Impact of Solvent and Protonation State on Rotational Barriers in [s]-Triazines.

Amine-substituted [s]-triazines display hindered rotation around the triazine-N bond. While this barrier, ΔG‡, has been measured to be between 15.1 and 17.7 kcal/mol for neutral triazines, the impacts that solvent and protonation state have not been addressed. Using a dimethylamine substituent as a reporter, ΔG‡ was measured to be 17.5-19.3 kcal/mol upon protonation across a range of solvents (D2O, DMSO-d6, MeCN-d3, MeOD-d4, tetrahydrofuran-d8, trifluoroethanol-d3). Furthermore, ΔG‡ increases as the solvent dielectric decreases (p < 0.01). This trend is consistent with the role that solvent plays in stabilizing the increased charge density on the triazine ring resulting from a loss of conjugation with the dimethylamine substituent. Across these solvents, ΔG‡ for the neutral molecule is smaller by ∼2-3 kcal/mol, ranging from 15.3-16.1 kcal/mol. In pyridine, ΔG‡ does not correlate with the solvent dielectric for the "protonated" model. The lower barrier is attributed to competitive protonation: the pKa of the protonated triazine (∼6) is similar to that of protonated pyridine-d5 (5.8). As additional acid is added, ΔG‡ increases. Adding additional acid to the protonated model in D2O or DMSO-d6 does not significantly affect ΔG‡.

Proton Affinity and Conformational Integrity of a 24-Atom Triazine Macrocycle across Physiologically Relevant pH.

For 24-atom triazine macrocycles, protonation of the heterocycle leads to a rigid, folded structure presenting a network of hydrogen bonds. These molecules derive from dynamic covalent chemistry wherein triazine monomers bearing a protected hydrazine group and acetal tethered by the amino acid dimerize quantitatively in an acidic solution. Here, lysine is used, and the product is a tetracation. The primary amines of the lysine side chains do not interfere with quantitative yields of the desired bis(hydrazone) at concentrations of 5-125 mg/mL. Mathematical modeling of data derived from titration experiments of the macrocycle reveals that the pKa values of the protonated triazines are 5.6 and 6.7. Changes in chemical shifts of resonances in the 1H NMR spectra corroborate these values and further support assignment of the protonation sites. The pKa values of the lysine side chains are consistent with expectation. Upon deprotonation, the macrocycle enjoys greater conformational freedom as evident from the broadening of resonances in the 1H and 13C NMR spectra indicative of dynamic motion on the NMR time scale and the appearance of additional conformations at room temperature. While well-tempered metadynamics suggests only a modest difference in accessible conformational footprints of the protonated and deprotonated macrocycles, the shift in conformation(s) supports the stabilizing role that the protons adopt in the hydrogen-bonded network.

Controlling Swing Rates in Macrocyclic Molecular Mortise Hinges.

Hinge motion is observed in macrocyclic, mortise-type molecular hinges using variable temperature NMR spectroscopy. The data is consistent with dynamic hinging from a folded-to-extended-to-folded enantiomeric state. Crystallographic and solution structures of the folded states are reported. Chemical shift predictions derived from crystallographic data corroborate fully revolute hinge motion. The rate of hinging is affected by steric congestion at the hinge axis. A macrocycle containing glycine, 1, hinges faster than one comprising aminoisobutyric acid, 2. The free energies of activation, ΔG≠ , for 1 and 2 were determined to be 13.3±0.3 kcal/mol and 16.3±0.3 kcal/mol, respectively. This barrier is largely independent of solvent across those surveyed (CD3 OD, CD3 CN, DMSO-d6 , pyridine-d5 , D2 O). Experiment and computation predict energy barriers that are consistent with disruption of an intramolecular network of hydrogen bonds. DFT calculations reveal a pathway for hinge motion.

A Model for the Rapid Assessment of Solution Structures for 24-Atom Macrocycles: The Impact of ß-Branched Amino Acids on Conformation.

Experiment and computation are used to develop a model to rapidly predict solution structures of macrocycles sharing the same Murcko framework. These 24-atom triazine macrocycles result from the quantitative dimerization of identical monomers presenting a hydrazine group and an acetal tethered to an amino acid linker. Monomers comprising glycine and the ß-branched amino acids threonine, valine, and isoleucine yield macrocycles G-G, T-T, V-V, and I-I, respectively. Elements common to all members of the framework include the efficiency of macrocyclization (quantitative), the solution- and solid-state structures (folded), the site of protonation (opposite the auxiliary dimethylamine group), the geometry of the hydrazone (E), the C2 symmetry of the subunits (conserved), and the rotamer state adopted. In aggregate, the data reveal metrics predictive of the three-dimensional solution structure that derive from the fingerprint region of the 1D 1H spectrum and a network of rOes from a single resonance. The metrics also afford delineation of more nuanced structural features that allow subpopulations to be identified among the members of the framework. Well-tempered metadynamics provides free energy surfaces and population distributions of these macrocycles. The areas of the free energy surface decrease with increasing steric bulk (G-G > V-V ∼ T-T > I-I). In addition, the surfaces are increasingly isoenergetic with decreasing steric bulk (G-G > V-V ∼ T-T > I-I).

Computational and Experimental Evidence for Templated Macrocyclization: The Role of a Hydrogen Bond Network in the Quantitative Dimerization of 24-Atom Macrocycles.

In the absence of preorganization, macrocyclization reactions are often plagued by oligomeric and polymeric side products. Here, a network of hydrogen bonds was identified as the basis for quantitative yields of macrocycles derived from the dimerization of monomers. Oligomers and polymers were not observed. Macrocyclization, the result of the formation of two hydrazones, was hypothesized to proceed in two steps. After condensation to yield the monohydrazone, a network of hydrogen bonds formed to preorganize the terminal acetal and hydrazine groups for cyclization. Experimental evidence for preorganization derived from macrocycles and acyclic models. Solution NMR spectroscopy and single-crystal X-ray diffraction revealed that the macrocycles isolated from the cyclization reaction were protonated twice. These protons contributed to an intramolecular network of hydrogen bonds that engaged distant carbonyl groups to realize a long-range order. DFT calculations showed that this network of hydrogen bonds contributed 8.7 kcal/mol to stability. Acyclic models recapitulated this network in solution. Condensation of an acetal and a triazinyl hydrazine, which adopted a number of conformational isomers, yielded a hydrazone that adopted a favored rotamer conformation in solution. The critical hydrogen-bonded proton was also evident. DFT calculations of acyclic models showed that the rotamers were isoenergetic when deprotonated. Upon protonation, however, energies diverged with one low-energy rotamer adopting the conformation observed in the macrocycle. This conformation anchored the network of hydrogen bonds of the intermediate. Computation revealed that the hydrogen-bonded network in the acyclic intermediate contributed up to 14 kcal/mol of stability and preorganized the acetal and hydrazine for cyclization.

Click Chemistry of Melamine Dendrimers: Comparison of "Click-and-Grow" and "Grow-Then-Click" Strategies Using a Divergent Route to Diversity.

Dendrimers are attractive macromolecules for a broad range of applications owing to their well-defined shapes and dimensions, highly branched and globular architectures, and opportunities for exploiting multivalency. Triazine dendrimers in particular offer advantages such as ease of synthesis, stability, well-defined spherical structure, multivalency, potential to achieve acceptable drug loadings, and low polydispersity. In this study, the potential utility of alkyne-azide "click" cycloadditions of first-, second-, and third-generation triazine dendrimers containing three or six alkynyl groups with benzyl azide was examined using copper catalysts. "Click-and-grow" and "grow-then-click" strategies were employed. For the first- and second- generation dendrimers, the desired triazole derivatives were obtained in high yields and purified by simple reprecipitation without column chromatography; however, some difficulties were observed in the preparation of third-generation dendrimers. The desired reaction proceeded under microwave irradiation as well as with simple heating. This click chemistry can be utilized for various melamine dendrimers that are fabricated with other amine linkers.

Two Decades of Triazine Dendrimers.

For two decades, methods for the synthesis and characterization of dendrimers based on [1,3,5]-triazine have been advanced by the group. Motivated by the desire to generate structural complexity on the periphery, initial efforts focused on convergent syntheses, which yielded pure materials to generation three. To obtain larger generations of dendrimers, divergent strategies were pursued using iterative reactions of monomers, sequential additions of triazine and diamines, and ultimately, macromonomers. Strategies for the incorporation of bioactive molecules using non-covalent and covalent strategies have been explored. These bioactive materials included small molecule drugs, peptides, and genetic material. In some cases, these constructs were examined in both in vitro and in vivo models with a focus on targeting prostate tumor subtypes with paclitaxel conjugates. In the materials realm, the use of triazine dendrimers anchored on solid surfaces including smectite clay, silica, mesoporous alumina, polystyrene, and others was explored for the separation of volatile organics from gas streams or the sequestration of atrazine from solution. The combination of these organics with metal nanoparticles has been probed. The goal of this review is to summarize these efforts.

Synthesis of Macrocycles Derived from Substituted Triazines.

A triazine ring derivatized with morpholine, an N-alkyl-N'-BOC-hydrazine (alkyl=isopropyl or benzyl) and the diethylacetal of glycinylpropionaldehyde undergoes spontaneous dimerization in good yields upon acid-catalyzed deprotection. The resulting 24-member macrocycles can be characterized by NMR spectroscopy, mass spectrometry, and single crystal X-ray diffraction. In the solid state, both homodimers adopt a taco-like conformation. Although each shows π-π stacking between the triazine rings, different patterns of hydrogen bonds emerge. The crystal structure of the isopropyl dimer shows that it includes two molecules of trifluoracetic acid per macrocycle. The trifluoroacetate anion charge balances the protonated triazines, which engage in bifurcated hydrogen bonds with the carbonyl acceptor of the distant glycine. This carbonyl also forms a hydrogen bond with the NH of the proximate glycine. The crystal structure of the benzyl derivative does not include trifluoracetic acid. Instead, two hydrogen bonds form, each between a glycine NH and the lone pair of the C=N nitrogen of the hydrazine group. In the solid state, both molecules present the alkyl side chains and morpholine groups in close proximity. A heterodimer is accessible in approximately statistical yields-along with both homodimers-by mixing the two protected monomers prior to subjecting them to deprotection.

Nanoparticle physicochemical properties determine the activation of intracellular complement.

The complement system plays an essential role in both innate and adaptive immunity. The traditional understanding of this system comes from studies investigating complement proteins produced by the liver and present in plasma to "complement" the immune cell-mediated response to invading pathogens. Recently, it has been reported that immune cells including, but not limited to, T-cells and monocytes, express complement proteins. This complement is referred to as intracellular (IC) and implicated in the regulation of T-cell activation. The mechanisms and the structure-activity relationship between nanomaterials and IC, however, are currently unknown. Herein, we describe a structure-activity relationship study demonstrating that under in vitro conditions, only polymeric materials with cationic surfaces activate IC in T-cells. The effect also depends on particle size and occurs through a mechanism involving membrane damage, thereby IC on the cell surface serves as a self-opsonization marker in response to the nanoparticle-triggered danger affecting the cell integrity.

Functionalization of a Triazine Dendrimer Presenting Four Maleimides on the Periphery and a DOTA Group at the Core.

A readily and rapidly accessible triazine dendrimer was manipulated in four steps with 23% overall yield to give a construct displaying four maleimide groups and DOTA. The maleimide groups of the dendrimer are sensitive to hydrolysis under basic conditions. The addition of up to four molecules of water can be observed via mass spectrometry and HPLC. The evolution in the alkene region of the ¹H-NMR--the transformation of the maleimide singlet to the appearance of two doublets--is consistent with imide hydrolysis and not the Michael addition. The hydrolysis events that proceeded over hours are sufficiently slower than the desired thiol addition reactions that occur in minutes. The addition of thiols to maleimides can be accomplished in a variety of solvents. The thiols examined derived from cysteine and include the protected amino acid, a protected dipeptide, and native oligopeptides containing either 9 or 18 amino acids. The addition reactions were monitored with HPLC and mass spectrometry in most cases. Complete substitution was observed for small molecule reactants. The model peptides containing nine or eighteen amino acids provided a mixture of products averaging between 3 and 4 substitutions/dendrimer. The functionalization of the chelate group with gadolinium was also accomplished easily.

Nanoparticle Effects on Human Platelets in Vitro: A Comparison between PAMAM and Triazine Dendrimers.

Triazine and PAMAM dendrimers of similar size and number of cationic surface groups were compared for their ability to promote platelet aggregation. Triazine dendrimers (G3, G5 and G7) varied in molecular weight from 8 kDa-130 kDa and in surface groups 16-256. PAMAM dendrimers selected for comparison included G3 (7 kDa, 32 surface groups) and G6 (58 kDa, 256 surface groups). The treatment of human platelet-rich plasma (PRP) with low generation triazine dendrimers (0.01-1 µM) did not show any significant effect in human platelet aggregation in vitro; however, the treatment of PRP with larger generations promotes an effective aggregation. These results are in agreement with studies performed with PAMAM dendrimers, where large generations promote aggregation. Triazine dendrimers promote aggregation less aggressively than PAMAM dendrimers, a factor attributed to differences in cationic charge or the formation of supramolecular assemblies of dendrimers.

Thermoregulated Coacervation, Metal-Encapsulation and Nanoparticle Synthesis in Novel Triazine Dendrimers.

The synthesis and solubility behaviors of four generation five (G5) triazine dendrimers are studied. While the underivatized cationic dendrimer is soluble in water, the acetylated and propanoylated derivatives undergo coacervation in water upon increasing temperature. Occurring around room temperature, this behavior is related to a liquid-liquid phase transition with a lower critical solution temperature (LCST) and is explained by differences in composition, notably, the hydrophobic nature of the terminal groups. Interestingly, the water solubility of the acetylated dendrimer is affected by the addition of selected metal ions. Titrating solutions of acetylated dendrimer at temperatures below the LCST with gold or palladium ions promoted precipitation, but platinum, iridium, and copper did not. Gold nanoparticles having diameters of 2.5 ± 0.8 nm can be obtained from solutions of the acetylated dendrimer at concentrations of gold less than that required to induce precipitation by treating the solution with sodium borohydride.

Triazine-Substituted and Acyl Hydrazones: Experiment and Computation Reveal a Stability Inversion at Low pH.

Condensation of a hydrazine-substituted s-triazine with an aldehyde or ketone yields an equivalent to the widely used, acid-labile acyl hydrazone. Hydrolysis of these hydrazones using a formaldehyde trap as monitored using HPLC reveals that triazine-substituted hydrazones are more labile than acetyl hydrazones at pH>5. The reactivity trends mirror that of the corresponding acetyl hydrazones, with hydrolysis rates increasing along the series (aromatic aldehyde

Light-fuelled transport of large dendrimers and proteins.

This work presents a facile water-based supramolecular approach for light-induced surface patterning. The method is based upon azobenzene-functionalized high-molecular weight triazine dendrimers up to generation 9, demonstrating that even very large globular supramolecular complexes can be made to move in response to light. We also demonstrate light-fuelled macroscopic movements in native biomolecules, showing that complexes of apoferritin protein and azobenzene can effectively form light-induced surface patterns. Fundamentally, the results establish that thin films comprising both flexible and rigid globular particles of large diameter can be moved with light, whereas the presented material concepts offer new possibilities for the yet marginally explored biological applications of azobenzene surface patterning.

TAT-mediated photochemical internalization results in cell killing by causing the release of calcium into the cytosol of cells.

BACKGROUND: Lysis of endocytic organelles is a necessary step in many cellular delivery methodologies. This is achieved efficiently in the photochemical internalization approach but the cell death that accompanies this process remains a problem. METHODS: We investigate the mechanisms of cell death that accompanies photochemical internalization of the fluorescent peptide TMR-TAT. RESULTS: TMR-TAT kills cells after endocytosis and light irradiation. The lysis of endocytic organelles by TMR-TAT causes a rapid increase in the concentration of calcium in the cytosol. TMR-TAT co-localizes with endocytic organelles containing calcium prior to irradiation and photochemical internalization leads to the release of the lumenal content of these organelles. Ruthenium red and cyclosporin A, inhibitors of calcium import in mitochondria and of the mitochondria permeability transition pore, inhibit cell death. CONCLUSIONS: TMR-TAT mediated photochemical internalization leads to a disruption of calcium homeostasis. The subsequent import of calcium in mitochondria is a causative factor of the cell death that accompanies photochemical internalization. General significance Understanding how the lysis of endocytic organelles affects cellular physiology and causes cell death is crucial to the development of optimal delivery methodologies.

Synthesis of large dendrimers with the dimensions of small viruses.

The dendrimer chemistry reported offers a route to synthetic target molecules with spherical shape, well-defined surface chemistries, and dimensions that match the size of virus particles. The largest target, a generation-13 dendrimer comprising triazines linked by diamines, is stable across ranges of concentration, pH, temperature, solvent polarity and in the presence of additives. This dendrimer theoretically presents 16,384 surface groups and has a molecular weight exceeding 8.4 MDa. Transmission electron and atomic force microscopies, dynamic light scattering, and computations reveal a diameter of ~30 nm. The target was synthesized through an iterative divergent approach using a monochlorotriazine macromonomer providing two generations of growth per synthetic cycle. Fidelity in the synthesis is supported by evidence from NMR spectroscopy, mass spectrometry, and high-pressure liquid chromatography.

Design, synthesis and biological assessment of a triazine dendrimer with approximately 16 Paclitaxel groups and 8 PEG groups.

The synthesis and characterization of a generation three triazine dendrimer that displays a phenolic group at the core for labeling, up to eight 5 kDa PEG chains for solubility, and 16 paclitaxel groups is described. Three different diamine linkers--dipiperidine trismethylene, piperazine, and aminomethylpiperidine--were used within the dendrimer. To generate the desired stoichiometric ratio of 8 PEG chains to 16 paclitaxel groups, a monochlorotriazine was prepared with two paclitaxel groups attached through their 2'-hydroxyls using a linker containing a labile disulfide. This monochlorotriazine was linked to a dichlorotriazine with aminomethylpiperidine. The resulting dichlorotriazine bearing two paclitaxel groups could be reacted with the eight amines of the dendrimer. NMR and MALDI-TOF confirm successful reaction. The eight monochlorotriazines of the resulting material are used as the site for PEGylation affording the desired 2:1 stoichiometry. The target and intermediates were amenable to characterization by (1)H and (13)C NMR, and mass spectrometry. Analysis revealed that 16 paclitaxel groups were installed along with 5-8 PEG chains. The final construct is 63% PEG, 22% paclitaxel, and 15% triazine dendrimer. Consistent with previous efforts and computational models, 5 kDa PEG groups were essential for making the target water-soluble. Molecular dynamics simulations showed a high degree of hydration of the core, and a radius of gyration of 2.8 ± 0.2 nm. The hydrodynamic radius of the target was found to be 15.8 nm by dynamic light scattering, an observation indicative of aggregation. Drug release studies performed in plasma showed slow and identical release in mouse and rat plasma (8%, respectively). SPECT/CT imaging was used to follow biodistribution and tumor uptake. Using a two component model, the elimination and distribution half-lives were 2.65 h and 38.2 h, respectively. Compared with previous constructs, this dendrimer persists in the vasculature longer (17.33 ± 0.88% ID/g at 48 h postinjection), and showed higher tumor uptake. Low levels of dendrimer were observed in lung, liver, and spleen (~6% ID/g). Tumor saturation studies of small prostate cancer tumors (PC3) suggest that saturation occurs at a dose between 23.2 mg/kg and 70.9 mg/kg.

Adaptation of Empirical Methods to Predict the LogD of Triazine Macrocycles.

Octanol/water partition coefficients guide drug design, but algorithms do not always accurately predict these values. For cationic triazine macrocycles that adopt a conserved folded shape in solution, common algorithms fall short. Here, the logD values for 12 macrocycles differing in amino acid choice were predicted and then measured experimentally. On average, AlogP, XlogP, and ChemAxon predictions deviate by 0.9, 2.8, and 3.9 log units, with XlogP overestimating lipophilicity and AlogP and ChemAxon underestimating lipophilicity. Importantly, however, a linear relationship (R2 > 0.98) exists between the values predicted by AlogP and the experimentally determined logD values, thus enabling more accurate predictions.

Experimental and computational evidence for an inversion in guest capacity in high-generation triazine dendrimer hosts.

The synthesis, characterization, and host-guest chemistry of high-generation triazine dendrimers are described. With pyrene and camptothecin as guests, experiments revealed that the guest capacity of odd-generation triazine dendrimers increased until generation 7 but decreased at generation 9. Molecular dynamics simulations conducted in explicit solvent showed a useful fingerprint for this behavior in radial distribution functions of water molecules penetrating the interior of the dendrimers. A linear relationship between the guest capacity of dendrimers measured experimentally and the number of water molecules within the interior determined computationally was observed.

Gadolinium MRI contrast agents based on triazine dendrimers: relaxivity and in vivo pharmacokinetics.

Four gadolinium (Gd)-based macromolecular contrast agents, G3-(Gd-DOTA)(24), G5-(Gd-DOTA)(96), G3-(Gd-DTPA)(24), and G5-(Gd-DTPA)(96), were prepared that varied in the size of dendrimer (generation three and five), the type of chelate group (DTPA or DOTA), and the theoretical number of metalated chelates (24 and 96). Synthesis relied on a dichlorotriazine derivatized with a DOTA or DTPA ligand that was incorporated into the dendrimer and ultimately metalated with Gd ions. Paramagnetic characteristics and in vivo pharmacokinetics of all four contrast agents were investigated. The DOTA-containing agents, G3-(Gd-DOTA)(24) and G5-(Gd-DOTA)(96), demonstrated exceptionally high r1 relaxivity values at off-peak magnetic fields. Additionally, G5-(Gd-DOTA)(96) showed increased r1 relaxivity in serum compared to that in PBS, which was consistent with in vivo images. While G3-(Gd-DOTA)(24) and G3-(Gd-DTPA)(24) were rapidly excreted into the urine, G5-(Gd-DOTA)(96) and G5-(Gd-DTPA)(96) did not clear as quickly through the kidneys. Molecular simulation of the DOTA-containing dendrimers suggests that a majority of the metalated ligands are accessible to water. These triazine dendrimer-based MRI contrast agents exhibit several promising features such as high in vivo r1 relaxivity, desirable pharmacokinetics, and well-defined structure.