Materials and Methods
HeLa cells were the generous gift of Dr. Audrey Minden (Department of Chemical Biology, Rutgers University). Petri dishes (35 mm) fitted with microwells (14 mm) and a No. 1.5 coverglass were from MatTek Corporation. Wheat germ agglutinin Alexa Fluor 594 conjugate, ProLong(R) Gold antifade reagent, Hoechst 34850, and DMEM (Dulbecco's Modified Eagle Medium) were from Invitrogen. Fetal bovine serum was from Intergen Company. C18 Sep-Pak® cartridges were purchased from Waters. Vydac 12-Well plates were from Corning Inc. Vydac 218TP54 and 218TP1010 columns were used for analytical and preparative RP-HPLC, respectively. All analytical and preparative RP-HPLC solvents, water and CH3CN, contained 0.10% TFA; and retention times (tR) are based on linear gradients (unless otherwise noted) starting from 100% H2O to 40% H2O:60% CH3CN over 60 min. All solvents were of HPLC grade. DIEA and TFA were of Sequalog/peptide synthesis grade from Fisher. Fmoc-Cys(Me)-OH was from Bachem and Fmoc-Lys(Dde)-OH was from Nova Biochem. PAL-PEG-PS was from Applied Biostems. Preloaded CLEAR-Acid resins were from Peptides International. All other reagents were from Sigma Aldrich. In the following procedures, unless determined by UV spectroscopy, actual peptide content has not been taken into account. All procedures involving fluorescent derivatives were protected from light as much as possible in order to avoid bleaching the fluorophore.
Ac, Acetyl; Acm, acetamidomethyl; BOC, t-butyloxycarbonyl; BOP, (benzotriazol-1-yloxy)tris-(dimethylamino)phosphonium hexafluorophosphate; t-Bu, tert-butyl; CLEAR, cross-linked ethoxylate acrylate resin; CPP, cell-penetrating peptide; DIC, N,N’-diisopropylcarbodiimide; Dde, 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl; DIEA, diisopropylethylamine; DIC, diisopropylcarbodiimide; DMEM, Dulbecco's Modified Eagle Medium, DMF, N,N-dimethylformamide; DTT, dithiothreitol; ESI-MS, electrospray ionization mass spectrometry; 5-Fam, 5-carboxyfluorescein; Far, farnesyl; FITC, fluorescein isothiocyanate; Fmoc, 9-fluorenylmethyloxycarbonyl; Ger, geranylgeranyl; HOBt, 1-hydroxybenzotriazole; Me, methyl; nL, norleucine; OAc, acetate; PAL, peptide amide linker; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; PEG-PS, polyethylene glycolpolystyrene graft copolymer; PMSF, phenylmethanesulfonyl fluoride; Pnt, penetratin; RP-HPLC, reversed-phase high pressure liquid chromatography; Scm, S-carbomethoxysulfenyl; SPPS, solid-phase peptide synthesis; TFA, trifluoroacetic acid; Trt, trityl.
Linear peptides were synthesized by Fmoc-based SPPS on either Applied Biosystems 433A or Pioneer automated peptide synthesizers according to manufacturer protocols. Peptides were cleaved with freshly prepared Reagent K17
(TFA-phenol-thioanisole-water-ethanedithiol 82.5:5:5:5:2.5) for 2 h, precipitated with Et2
O, and centrifuged to form a pellet which was washed with Et2
O. Crude peptides were purified by RP-HPLC.
General Procedure for Cysteine Alkylation: Prenylation of AcK(5-Fam)C(Acm)KKSRRC-NH2 (11) to produce 1a and 2a
(1.0 eq.) and Zn(OAc)2
O (5.0 eq.) were dissolved in DMF/1-butanol/0.10% aqueous TFA (2:1:1, v/v/v) (1.0 mL solvent per 2.0 mg peptide).18
Either farnesyl bromide or geranylgeranyl bromide (4.0 eq.) was then added. The reaction was monitored by RP-HPLC, and once judged complete (typically 2 h), was diluted with 10 volumes of 0.10% aqueous TFA, filtered, and purified by RP-HPLC.
Reaction scale: 0.014 mmol, yield: 13 mg (54%), purity by RP-HPLC: 95%, tR = 45 min, deconvoluted ESI-MS: calculated 1681.8, found 1681.8.
Reaction scale: 0.010 mmol, yield: 10.5 mg (62%), purity by RP-HPLC: 94%, tR = 58 min, deconvoluted ESI-MS: calculated 1973.1, found 1973.0.
Peptide 3a was synthesized by the above procedure, except Fmoc-Cys(Me)-OH was substituted for Fmoc-Cys(Trt)-OH during chain assembly on PAL-PEG-PS. Reaction scale: 0.025 mmol, yield: 19.9 mg (54%), purity by RP-HPLC: 99%, tR = 25 min, deconvoluted ESI-MS: calculated 1491.7, found 1491.9.
The geranylgeranyl group was introduced onto the appropriate peptide precursor as it was for 2a. Reaction scale: 0.012 mmol, yield: 12.7 mg (53%), purity by RP-HPLC: 98%, tR = 50 min, deconvoluted ESI-MS: calculated 1749.9, found 1750.3.
General Procedure for Conversion of Peptides with Cys(Acm) Side-chain Protection (1a, 2a and 3a) to Peptides with Cys(Scm) (1b, 2b and 3b)
CH3O(CO)SCl (5.0 μL) was added to CH3CN (0.20 mL) to give a 0.27 M stock solution. The Cys(Acm)-containing peptide was dissolved in DMF/CH3CN (1:1, v/v) (1.0 mL solvent per 1.0 mg of peptide) and the peptide concentration was determined by UV spectroscopy of the 5-Fam chromophore (ε = 79,000, 492 nm, pH 9.0). The reaction and stock solution were cooled in an ice bath and 1.0 eq. of CH3O(CO)SCl stock solution was added to the reaction. After 1 h, the reaction was analyzed by RP-HPLC. The percent conversion of Cys(Acm) to Cys(Scm) was calculated by comparing the peak areas of the product and starting peptide peaks (). Enough additional stock CH3O(CO)SCl was then added to complete the reaction, based on the percent conversion observed after addition of the first equivalent of CH3O(CO)SCl. The RP-HPLC monitoring and analysis was repeated after another 1 h of reaction time. If any precipitate of the product was observed, enough DMF was added to render the solution homogeneous, so as to ensure an accurate HPLC analysis of % conversion [Cys(Scm) is more hydrophobic than Cys(Acm), and hence less soluble]. When the reaction was judged complete, it was diluted with 10 volumes of 0.10% aqueous TFA, filtered, and purified by RP-HPLC. The pooled fractions of product from purification were used directly in the next step without concentration or lyophilization.
Figure 2 Reversed-phase HPLC analysis monitoring the progress of Acm to Scm conversion in a geranylgeranylated C-terminal CDC42 peptide. Gradient: 20−60% CH3CN over 60 min. Chromatogram (1): Peptide 2a (tR = 32.5 min) before the addition of CH3O(CO)SCl. (more ...)
Purity by RP-HPLC: 82%, tR = 46 min, deconvoluted ESI-MS: calculated 1700.8, found 1701.0.
Purity by RP-HPLC: 69%, tR = 51 min, deconvoluted ESI-MS: calculated 1768.8, found 1768.6.
The crude product was isolated and used in the synthesis of 3c without analytical characterization, tR = 27 min.
In early experiments to convert Cys(Acm) to Cys(Scm) in the Cys(Far)- and Cys(Ger)-containing peptides, excess CH3O(CO)SCl was used. A major early-eluting contaminant peak (tR = 30 min), observed on RP-HPLC, was isolated and identified as the bis(Scm) derivative, in which the Cys(prenyl) group had been cleaved by the excess CH3O(CO)SCl to yield Cys(Scm). This bis(Scm) derivative was completely stable in 0.10% aqueous TFA solutions for at least several weeks. Treatment of peptides containing prenylated cysteine residues with CH3O(CO)SCl to yield the corresponding Cys(Scm) derivative, followed by treatment with DTT or 2-mercaptoethanol, may prove useful as a mild method for removal of prenyl groups from cysteine. Deconvoluted ESI-MS: calculated 1586.6, found 1586.6.
General Procedure for Conjugation of AcK(5-Fam)C(Scm)KKSRRC(prenyl)-NH2 with Penetratin to Provide Mixed Disulfide Peptides (1c, 2c and 3c)
The concentrations of the Cys(Scm)-containing peptides were determined by UV spectroscopy of the 5-Fam chromophore (ε = 79,000, 492 nm, pH 9.0). Zn(OAc)2·2H2O (5.8 mg) was dissolved in 1.0 M sodium acetate (1.5 mL, pH 5.4) to give a 17 mM Zn2+ stock solution. Penetratin (12) was dissolved in 0.10% aqueous TFA (2.5 mg/mL, approximately 0.70 mM), and the exact concentration was determined by assaying the free thiol content with Ellman's reagent. Penetratin (1.0 eq.) was added from the stock solution to the pooled Cys(Scm)-containing fractions, the reaction mixture was cooled in an ice bath, and Zn(OAc)2 (5.0 eq.) from the stock solution was added. Next, enough sodium acetate (1.0 M, pH 5.4) was added to bring the overall acetate concentration to about 0.067 M, and the pH of the reaction mixture was adjusted to pH 5.0−5.2 with 0.10 N NaOH. After 15 min, the reaction was analyzed by RP-HPLC, and additional penetratin from the stock was added as necessary to drive the reaction to completion. When the reaction was judged complete by RP-HPLC analysis (), the pH was adjusted to the range of 2−3 by addition of neat TFA, the mixture was filtered, and purified by RP-HPLC. It is important to note that since this is a bimolecular reaction, the rate is highly dependent on concentration. For more dilute peptide solutions, one may have to wait longer than 15 min to analyze the extent of reaction. UV spectroscopic analysis underestimated the concentration of AcK(5-Fam)C(Scm)KKSRRC(prenyl)-NH2 relative to penetratin. This is possibly due to the instability of the former peptide at pH 9.0.
Figure 3 Reversed-phase HPLC analysis monitoring the progress of penetratin conjugation to a Scm-protected farnesylated C-terminal CDC42 peptide. Gradient: 0−60% CH3CN over 60 min. Chromatogram (1): Penetratin (12) (tR = 29.0 min) and peptide 1b (tR = (more ...)
Yield: 1.9 mg over 2 steps from 5.2 mg 1a (16%), Purity by RP-HPLC: 95%, tR = 40 min, deconvoluted ESI-MS: calculated 3925.1, found 3925.0.
Yield: 1.3 mg over 2 steps from 8.9 mg 1b (6%), Purity by RP-HPLC: 99%, tR = 48 min, deconvoluted ESI-MS: calculated 3993.2, found 3993.2.
Yield: 4.5 mg over 2 steps from 5.0 mg 1c (36%), Purity by RP-HPLC: 97%, tR = 31 min, deconvoluted ESI-MS: calculated 3734.9, found 3734.4.
The linear sequence was assembled on either PAL-PEG-PS or Rink-amide resin. DIC and HOBt were used for coupling to avoid racemization of cysteine.19
-terminal lysine side-chain was orthogonally protected with Dde. Acetic acid was coupled in the last instrument cycle to acetylate the N
-terminus. The Dde protecting group was removed selectively by treating protected peptide-resin (0.05 mmol) in a solid-phase reaction vessel with 5.0 mL of anhydrous NH2
/DMF (1:9, v/v), for 15 min, followed by washing with DMF (5 × 5.0 mL).20
5-Fam (38 mg, 0.10 mmol), DIC (16 mL, 0.10 mmol) and HOBt (13.5 mg, 0,10 mmol) were dissolved in DMF (2.0 mL) and then added to the peptide resin along with DMF washes (2 × 0.5 mL). The coupling reaction was allowed to tumble overnight, and then the peptide-resin was washed with DMF (3 × 5.0 mL) and CH2
(3 × 5.0 mL), and dried in vacuo
. The peptide was cleaved with Reagent K17
(TFA-phenol-thioanisole-water-ethanedithiol 82.5:5:5:5:2.5) for 2 h, precipitated with Et2
O, and centrifuged to form a pellet, which was washed with Et2
O. The crude peptide was purified by RP-HPLC. Yield: 18−30 mg (24−40%), purity by RP-HPLC: > 95%, tR
= 26 min, deconvoluted ESI-MS: calculated 1477.6, found 1477.4.
The linear peptide was synthesized as for compound 11 except acetic acid was not coupled in the last instrument cycle to acetylate the N-terminus. 5-Fam was coupled to the N-terminus of side-chain protected peptide-resin, using the same procedure described for fluorophore coupling used on compound 11. Purity by RP-HPLC: 97%, tR = 37 min, Deconvoluted ESI-MS: calculated 2674.4, found 2674.4.
AcK(5-Fam)AKKSRRCVLL (precursor to 4)
The linear peptide sequence was synthesized using CLEAR-Acid resin,21
and the 5-Fam fluorophore was coupled to the N
-terminal lysine side-chain of otherwise fully protected peptide resin as described for compound 11
. Purity by RP-HPLC: 99%, tR
= 34 min, deconvoluted ESI-MS: calculated 1700.9, found 1700.8.
CRNIKIWFQNRRnLKWKK (penetratin) (12)
Purity by RP-HPLC: 99%, tR = 29 min, Deconvoluted ESI-MS: calculated 2316.3, found 2316.0.
Geranylgeraniol (0.10 g, 0.69 mmol, 1.0 eq.) was dissolved in CH2Cl2 (4.2 mL). Next, polymer-supported PPh3 (0.45 g, 1.438 mmol, 2.0 eq.) was added, and the solution was gently shaken. After the addition, the heterogeneous solution turned a light brown color. After 30 min, CBr4 (0.27 g, 0.83 mmol, 1.2 eq.) was dissolved in 0.5 mL CH2Cl2 and added to the mixture via syringe. This solution was stirred for 6 h, after which the polymer-supported reagent was removed by filtration and the resulting solution was concentrated to yield 0.11 g of geranylgeranyl bromide (89% yield). This crude product was stored at −20° C. Just prior to use in the alkylation reaction, the calculated amount of geranylgeranyl bromide (4.0 eq. over peptide resin) was purified by solid phase extraction on a C18 Sep-Pak® cartridge. The cartridge was first equilibrated with 5% CH3CN in water that contained 0.10% TFA. The geranylgeranyl bromide was dissolved in 0.50 mL of DMF and applied to the column. The column was then washed with 10 mL of equilibration solvent and 10 mL of 50% CH3CN in water that contained 0.10% TFA. The geranylgeranyl bromide was then eluted from the column directly into the reaction vessel with 5.0 mL DMF. If this solid phase extraction procedure is not done, a large amount of disulfide impurity, rather than the desired geranylgeranyl alkylation product, is formed in the reaction. 1H NMR (300 MHz, CDCl3) δ = 1.62 (s, 6H), 1.70 (s, 3H), 1.77 (s, 3H), 1.79 (s, 3H), 2.00−2.16 (m, 12H), 4.04 (d, 1H, J=7.2 Hz), 5.11 (m, 3H) 5.54 (t, 1H, J=7.2 Hz). 13C NMR-DEPT (CDCl3) δ 16.0, 16.1, 16.2, 17.7, 25.8 (primary), 26.2, 26.7, 26.8, 29.8, 39.6, 39.7, 39.8, (secondary), 120.6, 123.5, 124.2, 124.4 (tertiary), 131.4, 135.1, 136.2, 143.7 (quaternary). HR-FAB-MS calculated for C20H33Br [M + NH4]+ 370.21, found 370.21.
Determination of Peptide Concentrations for Cell Studies
Stock peptide solutions were diluted using 20 mM Tris buffer, pH 9.0, and DMSO so that the final concentration of DMSO was no greater than 0.10% by volume. UV spectroscopy of the 5-Fam chromophore was used to determine the concentrations of filtered peptide stock solutions (ε492
= 79,000, 492 nm, 20 mM Tris·HCl, pH 9.0).23
HeLa cells were grown in DMEM supplemented with 10% fetal bovine serum at 37 °C with 5.0% CO2. For all experiments, 2.6 × 104 cells/cm2 were seeded in culture dishes and grown for 24 h to approximately 50% confluency.
HeLa cells were seeded in 35 mm glass bottom microwell dishes. Approximately 24 h after plating cells were washed twice with serum-free DMEM, and incubated for 1 h at 37 °C and 5.0% CO2
in serum-free DMEM containing 1.0 μM peptide. Hoechst 34580 was added to 1 μg/ml during the final 20 minutes of incubation, and wheat germ agglutinin Alexa Fluor 594 conjugate was added to 5 μg/ml during the final 10 minutes of incubation. For ATP depletion, cells were washed twice with glucose-free, serum-free DMEM and then incubated for 2 h at 37 °C and 5.0% CO2
in glucose-free, serum-free DMEM supplemented with 12 μM rotenone and 15 mM 2-deoxyglucose24
. Then peptides, Hoechst 34850 and wheat germ agglutinin AlexaFluor 594 conjugate were added and cells were incubated as described above. For peptide incubation at 4°C, cells were washed twice with chilled serum-free DMEM supplemented with 25 mM HEPES (pH 7.3) and incubated with peptide, Hoechst 34850 and wheat germ agglutinin Alexa Fluor 594 conjugate as described above at 4°C. After 1 h total incubation, all cells were washed twice with PBS. Cells were then either imaged live in serum-free DMEM, or cells were fixed for 10 min at room temperature in 3.7% formaldehyde in PBS. Coverslips were mounted with ProLong Gold Antifade Reagent on 1 mm glass microscope slides. Cells were imaged using an Olympus FluoView 1000 confocal microscope with a 60X objective with a N.A. of 1.42. The 5-Fam fluorophore was imaged using fluorescein settings, Hoechst 34850 was imaged using DAPI settings, and wheat germ agglutinin Alexa Fluor 594 conjugate was imaged using Alexa Fluor 594 settings. All fluorescence and brightfield images were acquired simultaneously.
Quantitation of Cellular Uptake of Fluorescent Peptides Using Cell Lysates
Peptide stock solutions in Tris (20 mM, pH 9.0) were prepared so that when the peptide (237 μL) was diluted into serum free DMEM (263 μL), the final concentrations of peptides would be 0.10, 0.30, 1.0, or 3.0 μM. The dilution kept the amount of Tris compared to DMEM constant, regardless of final peptide concentration. Dilution was required because of the differential solubility among peptides. HeLa cells (2.6 × 104 cells/cm2) were seeded in 12 well plates and grown for 20 h to approximately 50% confluency. Next, the media was removed and DMEM containing diluted peptide (500 μL) was added to each well. Cells were treated with each peptide in triplicate and were incubated for 1 h at 37 °C in 5.0% CO2. At this time, the peptide-containing media was removed, and the wells were washed with PBS (1.0 mL, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.3). Next, the cells were lysed with RIPA buffer (100 μL, 50 mM Tris·HCl, pH 7.4, 1.0% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1.0 mM EDTA) and scraped from the wells. The lysates were placed in 1.6 mL microcentrifuge tubes and centrifuged (1.0 min, 10,000 g). The fluorescence of the supernatant (490 nm excitation, 520 nm emission, 5 nm slit widths) was measured to quantitate the amount of internalized peptide. This measurement was then normalized against the amount of protein in the sample using the Bradford assay (BioRad) to measure the protein concentration. Each experiment was performed in triplicate and the results are expressed as the mean relative fluorescence ± standard deviation.
Quantitation of Cellular Uptake of Fluorescent Peptides Using Flow Cytometry
HeLa cells were seeded in 6-well plates and treated with peptides as described for microscopy. After treatment for 1, 2, 3, or 4 h, cells were washed twice with PBS, trypsinized for 5 minutes, and resuspended to a total volume of 2 mL with DMEM/10% FBS. Cells were centrifuged for 5 minutes at 100 × g and resuspended in PBS to a final volume of 2 mL. A total of 10,000 events for each sample were analyzed using a BD FACSCalibur (BD Biosciences). Each experiment was performed in triplicate and the results are expressed as the geometric mean fluorescence ± standard deviation.