Thesis advisor: Marc L. Snapper / Tandem processes can have several advantages over multiple single step processes. Non-metathesis transformations of ruthenium alkylidenes were studied and applied to tandem processes. Ruthenium catalyzed tandem RCM/hydroacylation that allows access to tricyclic ring systems from readily available substrates was developed. Mechanistic investigations indicated that this reaction may proceed through a mechanism involving [Ru]-H species. A Ru-catalyzed tandem RCM/olefin isomerization/C-H activation sequence that provides significant advantages in terms of rapid elaboration of simple reaction partners to more complex entities was developed. / Thesis (PhD) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

A Dissulfeto Isomerase Proteína (PDI) é uma chaperona redox essencial responsável pela inserção correta das ligações dissulfeto em proteínas nascentes no retículo endoplasmático. Nesta localização celular, bem como em outras regiões, como na superfície celular, a PDI atua na manutenção da homeostase redox e sinalização. Houve substanciosa evolução no conhecimento sobre a estrutura e funções da PDI, graças a estudos in vitro que utilizam a PDI purificada, quimeras ou seus domínios isolados. Nestas abordagens experimentais, as medidas das atividades redutase e chaperona da PDI são realizadas de forma relativamente simples. Entretanto, medir a atividade isomerase, que é a atividade autêntica da família das PDIs, é tecnicamente bastante complexo. Em células e tecidos, o papel da PDI tem sido descrito com base principalmente em estratégias experimentais de ganho e perda de função. Todavia, ainda há pouca informação na correlação entre os resultados funcionais com a medida das atividades da PDI. Este trabalho compila os principais métodos descritos para medir as quatro atividades da PDI: tiol redutase, tiol oxidase, tiol isomerase e chaperona, com ênfase na descrição de controles e interferentes críticos, como os tampões que contém surfactantes. Ainda, discutir-se-á criticamente os resultados obtidos quando da transposição destes métodos para amostras de homogenatos (celular ou tecidual) / Protein disulfide isomerase is an essential redox chaperone from endoplasmic reticulum, responsible for correct disulfide bond insertion in nascent proteins. At the endoplasmic reticulum and other locations including the cell surface, PDI accounts for redox homeostasis and signaling. Knowledge about PDI structure and function evolved substantially from in vitro studies using purified PDI and chimeras. In these experimental scenarios, PDI reductase and chaperone are readily approachable. However, isomerase activity, the hallmark of PDI family, is significantly complex. Assessment of PDI roles in cells and tissues mainly relies on gain- or loss-of-function experiments. However, there is limited information regarding correlation of these results with PDI activities. In this manuscript, we put together the main methods described for measuring the four PDI activities: thiol reductase, thiol oxidase, thiol isomerase and chaperone, with emphasis on controls and critical interferents, such as detergent-containing buffers. We also discuss the transposition of these methods from purified PDI to cellular or in vivo samples, with critical thoughts about the interpretation of results

Die Relaxation der Styrylfarbstoffe DCM und LDS-750 nach Photoanregung in flüssiger Phase wurde mittels zeitaufgelöster optischer Spektroskopie untersucht. Dabei wurde die Breitband-Pump-Probe-Technik angewandt. Zur Charakterisierung der Relaxation von DCM im elektronischen Grundzustand wurde außerdem die Breitband-Dump-Probe-Technik (stimuliertes Emissionspumpen) eingesetzt. Für die beobachtete schnelle Relaxation von DCM wurde eine annähernd lösungsmittelunabhängige Zeitkonstante von 0.23 (( 0.04) ps im elektronisch angeregten und von 0.28 (( 0.07 ps) im elektronischen Grundzustand gefunden. Sie wurde als Konforma tionsänderung mit nur geringer Ladungsverschiebung charakterisiert. Die weitere spektrale Entwicklung wird in polarer Lösungsmittelumgebung vorwiegend von der Solvatation bestimmt. Für das ionische Polymethin LDS-750 wurden nach der Anregung solvensabhängige Kinetiken beobachtet, die sich durch die Annahme dreier möglicher Konformationen im S1 erklären lassen. / Relaxation of the stryryl dyes DCM and LDS-750 after photoexcitation in the liquid phase has been investigated by means of time-resolved optical spectroscopy. For this purpose, the broadband pump-probe technique was used. To characterize the relaxation of DCM in the electronic ground state, additionally the broadband dump-probe technique (stimulated emission pumping) was applied. An approximately solvent-independent time constant was found typical for the observed fast relaxation of DCM, with values of 0.23 ((0.04) ps in the excited and 0.28 (( 0.07 ps) in the electronic ground state. The relaxation was characterized as conformational change with only a small amount of charge transferred. The further spectral evolution in polar solvents was dominated by solvation dynamics.For the ionic polymethine species LDS-750 solvent-dependent kinetics were found after photoexcitation. They could be accounted for by assuming the existence of three different conformers within the S1 state.

Uncaria tomentosa (Willd.) DC. (Rubiaceae), popularmente conhecida como cat’s claw ou “unha-de-gato”, é uma liana encontrada principalmente na região Amazônica assim como Uncaria guianensis (Aubl.) Gmel. (Rubiaceae), que é utilizada como substituinte ou adulterante em relação a U. tomentosa devido à sua maior abundância e menor valor comercial. A diferenciação de ambas pode ser realizada com base em aspectos morfoanatômicos, mas limita-se à composição química em derivados, como extratos fluidos e secos. As cascas do caule de U. tomentosa são compostas majoritariamente por derivados triterpênicos, polifenóis e alcaloides oxindólicos, aos quais são atribuídas as principais atividades biológicas da espécie. Contudo, o perfil de alcaloides oxindólicos é variável devido à ocorrência de quimiotipos e a elevada susceptibilidade dos mesmos à isomerização. Assim, a presente tese teve como objetivo avaliar a relevância dos alcaloides oxindólicos em U. tomentosa no que tange ao reconhecimento de adulteração na espécie, ocorrência de quimiotipos e isomerização desses compostos. Para isso, foram construídos modelos de classificação e regressão multivariada a partir das análises de CLAE-PDA, IV e UV destinados a diferenciação entre U. tomentosa e U. guianensis e ao reconhecimento de adulteração e determinação do percentual de adulterante em amostras de U. tomentosa. Os resultados obtidos demonstraram que os critérios farmacopéicos atualmente utilizados no controle de qualidade da matéria-prima vegetal e derivados de U. tomentosa baseados nos alcaloides oxindólicos são inefetivos em relação ao reconhecimento de adulteração. (Continuação A avaliação da atividade citotóxica dos diferentes quimiotipos baseados no perfil de alcaloides oxindólicos em U. tomentosa frente a leucócitos humanos e as células tumorias de bexiga (T24) e glioblastoma (U-251-MG) humanos, demonstrou que a seletividade frente às células tumorais é depentente do quimiotipo. Adicionalmente, a complexação dos alcaloides oxindólicos com sulfobutil-éter-β-ciclodextrina (SBE-βCD) minimizou a velocidade de isomerização sob condições de incubação (pH = 7,4; 37 ºC), sem contudo inibir o processo de isomerização. / Uncaria tomentosa (Willd.) DC. (Rubiaceae), popularly known as cat’s claw, is a liana found mainly in the Amazon rainforest as well as Uncaria guianensis (Aubl.) Gmel. (Rubiaceae) used as substituent or adulterant due to their higher wild population and lower market value. The differentiation among the raw material of both species can be performed from morphological and microscopic characteristics, but is limited in derivatives such as fluid and freeze-dried extracts. The stem bark from U. tomentosa is composed mainly by quinovic acid glycosides, polyphenols and oxindole alkaloids, to which have been assigned the major biological activities of the specie. However, the oxindole alkaloids profile in the U. tomentosa is variable due to chemotype occurrence and their susceptibility to isomerization. Thus, this study aimed to evaluate the relevance of oxindole alkaloids at U. tomentosa in relation to adulteration recognition, chemotype occurrence and oxindole alkaloids isomerization. Classification and multivariate regression models were built from HPLC-PDA, FT-IR and UV data to differentation between U. tomentosa e U. guianensis, as well as for adulteration recognition and determination of the adulterant level in the U. tomentosa. The current U.S. pharmacopeia monographs specifications for quality control of stem bark raw material from U. tomentosa, as well as for their derivatives, such as powdered dried extract, based on the oxindole alkaloids were ineffective for adulteration recognition. The cytotoxic activity evaluation of the different chemotypes, based on the oxindole alkaloid profile, against the human leukocytes and against human bladder cancer cell line (T24) and human glioblastoma cell line (U-251-MG) demonstrated that selectivity against the tumoral cells is dependent of the chemotype. In addition, the complexation of the oxindole alkaloids with Sulfobutyl ether β-cyclodextrin (SBE-βCD) minimize the isomerization rate under incubation conditions (pH = 7.4; 37 ºC) but without, however, inhibit the isomerization process.

Lewis acidic zeolites are silica-based, crystalline microporous materials containing tetravalent heteroatoms (M4+=Ti, Sn, Zr, Hf) substituted in framework locations, and have been reported to catalyze a wide range of reactions involving oxygenates and hydrocarbons. The synthetic protocols used to prepare Lewis acid zeolites determine the structures of the active sites and the reaction pockets that confine them, which in turn influences reactivity, product selectivity, and catalyst stability. Specifically, aqueous-phase reactions of biomass-derived molecules, such as glucose isomerization, are sensitive to the hydrophobicity of confining environments, leading to changes in turnover rates. As a result, precise evaluation of the structure and behavior of reaction environments and confined active sites among catalysts of varying provenance or treatment history requires quantitative descriptions of active Lewis acid site densities, of densities of surface functional groups that determine the polarity of microporous confining environments, and of the kinetic behavior of these catalytic materials.<div><br></div><div>Methods for quantifying Lewis acid sites and silanol defects are developed here by analyzing infrared (IR) spectra collected after Lewis base (CD3CN, pyridine) titrations of Lewis acidic zeolite surfaces and are compared to vapor-phase methanol and water adsorption isotherms. Additionally, IR spectra collected under ex situ (flowing vapor-phase water) and in situ (aqueous-phase, 373 K, 0-50 wt% glucose) conditions are used to compare co-adsorbed water densities and structures within hydrophobic (low silanol density) and hydrophilic (high silanol density) confining environments within M-Beta zeolites. Under reaction conditions relevant for sugar conversion in aqueous media (353-398 K, 1-50 wt% glucose), hydrophilic reaction pockets stabilize liquid-like extended water structures within microporous environments, while hydrophobic channels stabilize vapor-phase water at lower intraporous water densities. Higher aqueous-phase glucose isomerization rates (368-383 K, 1-50 wt% glucose, per kinetically relevant active site) are observed on hydrophobic Ti-Beta (~6-12x, per Lewis acidic Ti) and Sn-Beta (~50x, per Lewis acidic Sn in open configuration) zeolites over their hydrophilic analogs. Higher turnover rates on hydrophobic M-Beta zeolites reflect the absence of an extended, hydrogen-bonded network of waters, which entropically destabilizes kinetically relevant hydride shift transition states by reducing the flexibility of their primary solvation spheres. These findings suggest catalyst design strategies to minimize the generation of silanol groups within confining reaction environments would lead to increases in turnover rates.<br></div><div><br></div><div>The methods derived herein can be applied to understanding the role of the confining environment and the associated co-adsorbed water on zeolitic materials of different topology and Lewis acid site identity. For example, the transient formation of silanol defects under aqueous-phase operating conditions is primarily responsible for the deactivation of Sn-Beta catalysts observed during aqueous-phase glucose isomerization. Further, quantifying the role of the confining environment geometry and hydrophobicity on aqueous-phase glucose isomerization rates can be used as guidance for catalyst design to increase reaction rates and selectivities toward specific isomerization products. These findings show that both the active site identity and its confining environment, which vary with zeolite topology and micropore polarity, combine to influence reactivity, selectivity and stability for aqueous-phase glucose isomerization catalysis.<br></div>

Glucose isomerase (GI) catalyzes the isomerization of glucose to fructose and consequently is one of the bulkiest industrial enzyme for the manufacture of high fructose corn syrup and crystalline fructose. The GI is used in industry mainly in the form of immobilized enzyme. / In this work, the immobilization of GI had been studied by several methods: ion exchange adsorption, covalent binding, alginate cells entrapment and cells cross-linking. Three kinds of carrier support (ion exchange resin, epoxy resin and amino resin) have been used in the immobilization of cells-free enzyme; the whole cells immobilization of GI by cross-linking agents polyethyleneimid and glutaraldehyde were critically examined. The results show that the cells cross-linking is the best method to prepare the immobilized GI products, as it is high in specific activity and thermostability, and low the cost. The method is likely to make significant contribution to the field of immobilization, its application has expanding rapidly in many walks of the society, including environment protection, food and pharmaceutical industries. / Jin, Caike. / "August 2005." / Adviser: Jun Wang. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3521. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 125-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Currently, the major sources of fuel, energy, and chemicals are nonrenewable fossil resources such as petroleum, natural gas, and coal. Additionally, petroleum is used for the production of most transportation fuels and for the production of about 95% of organic chemicals. However, the production and use of non-renewable fossil fuels are unsustainable. For economic and environmental sustainability, there is a need to search for new and/or renewable resources and technologies for energy, fuels, and chemicals production that have the potential of effectively substituting fossil resources. In this context, lignocellulosic biomass is one of the candidates that meet these requirements due to its abundance and renewability. Lignocellulosic biomass-derived sugars can be chemically converted into 5-(hydroxymethyl)furfural (HMF), a versatile platform chemical that can used to generate intermediates for the production of biofuels and chemicals. In this dissertation novel catalytic processes for converting monosaccharides into HMF are described. In chapter one, a literature review of the significance of HMF and the chemical intermediates derived from HMF is presented. The chapter also describes some of recent developments in the catalytic production of HMF from lignocellulosic sugars using homogeneous and heterogeneous catalysts. Chapter two discusses the catalytic activity of dimethylaluminum complexes bearing (aminomethyl)phenolate ligands that I developed for converting glucose to HMF in ionic liquids. A systematic study on the effects of modification of the aluminum ancillary ligands on the efficiency of glucose conversion is presented. High HMF yield were obtained with substitution of an aryl substituent on the amino groups of the (aminomethyl)phenolate ligands. In an effort to improve HMF yield, the effects of modifying the ligands on the phenolate moiety of the (aminomethyl)phenolate ligands were investigated in chapter three. Using bulky ortho-phenoxide substituents achieved high HMF yields. The selectivity for HMF production with respect to fructose dehydration was also discussed, together with spectroscopic characterization of the polymeric humins produced from the dehydration reactions. In chapter four a study of the structural differences of poor vs. effective dimethylaluminum complexes bearing (aminomethyl)phenolate ligands is described. Insights from this study shows that different precatalyst intermediate could be formed depending on the aluminum complex used, which in turn affects HMF selectivity of dehydration reactions. The isomerization of glucose to fructose using aluminum complexes in N-methyl-2-pyrrolidone (NMP) is discussed in chapter five. Using NMR spectroscopy on isotopically labeled glucose, a mechanism for glucose isomerization to fructose is presented. Finally, chapter six gives a summary and describes potential future directions for the research detailed in this dissertation.

Disulfide bond formation is an essential process for the folding and biological activity of most extracellular proteins; however, it may become the limiting step when the production of these proteins is attempted in heterologous hosts such as Escherichia coli. The rearrangement of incorrect disulfide bonds between cysteines that do not normally interact in the native structure of a protein is carried out by disulfide isomerase enzymes. The disulfide isomerase present in the bacterial secretory compartment (the periplasmic space) is the homodimeric enzyme DsbC. The objective of this dissertation was to understand the key features of how DsbC catalyzes disulfide bond isomerization. Chimeric disulfide isomerases comprising of protein domains that share a similar function, or are homologous to domains of DsbC were constructed in an effort to understand the effect of the domain orientation in the dimeric protein, and the need for a substrate binding region in disulfide isomerases. We successfully created a series of fusion enzymes, FkpA-DsbAs, which catalyze in vivo disulfide isomerization with comparable efficiency to DsbC. These enzymes comprise of the peptide binding region of the periplasmic chaperone FkpA, which is functionally and structurally similar to the binding domain of DsbC but share no amino acid homology with it, fused to the bacterial oxidase DsbA. In addition, these chimeric enzymes were shown to assist in the initial formation of disulfide bonds, a function that is normally exhibited only by DsbA. Directed evolution of the FkpA-DsbA proteins conferred improved resistance to CuCl₂, a phenotype dependent on disulfide bond isomerization and highlighted the importance of an optimal catalytic site. The bacterial disulfide isomerase DsbC is a homodimeric V-shaped enzyme that consists of a dimerization domain, two α-helical linkers and two opposing catalytic domains. The functional significance of the existence of two catalytic domains of DsbC is not well understood yet. The fact that identical subunits naturally dimerize to generate DsbC has so far limited the study of the individual catalytic sites in the homodimer. In chapter 3 we discuss the engineering, in vivo function, and biochemical characterization chapter 3 we discuss the engineering, in vivo function, and biochemical characterization of DsbC variants covalently linked via (Gly3Ser) flexible linkers. We have either inactivated one of the catalytic sites (CGYC), or entirely removed one of the catalytic domains while maintaining the putative binding area intact. Our results support the hypotheses that dual catalytic domains in DsbC are not necessary for disulfide bond isomerization, but are important in terms of increasing the effective concentration of catalytic equivalents, and that the availability of a substrate binding region is a determining feature in isomerization. Finally, we have carried out initial studies to map the residues and sequence motifs that are recognized in substrate proteins that interact with DsbC. Although the main putative binding region of DsbC has been localized within the limits of the hydrophobic cleft that emerges from the interaction of the N-terminal domains of this enzyme, and, a few native substrates have already been identified, no information on the features of substrate proteins that are recognized by the enzyme has been reported. To address this problem, we have screened two different, 15 amino-acid random peptide libraries for binding to DsbC. We have successfully isolated several peptides with high affinity for the enzyme. Possible consensus binding motifs were identified and their significance in substrate recognition will be examined in future studies. / text

A series of cyclophanedienes (CPDs) with different internal functional groups were synthesized. Dicyano CPD 85, cyano methyl CPD 127 and phenylethynyl/methyl CPD 138 were synthesized from bis-bromomethyl aromatics via a thiacyclophane- thiomethylcyclophane route. Diformyl cyclophanediene 152 and bis(hydroxymethyl) CPD 159 were obtained by the functional group transformation of CPDs 85 and 152 respectively. Cyclophanedienes with internal olefinic groups were obtained by three different routes: the best was the functional group transformations of the dicyano mercaptomethylcyclophane 99 followed by a Hoffmann elimination. Using the best synthetic route, CPDs with substituted vinyl groups such as alkylvinyl (162, 163, 178 and 198), butadienyl (184, 185 and 186), styryl (202, 203 and 204), nitro-substituted styryl (210, 211 and 212), methoxy-substituted styryl (218, 219 and 220) and methyl-substituted styryl (226, 227 and 228) were synthesized. Cyclophanediene 235 with an internal ethynyl (alkynyl) group was also synthesized by a similar synthetic route; however, it gave two major interesting side products; vinyl-ethynyl CPD 237 and vinyl-styryl CPD 240. The cyclophanedienes except dicyano 85, cyano-methyl 127 and diformyl 152 were converted to their corresponding dihydropyrenes both thermally and photochemically. Dicyano CPD 85 and cyano-methyl CPD 127 were converted photochemically to the DHPs 86 and 128, respectively. Diformyl CPD 152 underwent decomposition in any attempt to transform it into the DHP 154 either thermally or photochemically. Diphenylethynyl DHPs 141 and 247 were obtained by the Sonogashira coupling of diethynyl DHP 236. The Eglinton coupling reaction was used to achieve butadiynyl DHPs 257 and 254. Naphthoyl DHPs 248 and 250 were synthesized by the Friedel-Crafts acylation reaction of DHPs 179 and 167, respectively. All compounds were characterized by NMR, IR, and UV spectroscopy and mass spectrometry. Dicyano CPD 85 was quite stable towards thermal isomerization to the dihydropyrene 86 and showed a calculated half life of ~ 36 years (three orders of magnitude higher than that of benzo CPD 53 i.e., 7.3 days) at room temperature, whereas CPDs 127 (cyano methyl), 138 (phenylethynyl/methyl) and 152 (diformyl) showed half lives less than a month at 20 oC. Cyclophanedienes with internal ethynyl and substituted vinyl groups were quite stable thermally and showed half lives of several years (1-16 years) at room temperature. CPDs with cis substituted internal vinyl groups were thermally more stable than their trans counterparts. Electron withdrawing substituent (NO2) at the para positions of the internal styryl groups accelerate, whereas electron donating groups (MeO, Me) decelerate the thermal return reaction. Naphthoyl CPDs 249 and 251 isomerized at rates about 6-12 times faster than their non naphthoylated analogues 178 and 166 respectively. DHPs with internal ethenyl (167, 238 and 241), substituted ethynyl (139, 141 and 247) and trans substituted vinyl (199, 207, 215, 223 and 231) groups failed to open under visible light irradiation. Dicyano DHP 86, diethynyl DHP 236 and the unsymmetrical isomers of internal olefinic CPDs (206, 214, 222 and 230) formed photostationary states (pss). Disubstituted vinyl (179) and cis substituted vinyl DHPs (164, 205, 213, 221 and 229) opened completely; however their opening rates although faster than the parent 43, were 4-6 times slower than the benzo DHP 47. Introduction of an electron withdrawing substituent on the internal styryl group decelerated the visible opening reaction whereas electron donating groups accelerated it. 2-Naphthoyl divinyl DHP 250 opened at rates quite comparable to those of benzo DHP 47 whereas 2-naphthoyl diisobutenyl 248 opened about 25 times faster than the benzo DHP. The [1,5]-sigmatropic rearrangement of the internal nitrile (DHPs 86 and 128) and formyl (DHP 153) groups was observed. The sigmatropic rearrangement of the nitrile group in 86 was quite favorable in CDCl3 (Eact = 23.4 + 0.7 kcal/mol) compared to benzene (Eact = 28.6 + 1.2 kcal/mol). Formyl groups showed a much higher migration aptitude and Eact is estimated to be < 20 kcal/mol in any solvent. In this study, the best switch pair obtained was naphthoyl diisobutyl 248/249 which in comparison with previously the best switch pair 47/53 (benzo) showed much higher stability of the cyclophanediene (two orders of magnitude); moreover, the dihydropyrene opened about 25 times faster as well and is one of the best new photochromes yet.

Cyclophilin A (CypA) is the well-studied member of a group of ubiquitous and evolutionarily conserved families of enzymes called peptidyl–prolyl isomerases (PPIases). These enzymes catalyze the cis-trans isomerization of peptidyl-prolyl bond in many proteins. The distinctive functional path triggered by each isomeric state of peptidyl-prolyl bond renders PPIase-catalyzed isomerization a molecular switching mechanism to be used on physiological demand. PPIase activity has been implicated in protein folding, signal transduction, and ion channel gating as well as pathological condition such as cancer, Alzheimer’s, and microbial infections. The more than five order of magnitude speed-up in the rate of peptidyl–prolyl cis–trans isomerization by CypA has been the target of intense research. Normal and accelerated molecular dynamic simulations were carried out to understand the catalytic mechanism of CypA in atomistic details. The results reaffirm transition state stabilization as the main factor in the astonishing enhancement in isomerization rate by enzyme. The ensuing intramolecular polarization, as a result of the loss of pseudo double bond character of the peptide bond at the transition state, was shown to contribute only about −1.0 kcal/mol to stabilizing the transition state. This relatively small contribution demonstrates that routinely used fixed charge classical force fields can reasonably describe these types of biological systems. The computational studies also revealed that the undemanding exchange of the free substrate between β- and α-helical regions is lost in the active site of the enzyme, where it is mainly in the β-region. The resultant relative change in conformational entropy favorably contributes to the free energy of stabilizing the transition state by CypA. The isomerization kinetics is strongly coupled to the enzyme motions while the chemical step and enzyme–substrate dynamics are in turn buckled to solvent fluctuations. The chemical step in the active site of the enzyme is therefore not separated from the fluctuations in the solvent. Of special interest is the nature of catalysis in a more realistic crowded environment, for example, the cell. Enzyme motions in such complicated medium are subjected to different viscosities and hydrodynamic properties, which could have implications for allosteric regulation and function.