Jamie M.
McCabe Dunn
*a,
Mikhail
Reibarkh
*a,
Edward C.
Sherer
b,
Robert K.
Orr
a,
Rebecca T.
Ruck
a,
Bryon
Simmons
a and
Ana
Bellomo
a
aDepartment of Process Research & Development, MRL, Merck & Co., Inc., Rahway, NJ 07065, USA. E-mail: Jamie.mccabe.dunn@merck.com; mikhail_reibarkh@merck.com
bDepartment of Modelling and Informatics, MRL, Merck & Co., Inc., Rahway, NJ 07065, USA
First published on 18th January 2017
The direct and chemoselective 3′-phosphoramidation, phosphorylation and acylation of nucleosides are described. Upon the discovery of a novel 3′-phosphorylamidation of therapeutic nucleoside analogues with DBU, we explored the mechanism of this rare selectivity through a combination of NMR spectroscopy and computational studies. The NMR and computational findings allowed us to develop a predictive computational model that accurately assesses the potential for 3′-functionalization for a broad range of nucleosides and nucleoside mimetics. The synthetic utility of this model was exemplified by demonstration on a broad scope of nucleosides and electrophiles yielding targets that were previously only accessible via a protection/deprotection sequence or an enzymatic approach.
Notably, enzymes are known to provide exquisite selectivity in functionalization reactions of biological molecules through a series of site-specific interactions between the protein and the substrate. For example, lipase from Pseudomonas cepacia (PCL) facilitates selective 3′-acylation of nucleosides. Hydrogen bonding interactions between the PCL and 5′-hydroxyl group of the nucleoside are believed to be responsible for inhibiting reactivity of the 5′-hydroxyl, while favouring reactivity at the 3′-hydroxyl (Fig. 1b).6,7 In this work, we report the non-enzymatic highly chemoselective 3′-functionalization of nucleosides. A combination of NMR spectroscopy and computational studies enabled development of a detailed mechanistic understanding of the selectivity. As a result we developed a predictive computational model that accurately assesses the potential for 3′-selectivity for a broad range of nucleosides and nucleoside mimetics.
Having identified DBU as the base that provided the ideal combination of excellent 3′-chemoselectivity and high phosphorus diastereoselectivity, we sought to find optimal reaction conditions to selectively phosphorylate nucleoside PSI-6206 (1a). We identified two key parameters for this reaction: solvent polarity and temperature. A moderately non-polar solvent, tetrahydrofuran (THF), was identified as the optimal solvent for chemo- and diastereoselectivity while polar aprotic solvents, such as NMP, led to diminution of the 3′-chemoselectivity. When salt additives such as MgBr2·Et2O or MgCl2 were used, minimal 3′-phosphorylation was observed while 5′-phosphorylation was the major product in a complex reaction mixture. Temperature also played a critical role in controlling the diastereoselectivity: as the temperature was increased, a corresponding decrease in diastereoselectivity was observed. Under optimized conditions, treatment of a mixture of PSI-6206 (1a) and phosphoramide 4 in THF at 0 °C with 1.0–1.05 equivalents of DBU provided the desired 3′-phosphorylated product 2a in an impressive 92% isolated yield and 95:
5 dr (Table 2, entry 1).10
While excellent experimental results were achieved, we sought to understand the molecular mechanism of this unprecedented non-enzymatic 3′-chemoselectivity in order to better apply this discovery to other nucleosides. An extensive NMR study of the DBU–nucleoside 1a binary system was initiated in order to determine what, if any, effect DBU had on the nucleoside. NMR titration experiments in which the 1H, 13C and 19F NMR chemical shifts of 1a were monitored in the independent presence of increasing amounts of DBU, or DIPEA as negative control, provided clues as to the role of DBU. As expected from the base screening results, systematic titration of up to 5 equivalents of DIPEA into a solution of 1a had no effect on its 1H and 13C NMR chemical shifts (Fig. 2A and C). In contrast, titrating the same molar amounts of DBU resulted in significant changes in the 1H and 13C NMR spectra of 1a (Fig. 2B and C).12
The 1H nuclei most sensitive to the DBU titration were both uracil protons, with Δδ of 0.40 ppm13 each, while the 1′ and 4′ protons showed a significant, albeit smaller, effect with Δδ of 0.12 ppm. The effect of DBU on 13C chemical shifts of 1a was far more pronounced: C-4′ exhibited a Δδ of 0.6 ppm, C-5 of uracil had Δδ of 2.4 ppm, and carbonyls C-2 and C-4 of uracil had Δδ of −5.7 ppm and −8.7 ppm, respectively. Since 13C chemical shifts are typically insensitive to the macro-environment, such strong changes suggested a specific interaction between DBU and the nucleoside 1a. To further probe this hypothesis, we conducted 1D NOE and 2D NOESY experiments on the DBU/1a mixture. Selective irradiation of the NH resonance yielded strong NOEs to the 6- and 9-methylene groups of DBU (Fig. 3A), providing direct evidence that the uracil NH of 1a is likely to be fully deprotonated by DBU. Furthermore, the 2D NOESY data (Fig. 3B) revealed unexpected intermolecular NOEs between the 2′-methyl of 1a and the 6- and 9-methylenes of DBU.
Taken collectively, the NOE data not only demonstrated proton transfer from the NH uracil, but revealed a stable acid–base complex formed between the nucleoside 1a and DBU.14 The observed effects of DBU and DIPEA on 1a in solution correlated well with the previously observed reactivities and suggest that deprotonation of the NH uracil is essential for reactivity since weak bases like DIPEA, which are unable to deprotonate the uracil, failed to promote reaction conversion (Table 1).
Once DBU binding and formation of an acid–base complex with 1a had been verified independently by NOE and diffusion data, further analysis of the DBU-induced 13C chemical shift changes of 1a was performed. While the largest changes (C-2, C-4 and C-6 of the uracil) were attributed to the deprotonation of the uracil NH, some additional effects were observed. In particular, significant Δδ of the 4′ carbon, which is not relevant to the uracil deprotonation, suggested that formation of a complex with DBU induces a conformational change of 1a. Additionally, a very large difference of 3 ppm between Δδ values of uracil carbonyls C-2 and C-4 was observed,15 suggesting that one of the carbonyls (likely O-2) was involved in an H-bonding interaction.
The discovery of the acid–base complex between DBU and 1a, as well as observation of a putative H-bonding interaction caused by DBU complexation, suggested that our observed nucleoside 3′-selectivity could arise via a similar pathway to the Pseudomonas cepacia lipase. To probe this hypothesis, we initiated computational studies aimed at augmenting the findings in the NMR studies. We developed a computational model to evaluate the solution state conformational distribution of nucleoside PSI-6206 (1a) using density functional theory (M06-2X/6-31+G** in vacuo or implicit THF). As expected, evaluating just the neutral form of nucleoside 1a revealed no conformational preference that would drive selectivity to afford the desired 3′-phosphorylated product, since low energy conformations identified the nucleoside base existed in both syn and anti forms (Fig. 4a).16 To the contrary, analysis of the conformational space of the deprotonated uridine suggested a dramatic change in conformational preference under these conditions, altering the distribution between the two main syn and anti conformations from a ∼20/80 to 100/0 ratio in vacuo.
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Fig. 4 (a) Relative ratios for syn and anti conformations. (b) Two views of the lowest energy DBU complex. |
In the dominant conformation of the anion, an intramolecular hydrogen bond is formed between the 5′-hydroxyl group and the O-2 of the uracil base. This result is consistent with the NMR spectroscopic observations and leads to a folded structure for the nucleoside (Fig. 4a). Furthermore, the lowest energy conformation of the DBU acid–base complex with nucleoside 1a places DBU on top of the nucleoside (Fig. 4b), consistent with experimentally observed intermolecular NOEs.
The agreement between the computational and NMR studies suggested the existence of three possible factors contributing to the experimentally observed selectivity: (1) conformational preferences (hydrogen bond) that cause the nucleoside to fold in such a way to effectively block the approach to the 5′-hydroxyl; (2) complexation of the DBU with the nucleoside that essentially blocks the approach to the 5′-hydroxyl; (3) or a combination of both conformation and complexation. Experimental data obtained during optimization supported that the hydrogen bond between the O-2 and 5′-hydroxyl is a contributing factor for the observed selectivity, since polar additives such as NMP or MgBr2 degraded 3′-selectivity.17 In an attempt to determine if the complexation of DBU played a role in the selectivity, we synthesized N-methyl-uridine 5.18 Although it cannot be deprotonated by DBU in the same fashion as 1a, a computational investigation of the conformational preference of N-methyl-uridine 5 revealed that the favoured conformation of the neutral state in implicit solvent maintained the intramolecular hydrogen bond to the O-5′. Exposure of nucleoside 5 and phosphoramide 4 to our optimized reaction conditions provided exclusively the 3′-phosphorylated material in 93% yield and excellent 3′-selectivity (98:
2) (Fig. 5). These results established the conformational preference as the sole driving force for the observed selectivity. Given this conclusion, we reasoned that 3′-selective functionalization of any nucleoside could be achieved if the conformational distributions energetically favoured the H-bonded conformation.
Computational analysis of a variety of custom nucleosides was employed to evaluate their Boltzmann conformational distributions and predict their corresponding selectivities (Table 2). For all 2′-doubly modified uridine nucleosides, the deprotonated distributions were 100% syn with intramolecular hydrogen bonds to the O-5′ hydroxyl and O-2 on the uridine.19 As predicted, changing from F to Cl still afforded the product with the desired 3′-selectivity in 89% yield (Table 2, entry 1 & 2). Furthermore, 2′-substitution with –CCH, –CN or –N3 also provided good yields of the 3′-phosphorylated products (Table 2, entries 3–5).
Entry | Nucleoside | Electrophile | Product | Yielde | 3′![]() ![]() |
---|---|---|---|---|---|
a Anions are expected to be formed and serve as the structures modelled in the conformational analysis.
b 2 equivalents of DBU used and temperature lowered to −15 °C.
c Temperature lowered to −15 °C.
d Based on recovered starting material (brsm).
e Isolated yields of pure 3′-phosphorated product and major p-epimer.
f Assay yield.
g 3′![]() ![]() |
|||||
1a |
![]() |
![]() |
![]() |
92 | 98![]() ![]() |
2a | (1b) R = Cl | 4 | (2b) R = Cl | 89 | 98![]() ![]() |
3a | (1c) R = –CCH | 4 | (2c) R = –CCH | 74 | 92![]() ![]() |
4a | (1d) R = N3 | 4 | (2d) R = N3 | 62 | 91![]() ![]() |
5a | (1e) R = CN | 4 | (2e) R = CN | 58 | 97![]() ![]() |
6 |
![]() |
4 |
![]() |
84 | ND |
7 |
![]() |
4 |
![]() |
85f | ND |
8a,b |
![]() |
4 |
![]() |
57 | 93![]() ![]() |
9 | 5 |
![]() |
![]() |
71 | 96![]() ![]() |
10c | 5 |
![]() |
![]() |
59 (68 brsm)d | 99![]() ![]() |
Analysis of non-uridine nucleosides revealed nucleoside-dependent H-bonding interactions that could direct 3′- or 5′-selectivity in that guanosine 7, cytidine 9 and inosine 11 all displayed a similar conformational preference to uridine 5: the 5′-hydroxyl group forms a hydrogen bond with the respective base.20 Furthermore, information gathered from the computational studies completely correlated with the experimental results. As predicted, the hydrogen bonding interaction between the N-4 of the guanosine and the 5′-hydroxyl group of guanosine 7 directs selective 3′-phosphorylation to give an 84% yield of the desired ProTide 8 (Table 2, entry 6). Likewise, the hydrogen bond conformation observed with the pyrimidin-1-one of cytidine 9 and N-4 purine of inosine 11 directed selective 3′-phosphorylation to give cytidine 10 and inosine 12 in 85% and 57% yields, respectively (Table 2, entries 7 and 8). It is remarkable that nucleosides that possess such diversity at the base are able to exhibit such exquisite selectivity for 3′-functionalization, providing a much broader substrate scope that would typically be expected through enzyme mediated reactions.
Conversely, analysis of α-thymidine (13) identified a 3′-hydroxyl group hydrogen bond with the thymidine base, leading to a prediction of a preference for 5′-phosphorylation. Consistent with our prediction, exposure of α-thymidine (13) to the same reaction conditions afforded the 5′-phosphorylation product 14 selectively in 53% yield (eqn (1)). Lastly, any nucleoside that possessed a 2′-hydroxyl group, such as cytidine (15) featured a preferred H-bonding interaction between the 2′-hydroxyl and the cytidine base. We envisioned that these 2′-hydroxyl nucleosides were unlikely to afford the desired 3′-selectivity in the phosphorylation; as expected, attempts to phosphorylate cytidine (15) yielded a complex mixture of phosphorylated products (eqn (2)).
![]() | (1) |
![]() | (2) |
Having established confidence in the predictability of the 3′-selectivity of the phosphorylation, we hypothesized that this selectivity should be independent of the partner electrophile and serve as a general strategy for selective 3′-functionalization of nucleosides. Changing from the phosphoramide 4 to phosphonate 16 had no effect on the selectivity: treatment of nucleoside 5 with phosphate 16 and DBU afforded a 71% yield of the desired 3′-phosphorylated product 17 (Table 2, entry 9). An even more exciting result was achieved by moving away from phosphonates completely. Reacting nucleoside 5 with isobutyric anhydride (18) in the presence of DBU afforded 59% yield (68% brsm21) of 3′-isobutyl ester 19 (Table 2, entry 10). This result demonstrates that selectivity of the nucleoside 3′-functionalization is driven exclusively by the ability of the 5′-hydroxyl to form an intramolecular H-bond with the nucleic base and is independent of the partner electrophile, and provides a novel, simple and general approach to the acylation of nucleosides that is complementary to the enzymatic approach.22
Footnotes |
† Electronic supplementary information (ESI) available. CCDC 1525736–1525737. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6sc05081f |
‡ Ross and co-workers observed trace 3′-phosphorylation and phosphorous epimerization upon treatment of nucleoside PSI-6206 (1a) with DBU and DMAP. |
This journal is © The Royal Society of Chemistry 2017 |