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ABSTRACT: Neighboring group participation is one of the fundamental interactions in organic reactions and can influence the reaction rate, stereoselectivity, and reaction pathway through transient carbon-carbon or carbon-heteroatom bond formation. The latter category includes cyclic three- and five-membered bromonium ions, wherein lone-pair electrons of the monovalent bromine atom stabilize a trigonal carbocation. Although similar nucleophilic interactions of monovalent halogen atoms with non-carbon atom-centered cations have long been predicted, we know of no experimental evidence of such an interaction. Here, we demonstrate a nucleophilic interaction of neighboring monovalent halogen to stabilize an imino sp(2) nitrogen cation. This interaction has an overwhelming impact on the reaction pathway, completely altering the migratory preference under acid-catalyzed Beckmann rearrangement conditions. In sharp contrast to the general case of anti-migration, peri-chloro- and peri-bromo-substituted O-tosyl oximes of 1-tetralone substructures and their derivatives undergo syn-migration under Beckmann rearrangement conditions (i.e., migration of the group on the syn side of the leaving group). The peri-chloro or peri-bromo neighboring group turned out to provide strong anchimeric assistance for syn-migration via transient formation of a cyclic five-membered imino-halonium cation with dissociation of tosylic acid. Thus, formation of the syn-migration products can be attributed to a reaction mechanism that is different from the conventional Beckmann rearrangement mechanism. That is, the positively charged imino nitrogen atom can be stabilized by, or interact with, a chloro or bromo group in close spatial proximity, and this interaction dramatically changes the reaction pathway, selectively affording regioisomeric lactams from closely related starting materials.
Proceedings of the National Academy of Sciences 02/2013; · 9.68 Impact Factor
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ABSTRACT: INTRODUCTION: IonWorks automated patch clamp systems are being widely used for ion channel drug discovery, but the perforated patch mode of these systems makes it difficult to obtain a steady intracellular Ca(2+) concentration ([Ca(2+)](i)). This difficulty prevents obtaining high-quality data regarding Ca(2+)-activated channels such as BK and SK channels. We examined the methods for stabilizing [Ca(2+)](i) in the IonWorks Quattro automated patch clamp system to evaluate BK channels. METHODS: Electrophysiological recordings were performed using the single-hole or population patch clamp mode of IonWorks Quattro. To increase [Ca(2+)](i), ionomycin was used. The variation in the BK current and the effect of BK channel modulators were examined in the presence and absence of an intracellular Ca(2+) chelator, BAPTA-AM (20μM). RESULTS: BK current activated by step pulses to +100mV in the presence of ionomycin exhibited large variation (ranging from 0.086 to 11nA). In individual cells, oscillation of the current amplitude was observed when five repetitive pulses were applied at 0.1Hz. Approximately 30% of cells exhibited current variation exceeding 20% when the variation was calculated using the first and third pulses. However, BAPTA-AM treatment before current measurement decreased the number of cells displaying large variation (>20%) to 5%. In the presence of BAPTA-AM, the BK channel modulators NS1619 and 12,14-dichlorodehydroabietic acid increased the BK current at concentrations of 10μM or more showing clear concentration dependency, whereas in its absence, the effect of both compounds was detected only at 30μM. DISCUSSION: The main finding of this study is that the [Ca(2+)](i) variation in the basal condition is very large and hinders the accurate evaluation of compounds in Ca(2+)-activated ion channels. The application of BAPTA-AM and ionomycin greatly improved the precision of BK channel screening, and this method should be applicable to other Ca(2+)-activated ion channels such as SK channels.
Journal of pharmacological and toxicological methods 11/2012; · 2.32 Impact Factor
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ABSTRACT: Although cations with three heteroatoms, such as monoprotonated guanidine and urea, are stabilized by Y-shaped conjugation and such Y-conjugated cations are sufficiently basic to be further protonated (or protosolvated) to dications in strongly acid media, only O-monoprotonated species have been detected in the case of carbamates even in magic acid. We found that the trifluoromethanesulfonic acid-catalyzed cyclization of arylethylcarbamates proceeds to afford dihydroisoquinolones in high yield. In strong acids, methyl carbamates are fully O-monoprotonated, and these monocations do not undergo cyclization even under heating. But, as the acidity of the reaction medium is further increased, the cyclization reaction of methyl phenethylcarbamates starts to proceed as a first-order reaction, with a linear relationship between rate and acidity. The sign and magnitude of the entropy of activation ΔS(⧧) were found to be similar to those of other A(Ac)1 reactions. These results strongly support the idea that further protonation of the O-protonated carbamates is involved in the cyclization, but the concentration of the dications is very low and suggests that the rate-determining step is dissociation of methanol from the diprotonated carbamate to generate protonated isocyanate, which reacts with the aromatic ring. Therefore, O-protonated carbamates are weak bases in sharp contrast to other Y-shaped monocations.
The Journal of Organic Chemistry 10/2012; 77(20):9313-28. · 4.45 Impact Factor
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Asuka Inoue,
Jun Ishiguro,
Hajime Kitamura,
Naoaki Arima,
Michiyo Okutani,
Akira Shuto,
Shigeki Higashiyama, Tomohiko Ohwada,
Hiroyuki Arai,
Kumiko Makide,
Junken Aoki
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ABSTRACT: A single-format method to detect multiple G protein-coupled receptor (GPCR) signaling, especially Gα(12/13) signaling, presently has limited throughput and sensitivity. Here we report a transforming growth factor-α (TGFα) shedding assay, in which GPCR activation is measured as ectodomain shedding of a membrane-bound proform of alkaline phosphatase-tagged TGFα (AP-TGFα) and its release into conditioned medium. AP-TGFα shedding response occurred almost exclusively downstream of Gα(12/13) and Gα(q) signaling. Relying on chimeric Gα proteins and promiscuous Gα(16) protein, which can couple with Gα(s)- and Gα(i)-coupled GPCRs and induce Gα(q) signaling, we used the TGFα shedding assay to detect 104 GPCRs among 116 human GPCRs. We identified three orphan GPCRs (P2Y10, A630033H20 and GPR174) as Gα(12/13)-coupled lysophosphatidylserine receptors. Thus, the TGFα shedding assay is useful for studies of poorly characterized Gα(12/13)-coupled GPCRs and is a versatile platform for detecting GPCR activation including searching for ligands of orphan GPCRs.
Nature Methods 09/2012; 9(10):1021-9. · 19.28 Impact Factor
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ABSTRACT: Large-conductance voltage- and Ca(2+)-activated K(+) (Slo1 BK) channels serve numerous cellular functions, and their dysregulation is implicated in various diseases. Drugs activating BK channels therefore bear substantial therapeutic potential, but their deployment has been hindered in part because the mode of action remains obscure. Here we provide mechanistic insight into how the dehydroabietic acid derivative Cym04 activates BK channels. As a representative of NS1619-like BK openers, Cym04 reversibly left-shifts the half-activation voltage of Slo1 BK channels. Using an established allosteric BK gating model, the Cym04 effect can be simulated by a shift of the voltage sensor and the ion conduction gate equilibria toward the activated and open state, respectively. BK activation by Cym04 occurs in a splice variant-specific manner; it does not occur in such Slo1 BK channels using an alternative neuronal exon 9, which codes for the linker connecting the transmembrane segment S6 and the cytosolic RCK1 domain--the S6/RCK linker. In addition, Cym04 does not affect Slo1 BK channels with a two-residue deletion within this linker. Mutagenesis and model-based gating analysis revealed that BK openers, such as Cym04 and NS1619 but not mallotoxin, activate BK channels by functionally interacting with the S6/RCK linker, mimicking site-specific shortening of this purported passive spring, which transmits force from the cytosolic gating ring structure to open the channel's gate.
Proceedings of the National Academy of Sciences 02/2012; 109(9):3552-7. · 9.68 Impact Factor
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Hajime Kitamura,
Kumiko Makide,
Akira Shuto,
Masaya Ikubo,
Asuka Inoue,
Kensuke Suzuki,
Yusuke Sato,
Sho Nakamura,
Yuko Otani, Tomohiko Ohwada,
Junken Aoki
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ABSTRACT: GPR34 is a G protein-coupled receptor belonging to the P2Y family. Here, we attempted to resolve conflicting reports about whether it is a functional lysophosphatidylserine (LysoPS) receptor. In HEK293 cells expressing human, mouse or rat GPR34 and Gα chimera between Gαq and Gαi1(Gq/i1), LysoPS quickly elevated intracellular Ca(2+) ion levels ([Ca(2+)](i)). LysoPS also stimulated alkaline phosphatase (AP)-tagged TGFα (AP-TGFα) release in GPR34-expressing HEK293 cells and induced the migration of CHO-K1 cells expressing GPR34. Other lysophospholipids did not induce these actions. Replacement of the serine residue of LysoPS abolished the reactivity of LysoPS with GPR34, indicating that GPR34 strictly recognizes the serine head group of LysoPS. Recombinant phosphatidylserine-specific phospholipase A(1) (PS-PLA(1)) that deacylates fatty acid at the sn-1 position of PS and produces 2-acyl-LysoPS, but not catalytically inactive mutant PS-PLA(1), stimulated the release of AP-TGFα from GPR34-expressing cells. Consistent with the result, LysoPS was detected in the cells treated with wild-type PS-PLA(1) but not with the mutant PS-PLA(1). PS treated with PLA(1) was much more effective at stimulating AP-TGFα release than PS treated with PLA(2). In addition, migration-resistant 2-acyl-1-deoxy-LysoPS, a 2-acyl-LysoPS analogue, was much more potent than 1-acyl-2-deoxy-LysoPS. The present studies confirm that GPR34 is a cellular receptor for LysoPS, especially with a fatty acid at the sn-2 position.
Journal of biochemistry 02/2012; 151(5):511-8. · 1.95 Impact Factor
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ABSTRACT: Although many organic/inorganic compounds that release nitric oxide (NO) upon photoirradiation (phototriggered caged-NOs) have been reported, their photoabsorption wavelengths mostly lie in the UV region, because X-NO bonds (X=heteroatom and metal) generally have rather strong π-bond character. Thus, it is intrinsically difficult to generate organic compounds that release NO under visible light irradiation. Herein, the structures and properties of N-pyramidal nitrosamine derivatives of 7-azabicyclo[2.2.1]heptanes that release NO under visible light irradiation are described. Bathochromic shifts of the absorptions of these nitrosamines, attributed to HOMO (n)-LUMO (π*) transitions associated with the nonplanar structure of the N-NO moiety, enable the molecules to absorb visible light, which results in N-NO bond cleavage. Thus, these compounds are innate organic caged-NOs that are uncaged by visible light.
Chemistry 12/2011; 18(4):1127-41. · 5.93 Impact Factor
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ABSTRACT: Nitrosamines are potent carcinogens and toxicants in the rat and potential genotoxins in humans. They are metabolically activated by hydroxylation at an α-carbon atom with respect to the nitrosoamino group, catalyzed by cytochrome P450. However, there has been little systematic investigation of the structure-mutagenic activity relationship of N-nitrosamines. Herein, we evaluated the mutagenicity of a series of 7-azabicyclo[2.2.1]heptane N-nitrosamines and related monocyclic nitrosamines by using the Ames assay. Our results show that the N-nitrosamine functionality embedded in the bicyclic 7-azabicylo[2.2.1]heptane structure lacks mutagenicity, that is, it is inert to α-hydroxylation, which is the trigger of mutagenic events. Further, the calculated α-C-H bond dissociation energies of the bicyclic nitrosamines are larger in magnitude than those of the corresponding monocyclic nitrosamines and N-nitrosodimethylamine by as much as 20-30 kcal/mol. These results are consistent with lower α-C-H bond reactivity of the bicyclic nitrosamines. Thus, the 7-azabicyclo[2.2.1]heptane structural motif may be useful for the design of nongenotoxic nitrosamine compounds with potential biological/medicinal applications.
Bioorganic & medicinal chemistry 03/2011; 19(8):2726-41. · 2.82 Impact Factor
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ABSTRACT: Oxime ether derivatives at the benzylic position of unsubstituted, dichloro, trichloro, and monobromo derivatives of the aromatic C-ring of dehydroabietic acid and podocarpic acid were synthesized and evaluated as BK channel openers in an assay system of CHO-K1 cells expressing hBKα channels. Detailed SAR analysis showed that the oximation was particularly effective in the cases of dehydroabietic acid derivatives, and some of these oxime derivatives showed more potent BK channel activities than the standard compound, NS1619. The present studies provide a new structural basis for development of efficient BK channel openers.
Bioorganic & medicinal chemistry 11/2010; 18(24):8642-59. · 2.82 Impact Factor
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ABSTRACT: Helical structures of oligomers of non-natural β-amino acids are significantly stabilized by intramolecular hydrogen bonding between main-chain amide moieties in many cases, but the structures are generally susceptible to the environment; that is, helices may unfold in protic solvents such as water. For the generation of non-hydrogen-bonded ordered structures of amides (tertiary amides in most cases), control of cis-trans isomerization is crucial, even though there is only a small sterical difference with respect to cis and trans orientations. We have established methods for synthesis of conformationally constrained β-proline mimics, that is, bridgehead-substituted 7-azabicyclo[2.2.1]heptane-2-endo-carboxylic acids. Our crystallographic, 1D- and 2D-NMR, and CD spectroscopic studies in solution revealed that a bridgehead methoxymethyl substituent completely biased the cis-trans equilibrium to the cis-amide structure along the main chain, and helical structures based on the cis-amide linkage were generated independently of the number of residues, from the minimalist dimer through the tetramer, hexamer, and up to the octamer, and irrespective of the solvent (e.g., water, alcohol, halogenated solvents, and cyclohexane). Generality of the control of the amide equilibrium by bridgehead substitution was also examined.
Journal of the American Chemical Society 09/2010; 132(42):14780-9. · 9.91 Impact Factor
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Tomohiko Ohwada
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ABSTRACT: Facial selections are reviewed to propose a new theory, orbital phase environment, for stereoselectivities of organic reactions. The orbital phase environment is a generalized idea of the secondary orbital interaction between the non-reacting centers and the unsymmetrization of the orbitals at the reacting centers arising from in-phase and out-of-phase overlapping with those at the neighboring non-reacting sites. In this context, the nucleophilic addition preferentially occurs on the face of the carbonyl functionality opposite to the better electron-donating orbital at the β position. In a similar manner to the carbonyl cases, the preferred reaction faces of olefins in electrophilic addition reactions are opposite to the better electron-donating orbitals at the β positions. The orbital phase environments in Diels-Alder reactions are also reviewed.
Topics in current chemistry 01/2010; 289:129-81. · 4.29 Impact Factor
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ABSTRACT: The generality and the effects of substituents on phenylation reactions of α-acylnitromethanes catalyzed by trifluoromethanesulfonic acid have been studied. α-Aroylnitromethanes afforded benzil monooximes in good yield. In the case of aliphatic α-acylnitromethane, a similar phenylation reaction proceeded, but the yield of the phenylated 1,2-dione monooxime was low. These phenylation reactions represent examples of the generation of carbocation electrophiles at the α-position of a nitro group.
Synthesis. 12/2009; 2009(24):4129-4136.
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ABSTRACT: The chemical features, such as substrate stability, product distribution, and substrate generality, and the reaction mechanism of Brønsted superacid-catalyzed cyclization reactions of aromatic ring-containing acetoacetates (beta-ketoesters) were examined in detail. While two types of carbonyl cyclization are possible, i.e., keto cyclization and ester cyclization, the former was found to take place exclusively. The reaction constitutes an efficient method to synthesize indene and 3,4-dihydronapthalene derivatives. Acid-base titration monitored with (13)C NMR spectroscopy showed that the acetoacetates are fully O(1),O(3)-diprotonated at H(0) = -11. While the five-membered ring cyclization of the arylacetoacetates proceeded slowly at H(0) = -11, a linear increase in the rate of the cyclization was found with increasing acidity in the high acidity region of H(0) = -11.8 to -13.3. Therefore, the O(1),O(3)-diprotonated acetoacetates exhibited some cyclizing reactivity, but they are not the reactive intermediates responsible for the acceleration of the cyclization in the high acidity region. The reactive cationic species might be formed by further protonation (or protosolvation) of the O(1),O(3)-diprotonated acetoacetates; i.e., they may be tricationic species. Thermochemical data on the acid-catalyzed cyclization of the arylacetoacetates showed that the activation energy is decreased significantly as compared with that of the related acid-catalyzed cyclization reaction of a compound bearing a single functional group, such as a ketone. These findings indicate that intervention of the trication contributes to the activation of the cyclization of arylacetoacetates in strong acid, and the electron-withdrawing nature of the O-protonated ester functionality significantly increases the electrophilicity of the ketone moiety.
Journal of the American Chemical Society 12/2009; 132(2):807-15. · 9.91 Impact Factor
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Masazumi Iwashita,
Kumiko Makide,
Taro Nonomura,
Yoshimasa Misumi,
Yuko Otani,
Mayuko Ishida,
Ryo Taguchi,
Masafumi Tsujimoto,
Junken Aoki,
Hiroyuki Arai, Tomohiko Ohwada
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ABSTRACT: In response to various exogenous stimuli, mast cells (MCs) release a wide variety of inflammatory mediators stored in their cytoplasmic granules and this release initiates subsequent allergic reactions. Lysophosphatidylserine (lysoPS) has been known as an exogenous inducer to potentiate histamine release from MCs, though even at submicromolar concentrations. In this study, through SAR studies on lysoPS against MC degranulation, we identified lysoPT, a threonine-containing lysophospholipid and its 2-deoxy derivative as novel strong agonists. LysoPT and its 2-deoxy derivative induced histamine release from MCs both in vitro and in vivo at a concentration less than one-tenth that of lysoPS. Notably, lysoPT did not activate a recently proposed lysoPS receptor on MCs, GPR34, demonstrating the presence of another undefined receptor reactive to both lysoPS and lysoPT that is involved in MC degranulation. Thus, the present strong agonists, lysoPT and its 2-deoxy derivative, will be useful tools to understand the mechanisms of lysoPS-induced activation of degranulation of MCs.
Journal of Medicinal Chemistry 10/2009; 52(19):5837-63. · 4.80 Impact Factor
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ABSTRACT: Oxime ether derivatives of the benzylic ketone of 12,14-dichlorodehydroabietic acid (diCl-DHAA, 4b) were synthesised, and their BK channel-opening activity was evaluated in an assay system of CHO-K1 cells expressing hBKalpha channels. Oxime ether structure on the B ring of diCl-DHAA significantly increased the BK channel-opening activity.
Bioorganic & medicinal chemistry letters 01/2009; 18(24):6386-9. · 2.65 Impact Factor
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ABSTRACT: X-ray crystallographic analysis showed that N-thiobenzoyl-7-azabicyclo[2.2.1]heptane displays marked nonplanarity of the thioamide (1a, alpha = 167.1 degrees and |tau| = 11.2 degrees) as compared with the corresponding monocyclic pyrrolidine thioamide (2a, alpha = 174.7 degrees and |tau| = 3.9 degrees). In a series of para-substituted or unsubstituted thioaroyl-7-azabicyclo[2.2.1]heptanes (1a-1h), the planarity of the thioamide depended significantly on the electronic nature of the substituent; for example, in the p-nitro-substituted compound, planarity was substantially restored (1h, alpha = 175.2 degrees and |tau| = 0.1 degrees). In solution, increasing electron-withdrawing character of the aromatic substituent was associated with a larger rotational barrier of the bicyclic thioamides, as determined by means of variable-temperature (1)H NMR spectroscopy and line shape analysis. The reduced rotational barrier, that is, reduced enthalpy of activation (DeltaH(double dagger)) for thioamide rotation, of 1a as compared with that of 2a in nitrobenzene-d5 is consistent with the postulate that 1a assumes a nonplanar thioamide structure in solution. These results indicate that the planarity of thioamides based on 7-azabicyclo[2.2.1]heptane is controlled by electronic factors in the solid phase and in solution.
The Journal of Organic Chemistry 11/2008; 73(22):9102-8. · 4.45 Impact Factor
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ABSTRACT: We found that the podocarpic acid structure provides a new scaffold for chemical modulators of large-conductance calcium-activated K(+) channels (BK channels). Structure-activity analysis indicates the importance of both the arrangement (i.e., location and orientation) of the carboxylic acid functionality of ring A and the hydrophobic region of ring C for expression of BK channel-opening activity.
Bioorganic & medicinal chemistry letters 09/2008; 18(19):5197-200. · 2.65 Impact Factor
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ABSTRACT: A series of dehydroabietic acid (DHAA, 2) derivatives was synthesized and evaluated as BK channel openers in an assay system of CHO-K1 cells expressing hBKalpha channels. Systematic modifications of the peripheral functionality of ring C of DHAA showed that the introduction of a nitro or (thio)urea group in ring C greatly enhanced the BK channel-opening activity.
Bioorganic & medicinal chemistry letters 09/2008; 18(19):5201-5. · 2.65 Impact Factor
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ABSTRACT: We present superacid-catalyzed intramolecular cyclization reactions of arylcyanopropionates to give cyclized five- and six-membered beta-enamino esters in moderate to high yields. Known intramolecular ring-closing reactions of protonated nitrile to aromatic carbon atom are limited to the 6-membered case. Interestingly, a significant synergistic increase of reactivity of the cyano functionality was observed, and the cyano nitrogen atom was converted into an amino group, when an ester group was present in a geminal arrangement. Deuterium exchange experiments excluded the involvement of deprotonation of the alpha-proton in the cyclization process. The acidity dependence of the cyclization reactions and (13)C NMR studies of a model compound, methyl cyanoacetate, in various acidic media were consistent with the involvement of the O, N-diprotonated dication of methyl cyanoacetate, a distonic dication, in strong acid, and this is considered to be the de facto electrophile in the present cyclization reaction of arylcyanopropionates.
The Journal of Organic Chemistry 07/2008; 73(11):4219-24. · 4.45 Impact Factor
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ABSTRACT: Fluorescent molecular thermometers showing temperature-dependent fluorescence lifetimes enable thermal mapping of small spaces such as a microchannel and a living cell. We report the temperature-dependent fluorescence lifetimes of poly(NIPAM-co-DBD-AA), which is a random copolymer of N-isopropylacrylamide (NIPAM) and an environment-sensitive fluorescent monomer (DBD-AA) containing a 4-sulfamoyl-7-aminobenzofurazan structure. The average fluorescence lifetime of poly(NIPAM-co-DBD-AA) in aqueous solution increased from 4.22 to 14.1 ns with increasing temperature from 30 to 35 degrees C. This drastic change in fluorescence lifetime (27% increase per 1 degrees C) is the sharpest ever reported. Concentration independency, one of the advantages of fluorescence lifetime measurements, was seen in average fluorescence lifetime (13.7 +/- 0.18 ns) of poly(NIPAM-co-DBD-AA) at 33 degrees C over a wide concentration range (0.005-1 w/v%). With increasing temperature, polyNIPAM units in poly(NIPAM-co-DBD-AA) change their structure from an extended form to a globular form, providing apolar and aprotic environments to the fluorescent DBD-AA units. Consequently, the environment-sensitive DBD-AA units translate the local environmental changes into the extension of the fluorescence lifetime. This role of the DBD-AA units was revealed by a study of solvent effects on fluorescence lifetime of a model environment-sensitive fluorophore.
The Journal of Physical Chemistry B 04/2008; 112(10):2829-36. · 3.70 Impact Factor
