T Rajkowski
email: trajkowski@3d-plus.com

F Saigné

V Pouget

F Wrobel

A Touboul

J Boch

P Kohler

P Dubus

P X Wang

Abstract-Power cycling of the Point-of-Load converter is observed during system level heavy ions tests. This event has low cross section and is observed for reduced supply voltage of device under test. Laser tests are used to reproduce this effect and show that it might be due to propagation of single event transients from the voltage reference to operational amplifier being part of the undervoltage protection circuit. Laser tests show that propagating transients are the ones with high enough positive peak and insignificant negative peak value whereas some transients with bigger maximum and/or peak to peak value do not propagate. SPICE simulation shows that in operational amplifier with low voltage difference between V+ and V-, there is difference in propagation of unipolar and bipolar transients from input to output of the amplifier. Analysis of the voltage controlled current source in the amplifier explains also difference in propagation of bipolar transients with negative peak followed by positive peak and with positive peak followed by negative peak.

Index Terms-single event transients, laser tests, heavy ions tests, transient propagation, system-level tests

I. INTRODUCTION

Single event transient (SET) generation and propagation in analog systems is a known problem, of a special concern in power supply components and systems. SETs on output of power supply may lead to damage or intermittent work of the device that is supplied [START_REF] Swonger | Radiation-Hardened Point-of-Load Buck Regulators for Space Applications[END_REF]. SET in the internal circuitry of power supply may lead to e.g. unexpected power shutdowns [START_REF] Johnston | Single-Event Transients in Voltage Regulators[END_REF].

The goal of this work is to investigate a phenomenon observed during heavy ion irradiation of a Point of Load (PoL) converter. We have shown in this paper that the effect observed is the power cycling of the device under test (DUT), induced by a SET. Analysis of the PoL converter schematics leads to the conclusion that the most probable source of the power cycling is due to triggering of UnderVoltage Protection (UVP) circuit. Complementary laser tests were then performed on voltage reference and main part of the UVP trigger circuit (operational amplifier, OpAmp), to understand the processes at play.

Using laser tests, we have shown that propagation of SET from voltage reference to OpAmp output is possible and it may lead to power cycling of the DUT in specific supply conditions. The worst case SET observed in our system is not the one with maximum amplitude, as is commonly described in the literature [START_REF] Buchner | Pulsed-laser testing for single-event effects investigations[END_REF]- [START_REF] Marec | Toward a single event transient hardness assurance methodology[END_REF]. In our results, SET with high enough positive peak and insignificant negative peak value are shown to be the only able to propagate up to the output of the circuit.

We then have used SPICE simulation to explain how changing voltage difference on inputs of OpAmp enable propagation of transients observed during laser tests. It also explains processes inside OpAmp chip leading to propagation or no propagation of different types of SET during laser tests and heavy ions tests.

II. EXPERIMENT DESCRIPTION

A. Device under test

DUT is the prototype board of PoL converter, electrically and functionally equivalent with 3DPM-0024 module from 3D-Plus. Block diagram of DUT is presented on Fig 1 . Main focus of this work was put on analysis of effects inside the UVP circuit and the reference voltage chip. The UVP circuit (Fig. 2) is based on OpAmp chip (IC2). Supply voltage (VIN) of the DUT is divided by voltage divider (R1 and R2) and connected to the non-inverting input of OpAmp. Reference voltage from VRef (IC1) is directly connected to the inverting input of OpAmp. In nominal operating conditions, the voltage value at non-inverting input of OpAmp is above VRef (and OpAmp output is driven high).

B. Heavy ion tests at CHARM facility

The DUT was tested with high energy (5.4 GeV/n), low Linear Energy Transfer (LET) (8.0 MeV•cm 2 /mg) and high flux Analysis of SET propagation in a system in package point of load converter T. Rajkowski, F. Saigné, V. Pouget, F. Wrobel, A. Touboul, J. Boch, P. Kohler, P. Dubus and P. X. Wang (~5x10 4 ions/cm 2 /s) Pb ions at CHARM facility (CERN) [START_REF] García Alía | Ultraenergetic heavy-ion beams in the CERN accelerator complex for radiation effects testing[END_REF] [START_REF] Fernández-Martínez | SEE Tests with Ultra Energetic Xe Ion Beam in the CHARM Facility at CERN[END_REF]. The beam flux was not constant -ions were delivered in spills with flux of about ~0.5-4 x10 6 ions/cm 2 /spill, with spill duration of about 200 ms, repeated every 5 to 60 seconds. Supply voltage used was 5.3 V, but due to setup characteristics (supply cables ~30 m long, leading to voltage drop and infeasibility of use of 4-wire connecting) the voltage at board level was ~4.6 V. Higher supply voltage was not used in order to not damage the DUT with too high voltage when switching on the device (for the short time after switching on the DUT, current draw is low, there is no significant voltage drop on cables and voltage at the board level is close to the supply voltage level).

One of the rare events observed during test was power cycling of the DUT: output voltage of the DUT was switching (Fig. 3). The calculated cross-section for this event is between 1.3x10 -8 and 2.3x10 -8 cm 2 . For comparison, the cross-section for SETs observed at output of voltage reference chip is in order of ~1x10 -5 cm 2 . The source of the power cycling event remained unknown after heavy ion test campaign. Analysis of the DUT schematics and of the output voltage switching sequence suggested that the most probable reason of the event is activation of the UVP circuit.

C. Laser tests

In order to better understand events observed during heavy ion tests, laser tests of VRef and OpAmp chips were performed with use of single photon laser facility of the IES PRESERVE platform of the University of Montpellier. Laser pulse energies between 150 pJ and 500 pJ were used. Both IC1 and IC2 were tested with laser, from the front side of the chip. Due to the low metal-coverage of those devices, this approach was assumed to be sufficient to reveal the mechanisms under investigation while reducing the sample preparation and board adaptation costs. Three types of signals were measured and recorded during laser tests: VRef output (with record of SETs), OpAmp output (UVP output from Fig. 2) and PoL outputthose measurement points were marked on Fig. 1 and Fig. 2.

Laser testing of IC2 was performed with laser energy of 500 pJ and the DUT was supplied with nominal voltage of 5 V (~4.6 V measured on board -this corresponds to the voltage during CHARM tests). No power cycling event was observed after scan of the whole chip die with laser pulses.

Similar scan with laser pulses was performed for IC1 chip, also no power cycling event was observed. At this point, supply voltage was reduced down to 4.87 V (4.43 V measured directly on the DUT board, which is close to the threshold of acceptable input voltage of the UVP, decreasing this value by few mV triggers the UVP). 4.43V voltage level is below specified supply voltage for the DUT, but is within supply voltage ranges specified for IC1 and IC2. Testing at this new supply voltage level let find areas of the IC1 chip die producing specific SETs, which were propagating through OpAmp and switching output of OpAmp, resulting in power cycling of the DUT (Fig. 4).

It was checked that propagation of those SETs was not correlated with the phase of operation of the PoL converterfor more than 10 laser pulses hitting single specific point of the die at random time, each of them was leading to power cycling of the DUT. The threshold laser energy to induce such SETs was measured at 150 pJ.

III. LASER TESTS RESULTS ANALYSIS AND DISCUSSION

To understand what kind of SETs are propagating from VRef through to OpAmp and result in power cycling of PoL converter output, different parameters of those SETs were calculated. All calculations presented in this section are based on data from tests with a laser energy of 300 pJ.

Figure 5 presents the laser mapping of maximum values of VRef output signals (measurement 1 on Fig. 1). Figure 6 presents the mapping of minimum values of the OpAmp output (measurement 2, blue curve in Fig. 4). Figure 7 presents the mapping of minimum values of the PoL output (measurement 3, red curve in Fig. 4).

During nominal operation of PoL, OpAmp output is close to supply voltage (4.43 V during this test) and PoL output is 2.5 V. Blue areas on Fig. 6 mean that OpAmp output signal was close to GND level, orange areas mean that OpAmp output signal was lower than nominally, but didn't reach less than Analysis of Fig. 5 shows that there are also many SETs generated at the output of the voltage reference by laser pulses hitting different parts of IC1 (all areas with color different than dark blue), w hich do not propagate to output of OpAmpthey were called "not propagating SETs".

As it was mentioned in the introduction of this paper, in the literature, the maximum value is commonly used to characterize worst case SETs. Comparison of Fig. 5 with Fig. 6 and Fig. 7 shows that SETs which propagate in our system are not the ones with highest peak value. Further analysis was performed to investigate this unexpected behavior.

Different parameters of SET signals (measurement 1) were calculated in order to characterize SETs of different groups: minimum, maximum, average values, widths of SET peaks, delays between positive and negative peaks. See Fig. 8 for description of those parameters. The most important results are presented below. not propagating SETs (blue dots) and propagating SETs (red circles), whereas plot on figure 10 presents data for not propagating SETs (blue dots) and partially propagating SETs (green circle s). According to these plots, propagating SETs (red circles) are the ones with maximum value of above ~1.4 V and minimum value usually not below 1.1 V (due to not strong minimum peak, their peak-to-peak value is usually low). Partially propagating SETs (green circles) usually have high maximum values and minimum values lower than propagating SETs (between 0.9 V and 1.1 V). However there is also small group of partially propagating SETs with parameters similar to propagating SETs. Not propagating SETs (blue dots) are of different types. There is big subgroup located in bottom right corner , that's area of SETs with small maximum and small minimum valuessmall positive peak and small negative peak. There is a big subgroup on left side of the plot -SETs with low minimum values and broad range of maximum values. There are also subgroups of not propagating SETs mixed with group of propagating SETs and group of partially propagating SETs.

Figures 11 and12 present plots with same parameters on axes X and Y: minimum voltage value of each signal from measurement 1 and its delay between maximum and minimum value. This delay represents delay between highest positive and lowest negative peak in the signal, delay less than 0 means that lowest negative peak is before highest positive peak. For better readability, only signals with maximum value higher than 1.35 V were plotted, this includes all propagating SETs, all partially propagating SETs and 57% of not propagating SETs.

According to figures 11 and 12, propagating SETs have their negative peak in different points before or after positive peak, but the common characteristics is -as it was already mentioned -that the negative peak is not very deep in this kind of SETs. Contrary, most of partially propagating SETs have their negative peak at specific point after positive peak and the negative peak is quite deep (down to ~0.85 V).

Figures 13 and14 present SET plots with maximum voltage value and positive peak width value of each signal from Simulations show that strong reduction of supply voltage, and resulting reduction of voltage difference on inputs of OpAmp, enable propagation of transients similar to observed during laser tests (SET sensitivit y of comparator working in similar conditions was described in [START_REF] Koga | Single event upset (SEU) sensitivity dependence of linear integrated circuits (ICs) on bias conditions[END_REF]). Analysis of the OpAmp SPICE model (OP484) shows that important in understanding this effect is the role of voltage controlled current source (VCCS) at output stage of OpAmp. This VCCS is modelling the transformation of voltage from first and second stage of the OpAmp and is directly connected to capacitance of output stage and indirectly controls output transistor of OpAmp. If difference between input voltages is big, then VCCS is fully charging or discharging that capacitance. But if this difference is small, then that capacitance is partially charged and the base voltage of the output transistor is close to the switching threshold between cut-off state and active state. In this case, even small SET might be enough to force VCCS to charge capacitance of the output stage and raise base voltage of the output transistor and switch it to active stateand thus switch the output of OpAmp.

SPICE simulation results showing propagation of SET are presented on figure 16. SET at VRef (green color) has maximum value of 1.7 V and the FWHM width is 0.625 μs. During the positive peak of SET, the VCCS starts to drive the current (orange color) and to charge capacitors on VCCS output (blue color). This is followed by increase of the voltage on output transistor base (black color), resulting in switching the output of OpAmp (magenta color). Propagating SETs with signature as on figure 16 (with positive peak only) were observed during laser te sts.

Figure 17 presents simulation results for not propagating SET with negative peak going to 0.7V (FWHM width 0.625 μs) and positive peak going to 1.7 V (FWHM width 0.625 μs). It was observed, that if the positive peak of SET is preceded by the strong negative part, the capacitance of OpAmp is first discharged by VCCS (during negative part of SET) and then it is recharged by VCCS during the positive part of SET. It takes some time until positive SET will charge capacitance to the initial value, and additional time would be needed to charge even more and finally to switch the output of OpAmp. That's why even very high positive peaks, if they are preceded by low enough negative peaks, will not switch the output of OpAmp. Not propagating SETs with such signature were observed during laser tests.

Figure 18 presents simulation results for partially propagating SET with positive peak going to 1.6 V (FWHM width 0.5 μs) and negative peak going to 0.7 V (FWHM width 0.625 μs). During positive part of SET, capacitors on VCCS output are charged, output transistor's base voltage increase and the output voltage of OpAmp starts to decrease. But simulation shows that if the negative peak is following closely the positive peak, the charging of capacitance is stopped and discharging starts, the base voltage of output transistor is decreasedtransistor stops conduction and the output comes back to high state. Partially propagating SETs with deep negative part following the positive part, were observed during laser tests.

V. COMPLEMENTARY HEAVY ION TESTS AT CYCLONE

FACILITY Additional heavy ion tests of PoL 2D board were performed in the end of June 2019 in the Cyclotron of Louvain la Neuve (CYCLONE) at Université catholique de Louvain (UCL) in order to better understand effects observed during previous test campaigns. PoL was tested in conditions of reduced supply votage: 4.5 V measured on board and it was several milivolts above the level of autonomous trigger of the undervoltage protection of the DUT. This level is different than measured during laser tests (4.43 V). This might be explained by higher external interference during heavy ion tests and the fact that supply cables were longer at UCL (~6 metres) than during laser tests (less than 4 metres), which gives higher voltage drop during each cycle of operation of the converter (PoL). LET values used during tests at UCL were 62.5 MeV•cm 2 /mg ( 124 Xe 35+ ion), 45.8 MeV•cm 2 /mg ( 103 Rh 31+ ion) and 20.4 MeV•cm 2 /mg ( 58 Ni 18+ ion). The maximum flux during test was 1.5x10 4 ions/cm 2 /s.

For LET equal to 62.5 MeV•cm 2 /mg and 45.8 MeV•cm 2 /mg, transient propagation and partial propagation was observed during direct irradiation of the voltage reference chip. Signatures of different types of SET had characteristics as observed in SPICE simulations or during laser tests. For LET equal to 20.4 MeV•cm 2 /mg, the propagation of SETs was not observed (fluence used for this test was 1x10 7 ions/cm 2 ).

Direct irradiation of the OpAmp chip during PoL test at UCL showed that for reduced supply voltage conditions, power cycling of PoL is also possible after ion hit at OpAmp. This effect was observed for LET values 62.5 MeV•cm2/mg, 45.8 MeV•cm2/mg and 20.4 MeV•cm 2 /mg. VI. DISCUSSION SPICE simulation explains results observed during laser tests but is also a strong background to propose a hypothesis on why propagation of specific SETs was observed also during heavy ions tests at CHARM facility. Most of the SETs observed during heavy ions tests had similar signature: negative peak followed by positive peak. But all the recorded SETs that did propagate in the system had different signature: negative peak was followed by much longer positive peak, or double positive peaks (see figure 19 for examples of SET waveforms captured during test campaign at CHARM). Most probably this longer positive part of SET was responsible for longer charging of OpAmp output stage capacitance, and finally for switching of the output. Specific signature of the propagating SETs gives the idea that pile-up of two SETs (due to two ions passing through voltage reference chip in close time) could give signature of the propagating SET. Facts that the flu x was high during CHARM tests and that cross section for SET propagation was low, do support this hypothesis.

To verify this hyphothesis, rough calculations with use of Monte-Carlo method were performed to estimate the probability of two SETs appearing at VREF close in time. Input data used was the SET cross-section (10 -5 cm 2 ), ion flux (according to the test log it was ~3-3.5x10 6 ions/cm 2 /spill during the period when power cycling was observed) and spill duration (200 ms). Resulting number of SETs at VREF output is 30-35 per spill. In proposed method, each spill was simulated as a group of 30 SETs, each SET having random time stamp in range from 0 to 200 ms. If in certain spill the difference between time stamps was smaller than 5 µs, it was marked as double-hit; time shift of 5 µs was chosen based on analysis of figure 19. According to simulation, for 10 4 simulated spills, number of double-hits is around 220. This means that for 3x10 5 simulated SETs there are around 220 double hits, so the difference is 3 orders of magnitude. This is compliant with experimental result showing that power cycling event cross-section is 3 orders of magnitude smaller than VREF SET cross-section. Uncertainty of proposed calculation is related to the choice of time shift between two SETs defined as double-hit (expected to lead to power cycling). Simulation for time shift of 10 µs (instead of 5 µs) gives result of around 420 double-hits per 10 4 simulated spills; whereas for 3 µs the result is around 120 double hits per 10 4 simulated spills. If we take number of SETs in spill equal to 35, for time shift of 5 µs we have result of around 305 doublehits per 10 4 simulated spills (3.5x10 5 SETs). These results show that there is still difference of around 3 orders of magnitude between number of simulated SETs and resulting number of double-hits.

Presented results show that the very high particle flux may lead to double (or multiple) SEEs in short time. This may lead to unexpected component-level or system-level effects, that would not be observed when testing with lower flux, and that may be very unlikely in target environment (like space). This leads to a conclusion that such over-testing is possible in the very high flux facility and it should be taken into account during test preparation phase or during results analysis. But it should be also reminded in this point, that although double SETs are supposed to generate power cycles during tests at CHARM, it was also possible to generate a power cycle by single SET (with different signature) during heavy ion tests at CYCLONE facility.

Synergistic effects between cumulative effects and analog SET were discussed e.g. in [START_REF] Roche | Impact of Cumulative Irradiation Degradation and Circuit Board Design on the Parameters of ASETs Induced in Discrete BJT-based Circuits[END_REF] [START_REF] Roche | Development of a new methodology to model the synergistic effects between TID and ASETs[END_REF]. In our work it was observed that power cycling event rate (due to SET propagation) was changing with time during the experiment. Figure 20 presents average number of power cycles per minute during last ~3 hours of the test at CHARM (15 minutes averaging period was used). It might be observed that number of power cycles is gradually increasing during the test (with local variations, as expected for a random process), although the flux (number of ions per spill) doesn't increase. This effect is expected to be result of degradation of PoL due to cumulative effects which lead to increase of current consumption and resulting decrease of the VIN voltage at level of the DUT (due to higher voltage drop on the supply cables). This lower VIN level lead to smaller difference between V+ and V-of OpAmp and this is supposed to have been the source of higher rate of power cycling events in time. Therefore, observed effect might be considered as a synergistic effect, but occuring at system level.

Unexpected power cycling of a power converter module is a very undesired situation from the user/application point of view. However, for the PoL 2D board (prototype version of the 3DPM-0024) which was the DUT during all the tests described, power cycling events were observed only for supply voltage below specified operating voltage range (4.75 V -5.25 V). Presented analysis shows that observed power cycling should not be considered as a threat when nominal supply voltage is applied.

From the more general point of view, SET propagation observed and described in this work can only appear when the voltage difference between V+ and V-inputs of operational amplifier is very small. (Behaviour verified for OP484, but expected for other amplifiers with similar internal structure , i.e. with output stage capacity being charged by internal current source which might be controlled by high enough transients on OpAmp input, when the voltage difference between V+ and Vinputs is small). It is important to note, that in the conditions described SET worst case signature is a unipolar, positive peak (for V+ nominally higher than V-) and therefore testing with bipolar only transients might not uncover sensitivity of the cicruit for propagation of unipolar transients with even lower amplitude.

Although in many applications, the difference between V+ and V-of operational amplifier will be higher than during tests presented in this work, the effect described in the paper might become important for a system end of life conditions: in this situation, parameters drift due to aging and/or radiation effects may lead to decrease of the V+ and V-voltages difference and increase of sensitivity to unipolar transients. In order to protect the PoL system against this effect, the Vref source (which is the input to the OpAmp) should be filtered to suppress SETs. For other systems/circuits where it is expected that OpAmp might work in the configuration of low V+ and V-difference, it should be verified if the IC/circuitry at input of OpAmp may generate unipolar SETs that could propagate through OpAmp. If such risk is not negligible, it should be also considered to use filters to suppress SETs.

Propagation of analog SETs was already broadly discussed in literature. In [START_REF] Buchner | Single-Event Transients in Bipolar Linear Integrated Circuits[END_REF] parameter influence on analog SETs in OpAmps is discussed. Different external factors (operating configuration, supply voltage, input voltage, output load, and gain) and internal factors (like compensating capacitor) are discussed and it is shown how they affect SET width, amplitude and sensitivity to SET propagation. It is also presented in [START_REF] Buchner | Single-Event Transients in Bipolar Linear Integrated Circuits[END_REF] that analog SETs are propagating from input to output of the SET-hardened IS-139ASRH voltage comparator only when the overdrive voltage is as low as 5.8 mV, but the propagation mechanism is different from the one discussed in this paper.

[7] describes use of macromodels (based on SPICE) and micromodels (involving 3-D device physics code with the layout geometry of the transistor) to model ion hit inside OpAmp and to verify if generated SET will produce any malfunction in the system. It is a complex tool using IC model created by the user of a IC, not by the manufacturer. However, the only SET parameters that are claimed to be important to define if there is SET propagation or not, are the SET height and width. [START_REF] Boulghassoul | System-Level Design Hardening Based on Worst-Case ASET Simulations[END_REF] presents how computer simulations can be used to develop system-level design hardening methodologies against SETs. Worst-case SET signatures are extracted from the simulations, confirmed with OpAmp heavy-ion test and used to propose mitigation techniques. Worst-case is defined here in terms of the maximum SET amplitude and width. However in [START_REF] Boulghassoul | Applicability of Circuit Macromodeling to Analog Single-Event Transient Analysis[END_REF], it is stressed that although macromodel of the ICs/circuit might be succesfully used to analyze system-level propagation of SET, for the analysis of SET inside of the IC, the device micromodel should be used.

In [START_REF] Roche | Investigation of Flip-Flop Effects in a Linear Analog Comparator-With-Hysteresis Circuit[END_REF], SET propagation in Schmitt trigger based on OpAmp is analysed. It is shown that SETs induced in this kind of circuit can change the state of the OpAmp output depending on the circuit design, the input voltage and the injected energy into the device. Particularly, it is presented that for lower difference of V+ and V-, lower laser energy is needed to produce the SET that will change the state of the output, but there is no analysis of SET signatures. [START_REF] Adell | Single Event-Induced Instability in Linear Voltage Regulators[END_REF] demonstrates that SETs can induce instability in another type of circuit: linear voltage regulators. Simulations and analysis with use of the OpAmp micromodel show that oscillation is initiated by a change in current flow between the current mirror of the differential input stage and the compensation capacitor of the OpAmp (SET is generated inside of the OpAmp and propagates to its output).

According to the best knowledge of authors of this paper, it is the first time to observe and present that in OpAmp with low voltage difference between V+ and V-, there is difference in propagation of unipolar and bipolar SETs from input to output of the OpAmp. It is also the first time to observe and explain (by analysis of VCCS in OpAmp) difference in propagation of bipolar SETs with negative peak followed by positive peak and with positive peak followed by negative peak.

VII. CONCLUSIONS

Laser tests are used to reproduce rare event observed during system level heavy ions tests: SEE provoking power cycling of the DUT (Point of Load converter) with reduced supply voltage. In the DUT analyzed, reduction of the supply voltage leads to reduction of voltage difference between V+ and Vinputs of OpAmp in the undervoltage protection circuit. Laser tests show that in these conditions, specific SETs generated at voltage reference chip might propagate to OpAmp output and up to the output of the DUT. SETs that do propagate are the ones with high enough positive peak and insignificant negative peak value whereas some transients with bigger maximum and/or peak to peak value will not propagate. SPICE simulation results show that important for understanding the observed effects is operation of the voltage controlled current source inside OpAmp, which is charging and discharging capacitance of the output stage of the OpAmp.

Fig. 1 .Fig. 2 .

 12 Fig. 1. DUT block diagram (3 measurement points refer to laser tests).

Fig. 3 .Fig. 4 .

 34 Fig. 3. Output voltage of DUT during an SET-induced power cycling observed under heavy-ions.

Fig. 5 .

 5 Fig. 5. Maximum values of VRef output (measurement 1) corresponding to laser pulse hits at different points of IC1 die.

  about 2.5 V. Comparison of this result with mapping for measurement 3 (Fig.7) shows that SET which propagated through OpAmp and forced it to reduce voltage to GND, also forced power cycling of PoL (low values on Fig.6(blue color) match low values on Fig.7(also blue color)). Such SET was called "propagating SET". SETs which propagated through OpAmp but didn't force it to reduce output voltage down to GND, didn't provoke PoL power cycling. Those SETs were called "partially propagating SETs" (orange area s in Fig.6corresponding points on Fig.7are dark red -PoL output value has nominal level).

Figures 9 Fig. 6 .

 96 Fig. 6. Minimum values of OpAmp output (measurement 2) corresponding to laser pulse hits at different points of IC1 die.

Fig. 7 .

 7 Fig. 7. Minimum values of PoL output (measurement 3) corresponding to laser pulse hits at different points of IC1 die.

Fig. 8 .

 8 Fig.8. Description of different parameters of SET that were calculated: Amaximum value, B-minimum value (measured from the level of 0V, not from average value of the waveform), C-delay between maximum and minimum peaks, D-SET width; average value was calculated basing on waveform range marked with letter E

Fig. 10 .

 10 Fig. 10. SET plot presenting minimum and maximum voltage value of each signal from measurement 1. Not propagating SETs (blue color) and partially propagating SETs (green color) are plotted.

Fig. 11 .Fig. 12 .

 1112 Fig. 11. SET plot presenting minimum voltage value of signals from measurement 1 and its delay between maximum and minimum value. Blue color corresponds to not propagating SETs, red to propagating SETs. For better readability, only signals with maximum value higher than 1.35V were plotted.

Fig. 13 .

 13 Fig. 13. SET plot presenting maximum voltage value and positive peak width value (full width at half maximum, FWHM) of signals from measurement 1. Not propagating SETs (blue color) and propagating SETs (red color) are plotted.

Fig. 15 Fig. 16 .

 1516 Fig. 15 Schematics of circuit model used for SPICE simulations.

Fig. 17 .

 17 Fig. 17. Plot with different waveforms obtained during SPICE simulation of not propagating SET: VRef voltage (green), OpAmp output voltage (magenta), VCCS output voltage -capacitance of the output stage (blue), OpAmp output transistor base voltage (black), VCCS current (orange).

Fig. 18 .

 18 Fig. 18. Plot with different waveforms obtained during SPICE simulation of partially propagating SET: VRef voltage (green), OpAmp output (magenta), VCCS output voltage -capacitance of the output stage (blue), OpAmp output transistor base voltage (black), VCCS current (orange).

Fig. 19 .

 19 Fig. 19. Example of SETs measured at VRef node, which caused power cycling (left) and which didn't cause power cycling (right) during PoL heavy ion test at CHARM facility.

Fig. 20 .

 20 Fig. 20. Averaged number of power cycles per minute during last ~3 hours of the test at CHARM (15 minutes averaging period was used).

VIII. ACKNOWLEDGMENTS

This work has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement number 721624.

Laser tests have been performed using the University of Montpellier PRESERVE Platform which has been funded thanks to the financial support of the Region Occitanie and the European Union via European Regional Development Fund.


Dimitrios Kapogiannis

Mohamad El Haj

The subjective experience of recollection and familiarity in Alzheimer's Disease

While the Integrative Memory model for Alzheimer's disease is interesting, it may benefit from incorporating the subjective experience of recollection. We therefore offer complementary lines of interpretation to explain how recollection and familiarity in Alzheimer's disease can be dissociated based not only on accounts of their neural correlates but, critically, on the subjective experience of memory in patients.

.

The decline of recollection in Alzheimer's disease can be attributed to decline in specific processes of the recollective experience. This decline has been pointed out by research demonstrating decline in the recollective experience during retrieval of past personal events in patients with Alzheimer's disease, including decline in subjective processes, such as

reliving, travel in time, remembering, realness, rehearsal, and visual imagery (El [START_REF] El Haj | Phenomenological Reliving and Visual Imagery During Autobiographical Recall in Alzheimer's Disease[END_REF]. Among these subjective processes, decline of visual imagery seems to play a key role in the decline of recollective experience in patients with Alzheimer's disease. The decline of visual imagery in Alzheimer's disease seems to deprive patients from the ability to retrieve and manipulate mental images during retrieval, and also deprive them from visual cues that mediate and/or accelerate their search through memory stores [START_REF] El Haj | Mental imagery and autobiographical memory in Alzheimer's disease[END_REF]El Haj, Moustafa, Gallouj, & Robin, 2019).

The relationship between decline of visual imagery and decline of the recollective experience in Alzheimer's disease can be understood by highlighting research using the Field/Observer paradigm, which has been widely used to assess the subjective experience of recollection and familiarity in general populations (Nigro & Neisser, 1983;Rice & Rubin, 2011). In one study in Alzheimer's disease, patients were invited to retrieve past personal events and, after a delay, provide a "Field" response, if they can visualize the event through their own eyes, or an "Observer" response if they can visualize themselves in the scene as a spectator would (El Haj, Moustafa, et al., 2019). Results demonstrated increased "Field" and decreased "Observer" responses in patients, suggesting a diminished ability of patients with Alzheimer's disease to construct vivid images when recollecting the past.

In summary, the decline of the recollective experience during retrieval can be associated with declines in several components of the subjective experience, such as reliving, travel in time, remembering, realness, and rehearsal. Critically, decline of visual imagery seems to deprive patients from the ability to construct mental images when recollecting the past. We propose that these processes should be taken into account by The Integrative Memory model, as proposed by Bastin et al., to better account for recollection deficits in patients with Alzheimer's disease. We believe that a decline in the subjective experience during retrieval in patients with Alzheimer's disease leads to a decline of recollection and, consequently, to the emergence of a general sense of familiarity that is typically associated with a sense of "having experienced this before". As pointed out by Bastin et al., the Integrative Memory model is not comprehensive and should evolve to incorporate other mechanisms. Therefore, we propose that the Integrative Memory model by Bastin et al is sufficiently flexible to include these subjective processes and may be enriched by this inclusion.

El Haj M, Moustafa AA, Gallouj K, & Robin F (2019) (2002). Episodic memory: from mind to brain. Annual Review of Psychology, 53, 1-25. doi: 10.1146/annurev.psych.53.100901.135114

  . Visual imagery: The past and future as seen by patients with Alzheimer's disease.Consciousness and Cognition, 68, 12-22. doi: 10.1016/ j.concog.2018.12.003 [PubMed: 30593998] Nigro G, & Neisser U (1983). Point of view in personal memories. Cognitive Psychology, 15(4), 467-482. doi: 10.1016/0010-0285(83)90016-6 Rice HJ, & Rubin DC (2011). Remembering from any angle: the flexibility of visual perspective during retrieval. Consciousness and Cognition, 20(3), 568-577. doi: 10.1016/j.concog.2010.10.013 [PubMed: 21109466] Tulving E

Behav Brain Sci. Author manuscript; available in PMC 2021 January 03.

Acknowledgments

This research was supported in part (DK) by the Intramural Research Program of the National Institute on Aging, NIH.


Kilian Caillaud
email: kilian.caillaud@gmail.com

Catherine Ladavière
email: catherine.ladaviere@univ-lyon1.fr

P V P Dos Santos

C M Ribeiro

F R Pavan

P P Corbi

F R G Bergamini

Y Zhang

J Chen

M Wei

H Li

Spectrochim Xin

Z He

Q Chen

X Mao

J Zhang

Soft L Zheng

N Kuhl

S Bode

R K Bose

J Vitz

A Seifert

S Hoeppener

S J Garcia

S Spange

A V Bogdanov

I F Zaripova

A D Voloshina

A S Strobykina

N V Kulik

L K Mustafina

A S Sapunova

V F Mironov

Russ J Gen

Chem

J M Dos

Santos Filho

D M A De Queiroz

T S Silva

H M P Macedo

D R M Teixeira

D Guilherme

J É Simonetti

L R S Folquitto

A C C Reis

L M M Silva

F C F Costa

A L P Brito

Miranda

Water-soluble (poly)acylhydrazones: Syntheses and Applications

Keywords: Acylhydrazone, polyacylhydrazone, dynamic combinatorial chemistry, dynamer, watersoluble polymers

Acylhydrazone-based (macro)molecules display an increasingly broad spectrum of applications, from small therapeutic molecules and metal-ions probes to self-healing films and biorelevant dynamic polymers. The acylhydrazone function is indeed of prime interest since its unique design combines hydrogen bonds, pH-dependent reversibility, ability to undergo molecular exchanges, and a possible polyvalency. However, a key parameter of its use in aqueous media is the associated water-solubility of the resulting (macro)molecule. This property can indeed become a limitation for applications requiring high concentrations or no organic cosolvent. This review firstly focuses on describing the acylhydrazone function, its properties, and its relevancy in dynamic combinatorial chemistry (DCC). Then it details the synthesis strategies (molecular design, cosolvent, reaction conditions) reported in literature to ensure a sufficient solubilization of both single-bond and multiple-bond acylhydrazone (macro)molecules, together with their various applications.

Introduction

In the past three decades, the increasing development of dynamic combinatorial chemistry (DCC) has led to new synthetic methodologies, to create even more diverse molecules in a minimum of steps. [1][2][3][4] Among the eligible covalent bonds required for DCC, the hydrazone ones (and by extension the acylhydrazone ones) attracted attention thanks to their reversibility and their biocompatibility 5,6 More, polymers made of acylhydrazone bonds have been found to have very interesting properties, by being able to undergo monomer exchanges through adaptation to the medium according to DCC. These properties can lead to relevant therapeutic molecules, ion probes, self-healing films, or dynamic polymers. 5,7,8 However, one of the major limitations for the elaboration of acylhydrazone-based (macro)molecules is their poor water-solubility. To circumvent this limitation, many synthesis strategies have been developed. They are different whether the (macro)molecule bears a single acylhydrazone bond or multiple bonds (Figure 1). This distinction will be explained in further parts, in which synthesis strategies of single-bond and multiple-bond acylhydrazone (also named polyacylhydrazones) will be discussed, as well as their associated applications.

But before exploring this, an introduction about this particular acylhydrazone function appears to us as pertinent. To this end, a description about its formation, its dynamic behavior, the catalysis of its formation, and a portfolio of (macro)molecular structures implying acylhydrazone bonds from organic solvent will be presented.

Acylhydrazone function

Description of the acylhydrazone function

Dynamic bonds (covalent or non-covalent) are known to continuously form and break, depending on the conditions of the medium. Perpetrated by reversible bonds, a dynamic library of molecules can be created by a thermodynamic equilibrium between building blocks, where the most stable constituents are in larger quantities. This equilibrium can then be shifted through the addition of external stimuli (e.g. pH, temperature, external molecule).

Hence, the amplification of some species informs about the products having the higher affinity toward the stimuli (Figure 2). The use of these reversible-bond building blocks to create polymers has also been exploited. The resulting dynamic polymers, whose length, constitution, and supramolecular arrangement strongly depend on the medium, are called dynamers. DCC of molecular or macromolecular species is of prime interest in many fields such as the drug discovery area. 4,6,[9][10][11] Figure 2 -Schematic representation of a DCC process and the reversible equilibrations of a building block library, depending on the presence of the target. From Frei et al. 9 Among the eligible dynamic functions to be used in DCC (which can be turned into supramolecular or covalent bonds), the aim of this review is to focus on the very interesting and well-known acylhydrazone family. Acylhydrazones can be synthetized though condensation between hydrazide and aldehyde moieties, displaying reversibility under mild conditions with acid catalysis. The condensation reaction to get acylhydrazones is presented on Figure 3, together with other imine bond types. 4,[11][12][13][14][15] Compared to other C=N imine-type dynamic bonds, such as imines, oximes or hydrazones, acylhydrazone functionality displays many attractive features, having the advantages of both imine and amide groups. 5,6 Indeed, hydrogen bonds can be performed thanks to C=O and NH groups, while C=N group provides reversibility. Furthermore, contrary to other types of imines, hydrazones are stable enough thanks to an enhanced electronic density on the carbon to analyze the resulting products. 9,16 Hydrazones are also less affected by hydrolysis due to their extra nitrogen (enhancing the stability in water by making the "imine" nitrogen less able to be protonized). 17 Finally, an additional electron-withdrawing group such as an acyl group (compared to oximes and hydrazones) makes the resulting acylhydrazones not too stable to be interestingly used in dynamic libraries. It also increases the possibility of hydrolysis, which can be an asset in some situations. 2,14,18 The understanding of acylhydrazone hydrolysis is very important since it leads to the reversibility of the reaction. The mechanism of hydrazone hydrolysis was thoroughly studied by Yildiz 19 through DFT calculations. The resulting conclusions are that the hydrazone (and by extension acylhydrazone) undergoes a water nucleophilic addition to give carbinolamine as intermediate species. Then, this carbinolamine is decomposed into hydrazide and carbonyl without any intermolecular proton transfer (Figure 4). The rate-determining step is the dissociation of the intermediate, but it was also demonstrated that the rate of hydrolysis depends on the ease of protonation of the "imine" nitrogen. 16

Figure 4 -Hydrolysis mechanism of hydrazone, proposed by Yildiz. 19 Note that another synthetic pathway was explored by Haugen et al. 20 , which implies a ketone linked to an electron-withdrawing group, and diazene compounds, catalyzed by a phosphine (Figure 5). The yields were found to be correct to excellent, depending on the reactants. Because the reaction conditions are harsh (THF, -78 °C to rt in 1h), this way of synthesis will not be explored in this review.

Figure 5 -Alternative acylhydrazone synthesis pathway, from Haugen et al. 20 Another remarkable property of the hydrazone bond (and by extension acylhydrazone) is its photosensitivity. Indeed, the configuration of its C=N double bond can be reversibly switched from (E)-isomer to (Z)-isomer upon UV irradiation (365 nm). [21][22][23][24] More, it was showed that UV irradiation can also cleave the acylhydrazone bond, leading to photodegradable polymers. 25 The "classic" synthesis of acylhydrazones, from aldehyde and hydrazide, is well known to be very slow in neutral medium. Therefore, many works have been focused on the ways to increase this reaction rate and will be presented in the next part.

Catalysis of the acylhydrazone formation

Acylhydrazone function displays a reversibility property under acid catalysis, at mild conditions. 2,5 This reversibility (also reachable by heating 5,26 ), allows hydrazide or aldehyde exchange (Figure 6B). Depending on the chemical nature of the molecule, the pH range of this reversibility state can vary from 3 to 7. 27 The rate of this exchange is indeed significantly decreased at pH > 7, 27 making the acylhydrazone a convenient and easily switchable bond through pH. This acid-sensitive property includes the possibility of developing pH-sensitive systems, usable for drug or fragrance delivery through the breaking of this acylhydrazone bond and the release of the drug under specific conditions. 25,[28][29][30] The mechanism of the hydrazone formation explaining this pH-catalysis is shown on Figure 6A. Fulton's group focused on the mechanistic difference between the acylhydrazone formation (from aldehyde and hydrazide) and the exchange (between two different acylhydrazones), because the ratedetermining step is, respectively, the β-elimination of the water and the nucleophilic attack on the protonated hydrazone. [31][32][33][34][35] Figure 6 -A) Mechanism of the hydrazone formation, according to Jencks and Kool. 31,32 B) Exchange properties of acylhydrazone in acidic medium.

Consequently, for dynamic biomedical applications that imply building blocks exchange, acylhydrazones are hardly usable in neutral biological buffers (pH=7.4) without catalysts, because of their low reversibility at this pH value. To enhance the acylhydrazone exchange velocity, the use of a trans-imination catalyst was developed, by using aniline (Figure 7). [36][37][38] The observed second-order rate constant at biological pH (7.4) was found to be around 14-fold higher for aniline than for uncatalyzed reaction. 39 Nevertheless, the use of aniline leads to many problems. Firstly, accelerating the reaction to reasonable time periods requires large amounts of catalyst (up to 10 000 eq).

Secondly, aniline is toxic towards cells, which can be a problem if the targeted system is living. 36,39 Finally, it was shown that the catalytic activity of aniline depends on the reactants, and therefore can become a bias for isoenergetic dynamic libraries. 41 Based on these observations, Blanden et al. 42 found that 4-aminophenylalanine (Figure 9B) was almost as effective as aniline, but with a superior compatibility with biological systems. Moreover, the reaction temperature can be at 0°C, which can be an asset in some biological experiments. Kool's group 39,43 then tried to improve the catalyst design based on aniline, for hydrazone formation and exchange instead of acylhydrazone, and have demonstrated that anthranilic acids can be a new class of catalysts for these functions.

Particularly, 5-methoxyanthranilic acid (Figure 9C) and 5-methyl-2-aminobenzenephosphonic acid (Figure 9D) showed a second-order rate constant over 6-fold and 8-fold, respectively, higher than the aniline's one. This new class of catalysts is likely to be less toxic than aniline.

Other types of catalysts have been studied at neutral pH, such as an artificial enzyme featuring an aniline residue. 44 More, an indoline-based catalyst, instead of an aniline-based one, has shown encouraging results, and the most performant one showed a 15-fold rate increase versus aniline (Figure 9E). 45 Morales et al. 46 used pyrrolidine (10 mol%) as a catalyst in water, obtaining a high yield, and therefore paving the way for more green-chemistrycompatible catalysts (Figure 9F).

Larsen et al. 47 developed a series of aqueous buffers that can also play the role of catalysts. The most performant molecules bear several amine groups, and reaction rates more than 200-fold rates of phosphate buffer alone were demonstrated. Furthermore, these catalyzing buffers showed a low toxicity, and therefore can be used for biological reactions of acylhydrazones.

Figure 9 -Different generations of catalysts to increase the reaction rate in neutral conditions.

A) Aniline 36 , B) 4-aminophenylalanine 42 , C) 5-methoxyanthranilic acid 39 , D) 5-methyl-2aminobenzenephosphonic acid 43 , E) 5-methoxylindoline 45 , F) Pyrrolidine 46 .

Since the use of an additional molecule as a catalyst can add complexity to the acylhydrazone reaction, Kool's group has developed a system of autocatalysis. Indeed, in order to accelerate the reaction rate at neutral pH, the design of hydrazines and aldehydes was tuned. It was shown that when these molecules bear a neighboring acid/base group, the formation of hydrazones is significantly accelerated at biological pH, if the molecule's pK A is close to the solution's pH. Furthermore, electron-deficient hydrazines, such as hydrazides, react more slowly than electron-rich ones, explaining their enhanced reversibility. 17,31,48 Beside, Higgs et al. 35 concluded that the judicial position of hydrogen bond acceptors near the carbonyl-derived moiety also increases the reaction-rate of the acylhydrazone formation. This was also demonstrated by both Gois' and Gao's groups [49][50][51][52] , who showed that 2formylphenylboronic acid (2-FPBA, Figure 10) promptly reacts with hydrazides, and allows a rapid exchange of hydrazones at pH 7.0 which can be reversibly paused by raising the pH up to 12.

Figure 10 -Chemical formula of 2-formylphenylboronic acid, reacting with hydrazides with a high rate (thanks to its ortho boronic acid moiety).

Portfolio of (supra)molecular structures implying acylhydrazone bonds, from organic-solvent syntheses

Using acylhydrazone bindings' reversibility and supramolecular properties, the elaboration and valorization of various innovative structures were described in literature.

These different structures can be either single-or multiple-acylhydrazone molecule. In this part, the (supra)molecular structures prepared in organic solvent and insoluble in water will be described, in order to show a portfolio of the applications of acylhydrazone bonds outside aqueous environment.

A common valorization of acylhydrazone puts forward its ability to bind with metal ions, as a Schiff base. These ligands are synthesized in organic solvents, and usable in aqueous solvents. Acylhydrazone derivatives have been firstly used as iron-chelating compounds. 53 Later, the same group synthesized acylhydrazones from three aldehydes and fourteen hydrazides in order to find the most suitable ligands to eliminate iron from the body, a consequence of long-term transfusion therapy. The structures of the complexes

[Fe(L) 2 •2H 2 O]
were determined, and it was shown that the acylhydrazone plays a key role in the complexation (Figure 11). 54 As stated in the first part, one of the most common uses of acylhydrazone is in the field of polymers, and particularly in dynamic polymers, but other structures were obtained by employing the acylhydrazone function, such as dendrimers 68 , [2]-catenanes 69 , organogels 70 , and hydrogels 71 . Finally, acylhydrazone polymers can also be valorized as films, with selfhealing properties (Figure 13), 72 or tunable Young modulus and T g . 73 Copolymers adaptable to their environment thanks to their reversibility properties, 11,26,74 , or "optodynamers", which are films displaying color and fluorescence by stacking with another one (Figure 14), were elaborated too . 75 In the next two parts of this paper, different strategies to design i) water-soluble single-bond acylhydrazone dimers/oligomers, or ii) polyacylhydrazones will be presented, such as using appended moieties to help their solubilization, synthetizing the targeted molecules in organic solvents (but with a water-solubility application), or using a mixed water/organic solvent.

Single-bond acylhydrazone synthesis strategies

As previously written, dimers of acylhydrazone (and by extension small oligomers) are different from polymers not only because of their molar mass, but also their number of acylhydrazone bonds per species. Throughout the multiple syntheses and uses of watersoluble single acylhydrazone bonds, two approaches stand out. The first one is the synthesis in organic solvents, most often in ethanol or DMF, with a water-soluble purpose for the resulting species, such as metal complexation or therapeutic action. The second one is the synthesis directly in water, or an organic/water solvent mixture, with the same goals.

Synthesis strategies in organic solvents with a water-soluble purpose

Despite being much less popular than other imines bonds (such as oximes) for bioconjugation, acylhydrazone have been used in this area in multiple ways by several groups. 17 Combining 1H-indole-2,3-dione (isatin) derivative and Girard's reagent, Bogdanov et al. 76,77 successfully obtained water-soluble acylhydrazone from a reaction in refluxed ethanol. Later, they combined two isatin derivatives and performed the same reaction (Figure 15). Antimicrobial activity tests were carried out, and some compounds showed a high activity toward S. aureus and B. cereus, compared to a control drug. Hemolytic activity experiments were also performed, and the same compounds were found to be less toxic than control drugs.

Synthesis strategies in aqueous medium

As for the syntheses in organic solvents, water-based syntheses can be used for the preparation of metallic complexes, leading to original DCLs. Goral et al. 63 elaborated compounds being "doubly-dynamic" due to their metal-ligand interactions and their acylhydrazone bonds. Exchange kinetic parameters (ligand or acylhydrazone) were measured at different conditions (pH, temperature, oxidation) in 30 % acetonitrile in 0.01 M aqueous NH 4 OAc, at pH = 7. Later, a triple-DCL was created from guanine-based components, through metal-ion binding, hydrogen bonds, and acylhydrazone bonds (Figure 18). The latter was used as a gelation factor, allowing the formation of a quartet assembly of guanines complexed by the metal ion, and consecutively, a gel. Thus, the gelation had interestingly the ability to redirect the acylhydrazone distribution, and macroscopic properties depending on the cation and the acylhydrazone structure. These reactions were performed in sodium acetate buffer. 87-89 The synthesis of acylhydrazone bonds in water can also lead to biologically active compounds. Recently, Dascalu et al. 90 prepared hydrazone-and acylhydrazone-based species using only water as a solvent, from aldehydes, hydrazines, and hydrazides (Figure 19). Many of all the hydrazone compounds showed an antifungal activity against a lot of the tested strains. Interestingly, switching to acylhydrazone compounds enhanced the activity of some of them, comparably to marked drugs, but also narrowed the spectrum of activity to the tested strains, while showing no cytotoxicity. The reaction was carried out in an aqueous buffer (pH = 6.2), with aniline as a catalyst. Using DCC, Hirsch's group 92,93 synthesized libraries from five hydrazides and five aldehydes to determine an inhibitor of the endiothiapepsin enzyme, in ammonium acetate buffer (pH = 4.6). Some potent molecules were identified, also showing that the protease selectively binds to the E isomers of acylhydrazone-based compounds.

Using water as the major solvent in their syntheses, Vicini et al. 94 prepared bioactive acylhydrazone molecules, with an anti-HIV-1 activity. The molecules were synthesized from hydrophobic aldehydes and water-soluble hydrazides in a water/ethanol (7:3 v:v) solvent, precipitated, filtered and washed with water. However, note that for the bioactivity studies, they were finally dissolved in DMSO and diluted in water.

Lehn's group 95,96 prepared DCLs of building blocks (hydrazides, monoaldehydes, and dialdehydes), obtaining water-soluble species. The synthesis solvent was a sodium acetate buffer (pH = 4) with the addition of CH 3 CN or DMSO to increase the solubility of some compounds. They were prepared from bioactive building blocks, and led to potent enzyme inhibitors, after the templating of the dynamic equilibrium (for acetylcholinesterase and HPr kinase enzymes). They later used the same approach, with DCLs prepared from sugar-based aldehydes, hydrazides, dihydrazides, and trihydrazides for the screening and the identification of active compounds able to bind to a carbohydrate-binding protein, Concanavalin A (Figure 21). Because of the sugar-based structure of the aldehydes, the building blocks and the resulting acylhydrazone species could easily be solubilized in an ammonium formate buffer solution (pH = 4.0) at the millimolar level, without the addition of a miscible organic solvent. 27,97 Through their studies on the catalysis of the acylhydrazone formation, Kool and

Crisalli 17,31,39,43,48 explored their synthesis from aldehydes and hydrazides. The main motivation of their work was to successfully prepare acylhydrazones in a neutral aqueous medium. So, they used a phosphate buffer saline (PBS, pH = 7.4) in all their experiments, with the addition of 10 v% of DMF to ensure a good solubilization of reactants and products.

Similarly, the other groups working on improving the catalysis of the acylhydrazone formation in water used water-soluble building blocks, and syntheses were made at room temperature in aqueous buffers. The pH of these buffers (phosphate or acetate) varies from 4.5 to 7.4. The addition of 10 and 20 v% DMF was necessary for Zhou et al. and Trausel et al., respectively, to improve the solubilization of both building blocks, and resulting compounds. 35,36,41,45,46 The use of acylhydrazone as a tool for time-controlled release of fragrance was explored by Levrand et al. 11,30 by binding a water-soluble hydrazide to a highly volatile aldehyde (Figure 22). The synthesis was carried out in a water/ethanol mixture (2/1, v/v), the aqueous phase being phosphate (pH = 2.47) or citrate (pH = 4.48) buffers.

Multiple-bond acylhydrazones synthesis strategies

Similarly to single-acylhydrazone molecules, polymers constituted of several acylhydrazone bonds display reversibility in their initial reactants in acidic conditions, and kinetical freeze under basic conditions. This property allows these kinds of polymers to be dynamic, giving "dynamers". The features of polyacylhydrazones are i) high yields in mild conditions, ii) dynamic covalent polymers, and iii) many hydrogen bond interactions.

Lehn 2,101 describes such polymers as "reversible polyamides", because of the similarity between the functions, but also because of their poor solubility in classic organic solvents and water, which complicates their synthesis and characterization.

According to literature, polyacylhydrazones can be classified in three categories. The first one is based on synthetic polymers with acylhydrazone functions in the main chain, obtained from either dihydrazides and dialdehydes, or monomers carrying both hydrazide and aldehyde moieties. Focusing on this kind of water-soluble polymers, a differentiation of their synthesis solvent (in organic or aqueous solvents) seems to be relevant in this review. Indeed, because of the intrinsic poor water-solubility of such polymers, the employed strategies will be different in those cases. 102,103 The second category corresponds to synthetic polymers with acylhydrazone functions in their side-chains They are basically (water-soluble or not) polymers bearing multiple pending aldehydes or hydrazides moieties, and able to undergo exchange with building blocks decorated with the complementary function, thermodynamically reaching an equilibrium. 102,103 A third category might as well be considered, not by its polymeric structure (i.e., the location of acylhydrazone function in the polymer), but by its composition. Indeed, this polymer family can bear the acylhydrazone functions in the main or side chains, but they are composed of water-soluble bio-based components, such as carbohydrates, amino acids, or nuclei acids. Thus, they can have interesting properties and many advantages compared to "classic" synthetic dynamers or biopolymers. 68,104 These three categories will be successively detailed in the next paragraphs.

Synthetic polymers with acylhydrazone functions in the main chain

Synthetic polyacylhydrazone with the functions in the main chain have several advantageous intrinsic structural properties compared to polymers whose acylhydrazone functions are located in the side chains. Beyond its dynamic behavior that was described above, it can be a homopolymer with a repeating unit bearing aldehyde and hydrazide functions, but also a copolymer with different repeating units bearing aldehyde or/and hydrazide functions. An interesting feature can be found when the repeating units are either dialdehydes or dihydrazides, because the resulting polymer is strictly alternating. 101

These water-soluble polyacylhydrazones can be prepared in an organic solvent for more convenient synthesis and purification, then diluted or dissolved in water (with the aim of using them in bioactivity applications). They can also be directly synthesized in water when the solubilities of both the monomers and the resulting polymer are high enough. Note that some syntheses, as described below, use organic co-solvents to enhance the solubility. 5,101

Synthesis strategies in organic solvents

The first polyacylhydrazone from dihydrazides and dialdehydes to our knowledge was synthesized by Michel and Murphey in 1963. 105 They reported many successful polymerizations in HMPA, DMF or DMSO, but struggled to re-dissolve the polymers once isolated from the solvent.

More recently, Cousins et al. 106 designed in 1999 one of the first DCLs based on these types of linkages, by mixing together four species in CH 2 Cl 2 (Figure 23). Through ESI-MS characterization, they observed all the combinations possible (more than 130 species) up to undecamers, with mainly cyclic compounds. The catalysis was performed with drops of TFA (also used for in situ deprotection of the aldehydes), and NEt 3 was chosen to freeze the equilibrium. Interestingly, some species were found to be amplified by the addition of

[18]crown-6, while the original distribution could be regenerated by adding KBr (3eq), which compete with the library components of the crown ether. Similarly, Voshell et al. 109 showed that a DCL made of two racemic compounds with the addition of a chiral template, (-)-adenosine, can result in the amplification of only one diastereoisomer species (Figure 24). In this study, the cyclic dimer (S,S) was amplified with an enantiomeric excess of 21% when templated by the (-)-adenosine, while no enantiomeric amplification was observed for the untemplated dynamic library. Ulrich's group prepared polyacylhydrazones from dihydrazides and dialdehydes, carefully designed to be able to complex plasmids, and vectorize them up to cell nuclei (Figure 25). The polycondensation was performed in methanol. It was demonstrated that the smaller macrocycles are preferentially formed at low concentration, whereas the linear polymers can be found at high concentrations. These dynamers effectively complexed dsDNA in aqueous buffers at biological pH. 110 Nevertheless, they also demonstrated that the addition of 4-hydroxybenzhydrazide can overcome these potential wells, and lead to an equilibrated final distribution.

Synthesis strategies in aqueous solvents

By using amphiphilic dialdehydes and dihydrazides, Frantz Folmer-Andersen et al. 113- 115 synthesized polyacylhydrazones in water, at pH = 2.0 (Figure 27A). These dynamers have the property to self-assemble into rods, because of hydrophobically-driven folding. The nucleation-growth effect can be observed, as hydrophobic interactions (and π-stacking) favor the addition of a monomer to the end of the polymer over the formation of smaller species.

More, the addition of co-solvents such as DMF or ACN prevents the formation of these secondary helical rod-like conformations. Surprisingly, the increase in temperature leads to an increase of the molar mass of the polymers (Figure 27B), and if the temperature is below LCST, the rod-like conformation is conserved. If the temperature is above LCST, the aggregation of polymers becomes too rapid, and large aggregates made of intertwined rods are created. dihydrazide and a dialdehyde containing poly(ethylene glycol) segment between phenyl groups (Figure 28A). The synthesis was interestingly performed only in MilliQ water (with drops of HCl) at 25 °C, yielding in a 28,000 g.mol -1 copolymer. Then, doxorubicin was added and attached to the hydrazide end-group thanks to its carbonyl moiety. These polymers were found to have a self-assembling behavior into spherical nanoparticles, which can be disrupted through the addition of acid (pH = 2.0) or light irradiation (254 nm, 0.1 mW.cm -1 ). These two stimuli can be combined in a synergistic manner to accelerate the disruption even more (Figure 28B). These experiments demonstrated that a hydrophobic drug, such as doxorubicin, can be covalently linked to a water-soluble polymer, and can be released under particular conditions at a useful location, showing the vector role potential of polyacylhydrazones. Hydrogen bonds can also be an asset for these polymers, as Daines et al. 7 demonstrated by using polyacylhydrazones in coatings. The synthesis was performed in water, from bis-functional levulinoyl esters and small water-soluble dihydrazides, yielding in a 50 % (w/v) final concentration, and a clear viscous solution for the case of adipic dihydrazide. Solutions showed linear and macrocycles species with degrees of polymerization around 6.

Water-soluble hyperbranched polymers were also developed. Indeed, Zhang et al. 117 synthesized them in an aqueous buffer at pH 4.0, then lyophilized. These dendritic hyperbranched polymers demonstrated precipitation upon heating, and re-solubilization upon cooling. Their cloud point depends on the solution pH. The authors also turned these dendrimers into hydrogels via cross-linking with dialdehydes at low pH (Figure 29). These hydrogels showed self-healing properties, no cytotoxicity, and good mechanical properties, making them good candidates for biomedical applications. Montenegro's group 68,118 also worked on amphiphilic dendrimers made of acylhydrazone linkages, by screening DCLs.

They developed dendrons able to transfect into cells thanks to their cationic charges, together with their hydrophobic tails which are able to complex functional DNA or siRNA, selfassembling into polyplexes as a means of vectorization.

Synthetic polymers with acylhydrazone functions in side chains

The introduction of acylhydrazone bonds on the side chain of polymeric structures can lead to complex dynamic polymers, by branching the pending aldehyde or hydrazide to the complementary function borne by grafting entities (Figure 30). A high variety of side-chain species can lead to multifunctional "brush-like" polymeric structures, providing many kinds of interactions, particularly useful for bioactive applications. Furthermore, this type of polymer can easily be converted into hydrogels by binding cross-linkers instead of functional species. 102,103 (1/1 v/v), the resulting amphiphilic polymer demonstrated self-assembling properties when combined with plasmid DNA or siRNA, forming polyplexes. These polyplexes demonstrated the ability to undergo transfection into human cells, hence releasing the plasmid in the cell (Figure 31). A higher-DP polymer was required to complex the plasmid DNA (> 4,500 bases) than siRNA (< 25 bases). The same strategy, with identical reaction conditions, was then applied to transfect mRNA. 121 Another approach was to replace the polymer scaffold by a peptidic scaffold instead of a "polyhydrazide" one, with pendant cationic and hydrazone moieties, branching it to hydrophobic aldehydes, for a successful transfection of plasmid DNA in human cells. 122 Fulton's group 123-125 also worked on polymer scaffolds, but with aldehyde moieties on pendant chains. Using different hydrazide residues, they developed a DCL in which the diversity point is the functionalization of the polymer scaffold (Figure 32). The resulting DCL demonstrated a high sensitivity to the surface potential of the templates (= biomacromolecules). The equilibrium condition being an aqueous buffer (pD = 4.5 in D 2 O), albumins (BSA and HSA), with pI = 5.5 (so positively charged in this buffer), displayed amplifications of anionic components over neutral, cationic or zwitterionic ones. On the other hand, pepsin, a negatively charged protein (pI = 2.2), displayed preference for functionalization with cationic residues. Interestingly, when all components except the cationic one are mixed with pepsin, the anionic one stand out, showing that the driving force of the amplification is more complex than the template surface potential alone.

Bio-based polymers with acylhydrazone functions in the main and side chains

As the previous polymers, bio-based polymers with acylhydrazone functions in the main and side chains (= biodynamers) can undergo a repeating unit exchange and continuous constitutional modifications. Thus, they have the particularity to be dynamic analogs of biopolymers. This statement implies that whereas the previously presented polymers must be based on synthetic repeating units with a hydrophilicity (and often bioactivity and biocompatibility) purpose, the biodynamers are made of biorelevant repeating units, most often with sufficient hydrophilicity. The challenges are therefore different, as it tends to find a synergistic manner to combine both the biorelevant characters of the monomers (and the biopolymer), and the dynamic bonds between them, to enhance the properties of the initial biomolecule. Due to their biocompatibility, acylhydrazone functions are one of the most used dynamic bonds for this kind of strategy. The applications for the three major families of biodynamers (glycopolymers, proteoids or analogs of proteins, and DyNA standing for dynamic DNA) will be described here. 68,104,130 The term "glycodynamers" can be applied to three different polymer designs. Dynamic proteoids, or analogs of proteins, with acylhydrazone reversible bonds has been studied by several groups. The use of amphiphilic dialdehydes, with pendant hexaglyme, together with amino acid hydrazides, was explored by Lehn's group. 8,104,130 These kinds of copolymers were qualified as "doubly" dynamic, because of the imine and acylhydrazone resulting bonds. In an acetate buffer (pD = 5 in D 2 O), they found that the polymerization is driven by the folding of these polymers, into spherical or cylindrical structures, and that the reaction rate and the final structure depends on the side-chain moieties on the amino hydrazides. Similarly, amino acids (Arg, His, Gly) have been turned into aminooxy and hydrazides terminations by Bouillon et al. 134,135 , and reacted with dialdehydes to form oxime and acylhydrazone bonds. The polymers were synthesized in DMSO but were then used in an aqueous buffer (pH = 7.0) with physiological saline conditions to effectively complex long DNA and siRNA, and to deliver siRNA in living cells (Figure 35). Cyclic peptides, functionalized with aldehyde moieties, have been used as a scaffold for the reaction with cationic hydrazides. These clusters were reported by Bartolami et al. 68,136,137 to be able to reversibly complex DNA and release it inside cells.

Reuther et al. 138,139 cleverly prepared peptides including some modified amino acids, bearing an aldehyde or hydrazide moiety as a side chain (Figure 36). Depending on the number of modified amino acids in the peptide, it became possible to cross-link the moieties to form loops, with dihydrazides and dialdehydes as cross-linkers, in a PBS/ACN (1/1 v/v) medium. These loops are used to change the quaternary structure of the peptides and can display an enhanced antimicrobial activity. Analogously to proteoids, polymers of amino acids analogs, the dynamic DNA (or DyNA) are polymers of nucleic acid analogs, with reversible covalent bonds such as acylhydrazone, while keeping a maximum of DNA properties. Lehn's group 88,104,140 prepared a polymer-scaffold with pendant nucleobases, in physiological pH medium, and showed their capacity to undergo monomer exchange and adaptability to external biological templates (Figure 37). These DyNA can also be synthesized using regular DNA as a template to obtain its complementary, similarly to the RNA.

Conclusion and Discussion

To conclude this review about water-soluble acylhydrazone (macro)molecules, the literature shows that the water-solubility of such molecules, depending on their molecular structure, is not trivial. Concerning single bond acylhydrazone molecules, the solubility can be predicted with physicochemical parameters such as logP. A common strategy is to synthesize the molecules in polar organic solvents (such as ethanol, DMSO, or acetonitrile), and to dilute them in water for the aimed use. The drawbacks of this approach are the inevitable presence of organic solvent in the final medium, and a relatively low final concentration, which can be obstacles for some valorizations. Parallelly, a direct synthesis in water can sometimes imply the requirement of organic co-solvents, as they can be used to ensure good solubilization of both the reactants and the products. However, if the goal is to have an organic-solvent-free solution, the structure of the reactants must be carefully designed, as the solubility of the final products mostly depends on them. As examples, carbohydrate-based molecules, or ionic ones, were found to be water-soluble without any organic solvent.

What is true for single acylhydrazone bond molecules becomes even more important for multiple acylhydrazone bond ones, or polyacylhydrazone, because the intra -and interhydrogen bonds become an obstacle when the number of acylhydrazone functions increases.

For these polymeric acylhydrazones, with functions in the main chain or the side chains, there are various applications in water medium. From the fundamental fields of rod-like structures to more applied domains such as enzyme inhibition or drug delivery, the dynamic aspect of these bonds provides great potency. In order to succeed in these applications, the polyacylhydrazones, whether synthesized in water or in organic solvents, need to be watersoluble enough despite the high number of hydrogen bonds.

Various strategies have been developed in the literature to ensure a satisfactory watersolubility such as: i) a solubilization in an organic solvent then a dilution in water, ii) a direct solubilization in a water-organic co-solvent, iii) a use of ionic monomers or pending moieties, iv) or the biopolymer development with the use of highly water-soluble monomers.

Despite being a major limitation for the development of polyacylhydrazones, the watersolubility is seldomly deeply analyzed in most of the papers cited in this review. In order to match the rapidly growing application field of these polymers, such studies would be welcome to help produce better-designed water-soluble polyacylhydrazones.

Figure 1 -

 1 Figure 1 -Schematic representation of the acylhydrazone function synthesis from hydrazide and aldehyde building blocks, leading to 1) single-bond or 2) multiple-bond acylhydrazones.

Figure 3 -

 3 Figure 3 -Condensation leading to different imine-type bonds. A) Hydrazide and aldehyde giving acylhydrazone. B) Hydrazine and aldehyde giving hydrazone. C) Aminooxy and aldehyde giving oxime. D) Amine and aldehyde giving imine.

Figure 7 -Figure 8

 78 Figure7-Mechanism of the formation of acylhydrazone using aniline as a catalyst.36

Figure 11 -

 11 Figure 11 -Example of an iron complex structure reached with acylhydrazone ligands, from Edward et al. 54

Figure 12 -

 12 Figure 12 -(A) Absorbance in presence of CN -and competing anions (L being the probe alone), and (B) the possible mechanism of the CN -sensing. Adapted from Hu et al. 65

Figure 13 -

 13 Figure 13 -Cross-linked film with self-healing properties. a) Polymer film, b) Film after scratch, c) Healed scratch after heating at 100°C for 64h. Adapted from Kuhl et al. 72

Figure 14 -

 14 Figure 14 -Visual color and fluorescence change after heating superimposed polymer films (P1 and P2). a) color before heating, b) color after heating, c) fluorescence after heating (at 365 nm). 75

Figure 15 -

 15 Figure 15 -Reaction conditions of the acylhydrazone synthesis by the combination of two isatin derivatives. Y = alkyl chains of different lengths. R = H or Br. NR 3 1 = NMe 3 or Py.From Bogdanov et al.77

Figure 16 -

 16 Figure 16 -Synthesis procedure of the biologically active acylhydrazone, from Guilherme et al. 82

Figure 17 -

 17 Figure 17 -Synthesis procedure of the tested compounds against several cancer cells, from Congiu & Onnis. Reaction conditions : (i) hydrazine hydrate, EtOH, reflux, 6 h ; (ii) EtOH, AcOH (cat), reflux, 2 h. 85

Figure 18 -

 18 Figure 18 -Reversible synthesis of acylhydrazone bonds (highlighted in dashed circles), allowing a quartet assembly of the guanosine hydrazides thanks to the addition of a metal ion (represented by the gray sphere). Adapted from Bulher et al. 89

Figure 19 -

 19 Figure 19 -Top: Synthesis of the acylhydrazone-based molecules, reaction steps and conditions: i) CH 3 SO 3 H, MeOH/ CHCl 3 , reflux, ii) hydrazine monohydrate, iii) aldehyde 1 equiv., H 2 O, rt, 1h. Bottom: R groups with reaction yields. Adapted from Dascalu et al. 90

Figure 20 -

 20 Figure 20 -Dynamic combinatorial library components for the identification of UDPgalactopyranose mutase inhibitors. The generation of the library was performed in an aqueous buffer, with aniline as a catalyst. Adapted from Fu et al. 91

Figure 21 -

 21 Figure 21 -Building blocks of the DCL generated in an ammonium formate buffer (pH = 4) without the use of an organic solvent. I -Sugar-based aldehydes. II -Mono-, di-and trihydrazides. Adapted from Ramström et al. 27

Figure 22 -

 22 Figure 22 -Structure of acylhydrazones obtained by the reaction of hydrazides 1-3 with volatile aldehydes a-c. The reactions were carried out in a water/ethanol mixture (2/1, v/v) at acidic pH. Adapted from Levrand et al.30

Figure 23 -

 23 Figure 23 -Monomers used by Sanders' group for the generation of their DCL. 106

Figure 24 -

 24 Figure24-Racemic DCL, yielding in a distribution of cyclic oligomers. The addition of (-)adenosine amplified the dimers proportions over the untemplated distribution, and the SS dimer was found with an enantiomeric excess of 21%.109

Figure 25 -

 25 Figure 25 -Monomers synthesized by Ulrich et al 110 , resulting in dynamers able to complex plasmids and vectorize them up to cell nuclei.

Furthering

  Michel and Murphey's work, Roberts and Thomas 111 synthesized polyacylhydrazones in DMF/H 2 O (1/1 v/v) at room temperature, from carbohydrate-based hydrazides and dicarbonyl compounds (Figure26). The obtained polymer from compounds 1a and 2,3-butanedione was insoluble in common solvents (except DMSO, boiling DMF, or water), but the addition of adipic dihydrazide to form a random copolymer led to watersoluble polymers at room-temperature.

Figure 26 -

 26 Figure 26 -Carbohydrate-based hydrazides and dihydrazides used for the synthesis of Roberts and Thomas' polyacylhydrazones. 111

Figure 27 -

 27 Figure 27 -A) Polymerization of dialdehyde 1 and dihydrazide 2. B) The heating of the rodlike structures (below LCST) reversibly aggregates them bigger rods. Adapted from Folmer-Andersen et al. 116

Figure 28 -

 28 Figure 28 -A) Water-soluble monomers for the synthesis of the polyacylhydrazones by Psarrou et al. B) Synergistic effect of the doxurobicin (DOX) release upon irradiation and medium pH (7.4 = blue triangles, 5.2 = green triangles, 2.0 = red squares). The black circles are the samples without irradiation at pH 2.0. Adapted from Psarrou et al. 25

Figure 29 -

 29 Figure 29 -Dendritic hyperbranched polymers developed by Zhang et al., that can be turned into hydrogels upon addition of dialdehyde cross-linkers. 117

Figure 30 -

 30 Figure 30 -Polymer-scaffolded dynamic bonds strategy, from Zhang et al. 103

Figure 31 -

 31 Figure31 -A) Polyplex formation process, from "polyhydrazide", named P, branched with cationic and hydrophobic aldehydes to complex mRNA. B) Microscopy images of Hek293

Figure 32 -

 32 Figure 32 -Polymer scaffold (P1) with pending aldehyde moieties, and hydrazide residues (from R1 to R4) with various charges. 125

Figure 35 -

 35 Figure 35 -Aminooxy-hydrazides monomers synthesized by Bouillon et al. 134 from amino acids and polymerized with dialdehydes to form a dynamer able to complex and deliver RNA in living cells.

Figure 36 -

 36 Figure36-Peptide sequence showing the modified amino acids bearing a hydrazide or aldehyde moiety (with the X). These pending moieties can then be cross-linked to create loops.139

Figure 37 -

 37 Figure 37 -Two different DyNAs prepared by Sreenivasachary et al., bearing pending nucleobases. 88

  4,107 Later, an equilibration of the same components with templates such as quinine, cinchonine, quinoline or quinuclidine led to surprising results. Indeed, diastereomeric templates such as quinine and quinidine led to a distribution of different oligomer macrocycles, amplifying the tetramer and the dimer respectively. 108 Lam et al.69 prepared a DCL from the PFm building block, and used acetylcholine as a template. Instead of giving a library made of macrocycles, as stated before,

	[2]-catenanes were obtained. As a matter of interest, only one diastereomer emerged. These
	results confirmed that chiral templates can differently modify DCL, and non-chiral templates
	can lead to chiral supramolecular structures. 69

  104,131 Firstly, they can be dynamers with modified carbohydrates, dynamically linked to the main chain.

	These types were studied by Ruff and Lehn 132 through polymerization with analogs of
	arabinofuranoside oligosaccharides, containing oxime bonds, but also by Roberts and
	Thomas 111 , whose polyacylhydrazone was already described above. Secondly, glycodynamers
	can also be made of carbohydrate-pending repeating units with acylhydrazone dynamic
	bonds, as studied by Kirker et al. 133 who cross-linked different types of
	aminopolysaccharides (hyaluronan and chondroitin) with poly(ethylene glycol)
	propionaldehyde, forming acylhydrazone bonds. They demonstrated that, once turned into
	films, these gels were able to accelerate the regeneration of wounded tissues. Ruff and
	Lehn 131 also worked on these types of glycodynamers, using dialdehydes and dihydrazides,
	both containing pendent carbohydrates (Figure 34), to obtain rod-like structures once the
	polymer was synthesized. They concluded that aromatic dialdehydes led to high molar mass

polymers, but with a slow reversibility, while aliphatic dialdehydes led to low molar mass polymers with a reversibility property under mild conditions. Both polymers can have applications in different fields, depending on if the preference goes to slow degradation or high adaptation. Finally, the last type of glycodynamers is made of a polymer scaffold with post-polymerization grafting of acylhydrazone-linked carbohydrate, as already studied before through the work of

Xu et al. 127

about the E. coli protein inhibition with galactose derivatives grafted on a polymer-scaffold.

Figure

34

-Carbohydrate-based monomers used by

Ruff and Lehn, with dihydrazide (1a, 1b)

and dialdehydes (2a, 2b, 3).

132

Acknowledgments

This work was financially supported by a grant from the French Ministry of Higher Education and Research.