Charles Guerin

Ludivine Jean-Gérard

Guillaume Octobre

Simon Pascal

Olivier Maury

Guillaume Pilet

Audrey Ledoux

Bruno Andrioletti

Bis-triazolyl BODIPYs: a simple dye with strong red-light emission

come

1

Please do not adjust margins

Bis-triazolyle BODIPYs: a simple dye with strong red-light emission

Charles Guérin, a Ludivine Jean-Gérard, a Guillaume Octobre, a Simon Pascal, b Olivier Maury, b Guillaume Pilet, c Audrey Ledoux, a and Bruno Andrioletti a,* a. Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS-UMR CNRS 5246), Université Claude Bernard Lyon 1, Bâtiment Curien (CPE) 43, Bd du 11 Novembre 1918, 69622 Villeurbanne cedex (France). b. Laboratoire de Chimie, UMR 5182, CNRS, Ecole Normale Supérieure de Lyon, Université de Lyon, 46 Allée d'Italie,69007 Lyon,c. Laboratoire des Multimatériaux et Interfaces (LMI-UMR CNRS 5615), Université Claude Bernard Lyon 1, bâtiment Chevreul, 43, Bd du 11 Novembre 1918, 69622 Villeurbanne cedex (France).

Abstract: The versatile synthesis of an unprecedented bis-triazolyle BODIPY dye displaying a high quantum yield even in water is described. X-Ray diffraction analyses and NMR measurements demonstrate that the simultaneous presence of the two triazole moieties helps stiffening the BF2 complex.

The development of highly fluorescent probes has witnessed a considerable development over the past two decades. 1 Indeed, fluorescence is a highly sensitive spectroscopy which is nowadays ubiquitous in numerous fields including -among others-medical diagnosis, material and analytical sciences and is demanding for even more performant systems. Within the myriads of fluorescent candidates, boron-dipyrromethene (BODIPY) dyes occupy a central position due to their intense and tunable absorption and emission properties. 2 The possibility to perform subtle chemical modifications on the BODIPY core allows to finely tune their photophysical properties or to functionalize the dye for bioconjugation purpose. 3 Nevertheless, the modifications of the BODIPY scaffold have to remain cost-effective for further applicative development. The wellknown CuAAc, Copper Catalyzed Azide-Alkyne cycloaddition approach is among the most appealing strategy for the efficient functionalization of substrates due to its efficiency, versatility and tolerance to many functional groups. [START_REF] Schoffelen | Modern Alkyne Chemistry[END_REF] Within the course of our investigations dealing with the development of 5,5'-diaryle dipyrromethenes, 5 we have been interested in the preparation of 5,5'-bis-triazolyle dipyrromethenes which extended -conjugated system could result in some promising photophysical properties. Interestingly, 5,5'-bis-triazolyle dipyrromethenes had not been reported yet, even if the monotriazolyle analogues had already been claimed very useful for Live Neuron Imaging or as "Megastokes" fluorescent dyes. 6 Herein, we report a very straightforward synthesis of 5,5'-bistriazolyle boron-dipyrromethenes dyes which exhibit remarkable fluorescent properties even in water and which were evaluated for cell imaging.

(bis)-Ethynyl dipyrromethenes being inherently unstable, we envisioned the synthesis of 5,5'-bis-triazolyle dipyrromethenes starting from the pyrrole-triazole building block 2. This synthon was readily prepared from 2-ethynyl-1-tosyl-1H-pyrrole 1 7 using a classical copper catalyzed 1,3-cycloaddition in the presence of sodium ascorbate and benzoic acid. 8 Pyrrole detosylation proceeded efficiently (90% yield) using aqueous NaOH in dioxane with NEt4Br as a phase transfer agent. The corresponding pyrrole-triazole moiety 3 was subsequently reacted with benzaldehyde using the so-called Lindsey's conditions 9 to afford the expected dipyrromethenes after oxidation with 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) in a satisfying 69% yield.

Scheme 1 Synthesis of the BODIPY dyes 4a-b. Reagents and conditions: (i) RN3, CuSO4, 5H2O, Na ascorbate, PhCO2H, tBuOH/H20, rt, 30 min; (ii) 5M NaOHaq, NEt4Br, dioxane, reflux, 40 hr; (iii) 1) PhCHO, BF3-Et2O, anhydrous CH2Cl2, rt, 24 hr, 2) DDQ, 2 hr, rt; (iv) BF3.Et2O, DIPEA, anhydrous CH2Cl2, rt, 2 hr.

Eventually, the targeted boron complex was isolated in 93% yield after reaction with BF3.Et2O using N,N-diisopropylethylamine (DIPEA) as a proton sponge (Scheme 1). Overall, 4a-b could be isolated in four steps with a remarkable 58% yield from the readily available ethynyltosylpyrrole 1. Aiming at developing water soluble adducts, 4b was further functionalized by reaction with K2Os(OH)2(H2O)2/NMO to afford the expected tetraol 5 in 67% yield (Scheme 2). The solubility of 5 in water still being poor, 5 was converted to the corresponding tetrasulfate sodium salt 6 in 93% yield using TEA.SO3 and NaOH in DMF (Scheme 2). The water solubility of 6 exceeds 10 mol.L -1 . Next the photophysical properties of the BODIPY dyes were investigated (Table 1). The chromophores display sharp and intense absorption transition (ε > 60,000 L.mol -1 .cm -1 ) centered in the orange-red spectral range with the typical shape of BODIPY dyes (Figure 1-S13, supp. information). The emission is localized in the red (570-590 nm) and is perfectly mirror compared with the absorption. Remarkably, the hexyl-derivative 4a revealed an optimal fluorescence quantum yield (> 0.95) in CH2Cl2. Similarly, the tetrasulfate sodium salt 6 exhibited a quantum yield of 0.91 in MeOH that is almost maintained in pure water (0.81). Thus, the quantum Please do not adjust margins yields of the bis-triazolyle BODIPYs family are excellent especially for red-emitting chromophores, independently of the nature of the alkyle chain on the triazole moieties and of the solvent. Last, rather long fluorescence lifetimes were measured with  values of about 7 ns. These exceptional photophysical properties account for a very rigid system exhibiting a very little loss of energy via non radiative pathway. This last point is confirmed by low non radiative kinetic constants compared with the radiative ones (Table 1). The rigidity of the molecules can be explained by i) the triazole rings, less bulky than the phenyl analogues often used that allow a better delocalization 10 and ii) the formation of intramolecular CH…F interactions (vide infra). 11 Scheme 2 Access to the highly water soluble dye 6. Reagents and conditions: (i) K2Os(OH)2(H2O)2, NMO, Me2CO/tBuOH/H2O, rt, 12 hr; (ii) 1) TEA.SO3, anhydrous DMF, 12 hr, rt, 2) NaOH, O°C.

Table 1 Photophysical properties of the dyes in various solvents

[a] Standard used for quantum yield measurements: cresyl violet ( = 0.55 in MeOH). [b] NanoLED excitation at 490 nm. [c] with kR=/ and kNR=(1-)/ The formation of intramolecular CH…F interactions was ascertained on the basis of the 13 C NMR analyses (Figure 1). Indeed, the triazole CH signals appeared as a triplet with a coupling constant of J TS = 11.3 Hz. This high "Through Space" coupling constant is similar to that measured by Burgess et al. [START_REF] Chen | 31H37BF2N8[END_REF] with "blocked" benzannulated BODIPY dyes (J TS = 12 Hz) and higher than values measured with rather planar systems like 10 Hence, the two triazole moieties act synergistically to stiffen the BF2 complex via strong intramolecular CH…F interactions Consequently the free rotation of the triazole is forbidden limiting non radiative desexcitations. Definitive proof for this hypothesis was brought by single-crystal Xray diffraction characterizations. Single-crystals suitable for X-ray diffraction analyses were obtained by slow diffusion of pentane in a concentrated solution of 4a in CHCl3 2). 4a crystallizes in the monoclinic system (P21/c centrosymmetric space group). 13 The X-ray crystal structure reveals a planar dipyrromethene (DPM) subunit with the triazoles lying approximatively in the same plane (tilt angle = -2.8° and +6.4°). The meso-phenyl substituent displays an angle of 49.1° vs the main DPM plane and the alkyle chains adopt the expected symmetrical zigzag conformations. The boron atom sits in the main plane with the fluorine atoms respectively on top and below. As expected after the NMR analyses, the CH of the triazoles point inward allowing stabilizing B-F•••H interactions with B-F•••H distances ranging from 2.287(2) Å to 2.571(3) Å. Next, the triazole-functionalized BODIPY dyes 4a and 6 were evaluated for cell imaging. HeLa cells (human fibroblasts) were chosen as models. In DMEM (Dulbecco's Modified Eagle Medium), 4a displayed a very efficient penetration as an equilibrium was observed after 10 min with a 10 nM solution of 4a. In the same conditions, 15 min were necessary for the penetration of 6, with an initial concentration of 10 µM. Interestingly, both dyes displayed very little cytotoxicity, as no cell death was evidenced even with concentrations as high as 1 mol.L -1 for 6. Thanks to the easy cell penetration of the dye and the highly fluorescent character of the bis-triazolyle-BODIPY dyes, very good quality images could be obtained after incubation. Two representative views highlight the high fluorescence observed (Figure 3). Both 4a and 6 displayed a cytoplasmic localization, with a diffuse signal in the case of 4a, while perinuclear punctuate spots were observed with 6. It should be noted that the optimal signal for 6 was observed with concentrations 3 orders of magnitude higher than those used for 4a, most probably due to the highly lipophobic nature of 6. In conclusion, we have developed the efficient synthesis of unprecedented bis-triazolyle BODIPY dyes. Straightforward access to both lipophilic and highly hydrophilic systems is described. The simultaneous presence of the two symmetrical triazole substituents allows for the establishment of B-F•••H interactions that help for stiffening of the system. Consequently, the non-radiative dissipation pathways are strongly limited and very high quantum yields were obtained. This high brightness conjugated with a low cytotoxicity allowed for cell imaging. After a short incubation time, good quality images could be collected stressing the promising interest of these new BODIPY derivatives for bio-imaging applications.

Work is currently underway in our Laboratory for the development of a functionalized version of the dye.

Notes and references

Fig. 1

 1 Fig. 1 13 C NMR spectrum of 4a revealing the B-F•••H-C a "Though Space" coupling of 11.3 Hz.

Fig. 2

 2 Fig. 2 (a) Crystal structure of 4a revealing (b) the planar Triazole-BODIPY substructure and short B-F•••H distances (orange dashed lines).

Fig. 3

 3 Fig. 3 Epifluorescence images of HeLa cells incubated for 15 min with 50 nM of in DMEM + DMSO 1%: a) Fluorescence signal in the red channel b) Bright field. Images of HeLa cells incubated for 15 min with 50 µM of 6 in DMEM: c) Fluorescence signal in the red channel d) Bright field.


Guillaume Castel

Claudia Filippone

Caroline Tatard

Jacques Vigan

Gauthier Dobigny

Role of Seaports and Imported Rats in Seoul Hantavirus Circulation, Africa

R odents are widespread, opportunistic, and compe- tent host reservoirs involved in the maintenance, circulation, and transmission of a wide panel of zoonotic pathogens [START_REF] Meerburg | Rodent-borne diseases and their risks for public health[END_REF]. Rodent-related zoonoses cause up to 400 million human infections worldwide each year [START_REF] Meerburg | Rodent-borne diseases and their risks for public health[END_REF][START_REF] Colombe | A new approach to rodent control to better protect human health: first international meeting of experts under the auspices of WHO and the Pan American Health Organization[END_REF]. Among zoonotic pathogens, hantaviruses (order Bunyavirales, family Hantaviridae, genus Orthohantavirus) are among agents considered most likely to emerge and have a global public health impact [START_REF] Grange | Expert panel; PREDICT Consortium; University of Edinburgh Epigroup members. Ranking the risk of animal-to-human spillover for newly discovered viruses[END_REF].

Hantaviruses are enveloped, negative, singlestranded RNA viruses with a tripartite genome comprised of large, medium, and small segments. Transmitted to humans via inhalation of aerosolized virus in contaminated rodent urine and feces, hantaviruses can cause hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome [START_REF] Jonsson | A global perspective on hantavirus ecology, epidemiology, and disease[END_REF]. Hantaviruses are generally carried by a rodent species host, and geographic distribution of the host can determine the area in which the associated disease occurs among humans. From this perspective, Seoul orthohantavirus (SEOV), identified in South Korea in 1982, deserves special attention because its cosmopolitan host, the Norwegian rat (Rattus norvegicus), also known as the brown rat, has been dispersed worldwide, resulting in a global distribution of the virus today [START_REF] Clement | Wild rats, laboratory rats, pet rats: global Seoul hantavirus disease revisited[END_REF]. Detection of SEOV is often considered anecdotal and speculated to be driven by sporadic introduction of infected brown rats via transportation but also by pet or laboratory rats [START_REF] Childs | A case study of two rodentborne viruses: not always the same old suspects[END_REF][START_REF] Dupinay | Detection and genetic characterization of Seoul virus from commensal brown rats in France[END_REF]. Diagnosing SEOV in humans remains a challenge due to milder and atypical HFRS pathology [START_REF] Clement | Clinical characteristics of ratborne Seoul hantavirus disease[END_REF]. However, mild symptoms can progress to acute renal disease associated with HFRS, in which patients experience low blood pressure, acute shock, and acute kidney failure, and the case-fatality rate is ≈1% [START_REF]FAQs: Seoul virus[END_REF].

History of Hantaviruses in Africa

Fifteen years ago, no indigenous hantavirus was known in Africa [START_REF] Kruger | Hantavirusesglobally emerging pathogens[END_REF]. Since then, few studies have investigated hantaviruses, including SEOV, in Africa and consequences for human health. The dearth of studies gives the appearance that SEOV is not a major public health threat on the continent because of the lack of local specific testing for SEOV among human serum samples [START_REF] Heyman | Seoul hantavirus in Europe: first demonstration of the virus genome in wild Rattus norvegicus captured in France[END_REF]. Nonetheless, suspicions of SEOV-like agents in humans and wild rats in 17 different countries in Africa are strong [START_REF] Clement | Wild rats, laboratory rats, pet rats: global Seoul hantavirus disease revisited[END_REF]. Until recently, immunofluorescence assays positive for Hantaan virus (HTNV), a closely related orthohantavirus in rats, was the only indication that SEOV probably was in Africa. Unfortunately, these serologic analyses were mainly based on cross-reactivity with better documented hantaviruses from Eurasia within the Murinae-associated hantavirus virus genera Seoul orthohantavirus (SEOV) is not considered a major public health threat on the continent of Africa. However, Africa is exposed to rodentborne SEOV introduction events through maritime traffic after exponential growth of trade with the rest of the world. Serologic studies have already detected hantavirus antibodies in human populations, and recent investigations have confirmed circulation of hantavirus, including SEOV, in rat populations. Thus, SEOV is a possible emerging zoonotic risk in Africa. Moreover, the range of SEOV could rapidly expand, and transmission to humans could increase because of host switching from the usual brown rat (Rattus norvegicus) species, which is currently invading Africa, to the more widely installed black rat (R. rattus) species. Because of rapid economic development, environmental and climatic changes, and increased international trade, strengthened surveillance is urgently needed to prevent SEOV dissemination among humans in Africa.

and did not enable identification of viruses at a finer specific level [START_REF] Diagne | Serological survey of zoonotic viruses in invasive and native commensal rodents in Senegal, West Africa[END_REF]. In addition, these analyses usually lacked confirmatory assays [START_REF] Witkowski | Hantaviruses in Africa[END_REF]. However, because of the high specificity of hantaviruses for their rodent hosts, positive serologic tests in rats could be ascribed to cross-reactions with SEOV or SEOV-like variants [START_REF] Clement | Wild rats, laboratory rats, pet rats: global Seoul hantavirus disease revisited[END_REF], as seen in Senegal [START_REF] Diagne | Serological survey of zoonotic viruses in invasive and native commensal rodents in Senegal, West Africa[END_REF][START_REF] Witkowski | Hantaviruses in Africa[END_REF]. Of note, older serologic studies in Africa, including regions in West Africa, have detected antibodies against hantaviruses in the human general population and in febrile patients with putative hantavirus disease [START_REF] Witkowski | Hantaviruses in Africa[END_REF][START_REF] Gonzalez | Serological evidence for Hantaan-related virus in Africa[END_REF][START_REF] Witkowski | Human seroprevalence indicating hantavirus infections in tropical rainforests of Côte d'Ivoire and Democratic Republic of Congo[END_REF]. Detecting putative hantavirus in febrile patients is a crucial public health issue in Africa, where fever of unknown etiology is very common. However, in the absence of differential diagnosis and further laboratory confirmation, we cannot be certain of the virus involved in these cases. We also cannot consider these initial observations exhaustive because of the lack of a proper epidemiologic approach and the limits of the methods used. Nonetheless, those reports might represent a primordial reflection of the health effects that hantavirus zoonoses could have in Africa.

Since 2006, a genus-reactive pan-hantavirus PCR has been available to search for new hantaviruses in small mammals [START_REF] Klempa | Hantavirus in African wood mouse, Guinea[END_REF][START_REF] Klempa | Novel hantavirus sequences in shrew, Guinea[END_REF]. This PCR led to the discovery of the 2 molecularly characterized endemic hantaviruses in mammals in Africa: Sangassou virus in the African wood mouse (Hylomyscus simus) and Tanganya virus in the Therese's shrew (Crocidura theresae) [START_REF] Witkowski | Hantaviruses in Africa[END_REF][START_REF] Klempa | Hantavirus in African wood mouse, Guinea[END_REF][START_REF] Klempa | Novel hantavirus sequences in shrew, Guinea[END_REF]. Since those discoveries, up to 10 indigenous hantaviruses have been identified in rodents, shrews, and even bats in Africa, making it the continent with the most recent scientific progress in hantavirus epizootiology and epidemiology [START_REF] Kruger | Hantavirusesglobally emerging pathogens[END_REF]. Recently, 2 studies using the pan-hantavirus PCR have molecularly assessed SEOV in rodents from southeastern Senegal [START_REF] Diagne | Seoul orthohantavirus in wild black rats, Senegal, 2012-2013[END_REF] and southern Benin [START_REF] Castel | Genetic characterization of Seoul virus in the seaport of Cotonou, Benin[END_REF], confirming that SEOV circulates in West Africa and could be a cause of hantavirus disease in humans (Figure). In both cases, phylogenic analyses grouped the retrieved viral sequences with SEOV strains from Asia but from 2 different genetic lineages [START_REF] Castel | Genetic characterization of Seoul virus in the seaport of Cotonou, Benin[END_REF]. Strains from Benin belonged to SEOV lineage 7, whereas lineages from Senegal belonged to SEOV lineage 3 or 4, depending on the genomic segment considered (Figure ); this difference could indicate different introduction events in these 2 countries [START_REF] Castel | Genetic characterization of Seoul virus in the seaport of Cotonou, Benin[END_REF]. transportation systems [START_REF] Arikawa | Epidemiology and epizootiology of hantavirus infection in Japan[END_REF][START_REF] Wu | Seaport as a source of hantavirus: a study on isolated isles[END_REF][START_REF] Lin | Migration of Norway rats resulted in the worldwide distribution of Seoul hantavirus today[END_REF]. In Japan, during the 1960s, brown rats captured in the Tokyo seaport area were shown to have a higher seroprevalence for an HTNV-like agent [START_REF] Clement | Wild rats, laboratory rats, pet rats: global Seoul hantavirus disease revisited[END_REF]. Since then, hantavirus has been detected in rats in other port areas in Asia, including China [START_REF] Wu | Seaport as a source of hantavirus: a study on isolated isles[END_REF][START_REF] Wang | Genetic and hosts characterization of hantaviruses in port areas in Hainan Province, P. R. China[END_REF], and in Taiwan, where hantavirus antibody prevalence was much higher (20%) in rodents trapped in international seaports than in rural regions (≈5%), thus suggesting that hantavirus in Taiwan mainly originated from sea transportation [START_REF] Chin | Hantavirus infection in Taiwan: the experience of a geographically unique area[END_REF]. The role of seaports as the source of hantavirus was further supported by an inverse correlation between the seropositive rate of reservoir host species and the distance of small mammal sampling sites to the seaport [START_REF] Wu | Seaport as a source of hantavirus: a study on isolated isles[END_REF]. Of note, SEOV seroprevalence in brown rats from small islands closed to Taiwan was similar to that retrieved in seaports in Taiwan, and the SEOV lineage identified was genetically closely related to SEOV strains from Taiwan. In addition, since 1949, the only channels to trade or travel with those islands has been by boat or airplane to and from Taiwan, pointing again toward the critical role of ship-mediated transportation of rats (rats are more likely transported by boat) in disseminating SEOV in this region [START_REF] Chin | Hantavirus infection in Taiwan: the experience of a geographically unique area[END_REF]. From this perspective, Africa is particularly exposed to future introduction events of rodentborne pathogens through maritime traffic due to the exponential increase of trade with the other continents. Increased maritime traffic potentially increases opportunities for ratborne pathogens, particularly SEOV, to expand their geographic range [START_REF] Diagne | Seoul orthohantavirus in wild black rats, Senegal, 2012-2013[END_REF]. Although one third of countries on the continent are landlocked, maritime trade constitutes Africa's main gateway to international trade with the global marketplace (25). Therefore, seaports in Africa can constitute a gateway for allochthonous rodentborne pathogens, notably from Europe and the Americas, the main regions with trading partners, but also from Asia, from which trade has been continuously increasing [START_REF] Ducruet | Maritime networks of Africa and Asia[END_REF]. Several rat species are well-known commensals to humans, among which brown rats live in close association with human infrastructure in many countries [START_REF] Heyman | Seoul hantavirus in Europe: first demonstration of the virus genome in wild Rattus norvegicus captured in France[END_REF]. This association could translate into the omnipresence of potential SEOV-carrying brown rats in human-made environments in Africa [START_REF] Clement | Wild rats, laboratory rats, pet rats: global Seoul hantavirus disease revisited[END_REF]. In addition, brown rats can be numerous in seaports located within coastal cities [START_REF] Voelckel | The distribution of R. norvegicus and R. rattus species in Douala [in French][END_REF][START_REF] Dossou | Fine-scale prevalence and genetic diversity of urban small mammal-borne pathogenic Leptospira in Africa: a spatiotemporal survey within Cotonou, Benin[END_REF], which provides opportunities for local SEOV infection among rats and port workers. Indeed, higher SEOV seroprevalence has been reported in workers in areas where seropositive urban rats were detected [START_REF] Lokugamage | Epizootiological and epidemiological study of hantavirus infection in Japan[END_REF].

Role of Seaports and Maritime Traffic in

Ratborne Hantavirus Transportation and Spread Via Maritime Traffic

Ratborne hantavirus dissemination through maritime traffic is not a new phenomenon and probably has been occurring since human navigation for migration and trade, involuntarily transporting rodents aboard vessels [START_REF] Lin | Migration of Norway rats resulted in the worldwide distribution of Seoul hantavirus today[END_REF]. In Madagascar, molecular evidence showed circulation of the variant Anjozorobe virus (ANJZV), belonging to the Thailand hantavirus (THAIV) species, in black rats (R. rattus) and in the indigenous Major's tufted-tailed rat (Eliurus majori) [START_REF] Reynes | Anjozorobe hantavirus, a new genetic variant of Thailand virus detected in rodents from Madagascar[END_REF]. THAIV is phylogenetically close to but distinct from SEOV, but the 2 viruses share a recent common ancestor [START_REF] Hugot | Genetic analysis of Thailand hantavirus in Bandicota indica trapped in Thailand[END_REF]. THAIV is associated with the greater bandicoot rat (Bandicota indica) in Thailand [START_REF] Hugot | Genetic analysis of Thailand hantavirus in Bandicota indica trapped in Thailand[END_REF]. In addition, THAIV strains Serang and Jurong have been found circulating in Asian house rats (R. tanezumi) in Indonesia and Singapore and in Cambodia in R. rattus rats [START_REF] Johansson | Molecular characterization of two hantavirus strains from different rattus species in Singapore[END_REF]. Detection of the ANJZV variant in Madagascar, far from its most probable areas of origin in South and Southeast Asia, is likely the result of black rat importation into Madagascar through the Arabian Peninsula 2,000-3,000 years ago, when humans colonized the island during a period of vast trading activity in the Indian Ocean [START_REF] Reynes | Anjozorobe hantavirus, a new genetic variant of Thailand virus detected in rodents from Madagascar[END_REF][START_REF] Brouat | Invasion genetics of a human commensal rodent: the black rat Rattus rattus in Madagascar[END_REF]. Serologic indication of hantavirus circulation in humans also was recently demonstrated in a large national population-based study in Madagascar [START_REF] Rabemananjara | Human exposure to hantaviruses associated with rodents of the Murinae subfamily, Madagascar[END_REF], confirming previous observations [START_REF] Rollin | Hemorrhagic fever with renal syndrome in Madagascar. First seroepidemiologic survey of rat populations[END_REF]. In another study conducted on nearby Mayotte Island, a novel hantavirus, Mayotte virus (MAYOV), which clustered within the THAIV clade, was detected in 18% (29/160) of captured black rats [START_REF] Filippone | Discovery of hantavirus circulating among Rattus rattus in French Mayotte Island, Indian Ocean[END_REF]. That finding also points to ship-transported virus by black rats from Southeast Asia via the Middle East during trade from Arabia thousands of years ago [START_REF] Reynes | Anjozorobe hantavirus, a new genetic variant of Thailand virus detected in rodents from Madagascar[END_REF][START_REF] Filippone | Discovery of hantavirus circulating among Rattus rattus in French Mayotte Island, Indian Ocean[END_REF].

No available studies describe similar putative human-mediated scenarios for the introduction and spread of hantaviruses within continental Africa. However, SEOV was recently detected in invasive rats in Senegal and Benin [START_REF] Diagne | Seoul orthohantavirus in wild black rats, Senegal, 2012-2013[END_REF][START_REF] Castel | Genetic characterization of Seoul virus in the seaport of Cotonou, Benin[END_REF], suggesting that human-mediated introductions have likely occurred.

Cross-Transmission from Brown Rats to Other Rodent Species

Although a strong virus-reservoir host specificity is globally accepted for hantaviruses, evidence of interspecies spillover among wild rodents exists, challenging the strict rodent-hantavirus coevolution and giving rise to fears of potential rodent host spectrum expansion [START_REF] Fang | Reservoir host expansion of hantavirus, China[END_REF]. In Madagascar, the indigenous Major's tufted-tailed rat was found to be infected by the ANJZV variant, pointing toward a spillover event among rodents from the Muroidea superfamily [START_REF] Reynes | Anjozorobe hantavirus, a new genetic variant of Thailand virus detected in rodents from Madagascar[END_REF]. In the same manner, spillover infection is the suspected cause of MAYOV and ANJZV acquisition by R. rattus rats from other hantavirus rodent reservoirs in Southeast Asia, such as B. indica for THAIV in Thailand and R. tanezumi for Jurong and Serang variants in Indonesia [START_REF] Filippone | Discovery of hantavirus circulating among Rattus rattus in French Mayotte Island, Indian Ocean[END_REF]. Another study also showed that, although hantaviruses have preferred host species, spillover events can occur between black rats and domestic mice (Mus musculus) [START_REF] Raharinosy | Geographical distribution and relative risk of Anjozorobe virus (Thailand orthohantavirus) infection in black rats (Rattus rattus) in Madagascar[END_REF]. Natural reassortment has already been documented for SEOV in brown rats and another hantavirus hosted by the striped field mouse (Apodemus agrarius) in Asia [START_REF] Zou | Genetic characterization of hantaviruses isolated from Guizhou, China: evidence for spillover and reassortment in nature[END_REF]. Furthermore, the unambiguous detection of SEOV, both molecularly and serologically, in black rats from Senegal [START_REF] Diagne | Seoul orthohantavirus in wild black rats, Senegal, 2012-2013[END_REF] shows that SEOV is not restricted to brown rats in Africa and can potentially jump to allied rat species via infected brown rats imported by ship (Figure ). This hypothesis has not yet been investigated, but it could have major consequences for SEOV ecology and epidemiology on the continent. Indeed, the brown rat is currently expanding its range across the continent [START_REF] Hima | Native and invasive small mammals in urban habitats along the commercial axis connecting Benin and Niger, West Africa[END_REF], which, by itself, might fuel SEOV dissemination in Africa. Even more, SEOV transmission and circulation in black rats could enhance geographic expansion because the R. rattus rat species was probably introduced centuries ago (41), is already widespread across the continent [START_REF] Hima | Native and invasive small mammals in urban habitats along the commercial axis connecting Benin and Niger, West Africa[END_REF], and is still propagating because of its substantial invasive ability [START_REF] Berthier | Black rat invasion of inland Sahel: insights from interviews and population genetics in South-Western Niger[END_REF][START_REF] Konečný | Invasion genetics of the introduced black rat (Rattus rattus) in Senegal, West Africa[END_REF]. When not dominated by other species, black rats are quite numerous in cities and live in close proximity to humans, including within households, especially in socioeconomically and environmentally degraded settlements where rat-to-human zoonotic spillover is possible (G. Dobigny et al., unpub. data, https://doi.org/10.5281/zenodo.6444777). Thus, if R. rattus rats are found to be a regular SEOV reservoir, the risk associated with this pathogenic but poorly documented virus in Africa could be even higher than is currently thought.

Discussion

Because of rapid economic development, environmental and climatic changes, and increased international trade, Africa urgently needs strengthened surveillance and timely rodent elimination in seaport areas, where rats can be numerous, to prevent transmission of rat-associated pathogens and potential disease outbreaks in humans [START_REF] Lin | Migration of Norway rats resulted in the worldwide distribution of Seoul hantavirus today[END_REF][START_REF] Muylaert | Hantavirus host assemblages and human disease in the Atlantic Forest[END_REF]. This strategy also represents an efficient way to limit the risk that newly introduced rodentborne viruses might disseminate further across the continent from seaports. To delineate the eco-epidemiology of hantaviruses and their associated risks in Africa, surveillance of viral genetic variability would provide valuable insights into pathogen transmission dynamics among animal reservoirs and the associated disease when human infection occurs. Low intrinsic genetic variability might reflect limited viral evolution and suggest recent colonization events from infected rats arriving via ships from a common source [START_REF] Arikawa | Epidemiology and epizootiology of hantavirus infection in Japan[END_REF][START_REF] Filippone | Discovery of hantavirus circulating among Rattus rattus in French Mayotte Island, Indian Ocean[END_REF]. This type of surveillance requires tools available on-site to amplify and characterize viral nucleic acid sequences from hantavirus-infected rodents or patients to unequivocally identify particular variants of SEOV or other hantaviruses, which is not possible with available serologic tests [START_REF] Klempa | Serological evidence of human hantavirus infections in Guinea, West Africa[END_REF].

Surveillance in Africa should initially be directed to seaports and seaport workers, which represent the front lines for contamination by newly introduced viruses. However, surveillance is also needed inland because of passive dissemination of the rodent hosts [START_REF] Lin | Migration of Norway rats resulted in the worldwide distribution of Seoul hantavirus today[END_REF][START_REF] Khan | Hantavirus: the next pandemic we are waiting for?[END_REF], especially if SEOV has already jumped to more widely distributed rodent species. Urban environments might further increase the risk for disease emergence because of close daily contact between humans and rodents, especially rats (47; G. Dobigny et al., unpub. data).

No effective approved hantavirus diseases treatment is available, and whole-virus inactivated vaccines are only licensed for use in South Korea and China but have uncertain protective efficacies [START_REF] Liu | Vaccines and therapeutics against hantaviruses[END_REF]. In addition, only supportive care is available to patients with Seoul virus disease [START_REF]FAQs: Seoul virus[END_REF]. Follow-up for rodent biologic invasion, particularly in seaports, is explicitly recommended by the World Health Organization International Health Regulations (2005) (49) and is critical for preventing future zoonotic emergence. Thus, seaports could play a role as sentinels of larger surveillance networks.

Conclusions

Because of associated risk for animal-to-human spillover of SEOV (3), prevention, detection, and healthcare personnel awareness of this often-misdiagnosed infection remain critical on the continent of Africa. Control of rats would require more effective and comprehensive collaboration between local authorities and the academic and research communities. This type of collaboration fits well with the World Health Organization 13th General Program of Work (49). Reducing the reservoir population by using a targeted pest management plan in areas where rodents are highly abundant and in frequent contact with humans could enable mitigation of rodent-related issues and the risk for human disease (K.R. Blasdell et al., unpub. data, https://doi.org/10.1101/2021.03.18.436089). However, eradication of rat populations in areas of the most concern likely constitutes a more ambitious and unattainable goal and can paradoxically have contrary effects [START_REF] Murray | Urban rat exposure to anticoagulant rodenticides and zoonotic infection risk[END_REF]. Thus, a surveillance rather than ripostebased strategy, combined with medical staff training and implementation of on-site diagnostic methods [START_REF] Witkowski | Hantaviruses in Africa[END_REF], could reduce SEOV outbreak risk among humans in Africa.

About the Author

Dr. Castel is a researcher in virology at Institut National de Recherche Pour l'Agriculture, l'Alimentation et l'Environnement, Paris, France. His research interests include using phylogenetic and phylogeographic approaches to analyze the diversity of hantaviruses and their evolutionary processes.

21 Figure .

 21. Figure.Seaports in which SEOV was detected in rats, West Africa. Detailed map shows localization of the 2 genetically characterized SEOV variants isolated from black rats (Rattus rattus)[START_REF] Diagne | Seoul orthohantavirus in wild black rats, Senegal, 2012-2013[END_REF] and brown rats (R. norvegicus)[START_REF] Castel | Genetic characterization of Seoul virus in the seaport of Cotonou, Benin[END_REF]. Red arrows indicate potential transmission between the rat species. Red question marks indicate current unknown SEOV infection status in the considered rat species. Inset shows the areas of interest on the continent of Africa. SEOV, Seoul orthohantavirus.

  This work was funded by The French National Research Program for Environmental and Occupational Health of Anses (ANSES-22-EST-090).

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 29, No. 1, January 2023w

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 29, No. 1, January 2023


Andrej Babič

Simon Pascal

Romain Duwald

Dimitri Moreau

Jérôme Lacour

Eric Allémann

[4]Helicene-squalene fluorescent nanoassemblies for specific targeting of mitochondria in live-cell imaging

Keywords: Nanoassemblies, squalene, fluorescence, cationic helicenes, red to near-IR imaging, mitochondria

come

INTRODUCTION

Self-assembled nanoparticles constitute an emerging domain in nanotechnology. [START_REF] Nie | Properties and emerging applications of selfassembled structures made from inorganic nanoparticles[END_REF] In this context, squalene based nanoassemblies (NAs) have arisen as a robust platform in theranostic nanomedicine. [START_REF] Arias | Squalene Based Nanocomposites: A New Platform for the Design of Multifunctional Pharmaceutical Theragnostics[END_REF] Squalene is a natural triterpene and biosynthetic precursor of steroids. It has been extensively used as an excipient for the preparation of lipid emulsions, in particular for the delivery of vaccines, various active pharmaceutical ingredients and genes. [START_REF] Reddy | Squalene: A natural triterpene for use in disease management and therapy[END_REF] Furthermore, by attaching covalently a squalene chain to a drug, in a so-called "squalenoylation" protocol, improved pharmacokinetics of hydrophilic cytostatics and improved therapeutic outcomes are observed, in particular in cancer chemotherapy. 4 Squalenoylation has also been found to enhance drug stability, lower toxicity, improve bioavailability and mean residence time of drugs in the systemic circulation. [START_REF] Lepeltier | Self-assembly of squalene-based nucleolipids: relating the chemical structure of the bioconjugates to the architecture of the nanoparticles[END_REF] This is perhaps best demonstrated by gemcitabine-squalene conjugate (Chart 1) which shows superior anti-cancer efficacy and better tolerability in vivo. [START_REF] Maksimenko | Therapeutic modalities of squalenoyl nanocomposites in colon cancer: an ongoing search for improved efficacy[END_REF] Further advantages of squalenebased self-assembled nano systems are simplicity of synthesis and reproducibility of nanoparticle formation. Very importantly, besides improving therapeutic indexes of hydrophilic drugs, the attachment of squalene moieties also allows water "solubilization" of poorly water-soluble molecules by inverted water in oil system, thus allowing biomedical applications of these compounds.

In another context, fluorescent nanoparticles have found applications in many fields ranging from biosensors [START_REF] Fan | Co-self-assembled nanoaggregates of BODIPY amphiphiles for dual colour imaging of live cells[END_REF] to bioimaging [START_REF] Zhang | Biocompatible pillararene-assembly-based carriers for dual bioimaging[END_REF] and theranostics. [START_REF] Lovell | Porphysome nanovesicles generated by porphyrin bilayers for use as multimodal biophotonic contrast agents[END_REF] They provide robust, reproducible and simple nanoplatforms with unique optical properties coming from their relatively simple building blocks. New far-red and near-infrared (NIR) nanoparticle dyes are highly sought in biomedicine since they allow better imaging resolution due to their absorption and fluorescence properties located in the transparency window of biological tissues around 650-1300 nm. [START_REF] Guo | Recent progress in the development of near-infrared fluorescent probes for bioimaging applications[END_REF] So far, helicenes -that are ortho-condensed polycyclic aromatic compounds -are rarely used in bioimaging, [START_REF] Shen | Helicenes: synthesis and applications[END_REF] even though their helical geometry (configuration) may be useful for selective DNA binding. [START_REF] Amemiya | Chiral recognition in noncovalent bonding interactions between helicenes: right-handed helix favors right-handed helix over left-handed helix[END_REF] In fact, their (high energy) fluorescence in the blue-green spectral range of visible light limits biological applications. In addition, as classical helicenes are extended polyaromatics, aggregation occurs in aqueous media, limiting thus solubility and modifying optical characteristics. However, aggregative properties can become assets, as demonstrated by Latterini and co-workers who observed emission at longer wavelengths due to aggregation of the N-methyl-aza- [5]helicenium dye in cytoplasm (Chart 1). 12f More recently, Lu, Wang, Chen et al. took advantage of the low water solubility of racemic tetrahydro [5]helicene derivatives to prepare self-assemblies by reprecipitation techniques (Chart 1). [START_REF] Selvaraju | Pre-clinical evaluation of [Ga-68]Ga-DO3A-VS-Cys(40)-Exendin-4 for imaging of insulinoma[END_REF] The nanoparticles displayed bright visible colorations, efficient staining of HeLa cells and low cytotoxicity. While these examples proved that helicenes can be used as efficient fluorophores in bioimaging applications, the poor configurational stability or racemic nature of the [5]helicene derivatives did not allow an insight on the influence of stereochemistry on the aggregation / nanoparticle self-assembly or on the chiroptical properties of the dyes.

Chart 1. Reported structures of gemcitabine-squalene conjugate and of [5] and [4]helicenes used for bioimaging (A); Diaza [4]helicene-squalene composites 2, 3, and 4 under the scope of this study (B).

In this context, helical dimethoxyquinacridium cations 1 (DMQA, Chart 1) 14 appeared as choice candidates for bioimaging applications since these cationic [4]helicenes possess high configurational and chemical stabilities, coupled with strong orange-red emission. [START_REF] Kel | Excited-state properties of chiral [4]helicene cations[END_REF] Moreover, DMQA derivatives can be synthesized as racemates in only two steps and straightforward resolution protocols permit the isolation of the left-and right-handed enantiomers, respectively of M and P configurations, in gram scale. [START_REF] Laleu | Resolution of [4]Heterohelicenium Dyes with Unprecedented Pummerer-like Chemistry[END_REF] Finally, by late stage functionalization and derivatizations, the tuning of solubility and of (chiro)optical properties is readily feasible, 17 making these versatile [4]helicenes suitable building blocks for supramolecular nanoconstructs.

Herein, the synthesis of [4]helicene-squalene constructs 2, 3 and 4 is presented using a composition of functionalized diaza [4]helicenes and squalene chains as nanoassembly scaffold. The new redemitting conjugates were comprehensively characterized in racemic and enantiopure forms for their (chir)optical properties as single molecules and as nanoconstructs. The nano-helicene supramolecular composites were tested on several cancer cell lines and demonstrated unique staining properties of mitochondria. To our knowledge, it is the first report of a squalene based nanoassembly system which displays selective subcellular accumulation in cancer cells.

RESULTS AND DISCUSSION

Synthesis

Preparations of [4]helicene cations 2, 3 and 4 from tris(2,6-dimethoxyphenyl)methylium tetrafluoroborate salt 5 are depicted in Scheme 1.. Briefly, the synthesis of DMQA cations is classically achieved by N-cyclizations of carbenium precursor 5 in presence of bridging nucleophiles such as aliphatic amines (typically n-propylamine). [START_REF] Laursen | Synthesis of a Triazatriangulenium Salt[END_REF][START_REF] Sørensen | dimethoxyquinacridinium[END_REF] Furthermore, DMQA derivatives can be post-functionalized by reactions with electrophilic reagents, such as Eschenmoser's CH2=N + Me2 iminium ion. 17a The resulting aldehyde adduct can be oxidized to a carboxylate functional group under Pinnick-Kraus conditions, affording zwitterionic diaza [4]helicene 6 (Scheme 1, A), which is a useful synthetic intermediate to access highly fluorescent dyes via esterification and amidation reactions. 17 Scheme 1. Synthetic routes to diaza [4]helicene-squalene composites. Starting from carbenium precursor 5 conjugation of squalene can be achieved through coupling of functionalized DMQA to squalene amine or alcohol (A) or integrated into the [4]helicene core by reacting carbenium precursor 5 with squalene amine 8 then closing the system with methylamine, affording construct 4 (B). Reference compounds 10 and 11 directly prepared from 6 by esterification (C).

In fact, carboxylate 6 was converted to the corresponding acyl chloride with SOCl2 and subjected to (slow) nucleophilic substitution reactions at 20 °C in presence of an excess of squalene alcohol 7 or amine 8 derivatives; these latter molecules being obtained from squalene using four and five step synthetic procedures, respectively. [START_REF] Ceruti | Synthesis and biological activity of new iodoacetamide derivatives on mutants of squalenehopene cyclase[END_REF] The corresponding ester and amide-functionalized [4]helicenes 2 and 3 were isolated after purification by flash column chromatography in 74% and 52% yield, respectively.

In a different approach (Scheme 1, B), it was possible to synthesize non-symmetrical diaza [4]helicene 4. First, acridinium-squalene intermediate 9 (34%) was prepared selectively by treatment at 20 °C of carbenium 5 with squalene amine 8. Then, at 90 °C, addition of an excess of methylamine (aqueous solution) afforded squalene-containing diaza [4]helicene 4 in 37% yield.

Compounds 2, 3 and 4 were synthesized as racemates and fully characterized by 1 H, [START_REF] Selvaraju | Pre-clinical evaluation of [Ga-68]Ga-DO3A-VS-Cys(40)-Exendin-4 for imaging of insulinoma[END_REF] C and [START_REF] Ceruti | Synthesis and biological activity of new iodoacetamide derivatives on mutants of squalenehopene cyclase[END_REF] F NMR, UV-vis, IR spectroscopies, and high-resolution mass spectrometry. In the case of 2, starting from either M or P building block 6, 17b it was possible to reproduce the synthesis in enantiopure series and hence investigate the physico-chemical properties of NAs made of rac-2, M-2 or P-2 respectively (Figure 1). Compared to racemic mixtures the enantiopure helicenes are known to interact differently with biological targets [START_REF] Kel | Chiral Selectivity in the Binding of [4]Helicene Derivatives to Double-Stranded DNA[END_REF] and display dissimilar aggregation properties [START_REF] Kel | Excited-state properties of chiral [4]helicene cations[END_REF] which are highly relevant in the context of self-assembled materials. Finally, reference compounds 10 and 11 (Scheme 1, C), containing linear aliphatic C8 and C18 chains on the ester group, were synthesized using an approach similar to that of 2. These relatively simple conjugates, synthesized in 92% and 74% yield respectively (see Supplementary information), will serve as controls in nanoassembly experiments.

Preparation and physico-chemical characterization of racemic and enantiopure nanoassemblies

The NAs were obtained by a controlled nanoprecipitation in ultrapure water from a mixture of acetone and ethanol (Figure S18). The organic to aqueous phase volume ratios, the rate of addition and rate of evaporation were all rigorously controlled to give maximum robustness and repeatability to the nanoassembly process. All of the squalene-helicenes constructs could be effectively formulated into nanosystems. The NAs were first analyzed by dynamic light scattering (DLS). In general the quasi-elastic light scattering showed monodisperse NAs around 100 nm with low to very low polydispersity indexes (PDI). Figure 2 presents the size distribution of nano-rac-2 (nanoassembly constructed from rac-2). The PDI was below 0.10 indicating excellent nanoassembly properties and monodisperse distribution. However, the NA size was influenced by the type of the building block. Nano-rac-2 showed the biggest average size (130±2 nm), followed by nano-rac-3 (115±5 nm) and nano-rac-4 being the smallest (100±5 nm) as presented in Figure 2 and S19-S22. This is in line with reported examples of squalene conjugates where each construct shows different size distribution and topology features depending on the molecule conjugated to the squalene moiety. [START_REF] Reddy | A new nanomedicine of gemcitabine displays enhanced anticancer activity in sensitive and resistant leukemia types[END_REF] Interestingly, nano-rac-4 formed the smallest NAs. In construct 4 the squalene moiety is integrated into the [4]helicene core which presumably allows better packing and smaller NA size. Reference diaza [4]helicene conjugates 10 and 11 containing C8 and C18 linear alkyl chains respectively, were used to test the building block requirements for good nanoassembly properties.

Using the same nanoprecipitation procedure, NAs nano-rac-10 and nano-rac-11 were obtained respectively. However, the yield of the nanoassembly process differed drastically. The shorter aliphatic chain in derivative 10 resulted in NAs in less than 5% yield with most of the material precipitating out of the polar medium. On the other hand, nano-rac-11 were obtained in excellent F yield with minimal losses comparable to the squalene series. As expected the apparent zeta potentials were strongly positive around +40 mV and the average size at 110 nm and PDI below 0.1, very similar to the squalene series nano-rac-2-4 (Figures S25 andS26). These experiments demonstrated the importance long lipophilic chains such as squalene or stearyl that induce nanoassembly.

TEM and cryo-TEM experiments confirmed the range of the monodisperse size distribution established by DLS and revealed perfectly spherical morphology of the nanoparticles. Cryo-TEM of the nano-rac-4 revealed a thin corona only a few nanometers in thickness around the nanoassembly visible at high magnifications (Figure 3). By cryo-TEM no higher order topologies such as lamellar [START_REF] Duhem | Self-assembling doxorubicin-tocopherol succinate prodrug as a new drug delivery system: synthesis, characterization, and in vitro and in vivo anticancer activity[END_REF] hexagonal liquid crystalline phase [START_REF] Couvreur | Discovery of new hexagonal supramolecular nanostructures formed by squalenoylation of an anticancer nucleoside analogue[END_REF] or cubosomes [START_REF] Spicer | Progress in liquid crystalline dispersions: Cubosomes[END_REF] could be observed with any the NAs, most likely due to the very high lipophilicity of the nanoassembly building block conjugates. In an attempt to gain further insight into the nature of the new NAs, the nanoprecipitation was performed in deuterated water. After evaporation of the deuterated organic solvent we observed dramatic changes in 1 H NMR spectra in D2O with water presaturation sequence. The peak broadening corroborated the presence of nanoassemblies in D2O. The squalene protons are predictably broad due to the supramolecular structures present in D2O (Figure 3D andS20).

For all NAs the apparent zeta potential was found positive at +37 ± 1 mV. The positive charge of the nanoparticles is a consequence of the positive charge of the helicene cation building block. The same apparent zeta potential is also partially responsible for the impressive stability of the nano systems. Figure 2E and 2F demonstrate stability over a period of 2 months. Both average size and PDI remained practically constant during this period. Nano-rac-3 and nano-rac-4 display similar stability profiles (Figures S21 andS22).

NAs were prepared using M-2 and P-2 and DLS analysis revealed that both enantiomers showed smaller size average diameter, with 90±1 nm and 99±5 nm for nano-M-2 and P-2, respectively (Figures S23 andS24). Compared to the size distribution of nano-rac-2, which showed a larger size average of 126±7 nm, these values indicate a better packing of enantiopure building blocks within the NAs. This observation is interesting in view of the stronger aggregation in water of racemic DMQAs 1 (R = Me, Pr) in comparison to their enantiopure equivalents. [START_REF] Kel | Chiral selectivity in the binding of [4]helicene derivatives to double-stranded DNA[END_REF]

Optical and chiroptical properties

The optical properties have been recorded for 2-4 single molecular entities (non-nanoassembled building blocks) in acetonitrile and for nano-rac-2-4 NA in water (Table 1). The absorption spectra of the NAs are presented in Figure 4A. They show intense absorption in the orange-red region of the visible spectrum, with maxima centered at 594, 608 and 618 nm, for ester-derivative 2, amide-derivative 3 and integrated squalene-DMQA composite 4, respectively. The fluorescence is centered in the red range ca. 660 nm for the three types of NAs, with average Stokes shifts of 1350-1550 cm -1 and cut-off emissions reaching the NIR region, up to 800 nm. The absorption and fluorescence profiles recorded for 2-4 as single molecules in acetonitrile or as NAs are quasisuperimposable, however a noticeable 8 nm red-shift is observed for nano-rac-4 emission band (Figures S15-16).

Table 1. Optical data of racemic diaza [4]helicenes (acetonitrile) and corresponding racemic NAs (water).

Compound λmax / nm

(εmax / L.mol -1 .cm -1 ) λem / nm Φ [a] τ1 / ns [b] τ 2 / ns [b] 2 a] Relative to cresyl violet (Φ = 0.54 in MeOH). [b] NanoLED excitation at 570 nm, composition indicated in parentheses.

In general, the fluorescence quantum yields are significantly impacted by the solvent and the organization of the dye. In the instance of 2, an honourable fluorescence efficiency of 0.39 is recorded in acetonitrile, which drops to 0.13 for the corresponding nano-rac-2 NAs in aqueous medium. Such quench of the fluorescence in polar solvent is classically accompanied with an enhancement of non-radiative de-excitation. It is furthermore worthy to underline that a progressive decrease of the fluorescence quantum yield and lifetime values occurs as the emission maxima are red-shifted (ϕ(2) > ϕ(3) > ϕ(4)), as previously observed. 17a Fluorescence time profiles reveal non-negligible emission lifetimes for organic red-fluorophores in acetonitrile, with values comprised between 14.7 ns and 4.6 ns, respectively for 2 and 4 in acetonitrile. Bi-exponential fits were necessary in order to determine the lifetimes of the NAs in water, which is the signature of two different emissive species, presumably corresponding to excited single molecules or excited aggregates that could partially quench the fluorescence. [START_REF] Kel | Excited-state properties of chiral [4]helicene cations[END_REF] The electronic circular dichroism (ECD) spectra of both enantiomers of 2 and nano-2 were respectively recorded in acetonitrile and milli-Q water, and the spectra are compared in Figure 4B (peak values are reported in the Supporting Information). Enantiomers M-2 and P-2 display mirror spectra characterized by strong Cotton effect in the UV region with peaks at 214 and 301 nm (│Δε│ca. 40 M -1 .cm -1 ) and less intense transitions in the visible range centered ca. 460 and 600 nm. The lower energy band remains unchanged in the case of enantiopure nano-M/P-2, whereas a distinct bathochromic shift of 10 nm is observed for the ECD signals around 310, 415 and 470 nm.

Interestingly, these red-shifted transitions are only slightly detected between the absorption spectra of enantiopure M/P-2 and nano-M/P-2 (Table S1, Figure S17).

Cell imaging

As mentioned, helicenes are rarely used in bioimaging due, in general, to poor water solubility, acute toxicity and suboptimal optical properties. However, constructs 2-4 seemed ideal to solve of these problems as (i) ester or amide groups on the [4]helicene core leads to brighter derivatives (ε.ϕ = 4'220 M -1 .cm -1 for 2) 17a and (ii) the linkage of squalene or other extended aliphatic chains prolongs further the fluorescence into the far-red windows and permits the formation of nanoassemblies. The latter properties allows the transfer of highly lipophilic dyes into aqueous media, thus greatly facilitating bioimaging applications. [START_REF] Wolfbeis | An overview of nanoparticles commonly used in fluorescent bioimaging[END_REF] Hence, all the prepared NAs could be easily screened by incubation with cancer cells using highthroughput screening platform. It became immediately apparent that the staining pattern was specific for a sub-cellular organelle in HeLa cells. Co-staining with MitoTracker Green revealed excellent overlap and confirmed the highly specific staining of mitochondria by NAs. As shown in Figure 6, this is the case for nano-rac-2 and both enantiopure nano-M-2 and nano-P-2 with small differences in staining pattern between these NAs. Similar specificity was observed for nano-rac-3, but interestingly, nano-rac-4 stained mitochondria with a lower affinity (Figure S27 and S28, respectively). The specific sub-cellular mitochondrial accumulation and staining is a consequence of the physico-chemical properties of the building blocks, the nature of the linkage that attaches the squalene chains to the helical core, but not of the stereochemical nature of the helicene (rac, M or P). Diaza [4]helicenes could be then potentially considered as a new member of structural class called delocalized lipophilic cations (DLC). They are known to accumulate in mitochondria due to high negative potential of the mitochondrial inner membrane caused by the electron transport chain and proton flux across the membrane. [START_REF] Weinberg | Targeting mitochondria metabolism for cancer therapy[END_REF] Only a handful of DLCs are routinely used to label mitochondria today namely, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol-carbocyanine iodide (JC-1), Rhodamine-123, and Mitotracker. [START_REF] Yousif | Targeting mitochondria with organellespecific compounds: strategies and applications[END_REF] Triphenyl phosphonium is non-fluorescent DLC and has been used as a mitochondrial targeting ligand. [START_REF] Dodani | A targetable fluorescent sensor reveals that copper-deficient SCO1 and SCO2 patient cells prioritize mitochondrial copper homeostasis[END_REF] The passive transport of DLCs into mitochondria is energetically favorable because of the charge delocalization creating a much greater ionic radius and the effectively lowering enthalpy of desolvation upon transfer into lipid

A B C
environment. The result can be several 100-fold higher concentrations of cations in the mitochondria compared to outside of the cells. [START_REF] Weinberg | Targeting mitochondria metabolism for cancer therapy[END_REF] Additional imaging experiments with several control compounds gave us more insight in the structure-activity relationship. As expected and previously reported, [START_REF] Kel | Chiral selectivity in the binding of [4]helicene derivatives to double-stranded DNA[END_REF] unfunctionalized DMQA cation 1 (Chart 1, A) displayed non-specific mostly nuclear DNA staining (Figure S32). On the other hand and importantly, the linear aliphatic conjugate DMQA-C18 11 that displayed good nanoassembly properties with similar average sizes and PDI, surprisingly did not display the same cell staining pattern as the squalene series. The staining of nano-rac-11 was weaker, spotty and mitochondria non-specific (Figure 7). The major difference between DMQA-C18 nano-11 and the squalene series nano-1-3 is the presence of multiple substituted double bonds in squalene which display the A 1,3 strain driving the squalene moiety into a folded conformation compared to the predominantly linear C18 chains. The mitochondrial specificity of the constructs is therefore inherent to the whole construct and not the individual squalene or helicene parts.

A B

LogP of nano-rac-2 and nano-rac-11 were calculated at 11.6 and 10.4 respectively, which is more than 10 orders of magnitude higher than logP of Rhodamine-123, a prototypical mitochondrial dye. [START_REF] Belostotsky | Mitochondrial targeting for photochemotherapy. Can selective tumor cell killing be predicted based on n-octanol/water distribution coefficients?[END_REF] It is very surprising that very high lipophilicity of the squalene NAs 2-4 did not bring about any considerable membrane accumulation as previously reported for other squalene NAs, such as squalenoyl gemcitabine (Chart 1). Squalene prodrugs normally distribute within cellular membranes, including those of organelles such as endoplasmatic reticulum. [START_REF] Bildstein | Transmembrane diffusion of gemcitabine by a nanoparticulate squalenoyl prodrug: an original drug delivery pathway[END_REF] It would be expected that with logP values of this order of magnitude the lipophilic partitioning overrides the delocalized cationic character of the compounds. However, this is clearly not the case for nano-rac-2 (Figure 7). Intracellular phenomena governing the distribution of this type of squalene NAs are not yet understood and deserve further investigations.

Internalization kinetics

Couvreur and co-workers presented evidence for another important aspect of squalene-based NAs. They demonstrated that squalenoyl gemcitabine NAs did not enter the cell through endocytosis but as single building block molecules by passive diffusion. [START_REF] Bildstein | Extracellular-protein-enhanced cellular uptake of squalenoyl gemcitabine from nanoassemblies[END_REF] This is perhaps the most striking difference of squalene based systems compared to other types of nanoparticles. [START_REF] Oh | Endocytosis and exocytosis of nanoparticles in mammalian cells[END_REF] The

internalization

Cytotoxicity

Cell viability of the most promising NAs nano-rac-2 and enantiopure nano-M-2 was determined to assess their biocompatibility. HeLa cells were incubated with 10, 1.0, 0.1 and 0.01 µM concentrations of NAs for 2 h, 6 h, and 24 h. Considerable cytotoxicity was detected only at 10 µM and 24h incubation time (Figure S33). It is noteworthy that nano-rac-11 control displayed lower cytotoxicity than nano-rac-2 at the same concentration.

CONCLUSIONS

In conclusion, this study presents the squalenoylation of lipophilic diaza [4]helicene dyes. In aqueous solution, the stable monodisperse NAs displayed effective brightness in the transparency window of biological media and impressive mitochondrial accumulation in several cell lines. To the best of our knowledge, this is the first example of squalene-based nanoassembly system that demonstrates the feasibility of specific sub-cellular targeting. In light of the emerging central role of mitochondria in the regulation of cellular signaling, homeostasis, and apoptosis with major implications for cancer and other diseases, novel nano-platforms with mitochondrial targeting properties show great promise in theranostic nanomedicine. Luminescence. Steady-state fluorescence spectra were measured using a Varian Cary 50 Eclipse spectrofluorimeter. All fluorescence spectra were corrected for the wavelength-dependent sensitivity of the detection. Fluorescence quantum yields Φ were measured in diluted solution with an optical density lower than 0.1 using the following equation:

METHODS

General

Φ 𝑥 Φ 𝑟 = ( A 𝑟 (𝜆) A 𝑥 (𝜆) ) ( 𝑛 𝑥 2 𝑛 𝑟 2 ) ( D 𝑥 𝐷 𝑟 )
where A is the absorbance at the excitation wavelength (λ), n the refractive index and D the integrated intensity. "r" and "x" stand for reference and sample. The fluorescence quantum yields were measured relative to cresyl violet in methanol (Φ = 0.54). Excitation of reference and sample compounds was performed at the same wavelength. Short luminescence decay was monitored with the TC-SPC Horiba apparatus using Ludox in distilled water to determine the instrumental response function used for deconvolution. Excitation was performed using a 570 nm NanoLED (peak wavelength: 573 nm; pulse duration: 1.5 ns), and the deconvolution was performed using the DAS6 fluorescence-decay analysis software.

Preparation of nanoassemblies Compound (1.00 µmol) was dissolved in a 50/50 V/V mixture acetone/ethanol (500 µL). The organic phase was added dropwise using a micro-syringe into MilliQ water (1.25 mL) at 100 µL/min under stirring. After 5 min under stirring the magnetic stir bar was removed and the organic solvents and the excess of water removed using a rotary evaporator at 40 °C. The evaporation program was the following: 5 min at 400 mbar, 5 min at 200 mbar, 10 min at 100 mbar and the remaining time at 80mbar. The final concentration of nanoassemblies was 1.00 mM.

Characterisation of Nanoassemblies.

Hydrodynamic diameters of nanoassemblies were measured by dynamic light scattering (DLS) using a NANO ZS instrument from Malvern (Worcestershire, UK) running the ZetaSizer 7.01 software. The analyses were performed with 4 mW He-Ne Laser (633 nm) at scattering angle of 173° at 25 °C in Polystyrene (PS) micro cuvette from Brand (Wertheim, Germany). Zeta potential (ZP) was determined using the same Nano ZS instrument from Malvern in folded capillary cells DTS 1070 from Malvern. Size distribution and size mean diameter were calculated from the data.

The morphological examination of nanoassemblies was performed using a transmission electron microscope (TEM) instrument TECNAI ® G 2 Sphera (FEI, Thermo Fisher Scientific) equipped with 2000 by 2000 pixel high resolution digital camera TCL (Gräfelfing, Germany). Samples were negatively stained with uranyl 2 % on a carbon film 200 Hex Mesh copper grid and run at 200 kV with a LaB6 cathode.

For the cryogenic transmission electron microscope (cryo-TEM) the vitrified-ice samples were prepared using the Virtobot cryo-plunger (FEI, Thermo Fisher Scientific). 2.0 µL of nanoassemblies (1.0 mM) were applied to Quantifoil Cu/Rh 200 mesh R3.5/1 grids (SPI, West Chester, USA) and vitrified using liquid ethane.

NMR of Nanoassemblies in Deuterated

Water. The nanoassemblies were prepared as described above except that deuterated solvents were used. The initial solution of the nanoassembly material in deuterated acetone was recorded at 500 MHz first. After the nanoprecipitation the spectra were recorded in D2O. The HDO signal was suppressed using presaturation before running the 1 H sequence.

Stability. The stability of nanoassemblies was monitored by visual control and by the change in particle size by dynamic light scattering upon storage at room temperature during a 2 months period in the dark. PDI and average size were recorded for each time point.

High-throughput imaging. HeLa cell maintenance and imaging were performed has described by Wallabregue et al. [START_REF] Wallabregue | Selective Imaging of Late Endosomes with a pH-Sensitive Diazaoxatriangulene Fluorescent Probe[END_REF]

and Chuard et al. (REF)

The automated IXM-XL microscope from molecular devices™ has been used to ensure consistent imaging conditions between all the samples. Fluorescent images were acquired with a 40X objective using the appropriate filter cube (405nm, 488nm and 594nm). Nanoassemblies were incubated at 1.0 µM concentration and equivalent DMSO solutions were prepared by diluting 2.0 mM DMSO solution to 1.0 µM with cell media.

Cell viability. The cell viability assay has been performed with the same automated IXM-XL microscope from molecular devices™. The HeLa cells have been treated for the time and concentration described in the manuscript in 96 well plate, 4 replicates per condition. Once fixed with PFA the nuclei have been stained with Hoechst. Using MetaXpress™ software each individual nucleus has been segmented and counted. The cell viability is directly reflect by the count of viable nuclei compared to control cells without NAs.

Confocal scanning microscopy. U87MG human glioblastoma cancer cells (ATTC ® HTB-14 TM , Manassas, Virginia) and PC3 human prostate cancer cells (ATTC ® CRL-1435 TM , Manassas, Virginia) were maintained in MEM (U87MG) and F12K nutrient mix (PC3). Culture media were supplemented with 10% fetal calf serum (CVFSVF00-01, Eurobio) and 100 µl/ml streptomycin and 100 IU/ml penicillin (15140-122, Thermo Fisher Scientific). The cells were seeded into ibidi µ-Slides, ibiTreat, 4 (80426-IBI) or 8 (80826-IBI) wells 24 h before the experiments. Hoechst 33342 (2 µg/mL, Thermo Fisher Scientific) and MitoTracker green (50 nM, M7514 Thermo Fisher Scientific) were freshly prepared in incomplete culture medium before imaging. Cell monolayers in each well were incubated at 37 °C with the solution of Hoechst and MitoTracker Green (700 µL) for 30 min. The cells were washed 2 times with DPBS. The cells were then incubated with nanoassemblies at 1.0 µM and imaged at regular time points at different locations within the wells. Control cells were incubated in complete culture medium under the same experimental conditions. Detection and localization of fluorescence in cells was done with the Zeiss LSM780 (Jena, Germany) inverted confocal microscope fitted with an objective lens Plan-Aprochromat 63 x/1.4 oil DIC M27. The images were taken using 405 nm, 488 nm and 590 nm excitation lasers and 430-470 nm, 500-550 nm, 680-800 nm emission band filters respectively. During the image acquisition time, slides were kept at 37 °C and 5% of CO2 in a humidified chamber (INUBTF-WSKM-F1, Tokai Hit, Gendoji-cho, Fujinomiya-shi, Shizuoka-ken, Japan). ZEN 2 software (Zeiss, Jena, Germany) was used for image processing.

Co-localization quantification. Quantification of co-localization between NAs (red) and MitoTracker ® Green was done by determining Pearson's coefficient of correlation (PCC) with Imaris x 64 8.0.0 software. PCC values may range between +1 (perfect correlation) and -1 (perfect negative correlation), where 0 means no correlation. [START_REF] Adler | Quantifying colocalization by correlation: the Pearson correlation coefficient is superior to the Mander's overlap coefficient[END_REF] Regions of interest (ROI) were defined and PCC was calculated. ROI was defined for each image separately by masking the dataset with red channel (NAs fluorescence). The value of PCC for each experiment is presented as means ± s.d. calculated from 3 separate images.

Statistical analysis. Statistical analysis were performed with Graphpad Prism 6.05 using the Student's t-test. Data with p < 0.05 were considered statistically significant.

ASSOCIATED CONTENT

Figure 1 .

 1 Figure 1. M (left) and P (right) enantiomers of 2.

Figure 2 .

 2 Figure 2. Characterization of nano-rac-2. TEM (A) and cryo-TEM image (scale 100 nm) (B) DLS size distribution showing (C), overlay of 1 H-NMR of 2 (orange) and nano-rac-2 (blue) in deuterated acetone and water, respectively (D), and size (E)and PDI stability (F).

Figure 3 .

 3 Figure 3. High magnification TEM (A) and cryo-TEM images (B) of nano-rac-4. Very thin corona measuring a few nm surrounding the nanoassemblies is visible in both images (scale 100 nm).

[

Figure 4 .

 4 Figure 4. Normalized absorption (plain lines) and fluorescence (dashed lines) spectra of racemic NAs in water (A) and UV-vis electronic circular dichroism spectra of M-2 (dashed lines) and P-2 (plain lines) single molecules (acetonitrile, red) and NAs (H2O, blue) (B).

Figure 6 .

 6 Figure 6. High-throughput imaging of racemic and chiral NAs on HeLa cells. NAs in water (left) and equivalent DMSO solutions (right). Racemic nano-2 (A), nano-M-2 (B) and nano-P-2 (C). Mitochondria labelled with MitoTracker (green) and NA (red). NA concentration 1 µM. Scale bar (10 µm).

Figure 7 .

 7 Figure 7. High-throughput imaging of [4]helicene-C18 nano-11 control (A) and racemic nano-2 (B) on HeLa cells. NAs (left) and equivalent DMSO solutions (right). NA concentration 1 µM. Scale bar (10 µm).

  studies usually require the use of fluorescent probes integrated into the nanoconstructs. Our nanosystems allowed the use of inherent NA fluorescence to follow and study internalization kinetics in several cell lines. In both U87MG and PC3 cell lines very fast internalization was observed. Confocal scanning microscopy revealed detectable amounts of NAs even after only 10 min of incubation at 37 °C and considerable amounts of the NAs in 30 min (Figure 8C and S29). The rapid kinetics of internalization and the absence of endosome-like vesicular structures confirms the passive internalization mechanism of single building blocks rather than the full NAs. This was further confirmed by preparing solutions of the building blocks 2, M-2 and P-2 in DMSO, diluting them in cell media and incubating them with HeLa cells. The mitochondrial staining was practically identical as with NAs. Active vs. passive internalization mechanism was further investigated by incubation of HeLa cells with nano-M-2 at 37 and 4 °C. The result shows fluorescence labeling of HeLa cells occurs at both temperatures, although the labeling is stronger at 37 °C. The experiment validates passive mechanism of internalization by diffusion of individual molecules into the cells. Confocal microscopy also allowed the quantification of co-localization. Pearson co-localization coefficient was determined for nano-M-2 at 0.79 for PC3 and U87MG cells at 30 min (Figures S30 and S31, respectively).

Figure 8 .

 8 Figure 8. Live-cell imaging by confocal laser scanning microscopy of nano-M-2 with PC3 prostate cancer cells (A) and U87MG glioblastoma cells (B) after 20 min of incubation. Left to right, cell nuclei (blue), mitochondria stained with MitoTracker (green), and nano-M-2 (red), and overlay (scale bar 10 µm). Cell internalization kinetics of nano-M-2 at 5, 10, 20, 30, 40 and 60 min of incubation in U87MG (C). Excellent mitochondrial staining upon rapid uptake of the probe was observed in both cell lines. NA concentration was 1 µM.

.

  Column chromatography was performed using Siliaflash P60 silicagel (40-63 µm, 60 Å) and Acros Brockmann I basic alumina (40-200 µm, 60 Å). Optical properties were recorded in analytical grade acetonitrile and Milli-Q water (specific resistance 18.2 MΩcm @ 25 °C). NMR spectra were recorded on Brucker Avance II+ AMX-500 and Varian Anova 500 MHz spectrometers at room temperature. NMR chemical shifts are given in ppm (δ) relative to Me4Si with solvent resonances used as internal standards (CD2Cl2: 5.32 ppm for 1 H and 53.8 for 13 C). IR spectra were recorded on a Perkin-Elmer 1650 FT-IR spectrometer using a diamond ATR Golden Gate sampling. Melting points (M.P.) were measured in open capillary tubes with a Buchi B-550 melting points apparatus and are uncorrected. UV-vis-NIR absorption spectra were recorded on a JASCO V-650 spectrophotometer at 20°C. Electrospray mass spectra were obtained on a Finnigan SSQ 7000 spectrometer QSTAR pulsar i (AB / MDS Sciex), ESI (TIS)/nanoESI/APCI-QqTof by the Mass Spectroscopy Platform of the Faculty of Sciences, University of Geneva. Optical rotation were measured on a Perkin Elmer 241 polarimeter at 20°C using a Hg lamp (365 nm). Electronic circular dichroism spectra were recorded on a JASCO J-815 spectrophotometer at 20°C. LogP was calculated using Chemicalize software and Marvin JS editor.

Acknowledgement

We thank Dr. Christoph Bauer and Jérôme Bosset from the Bioimaging platform at the University of Geneva for their input and help in live cell imaging. We are grateful to Dr. Laurence Marcourt of the School of Pharmaceutical Sciences Geneva-Lausanne for her contribution in NMR experiments. We thank the "Laboratoire de Chimie" at the ENS Lyon for access to NanoLED and TS-SPC equipment. We would also like to acknowledge Mass Spectrometry platform at the University of Geneva for the mass spectroscopy analysis. We thank Canton of Geneva, Switzerland and Swiss National Science Foundation for funding.

Funding Sources

Any funds used to support the research of the manuscript should be placed here (per journal style).

Notes

All of the authors declare no conflict of interest.

Supporting Information Available: AUTHOR INFORMATION

Corresponding Authors

*andrej.babic@unige.ch, jerome.lacour@unige.ch, eric.allemann@unige.ch.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.