Nitrobenzyl Moiety Research Articles

O-Nitrobenzyl-Based Caged exo-16,17-Dihydro-gibberellin A5-13-acetate for Photocontrolled Release of Plant Growth Regulators.

Caged plant growth regulators (caged PGRs) that release bioactive molecules under irradiation are critical in enhancing the efficacy and mitigating the negative environmental effects of PGRs. The synthetically derived plant growth inhibitor exo-16,17-dihydro-gibberellin A5-13-acetate (DHGA5) regulates the development and stress resilience of plants. We report here the conception of novel caged DHGA5 derivatives wherein the photoremovable protecting groups (PRPGs) serve not only to enable light-controlled release but also to protect the carboxyl group during chemical synthesis. Three o-nitrobenzyl-based caged DHGA5 derivatives with different substituents on the nitrobenzyl moiety were obtained and evaluated for their properties in vitro and in vivo. The photolysis half-life values of caged DHGA5 derivatives 7a, 7b, and 7c under a UV lamp were 15.6 h, 1.2 h, and 28.2 h, respectively. Experiments in vivo showed that 0.2 mM of the caged compounds significantly inhibited the growth of the model plant Arabidopsis thaliana and important crop rice in a precise photoactivated form.

Intracellular Crowding by Bio-Orthogonal Hydrogel Formation Induces Reversible Molecular Stasis.

To survive extreme conditions, certain animals enter a reversible protective stasis through vitrification of the cytosol by polymeric molecules such as proteins and polysaccharides. In this work, synthetic gelation of the cytosol in living cells is used to induce reversible molecular stasis. Through the sequential lipofectamine-mediated transfection of complementary poly(ethylene glycol) macromers into mammalian cells, intracellular crosslinking occurs through bio-orthogonal strain-promoted azide-alkyne cycloaddition click reactions. This achieves efficient polymer uptake with minimal cell death (99% viable). Intracellular crosslinking decreases DNA replication and protein synthesis, and increases the quiescent population by 2.5-fold. Real-time tracking of single cells containing intracellular crosslinked polymers identifies increases in intermitotic time (15 hvs 19 h) and decreases in motility (30 µm h-1 vs 15µm h-1 ). The cytosol viscosity increases threefold after intracellular crosslinking and results in disordered cytoskeletal structure in addition to the disruption of cellular coordination in a scratch assay. By incorporating photodegradable nitrobenzyl moieties into the polymer backbone, the effects of intracellular crosslinking are reversed upon exposure to light, thereby restoring proliferation (80% phospho-Rb+ cells), protein translation, and migration. Reversible intracellular crosslinking provides a novel method for dynamic manipulation of intracellular mechanics, altering essential processes that determine cellular function.

Near-Infrared Fluorescent Probe Activated by Nitroreductase for In Vitro and In Vivo Hypoxic Tumor Detection.

Tumor hypoxia is correlated with increased resistance to chemotherapy and poor overall prognoses across a number of cancer types. We present here a cancer cell-selective and hypoxia-responsive probe (fol-BODIPY) designed on the basis of density functional theory (DFT)-optimized quantum chemical calculations. The fol-BODIPY probe was found to provide a rapid fluorescence "off-on" response to hypoxia relative to controls, which lack the folate or nitro-benzyl moieties. In vitro confocal microscopy and flow cytometry analyses, as well as in vivo near-infrared optical imaging of CT26 solid tumor-bearing mice, provided support for the contention that fol-BODIPY is more readily accepted by folate receptor-positive CT26 cancer cells and provides a superior fluorescence "off-on" signal under hypoxic conditions than the controls. Based on the findings of this study, we propose that fol-BODIPY may serve as a tumor-targeting, hypoxia-activatable probe that allows for direct cancer monitoring both in vitro and in vivo.

A coelenterazine-type bioluminescent probe for nitroreductase imaging.

Novel coelenterazine-type bioluminescent probes have been designed and synthesized to detect nitroreductase (NTR) in hypoxic tumors. The design strategy is that NTR catalyzes the reduction of the nitrobenzyl moiety to the aniline group with an electron donor, thus resulting in 1,4 or 1,6-rearrangement-elimination to release coelenterazine analogues, which can be catalyzed by Renilla luciferase to emit bioluminescence. After careful evaluation, almost all probes had a 3-fold greater response for NTR over other biologically relevant substances at >100-fold dose more than NTR. In the dose-independent and selectivity study, probes A1, A2 and A5 presented a high selectivity in a dose-dependent manner. Overall, among all molecules, probe A5 showed high sensitivity, low cytotoxicity and good compatibility, so as to be successfully applied for assessing the hypoxic status in cellulo and in vivo as the first coelenterazine-type bioluminescent probe.

Photocleavage of Poly(methyl acrylate) with Centrally Located o-Nitrobenzyl Moiety: Influence of Environment on Kinetics

We characterized the kinetics of a photocleavable o-nitrobenzyl (ONB)-modified poly(methyl acrylate) in solution and in film. Using atom transfer radical polymerization, a single photolabile ONB group was incorporated into the center of the polymer chain, resulting in a clearly defined photoresponse. The photocleavage behavior was characterized in three difference solvents and in film, and it was fit to an exponential model. The dependence of the reaction rate constants and quantum efficiency on the interaction parameter between the solvent and the polymer (χ12) and the molecular weight was investigated. Interestingly, as χ12 decreased, the reaction rate increased and the quantum efficiency increased.

Photodegradable Gelatin-Based Hydrogels Prepared by Bioorthogonal Click Chemistry for Cell Encapsulation and Release.

In this study, we present a method for the fabrication of in situ forming gelatin and poly(ethylene glycol)-based hydrogels utilizing bioorthogonal, strain-promoted alkyne-azide cycloaddition as the cross-linking reaction. By incorporating nitrobenzyl moieties within the network structure, these hydrogels can be designed to be degradable upon irradiation with low intensity UV light, allowing precise photopatterning. Fibroblast cells encapsulated within these hydrogels were viable at 14 days and could be readily harvested using a light trigger. Potential applications of this new class of injectable hydrogel include its use as a 3D culturing platform that allows the capture and release of cells, as well as light-triggered cell delivery in regenerative medicine.

Abstract 4302: The development study of hypoxia responsive chemiluminescent probe for tumor hypoxia imaging

Abstract Introduction: The tumor microenvironment is recognized as a major factor that influences not only the response to conventional anticancer therapies but also the potential for malignant progression and metastasis. Therefore, it is considered that tumor hypoxia imaging is critical for cancer diagnosis and therapy. As optical modalities offer the advantages of non-invasive, easy-to-handle, and cost-effective imaging, much effort has been done to develop synthetic fluorescent imaging probes for tumor hypoxia. In particular, we focus our attention on chemiluminescence imaging which can avoid interference from tissue autofluorescence and scattering light. We therefore show the development of hypoxia responsive chemiluminescent probe, which has the advantage of lower detection limits and improved signal-to-noise ratio compared to existing fluorescent probes. Results and discussion: In our molecular design, luminescence is derived from 1,2-dioxetane cleavage triggered by hypoxia-selective enzymatic reduction of electron-deficient aromatics. At the start, nitrobenzyl moiety was chosen as a bioreductively cleavable protective group linked to 1,2-dioxetane moiety through phenyl ether linker to construct synthetic probe NB-Adm. The compound was synthesized from 2-adamantanone in 19% (overall yield, 6 steps). Then NB-Adm was attempted to be reduced by nitroreductase, however, no luminescence was observed. HPLC analysis of the reaction mixture indicated that our expected reduction did not occur, probably because bulky adamantane moiety prevented the enzymatic reaction owing to steric hindrance. Therefore, a self-immolative linker was employed to keep away 1,2-dioxetane moiety from enzyme recognition moiety. In this time, quinone moiety was chosen as a protective group which was expected to be better substrate than nitrophenyl one in terms of their redox potential. This newly designed compound, TMQ-Adm was synthesized from 2-adamantanone in 16% (overall yield, 6 steps). By the use of TMQ-Adm, we successfully observed luminescent emission from the reaction with nitroreductase. This implied that our bioredctively activatable probe can be applicable for chemiluminescence imaging for tumor hypoxia. Now we are investigating to develop chemiluminescent probe to realize in vivo imaging of tumor hypoxia by improving luminescent quantum yield under physiological condition. Citation Format: Kensuke Okuda, Toru Ban, Tasuku Hirayama, Hideko Nagasawa. The development study of hypoxia responsive chemiluminescent probe for tumor hypoxia imaging. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4302. doi:10.1158/1538-7445.AM2014-4302

Polymeric micelles based on photocleavable linkers tethered with a model drug

An amphiphilic block copolymer with photocleavable nitrobenzyl moieties in the side chain of the hydrophobic block was successfully synthesized by a combination of atom transfer radical polymerization (ATRP) and the Cu(I)-catalyzed 1,3-dipolar cycloaddition of azide and alkynes. 2-(Trimethylsilyloxy)ethyl methacrylate (HEMATMS) was polymerized from a poly(ethylene oxide) (PEO) macroinitiator via ATRP, leading to a well-defined block copolymer of PEO113-b-PHEMATMS45 with low polydispersity index (PDI = 1.09). After the polymerization, trimethylsilyl (TMS) groups were deprotected and then functionalized in-situ with 3-azidopropionic chloride to yield PEO-b-[2-(1-azidobutyryloxy)ethyl methacrylate] (PEO-b-PAzHEMA). Alkyne-functionalized pyrene with a photocleavable 2-nitrobenzyl moiety was added to the PEO-b-PAzHEMA backbone via click chemistry to produce the desired block copolymer with high fidelity. The resulting block copolymer was self-assembled in water to yield spherical micelles with an average diameter of 60 nm. Upon UV irradiation, 2-nitrobenzyl moieties were selectively cleaved, leading to the release of a model drug, 1-pyrenebutyric acid. Coumarin 102, another model drug that was physically encapsulated in the core of micelles during micellization in water, was also released at the same time. The general strategy presented herein can potentially be utilized for the preparation of polymeric vehicles that are capable of delivering multiple therapeutics under controlled individual release kinetics.

Facile preparation of photodegradable hydrogels by photopolymerization

Photodegradable hydrogels have emerged as a powerful material platform for studying and directing cell behaviors, as well as for delivering drugs. The premise of this technique is to use a cytocompatible light source to cleave linkers within a hydrogel, thus causing reduction of matrix stiffness or liberation of matrix-tethered biomolecules in a spatial-temporally controlled manner. The most commonly used photodegradable units are molecules containing nitrobenzyl moieties that absorb light in the ultraviolet (UV) to lower visible wavelengths (∼280–450 nm). Because photodegradable linkers and hydrogels reported in the literature thus far are all sensitive to UV light, highly efficient UV-mediated photopolymerizations are less likely to be used as the method to prepare these hydrogels. As a result, currently available photodegradable hydrogels are formed by redox-mediated radical polymerizations, emulsion polymerizations, Michael-type addition reactions, or orthogonal click chemistries. Here, we report the first photodegradable poly(ethylene glycol)-based hydrogel system prepared by step-growth photopolymerization. The model photolabile peptide cross-linkers, synthesized by conventional solid phase peptide synthesis, contained terminal cysteines for step-growth thiol-ene photo-click reactions and a UV-sensitive 2-nitrophenylalanine residue in the peptide backbone for photo-cleavage. Photolysis of this peptide was achieved through adjusting UV light exposure time and intensity. Photopolymerization of photodegradable hydrogels containing photolabile peptide cross-linkers was made possible via a highly efficient visible light-mediated thiol-ene photo-click reaction using a non-cleavage type photoinitiator eosin-Y. Rapid gelation was confirmed by in situ photo-rheometry. Flood UV irradiation at controlled wavelength and intensity was used to demonstrate the photodegradability of these photopolymerized hydrogels.

New ferrocenic pyrrolo[1,2-a]quinoxaline derivatives: Synthesis, and in vitro antimalarial activity – Part II

Following our search for antimalarial compounds, novel series of ferrocenyl-substituted pyrrolo[1,2-a]quinoxalines 1-2 were synthesized from ferrocene-carboxaldehyde and tested for their in vitro activity upon the erythrocytic development of Plasmodium falciparum strains with different chloroquine-resistance status. The ferrocenic pyrrolo[1,2-a]quinoxalines 1-2 were prepared in 6 or 9 steps through a Barton–Zard reaction. Promising pharmacological results against FcB1, K1 and F32 strains were obtained with ferrocenyl pyrrolo[1,2-a]quinoxalines 1j-l linked by a bis-(3-aminopropyl)piperazine linker substituted by a nitrobenzyl moiety.

Construction of Endo-Functionalized Two Dimensional Metallacycles via Coordination-Driven Self-Assembly

The synthesis of three endofunctionalized two-dimensional supramolecular metallacycles including two [2 + 2] rhomboids (5 and 6) and a [3 + 3] hexagon (7) is reported. The resulting self-assembled supramolecular structures, containing several nitrobenzyl moieties at their interior surface, have been fully characterized by multinuclear NMR ((31)P and (1)H) and electrospray ionization mass spectrometry. A significant C-H...O hydrogen bonding between the nitrobenzyl acceptor and the edge molecules of the supramolecular architecture is observed in the small rhomboid 5 and this interaction gradually decreases upon the enlargement of the resulting polygonal structures from a small rhomboid 5 through a large rhomboid 6 to a hexagon 7. Molecular modeling with the MMFF force field gives a possible conformation of each self-assembly in different solvents and shows that the hydrophilic nitrobenzyl moiety prefers to be buried in the cavity of the resulting polygonal structures in nonpolar solvents, thus forming hydrogen bonds with the peripheral component building units.

Synthesis and flow cytometric evaluation of novel 1,2,3,4-tetrahydroisoquinoline conformationally constrained analogues of nitrobenzylmercaptopurine riboside (NBMPR) designed for probing its conformation when bound to the es nucleoside transporter.

Novel regioisomers of conformationally constrained analogues of the potent es nucleoside transporter ligand, nitrobenzylmercaptopurine riboside (NBMPR), designed for probing its bound (bioactive) conformation, were synthesized and evaluated as es transporter ligands by flow cytometry. Purine 6-position 5, 6, 7, or 8-nitro-1,2,3,4-tetrahydroisoquinolylpurine ribosides, in which the nitrobenzyl moiety in NBMPR has been locked into the nitro-1,2,3,4-tetrahydroisoquinoline system, were synthesized by reaction of the appropriate nitro-1,2,3,4-tetrahydroisoquinoline with 6-chloropurine riboside. Flow cytometry was performed using 5-(SAENTA)-X8-fluorescein as the competitive ligand. A high degree of variation in the es transporter binding capacity of the target compounds was observed, with the K(i) values ranging from 0.45 nM for the most tightly bound compound (4) to 300 nM for the least tightly bound compound (5). The K(i) of NBMPR was 0.70 nM, a little higher than that of compound 4. Compound 4 is the isomer that has the nitro group in the best orientation at the es transporter binding site compared to the other three compounds, 2, 3, and 5.

Stereoselective Synthesis of over Two Million Compounds Having Structural Features Both Reminiscent of Natural Products and Compatible with Miniaturized Cell-Based Assays

Understanding the cellular function of a protein usually requires a means to alter the function. This is commonly done by mutating the gene encoding the protein (genetic approach). It can also be done by binding the protein directly with a small molecule ligand (chemical genetic approach). Such ligands, primarily natural products or synthetic variants of them, can either inactivate1 or activate2 protein function. For the chemical genetic approach to have its maximal impact, efficient methods of ligand discovery will be required to provide, in the limit, a small molecule partner for every gene product. Our laboratory has recently described miniaturized cell culture assays for screening large numbers of small molecule ligands, potentially on a genome-wide basis.3 However, despite its success in natural products synthesis, synthetic chemistry has not yet been applied to the synthesis of vast numbers of compounds with structures both reminiscent of natural products and compatible with miniaturized assays. These features of a synthesis will likely be required in order to discover nonnatural compounds having the binding affinities and specificities characteristic of natural products. The synthetic strategy we have undertaken is to develop highly efficient multistep syntheses of natural product-like compounds that include several coupling steps and to use split-pool techniques4 at these steps in order to generate diverse outcomes. This technique requires that one of the coupling substrates be immobilized on a solid support. Our syntheses are further constrained by the water-compatible photolabile supports required when carrying out large numbers (>105) of miniaturized “nanodroplet” cell culture assays aimed at identifying cell permeable ligands.3 This presents a considerable synthetic challenge since the structural elements that provide these properties (e.g., poly(ethylene glycol), nitrobenzyl moieties) are incompatible with numerous reagents used in conventional synthetic chemistry. With these considerations in mind, we first converted shikimic acid, 1, into both enantiomers of epoxycyclohexenol carboxylic acid 2,5 which were then coupled to a photocleavable linker on solid support by standard methods (Figure 1).6 Treatment of the resin-bound epoxycyclohexenol, 3, with various nitrone carboxylic acids,7 7a-f and 9b-d, under esterification conditions yielded tetracyclic compounds 4a-f and 5b-d with complete regioand stereoselectivity, presumably via tandem acylation/1,3-dipolar cycloaddition.8 No intermediate nitrone ester or carboxy isoxazolidine structures were observed. The iodoaryl tetracycles 4b-d and 5b-d were selected for further study since the supportbound aryl iodides could be modified with commercially available reagents, avoiding the need for solution-phase syntheses of numerous nitrone acids. Tetracycles 4b-d and 5b-d are rigid, densely functionalized compounds that can undergo further reactions to introduce a variety of functional groups around the central octahydrobenzisoxazole structure, notably, without the use of protecting groups. The iodoaryl groups can serve as substrates for palladium cross-coupling reactions. The electrophilic lactone and epoxide can react with nucleophiles while simultaneously unmasking alcohols for subsequent reactions. Furthermore, reductive N-O bond cleavage would provide two additional handles for functionalization. We developed several such reactions (Figure 2) with product purity ranging from ∼50 to g98% following photocleavage.9 Since purification is not practical in a large split-pool synthesis, only reactions generating products in g90% purity were acceptable. The most promising reactions were studied in detail to define the scope, limitations, and optimal conditions for each (Figure 3). Iodobenzyl tetracycles 4b-d were selected as scaffolds since (1) See http://www-schreiber.chem.harvard.edu on the Web. (2) Crabtree, G. R.; Schreiber, S. L. Trends in Biochem. Sci. 1996, 21, 418-422. (3) (a) Borchardt, A.; Liberles, S. D.; Biggar, S. R.; Crabtree, G. R.; Schreiber, S. L. Chem. Biol. 1997, 4, 961-968. (b) You, A. J.; Jackman, R. J.; Whitesides, G. M.; Schreiber, S. L. Chem. Biol. 1997, 4, 969-975. (4) (a) Furka, A.; Sebestyen, F.; Asgedom, M.; Dibo, G. Int. J. Pept. Protein Res. 1991, 37, 487-493. (b) Lam, K. S.; Salmon, S. E.; Hersh, E. M.; Hruby, V. J.; Kazmierski, W. M.; Knapp, R. J. Nature 1991, 354, 82-84. (5) (a) Wood, H. B.; Ganem, B. J. Am. Chem. Soc. 1990, 112, 89078909. (b) McGowan, D. A.; Berchtold, G. A. J. Org. Chem. 1981, 46, 23812383. (c) Mitsunobu, O. Synthesis 1981, 1-28. (6) The resin structure shown represents Tentagel S NH2, a poly(ethylene glycol)-polystyrene copolymer, loaded with a photocleavable 3-amino-3-onitrophenylpropionic acid linker. (Brown, B. B.; Wagner, D. S.; Geysen, H. M. Mol. DiV. 1995, 1, 4-12.). (7) Keirs, D.; Overton, K. Heterocycles 1989, 28, 841-848. (8) Tamura, O.; Okabe, T.; Yamaguchi, T.; Gotanda, K.; Noe, K.; Sakamoto, M. Tetrahedron 1995, 51, 107-118. (9) Photocleavage products were analyzed by 1H NMR, FAB-MS, HPLC, and TLC. Figure 1. Synthesis of tetracycles and nitrones. a: X ) H, b: X ) 2-I, c: X ) 3-I, d: X ) 4-I, e: X ) 4-CF3, f: X ) 3,4-(OMe)2.

5,17-Dinitro-11,23-diphthalimido-25,26,27,28-tetrapropoxycalix[4]arene

The aromatic phthalimido moieties of the title compound, C56H52N4O12, do not (mutually) stack intramolecularly nor intermolecularly, but rather intermolecularly with the nitrobenzyl moieties. The mol­ecules show twist-distorted C2v symmetry.

Nitrobenzyl-Based Photosensitive Phosphoramide Mustards: Synthesis and Photochemical Properties of Potential Prodrugs for Cancer Therapy.

Several nitrobenzyl-based photosensitive phosphoramide mustards were synthesized. The nitrobenzyl moiety was structurally varied to find the most promising prodrug candidates in respect to photorelease and activity of the alkylating species. The synthesis of these compounds proved to be applicable even in regard to compounds with additional functionalization. The target molecules 13a,b to 14 exhibited the expected red shift in their absorption spectra maximum compared to the parent nitrobenzyl moiety. As seen by UV and (31)P NMR spectroscopy, the phosphoramide mustard was quickly liberated upon irradiation with mercury arc lamps. Assaying the structurally different prodrugs on their alkylating activity showed that compounds 13b and 14, derived from secondary benzyl alcohols, are promising prodrug candidates. Their water solubility and the possibility of attaching macromolecules are encouraging vis-à-vis future investigations on their in vitro cytotoxicity.

Mass spectral analysis of peptides containing nitrobenzyl moieties

By means of MALDI-TOF analysis of peptides containing a nitrobenzyl moiety, we have observed that the predominant signal often corresponds to ions that have eliminated multiple oxygen atoms, [M−On+H]+, and not to the protonated molecular ion, [M+H]+. Matrix selection for handling these types of molecules appears to be important. The MALDI-TOF matrices, such as sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid) and α-cyano-4-hydroxycinnamic acid, resulted in the appearance of significant levels of the photodeoxygenated species, whereas 4-hydroxyazobenzene-2-carboxylic acid appeared to moderately and in some cases completely suppress the photodeoxygenation reaction. This phenomenon was independent of laser intensity. Photodeoxygenation was not observed with electrospray or fast atom bombardment ionization techniques. Therefore, use of MALDI should be avoided with peptides containing a nitrobenzyl moiety and similar moieties that are prone to reactive photochemistry.

Molecular Structure at 1·8 Å of Mouse Liver Class Pi Glutathione S-transferase Complexed with S-( p-Nitrobenzyl)glutathione and Other Inhibitors

The three-dimensional crystal structure of π class glutathione S-transferase YfYf from mouse liver complexed with the inhibitor S -( p-nitrobenzyl)glutathione has been determined at 1·8 Å resolution by X-ray diffraction. In addition two complexes with glutathione sulphonic acid and S-hexylglutathione have been determined at resolutions of 1·9 and 2·2 Å, respectively. The high resolution of the S-( p-nitrobenzyl)glutathione complex allows a detailed analysis of the active site including the hydrophobic (H-) subsite. The nitrobenzyl moiety occupies a hydrophobic pocket with its aromatic ring sandwiched between Phe8 and the hydroxyl group of Tyr108. An insertion of two residues Gly41 and Leu42; with respect to the pig enzyme, splits helix αB into an α-helix and a 3 10 helix. Water bridges between carbonyl oxygen atoms of the α-helix at its C terminus and the amide NH groups of the 3 10 helix at its N terminus provide structural continuity between these two secondary elements. Tyr7 appears to be the only residue close to the sulphur atom of glutathione, while three conserved water molecules lie in the surrounding area in all complexes. The enzyme mechanism is discussed on the basis of the structural analysis.