Search

The monitoring of diabetes mellitus, as it develops and becomes clinically evident, remains a major challenge for diagnostic imaging in clinical practice. Here we present a novel approach to beta-cell imaging by targeting the sulphonylurea receptor subtype 1 (SUR1), using multivalent derivatives of the anti-diabetic drug glibenclamide. Since glibenclamide has a high affinity for SUR1 but does not contain a suitable functional group to be linked to an imaging probe, we have synthesized 11 glibenclamide derivatives and evaluated their affinity to SUR1 in MIN6 cells. The most promising compound has been used to obtain multivalent glibenclamide-polyamidoamine (PAMAM) derivatives, containing up to 15 sulphonylurea moieties per dendrimer. The remaining functional groups on the dendrimers can consecutively be used for labeling with reporter groups for different imaging modalities, thus allowing for multifunctional imaging, and for the modification of pharmacokinetic properties. We synthesized fluorochrome-labeled multivalent probes, that demonstrate in cellular assays affinities to SUR1 in the nanomolar range, superior to native glibenclamide. The probes specifically label MIN6 cells, but not HeLa or PANC-1 cells which do not express SUR1. A very low cytotoxicity of the multivalent probes is demonstrated by the persistent release of insulin from MIN6 cells exposed to high glucose concentrations. Furthermore, the probes display positive labeling of beta-cells of primary mouse and human islet-cells ex vivo and of islets of Langerhans in vivo. The data document that multivalent probes based on glibenclamide derivatives provide a suitable platform for further developments of cell-specific probes, and can be adapted for multiple imaging modalities, including those that are now used in the clinics.

Polymeric photosensitizer prodrugs (PPPs) to be selectively activated by urokinase plasminogen activator (uPA) have been developed. They are composed of uPA-cleavable photosensitizer-peptide-conjugates covalently attached to a poly-lysine backbone (25 kDa). Phototoxicity of PPPs is efficiently reduced by energy transfer between closely positioned photosensitizers (PSs) on the polymeric backbone. Enzymatic cleavage by uPA, a serine protease that is up-regulated in certain types of cancer, leads to the release of PS-peptidyl-fragments and restoration of phototoxicity. In the current study, conjugates with different e-lysine side chain modifications of the polymer-backbone were synthesized and characterized with respect to in vitro properties such as fluorescence and reactive oxygen species (ROS) quenching, as well as enzymatic cleavage by uPA. Based on these in vitro results and a cell screening experiment for photo- and dark toxicity, two compounds, PEG20-N-uPA-PPP and N-uPA-PPP, were selected as the most promising candidates for further evaluation in two uPA-expressing cell lines, DU-145 and PC-3. Here, the pegylated compound PEG20-N-uPA-PPP showed enhanced selective prodrug activation by uPA in comparison to N-uPA-PPP, pinpointing the need for prolonged extracellular residence time of uPA-sensitive prodrugs. In preliminary PDT-experiments, a dose-dependent phototoxic effect of this compound was found in both tested cell lines. In conclusion, this study highlights the fact that backbone substitution units may impact not only photochemical and physicochemical properties of the PPPs, but play an important role for cellular prodrug localization and thus site selective enzymatic activation.

We have developed novel polymeric photosensitizer prodrugs (PPPs) for improved photodynamic therapy. In PPPs, multiple photosensitizer units are covalently coupled to a polymeric backbone via protease-cleavable peptide linkers. These initially non-photoactive compounds become fluorescent and phototoxic after specific enzymatic cleavage of the peptide linkers and subsequent release of the photosensitizer moieties. Tethering the photosensitizer via a short and easily modified amino acid sequence to the polymeric backbone allows for the targeting of a wide variety of proteases. Model compounds, sensitive to trypsin-mediated cleavage, with different pheophorbide a-peptide loading ratios and backbone net charges were evaluated with respect to their solubility, "self-quenching" capacity of fluorescence emission, and reactive oxygen species (ROS) generation. In addition, linker sequence impaired selectivity toward enzymatic cleavage was demonstrated either by incubating PPPs with different enzymes having trypsin-like activity or by introducing a single d-arginine mutant in the peptide sequence. In vitro cell culture tests confirmed dose-dependent higher phototoxicity of enzymatically activated PPPs compared to the nonactivated conjugate after irradiation with white light. These data suggest that similar compounds adapted to disease-associated proteases can be used for selective photodynamic therapy.

A targeting strategy based on the selective enzyme-mediated activation of polymeric photosensitizer prodrugs (PPP) within pathological tissue has led to the development of agents with the dual ability to detect and treat cancer. Herein, a detailed study of a simple model system for these prodrugs is described. We prepared "first-generation" PPP by directly tethering the photosensitizer (PS) pheophorbide a to poly-(L)-lysine via epsilon amide links and observed that by increasing the number of PS on a polymer chain, energy transfer between PS units improved leading to better quenching efficiency. Fragmentation of the PPP backbone by trypsin digestion gave rise to a pronounced fluorescence increase and to more efficient generation of reactive oxygen species upon light irradiation. In vitro tests using the T-24 bladder carcinoma cell line and ex vivo experiments using mouse intestines illustrated the remarkable and selective ability of these PPP to fluoresce and induce phototoxicity upon enzymatic activation. This work elucidated the basic physicochemical parameters, such as water solubility and quenching/activation behavior, required for the future elaboration of more adaptable "second-generation" PPP, in which the PS is tethered to a proteolytically stable polymer backbone via enzyme-specific peptide linkers. This polymer architecture offers great flexibility to tailor make the PPP to target any pathological tissue known to over-express a specific enzyme.

A novel 1,4-palladium migration between the o- and o'-positions of biaryls has been observed in organopalladium intermediates derived from o-halobiaryls. The organopalladium intermediates generated by this migration have been trapped either by a Heck reaction employing ethyl acrylate or by Suzuki cross-coupling using arylboronic acids. This palladium migration can be activated or deactivated by choosing the appropriate reaction conditions. Chemical and computational evidence supports the presence of an equilibrium that correlates with the C-H acidity of the available arene positions.

The correlation between β2-, β3-, and β2,3-amino acid-residue configuration and stability of helix and hairpin-turn secondary structures of peptides consisting of homologated proteinogenic amino acids is analyzed (Figs. 1–3). To test the power of Zn2+ ions in fortifying and/or enforcing secondary structures of β-peptides, a β-decapeptide, 1, four β-octapeptides, 2–5, and a β-hexadecapeptide, 10, have been devised and synthesized. The design was such that the peptides would a) fold to a 14-helix (1 and 3) or a hairpin turn (2 and 4), or form neither of these two secondary structures (i.e., 5), and b) carry the side chains of cysteine and histidine in positions, which will allow Zn2+ ions to use their extraordinary affinity for RS− and the imidazole N-atoms for stabilizing or destabilizing the intrinsic secondary structures of the peptides. The β-hexadecapeptide 10 was designed to a) fold to a turn, to which a 14-helical structure is attached through a β-dipeptide spacer, and b) contain two cysteine and two histidine side chains for Zn complexation, in order to possibly mimic a Zn-finger motif. While CD spectra (Figs. 6–8 and 17) and ESI mass spectra (Figs. 9 and 18) are compatible with the expected effects of Zn2+ ions in all cases, it was shown by detailed NMR analyses of three of the peptides, i.e., 2, 3, 5, in the absence and presence of ZnCl2, that i) β-peptide 2 forms a hairpin turn in H2O, even without Zn complexation to the terminal β3hHis and β3hCys side chains (Fig. 11), ii) β-peptide 3, which is present as a 14-helix in MeOH, is forced to a hairpin-turn structure by Zn complexation in H2O (Fig. 12), and iii) β-peptide 5 is poorly ordered in CD3OH (Fig. 13) and in H2O (Fig. 14), with far-remote β3hCys and β3hHis residues, and has a distorted turn structure in the presence of Zn2+ ions in H2O, with proximate terminal Cys and His side chains (Fig. 15).

Quinolines substituted in the 3-position by an iodo or phenylseleno group are readily prepared in good to excellent yields by the reaction of propargylic anilines with appropriate electrophiles under mild reaction conditions. [reaction: see text]

(S)-β2-Homoamino acids with the side chains of Asp, Glu, Asn, and Gln have been prepared and suitably protected (N-Fmoc, CO2tBu, CONHTrt) for solid-phase peptide syntheses. The key steps of the syntheses are: N-acylation of 5,5-diphenyl-4-isopropyl-1,3-oxazolidin-2-one (DIOZ) with succinic and glutaric anhydrides (Scheme 2), alkylation of the corresponding Li-enolates with benzyl iodoacetate and Curtius degradation (Scheme 4), and removal of the chiral auxiliary (Scheme 5). In addition, numerous functional-group manipulations (CO2H⇌CO2tBu, CO2Bn⇌CO2H, CbzNHFmocNH, CO2HCO2NH2CONHTrt; Schemes 2, 4, 5, and 6) were necessary, in order to arrive at the four target structures. The configurational assignments were confirmed by X-ray crystal-structure determinations (Scheme 2 and Fig. 3). The enantiomeric purities of a β2hAsn and of a β2hGln derivative were determined by HPLC on a Chiralcel column to be 99.7 : 0.3 and >99 : 1, respectively (Fig. 4). Notably, it took up to twelve steps to prepare a suitably protected trifunctional product with a single stereogenic center (overall yield of 10% from DIOZ and succinic anhydride)!

Quinolines substituted in the 3-position by an iodo or phenylseleno group are readily prepared in good to excellent yields by the reaction of propargylic anilines with appropriate electrophiles under mild reaction conditions.

The aim of this study was to assess the ability of commonly available red- or blue-light dental sources to generate reactive oxygen species (ROS) from photosensitive chemicals that might be useful for photodynamic antimicrobial chemotherapy (PACT).Although the use of red diode lasers is well documented, there is limited information on how useful blue-light sources might be for PACT in dental contexts.A diode laser (Periowave; see Table 1 for material and equipment sources) emitting red light (660-675 nm) was used to activate toluidine blue; riboflavin and pheophorbide-a polylysine (pheophorbide-a-PLL) were photoactivated using an Optilux 501 curing unit emitting blue light (380-500 nm). Ozone gas (generated by OzoTop, Tip Top Tips, Rolle, Switzerland), sodium hypochlorite, and hydrogen peroxide were used for comparison. ROS production was estimated using an iodine-triiodide colorimetric assay, and ROS levels were plotted versus concentration of chemicals to determine each chemical's efficiency in ROS production. One-way ANOVA with Tukey post hoc analysis (alpha = 0.05) was used to compare the efficiencies of ROS production for the various chemicals.Sodium hypochlorite, hydrogen peroxide, and ozone gas produced ROS spontaneously, whereas pheophorbide-a-PLL, riboflavin, and toluidine blue required light exposure. The efficiency of ROS production was higher for pheophorbide-a-PLL and toluidine blue than for ozone gas or riboflavin (p0.05). Hydrogen peroxide was the least efficient ROS producer.The results of the current study support the use of blue- or red-light-absorbing photosensitizers as candidates to produce ROS for clinical applications. Blue-light photosensitizers were as efficient as red-light photosensitizers in producing ROS and more efficient than the oxidant chemicals currently used for dental disinfection.

Objectives Disinfection of the tooth pulp-canal system is imperative to successful endodontic therapy. Yet, studies suggest that 30–50% of current endodontic treatments fail from residual bacterial infection. Photodynamic therapy using red-light chromophores (630 nm) to induce antimicrobial death mediated by generated reactive oxygen species (ROS) has been reported, but red-light also may thermally damage resident tissues. In the current study, we tested the hypothesis that several blue light chromophores (380–500 nm) generate intracellular reactive oxygen species but are not cytotoxic to mammalian cells. Methods THP1 monocytes were exposed to 10 μM of four chromophores (chlorin e6, pheophorbide-a, pheophorbide-a-PLL, and riboflavin) for 30 min before activation with blue light (27 J/cm 2 , 60 s). After activation, intracellular ROS were measured using a dihydrofluorescein diacetate technique, and cytotoxicity was determined by measuring mitochondrial activity with the MTT method. Results All photosensitizers produced intracellular ROS levels that were dependent on both the presence of the photosensitizer and blue light exposure. Riboflavin and pheophorbide-a-PLL produced the highest levels of ROS. Photosensitizers except riboflavin exhibited cytotoxicity above 10 μM, and all except pheophorbide-a-PLL were more cytotoxic after blue light irradiation. Significance The current study demonstrated the possible utility of blue light chromophores as producers of ROS that would be useful for endodontic disinfection.

Biologically interesting fluoren-9-one and xanthen-9-one derivatives have been prepared by a novel aryl to imidoyl palladium migration, followed by intramolecular arylation. The fluoren-9-one synthesis appears to involve both a palladium migration mechanism and a C-H activation process proceeding through an unprecedented organopalladium(IV) hydride intermediate. The results from deuterium labeling experiments are consistent with the proposed dual mechanism.

Early cancer diagnosis is of the most crucial factors determining proper patient management and long-term survival. Thus, there is considerable interest in developing more reliable methods for cancer diagnosis. Fluorescence diagnosis using metabolic alterations of neoplasia has emerged as a powerful new biomedical tool. It is characterized by superior sensitivity and selectivity over other methods, and it offers the possibility to obtain molecular information required to determine the state of the disease. Furthermore, important advancements have been made in generating deeper three-dimensional images in vivo by exploiting tissue transparency in the near-infrared region and fluorescence-mediated tomography, which might render fluorescence diagnosis less invasive. From basic principles in optics and molecular biology to the development of medical instrumentation and the launching of clinical trials, we have comprehensively compiled the basic information required to clearly understand fundamental aspects of fluorescence-based diagnosis.

The introduction of lipophilic derivatives of the naturally occurring heme precursor 5-aminolevulinic acid (5-ALA) into photomedicine has led to a true revival of this research area. 5-ALA-mediated photodynamic therapy (PDT) and fluorescence photodetection (FD) of neoplastic disease is probably one of the most selective cancer treatments currently known in oncology. To date, this method has been assessed experimentally for the treatment of various medical indications. However, the limited local bioavailability of 5-ALA has widely prevented its use in daily clinical practice. Although researchers were already aware of this drawback early during the development of 5-ALA-mediated PDT, only recently have well-established concepts in pharmaceutical science been adapted to investigate ways to overcome this drawback. Recently, two derivatives of 5-ALA, methylaminolevulinate (MAL) and hexylaminolevulinate (HAL), gained marketing authorization from the regulatory offices in Europe and Australia. MAL is marketed under the trade name Metvix for the treatment of actinic keratosis and difficult-to-treat basal cell carcinoma. HAL has recently been launched under the trade name Hexvix to improve the detection of superficial bladder cancer in Europe. This review will first present the fundamental concepts underlying the use of 5-ALA derivatives in PDT and FD from a chemical, biochemical and pharmaceutical point of view. Experimental evidences from preclinical data on the improvements and limits observed with 5-ALA derivatives will then be introduced. The state-of-the-art from clinical studies with 5-ALA esters will be discussed, with special emphasis placed on the process that led to the development of MAL in dermatology and to HAL in urology. Finally, we will discuss promising medical fields in which use of 5-ALA derivatives might potentially lead to further use of this methodology in photomedicine.

[reaction: see text] A variety of substituted polycyclic aromatics are readily prepared in good to excellent yields under very mild reaction conditions by the reaction of 2-(1-alkynyl)biphenyls with ICl, I(2), NBS, and p-O(2)NC(6)H(4)SCl. This methodology readily accommodates various functional groups and has been successfully extended to systems containing a variety of polycyclic and heterocyclic rings.

Novel palladium migration/arylation methodology for the synthesis of complex fused polycycles has been developed, in which one or more sequential Pd-catalyzed intramolecular migration processes involving C-H activation are employed. The chemistry works best with electron-rich aromatics, which is in agreement with the idea that these palladium-catalyzed C-H activation reactions parallel electrophilic aromatic substitution.

The history of peptide chemistry in our group is described. It all started with the cyclic undecapeptide cyclosporin, the immunosuppressive compound, which is commercialised as Sandimmune®/Neoral® by Sandoz/Novartis, and which has revolutionized transplant medicine. The discovery that cyclosporin can be deprotonated to a hexalithio derivative, and thus C-alkylated on a sarcosine moiety, led us into a research project on peptide modifications. We defined structural prerequisites for the use of peptide enolates and for electrolytic decarboxylation of peptides. Parallel to these activities, the group was engaged in developing synthetic methodologies aimed at stereoselective preparations of α-, β-, and γ-amino acid derivatives (cf. diastereoselective alkylations, self regeneration of stereogenic centers, axially chiral enolates). A third avenue into peptide chemistry originated from our investigations on the biopolymer PHB (poly-3-hydroxybutanoic acid); the question arose ‘what happens upon replacement of chain-bound O by NH in the polyester?’ A brief summary is given of the results obtained in our ensuing discovery tour of β-peptides built of homologated proteinogenic amino acids. They form secondary structures with short chain lengths and they have unexpected physiological properties, rendering them candidates for peptidic drugs. The synthesis of β3-peptides is straightforward, and in the meantime most of the Fmoc-protected building blocks are commercial. The β2-homoamino acids are less readily available. Their preparation and the assembly of a β2-eicosapeptide with the twenty proteinogenic side chains are discussed herein. The reasons for the chosen sequence and the strategy of what turned out to be a 159-step synthesis are described. Full experimental details are given for the preparation of the dimeric Fmoc-β2hXaa(PG)-β2hXaa(PG)-OH building blocks used, for their solid-phase coupling to two β2-decapeptide segments, for the thioligation, and for the purification, isolation and spectroscopic characterization of the resulting 20mer. An outlook to future projects in the exciting field of β- and γ-peptide chemistry and biology is given.

A novel palladium migration methodology for the synthesis of complex fused polycycles has been developed. This process involves 1,4-palladium alkyl to aryl migrations via through-space C−H activation, followed by intramolecular arylation or an intermolecular Heck reaction providing a very efficient way to synthesize fused ring systems.

A novel palladium migration/arylation methodology for the synthesis of complex fused polycycles has been developed, in which one or more sequential Pd-catalyzed intramolecular migration processes involving C-H activation are employed. The chemistry works best with electron-rich aromatics, which is in agreement with the idea that these palladium-catalyzed C-H activation reactions parallel electrophilic aromatic substitution.

A novel 1,4-palladium migration in organopalladium intermediates derived from o-iodobiaryls has been observed under modified Heck reaction conditions. This migration process can be switched “on” or “off” by simply choosing the appropriate reaction conditions.

The synthesis of various substituted fluoren-9-ones has been accomplished by the palladium-catalyzed cyclocarbonylation of o-halobiaryls. The cyclocarbonylation of 4'-substituted 2-iodobiphenyls produces very high yields of 2-substituted fluoren-9-ones bearing either electron-donating or electron-withdrawing substituents. 3'-Substituted 2-iodobiphenyls afford 3-substituted fluoren-9-ones in excellent yields with good regioselectivity. This chemistry has been successfully extended to polycyclic fluorenones and fluorenones containing fused isoquinoline, indole, pyrrole, thiophene, benzothiophene, and benzofuran rings.

The synthesis of various substituted fluoren-9-ones has been accomplished by a novel palladium-catalyzed cyclocarbonylation of o-halobiaryls. The cyclocarbonylation of 4'-substituted-2-iodobiphenyls produces very high yields of 2-substituted fluoren-9-ones bearing either electron-donating or electron-withdrawing substituents. 3'-Substituted 2-iodobiphenyls afford in excellent yields with good regioselectivity 3-substituted fluoren-9-ones. This chemistry has been successfully extended to polycyclic and heterocyclic fluorenones.

The synthesis of various substituted fluoren-9-ones has been accomplished by a novel palladium-catalyzed cyclocarbonylation of o-halobiaryls. The cyclocarbonylation of 4'-substituted-2-iodobiphenyls produces very high yields of 2-substituted fluoren-9-ones bearing either electron-donating or electron-withdrawing substituents. 3'-Substituted 2-iodobiphenyls afford in excellent yields with good regioselectivity 3-substituted fluoren-9-ones. This chemistry has been successfully extended to polycyclic and heterocyclic fluorenones.