Dendrimers based on melamine

Our group continues to pursue the synthesis and characterization of dendrimers based on melamine. These molecules serve as:

Interesting targets for macromolecular synthesis in solution or on the solid phase
Useful models for studying macromolecular properties including host-guest chemistry
Candidate vehicles for drug delivery

Our progress can be subdivided into these categories. This subdivision follows and is organized by the lesson learned.

Interesting targets for macromolecular synthesis in solution

Summary: We continue to make advances on the synthetic front. Over the last four years, we have identified more efficient routes based on better building blocks, established scalable processes (5g) and developed strategies for post-synthetic manipulation.

	*Lesson 1: Dendrimers based on melamine are tractable.* Investigator: Wen Zhang Manuscripts: Dendrimers Based on Melamine. Divergent and Orthogonal, Convergent Syntheses of a G3 Dendrimer. Zhang, W.; Simanek, E.E. Org. Lett. 2000, 2, 843-845.
	*Lesson 2: Specific substoichimetric numbers of reactive sites can be engineered on the surface.* Investigator: Wen Zhang Manuscripts: Orthogonal, Convergent Syntheses of Dendrimers Based on Melamine with One or Two Surface Sites for Manipulation. Zhang, W.; Nowlan, D.T.III; Thomson, L.M.; Lackowski, W.M.; Simanek, E.E. J. Am. Chem. Soc. 2001, 123, 8914-8922.
	*Lesson 3: Atom economy is a defining feature of this system.* Investigator: Mackay Steffensen Manuscript: Chemoselective building blocks for dendrimers from relative reactivity data. Steffensen, M.B.; Simanek, E.E. Org. Lett. 2003, 5, 2359-2361.

Using competition reactions with a model monochlorotriazine and mixtures of amine nucleophiles, the relative reactivity of amines has been determined (Figure 1). From this data, novel linkers have been designed by incorporating two groups with significantly different reactivities into the same molecule. We have moved from p-aminobenzylamine to aminomethylpiperidine, a group with similar reactivity differences, but better shelf-life. Compounds containing an aniline discolor over time. The result is that the only formal by-product of these syntheses is HCl. Future inquiry addresses chromatography-free syntheses and issues of scale.

Figure 1. Competition reactions reveal relative rates for substitution.

Lesson 4: Unprecedented structural diversity of the periphery can be obtained.
Investigator: Mackay Steffensen
Manuscript: Synthesis and Manipulation of Orthogonally Protected Dendrimers: BuildingBlocks for Library Synthesis. Steffensen, M.B.; Simanek, E.E. Angew. Chem. Int. Ed. 2004, In press.

We have completed the synthesis of dendrimers that display five or six orthogonally reactive sites by aping Wong's orthogonally protected carbohydrates. A free hydroxyl group is available for immediate acylation. Hydroxyl groups masked as tert-butyldiphenylsilyl ethers or levulic acid esters can be unmasked with tetrabutylammonium fluoride or hydrazine, respectively. The monochlorotriazine is available for nucleophilic aromatic substitution. The pyridyldisulfide is available for thiol-disulfide exchange. The BOC-protected amines offer a final site for manipulation. These targets are available on 5 g scale.

Lesson 5: Unprecedented structural diversity of the periphery can be obtained.
Investigator: Alona Umali
Manuscript: Preparation of Multivalent Dendrimers Through Thiol-disulfide Exchange. Umali, A.P.; Simanek, E.E. Org. Lett. 2003, 5, 1245-1247.

Interesting targets for solid-phase synthesis and application to environmental/materials challenges

Lesson 1: Interaction of dendritic surfactants with smectite clays is governed by the shape of the surfactant.
Investigator: Erick Acosta
Manuscript: Dendritic Surfactants Show Evidence for Frustrated Intercalation: A New Organoclay Morphology. Acosta, E.J.; Deng, Y.; White, G.N.; Dixon, J.B.; McInnes, K.; Senseman, S.A.; Frantzen, A.S.; Simanek, E.E. Chem. Mater.2003 15, 2903-2909.

We hypothesize that an inability to pack effectively leads to a new morphology, frustrated intercalation, in organoclay materials when the organic component is dendritic.

Lesson 2: The reactivity of triazines with constrained amine nucleophiles offers a strategy for contaminant sequestration.
Investigators: Erick Acosta, Mackay Steffensen, Emily Hollink
Manuscript: Acosta, E.J.; Steffensen, M.B.; Tichy, S.E.; Simanek, E.E. J. Agric. Food Chem. 2004, 52, 545-549.

Commercially available peptide synthesis resins with constrained secondary amines are show to covalently sequester atrazine.

Manuscript: Piperidine-Functionalized Supports as a Method to Sequester Atrazine from Solution. Hollink, E.; Tichy, S.E.; Simanek, E.E. Ind. Eng. Chem. Res. 2004, Submitted.

Janda amine-derivatized polystyrenes can be derivatized with isonipecotic acid. The resulting materials sequester atrazine.

Lesson 3: Dendrimers can be covalently anchored to silica gels and mesoporous silicas
Investigators: Erick Acosta and Sergio Gonzalez
Manuscript: Engineering Nanospace: Iterative Synthesis of Melamine-based Dendrimers on Amine-functionalized SBA-15 Leads to a Stepwise Reduction in Pore Volume. Acosta, E.; Carr, S.C.; Simanek, E.E.; Shantz. D.F.Adv. Mater. 2004, 16, 985-989.

Divergently prepared composites of mesoporous silica and triazine dendrimers prepared by iterative reactions of cyanuric chloride and piperazine off an amine-derivatized support lead to materials with decreasing pore volumes (and ultimately, occluded pores) based on BET analysis.

Lesson 4: Dendrimers can be covalently anchored to silica gels and mesoporous silicas
Investigators: Erick Acosta and Sergio Gonzalez
Manuscript: Synthesis, Characterization and Application of Melamine-Based Dendrimers Supported on Silica Gel. Acosta, E. J.; Gonzalez, S.O.; Simanek, E.E. J. Poly. Sci. A 2004, In press.

Dendrimers can be grown divergently (through iterative reaction) or convergently (by attaching the presynthesized unit) to silica gels. The material produced and its ability to sequester atrazine depends greatly on the strategy employed.

	*Lesson 5: Physisorbed materials may be useful for separations.* Investigators : Sergio Gonzalez with *Prof. David Ford's Lab** (lead group) Manuscript: Nanocomposite Membranes of Chemisorbed and Physisorbed Molecules on Porous Alumina for Environmentally Important Separations, Javaid, A.; Gonzalez, S. O.; Simanek, E. E.; Ford, D.M. Adv. Mater. Submitted.
	*Lesson 6: Derivatized thermomorphic polymers can sequester the pesticide atrazine.* Investigators : Sergio Gonzalez with Prof. David Bergbreiter's Laboratory (co-PI) Manuscript: Latent Solid-Phase Extraction Using Thermoresponsive Soluble Polymers . Gonzalez, S.O.; Furyk, S.; Li, C.; Tichy, S. E.; Bergbreiter, D. E.; Simanek, E. E. J. Poly. Sci. A. 2004 , In press.

A thermomorphic poly(N-isopropylacrylamide) polymer derivatized with aminomethylpiperidine groups sequesters atrazine and related monochlorotriazine analogues from solution upon heating.

Useful models for studying host-guest chemistry

	*Lesson 1: Dendrimers Based on melamine recognize small molecules and metal ions.* Investigator: Wen Zhang Manuscript: Synthesis and Characterization of Higher Generation Dendrons Based on p-Aminobenzylamine. Evidence for Molecular Recognition of Cu(II). Zhang, W.; Simanek, E.E.; Tetrahedron Lett. 2001, 42, 5355-5357.
	*Lesson 2: Surface and interior groups affect the aggregation state of dendrimers capable of hydrogen bonding.* Investigator: Wen Zhang and Sergio Gonzalez Manuscript: Structure-Activity Relationships in Dendrimers Based on Triazines: Gelation Depends on Choice of Linking and Surface Groups. Zhang, W.; Gonzalez, S.O.; Simanek, E.E. Macromolecules 2002, 35, 9015-9021.
	Lesson 3: The cleavage rates of bio-labile bonds within a dendrimer is dictated by bond position. Investigator: Wen Zhang Manuscript: Evaluation of Multivalent Dendrimers Based on Melamine: Kinetics of Thiol-Disulfide Exchange Depends on the Structure of the Dendrimer. Zhang, W.; Tichy,S.E.; Pérez, L.M.; Maria, G.; Lindahl, P.A.; Simanek, E.E. J. Am. Chem. Soc. 2003, 125.

The ability to control the position of functional groups within a dendrimer was exploited to append bio-labile disulfide bonds at varying distances from the core of the dendrimer. Disulfide reduction represents one opportunity for drug delivery because the intracellular concentration of thiols including cysteine, homocysteine, and glutathione are 100-1000x higher than that of the vasculature. We have found that dendrimers with mulitple disulfide-linked fluorophores undergo thiol-disulfide exchange with an exogenous thiol (dithiothreitol) at different rates.

The half-life of exchange for groups buried within the interior of the dendrimer (as predicted from a 2-D representation of the molecule) is 2.2x slower than groups on smaller dendrimers. Groups that appear accessible on larger dendrimers have half-lives 1.8x slower than those groups on smaller dendrimers. Interestingly, as the number of disulfides exchanged for a given dendrimer increases, the rate for exchange of remaining disulfides increases according to mathematical models derived from relative population data collected by mass spectometry. This work was published as cited below:

Candidate vehicles for drug delivery

	*Lesson 1: The toxicity of FDA approved drugs can be attenuated by coadministration with dendrimer.* Investigators: Michael Neerman and Hui-Ting Chen Manuscript: Attenuation of Drug Toxicity Using Dendrimers Based on Melamine, Candidate Vehicles for Drug Delivery . Neerman, M.F.; Chen, H.-T.; Parrish, A.R.; Simanek, E.E. Molec. Pharm. 2004, In Press
	*Lesson 2: Oligonucleotides can be covalently appended to dendrimers.* Investigators: Steve Bell and Megan McLean Manuscript: Synthesis and Characterization of DNA-Dendrimer Conjugates. Bell, S.A.; McLean, M.E.; Oh, S.-K.; Tichy, S.E.; Corn, R.M.; Crooks, R.M.; Simanek, E.E. Bioconj. Chem. 2003, 14 , 488-493.
	*Lesson 3: Initial biocompatibility studies of our first candidate, a polycationic dendrimer delivered i.p.* Investigator: Michael Neerman Manuscript: In vivo evaluation of a triazine dendrimer: a potential vehicle for drug delivery. F. Neerman, M.R.; Zhang, W.; Parrish, A.R.; Simanek, E.E. Int. J. Pharm. 2004 , 1 , 390-393.
	*Lesson 4 Hydrophobic drugs are sequestered.* Investigator: Wen Zhang Manuscript: Triazine Dendrimers for Drug Delivery: Evaluation of Solubilization Properties, Activity in Cell Culture, and in vivo Toxicity of a Candidate Vehicle. Zhang, W.; Jiang, J.; Qin, C.; Thomson, L.M.; Parrish, A.R.; Safe,S.H.; Simanek, E.E. Supramol. Chem. 2003, 15, 607.

A third-generation dendrimer comprising piperazine linking groups and cationic amines on the periphery can sequester hydrophobic guests in water. These guests include pyrene (0.2 molecules/dendrimer), 10-hydroxycamptothecin (4 molecules/dendrimer), and a bisindolemethane (5 molecules/dendrimer). We attribute sequestration to a "dendritic phase" that resembles organic solvent into which these drugs preferentially partition over water. This partitioning is reversible. In cell culture, the bisindolemethane delivered in DMSO (aq) has similar activity (upregulation of luciferase in a PPAR g gene expression construct) as the molecule delivered in the dendrimer. These preliminary experiments establish that molecular recognition can be employed.

Lesson 5 Preliminary examinations of these molecules in vitro and in vivo suggest that they may be suitable candidates for drug delivery.
Investigators: Michael Neerman and Hui-Ting Chen
Manuscript: Cytotoxicty, Hemolysis and Acute In Vivo Toxicity ofDendrimers Based on Melamine, Candidate Vehicles for Drug Delivery. Chen, H.-T.; Neerman, M.F.; Parrish, A.R.; Simanek, E.E. J. Am. Chem. Soc. 2004, 126 , 10044-10048.

In a series of experiments funded by the Center for Microencapsulation and Drug Delivery (TAMU), we established that these dendrimers may be suitable for the proposed long term goal, vehicles for drug delivery. A family of second generation dendrimers were prepared to address toxicity and hemolysis observed with the first dendrimer candidate (Chart 1. This target (1) contains AB 2 groups on the interior and AB 4 groups on the periphery to afford a generation three dendrimer with 48 surface groups that can be functionalized.

Cytotoxicity and hemolysis were probed with two cationic dendrimers, three anionic dendrimers and a PEGylated species. Hemolysis is significant in the cationic species, but markedly reduced in the anionic and neutral dendrimers (Figure 1)

Figure 1 The impact of surface modification on the hemotoxicity of 2-7 at 1 h (a) and 24 h (b) at doses that increase from left to right: 0.001 mg/mL; 0.01 mg/mL; 0.1 mg/mL; 1 mg/mL; 10 mg/mL.

Cell viability using normal cells (Clone 9) was also determined using the MTT assay and similar trends were observed (Figure 2).

Figure 2 Viability measured at concentrations that increase from left to right: 0.001 mg/mL; 0.01 mg/mL; 0.1 mg/mL; 1 mg/mL; 10 mg/mL.

In a CMDD sponsored study that acknowledges these NIH-supported data and syntheses, we examined the PEGylated target was evaluated in vivo. In single bolus injections to mice, we observe no anomolous blood chemistry using blood urea nitrogen (kidney damage indicator) or alanine transaminase levels (liver damage) at doses up to 2.6 g/kg i.p. or 1.3 g/kg delivered i.v. to the tail vein.

Lesson 6. Preliminary studies suggest peptide-dendrimer conjugates can elicit an immune response.
Investigator: Alona P. Umali
Manuscript: Biological Evaluations of Dendrimers Based on Melamine . Neerman, M.F.; Umali, A. P.; Chen, H. -T.; Waghela, S. D.; Parrish, A. R.; Simanek, E. E. J. Drug Del. Sci. Tech.. 2005, 15, 31-40.

Reviews of our drug delivery efforts:

	Dendrimers Based on Melamine: Vehicles for Drug Delivery? Simanek, E.E. A.C.S. Symposium Series. Submitted.
	Biological Evaluations of Dendrimers Based on Melamine . Neerman, M.F.; Umali, A. P.; Chen, H. -T.; Waghela, S. D.; Parrish, A. R.; Simanek, E. E. J. Drug Del. Sci. Tech.. 2005, 15, 31-40.