Supporting Information

Transcription

1 Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry. This journal is The Royal Society of Chemistry Supporting Information Convenient synthesis and application of versatile nucleic acid lipid membrane anchors in the assembly and fusion of liposomes Oliver Ries, Philipp M. G. Löffler, and Stefan Vogel University of Southern Denmark, Department of Physics, Chemistry and Pharmacy, Campusvej, Odense M, Denmark - -

2 Table of Content Characterization of oligonucleotides... Zeta Potential and DLS Experiments... Syntheses of compounds S-, a-c and -a-c... H, C, P NMR spectra of compounds S-S, a-c and -a-c... References

3 Characterization of oligonucleotides ON# Sequence ( -) R t [min] a) mass (calc.) mass (found) TX TGT GGA AGA AGT TGG TG. (A).. CAC CAA CTT CTT CCA CA XT. (B).9. TX CAC CAA CTT CTT CCA CA. (C) TGA AGA AGG TGT X TGT GGA AGA AGT. (A).. ACT TCT TCC ACA X ACA CCT TCT TCA. (A). 9. TX TGT GGA AGA AGT TGG TG XT. (C). 9. TX CAC CAA CTT CTT CCA CA XT 9. (C).9. CAC CAA CTT CTT CCA CA YT. (B) TY TGT GGA AGA AGT TGG TG. (B).. CAC CAA CTT CTT CCA CA ZT. (B).. TZ TGT GGA AGA AGT TGG TG. (B)..9 a) HPLC/UPLC method (flow, time [min], solvent gradient.m TEAA, ph. / ACN/H O :). Method A (UPLC): ml/min;, 9:;, :;, :. Method B (HPLC): ml/min;, 9:;, :;, :, 9, :. Method C (HPLC):.mL/min;, :;, :;, :;, : - -

4 Zeta Potential and DLS Experiments For zeta potential measurements POPC liposomes (mm, nm) were incubated for at least min with an appropriate amount of ON- in a total volume of µl HEPES buffer ([HEPES] mm, [Na + ] mm, [Cl - ] mm. Zeta potential was measured at C with a Beckman Coulter DelsaMax Pro equipped with a flowcell. Figure S: Zeta potential of POPC liposomes (nm) incubated with different oligonucleotide concentrations - -

5 For the experiment two liposome populations, A and B, either consisting of. mm DOPC/DOPE/cholesterol (::, molar ratio) and. µm ON- or. mm DOPC/DOPE/cholesterol (::, molar ratio) and. µm ON- were mixed and measured in DLS for h. Liposomes and lipid-dna conjugates were incubated prior to mixing for min at room temperature. The hydrodynamic diameters for populations A and B were. ±. nm and. ±. nm, respectively. Figure S: Hydrodynamic diameter over time for a fusion experiment - -

6 Syntheses of compounds S- and -a-c Cholesterylic acid (S) - H. Mg, EtBr, Et O. CO, Et O H Cl H H HO C Ethylbromide (. ml,.9 mmol,. eq) was added dropwise to magnesium turnings (. g, 9. mmol,. eq) in dry diethylether ( ml) under nitrogen atmosphere at room temperature. When gas formation started a solution of cholesteryl chloride (. g, 9. mmol,. eq) in dry diethylether ( ml) was added slowly over h and the reaction mixture was kept at refluxing. Stirring was continued for h under reflux. The reaction mixture was cooled in an ice-bath and a stream of CO was led through the solution for h at C. Sulfuric acid (%, ml) was added slowly and the aqueous layer was extracted with diethylether (x ml). The combined organic layers were washed with brine (x ml) and water (x ml) and dried over sodium sulfate. The solvent was removed under reduced pressure and the crude product was recrystallized from toluene to yield. g (. mmol, %) as a pale yellow solid. R f. (CH Cl /methanol 9:); H NMR ( MHz, CDCl ): δ. (s, H, -H ),. (d, H, J =., -H ),. (d, H, J =., -H ),.9 (d, H, J =., -H ),.9-. (m, 9H, chol-ch, chol-ch),. (s, H, 9-H ),.-. (m, H, chol-ch, chol-ch),.-. (m, H, -H); C NMR ( MHz, CDCl ): δ. (C),. (C), 9. (C9),. (chol-ch ),. (C),.9 (C),.,.,. (chol-ch ),. (chol-ch),.9 (chol-ch ),.9 (chol-ch),. (chol-ch ),. (chol-ch ),.9 (chol-ch),. (chol-ch ),. (chol-c),., 9., 9.9 (chol-ch ),. (chol-c),.,.,.,.9 (chol-ch),. (C),. (C-),. (-CO H); HRMS (ESI): calcd. for C H NaO m/z.9 [M+Na] +, found.. H S H (R,R)-Phytenic acid (S), HO. IBX, DCE. NaClO, sulfamic acid NaH PO, t BuOH, H O HO C A suspension of phytol (. ml,. mmol,. eq) and IBX (. g,. mmol,. eq) in,- dichloroethane ( ml) was stirred under reflux for. h and afterwards cooled in an ice-bath. The suspension was filtrated and the solvent was removed under reduced pressure. The residue was dissolved in a mixture of tert-butanol ( ml) and water ( ml) and sodium dihydrogen phosphate dihydrate (. g,. mmol,. eq), sulfamic acid ( mg,. mmol,. eq) and sodium chlorite ( mg,. mmol,. eq) were added. The reaction mixture was stirred for h at room temperature and diethylether ( ml) was added. The organic layer was extracted with water (x ml) and brine (x ml) and dried over magnesium sulfate. The solvent was removed under reduced pressure to yield. g (. mmol, 9 %) of a yellowish oil as mixture of two diastereomers and used without without further purification. - - S

7 R f.,. (petroleum ether/ethyl acetate :); H NMR ( MHz, CDCl ): δ.-.9 (m, H, x -CH, x -CH, x -H ),.-. (m, H, x -H, x -H, x -H, x -H --H, x - H, x -H --H, x -H),.9 (s, H, x -CH ),. (t, H, J =., x -H ),. (s, H, x - CH ),. (t, H, J =.9, x -H ),. (s, H, x -H),.9 (s, H, x -H); C NMR ( MHz, CDCl ): δ 9. (-CH ), 9., 9., 9., 9., 9., 9. (-CH, -CH, -CH ),.,. (phyt-ch ),.,.,. (phyt-ch ),. (-CH ),.99 (phyt-ch),.,.9,.,.,.,.9 (phyt-ch),. (x C),.,.,.,.9,.,., 9. (phyt-ch ),. (C),.,. (C),.,. (C),.,. (C); HRMS (ESI): calcd. for C H 9 O m/z.9 [M+H] +, found.9. (R,R)-Phytanic acid (S) HO C Pd/C (%), H, MeOH HO C S A solution of S (. g,. mmol,. eq) and Pd/C (%,. g,. mmol,. eq) in methanol ( ml) was evaporated x until boiling of the solvent and flushed with N. Afterwards the suspension was x evaporated and flushed with H. The reaction mixture was stirred under an atmosphere of H for h (TLC control). The mixture was dried over magnesium sulfate and filtered through a pad of celite (methanol). Solvent was removed under reduced pressure and the crude product was purified by column chromatography (SiO, petroleum ether/ethyl acetate :, % acetic acid) to yield. g (. mmol, %) of a clear oil as a mixture of two diastereomers. R f. (petroleum ether/ethyl acetate :); H NMR ( MHz, CDCl ): δ. (d, H, J =., x -CH, x -CH ),. (d, H, J =., x -H ),.9 (d, H, J =., x -CH ),.-. (m, H, x -H --H, x -H, x -H --H, x -H, x -H --H ),. (qq, H, J =., x - H),.9-. (m, H, x -H),. (dd, H, J =.9,.9, x -H a ),. (dd, H, J =.9,.9, x - H a ),. (dd, H, J =.9,., x -H b ),. (dd, H, J =.9,., x -H b ); C NMR ( MHz, CDCl ): δ 9., 9., 9., 9.9, 9.9 (-CH, -CH, -CH ),.9,. (C),.,.,.,.9,.9 (phyt-ch ),. (C),.,. (C),.9,.9,.9 (phyt-ch),.,.,.,.,.,.,.,.,.,.,., 9. (phyt-ch ),.,. (C), 9.9 (C); HRMS (ESI): calcd. for C H 9 O m/z.9 [M-H] -, found.9. S GENERAL PROCEDURE FOR ACYLATION (A-C, A-C). Cholesterylic acid and dihydrophytenic acid have been activated with thionyl chloride prior to the reaction. A solution of the acid in toluene was treated with thionyl chloride (. eq) and stirred under reflux for h. The solvent was removed under reduced pressure and the residue was used without further purification. The amine (. eq) was dissolved in a mixture of THF/water (:) and magnesium oxide (MgO,. eq) was added. The suspension was cooled to C and a solution of the acid chloride (. eq) in THF was added dropwise. Stirring was continued at C under TLC control. Except for a and a the reaction mixture was filtered through a short pad of celite and washed with hot THF. The solvent was removed under reduced pressure and the residue was purified by column chromatography (SiO ). - -

8 (R)-N-Palmitoyl--amino-,-propanediol (a). g (. mmol) (R)--amino-,-propanediol,. g (. mol) MgO in ml THF/water and. ml (. mmol) palmitoyl chloride in ml THF have been used. Reaction time: h. Celite ( g) and methanol ( ml) were added to the reaction mixture and the solvent was removed under reduced pressure. The residue was purified by dry column chromatography (SiO, CH Cl /methanol 9:). Yield:. g (. mmol, %) as white powder. R f. (CH Cl /methanol 9:); H NMR ( MHz, CDCl ): δ.9 (t, H, J =., palm--h ),.-.9 (m, H, palm--h --H ),.-. (m, H, palm--h ),. (t, H, J =., palm--h ),. (dd, H, J =.,., -H a ),. (dd, H, J =.,., -H b ),.-. (m, H, -H ),.-. (m, H, -H); C NMR ( MHz, CDCl ): δ. (palm-c),.,.,.,.,.,.,.9,.,. (palm-c-c),. (palm-c),.9 (palm-c),. (C-),. (C-),. (C-),.9 (palm-co); HRMS (ESI) calcd. for C 9 H 9 NNaO m/z. [M+Na] +, found.. (R)-N-(Cholest-'-en-'-carbonyl)--amino-,-propanediol (b) Activation:. ml (. mmol) thionyl chloride,. g (. mmol) S in ml toluene. mg (. mmol) (R)--amino-,-propanediol,. g (. mmol) MgO in ml THF/water and crude cholesteroyl chloride in ml THF have been used. Reaction time: h. Column chromatography: CH Cl /methanol 9:. Yield:.9 g (. mmol, %) as pale yellow solid. R f. (CH Cl /methanol 9:); H NMR ( MHz, CDCl ): δ. (s, H, chol--h ),. (d, H, J =., chol--h ),. (d, H, J =., chol--h ),.9-. (m, H, chol-ch, chol-ch, chol- CH a ),.9 (d, H, J =., chol--h ),. (s, H, chol-9-h ),. (t, H, J =., chol-ch b ),.-. (m, H, -H ),.-. (m, H, -H ),.9-.9 (m, H, -H),.9 (bs, H, OH),.-. (m, H, chol--h),. (t, H, J =., NH); C NMR ( MHz, CDCl ): δ. (chol-c),.9 (chol-c), 9.9 (chol-c9),. (chol-ch ),. (chol-c),.9 (chol-c),.,.,. (chol-ch ),. (chol-ch),.9 (chol-ch ),.9 (chol-ch),. (chol-ch ),. (chol-ch),.9,. (chol-ch ),. (chol-c),.9, 9., 9.9 (chol-ch ),.9 (C),. (chol-c),.,.,.,.9 (chol-ch),. (C-),. (C),. (chol-c),. (chol-c),.9 (chol-co); HRMS (ESI): calcd. for C H NNaO m/z.9 [M+Na] +, found.9. (R)-N-Phytanoyl--amino-,-propanediol (c) Activation:. ml (. mmol) thionyl chloride,. g (. mmol) S in ml toluene. mg (. mmol) (R)--amino-,-propanediol, mg (. mmol) MgO in ml THF/water and crude phytanoyl chloride in ml THF have been used. Reaction time: h. Column chromatography: CH Cl /methanol 9: 9:. Yield:. g (. mmol, 9 %). R f. (CH Cl /methanol 9:); H NMR ( MHz, CDCl ): δ. (d, H, J =., phyt--ch, phyt- -CH ),. (d, H, J =., x phyt--h ),.9 (d, H, J =., phyt--ch ),.9-. (m, H, phyt--h --H, phyt--h, phyt--h --H, phyt--h, phyt--h --H ),. (qqd, H, J =.,.,., phyt--h),.-. (m, H, phyt--h a, phyt--h),.-.9 (m, H, phyt--h b ),.-. (m, H, -H ),.9-. (m, H, -H ),.-. (m, H, -H),. (t, H, J =., NH); C NMR ( MHz, CDCl ): δ 9., 9., 9., 9., 9. (phyt--ch, phyt-'-ch ),.,. (phyt-c),.,.9,.,.9,.,.,.,.9,.,.,.9,., 9. (phyt-c-c'),.9 (phyt-c),. (C-),.,. (phyt-c),. (C-),.9 (C-),. (phyt-co); HRMS (ESI): calcd. for C H NNaO m/z. [M+Na] +, found.. - -

9 (R)-N-Hexadecyl-N-palmitoyl--amino-,-propanediol (a). g (. mmol) a,. g (. mmol) MgO in ml THF/water and. ml (. mmol) palmitoyl chloride in ml THF have been used. Reaction time: h. Dry column chromatography: CH Cl /methanol 9:. Yield:. g (. mmol, 9 %) as white solid. R f. (ethyl acetate); H NMR ( MHz, CDCl ): δ. (t, H, J =., x palm--h ),.9-. (m, H,x palm--h, x palm--h --H ),.-. (m, H, x palm--h, x palm--h ),. (t, H, J =., x palm--h ),.-. (m, H, x palm--h ),. (dd, H, J =.,., -H a ),. (dd, H, J =.9,., -H a ),. (dd, H, J =.,., -H b ),. (dd, H, J =.9,., -H b ),.-. (m, H, -H); C NMR ( MHz, CDCl ): δ. (palm-c),.,.9, 9., 9., 9., 9., 9., 9.9, 9., 9.,.,. (palm-c-c),., 9. (palm-c),. (palm-c), 9. (C),. (palm-ch N),. (C),. (C),. (palm-co); HRMS (ESI): calcd. for C H NNaO m/z. [M+Na] +, found.. (R)-N-(Cholest--en--carbonyl)-N-(cholest--en--meth-yl)--amino-,-propanediol (b) Activation:. ml (. mmol) thionyl chloride,. g (. mmol) S in ml toluene.. g (. mmol) b, mg (. mmol) MgO in ml THF/water and crude cholesteroyl chloride in ml THF have been used. Reaction time: h. Column chromatography: CH Cl /methanol 9:. Yield:. g (. mmol, %) as pale yellow solid. R f. (CH Cl /methanol 9:); H NMR ( MHz, CDCl ): δ. (s, H, x chol--ch ),. (d, H, J =., x chol--h ),. (d, H, J =., x chol--h ),.9 (d, H, J =., x chol--h ),.9-. (m, H, chol-ch, chol-ch),.9 (s, H, x chol-9-h ),. (s, H, x chol-9-h ),.-. (m, H, chol-ch ),.-. (m, H, chol-ch),.-. (m, H, chol-ch),. (dd, H, J =.,., chol-ch a N),. (dd, H, J =.,., chol-ch b N),.-. (m, H, -H, -H ),.-.9 (m, H, -H),.-. (m, H, x chol--h); C NMR ( MHz, CDCl ): δ. (chol- C),. (chol-c), 9., 9. (chol-c9),.,. (chol-ch ),. (chol-c),.9 (chol-c),.99,.,.,.9 (chol-ch ),. (chol-ch),. (chol-ch ),.9 (chol- CH),.9,.,.9 (chol-ch ),. (chol-ch),. (chol-ch ),. (chol-c),.,. (chol-ch ),.9 (chol-c), 9., 9., 9., 9.9, 9.9 (chol-ch ), 9. (C),.,. (chol-ch),. (chol-nch ),.,.,.9,. (chol-ch),.9 (C),. (C),.,. (chol-c),.,.9 (chol-c),. (chol-co); HRMS (ESI): calcd. for C 9 H NO m/z.9 [M+H] +, found.. (R)-N-Phytanyl-N-phytanoyl--amino-,-propanediol (c) Activation:. ml (. mmol) thionyl chloride, mg (. mmol) S in ml toluene. mg (. mmol) c, mg (. mmol) MgO in ml THF/water and crude phytanoyl chloride in ml THF were used. Reaction time: h. Column chromatography: CH Cl /methanol 9:. Yield: mg (. mmol, %) as pale yellow oil. R f. (CH Cl /methanol 9:); H NMR ( MHz, CDCl ): δ.-. (m, H, x phyt--ch, x phyt--ch, x phyt--ch ),.9-.9 (m, H, x phyt--ch ),.99-. (m, H, x phyt--h, x phyt--h, x phyt--h --H, x phyt--h, x phyt--h --H, x phyt--h, x phyt--h -- H ),. (qqd, H, J =.,.,., x phyt--h),.9-. (m, H, x phyt--h),.-. (m, H, x phyt--h a ),.-. (m, H, x phyt--h b ),.-. (m, H, -H, -H, phyt-ch N),.-. (m, H, -H); C NMR ( MHz, CDCl ): δ 9., 9., 9.9, 9., 9., 9.9, 9.9 (phyt-c-ch, phyt-c-ch ),.,. (phyt-c-ch ),.,.,.,.,.9,.9,.,.,.,.9,.,.,., 9.,.,. (phyt-c-c),.9-9 -

10 (phyt-c),., 9. (C-, phyt-ch N),. (C),.9,.9 (C),. (phyt-co); HRMS (ESI): calcd. for C H NO m/z.9 [M+H] +, found.. GENERAL PROCEDURE FOR AMIDE REDUCTION (A-C) A suspension of the amide (. eq) in dry THF under nitrogen atmosphere was cooled to C and a solution of lithium aluminium hydride (M in THF,. eq) was added dropwise. The solution was stirred for min at C and subsequently heated under reflux for h. The reaction was diluted with THF (- ml) and cooled to C. Then % sodium hydroxide solution (- ml) was added very slowly and stirring was continued until the mixture looked cloudy. The suspension was filtered through a short pad of celite and washed with hot THF. The solvent was removed under reduced pressure and the residue was used without further purification. (R)-N-Hexadecyl--amino-,-propanediol (a). g (. mmol) a,. ml (. mmol) lithium aluminium hydride solution in ml THF were used. Yield:. g (. mmol, 9 %) as white solid. R f.9 (CH Cl /methanol 9:); H NMR ( MHz, CD OD): δ.9 (t, H, J =., palm--h ),.-. (m, H, palm--h --H ),.-. (m, H, palm--h ),. (dd, H, J =.,., - H a ),.9 (t, H, J =., palm--h ),. (dd, H, J =.,., -H b ),. (dd, H, J =.,., - H ),.-. (m, H, -H); C NMR ( MHz, CD OD): δ. (palm-c),.,.9,.,.9,.,.,.,.,.,.,.,.,.9 (palm-c-c),.9 (palm-c),.9 (palm-c),. (C),. (C),.9 (C); HRMS (ESI): calcd. for C 9 H NO m/z. [M+H] +, found.. (R)-N-(Cholest--en--methyl)--amino-,-propanediol (b) 9 mg (. mmol) b,. ml (. mmol) lithium aluminium hydride solution in ml THF were used. Yield: mg (. mmol, 99 %) as pale yellow solid. R f.9 (CH Cl /methanol 9:); H NMR ( MHz, CDCl ): δ. (s, H, chol--h ),. (d, H, J =., chol--h ),. (d, H, J =., chol--h ),.9 (d, H, J =., chol--h ),.9-. (m, H, chol-ch, chol-ch),.9 (s, H, chol-9-h ),.-. (m, H, chol-ch ),.-. (m, H, chol-ch N),. (dd, H, J =.,., -H a ),.9 (dd, H, J =.,., -H b ),. (dd, H, J =.,., -H a ),.-. (m, H, -H b ),.-. (m, H, -H),.-. (m, H, chol--h); C NMR ( MHz, CDCl ): δ. (chol-c),. (chol-c), 9. (chol-c9),. (chol-ch ),. (chol-c),.9 (chol-c),.99,.,.9 (chol-ch ),. (chol-ch),.9 (chol- CH ), 9. (chol-ch),. (chol-ch ),.,.9 (chol-ch),.,.,., 9., 9. (chol-ch ), 9. (chol-ch), 9.9 (chol-ch ),. (chol-c),. (chol-ch),. (chol-c),. (chol-ch),. (chol-ch N),.9 (chol-ch),. (C), 9. (C),. (chol-c),. (chol-c); HRMS (ESI): calcd. for C H NO m/z. [M+H] +, found.. (R)-N-Phytanyl--amino-,-propanediol (c) mg (. mmol) c,. ml (. mmol) lithium aluminium hydride solution in ml were used. Yield: mg (. mmol, 99 %) as clear oil. H NMR ( MHz, CDCl ): δ. (d, H, J =., phyt--ch, phyt--ch ),. (d, H, J =., phyt--h ),. (d, H, J =., phyt--ch ),.9-. (m, H, phyt--h, phyt--h, phyt--h --H, phyt--h, phyt--h --H, phyt--h, phyt-- H --H ),. (qqd, H, J =.,.,., phyt--h),.-. (m, H, -H a, phyt--h ),. (dd, H, J =.,., x -H b ),. (dd, H, J =.,., x -H b ),. (bs, H, OH, NH),. (dd, H, J - -

11 =.,.9, -H a ),. (dd, H, J =.,., -H b ),.-. (m, H, -H); C NMR ( MHz, CDCl ): δ., 9., 9. (phyt--ch, phyt--ch ),.,. (phyt-c),.,.,.,.,.9,.9,.,.,.,.,.,., 9. (phyt-c-c),.9 (phyt-c),.9,.9 (phyt-c),.,. (C),. (C), 9., 9. (C); HRMS (ESI): calcd. for C H NO m/z. [M+H] +, found.. GENERAL PROCEDURE FOR DMTR-PROTECTION (A-C) A solution of dimethoxytrityl chloride (DMTrCl,. eq) in dry,-dichloroethane (DCE) was added to a solution of the alcohol (. eq), -dimethylamino pyridine (DMAP,. eq) and triethylamine (TEA,. eq) in dry DCE at room temperature. The reaction mixture was stirred at C and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (SiO ). (R)-N-Hexadecyl-N-palmitoyl--amino--(dimethoxytriphenylmethyloxy)--propanol (a). g (. mmol) DMTr chloride in ml DCE and. g (. mmol) a, 9 mg (. mmol) DMAP and.99 ml (. mmol) TEA in ml DCE were used. Reaction time: h. Column chromatography: petroleum ether/ethyl acetate : :,.% TEA. Yield:. g (. mmol, %) as yellow oil. R f.9 (petroleum ether/ethyl acetate :); H NMR ( MHz, CDCl ): δ. (t, H, J =., x palm--h ),.9-. (m, H, x palm--h, x palm--h --H ),.9-. (m, H, x palm-- H, x palm--h ),. (t, H, J =., x palm--h ).9 (dd, H, J = 9.,., -H a ),. (t, H, J =., palm--h ),. (dd, H, J = 9.,., -H b ),. (dd, H, J =.,., -H a ),.9 (dd, H, J =.,., -H b ),.9 (s, H, DMTr-OCH ),.9-. (m, H, -H),. (bs, H, OH),.9-. (m, H, DMTr-CH),.-. (m, H, DMTr-CH),.-. (m, H, DMTr-CH); C NMR ( MHz, CDCl ): δ. (palm-c),.,., 9., 9., 9., 9., 9., 9., 9., 9., 9.,. (palm-c, palm-c-c),., 9. (palm-c, palm-c),. (palm-c),. (palm-c),. (C),. (DMTr-OCH ),.9 (C),. (C),. (DMTr-CAr ),.,.,.,.9,.9,.,. (DMTr-CH),.,.,.9,. (DMTr-C),. (palm-co); HRMS (ESI): calcd. for C H 9 NNaO m/z. [M+Na] +, found.9. (R)-N-(Cholest--en--carbonyl)-N-(cholest--en--methyl)--amino--(dimethoxytriphenylmethyloxy)--propanol (b) mg (. mmol) DMTr chloride in ml DCE and. g (. mmol) b, mg (. mmol) DMAP and. ml (. mmol) TEA in ml DCE were used. Reaction time: h. Column chromatography: petroleum ether/ethyl acetate :,.% TEA. Yield:.9 g (.9 mmol, %) as pale yellow foam. R f. (petroleum ether/ethyl acetate :); H NMR ( MHz, CDCl ): δ. (s, H, chol--h ),. (s, H, chol--h ),. (d, H, J =., x chol--h ),. (d, H, J =., x chol--h ),.9 (d, H, J =., x chol--h ),.9-. (m, H, chol-ch, chol-ch),.99 (s, H, x chol-9- H ),. (s, H, x chol-9-h ),.-. (m, H, chol-ch ),.-. (m, H, chol-ch),.-. (m, H, chol-ch),.9 (dd, H, J = 9.,., -H a ),. (dd, H, J =.,., palm-ch a N),. (dd, H, J =.,., palm-ch b N),. (dd, H, J = 9.,.9, -H b ),.-. (m, H, -H a ),. (dd, H, J =.,., -H b ),. (s, H, DMTr-OCH ),.9 (s, H, DMTr-OCH ),.9-. (m, H, -H),. (bs, H, OH),.-. (m, H, x chol--h),.-. (m, H, DMTr-CH),.-. (m, - -

12 H, DMTr-CH),.-. (m, H, DMTr-CH); C NMR ( MHz, CDCl ): δ.99,. (chol- C),. (chol-c), 9.9, 9. (chol-c9),.99,. (chol-ch),. (chol-c),.9 (chol-c),.9,. (chol-ch ),.9,.9 (chol-c),. (chol-ch),. (chol-ch ),.9,.9,.99,. (chol-ch, chol-ch),. (chol-ch ),.9 (chol-ch),.,. (chol-ch ),.,. (chol-c),.9 (chol-ch ), 9. (chol-ch), 9., 9., 9.9, 9.9 (chol-ch ),. (chol-ch),.,. (chol-c),.,. (chol-ch),. (C),. (DMTr-OCH ),. (chol-ch N),.,.,.9,.9 (chol-ch),. (chol-c),. (C),. (C),. (DMTr-CAr ),.,.,. (DMTr-CH),.,.9 (chol-c),.9,.9,.9,., 9.,. (DMTr-CH),.9,. (DMTr-C),. (chol- C),.9,.,. (DMTr-C), 9. (chol-co); HRMS (ESI): calcd. for C H NNaO m/z 9. [M+Na] +, found 9.. (R)-N-Phytanyl-N-phytanoyl--amino--(dimethoxytriphenylmethyloxy)--propanol (c) mg (. mmol) DMTr chloride in ml DCE and 9 mg (.9 mmol) c, mg (. mmol) DMAP and.9 ml (. mmol) TEA in ml DCE were used. Reaction time: h. Column chromatography: petroleum ether/ethyl acetate :,.% TEA). Yield: 9 mg (. mmol, %) as pale yellow oil. R f. (petroleum ether/ethyl acete :); H NMR ( MHz, CDCl ): δ.-.9 (m, H, phyt-- CH, phyt--ch, phyt--ch, phyt--h ),.9-. (m, H, x phyt--h a, x phyt--h, x phyt- -H --H, x phyt--h, x phyt--h --H, x phyt--h, x phyt--h --H ),.-. (m, H, x phyt--h b ),. (qqt, H, J =.,.,., x phyt--h),.-. (m, H, x phyt--h a, x phyt--h),.9-. (m, H, x phyt--h b ),.-.9 (m, H, -H a ),.9-. (m, H, -H b, x phyt- -H ),.-. (m, H, -H ),. (s, H, DMTr-OCH ),.9 (s, H, DMTr-OCH ),.9-. (m, H, -H),. (d,.h, J =., OH),. (d,.h, J =., OH),.-. (m, H, DMTr-CH),.-. (m, H, DMTr-CH),.-. (m, H, DMTr-CH); C NMR ( MHz, CDCl ): δ 9., 9., 9. (phyt--ch, phyt--ch, phyt--ch ),.,. (phyt-c),.,.,.9,.,.,.,.,.9, 9. (phyt-c, phyt-c, phyt-c-c),. (phyt-c),. (phyt-c),. (phyt-c),. (phyt-c),. (C),.,. (DMTr-OCH ),. (C),. (C),.,.9 (DMTr-CAr ),.,.,.9,.,.,.,.9,.,.,., 9.,. (DMTr-CH),., 9.,.,.,.,. (DMTr-C),. (phyt-co); HRMS (ESI): calcd. for C H NNaO m/z 99. [M+Na] +, found

13 H, C, P NMR spectra of compounds S- and -a-c.... H-NMR spectrum of S ( MHz, CDCl, C) C-NMR spectrum of S ( MHz, CDCl, C) - -

14 H-NMR spectrum of S ( MHz, CDCl, C) C-NMR spectrum of S ( MHz, CDCl, C) - -

15 H-NMR spectrum of S ( MHz, CDCl, C) C-NMR spectrum of S ( MHz, CDCl, C) - -

16 H-NMR spectrum of a ( MHz, CD OD, C) C-NMR spectrum of a ( MHz, CD OD, C) - -

17 H-NMR spectrum of b ( MHz, CDCl, C) C-NMR spectrum of b ( MHz, CDCl, C) - -

18 H-NMR spectrum of c ( MHz, CDCl, C) C-NMR spectrum of c ( MHz, CDCl, C) - -

19 H-NMR spectrum of a ( MHz, CD OD, C) C-NMR spectrum of a ( MHz, CD OD, C) - 9 -

20 H-NMR spectrum of b ( MHz, CDCl, C) C-NMR spectrum of b ( MHz, CDCl, C) - -

21 H-NMR spectrum of c ( MHz, CD OD CDCl, C) C-NMR spectrum of c ( MHz, CD OD CDCl, C) - -

22 H-NMR spectrum of a ( MHz, CDCl, C) C-NMR spectrum of a ( MHz, CDCl, C) - -

23 H-NMR spectrum of b ( MHz, CDCl, C) C-NMR spectrum of b ( MHz, CDCl, C) - -

24 H-NMR spectrum of c ( MHz, CDCl, C) C-NMR spectrum of c ( MHz, CDCl, C) - -

25 H-NMR spectrum of a ( MHz, CDCl, C) C-NMR spectrum of a ( MHz, CDCl, C) - -

26 H-NMR spectrum of b ( MHz, CDCl, C) C-NMR spectrum of b ( MHz, CDCl, C) - -

27 H-NMR spectrum of c ( MHz, CD OD CDCl, C) C-NMR spectrum of c ( MHz, CD OD CDCl, C) - -

28 H-NMR spectrum of a ( MHz, CDCl, C) C-NMR spectrum of a ( MHz, CDCl, C) - -

29 P-NMR spectrum of a ( MHz, CDCl, C) H-NMR spectrum of b ( MHz, CDCl, C) - 9 -

30 C-NMR spectrum of b ( MHz, CDCl, C) P-NMR spectrum of b ( MHz, CDCl, C) - - -

31 H-NMR spectrum of c ( MHz, CD OD CDCl, C) C-NMR spectrum of c ( MHz, CD OD CDCl, C) - -

32 P-NMR spectrum of c (MHz, CDCl, C) - - -

33 References E. J. Corey and R. A. Sneen, J. Am. Chem. Soc., 9,,. W. J. Gensler and G. M. Sherman, J. Org. Chem., 9,,. S. M. Harwood, K. J. Toyne, J. W. Goodby, M. Parsley and G. W. Gray, Liquid Crystals,,,. J. D. More and N. S. Finney, Org. Lett.,,,. Y. Ikeda and J. Yamashita, EP A,. - -