Soy whey protein isolate (SWPI) – Fenugreek gum conjugates were developed and their molecular characteristics and emulsifying properties were investigated. SWPI was extracted from soy whey of tofu processing. SWPI exhibited excellent emulsifying properties comparable to soy protein isolate. However, to improve the emulsifying properties of SWPI for some applications, it was conjugated to fenugreek gum. The extent of conjugation was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Fourier transform infrared (FTIR) and High performance size exclusion chromatography (HPSEC). The SDS-PAGE of the conjugates showed polydispersed bands at the top of the separating gel in the conjugates suggesting the formation of high molecular weight products. Refractive index spectrum of HPSEC profiles showed a reduction of protein peak of unconjugated mixture and shifted a peak to higher molecular weight of the conjugates. Ultraviolet spectrum of HPSEC showed an increase of protein peak intensity at polysaccharide region. FTIR spectrum showed an amide band I and II were still observed in the conjugates after the unreacted proteins were removed. 1D NMR spectra showed that fenugreek gum was covalently bound to proteins through interaction between the reducing end of mannose residue and lysine. The protein solubility of SWPI – Fenugreek gum conjugates improved as compared to SWPI and SWPI – Fenugreek gum mixture when assessed in the pH range 3 to 8 at 22oC, especially at isoelectric point of protein (pl). A 1:3 and 1:5 ratio of SWPI – Fenugreek gum gave rise to better emulsion stabilization compared to 1:1 ratio. Particle size analysis revealed that conjugation of SWPI – Fenugreek gum at 60oC for 3 days was enough to produce relatively small droplet sizes in oil-in-water emulsions. SWPI – Unhydrolyzed fenugreek gum conjugates exhibited better emulsifying properties compared to SWPI – Partially hydrolyzed fenugreek gum conjugates. The conjugates improved emulsifying properties of SWPI, particularly around the pl of protein. The emulsifying properties were greatly increased by heating the conjugates before emulsification. The conjugates also improved emulsion stability at high salt concentration compared to SWPI. In summary, incorporation of SWPI into fenugreek gum improved emulsifying properties of SWPI near the pl of protein and at high salt concentration. / No / No

A targeted, stimuli-responsive, polymeric drug delivery vehicle has been developed to help alleviate the severe side-effects caused by narrow therapeutic window drugs. Doxorubicin, a commonly used chemotherapeutic agent has been conjugated to dextran by two different techniques. In the first method, doxorubicin and hepatocyte-targeting galactosamine were attached to dextran through amine bonds. Conjugation efficiency based on the amount loaded of each reactant varied from 1% to 50% for doxorubicin and from 2% to 20% for galactosamine, depending on various synthesis parameters. For the second conjugate, doxorubicin was attached to dextran through an acid-labile hydrazide bond. Fluorescence quenching indicated that all our conjugates can bind to DNA. The degree of binding was improved with increasing polymer molecular weight and substitution of doxorubicin, and also with hydrazide-bonded conjugate. In cell culture experiments, we have found that the uptake of conjugates was much lower than that of free doxorubicin. Lower uptake of conjugates decreased the toxicity of doxorubicin. Also, the uptake of non-galactosylated conjugate was lower than that of the galactosylated conjugate. Microscopy studies indicated that doxorubicin was localized almost exclusively at the nucleus, whereas the amine-bonded conjugates were present throughout the cell. Targeted amine-linked conjugates and hydrazide-bonded conjugates achieved greatly improved cytotoxicity. Following uptake, the doxorubicin was dissociated from the hydrazide conjugate in an endosomal compartment and diffused to the nucleus. The LC₅₀ values of non-targeted amine-linked, targeted amine-linked, and hydrazide-linked doxorubicin were 19.81 μg/mL, 7.33 μg/mL and 4.39 μg/mL, respectively. The amine-linked conjugates were also tested on a multidrug-resistant cell line; the LC₅₀ values of doxorubicin and the non-targeted amine-linked conjugate were 8.60 μg/mL and 36.02 μg/mL, respectively. / Singapore-MIT Alliance (SMA)

Hydroxycinnamoyl conjugates (HCC)s are an extremely diverse class of natural products that serve a wide variety of key functions in plant physiology, for example during wood formation, and in defence. They have diverse biological properties and act as antioxidants, antimicrobials, and antivirals. The biochemical basis of HCC diversity, however, has not yet been fully elucidated. Plants in the Populus genus are known to produce a particularly diverse range of HCCs and they constitute up to 5% of the leaf dry mass in some Populus species. HCCs can be formed by hydroxycinnamoyl transferases (HCTs) and distinct HCT isoforms in Populus may have distinct biological functions related to the synthesis of specific classes of HCCs. These can be identified on the basis of their evolutionary history and I show that many of the biochemically characterised HCTs belong to the BAHD superfamily of acyltransferases. My phylogenetic reconstruction of the BAHD superfamily has also defined a subclass containing most of the already-characterised HCTs, including nine potential HCT candidates in Populus. Caffeoyl-shikimate is a central precursor in the formation of lignin, a biopolymer (along with cellulose) that imparts mechanical stability to wood. Based on the transcript abundance of two candidate genes PtHCTA1 (Potri.001G042900)) and PtHCTA2 (Potri.003G183900) were hypothesised to be responsible for caffeoyl-shikimate formation in secondary xylem (i.e., wood). As part of this project, RNAi whole-plant knock-downs were generated for the xylem-associated PtHCTA1/2. The PtHCTA1/2 RNAi knock-downs have stunted growth, reminiscent of other mutants with impaired lignin biosynthesis. Based on thioacidolysis GC-MS, I found that the mutants produced a lignin with enriched hydroxyphenyl (H) subunits, which were derived from precursors upstream of the HCT-catalysed reaction and normally do not occur in Populus lignin. Interestingly, in one of the RNAi lines, the lignin phenotype was uncoupled from the developmental dwarfing phenotype. This is of high interest from a bioethanol perspective, since wood rich in H-lignin is more easily fermented than wood that is rich in guaiacyl (G) and syringyl (S) lignin. Another candidate gene (Potri.018G109900, HCT-E2) was linked to the formation of caffeoyl-spermidine in male catkins (which function in pollen coat formation), and one candidate gene (Potri.018G104700, HCT-C2) was associated with the formation of bioactive, soluble HCCs in leaves and roots. Since RNAi-mediated down-regulation proved ineffective, CRISPR-based gene knock-out methodology was developed and utilised for the Populus hairy root system. Targeted knock-out mutants for the leaf-associated HCT-C2 were generated. HCC identity was determined by metabolite purification and subsequent MS/MS/MS from leaf extracts, and the metabolite concentrations were determined by LC-MS. A decrease in chlorogenic acid concentration was apparent in CRISPR hairy-root knockouts of HCT-C2 indicating that HCTC2 is involved in HCC biosynthesis and can directly produce chlorogenic acid. Candidates for the HCTs involved in lignin biosynthesis, soluble ester biosynthesis, and pollen coat formation were identified and plant genetics confirmed the role of the lignin and soluble ester HCT candidates. / Graduate

The effect of peptide charge on the self-assembly and gelation behaviour of three octa-peptides: VEVKVEVK (VEK2), VKVKVEVK (VEK3) and VEVEVKVE (VEK1) has been investigated and characterised. The critical gelation concentration of each peptide was found to correlate with the charge modulus carried by the peptide and to be independent of the sign of the charge. Hydrogels formed were found to be transparent and stable when the peptide charge modulus is > 1. No differences in hydrogel structure or mechanical properties, as probed by TEM and SAXS and shear rheology, were found when the peptides were at the same concentration and carried the same charge modulus. These peptides were shown to form dense fibrillar network formed by β-sheet rich single fibre which lateral aggregation is controlled by the peptide charge modulus. The increase in fibre lateral aggregation with decreasing charge modulus was found to correlate with the increase in hydrogel mechanical properties, showing that fibre lateral aggregation pays a key role in controlling the mechanical properties of these hydrogels. The release profiles from VEK1 and VEK3 at pH 7 of two hydrophilic model drug molecules, namely napthol yellow (NY) and martius yellow (MY) was analysed using UV-Vis spectrophotometry. The incorporation of the guest molecules did not affect the self-assembly of the peptide at a molecular level but did affect the level of lateral fibre aggregation observed and therefore the mechanical properties of the hydrogels. The release of each of the model compounds was monitored over time and shown to be controlled by Fickian diffusion. The guest molecule diffusion rate D was dependent on the ratio between the overall effective charges carried by the peptide, i.e.: the fibrillar network, and the overall charges carried by the guest molecules but independent from the hydrogel concentration and mechanical properties, in the concentration and guest loading range investigated. This work shows that the rate of release of small drug molecules can be manipulated, not only by changing the charges on the guest molecules, but also by manipulating the charged state of the self-assembling peptide molecule and through it the charge state of the fribrillar network. Furthermore the VEK3 system was conjugated to a series of thermo-responsive synthetic polymers which imparted a significant change in mechanical properties, assembled structures and release profiles upon heating. Observed changes when above the polymers LCST include increased mechanical strength, fibre thickening and increased diffusion coeffcients. The synthesis, and subsequent characterisation, of these materials is the first time responsive hydrogels of OEGMA copolymers has been reported.

No description available.

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal common cancer in the United States: in 2017 there will be around 54,000 new cases and 43,000 patient deaths. (SEER, 2017) The high mortality of PDAC is related to late disease presentation and aggressiveness; nearly 52% of patients present with metastatic disease at the time of diagnosis. (SEER, 2017) Current treatments have marginal improvements on survival, with the most efficacious treatment, gemcitabine and nab-paclitaxel, having a median survival of 12.2 months. (Wu 2018) Failure of current PDAC treatments is attributed to the inefficacy of systemic chemotherapeutics and the development of resistance. (Rahib, 2014) The Dual Endothelin1/Signal PeptideVEGF receptor (DEspR), represents a promising therapeutic target for the treatment of PDAC: it is a highly expressed, specific tumor antigen, which is involved in tumor vasculogenesis and cancer stem cell (CSC) survival. DEspR is a developmentally crucial receptor, responsible for early angiogenesis and neural crest migration, with minimal expression in normal adult tissue. In vitro and in vivo studies of anti-DEspR therapy in PDAC have shown efficacy in decreasing CSC survival, tumor angiogenesis, and improving overall survival in xenograft models of PDAC, with anti-DEspR therapy being a promising candidate for clinical use. Furthermore, anti-DEspR therapy seems to augment chemotherapeutic therapy in vitro and in vivo, suggesting that a DEspR-targeted antibody drug conjugate (ADC) would be highly effective. ADCs are a re-emerging drug class with significant promise. Initial failures of ADCs in clinic were related to poor antigen specificity and failures in drug conjugation chemistry to minimally impact the antibody. To develop our ADC, I have developed a novel method of site-specific conjugation that relies on a novel method of supramolecular assembly. My system employs two specific protein sequences that do not self-interact, and tightly assemble through coulombic and hydrophobic interactions, allowing site-specific, stoichiometric self-assembly. To facilitate stable drug delivery, I have synthesized a novel enzymatically cleavable tyrosine-clickable linker, which prevents drug release prior to tumor delivery. Both further investigation into the efficacy of anti-DEspR therapy, and the development of a stoichiometric, site-specific, stable method for drug loading will provide an advancement in anti-cancer therapy.

Synthetic strategies for polysaccharide-protein conjugates, pH-responsive hydrogels, and amorphous solid dispersion (ASD) polymers were developed. Conjugating a polysaccharide to a protein drug via a covalent bond may improve its medical properties including solubility, stability, immunogenicity, circulation time, and targeting ability. Regioselectivity of conjugation is still challenging. We developed a strategy for regioselective conjugation of amino acid esters to polysaccharides, by employing 6-Br-polysaccharides in SN2 substitution reactions with amino acid esters. This work provides a good starting point for the regioselective conjugation of polysaccharides to proteins. Polysaccharides can also serve as hydrogel drug carriers. Most hydrogels employed in drug delivery work by incorporating the drug physically. We synthesized sustained and pH-responsive hydrogels using oxidized hydroxypropyl cellulose (Ox-HPC)/carboxymethyl chitosan (CMCS) crosslinked by imine bond. Phenylalanine as a model amine-containing drug was chemically bonded to the Ox-HPC hydrogel component and was observed to release faster at the pH of a tumor microenvironment. These hydrogels show promise as targeting cancer drug carriers. ASDs are polymeric systems to disperse poorly soluble drugs amorphously and enhance permeation from the gastrointestinal tract (GI tract) to the bloodstream. We synthesized potentially zwitterionic cellulose derivatives by reductive amination of Ox-HPC with ω-aminoalkanoic acids and obtained products with the degree of substitution (cation and anion) up to 1.6, which is difficult to attain using previous methods. The products showed manipulated amphiphilicity and excellent thermostability, exhibiting potential application in ASDs. We anticipate that these strategies will benefit future polysaccharide chemistry research and permit synthesis of a broad variety of more functional biomedical materials. / Doctor of Philosophy / Polysaccharides are long chains of individual sugars ("polymers"). Many natural-sourced polysaccharides are sustainable, biodegradable, and have low toxicity. Polysaccharide-based materials may improve the properties of current drugs, resulting in decreased cost, enhanced absorption efficiency, and continuous and/or targeted delivery. Protein drugs such as human insulin have a significant role in medicine. However, the residence time of a protein drug in the human body is short. To overcome this challenge, we designed a method to link polysaccharides to proteins at controlled reaction sites, and reported herein the first step of this route. The final polysaccharide-protein products will even have the ability to recognize and access target cells, like those of tumors. Tumor tissues are more acidic than normal tissue and can trigger faster release of drug from drug carriers. We developed polysaccharide-based hydrogels, which are gels that bind a great deal of water but won't dissolve in it, as acid-sensitive carriers. In addition, our hydrogels are also injectable, and can spontaneously repair themselves. These properties make our hydrogels promising as cancer targeting drug carriers. Most new drug candidates have poor water solubility and permeation through the gastrointestinal tract to reach the blood stream. Dispersing the insoluble drug into a properly designed polymer network can enhance dissolution, permeation, and absorption. We developed a new family of polymers designed for this purpose using two cheap starting materials. These polymers can interact with the drug, preventing it from forming crystals and simultaneously promoting slow drug release. Overall, we explored ways to modify polysaccharides to create harmless, effective medical materials. We aim to promote science and benefit human health via our research.

No / Two regioisomeric citrate-functionalized ciprofloxacin conjugates have been synthesized and their antimicrobial activities against a panel of clinically-relevant bacteria have been determined. Cellular uptake mechanisms were investigated using wild-type and ompF deletion strains of Escherichia coli K-12.

Es ADCs (Antibody-Drug Conjugates) correspondent à une nouvelle stratégie thérapeutique anti-tumorale particulièrement prometteuse. Néanmoins, les ADCs actuellement utilisés en clinique sont obtenus par conjugaisons chimiques, resultant en des mixtures hétérogènes impactant négativement leurs pharmacocinétiques et leurs performances in vivo.Récemment, différentes strategies de couplage site-spécifique ont été développées afin de réduire cette hétérogénéité. Dans cette thèse, nous rapportons le développement d’une nouvelle technologie CovIsoLink™ (Covalently Isopeptide Crosslinking) permettant la génération d’ADCs par utilisation de nouveaux peptides glutamine Q-Tag présentant des affinités optimisées par rapport à des peptides disponibles (ZQG, LLQG) pour une enzyme bactérienne la transglutaminase (mTG).La preuve de concept de cette technologie a été réalisée par insertion de ces peptides Q-Tag en C-ter de la région codant pour la chaine lourde des anticorps anti-HER2 (Trastuzumab). Nous avons ainsi pu démontrer la conjugaison homogène et reproductible de différentes drogues sans contamination par des chaines d’anticorps non conjuguées. Nous avons pu montrer que l’immunoréactivité et la capacité d’internalisation de ces ADCs n’étaient pas altérées par la conjugaison et qu’ils présentaient in vitro et in vivo, des propriétés de lyse de cellules tumorales similaires au Trastuzumab emtansine (Kadcyla®), actuellement en clinique. Par ailleurs, afin de généraliser notre technologie à différents formats d’anticorps nous avons générés des fragments Fab et scFv et évalué leur fonctionnalité. Ainsi, nous avons pu prouver que l’utilisation de nouveaux peptides optimisés Q-Tag substrat de la transglutaminase permettait une stratégie de couplage alternative plus homogène par couplage de différentes molécules sans espèce contaminante non couplée / Antibody-drug conjugates (ADCs) are a powerful class of therapeutic agents, demonstrating success in the treatment of several malignancies. The currently approved ADCs are produced by chemical conjugations and exist as heterogeneous mixtures that negatively influence the pharmacokinetics and in vivo performance. Recently many of site-specific conjugation technologies have been developed to reduce heterogeneity and batch-to batch variability. Microbial transglutaminase (mTG) has been demonstrated as efficient tool for site-specific conjugation. In this thesis we report the development CovIsoLink™ (Covalently Isopeptide Crosslinking) technology for the generation of homogenous immunoconjugates using a novel glutamine donor peptides (Q-tag) with improved affinity compared to the known peptides (ZQG, LLQG). As a proof of concept, the peptides sequences were engineered into the heavy chain C-terminal of Trastuzumab antibody. We demonstrated the reproducible and homogeneous conjugation of Q-tagged Trastuzumab with different payloads, without any unconjugated species. The ADCs were evaluated in series of in vitro and in vivo assays. We confirmed that the immunoreactivity and internalisation are not altered by the conjugation. Furthermore similar in vitro and in vivo tumor cell killing potency was demonstrated than Trastuzumab emtansine (Kadcyla®), which is already used in the clinic. Morover we extend our site-specific conjugation technology to antibody fragments (Fab and scFv), evaluating their functionality by conjugation with AlexaFluor488-cadaverine and in antigen binding assays. Thus, using novel glutamine donor peptides, our technology provides an alternative enzymatic conjugation strategy for the engrafment of different payloads resulting in homogeneous batches, without unconjugated species

Chapter 1: Facile entry into triazole fused tetrahydropyrazinones from amines and amino acids. In this chapter, A practical and high yielding regioselective synthesis of several new, enantiopure 4,5,6,7-tetrahydro[1,2,3]triazolo[1,5-a]pyrazin-6-ones is described starting from primary amines in a three step reaction sequence (alkylation, acylation, one-pot displacement with azide followed by cycloaddition) employing constrained intramolecular ‘click’ reaction as the key step. The method obviates chromatographic purification of products. This methodology was also extended to the synthesis of diverse triazole fused tetrahydropyrazinones derived from amino acids. The scope of this methodology was extended by varying the alkyl as well as acyl components which furnished other triazole fused novel heterocycles. Chapter 2: Facile entry into triazole fused heterocycles via sulfamidate derived azido-alkynes. Direct synthesis of condensed triazoles from diverse sulfamidates by ring opening of sulfamidates with sodium azide followed by one-pot propargylation and cycloaddtion furnished title compounds. The methodogy in general has been demonstrated on diverse sulfamidates derived from amino acids, amino acid derivatives to obtain a variety of triazole fused scaffolds. In one example, a condensed triazole containing amino acid has been synthesized by ring opening of a sulfamidate derivative with propargyl amine. This methodology has also been extended to the synthesis of condensed triazoles derived from D-glucose. Chapter 3: ‘Click Chemistry’ Inspired Synthesis of Novel Ferrocene-Amino acid, Peptide Conjugates. In this chapter synthesis of a wide range of ferrocene-amino acid and peptide conjugates in excellent yield is presented. Conjugation is established via copper catalyzed Huisgen 1,3-dipolar cycloaddition. Two complementary strategies were employed for conjugation, one involving cycloaddition of amino acid derived azides with ethynyl ferrocene and the other involving cycloaddition between amino acid derived alkynes with ferrocene derived azides. Labeling of amino acids at multiple sites with ferrocene is discussed. A new route to 1, 1’ unsymmetrically substituted ferrocene conjugates is reported. A novel ferrocenophane is accessed via bimolecular condensation of amino acid derived bis alkyne with azide. The electrochemical behavior of a few selected ferrocene conjugates has been studied by cyclic voltammetry. Chapter 4: Click Chemistry inspired Synthesis of Ferrocene Amino acids and other derivatives. This work reports the synthesis of a wide range of ferrocenyl-amino acids and other derivatives in excellent yield. Diverse amino acid containing azides were synthesized and ligated to ferrocene employing click reaction to access ferrocenyl amino acids. Chiral alcohols, esters, diols amines containing azido group were tagged to ferrocene via click reaction to generateferrocene derived chiral derivatives. A novel strategy for direct incorporation of ferrocene into a peptide and a new route to 1, 1’ disubstituted ferrocene amino acid derivative are reported. Synthesis of mono and disubstituted ferrocene derivatives employing ferrocene derived azides is also described. Chapter 5: Convenient synthesis of Ferrocene Conjugates mediated by Benzyltriethylammonium Tetrathiomolybdate in a multi-step tandem process. The synthesis of a wide range of ferrocene derived sulfur linked mono and disubstituted Michael adducts and conjugates mediated by benzyltriethylammonium tetrathiomolybdate in a tandem process is reported. New route to access acryloyl ferrocene and 1,1’-bis acryloyl ferrocene is discussed. Conjugation of amino acids to ferrocene is established via their Nand Ctermini and also via side chain employing conjugate addition as key step to furnish monovalent and divalent conjugates. This methodology has also been extended to access several ferrocene carbohydrate conjugates. The electrochemical behavior of a few selected ferrocene conjugates has been studied by cyclic voltammetry. Finally, 1,1’-bis acryloyl ruthenocene was synthesized and it was utilized for the preparation of ruthenocene-carbohydrate conjugate in good yield. Chapter 6: Formation of Intramolecular S-Se bond mediated by tetrathiomolybdate. In this chapter, we have disclosed our preliminary results on reactivity of tetrathiomolybdate towards compounds containing both thiocyanate and selenocyanate functionalities. Several such compounds have been synthesized from the corresponding dibromides in two steps. We have observed selective reductive dimerization of selenocyanate over thiocyanate. In all the cases we also obtained seleno-sulfides via disulfide diselenide exchange reaction upon addition of excess tetrathiomolybdate. In the case of substrates on benzene scaffold, disulfide and diselenide bridged macrocycles were obtained apart from seleno sulfides whereas in the case of ferrocene derived substrates, formation of macrocycles was not observed. A tentative mechanism for the formation of these novel seleno sulfides is also discussed.(For structural formula pl see the pdf file)