Solid-Supported Synthesis of Artificial Phospholipids

Abstract

A concise solid-supported synthesis of artificial phospholipids was developed. Functionalized phospholipids were prepared by introduction of a head group onto a solid-supported phospholipid framework in good overall yield and purity.

Key words

solid-phase synthesis - artificial phospholipid - Wang resin - Merrifield resin - oxime ligation

References and Notes

• For a recent review on artificial phospholipids, see:
• 1a Zumbuehl A. Chimia  2009,  63:  63 
• For examples on lipid-membrane protein and lipid-lipid interactions, see:
• 1b Prestwich GD. Acc. Chem. Res.  1996,  29:  503 
• 1c Ogawa Y. Hahn W. Garnier P. Higashi N. Massotte D. Metz-Boutigue M.-H. Rousseau B. Kodaka M. Sunamoto J. Ourisson G. Nakatani Y. Chem. Eur. J.  2002,  8:  1843 
• 1d Sugahara M. Uragami M. Regen SL. J. Am. Chem. Soc.  2003,  125:  13040 
• 1e Gubbens J. Ruijter E. de Fays LEV. Damen JMA. de Kruijff B. Slijper M. Rijkers DTS. Liskamp RMJ. de Kroon AIPM. Chem. Biol.  2009,  16:  3 
• For examples on membrane protein crystallization, see:
• 1f Drakopoulou E. Tsivgoulis GM. Mukhopadhyay A. Brisson A. Tetrahedron Lett.  2000,  41:  4131 
• 1g Thompson DH. Zhou M. Grey J. Kim H. Chem. Lett.  2007,  36:  956 
• 2 For a recent review on polymerizable lipids, see: Cashion MP. Long TE. Acc. Chem. Res.  2009,  42:  1016 
• 3a Frias JC. Williams KJ. Fisher EA. Fayad ZA.
  J. Am. Chem. Soc.  2004,  126:  16316 
• 3b Lampkins AJ. O’Neil EJ. Smith BD. J. Org. Chem.  2008,  73:  6053 
• 3c Huang Z. Jaafari MR. Szoka FC. Angew. Chem. Int. Ed.  2009,  48:  1 
• 3d Linderoth L. Peters GH. Madsen R. Andresen TL. Angew. Chem. Int. Ed.  2009,  48:  1823 
• 3e Mulder WJM. Strijker GJ. Van Tilborg GAF. Cormode DP. Fayad ZA. Nicolay K. Acc. Chem. Res.  2009,  42:  904 
• 4 Furuta T. Sakai M. Hayashi H. Asakawa T. Kataoka F. Fujii S. Suzuki T. Suzuki Y. Tanaka K. Fishkin N. Nakanishi K. Chem. Commun.  2005,  4575 
• 5 Furuta T. Mochizuki M. Ito M. Takahashi T. Suzuki T. Kan T. Org. Lett.  2008,  10:  4847 
• To the best of our knowledge, only one example of a solid-phase synthesis of phosphatidylethanolamines has been reported. In this case, the amino moiety of the head group was tethered to the resin for immobilization, see:
• 6a Tomoi M. Kimura Y. Tokuyama S. React. Polym.  1991,  15:  63 
• 6b For solid-supported synthesis of phospholipid-like bioactive molecules, see: Sasaki S. Ehara T. Alam MR. Fujino Y. Harada N. Kimura J. Nakamura H. Maeda M. Bioorg. Med. Chem. Lett.  2001,  2581 
• 7 The following conditions were unsuccessful: alkylation conditions with benzyl chloride in the presence of Cs₂CO₃; condensation with benzyl alcohol by DCC; and the mixed anhydride method with 2,4,6-triisopropylbenzenesulfonyl chloride in the presence of DMAP
• p-Anisidine was added as a catalyst for oxime bond formation, see:
• 8a Dirksen A. Hackeng TM. Dawson PE. Angew. Chem. Int. Ed.  2006,  45:  7581 
• 8b Dirksen A. Dawson PE. Bioconjugate Chem.  2008,  19:  2543 
• 10 Alfonta L. Zhang Z. Uryu S. Loo JA. Schultz PG.
  J. Am. Chem. Soc.  2003,  125:  14662 
• 11a García-Echeverría C. Tetrahedron Lett.  1997,  38:  8933 
• 11b Guillier F. Orain D. Bradley M. Chem. Rev.  2000,  100:  2091 
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• 12 Scalone M. Waldmeier P. Org. Process Res. Dev.  2003,  7:  418 

9

Experimental Procedure for 11c
Wang resin (80 mg, 1.2 mmol/g, 96 µmol) placed in a syringe reactor was solvated with CH₂Cl₂, and then CH₂Cl₂ was replaced with toluene. To the resin in toluene (1.0 mL), was added Ph₃P (25 mg, 96 µmol) and 5 (20 mg, 24 mmol). The suspension was shaken for 1 h at r.t., then DIAD (50 µL, 96 µmol) was added, and the suspension was shaken for 3 h at r.t. The resin was washed with toluene (3 × 3.0 mL), DMF (3 × 3.0 mL), CH₂Cl₂ (3 × 3.0 mL), and MeOH (3 × 3.0 mL), and dried in vacuo to give the resin 6.
To the resin 6 in toluene (1.0 mL), was added MeNHNH₂ (3.8 µL, 72 µmol). The suspension was shaken for 15 min at r.t., then the resin was washed with toluene (3 × 3.0 mL), DMF (3 × 3.0 mL), CH₂Cl₂ (3 × 3.0 mL), and MeOH (3 × 3.0 mL), and dried in vacuo to give a resin 8. This step was repeated twice to complete the deprotection.
To the resin 8 in CH₂Cl₂ (1.0 mL), was added a solution of 2,3,4-trihydroxybenzaldehyde (9c; 37 mg, 239 µmol) and p-anisidine (12 mg, 100 µmol). The suspension was shaken for 17 h at r.t., then the resin was washed with CH₂Cl₂ (3 × 3.0 mL), DMF (3 × 3.0 mL), CH₂Cl₂ (3 × 3.0 mL), and MeOH (3 × 3.0 mL), and dried in vacuo to give a resin 10c.
The resin 10c was treated with 1% TFA in CH₂Cl₂ (1.0 mL) for 30 min. The TFA solution was collected, the resin was washed with CH₂Cl₂ (3 × 3.0 mL), the washes were collected, and the combined solutions were dried in vacuo to give 11c (6.7 mg, 33% from 5).
Analytical Data of 11c: [α]_D ^²4 +6.4 (c 0.3, CHCl₃); ^¹H NMR (500 MHz, CDCl₃): δ = 8.07 (s, 1 H), 6.62 (d, J = 8.6 Hz, 1 H), 6.50 (d, J = 8.6 Hz, 1 H), 5.30-5.20 (m, 1 H), 4.45-4.10 (m, 8 H), 2.40-2.20 (m, 4 H), 1.70-1.50 (m, 4 H), 1.40-1.10 (m, 48 H), 0.88 (t, J = 6.9 Hz, 6 H); ^¹³C NMR (100 MHz, CDCl₃): δ = 173.7, 173.4, 152.2, 147.0, 145.2, 131.4, 122.5, 109.3, 107.8, 72.8, 69.5, 66.6, 65.1, 61.9, 34.1, 34.0, 31.9, 29.70, 29.66, 29.5, 29.4, 29.3, 29.12, 29.07, 24.8, 22.7, 14.1; IR (neat): 3460, 2918, 2851, 1740 cm^-¹; MS-FAB:
m/z = 843 [M - H]^-; HRMS-FAB: m/z [M - H]^- calcd for C₄₄H₇₇O₁₂NP: 842.5184; found: 842.5167.

13

Experimental Procedure for 11b: To Merrifield resin (3.6 g, 1.1 mmol/g, 4.0 mmol) in DMF (30 mL), were added 12 (2.0 g, 12 mmol), Cs₂CO₃ (3.8 g, 12 mmol), and NaI (588 mg, 3.9 mmol), and the mixture was stirred for 40 h at r.t. The resin was washed with DMF (3 × 15 mL), H₂O (3 × 15 mL), DMF (3 × 15 mL), CH₂Cl₂ (3 × 15 mL), DMF (3 × 15 mL), EtOAc (3 × 15 mL), CH₂Cl₂ (3 × 15 mL), and MeOH (3 × 15 mL), and then dried in vacuo to give resin 13 (4.1 g, 4.0 mmol, quant.).
The resin 13 (109 mg, 1.1 mmol/g, 120 µmol) placed in a syringe reactor was solvated with CH₂Cl₂, and then CH₂Cl₂ was replaced with toluene. To the resin 13 in toluene (1.0 mL), were added Ph₃P (25 mg, 96 µmol) and 5 (20 mg, 24 µmol). The suspension was shaken for 1 h then, DIAD (50 µL, 96 µmol) was added and the suspension was shaken for 3 h at r.t. The resin was washed with toluene (3 × 3.0 mL), DMF (3 × 3.0 mL), CH₂Cl₂ (3 × 3.0 mL), and MeOH (3 × 3.0 mL), and then dried in vacuo to give resin 14.
To the resin 14 in toluene (1.0 mL), was added MeNHNH₂ (3.8 µL, 72 µmol). The mixture was shaken for 15 min, then the resin was washed with toluene (3 × 3.0 mL), DMF (3 × 3.0 mL), CH₂Cl₂ (3 × 3.0 mL), and MeOH (3 × 3.0 mL), and dried in vacuo to give resin 15. This step was repeated twice to complete the deprotection.
To the resin 15 in CH₂Cl₂ (1.0 mL), was added p-bromo-benzaldehyde (9b; 31 mg, 239 µmol) and p-anisidine (12 mg, 100 µmol). The suspension was shaken for 21 h at r.t., then the resin was washed with CH₂Cl₂ (3 × 3.0 mL), DMF (3 × 3.0 mL), CH₂Cl₂ (3 × 3.0 mL), and MeOH (3 × 3.0 mL), and dried in vacuo to give the resin 16. To the resultant resin 16 in CH₂Cl₂ (1.0 mL), was added mCPBA (29 mg, 167 µmol) and the mixture was shaken for 1 h at r.t., and then washed with CH₂Cl₂ (3 × 3.0 mL), DMF (3 × 3.0 mL), CH₂Cl₂ (3 × 3.0 mL), and MeOH (3 × 3.0 mL), and dried in vacuo to give the sulfone resin 17. After treatment of resin 17 with a solution of Et₃N in CH₂Cl₂ (7%, 1.0 mL) for 16 h, the filtrate was collected and dried in vacuo to give 11b as a Et₃N salt (11 mg). For desalting and purification, TFA (10 µL) was added to a solution of 11b˙Et₃N salt in CHCl₃-MeOH (4:1, 0.5 mL) then the solution was dried in vacuo to give a residue, which was purified by preparative TLC on silica gel (CHCl₃-MeOH-TFA, 80:20:0.1) to give 11b (4.7 mg, 22% from 5).