Teja Šosterič

Consider the Worm

published or not. The documents may come

Consider the Worm

the situation today. I will argue that the advance of medical technology is creating a modern crisis that was impossible in early-modern England. This much is obvious; but contrary to what some might say, immortality projects are still being promoted as a better way of dealing with death. According to some texts, acceptance of physical death and decay of the body is a healthy way of accepting one's mortality. This is in line with how death is tackled in both Hamlet and Antony and Cleopatra, and is an indication that coming to terms with dying and a strong connection to the reality of death, decomposition of corpses (including getting eaten by worms) are still existent in today's society, forming an opposition to those who seek immortality with the aid of medicine and technology. To discuss this, I will review some recent texts that take different stances on the matter and show why it might be undesirable to strive for immortality. However, as I will argue, this does not mean that anxiety about death is not present; in fact, it is an essential part of life, and the drive behind most immortality projects.

In scene V of Act II of Antony and Cleopatra, a messenger arrives in Egypt and brings news of Antony, who had returned to Rome on urgent matters. Cleopatra is extremely anxious to receive the news, and worried about Antony. Before the messenger is even able to speak, she already assumes that the news is bad, and worries that some horrible fate has befallen her lover, exclaiming: "Antonius dead! -If thou say so, villain, / Thou kill'st thy mistress". 4 Two things are of significance here: firstly, Cleopatra is terrified of the possibility that Antony has perished. Considering the danger of sea journeys in the time in which the play is set, her worry is understandable: it is indicative of Antony's vulnerability. Regardless of his high social standing, Antony is as likely to suffer some horrible fate as a regular sailor, since his status and wealth would not protect him from potential shipwreck. Secondly, Cleopatra states that Antony's death would be the death of her. This might be meant metaphorically and dramatically, or refer to not actual, physical death, but rather the anguish his death would bring her; however, given how the play ends, one can assume that Cleopatra is being literal. Antony dying would mean that she too would die. But she doesn't say that she would kill herself in that case -she puts the blame of her death on the messenger. Later, she threatens to kill the messenger, saying "Rogue, thou hast lived too long". 5 Killing or shooting the messenger is a well-known metaphoric phrase dating from Plutarch; if nothing else, it shows how little human life was worth to those in power, the recipients of the news who could kill at will. Cleopatra, oscillating between wanting to shower the messenger in gold and wanting to torture and kill him, is exemplary of this.

We can see how in just those few short lines death and anxiety about death, symbolic and real death as well as precariousness of human life inform this play. One might argue that because of how different life was in Egyptian, Roman, and Shakespearian times compared to today, the relationship with death was also different. Modern medical advances sterilise our experience of death, which takes place not violently and brutally in front of our eyes, like Antony died in front of Cleopatra, but behind closed white curtains. The fragility of life in the time in which the play is set resulted in a relationship with death that was more organic, and some might say more realistic, than what we experience today. Indeed, it reaches beyond death, but how life itself was viewed by Egyptians. Randall Martin writes that Egyptians "[saw] themselves as physiologically embedded in the earth's organic cycles of material change, rebirth, and transmutation".6 Cleopatra's suicide is not sterile: it is a return to the earth. And here enter the worms. Martin turns to the edition of Antony and Cleopatra that contains notes by its editor, Michael Neill. Of course, the worm the Clown speaks of is the snake in the basket he carries, but as Martin points out, in Shakespeare, this word could mean other species of similar animals. The meanings become blurred when the Clown says "Give it nothing, I pray you, for it is not worth the feeding," and Cleopatra asks "will it eat me?"7 On the surface, they are talking about the snake, but Martin agrees with Neill, who notes that Cleopatra's question refers to the maggots in the grave. By asking that, she is expressing her anxiety of being eaten by maggots and her fear of bodily decomposition. On the other hand, she calls the bite of the worm/snake "sweet as balm, as soft as air",8 which indicates her acknowledgment of her physical mortality and her fate. She lets the worms bite her, and accepts the worms will eat her dead flesh.

But she is Cleopatra, and therefore must be aware that her reputation will not die with her -her "immortal longings" 9 indicate as much. Cleopatra's immortality project is her own position as queen, the fact that she has what we would now call a 'strong brand'. She knows she will not be forgotten. In fact, considering her propensity for the dramatic, I might conjecture that if she doubted her symbolic immortality, she would not have killed herself. Of course, her suicide is also an indication of her pride, because she does not want to be paraded in the streets of Rome as Caesar's conquest -to the Queen of Egypt, such a fate would be unacceptable, and suicide preferable. On the other hand, she presents her death as a return to Antony, her "husband", and as a ticket to immortality. Partially, refers to the afterlife in which she believes; partially, it's about the memory of her on Earth. To be sure, fame after death is a privilege of the higher classes, and there is no doubt that Cleopatra is not only in the highest position in her kingdom; her position was practically that of a goddess. But in Egypt, deities also die, at least as far as their physical form is concerned, that is something that both kings and beggars have in common. Death is the great social leveller, a position which is these days being challenged by the advance of medicine and technology.

Consequently, the idea that we will die, and be eaten by worms, is now shunned by many.

One might argue that even cremation is a way to sterilise death, since ashes are, after all, sterile. Embalming is another way we ward off decay; something we in fact have in common with the Egyptians, who mummified the most important members of their society. However, this is not something that is on Cleopatra's mind: she considers the worm. The exact reason behind this would be impossible to guess, but it might be related to Shakespeare's own ideas about death. Worms that eat flesh are after all not something unique to Antony and Cleopatra. In Hamlet, worms are referred to several times. In the scene where Claudius questions Hamlet about Polonius, Hamlet shrewdly declares that Polonius is at supper, "[n]ot where he eats, but where he is eaten. A certain convocation / of politic worms are e'en at him". 10 This, and the subsequent lines where he says "[y]our fat king and your lean beggar / is but variable service; two dishes, but to one table" 11 he shows no disgust at the idea of decomposition; in fact, he almost revels in it. To Hamlet, worms eating flesh are a part of the natural process of life. To be sure, he might be using the image of maggot-ridden flesh to make Claudius uncomfortable, but Claudius does not seem to be bothered by Hamlet's phrasing; he is more concerned with the fact that Hamlet has committed a murder.

Considering that cremation, embalming or such practices as Egyptian mummification were not available in those times, organic decomposition was much more accepted in society than 10 Hamlet, IV, iii, 2731IV, iii, -2732. 11 . 11 Hamlet, IV, iii, 2734-2735. it is today. That doesn't mean that it was not considered unpleasant; as Martin puts it, worms, or maggots (since worm in this case serves as an umbrella term) were "creatures exercising a trivial yet fearful agency in the world". 12 Being eaten by maggots was certainly not appetising, but was quite unavoidable.

Secondly, the answers Hamlet gives to Claudius are also political; this is clear from the use of the word 'politic'. Martin 13 and James Calderwood 14 both mention how this passage contains a reference to the Diet of Worms, the assembly at which Martin Luther responded to charges of heresy. This already invokes politics, but the political nexus does not end there: according to Martin, Hamlet's invocations of the power of the worm "dethrone humans as privileged consumers at the top of the food pyramid". 15 In a talk given at the Institute of Contemporary Arts, Steven Connor makes a similar point, saying that through this statement, "Hamlet's worms enact a radically egalitarian politics of the body that dissolve the imperious authority of the body politic". 16 Martin's point shows the error of perceived superiority of humans over other beings. The clever word play in the final sentence of Connor's speech links closer to politics, reminding us that decay is unavoidable for all things, even nations, states, and empires. But, as Connor is keen to point out in a different part of his talk, worms are not only agents of decomposition; they are invaluable in the circulation of organic matter, which fertilises the soil and promotes new life. In concrete terms, in the very unlikely event that immortality became possible for all humans, the planet might at some point reach full capacity, and the population would stagnate not only in keeping a stable number of individuals, but in literally keeping the very same individuals. It is unclear how that would affect our further progress as a species, but as a worst case scenario, it might result in a population that struggles with originality. From a personal perspective, I can imagine extreme dullness. It is only through accepting mortality and realising that death is a part of life that we can viably expect the formation of new life.

At least on the material level, there is constant recycling, death and life forming two indispensible parts of a whole. But why then do the grave-diggers, the clowns in Hamlet, 12 Martin, 141. 13 claim the opposite? The second clown asks: "Who builds stronger than a mason, a shipwright, or a / carpenter?" 17 The first clown does not know, but the second one replies: "when you are ask'd this / question next, say 'a grave-maker.' The houses he makes lasts / till doomsday". 18 This would seem in contrast with the previous idea, in which all things come to an end. However, the clown is not being literal; he does not believe that the grave itself will last forever. What he means is that regardless of the fact that bodies decay and all has an eventual end, death itself is eternal. Once they pass from life, the dead are dead -until the end of the world. The Christian undertones refer to a religious immortality project, by which the dead can enter new and better lands. We are also reminded of Hamlet's line in his famous soliloquy, where death is that "undiscover'd country, from whose bourn / No traveller returns". 19 At first glance, the finality of death invoked in this passage might seem at odds with the idea of an immortality project. However, the opposite is true: because death is the final and unavoidable end of our physical being, and because this is something that we fear, we become motivated to preserve some other part of ourselves for posterity, a memory or legacy that serves as symbolic continuation of life.

Immortality projects are one of the primary topics of Ernest Becker's book The Denial of Death, where he refers to them as causa sui. The book is premised on the idea that human civilisation is essentially built on the terror of death, and that one of the most important pursuits of humanity is the immortality project, which attempts to defy it. As Sam Keen lucidly puts it in his introduction, We achieve ersatz immortality by sacrificing ourselves to conquer an empire, to build a temple, to write a book, to establish a family, to accumulate a fortune, to further progress and prosperity, to create an information-society and global free market. 20 Becker also claims that each immortality project "is a lie that must take its toll as one tries to avoid reality", 21 because it is based on a fantasy, on an imagined heroism of the self that can cheat death. These premises are echoed in James Calderwood's book Shakespeare and the Denial of Death, the title of which was inspired by Becker's work and which not only builds 17 Hamlet, V, i, 3389-3390. 18 Hamlet, V, i, 3397-3399. 19 Hamlet, III, i, 1772III, i, -1773. 20 . 20 Sam Keen, introduction to The Denial of Death, by Becker (New York: The Free Press, 1973) on his ideas, but connects them to Shakespeare. About Hamlet, Calderwood writes that his immortality project was interrupted, as they often are, by the death of his father: this throws Hamlet into a spiral and "sets him off on a neurotically obsessive exploration of death in its various and most repellent forms". 22 The realisation of the lie of causa sui leads Hamlet to embrace and explore the more organic, tangible, gruesome truth of death: he considers the worm.

Immortality projects can change throughout a person's life, be destroyed or replaced, but presumably we all pursue some way of dealing with the terror of mortality. However, as Calderwood puts it, "the fate of all immortality projects [is] a demystifying return to the body and death". 23 This obvious, yet keen observation applies to the creator of the immortality project. Once dead, Cleopatra can no longer envision herself as the immortal queen of Egypt; any preservation of her person is left to those around her, who remember her … in her specific case, to this very day. That is how we learn that immortality projects can be successful. They provide comfort at the thought of death, because we know that some people managed to live on symbolically. For most of us, our goals are humbler that those of Cleopatra -often, we wish to be remembered by our families, or find comfort in having children. But Cleopatra found immortality in history books and in the works of Shakespeare, who matches her in fame, and whose symbolic existence also far exceeds his mortal life. It is easier to accept the idea of decay and decomposition of our human form when we have such examples of symbolic immortality.

In her book on the subject, Dr Muriel R. Gillick also invokes Becker's idea of immortality projects, and mentions how the "knowledge of our own mortality has shaped human culture". 24 She advocates for the acceptance of mortality, and directly attacks the modern medical pursuit of physical immortality I mentioned in the introduction. According to Gillick, chasing immortality and trying to drastically extend life is not only ineffective, but can also have dire social consequences. She writes that it would "create grave injustice if it is not equally available to all citizens". 25 She instead presents the immortality project as a healthy way of dealing with the knowledge of our inevitable death. I have previously implied 22 Calderwood, 117. 23 Calderwood,91. 24 Gillick,223. 25 Gillick,219. that physical immortality might result in decreased social productivity of the species as a whole: Gillick disagrees, instead saying that her "contention is not that it is good to be mortal because it promotes productivity, but rather that mortality is simply a reality". 26 In this, she echoes the telluric relationship demonstrated in Hamlet and Antony and Cleopatra, of death as a part of life which we might fear, but should embrace.

On the opposite end of this spectrum is Harris, who calls immortality "the Holy Grail". 27 In his chapter on the issue, he even mentions Shakespeare, saying that "Shakespeare's plays have all made such ideas familiar". 28 He is referring to fairies and magical creatures that feature in some of Shakespeare's plays, rather than immortality offered to humans, and he goes on to mention that many people would choose immortality if it were possible, even at cost of lower quality of life. But I would argue that the problem is not in whether we all want or do not want to live forever; it's whether or not it is ethical. Harris argues for human mental and physical enhancement through the use of gene manipulation and technology, but even though he mentions Karl Marx, 29 a person who most definitely concerned himself with class inequality, Harris neglects the class divide in these matters. He sees no problem with the fact that these enhancements and improvements would probably only be available to the selected and wealthy few. He even claims that justice should not enter into the picture. 30 For him, social inequality is the way of the world, something that always existed and always will; so why not perpetuate it. This is an idea that many, myself included, find unappetising. In her article on the matter, Ann Leckie heavily implies that increased longevity for some but not for all is immoral. However, her argument is naïve; she believes that those who want to be immortal and have the resources to do the research are "often thinking of their own, personal lives, and the lives of those they're surrounded by". 31 The truth is that they might be aware of the detrimental consequences of their actions on the underprivileged, and simply not care.

26 Gillick, 224. 27 John Harris, Enhancing Evolution: The Ethical Case for Making Better People (Princeton and Oxford: Princeton University Press, 2007) 59. 28 Harris, 59. 29 Harris, 186. 30 Harris, 62. 31 Ann Leckie, "Living to be 500 years old would be wonderful -but only for the rich," The Guardian, 16 Mar 2015, 16 July 2019 <https://www.theguardian.com/commentisfree/2015/mar/16/living-500-year-would-bewonderful-bill-maris> Harris certainly does not care about social equality, and does not think that creating "parallel populations of mortals and immortals" 32 is immoral; in fact, he relies on literature to make his point, claiming that such populations were "envisaged in literature and mythology". 33 However, the imaginary and magical immortals, such as fairies and elves, that do feature in literature, even Shakespeare's plays, are not the same as immortal humans. They are otherworldly: that's the point! Shakespeare's humans, regardless of their social position, are not immune to death. Both Hamlet and Cleopatra are arguably among the most powerful people in their respective polities, but in the end, they both perish. Their fates are indicative of the relationship with death of people in early modern England, where the relationship with the dead and the dying was much more direct. Of course, it would be impossible to predict how people in Shakespeare's time would feel about death if they had modern medicine at their disposal, but that is not the most important question to ask. It is whether physical immortality is even something we should be pursuing. Fear of death is natural, even evolutionary necessary, as it drives our survival instinct, but in order to achieve happiness in life we would perhaps be better off if we learned from Hamlet and Cleopatra, who did not ignore the inevitability of physical decay. Through their respective immortality projects, they learned to extend their symbolic existence. We too can learn to accept that our bodies will perish, and instead strive to improve the society in which we live for future generation, and so that they may remember us.

The fear of death is ever-present. It is most certainly true that most humans wish for some sort of immortality, some way of cheating death and prolonging life. However, chasing physical immortality is not only expensive and ineffective; it also increases social inequality that is already a problem in the modern world. The crisis of death which already significantly affects our perception of the gravely ill and disconnects us with the natural process of dying took on a new shape in the pursuit of physical immortality. While immortality would obviously mean the perpetual existence of the currently living humans, it would make no room for new life, new ideas, and perhaps even hinder further progress. If we consider the worm as not only the agent of decay, but also as the fertiliser of new life, and an agent of progress, we can more easily understand and accept that death is the physical end of an individual, but not of the species, and not of life in general. 32 Harris, 71. 33 Harris,71.

  Martin, 142. 14 James L. Calderwood, Shakespeare and the Denial of Death (Amherst University of Massachusetts Press, 1987) 17. 15 Martin, 142. 16 Steven Connor, "A Certain Convocation of Politic Worms," talk given at the Institute of Contemporary Arts, 23 April 2010.

Antony and Cleopatra II, v, 1082-1083.

Antony and Cleopatra II, v, 1144.

Randall Martin, Shakespeare and Ecology (Oxford: Oxford University Press, 2015) 137.

Antony and Cleopatra, V, ii,

Antony and Cleopatra, V, ii, 3776.

Antony and Cleopatra, V, ii, 3739.


Delphine M Allouche

Grégoire De Bisschop

Afaf Saaidi

Yann Ponty
email: yann.ponty@lix.polytechnique.fr

✉ Sargueil Bruno
email: bruno.sargueil@parisdescartes.fr

✉

RNA secondary structure modelling following the IPANEMAP workflow

come

It appeared that different probes provide complementary information that further improves RNA structure prediction. We therefore developed IPANEMAP, an experimental and computational workflow that models RNA secondary structure from different sets of RNA structure probing performed with different probes, and/or in different conditions and/or on mutants (Saaidi et al. Submitted). This workflow relies on medium or high throughput structure probing, and combines statistical sampling, clustering (Ding and Lawrence, 2003) and pseudo-potentials (Deigan et al, 2009). The method was shown to produce more accurate and stable predictions than other workflows developed to date, even when a single reactivity profile is available, while the availability of multiple reactivities was shown to increase robustness and, to a lesser extent, accuracy of the modeling (Saaidi et al. Submitted).

Below, we detail a whole IPANEMAP workflow, starting with experimental probing with DMS and/or CMCT and/or SHAPE reagent. Such probing can be carried out in various relevant conditions (varying température, Mg2+ concentration, introducing point mutations in the RNA to be modeled etc) (Saaidi et al. Submitted). Two versions of the experimental procedure (medium throughput and high throughput) are proposed, DMS and CMCT probing were adapted from Ehresmann et al. and Brunel et al. while the SHAPE probing is described in K. Weeks team publications (Karabiber et al., 2013;Low and Weeks, 2010a;Mortimer and Weeks, 2007;Smola et al., 2015a;Wilkinson et al., 2006Wilkinson et al., , 2008)). We then detail instructions for executing the IPANEMAP algorithm to obtain the RNA secondary structure model.

METHODS

1) RNA refolding

RNA purification protocol from cells or from in vitro transcription often include a denaturation step. In addition, RNA may also go through freeze and thaw cycles which also alter the native folding. In such cases, a denaturation-renaturation step, helps to homogenize RNA folding. In case a non denaturing protocol has been used to prepare the RNA this step may be skipped. Numerous refolding protocols have been proposed, their relative relevance may depend on the RNA considered (Chillón et al., 2015;Herschlag, 1995;Jaeger et al., 1993;Uhlenbeck, 1995) we propose below a "classical" protocol: a) Prepare two tubes with 6 pmol of RNA in 36 µl of water -Incubate for 5 minutes at 85°C. b) Add 4 µl of pre-warmed appropriate buffer (DMS, CMCT or SHAPE) and let cool down for 10 min at room temperature. Incubate for an additional 15 min at the probing temperature (30-37°C). Note: Reaction Buffers can be adapted to each situation, notably monovalent and divalent cations nature and concentrations may be changed to mimic at the best a physiological situation. MgCl2 can even be left out to destabilize tertiary structure. However, do not use Tris buffer with DMS because reacts with amine groups -Use potassium borate pH8 with CMCT and increase the buffer (any) concentration when using BzCN (SHAPE reagent) to compensate for the reagent acidification

2) RNA modifications:

a) With DMS:

• Add 4 µl of DMS (or 4 µl ethanol for the mock reaction) -Incubate 5 minutes at 37°C.

• Stop the reaction by addition of 400 mM of Tris pH7.5 (final) and immediately place on ice.

Or

b) With CMCT:

• Add 10 µl of CMCT (or 10 µl of H2O for the mock reaction) -incubate for 10 minutes at 37°C.

• Stop the reaction by adding 1 µl of tRNA (10 µg/µl). Put on ice.

Or c) With SHAPE Reagents:

• Add 5 µl of SHAPE reagent 10X (or 5 µl DMSO for the mock reaction). Note: In order to rapidly mix the solution, be careful to add the RNA solution onto the SHAPE reagent rather than the opposite

• Incubate for five reagent half-lives at 37°C (30 minutes for NMIA, 2 minutes for 1M7 or few seconds for BzCN) after which the shape compound is hydrolysed (no inactivation step required). Note: The solution will turn yellowish upon reaction with 1M7 or cloudy with BzCN.

Note 1: All these reagents are genotoxic and/or skin and/or eye irritant. They should be handled with care under a fume hood wearing nitrile gloves and safety glasses. Waste tips and dispose soiled materials in a saturated sodium hydroxide solution, or a 10% acetic acid solution for DMS and CMCT respectively. SHAPE reagents get hydrolysed relatively rapidly in water. Note 2: As the modification of one nucleotide may influence the rest of the structure, multiple modifications per RNA molecule should be avoided. As a rule of thumb we consider that such conditions are met when the amount of the full length product in at least 70% of that of that mock reaction. The modification conditions described above are pretty standard and work well for RNA between 200 and 500 nucleotides. They can be adjusted by modifying the incubation time and reagent concentration to get more or less modification (NB: concentration or time should be increased for shorter RNAs). This applies for DMS and CMCT, but not for SHAPE reagents which do not produce more than one modification per RNA under the above described conditions.

3) Ethanol precipitation

a) Add 1 µl of glycogen (20 mg/ml) or tRNA (10 mg/ml), 10% of ammonium acetate 5 M, 400 µl of 100% cold ethanol and incubate for at least 30 minutes at -20°C. b) Centrifuge for 30 minutes at 16 000 g at 4°C. Discard the supernatant. c) Wash with 400 µl of 70% cold ethanol. d) Centrifuge for 5 minutes at 16 000 g at 4°C. Discard the supernatant -repeat this step. e) Air dry the pellet and resuspend it in 10 µl of water.

STOP POINT: probed RNA can be stored at -20 °C for at least 6 months 4) Reverse transcription a) Transfer the resuspended RNAs to PCR tubes and prepare two PCR tubes with 6 pmol of RNA in 10 µl of water for the sequencing reactions. b) Add 1 µl of DMSO to the samples (modified RNA, mock reaction and sequencing reactions), denature for 3 minutes at 95°C then flash cool on ice. c) Add 3 µl of 2 µM D2-labelled primer to the sequencing reaction and 3 µl of D4-labelled primer to the modified / mock RNA. Incubate for 5 minutes at 65°C then anneal for 10 minutes at 35°C and store on ice. d) Add 4 µl of 5X RT buffer, 1 µl of the 10 mM deoxynucleotides mix and 1 µl of MMLV (H-) reverse transcriptase. For the sequencing reactions, also add 1 µl of 10 mM dideoxynucleotide. Incubate for 2 minutes at 35°C, 30 minutes at 42°C and 5 minutes at 55°C then store on ice. Note: For the sequencing reaction pick a nucleotide which is well represented all along the RNA sequence e) Add 5 µl of 5X RT Stop buffer. f) Combine one elongation from the modified RNA or the mock RNA with one sequencing reaction g) Add 250% (v/v) of 100% ethanol, incubate for at least 30 minutes at -20°C and precipitate the DNA as in 3.b-d). h) Air dry the pellet and resuspend in 40 µl of sample loading solution. Note: Resuspension will take time at room temperature. It can be accelerated by incubating the samples for 10 minutes at 50°C. STOP POINT: Elongated cDNA can be stored at -20 °C

5) Sequencing by capillary electrophoresis

a) Load side-by-side 20 µl of the elongated cDNA corresponding to the modified RNA and to the mock RNA. b) Run the capillary electrophoresis. c) Export the raw data as text files.

6) Data analysis with QuSHAPE

Different software such as QuSHAPE (Karabiber et al., 2013), RiboCAT (Cantara et al., 2017), CAFA (Mitra et al., 2008), FAST (Pang et al., 2011) andHiTRACE (Kim et al., 2013;Yoon et al., 2011) have been developed to analyse electropherogram. We mostly use QuSHAPE (Karabiber et al., 2013), please note that the experimental set-up might be slightly differ when using another analysis software. QuSHAPE can treat any type of chemical probing experiment after capillary electrophoresis separation of the RT products. Output files from ABi sequencer can be directly fed to QuSHAPE while those from CEQ8000 (AB Sciex) contains a header that needs to be removed beforehand (open the file with Excel and delete the header). For a description of QuSHAPE and its use please see (Karabiber et al., 2013) and the "help" of the software. As a result, QuSHAPE exports a tab delimited text file that contains the areas of the (+) ('RX') and (-) ('BG') reactions and their difference ("RX-BG") for each nucleotide, row-by-row. QuSHAPE also offers the possibility to normalize the results, however we have developed our own script for normalization including some specific treatment for DMS and CMCT, it also allows for the averaging of the reactivities issued from replicates (see next chapter). If using directly QuSHAPE output format the result file as described in "chapter XXX".

7) Normalization and averaging of the replicates

QuSHAPE data analysis yields raw reactivity values ("RX-BG") corresponding to the differences of the cognate area for the (+) and the (-) reactions for each nucleotide. Values corresponding to intrinsic reverse transcriptase stops need to be eliminated, the data are then normalized to allow the comparison between different experiments. To this end, two different approaches have been proposed in (Low and Weeks, 2010a), and should be used depending on the RNA length. a) Quantile normalization (RNA shorter than 300 bps) The 2% of the most reactive peaks of all reactivities (modified and unmodified condition) are removed, they empirically correspond to intrinsic stops (outlier). Then, the next 8 % of the most reactive peaks are averaged (M8) and finally all reactivities are normalized by the average (M8).

Or b) Parametric outlier removal (RNAs beyond 300 bps)

In this approach, peaks whose value is higher than the 75th percentile by at least 1.5 times the interquartile range are considered as outliers and removed. The 10% of the highest remaining reactivities (outliers removed) are averaged. All reactivities (outliers included) are normalized by this average value.

Typically, the probing of an RNA in a given condition is performed in triplicate before further analysis. To ease the normalization process as described above (Low and Weeks, 2010a), we have developed a python script (version 2.7) that allows the normalization, and yields for each nucleotide the average value of the replicates, and the standard deviation (The "Macro_CE" archive is available at https://github.com/afafbioinfo/Macro_CE ). The "SHAPE_Treat" folder contains:

• The Qu-shape-output folder: in which all the QuSHAPE.txt output files to be analysed should be put

Note:

The file title should be as follow: RNA_type-of-probe_replicate-number.txt (ex: TtLSU_DMS_2.txt)

• The CE_reactivity_profile folder: contains the resulting files (RNA_type_of_probe_number_of_triplicat.txt). This file contains three columns: nucleotide number; nucleotide type and normalized reactivity. • The Reactivity folder: contains normalized reactivities in a "RNA_probing_reagent.shape" file. Such file contains two columns: the first is the nucleotide number while the second is the corresponding SHAPE mean normalized value. • Macro_Qushape.py: python script • Macro_Qushape.config: This file that can be edited with any text editor, it contains all the parameters to be modified (nucleotides, lowervalue, threshold and Desactiv_threshold) for the normalization process.

The script can be customized for i) Parameters considered for the normalization and ii) Parameters considered for the final calculation of the reactivity i) Parameters for normalization:

• Nucleotides: SHAPE reagents hit any type of nucleotides, but reagents such as DMS and CMCT hit only two types of nucleotides. We therefore define the outlier on all nucleotides, but normalise the values considering the targeted nucleotides only. To configure this,enter the nature of the nucleotides targeted by the chemical agent (A,C,G,U for SHAPE reagents, A,C for DMS; G,U for CMCT). Note: Do not use any space after commas.

• Lowervalue: Reactivities are not supposed to be negative, notwithstanding, it happens that even after the scale factor correction some values are still negative. The negative threshold for which the reactivity will be set to zero can be configured. Default value = -0.1

ii) Parameters considered in the final analysis:

The averaged reactivity for each nucleotide is calculated from the replicates.

Rules for the analysis:

• If at least two reactivities are equal to -10 (outlier), the average is set to -10.

• If there is only one -10 in 3 sets of experiments, the mean is calculated with the two remaining reactivities.

• Threshold: This parameter has been added to characterize nucleotides which reactivity is variable and ensure the exclusive use of solid data. It is a maximal difference to the mean accepted for one of the replicate. Default value= 0.2. if the difference<0.2, for all the replicates, the average is calculated, in contrast if the difference >0.2, in one of the replicates, the reactivity for the nucleotide is set to -20. • Desactiv_threshold: It is the highest value for which the "threshold" correction will be applied (Default value= 0.6 ).

Once the parameters set, run the script by typing the following command line from the Qu-shape-output folder: > python2.7 Macro_Qushape.py

The script generates two output files: one in the "CE_reactivity_profile" folder that lists the normalized values for each reaction whereas in the "Reactivity" folder are found files with the normalized average reactivity for each nucleotide. This file has to be renamed to be fed into IPANEMAP (see below).

High throughput SHAPE probing: the SHAPE-MaP protocol (Smola et al., 2015b) Different protocols have been established for high-throughput SHAPE probing, the SHAPEseq on premature stops to detect the modified nucleotides (ref lucks), while the SHAPE-MaP rely on the property of the reverse transcriptase to make errors when copying a modified nucleotide in presence of Manganese ions (ref smola).

Although coupled to NGS, such techniques do not allow to probe a full transcriptome because each nucleotide should be read 5-20 000 times to obtain a significant reactivity. To overcome this limitation icSHAPE selects for modified RNA fragments before the reverse transcription steps, (spitale flynn).

Below is described the SHAPE-MaP protocol that we routinely use, note that it can be readily adapted to DMS (zubradt), or to any other probe.

BUFFERS AND SOLUTIONS

• RNA folding buffer (3X): 300 mM HEPES pH 8; 300 mM KOAc and 30 mM MgCl2.

• RNA denaturing buffer (10X): 500 mM HEPES and 40 mM EDTA.

• SHAPE-MaP pre-buffer (10X): 500 mM Tris-HCl pH8, 750 mM KCl and 100 mM DTT.

• Anhydrous DMSO.

• Multiplex Oligo (NEB E7335/E7500/E7710/E7730) • G50 sephadex (GE Healthcare) MATERIALS • Eppendorf 1.5 mL • PCR tubes (0.2 mL) • Mini column (Biorad)
• Ampure XP Beads (Beckman)

• Micro-volume spectrophotometer • Agilent Bioanalyser -Chip and Reagents for DNA analysis (usually available on sequencing platforms) • Thermocycler

RNA modifications with SHAPE reagents

Modification condition and negative control a) In PCR tubes add 10 pmol of RNA in 12 µl of water. Incubate for 3 minutes at 95°C, then immediately put on ice for 3 minutes. Note: In this protocol it is particularly important to use DNA-free RNA b) Flash spin before adding 6 µl of RNA folding buffer (3X) and incubate for 20 minutes at 37°C. Note: Other denaturation/renaturation protocol can be applied according to the RNA to be modified or any other specificity of the system studied. The Probing buffer can also be adapted (see above) c) In separate PCR-tubes add 1 µl of the SHAPE-probe at 100 mM (modification reaction) or 1 µl of DMSO (negative control). d) Add 9 µl of RNA folded directly on the SHAPE-probe and mix well by pipetting. Repeat this step for the negative control. e) Incubate for 6 minutes at 37°C for 1M7, and about 30 min for NMIA (approximately 5 times the half-life).

Store the PCR-tubes on ice.

Denaturing condition a)

In a PCR-tube add 5 pmol of RNA in 3 µl of water. Then, add 1 µl of RNA denaturing buffer (10X) and 50 % of formamide (v/v).

b) Incubate for 1 minute at 95°C. Note: For RNA with strong secondary structure may be incubated up to 5 minutes at 95°C. c) In another tube add 1 µl of SHAPE-probe at 100 mM. Make a short spin, then, quickly add the denatured RNA, mix well by pipetting and incubate for another minute at 95°C. Place the PCR-tube on ice.

RNA purification

Bring the final volume of each sample to 50 µl with water and place the tubes on ice time while preparing the G50 Sephadex mini-column. a) Place a mini column in a 1.5 mL Eppendorf and add 800 µl of G50 slurry. Spin for 30 seconds at 1000g. Discard the "eluate". b) Complete with G50 slurry and centrifuge for 30 seconds at 1000g. Discard the eluate. c) Repeat the centrifugation step twice. The first for 30 seconds, the second for 1 minute. d) Place the mini-column in a new 1.5 ml Eppendorf. Add RNA on the sephadex, spin for 2 minutes at 1000g.

Recover the eluate STOP POINT: RNA probed and denatured can be stored at -20 °C for at least 6 months.

cDNA synthesis by reverse transcription and purification

Prepare 5X SHAPE-MaP pre-buffer by combining an equal volume of SHAPE-MaP pre-buffer 10X and of dNTP stock at 5 mM. Then add an equal volume of 30 mM of MnCl2 to the SHAPE-MaP pre-buffer to obtain 2.5X SHAPE-MaP buffer. Note: It is important to prepare this buffer extemporaneously because, manganese oxidizes over time.

The reverse transcription can be performed with specific or random primers. We recommend specific primers for RNA up to 300-400 nt long.

Reverse transcription with specific primers (RNA up to 400 nt) a) In separate PCR tubes add 10 µl (corresponding to roughly 1 pmol) of the modified/unmodified/denatured RNA. Incubate for 3 minutes at 95°C then place on ice for at least 1 minute. b) Spin briefly, add 1 µl of 2 µM specific primers, incubate for 5 minutes at 65°C and cool down on ice. c) Add 8 µl of 2.5X SHAPE-MaP buffer, mix well by pipetting and incubate for 2 minutes at 50°C. d) Place the tube on ice and add 1 µl of SuperScript III reverse transcriptase. Flick the tube and spin briefly before incubating for 3 hours at 50°C. e) Inactivate the SuperScript III by heating samples at 70°C for 15 minutes. Place the samples on ice.

Or

Reverse transcription with random primers a) In separate PCR tubes add 10 µl modified/unmodified/denatured RNA. Incubate for 3 minutes at 95°C then place on ice for at least 1 minute. b) Spin briefly and add 200 ng/µl of random nonamer primers, incubate for 5 minutes at 65°C and cool down on ice. Note: Nonamers ensure a better sequence coverage than shorter or longer randomers. c) Add 8 µl of 2.5X SHAPE-MaP buffer, mix well by pipetting and incubate for 2 minutes at 25°C. d) Place the tube on ice and add 1 µl of SuperScript III reverse transcriptase. Flick the tube and spin briefly before incubating for 10 minutes at 25°C then for 3 hours at 50°C. Note: It is important to use a reverse transcriptase devoid of RNAse H activity to allow for the DNA second strand synthesis. e) Inactivate the SuperScript III by heating samples at 70°C for 15 minutes. Then, place samples on ice.

cDNA purification

Proceed as in "RNA purification" chapter "Medium throughput RNA structure probing with DMS, CMCT, or a SHAPE reagent" part 3, but do not bring the final volume to 50 µl. STOP POINT: cDNA can be stored at -20 °C for at least one year.

The steps described below are designed for Illumina sequencing. If using another type of NG sequencing the library preparation must be adapted. Also note that the ShapeMapper script will also have to be adapted to accept output files other than Illumina's. At least 3 different barcodes (libraries) need to be use, one for each condition: modified (BC1)/unmodified (BC2) and denatured (BC3). In each library, several RNA can be mixed if their sequence can be unambiguously distinguished. For RNA with identical or close sequences, use different barcodes for each condition.

Illumina library preparation and quality control Second strand synthesis (orange cap, E7770)

Prepare the samples on ice and follow the manufacturer instructions. Note: To improve the yield of DNA second strand synthesis, incubate for 2 hours instead of 1 hour as indicated in manufacturer's instructions.

dsDNA purification with Ampure XP beads a) Leave the beads on the bench for at least 30 minutes until they reach room temperature. Note: This step significantly increased the purification yield. b) In a 1.5 ml Eppendorf tube add 144 µl of the beads slurry on the "second strand synthesis" reaction, mix well by pipetting and incubate for 5 minutes at room temperature. c) Place the 1.5 ml Eppendorf on a magnetic rack and wait for 2 more minutes. Discard the liquid. d) Wash the beads with 200 µl of cold ethanol (80%). Note: Rotate the tube a quarter turn every 30 seconds to mix the beads and improve the wash. e) Discard the liquid and repeat the step. Spin briefly to discard all the liquid. f) Air dry the beads for 5-10 minutes. Note: Do not over dry the beads. The beads become mat when they are dry. g) Add 53 µl 0.1X TE, mix well and incubate for 2 minutes on magnetic rack. h) Remove 50 µl of purified DNA and place it in PCR tubes. STOP POINT: dsDNA can be stored at -20 °C for at least one year.

End-It Repair and Adaptors Ligation (green and red cap, E7770)

Samples are prepared of ice following the manufacturer instructions.

Ligated DNA purification with Ampure XP beads

Proceed as for "dsDNA purification" but add 85 µl of beads and elute with 15 µl of 0.1XTE. STOP POINT: dsDNA can be stored at -20 °C for at least one year.

Library enrichment (bleu cap from E7770 and barcode from E7335/E7500/E7710/E7730)

Follow the manufacturer's indications. Note: Typically, 8-10 PCR cycles are enough and avoid the formation of adaptor dimers.

Library purification with Ampure XP beads

Proceed as for "dsDNA purification" but add only 45 µl of beads and elute with 25 µl of 0.1X TE. Quantify the samples by OD260. STOP POINT: dsDNA library can be stored at -20 °C for at least one year.

Library verification with a Bioanalyzer (Agilent DNA 1000 Chip)

Incubate the gel dye-mix at least 30 minutes at room temperature before using. Follow manufacturer's indications. Notes: For a clear image load 5 ng sample in a final volume of 6 µl per well .

If in the elution profile you observed a thin peak at  85 bp it is due to the presence of primers. It is suitable to perform another purification with Ampure XP beads to improve the sequencing quality.

If in your elution profile you observed a thin peak at  120 bp, it is due to the presence of adaptor-dimer. To reduce this peaks dilute your sample ten times with 0.1X TE, verify the library quality on a new chip and use the diluted sample for sequencing.

Illumina sequencing

Notes: Separate projects or a project including multiple RNA can be loaded on the same Illumina sequencing. If loading separate projects with RNA of the same or similar sequences be careful to use different bar codes for each project (see above). As for the number of projects that can be loaded on the same run keep in mind that each nucleotide should be covered about 20 000 times (the significance threshold being 5000 reads).

Medium throughput RNA structure probing with DMS, CMCT, or a SHAPE reagent

	BUFFERS AND SOLUTIONS
	• Specific for DMS:
	o DMS (diluted in ethanol 1:12) CAS # 77-78-1
	o DMS buffer (10X) : 400 mM HEPES pH7.5, 1 M KCl, 10 mM MgCl2
	o DMS stop reaction: 400 mM Tris pH7.5
	o Sodium hydroxide
	• Specific for CMCT:
	o CMCT (42 mg/mL) CAS # 2491-17-0
	o CMCT buffer (10X) : 500 mM potassium borate pH8, 1 M KCl, 10 mM MgCl2
	o 10 % acetic acid
	• Specific for SHAPE
	o Shape reagent 10X in anhydrous DMSO : 40 mM 1M7 (CAS # 4693-01-0) or 40 mM NMIA
	(CAS # 10328-92-4) or 400 mM BzCN (CAS # 613-90-1)
	• Common to the three types of probing:
	o Ammonium Acetate 5M
	o 70 % and 100 % cold ethanol

o Dideoxynucleotide 10 mM, either ddTTP, ddATP or ddCTP o MMLV Reverse Transcriptase RNAse H (-) 200 u/µL (e.g Promega M1705) o MMLV RT buffer 5X (e.g Promega M5313) o Deoxynucleotide mix dATP, dTTP, dCTP and dITP, 10 mM each o Two differently labelled fluorescent primers at 2 µM in water, e.g. D2-PA and D4-PA (Sigma-Aldrich) if running the sequences on a CEQ8000 capillary sequencer. Note: Primer sequence should be chosen at least 50 to 75 nucleotides 3' (downstream) to the region of interest o RT Stop buffer 5X: 1.2 M sodium acetate pH5, 40 mM EDTA, 4 mg/mL glycogen. o Sample loading solution (deionised formamide) o Glycogen 20 mg/mL o Yeast tRNA 10 mg/ml MATERIAL • Usual disposable labware • Capillary electrophoresis (ABI or Ceq8000 AB Sciex) • Thermocycler • Dry bath • Refrigerated centrifuge • Fume hood

https://github.com/afafbioinfo/ReadsMap), but probably due to a higher error rate of this sequencing technique we found the results obtained less accurate. (Busan and Weeks, 2018) ShapeMapper2 generates four different output files:

Data analysis is carried out using ShapeMapper2 as described in

-Condition.map: contains a table composed of four columns: the first is the sequence position, the second contains SHAPE reactivities with the standard errors in the third column for each nucleotide (fourth column).

-Condition.shape: is a tab delimited text file with the of the nucleotide and the associate SHAPE reactivities. This file can be used as input for IPANEMAP software. Notes: A SHAPE reactivity egal to -999 corresponding to a nucleotide which isn't mapped 5000 times.

-Condition_histogram.pdf: is a graphical summary of the sequencing. Those histograms represents the mutation rate for each condition, the read depth and the reactivities analysis. In a successful experiment the mutation rate in the modified condition is higher than the untreated condition, the majority of nucleotides have read above 5 000 and finally reactivities are positive with a small standard errors.

-Condition_profiles.pdf: contains three graphs. The first graph represent SHAPE reactivity value for each nucleotide. The second graph correspond to the mutation rate for each condition (modified,unmodified and denatured) and for each nucleotide. The final graph shows the coverage for each condition. These graphs allow the experiment validation or are helpful for troubleshooting the problems.

Predicting structural models from one or several probing experiments using IPANEMAP Once reactivity profiles have been produced in one or several conditions, IPANEMAP can jointly integrate them and produce one or several secondary structure models that are well-supported by the experimental data.

SHAPE reactivities can be interpreted as follows:

• R= -20: Nucleotides with variable values (when multiple values available).

• R= -10: Removed outliers.

Note: Negative values are not interpreted by IPANEMAP

• Lowervalue < R < 0.4: Nucleotides with low reactivity.

• 0.4 < R < 0.7: Nucleotides with medium reactivity.

• 0.7 < R: Nucleotides with high reactivity.

Installing IPANEMAP.

IPANEMAP Note: Depending on your operating system, the above-mentioned software may be installed through some package manager, or using and environment manager such a Conda.

Downloading IPANEMAP.

The software can then be downloaded from: https://github.com/afafbioinfo/IPANEMAP Download a ZIP archive of the most recent version of the software and unzip it in a directory of your choice.

Setting up the analysis

Throughout the following, we assume that the name of the analyzed RNA is 'myRNA', and that the conditions are named 'Cond1', 'Cond2'... 1. Prepare a FASTA file named 'myRNA.fa' whose first line contains the FASTA header (eg '> myRNA') and the second line consists of the RNA sequence (eg 'CCCAAAGGG'); 2. Prepare reactivity files 'myRNACond1.txt', 'myRNACond2.txt'... (for instance 'HIVgRNADMS.txt', 'HIVgRNANMIA.text …) Files should be formatted to contain one line per nucleotide, each consisting of: i) the nucleotide number; and ii) the reactivity score, both separated by space/tabulations; 3. Copy/move reactivity files in 'Constraints_Probing' subfolder of the IPANEMAP folder.

Note: All names/paths can be changed to any other name (alphanumerical characters only

), but such modifications should be reflected in the file names. The README.md file also describes how to set up many additional options.

Running the analysis and retrieving the results

1) Open a terminal and move to the IPANEMAP installation folder 2) Execute the command: python2.7 IPANEMAP.py --RNA myRNA.fa --cond Cond1 Cond2… > Best.txt Note: Under some operating systems, "python2.7" may be safely replaced by "python" in the above command Note: Many additional options, including the possibility to specify user-defined hard constraints are available and documented in the `README.md` file found at the root of the installation.

Dominant conformations.

The main results are now located in the file 'Best.txt' featuring one or several dominant secondary structure models, along with additional information. For instance:

Structure dG #SupportingConditions BoltzmannProbability (((...)))...(((...)))

-4.3 1 0.546 indicates a single dominant conformer, associated with a secondary structure in dot-bracket notation. The Turner free-energy, number of conditions supporting the conformer, and the accumulated pseudo-Boltzmann probability across conditions, are also reported for each conformer Complete conformers list. The complete list of the main conformations across the pseudo-Boltzmann ensemble can be found in a file 'output/Centroids.fa'. The file is simply a list of sequence/structures, and includes conformers that are dominated, ie either less stable or less supported by experiments than the ones reported in the primary output. IPANEMAP workflow was extensively tested using different benchmark RNA and multiple probing conditions (Saaidi et al. Submitted). It appeared from that study that IPANEMAP provide more accurate and stable results than other existing workflow when using only one set of probing conditions. It is safer to use two conditions because if one of them is artifactual the second will correct the problem. However it is important to note that the prediction is never improved compared to the results obtained with the better of the two conditions. Modeling accuracy is when using at least three different conditions (Saaidi et al. Submitted).

ACKNOWLEDGMENTS:

Research in B.S laboratory is funded by the CNRS, the University of Paris and a grant from "la Fondation pour la Recherche Médicale" (FRM DBI20141423337) awarded to BS and YP laboratories. AS and DA were recipient of a phD fellowship from FRM (FRM DBI20141423337), GdB was recipient of a fellowship from the French Ministry for Education and Research (MESR).


Anaïs Coffinet

David Specklin

Laure Vendier

Michel Etienne

Antoine Simonneau

Frustrated Lewis Pair Chemistry Enables N2 Borylation by Formal 1,3-Addition of a B-H Bond in the Coordination Sphere of Tungsten

Keywords: bis(pentafluorophenyl)borane, dinitrogen, frustrated Lewis pairs, phosphines, tungsten

come

Frustrated Lewis Pair Chemistry Enables N 2 Borylation by Formal 1,3-Addition of a B-H Bond in the Coordination Sphere of Tungsten

Anaïs Coffinet, David Specklin, Laure Vendier, Michel Etienne, and Antoine Simonneau* [a] Dedicated to Professor Max Malacria on the occasion of his 70 th birthday.

Abstract:

The first example of a formal 1,3-B-H bond addition across the M-N≡N unit of an end-on dinitrogen complex 5 has been achieved. The use of Piers' borane HB(C6F5)2 1 was essential to observe this reactivity and it plays a triple role in this transformation: i) electrophilic N2-borylation agent, ii) Lewis acid in a frustrated Lewis pair-type B-H bond activation and iii) hydride shuttle to the metal center. This chemistry is supported by NMR and solid-state characterization of products and intermediates. The combination of chelate effect and strong  donation in the diphosphine depe ligand was mandatory to avoid phosphine dissociation that otherwise led to complexes where borylation of N2 occurred without hydride transfer.

The mild conditions under which the inert N2 molecule can be activated with metal complexes have sparked continual research efforts devoted to the fixation of nitrogen into N-containing compounds. [START_REF]Transition Metal-Dinitrogen Complexes: Preparation and Reactivity[END_REF] A distinct property of soluble transition-metal N2 complexes is that, beyond formation of N-H bonds of relevance for ammonia synthesis, [2][3][4] N-B, [START_REF] Ishino | [END_REF] N-Si [5c,6] and N-C bonds [7] can be formed by the reaction of metal-bound dinitrogen with various functionalizing agents, paving the way for the direct conversion of N2 into value-added nitrogen compounds. Thanks to the mild hydride donor reactivity and to the thermodynamic driving force offered by the formation of strong N-Si or N-B bonds, hydrosilanes and -boranes are attractive substrates for the synthesis of boryl-or silyl-amines or hydrazines. Indeed, they react across reactive M-N bonds of complexes incorporating a 4- [5b,6c-g] or 6e --reduced [8] N2 ligand (Scheme 1a). Diphenyldiazomethane (Ph2CN2), the formal addition product of N2 with diphenylcarbene, undergoes 1,1-hydroboration of the terminal N using Piers' borane HB(C6F5)2 (1). [9] Applications of these transformations towards the synthesis of nitrogen compounds have remained restricted to N2-derived nitrides. [8] We reported recently the silylation and borylation of a nonreduced N2 ligand using a hydro-silane or -borane. [5c] Our method took advantage of the formation of a frustrated Lewis pair (FLP) [10] between an end-on dinitrogen complex and B(C6F5)3 that could heterolytically cleave Si-H [11] and B-H bonds. [12] This allowed functionalization of the terminal N, while the hydride issued by the silane or borane was trapped in the inert anion [HB(C6F5)3] -(Scheme 1b). In the prospect of using this approach to realize a synthetic cycle, the absence of hydride transfer to the metal, due to the poor hydride donor ability of [HB(C6F5)3] -, appeared detrimental. Indeed, the reuptake of N2 by group 6 phosphine complexes upon photolysis of the corresponding hydrides [13] represents an attractive route to close a synthetic cycle. [14] This prompted us to examine the reactivity of HB(C6F5)2 ( 1), [15] speculating it could act both as a Lewis acidic borylation agent and as a more potent, [START_REF] Heiden | The hydride donor ability GH-of the [H2B(C6F5)2] anion is 61 kcal/mol vs. 65 kcal/mol for [HB(C6F5)3[END_REF] less sterically hindered boron-to-metal hydride shuttle in the form of the [H2B(C6F5)2] -anion that would be generated after the B-N bond formation/B-H bond activation event. Herein, we report on our studies of the reactivity of 1 and show that a judicious choice of phosphine ligands in Hidai-Chatt tungsten N2 complexes leads to the formal 1,3-B-H bond addition across a W-N≡N moiety (Scheme 1c).

Scheme 1. a) 1,2-E-H bond addition across M-N bonds in strongly activated N2 complexes (NPN = (PhNSiMe2CH2)2PPh, Cp'=1,2,4-trimethylcyclopentadi- enyl, Cp*=1,2,3,4,5-pentamethylcyclopentadienyl); [5b,6e,g,8b] b) FLP-inspired functionalization of N2; [5c] c) 1,3-B-H bond addition reported in this study.
We reacted cis-[W(N2)2(PMe2Ph)4] (2) [17] with 1 equivalent of 1 at room temperature in toluene-d8. Analysis of the reaction mixture by 31 P NMR showed diagnostic signals of the [HB(C6F5)2•PMe2Ph] adduct, characterized by a poorly resolved quartet at  -4.9 (JPB = 122 Hz, no 183 W satellites). [18,[START_REF] Takahashi | Complex 2 has been reported to lose a phosphine ligand upon treatment with HCl in dimethoxyethane[END_REF] To reach full and selective conversion of 2, we had to add up to three equivalents of 1. The 1 H NMR spectrum of the reaction mixture showed two broad signals ( 3.63, -0.75) that sharpened upon 11 B decoupling, suggesting the presence of B-H moieties. The deshielded signal characterizes [HB(C6F5)2•PMe2Ph] while the high field one may be assigned to that of protons interacting with the tungsten center in the new complex 3 (Scheme 2). The 31 P{ 1 H} NMR spectrum of 3 showed a A2X system and a 11 B NMR experiment revealed, in addition to the signal for 6. An X-ray diffraction study performed on crystals of 3 confirmed that one N2 ligand was borylated (Figure 1). [20] The N-N bond length is 1.280(4) Å, in the range of NN double bonds. [START_REF] Ogawa | 1.189[END_REF] Interestingly, the [H2B(C6F5)2] -anion coordinates to the metal center, now in the formal oxidation state +II. The two hydrogens occupy the coordination sites left vacant after the loss of one N2 and one PMe2Ph ligands. Prolonged, mild heating of the reaction mixture led to decomposition of 3 without evidence for the formation of metal hydrides.

Scheme 2. Reaction of N2-complex 2 with 1.

Next, we checked the reactivity of trans-[W(N2)2(dppe)2] (4) [22] [dppe = 1,2-bis(diphenylphosphino)ethane] and its depe [1,2-bis(diethylphosphino)ethane] analogue 5 [23] , with the assumption that chelation could prevent phosphine dissociation. This would force a modified geometry for the [H2B(C6F5)2] --W interaction that could promote W-H bond formation. The reaction of 4 with 3 equivalents of 1 yielded a product 6 very similar to 3 as revealed by NMR and confirmed by X-ray diffraction. [24] One phosphorus dissociated from tungsten and was trapped by 1. A [H2B(C6F5)2] -anion was formed concomitantly with the borylation of one N2 ligand (Scheme 3).

Under identical conditions, complex 5 quickly and selectively converted to a new compound 7 without phosphine dissociation, as a singlet was observed in the 31 P NMR spectrum ( 39.9 vs.  36.2 for 5). A hydride signal showing coupling to four equivalent phosphorus was found in the 1 H NMR spectrum (-4.47, quintet, 2 JHP = 38 Hz). Borane 1 was still present, but the anion [H2B(C6F5)2] -was not detected. Crystallization of 7 (84% yield) confirmed borylation of the N2 ligand as well as hydride transfer to the metal, realizing the targeted formal 1,3-B-H bond addition (Scheme 3, Figure 1). The thus-formed boryldiazenido ligand trapped another equivalent of 1, forming a Lewis pair. From the electron-withdrawing effect of the two boron groups ensues a noticeable N-N bond elongation [1.378( 4) Å [START_REF]This is comparable to the N-N bond[END_REF] ]. The electronrichness of the depe ligand combined with the chelate effect prevents phosphine dissociation, leading to effective hydride transfer. Since at least one equivalent of Piers' borane was in excess, we repeated the reaction with only 2 equivalents of 1. 31 P NMR analysis of the reaction mixture revealed selective transformation to a new compound 8 with equivalent phosphorus nuclei ( 32.4). Key spectral features for 8 were signals in the 1 H and 11 B NMR spectra assignable to the [H2B(C6F5)2] -anion ( 1 H{ 11 B}:  -1.6, singlet; 11 B:  -40.8, triplet, JBH = 71 Hz); shielding of its proton resonances was a clear indication of interaction with the W center. Thus, it can be proposed that 8 is an intermediate en route to 7 before hydride transfer (Scheme 4). 8 crystallized from the reaction mixture in 79% yield, allowing determination of its structure (Figure 1). As anticipated, the interaction of the W center with [H2B(C6F5)2] -formed after borylation of the N2 ligand is different from that in 3 and 6. In 8, the dihydroborate anion occupies a single coordination site trans to the boryldiazenido group, while in 3 and 6 the hydrides occupy one apical and one equatorial sites. The isolation of 8 allowed us to check the influence of 1 on the hydride transfer: indeed, addition of excess 1 to a solution of 8 quickly converted the latter into 7. Lewis pair formation of 1 with the boryldiazenido ligand in 8 might help trigger W-H bond formation by rendering the metal center more electrophilic. The formation of 8 confirms the triple role of Piers' borane: it acts as a N2-borylation agent, a Lewis acid able of B-H bond activation and a hydride shuttle. The two latter roles are performed by the second equivalent of 1, which is regenerated after the hydride transfer and subsequently trapped by the boryldiazenido ligand. This prompted us to verify whether a slight excess of 1 would be sufficient to perform the 1,3-B-H bond addition, this time affording the tungsten hydride 9.

Upon addition of 1.1 equivalent of 1 to a solution of 5, a mixture of 5, 8 and a third major compound ( 31 P NMR  35.6) was obtained after 10 min at room temperature. On the basis of 1 H{ 11 B}, [START_REF] Takahashi | Complex 2 has been reported to lose a phosphine ligand upon treatment with HCl in dimethoxyethane[END_REF][20][START_REF] Ogawa | 1.189[END_REF][22][23][24][START_REF]This is comparable to the N-N bond[END_REF][26][27][28][29][30][31][32], this compound was formulated as 10, the adduct of 5 with 1 (Scheme 4). When heated to 40 °C for 4 h, this mixture evolved into a single compound 9 isolated in 78% yield. In the 1 H NMR spectrum, a characteristic hydride quintet at  -2.62 ( 2 JHP = 27.6 Hz) was found. An X-ray diffraction study confirmed a structural analogy between 9 and 7, the difference lying in the absence of interaction of the boryldiazenido ligand with a second equivalent of 1. 9 could also be selectively prepared by mixing equimolar amounts of 5 and 8 for 10 d at room temperature. 9 reacted with one equivalent of 1 to give a roughly 1:1 mixture of 7 and 8 within 10 min, indicating that W-H bond formation was reversible in the presence of 1, as observed with hydrides of group 3 [26] and 4. [27] After 1 h, almost all 8 was converted to the tungsten hydride 7 (Figure 1, Scheme 4). In addition to 1,2-hydroboration of M-N bonds that has been reported for strongly activated N2 complexes, we have shown herein the possibility to achieve 1,3-B-H bond addition across a W-N≡N unit in which the N2 ligand is only weakly activated. This was made possible by taking inspiration from the FLP chemistry of hydroboranes [12] and from a rational experimental approach based on our previous findings. [9] The strong electrophilicity of 1 is essential for the B-H activation step: reactions of 5 with 9borabicyclo[3.3.1]nonane (9-BBN) or catecholborane were unproductive under the experimental conditions reported herein. We are currently exploring different metal-ligand combinations and other hydroboranes in order to study the generality of this transformation and apply it to the synthesis of boron-nitrogen compounds.

  , Dr. D. Specklin, Dr. L. Vendier, Prof. Dr. M. Etienne, Dr. A. Simonneau LCC-CNRS, Université de Toulouse, CNRS, UPS [HB(C6F5)2•PMe2Ph], two different resonances for 3 at  30.5 and -1.

Scheme 3 .

 3 Scheme3. Reactions of N2-complexes 4 and 5 with 1.

Figure 1 .

 1 Figure 1. Molecular structures of compounds 3 and 7-9 in the solid state. All hydrogens have been omitted except those bound to B or W. Selected distances (Å) for 3: W1-N1, 1.781(3); N1-N2, 1.280(4); N2-B1, 1.373(5); B2-H100, 1.15(4); B2-H101, 1.06(6); W1-H100, 2.05(5); W1-H101, 2.05(4). For 7: W1-N1, 1.807(2); N1-N2, 1.378(3); N2-B1, 1.392(5); N2-B2, 1.586(6); W1-H1, 1.91(4). For 8: W1-N1, 1.789(2); N1-N2, 1.256(3); N2-B1, 1.368(4); B2-H2C, 1.10(3); B2-H2D, 1.17(3); W1-H2C, 2.22(4); W1-H2D, 2.35(3). For 9: W1-N1, 1.807(2); N1-N2, 1.301(3); N2-B1, 1.369(3); W1-H1, 1.66(2).

Scheme 4 .

 4 Scheme 4. Complexes observed in the reactions of 5 with 1.

route de Narbonne, BP44099, F-31077 Toulouse cedex 4, France. E-mail: antoine.simonneau@lcc-toulouse.fr Supporting information for this article is available free of charge at: https://doi.org/10.1002/chem.201904084.

Acknowledgements A. S. would like to thank Dr. Nicolas Mézailles, Dr. E. Alessandra Quadrelli and Dr. Sylviane Sabo-Etienne for insightful discussions. A. C. is grateful to the French Ministry of National and Superior Education and Research (MENESR) for a Ph. D. fellowship. A. S. acknowledges the European Research Council (ERC) for funding (Grant Agreement 757501). All authors are indebted to the French National Center for Scientific Research (CNRS) for financial support.