Synthesis of photoactivatable analogues of lysophosphatidic acid and covalent labeling of plasma proteins

Abstract

[reaction: see text] Lysophosphatidic acids bearing a benzophenone group in either the sn-1 or sn-2 chain of an oleoyl-type ester or oleyl-type ether chain and (32)P in the phosphate group were synthesized. The benzophenone moiety was introduced by selective hydroboration of the double bond of enyne 11 at low temperature, followed by a Suzuki reaction with 4-bromobenzophenone. The key intermediates for the preparation of ester-linked lysophosphatidic acid (LPA) 1 and 3 were obtained in one pot by a modified DIBAL-H reduction of orthoformate intermediate 22. These probes were shown to covalently modify a single protein target in rat plasma containing albumin and several protein targets in rat plasma containing a low level of albumin.

Scheme 1

Retrosynthetic Strategy

Scheme 2. Synthesis of Long-Chain Alcohol 14

(a) LiNH(CH₂)₃NH₂, t-BuOK/H₂N(CH₂)₃NH₂; (b) DHP, PPTS, CH₂Cl₂; (c) i. n-BuLi, THF, −78 °C to rt; ii. allyl bromide, CuI, THF, −78 °C; (d) i. 9-BBN, THF, −15 °C to rt; ii. 4-bromobenzophenone, Pd(PPh₃)₄, K₃PO₄, THF/DMF, reflux, 2 h; (e) PPTS (cat.), MeOH; (f) H₂, Lindlar, MeOH.

Scheme 3. Synthesis of Glyceride 20 via FMOC-ester 18

(a) PDC/DMF, rt , 2 d; (b) (R)-(-)-2,2-dimethyl-1,3-dioxolane-4-methanol, DCC, DMAP, CH₂Cl₂, rt; (c) 0.4 N HCl/90% dioxane, rt; (d) FMOC-chloroformate, DMAP, CH₂Cl₂, −10 °C; (e) MOMCl, i-Pr₂NEt, CH₂Cl₂; (f) piperidine, CH₂Cl₂.

Scheme 4. Synthesis of 1 and 3

(a) (i). HC(OMe)₃, CSA (cat.), CH₂Cl₂, rt; ii. DIBAL-H, 0 °C; (b) 15, PPh₃, DIAD, THF, 0 °C-rt; (c) CAN, CH₃CN/H₂O (6:1); (d) i. DGK, [³²P]ATP, pH 6.6, 37 °C, 2 h; ii. TMSBr, CH₂Cl₂, 0 °C.

Scheme 5. Synthesis of Ether-linked Probes 2 and 4

(a) (i) CH₃SO₂Cl, Et₃N, 0 °C-rt; (ii) LiBr, THF, reflux; (b) (i) (n-Bu)₂SnO, CHCl₃/MeOH (10:1), reflux; (ii) 28, CsF, 18-crown-6, DMF, rt, 1 d; (c) MOMCl, i-Pr₂NEt, CH₂Cl₂; (d) CAN, CH₃CN/H₂O (6:1); (e) (i) DGK, [³²P]ATP; (ii) TMSBr, CH₂Cl₂, 0 °C.

Figure 1

Covalent modification of plasma proteins by benzophenone-containing LPA analogues. Plasma (5% in PBS) from Sprague-Dawley (19.4 mg albumin/mL) A or Nagase rats (0.4 mg albumin/mL) B was incubated with compounds 1, 2, 4, 20P, or 27P (50 nM, DMSO 10% final concentration) in the dark for 30 min prior to UV irradiation for up to 10 min on ice. Samples were diluted with 2 × sample loading buffer and separated by SDS-PAGE. Gels were subsequently dried and radioactive bands were visualized by autoradiography.

Figure 1

Covalent modification of plasma proteins by benzophenone-containing LPA analogues. Plasma (5% in PBS) from Sprague-Dawley (19.4 mg albumin/mL) A or Nagase rats (0.4 mg albumin/mL) B was incubated with compounds 1, 2, 4, 20P, or 27P (50 nM, DMSO 10% final concentration) in the dark for 30 min prior to UV irradiation for up to 10 min on ice. Samples were diluted with 2 × sample loading buffer and separated by SDS-PAGE. Gels were subsequently dried and radioactive bands were visualized by autoradiography.