Design and synthesis of a potential SH2 domain inhibitor bearing a stereodiversified 1,4-cis-enediol scaffold

Abstract

Synthesis of a potential Src family SH2 domain inhibitor incorporating a 1,4-cis-enediol scaffold is reported. The synthetic route offers straightforward and highly selective access to the enediol and its associated chiral centers. Key steps include stereocontrolled syn-aldol coupling, amide alkynylation, and asymmetric ketone reduction.

1. Introduction

Protein tyrosine kinase (PTK) signal transduction pathways control a host of important cellular functions, including metabolism, growth and proliferation, and apoptosis.¹ As a result, aberrant PTK signaling often results in disease, most notably cancer and autoimmune diseases.² The Src homology-2 (SH2) domain, composed of ~100 amino acids, is a non-catalytic phosphotyrosine binding domain found in cytosolic PTKs which, by virtue of its central role in mediating PTK signaling, has emerged as an important target for therapeutic intervention.^3,4 Small peptides containing a phosphotyrosine (pTyr) can effectively inhibit SH2 domain binding of cognate proteins in the signal cascade, and therefore provide a logical starting point for inhibitor design.^5,6 Among PTKs of the Src family, pTyr-containing substrates generally adopt a similar binding mode, as exemplified by Lck kinase binding to tetrapeptide Ac-pTyr-Glu-Glu-Ile, or pYEEI peptide.⁷ A highly-conserved pocket binds the pTyr, and a second pocket three residues away (the “pY+3” pocket) binds a hydrophobic substituent. The two pockets are connected by a narrow β-sheet region which protrudes into the solvent. This “plug-and-socket” binding mode can be used to derive a general model for inhibitor design (Fig. 1A).⁴ Despite the extensive structural data available, efforts towards the development of new and therapeutically-useful SH2 domain inhibitors have met with limited success, and progress remains slow.^4,8,9 One major challenge has been the intracellular penetration of a free phosphate group. This challenge has been addressed by various prodrug strategies, including a phosphoramidate-based prodrug developed in our laboratory.¹⁰ Further issues to be addressed include reducing peptide character, and expanding ligand diversity in order to improve potency and selectivity. An additional consideration is the role of conformational restraint in ligand binding.¹¹ In summary, an ideal inhibitor would be non-peptidic, allow for structural and stereochemical diversity, and provide a means to introduce conformational restraint.

Figure 1.

A. General ligand binding mode for Src family SH2 domains. Figure adapted from Machida and Mayer.⁴ B. Ribbon diagram of cis-enediol inhibitor docked to Lck SH2 domain (PDB 1LKK).

An enediol-based scaffold meets all of these criteria, and seemed to us an attractive choice for SH2 inhibitor design, especially given the report in the literature of a series of nanomolar mu-opioid receptor agonists based on a 1,5-enediol system.¹² We envisioned an inhibitor model that would incorporate a 1,4-cis-enediol core, a phosphotyrosine mimic, and a hydrophobic substituent. As the linking segment, the cis-alkene would provide a degree of conformational restraint and presumably serve to orient the ligands into their respective pockets by wrapping around the central β-sheet region. The associated chiral centers would allow for the systematic comparison of stereochemical preferences within the SH2 domain. A benzyl group was chosen as the pY+3 substituent based on previous reports in the literature highlighting its suitability.^13,14 The stereoisomer exhibiting the greatest binding affinity will ultimately be synthesized as its corresponding prodrug¹⁰ for cell-based and in vivo studies (Figure 2).

Figure 2.

Cis-enediol phosphoramidate prodrug.

3. Results and discussion

Computational docking of proposed inhibitors to the Lck SH2 domain (PDB 1Lkk) supported our reasoning and generated structures similar to pYEEI-bound Lck (Fig. 1B). Substituents at C-1 contributed little to binding, so they were not considered further.

A preliminary synthesis of compound 2 was carried out, using a published procedure to access the cis-enediol core via Grubbs ring-closing metathesis.¹⁵ We found the synthesis to be laborious and inefficient, but testing of 2 in a competitive fluorescence binding assay gave an IC₅₀ comparable to that of pYEEI peptide (Huang and Borch, unpublished). These results suggested that the cis-enediol scaffold could be used to generate high-affinity SH2 domain ligands, and encouraged us to develop an improved synthetic pathway to the proposed compounds.

Our new synthetic approach is focused on efficient conversions and installation of the three chiral centers with high selectivity. In addition, the synthesis allows for further modification of the linker region, which may be of use in future studies. Computational modeling studies showed the terminal amide of 2 directed outward into the solvent rather than interacting with the protein, so it was replaced with a hydroxyl group. The pathway to all eight stereoisomers is outlined in Scheme 1. 1,4-cis-enediol compounds 1 can be made by coupling a protected chiral alkyne and Weinreb amide. The alkyne can in turn be synthesized by Evans syn¹⁶ or Masamune anti¹⁷ stereocontrolled aldol coupling of TMS-propynal with the appropriate homobenzyl substituted chiral auxiliary. Herein we describe the synthesis of the (R,R,R)-stereoisomer.

Scheme 1.

Retrosynthetic analysis of modified1,4-cis-enediol compounds (PG = protecting group).

Synthesis of ynone 7 is shown in Scheme 2. Introduction of the chiral centers at carbons 5 and 6 was achieved by Evans asymmetric syn-aldol addition of trimethylsilylpropynal to chiral auxiliary (S,R)-3.¹⁸ Subsequent silyl cleavage with TBAF yielded aldol product 4, which was isolated with a diastereomeric ratio of 96:4 after column chromatography. Reductive cleavage of the chiral auxiliary with sodium borohydride and acetonide protection of the resulting diol afforded alkyne 5. The lithium alkynylide was then added to Weinreb amide 6 to give ynone 7 in 68% yield. An alkyne to amide ratio of 2:1 was necessary to obtain reasonable yields for this step, but unreacted alkyne was easily recovered. Zinc-mediated addition of the alkyne to Boc-glycinal was also tried under numerous conditions, but did not yield any product.

Scheme 2.

Preparation of ynone 7.

The next step required asymmetric reduction of ynone 7 to give the desired propargyl alcohol (Scheme 3). Reduction with methyl-CBS-oxazaborolidine gave poor selectivity, but asymmetric transfer hydrogenation with Noyori’s chiral ruthenium catalyst¹⁹ installed the third and final chiral center at carbon 2 with high yield and selectivity (dr 9:1). Stereochemical assignment was determined by Mosher analysis of the O-MTPA esters.²⁰ Propargyl alcohol 9 was then reduced to cis-alkene 10 via hydrogenation with Lindlar catalyst. At this point a protecting group exchange became necessary to ensure selective removal of the Boc group in the next step, so the 1,3-acetonide was cleaved and the resulting triol was protected by introduction of TBS groups to give protected aminotriol 11.

Scheme 3.

Synthesis of protected aminotriol 11.

Completion of (R,R,R)-1 is detailed in Scheme 4. tert-Butyl phosphoryl acid 13 was synthesized in two steps from commercially-available methyl 4-hydroxyphenyl-acetate.²¹ Boc cleavage of 11 with TBS-triflate followed by coupling to acid 13 with EDC/HOBt afforded acylation product 14 in good yield (Scheme 4). Global deprotection under acidic conditions completed the synthesis and gave (R,R,R)-1 in essentially quantitative yield.

Scheme 4.

Completion of (R,R,R)-1.

4. Conclusion

In conclusion, we have synthesized a novel Src family SH2 domain inhibitor based on a 1,4-cis-enediol core. Our synthesis offers straightforward and highly selective access to a privileged molecular scaffold, and will allow the study of stereochemical preferences within the SH2 domain. Synthesis of the remaining seven diastereomers is underway, and binding data for the entire series will be reported shortly.

5. Experimental

5.1 General procedures

All NMR spectra were recorded on a Bruker Avance DPX-300 spectrometer equipped with a multinuclear (³¹P, ¹³C, ¹H and ¹⁹F) probe. Chemical shifts are given in ppm with reference to tetramethylsilane. All ³¹P NMR spectra were acquired using broadband gated decoupling. ³¹P chemical shifts are reported in parts per million using 1% triphenylphosphine oxide in benzene-d₆ as the coaxial reference set at 0. Mass spectral data were obtained from the Purdue University Mass Spectrometry Service, West Lafayette, IN. IR spectra were recorded on a Perkin Elmer Spectrum I FTIR spectrometer using a NaCl plate. Optical rotations were measured on a Perkin Elmer 341 polarimeter with 0.5 dm path length cell. Analytical HPLC analysis was done using a Beckman System Gold equipped with a 168 detector set at 215 nm and a Zorbax Eclipse 5-micron analytical C18 column (4.6 × 250 mm) from Agilent Technologies Inc. Lck GST-SH2 protein was a kind gift from Dr. Robert Geahlen. All moisture-sensitive reactions were carried out under an atmosphere of argon, and all organic solvents were distilled prior to use unless otherwise specified. Flash chromatography using silica gel grade 60 (230–400 mesh) was carried out for all chromatographic separations. Thin layer chromatography was performed using Analtech glass plates precoated with silica gel (250 nm), and visualized using UV or phosphomolybdic acid stain. All reagents were purchased from Aldrich, Alfa-Aesar, or TCI America, unless stated otherwise.

5.2 Chemistry

(4S,5R)-3-((2S,3S)-2-benzyl-3-hydroxypent-4-ynoyl)-4-methyl-5-phenyloxazolidin-2-one (4)

(S,R)-3 (6.56 g, 21.2 mmol) was dissolved in 100 mL dry CH₂Cl₂ and cooled to 0°C. Dibutylboron triflate (23 mL, 1.0 M in CH₂Cl₂, 23.3 mmol) was added dropwise, and the light orange reaction mixture allowed to stir for 10 minutes at 0 °C. Diisopropylethylamine (4.42 mL, 25.4 mmol) was then added dropwise, and the resulting clear solution allowed to stir for 30 minutes at 0 °C. The mixture was then cooled to −78 °C, and a solution of trimethylsilylpropynal (3.1 mL, 21.0 mmol) in 30 mL CH₂Cl₂ at −78 °C was added via cannula. After 20 minutes stirring at −78 °C, the reaction mixture was warmed to 0 °C and allowed to stir for an additional 2 hours at that temperature, then quenched by dropwise addition of the following, in the order listed: 25 mL phosphate buffer (pH 7.4), 65 mL methanol, and 65 mL of a 2:1 solution of methanol and 30% aq. H₂O₂, cooled to 0 °C. The mixture was allowed to stir at 0 °C for one hour, then extracted 3X with CH₂Cl₂. The combined organic layers were washed with NaHCO₃ and brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was dissolved in 135 mL THF and 9 mL MeOH and cooled to −20 °C. Tetrabutylammonium fluoride (21 mL, 1.0 M in THF, 21.2 mmol) was added dropwise, and the mixture allowed to stir for two hours at 0 °C. The reaction was quenched with sat. NH₄Cl solution and extracted with CH₂Cl₂. The organic layer was washed with brine and dried over anhydrous Na₂SO₄, followed by removal of solvent under reduced pressure. The crude product was purified by column chromatography (20% ethyl acetate/hexane) to yield 5.8 g (60% over two steps) of major diastereomer (S,R)-4 as an off-white foam. TLC 50% ethyl acetate/hexane R_f = 0.7 visualized with PMA or UV. Diastereomeric ratio after flash column chromatography was 96:4, as determined by reverse phase HPLC. HPLC, Zorbax XDB-C18 (250 × 4.6 mm, 5 micron); Flow = 1.0 mL/min; λ= 215 nm; Isocratic (80:20 MeCN:ddH₂O); Major isomer R_t = 7.15 min. [α]_D²⁵ −34.0 (c 1.00, CHCl₃). ¹H NMR (CDCl₃, 300 MHz) δ 7.16–7.39 (m, 10H), 5.60 (d, J = 7.4 Hz, 1H), 4.66–4.80 (m, 2H), 4.64 (s, 1H), 3.18 (d, J = 6.4 Hz, 2H), 2.71 (b, 1H), 2.62 (d, 1H), 0.60 (d, J = 6.6 Hz, 3H). ¹³C NMR (CDCl₃, 75 MHz) δ 172.6, 152.8, 137.6, 133.0, 129.3, 128.7, 128.6, 128.3, 126.6, 125.6, 81.8, 78.7, 77.2, 75.0, 63.1, 54.7, 49.8, 34.5, 14.1. ESI (+) HRMS (m/z): [M+Na]⁺ calcd for C₂₂H₂₁NO₄ 386.1368; found, 386.1370.

(4S,5R)-5-benzyl-4-ethynyl-2,2-dimethyl-1,3-dioxane (5)

Aldol product 4 (415 mg, 1.14 mmol) was dissolved in 30 mL THF and cooled to 0 °C. NaBH₄ (173 mg, 4.56 mmol) was dissolved in 7 mL H₂O and added dropwise to the THF solution. The mixture was allowed to warm to room temperature and stirred for 1 hr, after which it was cooled to 0 °C and quenched with NH₄Cl. The mixture was again brought to room temperature and stirred for 1 hr. The layers were separated, and the aqueous layer extracted 3X with EtOAc. The combined organic layer was washed with NaHCO₃ and brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The chiral auxiliary was recovered by recrystallization of the crude residue with ethyl acetate/hexanes (~1:1). The remaining oil was then dissolved in 23 mL 2,2- dimethoxypropane. p-toluenesulfonic acid monohydrate (22 mg, 0.114 mmol) was added in one portion, and the mixture stirred for 1 hr at room temperature. The mixture was then diluted with CH₂Cl₂ and quenched with NaHCO₃ at 0 °C. The layers were separated and the aqueous layer extracted 3X with CH₂Cl₂. The combined organic layers were dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to yield 236 mg pure alkyne 5 as a light yellow oil (90% over two steps). TLC 30% ethyl acetate/hexane R_f = 0.9 visualized with PMA. [α]_D²⁵ +22.6 (c 1.00, CHCl₃). ¹H NMR (CDCl₃, 300 MHz) δ 7.21–7.34 (m, 5H), 4.92 (t, 1H), 3.80 (dd, J = 2.8, 12.1 Hz, 1H), 3.65 (dd, J = 3.3, 12.0 Hz, 1H), 2.99–3.11 (m, 2H), 2.62 (d, J = 2.4 Hz, 1H), 1.82–1.87 (m, 1H), 1.57 (s, 3H), 1.47 (s, 3H). ¹³C NMR (CDCl₃, 75 MHz) δ 140.0, 129.3, 128.4, 126.1, 99.4, 81.4, 75.2, 64.2, 60.8, 39.6, 31.6, 28.4, 20.8. CI (+) LRMS (m/z): 231 [M+H]⁺.

tert-Butyl 2-(methoxy(methyl)amino)-2-oxoethylcarbamate (6)

Weinreb amide 6 was synthesized from Boc-glycine as described by Morwick et al.^{22 1}H NMR (CDCl₃, 300 MHz) δ 5.29 (br, 1H), 4.01 (d, J = 4.0 Hz, 2H), 3.65 (s, 3H), 3.14 (s, 3H), 1.38 (s, 9H). ¹³C NMR (CDCl₃, 75 MHz) δ 170.1, 155.8, 79.5, 61.3, 41.6, 32.2, 28.2. CI (+) LRMS (m/z): 219 [M+H]⁺.

tert-Butyl-4-((4S,5R)-5-benzyl-2,2-dimethyl-1,3-dioxan-4-yl)-2-oxobut-3-ynylcarbamate (7)

Alkyne 5 (3.27 g, 14.2 mmol) was dissolved in 68 mL dry THF and cooled to −78°C. n-BuLi (8.37 mL, 1.78 M in hexane, 14.9 mmol) was added dropwise and the resulting mixture stirred at −78 °C for 1 hour. The mixture was then added via cannula to a solution of Weinreb amide 2 (1.55 g, 7.10 mmol) in 35 mmol THF cooled to −78 °C. The mixture was then stirred at 0 °C for 3 hours, cooled to −78 °C, and quenched by slow addition of sat. NH₄Cl solution. The resulting mixture was extracted 3X with Et₂O, and the combined organic layers washed with NaHCO₃ and brine and dried over anhydrous Na₂SO₄. Purification of the crude oil by column chromatography (10% → 20% ethyl acetate/hexane) yielded 1.87 g ynone 7 as a yellow oil (68%). 1.33 g (82%) of starting alkyne were also recovered. TLC 30% ethyl acetate/hexane R_f = 0.5 visualized with PMA. [α]_D²⁵ +34.0 (c 1.00, CHCl₃). ¹H NMR (CDCl₃, 300 MHz) δ 7.16–7.32 (m, 5H), 5.20 (b, 1H), 5.02 (d, J = 3.4 Hz, 1H), 4.15 (d, J = 5.3 Hz, 2H), 3.82 (dd, J = 3.0, 12.1 Hz, 1H), 3.64 (dd, J = 4.0, 12.1 Hz, 1H), 2.91–2.98 (m, 2H), 1.96–2.00 (m, 1H), 1.53 (s, 3H), 1.44 (s, 12 H). ¹³C NMR (CDCl₃, 75 MHz) δ 182.5, 155.4, 139.0, 129.2, 128.5, 126.3, 99.7, 91.9, 83.4, 80.2, 64.3, 60.9, 52.1, 39.1, 32.2, 28.2, 27.7, 21.5. ESI (+) HRMS (m/z): [M+Na]⁺ calcd for C₂₂H₂₉NO₅ 410.1943; found, 410.1946.

tert-Butyl-((R)-4-((4S,5R)-5-benzyl-2,2-dimethyl-1,3-dioxan-4-yl)-2-hydroxybut-3-yn-1-yl)carbamate (9)

To a stirred solution of ynone 7 (480 mg, 1.24 mmol) in 9.5 mL anhydrous dimethylformamide under argon atmosphere was added (S,S)-Noyori catalyst 8 (16 mg, 0.025 mmol) at room temperature. In a separate flask, diisopropylethylamine (208 mg, 1.61 mmol) was cooled to −10 °C, and 98% formic acid (188 mg, 4.09 mmol) was added dropwise, keeping the temperature below 0 °C. This solution was added dropwise to the ynone, and the reaction allowed to stir overnight at room temperature. The mixture was then cooled to 0 °C and quenched by addition of sat. NaHCO₃, extracted 3X with ethyl acetate, washed with brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude residue was purified by column chromatography (10% → 50% ethyl acetate/hexane) to give 420 mg of propargyl alcohol 9 as a light yellow oil (87%, dr 9:1). Stereochemical assignment of the alcohol configuration was determined by Mosher analysis of the (R) and (S)-MTPA esters.¹⁶ Diastereomeric ratio was determined by ¹⁹F NMR of the MTPA esters. TLC 30% ethyl acetate/hexane R_f = 0.4 visualized with PMA. [α]_D²³ +22.0 (c 1.00, CHCl₃). ¹H NMR (CDCl₃, 300 MHz) δ 7.15–7.29 (m, 5H), 5.21 (t, J = 5.5 Hz, 1H), 4.91 (s, 1H), 4.54 (b, 1H), 3.78 (dd, J = 2.6, 11.8 Hz, 1H), 3.59 (dd, J = 3.1, 12.0 Hz, 1H), 3.30–3.46 (m, 3H), 2.90–3.04 (m, 2H), 1.80 (m, 1H), 1.50 (s, 3H), 1.42 (s, 3H), 1.41 (s, 9H). ¹³C NMR (CDCl₃, 75 MHz) δ 156.6, 139.9, 129.3, 128.3, 126.0, 99.4, 85.7, 83.0, 79.8, 77.3, 64.3, 62.0, 60.9, 46.5, 39.6, 31.8, 28.4, 20.7. ESI (+) HRMS (m/z): [M+Na]⁺ calcd for C₂₂H₃₁NO₅, 412.2100; found, 412.2093.

tert-Butyl-((R,Z)-4-((4R,5R)-5-benzyl-2,2-dimethyl-1,3-dioxan-4-yl)-2-hydroxybut-3-en-1-yl)carbamate (10)

Propargyl alcohol 9 (271 mg, 0.693 mmol) was dissolved in 8.5 mL ethyl acetate and flushed with argon. Lindlar catalyst (5% palladium on calcium carbonate, poisoned with lead; 52 mg, 0.485 mmol) and quinoline (6 µL, 0.049 mmol) were added. The flask was flushed with argon again, and then flushed with H₂ gas 3X. A hydrogen balloon was then added, and the mixture allowed to stir overnight at room temp. The following day, the reaction mixture was filtered through Celite®, rinsing with ethyl acetate. The organic filtrate was washed with 1M HCl, satd. NaHCO₃, and brine, then dried over anhydrous Na₂SO₄ and concentrated to yield 273 mg alkene 10 as a light brown foam (quantitative). TLC 30% ethyl acetate/hexane R_f = 0.4 visualized with PMA. ¹H NMR (CDCl₃, 300 MHz) δ 7.14–7.28 (m, 5H), 5.59–5.62 (m, 2H), 5.15 (b, 1H), 4.97 (b, 1H), 4.54 (q, J = 5.3 Hz, 1H), 3.85 (d, J = 11.6 Hz, 1H), 3.53 (d, J = 11.9 Hz, 1H), 3.20–3.32 (m, 3H), 2.77–3.02 (m, 2H), 1.66 (d, J = 11.6 Hz, 1H), 1.49 (s, 6H), 1.43 (s, 9H). ¹³C NMR (CDCl₃, 75 MHz) δ 156.6, 140.5, 132.2, 131.3, 129.3, 128.3, 125.9, 99.0, 79.5, 70.0, 67.9, 61.6, 46.4, 39.9, 30.2, 29.7, 28.3, 19.1. ESI (+) HRMS (m/z): [M+Na]⁺ calcd for C₂₂H₃₃NO₅, 414.2256; found, 414.2273.

tert-Butyl-((2R,5R,6R,Z)-6-benzyl-2,5,7-tris((tert-butyldimethylsilyl)oxy)hept-3-en-1-yl)carbamate (11)

Alkene 10 (216 mg, 0.552 mmol) was dissolved in 3.5 mL THF and cooled to 0 °C. 85% phosphoric acid (566 µL, 8.28 mmol) was added dropwise, and the resulting solution stirred at room temp. for 3 hrs. The mixture was then cooled back down to 0 °C and diluted with H₂O. 50 wt% aq. NaOH was added dropwise to bring the solution to pH 8, and the solution was poured into brine and extracted 3X with EtOAc, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude oil was then dissolved in 9 mL anhydrous CH₂Cl₂ and cooled to −78 °C. 2,6-lutidine (322 µL, 2.76 mmol) was added dropwise, followed by TBS-trifluoromethanesulfonate (507 µL, 2.21 mmol). The reaction was stirred overnight at room temperature, after which it was quenched with MeOH at 0° C. The mixture was then poured into brine and extracted 3X with CH₂Cl₂, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude oil was purified by column chromatography (3% ether/pentane) to yield 190 mg pure tris-TBS aminotriol 11 as a clear, colorless oil (50% over two steps). TLC 10% ethyl acetate/hexane R_f = 0.7 visualized with PMA. ¹H NMR (CDCl₃, 300 MHz) δ 7.12–7.26 (m, 5 Hz), 5.50–5.57 (m, 1H), 5.19–5.26 (m, 1H), 4.80–4.83 (d, J = 8.4 Hz, 1H), 4.66 (b, 1H), 4.46–4.52 (m, 1H), 3.09–3.51 (m, 4H), 2.80 (dd, J = 4.6, 14.1 Hz, 1H), 2.43 (dd, J = 9.8, 14.0 Hz, 1H), 1.92 (b, 1H), 1.44 (s, 9H), 0.85–0.92 (m, 27H), −0.06–0.09 (m, 18H). ¹³C NMR (CDCl₃, 75 MHz) δ 155.6, 141.1, 134.9, 129.6, 128.9, 128.1, 125.6, 78.8, 68.1, 66.7, 61.7, 50.5, 46.4, 31.1, 28.3, 25.9, 18.1, −4.0, −4.1, −4.6, −4.9, −5.6. ESI (+) LRMS (m/z): 381 [M+Na]⁺.

Methyl-2-(4-((di-tert-butoxyphosphoryl)-oxy)phenyl)acetate (12)

To a stirred solution of imidazole (188 mg, 2.76 mmol) in 750 µL anhydrous THF at room temperature under argon atmosphere was added trifluoroacetic acid (210 µL, 2.76 mmol) dropwise. The resulting slurry was stirred at room temperature for 10 minutes, after which di-tert-butyl N,N-diisopropyl-phosphoramidite (285 µL, 0.90 mmol) was added dropwise. After stirring for an additional 10 minutes, a solution of methyl 4-hydroxyphenylacetate (100 mg, 0.60 mmol) in 300 µL anhydrous THF was added dropwise over 15 minutes. The solution was allowed to stir at room temperature for 30 minutes, then cooled to −40 °C. 14% aqueous tert-butyl hydroperoxide solution (1 mL) was added dropwise, and the mixture stored at 4°C overnight. The reaction was quenched with NaHCO₃ at 0 °C, poured into water, extracted 3X with EtOAc, dried over anhydrous Na₂SO₄, and concentrated in vacuo. The crude product was purified by column chromatography (5% → 10% ethyl acetate/CH₂Cl₂) to yield 200 mg pure phosphoryl methyl ester 12 as a clear, colorless oil (93%). TLC 20% ethyl acetate/CH₂Cl₂ R_f = 0.7 visualized with PMA. ¹H NMR (CDCl₃, 300 MHz) δ 7.08–7.17 (m, 4H), 3.61 (s, 3H), 3.52 (s, 2 H), 1.44 (s, 18 H). ¹³C NMR (CDCl₃, 75 MHz) δ 171.7, 150.5, 130.1, 129.7, 119.9, 83.5, 51.8, 40.2, 29.7. ³¹P NMR (CDCl₃) δ −40.8. ESI (+) LRMS (m/z): 716 [M+Na]⁺.

2-(4-((di-tert-butoxyphosphoryl)oxy) phenyl)acetic acid (13)

Methyl ester 12 (380 mg, 1.06 mmol) was dissolved in 5 mL H₂O and 13 mL THF, and cooled to 0 °C. A 0.3 M aqueous solution of LiOH (10.6 mL, 3.18 mmol) was added dropwise, and the mixture allowed to stir at 0 °C for 30 minutes. The reaction was acidified with 1 M aqueous HCl to a pH of 3.0, after which it was extracted 3X with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to yield 365 mg carboxylic acid 13 as a white solid (quantitative). ¹H NMR (CDCl₃, 300 MHz) δ 7.13–7.26 (m, 4H), 5.68 (b, 1H), 3.58 (s, 2H), 1.50 (s, 18H). ¹³C NMR (CDCl₃, 75 MHz) δ 175.5, 150.4, 130.3, 129.6, 119.9, 84.0, 40.2, 29.8. ³¹P NMR (CDCl₃) δ −41.1. ESI (+) HRMS (m/z): [M+Na]⁺ calcd for C₁₆H₂₅O₆P, 367.1286; found, 367.1277.

4-(2-(((2R,5R,6R,Z)-6-benzyl-2,5,7-tris((tert-butyldimethylsilyl)oxy)hept-3-en-1-yl)amino)-2-oxoethyl)phenyl di-tert butyl phosphate (14)

Protected aminotriol 11 (19 mg, 0.027 mmol) was dissolved in 500 µL anhydrous CH₂Cl₂ and cooled to −78 °C. TBS-trifluoromethanesulfonate (8 µL, 0.030 mmol) was added dropwise, and the reaction mixture stirred at 0 °C for 2 hrs. Diisopropylethylamine (0.030 mmol, 5 µL) was then added dropwise, and the solution poured into H₂O and extracted 3X with CH₂Cl₂. The organic layer was dried over anhydrous Na₂SO₄, concentrated in vacuo, and used immediately in the next step.

Phosphoryl acid 13 (11 mg, 0.032 mmol) was dissolved in 200 µL CH₂Cl₂ and stirred at 0 °C. EDC (6 mg, 0.032 mmol) was added, followed by HOBt hydrate (5 mg, 0.032 mmol) and DMAP (4 mg, 0.032 mmol). The resulting mixture was stirred 20 min. The crude amine was dissolved in 200 µL CH₂Cl₂ and added to the acid solution dropwise. The reaction mixture was warmed to room temperature and allowed to stir for two days. The solvent was then removed in vacuo and the crude residue redissolved in ethyl acetate and washed with the following: water, 5% aq. citric acid solution, satd. aq. NaHCO₃, and brine. The organic layer was then dried over anhydrous Na₂SO₄, concentrated, and purified by column chromatography (20% → 30% ethyl acetate/hexanes) to yield 12 mg 14 as a light yellow oil (50% over two steps). TLC 20% ethyl acetate/hexane R_f = 0.2 visualized with PMA. ¹H NMR (CDCl₃, 300 MHz) δ 7.09–7.26 (m, 9 H), 5.68 (b, 1H), 5.45–5.52 (m, 1H), 5.09–5.15 (m, 1H), 4.77 (d, J = 9 Hz, 1H), 4.46 (b, 1H), 3.34–3.58 (m, 5H), 3.02–3.11 (m, 1H), 2.74–2.81 (m, 1H), 2.37–2.45 (m, 1H), 1.88 (b, 1H), 1.50 (s, 27H), 0.90 (s, 9H), 0.83 (s, 9H), 0.77 (s, 9H), −0.08–0.10 (m, 18H). ¹³C NMR (CDCl₃, 75 MHz) δ 170.3, 150.7, 141.1, 134.8, 130.5, 129.2, 128.9, 128.1, 125.6, 125.2, 120.3, 83.7, 67.9, 66.6, 61.6, 50.5, 45.3, 43.1, 31.1, 30.3, 29.8, 25.9, 25.6, 18.1, 17.7, −3.7, −4.0, −4.7, −5.0, −5.6. ³¹P NMR (CDCl₃) δ −41.2. ESI (+) HRMS (m/z): [M+Na]⁺ calcd for C₄₈H₈₆NO₈PSi₃, 942.5277; found, 942.5297.

4-(2-(((2R,5R,6R,Z)-6-benzyl-2,5,7-trihydroxyhept-3-en-1-yl)amino)-2-oxoethyl)phenyl dihydrogen phosphate (1)

Acylation product 14 (23 mg, 0.025 mmol) was dissolved in 1 mL of a 1:1 mixture of anhydrous methanol and THF. The solution was cooled to 0 °C, and 230 µL of 3M HCl in MeOH were added. The reaction mixture was warmed to room temp. and allowed to stir overnight. Solvents were then removed in vacuo, and the product triturated with hexane and ether, and co-evaporated 2X with toluene to yield 12 mg cis-enediol phosphate 1 (quantitative). [α]_D²³ −20.6 (c 1.00, CH₃OH). ¹H NMR (CD₃OD, 300 MHz) δ 7.14–7.30 (m, 9H), 5.48–5.68 (m, 2H), 4.96 (b, phosphate), 4.52–4.56 (m, 2H), 3.20–3.66 (m, 10H), 2.59–3.21 (m, 2H), 1.79 (b, 1H). ¹³C NMR (CD₃OD, 75 MHz) δ 150.3, 140.7, 133.9, 131.8, 131.3, 129.9, 128.8, 127.9, 125.4, 119.9, 67.4, 65.7, 59.5, 48.7, 45.0, 41.4, 31.8. ³¹P NMR (CDCl₃) δ −30.1. ESI (+) HRMS (m/z): [M+H]⁺ calcd for C₂₂H₂₈NO₈P 466.1631; found, 466.1609.