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Quinolizinium as a new fluorescent lysosomotropic probe
Accepted Manuscript Quinolizinium as a new fluorescent lysosomotropic probe Emmanouil Zacharioudakis, Tatiana Cañeque, Raúl Custodio, Sebastian Müller, Ana M. Cuadro, Juan J. Vaquero, Raphaël Rodriguez PII: DOI: Reference:
S0960-894X(16)31239-2 http://dx.doi.org/10.1016/j.bmcl.2016.11.074 BMCL 24469
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Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
2 November 2016 22 November 2016 23 November 2016
Please cite this article as: Zacharioudakis, E., Cañeque, T., Custodio, R., Müller, S., Cuadro, A.M., Vaquero, J.J., Rodriguez, R., Quinolizinium as a new fluorescent lysosomotropic probe, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.11.074
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Quinolizinium as a new fluorescent lysosomotropic probe
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Emmanouil Zacharioudakis,a‡ Tatiana Cañeque,a* Raúl Custodio,b‡ Sebastian Müller,a Ana M. Cuadro,b Juan J. Vaquero,b Raphaël Rodrigueza
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com
Quinolizinium as a new fluorescent lysosomotropic probe Emmanouil Zacharioudakis,a‡ Tatiana Cañeque,a* Raúl Custodio,b‡ Sebastian Müller,a Ana M. Cuadro,b Juan J. Vaquero,b Raphaël Rodrigueza a Institut Curie, PSL Research University, Organic Synthesis and Cell Biology group, 26 rue d’Ulm, 75248 Paris Cedex 05, France; CNRS UMR3666, 75005 Paris, France; INSERM U1143, 75005 Paris, France. b Departamento de Química Orgánica y Química Inorgánica, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain.
ARTICLE INFO
ABSTRACT
Article history: Received Revised Accepted Available online
We have synthesized a collection of quinolizinium fluorescent dyes for the purpose of cell imaging. Preliminary biological studies in human U2OS osteosarcoma cancer cells have shown that different functional groups appended to the cationic quinolizinium scaffold efficiently modulate photophysical properties but also cellular distribution. While quinolizinium probes are known nuclear staining reagents, we have identified a particular quinolizinium derivative salt that targets the lysosomal compartment. This finding raises the question of predictability of specific organelle targeting from structural features of small molecules.
Keywords: Quinolizinium Heteroaromatic cations Fluorescent dyes Lysosomal probes Small molecules
Recent advances in fluorescence microscopy have permitted to study cellular processes providing unprecedented topological insights. This powerful imaging technology has been employed extensively to study organelle-specific biological events and the subcellular localization of biologically active small molecules, providing useful clues as to how drugs operate in cells. In particular, fluorescence microscopy is well suited for live cell imaging studies, potentially providing valuable temporal information, which can lead to a deeper appreciation of the dynamic nature of some biological events.1-3 Thus, others and we have investigated the design and synthesis of versatile molecular scaffolds with the aim to improve the specific targeting of a given cellular organelle. Among them, fluorescent dyes suitable for nuclear DNA recognition have been widely employed and these are generally based on heteroaromatic organic molecules (Figures 1). For example, 4',6-diamidino-2-phenylindole (DAPI, 1) binds tightly to A and T nucleobases within the DNA minor groove and represents one of the most universally employed probes to detect cell nuclei.4 The cell-permeant nucleic acid binding dye acridine orange (AO, 2) has also been widely employed for cell-cycle studies thanks to the distinctive fluorescence properties that characterizes this scaffold with a green fluorescence emission upon binding to dsDNA, and a red fluorescence when bound to ssDNA or RNA. Importantly, AO does not selectively stain the nucleus and therefore can also be employed to monitor pH variations in the lysosomal compartment.5-6 Cationic heteroaromatic dyes are broadly considered as classical DNA intercalators, whereas positively charged heterocycles play an important role in conferring fluorescence properties to molecular
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probes.7,8 As a result, a large number of positively charged substrates have been successfully developed as fluorescent probes for DNA detection, including propidium (3) and ethidium NH HN
N H
NH2
NH2
N
N
AO (2)
DAPI (1) Me I Et N Me
I N
Br
H2 N
NH2
NH2
Ethidium bromide (4) N
N
N
N
H 2N
Propidium iodide (3) O
N
N
N
O N
4I YOYO-1 (5) Me H N
OEt 3 Cl
H N
H N NH
N H Hoechst 33342 (6)
Figure 1. Representative examples of DNA binding fluorescent dyes.
halides (4), which are DNA stains with poor membrane permeability that are often used to differentiate between living and dead cells in cell culture.9-11 This also includes dyes from the cyanine family such as the bis-intercalator YOYO-1 (5) used for monitoring DNase activity, and quantitating the concentration of PCR products.12 It also includes the broadly used DNA dye Hoechst 33342 (6), a bis-benzo[d]imidazolium cation commonly used to visualize nuclear and mitochondrial DNA.13,14
to be the highest emitter, presumably reflecting the electronic nature of the indole moiety compared to pyrrole, thiophene and imidazole. We found that the fluorescence of the compounds is enhanced when binding to DNA (†ESI), as we previously reported for other quinolizinium analogues.17 Table 1. Photophysical properties of the quinolizinium derivatives: Maximum linear absorption and emission wavelength (λmax Abs and λmax Em). Compound
In order to broaden the scope of available organelle-targeting markers, we developed a library of quinolizinum-derived molecules: bridgehead quaternary nitrogen heterocycles (BQNH) harboring extended electronic networks that enable the finetuning of fluorescence properties (Scheme 1). Different quinolizinum-based compounds have been developed and extensively studied as molecular DNA dyes.15,16 We have previously shown that V-shaped quinolizinium chromophores with high emission quantum yields and environment sensitive fluorescence lifetimes are suitable probes for cell biology, taking advantage of nonlinear excitation (two photon absorption, TPA). In particular, fluorescence lifetime imaging allows for the acquisition of multicolor images and the detection of different cell structures including the cytosol, the nuclear membrane and the nucleus.17,18 These results prompted us to develop novel cationic fluorescent dyes to further improve the tropism for DNA, whilst being suitable for one-photon absorption. To this end, we synthesized a series of V-shaped chromophores derived from quinolizinium (D-π-A+-π-D) and explored their photophysical properties in order to establish the relevant structural features that may affect fluorescence with particular emphasis on the nature of electron-donating substituents and the extent of electronic delocalization. 2,8Dimethylquinolizinium hexafluorophosphate (7) was subjected to a double Knoevenagel condensation19 with diversely functionalized aromatic or heteroaromatic aldehydes to afford quinolizinium probes 8a-f in good to excellent yield (Scheme 1). It is noteworthy that the counter anion exchange in favor of BF4 was employed to enhance the water solubility of 8b, an important characteristic for cell-based studies. Me N PF6 7
OMe
8a
H R Piperidine CH3CN, reflux, 6h (32-95%) OMe H N
R=
8b
R
O
Me
8c
N
R
N X
X = PF6, BF4
NH
8d
8a-f
NH
8e
S
8f
Scheme 1. Synthesis of quinolizinium derivatives 8a-f.
Photophysical properties were monitored in methanol to maximize the solubility of the analogues in a cell-free environment, thereby enabling a direct comparison of each compound within this series. A summary of the results is presented in Table 1 and linear absorption and emission spectra are depicted Fig. 2. In general, a good correlation was found between the nature of the electron-donating substituents used and the stabilization energy for the different types of chromophores studied, compound 8b harboring a more extended electron delocalization framework, yielding the highest λmax value. In contrast, the heteroaryl indole-derived substituted 8c was found
λmaxAbs (nm)
λmax Em (nm)
8a
444
540
8b
463
620
8c
489
584
8d
430
527
8e
457
542
8f
432
529
Figure 2. Absorption (A) and emission (B) spectra of the quinolizinium derivatives 8a-f recorded in methanol at 25 °C.
With the linear optical data in hand, we next explored the cellular labeling of each dye in human U2OS osteosarcoma cancer cells using high-resolution fluorescence microscopy (Figure 3). All the molecules entered living cells upon treatment using a regular culture medium at the optimal concentration of 2 μM. A comparative analysis using the minor groove DNA binder 1, revealed a similar pan-nuclear staining pattern for 8b,d-f, which comes in agreement with the DNA intercalating properties of the quinolizinium core scaffold.20,21 In contrast, probes 8a,e concomitantly localized to the nucleus and the cytosol, sharing a pan-nuclear staining with 1 along with a more diffuse cytoplasmic staining. Strikingly, 8c stained small cytosolic vesicles in the green channel (†ESI). It is noteworthy that while these analogues are structurally close, the sub-cellular accumulation of 8c was different from that of the other analogues of the series, reflecting either the targeting of a different cellular component or a different cellular uptake mechanism (e.g. endocytosis vs passive diffusion). This prompted us to identify the nature of the vesicles where 8c accumulated. Thus, cells were co-treated with 8c (green staining) and other organelle specific reagents including the specific lysosomal marker LysoTracker® deep red (red staining). Remarkably, visual detection of colocalizing foci (i.e. yellow vesicles in merge image) outside of the nucleus was characteristic of the physical proximity of 8c with the lysosomal marker (Figure 4), consistent with the notion that 8c targets the lysosomal compartment, an unprecedented feature of a quinolizinium-derived small molecules. To test whether the nuclear staining of 1 influenced the staining of 8c, we performed an experiment in the presence and absence of 1 and found that the staining of 8c was not altered under these conditions in the green channel (†ESI). Lysosomes are cellular organelles involved in several cellular processes including protein degradation, cellular waste recycling, secretion, plasma membrane repair, signaling, metabolism and iron homeostasis,22,23 thereby conferring this organelle a pivotal role in the physiology of the cell and a relevant organelle to target with small molecules.24,25 Owing to the current challenge
to o develoop sm malll mo olecuules thatt sellectively y tarrget the lysoosom me too sttudyy orr im mpacct th he norm n mal funnctioons of this orgganeelle, wee becam me inteerested in i ccharaacterrizinng mech m haniismss annd sttructuraal feeatures thaat gove g ern accuumuulatioon of 8c in thee lyysoso omaal co omppartm mennt.
Figuree 3. Confo C ocal micr m oscoopy im magees shoowin ng DA API (pan( -nucllear blue) b ), 8aa,b,d d-f (ppan-nnucleear grreen)) andd 8c (greeen puunctaa) in U2O OS ceells. Scale S e barr, 100 µm m. Ceells w weree treaated with 2 µM µ 8a-f for f 30 miin annd thhen fixed f withh fo ormaaldehyyde. DAPII
8c c
Lyso otracke er
Merge e
Zoo om
In connclussion,, wee havve deve d elopeed a straaighhtforrwarrd syynthhetic rooute too gene g eratee a liibrarry of caationnic quiinoliiziniium m chhrom mophhorees abble to targget diffeerennt ceellullar com mparrtmeents, naamelly the t nuclleuss annd lyysossomees, throoughh suubtlee strructuural modifficattionss. Giivenn thaat quuino olizinnium m baased proobes are knoown NA inteercaalatoors, ourr finndings prov p videe un nantiicipaatedd to bee DN insighhts into how w sttructturall vaariatiions of the corre sccaffoold can modulate sub bcelllularr loccalizzatio on annd organ o nellee speecifiic taargetting wiith smal s ll molec m cules, a straategyy that may m be appl a licabble tto ottherr typpes of scaff s ffoldds. Inntereestinngly y, wee deetectted som s me feeint nuclearr acccum mulaation n of 8c with w redd fluo oresscencce (†ES SI), whil w le thhe grreen fluuoreescennce alloowedd thhe detecctionn of lyssosom mal acccumuulatiion, makinng 88c in i innterestinng dduall fluuoreescennt prob p be to o viisuaalize multtaneeouslly and a indeepennden ntly twoo ceellular orga o anellles. Forr sim instannce, this connstituutes a neew prob p be thhat can c be b used to stainn the lysosoomal co ompaartm ment in a diifferrent fluoorescentt channeel frrom m that of o D DNA A, perm p mittinng the usee off otther staains witth blue b fluuoreescennce in conj c juncctionn wiith 8c 8 to laabel thee nucleuus. T This featurre is impportaant give g en thhe neeed to t have toolls too lab bel ddifferrentt ceellulaar ccom mparttmennts witth disti d inct colors inn ceell biol b ogy exxperiimennts. Thhe sttructturall feaaturees thhat enab e ble 8a-f 8 f to enteer ceells rem main eluusivve and a requirre addi a ition nal invvestiggatioons to bee fully fu chharaccteriizedd. Thhis may m include receeptoor-m mediaated end docyytosiis orr bee linnkedd to the lipoophiilic natu ure of this t seriies oof comp c pounnds. M anissms undderlyyingg thhe uptak Mech u ke and a distribuution n of o thhese coompooundds inn ceells are undder in nvesstigaationn annd will w be b rreporrtedd in duee couursee.
C ocal micrroscoopy im magees shhowin ng coo-localizeed focci (yeellow w Figuree 4. Confo puunctaa) off 8c (green ( n pun ncta)) andd LyssoTraackerr deeep redd (redd punncta)) in U2OS U S ceells. DAP PI (ppan-nnuclear bllue). Thee yelllow bbox show ws a 6× m magnnificaationn. Scale bar, 10 µ µm. Cells C weree co-treatted with w 2 µM M 8c aand LysoT L Trackker for f 300 m min.
Too innvesstigaate the t acccumuulatiion of 8c in lysoosom mes, wee wondderedd whet w w ther 8a-f coul c d be b inter i rnaliized byy means m s of en ndoccytoosis or rath r er rreachhed their taargeet orrgannelle by passsivee d sionn thrrouggh thhe plasm diffu p ma mem mbrane.. Giiven thaat enndoccyticc proceesses arre signi s ificaantlyy im mpaiired at low w tem mpeeratuure (e.gg. 4°C),, U22OS cells were w treaated witth 2 µM M 8aa-f foor 30 min m at a 377 °C arteesum C annd 4 °C C. As A a conntroll expperim mennt, we w used u myciin, a n ral produ natur p uct hybr h rid of o marm m mycinn A prev viouusly shown to taargeet th his orga o anellle byy en ndocytossis.244,25 Inter I restiinglyy, we w obbserrved d thaat 8a-f and arteesum myciin beehavved similarlly where w e thee lysosoome wass taargeted whhen cellls werre iincuubateed at 37 °C.. H Howeeverr, in ncubbatinng cellss at a 4 °C C thhrouughoout the treeatm mentt peeriodd ab bolishedd lyssosoome targgetinng ass deffined by y thee abssencce off staainedd vesiccles in thhesee condittionss (Fiigurre 5,, †E ESI). This data d stro onglyy f r off a proocesss wher w reby y enndoccytosis of 8a-f arrguees iin favor d inatees the domi t sub-celllularr acccum mulaationn off thhese moolecu uless. S e lyysosomees have Since h e a low wer pH H thhan theeir surrroun ndingg organnellees, we w next n t invvestigated whe w etherr changees in pH H could c d allter thee fluuoresscen nce inteensitty oof 8cc in n vittro andd in cellluloo. L eringg thhe pH Lowe H diid not signiificaantlyy afffect the fluooresccencce of 8c (††ESII), arrguiing that t f rescencee off 8c observeed inn the greeen fluor ly enhhancced by a low ysossomees iis not n wer pH H. In I addi a tionn, bafiloomyycin A, chlo c oroquinee and N NH4C Cl, comppounnds thatt preevennt accidifficattion of lyso osom mes or raisse thhe lyso l som mal ppH, did d noot afffect thee fluuoresscen nce of o 8cc in lysoosom mes (†E ESI), sugggesting g thaat pH cchannges in this com mpaartmeent do not affeect the t sub-cellularr lo ocaliizatiion of this t mollecuule. T Thiss is in line l witth thhe iddea thatt, u ke chlor unlik c roquuinee, coomppounnd 8c doees not n acccumuulatee inn ly ysossomees ddue to t th he loow pH of this t celllularr coompaartm ment,, buut raather ennter ccell by endo e ocytoosis.
Figgure 5. 8cc enteers ccells iindeppend dentlyy of ppassiive diffus d sion. Fluooresceence miicroscopyy imaages of U U2OS S cellls shoowinng thee subbcelluular locallizatiion of o 8c (grreen)), artesum mycinn (exx. 5555 nm m; em m. 6055 nm m) annd DA API (blue ( e). Ceells were w inccubatted eeitherr withh 8c (10 µM, 30 min) m or arrtesuumyccin (110 µM M, 300 minn) att thee inddicateed tem mperraturee. Scale bar, b 10 1 µm m.
Lyysosoomootroppic pprobbes with h diifferrent proopertties havve bbeen deeveloopedd fo or in celllulo ex xperiimennts266-28. In thiis cont c text, coompooundd 8cc reepresentts ann in ntereestinng nnew proobe. Lysosoome tarrgetiing with h sm mall mollecuules has beeen shhown too be releevannt in the coontexxt of caancerr treeatm ment.28-300 Thuus, mole m ecullar prob p bes ssuch ass 8cc reppressent useeful toools to t study s y lyysossomee biiology andd lysosoome targgetinng annd may m offeer addvanntages comppared to knoown pic reag r gentss includ ding chllorooquinne, marrmyccin andd lysosoomootrop the coomm monlyy em mplooyedd LysoTrrackker sstainning reaggentts.
Au uthor info i rmaation Coorreespoondiing A Autthor * tatia t ana.ccaneequee-cobbo@ @currie.frr Au uthoor Cont C tribu utioons
‡These authors contributed equally.
Acknowledgments R.R. thanks the CNRS for funding. Research in R.R.’s laboratory is supported by the European Research Council (grant no. 647973). J.J.V. and A.M.C thank financial support from Spanish Ministerio de Ciencia y Competitividad (project MINECO/ CTQ2014-52488-R) and Feder founds from the Instituto de Salud Carlos III (ISCIII) RETIC REDINREN RD12/0021/0014.
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Supplementary Material Electronic Supplementary Information (†ESI) available: Synthesis procedure, NMR, MS and cell imaging data. Supplementary data associated with this article can be found, in the online version, at DOI: XXX.
S0960-894X(16)31239-2 http://dx.doi.org/10.1016/j.bmcl.2016.11.074 BMCL 24469
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Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
2 November 2016 22 November 2016 23 November 2016
Please cite this article as: Zacharioudakis, E., Cañeque, T., Custodio, R., Müller, S., Cuadro, A.M., Vaquero, J.J., Rodriguez, R., Quinolizinium as a new fluorescent lysosomotropic probe, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.11.074
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Quinolizinium as a new fluorescent lysosomotropic probe
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Emmanouil Zacharioudakis,a‡ Tatiana Cañeque,a* Raúl Custodio,b‡ Sebastian Müller,a Ana M. Cuadro,b Juan J. Vaquero,b Raphaël Rodrigueza
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com
Quinolizinium as a new fluorescent lysosomotropic probe Emmanouil Zacharioudakis,a‡ Tatiana Cañeque,a* Raúl Custodio,b‡ Sebastian Müller,a Ana M. Cuadro,b Juan J. Vaquero,b Raphaël Rodrigueza a Institut Curie, PSL Research University, Organic Synthesis and Cell Biology group, 26 rue d’Ulm, 75248 Paris Cedex 05, France; CNRS UMR3666, 75005 Paris, France; INSERM U1143, 75005 Paris, France. b Departamento de Química Orgánica y Química Inorgánica, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain.
ARTICLE INFO
ABSTRACT
Article history: Received Revised Accepted Available online
We have synthesized a collection of quinolizinium fluorescent dyes for the purpose of cell imaging. Preliminary biological studies in human U2OS osteosarcoma cancer cells have shown that different functional groups appended to the cationic quinolizinium scaffold efficiently modulate photophysical properties but also cellular distribution. While quinolizinium probes are known nuclear staining reagents, we have identified a particular quinolizinium derivative salt that targets the lysosomal compartment. This finding raises the question of predictability of specific organelle targeting from structural features of small molecules.
Keywords: Quinolizinium Heteroaromatic cations Fluorescent dyes Lysosomal probes Small molecules
Recent advances in fluorescence microscopy have permitted to study cellular processes providing unprecedented topological insights. This powerful imaging technology has been employed extensively to study organelle-specific biological events and the subcellular localization of biologically active small molecules, providing useful clues as to how drugs operate in cells. In particular, fluorescence microscopy is well suited for live cell imaging studies, potentially providing valuable temporal information, which can lead to a deeper appreciation of the dynamic nature of some biological events.1-3 Thus, others and we have investigated the design and synthesis of versatile molecular scaffolds with the aim to improve the specific targeting of a given cellular organelle. Among them, fluorescent dyes suitable for nuclear DNA recognition have been widely employed and these are generally based on heteroaromatic organic molecules (Figures 1). For example, 4',6-diamidino-2-phenylindole (DAPI, 1) binds tightly to A and T nucleobases within the DNA minor groove and represents one of the most universally employed probes to detect cell nuclei.4 The cell-permeant nucleic acid binding dye acridine orange (AO, 2) has also been widely employed for cell-cycle studies thanks to the distinctive fluorescence properties that characterizes this scaffold with a green fluorescence emission upon binding to dsDNA, and a red fluorescence when bound to ssDNA or RNA. Importantly, AO does not selectively stain the nucleus and therefore can also be employed to monitor pH variations in the lysosomal compartment.5-6 Cationic heteroaromatic dyes are broadly considered as classical DNA intercalators, whereas positively charged heterocycles play an important role in conferring fluorescence properties to molecular
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probes.7,8 As a result, a large number of positively charged substrates have been successfully developed as fluorescent probes for DNA detection, including propidium (3) and ethidium NH HN
N H
NH2
NH2
N
N
AO (2)
DAPI (1) Me I Et N Me
I N
Br
H2 N
NH2
NH2
Ethidium bromide (4) N
N
N
N
H 2N
Propidium iodide (3) O
N
N
N
O N
4I YOYO-1 (5) Me H N
OEt 3 Cl
H N
H N NH
N H Hoechst 33342 (6)
Figure 1. Representative examples of DNA binding fluorescent dyes.
halides (4), which are DNA stains with poor membrane permeability that are often used to differentiate between living and dead cells in cell culture.9-11 This also includes dyes from the cyanine family such as the bis-intercalator YOYO-1 (5) used for monitoring DNase activity, and quantitating the concentration of PCR products.12 It also includes the broadly used DNA dye Hoechst 33342 (6), a bis-benzo[d]imidazolium cation commonly used to visualize nuclear and mitochondrial DNA.13,14
to be the highest emitter, presumably reflecting the electronic nature of the indole moiety compared to pyrrole, thiophene and imidazole. We found that the fluorescence of the compounds is enhanced when binding to DNA (†ESI), as we previously reported for other quinolizinium analogues.17 Table 1. Photophysical properties of the quinolizinium derivatives: Maximum linear absorption and emission wavelength (λmax Abs and λmax Em). Compound
In order to broaden the scope of available organelle-targeting markers, we developed a library of quinolizinum-derived molecules: bridgehead quaternary nitrogen heterocycles (BQNH) harboring extended electronic networks that enable the finetuning of fluorescence properties (Scheme 1). Different quinolizinum-based compounds have been developed and extensively studied as molecular DNA dyes.15,16 We have previously shown that V-shaped quinolizinium chromophores with high emission quantum yields and environment sensitive fluorescence lifetimes are suitable probes for cell biology, taking advantage of nonlinear excitation (two photon absorption, TPA). In particular, fluorescence lifetime imaging allows for the acquisition of multicolor images and the detection of different cell structures including the cytosol, the nuclear membrane and the nucleus.17,18 These results prompted us to develop novel cationic fluorescent dyes to further improve the tropism for DNA, whilst being suitable for one-photon absorption. To this end, we synthesized a series of V-shaped chromophores derived from quinolizinium (D-π-A+-π-D) and explored their photophysical properties in order to establish the relevant structural features that may affect fluorescence with particular emphasis on the nature of electron-donating substituents and the extent of electronic delocalization. 2,8Dimethylquinolizinium hexafluorophosphate (7) was subjected to a double Knoevenagel condensation19 with diversely functionalized aromatic or heteroaromatic aldehydes to afford quinolizinium probes 8a-f in good to excellent yield (Scheme 1). It is noteworthy that the counter anion exchange in favor of BF4 was employed to enhance the water solubility of 8b, an important characteristic for cell-based studies. Me N PF6 7
OMe
8a
H R Piperidine CH3CN, reflux, 6h (32-95%) OMe H N
R=
8b
R
O
Me
8c
N
R
N X
X = PF6, BF4
NH
8d
8a-f
NH
8e
S
8f
Scheme 1. Synthesis of quinolizinium derivatives 8a-f.
Photophysical properties were monitored in methanol to maximize the solubility of the analogues in a cell-free environment, thereby enabling a direct comparison of each compound within this series. A summary of the results is presented in Table 1 and linear absorption and emission spectra are depicted Fig. 2. In general, a good correlation was found between the nature of the electron-donating substituents used and the stabilization energy for the different types of chromophores studied, compound 8b harboring a more extended electron delocalization framework, yielding the highest λmax value. In contrast, the heteroaryl indole-derived substituted 8c was found
λmaxAbs (nm)
λmax Em (nm)
8a
444
540
8b
463
620
8c
489
584
8d
430
527
8e
457
542
8f
432
529
Figure 2. Absorption (A) and emission (B) spectra of the quinolizinium derivatives 8a-f recorded in methanol at 25 °C.
With the linear optical data in hand, we next explored the cellular labeling of each dye in human U2OS osteosarcoma cancer cells using high-resolution fluorescence microscopy (Figure 3). All the molecules entered living cells upon treatment using a regular culture medium at the optimal concentration of 2 μM. A comparative analysis using the minor groove DNA binder 1, revealed a similar pan-nuclear staining pattern for 8b,d-f, which comes in agreement with the DNA intercalating properties of the quinolizinium core scaffold.20,21 In contrast, probes 8a,e concomitantly localized to the nucleus and the cytosol, sharing a pan-nuclear staining with 1 along with a more diffuse cytoplasmic staining. Strikingly, 8c stained small cytosolic vesicles in the green channel (†ESI). It is noteworthy that while these analogues are structurally close, the sub-cellular accumulation of 8c was different from that of the other analogues of the series, reflecting either the targeting of a different cellular component or a different cellular uptake mechanism (e.g. endocytosis vs passive diffusion). This prompted us to identify the nature of the vesicles where 8c accumulated. Thus, cells were co-treated with 8c (green staining) and other organelle specific reagents including the specific lysosomal marker LysoTracker® deep red (red staining). Remarkably, visual detection of colocalizing foci (i.e. yellow vesicles in merge image) outside of the nucleus was characteristic of the physical proximity of 8c with the lysosomal marker (Figure 4), consistent with the notion that 8c targets the lysosomal compartment, an unprecedented feature of a quinolizinium-derived small molecules. To test whether the nuclear staining of 1 influenced the staining of 8c, we performed an experiment in the presence and absence of 1 and found that the staining of 8c was not altered under these conditions in the green channel (†ESI). Lysosomes are cellular organelles involved in several cellular processes including protein degradation, cellular waste recycling, secretion, plasma membrane repair, signaling, metabolism and iron homeostasis,22,23 thereby conferring this organelle a pivotal role in the physiology of the cell and a relevant organelle to target with small molecules.24,25 Owing to the current challenge
to o develoop sm malll mo olecuules thatt sellectively y tarrget the lysoosom me too sttudyy orr im mpacct th he norm n mal funnctioons of this orgganeelle, wee becam me inteerested in i ccharaacterrizinng mech m haniismss annd sttructuraal feeatures thaat gove g ern accuumuulatioon of 8c in thee lyysoso omaal co omppartm mennt.
Figuree 3. Confo C ocal micr m oscoopy im magees shoowin ng DA API (pan( -nucllear blue) b ), 8aa,b,d d-f (ppan-nnucleear grreen)) andd 8c (greeen puunctaa) in U2O OS ceells. Scale S e barr, 100 µm m. Ceells w weree treaated with 2 µM µ 8a-f for f 30 miin annd thhen fixed f withh fo ormaaldehyyde. DAPII
8c c
Lyso otracke er
Merge e
Zoo om
In connclussion,, wee havve deve d elopeed a straaighhtforrwarrd syynthhetic rooute too gene g eratee a liibrarry of caationnic quiinoliiziniium m chhrom mophhorees abble to targget diffeerennt ceellullar com mparrtmeents, naamelly the t nuclleuss annd lyysossomees, throoughh suubtlee strructuural modifficattionss. Giivenn thaat quuino olizinnium m baased proobes are knoown NA inteercaalatoors, ourr finndings prov p videe un nantiicipaatedd to bee DN insighhts into how w sttructturall vaariatiions of the corre sccaffoold can modulate sub bcelllularr loccalizzatio on annd organ o nellee speecifiic taargetting wiith smal s ll molec m cules, a straategyy that may m be appl a licabble tto ottherr typpes of scaff s ffoldds. Inntereestinngly y, wee deetectted som s me feeint nuclearr acccum mulaation n of 8c with w redd fluo oresscencce (†ES SI), whil w le thhe grreen fluuoreescennce alloowedd thhe detecctionn of lyssosom mal acccumuulatiion, makinng 88c in i innterestinng dduall fluuoreescennt prob p be to o viisuaalize multtaneeouslly and a indeepennden ntly twoo ceellular orga o anellles. Forr sim instannce, this connstituutes a neew prob p be thhat can c be b used to stainn the lysosoomal co ompaartm ment in a diifferrent fluoorescentt channeel frrom m that of o D DNA A, perm p mittinng the usee off otther staains witth blue b fluuoreescennce in conj c juncctionn wiith 8c 8 to laabel thee nucleuus. T This featurre is impportaant give g en thhe neeed to t have toolls too lab bel ddifferrentt ceellulaar ccom mparttmennts witth disti d inct colors inn ceell biol b ogy exxperiimennts. Thhe sttructturall feaaturees thhat enab e ble 8a-f 8 f to enteer ceells rem main eluusivve and a requirre addi a ition nal invvestiggatioons to bee fully fu chharaccteriizedd. Thhis may m include receeptoor-m mediaated end docyytosiis orr bee linnkedd to the lipoophiilic natu ure of this t seriies oof comp c pounnds. M anissms undderlyyingg thhe uptak Mech u ke and a distribuution n of o thhese coompooundds inn ceells are undder in nvesstigaationn annd will w be b rreporrtedd in duee couursee.
C ocal micrroscoopy im magees shhowin ng coo-localizeed focci (yeellow w Figuree 4. Confo puunctaa) off 8c (green ( n pun ncta)) andd LyssoTraackerr deeep redd (redd punncta)) in U2OS U S ceells. DAP PI (ppan-nnuclear bllue). Thee yelllow bbox show ws a 6× m magnnificaationn. Scale bar, 10 µ µm. Cells C weree co-treatted with w 2 µM M 8c aand LysoT L Trackker for f 300 m min.
Too innvesstigaate the t acccumuulatiion of 8c in lysoosom mes, wee wondderedd whet w w ther 8a-f coul c d be b inter i rnaliized byy means m s of en ndoccytoosis or rath r er rreachhed their taargeet orrgannelle by passsivee d sionn thrrouggh thhe plasm diffu p ma mem mbrane.. Giiven thaat enndoccyticc proceesses arre signi s ificaantlyy im mpaiired at low w tem mpeeratuure (e.gg. 4°C),, U22OS cells were w treaated witth 2 µM M 8aa-f foor 30 min m at a 377 °C arteesum C annd 4 °C C. As A a conntroll expperim mennt, we w used u myciin, a n ral produ natur p uct hybr h rid of o marm m mycinn A prev viouusly shown to taargeet th his orga o anellle byy en ndocytossis.244,25 Inter I restiinglyy, we w obbserrved d thaat 8a-f and arteesum myciin beehavved similarlly where w e thee lysosoome wass taargeted whhen cellls werre iincuubateed at 37 °C.. H Howeeverr, in ncubbatinng cellss at a 4 °C C thhrouughoout the treeatm mentt peeriodd ab bolishedd lyssosoome targgetinng ass deffined by y thee abssencce off staainedd vesiccles in thhesee condittionss (Fiigurre 5,, †E ESI). This data d stro onglyy f r off a proocesss wher w reby y enndoccytosis of 8a-f arrguees iin favor d inatees the domi t sub-celllularr acccum mulaationn off thhese moolecu uless. S e lyysosomees have Since h e a low wer pH H thhan theeir surrroun ndingg organnellees, we w next n t invvestigated whe w etherr changees in pH H could c d allter thee fluuoresscen nce inteensitty oof 8cc in n vittro andd in cellluloo. L eringg thhe pH Lowe H diid not signiificaantlyy afffect the fluooresccencce of 8c (††ESII), arrguiing that t f rescencee off 8c observeed inn the greeen fluor ly enhhancced by a low ysossomees iis not n wer pH H. In I addi a tionn, bafiloomyycin A, chlo c oroquinee and N NH4C Cl, comppounnds thatt preevennt accidifficattion of lyso osom mes or raisse thhe lyso l som mal ppH, did d noot afffect thee fluuoresscen nce of o 8cc in lysoosom mes (†E ESI), sugggesting g thaat pH cchannges in this com mpaartmeent do not affeect the t sub-cellularr lo ocaliizatiion of this t mollecuule. T Thiss is in line l witth thhe iddea thatt, u ke chlor unlik c roquuinee, coomppounnd 8c doees not n acccumuulatee inn ly ysossomees ddue to t th he loow pH of this t celllularr coompaartm ment,, buut raather ennter ccell by endo e ocytoosis.
Figgure 5. 8cc enteers ccells iindeppend dentlyy of ppassiive diffus d sion. Fluooresceence miicroscopyy imaages of U U2OS S cellls shoowinng thee subbcelluular locallizatiion of o 8c (grreen)), artesum mycinn (exx. 5555 nm m; em m. 6055 nm m) annd DA API (blue ( e). Ceells were w inccubatted eeitherr withh 8c (10 µM, 30 min) m or arrtesuumyccin (110 µM M, 300 minn) att thee inddicateed tem mperraturee. Scale bar, b 10 1 µm m.
Lyysosoomootroppic pprobbes with h diifferrent proopertties havve bbeen deeveloopedd fo or in celllulo ex xperiimennts266-28. In thiis cont c text, coompooundd 8cc reepresentts ann in ntereestinng nnew proobe. Lysosoome tarrgetiing with h sm mall mollecuules has beeen shhown too be releevannt in the coontexxt of caancerr treeatm ment.28-300 Thuus, mole m ecullar prob p bes ssuch ass 8cc reppressent useeful toools to t study s y lyysossomee biiology andd lysosoome targgetinng annd may m offeer addvanntages comppared to knoown pic reag r gentss includ ding chllorooquinne, marrmyccin andd lysosoomootrop the coomm monlyy em mplooyedd LysoTrrackker sstainning reaggentts.
Au uthor info i rmaation Coorreespoondiing A Autthor * tatia t ana.ccaneequee-cobbo@ @currie.frr Au uthoor Cont C tribu utioons
‡These authors contributed equally.
Acknowledgments R.R. thanks the CNRS for funding. Research in R.R.’s laboratory is supported by the European Research Council (grant no. 647973). J.J.V. and A.M.C thank financial support from Spanish Ministerio de Ciencia y Competitividad (project MINECO/ CTQ2014-52488-R) and Feder founds from the Instituto de Salud Carlos III (ISCIII) RETIC REDINREN RD12/0021/0014.
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Supplementary Material Electronic Supplementary Information (†ESI) available: Synthesis procedure, NMR, MS and cell imaging data. Supplementary data associated with this article can be found, in the online version, at DOI: XXX.