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Saturation binding data

The applicability of radioligands for targeted endoradionuclide therapy is limited due to radiation-induced deleterious effects to healthy tissues. This applies in particular to the kidneys as primary organs of elimination, which requires dosimetric estimates to justify internal radionuclide therapy. In this context, the targeting of enzymes of the renal brush border membrane by cleavable linkers between target molecule and radiolabel that permit the formation of fast eliminating radionuclide-carrying cleavage fragments gains increasing interest. Herein, we synthesized a small library of ⁶⁴Cu-labeled cleavable linkers and quantified their substrate potentials toward neprilysin (NEP), a highly abundant peptidase at the renal brush border membrane. This allowed for the derivation of structure-activity relationships and selected cleavable linkers were attached to the somatostatin receptor subtype 2 ligand [Tyr³]octreotate. Subsequent radiopharmacological characterization revealed that a substrate-based targeting of NEP in the kidneys with small molecules or peptides entails a certain degree of premature cleavage in the blood circulation by soluble and endothelium-derived NEP. However, for a tissue-specific targeting of NEP in the kidneys, the additional targeting of albumin in the blood by albumin-binding moieties is highlighted.

Primärdaten zum FA-Assay, activity-based ELISA und ELISA

Objectives: The Ca2+-dependent transamidase activity of transglutaminase 2 (TGase2) is tightly regulated in healthy cells but can be utilized by various cancer cells to support their survival and progression. Therefore, molecules targeting this enzyme are promising candidates for the functional characterization of TGase2 in tumors. Recently, we developed an 18F-labeled irreversible inhibitor and highlighted its potential as radiometric tool for the in vitro characterization of TGase2. Herein, we report on the kinetic characterization of a 123I-labeled Nε-acryloyllysine, [123I]1, and its use for quantifying the functional expression of TGase2 in tissues Methods: [123I]1 was synthesized as recently presented [1]. The inhibitory potency of [123I]1 by means of its kinact/KI value was determined by a radio-TLC method using recombinant human TGase2. In vitro autoradiography was performed with fresh-frozen sections (12 µm) of several organs (heart, kidney, liver, spleen, and muscle), extracted from healthy NMRI nude mice. Binding experiments with [123I]1 were conducted at 0.7 MBq/mL in MOPS buffer at pH 7.4 containing 3 mM CaCl2 and 5 mM DTT. Non-specific binding was assessed in the presence of the TGase2 inhibitor Z006. Different molar activities (Am) were adjusted by the addition of compound 1. Results: [123I]1 was reliably obtained in high (radio)chemical purities of >99% and radiochemical yields of 79±6% (n=8). The Am was determined to be >6 TBq/µmol and the kinact/KI value to be 10,200 M-1s-1 (±1,000). Association (Figure 1) of n.c.a. [123I]1 at 37°C over 4 h to tissue sections furnished a high binding capacity and excellent ratios of total binding (TB) to non-specific binding (NSB). However, assessment of the quantitative TGase2 expression is limited as the inhibition rate at n.c.a. level is too low to achieve complete radioligand binding. Therefore, Am values of 70, 14, 7, and 1 GBq/µmol were adjusted to increase the association rates. A value of 7 GBq/µmol appeared to be optimal based on the extent of binding and the TB/NSB ratios. Higher Am values of 70 and 14 GBq/µmol still led to incomplete reaction and thus a lower apparent TGase2 concentration. In contrast, a Am value of 1 GBq/µmol resulted in extensive self-block, as indicated by an increased NSB (Figure 1). The highest TGase2 concentration has been observed in the heart and was lowest in muscle, with values of 1.3 and 0.2 pmol/mm3, respectively. Dissociation of [123I]1 under similar conditions proved the irreversible binding to TGase2 as only a minimal amount (

Polyamines are highly attractive vectors for tumor targeting, particularly with regards to the development of radiolabeled probes for imaging by positron emission (PET) and single-photon emission computed tomography (SPECT). However, the synthesis of selectively functionalized derivatives remains challenging owing to the presence of multiple amino groups of similar re-activity. In this work, we established a synthetic methodology for the selective mono-fluorobenz(o)ylation of various biogenic diamines and polyamines as lead compounds for the perspective development of substrate-based radiotracers for targeting polyamine-specific membrane transporters and enzymes such as transglutaminases. To this end, the polyamine scaffold was constructed by solid-phase synthesis of the corresponding oxopolyamines and subsequent re-duction with BH₃/THF. Primary and secondary amino groups were selectively protected using Dde and Boc as protecting groups, respectively, in orientation to previously reported proce-dures, which enabled the selective introduction of the reporter groups. For example, N¹-FBz-spermidine, N⁴-FBz-spermidine, N⁸-FBz-spermidine, and N¹-FBz-spermine and N⁴-FBz-spermine (FBz=4-fluorobenzoyl) were obtained in good yields by this approach. The advantages and disadvantages of this synthetic approach are discussed in detail and its suitability for radiolabeling was demonstrated for the solid-phase synthesis of N¹-[¹⁸F]FBz-cadaverine.

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 radiolabelled activity-based probes, will support the exploration of the enzyme’s function in various diseases. In this context, the radiosynthesis and detailed in vitro radiopharmacological evaluation of an 18F-labelled Nε-acryloyllysine piperazide is reported. Robust and facile detection of the radiotracer-TGase 2 complex by autoradiography of thin layer plates and polyacrylamide gels after chromatographic and electrophoretic separation owing to irreversible covalent bond formation was demonstrated for the isolated protein, cell lysates and living cells. Using this radiotracer, quantitative data on the expression profile of activatable TGase 2 in mouse organs and selected tumours were obtained for the first time by autoradiography of tissue sections.

Despite given high affinity and selectivity towards their distinct biological targets, theranostic radioligands can suffer strongly from fast blood clearance and metabolism, which limits the enrichment of the compounds in the tumour tissue. A viable strategy to increase the bioavailability of substances is the conjugation to human serum albumin (HSA). In this context, Nε-4-(4-iodophenyl)butanoyllysine (Nε-IPB-lysine) was recently discovered as potent albumin binder.[1] However, conjugation of Nε-IPB-lysine via its α-amino group to proteins, peptides or small molecules is challenging, as a free α-carboxyl group is required to maintain good binding to HSA. To allow the broad applicability and late-stage introduction of Nε-IPB-lysine we present a novel approach of Cu-catalyzed azide-alkyne cycloaddition (CuAAC) using Nε-IPB-lysine derivatives with azide/alkyne bearing moieties at the α-amino group. For small scales, a solid phase synthesis strategy starting from Fmoc-Lys(Alloc)-OH was developed. For upscaling, a 3-step synthesis starting from Boc-Lys-OH has been established. The HSA binding affinities were determined using microscale thermophoresis and a fluorescence-based competition assay. A library of azide/alkyne containing compounds with a binding affinity to HSA ranging from 0.4 µM to 100 µM could be obtained. To demonstrate the suitability of the novel albumin binders, Nα-5-azidopentanoyl-Nε-IPB-D-lysine (Kd = 8 µM) was coupled to the somatostatin receptor subtype 2 ligand NODAGA-Pra-O2Oc-TATE 1 by on-resin CuAAC. For radiolabelling, copper-64 was used. The resulting conjugate [64Cu]Cu-2 showed significantly improved binding to HSA compared to [⁶⁴Cu]Cu-1, [⁶⁴Cu]Cu-NODAGA-TATE, and [⁶⁴Cu]Cu-DOTA-TATE. All TATE derivatives were studied by PET imaging using a mouse phaechromocytoma (MPC)-allograft model highlighting an increased blood circulation time of [⁶⁴Cu]Cu-2 and higher tumor uptake at late time points (24 h) compared to the other radiotracers. Current studies aim to further optimise the biodistribution by selecting HSA binders of higher or lower binding affinity. Literature: [1] Dumelin et al. Angew. Chem. Int. Ed. Engl. 2008, 47(17), 3196-3201.

Files of NMR data for the different fluorobenz(o)ylated polyamines and radiosynthesis data

radio-TLC, radio-HPLC, radio-SDS, in vitro autoradiography

The development of radiotracers for transglutaminase 2 (TGase 2) based on irreversible inhibitors appears highly attractive to further uncover the role of that enzyme for the emergence and progression of various tumours. [1] In this context, the ¹⁸F-labelled analogue of Nε-acryloyl-L-lysine 1, developed in our lab, showed great potential as radiometric tool for in vitro investigations. However, application for in vivo imaging is strongly limited by its unfavourable pharmacokinetic properties such as fast blood clearance and metabolism. A common approach to improve biodistribution and blood circulation time of drugs is the reversible conjugation to human serum albumin (HSA). Recently, compound 2 was identified as HSA binder with good affinity (Kd = 3.2 µM). [2] Successful application of 2 was demonstrated for pharmacokinetic tuning of various radiolabelled compounds, but rather for hydrophilic than for hydrophobic molecules. [3] Moreover, compound 2 offers the possibility for radiolabelling with iodine-123. [4] For the purpose to develop radiotracers for TGase 2 with HSA binding capability, we designed compound 3 as lead structure containing the HSA binding moiety of 2 linked via a triazole ring as an amide bioisostere to the part of the TGase 2-inhibitor. Conjugation of alkyne-functionalised Nε-acryloyl-L-lysines and Boc-protected (R)-6-amino-2-azidohexanoic acid by copper-catalysed azide/alkyne cycloaddition followed by Boc deprotection and introduction of 4-(4-iodophenyl)butanoic acid via the respective N-hydroxysuccinimide ester gave access to 3 and two further derivatives with 3,5 and 2,5 substitution pattern at the pyridine ring. Evaluation of these compounds (and their alkyne precursors) by in-house assay methods revealed excellent inhibitory potencies towards TGase 2 (kinact/KI = 10,800 and 3,880 M-1s-1 for 3 and 1, respectively) and good binding affinities to HSA (Kd = 2.3 µM for 3). Overall, this indicates a promising basis for the application of this dual-targeting approach. Current studies focus on the radiolabelling of compound 3 with iodine-123 using the respective arylboronic acid as precursor which can be obtained via the same synthetic route. Literature: [1] Pietsch et al. Bioorg. Med. Chem. Lett. 2013, 23, 6528. [2] Dumelin et al. Angew. Chem. Int. Ed. 2008, 47, 3196. [3] Brandt et al. Nucl. Med. Biol. 2019, 70, 46. [4] Wen et al. Mol. Pharmaceutics 2019, 16, 816.

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-([18F]fluoromethyl)-1-[4-(methylsulfonyl)phenyl]-5-(p-tolyl)-1H-pyrazole ([18F]5a) was chosen as a lead compound having a reported high COX-2 inhibitory potency and a potentially low carbonic anhydrase binding tendency. The respective deuterated analog [D2,18F]5a and the fluoroethyl-substituted derivative [18F]5b were selected to study the influence of these modifications with respect to COX inhibition potency in vitro and metabolic stability of the radiolabeled tracers in vivo. COX-2 inhibitory potency was found to be influenced by elongation of the side chain but, as expected, not by deuteration. An automated radiosynthesis comprising 18F-fluorination and purification under comparable conditions provided the radiotracers [18F]5a,b and [D2,18F]5a in good radiochemical yields (RCY) and high radiochemical purity (RCP). Biodistribution and PET studies comparing all three compounds revealed bone accumulation of 18F-activity to be lowest for the ethyl derivative [18F]5b. However, the deuterated analog [D2,18F]5a turned out to be the most stable compound of the three derivatives studied here. Time-dependent degradation of [18F]5a,b and [D2,18F]5a after incubation in murine liver microsomes was in accordance with the data on metabolism in vivo. Furthermore, metabolites were identified based on UPLC-MS/MS.

Protease activity is increasingly drawn into the spotlight as a crucial modulator in cancer angiogenesis, invasion, and metastasis [1]. Elevated activity of multiple members of the family of cysteine cathepsins has been shown to correlate with increased metastasis and therapy resistance [2, 3]. Especially high expression levels of extracellular cathepsin B (CatB) indicate poor prognosis in neoplastic diseases, making CatB an interesting target for functional characterization of cancers by activity-based molecular imaging. It is our aim to develop such an imaging probe for CatB by combination of a polyarginine-based, activatable cell-penetrating peptide [4] (ACPP) and an optimised endopeptidase substrate for CatB. 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 CatB. We have generated a CatB-endoprotease substrate by C-terminally elongating the CatB carboxydipeptidase substrate Abz GIVR*AK(Dnp) OH [5] (Abz – amino-benzoyl, Dnp – dinitrophenyl, * – cleavage site) to the octapeptide Abz GIVR*AK(Dnp)GX CONH2, which could be used as activation site in the final ACPP. Introduction of any amino acid other than glycine at the P4’ position resulted in hysteretic kinetics for the CatB-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-ESI-MS-based analysis of serum-incubated substrates, the positions P1 and P3’ where 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 > 24 h under concomitant improvement of kinetic substrate efficiency towards CatB. Based on this optimised CatB-endopeptidase substrate, we have synthesised a fluorescently labelled ACPP with which we were able to demonstrate CatB-dependent uptake and subsequent nucleolar accumulation of the activated peptide in human U87 MG glioma cells. Radiolabelling of the probe with copper-64 was enabled by conjugating the ACPP to NODAGA as chelating moiety. Its evaluation in vivo using PET imaging is under current investigation. [1] Yang et al., Cancer Growth Metastasis 2009, 2, 13 [2] Aggarwal and Sloane, Proteomics Clin. Appl. 2014, 8, 427 [3] Löser and Pietzsch, Front. Chem. 2015, 3, article 37 [4] Jiang et al., PNAS, 2004, 101, 17867 [5] Cotrin et al., Anal. Biochem. 2004, 335, 244

Among the intertwined processes leading to cancer progression, protease activity plays an important role. Various attempts to develop molecular imaging probes have been made, as such probes can allow functional imaging and thus improve the understanding of tumour progression mechanisms and enable personalised cancer treatment. PET and SPECT tracers are particularly suitable for such applications. However, novel tracers have to overcome challenges such as stability, target efficiency and off-target effects. Multiple members of the cathepsin family have been demonstrated to be involved in tumour invasion, metastasis, and angiogenesis. Especially high expression levels of the cysteine cathepsins B, K, L, S, and X are correlated with an increased metastatic potential and poor prognosis [1]. Due to their high expression in a multitude of tumours, those enzymes represent promising targets for the therapy and imaging of tumours. Despite being virtually chemically inert, alkynes were shown to be able to irreversibly inhibit cysteine proteases: Both EKKEBUS et al. and SOMMER et al. independently described the unexpected inactivation of de-ubiquitinating enzymes by ubiquitin or ubiquitin-like modifiers bearing propargylamine in place of C-terminal glycine [2, 3]. We aimed to take advantage of those findings for designing alkyne-based cysteine cathepsin inhibitors suitable for radiolabelling with PET nuclides. The probes thus obtained would irreversibly bind to the target molecule without showing indiscriminate thiol reactivity. Based on a potent, highly selective dipeptidyl nitrile-based cathepsin B inhibitor reported by GREENSPAN et al. (left structure) [4], we designed dipeptide alkynes by isoelectronic replacement of the nitrile nitrogen atom by a methine group. To avoid partial enantiomerisation during the formation of the C-C triple bond as observed for the open-chain serine-derived alkyne, the synthesis was performed via Garner’s aldehyde. This ensured high stereochemical purity of the final compounds. The inhibitory potential was investigated against cathepsin B, S, L and K. To optimise the inhibitory potential and selectivity, we consecutively varied all moieties attached to the dipeptidic scaffold. We identified potent alkyne-based inhibitors for all tested cathepsins, with inactivation constants (kinact/KI) up to 10133 M-1s-1 and distinct selectivity profiles. We demonstrated irreversibility in a “jump-dilution” experiment and inhibitor reactivity in cell lysates and on living cells was exemplarily verified for cathepsin B. During our research, MONS et al. successfully demonstrated irreversible cathepsin K inhibition by alkyne-based small molecule inhibitors with no indiscriminate thiol reactivity [5], which indicates the viability of our concept. Among the tested inhibitors we identified two promising radiotracer candidates which are selective for cathepsin S and L. We successfully radiolabelled the cathepsin S-selective inhibitor with N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB). Radiopharmacological characterisation of the activity-based probe obtained by that approach is in progress. [1] Löser, R; Pietzsch, J.: Front. Chem., 2015, 3: 37. [2] Ekkebus et al.: J. Am. Chem. Soc., 2013, 135(8): 2867-2870. [3] Sommer et al.: Bioorg. Med. Chem., 2013, 21(9): 2511-2517. [4] Greenspan et al.: J. Med. Chem., 2001, 44(26): 4524-4534. [5] Mons et al.: J. Am. Soc. 2019, 141(8): 3507-3514.

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 tumors 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 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 behavior 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.

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 [11C]methyl iodide starting from cyclotron-produced [11C]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 non-radioactive 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 18F-fluoroethylated analogue and suggests the limited suitability of azadipeptide nitriles for the imaging of tumour-associated cysteine cathepsins

Even though the C-C triple bond is largely considered as a bioinert functional group, 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 ubiquitin or ubiquitin-like modifiers bearing propargylamine in place of C-terminal glycine by covalent targeting of the active-site cysteine residue [1, 2]. We intended to harness that finding for the design of inhibitor-based probes for the imaging of tumour-associated cysteine proteases. All 11 human cysteine cathepsins have been linked to tumour progression. Especially high expression levels of the cathepsins B, K, L, S and X are correlated with an increased metastatic potential and poor prognosis. [3] Therefore, those enzymes represent promising targets for the therapy and imaging of tumours. GREENSPAN et al. reported a potent, highly selective dipeptidyl nitrile-based cathepsin B inhibitor (1, structure shown above) [4]. Based on that lead compound, dipeptide alkynes were designed by isoelectronic replacement of the nitrile nitrogen atom by a methine group (2) and consecutive variation of the 2,4-difluorobenzoyl group and the amino acid-derived side chains. 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 to ensure high stereochemical purity of the final compounds. By investigating the inhibitory potential against cathepsin B, S, L and K potent alkyne-based inhibitors were identified for all tested cathepsins, with second-order inactivation constants (kinact/KI) up to 10133 M-1s-1 and interesting selectivity profiles. Based on these promising results and considering their absent indiscriminate thiol reactivity, dipeptidyl alkynes have the potential to be translated into activity-based probes for molecular imaging in vivo. In further studies, selected inhibitors will be labelled with suitable radionuclides such as fluorine-18, which will in turn enable further pharmacological evaluations. [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.

Tissue transglutaminase (TGase 2) is a multifunctional enzyme that catalyzes the formation of covalent crosslinks between protein-bound glutamine and primary amine substrates (transamidase activity) but also functions as a GTP-binding protein (Gh protein). These two functions are associated with an “open” and a “closed” conformation, respectively, being tightly regulated by Ca2+ and GDP/GTP levels. In recent years, several assays for the transamidase activity have been published [1], leaving the GTP-binding function virtually untouched. Here, we report a novel assay to quantify the GTP-binding activity of human TGase 2, which follows the increase in fluorescence anisotropy of an optimized fluorescein-labeled GTP probe upon binding to the protein. Validity of the assay was ensured by means of the (endogenous) ligands GTP, GTPγS and GDP showing inhibitory potencies (IC50) for displacement of the new probe comparable to reported values [2]. ATP, commonly not considered as being an inhibitor of TGase 2, was found to diminish binding of the probe to TGase 2 at unphysiologically high concentrations. The binding assay was then applied for the characterization of a small library of GDP and GTP analogs to obtain structure-activity relationships. In addition, assays quantifying the transamidase [3] and GTP-binding activities, respectively, were subjected to a titration with calcium chloride (Ca2+) to elucidate its influence on the conformation of TGase 2. Exclusive interaction of ligands/substrates with the GTP binding site and with the active site were found in the absence of Ca2+ and at [Ca2+] > 10 mM, respectively. Both assays exhibit an activity of ~60% at [Ca2+] = 0.5 mM, with this intermediate calcium concentration being applicable to identify ligands of both the active and the GTP-binding site at the same time. This finding was confirmed in both assays by means of GTPγS and recently reported N6-acryloyllysine piperazides [4,5] shown to irreversibly interact with the active-site cysteine residue. [1] Pietsch et al., Bioorg. Med. Chem. Lett. 2013, 23, 6528. [2] Schaertl et al., J. Biomol. Screen. 2010, 15, 478. [3] Hauser et al., Amino Acids 2017, 49, 567. [4] Wityak et al., ACS Med. Chem. Lett. 2012, 3, 1024. [5] Wodtke et al., J. Med. Chem. 2018, accepted.

Selected N-benzoylated piperazine derivatives were synthesized to study their conformational behavior using temperature-dependent 1H NMR spectroscopy. All investigated piperazine compounds occur as conformers at room temperature resulting from the reduced rotation of the partial amide double bond. Furthermore, a second conformational shape was observed for selected mono-N-benzoylated and unsymmetrically N,N’-substituted derivatives due to the limited change of the piperazine chair conformation. Therefore, two different coalescence points TC were determined and their resulting activation energy barriers ΔG# were calculated to be between 50 and 70 kJ/mol. In most of the cases, TC and ΔG# for the amide site appeared to be higher as for the amine site. Furthermore, benzoate moieties with electron withdrawing substituents like nitro show a higher rotational barrier compared to electron-releasing substitutents like methoxy. An additional aryl substituent connected at the amine site led to a reduced rotational barrier compared to the free secondary amine. To support and evidence the findings from the NMR analyses, single crystals of piperazines were obtained and XRD analyses were performed. To underline the results, two potential Tgase 2 inhibitors were investigated showing energy barriers with similar values.