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Gα proteins as part of heterotrimeric G proteins are molecular switches essential for G protein-coupled receptor- mediated intracellular signaling. The role of the Gα subunits has been examined for decades with various guanine nucleotides to elucidate the activation mechanism and Gα protein-dependent signal transduction. Several approaches describe fluorescent ligands mimicking the GTP function, yet lack the efficient estimation of the proteins' GTP binding activity and the fraction of active protein. Herein, we report the development of a reliable fluorescence anisotropy-based method to determine the affinity of ligands at the GTP-binding site and to quantify the fraction of active Gαi1 protein. An advanced bacterial expression protocol was applied to produce active human Gαi1 protein, whose GTP binding capability was determined with novel fluorescently labeled guanine nucleotides acting as high-affinity Gαi1 binders compared to the commonly used BODIPY FL GTPγS. This study thus contributes a new method for future investigations of the characterization of Gαi and other Gα protein subunits, exploring their corresponding signal transduction systems and potential for biomedical applications.

The applicability of radioligands for targeted endoradionuclide therapy is limited due to radiation-induced toxicity to healthy tissues, in particular to the kidneys as primary organs of elimination. The targeting of enzymes of the renal brush border membrane by cleavable linkers that permit the formation of fast eliminating radionuclide-carrying cleavage fragments gains increasing interest. Herein, we synthesized a small library of

The potential of designing irreversible alkyne-based inhibitors of cysteine cathepsins by isoelectronic replacement in reversibly acting potent peptide nitriles was explored. The synthesis of the dipeptide alkynes was developed with special emphasis on stereochemically homogenous products obtained in the Gilbert-Seyferth homologation for C-C triple bond formation. 23 dipeptide alkynes and 12 analogous nitriles were synthesized and investigated for their inhibition of cathepsins B, L, S and K. Numerous combinations of residues in the P1 and P2 positions as well as terminal acyl groups, allowed for the derivation of extensive structure-activity relationships, which were rationalized by computational covalent docking for selected examples. The determined inactivation constants of the alkynes at the target enzymes span a range of more than three orders of magnitude (3-10,133 M-1s-1). Notably, the selectivity profiles of alkynes do not necessarily reflect those of the nitriles. Inhibitory activity at the cellular level was demonstrated for selected compounds.

The intentional binding of radioligands to albumin gains increasing attention in the context of radiopharmaceutical cancer therapy as it can lead to an enhanced radioactivity uptake into the tumor lesions and thus, to a potentially improved therapeutic outcome. However, the influence of the radioligand’s albumin binding affinity on the time profile of tumor uptake depending on the particular albumin-binding affinity have been only partly addressed so far. Based on the previously identified Nε-4-(4-iodophenyl)butanoyl-lysine scaffold, we designed “clickable” lysine-derived albumin binders (cLABs) and determined their dissociation constants towards albumin by novel assay methods. Structure-activity relationships were derived and selected cLABs were applied for the modification of the somatostatin receptor subtype-2 ligand (Tyr³)octreotate (TATE). These novel conjugates were radiolabeled with copper-64 and subjected to a detailed in vitro and in vivo radiopharmacological characterization. Overall, the results of this study provide an incentive for further investigations of albumin binders for applications in endoradionuclide therapies.

The transamidase activity of transglutaminase 2 (TGase 2) is considered to be important for several pathophysiological processes including fibrotic and neoplastic tissue growth, whereas in healthy ...

Immunotherapy using CAR-T cells is a new paradigm technology for cancer treatment. To avoid severe side effects and tumor escape variants observed for conventional CAR-T cells approach, adaptor CAR technologies are under development, where intermediate target modules redirect immune cells against cancer. In this work, silicon nanowire field effect transistors are used to assist in the development of target modules for an optimized CAR-T cell operation. Focusing on a library of seven variants of E5B9 peptide that is used as CAR peptide epitope, we performed multiplexed binding tests in serum using nanosensor chips. Peptides have been immobilized onto the sensor to compare the signals of transistor upon titration with anti-E5B9 antibodies. Correlation analysis of binding affinities and sensitivities enabled a selection of best candidates for the interaction between CAR and target modules. Finally, cytotoxic functionality of CAR-T cells in combination with the selected target modules were successfully proven. Our results open the perspective for the nanobiosensorics to go beyond the early diagnostics in the field of clinical cancer research, and paves the way towards personalization and efficient monitoring of the immunotherapeutic treatment, where the quantitative analysis with the standard techniques is not an option.

Objective: The somatostatin receptor subtype 2 (SSTR2) is a suitable target for the diagnosis and therapy of neuroendocrine tumors using radiolabeled ligands [1]. BON-1S cells have been described to express SSTR2 receptors in vitro [2]. We therefore established 2D and 3D cell culture systems of BON-1S cells. For SSTR2 ligand binding, BON-1S cells cultured as monolayer or spheroids were incubated with an SSTR2 agonist (NODAGA-TATE) or an SSTR2 antagonist (NODAGA-JR11). Both were labeled with either 68Ga- or 64Cu and their respective binding and uptake were determined. Understanding the binding data is a prerequisite for using spheroids of BON-1S cells in a Micro Physiological System (MPS) [3]. Methods: Spheroids of SSTR2 containing BON-1S cells were created as follows: Freshly harvested cells were incubated with magnetic nanoparticles in RPMI medium overnight. After magnetic particle uptake, cells were split into a 24-well-plate (1.5 x 10-4 cells/well). In an incubator, the magnetic particles were moved via a magnetic drive for five days, to allow the formation of spheroids [4]. After incubation with the 68Ga- or 64Cu-labeled radioligands (RCY>95%), the BON-1S cells, either as monolayer or as spheroids, were washed and treated with acidic glycine buffer (pH 3.0) to remove the bound but not yet internalized molecules from the outer cellular membrane. After this washing step, the remaining radioligand molecules are assumed to represent internalized molecules [5]. In the monolayer, internalization was determined after 10 minutes, 1 hour and 3 hours after addition of the radioligand to the respective culture. Results: Saturation analysis of radiolabeled NODAGA-TATE as well as NODAGA-JR11 on BON-S1 monolayer yielded similar Kd values in the low nanomolar range (2.3 to 8.6 nM). Affinity of the respective radiolabeled ligand was much lower on BON-1S spheroids, with Kd values around 30 nM for both 64Cu-labeled ligands and almost 60 nM for the 68Ga-labeled ligands. The maximal binding capacity (Bmax) was higher on monolayers and spheroids for radiolabeled JR11 (NODAGA-TATE: monolayer 0.3 pmol/mg, spheroids 0.8 pmol/mg; NODAGA-JR11: monolayer and spheroids 1.2 pmol/mg). The internalization assay showed a ratio of internalized to surface bound radiolabeled NODAGA-TATE of about 80% to 20% for both forms of cell culture, for radiolabeled JR11 a ratio of 8% to 92% was determined, each after a 60 min-incubation time. The internalization after the three time points was ca. 10 times lower in case of radiolabeled JR11 (0.25% (TATE) versus maximal 0.03% (JR11) of administered activity amount/µg protein). Conclusions: Using monolayer or spheroids of BON-1S cells (the latter were generated by the iron nanoparticle method) we could estimate reliable and reproducible binding parameters for a radiolabeled agonist and antagonist of SSTR2. There are obvious differences in binding affinities of the examined radiolabeled conjugates in monolayer cell culture compared to the spheroids. Acknowledgements: The SSTR2 expressing BON-1S cells were kindly given by Prof. Dr. Wiedenmann, University Hospital Charité Berlin. References: [1] Eychenne, Molecules 2020, 25, 4012. [2] Exner, Front Endocrinol 2018, 9, 146. [3] Busek, J Sens Sens Syst 2016, 5, 228. [4] Noel, J Vis Exp 2017, 126, e56081. [5] Matzku, Cancer Res 1986, 46, 3848.

Tissue transglutaminase (TGase 2) is proposed to be important for biomaterial–tissue interactions due to its presence and versatile functions in the extracellular environment. TGase 2 catalyzes the cross-linking of proteins through its Ca2+-dependent acyltransferase activity. Moreover, it enhances the interactions between fibronectin and integrins, which in turn mediates the adhesion, migration, and motility of the cells. TGase 2 is also a key player in the pathogenesis of fibrosis. In this study, we investigated whether TGase 2 is present at the biomaterial–tissue interface and might serve as an informative biomarker for the visualization of tissue response toward gelatin-based biomaterials. Two differently cross-linked hydrogels were used, which were obtained by the reaction of gelatin with lysine diisocyanate ethyl ester. The overall expression of TGase 2 by endothelial cells, macrophages, and granulocytes was partly influenced by contact to the hydrogels or their degradation products, although no clear correlation was evidenced. In contrast, the secretion of TGase 2 differed remarkably between the different cells, indicating that it might be involved in the cellular reaction toward gelatin-based hydrogels. The hydrogels were implanted subcutaneously in immunocompetent, hairless SKH1-Elite mice. Ex vivo immunohistochemical analysis of tissue sections over 112 days revealed enhanced expression of TGase 2 around the hydrogels, in particular at days 14 and 21 post-implantation. The incorporation of fluorescently labeled cadaverine derivatives for the detection of active TGase 2 was in accordance with the results of the expression analysis. The presence of an irreversible inhibitor of TGase 2 led to attenuated incorporation of the cadaverines, which verified the catalytic action of TGase 2. Our in vitro and ex vivo results verified TGase 2 as a potential biomarker for tissue response toward gelatin-based hydrogels. In vivo, no TGase 2 activity was detectable, which is mainly attributed to the unfavorable physicochemical properties of the cadaverine probe used.

An O-methyltyrosine-containing azadipeptide nitrile was synthesised and investigated for its inhibitory activity towards cathepsins L, S, K, and B. Labelling with carbon-11 was accomplished by reaction of the corresponding phenolic precursor with [11 C]methyl iodide starting from cyclotron-produced [11 C]methane. Radiopharmacological evaluation of the resulting radiotracer in a mouse xenograft model derived from a mammary tumour cell line by small animal PET imaging indicates tumour targeting with complex pharmacokinetics. Radiotracer uptake in the tumour region was considerably lower under treatment with the nonradioactive reference compound and the epoxide-based irreversible cysteine cathepsin inhibitor E64. The in vivo behaviour observed for this radiotracer largely confirms that of the corresponding 18 F-fluoroethylated analogue and suggests the limited suitability of azadipeptide nitriles for the imaging of tumour-associated cysteine cathepsins despite target-mediated uptake is evidenced.

Digital, Immunotherapy using CAR-T cells is a new technological paradigm for cancer treatment. To avoid severe side effects and tumor escape variants observed for conventional CAR-T cells approach, adaptor CAR technologies are under development, where intermediate target modules redirect immune cells against cancer. In this work, silicon nanowire field-effect transistors are used to develop target modules for an optimized CAR-T cell operation. Focusing on a library of seven variants of E5B9 peptide that is used as CAR targeting epitope, we performed multiplexed binding tests using nanosensor chips. These peptides had been immobilized onto the sensor to compare the transistor signals upon titration with anti-La 5B9 antibodies. The correlation of binding affinities and sensor sensitivities enabled a selection of candidates for the interaction between CAR and target modules. An extremely low detection limit was observed for the sensor, down to femtomolar concentration, outperforming the current assay of the same purpose. Finally, the CAR T-cells redirection capability of selected peptides in target modules was proven successful in an in-vitro cytotoxicity assay. Our results open the perspective for the nanosensors to go beyond the early diagnostics in clinical cancer research towards developing and monitoring immunotherapeutic treatment, where the quantitative analysis with the standard techniques is limited., Ciencias Médicas y de la Salud

The transamidase activity of transglutaminase 2 (TGase 2) is considered to be important for several pathophysiological processes including fibrotic and neoplastic tissue growth, whereas in healthy cells this enzymatic function is predominantly latent. Methods that enable the highly sensitive detection of TGase 2, such as application of radiolabeled activity-based probes, will support the exploration of the enzyme's function in various diseases. In this context, the radiosynthesis and detailed

The inducible isoenzyme cyclooxygenase-2 (COX-2) is closely associated with chemo-/radioresistance and poor prognosis of solid tumors. Therefore, COX-2 represents an attractive target for functional characterization of tumors by positron emission tomography (PET). In this study, the celecoxib derivative 3-([

The cell surface receptor claudin-4 (Cld-4) is upregulated in various tumours and represents an important emerging target for both diagnosis and treatment of solid tumours of epithelial origin. The C-terminal fragment of the Clostridium perfringens enterotoxin cCPE290-319 appears as a suitable ligand for targeting Cld-4. The synthesis of this 30mer peptide was attempted via several approaches, which has revealed sequential SPPS using three pseudoproline dipeptide building blocks to be the most efficient one. Labelling with fluorine-18 was achieved on solid phase using N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) and 4-[18F]fluorobenzoyl chloride as 18F-acylating agents, which was the most advantageous when [18F]SFB was reacted with the resin-bound 30mer containing an N-terminal 6-aminohexanoic spacer. Binding to Cld-4 was demonstrated via surface plasmon resonance using a protein construct containing both extracellular loops of Cld-4. In addition, cell binding experiments were performed for 18F-labelled cCPE290-319 with the Cld-4 expressing tumour cell lines HT-29 and A431 that were complemented by fluorescence microscopy studies using the corresponding fluorescein isothiocyanate-conjugated peptide. The 30mer peptide proved to be sufficiently stable in blood plasma. Studying the in vivo behaviour of 18F-labelled cCPE290-319 in healthy mice and rats by dynamic PET imaging and radiometabolite analyses has revealed that the peptide is subject to substantial liver uptake and rapid metabolic degradation in vivo, which limits its suitability as imaging probe for tumour-associated Cld-4.

A reliable solution-phase synthesis of the water-soluble dipeptidic fluorogenic transglutaminase substrate Z-Glu(HMC)-Gly-OH is presented. The route started from Z-Glu-OH, which was converted into the corresponding cyclic anhydride. This building block was transformed into the regioisomeric α- and γ-dipeptides. The key step was the esterification of Z-Glu-Gly-OtBu with 4-methylumbelliferone. The final substrate compound was obtained in an acceptable yield and excellent purity without the need of purification by RP-HPLC. The advantage of this acyl donor substrate for the kinetic characterisation of inhibitors and amine-type acyl acceptor substrates is demonstrated by evaluating commercially available or literature-known irreversible inhibitors and the biogenic amines serotonin, histamine and dopamine, respectively.

Transglutaminase 2 (TGase 2)-catalyzed transamidation represents an important post-translational mechanism for protein modification with implications in physiological and pathophysiological conditions, including fibrotic and neoplastic processes. Consequently, this enzyme is considered a promising target for the diagnosis of and therapy for these diseases. In this study, we report on the synthesis and kinetic characterization of Ne-acryloyllysine piperazides as irreversible inhibitors of TGase 2. Systematic structural modifications on 54 new compounds were performed with a major focus on fluorine-bearing substituents due to the potential of such compounds to serve as radiotracer candidates for positron emission tomography. The determined inhibitory activities ranged from 100 to 10 000 M–1 s–1, which resulted in comprehensive structure–activity relationships. Structure–activity correlations using various substituent parameters accompanied by covalent docking studies provide an advanced understanding of the...

Objectives: Finding optimal 18F-fluorination conditions represents a critical and often time-consuming step in radiotracer development. Microfluidic or lab-on-a-chip devices are modern tools for that purpose but not accessible everywhere. Inspired by 18F-chemistry in low volumes based on a MAX/MCX-trapping technique for one complete 18F-batch,[1] we herein present an 18F-labeling approach using low volume aliquots (50 reactions/day) using minimal amounts of precursor. Utilizing mixtures of 2-3% water in acetonitrile for 18F-elution allowed for radiolabeling even without azeotropic drying in certain cases, which can further accelerate the entire labeling process. For more demanding radiolabeling reactions, drying of only 3 min with helium furnished highly reactive [18F]fluoride with comparable reactivity even several hours after the elution step. This optimization workflow was used successfully to identify high yielding reaction conditions for a variety of precursor molecules like aliphatic tosylates or mesylates as well as aromatic sydnones, boronic acids or trimethylammonium salts. As examples, optimization results for 18F-labeling of the FDG mannose triflate precursor and the tosylate precursor of celecoxib-derivative [18F]A[3] are shown in Figure 1. Transfer into isolated RCY at activity levels suitable for preclinical evaluations was achieved (e.g. for [18F]A: 25-30% after 18F-labeling in acetonitrile at 90°C and purification) and turned out to be comparable to results from conventional automated radiosyntheses. Conclusion: This microliter scale optimization and labeling procedure can easily be adapted and transferred to the synthesis of known and new radiotracers. The straightforward approach represents a valuable radiochemistry tool to enter rapidly into subsequent preclinical studies. References: [1] Iwata (2018), J.Labelled.Comp.Radiopharm., 61, 540; [2] Kniess (2017), Appl.Radiat.Isot 127, 260; [3] Laube (2020), RSC Advances, 10, 38601. Figure 1. Setup of HPLC vial experiments and representative results of optimization.

Selected

Tissue transglutaminase (TGase 2) is the most abundantly expressed enzyme of the transglutaminase family and involved in a large variety of pathological processes, such as neurodegenerative diseases, disorders related to autoimmunity and inflammation as well as tumor growth, progression and metastasis. As a result, TGase 2 represents an attractive target for drug discovery and development, which requires assays that allow for the characterization of modulating agents and are appropriate for high-throughput screening. Herein, we report a fluorescence anisotropy-based approach for the determination of TGase 2's transamidase activity, following the time-dependent increase in fluorescence anisotropy due to the enzyme-catalyzed incorporation of fluorescein- and rhodamine B-conjugated cadaverines 1-3 (acyl acceptor substrates) into N,N-dimethylated casein (acyl donor substrate). These cadaverine derivatives 1-3 were obtained by solid-phase synthesis. To allow efficient conjugation of the rhodamine B moiety, different linkers providing secondary amine functions, such as sarcosyl and isonipecotyl, were introduced between the cadaverine and xanthenyl entities in compounds 2 and 3, respectively, with acyl acceptor 3 showing the most optimal substrate properties of the compounds investigated. The assay was validated for the search of both irreversible and reversible TGase 2 inhibitors using the inactivators iodoacetamide and a recently published L-lysine-derived acrylamide and the allosteric binder GTP, respectively. In addition, the fluorescence anisotropy-based method was proven to be suitable for high-throughput screening (Z' factor of 0.86) and represents a non-radioactive and highly sensitive assay for determining the active TGase 2 concentration.

The cell surface receptor claudin-4 (Cld-4) is upregulated in various tumours and represents an important emerging target for both diagnosis and treatment of solid tumours of epithelial origin. The C-terminal fragment of the Clostridium perfringens enterotoxin cCPE

Transglutaminase 2 (TGase 2)-catalyzed transamidation represents an important post-translational mechanism for protein modification with implications in physiological and pathophysiological conditions, including fibrotic and neoplastic processes. Consequently, this enzyme is considered a promising target for the diagnosis of and therapy for these diseases. In this study, we report on the synthesis and kinetic characterization of N

Until recently, alkynes were considered bioinert. Thus, they are popular reaction partners in bioorthogonal click reactions in vitro and in vivo. Despite the virtual chemical inertness of the alkyne moiety, two research groups observed the irreversible inhibition of a cysteine protease by an alkyne functionalised substrate derivative: both EKKEBUS et al. and SOMMER et al. independently described the unexpected inactivation of de-ubiquitinating enzymes by propargylated ubiquitin or ubiquitin-like modifiers bearing propargylamine in place of C-terminal glycine [1, 2]. We intended to harness that finding for the design of inhibitor-based probes for the imaging of tumour-associated cysteine proteases. Cysteine cathepsins play an important role in tumour progression. In particular, cathepsin B is involved in a variety of tumour progression-related processes and an elevated extracellular levels are linked to increased malignancy and poor prognosis [3]. Therefore, this enzyme represents a promising target for the therapy and imaging of tumours. GREENSPAN et al. reported a potent and highly selective, dipeptidyl nitrile-based cathepsin B inhibitor (N-[2-[(3-Carboxyphenyl)methoxy]-1(S)-cyanoethyl]-3-methyl-N2-(2,4-difluorobenzoyl)-L-phenylalaninamide) [4]. Based on that lead compound, cathepsin B-targeting dipeptide alkynes were designed by isoelectronic replacement of the nitrile nitrogen atom by by a methine group and consecutive variation of the 2,4-difluorobenzoyl and (3-carboxybenzyl)oxymethyl residue. Formation of the C-C triple bond by reaction of the corresponding open-chain serine-derived aldehyde with the Bestmann-Ohira reagent was accompanied by partial enantiomerisation. Therefore, the synthesis was performed via Garner’s aldehyde, which accounted for high stereochemical purity of the final compounds. The inhibitory potential was investigated against cathepsins B, S, L and K. The most potent compound exhibited irreversible inhibition of cathepsin B with an inactivation constant (kinact/KI=771 M-1s-1). Values for cathepsins L, S and K were significantly lower; no irreverisible ihibition was observed for cathepsin K. In addition, inhibition of cathepsin B activity in human glioblastoma cell lysates and living cells has been demonstrated. Based on these promising results, dipeptidyl alkynes have the potential to become a valuable tool for imaging due to the expected low activity towards other cysteine proteases. In further studies, selected inhibitors for cathepsin B will be labelled with suitable radionuclides to obtain an inhibitor-based probe directed towards cathepsin B. [1] Ekkebus et al., J. Am. Chem. Soc., 2013, 135, 2867-2870 [2] Sommer et al., Bioorg. Med. Chem., 2013, 21, 2511-2517 [3] Löser and Pietzsch, Front. Chem., 2015, 3:37 [4] Greenspan et al., J. Med. Chem., 2001, 44, 4524-4534.

In our understanding of the many drivers of malignant progression and cancer metastasis, proteases are increasingly drawn into the spotlight as crucial agents in cancer angiogenesis, invasion, and metastasis [1]. Elevated activities of multiple members of the cathepsin family have been shown to correlate with increased metastasis and high therapy resistance [2, 3]. Especially high expression levels of extracellular cathepsin B (CTB) indicate poor prognosis in neoplastic diseases, making CTB an interesting target for activity-based molecular imaging in diagnostics as well as in therapy monitoring for personalised therapies. It is our aim to develop such a probe by combination of a polyarginine-based, activatable cell penetrating peptide (ACPP), as first described by R. Tsien, and an optimised endopeptidase substrate for CTB [4]. Substrate optimisation proofed to be challenging as two entirely opposite factors needed to be balanced – high stability against serum proteases to prevent premature cleavage of the activation sequence, while retaining efficient and specific endoproteolytic cleavability by CTB. We have generated a CTB-endoprotease substrate by C-terminally elongating the CTB carboxydipeptidase substrate Abz GIVR*AK(Dnp) OH (Abz - aminobenzoyl, Dnp – dinitrophenyl, * – cleavage site), described by Cotrin et al. in 2004, to the octapeptide Abz GIVR*AK(Dnp)GX CONH2, which could be used as activation site in the final ACPP [5]. Introduction of any amino acid other than glycine at the P4’ position resulted in hysteretic kinetics for the CTB-catalysed hydrolysis of the octapeptides, which might indicate the displacement of the occluding loop from the active site upon interaction with the substrates. Using LC-ES-MS-based analysis of serum-incubated substrates, the positions P1 and P3’ were determined to be primary determinants of serum stability. After suppression of the P3’ instability by Nα-methylation and optimisation within the positions P1-P3, we were able to increase serum half-life from < 5 min to > 1440 min under concomitant improvement of kinetic substrate efficiency towards CTB. Currently, the substrate is optimised towards CTB-specificity within the cathepsin protease family. Additionally, cell uptake studies of a fluorescently labelled ACPP using the optimised CTB-endoprotease substrate sequence are ongoing. Using this fluorescent probe, we plan to study cell uptake, in vivo stability and initial biodistribution. Furthermore, an ACPP conjugated to radiometal-chelating entities is currently prepared, which will allow for PET imaging in vivo. Literature (1) Yang et al., Cancer Growth Metastasis 2009, 2, 13-27 (2) Aggarwal and Sloane, Proteomics Clin. Appl. 2014, 8(5-6), 427-437 (3) Löser and Pietzsch, Front. Chem. 2015, 3, article 37 (4) Jiang et al., PNAS, 2004, 101(51), 17867-17872 (5) Cotrin et al., Anal. Biochem. 2004, 335, 244-252

A robust and versatile method for the synthesis of selected azide, alkyne and phosphane functionalized coumarin derivatives from the corresponding 7-amino and 7-hydroxy analogs for an application in bioorthogonal conjugation and labeling reactions like the Staudinger Ligation or the Huisgen-click reaction is presented. Further, the convenient preparation of modified coumarins was performed and subsequent regio- and chemo-selective labeling reactions delivered the desired biologically active derivatives in high yields. These new compounds may thus constitute attractive scaffolds for designing novel fluorescence building blocks for various challenging biolabeling applications.

Small glutamate-containing peptides bearing coumarin derivatives as fluorescent leaving groups attached to the γ-carboxylic acid group of the Glu residue were synthesised and investigated with regard to their potential to act as substrates for transglutaminase 2 (TGase 2). Their synthesis was accomplished by an efficient solid-phase approach. The excellent water solubility of the compounds enabled their extensive kinetic characterisation in the context of TGase 2-catalysed hydrolysis and aminolysis. The influence of the coumarin skeleton's substitution pattern on the kinetic properties was studied. Derivatives containing 7-hydroxy-4-methylcoumarin (HMC) revealed properties superior to those of their 7-hydroxycoumarin counterparts; analogous amides are not accepted as substrates. Z-Glu(HMC)-Gly-OH, which exhibited the best substrate properties out of the investigated derivatives, was selected for representative kinetic characterisation of acyl acceptor substrates and irreversible inhibitors.

The collagen telopeptides play an important role for lysyl oxidase-mediated crosslinking, a process which is deregulated during tumour progression. The DEKS motif which is located within the N-terminal telopeptide of the α1 chain of type I collagen has been suggested to adopt a βI-turn conformation upon docking to its triple-helical receptor domain, which seems to be critical for lysyl oxidase-catalysed deamination and subsequent crosslinking by Schiff-base formation. Herein, the design and synthesis of cyclic peptides which constrain the DEKS sequence in a β-turn conformation will be described. Lysine-side chain attachment to 2-chlorotrityl chloride-modified polystyrene resin followed by microwave-assisted solid-phase peptide synthesis and on-resin cyclisation allowed for an efficient access to head-to-tail cyclised DEKS-derived cyclic penta- and hexapeptides. An N(ε)-(4-fluorobenzoyl)lysine residue was included in the cyclopeptides to allow their potential radiolabelling with fluorine-18 for PET imaging of lysyl oxidase. Conformational analysis by (1)H NMR and chiroptical (electronic and vibrational CD) spectroscopy together with MD simulations demonstrated that the concomitant incorporation of a D-proline and an additional lysine for potential radiolabel attachment accounts for a reliable induction of the desired βI-turn structure in the DEKS motif in both DMSO and water as solvents. The stabilised conformation of the cyclohexapeptide is further reflected by its resistance to trypsin-mediated degradation. In addition, the deaminated analogue containing allysine in place of lysine has been synthesised via the corresponding ε-hydroxynorleucine containing cyclohexapeptide. Both ε-hydroxynorleucine and allysine containing cyclic hexapeptides have been subjected to conformational analysis in the same manner as the lysine-based parent structure. Thus, both a conformationally restricted lysyl oxidase substrate and product have been synthetically accessed, which will enable their potential use for molecular imaging of these important enzymes.

Tissue transglutaminase (transglutaminase 2) is a multifunctional enzyme with many interesting properties resulting in versatile roles in both physiology and pathophysiology. Herein, the particular involvement of the enzyme in human diseases will be outlined with special emphasis on its role in cancer and in tissue interactions with biomaterials. Despite recent progress in unraveling the different cellular functions of transglutaminase 2, several questions remain. Transglutaminase 2 features in both confirmed and some still ambiguous roles within pathological conditions, raising interest in developing inhibitors and imaging probes which target this enzyme. One important prerequisite for identifying and characterizing such molecular tools are reliable assay methods to measure the enzymatic activity. This digest Letter will provide clarification about the various assay methods described to date, accompanied by a discussion of recent progress in the development of inhibitors and imaging probes targeting transglutaminase 2.