Ethynyl benziodoxolones : Functional terminators for cell-penetrating poly ( disulfide ) s

Outperforming cell-penetrating peptides (CPPs), cell-penetrating poly(disulfide)s (CPDs) are attracting increasing interest. CPDs are accessible by ring-opening disulfide-exchange polymerization under mild conditions in neutral water. Initiation of the polymerization with thiols results in quantitative labeling of one CPD terminus with initiators of free choice. In contrast, labeling of the other terminus with iodoacetamides has so far been ineffective because of poor yields and the high excess of reagents needed. In this report, we introduce hypervalent iodine reagents as operational terminators of the synthesis of CPDs, also at high dilution. The power of the approach is exemplified with green-fluorescent initiators and ethynyl benziodoxolone terminators containing additional azides for CuAAC with red-fluorescent alkynes. The absorption spectra of the resulting CPDs demonstrate that stoichiometric application of ethynyl benziodoxolone terminators results in 46% incorporation. FRET between green-fluorescent initiators and red-fluorescent terminators demonstrates significant folding of CPDs in solution; it disappears upon reductive depolymerization. Substrates attached to the new termini are shown to enter into HeLa cells. Moreover, disappearance of FRET in the cytosol corroborated the reductive cleavage of CPDs upon internalization. Beyond the introduction of enthynyl benziodoxolones as operational terminators, these findings thus demonstrate also the compatibility of CuAAC with poly(disulfide)s and the usefulness of doubly-labeled CPDs for structural and mechanistic studies.


Materials and Methods
As in ref.S1, Supporting Information.Briefly, reagents for synthesis were purchased from Fluka, Sigma-Aldrich, TCI and Across, buffers and salts of the best grade available from Fluka or Sigma-Aldrich and used as received.Amicon Ultra 0.5 mL centrifugal filter units were purchased by Sigma-Aldrich.
Unless stated otherwise, column chromatography was carried out on silica gel 60 (Fluka,.Analytical TLC was performed on silica gel 60 (Fluka, 0.2 mm).Fluorescence measurements were performed with a FluoroMax-4 spectrofluorometer (Horiba Scientific) equipped with a stirrer and a temperature controller (25.0 ± 0.1 ºC).Fluorescence spectra were corrected using instrument-supplied correction factors.UV-Vis spectra were recorded on a JASCO V-650 spectrophotometer equipped with a stirrer and a temperature controller (25.0 ± 0.1 ºC) and are reported as maximal absorption wavelength  in nm (extinction coefficient  in M -1 cm -1 ).Gel-permeation chromatography (GPC) was performed using a JASCO LC-2000 Plus system equipped with a quaternary pump (JASCO PU-2089), photodiode array (JASCO MD-2018 Plus) and fluorescence (JASCO FP-2020 Plus) detectors.The chromatographic column used was a Superdex 75 10/300 GL (flow 0.4 mL/min, eluent: 30% ACN in 0.1 M acetate buffer pH = 6.5).Semi-preparative HPLC was performed using JASCO LC-2000 Plus system equipped with a quarternary pump (JASCO PU-2089) and UV/Vis detector (JASCO UV-2077 Plus).LC-MS were recorded using a Thermo Scientific Accela HPLC equipped with a Thermo C18 (5 cm x 2.1 mm, 1.9 µm particles) Hypersil gold column coupled with a LCQ Fleet three-dimensional ion trap mass spectrometer (ESI, Thermo Scientific) with a linear elution gradient from 95% H2O 0.01% TFA to 90% ACN 0.01% TFA in 4.0 minutes at a flow rate of 0.75 mL/min.pH values were measured with a Consort C832 multi-parameter analyzer equipped with a VWR glass membrane pH electrode calibrated with Titrisol solution from Merck at pH 4.00 and 7.00.IR spectra were recorded on a Perkin Elmer Spectrum One FT-IR spectrometer (ATR, Golden Gate) and are reported as wavenumbers ν in cm -1 with band intensities indicated as s (strong), m (medium), w (weak), br (broad). 1 H and 13 C NMR spectra were recorded (as indicated) on a Bruker 400 MHz spectrometer and are reported as chemical shifts (δ) in ppm relative to TMS (δ = 0).
Compound 1.This compound was prepared as reported in ref.S1.
Compound 2. This compound was prepared as reported in ref.S2.
Compound 3.This compound was prepared as reported in ref.S3.
Compound 7.This compound was prepared as reported in ref.S4.
Compound 10.This compound was prepared as reported in ref.S5.
Compound 11.This compound was prepared as reported in ref.

General Procedure
The procedure described in ref.

Purification and Characterization
Polymers were purified by GPC using Superdex 75 10/300GL (10×300 mm) chromatographic column and 30% ACN in 0.1 M acetate buffer (pH = 6.5) as eluent.100 L of sample were loaded on the column and the flow rate was set to 0.4 mL/min from 0 to 40 min, 0.6 mL/min from 41 to 80 min.Commercial molecular weight standards were used for calibration (Table S1, Fig. S1).Quantification of the polymers was achieved by UV-Vis measurements based on CF-initiator absorption.

CuAAC Reaction and Purification
Stock solutions of alkyne 5 and TBTA 11 were freshly prepared in THF.Stock solutions of sodium ascorbate and CuSO4 .5H2O were freshly prepared in water.
CuAAC reaction to obtain polymer 6 was carried out in an Eppendorf tube by mixing 73 L of polymer 4 (220 M in water) with 10 L of alkyne 5 solution (10 mM in THF) and then adding 10 L of sodium ascorbate solution (10 mM in water), 10 L of CuSO4 .5H2O solution (1 mM in water) and 2 L of TBTA solution (10 mM in THF).
CuAAC reaction to obtain polymer 9 was carried out in an Eppendorf tube by mixing 73 L of polymer 8 (45 M in water) with 10 L of alkyne 5 solution (10 mM in THF) and then adding 10 L of sodium ascorbate solution (10 mM in water), 10 L of CuSO4 .5H2O solution (1 mM in water) and 2 L of TBTA solution (10 mM in THF).
The resulting solutions were kept at 25 °C and vigorously agitated at 1000 rpm for 17h.
Purification of the mixtures was achieved by washing with water (12 x 200 L) using centrifugal filter units with a molecular weight cutoff of 3 kDa, yielding polymers 6 and 9 in water.The efficiency of the purification method was verified by LC-MS where no peak due to alkyne 5 and TBTA was visible.Moreover, GPC analysis of the purified polymer 6 confirmed that the polymer was not affected by CuAAC conditions (Fig. S2).Fluorescence in GPC detected at em = 582 nm (ex = 552 nm) confirmed that TAMRA was successfully conjugated to the terminator.

FRET Measurement and Depolymerization
Stock solutions of polymers 6 and 9 (8 M) in TEOA buffer (1 M, pH 7) were freshly prepared and their absorption measured by UV-Vis spectrometry.Fluorescence emission spectra were recorded of the same stock solutions by excitation at 488 nm (Fig. S3).The stock solution of DTT (1 M, water) was freshly prepared.Depolymerization was carried out on the same stock solutions by addition of 10 L of DTT (final concentration of 50 mM).The resulting solutions were kept at 25 °C and vigorously agitated at 1000 rpm.After 20 min, fluorescence emission spectra (ex = 488 nm) of the depolymerized mixtures were recorded (Fig. S3).

Confocal Microscopy
HeLa Kyoto cells were seeded at 1 x 10 5 cells/well on 35 mm glass-bottomed dishes (MatTek Corporation) and cultured overnight.After removing the medium, the cells were washed 3 times with Liebovitz's medium and treated with 1 mL of polymer 6 solution (500 nM in Liebovitz's medium).The cells were incubated for 15 min at 37 °C, then the media was removed by aspiration.Cells were washed 3 times with PBS containing 0.1 mg/mL heparin, twice with Liebovitz's medium and then kept in Liebovitz's medium.Distribution of fluorescent polymer was analyzed without fixing using a confocal laser scanning microscope (Leica SP5) equipped with 63x oil immersion objective lens.Ar laser was used as light source (4% laser power) with excitation wavelength 488 nm, emission 498 ~ 535 nm for CF and emission 571 ~ 650 nm for TAMRA (Leica HyD TM detector); DPSS laser (4%) with excitation wavelength 561 nm and emission 571 ~ 650 nm for TAMRA (Leica HyD TM detector).During CLSM analysis, the samples were kept at 37 °C.
S2 was adapted to obtain each polymer.Stock solutions of propagator 2, CF-initiator 1 and terminators 3 and 7 were freshly prepared.To 8 L of propagator solution (3 M in DMF) in an Eppendorf tube, 8 L of initiator solution (50 mM in DMF) and 64 L of TEOA buffer (1 M, pH = 7.0) were added.The sample was kept at 25 °C with vigorous agitation at 1000 rpm for 30 minutes.After polymerization, 10 L of terminator solution (40 mM in DMF) were added and the obtained solution was immediately purified by GPC.