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Supplemental Information
Dimeric Cationic Amphiphilic Polyproline Helices for Mitochondrial Targeting
Iris M. Geisler and Jean Chmielewski*
Synthesis of Dimer Linker (C5) 2-5
Scheme S1. Synthesis of dimer linker (C5) 2-5
Compound 2-1. Pentanediol (6.0 g, 0.058 mol) and imidazole (8.29 g, 0.12 mol) were
added to a oven dried round bottom flask and dissolved in dry (DMF) (30 mL) under
vigorous stirring. The reaction mixture was placed in an ice bath to cool to 0 oC.
Tetrabutyldimethylsilane chloride (TBDMS-Cl) was placed in a separate dry round
bottom flask and dry DMF (20 mL) was added by canulation and the mixture was stirred
until all TBDMS-Cl was completely dissolved. The TBDMS-Cl solution was added dropwise over a period of 30 min via canula to the cooled pentanediol/imidazole reaction
mixture under a constant stream of N2. Kept under N2, the reaction was allowed to warm
to room temperature and stirred overnight. The next day, a saturated aqueous lithium
chloride (LiCl) solution (75 mL) was added to the reaction mixture and the mixture was
transferred to a separatory funnel. The mixture was extracted with ether and the organic
fractions were combined, washed additionally with saturated LiCl and dried over
anhydrous MgSO4. Solvent was removed in vacuo. The product was purified by silica gel
chromatography (8:1 Hex:EA to 4:1 Hex:EA) to yield the desired compound 2-1 as a
colorless, clear, slightly viscous liquid in 73% yield.
MW=525.79 ESI+MS= 527.33 m/z (M + H+)
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H NMR (300 MHz, CDCl3): δ 5.25 (s, 1H), 3.61 (m, 4H), 1.55 (m, 4H), 1.41 (m, 2H),
0.88 (s, 9H), 0.04 (s, 6H).
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Compound 2-2. To a solution of 2-1 (1.9 g, 0.009 mol) in DCM (35 mL) at 0 oC was
added aqueous potassium bromide (KBr) (0.87 mL, 1 M) and 2,2,6,6tetramethylpiperidine-1-oxyl (TEMPO) (0.027 g, 0.174 mmol) and a freshly prepared 1:1
mixture of commercial bleach (17 ml) and saturated sodium bicarbonate (NaHCO3) (17
mL). The reaction was stirred vigorously for 2.5 hrs. The reaction mixture was extracted
with ether and washed with water and brine. The organic layer was dried over anhydrous
MgSO4 and concentrated in vacuo. The crude product was purified by silica gel
chromatography (4:1 Hex:EA ) to provide the desired compound 2-2 as a colorless clear,
slightly viscous liquid in 85% yield. The purified product was used immediately in the
next reaction due to poor product stability.
MW=216.39 ESI= 217 m/z (M + H+)
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H NMR (300 MHz, CDCl3): δ 9.8 (s, 1H), 3.63 (t, 2H), 2.42 (t, 2H), 1.65 (m, 2H), 1.27
(t, 2H), 0.90 (s, 9H), 0.05 (s, 6H).
Compound 2-3. 5-aminopentanol (2.1 g, 0.02 mol) was added to a solution of 2-2 (2 g,
0.009 mol) in dichloroethanol (DCE) (50 mL) and the mixture was stirred vigorously for
10 min. Sodium triacetoxyborohydride (NaBH(OAc)3) (2.09, 0.010 mol) was added after
10 min in one portion and the reaction was stirred at room temperature overnight. The
next day, DCE was removed in vacuo and the remaining residue was re-dissolved in ethyl
acetate and washed with water and brine. The organic layers were dried over anhydrous
MgSO4, filtered und concentrated in vacuo. The crude product was purified via silica gel
chromatography (93% ethyl acetate/7% MeOH/NH3) to provide the desired compound 23 as a clear, colorless oily residue in 25% yield.
MW=303.56 ESI+MS= 303.98 m/z (M+H+)
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H NMR (300 MHz, CDCl3): δ 3.63 (m, 4H), 2.62 (m, 4H), 1.53 (m, 4H), 1.38 (m, 4H),
0.9 (s, 9H), 0.05 (s, 6H).
Compound 2-4. To a solution of compound 2-3 (0.3 g, 1.58 mmol) in a 1:1 mixture of
MeOH (10 mL) and EtOH (10 mL) was added HCl (1.33 mL, 12 M) drop-wise. The
reaction mixture was allowed to stir for 1 h at room temperature. Excess solvent was
removed in vacuo and the product was used in the next step without further purification.
The residue was dissolved in H2O (20 mL) and sodium bicarbonate (0.810 g, 4.74 mmol)
was added to the solution. The reaction mixture was cooled to 0 oC and Fmoc-OSu
(0.588g, 1.74 mmol) dissolved in acetone (20 mL) was added drop-wise over a period of
30 min and the mixture was stirred for 12 hrs at room temperature. The reaction was
treated with 10% HCl to bring the pH to 2 and extracted with EtOAc. The organic layers
were dried over MgSO4 and solvent was removed in vacuo. The crude product was
purified by silica gel chromatography (100% EtOAc) to provide 2-4 as a colorless, clear
oil in 51% yield.
MW=411.24 ESI+MS = 411.18 m/z (M + H+)
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H NMR (300 MHz, CDCl3): δ 7.75 (m, 2H), 7.57 (m, 2H), 7.35 (m, 4H), 4.52 (d, 2H),
4.22 (t, 1H), 3.59 (m, 4H), 3.18 (t, 2H), 2.99 (t, 2H), 2.8 (s, 2H), 1.54 (m, 4H), 1.23 (m,
4H), 1.2 (m, 2H).
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Compound 2-5. To a solution of 2-4 (0.15 g, 0.37 mmol) in acetone (5 mL), cooled to
0oC was added freshly prepared Jones reagent (1 mL) drop-wise and the reaction mixture
was allowed to warm to room temperature. (The Jones reagent was prepared by
dissolving 25 g of Cr(VI)O3 in 25 mL of H2SO4 (conc.). The resulting solution is added
under stirring very slowly to 75 mL of cooled to (0oC) H2O.) The reaction was stirred at
room temperature for 2 hrs. Isopropanol (2 mL) and water (40 mL) were added to the
reaction mixture. The reaction mixture was extracted with EtOAc and the organic layer
was washed with a dilute mixture of NaHCO3, water and brine. The crude product was
purified by silica gel chromatography (93% DCM, 7% MeOH) to provide the desired
compound 2-5 as a white solid in 63% yield.
MW=439.5 MALDI-MS=438.37 m/z (M + H-)
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H NMR (300 MHz, CDCl3): δ 10.5 (s, 2H), 7.77 (m, 2H), 7.56 (m, 2H), 7.34 (m, 4H),
4.59 (d, 2H), 4.21 (t, 1H), 3.18 (t, 2H), 2.91 (t, 2H), 2.35 (t, 2H), 2.21 (t, 2H), 1.53 (m,
4H), 1.32 (m, 4H).
UV-Vis Analysis: All flourescein-labeled CAPHs were quantified by UV-vis
spectroscopy. The peptides were first dissolved in an appropriate amount of sterile, deionized water. To 1 mL of TRIS buffer pH 8 was added 5 μL of the peptide solution and
the absorbance of the solution was measured with the a Cary 100 Bio UV-Visible
spectrophotometer at 491 nm. Six absorbance readings were taken and averaged. A
sample with buffer alone was also analyzed to determine background fluorescence. The
peptide concentration was calculated using Beer’s Law (A=εbc). ε=60,000 M-1 cm-1, b=1
cm