Generation 3 PAMAM dendrimer TAMRA conjugates containing precise dye/dendrimer ratios.

The synthesis, isolation, and characterization of generation 3 poly(amidoamine) (G3 PAMAM) dendrimer containing precise ratios of 5-carboxytetramethylrhodamine succinimidyl ester (TAMRA) dye (n = 1-3) per polymer particle are reported. Stochastic conjugation of TAMRA dye to the dendrimer was followed by separation into precise dye-polymer ratios using rp-HPLC. The isolated materials were characterized by rp-UPLC, MALDI-TOF-MS, and 1H NMR spectroscopy, UV-vis, and fluorescence spectroscopies.

Isolation and characterization of precise dye/dendrimer ratios.

Fluorescent dyes are commonly conjugated to nanomaterials for imaging applications using stochastic synthesis conditions that result in a Poisson distribution of dye/particle ratios and therefore a broad range of photophysical and biodistribution properties. We report the isolation and characterization of generation 5 poly(amidoamine) (G5 PAMAM) dendrimer samples containing 1, 2, 3, and 4 fluorescein (FC) or 6-carboxytetramethylrhodamine succinimidyl ester (TAMRA) dyes per polymer particle. For the fluorescein case, this was achieved by stochastically functionalizing dendrimer with a cyclooctyne "click" ligand, separation into sample containing precisely defined "click" ligand/particle ratios using reverse-phase high performance liquid chromatography (RP-HPLC), followed by reaction with excess azide-functionalized fluorescein dye. For the TAMRA samples, stochastically functionalized dendrimer was directly separated into precise dye/particle ratios using RP-HPLC. These materials were characterized using (1)H and (19)F NMR spectroscopy, RP-HPLC, UV/Vis and fluorescence spectroscopy, lifetime measurements, and MALDI.

Selecting improved peptidyl motifs for cytosolic delivery of disparate protein and nanoparticle materials.

Cell penetrating peptides facilitate efficient intracellular uptake of diverse materials ranging from small contrast agents to larger proteins and nanoparticles. However, a significant impediment remains in the subsequent compartmentalization/endosomal sequestration of most of these cargoes. Previous functional screening suggested that a modular peptide originally designed to deliver palmitoyl-protein thioesterase inhibitors to neurons could mediate endosomal escape in cultured cells. Here, we detail properties relevant to this peptide's ability to mediate cytosolic delivery of quantum dots (QDs) to a wide range of cell-types, brain tissue culture and a developing chick embryo in a remarkably nontoxic manner. The peptide further facilitated efficient endosomal escape of large proteins, dendrimers and other nanoparticle materials. We undertook an iterative structure-activity relationship analysis of the peptide by discretely modifying key components including length, charge, fatty acid content and their order using a comparative, semiquantitative assay. This approach allowed us to define the key motifs required for endosomal escape, to select more efficient escape sequences, along with unexpectedly identifying a sequence modified by one methylene group that specifically targeted QDs to cellular membranes. We interpret our results within a model of peptide function and highlight implications for in vivo labeling and nanoparticle-mediated drug delivery by using different peptides to co-deliver cargoes to cells and engage in multifunctional labeling.

New porphyrins bearing pyridyl peripheral groups linked by secondary or tertiary sulfonamide groups: synthesis and structural characterization.

New pyridyl meso-tetraarylporphyrins (TArP, Ar = -C(6)H(4)-) of the general formula, T(R(1)R(2)NSO(2)Ar)P (R(1) = N-py-n-CH(2) (n = 2 or 4) and R(2) = CH(3)), have been synthesized by the versatile approach of utilizing meso-tetra(4-chlorosulfonylphenyl)porphyrin. After characterization by mass spectrometry and by visible absorption and (1)H NMR spectroscopy, the porphyrins were converted to various metalloderivatives, including Cu(II) and Zn(II). Treatment of T(N-py-4-CH(2)(CH(3))NSO(2)Ar)P (5) or TpyP(4) (meso-tetra(4-pyridyl)porphyrin) with CH(3)Co(DH)(2)H(2)O (DH = monoanion of dimethylglyoxime) afforded [CH(3)Co(DH)(2)](4)T(N-py-4-CH(2)(CH(3))NSO(2)Ar)P (6) and [CH(3)Co(DH)(2)](4)TpyP(4) (7). Typically, basic pyridines shift the axial methyl (1)H NMR signal of CH(3)Co(DH)(2)L upfield but leave the equatorial methyl signal unshifted. However, both signals for [CH(3)Co(DH)(2)](4)TpyP(4) are approximately 0.2 ppm more downfield than normal, suggesting perhaps an extremely non-basic pyridyl group. However, TpyP(4) forms CH(3)Co(DH)(2)py adducts with binding ability comparable to that of other pyridine ligands with normal basicity and to that of T(N-py-4-CH(2)(CH(3))NSO(2)Ar)P. Consequently, in 7 the deshielding of the methyl signals, even the axial Co-CH(3) signal, is attributed to anisotropy of the porphyrin core. The methyl signals for [CH(3)Co(DH)(2)](4)T(N-py-4-CH(2)(CH(3))NSO(2)Ar)P (6) have normal shifts. The absence of an anisotropic effect is attributable to the large distance of the CH(3)Co(DH)(2) moieties from the porphyrin core caused by the intervening linker in 6. Indeed, the separation led to only a slightly reduced (25%) fluorescence emission of 6 compared to 5, whereas that of 7 is considerably reduced (90%) compared to TpyP(4). The X-ray structures of 5, its Cu(II) complex, and [CH(3)Co(DH)(2)](4)TpyP(4) (7) (all of which have C(i) symmetry) support the spectroscopy. For example, the Co-N(ax) bond lengths of [CH(3)Co(DH)(2)](4)TpyP(4) (2.055(4) and 2.079(4) A) are comparable to that of CH(3)Co(DH)(2)py (2.068(3) A), consistent with the normal coordinating ability of TpyP(4). In an accompanying study, the new pyridylporphyrins have been converted to DNA-binding, water-soluble cationic porphyrins.

New porphyrins bearing positively charged peripheral groups linked by a sulfonamide group to meso-tetraphenylporphyrin: interactions with calf thymus DNA.

New water-soluble cationic meso-tetraarylporphyrins (TArP, Ar = 4-C(6)H(4)) and some metal derivatives have been synthesized and characterized. One main goal was to assess if N-methylpyridinium (N-Mepy) groups must be directly attached to the porphyrin core for intercalative binding of porphyrins to DNA. The new porphyrins have the general formula, [T(R(2)R(1)NSO(2)Ar)P]X(4/8) (R(1) = CH(3) or H and R(2) = N-Mepy-n-CH(2) with n = 2, 3, or 4; or R(1) = R(2) = Et(3)NCH(2)CH(2)). Interactions of selected porphyrins and metalloporphyrins (Cu(II), Zn(II)) with calf thymus DNA were investigated by visible circular dichroism (CD), absorption, and fluorescence spectroscopies. The DNA-induced changes in the porphyrin Soret region (a positive induced CD feature and, at high DNA concentration, increases in the Soret band and fluorescence intensities) indicate that the new porphyrins interact with DNA in an outside, non-self-stacking binding mode. Several new metalloporphyrins did not increase DNA solution viscosity and thus do not intercalate, confirming the conclusion drawn from spectroscopic studies. Porphyrins known to intercalate typically bear two or more N-Mepy groups directly attached to the porphyrin ring, such as the prototypical meso-tetra(N-Mepy)porphyrin tetracation (TMpyP(4)). The distances between the nitrogens of the N-Mepy group are estimated to be approximately 11 A (cis) and 16 A (trans) for the relatively rigid TMpyP(4). For the new flexible porphyrin, [T(N-Mepy-4-CH(2)(CH(3))NSO(2)Ar)P]Cl(4), the distances between the nitrogens are estimated to be able to span the range from approximately 9 to approximately 25 A. Thus, the N-Mepy groups in the new porphyrins can adopt the same spacing as in known intercalators such as TMpyP(4). The absence of intercalation by the new porphyrins indicates that the propensity for the N-Mepy group to facilitate DNA intercalation of cationic porphyrins requires direct attachment of N-Mepy groups to the porphyrin core.