Alkynyl-naphthalimide Fluorophores: Gold Coordination Chemistry and Cellular Imaging Applications

A range of fluorescent alkynyl-naphthalimide fluorophores has been synthesized and their photophysical properties examined. The fluorescent ligands are based upon a 4-substituted 1,8-naphthalimide core and incorporate structural variations (at the 4-position) to tune the amphiphilic character: chloro (<b>L1</b>), 4-[2-(2-aminoethoxy)­ethanol] (<b>L2</b>), 4-[2-(2-methoxyethoxy)­ethylamino] (<b>L3</b>), piperidine (<b>L4</b>), morpholine (<b>L5</b>), 4-methylpiperidine (<b>L6</b>), and 4-piperidone ethylene ketal (<b>L7</b>) variants. The amino-substituted species (<b>L2</b>–<b>L7</b>) are fluorescent in the visible region at around 517–535 nm through a naphthalimide-localized intramolecular charge transfer (ICT), with appreciable Stokes’ shifts of ca. 6500 cm^–1 and lifetimes up to 10.4 ns. Corresponding two-coordinate Au­(I) complexes [Au­(L)­(PPh₃)] were isolated, with X-ray structural studies revealing the expected coordination mode via the alkyne donor. The Au­(I) complexes retain the visible fluorescence associated with the coordinated alkynyl-naphthalimide ligand. The ligands and complexes were investigated for their cytotoxicity across a range of cell lines (LOVO, MCF-7, A549, PC3, HEK) and their potential as cell imaging agents for HEK (human embryonic kidney) cells and Spironucleus vortens using confocal fluorescence microscopy. The images reveal that these fluorophores are highly compatible with fluorescence microscopy and show some clear intracellular localization patterns that are dependent upon the specific nature of the naphthalimide substituent

The vertebral cartilage of aging fish is rich in chondroitin, but not keratan, sulfate.

<p><b>A</b>-<b>B</b>. Immunohistochemical labelling controls showing no non-specific binding of primary (mIg, ‘naive’ mouse immunoglobulin) or secondary antibody (PBS, phosphate buffered saline). <b>C</b>-<b>N</b>. Immunohistochemical labelling patterns of chondroitin/dermatan (C-0-S, C-4-S/DS, C-6-S) and keratan sulfate (KS) at 1, 2 and 3 years (left, middle and right panels, respectively). Note prominent pericellular labelling of CS/DS epitopes, particularly in 2 and 3 year samples. Unlike CS, KS occurs only within the notochordal tissue of the intervertebral disc (bottom left; arrowhead) and appears to diminish during aging. Scalebar represents 100 microns.</p

The vertebral cartilage of aged fish display changes in matrix ultrastructure and cell morphology.

<p><b>A</b>. Representative images showing the ultrastructure of vertebral cartilage, chondrocytes and pericellular matrix (top panel, middle panel and bottom panel, respectively) at 1, 2 and 3-years (left, middle and right panels, respectively). Chondrocytes display morphologies suggestive of programmed cell death at all stages. Note prominent lacunae in 2 and 3 year samples and increase in electron density of surrounding ECM. <b>B</b>. Graph showing mean lacunal area at each age. Note significant increase in 2 and 3-year samples, relative to 1 year samples tested by One-way ANOVA; 1 vs 2 year <i>P</i>=3.26E-07, 1 vs 3 year <i>P</i>=0.00945, 2 vs 3 year <i>P</i>=0.0512. <b>C</b>. Graph showing percentage area occupied by cell at each age. Note significant decrease in 2 and 3 year samples, relative to 1 year samples tested by One-way ANOVA; 1 vs 2 year <i>P</i>=1.13E-15, 1 vs 3 year <i>P</i>=1.61E-10, 2 vs 3 year <i>P</i>=0.357. <i>l</i>, lacunae; <i>v</i>.; intracellular vacuoles; <i>black </i><i>asterisks</i>, vesicular debris; <i>black </i><i>arrows</i> denote myelin figures; <i>white </i><i>arrows</i> show condensed nuclear material; <i>black </i><i>arrowhead</i> shows discontinuity of cell membrane. Scalebar in microns.</p

Aging zebrafish show changes in matrix organisation visible at the light microscopic level.

<p><b>A</b>. Schematic showing anatomical organisation of trunk vertebrae/intervertebral disc (mid-sagittal section plane). Vertebral bone depicted in pink; vertebral cartilage in blue; intervertebral discs shaded in black. <b>B</b>. Alcian blue staining of sulfated GAG in 1, 2, and 3 year spines. <i>Upper </i><i>panel</i>: low power showing discs and interjacent vertebral body. Boxed areas denote regions examined at high power in underlying panel. <i>Lower </i><i>panel</i>: Detail of Alcian blue staining within vertebral cartilage. Note prominent pericellular localisation of GAG and change in chondron morphology with age. <b>C</b>. Collagen birefringence (Picrosirius red staining under polarising optics) in 1, 2 and 3 year spines. <i>Upper </i><i>panel</i>: low power showing discs and interjacent vertebral body. Boxed areas denote regions examined at high power in underlying panel. <i>Lower </i><i>panel</i>: detail of collagen birefringence within vertebral cartilage. N.B. collagen birefringence occurs throughout the ECM at 1 and 2-years, but becomes increasingly organised within the pericellular matrix by 3-years. <i>iv</i>, intervertebral disc; <i>vb</i>, vertebral body; <i>n</i>, notochord-derived tissue; <i>asterisks</i>, cavities within cortical bone of vertebrae; <i>arrow-head</i>, notochordal tract running through cartilaginous inner facet of vertebral bodies; <i>block </i><i>arrow</i> denotes coalescence of adjacent chondrons in 3 year samples. Scalebar in microns.</p

HPLC reveals increases to total vertebral chondroitin sulfate levels during aging.

<p><b>A</b>. Total quantities of CS disaccharides recovered per mg dry weight. Note the significant increase in total CS with age tested by One-way ANOVA; 2 vs 3 year <i>P</i>=1.18E-03, 1 vs 3 year <i>P</i>=1.76E-04 <b>B</b>. Amounts of the different CS subtypes recovered. P values: for 4S 1 vs 3 year <i>P</i>=0.0486, for 6S 1 vs 3 year <i>P</i>=0.0458 C. Relative proportions of the different disaccharide species, shows no significant change to ratios of different CS subtypes. <b>D</b>. Types of sulfation observed. There is no significant change to the amount of sulfation of the disaccharides over the three ages. </p

Aging zebrafish show gross morphological changes to the vertebral column.

<p><b>A</b>. Graph showing incidence (%) of deformities by age. n=20 for each age group. <b>B</b>. Gross morphological appearance (left panel) and corresponding radiology (middle and left panels; left panels show detail of trunk and tail vertebrae) of zebrafish at 1, 2 and 3-years. Black arrows (in bottom panel) denote suspected dislocations of the spine. White arrowheads point to regions of increased bone density in vertebrae surrounding the dislocation. <b>C</b>. MicroCT images showing a representative single reconstructed vertebra (C5) from each age group, black arrows point to regions of bone erosion, white arrowheads point to bony outgrowths; <i>asterisk</i> denotes fracture. <b>D</b>. Graph of average bone mineral density shows no difference to bone density at the different ages, tested by One-way ANOVA; 1 vs 2 year <i>P</i>=0.80, 1 vs 3 year <i>P</i>=0.92, 2 vs 3 year <i>P</i>=0.79. n=3 for each age.</p

Fluorescent Rhenium-Naphthalimide Conjugates as Cellular Imaging Agents

A range of biologically compatible, fluorescent rhenium-naphthalimide conjugates, based upon the rhenium <i>fac</i>-tricarbonyl core, has been synthesized. The fluorescent ligands are based upon a N-functionalized, 4-amino-derived 1,8-naphthalimide core and incorporate a dipicolyl amine binding unit to chelate Re­(I); the structural variations accord to the nature of the alkylated imide with ethyl ester glycine (<b>L</b>^<b>1</b>), 3-propanol (<b>L</b>^<b>2</b>), diethylene glycol (<b>L</b>^<b>3</b>), and benzyl alcohol (<b>L</b>^<b>4</b>) variants. The species are fluorescent in the visible region between 505 and 537 nm through a naphthalimide-localized intramolecular charge transfer, with corresponding fluorescent lifetimes of up to 9.8 ns. The ligands and complexes were investigated for their potential as imaging agents for human osteoarthritic cells and protistan fish parasite <i>Spironucleus vortens</i> using confocal fluorescence microscopy. The results show that the specific nature of the naphthalimide structure serves to control the uptake and intracellular localization of these imaging agents. Significant differences were noted between the free ligands and complexes, with the Re­(I) complex of <b>L</b>^<b>2</b> showing hydrogenosomal localization in <i>S. vortens</i>

Fluorescent Rhenium-Naphthalimide Conjugates as Cellular Imaging Agents

A range of biologically compatible, fluorescent rhenium-naphthalimide conjugates, based upon the rhenium <i>fac</i>-tricarbonyl core, has been synthesized. The fluorescent ligands are based upon a N-functionalized, 4-amino-derived 1,8-naphthalimide core and incorporate a dipicolyl amine binding unit to chelate Re­(I); the structural variations accord to the nature of the alkylated imide with ethyl ester glycine (<b>L</b>^<b>1</b>), 3-propanol (<b>L</b>^<b>2</b>), diethylene glycol (<b>L</b>^<b>3</b>), and benzyl alcohol (<b>L</b>^<b>4</b>) variants. The species are fluorescent in the visible region between 505 and 537 nm through a naphthalimide-localized intramolecular charge transfer, with corresponding fluorescent lifetimes of up to 9.8 ns. The ligands and complexes were investigated for their potential as imaging agents for human osteoarthritic cells and protistan fish parasite <i>Spironucleus vortens</i> using confocal fluorescence microscopy. The results show that the specific nature of the naphthalimide structure serves to control the uptake and intracellular localization of these imaging agents. Significant differences were noted between the free ligands and complexes, with the Re­(I) complex of <b>L</b>^<b>2</b> showing hydrogenosomal localization in <i>S. vortens</i>