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ABSTRACT: Nevirapine is one of the most extensively prescribed antiretrovirals worldwide. The present analyses used data and specimens from two prior studies to characterize and compare plasma nevirapine phase I metabolite profiles following a single 200 mg oral dose of nevirapine in 10 HIV-negative African Americans, and at steady state with 200 mg twice-daily in 10 HIV-infected Cambodians. Nevirapine was assayed by HPLC. The 2-, 3-, 8- and 12-hydroxy and 4-carboxy metabolites of nevirapine were assayed by LC/MS/MS. Pharmacokinetic parameters were calculated by non-compartmental analysis. The metabolic index for each metabolite was defined as the ratio of metabolite AUC to nevirapine AUC. Every metabolite concentration was much less than the corresponding nevirapine concentration. The predominant metabolite after single dose and at steady state was 12-hydroxynevirapine. From single to steady state, the metabolic index increased for 3-hydroxynevirapine (p<0.01), but decreased for 2-hydroxynevirapine (p<0.001). The 3-hydroxynevirapine metabolic index was correlated with nevirapine apparent clearance (p<0.001). These findings are consistent with induction of CYP2B6 (3-hydroxy metabolite) and a possible inhibition of CYP3A (3-hydroxy metabolite), although these are preliminary data. There were no such changes in metabolic indexes for 12-hydroxynevirapine or 4-carboxynevirapine. Two subjects with the CYP2B6 *6*6 genetic polymorphism had metabolic indexes in the same range as other subjects. These results suggest that nevirapine metabolite profiles change over time under the influence of enzyme induction, enzyme inhibition, and host genetics. Further work is warranted to elucidate nevirapine biotransformation pathways, and implications for drug efficacy and toxicity.
Antimicrobial Agents and Chemotherapy 03/2013; · 4.84 Impact Factor
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Reda Z Mahfouz,
Ania Jankowska,
Quteba Ebrahem,
Xiaorong Gu,
Valeria Visconte,
Ali Tabarroki,
Pramod Terse,
Joseph Covey,
Kenneth Chan, Yonghua Ling,
Kory J. Engelke,
Mikkael A. Sekeres,
Ramon Tiu,
Jaroslaw Maciejewski,
Tomas Radivoyevitch,
Yogen Saunthararajah
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ABSTRACT: Purpose: The cytidine analogues 5-azacytidine and decitabine, used to treat myelodysplastic syndromes
(MDS), produce a molecular epigenetic effect, depletion of DNA-methyltransferase (DNMT1). This action is Sphase dependent. Hence, genetic factors that decrease the half-lives of these drugs could impact efficacy.
Documentation of such impact, and elucidation of underlying mechanisms, could lead to improved clinical
application.
Design: Cytidine deaminase (CDA) rapidly inactivates 5-azacytidine/decitabine. The effect of CDA SNP A79C
and gender on CDA expression, enzyme activity and drug pharmacokinetics/pharmacodynamics was
examined in mice and humans, and the impact on overall survival (OS) was evaluated in 5-
azacytidine/decitabine-treated MDS patients (n=90) and cytarabine-treated acute myeloid leukemia (AML)
patients (n=76).
Results: By HPLC, plasma CDA activity was decreased as expected in individuals with the SNP A79C.
Interestingly and significantly, there was an even larger decrease in females compared to males. Explaining
this decrease, liver CDA expression was significantly lower in female versus male mice. As expected,
decitabine plasma levels, measured by mass-spectrometry, were significantly higher in females. In
mathematical modeling, the detrimental impact of shorter drug half-life (e.g., in males) was greater in low
compared to high S-phase fraction disease (e.g., MDS versus AML), since in high S-phase fraction disease,
even a short exposure treats a major portion of cells. Accordingly, in multivariate analysis, OS was significantly
worse in male versus female MDS patients treated with 5-azacytidine/decitabine.
Conclusions: Increased CDA expression/activity in males contributes to decreased cytidine analogue half-life
and likely contributes to worse outcomes with 5-azacytidine or decitabine therapy.
Clinical Cancer Research 01/2013; · 7.74 Impact Factor
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Reda Z Mahfouz,
Ania Jankowska,
Quteba Ebrahem,
Xiaorong Gu,
Valeria Visconte,
Ali Tabarroki,
Pramod Terse,
Joseph M Covey,
Kenneth K Chan, Yonghua Ling,
Kory J Engelke,
Mikkael Sekeres,
Ramon Tiu,
Jaroslaw P Maciejewski,
Tomas Radivoyevitch,
Yogen Saunthararajah
[show abstract]
[hide abstract]
ABSTRACT: PURPOSE: The cytidine analogues 5-azacytidine and decitabine, used to treat myelodysplastic syndromes (MDS), produce a molecular epigenetic effect, depletion of DNA-methyltransferase (DNMT1). This action is S-phase dependent. Hence, genetic factors that decrease the half-lives of these drugs could impact efficacy. Documentation of such impact, and elucidation of underlying mechanisms, could lead to improved clinical application. Design: Cytidine deaminase (CDA) rapidly inactivates 5-azacytidine/decitabine. The effect of CDA SNP A79C and gender on CDA expression, enzyme activity and drug pharmacokinetics/pharmacodynamics was examined in mice and humans, and the impact on overall survival (OS) was evaluated in 5-azacytidine/decitabine-treated MDS patients (n=90) and cytarabine-treated acute myeloid leukemia (AML) patients (n=76). RESULTS: By HPLC, plasma CDA activity was decreased as expected in individuals with the SNP A79C. Interestingly and significantly, there was an even larger decrease in females compared to males. Explaining this decrease, liver CDA expression was significantly lower in female versus male mice. As expected, decitabine plasma levels, measured by mass-spectrometry, were significantly higher in females. In mathematical modeling, the detrimental impact of shorter drug half-life (e.g., in males) was greater in low compared to high S-phase fraction disease (e.g., MDS versus AML), since in high S-phase fraction disease, even a short exposure treats a major portion of cells. Accordingly, in multivariate analysis, OS was significantly worse in male versus female MDS patients treated with 5-azacytidine/decitabine. CONCLUSIONS: Increased CDA expression/activity in males contributes to decreased cytidine analogue half-life and likely contributes to worse outcomes with 5-azacytidine or decitabine therapy.
Clinical Cancer Research 01/2013; · 7.74 Impact Factor
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Amir Mortazavi, Yonghua Ling,
Ludmila Katherine Martin,
Lai Wei,
Mitch A Phelps,
Zhongfa Liu,
Erica J Harper,
S Percy Ivy,
Xin Wu,
Bing-Sen Zhou,
Xiyong Liu,
Deidre Deam,
J Paul Monk,
William J Hicks,
Yun Yen,
Gregory A Otterson,
Michael R Grever,
Tanios Bekaii-Saab
[show abstract]
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ABSTRACT: Purpose Prolonged exposure of cancer cells to triapine, an inhibitor of ribonucleotide reductase, followed by gemcitabine enhances gemcitabine activity in vitro. Fixed-dose-rate gemcitabine (FDR-G) has improved efficacy compared to standard-dose. We conducted a phase I trial to determine the maximum tolerated dose (MTD), safety, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary efficacy of prolonged triapine infusion followed by FDR-G. Experimental Design Triapine was given as a 24-hour infusion, immediately followed by FDR-G (1000 mg/m(2) over 100-minute). Initially, this combination was administered days 1 and 8 of a 21-day cycle (Arm A, triapine starting dose 120 mg); but because of myelosuppression, it was changed to days 1 and 15 of a 28-day cycle (Arm B, starting dose of triapine 75 mg). Triapine steady-state concentrations (Css) and circulating ribonucleotide reductase M2-subunit (RRM2) were measured. Results Thirty-six patients were enrolled. The MTD was determined to be triapine 90 mg (24-hour infusion) immediately followed by gemcitabine 1000 mg/m(2) (100-minute infusion), every 2 weeks of a 4-week cycle. DLTs included grade 4 thrombocytopenia, leukopenia and neutropenia. The treatment was well tolerated with fatigue, nausea/vomiting, fever, transaminitis, and cytopenias being the most common toxicities. Among 30 evaluable patients, 1 had a partial response and 15 had stable disease. Triapine PK was similar, although more variable, compared to previous studies using doses normalized to body-surface-area. Steady decline in circulating levels of RRM2 may correlate with outcome. Conclusions This combination was well tolerated and showed evidence of preliminary activity in this heavily pretreated patient population, including prior gemcitabine failure.
Investigational New Drugs 07/2012; · 3.36 Impact Factor
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Donald Lavelle,
Kestis Vaitkus, Yonghua Ling,
Maria A Ruiz,
Reda Mahfouz,
Kwok Peng Ng,
Soledad Negrotto,
Nicola Smith,
Pramod Terse,
Kory J Engelke,
Joseph Covey,
Kenneth K Chan,
Joseph Desimone,
Yogen Saunthararajah
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ABSTRACT: The deoxycytidine analog decitabine (DAC) can deplete DNA methyl-transferase 1 (DNMT1) and thereby modify cellular epigenetics, gene expression, and differentiation. However, a barrier to efficacious and accessible DNMT1-targeted therapy is cytidine deaminase, an enzyme highly expressed in the intestine and liver that rapidly metabolizes DAC into inactive uridine counterparts, severely limiting exposure time and oral bioavailability. In the present study, the effects of tetrahydrouridine (THU), a competitive inhibitor of cytidine deaminase, on the pharmacokinetics and pharmacodynamics of oral DAC were evaluated in mice and nonhuman primates. Oral administration of THU before oral DAC extended DAC absorption time and widened the concentration-time profile, increasing the exposure time for S-phase-specific depletion of DNMT1 without the high peak DAC levels that can cause DNA damage and cytotoxicity. THU also decreased interindividual variability in pharmacokinetics seen with DAC alone. One potential clinical application of DNMT1-targeted therapy is to increase fetal hemoglobin and treat hemoglobinopathy. Oral THU-DAC at a dose that would produce peak DAC concentrations of less than 0.2μM administered 2×/wk for 8 weeks to nonhuman primates was not myelotoxic, hypomethylated DNA in the γ-globin gene promoter, and produced large cumulative increases in fetal hemoglobin. Combining oral THU with oral DAC changes DAC pharmacology in a manner that may facilitate accessible noncytotoxic DNMT1-targeted therapy.
Blood 12/2011; 119(5):1240-7. · 9.90 Impact Factor
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Susan R Mallery,
Deric E Budendorf,
Matthew P Larsen,
Ping Pei,
Meng Tong,
Andrew S Holpuch,
Peter E Larsen,
Gary D Stoner,
Henry W Fields,
Kenneth K Chan, Yonghua Ling,
Zhongfa Liu
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ABSTRACT: Our oral cancer chemoprevention trial data implied that patient-specific differences in local retention and metabolism of freeze-dried components of black raspberries (BRB) affected therapeutic responsiveness. Subsequent studies have confirmed that anthocyanins are key contributors to BRB's chemopreventive effects. Consequently, functional assays, immunoblotting, and immunohistochemical analyses to evaluate levels and distribution of BRB anthocyanin-relevant metabolic enzymes in human oral tissues were conducted. Liquid chromatography/tandem mass spectrometry (LC/MS-MS) analyses of time course saliva samples collected following BRB rinses were conducted to assess local pharmacokinetics and compare the capacities of three different BRB rinse formulations to provide sustained intraoral levels of anthocyanins. Protein profiles showed the presence of key metabolic enzymes in all 15 oral mucosal tissues evaluated, whereas immunohistochemistry confirmed these enzymes were distributed within surface oral epithelia and terminal salivary ducts. β-Glucosidase assays confirmed that whole and microflora-reduced saliva can deglycosylate BRB anthocyanins, enabling generation of the bioactive aglycone, cyanidin. LC/MS-MS analyses showed retention of parent anthocyanins and their functional, stable metabolite, protocatechuic acid, in saliva for up to 4 hours after rinsing. Furthermore, postrinse saliva samples contained glucuronidated anthocyanin conjugates, consistent with intracellular uptake and phase II conversion of BRB anthocyanins into forms amenable to local recycling. Our data show that comparable to the small intestine, the requisite hydrolytic, phase II and efflux transporting enzymes necessary for local enteric recycling are present and functional in human oral mucosa. Notably, interpatient differences in anthocyanin bioactivation and capacities for enteric recycling would impact treatment as retention of bioactivated chemopreventives at the target site would sustain therapeutic effectiveness.
Cancer Prevention Research 05/2011; 4(8):1209-21. · 4.91 Impact Factor
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U V R Vijaya Saradhi,
Sneha V Gupta,
Ming Chiu,
Jiang Wang, Yonghua Ling,
Zhongfa Liu,
David J Newman,
Joseph M Covey,
A Douglas Kinghorn,
Guido Marcucci,
David M Lucas,
Michael R Grever,
Mitch A Phelps,
Kenneth K Chan
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ABSTRACT: A sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of the plant natural product silvestrol in mice, using ansamitocin P-3 as the internal standard. The method was validated in plasma with a lower limit of quantification of 1 ng/mL, accuracy ranging from 87 to 114%, and precision (coefficient of variation) below 15%. The validated method was used to characterize pharmacokinetics in C57BL/6 mice and metabolism in mouse, human and rat plasma, and liver microsomes. Mice were dosed with silvestrol formulated in hydroxypropyl-β-cyclodextrin via intravenous, intraperitoneal, and oral routes followed by blood sampling up to 24 h. Intraperitoneal systemic availability was 100%, but oral administration resulted in only 1.7% bioavailability. Gradual degradation of silvestrol was observed in mouse and human plasma, with approximately 60% of the parent drug remaining after 6 h. In rat plasma, however, silvestrol was completely converted to silvestric acid (SA) within 10 min. Evaluation in microsomes provided further evidence that the main metabolite formed was SA, which subsequently showed no cytotoxic or cytostatic activity in a silvestrol-sensitive lymphoblastic cell line. The ability of the analytical assay to measure tissue levels of silvestrol was evaluated in liver, brain, kidney, and spleen. Results indicated the method was capable of accurately measuring tissue levels of silvestrol and suggested it has a relatively low distribution to brain. Together, these data suggest an overall favorable pharmacokinetic profile of silvestrol in mice and provide crucial information for its continued development toward potential clinical testing.
The AAPS Journal 04/2011; 13(3):347-56. · 5.09 Impact Factor
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ABSTRACT: Curcumin and tetrahydrocurcumin (THC) have been found as potent DNMT1 inhibitors, but they suffer from low oral bioavailability and rapid metabolism in vivo. To circumvent these problems, two curcumin analogs: 1,7-bis(3,4-dimethoxyphenyl)-4,4-dimethyl-1,6-heptadiene-3,5-dione (TMC) and 1,7-bis(3,4-dimethoxyphenyl)-4-cyclohexyl-1,6-heptadiene-3,5-dione (DMCHC) have been synthesized to enhance their stability by blocking the two metabolic sites, the phenolic and C4 methylene moieties. Both compounds have shown inhibitory activity on M. SssI similar to that of curcumin and THC (Poster, M1114, AAPS, 2009). Preclinical pharmacokinetics has yet to be performed. In this paper, a simple liquid chromatography-tandem mass spectrometric method was developed for the determination of these four curcuminoids in cell medium and mouse plasma. The method showed linearity from 1 to 1000 ng/mL with the lower limit of quantification of 1 ng/mL in cell medium, and 5 ng/mL in mouse plasma for all test curcuminoids. The within-day coefficients of variation were found to be below 15% and the accuracy was in the range of 85-115%. This method was subsequently used to evaluate their stability in these matrices and a pilot pharmacokinetics of curcumin, DMCHC and TMC in mice after an intraperitoneal (i.p.) cassette dosing of 10mg/kg each. Curcuminoids degraded in two phases with terminal half lives of 186, 813, 724, and 2000 min for curcumin, THC, TMC, and DMCHC, respectively, in cell culture medium. In plasma, their respective half lives were 111, 232, 1202 and 3000 min. These data demonstrated that their stability is in the order curcumin<THC<TMC<DMCHC in both matrices. Following an i.p. cassette dose, both TMC and DMCHC showed the prolonged elimination half life (1.0, 1.0 h, respectively vs 0.4h for curcumin) and an increased drug exposure as described by the area under the curve (0.64, 0.98 μM h, respectively vs 0.4 μM h for curcumin).
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 09/2010; 878(30):3045-51. · 2.78 Impact Factor
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ABSTRACT: A highly sensitive and specific LC-MS/MS assay was developed and validated to quantify nevirapine (NVP) and its five metabolites [2-, 3-, 8-, 12-hydroxyl NVP (OHNVP) and 4-carboxyl NVP (CANVP)] simultaneously in baboon serum and the assay was used to characterize their pharmacokinetic studies of an oral-dose escalation study in baboon. The lower limit of quantification (LLOQ) for NVP and its four hydroxyl nevirapine metabolites was 1.0 ng/mL and for 4-CANVP was 5.0 ng/mL. The between-run and within-run precisions and accuracies at four quality control concentrations (1, 5, 50 and 500 ng/mL) were evaluated in baboon serum with less than 14% variation and 93-114% accuracies (n = 6), except for the LLOQ for 2-OHNVP, which had an accuracy of 115.8% for between-run validation. The pharmacokinetics of NVP and its five metabolites in non-pregnant baboons by a single-dose escalation study were also profiled. The major metabolites detected were 4-CANVP and 12-OHNVP. 3-OHNVP and 2-OHNVP were the minor metabolites with only a trace amount of 2-OHNVP detected in some pharmacokinetic samples. No 8-OHNVP was observed in all of the pharmacokinetic samples. In addition, the fragmentation for the four hydroxyl metabolite isomers is also discussed.
Biomedical Chromatography 11/2009; 24(7):717-26. · 1.97 Impact Factor
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ABSTRACT: Cyanidin 3-glucoside (C3GLU), cyanidin 3-rutinoside (C3RUT), cyanidin 3-sambubioside (C3SAM) and cyanidin 3-(2(G)-xylosyl) rutinoside (C3XRUT) are the four constituent black raspberry anthocyanins that contribute significantly to the chemopreventive effects of freeze-dried black raspberries (FBR). A highly sensitive and specific LC-MS/MS assay was developed and validated to simultaneously quantify these four FBR anthocyanins in human saliva, plasma and oral tissue homogenates. In saliva, the lower limit of quantification (LLOQ) for these anthocyanins was 1.0 ng/mL. The within-run and between-run coefficients of variations (CVs) at the quality control concentrations (1.0, 5.0, 50 and 500 ng/mL) were all <12%, except for C3SAM and C3RUT at the LLOQ, which showed a within-run CV of 18.3% and between-run CV of 16.0%, respectively. The accuracy values ranged from 87.5 to 110%. In plasma, the LLOQ for C3GLU and C3RUT was 1.0 ng/mL and for C3SAM 5.0 ng/mL. The CVs at the above concentrations were <15%, except for C3GLU at the LLOQ, which showed the between-run CV of 16.9%. The accuracy values ranged from 90.7% to 112.7% except for C3GLU at the LLOQ, which showed 119.3%. In tissue homogenates, the LLOQ for C3GLU and C3RUT was 2.0 ng/mL, and C3SAM 5.0 ng/mL. The CVs and accuracy values at concentrations (2.0, 5.0, 50 and 500 ng/mL) were similar to those in human plasma. This assay was subsequently used in a pilot pharmacology study to evaluate the effects of topical application of a 10% (w/w) FBR bioadhesive gel to selected mucosal sites in the posterior mandibular gingiva. Measurable saliva and tissue levels of the FBR anthocyanins confirmed that gel-delivered anthocyanins are readily distributed to saliva and easily penetrate human oral mucosa.
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 10/2009; 877(31):4027-34. · 2.78 Impact Factor
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ABSTRACT: Smaller members of water-soluble sulfonated calixarenes have been extensively explored in the context of host-guest complexation, supramolecular chemistry, and potential sensors. However, larger members especially eight-membered calixarene (CA[8]) has received much less attention because of its floppy nature and tendency to exist as a mixture of conformational isomers. Our continued interest in identifying molecules with an internal cavity as reaction vessels has led us to examine the host-guest complexation of CA[8] with photoactive bispyridyl ethylenes. We find that 4,4'-bispyridyl ethylene and 3,3'-bispyridyl ethylene upon complexation to CA[8] arrest the conformational equilibrium and force the latter to adopt a single conformation in solution. During complexation, bispyridyl ethylenes are protonated by the sulfonic acid groups of CA[8]. The host-guest complex is stabilized via an electrostatic interaction between the cationic bispyridyl ethylenes and anionic sulfonated calix[8]arene, and we propose the complex to have an inverted capsular structure. This model is also consistent with the electrochemical behavior of 4,4'-dimethylviologen included within CA[8]. Rigidification of bispyridyl ethylenes by the host has a consequence on the excited-state chemistry of the former. Generally, prevalent geometric isomerization of bispyridyl ethylenes are prevented by CA[8] upon complexation.
Langmuir 09/2009; 25(16):8982-92. · 4.19 Impact Factor
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ABSTRACT: The dicationic guest 2,7-dimethyldiazaphenanthrenium forms a fluorescent inclusion complex with the cucurbit[8]uril host, which can be used to effectively bind and detect indole derivatives, such as serotonin and tryptophan.
Chemical Communications 03/2007; · 6.17 Impact Factor
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ABSTRACT: The binding interactions in aqueous solution between the dicationic guest diquat (DQ(2+)) and the cucurbit[7]uril (CB7) and cucurbit[8]uril (CB8) hosts were investigated by (1)H NMR, UV/Vis, and fluorescence spectroscopy; mass spectrometry; single-crystal X-ray diffraction; and electrochemical techniques. The binding data were compared with previously reported results for the related paraquat guest (PQ(2+)). DQ(2+) was found to bind poorly (K=350 m(-1)) inside CB7 and more effectively (K=4.8 x 10(4) m(-1)) inside CB8. One-electron reduction led to increased binding affinity with both hosts (K(r)=1 x 10(4) m(-1) with CB7 and K(r)=6 x 10(5) m(-1) for CB8). While (1)H NMR spectroscopic data revealed that DQ(2+) is not fully included by CB7, the crystal structure of the CB8DQ(2+) complex-obtained from single-crystal X-ray diffraction-clearly establishes its inclusion nature. Overall, both diquat and its one-electron reduced radical cation are bound more effectively by CB8 than by CB7. In contrast to this, paraquat exhibits selectivity for CB7, but its radical cation forms a highly stable dimer inside CB8. These differences highlight the pronounced sensitivity of cucurbit[n]uril hosts to guest features such as charge, charge distribution and shape.
Chemistry 02/2007; 13(28):7908-14. · 5.93 Impact Factor
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ABSTRACT: The formation of highly stable inclusion complexes between the host cucurbit [7]uril (CB7) and the dicationic guests bis(diethylsulfonium)-p-xylylene (12+) and bis(tetrahydrothiophenium)-p-xylylene (22+) was demonstrated by mass spectrometric, NMR and UV−vis spectroscopic data. Although the inclusion complexes did not undergo Wessling polymerization, the monomers can be pre-polymerized to the polyelectrolyte stage and subsequently exposed to CB7 in aqueous solution. The CB7-treated polyelectrolyte developed conjugation more readily and at much lower temperatures than the untreated polyelectrolyte to yield poly(phenylenevinylene) (PPV). The formation of external complexes between CB7 and the cationic branches of the polyelectrolyte favored the cleavage of the branches. CB7 was also found to form a stable complex with diethyl sulfide (Et2S), the product of the elimination reaction that converts the polyelectrolyte to PPV. In contrast, the smaller analogue, the cucurbit [6]uril host (CB6) did not have any effect on the polyelectrolyte-to-PPV conversion. The CB7-treated polyelectrolyte showed enhanced luminescence both in solution and in precursor polymer films. Et2S@CB7 inclusion complexes located around the polymer chains seem to form a hydrophobic shell, which effectively protects the PPV chains from quenchers, such as vapor-phase dinitrotoluene molecules.
11/2006;
