The synthesis and cell interaction of statistical L-arginine â glycine â L

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The synthesis and cell interaction of statistical Larginine – glycine – L-aspartic acid terpolypeptides Siyasanga Mbizana, Lebohang Hlalele, Rueben Pfukwa, Andre du Toit, Dumisile Lumkwana, Benjamin Loos, and Bert Klumperman Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00620 • Publication Date (Web): 01 May 2018 Downloaded from http://pubs.acs.org on May 5, 2018

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Biomacromolecules

The synthesis and cell interaction of statistical L-arginine – glycine – L-aspartic acid terpolypeptides

Siyasanga Mbizana,a Lebohang Hlalele,a Rueben Pfukwa,a Andre Du Toit,b Dumisile Lumkwana,b Benjamin Loos,b and Bert Klumperman,a a

Stellenbosch University, Department of Chemistry and Polymer Science, Private Bag X1, Matieland 7602, South Africa

b

Stellenbosch University, Department of Physiological Sciences, Private Bag X1, Matieland 7602, South Africa

ABSTRACT Copolymerizations and terpolymerizations of N-carboxyanhydrides (NCAs) of glycine (Gly), Nδ-carbobenzyloxy-L-ornithine ((Z)-Orn) and β-benzyl-L-aspartate ((Bz)-Asp) were investigated. In situ 1H NMR spectroscopy was used to monitor individual comonomer consumptions during binary and ternary copolymerizations. The six relevant reactivity ratios were determined from copolymerizations of the NCAs of amino acids via nonlinear least squares curve fitting. The reactivity ratios were subsequently used to maximize the occurrence of the Asp-Gly-Orn (DGR′) sequence in the terpolymers. Terpolymers with variable probability of occurrence of DGR′ were prepared in the lab. Subsequently, the ornithine residues on the terpolymers were converted to L-arginine (R) residues via guanidination reaction after removal of the protecting groups. The resulting DGR terpolymers translate to traditional peptides and proteins with variable RGD content, due to the convention in nomenclature that peptides are depicted from N- to C-terminus, whereas the NCA ring-opening polymerization is conducted

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Biomacromolecules 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

from C- to N-terminus. The

L-arginine

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containing terpolymers were evaluated for cell

interaction, where it was found that neuronal cells display enhanced adhesion and process formation when plated in the presence of statistical DGR terpolymers.

Introduction Biodegradable polypeptides have potential for medical applications such as carriers for protein conjugates and in drug delivery systems.1 Copolypeptides resemble natural proteins in properties such as their degradability by proteolytic enzymes.1-2 However, amino acids in natural proteins are arranged in a well-defined sequence, which leads to characteristic functionalities and properties. Block copolypeptides are interesting for specific applications due to their ability to self-assemble.3 Statistical copolypeptides may deviate to a smaller or larger extent from natural proteins, with the former having a statistical or random distribution of amino acid residues and a broad distribution of chain lengths. Short sequences of amino acid residues within polypeptide chains can be targeted through the primary amine-initiated N-carboxyanhydride (NCA) ringopening copolymerization process.4 The incorporation of amino acid residues in a growing chain is controlled by the NCA monomers’ relative reactivities, resulting in their statistical distribution. Hence, the probability of occurrence of a certain monomer sequence can be varied by understanding NCAs’ copolymerization parameters, i.e. reactivity ratios. Wamsley et al., prepared terpolypeptides of L-leucine (L), L-aspartic acid (D) and L-valine (V), which they described by the copolymerization parameters and statistical probability equations.4a,

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Maximization of the occurrence of the LDV sequence was targeted. The LDV sequence is a ligand that recognizes the α4β1 integrin, and therefore has potential for drug delivery and cell recognition studies.6


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The microstructure of terpolypeptides can be measured by analysis of triad sequences in a manner similar to that of vinyl terpolymers.7 Although there are limitations, analytical methods such as 13C NMR and 15N NMR have previously been employed to determine triad distributions for terpolypeptides.4a, 8 In addition, the simulation of terpolymerization using statistical concepts provides a route to estimate/predict the relative occurrence of specific triad sequences.4a, 9 The latter approach relies on knowledge of reliable reactivity ratio values obtained from copolymerizations of the three monomers of interest. The procedure for determination of reliable reactivity ratios from copolymerization of respective NCA monomers, typically entails monitoring monomer consumption as a function of polymerization time. Two techniques that have been employed to monitor monomer concentration in NCA copolymerization are FTIR spectroscopy and HPLC.4b,

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In FTIR

spectroscopy, monitoring the decrease of the carbonyl peak intensity and also measuring the amount of evolved carbon dioxide provides overall monomer consumption. The drawback of using FTIR spectroscopy to monitor NCA monomer consumption in a copolymerization is that it is non-selective towards the individual NCAs.4a, 10 HPLC on the other hand is able to distinguish among individual monomers, however, the sample preparation and experimental methodology can be quite tedious.4b In the current contribution, in situ 1H NMR is employed to monitor monomer consumption during binary and ternary copolymerizations of NCAs. The in situ 1H NMR methodology is used to investigate kinetic features of the copolymerizations of Nδ-carbobenzyloxy-L-ornithine ((Z)Orn), β-benzyl-L-aspartate ((Bz)-Asp) and glycine (Gly) NCAs, and determine their reactivity ratios. Subsequently, these copolymerization parameters will be used with predetermined feed ratios to predict the relative occurrence of the (Bz)-Asp-Gly-(Z)-Orn sequence in terpolymer


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chains using probability equations. The Asp-Gly-Orn (DGR′) sequence is a precursor to Larginine – glycine – L-aspartic acid (RGD) sequence that is found in many glycoproteins and is recognized by a wide range of integrins in the extracellular matrix,11 and plays a vital role in cell adhesion and cell proliferation.12 Peptide sequences are by convention listed from the N-terminus to the C-terminus whilst the polymerization of amino acid NCAs via the normal amine mechanism, as adapted in this study, proceeds from C- to N-terminus, hence we will use DGR in italics when we refer to the C- to N-terminus sequence, and RGD in normal font when we speak about the normal N- to C-terminus sequence. The present contribution further reports the modification of ternary polypeptides with Asp-Gly-Orn sequences into Asp-Gly-Arg (DGR) sequences, and a study of their cell interaction. Experimental Material Glycine (99.6%, Sigma), L-aspartic acid (≥ 98%, Sigma), L-ornithine hydrochloride (≥ 99%, Sigma), (+)-α-pinene (98%, Aldrich), benzyl alcohol (≥99%, Sigma-Aldrich), benzyl chloroformate (95%, Aldrich), bis(trichloromethyl) carbonate (≥ 99%, Sigma-Aldrich), diphosphorus pentoxide (≥ 97%, Merk), trifluoroacetic acid (TFA, 99%, Sigma-Aldrich), deuterium oxide (D2O, 99.8%, Acros organics), pyridine (99.8%, Aldrich), glacial acetic acid (AcOH, 99.8%, SaarChem), ethylenediaminetetra-acetic acid disodium salt dehydrate (≥99%, SaarChem), copper(II)carbonate basic (≥ 95%, Sigma-Aldrich), trifluoroacetic acid-D (TFA-d, 99 Atom% D, Sigma-Aldrich), n-butylamine (≥ 99.5%, Sigma-Aldrich), DMSO-d6 (99.9% atom D, Aldrich), O-Methylisourea bisulfate (MIU, 99%, Sigma-Aldrich), Hydrobromic acid solution (33 wt. % in acetic acid) were used as received.


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Instrumentation. The online 1H NMR spectra were recorded with a 400 MHz Varian Unity Inova spectrometer in anhydrous DMSO-d6. The 1H NMR spectra were acquired with 3 µs (40°) pulse width and 4 s acquisition time. For the polymers that were prepared via bench polymerization, the 400 MHz Varian Unity Inova spectrometer was used for 1H NMR characterization, whilst the 600 MHz Varian Unity Inova spectrometer was used for

13

C NMR

characterization. D2O and TFA-d were used as solvents for the characterization of polymers prepared from bench polymerization. Online 1H NMR spectroscopy procedure. For the in situ experiments, measurements were carried out in a Wilmad quick pressure valve NMR tube. Degassing was conducted by three freeze-pump-thaw cycles. After the third cycle, samples were allowed to thaw, then inserted into the magnet at 25 °C and the magnet shimmed, the first spectrum collected served as a reference. Subsequent spectra were collected at 15 minute intervals for 14 hours. The NMR data were analyzed using ACD Labs 12.0

1

H NMR processor. Phase correction was performed

automatically whilst baseline correction and integration were performed manually. Monomer synthesis. The protected NCA precursors were prepared according to reported methods and the respective NCAs were prepared via the phosgenation method, as described in the SI.13

Scheme 1. General representation of a copolymerization reaction initiated by n-butylamine in DMSO at room temperature.


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Copolymerization of Gly NCA and Bz-Asp NCA monitored by in situ 1H NMR. Scheme 1 shows a general copolymerization reaction between two amino acid NCAs. In a typical copolymerization experiment, Gly NCA (7.1 mg, 0.07 mmol) and Bz-Asp NCA (157 mg, 0.63 mmol) were dissolved in DMSO-d6 (2.4 mL) to yield a 0.3 M solution. The NCA solution was subsequently transferred to a Wilmad quick pressure valve NMR tube with 0.6 µL n-butylamine. The reaction mixture was degassed by three freeze-pump-thaw cycles and left under vacuum after the final cycle, after which the progress of polymerization was monitored via in situ 1H NMR spectroscopy. In the preparation of the reaction mixture, care was taken to keep the temperature as low as practically possible, in order to minimize premature polymerization. Bench synthesis of statistical copolypeptides. The statistical copolypeptides were prepared in anhydrous DMSO with n-butylamine as initiator. The polymerization was allowed to proceed for 72 hours at room temperature with a target degree of polymerization DP = 70. In a typical experiment, n-butylamine (2 µL, 20.0 µmol), Z-Orn NCA (0.38 g, 1.3 mmol) and Gly NCA (0.057 g, 0.56 mmol), were dissolved in DMSO (7 mL) followed by three free-pump-thaw cycles. The copolymerization was allowed to proceed under vacuum for 72 hours at room temperature. The reaction mixture was precipitated from distilled water with several washings. The isolated copolymers were dried under vacuum at room temperature. The resulting copolymers were characterized via NMR spectroscopy. Deprotection of ornithine residues. The Orn side-chain protecting groups were removed by acidolysis with 33% HBr solution in AcOH and TFA as depicted in Scheme 2. In a typical experiment, 0.5 g of the protected poly(Gly-co-Z-Orn) was dissolved in TFA, then 4 equivalents of 33% HBr in AcOH relative to the Z-Orn groups were added slowly and the reaction mixture stirred at room temperature. After 24 hours, the reaction mixture was precipitated from diethyl


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ether and the white precipitate was isolated by filtration and dried under vacuum. The benzyl groups from the copolymers containing Bz-Asp residues were removed through an identical procedure.

Scheme 2. Acidolysis deprotection of Orn residues in poly(Z-Orn-co-Gly) and the subsequent guanidination to produce Arg residues in poly(Arg-co-Gly). Guanidination. The deprotected Orn residues were subsequently converted to Arg residues. The guanidination of δ-NH2 was achieved with MIU as a guanidylating agent as shown in Scheme 2 at pH 9.5 in aqueous medium.14 In a typical experiment, 0.5 g of the poly(Orn-co-Gly) was dissolved in distilled water (15.0 mL) and the pH was adjusted with NaOH and HCl to 9.5. In a separate vial, 4 equivalents of MIU to δ-NH2 were dissolved in distilled water (5.0 mL) and the pH was also adjusted to 9.5. The MIU solution was added to the copolymer solution and the reaction mixture was stirred at 40 °C for 72 hours. The pH of the reaction mixture was kept at 9.5, after 24 hours an additional 1 equivalent of MIU solution was added to the reaction mixture, which was stirred at room temperature for an additional 12 hours. The reaction mixture was dialysed against distilled water for 72 hours, where the water was replaced every 12 hours. Water was then removed at low pressure to yield a white powder that was analysed with 1H and

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C

NMR spectroscopy. Cell culture. Murine hypothalamic GT1-7 cells, a kind gift from Dr Craig Kinnear, Stellenbosch University were cultured in Dulbecco’s modified Eagle’s medium (DMEM),


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supplemented with 10% fetal bovine serum (FBS), 100 µg/mL penicillin and 100 µg/mL streptomycin.15 DMEM (41965062), PenStrep antibiotic (15140122) and trypsin (25200072) were purchased from Life Technologies. Fetal bovine serum (FBS, FBS-GI-12A) was supplied by Capricorn Scientific and 15 and 50 mL Falcon tubes were purchased from SPL Life Sciences (EF4661 and EF4663). The secondary antibody Alexa Fluor 488 donkey anti-mouse (A21202) was purchased from Cell Signaling; Hoechst 33342 (14533) was supplied by Sigma-Aldrich. Anti-acetylated α-tubulin (Santa Cruz 23950), Alexa Fluor 633 Phalloidin (Invitrogen, A22284) and CellTracker Green (Invitrogen, C2925) were utilized for fluorescence microscopy. GT1-7 cells were cultured at 37 °C within a humidified atmosphere containing 5% CO2. For immunofluorescence confocal analysis, cells were grown on 8-well borosilicate glass-bottomed chambers (Nunc, New York, USA). For life-cell imaging-based cell surface area analysis, cells were grown in 6 well plates (Greiner Bio One, Frickenhausen, Germany) to 70–80% confluency. Each well was treated with 2.5 µg fibronectin (35 µg/mL in 100 mM Na2CO3), PLL (0.1 mg/mL in dH2O), pre-activated (0.1 mg/mL in dH2O) and post-activated (0.1 mg/mL in dH2O) respectively. Chambers were left overnight at room temperature to dry. Next, wells were gently rinsed with culture medium to remove unbound substrate and cells were seeded at a density of 20 000 cells per well (8 well plates) or 100 000 cells per well (6 well plates). Epifluorescence and confocal microscopy. For confocal acquisition, cells were fixed for 10 min using ice cold methanol acetone 1:1, then rinsed with cold PBS and incubated for 30 min in 5% v/v donkey serum. Next, cells were subjected to the primary antibodies, and incubated overnight at 4 °C in PBS, after which Hoechst 33342 was added for 10 minutes. Chambers were rinsed 3× with PBS and mounted using Dako fluorescent mounting media. For life cell image acquisition, cells were loaded with the cell tracker and transferred to a wide field inverted


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Biomacromolecules

microscope (Olympus IX81, Olympus, Tokyo, Japan) equipped with a Xenon-Arc burner (Olympus Biosystems GMBH, Hamburg, Germany) and an F-view-II cooled CCD camera (Soft Imaging Systems, Olympus Corporation. The 360 nm DAPI and 488 nm excitation filter were used to capture micrographs for the acquisition of cytoplasmic cellular outline and nuclei, imaging 3-4 fields of view of 3 independent experiments. Emission was collected using a UBG triple-bandpass emission filter cube (Chroma) and micrographs acquired, processed and analyzed using the Olympus Cell^R (Hamburg, Germany) software. For cell morphological assessment, samples were acquired using the Zeiss LSM 780 system (Carl Zeiss Microimaging, Germany). In brief, z-stacks with 4-7 image frames and increments of ~0.5 µm step width were acquired, using an A LCI Plan-Apochromat 63×/1.4 Oil DIC M27, a Diode 405 nm CW/PS (pulsed), 488 nm laser, 633 nm laser and a GaAsP detector. Maximum intensity projections of z-stacks were processed and generated using Zeiss Zen Black Software (2012). Results and Discussion Monitoring NCA copolymerizations with 1H NMR spectroscopy. The objective of this study was to develop a method for the determination of NCA copolymerization reactivity ratios that can subsequently be utilized to optimize the corresponding terpolymerizations. The resulting terpolymers with variable fraction of DGR sequences would then be evaluated via cell interaction experiments. In this contribution we established a method to monitor copolymerizations and terpolymerizations of the NCAs of Z-Orn, Bz-Asp and Gly.


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Ha

A

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Ha'

Hg

14 hr 12 hr 10 hr

A'

8 hr 6 hr 4 hr 2 hr 0 hr

5.1

4.8

4.5

4.2

δ (ppm)

Figure 1. 1H NMR spectra at 2 hour intervals illustrating the enlarged region from 4.18 to 5.30 ppm during copolymerization of NCAs of Gly/Bz-Asp in DMSO-d6 for 14 hours at 25 °C started with fg0 = 0.10. Figure 1 shows an array of spectra of the enlarged region [4.18 ≤ δ (ppm) ≤ 5.30] during nbutylamine-initiated copolymerization of Bz-Asp/Gly NCAs, at an initial Gly fraction of fg0 = 0.10 in DMSO-d6 at 25 ºC. The peaks labelled Hg and Ha were used to monitor consumption of NCAs of Gly and Bz-Asp, respectively. The peaks A and A′ represent Bz-Asp in the copolymer, where A is the benzylic CH2, and A′ the αCH, which originates from Ha′, i.e. the αCH proton in the Bz-Asp monomer. Although peak Ha′ shows a decrease in intensity with time, it also overlaps with A′ and therefore, the benzylic CH2 (Ha) of Bz-Asp that was better resolved, was used in this study. The separation of these two resonance signals enabled us to follow the individual monomer consumptions during the Gly/Bz-Asp NCA copolymerization. Changes in intensities of Hg and Ha protons of Gly and Bz-Asp NCAs respectively, enabled calculations of overall conversion (Xt), instantaneous comonomer fractions (fi) and instantaneous


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copolymer compositions (Fi) using the equations S1, S2 and S3 in the Supporting Information (SI). Examples of results from copolymerizations between Gly NCA and Bz-Asp NCA are shown in Figure 2 and Figure 3. In the copolymerization systems where the Z-Orn NCA was used, its single α-proton was used for calculations. 0.6

0

fg = 0.1 0

fg = 0.3 0

fg = 0.6

glycine feed composition

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

0.4

0.2

0.0 0.0

0.2

0.4

0.6

0.8

overall conversion

Figure 2. Gly fractions in the reaction mixture as a function of overall monomer conversion from different copolymerizations of Gly/Bz-Asp in DMSO-d6 at 25 ºC for 14 hours. Figure 2 shows the fraction of Gly NCA as a function of overall monomer conversion during the copolymerization of Gly and Bz-Asp NCAs for three initial monomer feed compositions. The Gly NCA molar fractions in the reaction mixture decrease with increasing conversions. The relatively high reactivity of Gly NCA is shown by the higher Gly content in the copolymer in comparison to the Gly NCA fraction in the reaction mixture (Figure 3). For the other copolymerization systems, similar graphs have been constructed as shown in Figures S2 and S3. On the basis of the composition data, reactivity ratios (Table 1) were calculated using the Contour software developed by van Herk,16 which employs the nonlinear least squares


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Biomacromolecules

methodology in conjunction with the terminal unit model of copolymerization. A constant relative error was assumed during the fitting procedure. 1.0

0.8

0.6

Fg

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.4

0.2

0.0 0.0

0.2

0.4

0.6

0.8

1.0

fg

Figure 3. Gly fraction in the copolymer (Fg) versus Gly fraction in the reaction mixture (fg) from the copolymerizations of Gly NCA with Bz-Asp NCA. The solid curve is calculated from the terminal model, using the reactivity ratios for Gly NCA and Bz-Asp NCA (Table 1). The dashed line represents the diagonal and just serves as a guide to the eye. From the instantaneous comonomer fraction in the reaction mixture (fg), and the instantaneous copolymer composition (Fg) for Gly monomer, the reactivity ratios were calculated to be rGD = 2.66 and rDG = 0.412 for the Gly NCA and Bz-Asp NCA copolymerization. Table 1. Copolymerization reactivity ratios for the Gly/Bz-Asp, Gly/Z-Orn and Bz-Asp/Z-Orn systems. Gly and Bz-Asp

Gly and Z-Orn

Bz-Asp and Z-Orn

rGD

2.66

rGR′

4.66

rDR′

3.51

rDG

0.412

rR′G

0.195

rR′D

0.534


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1.0

Bz-Asp/Z-Orn Gly/Bz-Asp Gly/Z-Orn

0.8

0.6

r2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

0.4

0.2

0.0 2

4

6

8

10

r1

Figure 4. The 95% joint confidence intervals of reactivity ratios for the copolymerization systems studied. Bz-Asp (1)/Z-Orn (2), Gly (1)/Bz-Asp (2) and Gly (1)/Z-Orn (2) systems are as labelled. The numbers in parentheses relate to the assignments of r1 and r2. Reactivity ratios for the other copolymerization systems were determined in a similar manner and are displayed with their corresponding 95% joint confidence intervals in Figure 4 and listed in Table 1. Based on the determined reactivity ratios, the comonomers can be arranged in order of their reactivity as Gly>Bz-Asp>Z-Orn. The results obtained from the copolymerizations can be used to predict terpolymerization behaviour. As an illustration, the reactivity ratios together with statistical probability equations (Equation S4) can be used to predict the relative occurrence of certain monomer sequences in the terpolymers. In this study, the target was to vary the occurrence of the DGR′ sequence. The bench copolymerizations of amino acid NCAs were initiated with n-butylamine with a targeted DP = 70 under the conditions adopted from in situ copolymerizations. 1H NMR spectroscopy was used to characterize the copolymers before deprotection of the functional groups. Figure 5 shows the 1H NMR spectrum of poly(Z-Orn-co-Bz-Asp). The Z and Bz protecting groups from the copolymers were simultaneously removed by employing the


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acidolysis method with 33% HBr in acetic acid. The Orn residues were guanidinated after deprotection to afford L-arginine (Arg) residues. Figure 6 shows an overlay of 1H NMR spectra, where A is the spectrum of the deprotected copolymer poly(Orn-co-Gly) and B represent the guanidinated copolymer, i.e. poly(Arg-co-Gly). The guanidination reaction can be monitored by focusing on δ-CH2 protons which resonate at 3.10 ppm in Orn residues (Spectrum A, Figure 6) and resonate at 3.25 ppm upon guanidination (Spectrum B, Figure 6) i.e. in Arg residues as can be seen in Figure 6 , S5, S6 for the terpolymers and which is in agreement with the work of Vermeersch.17

I

I 2 6

3 4 5

ArH 5 ArCH2

DMSO-d6

2

NH 8

3,4 6 I

6

δ(ppm)

4

I

2

Figure 5. 1H NMR spectrum of poly(Z-Orn-co-Gly)


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O

O

H N

N H

H N

x

H 1-x n

δ NH2

A O

O

H N

N H

H N

H N

H 1-x

x NH2

n

δ NH

B

8

6

δ(ppm)

4

2

Figure 6. 1H NMR spectra of poly(Orn-co-Gly) (A) and poly(Arg-co-Gly) (B)

Ha

A

Ho

Ha'

Hg

14 hr 12 hr 10 hr 8 hr

6 hr 4 hr 2 hr 0 hr

5.2

5.0

4.8

4.6

4.4

4.2

4.0

δ (ppm)

Figure 7. 1H NMR spectra at 2 hour intervals illustrating the enlarged region from 4.0 to 5.2 ppm during terpolymerization of NCAs of Gly/Bz-Asp/Z-Orn in DMSO-d6 for 14 hours at 25 °C started with equal fractions of 0.33 for each NCA. In situ and bench terpolymerizations were carried out under identical conditions as those applied during binary copolymerizations. In situ 1H NMR spectroscopy was successfully employed to monitor the individual comonomer consumptions of Gly NCA, Bz-Asp NCA and ZOrn NCA during terpolymerization as shown in Figure 7. The instantaneous fractions of


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Biomacromolecules

monomers in the reaction mixture can be displayed as a function of overall conversion as shown in Figure 8. The first observation is that the experimental data points do not start at zero conversion. This is justified by the time it takes from preparing the reaction mixture to recording the first spectrum after shimming the NMR spectrometer. The Gly NCA’s high reactivity is illustrated in Figure 8 as its fraction decreases faster than the fraction of Bz-Asp NCA in the ternary mixture. As an indication of its low reactivity, the Z-Orn NCA fraction in the ternary mixture increases as conversion increases. Hence, the rate of individual comonomer consumptions during terpolymerization (Figure 8) corroborated the results obtained from the binary copolymerizations related to the reactivity of the individual NCA comonomers, as evidenced by the reasonable agreement between experimental data and predicted monomer fractions.

0.6

fGly

ternary composition

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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fAsp fOrn

0.4

0.2

0.0 0.0

0.2

0.4

0.6

0.8

overall conversion

Figure 8. Instantaneous fractions of monomers in the reaction mixture of the Gly NCA, Bz-Asp NCA and Z-Orn NCA terpolymerization as a function of overall conversion. The terpolymerization reaction was started with equal fractions of 0.33 for each NCA and monitored with 1H NMR spectroscopy in DMSO-d6 for 14 hours at 25 °C. The solid lines represent the


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theoretical feed compositions calculated with determined reactivity ratios and the symbols represent the experimental feed compositions. Equation 1. The Alfrey-Goldfinger terpolymerization equation.18 : :

+ + + + :

+ + + + :

+ + + + =

The reactivity ratios in Table 1 can be used to predict the relationship between the instantaneous ternary monomer mixture composition (fi) and the terpolymer composition (Fi) by using the Alfrey-Goldfinger equation (Equation 1).18 Figure 9 illustrates the relationship between terpolymer compositions and overall conversion. The solid lines represent the terpolymer composition calculated with Equation 1 and the symbols represent the experimental instantaneous terpolymer composition of the respective amino acid residues. The predicted terpolymer compositions are in good agreement with the experimentally determined terpolymer compositions, thus validating the applicability of the terminal model in describing the NCA binary and ternary copolymerizations. The terpolymers built from equimolar fractions, are rich with Gly residues at low conversion but as conversion increases, the Bz-Asp and Z-Orn residues dominate respectively.


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Biomacromolecules

FGly FAsp

0.6

FOrn terpolymer composition

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.4

0.2

0.0 0.0

0.2

0.4

0.6

overll conversion

Figure 9. Instantaneous terpolymer composition of the Gly, Bz-Asp and Z-Orn terpolymers as a function of overall conversion. The solid lines represent the predicted terpolymer compositions based on binary reactivity ratios. The good agreement between predicted and experimental terpolymer composition, allowed us to use the binary reactivity ratios to assess the occurrence of the DGR′ sequence in the terpolymer chains. The reactivity ratios, initial feed compositions and statistical probability equations can be used to calculate the various fractions of triad sequences. The experimental determination of triad fractions with

13

C NMR typically shows good agreement with predicted

triad fractions based on copolymerization statistics in the case of vinyl polymers.7, 9, 18-19 In this contribution, reactivity ratios obtained from copolymerizations were used in combination with initial monomer feed compositions and statistical equations (Equation S4) to predict the probability of occurrence of the Asp-Gly-Orn (DGR′) sequence in the terpolymers.


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1 = 0.25/0.25/0.5 D/G/R' 2 = 0.2/0.4/0.4 D/G/R' 3 = 0.1/0.8/0.1 D/G/R' 4 = 0.5/0.25/0.25 D/G/R'

0.06

DGR' probability

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

0.04

0.02

0.00 0.0

0.2

0.4

0.6

0.8

1.0

conversion

Figure 10. Illustration of predicted DGR′ probability fraction as a function of overall conversion, for the terpolymers, with initial D/G/R′ monomer fractions as displayed in the Figure. Figure 10 shows how the DGR′ probability varies as a function of conversion depending on the initial ternary composition, this implies that the DGR′ triads may vary in frequency at different positions along the terpolypeptide chain. For example, if we consider terpolymer 3, in Figure 10, the lowest DGR′ fractional probability is observed at low conversions, however, it increases as the terpolymerization reaction progresses, and composition drift takes place. This composition drift is demonstrated by the predicted evolution of the Gly NCA fraction, as a function of conversion, in the ternary mixture, as illustrated in Figure 11, which shows that the Gly NCA fraction is depleted early in the terpolymerization, resulting in composition drift at high conversions. For example, in Figure S11 and S12, the initial feed compositions of 0.1/0.1/0.8 D/G/R′ produced its highest DGR′ fractional probability (DGR′ = 0.079) at low conversion, but due to the rapid consumption of Gly NCA, the DGR’ probability rapidly declined. As a consequence, such an initial monomer feed was not considered experimentally, and we prepared terpolyemers based on the initial conditions illustrated in Figures 10, and used these for cell interaction studies.


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Biomacromolecules

1.0

1 = 0.25/0.25/0.5 D/G/R' 2 = 0.2/0.4/0.4 D/G/R' 3 = 0.1/0.8/0.1 D/G/R' 4 = 0.5/0.25/0.25 D/G/R'

0.8

0.6

fg

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0.4

0.2

0.0 0.0

0.2

0.4

0.6

0.8

1.0

conversion

Figure 11. Illustration of predicted Gly NCA fraction (fg) as a function of conversion during the preparation of different terpolymers with initial D/G/R′ displayed in the Figure respectively. Analysis with

13

C NMR spectroscopy shows the R′, G, and D centred triads displayed in

Figures 10, S8, S9 and S10. However, the

13

C NMR results show insufficient resolution to

quantify individual triad fractions. Examples of terpolypeptides of different composition are shown in Figure 12, where it is clear that the polymer with minimized R′ and D fractions (Figure 12A) has a narrower signal for the G centred triads than the one with the maximized R′ and D sequence (Figure 12B). The poor splitting in terpolypeptides’

13

C NMR determined triads was

also observed by Wamsley et al., where the authors cited the conformation of polypeptides to different secondary structures as the contributing factor.4a The deprotected Orn residues (R′) of the terpolymers (Figure S6) were guanidinated to Arg residues (R) (Figure S7) with MIU using conditions that were optimized on the basis of Orn copolypeptides. Terpolymers with the R residues were subsequently tested for cell interaction.


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B

G

R'

D

A G

R'

178

176

174

D

172

170

168

δ (ppm)

Figure 12.

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C NMR spectra of (Z-Orn-Gly-Bz-Asp) terpolymer with initial D/G/R′ ternary

compositions: A = 0.1/0.8/0.1 and B = 0.5/0.25/0.25 respectively

Figure 13. GT1-7 neuronal cells, stained with cytotracker green indicating cell morphology and degree of surface adhesion. Scale bar: 50 µm. Cell surface analysis of murine hypothalamic GT1-7 cells, as an indication for cellular process formation, was performed on the 4 different polypeptides that are already guanidinated, the


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probability of occurrence of the DGR′ sequence was varied in these polypeptides as illustrated in Figure 10. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) that was supplemented with 10% fetal bovine serum (FBS).

Figure 14. GT1-7 neuronal cells, mean surface area occupied in µm2. The plates were treated with poly-ʟ-lysine, fibronectin, collagen, post-activation (postguanidination) i.e. the four poly(Arg-co-Asp-co-Gly) guanidinated terpolymers and compared to non-treated control conditions (pure milli-q water). Poly-ʟ-lysine was used as a negative control for cell spreading, in addition fibronectin and collagen are compared to the terpolymers as they contain the natural RGD sequence, thus are used as positive control. Figure 13 illustrates the morphology of the cells during incubation after 48 hours, where it can be seen that the cells adhered in all the substrates except poly-ʟ-lysine. Quantification of cell surface analysis revealed a significant increase in the surface area occupied between non-treated control cells and guanidinated terpolymers as shown in Figure 14. In addition, the cells displayed high degree of adhesion on the surfaces treated with terpolymers 1 to 3, the cell spreading in these terpolymer-treated surfaces was higher than that of fibronectin as illustrated in Figure 14. As a negative control, poly-ʟ-lysine showed a decreased cell


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spreading after 48 hours compared to any of the substrates used in the study as can be seen in Figure 13. Conclusions Statistical terpolypeptides containing L-arginine, glycine, and L-aspartic acid were synthesized through N-carboxyanhydride (NCA) ring-opening polymerization of side-group protected NCAs of L-ornithine, glycine, and L-aspartic acid. The statistical character of the terpolymerization was assessed via a kinetic study of the three relevant copolymerization systems, of which the reactivity ratios were determined. On the basis of these reactivity ratios, we were able to predict the occurrence of the L-ornithine, glycine, and L-aspartic acid sequence (precursor of the celladhesive RGD sequence) in the terpolypeptide. After deprotection of the side-groups and guanidination of the L-ornithine residue, the DGR-containing terpolypeptides were subjected to cell interaction studies. It was found that despite the statistical nature of the monomer sequence distribution, the RGD-containing polypeptides perform as a good substrate for cell proliferation and neuronal process formation for GT1-7 neuronal cells. The potential advantage of this methodology is to circumvent the demanding synthesis of sequence-controlled polypeptides via solid phase synthesis. In addition, prepared terpolypeptides have potential application in assisting cellular regeneration processes.

ASSOCIATED CONTENT The structures of the three NCAs, characterization of the Bz-Asp NCAs, Gly NCA, Z-Orn NCA. The equations for the calculations of experimental overall comonomer conversion(Xt), instantaneous feed compositions(fg), instantaneous copolymer compositions(Fg) during the copolymerization of Gly NCA and Bz-Asp NCA. The copolymer compositions vs feed


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compositions for the Gly NCA/Z-Orn NCA and Bz-Asp NCA/Z-Orn NCA copolymerization systems, and cumulative terpolymer composition vs overall conversion. NMR spectroscopy information for the bench prepared terpolymers and

13

C NMR spectroscopy of the carbonyl

region. Images showing the morphologies of the neuronal cells on the different substrate groups after 24 and 48 hours.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]


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ACKNOWLEDGMENT This work is based on the research supported by the South African Research Chairs Initiative of the Department of Science and Technology (DST) and National Research Foundation (NRF) of South Africa (Grant No 46855).


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Table of content


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The synthesis and cell interaction of statistical L-arginine â glycine â L

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