1,2,4,5–Tetrazine derivatives have a high potential for biolo- gical activity, possessing a wide range of antiviral and anti- tumor properties, and these derivatives have been widely used in pesticides and herbicides (Sauer, 1996). In a continuation of our work on the structure-activity relationship of 1,2,4,5- tetrazine derivatives (Hu et al., 2002, 2004), we have obtained a yellow crystalline compound as the product of the reaction of ethyl chloroformate and 3,6-diphenyldihydro-1,2,4,5-tetra- zine. The structural identity of our product, (I), was resolved using single-crystal X-ray diffraction.
The molecular structure of (I) is illustrated in Fig. 1. Selected bond lengths and angles are listed in Table 1. Compound (I) crystallizes in the monoclinic space group C2/c with two symmetry-independent molecules having slightly different geometries (Table 1), both lying on twofold rotation axes. In both molecules, the central tetrazine ring adopts a boat conformation; atoms N2 (molecule 1) and N5 (molecule 2) and their symmetry equivalents deviate from the mean planes formed by the other four atoms of the tetrazine rings by 0.488 (8) and 0.470 (8) A ˚ , respectively.
3-tetra-O-acetyl–β-D-glucopyransoylimino,6(4-nitrophenyl) 1,2,4,5-tetrazine (IVa) was prepared by refluxing 4- nitrophenyl dihydroformazan (0.001mole) with glucosylimino isocyanodichloride (0.001mole) in chloroform medium for 4 hours. After completion of reaction, the reaction mixture was cooled; the solvent was distilled off to obtained pale yellow solid residue (IVa). It was recrystallized from ethanol (70%). m.p.132 0 C, yield 81%. 4a. IR: NH 3350cm -l , C=O 1715cm -l , -N-N- 1280cm -l , C-N 1160 cm -l , C=C 1560cm -l , CH 2 bend.1400cm -l . 1 HNMR: 12H
3,6-Di-2-pyridyl-1,2,4,5-tetrazine (Dptz) has been applied as a coordinative -acceptor moiety in the study of photophysical and redox properties of transition metal complexes, and a bridging building block for supramolecular assemblies (Campos-Fernandez et al., 1999; Bu et al., 2000). This type of aromatic compounds also exhibit proton-sponge properties (Staab & Saupe, 1988; Robertson et al., 1998), which can act as external proton acceptors through formation of NÐH Y hydrogen bonds. In the present paper, we report the crystal structure of the diprotonated salt of Dptz, namely 3,6-di-2- pyridinio-1,2,4,5-tetrazine, (I).
The molecular structure of (I) is illustrated in Fig. 1. Selected bond lengths and angles are listed in Table 1. In (I), atoms N4, C10, N6 and C9 are coplanar [deviations within 0.0347 (7) AÊ] and atoms N3 and N5 deviate from the plane by 0.412 (2) and 0.420 (2) AÊ, respectively, i.e. the central six- membered ring of (I), the tetrazine ring, has a boat conformation.
The title compound was obtained by adding dropwise phenyl chloroformate (10 mmol) to 3,6-bis(4-chlorophenyl)-1,4-dihydro- 1,2,4,5-tetrazine (5 mmol), using dichloromethane (40 ml) as solvent at 298 K. The precipitate was filtered off. A solution of the compound in ethanol was concentrated gradually at room temperature to afford colourless crystals (m.p. 491–493 K) suitable for X-ray diffraction.
A perspective view of the neutral molecule (I) including the atomic numbering scheme is shown in Fig. 1. The six- membered ring C6/N6/N5/C7/N8/N7 loses the aromaticity of the original tetrazine ring, and adopts a boat conformation, with N7 and N5 deviating by 0.388 (3) and 0.373 (2) AÊ, respectively, from the basal N8/C6/N6/C7 plane. The C6ÐN6 and C7ÐN8 bond distances are 1.265 (4) and 1.274 (4) AÊ, typical of C N double bonds. The other CÐN bonds (C6Ð N7 and N5ÐC7) and the two NÐN bonds have single-bond character. The pyridine rings A (C1ÐC5/N3), B (C8ÐC12/ N4), C (C20ÐC24/N1) and D (C13ÐC17/N2) are essentially planar. Plane A forms dihedral angles of 37.6 (2), 25.9 (2) and 20.9 (2) with planes B, C and D, respectively. Plane B forms
C8 0.0691 (15) 0.0523 (13) 0.0615 (14) −0.0030 (12) 0.0225 (12) −0.0006 (11) C9 0.0590 (14) 0.0578 (14) 0.0735 (16) 0.0066 (12) 0.0113 (12) −0.0004 (12) C10 0.0692 (18) 0.0720 (17) 0.097 (2) −0.0022 (14) 0.0126 (15) 0.0134 (16) C11 0.109 (3) 0.078 (2) 0.113 (3) −0.0036 (18) 0.039 (2) 0.0209 (19) C12 0.144 (3) 0.0643 (19) 0.077 (2) 0.019 (2) 0.033 (2) 0.0059 (15) C13 0.119 (3) 0.081 (2) 0.076 (2) 0.016 (2) −0.0080 (19) −0.0001 (17) C14 0.0735 (18) 0.0720 (18) 0.093 (2) 0.0008 (14) 0.0036 (15) −0.0019 (15) C15 0.113 (3) 0.186 (4) 0.098 (2) 0.020 (3) 0.043 (2) −0.019 (3) C16 0.239 (5) 0.093 (2) 0.088 (2) 0.038 (3) 0.047 (3) 0.021 (2) C17 0.0698 (16) 0.0717 (17) 0.0664 (16) 0.0028 (14) 0.0130 (13) −0.0194 (13) C18 0.0778 (19) 0.086 (2) 0.094 (2) −0.0177 (16) 0.0220 (16) −0.0237 (16) C19 0.091 (2) 0.136 (3) 0.128 (3) −0.035 (2) 0.028 (2) −0.058 (3) C20 0.095 (3) 0.179 (5) 0.127 (4) −0.017 (3) 0.008 (3) −0.076 (3) C21 0.119 (3) 0.173 (4) 0.079 (2) 0.003 (3) −0.012 (2) −0.038 (3) C22 0.099 (2) 0.111 (2) 0.0706 (19) 0.0045 (19) 0.0095 (17) −0.0152 (17) C23 0.0638 (15) 0.0607 (14) 0.0550 (13) −0.0024 (12) 0.0174 (11) −0.0013 (11) C24 0.0689 (16) 0.0671 (16) 0.0667 (15) −0.0022 (13) 0.0156 (12) 0.0036 (12) C25 0.0699 (18) 0.084 (2) 0.0808 (18) 0.0045 (15) 0.0153 (14) 0.0089 (15) C26 0.0659 (17) 0.111 (2) 0.0755 (18) −0.0056 (17) 0.0131 (14) 0.0070 (17) C27 0.0785 (19) 0.087 (2) 0.0841 (19) −0.0205 (16) 0.0219 (15) 0.0084 (16) C28 0.0769 (18) 0.0651 (16) 0.0738 (17) −0.0030 (14) 0.0219 (14) 0.0048 (13)
The s-tetrazine group occurs in about 90 reported structures. In most of these, the tetrazine core is highly substituted or else complexed. Many of the less substituted s-tetrazines prepared by Watson & Neilson (1975) and their students crystallize either as very ®ne needles or extremely thin plates. Attempts to collect data from these have not succeeded. However, the title compound, (I), yielded substantial plates which gave adequate X-ray data.
A crucial factor for the development of a successful pretargeted system is the elimination properties of the radiotracer. Renal clearance should be favoured over the hepatobiliary excretion in order to avoid unwanted, long-residing background radioactivity in the gastro- intestinal tract, as high radioactivity levels in the intestines hamper the delineation of nearby organs. The pharmaco- kinetic and elimination profile of a tetrazine can be al- tered, for example by the addition of a hydrophilic linker and/or changing the chelator . Comparison of the structures and biodistribution of the published tetrazines [2, 4, 9–13, 19, 20, 22] for pretargeted PET imaging shows that there are multiple factors that contribute to the phar- macokinetic profile. However, there might be one highly influencing factor, namely the introduction of positive charge to a radiopharmaceutical, which can increase the renal clearance [52, 53]. Despite the non-optimal elimin- ation kinetics of [ 18 F]TAF, our data shows that it can be used for the pretargeted PET imaging of antibodies. Given that this approach for pretargeting cetuximab and trastu- zumab is intended for the imaging of tumours outside the abdominal area, the hepatobiliary excretion would not interfere with the image analysis as much as it would in orthotopic colorectal tumour models. The three hydroxyl groups in the [ 18 F]TAF precursor, [ 18 F]-5-fluoro-5-deoxy- ribose ([ 18 F]FDR), lower the lipophilicity of the radiola- belled oxime product, which is desired for fast urinary elimination. However, [ 18 F]TAF has some uptake in the
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Earlier studies with HSV-1 and PRV indicated that glyco- proteins, capsids, and/or capsid-associated tegument proteins are transported separately from cell bodies to axon terminals (30, 33, 37, 38). Infection of primary cultures of fetal sympa- thetic ganglia neurons with the PRV-Us9 null mutant showed markedly reduced immunostaining for gE, gB, and gC in axons when compared to PRV wild-type infection (37). Additionally, PRV data suggested that Us9 plays a role in delivering vesicles containing envelope proteins, such as gE and gI, to the axonal terminals (37, 38). These observations indicated that some viral envelope proteins require Us9 for entry and transport in axons (37, 38). Our confocal microscopy data showed that anterograde axonal transport of the Us9 deletion mutant BHV-5 does not occur in vivo in the olfactory pathway. Rabbits infected intranasally with the Us9 deletion mutant of BHV-5 did not contain virus-specific antigens in the nerve processes of olfactory receptor neurons. While BHV-5 Us9 deletion mutant virus did enter and replicate efficiently in the olfactory receptor neurons and shed at the wild-type level, it failed to infect the olfactory bulb. In contrast, direct inoculation of Us9 deletion mutant virus into the bulb resulted in wild-type-level spread within the olfactory pathway. Taking together PRV in vitro data and our in vivo data, we believe that the lack of detectable viral antigens within the nerve processes of olfactory receptor FIG. 8. Confocal images of immunofluorescence-labeled viral an-
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in human platelets. For identification, UV spectrometry, matrix-assisted laser desorption/ionization, postsource de- cay matrix-assisted laser desorption/ionization mass spec- trometry, and enzymatic cleavage experiments were used. The adenosine(5 9 ) oligophospho-(5 9 ) guanosines act as va- soconstrictors and growth factors. The diguanosine poly- phosphates are potent modulators of growth in vascular smooth muscle cells, but do not affect vascular tone. ( J. Clin. Invest. 1998. 101:682–688.) Key words: dinucleoside polyphosphates • platelets • growth factors • cytosolic free
Self-assembly of metal cations with nitrogen heterocyclic bridging ligands is a central theme in supramolecular chemistry aimed at developing assemblies of electronically coupled metal centres. 6 These ligands can be used to bridge metal centres in various ways, allowing electron and charge transfer processes in the structures. 1,2,4,5-Tetrazines also have very interesting redox-behaviour, which are similar to quinones. 7 The very low-lying π* orbital localised at the four nitrogen atoms in tetrazines might allow intense low-energy charge valence transfer absorptions, electrical conductivity of coordination polymers, unusual stability of paramagnetic radicals or mixed valence intermediates. The ability of tetrazine π-ligand systems is well documented, 1,11a double coordination of metal fragments to such binucleating ligands are known to cause a particularly strong perturbation of the ligand π system as is evident from pronounced spectroscopic effects. 8
Abstract: Among extensive studies on click chemistry the inverse electron-demand Diels-Alder reaction between 1,2,4,5-tetrazine (Tz) and trans-cyclooct-2-en (TCO) has gained increasing attraction due to its exceptionally fast reaction kinetics and high selectivity for in vivo pretargeting applications including PET imaging. The facile two-step approach utilizing TCO-modified antibodies as targeting structures has not made it into clinic though as the increase in blood volume from mice to human seems to be the major limitation. This study aimed to show if the design of multimeric Tz-ligands by chelator scaffolding can improve the binding capacity and may lead to enhanced PET imaging with gallium-68. For this purpose we utilized the macrocyclic siderophore Fusarinine C (FSC) which allows to conjugate up to three Tz-residues due to three primary amines available for site specific modification. The resulting mono- di- and trimeric conjugates were radiolabelled with gallium-68 and characterized in vitro (logD, protein binding, stability, binding towards TCO modified rituximab (RTX)) and in vivo (biodistribution- and imaging studies in normal BALB/c mice using a simplified RTX-TCO tumour surrogate). The 68 Ga-labelled FSC-based Tz-
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To confirm the reactivity of s0-Tz, a small molecule test reaction was carried out in both water and DMF. Commercially available 5-norbornene-2-exo,3-exo-dimethanol was mixed with s0-Tz at a concentration of 50 μM and the reaction followed by HPLC. A clear peak shift was observed in both solvents (Supplementary Figure S38), confirming that the Tz retained its reactivity once conjugated to the DNA strand. The yield of the reaction was estimated by comparison of the areas under the peaks due to the Tz-Nb coupling product and unreacted DNA and found to be approximately 70% and 40% in water and DMF respectively. The difference in yield can possibly be attributed to degradation of the Tz group by free amines present in the DMF used.
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trans-cyclooctene (1) (Figure 1a).[23, 27-30] Analogs of these compounds have been shown to combine with rapid kinetics, thereby enabling probe construction for cancer imaging and diabetes monitoring within seconds.[28, 30] More recently, we have explored the probe 3 derived from diphenyl-s-tetrazine, which is less rapid but gives Diels-Alder adducts with improved in vivo stability relative to the first generation system. 18 F-labeled
Following the successful preparation of the 4a m series of tetrazine tripeptide linchpins, designated as (i, iþ 2) with regard to separation of Cys residues, we next sought to study larger macrocyclic congeners that inscribe the tetrazine ring. Standard Fmoc-based solid-phase peptide synthesiswasemployed to construct the(i, iþ 3) and (i, iþ 4) target peptides 9a,b and 9c respectively (Figure 6). With these model linchpins in hand, the photofragmentation reaction was again investigated. N anosecond flash photo- lysis 15 experiments revealed a dependence of the photo- chemical yield on the size of the macrocyclic peptide con- strained by the S,S-tetrazine. Peptide 4c, with an (i, iþ 2) relationship between the Cys residues, afford the highest photoproduct yield upon flash photolysis (> 25% ). The (i, iþ 3) relationship in 9b led to a decreasein photoproduct to > 15% ; a further decreaseto ∼10% wasobserved for the (i, iþ 4) peptide 9c. The observed photochemical yield dependence is likely related to the conformational strain exerted by the peptides on the S,S-tetrazine ring. Impor- tantly the photochemical yields are sufficient that popula- tion changes are observable by 2D I R, while adequate intact linchpin is maintained to enable spectroscopic data to be accumulated over an extended period of time. Figure 3. Tetrazine tripeptides synthesized in 7 30% overall
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Interestingly, during the synthesis of triazole 9p a precip- itate was formed and, when isolated, Wanzlick dimer 15 was obtained in 57% yield instead of the expected triazole (Scheme 4). Previously, photochemical for- mation of this intermediate had only been observed spectroscopically. 17 Further exposure to UV light yielded the desired product by rearrangement to 1,2,3- triazolium-1-aminide 16 and N-N bond cleavage. We suggest that formation of this Wanzlick dimer may occur through the carbenic resonance form 14 of the nitrile imine; which explains the tolerance of o- subtituents on the N-aryl ring, but not the C-aryl ring. 23 . Based on these observations, we also suggest that the tetrazine by-products previously reported during pyrazoline syntheses 15,16 may have been Wanzlick di- mers, as their NMR profile would appear similar. Fur- thermore, we did not observe tetrazine by-products dur- ing our syntheses of pyrazoline adducts.
3,6-Dichloro-1,2,4,5-tetrazine (1). In a single-neck, 4 L round bottom flask, dihydrazinyltetrazine (40) (0.318 mol, 38.5 g) was suspended in acetonitrile (500 mL). The flask was equipped with a stir bar and placed in an ice bath on a magnetic stirring plate. In a separate beaker, of trichloroisocyanuric acid (0.6517 mol, 151.3 g) is transferred to an addition funnel and slowly added to the suspension of dihydrazinyltetrazine, being careful to keep the internal temperature of the reaction mixture below room temperature. After the addition is complete and the temperature of the reaction mixture has stabilized, the flask is removed from the ice bath and the mixture is allowed to stir at room temperature for 30 minutes. The reaction mixture was vacuum filtered and the white solid precipitate was washed with acetonitrile. The solvent was stripped from the filtrate via rotovap (22°C, 70 torr). Dichlorotetrazine can sublimate and decomposes under high heat, so careful attention should be paid to keep temperature low and watch for the distinctive orange color of the product to collect in the cold trap. Removal of the acetonitrile results in the isolation of the crude product as a red-orange solid with a strong smell of chlorine. This solid was then dissolved in dichloromethane and passed through a celite plug. The resulting filtrate was concentrated in vacuo, again being cautious to prevent loss of volatile dichlorotetrazine. This filtration procedure was repeated 2 times until only 1 13 C carbon signal showed in the NMR spectrum and all
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Figure 1. a) Nanoparticles with an average diameter of 35 nm were fabricated from the amphiphilic block-co-polymer PEG-b-Dox, with the methacrylate–Doxorubicin conjugated segment forming the hydrophobic core. Upon reaction with tetrazine, doxorubicin (red spheres) is liberated, driven by the 1,6-elimination reaction of the self-immolative linker. b) The mechanism of tetrazine mediated vinyl ether decaging and cargo liberation (here RNH 2 ).