Dr D I Danylchuk

A S Klymchenko
email: andrey.klymchenko@unistra.fr

Dr S Moon

K Xu

Keywords: Fluorescent probes, solvatochromism, membranes, super-resolution microscopy, lipid order

Tailor-made switchable solvatochromic probes for live-cell superresolution imaging of plasma membrane organization

Dmytro I. Danylchuk, [a] ‡ Seonah Moon, [b], [c] ‡ Ke Xu, [b], [c]* Andrey S. Klymchenko [a]* Abstract: Visualization of the nanoscale organization of cell membranes remains challenging because of the lack of appropriate fluorescent probes. Here, we introduce a new design concept for super-resolution microscopy probes that combines specific membrane targeting, ON/OFF switching, and environment sensing functions. In this design, we propose a functionalization strategy for solvatochromic dye Nile Red that improves its photostability. The dye is grafted to a newly developed membrane-targeting moiety composed of a sulfonate group and an alkyl chain of varied lengths, which ensures high brightness and lipid-order sensitivity of the probes and tunes their binding/unbinding-induced ON/OFF switching required for single-molecule localization microscopy. While the longchain probe with strong membrane binding, NR12A, is suitable for conventional microscopy, the short-chain probe NR4A, due to the reversible binding, enables first nanoscale cartography of the lipid order exclusively at the surface of live cells. The latter probe reveals in plasma membranes the presence of nanoscopic protrusions and invaginations of lower lipid order, suggesting a subtle connection between membrane morphology and lipid organization.

The complex nature of cell plasma membranes raised intensive research in the last decades, stimulated by the hypothesis of membrane microdomains (lipid rafts). [1] In model membranes, these domains are identified in terms of lipid order, [2] where liquidordered phases (Lo) rich in sphingomyelin and cholesterol "swim" in a pool of disordered phase (Ld) formed by unsaturated lipids. The challenge to visualize lipid domains in cells is linked to their highly dynamic nature, nanoscopic size, and non-flat geometry of plasma membranes with protrusions and invaginations. [3] One approach to visualize membrane organization is fluorescent labeling of lipids, such as sphingomyelin [4] and ceramide, [5] but labeling may alter properties of lipids. [6] Another approach is to label proteins that specifically target sphingolipids, [7] although the effect of these large molecules on lipid order is still unclear. New possibilities are offered by the environment-sensitive probes, [8] sensitive to polarity (solvatochromic dyes), viscosity (molecular rotors), [9] and membrane tension (flippers). [10] The emission color of solvatochromic probes, like Laurdan, [2b] di-4-ANEPPDHQ, [11] and Nile Red, [12] directly reflects the lipid order, because it is linked with the local polarity and hydration, so that Lo and Ld phases can be distinguished in model membranes. Previously, based on Nile Red, we designed probe NR12S that reports lipid order selectively at the outer leaflet of cell plasma membranes [12] . Due to its high brightness and capacity to work at nanomolar concentrations, it became a common tool to study transmembrane asymmetry, [13] apoptosis, [14] maturation of endosomes, [15] membrane potential, [16] lipid trafficking, [17] etc. [8] The current challenge is to adapt this type of probes for super-resolution microscopy. [18] Super-resolution microscopy, including single-molecule localization microscopy (SMLM), is making a revolution in the structural and functional characterization at the subcellular level. [3,19] Numerous efforts have been made to develop fluorescent probes for super-resolution techniques, [20] where ON/OFF switching, required by SMLM, was implemented through photoswitching, [7a, 21] isomerization, [22] transient binding of dyes [23] and proteins [24] to the membrane, and DNA hybridization. [25] A largely underexplored direction, which can open a new dimension in SMLM, is to implement sensing function into this type of probes. It became possible with recently introduced spectrally resolved PAINT (SR-PAINT) for super-resolution imaging of biomembranes. [26] It exploits solvatochromic dye Nile Red, which (i) lights-up upon reversible binding to biomembranes [23a] and (ii) changes its emission color in response to the lipid order [6a, 8, 12, 26a, 26b] . However, this dye is not plasma membrane specific, as it binds all lipid components of cells. Here, based on Nile Red fluorophore, we designed the first probe combining together plasma membrane specificity, controlled ON/OFF switching, and sensing functions, which enabled imaging of both nanoscale morphology and lipid order in biomembranes.

To develop probes for SR-PAINT, we fundamentally changed the design strategy vs the original NR12S (Fig. 1a). First, we inverted the position of Nile Red modification and thus removed phenolic oxygen from the dye (NR12A and NR4A), which was hypothesized to cause lower photostability. Second, we propose here a new membrane-targeting moiety based on an anionic sulfonate head group and a lipophilic alkyl chain of varied lengths. We reasoned that dyes with long alkyl chains (NR12A and NR12S) should label plasma membranes quasi-irreversibly (Fig. 1b), which is suitable for conventional fluorescence microscopy. By contrast, a short alkyl chain (in NR4A) may enable reversible labeling (Fig. 1c) and thus the desired ON/OFF switching required for PAINT. [23a] Compounds NR12A and NR4A were thus synthesized in six steps starting from m-anizidine (Fig. S1). In organic solvents of varying polarity, the absorption and emission characteristics of NR4A and NR12A were close to Nile Red (Fig. S2, Table S1), indicating that the functionalization did not perturb the fluorophore properties. All studied probes showed fluorescence intensity increase (fluorogenic response) on binding to large unilamellar vesicles (LUVs) composed of DOPC, but the titration curves varied strongly (Fig. 1d). NR12A showed significantly higher affinity to LUVs than the parent Nile Red, displaying rapid intensity growth followed by a plateau at higher concentrations, similarly to NR12S. In sharp contrast, NR4A displayed a slow increase in the fluorescence intensity with liposomes concentration, even weaker than the parent Nile Red. These results confirmed that we obtained a weak (NR4A) and a strong (NR12A) binders of lipid membranes. Fluorescence spectra of both NR4A and NR12A (Fig. 1e,S3) exhibited blue shift by about 45-50 nm in Lo phase vesicles, composed of sphingomyelin/cholesterol (SM/Chol), compared to Ld phase vesicles, made of dioleoylphosphatidylcholine (DOPC). This behavior is similar to NR12S, [12] Nile Red (Fig. S3), and other solvatochromic probes, [2b, 11] reflecting lower local polarity/hydration in the Lo phase. [6a] Remarkably, the intensity ratio of blue to red part of emission band for both new probes was more sensitive than NR12S to lipid order (from Ld to Lo phase it increased 5.9-, 4.9-and 2.9-fold for NR4A, NR12A and NR12S, respectively), as their emission bands were narrower (Table S2). In phosphate buffer, NR12A displayed blue-shifted absorption compared to Nile Red (Table S1) and negligible fluorescence (Fig. 1e), similarly to NR12S, indicating that the NR12A forms selfquenched aggregates in water. By contrast, NR4A showed redshifted absorption (Table S1) and non-negligible fluorescence (Fig. 1e), similarly to Nile Red, suggesting that NR4A is well dissolved and does not aggregate in water. Importantly, NR12A and NR4A displayed high fluorescence quantum yields in LUVs (39-53%, Table S1). Moreover, in DOPC LUVs they were significantly more photostable than NR12S (Fig. 1f), confirming our hypothesis on the negative effect of the phenolic oxygen on the photostability of Nile Red fluorophore. In giant unilamellar vesicles (GUVs), using two-color confocal microscopy, NR12A and NR4A provided excellent contrast between Lo phase, characterized by high intensity ratio I550-600/I600-650 (in yellow, Fig. 1g,S4), and Ld phase with low I550-600/I600-650 (in blue). The ratio changes between the two phases were higher than that of NR12S (pseudo-color change from yellow to green), in line with the spectral data in LUVs. Single-molecule microscopy of the probes in supported DOPC lipid bilayers showed that the weak binders, NR4A and Nile Red, exhibited large number of single-molecule turn-on events that lasted well through >10 4 frames (Fig. 1hi, Movie S1), thanks to the continuous probe exchange between the solution and the membrane. By contrast, the strong binders, NR12A and NR12S, showed few turn-on events (Fig. 1hi), probably because most molecules were quasi-irreversibly bound to the membrane and then photobleached before single-molecule images could be recorded. Remarkably, single-molecule brightness histograms further showed that NR4A surpassed all other probes, including Nile Red, by the number of photons collected per molecule (Fig. 1j), in line with our photostability data in solution (Fig. 1f). Finally, single-molecule spectroscopy confirmed sensitivity of NR4A to lipid order in membranes (Fig. S5).

Next, the probes were incubated with HeLa cells and imaged using fluorescence microscopy (Fig. 2a). In contrast to Nile Red, showing intracellular fluorescence, NR12A exclusively stained the plasma membranes, being >2-fold brighter than NR12S. NR4A also showed specific staining for plasma membranes, but the signal was >10-fold lower, in line with its weak affinity to biomembranes. Among studied dyes only NR4A showed no signs of photobleaching (Fig. S6), which confirmed that it binds reversibly to cell membranes, so that bleached probe species are quickly replaced by intact ones, as required by PAINT. [23a] Overall, NR12A appears as a significantly improved analog of NR12S for conventional imaging, which enables, for instance, obtaining highquality three-dimensional (3D) confocal imaging of membrane surface (Fig. 2b). Meanwhile, NR4A, for its reversible binding capability, appears promising for PAINT. NR4A is also attractive for stimulated emission depletion microscopy, [19a] because exchangeable probes solve the problem of photobleaching. [27] We then performed 3D PAINT super-resolution imaging of live cells. With 10 nM dye in the imaging medium, NR4A reversibly bound to the plasma membrane, thus achieving high counts of single molecules over 5×10 4 frames (Fig. 2d), as in supported bilayers (Fig. 1h), hence good PAINT images within 1 min acquisition (Fig. 2ce and Fig. S7). In contrast, NR12S performed poorly with low counts of single molecules (Fig. 2d), and so resulted in very dim final PAINT images (Fig. 2c). Although Nile Red also allowed decent image quality, it strongly labeled internal membranes (Fig. S8), consistent with confocal data (Fig. 2a) and previous PAINT results. [26b] We also examined CM-DiI and DiD, two common plasma membrane probes for SMLM, [28] and found both to nonspecifically label internal membranes in live cells (Fig. S8). Owing to its high specificity, NR4A helps unveil rich nanoscale structural features of the plasma membrane (Fig. 2e-j). For HeLa cells, 3D PAINT showed numerous outward curves of the plasma membrane, including mildly bulging sites that raised up ~100 nm in height over ~1 µm lateral distances (Fig. 2fg), tube-like protrusions with higher curvatures (~100 nm radius; Fig. 2fh), as well as tall protrusions above the limit of our working z range (>400 nm above the focal plane), so that they appear dark at the center (Fig. 2fi). The observed nanoscale protrusions of the plasma membrane were highly dynamic, showing significant structural changes on the time scale of 1 minute (Fig. S8). Meanwhile, we also noted 150-200 nm-sized, pit-like invagination structures that dynamically moved into the cell on the time scale of minutes (Fig. 2j and Fig. S7), indicative of endocytosis. Similar membrane features were also observed in COS-7 cells (Fig S9). We next exploited solvatochromism of NR4A to examine the nanoscale distribution of local chemical polarity in plasma membranes, reflecting lipid order. To this end, we performed SR-PAINT, where the fluorescence spectrum of every single molecule of NR4A was measured together with its super-localized position. [26b, 29] The obtained spectral mean, calculated as the intensity-weighted average of the emission wavelengths of each probe molecule and presented in pseudo-color, allowed us to describe local lipid order with nanometer-scale spatial resolution and minute-scale temporal resolution [26b] . In live COS-7 cells, NR4A showed a variation of pseudo-color from blue to cyan (Fig. 3ab), with the averaged single-molecule spectra peaked at ~610 nm (Fig. 3d), similar to that of Nile Red in the plasma membrane. [26b] Interestingly, upon chemical fixation, NR4A became cell permeable and so labeled internal organelle membranes with a distinct ~20 nm redshift (Fig. 3cf), similar to the results obtained earlier with Nile Red, [26b] indicating lower lipid order for the organelle membranes. The high specificity of NR4A to the plasma membrane in live cells enabled nanoscale cartography of lipid order of cell surface, which has been a challenge so far as Nile Red exhibits strong background from internal membranes. Remarkably, for the tubelike nanoscale protrusions, in cyan pseudo-color, the locally averaged single-molecule spectra showed a ~4 nm redshift when compared to smooth parts of the plasma membrane, predominantly in blue (Fig. 3ad). This difference indicates slightly reduced local lipid order, possibly related to the dynamic structural changes of these protrusions under forces. More drastically, a strong redshift of ~20 nm, i.e., even lower lipid order, was observed for the ~200 nm-sized membrane pits (Fig. 3be), assigned above to endocytosis sites (Fig. 2j). Recent works [30] suggest that membrane curvature formation is closely related to the lipid composition. Our results above provide direct evidence of decreased local packing orders for the highly curved structures in the plasma membrane. In view of lipid rafts hypothesis, [1] our work suggests that nanoscopic domains of low lipid order can originate from protrusions and extensions at the cell surface. Thus, we the concept of a fluorescent probe for superresolution microscopy that combines target specificity, ON/OFF switching, and polarity sensing. It was realized based on solvatochromic dye Nile Red and a newly introduced low-affinity membrane binder that ensures reversible bind/unbinding events and targets specifically cell plasma membranes. The developed probe (NR4A) revealed lipid order heterogeneity at the cell surface and its connection to nanoscale membrane topology.

Entry for the Table of Contents

COMMUNICATION

Targeting, switching and sensing were combined together in one fluorescent probe to achieve nanoscale cartography of cell plasma membranes. The proposed design is based on a new functionalization strategy of solvatochromic dye Nile Red, sensitive to lipid order, and a special membrane-targeting moiety that controls binding/unbindinginduced ON/OFF switching for superresolution microscopy.

Figure 1 .

 1 Figure 1. Design of probes and their characterization in model membranes. (a) Chemical structures of NR12A and NR4A as well as the previously reported NR12S and Nile Red. (b,c) Design concept of the fluorogenic probes that irreversibly (b) or reversibly (c) bind to biomembranes due to the presence of long or short hydrophobic chains, respectively. (d) Titration of the four membrane probes (100 nM) with increasing concentrations of lipid vesicles: fluorescence intensity at 620 nm vs. lipid concentration. Errors are s.d.m. (n = 3). (e) Fluorescence spectra of NR12A (2 µM) in buffer (20 mM phosphate buffer, pH 7.4) and liposomes (LUVs) of different lipid composition (1 mM lipids). Excitation wavelength was 520 nm. All the spectra were corrected via dividing by absorbance at the excitation wavelength. (f) Fluorescence intensity of the different probes (400 nM) under continuous illumination vs. time in DOPC liposomes (200 µM). Excitation and emission wavelengths were 540 and 620 nm, respectively. (g) Ratiometric confocal microscopy of giant vesicles composed of DOPC/SM/Chol, 1/1/0.7, stained with NR12A, NR4A, NR12S, or Nile Red (200 nM). (h) Typical single-molecule raw images recorded at 9 ms integration time (110 frames per second) in DOPC supported bilayers, for the four probes (10 nM). (i) Time-dependent counts of single molecules detected per µm 2 per second for the four probes over the full camera view. (j) Distribution of singlemolecule brightness of probes in DOPC supported bilayers, expressed in terms of photon count after 50% splitting to the imaging channel. The average value for each probe is given in the legend.

Figure 2 .

 2 Figure 2. Cellular studies show NR12A and NR4A as superior probes for conventional and super-resolution microscopy, respectively. (a) Spinning disk confocal microscopy images of HeLa cells stained with NR12A, NR4A, NR12S, and Nile Red (20 nM) after 10 min incubation at r.t. (b) A 3D stacked image of KB cells stained with NR12A. (c) Zoom-out views of 3D PAINT results of COS-7 cells with NR4A (10 nM) and NR12S (20 nM). (d) Time-dependent counts of single molecules detected per µm 2 per second for the two probes in representative regions: 5×10 4 frames were recorded for >450 s at 110 frames per second. (e) 3D PAINT image of the top plasma membrane of a HeLa cell stained by NR4A. (f) Zoom-in of the white box in (e). (g-i) Virtual cross sections in the xz plane along lines 1-3 in (f). (j) Zoom-in in-plane (xy) image, and a time series of the vertical (xz) images of the endocytosis site marked by the arrow in (f). All 3D PAINT images are rendered with color to present the depth (z) information [color scale bar in (e)].

Figure 3 .

 3 Figure 3. Nanoscopic mapping of lipid order in cell membranes with NR4A. (a,b) SR-PAINT with NR4A for the plasma membrane of live COS-7 cells. Color represents single-molecule spectral mean (color scale bar). (c) NR4A SR-PAINT for a fixed COS-7 cell, showing staining for both the plasma membrane and internal membranes. (d) Averaged single-molecule spectra at smooth parts of the plasma membrane (black) vs. at the tube-like protrusions pointed to by the two arrows in (a) (magenta and green). (e) Averaged single-molecule spectra at smooth parts of the plasma membrane (black) vs. at the cluster structures pointed to by the two arrows in (b) (magenta and green). (f) Averaged single-molecule spectra at the plasma membrane of live (black) and fixed (blue) COS-7 cells, vs. at the internal membranes of fixed COS-7 cells (red).

Acknowledgements

This work was supported by the European Research Council ERC Consolidator grant BrightSens 648528 and the National Science Foundation (CHE-1554717). K.X. is a Chan Zuckerberg Biohub investigator. S.M. acknowledges support from a Samsung Scholarship. Bohdan Wasylyk is acknowledged for providing the KB cells.


Amélie Elmerich

Angélique Amelot

Shinji Maeda

Yves Laprie

Jean-François Papon

Lise Crevier- Buchman

F1 and F2 measurements for French oral vowel with a new pneumotachograph mask

Keywords: acoustics, French oral vowels, formants, pneumotachograph

Introduction:

In experimental phonetics, it's difficult to record different parameters on the same time. Indeed, combining acoustic and aerodynamic acquisitions is particularly challenging. In 1977, Rothenberg showed with a circumferentially vented pneumotachograph mask, that the vented mask had relatively low acoustic impedance. Several devices available to record aerodynamic data use different masks but it seems each time acoustic disturbance occurs; for instance, the rigid EVA TM mask [START_REF] Teston | A diagnostic and rehabilitation aid workstation for speech and voice pathologies[END_REF] acts as a low-pass filter which creates a strong resonance towards 1.4 kHz, an anti-resonance phenomenon at 3.5 kHz [START_REF] Ghio | Caractéristiques de la dynamique d'un Pneumotachographe pour l'étude de la production de la parole : aspects acoustiques et aérodynamique[END_REF] or a spectral attenuation in frequencies above 2 kHz. A pneumotachograph developed at Laboratoire de Phonétique et Phonologie (LPP) uses a mask made of synthetic fibers, instead of conventional rigid materials. This mask is acoustically almost transparent, and thus the radiated speech sound through the mask is almost free from acoustic distortions. The mask supplies a small resistance, as is necessary to measure airflow without perturbing much sound propagation [START_REF] Honda | Glottal-opening and airflow pattern during production of voiceless fricatives: a new non-invasive instrumentation[END_REF][START_REF] Vaissière | Multisensor Platform for Speech Physiology Research in a Phonetics Laboratory (< Feature Article> Methodology for Speech Physiology Research)[END_REF].

Objectives:

The goal of the current study is to validate that the acoustics of the fiber masks get closer to the acoustics of recording without a mask, that is to say without distortions.

Method:

Our corpus is composed of 3 parts: oral vowels in logatoms (n=872) "il a dit V: comme dans pV, pVp, pVp pVp tu n'as pas dit pVpVpVp mais pVpVpVp" with V= /i e ɛ a o u y ø/ and the first vowel (V:) as a sustained vowels (n=112) and the same vowels in the context of sentences in different position. For example: "Popo a posé le pot sur sa peau" (n= 877). Two female French native speakers repeat the corpus two times for each recording. Recordings took place in the soundproof chamber of the LPP. We did four types of recordings: 1) Acoustic-only microphone linked to a soundcard Protools, 2) Aerodynamic data recorded with the rigid EVA2 TM mask with oral mask and the microphone linked to an acquisition card (DT9003), these two first recordings were made with the microphone AKG C520L, 3) One recording with the fiber mask and 4) Last recording with the Rothenberg mask, these two recordings were made with the microphone Primo EMU-4520 linked to an acquisition card (DT9003). All data are resampling to 20000 Hz. For each recording, we extract F1 and F2 for all vowels: values were taken at the middle of the vowel.

Results: Statistical software R was used for Anova analysis; a value of p<0.05 was considered significant. We observed thanks to the vowel triangle a strong similarity between the formant structure of the fiber mask (Fiber_mask) and of our acoustic recording (micro) for every type of vowels. Indeed, we found no significant differences for the F2 for each sustained vowel. For the logatoms, we found significant differences for [o] (p<0,00001) and for the sentences (p<0,01). Regarding F1, we found significant differences for the sustained vowel [e] (p<0,01) between the fiber mask and the micro. Records with EVA2 TM (EVA_buccal) show a really distinct structure and a highly deformed triangle. Our results show a strong modification of F2 for the EVA recordings. F2 is probably influenced by the resonance at 1400 Hz [START_REF] Ghio | Caractéristiques de la dynamique d'un Pneumotachographe pour l'étude de la production de la parole : aspects acoustiques et aérodynamique[END_REF] and the anterior vowels are more influenced by the resonance. We found significant differences for F2 and the sustained vowels [i] (p< 0.00003) and [e] (p<0,05) between EVA and fiber mask recording. The significant differences between the EVA and the fiber mask recordings remain for the logatoms and sentences context for F2. We can observe that the vowel triangle of sentences is clearly different than the vowel triangles of the sustained vowels and logatoms. Certainly, speech is more natural in the context of sentences consequently the triangle will be less wide than sustained vowels and logatoms [START_REF] Lindblom | Spectrographic study of vowel reduction[END_REF]. F0 values remain constant between the 4 recordings for each speaker.

Figure 1 :

 1 Figure 1: Vowel triangle on the F1-F2 space. F1 and F2 average over two female speakers are indicated for four recording conditions: with the Rothenberg mask (in blue dash-dot line), microphone without a mask (in orange), with rigid EVA mask (in green dash-dot line) and with the fiber mask (in red). Sustained vowels, vowels in logatoms and in sentences are shown respectively in a), b) and c).


Alex Zhai
email: alexhezhai@gmail.com

Ronald Jenn
email: ronald.jenn@univ-lille.fr

Zheng Zhang
email: zzhang54@slb.com

Shelley Fisher Fishkin
email: sfishkin@stanford.edu

Amel Fraisse
email: amel.fraisse@univ-lille.fr

Pierre Zweigenbaum

TL-Explorer: A Digital Humanities Tool for Mapping and Analyzing Translated Literature

TL-Explorer is a digital humanities tool for mapping and analyzing translated literature, encompassing the World Map and the Translation Dashboard. The World Map displays collected literature of different languages, locations, and cultures and establishes the foundation for a variety of further analysis. It is comprised of three global maps for spatial and temporal interpretation. A further investigation into an individual node on the World Map -representing one edition or translation -leads to the Translation Dashboard. Collected translations are processed in order to build multilingual parallel corpora for a large number of under-resourced languages as well as to highlight the transnational circulation of knowledge.

Our first rendition of TL-Explorer was conducted on the well-traveled American novel, Adventures of Huckleberry Finn, by Mark Twain. The maps currently chronicle nearly 400 translations of this novel and the dashboard supports over 30 collected translations. However, the TL-Explore is easily extended to other works of literature and is not limited to type of texts, such as academic manuscripts or constitutional documents to name a few.

Introduction and Motivation

From a global perspective, human knowledge of culture and heritage has been shared, explored, and preserved for nearly centuries through translation. The art of translating texts is largely to thank for our ability to learn about and from other cultures, and vice versa. It is crucial to recognize that every person is shaped by their culture and identity. Hence, every body of knowledge, regardless of type of classification, is similarly impacted by specific historical, geopolitical, and sociocultural factors. TL-Explorer is created not only with this diversity in mind, but also as a tool to explore these nuances as they are reflected in translated literature.

TL-Explorer is designed to provide users with a feeling of continuity as they explore translated texts. The tool begins at a broad starting point -a global view of the entire collection of texts -and allows the user to smoothly zoom into a particular geographic region, individual editions or translations in that region, and specific chapters and paragraphs within the selected literature. The TL-Explorer uses a Geographic Information System (GIS) to create the World Map and NLP techniques to generate the Translation Dashboard.

Prior Work

Digital Humanities Mapping Tools

Hypercities [START_REF] Presner | Hypercities thick mapping in the digital humanities[END_REF] systems (GIS) such as Carto1 , Open Street Map (OpenStreetMap contributors, 2017), QGIS (QGIS Development Team, 2009), Harvard Worldmap (Guan et al., 2012), Spatial Data Explorer [START_REF] Kollen | Spatial data explorer: Providing discovery and access to geospatial data at the university of arizona[END_REF] and Unfolding [START_REF] Nagel | Unfolding-a library for interactive maps[END_REF] are also useful tools for digital humanities mapping.

Parallel Corpora Construction and Analysis Tools and Resources

There exist many construction and analysis tools for parallel corpora such as Uplug [START_REF] Tiedemann | Recycling Translations -Extraction of Lexical Data from Parallel Corpora and their Application in Natural Language Processing[END_REF], PENCIL [START_REF] Kakoyianni-Doa | A free online parallel corpus construction tool for language teachers and learners[END_REF] and The Sketch Engine [START_REF] Kilgarriff | The sketch engine: ten years on[END_REF], but there remain very little designed specifically for translated literature.

While the interpretive nature of literary translations has caused a lag in their adoption as a source for NLP development, multiple recent projects have developed parallel corpora based on well-known texts including the Harry Potter series and Le Petit Prince.

Alignment Visualization Tools

While there already exist alignment visualization tools such as ANNIS [START_REF] Druskat | corpus-tools. org: An interoperable generic software tool set for multi-layer linguistic corpora[END_REF], SWIFT Aligner (Gilmanov et al., 2014), Cario (Smith and[START_REF] Noah | Cairo: An alignment visualization tool[END_REF], VisualTCA [START_REF] Gomes | Visualtca: Uma ferramenta visual on-line para alinhamento sentencial de textos paralelos[END_REF] and MkAlign [START_REF] Fleury | Exploring translation corpora with mkalign[END_REF], most of them focus on word alignment. Further, even though some of these tools provide sentence alignment visualization, they are meant to be an intermediate step before the lexicon level. There are currently no other tools that allow users to explore data in a chapterparagraph-sentence/word, coarse-to-fine fashion. Moreover, these tools are not oriented towards literary texts, which is more challenging for alignment approaches. Though alignment should be as confident as possible [START_REF] Xu | Sentence alignment for literary texts[END_REF], this is complicated by the fact that a literary translation may include deliberate changes to the text inserted by the translator, and may not be a literal translation.

TL-Explorer

World Map Viewer

The World Map is the base tool in the TL-Explorer and provides a geographic display of collected translation information. It is separated into three maps: the Home Map, Heat Map, and Time Map, which display the same information in different views.

Home Map

The Home Map (see Figure 1) is the first map. It provides a global view of all the gathered texts, and each node represents one edition or translation. Nodes are placed at the location of publication, not based on the language of translation. Texts that are geographically close to each other are grouped into a cluster, represented by the light-yellow circles, with a number that reflects the number of texts in that cluster. A search function in the top left corner of the interface allows the user to search the map by title of the text. In the bottom left corner, a label identifies the number of texts being represented. The key informs the user of the types of languages represented on the map: English original, well-resourced language, medium-resourced language, and under-resourced language.

The World Map allows the user to zoom in from a global view to country view and even as close as specific streets. By clicking on a node, a pop-up displays the following information of the selected text: title, language, series/collection, edition, contributors (translators, editors, cover artists, and illustrators), date of publication, publisher, publisher city, and page count. If there is a digital version of the translation, the pop-up will include a link to it.

Time Map

The Time Map documents in chronological order developments of the literature of interest. The year is controlled by a scroll-bar below the map. As the years progress, nodes appear and accumulate. For example, Figure 1

Heat Map

The third map is the Heat Map, which displays translation count for each country and provides insight on which areas are more frequently represented (see Figure 1). It is aggregated using the data at a certain time, and changes as more data is added, such as a new translation or language.

Crowdsourcing for Data Curation

There are currently 386 points or translations on the maps. However, the TL-Explorer is immensely scalable. The TL-Explorer has a form where the user can submit all the information they have pertaining to an individual translation. After data-vetting, the point will be added as a new node to the maps. The field translation studies bridges comparative literature and corpus linguistics. While corpus linguists are likely to have at least basic programming skills and a broad familiarity with computational methods, the equivalent "digital humanities" training is less common in comparative literature. As a result, corpus linguists are more likely to focus on comparatively large-scale computational text analysis, and comparative literature scholars tend to conduct close examinations of a small number of texts.

Translation Dashboard

The Translation Dashboard is designed as an adjunctive tool for researchers in translation studies grounded in the comparative literature tradition. It provides a reading environment that could display the visualizations and text in parallel in order to allow scholars to easily see patterns of structural divergence between the source text and translations at different levels of granularity.

Text is aligned at the paragraph, sentence, and world level using Natural Language Processing algorithms, including the IBM Models 1 and 2 for Statistical Machine Translation [START_REF] Collins | Statistical machine translation: Ibm models 1 and 2[END_REF] and the Gale-Church Algorithm(Gale and Church, 1993).

Paragraph Count Analysis

After selecting a specific node on the World Map, the default view of the Translation Dashboard displays a per-chapter paragraph count, based on newlines and white space in that source text. The deviation in paragraph count between a source text and its translation is reflected in the color variation in the Heat Map within the table (see Figure 2, center image). An exceedingly high divergence from the source paragraph count alerts the scholar that there may be data cleaning issues (e.g. one instance where each line in a poem embedded in a narrative was treated as a new paragraph), but a moderate divergence can reflect the translator's deliberate stylistic choices about how the flow of the narrative should be rendered. A translation studies scholar in the literary tradition may use this information to select chapters for a close-reading analysis.

Paragraph Alignment

When the user selects an individual chapter in the text, they can view a display that presents both the original English chapter and the chapter in the translation. The tool displays the two parallel to each other to allow for easy comparison.

We divided chapters into 3 major categories based on the differences in their paragraph counts compared to the original English version: exact-match, large-difference, and small-difference. Different paragraph aligners may apply to different categories.

For exact-match chapters, our hypothesis is that their paragraphs were translated one to one. No further paragraph alignment methods are needed. This hypothesis has been confirmed for most of the exact-match cases by the human validation experiment.

Large-difference cases are normally caused by different ways of splitting quotations, so we provide a text pre-processing option before paragraph alignment when long quotations have been found under large-difference cases. This pre-processing option splits quotations into paragraphs according to the same standard in all translations. Experiments have shown that this action can significantly reduce differences in paragraph counts and sometimes move a chapter from the large-difference category to the smalldifference category.

For the majority small-difference cases, we applied the Gale-Church algorithm(Gale and Church, 1993). Here we treat paragraphs as sentences so as to feed them into this sentence alignment algorithm. The tool is easy to use, and thus easy for a native speaker to provide feedback on the accuracy of the alignments.

Conclusion

Encompassing both of the World Map and the Translation Dashboard, the TL-Explorer allows for analysis of translated literature at an exceptional range of specificity. The World Map provides a global view that can be shrunken into exact coordinates and streets and the Translation Dashboard allows for intense analysis of two texts from entire works to specific sentences. The TL-Explorer similarly serves a purpose of preservation and globalization, representing a large number of under-resourced languages and a transnational circulation of knowledge.

  introduced a digital humanities mapping tool for exploring and interacting with the layered histories of city and global spaces. Spatialization tools or geographic information * equal contribution This work is licensed under a Creative Commons Attribution 4.0 International License. License details: http:// creativecommons.org/licenses/by/4.0/.

  displays the translation of Adventures of Huckleberry Finn up to the year 1940.

Figure 1 :

 1 Figure 1: Home Map The width and height ratios of the maps have been changed to conserve space.

Figure 2 :

 2 Figure 2: Translation Dashboard, example with a Basque translation

https://carto.com/


Dr Gianni Cavallo

Dr Jean-Louis Clément

Dr Didier Gigmes

Dr Laurence Charles
email: laurence.charles@univ-amu.fr

Jean-François Lutz
email: jflutz@unistra.fr

Selective bond cleavage in informational poly(alkoxyamine phosphodiester)s

The collision-induced dissociation (CID) of sequence-defined poly(alkoxyamine phosphodiester)s was studied by electrospray ionization mass spectrometry. These informational polymers were synthesized using three different nitroxide building blocks, namely proxyl-, SG1-and TEMPO-derivatives. For a polymer containing TEMPO-and SG1based main chain alkoxyamines, it was found that both types of alkoxyamines break in CID tandem mass spectrometry (MS/MS). However, SG1-sites are preferentially cleaved and this predominance can be increased by reducing collision energy, even though selective bond fragmentation was not observed. On the other hand, for a polymer containing proxyl-and SG1alkoxyamines, selective bond cleavage was observed at all studied collision energies. The SG1alkoxyamines can be first cleaved in MS/MS conditions and secondly the proxyl-alkoxyamines in pseudo-MS 3 conditions. These results open up interesting new avenues for the design of readable, erasable or programmable informational polymers.

Introduction

Until very recently, macromolecular information storage was a property only exhibited by biopolymers. [1] Yet, starting about seven years ago, [2] synthetic information-containing macromolecules have been introduced as an exciting new class of functional polymers. [3][4] Just like biological polymers, these macromolecules store information in a defined comonomer sequence. [5][6][7] Hence, this new field of research opens up unprecedented applications of synthetic polymers in areas such as data storage, [8][9][10][11][12][13] cryptography, [14][15] traceability, plastic recycling, [16] anti-counterfeiting technologies, [17][18] and synthetic Life. [19] Overall, the synthesis of information-containing macromolecules is a topic that is increasingly studied and welldocumented. [4] Comparatively, another fundamental aspect that has been less investigated is the degradation of these macromolecules; even though it is a crucial parameter that influences storage, [20] erasure, [8] editing [21] and decryption of informational sequences. For instance, controlled bond cleavage is a crucial aspect in destructive sequencing methods such as collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) or chemicallyinduced depolymerization. [22][23][24][25] However, although all macromolecules are somehow decomposing over time, their degradation paths are not always controllable and useful. For example, the cleavage of DNA informational side-groups may occur in CID, thus prohibiting MS/MS sequencing. [26] In this context, guiding macromolecular degradation through molecular design is an important objective. This aspect is, of course, much easier to implement in synthetic polymers than in biological ones because the molecular structure of the former can be varied at will, whereas the one of the latter is often limited by biological constraints.

Among the wide variety of chemical reactions and building blocks that can be used to construct synthetic information-containing polymers, the chemistry of nitroxides [27] and alkoxyamines has been proven to be extremely useful. [8] It is well known that NO-C bonds, formed by radical-radical coupling of nitroxides with carbon-centered radicals, are thermolabile and dynamic. Thus, various types of dynamic polymer architectures have been prepared using nitroxide chemistry. [28] More recently, it was also shown that this chemistry can be used for the synthesis of digitally-encoded macromolecules such as poly(alkoxyamine amide)s [8,29] and poly(alkoxyamine phosphodiester)s. [30][31] Besides the fact that radical-radical coupling [32] is a fast and efficient reaction for stepwise orthogonal polymer synthesis, [33] the presence of alkoxyamine repeat units in the formed polymers enables chain erasing by thermal degradation [8] and favors sequencing because NO-C bonds are the first to break when subjected to CID. [30,34] Thus, alkoxyamines were afterwards intentionally included in other types of digital polymers to guide their CID dissociation and sequencing. [35] However, in all these works, the classic nitroxide 2,2,6,6-tetramethylpiperidinyloxy (TEMPO) was primarily used. Many other nitroxides exist and the stability of the corresponding alkoxyamines strongly depend on their molecular structure. [36][37][38] For example, 2,2,5,5-tetramethylpiperidinyloxy (proxyl) and Ntert-butyl-1-diethylphosphono-2,2-dimethyl propyl (SG1) [39] nitroxides lead to NO-C bonds, which are markedly different from TEMPO-based ones. [40] In the present work, TEMPO, proxyl and SG1 building-blocks were tested for the iterative synthesis of digital poly(alkoxyamine phosphodiester)s and the MS/MS sequencing of the formed coded copolymers was investigated. In particular, the CID of the different types of alkoxyamines was examined and compared.

Results and Discussion

Scheme 1 shows the route that was used in this work for the synthesis of the sequence-defined poly(alkoxyamine phosphodiester)s. As described earlier, [30] these polymers were prepared via an orthogonal iterative solid-phase approach involving two different types of bifunctional building blocks, namely a phosphoramidite monomer bearing an alkyl bromide and an hydroxyfunctional nitroxide, as well as two successive coupling reactions: (i) phosphoramidite coupling and (ii) radical-radical coupling. To include molecular information in the polymer chains, two different phosphoramidite monomers (0 and 1, Scheme 1) and three different nitroxides (P, S and T, Scheme 1) were used herein. In these syntheses, the radical-radical coupling step (ii) leads to the formation of main-chain alkoxyamines with the general structure -NHCOC(CH3)2-ON-. The bond dissociation energy (BDE) of these alkoxyamines depends on the molecular structure of the corresponding nitroxide. For instance, it has been reported that for alkoxyamines formed with the same alkyl fragment R= C6H5C(CH3), activation energy for the dissociation Ead at 293 K follows the trend R-SG1 < R-TEMPO < R-proxyl. [40] Based on these literature data, building blocks S and T were first considered for the construction of sequencedefined poly(alkoxyamine phosphodiester)s. The model polymer P1 with sequence α-0T0S0T was synthesized. Figure S1 shows the negative mode high-resolution electrospray ionization mass spectrometry (ESI-HRMS) spectrum recorded for this polymer. This measurement confirmed the synthesis of a uniform oligomer with a monoisotopic mass of 1574.8411 Da. The CID fragmentation of this polymer was then studied at different collision energies (Figure 1).

As previously observed, [30] alkoxyamines break preferentially in these structures, while other main-chain heteroatom-containing groups (i.e. amides and phosphates) remain stable. Yet, the fragmentation of the polymers strongly depends on collision energy (Figure 1a), with products of the SG1-based alkoxyamine (in blue) clearly formed from a lower energy threshold compared to those expected from C-ON bond homolysis at the TEMPO moiety. As a result, using very low collision energies allows almost selective homolysis of the SG1-based alkoxyamine bond. For example, in the MS/MS spectrum recorded for P1 at 9 eV (Figure 1b), the complementary products c2 •2-and y2 •-formed upon homolysis of the SG1-based alkoxyamine are observed with higher abundance than fragments c1 •1-and y3 •2-generated after cleavage of the first TEMPO C-ON bond (counting from left to right starting from the α terminus), while the c3 •3-ion expected at the second TEMPO-based alkoxyamine remains undetected. Raising collision energy up to 15 eV allows both SG1-and TEMPO-based alkoxyamines to undergo homolytic cleavage. Consistent with the lack of transition state for bond homolysis in gas phase ions, the higher dissociation rate of the SG1-based alkoxyamine compared to TEMPO accounts for the larger abundance of the c2 •2-/y2 •-ion pair compared to other fragments (Figure 1c). Nevertheless, these activation conditions allow all ci •i-fragments to form, hence enabling full sequence coverage by MS/MS according to decoding rules that have been previously established. [30] Overall, a highly-favored (but not truly selective) decomposition of SG1-based alkoxyamines was observed as compared to TEMPO-based alkoxyamines in P1.

A second model polymer P2, having the sequence α-1P1S0P1S, was then synthesized using the pair of building blocks S and P. ESI-HRMS (Figure S2) confirmed the formation of the targeted polymer with a mass of 2119.1089 Da. Figure S3 shows the MS/MS breakdown curves recorded for P2. At all studied collision energies, the cleavage of SG1-based alkoxyamines is orthogonal as compared to proxyl-based ones. Of note, the experiment presented in Figure S3 was limited to collision energies below 15 eV since activating P2 with higher energies induced secondary fragmentation via alternative rearrangement reactions.

Figure 2a shows the MS/MS spectrum of P2 recorded at a collision energy of 9 eV, corresponding to a center-of-mass energy (ECM = 0.470 eV) comparable to that used for P1 in Figure 1b (ECM = 0.473 eV) for the sake of comparison. All observed peaks indicate that primary fragmentation of P2 proceeds at the two SG1-based alkoxyamines. Indeed, the peak at m/z 309.1 assigned to c1 •1-clearly corresponds to a secondary fragment formed from c2 •2-(vide infra) because the complementary y4 •3-ion expected at m/z 602.0 in case of primary fragmentation of P2 is not detected. Data from Figure 2a show that other secondary dissociations selectively occur at SG1. This C-ON bond cleavage in c4 •4-yields c2 •2-(hence further contributing the peak intensity at m/z 399.7) together with the doubly charged internal fragment at m/z 502.7 (designated by a grey asterisk). The same m/z 502.7 internal fragment is also produced after the y3 •3-primary ion has eliminated the SG1-OH terminal radical moiety. Therefore, selective bond cleavage is more distinct for the pair SG1/proxyl than for SG1/TEMPO. These results are somehow in accordance with the homolysis rate constants reported for model alkoxyamines. [40] Yet, it shall be reminded that the CID experimental conditions studied in this work are markedly different from solution conditions that are typically used in nitroxide mediated polymerization or in ESR decomposition studies. Furthermore, the homolytic cleavage of proxyl-, SG1-and TEMPO-based alkoxyamines is influenced by the nature of the alkyl fragment (i.e. the amide moiety -NHCOC(CH3)2 in the present case), by the steric and electronic effects of nitroxide substituents and eventually by side reactions. [36-37, 41- 42] Nonetheless, in the studied experimental conditions (that is, for gas phase ions in vacuum), SG1-based alkoxyamines were selectively cleaved in MS/MS regime where homolysis of C-ON bonds at proxyl is not competitive. A second activation stage was required to induce cleavage of the proxyl-based alkoxyamines in primary fragments and hence recover the whole polymer sequence. To do so, ci •i-homolysis products of the two SG1 C-ON bonds were subjected to pseudo-MS 3 experiments, after being formed in-source upon raising the kinetic energy of their transfer through the interface of the mass spectrometer. In such conditions, CID of c2 •2-at m/z 399.2 led to c1 •1-at m/z 309.1 and y"1 •1-at m/z 490.3 (Figure 2b), indicating the α-1-P-1 starting block. Then, as illustrated in the inset dissociation scheme of Figure 2c, the MS/MS pattern of c4 •4-at m/z 451.2 permitted to complete the sequence as α-1-P-1-S-0-P-1, and subtracting its m/z value to the m/z 528.8 value of the [P2 -4H] 4-precursor confirmed the last building block was S-OH. Of note, selective homolysis of C-ON bonds at SG1 is also observed in the pseudo-MS 3 experiment reported in Figure 2c. Indeed, cleavage at the second proxyl group in c4 •4-is not a primary dissociation event since the c3 •3-fragment expected at m/z 438.2 is not detected. Instead, homolysis at this proxyl moiety occurs in y'2 •2-to produce y'1 •1- (at the same m/z 490.3 compared to the internal fragment released from c2 •2-to produce c1 •1-) together with the m/z 515.2 internal fragment (annotated with an asterisk in Figure 2c).

Conclusions

In summary, sequence-defined poly(alkoxyamine phosphodiester)s were synthesized using different nitroxide building blocks and their fragmentation was characterized by high-resolution electrospray ionization mass spectrometry. When the studied polymer contained both SG1-and TEMPO-based alkoxyamines, it was found that SG1 sites were predominantly (but not selectively) cleaved under CID conditions. Still, the preponderance of SG1-related fragments varied with collision energy. In case of a polymer containing both SG1-and proxyl-based alkoxyamines, selective bond cleavage was observed. First, the SG1-based sites were selectively cleaved in MS/MS and afterwards the proxyl alkoxyamines were cleaved in pseudo-MS 3 conditions. These findings open up interesting avenues for tuning the properties of digital polymers. For instance, they enable the design of informational macromolecules with programmed fragmentation; a strategy that has been recently validated for decoding long information sequences. [35] Alternatively, they may allow conception of blocky informational polymers, in which specific information blocks could be degraded and not others. Altogether, the results described in this communication further confirm that the properties of synthetic informational polymers can be finely tuned by macromolecular design. The grey asterisks designate internal fragments. Solid-phase iterative synthesis of the oligo(alkoxyamine phosphodiester)s. The protocol for the solid-phase iterative synthesis of oligo(alkoxyamine phosphodiester)s was reported in a previous publication. [1] In brief, the phosphoramidite coupling (step (i1) in Scheme 1) was performed using 3 eq. of monomer 0 or 1 for 30 minutes on 120 mg of modified resin (estimated loading of 0.7 mmol g -1 after modification). This reaction is always followed by an oxidation step using a 0.1 M iodine solution (step (i2) in Scheme 1). The radical-radical coupling step (step (ii) in Scheme 1) was performed using 5 eq. of nitroxide S, T or P for 40 minutes. Iterative cycles (i1), (i2) and (ii) were repeated a certain number of times until a desired chain-length and primary structure were reached. After synthesis, the oligomers were deprotected and cleaved using first a piperidine solution in acetonitrile followed by a treatment with a mixture of ammonia aqueous solution, methylamine aqueous solution and 1,4 dioxane (step (iii) in Scheme 1). Yield: 85% (P1), 70% (P2).

Electrospray Ionization Mass Spectrometry. High resolution MS, MS/MS and pseudo-MS 3 experiments were performed using a QqTOF mass spectrometer (QStar Elite, SCIEX, Concord, ON, Canada) with the ESI source operated in the negative mode (capillary voltage: -4200 V; cone voltage: -75 V for MS and MS/MS, -200 V for pseudo-MS 3 ). Ions were accurately mass measured in the orthogonal acceleration time-of-flight (oa-TOF) mass analyzer, using the precursor ions as internal standards to calibrate MS/MS data. In this instrument, air was used as nebulizing gas (10 psi) while nitrogen was used as curtain gas (20 psi) and collision gas.

Instrument control, data acquisition and data processing were achieved using Analyst software (QS 2.0) provided by Applied Biosystems. Polymer samples (a few mg) were first solubilized in methanol (SDS, Peypin, France) and then diluted (1/10 to 1/1000, v/v) in methanol supplemented with ammonium acetate (Sigma Aldrich, Saint Louis, MO) at a 3 mM concentration level, prior to injection in the ESI source at 10 µL/min using a syringe pump.

Figure 1 .

 1 Figure 1. MS/MS fragmentation of P1. (a) Breakdown curves recorded for the [P1-3H] 3-

Figure 2 .

 2 Figure 2. Successive MS/MS and pseudo-MS 3 fragmentation of P2. (a) MS/MS spectrum recorded for [P2-4H] 4-at a laboratory frame collision energy of 9 eV (0.478 eV in the centerof-mass frame). The grey asterisks designate internal fragments. (b) Pseudo-MS 3 spectrum recorded for the c2 •2-precursor ion at a 13 eV collision energy (laboratory frame). (c) Pseudo-MS 3 spectrum recorded for the c4 •4-precursor ion at a 10 eV collision energy (laboratory frame).

  chromatography and was stopped after 2.5 hours. The solvent was evaporated and purified by chromatographic column on silica gel (dichloromethane/methanol) (95/5). After purification, the OH-functional nitroxide P was recovered in 98% yield as a red yellow powder.ESI-HRMS [M+H]+ , m/z expected for C13H26N2O3•+ 258.1938, found 258.1936.

Acknowledgements

These results were obtained with the support of the French National Research Agency (ANR project 00111001, grant numbers ANR-16-CE29-0004-01 and ANR-16-CE29-0004-02) and of the H2020 program of the European Union (project Euro-Sequences, H2020-MSCA-ITN-2014, grant agreement n°642083). The PhD position of G.C. was supported by the ITN Euro-Sequences. L.C. acknowledges support from Spectropole, the Analytical Facility of Aix-Marseille University, by allowing a special access to the instruments purchased with European Funding (FEDER OBJ2142-3341).

were used as received. Anhydrous tetrahydrofuran (THF) was obtained on a dry solvent station GTS100. 5-((triisopropylsilyl)oxy)pentanoic acid was synthesized following a previously described procedure. [1] The nitroxides S, T and the phosphoramidite monomers 0, 1 were prepared as previously reported. [2][3] The phosphoramidite monomers were kept in the freezer at -18°C. Oligomers iterative syntheses were performed in an equipped argon/vacuum solid phase extraction glass tube (12 mL with frit 3) and stirred with an IKA HS 260 Basic shaker. They were performed on an aminomethylated-polystyrene resin (1.4 mmol g -1 , Merck) that was modified with 4-oxo-4-(3-(dimethoxy-trityloxy)propoxy)butanoic acid as reported earlier. [1] Synthesis of 5-hydroxy-N-proxyl-pentanamide. The nitroxide P was synthesized by reacting 3-amino-proxyl with 5-((triisopropylsilyl)oxy)pentanoic acid and subsequent deprotection of the triisopropylsilyl (TIPS) group. 5-((Triisopropylsilyl)oxy)pentanoic acid (190 mg, 0.66 mmol, 1 eq.), DIPEA (255mg, 1.98 mmol, 3 eq.) and 3-amino-proxyl (103 mg, 0.66 mmol, 1 eq.)

were dissolved in 20 mL of anhydrous DCM and the solution was cooled to 0°C. To the reaction mixture, PyBOP (515 mg, 0.99 mmol, 1.5 eq.) was added and the solution was allowed to reach room temperature and stirred overnight. The day after, the reaction was quenched with ammonium chloride saturated solution and the solvent was evaporated. The aqueous layer was extracted with EtOAc (3x20 mL), washed with brine (20 mL) and dried over MgSO4. Chromatographic column on silica gel (EtOAc/Cyclohexane) (30/70) gives the TIPS-protected product in 67% yield. ESI-HRMS [M+H] + , m/z expected for C23H46N2O3Si •+ 414.3272, found 414.3271. The TIPSprotected intermediate (353 mg, 0.85 mmol, 1 eq.) was then dissolved in10 mL of THF. To the solution, 3.0 mL of TBAF (1M in THF, 3.0 mmol, 3.5 eq.) was added. The reaction was monitored .