R E S E A R CH AR T I C L E

A cationic alternating copolymer composed of ornithineand glycine with an ordered sequence for enhancedbacterial activity

Fuqiang Liu1,2 | Nan Ma1 | Jun Liu1 | Qiongqiong Zhu1 | Ting Yue1 |

Junhui Ma1,2 | Yuan Wang1 | Wei Qu1 | Paul K. Chu3 | Yan Tang1 |

Wei Zhang1

1Technical Institute of Physics andChemistry, Chinese Academy of Sciences,Beijing, China2Chinese Academy of Sciences, Universityof Chinese Academy of Sciences, Beijing,China3Department of Physics, Department ofMaterials Science and Engineering, andDepartment of Biomedical Engineering,City University of Hong Kong, HongKong, China

CorrespondenceWei Zhang and Yan Tang, TechnicalInstitute of Physics and Chemistry,Chinese Academy of Sciences, Beijing100190, China.Email: weizhang@mail.ipc.ac.cn (W. Z.)and tangyan@mail.ipc.ac.cn (Y. T.)

Funding informationCity University of Hong Kong StrategicResearch Grant, Grant/Award Number:7005264; National Key Research andDevelopment Program, Grant/AwardNumber: 2016YFC1000900

Abstract

The chains and segments of unordered cationic polypeptides are complex and

may produce unexpected biological activities. Herein, the Ugi's 4CC reaction is

adopted to synthesize a cationic alternating copolymer comprising ornithine and

glycine (poly(Orn-alter-Gly)) with an ordered sequence for enhanced bacterial

resistance. In this technique, potassium isocyanate, 4-(N-carbobenzyloxyamino)-

1-butyraldehyde and 1-(4-Methoxyphenyl)ethylamine react to produce MPE-

substituted poly(Orn-alter-Gly) in one step without using a catalyst and then

poly(Orn-alter-Gly) is obtained by removing the N-(1-p-methoxyphenethyl)

(MPE) group. 1H NMR, Fourier transform infrared spectroscopy, and auto-

matic amino acid analysis confirm that ornithine and glycine are linked alter-

nately in the poly(Orn-alter-Gly) chains. Both MPE-substituted poly(Orn-alter-

Gly) and poly(Orn-alter-Gly) have excellent antibacterial activity against Staph-

ylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas

aeruginosa as well as excellent biocompatibility. The synthesis strategy and

materials provide new information on how to obtain ordered sequence cationic

polypeptides.

KEYWORD S

alternating copolymer, antibacterial activity, biocompatibility, cationic polypeptide, Ugi's 4CC

reaction

1 | INTRODUCTION

Cationic polypeptides prepared by chemical synthesis areimportant biomaterials for their antibacterial propertiesand biocompatibility.[1-6] They are generally composed ofcationic amino acids (ornithine, lysine, arginine, histi-dine) and other amino acids, and they are widely used ingene delivery,[7-12] drug delivery,[13-17] tissue engineering,scaffolds, and biomedical devices.[6,18-20] The biologicalactivity of cationic polypeptides depends on the structure

of the chains and segments.[6,19-22] However, the chainsand segments of cationic polypeptides synthesized by thetraditional NCA ring-opening polymerization or solid-phase process are complex and some unexpected biologicalactivities may result.[10,16,23,24] For example, the segmentsequence of the cationic block polypeptide copolymer con-sisting of lysine (K) and glycine (G) synthesized by NCAring-opening polymerization is unordered, for example,KKKKK, KKKKG, KKGGG, KGGGG, GGGGG, and soforth. Lysine and glycine are linked alternately in the

Received: 29 November 2020 Revised: 21 January 2021 Accepted: 21 January 2021

DOI: 10.1002/pen.25657

Polym Eng Sci. 2021;61:1405–1414. wileyonlinelibrary.com/journal/pen © 2021 Society of Plastics Engineers 1405

segments (KGKGKG) of the cationic alternating polypep-tide copolymer with lysine (K) and glycine (G). The cat-ionic alternating polypeptide copolymers have an orderedsequence and the structure of the chains and segmentsis similar. These alternating polypeptides are usuallysynthesized by stepwise polymerization with thecorresponding dipeptide (KG) as the raw materials. How-ever, there are limited applications due to the complexprocedures and high cost.[25] Therefore, a simpler andmore economical method is highly desirable for masssynthesis of cationic alternating polypeptide copolymerswith an ordered sequence.

Herein, a new technique based on Ugi's 4 componentcondensation (Ugi's 4CC) is described to synthesize the cat-ionic alternating polypeptide copolymer comprising orni-thine and glycine (poly(Orn-alter-Gly)) in large quantities.

To produce poly(Orn-alter-Gly) with the amino group onthe side chain, potassium isocyanate, 1-(4-methoxyphenyl)ethylamine, 4-(N-marbobenzyloxyamino)-1-butyraldehydeare the raw materials in polymerization (Figure 1(A)).Potassium isocyanate contains both isocyanate and carbox-ylic acid groups after acidification with trifluoromesulfonicacid and becomes glycine in the final copolymer.1-(4-methoxyphenyl)ethylamine contains the amino groupand becomes the side chain in the copolymer chains afterpolymerization. The N-(1-p-methoxyphenethyl) (MPE)group is attached to the amide nitrogen atoms on ornithineand removed finally. 4-(N-carbobenzyloxyamino)-1-butyraldehyde contains the aldehyde group and becomesthe side chain group of ornithine in the final copolymer,whereas the carbobenzyloxy group (CBZ) of 4-(N-carbobenzyloxyamino)-1-butyraldehyde is the protective

FIGURE 1 (A) Structure of the raw materials and corresponding cationic alternating copolymer composed of ornithine and glycine

based on Ugi's 4CC reaction; (B) reaction pathway

1406 LIU ET AL.

group of the amino group. After the aldehyde group in4-(N-carbobenzyloxyamino)-1-butyraldehyde is consumedby the amino group of 1-(4-methoxyphenyl)ethylamine,the amino group of 4-(N-carbobenzyloxyamino)-1-butyraldehyde is exposed. Since subsequent polymeriza-tion takes place in a strong acid, the CBZ group isremoved and the final polymer chains have the aminogroup on the terminal of the side chain. The cationicalternating polypeptide copolymer with ornithine andglycine arranged orderly is demonstrated to have goodantibacterial activity and biocompatibility.

2 | MATERIALS AND METHODS

2.1 | Materials

The main chemical reagents included ethyl iso-cyanoacetate (damas-beta), 4-amino-1-butanol (ShanghaiShaoyuan Co. Ltd.), benzyl chloroformate (Innochem),2.0 M oxalyl chloride in methylene chloride (Aladdin),N'N-dimethylformamide (Innochem) dried thoroughlywith molecular sieves and stored in re-sealable bottles,triethylamine (Macklin), 1-(4-methoxyphenyl)ethylamine(Accela), trifluoromethanesulfonic acid (damas-beta),methanesulfonic acid (damas-beta), and methyl thiazolyltetrazolium (MTT) (Solarbio). The four bacteria strains ofStaphylococcus aureus (S. aureus, ATCC 6538),Escherichia coli (E. coli, ATCC 25922), Klebsiellapneumoniae (K. pneumoniae, AS 1.1736), and Pseudomo-nas aeruginosa (P. aeruginosa, AS 1.2031) were obtainedfrom the American type culture collection and ChinaGeneral Microbiological Culture Collection Center. Themouse fibroblast cells (L929) were obtained from the rep-resentative culture preservation center of the UnitedStates.

2.2 | Measurements

The 1H NMR and 13C NMR spectra were recorded onthe Bruker (AVANCE III HD-400) and Bruker(AVANCE 600) spectrometers, the solvents includedD2O, CDCl3, and DMSO-d6 with tetramethylsilane or1, 4-dioxane as the internal standard. Fourier transforminfrared (FTIR) spectroscopy was carried out on theNicolet iS5 and glycine in the copolymer hydrolyzedfragments was measured by automatic amino acid anal-ysis (Hitachi High-Technologies Corporation, LA8080).The copolymers were hydrolyzed with hydrochloric acidat 110�C for 22 h and 440 nm and 570 nm light wasused to detect the amino acids when the degradationproducts were introduced into the sulfonic acid cationic

resin column. The relative molecular weight of thecopolymers was determined on the Agilent (1260 Infin-ity) instrument.

2.3 | Synthesis of poly(Orn-alter-Gly)

In the reaction, 1-(4-methoxyphenyl)ethylamine (0.33 g,2.69 mmol) was added to a solution of 4-(N-carbobenzyloxyamino)-1-butyraldehyde (0.6 g, 2.69 mmol)in chloroform (55 ml). The mixture was stirred at roomtemperature for 1 day and then the solvent was evaporatedat a low pressure to obtain the product (intermediate I).Trifluoromethanesulfonic acid (0.86 g, 5.38 mmol) wasintroduced to a solution of potassium isocyanate (0.33 g,2.69 mmol) in isopropyl alcohol (30 ml) at 0�C, then inter-mediate I was added to the solution. The mixture wasstirred for 6 days at room temperature, quenched withwater, and purified to obtain the MPE-substitutedpoly(Orn-alter-Gly).[25-28] MPE-substituted poly(Orn-alter-Gly) (0.3 g) was dissolved in methanesulfonic acid (5 g,3.4 ml) and stirred at 100�C for 16 h. The product was puri-fied to obtain the poly(Orn-alter-Gly).[25-28]

2.4 | The antibacterial test

In the antibacterial assessment, 4 ml of 0.03 mmol/L sterilephosphate buffered saline (PBS) solution, 0.5 ml of sterileaqueous solution containing different concentrations of thepolymer, and 0.5 ml of 0.03 mmol/L sterile PBS solutionwith 1.5 × 105 CFU bacteria were put in a centrifuge tube asthe experimental group. 4.5 ml of 0.03 mmol/L sterile PBSsolution and 0.5 ml of 0.03 mmol/L sterile PBS solution with1.5 × 105 CFU bacteria were put in a centrifuge tube as theblank group and three parallel samples were made for eachgroup. After centrifugation at a constant temperature at37�C for 24 h, the sample was taken out and diluted withnormal saline. 1 ml of the diluted solution and 15 ml of theculture medium were put on a petri dish, and the bacteriacolonies were counted after culturing for 24 h.

2.5 | The cytotoxicity test

In the cytotoxicity test, 100 μl of the culture medium with1 × 104 CFU mouse fibroblast cells were put on a 96-wellplate. They were cultured in a biochemical incubator under5% CO2 at 37�C. After 24 h, the culture medium wasremoved and 100 μl of the culture medium containing dif-ferent concentrations of the polymer were added. Six paral-lel samples were made for each group. After culturing for24 h, 50 μl of the MTT reagent was added to each well and

LIU ET AL. 1407

cultured for 2 h. The MTT reagent was removed and 100 μlof isopropanol were added to each well. The 96-well platewas agitated and the absorbance was determined at570 nm on the ELISA instrument.

2.6 | Statistical analysis

The experimental data of the antibacterial and cytotoxicitytests were analyzed statistically by the SPSS version 17.0(Chicago, IL) software. The difference (p) was calculated todetermine whether there were significant differencesbetween the copolymers in the antibacterial activity andcytotoxicity (p < 0.05 representing significant difference).

3 | RESULTS AND DISCUSSION

To prepare poly(Orn-alter-Gly), raw materials with thespecific structure were synthesized for polymerization.

Synthesis of potassium isocyanate and 4-(N-carbobenzyloxyamino)-1-butyraldehyde was described insupplementary materials S1 and 1-(4-methoxyphenyl)ethylamine was purchased. In the polymerization pro-cess, the ingredients were added as shown in Figure 1(B).4-(N-carbobenzyloxyamino)-1-butyraldehyde and 1-(4-methoxyphenyl)ethylamine react for 1 day to produceintermediate I with double-bond carbonic acid. After-ward, a solution of potassium isocyanate in isopropylalcohol is added with trifluoromethanesulfonic acid andintermediate I, and the one-pot reaction proceeds at roomtemperature. Trifluoromethanesulfonic acid reacts withpotassium isocyanate and the CBZ group of intermediateI is removed in this step prior to polymerization. Finally,the cationic alternating copolymer composed of ornithineand glycine with the MPE group (MPE-substitutedpoly(Orn-alter-Gly)) as the side chain is obtained. TheMPE group is attached to the amide nitrogen atom ofornithine in the copolymer. The MPE-substitutedpoly(Orn-alter-Gly) is exposed to methanesulfonic acid at

FIGURE 2 1H NMR spectra of (a) MPE-substituted poly(Orn-alter-Gly) and (B) poly(Orn-alter-Gly) with CDCl3 as the solvent; FTIR

spectra of (C) MPE-substituted poly(Orn-alter-Gly) and (D) poly(Orn-alter-Gly) at 1700–1200 cm−1

1408 LIU ET AL.

100�C for 16 h to obtain poly(Orn-alter-Gly). The synthe-sis is described in Figure 1(B).[25,26]

The 1H NMR spectra of MPE-substituted poly(Orn-alter-Gly) and poly(Orn-alter-Gly) are shown in Figure 2.There is no chemical shift of benzene-ring hydrogen ofthe CBZ group at 7.2 ppm as shown in Figure 2(A)suggesting that the CBZ group is removed completelyduring polymerization. There are no chemical shift peaksof aromatic hydrogen of the MPE group at 6.8 and7.4 ppm as shown in Figure 2(B) confirming that theMPE group is removed completely.

The FTIR spectra of MPE-substituted poly(Orn-alter-Gly) and poly(Orn-alter-Gly) are depicted in Figure 2.Figure 2(C) shows a wide absorption band of amide I at1619 cm−1 indicating that the MPE-substituted poly(Orn-alter-Gly) has a multiple secondary structure. The absorp-tion peaks of amide III at 1200–1350 cm−1 disclose thatthe copolymer has the β-sheet (1234 cm−1), random coil(1264 cm−1), and α-helix (1310 and 1333 cm−1) secondarystructures. Figure 2(D) shows a narrow absorption peakof amide I at 1633 cm−1 and small absorption peak ofamide III at 1220 cm−1 suggesting that the secondarystructure of poly(Orn-alter-Gly) is the β-sheet. The MPE-substituted poly(Orn-alter-Gly) and poly(Orn-alter-Gly)have different secondary structures maybe because theyhave different side chain groups. The molecular chains ofpoly(Orn-alter-Gly) can form the β-sheet structurebecause of hydrogen bond with the amino group of orni-thine in the poly(Orn-alter-Gly) chains. However, thenitrogen atom in the MPE-substituted poly(Orn-alter-Gly) is linked to a large quantity of the MPE group thusinterfering with hydrogen bond formation. Consequently,the MPE-substituted poly(Orn-alter-Gly) forms the ran-dom coil and α-helix structures.[29] It is noted that bothMPE-substituted poly(Orn-alter-Gly) and poly(Orn-alter-Gly) exhibit absorption peaks of amide I and amide III inthe FTIR spectra implying that they are polypeptides.[30]

Automatic amino acid analysis is conducted to mea-sure the amino acids in the degradation products of theMPE-substituted poly(Orn-alter-Gly) and poly(Orn-alter-Gly). As shown in the Figure 3, glycine is detected fromthe degradation products of both MPE-substituted

FIGURE 3 Voltage and time response of the degradation

products: (A) MPE-substituted poly(Orn-alter-Gly) and

(B) poly(Orn-alter-Gly) flowing in the sulfonic acid cationic resin

column as monitored by 440 nm light (gray line) and 570 nm light

(black line), respectively

FIGURE 4 Reaction pathway of a cycle of chain evolution in the polymerization process via the Ugi's 4CC reaction

LIU ET AL. 1409

poly(Orn-alter-Gly) and poly(Orn-alter-Gly) but ornithinecannot be detected by this method because of the lack ofthe ornithine standard. The raw materials react via theUgi's 4CC reaction without side reactions at room tem-perature, since only the amine group, aldehyde group,isocyanate group, and carboxyl group can participate inthe reaction. In addition, glycine is not added during thereaction. Analysis of the reaction mechanism and auto-matic amino acid analysis confirm that the cationic

alternating copolymer composed of ornithine and glycineis produced by the Ugi's 4CC reaction.

The chain evolution in the polymerization process bythe Ugi's 4CC reaction can be divided into four stages asshown in Figure 4.[26] The amino group of 1-(4-methoxyphenyl)ethylamine and aldehyde group of 4-(N-carbobenzyloxyamino)-1-butyraldehyde react to produceintermediate I with double-bond carbonic acid. The 1HNMR spectra of intermediate I in Figure 5 do not showany chemical shift of the aldehyde group at 9.8 ppm whilea big chemical shift of double-bond carbonic acid isobserved at 8.4 ppm showing that the aldehyde group isconsumed and intermediate I containing double-bond car-bonic acid is synthesized. In the second stage, nucleophilicaddition takes place between the isocyanate group of acid-ified potassium isocyanate and double-bond carbonic acidof intermediate I to produce intermediate II. In the thirdstage, the carboxylic acid group of acidified potassium iso-cyanate reacts with intermediate II to produce intermedi-ate III. Finally, intermediate III rearranges to formintermediate IV with the isocyanate group and carboxylacid group on the terminal. Intermediate IV continues totake part in nucleophilic addition with intermediate I andthe molecular chain becomes longer, finally the MPE-substituted poly(Orn-alter-Gly) is produced.

The relative molecular weights of the MPE-substituted poly(Orn-alter-Gly) and poly(Orn-alter-Gly) are determined by gel permeation chromatogra-phy using the 1.0 mg/ml polymer solutions in water at25�C and water as the mobile phase. Since there is nopolymer with a similar structure as the standard, poly-styrene is chosen to generate the corresponding stan-dard curve. Figure 6 shows the relative molecular

FIGURE 5 1H NMR spectrum of intermediate I with DMSO-

d6 as the solvent

FIGURE 6 Relationship between the RI response and time of

the MPE-substituted poly(Orn-alter-Gly) and corresponding

poly(Orn-alter-Gly) in gel permeation chromatography

TABLE 1 Polymerization reaction conditions in the Ugi's 4CC

reaction

Entry A:B:Ca TfOHb Timec

1 1:1:1 3 6

2 1:1:1 2 9

3 1:1:1 2 6

4 1:1:1 2 3

5 1d:1:1 2 3

6 1d:1:1 1 3

7 1d:1:1.5 1 3

a(A:B:C) represents the molar ratio of potassium isocyanate, 4-(N-Carbobenzyloxyamino)-1-butyraldehyde and 1-(4-Methoxyphenyl)ethylamine.bMolar ratio of trifluoromesulfonic acid and potassium isocyanate.cDays of polymerization.dPotassium isocyanate in isopropyl alcohol added to trifluoromesulfonic acidat room temperature for 1 day before intermediate I is added.

1410 LIU ET AL.

weights. The molecular weight of MPE-substitutedpoly(Orn-alter-Gly) is about seven times larger thanthat of poly(Orn-alter-Gly). This may be because thelong reaction strips the MPE group in the strong acid

at a high temperature thus breaking the molecularchains.

Since polymerization in the Ugi's 4CC reaction is cata-lyst free, the experimental conditions (Table 1) are changed

FIGURE 7 Effects of different reaction conditions on the polymerization yield and molecular weight of the MPE-substituted poly(Orn-

alter-Gly). Entries 1 and 3 are shown in (A), entries 2, 3, and 4 are shown in (B), entries 4 (marked “Normal”) and 5 (marked “advance”) areshown in (C), entries 5 and 6 are shown in (D), entries 6 and 7 are shown in (E). The molecular weights are determined using the 1.0 mg/ml

polymer solutions in DMSO at 25�C with the DMSO as the mobile phase

LIU ET AL. 1411

to explore the effects on the polymerization yield andmolecular weight of the MPE-substituted poly(Orn-alter-Gly). Potassium isocyanate, 4-(N-carbobenzyloxyamino)-1-butyraldehyde and 1-(4-methoxyphenyl)ethylamineare added in equal amounts but the quantity of

trifluoromesulfonic acid (TfOH) is different, becausetrifluoromesulfonic acid participates in acidificationof potassium isocyanate and removal of the CBZgroup. The polymerization yield and molecularweight of the MPE-substituted poly(Orn-alter-Gly)

TABLE 2 Minimal inhibitory concentration (MIC; ppm) values of MPE-substituted poly(Orn-alter-Gly) and poly(Orn-alter-Gly)

MIC (ppm) S. aureus E. coli K. pneumoniae P. aeruginosa

MPE-substituted poly(Orn-alter-Gly) 1000 1500 1000 1000

poly(Orn-alter-Gly) 1500 2500 2500 2000

FIGURE 8 Number of surviving bacteria on MPE-substituted poly(Orn-alter-Gly) and poly(Orn-alter-Gly): (A) S. aureus, (B) E. coli,

(C) K. pneumoniae, and (D) P. aeruginosa at concentrations of 100, 500, and 1000 ppm after culturing for 24 h (# in gray indicates that the

number of surviving bacteria is less than 20 CFU, *denoted 0.05 < p < 0.1, **denoted p < 0.05)

1412 LIU ET AL.

under different reaction conditions are shown inFigure 7.

Figure 7(A,B) show that over-acidification and a longreaction time adversely affect the evolution of the poly-mer chains and polymerization yield. Figure 7(C) showsthat when potassium isocyanate is acidified for 1 daybefore polymerization, polymerization proceeds fasterand the yield increases, but the molecular weight of thepolymer decreases. Figure 7(D) shows that the amount oftrifluoromesulfonic acid is not enough to satisfy polymer-ization and the yield drops greatly. Figure 7(E) revealsthat an excess amount of 1-(4-methoxyphenyl)ethylaminereduces the loss caused by instability of 4-(N-carbobenzyloxyamino)-1-butyraldehyde and improvesthe molecular weight of the polymer as well as polymeri-zation yield.

The molecular weights do not change significantlyunder different reaction conditions due to the MPE groupand CBZ group in the materials. The large-quantity groupnot only hinders attack of the isocyanate group, carboxylgroup, and double-bond carbonic acid to the molecularchains, but also restricts rearrangement of the molecularchains, thereby the continuous evolution of the molecu-lar chains is prevented.[26]

The antibacterial activity of the MPE-substitutedpoly(Orn-alter-Gly) and poly(Orn-alter-Gly) is evaluated.Although their molecular weights are not high, the MPE-substituted poly(Orn-alter-Gly) and poly(Orn-alter-Gly)exhibit low minimal inhibitory concentration (MIC) values(Table 2). Then we test the antibacterial activity of the syn-thesized cationic polymers cultured in bacterial solutionfor 24 h. Figure 8 shows that MPE-substituted poly(Orn-alter-Gly) has excellent antibacterial activity againstS. aureus, E. coli, K. pneumoniae, and P. aeruginosa with a99.99% antibacterial rate at a concentration of 100 ppm.The antibacterial activity increases with concentrationsand strong antibacterial activity against S. aureus andK. pneumoniae is demonstrated at 100 ppm. After remov-ing the MPE group, poly(Orn-alter-Gly) also shows goodantibacterial activity against these bacteria. The anti-bacterial activity increases with concentrations andpoly(Orn-alter-Gly) exhibits excellent antibacterial activityagainst S. aureus with a 97.61% antibacterial rate at a con-centration of 1000 ppm. After removing the MPE group ofMPE-substituted poly(Orn-alter-Gly), the antibacterialactivity of poly(Orn-alter-Gly) decreases because of thesmaller molecular weight. Figure 6 shows that the molecu-lar weight of MPE-substituted poly(Orn-alter-Gly) is aboutseven times greater than that of poly(Orn-alter-Gly). Poly-mers with different molecular weights have differentamounts of cationic charges on the molecular chains, andthe interaction between the anionic charge on the bacteriaand cationic charge of the molecular chains are different

giving rise to the different antibacterial activity.[31,32] All inall, the results demonstrate that both MPE-substitutedpoly(Orn-alter-Gly) and poly(Orn-alter-Gly) have excellentantibacterial activity.

To investigate the biocompatibility of MPE-substitutedpoly(Orn-alter-Gly) and poly(Orn-alter-Gly), the cytotoxic-ity to mouse fibroblast cells (L929) is assessed for differentconcentrations. The relative proliferation rates of cellsincubated with 1000 ppm of MPE-substituted poly(Orn-alter-Gly) and poly(Orn-alter-Gly) are 121.17% and117.00%, respectively (Figure 9). The results demonstratethat MPE-substituted poly(Orn-alter-Gly) and poly(Orn-alter-Gly) have good biocompatibility at high concentra-tions and promote cell reproduction consequently bodingwell for biomedical applications.

4 | CONCLUSION

A new technique to synthesize the cationic alternatingpolypeptide copolymer consisting of ornithine and gly-cine with an ordered sequence in large quantities basedon the Ugi's 4CC reaction is described. Ornithine and gly-cine are linked alternately in the poly(Orn-alter-Gly)chains and the yield of polymerization is 64.3%. Changesin the polymerization conditions do not alter the molecu-lar weights of MPE-substituted poly(Orn-alter-Gly) signif-icantly due to the large amounts of MPE and CBZ groupsin the materials. MPE-substituted poly(Orn-alter-Gly)exhibits excellent antibacterial activity against S. aureus,

FIGURE 9 Quantities of surviving L929 mouse fibroblast cells

after incubation with MPE-substituted poly(Orn-alter-Gly) and

poly(Orn-alter-Gly) at concentrations of 100, 500, and 1000 ppm for

24 h (*denoted 0.05 < p < 0.1, **denoted p < 0.05)

LIU ET AL. 1413

E. coli, K. pneumoniae, and P. aeruginosa with a 99.99%antibacterial rate at a concentration of 100 ppm.Poly(Orn-alter-Gly) also has good antibacterial activityagainst S. aureus with a 97.61% antibacterial rate at1000 ppm. The cytotoxicity test shows that both MPE-substituted poly(Orn-alter-Gly) and poly(Orn-alter-Gly)have good biocompatibility. The novel synthesis strategyand materials suggest large potential of cationic polypep-tide alternating copolymers in biology and medicine.

ORCIDWei Zhang https://orcid.org/0000-0003-3958-1423

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SUPPORTING INFORMATIONAdditional supporting information may be found onlinein the Supporting Information section at the end of thisarticle.

How to cite this article: Liu F, Ma N, Liu J, et al.A cationic alternating copolymer composed ofornithine and glycine with an ordered sequence forenhanced bacterial activity. Polym Eng Sci. 2021;61:1405–1414. https://doi.org/10.1002/pen.25657

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