Organolitnium compounds have been employed in synthetic worK for many years. However only during the last decade has much progress been made in establishing the mechanistic pathways for the reactions of these compounds.

Chemical modifications of oligonucleotides provide an important tool to understand how the natural substrate works as well as how to improve their biochemical and biological properties as potential therapeutics and diagnostics. Our carba-LNA (2',4'-carba-bridged Locked Nucleic Acid) modified oligo-DNA or -RNA have been found to be useful to modulate oligo-RNA and -DNA activity. This thesis is based on four papers: Paper I (J. Org. Chem. 2010, 75, 7112-7128) deals with the synthesis of 2',4'-propylene-bridged (Carba-ENA) thymidine and its analogues. These carba-ENA nucleosides have been subsequently incorporated into 15mer antisense oligodeoxynucleotides (AON), and their affinity toward complementary mRNA and DNA, as well as their nuclease resistance and RNase H recruitment capability have been investigated in comparison with those of the native and ENA counterparts. Paper II (J. Org. Chem. 2012, 77, 6855–6872) illustrates the synthesis of dimethylbicyclo[2.2.1]heptane and a diastereomeric mixture of oxabicyclo[2.2.1]heptanes by the free-radical ring-closure reaction approach. The role of steric factors for different chair- and the boat-like transition states was evaluated involving the 5-exo radical ring closure reaction to a tethered olefin. Paper III (J. Org. Chem. 2012, 77, 9747-9755) shows an unusual strain releasing reaction of 1-mesyloxy-8,7-dimethylbicyclo[2.2.1]heptane by a base-promoted substitution at the chiral C3 followed by spontaneous concerted ring opening involving the most strained C2-C3-C4 bonds (with bond angle 94°) and the C2 bridgehead leading to anti-endo elimination of the C1-mesyloxy group by the conjugate base of adenine or thymine to give two diastereomeric C3'(S) and C3'(R) derivatives of 1-thyminyl and 9-adeninyl cyclohexene, and a mechanistic rational has been formulated. Paper IV (J. Org. Chem. 2014, 79, 7266−7276) focuses on the diastereospecific synthesis of E/Z bicyclo[2.2.1]heptane-7- and oxabicyclo[2.2.1]heptane-8-oximes and their corresponding C-nitroso derivatives. The comparative kinetic and thermodynamic studies of the conversions of the C-nitroso side products to the required oximes have been delineated leading to the synthesis of desmethyl sugar derivatives.

This thesis examines the synthesis of conformationally constrained β-amino acids and β- peptides, and the electron transfer properties of the latter when immobilised on gold. Additionally, cross metathesis on gold was investigated as a method for surface functionalisation. Chapter One introduces the concepts of electron transfer in nature, how it is facilitated by the secondary structure in α-peptides, and why β-peptides might be useful for studying electron transfer. This is followed by a discussion of the properties of β-peptides, including the enhanced stability and variety of helical secondary structures and the greater potential for functionalisation of the peptide backbone when compared to α-peptides. Finally, the conformational constraints of ring-systems on cyclic amino acids is discussed, with reference to the stabilising effect of these compounds on peptide secondary structures. Chapter Two describes the electrochemical analysis of β-hexapeptides immobilised on gold. The chapter is prefaced by a discussion of the important electron transfer mechanisms for peptides, the fabrication of peptide-gold self-assembled monolayer (SAM) interfaces, and the electron transfer in helical α-peptides. β-Peptides containing an electroactive ferrocene moeity were immobilised on gold and studied using cyclic voltammetry and chronoamperometry. The latter method was used to examine the dependence of the electron transfer rate on overpotential, thereby determining the likely mode of electron transfer through the β-peptides SSβ₆Fc, Fcβ₆SS and SC₁₅β₆Fc. These peptides exhibited very weak dependence on overpotential, characteristic of electron transfer behaviour of an electron hopping mechanism (which is also thought to occur in helical α-peptides). Both the dipole moment of the peptides and the structure of the sulfurlinker group were found to be important in determining the rate of electron transfer. Conversely, the equivalent α-peptide SSα₆Fc exhibited electron transfer behaviour characteristic of the less efficient tunnelling mechanism, which is thought to operate in strand-like peptides. Chapter Three examines the application of cross metathesis, using a Grubbs' second generation catalyst, as a means to functionalise olefin-terminated self-assembled monolayers on gold. Abstract iv Firstly, an introduction into the limited published research on cross metathesis on both planar surfaces and nanoparticles is given. Olefin-terminated thiol 3.18, suitable for immobilisation on gold, and solution phase olefin-terminated ferrocene 3.10 were synthesised as reactants for cross metathesis studies. An analytical methodology was developed involving the cross metathesis of surface-immobilised 3.18 with ferrocene 3.10 in dichloromethane, whereby the concentration of electroactive cross metathesis product 3.22 was monitored electrochemically as a function of time. The concentration of surface-immobilised product 3.22 was determined by integration of the oxidation peak area and found to be highly dependent on both the concentration of immobilised olefin reactant 3.18 and reaction time. Furthermore, the surface concentration of ferrocenyl model disulfide 3.21 and thiol 2.18 decayed markedly upon addition of Grubb's catalyst, as revealed by the decrease in the oxidation peak area, which suggested that catalystmediated desorption was occurring. Chapter Four details the solution-phase synthesis of ferrocene- and thiol-functionalised β- hexapeptides used in both the electron transfer studies described in chapter two, and in the determination of secondary structure using circular dichroism and NMR techniques. The synthesis of simple model compounds 4.14, 4.16 and 4.18 established the incompatibility of the deprotection of methyl and benzyl ester protecting groups with protected-thiol and disulfide linkers, leading to the use of N-hydroxysuccinmide-activated sulfur-linkers 4.20 and 4.22 in further synthesis. A number of β-hexapeptides were synthesised by amide coupling of β- tripeptides functionalised at the N- and C-termini. Structural studies of the methanol soluble β- hexapeptide 4.60 suggested that the covalent attachment of ferrocene moeity to the C-terminus of a β-peptide did not disrupt the formation of a 14-helix in solution. β-peptides containing functionality at both the C- and N-termini (such as SSβ₆Fc, SSβ₆Et and acetyl-protected SC₁₅β₆Fc) were not suitable for solution phase structural studies; however, molecular modelling suggested that helical conformations are the most stable these β-peptides in solution phase. Chapter Five outlines the synthesis of novel cyclic β-amino acids by two different general synthetic routes. The first uses an efficient conjugate addition/fluorination reaction of α,β- unsaturated esters with lithiated chiral secondary amines to prepare the novel cyclopentyl- and cyclohexyl-based fluorinated β-amino acids 2.43a and 2.43b. The high diastereoselectivity of this reaction, which introduces two stereocentres into the achiral unsaturated esters, is directed by the configuration of the attacking amine. The second methodology utilizes the versatile ringclosing metathesis reaction in the synthesis of novel cyclic β-amino acids. A stereoselective Abstract v trans-alkylation of olefinic β-amino acids gave the required β-dienes 5.62 and 5.77. Optimised cyclisation yields were achieved with a Grubb's 2nd generation catalyst for diene 5.62 and Grubb's 1st generation catalyst for diene 5.77, to give the trans-cycloheptyl- and cyclooctylbased β-amino acids 5.63 and 5.78, respectively. The attempted synthesis of cyclononyl-based β-amino acid 5.87 using both catalysts yielded only cyclic dimer products 5.88 and 5.89. The trans configuration of the 5.62 diene was confirmed by x-ray crystallography. Chapter Six is an experimental chapter and outlines the electrochemical setup and analysis, and the synthesis, purification and characterisation of compounds described in this thesis.

A short oligonucleotide sequence as in a single-stranded antisense oligo nucleotides (AON) or in double-stranded small interfering RNAs (siRNA) can modulate the gene expression by targeting against the cellular mRNA, which can be potentially exploited for therapeutic purposes in the treatment of different diseases. In order to improve the efficacy of oligonucleotide-based drugs, the problem of target affinity, nuclease stability and delivery needs to be addressed. Chemical modifications of oligonucleotides have been proved to be an effective strategy to counter some of these problems. In this thesis, chemical synthesis of conformationally constrained nucleosides such as 7′-Me-carba-LNA-A, -G, -MeC and -T as well as 6′, 7′-substituted α-L-carba-LNA-T (Papers I-III) was achieved through a key free-radical cyclization. 1D and 2D NMR techniques were employed to prove the formation of bicyclic ring system by free-radical ring closure as well as to identify the specific constrained conformations in sugar moieties. These sugar-locked nucleosides were transformed to the corresponding phosphoramidites and incorporated into antisense oligonucleotides in different sequences, to evaluate their physicochemical and biochemical properties for potential antisense-based therapeutic application. AONs modified with 7′-Me-carba-LNA analogues exhibited higher RNA affinities (plus 1-4°C/modification) (Papers I & III), but AONs containing α-L-carba-LNA analogues showed decreased affinities (minus 2-3°C/ modification) (Paper II) towards complementary RNA compared to the native counterpart.  It has been demonstrated in Papers I-III that 7′-methyl substitution in α-L-carba-LNA caused the Tm drop due to a steric clash of the R-configured methyl group in the major groove of the duplex, whereas 7′-methyl group of carba-LNA locating in the minor groove of the duplex exerted no obviously negative effect on Tms, regardless of its orientation. Moreover, AONs containing 7′-Me-carba-LNA and α-L-carba-LNA derivatives were found to be nucleolytically more stable than native AONs, LNA modified AONs as well as α-L-LNA modified ones (Papers I-III). We also found in Paper II & III that the orientations of OH group in C6′ of α-L-carba-LNAs and methyl group in C7′ of 7′-Me-carba-LNAs can significantly influence the nuclease stabilities of modified AONs. It was proved that the methyl substitution in cLNAs which points towards the vicinal 3′-phosphate were more resistant to nuclease degradation than that caused by the methyl group pointing away from 3′-phosphate. Additionally, AONs modified with 7′-Me-carba-LNAs and α-L-carba-LNAs were found to elicit the RNase H mediated RNA degradation with comparable or higher rates (from 2-fold to 8-fold higher dependent upon the modification sites) as compared to the native counterpart. We also found that the cleavage patterns and rates by E. coli RNase H1 were highly dependent upon the modification sites in the AON sequences, regardless of the structural features of modifications (Papers II & III). Furthermore, we have shown that the modulations of Tms of AON/RNA duplexes are directly correlated with the aqueous solvation (Paper III).

This thesis examines the synthesis of conformationally constrained β-amino acids and β- peptides, and the electron transfer properties of the latter when immobilised on gold. Additionally, cross metathesis on gold was investigated as a method for surface functionalisation. Chapter One introduces the concepts of electron transfer in nature, how it is facilitated by the secondary structure in α-peptides, and why β-peptides might be useful for studying electron transfer. This is followed by a discussion of the properties of β-peptides, including the enhanced stability and variety of helical secondary structures and the greater potential for functionalisation of the peptide backbone when compared to α-peptides. Finally, the conformational constraints of ring-systems on cyclic amino acids is discussed, with reference to the stabilising effect of these compounds on peptide secondary structures. Chapter Two describes the electrochemical analysis of β-hexapeptides immobilised on gold. The chapter is prefaced by a discussion of the important electron transfer mechanisms for peptides, the fabrication of peptide-gold self-assembled monolayer (SAM) interfaces, and the electron transfer in helical α-peptides. β-Peptides containing an electroactive ferrocene moeity were immobilised on gold and studied using cyclic voltammetry and chronoamperometry. The latter method was used to examine the dependence of the electron transfer rate on overpotential, thereby determining the likely mode of electron transfer through the β-peptides SSβ₆Fc, Fcβ₆SS and SC₁₅β₆Fc. These peptides exhibited very weak dependence on overpotential, characteristic of electron transfer behaviour of an electron hopping mechanism (which is also thought to occur in helical α-peptides). Both the dipole moment of the peptides and the structure of the sulfurlinker group were found to be important in determining the rate of electron transfer. Conversely, the equivalent α-peptide SSα₆Fc exhibited electron transfer behaviour characteristic of the less efficient tunnelling mechanism, which is thought to operate in strand-like peptides. Chapter Three examines the application of cross metathesis, using a Grubbs' second generation catalyst, as a means to functionalise olefin-terminated self-assembled monolayers on gold. Abstract iv Firstly, an introduction into the limited published research on cross metathesis on both planar surfaces and nanoparticles is given. Olefin-terminated thiol 3.18, suitable for immobilisation on gold, and solution phase olefin-terminated ferrocene 3.10 were synthesised as reactants for cross metathesis studies. An analytical methodology was developed involving the cross metathesis of surface-immobilised 3.18 with ferrocene 3.10 in dichloromethane, whereby the concentration of electroactive cross metathesis product 3.22 was monitored electrochemically as a function of time. The concentration of surface-immobilised product 3.22 was determined by integration of the oxidation peak area and found to be highly dependent on both the concentration of immobilised olefin reactant 3.18 and reaction time. Furthermore, the surface concentration of ferrocenyl model disulfide 3.21 and thiol 2.18 decayed markedly upon addition of Grubb's catalyst, as revealed by the decrease in the oxidation peak area, which suggested that catalystmediated desorption was occurring. Chapter Four details the solution-phase synthesis of ferrocene- and thiol-functionalised β- hexapeptides used in both the electron transfer studies described in chapter two, and in the determination of secondary structure using circular dichroism and NMR techniques. The synthesis of simple model compounds 4.14, 4.16 and 4.18 established the incompatibility of the deprotection of methyl and benzyl ester protecting groups with protected-thiol and disulfide linkers, leading to the use of N-hydroxysuccinmide-activated sulfur-linkers 4.20 and 4.22 in further synthesis. A number of β-hexapeptides were synthesised by amide coupling of β- tripeptides functionalised at the N- and C-termini. Structural studies of the methanol soluble β- hexapeptide 4.60 suggested that the covalent attachment of ferrocene moeity to the C-terminus of a β-peptide did not disrupt the formation of a 14-helix in solution. β-peptides containing functionality at both the C- and N-termini (such as SSβ₆Fc, SSβ₆Et and acetyl-protected SC₁₅β₆Fc) were not suitable for solution phase structural studies; however, molecular modelling suggested that helical conformations are the most stable these β-peptides in solution phase. Chapter Five outlines the synthesis of novel cyclic β-amino acids by two different general synthetic routes. The first uses an efficient conjugate addition/fluorination reaction of α,β- unsaturated esters with lithiated chiral secondary amines to prepare the novel cyclopentyl- and cyclohexyl-based fluorinated β-amino acids 2.43a and 2.43b. The high diastereoselectivity of this reaction, which introduces two stereocentres into the achiral unsaturated esters, is directed by the configuration of the attacking amine. The second methodology utilizes the versatile ringclosing metathesis reaction in the synthesis of novel cyclic β-amino acids. A stereoselective Abstract v trans-alkylation of olefinic β-amino acids gave the required β-dienes 5.62 and 5.77. Optimised cyclisation yields were achieved with a Grubb's 2nd generation catalyst for diene 5.62 and Grubb's 1st generation catalyst for diene 5.77, to give the trans-cycloheptyl- and cyclooctylbased β-amino acids 5.63 and 5.78, respectively. The attempted synthesis of cyclononyl-based β-amino acid 5.87 using both catalysts yielded only cyclic dimer products 5.88 and 5.89. The trans configuration of the 5.62 diene was confirmed by x-ray crystallography. Chapter Six is an experimental chapter and outlines the electrochemical setup and analysis, and the synthesis, purification and characterisation of compounds described in this thesis.

Collagen is one of the most important and abundant proteins in mammals. It consists of three left-handed PPII helixes coiled along a common axis to form a very compact right-handed super helix. The primary structure is shown to be (Gly-Xaa-Yaa)n repeats with high content of prolyl residues at both Xaa and Yaa positions. <i>Cis-trans</i> isomerization of the prolyl amide bonds is one of the rate-limiting steps during collagen triple helix folding. The conformationally locked alkene isosteres Fmoc-Gly-Ψ[(E)CH=C]-Pro-Hyp(tBu)-OH and Fmoc-Pro-Ψ[(E)CH=C]-Pro-OH were designed and synthesized. The synthesis of the Gly-Pro isostere had no stereo-control, and the two diastereomers of the tripeptide isostere Fmoc-Gly-Ψ[(E)CH=C]-Pro-Hyp(tBu)-OBn were separated by normal phase HPLC. Although the stereoselectivity of the asymmetric reduction was not good for the Pro-Pro isostere, the resulting diastereomers was separable by flash chromatography, and the absolute stereochemistry of the two diastereomers was determined by Mosher's method. The Gly-Pro alkenyl peptides, and their control peptide Ac-(Gly-Pro-Hyp)8-Gly-Gly-Tyr-NH2 were synthesized and purified. All three peptides showed a maximum around 225 nm and a minimum close to 200 nm in the CD spectra, which indicated the formation of PPII helixes. The Tm value of the control peptide was determined to be 50.0 °C. The peptide with Gly-Ψ[(E)CH=C]-L-Pro-Hyp as the guest triplet formed a stable triple helix with a Tm value of 28.3 °C. The peptide with Gly-Ψ[(E)CH=C]-D-Pro-Hyp as the guest triplet showed a linear decrease in the ellipticity with increasing temperature, which indicated that no triple helix was formed. The Pro-Pro alkenyl peptide and its control peptide H-(Pro-Pro-Gly)₁₀-OH were synthesized and purified. The T_m value of control peptide was determined to be 31.6 °C by extrapolation to 0 M TMAO in PBS buffer, which was very close to the measured value of 31.5 °C. The Pro-Pro alkenyl peptide began to show a maximum around 225 nm in the CD spectra when the concentration of TMAO was higher than 2.5 M. After extrapolation to 0 M TMAO, the T_m value was determined to be –22.0 °C. These results indicate that the backbone inter-chain hydrogen bond is one of the major forces in stabilizing the collagen triple helix, while <i>cis-trans</i> isomerization has limited contribution. The intrinsic properties of the amide bond may have huge influence on the stability of the collagen triple helix. The helix-turn-helix motif is an important tertiary structure in DNA-binding proteins. Stepwise modifications of the Antennapedia HTH peptide (27-55) were performed to improve the helicity and stability. The peptide with more side-chain ion-pairs was over 4 times more helical than the native Antp peptide, while the Ala-based peptide was over 9 times more helical than the native peptide. A 12-membered ring, Fmoc-protected HTH-turn mimic was designed and synthesized, and was ready for solid phase peptide synthesis. The solubility of the cyclic peptide was very poor, and the purification of the final product was very difficult. The solubility problem might also affect solid phase peptide synthesis in the future. / Ph. D.

The conformation of paclitaxel in the bound state on the protein has been proposed to be the T-taxol conformation, and paclitaxel analogs constrained to the T-taxol conformation proved to be significantly more active than paclitaxel in both cytotoxicity and tubulin polymerization assays, thus validating the T-taxol conformation as the tubulin-binding conformation. In this work, eight compounds containing an aza-tricyclic moiety as a mimic of the baccatin core of paclitaxel have been designed and synthesized as water-soluble simplified paclitaxel analogs, among which 3.50-3.52 and 3.55 were conformationally constrained analogs designed to bind to the paclitaxel binding site of tubulin, based on their similarity to the T-taxol conformation. The open-chain analogs 3.41-3.43 and 3.57 and the bridged analogs 3.50-3.52 and 3.55 were evaluated for their antiproliferative activities against the A2780 cell lines. Analogs 3.50-3.52 and 3.55 which were designed to adopt the T-taxol conformation showed similar antiproliferative activities compared to their open-chain counterparts. They were all much less active than paclitaxel. In the second project, a series of paclitaxel analogs with various thio-containing linkers at C-2′ and C-7 positions were designed and synthesized in our lab. These analogs were attached to the surfaces of gold nanoparticles by CytImmune Sciences for the development of mutifunctional tumor-targeting agents. The native analogs and the gold bound analogs were evaluated for their antiproliferative activities against the A2780 cell line. All the compounds tested showed comparable or better activities than paclitaxel. Stability studies were performed for selected analogs in hydrolysis buffer, which showed that the analogs released paclitaxel in buffer over time. In the third project, the synthesis of a conformationally constrained paclitaxel analog which was designed to mimic the REDOR-taxol conformation was attempted. Two synthetic routes were tried and significant progress was made toward the synthesis of the conformationally constrained analog. However, both of the current synthetic routes failed to produce the key intermediate that would serve as the precursor for a ring-closing metathesis reaction to furnish the macrocyclic ring. / Ph. D.

This thesis is based on six original research publications describing synthesis, structure and physicochemical and biochemical analysis of chemically modified oligonucleotides (ONs) in terms of their potential diagnostic and therapeutic applications. Synthesis of two types of bicyclic conformationally constrained nucleosides, North-East locked 1',2'-azetidine and North locked 2',4'-aza-ENA, is described. Study of the molecular structures and dynamics of bicyclic nucleosides showed that depending upon the type of fused system they fall into two distinct categories with their respective internal dynamics and type of sugar conformation. The physicochemical properties of the nucleobases in the conformationally constrained nucleosides found to be depended on the site and ring-size of the fused system. The incorporation of azetidine modified nucleotide units into 15mer ONs lowered the affinity toward the complementary RNA. However, they performed better than previously reported isosequential 1',2'-oxetane modified analogues. Whereas aza-ENA-T modification incorporated into ONs significantly enhanced affinity to the complementary RNA. To evaluate the antisense potential of azetidine-T and aza-ENA-T modified ONs, they were subjected to RNase H promoted cleavage as well as tested towards nucleolytic degradation. Kinetic experiments showed that modified ONs recruit RNase H, however with lower enzyme efficiency due to decreased enzyme-substrate binding affinity, but with enhanced turnover number. Both, azetidine-T and aza-ENA-T modified ONs demonstrated improved 3'-exonuclease stability in the presence of snake venom phosphodiesterase and human serum compared to the unmodified sequence. Oligodeoxynucleotides (ODNs) containing pyrene-functionalized azetidine-T (Aze-pyr X) and aza-ENA-T (Aza-ENA-pyr Y) modifications showed different fluorescence properties. The X modified ODNs hybridized to the complementary DNA and RNA showed variable increase in the fluorescence intensity depending upon the nearest-neighbor at the 3'-end to X modification (dA &gt; dG &gt; dT &gt; dC) with high fluorescence quantum yield. However, the Y modified ODNs showed a sensible enhancement of the fluorescence intensity only with complementary DNA. Also, the X modified ODN showed decrease (~37-fold) in the fluorescence intensity upon duplex formation with RNA containing a G nucleobase mismatch opposite to the modification site, whereas a ~3-fold increase was observed for the Y modified probe.

Collagen is one of the most abundant human proteins. It exists as a right-handed superhelix called the triple helix. The triple helix consists of three left-handed polyproline type II (PPII helices) that intertwine around a common axis. Each PPII helix has the repeating peptide sequence (Gly–Xaa–Yaa)n with a high content of (2S)-proline (Pro) in the Xaa position (ca. 28%) and (2S,4R)-hydroxyproline (Hyp) in the Yaa position (ca. 38%). Unique to the prolyl amide is the ease of cis-trans isomerization. Since the triple helix necessitates that all peptide bonds be in the trans conformation, isomerization is the rate-limiting step in collagen folding. However, eliminating isomerization with a trans-locked alkene isostere destabilizes collagen-like peptides. Collagen is stabilized by electronic interactions, namely the n→π* interaction. Halo-alkene isosteres may be used to recapture these electronic interactions and stabilize a collagen-like peptide. An in-depth conformational analysis was conducted at the MP2/6-311+G(2d,p) level of theory to determine the viability of conformationally-locked halo-alkene isosteres. Fluoro-alkenes and chloro-alkenes were modeled at both the Gly–Pro and Pro–Pro (as a Pro–Hyp mimic) amide positions. Compared to the collagen crystal structure PDB ID: 1K6F, we found the fluoro-alkenes were closer geometric matches to both Gly–Pro and Pro–Pro than the corresponding chloro-alkenes. The chloro-alkene was predicted to have stronger n→π* interactions. The trans-locked proteo-alkene was also analyzed to understand why it destabilized the triple helix. We found that these models had other local minima close to the desired PPII geometry, likely leading to enhanced backbone flexibility. This deleterious flexibility was not predicted for either fluoro-alkene or chloro-alkene models. The conformationally-locked halo-alkene isostere Fmoc–Gly–Ψ[(Z)CF=C]-Pro–Hyp(tBu)–OH was designed and synthesized as a (Z)-fluoro-alkene Gly–Pro isostere. We used the chiral catalyst, L-Thr, for asymmetric aldol addition to cyclopentanone, which inadvertently enhanced the yield of the wrong enantiomer, in contrast with aldol addition to cyclohexanone. A Mg2+-promoted Horner-Wadsworth-Emmons reaction afforded the (Z)-fluoro-alkene over the (E)-fluoro-alkene in about a 2:1 ratio. The two diastereomers, Fmoc–Gly–Ψ[(Z)CF=C]-L-Pro–Hyp(tBu)–OH and Fmoc–Gly–Ψ[(Z)CF=C]-D-Pro–Hyp(tBu)–OH were separated by supercritical CO2 chromatography. The collagen-like peptides Ac–(Gly–Pro–Hyp)3–Gly–Ψ[(Z)CF=C]-L-Pro–Hyp–(Gly–Pro–Hyp)4–Gly–Gly–Tyr–NH2, Ac–(Gly–Pro–Hyp)3–Gly–Ψ[(Z)CF=C]-D-Pro–Hyp–(Gly–Pro–Hyp)4–Gly–Gly–Tyr–NH2, and the control peptide Ac–(Gly–Pro–Hyp)8–Gly–Gly–Tyr–NH2 were synthesized on solid-phase resin. The CD spectra of all three peptides showed the characteristic collagen triple-helix signature. The folding stability was determined by thermal melting (Tm). The peptide with the fluoro-alkene guest, Gly–Ψ[(Z)CF=C]-L-Pro–Hyp, was found to have a Tm value of 42.2 °C. The Tm of the control peptide was found to be 49.0 °C, a difference in stability of only ΔTm –6.8. Thus, the (Z)-fluoro-alkene as a Gly–Pro isostere forms a relatively stable triple helix. The peptide with the Gly–Ψ[(Z)CF=C]-D-Pro–Hyp guest was shown to have a linear relationship between ellipticity and temperature, indicating that a stable triple helix did not form. The enhanced stability of the (Z)-fluoro-alkene compared to the (E)-alkene Gly–Pro isostere (Tm = 28.3 °C) may be due to a stabilizing n→π* interaction, as determined by NMR deshielding of the 19F nucleus in the collagen-like peptide. In biological systems, isomerization of the prolyl amide is catalyzed by enzymes called PPIases. The PPIase Pin1 specifically catalyzes isomerization of the pSer–Pro sequence from the cis-conformation to the trans-conformation. Pin1 plays a crucial role in the G2→M transition of the cell cycle, implying the importance of cis-trans isomerization. The dipeptides H–Ser–Ψ[(Z)CH=C]-Pro–OH, H–Ser–Ψ[(E)CH=C]-Pro–OH and native H–Ser–Pro–OH were synthesized by literature methods, and activated for aminoacylation of tRNACUA for in vitro transcription-translation. Aminoacylation by chemical methods required the synthesis of a pdCpA dinucleotide. Formation of the dipeptide-dinucleotide complex was not completed because protection of the Ser side chain was problematic. On the other hand, conversion of the dipeptide into the 3,5-dinitrobenzyl ester conjugate allowed for enzymatic aminoacylation using the dFx flexizyme, an RNA enzyme. The native dipeptide was successfully coupled to tRNACUA and is ready for incorporation into a full-length Bora protein by in vitro transcription-translation. Both cis- and trans-locked alkene mimics have been converted to their respective 3,5-dinitrobenzyl ester conjugates. / Doctor of Philosophy / The proline amide (Xaa–Pro) in peptides and proteins is unique in that it allows for cis-trans isomerization. The triple-helix region of human collagen consists mostly of the repeating sequence (Gly–Pro–Hyp)n. Xaa–Pro amide-bond isomerization is rate-limiting for triple-helix formation. We eliminated isomerization at one position in a collagen-like peptide with a locked alkene mimic of Gly–Pro to attempt to stablize the triple-helix. Our computational results predicted that a fluoro-alkene Gly–Pro isostere would be a close geometric match for the native amide. Experimental results showed that a collagen-like peptide with a fluoro-alkene Gly–Pro isostere has an unfolding temperature that is 6.9 °C lower than the native control peptide. 19F NMR data of the collagen-like peptide shows a surprising deshielding of the fluorine nucleus, suggesting its participation in a stabilizing n→π* electronic interaction, similar to the native amide. Isomerization also plays a key role in proper cell division. We followed established methods to synthesize the cis- and trans-locked alkene mimics of Boc–Ser–Pro–OH and converted them into the 3,5-dinitrobenzyl ester conjugates. The 3,5-dinitrobenzyl ester is recognized by the dinitrobenzyl flexizyme (dFx) for enzymatic aminoacylation of tRNA. Once the alkene isosteres are aminoacylated, they will be incorporated into a full-length cell cycle regulatory protein called Bora to determine whether the cis- or trans-Pro state is necessary for healthy human mitosis, and which results in cancerous human mitosis.