be deshielded, this will be most downfield signal. The triplet at 3.69 ppm will therefore be HN. Since HO is next to a polar oxygen, it is more downfield compared to HQ, which is next to an amide. This allows for the assignment of all the triplets in Compound 20. For the two singlets, one is for the methyl group on the methanesulfonyl group HF, while the other singlet is the HR represent the BOC group.

Figure 60: The proton NMR for Compound 20 in D2O at 500 MHz.

The carbon NMR for Compound 20 (Figure 61) yielded nine distinct peaks (C9,

C15, C16, C17, C18, C19, C20, C21 and C22). C20 is the carbonyl carbon and will be the most downfield signal. C21 is a quaternary carbon which will be a shorter carbon signal due to not having any attached protons. C15, C16 and C17 are carbon signals next to oxygen. C15 should be the most downfield of these carbon signals since the methanesulfonyl group is adjacent. Since sulfur has a greater electron density compared to oxygen, C16 which is three bonds away from the methanesulfonyl group will be the second most downfield signal from C17. C9, C18 and C22 should be the most upfield signals, respectively. Since

C9 is next to the sulfonate, a more EWG will cause C9 to be more downfield compared to

C18 and C22. Finally, for C18 and C22, both will be the most upfield carbon signals. C22 will be the tallest carbons signals since there are three equivalent carbons, compared to

C18, which only has two protons.

Figure 61: The Carbon NMR for Compound 20 in CDCl3 at 125 MHz.

Synthesis of Compound 22, the short linker 67

The synthesis of BOC-(aminooxy)acetic acid was performed and was successful, yielding 80% of pure product (Compound 22, Figure 62). 67 This linker was synthesized in order to incorporate an aminooxy group, which will allow for the later addition of the sugars to the dendrimers. Figures 63 and 64 show the 1H and 13C NMRs, respectively.

The proton NMR spectrum yielded two distinct peaks, with the addition of two singlets, one at 4.51 ppm a methylene proton (HS), and the second at 1.52 ppm integrating for 9 protons, corresponding to the BOC group (HR). The carbon NMR spectrum yielded five distinct peaks (C20, C21, C22, C23 and C24). There are two carbonyl peaks in the carbon spectra, C20 and C24. C24 the carboxylic acid carbon signal, is the most downfield, and

C20 is the more upfield carbamate carbonyl carbon signal. Next, C21 corresponds to the tertiary carbon neighboring the oxygen and the three methyl groups, while C23 represents the secondary carbon immediately neighboring the carboxylic acid. Finally, C22 the tert- butyl carbons, result in the most upfield carbon signal.

Figure 62: The short aminooxy linker (Compound 22). 67

Figure 63: The proton NMR for Compound 22 in D2O at 500 MHz.

Figure 64: The Carbon NMR for Compound 22 in D2O with a methanol internal standard at 125 MHz.

Synthesis of Compound 23, the tetravalent core

After the addition of the BOC leaving group on Compound 20, its addition to

Core S can occur in order to achieve tetravalent branching (Compound 23, Figure 65).

Although purification of the tetravalent core was possible via flash chromatography in

9:1 chloroform: methanol, the yields were very low, around 15-20%. The low yield could result from many possibilities. The temperature for the reaction, or the number of equivalents used of the linker (Compound 20), could account for the low yield. The first

94 test to improve the yield in this process was to change the temperature of the reaction.

The original protocol called for a temperature of 85˚C in an oil bath. However, at this elevated temperature, the color of the reaction went from yellow to a mahogany brown.

This could be due to the linker reacting with itself, or decomposition of one or more of the reaction components. Thus, the reaction temperature was changed from 85oC to 45oC.

Unfortunately, the yield was lower at 10-14%, but the color of the reaction remained yellow. Since it seemed that temperature wasn’t the reason for the low yield, the number of equivalents of starting linker was changed. Most of the time, 18 equivalents of starting linker were used, which required flash chromatography to remove the excess linker. Once the number of equivalents was changed from 18 to 6 (1.5 per end group), the reaction yields increased to the high 80% range. The only purification needed was a simple extraction.

Figure 65: The synthesis of the tetravalent BOC core (Compound 23).

The proton NMR for Compound 23 (Figure 66) yielded seven distinct peaks

(HM, HN, HO HP, HQ, HR, HT and HU). In this case, the use of C6D6 helped identify signals better than CDCl3 since it allowed for splitting patterns to be better visualized. HP

a pentet pattern at 1.71 ppm, was equivalent to two protons. Both HN and HO are next to oxygens, which should make these the most downfield signals. Even though C6D6 should have resolved both signals, they were still overlapping. HM, HQ, HU and HT are all next to either a nitrogen or sulfur. HT is the most downfield of these signals since the sulfur is a greater EWG compared to the nitrogen, while HM , HQ and HU will be upfield. Finally, the signal at 5.13 ppm is the NH proton from the carbamate.

Figure 66: The proton NMR for Compound 23 in C6D6 at 500 MHz.

The carbon NMR for Compound 23 (Figure 67) yielded ten distinct peaks (C15,

C16, C17, C18, C19, C20, C21, C22, C25 and C26). C20 is the carbonyl carbon, and will be the

most downfield signal. C21, a quaternary carbon, will also give a small carbon signal due to not having any attached protons. C16 and C17 are both next to oxygen. Since C16 has a nitrogen two bonds away compared to C17, which has a carbon adjacent, C16 will be more downfield compared to C17. C15, C19 and C26 are each next to a nitrogen atom. Since C26 is also two bonds away from a sulfur atom this will be the most downfield of these signals. This is followed by C15 next to an amide, which will cause the signal to be more downfield compared C19. Finally, C18, C22 and C25 will be the most upfield of the carbons signals. C22 will be the tallest and most of the upfield carbon signals since there are three equivalent carbons. Finally, C18 and C25 can be assigned by knowing what is adjacent to them. Since C25 is next to a sulfur it will be more downfield compared to C18, which is adjacent to two carbons.

Figure 67: The Carbon NMR for Compound 23 in C6D6 at 125 MHz.

Synthesis of Compound 24, deprotection of the tetravalent core

The last step in functionalization of the tetravalent core was the deprotection of the tetravalent BOC-amine core (Figure 68). This is the last step in the functionalization to give Core S to the tetravalent dendrimer. Figure 69 shows the mass spectrum for

Compound 24 giving a M/Z of 557.38783 [M+H]+ with an error of 0.17ppm.

Figure 68: The deprotection of the tetravalent core (Compound 24).

Figure 69: HR-ESI+ of Compound 24.

The proton NMR for Compound 24 (Figure 70) yielded seven distinct peaks

(HM, HN, HO HP, HQ, HT and HU). The HP pentet pattern at 1.98 ppm, is equivalent to two protons. Since the short-range coupling constant for HP is J= 6.20 Hz, any of the triplets that have a similar coupling constant should be adjacent to HP. The triplet signals at 3.87 and 3.55 ppm must be next to each other since the coupling constants are similar

99 at 4.92 Hz and 5.01 Hz, respectively. Since the coupling constant does not correspond with the short-range coupling of HP, these triplets are not be adjacent to HP. These triplets must then be HN and HM. Since HN is next to an oxygen, which deshields the

CH2, making HN more downfield and HM more upfield since it’s next to nitrogen.

Finally, HO, HQ, HU and HV can be determined based on the last two multiplets. Since both HO and HQ are the protons on the linker they have a better splitting pattern. The final two apparent triplets but have coalesced so since HO and HT are next to electron withdrawing atoms oxygen and sulfur make them the most downfield triplets and HQ and

HU are next to electron donating atoms nitrogen making them the more upfield triplets.

100

Figure 70: The proton NMR for Compound 24 in D2O at 500 MHz.

The carbon NMR for Compound 24 (Figure 71) yielded seven distinct peaks

(C15, C16, C17, C18, C19, C25 and C26). C16 and C17, as carbons next to oxygen, will be the most downfield carbon signals. C16 is two bonds away from a nitrogen, and this would also deshield C16, making it the most downfield carbon followed by C17 which is just next to a carbon. C25 and C26 will be the next most downfield carbons due to the proximity of the disulfide bond from the core. Since C25 is next to a sulfur it will be more downfield compared to C26 which is next to nitrogen. C15 would be the most downfield of the signals to the left of the CD3OD signal. C18 and C19 will be the most upfield signals. C18

101 is adjacent to two carbons, causing a donating effect, this will make it the most upfield signal compared to C19.

Figure 71: The carbon NMR for Compound 24 in CD3OD at 125 MHz.

Synthesis of Compound 25 the octavalent core

After completing the tetravalent amine terminated core, the next step was to branch the core to create an octavalent core. The synthesis of the octavalent nitrile core

(Compound 25, Figure 72), consisted of a double Michael addition reaction. In a

Michael reaction, a nucleophile adds across a conjugated alkene or alkyne to produce the

1,4-addition product. For this Michael addition, different conditions were tried, from

102 running the reaction neat, to running the reaction solvated. For the reaction conducted in methanol, very little to no product was formed (0-25 % yield). However, when the reaction was run neat in acrylonitrile and DIPEA, the yield was increased to 98.1 %. The only purification process needed was dialysis, but the reaction product was slightly impure with leftover DIPEA. When trying to conduct NMR experiments on Compound

25, four addition signals would appear, a doublet, triplet, quartet and heptet from DIPEA, which would overlap with many of the core signals. Therefore, an IR was obtained in a solution of CHCl3 and Compound 25, which gave the resulting spectrum shown in

Figure 73. The signal at 2250 cm-1 confirms the presence of a nitrile attached to the core.

Figure 72: The Michael addition of acrylonitrile to synthesize the octavalent nitrile core (Compound 25).

103

Figure 73. IR of Compound 25, with a nitrile peak at 2250 cm-1.

Synthesis of Compound 26 the methyl ester of the octavalent core

After the addition of acrylonitrile to the core via Michael addition, the next step was to convert the nitrile to a carbonyl group on the core. The synthesis of the octavalent methyl ester core (Compound 26, Figure 74) was completed using Compound 25 and acetyl chloride. First, the right reaction conditions were determined using a nitrile linker.

Three conditions were tested: a neat reaction with just acetyl chloride (AcCl) and linker, a solvent system with ethyl acetate: methanol, AcCl and linker and just methanol, AcCl and linker. It was determined that the best conditions were methanol, acetyl chloride and linker. When running this reaction in an ice bath from 0˚C to room temperature, the reaction would turn green. This could be due to the acetyl chloride reacting with the stainless steel from the needle vent. Next, the reaction temperature was evaluated from

104 negative78oC (dry ice and acetone) to room temperature. By changing the temperature at the start of the reaction, the solution never turned green again. The purification for this reaction was accomplished via evaporation under reduced pressure, since all of the reaction components have high volatility. When attempting to run an NMR on the new core, the NMR yielded broad signals due to the presence of the methyl esters in

Compound 26, therefore, an IR was collected on the compound. The IR spectrum was obtained in a solution of methanol and Compound 26, which gave the resulting spectrum shown in Figure 75. The signal at 1708 cm-1 shows the C=O stretch, confirming the addition of a carbonyl group to the core. This, along with the signals at 1220 cm-1 and

1358 cm-1 show two unique C-O stretches. The signal at 1220 cm-1 represents the methyl

C-O stretch, while the 1358 cm-1 signal is the C-O stretch from the carbonyl group.

Finally, the C≡N at 2250 cm-1has vanished, showing full conversion of the nitrile to carbonyl.

105

Figure 74: The esterification synthesis of the octavalent methyl ester core (Compound 26).

Figure 75. IR of Compound 26, carbonyl stretch 1708 cm-1 and C-O stretch 1220 and 1358 cm-1.

Synthesis of Compound 28 the amine-terminated octavalent core

After esterification of the octavalent core, a primary diamine was added to allow conversion to the amine terminated octavalent core (Compound 28, Figure 76). With

Compound 27 already containing a methyl ester group, the addition of 1,3- diaminopropane was easy to complete under basic conditions. Since an excess of the diamine was used, RP-HPLC was used for purifcation. To help remove some of the excess 1,3-diaminopropane, an azeotrope with MeOH and toluene was used. The yield for this reaction ranged from 52-88 % yield.

106

Figure 76: The synthesis of the octavalent amine core (Compound 28).

The proton NMR for Compound 28 (Figure 77) shows nine signals, with many of them overlapping each other. This overlapping makes it difficult to identify each peak conclusively. The defining feature in the 1H NMR that identifies this as being amine- terminated core (Compound 28), and not just unreacted 1,3-diaminopropane and

Compound 24, is a shifting of both the starting core and new linker protons. Also, the addition of four new signalsfrom the linker lends confidence in the addition of the linker.

In the 1H NMR the circle area shows the peak of interest that confirms the addition of the new linker to the core representing HP. In Compound 24, the peak that was at 1.98 ppm

(Figure 70) has shifted to 1.87 ppm (Figure 77). Again, as it was with some of the other apparent triplets without coupling constants, it is not possible to identity these signals with complete confidence just based on the 1H spectrum.

107

Figure 77: The proton NMR for Compound 28 in D2O at 500 MHz.

The carbon NMR for Compound 28 (Figure 78) yielded ten distinct peaks. This overlapping makes it difficult to identify each peak conclusively. The defining feature in the 13C NMR that identifies this as being amine-terminated core (Compound 28) and not just unreacted 1,3-diaminopropane or Compound 24, is a shift on both the starting core and linker carbons. After the linker was added to Compound 24, there a new carbonyl signal (the circle). In the 13CNMR of Compound 24 most of the carbon signals are distinct (Figure 71), while Compound 28 the carbons becomes less distinct (Figure 77).

Again, it is not possible to identity these signals with complete confidence just based on

108 the 13C spectrum. Additionally, the use of 2D NMR technique HSQC and HMBC could have helped with the identification of all the carbon signals.

Figure 78: The carbon NMR for Compound 28 in D2O with a methanol internal standard at 125 MHz.

Synthesis of Compound 29, the aminooxy-terminated octavalent core

The last step illustrates the synthesis of the aminooxy octavalent core

(Compound 29, Figure 79). Unfortunately, it was not possible to determine if the synthesis went to completion fully substituting all eight amine groups. The NMR for this compound was 8 protons short. This could be the compound missing one arm of the core making it a heptavalent core, or alternately, by only adding one Compound 22 to the

109 core. Since Compound 22 is small and contains a bulky BOC group, the ability to couple the reaction proved to be difficult. Using O-(Benzotriazol-1-yl)-N,N,N’,N’- tetramethyluronium tetrafluoroborate (TBTU) as a coupling reagent seemed to give a better synthetic results compared to using N-(3-Dimethylaminopropyl)-N′- ethylcarbodiimide hydrochloride (EDCI) with 1-Hydroxybenzotriazole (HOBt) .

However, both pathways failed to show that octavalent core was successfully synthesized.

Figure 79: The synthesis of the octavalent aminooxy terminated core (Compound 29).

Synthesis of Compound 32, the t-butyl linker

Since the above syntheses all failed to deliver the desired hexavalent or octavalent dendrimers a new synthetic path was undertaken. A new link was synthesized to achieve

110 a easier pathway to achieve the desired core. The first part of the new synthesis involved the use of a linker which was synthesized as an undergraduate (Figure 80).68 Compound

32 adds an ether to the linker to allow for greater flexibility and hydrophilicity for the core later on.

Figure 80: The synthesis of the t-butyl linker (Compound 32)68

Synthesis of Compound 33 addition of succinimide to the linker

The first step in the alteration of the new carboxy linker was the addition of the succinimide group (Compound 33, Figure 81). The synthesis began via a Mitsunobu reaction with the t-butyl linker, where a succinimide was added to install and protect the aminooxy end. The succinimide group was chosen as previous syntheses showed that the purification was more facile and provided a cleaner product than the phthalimide approach.

Figure 81: The formation of mono succinimide of t-butyl linker (Compound 33).

111

The proton NMR for Compound 33 (Figures 82) yielded six distinct peaks with four triplets and two singlets (HA, HB, HC, HD, HE and HF). The identification of HE as the most downfield triplet is because of the proximity to the succinimide group. This will cause the CH2 protons to shift the more downfield and to have coupling constant of

J=2.07 Hz. To identify HB, HC and HD the coupling constants were used HB: J= 6.42 Hz,

HC: J= 6.44 Hz, HD: J= 2.40 Hz. Since HE already identified as the most downfield triplet

HD having a similar coupling constant made it the triplet at 3.78 ppm. Next, for HB and

HC the coupling constant shows they are adjacent to each other and since HC is next to an oxygen it will be more downfield compared to HB. Finally, the singlet HF from succinimide has four protons while the singlet HA from the t-butyl group has nine protons, allowing for their assignments to be easily made. Since the HF protons are next to a carbonyl group it will be the most downfield singlet compared to HA.

112

Figure 82: The proton NMR for Compound 33 in CD2Cl2 at 500 MHz.

The carbon NMR for Compound 33 (Figures 83) has nine distinct peaks. The addition of two new carbon signals at 171.71 (C8) and 28.36 (C9) ppm are from the succinimide group, confirming the attachment of the group to the linker. C9 is the tallest of the carbon signals, since the two methylene carbons are equivalent. C3 and C8 are the carbonyl signals. C8 is part of a ring structure that is more withdrawing on the carbonyl signal moving it more downfield. This is in contrast to the C3 carbonyl signal next to the t-butyl group, which will donate electron density moving C3 more upfield. As for C4-C7,

113

evaluating what is adjacent to these carbons will help identity each of them.C4 is only next to carbons, thus making it the most upfield of the remaining signal. C7 is next to the succinimide group, and therefore must be the most downfield signal. Finally, the assignment of C5 and C6 was more difficult, but C6 should be more downfield compared to C5 since C6 is closer to the succinimide group, deshielding C6 more. Next, C2 corresponds to the tertiary carbon neighboring the oxygen and the three methyl groups.

Finally, C1 represents the tert-butyl carbons, is the last upfield carbon signals not yet assigned.

Figure 83: The carbon NMR for Compound 33 in CD2Cl2 at 125 MHz.

114

Synthesis of Compound 34 deprotection of the carboxy linker

The last step in the synthesis of Compound 34 (Figure 84) was very simple and easy to complete. The deprotection was completed using 4 mL of TFA and 4 mL of

CH2Cl2. Upon evaporation under reduced pressure, the clear oil was slightly yellow in color. Figure 85 shows the mass spectrum for Compound 34 giving a M/Z of 270.37445

[M+K]+ with an error of 0.8ppm.

Figure 84: The deprotection of the mono-succinimide of t-butyl ester (Compound 34).

115

Figure 85: HR-ESI+ of Compound 34.

The proton NMR for Compound 34 (Figure 86) yielded four distinct peaks with two triplets, one muliplet and one singlet (HB, HC, HD, HE and HF). The identification HE as the most downfield triplet is due to its proximity to the succinimide group. This will cause a more downfield shift, and a larger coupling constant of J=3.94 Hz. After the removal of the t-butyl group, two of the triplets from Compound 34 coalesced into a multiplet at 3.79-3.76 ppm, for both HD and HC. Next, for HB, since the protons are next to a carboxylic acid, it is the most upfield proton signal, and a smaller coupling constant

116 of J= 5.92 Hz compared to the protected with a coupling constant of J= 6.40 Hz. Finally, the singlet can be identified as the ring methylene groups from the succinimide, HF.

Figure 86: The proton NMR for Compound 34 in D2O at 500 MHz.

The carbon NMR for Compound 34 (Figure 87) has seven distinct peaks. The disappearance of two carbon signals arises from the removal of the t-butyl group proves the removal of the protecting group. C9 is the tallest of the carbon signal, since the two methylene carbons are equivalent. C3 and C8 are the carbonyl signals and are now are very close to each other due to their similar chemical environments. C8 is part of a ring structure, and being more withdrawing on the carbonyl signal, it moved more downfield, while the C3 carbonyl signal is now a carboxylic acid but will be more downfield and was the shorter of the two carbonyl signals. To identify which signals correspond to C4-C7, it is necessary to examine what is adjacent to these carbons. C4 is only next to carbons, thus making it the most upfield of the remaining signals. C7 is adjacent to the succinimide

117

group, and therefore, must be the most downfield signal. Finally, assignment of C5 and

C6 was a little bit more difficult, but C6 should be more downfield compared to C5 since

C6 is closer to the succinimide group, deshielding C6 more.

Figure 87: The carbon NMR for Compound 34 in D2O with a methanol internal standard at 125 MHz.

Synthesis of Compound 38 hexavalent core

Since the original core syntheses were not successful, Compound 38 was as an alternative hexavalent core. Compound 38 was synthesized by another McReynolds groups member (Figure 88). While this core has been reacted with the short linker

(Compound 22), the reaction with the long linker has not been performed. This alternative core will be reacted with longer linker (Compound 34) Since the long linker

118 has an extra ether group, this will allow the dendrimer to have greater flexibility.

Figure 88: The synthesis of amine terminated hexavalent core (Compound 38).

Synthesis of Compound 39 through the addition of a longer linker to the hexavalent core

The synthesis of Compound 39 completes the aminooxy hexavalent core (Figure

89). Compound 34 and coupling agent TBTU were combined to create the hexavalent succinimide core. Because of the slight bulkiness of the succinimide group, the 1H NMR was not resolved sufficiently to use for structural confirmation. Therefore, the product was used in the second step of the synthesis without further characterization. In this step hydrazine monohydrate removed the succinimide group and leaves the aminooxy core.

119

After completion of the hydrazinolysis, the 1H NMR showed both DMSO and succinimide were present as impurities, thus an extraction was performed, followed by dialysis (500-1000 MWCO) followed by FPLC. This resulted in a 31% yield. Since the yield was so low, the reaction was performed again, however, this time no extraction was performed. This resulted in a 23% yield. Since the reaction times between these two reactions differed by 10 hours, is it unclear how the purification steps may have affected the yields. Another reaction was completed after 48 hours, using only FPLC and dialysis for purification. This resulted in a yield of 68%. By increasing the time, and using dialysis and FPLC to purify the reaction, the yield was improved from 31% to 68%.

Figure 90 shows the mass spectrum for Compound 39 giving a M/Z of 1702.079660 with an error of 0.04ppm.

120

Figure 89: The synthesis of aminooxy terminated hexavalent core (Compound 39).

121

Figure 90: HR-ESI of Compound 39.

The proton NMR for Compound 39 (Figure 91) yielded eleven distinct peaks with ten triplets and one pentet (HB, HC, HD, HE, HG, HI, HJ, HK, HL, HM, and HN). The identification of HE as the most downfield triplet was due to the proximity to the aminooxy group. This caused a more downfield shift with a coupling constant of J= 4.20

Hz. The identity of the signal at 3.69 ppm signal was HD, since the coupling constant is similar HE. Another signal that is facile to assign was HM, the pentet, where the coupling constant is J=6.87 Hz. The identity of the signals at 2.82 and 2.54 ppm with coupling

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constants of J= 6.93 and 6.03 HZ must be HL and HN, because the coupling constants between them and HM are similar, and they are in a similar chemical environment. Since

HC is a proton next to oxygen, it should be more downfield. The signal at 3.78 ppm has a coupling constant of J= 6.23 Hz and a signal at 2.54 ppm was coupling with HB protons.

HI, HJ and HG are next to nitrogen, while HK is next to a carbonyl, and since the nitrogen is more of an EWG compared to the carbonyl, HI, HJ and HG will be more downfield compared to HK. Also, since HI, HJ and HG are in similar chemical environments they have coalesced into one larger apparent quartet.

Figure 91: The proton NMR for Compound 39 in D2O at 500 MHz.

In the carbon NMR for Compound 39 (Figure 92) there should be thirteen separate carbon signals, however the NMR only shows nine carbon signals. This was due to a couple of the signals being equivalent. Since not all of the carbon signals were present it was difficult to assign each of the carbons with confidence.

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Figure 92: The carbon NMR for Compound 39 in D2O with a methanol internal standard at 125 MHz.

Synthesis of Compound 40 the cellobiose glycodendrimer

With the completion of the synthesis of the hexavalent aminooxy-terminated core oxime coupling with cellobiose was next undertaken (Figure 93). The reaction was conducted using two different microwaves. The first reactions were carried out at 28-30

% of 400 Watts with a 2.5 minute ramp to 65˚C and a hold for 30 minutes (CEM

MARS5, 4 total cycles). This microwave setting only allowed for a 32.8 % yield of the fully glycosylated core. The other microwave (Discover® SP) setting was 100 % of 7W to hold at 65˚C for 30 minutes. (5 total cycles) This microwave setting allowed for a 77.2 % yield of fully glycosylated core. Figure 94 shows the mass spectrum for Compound 40

124 giving a M/Z of 3647.73933 [M+H]+ with an error of 6.04ppm.

Figure 93: Oxime coupling with cellobiose and the hexavalent aminooxy terminated core (Compound 39).

125

Intens . 33266_DD6_69_F36_F46_MALDI_300_4000_pos_4M_15P_0_M2_000001.d: +MS x107 3647.73933

1.0

0.8

3620.80136

0.6

3494.69876 3662.84910

3717.01671

3519.61985 0.4

3588.92006 3772.05619 3577.90211

3546.74343

0.2 3862.95751

3746.01199

3800.04195 3834.90601

0.0 3500 3550 3600 3650 3700 3750 3800 3850 m/z

Figure 94: HR-MALDI-TOF of Compound 40.

Figure 95 shows the 1H NMR for Compound 40. Since the product is very large, the peaks were integrated for 1/3 of the full compound. Distinct peaks that are similar can be seen at 4.58 ppm representing equatorial protons from the anomeric carbon on cellobiose. There are also peaks at 7.66 and 6.99 ppm corresponding to the vinylic protons of the E and Z stereoisomers in the open form of the oxime linkage of the sugar ring. There are five broad triplets between 2.83-2.42 ppm integrating for 16 protons which correspond to the methylene protons from the linker and core. The broad pentet at

126

1.72 ppm integrates for 4 protons and corresponds to the methylene protons of the core

(HM).

Figure 95: The proton NMR for Compound 40 in D2O at 500 MHz.

The carbon NMR for Compound 40 (Figure 96) shows many distinct peaks. As it was with Compound 39, it’s very difficult to identity each of the individual carbon peaks.

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Figure 96: The carbon NMR for Compound 40 in D2O with a methanol internal standard at 125 MHz.

Synthesis of Compound 41 the dimer of colominic acid glycodendrimer

Oxime coupling between the dimer of colominic acid (Compound 13) and the hexavalent aminooxy terminated core (Compound 39) to produce a second hexavalent dendrimer (Compound 41) was next undertaken (Figure 97). The reaction was conducted using microwave irradiation (Discover® SP). The microwave setting was 100

% of 7W to hold at 65˚C for 30 minutes (5 total cycles). This approach allowed for a 79.2

% yield of fully glycosylated core Figure 98 shows the mass spectrum for Compound

40, which is inconclusive if fully substituted further work is need to find this out.

128

Figure 97: Oxime coupling with the dimer colomnic acid (Compound 13) and the hexavalent aminooxy terminated core (Compound 39) to yield Compound 41.

129

Intens . 33266_DD6_69_F27_F35_MALDI_pos_4M_100_6000_15P_0_M1_000001.d: +MS x108 1+ 2501.24783

1+ 1903.03988

1+ 2210.15724

1+ 2807.36007

1+ 3098.44649 2

1+ 1305.83962 1+ 1611.94325 1+ 1+ 2367.19303 2060.13369

1+ 1768.98420 1+ 3404.56297 1+ 3695.66213

0 1250 1500 1750 2000 2250 2500 2750 3000 3250 3500 3750 m/z

Figure 98: HR-MALDI-TOF of Compound 41.

The proton NMR of Compounds 41 (Figure 99) shows the addition of the dimer of colomnic acid and core peaks. Since the glycodendrimer compounds are very large, high proton counts can happen. This is due to the high proton count that comes from

Compound 41. Therefore, for this spectrum 1/3 of the total proton count confirmed the identity of the glycodendrimer. Due to the enormous size of glycodendrimer, the peaks associated with the core protons became more broadened, because the relaxation of the core protons is too slow for the NMR pulse sequence, and resolution is lost as a result.

Therefore, it was difficult to integrate all of the peaks and obtain accurate proton counts.

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The integral ratio of the number of the acetate peak protons associated with the dimer of colomnic acid residues and the number of the protons associated with the pentet and anomeric carbon peak dendrimers were utilized to determine the number of dimer residues added. For Compound 41, the expected ratio of (HY+HM)/(HX) protons was

8:12 for the fully substituted glycodendrimer and a ratio of 8:12 was observed, indicating fully substituted compound.

Figure 99: The proton NMR for Compound 41 in D2O at 500 MHz.

The carbon NMR for Compound 41 (Figure 100) shows well over many distinct peaks. As it was with Compound 39, it is very difficult to identity each of the carbon peaks. The peaks for sugar and core have been assigned.

131

Figure 100: The carbon NMR for Compound 41 in D2O with a methanol internal standard at 125 MHz.

132

Chapter 3:

Conclusions and Future Work:

HIV/AIDS has created a worldwide epidemic. An estimated 36.9 million people are infected with HIV.1 The current treatment for HIV is ART. As of now, ART can only slow the progression of the virus but doesn’t cure the disease. However, ART can have toxic side effects or become ineffective over time due to viral resistance. Therefore, finding a way to prevent HIV infections is vital. One area of research under development in recent years are potent anti-HIV drugs that can inhibit HIV entry. It has been established that sulfated molecules have an affinity to HIV through ionic interactions. In our research, the dendrimers terminate in aminooxy groups, which can then react chemoselectively with sugars to yield oxime-linked glycodendrimers. After the completion of the synthesis, these glycodendrimers are being used to determine the best structures that can ultimately inhibit HIV entry.

This study entailed the exploration of two failed and one effective route in synthesizing dendrimer cores. Unfortunately, the linker would not react with the hexavalent core so none of the desired product was obtained. Additionally, the aminooxy linker was unable added under carbamate coupling. Thus, not possible to achieve the desired octavalent aminooxy-terminated dendrimer core, so no glycodendrimers could be made by this route. For the successful dendrimer core synthesis, Compound 34, an aminooxy linker, with a yield of 99.8%. From the synthesis of the hexavalent aminooxy- terminated core (Compound 39), it was determined that purification could be accomplished using only dialysis and FPLC to achieve the best yield of 68%. Finally, for

133 both the cellobiose and dimer of colominic acid glycodendrimers (Compounds 40 and

41) the use of a smaller microwave cavity allowed for excellent yields of 77.8 and 79.2%, respectively. Further research needs to be conducted on this new core. The addition of different sugars and the optimization of the coupling to further increase the yield of the hexavalent aminooxy terminated core is needed.

In this research, two glycodendrimers, Compounds 40 and 41, were created.

Once sulfated, the glycodendrimers will be tested using an in-house ELISA. The ELISA is a competitive gp120 binding assay used to screen the glycodendrimers for binding affinity.56 If Compounds 40 and 41 exhibit positive binding affinity for gp120, they can be tested on active viral particles using a luciferase reporter gene assay to determine HIV inhibition of infectivity at Duke University.64 Finally, Compounds 40 and 41 will be compared to other glycodendrimer systems synthesized in our lab to see if there are differences between the enhanced flexibility of the core used here and inhibition of HIV.

While it is important to find treatments for HIV patients, it is also important to help find ways to protect uninfected individuals through the development of novel microbicide inhibitors.

134

Chapter 4:

Experimental

Methods and Materials

General

Identification of products was made by analyzing 1H NMR, spectra produced by

Bruker Advance III 500 MHz. NMR spectrometer in either CDCl3, D2O, C6D6 or

CD3OD. Reversed phase high pressure liquid chromatography (RP-HPLC) was conducted using an HP 1050 Series with a GRACE Prevail C18 5 µm x 250mm prep column with 0.1% TFA (v/v) in nanopure water with a linear gradient to 0.1% TFA (v/v) in acetonitrile. Fast pace liquid chromatography (FPLC) was completed in a 2.5 x 120 cm

Biogel P-10 column with 0.03 M NH4HCO3 with a Pharmacia LC Controller LCC-500

Plus and a P-500 pump. All microwave reactions were performed on a CEM MARS 5 or

Discover® SP microwave. The ESI-MS and MALDI-MS were obtained from Ohio State

University, Mass Spectrometry & Proteomics Facility.

135

Synthesis of Compound 2.A Compound 2.A was synthesized using Compound 1 (951.3 mg, 12.5 mmol), PPh3 (1.31 g, 5.00 mmol), and N-hydroxyphthalimide (830.7 mg, 5.00 mmol). To an oven-dried round-bottomed flask containing the above solids, 150 mL of THF was added until all of the solids were dissolved. Then, diisopropyl azodicarboxylate (DIAD, 0.983 mL, 5.00 mmol) was added dropwise. The reaction mixture was allowed to stir for 48 hours at room temperature, followed by evaporation under reduced pressure. The crude solid was purified via a flash column in 2:1 EtOAc: Hexanes. A slightly impure yellowish oil

1 (1.104 g, 99.9%) resulted. H NMR (500 MHz, CDCl3): d 7.80 (d, J = 2.20 Hz, 2H), 7.73

(d, J = 1.85 Hz, 2H), 4.35 (t, J = 5.40 Hz, 2H), 3.90 (t, J = 5.25 Hz, 2H), 2.80 (s, 1H),

13 1.98 (pentet, J =11.00, 5.50 Hz, 2H) C NMR (125 MHz, CDCl3): d 163.83,134.65,

128.83, 123.67, 76.02, 59.09, 30.74.

Synthesis of Compound 2B

Compound 2B was synthesized using Compound 1 (380.5 mg, 5.0 mmol), PPh3 (655.5 mg, 2.5 mmol), and N-hydroxysuccinimide (287.7 mg, 2.5 mmol). To an oven-dried round-bottomed flask containing the above solids, 150 mL of THF was added until all of the solids were dissolved. Then, diisopropyl azodicarboxylate (DIAD, 0.492 mL, 2.5 mmol) was added dropwise. The reaction mixture was allowed to stir for 24 hours at room temperature, followed by evaporation under reduced pressure. The crude oil was purified via liquid – liquid extraction by rinsing a CHCl3 layer 3 times with nanopure

136

water to remove any unreacted DIAD and PPh3. After the extraction, the aqueous layer was freeze-dried. A clear and colorless oil resulted (366.3 mg 84.07%). 1H NMR (500

MHz, CDCl3): d 4.23 (t, J = 5.40 Hz, 2H), 3.85 (t, J = 5.25 Hz, 2H), 2.70 (s, 1H), 1.90

13 (pentet, J =11.00, 5.50 Hz, 2H) C NMR (125 MHz, CDCl3): d 175.97, 75.03, 58.37,

49.14, 30.38, 25.62.

Synthesis of Compound 3A

Compound 3A was synthesized by adding 41.2 mg (0.186 mmol) of Compound 2A into

2.0 mL of CH2Cl2. Then, pyridine (0.0885 mL) was introduced to make the solution basic. Next, mesyl chloride (78.8 mg, 0.688 mmol) was added, and the reaction mixture was allowed to stir at room temperature for 24 hours, followed by evaporation under reduced pressure. An extraction was performed with 1M HCl and water to remove any unreacted mesyl chloride and pyridine, and the organic layer was dried with anhydrous

1 Na2SO4. A pure white solid compound was collected (36.7 mg, two-step 65.8% yield) H

NMR (500 MHz, CDCl3): d 7.82 (d, J =2.70 Hz, 2H) 7.76 (d, J = 2.75 Hz, 2H) 4.53 (t, J

= 5.80 Hz, 2H) 4.33 (t, J = 5.60 Hz, 2H) 3.11 (s, 3H) 2.20 (pentet, J = 11.40, 5.70 Hz,

13 2H) C NMR (125 MHz, CDCl3): d 163.53, 134.67, 128.84, 123.84, 73.80, 66.31, 37.14,

28.14.

Synthesis of Compound 3B

Compound 3B was synthesized by adding 183.15 mg (1.058 mmol) of Compound 2B into 15.0 mL of CH2Cl2. Then, pyridine (0.3406 mL) was introduced to make the solution basic. Next, mesyl chloride (484.9 mg, 4.233 mmol) was added, and the reaction mixture

137 was allowed to stir at room temperature for 24 hours, followed by evaporation under reduced pressure. An extraction was performed with 1M HCl and water to remove any unreacted mesyl chloride and pyridine, and the organic layer was dried with anhydrous

1 Na2SO4. A clear, colorless oil compound was collected (199.2 mg, 78.7%). H NMR (500

MHz, CDCl3): d 4.45 (t, J = 5.80 Hz, 2H) 4.34 (t, J = 5.60 Hz, 2H) 3.06 (s, 3H) 2.69 (s,

13 4H) 2.13 (pentet, J = 11.40, 5.70 Hz, 2H) C NMR (125 MHz, CDCl3): d 171.32, 77.48,

77.23, 76.97, 72.92, 66.48, 37.26, 28.28, 25.65.

Synthesis of Compound 5A

Compound 5A was synthesized using Compound 4 (975 mg, 5.00 mmol), PPh3 (1.31 g,

5.00 mmol), and N-hydroxyphthalimide (854.3 mg, 5.00 mmol). To an oven-dried, round-bottomed flask containing the above solids, 350 mL of THF was added and the mixture was stirred until all of the solids were dissolved. Next, DIAD (0.989 mL, 5.00 mmol) was added dropwise. The reaction mixture was allowed to stir for 24 hours at room temperature, followed by evaporation under reduced pressure. The crude solid was first purified via a flash column in 2:1 EtOAc: Hexanes and then a second flash column in 6:1 EtOAc: Hexanes. A slightly impure white solid (887.5 mg 75.2%) resulted. 1H

NMR (500 MHz, CDCl3): d 7.80 (d, J = 3.15 Hz, 2H) 7.71 (d, J =3.10 Hz, 2H) 4.35 (t, J

= 6.25 Hz, 2H) 3.86 (t, J =6.30 Hz, 2H) 3.73 (t, J = 5.75 Hz, 2H) 3.52 (t, J = 5.90 Hz,

13 2H) C NMR (125 MHz, CDCl3):d 163.81, 134.83, 129.27, 123.87, 71.78, 69.74, 42.95.

138

Synthesis of Compound 5B

Compound 5B was synthesized using Compound 4 (265.3 mg, 2.50 mmol), PPh3 (655.7 mg, 2.5 mmol), and N-hydroxysuccinimide (287.7 mg, 2.50 mmol). To an oven-dried, round-bottomed flask containing the above solids, 150 mL of THF was added and the mixture was stirred until all of the solids were dissolved. Next, DIAD (0.492 mL, 2.50 mmol) was added dropwise. The reaction mixture was allowed to stir for 24 hours at room temperature, followed by evaporation under reduced pressure. The crude oil was purified via liquid – liquid extraction by rinsing a CHCl3 layer 3 times with nanopure water to remove any unreacted DIAD and PPh3. After the extraction, the aqueous layer was freeze-dried. A clear and colorless oil resulted (861.3 mg, 84.7%) resulted. 1H NMR

(500 MHz, CDCl3): d 4.27 (t, J = 6.25 Hz, 2H) 3.77 (t, J =6.30 Hz, 2H) 3.71 (t, J = 5.75

13 Hz, 2H) 3.59 (t, J = 5.90 Hz, 2H) 2.70 (s, 4H). C NMR (125 MHz, D2O, internal MeOH std): d 175.67, 76.04, 71.91, 68.83, 60.44, 48.96, 25.27, 21.19

Synthesis of Compound 6A

Compound 6A was synthesized by adding 100 mg (0.398 mmol) of Compound 5A into

3.0 mL of CH2Cl2. Then, pyridine (0.128 mL) was introduced to make the solution basic.

Next, mesyl chloride (114.0 mg, 0.995 mmol) was added and the reaction mixture was allowed to stir at room temperature for 48 hours, followed by evaporation under reduced pressure. An extraction was performed with 1M HCl and water to remove any unreacted mesyl chloride and pyridine and the organic layer was dried with anhydrous Na2SO4. The crude solid was purified via a flash column in 2:1 EtOAc: Hexanes. A pure white solid

139

1 compound was collected (65.9 mg, 50.3%). H NMR (500 MHz, CDCl3): d 7.80 (d,

J=3.10 Hz, 2H) 7.73 (d, J =3.10 Hz, 2H) 4.33 (t, J= 4.20 Hz, 2H) 4.32 (t, J= 2.60 Hz, 2H)

3.83 (t, J = 4.20 Hz, 2H) 3.77 (t, J = 2.65 Hz, 2H) 3.03 (s, 3H) 13C NMR (125 MHz,

CDCl3): d 163.46, 134.66, 128.84, 123.62, 76.86, 69.27, 69.18, 69.17, 37.69.

Synthesis of Compound 6B

Compound 6B was synthesized by adding 522.8 mg (3.021 mmol) of Compound 5 into

50.0 mL of CH2Cl2. Then, pyridine (0.9719 mL) was introduced to make the solution basic. Next, mesyl chloride (1.384 g, 12.08 mmol) was added and the reaction mixture was allowed to stir at room temperature for 24 hours, followed by evaporation under reduced pressure. An extraction was performed with 1M HCl and water to remove any unreacted mesyl chloride and pyridine and the organic layer was dried with anhydrous

1 Na2SO4. A pure colorless oil was collected (898.2 mg, 84.02%). H NMR (500 MHz,

CDCl3): d 4.37 (t, J= 2.50 Hz, 2H), 4.28 (t, J= 2.00 Hz, 2H) 3.84 (t, J = 1.95 Hz, 2H),

13 3.75 (t, J = 2.50 Hz, 2H), 3.09 (s, 3H), 2.72 (s, 4H) C NMR (125 MHz, CDCl3): d 171.23, 77.48, 77.23, 76.97, 69.97, 69.31, 41.41, 37.96, 29.91, 25.69, 22.16.

Synthesis of Compound 768-69

Compound 7 was synthesized by adding 326 mg (3.24 mmol) of Core N into a dry round-bottomed flask. Next, 0.3 mL of NaOH (30 mg of NaOH, 0.3 mL of water) was added, followed by adding 5.24 mL (81 mmol) of acrylonitrile, dropwise. The reaction mixture was allowed to stir for 24 hours at 45°C. To stop the reaction, 6M HCl was

140 added to change the pH from 10 to 1. After acidifying the reaction, it was purified using an extraction with CHCl3 to remove any unreacted triethanolamine and acrylonitrile.

After the extraction, the aqueous layer was freeze-dried. This resulted in 775.0 mg

1 (77.5%) of a pure yellow oil product. H NMR (500 MHz, D2O): d 3.94 (t, J= 5.10 Hz,

2H) 3.80 (t, J= 5.80 Hz, 2H) 3.63 (t, J= 5.05 Hz, 2H) 2.82 (t, J= 5.85 Hz, 2H) 13C NMR

(125 MHz, D2O, internal MeOH std): d 115.35, 60.86, 60.62, 59.32, 13.60. HR-ESI: Calc

+ for C15H25N4O3 [M+H] 309.19265. Found: 309.19212.

Synthesis of Compound 8

Compound 8 was synthesized from Compound 7 (602.5 mg, 1.96 mmol). First

Compound 7 was dissolved in 20 mL of dry methanol. (BOC)2O (5.13 g, 23.5 mmol) and nickel chloride hexahydrate (373.2 mg, 0.8 mmol) were also added to the round- bottomed flask. Finally, sodium borohydride (1.56 g, 41.2 mmol) was added slowly to the solution. The reaction mixture was stirred at room temperature for 21 hours under nitrogen, then evaporated under reduced pressure. Next, an extraction was performed three times using CHCl3 and saturated NaHCO3. The organic layer was washed with brine and allowed to dry over anhydrous Na2SO4. A pure yellow oil (787.0 mg, 64.7 %)

1 resulted. H NMR (500 MHz, D2O): d 5.03 (s, 2H) 3.44 (s, 2H) 3.18 (s, 2H) 2.73 (s, 2H)

1.40 (s, 9H).

Synthesis of Compound 9

141

Compound 9 was placed in a dry 25 mL round-bottomed flask in the presence of 2 mL

CH2Cl2 and 2 mL of TFA and allowed to stir for two hours at room temperature. The solution was then evaporated under reduced pressure. A brown oil was collected (861.2

1 mg, 86.1 %) H NMR (500 MHz, D2O): d 3.75 (t, J= 4.65 Hz, 2H) 3.55 (t, J= 6.00 Hz,

2H) 3.45 (t, J= 4.70 Hz, 2H) 2.99 (t, J= 6.98 Hz, 2H) 1.85 (pentet, J= 13.70, 6.95 Hz,

13 2H) C NMR (125 MHz, D2O, internal MeOH std): d 68.46, 64.30, 53.55, 49.13, 37.53,

+ 26.91. HR-ESI: Calc for C15H37N4O3 [M+H] 321.28654. Found: 321.28597.

Synthesis of Compound 18 68-69

Compound 18 was synthesized by adding 23.45g (378 mmol) of ethylene glycol into a dry round-bottomed flask. Next, 0.3 mL of 10% (w/v) NaOH was added, followed by adding 6.25 mL (94.3 mmol) of acrylonitrile, dropwise. The reaction mixture was allowed to stir for 5 hours at 45°C. To stop the reaction, 6M HCl was added to change the pH from 10 to 7. After neutralization, the product was purified by flash chromatography in 9:1 ethyl acetate: hexanes. This resulted in a pure colorless oil product (9.13g,

1 95.2%). H NMR (500 MHz, D2O): d 3.80 (t, J= 6.0 Hz, 2H), 3.75 (t, J= 4.0 Hz, 2H), 3.69

13 (t, J= 3.1 Hz, 2H), 2.80 (t, J= 6.0 Hz, 2H) C NMR (125 MHz, D2O, internal MeOH std): d 120.09, 71.94, 65.49, 60.53, 18.30.

Synthesis of Compound 19

142

Compound 19 was synthesized using Compound 18 (500 mg, 4.34 mmol) in 40 mL of methanol. Tert-butyloxycarbonyl anhydride ((BOC)2O) (1892 mg. 8.67 mmol) and nickel chloride hexahydrate (103 mg, 0.434 mmol) were also added to the round-bottomed flask. Finally, sodium borohydride (1150 mg, 30.4 mmol) was added slowly to the solution. The reaction was stirred from 0°C to room temperature for 22 hours. Next, tris(2-aminoethyl) amine (0.650 mL, 4.43 mmol) was added and stirred for 30 minutes at room temperature before it was evaporated under reduced pressure. Then an extraction was performed three times using CHCl3 and saturated NaHCO3. The organic layer was washed with brine and allowed to dry over anhydrous Na2SO4 for an hour. A pure

1 colorless oil of 726.7 mg, 82.6 %, resulted. H NMR (500 MHz, D2O): d 3.63 (t, J= 4.5

Hz, 2H), 3.63-3.58 (m, 4H), 3.172 (t, J= 6.7 Hz, 2H), 1.78 (pentet, J= 13.1, 6.6 Hz, 2H),

13 1.45 (s, 9H) C NMR (125 MHz, D2O, internal MeOH std): d 158.36, 80.69, 71.81,

68.66, 60.79, 37.39, 29.35, 27.99.

Synthesis of Compound 20

Compound 20 was synthesized by adding 1.000 g (8.68 mmol) of Compound 19 into 20 mL of CH2Cl2. Next, pyridine (1.468 mL) was introduced to make the solution basic.

Mesyl chloride (114.0 mg, 0.995 mmol) was added and the reaction mixture was allowed to stir at room temperature for 48 hours, followed by evaporation under reduced pressure.

An extraction was performed with 1M HCl and water to remove any unreacted mesyl chloride and pyridine, and the organic layer was dried with anhydrous Na2SO4. Then, the product was purified by flash chromatography in 3:1 hexanes: EtOAc. A pure brown oil

1 compound was collected (1.1615 g, 50% yield). H NMR (500 MHz, D2O): d 4.76 (s, H)

143

4.35 (t, J= 2.05 Hz, 2H) 3.69 (t, J= 1.95 Hz, 2H) 3.56 (t, J= 5.60 Hz, 2H), 3.22 (t, J= 5.21

Hz, 2H) 3.06 (s, 3H) 1.77 (p, J= 10.50, 5.50 Hz, 2H) 1.44 (s, 9H) 13C NMR (125 MHz,

D2O, internal MeOH std): d 156.02, 69.51, 68.89, 68.64, 68.55, 38.38, 37.69, 29.81,

28.43.

Synthesis of Compound 2267

Compound 22 was synthesized by adding 1.092 g (9.99 mmol) of Compound 21 into a

50 mL solution of water/MeOH (3/2 v/v). Next, 1.12 g (20.0 mmol) of KOH was added to make the solution basic. 4.356 g (20.0 mmol) of (BOC)2O was then added, and the reaction mixture was allowed to stir at room temperature for 24 hours. The reaction was checked to see if the pH was around 10 over the day. If the pH fell below 9, more KOH was added until the pH was back to10. The solution was then evaporated under reduced pressure. The solid was taken up into 10 mL of nanopure water, placed in an ice bath, then 6 M HCl was added until the pH of the solution was 3. The precipitate was then

1 filtered off, yielding 800 mg (80%) of a white solid. H NMR (500 MHz, D2O): d 4.49 (s,

13 2H) 1.51 (s, 9H) C NMR (125 MHz, D2O, internal MeOH std): d 173.36, 158.43, 83.87,

72.88, 27.46.

Synthesis of Compound 24

144

Compound 24 was synthesized in a two-step process. Step one was conducted using 362.0 mg (1.22 mmol) of compound 20 and 45.0 mg (0.201 mmol) of Core S. The reaction occurred in the presence of K2CO3 (563.0 mg, 4.08 mmol), and 35 mL of acetonitrile, and was allowed to reflux under N2 (g) for 7 days. The pH was checked after

4 days, since the pH was at 7 more K2CO3 (515.0 mg) was added and the pH was about 9.

To stop the reaction, the solution was filtered through Celite to remove the solid K2CO3.

An extraction was next performed with water to remove any unreacted meyslated linker.

The organic layer was rotary evaporated under reduced pressure and 328.9 mg of a

1 slightly impure brownish oil was obtained. H NMR (500 MHz, C6D6): d 5.05 (s, 1H)

3.60 (t, J = 5.2 Hz, 4H) 3.18-2.74 (m, 4H) 1.71 (p, J = 12.3, 6.2 Hz, 2H) 1.40 (s, 9H). 13C

NMR (125 MHz, C6D6): d 156.43, 128.57, 128.37, 128.18, 78.69, 78.07, 69.59, 55.55,

54.78, 39.08, 30.61, 29.00. In the second step, the intermediate (328.9 mg, 0.333 mmol) was dissolved in 2 mL of TFA and 2 mL of CH2Cl2. The reaction mixture was allowed to stir for two hours. The solution was then evaporated under reduced pressure and

1 Compound 24. Yielding in 334.8 mg 96.8 % brown oil. H NMR (500 MHz, D2O): d

3.87 (t, J= 4.92 Hz, 2H), 3.69-3.65 (overlapping, 3H), 3.55 (t, J= 5.01 Hz, 2H), 3.15-3.09

13 (overlapping, 3H), 1.98 (p, J= 16.63, 6.20 Hz, 2H) C NMR (125 MHz, CD3OD): d

69.64, 65.93, 54.76, 50.00, 49.66, 49.49, 49.32, 49.15, 48.98, 48.81, 48.64, 38.71, 31.52,

+ 28.51. HR-ESI: Calc for C24H57N6O4S2 [M+H] 557.38824 . Found: 557.38763.

Synthesis of Compound 28

145

Compound 28 was synthesized in a three-step process. In the first step, 103.6 mg

(0.0993 mmol) of Compound 24 was placed into a round bottomed flask. Then 0.496 mL

(7.45 mmol) of acrylonitrile and 0.103 mL (0.598 mmol) of N,N-Diisopropylethylamine

(DIPEA) was added to make the solution basic. The reaction mixture was stirred at room temperature. After 72 hours, an additional 0.496 mL (7.45 mmol) of acrylonitrile was added. At 96 hours, the solution was rotary evaporated to remove any unreacted acrylonitrile and then dialyzed for 2 hours in 100 MWCO tubing against 1L of water. IR

-1 (CHCl3): 1133, 1199, 1671, 2250, 2994 and 3018 cm . In the second step, 42.5 mg

(0.0433 mmol) and anhydrous MeOH (2.0 mL) were combined in a round bottomed flask

(10 mL) and stirred in an ice bath. After the acetyl chloride (1.6 mL) was added, the reaction was stirred overnight at room temperature. After 24 hours, the reaction was stopped, then was evaporated. 81.7 mg (151% yield) of a brown oil was recovered after lyophilization. IR (acetone): 1220, 1358, 1419, 1708 and 3004 cm-1. In the third step, 1,3- diaminopropane (1.48 mL, 17.6mmol) was added and the reaction mixture was stirred at room temperature for 72 hours. The solution was then filtered through Celite and evaporated under reduced pressure to remove the excess1,3-diaminopropane. Compound

28 was purified via HPLC yielding 43.4 mg (85% three-steps) slightly impure brown oil

1 compound. H NMR (500 MHz, D2O): d 3.96 (bs, 2H), 3.84 (bs, 3H), 3.75-3.65 (overlap,

6H), 3.46-3.11 (overlap, 7H), 3.07 (t, J= 7.65 Hz, 4H), 3.00 (bs, 4H), 2.06 (d, J= 7.10

13 Hz, 4H), 1.87 (bs, 4H) C NMR (125 MHz, D2O, internal MeOH std): d 172.34, 50.04,

49.37, 37.54, 36.77, 29.12, 27.01, 25.30, 23.88, 18.74, 16.35.

Synthesis of Compound 3368

146

Compound 33 was synthesized by adding 10.312 g (166.1 mmol) of ethylene glycol into a dry round-bottomed flask. Next, 3.0 mL of 40% KOH (w/v) was added, followed by

6.39 mL (41.5 mmol) of tert-butyl acrylate, dropwise. The reaction mixture was allowed to stir for 5 hours at 45°C. To stop the reaction, approximately 20 drops of 6M HCl was added to change the pH from 10 to 7. After neutralizing the reaction mixture, an extraction was performed three times using CHCl3 and water. The organic layer was dried with anhydrous Na2SO4. The organic layer was next evaporated under reduced pressure and purified by flash chromatography in 2:1 EtOAc: hexanes. This resulted in 3.146g

1 (39.9%) of a colorless oil product. H NMR (500 MHz, CDCl3): d 3.59-3.54 (m, overlapping, 4H), 3.41 (t, J= 4.72 Hz, 2H), 3.21 (s, 1H), 2.36 (t, J= 6.31 Hz, 1H), 1.30 (s,

9H)

Synthesis of Compound 34

Compound 34 was synthesized by adding 2.531 g (13.3 mmol) of Compound 33, PPh3

(3.831 g, 14.6 mmol), and N-hydroxysuccinimide (1.680 g, 14.6 mmol). To an oven- dried, round-bottomed flask containing the above solids, 150 mL of THF was added and the mixture was stirred until all of the solids were dissolved. Next, DIAD (2.9 mL, 14.6 mmol) was added dropwise. The reaction mixture was allowed to stir for 24 hours at room temperature, followed by evaporation under reduced pressure. The crude solid was first purified via a flash column in 2:1 EtOAc: Hexanes. A pure colorless oil compound

1 was collected (3.401g, 89.9%). H NMR (500 MHz, CD2Cl2): d 4.19 (t, J= 4.40 Hz,2H),

3.71 (t, J= 2.40 Hz, 2H), 3.64 (t, J= 6.40 Hz, 2H), 2.64 (s, 4H), 2.43 (t, J= 6.40 Hz, 2H),

147

13 1.42 (s, 9H) C NMR (125 MHz, CD2Cl2): d 171.71, 171.12, 80.91, 76.31, 69.81, 67.54,

54.42, 54.20, 53.99, 53.77, 53.56, 36.77, 28.37, 26.04

Synthesis of Compound 35

Compound 35 was synthesized by adding 1.4108 g (4.91 mmol) of Compound 34 by placed in a dry 100 mL round-bottomed flask in the presence of 4 mL CH2Cl2 and 4 mL of TFA and allowed to stir for two hours at room temperature. The solution was then evaporated under reduced pressure. A clear-yellowish oil was collected (1.320 g, 99.8%)

1 H NMR (500 MHz, D2O): d 4.25 (t, J= 3.94 Hz, 2H), 3.79-3.76 (m, overlapping, 4H),

13 2.77 (s, 4H), 2.65 (t, J= 5.92 Hz, 2H) C NMR (125 MHz, D2O, internal MeOH std): d 176.17, 175.62, 75.90, 68.71, 66.44, 48.99, 34.38, 25.43. HR-ESI: Calc for C9H13KNO6

[M+K]+ 270.03798. Found: 270.37445.

Synthesis of Compound 39

Compound 39 was synthesized in a two-step process. Step one was conducted using 49.5 mg (0.0452 mmol) of Compound 38. Then 73.0 mg (0.316 mmol) of

Compound 34, was added in 2 mL of DMSO with 101.5 mg (0.316 mmol) of O-

(Benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate (TBTU) with 1 mL of DIPEA to adjust the pH to 9, and stirred at room temperature for 2 days. The reaction was then freeze-dried to remove DMSO. Next, 3 mL of MeOH was placed in a round- bottomed flask. Hydrazine hydrate (0.434 mL, 8.91 mmol) was then added dropwise. The reaction mixture was allowed to stir overnight at room temperature. Then reaction was purified by dialysis using a 500-1000 molecular weight for 4 hours, followed by FPLC. A

148 pure brown-white solid compound was collected with 52.4 mg (68.2%) yield. 1H NMR

(500 MHz, D2O): d 4.14 (t, J=4.31 Hz, 0.4H), 7.23 Hz. 3.85 (t, J= 4.20 Hz, 3H), 3.78 (t,

J= 6.23 Hz, 4H), 3.69 (t, J= 4.25 Hz, 4H), 3.61 (t, J= 5.02 Hz, 0.4H), 3.22 (t, J= 6.98 Hz,

8H), 2.82 (t, J= 6.93 Hz, 5H), 2.65 (s, 4H), 2.54 (t, J= 6.03 Hz, 4H), 2.43 (t, J= 7.22 Hz,

13 4H), 1.72 (p, J= 6.83, 13.68 Hz, 4H) C NMR (125 MHz, D2O, internal MeOH std): d 174.70, 174.04, 74.42, 68.57, 66.97, 49.40, 49.00, 36.88, 32.81, 28.10MALDI: Calc

+ for C72H145N22O24 [M+H] 1702.08012. Found: 1702.07960.

Synthesis of Compound 40

Compound 40 was synthesized using 13.0 mg (0.00764 mmol) of Compound 39 plus

20.2 mg (0.0590 mmol) of Compound 12. These were dissolved in 1.5 mL of 0.1 M ammonium acetate (NH4OAc) at a pH of 4.5, followed by 14.5 µL of aniline (0.150 M).

The reactions were conducted in either 400 W microwave (CEM MARS 5) at 25% power with a 2-minute ramp to temperature and a hold time of 30 minutes at a maximum temperature of 50˚C, or at7W (Discover® SP) with a 30 min hold time and 65 ˚C temperature. After the reaction was complete, the solutions were freeze-dried. Then reaction was purified by dialysis using a 500-1000 molecular weight for 4 hours, followed by FPLC. A pure brown-white solid compound was collected with 20.2 mg

1 74.2%. . H NMR (500 MHz, D2O): d 7.67 (d, J= 5.45 Hz, 0.5H), 7.00 (d, J= 5.70 Hz,

0.1H), 4.60-4.50 (m, 2H), 4.30-4.13 (m, 4H), 3.99-3.91 (m, 5H), 3.89-3.60 (m, 12H),

3.51-3.35 (m, 6H), 3.22 (t, J= 2.40 Hz, 8H), 2.82 (t, J= 7.25 Hz, 5H), 2.64 (s, 4H), 2.51

(t, J= 5.65 Hz, 4H), 2.43 (s, 4H), 1.72 (p, J= 6.35, 11.55 Hz, 4H) 13C NMR (125 MHz,

D2O, internal MeOH std): d 174.59, 173.86, 151.93, 102.25, 76.05, 75.85, 73.37, 73.38,

149

72.69, 71.59, 71.18, 69.39, 68.74, 66.96, 66.75, 62.12, 60.52, 60.41, 49.00, 48.91, 36.79,

+ 36.15, 32.71, 28.02. MALDI: Calc for C144H265N22O84 [M+H] 3647.71728.

Found:3647.73933.

Synthesis of Compound 41

Compound 41 was synthesized using 12.3 mg (0.00723 mmol) of Compound 39 plus

32.0 mg (0.0533 mmol) of Compound 13. These were dissolved in 1.5 mL of 0.1 M ammonium acetate (NH4OAc) at a pH of 4.5, followed by 14.5 µL of aniline (0.150 M).

The reactions were conducted at 7W (Discover® SP) with a 30 min hold time and 65˚C temperature. After the reaction was complete, the solutions were freeze-dried. Then reaction was purified by dialysis using a 500-1000 molecular weight for 4 hours, followed by FPLC. A pure brown-white solid compound was collected with 30.6 mg

1 (78.5%). H NMR (500 MHz, D2O): d 4.46-43 (m, 2H), 4.27 (t, J= 3.55Hz, 3H), 4.19-

4.12 (m, 2H), 4.09-3.55 (m, 37H), 3.40 (s, 1H), 3.39-2.82 (m, 23H), 2.79-2.43 (m, 20H),

13 2.09 (s, 12H), 1.77-1.69 (m, 8H) C NMR (125 MHz, D2O, internal MeOH std): d 175.22, 174.67, 174.17, 173.82, 171.62, 102.15, 74.64, 73.64, 73.03, 72.87, 72.05,

71.78, 69.12, 68.54, 68.36, 68.07, 67.82, 67.22, 66.71, 66.10, 62.97,61.44, 54.03, 53.74,

51.96, 50.23, 49.00, 40.39, 37.11, 36.39, 36.14, 28.92, 22.29

150

APPENDICES

151

APPENDIX A:

1H NMR Spectra

Compound 2A in CDCl3 at 500 MHz.

152

Compound 2B in D2O at 500 MHz

153

Compound 3A in CDCl3 at 500 MHz

154

Compound 3B in CDCl3 at 500 MHz

155

Compound 5A in CDCl3 at 500 MHz

156

Compound 5B in CDCl3 at 500 MHz

157

Compound 6A in CDCl3 at 500 MHz

158

Compound 6B in CDCl3 at 500 MHz

159

Compound 7 in D2O at 500 MHz

160

Compound 9 in D2O at 500 MHz

161

Compound 20 in D2O at 500 MHz

162

Compound 22 in D2O at 500 MHz

163

Compound 23 in C6D6 at 500 MHz

164

Compound 24 in D2O at 500 MHz

165

Compound 28 in D2O at 500 MHz

166

Compound 33 in CD2Cl2 at 500 MHz

167

Compound 34 in D2O at 500 MHz

168

Compound 39 in D2O at 500 MHz

169

Compound 40 in D2O at 500 MHz

170

Compound 41 in D2O at 500 MHz

171

APPENDIX B:

13C NMR Spectra

Compound 2A in CDCl3 at 125 MHz

172

Compound 2B in D2O with a methanol internal standard at 125 MHz.

173

Compound 3A in CDCl3 at 125 MHz

174

Compound 3B in CDCl3 at 125 MHz

175

Compound 5A in CDCl3 at 125 MHz

176

Compound 5B in D2O with a methanol internal standard at 125 MHz.

177

Compound 6A in CDCl3 at 125 MHz

178

Compound 6B in CDCl3 at 125 MHz

179

Compound 7 in D2O with a methanol internal standard at 125 MHz.

180

Compound 9 in D2O with a methanol internal standard at 125 MHz.

181

Compound 20 in CDCl3 at 125 MHz

182

Compound 22 in D2O with a methanol internal standard at 125 MHz.

183

Compound 23 in C6D6 at 125 MHz

184

Compound 24 in CD3OD 125 MHz.

185

Compound 28 in D2O with a methanol internal standard at 125 MHz.

186

Compound 33 in CD2Cl2 at 125 MHz

187

Compound 34 in D2O with a methanol internal standard at 125 MHz.

188

Compound 39 in D2O with a methanol internal standard at 125 MHz.

189

Compound 40 in D2O with a methanol internal standard at 125 MHz.

190

Compound 41 in D2O with a methanol internal standard at 125 MHz.

191

APPENDIX C:

IR Spectra

Compound 25: in CHCl3

192

Compound 26: in MeOH

193

APPENDIX D:

Mass Spectra

Compound 7: HR-ESI

194

Compound 9: HR-ESI

195

Compound 24: HR-ESI

196

Compound 34: HR-ESI

197

Compound 39: HR-MALDI-TOF

198

Compound 40: HR-MALDI-TOF m/z 3862.95751 3850 3834.90601 3800 3800.04195 33266_DD6_69_F36_F46_MALDI_300_4000_pos_4M_15P_0_M2_000001.d: +MS 33266_DD6_69_F36_F46_MALDI_300_4000_pos_4M_15P_0_M2_000001.d: 3772.05619 3750 3746.01199 3717.01671 3700 3662.84910 3650 3647.73933 3620.80136 3600 3588.92006 3577.90211 3550 3546.74343 3519.61985 3500 3494.69876 7 1.0 0.8 0.6 0.4 0.2 0.0 x10 Intens .

199

Compound 41: HR-MALDI-TOF m/z 3750 1+ 3695.66213 3500 1+ 3404.56297 3250 1+ 3098.44649 33266_DD6_69_F27_F35_MALDI_pos_4M_100_6000_15P_0_M1_000001.d: +MS 33266_DD6_69_F27_F35_MALDI_pos_4M_100_6000_15P_0_M1_000001.d: 3000 1+ 2807.36007 2750 1+ 2500 2501.24783 1+ 2367.19303 2250 1+ 2210.15724 1+ 2060.13369 2000 1+ 1903.03988 1+ 1750 1768.98420 1+ 1611.94325 1500 1+ 1305.83962 1250 8 6 4 2 0 x10 Intens .

200

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