Synlett 2017; 28(01): 108-112
DOI: 10.1055/s-0036-1588634
cluster
© Georg Thieme Verlag Stuttgart · New York

Spontaneous Phospholipid Membrane Formation by Histidine Ligation

Roberto J. Brea ‡, Ahanjit Bhattacharya ‡, Neal K. Devaraj*
  • Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, Building: Urey Hall, Room: 4120, La Jolla, CA 92093, USA   Email: ndevaraj@ucsd.edu
Further Information

Publication History

Received: 02 August 2016

Accepted after revision: 29 September 2016

Publication Date:
19 October 2016 (eFirst)

These authors contributed equally

Abstract

A major challenge for the construction of artificial lipid membranes is the development of simple and robust methods for mimicking natural phospholipid membrane generation. Here we describe a nonenzymatic and chemoselective approach that relies on histidine ligation to form phospholipids de novo from water-soluble amphiphilic precursors. The resulting phospholipids can spontaneously self-assemble into micron-sized vesicles and encapsulate biomacromolecules.

Supporting Information

 
  • References and Notes

  • 1 Holthuis JC, Menon AK. Nature (London, U.K.) 2014; 510: 48
  • 2 Brea RJ, Hardy MD, Devaraj NK. Chem. Eur. J. 2015; 21: 12564
  • 3 Jesorka A, Orwar O. Annu. Rev. Anal. Chem. 2008; 1: 801
    • 4a Hardy MD, Yang J, Selimkhanov J, Cole CM, Tsimring LS, Devaraj NK. Proc. Natl. Acad. Sci. U.S.A. 2015; 112: 8187
    • 4b Zhou CY, Wu H, Devaraj NK. Chem. Sci. 2015; 6: 4365
    • 4c Budin I, Devaraj NK. J. Am. Chem. Soc. 2012; 134: 751
    • 5a Brea RJ, Rudd AK, Devaraj NK. Proc. Natl. Acad. Sci. U.S.A. 2016; 113: 8589
    • 5b Cole CM, Brea RJ, Kim YH, Hardy MD, Yang J, Devaraj NK. Angew. Chem. Int. Ed. 2015; 54: 12738
    • 5c Brea RJ, Cole CM, Devaraj NK. Angew. Chem. Int. Ed. 2014; 53: 14102
  • 6 Zhang L, Tam JP. Tetrahedron Lett. 1997; 38: 3
  • 7 Tam JP, Xu J, Eom KD. Biopolymers 2001; 60: 194
  • 8 General Procedure for the Synthesis of Histidine-Functionalized Lysolipids: 1-Palmitoyl-2-(His)-sn-glycero-3-phosphocholine (1)A solution of 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (47.5 mg, 95.8 μmol), N-Boc-l-His(Trt)-OH (119.2 mg, 239.5 μmol), DMAP (70.2 mg, 574.8 µmol), and Et3N (46.7 μL, 335.3 μmol) in CDCl3 (7.5 mL) was stirred at r.t. for 10 min. Then, 2,4,6-trichlorobenzoyl chloride (TCBC, 97.3 μL, 622.7 μmol) was added. After 12 h stirring at r.t., H2O (250 μL) was added to quench the acid chloride, and the solvent was removed under reduced pressure to give a pale yellow solid. The corresponding residue was dissolved in MeOH (1 mL), filtered using a 0.2 μm syringe-driven filter, and the crude solution was purified by HPLC, affording 68.3 mg of 1-palmitoyl-2-[N-Boc-l-His(Trt)]-sn-glycero-3-phosphocholine (6) as a white foam [73%, t R = 10.9 min (Zorbax SB-C18 semipreparative column, 5% Phase A in Phase B, 15.5 min)]. 1H NMR (500.13 MHz, CDCl3): δ = 7.45–7.11 (m, 12 H, 12 × CHAr), 7.11–6.89 (m, 4 H, 4 × CHAr), 6.55 (s, 1 H, 1 × CHAr), 6.22 (m, 1 H, 1 × NH), 5.04–4.83 (m, 1 H, 1 × CH), 4.49–4.13 (m, 3 H, 1 × CH + 1 × CH2), 4.11–3.82 (m, 3 H, 1.5 × CH2), 3.80–3.62 (m, 1 H, 0.5 × CH2), 3.59–3.33 (m, 4 H, 2 × CH2), 3.03–2.79 (m, 2 H, 2 × CH2), 3.25 (s, 9 H, 3 × CH3), 2.23–1.78 (m, 2 H, 1 × CH2), 1.32 (s, 9 H, 3 × CH3), 1.25–0.94 (m, 24 H, 12 × CH2), 0.90–0.65 (m, 3 H, 1 × CH3). 13C NMR (125.77 MHz, CDCl3): δ = 173.2, 171.4, 155.9, 144.3, 138.9, 136.3, 128.3, 128.3, 128.2, 120.0, 79.5, 75.5, 72.3, 66.6, 64.0, 62.5, 59.5, 54.7, 53.9, 34.0, 32.1, 30.0, 29.9, 29.9, 29.8, 29.8, 29.7, 29.5, 29.4, 29.2, 28.6, 24.9, 22.8, 14.3. MS (ESI-TOF): m/z (%) = 976 (100) [MH]+. ESI-HRMS (TOF): m/z calcd for C54H80N4O10P [MH]+: 975.5607; found: 975.5603.A solution of 1-palmitoyl-2-[N-Boc-l-His(Trt)]-sn-glycero-3-phosphocholine (6, 15.0 mg, 15.4 μmol) in 2 mL of TFA/CH2Cl2/TES (0.9:0.9:0.2) was stirred at r.t. for 45 min. After removal of the solvent, the residue was dried under high vacuum for 3 h. Then, the corresponding residue was diluted in MeOH (250 μL), filtered using a 0.2 μm syringe-driven filter, and the crude solution was purified by HPLC, affording 7.0 mg of the lysolipid 1 as a colorless film [62%, t R = 8.0 min (Zorbax SB-C18 semipreparative column, 50% Phase A in Phase B, 5 min, and then 5% Phase A in Phase B, 10 min)]. 1H NMR (500.13 MHz, CD3OD): δ = 7.89–7.61 (m, 1 H, 1 × CHAr), 7.20–6.90 (m, 1 H, 1 × CHAr), 5.42–5.15 (m, 1 H, 1 × CH), 4.54–3.85 (m, 7 H, 3 × CH2 + 1 × CH), 3.71–3.51 (m, 2 H, 1 × CH2), 3.38–3.34 (m, 2 H, 1 × CH2), 3.23 (s, 9 H, 3 × CH3), 2.47–2.20 (m, 2 H, 1 × CH2), 1.71–1.48 (m, 2 H, 1 × CH2), 1.44–1.16 (m, 24 H, 12 × CH2), 0.90 (t, J = 6.6 Hz, 3 H, 1 × CH3). 13C NMR (125.77 MHz, CD3OD): δ = 174.9, 168.1, 136.9, 134.0, 117.2, 74.2, 67.8, 64.9, 63.1, 60.5, 54.7, 54.5, 34.9, 34.7, 33.1, 30.8, 30.8, 30.7, 30.7, 30.7, 30.6, 30.6, 30.5, 30.4, 30.2, 29.7, 29.6, 25.9, 23.7, 14.4. ESI-MS (TOF): m/z (%) = 633 (100) [MH]+. ESI-HRMS (TOF): m/z calcd for C30H58N4O8P [MH]+: 633.3987; found: 633.3986.

    • Sodium 2-mercaptoethanesulfonate (MESNA) was conveniently employed to synthesize the water-soluble long-chain acyl thioester 2. For references of MESNA, see:
    • 9a Mashiach E, Sela S, Weinstein T, Cohen HI, Shasha SM, Kristal B. Nephrol. Dial. Transplant. 2001; 16: 542
    • 9b Rise F, Undheim K. Acta Chem. Scand. 1989; 43: 489
  • 10 MESNA Oleoyl Thioester (2)A solution of oleic acid (189.2 mg, 670.0 μmol) in CH2Cl2 (5 mL) was stirred at 0 °C for 10 min, and then DMAP (7.4 mg, 60.9 μmol) and EDC·HCl (128.4 mg, 670.0 μmol) were successively added. After 10 min stirring at 0 °C, sodium 2-mercaptoethanesulfonate (MESNA, 100.0 mg, 609.1 μmol) was added. After 5 h stirring at r.t., the mixture was extracted with H2O (2 × 3 mL), and the combined aqueous phases were washed with EtOAc (3 mL). After evaporation of H2O under reduced pressure, the residue was washed with MeCN (5 mL), and then filtered to yield 194.7 mg of 2 as a white solid [75%]. 1H NMR (500.13 MHz, DMSO-d 6): δ = 5.36–5.27 (m, 2 H, 2 × CH), 3.05–2.99 (m, 2 H, 1 × CH2), 2.60–2.51 (m, 4 H, 2 × CH2), 2.02–1.92 (m, 4 H, 2 × CH2), 1.58–1.49 (m, 2 H, 1 × CH2), 1.34–1.18 (m, 20 H, 10 × CH2), 0.85 (t, J = 6.9 Hz, 3 H, 1 × CH3). 13C NMR (125.77 MHz, DMSO-d 6): δ = 198.7, 129.8, 129.7, 51.0, 43.4, 31.4, 29.2, 29.1, 28.9, 28.8, 28.7, 28.6, 28.5, 28.3, 26.7, 26.6, 25.1, 24.4, 22.2, 14.1. ESI-MS (TOF): m/z (%) = 429 (100) [MH]+. ESI-HRMS (TOF): m/z calcd for C20H38NaO4S2 [MH]+: 429.2104; found: 429.2105.
  • 11 Stafford RE, Fanni T, Dennis EA. Biochemistry 1989; 28: 5113
  • 12 General Procedure for the Synthesis of Histidine-Functionalized Phospholipids: 1-Palmitoyl-2-[His-(oleoyl)]-sn-glycero-3-phosphocholine (3)1-Palmitoyl-2-(His)-sn-glycero-3-phosphocholine (1, 2.00 mg, 3.16 μmol) and MESNA oleoyl thioester (2, 1.36 mg, 3.16 μmol) were dissolved in 633 μL of 100 mM HEPES buffer pH 7.5 and stirred under N2 at r.t. After 24 h, the corresponding mixture was filtered using a 0.2 μm syringe-driven filter, and the crude solution was purified by HPLC, affording 1.2 mg of the phospholipid 3 as a colorless film [43%, t R = 8.2 min (Zorbax SB-C18 semipreparative column, 100% Phase B, 15.5 min)]. ESI-MS (TOF): m/z (%) = 898 (100) [MH]+. ESI-HRMS (TOF): m/z calcd for C48H90N4O9P [MH]+: 897.6440; found. 897.6437.
  • 13 Monitoring the Progress of Phospholipid Formation12.5 μL (10 mM stock solution) of lysolipid 1 and 15 μL (10 mM stock solution) of thioester 2 were added to 22.5 μL of HEPES buffer (100 mM, pH 7.5) at r.t. Then, 7.5 μL of the reaction mixture were taken out, diluted with 92.5 μL MeOH and injected into HPLC at various time points (0, 2, 4, 8, 12, and 24 h). Method used: 50–95% Phase A in Phase B (8 min), and then 95% Phase A in Phase B (4 min). The progress of the reaction was monitored from the disappearance of peak corresponding to lysolipid 1 and appearance of peak corresponding to the phospholipid 3.
  • 14 Membrane Staining with Texas Red® DHPE5 μL of a 10 mM solution of phospholipid 3 in MeOH–CHCl3 (1:1) and 0.5 μL of a 0.1 mM solution of Texas Red® DHPE in EtOH were added to a glass vial, placed under a steady flow of N2, and dried for 10 min to prepare a lipid film. Then, 100 μL of H2O were added to the lipid film and briefly vortexed. The solution was tumbled at r.t. for 30 min. Afterwards, 5 μL of the vesicle solution were placed on a clean glass slide, secured by a greased cover slip and imaged on an Olympus BX51 microscope (DS Red Channel) to observe staining of membranes.
  • 15 Encapsulation of HPTS10 μL of a 10 mM solution of phospholipid 3 in MeOH–CHCl3 (1:1) were added to a glass vial, placed under a steady flow of N2, and dried for 10 min to prepare a lipid film. Then, 100 μL of 0.1 mM HPTS aqueous solution were added to the lipid film and briefly vortexed. The solution was tumbled at r.t. for 30 min. Afterward, the resulting cloudy solution was diluted with an additional 200 μL of H2O and transferred to a 100 kDa molecular weight cut-off (MWCO) centrifugal membrane filter and centrifuged for 3 min at 10,000 rcf (Eppendorf 5415C). The solution was similarly washed for additional 5× to remove any nonencapsulated dye. Then, 5 μL of the vesicle solution were placed on a clean glass slide, secured by a greased cover slip, and imaged on a spinning disc confocal microscope (488 nm laser) to observe encapsulation of HPTS.
  • 16 In situ Vesicle Formation12.5 μL (10 mM stock solution) of lysolipid 1 and 15 μL (10 mM stock solution) of thioester 2 were added to 22.5 μL of HEPES buffer (100 mM, pH 7.5) at r.t. Then, 5 μL of this solution were placed on a clean glass slide and secured by a greased cover slip. The solution was observed under optical microscope (Olympus BX51) using bright field with phase contrast objective (100X). Images of vesicle formation were taken at various time points.
  • 17 In situ Encapsulation of EGFP25.0 μL (10 mM stock solution) of lysolipid 1 and 30 μL (10 mM stock solution) of thioester 2 were added to 45.0 μL of HEPES buffer (100 mM, pH 7.5). Then, 2.0 μL of 220 μM solution of EGFP were added and the solution was kept at r.t. for 24 h. The resulting solution was diluted with an additional 200 μL of 45 mM HEPES buffer pH 7.5, transferred to a 100 kDa MWCO centrifugal membrane filter and centrifuged for 3 min at 10,000 rcf (Eppendorf 5415C). The solution was similarly washed for additional 4× to remove any nonencapsulated EGFP. Afterward, 5 μL of the vesicle solution were placed on a clean glass slide, secured by a greased cover slip and imaged on a spinning disc confocal microscope (488 nm laser) to observe encapsulation of EGFP in the in situ formed vesicles.