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Visualizing proteins in living cells without perturbing biological function remains a key challenge in chemical biology. A chemical approach to this problem is the synthesis of small molecule fluorophores that react specifically with a protein of interest (POI). We have developed a site-specific labelling method based on a Fluorogenic Addition Reaction (FlARe). The FlARe probe’s fluorescence is quenched until it undergoes thiol addition with a small, genetically encoded dicysteine peptide tag fused to the POI. Recent blue coumarin probes were shown to be highly selective for target proteins over other cellular thiols; however, fluorogens that can label in the red and green channels of the fluorescence microscope are more desirable for cellular imaging, as red light is lower in energy and therefore less photo-toxic. In the work presented herein, we use DFT calculations to guide the design of red-shifted, PeT-quenched BODIPY based dimaleimide fluorogens. Driven by the preliminary results of a FlARe probe (YC29) that emitted in the red channel, we attempted to prepare the hit compound through a new synthetic approach to further evaluate kinetics and in cellulo labelling. Given the time available, this compound was unable to be synthesized through an SNAr or Pd-catalyzed approach. Alternatives probes lacking the red-shifting substituent were synthesized and evaluated in vitro and in cellulo. The fluorescent enhancement and reaction kinetics of these probes were evaluated in detail, in order to determine the suitability of their application to cellular labelling. A green-BODIPY fluorogen was synthesized that exhibits suitable kinetics for labelling and a dramatic fluorescent enhancement of ~800-fold upon tagging. This probe was successfully applied to the specific, fluorescent labelling of a nuclear histone protein in cellulo.

Thesis (Ph. D.)--University of Florida, 1981. / Description based on print version record. Typescript. Vita. Includes bibliographical references (leaves 204-213).

Diploma thesis deals with preparation of polypropylene functionalized by maleimides, based on the reaction between maleic anhydride and amine. The overview of functionalization of polypropylene by maleic anhydride by reactive extrusion and routes for the synthesis and utilization of maleimides are contained in the theoretical part. Samples of maleimide-functionalized polypropylene were prepared by reactive extrusion using low molecular weight amines (aniline and 4-aminophenol) and hight molecular weight polyether monoamines. Functionalized polypropylene samples were prepared by several methods differing in the composition of the reaction mixture and performing a grafting reaction. Appropriate maleamic acids and maleimides were synthesized from low molecular weight amines and further used to functionalize the polypropylene. To characterize the samples, the degree of monomer conversion and melt flow index were determined and further analyses were performed by Fourier transform infrared spectroscopy, differential scanning calorimetry and thermogravimetric analysis. The synthesis of low molecular weight products was further monitored by thin layer chromatography and thermogravimetric analysis with evolved gas analysis.

This dissertation describes a new platform for the synthesis of thermally reversible polymers, based on Diels-Alder reactions of bis-cyclopentadienes (bis-CPDs) and bis-maleimides (bis-MIs), that meets two main objectives. First, the bis-CPD must resist characteristic self-coupling. Second, the CPD-MI adducts should undergo the retro-Diels-Alder (rDA) reaction (i.e., thermal depolymerization) in a temperature regime that is comparable or slightly higher than that of the freely reversible bis-furan/bis-MI polymers (rDA between 80 °C and 130 °C) but much lower than that of bis-CPD homopolymers (rDA above 160 °C). Structure-reactivity relationships gleaned from the literature and from related but as yet unpublished work in our own laboratories led to our main hypothesis that a CPD moiety bearing one sterically encumbering substituent such as isopropyl (𝑖Pr) or tert-butyl (𝑡Bu) and one electronwithdrawing substituent such as perfluoroaryl would have the desired reactivity and adduct stability in combination with an 𝑁-substituted maleimide. Synthetic considerations led to a bisCPD monomer design in which two alkylcyclopentadiene groups (alkyl = 𝑖Pr or 𝑡Bu) are connected by an octafluorobiphenylene linker. As an initial fundamental step (Chapter 3), 1-(nonafluorobiphenyl-4’-yl)-4-tertbutylcyclopentadiene (1) was synthesized to provide a monofunctional model for the proposed difunctional CPD monomer. Reactions of 1 and 𝑁-(4-fluorophenyl)maleimide (FMI) afforded up to five regio- and stereo-isomeric adducts (of fourteen possible). Variable-temperature reactivity studies combined with NMR spectroscopic analysis, X-ray crystallography, and computational modeling enabled product distributions to be understood according to a conventional kinetic-vs- iii thermodynamic framework. These studies also predicted the microstructure of polymers derived from the proposed bis-CPD monomer, which is structurally analogous to 1, and bis-MIs. Moreover, 1 does not undergo DA self-coupling under ordinary conditions (T < 180 °C). Thermolysis studies of the major adducts revealed that the rDA becomes observable on a laboratory timescale (hours) at about 140 °C, which is at the upper end of the temperature range reported for furan+MI adducts but well below that of CPD+CPD adducts. In contrast, adducts formed from either of the analogous monosubstituted cyclopentadienes (𝑡BuC₅H₅ and C₆F₅C₅H₅) do not undergo rDA below 180 °C. These results strongly support the proposed bis-CPD monomer design. In a second fundamental step (Chapter 4), the hypothesis that an electron-withdrawing CPD substituent would destabilize a CPD-MI adduct was further tested by reacting 𝑁-(4- fluorophenyl)maleimide with a series of triarylated cyclopentadienes (1,2,3-Ar₃C₅H₃ and 1,2,4- Ar₃C₅H₃, Ar = C₆F₅, C₆F₄CF₃, and Ar = C₅F₄N). The perfluorophenyl- and perfluorotolylsubstituted compounds were previously reported, but the perfluoropyridyl-substituted cyclopentadienes were prepared for this study using SNAr reactions of pentafluoropyridine and sodium cyclopentadienide. The least electron deficient cyclopentadiene in each series (Ar = C₆F₅) reacted the most quickly and with the highest ultimate equilibrium binding constant, confirming the electron-effects hypothesis as well as the underlying presumption that DA reactions of even relatively electron-poor CPDs with MI would behave according to normal-electron-demand principles. In the main section of this dissertation (Chapter 5) the proposed bis(cyclopentadiene)s reacted with a series of previously reported bis(maleimides) to form linear polymers having molecular weights (Mn) up to 40 kDa. Relationships among the length and flexibility of the bis-MI linker (C₆H₁₂, C₁₂H₂₄, C₆H₄OC₆H₄, and (C₂H₄O)₂), the identity of the CPD alkyl substitutent (CHMe₂, CMe₃ and CMe₂Ph) and the glass transition temperature (Tg) as measured by differential scanning calorimetry (DSC) were understood in terms of a general model of local segmental mobility and free volume. Solution thermolysis of a model polymer system (bis-MI linker = C₆H₁₂ (7), CPD alkyl substituent = 𝑡Bu) showed a rapid decrease in molecular weight at 160 °C as determined by size exclusion chromatography (SEC). Solution thermolysis in the presence of excess FMI (as a trap for free CPD moieties) revealed that the onset temperature for rDA on a laboratory time scale (hours) was as low as 120 °C. In the bulk, thermolysis above 250 °C under vacuum led to recovery of a small portion of the bis-CPD monomer, but bulk thermolysis at 200 °C did not reveal a change in molecular weight as determined by SEC. The current interpretation of these observations is that limited mobility in these glassy polymers prohibits retro-DA decoupling. These findings largely validate the main hypothesis of this dissertation. / Ph. D.

Type 1 diabetes affects one in every 400-600 children and adolescents in the US. Standard therapy with exogenous insulin is burdensome, associated with a significant risk of dangerous hypoglycemia, and only partially efficacious in preventing the long term complications of diabetes. Pancreatic islet transplantation has emerged as a promising therapy for type 1 diabetes. However, this cell-based therapy is significantly limited by inadequate islet supply (more than one donor pancreas is needed per recipient), instant blood-mediated inflammatory reaction, and loss of islet viability/function during isolation and following implantation. In particular, inadequate revascularization of transplanted islets results in reduced islet viability, function, and engraftment. Delivery of pro-vascularization factors has been shown to improve vascularization and islet function, but these strategies are hindered by insufficient and/or complex release pharmacokinetics and inadequate delivery matrices as well as technical and safety considerations. We hypothesized that controlled presentation of angiogenic cues within a bioartificial matrix could enhance the vascularization, viability, and function of transplanted islets. The primary objective of this dissertation was to enhance allogenic islet engraftment, survival and function by utilizing synthetic hydrogels as engineered delivery matrices. Polyethylene glycol (PEG)-maleimide hydrogels presenting cell adhesive motifs and vascular endothelial growth factor (VEGF) were designed to support islet activities and promote vascularization in vivo. We analyzed the material properties and cyto-compatibility of these engineered materials, islet engraftment in a transplantation model, and glycemic control in diabetic subjects. The rationale for this project is to establish novel biomaterial strategies for islet delivery that support islet viability and function via the induction of local vascularization.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22470号 / 工博第4731号 / 新制||工||1739(附属図書館) / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 大内 誠, 教授 秋吉 一成, 教授 竹中 幹人 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Octafluorobiphenylene-linked bis(cyclopentadienone) was prepared bearing one perfluoro-4-tolyl and one tert-butyl substituent on the terminal diene rings. Polymerizations with 1,4- and 1,3-diethynylbenzene afforded linear Diels-Alder polyphenylenes (DAPPs) having lateral tert-butyl and perfluoro-4-tolyl substituents. The perfluoro-4-tolyl-substituted DAPPs are thermally stable, glassy solids (Tg ~ 230 deg C) that could not be cast into stable films (Mn ~ 10kDa, DPn ~ 10). New compounds perfluoro(1-phenyl-1-octanone) and perfluoro(1,1-diphenyl-1-octanol) were prepared from pentafluorophenylmagnesium bromide and perfluorooctanoyl chloride by nucleophilic acyl substitution and addition reactions. Diels-Alder reactions of 1,2-bis(nonafluorobiphenyl-4-yl)-4-tert-butylcyclopentadiene (CPD-1) with N-(4-fluorophenyl)maleimide (FMI) were explored as models for cyclopentadiene-maleimide-based Diels-Alder polymerizations. Mixtures of five endo/exo adducts were obtained, dependent upon CPD-1 tautomers present at reaction temperatures. The thermodynamic adduct (B3LYP/6-31G* geometry optimizations) was found to be the exo DA adduct of FMI and 2,3-bis(nonafluorobiphenyl-4-yl)-5-tert-butylcyclopentadiene. Five of the six possible isomers were observed and characterized including two by single-crystal X-ray diffraction. Parallel reactions of FMI and 1,2-bis(pentafluorophenyl)-4-tert-butylcyclopentadiene yielded three crystallographically characterized isomers, and with 1H NMR and 19F NMR spectrometry, including 1-D NOE, allowed five isomeric products to be identified. Diene CPD-1 is reactive toward nucleophiles (such as potassium 4-methylphenoxide) at the 4-positions of the C12F9 groups. Using this reactivity pattern, CPD-1 was polymerized with bis(phenol) A (BPA) and bis(phenol-A-6F) (BPAF) to form linear poly(arylene ethers) (Mn ~35 kDa) containing backbone cyclopentadienes. These polymers are glassy solids (Tg ~ 220 deg C) with good thermal stability (Td ~ 290 deg C), and they form stable, creaseable films cast from chloroform solutions. Treatment with 1.5-5.0% of 1,6-bis(N-maleimido)dodecane in N,N-dimethylacetamide (DMAc) at 165 deg C gave insoluble, solvent-swellable networks confirmed using ATR-FTIR. CPD-1 was also used as a cyclopentadiene-based linking group for chain extension of phenol-terminated methyl-PEEK oligomers (PEEKMOHs) with Mn values of 2, 5, and 10 kDa. These polymers are glassy solids (Tg ~ 156 deg C) with good thermal stability (Td ~ 400 deg C), that form stable, creaseable films from chloroform. Segmented polymers were treated with FMI in NMP, and showed functionalization density of approximately 50% by 19F NMR. Segmented polymers were also cross-linked by reaction of 1,6-bis(N-maleimido)hexane (cyclopentadiene to maleimide functional group ratio of 1:1) in NMP at 140 deg C. / Ph. D.

Hydrogen storage remains a major technological barrier to the widespread adoption of hydrogen as an energy source. Organic polymers offer one potential route to useful hydrogen storage materials. Recently, Frechet and his coworkers described a series of hypercrosslinked polymers with high surface area and studied their surface properties and hydrogen storage capacities. McKeown and his coworkers studied a class of materials termed Polymers of Intrinsic Microporosity (PIMs) which are also based on a "hypercrosslinked" concept. We enchained N-substituted maleimide and functionalized stilbene alternating copolymers into a "hypercrosslinked system" to achieve high rigidity, high surface areas, high aromatic content and good thermal stability. Hypercrosslinked copolymers of N-(3-methylphenyl)maleimide (3MPMI), 4-methyl stilbene (4MSTBB), vinylbenzyl chloride (VBC) and divinyl benzene (DVB) were synthesized. Scanning electron micrographs (SEM) show the copolymers are porous and some examples have shown surface areas over 1200 m²/g. We have also found the incorporation of 3MPMI and 4MSTBB improves the thermal stability and raises the glass transition temperature of the copolymer. However, the incorporation of 3MPMI and 4MSTBB decreases the hypercrosslinking density and therefore causes a decrease in the copolymer surface area. The systematic study of styrene (STR) – vinylbenzyl chloride (VBC) – divinyl benzene (DVB) networks indicates that a low density of chloromethyl groups leads to a decrease in surface area. Therefore, we are continuing to investigate other monomers, such as N-substituted maleimide and functionalized stilbene containing chloromethyl groups, in order to enhance thermal stability while maintaining surface area. In order to increase the enthalpy of hydrogen adsorption and thus raise the temperature of hydrogen storage, the monomer N,N-dimethyl-N',N'-diethyl-4,4'-diaminostilbene (4,4'DASTB-3MPMI) which contains electron donating groups was incorporated into hypercrosslinked polymer particles. Hypercrosslinked polymer (4,4'DASTB-3MPMI)1.0(VBC)98.5(DVB).50 exhibits a surface area of 3257 m²/g. / Master of Science

This dissertation describes a general synthetic platform for segmented polymers that have main-chain reversible linkages based on cyclopentadiene-maleimide Diels-Alder chemistry. Research in the area of thermally reversible (self-healing) polymers has been an ever-expanding area of interest in the current scientific literature. However most of the emphasis has been on systems containing furan-maleimide linkages. While inexpensive and synthetically accessible, furan chemistry is mostly limited to crosslinked and hyperbranched architectures due to its relatively weak binding with maleimides at suitable propagation temperatures. Following a general review of the literature in this area (Chapter 1) the first stage of our research (Chapter 2) entails the synthesis of 2-substituted hydroquinones, which are needed as monomers in the later stages. The novelty of our hydroquinone synthesis stems from the use of allylic and other alkenyl ethers as the source of the ring substituent, and from the utilization of catalytic hydroboration to improve atom-efficiency. We showed that hydroquinones with widely varying functionality can be prepared efficiently by our method; these findings were published in the journal Tetrahedron in 2018. The second stage (Chapter 3) involves the use of the new hydroquinones in step-growth syntheses of hydroquinone-terminated telechelic and chain-extension of these telomers via Diels-Alder chemistry to form segmented polymers having thermally reversible linkages. The novelty of our approach rests with the use of cyclopentadiene-maleimide chemistry for the linkages, while the overall physical properties such as the glass transition temperature were established by using well-defined aromatic polymers — poly(ether ether ketones) or PEEK and poly(aryl ether sulfones) or PAES — as segments. This approach represents an important departure from earlier work in our group in which reversible linkages were present in every repeat unit of a step-growth Diels-Alder polymer that showed thermal reversibility in solution but not in the bulk, owing to glass transition temperatures that were too high. Using scratch-healing and mechanical (tensile) tests, we show that our new segmented polymers exhibit self-healing characteristics that are competitive with or superior to previously reported systems based on different Diels-Alder chemistry. The third stage (Chapter 4) aims to explore new application areas for some of the more novel functionalized hydroquinones reported in Chapter 2. First we developed an efficient synthesis of a PAES derivative bearing 5-phenoxypentyl groups on the hydroquinone moiety. Then we showed that the 5-phenoxy group can be cleanly cleaved, post-polymerization, to afford a PAES having 5-bromopentyl substituents. The promise of our method rests with the potential of the pendant electrophiles to undergo reactions with nucleophilic reagents to post-modify these polymers further. As proof of concept, we showed that substitution of the pendant bromides with furfuryloxy groups enabled thermally reversible crosslinking with a bis-maleimide reagent to form a polymeric material that demonstrates partial scratch healing. Finally we are exploring the synthesis of new ion-containing polymers by substituting the pendant bromides with tertiary amines. / PHD / This dissertation describes a new synthetic approach to polymeric materials that can heal themselves (for example, repair small cracks that may have formed due to stress or aging) simply by heating the damaged area. Our approach uses a thermally reversible chemical reaction (called the Diels-Alder reaction) to connect several shorter polymer segments into longer chains. Upon heating, the segments can come apart, diffuse into and through the damaged area, and then rejoin. The first chapter is a review of background in the published literature as well as previous not-yet-published work in our laboratory. The second chapter describes the creation of new building-block molecules (monomers) that will help control the temperature range necessary to induce self-healing after incorporation into the polymer segments. The third chapter details the process of forming the segments, the incorporation of self-healing functionalities on the ends of the segments, the joining of the segments into longer polymeric chains, and the testing of all of the physical properties of these new materials, including their self-healing capabilities. The fourth chapter represents a preliminary study of a new method of preparing ion-containing polymers. The latter materials have potential use in various membrane technologies including fuel cell devices for the harnessing of renewable energy.