Marie Devreux

Céline Henoumont

Fabienne Dioury

Sébastien Boutry

Olivier Vacher

Luce Vander Elst

Marc Port

Robert N Muller

Olivier Sandre

Sophie Laurent
email: sophie.laurent@umons.ac.be

Mn2+ Complexes with Pyclen-Based Derivatives as Contrast Agents for Magnetic Resonance Imaging: Synthesis and Relaxometry Characterization

Keywords: MRI, Contrast agent, Manganese ions, Pyclen

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

Introduction

Gadolinium complexes are currently the main contrast agents (CAs) authorized and used in the clinics for medical diagnosis by Magnetic Resonance Imaging (MRI). In fact, this powerful noninvasive imaging technique often requires the use of a CA to compensate its lack of sensitivity.

Different classes of CAs have been developed for MRI, yet gadolinium complexes are by far the most used in the clinical field thanks to their property to decrease the water longitudinal relaxation time of protons, highlighting on the images the areas where the CA is accumulated. 1 Nevertheless, some researches have demonstrated since more than a decade that the injection of gadolinium complexes to patients with kidneys failure can trigger apparition of a disease called nephrogenic systemic fibrosis (NSF). [2][START_REF] Beam | GBCAs and risk for nephrogenic systemic fibrosis: A literature review[END_REF][START_REF] Pan | Manganese-based MRI contrast agents: past, present and future[END_REF][START_REF] Botta | Mn(II) compounds as an alternative to Gd-based MRI probes[END_REF] This is particularly true for linear gadolinium complexes based on acyclic ligands such as Gd-DTPA (diethylenetriamine penta acetic acid gadolinium complex) that are less thermodynamically and kinetically stable than those based on macrocyclic complexes such as Gd-DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid gadolinium complex). Therefore, some marketed Gd-based CAs (GBCAs) are no longer recommended for these patients. 2,[START_REF] Beam | GBCAs and risk for nephrogenic systemic fibrosis: A literature review[END_REF][START_REF] Ramalho | Gadolinium-based contrast agent accumulation and toxicity: An update[END_REF] In addition, more recent studies have shown accumulation in the brain of gadolinium CAs in subjects with normal renal function. [START_REF] Kanda | Gadolinium-based contrast agent accumulates in the brain even in subjects without severe renal dysfunction: Evaluation of autopsy brain specimens with inductively coupled plasma mass spectroscopy[END_REF][START_REF] Gianolio | Chemical Insights into the Issues of Gd Retention in the Brain and Other Tissues Upon the Administration of Gd-Containing MRI Contrast Agents[END_REF] Further investigations were carried out on rats to compare macrocyclic (Gadoteridol ® ) with linear (Gadodiamide ® ) marketed agents and to quantify the Gd-species retained in the brain. It was concluded that intact and soluble Gd-species, whereas Gadodiamide ® was found mostly as insoluble species. [START_REF] Robert | T1-Weighted Hypersignal in the Deep Cerebellar Nuclei after Repeated Administrations of Gadolinium-Based Contrast Agents in Healthy Rats: Difference between Linear and Macrocyclic Agents[END_REF][START_REF] Gianolio | Gadolinium Retention in the Rat Brain: Assessment of the Amounts of Insoluble Gadolinium-containing Species and Intact Gadolinium Complexes after Repeated Administration of Gadolinium-based Contrast Agents[END_REF] This situation explains that scientists are much interested in finding a safer next generation of MRI CAs based on Gd-free alternatives, and one of them could be the use of manganese-based CAs. [START_REF] Drahoš | Manganese ( II ) Complexes as Potential Contrast Agents for MRI[END_REF][START_REF] Pan | Revisiting an old friend: Mn-based MRI contrast agents[END_REF] Manganese ions are essential and naturally present in the body under the high-spin form of Mn 2+ or Mn 3+ with 5 or 4 unpaired d-orbital electrons, respectively. The normal physiological concentration of manganese in the serum of healthy subjects is about 0.05-0.12 µg/dL (9-22 µM) [START_REF] Crossgrove | Manganese toxicity upon overexposure[END_REF] , and part of its important biological role in the organism is to act as a cofactor activating certain enzymes or as a constituent in metalloenzymes. Manganese ions also play a role in the development of the immune and nerve systems function and in the regulation of the amount of vitamins and sugar in the blood. [START_REF] Crossgrove | Manganese toxicity upon overexposure[END_REF][START_REF] O'neal | Manganese Toxicity Upon Overexposure : a Decade in Review[END_REF] The first use of manganese as MRI CA was an oral formulation containing liposome-encapsulated MnCl 2 salt (LumenHance ® ) indicated for gastrointestinal examinations. However, several studies have shown that too high doses of free manganese ions could induce a neurodegenerative disorder called manganism, a disease with symptoms similar to those of the Parkinson's disease. Despite toxicity of free Mn 2+ ions, manganese enhanced MRI (MEMRI) using MnCl 2 is used for preclinical studies in mice for brain [START_REF] Castets | In vivo MEMRI characterization of brain metastases using a 3D Look-Locker T1mapping sequence[END_REF] or lung [START_REF] Bianchi | Orotracheal manganese-enhanced MRI (MEMRI): An effective approach for lung tumor detection[END_REF] model tumors. To avoid adverse effects, manganese complexes have been developed, and the second manganese-based CA approved by the Food and Drug Administration (FDA) in 1997 was manganese dipyridoxyl diphosphate (Mn-DPDP, Teslascan ® , Figure 1) for use as a liver specific hepatobiliary CA. [START_REF] Pierre | Contrast agents for MRI: 30+ years and where are we going?[END_REF] This structure is based on a linear ligand and has low thermodynamic and kinetic stabilities with consequently a certain amount of free manganese ions released in vivo. The efficacy of the Mn-DPDP as a MRI CA is quite low because the ligand does not allow a coordination bond with one water molecule in the inner-sphere of metal coordination. The interest of such a candidate as CA comes therefore from the part of the released manganese ions whose relaxivity is 2.8 s -1 •mM -1 at 20 MHz and 40°C in aqueous solution; as a point of comparison, the relaxivity of Gd-DOTA which is a well-known commercialized macrocyclic CA is 3.5 s -1 •mM -1 at 20 MHz and 3.1 s -1 •mM -1 at 60 MHz and 37°C. [START_REF] Elizondo | paramagnetic Hepatobiliary Contrast Agent for MR Imaging[END_REF][START_REF] Laurent | Comparative study of the physicochemical properties of six clinical low molecular weight gadolinium contrast agents[END_REF] As a result and for safety concerns (potential toxicity of released free Mn 2+ ), Mn-DPDP is no longer commercialized for clinical use, so there is still need for Gd-free alternatives, with higher thermodynamic stability and kinetic inertness in order to avoid the release of free manganese ions in vivo, and with a higher efficacy to be competitive with GBCAs. [START_REF] Pan | Manganese-based MRI contrast agents: past, present and future[END_REF][START_REF] Botta | Mn(II) compounds as an alternative to Gd-based MRI probes[END_REF][START_REF] Drahoš | Manganese ( II ) Complexes as Potential Contrast Agents for MRI[END_REF][START_REF] Laurent | Comparative study of the physicochemical properties of six clinical low molecular weight gadolinium contrast agents[END_REF] As a possibility, researchers examined if Mn-porphyrins, where the manganese cation is the center of a heme ring, can be used as MRI CAs: they found very high longitudinal and transverse relaxivities, of 9.33 and 12.0 s -1 •mM -1 at 3T (20°C) respectively, thereby enabling MRI detection in vivo of Mn-labelled cells. [START_REF] Venter | A manganese porphyrin-based T1 contrast agent for cellular MR imaging of human embryonic stem cells[END_REF][START_REF] Alhamami | Manganese-porphyrin-enhanced MRI for the detection of cancer cells: A quantitative in vitro investigation with multiple clinical subtypes of breast cancer[END_REF] Porphyrins exhibit multiple advantages such as being able to stabilize manganese in its higher oxidation states Mn(III) or even Mn(V), needing to add a reducing agent in the medium such as sodium dithionite (Na 2 S 2 O 4 ) to insure the Mn(II) state. [START_REF] Aime | A p(O2)-Responsive MRI contrast agent based on the redox switch of manganese(II/III)-porphyrin complexes[END_REF] However, their synthesis is delicate in the perspective of scale-up production for an envisioned clinical use. Moreover, such compounds are highly light-sensitive, which is why they are intensively studied in a context of therapeutic agents for photodynamic therapy. Slightly larger ring-shaped ligands called texaphyrins complexing Mn 2+ were recently proposed as bimodal CAs (MRI and photoacoustic imaging). [START_REF] Ren | Manganese(II) Texaphyrin: A Paramagnetic Photoacoustic Contrast Agent Activated by Near-IR Light[END_REF] On the other hand, purely inorganic Mn-based nanoparticles (NPs) like antiferromagnetic MnO or paramagnetic Mn-doped ferrocyanide NPs (Prussian blues)

were also tested as potential CAs for MRI. [START_REF] Paul | MnII-containing coordination nanoparticles as highly efficient T1 contrast agents for magnetic resonance imaging[END_REF] In view of this effort of the community to develop macrocyclic ligands with high affinity for Mn(II) and high efficiency as MRI CAs [START_REF] Ndiaye | Unprecedented kinetic inertness for a Mn2+-bispidine chelate: a novel structural entry for Mn2+-based imaging agents[END_REF] , this report explores the potential of four Mn(II) 6 complexes based on the pyclen core (3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13triene) and carrying an additional site for the conjugation to a molecule of interest, for instance a chemotoxic drug or a polymeric vector. Pyclen is an interesting 12-membered macrocyclic structure that incorporates a N-pyridyl donor that rigidifies and pre-organizes the ligand coordinating groups (in particular rendering the 4 nitrogen atoms coplanar), an attribute that can improve the kinetic inertness of the resulting complex. Pyclen ligands have been reported

recently for coordination of luminescent lanthanide cations to develop optical probes for nearinfrared imaging. [START_REF] Hamon | Pyclen based Ln(III) complexes as highly luminescent bioprobes for in vitro and in vivo one-and two-photon bioimaging applications[END_REF] The pyridine subunit also provides enhanced degree of lipophilicity that can promote mixed renal and hepatobiliary clearances, another interesting attribute in the context of patients with reduced kidney function. The choice of pyclen-based chelating agents was also guided by their availability as "tailor-made" synthetic chelators prepared according to a versatile and potentially up-scalable strategy practiced by us and others which consists in assembling two synthons previously "dressed" with all the final functionalities required: the donor groups to satisfy the electronic demand of the paramagnetic ion, and an additional function to allow its conjugation to a molecule of interest. [START_REF] Dioury | Synthesis of a tricyclic tetraazatriacetic ligand for gadolinium(III) as potential contrast agent for MRI[END_REF][START_REF] Dioury | Synthesis of an hexadentate tricyclic tetraazadiacetic ligand as precursor for MRI contrast enhancement agents[END_REF][START_REF] Drahoš | Mn2+Complexes with 12-Membered pyridine based macrocycles bearing carboxylate or phosphonate pendant arm: Crystallographic, thermodynamic, kinetic, redox, and 1H/17O relaxation studies[END_REF][START_REF] Enel | Facile Access to the 12-Membered Macrocyclic Ligand PCTA and Its Derivatives with Carboxylate , Amide , and Phosphinate[END_REF][START_REF] Devreux | Bimodal Probe for Magnetic Resonance Imaging and Photoacoustic Imaging Based on a PCTA-Derived Gadolinium(III) Complex and ZW800-1[END_REF] Since Mn(II) complexes typically have coordination numbers of 6 or 7, ligands for Mn(II) cannot exceed hexadenticity to allow the exchange of at least one water co-ligand in its inner-sphere that is a crucial parameter to insure interesting relaxometric properties for MRI applications. Consequently, it was necessary to cleverly functionalize the pyclen derivative in order to limit to 6 its coordination bonds with Mn 2+ while providing a sufficiently high degree of coordination to preserve satisfying thermodynamic stability of the complex. , (C) PCTA, PC3AM H , PC3AM Gly , and PC3AM Pip , [START_REF] Garda | Effect of the nature of donor atoms on the thermodynamic, kinetic and relaxation properties of Mn(II) complexes formed with some trisubstituted 12-membered macrocyclic ligands[END_REF] and (D) PC2A, PC2A-EA, and PCTA [START_REF] Botár | Stable and inert Mn ( II ) -based and pH responsive contrast agents Stable and inert Mn ( II ) -based and pH responsive contrast agents[END_REF] The pyclen ( [12]PyN4) macrocyclic core is now recognized to form efficient chelators for the Mn 2+ cation complexation, and some studies can be found on the interest of such pyridinecontaining (PC) ligands (figure 1). Drahos et al. have studied the influence on the thermodynamic stability, the kinetic inertness, the redox potential, and the 1 H and [START_REF] Pierre | Contrast agents for MRI: 30+ years and where are we going?[END_REF] O relaxation rates by modifying the nature of the coordinating group on one arm of the macrocycle. They found that the mono-functionalized pyclens PCMA and PCMP with an acetate or a methyl phosphonate pendant arm give ternary hexacoordinate Mn 2+ complexes accommodated with one water co-ligand (Figure 1), both of them being rather stable. [START_REF] Drahoš | Mn2+Complexes with 12-Membered pyridine based macrocycles bearing carboxylate or phosphonate pendant arm: Crystallographic, thermodynamic, kinetic, redox, and 1H/17O relaxation studies[END_REF] With a similar purpose, Garda et al. studied the influence on the proton relaxometry, the thermodynamic stability and the kinetic inertness of the presence of a primary, a secondary or a tertiary amide instead of the carboxylate functions of the PCTA which is a well know pyclen derivative. [START_REF] Garda | Effect of the nature of donor atoms on the thermodynamic, kinetic and relaxation properties of Mn(II) complexes formed with some trisubstituted 12-membered macrocyclic ligands[END_REF] However, the corresponding Mn(II)-complexes show quite low relaxivities, less than 2 s - inert, with a maximum relaxivity at acidic pH of 3.5 s -1 •mM -1 at 20 MHz and 37°C. [START_REF] Botár | Stable and inert Mn ( II ) -based and pH responsive contrast agents Stable and inert Mn ( II ) -based and pH responsive contrast agents[END_REF] All these results show that much efforts is still to be done to try increasing the efficacy of Mn-complexes as MRI contrast agents (Figure 1).

In this work, it was decided to study a series of four N 4 O 2 hexadentate pyclen chelators including three structures carrying an additional function grafted onto the pyridine ring to allow conjugation to a molecule of interest (Figure 2). The "naked" complex MnL 1 H where -H designates absence of substituent in ortho position of the pyridine ring will be used as a reference to evaluate the impact of this additional subunit on the relaxometric properties of the corresponding Mn-complexes. Our previous work showed indeed that the functionalization of the carboxylate moiety into an amide bond on the pyridine arm of Gd-PCTA leads to a loss of one water molecule in the inner-sphere, giving q = 1 instead of q = 2. [START_REF] Devreux | Bimodal Probe for Magnetic Resonance Imaging and Photoacoustic Imaging Based on a PCTA-Derived Gadolinium(III) Complex and ZW800-1[END_REF] The syntheses are reported herein, together with the full relaxometric characterization of the corresponding Mncomplexes in order to have a complete understanding of their efficiency as MRI CAs (Figure 2).

Results and discussion

Synthesis of the pyclen-based Mn 2+ complexes

The synthetic route to triamine 5b is similar to that we described previously for the analogous compound bearing three acetate arms (Scheme 1). [START_REF] Devreux | Bimodal Probe for Magnetic Resonance Imaging and Photoacoustic Imaging Based on a PCTA-Derived Gadolinium(III) Complex and ZW800-1[END_REF] The selectively N,N''-protected As previously described for PCTA derivatives, the pyclen macro-ring formation was achieved by reacting the triamine 5b with a small excess of 2,6-dibromomethylpyridine 6a-b under heterogeneous conditions in refluxing acetonitrile at moderate dilution (0.01M) with sodium carbonate as scavenger (Scheme 2). [START_REF] Devreux | Bimodal Probe for Magnetic Resonance Imaging and Photoacoustic Imaging Based on a PCTA-Derived Gadolinium(III) Complex and ZW800-1[END_REF] In such conditions, the corresponding macrocycles 7a-b

diethylenetriamine
were isolated in 39 and 64% yields respectively.

Scheme 2. Synthesis of (d) Na 2 CO 3 , CH 3 CN, reflux

The macrocyclic pyclen skeleton built, successive step-by-step deprotections were necessary in order to allow selective grafting of different molecules of interest on the additional pendant group attached on the pyridine moiety and on the macrocycle (Scheme 3-5). subsequent conjugation to an amine function of different molecules of interest (Scheme 3). As previously shown by us for a bifunctional PCTA chelator prepared to complex Gd 3+ , [START_REF] Devreux | Bimodal Probe for Magnetic Resonance Imaging and Photoacoustic Imaging Based on a PCTA-Derived Gadolinium(III) Complex and ZW800-1[END_REF] the resulting amide bond can act as an additional donor group so that the overall denticity of the chelator is increased, and the coordination of water molecule as co-ligand in the inner-sphere of the paramagnetic center can be compromised which has a negative impact on the relaxivity of the chelate. Consequently, it was decided to study the impact of this additional amide bond on the relaxivity of the Mn 2+ complexes.

Both compounds 14 and 17 were designed with a dual purpose: to study the influence of an additional pendant amide bond on relaxometry, and to vary the function to allow subsequent conjugation for forthcoming studies: by click chemistry with Huisgen's 1,3-cyloaddition on the alkyne end-group, or by coupling the primary amine with carboxylic acid groups (Schemes 4-5).

On the one hand, propargylamine was grafted to compound 9 using the uronium-based On the other hand, the ethyl ester derivative 7b was submitted to aminolysis in ethylenediamine at room temperature for 1 hour and led to the expected amido derivative 15 in 58% yield.

Compound 15 was then submitted to acidolyses with hydrogen bromide in warmed acetic acid to cleave both the tert-butyl ester and the carbamate functions and led to the expected ligand 16a in 72% yield. It is noteworthy that upon treatment with hydrogen chloride in diethyl ether solution at room temperature, even repeatedly, only partial cleavage of the benzyloxycarbamate function was observed and gave a mixture of the desired compound 16a and of the intermediate 16b (as hydrochloride forms). Complexation with Mn 2+ finally proceeded very easily, in few minutes at room temperature (Scheme 5). In order to ensure that manganese is in the oxidation state +2, the complexations were performed under argon atmosphere and a reducing agent, sodium dithionite (approximately 1.3 eq Na 2 S 2 O 4 ), was added to the solutions with the pH maintained between 7 and 8. 22

Relaxometric characterization

Determination of the number of coordinated water molecules

The determination of the number of water molecules in the inner coordination sphere (q) is very important to estimate the efficacy of the complexes as MRI contrast agents, as the longitudinal proton relaxivity r 1 is directly related to the number of coordinated water molecules, in permanent exchange with those of the solvent. Two different techniques were used to determine this q parameter for the Mn(II) complexes, developed respectively by E. Gale et al.

water transverse relaxivity r 2 is measured as a function of temperature. [START_REF] Gale | Direct Measurement of the Mn(II) Hydration State in Metal Complexes and Metalloproteins through 17 O NMR Line Widths[END_REF] (Figure 3) The method is then based on the maximum [START_REF] Pierre | Contrast agents for MRI: 30+ years and where are we going?[END_REF] O transverse relaxivity, r 0 2max , measured in this case at low temperature (equation 1). This equation is obtained by different approximations, as explained by E. Gale et al., and the r 0 2max measured at 11.75 T lead to the results shown in Table 1. The number q is approximately the same for all complexes and close to one. [START_REF] Gale | Direct Measurement of the Mn(II) Hydration State in Metal Complexes and Metalloproteins through 17 O NMR Line Widths[END_REF] To confirm these results, a second technique was used, based on the measurement of NMRD profiles (i.e. the water proton longitudinal relaxivity r 1 as a function of magnetic field or proton resonance frequency) at 25°C, developed by J. Peters and C. Geraldes. [START_REF] Peters | A Semi-Empirical Method for the Estimation of the Hydration Number of Mn(II)-Complexes[END_REF] (Figure 4) This is based on another equation related to the proton longitudinal relaxivity at low field (on the plateau of the NMRD profile) and the formula weight (FW) of the complex (equation 2). The results (Table 1) confirm the presence of one coordinated water molecule in the inner sphere for the four synthesized complexes. The slight difference between the results coming from the two techniques can be explained by the fact that both methods rely on different approximations. The presence of one coordinated water molecules is an interesting result since it shows that the presence of the arm on the pyridine moiety, with or without amide bond, does not change the number of coordinated water molecules, meaning that this appended functionality does not participate to the coordination bounds with the manganese ion. It will thus allow further grafting of molecules of interest in a forthcoming study.

16 from Formula (1) derived in reference 34 as a function of the maximum value r 0 2max .

𝑞𝑞 = 𝑟𝑟 2max 0 [H 2 O] ⎝ ⎛ 2
� (𝑆𝑆(𝑆𝑆 + 1) 3 𝐴𝐴 0 ℏ ⎠ ⎞ ≅ 𝑟𝑟 2max 0 510 (1)
𝑞𝑞 = 𝑟𝑟 1p 9.16 �1-e (-2.97 × FW × 10 -3 ) � (2)

Determination of the exchange rate of the coordinated water molecule

The technique which allows to determine the lifetime of the coordinated water molecule was developed by T. Swift and R. Connick. [START_REF] Swift | NMR Relaxation Mechanisms of O17 in Aqueous Solutions Sphere[END_REF] It is based on the measurement of the [START_REF] Pierre | Contrast agents for MRI: 30+ years and where are we going?[END_REF] O transverse relaxation rate as a function of temperature, as for the determination of q for the Mn(II) complexes. (Figure 5). The results (Table 2) show that the coordinated water molecule is in a fast exchange regime for the four complexes studied since residence times τ M of the order of 6 ns are obtained. 2.

Table 2. Determination of the residence time at 37°C and 11.75 T of the coordinated water molecule (𝜏𝜏 M ), as well as the other parameters characterizing this exchange : A/ħ, the hyperfine coupling constant between the oxygen nucleus of the bound water molecule and the Mn 2+ ion; τ v , the correlation time modulating the electronic relaxation of Mn 2+ ; E v , the activation energy related to τ v ; B, related to the mean-square value of the zero-field splitting energy ∆ (B = 2.4∆ 2 );

and ∆H ≠ and ∆S ≠ the enthalpy and entropy of activation, respectively, of the water exchange process. The number of coordinated water molecules was set to q = 1.

𝜏𝜏 𝑀𝑀 (ns) ∆𝐻𝐻 ≠ (kJmol -1 ) ∆𝑆𝑆 ≠ (Jmol -1 K -1 )
A/ħ (10

Relaxometric measurements and NMRD profiles to assess the efficiency as MRI CA

As a reminder, the relaxivity is defined as the increase of the water proton relaxation rate induced by one mmol per liter of paramagnetic complexes. Longitudinal (r 1 ) relaxivities were measured at 37°C and respectively 0.47 T (20 MHz) and 1.41 T (60 MHz) (Table 3). They are lower than that of the commercially available gadolinium complexes (the data for Gd-DOTA are, for comparison, 3.5 s -1 •mM -1 at 20 MHz and 3.1 s -1 •mM -1 at 60 MHz and 37°C), as expected part due to the intrinsic lower number of unpaired electrons for manganese (5) compared to that for gadolinium (7). The relaxivities could however be increased by grafting of the complexes on macromolecular entities that would reduce the tumbling rate of the complex, which enhances the interaction time between the nuclear magnetic moments of the water protons and the orbital one of the paramagnetic complex during an echo time of the NMR radiofrequency field pulse sequence, and therefore the spin relaxation mechanism. [START_REF] Laurent | Comparative study of the physicochemical properties of six clinical low molecular weight gadolinium contrast agents[END_REF] This is envisaged as outlook of this work in a near future.

NMRD profiles were performed for the four pyclen-based manganese complexes in order to evaluate the efficacy of each complex according to the magnetic field or frequency used. The fitting of these profiles according to the theory of Solomon and Bloembergen allows extracting important parameters to understand this efficacy such as the rotational correlation time (𝜏𝜏 R ) or 20 "tumbling time", the electronic relaxation time at zero field (𝜏𝜏 SO ) and the correlation time that modulates electronic relaxation (𝜏𝜏 V ). [START_REF] Solomon | Relaxation Processes in a System of Two Spins[END_REF][START_REF] Bloembergen | Proton Relaxation Times in Paramagnetic Solutions[END_REF][START_REF] Bloembergen | Proton Relaxation Times in Paramagnetic Solutions. Effects of Electron Spin Relaxation[END_REF] Among the four complexes studied, MnL 1 H, has a r 1 NMR dispersion profile below the other curves, especially at low field. This difference can be ascribed to the electronic properties of the complex which are slightly less favorable than for the other ones: 𝜏𝜏 SO is indeed lower compared to the values of the three other complexes. This is again encouraging for the future studies where we plan to graft other molecules thanks to the arm present on the pyridine moiety. (Figure 6) Solomon's theory using the MINUIT minimization software. Parameters, such as the distance of closest approach (d NMR ) and the water diffusion coefficient (D) governing the outer sphere mechanism, or the distance between the coordinated proton of the water molecule and the manganese ion (r) related to the inner sphere mechanism, can be set constant during the fitting procedure thank to their prior determinations along the study of the manganese complex properties. [START_REF] Solomon | Relaxation Processes in a System of Two Spins[END_REF][START_REF] Bloembergen | Proton Relaxation Times in Paramagnetic Solutions[END_REF][START_REF] Bloembergen | Proton Relaxation Times in Paramagnetic Solutions. Effects of Electron Spin Relaxation[END_REF] The values of q and τ M used were also predetermined thanks to their separate evaluation described above, thereby allowing to evaluate the rotational correlation time (𝜏𝜏 R ), the electronic relaxation time to ground level at zero magnetic field (𝜏𝜏 SO ) and the correlation time that modulates electronic orbital relaxation (𝜏𝜏 V ) at 37°C.

q [a] 1 1 1 1 r 1 at 20MHz (s -1 •mmol -1 •L)
2.6 2.7 3.2 2.9

r 1 at 60MHz (s -1 •mmol -1 •L) 2.4 2.6 3.3 2.4
[a] Parameters pre-set at constant values during the fitting procedure [b] Parameters obtained by the fitting of NMRD profiles with Bloembergen and Solomon's model.

Transmetalation evaluation

It is now well-established that the kinetic inertness of a complex is a more important parameter than its intrinsic thermodynamic stability for in vivo applications. In order to define the stability of the manganese complexes against the most important endogenous divalent metal cations present in body fluids (Zn 2+ , Cu 2+ , Ca 2+ , Mg 2+ ), a transmetalation study was performed in the presence of Zn(II) as exchanging metal cation. The choice of Zn 2+ as challenger to assess the in vivo transmetalation behavior of the Mn(II) complexes was governed by the following considerations: [START_REF] Merbach | 4.3.5 Mn2+-based contrast agents[END_REF][START_REF] Laurent | How to measure the transmetallation of a gadolinium complex[END_REF] i) none of the four competitive endogenous cations is paramagnetic and therefore does respond to NMR; ii) Zn 2+ is the second most abundant transition metal in the human body (33 ppm) after Fe 3+ (60 ppm); iii) for a given exogenous ligand, complexes of Zn 2+ as well as Cu 2+ have generally higher stability than the corresponding Mn 2+ complex as ascribed to LFSE effect (ligand field stabilization energy), whereas alkaline earth ions Ca 2+ and Mg 2+ , although present at higher concentrations in blood plasma than Zn 2+ , form generally less stable complexes with the exogenous ligands generally studied for Mn 2+ due to their absence of d electronic orbitals; iv) the concentration of Zn 2+ in blood is higher compared to that of Cu 2+ (5 to 100 times higher). [START_REF] Hare | Imaging metals in biology: Balancing sensitivity, selectivity and spatial resolution[END_REF] As previously described by us, [START_REF] Laurent | How to measure the transmetallation of a gadolinium complex[END_REF][START_REF] Laurent | Stability of MRI paramagnetic contrast media: A proton relaxometric protocol for transmetallation assessment[END_REF] the transmetalation rate was assessed by measuring the progress of the proton relaxivity at 37°C and 20 MHz. The method consists in using a solution of the Mn-complexes at concentration 2.5 mM in phosphate buffer in the presence of an equimolar amount of zinc chloride. The proton longitudinal relaxation time (T 1 ) is then measured in order to determine paramagnetic relaxation rate R 1 p as a function of time.

Indeed, when the exchange between Mn 2 and Zn 2+ takes place, a precipitate of manganese phosphate (Mn 3 (PO 4 ) 2 ) is formed. This process leads to increase of T 1 and thus decrease of R 1 p compared to its initial value R 1 p (t=0), as ascribed to decrease of paramagnetic ion concentration in solution.

The results (Figure 7) are similar for all the Mn complexes studied and show, during the first day, a small fast yet transient increase of the ratio R 1 p /R 1 p (t=0), which could be due to the presence of transient polynuclear species coordinated to both Mn 2+ and Zn 2+ . This step is followed by a decrease of about 50% of the relaxation rate due to the formation of the insoluble solid as explained previously. This decrease is however quite slow (t 1/2 half-life of approximately 23 1350 min) and we can notice that at the first measurement of the second day, the relaxation rate reached 80% of the initial value of relaxation rate. As a conclusion, even though this study highlights a lack of kinetic stability towards the transmetalation with zinc ions after a long time, we can be confident for the use of those complexes in vivo given their expected fast clearance from the organism. Further in vitro toxicity and in vivo pharmacokinetic studies will however be necessary in the future to definitively demonstrate the biosafety of these paramagnetic MRI CAs. Experiment was performed during 5 days in phosphate buffer at 20 MHz and 37°C. The concentration of Mn-complexes and of competing Zn(II) salt used is 2.5 mM, which is much higher than its normal physiological level (∼10 µM). [START_REF] Wessells | Plasma Zinc Concentration Responds Rapidly to the Initiation and Discontinuation of Short-Term Zinc Supplementation in Healthy Men[END_REF] Phantom images MRI images were recorded both at preclinical (9.4 T) and clinical magnetic field (1 T), for each manganese complex at a concentration of 0.5 mM and compared with Gd-DOTA at the same concentration and with water (Figure 8). As expected, the images are highlighted for the four Mn-complexes compared to water but their contrast is less enhanced than with Gd-DOTA.

Among the Mn-complexes, we can also notice a smaller highlighting effect for MnL 1 H, which can be related to its smaller relaxivity because of the absence of the arm on the pyridine structure (Table 3). (1 T and 9.4 T) of each Mn 2+ -complex at a concentration of 0.5 mM compared with a commercial Gd-complex, Gd-DOTA, at the same concentration, and with pure water.

Conclusions and perspectives

Four novel pyclen-based complexing agents were synthesized and the molecular structure of intermediated compounds after each step of the synthesis was characterized. The corresponding manganese complexes were prepared and fully characterized by NMR relaxometry methods to evaluate the parameters governing their properties and efficacy as paramagnetic MRI contrast agents. This characterization is important in order to evaluate if the complexes have an efficacy close to the commercial gadolinium complexes, and could potentially replace them on market, if they pass further evaluation of their bio-safety.

Although these Mn-based compounds have been shown to be slightly less effective than the marketed Gd-based compounds as ascribed to lower magnetic moment of the metal, the potential of these pyclen-based chelating compounds remains high. Their relaxivity could indeed be improved by different ways. Firstly, it will be possible to play on the rotational correlation time of the complex by blocking them in a nanostructure either organic (e.g. micelle or vesicle) or inorganic (e.g. silica nanoparticle), while keeping sufficiently small hydrodynamic size and stealthness to allow rapid clearance when injected in systemic circulation (which is a prerequisite for CAs used in clinics). Moreover, the additional chemical functions on the pyridine moiety will allow conjugation to biologically relevant vectors such as peptides or antibodies in order to target specific pathologies. Finally, on the basis of reported results, further improved kinetic inertness of the Mn 2 -complexes might be obtained in future by replacing the two acetate complexing groups by non-ionizable groups such as amides; in this case, the positive charge on the resulting cationic Mn 2 complexes could be protective against acid-assisted dissociation, although grafting of neutral macromolecules might be necessary in that case to insure stealthness against of those amide functions does not increase too much the residence time in the inner sphere of the coordinated water molecule. As a conclusion, these preliminary results show very encouraging properties of pyclen derivatives for MRI contrast properties, although further study is necessary to demonstrate that they can replace gadolinium complexes in clinical diagnosis of diseases (cancers, strokes…) by MRI.

Experimental section

Materials

Chemical and physical measurements:

The NMR spectra ( 1 H, [START_REF] Crossgrove | Manganese toxicity upon overexposure[END_REF] C, COSY, DEPT) were recorded at 298K and reported in ppm.

Chemical material and methods:

The flash chromatography was performed on Biotage® flash chromatography using prepacked cartridges. The collection is based on absorption at 254 and 270 nm by UV detection. Overnight, the solution is concentrated and washed two times with diethylether (4 mL) to give the desired compound 10a (12 mg, 0.018mmol). Yield 79.8 % (determined by weighing). triplet that can be attributed to a doublet of doublet with similar 3 J (8.4 Hz, vicinal coupling) and [START_REF] Pan | Manganese-based MRI contrast agents: past, present and future[END_REF] J coupling constants; the unusually high long-range 4 J coupling constant (≈ 8.4 Hz) may be due to a pseudo W-conformation of the four sigma bonds between the two coupled 1 H. [START_REF] Crossgrove | Manganese toxicity upon overexposure[END_REF] C NMR (CDCl 3 , 100 MHz) δ (ppm): Caution: Some of the [START_REF] Crossgrove | Manganese toxicity upon overexposure[END_REF]

Methods

Synthesis of the lower part

Determination of Mn 2+ concentration for relaxometric and 17 O measurements

The detection of free manganese ions is performed with a test of xylenol orange: 300 µL of phosphate or acetate buffer, one drop of pyridine, one drop of the compound solution and one drop of xylenol orange (0.1 g per 100 mL). The color is pink or orange for phosphate or acetate buffer respectively without manganese ions and gets purple when free manganese ions are present. The solution is then treated with a Chelex ® 100 resin (sodium form) to eliminate those free ions.

The Mn 2+ concentration determination is performed either by relaxometry and ICP-AES, on samples digested with nitric acid. The first method consists in measuring the water paramagnetic longitudinal relaxation rate of the digested solution at 20 MHz. It is then compared to the paramagnetic relaxation rate of a 1 mM solution of free manganese ions (equal to 8 s -1 at 20

MHz and 37°C) in order to extract the manganese concentration. [START_REF] Pan | Manganese-based MRI contrast agents: past, present and future[END_REF]

Relaxometry measurement

The longitudinal relaxation times (T 1 ) are determined at 20 MHz and 60 MHz on Bruker® Minispec mq20 and mq60 relaxometers. These measurements allow to obtain the relaxivity of the complexes according to the following equation 𝑟𝑟 1 =

Temperature dependent 17 O NMR measurements

The transverse [START_REF] Pierre | Contrast agents for MRI: 30+ years and where are we going?[END_REF] Transverse relaxation times (T 2 ) are obtained by the measurement of the signal width at midheight. For the determination of τ M , the data are presented as the reduced transverse relaxation rate R 2 R =1/T 2 R = 55.55R 2 P /([Mn complex ]•q), where [Mn complex ] is the molar concentration of the complex, q is the number of coordinated water molecules and R 2 P is the paramagnetic transverse relaxation rate obtained after subtraction of the diamagnetic contribution from the observed relaxation rate. The treatment of the experimental data is performed as already described and the fitting of the data is obtained with a home-made program. [START_REF] Laurent | Synthesis and characterization of various benzyl diethylenetriaminepentaacetic acids (dtpa) and their paramagnetic complexes, potential contrast agents for magnetic resonance imaging[END_REF] For the determination of the number of coordinated water molecules, the data are presented as the transverse relaxivity r 2 = R 2 P /[Mn complex ] where the concentration of the Mn complex is expressed in millimolar (mmol•L -1 ).

Transmetalation

The transmetalation is performed in a phosphate buffer solution (pH 7.06, salt concentrations below) and on the Bruker ® Minispec mq20 relaxometer. 300 µL of a solution of each complex at a concentration of 2. induced by the presence of zinc cations, which leads to a release of manganese ions and their precipitation by phosphates. This release induces formation of a precipitate of Mn 3 (PO 4 ) 2 , which leads to a decrease of the paramagnetic relaxation rate. [START_REF] Laurent | How to measure the transmetallation of a gadolinium complex[END_REF]

Table of contents Graphic Table of contents synopsis

Step-by-step synthesis of manganese complexes based on the pyclen structure with a functionalized arm present on the pyridine moiety, as an alternative to gadolinium complexes as MRI contrast agents. This functionalized arm will allow the coupling of different molecules of interest for specific applications. A complete physicochemical characterization of the synthesized molecules is presented.

Figure 1 .

 1 Figure 1. Structures of the ligands discussed in this work : (A) DPDP 17 , (B) PCMA and PCMP 29

Figure

  Figure 2. Structure of the four Mn-complexes synthesized and studied in this work MnL 1 H:

Scheme 4 .

 4 Scheme 4. Synthesis of (j) HBTU, NH 2 CH 2 CCH, DIPEA, CH 2 Cl 2 , r.t. 4h30 (k) TFA, CH 2 Cl 2 ,

Figure 3 .

 3 Figure 3. Measurements of the 17 O water transverse relaxivity versus inverse of temperature,

Figure 4 .

 4 Figure 4. NMRD profiles at 25°C in order to determine the number of inner sphere water

Figure 5 .

 5 Figure 5. Measurement of the 17 O water transverse relaxation rate as a function of the inverse of

Figure 6 . 1 H

 61 Figure 6. 1 H NMRD profiles at 37°C for Mn 2+ -complexes, showing the longitudinal relaxivity at

Figure 7 .

 7 Figure 7. Evolution of the normalized paramagnetic longitudinal relaxation rate in order to evaluate the transmetalation kinetics between manganese and zinc ions for the four Mn 2+complexes: MnL 1 H (triangles), MnL 2 COO -(stars), MnL 3 CH (circles) and MnL 4 NH 3 + (squares).

Figure 8 .

 8 Figure 8. Phantom images recorded at approximately 37°C under two different magnetic fields

(

  Bruker® AVANCEII-500 at 500 MHz, Bruker® AVANCE NEO at 400 MHz or Bruker® AVANCE NEO at 600 MHz). The multiplicity of the peaks is defined as s (singlet), d (doublet), t (triplet) and m (multiplet). The reactions were monitored by mass spectrometry (Waters®, ZQ-2000) and all the compounds were characterized by mass spectrometry (Waters® ZQ-2000, Waters® QDa, Waters® Quattro Premier or Waters® QTofUs). The ESI-LRMS was performed on 3200 QTrap (AB Sciex). The pH was controlled by Mettler Toledo® fiveEasy pH/mV. The relaxometry and NMRD profiles measurements were performed on Bruker® MiniSpec at 20 and 60 MHz at 37°C and on a fast field cycling (FFC) relaxometer between 0.02 and 40 MHz (Spinmaster, Stelar®, Italy). 17 O NMR measurements were performed on a Bruker® AVANCEII-500 at 67.8 MHz. The MRI measurements were performed on 1T (Bruker® ICON) and 9.4T (Bruker® Biospec) with a Rapid Acquisition with Relaxation Enhancement sequence (RARE) (TE = 12 ms, TR = 350 ms, resolution = 156 x 146 microns, RARE factor = 2, 2 or 4 averages, slice thickness = 1.25 mm).

1 H

 1 NMR (CD 3 OD, 500 MHz) 𝛿𝛿 (ppm): 3.11 (broad t, J 5.21 Hz, 4H, -N-CH 2 -CH 2 -N-); 3.21 (broad t, J 5 Hz, 4H, -N-CH 2 -CH 2 -N-); 3.62 (s, 4H, -N-CH 2 -N-); 4.12 (s, 4H, -CH 2 -CO 2 tBu); 7.25 (d, J 7.71 Hz, 2H, CH=CH-CH=); 7.79 (t, J 7.7 Hz, 1H, -CH=CH-CH=). 2-(12-{[(2-aminoethyl)carbamoyl]methoxy}-9-(carboxymethyl)-3,6,9,15tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-trien-3-yl)acetic acid manganese compound 11a: A solution of MnCl 2 .4H 2 O (7 mg, 0.035 mmol) in water (0.5 mL) is added dropwise to a solution of compound 10a (12 mg, 0.018 mmol) in water (1 mL). The pH is adjusted and maintained to 5 < pH < 6 with 0.1 M aqueous solution of NaOH. The mixture is warmed at 40°C overnight. The presence of free Mn 2+ is revealed by the colorimetric test with orange xylenol. The aqueous mixture is treated with Chelex resin, then freeze-dried to give the desired compound 11a. ESI-MS (C 15 H 20 MnN 4 O 4 ): m/z 398.09 [M+Na] + .Synthesis of MnPy(COO-) 2 -OCH 2 CO 2 -(Compound MnL 2 COO -) Benzyl 3,9-bis(2-tert-butoxy-2-oxo-ethyl)-12-(2-ethoxy-2-oxo-ethoxy)-3,6,9,15tetrazabicyclo[9.3.1]pentadeca-1(14),11(15),12-triene-6-carboxylate compound 7b: Solid Na 2 CO 3 (466 mg, 4.40 mmol, 4 eq) is added to a solution of triamine lower part 5b (513 mg, 1.10 mmol) with dibromomethyl compound 6b (444 mg, 1.21 mmol, 1.1 eq) in acetonitrile (110 mL). The suspension is refluxed until completion of the reaction (MS and/or TLC monitoring; 1 hour). The insoluble matter is filtered off, and the filtrate is concentrated to dryness. The crude material obtained is purified by chromatography on silica gel (CH 2 Cl 2 /MeOH, 100/0 to 98/2 v/v) to give the desired compound 7b as a thick oil (471 mg, 0.702 mmol). Yield 64%. 1 H NMR (CDCl 3 , 400 MHz) δ (ppm): 1.27 (t, 3H, J 7.1 Hz, -Et); 1.45 (s, 9H, -tBu); 1.48 (s, 9H, -tBu); 2.68-3.01 (m, 4H, -N-CH 2 -CH 2 -N-); 3.20-3.55 (m, 8H, -N-CH 2 -CH 2 -N-(4H) and -N-CH 2 -); 3.95 (d, 2H, J 10.2 Hz, -N-CH 2 -); 4.19 (d, 2H, J 7.3 Hz, -N-CH 2 -); 4.24 (d, 2H, J 7.1 Hz, -Et); 4.66 (s, 2H, -O-CH 2 -CO 2 -Et); 5.04 (s, 2H, -CH 2 -Ph); 7.04 (d, J 8.4 Hz, 1H, -CH 2 -C(N)-CH=CH-C(OR) -C); 7.18 (broad dd, ££ 4 J ≈ 3 J 8.4 Hz, 1H, -CH 2 -C(N)-CH=CH-C(OR) -C); 7.24-7.37 (m, 5H, -Ph). ££ That resonance looks like a broad unsymmetrical

  •mM -1 (3) where 𝑅𝑅 1 (water) d = 0.2826 s -1 at 37°C. The NMRD profiles were recorded at 37°C and 25°C on a Stelar ® fast field cycling (FFC) relaxometer (Stelar, Mede, Italy) and the least-squares fitting of the data was performed using a home-made program (Fitting2000) and equations from the Solomon and Bloembergen (SBM) theory.

  5 mM is prepared in phosphate buffer solution ([NaH 2 PO 4 ] = 0.026 mol/L et [Na 2 HPO 4 ] = 0.041 mol/L) and 3 μL of a solution 250 mM of ZnCl 2 (1 eq) is added in the studied Mn-complexes solutions. Then, the longitudinal relaxation time (T 1 ) is measured as a function of time to obtain the evolution of R 1 p following the equation: is determined at regular intervals to evidence the transmetalation kinetics

Table 1 .

 1 Evaluation of the number of coordinated water molecules in the inner sphere for all complexes by two different techniques

		r 0 2max (s -1 •mM -1 )	q (Eq. 1)	r 1p (s -1 •mM -1 )	q (Eq. 2)
		(67.8 MHz)		(25°C and 0.02 MHz)
	MnL 1 H	419.2	0.82 ± 0.2	7.7	1.25 ± 0.4
	MnL 2 COO -	458.4	0.90 ± 0.2	8.3	1.30 ± 0.4
	MnL 3 CH	473.4	0.93 ± 0.2	9.4	1.35 ± 0.4
	MnL 4 NH 3 +	478.6	0.94 ± 0.2	10.0	1.42 ± 0.4

Table 3 .

 3 Results of the theoretical fitting of1 H NMRD profiles at 37°C withBloembergen and

Table 3 .

 3 Values of longitudinal relaxivity calculated from the water proton relaxation time T 1 measured at approximately 37°C under a field of 9.4T (400 MHz).

		MnL 1 H	MnL 2 COO -	MnL 3 CH	MnL 4 NH 3	+	Gd-DOTA
	r 1 (s -1 •mM -1 )	2.8 ± 0.1	2.7 ± 0.1	3.1 ± 0.1	2.95 ± 0.1	3.2 ± 0.1
	9.4T, 37°C

Synthesis of MnPy(COO -) 2 -OCH 2 CO 2 NHCH 2 CCH (Compound MnL 3 CH)

  ) and put under stirring followed by the addition of trifluoroacetic acid (1 mL). The solution is stirred overnight. It is then concentrated and washed two times with diethyl ether (4 mL) to give the desired compound 10b as TFA salt. The crude product is directly engaged in the next synthesis step.1 H NMR (CD 3 OD, 500 MHz) 𝛿𝛿 (ppm): 3.08-3.14 (broad s, 4H, -N-CH 2 -CH 2 -The solution is stirred during few hours at room temperature then the middle is washed three times with water (5 mL) to give the desired compound. Yield (crude) >90% (determined by weighing). The crude product is directly engaged in the next synthesis step.1 H NMR (CDCl 3 , 500 MHz) 𝛿𝛿 (ppm): 1.45 (s, 18H, -tBu) 2.22 (t, J (8 mg, 0.01 mmol) in water (1 mL). The pH is adjusted and maintained to 5 < pH < 6 with 0.1 M aqueous solution of NaOH. The mixture is warmed at 40°C overnight. The presence of free Mn 2+ ions is revealed by the colorimetric test with orange xylenol. The aqueous mixture is treated with Chelex resin, then freeze-dried to give the desired compound 14. ESI-MS (C 20 H 25 MnN 5 O 6 ): m/z 509.14 [M+Na] + .

	C are doublet due to the presence of rotamers. 14.2 (CH 3 _Et); 28.3 (CH 3 _2 -tBu); 44.6, 45.0, 45.5, 46.1 $$ (2 -N-CH 2 -CH 2 -N-); 51.3, 51.8, 52.0, 52.4 $$ (2 -N-CH 2 -CH 2 -N-); 54.5, 54.6 $ (-N-CH 2 -); 58.7, 58.9, 59.5 (3 -N-CH 2 -); 61.6 (CH 2 _Et); 65.8 (-O-CH 2 -CO 2 -Et); 66.8 (-CH 2 -Ph); 81.0, 81.2 (2 C quat tBu); 119.8, 119.9 $ (CH Ar Pyr); 123.8 (CH Ar Pyr); 127.6, 127.8, 128.5 (3 CH Ar Ph); 137.1 (C quat Ph); 147.6, 151.8 £ , 156.2 (NCO 2 , and 3 C quat Pyr); 166.4, 168.3, 170.7-170.9 & (3 CO 2 ). $ Double signal for the assigned 13 C (rotamers); $$ Double signal for each of the assigned 13 C; & Broad and small signal; £ Higher signal than the adjacent one; may account for several 13 C. Ethyl 2-({3,9-bis[2-(tert-butoxy)-2-oxoethyl]-3,6,9,15-tetraazabicyclo[9.3.1] pentadeca-N-); 3.18-3.26 (broad m, 4H, -N-CH 2 -CH 2 -N-); 3.62 (s, 2H, -N-CH 2 -N-); 3.69 (s, 2H, -N-CH 2 -N-); 4.06 (s, 2H, -N-CH 2 -CO 2 H); 4.27 (s, 2H, -N-CH 2 -CO 2 H); 4.82 (s, 2H, -O-CH 2 -CO 2 H); 7.19 (d, 1H, J 8.29 Hz, -O-C=CH-CH=C-); 7.34 (d, 1H, J 8.43 Hz , -O-C=CH-CH=C-). 2-(12-{[(2-aminoethyl)carbamoyl]methoxy}-9-(carboxymethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-trien-3-yl)acetic acid manganese compound 11b: A solution of MnCl 2 .4H 2 O (6 mg, 0.03 mmol) in water (0.5mL) is added dropwise to a solution of compound 10b (11 mg, 0.016 mmol) in water (1 mL). The pH is adjusted and maintained to 5 < pH < 6 with 0.1 M aqueous solution of NaOH. The mixture is warmed at 40°C overnight. The presence of free Mn 2+ is revealed by the colorimetric test with orange xylenol. The aqueous mixture is treated with Chelex resin, then freeze-dried to give the desired compound 11b. ESI-MS (C 17 H 22 MnN 4 O 7 ): m/z 472.10 [M+Na] + . Tert-butyl 2-{9-[2-(tert-butoxy)-2-oxoethyl]-12-{[(prop-2-yn-1-yl)carbamoyl] methoxy}-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-trien-3-yl}acetate compound 12: Compound 9 (10 mg, 0.019 mmol) and HBTU (15 mg, 0.04 mmol, 2.1 eq) are solubilized in CH 2 Cl 2 (2 mL) followed by the addition of propargylamine (2 µL, 0.031 mmol, 1.6 eq) and tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-trien-3-yl]acetic acid compound 13: Compound 12 (119 mg, 0.218 mmol) is solubilized in CH 2 Cl 2 (3 mL) and stirred followed by the addition of trifluoroacetic acid (3 mL). Overnight, the solution is concentrated and washed two times with diethyl ether to give the desired compound 13 as TFA salt. The crude product is directly engaged in the next synthesis step. 1 H NMR (CD 3 OD, 500 MHz) 𝛿𝛿 (ppm): 2.60 (t, J 2.54 Hz, 1H, -C≡CH); 3.10-3.16 (m, 4H, N-CH 2 -CH 2 -NH-); 3.19-3.25 (m, 2H, N-CH 2 -CH 2 -NH-); 3.25-3.30 (m, 2H, N-CH 2 -CH 2 -NH-); 3.69 (s, 2H, -N-CH 2 -C-); 3.80 (s, 2H, -N-CH 2 -C-); 4.03 (d, J 2.56 Hz, 2H, -NH-CH 2 -C); 4.15 (s, 2H, -N-CH 2 -COOH); 4.38 (s, 2H, -N-CH 2 -COOH); 4.71 (s, 2H, -O-CH 2 -CO-); 7.28 (d, J 8.49 Hz, 1H, -O-C=CH-CH=C-); 7.41 (d, J 8.49 Hz, 1H, -O-C=CH-CH=C-). 2-(12-{[(2-Aminoethyl)carbamoyl]methoxy}-9-(carboxymethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-trien-3-yl)acetic acid manganese compound 14: A solution of MnCl 2 .4H 2 O (4 mg, 0.02 mmol) in water (0.5 mL) is added dropwise to a solution of DIPEA (8 µL, 0.047 mmol, 2.47 eq). 2.56 Hz, 1H, -C≡CH); 3.03-3.11 (m, 4H, -N-CH 2 -CH 2 -NH-); 3.21-3.28 (m, 4H, -N-CH 2 -CH 2 -NH); 3.43 (s, 2H, -C-CH 2 -N-); 3.47 (s, 2H, -C-CH 2 -N-); 3.92 (s, 2H, -N-CH 2 -CO-); 4.08-4.16 (m, 4H, -N-CH 2 -CO-and -NH-CH 2 -C-); 4.54 (s, 2H, -O-CH 2 -CO-); 7.02 (d, J 8.39 Hz, 1H, -O-C=CH-CH=C-); 7.07 (d, J 8.39 Hz, 1H, -O-C=CH-CH=C-). 2-[9-(Carboxymethyl)-12-{[(prop-2-yn-1-yl)carbamoyl]methoxy}-3,6,9,15-compound 13 Synthesis

1

(15)

,11,13-trien-12-yl}oxy)acetate compound 8b: Compound 7b (55 mg, 0.082 mmol, 1 eq) is

(1 mL

of MnPy(COO -) 2 -OCH 2 CO 2 NHCH 2 CH 2 NH 2 (Compound MnL 4 NH 3 + )

  Double signal due to two conformers (double-bond character of the amide bond: cis and trans conformations) or two rotamers (free rotation obstruction due to Cbz steric hindrance). The dark orange supernatant is removed by suction and the solid material is washed several times with Et 2 O until a clear wash is obtained. The organic solvents are completely removed first under reduced pressure and then by freeze-drying after solubilization in water to give the desired compound as a brownish solid material (200 mg, 0.288 mmol on the basis on an estimated PM 693.38 for 3HBr/4HBr hydrobromide forms in 85/15 mol/mol at The pH is carefully adjusted again to ~ 5.7 with aqueous NaOH. The resulting mixture is stirred at room temperature for 5 minutes. A monitoring by MS indicated a total conversion to the desired manganese complex with no trace of free ligand 8. The pH is then raised up to ~ 7.5 (NaOH aq ) and stirring maintained overnight. The fine brown precipitate formed is filtered off over a 0.2 µm membrane (PALL ® , Acrodisc ® ). The filtrate is concentrated by freeze-drying. The

	(m, 6H, -CH 2 -C(N)-CH=CH-C(OR)-C, and Ph); 8.15, and 8.25 $ (2 broad t, 1H (≈53/47), -CO-
	Benzyl 12-[2-(2-aminoethylamino)-2-oxo-ethoxy]-3,9-bis(2-tert-butoxy-2-oxo-ethyl)-3,6,9,15-tetrazabicyclo[9.3.1]pentadeca-1(14),11(15),12-triene-6-carboxylate compound 15: A solution of the previously prepared ethyl ester 7b (470 mg, 701 mmol) in ethylenediamine (2.3 mL, 34.4 mmol, 50 eq) is stirred at room temperature until completion of the reaction (MS monitoring; 1 hour). The crude medium is diluted in CH 2 Cl 2 (15 mL) and is washed with water (3 mL). The organic layer is dried then concentrated to dryness. The resulting material is purified by chromatography on silica gel (CH 2 Cl 2 /MeOH/NH 3 7N in MeOH 95/2.5/2.5 to 90/5/5 v/v/v) and led to the desired product 15 as a beige solid (279 mg, 0.407 mmol). Yield 58%. 1 H NMR (CDCl 3 , 400 MHz) δ (ppm): 1.34-1.53 (m, 18H, -tBu); 1.94 (broad s, 2H, -NH 2 ); 2.55-2.66 (m, 2H, -N-CH 2 -CH 2 -N-); 2.66-2.88 (m, 4H, -N-CH 2 -CH 2 -N(2H) and -CH 2 -NH 2 ); 2.96-3.09 (m, 2H, -N-CH 2 -CH 2 -N-); 3.28-3.50 (m, 8H, -CO-NH-CH 2 -, -N-CH 2 -(4H), -N-CH 2 -CH 2 -N-(2H)); 3.89 and 3.93 (2 br s $ , 2H, -N-CH 2 -); 4.02 and 4.05 (2 br s $ , 2H, -N-CH 2 -); 4.56 (broad s, 2H, -O-CH 2 -); 5.01 (s, 2H, -CH 2 -Ph); 7.16 (d, J 8.4 Hz, 1H, -CH 2 -C(N)-CH=CH-C(OR)-C); 7.21-7.33 NH-). 13 C NMR (CDCl 3 , 100 MHz) δ (ppm): Caution: As previously observed for the precursor 7b, nearly all of the 13 C are doublet due to the presence of rotamers (double bond character of the amide bond and/or free-rotation obstruction of the carbamate bond). 28.18, 28.23 (CH 3 _2 -tBu); 41.8, 42.1, 42.2, 42.3 (-CO-NH-CH 2 -CH 2 -NH 2 ) # ; 44.9, 45.4 (-N-CH 2 -CH 2 -N-) # ; 50.9, 51.0, 51.6, 51.8 (2 -N-CH 2 -CH 2 -N-) # ; 54.2, 54.5 (-N-CH 2 -) # ; 58.8, 59.0, 59.1, 59.4 (-N-CH 2 -CH 2 -N-, and 3 -N-CH 2 -); 66.9 (-CH 2 -Ph); 67.2 (-O-CH 2 -); 81.2, 81.47, 81.52 (2 C quat tBu) $ ; 119.65, 119.68 (-CH 2 -C(N)-CH=CH-C(OR)-C) # ; 124.97, 125.01 (-CH 2 -C(N)-CH=CH-C(OR)-C) # ; 127.57, 127.65, 127.83, 127.90, 128.46 (3 CH Ar Ph) $ ; 136.85, 136.94 (C quat Ph) # ; 145.9, 146.1, 149.2, 149.7, 151.7, 151.8, 155.9, 156.0 (-N-CO 2 , and 3 C quat Pyr) # ; 168.21, 168.24, 170.4, 170.6, 170.7, 170.8 (2 CO 2 , and CONH) # . # Double signal for (each of) the assigned 13 C (rotamers); $ Double signal for at least one of the assigned 13 C. 2-(12-{[(2-Aminoethyl)carbamoyl]methoxy}-9-(carboxymethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-trien-3-yl)acetic acid compound 16a.xHBr: A concentrated solution of the fully protected precursor 15 previously prepared (210 mg, 0.307 mmol) in CH 2 Cl 2 (0.250 mL) is diluted in acetic acid (2 mL). A solution of hydrogen bromide in acetic acid (33% w/w, 0.500 mL) is then added and a white precipitate appeared instantly. The mixture is vigorously stirred and heated at 70°C for 7 hours and then at room temperature overnight. pH 0.7 47 ). Yield (crude) 94%. The crude material can be purified by chromatography on RP18 (CH 3 CN/H 2 O 9/1 v/v) after raising the pH from 0.7 to about 4.5 (NaOH aq ) and leads to the desired product as a beige solid (115 mg, 0.221 mmol on the basis on an estimated PM 516.97 for .1HBr/.0HBr/.2HBr hydrobromide forms in 73/15/12 mol/mol/mol at pH 4.5 47 ). Yield 72%. 1 H NMR (D 2 O, pH<1) δ (ppm): 3.14 (broad t, 2H, -CH 2 -NH 2 -); 3.20-3.38 (m, 6H, -N-CH 2 -CH 2 -NH-); 3.44 (broad s, 2H, -N-CH 2 -CH 2 -N-); 3.56 (broad t, 2H, -CO-NH-CH 2 -); 4.00 (s, 2H, -N-CH 2 -); 4.06 (s, 2H, -N-CH 2 -); 4.47 (s, 2H, -N-CH 2 -); 4.65 (s, 2H, -N-CH 2 -); 4.80 13 C NMR (D 2 O, pH<1): 36.6 (-CO-NH-CH 2 -); 39.0 (-CH 2 -NH 2 -); 43.7, 43.9 (2 -N-CH 2 -CH 2 -NH-); 51.0, 52.2 (2 -N-CH 2 -CH 2 -NH-); 54.0, 56.7, 57.1, 57.4 (4 -N-CH 2 -); 67.3 (-O-CH 2 -CO-N-); 125.0 (2 CH Ar ); 142.1, 144.3, 152.0 (3 C quat Pyr); 170.6, 172.2, 172.3 (2 CO 2 , and -CO-NH-). 2-(12-{[(2-aminoethyl)carbamoyl]methoxy}-9-(carboxymethyl)-3,6,9,15-tetra azabicyclo[9.3.1]pentadeca-1(15),11,13-trien-3-yl)acetic acid manganese compound 17: A solution of the previously prepared ligand 16a.xHBr (~0.17 mmol) in deionized H 2 O (3 mL) is adjusted to pH ~ 5.5 with an aqueous solution of NaOH. Manganese(II) chloride tetrahydrate (33 mg, 0.168 mmol) is added in one portion which immediately induced a decrease of pH down to the desired product 17 as a beige solid (108 mg, x H 2 O.y HCl or NaCl probable form(s)). ESI-to 2.2. solid obtained (113 mg) is purified by chromatography on RP18 (MeOH/H 2 O 9/1 v/v) and leads LRMS m/z: 492.2 [M+H] + , 514.1 [M+Na] + .

$ * (2H, -O-CH 2 -CO-N-); 7.57 (d of AB system, J 8.4 Hz, 1H, CH Ar );7.71 (d of AB system, J 8.4 Hz, 1H, CH Ar ). * Fully superimposed with the deuterated solvent (deduced from corr 1 H/ 13 C 2D experiment).

  To ensure the results, ICP-AES measurements are also performed on Varian Liberty Series II, based on a Mn 2+ calibration curve. The concentrations obtained respectively from ICP-AES or relaxometry methods are 40 respectively 1.80 and 1.76 mM (MnL 1 H), 5.09 and 5.66 mM (MnL 2 COO -), 7.85 and 8.05 mM (MnL 3 CH), and 2.47 and 2.35 mM (MnL 4 NH 3 + ).

  O relaxation rates (R 2 =1/T 2 ) are measured in aqueous solutions of each manganese complex (MnL 1 H; 2.06 mM, MnL 2 COO -; 2.58 mM, MnL 3 CH: 2.05 mM and MnL 4 NH 3 + : 1.21 mM) in the temperature range 7 -81°C, on a Bruker ® AvanceII-500 (11.75 T, 67.8 MHz) spectrometer. The temperature is calculated according to a previous calibration with ethylene glycol and methanol. Proton decoupling is applied during all the acquisitions.

Acknowledgements

This work was performed with the financial support of SIRIC BRIO (COMMUCAN project https://siric-brio.com/commucan/), ARC the Walloon Region (Gadolymph, Prother-Wal and Interreg projects), FNRS, and the COST actions TD1004, TD1007, TD1402 and CM1006. The Bioprofiling platform, supported by the European Regional Development Fund and the Walloon Region, Belgium, is also acknowledged. Authors thank the Center for Microscopy and Molecular Imaging (CMMI, supported by European Regional Development Fund and Wallonia), Professor Gerbaux P., Dr De Winter J. and PhD student Weber P. from S 2 Mos laboratory for the mass spectrometry analysis and Pierart C. for the NMRD measurements.

Conflicts of interest

The authors declare no conflicts of interest


Placide Uwizeyimana

Marianne Perrin

Florent Eyma

CONTINUOUS MEASUREMENT OF MOISTURE CONTENT IN GLULAM STRUCTURES

Keywords: Moisture content measurement, Glulam structures, Resistive method

Introduction:

Today, a strong expansion of timber bridges is promoted by using glulam. The glulam bridges are widespread in Nordic countries but also in France. However, durability issues related to moisture content (MC) variation or wetting/drying cycles limit their development. For this reason, timber bridges supervisors have expressed their needs on continuous MC monitoring. The MC of wood is commonly determined by measuring the electrical resistance within pin electrodes inserted in the wood. To monitor locally MC between the lamellas of glulam, a previous study carried out in our laboratory highlighted the feasibility of resistive measurements using surface sensors embedded in glulam joints. Nevertheless, many parameters influence these measurements and need to be explored.

Developments:

In the present study, a series of experiments was conducted to highlight main important parameters influencing on MC measurement with surface sensors. This study focuses on "Douglas fir" which is the most used species in glulam bridges in France due to its good mechanical resistance and natural durability. Firstly, the influence of sensor-size and spacing between sensor and specimen-edge was studied. Then, the influence of temperature and MC variation on the electrical resistance of wood has been evaluated. Finally, the influence of embedded sensors inside lamellas on mechanical properties of glulam beams was also investigated.

Remarks and Conclusion:

The results obtained highlighted the most impacting parameters on MC measurement with surface sensors. Moreover, a calibration curve expressing the MC as a function of the electrical resistance of wood was obtained. The influence of embedded sensors on mechanical properties was also studied to validate the instrumentation of glulam beams. Future work will focus on the influence of wetting/drying cycles on mechanical integrity of glulam beams.

INTRODUCTION

In recent decades, the use of timber in civil engineering has become an interesting solution to the issues of sustainable development. Advantageous properties such as renewability, sustainability and high ratio strength to weight make the timber an excellent material to use in sustainable construction. The use of this material has been steadily increasing in recent times due to advances in engineered wood, especially the manufacturing of glue-laminated (glulam) timber.

However, durability issues related to moisture content (MC) variation or wetting/drying cycles limit the development of timber bridges. Indeed, the wood is a hygroscopic material, his behavior is affected by a change of temperature and moisture content. For the glulam beams, the problems of delaminations, cracks, dimensional stability due to swelling/ shrinkage have been observed [START_REF] Dietsch | Monitoring Building Climate and Timber Moisture Gradient in Large-Span Timber Structures[END_REF][START_REF] Angst-Nicollier | Moisture Induced Stresses in Glulam[END_REF][START_REF] Setra | Les Ponts en Bois: Comment Assurer leur Durabilité. (Timber Bridges: How to Ensure their Durability)[END_REF]. For this reason, timber bridges supervisors have expressed their needs on continuous MC monitoring [START_REF] Kasal | Assessment, Reinforcement and Monitoring of Timber Structures-COST FP1101[END_REF]. The aim of MC monitoring is to inform the bridges supervisors as early as possible of the damage risk so that appropriate maintenance operations and reinforcement can be planned. Recent studies have shown a great potential of resistive methods for MC measurement using pins electrodes inserted in wood [START_REF] Dietsch | Methods to Determine Wood Moisture Content and their Applicability in Monitoring Concepts[END_REF][START_REF] Bjorngrim | Moisture Content Monitoring of a Timber Footbridge[END_REF][START_REF] Koch | Monitoring of Moisture Content of Protected Timber Bridges[END_REF]. A previous study carried out in our laboratory highlighted also the feasibility of resistive measurement using surface electrodes embedded in glulam joints for MC monitoring insides lamellas [START_REF] Li | Moisture Content Monitoring in Glulam Structures by Embedded Sensors via Electrical Methods[END_REF].

Nevertheless, many parameters such as the size of electrodes, spacing between electrode to specimen-edge and temperature can influence MC measurements. However, none have been specifically determined for surface electrodes. Knowing that the electrical resistance of wood is related to its MC, in this paper, a series of experiments was designed to investigate the main important parameters influencing on electrical resistance of wood and MC monitoring using surface electrodes. It can be noted that the magnitude of electrical resistance depends not only the type of electrode, but also on wood species [START_REF] James | Electric Moisture Meters for Wood[END_REF][START_REF] Brischke | Measurement System for Long-Term Recording of Wood Moisture Content with Internal Conductively Glued Electrodes[END_REF]. This study focuses on "Douglas fir" which is the most used species in glulam bridges in France due to its good mechanical resistance and natural durability [START_REF] Setra | Les Ponts en Bois: Comment Assurer leur Durabilité. (Timber Bridges: How to Ensure their Durability)[END_REF].

The first test was performed to determine the influence of electrode-size on the measured resistance. Then, once the size of the electrodes determined, the influence of spacing between electrode and specimen-edge on the electrical resistance was evaluated. The influence of temperature on the measured resistance has then been investigated. Finally, a calibration curve was obtained to correlate electrical resistance to the MC of wood. In the light of further work on durability of glulam beams, the effect of electrodes between lamellas on mechanical properties of glulam beams was also evaluated.

MATERIALS AND METHODS

This study was based on glulam specimens made of three lamellas. The latter were cut from the same board of Douglas fir (Pseudotsuga menziesii) to reduce the wood variability. The glulam specimens were glued using polyurethane glue (KLEIBERT PUR 510 FiberBond) and pressed with a bonding pressure of 10 bars during 8 hours. The surface electrodes "copper patches" embedded between lamellas were used as MC sensors. They were stuck directly with their own adhesive layers on the timber. The cables (stripped at the end) for resistance measurements were then welded to the centre of the electrodes. The details of instrumentation are shown in Figure 1. A measurement device capable of measuring electrical resistance up to several tens of Giga-Ohms has been developed and used for resistance measurements. In fact, a voltage is applied across two electrodes stuck on the timber and the electrical resistance is measured in the tangential direction of wood.

.. L, R and T indicate the anatomical directions of wood, respectively longitudinal, radial and tangential b x h is the cross section and l represents the length of specimen e indicates the spacing between electrode and specimen-edge

INFLUENCE OF SENSORS-SIZE ON ELECTRICAL RESISTANCE

Special conditions of the experiment

Five electrode diameters from 10 mm to 50 mm have been tested and three specimens have been prepared for each diameter. Spacing between electrode and specimen-edge was 10 mm for all specimens to have the same boundary and limit conditions. The electrodes sizes and specimens' dimensions are presented in Table 1. The experiment was then performed at the equilibrium MC of 55% and 98% relative humidity (RH) and a temperature of 35°C. The specimens were considered to have attained the equilibrium MC when the mass was stable according to standard NF B 51-003 [START_REF] Afnor | NF B 51-003, General Requirements for Physical and Mechanical Tests[END_REF].

Results and discussion

The results show that the electrical resistance decreases as the diameter of the electrode increases (Figure 2). For the two boundary conditions studied (55% RH and 98% RH), the trends are identical. These results are consistent with theoretical equations that relate electrical resistance to electrode dimensions [START_REF] Menguy | Mesure des Caractéristiques des Matériaux Isolants Solides[END_REF]. On the other hand, it is interesting to note that, overall, the smaller the diameter of the electrode, the lower the standard deviation of the measurements. This can be explained by the effect of wood heterogeneity on the resistance measurements. This test allowed choosing the diameter of the electrode for the instrumentation of glulam beams. Therefore, the electrodes of diameter 10 mm will be used in the following tests.

INFLUENCE OF SPACING BETWEEN ELECTRODE AND SPECIMEN-EDGE

Special conditions of the experiment

The effect of distance between electrode and specimen-edge was studied from 5 mm to 30 mm by increments of 5 mm. The diameter of electrode was 10 mm for all specimens. The details of the specimens are presented in Table 2. The experiment was performed at the equilibrium MC of 55% RH and 98% RH and the temperature of 35°C.

Results and discussion

In Figure 3, the electrical resistance decreases with increasing spacing between the electrode and the specimen-edge. The hypothesis that is made here is that the equipotential curves are influenced by the quantity of material available between the electrode and the specimenedge. So, more the quantity of material is limited, more the equipotential curves are concentrated and the greater the resistance, as it can be observed from the results for two different humidity levels (55% RH and 98% RH). It can also be seen that the MC has an effect on the distribution of the equipotential curves. Indeed, the higher the MC, the lower the resistivity of the material and the less material between the specimen-edge and the electrode impacts the measurements. Regarding the standard deviation, it seems that the latter increases when the quantity of material between the specimen-edge and the electrode increases. This is certainly related to the heterogeneity of the material and the eventual MC gradients. So, for the rest of this work, a distance of 10 mm between the specimen-edge and the electrode will be used.

INFLUENCE OF TEMPERATURE ON ELECTRICAL RESISTANCE

Special conditions of the experiment

This test was performed to investigate how the variation of temperature affects the electrical resistance. Five specimens measuring 30 x 30 x 30 mm 3 were used. Each specimen was equipped with surface electrodes for resistance measurement and with a thermocouple for temperature (Figure 4). The specimens were conditioned to 65% RH and then insulated with protective coating (M-Coat C Silicone Rubber) to avoid drying when temperature is changed. They were then placed in climate chamber, and the temperature was increased from -10°C to 40°C by increments of 10°C. The temperature inside specimen was controlled with the thermocouple, and the electrical resistance was measured when the temperature in wood was equal to the climate chamber. It can be noted that the mass of specimens was controlled to ensure no MC modification.

Results and discussion

Very often in the literature, studies concerning the influence of temperature on the resistance measurements are carried out without considering the MC variation induced by the variation of temperature. In order to solve this problem, tests were carried out by isolating the specimens with a resin (M-COAT C Silicone Rubber) allowing keeping a constant MC in the wood and allowing the temperature transfer. The results of this study show clearly a logarithmic decrease in electrical resistance as temperature increases (Figure 5). This effect has already been observed by several authors [START_REF] Davidson | The Effect of Temperature on the Electrical Resistance of Wood[END_REF][START_REF] Clark | The Electrical Conductivity of Commercial Dielectrics and its Variation with Temperature[END_REF] on different types of materials. They showed Thermocouple Specimen with coating ( Cable welded on the electrodes that the conduction of electric charges is activated by the increase of the temperature. As result, the resistance to the electric current decreases. The curve obtained for this test will therefore allow us to make a correction to the calibration curve established at a constant temperature with surface electrodes. Moreover, it can be noted that the standard deviations decrease with the increase of temperature.

CALIBRATION CURVE MC VERSUS ELECTRICAL RESISTANCE

Special conditions of the experiment

The electrical resistance of wood decreases with increasing MC. This has been known for long time [START_REF] Stamm | The Electrical Resistance of Wood as a Measure of its Moisture Content[END_REF][START_REF] Skaar | Some Factors Involved in the Electrical Determination of Moisture Gradients in Wood[END_REF][START_REF] Dai | Long-Term Monitoring of Timber Moisture Content Below the Fiber Saturation Point using Wood Resistance Sensors[END_REF]. Many investigations have been done over the years to establish a relationship between electrical resistance and MC. However, very few of these were made with surface electrodes. This test was performed to determine the calibration curve on Douglas fir with surface electrode of diameter 10 mm embedded in a center of specimen. Ten specimens measuring 60 mm long and section of 30 x 30 mm² were used. The experiment was performed in climate chamber with six climate levels from 50% RH to 98% RH at 35°C (Table 3). From level to another, the mass and the resistance were measured every 24 hours up to the stabilization of mass according to the standard EN ISO 12571 [START_REF] Afnor | EN ISO 12571, Hygrothermal Performance of Building Materials and Products --Determination of Hygroscopic Sorption Properties[END_REF].

Results and discussion

The MC of specimens are calculated after determination of the anhydrous mass according to standard EN 13183-1 [START_REF] Afnor | EN 13183-1, Moisture content of a piece of sawn timber -Part 1: determination by oven dry method[END_REF] (Figure 6 (a)). The measurements of electrical resistance made at the equilibrium MC for each level were used to determine the calibration curve. The curve presented in Figure 6 (b) shows the evolution law of the resistance as a function of the MC of the type log (R) = b log (MC) + a. It can also be noted that the standard deviations are less important for low MC than for high MC. The calibration curve obtained will be used in future works to estimate MC from the resistance measurements with surface electrodes.

INFLUENCE OF EMBEDDED SENSORS ON MECHANICAL PROPERTIES OF GLULAM BEAMS

Special conditions of the experiment

The objective of this test was to evaluate the effect of embedded sensors on the bending strength (BS) and the modulus of elasticity in bending (MOE) of glulam. To achieve that, four-point bending test was conducted on ten specimens with surface electrodes of diameter 10 mm (Figure 7) and ten specimens without electrodes. Each glulam specimen was made of three lamellas and measuring 480 mm long with cross-section of 30 x 30 mm². They were then conditioned at 65% RH and 20°C for two weeks to have equilibrium MC before bending tests. The experiment consisted of carrying out four-point bending tests arranged in accordance with EN 408 [START_REF] Afnor | NF EN 408, Timber Structures -Structural Timber and Glued Laminated Timber -Determination of Some Physical and Mechanical Properties[END_REF] using the testing machine MTS with a cell capacity of 100 kN. The test was conducted at a speed of 5.4 mm/min, and the deflection allowing to calculate the MOE was measured with a Solatron LVDT displacement sensor (Figure 8). The BS and MOE were determined according to the standard EN 408 [START_REF] Afnor | NF EN 408, Timber Structures -Structural Timber and Glued Laminated Timber -Determination of Some Physical and Mechanical Properties[END_REF].

Results and discussion

Figure 9 shows the comparison of the mechanical properties (BS and MOE) between the specimens with and without sensors. The average values obtained are in agreement with the literature. According to Gerard et al. [START_REF] Gérard | Tropix 7 (Version 7.5.1)[END_REF], the BS of Douglas is between 70 and 100 MPa and the MOE between 11 and 16.8 GPa. Considering the average values and the standard deviations obtained on the specimens with and without sensors, it is observed that there is no significant difference for these two configurations. Consequently, the embedded sensors have no influence on the mechanical properties of the instrumented glulam beams. For information, the total section of the three electrodes used represents 1.64% of bonding section.

CONCLUSIONS

This study was carried out to highlight the main parameters influencing the measurements of electrical resistance and as a result of MC in glulam using surface electrodes. The experiments highlighted the following conclusions:

1. The size of the electrodes and the distance between the electrode and the specimen-edge affect the measurements of electrical resistance. It should therefore be noted that the calibration curve of resistance versus MC, must then mention the size of electrodes used. Moreover, the findings might not be transferable to other size without correction. 2. The electrical resistance is highly decreases when the wood temperature increases.

Therefore a correction is necessary for the MC measurements in the climate of variable temperature. 3. A calibration curve is defined to obtain the MC of Douglas fir from the electrical resistance measured using the surface electrodes.

LVDT sensor 4. The surface electrodes embedded between lamellas do not affect the mechanical resistance of glulam beams. This study allowed validating the use of surface electrodes as a reliable system for MC measurement between lamellas of glulam beams. Future work will focus on the influence of wetting/drying (W/D) cycles on the mechanical integrity of glulam beams with embedded sensors for MC monitoring. Some accelerated W/D cycles and tests in an outdoor environment will be carried out. Subsequently, the influence of aging on the mechanical properties will be evaluated. Currently, a wireless system able to send MC data to the acquisition center is developed. This technology will then be used in timber bridges monitoring.

Figure 1 :

 1 Figure 1: Instrumentation of glulam specimen with surface electrodes (example of cube specimen 30 x 30 x 30 mm 3 )

Figure 2 :

 2 Figure 2: Influence of electrode diameter on electrical resistance of wood: (a) at 55% RH and 35°C; (b) at 98% RH and 35°C

Figure 3 :

 3 Figure 3: Electrical resistance as a function of spacing between electrode and specimen-edge: (a) at 55% RH and 35°C; (b) at 98% RH and 35°C

Figure 4 :

 4 Figure 4: Specimen instrumentation for influence of temperature

Figure 5 :

 5 Figure 5: The electrical resistance as a function of temperature at 65% RH

Figure 6 :

 6 Figure 6: (a) Equilibrium MC for calibration levels; (b) logarithm of electrical resistance as a function of logarithm of MC

Figure 7 :

 7 Figure 7: Electrodes positions between two lamellas for instrumented specimens

Figure 8 :

 8 Figure 8: Four-point bending test and LVDT sensor

Figure 9 :

 9 Figure 9: Comparison of the BS and MOE for specimens without sensors and specimens with sensors

Table 1 :

 1 Sizes of electrodes and dimensions of specimens

Electrode di- ameter [mm] Spacing electrode to specimen-edge [mm] Dimensions of specimens [mm]

			L	R	T
	10	10	30	30	30
	20	10	40	40	30
	30	10	50	50	30
	40	10	60	60	30
	50	10	70	70	30

Table 2 :

 2 Spacing between electrode and specimen-edge, and dimensions of specimens

	Electrode di-	Spacing electrode to	Dimensions of specimens [mm]
	ameter [mm]	specimen-edge [mm]	L	R	T
	10	5	20	20	30
	10	10	30	30	30
	10	15	40	40	30
	10	20	50	50	30
	10	25	60	60	30
	10	30	70	70	70

Table 3 :

 3 Conditioning levels in climate chamber for calibration curve

	Relative humidity [%] Temperature [°C]
	50	35
	60	35
	70	35
	80	35
	90	35
	98	35

SHATiS'19 -Structural Health Assessment of Timber Structures

ACKNOWLEDGEMENTS

This work has been funded by la Région Occitanie, la Communauté d'Agglomération Tarbes-Lourdes-Pyrénées and IUT de Tarbes. The authors would also like to thank the Department GEII (electrical engineering and industrial computing), in particular, Emmanuel Laügt for his contribution in developing the measurement devices. The contribution of Frédéric Leroy in experimentation is acknowledged.