Poly(S-ethylsulfonyl-l-homocysteine): An Î±-Helical Polypeptide for

Article Cite This: Macromolecules XXXX, XXX, XXX−XXX

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Poly(S‑ethylsulfonyl‑L‑homocysteine): An α‑Helical Polypeptide for Chemoselective Disulfide Formation Christian Muhl,† Olga Schäfer,† Tobias Bauer,†,§ Hans-Joachim Räder,‡ and Matthias Barz*,† †

Institute of Organic Chemistry, Johannes Gutenberg University Mainz, Duesbergweg 10-14, 55128 Mainz, Germany Max-Planck-Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany § Graduate School MAterials Science IN mainZ (MAINZ), Staudingerweg 9, 55128 Mainz, Germany ‡

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S Supporting Information *

ABSTRACT: Homocysteine and cysteine are the only natural occurring amino acids that are capable of disulfide bond formations in peptides and proteins. The chemoselective formation of asymmetric disulfide bonds, however, is chemically challenging and requires an activating group combining stability against hard nucleophiles, e.g., amines, with reactivity toward thiols and soft nucleophiles. In light of these considerations, we introduced the Salkylsulfonyl cysteines in our previous work. Here, we present the synthesis and ring-opening polymerization of S-ethylsulfonyl-L-homocysteine N-carboxyanhydrides. We demonstrate that the polymerization leads to narrowly distributed polypeptides (Đ = 1.1−1.3) with no detectable side reactions in a chain length regime from 11 to 165. In contrast to the already reported cysteine derivatives, poly(S-ethylsulfonyl-L-homocysteine)s do not form β-sheets, which reduce solubility and limit the degree of polymerization of poly(S-ethylsulfonyl-L-cysteine)s to 50. Instead, these polymers form α-helices as confirmed by circular dicroism (CD) experiments and infrared spectroscopy (FT-IR). In comparison to the cysteine derivatives, the α-helix formation leads to slightly faster polymerization kinetics (rate constants from 1.44 × 10−5 to 5.29 × 10−5 s−1). In addition, the ability for the chemoselective formation of asymmetric disulfides is preserved as monitored via 1H NMR experiments. Consequently, this new polypeptide overcomes the chain length limitations of poly(Sethylsulfonyl-L-cysteine)s and thus provides convenient access to reactive poly(S-ethylsulfonyl-L-homocysteine)s for chemoselective disulfide formation.

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was described by Deming et al. via alkylation of thioethers.19,29 However, when it comes to biomedical applications, it is often preferred to keep the linkage addressable by an external stimulus, which can lead to an enhanced release of cargo or a possible degradation of the material.30 Therefore, commonly employed moieties are redox-sensitive disulfide moieties or acid-labile acetal or hydrazone linkages, which can be cleaved due to the differences of intra- and extracellular conditions, such as glutathione concentrations or pH-values. Since differences of intra- and extracellular levels of glutathione can be up to 100-fold higher than differences in proton concentrations (pH-value), disulfide bonds are often chosen to combine extracellular stability with intracellular degradability, which is why we have focused on the development of functional groups for chemoselective disulfide formation.22,23,31,32 In a previous study, a range of activated cysteine NCAs were synthesized and polymerized under various conditions.33,34 However, all of these compounds showed a tremendous amount of side reactions during monomer synthesis and polymerization due to unbalanced electron densities of the

INTRODUCTION The synthesis of multifunctional peptide-based materials via ring-opening polymerization (ROP) of N-carboxyanhydrides (NCAs) has attracted increasing attention in the last decade.1−6 Because of their unique chemical, physical, and biological properties, peptides, polypeptides, and polypept(o)ides are promising candidates for bio-inspired nano- or macroscopic materials, such as nanosized drug-delivery systems (nano-DDS) or scaffolds for tissue engineering.7−−18 For these purposes it is important to introduce functionality into polypeptides, which is commonly done by postpolymerization modification chemistry and thus generates a need for reactive groups within the polypeptide, which are stable during NCA polymerization but can be chemoselectively addressed in the final material.19−24 Several reactive groups, which ensure the addressability via PPM, have already been successfully established and described in the literature, such as azides (copper(I)-catalyzed azide− alkyne cycloaddition (CuAAC)21), alkynes (CuAAC,25 thiol− yne26), and double bonds (thiol−ene,26 Michael addition,27 olefin metathesis28). Nevertheless, all of these methods have a potential major drawback: the resulting covalent bonds (triazines, carbon−carbon and carbon−sulfur bonds) are irreversible and therefore lead to permanent modification of the polymer. An alternative route for reversible modification © XXXX American Chemical Society

Received: July 6, 2018 Revised: September 12, 2018

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DOI: 10.1021/acs.macromol.8b01442 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules

Synthesis of Ethanesulfinic Acid Sodium Salt. A solution of sodium sulfite (391.98 g, 3.11 mol) in 800 mL of water was heated to 80 °C. Ethanesulfonyl chloride (147.40 mL, 199.30 g, 1.55 mol) and sodium carbonate (329.63 g, 3.11 mol) were added simultaneously while significant quantities of CO2 evolved. The reaction mixture was stirred for 1 h at 80 °C, and afterward the water was removed in vacuo at 60 °C. The resulting solid was suspended in methanol (degassed) and filtrated. Evaporation of methanol gave ethanesulfinic acid sodium salt (153.54 g, 1.32 mol, 85%) as a colorless solid. 1H NMR (400 MHz, D2O/[D1]TFA) δ [ppm] = 2.67 (q, 3J = 7.6 Hz, 2H, −CH2−), 1.15 (t, 3J = 7.6 Hz, 3H, −CH3). Synthesis of L-Homocysteine (Hcy). L-Methionine (5.62 g, 37.67 mmol) was placed in a three-necked round-bottom flask and cooled to −60 °C (acetone/dry ice). Ammonia gas was condensed into the reaction flask through a gas injection apparatus to solve the Lmethionine. With the addition of sodium (2.69 g, 116.78 mmol) in small portions the colorless solution turned dark blue. The blue color vanished after a view minutes, and a colorless precipitate formed. After complete addition of the sodium the solution stayed dark-blue and was stirred for an additional 30 min. After this, ammonium chloride was added in small portions until the blue color disappeared completely.38 The cooling bath (acetone/dry ice) was removed, and a slight stream of nitrogen was passed through the apparatus to remove the ammonia. The dry residue was taken up with degassed water. Concentrated hydrochloric acid was added slowly until a pH of 5.5 was reached. The solution was degassed via freeze−pump−thaw and filtrated afterward. The filtrate was stored at 4 °C until the product crystallized to yield 4.76 g (34.98 mmol, 93%) of L-homocysteine. 1H NMR (400 MHz, D2O) δ [ppm] = 4.02 (dd, 3J = 7.2, 5.6 Hz, 1H, −CH−), 2.84−2.72 (m, 2H, HS-CH2−), 2.36−2.20 (m, 2H, HS− CH2−CH2−). 13C NMR (101 MHz, D2O): δ [ppm] = 174.41 (COOH), 53.74 (NH2−CH−COOH), 34.69 (HS−CH2−CH2−), 20.06 (HS−CH2). ESI-MS: Hcy (calcd. 135.18): 136.06 ([M+H]+). Synthesis of S-Ethylsulfonyl-L-homocysteine (Hcy(SO2Et)). An ice-cold solution of sodium nitrite (1.62 g, 23.5 mmol) in degassed water (8 mL) was added slowly to a cooled solution of Lhomocysteine (3.2 g, 23.5 mmol) in 25 mL of degassed water and 22.5 mL of hydrochloric acid (2 M). It is crucial to keep the solution temperature around 0 °C during the whole addition to prevent the formation of nitrous gases. After complete addition, the deep-red solution was stirred for an additional 30 min at 0 °C. A solution of ethanesulfinic acid sodium salt (6.8 g, 58.75 mmol) in 10 mL of degassed water and 22.5 mL of hydrochloric acid (2 M) was added, and the reaction mixture was stirred overnight at 0 °C with a gentle flow of nitrogen over the reaction mixture. The colorless solution was stored at 4 °C for several days, and eventually S-ethylsulfonyl-Lhomocysteine (3.6 g, 15.6 mmol, 66%) precipitated. If no precipitation occurred, the water was removed in vacuo, and the residue was suspended in degassed ethanol and filtered. Ethanol was removed from the filtrate in vacuo, resulting a slightly yellow oil, which contains the product as well as byproducts. The product was isolated via semipreparative HPLC (Column: Phenomenex Luna C18(2); size: 250 × 30 mm2; eluent: water/MeOH with the following gradient: 1 min: 90/10; 16 min: 40/60; 17 min: 0/100; 20.5 min: 0/ 100; 21 min: 90/10). The product peak was collected in several runs, methanol was removed in vacuo, and the solution was freeze-dried. After lyophilization 546 mg (2.4 mmol, 10%) of S-ethylsulfonyl-Lhomocysteine was obtained as a sticky, colorless solid. For further characterization, a small amount of the product was recrystallized from water and analyzed via X-ray crystallography. Crystal structure: CCDC-1858027. 1H NMR (400 MHz, D2O) δ [ppm] = 3.87 (t, 1H, −CH−), 3.62 (q, 3J = 7.28 Hz, 2H, CH3−CH2−SO2−), 3.40−3.27 (m, 2H, −S−CH2−), 2.42−2.30 (m, 2H, −S−CH2−CH2−), 1.44 (t, 3 J = 7.28 Hz, 3H, CH3−CH2−SO2−). 13C NMR (101 MHz, D2O): δ [ppm] = 173.33 (−COO), 56.42 (−CH2−SO2−), 53.24 (−CH−), 31.42 (−S−CH2−), 31.12 (−CH−CH2−CH2−), 7.58 (−CH3). ESIMS: Hcy(SO2Et) (calcd. 227.03): 228.04 (M + H+), 250.02 ([M +Na]+). Synthesis of S-Ethylsulfonyl-L-homocysteine N-Carboxyanhydride (Hcy(SO2Et)-NCA). Dried S-ethylsulfonyl-L-homocysteine

thiol protective group. Only recently we were able to realize Salkylsulfonyl protecting groups for cysteine-based polypeptides, which fulfill the requirements mentioned above.31,32 It has been shown that the thiol in the side chain is activated toward the reaction with other thiols under formation of asymmetric disulfides and is simultaniously deactivated against the reaction with amines, which ensures stability not only during NCA synthesis, but also under polymerization conditions or solid phase peptide synthesis (SPPS).31,32 Because of the balanced reactivity profile of the Sethylsulfonyl protecting group, well-defined homopolymers could be synthesized with dispersity indices lower than 1.2 and chain lengths of up to 50.31 The limitation regarding the maximum achievable molecular weight is attributed to the formation of β-sheets, which causes aggregation and precipitation of the growing polycysteine chains.35 By using L-homocysteine instead of L-cysteine, this limitation may be resolved, since polyhomocysteine is known to form α-helical secondary structures, which prevents the growing chains from precipitation.36 Therefore, polymerization may be carried out to higher degrees of polymerization without a loss in polymerization control. This work describes the synthesis of S-ethylsulfonyl-Lhomocysteine NCA (Hcy(SO2Et)-NCA) out of L-methionine, its nucleophilic ring-opening polymerization, and the postpolymerization modifications of the corresponding polypeptides. We report on the polymer properties, as well as the reaction kinetics under various conditions, and relate the results to the analogue polycysteine derivatives. Finally, the PPM of the synthesized polymers will be presented, which displays chemoselective formation of asymmetric disulfides.

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EXPERIMENTAL SECTION

THF, n-hexane, and diethyl ether were distilled from Na. DMF (99.8%, extra dry over molecular sieve (4 Å) with AcroSeal) was purchased from Acros. Prior to use, DMF was degassed in vacuo to remove traces of dimethylamine. HFIP was purchased from Fluorochem. Millipore water was prepared by a MILLI-Q Reference A+ System. Neopentylamine was purchased from TCI Europe, dried over NaOH for several days, and fractionally distilled before use. LMethionine was purchased from OPREGEN and used as received. Diphosgene was purchased from Alfa Aesar and deuterated solvents from Deutero GmbH. Other chemicals were purchased from SigmaAldrich and used as received unless otherwise stated. 1H and 13C NMR spectra were recoreded on a Bruker AC 400 at a frequency of 400 and 101 MHz, respectively. 1H NMR spectra were also recorded on a Bruker Avance III HD 300 at 300 MHz. Two-dimensional NMR spectra such as DOSY, COSY, HSQC, and HMBC were recorded on a Bruker Avance III HD 400 at 400 MHz and 101 MHz. All spectra were recorded at room temperature (25 °C) and calibrated using the solvent signals.37 Melting points were measured using a Mettler FP62 melting point apparatus at a heating rate of 1 °C min−1. Gel permeation chromatography (GPC) was performed with hexafluoroisopropanol (HFIP) containing 3 g L−1 potassium trifluoroacetate (KTFA) as eluent at 40 °C and a flow rate of 0.8 mL min−1. The columns were packed with modified silica (PFG columns particle size: 7 μm; porosity: 100 and 1000 Å). Poly(methyl methacrylate) standards (PMMA, Polymer Standards Services GmbH) were used for calibration, and toluene was used as the internal standard. A refractive index detector (G1362A RID) and an UV/vis detector (at 230 nm unless otherwise stated, Jasco UV-2075 Plus) were used for polymer detection. Infrared (IR) spectroscopy was performed on a Jasco FT/IR-4100 with an ATR sampling accessory (MIRacle, Pike Technologies), and Spectra Manager 2.0 (Jasco) was used for integration. B


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Figure 1. (A) Synthesis of Hcy(SO2Et) NCA. (B) 1H NMR of Hcy(SO2Et) with corresponding crystal structure. (C) 1H NMR of Hcy(SO2Et) NCA with corresponding crystal structure. stronger NCA carbonyl peak at 1788 cm−1 was used to correlate the integral of the peak to the NCA concentration. Polymerizations performed at higher initial monomer concentrations exhibited faster rate constants. Circular Dichroism (CD) Spectroscopy. CD experiments were performed on a Jasco J-815 spectrometer at room temperature (25 °C), and Spectra Manager 2.0 (Jasco) was used to analyze the spectra. A cell with a path length of 2 mm was used. Spectra were recorded at a concentration of 0.25 g L−1 polymer in HFIP. ΘMR was calculated using the following equation with MRepeating Unit = 209.3 g mol−1, cM = 0.25 g L−1, and l = 0.2 cm.

(2.43 g, 10.703 mmol) was suspended in absolute THF (200 mL) at room temperature (25 °C) in a nitrogen atmosphere. Diphosgene (1.034 mL, 1.706 g, 8.563 mmol) was added at once, and the suspension became clear after 1 min. The colorless solution was stirred for an additional 5 min before THF, excess diphosgene and HCl were removed in vacuo. The residue was dissolved in 30 mL of absolute ethyl acetate, and any insoluble compounds were removed by filtration, avoiding contact with air. The NCA was precipitated by adding a mixture of 150 mL of absolute n-hexane and 150 mL of absolute diethyl ether. Filtration in nitrogen atmosphere yielded Sethylsulfonyl-L-homocysteine NCA (1.2 g, 4.74 mmol, 44%) as a colorless powder; mp 95 °C. For further characterization, a small amount of the product was recrystallized from dry THF and analyzed via X-ray crystallography. Crystal structure: CCDC-1858028. 1H NMR (300 MHz, [D6]DMSO) δ [ppm] = 9.14 (s, 1H, NH), 4.53 (ddd, 3J = 7.77, 5.52, 1.08 Hz, 1H, −CH−), 3.57 (q, 3J = 7.26 Hz, 2H, CH3−CH2−SO2−), 3.29−3−18 (m, 2H, −S−CH2−), 2.24−2.07 (m, 2H, −S−CH2−CH2−), 1.30 (t, 3J = 7.26 Hz, 3H, CH3−CH2− SO2−). 13C NMR (101 MHz, [D6]DMSO): δ [ppm] = 171.44 (−COO), 152.27 (−CNOO), 56.33 (−CHN−), 56.07 (−CH− CH2−CH2−), 31.8 (−S−CH2−), 31.5 (−SO2−CH2−), 8.7 (−CH3). Ring-Opening Polymerization of Hcy(SO2Et)-NCA. Hcy(SO2Et)-NCA (59.4 mg, 0,235 mmol) was transferred into a Schlenk tube under dry nitrogen counterflow. Dry DMF (2 mL) and absolute neopentylamine (0,37 μL, 0.237 mg, 0.003 mmol) were added, and the vessel was cooled to −10 °C. A steady flow of dry nitrogen was sustained during the polymerization, preventing any impurities from entering the Schlenk tube, while ensuring the escape of produced CO2. The progress of the polymerization was monitored via IR spectroscopy by the disappearing intensities of the NCA carbonyl peaks at 1858 and 1788 cm−1. Samples were taken using a nitrogen flushed syringe through a septum. The polymer was precipitated after complete conversion in cold diethyl ether. The suspension was centrifuged (4500 rpm, 10 min, 0−5 °C) and decanted. This procedure was repeated three times yielding poly(S-ethylsulfonyl-Lhomocysteine) (45 mg, 71%) as a colorless solid. 1H NMR (400 MHz, [D6]DMSO) δ [ppm] = 8.35 (m, 1n H, NH), 4.35 (m, 1n H, α-H), 3.56 (q, 2n H, −CH2−), 3.14 (m, 2n H, −CH2−), 2.01 (m, 2n H, −CH2−), 1.30 (t, 3n H, −CH3), 0.85 (s, 9H, −(CH3)3). Kinetic Measurements. Polymerizations were analyzed until complete conversion was reached using a nitrogen flushed syringe through a septum. The decreasing NCA carbonyl peaks at 1858 and 1788 cm−1 were monitored. For the kinetic evaluations only the

θMR =

θMRepeating Unit 10cMl

[deg cm 2 dmol−1]

SH Reactivity. To investigate the ability of p(Hcy(SO2Et)) to react with thiols in a postpolymerization modification (PPM) reaction, a mixture of p(Hcy(SO2Et)) and benzyl mercaptan in [D6]DMSO was monitored by 1H NMR. Therefore, p(Hcy(SO2Et))11 (P1, 7 mg, 2.93 μmol, 1.0 equiv) was dissolved in [D6]DMSO (0.6 mL), and benzyl mercaptan (4.404 mg, 35.45 μmol, 1.1 equiv with respect to Xn Hcy) in [D6]DMSO (0.1 mL) was added immediately prior to the measurement. The shift of the CH3 signal from the sulfonyl group (−SO2Et) was used to monitor the conversion. Additionally, the conjunction of the relevant signals to a polymeric species was confirmed by diffusion-ordered 1H NMR experiments (1H DOSY NMR).

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RESULTS AND DISCUSSION Monomer synthesis was performed using L-methionine as the starting material, which was reduced with elemental sodium in liquid ammonia (analogue to the Birch reaction39,40). After evaporation of ammonia, the crude product was dissolved in degassed water, and the pH value was carefully adjusted to the isoelectric point of L-homocysteine (pH = 5.5). The product (L-homocysteine) could be isolated in near-quantitative yields (>90%) in high purity by recrystallization from water. In the next step, the protection of the thiol group was performed in analogy to the already synthesized cysteine derivative31,32 via umpolung of the thiol group, which was performed in a nitrosation reaction with sodium nitrite followed by reaction with sodium ethanesulfinate.41 C


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Figure 2. (A) Polymerization of Hcy(SO2Et) NCA with neopentylamine as initiator. (B) HFIP GPC traces and CD spectra (C) of p(Hcy(SO2Et)) at various degrees of polymerization (see also Table 1).

Table 1. Comparison of Degrees of Polymerization (Calculated via 1H NMR End-Group Analysis) and Molecular Weights (Calculated via 1H NMR End-Group Analysis and Detected by HFIP-GPC) P1 P2 P3 P4 P5 P6

M/I

Xn(NMR)a

Mn(calcd)

Mn(NMR)a

Mn(SEC)b

Đ(SEC)b

10 15 25 50 100 150

11 19 35 45 80 165

2180 3227 5320 10552 21017 31482

2390 4064 7413 9505 16831 34621

3507 5166 7534 8320 14694 20856

1.34 1.24 1.18 1.20 1.18 1.12

a

Determined by 1H NMR in DMSO-d6. bDetermined by HFIP-SEC using PMMA standards.

polymerizations can be performed to full conversion displays a major difference between poly(S-ethylsulfonyl-L-homocysteine) (p(Hcy(SO2Et))) and poly(S-ethylsulfonyl-L-cysteine) (p(Cys(SO2Et))). Assessment of the degree of polymerization was performed by 1H NMR, since the singlet at 0.83 ppm, which originates from the nine methyl protons of the initiator (neopentylamine), can be related to the protons of the polymer backbone as well as to the ones of the side chain (Figure S3). To confirm that all the peaks originate from one polymer species 1H DOSY NMR experiments were carried out. The DOSY spectra in Figure S4 show two diffusing species of which one is the solvent (DMSO-d6 and HDO) while all other signals correspond to p(Hcy(SO2Et)). This validates the determination of the degree of polymerization by the use of end-group analysis. The stability of the S-ethylsulfonyl protecting group during the polymerization can be verified by comparing the protons related to the backbone, i.e., the α-proton at 4.35 ppm, with the side chain protons at 3.56 and 1.31 ppm, respectively (Figure S3). These findings are in line with the HFIP-GPC traces (Figure 2B) since no species at lower elution volume is detectable, which would originate from aggregate formation or crosslinked structures due to protective group cleavage. If the protective group is cleaved during polymerization, the resulting thiol in the side chain will undergo a nucleophilic attack of another side chain or the NCA monomer, which would ultimately lead to polymeric species with higher molecular weight. In Table 1 it can be seen that all homopolymers (except P1 + P2) display dispersity indices below 1.2, which underlines with the symmetric GPC plots the controlled living nature of the polymerization. The low-molecular-weight shoulders of P1 and P2 can be explained with coexistence of two secondary structures, namely random coil and α-helix, which differ substantially in

In contrast to the cysteine-based derivatives, numerous side reactions occurred, likely leading to the formation of ethanesulfinate and −sulfonate, as well as ethanesulfohydroxamic acid, which requires the purification of Sethylsulfonyl-L-homocysteine by semipreparative high-pressure liquid chromatography (semiprep HPLC, Figure S1). The protected homocysteine derivative (S-ethylsulfonyl-L-homocysteine) was then transferred to the corresponding NCA by the Fuchs−Farthing method using diphosgene42−44 and could be isolated by recrystallization from ethyl acetate and nhexanes/diethyl ether (1:1) (melting point: 95 °C) in 44% yield. The NCA crystals have been characterized by X-ray crystallography to ensure that the absolute configuration of the amino acid was preserved during the three-step synthesis. Corresponding crystal structures are displayed in Figure 1, which also proves that the information about the stereogenic center (L-configuration) is kept over all three steps and no racemization occurs. Moreover, an interesting effect can be observed in the arrangement of the individual NCA molecules within the crystal. In Figure S2 it can be seen that the NCA molecules already tend to form an α-helix in the crystal lattice, which is the proposed secondary structure of the corresponding polymer and will be further discussed later. The polymerization of the S-ethylsulfonyl-L-homocysteine NCA was performed in absolute DMF using previously dried and freshly distilled neopentylamine as the initiator (Figure 2A). The polymerization was carried out to full conversion was ensured via IR spectroscopy by monitoring the NCAs carbonyl stretching frequencies at 1786 and 1854 cm−1, since the decreasing intensity is directly related to the conversion of the monomer. For quantitative determination, the peak at 1786 cm−1 was used. As soon as full conversion was observed (Figure 4A), the polymer was precipitated in diethyl ether. The fact that D


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symmetric distribution of the polymer (P3). The degree of polymerization determined via 1H NMR (Xn,NMR = 35) varies from the one that is displayed in the mass spectrum (Xn,MALDI‑ToF = 24). This indicates the presence of pronounced mass discrimination effects, which originate from the effect that heavier polymer chains are transferred to the gas phase with lower efficiency than shorter chains, and therefore the mass spectra are usually biased toward lower molecular weights. Already at DP of around 30, the distribution is not perfectly symmetric and starts to shift toward lower m/z ratios. Nevertheless, the performed MALDI-ToF MS experiments clearly demonstrate that each polymer chain is equipped with the initiator (neopentylamine) and the amine end group. Moreover, no products of side reactions (e.g., initiation by impurities or water) can be detected. The most prominent signals are sodium (+23) and potassium (+39) adducts. The additional peaks with a m/z difference of 114 can be assigned to species where the amine end group is quarternized with trifluoracetic acid (TFA), which is a result of the sample preparation, since ionization was done by adding NaTFA to the sample (Supporting Information). Further magnification of the main peak displays the broadening of the peaks, which is a result of the isotopic distribution of sulfur and carbon atoms. Because every repeating unit contains two sulfur atoms each with two prominent isotopes (32S and 34S), the signals for each species are broadened with a distinct distribution. The signal broadening, however, is perfectly in line with a simulated spectra of p(Hcy(SO2Et)) + Na+ with Xn = 24 taking the isotopic pattern into account (Figure 3B). In summary, MALDI-ToF analysis clearly illustrates the absence of side reactions and verifies the incorporation of initiator in all polymer chains, but it also shows that the determination of Xn at higher degrees of polymerization is not comparable with NMR end-group analysis due to mass discrimination and sample preparation. To underline the living character of the polymerization furthermore, synthesis of a diblock copolymer has been conducted. For this purpose dried P3 has been dissolved in dry DMF to use it as an initiator for the polymerization of sarcosine NCA. In the GPC plot of P3 and the diblock copolymer (Figure S8) it can be clearly seen that the block copolymer shifts to higher molecular weights. The number of sarcosine units was determined via NMR end-group analysis (Figure S6), and an additional DOSY experiment (Figure S7)

hydrodynamic volume. It has been already shown that i.e. p(Lys(Z)) and p(Lys(TFA)) have a transition in their secondary structure from random coil to α-helix and therefore show a bimodal distribution in the GPC of comparable DPs.45 This effect vanishes with higher degrees of polymerization because at some point all polymer chains are in the same secondary structure (i.e., α-helix). In case of p(Hcy(SO2Et)) the transition range seems to be around DP 20, since polymers with higher degrees of polymerization show exclusively monomodal distributions. It is worth to point out the polymer P6 which possesses besides a low dispersity of 1.12 additionally a high degree of polymerization of 165, as determined via NMR end-group analysis, which is 4-fold higher than the highest degree of polymerization of the previously reported polycysteines.32 It has to be noted that the molecular weights obtained by GPC have to be considered as relative values not as absolute and can only be compared with each other, since the calibration was performed with PMMA standards. Recent studies have shown that the Rh values of PMMA in HFIP are anomalously high compared to other solvents such as acetone, leading to an underestimation of molecular weights of polypeptides.46,47 Further characterization of the p(Hcy(SO2Et)) polypeptides by MALDI-ToF MS analysis underlines the absence of side reactions. As can be seen in Figure 3A, the spectrum shows a

Figure 3. (A) MALDI-ToF spectrum of p(Hcy(SO2Et)) with Xn,NMR = 35 (P3). (B) Overlay of simulation and measurement of the isotopic pattern of p(Hcy(SO2Et)) with Xn = 24.

Figure 4. (A) Generic FT-IR spectra of a p(Hcy(SO2Et)) polymerization at different reaction times (in DMF, liquid film). Polymerization was performed with a cNCA = 0.1 mol/L and M/I = 75. (B) Kinetic plot of p(Hcy(SO2Et)) polymerizations at varying monomer concentrations with constant monomer-to-initiator ratio (M/I = 50) and (C) kinetic plots at varying monomer-to-initiator ratios and constant monomer concentrations (cNCA = 0.1 mol/L). Full conversion was ensured in all cases via FT-IR monitoring. E


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Macromolecules ensured that all signals of the proton spectrum correspond to one single diffusing polymeric species. Both the DOSY spectrum and the GPC plot verify that initiation of water or other impurities can be dismissed, since no sarcosine homopolymer is detectable. Analysis of the secondary structure formation in solution has been performed via CD spectroscopy to confirm the α-helix conformation, which can be seen in Figure 2C. The measurements underline that the protecting group (S-ethylsulfonyl) does not alter the secondary structure, since poly(Lhomocysteine) is already known to form α-helical secondary structures.48 Additionally, it can be seen that the α-helical character gets more pronounced with higher degrees of polymerization. Furthermore, solid-state IR spectroscopy was performed (Supporting Information), which also confirms these findings by the appearance of two strong amide bands at 1546 and 1650 cm−1.49−51 For the determination of polymerization kinetics samples were taken at different time points and IR spectroscopy was used to monitor monomer consumption by the decrease in carbonyl peak intensity at 1854 and 1786 cm−1. All samples were taken with previously N2 flushed syringes to prevent contamination. For the calculation of the rate constants only the prominent peak at 1786 cm−1 was used, since the peak at 1854 cm−1 already vanished completely before full conversion was reached (see Figure 4A). The area under the curve was used for each individual time point and compared with the value, which was determined right after the initiation (M0). Since most NCA polymerizations follow pseudo-first-order kinetics, the rate law is given by the following equation: −

d[M] = k[I][M] dt

Table 2. Apparent Rate Constants of Polymerizations Determined via FT-IR Spectroscopy with Monomer-toInitiator Ratios (M/I) of 50a amino acid NCA

concentration [mol/L]

kapp [s−1]

Hcy(SO2Et)-NCA

0.1 0.3 0.5 0.17 0.43 0.59

1.44 × 10−5 4.33 × 10−5 5.29 × 10−5 3.43 × 10−6 8.09 × 10−6 11.31 × 10−6

Cys(SO2Et)-NCA

All values are determined at −10 °C with different NCA concentrations. a

To gain further insight into the kinetic behavior of the polymerization of Hcy(SO2Et)-NCA, two more variations of the reaction conditions have been performed. In Figure 4C, rate constants are determined for different M/I ratios, while the initial monomer concetration was maintained. As can be seen in Figure 4C,the rate constant rises from 14.4 × 10−6 s−1 at M/I = 50 to 1.10 × 10−6 s−1 at M/I = 100 (see Figure 4C) was obtained, which is in line with the rate law (1). Additionally, the influence of different temperatures on the polymerization was studied (Figure 5). A higher temperature may lead to a higher reaction rate but may vice versa lead to side reactions (Figure 5B). The polymerizations performed at −10 and 0 °C show a symmetric distribution in the GPC, while the polymerization at 10 °C shows a shoulder at lower molecular weights. This is due to protective group cleavage or

(1)

with [I] = initiator concentration, [M] = monomer concentration, and k = rate constant. As the concentration of active chain ends corresponds to the amount of initiator used and thus remains constant over the whole time period of the polymerization, k[I] can be reduced to an apparent value kapp. Integration of the formula yields iM y ln(M 0) − ln(M ) = lnjjj 0 zzz = kappt kM{

(2)

M0 M

( ) versus the reaction time (t) should

Therefore, plotting ln

lead to a linear relation, which indeed can be observed and is illustrated in Figure 4B,C. According to eqs 1 and 2, the velocity of the polymerization is dependent from the starting monomer concentration (M0), which was verified by using different monomer concentrations while keeping the monomer to initiator ratio (M/I) constant for all three performed polymerizations (Figure 4B). Here, an increase of the apparent rate constant from 1.44 × 10−5 to 5.29 × 10−5 s−1 can be seen by rising the initial monomer concentration from 0.1 to 0.5 mol/L. In comparison to the Cys(SO2Et)-NCA, the polymerization of the Hcy(SO2Et)-NCA is up to 5 times faster (values are displayed in Table 2), which may originate from the different secondary structures as reported by Weingarten et al.52 With the formation of α-helices the addition of another NCA is more favorable, and therefore the reaction rate of p(Hcy(SO2Et)) is enhanced compared to the formation of βsheets in the case of p(Cys(SO2Et)).53

Figure 5. (A) Kinetic plot of the p(Hcy(SO2Et)) polymerizations at varying temperatures and corresponding HFIP-GPC traces. (B) Monomer-to-initiator ratios and monomer concentrations were the same at all measurements (M/I = 50, cNCA = 0.3 mol/L). Full conversion was ensured in all cases via FT-IR. Large extrapolation of the curve at 0 °C was made to clarify the difference to the curve at −10 °C. F


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Figure 6. Scheme of the reaction of p(Hcy(SO2Et)11) (P1) with benzyl mercaptan. (B) 1H NMR spectrum of p(Hcy(SO2Et)11) (P1) prior to conversion. (C) 1H NMR spectrum after conversion with benzyl mercaptan. (D) Enlarged comparison of both 1H NMR spectra emphasizing the quantitative conversion (disappearance of polymer associated protecting group signal at 1.31 ppm and emerging signal at 1.08 ppm of the fully converted protecting group). (E) 1H DOSY NMR spectrum of p(Hcy(SO2Et)11) (P1) prior to conversion. (F) 1H DOSY NMR spectrum after conversion with benzyl mercaptan showing one single diffusing polymeric species.

which corresponds to the polymer bound protective group (f), to 1.08 ppm, which is originating from the free ethanesulfinate (5). All signals associated with the polymer backbone (a−d) and the end group (g) remain present. Due to the slight excess of benzyl mercaptan, signals of the free species (1, 2) and polymer bound mercaptan (e′, f′) are present in the proton spectrum as well as in the DOSY spectrum. Here,the peaks of the aromatic protons (1, f′) at 7.29 ppm overlap and cannot be distinguished in contrast to the protons in the benzylic position (2, e′). The DOSY spectrum after conversion (Figure 6E,F) shows the association of the aromatic and benzylic protons to the diffusing polymer species. Additionally, free ethanesulfinate, benzyl mercaptan, and traces of solvents resulting from the polymerization (DMF), as well as the DMSO signal can be seen. All NMR data underline that the S-ethylsulfonyl protective group is highly reactive toward free thiols without generating any detectable side products, which establishes the polymer as a valuable material for the chemoselective formation of asymmetric disulfides.

overlay of activated monomer mechanism with the normal amine mechanism in NCA polymerization at elevated temperatures, as reported by Vayaboury et al.54 and Habraken et al.,55 which ultimately leads to unsymmetrical or multimodal GPC plots. In summary, we can conclude that the polymerization of Hcy(SO2Et)-NCA can be performed under controlled living conditions at −10 or 0 °C, leading to the formation of α-helical polymers. In comparison to the polymerization of Cys(SO2Et)NCA not only reaction rates can be enhanced significantly but most importantly higher degrees of polymerization of up to 165 can be achieved by amine initiated ring-opening polymerization. In the last part of this work, the ability of the polymer to undergo chemoselective disulfide formation with thiols was investigated by 1H NMR and 1H-DOSY NMR experiments. For this purpose, the polymer P1 was dissolved in d6-DMSO, and benzyl mercaptan was added prior to the measurement in slight stoichiometric excess to ensure that each homocysteine residue can be fully converted to the corresponding disulfide. Benzyl mercaptan engages with the protective group in the side chain in a nucleophilic substitution reaction where the asymmetric disulfide is formed and ethanesulfinate is liberated (Figure 6A). The reaction was performed in the NMR tube and reached quantitative conversion after ∼180 s (Figure 6B,C). With the assumption of first-order reaction kinetics for the nucleophilic substitution reaction a reaction, rate of kapp > 1.33 L s−1 mol−1 can be estimated. Comparable fast reaction kinetics could already be observed for the previously reported cysteine derivatives and suggest similar reactivities toward thiols. Figure 6D shows the shift of the triplet from 1.31 ppm,

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CONCLUSION

In this work, we have presented the successful synthesis and ROP of S-ethylsulfonyl-L-homocysteine NCA under various conditions. We could show that the polymerization at −10 and 0 °C proceeds in a controlled living fashion with rate constants at kapp = 4.33 × 10−5 (−10 °C, c = 0.3 mol/L) to kapp = 5.29 × 10−5s−1 (0 °C, c = 0.3 mol/L), in which side reactions cannot be detected. The absence of side products has been ensured by MALDI-ToF MS analysis. Moreover, HFIP-GPC traces display monomodal, symmetrical, and narrow distributed polypepG


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(5) Bonduelle, C.; Lecommandoux, S. Synthetic Glycopolypeptides as Biomimetic Analogues of Natural Glycoproteins. Biomacromolecules 2013, 14 (9), 2973−2983. (6) Kricheldorf, H. R. α-Aminoacid-N-Carboxy-Anhydrides and Related Heterocycles: Syntheses, Properties, Peptide Synthesis, Polymerization; Springer: Berlin, 1987. (7) Nitta, S. K.; Numata, K. Biopolymer-Based Nanoparticles for Drug/Gene Delivery and Tissue Engineering. Int. J. Mol. Sci. 2013, 14 (1), 1629−1654. (8) Li, Y.; Rodrigues, J.; Tomás, H. Injectable and Biodegradable Hydrogels: Gelation, Biodegradation and Biomedical Applications. Chem. Soc. Rev. 2012, 41 (6), 2193−2221. (9) Duro-Castano, A.; Conejos-Sánchez, I.; Vicent, M. J. PeptideBased Polymer Therapeutics. Polymers (Basel, Switz.) 2014, 6 (2), 515−551. (10) Zhang, R.; Song, Z.; Yin, L.; Zheng, N.; Tang, H.; Lu, H.; Gabrielson, N. P.; Lin, Y.; Kim, K.; Cheng, J. Ionic α-Helical Polypeptides toward Nonviral Gene Delivery. Wiley Interdiscip. Rev. Nanomedicine Nanobiotechnology 2015, 7 (1), 98−110. (11) Fleige, E.; Quadir, M. A.; Haag, R. Stimuli-Responsive Polymeric Nanocarriers for the Controlled Transport of Active Compounds: Concepts and Applications. Adv. Drug Delivery Rev. 2012, 64 (9), 866−884. (12) Liu, M.; Du, H.; Zhang, W.; Zhai, G. Internal StimuliResponsive Nanocarriers for Drug Delivery: Design Strategies and Applications. Mater. Sci. Eng., C 2017, 71, 1267−1280. (13) Shim, M. S.; Kwon, Y. J. Stimuli-Responsive Polymers and Nanomaterials for Gene Delivery and Imaging Applications. Adv. Drug Delivery Rev. 2012, 64 (11), 1046−1059. (14) Birke, A.; Ling, J.; Barz, M. Polysarcosine-Containing Copolymers: Synthesis, Characterization, Self-Assembly, and Applications. Prog. Polym. Sci. 2018, 81, 163−208. (15) Magnusson, J. P.; Saeed, A. O.; Fernández-Trillo, F.; Alexander, C. Synthetic Polymers for Biopharmaceutical Delivery. Polym. Chem. 2011, 2 (1), 48−59. (16) Canal, F.; Sanchis, J.; Vicent, M. J. Polymer-Drug Conjugates as Nano-Sized Medicines. Curr. Opin. Biotechnol. 2011, 22 (6), 894− 900. (17) Deming, T. J. Synthetic Polypeptides for Biomedical Applications. Prog. Polym. Sci. 2007, 32 (8−9), 858−875. (18) Deng, C.; Wu, J.; Cheng, R.; Meng, F.; Klok, H. A.; Zhong, Z. Functional Polypeptide and Hybrid Materials: Precision Synthesis via α-Amino Acid N-Carboxyanhydride Polymerization and Emerging Biomedical Applications. Prog. Polym. Sci. 2014, 39 (2), 330−364. (19) Kramer, J. R.; Deming, T. J. Preparation of Multifunctional and Multireactive Polypeptides via Methionine Alkylation. Biomacromolecules 2012, 13 (6), 1719−1723. (20) Quadir, M. A.; Martin, M.; Hammond, P. T. Clickable Synthetic Polypeptides - Routes to New Highly Adaptive Biomaterials. Chem. Mater. 2014, 26 (1), 461−476. (21) Rhodes, A. J.; Deming, T. J. Soluble, Clickable Polypeptides from Azide-Containing N-Carboxyanhydride Monomers. ACS Macro Lett. 2013, 2 (5), 351−354. (22) Schäfer, O.; Barz, M. Frontispiece: Of Thiols and Disulfides: Methods for Chemoselective Formation of Asymmetric Disulfides in Synthetic Peptides and Polymers. Chem. - Eur. J. 2018, DOI: 10.1002/ chem.201884763. (23) Klinker, K.; Schäfer, O.; Huesmann, D.; Bauer, T.; Capelôa, L.; Braun, L.; Stergiou, N.; Schinnerer, M.; Dirisala, A.; Miyata, K.; et al. Secondary-Structure-Driven Self-Assembly of Reactive Polypept(o)Ides: Controlling Size, Shape, and Function of Core Cross-Linked Nanostructures. Angew. Chem., Int. Ed. 2017, 56 (32), 9608−9613. (24) Schäfer, O.; Klinker, K.; Braun, L.; Huesmann, D.; Schultze, J.; Koynov, K.; Barz, M. Combining Orthogonal Reactive Groups in Block Copolymers for Functional Nanoparticle Synthesis in a Single Step. ACS Macro Lett. 2017, 6 (10), 1140−1145. (25) Huang, J.; Habraken, G.; Audouin, F.; Heise, A. Hydrolytically Stable Bioactive Synthetic Glycopeptide Homo-and Copolymers by

tides. In addition, even at high degrees of polymerization of Xn = 165, aggregation of polypeptides cannot be detected, which is of major importance, since aggregation limits the chain lengths to around 50 for previously published S-alkylsulfonyl-Lcysteine derivatives. Further, 1H NMR experiments demonstrate also that p(S-ethylsulfonyl-L-homocysteine)s can be used for chemoselective disulfide formation, wherewith the polypeptide has the potential of reversibly cross-linking and/ or binding of peptides or small molecules, such as drugs or dyes. In summary, all these features make the S-ethylsulfonyl-Lhomocysteine polymer a valuable reactive polypeptide, which nicely adds up to the already established platform of reactive polypept(o)ides for reversible disulfide cross-linking and bioconjugation.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.8b01442. Materials, experimental information, Figures S1−S10, Table S1, GPC and NMR spectra (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(M.B.) E-mail: [email protected]. ORCID

Hans-Joachim Räder: 0000-0002-7292-4013 Matthias Barz: 0000-0002-1749-9034 Funding

The SFB 1066-2 is acknowledged for financial support. O.S. acknowledges support by the “Evangelisches Studienwerk e.V. Villigst”. C.M. acknowledges support by the “EU”. T.B. acknowledges financial support of the HaVo Foundation and the graduate school MAINZ. Notes

The authors declare the following competing financial interest(s): Patent WO2015169908A1, Thiol-protected amino acid derivatives and uses thereof.

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ACKNOWLEDGMENTS We thank Dr. D. Schollmeyer for X-ray crystal structure analysis, Prof. Pol Besenius for providing access to CD spectroscopy, and S. Türk for MALDI−ToF MS measurements.

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REFERENCES

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Poly(S-ethylsulfonyl-l-homocysteine): An Î±-Helical Polypeptide for

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