A novel function for transglutaminase 1: Attachment of long-chain omega -hydroxyceramides to involucrin by ester bond formation -- Nemes et al. 96 (15): 8402 Data Supplement - Supplemental Materials and Methods -- Proceedings of the National Academy of Sciences

Supplementary material for Nemes et al. (1999) Proc. Natl. Acad. Sci. USA 96(15), 8402-8407.

Synthesis of Lipid Z

16-Hydroxyhexadecanoic acid [5 g (18 mmol); Aldrich] was added to a mixture of 0.75 ml of 85% phosphoric acid, 2.525 g of 100% phosphoric acid, and 6.1 g of KI in a pressure tube, sealed, and incubated overnight at 120°C. The reaction mixture was taken up in chloroform; washed with 0.1 M sodium thiosulfate, 0.1 M HCl, and water; and dried with Na₂SO₄. After the evaporation of the solvent, the resulting oil was taken up in 15 ml of chloroform/methanol (95:5) and chromatographed on 150 g of Kieselgel 60 (Merck) by washing with 250 ml and eluting with 300 ml of the same solvent. After evaporation of the solvent, 2.1 g of 16-iodohexadecanoic acid was obtained (31%).

1,16-Hexadecanediol [1 g (3.9 mmol); Aldrich] was fully converted to its sodium dialcoholate by adding 200 mg of NaH in 20 ml of dry tetrahydrofuran.

16-Iodohexadecanoic acid [1.48 g (3.9 mmol)] was added to this stirred slurry under argon and refluxed for 6 h. The solvent was evaporated, and the product was dissolved in a warm mixture of chloroform and 1 M HCl, washed with water until neutral, and dried with Na₂SO₄. This mixture of the initial organic reagents, 16,16¢-[1,16-hexadecanediylbis(oxy)]bis[hexadecanoic acid] and the product 16-(16-hydroxyhexadecanoyl)oxyhexadecanoic acid, was separated by chromatography on 100 g of Kieselgel 60 in chloroform/methanol (95:5) (R_f » 0.4). After evaporation of the solvent, 540 mg of 16-(16-hydroxyhexadecanoxyhexadecanoic acid was obtained (55%), which was then acetylated, coupled to sphingosine base (Calbiochem), and saponified as described (44). The resulting lipid Z (yield, 61%) was purified by preparative Kieselgel 60 TLC developed with chloroform/acetone/methanol/acetic acid/water (10:4:3:2:1) (R_f = 0.65).

Chemical Analysis

16-Iodohexadecanoic Acid. mp: 87-88°C (from petroleum ether). ¹H NMR: (Bruker 250-MHz, CDCl₃) d 1.2-1.40 and 1.45-1.7 (m, 24H + 6H, -CH₂-), 2.4 (t, 2H, 8 Hz, -CH₂-CO-), 3.17 (t, 2H, J = 7 Hz, -CH₂-I). MS [fast atom bombardment (FAB), mb]: M+H⁺ = 383.3 m/z. Analysis: Calc. for C₁₆H₃₁IO₂ (382.32): C, 50.27; H, 8.17; I, 33.19. Found: C, 50.29; H, 8.12; I, 33.4.

16-(16-Hydroxyhexadecyl)oxyhexadecanoic Acid. mp: 91°C (from petroleum ether). ¹H NMR: MHz, CDCl₃) d 1.2-1.4 and 1.45-1.7 (m, 44 + 10H, -CH₂-), 2.25 (t, 2H, J = 7.8 Hz, -COCH₂-), 3.34 (t, 4H, J = 6.9 Hz, -CH₂-O-CH₂-), 3.56 (t, 2H, J = 7 Hz, -CH₂-OH). MS (FAB, mb): M+H⁺ = 513.8 m/z. Analysis: Calc. for C₃₂H₆₄O₄ (512.85): C, 0.75; H, 12.5. Found: C, 74.74; H, 12.45.

N-[16-(16-Hydroxyhexadecyl)oxyhexandecanoyl]-D-sphingosine. mp: 96-97°C (from petroleum ether). ¹H NMR (250 MHz, CDCl₃) d 0.88 (t, 3H, Me), 1.0-1.4 and 1.45-1.7 (m, 68H + 12H, -CH₂-), 2.05 (»q, 2H, J_5,6 = J_6,7 = »7 Hz, -CH = CH-CH₂-), 2.24 (t, 2H, J = 7.8 Hz, -COCH₂-), 3.34 (t, 4H, J = 6.9 Hz, -CH₂-O-CH₂-), 3.5-3.7 (m, 5H, H-1, and CH₂OH), 3.75-3.9 (m, 2H, H-1¢, 2), 4.3 (»t, 1H, J_2,2 » 5, J_3,4 = 6.6 Hz, H-3), 5.55 (»dd, 1H, J_4,5 = 15.5 Hz, H-4), 5.84 (m, 1H, J_5,6(6) » 7 Hz, H-5). IR: (Perkin--Elmer IR 1,600, KBr) n 3,600-3,100 (OH, NH), 1,650 and 1,550 (amide), and 1,240 cm^-1 (ether). High-resolution FAB MS: 794.3464 m/z (calc. for C₅₀H₉₉NO₅: 794.3459). Analysis: Calc. for C₅₀H₉₉NO₅: C, 75.60; H, 12.56; N, 1.76. Found: C, 75.37; H, 12.51; N, 1.74.