Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references. / The spatial organization of RNA within cells is crucial for the regulation of a wide range of biological functions, spanning all kingdoms of life. However, a general understanding of RNA localization has been hindered by a lack of simple, high-throughput methods for mapping the transcriptomes of subcellular compartments. Here, we developed two methods, termed APEX-RIP and APEX-Seq. APEX-RIP combines peroxidase-catalyzed, spatially restricted in situ protein biotinylation with RNA-protein chemical crosslinking, while APEX-Seq utilizes peroxidase-catalyzed in situ biotinylation on RNA. We demonstrated that APEX-RIP can isolate RNAs from a variety of subcellular compartments, including the mitochondrial matrix, nucleus, bulk cytosol, and endoplasmic reticulum (ER) membrane, with higher specificity and coverage than conventional approaches. We furthermore identified candidate RNAs localized to mitochondria-ER junctions and nuclear lamina, two compartments that are recalcitrant to classical biochemical purification. Similarly, APEX-Seq can isolate RNAs from mitochondrial matrix, ER-associated RNAs, OMM-associated RNAs, and potentially other non-membrane bound compartments. We also revealed many non-coding RNA candidates at these sites. Since APEX-RIP and APEX-Seq are simple, versatile, and do not require special instrumentation, we envision their broad applications in a variety of biological contexts. / by Pornchai Kaewsapsak. / Ph. D.

Thesis: Ph. D. in Biological Chemistry, Massachusetts Institute of Technology, Department of Chemistry, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 231-249). / Fluorogenic probes, small-molecule sensors that unmask brilliant fluorescence upon exposure to specific stimuli, are essential tools for chemical biology. Probes that detect enzymatic activity can be used to illuminate the complex dynamics of biological processes at a level of spatiotemporal detail and sensitivity unmatched by other techniques. This dissertation describes the development of new fluorophore chemistries to expand our current fluorogenic probe toolkit and the subsequent application of these probes to study dynamic cell transport processes. Chapter 1. Enzyme-Activated Fluorogenic Probes for Live-Cell and In Vivo Imaging. Chapter 1 reviews recent advances in enzyme-activated fluorogenic probes for biological imaging, organized by enzyme classification. This review surveys recent masking strategies, different modes of enzymatic activation, and the breadth of current and future probe applications. Key challenges, such as probe selectivity and spectroscopic requirements, are described in this chapter along with therapeutic and diagnostic opportunities that can be accessed by surmounting these challenges. Chapter 2. Electronic and Steric Optimization of Fluorogenic Probes for Biomolecular Imaging. In many fluorogenic probes, the intrinsic fluorescence of a small-molecule fluorophore is masked by ester masking groups until entry into a cell, where endogenous esterases catalyze the hydrolysis of esters, generating fluorescence. The susceptibility of masking groups to spontaneous hydrolysis is a major limitation of these probes. Previous attempts to address this problem have incorporated auto-immolative linkers at the cost of atom economy and synthetic adversity. In this chapter, I report on a linker-free strategy that employs adventitious electronic and steric interactions in easy-to-synthesize probes. I find that halogen-carbonyl n-->[pi]* interactions and acyl group size are optimized in 2',7'-dichlorofluorescein diisobutyrate. This probe is relatively stable to spontaneous hydrolysis but is a highly reactive substrate for esterases both in vitro and in cellulo, yielding a bright, photostable fluorogenic probe with utility in biomolecular imaging. Chapter 3. Cellular Uptake of Large Monofunctionalized Dextrans. Dextrans are a versatile class of polysaccharides with applications that span medicine, cell biology, food science, and consumer goods. In Chapter 3, I apply the electronically stabilized probe described in Chapter 2 to study the cellular uptake of a new type of large monofunctionalized dextran that exhibits unusual properties: efficient cytosolic and nuclear uptake. This dextran permeates various human cell types without the use of transfection agents, electroporation, or membrane perturbation. Cellular uptake occurs primarily through active transport via receptor-mediated processes. These monofunctionalized dextrans could serve as intracellular delivery platforms for drugs or other cargos. Chapter 4. Paired Nitroreductase-Probe System to Quantify the Cytosolic Delivery of Biomolecules. Cytosolic delivery of large biomolecules is a significant barrier to therapeutic applications of CRISPR, RNAi, and biologics such as proteins with anticancer properties. In Chapter 4, I describe a new paired enzyme-probe system to quantify cytosolic delivery of biomolecules-a valuable resource for elucidating mechanistic details and improving delivery of therapeutics. I designed and optimized a nitroreductase fusion protein that embeds in the cytosolic face of outer mitochondrial membranes, providing several key improvements over unanchored reporter enzymes. In parallel, I prepared and assessed a panel of nitroreductase-activated probes for favorable spectroscopic and enzymatic activation properties. Together, the nitroreductase fusion protein and fluorogenic probes provide a rapid, generalizable tool that is well-poised to quantify cytosolic delivery of biomolecules. Chapter 5. Future Directions. This chapter outlines several future directions for expanding the scope of fluorogenic probes and developing new biological applications. Additionally, Chapter 5 is followed by an appendix describing a tunable rhodol fluorophore scaffold for improved spectroscopic properties and versatility. Overall, the work described in this thesis illustrates the power of enzyme-activated fluorogenic probes to provide fresh insight into dynamic biological processes, with direct implications for improved therapeutic delivery. / by Wen Chyan. / Ph. D. in Biological Chemistry

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2009. / This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Includes bibliographical references (p. 141-158). / We study electronic conductance through single molecules by subjecting a molecular junction to a time dependent potential and propagating the electronic state in real time using time-dependent density functional theory (TDDFT). This is in contrast with the more common steady-state nonequilibrium Green's function (NEGF) method. We start by examining quantum scale conductance methods in both the steady state and real-time formulations followed by a review of computational quantum chemistry methods. We then develop the real-time density functional theory and numerical solution techniques and use them to examine transport in a simple trans-polyacetylene wire. The remaining chapters are devoted to examining real-time transport behavior of various systems and model chemistries. Open-shell calculation of the polyacetylene wire reveal that, in agreement with various correlated model calculations, charge and spin behave as separate quasiparticles with different rates of transport. However, the transport of charge, and especially spin are highly dependent upon the amount of exact exchange included in the approximate exchange-correlation energy functional. This functional dependence is further illustrated when we demonstrate that the conductance gap of a device imperfectly coupled to wires varies based upon the non-local exchange and correlation. We also study the dynamic transport behavior of benzene-1,4-dithiol (BDT) coupled to gold leads and find that both the transient current and device charge density fluctuate with time,. This suggests that the steady-state assumption of the NEGF method may not be accurate. / by Jeremy Scott Evans. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1994. / Includes bibliographical references (leaves ). / by Shannon Storm Blanchard. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1995. / Vita. / Includes bibliographical references. / by Katherine E. Liu. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1997. / Includes bibliographical references. / by Brenda Lynn Palucki. / Ph.D.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2002. / Vita. / Includes bibliographical references. / Scope. The body of work described in this thesis focuses on the synthesis of donor-acceptor architectures of the pseudorotaxane, rotaxane, and catenane genres. The binding constants of thiophene and phenylene-ethynylene based crown ethers are determined via fluorescence quenching titrations, in an attempt to correlate structure with binding affinities. The insight obtained from the binding constant determinations allows for the proper choice of crown ether for the formation of [2]-catenanes and [2]-rotaxanes. The electronic properties of these complexes are then probed by electrochemical, spectroelectrochemical, and conductivity measurements. The potential of a poly([2]-catenane) as a photoconductive polymer is also investigated. The obstacles encountered in synthesizing poly([2]-rotaxanes) is also discussed, with emphasis on end-group strategies that aid in thwarting electrochemically induced dethreading. Chapter 1. This chapter provides a broad overview of the physical methods utilized in this thesis. Electrochemical methods are covered, with particular emphasis on oxidative polymerization of heteroaromatic monomers. Correlation between electrode phenomena and cyclic voltammogram waveshapes is included, in order to reinforce an understanding of the various electrochemical processes. Electroactive catenane and rotaxane supramolecules feature prominent in the literature, and a brief overview of significant examples is given. Advanced electrochemical techniques regarding in-situ conductivity measurements and spectroelectrochemistry are discussed. The elucidation of binding affinities for hosts and guest via fluorescence spectroscopy, a major theme in Chapter 2, is presented with focus on interpreting Stern-Volmer plots. / (cont.) A lengthy discussion of photovoltaic devices emphasizing I-V, C-V, and photoaction spectra interpretation is presented with relevant literature examples. Chapter 2. The synthesis of halo-functionalized crown ethers and efforts toward improving reaction yields for the macrocyclization are presented. Subsequent functionalization of the crown ethers with acetylide and thiophene groups via cross-coupling reactions, provides a wealth of receptors for the analyte paraquat. The affinities of the macrocycles for paraquat are determined through Stern-Volmer fluorescence quenching experiments. Correlations between structure and binding constants indicate that a-stacking interactions in two parallel planes, as seen for thiophene derivatized crown ether 33, affords the greatest complimentarity with paraquat, Ksv = 9665 +/- 179M-1. Acetylide functionalized crown ethers tend to have the weakest binding affinities, attributed to the poorer - extension. Increasing the electron-donating nature of the thiophene function as seen for 23 versus 15 leads to the doubling of the binding constant. Multiple crown ether receptor 29 does not display a non-linear Stern-Volmer plot, indicating that a single binding event occurs for this molecule. Relative to its monocrown analog 25, the binding constant for 29 is 1.2x larger, hinting at cooperativity between crown ether units. Chapter 3. Electrochemical studies on [2]-catenane 38, its non-catenated analog 23, and their respective polymers are undertaken in an effort to quantify the electronic disparity between the differing architectures ... / by Davide Louis Simone. / Ph.D.

Thesis (M.S.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1997. / Includes bibliographical references (p. 34). / by Christopher L. May. / M.S.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2007. / Vita. / Includes bibliographical references. / Chapter 1. Three monofunctional mixed alkyl hafnium complexes containing the (MesNpy)2 ligand ([(MesitylNCH2)2CMe(2-CsH4N)]2) were synthesized. (MesNpy)Hf(Neo)R ((2b), R = Me; Neo = CH2CMe2Ph) and (MesNpy)Hf(CH2TMS)(R), ((3b), R = Me and (4b), R = i-Bu)) were activated with {Ph3C} {B(C6F5)4} and one alkyl group was selectively removed in all cases. The kinetics of activated 4b were studied and the resulting complex was an initiator for the living polymerization of 1-hexene, with the exception of a slight initiation effect. Five linker molecules were investigated for the preparation of bifunctional hafnium mixed alkyl complexes. One complex synthesized, disilyl[(MesNpy)HfMe]2 (disilyl(X)2 = X-CH2SiMe2(CH2)5SiMe2CH2-X) was activated with {Ph3C} {B(C6F5)4} and the methyl groups were successfully removed to form {disilyl[(MesNpy)Hf]2} {B(C6F5)4}2. This complex was also a living catalyst for the polymerization of a-olefins. / Chapter 2. Addition of p-divinylferrocene to Mo(CHCMe2Ph)(NAr)(ORF6)2 (ORF6 = OCMe(CF3)2; Ar = 2,6-diisopropylphenyl) produced the bimetallic species [Mo(NAr)(ORF6)2(=CHC5H4)]2Fe (6), which upon treatment with lithium tert-butoxide produced the related tert-butoxide complex (7). X-ray crystallography studies of 6 and 7 showed them to be "syn/syn " bimetallic species. In solution, two resonances can be observed in 'H NMR spectra in the alkylidene region for the "syn/anti" isomer of 6 and 7; the total amounts were 4 and 8%, respectively. Bimetallic species 6 and 7 have been shown to initiate competitively at both ends and to produce homopolymers of 2,3-dicarbomethoxynorbornadiene (DCMNBD) and methyltetracyclododecene (MTD) in a living fashion. MALDI-TOF mass spectra of ferrocene-containing species were obtained and are consistent with the polymerization process being living. Triblock copolymers (poly[(MTD)x/2(DCMNBD)y(MTD)y(MTD)x/2]) were prepared by adding y equivalents of DCMNBD to the bimetallic initiators followed (after consumption of DCMNBD) by x equivalents of MTD. These triblocks were shown to be of high purity (free of homopolymer and diblock copolymers) and to have relatively low PDIs (< 1.2). / Chapter 3. A series of monomers suitable for ring opening metathesis polymerization (ROMP) containing a side chain liquid crystal mesogen were synthesized, where the mesogen was biphenyl-4-carboxylic acid 4-(1-butoxycarbonyl-ethoxy)-phenyl ester (abbreviated as BPP4). Two of the monomers have BPP4 attached to norbornene with a six or ten carbon spacer ((R)-4'- (5-bicyclo[2.2.1 ]hept-5-en-2-yl-pentyloxy)-BPP4, NB6wBPP4, and (R)-4'-(10- bicyclo[2.2.1]hept-5-en-2-yl-decyloxy)-BPP4, NB10OwBPP4, respectively), and the third monomer has BPP4 attached to norbornadiene via a ten carbon spacer ((R)-4'-(10- bicyclo[2.2.1]hepta-2,5-dien-2-yl-decyloxy)-BPP4, NBD10wBPP4). The monomers were polymerized by ROMP using the bimetallic initiator, [Mo(NAr)(ORF6)2(=CHCsH4)]2Fe (Ar = 2,6-diisopropylphenyl; ORF6 = OCMe(CF3)2). ABA triblock copolymers were also synthesized where the B block is NBD10OwBPP4 or NBnwBPP4 (n = 6, 10) and the A block is methyltetracyclododecene (MTD). All polymerizations are living with isolated yields greater than 90% and PDI values ranging from less than 1.05 to 1.31. The triblock copolymers were found to have glass transition temperatures of the inner block at approximately 20 'C and smectic to isotropic transitions around 150 'C. Small angle X-ray scattering (SAXS) indicated that the triblock copolymers exhibit phase segregation. SAXS data demonstrated that the triblock copolymer containing NB6wBPP4 forms smectic C* monolayers while the triblock copolymers that contain NB10OwBPP4 and NBD10wBPP4 form smectic C* bilayers at room temperature. Dynamic mechanical analysis of the triblock copolymers reveals an elastic plateau above the glass transition temperature of the center block, suggesting these systems may be interesting materials for shape-memory elastomers. / Chapter 4. The reaction of [Mo(NAr)(ORF6)2]2DVF (ORF6 = OCMe(CF3)2; Ar = 2,6- diisopropylphenyl; DVF = divinylferrocene) with lithium pyrrolide (LiPyr) or lithium 2,5- dimethylpyrrolide (LiMe2Pyr) formed the complexes [Mo(NAr)(Pyr)2(=CHCsH4) 2Fee2Li(Pyr) (8) and Mo(NAr)(Me2Pyr)2(=CHCsH4)]2Fe (9), respectively. X-ray crystallography studies of 8 reveal it to be a dimer of imetallic complexes while 9 is a monomer in the solid state. The reaction of [(DME)Mo(NAr)(ORF6)2]2DVB (DVB = ivinylbenzene; DME = dimethoxyethane) with LiMe2Pyr formed [Mo(NAr)(Me2Pyr)2(=CH)]2(1,4-C6H-4) (10). The reactivities of 8, 9, and 10 with various diols were studied. Three compounds, namely, [Mo(NAr)(Biphen)(Biphen)(=CHC5H4)2Fe (11, H2[Biphen] = 3,3'-di-tert-butyl-5,5',6,6'- tetramethylbiphenyl-2,2'-diol), [Mo(NAr)(Benz2Bitet)(=CH)]2(1,4-C6H4) (12, (R)- H2[Benz2Bitet] = (R)-3,3'-dibenzhydryl-5,5',6,6',7,7',8,8'-octahydro-1,1'-binaphthyl-2,2'-diol) and [Mo(NAr)(MesBitet)(=CH)]2(1,4-C6H4) (13, (R)-H2[MesBitet] = (R)-3,3'-dimesityl- 5,5',6,6',7,7',8,8'-octahydro-1,1'-binaphthyl-2,2'-diol) were found to be isolable compounds and could be generated cleanly in situ. 2,3-Dicarbomethoxynorbomadiene (DCMNBD) and 2,3- bis(trifluoromethyl)norbornadiene (NBDF6) were polymerized using initiators 11, 12, and 13, both isolated and in situ generated. Poly(DCMNBD) and poly(NBDF6) prepared with 12 and 13 were found to be highly cis and highly tactic. Triblock copolymers were also prepared, and the tacticity of the polymer was maintained. / Appendix A. Various monomers suitable for ROMP were synthesized for optimized application in thermoplastic liquid crystal elastomers. Two new inner block monomers, CBEwBPP4 [4-(1- butoxy-l-oxopropan-2-yloxy)phenyl 4'-(2-(2-(cyclobut-2-enylmethoxy)ethoxy)ethoxy)biphenyl- 4-carboxylate] and ONBwMPOB [4-methoxyphenyl 4-(4-(7-oxabicyclo[2.2.1]hept-5-en-2- ylmethoxy)butoxy)benzoate] were polymerized and the thermal properties of the polymers were studied. Poly(CBEwBPP4) exhibited a glass transition temperature (Tg) of -10 oC but no liquid crystal (LC) transition was observed. Poly(ONBwMPOB) exhibited a Tg of 15 oC and a broad LC transition was observed around 40 TC. Three new outer block monomers, exo-4-cyclohexyl- 4-aza-tricyclo[5.2.1.02,6]dec-8-ene-3,5-dione (NBwNCy), [3-(4-fluoro-benzoyl)- bicyclo[2.2.1 ]hept-5-en-2-yl]-(4-fluoro-phenyl)-methanone, (NBwCOPhF), and bicyclo[2.2.1]hept-5-en-2-yl-phenyl-methanone (NBwCOPh) were also polymerized and their thermal properties were studied. / by Andrea Jennifer Gabert. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2015. / Cataloged from PDF version of thesis. / Includes bibliographical references. / This thesis describes the development and application of chemical methodologies for the use of 2-pyridyl and other aryl and heteroaryl organometallic nucleophiles. Aiming for a practical and effective alternative to 2-pyridylboron reagents, a solid 2-pyridylzinc compound was prepared and is described in Chapter 1. Chapter 2 details a new approach to the synthesis of sulfonamides using aryl and heteroaryl organozine nucleophiles. Chapter 3 describes ongoing collaborative efforts to discover new malaria therapeutics where the synthetic method developed in Chapter 2 facilitated access to targeted compounds. Chapter 1. The Synthesis of a Solid, Air-Stable 2-Pyridylzine Reagent and its Use in Negishi Cross-Coupling Reactions. As an alternative to unstable or unreliable 2-pyridylboron reagents, a solid, air-stable 2- pyridylzinc reagent was developed. Using 1,4-dioxane as an additive enabled a 2-pyridylzinc concentrate as a free-flowing solid that was not deliquescent. The reagent can be manipulated in air and is a competent nucleophile in Negishi cross-coupling reactions. The reagent can also be stored in paraffin wax capsules for significant added stability. Chapter 2. Synthesis of Heteroaryl Sulfonamides from Organozinc Reagents and 2,4,6- Trichlorophenyl Chlorosulfate. A method for the preparation of aryl and heteroaryl sulfonamides using 2,4,6-trichlorophenyl chlorosulfate (TCPC) is described. The reaction of 2-pyridylzinc reagents with TCPC resulted in 2,4,6-trichlorophenyl (TCP) pyridine-2-sulfonates, and the parent pyridine-2-sulfonate was shown to react with amines. Less electron-rich aryl- and heteroarylzinc reagents reacted with TCPC to afford sulfonyl chlorides that were converted in situ to sulfonamides. Chapter 3. Structure-Activity Relationship Studies on Selective Inhibitors of Plasmodium Falciparum Growth. Two classes of compounds that selectively inhibit P. Falciparum were identified by the Lindquist Lab at MIT through the MLPCN compound library and high-throughput screening facilities at the Broad Institute. Using the sulfonamide synthesis methodology described in Chapter 2 and other approaches, analogs of these compounds were prepared and tested using a yeast assay in collaboration with the Lindquist lab at MIT and a P. falciparum assay in collaboration with the Wirth lab at the Harvard T.H. Chan School of Public Health. / by James Robert Colombe. / Ph. D.