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.

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.

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.

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.

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).

Well-Tempered Metadynamics Simulations Predict the Structural and Dynamic Properties of a Chiral 24-Atom Macrocycle in Solution.

Inspired by therapeutic potential, the molecular engineering of macrocycles is garnering increased interest. Exercising control with design, however, is challenging due to the dynamic behavior that these molecules must demonstrate in order to be bioactive. Herein, the value of metadynamics simulations is demonstrated: the free-energy surfaces calculated reveal folded and flattened accessible conformations of a 24-atom macrocycle separated by barriers of ∼6 kT under experimentally relevant conditions. Simulations reveal that the dominant conformer is folded-an observation consistent with a solid-state structure determined by X-ray crystallography and a network of rOes established by 1H NMR. Simulations suggest that the macrocycle exists as a rapidly interconverting pair of enantiomeric, folded structures. Experimentally, 1H NMR shows a single species at room temperature. However, at lower temperature, the interconversion rate between these enantiomers becomes markedly slower, resulting in the decoalescence of enantiotopic methylene protons into diastereotopic, distinguishable resonances due to the persistence of conformational chirality. The emergence of conformational chirality provides critical experimental support for the simulations, revealing the dynamic nature of the scaffold-a trait deemed critical for oral bioactivity.

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.

Efficient syntheses of macrocycles ranging from 22-28 atoms through spontaneous dimerization to yield bis-hydrazones.

Acid treatment of a triazine displaying both a tethered acetal and BOC-protected hydrazine group leads to spontaneous condensation to yield macrocyclic dimers in excellent yields and purity. The bis-triazinyl hydrazones that form are characterized by 1H-NMR, 13C-NMR, 1H-COSY spectroscopy, X-ray diffraction, and mass spectrometry. By varying the length of the tether-the condensation product of an amino acid and amino acetal-rings comprising 22-28 atoms can be accessed. Glycine and ß-alanine were used for the amino acid. The amino acetal comprised 2, 3 or 4 carbon atoms in the backbone. High-performance liquid chromatography (HPLC) was employed to assess purity as well as to fingerprint the six homodimeric products. By combining the protected monomers and subjecting them to acid, mixtures of homodimers and heterodimers are obtained. When all six protected monomers are combined, at least 14 of the 21 theoretical dimeric products are observed by HPLC. Single crystal X-ray diffraction and solution NMR studies reveal the diversity of shapes available to these molecules.

Tumor Uptake of Triazine Dendrimers Decorated with Four, Sixteen, and Sixty-Four PSMA-Targeted Ligands: Passive versus Active Tumor Targeting.

Various glutamate urea ligands have displayed high affinities to prostate specific membrane antigen (PSMA), which is highly overexpressed in prostate and other cancer sites. The multivalent versions of small PSMA-targeted molecules are known to be even more efficiently bound to the receptor. Here, we employ a well-known urea-based ligand, 2-[3-(1,3-dicarboxypropyl)-ureido] pentanedioic acid (DUPA) and triazine dendrimers in order to study the effect of molecular size on multivalent targeting in prostate cancer. The synthetic route starts with the preparation of a dichlorotriazine bearing DUPA in 67% overall yield over five steps. This dichlorotriazine reacts with G1, G3, and G5 triazine dendrimers bearing a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) group for 64Cu-labeling at the core to afford poly(monochlorotriazine) intermediates. Addition of 4-aminomethylpiperidine (4-AMP) and the following deprotection produce the target compounds, G1-(DUPA)4, G3-(DUPA)16, and G5-(DUPA)64. These targets include 4/16/64 DUPA groups on the surface and a DOTA group at the core, respectively. In vitro cell assay using PC3-PIP (PSMA positive) and PC3-FLU (PSMA negative) cells reveals that G1-(DUPA)4 has the highest PC3-PIP to PC3-FLU uptake ratio (10-fold) through the PSMA-mediated specific uptake. While G5-(DUPA)64 displayed approximately 12 times higher binding affinity (IC50 23.6 nM) to PC3-PIP cells than G1-(DUPA)4 (IC50 282.3 nM) as evaluated in a competitive binding assay, the G5 dendrimer also showed high non-specific binding to PC3-FLU cells. In vivo uptake of the 64Cu-labeled dendrimers was also evaluated in severe combined inmmunodeficient (SCID) mice bearing PC3-PIP and PC3-FLU xenografts on each shoulder, respectively. Interestingly, quantitative imaging analysis of positron emission tomograph (PET) displayed the lowest tumor uptake in PC3-PIP cells for the midsize dendrimer G3-(DUPA)16 (19.4 kDa) (0.66 ± 0.15%ID/g at 1 h. p.i., 0.64 ± 0.11%ID/g at 4 h. p.i., and 0.67 ± 0.08%ID/g at 24 h. p.i.). Through the specific binding of G1-(DUPA)4 to PSMA, the smallest dendrimer (5.1 kDa) demonstrated the highest PC3-PIP to muscle and PC3-PIP to PC3-FLU uptake ratios (17.7 ± 5.5 and 6.7 ± 3.0 at 4 h p.i., respectively). In addition, the enhanced permeability and retention (EPR) effect appeared to be an overwhelming factor for tumor uptake of the largest dendrimer G5-(DUPA)64 as the uptake was at a similar level irrelevant to the PSMA expression.

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.

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.

A new triazine bearing a pyrazolone group capable of copper, nickel, and zinc chelation.

Interests in inorganic applications of triazines is growing. In this report, metal complexes of copper(II), nickel(II), and zinc(II) and a novel class of chelates comprising a triazine ring substituted with a hydrazine group and pyralozone are evaluated using spectrophotometric methods, single crystal X-ray diffractometry, and electrochemistry. Complexes with copper(II) include a single chelate and two chloride atoms to satisfy a trigonal bipryamidal coordination sphere. The nickel(II) and zinc(II) complexes are comprised of two chelating groups that adopt an octahedral geometry around the metal ion. Irreversible redox activity was observed with the copper(II) complex but no redox activity was observed with the ligand alone or zinc(II) and nickel(II) complexes. Use of the coumarin carboxylic acid assay shows that the ligand motif is capable of preventing redox cycling of copper in biological conditions and could thus serve as an antioxidant preventative agent. Cellular toxicity studies show that the new triazine molecule could have therapeutic applications in the µM concentration range based on the measured EC50=1.183±2 mM. Altogether this work shows that by merging triazine chemistry into inorganic compounds, there is potential to explore a range applications thanks to the new architecture.

Correction: Facile synthesis of stable, water soluble, dendron-coated gold nanoparticles.

Correction for 'Facile synthesis of stable, water soluble, dendron-coated gold nanoparticles' by Alan E. Enciso, et al., Nanoscale, 2017, 9, 3128-3132.

Facile synthesis of stable, water soluble, dendron-coated gold nanoparticles.

Upon reduction with sodium borohydride, diazonium tetrachloroaurate salts of triazine dendrons yield dendron-coated gold nanoparticles connected by a gold-carbon bond. These robust nanoparticles are stable in water and toluene solutions for longer than one year and present surface groups that can be reacted to change surface chemistry and manipulate solubility. Molecular modeling was used to provide insight on the hydration of the nanoparticles and their observed solubilties.

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.

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.

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.

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