Rapid Synthesis of Nucleoside Triphosphates and Analogues

Nucleoside triphosphates (NTPs) are essential biomolecules involved in almost all biological processes, and their study is therefore critical to understanding cellular biology. Here, we describe a chemical synthesis suitable for obtaining both natural and highly modified NTPs, which can, for example, be used as surrogates to probe biological processes. The approach includes the preparation of a reagent that enables the facile introduction and modification of three phosphate units: cyclic pyrophosphoryl P‐amidite (c‐PyPA), derived from pyrophosphate (PV) and a reactive phosphoramidite (PIII). By using non‐hydrolyzable analogues of pyrophosphate, the reagent can be readily modified to obtain a family of non‐hydrolyzable analogues containing CH2, CF2, CCl2, and NH that are stable in solution for several weeks if stored appropriately. They enable the synthesis of NTPs by reaction with nucleosides to give deoxycyclotriphosphate esters that are then oxidized to cyclotriphosphate (cyclo‐TP) esters. The use of different oxidizing agents provides an opportunity for modification at P‐α. Furthermore, terminal modifications at P‐γ can be introduced by linearization of the cyclo‐TP ester with various nucleophiles. © 2020 The Authors.


INTRODUCTION
(Deoxy)nucleoside triphosphates are building blocks for essential biomacromolecules (DNA and RNA), and furthermore have fundamental importance in signaling processes and metabolic pathways (Hollenstein, 2012;Roy, Depaix, Périgaud, & Peyrottes, 2016). Chemical syntheses of natural and unnatural/modified NTPs provide many significant opportunities for the study of their versatile functions (Dutta, Captain, & Jessen, 2017;Ermert, Marx, & Hacker, 2017;Hocek, 2014). Yet, despite impressive progress over the years (Liao, Bala, Ngor, Yik, & Chaput, 2019;Shepard, Windsor, Raines, & Cummins, 2019;, novel, easy-to-implement synthetic methods are still very much needed (Liao et al., 2019). In this context, we recently reported a family of reagents, the so-called cyclic pyrophosphoryl P-amidites (c-PyPA) (Singh, Steck et al., 2019), that allow an easy and straightforward synthesis of α,βand γ-modified NTPs in a one-flask operation . Due to the selectivity of the initial phosphitylation reaction, nucleosides can be customized without protecting groups, although placing protecting groups on the nucleosides will increase yield and facilitate purification. Figure 1 illustrates the syntheses of NTPs by this approach, highlighting the possible modifications that can be introduced. c-PyPA can be prepared before NTP synthesis and stored, thereby enabling the synthesis of several analogues in multiple reactions. The reactions of different pyrophosphate derivatives (such as pyrophosphate, imidodiphosphate, methylene diphosphonate, dichloromethylene diphosphonate, and difluoromethylenediphosphonate; (1)) with diisopropylphosphoramidous dichloride (2) allow access to a variety of c-PyPA-type reagents (3). The resulting c-PyPA and its analogues (c-Py NH PA, c-Py CH2 PA, c-Py CCl2 PA, c-Py CF2 PA) are stable for at least 4 weeks under an argon atmosphere at -20°C, except for the imidodiphosphate analogue.
The c-PyPA reagents can be used directly in solution without purification for NTP synthesis and, in the presence of an acidic activator, will react preferentially with the sterically least hindered alcohol of the nucleoside. Thus, there is a pronounced preference for primary over secondary alcohols. Due to this selectivity, nucleosides can be used without protecting groups provided they have some solubility in polar organic solvents (such as DMF, DMSO, MeCN, or ionic liquids). After the coupling step, the deoxycyclo-TP ester (5) is oxidized and then linearized through a nucleophilic attack on the cyclo-TP intermediate (6), which generally results in linearization and places the incoming nucleophile in the γ position. This pathway was validated both experimentally and by computation . Variations of the oxidizing reagent and the nucleophile for linearization provide diverse options for modification and can be mutually combined. For instance, using imidazole or morpholine as nucleophiles enables the synthesis of well-known activated NTPs (γ-morpholidates, γ-imidazolides; Wanat et al., 2015;Warminski, Sikorski, Kowalska, & Jemielity, 2017), which can be used to generate long oligophosphate chains. As another example, the use of propargylamine as nucleophile yields precursors for azide-alkyne cycloadditions ("click chemistry"; Azevedo et al., 2018;Rostovtsev, Green, Fokin, & Sharpless, 2002;Serdjukow, Kink, Steigenberger, Tomás-Gamasa, & Carell, 2014;Wanat et al., 2015).
This protocol is designed to guide users in applying c-PyPA reagents and benefiting from their versatility. We describe potential pitfalls and how to avoid them, so that high yields and easy purifications can be achieved in every laboratory that wants to include these novel reactions in their NTP synthesis portfolio.
Basic Protocol 1 presents a systematic procedure for the synthesis of c-PyPA deriving from pyrophosphate. This protocol can also be applied to the rest of this reagent family (c-Py NH PA, c-Py CH2 PA, c-Py CCl2 PA, c-Py CF2 PA) by using the corresponding pyrophosphate analogues. However, for the synthesis of c-Py NH PA, c-Py CH2 PA, c-Py CCl2 PA, and c-Py CF2 PA, some minor deviations, as compared to the constitutive c-PyPA protocol detailed here, need to be considered. These deviations are indicated directly after the respective steps. In Basic Protocol 2, the use of c-PyPA for the synthesis of 3 -azidothymidine (AZT) triphosphate (AZTTP) and its modification on P-α using different oxidizing agents is discussed. Basic Protocol 3 provides further insights into the triphosphorylation of unprotected 2 -deoxynucleosides, with discussion of selectivity issues and separation of regioisomers. The triphosphorylation of adenosine as a prototype ribonucleoside is highlighted in Basic Protocol 4. Finally, in Basic Protocol 5 we discuss the application of c-PyPA analogues to generate non-hydrolyzable NTPs with modifications between the β-γ phosphates. Finally, a Support Protocol describes the synthesis of diisopropylphosphoramidous dichloride, a necessary reagent for the preparation of cyclic pyrophosphoryl P-amidite (c-PyPA) and its analogues in Basic Protocol 1.
IMPORTANT NOTE: Several steps of the following procedures require anhydrous conditions. These steps should be carried out under a dry argon atmosphere in oven-dried glassware and anhydrous solvents. The anhydrous solvents can be purchased from commercial suppliers unless otherwise noted.
IMPORTANT NOTE: To guarantee a strictly dry, inert atmosphere during reactions, use the Schlenk technique. In addition, dry the argon gas by passing it through a column containing phosphorous pentoxide-coated boiling stones. Transfer the liquid chemical reagents by using a syringe and needles under a flow of dry argon. An argon-filled balloon can provide a dry argon atmosphere in a septum-sealed flask.
IMPORTANT NOTE: Both syringes and needles should be purged with argon three times before filling with chemicals.
IMPORTANT NOTE: Be careful not to contaminate chemical bottles while transferring liquid reagents. Be aware that chemical containers are not under pressure; therefore, slight positive pressure is required. This protocol describes the synthesis of cyclic pyrophosphoryl P-amidite (c-PyPA) and its analogues (c-Py NH PA, c-Py CH2 PA, c-Py CCl2 PA, and c-Py CF2 PA) (3a-e) deriving from the reaction of diisopropylphosphoramidous dichloride (2) with the corresponding pyrophosphate analogues (1; Fig. 2). Pyrophosphate and the other analogues, which are commercially available as sodium salts (8), must first be converted into the corresponding tetrabutylammonium (TBA) salts. The resulting bis-TBA pyrophosphate, imidodiphosphate, methylene diphosphonate, dichloromethylene diphosphonate, and difluoromethylenediphosphonate, now soluble in polar organic solvents, then react with 2 in the presence of a tertiary amine base (trimethylamine or diisopropylethylamine) under strictly anhydrous conditions. After quantitative conversion into the desired reagents (3a-e), monitored by 31 P-NMR (diagnostic triplet at 130-125 ppm for P III atom), the reaction mixture can be directly used for the triphosphorylation of nucleosides.
The synthesis of c-PyPA (3a), c-Py CH2 PA (3c), c-Py CCl2 PA (3d), and c-Py CF2 PA (3e) starting from pyrophosphate or phosphonate analogues is readily implemented, whereas c-Py NH PA (3b) is much more difficult to handle and proved to be unstable in the reaction mixture. This is further complicated by the fact that the commercially available salt is impure. Therefore, 3b must be used directly for the coupling reactions, as it cannot be stored, and yields are typically lower. Because c-PyPA (3a), c-Py CH2 PA (3c), c-Py CCl2 PA (3d), and c-Py CF2 PA (3e) are stable over extended periods of time if stored appropriately (argon, anhydrous conditions), the preparation of more significant amounts of these compounds is recommended, as this facilitates the correct weighing of reagents.

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Current Protocols in Nucleic Acid Chemistry The reagents (3a-d) can be prepared less concentrated (0.075 M) in anhydrous MeCN or more concentrated (0.5 M) in anhydrous DMF, depending on the solubility of the nucleoside substrate.

Materials
Starting reagent, i.e., one of the following:

Synthesis of tetrabutylammonium pyrophosphate salt
IMPORTANT NOTE: Confirm the quality of the pyrophosphate and derivates by 31 P-NMR before use, as high quality of the material is essential for reliable results (>99%). Pure sodium dihydrogen diphosphate resonates in D 2 O as a singlet at -10 ppm. Monophosphate, a common impurity, generates a singlet at 0 ppm in D 2 O.
The procedure is optimized for pyrophosphate and its diphosphonate analogues. Because imidodiphosphate sodium salt is already unstable during cation exchange on the Dowex column and also later in the reaction mixture, the procedure will yield less reliable results for this reagent.
1. Weigh 3.0 g sodium dihydrogen diphosphate (13.5 mmol, 1.0 eq.) into a 10-ml beaker and dissolve in 20 ml deionized water. 3. Elute the target compound with ∼300-350 ml deionized water, collecting it as one fraction in a 500-ml flask. Monitor the elution of the pyrophosphoric acid by checking the pH value using universal indicator paper.
A pH of 2 indicates the presence of pyrophosphoric acid during elution.
4. The elution of the target compound into the 500-ml flask will be complete as soon as the pH value of the eluate is neutral again.
Alternatively, use commercially available 1.5 M TBAOH solution and add 27.0 mmol.
IMPORTANT NOTE: When using methylene bisphosphonic acid or difluoromethylene bisphosphonic acid as a reagent, skip steps 1-4 and start directly with step 5. Dissolve the acid in deionized water and add 2.0 eq. TBAOH.
6. Attach the flask to a rotary evaporator and replace the heating bath with a dewar filled with acetone and dry ice (−78°C). Rotate slowly to freeze the water inside the flask.
Make sure that the volume of the flask is sufficient so that the volume of ice in the flask is less than half of the total volume to avoid cracks during freeze-drying (step 7). 7. Remove water by lyophilization on a freeze dryer overnight. 9. Dissolve the solidified product in 30 ml anhydrous MeCN and transfer the liquid into a precisely weighed 100-ml single-neck round-bottom flask. Wash the 500-ml flask with 20 ml anhydrous MeCN to complete the transfer into the 100 ml flask.
10. Evaporate the solvent under reduced pressure by using a rotary evaporator.
11. Repeat step 10 twice with 10 ml anhydrous MeCN and dry the obtained solid (solidified oil) further under high vacuum overnight.
Ripp et al.

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Current Protocols in Nucleic Acid Chemistry 12. Weigh out the flask and calculate the exact yield (in millimoles) of the respective TBA pyrophosphate salt in consideration of the already determined number of equivalents of TBA cation.

Synthesis of cyclic pyrophosphoryl P-amidite
The amount of TBA PPi salt given is that obtained from step 12. However, there can be batch to batch variations, so recalculate the amounts of the additional reagents as appropriate with regard to equivalents and molarities.
15. Seal the flask with a rubber septum.
16. Exchange the atmosphere three times with dry argon, and dissolve TBA PP i in 40 ml anhydrous MeCN (13.3 ml per mmol TBA PP i ).
17. Let the solution stand for at least 4 h to enable efficient removal of residual water.
Alternatively, use anhydrous DIPEA as a base.
20. Monitor progress of the reaction by 31 P-NMR: Mix 250 μl of the reaction mixture and 250 μl CDCl 3 in an NMR tube under argon atmosphere. Completion of the reaction is revealed by the disappearance of a singlet at +170 ppm (resonance of (iPr) 2 N-PCl 2 ) and the appearance of a triplet at +130 ppm (resonance of P III of the c-PyPA).
21. Seal the reaction flask with parafilm and store it at −20°C.
22. Use the clear solution obtained (approximate molarity 0.075 M) directly for the coupling reactions without further manipulations (calculate the molarity of the solution by dividing the initial amount of TBA PP i by the added amount of anhydrous MeCN, assuming quantitative conversion.) IMPORTANT NOTE: During the preparation of the c-PyPA reagent, the formation of a colorless precipitate indicates that the reaction was unsuccessful; in this case, an inappropriate amount of reagent was likely used in the synthetic procedure.
23. Using an aliquot of the reaction mixture, characterize the product, 3a-e, by 31 P-NMR and HR-MS.

SYNTHESIS OF 3 -AZIDOTHYMIDINE 5 -γ-P-PROPARGYLAMIDO TRIPHOSPHATES AND ANALOGUES
In the following, the synthesis of 3 -azidothymidine (AZT) 5 -γ-P-propargylamido triphosphate and P-α analogues is described in detail (Fig. 3). Under dry conditions and in the presence of an acidic activator, c-PyPA reacts with AZT (8), forming the deoxycyclo-TP ester (9). Three options are described for the subsequent oxidization of the ester: Oxidation of 9 with mCPBA transfers an oxygen atom, while oxidation with Beaucage's reagent or KSeCN transfer S or Se, respectively. The obtained cyclo-TP esters (10a-c) are then linearized by a nucleophile. By using propargylamine as the nucleophile,

Part 1: Preparation of deoxycyclo-TP ester (9)
1. Perform the reaction in an oven-dried 25-ml pear-shaped single-neck flask equipped with a triangular-shaped magnetic stirring bar.
2. Seal the flask with a rubber septum; heat it with a heat gun under high vacuum.
9a. Stir the reaction mixture for 5-10 min and monitor complete oxidation to the cyclo-TP ester 10a via 31 P-NMR: a triplet at around -25 ppm should be observed.
Complete oxidation with mCPBA is usually achieved within 3-5 min.
IMPORTANT NOTE: Track the linearization of the triphosphate by analyzing an aliquot of the reaction mixture by 31 P{ 1 H}-NMR, which will show the consumption of cyclo-TP ester 10a (disappearing triplet at -25 ppm) and the formation of the linearized product by its characteristic γ -P phosphoramidate resonance (doublet at -2 ppm).
13a. Distribute the reaction mixture in equal parts into the two centrifuge tubes and shake them. The sodium salt of the triphosphate (11a) will precipitate.
15a. Dispose of the organic layer in the beaker.
Alternatively, to recover the crude product from the organic layer, keep the organic layer in a beaker and let it stand overnight to allow evaporation of the solvent. Add acetone to the residual solid in the beaker. Collect the precipitate by centrifugation again and perform steps 16a and 17a with the collected solids. This can improve the yield but is only necessary if the precipitation was not complete.
16a. Resuspend the precipitate in 30 ml acetone, centrifuge again, and dispose of the supernatant. Repeat this washing step once.

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Current Protocols in Nucleic Acid Chemistry

Part 2b: Oxidation of deoxycyclo-TP ester (9) with Beaucage's reagent (α = S)
IMPORTANT NOTE: In part 1, deoxycyclo-TP ester (9) was prepared on a smaller scale (0.11 mmol). The described oxidation was optimized for this scale, but scaling up should be straightforward.
8b. Cool the solution from part 1 (being careful with quantities, as the scale of the experiment is different) to 0°C and add 4.5 mg Beaucage's reagent (0.22 mol, 2.0 eq.) in one portion.
9b. Stir the reaction mixture for 5 min and control the formation of α-(S)-cyclo-TP ester (10b) via 31 P-NMR. A characteristic signal at ∼43 ppm should appear.
Adjust the volume of the solutions of 0.5 M NaClO 4 in acetone to your reaction scale.
11b. Dry the obtained solid under high vacuum and determine the yield of AZT 5 -γ propargylamido triphosphate 11b.
The compounds are obtained as a mixture of diastereoisomers in ∼1:1 ratio.
8c. Cool the solution from part 1 (being careful with quantities, as the scale of the experiment is different) to 0°C and carefully add 650 mg KSeCN (4.51 mmol, 12.5 eq.) in one portion.
10c. To achieve complete precipitation, add 30 ml acetone to the flask, collect the precipitate by centrifugation (5-7 min at 7700 rcf), and analyze an aliquot of the precipitate by 31 P-NMR.
12c. Perform precipitation of the product by following steps 10a-16a of this protocol, adjusting the volume of the solution of 0.5 M NaClO 4 in acetone to your reaction scale.

Part 1: Preparation of deoxycyclo-TP esters (13)
1. Perform the reaction in a 50-ml oven-dried pear-shaped single-neck flask equipped with a triangular-shaped magnetic stirring bar.
2. Seal the flask with a rubber septum; heat it with a heat gun under high vacuum.
4. Co-evaporate the solids with 10 ml MeCN as described in Basic Protocol 2, step 4. 5. Dry the solids for at least 30 min under high vacuum and purge three times with argon.
7. Monitor the formation of the deoxycyclo-TP ester (13) by 31 P-NMR. A shift of the P III resonance from +130 ppm toward +100 ppm should be observed.
IMPORTANT NOTE: Track the linearization of the triphosphate by analyzing an aliquot of the reaction mixture by 31 P{ 1 H}-NMR, which will show the consumption of cyclo-TP ester 10a (disappearing triplet at -25 ppm) and the formation of the linearized product with its characteristic γ -P phosphoramidate resonance (P-N) (doublet at -2 ppm).
11. Isolate the sodium salt of the product triphosphate by NaClO 4 precipitation as described in Basic Protocol 2, steps 12a-16a.
12. Dry the obtained solid under high vacuum.
13. Analyze an aliquot on a reverse-phase analytical HPLC to identify conditions for the separation of the 5 -and 3 -regioisomer on a preparative scale (an exemplary chromatogram is shown in Fig. 5). Use the following HPLC conditions:

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Current Protocols in Nucleic Acid Chemistry 14. For large-scale separation of the regioisomers, use an MPLC system. Dissolve half of the sample (to avoid column overload) in 1 ml deionized water and load it onto a C18 column. Use the following separation conditions: Column: F0012, 20 g; particle size: 30 μm, PF-C18-AQ 15. Evaporate the collected fractions of the 5 -and 3 -regioisomers under reduced pressure and isolate the regioisomers by NaClO 4 precipitation (Basic Protocol 1, step 3a, 10-16).

SYNTHESIS OF ADENOSINE 5 -γ-P-AMIDO TRIPHOSPHATE (19) AND ADENOSINE 5 -γ-P-PROPARGYLAMIDO TRIPHOSPHATE (20)
The synthesis of P-γ modified 5 -adenosine triphosphates (19, 20) is described in the following procedure (Fig. 6). Adenosine (18), as a representative of the ribonucleosides, has one primary alcohol moiety at the 5 position and two secondary alcohol moieties in the 2 and 3 positions. A triphosphorylation with c-PyPA reagent leads to the desired

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Current Protocols in Nucleic Acid Chemistry 5 -phosphorylated product 19 or 20, depending on the nucleophile used for linearization, as a major component and 2 ,3 -cyclic AMP (2 ,3 -cAMP) as a byproduct. 2 ,3 -cAMP, which generates a characteristic singlet at ∼20 ppm in the 31 P-NMR, can be easily removed by ion-exchange chromatography given its significant difference in overall charge. To prevent precipitation during the coupling step with c-PyPA, since adenosine is only soluble in DMF, a concentrated (0.5 M) c-PyPA mixture in DMF is preferred to a less concentrated (0.075 M) c-PyPA mixture in MeCN. 10 -3 mbar) 0.1-ml NMR tube 20-or 50-ml Falcon tubes Centrifuge Strong anion-exchange chromatography (SAX) system (e.g., ÄKTApure TM system and HiTrap TM column) Lyophilizer (Alpha 1-4 LD plus Freeze Dryer from Christ)
2. Seal the flask with a rubber septum; heat it with a heat gun under high vacuum.
4. Co-evaporate the solids with 10 ml MeCN as described in Basic Protocol 2, step 4.

5.
Purge three times with argon.
7. Monitor the formation of the deoxycyclo-TP ester (17) by 31 P-NMR. A shift of the P III atom from +130 ppm toward +104 ppm should be observed.

Part 3: Linearization of cyclo-TP ester (18) to obtain adenosine 5 -γ-modified triphosphates (19, 20) as their ammonium salts
IMPORTANT NOTE: The process of ring opening with different nucleophiles is performed by combining the reaction mixture in an NMR tube also containing the nucleophile and D 2 O.
10. Add 0.5 ml (∼25 μmol) of the reaction mixture, with a calculated maximal concentration of 0.049 M cyclo-TP ester, to a 0.1-ml NMR tube containing D 2 O and 0.1 ml of either conc. aq. NH 3 , to give 19, or propargylamine, to give 20.
A 31 P-NMR analysis will show the formation of the desired products, but also of 2 ,3 -cAMP and PP i (modified ATP:2 ,3 -cAMP = 1.0:0.6).
12. Dry the obtained solid under high vacuum.
The crude product contains impurities, which we identified as 2 ,3 -cAMP and PP i . These can be easily removed by strong ion-exchange chromatography.
The second cycle of freeze-drying helps to remove residual buffer. Another repetition might be required.

SYNTHESIS OF d4T 5 -γ-PROPARGYLAMIDO β,γ-(DIFLUOROMETHYLENE)TRIPHOSPHATE
This procedure highlights the synthesis of β,γ non-hydrolyzable nucleotides by utilizing the family of c-PyPA reagents, e.g., c-Py CH2 -PA and c-Py CF2 -PA. The protocol details the synthesis of d4T 5 -γ-propargylamido β,γ-(difluoromethylene)triphosphate as an example. This protocol can be easily adopted to generate a range of other compounds, including nucleosides, and by combination with the other protocols in regard to, for example, the oxidation or linearization steps. Several examples are discussed in the primary publications . Figure 7 summarizes the possibilities of this approach.

Part 1: Preparation of deoxycyclo-TP ester (22)
1. Perform the reaction in a 25 ml oven-dried, pear-shaped single-neck flask equipped with a triangular-shaped magnetic stirring bar.
2. Seal the flask with a rubber septum; heat it with a heat-gun under high vacuum.
4. Co-evaporate the solids with 8 ml MeCN as described in Basic Protocol 2.
5. Purge three times with Ar.
6. Add 1 ml anhydrous DMF to dissolve the solids.
8. The reaction results in the formation of a deoxycyclo-TP ester (22), which can be monitored by 31 P-NMR. A shift of the P III atom from +130 ppm toward +108 ppm can be observed.
10. Stir the reaction mixture for 3 min and monitor complete oxidation to the cyclo-TP ester (23) via 31 P-NMR. A triplet will appear at around -25 ppm.

Part 3: Linearization of cyclo-TP ester (23) to obtain d4T 5 -γ-propargylamido β,γ-(difluoromethylene)triphosphate (24) as its sodium salt
IMPORTANT NOTE: The ring opening is performed similarly to that in Basic Protocol 4: The reaction mixture is added to an NMR tube containing the nucleophile and D 2 O.
Monitor the linearization by 31 P-NMR to observe the complete consumption of cyclo-TP ester 10a (triplet at -25 ppm) and the formation of the desired product (resonance for γ -P phosphoramidate bond (P-N) at -6 ppm).
12. Isolation the sodium triphosphate salt by the NaClO 4 precipitation method as described in Basic Protocol 2 steps 12-16.
13. Dry the obtained solid under high vacuum.

SYNTHESIS OF DIISOPROPYLPHOSPHORAMIDOUS DICHLORIDE
This protocol describes the synthesis of diisopropylphosphoramidous dichloride (2), necessary as a reagent for the preparation of the cyclic pyrophosphoryl P-amidite (c-PyPA) and its analogues (c-Py NH PA, c-Py CH2 PA, c-Py CCl2 PA, c-Py CF2 PA) (3a-e) (Basic Protocol 1). Although diisopropylphosphoramidous dichloride (2) is commercially available, we recommend performing an in-laboratory synthesis when it is used on a larger scale. The preparation we describe here is based on published synthesis from 1985 using anhydrous diethyl ether as solvent (King & Sadanani, 1985). We also found another synthetic approach for this compound, reported by Amigues et al., to be very suitable (Amigues, Hardacre, Keane, & Migaud, 2008). By using an ionic liquid as a solvent, the pure product could be easily obtained by distillation directly out the reaction mixture, and recycling of the ionic liquid is possible.