In situ generation of a Zwitterionic fluorescent probe for detection of human serum albumin protein.

In this article, a new approach for human serum albumin selective fluorophore design has been reported. The fluorophore reported here comprises a substituted phenol donor and a cationic benzo[e]indolium acceptor connected with a π bond. Originally, the cationic fluorophore did not bind with human serum albumin. Upon deprotonation of the phenolic-OH by a water molecule the cationic form was transformed into an active zwitterionic form. Spectroscopic studies and theoretical calculations revealed that the new active form remained in a zwitterionic state in neutral aqueous solution, and it formed a strong supramolecular complex with human serum albumin. The spontaneous complexation resulted multi-fold increase of fluorescence intensity which increased linearly with the concentrations of the protein, thus giving an analytical tool to monitor human serum albumin in aqueous samples. We believe, this simple strategy applied on appropriate fluorogenic scaffolds would prove useful to develop new and improved turn-on fluorescent probes for pH regulated biological applications.

Evaluating the Merit of a Syringol Derived Fluorophore as a Charge Transfer Probe for Detection of Serum Albumins.

In this article a syringol-π-benz[e]indolium based donor-acceptor fluorophore has been reported. The fluorophore shows a solvent polarity dependent change in the absorption and emission spectra in solution. A combined spectroscopic and time dependent density functional theory (TDDFT) studies reveal higher dipole moment of the fluorophore in the excited state, resulting positive solvatochromism. In physiological pH, the phenol group in the fluorophore is easily deprotonated owing to the electron pulling effect of the substituents. Consequently, the phenolate (PhO-) becomes a strong active donor in the new donor-acceptor pair. In aqueous solution, the new phenolate fluorochrome shows negligible fluorescence due to energy loss via non-radiative pathways from the low-lying polar excited states. The fluorochrome can detect human and bovine serum albumins in physiological buffer solution with high selectivity. The underlying mechanism of human serum albumin (HSA) detection was estimated to be strong (1.46 × 105 M-1, ΔG = -7.05 kcal/mol) supramolecular complexation between the fluorophore and albumin in hydrophobic binding site III-B. The linear relationship between fluorescence intensity and HSA concentration extends from 40 mg/L to an impressive upper limit (540 mg/L), thereby opening an opportunity for albumin detection in a broad range of health conditions. The practical applicability of the fluorophore was tested in spiked urine samples and a good correlation was observed between fluorescence intensity and the concentration of human serum albumin in neutral aqueous samples.

A Singlet Oxygen Priming Mechanism: Disentangling of Photooxidative and Downstream Dark Effects.

Airborne singlet oxygen obtained from photosensitization of triplet dioxygen is shown to react with an alkene surfactant (8-methylnon-7-ene-1 sulfonate) leading to "ene" hydroperoxides that in the dark inactivate planktonic Escherichia coli (E. coli). The "ene" hydroperoxide photoproducts are not toxic on their own, but they become toxic after the bacteria are pretreated with singlet oxygen. The total quenching rate constant (kT) of singlet oxygen of the alkene surfactant was measured to be 1.1 × 106 M-1 s-1 at the air/liquid interface. Through a new mechanism called singlet oxygen priming (SOP), the singlet oxygen leads to hydroperoxides then to peroxyl radicals, tetraoxides, and decomposition products, which also promote disinfection, and therefore offer a "one-two" punch. This offers a strong secondary toxic effect in an otherwise indiscernible dark reaction. The results provide an insight into assisted killing by an exogenous alkene with dark toxicity effects following exposure to singlet oxygen.

Dual luminescent charge transfer probe for quantitative detection of serum albumin in aqueous samples.

In diagnostic medicine serum albumin is considered as an important biomarker for assessment of cardiovascular functions and diagnosis of renal diseases. Herein, we report a novel donor-π-π-acceptor fluorophore for selective detection of serum albumin in urine samples. In our design, a phenolic donor was conjugated with a tricyanofuran (TCF) acceptor through a dimethine bridge via a simple condensation reaction. The stereoelectronic effects of the incorporated methoxy (-OCH3) groups and the TCF moiety-in conjunction with the extended π-electron conjugation-led to dual red and NIR-I absorption/emission in water. Moreover, due to superior electron transfer between a phenolate donor and the TCF acceptor and the subsequent energy decay from the charge transfer states, the fluorophore displayed negligible fluorescence emission in water and other polar solvents. Consequently, we have been able to utilize the fluorophore for quantitative estimation of serum albumin both in the red (<700 nm) and NIR-I (700-900 nm) regions of the electromagnetic spectrum with excellent reproducibility. The fluorophore selectively recognized human serum albumin over other proteins and enzymes with a limit of detection of 10 mg/L and 20 mg/L in simulated urine samples at red and NIR-I emission window of the spectrum, respectively. By molecular docking analysis and experimental displacement assays, we have shown that the selective response of the fluorophore toward human serum albumin is due to tighter supramolecular complexation between the fluorophore and the protein at subdomain IB, and the origin of the NIR-I (780 nm) emission was attributed to a twisted conformer of phenolate-π-π-TCF system in aqueous solution. These findings indicate that the fluorophore could be utilized for quantitative detection of human serum albumin in urine samples for clinical diagnosis of albuminuria.

Selective Detection of Human Serum Albumin by Near Infrared Emissive Fluorophores: Insights into Structure-property Relationship.

Two donor-acceptor fluorophores were prepared and tested for quantitative determination of HSA in aqueous samples. Fluorophores were non-emissive in polar solvents due to energy loss via non-radiative decays. Complexation of the fluorophores with HSA resulted multi-fold enhancement of emission in the red-near infrared (NIR) region. The emission intensity was linearly correlated to the amount of protein in the solution, which enabled us to develop calibration graphs for quantitative estimation of HSA in synthetic urine samples. Between the two fluorophores, the methoxy substituted fluorophore 1 selectively recognized HSA. It exhibited remarkable fluorescence enhancement with HSA over bovine serum albumin (BSA) and other globular proteins. The selective sensing aptitude of 1 was attributed to its restricted motions in the protein's microenvironment due to multiple non-covalent interactions, preventing energy loss by radiationless decay. The different recognition properties of the fluorophores were estimated by the steady-state fluorescence and molecular docking studies. These findings indicate that this class of fluorophores can be useful for quantitative estimation of HSA in biological urine and blood samples in clinical practice.

Multiplex PCR from Menstrual Blood: A Non-Invasive Cost-Effective Approach to Reduce Diagnostic Dilemma for Genital Tuberculosis.

AIM: Genital tuberculosis (GTB) is a potent contributor to irreversible damage to the reproductive system and infertility in females. As no gold standard diagnostic tool is yet available, clinical suspicion and relatively insensitive approaches such as histopathology, laparoscopy and hysterosalpingogram are currently critical determinants in the diagnosis of GTB. Although a polymerase chain reaction (PCR)-based assay using endometrial tissue seems promising, sampling does require an invasive procedure. OBJECTIVE: We hypothesized that menstrual blood may provide an alternate non-invasive source of samples for PCR-based GTB diagnosis. METHODS: We enrolled 195 women with primary infertility in whom GTB was suspected. We obtained ethics committee approval from our institution and written informed consent from subjects. Endometrial tissue and menstrual blood was collected from the subjects and culture, histopathology, and multiplex PCR with both sample type was performed for each subject. RESULTS: The sensitivity and specificity of multiplex PCR was, respectively, 90.2 and 86.1% for menstrual blood, 95.8 and 84.3% for endometrial tissue, and 64.8 and 93.2% for histopathology staining. CONCLUSIONS: A strong clinical suspicion aided with multiplex PCR using menstrual blood may significantly reduce the diagnostic dilemma for GTB diagnosis in a non-invasive, sensitive, rapid, and cost-effective manner.

Ultrafast Electron Transfer across a Nanocapsular Wall: Coumarins as Donors, Viologen as Acceptor, and Octa Acid Capsule as the Mediator.

Results of our study on ultrafast electron transfer (eT) dynamics from coumarins (coumarin-1, coumarin-480, and coumarin-153) incarcerated within octa acid (OA) capsules as electron donors to methyl viologen dissolved in water as acceptor are presented. Upon photoexcitation, coumarin inside the OA capsule transfers an electron to the acceptor electrostatically attached to the capsule leading to a long-lived radical-ion pair separated by the OA capsular wall. This charge-separated state returns to the neutral ground state via back electron transfer on the nanosecond time scale. This system allows for ultrafast electron transfer processes through a molecular wall from the apolar capsular interior to the highly polar (aqueous) environment on the femtosecond time scale. Employing femtosecond transient absorption spectroscopy, distinct rates of both forward (1-25 ps) and backward eT (700-1200 ps) processes were measured. Further understanding of the energetics is provided using Rehm-Weller analysis for the investigated photoinduced eT reactions. The results provide the rates of the eT across a molecular wall, akin to an isotropic solution, depending on the standard free energy of the reaction. The insights from this work could be utilized in the future design of efficient electron transfer processes across interfaces separating apolar and polar environments.

Air-Water Interface Effects on the Regioselectivity of Singlet Oxygenations of a Trisubstituted Alkene.

The regioselective synthesis of allylic hydroperoxide sulfonates by singlet oxygenation at the air-water interface has been found to depend on the concentration of the alkene sulfonate and added calcium salt. The regioselectivity is proposed to originate from an orthogonal alkene relative to the water surface for preferential methyl hydrogen abstraction by airborne singlet oxygen in an ene reaction. The findings hint that the air-water interface is a locale for synthetic reactions.

Photoisomerization and photooxygenation of 1,4-diaryl-1,3-dienes in a confined space.

Geometric isomerization of light-activated olefins plays a significant role in biological events as well as in modern materials science applications. In these systems, the isomerization occurs in highly confined spaces, and concepts derived from solution investigations are only partially applicable. This study makes contributions in understanding the excited-state behavior of olefins in confined spaces by investigating the excited-state behavior of 1,4-diphneyl-13-butadiene (DPB) and 1,4-ditolyl-1,3-butadiene (DTB) encapsulated in a well-defined organic capsule made up of the octa acid (OA) host. Both of these dienes that exist in three isomeric forms (trans,trans; trans,cis; and cis,cis) formed 1:2 guest-host complexes with OA in aqueous borate buffer. Competition experiments monitored by (1)H NMR signals revealed that among the three isomers the cis,cis isomer of DPB and DTB formed the most stable complex with OA. Molecular modeling studies suggested that all six isomers of DPB and DTB preferred the cisoid conformation within the OA capsule. Irradiation (>280 nm) of the diene-OA complex (diene@OA2) resulted in geometric isomerization, and the photostationary state consisted of cis,trans isomer as major and cis,cis as minor products. The photostationary state could be enriched with the cis,cis isomer in yields close to 70% with proper cutoff filters because the cis,cis isomer absorbs at shorter wavelength than the other two isomers. Consistent with the MD simulation prediction that trans,trans-DPB and trans,trans-DTB existed in cisoid conformation within OA capsule, the generation of singlet oxygen in the presence of OA encapsulated DPB or DTB resulted in facile [4 + 2] addition between the diene and the singlet oxygen.

Synthetic versus natural receptors: supramolecular control of chemical sensing in fish.

The encapsulation of odorants by the synthetic receptor cucurbit[7]uril (CB[7]) reduces the response of olfactory receptors in Mozambique tilapia (Oreochromis mossambicus) in vivo. For example, the olfactory receptor response to the odorant adamantan-1-amine, as measured by electro-olfactography, was suppressed by 92% in the presence of CB[7]. A reduction in olfactory response of 88% was observed for pentane-1,5-diamine (cadaverine), an odorant associated with carrion avoidance in some fish. The results reveal how the association constants and the concentrations of natural and synthetic receptors play a determinant role and show that synthetic receptors can be used to remove bioactive molecules from fish olfaction.

Synergism between airborne singlet oxygen and a trisubstituted olefin sulfonate for the inactivation of bacteria.

The reactivity of a trisubstituted alkene surfactant (8-methylnon-7-ene-1 sulfonate, 1) to airborne singlet oxygen in a solution containing E. coli was examined. Surfactant 1 was prepared by a Strecker-type reaction of 9-bromo-2-methylnon-2-ene with sodium sulfite. Submicellar concentrations of 1 were used that reacted with singlet oxygen by an "ene" reaction to yield two hydroperoxides (7-hydroperoxy-8-methylnon-8-ene-1 sulfonate and (E)-8-hydroperoxy-8-methylnon-6-ene-1 sulfonate) in a 4:1 ratio. Exchanging the H2O solution for D2O where the lifetime of solution-phase singlet oxygen increases by 20-fold led to an ∼2-fold increase in the yield of hydroperoxides pointing to surface activity of singlet oxygen with the surfactant in a partially solvated state. In this airborne singlet oxygen reaction, E. coli inactivation was monitored in the presence and absence of 1 and by a LIVE/DEAD cell permeabilization assay. It was shown that the surfactant has low dark toxicity with respect to the bacteria, but in the presence of airborne singlet oxygen, it produces a synergistic enhancement of the bacterial inactivation. How the ene-derived surfactant hydroperoxides can provoke (1)O2 toxicity and be of general utility is discussed.

Deep-cavity cavitand octa acid as a hydrogen donor: photofunctionalization with nitrenes generated from azidoadamantanes.

1-azidoadamantane and 2-azidoadamantane form a 1:1 complex with hosts octa acid (OA) and cucurbit[7]uril (CB7) in water. Isothermal titration calorimetric measurements suggest these complexes to be very stable in aqueous solution. The complexes have been characterized by (1)H NMR in solution and by ESI-MS in gas phase. In both phases, the complexes are stable. Irradiation of these complexes (λ > 280 nm) results in nitrenes via the loss of nitrogen from the guest azidoadamantanes. The behavior of nitrenes within OA differs from that in solution. Nitrenes included within octa acid attack one of the four tertiary benzylic hydrogens present at the lower interior part of OA. While in solution intramolecular insertion is preferred, within OA intermolecular C-H insertion seems to be the choice. When azidoadamantanes included in CB7 were irradiated (λ > 280 nm) the same products as in solution resulted but the host held them tightly. Displacement of the product required the use of a higher binding guest. In this case, no intermolecular C-H insertion occurred. Difference in reactivity between OA and CB7 is the result of the location of hydrogens; in OA they are in the interior of the cavity where the nitrene is generated, and in CB7 they are at the exterior. Reactivity of nitrenes within OA is different from that of carbenes that do not react with the host.

Hydrocarbons depending on the chain length and head group adopt different conformations within a water-soluble nanocapsule: 1H NMR and molecular dynamics studies.

In this study we have examined the conformational preference of phenyl-substituted hydrocarbons (alkanes, alkenes, and alkynes) of different chain lengths included within a confined space provided by a molecular capsule made of two host cavitands known by the trivial name "octa acid" (OA). One- and two-dimensional (1)H NMR experiments and molecular dynamics (MD) simulations were employed to probe the location and conformation of hydrocarbons within the OA capsule. In general, small hydrocarbons adopted a linear conformation while longer ones preferred a folded conformation. In addition, the extent of folding and the location of the end groups (methyl and phenyl) were dependent on the group (H(2)C-CH(2), HC═CH, and C≡C) adjacent to the phenyl group. In addition, the rotational mobility of the hydrocarbons within the capsule varied; for example, while phenylated alkanes tumbled freely, phenylated alkenes and alkynes resisted such a motion at room temperature. Combined NMR and MD simulation studies have confirmed that molecules could adopt conformations within confined spaces different from that in solution, opening opportunities to modulate chemical behavior of guest molecules.

Gold nanoparticles functionalized with deep-cavity cavitands: synthesis, characterization, and photophysical studies.

In this report, we present methods of functionalization of AuNP's with deep-cavity cavitands that can include organic molecules. Two types of deep-cavity cavitand-functionalized AuNP's have been synthesized and characterized, one soluble in organic solvents and the other in water. Functionalized AuNP soluble in organic solvents forms a 1:1 host-guest complex where the guest is exposed to the exterior solvents. The one soluble in water forms a 2:1 host-guest complex where the guest is protected from solvent water. Phosphorescence from thiones and benzil included within heterocapsules attached to AuNP was quenched by gold atoms present closer to the guests included within deep-cavity cavitands. During this investigation, we have synthesized four new deep-cavity cavitands. Of these, two thiol-functionalized hosts allowed us to make stable AuNP's. However, AuNP's protected with two amine-functionalized cavitands tended to aggregate within a day.

Cucurbituril adamantanediazirine complexes and consequential carbene chemistry.

Adamantanediazirines, precursors of adamantanylidenes, form 1:1 complexes (guest to host) with cucurbit[7]uril and cucurbit[8]uril and a 3:1 complex with a Pd nanocage in water. (1)H NMR spectra suggested that these complexes are stable in water on the NMR time scale. While photolysis of adamantanediazirines in water gave mainly adamantanone and adamantanol via adamantanylidene as intermediate, the 1:1 complexes of adamantanediazirine with cucurbiturils gave intramolecular C-H insertion products of adamantanylidene in >90% yield. The study establishes that significant control of carbene reactivity can be achieved when the precursor is encapsulated within a tight inert cavity. While the general characteristics of molecular containers can be understood on the basis of concepts such as "confinement" and "weak interactions", each one is unique and deserves careful scrutiny.

Photochemical generation and reactivity of carbenes within an organic cavitand and capsule: photochemistry of adamantanediazirines.

Chemical behavior of carbenes (adamantylidenes) generated by photolysis of adamantanediazirines has been investigated while they were incarcerated within an organic container in water and on silica surfaces. Confined carbenes behave differently from the free ones, and their behavior is dictated by the nature and the structure of the host-guest complexes. The substituent present on the adamantyl skeleton controls the stoichiometry (1:1 or 2:2) and the orientation of guest molecules within the host.

Restricted rotation due to the lack of free space within a capsule translates into product selectivity: photochemistry of cyclohexyl phenyl ketones within a water-soluble organic capsule.

The rotational mobility of organic guest molecules when included within a confined capsule is restricted and this feature could be translated into product selectivity as established with the photochemical behavior of cyclohexyl phenyl ketones.

Guest rotations within a capsuleplex probed by NMR and EPR techniques.

With the help of (1)H NMR and EPR techniques, we have probed the dynamics of guest molecules included within a water-soluble deep cavity cavitand known by the trivial name octa acid. All guest molecules investigated here form 2:1 (host/guest) complexes in water, and two host molecules encapsulate the guest molecule by forming a closed capsule. We have probed the dynamics of the guest molecule within this closed container through (1)H NMR and EPR techniques. The timescales offered by these two techniques are quite different, millisecond and nanosecond, respectively. For EPR studies, paramagnetic nitroxide guest molecules and for (1)H NMR studies, a wide variety of structurally diverse neutral organic guest molecules were employed. The guest molecules freely rotate along their x axis (long molecular axis and magnetic axis) on the NMR timescale; however, their rotation is slowed with respect to that in water on the EPR timescale. Rotation along the x axis is dependent on the length of the alkyl chain attached to the nitroxide probe. Overall rotation along the y or z axis was very much dependent on the structure of the guest molecule. The guests investigated could be classified into three groups: (a) those that do not rotate along the y or z axis both at room and elevated (55 degrees C) temperatures, (b) those that rotate freely at room temperature, and (c) those that do not rotate at room temperature but do so at higher temperatures. One should note that rotation here refers to the NMR timescale and it is quite possible that all molecules may rotate at much longer timescales than the one probed here. A slight variation in structure alters the rotational mobility of the guest molecules.