Sergiy Litvinenko

Pavlo Lishchuk
email: pavel.lishchuk@univ.kiev.ua

Vladimir Lysenko

Mykola Isaiev

Bi-modal photothermal/optical microscopy for complementary bioimaging with high resolution and contrast

Keywords: photothermal imaging, photoacoustic microscopy, thermal wave method

In the paper, combined bimodal microscopic visualization of biological objects based on reflection-mode optical and photoacoustic measurements was presented. Gasmicrophone configuration was chosen for the registration of photothermal response.

Precise positioning of the scanning laser beam with modulated intensity was performed employing acousto-optic deflectors. Photoacoustic images are shown to give complementary details to the optical images obtained in the reflected light. Specifically, the photoacoustic imaging mode allows much better visualization of the features with enhanced heat localization due to the reduced heat outflow. For example, the application of the photoacoustic imaging mode was especially successful to visualize Drosophila fly's micro-hairs. Furthermore, the photoacoustic image quality was shown to be adjusted through modulation frequency.

Introduction

The photoacoustic (PA) effect is a generation of acoustic waves due to absorption of electromagnetic radiation by a media. This phenomenon was observed for the first time by A.G. Bell 140 years ago [1], and it was started to be widely used after the work of Rosencwaig and Gersho [2], where they developed a quantitative theory of PA response formation in solids based on the thermal wave formalism. From the 90s, the PA effect was considered a promising tool for various biomedical applications.

Specifically, the main advantage of the approach is the possibility to use the same laser source as for the excitation of PA response for diagnostics of diseases as well for its treatment [3,4].

Since that time, various methods for material diagnostics, based on the PA effect, have been developed qualitatively and quantitively. Among the PA-based techniques, we should pay attention to methods that can be used directly for measuring the thermophysical, mechanical, and optical properties of materials [5][6][7][8][9][10][11][12]. The various photoacoustic techniques have been developed to provide innovative solutions for spectroscopy [13,14], microscopy [3,15,16] and tomography [3,[17][18][19][20][21][22][23][24][25]. Specifically, the latter is widely used in fundamental life science and medical practice for the visualization over a wide range of length scales covering from organelles and single cells [17,26,27] to tissues and organs [20][21][22][23]25,28].

The use of PA-based approaches in microscopy gives the possibility to overcome the optical limit, for instance, by localization of the heat due to the use of short pulses [29]. Thus, PA imaging techniques could be used noninvasively "in vivo" and provide useful information in anatomy imaging [30][31][32], neurology [33,34], cardiology [35,36], dermatology [3,19,28], oncology [3,21,28,37], and other preclinical and clinical applications for both humans and animals. However, utilization of the pulsed laser radiation with the power required for the characterization of the object situationally can lead to its significant overheating.

Therefore, the frequency-domain PA microscopy with respectively low intensity of radiation is preferable in numerous applications [5,10,38]. It should be noted that in this case, the level of signal can be small formed based on the deformations of a piezoelectric transducer in traditional configurations. From this point of view, the use of the gas-microphone (GM) PA approach is promising. As an example, the GM PA method has already been applied for the imaging of blood cells, and it was revealed its good potential in biology [39]. However, the authors used a resonance PA cell, and its application for the object with complex three-dimensional morphology is problematic.

In this article, we present the results of PA microscopy in GM configuration. For the modulation of the laser radiation and its positioning an electro-optical modulating system was used. A front configuration GM PA cell [40] with flat amplitude-frequency characteristic (out of resonance) in considered frequency range was used. It allowed us to obtain PA images for several frequencies of laser modulation and to visualize the impact of the modulation frequency on the contrast of the image. A fruit fly (drosophila melanogaster) was used as bio-object for imaging with the PA microscope.

Experimental details

Fig. 1(a) presents a schematic sketch view of the experimental setup, which consists of the three main blocks: (i) system for formation and deviation of a laser beam (previously reported in [41,42]); (ii) system for signal formation (PA cell coupled with a photodetector of the reflected light, schematically depicted in Fig. 1(b)); and (iii) system for signal detection and image formation.

System for formation and deviation of a laser beam (i).

The block (i) includes an operational power source, laser source (λ = 532 nm, with output optical power on a scanning area ~ 5 mW), acousto-optic deflectors coupled with the optical system (inbuild on equipment) for focusing of the laser beam on a scanned area with the ability to achieve down to μm-scale lateral resolution (both the spot and the minimal scanning step size are 10 µm), a backing stage for positioning and fixation of the device on the microscope. A significant advantage of the use of the acousto-optic deflectors to control the deviation of the laser beam is the possibility of prevention of the motion of a PA cell during the scanning process. Beam focusing was carried out by placing the plane of the object in the focal plane of the optical system using the adjustment probe method in the reflected light mode. The lateral resolution analysis of the system was carried out with the calibration on optically transparent plates that contain absorbing rings with different scales.

System for signal formation (ii). The non-resonant GM PA cell in a front configuration [7,9,40] was used for signal formation by the microscope. The material of the PA cell is aluminium, the maximum scanning area is 5x5 mm 2 . Calibration analysis of samples surface with homogeneous thermophysical and optical properties indicated no angular dependence of light incidence within the scanned area for both configurations (optical and photoacoustic). The inner working space of the PA cell was isolated from the external environment and a studied object can be scanned through the optically transparent glass window. The photothermally induced pressure oscillations coming from the object localized inside the PA cell were recorded by an electret microphone PX-53 built-in into the cell. An air channel between the laser illumination area and the microphone membrane has, respectively, a length and diameter of 2 mm.

Results and discussions

A small fruit fly, Drosophila melanogaster (see Fig. 3(a)), was used as a studied object for demonstration of the performances of such bimodal 2D imaging microscopy.

The size of an adult Drosophila fly normally does not exceed a few millimetres. Fig. First, it should be noted that the PA mode gives better visualization quality of small features from which the heat outflow is difficult. Thus, when the optical microscopy is complicated, for example, through the poor reflectivity of the studied object, PA imaging mode becomes an attractive alternative, because the PA signal depends not only on the object's optical properties but also on its thermophysical properties in the scanning area. For example, hairs on the surface of an insect that during the fly`s life are served as tiny 'feelers' and heat regulators can be more clearly seen in the images obtained by the PA approach. A comparison of the PA images obtained under different modulation frequencies shows, that the increase of the frequency values leads to an increase in image sharpness.

The latter may be explained by a frequency-dependent size of the heat localization region:

𝑙~√ 𝑐𝜌𝜔 𝜅 ,
where 𝑙 is the linear size of the region, 𝑐 and 𝜌 are, respectively, specific heat capacities and density of the media, 𝜔 is the cyclical frequency of modulation, 𝜅 is the thermal conductivity of the media. In the case of a complex heterogeneous media, one can consider all the parameters mentioned above as effective ones. Thus, there is the possibility to adjust the image quality by modulation frequency.

Additionally, an increase of the modulation frequency leads to a decrease in the relaxation time to reach a study state regime. Therefore, it gives the possibility to significantly optimize a scan rate. On another hand, the increase of the modulation frequency induces a decrease of the PA signal level, thus a satisfying balance should be found. when viewed from the surface, look like hexagons. Between each ommatidia unit, fruit flies possess evenly spaced hair-like structures called inter ommatidia bristles [START_REF] Paul | Fruit Flies[END_REF][START_REF] Garg | [END_REF].

These ocular hairs reduce airflow at the eye surface by up to 90% crucially reducing particle deposition and damage probability of the eye during the insects fly. At the same time, these micro-hairs minimally prevent the incoming light on the eye thereby, provide good vision [45]. The outer lenses of ommatidia can be clearly seen in an optical microscope, while ommatidia bristles can be observed qualitatively only in images obtained by a scanning electron microscope. Nevertheless, the 1D limitation of the ocular hairs structure complicates heat dissipation when it is excited by laser irradiation. This important factor allows to clearly see the hairs on the eye of the fly in PA mode. Thus, the image of ommatidia hexagons, which was obtained in the reflected light scanning mode, is complemented by the image of ommatidia hairs from the PA mode, creating the integrity of information about the structure of the fly's (see the markers 2 on Fig. 4).

Conclusion

In this work, the application of the photoacoustic technique for microscopy of biological objects is reported. A photoacoustic method with gas-microphone registration was chosen for detections of laser-induced thermal perturbations. Scanning of the modulated laser beam was performed with the use of acousto-optic deflectors.

The photothermal microscopy of a fruit fly was used to demonstrate the contrast and resolution levels of the obtained images. The dependence of these parameters on the modulation frequency is demonstrated. Photoacoustic images are shown to give complementary details to the optical images obtained in the reflected light. In particular, it was revealed that contrary to the optical image, the PA scanning mode allows to clearly see the hairs on the body and legs of the fruit fly, as well as ommatidia bristles, which are the structural units of the structure of the insect's eye. Thus, bimodal microscopy based on both visualization approaches can be applied in different fields where the use of a single imaging mode is not satisfying.

Fig. 1 .Fig. 2 .

 12 Fig. 1. Experimental set-up for the bimodal photoacoustic/optical reflection microscopy (a), the used PA cell (b)

3

  (b) presents the image of the fly in the reflected light, and it correlates very well with the optical image in Fig. 3(a).

Fig. 3 (

 3 Fig. 3(c) and 3(d) show the images obtained with the use of the PA technique at different modulation frequencies (75 Hz and 735 Hz respectively). The PA signal levels were up to 1 mV at 75 Hz and up to 100 µV at 735 Hz, respectively. The maximal PA signals correspond to bright colour on the PA images. First, it should be noted that

Fig. 3 .

 3 Fig. 3. Images of Drosophila fly: a) obtained as a reference picture by a portable optical microscopy for general visualization of the object; b) in the reflected light mode provided by our experimental set-up; in the PA mode under 75 Hz (c) and 735 Hz (d) modulation frequencies. Scale grades of the response signal are applicable to figures (b) -(d).

Fig. 4

 4 Fig.4presents images of the Drosophila fly close to the eye region. One can see that the reflection-mode optical imaging gives better visualization of the body of the insect, while the image obtained in the PA mode microscopy gives better visualizations of the regions where photo-induced thermal energy is strongly localized. Specifically, it is known that the eyes of a fly are composed of separate structural units called ommatidia. The outer lenses of ommatidia are closely spaced among themselves and,

Fig 4 .

 4 Fig 4. Images of Drosophila fly eye obtained in the reflected light mode (a) and in PA mode under 340 Hz modulation frequency (b). The markers in the images indicate the examples of characteristic differences in the visualization of the object that is scanned bi-modally. Marker (1) indicates the difference in signal from the body of the haircovered insect; marker (2) indicates the discrepancy in the visualization of the eye of the object.

Acknowledgement

This research work was carried out in frames of the CARTHER project (proposal #690945) of Marie Skłodowska-Curie Research and Innovation Staff Exchange program. The publication contains the results obtained in the frames of the research work "Features of photothermal and photoacoustic processes in low-dimensional silicon-based semiconductor systems" (Ministry of Education and Science of Ukraine, state registration number 0118U000242).


Thomas Cheviet

Suzanne Peyrottes
email: suzanne.peyrottes@umontpellier.fr

Synthesis of amino-methylene-gem-bisphosphonates containing an aziridine motif: Studies of the reaction scope and insight into the mechanism

A broad range of N-carbamoyl aziridines were obtained and then treated by the diethyl phosphonate anion to afford α-methylene-gem-bisphosphonate aziridines. Study of the reaction's scope and additional experiments indicate that the transformation proceed via a new mechanism involving the chelation of lithium ion. This last is crucial for the reaction to occur and disfavors the aziridine ringopening. A phosphonate-phosphate rearrangement from a -hydroxybisphosphonate aziridine intermediate is also proposed for the first time. This reaction provides a simple and convenient method for the synthesis of highly functionalized phosphonylated aziridine motif.

INTRODUCTION

Aziridines are widely studied as key intermediates in organic syntheses and represent a valuable source of chiral centers because of their regio-and stereo-selectivity in ring-opening reactions. Their reactivity has recently been reviewed. 1 When combined with a phosphonate moiety (widely used in medicinal chemistry as bio-isoster of organic phosphate and insensitive to enzymatic cleavage), they are useful building blocks for the synthesis of amino-phosphonates that can be considered as analogues of -aminoacids. As example, the synthesis of aziridine-2-phosphonates and aziridine-1-ylmethylphosphonates have been well described in the literature. [2][3][4][5] However, the association of a chiral aziridine with several phosphonate groups is rather limited and may lead to promising scaffolds for obtaining biomolecules of interest. Indeed, bisphosphonates (or BPs, Figure 1) have been developed

for their coordination abilities and their structural analogy with inorganic pyrophosphate (the P-C-P bridge replacing the P-O-P bonds is not sensitive to enzymatic cleavage), making them suitable for the treatment of bone diseases or drug delivery. [6][7][8][9][START_REF] Aoun | A Novel Hydroxy-Bisphosphonic Acid Prodrug as a Candidate for the Delivery of Ibuprofen to Bone[END_REF][START_REF] Farrell | Bisphosphonate Conjugation for Bone Specific Drug Targeting[END_REF] N-BPs are nitrogen-containing bisphosphonates, targeting enzymes of the mevalonate pathway and exhibiting antiosteoporosis and antiproliferative activities. [START_REF] Simoni | Synthesis, and Biological Evaluation of Novel Aminobisphosphonates Possessing an in Vivo Antitumor Activity through a Gammadelta-T Lymphocytes-Mediated Activation Mechanism[END_REF]13 For example, the incadronate (Figure 1) is an anti-osteoporosis drug with a 1aminobisphosphonate motif. Syntheses of 1-aminobisphosphonates (or aminomethylene-gem-bisphosphonates) are described through numerous methods, 14, namely the condensation of ethyl orthoformates and phosphites, [START_REF] Bálint | Microwave-Assisted Synthesis of (Aminomethylene)Bisphosphine Oxides and (Aminomethylene)Bisphosphonates by a Three-Component Condensation[END_REF][START_REF] Dąbrowska | Insight into the Mechanism of Three Component Condensation Leading to Aminomethylenebisphosphonates[END_REF] the Beckmann rearrangement of oximes, [START_REF] Yokomatsu | Simple and Efficient Method for Preparation of Conformationally Constrained Aminomethylene Gem-Diphosphonate Derivatives via Beckmann Rearrangement[END_REF][START_REF] Wu | Simple, Efficient and One-Pot Method for Synthesis of Aminomethylene Gem-Diphosphonic Acid Derivatives from Ketones via Beckmann Rearrangement[END_REF] or the bisphosphorylation of amides, [START_REF] Qian | The Synthesis and Reactivity of Alkyl-Aminosubstitutedmethylenediphosphonates[END_REF][START_REF] Wang | General and Chemoselective Bisphosphonylation of Secondary and Tertiary Amides[END_REF] imines, [START_REF] Prishchenko | Synthesis of New Types of Aminomethylenediphosphorus-Containing Acids and Their Derivatives[END_REF] isonitriles, [START_REF]получения =фениламинотетралкилдифосфонметана -SU 445675[END_REF][START_REF] Goldeman | The Preparation of N-Substituted Aminomethylidenebisphosphonates and Their Tetraalkyl Esters via Reaction of Isonitriles with Trialkyl Phosphites and Hydrogen Chloride. Part 1[END_REF][START_REF] Kurzak | Synthesis of N-Methyl Alkylaminomethane-1,1-Diphosphonic Acids and Evaluation of Their Complex-Formation Abilities towards Copper(II)[END_REF] or nitriles. [START_REF] Kaboudin | ZnCl2-Mediated Double Addition of Dialkylphosphite to Nitriles for the Synthesis of 1-Aminobisphosphonates[END_REF] To our knowledge, such derivatives when containing an aziridine motif are not reported in the literature. Thus, we propose an original synthetic route involving lithiated diethylphosphite for the valorization of these three-membered rings in phosphorus chemistry (Scheme 1). We use N-carbamoyl aziridines as starting material for the preparation of brand new phosphonylaziridine compounds, such as aziridine α-methylene-gem-bisphosphonates (abbreviated AzbisPs). To investigate the scope and limitations of the method, a library of N-carbamoyl aziridines is obtained beforehand. In addition, complementary experiments are performed to propose a mechanism for the formation of such derivatives. Scheme 1. Synthesis of aziridine -methylene-gem-bisphosphonates (AzbisPs) from Ncarbamoyl aziridines.

RESULTS AND DISCUSSION

In the course of a medicinal project, we envisaged the preparation of β-aminophosphonates through the ring-opening of aziridines with phosphites, [START_REF] Cheviet | β-Hydroxy-and β-Aminophosphonate Acyclonucleosides as Potent Inhibitors of Plasmodium Falciparum Growth[END_REF] a methodology poorly reported in the literature. [START_REF] Stamm | Reactions with Aziridines XXI The (Michaelis-)Arbusov Reaction with N-Acyl Aziridines and Other Amidoethylations at Phosphorus[END_REF][START_REF] Stamm | Reaktionen mit Aziridinen, 29. N-Acylierte bzw. Nsulfonierte 2-Aminoethylphosphonsäureester durch basenkatalysierte Amidoethylierung von Phosphorigsäurediestern mit aktivierten Aziridinen[END_REF] When such conditions (DEP/LiHMDS in THF) are applied to a N-Cbz aziridine as substrate, we observed the formation of an aziridine α-methylene-gem-bisphosphonate (AzbisP), instead of the expected ring-opened product. To study the scope of this reaction, we examined the behavior of several N-Boc or N-Cbz-protected aziridines.

With this aim, 14 substrates (Table 1, compounds 2a-n) were synthesized starting from commercially available amino-acids and amino-alcohols, through protection/reduction steps and then cyclization. Among them, nine N-carbamoyl aziridines (2a, 2d, 2e, 2f, 2g, 2i, 2j, 2l and 2n) were hitherto unknow compounds, that may be viewed as valuable building blocks. According to the nature of the substituents, either Mitsunobu conditions (route A) or intramolecular substitution (route B) is used. Likely due to interactions/steric hindrance between the tosylate group and the phenyl moiety of the Cbz protecting group, the one-pot cyclisation of N-Cbz aziridines usually led to lower yields than under Mitsunobu conditions. Except in the case of the cyclohexyl aziridine 2k (Table 1, entry 11), where the 3D conformation of the carbocycle is supposed to reduce the possible interactions between the two groups. We observed lower yields for compounds 2l and 2m (obtained as invertomers because of the high-energy barrier of the nitrogen inversion) and this may be associated with the formation of a highly constrained aziridine ring. In the Mitsunobu conditions, no reactivity is observed.

Table 1. Preparation of N-carbamoyl aziridines (as substrates) from commercially available amino acids and amino-alcohols.

Entry

Starting Material Synthetic route a Product Yield b 1 1a [c] A 2a 76%

2 N-Boc-L-Phenylalaninol [d] B 2b 75%

3 N-Cbz-L-Phenylalaninol [d] A 2c 67% 4 1d [c] A 2d 49% 5 1e [c] A 2e 53% 6 L-Aspartic acid [d] 5 steps 29,[c] 2f 22% (overall)

7

N-Boc-L-Isoleucinol [d] B 2g 97% 8 Cbz-L-Asp(OtBu)-OH [d] 2 steps [START_REF] Jung | Synthesis of Threo-β-Aminoalcohols from Aminoaldehydes via Chelation-Controlled Additions. Total Synthesis of l-Threo Sphingosine and Safingol[END_REF][START_REF] Aaseng | Synthesis of Substituted (S)-2-Aminotetralins via Ring-Opening of Aziridines Prepared from l-Aspartic Acid β-Tert-Butyl Ester[END_REF] 2h 37% (overall)

9 1i [c] A 2i 95% 10 1j [c] B 2j Quantitative 11 1k [c] B 2k 32 97% 12 1l [c] B 2l 16% 13 1m [c] B 2m 31% 14 1n [c] A 2n 76%
[a] Reaction conditions: Mitsunobu (route A) PPh3, DEAD, THF, 0°C to rt, overnight; One-pot cyclisation (route B) p-TsCl, KOH, Et2O, reflux, overnight.

[b] Isolated yield after purification by column chromatography.

[c] Preparation of starting material 1a, 1d, 1e and 1i-n is available in the experimental section. [d] Commercially available.

Having established the suitable reaction conditions (data not shown), aziridines 2a-n were treated with 6.1 equivalents of diethylphosphite and 6 equivalents of LiHMDS (1M in THF), in anhydrous THF, at -78°C. Then, the reaction mixture was heated at the indicated temperature until completion of the reaction (TLC monitoring). According to our results, formation of AzbisPs occurs both in the presence of N-Boc and N-Cbz protecting groups, with an enhanced reactivity of the latter (Table 2, comparison between substrates 2b and 2c, 2l and 2m). Three substrates did not lead to the expected derivatives. For the bicyclic compounds 2l and 2m, the lack of reactivity is mostly associated with the highly constrained conformation of the aziridine-ring, and the reduced product 3l is isolated in high yields. For the bi-aryl aziridine 2n, the starting material is almost entirely recovered even after 4 days at reflux. Thus, the high steric hindrance of the heterocycle appears to prevent the formation of the AzbisP derivative.

Ring-opened products are observed as byproducts for a few aziridines (2f, 2h and 2i), highlighting the competition between the formation of aziridine α-methylene-gem-bisphosphonates and β-aminophosphonates in the presence of electron-withdrawing substituent, which enhances the electrophilicity of the aziridine ring. In the case of aryl or alkyl substituents, no byproducts are isolated.

As expected, we obtained better yields at higher temperatures (from rt to 55°C) for derivatives 3b and 3f, whereas some aziridines (2a, 2c and 2k) appeared unstable upon heating. Temperature has also an effect on the competition between ring-opening and AzbisP formation (no byproduct is isolated from 2f after refluxing the reaction mixture at 80°C, whereas the ring-opened product is recovered, in 5% and 16% yield, respectively at room temperature and 50°C) indicating that the AzbisP is the thermodynamic product of the reaction. In addition, the studied reaction does not lead to the epimerization of the aziridine ring, since compound 3g is not isolated as a mixture of diastereoisomers. The reaction does not occur on N-Boc protected primary amines (Table 2, side chain of the lysine derivatives 2i and 2j). This difference of reactivity may be explained by the pKaH values of aziridine and aliphatic amine (7-8 vs 9-11 in H2O, respectively), which has an impact on the electrophilicity of the carbonyl moiety. The N-carbamoyl aziridine moiety is more reactive in our conditions, thus allowing the selective formation of the AzbisP, even in the presence of another carbamate group. To validate this hypothesis, we examined the behavior of few analogues of the N-carbamoyl aziridine scaffold with higher pKaH values, such as N-Cbz-azetidine (10-11) N-Cbz-pyrrolidine (10.8) and N-Cbz-piperidine (10) derivatives (pKaH values have been estimated using the MarvinSketch software), [START_REF]ChemAxon -Software Solutions and Services for Chemistry & Biology[END_REF] as well as a well-known rearrangement product of N-carbamoyl aziridines in the presence of azaphilic Lewis acids, the oxazolidine-2-one 4 (Scheme 2). [START_REF] Cardillo | Influence of Lewis Acids on the Regioselectivity of N-Boc-Aziridine-2-Carboxylate Microwave-Assisted Rearrangement[END_REF] In all cases, the pKaH of the nitrogen is higher than the one of the aziridine scaffold, and no reactivity is observed in our conditions (DEP/LiHMDS).

Scheme 2. Synthesis of the oxazolidine-2-one 4 from 2b using previously published procedure [START_REF] Cardillo | Influence of Lewis Acids on the Regioselectivity of N-Boc-Aziridine-2-Carboxylate Microwave-Assisted Rearrangement[END_REF] and its lack of reactivity towards nucleophilic attack of diethylphosphite. The chelation model and the proposed mechanism for the ring expansion of N-Boc-aziridines into oxazolines catalyzed by azaphilic Lewis acids is well-known in the literature. [START_REF] Tomasini | Novel Synthesis of 4-Carboxymethyl 5-Alkyl/Aryl Oxazolidin-2-Ones by Rearrangement of 2-Carboxymethyl 3-Alkyl/Aryl N-Tert-Butoxycarbonyl Aziridines[END_REF][START_REF] Ferraris | Orthogonal" Lewis Acids: Catalyzed Ring Opening and Rearrangement of Acylaziridines[END_REF] The structures of bisphosphonate esters (compounds 3a-b, 3d-i and 3k) were readily identified by analysis of the NMR data, including bidimensional techniques, as well as mass spectrometry, and compared to their corresponding N-carbamoyl aziridines used as starting materials (see SI). In all cases, 1 H NMR spectra shows a triplet in the range of 2.4 to 2.8 ppm with a coupling constant value of ~18 Hz and corresponding to a single proton. The 2D spectral data (COSY 1 H/ 1 H and HSQC 1H/13C) demonstrate that this proton is not coupling with other protons of the molecules and is associated with a carbon atom, which signal is also a triplet at ~ 65 ppm with a wide coupling constant of ~150 Hz. This observation is consistent with the proposed structure including two phosphonate groups attached to the same carbon (P-CH-P). As example, detailed study of 1D and 2D RMN spectra (see SI) for two AzbisPs (compounds 3d and 3g) and their corresponding N-carbamoyl-aziridines (compounds 2d and 2g) highlights the characteristic signals of these compounds and peak attribution.

The aziridine structure is clearly recognizable by the presence of 2 doublets at ~ 2 ppm in 1 H NMR (corresponding to the methylene of the aziridine ring) in the AzbisPs, which are also present in 1 H NMR spectrum of the starting material, supporting that the ring is kept intact at the end of the reaction.

In addition, COSY and 13 C spectra also demonstrate the presence of the aziridine ring, the 2 doublets in 1 H NMR are associated to the same carbon according to HSQC experiment. The signal of the methylene protons is coupling only with the methyne proton of the aziridine ring in the COSY experiment.

Then, we get interested in deciphering the mechanism of the reaction. Because the aziridine 2g does not show any epimerization, we exclude the ring opening of the aziridine and its subsequent ring closing. Thus, our first hypothesis consisted in the nucleophilic attack of one equivalent of diethylphosphite anion on the carbonyl group, leading to the elimination of the alkoxide and the formation of an aziridine N-carbamoyl phosphonate (scheme 3A). Differences of reactivity between N-Boc and N-Cbz substrates support this assumption as the benzyloxide ion may be considered as a better leaving group than the tert-butyloxide one. These kind of addition/elimination occurring on a carbamate moiety has already been reported in the literature. [START_REF] Shtrumfs | Unnatural Amino Acids. 2. Simple Method of Obtaining Esters of Aziridine-2-Carboxylic Acids by a Transesterification Reaction[END_REF] In addition, an acyl derivative of the 2benzyl-aziridine treated in our conditions (data not shown) did not lead to the formation of the corresponding AzbisP, which indicated the key role of the carbamoyl moiety.

This addition/elimination step may be competing with the nucleophilic attack on the aziridine ring that is also an electrophilic center. Here, we assumed that the chelation of the lithium with the ester moiety enhanced the reactivity of the carbamate, since lithium is considered as a better Lewis acid than the other alkali ions (sodium and potassium). Corresponding models and rules are presented in Scheme 3A. Herein, we proposed a different site of chelation for the Lewis acid (than the one drawn in scheme 2) on the basis of the work of T. Lectka et al., [START_REF] Ferraris | Orthogonal" Lewis Acids: Catalyzed Ring Opening and Rearrangement of Acylaziridines[END_REF] indicating that coordination to the carbonyl may be better at activating the substrate toward external nucleophilic attack.

In To go further in the study of the impact of the lithium ion in the proposed mechanism and rearrangement as well as the nature of the base, we envisaged additional experiments (Table 3). Thus, various bases susceptible to deprotonate the diethylphosphite were tested using aziridine 2f as substrate. In the presence of tBuOK (entry 1), only 3% of AzbisP is recovered and we mainly observed the formation of ring-opened product (47% yield). When using NaHMDS (entry 2) or LiHMDS (entry 3) with similar pKa values, no AzbisP is observed for NaHMDS and only 9% of the ring-opened product is recovered. These results are in agreement with previous data from the literature, comparing the effect of various [M]HMDS bases on the double addition of DEP on acid chlorides, [START_REF] Ruel | Single-Step Preparation of 1-Hydroxybisphosphonates via Addition of Dialkyl Phosphite Potassium Anions to Acid Chlorides[END_REF] and the recent mechanistic study of the role of the lithium ion in the [1,2]-phospha-Brook rearrangement. [START_REF] Ranga | Mechanistic Insights into N-BuLi Mediated Phospha-Brook Rearrangement[END_REF] When a stronger base such as NaH was used (entry 4), only traces of ring-opened product were observed. Accordingly, the lithium is conjugated to the hydroxylate anion generated after the second nucleophilic attack of DEP on the acylphosphonate moiety (scheme 3C). Lithium acts as a Lewis acid as well as a conjugated cation. It is supposed to chelate the phosphonate group I, to facilitate the formation of the oxaphosphirane II, and to stabilize the carbanion III with the formation of a strong ion pair. The proposed model (Scheme 3B) is hypothesized from the mechanism of the reverse reaction, the phosphate-phosphonate rearrangement, in the presence of strong lithiated base [START_REF] Hammerschmidt | The Phosphonate-Phosphate-and Phosphate-Phosphonate Rearrangement and Their Applications, 4. Deprotonation of Secondary Benzylic Phosphates -Configurationally Stabile Benzylic Carbanions with a Diethoxyphosphoryloxy Substituent and Their Rearrangement to Optically Active Tertiary Α-Hydroxyphosphonates[END_REF][START_REF] Hammerschmidt | Metallation of Phosphorylated Aliphatic Alcohols to Configurationally Stable α-Oxyalkyllithium Compounds -Use of the Phosphoryl Group as an Activating Group and Electrophile[END_REF] and on the basis of the work of Ranga et al. [START_REF] Ranga | Mechanistic Insights into N-BuLi Mediated Phospha-Brook Rearrangement[END_REF] This point further reinforces the crucial role of lithium in the stabilization of the transition states. The [1,2]-phospha-Brook rearrangement likely ends by the lithium-proton exchange between DEP and the transition state III.

In the last step, we assumed that the newly formed phosphate group may be substituted by a third equivalent of deprotonated diethylphosphite, still with the assistance of the lithium as a Lewis acid, which chelate the phosphate group and leading to the AzbisP product (Scheme 4). A similar substitution reaction is observed by Fitch and Moedritzer on a phosphonylphosphate compound, in the presence of PCl5. [START_REF] Fitch | NMR Study of the P-C(OH)-P to P-C-O-P Rearrangement: Tetraethyl 1-Hydroxyalkylidenediphosphonates[END_REF] It has to be specified that the diethylphosphoester moiety (in red, intermediate IV, Scheme 4), as well as the phosphonate group, certainly increase the electrophilicity of the vicinal carbon atom (in green, intermediate IV, Scheme 4) assisting consequently the nucleophilic attack of DEP (in blue, Scheme 4) on this position. As for the previous steps, the aziridine ring is supposed to participate in the same way because of the low pKa value of its nitrogen atom. This last step is thought to be fast, in order to consume quickly the rearranged phosphorylated compound and thus preventing the reverse reaction.

Scheme 4. Substitution of the phosphate group by a third nucleophilic attack of DEP.

Furthermore, several attempts to isolate intermediates during the reaction and to synthesize the Ncarbamoyl phosphonate intermediates were unsuccessful, thus demonstrating both high reactivity and instability of these species. When using lower equivalents of DEP/base (Table 4), total consumption of the starting N-carbamoyl aziridine is observed but the yields of the recovered AzbisP remains below the one obtained with 6 equivalents of DEP/LiHMDS, and yet no intermediates is isolated. The similar yields obtained for 2 and 3 equivalents of the reactants (Table 4, Entry 1 and 2) may suggest that the reaction requires at least 3 equivalents of DEP/LiHMDS to be in stoichiometric conditions.

CONCLUSION

N-carbamoyl aziridines are efficiently converted into their corresponding methylene-gembisphosphonylated derivatives in the presence of LiHMDS and DEP. To our knowledge, it is the first time that such reaction is reported in the literature. This transformation preferably occurs with N-Cbz aziridines, and competes with the aziridine ring-opening reaction. A broad range of N-Cbz and N-Boc aziridines were synthesized to study the scope of the reaction and afforded the desired phosphonylated compounds with mild to good yield. Influence of the nature of the base, the nature of the aziridine protecting group, amount of reactants, and the aziridine substituent were studied.

According to the reaction scopes and additional experiments, we proposed a mechanism and highlighted the essentiality of the lithium ion and the basicity of the nitrogen. The latter involved a phosphonate-phosphate rearrangement newly described from a lithiated α-hydroxy-bisphosphonate species.

EXPERIMENTAL SECTION

General Information

Reagents were from the followed suppliers: Sigma-Aldrich, Alfa Aesar, Acros or TCI. Anhydrous solvents (sealed flasks and stored on molecular sieves) were from Sigma-Aldrich (acetonitrile, MeOH, dichloromethane, THF, DMF, Et2O), or distillated beforehand on P2O5 or CaCl2 (dichloromethane), sodium and benzophenone (THF), according to protocols by Armarego et Perrin. [START_REF] Armarego | Purification of Laboratory Chemicals[END_REF] Diethylphosphite (Acros) was distilled on KOH under reduced pressure and stored in a sealed round-bottom flask under argon atmosphere and in the dark. Reactions that required heating were performed in hot plate/heating block apparatus with internal temperature control.

Microwave conditions reactions were performed on Anton-Paar Monowave instrument and using sealed tubes, the reaction temperature was monitored by external surface sensor.

Thin-layer chromatography (TLC) was performed on precoated aluminum sheets of silica 13 C NMR spectra, and 162 or 202 MHz for 31 P NMR), at room temperature (20°C). Chemical shifts were reported in ppm (parts per million) and determined according to the solvent peak used as internal reference and relatively to the trimethylsilyl peak (TMS) for 1 H and 13 C NMR, and according to an external reference for 31 P NMR. Used solvents were CDCl3, D2O, MeOD-d4 and DSMO-d6 (Sigma-Aldrich). COSY ( 1 H 2D), HSQC 1 H- 13 C, and HMBC 1 H- 13 C were obtained to interpret and confirm 1 H and 13 C NMR spectra. 1 H NMR data are reported as follows: chemical shift (ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad signal), coupling constants and integration. High-resolution mass spectra (HRMS) were recorded on a Q-TOF Synapt-G2-S instrument using electrospray ionization (ESI) in positive or negative ion polarity mode.

The pKa and pKaH values calculations were performed on Marvin Sketch software (ChemAxon, version 19.13.00) [START_REF]ChemAxon -Software Solutions and Services for Chemistry & Biology[END_REF] from the compounds structures. Acidic pKa value was leveled up to 50 for the calculations.

General procedure for the preparation of N-Boc and N-Cbz aziridines using the Mitsunobu reaction (Table 1, route A)

N-Cbz or N-Boc amino-alcohol (1.0 eq.) is dissolved under argon atmosphere in anhydrous THF (13.2 mL/mmol) at 0°C. Triphenylphosphine (1.5 eq.) is added in one portion, followed by diethylazodicarboxylate (1.64 eq.) dropwise, at 0°C. The reaction mixture is allowed to warm to room temperature and kept stirring until completion of the reaction (TLC monitoring). Solvents are removed in vacuum, and the crude is purified by silica gel flash chromatography (petroleum ether/ethyl acetate ) to obtain the desired compound.

Benzyl (S)-2-phenylaziridine-1-carboxylate (2a)

The compound (S)-2a (1.42 g, 5.61 mmol, 76 % yield) was obtained as an oil following the procedure described above (overnight reaction) from compound 1a (2.00 g, 7.38 mmol).

Rf petroleum ether/EtOAc (7/3, v/v): 0.90.

Benzyl (S)-2-benzylaziridine-1-carboxylate (2c)

The compound (S)-2c (626 mg, 2.34 mmol, 67% yield) was obtained as a white solid following the procedure described above (overnight stirring) from commercially available N-Cbz-L-phenylalanilol

(1.00 g, 3.50 mmol).

Rf hexane/EtOAc (7/3, v/v): 0.80. 1

Benzyl (S)-2-((1H-indol-3-yl)methyl)aziridine-1-carboxylate (2d)

The compound (S)-2d (550 mg, 1.80 mmol, 49% yield) was obtained as an oil following the procedure described above (overnight stirring) from compound 1d (1.20 g, 3.70 mmol).

Rf petroleum ether/EtOAc (7/3, v/v): 0.43.

tert-Butyl (S)-2-(4-(benzyloxy)benzyl)aziridine-1-carboxylate (2e)

The compound (S)-2e (766 mg, 2.26 mmol, 53% yield) was obtained as an oil following the procedure described above (6h stirring) from compound 1e (1.51 g, 4.23 mmol).

Rf petroleum ether/EtOAc (9/1, v/v): 0.42.

Benzyl (S)-2-(4-((tert-butoxycarbonyl)amino)butyl)aziridine-1-carboxylate (2i)

The compound (S)-2i (900 mg, 2.59 mmol, 95% yield) was obtained as an oil following the procedure described above (overnight stirring) from compound 1i (1.00 g, 2.73 mmol).

Rf petroleum ether/EtOAc (7/3, v/v): 0.51.

General Procedure for the Preparation of N-Boc and N-Cbz aziridines from amino-alcohols via onepot cyclization (Table 1, route B)

N-Cbz or N-Boc amino-alcohol (1 eq.) is dissolved under argon atmosphere in anhydrous diethyl ether (15 mL/mmol) at room temperature. Dry para-toluenesulfonyl chloride (2.2 eq.) is added, followed by the addition of KOH grinded pellets (9.0 eq.) A drying tube filled with calcium chloride (CaCl2) seals the condenser. The reaction mixture is heated to 40°C and stirred until completion of the reaction. Solvents are evaporated in vacuum, and the crude dissolved in ethyl acetate. The salts are filtered and the filtrate concentrated to obtain the desired compound. Purification step using silica gel flash chromatography (petroleum ether/ethyl acetate gradient) was only performed for compounds 2b, 2l and 2m.

tert-Butyl (S)-2-benzylaziridine-1-carboxylate (2b)

The compound (S)-2b (1.39 g, 5.96 mmol, 75 % yield) was obtained as a pale orange oil following the procedure described above (overnight stirring) from commercially available N-Boc-L-phenylalanilol

(2.00 g, 7.96 mmol).

Rf petroleum ether/EtOAc (9/1, v/v): 0.75.

tert-Butyl (S)-2-((S)-sec-butyl)aziridine-1-carboxylate (2g)

The compound (S)-2g (891 mg, 4.47 mmol, 97% yield) was obtained as an oil following the procedure described above (overnight stirring) from commercially available N-Boc-L-isoleucinol (1.00 g, 4.60 mmol).

Rf hexane/EtOAc (7/3, v/v): 0.85.

tert-Butyl (S)-2-(4-((tert-butoxycarbonyl)amino)butyl)aziridine-1-carboxylate (2j)

The compound (S)-2j (284 mg, 0.90 mmol, quantitative yield) was obtained as an oil following the procedure described above (4h stirring) from compound 1j (300 mg, 0.90 mmol).

Rf Dichloromethane/MeOH (95/5, v/v): 0.70. 1

Benzyl (1R,6S)-7-azabicyclo[4.1.0]heptane-7-carboxylate (2k)

The compound (S)-2k (789 mg, 3.41 mmol, 97% yield) was obtained as a pale orange oil following the procedure described above (5h stirring) from compound 1k (1 g, 3.5 mmol).

Rf petroleum ether/EtOAc (9/1, v/v): 0.50. ). Data in accordance with the literature. [START_REF] Mcghee | Stereoselective Synthesis of 2,5-Disubstituted Morpholines Using a Palladium-Catalyzed Hydroamination Reaction[END_REF] Weakly ionizable compound, molecular peak undetected in MS analysis, neither in positive or negative mode.

Benzyl (1aR,6aS)-6,6a-dihydroindeno[1,2-b]azirine-1(1aH)-carboxylate (invertomers) (2l)

The compound (S)-2l (150 mg, 0.57 mmol, 16% yield) was obtained as an oil following the procedure described above (overnight stirring) from compound 1l (1.00 g, 3.53 mmol).

Rf hexane/EtOAc (7/3, v/v): 0.30.

Synthesis of aziridine 2f tert-Butyl (S)-(1,4-dihydroxybutan-2-yl)carbamate (5)

The protocol is adapted from Jorrës et al. [START_REF] Jörres | Asymmetric Michael Additions of α-Nitrocyclohexanone to Aryl Nitroalkenes Catalyzed by Natural Amino Acid-Derived Bifunctional Thioureas[END_REF] Compound 5 was isolated as oil (6.54 g, 31.80 mmol, 73% yield) from L-aspartic acid. Data in accordance with the literature. 29

tert-Butyl (S)-2-(2-(tosyloxy)ethyl)aziridine-1-carboxylate (6)

The procedure is adapted from Aaseng et al. [START_REF] Aaseng | Synthesis of (S)-2-Amino-7-Methoxytetralin and Isoindolo[1,2a]Isoquinolinone Derivatives from l-Aspartic Acid[END_REF] The compound 5 (2.96 g, 14.40 mmol, 1 eq.) was dissolved in diethyl ether (15 mL/mmol) under argon atmosphere at room temperature. Paratoluenesulfonyl chloride (2.2 eq.) was added, followed by grinded KOH pellets (6 eq.).

Benzyl (R)-2-(2-(tert-butoxy)-2-oxoethyl)aziridine-1-carboxylate (2h)

The procedure is adapted from Jung et al. [START_REF] Jung | Synthesis of Threo-β-Aminoalcohols from Aminoaldehydes via Chelation-Controlled Additions. Total Synthesis of l-Threo Sphingosine and Safingol[END_REF] Cbz-L-Asp(OtBu)-OH (882 mg, 2.73 mmol, 1 eq.) was dissolved in anhydrous THF (3.2 mL/mmol) under argon atmosphere, at 0°C. BH3.THF (1M in THF, 5 eq.) was added portion wise. The mixture was stirred for 1h at 0°C then allowed to warm at room temperature and quenched by slow addition of methanol. The mixture is concentrated in vacuum. The crude was purified on silica gel by flash chromatography (dichloromethane/ethyl acetate gradient) to obtain the amino-alcohol [START_REF] Jung | Synthesis of Threo-β-Aminoalcohols from Aminoaldehydes via Chelation-Controlled Additions. Total Synthesis of l-Threo Sphingosine and Safingol[END_REF] as oil (508 mg, 1.64 mmol, 60% yield). For the next step, the protocol from Aaseng et al. [START_REF] Aaseng | Synthesis of Substituted (S)-2-Aminotetralins via Ring-Opening of Aziridines Prepared from l-Aspartic Acid β-Tert-Butyl Ester[END_REF] was adapted. The reduced compound (508 mg, 1.64 mmol, 1 eq.) was dissolved under argon atmosphere in anhydrous THF (13.2 mL/mmol) à 0°C. Triphenylphosphine (1.5 eq.) was added, followed by diethylazodicarboxylate (1.64 eq.) dropwise, at 0°C. The mixture was allowed to warm to room temperature for 2.5h. Solvents were evaporated in vacuum and the crude was purified by silica gel flash chromatography (petroleum ether/ethyl acetate gradient) to obtain the desired compound (296 mg, 1.02 mmol, 62% yield) as an oil.

(S)-4-Benzyloxazolidin-2-one (4)

The protocol is adapted from Cardillo et al. [START_REF] Cardillo | Influence of Lewis Acids on the Regioselectivity of N-Boc-Aziridine-2-Carboxylate Microwave-Assisted Rearrangement[END_REF] The compound 2b (160 mg, 0.68 mmol, 1 eq.) was dissolved in anhydrous toluene (35.7 mL/mmol), at room temperature under argon atmosphere, in a microwave tube. BF3.Et2O (1 eq.) was added, and then the tube was sealed and placed under microwave radiations (55°C for 10min, 100W, and 1000 rpm). The reaction mixture was diluted with ethyl acetate and water. The aqueous layer was extracted twice with ethyl acetate, and the organic layers were combined, dried on MgSO4, then filtered, concentrated in vacuum, and the crude was purified by silica gel flash chromatography (dichloromethane/MeOH gradient) to afford the title compound (88 mg, 0.49 mmol, 73% yield) as a white solid.

Benzyl (S)-(2-hydroxy-1-phenylethyl)carbamate (1a)

The title compound (3.76 g, 13.87 mmol, 95% yield) was obtained as a white powder following the procedure described by Sultane et al. [START_REF] Sultane | A Convenient Protocol for the Deprotection of N-Benzyloxycarbonyl (Cbz) and Benzyl Ester Groups[END_REF]

Benzyl (S)-(1-hydroxy-3-(1H-indol-3-yl)propan-2-yl)carbamate (1d)

The title compound (720 mg, 2.22 mmol, 42% yield) was obtained as a white foam following the procedure from Yeung et al., [START_REF] Yeung | Spirotetrahydro β-Carbolines (Spiroindolones): A New Class of Potent and Orally Efficacious Compounds for the Treatment of Malaria[END_REF] from the commercially available L-tryptophanol (1.00 g, 5.26 mmol).

Rf petroleum ether/EtOAc (6/4, v/v): 0.10.

tert-Butyl (S)-(1-(4-(benzyloxy)phenyl)-3-hydroxypropan-2-yl)carbamate (1e)

The protocol was adapted from Jung et al. [START_REF] Jung | Synthesis of Threo-β-Aminoalcohols from Aminoaldehydes via Chelation-Controlled Additions. Total Synthesis of l-Threo Sphingosine and Safingol[END_REF] Boc-Tyr(OBn)-OH (1.00 g, 2.69 mmol, 1 eq.) was dissolved in anhydrous THF (2.3 mL/mmol) at 0°C under argon atmosphere. BH3.THF (1M in THF, 2.5 eq.) was added dropwise over 20 minutes (exothermic reaction) to the solution and the reaction mixture was stirred 1h at 0°C, then 3h at room temperature. Methanol was slowly added at 0°C to quench the reaction. Volatiles were removed in vacuum and the crude was dissolved in methanol then concentrated several times. The crude was purified by flash chromatography on silica gel (dichloromethane/MeOH gradient) to obtain the title product as a white solid (958 mg, 2.68 mmol, 99% yield).

Rf Dichloromethane/MeOH (95/5, v/v): 0.55. 1 (s, 9H, 3CH3, Boc). Data in accordance with the literature. [START_REF] Jung | Synthesis of Threo-β-Aminoalcohols from Aminoaldehydes via Chelation-Controlled Additions. Total Synthesis of l-Threo Sphingosine and Safingol[END_REF][START_REF] Cui | α-and β-Substituted Phosphonate Analogs of LPA as Autotaxin Inhibitors[END_REF] Benzyl tert-butyl (6-hydroxyhexane-1,5-diyl)(S)-dicarbamate (1i)

The L-Nα-Z,Nε-BocLys-OH (2.00 g, 5.26 mmol, 1 eq.) was dissolved in anhydrous THF (11.0 mL/mmol) under argon atmosphere. N-methylmorpholine and ethyl chloroformate (1 eq.) were added at -10°C and the reaction mixture was stirred for 30 minutes at -10°C. LiAlH4 (2 eq.) was then slowly added dropwise and the reaction was allowed to warm at room temperature. The reaction mixture was stirred overnight at room temperature. The reaction was quenched by the slow addition of a 4.5%

NaOH solution in water, at 0°C. After stirring for 1h30, the reaction mixture was filtered on a glass filter with dichloromethane. The filtrate was concentrated, dissolved in ethyl acetate and washed with water. Organic layer was dried over MgSO4, filtered and concentrated in vacuum. The resulting crude was purified by silica gel flash chromatography (dichloromethane/MeOH gradient) to obtain the title compound as an oil (1.53 g, 4.18 mmol, 80% yield).

Rf dichloromethane/MeOH (95/5, v/v): 0.40.

Di-tert-butyl (6-hydroxyhexane-1,5-diyl)(S)-dicarbamate (1j)

The L-Nα,Nε-bisBoc-Lys-OH dicyclohexylammonium (600 mg, 1.14 mmol, 1 eq.) was dissolved in anhydrous THF (11 mL/mmol) under argon atmosphere. N-methylmorpholine and ethyl chloroformate (1 eq.) were added at -10°C and the reaction mixture was stirred for 30 minutes at -10°C. LiAlH4 (2.0 eq.) was then slowly added dropwise and the reaction was allowed to warm at room temperature. The reaction mixture was stirred overnight and then quenched by the slow addition of a 4.5% NaOH solution in water, at 0°C. After stirring for 1h30, the reaction mixture was filtered on a glass filter. The filtrate was concentrated in vacuum, dissolved in ethyl acetate and washed with water. Organic layer was dried over MgSO4, filtered and concentrated in vacuum. The crude was purified by silica gel flash chromatography (dichloromethane/MeOH gradient) to obtain the title compound as a colorless oil (300 mg, 0.90 mmol, 80% yield).

Rf dichloromethane/MeOH (95/5, v/v): 0.25.

Benzyl ((1R,2R)-2-hydroxycyclohexyl)carbamate (1k)

The protocol was adapted from Luna et al. [START_REF] Luna | Enzymatic Resolution of (±)-Cis-2-Aminocyclopentanol and (±)-Cis-2-Aminocyclohexanol[END_REF] The (1R,2R)-trans-2-aminocyclohexanol hydrochloride (2.00 g, 13.19 mmol, 1 eq.) was dissolved in distilled water (1.7 mL/mmol) and K2CO3 (1.2 eq.) was added. Then benzyl chloroformate (CbzCl, 1.2 eq.) was added dropwise at 0°C. A white precipitate was formed. The reaction mixture was allowed to warm to room temperature and stirred overnight.

Dichloromethane was added to the reaction mixture and the aqueous layer was extracted with dichloromethane. Organic layers were combined, dried on MgSO4, filtered and concentrated in vacuum. The crude was purified on silica gel (petroleum ether/ethyl acetate gradient) to afford the title compound (3.72 g, 13.00 mmol, 98% yield) as a white solid.

Rf petroleum ether/EtOAc (7/3, v/v): 0.20. The (1R,2R)-(-)-1-amino-2-indanol (1.00 g, 6.70 mmol, 1 eq.) was dissolved in anhydrous dichloromethane (1 mL/mmol) at 0°C under argon atmosphere. Benzyl chloroformate (CbzCl, 1 eq.) was added at 0°C, followed by pyridine (1.5 eq.), and the reaction mixture was stirred at room temperature overnight. The reaction was quenched by addition of brine and water was added to dissolve the salts. The mixture was extracted with dichloromethane and the resulting emulsion was filtered on a hydrophobic filter cartridge. Organic layers were recovered, dried on MgSO4, filtered and concentrated in vacuum. The crude was purified by silica gel flash chromatography (petroleum ether/ethyl acetate gradient) to obtain the title compound (1.20 g, 4.24 mmol, 63% yield) as a white solid.

Rf hexane/EtOAc (66/34, v/v): 0.30. The (1R,2R)-(-)-1-amino-2-indanol (1.00 g, 6.70 mmol, 1 eq.) was dissolved in anhydrous dichloromethane (1.8 mL /mmol) at room temperature under argon atmosphere. Triethylamine (Et3N, 1.5 eq.) was added, followed by Boc2O (di-tert-butyl dicarbonate, 1.2 eq.). The reaction was stirred at room temperature for 6h. Reaction was quenched by addition of an aqueous solution of saturated

General Procedure for the Preparation of N-methylene-gem-bisphosphonates aziridines from Ncarbamoyl aziridines

Freshly distilled diethylphosphite (6.1 eq.) was dissolved in anhydrous THF (0.5 mL/mmol of diethylphosphite) under argon atmosphere, and cooled to -78°C. Then LiHMDS (1M in THF, 6 eq.)

was added and the reaction mixture was stirred for 30 minutes at -78°C. A solution of the N-carbamoyl aziridine (1 eq.) in anhydrous THF (2.8 mL/mmol of aziridine) was added dropwise to the reaction, which was then allowed to warm to room temperature. The reaction mixture was stirred at temperature T° until completion of the reaction (TLC monitoring). The reaction was quenched by addition of a saturated aqueous solution of NH4Cl until dissolution of the salts formed. The mixture was extracted twice with ethyl acetate. Organic layers were dried on MgSO4, filtered and concentrated in vacuum and the crude was purified by silica gel flash chromatography (dichloromethane/MeOH gradient) to afford the desired product.

Tetraethyl ((2-phenylaziridin-1-yl)methylene)(S)-bis(phosphonate) (3a)

The title compound (100 mg, 0.39 mmol, 45 % yield) was obtained as a colorless oil following the procedure described above, after stirring 1h at room temperature, from compound 2a (73 mg, 0.18 mmol).

Rf dichloromethane/MeOH (95/5, v/v): 0.30.

Figure 1 .

 1 Figure 1. Structures of BPs and N-BPs clinically used or of biological interest.

-

  123.3 (m, CHAr), 82.1 (s, CCHAz, A), 75.0 (s, CCHAz, B), 62.0 (s, CH2CHAz, A), 54.8 (s, CH2CHAz, B), 37.0 (s, CH2, B), 36.2 (s, CH2, A), 28.5 (s, 3C, CH3, Boc). HRMS (ESI/Q-TOF) m/z: [M+H] + Calcd for C14H18NO2: 232.1332; found: 232.1329.

  Rf dichloromethane/MeOH (95/5, v/v): 0.30.1 H NMR (500 MHz, CDCl3) δ 7.42 -7.13 (m, 5H, CHAr), 5.38 (d, J = 4.0 Hz, 1H, NH), 4.87 (dq, J = 8.0, 6.7 Hz, 1H, CH), 3.58 (t, J = 8.4 Hz, 1H, OCH2), 3.34 (dd, J = 8.5, 6.9 Hz, 1H, OCH2), 3.15 (dd, J = 14.0, 6.2 Hz, 1H, CH2Ph), 2.95 (dd, J = 14.0, 6.8 Hz, 1H, CH2Ph).13 C{ 1 H} NMR (126 MHz, CDCl3) δ 159.7 (s, C(O)), 135.3 (s, CAr), 129.5 (s, CHAr), 128.9 (s, CHAr), 127.3 (s, CHAr), 77.2 (s, CH), 45.1 (s, OCH2), 40.7 (s, CH2Ph). HRMS (ESI/Q-TOF) m/z: [M+H] + Calcd for C10H12NO2: 178.0868; found: 178.0868.

  H NMR (400 MHz, CDCl3) δ 7.47 -7.29 (m, 5H, CHAr), 7.12 (d, J = 8.5 Hz, 2H, CHAr), 6.92 (d, J = 8.5 Hz, 2H, CHAr), 5.05 (s, 2H, CCH2Ph), 3.82 (m, 1H, CH), 3.60 (ddd, J = 43.3, 11.3, 5.4 Hz, 2H, CHCH2Ph), 2.78 (d, J = 7.2 Hz, 2H, CH2OH), 1.42

60 Benzyl

 60 1 H NMR (400 MHz, CDCl3) δ 7.40 -7.28 (m, 5H, CHAr), 5.20 -5.04 (m, 2H, CH2Ph), 4.72 (br s, 1H, NH), 3.40 (m, 1H, CH), 3.32 (m, 1H, CH), 2.80 (br s, 1H, OH), 2.11 -1.93 (m, 2H, CH2), 1.71 (m, 2H, CH2), 1.40 -1.09 (m, 4H). Data in accordance with the literature.

31 P{ 1 13 C{ 1

 311131 H} NMR (202 MHz, MeOD) δ 18.2 (d, JPP = 5.1 Hz), 18.1 (d, JPP = 5.1 Hz). HRMS (ESI/Q-TOF) m/z: [M+H] + Calcd for C15H34NO6P2: 386.1861; found: 386.1860.tert-Butyl (S)-2-(1-(bis(diethoxyphosphoryl)methyl)aziridin-2-yl)acetate (3h)The title compound (30 mg, 0.07 mmol, 20 % yield) was obtained as a colorless oil following the procedure described above (stirring for 2h at room temperature) from compound 2h (100 mg, 0.34 mmol).Rf dichloromethane/MeOH 95/5 (v/v): 0.22. 1 H NMR (500 MHz, CDCl3) δ 4.30 -4.16 (m, 8H, 4CH2CH3), 3.01 (dd, J = 15.9, 3.1 Hz, 1H, CH2C(O)), 2.38 (t, J = 18.1 Hz, 1H, PCHP), 2.11 -2.04 (m, 1H, CHAz), 1.98 (dd, J = 15.9, 9.3 Hz, 1H, CH2C(O)), 1.95 (d, J = 3.8 Hz, 1H, CH2, Az), 1.81 (d, J = 6.5 Hz, 1H, CH2, Az), 1.43 (s, 9H, 3CH3, tBu), 1.39 -1.31 (m, 12H, 4CH2CH3). 13 C{ 1 H} NMR (126 MHz, CDCl3) δ 170.8 (s, C(O)tBu), 80.8 (s, C(CH3)3, tBu), 66.0 -63.5 (t, J = 150.3 Hz, PCHP), 63.4 -63.0 (m, 4C, 4CH2CH3), 39.0 (s, CH2C(O)), 38.4 (d, J = 15.6 Hz, CHAz), 36.2 (d, J = 15.3 Hz, 2C, CH2, Az), 28.2 (s, 3C, 3CH3, tBu), 16.6 (s, 4C, 4CH2CH3). 31 P{ 1 H} NMR (162 MHz, CDCl3) δ 17.8 (d, JPP = 3.3 Hz, 1P), 17.7 (d, JPP = 3.3 Hz, 1P). HRMS (ESI/Q-TOF) m/z: [M+H] + Calcd for C17H36NO8P2: 444.1911; found: 444.1913.tert-Butyl (S)-(4-(1-(bis(diethoxyphosphoryl)methyl)aziridin-2-yl)butyl)carbamate (3i)The title compound (115 mg, 0.23 mmol, 40 % yield) was obtained as colorless oil following the procedure described above, (stirring for 4h at room temperature) from compound 2i (200 mg, 0.58 mmol). Rf dichloromethane/MeOH (95/5, v/v): 0.26. 1 H NMR (600 MHz, CDCl3) δ 4.71 (s, 1H, NH), 4.31 -4.16 (m, 8H, 4CH2CH3), 3.15 -3.06 (m, 2H, NHCH2), 2.34 (t, J = 18.2 Hz, 1H, PCHP), 1.83 -1.79 (m, 1H, CHAz), 1.79 (d, J = 4.3 Hz, 1H, CH2, Az), 1.66 (d, J = 6.4 Hz, 1H, CH2, Az), 1.56 -1.43 (m, 6H, 3CH2), 1.43 (s, 9H, CH3, Boc), 1.39 -1.34 (m, 12H, 4CH2CH3). 13 C{ 1 H} NMR (151 MHz, CDCl3) δ 156.2 (s, C(O)Boc), 65.3 (t, J = 150.3 Hz, PCHP), 63.3 -62.8 (m, 4C, CH2CH3), 42.0 (d, J = 15.3 Hz, CHAz), 40.7 (s, CH2NH), 36.7 (d, J = 16.3 Hz, CH2, Az), 29.9 (s, 3C, 3CH2), 28.6 (s, 3C, CH3, Boc), 18.0 -15.6 (m, 4C, CH2CH3). 31 P{ 1 H} NMR (162 MHz, CDCl3) δ 18 (d, JPP = 3.2 Hz), 17.9 (d, JPP = 3.2 Hz). HRMS (ESI/Q-TOF) m/z: [M+H] + Calcd for C20H43N2O8P2: 501.2495; found: 501.2497.Tetraethyl (((1R,6S)-7-azabicyclo[4.1.0]heptan-7-yl)methylene)bis(phosphonate) (3k)The title compound (120 mg, 0.31 mmol, 72 % yield) was obtained as a colorless oil following the procedure described above (stirring for 2h at room temperature) from compound 2k (100 mg, 0.43 mmol). Rf dichloromethane/MeOH 95/5 (v/v): 0.25. 1 H NMR (500 MHz, CDCl3) δ 4.32 -4.16 (m, 8H, 4CH2CH3), 2.39 (t, J = 18.0 Hz, 1H, PCHP), 1.94 -1.87 (m, 4H, 2CH2, 2CHAz), 1.77 -1.70 (m, 2H, CH2), 1.35 (td, J = 7.1, 5.9 Hz, 12H, 4CH2CH3), 1.34 -1.30 (m, 2H, 2CH2), 1.20 -1.11 (m, 2H, 2CH2). H} NMR (126 MHz, CDCl3) δ 64.7 (t, J = 149.8 Hz, PCHP), 63.5 -62.4 (m, 4C, 4CH2CH3), 41.3 (d, J = 16.4 Hz, 2C, 2CHAz), 24.1 (s, 2C, 2CH2), 20.5 (s, 2C, 2CH2), 17.0 -16.3 (m, 4C, 4CH2CH3). 31 P{ 1 H} NMR (202 MHz, CDCl3) δ 18.2 (s, 2P). HRMS (ESI/Q-TOF) m/z: [M+H] + Calcd for C15H32NO6P2: 384.1705; found: 384.1705. The title compound (57.7 mg, 0.44 mmol, 97% yield) was obtained as a solid following the procedure described above (overnight stirring at room temperature) starting from compound 2l (N-Cbz, 120 mg, 0.45 mmol); or from compound 3m (N-Boc, 120 mg, 0.52 mmol)) with 84% yield (57 mg, 0.43 mmol). Rf dichloromethane/MeOH (95/5, v/v): 0.50. 1 H NMR (500 MHz, CDCl3) δ 7.50 -7.45 (m, 1H, CHAr), 7.36 -7.24 (m, 2H, CHAr), 7.22 (d, J = 7.5 Hz, 1H, CHAr), 6.84 (s, 1H, NH), 5.93 (d, J = 7.5 Hz, 1H, CH), 4.64 (t, J = 7.0 Hz, 1H, CH), 3.21 (dd, J = 17.1, 6.5 Hz, 1H, CH2), 3.03 (d, J = 17.0 Hz, 1H, CH2).

Table 2 .

 2 Substrate scope for the formation of aziridine α-methylene-gem-bisphosphonates

	Isolated yield after purification by column chromatography. Abbreviation o.n. means overnight. [a] Total
	conversion indicated by TLC. [b] Starting material recovered. [c] Corresponding ring-opened byproduct was
	also isolated (from 2f, 5% at rt and 16% at 55°C) or detected (from 2h and 2i).

Table 3 .

 3 Influence of the nature of the base on the formation of AzbisPs.

	Entry	Base (6 eq.)	pKa	Yield (%) AzbisP 3f	β-aminoP
	1	tBuOK	17	3%	47%
	2	NaHMDS	30	0%	9%
	3	LiHMDS	30	30%	5%
	4	NaH	35	0%	5%

Table 4 .

 4 Influence of the amounts of DEP/LiHMDS on the yield of the reaction.

	Entry	DEP (eq.)	LiHMDS (eq.)	t (h)	Yield 3b (%)
	1	2.1	2	6	13
	2	3.1	3	4.5	14
	3	4.1	4	4	29

  Hz, 1H, CH2, Az), 1.98 (d, J = 3.8 Hz, 1H, CH2, Az), 1.55 -1.45 (m, 6H, 3CH2), 1.44 (s, 9H, 3CH3, Boc). 13 C{ 1 H} NMR (151 MHz, CDCl3) δ 163.6 (s, C(O)Cbz), 156.1 (s, C(O)Boc), 136.0 (s, CAr), 128.7 (s, CHAr), 128.5 (s, CHAr), 128.3 (s, CHAr), 68.2 (s, OCH2Ph), 40.6 (s, CH2NH), 38.3 (s, CHAz), 32.0 (s, CH2, Az), 31.9 (s, CH2), 28.6 (s, 3C, 3CH3, Boc), 24.2 (s, 2C, CH2). HRMS (ESI/Q-

	TOF) m/z: [M+Na] + Calcd for C19H28N2O4Na: 371.1947; found: 371.1945.
	Benzyl (2S,3S)-2,3-diphenylaziridine-1-carboxylate (2n)
	The compound (S)-2n (724 mg, 2.20 mmol, 76% yield) was obtained as an oil following the procedure
	described above (overnight stirring) from compound 1n (1.00 g, 2.88 mmol).
	Rf petroleum ether/EtOAc (7/3, v/v): 0.90. 1 H NMR (600 MHz, CDCl3) δ 7.29 -7.15 (m, 13H, CHAr),
	6.94 (dd, J = 7.8, 1.6 Hz, 2H, CHAr), 4.91 (dd, J = 65.4, 12.1 Hz, 2H, OCH2Ph), 3.71 (s, 2H, CHAz).

1 H NMR (600 MHz, CDCl3) δ 7.38 -7.30 (m, 5H, CHAr), 5.12 (s, 2H, OCH2Ph), 4.49 (s, 1H, NH), 3.10 (m, 2H, CH2NH), 2.46 -2.38 (m, 1H, CHAz), 2.34 (d, J = 6.1 13 C{ 1 H} NMR (151 MHz, CDCl3) δ 160.5 (s, C(O)Cbz), 135.5 (s, CAr), 129.7 -125.5 (m, CHAr), 68.3 (s, OCH2Ph), 48.5 (s, CHAz). HRMS (ESI/Q-TOF) m/z: [M+H] + Calcd for C22H20NO2: 330.1494; found: 330.1491.

  from the commercially available L-phenylglycinol (2.00 g, 14.58 mmol).

	Rf petroleum ether/EtOAc (6/4, v/v): 0.26. 1 H NMR (400 MHz, CDCl3) δ 7.42 -7.23 (m, 10H, CHAr),
	5.47 (s, 1H, NH), 5.11 (q, J = 12.2 Hz, 2H, OCH2Ph), 4.86 (s, 1H, CH), 3.95 -3.80 (m, 2H, CH2OH).
	Data in accordance with the literature. 54

  NaHCO3 and extracted with dichloromethane. The emulsion was filtered on a hydrophobic filter cartridge. The organic layer was dried over MgSO4, filtered and concentrated in vacuum to obtain the title compound (1.67 g, 6.7 mmol, quantitative yield) as a white solid. CbzCl, 1.2 eq.) dropwise. The mixture was stirred overnight at room temperature until completion of the reaction (TLC monitoring). The reaction was quenched by addition of water and extracted with ethyl acetate. Organic layers were acidified with HCl 1N, washed with brine, dried on MgSO4, filtrated and concentrated in vacuum. The crude was purified by silica gel flash

	Rf Hexane/EtOAc (66/34, v/v): 0.50. 1 H NMR (600 MHz, CDCl3) δ 7.30 -7.14 (m, 4H, CHAr), 5.06
	(s, 1H, NH), 4.90 (t, J = 5.9 Hz, 1H, CHNH), 4.41 (dd, J = 14.2, 7.8 Hz, 1H, CHOH), 4.30 (s, 1H,
	OH), 3.28 (dd, J = 15.8, 7.7 Hz, 1H, CH2), 2.91 (dd, J = 15.7, 8.1 Hz, 1H, CH2), 1.50 (s, 9H, CH3,
	Boc). 13 C{ 1 H} NMR (151 MHz, CDCl3) δ 157.6 (s, C(O)Boc), 140.3 (s, CArCH2), 139.5 (s,
	CArCHNH), 128.6 (s, CH), 127.3 (s, CH), 125.3 (s, CH), 123.1 (s, CH), 82.2 (s, CHOH), 80.6 (s,
	C(CH3)3), 64.2 (s, CHNH), 38.5 (s, CH2), 28.6 (s, CH3, Boc). HRMS (ESI/Q-TOF) m/z: [M+H] + Calcd
	for C14H20NO3: 250.1437; found: 250.1434.
	Benzyl ((1S,2R)-2-hydroxy-1,2-diphenylethyl)carbamate (1n)
	The (1R,2S)-2-amino-1,2-diphenylethanol (2.00 g, 9.38 mmol, 1 eq.) was dissolved in anhydrous THF
	(2.77 mL/mmol) at 0°C under argon atmosphere. Solid NaHCO3 (2 eq.) was then added, followed by
	benzyl chloroformate (chromatography (dichloromethane/MeOH gradient) to obtain the title compound (3.25 g, 9.38 mmol,
	quantitative yield) as a white powder.
	Rf petroleum ether/EtOAc (7/3, v/v): 0.50. 1 H NMR (400 MHz, CDCl3) δ 7.41 -7.28 (m, 5H, CHAr),
	7.24 -7.19 (m, 6H, CHAr), 7.04 (m, 4H, CHAr), 5.59 (br s, 1H, NH), 5.15 -4.99 (m, 4H, CHOH,
	CHNH, CH2Ph), 2.39 (br s, 1H, OH). Data in accordance with the literature. 61

  The title compound (121 mg, 0.31 mmol, 41% yield) was obtained as a colorless oil following the procedure described above (stirring for 2h at 80°C) from compound 2g (150 mg, 0.75 mmol).Rf petroleum ether/EtOAc (7/3, v/v): 0.10.1 H NMR (500 MHz, MeOD) δ 4.31 -4.15 (m, 8H, 4CH2CH3), 2.71 (t, J = 18.8 Hz, 1H, PCHP), 1.87 (tdd, J = 6.3, 4.5, 1.7 Hz, 1H, CHAz), 1.84 -1.81 (m, 1H, CH2, Az), 1.76 -1.67 (m, 1H, CHCH2CH3), 1.66 (dd, J = 6.5, 1.3 Hz, 1H, CH2, Az), 1.44 -1.39 (m, 1H, CHCH3), 1.37 (tdd, J = 7.1, 2.5, 1.2 Hz, 12H, 4 CH2CH3), 1.31 -1.19 (m, 1H, CHCH2CH3), 0.94 (t, J = 7.5 Hz, 3H, CH3CH2CH), 0.81 (d, J = 6.9 Hz, 3H, CH3CH).13 C{ 1 H} NMR (126 MHz, MeOD) δ 65.0 (t, J = 152.0 Hz, PCHP), 64.6 (ddd, J = 17.5, 13.8, 6.8 Hz, 4C, CH2CH3), 47.7 (dd, J = 13.7, 5.2 Hz, CHAz), 37.9 (s, CHCH3), 35.3 (d, J = 14.6 Hz, CH2 Az), 28.6 (s, CHCH2CH3), 16.8 (dd, J = 10.8, 4.8 Hz, 4C, CH2CH3), 14.9 (s, CHCH3), 11.9 (s, CHCH2CHy).

	(s, CAr), 122.9 (s, CAr), 122.2 (s, CAr), 120.6 (s, CAr), 109.8 (s, CAr), 64.9 (t, J = 150.5 Hz, PCHP),
	63.4 (dd, J = 26.4, 6.4 Hz, 4C, CH2CH3), 42.8 (s, CH2CH2N), 39.3 (d, J = 20.5 Hz, CHN), 35.9 (d, J =
	15.0 Hz, CHCH2N), 33.4 (d, J = 40.2 Hz, CH2CH2N), 16.7 (s, 4C, CH3CH2). 31 P{ 1 H} NMR (162 MHz,
	CDCl3) δ 17.8 (s, 1P), 17.7 (s, 1P). HRMS (ESI/Q-TOF) m/z: [M+H] + Calcd for C20H34N3O6P2:
	474.1923; found: 474.1932.
	Tetraethyl (((S)-2-((S)-sec-butyl)aziridin-1-yl)methylene)bis(phosphonate) (3g)
	1 H NMR (400 MHz, CDCl3) δ 7.31 -7.24 (m, 4H,
	CHAr), 7.21 (m, 1H, CHAr), 4.37 -4.24 (m, 4H, 2CH2CH3), 4.21 -3.98 (m, 4H, 2CH2CH3), 2.84
	(ddd, J = 6.1, 4.1, 1.7 Hz, 1H, CHAz), 2.61 (t, J = 18.0 Hz, 1H, PCHP), 2.19 (d, J = 3.9 Hz, 1H, CH2,
	Az), 2.14 (dd, J = 6.7, 1.8 Hz, 1H, CH2, Az), 1.39 (td, J 7.1, 4.1 Hz, 6H, 2CH2CH3), 1.18 (dt, J =
	14.2, 7.1 Hz, 6H, 2CH2CH3). 13 C{ 1 H} NMR (126 MHz, CDCl3) δ 139.3 (s, CAr), 128.2 (s, CHAr),
	127.2 (s, CHAr), 126.5 (s, CHAr), 65.1 (t, J = 149.3 Hz, PCHP), 63.6 (d, J = 7.1 Hz, 2C, CH2CH3),
	63.4 -63.1 (m, 2C, CH2CH3), 43.6 (d, J = 15.6 Hz, CHAz), 40.2 (d, J = 15.9 Hz, CH2, Az), 16.8 -
	16.6 (m, 2C, 2CH2CH3), 16.4 (d, J = 6.8 Hz, 2C, 2CH2CH3). 31 P{ 1 H} NMR (162 MHz, CDCl3) δ 17.6
	The title compound (264 mg, 0.63 mmol, 84 % yield) was obtained as a colorless oil following the
	procedure described above (overnight stirring at room temperature) from compound 2c (200 mg, 0.75
	mmol) or from compound 2b (stirring 2h at 80°C, 500 mg, 2.14 mmol) with 69% yield (630 mg, 1.50
	mmol).
	Rf dichloromethane/MeOH (95/5, v/v): 0.35. 1 H NMR (500 MHz, CDCl3) δ 7.30 -7.18 (m, 5H,
	CHAr), 4.32 -4.18 (m, 8H, CH2CH3), 3.33 (dd, J = 14.1, 3.6 Hz, 1H, CH2Ph), 2.40 (t, J = 18.1 Hz,
	1H, PCHP), 2.40 (dd, J = 13.4, 9.4 Hz, 1H, CH2Ph), 2.11-2.00 (m, 1H, CHAz), 1.93 (d, J = 3.9 Hz,
	1H, CH2, Az), 1.72 (d, J = 8.3 Hz, 1H, CH2, Az), 1.38 (t, J = 7.1 Hz, 6H, CH2CH3), 1.35 (dt, J = 7.0,
	4.4 Hz, 6H, CH2CH3). 13 C{ 1 H} NMR (126 MHz, CDCl3) δ 138.8 (s, CAr), 129.1 (s, 2C, CHAr), 128.5
	(s, 2C, CHAr), 126.4 (s, CHAr), 64.9 (t, J = 150.1 Hz, PCHP), 63.3 (d, J = 10.6 Hz, 2C, CH2CH3),
	63.1 (d, J = 6.9 Hz, 2C, CH2CH3), 43.1 (d, J = 16.2 Hz, CHAz), 38.9 (s, CH2Ph), 36.5 (d, J = 17.0 Hz,
	CH2 Az), 16.7 (d, J = 10.3 Hz, 2C, CH2CH3), 16.6 (d, J = 10.1 Hz, 2C, CH2CH3). 31 P{ 1 H} NMR (202
	MHz, CDCl3) δ 18.0 (s, 1P), 17.9 (s, 1P). HRMS (ESI/Q-TOF) m/z: [M+H] + Calcd for C18H32NO6P2:
	420.1705; found: 420.1709.

(d, JPP = 2.6 Hz), 17.4 (d, JPP = 2.6 Hz). HRMS (ESI/Q-TOF) m/z: [M+H] + Calcd for C17H30NO6P2: 406.1548; found: 406.1549.

Tetraethyl ((2-benzylaziridin-1-yl)methylene)(S)-bis(phosphonate) (3b) Tetraethyl ((2-((1H-indol-3-yl)methyl)aziridin-1-yl)methylene)(S)-bis(phosphonate) (3d)

The title compound (53 mg, 0.11 mmol, 45 % yield) was obtained as a colorless oil following the procedure described above (4h stirring at room temperature) from compound 2d (78 mg, 0.25 mmol).

Acknowledgments

We are grateful to the CNRS (National Scientific Research Council) for financial support

Supporting Information.

Copies of 1 H, 13 C and eventually 31 P NMR spectra of all new compounds described in the experimental section, copies of 1 H NMR spectra for known compounds or intermediates. NMR comparative study for compounds 2d-3d and 2g/3g.