Synthesis of Mixed Dinucleotides by Mechanochemistry

We report the synthesis of vitamin B1, B2, and B3 derived nucleotides and dinucleotides generated either through mechanochemical or solution phase chemistry. Under the explored conditions, adenosine and thiamine proved to be particularly amenable to milling conditions. Following optimization of the chemistry related to the formation pyrophosphate bonds, mixed dinucleotides of adenine and thiamine (vitamin B1), riboflavin (vitamin B2), nicotinamide riboside and 3-carboxamide 4-pyridone riboside (both vitamin B3 derivatives) were generated in good yields. Furthermore, we report an efficient synthesis of the MW+4 isotopologue of NAD+ for which deuterium incorporation is present on either side of the dinucleotidic linkage, poised for isotopic tracing experiments by mass spectrometry. Many of these mixed species are novel and present unexplored possibilities to simultaneously enhance or modulate cofactor transporters and enzymes of independent biosynthetic pathways.


Introduction
Together, the vitamin B1-thiamine, B2-riboflavin and B3-niacin/nicotinamide derived cofactors include but are not limited to thiamine monophosphate, pyrophosphate and triphosphate, thiamine adenine dinucleotide (ThMP, ThDP, ThTP and ThAD), riboflavin monophosphate and riboflavin adenine dinucleotide (FMN and FAD) and nicotinamide adenine dinucleotide (NAD + ), respectively. The recent resurgence of basic and biomedical research effort that seeks to probe the mechanisms that these cofactors regulate in cells, tissues and whole organisms [1] has stemmed from new capabilities to manipulate and trace their intracellular abundance; e.g., [2,3]. Methods to boost cofactor levels include the use of specific precursors and the manipulation of the biosynthetic pathways that lead to their conversion to the phosphate-containing cofactors [4,5]. Precursors and cofactors that are modified with stable isotopes are used in metabolomic tracing and flux experiments to inform on the (de)regulation of biosynthetic pathways, and cellular or tissues distribution of these cofactors; e.g., [6,7]. These strategies help better understand why these vitamins and their related cofactors are so central to metabolism and cellular machinery.
Although much progress has been made on defining the biology of the multiple derivatives of B-vitamins that constitute the B-vitaminome, much remains to investigate regarding how cells transport vitamin B1, B2 and B3 derived cofactors and cofactor precursors across membranes. Specific transporters on the cell surface recognize and import the non-charged water-soluble vitamins that are then rapidly phosphorylated in the cytosol or exported back in the extracellular space by equilibrative transporters [8][9][10][11]. While transporters of extracellular phosphorylated cofactor precursors remain under intense scrutiny, specific mitochondrial membrane carriers, SLC25A19, SLC25A32, and SLC25A51, have been shown to import across the mitochondrial membrane from the cytosol ThDP, NAD and FAD, respectively, and support the sub-cellular distribution of each cofactors [12][13][14] in this

Synthesis of Monophosphates
Several chemical and chemoenzymatic methods are available to access the monophosphoester of nucleosides [32] such as adenosine, and of thiamine, riboflavin, and nicotinamide riboside [33], abbreviated AMP, ThP, FMN and NMN, respectively. However, the water content of the starting materials often compromises the efficacy of the chemical conversions, regardless of the method employed. One approach to overcome this limitation and phosphorylate the C5' position of canonical nucleosides without prior protection of the C2', C3' riboside hydroxyls is the Yoshikawa phosphorylation [34,35]. Trimethyl and triethylphosphate are often used as co-solvents along with a molar excess of POCl3 to bring polar nucleosidic reagent in solution, and preferentially activate the C5' hydroxy position to favor a regioselective phosphorylation. Alternatively and to limit polyphosphorylation of the nucleoside, the use of a mixture of excess POCl3, pyridine and water has also been implemented [35]. However, this approach can lead to the partial decomposition of starting materials. Removal of excess phosphotriester solvents is often the plight of synthetic chemists seeking to use the resulting phosphomonoester product in a pyrophosphate bond formation. The inorganic phosphate must also be comprehensively removed, as its presence undermines the subsequent chemistry. Having already established that nicotinamide riboside was particularly amenable to regiospecific phosphorylation at the C5' position, under mechanochemical conditions that minimized nucleosidic bond breakage upon work up [27], we sought to apply these phosphorylation conditions to thiamine, riboflavin, nicotinamide riboside, 3-carboxamide 4-pyridone riboside and adenosine ( Table 1).
The outcomes of the phosphorylation of thiamine (Th), riboflavin (RF), nicotinamide riboside (NR), 3-carboxamide 4-pyridone riboside (PYR) and adenosine (Ade) conducted in solution were compared with the outcomes of phosphorylation conducted under milling conditions (Table 1). Scheme 1 summarizes the chemistry applied to the nucleoside scaffolds described in Table 1. Under mechanochemical ball-milling conditions (BM), POCl3 (4 eq.) was the sole reagent utilized in the preparation of these monophosphates. The progress of the milling reactions was initially monitored by 31 P-

Synthesis of Monophosphates
Several chemical and chemoenzymatic methods are available to access the monophosphoester of nucleosides [32] such as adenosine, and of thiamine, riboflavin, and nicotinamide riboside [33], abbreviated AMP, ThP, FMN and NMN, respectively. However, the water content of the starting materials often compromises the efficacy of the chemical conversions, regardless of the method employed. One approach to overcome this limitation and phosphorylate the C5' position of canonical nucleosides without prior protection of the C2', C3' riboside hydroxyls is the Yoshikawa phosphorylation [34,35]. Trimethyl and triethylphosphate are often used as co-solvents along with a molar excess of POCl 3 to bring polar nucleosidic reagent in solution, and preferentially activate the C5' hydroxy position to favor a regioselective phosphorylation. Alternatively and to limit polyphosphorylation of the nucleoside, the use of a mixture of excess POCl 3 , pyridine and water has also been implemented [35]. However, this approach can lead to the partial decomposition of starting materials. Removal of excess phosphotriester solvents is often the plight of synthetic chemists seeking to use the resulting phosphomonoester product in a pyrophosphate bond formation. The inorganic phosphate must also be comprehensively removed, as its presence undermines the subsequent chemistry. Having already established that nicotinamide riboside was particularly amenable to regiospecific phosphorylation at the C5' position, under mechanochemical conditions that minimized nucleosidic bond breakage upon work up [27], we sought to apply these phosphorylation conditions to thiamine, riboflavin, nicotinamide riboside, 3-carboxamide 4-pyridone riboside and adenosine ( Table 1).
The outcomes of the phosphorylation of thiamine (Th), riboflavin (RF), nicotinamide riboside (NR), 3-carboxamide 4-pyridone riboside (PYR) and adenosine (Ade) conducted in solution were compared with the outcomes of phosphorylation conducted under milling conditions (Table 1). Scheme 1 summarizes the chemistry applied to the nucleoside scaffolds described in Table 1. Under mechanochemical ball-milling conditions (BM), POCl 3 (4 eq.) was the sole reagent utilized in the preparation of these monophosphates. The progress of the milling reactions was initially monitored by 31 P-NMR at 30 min intervals. Milledreactions were stopped after 60 min while solution reactions were pursued for 24 h if deemed incomplete after the first 60 min. Table 1 reveals that mechanochemical conditions (@30 Hz) were well suited for the conversion of PYR, NR, and thiamine (e.g., Scheme 2) to the respective monophosphates in good, isolated yields, but unsuitable for riboflavin, that remained unreactive. 1 H-NMR analyses of the phosphorylation of adenosine under mechanochemical conditions revealed a mixture of unreacted adenosine (64%) and product AMP (36%), with better yields consistently achieved by solution phase chemistry (Table 1). reveals that mechanochemical conditions (@30 Hz) were well suited for the conversion of PYR, NR, and thiamine (e.g., Scheme 2) to the respective monophosphates in good, isolated yields, but unsuitable for riboflavin, that remained unreactive. 1 H-NMR analyses of the phosphorylation of adenosine under mechanochemical conditions revealed a mixture of unreacted adenosine (64%) and product AMP (36%), with better yields consistently achieved by solution phase chemistry (

Synthesis of Pyrophosphates
We originally reported [26] the two-step synthesis of unsymmetrical and symmetrical dinucleotides, including NAD (20) and Ap2A (21). Since then, several vibrational or mechanical ball milling and solution phase procedures that enable the generation of symmetrical and non-symmetrical dinucleotides have been described and reviewed [36][37][38][39]. This chemistry employed morpholidate precursors generated via solution phase chemistry and purified before use in pyrophosphate bond formations enabled by mechanochemistry. While seeking to generate the morpholidate intermediates of AMP, NMN, PYRMP, ThP and FMN, necessary for the P-O-P bond forming process developed for milling conditions, we rapidly identified several issues that promoted the homodimerization of the phosphate monoesters to the symmetrical pyrophosphates upon activation. It became clear that to access unsymmetrical pyrophosphates in good yields, an alternative procedure had to be developed. More recently, Appy et. al. reported the efficient synthesis of symmetrical dinucleotides in good yields in a two step-one pot approach employing milling and in situ activation of nucleotides by CDI in the presence of imidazole [37]. However, these conditions were not suited to the generation of unsymmetrical dinucleotides. Therefore, we sought conditions that generated the imidazolate of each phosphoester quantitatively, so that these intermediates could then be applied in the syntheses of asymmetrical pyrophosphates in a step-wise one-pot process. Ball-milling conditions proved particularly amenable to this process.
In this approach, individual monophosphates (1 eq.) were ball-milled with CDI (4 eq.) for 1 h at 30 Hz in a stainless-steel jar in the presence of acetonitrile (0.3 L/mg of monophosphate). 31 P-NMR spectra of the reaction mixture showed single peaks in the region spanning between −8.5 and −9.7 ppm, indicative of phosphoimidazolate. This conversion was quasi quantitative for all phosphate monoesters, except for FMN that Scheme 2. Synthesis of thiamine monophosphate and its imidazolate.

Synthesis of Pyrophosphates
We originally reported [26] the two-step synthesis of unsymmetrical and symmetrical dinucleotides, including NAD (20) and Ap 2 A (21). Since then, several vibrational or mechanical ball milling and solution phase procedures that enable the generation of symmetrical and non-symmetrical dinucleotides have been described and reviewed [36][37][38][39]. This chemistry employed morpholidate precursors generated via solution phase chemistry and purified before use in pyrophosphate bond formations enabled by mechanochemistry. While seeking to generate the morpholidate intermediates of AMP, NMN, PYRMP, ThP and FMN, necessary for the P-O-P bond forming process developed for milling conditions, we rapidly identified several issues that promoted the homodimerization of the phosphate monoesters to the symmetrical pyrophosphates upon activation. It became clear that to access unsymmetrical pyrophosphates in good yields, an alternative procedure had to be developed. More recently, Appy et. al. reported the efficient synthesis of symmetrical dinucleotides in good yields in a two step-one pot approach employing milling and in situ activation of nucleotides by CDI in the presence of imidazole [37]. However, these conditions were not suited to the generation of unsymmetrical dinucleotides. Therefore, we sought conditions that generated the imidazolate of each phosphoester quantitatively, so that these intermediates could then be applied in the syntheses of asymmetrical pyrophosphates in a step-wise one-pot process. Ball-milling conditions proved particularly amenable to this process.
In this approach, individual monophosphates (1 eq.) were ball-milled with CDI (4 eq.) for 1 h at 30 Hz in a stainless-steel jar in the presence of acetonitrile (0.3 µL/mg of monophosphate). 31 P-NMR spectra of the reaction mixture showed single peaks in the region spanning between −8.5 and −9.7 ppm, indicative of phosphoimidazolate. This conversion was quasi quantitative for all phosphate monoesters, except for FMN that remained unreactive ( Table 2). The individual imidazolates still contained in the milling jar were then used in the pyrophosphate bond formation step (Scheme 1, step 3). Two methods were then explored for the pyrophosphate bond formation, Method A [25] and Method B [37]. According to Method A, the second monophosphate (1 eq.) unit was immediately added to the jar containing the imidazolate intermediate, along with MgCl 2 .6H 2 O (1.5 eq.), tetrazole (2 eq.), and H 2 O (6 eq.). The jar was then vibrated for 90 min, while according to Method B, the imidazolate containing jar was charged with the second monophosphate in the free acid form with ACN (0.6-0.95 µL/monophosphate) and vibrated for 2 h. The reaction progression was monitored by 31 P-NMR. Overall, the crude yields obtained for the dinucleotides synthesized using Method B were relatively low, and in most cases, unreacted imidazolate starting materials were recovered. When using Method A, the same dinucleotides were obtained with good yields. The imidazolate intermediates were either converted to the symmetrical or asymmetrical dinucleotides. The crude pyrophosphate reaction mixtures generated using this process were lyophilized, diluted in the least amount of water possible, adsorbed on silica gel, and purified using reversed-phase chromatography (Teledyne) with a C18 column and acetonitrile: pH 7 buffer, 100 mM ammonium acetate (2:98 to 10:90, v:v). Isolated yields ranged between 12% and 63%. Overall, combining the in-situ generation of imidazolate by milling with a stepwise activation of the imidazolate intermediate for conjugation with a free monophosphate provided a versatile approach to unsymmetrical pyrophosphates (Table 2). Crucially, the reactivity of the individual imidazolate towards other monophosphate partners and the outcomes of the pyrophosphate forming reaction could be manipulated by choosing to activate one phosphate over the other. Thus, we generated nicotinamide adenine dinucleotide (NAD) (20), 3-carboxamide-4-pyridone adenine dinucleotide (4-ox-NAD) (23), adenosine and nicotinamide thiamine diphosphate (AThDP and NRThDP (30a & 31), respectively, Scheme 3), and nicotinamide riboside riboflavin diphosphate (NMN-FMN) (33) (Scheme 4). Finally, by using TDP (36) instead of AMP (11), we also accessed adenine thiamine triphosphate (AThTP) (37), in good, isolated yields (Scheme 5). Isolated symmetrical byproducts included diadenosine pyrophosphate (AP 2 A) (21), dinicotinamide riboside pyrophosphate (NMN-NMN) (22), dithiamine pyrophosphate (ThP 2 Th) (30b), and di-3-carboxamide-4-pyridone riboside pyrophosphate (di-4-PYR-MP) (24). However, even under the conditions that proved to be most successful for the formation of pyrophosphates, FMN (32) did not yield riboflavin containing dinucleotides, but instead afforded the cyclicriboflavin monophosphate, cFMN (34) [40,41], as the major product. The versatility of this approach proved to be highly valuable when seeking to generate an isotopically labeled NAD in good yields.
Method A Scheme 5. Synthesis of thiamine adenosine triphosphate (37). (25) Recent efforts towards understanding how the vitamin B3 derived cofactors are transported across membranes based on mass spectrometry enabled isotopic tracing metabolomics has required the synthesis of stable isotopically labeled NAD (25) [2] . Such species informed on whether NAD was transported intact or was first metabolized to a non-phosphorylated species prior to transport from one organelle to another [2,42]. In the latter case, a NAD isotopologue possessing heavy atoms on either side of The versatility of this approach proved to be highly valuable when seeking to generate an isotopically labeled NAD in good yields.

Synthesis of Labeled NAD (25)
Recent efforts towards understanding how the vitamin B3 derived cofactors are transported across membranes based on mass spectrometry enabled isotopic tracing metabolomics has required the synthesis of stable isotopically labeled NAD (25) [2]. Such species informed on whether NAD was transported intact or was first metabolized to a non-phosphorylated species prior to transport from one organelle to another [2,42]. In the latter case, a NAD isotopologue possessing heavy atoms on either side of the pyrophosphate bonds, for example on each riboside unit, would lead to NAD species possessing lower molecular weight as scrambling would occur, while an NAD transporter would enable the detection of the synthesized NAD isotopologue. While the approach demonstrated that a mitochondrial transporter was indeed responsible for the NAD import in the mitochondrion from the cytoplasm, the synthetic route to the isotopologue was low yielding. Therefore, we sought an approach that could be easily applied to other NAD isotopologues while also useful for a wider range of nucleotides. Crucially, the synthetic commitment to the introduction of isotopes across the building blocks requires the final pyrophosphate formation step to be high yielding, and the synthetic sequence enabled by mechanochemistry offered such prospect.
Thus, we generated the MW+2 adenosine (48) for which deuterium isotopes have been introduced on the C5 position of D-ribose, and the MW+2 nicotinamide riboside isotopologue (54) with 18 O modified nicotinamide. The C5-deuterated adenosine monophosphate was produced from D-ribose (38). Following a 2,3-O-isopropyledene protection (39), followed by oxidation of the C5-primary alcohol to the carboxylic acid and methyl esterification, the ester (41) (Scheme 6) was isolated in 51% yield following chromatographic purification. Two distinct approaches were explored to introduce deuterium at the C5 position. Reduction of the methyl ester with LiAlD 4 in diethyl ether occurred in 35% yield, whereas reduction with NaBD 4 in D 2 O (99.9%) yielded the MW+2 riboside (42) in 64% isolated yield. Following the aq. TFA. deprotection of the isopropyledene and removal of excess reagents, the crude oily 2 H 2 C5-ribose (43) was treated with methanol in the presence of conc. H 2 SO 4 . The resulting 2 H 2 C5-C1-O-methyl ribofuranoside (44) was converted to the deuterated acetylated ribofuranoside (46) with acetic anhydride in glacial acetic acid in the presence of a catalytic amount of concentrated sulfuric acid initially at 0 • C and then room temperature. Longer reaction time increased the formation of pyranoside byproducts. The anomeric mixture of C5 deuterated riboside tetraacetate (46) was obtained in 69% isolated yield and did not require any chromatographic purification. Glycosylation of riboside (46) with adenine under Vorbrüggen conditions produced the C5-deuterated triacetate adenosine (47) in 60% isolated yields. The nucleoside was then treated with a 2:1 combination of MeOH and aq. NH 4 OH for 24 h at 8 • C. After complete deacetylation, the mixture was placed at −20 • C for a week to precipitate the beta isomer of the C5-deuterated adenosine (48) in a 25% yield as a white powder. The filtrate was concentrated and stored for additional purification. The phosphorylation of the beta-anomer of C5-deuterated adenosine (48) was conducted under solution phase conditions using POCl 3 in (MeO) 3 PO at −20 • C for 24 h. Upon reaction completion and addition of water, the pH of the solution was raised to 7 by adding triethylamine. The C5-deuterated adenosine monophosphate triethylammonium salt (49) (80% yield) was then isolated upon removal of the water by freeze-drying. The MW+2 18 O labeled NR (54) was synthesized according to described procedures [3] and 18 O-NMN (15), prepared by phosphorylation of 18 O-NR, was activated with CDI under milling conditions. C5-deuterated adenosine monophosphatetriethyl amine salt (49) was added to the resulting NMN-derived imidazolate (19) and milled to yield a mixture of MW+4 labeled NAD (25) and MW+4 labeled double-NMN (26) (Scheme 7). According to the 31 P NMR spectra of the crude composition, mixture of NAD (25) and double NMN (26) represented 29%, with 71% of remaining labeled AMP and NMN that could be recycled. As such, the crude material was diluted in a small amount of water, adsorbed in silica, and purified by reverse phase chromatography to yield pure M+4 NAD (25) and pure M+4 double NMN (26) in 8% and 11% isolated yields, respectively, as quantified by UV spectrometry, while the mixture of unreacted labeled AMP (14) and NMN (15) was recovered. The MW+2 18 O labeled NR (54) was synthesized according to described procedures [3] and 18 O-NMN (15), prepared by phosphorylation of 18 O-NR, was activated with CDI under milling conditions. C5-deuterated adenosine monophosphatetriethyl amine salt (49) was added to the resulting NMN-derived imidazolate (19) and milled to yield a mixture of MW+4 labeled NAD (25) and MW+4 labeled double-NMN (26) (Scheme 7). According to the 31 P NMR spectra of the crude composition, mixture of NAD (25) and double NMN (26) represented 29%, with 71% of remaining labeled AMP and NMN that could be recycled. As such, the crude material was diluted in a small amount of water, adsorbed in silica, and purified by reverse phase chromatography to yield pure M+4 NAD (25) and pure M+4 double NMN (26) in 8% and 11% isolated yields, respectively, as quantified by UV spectrometry, while the mixture of unreacted labeled AMP (14) and NMN (15)  We presented in detail how a two-step one pot approach could be applied to the generation of unsymmetrical canonical and non-canonical dinucleotides, obtained in isolated, UV-quantified yields that compare well with the yields of the current protocols of simpler dinucleotides. With milling, we gathered a greater understanding of the phosphorylation step of the primary alcohol of each synthetic components and altered our approach to the pyrophosphate bond formation to minimize homodimerization. Crucially, we observed that thiamine, a vitamin particularly notorious for its poor solubility in most solvents, was particularly amenable to mechanochemical conditions, both for phosphorylation and for further conversions. Alternatively, we observed that the mechanochemical conditions we explored were not well suited for the synthesis of FMN (32) and FAD (35) from FMN (32), as riboflavin proved particularly unreactive towards POCl3, and divalent cations promoted cyclic-FMN (34) [41] formation in very good yields, instead. Finally, while the challenges of achieving good isolated yields following chromatography purifications still remain, we observed that conditions where nucleosides and nucleotides could be exhaustively dried offered better outcomes. We had made similar observations when handling P(III) reagents with nucleosides [43,44]. Yet, when we applied this synthetic approach to isotopically labeled NAD nucleoside precursors, we were able to generate an NAD isotopologue in good isolated yields; in quantities that would be well suited to inform on exogenous NAD turn-over, including NAD's use by sirtuins, PARP enzymes, glycohydrolases, as well as reductases, in cell based-assays by mass spectrometry. (11-15 & 28)

By Mechanochemical Ball-Milling
A mixture of nucleoside (1 eq.) and POCl3 (4 eq.) was introduced into a stainlesssteel jar (1.5 mL) along with one stainless steel ball (20 mm in diameter). The reaction vessel, along with another identical empty vessel, was closed and fixed on the vibration arms of a Retsch MM400 miller and vibrated at 30 Hz at room temperature for one We presented in detail how a two-step one pot approach could be applied to the generation of unsymmetrical canonical and non-canonical dinucleotides, obtained in isolated, UV-quantified yields that compare well with the yields of the current protocols of simpler dinucleotides. With milling, we gathered a greater understanding of the phosphorylation step of the primary alcohol of each synthetic components and altered our approach to the pyrophosphate bond formation to minimize homodimerization. Crucially, we observed that thiamine, a vitamin particularly notorious for its poor solubility in most solvents, was particularly amenable to mechanochemical conditions, both for phosphorylation and for further conversions. Alternatively, we observed that the mechanochemical conditions we explored were not well suited for the synthesis of FMN (32) and FAD (35) from FMN (32), as riboflavin proved particularly unreactive towards POCl 3 , and divalent cations promoted cyclic-FMN (34) [41] formation in very good yields, instead. Finally, while the challenges of achieving good isolated yields following chromatography purifications still remain, we observed that conditions where nucleosides and nucleotides could be exhaustively dried offered better outcomes. We had made similar observations when handling P(III) reagents with nucleosides [43,44]. Yet, when we applied this synthetic approach to isotopically labeled NAD nucleoside precursors, we were able to generate an NAD isotopologue in good isolated yields; in quantities that would be well suited to inform on exogenous NAD turn-over, including NAD's use by sirtuins, PARP enzymes, glycohydrolases, as well as reductases, in cell based-assays by mass spectrometry.

By Mechanochemical Ball-Milling
A mixture of nucleoside (1 eq.) and POCl 3 (4 eq.) was introduced into a stainless-steel jar (1.5 mL) along with one stainless steel ball (20 mm in diameter). The reaction vessel, along with another identical empty vessel, was closed and fixed on the vibration arms of a Retsch MM400 miller and vibrated at 30 Hz at room temperature for one hour. After completion of the reaction, the jar was allowed to cool down to room temperature and the contents of the jar were dissolved in a minimum amount of water and stirred at room temperature for 3-4 h for the complete hydrolysis of dichloromonophosphate to dihydroxy monophosphate. The percentage conversion was monitored by 1 H-NMR (Table 1).

By Solution-Phase Synthesis
A solution of a specific nucleoside (1 eq.) in trimethyl phosphate (5 mL) was mixed with POCl 3 (3 eq.) at −5 • C. The mixture was stirred at the same temp for 1 hour and then was kept in the −20 refrigerator for the next 48 h. The reaction progress was monitored by 1 H and 31 P-NMR analysis. Upon completion of the reaction, the crude material was treated with MeCN-Et 2 O (1:3), and the white precipitate was separated from the liquor. Once isolated, the solid was stirred with a minimum amount of water overnight for complete hydrolysis of the C5-dichloromonophosphate to the C5 dihydroxymonophosphate [1]. This crude was further purified through the Dowex 50WX8 resin (H+ form) column by using water and formic acid (100%:0% to 90%:10%) as an eluent to give the monophosphate in the isolated yield presented in Table 1. The C5 deuterated adenosine (14) was converted into its TEA salt (49) and used for the next step without further purification. Among the series, the formation of AMP (11), C5 deuterated adenosine (14), O 16

Synthesis of FMN (32)
In a round-bottom flask, POCl 3 (16 mL) was added to 15 mL of anhydrous methanol at 0 • C in a dropwise manner, according to the reported literature [36]. The resulting mixture was left at room temperature for 16 h with slow stirring. The round bottom flask was covered with a septum containing an empty balloon to control the internal pressure of the HCl gas which was evolved during the reaction. After 16 h, riboflavin (3.19 g, 8.50 mmol) was added, and the resulting solution was stirred at RT for a further 16 h. A dark brown, thick, oily syrup was formed, which was diluted with 150 mL of water and then heated at 85-90 • C for 30 minutes. Upon cooling to room temperature with stirring, orange colored FMN (32) precipitated (yield 67%) [45]. 31

General Synthetic Procedure for the Preparation of Monophospho-Imidazolates (16-19 & 29) by Mechanochemical Ball-Milling
Activation of monophosphates (11-13, 15 & 28) by using 1,1'-carbonyldiimidazole (CDI): A mixture of monophosphate (1 eq.), 1,1'-carbonyldiimidazole (CDI) (4 eq.) and anhydrous acetonitrile (0.3 µL/mg of monophosphate) together with 1 stainless steel ball (20 mm in diameter) was introduced into a stainless-steel jar (1.5 mL). The reaction vessel, along with another identical empty vessel, was closed and fixed on the vibration arms of a Retsch MM400 miller and vibrated at 30 Hz at room temperature for one hour. The reaction progress was monitored by 31 P-NMR analysis ( Table 2). After completion of the reaction, the resulting mixture was used for the next step without purification. Among the series of monophosphates (11-13, 15 & 28), only compound 32 was unreactive under these conditions [36]. As an example, the sequence implemented for thiamine is presented Scheme 2.
Adenosine monophosphate imidazolate (AMP-IM) (16): 31  The metallic jar was charged with monophosphate imidazolate (0.144 mmol, 1 eq.). The coupling monophosphate (0.144 mmol, 1 eq.) was added, along with MgCl 2 .(H 2 O) 6 (0.216 mmol, 43.91 mg, 1.5 eq.), tetrazole (0.288 mmol, 20.17 mg, 2 eq.), H 2 O (0.864 mmol, 15.6 µL, 6 eq.) and a 20 mm stainless steel ball. The ball mill was set to vibrate at a frequency of 30 Hz for 90 minutes [3], after which time the jar was left to cool down to room temperature. Once the jar was opened, the product was dissolved in water, adsorbed on silica gel, and purified. Automated flash column chromatography was performed using a Teledyne ISCO CombiFlash Companion system with a RediSepRf reverse-phase C18 column (cv = 133 mL), packed with 130 g silicagel (average particle size: 40-63 microns, 230-400 mesh, 60 A o pore size ) by using 100 mM ammonium acetate: acetonitrile (98%:2% to 85%:15%) as an eluent with a flow rate of 5 mL/min [27,36].   SO 4 was then added in a dropwise manner with stirring. Upon completion of the addition, the ice bath was removed, and the resulting mixture was stirred at room temperature for 3 h. It is important to note that longer stirring time facilitates the formation of the pyranose form, yielding a mixture of pyranose and furanose forms of C-5 deuterated ribose tetraacetate. After 3 h, a 1 H-NMR analysis of the crude showed that the desired furanoside had formed preferentially. Crushed ice was then added, and the quenching reaction mixture was stirred for 1 hour to allow for the complete hydrolysis of excess Ac 2 O to acetic acid. After that time, 15 mL of CHCl 3 was added to the mixture, which was stirred for 30 minutes (repeated 3 times). Upon extraction with ethyl acetate, the organic layers were combined and stirred with 50 mL of a saturated NaHCO 3 solution for 1 hour and then separated. The organic layer was dried over anhydrous Na 2 SO 4 , concentrated under a reduced vacuum to afford the desired product, 46 as an α/β mixture. The product was used in the next step without purification. Yield 69%. 1  Synthesis of C-5 deuterated adenosine (48): Step-1: A 25 cm 3 PTFE ball milling vessel was charged with crude C-5 deuterated ribose tetraacetate sugar 46 (0.30 g, 0.94 mmol), TMSOTf (0.42 g, 1.87 mmol), adenine (0.13 g, 0.94 mmol) and a PTFE ball bearing. The vessel was shaken with a Retsch MM 400 mixer mill for 35 min at 30 Hz. The crude material was dissolved in a mixture of MeOH: DCM (1:1), adsorbed on silica gel, and purified by flash column chromatography using n-hexane: DCM (1:4 to 0:1). After purification, an α/β mixture of the triacetylated nucleoside intermediate (47) was isolated.
Step 2: The intermediate isolated in step 1 (0.37 g, 0.94 mmol) was dissolved in a 9 mL mixture of MeOH and NH 4 OH (2:1) and stored in the refrigerator at 8 • C for 24 h. After that time, a 1 H-NMR of the crude was recorded in D 2 O to ensure that the complete removal of all three acetates had occurred. Upon completion, the solvent was removed under reduced pressure and the crude was treated with 2 mL of MeOH and kept at −20 • C for a week to precipitate the β-isomer as a white powder (48). Yield 25% (β-isomer) The supernatant was concentrated under reduced pressure and stored for future use. 1  Synthesis of C-5 deuterated adenosine monophosphate triethylamine salt (49): Using a heat gun, C-5 bis-deuterated adenosine (48) (0.07 g, 0.26 mmol) was dissolved in 1 mL of (MeO) 3 PO. The resulting solution was cooled and stirred at −20 • C for 20 min. POCl 3 (72 µL) was added. The resulting mixture was stirred at −20 • C for a few min more before being stored at −20 • C in a refrigerator for 24 h, after which time 50 µL of the crude was dissolved in 450 µL of D 2 O and stirred at RT for 1 hour before being analyzed by 1 Hand 31 P-NMR to ensure complete phosphorylation of adenosine 48. Upon confirmation, 1.5 mL of water was added to the reaction mixture. The resulting solution was stirred for 2 h at RT. The pH of the solution was subsequently adjusted to 7 by adding Et 3 N. The solution was freeze-dried to afford the triethylammonium salt of C-5 bis-deuterated adenosine monophosphate (49) as a white solid. 1 H-and 31 P-NMR showed some amount of unreacted M+2 adenosine (8%) with C-5 bis-deuterated adenosine monophosphate (49). The crude material was used directly for the next step without further purification. Yield 80% (49, β-isomer);

Conclusions
A novel two-step one pot approach to the generation of unsymmetrical canonical and non-canonical dinucleotides in isolated, UV-quantified yields that are in par and even exceed that of the current protocols of simpler dinucleotides has been demonstrated. It offers means to generate valuable, new chemical tools, at scale that enable more bold biological investigations for which milligrams rather than microgram of labeled materials of dinucleotides would be required to pursue functional investigations. Studies related to bio-distributions in animal models, transporters of extracellular and intracellular (di)nucleotides, and identifications of novel biosynthetic pathways that rely on traceable chemical tools could greatly benefit from this versatile chemistry. Crucially, the reaction time reduction and the ease of chemical handling that this approach offers, provide scope for implementation in settings seeking to perform radio-emitting isotopic labelling.