Tiago Gräf

Cynthia Vazquez

Marta Giovanetti

Fernanda De Bruycker-Nogueira

Vagner Fonseca

Ingra Morales Claro

Jaqueline Goes De Jesus

Andrea Gómez

Joilson Xavier

Marcos Cesar Lima De Mendonça

Shirley Villalba

Juan Torales

Maria Liz Gamarra

Julien Thézé

Ana Maria Bispo De Filippis

Vasco Azevedo

Tulio De Oliveira

Leticia Franco

Carlos F Campelo De Albuquerque

Sandra Irala

Edward Charles Holmes

Jairo Andrés

Luiz Méndez Rico

Carlos Junior

Alcantara Research

Oswaldo Cruz

J G De Jesus

Fundaç

M C L De Mendonça

L C J Alcantara

Keywords:

C hikungunya virus (CHIKV), dengue virus (DENV), and Zika virus (ZIKV) are 3 of the most common arthropod-borne viruses (arboviruses) that infect humans. All are transmitted by the anthropophilic and urban-adapted Aedes aegypti and Ae. albopictus mosquito vectors (1). Driven by human movement and climate trends, the distribution of these mosquitoes is expanding along with the arboviruses they transmit (2). In Latin America, CHIKV and ZIKV have emerged since the mid-2000s, joining DENV, which is already endemic there (3). In this region, only Uruguay and Chile did not report autochthonous transmissions of one of these arboviruses during 2014-2019, highlighting the current state of endemicity (4).

Paraguay is a landlocked country in the center of South America; it borders Bolivia, Brazil, and Argentina. DENV is endemic to Paraguay, and all 4 serotypes (DENV-1-4) have been detected there; in some seasons, multiple serotypes co-circulate (5,6). Phylogenetic analysis has shown that DENV genetic diversity in Paraguay is closely related to that in neighboring countries, particularly Brazil (7,8). However, more genomic surveillance studies in Paraguay are needed to learn more about this epidemiologic pattern.

Paraguay has been severely aff ected by emergent Zika and chikungunya viruses, and dengue virus is endemic.

To learn more about the origins of genetic diversity and epidemiologic history of these viruses in Paraguay, we deployed portable sequencing technologies to strengthen genomic surveillance and determine the evolutionary and epidemic history of arthropod-borne viruses (arboviruses). Samples stored at the Paraguay National Central Laboratory were sequenced and subjected to phylogenetic analysis. Among 33 virus genomes generated, we identifi ed 2 genotypes of chikungunya and 2 serotypes of dengue virus that circulated in Paraguay during 2014-2018; the main source of these virus lineages was estimated to be Brazil. The evolutionary history inferred by our analyses precisely matched the available travel history of the patients. The genomic surveillance approach used was valuable for describing the epidemiologic history of arboviruses and can be used to determine the origins and evolution of future arbovirus outbreaks.

of chikungunya fever in Paraguay were first reported in June 2014; autochthonous cases were first detected in 2015, and CHIKV caused seasonal outbreaks every year until 2018. Zika was first detected in November 2015, and autochthonous infections were confirmed soon after (9). To date, however, little is known about the genetic diversity of CHIKV and ZIKV that circulate in Paraguay.

The potential triple epidemic scenario (i.e., CHIKV, DENV, ZIKV) in Paraguay could pose serious public health and economic burdens. Arbovirus surveillance is critical for assisting health services with preparedness, providing key information about the seasonality of infections and diversity of circulating viral lineages. When resources allow, such surveillance can now involve genomic surveillance via portable sequencing technologies. For example, this approach was successfully used to study the ZIKV epidemic in the Americas [START_REF] Faria | Establishment and cryptic transmission of Zika virus in Brazil and the Americas[END_REF][START_REF] Giovanetti | Genomic and epidemiological surveillance of Zika virus in the Amazon Region[END_REF], the reemergence of yellow fever virus in Brazil [START_REF] Faria | Genomic and epidemiological monitoring of yellow fever virus transmission potential[END_REF][START_REF] Giovanetti | Yellow fever virus re-emergence and spread in Southeast Brazil, 2016-2019[END_REF], and recurrent outbreaks of CHIKV in several regions of Brazil [START_REF] Naveca | Genomic, epidemiological and digital surveillance of chikungunya virus in the Brazilian Amazon[END_REF][START_REF] Gusmão | Return of the founder chikungunya virus to its place of introduction into Brazil is revealed by genomic characterization of exanthematic disease cases[END_REF][START_REF] Xavier | Circulation of chikungunya virus East/ Central/South African lineage in Rio de Janeiro, Brazil[END_REF].

To help reinforce arbovirus surveillance in Paraguay, we performed portable genome sequencing under the scope of the ZIBRA project (http://zibraproject.org) at the Laboratorio Central de Salud Publica in Asunción, Paraguay. During July 16-20, 2018, a team of molecular biologists from Brazil and Paraguay worked on a group of samples selected to determine the recent history of arboviruses in the country, generating 33 viral genomes and building capacity skills among the local laboratory staff. We report the analysis of the origins and spread of CHIKV in Paraguay as well as the current dynamics of DENV. The project was reviewed and approved by the Comissão Nacional de Ética em Pesquisa (CONEP) from the Brazilian Ministry of Health as part of the arbovirus genomic surveillance efforts within the terms of CONEP Resolution 510/2016 by the Pan American Health Organization Ethics Review Committee (PAHO-2016-08-0029) and by the Paraguayan Ministry of Public Health and Social Welfare (MSPyBS/ S.G. no. 0944/18).

Methods

Sample and Data Collection

This study was necessarily based on convenience sampling; de-identified samples were obtained from material exceeding the routine number of arbovirus diagnoses and stored at the Laboratorio Central de Salud Publica de Asunción, Paraguay, which concentrates biological samples collected throughout the country. On the basis of resources and time availability, we selected 50 acute-phase serum samples that were positive for DENV or CHIKV with PCR cycle threshold (C t ) values <28 at the time of diagnosis. Using epidemiologic data, we chose samples to represent geographic departments in Paraguay with the highest number of cases. ZIKV-positive samples with low C t and historical samples for DENV were unavailable; hence, for DENV, we studied only the 2018 epidemic. The Direccion General de Vigilancia de la Salud of Paraguay provided temporal data on the incidence of chikungunya and dengue cases by department within Paraguay.

Virus Amplification and Whole-Genome Sequencing

We extracted viral RNA from the selected samples by using the QIAamp Viral RNA Mini Kit (QIAGEN, https://www.qiagen.com) and subjected the RNA to real-time reverse transcription quantitative PCR to detect CHIKV and DENV serotypes 1-4 as described previously [START_REF] Lanciotti | Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007[END_REF][START_REF] Lanciotti | Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptasepolymerase chain reaction[END_REF][START_REF] Lanciotti | Chikungunya virus in US travelers returning from India, 2006[END_REF]. To increase the genome coverage, we selected only samples with C t <35 for sequencing. Extracted RNA was converted to cDNA by using the Protoscript II First Strand cDNA Synthesis Kit (New England Biolabs, Inc., https://www.neb.uk.com) and random hexamer priming. We attempted wholegenome amplification by multiplex PCR as previously described [START_REF] Quick | Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples[END_REF].

We purified amplicons by using 1x AMPure XP Beads (Beckman Coulter, https://www.beckman. com) and quantified them on a Qubit 3.0 fluorimeter by using a Qubit dsDNA HS Assay Kit (Thermo-Fisher Scientific, https://www.thermofisher.com). We performed DNA library preparation by using a Ligation Sequencing Kit and Native Barcoding Kit (NBD103; Oxford Nanopore Technologies, https:// nanoporetech.com). We generated sequencing libraries from the barcoded products by using the Genomic DNA Sequencing Kit SQK-MAP007/SQK-LSK208 and loaded them into an R9.4 flow cell (Oxford Nanopore Technologies).

Generation of Consensus Sequences

We base-called raw files by using Albacore software, demultiplexed and trimmed by using Porechop software (https://github.com) and then mapped with Burrows-Wheeler Aligner software to a reference genome. On the basis of PCR analyses, we used Gen-Bank accession nos. KP164568 for CHIKV, KF672760 for DENV-1, and JN559741 for DENV-4 as reference sequences. To detect single-nucleotide variants to the reference genome, we applied Nanopolish software variant calling (https://nanoporetech.com) to the assembly. Nonoverlapped primer binding sites and sites for which coverage was <20× were replaced with ambiguity code N.

Phylogenetic Analyses

We first investigated sequence genotypes by using the arbovirus genotyping tool [START_REF] Fonseca | A computational method for the identification of dengue, Zika and chikungunya virus species and genotypes[END_REF]. To investigate the origins and spatial dynamics of arboviruses in Paraguay, we downloaded all sequences assigned as CHIKV, DENV-1, and DENV-4 from GenBank. We excluded sequences without sampling date and location and sequences covering <50% of the virus genome. Sequence alignment was performed by using MAFFT [START_REF] Katoh | MAFFT version 5: improvement in accuracy of multiple sequence alignment[END_REF] (FFT-NS-2 algorithm) and visually inspected in Aliview [START_REF] Larsson | AliView: a fast and lightweight alignment viewer and editor for large datasets[END_REF]. We estimated maximum-likelihood phylogenies in IQ-TREE (24) by using the best-fit model of nucleotide substitution as indicated by the ModelFinder application (implemented in IQ-TREE). Branch support was assessed by the SH-like approximate-likelihood ratio test, and we submitted highly supported (>0.9) clades containing the DENV genomes from Paraguay (Appendix 1 Figure 1, https:// wwwnc.cdc.gov/EID/article/27/5/20-4224-App1. pdf) and the clades of CHIKV from the Americas to TempEst [START_REF] Rambaut | Exploring the temporal structure of heterochronous sequences using TempEst (formerly Path-O-Gen)[END_REF] to assess the strength of temporal signal in these data.

Time-scaled phylogenetic trees were inferred by using the BEAST package [START_REF] Suchard | Bayesian phylogenetic and phylodynamic data integration using BEAST 1.10[END_REF]. We chose the uncorrelated relaxed molecular clock model as indicated by the marginal likelihood estimation model test procedure. We also used the codon-based SRD06 model of nucleotide substitution and the nonparametric Bayesian Skygrid coalescent model. A discrete phylogeographical model [START_REF] Lemey | Bayesian phylogeography finds its roots[END_REF] was used to reconstruct the spatial diffusion of the virus across the compiled dataset sampling locations (Appendix 2, https://wwwnc.cdc. gov/EID/article/27/5/20-4224-App2.xlsx). Phylogeographic analyses were performed by applying an asymmetric model of location transitioning coupled with the Bayesian stochastic search variable selection procedure. We complemented this analysis with Markov jump estimation that counts location transitions per unit time along the tree. We ran Monte Carlo Markov chains long enough to ensure stationarity and an adequate effective sample size of >200.

Results

Of the 50 samples tested, 25 were positive for CHIKV, 14 for DENV-1, and 11 for DENV-4. For positive samples, the average PCR C t value was 26.36 (range . From the 50 samples, we were able to generate 33 complete or near-complete genome sequences (17 CHIKV and 16 DENV genomes) (Table ). The GenBank accession numbers of newly generated sequences are MT038393-409 (CHIKV) and MT040672-87 (DENV). The collection dates of the samples sequenced were November 3, 2014, through July 10, 2018, and locations covered 15 municipalities and 8 departments (the first-level administrative subdivisions) of Paraguay (Figure 1). Women accounted for 58% of the samples, and the median patient age was 34 years. A TempEst analysis of all arbovirus lineages found here revealed a strong correlation between the sampling time and the root-to-tip divergence (Appendix 1 Figure 2).

The CHIKV Epidemic in Paraguay during 2014-2018

Of the 17 CHIKV genomes, 12 were classified as Asian genotype (sampled during 2014-2016) and 5 as East/Central/South African (ECSA) genotype (sampled during 2018). The oldest CHIKV sample analyzed (patient PY45) was obtained in November 2014 from the department of Amambay and was identified as an Asian genotype (Figure 1). However, autochthonous transmission of CHIKV was not confirmed until February 2015, followed by an increased number of reported infections (9) (Figure 2, panel A). Phylogeographic analysis revealed that the most likely origin of PY45 was Central or South America (Figure 3, panel A). The travel history for PY45 is in agreement with these results because the patient reported having visited Panama and San Andrés, a Colombian cluster of islands in the Caribbean region. In November 2014, another introduction of CHIKV in Paraguay was detected in the Central Department. That sequence (patient PY44) clustered with high support (posterior probability [PP] = 1) among sequences from Colombia and 1 sequence from Nicaragua, and the ancestral state of the most recent common ancestor (MRCA) of this clade was South America (PP = 0.8). Patient PY44 reported traveling to Cartagena, Colombia, supporting the origin estimated by the phylogeographic analysis.

In 2015, a large CHIKV epidemic occurred in Paraguay, resulting in ≈10,000 cases (suspected and confirmed); the main affected departments were Central, Asunción, and Paraguarí (Figure 2, panel A). All genomes generated from the 4 samples from 2015 were classified as members of the Asian genotype and grouped together with high support (PP = 1) (Figure 3 CHIKV infection in Paraguay is believed to be the housemaid of a family returning from Puerto Rico in October 2014, who sought healthcare services for symptoms of chikungunya fever. CHIKV infections were confirmed for the housemaid and the family, and our phylogenetic analysis confirmed this epidemiologic history.

The time distributions of CHIKV infection cases from 2016 were very similar to those in 2015 (Figure 2, panel A), although they did reveal that at least 2 lineages were circulating in the country (Figure 3, panel A). A new introduction is likely to have occurred in the Paraguarí Department at the mean time point of November 2015 (95% HPD June 2015 to January 2016); the most likely place of origin was Central America (PP = 0.76) (Figure 3, panel A). However, the sequence from Amambay Department, isolated in 2014, is positioned basally to the Paraguarí cluster, suggesting that the same variant persisted in the country up to 2016. Year-round persistence of a CHIKV strain is clearly observed in the Central/ Asunción Department cluster in which the genome We also summarized all geographic locations that had significantly (Bayes factor >3) seeded new CHIKV lineages to Paraguay and superimposed it onto the tMRCA of the 3 main CHIKV clusters detected there (Figure 2, panel B). Most transitions in the Asian genotype occurred in 2014, when CHIKV was spreading rapidly through the Americas. Far fewer transitions were estimated to have occurred in 2015, which accords with the hypothesis that the 2016 outbreak in Paraguarí was caused by a lineage already circulating in the country. For the ECSA genotype of CHIKV, the importations from Brazil were widespread between the middle of 2015 and the beginning of 2019. These widespread importations result from the long branch connecting the Paraguay ECSA cluster to the Brazilian sequences (Figure 3, panel B), increasing uncertainty in the relevant parameter estimates.

Genetic Diversity of DENV in 2018

The number of DENV cases in Paraguay during 2015-2018 shows a very similar pattern to that for CHIKV (Figure 2, panel A; Figure 4,panel A). Case numbers were higher at the beginning of each year, except for 2017 when the DENV season was atypically mild. Our sampling from 2018 captured the 2 DENV serotypes (DENV-1 and DENV-4) circulating in the country (9), and molecular clock analyses estimated that DENV-4 was introduced in Paraguay just before the beginning of the 2018 outbreak, whereas DENV-1 was already circulating in 2017 (Figure 4, panel B).

DENV-1 and DENV-4 sequences from Paraguay clustered together with high support (PP>0.9) and belonged to genotypes V and II, respectively, which predominate in Latin America (Figure 5). The most likely origin of the DENV-1 strain circulating in Paraguay in 2018 was estimated to be Brazil (PP = 0.75) (Figure 5

Discussion

The first CHIKV outbreaks in the Americas (the Asian genotype) were reported for the French Caribbean islands of Saint Martin and Martinique in December 2013 [START_REF] Leparc-Goffart | Chikungunya in the Americas[END_REF]. The virus rapidly spread throughout the Caribbean and Central America in 2014, and autochthonous transmissions were reported in almost all countries/territories of these regions. In 2014, Paraguay reported imported cases only, mostly in persons with a history of travel to Central America or the Caribbean. Our analysis of virus sequences from 2 of these persons with imported cases, and the virus phylogenetic relatedness to foreign viruses, matched the travel history with precision. In addition, the estimated origin of the first outbreak of CHIKV in Paraguay in 2015 agreed both in time (October 2015) and location (Puerto Rico) with the travel data collected by the Paraguay surveillance system.

The CHIKV epidemic in Paraguay in 2016 was very similar to that in 2015, when the most affected departments were Central, Asunción, and Paraguarí. These neighboring departments are located in the most densely populated region of Paraguay, which might lead to consecutive outbreaks. In our sampling, most sequences from 2016 were from the Paraguarí Department and formed a separate clade In Brazil, 2 distinct lineages of CHIKV were detected at the end of 2014, the ECSA genotype in the Northeast Region and the Asian genotype in the North Region [START_REF] Nunes | Emergence and potential for spread of chikungunya virus in Brazil[END_REF]. Whereas the CHIKV outbreak of the Asian genotype remained restricted to a small number of cases, the ECSA lineage spread throughout Brazil. In this study, we determined that the ECSA genotype was the causative agent of a CHIKV outbreak in Amambay (a Paraguay department on the Brazil border) in 2018. To our knowledge, these are the only 2 countries in the Americas to report outbreaks of both the Asian and ECSA genotypes to date. Our analysis revealed a mean time of entry into Paraguay of around December 2017, most likely from the Northeast Region in Brazil. However, because of the small number of samples from states in Brazil that border Paraguay, all such inferences of geographic origins should be interpreted with caution. It is possible that previous exposure to the CHIKV Asian genotype may have created some population immunity that restricted ECSA circulation in other parts of Paraguay (e.g., Central and Asunción Departments) in 2018. Unfortunately, no data on CHIKV seroprevalence in Paraguay are available to test this hypothesis. Amambay, on the other hand, reported few CHIKV cases during the epidemics of 2015 and 2016 (Figure 2, panel A), potentially enabling the emergence of the ECSA genotype in 2018.

The 2018 dengue season in Paraguay was dominated by DENV-1, reported in all departments (9). Three departments (Central, Alto Paraná, and Guaira) also reported the circulation of DENV-4. Our analysis suggests that the origin of both serotypes in Paraguay is Brazil, supporting findings of previous studies [START_REF] De Bruycker-Nogueira | DENV-1 genotype V in Brazil: spatiotemporal dispersion pattern reveals continuous co-circulation of distinct lineages until 2016[END_REF]. Although our sampling was restricted to 2018, we observed that the tMRCA of DENV-1 was much earlier (October 2016), suggesting that this lineage may have persisted during the 2017 and 2018 seasons, when it may have been responsible for most cases (9). DENV-4, on the other hand, was introduced in September 2017, just before the start of the dengue season, and was responsible for few infections until the 2019-20 season, when it dominated the epidemic (32, 33).

Despite screening all publicly available (Gen-Bank) sequences of DENV from the Americas, we found that the DENV datasets were sparsely distributed in time (DENV-4) or contained large temporal gaps (DENV-1) (Appendix 1 Figure 2), potentially biasing our results. The paucity of available DENV complete genomes in South America constrains the applicability of phylogenetic tools for studying virus population dynamics. It also highlights the value of intensifying sequencing efforts in line with the genomic surveillance approach and for real-time generating and sharing of data. The CHIKV datasets were much more comprehensive; for instance, the Asian lineage dataset analyzed included 291 genomes sampled during 2014-2018, representing 38 countries/territories (Appendix 2). The more comprehensive CHIKV datasets reflect the fact that CHIKV emerged in the Americas in the era of next-generation sequencing, when the development of numerous platforms reduced the cost and shortened the time from sample preparation to data generation. Hence, this increased availability of CHIKV virus genomes allowed a more detailed analysis of the virus's spatiotemporal history in Paraguay. However, another study limitation is the convenience sampling used, with a narrow breadth, potentially biasing molecular clock dating and location ancestral reconstruction. The limited availability of stored samples from years before 2018, and their possibly limited RNA integrity, impaired genome sequencing from previous outbreaks. Nevertheless, we suggest that the 33 genomes generated here are representative of the current DENV diversity and the recent CHIKV evolutionary history in Paraguay.

Of note, the mean tMRCAs for the 3 clusters of CHIKV and the 2 clusters of DENV were estimated in the last trimester of the year (September-December), the start of arbovirus season in many tropical and subtropical regions in the Southern Hemisphere (e.g., Paraguay). A previous study [START_REF] Obolski | MVSE: an R-package that estimates a climatedriven mosquito-borne viral suitability index[END_REF] has modeled the timing and scale of arbovirus transmission potential and found that in many cities in Brazil with climates similar to that of Paraguay, transmission starts to increase around September. Thus, the tMRCAs estimated here probably reflect the onset of the arbovirus season, which peaks during January-March. Oddly, the 2017 season was marked by a noticeable reduction in cases of both DENV and CHIKV in Paraguay (Figure 2, panel A; Figure 5, panel A). This pattern was observed for dengue throughout the Americas, where cases decreased by 73% in 2017 compared with 2016 [START_REF] Perez | The decline of dengue in the Americas in 2017: discussion of multiple hypotheses[END_REF]. It is possible that the mild season in 2017 might be explained by a transient strengthening of vector control interventions, implemented after the arrival of CHIKV and ZIKV in the Americas and the consequent public health emergency triggered by these pathogens. In addition, cross-immunity between ZIKV and DENV has been observed in the laboratory [START_REF] Priyamvada | Human antibody responses after dengue virus infection are highly cross-reactive to Zika virus[END_REF]. Hence, population immunity to ZIKV after the 2015-2016 epidemic may have provided some transitory protection against DENV, resulting in lower incidence in 2017 [START_REF] Pérez-Guzmán | Time elapsed between Zika[END_REF]38), although this protection alone would not explain the decreased CHIKV cases in 2017 in Paraguay.

In conclusion, our study reveals a complex pattern of arbovirus circulation in Paraguay. We identify Brazil as a source of CHIKV and DENV lineages and show that other countries from South America and the Caribbean, mainly tourist destinations, were also hubs of virus spread toward Paraguay. Our sequencing and phylogenetic analyses proved to be powerful tools for revealing the transmission dynamics between the sampled locations and matched, with striking precision, available patient travel history. With support from the Pan American Health Organization, this project developed capacity-building skills in Paraguay, which can be applied in future arbovirus outbreaks.

  , panel A) in a clade for which time to MRCA (tMRCA) was October 2014 (95% highest posterior density [HPD] May 2014 to November 2014) (Figure 2, panel B). From our analysis, we estimated that the geographic origin of the variant circulating in Paraguay in 2015 was Puerto Rico (PP = 0.98) (Figure 3, panel A). The first patient with autochthonous

  isolated in 2016 clustered with the genomes isolated in 2015 (Figure 3, panel A). Besides the Asian genotype of CHIKV, an outbreak of the ECSA genotype occurred in Paraguay in 2018. After a year of very few CHIKV infections in 2017 (Figure 2, panel A), a new outbreak was observed in 2018 (although milder than that of 2015 and 2016), and the main affected department was Amambay. Five genomes from this department revealed circulation of the CHIKV ECSA genotype in 2018, and the mean tMRCA of this cluster was December 2017 (95% HPD July 2017 to April 2018) (Figure 2, panel B). The source of introduction of this new CHIKV lineage in Paraguay was estimated to be Brazil, most likely the Northeast Region (PP = 0.64), or perhaps the North Region (PP = 0.32) (Figure 3, panel B).

Figure 1 .

 1 Figure 1. Geopolitical map of Paraguay showing locations of sampling for dengue virus (DENV) and chikungunya virus (CHIKV). Circle sizes are scaled to represent the number of genomes isolated in each municipality. Numbers inside triangles indicate sampled departments: 1, Amambay; 2, San Pedro; 3, Alto Paraná; 4, Itapúa; 5, Guairá; 6, Paraguarí; 7, Central; 8, Asunción. Callout map shows the Central and Asunción Departments of Paraguay; inset map shows the location of Paraguay in South America.

  , panel A), and the mean tMRCA was estimated to be October 2016 (95% HPD February 2016 to May 2017) (Figure 4, panel B). DENV-4 was also estimated to have an origin in Brazil; the best-supported regions of origin were Central-West (PP = 0.64) and North (PP = 0.34) (Figure 5, panel B). The mean tMRCA of DENV-4 was September 2017 (95% HPD April 2017 to February 2018), ≈1 year later than DENV-1. Examining the DENV-4 cluster in Paraguay in more detail revealed that sequences from the Alto Paraná Department are basal and that sequences from the Central and Guairá Departments group together in a highly supported (PP>0.9) and distal cluster (Figure 5, panel B). Alto Paraná borders Brazil, and although not formally tested because of the small number of sequences, Alto Paraná could be the point of introduction of the current DENV-4 lineage into Paraguay.

Figure 2 .

 2 Figure 2. Chikungunya virus (CHIKV) outbreaks in Paraguay during 2014-2018 and the spatial-temporal history of virus diffusion. A) Total number of cases of CHIKV infection reported by epidemiologic week in the departments from which genome sequences were available. B) Location transitions to Paraguay inferred from the posterior distribution of phylogenetic trees by the Markov jump approach, and the time to most recent common ancestor for the CHIKV clusters detected in the country. Lines are colored according to the origin of the estimated transition toward Paraguay. Violin plots show 95% CIs with internal boxplots showing median and interquartile ranges. Brazil-N, Brazil North Region; Brazil-NE, Brazil Northeast Region; ECSA, East/Central/South African genotype.

Figure 3 .

 3 Figure 3. Time-scaled phylogenetic trees of chikungunya virus (CHIKV) genomes isolated in the Americas. A) CHIKV Asian genotype; B) CHIKV East/Central/South African genotype from Brazil. Tips and internal branches are colored according to the most likely geographic location, and ancestral states were estimated by phylogeographic methods. Clusters relevant to the epidemic in Paraguay are shown in detail where the most likely ancestral state estimation is annotated. Asterisks indicate highly supported clusters (posterior probability >0.9).

  from the 2015 epidemic, suggesting a new introduction event. However, because of the co-clustering of sequences from the Asunción and Central Departments isolated in 2015 and 2016, we cannot exclude year-round persistence of the virus. Models of Aedes spp. mosquito competence for DENV transmission have shown that the Paraguay climate is conducive to year-round persistence (29).

Figure 4 .

 4 Figure 4. Dengue virus (DENV) outbreaks in Paraguay during 2015-2018 and tMCRA of serotypes 1 and 4. A) Total cases of DENV infections reported by epidemiologic week in the departments from which genome sequences were available. The black bars in 2018 delimit the sampling time range for the DENV genomes. B) tMRCA for DENV-1 and DENV-4 in the same timescale as the number of cases reported. Violin plots show 95% CIs; internal boxplots show medians and interquartile ranges. tMCRA, time to most recent common ancestor.

Figure 5 .

 5 Figure 5. Time-scaled phylogenetic trees of dengue virus (DENV) serotypes 1 and 4 genomes isolated in Paraguay and related sequences. A) DENV-1; B) DENV-4. Tips and internal branches are colored according to geographic location, and ancestral states were estimated by phylogeographic methods. Clusters relevant to the epidemic in Paraguay are shown in detail where the most likely ancestral state estimation is annotated. Asterisks indicate highly supported clusters (posterior probability >0.9).

Table .

 . Patient demographic and virus sequencing data for samples from the Laboratorio Central de Salud Publica de Asunción, Paraguay, 2014-2018*

					Patient age,			Genome
	Sample	Virus	Department, municipality	Collection date	y/sex	Ct	Reads	coverage, %
	PY02	CHIKV-Asian	Paraguarí, Yaguarón	2016 Jan 20	40/M	23.6	204,763	88.1
		genotype
	PY03	CHIKV-Asian	Paraguarí, Yaguarón	2016 Jan 21	67/M	16.9	215,137	87.0
		genotype
	PY06	CHIKV-Asian	Paraguarí, Yaguarón	2016 Feb 16	9/M	26.9	282,182	88.3
		genotype
	PY07	CHIKV-Asian	Paraguarí, Yaguarón	2016 Fab 19	34/F	29.7	267,784	87.2
		genotype
	PY08	CHIKV-Asian	Paraguarí, Yaguarón	2016 Feb 22	39/F	30.2	142,555	87.1
		genotype
	PY09	CHIKV-Asian	Asunción, Asunción	2016 Mar 21	27/F	28	265,596	87.9
		genotype
	PY12	CHIKV-ECSA/BR Amambay, Pedro Juan Caballero	2018	21/M	29	236,285	84.8
	PY13	CHIKV-ECSA/BR Amambay, Pedro Juan Caballero	2018 Jun 25	35/F	28	175,112	85.3
	PY15	CHIKV-ECSA/BR	Amambay, Bella Vista Norte	2018 Jun 29	40/F	34	17,030	70.9
	PY17	CHIKV-ECSA/BR	Amambay, Bella Vista Norte	2018 Jul 3	22/M	34	320,142	86.8
	PY18	CHIKV-ECSA/BR	Amambay, Bella Vista Norte	2018 Jul 10	57/M	29	315,588	86.0
	PY19	DENV-4	Guairá, Villarrica	2018 Apr 23	38/F	23	22,041	82.3
	PY21	DENV-4	Guairá, Villarrica	2018 Apr 26	68/F	22	21,042	96.0
	PY22	DENV-4	Guairá, Villarrica	2018 Apr 27	52/M	22	13,213	96.0
	PY23	DENV-4	Central, San Lorenzo	2018 May 6	19/M	27	11,548	74.6
	PY24	DENV-1	San Pedro, San Pedro De	2018 May 4	29/F	20	7,265	89.1
			Ycuamandyju
	PY25	DENV-4	Central, San Lorenzo	2018 May 5	38/F	21	17,299	96.0
	PY27	DENV-4	Alto Paraná, Domingo Martinez	2018 May 9	27/F	19	21,188	96.0
			De Irala
	PY28	DENV-4	Alto Paraná, Hernandarias	2018 May	30/M	21	22,800	86.6
	PY31	DENV-4	Alto Paraná, Hernandarias	2018 May 22	14/M	32	8,770	95.9
	PY32	DENV-4	Central, San Lorenzo	2018 May 31	28/M	28	6,907	96.0
	PY33	DENV-1	Asunción, Asunción	2018 May 31	3/F	22	9,846	76.8
	PY34	DENV-4	Alto Paraná, Juan Leon	2018 May 28	47/F	26	7,945	96.0
			Mallorquin
	PY35	DENV-1	Itapúa, Encarnacion	2018 Jun 7	62/F	23	119,293	89.1
	PY36	DENV-1	Itapúa, Encarnacion	2018 Jun 8	61/M	23	6,448	76.8
	PY38	DENV-1	Itapúa, Cambyreta	2018 Jun 8	6/F	25	111,057	89.1
	PY43	DENV-1	Guairá, Villarrica	2018 Jun 4	53/F	25	8,779	89.1
	PY44	CHIKV-Asian	Central, Luque	2014 Nov 30	33/F	24	13,687	85.1
		genotype
	PY45	CHIKV-Asian	Amambay, Pedro Juan Caballero	2014 Nov 3	54/M	24	12,214	86.6
		genotype
	PY47	CHIKV-Asian	Central, Guarambare	2015 Apr 26	25/M	17	9,536	85.2
		genotype
	PY48	CHIKV-Asian	Central, Fernando De La Mora	2015 Apr 28	50/F	18	6,002	86.6
		genotype
	PY49	CHIKV-Asian	Central, Fernando De La Mora	2015 May 3	12/F	27	53,928	86.4
		genotype
	PY50	CHIKV-Asian	Central, Fernando De La Mora	2015 May 6	25/F	16	49,813	86.8
		genotype

*CHIKV, chikungunya virus; Ct, cycle threshold; DENV, dengue virus; ECSA/BR, East/Central/South African genotype from Brazil. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 27, No. 5, May 2021 1397 History of Chikungunya and Dengue, Paraguay

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 27, No. 5, May 2021

This work was supported by Decit/SCTIE/MoH and CNPq (440685/2016-8 and 440856/2016-7); by CAPES (88887.130716/2016-00, 88881.130825/2016-00, and 88887.130823/2016-00), and by the EU Horizon 2020 Programme through ZIKAlliance (PRES-005-FEX-17-4-2-33). M.G. is supported by the Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro-FAPERJ. E.C.H. is supported by an Australian Research Council Australian Laureate Fellowship (FL170100022). A.M.B.F. was supported by International Development Research Centre, Canada (grant 108411-001) and by Horizon 2020 through ZikaPlan and ZikAction (grant agreement nos. 734584 and 734857).

About the Author

Dr. Gräf is a public health researcher at the Gonçalo Moniz Institute of the Oswaldo Cruz Foundation, Salvador, Brazil. His research interests focus on molecular epidemiology and the application of phylogenetics to study viral evolution and epidemic spread.


Paul Bourbon

Emeline Appert

Agnes Martin-Mingot

Bastien Michelet

Sébastien Thibaudeau

Complementary Site-Selective Sulfonylation of Aromatic Amines by Superacid Activation

Under superacidic conditions, aniline and indole derivatives are sulfonylated at low temperature with easy-to-access arenesulfonic acids or arenesulfonyl hydrazides. By modification of the functional-group directing effect through protonation, this method allows nonclassical site functionalization by overcoming the innate regioselectivity of electrophilic aromatic substitution. This superacid-mediated sulfonylation of arenes is complementary to existing methods and can be applied, through protection by protonation, to the late-stage site-selective functionalization of natural alkaloids and active pharmaceutical ingredients.

A romatic sulfones are important scaffolds in organic synthesis and have found many applications in the medicinal, agrochemical, and materials fields (Scheme 1). 1

Over the past decades, numerous methods have been developed for their synthesis. 1,2 The most classical approaches rely on oxidation of the corresponding sulfides or sulfoxides, addition of sulfinates to unsaturated bonds, or metal-catalyzed cross-coupling reactions from sulfinates or sulfonyl halides. 1 Although these methods proved their efficiency to generate a wide variety of aromatic sulfones, they are still strongly limited by the necessity of prefunctionalizing the aromatic starting material. In this context, great efforts have recently been made to develop new methods for direct C(sp 2 )-H sulfonylation, 2a-c mainly through metal-catalyzed C-H activation (Scheme 2a) 3 or oxidative C-H functionalization of quinolines, 4 anilines, 5 indoles, 6 or phenol derivatives. 7 However, these strategies necessitate the preinstallation of a directing group and/or are limited to specific substrates, narrowing their

scope of application. Moreover, although it is highly sitespecific, directed C-H sulfonylation of arenes occurs mostly at the position ortho to the directing group. 2c,8 On the other hand, one of the simplest and oldest strategies to substitute an aromatic hydrogen atom by a sulfonyl group is probably aromatic electrophilic substitution, akin to Friedel-Crafts acylation (Scheme 2b). [START_REF] Olah | Friedel-Crafts Sulfonylation of Benzene and Toluene with Alkyl-and Arylsulfonyl Halides and Anhydrides[END_REF] This strategy benefits from ease of setup, a large variety of accessible catalysts, and a wide range of commercially available reagents. Nevertheless, in most cases Friedel-Crafts-type sulfonylation is applicable only to lowweight benzene derivatives and suffers from poor regioselectivity dictated by the innate orientation effect of the substituents.

We previously demonstrated 10 that under superacid conditions, [START_REF] Olah | Superacid Chemistry[END_REF] aromatic amines (or amides)despite being fully protonatedcan react with an electrophile provided that the latter is further activated by the superacidic medium (superelectrophilic activation). [START_REF] Liu | Synthesis of Sulfones via Selective C-H-functionalization[END_REF] In particular, under these conditions the orientation effect of the amine substituent is no longer effective, and the site selectivity of the reaction is dictated by the secondary substituent, leading to nonclassical regioselectivity. Motivated by these previous results and by the absence of general procedures for direct electrophilic sulfonylation of aromatic amines, [START_REF] Olah | Friedel-Crafts Sulfonylation of Benzene and Toluene with Alkyl-and Arylsulfonyl Halides and Anhydrides[END_REF] we aimed at developing a complementary site-selective method that would allow the direct functionalization of aniline and indole derivatives from readily available sulfone precursors by taking advantage of superelectrophilic activation (Scheme 2c).

We started our investigations with p-methylacetanilide as model substrate and benzenesulfonic acid A a as a cheap and readily available sulfonyl source (Table 1). With neat HF/SbF 5 (7/1) superacid, the desired product 1a was obtained after 2 h at -20 °C in an encouraging 30% yield (Table 1, entry 1). Delightfully, the substrate was selectively functionalized at the meta position (with respect to the acetamido group), confirming our initial hypothesis. The peculiar regioselectivity of this transformation was also confirmed by X-ray analysis of collected crystals of 1a (see the Supporting Information (SI)). We next evaluated the impact of the acidity on the reactivity by modulating the concentration of SbF 5 (Table 1, entries 1-4) and found that the optimal conditions were reached with a 2/1 HF/SbF 5 mixture (73% yield; Table 1, entry 3). Increasing the amount of benzenesulfonic acid to 2 equiv did not improve the yield (Table 1, entry 5). While decreasing the temperature to -40 °C completely inhibited the reaction (Table 1, entry 6), performing the reaction at higher temperature led to a poor yield and to the formation of the undesired regioisomer 1a′ (Table 1,entry 7). The formation of 1a′ can be explained by an initial intramolecular rearrangement within the arenium ion generated by the protonation of p-methylacetanilide to the more stable m-methylacetanilide, which then reacts with benzenesulfonic acid. [START_REF] Olah | m-Methylacetanilide was observed in some cases in the crude reaction mixture but was never isolated. Such isomerization is a known drawback of Friedel-Crafts alkylations[END_REF][START_REF] Michelet | XXXX, XXX, XXX-XXX E Pure Benzosultams by Superelectrophilic Activation[END_REF] No reaction occurred using the weaker superacid TfOH (Table 1, entry 8), a result that supports our initial hypothesis and the necessity to use superelectrophilic activation to perform this transformation. As expected, the use of excess of Lewis acids in dichloromethane did not afford the desired product even at room temperature with a prolonged reaction time (Table 1, entries 9-14). We next evaluated the reactivity of p-methylacetanilide with other sulfonyl precursors (Table 1, entries 15-23). Methyl sulfonate B a was found to be totally unreactive under these conditions (Table 1, entry 15). Although good conversion of the starting material was obtained with sulfonyl chloride C a , 1a was obtained in a low 10% yield accompanied by the formation of 3-chloro-4-methylacetanilide in 50% yield (Table 1,entry 16). In this case, reagent C is a source of chloride ions, which can be oxidized in situ by Sb V . The generated halenium ions then react with the aromatic nucleophile to furnish the chlorinated product, as previously observed. 10b In order to avoid this competitive process, sulfonyl fluoride D a was evaluated as the reaction partner (Table 1, entry 17). 15 However, the reaction was very slow in this case, and 1a was isolated in only 12% yield. We next turned our attention to sulfonyl reagents derived from sulfonamide. While N,N-dimethylsulfonamide E a and N-acetylsulfonamide F a were found to be unreactive (Table 1, entries 18 and 19), using the symmetrical sulfonimide G a afforded 1a in a good 68% yield (Table 1, entry 20). On the other hand, only traces of 1a were obtained when the reaction was performed with imidazole derivative H a (Table 1, entry 21). Sulfonyl azide I a and sulfonyl hydrazide J a were revealed to be also very efficient in this transformation, broadening the panel of reagents with the ability to sulfonate aromatic amines in superacid. Under these conditions, 1a was obtained in 86% and 72% yield from I a and J a , respectively (Table 1, entries 22 and 23). With these optimized conditions in hand, we next moved to the study of the scope and limitations of this transformation, starting with the screening of various arenesulfonyl precursors (Scheme 3). At this stage, we decided to evaluate the reactivity of both sulfonic acids and sulfonyl hydrazides. We chose to discard G-type sulfonimides (as they would be less accessible once functionalized) and sulfonyl azides (for safety reasons). It appeared that sulfonic acids, despite their apparent simplicity, are not ideal reagents: they are surprisingly less commercially available than the sulfonyl chloride analogues (from which they were prepared in some cases), sometimes difficult to synthesize, and most of all highly hygroscopic, leading to poorly reproducible results. On the contrary, sulfonyl hydrazides 2d are easy-to-handle, are readily prepared from the corresponding sulfonyl chlorides and hydrazine hydrate, and gave better results than their sulfonic acid counterparts in most of the cases. Thus, diaryl sulfones 1a-d were synthesized from alkyl-substituted arenesulfonyl precursors in good yields with, again, full meta selectivity. The reaction proceeded equally well with an electron-rich methoxy-substituted arenesulfonyl hydrazide, affording compound 1e in 61% yield. Naphthalene derivatives 1f and 1g could also be generated, although in low yields due to the extensive formation of degradation byproducts. Halo-substituted arenesulfonyl hydrazides were also compatible with these conditions, affording fluoro-, chloro-, bromo-, and iodo-substituted diaryl sulfones 1h-k. This method also afforded diaryl sulfone 1l from an electron-poor trifluoromethyl-substituted arenesulfonyl hydrazide. However, strongly deactivated arenesulfonyl precursors, such as nosyl or 3-pyridinesulfonyl derivatives, and alkanesulfonyl reagents were found to be unreactive under these conditions.

We next evaluated the scope of the superacid-mediated sulfonylation on various aromatic amines (Scheme 4). With pfluorobenzenesulfonyl hydrazide J h , unsubstituted acetanilide reacted as expected at the para position, affording sulfone 2 in 65% yield. The meta-sulfonylation was again observed starting from 2-chloroacetanilide to afford compound 3, the chloro substituent orientating the substitution in this case despite its innate deactivating nature. The reaction could also be performed on unprotected 4-methylaniline to afford product 4, albeit with moderate efficiency. On the other hand, the sulfonylation was found very efficient on unprotected indoline, tetrahydroquinoline, and tetrahydrocarbazole, affording the corresponding compounds 5-7 in very good yields. In all three cases, the reaction occurred meta to the nitrogen atom and para to the alkyl chain. The unnatural regioselectivity of this reaction is even more striking with 2-methylindole as the starting material. Indoles are known to be excellent nucleophiles, usually reacting at C3. [START_REF] Bandini | Catalytic Functionalization of Indoles in a New Dimension[END_REF] In our case, the C6sulfonylated compound 8 was exclusively obtained and represents, to the best of our knowledge, the first example of non-directed C-H sulfonylation at C6 from a C3-unsubstituted indole. 3b This unusual selectivity for an electrophilic substitution on indole derivatives can be attributed to the formation of an iminium ion after protonation of the pyrrole ring in superacid. Oxindoles tend to react at C5, as exemplified by the preparation of sulfones 9 and 10 in good to very good yields. This superacid-mediated sulfonylation could also be applied to the late-stage functionalization of more complex and valuable substrates. The local anesthetic lidocaine was efficiently sulfonylated at C3 to furnish compound 11. The vinca alkaloid vinburnine was functionalized at C6 of its indole core to give 12 in good yield. Protected phenylalanine was also found to be compatible with our conditions and was transformed into optically pure sulfone 13 in very good yield. [START_REF]As far as we are aware[END_REF] To gain better insight into the mechanism of this transformation and explore the transient formation of an activated sulfonyl intermediate, we next decided to evaluate the behavior of benzenesulfonic acid A a in HF/SbF 5 by lowtemperature in situ NMR analysis (see the SI). The 1 H NMR spectrum of this mixture displayed two sets of signals corresponding to two different species in a 4.6 to 1 ratio. Interestingly, in the [START_REF] Dhakal | Trifluoromethanesulfonate Anion as Nucleophile in Organic Chemistry[END_REF] F NMR spectrum, a new signal appeared at 58.0 ppm that was attributed to the formation of a sulfonyl fluoride derivative, as previously observed by Olah under similar conditions. 18 This was confirmed by the NMR analysis of a solution of benzenesulfonyl fluoride D a in HF/SbF 5 , which provided the same set of 1 H NMR signals as for the major species observed from benzenesulfonic acid A a . The formation of the sulfonyl fluoride was also confirmed by [START_REF] Dhakal | Trifluoromethanesulfonate Anion as Nucleophile in Organic Chemistry[END_REF] F and 13 C NMR analyses. We also submitted reagent A a to the softer superacid TfOH and obtained a very clean 1 H NMR spectrum displaying the same signals as for the minor species observed in HF/SbF 5 . More importantly, no evidence for the eventual addition of triflate to benzenesulfonic acid was obtained, despite the stronger nucleophilicity of triflate anion compared with fluoroantimonates. [START_REF] Dhakal | Trifluoromethanesulfonate Anion as Nucleophile in Organic Chemistry[END_REF] This observation suggests that under HF/SbF 5 conditions, benzenesulfonic acid is transformed into a highly activated electrophilic species. Although this species could not be directly observed because of its rapid transformation into sulfonyl fluoride, we can postulate the formation of a benzenesulfonylium ion, as has been previously suggested. 18 In the presence of an aromatic nucleophile, the transient generation of this key intermediate would trigger the electrophilic aromatic substitution (Scheme 5, path a), while in the absence of a sufficiently nucleophilic partner, the latter sulfonylium ion would collapse to the more stable sulfonyl fluoride, [START_REF]The arenesulfonyl fluoride is most likely in a protonated form in HF/SbF 5 , as suggested by the in situ 13 C and 19 F NMR analyses (see the SI). (21) Benzenesulfonyl fluoride is protonated on the oxygen atom in FSO 3 H/SbF 5 /SO 2 ClF superacidic solution and remains stable up to room temperature[END_REF]21 which would react very slowly because of its very high stability in superacidic media 21 (Scheme 5, path b).

To confirm the generality of this mechanistic hypothesis, we evaluated the reactivity of the benzenesulfonyl sources listed in Table 1 by in situ NMR analysis in HF/SbF 5 without nucleophilic partners (see the SI). As expected, the effective reagents C a , G a , I a , and J a were almost instantaneously transformed into sulfonyl fluoride similarly to A a , while the ineffective sulfonyl derivatives remained stable or were transformed very slowly. Likewise, the low reactivity of alkanesulfonic acids under these conditions could be explained by the very high stability of their protonated forms in superacid. 18 To conclude, taking advantage of the superelectrophilic activation of simple arenesulfonic acids and hydrazides, we have developed a direct and regioselective C-H sulfonylation of arenes at low temperature. These conditions allow the electrophilic sulfonylation of functionalized arenes with excellent regioselectivity. Furthermore, aniline and indole derivatives are functionalized with nonclassical regioselectivity due to deactivation of their nitrogen substituent by protonation. In a context where new approaches are required to address unmet selectivity challenges in the field of late-stage C-H functionalization, [START_REF] Börgel | Late-Stage Functionalization[END_REF] this new method provides access to diaryl sulfones with site selectivity complementary to that of existing methods.

■ ASSOCIATED CONTENT

* sı Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.orglett.1c00994.

General experimental procedures, reaction setup, characterization data, low-temperature NMR spectra, and spectra for all key compounds (PDF)

Accession Codes CCDC 2081182 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, U.K.; fax: +44 1223 336033.

■ AUTHOR INFORMATION Corresponding Authors

Sébastien Thibaudeau -Université de Poitiers, UMR-CNRS 7285, IC2MP, Superacid Lab -Organic Synthesis Team, 86073 Poitiers, France; orcid.org/0000-0002-6246-5829; Email: sebastien.thibaudeau@univ-poitiers.fr Bastien Michelet -Université de Poitiers, UMR-CNRS 7285, IC2MP, Superacid Lab -Organic Synthesis Team, 86073 Poitiers, France; Email: bastien.michelet@univ-poitiers.fr

Scheme 3 .a

 3 Scheme 3. Scope and Limitations of the Superacid-Promoted Sulfonylation of p-Methylacetanilide Using Various Sulfonylation Reagents

Table 1 .

 1 Optimization of the Superacid-Promoted Benzenesulfonylation of p-Methylacetanilide

	Yield [%] b

a Used as the solvent unless stated otherwise. b Yields of isolated products. c 58% yield after 1 h.

d

The reaction time was extended to 20 h. e 2 equiv in CH 2 Cl 2 . f Concomitant formation of 3-chloro-4methylacetanilide in 50% yield.

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Authors

Paul Bourbon -Université de Poitiers, UMR-CNRS 7285, IC2MP, Superacid Lab -Organic Synthesis Team, 86073 Poitiers, France Emeline Appert -Université de Poitiers, UMR-CNRS 7285, IC2MP, Superacid Lab -Organic Synthesis Team, 86073 Poitiers, France; @rtMolecule, 86000 Poitiers, France Agnes Martin-Mingot -Université de Poitiers, UMR-CNRS 7285, IC2MP, Superacid Lab -Organic Synthesis Team, 86073 Poitiers, France

Complete contact information is available at: https://pubs.acs.org/10.1021/acs.orglett.1c00994

Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

We gratefully acknowledge the Région Nouvelle-Aquitaine (Ph.D. grant to P.B.), ANRT and the @rtMolecule Society (Ph.D. grant to E.A. and financial support through the ISOTOP Project). We also acknowledge the University of Poitiers, the Centre National de la Recherche Scientifique


Emanuela Berrino

Bastien Michelet

Agnès Martin-Mingot

Fabrizio Carta

Claudiu T Supuran

Sébastien Thibaudeau

Modulating the Efficacy of Carbonic Anhydrase Inhibitors through Fluorine Substitution

Introduction

Enhancement of therapeutic efficacy and improvement of pharmacological properties of a drug can be tuned through fluorine atom(s) insertion. [START_REF] Bégué | Chimie Bioorganique et Médicinale Du Fluor[END_REF] This strategy is now well-established, [START_REF] Wang | [END_REF] as revealed by the increasing number of fluorine-containing compounds currently in phase II-III clinical trials. [3] The insertion of fluorine atom(s) into a scaffold can affect its physico-chemical properties, [4] with evident consequences on the drug biological features. [5] Overall these benefits make the fluorine as "the second-favorite heteroatom" after nitrogen in drug design. [6] Fluorine is commonly used to tune amine groups basicity which in turn affects biomembrane penetration and bioavailability of drugs or even to protect them from metabolic transformations. [7,8] State of-the-art application also accounts for the use of fluorine to induce preferential molecular conformations as a relevant strategy for molecular design in medicinal chemistry. [8][9][10][11][12][13][14][15][16] Here we set the research topic by scrutinizing the most relevant studies referred to fluorinated inhibitors of the metalloenzymes Carbonic Anhydrases (CAs; EC 4.2.1.1) and deciphering the effect that fluorine insertion could induce. Recent reports on 19 F NMR based approaches to decipher ligand-enzyme interactions within CAs are also discussed.

CAs are virtually expressed in every living organism being encoded by 8 genetically unrelated families (i.e.

and-).

The class is expressed in humans, up to 15 isoforms with diverse cellular and tissue distribution have been identified so far. These enzymes are highly efficient in catalyzing the equilibrium in Eq. 1, essential in sustaining the cellular metabolic pathways, [17][18][19][20] through a well-defined catalytic mechanism which actively involves a Zn (II) ion. [17,18,21] (Eq. 1)

As peculiar structural feature, CAs have their active site split into a hydrophilic and a hydrophobic halves facing each other (Figure 1). Such an architecture contributes itself to the enzymatic efficiency by creating preferential ways for the substrate (CO2) to feed in and for the products (H + , HCO3 -) to be expelled out of the enzyme. [21][22][23] Large series of compounds have been explored and/or developed as CA inhibitors (CAIs), [21,24] and many were proved to exert their activity through 4 distinct mechanisms (Figure 1). [21,[25][26][27][28][29][30][31][32][33] The hydrophobic adjacent pocket where inhibitors bind outside the active site is shown in yellow. [21,34] Using CA II isoform as model enzyme target, this review will focus on metal binding compounds bearing the sulfonamide group, which constitute the wider and the most studied class of inhibitors to date. [31,35] Such a moiety coordinates in the deprotonated form, acting as fourth ligand to the enzymatic metal ion upon displacement of the water/OH -molecule according to slightly distorted tetrahedral geometry. [21] Metal chelation with auxiliary hydrogen bonds represent a highly conserved binding cluster which confers to such a moiety unique CA inhibitory potencies but also indiscriminate activities against the human expressed isoforms (Figure 2). [21] Most of the side effects of the sulfonamide-based CA inhibitors are due to the inhibition of the cytosolic isoform CA II, abundant in many tissues/organs, and involved in numerous physiological functions (i.e. pH homeostasis, secretion of electrolytes, transport of anions). [21,24,36] Without claiming completeness, this study would provide key statements on why and how fluorine atom(s) should be incorporated in this type of CA inhibitors affecting their potency or selectivity towards specific isoforms, with key examples of fluorinated inhibitors targeting the tumor associated isoform CA IX and the central nervous system (CNS) abundantly expressed CA VII selected at this aim. [37,38] Figure 2. Key interactions between a generic sulfonamide within the CA II active site. [21] Manipulations on R section preferentially affect the ligand potency whereas on the tail (T) major relevance for isoform selectivity are observed. [21,39,40]

Fluorinated sulfonamides as CAI

Clear impact of the fluorine on CA II inhibition potency of sulfonamide was first offered by the CF3-SO2NH2 13 when compared to its non-halogenated counterpart 14. In vitro significative IC50 lowering values (5 orders of magnitude) were attributed to the sulfonamide pKa reduction as a consequence of the CF3 electronwithdrawing effect (Figure 3a). [41] ). [42] a) b)

CF3-SO2NH2 13 revealed itself also an adequate model to investigate the influence of the fluorine on the orientation of the ligand within the enzymatic cavity. Structure superposition of 13-CA II and 15-CA II adducts showed the hydrophobic CF3 moiety oriented inwards the active site and engaged in van der Waals interactions with the hydrophobic section of the cleft, thus opposite to the usually observed orientation of the R moiety as for classical aromatic/heteroaromatic sulfonamides (Figure 3b). [42] As a consequence the sulfonamide in 13-CA II was rotated 180° around the sulfur-nitrogen bond with respect to 15-CA II.

Replacement of the CH2 in 16 with a CF2 instead (i.e. as in 17) determined a CA II KI lowering up to 8fold. [43] Again, the inhibition data were supported by different orientation of the ligands within the CA II cavity (Figure 4). [44] Figure 4. CA II in complex with 16 (magenta, PDB 3BL0) and (B) 17 (gray, PDB 2EU3). [44] The thiadiazole moiety in 17 was found pointing towards the hydrophilic cavity section and hold by a -stacking interaction with His94 and a H-bond of the amine with Asn62 by means of a water bridge (not shown) and Asn67. Further stabilization is given by the interaction occurring between the CF2 with Thr200.

The role of fluorine in the binding of 17 is therefore attributable to the withdrawing effect on the sulfonamide moiety, as in agreement with the example before stated, and to the direct interaction of the CF2 moiety with an aminoacidic residue (Figure 4). Detailed analysis of 16 and 17 poses reveals that the primary amine in the latter makes use of hydrogen bonds to lock the ligand into a favorable orientation for the fluorine interaction to occur. (Figure 4). [44] The discussed examples afford direct evidences on the role of fluorine(s) on ligand-CA interactions, but also prove the difficulty to dissect the effective overall contribution as: i) the sulfonamide itself accounts up to the 65% of the free energy binding, and thus it scales down the other interactions (i.e. hydrogen bonds and hydrophobic contacts) to mere "accessories" difficult to predict and/or to calculate; [45] ii) the controversial nature of fluorine and features of the C-F bond need to be properly considered as well as the position of fluorine within the ligand molecule. Below, we present a selection of examples indicative of the effects on ligand binding due to the introduction of fluorine(s) within CAIs of the arylsulfonamide type together with related advantages for biomedical purposes (Figure 5). [4,46] Figure 5. Fluorine insertions on CAIs; R= ring and T=tail.

Fluorine on the "ring"

Considering the simplest benzene sulfonamide inhibitor 15 that could be tested and compared, the impact of the fluorine substitution and its comparison with related halogenated congeners can be considered as a preliminary important study. Single fluorine insertion as in CAI 20 induced far superior free binding energy (∆G°) and affinity (Kd) values for CA II when compared to the unsubstituted 15, its related fluoro isomer 19 or chloro analogue 18 (Figure 6). [47] Particularly relevant are the diverse ∆H° and ∆S° contribution values observed for the ortho-19 and meta-20 fluoro substituted compounds, being the affinity increase for the former mainly related to a significant enthalpic gain, whereas for the latter a large entropic contribution was observed. Evaluation of X-ray crystal structures of 15, 18-20 in adduct with CA II allowed to decipher these thermodynamic features (Figure 6). [47] The interaction of the fluorine in 19 with Thr200 was found responsible for the observed enthalpic gain. Such a contribution was missing in the chloro analogue 18, as the larger halogen forced the molecule to assume different orientation within the enzyme active site (Figure 6). As for the meta-F derivative 20, favorable dispersive interactions between the halogen and the hydrophobic wall of the CA justify the entropic gain and the favorable Kd values observed. [47] These findings were further sustained by kinITC and high-resolution X-ray crystallographic data (Figure 6). [48] ) [47] and kinITC data of 15 and fluorinated compounds 19 and 20 bound to CA II. [48] Kinetic data revealed fluorine in 20 to induce modest dissociation (koff) and large association (kon) values, which are highly influenced by the hydration state of the enzyme cavity [47][48][49][50] and by the hydrophobic features of the ligand, [51,52] respectively. As for the former, a striking example is the introduction ad hoc of fluorine substitutions within the benzothiazole scaffold of 21 for assessment of the ∆H/∆S compensation phenomenon on CA II. [49][50][51] (Figure 7). (compared to H4BTA) where J =G (blue), H (green), and S (red), obtained from ITC measurements at 298.15 K. To account for differences in the pKa of each ligand, the measured thermodynamic parameters were corrected to represent the binding of the sulfonamide anion (Ar-SO2NH -) to hCA (hCA-ZnII-OH 2+ ), allowing to compare the thermodynamic binding parameters of different ligands in a scheme that is independent of their pKa values (pKa -corrected thermodynamic results). The gray region demarcates the 95% confidence interval (i.e., two standard deviations) of ΔΔG° bind for H4BTA 21; D) WaterMap calculations for ΔG° bind. [49] As expected, the binding geometry as well as the free binding energy for 21-26 and 28, 30 were found relatively unchanged and were all ascribed to the compensation effect of the thermodynamic features from the ligands surrounding waters (Figure 7C,D). The higher CA II binding affinity of 27 was justified with a remarkable entropic contribution due to the ligand desolvation effect. Overall, such an approach is by far the best tool for koff determining parameters as it allows to alter considerably the ligand dipole moment without affecting its size, and this in turn heavily affects its interactions with surrounding residues, including waters. [49] As for the kon regulating features, combination of experimental and in silico techniques (i.e. SPR, X-ray crystallography, metadynamics, DFT calculations) gave evidence for a "pre-binding" stage which benefits from favourable interactions established between the apolar portions of the ligand with the CA II hydrophobic wall section. [51,52] Such a mechanism also applies to the primary sulfonamide which is being deprotonated only when the distance between its terminal NH2 and the enzymatic zinc (II) ion is in the 2.50-2.75 Å range.

Thus skilful introduction of fluorine(s) within benzene sulfonamide CAIs represents unique means to properly tune kon/off ligand parameters towards CA isoforms worth of biomedical consideration. [48,52,53] A remarkable application of this strategy was conducted by Ilies et al on a series of mono/di-substituted sulfanilamide derivatives 30-40 kinetically profiled on both CA II and IX isoforms. (Figure 8). [54] Figure 8. Mono/di-halogenated sulfonamides 30-40 and KI values against CA II and IX. [54] As previously observed for compound 20, the insertion in 30 of a fluorine in meta position significantly showed slightly enhanced differences among each other when compared to their monosubstituted precursors 32-34. [54] Such an outcome can be explained invoking the lower cost for desolvation upon binding of fluorinated ligands when compared to either hydrogen or differently halogenated compounds, more prone to establish halogen bonds when compared to fluorine. [55,56] Moreover, the introduction of one fluorine atom within an halogenated compound involved in an halogen bond interaction is reported to enhance the strength of such bond, increasing the whole binding affinity. [57] The lack of crystallographic data for these compounds makes not trivial the elucidation of the possible contributions to the binding played by fluorine.

Further increase of molecular complexity was reported by Scott et al. which extended the ortho-19 and meta-20 by appending a 4-benzylamide tail. (Figure 9). [47] ). [47] In this case, thermodynamic signatures revealed that fluorine insertion in meta position (43) led to a weaker CA II inhibitor (Kd = 20 nM) when compared to its ortho regioisomer 42 (Kd = 5.7 nM). The higher affinity showed by the latter was ascribed to a strong enthalpic gain, likely due to favorable contacts between the halogen and the Thr200 residue, as previously described for 19.

Such an example, although restricted to the CA II isoform, is quite valuable as it closely resembles the synthetic evolution of CAIs usually reported in medicinal chemistry. The use of fluorine to afford an enthalpically dominated chemical precursor (i.e. the ortho-F 19) is only apparently misleading as it turns to the desired thermodynamically optimized ligand as chemical manipulation is pursued which contributes to enrich the entropic gain. [47,58] Extension of such an approach and its application to drive ligands toward specific (cyan). [59] X-Ray crystal structures resolution of their CA II adducts showed the halogen in 45 induces a nearly 180° torsion of the tail carbonyl oxygen to point towards Pro202. The thiophene ring was consequently rotated and located in proximity of Pro202 and Val135 (Figure 10). [59] However, the CA II unfavoured fluoro induced conformation in 45 was not detected for the abundantly expressed CNS CA VII since its KI value was almost superimposable to the non-halogenated 44 for the same isoform. This makes such a compounds particularly suitable for the development of CA VII selective inhibitors with biomedical applications. [59] Overall, insertion of fluorine atom(s) on the aromatic/heteroaromatic "head" of sulfonamides CAIs

Fluorine on "aliphatic tail"

CAIs bearing fluoroalkyl tails, as in Figure 11, generally show slightly enhanced CA affinities (5.6 to 70 nM variation of Kd) when compared to their corresponding hydrocarbon analogues. Such an effect was mainly ascribed to larger hydrophobic surface areas desolved upon ligand binding. [60,61] Compounds of this kind still preserve the canonical binding mode within the CA II, with the tail being associated with the enzymatic hydrophobic wall section (Figure 11C). Thus thermodynamic signatures between alkyl and fluoroalkyl derivatives are very close, with the only exception being the longest fluoroalkyl 46, (X = F, n= 4) which showed the smallest ΔH value among the fluorinated ligands. [61] In this compound, the terminal CF3 induced the Gln136 side chain to adopt a gauche conformation (Figure 11D). Overall, the desolvation effects on tails (greasy or not) and the hydrophobic interaction with the enzymatic wall were found the main determinants in thermodynamics of binding. Molecular basis for tuning up the ligand affinity by means of surface area increase of the tail is therefore similar for alkyl and fluoroalkyl CAI series, thus substituents other than fluorine may be potentially considered. [61] Considering the recent studies conducted by Klebe's group on alkyl substituted CAIs, [52,53] it can be presumed that the insertion of a perfluoroalkyl tail on the benzenesulfonamide scaffold to give compounds of the type in Figure 11 could be favourable for the binding kinetics, ensuring more contacts with the hydrophobic patch in the "pre-binding" stage, but experimental data are needed to support this hypothesis. [61] Figure dashed line with its length labelled in angstroms. [61] Perfluorooctanoic acid 47 (PFOA), perfluorooctanesulfonic acid 48 (PFOS) and perfluorooctanesufonamide 49 (PFOSA) were investigated in vitro for their inhibitory activity against various CAs and their binding affinities (for CAs I and II) were also assessed by means of native mass spectrometry (MS). The binding modes of 47-49 within CA II and IX were investigated in silico (Figure 12). [62] Figure 12. KI values against CA II and IX for compounds 47-49 and proposed binding mode of perfluorooctanesulfonamide 49 in CA II (A) and CA IX (B). [62] Calculations showed the PFOSA 49 perfluoroalkylated tail oriented differently within the CA clefts, with a more favorable accommodation for the isoform II. However such differences didn't suit the experimental inhibition potencies (KIs of 2 and 4 nM for CA II and IX respectively) as the sulfonamide accounted for the main contribution to the overall binding potency. [62] A better agreement between in silico structural analysis and experimental kinetic data were obtained for the "less efficient" CA binders 47 and 48

as the binding contribution from the chain resulted more appreciable. This opens a promising approach to differentiate CAs by exploiting fluorinated tails with non-zinc binding inhibitors. In this context a selection of primary, secondary and tertiary benzenesulfonamides containing fluorinated moieties on aliphatic chains were investigated in vitro as potential CAIs (compounds 50-62 in Figure 13). [63,64] The -fluoro aniline in 51 strongly enhanced the inhibition potency of its allyl precursor 50 against CA II and IX and also resulted fairly more potent than the cyclic derivative 52. [63] Among the secondary sulfonamides, the β-fluorine moiety showed appreciable inhibition data only for 55 and the gem-fluorinated 57 with KI values in the medium nanomolar range. Interestingly, gem-fluorinated showed to be largely more potent than the fluoro-chloro analogue 56.

Translocation of the halogen(s) substituents to the adjacent -position (i.e. 58, 59) brought the KI values up the micromolar range. [63] Even more interesting are the tertiary β-fluorinated sulfonamide 61-62 as showed good potency and selectivity for the tumor associated isoform IX over the widespread isoform CA II. All such examples gave first evidences of the β-fluorinated amine/sulfonamide moiety on CAIs and its effects on kinetics also when inserted within tertiary sulfonamides which mode of inhibition still remains to be determined. [4,65] At this stage, it may be suggested that a gauche conformational effect of this unique βfluorinated (or difluorinated) amine/sulfonamide core could account for a privileged orientation of the molecule in the active site.

Figure 13. CA II and IX and inhibition values of sulfonamides 50-62. KI values are in nM. [63,64]

Fluorine on "aromatic tail"

Binding and structural studies using the N-(4-sulfamylbenzoyl)benzylamine (SBBs) bearing fluorinated aromatic tails as model, allowed to explore the different interactions established within the CA II cleft and F131V-CA mutant (Figure 14). Electron density calculations on the unsubstituted benzyl tail in SBB 41 showed a negative charge distribution on both sides of the ring plane, with a positive electrostatic potential contouring its peripheral area. This allows a quadrupole-quadrupole interaction between any perfluorinated benzyl ring and the Phe131 in CA II to be studied. Aromatic moieties of reversed quadrupoles prefer to interact with each other in face-face stacked geometries. [66] A known example is the stacking of benzene and perfluorobenzene which displays nearly identical values in their quadrupole moments, but with opposite signs. [67,68] The small molecule 1:1 co-crystal structures of benzene and hexafluorobenzene exhibit a kind of edge-to-face arrangement alternating parallel stacking of benzene and hexafluorobenzene molecules with their planes separated by about 3.5 Å [69,70] due to the orientation of bond dipoles towards the quadrupole moments. [71,72] The impact of temperature on these interactions and the ability to finely model these interactions are still under discussions. [68,73] Thus, one could have expected that the insertion of the electronegative fluorine(s) in 63-66 could have provoked similar interactions of Phe131 and the fluorinated ligand benzyl tail.

Kd values of the fluorinated derivatives were progressively lower thus indicating that tighter bindings were established (Figure 14). [74,75] Intercentroid Separation Distances (Å) for each of the SBB-Phe-131 and SBB-Pro-202 complexes 63-66. [74] However, X-Ray resolution of CA II/63-66 complexes showed an edge-to-face interaction between the centroid of Phe131 and the fluorinated benzyl ring of the ligands (with the exception of 65). The distances were gradually decreased upon fluorination (Figure 14 B andC). For the difluorinated compound 65, the aromatic ring was rotated around the C-phenyl bond to place the fluorine at 2-position close to Phe131, and thus establishing favorable electrostatic interactions with its partially positively charged peripheral area. [74][75][76][77] Dispersive forces engaged the ligands aryl rings and Pro202 , with the 2-fluorine 63 being the most involved (Figure 14 C). [74] Such evidences pointed out the important role of dispersive interactions within the CA II other than the canonical one with Phe131. This was more evident in the F131V CA mutant which lacks that residue (Figure S1 A andB). [77] Here, pairwise decomposition of residue interaction energies, accounted for the distances between the Thr199 residue and the sulfonamide moiety, being the real determinant for diverse Kd values within the fluorinated series (Figure S1 C). [77] Such an effect is integrated with pKa lowering effects in fluorine substituted ligands, which further contributes to the inhibitory potency. [75] These results clearly evidence the ability of (per)fluorinated aromatic tail to strongly impact on potency and selectivity for these inhibitors through a combination of pKa lowering effect and finely tuned dispersive interactions.

The influence of aromatic fluorinated tails on ligand's physicochemical features has also been widely explored, as in the case of AAZ 1 and BZA 3. [78,79] Although the perfluoroarylsulfonyl/carbonyl aromatic tails did not affect the in vitro inhibitory profiles of such compounds when compared to AAZ and BZA, they proved to be potent IOP-lowering agents due to proper LogPs. [78] X-ray crystallography of the CA II/67 adduct revealed stacking interactions between the perfluorophenyl ring of the ligand and Phe 131 (Figure 15). [80] Moreover, an ortho fluorine atom of the perfluorobenzoyl tail was involved in a complex H-bond network also including the exocyclic nitrogen atom of the ligand and two water molecules (Wat 1194 and Wat 1199). These additional interactions contributed to the stabilization of the inhibitor within the enzymatic site, thus fitting the lower KI values observed for PFMZ 67 when compared to MZA (KI=1.5 and 14 nM respectively). ). [21,80] The strong electrophilic character of the para sp 2 -carbon bearing the fluorine makes such a position a preferential site for nucleophilic substitutions to take place, and opened the way for substrates lacking the halogen (i.e. 68, 69, Figure 16) or bearing appropriate linkers with the intent to disfavor undesired tautomeric structures (i.e. 70, 71, Figure 16). [79,[81][82][83] In vitro inhibition profiling revealed these compounds as very potent inhibitors of the tumor associated CA IX, particularly for the ureido derivative 71. [82,84] Figure 16. Fluorine-containing inhibitors devoid of enhanced reactivity with [79,[81][82][83]85] Compound 71 is in Phase 1b/II clinical trials as CA IX inhibitor in association with gemcitabine for the treatment of hypoxic solid tumors. The role of fluorine is mainly to endow the ligand with proper pharmacokinetic and pharmacodynamic features rather than driving CA inhibitory potency or isoform selectivity, although X-Ray studies showed the larger cavity of CA IX to better accommodate 71 tail within the hydrophobic pocket created by Val131 and Gly132, thus leading to more efficient hydrophobic interactions to take place when compared to CA II (Figure S2). [85][86][87][88][89] 3. CAIs and 19 F NMR 19 F Nuclear Magnetic Resonance (NMR) spectroscopy is a very reliable and sensitive technique to study the protein-ligand binding features. [90,91] Since this element is rare in biological systems it offers several advantages when compared to usually explored nuclei (i.e. 1 H, 15 N and 13 C) and perfectly suits as bioorthogonal element when retrieving molecular interactions. [90,92,93] In particular 19 F-NMR possesses: i) high sensitivity (83% relative to 1 H-NMR) and 100% isotopic natural abundance; ii) wide dispersion chemical shifts (> 300 ppm range) and iii) large Chemical Shift Anisotropy (CSA). Overall 19 F-NMR makes use of the high sensitivity of the nucleus to electronic and magnetic environmental changes coupled to wide signals spreading in the ppm scale. [90,[92][93][94] Noteworthy is the "rule of shielding" which correlates 19 F chemical shift values, to the nature of contacts established (i.e. hydrogen bond/dipolar interactions or hydrophobic contacts) (Figure S3). [95] Two complementary approaches are largely used in NMR spectroscopy: 1) the ligand based and 2) the protein based methods (Figure S4 a andb). [92,93] Ligand-observed NMR is a fast and easy technique, which doesn't require protein labelling neither high concentrations of ligand (i.e. <10 M) and allows to evaluate various inhibitors at the same time. [45,92] An example of protein-observed NMR with CA was reported by Gerig, using fluorophenylalanine as modified residue (Figure S4 b). [96] A recent and successful example of selective aminoacidic chemical modification on CAs applied to NMR protein detection is referred as the ligand-directed tosyl (LDT) approach, aimed to selectively label a protein with a desired probe within its native environment (Figure 17). [97] The same authors also performed CA I labeling within Red Blood Cells (RBC) thus opening new opportunities for the in cell NMR screening of CAIs. [98] Figure 17. A) Scheme of the self-assembly, hydrolysis and labelling processes in 72-mediated CA I labelling.

B) 19 F-NMR spectra of a reaction mixture during the labelling process; (X) 3,5-bis(trifluoromethyl)benzene derivative (FB) -containing fragment (FBOH). [97] An unconventional approach to gather structural information on ligand protein interactions is represented by 19 F-NMR pseudocontact shift (PCS) analysis. [99] When compared to NOE experiments, PCS drastically reduces the measurement times, also eliminating the need for isotope labelled protein as well as chemical modification of the ligand. As example of 19 F-NMR-PCS is reported the use of lanthanide chelating tags bound site-specifically to single cysteine mutants to determine the anisotropy tensors. The measurement of one-dimensional 19 F-NMR experiments and analysis of the obtained 19 F-PCS over a distance range of 22-38 Å, allowed to determine the binding of the fluorinated compounds 64 and 73 within CA II (Figure 18). [99] light blue sticks: fluorine atoms, blue sphere: Zn 2+ ion. [99]

Conclusions

By selecting specific examples from a wide range of available data on fluorinated CAIs of the sulfonamides type, this review attempts to give a rationale why should we use fluorine in CA ligands and where does such a substitution pattern provide any advantage, but also, where it is detrimental for binding and selectivity. Crystallographic and thermodynamic studies conducted on simple scaffolds helped to rationale how wisely insert fluorine atom(s) within a CAI moiety, in order to precisely enhance or disrupt ligand protein interactions, depending on the location the fluorine substitution is targeted on the ligand. Using benzenesulfonamide CA inhibitors, exhibiting different fluorinated patterns (fluorine atom(s) located on the ring, on the aliphatic or aromatic tails), some "general guidelines" could be summarized as following: 1) For ring-substituted benzenesulfonamide inhibitors, the small size of the fluorine atom and its electronically rich nature allows really specific interactions with a Thr200 residue close the zinc atom when the aromatic ring is ortho substituted with fluorine atom(s), as for e.g. compound 19. Large association and small dissociation constants calculated for meta-F substituted derivatives (i.e. 20) highlighted a special role of fluorine for the binding kinetics when inserted in such position, ensuring an optimal placement and fit of the ligand within the binding pocket. Multiple addition of fluorine atoms did not provide any advantage in terms of free binding energy and thermodynamic signatures, probably because the scaffold of these compounds is too simple and they can adopt a variety of orientation within the active site, "compensating" for the introduction of additional fluorine atoms. [47,48,52,53] Another very interesting impact that can have fluorine insertion in meta positions of the aromatic ring is on the discrimination between hCA isoforms, as shown for compound 45 showing high selectivity for the CNS expressed CA VII over the widespread isoform II; [59] 2) In analogy with alkyl tails, perfluorinated aliphatic tail interaction with the hydrophobic pocket may favor binding kinetics in the prebinding stage and can be envisaged as a strategy to enhance inhibitors potency. [53,61] However, the increased affinity due to the large hydrophobic surface areas desolved upon ligand binding can be potentially obtained with substituents other than fluorine (i.e. alkyl groups). [61] β-fluorinated amines and sulfonamides can also be seen as a promising specific pharmacophore to specifically interact with one selected isoform, although for this case, the reason for this selectivity remained unclear (e.g. compound 61). [START_REF] Thibaudeau | Mod. Synth. Process. React. Fluorinated Compd[END_REF] 3) Fluorination of the aromatic tail also strongly impacts the potency and selectivity of the studied inhibitors. This comes from mainly a combination of pKa lowering effect and finely tuned dispersive interactions. Moreover, perfluroaromatic tail insertion may confer a balanced hydro/lipophilic profile to CAIs, improving their pharmacokinetic properties.

It is the case of the anti-glaucoma agent 67, which showed a very high penetrability through the cornea and a slow elimination, ensuring a long-lasting IOP lowering effect. [78,79,[81][82][83] Overall, the insertion of fluorine on CAIs of the sulfonamide type accounts for a multitude of effects. The increase of molecular complexity of the scaffolds along with the strength of the sulfonamide-zinc coordination, which represents the main contribution to the binding, may shade other interactions mediated by the fluorine atom(s). In this context, multiple approaches such as ITC, kinetic and inhibition studies, X-ray crystallography and NMR are useful in dissecting single binding contribution to the overall observed effect. [92,[START_REF] Gimeno | [END_REF]102] It makes no doubt that the exploitation of innovative directions made in the field of protein and ligand-based fluorine NMR screening [92,103] will allow to avoid misconduct and finely tune the exploitation of the selective fluorine atom insertion in the future.

Figure 1 .

 1 Figure 1. Solvent accessible surfaces of CA II: residues delimiting the hydrophobic half are in red, the hydrophilic ones are in blue. Main inhibition mechanisms of the CAIs (PDBs 3HS4, 3F8E, 4QY3). a) Acetazolamide (AAZ) 1, and other metal binding moieties 2-9; b) compounds anchoring the zinc-bound water molecule, i.e. phenol 10, c) compounds occluding the entrance of the active site, i.e. 2-hydroxy-cinammic acid 11 d) out of the binding site inhibitors, i.e. benzoic acid 12. The hydrophobic adjacent pocket where inhibitors

Figure 3 .

 3 Figure 3. a) Structures of trifluoromethanesulfonamide 13, methanesulfonamide 14 and benzenesulfonamide 15; b) Structural superposition of CA II/13 (yellow, PDB 1BCD) and CA II/15 (red, PDB 2WEJ).[42]

Figure 6 .

 6 Figure 6. Superposition of CA II/inhibitor adducts: 15 (magenta, PDB 2WEJ), 18 (yellow, PDB 2WEH), 19 (gray, PDB 2WEG) and 20 (blue, PDB 2WEO). Residues delimiting the hydrophobic half are in red, the hydrophilic ones are in blue. Calorimetric data of 15, 18-20 ([a]=kcal mol -1 )[47] and kinITC data of 15 and

Figure 7 .

 7 Figure 7. A) Diagram of the amino acid residues of CA forming contacts with the benzothiazole (H4BTA, 21); B) perfluorobenzothiazole (F4BTA, 29). Favorable ligand-protein interactions are represented with a blue

  impact ligand/protein interaction as shown by lower KI values against the CA II for compound 31. However, either Br(33) or I (34) were able to induce similar KI reduction on CA II, thus meaning the halogen impact on the binding of such ligands is not limited to fluorine. Less differences between the unsubstituted 31 and monohalogenated 31-34 were observed for inhibition of the tumor associated CA IX. Introduction of an additional halogen in position 5 of the aryl moiety (i.e. 35-40) determined remarkable reduction of whole KI values with major effects on the isoform IX. The most potent CA IX inhibitor was the 3-F,5-Cl (35) with a KI of 12 nM, thus 20.4-fold more potent when compared to the mono halogenated precursor 31 (KI 245 nM). Specifically, the impact of the fluorine on CA II was clearly observed in 35-37 as lower and close matching KI values were obtained when compared to those of 38-40 lacking such an element. As for the CA IX, compounds 35-37

Figure 9 .

 9 Figure 9. Structural extension of 15, 19 and 20 to give 41, 42 and 43 and their CA II thermodynamic binding parameters ([a]=kcal mol -1).[47]

  isoforms is well represented by CAIs 44 and 45. The former resulted far more potent as a CA II inhibitor than the fluoro containing one (KIs of 50 and 390 nM respectively).

Figure 10 .

 10 Figure 10. Stick representation of CA II active site in adduct with (A) 44 (green, PDB 3R16) and (B) 45 (cyan, PDB 3R17). The active-site zinc is depicted as a gray sphere. The electron density is represented by a 1.2 weighted 2Fo-Fc Fourier map (blue mesh). (C) Superposition of CA II complexed with 44 (green) and 45

  determined multiple effects on binding as it primarily affects the sulfonamide pKa and the dipole moment of the molecule, with consequences on the binding affinities as well as on the molecule desolvation event within the binding site. Privileged position is the meta, as interactions of the fluorine with the hydrophobic pocket of the active site are thus favored, which in turn lower the KI values. The ortho fluorine regioisomers resulted particularly advantageous for ensuring high inhibitory potency for ligands developed during the optimization process by means of tail chemical manipulation. In this case, the halogen resulted particularly effective in increasing the enthalpic gain. Skillful insertion of the fluorine within the ligands aromatic head represents a validated strategy for driving the ligand toward preferential CA isoforms as usually multiple halogenation fails to confer enhanced features.

11 .

 11 Figure 11. Dependence of ΔG° for binding of ligands with alkyl (○) and fluoroalkyl (•) tails (A) and on their solvent-accessible surface area in the fully extended conformation (B); C) Aligned structures for 10 ligands determined by X-ray crystallography; D) Crystal structures of fluorinated "greasy tails" complexed with CA

Figure 14 .

 14 Figure 14. A) Superposition of CA II-inhibitor complexes. Inhibitors are color coded as follows: 2-fluoro-SBB 63 (green), 2,3-difluoro-SBB 64 (blue), 2,6-difluoro-SBB 65 (red), and 2,3,4,5,6-pentafluoro-SBB 66 (yellow); B) Interaction between Phe131 and 63-66 within CA II; C) Interaction between Pro202 and 63-66 within CAII; A) Fluorine insertion on SBB (41) benzyl group, to give 63-66; Binding energies (G) and

Figure 15 .

 15 Figure 15. A) CA II/67 complex showing the zinc ion coordination, hydrogen bonds and residues participating in the van der Waals interactions with the inhibitor scaffold (distance <4.5 Å). B) Schematic representation of PFMZ 67 binding to the CA II active site (numbers represent distances in Å ).[21,80]

Figure 18 .

 18 Figure 18. A) X-ray structure of CA II (PDB 3KS3). Red: selected serine to cysteine mutation sites, yellow: native Cys206, blue: leucine residues and orange: zinc ion.; B) Selected ligands for proof-of-principle study;