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Synthesis of novel cysteine Peptide Nucleic Acid (CPNA)


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Synthesis of novel cysteine Peptide Nucleic Acid (CPNA)
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Ajmera, Mehul J
University of South Florida
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Tampa, Fla
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Cytosine acetic acid
Dissertations, Academic -- Chemistry -- Masters -- USF   ( lcsh )
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ABSTRACT: Many diseases are caused due to abnormalities in production of specific protein. Across this protein the conventional lock and key mechanism shows binding at the specific cites of protein. However use of antisense technology can prevent formation of protein. It does so by binding to mRNA and prevents transcription. The structural modifications lead to synthetic molecules with 18-mer units which show significant improvement in binding properties, this gives birth to a new class of oligomers called Peptide Nucleic Acid (PNA). We herein report cysteine based PNA called CPNA.
Thesis (M.S.)--University of South Florida, 2008.
Includes bibliographical references.
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by Mehul J. Ajmera.
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Document formatted into pages; contains 61 pages.

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Synthesis of novel cysteine Peptide Nucleic Acid (CPNA)
h [electronic resource] /
by Mehul J. Ajmera.
[Tampa, Fla] :
b University of South Florida,
Title from PDF of title page.
Document formatted into pages; contains 61 pages.
Thesis (M.S.)--University of South Florida, 2008.
Includes bibliographical references.
Text (Electronic thesis) in PDF format.
3 520
ABSTRACT: Many diseases are caused due to abnormalities in production of specific protein. Across this protein the conventional lock and key mechanism shows binding at the specific cites of protein. However use of antisense technology can prevent formation of protein. It does so by binding to mRNA and prevents transcription. The structural modifications lead to synthetic molecules with 18-mer units which show significant improvement in binding properties, this gives birth to a new class of oligomers called Peptide Nucleic Acid (PNA). We herein report cysteine based PNA called CPNA.
Mode of access: World Wide Web.
System requirements: World Wide Web browser and PDF reader.
Advisor: Mark L. McLaughlin, Ph.D.
Cytosine acetic acid.
Dissertations, Academic
x Chemistry
t USF Electronic Theses and Dissertations.
4 856


Synthesis of Novel Cysteine Peptide Nucleic Acid (CPNA) by Mehul J. Ajmera A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Chemistry College of Arts and Sciences University of South Florida Major Professor: Mark L. Mclaughlin, Ph.D. Roman Manetsch, Ph.D. Abdul Malik, Ph.D. Date of Approval: December 3, 2007 Keywords: Antisense, Antigene, Antibacterial, Antiviral, Cytosine acetic acid. Copyright 2008, Mehul Ajmera


ACKNOWLEDGEMENTS I would like to thank my parents and my sist er for encouragement. I would like to thank my mentor Dr McLaughlin for his guidance; he is a great scientist and very good person. I am thankful to my committee members Dr Manetsch and Dr Malik for their support. I thank Dr Ted Gauthier and all my friends colleagues Raj, Kiran, Woogie, David, Priyesh, Sridhar, Mingzhou, Laura, Missy, Sumedh, Kirti.


i TABLE OF CONTENTS LIST OF FIGURES iii LIST OF ABBREVATIONS v LIST OF SPECTRA vi ABSTRACT vii CHAPTER 1. INTRODUCTION 1 1.1 Introduction to Peptide Nucleic Acid 1.2 PNA as Antisense Oligonucleotide 1.3 Mechanism of Antisense Inhibition CHAPTER 2. STRUCTURAL MODIFICATIONS 2.1 Antisense Oligonucleotide Stru ctural Modification 2.2 Antiviral PNAs 2.3 Antibacterial PNA 2.4 PNA as Antigene 2.5 Structural Modification of Peptide Nucleic Acid 2.6 Cellular Uptake of PNA CHAPTER 3. STRUCTURE AND S YNTHESIS OF MODIFIED PNA 3.1 Design of Potent Antisense Drug 3.2 Results & Discussions 1 5 9 11 11 14 15 16 16 20 22 22 23


ii 3.3 Synthesis of Monomer 3.4 Experimental Procedures 3.4.1 Synthesis of Compound 2a 3.4.2 Synthesis of Compound 3a 3.4.3 Synthesis of Compound 4a 3.4.4 Synthesis of Compound 5a 3.4.5 Synthesis of Compound 6a 3.4.6 Synthesis of Compound 7a 3.4.7 Synthesis of Compound 8a 3.4.8 Synthesis of Compound 9a 3.4.9 Synthesis of Compound 10a 3.4.10 Synthesis of Compound 11a 3.4.11 Synthesis of Compound 12a 3.4.12 Synthesis of Compound 13a 3.4.13 Synthesis of Compound 15a 3.4.14 Synthesis of Compound 16a 3.4.15 Synthesis of Compound 17a REFRENCES APPENDIX A:1H AND 13C NMR SPECTRA 25 30 30 30 31 32 32 33 34 34 35 36 37 38 39 39 40 41 46


iii LIST OF FIGURES Figure 1. DNA and PNA Backbones 2 Figure 2. Thermal Stability plot of PNA/DNA and DNA/DNA Duplexes 4 Figure 3. Duplexes of PNA with DNA or RNA 4 Figure 4. Triplex Between PNA-DNA2 5 Figure 5. Difference Between Normal Cells, An tisense and Antigene Inhibited Ones 7 Figure 6. Different Phosphate Linkers 8 Figure 7. Possible Modifi cation Sites for DNA 12 Figure 8. Various Phosphate Linkage Modifications 13 Figure 9. Modified Antisense Oligonucleotides 14 Figure10. Phosphonate PNA 18 Figure 11. PNA with Ether Linkages 18 Figure 12.GPNA 19 Figure 13. Retro-Inverso, Peptoid, Heterodimeric Analog of PNA 19 Figure 14. PNA with Modified Backbone 20 Figure 15. Structure of Cyst eine Based PNA (CPNA) 23 Figure 16. Structure of Modified CPNA 24 Figure 17. Synthesis of S-alkylated Compounds Figure 18. Synthesis of S-Methyl Monomer 25 26


iv Figure 19. Synthesis of S-Alkylated Monomer Figure 20. Synthesis of Cytosine Acetic Acid 27 29


v LIST OF ABBREVIATIONS Boc = tert -Butoxycarbonyl. Cbz = Benzyloxycarbonyl. DCC = Dicyclohexylcarbodiimide. DIEA = Diisopropylethylamine. DMAP = 4-Dimethylaminopyridine. DMF = N, N-Dimethylformamide. EDC = 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. Fmoc = 9-Fluorenymethoxycarbonyl. HOBT = 1-Hydroxybenzotriazole. Ph = Phenyl. TBAF = Tetrabutylammonium fluoride. TES = Triethylsilane. TFA = Trifluoroacetic acid.


vi LIST OF SPECTRA Spectrum 4.01 47 Spectrum 4.02 48 Spectrum 4.03 49 Spectrum 4.04 50 Spectrum 4.05 51 Spectrum 4.06 52 Spectrum 4.07 53 Spectrum 4.08 54 Spectrum 4.09 55 Spectrum 4.10 56 Spectrum 4.11 57 Spectrum 4.12 58 Spectrum 4.13 59 Spectrum 4.14 60 Spectrum 4.15 61


vii SYNTHESIS OF NOVEL CYSTEINE PEPTIDE NUCLEIC ACID (CPNA) MEHUL J. AJMERA ABSTRACT Many diseases are caused due to abnormalities in production of specific protein. Across this protein the conventional lock and key mechanism shows binding at the specific cites of protein. However use of an tisense technology can prevent formation of protein. It does so by binding to mRNA and prevents transcription. The structural modifications lead to synthetic molecule s with 18-mer units which show significant improvement in binding properties, this give s birth to a new class of oligomers called Peptide Nucleic Acid (PNA). We herein report cysteine based PNA called CPNA.


1 CHAPTER 1 INTRODUCTION 1.1-Introduction to Peptide Nucleic Acid One of the most important processes in the human body is the controlled production of specific proteins. The under or over production of these proteins at wrong time in cell cycle can lead to many different types of diseases. One way to modify this process is by antigene or antisense manipul ation of protein production. In order to accomplish this task, it is vital to know the DNA sequence for each protein of interest so as to provide targets for new drugs. This quest for novel treatments opened the doors to the concept of Peptide Nucleic Acid (PNA).1 PNA is an excellent mimic of DNA wh ile maintaining cer tain substantial differences in its basic chemical structure th at provide it certain immunities from the cell’s standard degradation mech anisms. Also, it has displaye d high affinity binding with the double stranded DNA via Watson-Crick base pairing and from Hoogstein type base pairing in the major groove. Discovered by Nielsen et al. in 1991, peptid e nucleic acids are chemically similar to DNA or RNA. However, because PNA does not occur naturally in the body, it must be chemically synthesized for all biological re search needs. In a comparison of their basic structures, the backbone of DNA is co mprised of deoxyribose sugar units while


2 RNA uses ribose sugars for its backbone. On the other hand, the backbone of PNA is comprised of N-(2-aminoethyl )-glycine units which are li nked by peptide bonds as shown in Figure 1.1 Figure 1 – DNA and PNA Backbones PNA’s are acyclic, achiral neutral oligom ers that are designed to resemble the backbone structure of DNA and RNA. However, since PNA consists of a string of amino acid residues it begins with an N-terminus inst ead of a 5’ end and ends with a C-terminus instead of a 3’ end. Anothe r difference in structure is th e fact that the purine and pyrimidine bases are linked to the PNA b ackbone via a methylen e carbonyl group. Additionally, it has been shown that the bi nding between PNA/DNA is much stronger


3 than the binding seen between two antiparallel strands of DNA. This is due to the electrostatic repulsions between the phosphate groups on each strand of DNA. When this repulsive force is removed (as seen with the DNA/PNA binding), the two strands bind much more tightly. In recent years, peptide nucleic acids have allowed scientists to make significant advancements in molecular biology procedures, diagnostic assays and antisense therapy. When a PNA sequence is designed, it is ex tremely important to ensure high binding specificity with the complementary DNA stran d. This is because a mismatch between the PNA and DNA results in a much larger destabilizing force than a similar mismatch in the DNA/DNA pair. This binding strength an d specificity also applies to PNA/RNA duplexes. Some of the early studies preformed by Nielsen et al. have demonstrated that a PNA/DNA mismatch is more destabilizing than a DNA/DNA duplex mismatch. When measuring the stability of any duplex (DNA/DNA, PNA/DNA etc.), one of the most common methods is to observe the melting temperature (Tm).2 For the DNA/DNA duplex, the average Tm was about 110C while the average Tm for the PNA/DNA duplex was 150C. This higher value of Tm for the PNA/DNA complex indicates that it does in fact have a greate r degree of stability than the DNA/DNA duplex. The salt concentration’s effect on Tm was also observed for both sets of duplexes. It can be seen in Figure 22 that the Tm for the PNA/DNA duplex (shown as blue circles) is not dependent on the salt concentration while the Tm for the DNA/DNA duplex (shown as pink squares) is highly depende nt on the salt concentration.


4 Thermal Stability Plot20 30 40 50 60 70 80 1101001000 NaCl concentration (mM)Tm (C) Figure 2 Thermal Stability Plot of PNA/DNA and DNA/DNA Duplexes When PNA hybridizes with DNA or R NA, it does so via Watson-Crick base pairing as shown in Figure 3.3 Figure 3 Duplex of PNA with DNA or RNA When binding to DNA or RNA, it is po ssible for PNA to bind in either the parallel or the antiparallel or ientation. If it bi nds in the parallel orientation, the amine terminus of PNA binds to the 5’ end of either the DNA or RNA sequence, whereas binding in the antiparallel orie ntation consists of the 3’ end of either the DNA or RNA


5 sequence binding to the amine terminus of the PNA. When the PNA oligonucleotide binds to double stranded DNA, it first i nvades the double stra nded helix and then hybridizes to the target se quence thereby displacing the second DNA strand into a ‘D’ loop as shown in Figure 4.4,5,6. Figure 4 Triplex between PNA-DNA2 1.2-PNA as an Antisense Oligonucleotide During the past decade, many Biotech comp anies have displayed an interest in antisense oligonucleotides fo r the treatment and prevention of disease. Antisense oligonucleotides are synthetic molecules that resemble pieces of single stranded DNA and incorporate any and/or all of the four base s. These oligonucleotides can then be used to either inhibit or promote gene expressi on by binding directly to target DNA or RNA. Thus a particular sequence of antisense o ligonucleotide could be designed specifically around a known mRNA transcript a nd then allowed to bind with that mRNA in order to control the production of a specific protein. Also, a plethora of information about any gene could be obtained by synt hesizing a vast library of complementary oligonucleotide sequences. This is because of the hydrogen bonding that exists between the


6 complementary base pairs; it can provide ne cessary information for the design of an oligonucleotide which will target any gene w ith a known sequence. Greater control over binding specificity could be obtai ned as a result of targeting a defective gene with such oligonucleotides which in turn would give birth to a new strategy of drug design. Antisense RNA can be described as a na turally occurring phenomenon in which the cell transcribes a strand of RNA which is complementary to a specific segment of mRNA.7,8 These antisense RNA strands hybridized to the mRNA thereby inhibiting gene expression. As a way to keep these antisen se RNA strands in check, certain enzymes have important key roles in th e regulatory process of these antisense RNA strands. It has been shown that studying a small portion of any gene can reveal its sequencing information thus providing scientists with the antisense sequence. The inhibition of gene expression through a natural process is already known for antisense RNA strands. However, a simila r natural system involving antisense DNA is unknown at this time. In theory, the inhi bition of a single gene target could be accomplished through the use of an oligomer 15 nucleotide bases in length that binds complementary to the mRNA strand in question. In 1978, Zamecnik and Stephenson discovere d a new class of therapeutic agents called antisense oligonucleotide s which specifically targeted mRNA. This targeting can be seen in Figure 5.9


7 Figure 5 – Differences between Normal Cells, Antisense and Antigene Inhibited Ones These oligonucleotides bind to the mRNA strands via the Watson and Crick base pairing methods. In doing so, this pairing inhibits th e translation of diseas e causing proteins. This marked the beginning of a new strategy for the prevention of viral replication. Using this method, novel antivir al drug candidates could be designed to target and specifically bind to the viral RNA. This ne w strategy can be seen as a combination of two current strategies. The first being gene therapy which involves the introduction of a new gene into the affected individual, the second strategy is the conventional lock and key mechanism for drug discovery. In their research, Zamecnik and Stephens on successfully showed that antisense could be used to inhibit viral replication.10 using an antisense phosphodiester oligodeoxynucleotide (ODN), they observed that it bound to the mRNA strands of the Rous sarcoma virus which in turn inhibited re plication. Early studie s into the mode of this type of inhibition prove that the bi nding between the mRNA strands and the ODN’s follow the antisense mechanism. These st udies used two types of ODN’s, control ODN’s, which were sequences that were not complementary for the target mRNA and


8 complementary ODN’s. The specificity of binding was compared between the complementary ODNs and controls. There were three different types of phosphate linkers used, one for each control stra nd and its complementary counterpart; phosphorothioate, phosphodiester and methyl phos phonate linkers. These can be seen in Figure 6 .11 O HO B O P O O O O B OH O HO B O P O O S O B OH O HO B O P O O H3C O B OH Phosphodiester Phosphorothioate Methylphosphonate Figure 6 – Different Phosphate Linkers Due to the fact that cellu lar RNA’s can have complex secondary and tertiary structures somewhat like prot eins, binding complexities can been seen when the target antisense sequence is buried well within the s econdary (or tertiary) structure. One way to improve the binding specificity between an oligonucleotide and its target is to increase the number of complementary ba se pairs on the oligonucleotide.11,12,13. In doing so, the length of the oligonucle otide increases which can then present its own problems. When


9 the oligonucleotide reaches a certain point, its binding specificity can be compromised due to its length. 1.3-Mechanism of Antisense Inhibition In order for the oligodeoxynuc leotide to inhibit any cellular process, it must bind to the mRNA. This can occur in either the nucleus or the cytoplasm. The inhibition of any gene can take place by two different mech anisms. The first mechanism of inhibition involves the mRNA/DNA duplex. In side the nucleus, mRNA can be the initial sequence on which DNA is built. Then, the RNase H enzyme moves in to remove the mRNA strands by degrading them which allows for the complementary DNA base pairs to be added to this newly formed strand t hus providing you with double stranded DNA.13,14,15 However, if ODN’s are present in the nucle us, then they will bi nd to the mRNA and prevent it from forming the new single strand ed DNA. This type of mechanism has the advantage that a single ODN can target many mRNA thus the therapeutic amount of this drug would be very small. The second mechanism of inhibition involves the formation of the ODN/RNA duplex. Here, the single stra nded ODN binds to the single stranded mRNA. Since the ODN binds to the mRNA with such a high affi nity, it sterically blocks any type of translation.16 when this occurs in the nucleus, transport across the nuclear membrane into the cytoplasm can be hindered due to the incr eased amount of sterics within this larger molecule. If this occurs in the cytoplasm, the ODN’s will block the translation process thus preventing the formation of proteins. However, the limitation to this mechanism is


10 that the ODN will have to physically bind to the mRNA molecule continuously. In order to accomplish this task, three primary obstacl es must be overcome. First, the ODN must have enough cell permeability so as to cross the cell (and nuclear) membrane so that it can then bind to the mRNA. Second, the ODN/RNA duplex must have a sufficiently long lifetime so as to prevent the transla tion of proteins. Fi nally, the ODN should not disrupt the expression of non-ta rgeted genes. Since a non -targeted sequence will have some base pair mismatches, complexes with at least one mismatch should have a short half-life.


11 CHAPTER 2 STRUCTURAL MODIFICATIONS 2.1-Antisense Oligonucleotide Structural Modification The antisense ODN’s are molecules whose length is generally between 18 and 20 base pairs long and possess an overall negative charge for every phosphate or phosphorothioate. In order fo r the ODN’s to be effective drug molecules, they must display good pharmacokinetics as well as good pharmacodynamics. The biggest challenge lies with the cellular uptake of thes e drug molecules because if these oligomers cannot readily enter the cells through normal processes (i .e. endocytosis, phagocytosis etc.), then they maybe useless as a novel drug. Additional, there are other issues that must be addressed if these types of oligom ers are to be considered successful drug candidates. Such as the fact that they should be slowly degraded in the body, they should be highly bioavailable and once they have done their function they should be easily cleared from the body.17,18,19,20 With these facts in mind, studies have been preformed with various structural m odifications of the ODN’s in order to determine how the modifications affect the oligomer’s properties. Some of these structural changes include modification of the sugar backbone as well as modification of the phosphate linkages. Figure 7 shows the backbone structure of an ol igonucleotide as well as the potential modification sites.


12O B O R P O O O O R O B HeterocyclicbasemodificationSugarmodification 2'modification Phosphatemodification 5'modification 3'modification Figure 7 Possible Modification Sites for DNA Over the past few decades, the modified antisense ODN’s have shown remarkable improvement in their cellular uptake as we ll as an increase in their specificity accompanied by a decrease in th eir toxicity. Several new cl asses of ODN’s have been developed by modifications of the phosphate bonds, modifying the way the bases are attached and also by modifying the oligonuc leotide’s sugar backbone. These resulting molecules have shown tremendous improvement in biological prop erties over their predecessors. One such example of a curr ent antisense ODN drug is Vitravene, which has been modified to include phosp hothioate linkages in its backbone.21 However, there are other types of phosphate li nkage modifications that can be incorporated into these oligonucleotides. Some of these include phosphorothioates, phosphoramidates and phosphotriesters and can be seen in Figure 8


13 O B HN P O O O O B O P O O O O B O P O S O Phosphoroamidate Phosphotriester Phosphorothioate Figure 8 – Various Phosphate Linkage Modifications When the sugar backbone was modified to include carbocycles or hexose,22 the oligomers did not show any significant im provement in their binding to mRNA. However, an increase in cell permeability ha s been observed for oligomers whose sugar backbone has been replaced with morpholine monomers.23 These monomers are linked with either a neutra l carbamate-type linkage24 or with a phosphoromidate linkage. Locked Nucleic Acids (LNA)25 are another class of ODN’s that have shown good water solubility as well as excellent binding properties with comple mentary DNA. Structurally, they contain one or more 2’-O and 4’-C-methylene -D-ribofuranosyl nucleotide(s). Additionally, the 5 membered sugar backbone ring can be replaced with a six membered ring. Doing so produces thre e different types of monomer backbones; cyclohexene nucleic acid, hexitol nucleic aci d and cyclohexanyl nucleic acid.26 The hexitol nucleic acid can form enzymatically stable comple xes with both DNA and RNA. These various types of backbone modifications are shown in Figure 9


14 O O O P O O O B O N O B O O P O N O Locked nucleic acid Cyclohexene nucleic acid Morpholine oligonucleotide O O O P O O B Cyclohexanyl nucleic acid Figure 9 Modified Antisense Oligonucleotides 2.2-Antiviral PNAs Many antisense oligonucleotides have been shown to inhibit viruses; the most commonly inhibited ones being HIV27, HSV1 & 228 and influenza. This is because the viral genetic sequences are unique with re spect to the host’s genetic sequences. Therefore, it should possible to design sp ecific oligonucleotid es that will bind preferentially and specifically to the vi ral mRNA without specific binding of the host mRNA. PNA has already shown its antiviral properties by selec tively inhibiting the reverse transcriptase enzyme, which is a key en zyme in the life cycle of HIV. When the PNA oligomer binds to the RNA template, it prevents the reverse transcription process thereby preventing HIV infection. This inhibition of the HIV virus’s replication cycle in


15 cultured cells has increased hopes that PNA can be used as an antiviral drug. However, there are still obstacles to be overcome before PNA can be effectively used as an antiviral drug. One such obstacle is the fact that the c oncentration of PNA that is required for this antiviral process is extremely high which indi cates the need for either an alternate cell delivery system or for structur al modifications of the PNA. 2.3 Antibacterial PNA The scope of PNA is not just limited to an tiviral drugs; it can also be used in for many antibacterial applications. This is because PNA can be specifically designed to enter any given bacteria and control its gene expression. This gives PNA tremendous scope as antibacterial drug candidate. For in stance, antibacterial gene expression could be studied using antisense PNA. The use of antisense technology as a tool for the creation of antibacterial drugs has gained momentum in the past decade. Since the genome of any given bacteria is so small, it is easier to design and modify PNA so that it has improved binding and gene specific properties. Structurally, bacteria are easier targets than eukaryotic cells because they lack internal organelles. Therefore, since cellu lar distribution is not restricted and the bacterial genes are less complex, the PNA targ et is easier to access thus bacteria gene control should be easier to accomplish. However, the PNA must first enter the bacteria which can be a problem due to the fact that bacteria protect themselves from their environment via a cell wall. If the antisen se PNA with specific binding properties has chemical permeability issues, it might not be able to penetrate the cell wall thus rendering


16 the PNA useless. With this proble m in mind, when mutant strains of E coli cells with a defective lipopolysaccharide (LPS) layer were us ed, they showed a drastic increase in cellular uptake of PNA. Add itionally, both the binding properti es and the length of the oligomer are key areas which affect the cell permeability. Finally, the use of cell wall active peptides (KFFKFFKFFK) attached to the PNA can increase the cellular uptake.29 Use of these peptides also shows antibac terial properties at low concentrations. 2.4PNA as Antigene PNA has also been shown to form a st able triplex complex with double stranded DNA as well as forming a strand displacem ent complex with double stranded DNA. When PNA is bound to DNA to form the tr iplex complex, the RNA polymerase cannot bind to create mRNA which in turn prevents the creation of a protein. This type of inhibition of protein formation is called antigene inhibition.30,31 The binding efficiency of the gene targeting PNA must be high whic h leads to complete distortion of the DNA helix. 2.5-Structural modification of Peptide Nucleic Acid As PNA’s applications as a drug began increasing, there was an increasing need to modify its structure to fit specific requi rements for each unique application. PNA has certain inherent limitations, two of which ar e cell permeability and solubility. In order to circumvent these limitations, it was necessary to create structural modifications in the basic PNA monomer. Two of the more comm on structural modifica tions are either a


17 change of ethylene diamine in the backbone or a nucleobase alteration. The approach also includes introducing a chir al backbone from a conventional achiral one. Doing so improves the binding selectivity between th e PNA and the DNA as we ll as entropically favoring the duplex formation. When a chiral backbone is used, the duplex gains stability from the conformational rigidity that is imparted from the chiral center. As a way to increase water solubility, positively charged species are put onto the PNA. The location of this positively charged species can be at either the C terminus or the N terminus. Generally, an oligomer’s water solubility can be increase by either incorporating a charged species or a func tional group thereby increasing its overall polarity. When negatively charged species such as phosphate analogs are incorporated, the oligomer’s water solubil ity generally increases. Ho wever, strong electrostatic repulsions between the nega tively charged oligomer and the negatively charged phosphates of the complementary DNA signifi cantly reduce its binding affinity. Figure 10 shows two examples of phosphonate PNA’s.32


18 O N P O N P Base O O O Base O O O N H N P N H N P Base O O O Base O O O Figure 10 Phosphonate PNA There have been several struct ural modifications done to PNA in order to obtain different desired properties; some of these include:Introducing the ether linkages within the PNA backbone.33,34 Doing so has shown to increase water solubility. Exampl es of these linkages are shown in Figure 11 O OH Base H2N O O OH H2N O Base H2N O OH O Base Figure 11 PNA with Ether Linkages Use of guanidium functional group. The in corporation of this functional group has shown remarkable improvement in both water solubility and cell pe rmeability. Also, the binding efficiency with the complementary DN A is not compromised. This has lead to various cationic PNA’s (DNG/ PNA) which bind to comple mentary DNA and RNA. One example is shown in Figure 12 .35


19H2N N NH NH2 O Base O H2N Figure 12 GPNA Another type of modification was made fo r the formation of heterodimeric analogs,36 peptoid37 and retro-inverse.38 Structurally these compounds differ in their positioning of the carbonyl (CO) and amine (NH) groups as shown in Figure 13 H2N N OH Base O O H2N N OH O Base O H2N N N Base O O OH O Base O Figure 13 Retro-inverso Peptoid Heterodimeric analog of PNA A decrease in PNA/DNA hybridization wa s observed with both the peptoid and retroinverse type of structur es. The difference in the position of the CO group lead to geometrical modifications of the structures and proved less effective. However in the case of the heterodimeric PNA, an improvement in the duplex formation was observed. Modifications were also preformed by a dding a methylene group in the aminoethyl part of backbone and al so in the nucleobases.39 Examples of these modifications are shown in Figure 14


20H2N N OH O Base O H2N N OH O O Base Figure 14 PNA with Modified Backbone Unfortunately, these structures have shown poor binding effi ciency with complementary DNA and reduced thermal stability of the PNA/DNA duplex. 2.6-Cellular uptake of PNA Peptide nucleic acid has a neutral backbone. As a result, it cannot be complexed with cationic lipids such as DNA or RNA. Therefore, PNA’s cell permeability is decreased which in turn require s that a very high concentratio n of oligomer be used when treating a disease. However, Corey and co-w orkers have shown th at the in-vitro cell delivery method of PNA is possible when using a cationic lipid.40 This is because of the electrostatic force of attraction between the positively charged lipids and negatively charged phosphodiester of DNA and RNA. When this cationic lipid fuses w ith the cell it allows the oligonucleotide to en ter the cell. This phenomenon is then used to inject the PNA into cell. First, the PNA is allowed to hybridize to overlapping oligonucleotide, and then the cationic liquid is mixed with the hybridized complex which allows the PNA enter the cell. Using this type of method, the desired cell permeability could be achieved by attaching certain peptide chains to the PNA.


21 Another approach to import PNA’s into th e cell is by using transport vehicle like liposome’s. At lower concentrations, PNA’ s have demonstrated a certain ability to penetrate through the cell membrane.41,42 Finally, an increase in the intracellular PNA concentration has been shown to increase PNA’s cytotoxicity.


22 CHAPTER 3 STRUCTURE AND SYNTHESIS OF MODIFIED PNA 3.1-Design of potent Antisense drug To design drug candidate’s using an tisense mechanisms, it is important to consider the following hurdles. The first being poor cellular uptake. In order to reach to the cytoplasm or the nucleus the candidate mu st be able to cross the cell membrane. Secondly there should not be si gnificant degradation once inside the cell. To inhibit a specific gene expression the candidate must have a high affinity and specificity to bind at the required site. To enhance specific binding properties, informati on regarding nucleic acid structure and thermodynamics is very impo rtant. A variety of structures could be obtained by changing position of the nuc leobase, phosphodiester backbone and sugar. However for any of the designed molecules, the binding properties are governed by the Watson-Crick hydrogen bonding model. To increas e the cell permeability, modifications of the phosphodiester backbone has been a very useful strategy. By the aid of molecular modeling, Dr Mark McLaughlin has an appro ach to replace the phosphodiester and sugar backbone with a chiral cysteine backbone intr oducing charged species to enhance cellular uptake.


23 3.2-Results & Discussions The peptide designed by Dr McLaughlin has positively charge d guanidine units as side chains. This species is introduced at the thiol by SN2 reaction. For the synthesis of PNA either Boc or Fmoc can be used as protecting group for the amine. We herein report Fmoc as protecting group for the N-terminus of the CPNA monomer with Cbz as the protecting group for the nucleobase and t-butyl ester for the ca rboxylic acid. This type of orthogonal protecting group strategy is require d for the selective deprotection at the Cterminus and N-terminus during solid phase synthesis. N N N N N N N N S R S R S R S R B B B B O O O O O O O H H H H R= Me, N NHCbz O H B= Nucleobases (Adenine, Guanidine, Cytosine, Thymine) O Figure 15Structure of Cysteine based PNA (CPNA)


24 Modification of this struct ure leads to formation of new a CPNA as shown below. N N N N N N N N B B B B O O O O O O O H H H H O S S S S R R R R R= Me, N NHCbz O H B= Nucleobases (Adenine, Guanidine, Cytosine, Thymine) Figure 16Structure of modified CPNA


25 3.3-Synthesis of the monomer BocHN CO2H S Ph Ph Ph BocHN CONH2 S Ph Ph Ph 1a 2a BocHN S Ph Ph Ph NH2 BocHN S Ph Ph Ph NHFmoc 3a 4a BocHN HS NHFmoc 5a BocHN S NHFmoc R R=Me, R= N NHCbz O H 6a 10a DCC, HOBT, NH4OH THF BH3.THF THF Fmoc-Cl, DIEA DCM TFA, TES DCM EtOH R-X, NaOH Figure 17Synthesis of S-alkylated compounds


26 BocHN S NHFmoc ClH.H2N S NHFmoc N S NHFmoc O H O N S NHFmoc O O N N O O H O 6a 7a 8a 9a HCl gas 1,4-Dioxane BrCH2CO2t-Bu, DIEA DMF Thymine acetic acid, SOCl2, Reflux DIEA, DMF Figure 18Synthesis of S-methyl monomer


27BocHN S NHFmoc N CbzHN O H ClH.H2N S NHFmoc N CbzHN O H H N S NHFmoc N CbzHN O H O O N S NHFmoc N CbzHN O H O O N N O O O H HCl gas 1,4-Dioxane BrCH2CO2t-Bu, DIEA DMF Thymine acetic acid, SOCl2 Reflux DIEA, DMF 10a 11a 12a 13a Figure 19Synthesis of S-alkylated monomer Synthesis of CPNA monomer wa s done using commercially av ailable starting material Boc(Cys)Trt-COOH. Literature survey rev eals reductive amination as most common route for many scientists, but th is route can lead to partia l racemization. We avoid this potential problem. Compound 2a was made by using dicyclohex yl carbodiimide to activate the carboxylic acid. When all the carboxylic acid was activated, ammonium hydroxide was added to the reaction at 0 0C. Here the byproduct is N-N’-DCC urea. Other activating agent like DIC and EDC were also used. Triturating the crude reaction mixture gives pure compound but better yiel ds were obtained by using column chromatography. Reduction of amide 2a using BH3-THF complex gave amine 3a. Methanol was used as solvent to break borane complex at the end of reaction. The crude mixture was subjected


28 to water workup at pH=8.5. The free amine 3a was protected with Fmoc group by using 1eq of Fmoc-Cl and 1.05eq of DIEA as base Addition of Fmoc-Cl and base was done at 0 0C and the reaction was warmed to room temperature. Although the reaction is straightforward purification us ing column chromatography was difficult, even flash silica gel could not enable separation. However tritu ration with ethyl acetate and hexane proved to be best solution for this problem and afforded compounds in good yield. In order to synthesi ze compound 6a and 10a, TFA was used under dilute conditions at 0 0C. The Boc and trityl groups are acid labile However in dilute conditions, the trityl group is selectively removed. To this thiol 1eq of base was added followed by the alkylating agent. The Boc group was remove d before N-alkylation; this was done by passing anhydrous HCl gas into the reaction mixture containi ng 1, 4-dioxane as solvent. The alkylation was done using tert-butyl br omoacetate and DIEA. The alkylating agent must be exactly 1eq as even a slight excess leads to formation of dialkylated product. This reaction was tried with different solv ents but DMF proved to be the best keeping 1Molar concentration of st arting material in solution. Monomer was synthesized from sub-monomer by base coupling reaction. Coupling of nucleobase using HATU and TEA as base did not work. Another strategy had to be applied wherein thymine acetic acid was refluxed at 55 0C in thionyl chloride for 2hr followed by concentration and sub-monomer 8a, 12a were added in DMF followed by TEA. This afforded successful base c oupling reaction for thymine acetic acid.


29N N NH2 O H N N NHCbz O H N N NHCbz O O O N N NHCbz O OH O 14a 15a 16a 17a Cbz-Cl, DMAP Pyridine BrCH2CO2t-Bu, K2CO3, Cs2CO3DMF 4N HCl/ 1,4-Dioxane DCM Figure 20Synthesis of Cytosine acetic acid. Modified nucleobase was used to synthesi ze the monomer. The free amine is protected using Cbz group as it may cause side reaction s during solid phase synthesis. Once the oligomer is synthesized the Cbz groups will be removed. The modification of nucleobase cytosine was done in three steps. In the first step, the NH2 was protected using Cbz-Cl in pyridine as solvent. The reaction was stirred for 3 days and filtered, the filtrate was the product. Compound 16a was synthesized from 15a by using tert-butyl bromoacetate and potassium carbonate. Compound 17a was synthe sized by deprotection of t-butyl group using HCl in 1, 4-Dioxane.43


30 3.4Experimental Procedures 3.4.1. Carbamic acid, [(1R)-2-amino-2-o xo-1-[[triphenylmet hyl)thio]methyl] ethyl]-1,1-dimethylethyl ester, 2a To a solution of 1a (2 g, 4.31 mmol), HOBT (700 mg, 5.18 mmol) and DCC (1.07 g, 5.18 mmol) in THF (10 ml) was added 28 % NH4OH (0.78 ml, 6.46 mmol) at 0 C. After 2hr of st irring at 0 C, the re action mixture was filtered through celite and filtrate was concentrated, diluted with EtOAc, and washed with water and brine. Organic layer was then dr ied over sodium sulfate, concentrated and subjected to flash column chromatography to give 2a (1.95 g, 98 %) as a white solid. Rf = 0.51 (1:1 :: Ethyl acetate/ Hexane ). IR (Nicolet Avatar 320 FTIR) max 3306.45, 2977.8, 1676.78, 1490.10, 1392.02, 1366.78, 1249.95, 1164.94, 740.79, 699.77 cm-1. 1H NMR (250MHz, CDCl3) 7.46-7.38 (d, 6H), 7.23 (ddd, J=10.62, 5.16, 9H), 6.05 (s, 1H), 5.95-5.82 (m, 1H), 4.94 (t, J=6.90Hz, 1H), 3.95-3.85 (m, 1H), 2.67 (dd, J=12.62 Hz), 2.54 (dd, J=12.84 Hz, 1H), 1.41 (s, 9H). 13C (250MHz, CDCl3) 173.8, 155.42, 144.42, 130.04, 129.60, 128.10, 126.93, 80.34, 67.24, 53.22, 33.80, 28.33. m/z 485.16 (M+Na)+ 3.4.2. (2-Amino-1-tritylsulfanylmethyl-ethyl)-carbamic acid tert-butyl ester 3a To solution of 2a (1 g, 2.16 mmol) in anhy THF(5 ml ) was added 1 M BoraneTHF complex(5.4 ml, 5.4 mmol) at 0 C and after st irring at 0 C for 3 hrs, the reaction mixture was then heated to 60C and refluxed for 12 hrs. The reaction mixture was cooled to 0 C and quenched by MeOH, the reaction mixture was then concentrated and diluted with MeOH and concentrated again. Th e concentrated oil was then subjected to


31 water work-up pH-8.5 followed by flash column chromatography to afford 3a (0.58 mg, 60 %). Rf = 0.28 (100% Ethyl acetate). IR (Nicolet Avatar 320 FTIR) max 3048.9, 2360.69, 1706.97, 1489.75, 1445.10, 1365.46, 1265.58, 1167.82, 742.71, 700.29, 686.87 cm-1. 1H NMR (250MHz, CDCl3) 7.40 (d, J=7.27 Hz, 6H), 7.19 (ddd, J=14.29, 5.76 Hz, 9H), 5.27-4.85 (br, 1H), 3.55 (br, 2H), 2.35 (tt, J=17.88, 8.99 Hz, 2H), 2.63 (d, J=5.68 Hz, 2H), 1.39 (s, 9H). 13C (250MHz, CDCl3) 155.57, 144.65, 79.35, 66.79, 64.35, 60.39, 44.40, 34.37. m/z 449.22 (M+H)+ 3.4.3. [2-(9H-Fluoren-9-ylmethoxycarbonyla mino)-1-tritylsulfanyl)methylethyl]-carbamic acid tert-butyl ester, 4a. To a solution of 3a (0.1168 gm, 0.26 mmol) in DC M, Fmoc-Cl(70.7 mg, 0.273 mmol) was added followed by addition of DM AP(3.18 mg, 0.026 mmol) and DIPEA(0.043 ml, 0.26 mmol)was added at 0 C and stirred for 3 h at room temperature. The reaction mixture was concentrated to give crude oil. The product 4a was obtained by triturating the crude oil with ethyl acetat e and hexane (0.147 gm, 85 %). Rf = 0.5 (1:2 :: Ethyl acetate/ Hexane ). IR (Nicolet Avatar 320 FTIR) max 3300.13, 2977.61, 1697.26, 1518.36, 1447.66, 1366.39, 1247.71, 1164.98, 758.74, 739.49, 701.05 cm-1. 1H NMR (250MHz, CDCl3) 7.74 (d, J=7.23 Hz, 2H), 7.55 (d, J=7.39 Hz, 2H), 7.39 (t, J=7.59 Hz, 8H), 7.30-7.13 (m, 11H), 4.79-4.68 (m, 2H), 4.36 (d, J=6.64 Hz, 2H), 4.17 (d, J=6.65 Hz, 1H), 3.65-3.48 (m, 1H), 3.12 (d, J=5.18 Hz, 2H), 2.30 (d, J=6.21 Hz, 2H), 1.42 (s, 9H). 13C (250MHz, CDCl3) 156.89, 155.73, 144.56, 144.00, 143.96,


32 129.62, 128.06, 127.13, 125.10, 120.05, 79.70, 50.41, 47.31, 44.32, 34.05, 28.45. m/z 671.28 (M+H)+ 3.4.4. [2-(9H-Fluoren-9-ylmethoxycar bonylamino)-1-mercaptomethylethyl]-carbamic acid tert-butyl ester, 5a To a solution of 4a (62.9 mg, 0.093 mmol) in 10ml DCM was added TES (12 mg, 0.103 mmol). The reaction mixture was cooled to 00 C. TFA 0.5 ml was added dropwise. The reactio n mixture was concentrated and subjected to flash column chromatography to afford 5a (39.5 mg, 98.8 %). Rf = 0.42 (1:2 :: Ethyl acetate/ Hexane). IR (Nicolet Avatar 320 FTIR) max 3341.02, 2977.59, 1685.18, 1529.01, 1450.07, 1392.13, 1367.11, 1318.53, 1252.73, 1166.74, 1006.71, 758.84, 740.47 cm-1. 1H NMR (250MHz, CDCl3) 7.75 (d, J=7.35 Hz, 2H), 7.57 (d, J=7.39 Hz, 2H), 7.33 (ddd, J=18.25, 5.95 Hz, 4H), 5.29 (d, J=6.53 Hz, 2H), 4.40 (d, J=6.52 Hz, 2H), 4.18 (t, J=6.66 Hz, 1H), 3.78 (d, J=4.61 Hz, 1H), 3.36 (dd, J=10.96 Hz, 2H), 2.77-2.48 (m, 2H), 1.56 (t, J=8.75, 1H), 1.43 (s, 9H). 13C (250MHz, CDCl3) 157.19, 155.94, 143.83, 141.34, 127.75, 127.09, 126.31, 120.02, 80.07, 66.91, 52.46, 47.24, 43.07, 28.36. m/z 429.18 (M+H)+ 3.4.5. [2-(9H-Fluoren-9-ylmethoxycarbonyla mino)-1-methylsulfanylmethylethyl]-carbamic acid tert-butyl ester, 6a To a solution of 5a (0.8 gm, 1.86 mmol) in ethanol was added TBAI (6.9 mg, 0.186 mmol) at 0 0C. From 2N NaOH (0.93 ml, 1.86 mmol) was added drop wise followed by drop wise addition of Me-I (0.1746 ml, 2.79 mmol) in ethanol. The reaction mixture was st irred for 3 hr and concentrated followed by


33 water work-up at pH=8.5. The organic la yer was dried over anhydrous sodium sulfate and concentrated followed by flash column chromatography gave 6a (0.727 gm, 88. %). Rf = 0.35 (1:2 :: Ethyl acetate/ Hexane). IR (Nicolet Avatar 320 FTIR) max 3343.45, 2980.49, 1682.50, 1532.49, 1445.29, 1317.49, 1287.41, 1264.77, 1172.02, 1004.03, 757.59, 740.41 cm-1. 1H NMR (250MHz, CDCl3) 7.75 (d, J= 7.35 Hz, 2H), 7.58 (d, J=7.26 Hz, 2H), 7.33 (ddd, J=17.60, 5.17 Hz, 4H), 5.36-5.26 (m, 1H), 5.15 (d, J=7.51 Hz, 1H), 4.40 (d, J= 6.76 Hz, 2H), 4.21 (d, J=6.52 Hz, 1H), 3.89-3.72 (m, 1H), 3.51-3.26 (m, 2H), 2.71-2.50(m, 2H), 2.13 (s, 2H), 1.43 (s, 9H). 13C (250MHz, CDCl3) 157.17, 143.90, 141.33, 127.73, 127.09, 125.05, 120.0, 79.85, 66.88, 47.25, 44.09, 36.51, 28.39, 16.16. m/z 465.18 (M+Na)+ 3.4.6. (2-Amino-3-methylsulfanyl-propyl )-carbamic acid 9H-fluoren-9ylmethyl ester, 7a To a solution of 6a (0.727 gm, 1.64 mmol) in 1,4-dioxane at 0 oC hydrogen chloride gas was passed for 15 min and reaction was stirred at room temperature for 30 min. The reaction mixture wa s concentrated and kept in vacuum for drying gave 7a (0.562 gm, 100 %). IR (Nicolet Avatar 320 FTIR) max 2918.21, 1694.85, 1532.71, 1449.60, 1254.57, 758.78, 737.48 cm-1. 1H NMR (250MHz, CDCl3) 7.65 (d, J=6.61 Hz, 4H), 7.33-7.17 (m, 4H), 4.33-4.06 (m, 5H), 3.99-3.93 (m, 1H), 3.08-2.58 (m, 3H), 2.03 (s, 4H). 13C (250MHz, CDCl3) 157.28, 143.79, 141.20, 127.72, 127.15, 125.15, 125.41, 119.93, 67.35, 51.91, 47.05, 42.43, 34.32, 15.95. m/z 343.14 (M+H)+


34 3.4.7. [2-9H-Fluoren-9-ylmethoxycarbonny lamino)-1-methylsulfanylmethylethylamino]-acetic acid tert-butyl ester, 8a To a solution of 7a (0.562 gm, 1.486 mmol) in DMF was added tert-butyl bromoacetat e (0.23 ml, 1.56 mmol) at 0C followed by addition of DIPEA (0.515 ml 3.12mmol) and tetra butyl ammonium iodide(55 mg, 0.148 mmol). The reaction mixture was stirred fo r 3 hr. The reaction mixture was then concentrated and subjected to water wo rk-up at pH=8.5 fo llowed by drying with anhydrous sodium sulfate. Flash column chromatography gave 8a (0.449 gm, 60 %). Rf = 0.18 (1:2 :: Ethyl acetate/ Hexane). IR (Nicolet Avatar 320 FTIR) max 3313.15, 2976.17, 1727.23, 1518.20, 1477.76, 1449.95, 1367.62, 1246.23, 1153.07, 759.28, 741.64 cm-1. 1H NMR (400MHz, CDCl3) 7.76(d, J=7.48 Hz, 2H), 7.62 (d, J=7.44 Hz, 2H), 7.39 ( t, J=7.28 Hz, 2H), 7.31 (t, J=7.45 Hz, 2H), 7.26 (s, 1H), 5.57 (s, 1H), 5.29 (s, 1H), 4.424.36 (m, 2H), 4.23 (d, J=7.02 Hz, 1H), 3.36 ( s, 2H), 3.33-3.25 (m, 2H), 2.82 (d, J=5.01 Hz, 1H), 2.58 (dd, J=16.81 Hz, 2H), 2.11 (s, 2H), 1.48 (s, 9H). 13C (400MHz, CDCl3) 172.03, 156.97. 144.23, 141.52, 127.86, 127.24, 125.35, 120.16, 81.82, 66.93, 49.43, 43.28, 28.33, 16.21. m/z 457.21 (M+H)+ 3.4.8. {[2-9H-Fluoren-9-ylmethoxycarbony lamino)-1-methylsulfanylmethylethyl]-[2-(5-methyl-2,4-dioxo-3,4-dihydro-2H pyrimidin-1-yl-acety l]-amino}-acetic acid tert-butyl ester, 9a. To a solution of thionyl chloride 1.5 ml was added thymine acetic acid (0.173 gm, 0.939mmol) the solution was refluxed at 60 oC for 3 hr under an atmosphere of nitrogen. The solution was concentrated and compound 8a (0.1435 gm, 0.314mmol) dissolved in


35 0.5 ml of DMF, followed by addition of DI EA (0.078 ml, 0.471mmol). The solution was refluxed at 55 oC for 2hr. The solution was concentrated and subjected to water workup with water and ethyl acetate. The organic laye r was concentrated and subjected to flash column chromatography to give compound 9a (97.8 mg, 50%). Rf = 0.15 (1:1 :: Ethyl acetate/ Hexane). IR (Nicolet Avatar 320 FTIR) max 3060.05, 2926.15, 1672.01, 1522.05, 1450.64, 1370.04, 1324.59, 1246.16, 1152.04, 1080.95, 910.32, 760.31, 740.28, 702.03 cm-1. 1H NMR (400MHz, CDCl3) 9.37-9.36 (m, 1H), 7.76 (d, J=7.43 Hz, 2H), 7.63 (dd, J=7.20 Hz, 2H ), 7.40 (t, J=7.41 Hz, 2H), 5.30 (s, 1H), 4.70-4.62 (m, 1H), 4.36 (s, 2H), 4.28-4.20 (m, 4H), 4.16-4.10 (m, 3H), 4.06 (s, 2H), 3.513.42 (m, 1H), 2.63 (d, J=7.06 Hz, 2H), 2.19 (s, 2H), 2.04 (s, 3H), 1.84 (s, 9H). 13C (400MHz, CDCl3) 175.33, 169.29, 156.71, 150.53, 144.09, 143.78, 141.46, 127.85, 125.29, 120.01, 110.51, 83.99, 82.09, 57.70, 55.76, 42.39, 41.46, 35.86, 33.31, 21.19, 19.12, 13.70. m/z 623.25 (M+H)+ 3.4.9. (1-[(3-Benzyloxycarbonylamino-propylcarbamoyl)methylsulfanylmethyl]-2-(9H-fluoren-9ylmethoxycarbonyl amino)-ethyl]-carbamic acid tert-butyl ester, 10a To a solution of 5a (31.2 mg, 0.0728 mmol) in ethanol was added TBAI (2.7 m g, 00728 mmol) at 0 oC. From 2N NaOH solution (0.036 ml, 0.0728 mmol) was added drop-wise followed by dropwise addition of R-Br (25.26 mg, 0.0765 mmol) in ethanol. The reaction mixture was st irred for 3 hr and concentrated followed by water work-up at pH=8.5. The organic la yer was dried over anhydrous sodium sulfate


36 and concentrated followed by flas h column chromatography gave 10a (0.0435 mg, 88.7 %). Rf = 0.53 (100% Ethyl acetate). IR (Nicolet Avatar 320 FTIR) max 3320.81, 3064.27, 2930.73, 1678.66, 1651.12, 1533.07, 1448.70, 1391.80, 1366.94, 1249.57, 1150.65, 1065.28, 1003.67, 756.99, 736.50 cm-1. 1H NMR (250MHz, CDCl3) 7.74 (d, J=7.37 Hz, 2H), 7.56 (d, J=7.33, 2H), 7.42-7.21 (m, 9H), 5.73 (dd, J=8.46 Hz, 1H), 5.51 (t, J=5.76 Hz, 1H), 5.07 (d, J=7.26 Hz, 2H), 4.37 (d, J= 6.79 Hz, 2H), 4.16 ( t, J=6.51 Hz, 1H), 3.393.13 (m, 8H), 2.82-2.55(m, 2H), 2.21 (s, 1H), 2.03 (s, 1H), 1.70-1.60 (m, 2H), 1.40 (s, 9H). 13C (250MHz, CDCl3) 169.40, 157.45, 143.86, 141.31, 136.51, 128.52, 128.12, 128.05, 127.75, 127.08, 125.04, 120.01, 79.88, 66.88, 51.36, 47.21, 43.66, 37.87, 36.81, 36.46, 35.21, 29.89, 29.09, 28.40. m/z 677.29 (M+H)+ 3.4.10. {2-Amino-3-[(3-benzyloxycarbonylamino-propylcarbamoyl)methylsulfanyl]-propyl}-carbamic ac id9H-fluoren-9-ylmethyl ester, 11a To a solution of 10a (1.16 g, 1.715 mmol) in 1,4-dioxane at 0 C hydrogen chloride gas was passed for 15 min and reaction was stirred at room temperature for 30 min. The reaction mixture was concentrated and kept in vacuum for drying. 11a (1.05 gm, 100 %). IR (Nicolet Avatar 320 FTIR) max 3053.06, 1701.50, 1653.13, 1523.91, 1450.51, 1264.47, 1120.71, 729.93, 700.64 cm-1. 1H NMR (250MHz, CDCl3) 8.64-8.38 (m, 2H), 8.08-7.88 (m, 1H), 7.59 (s, 4H), 7.37-7.09 (m 10H), 5.96-5.69 (m, 1H), 5.07-5.04 (m, 1H), 4.96-4.89 (m,1H), 4.58-4.52 (m,1H), 4.29-3.88 (m, 4H), 3.31-3.01 (m, 5H), 1.681.52 (m, 2H). 13C (250MHz, CDCl3) 170.88, 166.77, 157.51, 157.01, 143.73, 141.14,


37 137.49, 128.74, 128.59, 128.49, 128.40, 128.13, 127.87, 127.32, 119.96, 67.07, 66.74, 52.19, 46.97, 46.29, 42.73, 38.37, 37.72, 36.63, 33.08. m/z 577.24 (M+H)+ 3.4.11. [1-[(3-Benzyloxycarbonylamino-propylcarbamoyl)methylsulfanylmethyl)-2-(9H-fluoren9-ylmethoxycarbonylamino)-ethylamino]acetic acid tert-butyl ester, 12a To a solution of 11a (1.05 gm, 1.715 mmol) in DMF was added tert-butyl bromoacetate (0.267 ml, 1.8 mmol) at 0C followed by addition of DIPEA (0.595 ml, 3.6 mmol) and tetra butyl ammonium iodide(63.34 mg, 0.171 mmol). The reaction mixture was stirred for 3 hr. Th e reaction mixture was then concentrated and subjected to water work-up at pH=8.5 followed by drying with anhydrous sodium sulfate, flash column chromatography to obtain 12a (0.71 gm, 60 %). Rf = 0.35 (100 Ethyl acetate). IR (Nicolet Avatar 320 FTIR) max 3332.82, 3068.60, 1716.22, 1655.08, 1528.32, 1450.37, 1367.88, 1249.77, 1151.09, 1020.30, 759.39, 740.81 cm-1. 1H NMR (400MHz, CDCl3) 7.74 (d, J=7.51 Hz, 2H), 7.59 (d, J=7.44 Hz, 2H), 7.38 (t, J=7.45 Hz, 2H), 7.28 (dd, J=12.39 Hz, 6H ), 5.86-5.78 (m, 1H), 5.44 (s, 1H), 5.06 (s, 2H), 4.37 (d, J=6.91 Hz, 2H), 4.20 (d, J= 6.88 Hz, 1H), 4.11 (d, J=7.17 Hz, 1H), 3.363.24 (m, 6H), 3.22-3.17 (m, 4H), 2.87-2.78 (m, 1H), 2.75-2.56 (m, 2H), 2.03 (d, J=4.13 Hz, 2H), 1.70-1.61 (m, 2H), 1.45 (s, 9H). 13C (400MHz, CDCl3) 172.22, 169.47, 157.26, 144.15, 141.51, 136.75, 128.70, 128.28, 128.22, 127.89, 127.26, 125.31, 120.18, 81.94, 66.92, 60.61, 49.37, 47.48, 43.27, 38.05, 36.64, 36.5, 36.26, 28.32. m/z 691.31 (M+H)+


38 3.4.12. {[1-[(3-Benzyloxycardonylamino-propylcarbamoyl)methylsulfanylmethyl]-2-(9H-fluoren9-ylmethoxycarbonylamino)-ethyl-2-[(5methyl-2,4-dioxo-3,4-dihydro-2H pyrimidin-1-yl)-acetyl]-ami no} acetic acid tertbutyl ester, 13a. To a solution of thionyl chloride 1.5 ml was added thymine acetic acid (0.104 gm, 0.564mmol) the solution was refluxed at 60 oC for 3 hr under atmosphere of nitrogen. The solution was concentrated and compound 12a (0.1035 gm, 0.1499mmol) dissolved in 0.5 ml of DMF was added, followed by add ition of DIEA (0.037 ml, 0.224mmol). The solution was refluxed at 55 oC for 2hr. The solution was con centrated and subjected to water workup with water and ethyl acetate. The organic layer was concentrated and subjected to flash column ch romatography to give compound 13a (64.2 mg, 50%). Rf = 0.5 (100% Ethyl acetate). IR (Nicolet Avatar 320 FTIR) max 3325.99, 2361.07, 1700.50, 1673.81, 1533.16, 1452.49, 1372.57, 1250.96, 1152.64, 760.57, 740.24 cm-1. 1H NMR (400MHz, CDCl3) 9.59-9.52 (m, 1H), 7.81-7.73 ( m, 2H), 7.67-7.56 ( m, 2H), 7.43-7.28 (m, 9H), 6.87-6.84 ( m, 1H), 6.666.59 (m, 1H), 5.45-5.39 (m, 1H), 5.31-5.27 (m, 1H), 5.15-5.01 (m, 3H), 4.44-4.35 (m, 1H), 4.26-4.19 (m, 1H), 4.12 (d, J=7.15 Hz, 2H), 3.23 (d, J=6.12 Hz, 8H), 2.77-2.71 (m, 1H), 2.04 (s, 2H), 1.77 (s, 4H), 1.75 (s, 9H). 13C (400MHz, CDCl3) 171.15, 169.50, 164.22, 157.10, 155.60, 151.38, 128.77, 128.50, 128.05, 127.97, 127.71, 127.67, 120.01, 112.06, 83.14, 67.01, 66.72, 61.02, 59.04, 47.20, 44.64, 42.22, 37.91, 33.08, 30.79, 27.97, 14.22. m/z 857.35 (M+H)+


39 3.4.13. 4-N-(Benzyloxycarbonyl)cytosine, 15a. Cytosine 14a (1 g, 9mmol) was added to pyridine (16 ml) along with DM AP (0.22 g, 1.8mmol). The reaction mixture was stirred for 5 min and benzyl chloro formate (2.83 ml, 19.8mm ol) was added drop wise. After stirring at room temp for 3 days, 35 ml of ice cold water was added into the reaction mixture and stirred for 15 min a nd filtered and dried in vacuum at 50 oC to give white crystalline powder of 15a (1.21 gm, 55 % ) IR (Nicolet Avatar 320 FTIR) max 1742.59, 1686.42, 1630.85, 1588.95, 1513.38, 1472.37, 1320.0, 1230.8, 1202.91, 1076.28, 1005.71, 807.30, 742.07, 696.13 cm-1 1H NMR (250MHz, CDCl3) 11.18-11.00 (br, 1H), 7.80 (d, J=7.08 Hz, 1H), 7.39 (s, 5H), 6.92 (d, J=7.02 Hz, 1H), 5.17 (s, 2H). 13C (400MHz, DMSO) 164.29, 156.50, 154.12, 147.37, 136.7, 129.12, 128.78, 128.56, 94.18, 67.06. m/z 246.08 (M+H)+ 3.4.14. 1-(tert-Butoxycarbonylmethyl)-4 -N-(benzyloxycarbonyl)cytosine, 16a. To a suspension of 15( 3 g, 12.25mmol) in anhydrous DMF (50 ml) was added anhydrous K2CO3 (1.69 gm, 12.22mmol) and anhydrous Cs2CO3 (0.4 gm, 1.22mmol). After stirring for 10 min tert-butyl bromoacetate (2.08 ml, 12.9mmol) was added dropwise and the mixture was stirred for 2 days. The resulting suspension was filtered, concentrated and subjected to a flash column chromatography to give 16a (1.625 gm, 37 %). Rf = 0.35 (3:1 :: Ethyl acetate/ Hexane). IR (Nicolet Avatar 320 FTIR) max 2983.2, 2360.1, 1745.47, 1662.13, 1628.07, 1544.52, 1498.38, 1367.94, 1213.75, 1149.50, 1062.56, 800.77, 785.62, 738.67, 694.04 cm-1. 1H NMR (250MHz, CDCl3) 8.42 (s, 1H), 7.62 (d, J=7.27 Hz, 1H), 7.34 (s, 5H), 7.24 (d, J=6.66 Hz, 1H), 5.17 (s, 2H), 4.51 (s, 2H),


40 1.44 (s, 9H). 13C (250MHz, CDCl3) 166.66, 162.96, 155.59, 152.50, 149.39, 135.17, 128.60, 128.26, 95.05, 83.11, 67.76, 51.49, 28.25. m/z 360.15 (M+H)+ 3.4.15. 1-(Carboxymethyl)-4-N-(benzy loxycarbonyl)cytosine, 17a. To a suspension of 16 (1.5 gm, 2.925mmol ) in anhydrous CH2Cl2 (5 ml) was added 4 N HCl in 1,4-dioxane (10 ml). Th e reaction was stirred for 16 hr s and partially concentrated in vacuum. Hexane (10 ml) was added to th e mixture and the suspension was filtered. The residue was dried in vacuum at 60 0C to obtain 17a (1.265 gm, 100%) IR (Nicolet Avatar 320 FTIR) max 3081.49, 2505.5, 1714.89, 1633.28, 1611.12, 1547.94, 1346.99, 1209.08, 1183.75, 1066.34, 847.20, 781.23, 726.24, 695.27 cm-1. 1H NMR (250MHz, CDCl3) 12.10-11.93 (br, 1H), 8.19 (d, J=6.73 Hz, 1H), 7.38 (d, J=11.65 Hz, 5H), 7.06 (d, J=6.60 Hz, 1H), 5.21 (s, 2H), 4.60 (s, 2H). 13C (250MHz, DMSO) 168.98, 162.45, 153.33, 152.67, 151.59, 135.63, 128.79, 128.47, 128.01, 94.00, 66.90, 50.59. m/z 304.09 (M+H)+


41 REFRENCES 1) P. E. Nielsen; M.Eghoim; R.H. Berg; O. Buchardt, Sequence selective recognition of DNA by Strand displacement with thymine-substituted polyamide, Science 1991, 254, 1497-1500. 2) Peptide Nucleic Acids Protocols an d application by Peter E. Neilsen. 3) S. C. Brown, S. A. Thomson, J. M. Veal, D. G. Davis, NMIR solution structure of peptide nucleic acid complexed with RNA, Science 1994 265 777-780. 4) P. Wittung, P. E. Nielsen, O. Buchardt, M. Eghoim, B. Norden, DNA-like double helix formed by peptide nucleic acid, Nature 1994 368 561-563. 5) M. Egholm; O. Buchardt; L. Christensen; C. Bebrens; S. M. Freier; D. A. Driver; R. H. Berg; S.K. Kim; B. Nordon; P. E. Nielsen. Nature 1993 365 566-568. 6) P. E. Nielsen; M. Egholm; O. Buchardt. J. Mol. Recognit 1994 7 165-170. 7) Weintraub, H. M. Antisense RNA and DNA. Sci. Am 1990 262 40-46. 8) Helen C.; Toulme, J. Specific Regulation of Gene Expression by Antisense, Sense and Antigene Nucleic Acids. Biochim. Biophys. Acta 1990 1049 99-125. 9) Birgitte, H.; Nielsen, P. E. Bioorganic & Medicinal Chemistry 4 5-23. 10) Zamecnik, P. C.; Stephenson, M. L. Inhibition of Rous sarcoma virus replication and cell transformation by specific oligodeoxynucleotide. Proc. Natl. Acad. Sci 1978 75 280-284.


42 11) Cohen, J. S. Atisense Oligodeoxynuc leotides as Antiviral Agents. Antivir. Res 1991 16 121-133. 12) Calabretta, B. Inhibition of Protooncogene Expression by Antisense Oligonucleotides. Cancer Res 1991 51 4505-4510. 13) Cazenave, C.; Chevrier, M.; Thoung, N, T.; Helene, C. Nucleic Acids Res 1987 15 10507-10521. 14) Dash P.; Lotan, I.; Knapp, M.; Kandel, E. R.; Geolet, P. Proc. Natl. Acad. Sci 1987 84 7896-7900. 15) Dagle, J. M.; Walder, J. A.; Weeks, D. L. Nucleic Acids Res 1990 18 4751-4757. 16) Boiziau, C.; Kurfurst, R.; Cazenave, C. ; Roig, V.; Thoung, N. T.; Toulme, J. Nucleic Acids Res 1991 19 1113-1119. 17) Mickelfield, J. Backbone modification of nucleic acids: Synthesis, structure and therapeutic applications. Current Med. Chem 2001 8 1157-1179. 18) Holt, J. T.; Render, R. L.; Nienhuis, A. W. An oligomer complementary to C-myc RNA inhibits proliferation of HL-6 0 promyelocytic cells and induces differentiation. Mol. Cell. Biol. 1988 8 963-973. 19) Woolf, T.; Jennings, C.; Rebagliati, M.; Melton, D. The stability, toxicity and effectiveness of unmodified and phosphor othioate antisense oligodeoxynucleotides in xenopous oocytes and embryos. Nucleic Acid res 1990 18 1763-1769. 20) Woolf, T. M.; Melton, D. A.; Jennings, C. Specificity of antisense oligonucleotides in vivo. Proc. Natl. Acad. Sci. 1992 89 7305-7309.


43 21) Marwick, C. First “antisense” drug will treat CMV retinis. J. Am. Med. Assoc 1998 280 871. 22) Miculka, C.; Windhab, N.; Escenmoser, A.; Sh erer, S; Quinkert, G. PCT Int. Appl. WO 9915509 A2 1999 pp43. 23) Summerton, J.; Weller, D. Morpholine anti sense oligomer: design, preparation and properties. Antisense Nucleic Drug Dev 1997 7 187-190. 24) Stirchak, E. P.; Summerton, J. E.; Weller, D. D. Uncharged stereoregular nucleic acid analogs Nucleic Acids Res 1989 17 6129-6141. 25) Petersen, M.& Wengel, J. LNA: a vers atile for therapeutics and genomics. Trends in Biotechnology 2003 21 74-81. 26) Maurinsh, Y.; Rosemetr, R.; Esnouf, R.; Wa nng, J.; Ceulemans, G.; Lescrinier, E.; Hendrix, C.; Busson, R.; Seela, F.; VanAer schot, A.; Herdewinjn, P. Synthesis and properties of oligonucleotides c ontaining 3-hydroxy-4-hydroxymethyl-1cyclohaxananyl nucleosides. Chem. Eur. J 1999 5 2139-2150. 27) Mitsuya, H.; Looney, D. J.; Kuno, S.; Ueno, R.; Wong-Staal, F.; Border, S. Science 1988 240 646-649. 28) WuDunn, D.; Spear, P. G. Initial Interaction of Herpes Simplex Virus with Cells is Binding to Heparan Sulfate. J. Virol 1989 63 52-58. 29) Good, L.; Awasthi, S. K.; Dryselius, R.; Larsson, O.; Nielsen, P. E. Bactericidal antisense effects of peptide-PNA conjugates, Nat. Biotechnol 2001 19 360-364.


44 30) Hanvey, J. C.; Peffer, N.J.; Bissi, J.E.; Thomson, S. A.; Cadilla, R.; Josey, J. A.; Ricca, D. J.; Hassman, C. F.; Bonham, M. A.; K. G. Antisense and antigene properties of peptide nucleic acids. Science 1992 258 1481-1485. 31) Knudsen, H. & Nielsen P. E. Antisense properties of duplex and triplex forming Peptide nucleic Acids. Nucleic Acid Res 1996 24 494-500. 32) Altmann, K. H.; Chiesi, S.; Echevevria, C. Bioorg. Med. Chem. Lett 1997 7 11191122. 33) Efimov, V. A.; Choob, M. V.; Buryakova, A. A.; Chakhmakhcheva, O. G. Synthesis and binding study of phosphonate analogues of PNAs and their hybrib with PNA. Nucleosides Nucleotides 1998 17 1671-1679. 34) VanderLaan, A. C.; Stromberg, R.; Va nBoom J. H.; Kuyl-Yeheskiely, E.; Chakhmakhcheva, O. G. Tet. Lett 1996 37 7857-7860. 35) Zhou, P.; Wang, M.; Du, L.; Fischer, G. W.; Waggoner, A.; Ly, W. H. J.Am. Chem. Soc 2003 125 6878-6879. 36) Lagriffoule, P. H.; Egholm, M.; Niel sen, P. E.; Berg, R.H.; Buchardt, O. Bioorg. Med. Chem. Lett 1994 4 1081-1082. 37) Almarison, O.; Bruice, T. C. Proc. Natl. Acad. Sci 1993 90 9542-9546. 38) Krotz, A. H.; Buchardt, O.; Nielsen, P. E. Tetrahedron Lett 1995 36 6937-6940. 39) Gangamani, B. P.; Kumar, V. A.; Ganesh, K. N. Tetrahedron 1996 52 1501715030. 40) Hamilton, S .E.; Simmons, C. G.; Kathiriya, I. S.; Corey, D. R. Chem Biol ., 1999 6 343-351.


45 41) Uhlamann, E.; Will, D. W.; Breiphol, G.; Langer, D.; Ryte, A. Angew Chem 1996 85 2632-2635. 42) Uhlamann, E.; Will, D. W.; Breiphol, G.; Langer, D.; Ryte, A. Angew Chem. Int. Ed. Engl 1996 85 2632-2635. 43) Thomson, S A.; Josey, J. A.; Cadilla, R.; Ga ul, M. D.; Hassman, C. F.; Luzzio, M. J.; Pipe, A. J.; Reed, K. L.; Ricca, D. J.; Wiethe, R. W.; Noble, S. A. Tetrahedron 1995 51 6179-6194.




47 Spectrum 4.01 BocHN CONH2 S Ph Ph Ph 2a1H NMR (CDCl3, 250 MHz) of 2a ppm (f1) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 0 500 100 150 6.052 4.941 3.894 2.662 2.576 1.407 13C NMR (CDCl3, 250 MHz) of 2a ppm (f1) 50 100 150 0 500 100 150 200 250 300 350 173.172 155.436 144.414 80.417 67.243 53.234 33.810


48 Appendix A (Continued) Spectrum 4.02 BocHN S Ph Ph Ph NH2 3a 1H NMR (CDCl3, 250 MHz) of 3a ppm (f1) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 0 500 100 150 5.034 3.544 2.694 2.350 1.394 13C NMR (CDCl3, 250 MHz) of 3a ppm (f1) 50 100 150 0 10 20 30 40 50 60 155.470 144.649 79.286 66.799 60.407 44.413 34.384


49 Appendix A (Continued) Spectrum 4.03 BocHN S Ph Ph Ph NHFmo c 4a 1H NMR (CDCl3, 250 MHz) of 4a ppm (f1) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 0 500 100 150 200 4.736 4.372 4.157 3.590 3.129 1.415 13C NMR (CDCl3, 250 MHz) of 4a ppm (f1) 50 100 150 0 10 20 30 40 50 60 70 80 90 156.893 155.677 144.568 120.027 79.779 28.563


50 Appendix A (Continued) Spectrum 4.04 BocHN HS NHFmoc 5a 1H NMR (CDCl3, 250 MHz) of 5a ppm (f1) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 0 500 100 150 200 250 300 350 5.275 4.390 4.179 3.788 3.350 1.557 1.428 13C NMR (CDCl3, 250 MHz) of 5a ppm (f1) 50 100 150 0 50 157.195 155.853 143.993 141.343 80.044 67.003 52.444 47.182 43.092 28.380


51 Appendix A (Continued) Spectrum 4.05 BocHN S NHFmo c 6a 1H NMR (CDCl3, 250 MHz) of 6a ppm (f1) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 0 500 100 150 200 250 300 5.3 11 5.1 33 4.3 85 4.1 93 2.1 26 1.4 31 13C NMR (CDCl3, 250 MHz) of 6a ppm (f1) 50 100 150 0 50 10 15 7 .1 76 14 4 .6 01 14 1 .3 28 79 .86 2 66 .89 3 47 .26 6 28 .41 0 16 .11 3


52 Appendix A (Continued) Spectrum 4.06 ClH.H2N S NHFmoc 7a 1H NMR (CDCl3, 250 MHz) of 7a ppm (f1) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 0 10 20 30 40 4.2 38 2.0 26 13C NMR (CDCl3, 250 MHz) of 7a ppm (f1) 50 100 150 0 100 200 300 400 500 15 7 .2 23 14 3 .7 88 14 1 .2 00 67 .09 2 51 .62 8 47 .06 2 42 .96 9 34 .33 8 15 .97 3


53 Appendix A (Continued) Spectrum 4.07 N S NHFmoc O H O 8a 1H NMR (CDCl3, 400 MHz) of 8a ppm (f1) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 0 500 100 150 200 250 300 5.569 4.381 4.238 3.361 3.287 2.113 1.480 13C NMR (CDCl3, 400 MHz) of 8a ppm (f1) 50 100 150 0 100 0 200 0 300 0 400 0 500 0 172.181 156.831 144.205 141.472 81.789 66.927 43.285 28.333 16.216


54 Appendix A (Continued) Spectrum 4.08 N S NHFmoc O O N N O O H O 9a 1H NMR (CDCl3, 400 MHz) of 9a ppm (f1) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 0 500 100 13C NMR (CDCl3, 400 MHz) of 9a ppm (f1) 50 100 150 0 100 200 300 400 500


55 Appendix A (Continued) Spectrum 4.09 BocHN S NHFmoc N CbzHN O H 10a 1H NMR (CDCl3, 250 MHz) of 10a ppm (f1) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 0 500 100 5.698 5.511 5.057 3.199 2.208 2.029 1.654 1.403 13C NMR (CDCl3, 250 MHz) of 10a ppm (f1) 50 100 150 0 500 100 150 169.413 157.458 143.807 141.313 136.613 80.034 66.777 51.389 47.225 43.685 28.418


56 Appendix A (Continued) Spectrum 4.10 ClH.H2N S NHFmoc N CbzHN O H 11a 1H NMR (CDCl3, 250 MHz) of 11a ppm (f1) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 0 50 13C NMR (CDCl3, 250 MHz) of 11a ppm (f1) 50 100 150 0 500 100 170.880 166.771 143.734 141.146 136.523 119.966 36.886 29.864


57 Appendix A (Continued) Spectrum 4.11 H N S NHFmoc N CbzHN O H O O 12a 1H NMR (CDCl3, 400 MHz) of 12a ppm (f1) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 0 500 100 150 200 250 5.816 5.436 5.055 4.361 4.101 2.031 1.653 1.460 13C NMR (CDCl3, 400 MHz) of 12a ppm (f1) 50 100 150 0 500 0 100 0 172.222 169.466 157.196 144.162 141.650 137.016 120.103 81.980 66.825 60.613 56.507 36.639 28.325


58 Appendix A (Continued) Spectrum 4.12 N S NHFmoc N CbzHN O H O O N N O O O H 13a 1H NMR (CDCl3, 400 MHz) of 13a ppm (f1) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 0 500 0 100 0 150 0 13C NMR (CDCl3, 400 MHz) of 13a ppm (f1) 50 100 150 0 500 0 100 0


59 Appendix A (Continued) Spectrum 4.13 N N NHCbz O H 15a 1H NMR (d6DMSO, 250 MHz) of 15a ppm (f1) 5.0 10.0 0 10 20 30 40 50 11 .10 6 7.7 81 7.3 92 6.9 33 5.1 74 13C NMR (d6DMSO, 400 MHz) of 15a ppm (f1) 50 100 150 0 50 10 15


60 Appendix A (Continued) Spectrum 4.14 N N NHCb z O O O 16a 1H NMR (CDCl3, 250 MHz) of 16a ppm (f1) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 0 500 100 150 200 250 300 8.423 7.632 7.338 7.225 5.167 4.509 1.444 13C NMR (CDCl3, 250 MHz) of 16a ppm (f1) 50 100 150 0 100 200 300 400 500 600 166.746 162.965 155.594 152.502 95.062 83.121 67.775 51.506 27.971


61 Appendix A (Continued) Spectrum 4.15 N N NHCbz O OH O 17a 1H NMR (d6DMSO, 250 MHz) of 17a ppm (f1) 5.0 10.0 0 10 20 30 40 50 60 11 .99 6 8.2 03 7.4 08 7.0 70 5.2 14 4.5 99 13C NMR (d6DMSO, 250 MHz) of 17a ppm (f1) 50 100 150 0 500 100 168.983 162.455 153.334 152.675 151.588 135.638 94.071 66.912 50.914