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. 2020 Jul 23;5(30):19020–19028. doi: 10.1021/acsomega.0c02326

Bifunctional Peptide–Polymer Conjugate-Based Fibers via a One-Pot Tandem Disulfide Reduction Coupled to a Thio-Bromo “Click” Reaction

Sonu Kumar †,‡, Gerd Hause §, Wolfgang H Binder †,*
PMCID: PMC7408259  PMID: 32775904

Abstract

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In view of the potential applications of fibers in material sciences and biomedicine, an effective synthetic strategy is described to construct peptide-based bifunctional polymeric conjugates for supramolecular self-association in solution. A direct coupling method of an α-acyl-brominated peptide Phe-Phe-Phe-Phe (FFFF) with a disulfide-bridged polymeric scaffold of poly(ethylene glycol) (PEG) (Mn,GPC = 8700 g mol–1, Đ = 2.02) is reported to readily prepare the bi-headed conjugate FFFF-PEG-FFFF (Mn,GPC = 3800 g mol–1, Đ = 1.10) via a one-pot, tandem disulfide reduction (based on tris(2-carboxyethyl)phosphine hydrochloride (TCEP)) coupled to a thio-bromo “click” reaction. The conjugate was investigated via transmission electron microscopy to exploit supramolecular fibril formation and solvent-dependent structuring into macroscale fibers via fibril–fibril interactions and interfibril cross-linking-induced bundling. Circular dichroism spectroscopic analysis is further performed to investigate β-sheet motifs in such fibrous scaffolds. Overall, this synthetic approach opens an attractive approach for a simplified synthesis of PEG-containing peptide conjugates.

Introduction

Peptide/protein-based self-assembled fibrils/fibers have emerged as promising structured biomaterials with improved mechanical properties and/or functionalities.1−4 Such fibril-based higher-ordered supramolecular structures are considered as building blocks for the design of biohybrid materials for applications in biomedicine, such as drug delivery, tissue engineering, and biosensors.5−8 Rieger and Stoffelbach et al. recently documented an amphiphilic supramolecular-system for the direct access to polymer decorated fiber morphologies by polymerization-induced self-assembly in water.9 Amphiphilic peptide systems in particular have been found to be advantageous in controlling, directing, and using artificial fibers for biomedical purposes, and have been earlier reviewed by several groups.10−12 Stupp and co-workers pioneered the field by demonstrating that various peptide-amphiphile allowed their supramolecular arrangement via induced self-assembly into one-dimensional nanofibers,10,13,14 and investigated pathway-dependent morphology and internal dynamics of such nanostructures to hold significant promise as advanced hybrid materials for biofunctions.15,16 Furthermore, peptidic fibrils have also been found to be associated with several neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and type II diabetes.17−19 Considering the unique and versatile properties of polymers for advanced applications,20,21 there has been increased scientific interest in the design of peptide-based synthetic polymer conjugates able to fold into supramolecular fibrillar structures,22,23 in particular β-sheet-enriched fibrils,24,25 also as important structural motifs in amyloids19 and silks.26 Peptide-based, polymeric α-helical amphiphiles can act as β-sheet breakers and thus inhibit fibrillation of amyloids.27 However, the efficient synthetic methodology development for such β-sheet-based hierarchical fibrillar bioconjugates28,29 still remains a fundamental challenge and therefore is highly desirable, particularly in the design of biomimetic materials based on such amyloid-like fibrils to explore their associated molecular structuring and mechanism.30,31 Critical in this endeavor is the efficient linkage of fibrillating peptides onto synthetic polymers, able to achieve the chemical connection under conditions where fibrillation does not interfere with the chemical reaction. Especially in aqueous solutions, amphiphilic polymer/peptide conjugates are prone to aggregation, thus often preventing an efficient linkage between the peptide and the (usually) larger-sized polymer. Important thereto is the proper choice of a common solvent for both, the peptide and the polymer, as well as mild reaction conditions, in line with the subsequent potential for biological application. We have previously described32,33 a synthetic methodology to prepare monofunctional polymer–peptide conjugates, taking advantage of poly(ethylene glycol) (PEG)34,35 and the phenylalanine-based hydrophobic tetrapeptide FFFF,36 inspired by the amyloid-β(Aβ)protein core sequence FF (Aβ19–20) explored by Gazit and co-workers.37−39 Our focus in this work is on the synthetic methodology development and the preparation of α- and ω-chain-ends-modified PEGylated peptidic conjugates FFFF-PEG-FFFF (with C-terminal methyl ester units) that are able to self assemble into β-sheet-enriched fibrous structures.40−44 The synthesis of such β-sheet fibril-based bifunctional conjugates and peptide-PEG-peptides has been previously reported by Hamley et al. for the dipeptide YY based conjugate YY-PEG-YY (with N-terminal NH2 or Fmoc units)45 and by Besenius et al. for the pentapeptide FHFHF based conjugate FHFHF-PEG-FHFHF (with acetylated N-termini), employing conventional amide coupling chemistry.46 We here exploit the less explored metal-free thio-bromo based “click” chemistry approach32,47−50 (compared to various other types of thiol-based click reactions, such as the thiol-ene/yne/maleimide/-conjugation51−53) for the synthesis of peptidic polymer conjugates, which plausibly is more promising for bioapplications than the utilization of, e.g., the copper(I) metal-catalyzed azide-alkyne cycloaddition (CuAAC).54,55 The methodology developed here can lead to the widening of the applicability of such unexplored click approach because of its following significant advantages: (1) It represents the first example of the implementation of thio-bromo click reaction directly coupled to a one-pot tandem disulfide reduction of disulfide-bridged PEG polymers (thus improving the ease of implication of such polymers by unveiling this critical issue of disulfide formation). (2) The illustrated coupling reaction commenced in aqueous solution for the synthesis of conjugates (therefore, the strategy can be conveniently adopted for the polymer conjugation with various other bioimolecules such as proteins, mRNAs, etc.)56,57 Conceptually, we focused on an in situ thio-bromo click reaction of the peptide (5) to an in situ generated poly(PEG-disulfide) (4) to expand the utilization of such effective thio-bromo click reaction approach to design structured tailor-made functional polymers for materials and biomedicine.58,59

Results and Discussion

For the preparation of peptide–polymer conjugate, we started from the readily prepared 2-bromo-acyl-derivative of tetraphenylalanine peptide, Br-FFFF-OMe (5), documented in our recent article32 for conjugation to yield the PEGylated conjugate MeO-FFFF-PEG-FFFF-OMe (6). The intermediate peptidic derivatives (Figures S1–S6, Supporting Information) and the final bromo-peptide 5 (Figures S7 and S8, Supporting Information) were synthesized via conventional solution-phase peptide synthesis (Scheme S1, Supporting Information).60 The commercially available PEG polymer, HO-PEG-OH (1), was transformed via mesylation to afford the polymer MsO-PEG-OMs (2) (Scheme 1) (see also 1H and 13C NMR spectra, Figures S9 and S10, Supporting Information), and further via chain-end modification using potassium thioacetate (KSAc) to yield the bis-thioacetate capped, PEGylated polymer AcS-PEG-SAc (3). Proof of the complete transformation was obtained by the appearance of the characteristic proton and carbon signals for the −SCOCH3 moiety at δ 2.33 and 28.83 ppm in the 1H and 13C NMR spectrum, respectively (Figures 1A and S11, Supporting Information.)61

Scheme 1. Synthesis of the FFFF-Peptide-Based Bifunctional Polymer Conjugate 6 via a One-Pot Disulfide Reduction/Thio-Bromo Click Reaction.

Scheme 1

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Figure 1.

Figure 1

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1H NMR spectra of (A) thioacetate PEGylated polymer 3, (B) disulfide-bridged PEGylated polymer 4, and (C) peptide-based bifunctional PEGylated polymeric conjugate 6 (encircled sections represent major peaks from a peptidic constituent, and * denote the solvent resonances).

Electrospray ionization time-of-flight mass spectroscopy (ESI-TOF MS) spectra for polymer 3 further confirmed the molecular structure by illustrating the appearance of peaks with two different series of 1+ and 2+ charged states, where the observed peaks (e.g., the isotopic peak maxima at m/z = 1565.9513 g mol–1 for [M + Na]+ with n = 30, and at m/z = 860.5184 g mol–1 for [M + 2Na]2+ with n = 33, respectively), match with the simulated isotopic patterns of the proposed ions (Figure S12, Supporting Information).47

For the required de-acylation of 3, sodium methoxide (NaOMe) solution was utilized with the initial aim to prepare the free thiol carrying PEG polymer HS-PEG-SH (7).62 However, although great care was taken to perform the deprotection reaction under inert condition with dry reaction solvent, the obtained polymer product was found to form the oligomerized disulfide-bridged PEGylated polymer, poly(PEG-disulfide) (4),63 via instantaneous formation of the disulfide bond linkage upon in situ generation of free thiol chain ends.64 The significantly higher molecular weight of the obtained product 4 was initially assessed by analytical gel permeation chromatography (GPC) based studies (Mn,GPC = 8700 g mol–1, Đ = 2.02), which demonstrated a significant shift in its refractive index (RI) trace, with a peak maximum at a retention volume (Rv) position ∼6.81 mL as compared to the native polymer reactant 3 (Rv = 8.02 mL, Mn,GPC = 2500 g mol–1, Đ = 1.07). The 1H NMR spectrum (Figure 1B) further suggested the conversion of the chain elongated polymer 3 to the disulfide polymer 4 by the disappearance of the methyl protons peak of −SCOCH3 at δ 2.33 ppm, along with the upfield shifted methylene proton peak of −SCH2CH2– to δ 2.86 ppm value.65

With the bromo-peptide 5 and the stable (poly)sulfide polymer product 4 in hand, the synthesis of the peptide–polymer conjugate 6 was envisioned. To this endeavor, we here use a new method by combining the thio-bromo -based click reaction via in situ generation of the free thiol chain-end containing PEG polymer by disulfide reduction of the disulfide-bridged polymer 4. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) as a reducing agent was added to polymer 4 in an aqueous solution, and the disulfide bond cleavage was observed by analyzing the GPC spectrum of the samples taken periodically during the reaction.66Figure 2 reveals a shift in the RI trace of polymer 4 toward lower average molecular weight upon the progression of the reaction, and the final RI trace for the completely reduced bis-thiol-polymer (7) was observed after 1 h, being identical to the Rv value (8.02 mL) matching with polymer 3.67 After confirming the complete disulfide reduction, to the same reaction the brominated peptide 5 was added under inert conditions while changing the final reaction solvent to an aqueous binary mixture H2O/THF with a ratio 2:1 (v/v), additionally adding triethylamine (Et3N) base to enable catalysis of the thio-bromo click reaction. Under these conditions, the reaction completed within 2 h and the final product, the bifunctional peptidic conjugate 6, was obtained and subsequently purified further in a yield of 62% via preparative GPC (Figure 3A), a method recently exploited by our group.32,68 Note that in the view of synthetic hurdles due to purification struggles associated with the standard purification methods, which has vexed scientists for decades, the herein exemplified preparative GPC-based method is found to be trivial for complex peptide–polymer conjugates (such as here due to the high susceptibility of the amyloidogenic peptide Aβ17–20 to supramolecular aggregation in solution), in turn increasing the yield of the conjugate highly effective in terms of production time and cost. Figure 3C for the analytical GPC chromatograms evidenced the appearance of a monomodal RI trace for the peptide conjugate 6 with Rv ∼ 7.65 mL (Mn,GPC = 3800 g mol–1, Đ = 1.10), which displayed a higher molecular weight compared to that of peptide 5 (Rv ∼ 8.94 mL, Mn,GPC = 750 g mol–1, Đ = 1.06) and the in situ generated free thiolated PEG polymer 7 (Rv ∼ 8.02 mL, Mn,GPC = 2400 g mol–1, Đ = 1.07) but lower compared to that of the intermediate polymer 4 (Rv ∼ 6.86 mL, Mn,GPC = 9350 g mol–1, Đ = 1.62). The successive formation of the desired conjugate 6 was further confirmed by HPLC studies (Figure S13, Supporting Information)47 and via structural analysis by 1H NMR spectroscopy (Figure 1C), which exhibited the characteristic major proton signals for the peptidic constituent FFFF (at δ 7.29–7.05 ppm for four phenyl ring protons and at δ 3.16–2.60 ppm of four benzyl group protons, of tetraphenylalanine) and for the PEGylated polymer segment (at δ 3.54 ppm for their repeating unit −OCH2CH2−) (see also Figure S14, Supporting Information, for the 13C NMR spectrum). Moreover, ESI-TOF MS investigations were undertaken to fully prove the chemical composition of conjugate 6 (Figure 4), where the observed major isotopic peak maximum of 1+ charged series, as a typical example at m/z = 2819.1678 g mol–1, corresponds to [M + Na + CH3OH]+ with a degree of polymerization (n = 29) successfully simulated to match the desired ion.32

Figure 2.

Figure 2

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Normalized RI traces by the analytical GPC studies of thioacetate polymer 3, and time-dependent disulfide reduction reaction of polymer 4 to in situ generate polymer 7 after 60 min (as illustrated by a cartoon representation).

Figure 3.

Figure 3

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Analytical GPC studies based normalized RI trace for the (A) peptide–polymer conjugate 6 based crude product just after the one-pot click reaction. Also shown is the additional minor peak for the excess unreacted peptide 5, and (C) the corresponding purified conjugate 6 achieved from preparative GPC-based purification technique, and peptide 5, polymer 7, and the intermediate polymer 4. (B) Cartoon representation of the coupling reaction.

Figure 4.

Figure 4

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ESI-TOF MS spectra of the FFFF-based conjugate 6 in the binary solvent CH3OH/H2O = 9/1 (v/v). Inset illustrates the observed and simulated isotopic pattern.

The structural assembly of the bifunctional PEGylated peptidic conjugate 6 in an aqueous solution was further investigated via transmission electron microscopy (TEM). Figure 5 demonstrates that the conjugation of hydrophilic PEG polymer to the hydrophobic peptide residues FFFF transformed the self-assembled structure of undefined spherical aggregates of native peptide NH2-FFFF-OMe (8), as reported in our recent article,32 to micrometer long defined fibrillar structures with a diameter of approximately 10 nm for

Figure 5.

Figure 5

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TEM images of peptide–polymer conjugate 6 (A) in H2O/THF (8/2, v/v) (concentration (c) = 0.7 mg mL–1) and (B, C) for the dialyzed sample in H2O (c = 0.4 mg mL–1) with different magnifications. (D) Cartoon representation of the self-assembly of conjugate 6 to fibers.

conjugate 6 (Figure 5A) in a binary solvent mixture (H2O/THF = 8/2, v/v), attributed to the amphiphilic nature of the conjugate. It was noted that such a fibrilar morphology is retained for both the FFFF-based bifunctional conjugate 6 and the monofunctional conjugate mPEG-FFFF-OMe reported in our recent article published elsewhere,32 as the amphiphilicity of both conjugates, although structurally different (utilizing native PEG polymer 1 and mPEG-OH having average Mw = 1500 and 750 g mol–1, respectively), displays similar hydrophobic/hydrophilic ratio. The TEM images of the extensively dialyzed sample of conjugate 6 against water for 2 days using a dialysis membrane of molecular weight cutoff with ∼1 kDa revealed the formation of fibers with a larger average diameter of approximately 50 nm (Figure 5B,C),44 visible as bundling of the generated individual fibrils due to the supramolecular fibril–fibril interactions.15,69,70 Presumably, also interfibril cross-linking due to such bifunctional peptides (Figure 5D) can be proposed.45,71,72 Herein, such fibrillar or fibrous aggregates of the above amphiphilic conjugate can be attributed to its core–shell-like assembly, with a hydrophobic peptidic core along with a hydrophilic PEGylated shell. The existence of a secondary conformation of the conjugate 6 in the above fibrous scaffold was further investigated by circular dichroism (CD) spectroscopic studies (Figure 6), which exhibited a positive CD signal maxima at 196 and ∼217 nm. Herein, the appearance of a broad peak at around 217 nm, corresponding to the aromatic stacking (of π–π interaction) of the peptide constituents,41,43 is plausibly overlapping with the region of the typical characteristic of β-sheets negative minima (typically at 218 nm) as previously observed by Adams and Topham et al.44 and Besenius et al.46 At this particular wavelength, the existence of the relatively higher CD value of conjugate 6 as compared to the CD spectrum of peptide 8 (reported in our recent article32), suggests increased π–π interactions in the core of fibrous assembly for 6 in comparison to the aggregated morphology of 8. It is noted that the CD investigation for conjugate in the binary solvent H2O/THF (8/2, v/v) could not be obtained with a presentable spectrum, possibly due to the unavoidable light scattering therein.

Figure 6.

Figure 6

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CD spectrum of peptide–polymer conjugate 6 (c = 0.2 mg mL–1) for the dialyzed sample in H2O.

Conclusions

In summary, a one-pot synthetic approach has been developed to prepare the tetrapeptide-based bifunctional PEGylated conjugated MeO-FFFF-PEG-FFFF-OMe via a tandem-reaction based on a TCEP-mediated disulfide reduction, followed by thio-bromo click reaction with a bromo-acylated peptide in an aqueous binary solvent mixture. The synthesized conjugate was extensively purified by preparative GPC and further characterized to evidence its successful functionalization. The conjugate showed self-association to form well-defined fibrillar structures with an average diameter of 10 nm. In contrast, the aqueous dialyzed sample formed supramolecular fibers with an average diameter of 50 nm via bundling of thin fibrils, presumably due to fibril–fibril interactions and further cross-linking. Studies are underway to further apply the above synthetic methodology involving thio-bromo click chemistry to effectively prepare more complex hierarchical multifunctional polymeric biomaterials with advanced physical properties for various applications.

Experimental Procedures

Materials

L-Phenylalanine methyl ester hydrochloride (HCl·H2N-F-OMe, 98%) was purchased from Carbolution Chemicals. Boc-L-phenylalanine monohydrate (Boc-F-OH·H2O, 99%) was received from TCI chemicals. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 98%), 1-hydroxybenzotriazole hydrate (HOBt, 97%), trifluoroacetic acid (TFA, 99%), poly(ethylene glycol) (HO-PEG-OH, average Mw = 1500 g/mol), potassium thioacetate (KSOAc, 98%), triethylamine (Et3N, 99%), sodium methoxide solution (NaOMe, 25 wt % in methanol), and 2-bromopropionyl bromide (97%) were purchased from Sigma-Aldrich and used as received. Mesylchloride (MsCl, 99%) and N,N-dicyclohexylcarbodiimide (DCC, 99%) were received from Merck and Alfa Aesar, respectively. Br-FFFF-OMe was adopted from our recent work published elsewhere.32 Reaction solvents were dried and distilled according to the standard procedures.

Instrumentation and Measurements

Gel permeation chromatography (GPC) spectra for polymers were recorded using a Viscotek GPC max VE 2001 from Viscotek equipped with a column set of a HHR-H-Guard-17369 and a GMHHR-N-18055 column and in a solution of 10 mM LiNTf2 in dimethylformamide (DMF) at 60 °C (flow rate 1.0 mL min–1). Polystyrene (PS) standards were used for the external calibration, and data analysis was performed using OmniSEC software (V 4.5.6). Preparative gel permeation chromatography (preparative GPC) studies were performed using a VWR HITACHI Chromaster equipped with a column KD-2002.5 from Shodexin a solution of tetrahydrofuran (THF) solvent with a flow rate of 1 mg mL–1 at 30 °C. The detection of the injected polymeric sample solutions (c = 20 mg mL–1) was carried out using a refractive index (RI) detector from VWR at 30 °C and EZChrom Elite (version 3.3.2 SP2) software was used to analyze the obtained data. Analytical high-performance liquid chromatography (HPLC) studies were performed on a Chromaster by Hitachi VWR equipped with a column oven (L-5310) having temperature control, a pump (L-5160), a diode array detector (DAD; L-5430), an autosampler (L-5260), and a degasser. A reversed-phase column (RP C-18) Waters Atlantis-T3 (5 μm, 100 Å, 4.6 mm × 250 mm) was used to record the spectra. The mobile phase system (at temperature 30 °C) was applied with a solvent mixture of methanol (MeOH) and water in the ratio of 9:1 (v/v) and the sample was injected with 10 μL volume (c = 1 mg mL–1) at the flow rate of 0.4 mL min–1. Chromaster software manager version 1.1 was used to record DAD signals with an operating wavelength range from 190 to 900 nm. More instrumentation details have been provided in the Supporting Information. Nuclear magnetic resonance (NMR) studies for 1H and 13C NMR spectroscopy (in solution state) was performed on a Varian Gemini 2000 (400 MHz) or a Varian Unity Inova 500 (500 MHz) NMR spectrometer using THF-d8, CDCl3, or DMSO-d6 as solvent at 27 °C. NMR data analyses were accomplished using MestRec-C software (version 4.9.9.6). Electrospray ionization time-of-flight mass spectroscopy (ESI-TOF MS) studies were carried out using a Focus Micro TOF of Bruker Daltonics. Measurements were undertaken under positive mode by applying a 4.5 kV accelerator voltage and a transfer line with 190 °C at the spectral rate of 1 Hz. Brucker Daltonic ESI compass 1.3 for microTOF (data analysis 4.0) was used to process the obtained spectra. Circular dichroism (CD) measurements were performed on a JASCO Corp., J-810, Rev.1.00 system at 20 °C for the polymer solutions filled in a cuvette with a space length of 1 mm. The spectra were obtained with a scan rate of 1 nm per second and in the range of wavelength from 250 to 190 nm while maintaining the absorbance of <2 at any measured point. The final CD spectra were acquired after the subtraction of the blank solvent measurement from the obtained sample spectra. Transmission electron microscopic (TEM) studies were carried out using an EM 900 transmission electron microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) for investigating self-assembled morphologies acquired by the polymers in solution. The droplet of the prepared sample solution was placed over a Cu grid coated with a carbon film, stained with uranyl acetate solution (2%), followed by air drying, and further utilized to record their micrographs.

Synthesis of Thioacetylated Polymer AcS-PEG-SAc (3)

To a stirred solution of 2 g (0.8 mmol) of MsO-PEG-OMs, 2 (Mn,GPC = 2500 g mol–1, Đ = 1.06) in 60 mL of dry DMF solvent, 1.74 g of potassium thioacetate (15.24 mmol) was added portion-wise at 75 °C under N2 atmosphere. The reaction mixture was allowed to further stir for 48 h. Then, the reaction mixture was filtered while hot, and the solution and the filtrate were dried via a rotary evaporator. Two hundred milliliters of CH2Cl2 was added to this crude, and the organic layer was washed with 100 mL of water and brine solution and dried over anhydrous Na2SO4, followed by evaporation in vacuum. The crude product was further purified by silica gel column chromatography using CH2Cl2/methanol (10:1) as eluent, resulting in thioacetylated polymer AcS-PEG-SAc (3) with a yield of 78%. The obtained pure polymer was characterized by 1H and 13C NMR and ESI-TOF MS (Figures 1A, S11, and S12, Supporting Information).

Synthesis of Disulfide-Bridged Polymer Poly(PEG-disulfide) (4)

One gram of the as-synthesized thioacetylated polymer AcS-PEG-SAc, 3 (0.4 mmol, Mn,GPC = 2500 g mol–1, Đ = 1.06) was dissolved in 10 mL of dry methanol solvent, and the solution was purged thoroughly under N2 atmosphere for 20 min, followed by addition of 1.27 mL of NaOMe solution (5.9 mmol). The reaction mixture was allowed to stir at room temperature for 10 h and then dried under the rotary evaporator. Fifty milliliters of dry CH2Cl2 were added to this crude mixture, and the organic layer was thoroughly washed with 50 mL of a degassed aqueous solution of 1 N HCl (×3), and finally dried over anhydrous Na2SO4 followed by evaporation in rotary and vacuum pump. The obtained disulfide-bridged PEG polymer poly(PEG-disulfide) (4) in a yield of 75% was characterized by 1H NMR and GPC (Figures 1B and 2).

Synthesis of Peptide–Polymer Conjugate 6 via Tandem Disulfide Reduction and Thio-Bromo Click Reaction

To a degassed solution of 100 mg of disulfide-bridged polymer poly(PEG-disulfide), 4 (0.012 mmol, Mn,GPC = 8700 g mol–1, Đ = 2.02), dissolved in 2 mL of deionized (DI) water, 40 mg of TCEP (0.14 mmol) was added and this reaction mixture was degassed again by purging with dry N2 for 10 min and stirred for 1 h at room temperature. Around 0.05 mL of the reaction mixture was taken out periodically by a N2 purged syringe for GPC analysis to determine their number-average molecular weight (Mn,GPC) to confirm the complete disulfide reduction (via in situ generation of polymer 7). Subsequently a predegassed solution of 37 mg of Br-FFFF-OMe, 5 (0.05 mmol), and 17 mg of Et3N (0.17 mmol, 24.1 μL) in 1 mL of THF were added and the final reaction mixture was thoroughly degassed by applying three consecutive cycles of freeze–pump–thaw and allowed to stir under N2 at room temperature for 2 h. The crude reaction product was then transferred to a dialysis membrane bag (molecular weight cutoff (MWCO) of 1 kDa) and extensively dialyzed against a solvent mixture of THF/MeOH (1:1) for 48 h to remove the unreacted residues. The conjugate MeO-FFFF-PEG-FFFF-OMe (6) was obtained by evaporating the dialyzed solution and further subjected for preparative GPC-based purification for achieving the pure product in 62% yield (where the low yield can be attributed to the loss of compound during such purification processes), and finally, the conjugate was dried in a high vacuum pump and characterized via 1H NMR and GPC (Figures 1D and 2B, respectively).

Acknowledgments

Financial support of this work by the SFB-TRR 102 (German Research Foundation Project ID 189853811, Projects A03 and A12) is greatly acknowledged. S.K. thanks Alexander von Humboldt Foundation for the research fellowship and the Punjab Engineering College (Deemed to be University), Chandigarh, for research initiation grant.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c02326.

  • Synthetic details of peptides, PEGylated polymers, and peptide–polymer conjugate,1H and 13C NMR studies, ESI-TOF MS spectra, and HPLC spectra (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao0c02326_si_001.pdf (700.4KB, pdf)

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