Amélie Banc

Pierre Bauduin

Bernard Desbat

Isabelle Ly

Olivier Diat

Ion extraction mechanism studied in a lyotropic lamellar phase

In this paper we used a surfactant-stabilized lyotropic lamellar model system to study the interfacial behaviour of an ion-extracting agent: N 1 ,N 3 dimethyl-N 1 , N 3 -dibutyl-2-tetradecyl malonamide (DMDBTDMA). An analysis of small angle X-ray scattering (SAXS) and polarized Attenuated Total Reflectance -Fourier Transform Infra Red (ATR-FTIR) data enabled to describe the distribution of the malonamide extractant within the bilayers and its complexation state at the equilibrium. The lamellar phase was diluted with salt water containing varying amount of complexing salt, and each structural state measured was described using a thermodynamic model based on three elementary equilibria: (i) partition of the extractant polar heads between the core and the polar shell of the bilayers, (ii) the complexation of ions by extractants at the bilayers surfaces, and (iii) the partition of bonded extractants between the core and interfaces of bilayers. This model enabled to compare the energy cost of each step.

INTRODUCTION

Reactions at liquid-liquid interfaces are of great fundamental interest because they play a key role in many important chemical and biological systems [START_REF]Liquid interfaces in chemical, biological, and pharmaceutical applications[END_REF] , such as phasetransfer catalysis [START_REF] Fiamegos | [END_REF] , liquid-liquid extraction, micellar catalysis 3 , biomembrane activity, enzymatic reactions on fat lipases 4 . Systems in which such reactions take place are difficult to study because their global behaviour is the result of reactants diffusion in bulk and interfacial mechanisms.

To study specifically liquid interfaces, different experimental systems were developed: systems with a unique and controlled interface between two immiscible liquids (interface of pendant drops 5 , interface developed for reflectance measurements 6 , interface created into a microchannel 7 , nanoscopic interface supported by nanopipette 8 , and systems with interfaces stabilized by amphiphilic molecules (vesicles [START_REF] Mircheva | Colloids Surf[END_REF] , micelles and microemulsions 10 ). In the present work, we chose to use lyotropic lamellar phases, periodical stacking of oily layers (made by the surfactant tails) diluted by salt water. These systems with multiple interfaces, in thermodynamic equilibrium, are structurally and mechanically responsive to weak effects, such as the Hofmeister ion effect 11,12,13 or the partitioning of host molecules within bilayers 14,15,16,17 . In a previous paper [START_REF] Banc | Advanced separation techniques for nuclear fuel reprocessing and radioactive waste treatment[END_REF] we described and characterized the insertion of a lipophilic ion-complexing agent, N 1 ,N 3 dimethyl-N 1 , N 3 dibutyl-2-tetradecyl malonamide (DMDBTDMA), in a lamellar phase stabilized by a non-ionic surfactant: pentaethyleneglycol dodecyl ether (C 12 E 5 ). DMDBTDMA is an extractant molecule used in nuclear industry to separate minor actinides from high level radioactive liquid wastes by liquid-liquid extraction processes 19 (DIAMEX process) or in more conventional hydrometallurgy for the rare earths elements recycling 20,21 . We showed that the structural parameters of an ideal lamellar system enabled to describe the partition of the extractant molecules between the core and the interface of bilayers. This simple approach was based on the evolution of interfacial area with the addition of extractants, which was measurable thanks to the multitude of interfaces in lamellar phases, as for microemulsion systems [START_REF] Tchakalova | Colloids Surf[END_REF] . In the present paper, we studied the effect of ion complexation on the system. First, we characterized the system using Small Angle X-Ray Scattering (SAXS) and transmission electron microcopy (TEM) coupled with freeze fracture to follow structural parameters, and polarized Attenuated Total Reflectance -Fourier Transform Infra Red (ATR-FTIR) spectroscopy to measure orientation of dipoles and to quantify the ion complexation phenomenon. In a second part, a thermodynamic approach of liquid-liquid extraction was developed on the basis of the experimental data by considering the ion extraction process as the sum of three equilibria (Scheme 1): (1) Positioning of free extractants polar head areas at interfaces (2) Complexation of cations with extractants at interfaces (3) Burying of bonded extractants within the bilayers Hence, the free energy contributions of each step was estimated and compared. This allowed to elucidate the precise mechanism of extraction by DMDBTDMA in this model system.

EXPERIMENTAL SECTION

C 12 E 5 was obtained from Nikko Ltd. (high purity grade > 99%) and used as received. DMDBTDMA was obtained from Panchim and was purified on an alumina column to eliminate traces of surface active impurities. Aqueous solutions were prepared using deuterated water (99.9%, Eurisotop), anhydrous LiNO 3 salt (purity>99%) from Strem, and Nd(NO 3 ) 3 ,6H 2 O salt (purity>99.9%) from Aldrich. Adsorption of extractants from the organic phase to the interface (K 1 ), complexation of cations with extractants which are at the interface (K 2 ), and desorption of ionextractant complex from the interface to the organic phase (K 3 ). To determine conditions of homeotropic orientation of lamellar samples (bilayers oriented perpendicular to the optical axis), different thicknesses of spacers between glass slide and cover slip were tested. It was observed that samples sheared between slide and cover slip spaced by 10µm thick mica sheets, display homeotropic orientation after few minutes (Figure 1). For freeze-fracture measurements, a drop of sample was placed on a gold holder and frozen by plunging into liquid propane. Fracture and replication were carried out in a freeze-fracture apparatus (Leica EM BAF060) at the temperature of -150 °C under vacuum. Pt/C was deposited at an angle of 45° and C at 90°. Replicas were recovered at room temperature by specimen dissolution in ethanol and water. Then, replicas were collected onto ATHENE copper 400 mesh grids to be examined on an Hitachi H-600 75kV's transmission electron microscope equipped with an AMT's CCD camera (DVC 1 megapixels).

Small angle X-ray scattering (SAXS) measurements using Mo-radiation (λ=0.71 Å) were performed on a bench built by XENOCS. The scattered beam was recorded using a large online scanner detector (diameter: 345 mm, from MAR Research) located at 750 mm from the sample stage.

A large Q range (2.10 -2 -2.5Å -1 ) was covered thanks to an off centre detection. The collimation was applied using a 12:∞ multilayer Xenocs mirror (for Mo radiation) coupled to two sets of scatterless FORVIS slits 23 providing a 0.8 x 0.8 mm x-ray beam at the sample position. Pre-analysis of data was performed using FIT2D software, taking into account the electronic background of the detector (the flatfield is homogeneous) and the empty cell subtraction. The scattered intensities were expressed versus the magnitude of scattering vector Q = [(4π)/λ].sin (θ/2), where λ is the wavelength of incident radiation and θ the scattering angle. Experimental resolution was ∆Q/Q=0.05.

Silver behenate in a sealed capillary was used as the scattering vector calibration standard. The lamellar periodicity of samples (d) was deduced from the first Bragg peak position Q 0 :

0 2 Q d Π = (1)
Surface tension experiments at the oil-water interface were performed on pendant drops using a drop shape analyser (Kruss DSA 100). The aqueous solution was either LiNO 3 1M or Nd(NO 3 ) 3 0.33M, and organic solutions were DMDBTDMA solubilized into dodecane (from 0.005 to 1M). Before tensiometry measurements, each organic solution was emulsified with the aqueous solution of interest (50/50 v/v) by mechanical stirring for 2 minutes and then isolated after centrifugation. Densities of solutions were measured at 20°C using a vibrating tube densimeter, DSA 5000 from Anton Paar. After injection of the organic drop into the aqueous solution, surface tension was measured versus time. The values at equilibrium (<1% evolution) were selected for this study. The Gibbs equation was used to calculate the superficial excess of extractant (Γ):

P T C RT , ln 1       ∂ ∂ - = Γ γ (2)
where γ is the surface tension and C is the concentration of the adsorbing specie (in the concentration region below cmc). Hence, the polar head area (A) was determined using:

Γ = a N A 1 (3)
where N a is the Avogadro constant. Polarized ATR-FTIR experiments were performed on a Perkin Elmer Spectrum-One Fourier Transform Infrared system equipped with an attenuated total mono reflection (ATR) device, using a diamond crystal. Lamellar phase samples, spread on the ATR crystal, were confined under a glass coverslip, 10µm-spaced from the crystal (Figure 1). This confinement ensured the orientation of the lamellar phase (bilayers parallel to the crystal) as demonstrated by polarized light microscopy and a sample thickness higher than the penetration depth of the evanescent electromagnetic waves, d p , defined by 24 :

) (sin 2 where Φ is the incidence angle, λ is the wavelength, n 1 is the refractive index of the ATR crystal, n 2 the refractive index of the sample, and n 21 =n 2 /n 1 . In our case, d p varied between 0.2 and 1µm (n 1 =2.4, n 2 =1.45 and Φ=45°). For each sample, 64 scans spectra, with a resolution of 4 cm -1 , were obtained for two polarizations of the incident beam: parallel (p) and perpendicular (s) to the incidence plane. A linear baseline correction was performed on each spectrum. Two kinds of analysis were performed with FTIR data: first, to obtain information about orientation of dipoles into samples, and second to quantify bonded extractants. Orientation information of dipoles is contained in the dichroic ratio R ATR which is the ratio of the absorbance of the corresponding band measured with a parallel polarization of the incident light A p to the absorbance measured with a perpendicular polarization A s :

R ATR = s p A A (5)
The dichroic ratio is related to the dipole order parameter S, according to:

) 2 ( ) ( 2 2 2 2 2 2 z y ATR x z y ATR x E E R E E E R E S - - + - = (6)
where E x ², E y ² and E z ² are the time-averaged square electric field amplitudes of the evanescent wave in the sample at the crystal/sample interface. The electric field amplitudes on the crystal-sample interface were calculated using the thick film model of Harrick 25 , valid when the thickness of the sample is very large compared to the penetration depth:

( ) [ ] 2 21 2 21 2 21 2 21 2 2 2 ² sin 1 ) 1 ( ) .(sin cos 4 n n n n E x - Φ + - - Φ Φ = (7) ) 1 ( ² cos 4 2 21 2 n E y - Φ = (8) ( ) [ ] 2 21 2 21 2 21 2 ² sin 1 ) 1 ( ² sin ² cos 4 n n n E z - Φ + - Φ Φ = (9)
In our case, E x =1.157, E y =1.774 and E z =2.227. The order parameter S contains the information on the mean orientation of the transition dipole and on the distribution of the dipole orientations at the mean value:

2 1 ² cos 3 - = θ S ( 10
)
where θ is the angle between the dipole and the normal of the ATR plate, and θ ² cos is the mean value of cos²θ for a distribution of transition moment in the film. To perform quantitative analysis of bonded extractants, the isotropic spectrum of samples was calculated from the polarized spectra. The isotropic sprectrum can be defined as:

S iso =S x +S y +S z (11) where S i is the spectrum in the direction i. The sample being uniaxial, S x =S y . Polarized spectra can be defined as follow: S s =E x .S x and S p =E y .S y +E z .S z (12) Hence using our parameters, S iso =0.8345.S s +0.449.S p (13) The second analysis was related to the ion-bonding of extractants and was studied using the carbonyl band positioned around 1650 cm -1 . This band was fitted using two Gaussian curves centred at 1640 and 1615 cm -1 assigned to free carbonyls (not complexed) and bonded carbonyls (complexed with Nd 3+ ) respectively. Fitting procedure was realised with the Omnic software which uses a Fletcher-Powell-McCormick algorithm as convergence routine. To obtain the area of each contribution, positions and the full widths at half height were fixed and maintained constant for all samples. The evolution of the free carbonyl absorbance (area) in isotropic spectra enabled to quantify the complexation into samples. According to the Beer-Lambert law:

A=ε.l.C (14) where A is the absorbance, l the length of the pathway, C the concentration, and ε the extinction coefficient.

Considering that for a vibration mode ε is constant, and that l is constant in our samples (the sample thickness is higher than the penetration depth which is constant for given wavelength and setup), the following relationship can be established:

(A 0 -A)/A 0 =(C 0 -C)/C 0 (15)
Hence, the fraction of free carbonyl consumed (bonded carbonyls created) was determined.

RESULTS

Preliminary experiments were carried out to identify the boundaries of the monophasic lamellar phase in the ternary phase diagram C 12 E 5 /DMDBTDMA/aqueous solution (1M). Macroscopic and microscopic (optical microscopy with crossed polarizers) observations performed on some formulations, derived from the lamellar domain of the surfactant/water system 26 , enabled to roughly determine the lamellar region in the ternary system (Fig. 2). In the present work, we choose to work with a formulation located at the center of this domain: a premix C 12 E 5 /DMDBTDMA 80/20 molar hydrated with 40 wt% of aqueous solution. This composition lies in the concentrated region of the lamellar phase (aqueous layers are about 2 nm thick) mainly stabilized by the interplay of the attractive van der Waals force and the steric repulsion of the hydrated head groups (repulsive undulation forces are negligible) 27 . The effect of complexation was studied varying the nature of the aqueous solution from LiNO 3 1M to Nd(N0 3 ) 3 0.33M to keep 1 molar of ionic strength (range of ionic strength applied in extraction systems) in the different samples. The lamellar phases obtained with such formulations were characterized by freeze-fracture (see Figure 3). A characteristic lamellar structure with periodical steps, was obtained for both LiNO 3 or Nd(NO 3 ) 3 hydrated extractantcontaining lamellar phases, like for the binary system. No structural change e.g. topological defects 28 was detected after addition of extractant or salt to the lamellar phase. The periodicity (d) of samples was characterized using SAXS. Assuming an ideal lamellar structure (flat rigid bilayers without defect), the hydrocarbon thickness of bilayers (δ HC ) was determined using the following dilution law: .906 (measured). The interface was defined as being located between the aliphatic chains of surfactants, and the polar layer comprising the polar heads of surfactants and hydration water. Figure 4 displays the evolution of bilayer thickness versus the neodymium percentage in the hydration solution for C 12 E 5 and C 12 E 5 /DMDBTDMA lamellar phases. By increasing the neodymium content, the bilayer thickness remains constant for the reference C 12 E 5 samples, whereas it continuously decreases from 18.8 to 18.3 Å for samples containing DMDBTDMA. In our previous paper [START_REF] Banc | Advanced separation techniques for nuclear fuel reprocessing and radioactive waste treatment[END_REF] , we demonstrated in analysing the bilayer thicknesses that the extractant molecule can be considered either as a co-surfactant or as a solvent molecule distributed within the aliphatic domain of the bilayers, both configurations being in equilibrium for a given surfactant/extractant ratio. Assuming an ideal mixing of extractant and surfactant, and constant polar areas per molecule at the hydrophilic/hydrophobic interface, the bilayer thickness was expressed as a function of the extractant repartition between the interfaces and the bulk of the bilayers: (17) where V i are molecular volumes determined from density values, A i are polar head areas, x is the molar fraction of DMDBTMA among the organic components and κ the fraction of DMDBTDMA participating to the interfacial area. The two extreme cases, κ=0 and κ=1, are plotted on Figure 5. The experimental points are intermediate (0<κ<1) and indicate roughly an equi-partitioning of extractant molecules between the core of the bilayers and the interfaces. The decrease of δ HC with the percentage of complexing ions can be thus interpreted as an evolution of extractants partition within the bilayers; qualitatively, the interaction between extractant molecules and ions that can be complexed, pull the lipophilic extractants towards the interface. [START_REF] Banc | Advanced separation techniques for nuclear fuel reprocessing and radioactive waste treatment[END_REF] . The dotted line display the case for which extractants are fully embedded within bilayers, whereas the dashed line represents the case for which extractants fully participate to the interfacial area as the surfactant molecules. Full dots are experimental points obtained for a system hydrated with LiNO 3 1M.The arrow indicates the decrease of the bilayers thickness by exchanging lithium with neodymium cations at constant DMDBTDMA percentage.

δ HC =d.Φ HC ( 16)
To estimate the contribution of extractants to the interfacial area we measured their polar head area at model oil-water interfaces: dodecane-LiNO 3 1M and dodecane-Nd(NO 3 ) 3 0.33M, to consider free and bonded extractants respectively. Dodecane was chosen to mimic the C 12 chain of surfactants which constitute the oily domains. Figure 6 displays the evolution of interfacial tensions versus DMDBTDMA concentration for both cases. Critical aggregation concentrations around 0.1M can be noticed, in accordance with literature 30,31 . Using the Gibbs equation (2), we obtained A free DMDBTDMA =80±5 Ų/molecule and A bonded DMDBTDMA =100±5 Ų/molecule. These values do not represent only a geometrical polar area but takes into account all the lateral interactions within the bilayers. Ion complexation charges the extractant molecules and induces additional electrostatic lateral repulsions that consequently increase the apparent extractant polar head area. DMDBTDMA is studied using an aqueous solution of LiNO 3 1M, whereas bonded DMDBTDMA is studied using a 0,3M Nd(NO 3 ) 3 solution.

DMDBTDMA E C DMDBTDMA C HC A x A x xV V x A V κ δ + - + - = =
Figure 7 displays the characteristic polarized ATR-FTIR spectra of oriented samples. We can notice the large OD elongation vibrations around 2500 cm -1 , the OH elongation vibrations attributed to the polar head of surfactants around 3400 cm -1 , the CH 2 vibration modes around 2900 cm -1 , and the amide I (mostly the C=O elongation vibration) around 1650 cm -1 . The symmetric vibration mode of CH 2 , positioned at 2855 cm -1 ( S 2 CH ν ), was used to probe the order of aliphatic chains into bilayers, whereas the C=O elongation vibration mode was used to probe extractant molecules. This latter was decomposed into two main contributions centred at 1640 and 1615 cm -1 attributed to free ( . These ratios are constant whatever the neodymium percentage and the mean values are displayed in Table 1. As the transition moment for the S 2 CH ν lies perpendicular to the chain axis, the order parameter of aliphatic chains was calculated using the following relationship:

) 2 )( 1 cos 3 ( ) ( 2 2 2 2 2 2 2 2 z y ATR x z y ATR x E E R E E E R E S - - - + - = α ( 18
)
where α is the angle between the chain axis director and the transition dipole (α=90°). The low value obtained (S=0.1) is explained by the fluid character of the L α bilayers and is consistent with literature results for C 12 E 5 /water lamellar phases 32 . The zero value of S for free carbonyls indicates an isotropic orientation of these bonds, as previously measured using polarized microRaman spectroscopy [START_REF] Banc | Advanced separation techniques for nuclear fuel reprocessing and radioactive waste treatment[END_REF] , contrary to bonded carbonyls which are characterized by a value close to that of aliphatic chains. This result must be analysed considering that the experimental order parameter measured from R ATR , denoted S exp , is the product of several order parameters related to a set of nested, uniaxial symmetric distributions 33,34 :

S exp =S membrane S molecule S dipole (19)
where S memb describes the distribution function of the membrane patches with respect to the internal reflection element, S molecule describes the orientation of the molecules within the membrane plane, and S dipole describes the orientation of the dipole relative to the molecule. Providing the good orientation of the samples sheared between 10µmspaced flat surfaces, we can estimate that S membrane is close to 1. Hence, the variation of order parameter with complexation can be attributed either to a change of molecule orientation inside bilayers (S molecule ) or a modification of carbonyls orientation with respect to the molecule axis (S dipole ). It is difficult to consider a higher order parameter for the extractant compared to the one of the surfactant (S=0.1). Then, with S C=Obonded =0.096 and considering S memb ~1 and S molecule ≤0.1, S dipole-bonded must be close to 1. This means that in a bonded malonamide, both carbonyls bonds are parallel. For free malonamides, S C=Ofree =0 is explained either by the trans/gauche conformation of carbonyl groups or by the isotropic orientation of molecules within bilayers. Using a Quantum Mechanics method, Boehme et al 35 showed that free malonamides, in gas phase, roughly adopt trans conformation (β, the angle between the two carbonyl dipoles is 162°). The monodentate mode, rarely observed, displays a gauche conformation (β is about 90°). Both conformations would lead to S dipole <<1. However, the bidentate coordination state with lanthanides induces an alignement of carbonyl bonds i.e. cis conformation with 30°<β<60° according to the stoichiometry considered. This latter conformation can be considered as the most probable for ion bonded extractants in our system. The evolution of the carbonyl band shape with the addition of neodymium (figure 8a) enabled to follow quantitatively the complexation. Figure 8b display the fraction of bonded carbonyls versus the percentage of neodymium salts in the aqueous solution. In 0.33M neodymium samples (100% of Li + replaced by Nd 3+ at constant ionic strength), the DMDBTDMA/Nd ratio is 2 and only 30% of carbonyls are bonded. Assuming a unique denticity of the coordination complex, the fraction of bonded carbonyls is equal to the fraction of bonded extractants.

ANALYSIS -DISCUSSION

The liquid-liquid extraction of neodymium by DMDBTDMA is usually described by the following solvation reaction 36 , characterized by an equilibrium constant K T :

x DMDBTDMA + Nd 3+ + 3NO 3 - (DMDBTDMA) x Nd(NO 3 ) 3
where upperlined species are solubilized in the organic phase and the other species are solubilized in water. Usually, above a given bulk concentration in organic phases the extractant forms reverse aggregates which are characterized by their aggregation number (from 4 to 10 37,38 , depending on concentration, pH, type of salt…). Within these aggregates the coordination number, x, is still under debate since the molecular conformation is not well defined, but it is established that 2<x<4. However, at a flat interface x was never determined, neither experimentally nor theoretically. Usually the aggregate size varies between 2 and 3nm 37,38 . In our case, it is difficult to consider extractant aggregates within the bilayers as their formation would have induced topological defects that we did not observe by electron or optical microscopies. Moreover, a lower order parameter (closer to zero than 0.1) of bonded carbonyls would have been measured with the formation of extractant aggregates. As a consequence, it appears reasonable to consider a 1:1 stoechiometry (x=1) with a bidentate coordination between the ion and the extractant as demonstrated previously. We propose to describe the solvation reaction in the present system as the sum of three equilibria (Scheme 1): (1) Positioning of free extractants polar heads at interfaces (3) Burying of bonded extractants from interfaces (DMDBTDMA)Nd(NO 3 ) 3 (DMDBTDMA)Nd(NO 3 ) 3 K 3 where dash upperlined species are localized at interfaces, and K i are the equilibrium constants. In the next part, we will show that these equilibria can be quantified knowing the concentration of each species. where y is the molar fraction of bonded extractants deduced from the FTIR analysis, κ 1 the fraction of free extractants at interfaces and κ 3 the fraction of bonded extractants at interfaces. Each polar head areas was considered constant and was evaluated by tensiometry for DMDBTDMA, and taken from the literature for C 12 E 5 (A C12E5 =50Ų/molecule) 39,[START_REF] Banc | Advanced separation techniques for nuclear fuel reprocessing and radioactive waste treatment[END_REF] . Figure 9 displays the evolution of the bilayers thickness as a function of the fraction of bonded extractant, which was adjusted by the equation (20) giving κ 1 =0.43±0.01 and κ 3 =0.55±0.02 (correlation coefficient: 0.8). Approximating extractant activities to molar concentrations, the apparent related equilibrium constants can be calculated using: values must be used with care. However, this result indicates that bonded extractants, i.e. extractant forming a complex with Nd 3+ are more amphiphilic than free extractants. The complexation must charge the lipophilic extractant and should make the head more polar. This is in accordance with tensiometry measurements which display a more important and faster reduction of surface tension with DMDBTDMA in presence of neodymium salt (bonded molecules) than with DMDBTDMA in presence of lithium salt (free extractant). The complexation of neodymium by DMDBTDMA at the interfaces (K 2 ) can be described in a first approach as a cation adsorption isotherm at fluid interfaces. Thus, the Langmuir model can be used: (22) where θ is the relative occupancy of sites, each site being the polar head of an extractant at interface, K 2 the adsorption constant, and a the complexing ion activity . The relative occupancy of sites, defined as the ratio between bonded extractants over the total number of extractants at the interface, was determined using FTIR data (y), κ 1 and κ 3 : This definition assumes that the saturation limit of the ion isotherm is Nd/DMDBTDMA=1/1 molar ratio. As previously, the molar concentration was used instead of the ion activity. As a consequence, an apparent adsorption constant K' 2 was obtained:

γ 2 ' 2 K K = (24)
where γ is the molar activity coefficient of the complexing ion (a=γC where C is the concentration). Figure 10 shows that experimental data were well fitted with such model, using K' 2 =1.85±0.3. The apparent equilibrium constants characterizing the system (K' 1 , K' 2 , K' 3 ) deduced from experimental data enabled to calculate the apparent free energy (∆G') corresponding to the different equilibrium:

' ln ' K RT G - = ∆ (25)
The different contributions to the free energy are displayed in a table and illustrated by an energy diagram in figure 11. We can observe that the free energy of the total reaction, the ion extraction from the aqueous domains towards the bilayers, is negative due to the complexation (2) reaction which is spontaneous. However this effect is reduced by the energy cost to position the free extractant at the interfaces (1) and to desorb bonded extractant from the interfaces (3), both being energetically unfavourable. The thermodynamics of liquid-liquid extraction by diamides was previously studied in classic biphasic systems. Taking into account the formation of supramolecular aggregates of extractant in the organic phase 37,38 and their associated potential surfaces on which ions can adsorb, Zemb et al 40,41 have obtained thermodynamical extraction parameters assuming a sum of Langmuir isotherms for each state of aggregation.

This view enabled to associate thermodynamical values to the different aggregation states of extractants. Free energies of about few k B T were deduced, slightly higher than our values. However, in our model system, we cast off aggregation and only elementary mechanisms at interfaces were studied. Consequently, adsorption constants are not fully comparable. Nevertheless the complexation free energy value can be evaluated looking at the complexation values obtained for hydrosoluble malonamides. Couston et al. 42 found an 1:1 complexation equilibrium constant K 1:1 equals to 1.3 for tetraethylmalonamides with europium in water. The corresponding free energy value (∆G=-0.26k B T) has the same order of magnitude as ∆G' 2 . In our system we use surfactant molecules which must be in competition with extractants for the interfacial adsorption, like in model micellar systems 10 . Thus, the results are certainly function of the surfactant used, but for a given system, parameters such as the nature of extracted cations, the nature of extractant, the ionic force can be studied comparatively. Nevertheless, from these data, the amphiphilic property of extractant could be considered as the limiting step for extraction.

In future studies, the extractant aggregation could be eventually introduced in the lyotropic system releasing sterical constraints by swelling bilayers with oil. In addition, the dehydration of cations, which is included in the mechanism of cation adsorption at interfaces, could be modulated decreasing the water thickness between bilayers. Indeed, the water activity in such confined environments was shown to be modulated by the distance between bilayers 43 . The cation desolvation energy could be thus studied using the lamellar phase.

CONCLUSIONS

In this work we proposed a lyotropic lamellar phase to study interfacial mechanisms of liquid-liquid extraction. Due to the numerous oriented interfaces and using a combination of SAXS and FTIR techniques, we determined the concentrations of free and bonded extractants molecules either buried into bilayers or localized and oriented at interfaces,. Hence, apparent free energies associated to each interfacial elementary mechanism were estimated. Results indicate that extraction by DMDBTDMA is partially controlled by the variation of amphiphilicity of the molecule during the ion complexation. Between bonded and free extractant, the former appears to be a more favourable state at interfaces. This observation could be considered as a bridle to an ideal extraction kinetics. With this study we demonstrated that a lyotropic lamellar phase can be a meaningful system to model reactive liquidliquid interfaces. In the future, other studies could be engaged varying parameters such as the nature of reactants or layers thicknesses.

ACKNOWLEDGMENTS

We thank Thomas Zemb, Laurence Berthon and Marie-Christine Charbonnel for stimulating discussions, David Maurin for his assistance during polarized FTIR-ATR experiments, Olivier Mondain Monval for his expertise on cryofracture observations, Jérôme Maynadié for his help during deuteration of neodymium salts and Gaëlle Gassin-Martin for fruitful discussions concerning tensiometry measurements. We also thank Daniel Meyer for fruitful discussion on ion coordination with extractant molecules.

Energy

This work was supported by the European ACSEPT network.

Scheme 1 .

 1 Scheme 1. Schematic view of cation extraction by extractant molecules. The global extraction reaction (K T ) can be divided into three elementary mechanisms which are characterized by thermodynamic equilibrium constants:Adsorption of extractants from the organic phase to the interface (K 1 ), complexation of cations with extractants which are at the interface (K 2 ), and desorption of ionextractant complex from the interface to the organic phase (K 3 ).

Figure 1 .

 1 Figure 1. Polarized FTIR-ATR. (a) Scheme of the experimental setup. The sample was confined between the ATR crystal and a glass cover slip to ensure an homeotropic orientation of the lamellar phase. Polarized light microscopy pictures of samples observed 1 minute (b) and 5 minutes (c) after shearing between flat surfaces separated by 10µm.

Figure 2 .

 2 Figure 2. Molecular structures of C 12 E 5 (a) and DMDBTDMA (b). (c) Ternary phase diagram of C 12 E 5 /DMDBTDMA/aqueous solution indicating in grey the monophasic lamellar phase domain at room temperature. The salty aqueous solution contains LiNO 3 and Nd(NO 3 ) 3 salts in a 1 molar ionic strength. Crosses indicate experimental points and the star indicates the composition of samples studied in this paper (C 12 E 5 /DMDBTDMA = 80/20 molar, 40% hydration).

Figure 4 .

 4 Figure 4. Evolution of bilayer thickness (δ HC ) with the percentage of Nd(NO 3 ) 3 in the aqueous solution. Empty dots display the reference system (C 12 E 5 ) whereas full dots display the mixed system C 12 E 5 /DMDBTDMA (80/20 molar).

Figure 3 .

 3 Figure 3. TEM pictures of freeze-fracture replica: (a) C 12 E 5 lamellar phase hydrated with 30% LiNO 3 1M, (b) C 12 E 5 /DMDBTDMA lamellar phase hydrated with 40% LiNO 3 (1M), and (c) C 12 E 5 /DMDBTDMA lamellar phase hydrated with 40% Nd(NO 3 ) 3 (0.33M).where φ HC    is the volume fraction of DMDBTDMA and alkyl chains of C 12 E 5 in the sample. The following density values were used: d C12 =0.80329 , d C12E5 =0.963 (from supplier) and d DMDBTDMA =0.906 (measured). The interface was defined as being located between the aliphatic chains of surfactants, and the polar layer comprising the polar heads of surfactants and hydration water. Figure4displays the evolution of bilayer thickness versus the neodymium percentage in the hydration solution for C 12 E 5 and C 12 E 5 /DMDBTDMA lamellar phases. By increasing the neodymium content, the bilayer thickness remains constant for the reference C 12 E 5 samples, whereas it continuously decreases from 18.8 to 18.3 Å for samples containing DMDBTDMA. In our previous paper[START_REF] Banc | Advanced separation techniques for nuclear fuel reprocessing and radioactive waste treatment[END_REF] , we demonstrated in analysing the bilayer thicknesses that the extractant molecule can be considered either as a co-surfactant or as a solvent molecule distributed within the aliphatic domain of the bilayers, both configurations being in equilibrium for a given surfactant/extractant ratio. Assuming an ideal mixing of extractant and surfactant, and constant polar areas per molecule at the hydrophilic/hydrophobic interface, the bilayer thickness was expressed as a function of the extractant repartition between the interfaces and the bulk of the bilayers:

Figure 5 .

 5 Figure 5. Evolution of bilayer thickness as function of the percentage of DMDBTDMA in the C 12 E 5 /DMDBTDMA mixture[START_REF] Banc | Advanced separation techniques for nuclear fuel reprocessing and radioactive waste treatment[END_REF] . The dotted line display the case for which extractants are fully embedded within bilayers, whereas the dashed line represents the case for which extractants fully participate to the interfacial area as the surfactant molecules. Full dots are experimental points obtained for a system hydrated with LiNO 3 1M.The arrow indicates the decrease of the bilayers thickness by exchanging lithium with neodymium cations at constant DMDBTDMA percentage.

Figure 6 .

 6 Figure 6. Determination of polar-head areas of free and bonded DMDBTDMA by tensiometry measurements. FreeDMDBTDMA is studied using an aqueous solution of LiNO 3 1M, whereas bonded DMDBTDMA is studied using a 0,3M Nd(NO 3 ) 3 solution.

Figure 7 .Figure 8 .

 78 Figure 7. Polarized ATR-FTIR spectra of samples. The carbonyl band was decomposed into two mains contributions centred at 1640 cm -1 and 1615cm -1 (in inset, yellow line : experimental spectrum, full dark line: fitted spectrum).

  carbonyls and aliphatics chains. (*Dichroïc ratio of CH 2 and order parameter of aliphatic chains).

Figure 9 .

 9 Figure 9. Evolution of bilayer thicknesses (δ HC ) versus the fraction of bonded extractant (y). Experimental points are adjusted with equation (20), using κ 1 = 0.43±0.01 et κ 3 = 0.55±0.02.The variation in the bilayer thickness can be analysed considering the partition of extractants between the core (as the solvent molecule) and the interface of bilayers (as a cosurfactant), using the following relationship derived from (17):

  Hence, K' 1 =0.75±0.3 and K' 3 =0.82±0.7. Because of the dispersion of experimental points in the studied scale,

Figure 10 .

 10 Figure 10. Fraction of bonded extractant at the interface (θ) as a function of the Nd(NO 3 ) 3 concentration in the aqueous solution. Experimental points are well fitted with a Langmuir isotherm using K 2 =1.85±0.3.

Figure 11 .

 11 Figure 11. (a) Energy diagram of the extraction reaction divided into three elementary equilibrium: positioning of free extractants at interfaces (1), complexation of extractants with cations at interfaces (2), burying of bounded extractants from interfaces (3). (b) Free energy values associated to each elementary steps and to the global extraction reaction.

Table 1 .

 1 Dichroïc ratios of

f O C=


Séverine Zirah
email: szirah@mnhn.fr

Carlos Afonso
email: carlos.afonso@upmc.fr

Uwe Linne
email: linne@chemie.uni-marburg.de

Thomas A Knappe

Mohamed A Marahiel
email: marahiel@chemie.uni-marburg.de

Sylvie Rebuffat
email: rebuffat@mnhn.fr

Jean-Claude Tabet
email: jean-claude.tabet@upmc.fr

Topoisomer Differentiation of Molecular Knots by FTICR MS: Lessons from Class II Lasso Peptides

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structural characterization of peptide topoisomers, as well as the effect of conformation on hydrogen abstraction subsequent to ECD.

INTRODUCTION

Molecular knots constitute original structural motifs in biomolecules and fascinating topological objects. [1][2] Bioactive peptides with knotted structures, such as lasso peptides and cyclotides, share a knot topologically defined by covalent crosslinks (lactam ring and/or cysteine bridges) and display very compact and stable structures. Such structures therefore constitute valuable scaffolds with potential applications in peptide-based drug design. [3][4] Lasso peptides form a class of bacteria-produced bioactive peptides with a threaded structure involving an 8/9-residue ring resulting from an amide bond between the N-terminus and the carboxylic group of an Asp/Glu residue, crossed by the C-terminal tail. 5 They form three classes depending on the absence (class II) or presence of one (class III) or two (class I) disulfide bridges that further stabilize the structure. 6 They exhibit a variety of biological activities, involving enzyme inhibition, receptor antagonism, antimicrobial or anti-HIV activities (see Table 1 for class II lasso peptides). 5,[7][8][9][10][11][12] Their compact structure confers to lasso peptides a great resistance to denaturing conditions and certain proteases. The structural characterization of lasso structures and differentiation from their unthreaded topoisomers is not trivial and generally requires the use of complementary biochemical and spectroscopic methods.

Microcin J25 (MccJ25

) is a lasso peptide secreted by Escherichia coli AY25 that exerts potent antibacterial activity against Escherichia and Salmonella species, through import in the target bacteria upon interaction with the iron-siderophore receptor FhuA 13 and inhibition of the RNA polymerase. 14 Its C-terminal (18-21 SFYG) segment is sterically entrapped into the macrolactam ring by F19 and Y20 located on each side of the ring. Cleavage within the Y9-I18 region, either in solution by enzymatic cleavage or acidic hydrolysis, or in the gas-phase upon collision-induced dissociation (CID), generates two peptide complexes associated through the steric hindrance provided by the side chains of F19 and Y20. [15][16][17] A particular gas-phase fragmentation pattern upon CID, as compared to the corresponding synthetic peptide encompassing a macrolactam ring, has also been reported for the lasso peptide RES 701-1, 18 but generally only NMR could unambiguously characterize the threading of the C-terminal tail into the macrolactam rings. [7][8]19 Capistruin is a lasso peptide secreted by Burkholderia thailandensis E264 discovered by genome mining, which exhibits antibacterial activities against closely related Burkholderia and Pseudomonas species. 8 It showed a low overall fragmentation pattern upon CID.

However, no two-peptide complexes unequivocally proving a lasso structure were observed. 8 Comparison of the three-dimensional structures of MccJ25 and capistruin shows that MccJ25 displays a large loop involving a short antiparallel -sheet and a very short C-terminal segment below the ring, while capistruin displays a very tight loop and a much longer C-terminal segment below the ring (Figure 1). In both peptides, the steric entrapping of the C-terminal tail in the macrolactam ring relies on the presence of a bulky amino acid located below the ring: Y20 for MccJ25 20 and R15 for capistruin. 21 In addition, both peptides contain a bulky amino acid located above the ring: F19 for MccJ25 and R11 for capistruin. However, in capistruin the two bulky amino acids, R11 and R15, are not adjacent.

Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry has shown a great capability for the analysis of peptides and proteins, in particular for the characterization of posttranslational modifications. The main advantages of this method are the high resolution provided by the ICR cell together with the multiple excitation modes available on this instrument, such as (i) collisioninduced dissociation (CID) in the external collision cell or in the ICR cell through sustained off resonance irradiation (SORI) under CID conditions, 22 (ii) infrared multiphoton dissociation (IRMPD) 23 and (iii) for positive ions, electron-capture dissociation (ECD). [24][25] The latter has revealed particularly appealing for the structural characterization of peptides and proteins 26 , given the ability to cleave the peptide backbone while leaving intact labile side-chain modifications, or without disrupting the noncovalent interactions involved in the three-dimensional structure of proteins. [27][28] In order to develop an analytical method to unambiguously characterize topoisomeric peptides by mass spectrometry, the fragmentation patterns MccJ25, capistruin, and their corresponding non-lasso topoisomers MccJ25-lcm and capistruin-lcm, were analyzed by ESI-FT-ICR-MS upon CID, SORI-CID, IRMPD and ECD.

EXPERIMENTAL SECTION

Peptides

MccJ25 was produced from a culture of E. coli MC4100 harboring the plasmid pTUC202, cultivated for 16 h in M63 medium supplemented with 1 mg/mL vitamin B 1 , 0,02% MgSO 4 , 0,02% glucose and 1 g/L casamino acids. MccJ25 was purified from the culture supernatant by solid phase extraction on a SepPak C18 35 cc cartridge and by semi-preparative RP-HPLC on a C18 μBondapak column (300 mm × 3.9 mm, 10 μm, Waters).

Capistruin was produced from a culture of Burkholderia thailandensis E264 incubated for 24 h at 42 °C in M20 medium containing gentamycin (8 μg/mL), as described previously. 8 The purification was carried out by solid phase extraction followed by preparative RP-HPLC. 8 The synthetic cyclic branched peptides, MccJ25-lcm and capistruin-lcm, were obtained from Genepep (Montpellier, France).

FT-ICR mass spectrometry

Mass spectrometry experiments were performed on a hybrid Qh-FT/ICR (Bruker Daltonics, Bremen, Germany) equipped with an off-axis Apollo II ESI source operated in positive ion mode. A syringe pump was used to infuse the peptide solution (10 µM in H 2 O/CH 3 CN + 0.1% (v/v) formic acid) with a gas-tight syringe at a flow rate of 120 µL/h. CID experiments were performed in the collision cell or upon SORI-CID in the ICR cell using argon as collision gas. For SORI-CID, the ions were activated using a -1000 Hz frequency offset for 250 ms with a 3.3 Vp-p excitation amplitude. A pumping delay of 2 s was applied prior to ion analysis. When necessary, MS 3 experiments were conducted using CID in the collision cell in conjunction with either SORI-CID or IRMPD. ECD experiments were performed with an indirectly heated hollow cathode set to 1.7 A. Electrons emitted during 0.1 s were injected into the ICR cell with a 1.2 V bias.

Nomenclature of the product ions -6 -

To avoid any ambiguity, the nomenclature introduced by Roepstorff and Folman, 29 further modified by Biemann and coworkers, 30 was adopted to describe the product ions. For internal fragment ions, the general presentation form is (b r y s ) (r+s-t) , where r, s and t indicate the number of amino acids cleaved counting from the N-terminus, the number of amino acids cleaved from the C-terminus and the total number of amino acid residues in the peptide, respectively. The subscript (r+s-t) thus accounts for the number of residues in the internal fragment. For ECD fragmentations, similar notation using c/z codes were used. The charge states were omitted for singly-charged species and indicated for doubly-and triply-charged species.

RESULTS AND DISCUSSION

Fragmentation patterns of MccJ25 and capistruin and their non-lasso topoisomers generated by CID, SORI-CID and IRMPD

The CID spectra of MccJ25, capistruin, and their corresponding synthetic non-lasso topoisomers,

MccJ25-lcm and capistruin-lcm, which contain the macrolactam ring but without steric entrapping of the C-terminal tail inside, were obtained through CID in the external collision cell or SORI-CID in the ICR cell. The spectra provided similar information, therefore only SORI-CID spectra are presented, since these experiments permit to avoid m/z discrimination during the ion transfer between the collision cell and the ICR cell.

The SORI-CID spectrum of the M+3H 3+ species of MccJ25 (m/z 703.0) showed a complicated fragmentation pattern, involving two competitive dissociation processes (Figure 2A, supplementary Table S1). On the one hand, extensive fragmentations in the Y9-I17 loop were detected, yielding typical two-peptide product ions resulting from the steric entrapping of the (SFYG) C-terminal segment in the macrolactam ring, as already reported. 15,17 The main product ions consisted of ( series, together with the y 2 , y 3 , y 4 and y 6 series (supplementary Table S1). These series indicated a release of the (SFYG) C-terminal segment and therefore that fragmentation in the macrolactam ring occurred. Alternatively, such product ions could result from consecutive dissociation from b 20 3+ . Indeed, the loss of the C-terminal G21 residue may release the C-terminal tail.

Finally, consecutive H 2 O and CO losses from the precursor ion were observed (M+3H-H 2 O 3+ being the base peak of the spectrum). The SORI-CID spectrum of the M+2H 2+ species of MccJ25 displayed a weak dissociation profile, with M+2H-H 2 O 2+ as base peak (data not shown). The M+3H 3+ species of MccJ25-lcm showed a more specific fragmentation pattern under SORI-CID than that of its lasso topoisomer (Figure 2B, supplementary Table S2). The IRMPD spectrum of the [M+3H] 3+ species of MccJ25 (m/z 703.0) revealed to be less complex than the corresponding SORI-CID spectrum (Figure 3A, supplementary Table S3). The (m/z 567.3) (Figure 2B, 3B). In this regard, the SORI-CID spectrum presents more information about the existence of the lasso form, given the presence of the two-peptide product ions and although in this case the dissociation efficiency is lower as loss of water dominates the product ion spectrum. Thus, resonant activation mode that limits consecutive dissociation is more adequate for the structural characterization of lasso peptides as it allows here to conserve intact two-peptide ions that are a direct proof of the threaded structure.

The SORI-CID spectra of capistruin and capistruin-lcm (supplementary Figure S1) were poorly informative, as they showed mainly consecutive H 2 O and NH 3 losses from the precursor ion. In addition, capistruin showed very weak y/b product ions resulting from cleavages in the C-terminal tail.

The IRMPD spectra of capistruin and capistruin-lcm (Figure 4) also revealed mainly neutral losses, together with weak y/b series corresponding to cleavages in the C-terminal tail that were also affected by extensive neutral losses, and internal product ions. Many common product ions were detected for both topoisomers, but with different relative abundance and neutral loss patterns. As an example, the its non-lasso topoisomer, the peptides capistruin and capistruin-lcm were not distinguished by CID, which was completely dominated by the water loss, but gave different product ion abundance profiles upon IRMPD, which suggests the formation of two-peptide product ions for the lasso topoisomer. This weak fragmentation behavior upon ergodic dissociative processes is attributed to the presence of R11 and R15 residues in the tail region, which most probably sequester the protons. The lasso peptides

MccJ25 and capistruin displayed a very extensive or very weak fragmentation extent, respectively, which illustrates the influence basic amino acids on ergodic dissociation, in agreement with the mobile proton model. 31

Fragmentation patterns of MccJ25 and capistruin and their non-lasso topoisomers generated by ECD

The and (ii) ECD in the tail yielding a c/z • series. As expected, the two-peptide product ions, characteristic of the lasso structure, were absent. Note that the dissociation efficiency of the charge-reduced species was significantly higher for the non-lasso peptide as compared to the lasso topoisomer. This illustrates the high stability of the lasso structure, as observed by CID. MccJ25-lcm, which are both cyclic-branched peptides, the dissociation appear more complex, with both radical-driven and charge-driven bond cleavages. Such a competition between these two processes has been described recently for arginine-containing peptides, and the absence of basic residues is proposed to favor charge-driven fragmentations while arginine would promote radical-driven fragmentations. 33 The absence of basic residues in the tail of MccJ25 supports this tendency.

For both CID and ECD, MccJ25 showed a fragmentation pattern typical of the lasso structure, which permits to assign the lasso character directly, even without a comparison to the non-lasso topoisomer. The formation of the typical two-peptide product ions is particularly favorable for MccJ25, as compared to capistruin given the large size of the loop above the ring and the vicinity of the two bulky amino acids that maintain the C-terminal tail entrapped within the ring. Many two-peptide product ions show y 4 (SFYG) as minimal entrapped segment.

The dissociation of the [M+3H] 3+ and [M+2H] 2+ species of capistruin and its topoisomer capistruin-lcm were also investigated under ECD conditions. The ECD spectra of the [M+2H] 2+ species of capistruin and capistruin-lcm (supplementary Figure S2) revealed similar fragmentation patterns, with a c 16 to c 18 series. The ECD spectrum of the [M+3H] 3+ species of capistruin (Figure 6A) showed an abundant charge-reduced species [M+3H] •2+ , together with product ions resulting from neutral losses and a series of c and z • ions. The product ion at m/z 996 corresponded to a loss of CH 5 N 3 from the arginine side chain, a usual side chain cleavage in ECD. 34 The z 5 • to z 9

• product ions corresponding to cleavages within the tail were fairly abundant. The complementary c 11 to c 18 were produced as singly-

-12 -
charged, but also as doubly-charged species for the larger product ions (c 15 to c 18 ). c 11 , the smallest c ion detected, corresponded to the cleavage after R11. The z 10 • and z 9

• product ions were detected, but in very low abundance. It should be noted that none of these main cleavages involves dissociation of the ring. This indicates that an initial cleavage produced after electron capture occurred within the tail. This is most likely due to the presence of the two basic residues (R11 and R15) in this region, which promotes local c/z • fragmentations in ECD. However, low abundance product ions were attributed to internal cleavages within the ring. The ECD spectrum of the [M+3H] 3+ species of the capistruin-lcm topoisomer revealed a similar fragmentation pattern (Figure 6B), but with significant differences in the relative abundance of the product ions. In particular, the z 10 • and z 9

• product ions were detected in a much larger abundance as compared to capistruin. This trend suggests that when these cleavages occur for the lasso topoisomer capistruin, the two peptidic entities constitute two-peptide product ions as for

MccJ25, due to the steric hindrance provided by R11 and R15 (as already suggested for CID experiments). 8 In spite of this specific profile of product-ion abundances, no strong evidence of the lasso structure could be detected.

Interestingly, part of the product ions corresponded to c and z • , but several c • and z were also detected. Such a trend is relatively frequent during ECD dissociation of peptides and it has been proposed that the c • and z species are produced from a hydrogen transfer occurring in a long life c/z • complex. [35][36][37] For capistruin and capistruin-lcm topoisomers, the c • ions revealed particularly abundant for cleavages of the tail nearby the ring. In addition, the relative abundance of c • /z species was strongly reduced for the non-lasso topoisomer. This behavior is illustrated in the spectra enlargement in Figure 6C. In the non-lasso peptide, the normal c 11 was mainly detected, while a significant amount of c 11

• species was detected for the lasso topoisomer. The same behavior was observed for z 9 , which was mostly detected as z 9

• for the capistruin-lcm and as a mixture z 9

• /z 9 for capistruin. Only a comparison with the non-lasso topoisomer permitted to assign the lasso structure of capistruin, with regard to the relative abundance of the product ions and the extent of hydrogen abstraction. Therefore, ECD appeared as more powerful in differentiating the two topoisomers capistruin and capistruin-lcm as compared to ergodic dissociation and generated mainly c/z • product ions within the C-terminal tail. The absence of y/b product ions suggested that electron capture occurred within the tail and yielded a free radical reaction cascade resulting in multiple backbone cleavages.

CONCLUSIONS

Our data showed that characterization of lasso peptides from gas-phase dissociation can be more or less complex depending on several parameters (Figure 1

FIGURES AND TABLES

Table 1

Sequences and biological activities of type-II lasso peptides a

Name

Y V L E Q S G W I V N S K H G V P P G R Y V L E Q S G W I V N S K H G V P P G R Y V L E Q S G W I V N S K H G V P R Y V L E Q S G W I V N S K

  As an example, the internal fragment GIGTPI of MccJ25 has r = 17, s = 10 and t = 21 and is denoted (b 17 y 10 ) 6 . The product ions resulting from fragmentation in the loop of the lasso structure and where the C-terminal tail is threaded into the macrolactam ring were denoted [(b r )*(y s )], where r and s indicate the number of amino acids in the non-covalently bound Nterminal and C-terminal peptide segments. As an example, the product ion [MccJ25-{GIGTPI}] is denoted [(b 11 )*(y 4 )].

  b 16 )*(y 4 ) 3+,2+ and (b 15 )*(y 4 ) 3+,2+ , with consecutive H 2 O and CO neutral losses, together with (b 11 )*(y 6 ) 2+ and (b 11 )*(y 4 ) 2+ . Several internal product ions, complementary to the two-peptide product ions, were also detected: (b 15 y 10 ) 4 , (b 17 y 10 ) 6 , (b 19 y 8 ) 6 , (b 16 y 9 ) 4 … On the second hand, complementary y/b series corresponding to fragmentations in the C-terminal tail were detected. They consisted of the b 8 , b 9 , b 10 ,

  As for MccJ25, the base peak was M+3H-H 2 O 3+ at m/z 697.0. The spectrum was dominated by the b series (singly-charged b 9 to b 13 , doubly-charged b 11 2+ to b 15 2+ , b 17 2+ , b 19 2+ , triply-charged b 20 3+ ). Intense product ions corresponding to one or two H 2 O losses were observed for b 17 2+ , b 19 2+ and b 20 3+ . This behavior is most likely due to the presence of the T15 and S18 residues in the vicinity of the charge. The complementary y 2 , y 4 , y 6 , y 7 and y 8 -H 2 O were also observed. Finally, only few internal product ions were detected, such as (b 19 y 8 ) 6 -2H 2 O (m/z 567.3). As expected, two-peptide product ions were not detected.

  spectrum was dominated by the b series (b 8 to b 13 , b 16 2+ , b 17 2+ and b 19 2+ with one or two H 2 O losses). The y series consisted in y 2 , y 3 , y 4 , y 6 . Only few low abundance two-peptide product ions were detected: [(b 16 )*(y 4 )-H 2 O] 3+,2+ (m/z 659.3, 988.5) and [(b 15 )*(y 4 )-H 2 O] 2+ (m/z 939.9). This result contrasts with the -8 -SORI-CID spectrum where these two peptide ions are produced in high abundance. In addition, several internal product ions were observed, in particular (b 10 y 13 ) 2 and/or (b 20 y 3 ) 2 (m/z 311.1), together with (b 17 y 10 ) 6 -H 2 O (m/z 521.3). This fragmentation pattern illustrates that IRMPD triggers consecutive dissociation that result in multiple neutral losses and in the opening of the macrolactam ring, and therefore in the release of the entrapped C-terminal segment. A similar behavior towards IRMPD was observed for the [M+2H] 2+ species of MccJ25 (data not shown). The IRMPD spectrum of the [M+3H] 3+ species of MccJ25-lcm showed a y/b fragmentation pattern close to that of MccJ25, but with a significantly higher fragmentation efficiency (Figure 3B, supplementary Table S4). While for the lasso peptide MccJ25 the precursor ion dominated the IRMPD spectrum, for the non-lasso topoisomer, the b 13 product ion was the most intense ion. The IRMPD spectra of MccJ25 and MccJ25-lcm were dominated by b/y series corresponding to cleavages within the C-terminal tail but were fairly different in terms of product ion relative abundance. The b 17 to b 20 product ions, corresponding to cleavages within the (SFYG) C-terminal segment, were favored for the lactame topoisomer as compared to the lasso. This can be explained by the burring of this segment in the macrolactame ring, which may strengthen the peptidic bonds in this region. Only the minor two-peptide product ions characteristic of the lasso topoisomer, i.e., [(b 16 )*(y 4 )-H 2 O] 3+,2+ and [(b 15 )*(y 4 )-H 2 O] 2+ , could allow to unambiguously distinguish the two structures. SORI-CID is an "almost" resonant activation mode where only the precursor ion is effectively activated at its cyclotron frequency. With IRMPD, both the precursor ion and product ions present in the laser beam path are activated, which tend to enhance consecutive dissociation. 23 Thus, the [(b r )*(y s )] two-peptide product ions can dissociate, yielding the conventional y s or b r product ions. Consecutive dissociation can also explain the high abundance of some internal product ions such as (b 19 y 8 ) 6 -2H 2 O

[b 15 -

 15 H 2 O] 2+ , [b 16 -H 2 O] 2+ , [b 17 -H 2 O] 2+ , [b 18 -H 2 O] 2+ series dominated the m/z 700-1000 region in the IRMPD spectrum of capistruin, while this region was dominated by y 6 -NH 3 , y 7 -NH 3 , y 8 -NH 3 for capistruin-lcm. This trend suggests that the product ions [b 15 -H 2 O] 2+ to [b 18 -H 2 O] 2+ , more intense for the lasso topoisomer, correspond to lasso-structured product ions, maintained through R11 and R15 sidechain located above and below the ring. Thus, as for MccJ25, the ergodic gas-phase fragmentations of capistruin appear to result in two dissociative processes, (i) the former preserving the lasso structure and yielding the [b 15 -H 2 O] 2+ to [b 18 -H 2 O] 2+ product ions, and (ii) the latter yielding y/b series corresponding to cleavages in the C-terminal tail, which indicates a previous precursor ion isomerization through opening of the macrolactam ring. However, the short sequence of the loop did not permit to generate two-peptide product ions, which require two bond cleavages in the loop region. Contrary to MccJ25 and

  ECD spectra of MccJ25 revealed different fragmentations patterns for the [M+2H] 2+ and [M+3H] 3+ species (Figure 5 A,B). The doubly-charged species generated mainly a c series (c 17 , c 18 , c 19 , c 20 ), together with two-peptide product ions: [(c 9 )*(y 7 )-H 2 O], [(c 10 )*(y 7 )-H 2 O] and [(c 11 )*(y 7 -H 2 O)]. Two relatively intense product ions at m/z 1298.7 and 1341.7 could not be assigned. The triply-charged species yielded a y series (y 4 , y 6 , y 8 ) and b series (b 10 , b 11 , b 13 , b 15 ) corresponding to + 1 u mass shifts, noted b' • n (Figure 5B). Almost no c/z • ions were detected, with the exception of c 18 (m/z 1738.8) and c 19 2+ (m/z 943.5). In addition, in a lower extent, two-peptide product ions such as [(b 8 )*(y 12 )] 2+ and the series [(c 8 )*(y 4 )], [(c 9 )*(y 4 )] and [(c 10 )*(y 4 )] were present. Finally, c/y or b/y internal product ions corresponding to tail fragments, i.e., (c 19 y 4 ) 2 , (c 20 y 4 ) 3 , (c 19 y 6 ) 4 , (c 19 y 8 ) 6 , …, were detected. The non-lasso topoisomer MccJ25-lcm, dissociated upon ECD, showed certain trends common to its lasso topoisomer: a c 11 to c 20 series for the doubly-charged species (Figure 5C), and two competitive dissociation processes for the triply-charged species (Figure 5D): (i) ECD in the ring and consecutive y/ b' • series

  The almost complete absence of c/z • ions for the [M+3H] 3+ species of MccJ25 and MccJ25-lcm is quite unusual and should indicate a particular dissociation pathway. Indeed, electron capture is expected to yield charge-reduced species [M+3H] •2+ that consecutively undergo c/z • cleavage. The b' • series and bipeptic product ions permitted to propose a competitive fragmentation pattern involving two isomeric charge-reduced species [M+3H] •2+ (m/z 1054.5) (Scheme 1). Most likely, the initial ECD occurred within the macrolactam ring, which is consistent with the presence of the basic histidine residue at position 5 (pathway a). The excess of energy in the [M+3H] •2+ charge-reduced species, due to electron capture, would yield consecutive dissociation involving a conventional y/b cleavage within the C-terminal tail (b series corresponding to + 1 u mass shifts). On the other hand, an initial ECD in the Cterminal tail (pathway b) would result in a two-peptide charge-reduced species [M+3H] •2+ , further dissociated into two-peptide and internal product ions. Such pathways result from the presence of both a cyclic and a linear region in the peptide. Multiple backbone cleavages in cyclic peptides have been proposed to result from a free radical reaction cascade in which the -carbon radical formed by electron capture propagates along the peptide backbone by free radical rearrangements. 32 For MccJ25 and

  ):(1) the size of the loop above the macrolactam ring, (2) the vicinity of the bulky amino acids in the C-terminal tail located above and below the macrolactame ring, (3) the presence of basic residues within the ring and the tail (that limits the fragmentation extent in ergodic dissociation and promote local fragmentations in ECD). ECD appears particularly promising, since it provides evidence of the lasso structure for both MccJ25 and capistruin.It generates competition between charge-driven and radical-driven bond cleavages, which orientation was strongly influenced by the presence or absence of arginines. The presence of two-peptide product ions is the main trend typical of the lasso structure and was clearly observed upon CID and ECD of MccJ25. For capistruin, the absence of double cleavage between D9 and R11 segment did not permit to detect it.The different hydrogen abstraction extent for the two topoisomers capistruin and capistruin-lcm is the first evidence of the influence of topology on this process, which was mainly interpreted with regard to the amino acid sequences. It suggests potential applications in the characterization of peptide or protein conformations, topology or non-covalent associations.ACKNOWLEDGEMENTThis work was supported by the ANR project no. BLAN_NT09_692063 and the Deutsche Forschungsgemeinschaft (DFG MA 811-25/1).

Figure 1 .

 1 Figure 1. Three-dimensional structures of MccJ25 17 (A) and capistruin 8 (B). Scheme representing

Figure 2 .

 2 Figure 2. SORI-CID spectra of the [M+3H] 3+ species of (A) MccJ25 and (B) MccJ25-lcm (m/z 703.0).

Figure 3 .

 3 Figure 3. IRMPD spectra of the [M+3H] 3+ species of (A) MccJ25 and (B) MccJ25-lcm (m/z 703.0). The

Figure 4 .

 4 Figure 4. IRMPD spectra of the [M+3H] 3+ species of (A) capistruin and (B) capistruin-lcm (m/z 683.7).

Figure 6 .

 6 Figure 6. ECD spectra of the [M+3H] 3+ species of (A) capistruin (B) and capistruin-lcm (m/z 683.7). Typical product ions are displayed. (C) c 11 /c 11 • isotopic profiles obtained experimentally for capistruin and capistruin-lcm (on the left), and expected for c 11 and c 11 • (on the right).

a The sequences are represented unthreaded for clarity.b The lasso structure (threading of the tail into the macrolactame ring) is hypothesized but has not been shown.