Second Generation G-Quadruplex Stabilizing Trimethine Cyanines.

G-Quadruplex DNA has been recognized as a highly appealing target for the development of new selective chemotherapeutics, which could result in markedly reduced toxicity toward normal cells. In particular, the cyanine dyes that bind selectively to G-quadruplex structures without targeting duplex DNA have attracted attention due to their high amenability to structural modifications that allows fine-tuning of their biomolecular interactions. We have previously reported pentamethine and symmetric trimethine cyanines designed to effectively bind G-quadruplexes through end stacking interactions. Herein, we are reporting a second generation of drug candidates, the asymmetric trimethine cyanines. These have been synthesized and evaluated for their quadruplex binding properties. Incorporating a benz[c,d]indolenine heterocyclic unit increased overall quadruplex binding, and elongating the alkyl length increases the quadruplex-to-duplex binding specificity.

Tissue-Specific Near-Infrared Fluorescence Imaging.

Near-infrared (NIR) fluorescence light has been widely utilized in clinical imaging by providing surgeons highly specific images of target tissue. The "NIR window" from 650 to 900 nm is especially useful due to several special features such as minimal autofluorescence and absorption of biomolecules in tissue, as well as low light scattering. Compared with visible wavelengths, NIR fluorescence light is invisible, thus allowing highly sensitivity real-time image guidance in human surgery without changing the surgical field. The benefit of using NIR fluorescence light as a clinical imaging technology can be attributed to its molecular fluorescence as an exogenous contrast agent. Indeed, whole body preoperative imaging of single-photon emission computed tomography (SPECT) and positron emission tomography (PET) remains important in diagnostic utility, but they lack the efficacy of innocuous and targeted NIR fluorophores to simultaneously facilitate the real-time delineation of diseased tissue while preserving vital tissues. Admittedly, NIR imaging technology has been slow to enter clinical use mostly due to the late-coming development of truly breakthrough contrast agents for use with current imaging systems. Therefore, clearly defining the physical margins of tumorous tissue remains of paramount importance in bioimaging and targeted therapy. An equally noteworthy yet less researched goal is the ability to outline healthy vital tissues that should be carefully navigated without transection during the intraoperative surgery. Both of these paths require optimizing a gauntlet of design considerations to obtain not only an effective imaging agent in the NIR window but also high molecular brightness, water solubility, biocompatibility, and tissue-specific targetability. The imaging community recognizes three strategic approaches which include (1) passive targeting via the EPR effect, (2) active targeting using the innate overall biodistribution of known molecules, and (3) activatable targeting through an internal stimulus, which turns on fluorescence from an off state. Recent advances in nanomedicine and bioimaging offer much needed promise toward fulfilling these stringent requirements as we develop a successful catalog of targeted contrast agents for illuminating both tumors and vital tissues in the same surgical space by employing spectrally distinct fluorophores in real time. These tissue-specific contrast agents can be versatile arsenals to physicians for real-time intraoperative navigation as well as image-guided targeted therapy. There is a versatile library of tissue-specific fluorophores available in the literature, with many discussed herein, which offers clinicians an array of possibilities that will undoubtedly improve intraoperative success and long-term postoperation prognosis.

Full-length nucleotide sequence of ERMAP alleles encoding Scianna (SC) antigens.

BACKGROUND: Scianna (SC) blood group system comprises two antithetical antigens, Sc1 and Sc2, and five additional antigens. The antigens reside on a glycoprotein encoded by the erythroblast membrane-associated protein (ERMAP) gene. For the common ERMAP alleles, we determined the full-length nucleotide sequence that encodes the Scianna glycoprotein. STUDY DESIGN AND METHODS: Blood donor samples from five populations were analyzed including 20 African Americans, 10 Caucasians, 10 Thai, five Asians, and five Hispanics for a total of 100 chromosomes. An assay was devised to determine the genomic sequence of the ERMAP gene in one amplicon, spanning 21.4 kb and covering Exons 2 to 12 and the intervening sequence (IVS). All alleles (confirmed haplotypes) were resolved without ambiguity. RESULTS: Among 50 blood donors, we found 80 single-nucleotide polymorphisms (SNPs), including six novel SNPs, in 21,308 nucleotides covering the coding sequence of the ERMAP gene and including the introns. The noncoding sequences harbored 75 SNPs (68 in the introns and seven in the 3'-UTR). No SNP indicative of a nonfunctional allele was detected. The nucleotide sequences for 48 ERMAP alleles (confirmed haplotypes) were determined by allele-specific polymerase chain reaction and sequencing in 100 chromosomes. CONCLUSIONS: We documented 48 ERMAP alleles of 21,308 nucleotides each. The two nucleotide sequences available in GenBank for ERMAP alleles of similar length have not been found in our 100 chromosomes. Alleles determined without ambiguity can be used as templates to analyze next generation sequencing data, which will enhance the reliability in clinical diagnostics.

Cartilage-Specific Near-Infrared Fluorophores for Biomedical Imaging.

A novel class of near-infrared fluorescent contrast agents was developed. These agents target cartilage with high specificity and this property is inherent to the chemical structure of the fluorophore. After a single low-dose intravenous injection and a clearance time of approximately 4 h, these agents bind to all three major types of cartilage (hyaline, elastic, and fibrocartilage) and perform equally well across species. Analysis of the chemical structure similarities revealed a potential pharmacophore for cartilage targeting. Our results lay the foundation for future improvements in tissue engineering, joint surgery, and cartilage-specific drug development.

Hydroxylated near-infrared BODIPY fluorophores as intracellular pH sensors.

In this study, a series of new, highly sensitive BF2-chelated tetraarylazadipyrromethane dyes are synthesized and analyzed to be suitable as on/off photo-induced electron transfer modulated fluorescent sensors for determination of intracellular pH. The ethanolic solutions of the new indicators feature absorption maxima in the range of 696-700 nm and a fluorescence emission maximum at 720 nm. Molar absorptivity and fluorescence quantum yield data were determined for the studied set of aza-BODIPY indicators. These indicators have high molar absorption coefficients of ∼80,000 M(-1) cm(-1) and quantum yields (up to 18%). Corresponding pKa values of indicators are determined from absorbance and fluorescence measurements and range from 9.1 to 10.8, depending on the selective positioning of electron-donating functionalities. The excellent photostability of the aza-BODIPY indicators makes them particularly suitable for long duration measurements. The in vitro cellular staining of living tissues in PC3 cells based on the isosbestic point at pH 7.8 and pH 9.3 has been employed which shows an increase in fluorescence intensity at 800 nm with increase in pH for certain compounds and fluorescence intensity decreases at 700 nm. Therefore, the new indicators are suitable for exploitation and adaptation in a diverse range of analytical applications.

Oxidative cleavage of DNA by pentamethine carbocyanine dyes irradiated with long-wavelength visible light.

Here we report the synthesis of seven symmetrical carbocyanine dyes in which two nitrogen-substituted benz[e]indolium rings are joined by a pentamethine bridge that is meso-substituted with chlorine or bromine versus hydrogen. The heteroatom of benz[e]indolium is modified with a phenylpropyl, methyl, or cationic quaternary ammonium group. In reactions containing micro molar concentrations of halogenated dye, irradiation at 575, 588, 623, or 700nm produces good photocleavage of plasmid DNA. UV-visible spectra show that the carbocyanines are in their H-aggregated and monomeric forms. Scavenger experiments point to the involvement of singlet oxygen and hydroxyl radicals in DNA photocleavage.

Selective G-quadruplex DNA recognition by a new class of designed cyanines.

A variety of cyanines provide versatile and sensitive agents acting as DNA stains and sensors and have been structurally modified to bind in the DNA minor groove in a sequence dependent manner. Similarly, we are developing a new set of cyanines that have been designed to achieve highly selective binding to DNA G-quadruplexes with much weaker binding to DNA duplexes. A systematic set of structurally analogous trimethine cyanines has been synthesized and evaluated for quadruplex targeting. The results reveal that elevated quadruplex binding and specificity are highly sensitive to the polymethine chain length, heterocyclic structure and intrinsic charge of the compound. Biophysical experiments show that the compounds display significant selectivity for quadruplex binding with a higher preference for parallel stranded quadruplexes, such as cMYC. NMR studies revealed the primary binding through an end-stacking mode and SPR studies showed the strongest compounds have primary KD values below 100 nM that are nearly 100-fold weaker for duplexes. The high selectivity of these newly designed trimethine cyanines for quadruplexes as well as their ability to discriminate between different quadruplexes are extremely promising features to develop them as novel probes for targeting quadruplexes in vivo.

Comparison of echocardiographic measurements and cardiac biomarkers in healthy dogs eating nontraditional or traditional diets.

BACKGROUND: There has been a recent association between nontraditional diets and development of diet-associated dilated cardiomyopathy (DCM) in dogs. HYPOTHESIS/OBJECTIVES: To compare echocardiographic measurements and cardiac biomarkers between healthy dogs eating nontraditional vs traditional diets. We hypothesized that dogs eating nontraditional diets would have lower measures of systolic myocardial performance compared to dogs eating traditional diets. ANIMALS: Forty-six healthy dogs: 23 eating nontraditional diets and 23 eating traditional diets. METHODS: Prospective, cross-sectional study. Dogs were divided into groups based on diet ingredients. Dogs underwent 2-dimensional (2D), 3-dimensional (3D), and Doppler echocardiographic examinations and analysis of plasma N-terminal prohormone of B-type natriuretic peptide, serum cardiac troponin I, and whole blood and plasma taurine concentrations. RESULTS: Mean 2D ejection fraction (EF) was lower for dogs eating nontraditional diets (48.65 ± 7.42%) vs dogs eating traditional diets (56.65 ± 4.63%; P < .001; mean difference 8.0% [4.0%-12.0%] 95% confidence interval [CI]). Mean 3D EF was lower for dogs eating nontraditional diets (45.38 ± 7.35%) vs dogs eating traditional diets (57.58 ± 4.84%; P < .001; 12.0% [8.0%-16.0%] 95% CI). Mean 2D left ventricular end-systolic volumes, indexed to body weight, were significantly higher in dogs eating nontraditional diets (1.46 ± 0.08 mL/kg) vs dogs eating traditional diets (1.06 ± 0.08 mL/kg; P = .002; 0.4 mL/kg [0.18-0.62 mL/kg] 95% CI). CONCLUSIONS AND CLINICAL IMPORTANCE: Healthy dogs eating nontraditional diets had lower indices of systolic function and larger left ventricular volumes compared to dogs eating traditional diets. Screening of apparently healthy dogs eating nontraditional diets might allow for early detection of diet-associated DCM.

Near infrared active heptacyanine dyes with unique cancer-imaging and cytotoxic properties.

Three near-infrared fluorescent heptacarbocyanine dyes have been synthesized using a facile one-pot synthetic approach. The reaction methodology afforded a mixture of three symmetric and unsymmetric heptacyanines containing various N-indolenine substituents, a dicarbocyclic acid (DA), a monoester (ME), and a diester (DE). These compounds were isolated, purified, characterized and biologically investigated for tumor cell cytotoxicity and uptake selectivity. Using cell viability and in vitro proliferation assays, we found that the esterified dyes (monoester, ME and diester, DE) were selectively cytotoxic to cancer cells and spared normal fibroblast cells. Additionally, confocal fluorescence imaging confirmed selective uptake of these dyes in cancer cells, thus suggesting tumor cell targeting.

Halogenated pentamethine cyanine dyes exhibiting high fidelity for G-quadruplex DNA.

Design and optimization of quadruplex-specific small molecules is developing into an attractive strategy for anti-cancer therapeutics with some promising candidates in clinical trials. A number of therapeutically favorable features of cyanine molecules can be effectively exploited to develop them as promising quadruplex-targeting agents. Herein, the design, synthesis and evaluation of a series of dimethylindolenine cyanine dyes with varying halogen substitutions are reported. Their interactions with telomeric and c-myc quadruplexes as well as a reference duplex sequence have been evaluated using thermal melting, biosensor-surface plasmon resonance, circular dichroism, isothermal titration calorimetry and mass spectrometry. Thermal melting analysis indicates that these ligands exhibit significant quadruplex stabilization and a very low duplex binding, with the dimethyl incorporation of paramount importance for decreased duplex affinity. Circular dichroism studies showed that the interaction of cyanines with quadruplex structures are primarily through stacking at one or both ends of the terminal tetrads with the two (trimethylammonium)propyl groups interacting in the accessible quadruplex grooves. Surface plasmon resonance and mass spectral studies shows the formation of an initial strong 1:1 complex followed by a significantly weaker secondary binding. Isothermal calorimetry studies show that the interaction of cyanines is predominantly entropy driven. In line with the design principles, this work provides new insights for further developing potent, highly selective cyanines as promising quadruplex-specific agents.

What Is Your Diagnosis?

In collaboration with the American College of Veterinary Radiology.

Comparison of left and right atrial volumes determined by two- and three-dimensional echocardiography with those determined by multidetector computed tomography for healthy dogs.

OBJECTIVE: To compare left atrial volume (LAV) and right atrial volume (RAV) determined by 2-D and 3-D echocardiographic methods with the LAV and RAV determined by ECG-gated multidetector CT (MDCT) for healthy dogs. ANIMALS: 11 healthy purpose-bred young adult hound-type dogs. PROCEDURES: Each dog was anesthetized and underwent MDCT and a complete echocardiographic examination. Modality-specific software was used to measure the respective atrial volumes at ventricular end systole, and LAV and RAV measurements were subsequently indexed to body weight and compared among imaging modalities. RESULTS: The LAV determined by echocardiographic methods did not differ significantly from the LAV determined by MDCT. However, the RAV determined by 3-D echocardiography and 2-D echocardiography via the left apical and left cranial windows differed significantly from the RAV determined by MDCT. Bland-Altman analyses indicated that the indexed LAV and RAV determined by echocardiographic methods were systematically underestimated, compared with MDCT measurements, but the bias was much smaller for LAV than for RAV. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that, for dogs, echocardiography might be an acceptable alternative to MDCT for measurement of LAV but not for measurement of RAV. However, the study population was small and homogenous in terms of breed, age, and weight. These findings need to be validated in a larger, more varied population of dogs with and without cardiac disease.

Benefits and applications of microwave-assisted synthesis of nitrogen containing heterocycles in medicinal chemistry.

Nitrogen containing heterocycles are of immense research interest because they are often found as naturally occurring bioactive compounds. The prominence of N-heterocycles makes it vital to develop methods to increase their synthetic efficiencies and probe the effects of their modifications on biological efficacy. Medicinal chemists have exploited microwave-assisted organic synthesis (MAOS) to facilitate the development of complex heterocyclic structures. MAOS is a growing synthetic methodology among medicinal chemists and has proven to be more efficient in terms of reaction yield, reaction time, product purity and environmental friendliness for many reactions when compared to conventional thermal methods for cycloaddition and selective functionalization. The importance of nitrogen containing ring systems in medicine cannot be understated, as such ring systems have shown to be applicable in compounds such as vitamins, herbicides, anti-fungal agents, anti-bacterial agents and anti-cancer agents, among other things. The significance of these applications has created an unprecedented need for more efficient synthetic methods. This review presents an overview of MAOS and its role in recent and pressing advancements for the synthesis of small- and medium-sized nitrogen containing heterocycles, including pyrroles, indoles, pyridines, pyrrolidines, imidazoles, pyrazoles, pyrazolines, lactams, and 1,2,3-triazoles, which are significant scaffolds for compounds with medicinal uses.

Ultrabright and Serum-Stable Squaraine Dyes.

Highly stable symmetric and asymmetric squaraine fluorophores have been synthesized featuring an internal salt bridge between a quaternary ammonium cation and the central oxycyclobutenolate ring of the chromophore. Some of our newly synthesized symmetric and asymmetric compounds display increased molar absorptivity, quantum yield in serum, and thermal/photochemical stability over previously reported squaraine-based dyes. Consequently, both classes show great promise in resurfacing the normal environment-labile squaraine dyes as novel imaging agents and scaffolds for fluorescence sensing. Furthermore, incorporating a covalent attachment point away from the conjugated system allows for biological tagging applications without disturbing the optimum optical characteristics of the newly designed fluorophore.

Management of vascular graft infections with soft tissue flap coverage: improving limb salvage rates--a veterans affairs experience.

Graft infections are one of the most challenging issues in surgery with an incidence of 0.7 to 7 per cent, with femoral site infections being the most common (13% incidence). The gold standard treatment has been graft removal, wide débridement, and extra-anatomical bypass. Routine excision of infected peripheral arterial grafts and vascular reconstruction with extraanatomic conduits are associated with mortality rates ranging from 10 to 30 per cent and amputation rates of up to 70 per cent. As a result of the high morbidity and mortality associated with this approach, selective graft preservation techniques have been developed. Newer treatment plans discuss preservation of the graft with débridement and coverage of the infected region. Better wound care, nutrition optimization, and robust flap coverage have led to significantly improved graft salvage, lower amputation rates, and improved outcomes. The objective of this study was to evaluate the Veterans Affairs (VA) experience with flap coverage for femoral vascular graft infections. A retrospective review was conducted of all VA data from 1997 to 2008 with inclusion criteria of patients with deep groin wound infections requiring flap coverage after femoral bypass surgery. Eleven such patients were identified with a mean age of 73 years and with multiple comorbidities (hypertension, malnutrition, diabetes mellitus, chronic obstructive pulmonary disease, coronary artery disease, chronic renal insufficiency). Patients presented with wound drainage, exposed graft, hematoma, perigraft fluid collection, and pseudoaneurysm. Treatment protocol included: 1) aggressive débridement of the wound bed; 2) early soft tissue (flap) coverage; 3) wound vacuum assisted closure device or frequent dressing changes; and 4) skin graft once the bed was prepared. Eighty-two per cent of wounds had positive cultures with equal numbers of patients with Staphylococcus epidermidis, Pseudomonas, Escherichia coli (22%), and higher methicillin-resistant Staphylococcus aureus (33%), whereas in the literature Staphylococcus is the most common (greater than 50%). Average hospital length of stay was 94 days with average follow up at 10 months. Fifty-five per cent graft salvage (one Dacron [50%], two polytetrafluoroethylene [33%], two saphenous vein graft [100%], one cryovein [100%]) was achieved with 91 per cent limb salvage. Complications included graft blowout (two) requiring partial flap loss (one), retroperitoneal hematoma (one), limb loss (one), sepsis (one), and death (one). Infected vascular grafts remain a challenging problem requiring multidisciplinary care. Careful débridement and aggressive wound care followed by selective flap coverage appears to decrease morbidity and increase graft and limb salvage.

Near-Infrared Illumination of Native Tissues for Image-Guided Surgery.

Our initial efforts to prepare tissue-specific near-infrared (NIR) fluorescent compounds generated successful correlation between physicochemical properties and global uptake in major organs after systemic circulation and biodistribution. Herein, we focus on the effects on biodistribution based on modulating electronic influencing moieties from donating to withdrawing moieties at both the heterocyclic site and through meso-substitution of pentamethine cyanine fluorophores. These selected modifications harnessed innate biodistribution pathways through the structure-inherent targeting, resulting in effective imaging of the adrenal glands, pituitary gland, lymph nodes, pancreas, and thyroid and salivary glands. These native-tissue contrast agents will arm surgeons with a powerful and versatile arsenal for intraoperative NIR imaging in real time.

700-nm Zwitterionic Near-Infrared Fluorophores for Dual-Channel Image-Guided Surgery.

PURPOSE: The purpose of this study was to develop a family of 700-nm zwitterionic pentamethine indocyanine near-infrared fluorophores that would permit dual-channel image-guided surgery. PROCEDURES: Three complementary synthetic schemes were used to produce novel zwitterionic chemical structures. Physicochemical, optical, biodistribution, and clearance properties were compared to Cy5.5, a conventional pentamethine indocyanine now used for biomedical imaging. RESULTS: ZW700-1a, ZW700-1b, and ZW700-1c were synthesized, purified, and analyzed extensively in vitro and in vivo. All molecules had extinction coefficients ≥199,000 M(-1) cm(-1), emission ≥660 nm, and stability ≥99 % after 24 h in warm serum. In mice, rats, and pigs, ≥80 % of the injected dose was completely eliminated from the body via renal clearance within 4 h. Either alone or conjugated to a tumor targeting ligand, ZW700-1a permitted dual-channel, high SBR, and simultaneous imaging with 800-nm NIR fluorophores using the FLARE® imaging system. CONCLUSIONS: Novel 700-nm zwitterionic NIR fluorophores enable dual-NIR image-guided surgery.

Tailored near-infrared contrast agents for image guided surgery.

The success of near-infrared (NIR) fluorescence to be employed for intraoperative imaging relies on the ability to develop a highly stable, NIR fluorescent, nontoxic, biocompatible, and highly excreted compound that retains a reactive functionality for conjugation to a cancer-recognizing peptide. Herein, systematic modifications to previously detailed fluorophore ZW800-1 are explored. Specific modifications, including the isosteric replacement of the O atom of ZW800-1, include nucleophilic amine and sulfur species attached to the heptamethine core. These novel compounds have shown similar satisfactory results in biodistribution and clearance while also expressing increased stability in serum. Most importantly, all of the synthesized and evaluated compounds display a reactive functionality (either a free amino group or carboxylic acid moiety) for further bioconjugation. The results obtained from the newly prepared derivatives demonstrate that the central substitution with the studied linking agents retains the ultralow background in vivo performance of the fluorophores regardless of the total net charge.

NIR fluorescent small molecules for intraoperative imaging.

Recent advances in bioimaging and nanomedicine have permitted the exploitation of molecular optical imaging in image-guided surgery; however, the parameters mediating optimum performance of contrast agents are not yet precisely determined. To develop ideal contrast agents for image-guided surgery, we need to consider the following criteria: (1) excitation and emission wavelengths in the near-infrared (NIR) window, (2) optimized optical characteristics for high in vivo performance, (3) overcoming or harnessing biodistribution and clearance, and (4) reducing nonspecific uptake. The design considerations should be focused on optimizing the optical and physicochemical property criteria. Biodistribution and clearance should first be considered because they mediate the fate of a contrast agent in the body such as how long after intravenous injection a contrast agent reaches the peak signal-to-background ratio (SBR) and how long the signal lasts (retention).