Aurélie Claraz

Géraldine Masson

Asymmetric iodine catalysis-mediated enantioselective oxidative transformations

The implementation of chiral iodine catalysis has tremendously been developed in the field of asymmetric synthesis during the last decade. It enables the stereoselective creation of C-O as well as C-C, C-N and C-X (X = halogen) bonds through oxidative transformations. Thanks to the low toxicity and ease of handling of iodine compounds, this strategy offers many advantages over classical metal-catalyzed oxidations with chiral ligands. The approaches rely on iodine (I/III) or (-I/+I) catalysis by using a chiral aryliodine or ammonium iodide respectively in combination with a suitable terminal oxidant. As such, the design of iodine compounds with central, axial or even planar chirality has allowed to achieve high enantioselectivities. The goal of this review is to cover the different chiral iodine compounds-catalyzed oxidative transformations including -functionalization of carbonyl compounds, dearomatization of phenol derivatives and difunctionalization of alkenes which should demonstrate that iodine catalysis has now found its place in the realm of asymmetric organocatalysis.

Introduction

Iodine catalysis has emerged as a versatile and eco-friendly strategy to perform oxidative transformations allowing for instance couplings with formation of a C-O, C-N, C-C or C-X (X = halogen) bond. 1 In order to describe mechanisms of such reactions, synthetic chemists frequently borrow the terms of oxidative additions, ligand exchanges and reductive eliminations to the field of metal-mediated processes. Nevertheless, iodine compounds are mild, stable, easy to handle and eco-compatible reagents, advantageously competing with heavy metals. As such, it is not surprisingly that iodine catalysis has attracted the interest of the synthetic community in the field of asymmetric synthesis with the aim to develop a greener and more environmentally benign chemistry. [START_REF] Berthiol | [END_REF] Up to now, asymmetric iodine catalysis-mediated oxidative transformations rely on the catalytic use of either hypervalent organoiodine (III) reagents (also called  3 -iodanes) or organic hypoiodites (I) as chiral oxidants. In the first case, chiral organoiodanes (III) 3 are generated in situ from aryliodine precatalysts and a stoichiometric amount of achiral co-oxidant (Figure 1). The first enantioselective catalytic transformation was reported in 2007 by Wirth et al. for the -oxytosylation of ketones by using an aryliodine species bearing a chiral moiety ortho to the iodine atom in the aromatic ring. 4 Since this original report, numerous chiral aryliodoarene-mediated enantioselective oxidative transformations through iodine (I/III) catalysis have been investigated. 5 Besides cost reductions and atom economy, the development of catalytic processes allows to avoid the preliminary preparation and isolation of the chiral hypervalent orgaonoiodine (III) reagent, since this reactive species is generated in situ from an aryliodine. Obviously, this feature can be advantageous during the design of new chiral precatalysts, especially for unstable hypervalent organoiodine (III) derivatives. The currently known enantiopure precatalysts of hypervalent organoiodine (III) compounds exhibit either C 1or C 2 -symmetry with central, axial or even planar chirality (Figure 1). On the other hand, chiral hypoidites (I) are generated in situ from chiral organic iodides (-I) (typically chiral ammonium iodides that are traditionally used in asymmetric phase transfer catalysis), 6 and a stoichiometric amount of achiral co-oxidant (Figure 2). In both catalytic cycles depicted above, (re)oxidation of the iodine atom of a precatalyst in low valent state to higher oxidation state is the key step. 8 The choice of this stoichiometric achiral oxidant is critical for the success of the reaction. In an ideal fashion, it should not interact with the starting material or, at least, reoxidation of the precatalyst should be faster than the rates of undesired side reactions. In asymmetric processes, peroxides are the most commonly used oxidants that satisfy with more or less success these requirements.

To get cleaner reactions along with highest levels of enantiocontrol, a careful optimization of other classical reaction parameters such as solvent, temperature and the use of additives must be undertaken.

The focus of this review is to summarize the use of chiral iodine compounds in catalytic enantioselective oxidative processes with the aim of highlighting both catalysts design and synthetic applications. As such, we hope that this survey will help the reader in designing new chiral iodine precatalysts and applying those to new catalytic processes. For the sake of clarity, this review has been divided into different sections according to the kind of oxidative transformation considered: a. -functionalization of carbonyl compounds, b. dearomatization of phenol derivatives, c. functionalization of alkenes.

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-Functionalization of carbonyl compounds

Hypervalent iodine compounds enable the introduction of a nucleophile at the -position of a carbonyl compound. As such, numerous efforts have been made towards the development of enantioselective versions to take advantage of this inversion of polarity (umpolung reaction).

-Oxytosylation

The enantioselective -oxytosylation of ketones mediated by chiral hypervalent iodines gives access to synthetically useful enantioenriched carbonyl compounds. along with the regeneration of aryliodine I. Interestingly, the reaction is generally clean and the -oxybenzoyl ketones which would result from the nucleophilic addition of the mCBA by-product were not formed. Importantly, the authors pointed out the need of a chelating group at the benzylic position on the ortho substituent of the iodine atom in I to get the highest possible levels of enantioselectivity. Based on this observation, they synthesized compound II, having a chiral ester moiety as the chelating function at the benzylic position instead of the methoxy group, and resulting in an increase of enantioselectivity up to 39% during the -oxytosylation of propiophenone (Scheme 2). 11 In 2010, the same group prepared derivative III, bearing a chiral ester at the ortho position of the iodine, and leading to lower enantioselectivity. 12 In 2012, the group of Legault et al. developed chiral aryliodine IVa bearing a chiral oxazoline as a coordinating motif and a methyl at the ortho and ortho'-positions of the iodine. 13 Premixing catalyst, substrate, and TsOH, followed by slow addition of mCPBA slightly enhanced both yield and selectivity during the -oxytosylation of propiophenone. According to computational analyses, this procedure would favor the formation of the key reactive iodane 5 by minimizing Noxidation of the oxazoline moiety through N-protonation. 14 Noteworthily, corresponding aryliodine IVb without the ortho'-methyl substituent is not able to promote the reaction as protonation of IVb is much more difficult. The best selectivity was finally obtained after the introduction of a chlorine atom para to the iodine (IVc). The enantiopure spirobiindane scaffold has also been exploited as a source of chirality in the -oxytosylation of carbonyl compounds. In 2011, Zhang et al. have synthesized chiral iodoarene Va in 7 steps (28% yields) from expensive commercially available (S)-1,10-spirobiindane-7,7'-diol. 15 In the presence of mCPBA as stoichiometric oxidant in acetonitrile, the -oxytosylation of propiophenone 1 afforded product 2 in 65% yield and 30% ee. Enantioselectivity has been improved to 53% ee by simply switching solvent from acetonitrile to ethyl acetate (Scheme 3). In contrast to Wirth's report, slight racemization of the product was observed in the reaction medium. It is worth mentioning that lower enantioselectivity was obtained with C 2symmetrical chiral diiodoarene Vb. Introduction of other chelating and/or sterically demanding substituents on the spirobiindane scaffold close to the iodine atom (4-, 6-, and 7'positions) did not improve enantioselectivity. This reaction scope has been extended to other aromatic ketones and sulfonic acids, yielding corresponding -functionalized ketones in up to 56% ee.

During their quest for a suitable precatalyst in the catalytic enantioselective -oxytosylation of propiophenone, Wirth et Despite the great advances which have been made, the direct -oxytosylation of ketones still suffers from moderate enantioselectivities. The competition between the two different routes depicted in scheme 1 might be responsible of this limited enantiocontrol. Especially, the great distance between the iodoarene group in O-iodonium enolate 3 and the forming stereocenter can make efficient stereoinduction very Please do not adjust margins Please do not adjust margins difficult. 20 The lack of control in the geometry of enol 3 might be also problematic. In 2016, Xiong, Coeffard et al. investigated the oxytosylation of -substituted -ketoesters 7 by using C 2symmetric iodoarene IX as chiral precatalyst in combination with mCPBA as stoichiometric oxidant at -20 °C in chloroform (Scheme 6). 22 Corresponding products 8 with a quaternary stereogenic center were obtained in poor yields albeit respectable enantiomeric excesses (up to 37% yield and 62% ee). Interestingly, 3 equivalents of trifluoroacetic acid (TFA) as additive were able to increase enantioselectivity. 23 Scheme 6 -oxytosylation of -substituted -ketoesters.

Intramolecular -oxygenation

The hypervalent iodine catalyzed -oxygenation of carbonyl compounds has been extended by Moran et al. to an intramolecular version via lactonization of 5-oxo-5phenylpentanoic acid 9 (Scheme 7). 24 Inspired by the works of Legault et al., 13 the authors synthesized ortho-tolyliodine XI bearing a pseudoephedrine-based chiral amide as chelating Lewis agent. In the presence of substoichiometric amount of mCPBA and pTsOH, 5-benzoyldihydrofuran-2(3H)-one 10 was obtained with moderate yield and enantioselectivity.

Scheme 7 Oxidative cyclization of 5-oxo-5-phenylpentanoic acid.

In 2010, an elegant catalytic enantioselective oxidative cyclization of ketophenols 11 to the five-membered ring products 2-acyl-2,3-dihydrobenzofurans 12 has been developed by Ishihara et al. (Scheme 8a). Excellent yields and enantioselectivities at very low catalyst loading were obtained Their approach relied on the use of a in situ generated chiral hypoiodite [IO] -catalyst from the corresponding quaternary ammonium iodide precatalyst XII with hydrogen peroxide or tert-butyl hydroperoxide (TBHP) as environmentally benign cooxidants. 25 The unstable catalytic active species hypoiodite has been detected by a series of Raman spectra unveiling an unprecedent I(-I/+I) asymmetric catalysis. 26 This method has been extended to the less favorable six-membered ring oxidative cyclization to access 2-acyl-chromane derivatives. 26 In a similar fashion, highly enantioenriched 2-acyl tetrahydrofuran derivatives 14 have been obtained via cycloetherification of corresponding δ-hydroxyketones 13 using cumene hydroperoxide (CHP) as stoichiometric co-oxidant (Scheme 8b). 27 Scheme 8 In situ generated chiral hypoiodite-catalyzed oxidative cyclization. This journal is © The Royal Society of Chemistry 20xx

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-Fluorination

A chiral iodoarene-catalyzed asymmetric nucleophilic fluorination of indanone-based -ketoesters 15 has been initially reported by Kita, Shibata et al. using C 2 -symmetric axially chiral precatalyst VI in combination with mCPBA as a stoichiometric oxidant and nHF•pyridine as fluorine source (Scheme 9). 28 Moderate enantioselectivity was obtained with methyl ester 15a, whereas sterically demanding adamantyl ester 15b afforded a higher enantiomeric excess (56% ee vs. 25% ee). A breakthrough has been very recently accomplished by Rueping et al. by using chiral iodoarenes derived from 2iodoresorcinol and lactate scaffolds with triethylamine pentahydrofluoride as the fluorine source. 29 More particularly, the authors developed the flexible C 2 -symmetrical menthol ester derivative XIII bearing a 3,5-bis(trifluoromethyl)-phenyl group para to the iodine providing fluorinated product 16 in good yields and with high enantioselectivities. Chiral iodoresorcinol derivative lacking C 2 -symmetry proved to be inferior. In parallel to Rueping's work, Lu, Zheng et al. have designed a novel planar chiral iodoarene XIV based on [2,2]paracyclophane and bearing a benzoyl group as potentially coordinating function of the ortho-iodine atom. Triethylamine trihydrofluoride (TEA•3HF) was selected as fluorine source. It is worth mentioning that a positive effect on the enantioselectivity was observed with the introduction of methoxy-groups on the phenyl ring of the benzoyl. 30 In line with experimental observations, these three research groups assumed in situ generation of Ar*-I-F 2 from Ar*-I, mCPBA and HF along with activation of this key chiral reactive species by an excess of HF. Based on DFT calculations, Rueping et al. proposed a mechanism with formation of an O-bonded hypervalent iodine intermediate 17. 29 Migration of the aryl iodonium from the O-ester enolate to the -C atom would lead to intermediate 18 via transition state 17→18 and yield the -fluorinated product through reductive elimination. The competitive hydroxylation of the carbonyl compounds is responsible of the moderate yield. Transition state 17→18 is anticipated to be the enantioselectivity-determining step. According to it, formation of (R)-18 is favored as transition state (pro-R) 17→18 was found 1.4 kcal/mol lower in energy than transition state (pro-S) 17→18. Reductive elimination would afford -fluorinated (R)product 16, which is in agreement with experimentally observed stereochemistry. Unfortunately, the authors did not provide any calculations concerning the energy of a transition state corresponding to a pseudo-intramolecular S N 2' type reductive elimination of an O-iodonium keto-enolate, as it was proposed for the -oxytosylation of carbonyl compounds by Legault et al. (scheme 1, intermediate 3, L = F). 20 Scheme 9 -fluorination of indanone derivatives.

Spirocyclization through C(sp 2 )-C(sp 3 ) coupling

Although most hypervalent iodine (III) catalyzed-asymmetric oxidative coupling protocols enable the formation of C-X bonds (X = O, N, F), an elegant intramolecular C(sp 2 )-C(sp 3 ) coupling reaction of N 1 ,N 3 -diphenylmalonamide 19 has been achieved by Gong et al. in 2014 (Scheme 10). 31 This transformation provided access to biologically important spirobisoxindoles 20 with a quaternary carbon stereogenic center in good yields and with good enantiomeric excesses. The best reaction conditions involved the use of ethaneperoxoic acid (MeCO 3 H) as the stoichiometric oxidant in nitromethane with C 2 -symmetric iodoresorcinol derivative XV as precatalyst. Notably, the tertiary proline amide on XV (at the end of the resorcinol arms) played key role in inducing the highest enantioselectivities compared to secondary amides, carboxylic acids or esters.

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Please do not adjust margins Moreover, the addition of 2 equivalents of trifluoroacetic acid enhanced the catalytic performances in terms of both yield and enantioselectivity. To shed light on the reaction mechanism and the origin of the stereoselectivities, computational studies have been undertaken by Sunoj et al. 32 Based on DFT calculations, the authors proposed a mechanism during which the in situ generation of the key chiral hypervalent iodine intermediate Ar*-I(TFA) 2 was the initial step. Ligand exchange with the nitrogen atom of incoming dianilide substrate 19 followed by pseudo-intramolecular deprotonation of 21 by the leaving trifluoroacetate anion generated O-iodonium enolate 22. The ensuing O to C-aryliodonium migration leads to C-iodonium enolate 23 with a lower energy. Intramolecular Friedel-Craft reaction assisted by the trifluoroacetate anion allowed the formation of the first oxindole ring 24 with concomitant regeneration of precatalyst XV. The second ring formation would be enabled by a similar reaction sequence yielding spirobisoxindole 19. It is worth mentioning that direct ring closings from O-iodonium 22 and 26 have been calculated to be of higher energy, indicating that formation of C-iodonium enolates 23 and 27 might occur. As chiral information of intermediate 24 is lost during the second catalytic cycle, stereoselectivity of the reaction is solely governed during the second ring closure. More precisely, the 1,3-migration of the chiral aryl iodonium from O to C-enolate (26→27) is the stereocontrolling step of the reaction. The transition states leading to (S)-product 19 were found to be 2.1 kcal/mol lower than those leading to the (R)-product, which is in good agreement with the experimentally obtained enantioselectivity. Noteworthily, in the preferred conformer of key chiral hypervalent iodine intermediate Ar*-I(TFA) 2 , resorcinol arms were found to organize in a C 2 -symmetric helical assembly with a right-handed P helicity in which the two trifluorooacetate ligands form a network of nonconvalent interactions with the chiral arms.

Scheme 10 Spirocyclisation through two sequential C-C cyclizing couplings.

Dearomatization of phenol derivatives

The asymmetric dearomatization of phenol derivatives is of particular interest for the synthetic community as it can be a pivotal step in the total synthesis of natural products. Chiral hypervalent organoiodines reagents are organo-oxidants of choice to perform this transformation enantioselectively. 33 They can be generated in situ from a catalytic amount of chiral iodoarene and mCPBA as a stoichiometric oxidant. Ligand exchange between the phenol and in situ generated iodine (III) would furnish phenoxy- 3 -chiral aryliodarene 28 (Figure 3). Subsequently, two mechanisms are generally considered: direct attack of the nucleophile via a S N 2'-type substitution (associative path) or formation of phenoxenium ion 29, which is trapped by the nucleophile (dissociative path). The key feature to successively achieve asymmetric induction is to find reaction conditions (catalyst, solvent, additive…) capable of favorizing the associative mechanism. This journal is © The Royal Society of Chemistry 20xx

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Intramolecular transformations

The intramolecular dearomatization of phenol derivatives provides a straightforward way to spirocyclic compounds. The asymmetric dearomatization of propanoic acid substituted 1naphthol 30 to spirolactone 31 has been intensively studied as a model reaction for the design of new aryliodine precatalysts. Indeed, precatalysts which exhibits either C 1 -or C 2 -symmetry and central or axial-chirality have been developed to achieve the highest enantioselectivity (Scheme 11).

In 2017, Nachtsheim and Pericàs et al. reported a new C 1symmetric triazole-based chiral iodoarenes (XVI). This catalyst was prepared thanks to an enzymatic kinetic resolution followed by a copper-catalyzed azide-alkyne cycloaddition and provided spirolactone 31a with moderate yield and good enantioselectivity.

Ishihara et al. developed conformationally flexible C 2symmetric chiral iodoarenes XVII derived from lactate as a source of chirality affording spirolactone 31a in good yield and with high enantioselectivity (55%, 92% ee). 34 More recently, an enhancement of the yield and the enantioselectivity was achieved in the same group by using catalyst XVIII derived from chiral amino-alcohol at low loadings. 35 X-ray and NMR analyses of in situ generated organoiodine(III) species from XVIII showed that intramolecular hydrogen-bonding interactions between acidic amido protons and ligands of iodine(III) allowed the construction of a suitable chiral environment around the iodine(III) center. 36 An original C 2 -symmetric iodoarene XIX with central chirality based on the rigid all-carbon anti-dimethanoanthracene framework displayed lower activity and selectivity. 37 However, a rigid C 2 -symmetric aryiliodine Vb based on the axially chiral spirobiindane scaffold has been disclosed by Kita et al. in 2008. Cyclized product 31a has been obtained in good enantioselectivity with this catalyst. 38 An enhancement of the enantioselectivity was observed with Vc via introduction of ethyl-substituents at the ortho-positions of the iodine atoms, presumably thanks to an extension of surroundings around the reactive sites. 39 In 2016, Masson et al. explored axially chiral 1,1'binaphtalene framework and derivatives for the development of novel chiral iodoarenes prepared from commercially available (R)-1,1'-binaphtyl-2,2'-diamine (BINAM). 40 Spirolactone 31a was obtained with little enantiomeric excess with H 8 -BINI XXa. Introduction of 1-naphtyl substituents at the 3,3'-positions (XXb) exerted a slightly beneficial effect on enantioselectivity. Interestingly, a non C 2 -symmetric iodoarene precatalyst (XXI) harboring an amide group at the 2'-position afforded spirolactone 31a in poor enantioselectivity but with an inversion of configuration. By analogy with Ishihara and Wirth's catalysts, additional non-covalent interactions between the amide group and the iodine (III) center or its ligands may be responsible of this reversal through a different transition state. Kita et al. designed a new type of chiral 1,1'-binaphthalene (XXII) substituted with the iodine atoms at the 8,8'-positions which dramatically improved enantioselectivity, but at the price of a high catalyst loading (0.55 equiv.). 41 This precatalyst bearing the iodine atoms within the major groove of the naphtalene rings has been synthesized by a nickel-catalyzed reductive homocoupling, enantiomeric resolution of the obtained racemic diamine on preparative HPLC with chiral stationary phase column, followed by Sandmeyer-type iodination.

Please do not adjust margins Please do not adjust margins Scheme 11 Asymmetric dearomatization of propanoic acid substituted 1-naphthol 30a.

In 2015, Ishihara et al. reported application of their strategy of in situ generated-chiral ammonium hypoiodite from the corresponding chiral ammonium iodide and hydrogen peroxide as stoichiometric oxidant to the catalytic spirolactonization of 1-naphtol derivatives with good yields and good enantioselectivities at room temperature (Scheme 12). 42 The use of hydrogen-peroxide as stoichiometric oxidant offers the advantage of generating only water as a by-product instead of mCBA, but limited the scope of the transformation to 3 or 4substituted naphtols.

Scheme 12 Hypoiodite-catalyzed asymmetric dearomatization of 3 or 4 substituted 1naphthol derivatives 30.

In 2013, Ishihara et al. investigated oxidative cyclizative dearomatization of phenol derivatives 32 by using precatalyst XVIII (Scheme 13). 36 In the case of poorly reactive electrondeficient phenols, the addition of 50 equivalent of 1,1,1,3,3,3hexafluoroisopropanol (HFIP) was required and dramatically enhanced efficiency of the process. [START_REF] Colomer | Mechanistic studies on the activation of iodine (III) derivatives with fluorinated alcohols: I[END_REF] In contrast, addition of 25 equivalents of methanol was necessary in the case of highly reactive electron-rich phenols to presumably ensure a ligand exchange from acyloxyphenoxyiodine (III) 34. This protocol would slow down the racemic dissociative pathway and would favor the enantioselective associative pathway as methoxy ligand is a less good leaving group than carboxylate ligand. Under these conditions, high yields and excellent enantioselectivities were obtained in both cases. Noteworthily, this protocol has proved to be also very efficient for the spirolactonization of poorly reactive 2-naphtol derivatives 35 and for dearomatization of ortho-and para-hydroquinone derivatives allowing the construction of ortho-and parabenzoquinones. 44 Scheme 13 Spirocyclization of phenols. This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins In 2017, Ciufolini et al. described the hypervalent-iodine catalyzed enantioselective oxidative cycloetherification reaction of naphtolic alcohols 35 (Scheme 14). 45 The design of a new precatalyst was required as these substrates are less reactive than the corresponding carboxylic acids. In this context, a variant of the Ishihara's catalyst was examined. Considering that intramolecular H-bonding between the secondary amide NH group and the ligand of the iodine III is the key feature in these type of catalysts, the authors relocated the stereogenic center closer to the iodine atom. As a result, XXIII provided spiro compounds 36 in high yields and with excellent enantioselectivities. This procedure has been extended to oxidative cyclization of naphtolic sulfonamides with good enantioselectivity.

Scheme 14 A modification of Ishihara's type catalysts for the spirocyclization of naphtolic alcohols.

In their continuous efforts to exploit chiral hypervalent iodine catalysis for the creation of C-C bonds, Gong et al. have disclosed a highly enantioselective dearomatizative spirocyclization of derivatives 37, providing access to spiroxindoles derivatives 38 with good yields and enantioselectivity. 46 Precatalyst XVII was the aryliodine of choice for this transformation the presence of mCPBA as a stoichiometric oxidant. The reaction would proceed through the formation of phenoxy- 3 -iodine species 39, which would undergo intramolecular S N 2'-like Friedel-Crafts substitution. The addition of a mixture of trifluoroethanol and water was critical for the improvement of both yields and enantioselectivity (Scheme 15). [START_REF] Colomer | Mechanistic studies on the activation of iodine (III) derivatives with fluorinated alcohols: I[END_REF] As previously suggested by Ishihara et al., these additives would facilitate ligand exchanges to favor the enantioselective associative pathway over the racemic dissociative pathway.

Scheme 15

Oxidative C-C coupling-enabled naphtols dearomatization.

Intermolecular transformations

Asymmetric intermolecular phenol dearomatization is a highly challenging task as this transformation can easily proceed through a racemic dissociative pathway. 47 Quideau et al. have pioneered catalytic enantioselective hydroxylative phenol dearomatization. 48 Axially chiral monoiodobinaphtyl XXIV was used in the presence of a stoichiometric amount of mCPBA to convert 2-methylnaphtol 40 into epoxy ortho-quinol 41 in good yield and with moderate enantioselectivity. Unfortunately, epoxidation of the ortho-quinol by the stoichiometric oxidant cannot be avoided. Secondary I … O interactions between the iodine (III) atom and the methoxy group and/or the carboxylic acid function were assumed, but the real nature ( 3 or  5 ) of reacting hypervalent iodine involved in this transformation could not be determined with certainty.

Scheme 16 Intermolecular naphtol dearomatization.

Hypervalent

iodine-catalyzed intermolecular dearomatization of phenol can also enable introduction of a nucleophile at the para position of the hydroxyl group. Designing an enantioselective version of such a reaction seems to be even more arduous, as the creation of the stereogenic center occurs quite far away from the chiral environment (namely the phenoxy- 3 -chiral aryliodane). To address this challenge, Harned et al. designed C 1 -symmetric chiral iodine XXV derived from 8-iodotetralone and tartaric acid for the 4-Please do not adjust margins Please do not adjust margins hydroxylation of phenols 42 in the presence of mCPBA as the stoichiometric oxidant (Scheme 17). 49 The resulting 2,5cyclohexanedienones 43 were obtained in moderate yields and with enantioselectivity up to 60% ee. In 2017, Muñiz et

Functionalization of alkenes

Hypervalent iodine-mediated asymmetric difunctionalization of alkenes is a useful strategy for the preparation of molecules with the creation of one or two vicinal stereogenic centers from either two identical or different nucleophiles. 52

Intramolecular version

In its intramolecular version, such a reaction enables the construction of polysubstituted heterocycles in one step.

Lactonization.

The first example of a catalytic difunctionalization of alkenes mediated by chiral hypervalent iodine has been reported by Fujita et al. In their original reports, the authors disclosed an oxidative double cyclization of 2-(4hydroxybut-1-enyl)benzoates 44 (Scheme 18). 53 By using precatalyst XXVIII, mCPBA as the terminal oxidant and trifluoroacetic acid as an activator, the dihydrofuran-fused isochromanones 45 were obtained in moderate yields and enantioselectivities up to 91% ee. The reaction would proceed through the diastereofacial attack of the chiral iodane on the double bond to form iodonium 46. The latter would undergo nucleophilic substitutions by the internal hydroxy and carboxymethyl groups with inversion of configuration. The competitive direct oxidation of the double bound by mCPBA is responsible of the moderate yields.

Scheme 18 Oxidative double cyclization.

The background oxidation with mCPBA has been well identified by the same research group in the hypervalent-iodine catalyzed trifluoroacetoxylactonisation of ortho-alk-1enylbenzoates 47 (Scheme 19). 54 In this process, the catalytic oxidation mediated by chiral hypervalent iodine(III) species gives enantioenriched syn-products 48a and 48b via ring opening of iodonium 49, while the direct oxidation with mCPBA delivers racemic anti-products 48c and 48d via opening of epoxide 50. Good 6-endo regioselectivity and high enantioselectivity were obtained on cis-isochromanone 48a with precatalyst XXIX. Unfortunately, the competitive background oxidation with mCPBA of the electron-rich internal double bound significantly affected the yield. This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins Scheme 19 Oxy-lactonization of ortho-alk-1-enylbenzoates 47.

In 2017, Masson et al. reported a tosyloxy-and phosphoryloxy-lactonization of flexible 4-pentenoic acid derivatives 51 (Scheme 20). 55 By using XVII and a stoichiometric amount of mCPBA in the presence of sulfonic or phosphoric acid, excellent exo selectivity was observed, providing access to valuable y-butyrolactones 52 with good yields and good enantioselectivities. To minimize competitive direct oxidation of the terminal double bound by mCPBA, the reactions needs to be carried out at 0 °C. Addition of 2,2,2-trifluoroethanol (TFE) as a cosolvent was required to improve the yield of the phosphoryloxycyclization. [START_REF] Colomer | Mechanistic studies on the activation of iodine (III) derivatives with fluorinated alcohols: I[END_REF] From a mechanistic point of view, the authors proposed in situ generation of intermediate 53 from precatalyst XVII, mCPBA and the acid. Electrophilic attack of the double bound by this chiral  3 -aryliodane would furnish chiral aryliodonium 54. Intramolecular nucleophilic attack of the carboxy group with exo selectivity followed by substitution of the iodonium with the sulfonyl or phosphoryl group would afford y-lactone 52.

Scheme 20 Tosyloxy-and phosphoryloxy-lactonization of flexible 4-pentenoic acid 51.

The hypervalent iodine catalyzed fluorolactonization of ortho-alk-1-enylbenzoates 55 has been investigated by Jacobsen et al. (Scheme 21). 56 This method used HF•pyridine as a nucleophilic fluoride source with chiral precatalyst XXX and mCPBA, affording 3-alkyl-4-fluoroisichromanones 56 as a single syn diastereoisomer with moderate yields and high enantioselectivities. Noteworthily, the obtained regioselectivity (formation of the 6-membered ring) is complementary to that established in asymmetric fluorolactonization with an electrophilic fluoride source. This regioselectivity is explained by activation of the alkene by hypervalent iodine followed by addition of the fluoride anion onto the benzylic carbon, furnishing anti-vicinal fluoroiodonium intermediate 57. The latter would undergo intramolecular nucleophilic substitution by the carboxymethyl group with inversion of configuration providing syn-diastereoisomer 56.

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Please do not adjust margins 28 This transformation enabled the formation of 2-fluoropiperidine 59 with moderate yield and enantioselectivity. According to the observed 6-endo selectivity of the process, the aminofluorination would proceed through oxidation of the sulfonamide by in situ generated ArIF 2 , furnishing electrophilic species 60. The latter would react with the double bond to release the precatalyst and concomitantly form intermediary aziridinium 61. The latter would undergo nucleophilic attack of the fluoride on the tertiary carbon to deliver endo-type cyclized product 59. The best aryliodine precatalyst evaluated was C 2 axially chiral BINI VI. Strikingly, while a stoichiometric of chiral aryliodonium difluoride XXXIb has been reported to give 59 with 81% ee, 57 no enantioselectivity was observed with parent aryliodine XXXIa under catalytic conditions. This observation suggests different type of mechanisms between the catalytic and stoichiometric processes. Very recently, the highly enantioselective syn-fluoroaziridination of cynnamyltosylamide 62 has been disclosed by Jacobsen et al. (Scheme 23). 58 The optimal reaction conditions are similar to those previously reported by the group for fluorolactonization (see section 4.1.1) with XXXII as the precatalyst of choice. In contrast with the above fluoropiperidination reported by Kita, Shibata et al., the reaction would be initiated by oxidation of the electron-rich internal alkene by the in situ generated ArIF 2 furnishing an electrophilic iodonium. Nucleophilic attack by the excess of fluoride would generate anti-vicinal fluoroiodonium 63, which would be intramolecularly trapped by the sulfonamide to give fluoroaziridines 64. This reaction sequence accounts for the total syn-selectivity. The process is limited to electron deficient cinnamyl tosylamides since substrates lacking electron withdrawing substituents gave rise to 1,1-difluoromethylated products 66 via phenonium ion 65 (Scheme 23, R = H, EDG). Carbonyl-based protected amines (carbamates, amides) led to the formation of 1,2-oxy-fluorinated products via intramolecular nucleophilic attack of the carbonyl oxygen onto the fluoroiodonium rather than the nitrogen. This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins In 2014, Wirth et al. reported an elegant intramolecular diamination of homoallylic guanidine and diaminosulfone derivatives 67. 59 In this transformation, the two attacking nucleophiles are on the substrate. Activation of the double bound by the hypervalent iodine triggered the first nucleophilic attack of one nitrogen to the more substituted carbon. S N 2-type substitution of the resulting hypervalent moiety (intermediate 68) delivered bicyclic molecules 69. While poor enantioselectivity was obtained with the flexible C 2 -symmetric Ishihara's type catalyst, the design of a more rigid catalyst XXXIII featuring a pyridine moiety attached to a chiral benzylic center afforded the diamines with up to 86% ee. The pyridine nitrogen would coordinate iodine (III). The authors did not rule out also a possible coordination of the oxygen atom belonging to the methoxy group to iodine (III). It is worth pointing out that sodium perborate in the presence of acetic acid as an activator was the stoichiometric oxidant of choice with this catalyst instead of the commonly employed mCPBA. 60 By using C 2 -symmetric bislactamide XXXIV, peracetic acid as the terminal oxidant and triflic acid as a Brønsted acid co-catalyst in acetic acid, vicinal diacetoxylation of terminal styrenes 70 took place in good yields with up to 94% ee (Scheme 25). X-ray analyses and 1 H-NMR spectroscopy of the corresponding diacetate iodonium to XXXIV unveiled hydrogen bonding between the amide NH groups of the arms and ligand acetoxy, resulting in the creation of a supramolecular helical chirality around the central iodine (III) atom. From a mechanistic point of view, the catalytic cycle would start with the oxidation of XXXIV by peracetic acid in acetic acid to generate intermediary diacetoxyiodonium 71. Addition of triflic acid would allow the formation of a more reactive cationic catalyst state 72 61 63 In combination with mCPBA as stoichiometric oxidant and pyridine•9HF as nucleophilic fluorine source, the in situ generated reactive iodoarene difluoride would activate the internal alkene. Nucleophilic fluorination at the benzylic carbon would lead to anti-fluoroalkyl iodonium (III) intermediate 78. Anchimeric nucleophilic assistance by the neighboring amide group (red arrows) would allow the second fluorination to take place via intermediate 79 with an overall total anti-diasterosectivity to afford 1,2-difluorinated product 80. In the case of disubstituted cinnamide derivatives (R 2 = H), fluoroalkyl iodonium (III) intermediate 78 would preferentially undergo aryl migration (blue arrows) via phenonium ion 81 to afford gem-difluoro compounds 82 in good yields and with excellent enantioselectivities using XXXII (Scheme 27, right part). 64 This elegant migratory geminal difluorination has been extended to secondary and tertiary cinnamate ester derivatives. It is worth mentioning that, in both transformations, higher enantioselectivities were obtained with aryl iodine bearing benzylic versus aliphatic substituents at the stereogenic centers probably due to attractive -cation interactions in the transition states.

Diamination

In 2017, Muñiz et al. reported an iodine (I/III)-catalyzed enantioselective vicinal diamination of internal and external styrenes using mCPBA as a terminal oxidant and bismesylimide as a nitrogen source (Scheme 29). 66 The undesired epoxidation background reaction was advantageously reduced when MTBE/HFIP or MTBE/TFE solvent combination were used at low temperature. Tertiary amide iodoresorcinol derivative XXXVII was the catalyst of choice to provide diamines 85 in good yields and with excellent enantioselectivity. Since the previously observed supramolecular arrangement of resorcinol derivatives with secondary amides through intramolecular hydrogenbonding is not directly possible with this tertiary amide version, the intervention of water for the formation an analogous helical framework through intermolecular hydrogen-bonding has been proposed. From a mechanistic point of view, the authors This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins suggested the initial formation of key reactive catalyst diaminoiodonium 86 which would activate the alkene. Subsequent nucleophilic addition of the bismesylimide on the benzylic carbon would give anti-vicinal aminoalkyliodonium (III) 87. Intramolecular displacement of the iodonium by the bisulfonimide with inversion of configuration would liberate the catalyst and generate cyclic intermediate 88. Ring opening of this intermediate would allow the second C-N bond formation with second inversion of configuration, which would account for the anti-diastereoselectivity observed on diamines products 85.

Scheme 29 Vicinal diaminations of alkenes.

Conclusions

The blossoming research field of asymmetric iodine catalysis has been summarized. Since the pioneering iodoarenes of Wirth et al. with a chiral coordinating group ortho to the iodine atom, we have seen that a handful of chiral aryliodines has been developed and used efficiently as precatalyst of hypervalent iodine (III) to perform oxidative transformations with high enantiocontrol. Although the flexible and easily tunable C 2symmetric resorcinol derivative seems to be a privileged structure in many transformations, other compounds with either central, axial or planar chirality have appeared to be also suitable precatalysts. Chiral quaternary ammonium iodides have been also ingeniously employed as precursors of hypoiodites for iodine (-I/+I) catalysis. Both such catalytic systems have proven to be ideal methods for functionalization of carbonyl compounds, dearomatization of phenol derivatives and functionalization of alkenes with different type of nucleophiles (in an intra-or intermolecular way) in high enantioselectivities. Moreover, recent mechanistic investigations via X-Ray, NMR and computational analysis have allowed to shed on light on the structure of the reactive catalysts and the involved intermediaries in the catalytic cycles. These insights are expected to contribute in the near future to the design of new chiral iodine coupounds and their applications in new asymmetric reactions.

Figure 1

 1 Figure 1 Concept of iodine (I/III)-enabled enantioselective oxidative transformations and general structure of chiral ayliodine precatalysts.

7

 7

Figure 2

 2 Figure 2 Concept of iodine (-I/I)-enabled enantioselective oxidative transformations and general structure of chiral ammonium iodide precatalysts.

Scheme 1 2

 12 Scheme 1 First catalytic enantioselective -oxytosylation of ketones.

Figure 3

 3 Figure 3Proposed mechanisms for organoiodane-mediated phenol dearomatization.

Scheme 21

 21 Scheme 21 Hypervalent iodine catalysed asymmetric 6-endo-type-fluorolactonization.

Scheme 22

 22 Scheme 22Access to enantioenriched 3-aminopiperidine 59.

Scheme 23

 23 Scheme 23Synthesis of syn--fluoroaziridnes and competitive rearrangement.

Scheme 24

 24 Scheme 24 Intramolecular diamination leading to dicyclized product.

4. 2 .

 2 Intermolecular version 4.2.1. Dioxygenation. In 2016, Ishihara and Muñiz et al. reported the first intermolecular asymmetric alkene dioxygenation under hypervalent iodine catalysis.

Scheme 27 65 Scheme 28

 276528 Scheme 27Vicinal and geminal difluorination of alkenes.

  As mentioned above, the first catalytic version of this reaction was reported in 2007 by Wirth et al. Inspired by the works of Togo et al. who demonstrated a catalytic racemic version of this reaction,9 Wirth et al. employed organoiodane I with central chirality as the chiral inducer, meta-chloroperoxybenzoic acid (mCPBA) as the stoichiometric oxidant and p-toluenesulfonic acid monohydrate (TsOH•H 2 O) as a source of tosylate nucleophiles (Scheme 1).4 Contrarily to the process under stoichiometric conditions, 10 the catalytic transformation needs to be carried out at room temperature, which suggests that oxidation of the iodoarene is the rate determining step in the catalytic cycle.

Noteworthily, no racemization of the product in the reaction medium was observed. Nevertheless, only moderate enantioselectivities were obtained in this difficult transformation. From a mechanistic point of view, the authors proposed the in situ generation of a chiral Koser's-type reagent from I, mCPBA and TsOH•H 2 O, furnishing either intermediate 3 or 4. Subsequent nucleophilic addition of a tosylate anion on 3 (path A) or 4 (path B) via S N 2' and S N 2 type mechanisms would both provide enantioenriched ketone

  Spirobiindane scaffold in the -oxytosylation of ketones.

		67% ee, which is the best enantioselectivity obtained up to date
		from 1a under catalytic conditions. 18 Noteworthily, this catalyst
		is easily prepared in few steps involving a Palladium-catalyzed
		stereoselective cross-coupling. 19
	al. have tested 2,2'-diiodo-1,1'-binaphthyl (BINI) VI.Erreur ! Signet non défini. Poor enantioselectivity was obtained indicating that further tuning of this axially chiral backbone was required. In	Scheme 3 Scheme 4 Axially chiral biaryl precatalysts in the -oxytosylation of ketones.
	2013, Berthiol et al. introduced new C 2 -symmetric 3,3'-diiodo-
	BINOL-fused maleimide derivatives VIIa 16 and VIIb 17 that
	showed a stereochemical efficiency comparable to the best
	results previously reported (Scheme 4). In 2017, Masson et al.
	reported an axially chiral, non C 2 -symmetric iodoarene VIII
	bearing a sulfone substituent as a potentially chelating group of
	the iodine atom. This catalyst afforded (R)-2a in 75% yield and

  To avoid the passage through the Obonded intermediate 3 and favor the C-bonded intermediate 4,

	Legault et al. considered -oxytosylation of enolacetate 6
	(Scheme 5). With such a strategy, C 2 -symmetric 2-
	idodoresorcinol derivative IX offered the -oxytosylated ketone
	2 in good yield and with high enantioselectivity. 21 It is worth
	pointing out that very poor enantioselectivity was obtained
	during the direct -oxytosylation of ketone 2 with the same kind
	of catalyst. This observation supports two different mechanisms
	between the α-oxytosylation of ketone and enol acetate: S N 2'-
	type reductive elimination of a O-enolate intermediate in the
	former case providing low enantiocontrol in the former case
	and highly enantioselective S N 2 substitution of a C-enolate in
	the latter case. 20
	Scheme 5 -oxytosylation of enol acetates.

  which no longer exhibits C 2 -symmetry. Upon oxidation of the styrene and subsequent nucleophilic attack of acetate, intermediate 73 would be formed. Intramolecular nucleophilic addition of the acetate would regenerate catalyst XXXIV along with the formation of dioxolonium 74. The latter would undergo ring opening by acetate or hydrolysis to provide vicinally dioxygenated product 75 upon treatment with acetic anhydride.Muñiz et al. have also demonstrated the use of bislactateXXXV to efficiently catalyze vicinal diacetoxylation of external and internal styrenes with up to 96% ee (Scheme 26).62 This protocol relies on the use of Selectfluor as the terminal oxidant Please do not adjust margins Please do not adjust margins (to avoid epoxidation of the alkene observed with the commonly employed peracids) and a stoichiometric amount of trimethylsilyltriflate as an iodonium co-activator. In 2016, Jacobsen et al. described asymmetric vicinal difluorination of trisubstituted cinnamide derivative 76 in 93% ee and with excellent antidiastereoselectivity using resorcinol-based diester XXXVI (Scheme 27, left part).

	Scheme 26 Diacetoxylation of styrene with bis-lactate XXXV and selectfluor.
	4.2.2. Difluorination.
	Scheme 25 Diacetoxylation of styrene.

Institut de Chimie des Substances Naturelles, CNRS UPR 2301Université Paris-Sud, Université Paris-Saclay 1 av. de la Terrasse, 91198 Gif-sur-Yvette (France) e-mail: aurelie.claraz@cnrs.fr; geraldine.masson@cnrs.fr homepage: http://www.icsn.cnrs-gif.fr/spip.php?article848&lang=en

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 5

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 11

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 13

Acknowledgements

The CNRS is gratefully acknowledged for financial supports.

Notes and references

Conflicts of interest

There are no conflicts to declare.


Franck Vandermoere

Ikram El

Christian Slomianny

Yohann Demont

Gabriel Bidaux

Eric Adriaenssens

Jérôme Lemoine

Hubert Hondermarck
email: hubert.hondermarck@univ-lille1.fr

Ikram El Yazidi-Belkoura

The Valosin-containing Protein (VCP) Is a Target of Akt Signaling Required for Cell Survival

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

The serine/threonine kinase Akt is a key mediator of cell survival and growth, but its precise mechanism of action and more specifically the nature of its signaling partners largely remains to be elucidated. Here we show, using a proteomics-based approach, that the valosin-containing protein (VCP), a member of the AAA (ATPases associated with a variety of cellular activities) family, is a target of Akt signaling. SDS-PAGE of Akt coimmunoprecipitated proteins obtained from MCF-7 breast cancer cells revealed the increase of a 97 kDa band under Akt activation. Mass spectrometry analysis allowed the identification of VCP and we have shown a serine/threonine phosphorylation on a Akt consensus site upon activation by growth factors. Site-directed mutagenesis identified Ser351, Ser745, and Ser747 as Akt phosphorylation sites on VCP. Confocal microscopy indicated a co-localisation between Akt and VCP upon Akt stimulation. Interestingly, siRNA against VCP induced an inhibition of the growth factor-induced activation of NF-kB and a potent proapoptotic effect. Together, these data identify VCP as an essential target of Akt signaling.

The valosin-containing protein (VCP) belongs to the AAA (ATPases associated with various cellular activities) family, whose members are characterized by having highly conserved ATPase domain(s) with high sequence similarities and ring structures consisting of homo-oligomers (1,2). VCP, also known as VAT in archaebacteria, CDC48p in yeast, TER94 in Drosophilia and p97 in Xenopus, is one of the most evolutionarily conserved proteins that is ubiquitous and abundant in cells, accounting for more than 1% of total cellular proteins. Like other AAA proteins, VCP has been shown to be involved in a wide variety of ATP-dependent cellular processes such as ubiquitin-mediated proteolysis, DNA repair, membrane fusion and dynamic of subcellular compartments, gene expression and cell growth. Admittedly, it acts as a molecular chaperone that unfolds or unwinds proteins, although the detailed mechanisms of VCP function remain to be determined, as well as its regulation mechanisms in both physiological and pathological conditions.

During the course of studying the molecular partners of the serine/threonine kinase Akt, a key regulator of cell survival that is activated by growth factors, we detected VCP in Akt coimmunoprecipitated material. The interaction between Akt and VCP was further studied and from the functional point of view, we showed that disruption of VCP using siRNA impaired the cell survival signaling mediated by Akt. We propose that VCP is an important player in the Akt-mediated signaling of cell survival.

EXPERIMENTAL PROCEDURES

Materials-Cell culture reagents were purchased from Biowhittaker (France). Recombinant human fibroblast growth factor-2 (FGF-2) was from R&D systems (France). Wortmannin (PI3K inhibitor) was from Calbiochem (France). C2 ceramide analogue (N-acetyl-D-sphingosine), 5-Fluorouracil, camptothecin, etoposide, Hoechst 33258, G250 brilliant blue, DMSO and peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G antibody were from Sigma (France). Dynabeads M-280 tosylactivated were from Dynal Biotech (France). Constitutively active Akt and Wild-type Akt pUSE encoding plasmids were from Upstate (NY).

Cell culture, NF-κB activation, and induction of apoptosis-Breast cancer cell lines MCF-7, T-47D, BT-20 were obtained from the American Type Culture Collection and routinely grown as monolayers as previously described (3). Apoptosis of MCF-7 cells was induced by treatment with ceramide analogue C2 (10 µM for 24 h), previously described as a pro-apoptotic agent for human breast cancer cells (4). The determination of both the NF-κB activation (luciferase reporter assay) and the proportion of cells in apoptosis (Hoechst staining) were performed as described (3). For Apoptosis induction by chemotherapeutic agents, MCF-7 breast cancer cells were treated 3h with 5-Fluorouracil (100 µM), camptothecin (0,1 nM) or etoposide (10 µM) and replaced in new culture medium. After 24 h, cells were fixed with cold methanol (-20°C) for 10 min and washed twice with phosphate-buffered saline (PBS) before staining with 1µg/mL Hoechst 33258 and apoptosis quantification.

Immunoprecipitation of Akt and interacting

proteins-MCF-7 cells were rinsed by phosphatebuffered saline (PBS) pH 7.5, serum-starved for 3h in minimum essential medium (MEM-Earle's salts) and treated for 24 h by 10 µM C2 ceramide in fresh minimal essential medium. Proteins were extracted from cells after 10 min of 5 ng/mL FGF-2 stimulation in 100 µM pervanadate. To inhibit PI3K/Akt, cells were pretreated for 3 h by 100 nM wortmannin before FGF-2 stimulation. Then, cells were rinsed with PBS pH 7.5 and scratched in lysis buffer (150 mM NaCl, 50 mM Tris pH 7.5, 0.1% SDS, 1% Nonidet P-40, 100 µM sodium orthovanadate). After centrifugation (10, 000 g, 5 min), proteins were quantified using Bradford's method (Bio-Rad). Akt and interacting proteins were coimmunoprecipitated from equal quantities of proteins using antibodies covalently bound to magnetic beads, according to Dynal biotech protocole using 2.5 µg of anti-Akt for 500 µg of total proteins. Immunoprecipitated material was rinsed three times by PBS pH 7.5 before a 5 min boiling in Laemmli buffer 1X.

Gradient

gel electrophoresis-Akt immunoprecipitated material was solubilized in SDS solution (0.3% SDS and 1% ß-mercaptoethanol) before SDS-PAGE migration and colloidal blue staining (G250 brilliant blue).

Protein identification by mass spectrometry-Protein identification was performed by Matrix Assisted Laser Desorption Ionisation-Time Of Flight (MALDI-TOF) and tandem mass spectrometry (MS-MS). The protein band of interest was trypsin digested as previously described (5). MALDI-TOF and MS-MS analysis of the trypsin digests were performed on a Voyager reflector instrument (Applied biosystems) and a Q-STAR (Perseptive biosystems) in positive ion mode. For MALDI-TOF, mass measurements were made after peak smoothing and internal calibration using the average mass of the two autolysis trypsin fragment ions at m/z 842.510 and 2211.105. Protein sequence database searching was performed with MS-Fit (http://prospector.ucsf.edu/ucsfhtml4.0/msfit.htm ) and Mascot (http://www.matrixscience.com/).

Western-blotting-Co-immunoprecipated proteins were separated by 10% SDS-PAGE and Western blotted with anti-VCP (1/2000), anti-Akt and anti-phospho-(Ser/Thr) Akt substrate antibodies in experimental conditions described in (3).

Confocal microscopy-After Akt activation using FGF-2, cells were successively rinsed with PBS pH 7.5, fixed 20 min with paraformaldehyde 4%, permeabilized for 20 min at room temperature with PBS pH 7.5; 0.05% saponine; 50 mM ammonium chloride and blocked for 30 min with PBS pH 7.5; 0.05% saponine; 2% bovine serumalbumine. Then, cells were incubated for 2h at room temperature in a blocking solution containing 1/50 rabbit phospho-Akt antibody (cell Signaling, UK) and 1/50 mouse VCP antibody (Abcam, UK), rinsed with PBS pH 7.5, incubated for 1h at 37°C in a blocking solution containing 1/4000 alexa fluor 546 goat antirabbit IgG and 1/3000 alexa fluor 488 goat antimouse IgG. Cells were rinsed with PBS and mounted under mowiol.

VCP RNA interference-VCP siRNA was designed and validated as described by Wójcik et al. (6). Corresponding sequences are Forward 5′-TAAGACGCTGCTGGCCAA-3′ and Reverse 5′-CATCGCGGAGTTCTGATTTT-3′. The siRNAs were synthesized using the Silencer® siRNA construction kit (Ambion, Austin, TX) according to manufacturer's instructions and transfection was performed as previously described (7).

Site-directed mutagenesis of VCP-CDNA of VCP was obtained by PCR on MCF-7 cells cDNA bank using following primers that incorporatd Knp1 site and Not1 site at respectively 5' and 3' ends of the cDNA of VCP: « Vcp-dir+KpnI » 5'-ATCGGTACCGCTCGTAGCCGTTACC-3', « Vcp-rev+NotI » 5'-ATGCGGCCGCTGAACTGTTCAGACTGGA G-3'. PCR product was inserted and in topoA vector and amplified in E. coli bacteria. Then double digestion by Knp1 and Not1 was used to insert VCP cDNA in CMVTNT vector that allowed expression in mammalian cells. VCP serine 352 mutation to alanine S351A, S745A, S747A, and a triple mutant were carried out using PCR with mutant primers. The primers used to generate the mutants were as follows: S351A:5'GACCCAACGCCATTGACCC3', S745A:5'GCCCGACGTGCTGTCAGCG-3', S747A:5'CGACGTTCTGTCGACGATAATGA C-3', S745AS747A:5'CGACGTGCTGTCGC CGATAATG-3'. Cloning and mutation were confirmed by DNA sequencing. Expression of pCMVTNT-VCP, pCMVTNT-VCPS351A, pCMVTNT-VCPS745A, pCMVTNT-VCPS747A, and pCMVTNT-VCPS745AS747A plasmids was carried out by transfection in MCF-7 cells.

RESULTS

VCP is co-immunoprecipitated with activated

Akt. The protocole used for identification of VCP in Akt co-immunoprecipitates is summarized in Fig. 1A. Immunoprecipitates obtained with Akt antibodies were resolved on SDS-polyacrylamide gel. Two experimental conditions were compared. In the first, Akt was not activated and in the second, Akt was activated by cell stimulation with FGF-2. Silver staining or Coomassie blue staining of the gel revealed the appearance of a 97 kDa band in the Akt stimulated condition (Fig. 1B). The equiloading was assessed by detection of Akt (60kDa) and IgG (70 kDa). The 97 kDa band was cut out and trypsin digested before mass spectrometry analysis. Mass spectra from MALDI-TOF (Fig. 1C) and MS-MS (Fig. 1D) allowed the establishment of a peptide mass fingerprint and aminoacid sequence respectively, thereby leading to the identification of VCP.

From the peptide fingerprint, a total of 31 experimental masses matched theoretical masses from VCP, leading to a protein sequence covering of 23% and this identification was reinforced by the sequencing of 12 consecutive amino acids (Table 1).

The interaction between Akt and VCP is dependent on Akt activation and leads to VCP phosphorylation. The interaction between Akt and VCP was further confirmed by confocal microscopy showing a cellular co-localization of VCP and activated Akt (pAkt) upon growth factor stimulation (Fig. 2A). Importantly, reverse co-immunoprecipitation, with anti-VCP antibody, followed by anti-Akt Western-blotting led to the same result (Fig. 2B), hence confirming the interaction between Akt and VCP upon fibroblast growth factor-2 stimulation. The interaction was further confirmed in different breast cancer cell types and under stimulation by insulin-like growth factor 1 and epidermal growth factor (Fig. 2C). Moreover, the interaction between VCP and Akt was abolished by treatment of the cells with the PI3K/Akt pharmacological inhibitor wortmannin (Fig. 2B), thus indicating the requirement of Akt activation for interaction with VCP. Using anti-phospho-Akt substrate (PAS) antibody, that recognizes phosphorylated serine or threonine in a consensus site for Akt, we have been able to show that Akt activation resulted in VCP phosphorylation (Fig. 2B). This Western-blotting experiment showed that VCP contained a serine or a threonine phosphorylated, upon growth factor stimulation, on a site corresponding to Akt phosphorylation consensus. Interestingly, treatment by wortmannin resulted in a decrease in VCP phosphorylation, confirming that VCP is a target of Akt signaling.

Identification of Akt phosphorylation sites on VCP.

In our study, we demonstrated that FGF-2 stimulates both the interaction between Akt and VCP and also the phosphorylation of VCP on a site recognized by PAS antibody. The Aktphosphorylated consensus site is commonly defined as RXRXX(S/T) (8,9). We performed bioinformatics search to identify putative Akt phosphorylation site on VCP using the Scansite software (http://scansite.mit.edu/motifscanner). The putative Akt phosphorylation sites on VCP are AATNRPNS351, AMRFARRS745 and RFARRSVS747. To test the involvement of S351, S745, and S747 we performed site-directed mutagenesis and we generated pCMVTNT-VCP S351A , pCMVTNT-VCP S745A , and pCMVTNT-VCP S747A and pCMVTNT-VCP S351AS745AS747A . These VCP wild type and mutant cDNAs were transfected in MCF-7 cells in parallel with Akt constitutively activated (AktCA) and after immunoprecipitation, phosphorylation on Akt consensus site was detected with PAS antibody. The results (Fig. 2D) show that wild type VCP was phosphorylated on PAS consensus to a greater extent as compared to S351A, S745A and S748A mutants. The triple mutant S351A-S745A-S747A was phosphorylated to an even lesser extent as no phosphorylation was detected. These results indicate that S351, S745 and S747 are Akt phosphorylation sites on VCP.

VCP is functionally involved in Akt antiapoptotic signaling and is required for cell survival. The functional involvement of VCP in Akt signaling was shown by the treatment of MCF-7 cells with siRNA directed against Akt or VCP. Treatment by 100 nM siRNA directed against Akt or VCP resulted in a strong reduction of the pro-survival effect of FGF-2 for breast cancer cells (Fig. 3A). Similarly, transfection with the triple VCP mutant S351A-S745A-S747A resulted in a reduction of FGF-2 antiapoptotic activity confirming the functional involvement of the detected phosphorylation site. Negative dominant Akt also resulted in the inhibition of FGF-2 survival effect, in contrast with constitutively activated Akt. It has been previously demonstrated that the Akt-mediated anti-antiapoptotic signaling in breast cancer cells requires the activation of the nuclear factor kappaB (NF-κB) (7). We have evaluated the impact of VCP on NF-kB activation induced by Akt. Treatment with 100 nM siRNA directed against Akt or VCP, as well as transfection with dominant negative Akt or VCP mutant S351A-S745A-S747A resulted in a strong inhibition of NF-κB activation by FGF-2 (Fig. 3B). The efficiency of cell treatment by siRNA directed against Akt and VCP was demonstrated by Western-blotting showing the disappearance of Akt and VCP with increasing concentrations of siRNA (Fig. 3C). Together, these data show that VCP is involved in the growth factor-stimulated antiapoptotic signaling mediated by Akt.

Interestingly, high concentrations of siRNA against VCP were shown to induce a potent apoptosis in breast cancer cells that was not rescued by overexpression of a dominant-active Akt mutant (Fig. 4A). Cell treatment with increasing concentration of siRNA against VCP resulted in increase of breast cancer cell death, and it is worth noting that for identical concentrations, no apoptosis was induced by the control siRNA. For example, 50% of apoptosis was observed for 150 nM siRNA against VCP after a 48 h treatment, whereas no induction of cell death was detected with control siRNA. In conclusion, VCP depletion dramatically induced, by itself, a massive apoptosis in breast cancer cells, demonstrating the crucial role played by this protein in cell survival. Interestingly, siRNA directed against VCP were found to induce a decrease in the level of phospho-IκBα (Fig. 4B). This is consistent with the decrease of NF-κB activation that was also observed when cells were treated with siRNA against VCP (Fig. 3B). Moreover, we have shown that depletion in VCP resulted in a potentiation of the proapoptotic effet of various chemotherapeutic drugs (Fig. 4C). The proapototic effect of 5FU, camptothecine and etoposide obtained with MCF-7 cells was potentiated by siRNA directed against VCP (at the 100nM dose that has no apoptotic effect by itself). This demonstrates the significance of VCP under conditions of apoptosis induced by anti-cancer treatment drugs.

DISCUSSION

The serine/threonine kinase Akt is increasingly appearing as a general mediator of cell survival signaling, with high potentialities as a drug target in various diseases characterized by a deregulation of the balance between cell apoptosis and cell growth, such as cancer (10). Akt is mainly, but not exclusively, activated through the phosphatidylinositol 3-kinase (PI3-K) pathway that is stimulated by a wide range of tyrosine kinase-growth factor receptors. Although Akt signaling remains incompletely known, it noticeably promotes cell survival by indirectly activating the pro-survival transcription factor NF-κB, through the stimulation of the inhibitor I-kappaB kinase (IΚΚ). Akt activation of IKK results in the phosphorylation and subsequent degradation of IκB through the ubiquitin-mediated proteasome proteolysis, allowing NF-κB to translocate into the nucleus where it stimulates target gene expression (11). Due to its involvement in several pathologies such as cancer, the signaling initiated by Akt is the subject of increasing attention and we indicate here the involvement of VCP in the growth factor-induced antiapoptotic signaling mediated by the Akt/NF-kB pathway. In contrast to classical approaches to studying signal transduction (based on the use of specific antibodies to identify signaling partners), proteomic approaches have the advantage of allowing the identification of unexpected partners and this is of considerable potential for defining new therapeutic targets in pathologies such as breast cancer (12). Interestingly, VCP has previously been shown to associate directly with the alpha form of IκB (IκBα) and is co-purified with the mammalian 26S proteasome involved in the ubiquitindependent proteasome degradation pathway of IκBα (13,14). In breast cancer cells, growth factors such as fibroblast growth factor-2 (FGF-2) and nerve growth factor (NGF) have been shown to stimulate cell growth and survival through the activation of a PI3-K/Akt/NFκB mediated pathway (7,15,16). In this context, our results indicate that the NF-κB activation and the subsequent effects on cell survival are functionally regulated through the recruitment and phosphorylation of VCP by the Akt pathway.

The Akt-phosphorylated consensus site is commonly defined as RXRXX(S/T) (8,9). In fact, the primary sequence of VCP does not contain the precise consensus sequence predicted for an Akt substrate: the arginine at position -5 is lacking. However, VCP encodes a peptide sequence that is consistent with a pattern of other Akt-phosphorylated targets, where Arginine at position -5 is not present as in insulin-response element-binding protein 1 (KERCQS1036) (17), CREB LSRRPS133Y (18) and ATP-citrate lyase (TPAPSRTASF) (19). The bioinformatic analysis performed with scansite indicated three potential Akt phosphorylation site in VCP and we have used site-directed mutagenesis to confirm their involvement. We demonstrated that S351, S745 and S747 on VCP are phosphorylated on a site recognized by PAS antibody corresponding to Akt consensus site. The phosphospecific Akt substrate antibody (PAS) that we have used here was raised against Akt RXRXX(pS/pT) consensus sequence, but does not appear to strictly require arginine in the -5 position for phosphoprotein recognition, as was obtained for ATP-citrate lyase (19). Interestingly it has very recently been shown, in rat hippocampal tissue, a functional interation between Akt and VCP that also leads to VCP phosphorylation on the same corresponding serine residues (20). Therefore, these three serine residues appear to be generally crucial for VCP regulation by Akt.

VCP is known to interact with other adaptor proteins, which are proposed as functionning in various cellular pathways. The list of proteins known to interact with VCP includes clathrin (21), the p47 membrane fusion required for organelle biogenesis (22), the VCP p97/p47 complex-interacting protein (VCPIP135) (23), the small VCP-interacting protein (SVIP) (24) and the breast/ovarian cancer susceptibility gene product (BRCA1) (25). BRCA1 is of particular interest in the context of breast cancer, as this protein is one of the most prominent players identified in breast tumorigenesis, with 50% of familial breast tumors exhibiting BRCA1 mutations (26). BRCA1 presumably acts as a DNA unwinding factor in DNA damage repair, although VCP is predominantly found in the cytoplasm and less abundantly in the nucleus. However, VCP can be translocated to the nucleus and in vitro studies have revealed that VCP, via its N-terminal region, binds to amino acid residues 303-625 in the BRCA1 protein (25). Interestingly, the cell growth regulator heregulin has been shown to induce phosphorylation of BRCA1 through PI3-Kinase/Akt in breast cancer cells (27), and therefore our data suggest that VCP might participate in the interaction between Akt and BRCA1, leading to DNA damage-repair function and an antiapoptotic effect under growth factor stimulation. However, our study also suggests that other known biological activities of VCP, such as those related to intracellular trafficking, ubiquitin mediated proteolysis and activation of transcription ( 28) might be regulated by Akt through the activation of VCP. In our study, a potent induction of apoptosis was observed in breast cancer cells treated with siRNA against VCP, thereby demonstrating the crucial role played by this protein in the survival of cancer cells. VCP has been described as an important regulator of protein ubiquitination and interestingly it was reported before that VCP is involved in the ubiquitin-proteasome-mediated degradation of IκB (13). In our study, we demonstrated that siRNA directed against VCP induced a decrease in the level of phospho-IκB. As the phosphorylation of IκB is the germane event leading to the proteolytic proteasome-mediated degradation of IκB responsible for the release and nuclear translocation of NF-κB, the decrease in phospho-IκB that we observe with siRNA directed against VCP is consistent with the concomitant inhibition of NF-κB and provides a biochemical basis for the cell survival activity of VCP. In addition, the potential of VCP as a therapeutic target is particularly well illustrated by the potentiation of the proapototic effect of chemotherapeutic drugs that we have obtained with anti-VCP siRNA. Hence, VCP targeting appears as a rational approach to sensitize breast cancer cells to chemotherapy.

In conclusion, we identified VCP as an essential new target in the Akt signaling pathway that is necessary to its related antiapoptotic effect and therefore, this protein appears as a new player to be considered in cancer cell survival, with a correlative potential as a target for cancer therapy. Activated Akt appears in red and VCP in green. B, Reverse co-immunoprecipitation with anti-VCP was separated by SDS-PAGE before Western-blotting using anti-Akt. A band of 60 kDa, corrresponding to Akt was found in Akt activated conditions, confirming the interaction between Akt and VCP. The PI3K/Akt pharmacological inhibitor wortmannin inhibited the interaction between Akt and VCP. In addition, immunoprecipitated VCP was resolved by SDS-PAGE before Western-blotting using PAS (phospho-Akt substrate) antibody. The 97 kDa band corresponding to VCP was found in Akt-activated conditions, confirming that interaction between Akt and VCP correlated with phosphorylation of VCP on an Akt consensus site. The PI3K/Akt pharmacological inhibitor wortmannin inhibited this phosphorylation. C, The same co-immunoprecipitation experiment was reproduced with stimulation of Akt by three different growth factors: fibroblast growth factor-2 (FGF-2), insulin-like growth factor-1 (IGF-1) and epidermal growth factor (EGF). D, MCF-7 cells were transfected with pCMVTNT plasmids encoding VCP wild type, VCP S351A, VCP S745A, or VCP S351AS745AS747A and cotransfected with empty vector or vector encoding constitutively active Akt (AktCA). Immunoprecipitated VCP was then resolved by SDS-PAGE before Western-blotting using PAS antibody. on breast cancer cell apoptosis. MCF-7 cells were transfected with increasing amounts (0, 50, 100, 150, 200 nM) of siRNA directed against VCP (or control siRNA) and cotransfected with plasmid encoding Akt constitutively active (AktCA). After Hoescht staining, apoptotic cells were counted and results are expressed as the mean +/-SD of five independent experiments. B, Western-blotting detection of phospho-IκBα in MCF-7 cells treated with increasing concentrations (same as before) siRNA directed against VCP. C, MCF-7 cells were transfected with 100 nM siRNA against VCP or with nontargeting siRNA and were treated with C2, 5-Fluorouracil (5FU), camptothecin (campto.) or etoposide (eto.) before serum deprivation. Percentage of apoptotic cells was determined after Hoescht staining.

FGF-2 control

Figure 1AB

Vandermoere et al.

Figure 1D

Vandermoere et al.

Figure 1 .

 1 Figure1. VCP is co-immunoprecipitated with activated Akt. A, Akt co-immunoprecipitated material was resolved on SDS-polyacrylamide gel. Two experimental conditions were compared: Akt activated by cell stimulation with FGF-2 for 15 min versus the control in which Akt was not activated. The 97 kDa protein band was analyzed by mass spectrometry (MALDI-TOF and MS/MS) after trypsin digestion. B, Silver staining of the SDS-PAGE gel revealed the appearance of a 97 kDa band in the Akt stimulated condition. The levels of both immunoprecipitated-Akt and IgG were identical under the two conditions. C, Peptide mass fingerprint of the trypsin-digested 97 kDa band obtained by MALDI-TOF analysis. D, ESI-MS-MS spectrum, corresponding to the 1328 Da peptide, allowing the determination of a 12 amino-acid sequence corresponding to VCP.

Figure 2 .

 2 Figure 2. Interaction between Akt and VCP is dependent on Akt activation. A, Confocal microscopy revealed the co-localization of VCP and Akt upon FGF-2 induced stimulation of Akt.Activated Akt appears in red and VCP in green. B, Reverse co-immunoprecipitation with anti-VCP was separated by SDS-PAGE before Western-blotting using anti-Akt. A band of 60 kDa, corrresponding to Akt was found in Akt activated conditions, confirming the interaction between Akt and VCP. The PI3K/Akt pharmacological inhibitor wortmannin inhibited the interaction between Akt and VCP. In addition, immunoprecipitated VCP was resolved by SDS-PAGE before Western-blotting using PAS (phospho-Akt substrate) antibody. The 97 kDa band corresponding to VCP was found in Akt-activated conditions, confirming that interaction between Akt and VCP correlated with phosphorylation of VCP on an Akt consensus site. The PI3K/Akt pharmacological inhibitor wortmannin inhibited this phosphorylation. C, The same co-immunoprecipitation experiment was reproduced with stimulation of Akt by three different growth factors: fibroblast growth factor-2 (FGF-2), insulin-like growth factor-1 (IGF-1) and epidermal growth factor (EGF). D, MCF-7 cells were transfected with pCMVTNT plasmids encoding VCP wild type, VCP S351A, VCP S745A, or VCP S351AS745AS747A and cotransfected with empty vector or vector encoding constitutively active Akt (AktCA). Immunoprecipitated VCP was then resolved by SDS-PAGE before Western-blotting using PAS antibody.

Figure 3 .

 3 Figure 3. VCP is functionally involved in Akt mediated antiapoptotic signaling. A, Quantification of apoptosis with Hoechst staining revealed that siRNA against VCP or Akt (100 nM) inhibited the antiapoptotic effect of FGF-2. B, Transfection of MCF-7 cells with siRNA directed against Akt or VCP or transfection with plasmid encoding a negative dominant Akt, resulted in a strong reduction of NF-κB activation by FGF-2. In contrast, transfection with plasmid encoding a constitutively active form of Akt, mimicked the stimulation of NF-kB induced by FGF-2. C, Western-blotting detection of Akt and VCP levels in cells treated by increasing concentrations of siRNA. The efficiency of siRNA was tested 48 h after cell transfection.

Figure 4 .

 4 Figure 4. VCP is required for breast cancer cells survival. A, Dose-effect of siRNA against VCP

  Figure 4CVandermoere et al.

Table 1 .

 1 Identification of VCP, from MALDI-TOF obtained peptide masses. The amino acid sequence from the Swiss-Prot database of each matching peptide, the delta mass between measured and matched masses and the number of cleavages missed by trypsin are reported. The identification of VCP was comforted by the full MS/MS sequencing of the underlined peptide.

	MH+ submitted MH+ matched delta mass (Da) Miss peptide sequence consistant with mass
	783.42	783.36	0.06	0 506FGMTPSK512 Oxidation (M)
	797.40	797.40	0.00	1 360FGRFDR365
	819.47	819.46	0.01	0 149GDIFLVR155
	837.52	837.52	-0.01	0 232AIGVKPPR239
	947.58	947.56	0.02	1 148KGDIFLVR155
	951.50	951.47	0.03	1 288KAFEEAEK295
	986.54	986.53	0.02	0 218EMVELPLR225
	1033.54	1033.52	0.02	1 746SVSDNDIRK754
	1034.46	1034.43	0.03	0 734DHFEEAMR741
	1049.55	1049.54	0.01	0 669DVDLEFLAK677
	1050.48	1050.42	0.06	0 734DHFEEAMR741 Oxidation (M)
	1051.53	1051.52	0.01	0 46MDELQLFR53
	1067.53	1067.51	0.01	0 46MDELQLFR53 Oxidation (M)
	1075.55	1075.53	0.02	0 278LAGESESNLR287
	1118.60	1118.57	0.02	1 701ESIESEIRR709
	1172.68	1172.66	0.02	0 240GILLYGPPGTGK251
	1190.58	1190.53	0.05	1 733RDHFEEAMR741
	1203.65	1203.63	0.02	1 278LAGESESNLRK288
	1206.59	1206.52	0.06	1 733RDHFEEAMR741 Oxidation (M)
	1329.71	1329.68	0.03	0 454WALSQSNPSALR465
	1629.84	1629.80	0.04	1 754KYEMFAQTLQQSR766
	1645.82	1645.79	0.03	1 754KYEMFAQTLQQSR766 Oxidation (M)
	1660.83	1660.82	0.01	1 363FDREVDIGIPDATGR377
	1667.85	1667.94	-0.09	1 212QLAQIKEMVELPLR225
	1695.87	1695.82	0.04	0 600VINQILTEMDGMSTK614 Oxidation (M)
	1711.87	1711.82	0.05	0 600VINQILTEMDGMSTK614 2 Oxidation (M)
	1823.95	1823.92	0.03	1 600VINQILTEMDGMSTKK615 Oxidation (M)
	1839.93	1839.91	0.02	1 600VINQILTEMDGMSTKK615 2 Oxidation (M)
	2156.00	2155.97	0.03	0 714QTNPSAMEVEEDDPVPEIR732
	2312.10	2312.07	0.02	1 714QTNPSAMEVEEDDPVPEIRR733
	2328.08	2328.07	0.01	1 714QTNPSAMEVEEDDPVPEIRR733 Oxidation (M)

Table 1 .

 1 Identification of VCP, from MALDI-TOF obtained peptide masses. The amino acid sequence from the Swiss-Prot database of each matching peptide, the delta mass between measured and matched masses and the number of cleavages missed by trypsin are reported. The identification of VCP was comforted by the full MS/MS sequencing of the underlined peptide.

FOOTNOTES

This work is supported by grants from the Ligue Nationale Contre le Cancer, the Association pour la Recherche sur le Cancer (Contract 3305), the Fondation pour la Recherche Médicale (FRM comité du Nord), the Institut Universitaire de France, the Region Nord-pas-de-Calais and the Genopole of Lille. F V was a recipient of a fellowship from the French Ministry for Research and Education and the Ligue National Contre le Cancer.

A.

Figure 3AB

Vandermoere et al.

B.

Normalized NF-κB activity


Jessica El Khoury
email: jessica.el-khoury@u-bourgogne.fr

The impact of noise estimation on dehazing

Keywords: dehazing, denoising, image quality, color

Under haze or fog, the quality of the images is degraded due to the atmosphere, causing the details of the images to be difficult to identify by observers and computer vision systems. Such images contain noise, which is mainly due either to environment (extrinsic noise) or sensor (intrinsic noise). As the transmission of light coming from the scenes' objects is exponentially attenuated and comes quickly down to zero in presence of haze, the noise is greatly amplified at high haze densities and long distances. In order to investigate the importance of the accurate estimation and the removal of noise from hazy images, we used the CHIC (Color Hazy Image for Comparison) database, which provides, for a given scene, the haze-free image and a set of images with different haze densities. For each scene, several parameters are available like the distance from the camera of known objects such as Macbeth Color Checkers, their radiance, and the haze level through transmittance. At two levels of haze, we added some Gaussian noise. We first applied dehazing without considering the induced noise. Later, we applied it with including the accurate value of added noise and finally by using biased values. This study shows the importance to estimate as accurately as possible the noise in order to remove it and guarantee a high quality and a good recovery of images' features after dehazing.

INTRODUCTION

Bad visibility can be caused by the presence of particles in the atmosphere. The nature of these particles is determined by the causing phenomenon: it could be haze, fog, smoke or pollution. Although these phenomena have different natures, they all degrade severely the atmospheric visibility, depending on their density.

To deal with these phenomena produced either by human activities or nature, a wide number of dehazing approaches have been developed in the last decade to increase visibility. Earlier methods favored multiple data acquisition to break down the ill-posedness of the formulation of visibility degradation (Equation 1). Besides the RGB hazy image, the additional data could be a near infrared image, a differently polarized image or a depth map [START_REF] Srinivasa | Contrast restoration of weather degraded images[END_REF][START_REF] Schechner | Polarization based vision through haze[END_REF]Kopf et al. 2008). With the multiplication of computer vision applications for driving assistance, air navigation and outdoor surveillance, researchers have been focusing on single image dehazing for several years. This is because single image dehazing requires only the degraded image provided by a simple and an affordable imaging system.

Several dehazing approaches are currently available. In some of them, haze is considered as a contrastreducing veil. Thus, classical contrast enhancement methods have been applied on hazy images [START_REF] Xu | Fog removal from color images using contrast limited adaptive histogram equalization[END_REF]. Other approaches consider the physical aspect of haze, through the estimation of the physical parameters of the haze model. The estimation of parameters is usually based on hypotheses made upon observations on hazy images. The most known dehazing approach is based on dark channel prior. It mostly considers daytime outdoor images that have some very dark pixels, which provide a good estimate of haze [START_REF] He | Single image haze removal using dark channel prior[END_REF]. The methods, which are recently proposed, mostly adopt machinelearning approaches [START_REF] Cai | Dehazenet: An end-to-end system for single image haze removal[END_REF]). Among these numerous approaches, only a few methods consider the removal of noise [START_REF] Matlin | Removal of haze and noise from a single image[END_REF][START_REF] Liu | Efficient single image dehazing and denoising: An efficient multi-scale correlated wavelet approach[END_REF], which is inevitable in natural images and when it is ignored, it degrades remarkably the quality of dehazed images.

In the following sections, we introduce the noise and its modelling in hazy images. We then describe the experiment we performed to demonstrate how the bad estimate of noise in hazy images impact the image quality after dehazing and the recovery of colors. We conclude the article by a brief discussion of the results.

NOISE IN HAZY IMAGES

According to Kochmieder [START_REF] Koschmieder | Theorie der horizontalen Sichtweite[END_REF], the formation of hazy images can be modelled by the following equation:

𝑰(𝒙) = 𝑱(𝒙)𝒕(𝒙) + 𝑨 ∞ (𝟏 -𝒕(𝒙)) (1)
where 𝐼(𝑥) is the hazy image captured by the camera's sensor. It is formed by the scene radiance 𝐽(𝑥) and the atmospheric light 𝐴 ∞ weighted by the light transmission factor 𝑡(𝑥), which depends on the scene depth 𝑑 and the scattering coefficient 𝛽 of the haze, such that 𝑡(𝑥) = 𝑒 -𝛽𝑑(𝑥) .

The equation 1 represents the optimistic model of hazy images free of noise. However, the imperfection of the environment conditions and the sensors induces noise 𝑛(𝑥) in digital images, which is usually amplified through dehazing. 𝑛(𝑥) is the noise contribution, assumed to be independent and identically distributed with zero mean and variance 𝛿 2 :

𝑰(𝒙) = 𝑱(𝒙)𝒕(𝒙) + 𝑨 ∞ (𝟏 -𝒕(𝒙)) + 𝒏(𝒙) (2)
There are two main approaches to deal with haze and noise. The first one consists of dehazing followed by denoising as two separate processing steps. The second one is the application of dehazing and denoising simultaneously. Noise can be over-estimated or under-estimated, depending on the applied approach. When it is over-estimated, this means the hazy image will be over-denoised and therefore smoother. This induces the loss of fine useful details such as edges and textures. However, it produces results of better quality than the under-estimation of noise, since the remaining amount of noise is noticeably amplified after dehazing [START_REF] Matlin | Removal of haze and noise from a single image[END_REF].

EXPERIMENTAL SETUP

In order to show how much the quality and the recovered colors of dehazed images are affected when the noise is ignored or not accurately estimated, we used, as shown in figure 1, two images of the same scene with two densities of haze, H1 and H2, from CHIC database [START_REF] El Khoury | A color image database for haze model and dehazing methods evaluation[END_REF]. From the entire image, we only considered the Macbeth ColorChecker placed at the back of the scene, since the parameters forming equation 1 are provided in the database at this position. To both images cropped from H1 and H2, we added some gaussian noise with 𝛿 = 0.01 (figure 2). To investigate the importance of estimating accurately the noise in images, at two levels of haze H1 and H2, we performed dehazing by inverting the equation 1 and calculating 𝐽(𝑥) through four ways: in the first we dehazed the image without considering noise. In the second, while dehazing we removed the accurate amount of added noise. In the third and fourth, the dehazing was performed without any accurate estimation of noise by using slightly higher and lower values of the real 𝛿 denoted by 𝛿 𝑒 . The corresponding results are shown in Figures 3 and4. To quantify the quality of images after the application of dehazing and denoising, we calculated the scores of the classical metrics that are often used to assess the performance of dehazing methods when the haze-free image is available, PSNR and SSIM [START_REF] Wang | Image quality assessment: from error visibility to structural similarity[END_REF]. A higher quality is indicated by a higher value of PSNR and a SSIM value closer to 1. We also calculated the color difference parameter ∆𝐸 𝑎𝑏 as an indicator to report the inaccuracy in the recovery of colors. All these indicators have been calculated between the dehazed images and the haze-free image considered as the reference image with optimal quality.

DISCUSSION

Considering the visual assessment of the images presented in Figures 3 and4, the dehazed images of H1, are strikingly affected by noise. With the same quantity of noise, the quality of these images is more affected than in the images of H2, which has a lower density of haze. This observation is in line with the scores of indicators in Table 1. PSNR and SSIM indicate that all dehazed images of H2 have a higher quality than the corresponding images of H1. Likewise, ∆𝐸 𝑎𝑏 underlines a higher similarity between dehazed images and the haze-free image.

According to the scores in Table 1, the accurate estimate of noise is always beneficial no matter what the level of haze is. Contrary to what might be expected, ignoring completely the induced noise seems to provide a better outcome than when it is inaccurately estimated. This is valid for both levels of haze either when the noise is overestimated or underestimated. However, we should mention that when 𝛿 𝑒 is lower than the real 𝛿, the resulting image is smoother as some noise are eliminated. Therefore, the dehazed image in this case is closer to the reference image. 3 and4.

Dehazing without denoising

Dehazing

CONCLUSIONS

In this paper, we have investigated the importance of noise estimation accuracy and how this impacts the features of images. For this, we used some quality and color difference metrics. This study showed that when the noise is not accurately estimated, it is better to ignore it. And when it is accurately estimated, the resulting image has the highest quality.

To the best of our knowledge, the quantification of color and quality degradation caused by the lack of the accuracy in noise estimation, has been timidly addressed. Through this study, we pointed out the importance to stimulate the awareness of researchers of the need to include denoising as a mandatory treatment into dehazing and to focus on the proper modelling of noise that will guarantee an accurate estimation of it.

Figure 1 :

 1 Figure 1: Images taken from CHIC database. From left to right: Haze-free image, hazy image H1, hazy image H2.

Figure 2 :

 2 Figure 2: Macbeth ColorChecker cropped from images H1 and H2.

Figure 3 :

 3 Figure 3: The haze-free and the denoised images of the Macbeth ColorChecker placed at the back of the hazy image H1.

Figure 4 :

 4 Figure 4: The haze-free and the denoised images of the Macbeth ColorChecker placed at the back of the hazy image H2.

PSNR SSIM ∆𝑬 𝒂𝒃 PSNR SSIM ∆𝑬 𝒂𝒃 PSNR SSIM ∆𝑬 𝒂𝒃 PSNR SSIM ∆𝑬 𝒂𝒃

	H1 16.89 0.40 25.32 17.64 0.52 21.15 15.67 0.30 31.88 16.74 0.39 26.21
	H2 19.06 0.67 18.55 19.96 0.78 13.35 17.65 0.56 25.11 18.90 0.65 19.39

Table 1 :

 1 The values of PSNR, SSIM and ∆𝐸 𝑎𝑏 of the images presented in Figures