Evangelos Balanikas

Akos Banyasz

Gérard Baldacchino

Dimitra Markovitsi
email: dimitra.markovitsi@cea.fr

Deprotonation dynamics of guanine radical cations

INTRODUCTION

Guanine radical cations (G + )  are precursors to DNA damage under oxidative stress (1). These species are unstable in neutral aqueous solutions and tend to lose a proton, giving rise to deprotonated guanine radicals (G-H)  (2). Alternatively, (G + )  may follow other reaction path; for example, undergo hydration, which subsequently leads to the well-known oxidation marker 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) [START_REF] Cadet | Oxidatively Generated Damage to Cellular DNA by UVB and UVA Radiation[END_REF]. The relative yields of the final lesions resulting from various reactions in competition depend, among others, on the dynamics of the associated reactions. In other terms, the faster the reaction rate, the higher the probability to observe lesions resulting from this reaction (3).

Information about the deprotonation dynamics of (G + )  in room temperature solutions has been obtained by time-resolved techniques, pulsed radiolysis [START_REF] Kobayashi | Direct observation of guanine radical cation deprotonation in duplex DNA using pulse radiolysis[END_REF][START_REF] Choi | Proton Transfer of Guanine Radical Cations Studied by Time-Resolved Resonance Raman Spectroscopy Combined with Pulse Radiolysis[END_REF] and flash photolysis [START_REF] Wu | Direct Observation of Guanine Radical Cation Deprotonation in G-Quadruplex DNA[END_REF][START_REF] Banyasz | Absorption of Low-Energy UV Radiation by Human Telomere G-Quadruplexes Generates Long-Lived Guanine Radical Cations[END_REF][START_REF] Balanikas | Populations and Dynamics of Guanine Radicals in DNA strands: Direct versus Indirect Generation[END_REF][START_REF] Banyasz | Radicals generated in tetramolecular guanine quadruplexes by photo-ionization: spectral and dynamical features[END_REF][START_REF] Zhang | Deprotonation of Guanine Radical Cation G(center dot+) Mediated by the Protonated Water Cluster[END_REF][START_REF] Behmand | Potassium Ions Enhance Guanine Radical Generation upon Absorption of Low-Energy Photons by G-quadruplexes and Modify Their Reactivity[END_REF][START_REF] Balanikas | Guanine Radicals Generated in Telomeric G-quadruplexes by Direct Absorption of Low-Energy UV Photons: Effect of Potassium Ions[END_REF][START_REF] Balanikas | Guanine Radicals Induced in DNA by Low-Energy Photoionization[END_REF], probing the absorption of guanine radicals on the UV-visible spectral domain. Depending on the way in which the (G + )  are generated, we can distinguish two main experimental approaches. On the one hand, (G + )  may be produced by electron abstraction from DNA through a reaction with an external oxidant; this approach is named hereafter "indirect". On the other, (G + )  may result from photoionization of DNA, without mediation of other molecules, which we coin "direct" approach.

In 1992, Candeias and Steenken observed that radicals of monomeric guanosine derivatives generated by photoionization in neutral aqueous solutions are already deprotonated in 200 ns [START_REF] Candeias | Ionization of purine nucleosides and nucleotides and their components by 193-nm laser photolysis in aqueous solution: model studies for oxidative damage of DNA[END_REF]. The first determination of (G + )  deprotonation dynamics was achieved a decade later by Kobayashi and Tagawa (4). This study, as well as subsequent ones performed for different types of DNA multimers, revealed that the (G + )  deprotonation rate strongly depends on the secondary structure [START_REF] Kobayashi | Direct observation of guanine radical cation deprotonation in duplex DNA using pulse radiolysis[END_REF][START_REF] Wu | Direct Observation of Guanine Radical Cation Deprotonation in G-Quadruplex DNA[END_REF][START_REF] Banyasz | Absorption of Low-Energy UV Radiation by Human Telomere G-Quadruplexes Generates Long-Lived Guanine Radical Cations[END_REF][START_REF] Balanikas | Populations and Dynamics of Guanine Radicals in DNA strands: Direct versus Indirect Generation[END_REF][START_REF] Banyasz | Radicals generated in tetramolecular guanine quadruplexes by photo-ionization: spectral and dynamical features[END_REF][START_REF] Behmand | Potassium Ions Enhance Guanine Radical Generation upon Absorption of Low-Energy Photons by G-quadruplexes and Modify Their Reactivity[END_REF][START_REF] Balanikas | Guanine Radicals Generated in Telomeric G-quadruplexes by Direct Absorption of Low-Energy UV Photons: Effect of Potassium Ions[END_REF][START_REF] Balanikas | Guanine Radicals Induced in DNA by Low-Energy Photoionization[END_REF]. Despite the qualitative agreement of the findings published by various groups, important discrepancies regarding the reported rate or time constants appeared, mostly in the case of guanine quadruplexes (G-Quadruplexes). These four-stranded DNA structures are formed by folding and/or association of guanine rich DNA/RNA strands in the presence of metal cations (Figure 1); the latter affect both the folding geometry and the stability of the resulting scaffold [START_REF] Hud | The role of cations in determining quadruplex structure and stability[END_REF]. G-Quadruplexes are extensively studied because of their important role in numerous biological functions [START_REF] Rhodes | G-quadruplexes and their regulatory roles in biology[END_REF][START_REF] Kharel | Properties and biological impact of RNA G-quadruplexes: from order to turmoil and back[END_REF] and their potential applications in biosensors [START_REF] Xiang | Label-free and dye-free detection of target DNA based on intrinsic fluorescence of the (3+1) interlocked bimolecular G-quadruplexes[END_REF][START_REF] Liu | Intercalative methylene blue as an efficient signal molecule of tremella-like Bi 2 WO 6: Toward high performance photoelectrochemical biosensing[END_REF][START_REF] Gu | Homogenous cathodic photoelectrochemistry for DNA binding protein analysis[END_REF].

>Figure 1<

The objective of the present review is to (i) highlight the major outcomes on the dynamics of (G + )  deprotonation in neutral aqueous solutions reported from different groups, (ii) compare the conclusions reached following different experimental methodologies and (iii) discuss the origins for the observed inconsistencies. As experiments providing information on reaction dynamics require the knowledge of the absorption spectra of the radicals, we start our review by presenting their features. In a next step, the basic principles underlying direct and indirect (G + )  generation are outlined. Then, we present the main results acquired by both indirect and direct approaches. The latter were obtained in our group exploiting a recently discovered unexpected phenomenon: absorption directly by DNA of single photons with energies significantly lower compared to the vertical ionization potential of nucleobases (21) may provoke electron ejection (22-24, 13, 25). The photoionization process as well as the general methodology adopted for the quantification of the ejected electrons and guanine radicals have been recently highlighted [START_REF] Balanikas | Guanine Radicals Induced in DNA by Low-Energy Photoionization[END_REF]. Here, we insist on quantitative aspects related to the deprotonation dynamics, enriching the existing picture with a novel methodology for the determination of the (G + )  survival probability in G-Quadruplexes. Finally, after comparing the various findings, we discuss the occurrence of non-constant reaction rates involving DNA.

ABSORPTION SPECTRA: A PREREQUISITE FOR DYNAMICAL

STUDIES

The absorption spectra of both (G + )  and the resulting deprotonated radicals were first determined by Candeias and Steenken (26). In the case of the nucleoside (dG) and nucleotide (dGMP), they found that the proton in position 1 of the guanine is lost upon deprotonation; but if the release of this proton is not possible, as happens in the case of 1-methyl guanine, the proton in position 2 of the amino group takes over (Figure 2, insets). The ensued deprotonated radicals, noted (G-H1)  and (G-H2)  , respectively, exhibit quite different absorption spectra.

As the relevant feature for the determination of the deprotonation dynamics is the spectral change occurring during this process, we present in Figure 2 the differential spectra corresponding to (G + )   (G-H1)  and (G + )   (G-H2)  deprotonations.

>Figure 2<

The spectral features of the various guanine radicals were recently rationalized by quantum chemistry calculations [START_REF] Kumar | Excited States of One-Electron Oxidized Guanine-Cytosine Base Pair Radicals: A Time Dependent Density Functional Theory Study[END_REF][START_REF] Martinez-Fernandez | Studying the excited electronic states of guanine rich DNA quadruplexes by quantum mechanical methods: main achievements and perspectives[END_REF]. In addition, these studies showed, that their main characteristics in the visible spectral domain are maintained upon hydrogen-bonding and basestacking. Computations also indicated subtle differences in duplex spectra depending on whether the H1 proton is transferred either to the bulk water or to the cytosine (Figure 3a). However, the sensitivity of time-resolved measurements does not allow a clear distinction between these two transfers.

Globally, the spectra of deprotonated radicals in DNA multimers match quite well those of monomeric guanosine in the visible spectral domain [START_REF] Banyasz | Radicals generated in tetramolecular guanine quadruplexes by photo-ionization: spectral and dynamical features[END_REF][START_REF] Balanikas | Guanine Radicals Induced in DNA by Low-Energy Photoionization[END_REF]. But moving towards the UV, the agreement may be less good, because of the contribution of the "initial" multimer absorption (see Figure 9 in reference ( 7)). As a matter of fact, due to the electronic coupling among nucleobases, the Franck-Condon excited states of DNA multimers differ from those of noninteracting monomers [START_REF] Emanuele | Exciton states of dynamic DNA double helices: alternating dCdG sequences[END_REF][START_REF] Nogueira | Electronic delocalization, charge transfer and hypochromism in the UV absorption spectrum of polyadenine unravelled by multiscale computations and quantitative wavefunction analysis[END_REF][START_REF] Martinez-Fernandez | Studying the excited electronic states of guanine rich DNA quadruplexes by quantum mechanical methods: main achievements and perspectives[END_REF][START_REF]nche -urcia, . ai, . ogueira and . on le (2020) Excimer Intermediates en Route to Long-Lived Charge-Transfer States in Single-Stranded Adenine DNA as Revealed by Nonadiabatic Dynamics[END_REF] and this is reflected in their time-resolved absorption spectra.

>Figure 3<

The absorption spectra of (G + )  in certain G-Quadruplexes were determined experimentally for solutions with pH 3, where deprotonation is known to be hindered for monomeric guanosine derivatives [START_REF] Candeias | Stucture and acid-base properties of one-electronoxidized deoxyguanosine, guanosine, and 1-methylguanosine[END_REF]. It was observed that the spectral intensity below 450 nm is significantly lower than that of the monomeric analogue [START_REF] Banyasz | Absorption of Low-Energy UV Radiation by Human Telomere G-Quadruplexes Generates Long-Lived Guanine Radical Cations[END_REF][START_REF] Banyasz | Radicals generated in tetramolecular guanine quadruplexes by photo-ionization: spectral and dynamical features[END_REF]. This finding is supported by quantum chemistry computations [START_REF] Banyasz | Absorption of Low-Energy UV Radiation by Human Telomere G-Quadruplexes Generates Long-Lived Guanine Radical Cations[END_REF][START_REF] Martinez-Fernandez | Studying the excited electronic states of guanine rich DNA quadruplexes by quantum mechanical methods: main achievements and perspectives[END_REF], which also showed that the electron hole may be delocalized over parallel stacked guanines [START_REF] Adhikary | Direct Observation of the Hole Protonation State and Hole Localization Site in DNA-Oligomers[END_REF][START_REF] Adhikary | Prototropic equilibria in DNA containing one-electron oxidized GC: intra-duplex vs. duplex to solvent deprotonation[END_REF][START_REF] Kumar | Density Functional Theory Studies of the Extent of Hole Delocalization in One-Electron Oxidized Adenine and Guanine Base Stacks[END_REF][START_REF] Livshits | Long-range charge transport in single G-quadruplex DNA molecules[END_REF][START_REF] Capobianco | Delocalized Hole Domains in Guanine-Rich DNA Oligonucleotides[END_REF][START_REF] Martinez-Fernandez | Studying the excited electronic states of guanine rich DNA quadruplexes by quantum mechanical methods: main achievements and perspectives[END_REF]. Such a delocalization could influence the deprotonation rate. However, its occurrence has not been clearly established on the time-scales and temperatures discussed here.

GENERATION OF RADICAL CATIONS

All the studies that reported deprotonation dynamics following the indirect approach, used (SO 4 -) • as external oxidant to produce (G • ) + radicals.

(SO 4 - ) • + G SO 4 2 -+ (G • ) + (reaction I).
The oxidant itself is formed from a peroxodisulfate salt via a two-step or one-step process. In pulsed radiolysis [START_REF] Kobayashi | Direct observation of guanine radical cation deprotonation in duplex DNA using pulse radiolysis[END_REF][START_REF] Choi | Proton Transfer of Guanine Radical Cations Studied by Time-Resolved Resonance Raman Spectroscopy Combined with Pulse Radiolysis[END_REF], first, the interaction of an electron beam with water gives rise to hydrated electrons e hyd -and HO • radicals. The latter are scavenged by tert-butyl alcohol, which is also added in the solution so that to avoid their reaction with DNA. Then, hydrated electrons react with peroxodisulfate anions:

e hyd -+ S 2 O 8 2 - SO 4 2 -+ (SO 4 - ) • (reaction II).
If laser irradiation is used instead of an electron beam, the oxidant is generated by a single photochemical reaction (6, 10):

hv + S2O8 2 - 2(SO 4 - ) • (reaction III).
In the low-energy photoionization studies, (G • ) + generation takes place at 266 nm (13). The key point of this approach is the use of laser intensities (≤ 2×10 6 Wcm -2 ) significantly lower compared to those used to study final lesions [START_REF] Cadet | Biphotonic Ionization of DNA: From Model Studies to Cell[END_REF]. Under these conditions, the e hyd -originate only from DNA, without interference from solvent ionization. Consequently, they can be quantified and compared with the radical concentration through the corresponding molar absorption coefficients () as explained in reference [START_REF] Balanikas | Guanine Radicals Induced in DNA by Low-Energy Photoionization[END_REF]. For the experiments reported here, DNA was dissolved in the phosphate buffer, composed of an equimolar mixture of NaH 2 PO 4 /Na 2 HPO 4 or KH 2 PO 4 /K 2 HPO 4 in concentrations of 0.015 -0.15 mol•L -1 each. Under these conditions, e hyd -decay in less than 2 µs, reacting with H 2 PO 4 -2 (13).

We remark that, going from pulse radiolysis to photosensitized electron abstraction and further to photoionization, both the number of reaction steps necessary to generate (G • ) + and the number of chemicals present in the solution, decrease. The counterpart is that, in parallel, the intensity of the transient absorption signals decreases, typically by two orders of magnitude (compare, for example, Figure 5 in reference (4) and Figure 4 in this manuscript). Therefore, low-energy photoionization studies require long measurement times. In addition, both DNA and the buffer ingredients need to be purified so that to avoid spurious signals from impurities and/or their interaction with radicals.

We stress that the initial electron holes generated by either the indirect or the direct method are not necessarily located on guanine sites. On the one hand, (SO 4 -) • may oxidize other nucleobases [START_REF] Candeias | Ionization of polynucleotides and DNA in aqueous solution by 193 nm pulsed laser light: identification of base derived radicals[END_REF][START_REF] Candeias | Electron transfer in di(deoxy)nucleoside phosphates in aqueous solution: rapid migration of oxidative damage (via adenine) to guanine[END_REF], and, on the other, low-energy photoionization takes place also in DNA multimers devoid of guanines [START_REF] Banyasz | UV-induced Adenine Radicals Induced in DNA A-tracts: Spectral and Dynamical Characterization[END_REF][START_REF] Banyasz | Adenine radicals generated in alternating AT duplexes by direct absorption of low-energy UV radiation[END_REF]. But, as guanine has the lowest oxidation potential among nucleobases [START_REF] Palecek | Electrochemistry of Nucleic Acids[END_REF], the electron hole is rapidly trapped by guanines following a charge transport process [START_REF] Kanvah | Oxidation of DNA: Damage to Nucleobases[END_REF][START_REF] Douki | UV laser photolysis of DNA: effect of duplex stability on charge-transfer efficiency[END_REF][START_REF] Meggers | Sequence dependent long range hole transport in DNA[END_REF][START_REF] Saito | Mapping of the hot spots for DNA damage by one-electron oxidation: Efficacy of GG doublets and GGG triplets as a trap in long-range hole migration[END_REF][START_REF] Genereux | Mechanisms for DNA Charge Transport[END_REF][START_REF] Kawai | Hole Transfer Kinetics of DNA[END_REF][START_REF] Lewis | Dynamics of photoinduced charge transfer and hole transport in synthetic DNA hairpins[END_REF] and only guanine radicals are observed in the experiments described here.

MAIN FINDINGS: INDIRECT APPROACH

In their seminal work, Kobayashi and Tagawa (4) performed pulse radiolysis and probed the transient absorption at 625 nm (see Figure 2a). They found that (G + )   (G-H1)  deprotonation in dG proceeds with a rate constant of 1.8×10 7 s -1 (56 ns). Recent flash photolysis measurements yielded a similar rate constant (1.5×10 7 s -1 / 67 ns) (10).

Kobayashi and Tagawa also studied the deprotonation dynamics of (G + )  in a series of model duplexes with 12-13 base-pairs containing G, GG and GGG steps at different positions (4). In this case, the fit of the transient signals required two-exponential functions, provided rate constants of 1.3×10 7 s -1 (78 ns) and 3×10 6 s -1 (330 ns). The fastest one was attributed to guanines located close to the ends of the duplex, while the slowest was assigned to internal guanine sites.

The first study on G-Quadruplexes was performed by Su and co-workers, using photosensitized electron abstraction [START_REF] Wu | Direct Observation of Guanine Radical Cation Deprotonation in G-Quadruplex DNA[END_REF]. They examined four different sequences forming monomolecular, bimolecular and tetramolecular structures (Figure 1). They came to two important conclusions. First, they showed that, in contrast to what was observed for duplexes, (G + )  deprotonation in these systems does not involve release of the H1 proton but of the H2 proton (Figure 1b). This conclusion was attested by the appearance in the transient absorption spectra of a band peaking in the red, which is typical of the (G-H2)  radicals [START_REF] Candeias | Stucture and acid-base properties of one-electronoxidized deoxyguanosine, guanosine, and 1-methylguanosine[END_REF][START_REF] Chatgilialoglu | The Two Faces of the Guanyl Radical: Molecular Context and Behavior[END_REF]. It was explained by the fact that H1 protons are engaged in the Hoogsteen hydrogen bonds connecting the guanines within the guanine tetrads (Figure 3b). Later, quantum chemistry calculations found that, indeed, (G-H2)  radicals in G-quadruplexes are more stable than the (G-H1)  analogues (9). The second conclusion was that (G + )   (G-H2)  deprotonation in G-Quadruplexes is slower compared to (G + )   (G-H1)  in duplexes. Rate constants ranging from 1.4×10 6 s -1 (0.7 µs) and 2×10 5 s -1 (5 µs) were reported.

A subsequent study on two G-quadruplexes, also performed by photosensitized electron abstraction reached a different conclusion [START_REF] Merta | Generation of 8-oxo-7,8dihydroguanine in G-Quadruplexes Models of Human Telomere Sequences by One-electron Oxidation[END_REF]. The transient absorption spectra recorded at 50 µs were attributed to (G-H1)  deprotonated radicals but the discrepancy with previously reported studies (6, 7) was not discussed.

MAIN FINDINGS: DIRECT APPROACH

In photoionization studies, a full spectral characterization of the radicals is possible only after 2-3 µs. This is due, on the one hand, to the low intensity of the transient absorption signals, which are noisier on shorter times, and, on the other, to the presence of hydrated ejected electrons, whose broad and intense absorption is extended over the whole visible domain ( 13).

Yet, some delicate measurements were performed on the sub-microsecond time-scale by adding in the solution efficient electron scavengers, N 2 O or NO 3 -(50).

For all the DNA systems studied by this method, the guanine radical concentration at 2-3 µs was found to be equal to that of the hydrated ejected electrons [e hyd -] 0 , determined by independent experiments on the sub-microsecond time-scale (13). Thus, it was possible to quantitatively follow the entire radical population during the time. We note that these "initial" concentrations correspond to electron and holes that escaped recombination (51) and may provoke DNA damage.

The differential absorption spectra recorded on the microsecond time-scale for model duplexes and purified calf thymus DNA (CT-DNA) [START_REF] Banyasz | Radicals generated in alternating guanine-cytosine duplexes by direct absorption of low-energy UV radiation[END_REF][START_REF] Balanikas | Populations and Dynamics of Guanine Radicals in DNA strands: Direct versus Indirect Generation[END_REF][START_REF] Balanikas | Guanine Radicals Induced in DNA by Low-Energy Photoionization[END_REF] correspond to that of (G-H1)  radicals,

showing that (G + )   (G-H1)  deprotonation takes place earlier. The deprotonation dynamics was determined in the case of CT-DNA by recording transient absorption signals at 700 nm.

The latter exhibit a rise, which can be described by a mono-exponential function with a time constant of 320 ± 30 ns ns; after 2µs, the signal starts decreasing slowly (Figure 4) and subsequently it decays on the millisecond time-scale (13).

>Figure 4<

A more complex picture was found in the case of G-Quadruplexes. The study of 7 different systems showed that a significant population of the initially generated (G + )  is still present at 3 µs (Table 1). Depending on the G-quadruplex type, the concentration of this "long-lived" (G + )  population varies from 25% to 60% of [e hyd -] 0 while the remaining part corresponds to (G-H2)  radicals.

>Table 1<

The percentages presented in Table 1 were determined by reconstructing the transient absorption spectra obtained for the studied G-Quadruplexes by linear combinations of the spectra reported for monomeric guanosine radicals the (G + )  and (G-H2)  . This reconstruction takes into account not only the spectral shape but also the spectral intensities. To this end, the differential absorption A, observed over an optical path-length of 1 cm, was divided by the concentration of hydrated ejected electrons, while the monomer spectra were considered with their  (26). An example is given in Figure 5a where the transient absorption spectrum of OXY/Na + at 3 µs is presented together with that corresponding of a linear combination of (G + )  and (G-H2)  radicals at a ratio of 45/55 while the sum of their concentrations is equal to the detected [e hyd -] 0 .

>Figure 5<

For some G-Quadruplexes, deprotonation toward (G-H2)  becomes much more obvious on later times. For example, the spectrum of (TG 4 T) 4 /Na + at 50 µs (Figure 5 in reference ( 9)) strongly resembles that of monomeric (G-H2)  . At even longer times, (G-H2)   (G-H1)  tautomerization, which is in competition with other reaction paths, may be observed [START_REF] Balanikas | Guanine Radicals Induced in DNA by Low-Energy Photoionization[END_REF]. But this aspect is out of the scope of the present review.

>Figure 6<

For a few cases, the early deprotonation dynamics could be resolved. This is presented in Figure 6 for TEL21/K + , OXY/Na + and OXY/K + : the transient signals recorded at 620 nm exhibit a rise, corresponding to the appearance of (G-H2)  radicals (see Figure 2b). Although these signals are very noisy, it is clear that a fast deprotonation process takes place in less than 1 µs; the time constants, derived from fits with mono-exponential functions (150 ± 15 ns in Figure 6a and 200 ± 30 ns in Figure 6b), are smaller than that found for CT-DNA (320 ± 30 ns, Figure 4).

Coming to the slow deprotonation process, it could be visualized directly by transient absorption signals only in the case of tetramolecular G-quadruplexes [START_REF] Banyasz | Radicals generated in tetramolecular guanine quadruplexes by photo-ionization: spectral and dynamical features[END_REF][START_REF] Behmand | Potassium Ions Enhance Guanine Radical Generation upon Absorption of Low-Energy Photons by G-quadruplexes and Modify Their Reactivity[END_REF]. For these systems, the (G-H2)  population at 50 µs is still equal to that of the total radical population at 3 µs. Thus, the decay of the differential absorbance at 500 nm and the rise at 600 nm reflect directly the deprotonation dynamics. This is shown in Figure 7a for (TG 4 T) 4 /Na + , where both the decay and the rise are approximated by single exponential functions, with the same time constant of 6 ± 1 µs (9). The deprotonation is practically completed at 20 µs. For other systems, the total radical population, determined by the transient absorption decays at 510-515 nm as explained in reference [START_REF] Balanikas | Guanine Radicals Induced in DNA by Low-Energy Photoionization[END_REF], starts decreasing before completion of the deprotonation processes. For this reason, a different method was adopted in order to determine the reaction dynamics of (G + )  . Several time-resolved spectra were recorded on the microsecond time-scale and, subsequently, reconstructed in the same way as that followed for the 3 µs spectra. As example is presented in Figure 5b the reconstruction of the spectra recorded for TEL21/Na + at 25 µs. At this time, the total radical concentration corresponds to 85% of the hydrated ejected electrons; the missing part was associated with formation of 8-oxodG which was detected by analytical methods [START_REF] Banyasz | Absorption of Low-Energy UV Radiation by Human Telomere G-Quadruplexes Generates Long-Lived Guanine Radical Cations[END_REF]. Finally, the (G + )  survival probability, determined as a percentage of the initially generated electron holes that have escaped recombination, equal to [e hyd -] 0 , was plotted. Figures 7b and7c show the results derived from such an analysis performed for the OXY/Na + and TEL21/Na + . At times longer than 50 µs for the former system and 40 µs for the latter, the errors related with the spectral analysis increase to provide reliable data. At those times, about ~10% of the initial population is still present in the G-Quadruplex.

>Figure 7<

COMMON CONCLUSIONS AND DISCREPANCIES

The above described studies carried out by different groups using different approaches lead to some common conclusions regarding the (G + )  deprotonation dynamics.

First, similar rate constants were found for deprotonation in dG via the indirect method using either pulse radiolysis (4) and flash photolysis (6). Second, the characteristic time determined by photoionization of CT-DNA (320 ± 30 ns; Figure 4) (13) matches well the longest time constants (330 ns) derived from the study of model duplexes by pulsed radiolysis (4). The shortest time constants reported in the latter study had been correlated with guanines located close to the ends of the duplexes and it is normal that they are not detected for genomic DNA which is very long macromolecule.

Third, the specificity of (G + )  deprotonation towards (G-H2)  in G-Quadruplexes, initially deduced from photosensitized electron abstraction studies, carried out by Su and co-workers [START_REF] Wu | Direct Observation of Guanine Radical Cation Deprotonation in G-Quadruplex DNA[END_REF], was largely confirmed by our photoionization studies [START_REF] Behmand | Potassium Ions Enhance Guanine Radical Generation upon Absorption of Low-Energy Photons by G-quadruplexes and Modify Their Reactivity[END_REF][START_REF] Balanikas | Guanine Radicals Induced in DNA by Low-Energy Photoionization[END_REF][START_REF] Balanikas | Guanine Radicals Generated in Telomeric G-quadruplexes by Direct Absorption of Low-Energy UV Photons: Effect of Potassium Ions[END_REF][START_REF] Banyasz | Radicals generated in tetramolecular guanine quadruplexes by photo-ionization: spectral and dynamical features[END_REF][START_REF] Balanikas | Populations and Dynamics of Guanine Radicals in DNA strands: Direct versus Indirect Generation[END_REF][START_REF] Banyasz | Absorption of Low-Energy UV Radiation by Human Telomere G-Quadruplexes Generates Long-Lived Guanine Radical Cations[END_REF]. And it is important to note that oxidatively generated lesions to DNA resulting from (G-H2)  radicals have not been documented so far. Yet, a study Merta et al. using, as the Su group, photosensitized electron abstraction (reaction I), failed to detect the presence of (G-H2)  in telomeric G-Quadruplexes [START_REF] Merta | Generation of 8-oxo-7,8dihydroguanine in G-Quadruplexes Models of Human Telomere Sequences by One-electron Oxidation[END_REF].

Another common conclusion drawn by the Su group and our work is that (G + )  radicals in G-Quadruplexes are still observed on the microsecond time-scale. But quantification of (G + )  population, possible only through photoionization studies, revealed that these "long-lived" species, whose decays were characterized in the 3 -50 µs interval (Figure 7), represent only part of the initially generated electron holes (see Table 1). As shown in Figures 6a and6b, an important part of (G + )  deprotonates on much shorter times, even faster compared to CT-DNA (Figure 4).

REACTION RATES IN ANISOTROPIC STRUCTURES

In order to understand the discrepancies regarding the rate constants determined for (G + )  deprotonation in G-Quadruplexes, a closer look at the reaction mechanism underlying their determination may be informative. Proton transfer from (G + )  to water is considered to be a second order reaction. Given that the bulk concentration of water molecules is much higher than that of (G + )  , this second order reaction is approximated by a pseudo-first order one, as indicated by the fact that the rate constants are expressed in s -1 (4, 6, 10). However, the validity of such an approximation requires certain conditions to be fulfilled. The reaction rate needs to be controlled by diffusion of the reactants and the probability of their encounter should be uniform over the three-dimensional space over the time-window of the experimental measurements [START_REF] Blumen | Models for reaction dynamics in glasses[END_REF]. This is the model of a well-stirred reactor.

The conditions corresponding to the simplified pseudo-first order reaction kinetics are far from valid in G-Quadruplexes. As a matter of fact, analysis of several crystal structures revealed the existence of well-defined water networks, whose properties depend on the topology of G-Quadruplex; the primary water molecules may be hydrogen bonded to phosphate and/or sugar groups and even to guanines at the edges of the G-quadruplexes (54).

Heterogeneities are encountered not only in the static arrangement of the DNA hydration shell but also on the lability of its water molecules which may be modulated by slow conformational motions of the nucleic acids [START_REF] Duboue-Dijon | Dynamical Disorder in the DNA Hydration Shell[END_REF]. Interestingly, the hydration cell has been found to be thicker around G-quadruplexes than duplexes and more difficult to be disrupted by solute molecules [START_REF] Nakano | Thermodynamic properties of water molecules in the presence of cosolute depend on DNA structure: a study using grid inhomogeneous solvation theory[END_REF]. According to two-dimensional NMR experiments, the residence times of the water molecules belonging to the hydration shell of model duplexes do not exceed 1 ns at room temperature [START_REF] Phan | Determination of the residence time of water molecules hydrating B '-DNA and B-DNA, by one-dimensional zero-enhancement nuclear Overhauser effect spectroscopy[END_REF][START_REF] Denisov | Kinetics of DNA hydration[END_REF]. Consequently, these effects should be averaged on the time-scale on which (G + )  deprotonation dynamics is detected for CT-DNA by transient absorption (Figure 4). In contrast, longer water rotation and residence times, extended on the microsecond timescale, have been reported for OXY/Na + G-Quadruplexes [START_REF] Snoussi | Internal Sodium Ions and Water Molecules in Guanine Quadruplexes: Magnetic Relaxation Dispersion Studies of d(G(3)T(4)G(3)) (2) and d(G(4)T(4)G(4)) (2)[END_REF], explaining, at least partly, the observed slower (G + )  deprotonation.

Second order kinetics in systems with spatial and dynamical heterogeneity may give rise to multiscale survival probabilities of the reactants [START_REF] Benichou | Geometrycontrolled kinetics[END_REF]. Such reaction patterns artificially appear to be mono-exponential if the observation and/or analysis of the time-resolved signals is restricted over a small time-window. In addition, different time-windows result to different time constants. It is worth-noticing that multiscale dynamics characterizes also the excited state relaxation in DNA, where electronic coupling, affected by conformational motions among nucleobases [START_REF] Bouvier | Influence of conformational dynamics on the exciton states of DNA oligomers[END_REF], is operative (62).

Multiscale reaction rates may also concern (G + )  generation in DNA multimers using (SO 4 -) • .

Reaction I takes place between negatively charged electron acceptors and spatially correlated electron donors located on a polyelectrolyte. It is possible that the rate constants determined for the oxidation process reflect only part of the reaction. In this case, a simple change in the concentration ratios between DNA and the oxidant or the duration of the laser pulse may modify the oxidation rates and, hence, those of deprotonation. This could provide an explanation why (G-H2)  radicals have gone undetected in one study [START_REF] Merta | Generation of 8-oxo-7,8dihydroguanine in G-Quadruplexes Models of Human Telomere Sequences by One-electron Oxidation[END_REF] while they were readily detected in another [START_REF] Wu | Direct Observation of Guanine Radical Cation Deprotonation in G-Quadruplex DNA[END_REF].

Focusing on the deprotonation process determined by the photoionization method, the time constants derived from the fits with exponential functions (Figures 4, 6

ACKNOWLEDGMENTS:

  and 7) do not necessarily correspond to reaction rate constants; they are simply useful for a phenomenological comparison of the transient absorption traces on a given time-window. In this respect, the first photoionization study of G-Quadruplexes reported that (G + )  survive in TEL21/Na + on the millisecond time-scale signals[START_REF] Banyasz | Absorption of Low-Energy UV Radiation by Human Telomere G-Quadruplexes Generates Long-Lived Guanine Radical Cations[END_REF]. This conclusion was based on time constants derived from exponential fits of the transient absorption signals which, following the above reasoning, may lead to erroneous results. The methodology presented here for the determination of the (G + )  survival probability (Figure5b), based on the transient absorption spectra, provided shorter lifetimes. Therefore, we conclude that deprotonation in G-Quadruplexes spans over three orders of magnitude of time, from at least 30 ns to over 50 µs, the limits being determined by the time resolution and the sensitivity of the experimental set-up. This anisotropic deprotonation dynamics can be also viewed as the occurrence of dynamically anisotropic pKa values.As analytical formulas derived from simple reaction patterns cannot describe correctly the (G + )  deprotonation dynamics, specific theoretical developments are needed blatantly. A first step toward this direction was made in a recent quantum chemical study: it was shown that a cluster of four water molecules must be taken into account to correctly reproduce the temperature dependence of the reaction rates (G + )   (G-H1)  in dG determined experimentally as a function of temperature[START_REF] Zhang | Deprotonation of Guanine Radical Cation G(center dot+) Mediated by the Protonated Water Cluster[END_REF]. Regarding DNA multimers, theoretical studies should encompass, in addition, molecular dynamics simulations on tens of microseconds, already used to describe interaction of DNA with biomolecules[START_REF] Dai | Revealing atomic-scale molecular diffusion of a plant-transcription factor WRKY domain protein along DNA[END_REF][START_REF] Armeev | Histone dynamics mediate DNA unwrapping and sliding in nucleosomes[END_REF]. In this way, it should be possible to establish a correlation between DNA topology and (G + )  dynamics. This work has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 765266 (LightDyNAmics).

Table 1 .

 1 photoionization, expressed as percentage of [e hyd -] 0 ; error: ≤±5%.

Figure 1 .

 1 Figure 1. Schematic illustration of the G-Quadruplexes discussed in this review: (a) monomolecular, formed by folding of a single strand, (b) bimolecular, formed by folding and association of two single strands and (c) tetramolecular, formed by association of four single strands; red spheres represent metal cations (Na + , K + ) located in the central cavity. Guanines are shown in violet and the backbone in grey.

Figure 2 .

 2 Figure 2. Spectral differences corresponding to (G + )   (G-H1)  (a) and (G + )   (G-H2)  (b) deprotonation, obtained by subtracting the spectrum of the radical cation from those of deprotonated radicals determined for monomeric guanosine derivatives (2, 48). The position of H1 and H2 hydrogen atoms is indicated in the insets.

Figure 3 .

 3 Figure 3. Location of the hydrogen atoms H1 and H2 in (a) a Watson-Crick guanine-cytosine pair; (b) a tetrad composed of four guanines interconnected via Hoogsteen hydrogen bonds.

Figure 4 . 29 Figure 5 .

 4295 Figure 4. Transient absorption signals (black) recorded for N 2 O-saturated solutions of CT-DNA at 700 nm. The red line in (a) is derived from a fit with the model function A 1 (1-exp(t/τ 1 ))+A 0 with τ 1 = 320 ± 30 ns. Adapted from reference 9 with ACS permission.

Figure 6 .

 6 Figure 6. Deprotonation of in G-Quadruplexes on the sub-microsecond time scale. Transient absorption signals recorded for N 2 O-saturated solutions of TEL21/K + (red) (12), OXY/Na + (green) and OXY/K + (pink) (13). Yellow lines correspond to fits with mono-exponential functions, A 1 (1-exp(-t/τ))+A 0 ; τ = 150 ± 15 ns (a) and 200 ± 30 ns (b; adapted from reference (13) with ACS permission).

Figure 7 .

 7 Figure 7. Reaction dynamics of (G + ) • in G-Quadruplexes on the microsecond time-scale. (a) transient absorption signals recorded at 500 nm (green) and 600 nm (red) for (TG 4 T) 4 /Na + ; their intensity has been arbitrarily scaled; reproduced from reference 24 with ACS permission. Survival probability of the (G + ) • population (circles) in OXY/Na + (b) and TEL21/Na + (c), determined from reconstruction of the time-resolved spectra, as shown in Figure 5. Black lines correspond to fits with exponential functions A 1 (1-exp(t/τ 1 ))+A 0 (rise) and A 1 exp(-t/τ 2 )+A 0 (decays) with time constants: 6 ± 1 µs (a); 22 ± 2 µs (b) and 7 ± 0.5 µs (c).


Quentin Saby

Jean-Yves Buffière

Eric Maire

Thomas Joffre

Julien Bajolet

Stéphane Garabédian

Peter Vikner

Xavier Boulnat

Laser Powder Bed Fusion printability of cobalt-free steel powders for manufacturing injection molds

Keywords: Laser Powder Bed Fusion, Conformal Cooling, Martensitic Steels, Mechanical Properties, X-ray Tomography

Laser Powder Bed Fusion (LPBF) printing is used for manufacturing conformal cooling injection molds with internal cooling channels. Currently, the steel used to manufacture such molds by LPBF is the maraging 18Ni300. However, this steel is softer at high temperatures and less resistant to corrosion than the reference steel, the AISI H11. Moreover, 18Ni300 contains cobalt, an element that can be harmful to the health of people handling powders made of this steel. In order to find a cobalt-free alternative to 18Ni300, the printability of three different types of steel is studied: maraging stainless steels (CX, L40), martensitic non-stainless steels (42CD4, H11, H13, PM2012) and martensitic stainless steels (X15TN, PM420). The printability of those eight steels is studied through their mechanical properties (Rockwell hardness, Charpy impact test), their internal defects and surface roughness (X-ray tomography), the presence of cracks (optical microscope), and also through their microstructure (X-ray diffraction) in the as-built state. The results show that the stainless steels (PM420, X15TN, L40 and CX) exhibit interesting as-built properties allowing them to be considered as a possible alternative to the maraging steel 18Ni300 to produce LPBFed injection molds. A conformal cooling injection mold was then printed as a demonstrator.

Introduction

The development of Additive Manufacturing technologies (AM), which has accelerated sharply in recent years, offers the possibility of designing parts and sets of parts that were previously impossible to achieve with traditional manufacturing processes such as milling, drilling or turning. In the case of traditional machining, parts are made by removing material, whereas in AM, parts are made by successive additions of layers of material and this allows new design possibilities, such as parts containing lattice structures or containing internal cooling channels [START_REF] Han | Abrasive flow machining (AFM) finishing of conformal cooling channels created by selective laser melting (SLM)[END_REF][2] [START_REF] Mazur | Numerical and experimental evaluation of a conformally cooled H13 steel injection mould manufactured with selective laser melting[END_REF].

Metallurgical issues encountered during welding can also be found in LPBF printing since this method consists of creating weld beds horizontally and then superimposing them vertically. Therefore, the most commonly used steels are those whose good weldability is known, mainly low-carbon steels such as 18Ni300 or 316L steels [START_REF] Kempen | Microstructure and mechanical properties of Selective Laser Melted 18Ni-300 steel[END_REF] [START_REF] Kang | Selective laser melting of tungsten carbide reinforced maraging steel composite[END_REF][6] [START_REF] Yan | A Comprehensive Study of Steel Powders (316L, H13, P20 and 18Ni300) for Their Selective Laser Melting Additive Manufacturing[END_REF][8] [START_REF] Sun | Sliding Wear Characteristics and Corrosion Behaviour of Selective Laser Melted 316L Stainless Steel[END_REF].

In the plastic industry, LPBF printing enables the design of injection molds with internal 'conformal cooling' channels that fit the mold shape as closely as possible and ensure homogeneous and quick cooling of the part before it is ejected from the mold thus increasing the cost and energy efficiency of the process (see Figure 1) [START_REF] Mazur | SLM additive manufacture of H13 tool steel with conformal cooling and structural lattices[END_REF] [START_REF] Narvan | Process-Structure-Property Relationships of AISI H13 Tool Steel Processed with Selective Laser Melting[END_REF].

∼90 mm

Figure 1: Mold printable by LPFB with conformal cooling channels as close as possible to the molding shape to ensure quick and homogeneous cooling of the injected part (courtesy of IPC and Smoby) AISI H11 and AISI H13 tool steels are the reference alloys for manufacturing respectively plastic injection molds and die casting molds by traditional machining. Tool steels have a carbon content between 0.3 and 0.6 wt.%, which confers high hardness and low ductility after quenching, making them difficult to weld and therefore to print. The compressive and tensile stresses which appear during the heating and cooling cycles of printing cause the most brittle zones to break in tool steels [START_REF] Narvan | Process-Structure-Property Relationships of AISI H13 Tool Steel Processed with Selective Laser Melting[END_REF] [START_REF] Damon | Process development and impact of intrinsic heat treatment on the mechanical performance of selective laser melted AISI 4140[END_REF]. A tempering or double tempering heat treatment is necessary to lower the hardness and improve the ductility. For the reasons stated above, tool steels are difficult to print with LPBF. Therefore, numerous studies have been carried out on the printing of molds from such steels [START_REF] Casati | Microstructure and mechanical behavior of hot-work tool steels processed by Selective Laser Melting[END_REF][14] [START_REF] Džugan | Thermo-physical properties investigation in relation to deposition orientation for SLM deposited H13 steel[END_REF][16] [START_REF] Laakso | Optimization and Simulation of SLM Process for High Density H13 Tool Steel Parts[END_REF]. However neither H11 nor H13 large-scale molds printed by LPFB have been found in literature yet.

So far the strategy for printing injection molds has been to use maraging steels whose good printability is known. Printability is the ability of a steel to form parts without cracks, and with the fewest defects (pores, roughness) which could be areas of stress concentration and lead to a degradation of the mechanical properties. Low-carbon maraging steels exhibit good printability thanks to a soft martensitic structure that is further hardened by precipitation hardening. A good example of such steel is the 18Ni300 alloy, its high nickel content (about 18 wt.%) allows the formation of martensite by cooling in ambient air. The hardening of 18Ni300 is mainly due to the formation of Ni 3 Ti precipitates created during an ageing treatment, it is accompanied with a decrease in ductility [START_REF] Sha | Phase chemistry and precipitation reactions in maraging steels: Part II. Co-free T-300 steel[END_REF]. The major drawbacks of 18Ni300 is that its hardness is lower than that of tool steels at high temperatures, its price is high due to its high-nickel content [START_REF] Mazur | SLM additive manufacture of H13 tool steel with conformal cooling and structural lattices[END_REF] and its corrosion resistance is low due to the absence of chromium in its composition.

For all these reasons, in spite of their potential, none of those steels (tool steels and maraging steels) fulfills perfectly the manifold requirements for producing an injection mold by LPFB. The topic of this study is therefore to extend the search for good printability, by studying several kinds of steels: maraging stainless steels, martensitic non-stainless steels (traditional tool steels), and martensitic stainless steels.

As cobalt has detrimental health impact and as it is a rare and expansive alloying element, all steels analysed in this study are cobalt-free [START_REF] Lauwerys | Health risks associated with cobalt exposure -an overview[END_REF] [START_REF] Boeck | Cobalt and antimony: genotoxicity and carcinogenicity, Mutation Research/Fundamental and Molecular Mechanisms of[END_REF].

In this work, printability is assessed by examining the mechanical properties (Rockwell hardness, Charpy impact test), defects and roughness (X-ray tomography, optical microscopy), and also through their microstructure (X-ray diffraction) in the as-built state.

Experimental

Atomised Powders

X15TN, PM420, PM2012 and 42CD4 powders were provided by Aubert & Duval; L40 powder was provided by Nanosteel; H11 and H13 powders were provided by LPW; CX powder was provided by EOS. All powders had a particle size between 20 and 53 µm and were gas atomised. Chemical compositions of the steels used are given in Table 1 and were found in the technical documentation of the suppliers, except for L40 whose composition was found in the patent [START_REF] Tuffile | 3d printable hard ferrous metallic alloys for powder bed fusion[END_REF] and for PM420 whose composition was provided by Aubert & Duval.

Laser powder bed fusion

The samples were printed with an EOS INT M270 machine. The melting parameters used are listed in Table 2. To compare the printability of the different steels with respect to their chemical composition, average melting parameters were used with two different energy densities. Previous internal experiments, had shown that stainless steels require higher energy density than the one used to print non-stainless steels, respectively 93±11 J/mm 3 and 60±7 J/mm 3 [START_REF] Limousin | Heat Treatment Optimization of a High Nitrogen Stainless Steel Manufactured by Laser Beam Melting[END_REF] [START_REF] Maillol | Impact of Exposure Parameters on Tooling Steel Properties and Performance[END_REF]. Consequently two sets of parameters have been used : one set for PM2012, 18Ni300, 42CD4, H11 and H13 steels on the hand, and another set for the PM420, L40, CX and X15TN stainless steels on the other hand (Table 2). The laser power and the hatch distance were set at 195 W and 100 µm respectively, the layer thickness was 40 µm for the steels and 30 µm for the stainless steels. A layer thickness of 40 µm was chosen for martensitic non-stainless steels to reduce residual stresses during printing. Studies have shown that increasing the layer thickness can reduce residual stresses by reducing the thermal gradient in the printed layer [START_REF] Van Belle | Investigation of residual stresses induced during the selective laser melting process[END_REF][25] [START_REF] Zaeh | Investigations on residual stresses and deformations in selective laser melting[END_REF]. The orientation of hatching was 67 • per layer. The scan speed v s (mm/s) was determined using the formula

v s = P E d × h × d
where E d is the energy density (J/mm 3 ), P is the laser power (W), h is the hatch distance (mm) and d is the layer thickness of the powder bed (mm).

Mechanical properties characterization

Hardness measurements were performed with a Wolpert R-Testor 643R. Before indentation, the specimen surfaces were polished with P320 sandpapers to remove the initial roughness.

Charpy impact tests were carried out with a Zwick Roell Charpy impact test machine. The Charpy specimen dimensions were 5 × 10× 55 mm 3 and had 5 mm U-shaped notches, in accordance with the NF EN ISO 148-1 (2017) standard. The external surfaces were milled and the notches were made by Electrical Discharge Machining (EDM).

Defects characterization

Optical observations were carried out using a Hirox RH-2000 optical microscope. SEM observations were made with a Zeiss SUPRA 55 field emission gun microscope.

X-ray tomography was performed with a v|tome|x s tomograph made by General Electric TM, using a voxel size of 3 µm. A voltage of 140 kV and an intensity of 80 µA were used to scan cylindrical samples with a 3 mm diameter and 10 mm length. 1200 projections were made per scan and the scan time was approximately 20 min. A thin copper foil (0.3 mm) was used as filter. Phoenix datos x software was used for the 3D image reconstruction and data were analysed with the software ImageJ [START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF]. The thresholding was performed automatically using an IsoData algorithm. Pores with a volume smaller than 9 voxels were filtered out and therefore, the smallest pore equivalent diameter that can be detected was about 7,7 µm. The reconstructed 3D samples were used to analyse internal defects and surface roughness as illustrated in Figure 2. The calculation of the relative density of the parts was performed from the 3D images, by computing the ratio between the volume of the sample (without pores) and the total volume of pores. In order to characterize the surface quality of the as-built samples, lateral surface profiles were extracted from the X-ray scans as shown in Figure 2c) using a method developed in [START_REF] Persenot | Fatigue performances of chemically etched thin struts built by selective electron beam melting: Experiments and predictions[END_REF] [START_REF] Persenot | Fatigue of Ti-6Al-4V thin parts made by electron beam melting[END_REF]. The roughness is characterized with the R a and R t indicators defined by the equations :

R a = 1 N N i=0 |f n | R t = max(f n ) + |min(f n )|
where f n is the height of peaks or depths obtained from the profiles extracted from the reconstructed 3D samples at N positions along the extracted profiles. As f n values were obtained from to X-ray scans, it was possible to observe thin defects and cavities impossible to detect with profilometer diamond stylus or confocal microscope. The sphericity of the pores was calculated by carrying out the ratio between the real surface of the pore and the surface of a sphere of volume equivalent to that of the pore. Sphericity values range between 0 and 1, the latter being the sphericity of a perfect spherical pore.

Pore projection was done using minimum intensity projection in ImageJ. Minimum Intensity projection creates a 2D (X,Y) image where each pixel has the minimum value over all images in the Z (build direction) stacks at the (X,Y) position of the pixel as explained in [START_REF][END_REF]. In our case, pores had the minimum values after thresholding, and projections were made along the construction axis. Therefore, pores at different heights in the sample can be seen in a 2D view.

Characterization of the crystallographic structure

A D8 Advance Bruker AXS diffractometer was used to perform X-ray diffraction (XRD) at 4 kV and 40 mA with CuK α radiation (1.54 Å). Scans were acquired in a θ -θ configuration from 30 • to 130 • (2θ) with a step size of 0.05 • and a step time of 1 s. Austenite quantification was performed using TOPAS software.

Results and discussion

Cracks, pores and surface roughness

The presence of cracks, pores and relatively large surface roughness is an inherent problem encountered in LPBFed parts. They have detrimental consequences on the fatigue behaviour of the parts because they can lead to stress concentration and consequently to failure [START_REF] Narvan | Process-Structure-Property Relationships of AISI H13 Tool Steel Processed with Selective Laser Melting[END_REF][31][32] [START_REF] Andreau | A competition between the contour and hatching zones on the high cycle fatigue behaviour of a 316L stainless steel: Analyzed using X-ray computed tomography[END_REF].

Cracks are observable in martensitic non-stainless steels Charpy specimens (42CD4, H11, H13, PM2012) as illustrated Figure 3. The cracks are mainly located at the base of the samples, i.e. close to the fabrication plate, this phenomenon has already been observed in [START_REF] Zaeh | Investigations on residual stresses and deformations in selective laser melting[END_REF] [START_REF] Royakkers | Selectief Laser Smelten van AISI M2 Snelstaal poeder[END_REF]. It was shown in [START_REF] Van Belle | Comparisons of numerical modelling of the Selective Laser Melting[END_REF] that the maximum stresses are found at the connection between the specimen and the fabrication plate. The propagation of a crack in an H13 specimen is visible in the Figure 4. The crack appears to start at a surface defect. It propagates initially horizontally at the interface between the fusion layers, resulting from delamination. It then changes direction probably due to the state of stress. Other steels did not crack, even martensitic stainless steels, which contain as much carbon as martensitic non-stainless steels. No cracks were observed in the cylinders used for tomography, except in the PM2012 cylinders as illustrated Figure 5. The absence of cracks in the cylinders is probably related to the small size of these parts compared to the Charpy specimens and therefore to their lower stress concentration. Concerning internal defects, two different types of pores can be observed: spherical pores, resulting from the formation of keyholes or gas inclusions in powders; and non-spherical pores, resulting from a lack-of-fusion as shown in Figure 2.b. and Figure 6. Table 3 shows that all the printed steels are nearly fully dense. Relative densities are all higher than 99.8% except for 42CD4 (99.7%). Stainless steels are slightly denser than the non-stainless steels, this may result from the different sets of parameters used. It was found that the relative density does not vary along the construction axis for all steels. For instance, Figure 7 shows the relative density of a 42CD4 sample along the construction axis Z. (BD) However, the amount of pores increases close to the surface in all samples. This can be observed when the pore projection is done without showing the volume of the part as illustrated Figure 8.a) for PM420. Such pores are not visible on Figure 6 as their position near the surface makes them less visible because of the black background.

Such pores might be caused by the acceleration and deceleration of the laser beam at the edges of the samples resulting in locally higher energy densities and therefore in pore formations as illustrated Figure 8.b). In our case, this hypothesis is unlikely as the EOS M270 machine used for printing has a "sky-writing" mode which allows the mirrors to reach the scanning speed before switching on the laser beam [START_REF] Tian | Influences of processing parameters on surface roughness of Hastelloy X produced by selective laser melting[END_REF][37][38] [START_REF] Mancisidor | Reduction of the Residual Porosity in Parts Manufactured by Selective Laser Melting Using Skywriting and High Focus Offset Strategies[END_REF]. A second hypothesis is that at the beginning and at the end of the printing of a cylindrical layer the length of the weld beads is shorter, and therefore the beads have less time to cool down between each laser run than at the central part of the layer as shown Figure 8.c). Since each layer is printed with an angle of 67 • with the previous layer, the presence of pores is detectable all around the sample. A concentric scanning strategy If the laser accelerates at the beginning of the weld bead formation to reach the required scanning speed and decelerates at the end, the energy density will locally be more important. This higher energy density may be responsible for the formation of pores at the edge of the specimens by keyholing. c) Second hypothesis on the greater presence of pores at the edge of the samples. The grey circle represents the layer of the sample being printed and the arrows represent the weld beads. The distance the laser has to travel from point A to point B (yellow lines) is smaller than from point C to point D (blue lines). This means that the material has less time to cool down when the laser fuses the bead from point B than when it fuses the bead from point D. Also, the closer the laser is to an edge, the slower the bead will cool down since the thermal conductivity of the powder bed is lower than that of the printed part. For these reasons, as the material gets warmer, it is possible that pores will appear in the areas highlighted in pink by keyholing. Since each layer is printed with an angle of 67 • from the previous one, pores are observable over the whole height of the sample.

could be tried to avoid a localized higher energy density. Median values are presented in Table 3 for all steels. They range between 0.67 and 0.82 which means that more than half of the pore volume is composed of quasi-spherical pores. A typical illustration of the pores 3D shape are given in Figure 6. The pore diameter median values given in Table 3 show that the median diameter sizes for all steels range between 18 and 39 µm. So, globally, pores are very similar in size and shape for all steels.

Concerning surface roughness, melting parameters can lead to roughness by causing lack of powder fusion and balling effect [START_REF] Narvan | Process-Structure-Property Relationships of AISI H13 Tool Steel Processed with Selective Laser Melting[END_REF].

The results of roughness measurements are shown in Table 3. Overall, both R a and R t values remain constant for the different materials (R a ∼ 14 µm, R t ∼ 120 µm). Those values are similar to the ones reported by [11][40] for 316L and AISI H13 materials.

Consequently, the tomographic analysis shows that the different steels are comparable in terms of pore characteristics and roughness, regardless of the melting parameters used to print them, except for 42CD4 that presents a higher porosity.

Microstructure analysis

XRD analyses were performed on all steels, patterns are given in Figure 9.a) and the amount of austenite in each steel is given in Figure 9.b).

Retained austenite is due to incomplete martensitic transformation, i.e. when the martensite finish temperature M f is not reached because M f and M s (the martensite start temperature) are low. These temperatures depend on the concentrations of the alloying elements and can be estimated using empirical equations [START_REF] Nehrenberg | The temperature range of martensite formation[END_REF][42] [START_REF] Andrews | Empirical formulae for the calculation of some transformation temperatures[END_REF]. It has been shown in [START_REF] Kung | An examination of the validity of existing empirical formulae for the calculation of ms temperature[END_REF] that the Steven & Haynes equation [START_REF] Steven | The Temperature of Formation of Martensite and Bainite in Low-Alloy Steels[END_REF] :

M s = 561 -474C -33M n -17N i -17Cr -21M o
and Andrews equation [START_REF] Andrews | Empirical formulae for the calculation of some transformation temperatures[END_REF] : As-built steels a) b)

M s = 539 -423C -30, 4M n -17, 7N i -12, 1Cr -7, 5M o
Figure 9: XRD analysis : a) XRD patterns of the as-built steels. All steels present iron-α and iron-γ peaks, except the X15TN which only shows iron-γ peaks, meaning is mainly composed of austenite after melting ; b) Austenite fraction from the XRD pattern. Martensitic stainless steels contained an important amount of austenite in the as-built state.

are particularly efficient for estimating the M s temperatures of low-and high-alloy steels. However, the Andrews equation overestimates the M s of high Cr steels while the Steven & Haynes equation underestimates the M s of high Mo steels [START_REF] Kung | An examination of the validity of existing empirical formulae for the calculation of ms temperature[END_REF]. These equations highlight the impact of the elements C, Mn, Ni, Cr and Mo on the reduction of M s temperature. The M s temperatures calculated for the eight steels are given in the Table 4. The percentage of austenite detected in relation to the M s temperature is shown in the Figure 10. X15TN is the steel with the lowest M s temperature and it is also the steel with the highest austenite content after melting. This is due to its higher concentration of alloying elements. 42CD4, H11, H13 and L40 have the highest M s temperature and also the lowest austenite content. PM420, PM2012 and CX steels have similar M s temperatures. The higher concentration of austenite in PM420 is probably related to a reversion of austenite, which is caused by the tempering of the lower layers during printing of the upper layers. This phenomenon was highlighted in AISI 420 in [START_REF] Krakhmalev | In situ heat treatment in selective laser melted martensitic AISI 420 stainless steels[END_REF] and results from a local increase in carbon content at either retained austenite grains interfaces or at interfaces between martensite and austenite [START_REF] Yuan | Nanoscale austenite reversion through partitioning, segregation and kinetic freezing: Example of a ductile 2GPa Fe-Cr-C steel[END_REF]. The lower fraction of austenite in PM2012 is probably related to the lower energy density used for printing, resulting in less tempering of the lower layers and therefore less reversion. For CX, no reversion could take place since it does not contain carbon.

It is possible that the crack-free printing of high carbon martensitic stainless steels is related to this large amount of austenite after LPBF printing. Austenite is much softer than martensite, can be highly deformed, and is therefore less prone to cracking. Stainless maraging steels have a lower quantity of austenite after melting, probably because of their lower carbon content which is a strong stabilizing element for austenite. = M a r a g i n g s t a i n l e s s s t e e l s = M a r t e n s i t i c n o n -s t a i n l e s s s t e e l s = M a r t e n s i t i c s t a i n l e s s s t e e l s

Mechanical properties

Hardness and Charpy impact tests show a significant heterogeneity between the different kinds of steels (Figure 11). As could be expected, martensitic non-stainless steels and martensitic stainless steels are the hardest (∼ 53 HRC) and most brittle (∼ 15 J/cm 2 ), and maraging the softest (∼ 40 HRC) and most ductile (∼ 100 J/cm 2 ). This is likely to be related to the lower carbon content in the maraging steels than in the martensitic steels (Table 1). However the carbon content is not the only factor influencing the mechanical properties of LPBFed parts. Figure 12.a) shows that 42CD4 and X15TN steels have lower hardness than H11 and H13 steels, although their carbon content is similar. 42CD4 is probably softer due to its lower relative density compared to other steels, while X15TN is probably softer due to its high austenite content after melting (Figure 12.b)).

Surface roughness, presence of pores and cracks, loss of alloying elements and residual stresses are inherent defects in AM affecting the mechanical properties of the fused parts [START_REF] Debroy | Additive manufacturing of metallic components -Process, structure and properties[END_REF]. It has been shown previously that internal defects and roughness are similar for all steels. Concerning the residual stresses, X-ray diffraction measurements in accordance with the NF EN 15305 standard have been carried out but the results were very scattered.

Maraging steels are the softest and most ductile because they are mainly composed of soft martensite i.e. a martensite with relatively low carbon content.

The X15TN is surprisingly soft for a steel with high carbon content (Figure 12.a)). It is also slightly less brittle than martensitic non-stainless steels. This is probably because it is mainly composed of austenite after fusion (Figure 12.b)).

Regarding the PM420, its hardness is comparable to the martensitic non-stainless steels and it has a better ductility than the X15TN and the martensitic non-stainless steels. This is possibly due to the presence of austenite in the PM420 (34%) after melting, higher than in the AISI H13 (19%).

Figure 13 shows that maraging (represented by CX) and martensitic (represented by PM420) have different failure modes. A strong plastic deformation of the maraging before fracture is observed Figure 13a) with, on a microscopic scale, the formation of dimples as shown in Figure 13c), proving the ductile nature of the fractures. Concerning the fracture surface of martensitic steels, the plastic deformation of the specimens before fracture is negligible (Figure 13b)) and no ductile dimples are detected (Figure 13d)), showing a brittle fracture. No defects could be identified as the initiator of the fracture. Martensitic stainless steels are softer and tougher than the martensitic non-stainless steels, this may be due to their higher content in austenite after melting. Maraging steels are soft and resilient after melting.

Assessment of complex mold printing

After microstructural and mechanical characterization, the manufacturing of complex conformal cooling mold was assessed (Figure 14). This first attempt was performed using PM420 powder, this steel showed an excellent strength/ductility compromise.

Conclusion

In this study, the printability of eight different steels was investigated through their mechanical properties, their defects, and their microstructure in the as-built state. The results show that:

• All martensitic non-stainless steels cracked during printing. The martensitic stainless steels did not crack even if they contain the same quantity of carbon than the martensitic non-stainless steels. This good printability of martensitic stainless steels may originate from a large amount of austenite, which can accept plastic deformation during printing.

• The melting parameters used in this study allow to obtain nearly fully dense parts. The internal defects are similar between the different steels except for 42CD4, which has a lower density than other steels.

• For all steels, relative density does not vary along the build direction but the amount of pores increases close to the surface.

• The median sizes of the pores observed are less than 50 µm. The median sphericities are between 0.67 and 0.82, indicating that more than half of the pore volume is composed of quasi-spherical pores.

• As maraging stainless steels are the softest and the less brittle steels because they form a relatively "soft" martensite after melting, they have the best printability.

• In spite of the very high density achieved, martensitic non-stainless steels have the lowest printability due to the high hardness of the martensite formed during printing.

• PM420 did not crack and has a good compromise between hardness and ductility. It is comparable in hardness to martensitic non-stainless steels and is more resilient than martensitic steels and X15TN, probably because of its high austenite content after melting (34%).

• X15TN did not crack neither, even though it contains as much carbon as martensitic non-stainless steels. This is probably because it is mainly composed of austenite after fusion (96%). It is softer than the other martensitic steels and it is slightly less brittle than martensitic non-stainless steels but is more brittle than PM420.

• PM420, X15TN, CX and L40 are possible alternatives to the 18Ni300 maraging steel to print molds by LPBF as they are printable, cobalt-free, and stainless steel.

• In order to determine whether the steels identified as potential alternatives to 18Ni300 can really be, they should be heat-treated. Indeed, maraging steels are designed to be aged and martensitic steels are generally tempered. A dedicated study on the effect of heat treatment on the properties of printed parts is on-going.

Figure 2 :

 2 Figure 2: X-ray tomography analysis; a) 3D sample reconstruction in ImageJ; b) Relative density, pore diameter fraction, pore sphericity; c) Roughness analysis based on a method developed in [28][29]

Figure 3 :

 3 Figure 3: Cracks on martensitic non-stainless Charpy specimens. Cracks are mainly located at the base of the specimens where the residual stresses are the highest.

Figure 4 :BDFigure 5 :

 45 Figure 4: A crack in an H13 sample observed after a chemical etching with Fry's reagent. Delamination is visible in the red circle.

Figure 6 :

 6 Figure 6: Pore projection following the manufacturing axis showing the part volume, and pore shape. Some pores may overlap due to projection creating an apparent irregular shape.

Figure 7 :

 7 Figure 7: Relative density as a function of height Z in 42CD4

Figure 8 :

 8 Figure 8: Projection showing the more important amount of pores close to the surface and hypotheses to explain this phenomenon. a) Projection of the pores in PM420 within the volume of the sample. The amount of pores increases close to the surface of the samples. Same observations were made in all the samples ; b) First hypothesis on the higher presence of pores at the edge of the samples. The grey circle represents the layer of the sample being printed and the arrows represent the weld beads.If the laser accelerates at the beginning of the weld bead formation to reach the required scanning speed and decelerates at the end, the energy density will locally be more important. This higher energy density may be responsible for the formation of pores at the edge of the specimens by keyholing. c) Second hypothesis on the greater presence of pores at the edge of the samples. The grey circle represents the layer of the sample being printed and the arrows represent the weld beads. The distance the laser has to travel from point A to point B (yellow lines) is smaller than from point C to point D (blue lines). This means that the material has less time to cool down when the laser fuses the bead from point B than when it fuses the bead from point D. Also, the closer the laser is to an edge, the slower the bead will cool down since the thermal conductivity of the powder bed is lower than that of the printed part. For these reasons, as the material gets warmer, it is possible that pores will appear in the areas highlighted in pink by keyholing. Since each layer is printed with an angle of 67 • from the previous one, pores are observable over the whole height of the sample.

  r a g i n g s t a i n l e s s s t e e l s = M a r t e n s i t i c n o n -s t a i n l e s s s t e e l s = M a r t e n s i t i c s t a i n l e s s s t e e l s A u s t e n i t e f r a c t i o n ( % ) M s ( °C ) [ S t e v e n & H a y n e s ] a ) b ) A u s t e n i t e f r a c t i o n ( % ) M s ( °C ) [ A n d r e w s ]

Figure 10 :

 10 Figure 10: Austenite fraction detected by XRD depending on Ms temperature : a) According to Steven & Haynes[START_REF] Steven | The Temperature of Formation of Martensite and Bainite in Low-Alloy Steels[END_REF] ; b) According to Andrews[START_REF] Andrews | Empirical formulae for the calculation of some transformation temperatures[END_REF]

Figure 11 :

 11 Figure11: As-built hardness and impact energy. Martensitic non-stainless steels are the hardest and the more brittle materials. Martensitic stainless steels are softer and tougher than the martensitic non-stainless steels, this may be due to their higher content in austenite after melting. Maraging steels are soft and resilient after melting.

Figure 12 :

 12 Figure 12: Hardness depending on a) carbon content and b) austenite fraction

Figure 13 :

 13 Figure 13: Fracture surfaces of CX and PM420 steels observed by optical (a-b) and SEM (c-d)

Figure 14 :

 14 Figure 14: Examples of molds printed in PM420. The dimensions of the mold are 100x100x20 mm for the platform and 50x50x20 mm for the molding surface. b) highlights in red the lattice structure in the mold and in blue the cooling channels.

Table 1 :

 1 Chemical compositions of the steels in wt.% based on the supplier documentation

	Steel types	Steel names	C	Mn	Cr	Mo	V	Ni	Cu	Si	Al	Ti	W	N	Fe
	Martensitic	PM420 (X30Cr13 -AISI 420)	0.35 0.28 13.7 0.028		0.109 0.014 0.36		0.003 0.03		//
	stainless steels	X15TN (X40CrMoVN16-2)	Min 0.37 Max 0.45 0.60 16.50 1.90 0.40 0.30 -15.00 1.50 0.20 -		-0.60				0.16 // 0.25
		42CD4 (42CrMo4)	Min 0.38 0.6 Max 0.45 0.9	0.9 1.2	0.15 0.3				-0.4					//
	Martensitic steels	AISI H11 (X37CrMoV5-1) AISI H13 (X40CrMoV5-1)	Min 0.35 0.20 4.75 Max 0.45 0.60 5.50 Min 0.32 0.20 4.75 Max 0.45 0.50 5.50	1.10 0.30 1.60 0.60 1.10 0.80 1.75 1.20 0.30 -	0.80 1.25 0.80 0.25 1.20 -				-0.1	// //
		PM2012 (HS 2-2-2)	0.6	0.3	4.0	2.0	1.5			1.0			2.1		//
	Maraging	L40	0.16		10.64 1.48		1.96	0.54 0.19				0.07 //
	stainless steels	CX	Min Max 0.05 0.40 13.00 1.70 --11.00 1.10		8.40 10.00		-0.40 2.00 1.20				//

Table 2 :

 2 Melting parameters : two different sets of parameters were used to print the stainless steels and the non-

stainless steels Non-stainless steels Stainless steels P -Laser power

	[W]	195	195
	v s -Scan speed [mm.s -1 ]	818	699
	d -Layer thickness [µm]	40	30
	h -Hatch distance [µm]	100	100
	E d -

Energy density[J/mm 3 ] 60 93

Table 3 :

 3 Porosity, pore size and roughness determined by X-Ray tomography. Internal defects and roughness are homogeneous between the steels. Only 42CD4 presents a lower relative density than the other steels.

		Steel types	Martensitic stainless steels		Martensitic non-stainless steels	Maraging stainless steels
		Steel names	PM420 X15TN 42CD4 AISI H11 AISI H13 PM2012	L40	CX
		Relative density (%)	99.994	99.994	99.734	99.980	99.983	99.990	99.993 99.998
		Sphericity median value	0.70		0.72	0.82	0.77	0.69	0.77	0.67	0.76
	Pore diameter median value (µm)	29		27	39	25		33	24	30	18
		R a (µm)	15.1		14.1	14.1	13.7	14.5	14.2	13.1	12.4
		R t (µm)	121.1		110.4	116.3	116.4	129.9	116.3	112.7	96.3
	Relative density (%)	99,5 100,0			42CD4				Z
		99,0
		0	500	1000	1500	2000	2500	3000	3500
					Z (µm)

Table 4 :

 4 Ms temperatures of the steels estimated using Stevens & Haynes and Andrews empirical formulas

	Steel types	Martensitic stainless steels		Martensitic non-stainless steels		Maraging stainless steels
	Steel names	PM420 X15TN 42CD4 AISI H11 AISI H13 PM2012 L40	CX
	M s ( • C) Steven & Haynes	151	51	313	243	272	157	240	153
	M s ( • C) Andrews	215	150	322	285	311	213	297	204