Organocatalytic Phosphorylation of Alcohols Using Pyridine-N-oxide

Abstract

Phosphorylation of alcohols by phosphoryl chlorides catalysed by pyridine-N-oxide is reported. The utility of this method is demonstrated through phosphorylation of primary, secondary and a tertiary alcohol as well as phenols under mild reaction conditions and with low catalyst loading (5 mol%).

Phosphorylation of alcohol groups in e.g. peptides, proteins, inositols, glycerols and steroids plays a pivotal role in many physiologically important processes.[1] Disease states including cancers and immune system disorders are often characterised by perturbation of these processes.[2] [3] Phosphate derivatives are also commonly prepared in order to increase the aqueous solubility of alcohols e.g. in pro-drugs.[4–6] The development of synthetic methods for the efficient, cost-effective and scalable phosphorylation of alcohol derivatives is therefore an important goal.

Alcohol phosphorylation (i.e. phosphate monoester formation) by direct acid-catalysed condensation with phosphoric acid is generally difficult to control for structurally diverse alcohols.[7] Consequently, phosphorylation is more generally achieved either by phosphitylation then oxidation/deprotection [i.e., using a P(III) reagent such as a phosphoramidite] or by direct phosphorylation then deprotection [i.e., using a P(V) reagent such as a dialkoxychlorophosphate].[8] Phosphitylation reactions tend to be faster than phosphorylation reactions but require the substrate to display stability to the subsequent oxidation. Direct phosphorylation can be achieved with Brønsted/Lewis acid or nucleophilic catalysis. For example, Jones et al. have used Ti(Ot-Bu)₄, TiCl₄ or Cu(OTf)₂ with phosphoryl chlorides[9] [10] and N-phosphoryl oxazolidinones,[11–13] and more recently, Sculimbrene et al. have reported the use of Ti(Ot-Bu)₄ with pyrophosphates.[14,15] Pyridine,[16] 4-DMAP[17] and DABCO[18] have been used as super-stoichiometric nucleophilic promotors of phosphorylation and Miller et al. have deployed N-methylimidazole (NMI)[19] and 4-DMAP[20] substoichiometrically with phosphoryl chlorides (Scheme [1]).

Whilst the Lewis acid catalysed methods have demonstrated good substrate scope and are moderate- to high-yielding, separation of the metal catalysts from the products can be problematic and costly e.g. in late stage pharmaceutical synthesis.[21] [22] [23] [24] The existing organonucleophile-catalysed processes do not appear to have been systematically investigated but our experience indicates that their scope/efficacy is inferior to the procedure described herein.

Pyridine-N-oxides have been used extensively as nucleophilic catalysts for the coupling of 3′-phosphorylated nucleotides with 5′-OH nucleotides in the synthesis of oligonucelotides[25] [26] and we have recently reported the use of 2-aryl-4-(dimethylamino)pyridine-N-oxides as catalysts for the chemoselective phosphorylation of α-amino acid derivatives, polyols and peptides.[27] During the course of these studies, it was noted that pyridine-N-oxide itself provided significant rate enhancement in alcohol phosphorylation reactions using phosphoryl chlorides. Herein, we report the use of pyridine-N-oxide as a competent nucleophilic catalyst for the phosphorylation of hydroxyl-containing substrates using phosphoryl chlorides.

Our initial studies focused on evaluation of bases for the phosphorylation of 4-phenyl-1-butanol (1) by diphenyl phosphoryl chloride (DPPCl) using pyridine-N-oxide (2) as a catalyst (Table [1]).

Table 1 Phosphorylation of 4-Phenyl-1-butanol

Entry	Catalyst	Base	Conv. (%)a
1	none	none	0
2	2	none	0
3	2	PPO	47
4	2	PS®	>99 (98)b
5	2	Et3N	87
6	2	PMP	>99
7	none	PS®	0

^a Conversion to product 3 as determined by ¹H NMR of the crude reaction mixture.

^b Isolated yield after chromatographic purification. PPO = propylene oxide, PS^® = 1,8-bis(NMe₂)-naphthalene (Proton-sponge^®), PMP = 1,2,2,6,6-pentamethylpiperidine.

In the absence of base and/or catalyst no reaction was observed (entries 1, 2 and 7, Table [1]). Whilst propylene oxide provided moderate levels of conversion (entry 3, Table [1]), both Proton-sponge^® and PMP provided quantitative conversion within two hours (entries 4 and 6, Table [1]). With the aim of developing a general method for the economic and scalable phosphorylation of alcohols, Proton-sponge^® was identified as optimal and these conditions were applied to a variety of alcohol substrates (Table [2]).

Table 2 Pyridine-N-oxide-Catalyzed Phosphorylation: Substrate Scope

Entry	Product	Compound	Time (h)	Yield (%)a
1		3b	2	98
2		3c	8	94
3		3d	8	ca. 81b
4		3e	24	57
5		3f	1	95 (17)c
6		3g	1	97 (21)c
7		3h	1	91 (14)c
8		3i	1	82 (7)c

^a Isolated yield after chromatographic purification.

^b Product 3d decomposes on silica gel (see refs. 9 and 14), impurities present, see Supporting Information.

^c Conversion to product 3 as determined by ¹H NMR of the crude reaction mixture of the uncatalyzed reaction.

Benzyl alcohol was phosphorylated in good yield within two hours (entry 1, Table [2]). Secondary alcohols and tert-butyl alcohol were also phosphorylated with moderate to good yields, albeit with increased reactions times (entries 2–4, Table [1]). Other tertiary alcohols (e.g., α-terpineol and 2-phenyl-2-propanol) were unreactive to these conditions. Phenols were converted to phosphorylated products in high yields and short reaction times (1 h), irrespective of the electronics of other aryl substituents (entries 5–8, Table [2]). Although these phenols are also phosphorylated in the absence of catalyst, rates are significantly increased in the presence of the pyridine-N-oxide catalyst.

Having established reaction conditions that appeared general for the phosphorylation of a range of hydroxy-containing substrates, including the preparation of phosphoryl derivatives known to be highly sensitive to adventitious nucleophiles (e.g., compounds 3b and 3d), phosphorylation of a selection of biologically relevant molecules and phosphate ester prodrugs was evaluated (Table [3]).

The allylic alcohol geraniol was phosphorylated in good yield within two hours (entry 1, Table [3]). Steroid derivatives containing phenolic and primary alcohols were also phosphorylated in good yield and with chemoselectivity over secondary and tertiary hydroxyl functionalities (entries 2 and 3, Table [3]). Phosphorylation of the primary 5′-OH group of adenosine in the presence of the 2′- and 3′-OH groups was also achieved (entry 4, Table [3]); phosphorylation of the 2′- and 3′-OH groups was not detected (by ³¹P NMR). Phosphorylation of protected amino acid residues Ser, Thr and Tyr was also achieved in moderate to good yields (entries 5–7, Table [3]). Phosphorylation of Boc-Ser(OH)-OMe was also conducted on a 20 mmol (5 g) scale with comparable yield in the same timescale (entry 5, Table [3]).

For the phosphorylation of the phenolic substrates,[28] o-xylenyl phosphoryl chloride (o-XPCl) was also employed in place of DPPCl (entries 2 and 7, Table [3]).[29] This allows for selective deprotection by hydrogenolysis (H₂, Pd/C)[27] [30] or with acid (e.g., HBr, AcOH),[31] which cannot be achieved for phenyl phosphate esters (or for products containing other phenyl moieties).[31–33] Thus, deprotection of phosphates 5b and 8 by hydrogenolyis yielded the corresponding phosphates quantitatively (Scheme [2]).

To probe the mechanism of catalysis operating in the phosphorylation reactions, a 1:1 mixture of pyridine-N-oxide and DPPCl was analysed by NMR in CDCl₃. The ¹H NMR and ³¹P NMR spectra of this mixture revealed complete conversion of both starting materials to a new species in which the pyridyl C2/C6 protons had shifted by Δδ_H = +0.28 ppm and the phosphorus signal by Δδ_P = –20.32 ppm, suggesting the formation of an O-phosphorylated salt as expected during nucleophilic catalysis (Figure [1]).

Subsequent treatment of this salt with an alcohol results in rapid conversion to the phosphate monoester product. Our attempts to obtain crystals of the intermediate salt, suitable for single-crystal X-ray structure determination, have however so far been unsuccessful.

Table 3 Phosphorylation of Biologically Relevant Molecules and Phosphate Prodrugs

Entry	Product	Compound	Time (h)	Yield (%)a
1		4	2	85
2		5a R = Ph 5b R–R = o-XP	1 1	74 (27)b 71 (15)b
3		6	2	83
4		7	2	61
5		8	2	77 (74)c
6		9	8	54
7		10a R = Ph 10b R–R = o-XP	1 1	91 (59)b 93 (21)b

^a Isolated yield after chromatographic purification.

^b Conversion to product as determined by ¹H NMR of the crude reaction mixture of the uncatalyzed reaction.

^c Yield of reaction conducted on 20 mmol scale with respect to alcohol.

In conclusion, we have demonstrated the use of pyridine-N-oxide as an effective and economical organocatalyst for the phosphorylation of primary and secondary alcohols and phenols, including several biologically relevant molecules. The reaction times and yields are comparable to those described for Lewis acid catalysed conditions and superior to those of previously described simple nucleophilic organocatalysts (e.g. 4-DMAP and NMI). This method therefore provides a complimentary approach to catalytic phosphoryl transfer that obviates the need for metal catalysts and which may be of particular utility in the late-stage production of phosphate esters for biological applications, e.g. in the preparation of prodrugs. For difficult alcohol phosphorylation cases, for example the site-selective phosphorylation of polyols, the use of a 2-aryl-4-DMAP-N-oxide catalyst will likely be warranted,[27] but the commercial availability and low cost of pyridine-N-oxide makes it an attractive first choice catalyst for evaluation.

Acknowledgment

We thank the EPSRC and the Imperial College Institute of Chemical Biology for funding. J.I.M. is also grateful to the Society of Chemical Industry for a Messel Scholarship.

• References and Notes

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• 27 Murray JI, Woscholski R, Spivey AC. Chem. Commun. 2014; 50: 13608
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• 28 The o-XP group can also be introduced onto non-phenolic alcohols (see ref. 27), where its ease of removal can also be advantageous for certain applications.
• 29 General Method: To a solution of alcohol component (0.9 mmol), pyridine-N-oxide (0.045 mmol, 5 mol%) and Proton-sponge^® (1.8 mmol) in CH₂Cl₂ (0.2 M) was added the specified phosphoryl chloride (1.0 mmol). The reaction mixture was stirred at r.t. for the time specified before the addition of MeOH (2 mL). The reaction mixture was then concentrated in vacuo and the crude material was purified by flash chromatography to afford the product. 17-β-3-xylenylphosphoryl estradiol (5b): According to the general method, 17-β-estradiol (246 mg, 0.9 mmol), pyridine-N-oxide (5 mg, 5 mol%), Proton-sponge^® (392 mg, 2.0 mmol) and xylenyl phosphoryl chloride (217 mg, 1.0 mmol) followed by flash chromatography (eluent: 0–50% EtOAc–hexanes) afforded xylenylphosphate 5b as a white solid (290 mg, 0.64 mmol, 71%); mp 146–147 °C; [α]_D ¹⁹ –87 (c = 0.99, CHCl₃). IR: 3314, 3033, 3001, 2936, 1707, 1612, 1536, 1286, 1211, 1097, 875, 736, 467 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ = 7.41–7.43 (m, 2 H), 7.34–7.36 (m, 2 H), 7.28–7.30 (m, 1 H), 7.05–7.08 (m, 1 H), 5.38–5.44 (m, 2 H), 5.19–5.28 (m, 2 H), 3.76 (t, J = 8.5 Hz, 1 H), 2.88 (m, 2 H), 2.31–2.39 (m, 1 H), 2.11–2.26 (m, 2 H), 1.99 (dt, J = 12.5, 3.3 Hz, 1 H), 1.89–1.94 (m, 1 H), 1.69–1.77 (m, 1 H), 1.18–1.55 (m, 9 H), 0.81 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃): δ = 138.8, 135.2, 129.4, 129.1, 126.8, 119.6, 119.5, 116.8, 114.2, 81.9, 68.9, 50.1, 44.1, 43.2, 38.5, 36.7, 30.6, 29.6, 27.0, 26.2, 23.1, 11.1. ³¹P NMR (161 MHz, CDCl₃): δ = –6.94. HRMS (ES): m/z [M + H⁺] calcd for C₂₆H₃₂O₅P: 455.1987; found: 455.1988. (S)-Methyl 2-[(tert-butoxycarbonyl)amino]-3-[(diphenoxyphosphoryl)oxy]propanoate (8): According to the general method, Cbz-Ser(OH)-OMe (200 mg, 0.9 mmol), pyridine-N-oxide (5 mg, 5 mol%), Proton-sponge^® (392 mg, 2.0 mmol) and diphenyl phosphoryl chloride (0.21 mL, 1.0 mmol) followed by flash chromatography (eluent: 0–10% EtOAc–hexanes) afforded diphenylphosphate 8 as a white solid (311 mg, 0.69 mmol, 77%); mp 61–63 °C; [α]_D ¹⁹ + 161 (c = 0.96, CHCl₃). IR: 3313, 3067, 3039, 2957, 1721, 1590, 1514, 1488, 1456, 1288, 1185, 1163, 948, 754, 688 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ = 7.20–7.40 (m, 10 H), 5.40 (d, J = 7.9 Hz, 1 H), 4.65–4.67 (m, 1 H), 4.51–4.60 (m, 1 H), 3.71 (s, 3 H), 1.46 (s, 9 H). ¹³C NMR (100 MHz, CDCl₃): δ = 169.4, 155.1, 150.4, 129.9, 129.8, 125.6, 120.1, 120.0, 80.4, 68.8, 53.8, 52.8, 28.3. ³¹P NMR (161 MHz, CDCl₃): δ = –12.34. HRMS (ES): m/z [M + Na] calcd for C₂₁H₂₆NO₈PNa: 474.1294; found: 474.1300.
• 30 Yamago S. US Patent US2008177049A1, 2008
• 31 Toth G, Kele Z, Varadi G. Curr. Org. Chem. 2007; 11: 409
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• 32 Jarowicki K, Kocienski P. J. Chem. Soc., Perkin Trans. 1 1998; 1: 4005
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• 33 McMurray JS, Coleman DR, Wang W, Campbell ML. Biopolymers 2001; 60: 3
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• References and Notes

• 1 Corbridge DE. C. J. Am. Chem. Soc. 2000; 122: 10742
  Search in Google Scholar
• 2 Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S. Science 2002; 298: 1912
  CrossrefSearch in Google Scholar
• 3 Jessen HJ, Ahmed N, Hofer A. Org. Biomol. Chem. 2014; 3526
• 4 Schultz C. Bioorg. Med. Chem. 2003; 11: 885
  CrossrefSearch in Google Scholar
• 5 Zawilska JB, Wojcieszak J, Olejniczak AB. Pharmacol. Reports 2013; 65: 1
  Search in Google Scholar
• 6 Rautio J, Kumpulainen H, Heimbach T, Oliyai R, Oh D, Järvinen T, Savolainen J. Nat. Rev. Drug Discovery 2008; 7: 255
  CrossrefPubMedSearch in Google Scholar
• 7 Uphues G, Ploog U. US Patent US4921990A, 1990
• 8 Beaucage SL, Iyer RP. Tetrahedron 1993; 49: 1925
  CrossrefSearch in Google Scholar
• 9 Jones S, Selitsianos D. Org. Lett. 2002; 4: 3671
  CrossrefSearch in Google Scholar
• 10 Jones S, Selitsianos D, Thompson KJ, Toms SM. J. Org. Chem. 2003; 68: 5211
  CrossrefSearch in Google Scholar
• 11 Jones S, Smanmoo C. Org. Lett. 2005; 7: 3271
  CrossrefSearch in Google Scholar
• 12 Jones S, Selitsianos D. Tetrahedron: Asymmetry 2005; 16: 3128
  CrossrefSearch in Google Scholar
• 13 Crook S, Parr NJ, Simmons J, Jones S. Tetrahedron: Asymmetry 2014; 25: 1298
  CrossrefSearch in Google Scholar
• 14 Fenton OS, Allen EE, Pedretty KP, Till SD, Todaro JE, Sculimbrene BR. Tetrahedron 2012; 68: 9023
  CrossrefSearch in Google Scholar
• 15 Coppola KA, Testa JW, Allen EE, Sculimbrene BR. Tetrahedron Lett. 2014; 55: 4203
  CrossrefSearch in Google Scholar
• 16 Mora N, Lacombe JM, Pavia AA. Tetrahedron Lett. 1993; 34: 2461
  CrossrefSearch in Google Scholar
• 17 Sabesan S, Neira S. Carbohydr. Res. 1992; 223: 169
  CrossrefSearch in Google Scholar
• 18 Pisarek S, Bednarski H, Gryko D. Synlett 2012; 23: 2667
  Thieme ConnectSearch in Google Scholar
• 19 Sculimbrene BR, Miller SJ. J. Am. Chem. Soc. 2001; 123: 10125
  CrossrefSearch in Google Scholar
• 20 Sculimbrene BR, Morgan AJ, Miller SJ. J. Am. Chem. Soc. 2002; 124: 11653
  CrossrefSearch in Google Scholar
• 21 Garrett CE, Prasad K. Adv. Synth. Catal. 2004; 346: 889
  CrossrefSearch in Google Scholar
• 22 Kucznierz R, Dickhaut J, Leinert H, Von Der Saal W. Synth. Commun. 1999; 29: 1617
  CrossrefSearch in Google Scholar
• 23 Guillaume M, Cuypers J, Dingenen J. Org. Process Res. Dev. 2007; 11: 1079
  CrossrefSearch in Google Scholar
• 24 Reeves JT, Tan Z, Han ZS, Li G, Zhang Y, Xu Y, Reeves DC, Gonnella NC, Ma S, Lee H, Lu BZ, Senanayake CH. Angew. Chem. Int. Ed. 2012; 51: 1400
  CrossrefSearch in Google Scholar
• 25 Efimov VA, Chakhmakhcheva OG, Ovchinnikov YA. Nucleic Acids Res. 1985; 13: 3651
  CrossrefSearch in Google Scholar
• 26 Efimov VA, Buryakova AA, Dubey IY, Polushin NN, Chakhmakhcheva OG, Ovchinnikov YA. Nucleic Acids Res. 1986; 14: 6525
  CrossrefSearch in Google Scholar
• 27 Murray JI, Woscholski R, Spivey AC. Chem. Commun. 2014; 50: 13608
  CrossrefSearch in Google Scholar
• 28 The o-XP group can also be introduced onto non-phenolic alcohols (see ref. 27), where its ease of removal can also be advantageous for certain applications.
• 29 General Method: To a solution of alcohol component (0.9 mmol), pyridine-N-oxide (0.045 mmol, 5 mol%) and Proton-sponge^® (1.8 mmol) in CH₂Cl₂ (0.2 M) was added the specified phosphoryl chloride (1.0 mmol). The reaction mixture was stirred at r.t. for the time specified before the addition of MeOH (2 mL). The reaction mixture was then concentrated in vacuo and the crude material was purified by flash chromatography to afford the product. 17-β-3-xylenylphosphoryl estradiol (5b): According to the general method, 17-β-estradiol (246 mg, 0.9 mmol), pyridine-N-oxide (5 mg, 5 mol%), Proton-sponge^® (392 mg, 2.0 mmol) and xylenyl phosphoryl chloride (217 mg, 1.0 mmol) followed by flash chromatography (eluent: 0–50% EtOAc–hexanes) afforded xylenylphosphate 5b as a white solid (290 mg, 0.64 mmol, 71%); mp 146–147 °C; [α]_D ¹⁹ –87 (c = 0.99, CHCl₃). IR: 3314, 3033, 3001, 2936, 1707, 1612, 1536, 1286, 1211, 1097, 875, 736, 467 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ = 7.41–7.43 (m, 2 H), 7.34–7.36 (m, 2 H), 7.28–7.30 (m, 1 H), 7.05–7.08 (m, 1 H), 5.38–5.44 (m, 2 H), 5.19–5.28 (m, 2 H), 3.76 (t, J = 8.5 Hz, 1 H), 2.88 (m, 2 H), 2.31–2.39 (m, 1 H), 2.11–2.26 (m, 2 H), 1.99 (dt, J = 12.5, 3.3 Hz, 1 H), 1.89–1.94 (m, 1 H), 1.69–1.77 (m, 1 H), 1.18–1.55 (m, 9 H), 0.81 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃): δ = 138.8, 135.2, 129.4, 129.1, 126.8, 119.6, 119.5, 116.8, 114.2, 81.9, 68.9, 50.1, 44.1, 43.2, 38.5, 36.7, 30.6, 29.6, 27.0, 26.2, 23.1, 11.1. ³¹P NMR (161 MHz, CDCl₃): δ = –6.94. HRMS (ES): m/z [M + H⁺] calcd for C₂₆H₃₂O₅P: 455.1987; found: 455.1988. (S)-Methyl 2-[(tert-butoxycarbonyl)amino]-3-[(diphenoxyphosphoryl)oxy]propanoate (8): According to the general method, Cbz-Ser(OH)-OMe (200 mg, 0.9 mmol), pyridine-N-oxide (5 mg, 5 mol%), Proton-sponge^® (392 mg, 2.0 mmol) and diphenyl phosphoryl chloride (0.21 mL, 1.0 mmol) followed by flash chromatography (eluent: 0–10% EtOAc–hexanes) afforded diphenylphosphate 8 as a white solid (311 mg, 0.69 mmol, 77%); mp 61–63 °C; [α]_D ¹⁹ + 161 (c = 0.96, CHCl₃). IR: 3313, 3067, 3039, 2957, 1721, 1590, 1514, 1488, 1456, 1288, 1185, 1163, 948, 754, 688 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ = 7.20–7.40 (m, 10 H), 5.40 (d, J = 7.9 Hz, 1 H), 4.65–4.67 (m, 1 H), 4.51–4.60 (m, 1 H), 3.71 (s, 3 H), 1.46 (s, 9 H). ¹³C NMR (100 MHz, CDCl₃): δ = 169.4, 155.1, 150.4, 129.9, 129.8, 125.6, 120.1, 120.0, 80.4, 68.8, 53.8, 52.8, 28.3. ³¹P NMR (161 MHz, CDCl₃): δ = –12.34. HRMS (ES): m/z [M + Na] calcd for C₂₁H₂₆NO₈PNa: 474.1294; found: 474.1300.
• 30 Yamago S. US Patent US2008177049A1, 2008
• 31 Toth G, Kele Z, Varadi G. Curr. Org. Chem. 2007; 11: 409
  CrossrefSearch in Google Scholar
• 32 Jarowicki K, Kocienski P. J. Chem. Soc., Perkin Trans. 1 1998; 1: 4005
  Search in Google Scholar
• 33 McMurray JS, Coleman DR, Wang W, Campbell ML. Biopolymers 2001; 60: 3
  CrossrefSearch in Google Scholar

• Supplementary Material