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UNLABELLED: Fluorescence-guided surgery is set to play a pivotal role in the intraoperative management of pediatric tumors. Shortwave infrared imaging (SWIR) has advantages over conventional near-infrared I (NIR-I) imaging with reduced tissue scattering and autofluorescence. Here, two NIR-I dyes (IRDye800CW and IR12), with long tails emitting in the SWIR range, were conjugated with a clinical-grade anti-GD2 monoclonal antibody (dinutuximab-beta) to compare NIR-I and SWIR imaging for neuroblastoma surgery. A first-of-its-kind multispectral NIR-I/SWIR fluorescence imaging device was constructed to allow an objective comparison between the two imaging windows. Conjugates were first characterized in vitro. Tissue-mimicking phantoms, imaging specimens of known geometric and material composition, were used to assess the sensitivity and depth penetration of the NIR-I/SWIR device, showing a minimum detectable volume of ∼0.9 mm3 and depth penetration up to 3 mm. In vivo, fluorescence imaging using the NIR-I/SWIR device showed a high tumor-to-background ratio (TBR) for both dyes, with anti-GD2-IR800 being significantly brighter than anti-GD2-IR12. Crucially, the system enabled higher TBR at SWIR wavelengths than at NIR-I wavelengths, verifying SWIR imaging enables high-contrast delineation of tumor margins. This work demonstrates that by combining the high specificity of anti-GD2 antibodies with the availability and translatability of existing NIR-I dyes, along with the advantages of SWIR in terms of depth and tumor signal-to-background ratio, GD2-targeted NIR-I/SWIR-guided surgery could improve the treatment of patients with neuroblastoma, warranting investigation in future clinical trials. SIGNIFICANCE: Multispectral near-infrared I/shortwave infrared fluorescence imaging is a versatile system enabling high tumor-to-background signal for safer and more complete resection of pediatric tumors during surgery. ispartof: Cancer Res vol:83 issue:12 pages:2077-2089 ispartof: location:United States status: published

There is no established method to assess the PD-L1 expression in brain tumours. Therefore, we investigated the suitability of affibody molecule (ZPD-L1) radiolabelled with F-18 (Al18F) and Ga-68 to measure the expression of PD-L1 in xenograft mouse models of GBM. Mice bearing subcutaneous and orthotopic tumours were imaged 1 h post-radioconjugate administration. Ex vivo biodistribution studies and immunohistochemistry (IHC) staining were performed. Tumoural PD-L1 expression and CD4+/CD8+ tumour-infiltrating lymphocytes were evaluated in human GBM specimens. ZPD-L1 was radiolabelled with radiochemical yields of 32.2 ± 4.4% (F-18) and 73.3 ± 1.8% (Ga-68). The cell-associated radioactivity in vitro was consistent with PD-L1 expression levels assessed with flow cytometry. In vivo imaging demonstrated that 18F-AlF-NOTA-ZPD-L1 can distinguish between PD-L1 high-expressing tumours (U87-MGvIII) and PD-L1-negative ones (H292PD-L1Ko). The radioconjugate was quickly cleared from the blood and normal tissues, allowing for high-contrast images of brain tumours as early as 1 h post-injection. 68Ga-NOTA-ZPD-L1 showed heterogeneous and diffuse accumulation that corresponded to the extensively infiltrating GCGR-E55 tumours involving contiguous lobes of the brain. Lastly, 39% of analysed GBM patient samples showed PD-L1+ staining of tumour cells that was associated with elevated levels of CD4+ and CD8+ lymphocytes. Our results suggest that the investigated radioconjugates are very promising agents with the potential to facilitate the future design of treatment regimens for GBM patients.

Representative 3D-rendering of segmented MCF-7, MDA-MB-468 and MDA-MB-231 xenografts following PET/CT image acquisition 3 h after 89Zr-DFO-ZHER3:8698 injection.

Purpose: Recent studies have highlighted a role of HER3 in HER2-driven cancers (e.g., breast cancer), implicating the upregulation of the receptor in resistance to HER-targeted therapies and Hsp90 inhibitors (e.g., AUY922). Therefore, we have developed an affibody-based PET radioconjugate that quantitatively assesses HER3 changes induced by Hsp90 inhibition in vivo.Experimental Design: ZHER3:8698 affibody molecules were conjugated via the C-terminus cysteine to DFO-maleimide for 89Zr radiolabeling. The probe was characterized in vitro and in vivo in a panel of human breast cell lines and xenograft models with varying HER3 receptor levels. In addition, the radioconjugate was investigated as a tool to monitor the outcome of AUY922, an Hsp90 inhibitor, in an MCF-7 xenograft model.Results: We demonstrated that 89Zr-DFO-ZHER3:8698 can track changes in receptor expression in HER3-positive xenograft models and monitor the outcome of AUY922 treatment. Our in vitro findings showed that MCF-7 cells, which are phenotypically different from BT474, develop resistance to treatment with AUY922 through HER3/IGF-1Rβ–mediated signaling. Of note, the lack of response in vitro due to HER3 recovery was confirmed in vivo using 89Zr-DFO-ZHER3:8698–based imaging. Upon AUY922 treatment, higher radioconjugate uptake was detected in treated MCF-7 xenografts, correlating with an AUY922-induced HER3 upregulation concomitant with an increase in IGF-1Rβ expression.Conclusions: These data underline the potential of HER3-based PET imaging to noninvasively provide information about HER3 expression and to identify patients not responding to targeted therapies due to HER3 recovery. Clin Cancer Res; 24(8); 1853–65. ©2018 AACR.

Materials and Methods Fig. S1. Time course of AUY922 treatment in BT-474 and MCF-7 cells. Fig. S2. Effects of AUY922 and PPP treatment in low and high HER2-expressing breast cancer cell lines. Fig. S3. Effect of HRG stimulation in AUY922 and PPP treated MCF-7 cells. Fig. S4. AUY922 and PPP treatment effect on the proliferative and invasive phenotype of BT-474 and MCF-7 breast cancer cells. Fig. S5. HPLC chromatograms of purified DFO-affibody conjugates. Fig. S6. MALDI-MS spectra of purified DFO-ZHER3:8698 (A) and DFO-ZTAQ (B). Fig. S7. Representative radio-ITLC of the crude radiolabelling mixture of 89Zr-DFO-ZHER3:8698. Fig. S8. Representative radio-ITLC of the 89Zr-DFO-ZHER3:8698 serum stability at 0, 3 and 24 h. Fig. S9. In vivo 89Zr-DFO-ZHER3:8698 tumour uptake quantification by image analysis. Fig. S10. Ex vivo biodistribution of 89Zr-DFO-ZHER3:8698 following AUY922 treatment in MCF-7 xenografts. Fig. S11. Microvessel density analysis in response to AUY922 treatment in MCF-7 xenografts. Fig. S12. Tumour volume estimation in response to AUY922 treatment in MCF-7 xenografts. Fig. S13. AUY922 treatment response monitoring in BT-474 xenografts by 89Zr-DFO-ZHER3:8698 PET/CT imaging. Fig. S14. Texture analysis of 89Zr-DFO-ZHER3:8698 PET imaging data following AUY922 treatment in MCF-7 xenografts. Fig. S15. Correlation between %ID/g ratios obtained from 89Zr-DFO-ZHER3:8698 PET images and HER3 protein expression per control and AUY922-treated MCF-7 xenografts. Fig. S16. HER3 intra-tumoral heterogeneity highlighted by histopathological analysis of control and AUY922-treated MCF-7 xenografts. Fig. S17. HER2-HER3 interaction in BT-474 and MCF-7 cells following AUY922 treatment. Fig. S18. Scheme illustrating the proposed effect of AUY922-induced Hsp90 inhibition in MCF-7 breast cancer xenografts. Table S1. Densitometric analysis of Western blots related to the treatment of BT-474, MCF-7, MCF-7 HER2+++ (-dox), and MCF-7 HER2+ (+dox) following 48 h treatment with AUY922 and PPP alone, or in combination. Table S2. Biodistribution results for 89Zr-DFO-ZHER3:8698 and 89Zr-DFO-ZTAQ (3 µg, 7.2-8.1 MBq/mouse) at 3 h after injection. Table S3. Biodistribution results for 89Zr-DFO-ZHER3:8698 at 3 h and 24 h after injection in MCF-7 and MDA-MB-468 tumour-bearing mice. Table S4. Tumour/organ ratios for 89Zr-DFO-ZHER3:8698 at 3 h and 24 h after injection in MCF-7 and MDA-MB-468 tumour-bearing mice. Table S5. HER3 and IGF1R co-occurrence in breast invasive carcinoma samples. Table S6. HER3 and IGF1R alterations lead to a shorter overall survival in breast invasive carcinoma samples. Table S7. HER3 and IGF1R alterations lead to a shorter disease free survival in breast invasive carcinoma samples. Table S8. HER3 and IGF1R co-occurrence in HER2 up-regulated breast invasive carcinoma samples.

A large number of applications for fibroblast activation protein inhibitors (FAPI)-based PET agents have been evaluated in conditions ranging from cancer to non-malignant diseases such as myocardial infarction. In particular

BackgroundSurgical resection followed by chemo-radiation postpones glioblastoma (GBM) progression and extends patient survival, but these tumours eventually recur. Multimodal treatment plans combining intraoperative techniques that maximise tumour excision with therapies aiming to remodel the immunologically cold GBM microenvironment could improve patients’ outcomes. Herein, we report that targeted photoimmunotherapy (PIT) not only helps to define tumour location and margins but additionally promotes activation of anti-GBM T cell response.MethodsEGFR-specific affibody molecule (ZEGFR:03115) was conjugated to IR700. The response to ZEGFR:03115-IR700-PIT was investigated in vitro and in vivo in GBM cell lines and xenograft model. To determine the tumour-specific immune response post-PIT, a syngeneic GBM model was used.ResultsIn vitro findings confirmed the ability of ZEGFR:03115-IR700 to produce reactive oxygen species upon light irradiation. ZEGFR:03115-IR700-PIT promoted immunogenic cell death that triggered the release of damage-associated molecular patterns (DAMPs) (calreticulin, ATP, HSP70/90, and HMGB1) into the medium, leading to dendritic cell maturation. In vivo, therapeutic response to light-activated conjugate was observed in brain tumours as early as 1 h post-irradiation. Staining of the brain sections showed reduced cell proliferation, tumour necrosis, and microhaemorrhage within PIT-treated tumours that corroborated MRI T2*w acquisitions. Additionally, enhanced immunological response post-PIT resulted in the attraction and activation of T cells in mice bearing murine GBM brain tumours.ConclusionsOur data underline the potential of ZEGFR:03115-IR700 to accurately visualise EGFR-positive brain tumours and to destroy tumour cells post-conjugate irradiation turning an immunosuppressive tumour environment into an immune-vulnerable one.

Although nanomaterial-mediated phototherapy has been extensively studied, the major antitumor success is limited to treating subcutaneous tumor on nude, lacking of clinically-relevant big animal study. Therefore, it is urgent to make further investigation on the typical big model, which is more closely related to the human body. In this work, niobium carbide (NbC) was selected as photoactive substance in virtue of its outstanding near infrared (NIR) absorption properties and resultantly NIR-triggered hyperthemia and reactive oxygen species generation for the synergetic photothermal and photodynamic effect. Moreover, macrophage was used as bio-carrier for the targeted delivery of NbC and the phagocytosis of macrophages was proved to be able to retain the photothermal/photodynamic effect of NbC. Resultantly, macrophage loaded NbC could realize complete removal of solid tumor on both of nude mice and big animal of rabbits. Meanwhile, two-dimensional ultrasound, shave wave elastography (SWE) and contrast-enhanced ultrasound (CEUS) have been applied for monitoring the physiological evolutions of in vivo tumor post treatment, which clearly disclosed the photoablation process of tumor and provided a new way for the surveillance of tumor on the big animal study. Hence, large animal model study in this work presented higher clinical significance than the previous studies.

In radiopharmaceutical syntheses, maleimide is commonly used for linking thiol-bearing bioactive molecules to metal-complexing ligands (chelators). However, due to instability of the resulting linkage, phenyloxadiazolyl methylsulfone (PODS) was developed as an alternative to maleimide. This coupling strategy has never been attempted with HBED which is a powerful chelator for gallium-radiolabeling especially at ambient temperature. Here we present HBED-CC-PODS as a bifunctional chelator scaffold for the site-selective conjugation of thiol-bearing vectors and [68Ga]Ga-radiolabeling.

There is an urgent need to develop therapeutic approaches that can increase the response rate to immuno-oncology agents. Photoimmunotherapy has recently been shown to generate anti-tumour immunological responses by releasing tumour-associated antigens from ablated tumour cell residues, thereby enhancing antigenicity and adjuvanticity. Here, we investigate the feasibility of a novel HER2-targeted affibody-based conjugate (ZHER2:2395-IR700) selectively to induce cancer cell death in vitro and in vivo. The studies in vitro confirmed the specificity of ZHER2:2395-IR700 binding to HER2-positive cells and its ability to produce reactive oxygen species upon light irradiation. A conjugate concentration- and light irradiation-dependent decrease in cell viability was also demonstrated. Furthermore, light-activated ZHER2:2395-IR700 triggered all hallmarks of immunogenic cell death, as defined by the translocation of calreticulin to the cell surface, and the secretion of ATP, HSP70/90 and HMGB1 from dying cancer cells into the medium. Irradiating a co-culture of immature dendritic cells (DCs) and cancer cells exposed to light-activated ZHER2:2395-IR700 enhanced DC maturation, as indicated by augmented expression of CD86 and HLA-DR. In SKOV-3 xenografts, the ZHER2:2395-IR700-based phototherapy delayed tumour growth and increased median overall survival. Collectively, our results strongly suggest that ZHER2:2395-IR700 is a promising new therapeutic conjugate that has great potential to be applicable for photoimmunotherapy-based regimens.

In head and neck squamous cell cancer, the human epidermal growth factor receptor 1 (EGFR) is the dominant signaling molecule among all members of the family. So far, cetuximab is the only approved anti-EGFR monoclonal antibody used for the treatment of head and neck squamous cell cancer, but despite the benefits of adding it to standard treatment regimens, attempts to define a predictive biomarker to stratify patients for cetuximab treatment have been unsuccessful. We hypothesized that imaging with EGFR-specific radioligands may facilitate noninvasive measurement of EGFR expression across the entire tumor burden and allow for dynamic monitoring of cetuximab-mediated changes in receptor expression. Methods: EGFR-specific Affibody molecule (ZEGFR:03115) was radiolabeled with 89Zr and 18F. The radioligands were characterized in vitro and in mice bearing subcutaneous tumors with varying levels of EGFR expression. The protein dose for imaging studies was assessed by injecting 89Zr-deferoxamine-ZEGFR:03115 (2.4–3.6 MBq, 2 μg) either together with or 30 min after increasing amounts of unlabeled ZEGFR:03115 (1, 5, 10, 15, and 20 μg). PET images were acquired at 3, 24, and 48 h after injection, and the image quantification data were correlated with the biodistribution results. The EGFR expression and biodistribution of the tracer were assessed ex vivo by immunohistochemistry, Western blot, and autoradiography. To downregulate the EGFR level, treatment with cetuximab was performed, and 18F-aluminium fluoride-NOTA-ZEGFR:03115 (12 μg, 1.5–2 MBq/mouse) was used to monitor receptor changes. Results: In vivo studies demonstrated that coinjecting 10 μg of nonlabeled molecules with 89Zr-deferoxamine-ZEGFR:03115 allows for clear tumor visualization 3 h after injection. The radioconjugate tumor accumulation was EGFR-specific, and PET imaging data showed a clear differentiation between xenografts with varying EGFR expression levels. A strong correlation was observed between PET analysis, ex vivo estimates of tracer concentration, and receptor expression in tumor tissues. Additionally, 18F-aluminium fluoride-NOTA-ZEGFR:03115 could measure receptor downregulation in response to EGFR inhibition. Conclusion: ZEGFR:03115-based radioconjugates can assess different levels of EGFR level in vivo and measure receptor expression changes in response to cetuximab, indicating a potential for assessment of adequate treatment dosing with anti-EGFR antibodies.

Site-selective bioconjugation of cysteine-containing peptides and proteins is currently achieved via a maleimide&ndash, thiol reaction (Michael addition). When maleimide-functionalized chelators are used and the resulting bioconjugates are subsequently radiolabeled, instability has been observed both during radiosynthesis and post-injection in vivo, reducing radiochemical yield and negatively impacting performance. Recently, a phenyloxadiazolyl methylsulfone derivative (PODS) was proposed as an alternative to maleimide for the site-selective conjugation and radiolabeling of proteins, demonstrating improved in vitro stability and in vivo performance. Therefore, we have synthesized two novel PODS-bearing bifunctional chelators (NOTA-PODS and NODAGA-PODS) and attached them to the EGFR-targeting affibody molecule ZEGFR:03115. After radiolabeling with the aluminum fluoride complex ([18F]AlF), both conjugates showed good stability in murine serum. When injected in high EGFR-expressing tumor-bearing mice, [18F]AlF-NOTA-PODS-ZEGFR:03115 and [18F]AlF-NODAGA-PODS-ZEGFR:03115 showed similar pharmacokinetics and a specific tumor uptake of 14.1 &plusmn, 5.3% and 16.7 &plusmn, 4.5% ID/g at 1 h post-injection, respectively. The current results are encouraging for using PODS as an alternative to maleimide-based thiol-selective bioconjugation reactions.

RATIONALE The consumption of red meat is known to enhance the endogenous formation of N-nitroso compounds (NOCs), which are potent carcinogens. DNA damage related to NOCs, and hence, red meat has been detected in colorectal cells and in blood. We proposed to extend previous studies to a non-invasive approach for the detection of O6-carboxymethyl-guanine (O6CMG) and O6-carboxymethyl-2’-deoxiguanosine (O6CMdG) in urine in relation to red meat intake using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The presence of the adduct in urine samples either as free base or as 2’-deoxynucleoside could help in determining the repair mechanism involved when such lesions are produced. A non-invasive assessment of DNA adducts could also allow for large scale analyses in the population and cancer prevention dietary strategies. METHODS An LC-MS/MS method for quantitation of O6CMG and the corresponding nucleoside O6CMdG was developed. 24 hours urine samples, collected from healthy volunteers (N=12) on red meat and vegetarian diets, were analysed either by direct injection or after purification by solid phase extraction (SPE). A separate LC-MS/MS method for the quantitation of O6-methyl guanine (O6MeG) and O6-methyl-2’-deoxyguanosine (O6MedG), which are possible hydrolysis products forming during the samples pre-treatment, was also developed. RESULTS The developed LC-MS/MS method allowed the simultaneous measurement of O6CMG and O6CMdG. The limit of detection was 0.38 ng/mL for O6CMG and 0.18 ng/mL for O6CMdG. The direct injection analysis of the clinical samples showed low sensitivity due to high background signal that was improved by SPE purification. However, the concentrations of the adducts were still found to be below the LOD. CONCLUSIONS Two novel, reproducible, and accurate LC-MS/MS methods were developed for the determination of O6CMG and O6CMdG, and O6MeG and O6MedG in urine. Clinical samples from volunteers on different diets were analysed. Further studies are required to discover a link between the presence of these biomarkers in urine and red meat consumption.

The human epidermal growth factor receptor 3 (HER3) is overexpressed in several cancers, being linked to a more resistant phenotype and hence leading to poor patient prognosis. Imaging HER3 is challenging owing to the modest receptor number (50000 receptors/cell) in overexpressing cancer cells. Therefore, to image HER3 in vivo, high target affinity PET probes need to be developed. This work describes two different [(18)F]AlF radiolabeling strategies of the ZHER3:8698 affibody molecule specifically targeting HER3. The one-pot radiolabeling of ZHER3:8698 performed at 100 °C and using 1,4,7-triazanonane-1,4,7-triacetate (NOTA) as chelator resulted in radiolabeled products with variable purity attributed to radioconjugate thermolysis. An alternative approach based on the inverse electron demand Diels-Alder (IEDDA) reaction between a novel tetrazine functionalized 1,4,7-triazacyclononane-1,4-diacetate (NODA) chelator and the trans-cyclooctene (TCO) functionalized affibody molecule was also investigated. This method enabled the radiolabeling of the protein at room temperature. The [(18)F]AlF-NOTA-ZHER3:8698 and [(18)F]AlF-NODA-ZHER3:8698 conjugates showed a specific uptake at 1 h after injection in high HER3-expressing MCF-7 tumors of 4.36 ± 0.92% ID/g and 4.96 ± 0.65% ID/g, respectively. The current results are encouraging for further investigation of [(18)F]AlF-NOTA-ZHER3:8698 as a HER3 imaging agent.

Aptamers are characterized by a rapid renal clearance leading to a short in vivo circulating half-life. In order to use aptamers as anticancer therapeutic agents, their exposure time to the tumor has to be enhanced via increasing residency in the bloodstream. A way to achieve this goal is by conjugating the aptamer to poly(ethylene glycol) (PEG). Herein, we present the conjugation of a bifunctionalized anti-MUC1 aptamer (NH(2)-AptA-SR) with the (99m)Tc coordinating moiety MAG2 and either a conventional branched PEG or the comb-shaped PolyPEG via a two-step synthesis. The isolated products were radiolabeled with (99m)Tc and their biodistribution and tumor-targeting properties in MCF-7 tumor bearing mice were analyzed and compared.

The synthesis of a benzooxazol-5-yl acetic acid derivative (9) with strong heparanase and angiogenesis inhibitory activity, and thus possible commercial interest, is described in detail.

Aptamers are known for their short in vivo circulating half-life and rapid renal clearance. Their conjugation to poly(ethylene glycol) (PEG) is a way to improve their residence in the body. Two aptamers (AptD and AptF), having a disulfide protected thiol modification on the 3′ end, have been conjugated to maleimide activated PEGs of various molecular weights and structures (linear PEG20; branched PEG20 and 40; PolyPEG17, 40, and 60 kDa). The high yield coupling (70−80% in most of the cases) could be achieved using immobilized tris[2-carboxyethyl]phosphine hydrochloride (TCEP) as reducing agent at pH 4. The affinity of PEGylated AptD for its target was reduced by conjugation to linear PEG20 and branched PEG40, but not to branched PEG20 and PolyPEGs. This work demonstrates an alternative approach to PEGylation of aptamers, and that the effect of PEG on the affinity for the target varies according to the structure and conformation of the synthetic polymer.

The synthetic access to 1,4,7,10,13-pentaazacyclohexadecane-14,16-dione was significantly improved from an overall yield of 1% to 33%. The procedure involved the use of COCF 3 or DDE as highly selective protecting groups for primary amines and of Boc to protect the secondary amine functions. The reaction of a tris-Boc protected tetraethylenepentamine with malonic acid gave the macrocycle in a 44% yield, the same reaction with malonyl chloride, however, yielded only 3.6% of the desired product. Boc deprotection and removal of the trifluoroacetate salts gave access to the final product, 1,4,7,10,13-pentaazacyclohexadecane-14,16-dione, in an overall yield of 33%.

X-ray irradiation of plasmid DNA in presence of platinum (II)-based compounds was carried out in order to assess the radiosensitization capabilities of these drugs. In present investigations pBR322 plasmid DNA was used to monitor the effectiveness of chosen compounds in inducing strand breaks. Samples were incubated in the presence of potential radiosensitisers: platinum (II) bromide and cis-diamminedibromoplatinum (II). The results were examined against a common cancer chemotherapy drug cis-diamminedichloroplatinum (II). It was found that platinum (II) bromide can greatly increase the levels of single- and double-strand break formation observed in the irradiated samples with respect to the samples containing platinum as a radiosensitizer only, possessing very little chemotherapeutic activity. The suggested drugs exhibit much higher level of radiosensitivity than widely used cisplatin and thus may be good candidates for cancer treatment.

The consumption of red meat is known to enhance the endogenous formation of N-nitroso compounds (NOCs), which are potent carcinogens. DNA damage related to NOCs, and hence red meat, has been detected in colorectal cells and in blood. We proposed to extend previous studies to a non-invasive approach for the detection of O(6)-carboxymethylguanine (O(6)CMG) and O(6)-carboxymethyl-2'-deoxyguanosine (O(6)CMdG) in urine in relation to red meat intake using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The presence of the adduct in urine samples either as the free base or as 2'-deoxynucleoside could help in determining the repair mechanism involved when such lesions are produced. A non-invasive assessment of DNA adducts could also allow for large-scale analyses in the population and cancer prevention dietary strategies.An LC/MS/MS method for the quantitation of O(6)CMG and O(6)CMdG was developed. Urine samples collected from healthy volunteers on red meat and vegetarian diets were analysed either by direct injection or after purification by solid-phase extraction (SPE). A separate LC/MS/MS method for O(6)-methylguanine (O(6)MeG) and O(6)-methyl-2'-deoxyguanosine (O(6)MedG), which are possible hydrolysis products forming during the sample pre-treatment, was also developed.The developed LC/MS/MS method allowed the simultaneous measurement of O(6)CMG and O(6)CMdG. The limits of detection (LODs) were 0.38 ng/mL for O(6)CMG and 0.18 ng/mL for O(6)CMdG. The direct injection analysis of the clinical samples showed low sensitivity due to high background signal that was improved by SPE purification. However, the concentrations of the adducts in clinical samples were still found to be below the LOD.Novel, reproducible, and accurate LC/MS/MS methods were developed for the determination of the urinary content of O(6)CMG and O(6)CMdG, and of the possible formation of O(6)MeG and O(6)MedG by decarboxylation. Clinical samples from volunteers on different diets were analysed. Further studies are required to discover a link between the presence of these biomarkers in urine and red meat consumption.

The synthesis of the novel Co(III)(15,15-difluoro-1,4,7,10,13-pentaazacyclohexadecane-14,16-dione) acetate complex from different cobalt sources is described. Furthermore, the corresponding nickel complex and its new analog with the 15-fluoro-15-methyl-1,4,7,10,13-pentaazacyclohexadecane-14,16-dione ligand were studied in great detail including cyclic voltammetry and single crystal analyses of a total of 6 complexes. The results for the nickel compounds showed that depending upon ligand substitution and solvent, an N3 or, after a double deprotonation, an N5 coordination mode could be observed and characterized in the solid state and in solution. Moreover, cyclic voltammetric analyses were performed revealing that the Ni(II/III) oxidation potentials are becoming more positive with the increasing number of fluorine atoms. This effect is due to the electron withdrawing property of fluorine that is reducing the electron richness of the metal ion.

The synthesis of a benzooxazol-5-yl acetic acid derivative (9) with strong heparanase and angiogenesis inhibitory activity, and thus possible commercial interest, is described in detail.

Metal ions bind to nucleic acids at various positions. This binding can be modulated by using metal complexes with appropriate ligands. Novel mono- and especially dinuclear metal complexes could be a powerful tool to detect rare, but still physiologically relevant, forms of DNA, e.g. the left-handed Z-DNA. In this review, our recent research activities in this area of bioinorganic chemistry are summarized. A special emphasis is laid on the synthetic challenges that arose upon the synthesis of the polyamine ligands. Further, some rather unusual approaches to elucidate the solution structure of copper bound to guanosine monophosphate with the help of pulsed EPR techniques like ENDOR and HYSCORE are described.

Metal ions bind to nucleic acids at various positions. This binding can be modulated by using metal complexes with appropriate ligands. Novel mono- and especially dinuclear metal complexes could be a powerful tool to detect rare, but still physiologically relevant, forms of DNA, e.g. the left-handed Z-DNA. In this review, our recent research activities in this area of bioinorganic chemistry are summarized. A special emphasis is laid on the synthetic challenges that arose upon the synthesis of the polyamine ligands. Further, some rather unusual approaches to elucidate the solution structure of copper bound to guanosine monophosphate with the help of pulsed EPR techniques like ENDOR and HYSCORE are described.

The new macrocyclic ligand 15-fluoro-15-methyl-1,4,7,10,13-pentaazacyclohexadecan-14,16-dione (2) was synthesised and its crystal structure determined together with the ones of the known analogues of 2, 15-fluoro-1,4,7,10,13-pentaazacyclohexadecan-14,16-dione (1) and 15,15-difluoro-1,4,7,10,13-pentaazacyclohexadecan-14,16-dione (3). The binding behaviour of all three ligands to copper and zinc was studied in the solid state. They can bind to the metal centre by either triple coordination (N3) with all secondary amines or after double deprotonation of the two amides with all five nitrogen atoms (N5). The N5 coordination mode is favoured by the presence of one or two fluorine substituents at the C(15) position and by a high pH in the case of aqueous solutions. Circular dichroism titrations of poly d(GC) with the metal complexes showed that only 4 and 5, that is the copper complexes of 1 and 2, induced a complete B- to Z-DNA transition. The degree of cooperativity of the transition was found to be 3.4 and 7.3 for 4 and 5 respectively. As a possible hypothesis to explain this difference, the additional methyl group in 5 compared with 4 may be involved in a hydrophobic interaction with the DNA. Ligand 2, the copper complex 6 of the bis fluoro substituted ligand 3, and the zinc complex 7 of ligand 1 did not induce any change in the direction of Z-DNA. In the case of 6, the CD spectrum of the DNA actually showed no change at all, indicating that the complex was even not interacting with the B form of DNA. Therefore it is assumed that the bis fluoro substitution is causing the complex to be in the neutral N5 coordination mode at the experimental conditions of pH 7. The electrostatic contribution together with the shielding effect of the ligand might explain the absence of any interaction with the DNA.