Lauric acid

PAs were synthesized using standard Fmoc solid-phase synthesis conditions. Coupling reactions were performed using Fmoc-amino acids (4 equiv), HBTU (3.95 equiv) and diispropylethylamine (DIEA) (6 equiv) in dimethylformamide (DMF). Synthesis of L-KLAK PA and D-KLAK PA was achieved using L-amino acids and D-amino acids, respectively. For the hydrophobic tail of PEG PA (sequence K(C₁₂)A₄G₃E₃-PEG), lauric acid was attached to the εamine of a lysine, which was deprotected by selective removal of the Mtt (Mtt = 4-methyltrityl) group using 4% trifluoroacetic acid (TFA) + 5% triisopropylsilane (TIPS) in CH₂Cl₂. CH₃O-PEG-COOH (MW 2000) was prepared as previously described⁴⁴ and attached on resin at the N-terminus of the PEG PA. The alkyl tail of the KLAK PA was formed by reacting the N-terminus with palmitic acid (4 equiv), HBTU (3.95 equiv) and DIEA (6 equiv) in DMF. Following cleavage using a TFA/TIPS/H₂O mixture (95:2.5:2.5), PAs were purified by high-performance liquid chromatography (HPLC).
Purification by preparative-scale HPLC was carried out on a Varian Prostar 210 HPLC system, eluting with 2% acetonitrile (ACN) to 100% ACN in water on a Phenomenex C18 Gemini NX column (150 × 30 mm) with 5 μm pore size and 110Å particle size. 0.1% NH4OH or 0.1% trifluoroacetic acid, for acidic or basic PAs, respectively, were added to both mobile phases to aid PA solubility. Product-containing fractions were confirmed by ESI mass spectrometry (Agilent 6510 Q-TOF LC/MS), combined, and lyophilized after removing ACN by rotary evaporation. Amino acid analyses were performed by Commonwealth Biotechnologies (Richmond, VA).
Co-assembly of the PAs was achieved by dissolving the KLAK PA and PEG PA separately in hexafluoroisopropanol (HFIP), an organic solvent known to disrupt hydrogen bonds,^{45 (link)–47 (link)} and then mixed together for at least 15 minutes. Samples were lyophilized to dryness to form a powder, as previously reported by our group.⁴⁸ After lyophilization in HFIP, samples were dissolved in water, aliquoted, and lyophilized again. A KLAK PA-AlexaFluor 700 conjugate was synthesized by reacting a 5 molar excess of KLAK PA with NHS-AlexaFluor 700 (Invitrogen). KLAK PA was dissolved in DMSO at a concentration of 0.5 mM with 1% triethylamine (TEA), and AlexaFluor 700 was added drop-wise to a stirring solution and reacted overnight (the reaction was confirmed using ESI mass spectrometry). The KLAK PA-AlexaFluor 700 conjugate was dialyzed in H₂O overnight to remove any unreacted dye and subsequently lyophilized. KLAK PA-AlexaFluor 700 and PEG PA mixtures were co-assembled in HFIP, as described above.
Specimens for conventional transmission electron microscopy (TEM) were prepared by drop-casting samples on carbon type B copper grids (Ted Pella) followed by staining with a 2% uranyl acetate aqueous solution. Cryogenic TEM (cryo-TEM) specimens were prepared using an FEI Vitrobot by blotting in 95% humidity and subsequently plunging grids into liquid ethane. Images were taken for both conventional and cryo-TEM using a JEOL 1230 transmission electron microscope operating at 100 keV equipped with a Gatan camera.
Small angle X-ray scattering (SAXS) experiments were performed at the Advanced Photon Source, Argonne National Laboratory. The X-ray energy (15 keV) was selected using a double-crystal monochromator, and the SAXS CCD camera was offset in order to achieve a wide range of scattering angles. Samples were dissolved at a concentration of 2 mM and placed in 1.5 mm quartz capillary tubes. The typical incident X-ray flux on the sample was ~1×10¹² photons/s with a 0.2×0.3 mm² collimator, estimated by a He ion channel, and samples were irradiated for 5 s. The 1D scattering profiles were obtained by radial integration of the 2D patterns, with scattering from the capillaries subtracted as background. Scattering profiles were then plotted on a relative scale as a function of the scattering vector q = (4π/λ) sin(θ/2), where θ is the scattering angle.
1H-Diffusion Ordered Spectroscopy (DOSY) was performed using a Bruker Avance 600 MHz spectrometer at ambient temperature. For these experiments samples were dissolved at a constant total concentration of PA (5 mM KLAK PA alone or 2.5 mM KLAK PA and 2.5 mM PEG PA in the mixed case) in 99.9% D₂O (Sigma), and 32 points were measured with a 7 μs 90 degree pulse. Diffusion data were processed and analyzed using DOSY Toolbox.^{49 (link)}