Gilles Dour
email: gilles.dour@applusrtd.com.au

Farid Medjedoub
email: farid.medjedoub-renexter@renault.com

Gabriel Diaconu
email: gabita.diacou@yahoo.com

Denis Girardin
email: denis.girardin@saint-gobain.com

Abderrahim Michrafy
email: michrafy@enstimac.fr

Tahar M Loulou
email: tahar.loulou@univ-ubs.fr

Farhad Rezai-Aria

F Rézaï-Aria

Size effect in transient thermal fatigue testing and thermo-mechanical screening of coatings

Keywords: hot forming processes, shaping processes, hot work tool steels, Thermal Fatigue, TF, thermo-mechanical, thermal stresses, heat checking

Introduction

Forming materials at high temperature is a common process found in industry. Many classical examples can be given, such as permanent die casting, forging, extrusion, glass blowing in dies, glass pressing, etc. The trend in industry seems to be getting more and more applications of hot forming (e.g., deep drawing of martensitic steels), if not using higher temperatures in classical processes (e.g., isothermal forging and high-pressure die casting of copper alloys). The common feature of all the hot-forming processes is that the tools never last forever. Usually the lifetime is limited by the formation of too large or too dense or too deep heat-checking cracks. The criteria determining the end of life of tools can vary extremely from one industrial sector to another. Heat-checking cracks are characterised by a network of cracks on the mating surface of the tools (where contact with the parts occurs). The depth of the cracks is usually limited, although sometimes one or a few of the cracks take the opportunity of some stresses to propagate deeply into the tool. Heat checking is the result of a Thermal Fatigue (TF) phenomenon: the mating surface is heated by the brief contact with the hot part being formed, while the bulk of the tool remains colder. The dilatation of the hot surface of the tool is restricted by the bulk, leading to some thermo-mechanical loadings. Depending on the process and on the tool surface requirements, the lifetime of such tools rank from 10 000 to several 100 000 cycles. It rarely extends to high-cycle fatigue lifetimes. Because tools are expensive and because their lifetime tends to get shorter with higher productivity, the Western industries seeking higher productivity need to improve their tools so that productivity gains will not be lost by higher tools costs. It is the reason why many investigations have been launched loadings in industrial tools to laboratory specimen, a normalisation of the thermo-mechanical equations is proposed. A size effect in those thermal mechanical problems is justified and used to set an original rule to design transient TF tests. In a second example related to coating tools or bi-materials tools, the concept of thermo-mechanical screening is explained: it corresponds to the optimum combination of materials properties leading to the vanishment of thermal stresses at the mating surface of a tool. An example is given that shows some conditions in which this can happen, depending on the combination of material properties. An example of application is then proposed.

on the understanding of TF and on the improvement of lifetime of tools. Many TF experiments have been developed in the USA [START_REF] Benedyck | Thermal fatigue behavior of die materials for aluminium die casting[END_REF] and more recently in Europe [START_REF] Jean | An investigation on heat checking of hot work tool steels[END_REF][START_REF] Pellizzari | Thermal fatigue resistance of plasma duplex-treated tool steel[END_REF][START_REF] Persson | Evaluation of heat checking damage in die casting[END_REF][START_REF] Siller | Numerical modelling and physical simulation of the softening behaviour of hot work tool steels during thermal fatigue[END_REF]. American standard tests for foundries are based on the alternative dipping of a specimen in a molten alloy followed by external water spraying [START_REF] Benedyck | Thermal fatigue behavior of die materials for aluminium die casting[END_REF]. Many materials, particularly tool steels, have been tested. Other investigations took inspiration in such a test with more or less success [START_REF] Mitterer | Application of hard coatings in aluminium die casting -soldering, erosion and thermal fatigue behaviour[END_REF]. The other TF tests conceived are based on the heating of the surface of specimen by an external source of heat such as LASER, inductive heating, etc. The advantage of such techniques is that one can monitor [START_REF] Persson | Evaluation of heat checking damage in die casting[END_REF] the deformation of the tool surface, or control the heating rate of the surface [START_REF] Medjedoub | Damage to die casting dies through crazing by thermal fatigue: origins, mechanisms and approaches[END_REF]. Up to now, no clear trends in lifetime extension can be found yet from all these tests when applied to tool steels. Another trend that we can see in industry is the temptation to apply coating on the surface of the tools in order to extend their lifetime. If the effects on chemical interaction are quite certain in high-pressure die casting with PVD coatings, improvement in the TF resistance is not as clear [START_REF] Persson | Thermal fatigue cracking of surface engineering hot work tool steels[END_REF] depending on the criteria used to define the improvement.

The present paper intends to address both the TF of massive tools and the effect of coatings. The understanding of thermal fatigue is first summarised in the principle of the two driving forces of the damage process under thermal fatigue conditions. In order to improve the design of coating or of thermal fatigue tests, we propose to exploit normalisation techniques to the multiphysical problem of thermo-mechanical loading. It is then used as an example to design a TF test based on the two driving forces principle and to the size effect in thermo-mechanical problems. As a last example, the normalisation technique is applied to bi-material tools. A condition called thermo-mechanical screening (i.e., the condition to reach in order to reduce to zero the thermal stresses at the surface of the tool) is sought when changing the thermo-physical properties of the coating for a given processing condition.

The principle of the two driving forces of damage by thermal fatigue

A given tool made of a given material is the centre of a number of loadings. Basically it feels some loadings from its surface and mainly from the surface in contact with the part being produced -the mating surface. The boundary conditions are then essential to understanding the way a tool has to work: sliding of the part and shear stresses, filling of the tool under high pressure and compression stresses, high temperature of the part and thermal stresses. In hot-forming processes the thermal loading is fundamental [START_REF] Dusserre | Thermo-mechanical stresses in cast steel dies during glass pressing process[END_REF]. As seen from the tools, the essential thermal loading is the density of heating power it receives from the mating surface, no matter what the process is. This density is known in the heat transfer community as the heat flux density, measured in W/m 2 . A common order of magnitude in hot-forming processes is the MW/m 2 . There exist other boundary conditions, such as mechanical ones: the pressure felt by the die or the matrix, the tangent loads caused by the friction with the part being produced or restraints caused by the bolting of the tool in a machine. All these, plus the geometry of the tool, are the necessary information one needs to know if ever one intended to understand the working conditions of a tool (e.g., see [START_REF] Dour | Development of a non-intrusive heat transfer coefficient gauge and its application to high pressure die casting[END_REF] for heat flux density measurement). Any numerical FE packages could be used to evaluate the temperatures and stresses in the tool. In cases where the thermal loading is the main restricting load, one may wonder which behaviour law should be considered for the tool material. To illustrate the difficulty let us imagine two new fresh tools, one hard and the other one soft. The soft tool steel has then a much smaller yield stress compared with the hard tool. Nevertheless most of the other properties are the same: elastic properties, thermal properties, expansion coefficient. The two tools also see the same boundary conditions while in production. As often the lifetime of hot-forming tools range is in the Low Cycle Fatigue category (less than 100 000 cycles), lifetime can be related to plastic deformation (Manson-Coffin analysis) instead of stresses (Basquin analysis). In such a case, comparing the two materials fatigue properties would require that one apply the same plastic deformation to each of the specimen during the test. Because they have different yield stresses, the thermal fatigue test should then be performed at different thermal loadings (higher heat flux for the harder material). This does not reflect what actually happens to the tools when in service: the harder tool is thermally loaded with the same thermal gradients that lead to the same thermo-mechanical loadings, and then to lower plastic deformation than the soft tool. In order to replicate the service conditions, it is better to test the two steels in exactly the same thermal conditions: same temperature, same heat flux density and same geometry. The two materials will then react to the thermal loading according to their own behaviour. This will eventually lead to different TF resistances as the softer steel will deform plastically more than the other one.

The same question arises when one wishes to compare results from several laboratory TF tests. It is obvious the temperature should remain the same in all geometries, because any thermally activated phenomenon strongly depends on it. However, if the same heat flux density were used, the thermal stresses in the two tests would be different, leading necessarily to two different TF lifetimes. If one expects to compare these two tests, something has to remain constant. The previous example of hard and soft steels suggests that the Elastic Body Thermal Stresses (EBTS) would be the parameter to keep constant; it corresponds to the thermal stresses as can be evaluated when considering that the materials is perfectly elastic. The reason behind this choice of EBTS comes from the consideration that the origin of all the stresses and mechanical strain during thermal loading is the incompatibility of thermal expansion when a part is heated or cooled with a non-uniform temperature field. It is well known that the thermal expansion is linked to the lattice bulging of the crystalline network. Any material can accommodate the incompatibility of thermal expansion with another crystalline lattice distortion. This one is called elastic deformation, or Elastic Body (EB) deformation. When it is induced by heterogeneous thermal field, the lattice distortion is what we call EBTS. This EBTS response of the material to thermal heterogeneity is reversible and immediate. However, the EBTS are often high enough to push dislocations to motion, inducing plastic deformation. This second class of response of a material to thermal loadings is evidently irreversible and its kinetics is usually slow and temperature-dependent. As a consequence, the elastic body thermal stresses EBTS and the temperature taken together can be seen as the potential that push materials to transform irreversibly when thermally loaded. With the accumulation of irreversible deformation leading to failure, the couple (T, EBTS) also potentially leads to TF when thermal loads are repeated cyclically. As a consequence, TF tests performed with the same materials can be compared only if the same couple (T, EBTS) is applied to the specimen. Most of the time, T may be the same, but certainly not the EBTS.

In a multiphysical problem such as the thermo-mechanical problem that is faced by tools, the number of parameters is tremendous. Typically EBTS are controlled by the thermal gradients in the bulk of the tool, which in turn is controlled by the thermal properties (conductivity, diffusivity) and the thermal loading (heat flux density). Of course the expansion coefficient and elastic properties will have their words to say, as well as the geometry of the specimen. Our aim here is to describe the relationship between the thermal loading, material properties and a characteristic dimension of the tool for a simple geometry (e.g., slab or hollow cylinder). Nevertheless the number of parameters remains too large to study the influence of each of them separately. It is necessary to reduce the number of parameters. The best way to do so is to normalise the equations of the problem. Such a normalisation had been performed by Landau and Paschkis (LP, 1957) in the 1950s for a slab dipped in a liquid bath at constant temperature (quenching condition). Figure 1 summarises the whole set of results. The parameters of this problem are: the temperature difference T a between the bath (constant and set to 0) and the initial temperature of the slab; the heat transfer coefficient between the slab and the liquid h; the thickness of the slab 2L; the thermal conductivity of the slab materials k; its thermal Source: [START_REF] Boley | Theory of Thermal Stresses[END_REF] In the problem of tools in hot-forming processes, the problem is slightly different from the LP quenching problem. First of all, the problem is not symmetrical as one face is heated up by contact with the hot part being shaped, and the second face is usually cooled down by a cold fluid (air, water or oil). A second difference is that the temperature of the part being shaped varies during the process (unlike the liquid in the quenching problem of LP), as well as the tool/part heat transfer coefficient. As a consequence, the heat transfer coefficient is not a relevant parameter for the mating surface of the tool. As the key information to describe the thermal loading of a tool remains the heat flux density, we will prefer it to a description with a heat transfer coefficient. The heat flux density usually varies dramatically during the fabrication process. In the following we will model the thermal loading by a step function of the heat flux density characterised by a maximum value φ max and a duration τ. For the back surface of the slab, the liquid cooling is characterised, like in LP by a heat transfer coefficient h and the temperature of the liquid (set to zero). Taking all this into account, the normalisation of the problem for a 2L-thick slab can be performed with the following set of equations (see Diaconu (2004) and [START_REF] Diaconu | Heat transfer and thermal stresses in a centrifugal casting die[END_REF] for the details of the equations):

2 t t L κ + = (1) z z L + = (2) h L h k + = (3) max ( ) ( ) t t φ φ φ + + = (4) max ( , ) ( , ) T z t T z t L k φ + + + = (5) max ( , ) ( , ) 1 z t z t L E k σ σ φ α ν + + + = - (6)
where the parameters k, κ, E, α and ν are the same as in the LP problem and z is the coordinate in the slab thickness direction (zero being at the mating face). All the new parameters (noted with + ) are dimensionless.

Application to thermal fatigue: size effect and towards the design of a TF test

The tabulation performed by LP demonstrates clearly that two slabs of thickness L and L′ (L′ > L) made of the same materials (same E, α and ν) and quenched in the same conditions (same T a and same h) develop different EBTS. They indeed have a different

Biot number m and m′ (m′ > m). The resulting maximum EBTS will be different with the larger slab being the most stressed, even though the thermal gradients are the same. This effect is hereby named the 'size effect in EBTS problems'. Back again to the TF tests for tools, two questions arise from the size effect: How do the results from two TF tests compare if they have been performed on the same geometry but different sizes? How does a TF test result compare to the TF of a real industrial tool, knowing that the dimensions are an order of magnitude different? In order to address these two questions, it is necessary to consider the previous remarks on the driving forces and the normalisation of the problem. When one designs a TF test for transient thermal load, one should try to create on the surface of the lab specimen the same driving forces (T max and EBTS σ max ) in the same time t as what could be seen in a real industrial tool.

Let us assume that measurements on the industrial tool and numerical simulations provide the necessary information for the most critical area of the tool. It would be easy to reach the same temperature in the same time as for the tool. It is just a matter of adapting the heating power density (inductive heating, IR lamps, etc.) to heat up the specimen at the right rate and to the same temperature. However, there is no warranty that the EBTS will be the same as in the tool. In order to make it for sure, a proper design of the test has to be performed. We have modelled the EBTS in a hollow cylindrical specimen. The heat conduction and the thermo-elastic set of equations is given below:

1 1 0 T T r r r r t ∂ ∂ ∂ ∂ ∂ κ ∂ ⎛ ⎞ - = ⎜ ⎟ ⎝ ⎠ (7) 2 1 1 2 2 1 2 2 2 2 2 1 ( ) 1 ( , ) ( , ) ( , ) (1 )
(1 ) r R rr R R r R E E r t T r t r dr T r t r dr r R R r α α σ ν υ - ⋅ ⋅ = - ⋅ ⋅ - ⋅ ⋅ - -⋅ - ∫ ∫ (8) 0 2 1 2 2 2 1 2 2 2 2 1 ( , ) ( , ) ( , ) 1 (1 ) ( ) 1 ( , ) (1 ) r R R R E E r t T r t r dr T r t r r R E T r t r dr R R r θθ α α σ υ υ α ν ⋅ = ⋅⋅ -- -⋅ + ⋅ + ⋅ ⋅ - - ∫ ∫ (9)
where R 1 and R 2 are the inner and outer radius of the cylinder, respectively. The thickness Ep is equal to R 2 -R 1 .

In the TF test, usually the outer surface is heated up by the application of LASER or by inductive heating with a step-type of heating power. The inner tube is usually cooled down with a flow of fluid. When normalised with the set of Equations (1 to 6), L being replaced by R 2 for instance and z being replaced by r, the set of EBTS expression transforms s into [START_REF] Dour | Normalized thermal stresses analysis to design a thermal fatigue experiment[END_REF]: Compared to the situation described by LP, the normalised problem has a set of two parameters: the biot number h + characterising the heat transfer with a cooling liquid in the inner tube and the normalised thickness Ep + of the cylinder. Figure 2(a) shows the thermo-mechanical path σ + θθ versus T + for the heated surface for cylinders with various

1 2 2 2 2 1 1 1 (1 ) ( , ) ( , ) ( , ) (
+ + + + + - - ⎡ ⎤ - - = - + ⎢ ⎥ - - ⎢ ⎥ ⎣ ⎦ ∫ ∫ (10) 1 2 2 2 1 2 1 (1 ) ( , ) ( , ) 1 ( , ) (
Ep + and h + . Normalised time appears in the chart in the sense that each dot corresponds to a normalised time increment of ∆t + =0.02. It shows in particular that high biot numbers situation is very different from that with low biot numbers: for large times the low biot numbers converge to a steady EBTS, whereas temperature keeps increasing, which is contrary to a high biot number situation where both stress and temperature tend to converge in the same time to a definite value. Figure 2(b) represents the evolution with time of the ratio between T and EBTS in the normalised form. As expected from the similarity of the curves 2a in the first times, the ratio tends to be 1 for all cases. It is only for longer times that the curves diverge.

The results shown in Figure 2(a) can be used to choose the proper dimensions of the TF test specimen [6]. In order to do so, the following method is proposed:

• Measure or collect information about the material's key parameters (E, α, ν, κ, k).

• It is assumed that the working conditions of the reference tool have been determined.

In particular the maximum EBTS σ max and maximum temperature T max are recorded to occur at the time τ.

• From Equations ( 5) and ( 6), Equation ( 13) is obtained. This equation can be applied for r = R 2 at the surface of the TF specimen (i.e., r + =1) and for the time τ (the same as identified on the reference tool) when the driving forces must be at their peak values. The non-normalised stresses and temperature should then be T max and σ max as identified on the reference tool.

• Identify the ratio T + max σ θθ + max on the chart in Figure 2(b) (see the horizontal line) using the second member of Equation ( 8) with the maximum temperature, maximum stresses, and the materials data.

• At that point it is necessary to choose the normalised thickness Ep + of the tube and the relative heat transfer coefficient h + . Where the chosen curve crosses the horizontal line, the time τ + reads the normalised time that should be used on the lab specimen to reproduce the real time τ on the industrial tool.

• In order to obtain the same time on the lab specimen as on the actual part, the reference length R 2 must satisfy Equation (1), which leads to Equation (15). • From the chart found in Figure 2a, it is now possible to determine T + max and σ θθ + max at the instant τ + .

• The heat flux density that is needed to perform such a TF test is given by Equation ( 16) and the power supply that is needed for a tube of length Z is given by Equation ( 17):

( , ) ( , ) ( , ) 1 ( , ) T r t T r t E r t r t α σ ν σ + + + + + + = - ( 13
) max max max max 1 T T E θθ α σ ν σ + + = - (14) 2 R κ τ τ + = (15) max max 2 max T k R T φ + = (16) 2 m a x 2 . P R Z π φ = (17)
If one follows the method step by step, one will be sure that the two driving forces of the TF damage phenomenon will be the same on a lab specimen as on a real tool. This is the necessary condition if one expects to reproduce the same TF and to find comparative results.

Application to coatings: the thermo-mechanical screening

In order to improve the wear resistance of tools or to prevent them from corroding or to isolate them thermally, it becomes more and more common to cover tools with a coating. We are not going to discuss the efficiency of the coating with regard to the primal function it probably fulfils. The issue that we consider in the following is the effect that coating has on the EBTS. In other words, the question is: does a coating improve or worsen the EBTS build-up and consequently does it affect the TF resistance of a hot forming tool?

In an attempt to understand these effects, we developed precise semianalytical calculations of the EBTS in a coated cylinder representing the die of centrifugal casting [START_REF] Diaconu | A normalised approach to optimize the coating/substrate couples with regards to thermal stresses: conditions for thermo-mechanical screening[END_REF]. The hollow cylindrical die is filled with cast iron in the inner radius, while the die rotates at high speed. The molten metal is then forced into contact with the inner radius of the die and solidifies to take the dimensions and the shape of the inner cylinder. Of course the die is heated up from its inner radius and cooled down by water flow at the outer radius. Figure 3(a) shows the temperature field that should develop during casting for a 4 mm thick steel layer coated on a copper substrate (20 mm thick). Copper is well known to be highly thermally conductive. It also has a higher expansion coefficient, but a lower Young modulus than steel has. One can notice that the highly conductive copper shows very little temperature gradients compared to steel. During the heating process, the temperature gap at the interface between the two materials builds up owing to a heat contact resistance (RTC is taken to be 10 -5 m 2 KW -1 ).

Figure 3(b) shows the resulting hoop EBTS σ θθ through the bi-materials die. At the beginning of the cycle (T uniform at 200°C) the stresses are not null. This is due to the heterogeneous dilatation from 0°C to 200°C of the two materials (0°C being considered here as the temperature of deposition free of stress). The steel (respectively copper) is always in tensile (respectively compressive) stresses at that stage, because it dilates the least (respectively the most). When the thermal loading starts, the coating EBTS tends towards negative values. This means lowering the already tensile stress. The resulting Von Mises stress tends to smaller values at the mating surface, which faces less risk of yielding, and hence of TF failing. However, the interface remains at large stresses. The simulation of other hypothetical combinations of materials showed that extra care should be taken when choosing a material to coat a tool substrate. In order to investigate which combination of thermo-mechanical properties would avoid an overstressed situation, it is necessary to normalise the EBTS equations. As previously done, we chose to normalise the equation with the heat flux density, the inner radius of the shell and the thermo-mechanical properties of the substrate. The main difficulty here is that the two layers have different properties, so that ratios of properties will influence the results. Typically, three more parameters have to be set in the problem: the ratio of Young modulus E 1 /E 2 , one referring to the coating and two to the substrate, of expansion coefficient α 1 /α 2 and of heat conduction properties λ 1 /λ 2 . As an example of our results,

R 1 + = 1.0006, λ 1 / λ 2 = 2
Figure 4 gives the maximal EBTS seen at the surface of the coating in conditions where the thickness of the coated layer and of the tool, the duration τ of the heating and the Biot number for the fluid cooling at the outer radius are fixed. The ratio of conductivity in the example is set to 2 (coating twice more conductive than the tool). What the diagrams tell us is that the sign of the EBTS depends on the value of the expansion coefficient ratio, no matter what the Young modulus ratio is. It appears that above α 1 /α 2 = 0.3 the stress is compressive (see Figure 4b). Below that α-ratio value, the EBTS at the mating surface of the coating is positive (see Figure 4a). It also tells us that the larger the E ratio is (when the coating is much stiffer than the substrate) the larger the EBTS tend to be in absolute value.

At the critical value of the α-ratio, the EBTS is zero whatever E 1 /E 2 is. This is what we call the 'condition of thermo-mechanical screening'; it is the condition in which the combination of materials properties between the coating and the substrate have been genuinely chosen so that the coating can be thermally loaded without any build-up of EBTS. The fact that the zero-stress condition is obtained for the α-ratio at 0.3 means that the coating should have a lower dilation coefficient so that the compression resulting from the thermal gradients in the substrate balances exactly the extensions to follow the dilatation of the substrate. The important result is that the condition for thermo-mechanical screening does not depend on the E-ratio, but the critical value must depend on R 1 + , on t + and on the λ-ratio. Nevertheless a less rigid coating material should be preferred to lower thermal stresses in order to reduce EBTS. It is the opposite in most trials (ceramics, hard coatings, etc.) where stiff materials are usually used for coating.

Conclusion and perspectives

The paper discussed the important issue of the two driving forces responsible for the thermal fatigue of hot-forming tools. From this principle it appears obvious that thermal fatigue tests should reproduce the same driving forces if one intends to compare them together or with the actual industrial tools. From that comes the conclusion that the size effect of thermal stresses problem should be addressed. A method is proposed to warranty a proper design of a TF test. The method uses the normalisation of the variables involved in the thermal stresses problem. In the past, TF tests have been conducted with the same materials, same geometry, same maximum temperature but different heating conditions (different heat flux density applied). The conclusions were that the EBTS and the TF resistance were very different REF, confirming the importance to use the couple (T, EBTS) to characterise the TF resistance of materials. An important perspective of this work is the idea of performing a benchmark of all the TF tests that exist in order to improve comparison between all the tests. The two driving forces could be the base for the analysis. Particularly, thermal fatigue results could be plotted in a diagram accounting for the two driving forces for the same steel tested in different manners.

The same method is employed to determine the condition of thermo-mechanical screening of a coated tool: in a given working condition, a specific ratio of expansion coefficient can be chosen so that one can perfectly balance the thermal stresses in the coating caused by the thermal gradient along with the thermal stresses caused by the mismatch at the coating/tool interface, no matter what the Young modulus may be. A rational choice of coating materials for hot-forming tools subjected to thermal stresses can be applied. Material selection software could be used to help in such an attempt. When doing so with the condition of calculation shown in the paper example, some interesting materials, such as Si 3 N 4 and INVAR, appear because they have expansion coefficient close to 0.33 times that of steels.

  diffusivity κ; the thermo-elastic properties of the slab materials (Young modulus E, Poisson coefficient ν, thermal expansion α) and time t. When writing the non-transient conduction equation coupled with the thermo-elastic balance equation (EBTS problem), LP managed to reduce the set of seven parameters to four non-dimensional numbers: the Biot number m=hL/k; the normalised time t + =κt/L 2 the normalised temperature T + =T/T a and the normalised EBTS σ + =-σ/[αET a /(1-ν)]. Figure 1 shows how the normalised EBTS σ + evolves with the normalised time t + , as a function of the Biot number m. If one has to determine the real EBTS for a given slab in some quenching condition, one has just to determine which curve best describes its problem and then to renormalise the EBTS and time parameters.

Figure 1

 1 Figure 1 Tabulation of EBTS in a slab quenched in a liquid pool

Figure 2

 2 Figure 2 Normalised EBTS in a cylindrical TF test specimen, hoop stress σ θθ +

Figure 3 Figure 4

 34 Figure 3 The temperature and hoop stress fields at different instants for the assembly steel/copper (for colour please see online version)


Paulina Gonzalez

Karolina Bossak

Ewelina Stefaniak

Christelle Hureau

Laurent Raibaut

Wojciech Bal

Peter Faller

N-terminal Cu Binding Motifs Xxx-Zzz-His (ATCUN) and Xxx-His and their derivatives: Chemistry, Biology and Medicinal Applications

Peptides and proteins with N-terminal amino acid sequences NH2-Xxx-His (XH) and NH2-Xxx-Zzz-His (XZH) form well established high affinity Cu II -complexes. Key examples are Asp-Ala-His (in serum albumin) and Gly-His-Lys, the wound healing factor. This opens a straightforward way to add a high affinity Cu II -binding site to almost any peptide or protein, by chemical or recombinant approaches. Thus, these motifs, NH2-Xxx-Zzz-His in particular, have been used to equip peptides and proteins with a multitude of functions based on the redox activity of Cu, including nuclease, protease, glycosidase, or oxygen activation properties, useful in anticancer or antimicrobial drugs. More recent research suggests novel biological functions, mainly based on the redox inertness of Cu II in XZH, like PET imaging (with 64 Cu), chelation therapies (for instance in Alzheimer's disease and other types of neurodegeneration), antioxidant units, Cu transporters and activation of biological functions by strong Cu II binding. This review gives an overview on the chemical properties of Cu-XH and -XZH motifs and discusses the pros and cons for the vast and different biological applications, and how they could be improved depending on the application. Kinetic Stability (of Cu II ) Chemical stability in reducing Intracellular environment Nuclease (DNA/RNA) high high high high Protease high high high high Carbohydratase high high high mostly extracellular Antimicrobial and host-defense peptides high, Cu as ROS producer, or none, Cu structural role high high high (when intracellular activity) Chelator to redox silence (antioxidant) none high high high (when intracellular) Imaging (PET) none high high high (when intracellular) Cu-sensing none medium to high Low to medium high (when intracellular)

Concerning the redox activity of the Cu II / III couple, Cu III can form a stable complex with XZH, without a large rearrangement compared to Cu II . Therefore, the redox based ROS production is potentially very efficient. However, this is limited by the high redox potential (~1V NHE), a value particularly difficult to achieve in the rather reducing environment of the cell.

Introduction

Peptides and proteins with N-terminal amino acid sequences Xxx-His (XH) and Xxx-Zzz-His (XZH) bind Cu II with a high affinity. Adding XH or XZH by chemical or recombinant approaches is a straightforward way to add a high affinity Cu II binding site to almost any peptide or protein. Thus, these motifs have been used to equip peptides and proteins with a multitude of functions. In the present review, we focus on the applications relevant for biology and medicine, which include nuclease, protease, glycosidase, or oxygen activation, properties useful in anticancer or antimicrobial drugs, imaging, chelation, design of antioxidant units and Cu transporters.

2. The two motifs and metal ions they bind.

Short description of the motifs:

The two motifs considered here, H2N-Xxx-His (XH) and H2N-Xxx-Zzz-His (XZH), are N-terminal sequences of peptides and proteins which have a His residue at the second or third position with respect to the free N-terminal amine. Cu II can bind to XH in a tridentate fashion, with three nitrogens from the N-terminal amine (NH2), the first amide (N -) and the imidazole nitrogen at the delta ring position (N δ ) being involved (Figure 1, left). The fourth equatorial binding site is occupied by an external molecule such as water, buffer, etc. In the case of the XZH motif, Cu II binds to four nitrogens: the N-terminal amine, the first two amides and the N δ imidazole nitrogen (Figure 1, right). Such coordination modes are denoted as 3N (NH2; N -; Im) and 4N (NH2; 2×N -; Im), respectively, throughout the text. Their structures are known from several X-ray studies [START_REF] Camerman | [END_REF][2][3] and from the analysis of their spectroscopic properties (Table 1)

Spectroscopic characteristics of 3N and 4N motifs

The Cu II complexes of XH and XZH motifs can be readily identified upon their spectroscopic properties, collected in Table 1. The split pattern of bands in the visible range of CD spectra is particularly characteristic for 4N complexes of XZH peptides. This split is less visible for the XH pattern, where the higher energy negative band is very weak and not always visible.

Significance of copper complexes, and XH and XZH motifs

a) Identity of the amino acid: The Cu II ion exhibits high affinity towards nitrogen donors, and is one of very few metal ions able to deprotonate and coordinate the peptide bond nitrogen. [4][5][6][7] Moreover, binding with the N δ of the His side chain creates a stable 6-membered chelate ring with the preceding amide nitrogen. Such rings are also achievable via Asp and Cys residues. Asp is a hard acid, and therefore it binds Cu II weaker than His. Cys, on the other hand, can form strong bonds similarly to His, but due to its redox reactivity Cu II undergoes reduction to Cu I by the thiol group, which is oxidized to a disulfide, resulting in a motif loss. Other amino acids (Glu, Met, etc.) would form less favored, larger rings. Therefore, in terms of Cu II binding, the His residue provides the strongest bond and the most stable complex. b) Influence of His position on metal binding: With a His residue at position 4 and beyond, the advantage of the formation of successive 5 or 6 chelate-rings cannot occur. Thus the Cu IIbinding becomes much weaker. [7] His at position 1 provides a stable bidentate ligand (N-terminal amine and N δ of His). The coordination sphere can be completed by another His-1 peptide molecule, resulting in a 2:1 4N complex (2 x NH2; 2 × Im), able to provide high affinity binding only at a high concentration. At lower concentrations and ratios ~1:1, XH and XZH motifs bind Cu II much stronger. Notably, His analogues ((4-thiazolyl)-Lalanine, (2-pyridyl)-L-alanine, and (pyrazol-1-yl)-L-alanine) can also form stable 4N motifs with affinities similar to His. [7] c) Significance of Cu II : In order to form Cu-XH and Cu-XZH complexes, one or two amides have to be deprotonated, respectively. Thus, a strong Lewis acid, and a metal ion that favors three or four ligands in an equatorial plane are needed (the latter condition stems from structural properties of peptides composed of α-amino acids). Those requirements are met well by Cu II and somewhat poorer (at higher pH) by N III ions. Pd II , Au III and Co II also bind ATCUN motifs, although for Pd II and Au III the kinetics is slow and those metals are less important biochemically. Co II requires high pH for efficient binding, while Zn II seems not to be able to support the 4N form (at least not at pH below 9), due to its lower Lewis acidity and its preference for tetrahedral geometry. It was reported that Zn II can bind to the 3N form in XH at higher pH. [7][8][9] d) Influence of the neighboring His residues: In principle, these amino acids can be any, but Pro as Zzz. Pro is a secondary amine, hence its nitrogen does not carry a dissociable H that could be replaced by the metal ion, when it participates in the peptide bond. Cys is easily oxidized (2Cu II + 2CysS ---> 2Cu I + CysS-SCys), but the disulfide formation does not preclude its participation in Xxx-His and Xxx-Zzz-His motifs.

2.4.

Relative affinities of the two motifs and its pH dependence:

Comparison of XZH and XH:

Cu(XH) 3N complexes recruit one external ligand to complete equatorial coordination sphere, whereas the Cu(XZH) 4N complexes have it already completed (Figure 1). This produces an affinity gain in Cu(XZH) due to the entropic chelate effect, caused by the additional amide donor. However, the entropic gain by the chelate effect of an additional coordination site is counteracted by the enthalpic cost of deprotonating the amide at pH 7.4. Table 2 presents a selection of conditional stability constants at pH 7.4 ( c K7.4) for both motifs, calculated from the original using the competitivity index (CI) method. [10,11] These data serve to illustrate the general ranges of affinities, K of 10 11.3 -10 13.2 M -1 for 3N XH complexes and 10 12 -10 14.7 M -1 for 4N XZH complexes. Thus, despite some overlap, Cu(XZH) complexes are typically about one order of magnitude stronger at physiological pH than Cu(XH) complexes. Moreover, these affinities are generally lower than the affinities reported for Cu(II)-binding enzymes, such as superoxide dismutase. [12]

The pH dependence:

The metal-binding affinities of the XZH and XH motifs are pH dependent. Increasing the pH increases the affinity, because in order to bind, the metal ion must replace peptide's hydrogen ions. For Cu II this process occurs at pH as low as ~3-4 for XH motifs and at slightly higher pH for XZH, due to the higher number of amides involved. This means that below a certain pH the 3N Cu(XH) complexes become more stable than the 4N Cu(XZH) complexes. The coordination process is very cooperative. The intermediate complexes with the less than maximal number of nitrogen donors are virtually absent for XH and seldom and minor if present for XZH [13] Table 2. Conditional affinity constants for Cu II complexes of XH and XZH motifs, obtained by recalculation of original potentiometric data by the CI method [10,11] or by calorimetric (a) or spectroscopic (b) GGH-COO - GGH 12.4 [21] des-angiotensinogen N-term VIH 13.0 [4] Aβ4-16 RFH 13.5 [22] DAHK-amide DAH 13.8 [4] YYH-COO -YYH 14.4 [23] HP2 N-term RTH 14.5 [13] Endostatin N-term HSH 14.5 [24] Hepcidin N-term DTH 14.7 [25] * this apparent discrepancy may be due to different background salts used, KNO3 in ref. 15 and Cl -in ref. 16, the latter being a possible auxiliary Cu II ligand.

Influence of Xxx/Zzz:

Side chains of amino acids surrounding His residues within XH and XZH motifs have an impact on the Cu II affinity, although they do not directly participate in a metal ion coordination. [6,[26][27][28] For the XH motif, residues located further away from the N-terminus or coming from another molecule can bind to the vacant fourth coordination site, but this case will not be discussed here. A systematic analysis by Kozłowski et al. indicated that amino acid residues at positions 1 and 2 have the significant influence on Cu II coordination of the XZH motif, with partial linear correlations between the pKa values of amine nitrogens of the amino acids and log K of the 4N complex formation. [29] Recently, Miyamoto et al. compared several XZH complexes (X = Z = Asn, Phe, Arg, Lys, Val, Asp, Glu, Tyr, Thr) and found that the pKa of the Nterminal amine is the most determinant factor in this series, conforming this view. [23] The pKa varied from 6.83 (Asn) to 8.18 (Asp). An explanation for Asp having the highest pKa is due to the stabilization of the protonated amine by the H-bond from the carboxylate side chain (formation of a 6-membered ring) (Figure 2A). As Cu II -binding involves the deprotonation of this amine, the Cu II affinity will decrease, since Cu II must compete with the stabilized amine proton. In contrast, Asn could stabilize the deprotonated form by an H-bond, by donating a H-bond from its amide, thus enhancing Cu II binding (Figure 2B). Also, pKa of His residue changed with the sequence (pKa change from 5.19 to 6.64) but this had little correlation with the thermodynamic stability. This seems reasonable as His is already deprotonated at pH 7.4. It is also in line with the small effect of pKa of His and its analogues on the stability of DSUAK-amide pentapeptides derived from histatin-5 (U = His, 4-thiazolyl)-L-alanine, (2pyridyl)-L-alanine, and (pyrazol-1-yl)-L-alanine). [30] Figure As indicated above, [29] the pKa of the coordinating peptide bond nitrogens has a significant effect on Cu II affinity (assuming that the amide pKa is strongly correlated with the amine pKa of the source amino acid). However, due to the high amide pKa values (>15) they are not measurable by experimental titration. [31] Nevertheless, based on the discussion presented above, one may propose that positively charged and/or H-bond donor side chains could interact with the amide carbonyl oxygen, hence decreasing the peptide group pKa (Figure 2C). Thus, side chains like Lys and Arg at positions 1 and 2 should increase the affinity of Cu II . In contrast, negatively charged and H-bond acceptors could interact (via H-bond) with the H-N of the amide bond, increasing its pKa (Figure 2D), and consequently decreasing its affinity for Cu II . For example, Asp is more efficient than Glu in this respect, as the H-bond created forms a 7-membered vs. a 9-membered ring. Moreover, hydrophobic amino acids may increase the stability of complexes of XZH and XH motifs through a hydrophobic fence formation. Although the Cu II complex of Val-Ile-His-Asn is just 4fold stronger than that of Gly-Gly-His, the effect for the structurally related N III complex is 60-fold. [4][5][6]32] However, the increased stability could be partially due to a sequence difference at position 4 (which is absent in GGH). Additionally, aromatic side chains were proposed to increase Cu II complex stability with XZH and XH motifs due to their direct d-π interaction. [27] With all these observations and correlations listed, an inspection of Table 2 reveals a disturbing fact that essentially the same XZH motif yields complexes placed all over the affinity range: the Cu(DAH) complex in HSA is among the weakest known, the same sequence when isolated in a tri-or tetrapeptide provides a 100-fold higher stability, while a similar Cu(DTH) complex (also present in several mammalian albumins) formed by the hepcidin N-terminal hexapeptide is the most stable one known so far. [25] The reasons for this effect remain unknown and there is not enough data to even speculate responsibly in this respect.

Cyclisation:

Kritzer and coworkers [33,34] recently investigated cyclic XZH motifs, by linking the side chain of Lys at position 1 with the Cterminus of His3 via a -peptide bond. For such structure, the Cu II complex formation was recorded at a much higher pH compared to the linear XZH peptide, i.e. with a midpoint near 6.0 vs. 4.5. This indicates that the cyclisation decreases thermodynamic stability, although a kinetic effect cannot be excluded (not studied). Plausibly, perturbation of square planar arrangement within a ring created a conformation unfavorable for equatorial Cu II ion coordination.

Chelate ring size:

Different chelate ring sizes in the XZH motif have been studied in the past, but the impact of the ring size was recently studied more systematically, by introducing β-Ala at different positions. [35,36] The 4N coordination in Cu-XZH consists of three chelate rings (5,5, and 6-membered) as shown in the right-side panel of Figure 2. Thermodynamic stability ranked the natural form (without β-Ala) as a most stable. The general ranking of chelate ring sizes was: 5,5,6 > 6,5,6 (β-Ala in position 1) > 5,6,6 (β-Ala in position 2) ≫ 6,6,6 (β-Ala in both positions).

Kinetic stability

In contrast to the thermodynamic stability, still little is known about the kinetic stability of XH and XZH complexes. Recently, the lifetime in the blood plasma was measured for 64 Cu complexes of XXH tripeptides mentioned above (X = Asn, Phe, Arg, Lys, Val, Asp, Glu, Tyr, Thr). [START_REF] Camerman | [END_REF]23] It was concluded that the kinetic stability depends mostly on the bulkiness and hydrophobicity of the side chain, with YYH being the most stable, followed by VVH. The orders of kinetic and thermodynamic stabilities were significantly different in this series of peptides. This finding raises a barely explored issue about the reaction pathways in formation and decomposition of these complexes, casting a shadow on the general assumption that the kon rates are similarly high, near the diffusion limit, and the stability of Cu II peptide complexes is controlled by koff rates. [29] On the other hand, we measured the rate of Cu II transfer from HSA to the hepcidin N-terminal hexapeptide which supports the nearly diffusion-controlled formation of the Cu(HSA) complex. [25] NMR studies compared the self-exchange of Cu II ions bound to canonic XZH and XH peptides, DAHK and GHK. The Cu II exchange from Cu(DAHK) to DAHK (at a substoichiometic Cu:DAHK ratio) was slower than the NMR time scale, i.e. of the order of minutes or slower. In contrast, substoichiometric Cu II was exchanged between GHK molecules very rapidly, i.e. within ms or faster. The amide (de)protonation can be quite slow, [37] thus the Cu II binding and release from a peptide is very dependent on the number of amides involved in coordination, stipulating a higher kinetic stability for XZH peptides. The incomplete coordination sphere of GHK, and its ability to form self-ternary GHK-Cu-GHK complexes under substoichiometric Cu:GHK ratios was suggested to command rate exchange acceleration. [2] This clearly shows that although the thermodynamic affinities of Cu II complexes of XZH and XH motifs are quite similar at pH 7.4, the complexes exhibit divergent kinetic features.

Redox chemistry 3.1 Electrochemical studies

Cu II -XH and Cu II -XZH exhibit very different properties in cyclic voltammetry (CV), mirroring the different Cu II coordination site in the two kinds of peptides. Cu II -XZH can be electrochemically oxidized to Cu III with anodic potential ranging from 0.87 to 1.07 V vs. NHE depending on the nature of X and Z. The reversibility is also dependent on the nature of the X and Z and of other amino-acid residues present upstream of the ATCUN motif. [2,22,34,36,38- 40] Cu-XZH could not be electrochemically reduced down to -1.0 V vs. NHE. [2] In contrast, Cu II -XH complexes cannot be oxidized up to 1.2 V vs. NHE, but can be reduced at a cathodic potential ~ -0.2 V vs. NHE. [2,41] The reduction is electrochemically irreversible leading to two anodic peaks on the reverse scan at ~ 0.1 and 0.3 V. vs. NHE, corresponding to oxidation of uncoordinated Cu I and of Cu I bound to two His residues, respectively. In the presence of more than one His or with an exogenous imidazole source, the first cathodic peak is no more observed. The electrochemical properties of both systems are summarized in Figure 3, where they are compared with the chemical redox properties of both system.

Chemical redox reactivity

Most of the non-electrochemical redox reactivity studies of Cu II i) Cu-XZH: In line with the redox potentials evaluated by electrochemistry, the addition of excess of ascorbate is not able to diminish the intensity of the d-d band of Cu II -DAHK complex , [42] supporting that ascorbate is not able to reduce Cu II in DAHK effectively. The same was observed for the complex of peptide Aβ4-16 containing the FRH motif. [43] This agrees with most of the reported studies showing that Cu-XZH complex are not able to catalyze the oxidation of ascorbate efficiently. This is confirmed by the measurements of the HO° production by Cu II -XZH in the presence of ascorbate/O2: compared to Cu II in buffer, or Cu-peptide complexes like Cu II -Aβ1-16 or Cu II -α-synuclein, the HO° production by Cu II -XZH was virtually absent. [2,22,39,42,[44][45][46] However, introducing a His-His motif nearby the ATCUN site can help Cu II -reduction by ascorbate, via coordination and stabilization of Cu I in the His-His motif. [47][48][49] Specifically, in the N-terminal peptide of the Ctr1 membrane copper receptor, containing an XZH motif followed by a HH couple (MDHSHHMG…), the addition of stoichiometric amounts of ascorbate resulted in a partial reduction of the d-d band intensity. This effect was assigned to the HH motif, which is able to bind Cu I , and hence to stabilize it after reduction. [43,[50][51][52] Thus ascorbate is able to reduce the Cu II -XZH only very slowly and hence it is not clear if the Cu II can be reduced in the tight 4N coordination, or if a very low populated, different Cu IIcoordination sphere (like Cu with one more ligand(s) uncoordinated) undergoes the reduction. Also Cu II bound to human serum albumin (HSA), bearing the DAHK motif could be slowly and only partially reduced by ascorbate. [42] The reason why HSA acts differently from DAHK is not known, but indeed might be due to a presence of Cu I stabilizing coordination site elsewhere in the HSA molecule, analogously to the mechanism postulated for the Ctr1 peptide.

Cowan and coworkers analyzed several Cu II -complexes (including XZH) for their ascorbate oxidation capacity in the presence of O2 or H2O2. [53] They found that only Cu-complexes with reduction potentials between -250 and +500 mV (NHE) were able to oxidize ascorbate efficiently. Cu II -XZH complexes do not fall into this range (having their potentials between 0.87 -1.07 V, see above), and hence ascorbate oxidation was slow and could only be observed in presence of H2O2. This correlates with previous results by Margerum and coworkers, [54] and more recent results on the N-terminal peptide of the Ctr1 membrane copper receptor. [47] It was proposed that Cu-XZH induces the formation of alkene peptides via an intermediate Cu III species, the nature of which is not known yet. However, even when H2O2 was added, the ascorbate oxidation was slow compared to other Cu-peptides of certain inorganic Cu complexes (see above).

ii) Cu-XH: this motif has been less investigated compared to the XZH one. Tested Cu-XH complexes were not able to perform ascorbate oxidation and associated HO° production efficiently. [2,46] Although the reduction to Cu I is accessible as shown in the electrochemistry, Cu I will bind differently to XH (does not bind to amidates) and with a much lower affinity. A multitude of proteins and peptides have the N-terminal motif XZH. This includes serum albumins of humans and most mammals, salivary histatins, Ctr1, neurokinin B, [55] hepcidin, [56,57] sperm protamine 2 of humans and several other mammals, [13] and others. However, in humans only serum albumin (HSA) clearly binds Cu II under physiological conditions. For other proteins and peptides, evidence for in vivo Cu II -binding is mostly indirect. HSA is the most abundant protein in the blood, present at a concentration of about 0.6 mM (40 mg/ml). Only a minor fraction, about 0.5-1%, is loaded with Cu II . Whether other peptides can bind Cu II in vivo is still not clear, requiring further research. Factors to consider include: i) Cu II -binding affinity and concentration: the higher, the more likely. If the peptide/protein is present/exposed to the blood, they are in a Cu II -binding competition with the highly abundant HSA (containing the DAH motif). ii) Localization: intracellular Cu is mostly in the reduced Cu I state, thus Cu II binding is more likely to occur extracellularly, or in more oxidative compartments, as a result kinetic stability may be more important than thermodynamic stability for intracellular Cu II binding.

iii) Cu II availability: It is also possible that XH or XZH motifs do not bind Cu II under regular conditions, but do that when Cu II concentrations increase, like due to Cu-intoxication, Cumisregulation etc.

Cu-XH

As for XZH, XH occurs in a multitude of proteins/peptides, e.g. in the Immunosuppressant Protein from tick, [58] and in Saccharomyces cerevisiae mating α-factor. [59] When or whether they bind Cu II in vivo is not certain. The most prominent peptide of this class is GHK, present in blood plasma at submicromolar concentrations. Indication that GHK binds Cu in vivo comes from reports stating that GHK is isolated at least partially as a Cu II complex and that Cu II is required for its biological activity. [60,61] Likewise, α-factor, which has a WHW N-terminal sequence, was co-isolated from cell culture media as Cu II complex, [59] and its Cu II affinity indicates that its Cu II complexed form may be the actual biological species. [17]

Applications

XZH and XH motifs have been used for a vast variety of applications. Here, we focus on the relevant biological and medicinal applications, which are the most abundant ones. For other applications, particularly regarding very intriguing nonbiological catalytic properties see e.g. [62] or [63] for similar motifs.

Catalytic activity: cleavage of biomolecules: DNA, RNA, proteins and sugars

Studies on catalytic and stoichiometric reactivity of XZH and XH complexes have been largely inspired by biology. Initial research was focused on potential mechanisms of metal toxicity, with particular attention turned to oxidative DNA damage catalyzed by redox active central metal ions. [64] Quite naturally, the flip side of this research was testing the application of this newly discovered reactivity to create artificial nucleases. More recent research is largely inspired by attempts to develop novel metal bearing antimicrobial peptides (AMPs) and artificial proteases.

DNA and RNA cleavage by ATCUN complexes

A remarkable 1983 investigation by Pauling and coworkers [65] of antitumor activity of the Cu II -GGH complex against Ehrlich ascites tumor cells [65] raised interest in ATCUN motifs as DNA cleavage agents with a therapeutic potential. Since then several groups have been working towards a better understanding of Cu II -XZH nuclease activity, often accompanied by parallel studies of Ni II complexes. [66] In general, metal complexes with oligopeptides are attractive candidates for metallodrugs because they are easy to synthesize in high quantities and can be easily tuned towards specific DNA fragments by ATCUN amino acid substitutions or addition of DNA recognition motifs.

Fragments of proteins or peptides that were already known to exhibit specific DNA binding abilities, such as Hin recombinase, [67] leucin zipper Fos, [68,69] histatin-5, [70] or Buforin II [71,72] have been applied in such a role. Non-protein modifiers or scaffolds can be also used as nucleic acid recognition motifs. Literature includes bis-PNA [73] and acridine [74] as examples. Long et al. proposed a "tandem array" with GGH motifs incorporated within repetitive linear sequences through the Gly-to-δ-Orn substitution. [75] Neupane et al. [34,76] embedded an ATCUN motif inside a peptidic macrocycle created by coupling Lys and Asp side chains together. Such tight structure exhibited more efficient DNA cleavage than its linear counterpart. [34] The conjugates of Cu II -XZH complexes exhibiting nuclease activity are listed in Table 3. KD-D-His Leu Macrocyclic complex [34] Even though the other part of such conjugate may provide a crucial selectivity in DNA recognition, the ATCUN motif is essential for the nuclease activity. It was proposed that the complexed metal ion is a redox center capable of formation of metal-bound hydroxyl radicals that act as intermediate reactive species, causing the H-atom abstraction from the C4 of the 2deoxyribose, which ultimately leads to a DNA strand break. [38,80] Although historically the Ni(GGH) complex was the first one studied as a potential nuclease, [67,80] most recent studies focused on Cu II complexes. Jin and Cowan proposed that the redox activity of Cu II /Cu III coordinated to the XZH motif under physiological conditions could generate reactive oxygen species (ROS) that contribute to the localized DNA damage. [81] This reaction required ascorbate in addition to molecular oxygen to proceed and a Fenton-like mechanism was proposed for it. Amino acid replacement, including the usage of D-amino acids in the X and Z positions of the motif improved the binding and recognition of the DNA minor groove (usually within A/T rich regions), and eventually the nuclease activity. [72] Cu II -XZH complexes generated not only single, but also double-strand DNA breaks. This activity was attributed to a long residency time at DNA and a diffusible nature of ROS generated by the complex. [38,72] Cleavage of RNA was studied mostly for the genetic material of Human Immunodeficiency (HIV) [45,[81][82][83] and hepatitis C (HCV) viruses, [33,84] and yeast tRNAPhe. [85,86] Cu II (GGH) and Cu II (KGHK) motifs conjugated with Rev1 and Rev2 domains were demonstrated to target their cognate Rev Response Element (RRE) in HIV virus RNA in the presence of ascorbate. The site-selectivity rather than diffused cleavage was attributed to the formation of copper-bound non-diffusible ROS. [82] The activity was preserved under physiological (cellular) conditions in E. Coli and mammalian Jurkat cells. Both the ATCUN and the Rev motifs were necessary for the activity. [81] Later studies expanded the knowledge concerning the cleavage chemistry showing: (i) irreversibility of RRE RNA cleavage, (ii) possibility of regeneration of reduced Cu II -ATCUN-Rev, and (iii) identification of cleavage products attributable to the catalyst-mediated oxidative hydrogen abstraction. [45,83] The cleavage of HCV RNA was achieved by adopting an analogous strategy, using the Cu II complex of GGH as the cleaving unit and assuring RNA sequence recognition by specific peptides identified in separate studies. The IC50 of these complexes in an HCV cellular replicon assay was in the ~1 μM range, yielding promise for further development as anti HCV drugs. [33,84] The Cu(KGHK) complex was highly selective towards isolated tRNAPhe, cleaving it within D and TψC loops [86] with a higher specificity than N III -XZH nucleases, tested previously against the same target. [85] Both DNA and RNA nuclease activities require further studies since many DNA cleaving agents work in vitro, but not under physiological conditions. The underlying redox activity is quite low, due to the high stability of the Cu II complexes. Moreover, there are ubiquitous natural competitors for Cu II ions and reducing agents (such as GSH), that could potentially "deactivate" metallopeptidic nuclease by reducing and/or pulling the metal out of the XZH complex. Therefore, the design of such molecules should focus also on the complex kinetic stability in order to reach the target DNA cleavage site in an active form.

Protein cleavage by ATCUN complexes

Cowan et al. used the concept of XZH motif attached at the Nterminus of a ligand-binding domain, presented above in the context of nuclease activity, to design artificial proteases. Their first study reported inactivation of human angiotensin converting enzyme (ACE, peptidyl dipeptidase A) by the Cu(KGHK) + motif, with redox cycling provided by ascorbate. The observed rate constant kobs of 2.9 ± 0.5 × 10 -2 min -1 (t1/2 ~24 min.). This was much slower than compared to the native enzyme ascorbate oxidase with a rate of 1.7 10 4 M -1 s -1 . [START_REF] Kroneck | Advances in Chemistry, Ascorbic Acid:Chemistry, Metabolism, and Uses[END_REF] Thus, further development of this novel class of catalytic enzyme inactivators is warranted. [68] Interestingly, the Cu II and Co III (NH3)2 complexes of KGHK inhibited the enzyme ACE competitively in the absence of the redox agent (ascorbate). In a follow-up study the same authors tested several XZH motifs to select the one best suited for inactivation of rabbit angiotensin-converting enzyme (rACE) and human endothelin-converting enzyme (hECE-1). These results are proposed as a novel strategy for the development of antihypertension agents. [69] Analogous constructs have also been tested as irreversible inactivators of activity of SrtA, a transpeptidase that anchors surface adherence proteins to the bacterial cell wall, thus being a target for novel antibiotics. [65] The GGH motif was thus linked to amidated peptides LPET and LPETG, cell wall sorting signals which act as sortase-targeting moieties. The LC-MS/MS analysis showed oxidation of Cys-184 and Arg-197 residues at the SrtA active site as the mechanism of inactivation. This result provides a link to section 4.2, dealing with peptides designed to combat pathogenic microorganisms.

Sugar cleavage by ATCUN complexes

A recent study by Yu et. al proposed a novel artificial fucosidase (Cu-GGH-tOL), in the form of a conjugate of the Cu II -GGH complex with a truncated form of odorranalectin (tOL: KCFRYPNGVLACT) to produce artificial Bombay phenotype blood (lacking the functional H2 antigen, also known as Oh, or h/h). Their study showed that such conjugate cleaves L-fucose more efficiently than D-glucose. Therefore Cu-GGH-tOL can act as a potential agent that could specifically regress the polysaccharide Fucα1-2Galβ1-4GlcNAc, a residue of regular glycolipids and glycoproteins present on the surface of erythrocytes, into a disaccharide Galβ1-4GlcNAc, thus forming a H2 antigen depleted analogue present in a Bombay phenotype blood. Cleavage was observed upon addition of mild reducing agents (ascorbate or hydrogen peroxide) that are natural agents under physiological conditions. The importance of this research is driven by the rarity of such blood phenotype and its limited supply in a blood bank reserves. [74]

Combination with antimicrobial and antifungal peptides

The inspection of Antimicrobial Peptide Database (APD) revealed the presence of nearly 50 antimicrobial peptides (AMP) that contain the N-terminal XZH motif within a total of 2813 AMPs. Only a few example (<5) of AMP containing the Nterminal XH metal-binding motif were listed. Copper has been long known for its antimicrobial activity. Indeed, a few cases of an increase in activity of AMPs with a native XZH motif (such as hepcidin or ixosin [START_REF] Libardo | [END_REF] ) by addition of Cu II have been reported towards several microorganisms. [71] Although the mechanisms of activity enhancement by copper have not been well understood, it has been suggested to include Cu-catalyzed ROS production leading to DNA cleavage [89] or other oxidative degradation of biomolecules, like phospholipids. [START_REF] Libardo | [END_REF] Based on these results, XZH motifs were also grafted to short AMPs (such as anoplin or buforin), which act by interacting with the cell membrane [71] or inhibiting intracellular targets. [90] This can be obtained by elongation of the AMP N-terminus with the XZH sequence or by mutating the 3 rd amino acid to His. N-terminal elongation by XZH of the AMP anoplin, pro-apoptotic peptide, and sh-buforin showed an about 2-8 fold increase in activity (without coordinated Cu II ), but the pre-formed Cu II -XZH-AMP complexes did not show any increase in activity compared to XZH-AMP. [71] Very recently, the impact of Cu-binding to histatin-5, a salivary peptide with a XZH motif, on the fungus Candida albicans was tested. A five-fold decrease of the EC50 from ~5 to ~1 µM was obtained by the Cu-histatin 5 compared to the apo-form. The presence of a bis-His site (two adjacent His) was crucial for the activity and underlined the importance of a Cu I -binding site (different from XZH) for the increased activity. [49]

XZH and XH as chelators to remove and/or redoxsilence Cu

Based on the high stability of Cu II (XZH) complexes towards oxidation and reduction described above, the motif has been used to redox silence copper. In aqueous solutions Cu II is a very efficient catalyst of ascorbate oxidation in the presence of dioxygen and hence produces efficiently ROS like H2O2 and HO°. Thus, addition of peptides bearing XZH motifs abolished almost completely this ROS production by Cu. An early example of such concept is provided by quenching of deleterious oxidative chemistry of the Cu II /H2O2 couple by the N-terminus of human protamine 2 (HP2). Protamines are peptides replacing histones in sperm cells. Notably, impairment of HP2 production is one of major causes of male infertility in humans. [13,91,92] A similar protection against Cu catalyzed ROS production was shown against the complex of Cu with the Aβ1-x peptide. Aβ1-x forms aggregates containing a high Cu content in Alzheimer's disease brains and can quite efficiently catalyze the production of ROS. Addition of XZH to Cu(Aβ1-x) resulted in a Cu transfer from Aβ1-x to XZH and a concomitant abolishment of ROS production and related cell toxicity. [2,42,93,94] Moreover, as such, XZH was also able to suppress Cu-induced Aβ1-x aggregation. It is tempting to speculate that protection from copper toxicity is a biological function of some endogenous XZH motifs for which no other function has been found. Following the idea, Franz [48] and co-workers designed a prochelator peptide that gets activated enzymatically. The betasecretase enzyme cleaves the pro-chelator in a designed site, yielding the XZH motif at the site of Aβ production and a supposed Cu-induced oxidative stress. On the other hand, a strong critique of the chelation approach to treat Alzheimer's disease was presented recently. [95]

Cu II -XZH for biological imaging

Miyamoto et al. [23] reported the imaging of tumor-bearing mouse by using the N-terminal sequence YYH attached to the peptide octreotide, a somatostatin mimic that binds to the somatostatin receptor. The latter is expressed at relatively high levels in many tumors. The YYH-Octreotide was labelled with 64 Cu, an isotope emitting a positon, and hence useful for positon emission tomography (PET). The YYH motif was selected based on its highest kinetic stability of the tested XXH peptides. Further studies are needed to see if the kinetic stability of XZH complexes can be further exploited to find a lead for medicinal applications.

Cu-sensing

In the past two decades, different sensors have been developed to serve as tools to study the homeostasis and transport of metals in biology. Different strategies have been used but the most common involve the use of fluorescence. Generally, a "turn-on" switch is favorable, in other words, the fluorophore would not be active until it binds the desired metal ion. Unfortunately, this is very difficult to reach for Cu II , due to its paramagnetic character resulting in a universal fluorescence quenching. Thus, most of the sensors reported in the literature have a "turn-off" character. One of the first sensors that used an XZH motif for selective Cu II sensing was designed by Torrado et.al, [96] constituting a series of pentapeptides based on the XZH motif with the introduced a Dns fluorophore (5-(dimethylamino)naphthalene-1-sulfonamide) at the X position.

Cu II binding resulted in a strong and selective fluorescence quenching. The selectivity of this sensor was assured by the Cu II ability to deprotonate peptide nitrogens, and the lack of quenching by the isostructural, but diamagnetic N III complex.

Using the same premise Jung and Chung [97] designed a Cu II selective reporter protein using the Gly-Gly-His sequence added next to the fluorophore in the green fluorescent protein (GFP).

The GFP fluorescence was quenched by about 85% upon the Cu II binding, thus providing live imaging of Cu II fluctuations on the surface of HeLa cells. Utilizing the backbone of Imperiali and co-workers, Kulak group took the compound R-NH2-Dap-β-Ala-His-Ser-Ser-CONH2 and attached different fluorophores (R= fluorophore = Rhodamine B, dansyl chloride, fluorescein isothiocyanate) to it . [98] They were able to show that their analogs cleaved DNA efficiently with a minimal concentration of 25 µM, but only in the presence of a reducing agent like ascorbic acid. The attached fluorophore allowed them to monitor the reduction of Cu II to Cu I , via the fluorescence turn-on, as Cu I is diamagnetic and hence does not quench the fluorophore like Cu II does.

Conclusions

The [99] ii) High stability and redox inertness of Cu II -XZH complexes empowers usage of XZH peptides to redox silence loosely bound Cu II , and to image and sense Cu II . These two categories are somehow contradictory, as on one side a highly efficient ROS production is warranted and on the other hand is the lowest possible. It seems that variations in the sequence can modulate the redox activity, however, these changes impact only the second coordination sphere and hence are limited. The XZH motif is very well suited for a stable Cu II coordination, but is a weak ligand for Cu I . Thus, the Cu I / II redox activity is quite inefficient and a risk exists that during the redox cycle the Cu I is released form the peptide. This is particularly the case intracellularly, where strong Cu I chelators are present. The motif XH has been much less studied. A reason is that Cu-XH is generally less stable, particularly kinetically, but at pH 7.4 (and higher pH) also thermodynamically. Moreover, from the electrochemical studies, reduction of Cu II -GHK seems accessible by physiological reductants (like superoxide, NADH etc.). This might seem to be of interest for catalytically activity, however, Cu I binds very weakly to XH and hence such a Cu I -XH complex would not be stable in a biological medium due to the presence of quite strong Cu I ligands (like glutathione, methionine etc.).

Outlook

As pointed out, there are two different general approaches for the use of XH and XZH, one based on the redox inertness and the other on the redox cycling. Gathering all the results discussed above, it seems that these motifs are rather prone to redox inertness, including the application like detoxification, redox silencing, PET etc. For such application the motif XZH is more suited than XH, due to its higher thermodynamic and kinetic stability. It would be worthwhile to explore variants of XZH to improve both stabilities. There are a lot of thermodynamic binding constants known for XZH, but as discussed above there is no broad rationalization reported that would explain the differences and allow predictions of affinity constants. Up to now, the conditional Kd values at pH 7.4 span from log 12 -14.7. There might be room to increase the affinities, although likely limited, because the first equatorial coordination sphere cannot be changed or expanded. Nonetheless, rationalization of the relation of sequence with Kd seems a primordial aim for the future.

In contrast to the thermodynamic stability, little work has been done on the kinetic inertness. This seems to be another important question to tackle in the future. For PET imaging and redox silencing kinetic stability is warranted, however for Cu II sensing, faster kinetic might be of interest as well, hence making the XH motif of potential high interest.

For applications based on the stability and inertness of Cu-XZH, it is very important not only to explore stability against a competing chelator, but also the redox activity. As the Cu II /Cu III potential is very high and Cu III binds strongly to XZH, the key point is the Cu II /Cu I couple. Cu I cannot bind in the same coordination as Cu II in XZH, and Cu I binds only weakly to XZH. Thus it is very likely that once Cu II is reduced to Cu I , it will very rapidly be bound by other biomolecules in vivo and the Cu-XZH motif will be lost. Indeed very recently Santoro et al. showed that glutathione and cysteine can pull out Cu II from Cu-Aβ4-16 (with XZH) via a reduction to Cu I followed by Cu I transfer to metallothionein (an abundant Zn II and Cu I -binding protein). [100] This reaction is a potential disrupter for all Cu-XH/XZH, in particular intracellularly where mM concentrations of glutathione are present. This type of reactions are defined so far and merit a more detailed analysis.

In potential application based on the redox reactions of Cu II -XH/XZH, mainly cleavage of biomolecules (DNA, RNA, proteins etc.) it is still not very clear which redox couple of Cu is involved, although the Cu I / II is generally favored. The Cu II / III couple has a redox potential above the range for efficient ascorbate oxidation, but has an advantage of retaining the coordination sphere. Thus, the reorganization energy is low and hence redox activity could be very efficient. For this reason, an aim would be to try to reduce the redox potential, by exploring various X, Z and R in the XZHR sequence. However, as this influences mainly the secondary coordination sphere of a quite rigid first equatorial sphere, it might be not possible to lower the redox potential enough. One should also consider that the electrochemical determination of the redox potential does not include the interaction of the substrates (such as O2 and ascorbate) and their binding could change the redox activity. The other relevant redox couple is Cu I / II , which is generally more available in a biological medium due to the presence of quite strong reducing agents. Here the inherent problem is that the coordination chemistry is very different for Cu I and Cu II , and XH/XZH is well suited for Cu II , but not for Cu I . Two problems are involved here: i) Cu I binds very differently to XH/XZH (does not bind amidate) and hence a large reorganization has to occur, meaning that the redox reaction in not efficient. Indeed this is what is observed. This should be somehow improved. A step into this direction is the presence of a Cu I binding site nearby, such as a His-His (see above). However, still the reorganization is quite important and it is questionable if this problem can be efficiently solved in the framework of XZH alone.

ii) The Cu I affinity of XH/XZH is quite low. Thus there is a high risk that upon reduction of Cu II -XH/XZH, the Cu I is fast chelated by biomolecules. This is clearly expected as discussed above, and could only be avoided if the Cu I is reoxidized faster than the complexation of Cu I by a competing biomolecule. This is particular relevant for all intracellular application such as cleavage of DNA, RNA and cytosolic proteins, due to the high concentration of glutathione and other strong reducing agents (Table 4). Hence we consider it as outmost important that for such application their activity in the presence of glutathione is determined and considered as a standard experiment. Apart from the here discussed improvements and understanding of the motif, concerning the already known application, one can also expect new applications in the future. Most of the work has been done with XZH, which seems to be the most suitable motif for application which needs high thermodynamic and kinetic stabilities (Table 4). However, the motif XH deserves more attention, not only for applications that would need more labile complexation, but also because Cu-XH can form ternary complexes that could change its chemical properties. So far the cleavage of biomolecules has not been applied to lipids. Another interesting and so far unexplored application would be Cutransporters. So far only solidly confirmed function of Cu-XZH in humans is Cu transport by serum albumin. Hence the motif could be useful for Cu-transporting application concerning distortions in Cu-metabolism such as Wilson's or Menkes disease. [101,102] Moreover, XH/XZH bind also other metals including medicinally relevant ones, such as Au III , Pt II , Pd II that might also expand the applications.

Figure 1 :

 1 Figure 1: Scheme of the equatorial Cu II -coordination of the two motifs, Xxx-His (left) and Xxx-Zzz-His (right). L depicts an external ligand, could be solvent or other ligand.

Figure 3 . 4 .

 34 Figure 3. Scheme of the electrochemical (blue arrow) and chemical (orange arrow) redox properties of Cu-XZH and Cu-XH.3. Occurrence and Possible Roles in Biology

Table 1 .

 1 Spectroscopic features of Cu II -XH and Cu II -XZH complexes

		Absorption	CD			EPR
	Motif	d-d transition d-d transition	Charge transfer	gǁ	g	Aǁ (MHz)
	XH	~ 600 nm	~590 nm (+) ~490 nm (-)*	~300 nm, ~330 nm	~ 2.22	~ 2.05	~ 560 [1.8 x 10 -2 cm -1 ]
	XZH	~520 nm	~480 nm (+) ~560 nm (-)	~310 nm	~ 2.19	~ 2.04	~ 600 [2.0 x 10 -2 cm -1 ]
	* weak band, not always discernible

  titration.

	peptide/protein	Cu II binding sequence	log c K7.4	ref. original data	to
	XH
	GH-COO -	GH	12.3 11.3	[14][15]*
	RHA-COO -	RH	12.6	[16]
	α-factor model	WH	12.8	[17]
	GHK-COO -	GH	12.7 13.2 a	[18][19]
	XZH
	HSA	DAH	12.0 b	[20]

  -XH and Cu II -XZH complexes concern reactions with ascorbate and/or O2 or H2O2 as substrates. The main driving force for these studies was the idea to induce cleavage of DNA and other biomolecules by Cu II -XZH moieties, as described below. As indicated by CV studies, Cu II -XZH can support the Cu II /Cu III , but not the Cu I /Cu II couple, and conversely, the Cu II /Cu III couple is not accessible for Cu II -XH, but the Cu II /Cu I couple is.

Table 3 .

 3 List of conjugates of CuII-XZH complexes exhibiting nuclease activity

	ATCUN	Conjugate sequence	Comment

  XH and XZH motifs have been studied concerning their metal-ion coordination chemistry, on the one hand due to the potential physiological role and on the other hand for different applications, e.g. in material science, catalysis, biology and medicine of complexes with Cu II , N II I, Co II , Au III , and Pd II ions). For medicinal purposes, Cu II -XH/XZH complexes have been studied most avidly, because out of all metals listed above, copper is the only essential metal ion in humans potentially able to bind (Co II is present only in cobalamin). A very convenient advantage of these two motifs is that they are very small (2 or 3 amino acids) and can be very easily added by either recombinant methods or chemical synthesis to any wanted peptide or protein.The ability of XH and XZH peptides to bind Cu II has gained versatile interest in the literature, regarding mainly XZH peptides. The applications can be divided into two categories (Table4). i) Cu II -XZH complexes are used to produce ROS to cleave biological targets such as proteins, DNA, RNA, lipids or sugars. The efficiency of the cleavage is based on the redox-reactivity of the Cu II -XZH complex, but whether it involves CuI/Cu II or Cu II /Cu III couple is not clear. (In a study of Cu II in solution and H2O2 , the redox couple Cu I /Cu III was suggested)

Table 4 .

 4 List of applications of Cu II -XZH/XH applications and their chemical properties

	Application	Redox activity	Thermodynami
			c stability (of Cu II )

Acknowledgements

This work was sponsored by University of Strasbourg Institute for Advanced Study (USIAS), Strasbourg, France (P.F. and C.H.), installation grant of the Frontier Research in Chemistry Foundation, Strasbourg (P.F.), the ERC StG-638712 (C.H.), and the National Science Centre of Poland grant Nos. 2013/09/B/ST5/03398 and 2016/23/B/ST5/02253 (W.B.)